Combination therapy with substance P for hematopoietic stem cell mobilization

Abstract

The present invention relates to a combination therapy and combination product for mobilizing large numbers of hematopoietic stem cells in the blood, comprising substance P and AMD3100 or a pharmaceutically acceptable salt thereof; a method for collecting large numbers of mobilized hematopoietic stem cells, comprising a step of separating a blood fraction containing mobilized hematopoietic stem cells from peripheral blood obtained from an individual to whom substance P and AMD3100 or a pharmaceutically acceptable salt thereof have been sequentially administered; and the use of a formulation comprising substance P and AMD3100 or a pharmaceutically acceptable salt thereof for mobilizing large numbers of hematopoietic stem cells in the blood.

Description

Technical Field

[0001] The present invention relates to a combination therapy and a combination product for mobilizing a large number of hematopoietic stem cells in the blood, comprising substance P and AMD3100 or a pharmaceutically acceptable salt thereof; a method for collecting a large number of mobilized hematopoietic stem cells, comprising the step of separating a blood fraction containing mobilized hematopoietic stem cells from peripheral blood obtained from an individual administered substance P and AMD3100 or a pharmaceutically acceptable salt thereof sequentially; and the use of a formulation comprising substance P and AMD3100 or a pharmaceutically acceptable salt thereof for mobilizing a large number of hematopoietic stem cells in the blood.

Background Art

[0002] Transplanting hematopoietic stem cells (HSCs) mobilized or migrated from bone marrow to peripheral blood has replaced conventional bone marrow transplantation for patients suffering from blood diseases. Some clinical practices for mobilizing hematopoietic stem cells from bone marrow to peripheral blood are achieved by a therapy of administering recombinant granulocyte colony stimulating factor (G-CSF) (Granulocyte colony stimulating factor), which is thought to stimulate the production of proteases that disrupt the CXCR4 / SDF-1 interaction, continuously for at least 5 days. Granulocyte colony stimulating factor (G-CSF) is a glycoprotein that stimulates the survival, proliferation, differentiation and function of neutrophil granulocyte progenitor cells and mature neutrophils.

[0003] However, G-CSF has been reported to be ineffective in large patient cohorts and to be associated with several side effects, such as bone pain, splenomegaly, and rarely, splenic rupture, myocardial infarction, and cerebrovascular disease. Furthermore, G-CSF has been shown to induce a strong inflammatory response and to induce the recruitment of various types of blood cells, in addition to stem cells, into the peripheral blood, and there is a reported risk of thrombotic complications with long-term administration. In particular, G-CSF requires subcutaneous injection for at least 5 consecutive days, during which the patient experiences thigh pain, so caution is necessary when using G-CSF clinically. The main mechanism of action of G-CSF is the promotion of neutrophil production, and the treatment of neutropenia is a primary target.

[0004] The inherent shortcomings of G-CSF have led to efforts to identify alternative recruitment methods based on small molecules. For example, the FDA-approved CXCR4 antagonist AMD3100 (Plerixafor; Mozobil®) has limited toxicity issues and is known to rapidly recruit hematopoietic stem cells. However, clinical recruitment using AMD3100 has been shown to be effective only when used in combination with G-CSF. The current clinical dosage of combination therapy with G-CSF and AMD3100 involves subcutaneous injection of G-CSF once daily for five consecutive days, followed by blood collection one hour after AMD3100 administration, at which point hematopoietic stem cell migration into the peripheral blood is known to be maximized.

[0005] However, responses to combination therapy with G-CSF and ADM3100 vary among patients, and it is known that some patients do not show any change in hematopoietic stem cell levels in their blood (non-responders). Furthermore, even though G-CSF is the most widely used migrator clinically, it has further drawbacks (e.g., potential toxic side effects, induction of strong inflammatory responses, a relatively long treatment course (e.g., 5-7 days of continuous injections), and variability in responses among patients).

[0006] Therefore, the search for rapid, selective, and G-CSF-independent mobilization methods remains a clinical concern. Research and development of alternative mobilizers to G-CSF is currently underway.

[0007] Shun He et al. reported that co-administration of FLT3 ligand (FLT3L) and AMD3100 in mice increased hematopoietic stem cell recruitment (Non-Patent Document 1).

[0008] Furthermore, MP Rettig et al. have disclosed a method for recruiting hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HSPCs) by co-administering an integrin α4β1 (very slow antigen 4 (VLA-4)) inhibitor and a CXCR4 inhibitor. In particular, it has been reported that co-administration of the VLA-4 antagonists BIO5192 and AMD3100 in mice has an additive effect on the recruitment of hematopoietic progenitor cells (HPSCs) (Non-Patent Literature 2).

[0009] Furthermore, Kasia Mierzejewska et al. reported that co-administration of phenylhydrazine (PHZ) and the CXCR4 inhibitor AMD3100 increased hematopoietic stem cell recruitment in mice (Non-Patent Literature 3).

[0010] Furthermore, Patent Document 1 discloses a method for detaching and releasing HSCs using an α9 (alpha-9) integrin antagonist and a CXCR4 antagonist.

[0011] However, despite research and development into alternative mobilizers to G-CSF, the combination therapy of G-CSF and AMD3100 remains the only recognized standard treatment.

[0012] On the other hand, substance P is an 11-amino acid neuropeptide expressed in sensory neurons, macrophages, eosinophils, endothelial cells, epithelial cells, corneal cells such as corneal stromal cells, and granulation tissue. Several reports suggest that substance P is involved in neuroimmunological communication for hematopoietic regulation. The nerve fibers of substance P neurons are distributed in the bone marrow matrix, and substance P transmits signals via the surface receptor NK-1 on bone marrow matrix cells, stimulating these cells to produce stem cell factors and interleukin-1, which are favorable to promoting hematopoiesis as suppliers.

[0013] In relation to the role of substance P in the migration and repopulation of mesenchymal stem cells in the aforementioned research, the inventors have clarified in Patent Document 2 the use of substance P as a wound healing agent or wound healing promoter, the use of substance P as a mesenchymal stem cell release or release promoter, the use of substance P as a mesenchymal stem cell proliferation or proliferation promoter, and the use of the same as a wound healing agent or wound healing promoter.

[0014] However, nothing had been known about the combination therapy of Substance P and AMD3100 until now. [Prior art documents] [Patent Documents]

[0015] [Patent Document 1] Korean Published Patent Gazette No. 10-2017-0105514 [Patent Document 2] Korean Patent No. 10-593397 [Non-patent literature]

[0016] [Non-Patent Document 1] Shun He et al., "FLT3L and Plerixafor Combination Increases Hematopoietic Stem Cell Mobilization and Leads to Improved Transplantation Outcome," Biol Blood Marrow Transplant (2014), 20(3): 309-313 [Non-Patent Document 2] MP Rettig et al., "Mobilization of hematopoietic stem and progenitor cells using inhibitors of CXCR4 and VLA-4," Leukemia (2012), 26: 34-53 [Non-Patent Document 3] Kasia Mierzejewska et al., "Sphingosine-1-phosphate-Mediated Mobilization of Hematopoietic Stem / Progenitor Cells during Intravascular Hemolysis Requires Attenuation of SDF-1-CXCR4 Retention Signaling in Bone Marrow," Hindawi Publishing Corporation, BioMed Research International (2013), Article ID 814549, p. 5 [Overview of the project] [Problems that the invention aims to solve]

[0017] The inventors of this invention aim to provide a combination therapy that can replace conventional G-CSF and ADM3100 combination therapy for mobilizing hematopoietic stem cells in the blood, given that G-CSF requires a relatively long treatment course (e.g., continuous injections for 5 to 7 days) and has the side effect of inducing a strong inflammatory response. [Means for solving the problem]

[0018] As a result of repeated studies to solve the above problems, the inventors of the present invention found that when substance P is used in combination with AMD3100 instead of G-CSF, it has the same effect as mobilizing hematopoietic stem cells into peripheral blood by a single intravenous injection of substance P. Since substance P does not have the function of a powerful neutrophil mobilizer that G-CSF has, the side effects of the inflammatory reaction are much less, and it is found that there is a possibility of mobilizing more hematopoietic stem cells into the blood as compared with the conventional combined therapy of G-CSF and ADM3100, and the present invention has been completed.

[0019] Therefore, an object of the present invention is to provide a combined therapy for mobilizing a large amount of hematopoietic stem cells in blood, which comprises substance P and AMD3100 or a pharmaceutically acceptable salt thereof.

[0020] Another object of the present invention is to provide a combination product for mobilizing a large amount of hematopoietic stem cells in blood, which comprises substance P and AMD3100 or a pharmaceutically acceptable salt thereof.

[0021] Another object of the present invention is to provide a method for collecting a large amount of mobilized hematopoietic stem cells, which comprises a step of separating a blood fraction containing mobilized hematopoietic stem cells from peripheral blood obtained from an individual administered with substance P and AMD3100 or a pharmaceutically acceptable salt thereof in sequence.

[0022] Another object of the present invention is to provide the use of a formulation containing substance P and AMD3100 or a pharmaceutically acceptable salt thereof for mobilizing a large amount of hematopoietic stem cells in blood.

Effects of the Invention

[0023] The combination therapy of the present invention, comprising Substance P and ADM3100 or a pharmaceutically acceptable salt thereof, activates hematopoietic stem cells in the peripheral blood with only a single intravenous injection of Substance P, significantly reducing the burden on patients during treatment compared to conventional G-CSF and ADM3100 combination therapy, which requires a long treatment course of continuous injections for 5 to 7 days. Furthermore, because Substance P does not possess the potent neutrophil mobilizer function of G-CSF, it has far fewer inflammatory side effects and may be able to activate more hematopoietic stem cells in the blood compared to conventional G-CSF and ADM3100 combination therapy. In addition, since the blood fraction collected by the collection method of the present invention contains a large amount of mobile hematopoietic stem cells, it can be transplanted or reinjected into patients suffering from blood disorders and used for the prevention or treatment of various diseases requiring hematopoietic stem cells. [Brief explanation of the drawing]

[0024] [Figure 1] This graph shows the results of comparing the levels of hematopoietic stem cells in the blood of rabbits after intravenous injection of Substance P (SP) and G-CSF, respectively, over a period of three days. [Figure 2] This graph shows the results of comparing the ratio of immune cells in the blood of rabbits after intravenous injection of Substance P (SP) and G-CSF, respectively, for three days (neutrophil: neutrophil, monocyte: monocyte, lymphocyte: lymphocyte, B+E (basophil + eosinophil): basophil + eosinophil). [Figure 3] This is a schematic diagram showing the administration schedule for conventional combination therapy with G-CSF and ADM3100, and the administration schedule for combination therapy with Substance P and ADM3100 according to the present invention. [Figure 4] This graph shows the results of comparing the levels of hematopoietic stem cells in collected blood after administering the conventional combination therapy of G-CSF and ADM3100 to mice according to the administration schedule of the combination therapy of Substance P and ADM3100 of the present invention, respectively. [Modes for carrying out the invention]

[0025] The inventors found that when substance P is used in combination with AMD3100 instead of G-CSF, substance P exhibits a similar effect, activating hematopoietic stem cells into the peripheral blood with only a single intravenous injection. Furthermore, because substance P does not possess the potent neutrophil mobilizer function of G-CSF, it has far fewer inflammatory side effects and has the potential to activate more hematopoietic stem cells into the bloodstream compared to conventional combination therapy of G-CSF and ADM3100.

[0026] Accordingly, the present invention provides a combined therapy for mobilizing a large number of hematopoietic stem cells in the blood, comprising substance P and AMD3100 or a pharmaceutically acceptable salt thereof.

[0027] The present invention also provides a combination product for mobilizing large amounts of hematopoietic stem cells in the blood, comprising substance P and AMD3100 or a pharmaceutically acceptable salt thereof.

[0028] The present invention also provides a method for collecting a large quantity of mobile hematopoietic stem cells, comprising the step of separating a blood fraction containing mobile hematopoietic stem cells from peripheral blood obtained from individuals sequentially administered with substance P and AMD3100 or a pharmaceutically acceptable salt thereof.

[0029] The present invention also provides the use of a formulation comprising Substance P and AMD3100 or a pharmaceutically acceptable salt thereof for mobilizing a large number of hematopoietic stem cells in the blood.

[0030] As used herein, the term "substance P" is used interchangeably with "substance P" or "SP".

[0031] As used herein, the term "hematopoietic stem cells" is used interchangeably with "hematopoietic stem cells" or "hematopoietic stem cells and progenitor cells."

[0032] As used herein, the terms "migration," "migration," and "mobilization" all have the same meaning.

[0033] As used herein, the term "combined therapy" is used interchangeably with "combination." "Combined therapy" means the use of two or more drugs together. Therefore, the two or more drugs do not need to be mixed as a composition; they may be used as individual formulations. Thus, "combined therapy" includes individual formulations of two or more drugs, allowing for simultaneous, individual, or sequential administration of the two or more drugs.

[0034] As used herein, the term “combination product” means the finished product form of the “combination therapy.” For example, if there are two or more drugs, the combination product means the finished product containing these drugs in the form of a combination. The two or more drugs may be packaged as a single formulation within the finished product and administered simultaneously, or they may be packaged as two individual formulations within the finished product and administered simultaneously, separately, or sequentially. An example of such a finished product is a “kit-of-parts.”

[0035] As used in this invention, the term "individual" refers to an animal to which the method of mass harvesting of mobile hematopoietic stem cells of this invention can be applied, preferably a vertebrate, more preferably a mammal, and including, for example, humans, monkeys, dogs, cats, goats, pigs, rats, guinea pigs, hamsters, chimpanzees, or gorillas. Most preferably, the individual is a human.

[0036] In one embodiment of the present invention, in a combined therapy for mobilizing a large amount of hematopoietic stem cells in the blood, containing substance P and AMD3100 or a pharmaceutically acceptable salt thereof, substance P and AMD3100 can be administered sequentially. Specifically, substance P is administered first, followed by AMD3100. More specifically, substance P is administered once or twice over two consecutive days, and AMD3100 is administered once approximately 1 to 2 days later.

[0037] In one embodiment of the present invention, a combination product for mobilizing a large amount of hematopoietic stem cells in the blood, comprising substance P and AMD3100 or a pharmaceutically acceptable salt thereof, allows for sequential administration of substance P and AMD3100. Specifically, substance P is administered first, followed by AMD3100. More specifically, substance P is administered once or twice, or twice over two consecutive days, followed by a single administration of AMD3100 approximately 1-2 days later.

[0038] In one embodiment of the present invention, the administration method of substance P and AMD3100 is not particularly limited, and they can preferably be administered parenterally by injection or the like. Specifically, substance P is administered intravenously, and AMD3100 is administered intraperitoneally. Furthermore, depending on the type and condition of the recipient, substance P and AMD3100 can be administered once or several times in an amount sufficient to mobilize hematopoietic stem cells into the peripheral blood.

[0039] In one embodiment of the present invention, substance P can be administered once or twice over two consecutive days at a dose of 0.1 to 100 μg / kg, preferably 1 to 20 μg / kg, and AMD3100 can be administered once or several times at a dose of 100 to 500 μg / kg, preferably 250 μg / kg. However, the administration method, number of administrations, and dosage can be appropriately changed at the discretion of those skilled in the art.

[0040] In one embodiment of the present invention, a combination product for mobilizing a large number of hematopoietic stem cells in the blood, comprising substance P and AMD3100 or a pharmaceutically acceptable salt thereof, may be in kit-of-parts form.

[0041] In one embodiment of the present invention, a method for collecting a large quantity of mobile hematopoietic stem cells includes a step of separating a blood fraction containing mobile hematopoietic stem cells from peripheral blood obtained from an individual that has been sequentially administered substance P and AMD3100 or a pharmaceutically acceptable salt thereof, wherein the step of separating the blood fraction can be performed using apheresis.

[0042] Substance P is a neuropeptide containing 11 amino acids that is expressed in sensory neurons, macrophages, eosinophils, endothelial cells, epithelial cells, corneal cells such as corneal stromal cells, and granulation tissue. The substance P used in this invention can be obtained through various routes, including synthesis from known amino acid sequence information, isolation from the organism used, and acquisition from commercially available formulations. For example, commercially available substance P includes substance P acetate hydrate (Sigma), substance P (Anygen), and bustans P (Merck Millipore).

[0043] AMD3100, also known as Plerixafor, is a compound whose name is 1,1'-[1,4-phenylenebis(methylene)]bis[1,4,8,11-tetraazacyclotetradecane]phosphorus compound and is known to function as an antagonist of the chemokine receptor CXCR4 (see U.S. Patent No. 5,612,478).

[0044] The AMD3100 used in this invention can be obtained through various routes, such as chemical synthesis methods known in the art and commercially available formulations. For example, commercially available MD3100s include Mozobil® (manufactured by Sanofi-Aventis).

[0045] Furthermore, AMD3100 used in the present invention can be used in the form of a pharmaceutically acceptable salt. For example, suitable acid addition salts may include salts of biocompatible inorganic acids (e.g., HCl, HBr, sulfuric acid, and phosphoric acid), organic acids (e.g., acetic acid, propionic acid, and butyric acid), and acids containing one or more carboxyl groups (e.g., oxalic acid, glutaric acid, adipic acid).

[0046] Furthermore, AMD3100 used in the present invention can be manufactured and used in the form of a prodrug, that is, in a protective form that releases the compound of the present invention after administration to the target organism. In addition, when manufactured in a purified form, the compound can crystallize as a hydrate.

[0047] A method for collecting a large number of mobile hematopoietic stem cells using a combination therapy comprising substance P and AMD3100 or a pharmaceutically acceptable salt thereof according to the present invention may specifically include (a) separating a blood fraction from the peripheral blood of an individual to which substance P and AMD3100 or a pharmaceutically acceptable salt thereof has been sequentially administered; and (b) concentrating the blood fraction.

[0048] In one embodiment of the present invention, the individual may be a vertebrate, more preferably a mammal including a human. In the present invention, the hematopoietic stem cells collected in large quantities are used for the prevention or treatment of vascular diseases, and therefore it is most preferable to obtain them from a patient being treated.

[0049] In one embodiment of the present invention, in the sampling method, substance P was administered first, then AMD3100 was administered, and blood was collected approximately 1 to 5 hours after the administration of AMD3100. More specifically, substance P was administered once, or twice for two consecutive days, and then AMD3100 was administered once approximately 1 to 2 days later, and blood was collected approximately 1 to 3 hours after the administration of AMD3100.

[0050] The separation of blood fractions from peripheral blood in step (a) above can be carried out by conventionally known methods for collecting blood components, such as collecting peripheral blood by passing it through an apheresis machine. Examples of apheresis machines widely used in the art include the MCS 3p, CS-3000 plus, and COBE Spectra, but the present invention is not limited to these. Once blood enters the apheresis machine via a central venous catheter, the blood fractions are collected by the machine, and the remaining blood can be returned to the body via the catheter. This process may take approximately 4 to 10 hours and may be repeated continuously for 2 to 10 days to collect a sufficient cell fraction for therapeutic use.

[0051] In one embodiment of the present invention, step (b) is a step of concentrating the blood fraction separated in step (a). Preferably, the concentration of the blood fraction can be carried out using a blood centrifuge. The centrifugation conditions can be adjusted as appropriate by those skilled in the art to remove the plasma component and obtain a concentrate (blood cell layer fraction) consisting of monocyte components excluding red blood cells. In a particular embodiment of the present invention, the blood fraction was placed on a Ficoll layer and centrifuged for 20 minutes (813g, Megafuge 8R; Thermo Scientific) to obtain a total of four layers. The top layer was plasma, the second layer was the blood cell layer excluding red blood cells, the third layer was Ficoll, and the bottom layer was the red blood cell layer. Of these, the blood cell layer was obtained as a concentrate containing a large amount of mobile hematopoietic stem cells, but the present invention is not limited thereto.

[0052] The configuration and effects of the present invention will be described in more detail below using examples. These examples are merely illustrative to aid in understanding the present invention, and the scope of the present invention is not limited to them.

[0053] Example 1: Mobilization of hematopoietic stem cells using SP alone. To confirm the effect of the drug when administered only in animals under normal physiological conditions, the effect of SP was tested using a normal group of uninjured animals. This is because, in injured animals, there are many other factors secreted from the wound, making it difficult to distinguish such factors from the effect on animals that have been administered the drug, especially since both SP and G-CSF are endogenous substances in the body.

[0054] To confirm the hematopoietic stem cell mobilization effect of SP alone, five rabbits were prepared for each group. SP (6.7 μg / kg, Sigma Aldrich) was administered twice via ear vein, and the levels of hematopoietic stem cells in the blood of the same animals were compared for three days. G-CSF (10 μg / kg, JW Pham) was used at conventional clinical doses, but was administered in the same manner as SP (i.e., intravenous injection). After drug injection, 5 mL of blood was collected once daily, added to a 15 mL tube (Falcon), and diluted 2-fold with PBS (Welgene). To separate blood cells, 5 mL of Ficoll-paque (Amersham) was added to a new tube, and the diluted blood was placed on top. Care was taken in handling the tubes to prevent mixing of the Ficoll layer and the blood layer, and after the blood layer was completely in the tube, centrifugation (813 g, Megafuge 8R, Thermo Scientific) was performed for 20 minutes. After centrifugation, the layers were separated, and a total of four layers were obtained. The top layer was plasma, the second layer was the blood cell layer excluding red blood cells, the third layer was Ficol, and the bottom layer was the red blood cell layer. Of these, only the blood cell layer was separated, placed in a tube, and centrifuged with PBS. 5 × 10 6 Individual blood cells were placed in 35m plates and cultured in HSC-CFU medium (Hematopoietic stem cells-colony forming unit media, purchased from Miltenyi). The cells were cultured for two weeks in the initial state without changing the medium, and the number of colonies was observed daily and finally counted. The results are shown in Figure 1.

[0055] As shown in Figure 1, hematopoietic stem cell mobilization was highest on day 2 after SP injection and was maintained until day 3. On the other hand, G-CSF administered by the same method (intravenous injection) showed only a slight effect on hematopoietic stem cell mobilization. In particular, it appeared to increase on day 1 while maintaining a similar level to day 0 (immediately before administration), and did not increase further. Specifically, based on 1X on day 0, SP showed 3X (3 times) and CSF showed 2X (2 times) on day 1; SP showed 7X (7 times) and CSF showed 2.5X (2.5 times) on day 2; and SP showed 6X (6 times) and CSF showed 0.6X (0.6 times) on day 3.

[0056] The main mechanism of action of G-CSF is the stimulation of neutrophil production. When comparing the degree to which hematopoietic stem cells are mobilized into the bloodstream by G-CSF at concentrations effective in mobilizing neutrophils into peripheral blood with that of SP, it was found that SP induces the mobilization of hematopoietic stem cells in the bloodstream more efficiently than G-CSF.

[0057] Example 2: Effects of SP on blood immune cells To confirm the effect of SP on the ratio of immune cells in the blood, five rabbits were prepared in each group. After administering SP (6.7 μg / kg) twice via ear vein, the ratio of immune cells in the blood of the same animals was compared for three days. G-CSF (10 μg / kg, JW Pham) was used at the conventional clinical dose and administered in the same manner as SP (intravenous injection). After administration, 1 mL of blood was collected at one-day intervals, transferred to a tube containing EDTA, and analyzed using diagnostic equipment.

[0058] The ratios of lymphocytes, monocytes, neutrophils, basophils, and eosinophils in total white blood cells in the blood were analyzed using CBC Count, and the results are shown in Figure 2.

[0059] As shown in Figure 2, SP administration did not significantly affect the distribution of immune cells in the blood, but G-CSF increased the proportion of neutrophils, a representative inflammatory cell in the blood, from day 1 after administration, and increased to approximately 65% ​​by day 2.

[0060] These are results from a healthy group with no injuries, suggesting that patients who already have high levels of inflammation may experience greater side effects from using G-CSF.

[0061] Example 3: Mobilization of hematopoietic stem cells by combination therapy with SP and AMD3100 To compare the effects of G-CSF and SP based on clinical dosage / administration, five 8-week-old C57Bl / 6 mice were prepared in each group. They were divided into three groups: a vehicle (physiological saline) administration group, a G-CSF + AMD3100 administration group, and an SP + AMD3100 administration group.

[0062] Mice were administered either G-CSF (250 μg / kg, subcutaneously, STEM CELL) or SP (6.7 μg / kg, intravenously, Sigma Aldrich). G-CSF was administered subcutaneously for 5 consecutive days according to a conventional clinical schedule, and SP was administered intravenously for 2 consecutive days. AMD3100 was administered on the last day or one day after each administration according to a normal clinical schedule, and blood was collected 1 hour after administration.

[0063] Figure 3 is a schematic diagram illustrating the administration schedule for conventional combination therapy with G-CSF and ADM3100, and the administration schedule for combination therapy with substance P and ADM3100 according to the present invention.

[0064] After administering AMD3100, 1 mL of whole mouse blood was drawn using a syringe (containing heparin). The blood was placed in a 15 mL tube and diluted 2-fold with PBS (Welgene). To separate the blood cells, 3 mL of Ficoll-paque (Amersham) was placed in a new tube, and the diluted blood was placed on top of it. Care was taken to handle the tube to prevent the Ficoll layer and the blood layer from mixing. After the blood layer was completely in the tube, centrifugation (813 g, Megafuge 8R, Thermo Scientific) was performed for 20 minutes. After centrifugation, the layers were separated, yielding a total of four layers. The top layer was plasma, the second layer was the blood cell layer excluding red blood cells, the third layer was Ficoll, and the bottom layer was the red blood cell layer. Of these, only the blood cell layer was separated, placed in a tube, and centrifuged with PBS. 5 × 10 6 Individual blood cells were placed in 35m plates and cultured in HSC-CFU medium (Hematopoietic stem cells-colony forming unit media, purchased from Miltenyi). The cells were cultured for two weeks in the initial state without changing the medium, and the number of colonies was observed daily and finally counted. The results are shown in Figure 4.

[0065] As shown in Figure 4, the combination of G-CSF and AMD3100 promoted the migration of many hematopoietic stem cells into the bloodstream, as clinically demonstrated. However, the SP+AMD3100 combination showed even greater migration of hematopoietic stem cells. In particular, in the experiments of this invention, G-CSF was administered 5 times and SP was administered 2 times, and it was found that the SP+AMD3100 combination had a greater effect on mobilizing hematopoietic stem cells in the bloodstream, depending on the dose, compared to the G-CSF+AMD3100 combination. Embodiments of the present invention are further described in the following sections: [Section 1] A combination therapy containing Substance P and AMD3100 or a pharmaceutically acceptable salt thereof to mobilize a large number of hematopoietic stem cells in the blood. [Section 2] The combination therapy described in item 1 above, comprising sequentially administering substance P and AMD3100. [Section 3] The combination therapy described in item 1 above, in which Substance P is administered once, followed by AMD3100 administered once approximately 1 to 2 days later. [Section 4] The combination therapy described in item 1 above, in which Substance P is administered twice on two consecutive days, followed by a single dose of AMD3100 approximately 1 to 2 days later. [Section 5] The combination therapy described in any one of the above items 1 to 4, wherein substance P is administered by intravenous injection. [Section 6] A combination product containing Substance P and AMD3100 or a pharmaceutically acceptable salt thereof for mobilizing a large number of hematopoietic stem cells in the blood. [Section 7] The combination product described in item 6 above, wherein Substance P and AMD3100 are administered sequentially. [Section 8] The combination product described in item 6 above, in which Substance P is administered once, followed by AMD3100 administered once approximately 1 to 2 days later. [Section 9] The combination product described in item 6 above, in which Substance P is administered twice on two consecutive days, followed by a single dose of AMD3100 approximately 1 to 2 days later. [Section 10] A combination product as described in any one of items 6 to 9 above, in which substance P is administered by intravenous injection. [Section 11] A combination product as described in any one of items 6 to 9 above, wherein the combination product is in kit-of-parts form. [Section 12] A method for collecting a large quantity of mobile hematopoietic stem cells, comprising the step of separating a blood fraction containing mobile hematopoietic stem cells from peripheral blood obtained from an individual that has been sequentially administered substance P and AMD3100 or a pharmaceutically acceptable salt thereof. [Section 13] The method described in item 12 above, wherein substance P is administered first, then AMD3100 is administered, and blood is collected approximately 1 to 5 hours after the administration of AMD3100. [Section 14] The method according to item 12 above, wherein the step of separating blood fractions is performed using apheresis.

Claims

1. A composition for a method of treating an individual suffering from a disease requiring hematopoietic stem cells, wherein the composition comprises substance P or AMD3100 or a pharmaceutically acceptable salt thereof, and the method is A step of sequentially administering substance P and AMD3100 or a pharmaceutically acceptable salt thereof to an individual in the absence of G-CSF; A step of separating a blood fraction containing mobile hematopoietic stem cells from peripheral blood obtained from the aforementioned individual; A step of concentrating the blood fraction to obtain a concentrate containing mobile hematopoietic stem cells; and A step of injecting the concentrate containing mobile hematopoietic stem cells into the individual. A composition containing the following:

2. A kit for a method of treating an individual suffering from a disease requiring hematopoietic stem cells, the kit comprising a first composition comprising substance P and a second composition comprising AMD3100 or a pharmaceutically acceptable salt thereof, the method being: A step of sequentially administering substance P and AMD3100 or a pharmaceutically acceptable salt thereof to an individual in the absence of G-CSF; A step of separating a blood fraction containing mobile hematopoietic stem cells from peripheral blood obtained from the aforementioned individual; A step of concentrating the blood fraction to obtain a concentrate containing mobile hematopoietic stem cells; and A step of injecting the concentrate containing mobile hematopoietic stem cells into the individual. A kit that includes this.

3. The composition according to claim 1 or the kit according to claim 2, wherein in the method, substance P is administered first, and then AMD3100 or a pharmaceutically acceptable salt thereof is administered.

4. The composition according to claim 1 or the kit according to claim 2, wherein, in the method described above, substance P is administered once or twice on two consecutive days, and then AMD3100 or a pharmaceutically acceptable salt thereof is administered once one to two days later.

5. The composition according to claim 1 or the kit according to claim 2, wherein, in the method, substance P is administered twice on two consecutive days, and then AMD3100 or a pharmaceutically acceptable salt thereof is administered once one to two days later.

6. The composition according to any one of claims 1 and 3 to 5 or the kit according to any one of claims 2 to 5, wherein the substance P is administered by intravenous injection in the method described above.

7. The composition according to any one of claims 1 and 3 to 6, or the kit according to any one of claims 2 to 6, wherein in the method, substance P is administered first, then AMD3100 or a pharmaceutically acceptable salt thereof is administered, and blood is collected 1 to 5 hours after the administration of AMD3100 or a pharmaceutically acceptable salt thereof.

8. The composition according to any one of claims 1 and 3 to 7 or the kit according to any one of claims 2 to 7, wherein the step of separating the blood fraction is performed using apheresis.

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