JP2006271376A - Method for screening human platelet count-controlling medicine - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a screening method for evaluating effect of a human platelet count-controlling medicine on human platelet in an animal body. <P>SOLUTION: It is made possible to built a human megakaryocyte-platelet hematopoiesis system in an experimental animal mouse body by using an NOG mouse in which a human stem cell is transplanted and it is made possible to monitor control of platelet count by administering a compound, an antibody, a peptide, a protein, etc., which are expected to have action on human megakaryocyte-platelet hematopoiesis to the NOG mouse to which human stem cell is transplanted. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for screening a factor such as a compound, antibody, peptide or protein that constantly produces human platelets in a mouse body and regulates the number of human platelets.

  Platelets are important cells that control hemostasis in blood. Its life span in vivo is short, and platelets are produced daily from hematopoietic stem cells via megakaryocytes. This in vivo system for producing platelets is called megakaryocyte / platelet hematopoiesis. It is known that humoral factors such as thrombopoietin (TPO), interleukin 11, interleukin 3, and stromal cell-derived factor 1 (SDF1) are involved in megakaryocytes and platelet hematopoiesis (Non-patent Documents 1-2) . In particular, TPO is known to play an important role in megakaryocytes and platelet hematopoiesis. TPO promotes proliferation and differentiation from hematopoietic stem cells to megakaryocytes via its receptor, Mpl protein, and produces platelets (Non-patent Document 1). Activation of Mpl protein due to the action of TPO is expected to be useful as a treatment method that increases platelets and avoids the risk of bleeding due to thrombocytopenia. For this reason, for example, compounds and antibodies that activate the Mpl protein have been screened (Patent Document 1).

  On the other hand, if platelets are present in an excessive amount in the blood, the risk of forming a thrombus increases. Therefore, there is a need for a treatment that reduces excessive amounts of platelets. As such a treatment method, for example, administration of drugs such as anagrelide and interferon alpha has been attempted (Non-Patent Documents 3 to 4).

  In addition, interferon is used as a therapeutic agent for hepatitis B or C disease and the like, but it is known to induce thrombocytopenia as the main side effect. Therefore, development of drugs such as interferon that do not induce thrombocytopenia is desired.

  In general, in screening for substances such as compounds, antibodies, peptides or proteins (hereinafter referred to as compounds) as drugs, for example, a protein having the same amino acid sequence as that of humans is prepared using molecular biological techniques. The method of measuring the activation ability and the inhibition ability is taken in vitro. Promising compounds screened by such a method are subjected to animal tests (in vivo) in order to confirm the effects in vivo. However, in most cases, the amino acid sequence of the target protein is not identical between humans and experimental animals. Therefore, the activity value of the target protein having the amino acid sequence of the experimental animal species used and the target protein of the human amino acid sequence often deviate from each other. In some cases, it is promising only for amino acid sequence target proteins of very limited species. Some compounds may show activity.

  Thus, in the case of exhibiting species-specific activity, an animal with a modified gene can be used as an animal test. For example, a transgenic mouse into which a gene that expresses a human amino acid sequence is introduced, a knock-in mouse into which a gene is introduced into a specific region, and the like can be used. However, in such transgenic animals, only the target protein is replaced by the target species (eg, human), and therefore the function expressed in the interaction between the protein and the protein shows an unusual reaction. There is a possibility. For this reason, sufficient knowledge may not be obtained in the original meaning of animal tests for predicting human effects of promising compounds.

  Therefore, it is necessary to construct not only the target protein but also the target megakaryocyte / platelet hematopoietic system itself in the experimental animal body.

  In order to analyze the proliferation and differentiation of human cells in an animal body, it is necessary to transplant human cells into an immune-tolerant animal. As such immunotolerant animals, sheep fetuses and immunodeficient mice are known. Immunity-deficient mice include nude mice and CB-17-scid mice, and animals that are easier to engraft human cells include NOD / shi-Scid mice, NOD / LtSz-scid mice, β2m (null) NOD / LtSz- Modified animals such as scid mice have been produced. By transplanting these modified mice with CD34-positive cells called human hematopoietic stem cells / progenitor cells, measurement of transplantation efficiency and hematopoietic remodeling ability using human CD45-positive cells as indicators is widely performed. However, in the chimera rate using human CD45 as an indicator, many cells are CD19-positive human B cell lineages, and the appearance rate of human myeloid cells is considered to be low (Non-patent Document 5). ). In particular, in megakaryocytes and platelet hematopoiesis, it is known that human megakaryocytes (CD41 positive cells) are present in mouse bone marrow, but little is known about the dynamics of human platelets present in mouse peripheral blood. .

  In previous reports, when human peripheral blood CD34 positive cells were transplanted into NOD / LtSz-scid mice or β2m (null) NOD / LtSz-scid mice, humans were injected into the peripheral blood of mice 2-3 weeks after transplantation. It is known that platelets appear. However, there are individuals in which human platelets are not detected, and there are variations in engraftment. Further, after 4 weeks, the number of human platelets has been remarkably decreased and disappeared (Non-patent Documents 6-8). Furthermore, it is known that administration of cytokines containing TPO to NOD / LtSz-scid mice transplanted with human peripheral blood CD34-positive cells significantly inhibits human megakaryocytes and platelet hematopoiesis (Non-patent Document 6). ). According to a report by Perez L.E. et al., When human umbilical cord blood-derived mononuclear cells are transplanted into NOD / LtSz-scid mice, human platelets appear up to 13 weeks, but the long term thereafter is unclear. In addition, by infecting the mouse with an SDF-1-expressing adenovirus, a transient increase in human platelets is observed, but no increase effect is observed at the initial stage of transplantation (3 weeks). Furthermore, when the model mouse is infected with a TPO-expressing adenovirus, no increase in human platelets is observed (Non-patent Document 9). For these reasons, human megakaryocyte / platelet hematopoiesis analysis using immunodeficient mice so far has not been used as a screening system for compounds that regulate the number of human platelets.

  Further, according to the description of WO 2002/062775 (Patent Document 2), it is confirmed that a compound having an agonistic action on Mpl protein has a platelet increasing action in a mouse in which human platelet production is observed. Is unknown.

  According to the description of WO 2002/043477 (Patent Document 3), when human umbilical cord blood-derived CD34 positive cells are transplanted into immunodeficient mice (NOG mice), human platelets appear until the 12th week. However, when human peripheral blood CD34 positive cells were transplanted into NOD / LtSz-scid mice or β2m (null) NOD / LtSz-scid mice as described above, the number of human platelets decreased markedly after 4 weeks and disappeared. Therefore, it is unclear whether human platelets appear stably in the NOG mouse for a long period after 12 weeks. In addition, in NOD / LtSz-scid mice as described above, administration of cytokines containing TPO significantly inhibits human megakaryocytes and platelet hematopoiesis, or humans when infected with TPO-expressing adenovirus. As there is a report that the increase in platelets is not observed, whether or not it can be used for screening systems such as compounds that regulate the number of human platelets only by the knowledge that human platelets appear in immunodeficient mice It was unpredictable.

WO 2004/108683 WO 2002/062775 WO 2002/043477 Proc. Natl. Acad. Sci. U. S. A., 1995; 92, p. 3234-3238 Nat. Med., 2004; 10, p. 64-71 Cancer, 2003; 98, p. 100-109 Cancer, 2004; 101, p. 2239-2246 Leukemia, 2003; 17, p. 960-964 Blood, 2001; 97, p. 1635-1643 Exp. Hematol., 2003; 31, p. 413-420 Transfusion, 2004; 44, p. 555-566 Exp. Hematol., 2004; 32, p. 300-307

  The problem to be solved is that the conventional megakaryocyte / platelet hematopoietic system itself cannot be constructed in the experimental animal body. In other words, human megakaryocyte / platelet hematopoiesis is observed in all cases when a considerable number of human hematopoietic stem cells are transplanted, and human platelets are produced over a long period (6 months or more), which acts on the human megakaryocyte / platelet hematopoietic system. It has been strongly desired that the effects on fluctuations in the number and / or function of human platelets such as antibodies, peptides or proteins can be monitored immediately after transplantation.

  The present invention solves the above-mentioned problems, and constantly produces human platelets in the mouse body and screens for factors (hereinafter, compounds, etc.) such as compounds, antibodies, peptides or proteins that regulate the number and / or function of human platelets. It aims to provide a way to do.

  As a result of intensive studies in order to solve the above problems, the present inventors have used human NOC mice described in WO 2002/043477 as recipient animals, and transplanted human stem cells to obtain human megakaryocytes, We found that platelet hematopoiesis was maintained over a long period (over 6 months). Furthermore, administration of compounds or proteins expected to have an effect on human megakaryocytes / platelet hematopoiesis to NOG mice transplanted with human stem cells has made it possible to regulate the number of human platelets. Moreover, the knowledge that the number of human platelets returns to the pre-dose value by stopping the administration was obtained. Furthermore, the present inventors have found that the administration start time when monitoring the effect of regulating the number of human platelets by a compound is not affected by the number of days after transplantation. From these facts, a method for screening a compound expected to have an action on human megakaryocytes / platelet hematopoiesis in an animal body has been completed. That is, the embodiments of the present invention are as follows.

(1) Platelet number regulation comprising administering a candidate substance to a NOG (NOD / Shi-scid, IL-2Rγ KO) mouse transplanted with human stem cells and measuring the number and / or function of human platelets in the mouse Screening method for drugs or drugs that do not affect platelet count.
(2) The screening method according to (1), wherein the platelet number regulator is a platelet-increasing drug.
(3) The screening method according to (1), wherein the platelet number regulator is a thrombocytopenic.
(4) The screening method according to (1), wherein the platelet number regulator is a drug for maintaining, enhancing or decreasing platelet function.
(5) The screening method according to (1), wherein the drug having no effect on platelet count is a non-platelet increasing drug.
(6) The screening method according to (1), wherein the platelet number non-influencing drug is a non-platelet reducing drug.
(7) The screening method according to (1), wherein the drug having no effect on platelet count is a drug for maintaining, enhancing or decreasing platelet function.
(8) A method for producing a mouse model having a human megakaryocyte / platelet hematopoietic system, comprising transplanting human stem cells into NOG (NOD / Shi-scid, IL-2Rγ KO) mice.
(9) NOG (NOD / Shi-scid, IL-2Rγ KO) mouse model in which human stem cells are transplanted and has a human megakaryocyte / platelet hematopoietic system in the body.

  Using a NOG mouse transplanted with human stem cells, a human megakaryocyte / platelet hematopoietic system is constructed, and a compound, antibody, peptide, or protein that is expected to have an effect on human megakaryocyte / platelet hematopoiesis is administered to the mouse. Thus, there is provided a screening method for evaluating the effect in the form of increase / decrease in the number of platelets and / or maintenance, enhancement or decrease of function.

Creation of NOG (NOD / Shi-scid, IL-2Rγ KO) mice
NOG mice can be produced by the method described in WO 2002/043477 International Publication Pamphlet, and can also be obtained by transfer from the Central Institute for Experimental Animals.
According to the method described in WO02 / 043477, NOG mice can be created as follows.

NOG mice can be created by backcrossing B mice to A mice shown below.
A: A mouse obtained by backcrossing a CB-17 / Icr-scid (also called CB-17-scid or CB17 / SCID mouse) mouse to a NOD / Shi mouse,
B: Mice knocked out of the interleukin 2 receptor γ chain gene.

  Examples of the heterologous cells include cells and tissues derived from mammals such as mice and rats including humans, particularly human stem cells, lymphocytes and tumor cells, but are not limited thereto.

  Backcrossing of CB-17 / Icr-scid mice to NOD / Shi mice in A mice can be accomplished by techniques known to those skilled in the art, such as backcrossing by the Cross Intercross method (Inbred Strains in Biomedical Research, MFWFesting, 1979, ISBN). 0-333-23809-5, The Macmillan Press, London and Basingstoke), a CB-17 / Icr-scid mouse and a NOD / Shi mouse were mated and the F1 mice obtained by further mating the F1 mice Measure the amount of immunoglobulin in serum and select mice that cannot be detected. This mouse is again mated with a NOD / Shi mouse. This operation can be carried out by repeating it 9 times or more (Cross Intercross method).

  Both NOD / Shi mice and C.B-17 / Icr-scid mice are sold by Clea Japan. Further, as a mouse obtained by crossing these mice, for example, there is a NOD-scid mouse (Japanese Patent Laid-Open No. 9-94040), which is obtained from Nippon Claire Co., Ltd. and used directly as a mouse of A. it can.

  In addition, knockout of the interleukin 2 receptor γ chain gene in B mice can be performed by techniques known to those skilled in the art, for example, homologous recombination techniques using mouse ES cells (Capecchi, MR, Altering the genome by homologous recombination, According to Science, (1989) 244, 1288-1292), a specific gene derived from a mouse and a homologous gene containing a drug resistance gene such as neomycin are replaced at the ES cell stage, and then the ES cell is injected into a fertilized egg Can be implemented.

Specifically, for example, a gene clone containing mouse IL-2Rγ was isolated from a genomic library of 129 / SV mouse using human IL-2Rγ cDNA as a probe, and among these clones, a size of 8.6 kb containing the full length IL-2Rγ. A targeting vector is prepared using this fragment. That is, pMC1-neo poly A expressing a neomycin resistance gene is inserted between exon 7 and 8 of IL2R of the above fragment, and diphtheria toxin-A gene is placed 3 ′ from exon 8 at a distance of 1 kb. Next, the vector is linearized and introduced into 1 × 10 ⁷ E14 ES cells by electroporation. Then, select ES clones that have undergone homologous recombination in a culture medium containing G418 (confirmed by PCR or Southern method), and inject the ES clones into blastocysts of C57BL / 6 mice, Transplanted into the uterus. A chimeric mouse born from this foster parent mouse can be further crossed with a C57BL / 6 mouse to obtain an IL2RγKO hetero mouse that has been transmitted to the germ line.

  Alternatively, an already established interleukin 2 receptor γ chain gene knockout mouse strain may be directly obtained and used. For example, the IL2RγKO mouse strain (Professor Kazuo Tsujimura [Tohoku University School of Medicine, Department of Bioprotection)・ Interleukin-2 receptor gamma chain knockout mice (Ohbo K, Suda T, Hashiyama M, et al., Modulation of hematopoiesis in mice with a truncated mutant of the interleukin-2 receptor gamma chain. Blood 1996; 87 (3): 956-67)). IL2RγKO mice are now preserved in the embryo preservation bank of the Central Institute for Experimental Animals, under the commission of Prof. Sugawara, the creator, and can be used as frozen embryos or thawed mice whenever necessary. It can be obtained.

  Further, backcrossing of the B mouse to the A mouse is performed in the same manner as described above according to a method known to those skilled in the art, for example, the above-mentioned backcrossing, that is, NOD-scid mouse and IL2RγKO mouse are crossed, and the F1 This can be done by backcrossing mice to NOD-scid mice.

Construction of the human megakaryocyte / platelet hematopoietic system of the present invention The age of the mouse used is preferably 8 to 14 weeks old, but is not limited thereto, for example, a mouse 1 to 3 days after birth may be used. Is possible. Since mice are immunodeficient, it is desirable to keep them in a breeding facility capable of protecting against infection with microorganisms.

  In order to improve the transplantation efficiency, a mouse bone marrow destruction operation may be performed. The mouse bone marrow destruction operation can be performed, for example, by irradiation or administration of an anticancer agent. Radiation is applied to the mouse whole body using gamma rays or X-rays. The radiation dose is 3.4 gray or less, preferably 1.0 gray to 3.0 gray, but is not limited thereto. The dose rate of radiation is preferably 0.1 gray / minute to 0.8 gray / minute, more preferably 0.2 gray / minute to 0.6 gray / minute, but is not limited thereto. In setting the voltage and current when using X-rays, the voltage is preferably from 100 kilovolts to 150 kilovolts, more preferably from 125 kilovolts to 140 kilovolts, and the current is preferably from 2 milliamperes to 20 milliamperes, and Preferably 5 to 10 milliamps is used, but is not limited thereto.

  Here, human stem cells mainly mean human hematopoietic stem cells, but can also be mixed with cells that support the human hematopoietic system, such as human mesenchymal stem cells and human vascular endothelial progenitor cells.

  Human hematopoietic stem cells can be identified, for example, as CD34 positive cells or as CD133 positive cells by cell surface markers associated with specific epitope sites identified by antibodies (Stem Cells, 1998; 16, p. 387-396, Ann. NY Acad. Sci., 1999; 872, p. 25-39). Further, for example, a partial population in CD34 positive cells or CD133 positive cells can be identified as, for example, CD38 negative cells, CD33 negative cells, or a combination thereof based on the presence or absence of a cell surface marker (Bone Marrow Transplant). ., 2002; 30, p. 851-860). The identified human hematopoietic stem cells can be separated by, for example, a sorting technique using a flow cytometer or a technique using an antibody bound to a magnetic bead, or, for example, human CD34-positive cells or CD133-positive cells can be separated by Cambrex Bio Science. It can be purchased from Walkersville, Inc. (MD, USA) or All Cells, LLC (CA, USA). As the origin of the cell, for example, it can be collected from human bone marrow, human umbilical cord blood or human peripheral blood, or after being collected from human bone marrow, human umbilical cord blood or human peripheral blood, it is amplified in a test tube. It can be collected or differentiated from human ES cells in vitro (Blood, 2004; 104, Abstract # 564), or the obtained human hematopoietic stem cells can be used in sheep fetuses and immune cells. It can also be collected after being transplanted into an immunotolerant animal such as a deficient mouse and amplified in the animal body. The collected cells can also be used after introducing the gene exogenously using a viral vector, electroporation, liposome, gene gun or the like.

  Cells that support the human hematopoietic system include human stromal cells that adhere and proliferate in vitro and cell surface markers, such as human-like cells identified as CD105 positive, CD166 positive, CD29 positive and CD44 positive cells. Stem cells can be used (Exp Hematol., 2004; 32, p. 657-664). Alternatively, for example, human vascular endothelial progenitor cells identified as CD34 positive and vascular endothelial growth factor receptor-2 (KDR) positive cells can be used (Blood. 2005; 105, p. 199-206).

  In the method of the present invention, human stem cells identified or isolated as described above or a mixture of human stem cells and cells supporting the human hematopoietic system may be transplanted into NOG mice.

  The number of transplanted human stem cells is preferably 1,000 to 10,000,000 cells, more preferably 20,000 to 200,000 cells per mouse, but is not limited thereto. Regarding the time for transplantation of human stem cells, the time after irradiation is preferably within 96 hours immediately after irradiation, more preferably within 4 hours after 72 hours, but is not limited thereto.

  As a method for transplanting human stem cells, for example, in the case of a mouse aged 8 to 14 weeks, a 27 gauge needle is used to transplant the mouse into the tail vein, orbital venous sac, or femur. For example, in the case of a mouse 1 to 3 days after birth, it can be implanted into the abdominal cavity, liver or temporal vein of the mouse using a 27 or 30 gauge injection needle. However, it is not limited to these.

  As a method for measuring human platelets present in mouse blood after transplantation, for example, a method using a flow cytometer is possible. Blood is collected from the tail vein, orbital venous sac, or heart as EDTA, citrate, or heparinized blood, and whole blood or platelet-rich plasma (PRP) is prepared. Next, staining is performed using species-specific antibodies having different fluorescent colors against surface markers that are specifically expressed by platelets, such as CD41a, CD61, or CD42b. Using a flow cytometer, cells that react with either human-specific antibodies or mouse-specific antibodies are recognized as human platelets or mouse platelets after gating on the platelet region with forward and side scattered light, respectively. The chimera rate can be calculated from the ratio of the number of platelets of each species. In addition, the absolute number of human platelets can be calculated, for example, by multiplying the total platelet count measured by an automatic hemocytometer or the like with the previously calculated chimera rate.

  As a method for measuring the function of human platelets present in mouse blood after transplantation, for example, a method using a flow cytometer is possible. Blood is collected from the tail vein, orbital venous sac, or heart of the transplanted mouse as EDTA, citrate or heparinized blood, and whole blood or platelet-rich plasma (PRP) is prepared. Next, whole blood collected from mice or platelets in PRP is activated by a platelet activating factor such as adenosine diphosphate (ADP), thrombin, thrombin receptor activating peptide (TRAP) or epinephrine. At that time, a fibrin polymerization inhibitor or the like may be added. Next, a platelet-specific surface marker such as CD41a, CD61 or CD42b, a species-specific antibody having a different fluorescent color, and an activated platelet-specific surface marker such as activated integrin (gpIIb / IIIa) or Stain using antibodies with different fluorescent colors against CD62p. Using a flow cytometer, cells that react with either human-specific antibodies or mouse-specific antibodies are recognized as human platelets or mouse platelets after gating on the platelet region with forward and side scattered light, respectively. After that, the ability to activate human platelets can be measured by measuring the expression level of activated gpIIb / IIIa or CD62p.

  The present invention relates to a method for producing a NOG mouse having a human megakaryocyte / platelet hematopoietic system in which human megakaryocytes / platelets are formed in the body, including transplanting human hematopoietic stem cells into NOG mice, and a method obtained by the method. It also includes NOG mice that have human megakaryocytes / platelet hematopoietic system.

Screening method for evaluating the effects of candidate compounds in the form of increase / decrease and / or retention, enhancement or decrease of function of human platelets of the present invention, which acts on human megakaryocytes / platelet hematopoiesis in NOG mice transplanted with human stem cells Administer substances (compounds, etc.) such as candidate compounds, antibodies, peptides or proteins expected to have or not. The compound or the like is dissolved or suspended in a suitable solvent such as physiological saline, phosphate buffered saline or methylcellulose solution. At that time, a solubilizer such as polyethylene glycol or ethanol, or a stabilizer such as bovine serum albumin may be added. As an administration route, administration can be performed subcutaneously, abdominally, tail vein, orally. Alternatively, the osmotic pump may be filled with a compound or the like and placed in the mouse subcutaneously. The dose can be appropriately determined depending on the type of compound to be administered.

  As for the number of cases in one group, the effect can be confirmed in two or more cases. As the administration period, single administration or continuous administration is selected depending on the properties of the compound and the like. There is no limitation on the duration of administration for one NOG mouse in which a human megakaryocyte / platelet hematopoietic system has been constructed, and there is no limitation on the type of compound administered, etc. Can be evaluated.

  After administering a compound, etc. to a mouse, by measuring the number and / or function of human platelets in the mouse body, it is determined whether the compound has an effect as a platelet number regulator or a platelet number non-influencing drug can do. For example, a drug having a platelet number fluctuation action may be selected as a platelet number regulator, or a drug having no platelet fluctuation action may be selected as a platelet number non-influence drug. For example, when NOG mice administered with human stem cells are divided into two groups, a compound or the like is administered to one group (compound administration group), and a solvent is administered to the other group (solvent administration group), the compound administration group When the number of human platelets in mice is significantly higher or lower than that in the solvent-administered group, it can be determined that the compound or the like has an effect of varying the number of platelets as a platelet number regulator. Alternatively, when there is no significant difference in the number of human platelets, it can be determined that the compound or the like does not have an effect of varying the number of platelets as a platelet number non-influencing drug. At this time, not only the number of platelets but also whether or not the function of human platelets in the mouse body is maintained, enhanced, or decreased can be a criterion for determination.

  Candidate compounds and the like that are confirmed to be effective by screening are platelet-regulators, that is, thrombocytopenia as a therapeutic agent for thrombocytopenia, thrombocytopenia as a therapeutic agent for thrombocytosis, and / or hypoplatelet function , Or can be used as a platelet hypersensitivity drug as a therapeutic agent for platelet hyperfunction, or a platelet function lowering drug, or as a platelet count non-influence drug, that is, thrombocytopenia, or platelet count increase, In addition, it can be used as a non-platelet effect drug that does not induce hypoplatelet function or platelet hyperfunction. Thrombocytopenia includes, for example, idiopathic thrombocytopenic purpura, or myelodysplastic syndrome, aplastic anemia, chronic hepatitis, cirrhosis, thrombocytopenia due to disseminated intravascular coagulation, or thrombocytopenia associated with bleeding Or thrombocytopenia caused by chemotherapy, hematopoietic stem cell transplantation or radiation therapy, but is not limited thereto. Examples of thrombocytosis include essential thrombocythemia, thrombocytosis caused by chronic myelogenous leukemia, myeloproliferative syndrome, myelofibrosis or polycythemia, or thrombocytosis occurring after liver transplantation, etc. Is not limited to this.

  Here, the platelet number non-influence drug is a substance that does not induce hypoplatelet function or hypertension, and includes a non-platelet increasing drug that does not increase platelets and a non-platelet reducing drug that does not decrease platelets. For example, certain cytokines, anticancer agents, antibiotic preparations, and central nervous system agents have the side effect of increasing platelets or decreasing platelets. According to the present invention, it is possible to modify a drug that affects the number and function of platelets, and to screen for drugs that have only reduced effects on platelets without reducing the original effects of the drug. For example, in the case of a protein such as interferon, an interferon in which some amino acids are substituted, deleted or added, or modified is prepared, and whether or not it affects the platelet count or function is measured by the method of the present invention. That's fine. In the case of a compound, the group of the compound may be modified to determine whether it similarly affects the platelet count or function. Drugs that affect platelet count or function include cytokines such as interferon alpha, interferon beta, interferon gamma, sermoleukin, and teseleukin, various anticancer agents such as busulfan, cyclophosphamide, doxorubicin, cisplatin, and heparin. A pharmaceutical derivative that affects the platelet count or function by the method of the present invention and that does not affect the platelet count, for example, a pharmaceutical derivative that has the side effect of increasing or decreasing the platelet count. Thus, it is possible to screen for a drug that maintains the pharmaceutical effect but has reduced side effects of increasing or decreasing the platelet count. A specific example of such a medicament is an interferon derivative that has a reduced function of changing the platelet count.

  X-rays (2.4 gray, 0.6 gray / min; 125 kV, 10 mA; using Hitachi Medical MBR-1520R-3) were irradiated to NOG mice (12 weeks old, female; obtained from the Institute for Experimental Animal Research) Four hours later, 115,000 human umbilical cord blood-derived CD34 positive cells (obtained from AllCells, LLC) per mouse were transplanted from the tail vein. Surface markers of the transplanted cells were stained with PE-labeled anti-human CD34 antibody, FITC-labeled anti-human CD33 antibody and PE-Cy5-labeled anti-human CD38 antibody (purchased from BD Pharmingen), and flow cytometer (Beckman Coulter) We analyzed with. As a result, 83% of the cells were CD34 positive, and 49% of the cells were CD34 positive, CD38 negative, and CD33 negative.

  At 10 weeks after transplantation, the chimeric rate of human platelets was measured using a flow cytometer. Blood was collected from the tail vein or orbital venous sac or heart as EDTA or 0.38% citrated blood. Using 2 to 10 microliters of whole blood, antibodies against CD41a, a surface marker that specifically expresses platelets (PE-labeled anti-human CD41a antibody and FITC-labeled anti-mouse CD41 antibody, both purchased from BD Pharmingen) Stained. Using a flow cytometer, cells that react with either PE-labeled anti-human CD41a antibody or FITC-labeled anti-mouse CD41 antibody were humanized using a gate pre-applied to the platelet region by forward scattered light and side scattered light. Recognized as platelets or mouse platelets, the chimera rate was calculated from the ratio of the number of platelets of each species. In addition, the absolute number of human platelets was calculated by multiplying the total platelet count measured by an automatic hemocytometer or the like with the previously calculated chimera rate. As a result, human platelets were confirmed in 6 out of 6 cases, the average and standard deviation of the chimera rate of human platelets was 0.47 ± 0.37%, and the average and standard deviation of the absolute number of human platelets per microliter of blood Was 2,500 ± 1,800.

  These mice are divided into two groups of three and three, and 5-[(2- {1- [5- (3,4-) having an agonistic effect on the Mpl protein described in WO 2004/108683. Dichlorophenyl) -4-hydroxy-3-thienyl] ethylidene} hydrazino) carbonyl] -2-thiophenecarboxylic acid potassium salt (hereinafter referred to as Compound A) administration group and solvent administration group. As the solvent, a solution (PBS-PE) in which 1.6% polyethylene glycol 400 and 2.4% ethanol were added to phosphate buffered saline (PBS) was used. Compound A was injected subcutaneously at 30 milligrams per kilogram of mouse body weight. Administration was performed once a day from the 10th week after transplantation, 8 times. As shown in Fig. 1, the number of human platelets increased significantly by 4.5 times by the second week, and then the number of human platelets that increased was almost the same as the value before administration and the value of the solvent administration group . At this time, mouse platelets were not affected.

  NOG mice (11 weeks old, female) were irradiated with X-rays (2.4 gray, 0.6 gray / min; 125 kV, 10 mA), and 4 hours later, 60,000 human umbilical cord blood-derived CD34 positive cells (AllCells, (Obtained from LLC) was transplanted from the tail vein. As a result of analyzing the surface marker of the transplanted cells with a flow cytometer, 79% of the cells were CD34 positive, and 56% of the cells were CD34 positive, CD38 negative and CD33 negative.

  At 10 weeks after transplantation, the chimeric rate of human platelets was measured using a flow cytometer, and the absolute number was calculated. As a result, human platelets were confirmed in 6 out of 6 cases, the average and standard deviation of the chimera rate of human platelets was 0.42 ± 0.13%, and the average and standard deviation of the absolute number of human platelets per microliter of blood The number was 2,800 ± 800.

  These mice were divided into two groups of 3 and 3 mice, which were divided into a compound A administration group and a solvent administration group. PBS-PE was used as the solvent. Compound A was injected subcutaneously at 30 milligrams per kilogram of mouse body weight. Administration was performed once a day from the 10th week after transplantation for 5 weeks and 27 times. As shown in FIG. 2, the number of human platelets significantly increased by 4.5 times by administration up to 2 weeks, and significantly increased by 6.4 times by administration by 5 weeks. In addition, when the administration was discontinued from the 5th week and the number of human platelets was measured again from the first administration to the 10th week, the increased number was almost equal to the value before administration and the value of the solvent administration group. Indicated.

  NOG mice (10 weeks old, female) were irradiated with X-rays (3.0 grey, 0.2 grey / min; 145 kilovolts, 5 milliamps), and after 4 hours, 85,000 human umbilical cord blood-derived CD34 positive cells (AllCells, (Obtained from LLC) was transplanted from the tail vein.

  At 19 weeks after transplantation, the chimeric rate of human platelets was measured using a flow cytometer, and the absolute number was calculated. As a result, human platelets were confirmed in 4 out of 4 cases, the average and standard deviation of the chimera rate of human platelets was 1.86 ± 1.39%, and the absolute number of human platelets was the average and standard deviation per microliter of blood There were 9,700 ± 7,200 pieces.

  These mice were divided into two groups of 3 and 1 group, which were divided into a compound A administration group (3 animals) and a solvent administration group (1 animal). PBS-PE was used as the solvent. Compound A was injected subcutaneously at 30 milligrams per kilogram of mouse body weight. Administration was performed once a day for 2 weeks and 14 times from the 20th week after transplantation. As shown in FIG. 3, the number of human platelets significantly increased to 4.8 times after administration up to the second week.

  Six mice used in Example 2 were withdrawn for 2 months and divided into two groups of 3 and 3 mice, which were divided into a compound A administration group and a solvent administration group. PBS-PE was used as the solvent. Compound A was injected subcutaneously at 30 milligrams per kilogram of mouse body weight. The administration was performed once a day from the 26th week after transplantation for 17 times for about 2.5 weeks. The number of human platelets administered up to day 17 was 4.1 ± 0.6 times in the compound A administration group and 1.5 ± 0.5 times (mean ± standard error) in the solvent administration group, which was significant (p value <0.05; Student) t-test).

  NOG mice (10 weeks old, female) were irradiated with X-rays (2.4 grey, 0.2 grey / min; 145 kilovolts, 5 milliamps), and 4 hours later, 120,000 human umbilical cord blood-derived CD34 positive cells (Cambrex Bio) Science Walkersville, Inc.) was transplanted from the tail vein. As a result of analyzing the surface markers of the transplanted cells with a flow cytometer, 76% of the cells were CD34 positive, and 47% of the cells were CD34 positive, CD38 negative and CD33 negative.

  Immediately after transplantation, the mice were divided into two groups of 5 and 5 groups, which were a compound A administration group and a solvent administration group. PBS-PE was used as the solvent. Compound A was injected subcutaneously so that 30 milligrams per kilogram of mouse body weight was administered. The administration was performed once a day from the first day after transplantation for about 2 weeks and 11 times.

  On the 13th day after transplantation, the chimeric rate of human platelets was measured using a flow cytometer, and the absolute number was calculated. As a result, human platelets were confirmed in 10 of 10 cases. The mean and standard deviation of the absolute number of human platelets per microliter of blood in the solvent-administered group is 2,500 ± 1,000, and the average and standard deviation of absolute numbers per microliter of human platelets in the compound A-administered group are The number was 5,600 ± 1,200 (p value = 0.02; Student t-test), and a significant increase was observed.

  It was examined that the human platelets monitored with a flow cytometer were functional platelets. On day 16, one animal was sacrificed from the compound A administration group and the solvent administration group, and 0.38% citrated blood was obtained from the heart. Platelet rich plasma (PRP) is obtained by centrifugation, stimulated with 10 micromol / liter ADP for 15 minutes, and antibodies (PE-labeled anti-human CD62p antibody, PE-Cy5-labeled anti-human CD41a antibody and FITC-labeled anti-mouse CD41 antibody or The cells were stained with PE-labeled anti-human CD42b antibody and FITC-labeled anti-human activated CD41a antibody (clone name PAC-1); both purchased from BD Pharmingen. Stimulation was stopped with 0.5% paraformaldehyde fixation. Using a flow cytometer, cells that respond to either PE-Cy5-labeled anti-human CD41a antibody or FITC-labeled anti-mouse CD41 antibody using a gate pre-applied to the platelet region by forward scattered light and side scattered light Each was recognized as human platelet or mouse platelet and gated to examine the reactivity of PE-labeled anti-human CD62p antibody. As a result, in the PRP derived from both the compound A-administered group and the solvent-administered group, an increase in cell surface expression of CD62p antigen by ADP stimulation with about 50% human platelets was observed. In addition, it was recognized as human platelets with PE-labeled anti-human CD42b antibody and gated to examine the reactivity of FITC-labeled PAC-1 antibody. As a result, in the PRP derived from the mice in both the compound A administration group and the solvent administration group, an increase in activated CD41a antigen by ADP stimulation was observed in about 70% of human platelets. On the other hand, the positive rate of CD62p antigen or activated CD41a antigen when ADP-unstimulated PRP was used was 10% or less. From the above, it was found that human CD41-positive platelets present in NOG mice transplanted with human umbilical cord blood-derived CD34-positive cells retain their functions.

  NOG mice (36-48 hours after birth, male and female) were irradiated with X-rays (1.0 gray, 0.6 gray / min; 125 kilovolts, 10 milliamps), and after 4 hours, 110,000 human umbilical cord blood-derived CD34 positive cells per mouse ( Cambrex Bio Science Walkersville, Inc.) was transplanted from the peritoneal cavity. As a result of analyzing the surface markers of the transplanted cells with a flow cytometer, 62% of the cells were CD34 positive, and 45% of the cells were CD34 positive, CD38 negative and CD33 negative.

  At 27 weeks after transplantation, the chimeric rate of human platelets was measured using a flow cytometer, and the absolute number was calculated. As a result, human platelets were confirmed in 4 out of 4 females, and the average and standard deviation of the chimera rate of human platelets was 5.21 ± 3.44%. The average and standard of the absolute number of human platelets per microliter of blood The deviation was 30,000 ± 17,000.

  In addition, the chimera rate of human platelets was measured using a flow cytometer 45 weeks after transplantation of a female individual different from the above, and the absolute number was calculated. As a result, human platelets were confirmed in 4 out of 4 cases, the average and standard deviation of the chimera rate of human platelets was 4.44 ± 3.40%, the average and standard deviation of the absolute number of human platelets per microliter of blood The number was 31,000 ± 26,000.

  The mice 45 weeks after transplantation were divided into two groups of 2 and 2 animals, and they were divided into an interferon alpha-2b (trade name Intron A, Schering-Plough Co., INF) administration group and a solvent administration group. As the solvent, a solution in which 0.1% bovine serum albumin was added to PBS was used. INF was injected subcutaneously so that 1 million international units were administered per kilogram of mouse body weight. Administration was once a day, 9 times. The number of human platelets is 0.4 ± 0.2 times in the INF-administered group and 1.1 ± 0.1 times (mean ± standard deviation) in the INF-administered group up to the 10th day, and decreases significantly (p value <0.05; Student t-test) did. At this time, mouse platelets were not affected.

The degree of change in human platelet count when compound A was administered from the 10th week after transplantation of human cord blood CD34 positive cells was shown. Administration was performed 8 times a week. The vertical axis shows the degree of change (multiplier) when the number of human platelets before administration is 1 in each individual. The horizontal axis indicates the number of days from the administration start date (1 day). ■ indicates the average value of the compound A administration group (3 cases), and ○ indicates the average value of the solvent administration group (3 cases). Error bars indicate standard deviation. * Indicates p value <0.05 (Student t-test). In the second week, Compound A showed a significant increase in human platelets compared to the solvent-administered group. The degree of change in human platelet count when compound A was administered from the 10th week after transplantation of human cord blood CD34 positive cells was shown. Administration was carried out 27 times for 5 weeks. The vertical axis shows the degree of change (multiplier) when the number of human platelets before administration is 1 in each individual. The horizontal axis indicates the number of days from the administration start date (1 day). ■ indicates the average value of the compound A administration group (3 cases), and ○ indicates the average value of the solvent administration group (3 cases). Error bars indicate standard deviation. * Indicates p value <0.05, ** indicates p value <0.01 (Student t-test). In the first week, Compound A showed a significant increase in human platelets compared to the solvent-administered group. The changes in the number of human platelets when compound A was administered from the 20th week after transplantation of human cord blood CD34 positive cells were shown. Administration was carried out 14 times for 2 weeks. The vertical axis shows the degree of change (multiplier) when the number of human platelets before administration is 1 in each individual. The horizontal axis indicates the number of days from the administration start date (1 day). ■ shows the average value of the compound A administration group (3 cases), and □ shows the solvent administration group (1 case). Error bars in the compound A administration group indicate standard deviation. * Indicates p-value <0.05 (Paired t-test). In the second week, Compound A showed a significant increase in human platelets compared to the solvent-administered group.

Claims (9)

  A platelet number regulator or platelet comprising administering a candidate substance to a NOG (NOD / Shi-scid, IL-2Rγ KO) mouse transplanted with human stem cells and measuring the number and / or function of human platelets in the mouse How to screen for non-influential drugs.   The screening method according to claim 1, wherein the platelet number regulator is a thrombocytosis drug.   The screening method according to claim 1, wherein the platelet number regulator is a thrombocytopenia.   The screening method according to claim 1, wherein the platelet number regulator is a platelet function maintaining, enhancing or decreasing agent.   The screening method according to claim 1, wherein the platelet number non-influencing drug is a non-platelet increasing drug.   The screening method according to claim 1, wherein the platelet number non-influencing drug is a non-platelet reducing drug.   The screening method according to claim 1, wherein the platelet number non-influencing drug is a drug for maintaining, enhancing or decreasing platelet function.   A method for producing a mouse model having a human megakaryocyte / platelet hematopoietic system, comprising transplanting human stem cells into NOG (NOD / Shi-scid, IL-2Rγ KO) mice.   NOG (NOD / Shi-scid, IL-2Rγ KO) mouse model in which human stem cells are transplanted and has a human megakaryocyte / platelet hematopoietic system.

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