WO2014172705A1 - Méthodes et compositions pour réguler la thrombocytopoïèse - Google Patents

Méthodes et compositions pour réguler la thrombocytopoïèse Download PDF

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WO2014172705A1
WO2014172705A1 PCT/US2014/034801 US2014034801W WO2014172705A1 WO 2014172705 A1 WO2014172705 A1 WO 2014172705A1 US 2014034801 W US2014034801 W US 2014034801W WO 2014172705 A1 WO2014172705 A1 WO 2014172705A1
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lpar
pathway
thrombocytes
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activity
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Hsinyu Lee
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Hsinyu Lee
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    • 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
    • A61K31/66—Phosphorus compounds
    • A61K31/683—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols
    • A61K31/685—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols one of the hydroxy compounds having nitrogen atoms, e.g. phosphatidylserine, lecithin
    • 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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42—Oxazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid

Definitions

  • the present invention relates to methods and compositions for regulating thrombocytopoiesis.
  • the present invention relates to methods and compositions for regulating thrombocytopoiesis and/or megakaryopoiesis with a compound modulating the activity of lysophosphatidic acid receptor subtype 3 (LPAR 3 ) pathway.
  • LPAR 3 lysophosphatidic acid receptor subtype 3
  • Lysophosphatidic acid is a small glycero-phospholipid which derived from cell membrane phospholipid by autotoxin catalyzation.
  • LPA is known as an important regulator of numerous physiological processes through binding to various trans-membrane LPA receptors (LPARs). So far, there are six LPARs that had been identified. By binding to different receptors, LPA exerts various physiological and pathological functions.
  • the expression of LPA receptors is found in multi-potent stem cells in mouse and human, such as hematopoietic stem cells, which can differentiate into all blood lineages. It was previously reported that the embryonic stem cells (ESCs) proliferation and differentiation were induced by LPA, which influenced the c-Myc expression and Ca 2+ signaling.
  • ESCs embryonic stem cells
  • thrombocytopoiesis/megakaryopoiesis process of hematopoietic stem cells can be regulated through the inhibition or enhancement of the activity of LPAR 3 pathway.
  • thrombocytes an abnormal level of platelets
  • a method for regulating thrombocytopoiesis in a subject comprising administering to the subject a therapeutically effective amount of a compound modulating the activity of LPA receptor subtype 3 (LPAR 3 ) pathway.
  • LPAR 3 LPA receptor subtype 3
  • a pharmaceutical composition for regulating thrombocytopoiesis comprising a therapeutically effective amount of a compound modulating the activity of LPAR 3 pathway in association with a pharmaceutically acceptable carrier.
  • a compound modulating the activity of LPAR 3 pathway for manufacturing a medicament for regulating thrombocytopoiesis.
  • a pharmaceutical composition for regulating megakaryopoiesis comprising a therapeutically effective amount of a compound modulating the activity of LPAR 3 pathway in association with a pharmaceutically acceptable carrier.
  • a pharmaceutical composition for regulating megakaryopoiesis comprising a therapeutically effective amount of a compound modulating the activity of LPAR 3 pathway in association with a pharmaceutically acceptable carrier.
  • thrombocytes in a subject comprising administering to the subject a therapeutically effective amount of a compound modulating the activity of LPAR 3 pathway.
  • thrombocytes comprising a therapeutically effective amount of a compound modulating the activity of LPAR 3 pathway in association with a pharmaceutically acceptable carrier. And, it is to provide the use of a compound modulating the activity of LPAR 3 pathway for manufacturing a medicament for treating or preventing a disorder or a condition associated with an abnormal level of thrombocytes.
  • a method for treating or preventing a disorder or a condition associated with an abnormally low level of thrombocytes in a subject comprising administering to the subject a therapeutically effective amount of a compound inhibiting the activity of LPAR 3 pathway.
  • a pharmaceutical composition for treating or preventing a disorder or a condition associated with an abnormally low level of thrombocytes comprising a therapeutically effective amount of a compound inhibiting the activity of LPAR 3 pathway in association with a pharmaceutically acceptable carrier.
  • a method for treating or preventing a disorder or a condition associated with an abnormally high level of thrombocytes in a subject comprising administering to the subject a therapeutically effective amount of a compound enhancing the activity of LPAR 3 pathway.
  • a pharmaceutical composition for treating or preventing a disorder or a condition associated with an abnormally high level of thrombocytes comprising a therapeutically effective amount of a compound enhancing the activity of LPAR 3 pathway in association with a pharmaceutically acceptable carrier.
  • the invention provides a method for inducing the proliferation and/or differentiation to megakaryocytes/thrombocytes from
  • hematopoietic stem cells comprising contacting hematopoietic stem cells with an effective amount of a compound inhibiting the activity of LPAR 3 pathway.
  • a pharmaceutical composition for inducing the proliferation and/or differentiation to thrombocytes from hematopoietic stem cells comprising an effective amount of a compound inhibiting the activity of LPAR 3 pathway in association with a
  • the invention provides a method for reducing the proliferation and/or differentiation to megakaryocytes/thrombocytes from hematopoietic stem cells comprising contacting hematopoietic stem cells with an effective amount of a compound enhancing the activity of LPAR 3 pathway. Then, it is also to provide a method for reducing the proliferation and/or differentiation to thrombocytes from hematopoietic stem cells in a subject comprising administering the subject with a therapeutically effective amount of a compound enhancing the activity of LPAR 3 pathway. On the other hand, it is to provide a pharmaceutical composition for inhibiting the proliferation and/or differentiation to megakaryocytes from
  • hematopoietic stem cells comprising an effective amount of a compound enhancing the activity of LPAR 3 in association with a pharmaceutically acceptable carrier.
  • the compound modulating the activity of LPAR 3 pathway is selected from the group consisting of LPAR 3 agonists and antagonists, and other agents which are sufficiently structurally similar to LPA to elicit or suppress the LPAR 3 activity.
  • the compound modulating the activity of LPAR 3 pathway is selected from the group consisting of lysophosphatidyl choline (LPC),
  • sphingosylphosphorylcholine l-oleoyl-2-O-methyl-rac-glycerophosphothionate (OMPT), (3S)-l-fluoro-3-hydroxy-4-butyl-l-phosphonate (XY-17), T13, T10, VPC12086, VPC31143, mono-fluorinated analogs of LPA, 3-[( ⁇ 4-[4-( ⁇ [l-(2-chlono- 4-methylphenyl)ethoxy]carbonyl ⁇ amino)-3-methyl-l ,2-oxazol-5- yl]phenyl ⁇ methyl)sulfanyl]propanoic acid (Kil6425), diacylglycerol pyrophosphate (DGPP), VPC12249, dioctyl-PA, 1-0-acyl-a-fluoromethylenephosphonate, and a- hydroxymethylenephosphonate LPA, analogues thereof, and pharmaceutically acceptable salts thereof.
  • DGPP
  • Figure 1A provides the quantification of CD41gene expression by qPCR in LPAR 3 MO morphant injected zebrafish embryos;
  • Figure IB shows that CD41 expression was enhanced by Ki 16425 -treatment;
  • Figure 1C shows that CD41 expression was decreased by OMPT-treatment; and
  • Figure ID shows that there is no effect on CD41 expression in LPARi knockdown morphant injected zebrafish embryos.
  • Figures 2A-2C show that LP A receptors profiling during TPO/SCF induction period of human stem cells; wherein that the mRNA expression of LPARi had no significant change under cytokine induction (Figure 2A), and on the other hand, LPAR 2 increased during MK differentiation ( Figure 2B) while LPAR 3 mRNA expression was decreased ( Figure 2C).
  • Figure 3 shows the effect of MDP on MK differentiation which provides the quantitative data of flow cytometry results showing that MDP, a LPAR 2 agonist, had no significant effects on megakaryopoiesis.
  • Figure 4 shows the effects of OMPT on MK differentiation, which provides the quantitative data of flow cytometry results showing that OMPT, a LPAR 3 agonist, inhibited MK.
  • Figure 5 shows that the number of GFP positive cells in CD41-GFP embryos was increased in LPAR 3 morphant injected zebrafish embryos.
  • an antisense zLPAR 3 -morpholino oligonucleotide was used to knock down LPAR 3 in zebrafish as a model to observe
  • LPAR 3 is an important regulator for thrombocytopoiesis/megakaryo-poiesis process of hematopoietic stem cells.
  • a LPAR 3 antagonist induces thrombocytopoiesis/megakaryopoiesis, but a LPAR 3 agonist inhibits
  • the LP AR 3 -mediated signaling pathway influences the thrombocytopoiesis/ megakaryopoiesis process, suggesting that modulating the activity and/or quantity of the members present in the LPAR 3 -mediated signaling pathway in a subject that possesses the signaling pathway would lead to a change in thrombocytopoiesis/ megakaryopoiesis in the subject.
  • the members in the LP AR 3 -mediated signaling pathway encompass bio-molecules that involve in the biosynthesis and biodegradation of ligands of LPAR 3 and the ligands thus synthesized, such as LPA and the enzymes and intermediates for LPA synthesis or degradation, or in the signal transduction cascades of LPAR 3 , such as LPAR 3 itself and its downstream effectors.
  • the members include those described in Hama K. and Aoki J., LPAR 3 , a unique G protein-coupled receptor for lysophosphatidic acid. Progress in Lipid Research 49:335-342 (2010); and Aoki J., Inoue A. and Okudaira S., Two pathways for lysophosphatidic acid production.
  • the object of regulating thrombocytopoiesis/ megakaryopoiesis in a subject can be achieved by bringing a member of the LPAR 3 signaling pathway in contact with a compound modulating the activity of said member involved in the LPAR 3 pathway, e.g. an inhibitor or a stimulator of LPAR 3 pathway.
  • the invention provides a novel method for regulating thrombocytopoiesis/megakaryopoiesis in a subject.
  • the method comprises administering to the subject a therapeutically effective amount of a compound modulating the activity of LPAR 3 pathway.
  • the present invention provides a method for regulating thrombocytopoiesis/megakaryopoiesis in a subject, comprising contacting a member of the LPAR 3 pathway with an inhibitor or a stimulator of said member.
  • a pharmaceutical composition for regulating thrombocytopoiesis/megakaryopoiesis comprising a therapeutically effective amount of a compound modulating the activity of LPAR 3 pathway in association with a pharmaceutically acceptable carrier; and the use of a compound modulating the activity of LPAR 3 pathway for manufacturing a medicament for regulating thrombocytopoiesis/megakaryopoiesis.
  • thrombocytes refers to cells lacking major organelles that play a key role in blood clotting, aggregating with thrombin; in particular, mammalian thrombocytes are anucleate cells, also called platelets.
  • thrombocytopoiesis refers to a process of proliferation and differentiation of hematopoietic stem cells (HSCs) and/or hematopoietic progenitor cells, leading to the production of thrombocytes.
  • HSCs hematopoietic stem cells
  • regulating thrombocytopoiesis refers to the action of enhancing, stimulating or inhibiting the thrombocytopoiesis process. More specifically, the action of "regulating thrombocytopoiesis” means altering the quantity of hematopoietic progenitor cells to be proliferated and differentiated into
  • thrombocytes with a composition or by a method, so that the quantity of functional thrombocytes in blood is restored to a normal level in a healthy subject required for normal hemostasis.
  • megakaryocytes refers to bone marrow cells responsible for the production of thrombocytes.
  • the level of megakaryocytes is related to the production of thrombocytes.
  • the term “megakaryocytopoiesis” as used herein refers to a process by which bone marrow progenitor cells develop into mature megakaryocytes, which in turn produce thrombocytes required for normal hemostasis.
  • regulating megakaryocytopoiesis refers to the action of enhancing, stimulating or inhibiting the megakaryocytopoiesis process.
  • the action of "regulating megakaryocytopoiesis” means altering the quantity of bone marrow progenitor cells to be proliferated and differentiated into megakaryocytes with a composition or by a method, so that the quantity of functional megakaryocytes is restored to a normal level in a healthy subject required for normal hemostasis.
  • lysophosphatidic acid refers to the compound having an IUPAC name of (2-hydroxy-3- phosphonooxypropyl) (Z)-octadec-9-enoate, which is known to be capable of binding to and activating G-protein-coupled receptors including LPARi, LPAR 2 and LPAR 3 (also known as EDG2, EDG4, and EDG7), and LPAR 4 (p2y9/GPR23) and LPAR 5 (GPR92).
  • LPARi 2, LPAR 2 and LPAR 3
  • EDG2, EDG4, and EDG7 also known as EDG2, EDG4, and EDG7
  • LPAR 4 p2y9/GPR23
  • LPAR 5 GPR92
  • LPA receptor subtype 3 refers to a cellular receptor which was found to interact with LPA and other lysophospholipids to manifest physiological or pathophysiological effects of LPA (Bandoh et al., Molecular cloning and characterization of a novel human G-protein- coupled receptor, EDG7, for lysophosphatidic acid. J. Biol. Chem. 274 (39): 27776- 85, September, 1999).
  • Human LPAR 3 has a nucleotide sequence deposited in the GenBank database with the Accession No. NM-012152.
  • a compound modulating the activity of LPAR 3 pathway refers to a compound capable of altering the activity of a member of the LPAR 3 signaling pathway to consequently result in enhancement, stimulation or inhibition of thrombocytopoiesis/megakaryocytopoiesis process.
  • a compound modulating the activity of LPAR 3 pathway may be an inhibitor or a stimulator of LPAR 3 pathway.
  • the compound is one capable of mediating the receptor activity of LPAR 3 .
  • a compound capable of mediating the activity of LPAR 3 and "LP AR 3 -mediating compound” are used interchangeably herein to refer to a compound that binds to LPAR 3 to induce a detectable increase or decrease in receptor activity in vivo and in vitro (for example, at least a 10% increase or decrease in receptor activity).
  • the compounds include, but are not limited to LPAR 3 agonists and antagonists, and other agents, which, either known in the art or to be developed, are sufficiently structurally similar to LPA to elicit or suppress the biological pathways mediated by LPAR 3 .
  • Some of the LPAR 3 agonists/antagonists that may be useful for the purposes of the invention are disclosed in, for example, US7,169,818;
  • LPAR 3 agonists include, but are not limited to lysophosphatidyl choline (LPC), sphingosylphosphoryl- choline, l-oleoyl-2-O-methyl-rac-glycerophosphothionate (OMPT), (3S)-l-fluoro-3- hydroxy-4-butyl-l-phosphonate (XY-17), T13 and T10 (see, for example, Tamaruya Y. et al., Angew. Chem. (Int. Ed. Engl.) 43: 2834 (2004)), VPC12086 and VPC31143 (see, for example, Heise C.E. et al., Mol. Pharmacol.
  • LPAR 3 antagonists include, but are not limited to, 3-[( ⁇ 4-[4-( ⁇ [l-(2- chloro-4-methylphenyl)ethoxy]carbonyl ⁇ amino)-3-methyl- 1 ,2-oxazol-5- yl]phenyl ⁇ methyl)sulfanyl]propanoic acid (Kil6425), diacylglycerol pyrophosphate (DGPP), VPC12249 (see Heise C.E.
  • LPAR 3 agonists/antagonists as well as their chemical structures, are generally described in, for example, Kano K., et al., LPA and its analogs- attractive tools for elucidation of LPA biology and drug development. Curr. Med. Chem. 15: 2122-2131 (2008); Noguchi K., et al., Lysophosphate acid (LPA) and its receptors. Curr. Opin. Pharmacol. 9: 15-23 (2009); and Hama K. and Aoki J., Progress in Lipid Research 49:335-342 (2010).
  • the LP AR 3 -mediating compound used is selective for LPAR 3 , i.e., exhibits higher binding affinity and specificity for LPAR 3 than for the other LPA receptor subtypes.
  • the compounds selective for LPAR 3 include LPA derivatives having a phosphonate or thiophosphate group in place of the phosphate group, such as OMPT, 1-O-acyl-a-fluoromethylenephos- phonate, a-hydroxymethylenephosphonate LPA analogues, and XY-17, T13 and T10.
  • salts with inorganic bases such as sodium, potassium, magnesium and calcium and ammonium salts
  • salts with organic bases such as lysine, N,N- dibenzylethylenediamine and angelic acid.
  • organic bases such as lysine, N,N- dibenzylethylenediamine and angelic acid.
  • Many of the compounds are commercially available from, for example, Avanti Polar Lipids Inc. of Alabaster, AL.
  • Autotoxin is responsible for the conversion of lysophospholipids to LPA by functioning as a lysophospholipase D.
  • the compound capable of modulating the enzymatic activity of ATX is employed, it is preferably a compound that exhibits an ATX inhibitory activity, such as ccPA, L-histidine, VPC8a202, Darmstoff analogs and thiophosphoric acid O-octadec-9-enyl ester, or an ATX enhancing activity.
  • ATX inhibitory activity such as ccPA, L-histidine, VPC8a202, Darmstoff analogs and thiophosphoric acid O-octadec-9-enyl ester, or an ATX enhancing activity.
  • the compound modulating the nuclear translocation of ⁇ - catenin can be employed in the invention.
  • One example is a compound that suppresses the translocation of ⁇ -catenin, such as quercetin, epigallocatechin-3-gallate (EGCG), curcumin or resveratrol, or that elicits the translocation.
  • EGCG epigallocatechin-3-gallate
  • curcumin curcumin
  • resveratrol resveratrol
  • the term "subject” refers to any organism having thrombocytes in blood required for normal hemostasis.
  • the term “subject” encompasses human or non-human mammal or animal.
  • Non-human mammals include livestock animals, companion animals, laboratory animals, and non-human primates.
  • Non-human subjects also include, without limitation, horses, cows, pigs, goats, dogs, cats, mice, rats, guinea pigs, gerbils, hamsters, mink, rabbits and fish. It is understood that the preferred subject is a human.
  • the term "subject” refers to a biological sample as defined herein, which includes but is not limited to a cell, tissue, or organ that is isolated from an organism having thrombocytes required for normal hemostasis. Accordingly, the methods, uses and compositions disclosed herein are intended to be applied in vivo as well as in vitro.
  • the invention provides a method for treating or preventing a disorder or a condition associated with an abnormal level of
  • the invention provides the use of a compound modulating the activity of LPAR 3 pathway for the manufacture of a medicament for treating or preventing a disorder or a condition associated with an abnormal level of thrombocytes in a subject.
  • the invention provides a pharmaceutical composition for treating or preventing a disorder or a condition associated with an abnormal level of
  • thrombocytes in a subject comprising a compound modulating the activity of LPAR 3 pathway.
  • the compound includes those defined above.
  • the invention provides a method for treating or preventing a disorder or a condition associated with an abnormally low level of thrombocytes in a subject, comprising administering to the subject a therapeutically effective amount of a compound inhibiting the activity of LPAR 3 pathway.
  • the invention provides a method for treating or preventing a disorder or a condition associated with an abnormally high level of thrombocytes in a subject, comprising administering to the subject a therapeutically effective amount of a compound enhancing the activity of LPAR 3 pathway.
  • the term “administering to a subject” includes dispensing, delivering or applying a compound modulating the activity of LPAR 3 pathway in a pharmaceutical formulation to a subject by any suitable route for delivery of the compound to the desired location in the subject to contact the compound with a target member of the LPAR 3 pathway.
  • the term “preventing” relates to the reduction of the risk of developing a prevailing disease
  • the term “treating” relates to the amelioration of the symptoms of a prevailing disease condition, deceleration of the course of disease.
  • a disorder or a condition associated with an abnormal level of thrombocytes in a subject used in the context of the invention includes those associated with a decrease or an increase in number or function of thrombocytes in the blood relative to normal levels, such as thrombocytopenia and thrombopenia.
  • the disorder or condition associated with an abnormal level of thrombocytes is called a thrombocytopathy, which could be either an abnormally low level of thrombocytes such as thrombocytopenia, a decrease in function of thrombocytes such as thrombasthenia, or an abnormally high level of thrombocytes such as thrombocytosis.
  • the disorder or condition associated with an abnormally low level of thrombocytes may be thrombocytopenia or thrombopenia, which refers to a relative decrease of thrombocytes in blood, and is defined by a thrombocytes count below 50,000 per microlitre.
  • Examples of the disorder or condition associated with an abnormally low level of thrombocytes also include, but are not United to, heparin- induced thrombocytopenia (HIT) or thrombotic thrombocytopenic purpura (TTP) that typically cause thromboses, or clots, instead of bleeding.
  • HIT heparin- induced thrombocytopenia
  • TTP thrombotic thrombocytopenic purpura
  • LPAR 3 is an important regulator for thrombocytopoiesis/megakaryocytes process of hematopoietic stem cells. Therefore, the invention provides a method for inducing the proliferation and/or differentiation to megakaryocytes/thrombocytes from hematopoietic stem cells comprising contacting hematopoietic stem cells with an effective amount of a compound inhibiting the activity of LPAR 3 pathway.
  • a method for reducing the proliferation and/or differentiation to megakaryocytes/thrombocytes from hematopoietic stem cells comprising contacting hematopoietic stem cells with an effective amount of a compound enhancing the activity of LPAR 3 pathway.
  • the invention provides a method for reducing the proliferation and/or differentiation to thrombocytes from hematopoietic stem cells in a subject comprising administering the subject with a therapeutically effective amount of a compound enhancing the activity of LPAR 3 pathway.
  • a pharmaceutical composition for reducing/inducing the proliferation and/or differentiation to thrombocytes/megakaryocytes from hematopoietic stem cells comprising an effective amount of a compound enhancing/inhibiting the activity of LPAR 3 pathway in association with a pharmaceutically acceptable carrier.
  • the compound modulating the activity of LPAR 3 pathway may be formulated with an agent for enhancing/reducing the number or function of thrombocytes/megakaryocytes.
  • an effective dose of the compound modulating the activity of LPAR 3 pathway is used, in addition to a physiologically acceptable carrier, diluent, adjuvant and/or excipient for producing a pharmaceutical composition.
  • the compound may conveniently be formulated in unit dosage form by conventional pharmaceutical techniques. Such techniques include the step of bringing into association the compound and the physiologically acceptable carrier, diluent, adjuvant and/or excipient.
  • the formulations are prepared by uniformly and intimately bringing into association the compound with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • the dose thereof can vary depending on the route of administration, the age and weight of the subject to be treated, the nature and severity of the diseases to be treated and similar factors.
  • the dose, when administered to a subject, such as cells or a human subject, is sufficient to produce a selected effect.
  • the amount of the LP AR 3 -mediating compound to be administered is an amount that leads to a physiologically meaningful increase or decrease in the receptor activity of LPAR 3 .
  • the daily dose ranges between 0.1-500 mg/kg body weight, preferably 0.1-100 mg/kg body weight, more preferably 0.1-10 mg/kg body weight, and most preferably 0.1-1.0 mg/kg body weight, which can be formulated as a single dose to be administered once per day, or be subdivided into two or more daily doses.
  • Suitable administration forms include, but are not limited to, topical, oral, rectal, intraperitoneal or parenteral (e.g., intravenous, subcutaneous or intramuscular) preparations. Preferred are oral and parenteral preparations.
  • the customary galenic preparation forms such as tablets, sugar-coated tablets, capsules, dispersible powders, granulates, aqueous solutions, alcohol-containing aqueous solutions, aqueous or oily suspensions, syrups, juices or drops, may also be used.
  • Solid medicinal forms can comprise inert components and carrier substances, such as calcium carbonate, calcium phosphate, sodium phosphate, lactose, starch, mannitol, alginates, gelatine, guar gum, magnesium stearate, aluminium stearate, methyl cellulose, talc, highly dispersed silicic acids, silicone oil, higher molecular weight fatty acids, (such as stearic acid), gelatine, agar or vegetable or animal fats and oils, or solid high molecular weight polymers (such as polyethylene glycol); preparations which are suitable for oral administration can comprise additional flavourings and/or sweetening agents, if desired.
  • carrier substances such as calcium carbonate, calcium phosphate, sodium phosphate, lactose, starch, mannitol, alginates, gelatine, guar gum, magnesium stearate, aluminium stearate, methyl cellulose, talc, highly dispersed silicic acids, silicone oil, higher molecular weight
  • Liquid medicinal forms can be sterilized and/or, where appropriate, comprise auxiliary substances, such as preservatives, stabilizers, wetting agents, penetrating agents, emulsifiers, spreading agents, solubilizers, salts, sugars or sugar alcohols for regulating the osmotic pressure or for buffering, and/or viscosity regulators.
  • auxiliary substances such as preservatives, stabilizers, wetting agents, penetrating agents, emulsifiers, spreading agents, solubilizers, salts, sugars or sugar alcohols for regulating the osmotic pressure or for buffering, and/or viscosity regulators.
  • additives examples include tartrate and citrate buffers, ethanol and sequestering agents (such as ethylenediaminetetraacetic acid and its non-toxic salts).
  • High molecular weight polymers such as liquid polyethylene oxides, microcrystalline celluloses, carboxymethyl celluloses, polyvinylpyrrolidones, dextrans or gelatine, are suitable for regulating the viscosity.
  • solid carrier substances examples include starch, lactose, mannitol, methyl cellulose, talc, highly dispersed silicic acids, high molecular weight fatty acids (such as stearic acid), gelatine, agar, calcium phosphate, magnesium stearate, animal and vegetable fats, and solid high molecular weight polymers, such as polyethylene glycol.
  • Preparations for parenteral administration can be present in separate dose unit forms, such as ampoules or vials. Use is preferably made of solutions of the active compound, preferably aqueous solution and, in particular, isotonic solutions and also suspensions. These injection forms can be made available as ready-to-use preparations or only be prepared directly before use, by mixing the active compound, for example the lyophilisate, where appropriate containing other solid carrier substances, with the desired solvent or suspending agent.
  • the compound modulating the activity of LPAR 3 pathway such as a LP AR 3 -mediating compound, may be administered as a combination therapy with further active agents, useful in the treatment of
  • thrombocytopenia and associated disorders and conditions thrombocytopenia and associated disorders and conditions.
  • the suitable doses of these active agents are known by those skilled in the art.
  • the active ingredients may be formulated as compositions containing several active ingredients in a single dose form and/or as kits containing individual active ingredients in separate dose forms.
  • the active ingredients used in combination therapy may be co-administered or administered separately.
  • Embryos were collected from natural spawning, washed, and incubated in 0.3x Danieau's buffer at 28°C until being used. Each stage of fish embryos was determined from their morphology. Embryos at 12 hour post- fertilization (hpf) were treated with 0.2 mM l-phenyl-2-thiourea (PTU) (Sigma, St. Louis, MO, USA) in 0.3x Danieau's buffer to suppress melanization. Dechorionation of embryos was carried out with 0.01 g/ml protease (Sigma).
  • Kil6425 (Cayman Chemical, Ann Arbor, MI, USA) powder was diluted in dimethyl sulfoxide (DMSO) at a concentration of 40 mM as the stock. HSCs were treated with Kil6425 at a final concentration of 20 ⁇ .
  • DMSO dimethyl sulfoxide
  • RNA extraction was isolated using Trizol Reagent (Invitrogen, Carlsbad, CA, USA).
  • Complementary (c) DNA was synthesized from 1 ⁇ g total RNA with a reverse-transcription polymerase chain reaction (RT-PCR) using CD41, LPARi, LPAR 2 and LPAR 3 primers.
  • RT-PCR reverse-transcription polymerase chain reaction
  • a real-time PCR with the mixture reagent SYBR-Green I (Thermo Scientific, San Diego, CA, USA) was carried out on an iCycler iQ real-time detection system (Bio-Rad, Hercules, CA, USA). The specificity of the primers was confirmed from a single peak of the melting curve. Each target mRNA level was evaluated from the real-time threshold cycle and compared to the GAPDH amount as an internal control.
  • HSC cultures were collected, washed, and stained either with anti-CD41a- PE and anti-CD61-FITC for 30 minutes (BD Biosciences Pharmingen, San Diego, CA, USA). Stained cells were washed and resuspended in PBS. Non-stained and single-stained samples were prepared for fluorescent compensation. For all experiments, analysis of cells used a FACSCalibur instrument (BD Biosciences) and FCS Express software (De Novo, Los Angeles, CA, USA).
  • Tg(CD41 :GFP) zebrafish embryos were injected with either zlpa 3 morpholinos or control morpholinos.
  • Thrombocyte of 96 hpf embryos were examined by a DMIL epifluorescent microscope (Leica, Wetzlar, Germany). The images were acquired using the EOS Utility program (images not shown) and the amount of fluorescent thrombocytes were quantified and analyzed.
  • Example 2 LPA Receptors Profiling
  • LPA receptors profiling during TPO/SCF induction period was investigated.
  • Figure 2A the mRNA expression of LPARi had no significant change under cytokine induction
  • Figures 2B and 2C LPAR 2 increased during MK differentiation
  • Figure 2C LPAR 3 mRNA expression was decreased
  • Example 5 Expression of GFP in the CD41-GFP Embryos was Increased in zlpa3 Morphant
  • CD41-GFP expression in transgenic embryos is presented in the tail (images not shown). At 4 dpf, the numbers of CD41-GFP cells are increased in zebrafish after zlpa 3 tMOl injection compared to the control (see Figure 5).

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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne une méthode ou une composition pharmaceutique pour réguler la thrombocytopoïèse/les mégacaryocytes chez un sujet, laquelle méthode consiste à administrer au sujet une quantité thérapeutiquement efficace d'un composé modulant l'activité de la voie de passage (LPAR3) de sous-type 3 du récepteur LPA.
PCT/US2014/034801 2013-04-19 2014-04-21 Méthodes et compositions pour réguler la thrombocytopoïèse WO2014172705A1 (fr)

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
WO2021107125A1 (fr) 2019-11-29 2021-06-03 小野薬品工業株式会社 Composé ayant une activité agoniste du récepteur de l'acide lysophosphatidique et utilisation pharmaceutique dudit composé

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120270780A1 (en) * 2011-03-14 2012-10-25 Hsinyu Lee Method and composition for modulating erythropoiesis

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
US20120270780A1 (en) * 2011-03-14 2012-10-25 Hsinyu Lee Method and composition for modulating erythropoiesis

Non-Patent Citations (1)

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Title
LI, MW ET AL.: "Study of the roles of LPA3 on erythropoiesis and thromobocytopoiesis process in zebrafish", THE FASEB JOURNAL, 1 April 2013 (2013-04-01) *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021107125A1 (fr) 2019-11-29 2021-06-03 小野薬品工業株式会社 Composé ayant une activité agoniste du récepteur de l'acide lysophosphatidique et utilisation pharmaceutique dudit composé
EP4066895A1 (fr) 2019-11-29 2022-10-05 ONO Pharmaceutical Co., Ltd. Composé ayant une activité agoniste du récepteur de l'acide lysophosphatidique et utilisation pharmaceutique dudit composé

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