Use of a cationic compound in the preparation of a hematopoietic stem cell or progenitor cell mobilizer

Abstract

The present invention discloses the use of a cationic compound in the preparation of a hematopoietic stem cell or progenitor cell mobilizer; the cationic compound is hexamethonium bromide (HEX) or protamine sulfate (PRO). In the present invention, the cationic compounds HEX or PRO are used to prepare a hematopoietic stem / progenitor cell mobilizer, and the mobilization levels of hematopoietic stem cells and progenitor cells in peripheral blood are both significantly enhanced, with good mobilization effects. The present invention provides a hematopoietic stem cell and / or progenitor cell mobilization strategy option with clinical transformation potential, enabling more patients to benefit from the treatment.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology. Specifically, it relates to the application of a cationic compound in the preparation of a hematopoietic stem cell or progenitor cell mobilizer. Background Art

[0002] Hematopoietic stem cell transplantation is an effective treatment method for certain malignant or non-malignant diseases, saving thousands of lives every year. Despite great progress in recent years, the shortage of the source of supply remains one of the important challenges in the field. Clinically, mobilized hematopoietic stem cells are widely used in hematopoietic stem cell transplantation, and compared with bone marrow-derived hematopoietic stem cells, their transplantation effect is better. At present, although the mainstream mobilizer such as granulocyte colony-stimulating factor (G-CSF) has good effects, its onset is slow, and there are many G-CSF-resistant patients or donors. Therefore, exploring new rapid and efficient stem cell mobilization methods has very important clinical value and scientific significance. Summary of the Invention

[0003] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide the application of a cationic compound in the preparation of a hematopoietic stem cell or progenitor cell mobilizer. It provides a hematopoietic stem cell and / or progenitor cell mobilization strategy option with clinical transformation potential, enabling more patients to benefit from the treatment.

[0004] The present invention provides the application of a cationic compound in the preparation of a hematopoietic stem cell or progenitor cell mobilizer, and the cationic compound is hexadimethrine bromide (HEX) or protamine sulfate (PRO). Hexadimethrine Bromide (HEX), CAS No.: 28728-55-4, Protamine sulfates (PRO), CAS No.: 9009-65-8.

[0005] Preferably, the hematopoietic stem cell or progenitor cell mobilizer contains at least one cationic compound.

[0006] Preferably, the dosage form of the hematopoietic stem cell or progenitor cell mobilizer is an injection, which is introduced into the human body by subcutaneous injection or intravenous injection.

[0007] Preferably, the application direction of the hematopoietic stem cell or progenitor cell mobilizer is diseases involving stem cell therapy, including malignant and non-malignant blood diseases, immunodeficiency diseases, and metabolic disorder diseases.

[0008] Preferably, the hematopoietic stem cell or progenitor cell mobilizer is used for bone marrow transplantation.

[0009] The present invention has conducted experiments related to hematopoietic stem / progenitor cell mobilization. Chemotaxis of hematopoietic stem / progenitor cells extracted from human umbilical cord blood was applied with relevant drugs. The results showed that administration of HEX and PRO could significantly improve the migration ability of CD34-positive hematopoietic stem cells and progenitor cells. As cationic compounds, HEX and PRO could significantly reduce the expression level of CXCR4 on the surface of hematopoietic stem cells and progenitor cells, thereby weakening their chemotactic ability. Therefore, HEX and PRO can be applied to regulate the function of human hematopoietic stem cells.

[0010] The present invention further provides a method for inducing hematopoietic stem / progenitor cell mobilization by applying HEX and PRO in mice. The method includes administering a certain amount of PRO to an object, thereby enhancing the mobilization of hematopoietic stem cells or progenitor cells in the object. In addition, the present invention proposes to apply an optimized dose of PRO in cynomolgus monkeys, a non-human primate, thereby mobilizing hematopoietic stem cells and progenitor cells in the object. Furthermore, the present invention proposes to transplant hematopoietic stem cells or progenitor cells mobilized by PRO in mice, thereby evaluating the potential of bone marrow reconstitution in the object. The administered drug contains at least one cationic compound, including but not limited to compounds, proteins, nucleic acids, natural products, etc., applied alone or in combination. The administration methods include but are not limited to single use and any combination thereof. The administration routes include but are not limited to subcutaneous injection, intravenous injection, etc., and the administration models include but are not limited to mice, primates, etc. Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention uses the cationic compounds HEX and PRO to prepare hematopoietic stem cell mobilizing agents, and the mobilization levels of hematopoietic stem cells and progenitor cells in peripheral blood are both significantly enhanced, with a fast onset and good mobilization effect. Description of the Drawings

[0011] Figure 1 A-1C shows the down-regulation of the expression of the surface receptor CXCR4 of human umbilical cord blood CD34+ cells by applying the cationic compounds HEX and PRO; Figure 1 D-1F shows the down-regulation of the expression of the surface receptor CXCR4 of human hematopoietic stem cells by applying the cationic compounds HEX and PRO.

[0012] Figure 2 A shows that applying the cationic compounds HEX and PRO significantly down-regulates the chemotaxis ability of human umbilical cord blood CD34+ cells towards stromal cell-derived factor-1 (SDF1), and this phenomenon can be reversed by applying the anionic compound poly(acrylic acid sodium) (PAA, CAS No.: 9003-04-7); Figure 2 B shows that applying the cationic organic compounds HEX and PRO significantly down-regulates the chemotaxis ability of human hematopoietic stem cells towards SDF1, and this phenomenon can be reversed by the anionic compound PAA.

[0013] Figure 3 A shows that the application of cationic compounds HEX and PRO significantly enhances the fugetaxis ability of human umbilical cord blood CD34 + cells to SDF1, and this phenomenon can be reversed by the application of anionic compound PAA; Figure 3 B shows that the application of cationic compounds HEX and PRO significantly down-regulates the fugetaxis ability of human umbilical cord blood hematopoietic stem cells to SDF1, and this phenomenon can be reversed by the application of anionic compound PAA.

[0014] Figure 4 A is an in-vivo experiment on mice. The results of a single transplantation experiment show that both cationic compounds HEX and PRO can significantly up-regulate the stem cell mobilization ability, manifested as a significant increase in the number of LSK cells in peripheral blood. Figure 4 B is to detect the colony-forming ability of peripheral blood after transplantation. The results show that compared with the control group (VEH), both cationic compounds HEX and PRO can significantly enhance the colony-forming ability of peripheral blood stem cells in transplanted mice.

[0015] Figure 5 A is an in-vivo experiment confirming that the application of cationic compound PRO significantly enhances the mobilization ability of mouse hematopoietic stem and progenitor cells, and this effect can be weakened by the application of anionic compound PAA; Figure 5 B is an in-vivo experiment confirming that the application of cationic compound PRO significantly enhances the mobilization ability of mouse hematopoietic stem cells, and this effect can be weakened by the application of anionic compound PAA; Figure 5 C is that in-vivo application of cationic compound PRO significantly enhances the colony-forming ability of hematopoietic stem / progenitor cells in the mobilized peripheral blood of mice, and this phenomenon can be reversed by the application of anionic compound PAA.

[0016] Figure 6 A-6E shows that the application of cationic compound PRO significantly enhances the mobilization ability of hematopoietic stem / progenitor cells in a non-human primate model - cynomolgus monkeys, as well as the colony-forming ability of hematopoietic stem / progenitor cells in the mobilized peripheral blood. Detailed implementation mode

[0017] The technical solutions of the present invention will be elaborated in detail below with reference to the accompanying drawings and embodiments.

[0018] Example 1 Regulation of the migration ability of human umbilical cord blood hematopoietic stem / progenitor cells by HEX and PRO

[0019] Experimental method: Human umbilical cord blood hematopoietic stem / progenitor cells were extracted and each sample was evenly divided into three groups. The control group (VEH) was treated with PBS, and the experimental groups were treated with HEX (10 μg / mL) or PRO (20 μg / mL) respectively. Cells were collected to detect the migratory phenotype of the cells. Considering the core role of the SDF1-CXCR4 axis in the directed migration of hematopoietic stem cells, we focused on detecting the changes in the expression level of CXCR4 on the cell surface and its related cell migration ability.

[0020] Experimental results: As Figure 1 shown, the expression levels of CXCR4 on CD34 + cells and hematopoietic stem cells HSC in the experimental group-treated cells were significantly decreased, manifested as a significant decrease in both the mean fluorescence intensity (MFI) and the number of CXCR4 hi cells. As Figure 2 , Figure 3 shown, the ability of CD34 + cells and hematopoietic stem cells HSC to migrate towards SDF-1 was significantly weakened, and the related phenomenon could be reversed by the combined application of the anionic compound PAA.

[0021] Example 2 HEX and PRO can mobilize functional hematopoietic stem / progenitor cells in mice

[0022] Experimental method: Nine C57 mice were randomly divided into 3 groups. Drugs were administered by subcutaneous injection. Among them, the control group (VEH) was treated with PBS, and the experimental groups were treated with HEX (10 mg / kg) and PRO (10 mg / kg) respectively. Mouse peripheral blood was collected for flow cytometry to detect the enrichment of Lin - Sca-1 + c-Kit + (LSK) cells. In addition, cells were collected for colony formation assay to analyze the colony formation ability of hematopoietic stem / progenitor cells in the mobilized peripheral blood after drug treatment.

[0023] Experimental results: As Figure 4 shown, the mobilization levels of LSK cells in the peripheral blood of mice in the HEX and PRO treatment groups were significantly enhanced. The colony formation ability was significantly improved one week later.

[0024] Example 3 PRO mobilizes functional hematopoietic stem / progenitor cells in mice depending on its cationic properties

[0025] Experimental method: Twenty-four C57 mice were randomly divided into 4 groups. Drugs were administered by subcutaneous injection. Among them, the control group (VEH) was treated with PBS, and the experimental groups were treated with PRO (40 mg / kg), PAA (40 mg / kg), and PRO (40 mg / kg) + PAA (40 mg / kg) respectively. Mouse peripheral blood was collected for flow cytometry to detect Lin- Sca-1 + c-Kit + Enrichment of (LSK) cells and hematopoietic stem cell (HSC) cells. In addition, cells were collected for colony formation assay to analyze the colony formation ability of hematopoietic stem / progenitor cells in mobilized peripheral blood after drug treatment.

[0026] Experimental results: As Figure 5 shown, the mobilization levels of LSK cells and hematopoietic stem cell (HSC) cells in the peripheral blood of PRO-treated mice were significantly enhanced. The colony formation ability was significantly improved one week later. However, there was no significant difference between the PRO+PAA treatment group and the control group (VEH).

[0027] Example 4 PRO can mobilize functional hematopoietic stem / progenitor cells in non-human primates

[0028] Experimental method: Four cynomolgus monkeys were administered by intravenous injection (10 mg / kg). Peripheral blood of cynomolgus monkeys was collected at a total of 4 time points before and after PRO treatment, and flow cytometry was used to detect the enrichment of CD34 + cells. In addition, peripheral blood cells were collected at a total of 4 time points before and after PRO treatment for colony formation assay to analyze the colony formation ability of hematopoietic stem / progenitor cells in mobilized peripheral blood after PRO treatment.

[0029] Experimental results: As Figure 6 shown, the number of CD34 + in the peripheral blood of cynomolgus monkeys was significantly increased at 1 h and 2 h after PRO treatment compared with that before treatment, but there was no significant increase at the 4 h time point. In addition, the colony formation ability of peripheral blood cells collected at all time points was significantly improved one week later compared with that before treatment.

Claims

1. Use of a cationic compound in the preparation of a hematopoietic stem cell or progenitor cell mobilizer, characterized in that, the cationic compound is hexamethonium bromide HEX.

2. The use according to claim 1, characterized in that, the dosage form of the hematopoietic stem cell or progenitor cell mobilizer is an injection, which is introduced into the human body by subcutaneous injection or intravenous injection.

3. The use according to claim 1, characterized in that , the application direction of the hematopoietic stem cell or progenitor cell mobilizer is diseases related to stem cell therapy.

4. The use according to claim 3, characterized in that, the diseases treated by stem cell therapy include malignant and non-malignant blood diseases, immunodeficiency diseases and metabolic disorders.

5. The use according to claim 1, characterized in that , the hematopoietic stem cell or progenitor cell mobilizer is used for bone marrow transplantation.

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