WO2004103406A1

WO2004103406A1 - Therapeutic composition for disease accompanied by decrease or increase of platelet

Info

Publication number: WO2004103406A1
Application number: PCT/JP2004/007566
Authority: WO
Inventors: Yuka Nagata; Kazuo Todokoro
Original assignee: Japan Science And Technology Agency; The Institute Of Physical And Chemical Research (Riken); Mochida Pharmaceutical Co., Ltd.
Priority date: 2003-05-26
Filing date: 2004-05-26
Publication date: 2004-12-02

Abstract

A therapeutic composition for diseases accompanied by decrease or increase of platelets, which is capable of selective decrease or increase of platelets, having less influence on other hematocyte components and which does not involve any of extracorporeal circulation, whole blood transfusion and blood component transfusion, ensuring less side effects. In particular, a therapeutic composition for diseases accompanied by decrease of platelets or diseases accompanied by increase of platelets, comprising a substance exhibiting 3β-hydroxydehydrogenase activating activity or inhibiting activity.

Description

Description: COMPOSITION FOR THE TREATMENT OF DISEASES ASSOCIATED WITH DURING OR INCREASING PLATEL

The present invention relates to a novel megakaryocyte-containing platelet production containing a substance having an activity of activating or inhibiting 3] 3-hydroxydehydrogenase (hereinafter sometimes abbreviated as “3j3 HSD”). Of the regulator.

In addition, the present invention relates to a novel megakaryocyte-mediated blood vessel growth regulator comprising an estrogen receptor agonist or an estrogen receptor antagonist. The present invention also relates to a novel composition for treating a disease associated with a decrease in platelets, comprising the above-mentioned regulator as an active ingredient.

The present invention also relates to a novel composition for treating a disease associated with an increase in platelets, comprising the above-mentioned regulator as an active ingredient.

The present invention also relates to a novel method for regulating the production of platelets from megakaryocytes using the above-mentioned regulator.

The present invention also relates to a novel screening method for the above-mentioned therapeutic composition. Background art

Platelets, like other blood cells, are derived from pluripotent hematopoietic stem cells in the bone marrow. After hematopoietic stem cells have matured and differentiated into megakaryocytic progenitor cells, megakaryoblasts, prokaryotic cells, and megakaryocytes, and after completing cytoplasmic maturation such as formation of a separation membrane, elongated endoplasmic reticulum resembling neurites (Prop. 1 ate 1 etformation. Hereafter, it may be abbreviated as “PPF.”), And the rupture releases many platelets into the blood. This mechanism of platelet production is thought to be common in mammals. However, its molecular mechanism is still unknown.

Knockout of megakaryocyte-specific transcription factor NF-E2 (hereinafter sometimes abbreviated as "NF-E2-1"). Mice become embryonic or neonatal due to a significant decrease in platelets. (Shi V dasani, RA), et al., Cell (Ce11), (USA), 1995, 81, p. 695-70). Similarly, NF-E2-z-mouse-derived embryonic stem cells (embry on icst emce 11; hereinafter, sometimes abbreviated as "ES cells") are differentiated into megakaryocytes and platelets in vitro. When induced, megakaryocytes differentiate normally to some extent, but no PPF is seen and no platelets are produced.

Factors that enhance production by NF-E2 include β1-tupurin (Lecine, P.), Blood, (USA), 2000, 96, p. 1366-1373) and thrompoxane synthase (Deveaux, S.), et al., EMBO Journal, (UK), 1999, Volume 16, p. 56 54- 5661), but none of them can restore PNF and platelet production from NF-E2_z-mouse ES cells.

Known diseases associated with thrombocytopenia include thrombocytopenic purpura, aplastic anemia, and the like. In these diseases, purpura on the skin and visible mucous membranes Frequent, bleeding or anemia. Treatment is based on corticosteroids. Splenectomy, high-dose immunoglobulin administration, and platelet transfusion are also used in cases where the effects of steroids are low or those with strong side effects. However, there are some cases that are ineffective and there are side effects or risk of infection, and at present, there is no satisfactory treatment for a disease associated with thrombocytopenia.

On the other hand, as diseases associated with an increase in platelets, for example, thrombocytosis, thrombocythemia and the like are known. In these diseases there is a risk of bleeding due to thrombosis or abnormal platelet function. The basic treatment is to reduce the platelet count. In an emergency, platelet exchange removal (platelet phlesis) and myelosuppressants (6-mercaptoprine, busulfan, melphalan, nitrosopera, hydroxyperea, etc.), anticancer agents (ranimustine, etc.) However, it is necessary to take measures against circulatory diseases due to the movement of blood to the extracorporeal circulation system, and to pay attention to other side effects of blood cell component reduction. In addition, antiplatelet agents (aspirin, ticlopidine, dipyridamole, etc.) are administered as coping therapy, but this is not necessarily a fundamental treatment. At present, there are no satisfactory treatments for diseases associated with an increase in platelets. Disclosure of the invention

Therefore, an object of the present invention is to provide high efficacy, selectively reduce or increase platelets, have little effect on other blood cell components, and do not involve extracorporeal circulation, whole blood transfusion or component transfusion in view of the above situation. Platelets with less side effects Another object of the present invention is to provide a composition for treating a disease accompanied by an increase.

In addition, in studying the mechanism of platelet production from megakaryocytes, modulators that have the effect of promoting or inhibiting platelet production from megakaryocytes are required. Therefore, an object of the present invention is to provide such a regulator and a method for regulating platelet production from megakaryocytes using the regulator.

Another object of the present invention is to provide a method for screening a composition for treating a disease associated with an increase or decrease in platelets.

In order to achieve the above object, the present invention provides the following.

3] A modulator comprising a substance having an action of activating 3-hydroxydehydrogenase and having an action of increasing platelet production from megakaryocytes.

Use of a substance having an activity of activating 3β-hydroxy dehydrogenase for producing an agent having an effect of increasing platelet production from megakaryocytes (use).

A modulator containing an estrogen receptor agonist and having an effect of increasing platelet production from megakaryocytes.

Use of an estrogen receptor agonist for producing an agent having an effect of increasing platelet production from megakaryocytes (use).

Further, a composition for treating a disease associated with a decrease in platelets, comprising the above-mentioned regulator as an active ingredient.

In the above-mentioned therapeutic composition, the active ingredient is preferably at least one selected from the group consisting of liothyronine, estradiol and genistein.

Further, in the above therapeutic composition, the disease associated with the decrease in platelets is blood Platelet-reduced purpura, aplastic anemia, leukemia, hemolytic uremic syndrome, disseminated intravascular coagulation, radiation, anticancer drugs, and side effects of myelosuppressive drugs Is preferred.

3 A method for increasing megakaryocyte platelet production by bringing a substance having an activator of ^ -hydroxy dehydrogenase into contact with megakaryocytes in vitro.

A method for increasing megakaryocyte platelet production by bringing an estrogen receptor agonist into contact with a megakaryocyte in vitro.

Also, a modulator containing a substance having an inhibitory action on 33-hydroxy dehydrogenase, and having an action of reducing platelet production from megakaryocytes.

Use of a substance having a 33-hydroxy dehydrogenase inhibitory activity for producing an agent having an effect of reducing platelet production from megakaryocytes (use).

Also, a modulator comprising an estrogen receptor antagonist and having an action of reducing platelet production from megakaryocytes.

Use of an estrogen receptor antagonist for producing an agent having an effect of reducing platelet production from megakaryocytes (use).

A composition for treating a disease associated with an increase in platelets, comprising the above-mentioned regulator as an active ingredient.

In the above-mentioned therapeutic composition, the active ingredient is at least one selected from the group consisting of trilostane, mitotane, medroxyprogesterone, tamoxifen, metipiostan, toremifene, clomiphene, and ICI 18280. Preferably, there is.

More preferably, the active ingredient is trilostane. Further, in the above therapeutic composition, the disease associated with an increase in platelets is any one selected from the group consisting of thrombocytosis, thrombocythemia, chronic myelogenous leukemia, and chronic myeloproliferative syndrome. It is preferable that

3 A method of reducing megakaryocyte platelet production by bringing a substance having i3-hydroxy dehydrogenase inhibitory activity into contact with megakaryocytes in vitro.

A method of reducing megakaryocyte platelet production by bringing an estrogen receptor antagonist into contact with a megakaryocyte in vitro.

The present invention also includes, as an active ingredient, a substance having an activity of activating or inhibiting 3-hydroxydehydrogenase, which comprises the following step (A) or (A) and (B): A method for screening a composition for treating a disease associated with a decrease or increase in platelets characterized by the following.

(A) 3] a step of adding a substance serving as a substrate and a test substance to 3_hydroxy dehydrogenase and measuring the activity of 3 -hydroxy dehydrogenase;

(B) a step of adding a test substance to the cultured megakaryocytes and measuring the formation of endoplasmic reticulum. BRIEF DESCRIPTION OF THE FIGURES

1A to 1C are micrographs of megakaryocytes derived from mouse ES cells in Experimental Example 1, and FIG. 1A is a micrograph of NF-E2 ^{+ / +} ES cells on day 10 of differentiation induction (magnification). FIG. 1B is a photomicrograph (× 600) of NF—E2 _ / — ES cells on day 12 of differentiation induction, and FIG. 1C is 3 / 3HS on day 10 of differentiation induction. It is a micrograph (600 times magnification) of NFE2 ES cells into which the D gene has been introduced. FIG. 2 is a graph showing the measurement results of PPF in Example 1, in which the PPF of the control is set as 100%.

FIG. 3 is a graph showing the measurement results of PPF in Example 2, in which the PPF of the control is shown as 100%.

FIG. 4 is a graph showing the PFF measurement results when culturing with the addition of estrogen receptor agonist estradiol in Example 3, wherein the control PPF is shown as 100%. .

FIG. 5 is a graph showing the measurement results of the platelet count in the peripheral blood of the mouse in Example 4, wherein the platelet count in the control is shown as 100%. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in more detail.

The present inventors have conducted intensive studies on the mechanism of platelet production from megakaryocytes. As shown in Experimental Examples 1 and 2 below, 3; 8-hydroxydehydration was involved in the formation of vesicular processes (PPF) and platelet production in megakaryocytes. That the enzymatic enzyme (3 / 3HSD) plays an important role, and that the 33HSD gene is transferred to the embryonic stem (ES) cells of NF-E2 knockout (NF-E2 _ / _) mice It was found that when differentiation was induced, PPF occurred and platelet production ability was restored.

(Experimental Example 1) Isolation and identification of factors involved in platelet production from megakaryocytes Sidasa, R. A. (Shi V dasani, RA) et al. (Cell (1995), p. 81, p. 695— NF produced by 704— Heterozygous E2 (hereinafter sometimes abbreviated as "NF-E2 ^{+ /} -J") Mouse (obtained from Jackson Laboratory) was crossed to obtain normal blastocyst on day 4 of development (Hereinafter sometimes abbreviated as “NF-E2 ^{+ / +} ”) and NF-E2-/ _ ES cells were established. Each ES cell was cultured in a culture medium containing α-MEM (Gibco — BRL, Grand Island (Gr and Isl), New York, USA) containing 20% fetal serum OP 9 cells (Nakano et al., Science) (Science), 1994, vol. 265, p. 1098-1101) at 37 ° C. for 3 days, and then recombinant mouse thrombopoietin (hereinafter sometimes abbreviated as “TP〇”). (50 units Zm 1) and cultured for 8 days to induce differentiation into megakaryocytes. Mouse recombinant TPO is described in Nagata, Y., et al., Journal of Biological Chemistry, 1995, Vol. 270, p. 19673— Prepared from the culture supernatant of COS-7 cells transformed with pME18 into which the cDNA encoding mouse TPO was inserted, as described in 19675. The total RNA was isolated using RN easy Mini Kit (Qiagen, Valencia, California, USA) according to the manual. Next, the subtractive PCR method was performed using PCR—selectc DNA hybridization kit (Clonetech (c1onetech), Palo Alto (Pa1oA1to), California, USA). Following that manual, we isolated factors that could be downstream of NFE2 and regulate PPF. As a result, the cDNA fragment encoding 3 / 3HSD was detected most frequently (about 16%). 3) The 3HSD cDNA was inserted into the pcDNA3.1 expression vector (Invitrogen). Using Northern plot and immunoblot, 3 i8HSD was expressed in NF-E 2 ^{+ / +} ES cell-derived megakaryocytes and bone marrow-derived megakaryocytes, whereas NF-E 2 It was confirmed that 3/3 HSD was not expressed in cell-derived megakaryocytes.

(Experimental Example 2) 3 Effect of platelet production by introducing J3HSD

The 3J3HSD gene was introduced into NF-E2-Z- ES cells by electroporation to obtain a 3iSHSD-expressing clone. NF-E 2 ^{+ / +} ES cells, NF-E 2 _ ES cells and 3/3 HSD gene-introduced NF-E 2 — and one ES cell were each used in 20% fetal MEM containing fetal serum. Co-cultured with OP 9 tumor cells in culture medium at 37 ° C for 3 days, and then cultured for 8 days with addition of mouse recombinant TP O (50 units / ml). The number of cells causing F was counted, and the ratio was determined. Micrographs of NF-E2 ^{+ /} ⁺ ES cells and NF-E2- ^/ -ES cells transfected with 3i8HSD gene were taken on day 10 of culture (day 10 of differentiation induction), and NF-E2 For —Z- ES cells, microscopic photographs were taken on day 12 of culture (day 12 of induction of differentiation), the number of cells causing PPF was similarly counted, and the percentage was determined.

FIG. 1A is a photomicrograph (× 600) of NF-E 2 ^{+ / +} ES cells on day 10 of differentiation induction, and FIG. 1B is a micrograph of NF-E2− ES cells on day 12 of differentiation induction. The micrograph (magnification: 600x) is shown in Fig. 1C. It is a microscope picture (600 times magnification) of NF-E2-/-ES cells transfected with the 3; 8 HSD gene of the eye. As is clear from FIG. 1A, in megakaryocytes induced to differentiate from NF-E2 ^{+ / +} ES cells, PPF was observed on day 10 of differentiation induction, but as is clear from FIG. In megakaryocytes induced to differentiate from NF-E2 / one ES cells, PPF was not observed even on day 12 of induction of differentiation. On the other hand, as is clear from FIG. 1C, in the NF-E2- ES cells into which the 3J3HSD gene was introduced, PPF was observed on day 10 of the differentiation induction.

In addition, the PPF measured on the 8th day of differentiation induction did not show any PPF in megakaryocytes induced to differentiate from NF-E2- ES cells, but NF-E2- It was confirmed that most of megakaryocytes induced to differentiate from ES cells induced PPF, and the introduction of the 3/3 HSD gene restored the platelet-producing ability of megakaryocytes.

3 i3HSD is a key enzyme in the synthesis of all steroid hormones, including estradiol, in mammals, and is derived from Δ5-predanenolone to p-gesterone and 17-hydroxypredanenolone to 170; It is known as an enzyme that converts oral gesterone and dehydroepiandrosterone to Δ4-androstenedione.

Based on the above findings, the present inventors added a substance having a 3j8 HSD activating action or a substance having a 3] 3 HSD inhibitory action to megakaryocytes derived from mouse bone marrow cells. It was found that when a substance having an action was added, PFF from megakaryocytes was promoted, while 3) when a substance having an inhibitory action on 3HSD was added, PPF from megakaryocytes was inhibited. In addition, the present inventors have found that the addition of estradiol, an estrogen receptor agonist, to megakaryocytes derived from mouse bone marrow cells promotes megakaryocyte PFF.

In addition, the present inventors have found that when mice were administered estrogen receptor antagonists moxifen or ICI 182780 by in vivo, the platelet count in peripheral blood of the mice decreased.

A first aspect of the present invention is a modulator containing a substance having a 3jSHSD activating action and having an action of increasing platelet production from megakaryocytes.

Here, the “substance having a 3jSHSD activating effect” is a substance that promotes the enzyme activity of 3j3HSD. Specific examples of such a substance include liothyronine.

A second aspect of the present invention is a modulator containing a substance having an inhibitory action on 3 HSD and having an action of reducing platelet production from megakaryocytes.

Here, the “substance having a 3jSHSD inhibitory action” is a substance that inhibits the enzyme activity of 3 ^ HSD. Such substances include, for example, trilostane, mitotane, medroxyprogesterone.

It is known that the substances exemplified as the substances contained in the modulators of the first and second embodiments have a 3/3 HSD activating or inhibiting action. For example, the above-mentioned liothyronine (InM) or trilostane (100 ιι 促進) promotes or suppresses progesterone secretion by bush luteal cells (Gregoraschuk, ii. Elire. (Grego raszczuk, EL), etc.) End Klein Regulations (En dcrine Re gu lation s), 1999, Vol. 33, p. 155-160), that trilostane specifically inhibits 3/3 HSDs of rat or pea adrenal cortex mitochondria (Km = 2 M) (Pots, G. (Potts, GO) et al., Steroids, 1978, Volume 32, p. 257-267, and J. A. Bios. Bioc hemi stry, 1985, 98, p. 1519-1526), that mitotane inhibits 3 j3HSD (50% inhibitory concentration 8 M) (Ojima, Jem, Journal of the Japan Endocrine Society, 1985, 61, p. 168-178), and that medroxyprogesterone acetate inhibits human 3 iSHSD (K i = 3 tM) (Lee, TC). The Journal of Clinical Endocrinology and Metapolis (The Journal of Clinical Endo crinol ogy and Metabo li sm), 1999, 84 p. 2104-2110) it is has been reported.

However, 3; it has not been known that 8HSD plays an important role in the production of PPF and platelets from megakaryocytes, and it is a finding newly found by the present inventors. It has been found for the first time in the present invention that a substance having an activity of activating 3 HSD or a substance having an inhibitory action of 3 i3HSD acts as a regulator of the promotion or inhibition of platelet production from megakaryocytes. is there.

The substance having 3 HSD activity contained in the modulator of the first embodiment A commercially available product may be used. For example, liothyronine is marketed in Japan in the form of a sodium salt, such as thyronine (Taisho Pharmaceutical Co., Ltd.) for treating hypothyroidism and the like. Similarly, a commercial product may be used as the substance having a 3/3 HSD inhibitory action contained in the modulator of the second embodiment. For example, trilostane is marketed in Japan as Desopan Tablets ^TM (Mochida Pharmaceutical Co., Ltd.) for the treatment of idiopathic aldosteronism, etc., and modo stane ™ (modo rastane ™), a treatment for Cushing's syndrome, (SANOF I ))), Is marketed and developed overseas as a drug for the treatment of lung cancer, modrenal (modrenal ^™ ) (bioenVision). Mitotane is, as adrenal cancer and the like of the therapeutic agent Opepurimu ^TM (Co., Ltd. Yakult), has been巿sales in Japan. Medroxyprogesterone is marketed in Japan in the form of acetate, such as Hythron ^™ (Kyowa Hakko Kogyo Co., Ltd.), a therapeutic agent for amenorrhea. All of the above drugs are highly safe drugs and are available. The modulators of the first and second embodiments are useful as experimental modulators for studying the mechanism of platelet production from megakaryocytes. Therefore, the present invention provides, as a third aspect, a method for regulating the production of platelets from megakaryocytes using the modulator of the first aspect or the second aspect. According to the regulation method of the present invention, when culturing megakaryocytes of a human or a mammal such as a mouse, the regulator of the first aspect or the second aspect is added to the culture environment, When the regulator of the embodiment is added, PPF from megakaryocytes after culture is increased to increase platelet production. On the other hand, when the regulator of the second embodiment is added, PPF from megakaryocytes after culture is reduced, and platelet production is reduced. That is, the book According to the regulation method of the present invention, platelet production from megakaryocytes can be selectively increased or decreased.

Further, the modulators of the first and second embodiments can be applied to treatment and research for diseases associated with thrombocytopenia or increase, and these modulators can be used for diseases associated with reduction or increase of platelets. Therapeutic applications are a preferred use of the modulators of the invention.

That is, a fourth aspect of the present invention is a method according to the first aspect of the present invention, which comprises a substance having an activity of activating 3] 3 HSD and having an effect of increasing platelet production from megakaryocytes. A fifth aspect of the present invention is a composition for treating a disease accompanied by a decrease in platelets, which comprises a substance having a 3i3 HSD inhibitory activity, and reduces platelet production from megakaryocytes. A composition for treating a disease associated with an increase in platelets, comprising the modulator of the second aspect of the present invention having an effect of causing the effect of the present invention as an active ingredient.

As used herein, the term "associated with a decrease in platelets" refers to a state in which the platelet count in blood is below the normal value, regardless of the cause, and in humans, for example, decreased to 100,000 or less. State. Examples of "disorders associated with thrombocytopenia" include thrombocytopenic purpura, aplastic anemia, leukemia, multiple myeloma, malignant lymphoma, hemangiomas, myelofibrosis, hemolytic uremic syndrome, paroxysmal nocturnal Hemoglobinuria, disseminated intravascular coagulation, sarcoidosis, portal hypertension, cirrhosis, megaloblastic anemia, megakaryocyte hypoplasia (Fanco-II syndrome, thrombopoietin deficiency, neonatal rubella), radiation and Side effects of drugs such as anticancer drugs and myelosuppressants, sepsis, tuberculosis, viral infections, autoimmune hemolytic anemia Complications, systemic erythematosus, lymphoproliferative disorders, hypersplenism, congenital thrombocytopenia, massive bleeding, purpura after blood transfusion or extracorporeal circulation. Diseases with thrombocytopenia are prone to bleeding, increasing the risk of trauma with massive bleeding and organ bleeding. In addition, frequent plaques on the visible mucosa and anemia are present.

The term "with increased platelets" refers to a condition in which the number of platelets in the blood has increased above normal levels, regardless of the cause, and in humans, for example, has increased to more than 400,000 ZL. Examples of "diseases associated with an increase in platelets" include thrombocytosis, increased hematopoiesis such as iron deficiency anemia, cancer, thrombocythemia, chronic myelogenous leukemia, myelofibrosis, and essential thrombocytosis. And Erythrocytosis or chronic myeloproliferative syndrome.

Diseases with increased platelets are prone to thrombosis, increasing the risk of cerebral thrombosis and myocardial infarction. On the other hand, there are cases where bleeding symptoms are accompanied by abnormal platelet function despite high platelet count. Also, transient cerebral ischemic attacks, numbness of limbs or angina may occur.

Diseases associated with platelet depletion or increase can be easily diagnosed by counting the number of platelets in blood using a hemocytometer such as Coulter Counsel or a microscope.

The “therapeutic composition” is not limited to the case where it is administered in a state where platelets are already decreased or increased, but includes a composition which is prophylactically administered in a state where platelets are expected to be decreased or increased. Moreover, since the mechanism of platelet production is considered to be common in mammals, the therapeutic composition of the present invention is not limited to humans, It can be used for mammals such as cats, porches, pomas, pigs, sheep, sheep, goats, magpies, and rats.

The therapeutic compositions of the fourth and fifth aspects are characterized in that the active ingredient is administered alone as a compound (which may contain other components unavoidably included in the purification), or Suitable carriers or vehicles commonly used for compounds, for example, excipients, binders, lubricants, coloring agents, flavoring agents, and, if necessary, sterile water or vegetable oils, and also non-toxic organic solvents or harmless Solubilizers (eg, dariserin, propylene glycol), emulsifiers, suspending agents (eg, Tween 80, gum arabic solution), isotonic agents, pH adjusters, stabilizers, soothing agents, etc. Can be appropriately selected and combined to prepare an appropriate pharmaceutical preparation. In addition, commercially available preparations exemplified for the modulator can also be administered.

In addition, in the case of diseases with decreased platelets, corticosteroids, splenectomy, transfusion of immunoglobulins and platelets, and megakaryocytes such as erythropoietin and TPO, and Z or inulin leukin 3, 6, 7, or 11 Can be used in combination with substances that promote differentiation and maturation. On the other hand, in the case of diseases accompanied by an increase in platelets, it can be used in combination with myelosuppressants such as 6-mercaptopurine, busulfan, melphalan, nitrosodia, hydroxydialea, ranimustine, and antiplatelet agents such as aspirin, ticlovidine, dipyridamole . The term “combination” refers to a compound that is an active ingredient of a composition for treating a disease associated with a decrease or increase in platelets of the present invention and a substance that promotes differentiation and maturation of megakaryocytes, or a myelosuppressant and / or an antiplatelet And two or more drugs, each of which is separately contained, and these drugs can be administered simultaneously or sequentially, or a mixture of these In the form of co-administration.

Pharmaceutical dosage forms include tablets, capsules, microcapsules, granules, fine granules, powders, oral liquid preparations, suppositories, syrups, inhalants, eye drops, ointments, injections (emulsions , Suspension, non-aqueous) or in the form of a solid injection used in the form of an emulsion or suspension at the time of use, whether orally and intravenously or intraarterially, inhalation, eye drops, rectum, vagina or external use To be administered. Oral administration is preferred when a prophylactic effect is desired, and intravenous or intraarterial administration is preferred when relatively rapid efficacy is desired. It can also be added to extracorporeal blood circulation, or administered using a known nebulizer as an inhalant.

The dose of the composition for treating a disease associated with a decrease or increase in platelets of the present invention is an amount sufficient to exhibit the target effect, but its active ingredient, dosage form, administration method, daily administration The number can be adjusted as appropriate according to the frequency, degree of symptoms, weight, age, gender, conditions for using other drugs.

For example, in the case of oral administration of liothyronine sodium, for adults, 0.1 gZ days to 50 O gZ days, preferably 5 gZ days to 75 gZ days should be administered once or divided into 2 to 3 times. Is preferred. In the case of oral administration of trilostane, for adults, it is preferable to administer 1 OmgZ day to 500 OmgZ day, preferably 24 OmgZ day to 48 OmgZ day once or in 2 to 4 divided doses. In the case of oral administration of mitotane, for adults, it is preferable to divide the dose into 10 OmgZ days to 1 Og / day, preferably 1.5 gZ days to 3 g / day once or 2-3 times. . For oral administration of medroxyprogesterone acetate, 0.1 mg mg to 50 mg / day for adults, preferably 2.5 m It is preferable to administer the gZ day to 15 mg gZ day once or in 2 to 3 divided doses. ^ When estradiol valerate is injected intramuscularly, lmg to 5 Omg is preferable for an adult, preferably It is preferred to administer 5 mg to 10 mg every 1 to 4 weeks.

In any case, it is preferable to adjust the dose, frequency and period while monitoring the blood platelet count.

The therapeutic composition according to the fourth aspect of the present invention can be used in the following form (a). (A) adding the therapeutic composition to a culture environment of cells containing any one of hematopoietic stem cells, megakaryocyte progenitor cells, megakaryoblasts, prokaryotic or megakaryocytes isolated from an individual, and inducing platelets in vitro. Increase production, collect the produced platelets, and transfuse platelets to the same individual or another individual with a disease involving platelet depletion. In this culture environment, interleukin 3, 6, 7 or 11, or a substance that promotes the differentiation and maturation of megakaryocytes such as erythropoietin and TPO can be further added to culture.

The amount of the therapeutic composition to be added in the above-mentioned usage form is sufficient to exhibit the target effect, but can be appropriately increased or decreased depending on the active ingredient, culture environment, conditions for using other drugs, and the like. It is preferable to adjust the amount of addition while monitoring the state of formation and the number of platelets produced.

A sixth aspect of the present invention is a modulator containing an estrogen receptor agonist and having an action of increasing platelet production from megakaryocytes.

Here, examples of the estrogen receptor agonist include estradiol and genistein. A seventh aspect of the present invention is a modulator containing an estrogen receptor antagonist, and having an action of reducing platelet production from megakaryocytes.

Here, examples of the estrogen receptor antagonist include moxifen evening, moxifen evening citrate, metipiostan, toremifene, clomiphene, and ICI 187280.

The estrogen receptor agonist contained in the modulator of the sixth embodiment of the present invention and the estrogen receptor antagonist contained in the modulator of the seventh embodiment of the present invention are commercially available products. You may. For example, estradiol, an estrogen receptor agonist, is commercially available in Japan as peranine ^TM (Mochida Pharmaceutical Co., Ltd.), a therapeutic agent for amenorrhea, etc., in the form of an ester with valeric acid. Have been. On the other hand, Kuen acid tamoxifen estrogen receptor-en evening agonist is commercially available in Japan as an anti-breast cancer agents Norupa Tex ^TM and Norubatekkusu ^TM D (Asutorazeneka Ltd.). In addition to this, Mepichiosutan is, as an anti-breast cancer agents Chioderon ^TM (salt Nogi Pharmaceutical Co., Ltd.), toremifene in the form of Kuen salt anti postmenopausal breast cancer agent Feasuto ^TM (Japan Department of drug Co., Ltd.), clomiphene Kuen acid It is marketed in Japan as a salt form, Ovulation inducer for infertility, Supacromin ^™ (Kasayaku Co., Ltd.), and ICI 182,780 is the anti-breast cancer drug Flubastran (Fas1odex ^™ , Astra Zeneca) is commercially available in the United States.

The modulator according to the sixth and seventh aspects of the present invention, like the modulator according to the first and second aspects of the present invention, is used for an experimental study of the mechanism of platelet production from megakaryocytes. It is useful as a regulator. Therefore, the present invention provides a sixth aspect The present invention provides, as an eighth aspect, a method for regulating platelet production from megakaryocytes using the modulator of any one of the first to seventh aspects and the seventh aspect. The specific adjustment method is the same as that described in the third embodiment of the present invention. That is, by adding the modulator of the sixth aspect or the seventh aspect to the culture environment of megakaryocytes of a mammal such as a human or a mouse, when the modulator of the sixth aspect is added, Can increase platelet production by increasing PPF from megakaryocytes. On the other hand, when the regulator of the seventh embodiment is added, PPF from megakaryocytes after culture can be reduced, and platelet production can be reduced.

Further, the modulators of the sixth and seventh aspects of the present invention can be applied to treatment, research, and the like for diseases associated with thrombocytopenia or increase. Therapeutic applications for diseases with an increase are preferred uses of the modulators of the invention.

That is, a ninth aspect of the present invention is characterized in that it contains an estrogen receptor agonist and contains, as an active ingredient, the modulator of the sixth aspect of the present invention, which has an effect of increasing platelet production from megakaryocytes. A tenth aspect of the present invention is a composition for treating a disease accompanied by a decrease in platelets, which comprises an estrogen receptor antagonist, and has an action of reducing platelet production from megakaryocytes. A composition for treating a disease associated with an increase in platelets, which comprises the regulator of any one of the above embodiments as an active ingredient.

The use forms of the therapeutic compositions of the ninth and tenth aspects of the present invention are the same as the use forms of the therapeutic compositions of the fourth and fifth aspects of the present invention, respectively. According to an eleventh aspect of the present invention, there is provided a composition for treating a disease associated with thrombocytopenia containing a substance having a 3jSHSD activating action as an active ingredient or a substance having a 30HSD inhibitory action as an active ingredient. A method for screening a composition for treating a disease associated with an increase in platelets, characterized by the following.

In the screening method of the eleventh aspect of the present invention, a substance serving as a substrate and a test substance are added to 3j3HSD, and the enzyme activity of 3βΗSD is measured.

The enzyme activity of 3 / 3HSD is determined by measuring the substrate and nicotine adenine dinucleotide (hereinafter sometimes abbreviated as “NAD +”) or nicotinade in a sample containing 33 HSD such as purified 3jSHSD or adrenal microsomes. Nindinucleotide phosphate (hereinafter sometimes abbreviated as “NADP +”) is added and reacted, and the amount of dehydrogenated and converted substance or reduced nicotine adenine dinucleotide (hereinafter abbreviated as “NADH”) ) Or reduced nicotine adenine dinucleotide phosphate (hereinafter sometimes abbreviated as “NADPH”) by measuring absorbance or the like.

3/3 HSD can be purified from common adrenal microsomes by conventional methods, for example. In addition, homogenates of mouse and rat adrenal microsomes can also be used. Combinations of 30HSD substrate and dehydrogenated and converted material include, for example, Δ5-predanenolone to progesterone, 17-hydroxypredanenolone to 17α-hydroxyprogesterone and dehydroepiandrosterone to Δ 4—Androstension. For quantification of NADH or NADPH, for example, a wavelength of 260 nm Alternatively, it can be calculated by measuring the absorbance at 340 nm.

In the screening method according to the eleventh aspect of the present invention, a change in PPF can be measured by adding a test substance to a megakaryocyte culture system of mammals such as humans, mice, and rats. For example, in a megakaryotic culture system, the PPF from megakaryocytes is set to 100% .At that time, substances that promote the differentiation and maturation of megakaryocytes, such as interleukin 3, 6, 7 or 11, erythropoietin and TP〇 It can also be added.

By measuring and evaluating the change in 3SD-HSD activity or the change in PPF when a test substance is added to these measurement systems, a substance having a 3 ^ -hydroxy dehydrogenase activating or inhibitory action can be obtained. Can be screened for a composition for treating a disease associated with a decrease or increase in platelets, characterized in that it contains as an active ingredient.

For example, by adding ImM or less, preferably 0.0 ImM or less, more preferably 0.0 ImM or less among the test substances, the enzyme activity of the control 3HSD is 10% or more, preferably 20% or more. More preferably, a test substance that activates or inhibits 50% or more, more preferably 70% or more, is selected, and the substance is contained as an active ingredient for the treatment of a disease associated with platelet decrease or increase. The composition can be screened. Further, among the test substances selected above, the addition of 10 mM or less, preferably ImM or less, more preferably 0. ImM or less makes PPF from megakaryocytes 10% or more, preferably 20%. A test substance that activates or inhibits the above, more preferably 30% or more, and even more preferably 50% or more is selected, and the substance is included as an active ingredient. It is possible to screen for a composition for treating a disease associated with a decrease or increase in platelets characterized by having

(Example)

Hereinafter, the present invention will be described more specifically with reference to Examples. However, the present invention is not limited to these.

(Example 1) 3 Effect of a modulator containing a substance having J3HSD activating action on platelet-producing ability in mouse megakaryocytes

Bone marrow cavities of normal mice (BDF 1 mouse, 6-8 weeks old) were treated with CATCH medium (ImM adenosine (Sigma, St. Louis, Missouri, USA), 2 mM theophylline (ヮ Koichi). Hank's balanced salt solution (Gibco) containing 13.6 mM sodium citrate hydrate (Nacalai Task, Kyoto, Japan) and Hank 'sbal anc edsaltsolution (Gibco—BRL, Grand) Bone marrow cells were obtained by washing on an island (Gr and Is and), New York, USA) to prepare megakaryocytes.3 / 3 HSD activation was performed on the prepared megakaryocytes (3 x 10 ³ ml). A modulator containing liothyronine (100 pM) as an active substance was added, and 37 ° C in serum-free S clone medium containing 1% serum albumin (S—c 1one, Sanko Ichi, Japan) Incubate for 24 hours, take micrographs, raise PPF Similarly, mouse megakaryocytes were cultured for 24 hours without adding liothyronine, and micrographs were taken to determine the number of cells causing PPF. It was measured and the ratio was determined (control). FIG. 2 is a graph showing the PPF measurement results, with the PPF of the control being 100%. As shown in Fig. 2, in mouse megakaryocytes cultured with the addition of a modulator containing liothyronine as a substance having a 3HSD activating effect, PPF was increased by about 50% and platelet production was promoted. This was confirmed.

(Example 2) Effect of a modulator containing a substance having a 3/3 HSD inhibitory effect on the platelet-producing ability of mouse megakaryocytes

The bone marrow cavity of normal mice (BDF 1 mouse, 6 to 8 weeks old) was washed with CATCH medium to obtain bone marrow cells, and megakaryocytes were prepared. To the prepared megakaryocytes ( ³ × 10 ³ Zml), a regulator prepared by dissolving trilostane at a concentration of 0.09 mM, 0.45 mM and 0.9 mM in a solvent DMS O as a substance having a 33HSD inhibitory action was added. The cells were cultured for 24 hours at 37 ° C in serum-free S-clonal medium containing 1% serum albumin, and micrographs were taken to determine the number and percentage of cells causing PPF. Similarly, micrographs were taken of a mouse megakaryocyte cultured for 24 hours without adding trilostane (control) and a mouse cultured for 24 hours with only DMSO added (solvent), and PPF was taken. The number of arising cells and the ratio were measured.

Figure 3 is a graph showing the PPF measurement results, with the control PPF being 100%. As shown in Fig. 3, 3) in mouse megakaryocytes cultured with the addition of a modulator containing trilostane as a substance with 8 HSD inhibitory activity, PPF decreases in a concentration-dependent manner, The inhibition was confirmed. In the case of culturing with only DMSO added (solvent), It was confirmed that the PPF had almost the same level as the PPF.

(Example 3) Effect of steroid on platelet-producing ability in megakaryocytes of mice

Megakaryocytic progenitor cells, c-kit + CD41 + cells, were analyzed by Oda, M., et al., Jeans to Cells (2003), Vol. 8, p. 9—16. It was prepared from normal mouse bone marrow according to the method described in (1). BDF-1 mouse bone marrow cells were conjugated with a fluorescent isocyanate-conjugated anti-mouse CD41 antibody (BD Pharmamin, San Diego, California, USA) and an APC-conjugated anti-mouse CD The antibody was incubated with the 117 (c-kit) antibody (BD Pharmingen, San Diego, California, USA) for 1 hour on water. Cell, 0. 05% N a N ₃ and 2% © shea fetal serum-containing phosphate-buffered raw physiological saline of washed twice, c one kit ⁺ CD41 + cells FACS Va t ag e SE (Beady one Pharmingen (BD Pharmaceuticals), San Diego (San Diego, CA, USA).

The prepared c-kit + CD41 + cells (2 × 10 ³ Zm 1) were concentrated at the concentrations of estradiol, testosterone or progesterone, which are steroids formed in the metabolic pathway of corticosteroids, or dexamethasone, which is a synthetic steroid. 2.3%, 23nM, 230nM, and 2.3M were added to the S-cut containing 1% fetal serum and mouse TPO (50 units / ml). The cells were cultured in a lawn medium at 37 ° C for 5 days, micrographs were taken, the number of cells causing PPF was counted, and the ratio was determined. Similarly, a mouse megakaryocyte cultured for 24 hours without the addition of steroids was photographed with a micrograph, the number of cells causing PPF was counted, and the ratio was determined. .

As a result of measurement of PPF, it was confirmed that the culture cultured with the addition of the estrogen receptor agonist estradiol showed an increase in the PPF compared to the control, but the culture cultured with the addition of other steroids did not. There were no significant differences from the control.

FIG. 4 is a graph showing the PPF measurement results in the case where estradiol was added, in which the PPF in the control was set as 100%. As shown in Fig. 4, in the case of culturing with added estradiol, PPF increased in a concentration-dependent manner with respect to the added estradiol, confirming that the platelet-producing ability was promoted. Was.

(Example 4) Effect of in vivo administration of estrogen receptor angonist on platelet count in peripheral blood of mice

Estrogen receptor angiotensin, as evening moxifen (Sigma, St. Louis, Missouri, USA) or ICI 182780 (To cris), Bristol (Bristo1, UK) Was dissolved in a solvent (sesame oil: ethanol = 19: 1) at a concentration of 2.5 mg / mL. 100 L was intraperitoneally injected into a normal mouse (BDF I mouse, 8 weeks old, male, n = 7) for 1 day. One dose was administered for 9 consecutive days. After administration, tail vein Blood was collected from the blood (2 L) and the platelet count in the peripheral blood was measured by the Brecher-Clonite method (0, 2, 4, 6, 8, 11, 14 and 20). Day (once a day)). As a control, BDF I mice were intraperitoneally administered only 100 L of a solvent (sesame oil: ethanol = 19: 1) intraperitoneally once a day for 9 days continuously instead of the estrogen receptor antagonist, and were administered through the tail vein. Blood samples were collected (2 ^ L) and the number of platelets in peripheral blood was measured by the Brecher-Cronite method (0, 2, 4, 6, 8, 11, 14 and Day 20 (once a day)).

FIG. 5 is a graph showing the measurement results of the platelet count in peripheral blood, where the control platelet count measurement value is 100%.

As is clear from FIG. 5, the platelet count began to decrease 5 to 6 days after the start of administration, and the platelet count further decreased as long as the estrogen receptor antagonist was continued to be administered. Then, 8 to 11 days after the start of administration, the platelet count decreased most. Platelet counts were reduced by up to 45% in those treated with evening moxifen compared to controls, and by up to 25% in those treated with ICI 182780 compared to controls. Within 5 days after the administration, the platelet count returned to the original level. Since the half-life of mouse platelets is 5 to 6 days, it took 5 to 6 days from administration of estrogen receptor antagonist to decrease in the number of platelets in peripheral blood, indicating that estrogen receptor antagonis This shows that the administration of the mouse suppressed the release of newly produced platelets from megakaryocytes into peripheral blood. From these results, it was confirmed that platelet production was suppressed when the estrogen receptor antagonist was administered in vivo. (Example 5) Screening of a composition for treating a disease associated with a decrease or increase in platelets, characterized by containing a substance having a 33 HSD activating or inhibiting action as an active ingredient

Normal rats (Sprague—Daw 1 ey, male) Using homogenates of adrenal microsomes, Potts, G. ot. (Po tts, GO), etc., Steroids, 32 vol., 257—267, 1978 By measuring the effect of the test substance on 3i3HSD activity in accordance with the method described in 1975, a substance having a 3HSD activating or inhibitory action is contained as an active ingredient. A composition for treating a disease accompanied by a decrease or increase in platelets that develops is screened.

That is, 75 mM glycine buffer containing 70 mM sucrose, 11.5 mM NaC15.4 mM KC1, 5 mgZml ゥ serum albumin, 0.84 mM NAD +, 8.5-68 / ΜΔ5-predanenolone (p H9.5) Add homogenate of rat adrenal microsomes (equivalent to 6.7 mg of adrenal gland) and test substances (0.1 M, 10 M and ImM) to lm1, and incubate at room temperature while incubating. The change in absorbance at 340 nm is measured, and the intensity of the activating or inhibiting effect is calculated by comparing the result with the measurement result of a control to which no test substance is added.

As a result, 3) 3HSD activating action was confirmed for liothyronine sodium salt and 3) 3HSD inhibitory action for avian stan, mitotane and medroxyprogesterone acetate. Furthermore, in the same procedure as the method used in Example 2, the test substance was added to the cultured megakaryocytes, and the results obtained by measuring the PPF were evaluated. As a result, the test substance contains a substance having a 3j8 HSD activating or inhibitory action of the present invention as an active ingredient, thereby providing a composition for treating a disease associated with a decrease or increase in platelets. Screening for what can be done can be done.

Hereinafter, in Examples 6 to 9, the composition for treating a disease associated with decrease or increase of platelets, which comprises a substance having an activity of activating or inhibiting 3 iS HSD of the present invention as an active ingredient. The production example which produced the pharmaceutical preparation is shown. (Example 6) Riothyronine sustained release tablet

Liothyronine sodium 0.005 g

Methocel K 100M (within granules) 16. 125 g

Dicalcium phosphate dihydrate 181.670 g

Methocel K 100M (between granules) 16. 125 g

Magnesium stearate 1.075 g

In this example, tablets of a composition for treating a disease associated with a decrease in platelets, comprising liothyronine as a substance having a 3/3 HSD activating action, were produced. The actual procedure involves pouring an ethanolic solution of liothyronine sodium into dicalcium phosphate dihydrate and drying, along with Methocel K100M (intragranular), Methocel K100M (intergranular) and magnesium stearate. Each component was mixed in the above amounts. This was compressed to produce 215 mg tablets.

(Example 7) Trilostane tablet

Trilostane 6.0 g Lactose 6.0 g

Polyvinylpyrrolidone 0.4 g

Magnesium stearate 0.1 g

In this example, tablets of a composition for treating a disease associated with an increase in platelets, characterized by containing trilostane as a substance having a 3/3 HSD inhibitory action, were produced. As an actual procedure, each of the above components was weighed, mixed and granulated by a wet granulator. This was used to produce 125 mg tablets using a tableting machine.

(Example 8) Mitotane capsule

In this example, capsules of a composition for treating a disease associated with an increase in platelets, which contains mitotane as a substance having a 3/3 HSD inhibitory action, were produced. As an actual procedure, 50 Omg of mitotane was weighed and encapsulated in No. 0 gelatin capsule to produce a capsule.

(Example 9) Medroxyprogesterone acetate tablet

0.5 g of medroxyprogesterone acetate

Corn starch 1.5 g

Hydroxypropyl cellulose 0.3 g

Magnesium stearate 0.2 g

Lactose 12.5 g

In this example, tablets of a composition for treating a disease associated with an increase in platelets, comprising medroxyprogesterone acetate as a substance having a 3j3HSD inhibitory action, were produced. As an actual procedure, each of the above components was weighed, mixed, and granulated by a wet granulator. This was used to produce 15 Omg tablets using a tableting machine. Was. Industrial applicability

The modulator of the present invention characterized by increasing or decreasing the amount of platelet production from megakaryocytes is useful as an experimental reagent for studying the mechanism of platelet production from megakaryocytes. Further, the modulator of the present invention can be applied to treatment and research on diseases associated with thrombocytopenia or increase.

The composition for treating a disease associated with a decrease or increase in platelets, which contains the modulator of the present invention as an active ingredient, is a disease requiring a normalization of platelet count in humans and animals, for example, thrombocytopenic purpura, regeneration For adverse anemia, acute leukemia, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, thrombocytosis, thrombocythemia, chronic myeloproliferative syndrome, etc. Less useful as a therapeutic agent.

Further, the method for screening a composition for treating a disease associated with a decrease or increase in platelets, which comprises a substance having a 3 jS HSD activating or inhibiting action according to the present invention as an active ingredient, comprises: It is useful as a method for efficiently identifying therapeutic agents for various diseases and substances for use in treatment and research for diseases associated with thrombocytopenia or increase.

Claims

The scope of the claims

1.3 / 3-A regulatory agent containing a substance having an activity of activating hydroxy dehydrogenase and having an effect of increasing platelet production from megakaryocytes.

2. 3 / 3-A modulator containing a substance having an inhibitory effect on hydroxy dehydrogenase and having an action of reducing platelet production from megakaryocytes.

3. A composition for treating a disease accompanied by a decrease in platelets, comprising the modulator according to claim 1 as an active ingredient.

4. The therapeutic composition according to claim 3, wherein the active ingredient is liothyronine.

5. The above-mentioned diseases accompanied by the decrease in platelets are caused by thrombocytopenic purpura, aplastic anemia, leukemia, hemolytic uremic syndrome, disseminated intravascular coagulation syndrome and radiation, anticancer drugs, and side effects of bone marrow depressants. 5. The therapeutic composition according to claim 3, wherein the composition is any one selected from the group consisting of:

6. A composition for treating a disease associated with an increase in platelets, comprising the agent according to claim 2 as an active ingredient.

7. The therapeutic composition according to claim 6, wherein the active ingredient is at least one selected from the group consisting of trilostane, mitotane and medroxyprogesterone.

8. The therapeutic composition according to claim 6, wherein the active ingredient is trilostane.

9. The diseases with increased platelets include thrombocytosis, thrombocythemia, and chronic myeloid white The therapeutic composition according to any one of claims 6 to 8, wherein the composition is any one selected from the group consisting of blood disease and chronic myeloproliferative syndrome.

10. It is characterized by including the following steps (A) or (A) and (B). 3) 3 It is required that a substance having an activity of activating or inhibiting monohydroxy dehydrogenase is contained as an active ingredient. A method for screening a composition for treating a disease associated with a characteristic decrease or increase in platelets.

(A) Step of measuring the activity of 3 | S-hydroxy dehydrogenase by adding a substrate substance and a test substance to 3-hydroxy dehydrogenase

(B) a step of adding a test substance to cultured megakaryocytes and measuring the formation of endoplasmic reticulum