Use of integrin alpha 4 beta 1 agonists in the preparation of a product for increasing the success rate of hematopoietic stem cell engraftment

Abstract

The present application relates to the technical field of medicine, in particular to the use of an Integrin alpha4 beta1 agonist in the preparation of a product for improving the success rate of hematopoietic stem cell implantation, wherein the Integrin alpha4 beta1 agonist is selected from a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a compound of formula (II) or a pharmaceutically acceptable salt thereof. The Integrin alpha4 beta1 agonist of the present application can improve the hematopoietic stem cell implantation capacity by agonizing the Integrin alpha4 beta1 on the surface of hematopoietic stem cells, promote the implantation of hematopoietic stem cells, thereby improve the success rate of implantation, and provide a new prevention and treatment scheme for patients with a large number of implantation failures.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the use of Integrin α4β1 agonists in the preparation of products that improve the success rate of hematopoietic stem cell engraftment. Background Technology

[0002] Allogeneic hematopoietic stem cell transplantation (Allo-HSCT) is an important treatment for both benign and malignant hematological diseases. In recent years, an increasing number of patients with non-malignant diseases, as well as those who cannot tolerate conventional myeloablative conditioning due to age or other comorbidities, often opt for Allo-HSCT with non-myeloablative conditioning or reduced-intensity conditioning, primarily involving immunosuppression. Consequently, the proportion of recipients with mixed chimerism of donor cells after transplantation has increased, with an incidence rate as high as 28-44%. Mixed chimerism increases the engraftment failure rate, weakens the graft-versus-leukemia (GVL) effect, and increases the risk of disease relapse, which are significant factors affecting the survival of Allo-HSCT patients. Donor lymphocyte infusion (DLI) is one of the main methods to prevent a decline in chimerism after Allo-HSCT, aiming to promote hematopoietic stem cell (HSC) engraftment and improve prognosis by infusing donor lymphocytes into hematopoietic stem cells. However, this therapy is limited by the availability of donor lymphocytes, and it may cause serious complications such as hematopoietic suppression and severe acute graft-versus-host disease (GVHD). Summary of the Invention

[0003] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide the use of Integrin α4β1 agonists in the preparation of products that improve the success rate of hematopoietic stem cell engraftment. Integrin α4β1 agonists can effectively enhance the engraftment ability of hematopoietic stem cells in mouse allogeneic bone marrow transplantation models, promote the engraftment of donor hematopoietic stem cells, and can be developed into a clinical drug to assist hematopoietic stem cell engraftment, or a drug for early intervention or treatment of mixed chimerism after transplantation. For example, in non-myeloablative transplantation, the agonist is applied simultaneously with the transplantation to promote stem cell engraftment, thereby solving the problems in the prior art.

[0004] To achieve the above and other related objectives, the first aspect of the present invention provides the use of Integrin α4β1 agonist in the preparation of products that improve the success rate of hematopoietic stem cell engraftment.

[0005] In some embodiments of the invention, the Integrin α4β1 agonist is selected from the compound of formula (I) or a pharmaceutically acceptable salt thereof, or the compound of formula (II) or a pharmaceutically acceptable salt thereof;

[0006]

[0007] In some embodiments of the invention, Integrin α4β1 agonists improve the success rate of hematopoietic stem cell engraftment by increasing the expression or activity of Integrin α4β1 on the surface of hematopoietic stem cells.

[0008] In some embodiments of the invention, Integrin α4β1 agonists enhance the engraftment capacity of hematopoietic stem cells by increasing the expression or activity of Integrin α4β1 on the surface of hematopoietic stem cells, thereby promoting stem cell engraftment and improving the success rate of hematopoietic stem cell engraftment.

[0009] In some embodiments of the invention, the pharmaceutically acceptable salts of the compounds of formula (I) or formula (II) are selected from pharmaceutically acceptable inorganic acid salts or organic acid salts.

[0010] In some embodiments of the invention, the molecular formula of the compound of formula (I) is C. 12 H 14 N2O3; molecular weight 234.25; CAS number 73096-22-7.

[0011] In some embodiments of the invention, the molecular formula of the compound represented by formula (II) is C. 13 H 14 N2O4, with a molecular weight of 262.26 and CAS number 2023788-32-9.

[0012] In some embodiments of the invention, the hematopoietic stem cells are allogeneic hematopoietic stem cells.

[0013] In some embodiments of the invention, the product for improving the success rate of hematopoietic stem cell implantation includes a drug.

[0014] In some embodiments of the invention, the product is a product that is administered by injection or orally.

[0015] A second aspect of the present invention provides a drug for improving the success rate of hematopoietic stem cell engraftment, wherein the active ingredient of the drug includes an Integrin α4β1 agonist, or a pharmaceutically acceptable carrier or excipient.

[0016] In some embodiments of the present invention, the Integrin α4β1 agonist is selected from the compounds of formula (I) or pharmaceutically acceptable salts thereof, or the compounds of formula (II) or pharmaceutically acceptable salts thereof:

[0017]

[0018] In some embodiments of the present invention, the pharmaceutically acceptable salts of the compounds of formula (I) or formula (II) are selected from pharmaceutically acceptable inorganic acid salts or organic acid salts.

[0019] In some embodiments of the present invention, the molecular formula of the compound of formula (I) is C 12 H 14 N2O3; molecular weight 234.25; CAS number 73096-22-7.

[0020] In some embodiments of the present invention, the molecular formula of the compound represented by formula (II) is C. 13 H 14 N2O4, with a molecular weight of 262.26 and CAS number 2023788-32-9.

[0021] A third aspect of the present invention provides a method of using a drug to improve the success rate of hematopoietic stem cell implantation, the method comprising injecting the drug.

[0022] As described above, the use of the Integrin α4β1 agonist of the present invention in the preparation of products that improve the success rate of hematopoietic stem cell engraftment has the following beneficial effects:

[0023] (1) This application opens up new clinical application areas for known Integrin α4β1 agonists.

[0024] (2) This application finds that Integrin α4β1 plays an important role in promoting the engraftment of hematopoietic stem cells after allogeneic hematopoietic stem cell transplantation in humans or mice.

[0025] (3) This application is to discover that Integrin α4β1 agonists promote or enhance the implantation of donor grafts into recipients after hematopoietic stem cell transplantation. Attached Figure Description

[0026] Figure 1 The diagram illustrates the sample delivery process for patients. Bone marrow mononuclear cell samples in a mixed chimeric state are taken approximately 2 weeks after the patient's allogeneic hematopoietic stem cell transplantation, and bone marrow mononuclear cell samples in a fully chimeric state are taken approximately 4 weeks after the transplantation. These samples are then sent for single-cell sequencing and analysis.

[0027] Figure 2 According to Figure 1 The schematic diagram shows that after sample delivery, single-cell sequencing analysis was performed to obtain the quantitative results of the mRNA levels of Itga4 and Itgb1 genes in HSC cell subsets under different chimeric states after allogeneic hematopoietic stem cell transplantation.

[0028] Figure 3To illustrate the construction of mixed chimeric and complete chimeric mouse models through allogeneic bone marrow transplantation, bone marrow mononuclear cells were collected from mixed chimeric mice on day 14 post-transplantation and from complete chimeric mice on day 28 post-transplantation. These cells were then sent for single-cell sequencing and analysis.

[0029] Figure 4 According to Figure 3 The schematic diagram shows that after sample delivery, single-cell sequencing analysis was performed to obtain the quantitative results of the mRNA levels of Itga4 and Itgb1 genes in HSC cell subsets under different chimeric states in mice after allogeneic hematopoietic stem cell transplantation.

[0030] Figure 5 According to Figure 3 During the process of obtaining hybrid chimeric mice, the mice were administered solvent and three Integrin α4β1 agonists daily from day 1 to day 10 post-transplantation. On day 14 post-transplantation, the donor engraftment level in the peripheral blood of each group of mice was measured.

[0031] Figure 6 According to Figure 3 During the process of obtaining hybrid chimeric mice, the mice were administered solvent and three Integrin α4β1 agonists daily from day 4 to day 12 post-transplantation. On day 14 post-transplantation, the donor engraftment level in the peripheral blood of each group of mice was measured.

[0032] Figure 7 According to Figure 3 During the process of obtaining hybrid chimeric mice, the mice were administered solvent and three Integrin α4β1 agonists daily from day 6 to day 12 post-transplantation. On day 14 post-transplantation, the donor engraftment level in the peripheral blood of each group of mice was measured.

[0033] Figure 8 According to Figure 3 During the process of obtaining hybrid chimeric mice, the mice were administered solvent and Integrin α4β1 agonist No. 1 and agonist No. 2 daily from day 8 to day 14 post-transplantation. The donor engraftment level in the peripheral blood of each group of mice was measured on day 14 post-transplantation.

[0034] Figure 9 According to Figure 3 During the process of obtaining hybrid chimeric mice, the mice were administered solvent and Integrin α4β1 agonist No. 1 and agonist No. 2 daily from day 8 to day 28 post-transplantation. The donor engraftment level in the peripheral blood of each group of mice was measured on day 28 post-transplantation.

[0035] Figure 10 According to Figure 3During the process of obtaining hybrid chimeric mice, the mice were administered solvent and Integrin α4β1 agonist No. 2 daily on days 8 to 14, 10 to 14, and 12 to 14 post-transplantation. The donor engraftment level in the peripheral blood of each group of mice was measured in the second week post-transplantation.

[0036] Figure 11 According to Figure 3 During the process of obtaining hybrid chimeric mice, the mice were administered solvent and Integrin α4β1 agonist No. 2 daily from day 8 to day 28, day 10 to day 28, and day 12 to day 28 post-transplantation. The donor engraftment level in the peripheral blood of each group of mice was measured at week 4 post-transplantation.

[0037] Figure 12 According to Figure 3 During the process of obtaining hybrid chimeric mice, the mice were administered solvent and Integrin α4β1 agonist No. 2 daily from day 8 to day 28, day 10 to day 28, and day 12 to day 28 post-transplantation. The donor engraftment level in the peripheral blood of each group of mice was measured at week 8 post-transplantation.

[0038] Figure 13 For the continuation Figure 12 In a mouse model, the level of donor cell lineages in the peripheral blood of mice in each group was examined at week 8 post-transplantation.

[0039] Figure 14 To continue Figure 12 In a mouse model, the engraftment levels of various hematopoietic stem / progenitor cell donors in the bone marrow of mice in the group that started receiving agonist 2 on day 8 post-transplantation were measured at week 8.

[0040] Figure 15 To continue Figure 12 In a mouse model, the results of bone marrow cell clone formation in mice that started receiving agonist 2 on day 8 post-transplantation were detected at week 8. Detailed Implementation

[0041] The inventors of this application, through single-cell transcriptome sequencing analysis of bone marrow cells in both human and mouse hybrid chimeras and fully implanted states, discovered that the Integrin α4β1 receptor on the surface of hematopoietic stem cells is a key regulatory molecule affecting allogeneic hematopoietic stem cell implantation. The inventors of this application found that Integrin α4β1 compound (I) (agonist 1) and compound (II) (agonist 2) can promote hematopoietic stem cell implantation and increase donor chimerism by activating Integrin α4β1 on the surface of hematopoietic stem cells, and that the effects of the two agonists differ under different administration conditions. Based on these findings, this application was completed.

[0042] This invention provides the use of Integrin α4β1 agonists in the preparation of products that improve the success rate of hematopoietic stem cell engraftment.

[0043] In the use of the Integrin α4β1 agonist provided by this invention in the preparation of products that improve the success rate of hematopoietic stem cell engraftment, the Integrin α4β1 agonist improves the success rate of hematopoietic stem cell engraftment by enhancing the ability of hematopoietic stem cells to engraft.

[0044] In the use of the Integrin α4β1 agonist provided by this invention in the preparation of products that improve the success rate of hematopoietic stem cell engraftment, the Integrin α4β1 agonist improves the success rate of hematopoietic stem cell engraftment by increasing the expression or activity of Integrin α4β1 on the surface of hematopoietic stem cells.

[0045] In the use of the Integrin α4β1 agonist provided by this invention in the preparation of products that improve the success rate of hematopoietic stem cell engraftment, the Integrin α4β1 agonist enhances the engraftment ability of donor hematopoietic stem cells and improves the success rate of hematopoietic stem cell engraftment by stimulating the expression or activity of Integrin α4β1 on the surface of hematopoietic stem cells.

[0046] This invention provides an Integrin α4β1 agonist. The Integrin α4β1 agonist improves the success rate of hematopoietic stem cell engraftment by increasing donor chimerism. The success rate of engraftment can be assessed using the donor chimerism rate.

[0047] In the use of the Integrin α4β1 agonist provided by this invention in the preparation of products that improve the success rate of hematopoietic stem cell engraftment, the Integrin α4β1 agonist improves the success rate of hematopoietic stem cell engraftment by stimulating the expression or activity of Integrin α4β1 on the surface of hematopoietic stem cells, thereby increasing the donor chimerism rate.

[0048] The Integrin α4β1 agonist is selected from the compound of formula (I) or a pharmaceutically acceptable salt thereof, or the compound of formula (II) or a pharmaceutically acceptable salt thereof;

[0049]

[0050] In the use of the Integrin α4β1 agonist provided in this invention in the preparation of products that improve the success rate of hematopoietic stem cell engraftment, the compound of formula (I) is named Agonist No. 1, and the molecular formula of Agonist No. 1 is C 12 H 14N2O3, with a molecular weight of 234.25 and CAS number 73096-22-7, is a compound that currently has no common name. It can be used as a dual-action probe in the Huntington's cell model, but its therapeutic applications are not yet clearly defined.

[0051] In the use of the Integrin α4β1 agonist provided in this invention in the preparation of products that improve the success rate of hematopoietic stem cell engraftment, the compound of formula (II) is named agonist 2, and the molecular formula of agonist 2 is C 13 H 14 N2O4, with a molecular weight of 262.26 and CAS number 2023788-32-9, currently has no generic name and no known therapeutic applications.

[0052] In the use of the Integrin α4β1 agonist provided by this invention in the preparation of products that improve the success rate of hematopoietic stem cell engraftment, the pharmaceutically acceptable salts of the compounds described in formula (I) or (II) are selected from pharmaceutically acceptable inorganic or organic acid salts. Inorganic acid salts may be, for example, sodium salts, calcium salts, etc. Organic acid salts may be, for example, lysine salts, aminobutanetriol salts, etc.

[0053] "Pharmaceutical acceptable" means that when the molecular basis and the composition are properly administered to animals or humans, they do not produce adverse, allergic, or other adverse reactions.

[0054] In the application of the Integrin α4β1 agonist provided in this invention in the preparation of products that improve the success rate of hematopoietic stem cell engraftment, this invention provides the application of Integrin α4β1 agonist administered at different time points after mouse hematopoietic stem cell transplantation to improve the donor chimerism rate in mice. The hematopoietic stem cell transplantation is an allogeneic hematopoietic stem cell transplantation. Specifically, this invention targets mouse models in a mixed chimerism state after allogeneic hematopoietic stem cell transplantation, where the Integrin α4β1 agonist can effectively improve the chimerism rate and partially achieve complete chimerism. More specifically, administering Integrin α4β1 agonist No. 2 starting on day 8 after mouse transplantation maximizes the donor chimerism rate in mice.

[0055] Recipients with 5%-95% donor-derived cells are defined as mixed chimeras, while non-mixed chimeras include complete implantation with >95% donor-derived cells and complete rejection with <5% donor-derived cells.

[0056] In the use of the Integrin α4β1 agonist provided by this invention in the preparation of products that improve the success rate of hematopoietic stem cell engraftment, there are no special restrictions on the form of the products that improve the success rate of hematopoietic stem cell engraftment, and they can be in various forms such as solid, liquid, gel, semi-liquid, and aerosol.

[0057] The product can be a single-component substance or a multi-component substance.

[0058] The product used to improve the success rate of hematopoietic stem cell engraftment is a drug. In some embodiments, the drug may be administered by injection or orally.

[0059] In this application, the Integrin α4β1 agonist can effectively improve the donor chimerism rate after allogeneic hematopoietic stem cell transplantation in mice and promote the engraftment of donor hematopoietic stem cells. It can be developed into a drug for early intervention or treatment of mixed chimerism after transplantation.

[0060] A second aspect of this invention provides a drug that improves the success rate of hematopoietic stem cell implantation.

[0061] Furthermore, the active ingredient of the drug includes an Integrin α4β1 agonist, or a pharmaceutically acceptable carrier or excipient.

[0062] The Integrin α4β1 agonist is selected from the compound of formula (I) or a pharmaceutically acceptable salt thereof, or the compound of formula (II) or a pharmaceutically acceptable salt thereof:

[0063]

[0064] In the drug for improving the success rate of hematopoietic stem cell engraftment described in this application, the molecular formula of the compound of formula (I) is C. 12 H 14 N₂O₃; molecular weight 234.25; CAS number 73096-22-7; the molecular formula of the compound represented by formula (II) is C₂O₃. 13 H 14 N2O4, with a molecular weight of 262.26 and CAS number 2023788-32-9.

[0065] In the drug for improving the success rate of hematopoietic stem cell engraftment described in this application, the pharmaceutically acceptable salts of the compound described in formula (I) or the pharmaceutically acceptable salts of the compound described in formula (II) are all selected from pharmaceutically acceptable inorganic acid salts or organic acid salts.

[0066] "Pharmaceutical acceptable" means that when the molecular basis and the composition are properly administered to animals or humans, they do not produce adverse, allergic, or other adverse reactions.

[0067] Furthermore, the pharmaceutically acceptable drug carrier comprises one or more of the following: enteric-coated formulations, capsules, microspheres / capsules, liposomes, microemulsions, complex emulsions, nanoparticles, magnetic particles, gelatin, or gels.

[0068] Furthermore, pharmaceutically acceptable excipients should be compatible with the active ingredient, meaning they can be miscible with it without significantly reducing the drug's efficacy under normal circumstances. Specific examples of substances that can serve as pharmaceutically acceptable carriers or excipients include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methylcellulose, ethylcellulose, and methylcellulose; tragacanth gum powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter; polyols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; colorants; flavoring agents; tableting agents; stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic salt solutions; and phosphate buffers, etc. These substances are used as needed to help stabilize the formulation or to improve its activity or bioavailability or to produce an acceptable taste or smell when taken orally.

[0069] The drug may be, for example, an injectable drug or an oral drug.

[0070] In this invention, unless otherwise specified, the drug dosage form is not particularly limited and can be formulated as injections, oral solutions, tablets, capsules, pellets, sprays, inhalers, etc., and can be prepared by conventional methods. The choice of drug dosage form should be matched with the route of administration.

[0071] The drug can effectively improve the donor chimerism rate after allogeneic hematopoietic stem cell transplantation in mice and promote the implantation of donor hematopoietic stem cells, thereby preventing or treating post-transplant mixed chimerism.

[0072] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0073] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0074] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0075] The structural formulas of agonist 1 and agonist 2 in the Integrin α4β1 agonist in the following examples are as follows:

[0076]

[0077] And for comparison, the structural formula of THI0019 (CAS number 1378532-99-0) is as follows:

[0078]

[0079] Example 1:

[0080] Take patient specimens and send samples

[0081] 1. Experimental Materials

[0082] (1) Bone marrow specimen: derived from bone marrow of patients after allogeneic hematopoietic stem cell transplantation (all patients were aware of this and signed informed consent forms).

[0083] (2) Main reagents: phosphate buffered saline (PBS) (Gibco, USA), fetal bovine serum (Gibco, USA), serum-free culture medium SFEM (StemCell, Canada), lymphocyte separation medium (StemCell, Canada).

[0084] (3) Instruments: centrifuge (Eppendorf, Germany), 10x Genomic single-cell sequencer.

[0085] 2. Experimental Methods:

[0086] (1) Preparation of bone marrow mononuclear cells: After mixing bone marrow sample with an equal volume of PBS containing 2% serum, slowly add lymphocyte separation medium (volume ratio: 1:1:1); centrifuge at 3000 rpm for 30 min (turn off the centrifuge brake or reduce the speed to the lowest setting); aspirate the central thin film layer (the upper layer is serum and the lower layer is granulocytes, try to aspirate the entire thin film layer); centrifuge at 2000 rpm for 10 min, discard the supernatant (do not pour out); resuspend the cells in serum-free SFEM medium, count the cells to calculate the cell concentration, and prepare a single-cell suspension.

[0087] (2) Single-cell sequencing: The prepared single-cell suspension was first subjected to quality inspection. After passing the quality inspection, it was loaded into the 10xGenomic single-cell capture system to construct a cDNA library with 10x tags and then short-read NGS sequencing was performed on an Illumina sequencer.

[0088] 3. Experimental Results:

[0089] The experimental flowchart can be found here. Figure 1 .

[0090] Example 2: Detection of mRNA levels of Itga4 and Itgb1 genes in patients with different chimeric states

[0091] 1. Experimental Methods: Bone marrow cells from transplanted patients, including mixed chimeric and fully chimeric cells, were collected and sequenced using a 10XGenomics platform based on microfluidic technology. Cell Ranger was used to perform data quality analysis on the raw data, which was then aligned to a reference genome in the Ensembl database to obtain a two-dimensional matrix of gene expression in the cells. Cell filtering was then performed to remove double-celled and dead cells, resulting in high-quality single cells for subsequent analysis. Multiple samples were pooled for analysis to correct for batch effects and other technical factors. For high-quality single cells and the number of UMI sequences of genes within the sample, the total number of UMIs per cell and their ratio to a scaling factor of 10,000 were calculated. Normalization was performed to correct for sequencing depth. Dimensionality reduction clustering analysis was conducted on high-variability genes based on diffusion coefficients. Cells in the high-quality cell populations were annotated using known cell marker genes. The FindMarkers function in the Seurat package was used to identify differentially expressed genes in each cell population under mixed chimeric and fully chimeric states. Itga4 and Itgb1 genes were extracted for plotting and statistical testing.

[0092] 2. Experimental Results:

[0093] The experimental results are shown in Figure 2 .

[0094] Conclusion: After allogeneic hematopoietic stem cell transplantation patients transformed from mixed chimeras (MDC) to complete chimeras (FDC), the mRNA expression levels of Itga4 and Itgb1 genes in the hematopoietic stem cell subsets of the patients were significantly increased (p values ​​were 0.0031 and 0.0054, respectively).

[0095] Example 3:

[0096] Construct and deliver hybrid chimeric and complete chimeric mouse models.

[0097] 1. Experimental Materials

[0098] (1) Mice: The donor mice were inbred SPF grade C57BL / 6J(H-2K) mice. b Male, 6-8 weeks old; recipient mice were SPF grade Balb / c (H-2K) mice. d Female, 6-8 weeks old, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0099] (2) Main reagents: Phosphate-buffered saline (PBS) (Gibco, USA), fetal bovine serum (Gibco, USA), RPMI 1640 medium (Gibco, USA), FACS buffer (StemCell, Canada), serum-free SFEM medium (StemCell, Canada), lymphocyte separation medium (StemCell, Canada), PE-H-2K d Monoclonal antibody (BD Pharmingen, USA), APC-H-2K b Monoclonal antibody (BD Pharmingen, USA), cyclophosphamide (Sigma-Aldrich, USA), EDTA anticoagulant (Gibco, USA), red blood cell lysis buffer (China Sangon Biotech).

[0100] (3) Instruments: Small animal irradiation instrument (Kubtec, USA), centrifuge (Eppendorf, Germany), 10xGenomic single-cell sequencer.

[0101] 2. Experimental Methods

[0102] (1) Preparation of mouse bone marrow single-cell suspension: After euthanasia, the femurs of both sides of the mice were placed in FACS buffer. Holes were cut at the edges of the intact bones with scissors. Then, the bone marrow cells were flushed out into a 15mL centrifuge tube with FACS buffer through a 1mL syringe. The cells were centrifuged at 300g and 4℃ for 5min. The supernatant was discarded. 1mL of red blood cell lysis buffer was added and mixed by pipetting. The cells were lysed at room temperature for 5min. 5mL of FACS buffer was added. The cells were centrifuged at 300g and 4℃ for 5min. The supernatant was discarded. The cells were washed once with FACS buffer. The cells were then resuspended in 1mL of FACS buffer and filtered through a 40μm filter membrane to obtain a single-cell suspension.

[0103] (2) Obtaining mouse spleen cells: After euthanasia, the spleen of the mouse was placed in a 6 cm culture dish with FACS buffer. Then, the spleen was placed on a 40 μm filter membrane and ground into a single cell suspension with a syringe plunger. The suspension was then transferred to a 15 mL centrifuge tube and centrifuged at 300 g for 5 min at 4 °C. The supernatant was discarded, and 2 mL of red blood cell lysis buffer was added and mixed by pipetting. The cells were lysed at room temperature for 5 min. Then, 10 mL of FACS buffer was added, and the suspension was centrifuged at 300 g for 5 min at 4 °C. The supernatant was discarded, and the cells were washed with FACS again. The cells were then resuspended in 1 mL of FACS buffer and filtered through a 40 μm filter membrane to obtain a single cell suspension.

[0104] (3) Obtaining peripheral blood cells from mice: Blood was collected from the orbital cavity of mice and placed in a centrifuge tube containing EDTA anticoagulant. The blood was gently tapped to mix. An appropriate amount of peripheral blood was transferred to a new centrifuge tube, 1 mL of erythrocyte lysis buffer was added, and the cells were lysed at room temperature for 10 min. After centrifugation at 300 g and 4 °C for 5 min, the supernatant was discarded, the cells were washed once with FACS buffer, and then resuspended in an appropriate amount of FACS buffer for subsequent operations.

[0105] (4) Chimerism level detection: Peripheral blood, bone marrow and spleen cell suspensions of recipient mice were collected periodically and PE-H-2K was added. d and APC-H-2K b Monoclonal antibodies were used to distinguish between donors and recipients, and flow cytometry was used to analyze the proportion of donor-derived nucleated cells in the bone marrow, spleen, and peripheral blood of recipient mice. Recipients with 5%–95% donor-derived cells were defined as mixed chimeras, while non-mixed chimeras included complete engraftment of >95% donor-derived cells and complete rejection of <5% donor-derived cells.

[0106] (5) Establishment of a hybrid chimeric mouse model: On the day of transplantation, recipient mice were given a sublethal dose of whole-body irradiation (2-3 Gy X-ray, 120 cGy / min), and 4 hours later, 2 × 10⁻⁶ cGy / min was infused via the tail vein. 7Bone marrow nucleated cells / mouse. On day 3 post-transplantation, mice were injected intraperitoneally with cyclophosphamide 200 mg / kg (cyclophosphamide dissolved in PBS, final concentration 8 mg / ml). Mice were randomly divided into experimental and control groups.

[0107] (6) Donor lymphocyte infusion (DLI): The obtained spleen cell suspension was counted, and the cell concentration was adjusted to 2 × 10⁶ cells / mL using RPMI 1640 medium. 8 / ml for later use. On day 15 post-transplantation, mice in the experimental group were injected with nucleated spleen cells via the tail vein, 5×10⁹ cells per mouse. 7 One cell, the system is 250ul.

[0108] (7) Single-cell sequencing: The prepared mouse bone marrow single-cell suspension was first subjected to quality inspection. After passing the quality inspection, it was loaded into the 10x Genomic single-cell capture system to construct a cDNA library with 10x tags and then performed short-read NGS sequencing on an Illumina sequencer.

[0109] 3. Experimental Results:

[0110] The experimental flowchart can be found here. Figure 3 .

[0111] Conclusion: Using this model, a mixed chimeric (MDC) mouse model of allogeneic bone marrow transplantation can be successfully obtained on day 14 post-transplantation. DLI intervention in the experimental group on day 15 can successfully obtain a complete chimeric (FDC) mouse on day 28 post-transplantation, while the control group without DLI intervention failed to implant.

[0112] Example 4:

[0113] Detecting the mRNA levels of Itga4 and Itgb1 genes in mice under different chimeric states

[0114] 1. Experimental Methods: Mice with mixed chimerism and complete chimerism were harvested and single-cell sequenced using the 10XGenomics platform based on microfluidic technology. Cell Ranger was used to perform data quality analysis on the raw data, which was then aligned to the reference genome in the Ensembl database to obtain a two-dimensional matrix of gene expression in cells. Cell filtering was then performed to remove double-cell and dead cells, resulting in high-quality single-cell sequences for further analysis. Multiple samples were merged for analysis to correct for batch effects and other technical factors. For high-quality single cells and the number of UMI sequences of genes within each sample, the total number of UMIs per cell and their ratio to a scaling factor of 10,000 were calculated. Normalization was performed to correct for cell sequencing depth. Dimensionality reduction and clustering analysis were conducted on high-variability genes based on diffusion coefficients. Cells in the high-quality cell populations were annotated using known cell marker genes. The FindMarkers function in the Seurat package was then used to identify differentially expressed genes in each cell population under mixed chimerism and complete chimerism. Itga4 and Itgb1 genes were extracted for plotting and statistical testing.

[0115] 2. Experimental Results:

[0116] The experimental results are shown in Figure 4 .

[0117] Conclusion: After mice with allogeneic hematopoietic stem cell transplantation were transformed from mixed chimeras (MDC) to complete chimeras (FDC), the mRNA expression levels of Itga4 and Itgb1 genes in the mouse hematopoietic stem cell subsets were significantly increased (p values ​​were 0.0023 and 0.0082, respectively).

[0118] Example 5:

[0119] The model mice were administered the drug on days 1 to 10 post-transplantation, and the peripheral blood chimerism rate of each group of mice was measured on day 14 post-transplantation.

[0120] 1. Experimental materials:

[0121] (1) Mice: The donor mice were inbred SPF grade C57BL / 6J(H-2K) mice. b Male, 6-8 weeks old; recipient mice were SPF grade Balb / c (H-2K) mice. d Female, 6-8 weeks old, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0122] (2) Integrin α4β1 agonists: agonist 1 (Activator-1), agonist 2 (Activator-2) and THI0019.

[0123] (3) Main reagents: Phosphate-buffered saline (PBS) (Gibco, USA), fetal bovine serum (Gibco, USA), RPMI 1640 medium (Gibco, USA), FACS buffer (StemCell, Canada), serum-free SFEM medium (StemCell, Canada), lymphocyte separation medium (StemCell, Canada), PE-H-2K d Monoclonal antibody (BD Pharmingen, USA), APC-H-2K b Monoclonal antibody (BD Pharmingen, USA), cyclophosphamide (Sigma-Aldrich, USA), EDTA anticoagulant (Gibco, USA), red blood cell lysis buffer (China Sangon Biotech), DMSO (Sigma-Aldrich, USA).

[0124] (4) Instruments: Small animal irradiation instrument (Kubtec, USA), centrifuge (Eppendorf, Germany), flow cytometer (BD Pharmingen, USA).

[0125] 2. Experimental Methods:

[0126] (1) Establishment of a hybrid chimeric mouse model: On the day of transplantation, recipient mice were given a sublethal dose of whole-body irradiation (2-3 Gy X-ray, 120 cGy / min), and 4 hours later, 2 × 10 cGy was infused via the tail vein. 7 Nucleated bone marrow cells / animal. Cyclophosphamide 200 mg / kg was administered intraperitoneally on day 3 post-transplantation. (See Example 3 for other experimental details.)

[0127] (2) Drug dissolution: All three Integrin α4β1 agonist powders were first dissolved in DMSO solution to a final concentration of 100 mg / ml, and then dissolved in PBS solution to a final concentration of 1 mg / ml. That is, the drug was dissolved in PBS solution containing 1% DMSO.

[0128] (3) Mouse administration: Mice were randomly divided into a control group and three experimental groups. The mice in the three experimental groups were injected intraperitoneally with three Integrin α4β1 agonists, namely agonist-1, agonist-2 and agonist-3, from day 1 to day 10 after transplantation. The dosage was 5 mg / kg per mouse. The mice in the control group were injected intraperitoneally with the same amount of solvent at the same time.

[0129] (4) Chimerism level detection: Peripheral blood cell suspension from recipient mice was collected on day 14 post-transplantation and PE-H-2K was added. d and APC-H-2Kb Monoclonal antibodies were used to distinguish between donors and recipients, and flow cytometry was used to analyze the proportion of donor-derived nucleated cells in the peripheral blood of recipient mice. (For other experimental details, see Example 3 for experimental methods.)

[0130] 3. Experimental Results:

[0131] The experimental results are shown in Figure 5 .

[0132] Conclusion: Integrin α4β1 agonist was administered to mixed chimeric mice from day 1 to day 10 post-transplantation. The chimerism rate of peripheral blood cells was measured 2 weeks post-transplantation. The mean chimerism rate of the three agonist groups was higher than that of the control group. Only agonist-1 showed a statistically significant difference (P = 0.011).

[0133] Mean ± standard error of peripheral blood chimerism rate (%) in each group of mice: Control: 77.16±1.645; Activator-1: 83.39±1.225; Activator-2: 81.70±1.257; THI0019: 78.85±3.475.

[0134] Example 6:

[0135] The model mice were administered the drug on days 4 to 12 post-transplantation, and the peripheral blood chimerism rate of each group of mice was measured on day 14 post-transplantation.

[0136] 1. Experimental materials:

[0137] (1) Mice: The donor mice were inbred SPF grade C57BL / 6J(H-2K) mice. b Male, 6-8 weeks old; recipient mice were SPF grade Balb / c (H-2K) mice. d Female, 6-8 weeks old, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0138] (2) Integrin α4β1 agonists: agonist 1 (Activator-1), agonist 2 (Activator-2) and THI0019.

[0139] (3) Main reagents: Phosphate-buffered saline (PBS) (Gibco, USA), fetal bovine serum (Gibco, USA), RPMI 1640 medium (Gibco, USA), FACS buffer (StemCell, Canada), serum-free SFEM medium (StemCell, Canada), lymphocyte separation medium (StemCell, Canada), PE-H-2K dMonoclonal antibody (BD Pharmingen, USA), APC-H-2K b Monoclonal antibody (BD Pharmingen, USA), cyclophosphamide (Sigma-Aldrich, USA), EDTA anticoagulant (Gibco, USA), red blood cell lysis buffer (China Sangon Biotech), DMSO (Sigma-Aldrich, USA).

[0140] (4) Instruments: Small animal irradiation instrument (Kubtec, USA), centrifuge (Eppendorf, Germany), flow cytometer (BD Pharmingen, USA).

[0141] 2. Experimental Methods:

[0142] (1) Establishment of a hybrid chimeric mouse model: On the day of transplantation, recipient mice were given a sublethal dose of whole-body irradiation (2-3 Gy X-ray, 120 cGy / min), and 4 hours later, 2 × 10 cGy was infused via the tail vein. 7 Nucleated bone marrow cells / animal. Cyclophosphamide 200 mg / kg was administered intraperitoneally on day 3 post-transplantation. (See Example 3 for other experimental details.)

[0143] (2) Drug dissolution: All three Integrin α4β1 agonist powders were first dissolved in DMSO solution to a final concentration of 100 mg / ml, and then dissolved in PBS solution to a final concentration of 1 mg / ml. That is, the drug was dissolved in PBS solution containing 1% DMSO.

[0144] (3) Mouse administration: Mice were randomly divided into a control group and three experimental groups. The mice in the three experimental groups were injected intraperitoneally with three Integrin α4β1 agonists, namely agonist-1, agonist-2 and agonist-3, from day 4 to day 12 after transplantation. The dosage was 5 mg / kg per mouse. The mice in the control group were injected intraperitoneally with the same amount of solvent at the same time.

[0145] (4) Chimerism level detection: Peripheral blood cell suspension from recipient mice was collected on day 14 post-transplantation and PE-H-2K was added. d and APC-H-2K b Monoclonal antibodies were used to distinguish between donors and recipients, and flow cytometry was used to analyze the proportion of donor-derived nucleated cells in the peripheral blood of recipient mice. (For other experimental details, see Example 3 for experimental methods.)

[0146] 3. Experimental Results:

[0147] The experimental results are shown in Figure 6 .

[0148] Conclusion: Integrin α4β1 agonist was administered to mixed chimeric mice from day 4 to day 12 post-transplantation. The chimerism rate of peripheral blood cells in mice was measured 2 weeks post-transplantation. Among the three agonist-administered groups, only agonist-1 had a higher mean chimerism rate than the control group.

[0149] Mean ± standard error of peripheral blood chimerism rate (%) in each group of mice: Control: 78.20 ± 3.328; Activator-1: 85.34 ± 2.093; Activator-2: 77.17 ± 3.857; THI0019: 76.54 ± 5.212.

[0150] Example 7:

[0151] The model mice were administered the drug on days 6 to 12 post-transplantation, and the peripheral blood chimerism rate of each group of mice was measured on day 14 post-transplantation.

[0152] 1. Experimental materials:

[0153] (1) Mice: The donor mice were inbred SPF grade C57BL / 6J(H-2K) mice. b Male, 6-8 weeks old; recipient mice were SPF grade Balb / c (H-2K) mice. d Female, 6-8 weeks old, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0154] (2) Integrin α4β1 agonists: agonist 1 (Activator-1), agonist 2 (Activator-2) and THI0019.

[0155] (3) Main reagents: Phosphate-buffered saline (PBS) (Gibco, USA), fetal bovine serum (Gibco, USA), RPMI 1640 medium (Gibco, USA), FACS buffer (StemCell, Canada), serum-free SFEM medium (StemCell, Canada), lymphocyte separation medium (StemCell, Canada), PE-H-2K d Monoclonal antibody (BD Pharmingen, USA), APC-H-2K b Monoclonal antibody (BD Pharmingen, USA), cyclophosphamide (Sigma-Aldrich, USA), EDTA anticoagulant (Gibco, USA), red blood cell lysis buffer (China Sangon Biotech), DMSO (Sigma-Aldrich, USA).

[0156] (4) Instruments: Small animal irradiation instrument (Kubtec, USA), centrifuge (Eppendorf, Germany), flow cytometer (BD Pharmingen, USA).

[0157] 2. Experimental Methods:

[0158] (1) Establishment of a hybrid chimeric mouse model: On the day of transplantation, recipient mice were given a sublethal dose of whole-body irradiation (2-3 Gy X-ray, 120 cGy / min), and 4 hours later, 2 × 10 cGy was infused via the tail vein. 7 Nucleated bone marrow cells / animal. Cyclophosphamide 200 mg / kg was administered intraperitoneally on day 3 post-transplantation. (See Example 3 for other experimental details.)

[0159] (2) Drug dissolution: All three Integrin α4β1 agonist powders were first dissolved in DMSO solution to a final concentration of 100 mg / ml, and then dissolved in PBS solution to a final concentration of 1 mg / ml. That is, the drug was dissolved in PBS solution containing 1% DMSO.

[0160] (3) Mouse administration: Mice were randomly divided into a control group and three experimental groups. The mice in the three experimental groups were injected intraperitoneally with three Integrin α4β1 agonists, namely agonist-1, agonist-2 and agonist-3, from day 6 to day 12 after transplantation. The dosage was 5 mg / kg per mouse. The mice in the control group were injected intraperitoneally with the same amount of solvent at the same time.

[0161] (4) Chimerism level detection: Peripheral blood cell suspension from recipient mice was collected on day 14 post-transplantation and PE-H-2K was added. d and APC-H-2K b Monoclonal antibodies were used to distinguish between donors and recipients, and flow cytometry was used to analyze the proportion of donor-derived nucleated cells in the peripheral blood of recipient mice. (For other experimental details, see Example 3 for experimental methods.)

[0162] 3. Experimental Results:

[0163] The experimental results are shown in Figure 7 .

[0164] Conclusion: Integrin α4β1 agonist was administered to mixed chimeric mice from day 6 to day 12 post-transplantation. The chimerism rate of peripheral blood cells in mice was measured 2 weeks post-transplantation. The mean chimerism rate of the three agonist groups was higher than that of the control group, but only agonist-2 showed a statistically significant difference (P = 0.048).

[0165] Mean ± standard error of peripheral blood chimerism rate (%) in each group of mice: Control: 78.20 ± 3.328; Activator-1: 84.16 ± 2.156; Activator-2: 86.66 ± 1.537; THI0019: 85.24 ± 2.073.

[0166] Example 8:

[0167] The model mice were administered the drug from day 8 to day 14 post-transplantation, and the peripheral blood chimerism rate of each group of mice was measured on day 14 post-transplantation.

[0168] 1. Experimental materials:

[0169] (1) Mice: The donor mice were inbred SPF grade C57BL / 6J(H-2K) mice. b Male, 6-8 weeks old; recipient mice were SPF grade Balb / c (H-2K) mice. d Female, 6-8 weeks old, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0170] (2) Integrin α4β1 agonists: agonist-1 and agonist-2.

[0171] (3) Main reagents: Phosphate-buffered saline (PBS) (Gibco, USA), fetal bovine serum (Gibco, USA), RPMI 1640 medium (Gibco, USA), FACS buffer (StemCell, Canada), serum-free SFEM medium (StemCell, Canada), lymphocyte separation medium (StemCell, Canada), PE-H-2K d Monoclonal antibody (BD Pharmingen, USA), APC-H-2K b Monoclonal antibody (BD Pharmingen, USA), cyclophosphamide (Sigma-Aldrich, USA), EDTA anticoagulant (Gibco, USA), red blood cell lysis buffer (China Sangon Biotech), DMSO (Sigma-Aldrich, USA).

[0172] (4) Instruments: Small animal irradiation instrument (Kubtec, USA), centrifuge (Eppendorf, Germany), flow cytometer (BD Pharmingen, USA).

[0173] 2. Experimental Methods:

[0174] (1) Establishment of a hybrid chimeric mouse model: On the day of transplantation, recipient mice were given a sublethal dose of whole-body irradiation (2-3 Gy X-ray, 120 cGy / min), and 4 hours later, 2 × 10 cGy was infused via the tail vein. 7 Nucleated bone marrow cells / animal. Cyclophosphamide 200 mg / kg was administered intraperitoneally on day 3 post-transplantation. (See Example 3 for other experimental details.)

[0175] (2) Drug dissolution: Both Integrin α4β1 agonist powders were first dissolved in DMSO solution to a final concentration of 100 mg / ml, and then dissolved in PBS solution to a final concentration of 1 mg / ml. That is, the drug was dissolved in PBS solution containing 1% DMSO.

[0176] (3) Mouse administration: Mice were randomly divided into a control group and two experimental groups. Two Integrin α4β1 agonists, namely agonist-1 and agonist-2, were injected intraperitoneally daily from day 8 to day 14 after transplantation. The dosage was 5 mg / kg per mouse. Mice in the control group were injected intraperitoneally with the same amount of solvent at the same time.

[0177] (4) Chimerism level detection: Peripheral blood cell suspension from recipient mice was collected on day 14 post-transplantation and PE-H-2K was added. d and APC-H-2K b Monoclonal antibodies were used to distinguish between donors and recipients, and flow cytometry was used to analyze the proportion of donor-derived nucleated cells in the peripheral blood of recipient mice. (For other experimental details, see Example 3 for experimental methods.)

[0178] 3. Experimental Results:

[0179] The experimental results are shown in Figure 8 .

[0180] Conclusion: Integrin α4β1 agonist was administered to mixed chimeric mice from day 8 to day 14 post-transplantation. The chimerism rate of peripheral blood cells in mice was measured 2 weeks post-transplantation. The mean chimerism rate of both agonist-treated groups was higher than that of the control group, but only agonist-2 showed a statistically significant difference (P = 0.0005).

[0181] Mean ± standard error of peripheral blood chimerism rate (%) in each group of mice: Control: 56.61 ± 3.601; Activator-1: 63.17 ± 3.715; Activator-2: 75.76 ± 3.185.

[0182] Example 9:

[0183] The model mice were administered the drug from day 8 to day 28 post-transplantation, and the peripheral blood chimerism rate of each group of mice was measured on day 28 post-transplantation.

[0184] 1. Experimental materials:

[0185] (1) Mice: The donor mice were inbred SPF grade C57BL / 6J(H-2K) mice. b Male, 6-8 weeks old; recipient mice were SPF grade Balb / c (H-2K) mice. d Female, 6-8 weeks old, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0186] (2) Integrin α4β1 agonists: agonist-1 and agonist-2.

[0187] (3) Main reagents: Phosphate-buffered saline (PBS) (Gibco, USA), fetal bovine serum (Gibco, USA), RPMI 1640 medium (Gibco, USA), FACS buffer (StemCell, Canada), serum-free SFEM medium (StemCell, Canada), lymphocyte separation medium (StemCell, Canada), PE-H-2K d Monoclonal antibody (BD Pharmingen, USA), APC-H-2K b Monoclonal antibody (BD Pharmingen, USA), cyclophosphamide (Sigma-Aldrich, USA), EDTA anticoagulant (Gibco, USA), red blood cell lysis buffer (China Sangon Biotech), DMSO (Sigma-Aldrich, USA).

[0188] (4) Instruments: Small animal irradiation instrument (Kubtec, USA), centrifuge (Eppendorf, Germany), flow cytometer (BD Pharmingen, USA).

[0189] 2. Experimental Methods:

[0190] (1) Establishment of a hybrid chimeric mouse model: On the day of transplantation, recipient mice were given a sublethal dose of whole-body irradiation (2-3 Gy X-ray, 120 cGy / min), and 4 hours later, 2 × 10 cGy was infused via the tail vein. 7 Nucleated bone marrow cells / animal. Cyclophosphamide 200 mg / kg was administered intraperitoneally on day 3 post-transplantation. (See Example 3 for other experimental details.)

[0191] (2) Drug dissolution: Both Integrin α4β1 agonist powders were first dissolved in DMSO solution to a final concentration of 100 mg / ml, and then dissolved in PBS solution to a final concentration of 1 mg / ml. That is, the drug was dissolved in PBS solution containing 1% DMSO.

[0192] (3) Mouse administration: Mice were randomly divided into a control group and two experimental groups. Two Integrin α4β1 agonists, namely agonist-1 and agonist-2, were injected intraperitoneally daily from day 8 to day 28 after transplantation. The dosage was 5 mg / kg per mouse. Mice in the control group were injected intraperitoneally with the same amount of solvent at the same time.

[0193] (4) Chimerism level detection: Peripheral blood cell suspension from recipient mice was collected on day 28 post-transplantation and PE-H-2K was added. d and APC-H-2K b Monoclonal antibodies were used to distinguish between donors and recipients, and flow cytometry was used to analyze the proportion of donor-derived nucleated cells in the peripheral blood of recipient mice. (For other experimental details, see Example 3 for experimental methods.)

[0194] 3. Experimental Results:

[0195] The experimental results are shown in Figure 9 .

[0196] Conclusion: Integrin α4β1 agonist was administered to mixed chimeric mice from day 8 to day 28 post-transplantation. The chimerism rate of peripheral blood cells in mice was measured 4 weeks post-transplantation. The mean chimerism rate of both agonist-treated groups was higher than that of the control group, but only agonist-2 showed a statistically significant difference (P = 0.0028).

[0197] Mean ± standard error of peripheral blood chimerism rate (%) in each group of mice: Control: 46.88 ± 4.323; Activator-1: 48.47 ± 4.162; Activator-2: 71.34 ± 5.596.

[0198] Example 10:

[0199] The model mice were administered the drug on days 8, 10, 12 to 14 post-transplantation, and the peripheral blood chimerism rate of each group of mice was measured on day 14 post-transplantation.

[0200] 1. Experimental materials:

[0201] (1) Mice: The donor mice were inbred SPF grade C57BL / 6J(H-2K) mice. b Male, 6-8 weeks old; recipient mice were SPF grade Balb / c (H-2K) mice.d Female, 6-8 weeks old, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0202] (2) Integrin α4β1 agonist: Activator-2.

[0203] (3) Main reagents: Phosphate-buffered saline (PBS) (Gibco, USA), fetal bovine serum (Gibco, USA), RPMI 1640 medium (Gibco, USA), FACS buffer (StemCell, Canada), serum-free SFEM medium (StemCell, Canada), lymphocyte separation medium (StemCell, Canada), PE-H-2K d Monoclonal antibody (BD Pharmingen, USA), APC-H-2K b Monoclonal antibody (BD Pharmingen, USA), cyclophosphamide (Sigma-Aldrich, USA), EDTA anticoagulant (Gibco, USA), red blood cell lysis buffer (China Sangon Biotech), DMSO (Sigma-Aldrich, USA).

[0204] (4) Instruments: Small animal irradiation instrument (Kubtec, USA), centrifuge (Eppendorf, Germany), flow cytometer (BD Pharmingen, USA).

[0205] 2. Experimental Methods:

[0206] (1) Establishment of a hybrid chimeric mouse model: On the day of transplantation, recipient mice were given a sublethal dose of whole-body irradiation (2-3 Gy X-ray, 120 cGy / min), and 4 hours later, 2 × 10 cGy was infused via the tail vein. 7 Nucleated bone marrow cells / animal. Cyclophosphamide 200 mg / kg was administered intraperitoneally on day 3 post-transplantation. (See Example 3 for other experimental details.)

[0207] (2) Drug dissolution: Both Integrin α4β1 agonist powders were first dissolved in DMSO solution to a final concentration of 100 mg / ml, and then dissolved in PBS solution to a final concentration of 1 mg / ml. That is, the drug was dissolved in PBS solution containing 1% DMSO.

[0208] (3) Mouse administration: Mice were randomly divided into control group and experimental group. Activator-2 was injected intraperitoneally daily from day 8 to day 14, day 10 to day 14, and day 12 to day 14 after transplantation. The dosage was 5 mg / kg per mouse. The control group mice were injected intraperitoneally with the same amount of solvent at the same time.

[0209] (4) Chimerism level detection: Peripheral blood cell suspension from recipient mice was collected on day 14 post-transplantation and PE-H-2K was added. d and APC-H-2K b Monoclonal antibodies were used to distinguish between donors and recipients, and flow cytometry was used to analyze the proportion of donor-derived nucleated cells in the peripheral blood of recipient mice. (For other experimental details, see Example 3 for experimental methods.)

[0210] 3. Experimental Results:

[0211] The experimental results are shown in Figure 10 .

[0212] Conclusion: In mixed chimeric mice, administration of agonist 2 after transplantation and detection of peripheral blood cell chimerism rate 2 weeks post-transplantation resulted in different experimental results depending on the timing of administration. Compared with other administration times, the optimal time was to start administration on day 8, with a significantly higher mean donor chimerism rate than the control group, showing a statistically significant difference (P = 0.0199).

[0213] Mean ± standard error of peripheral blood chimerism rate (%) in each group of mice: Control: 74.54 ± 1.586; Activator2-d8: 80.18 ± 1.370; Activator2-d10: 72.32 ± 3.172; Activator2-d12: 77.01 ± 2.579.

[0214] Example 11:

[0215] The model mice were administered the drug on days 8, 10, 12 to 28 post-transplantation, and the peripheral blood chimerism rate of each group of mice was measured on day 28 post-transplantation.

[0216] 1. Experimental materials:

[0217] (1) Mice: The donor mice were inbred SPF grade C57BL / 6J(H-2K) mice. b Male, 6-8 weeks old; recipient mice were SPF grade Balb / c (H-2K) mice. d Female, 6-8 weeks old, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0218] (2) Integrin α4β1 agonist: Activator-2.

[0219] (3) Main reagents: Phosphate-buffered saline (PBS) (Gibco, USA), fetal bovine serum (Gibco, USA), RPMI 1640 medium (Gibco, USA), FACS buffer (StemCell, Canada), serum-free SFEM medium (StemCell, Canada), lymphocyte separation medium (StemCell, Canada), PE-H-2K d Monoclonal antibody (BD Pharmingen, USA), APC-H-2K b Monoclonal antibody (BD Pharmingen, USA), cyclophosphamide (Sigma-Aldrich, USA), EDTA anticoagulant (Gibco, USA), red blood cell lysis buffer (China Sangon Biotech), DMSO (Sigma-Aldrich, USA).

[0220] (4) Instruments: Small animal irradiation instrument (Kubtec, USA), centrifuge (Eppendorf, Germany), flow cytometer (BD Pharmingen, USA).

[0221] 2. Experimental Methods:

[0222] (1) Establishment of a hybrid chimeric mouse model: On the day of transplantation, recipient mice were given a sublethal dose of whole-body irradiation (2-3 Gy X-ray, 120 cGy / min), and 4 hours later, 2 × 10 cGy was infused via the tail vein. 7 Nucleated bone marrow cells / animal. Cyclophosphamide 200 mg / kg was administered intraperitoneally on day 3 post-transplantation. (See Example 3 for other experimental details.)

[0223] (2) Drug dissolution: The Integrin α4β1 agonist powder was first dissolved in DMSO solution to a final concentration of 100 mg / ml, and then dissolved in PBS solution to a final concentration of 1 mg / ml. That is, the drug was dissolved in PBS solution containing 1% DMSO.

[0224] (3) Mouse administration: Mice were randomly divided into control group and experimental group. Activator-2 was injected intraperitoneally daily from day 8 to day 28, day 10 to day 28, and day 12 to day 28 after transplantation. The dosage was 5 mg / kg per mouse. The control group mice were injected intraperitoneally with the same amount of solvent at the same time.

[0225] (4) Chimerism level detection: Peripheral blood cell suspension from recipient mice was collected on day 28 post-transplantation and PE-H-2K was added. d and APC-H-2K bMonoclonal antibodies were used to distinguish between donors and recipients, and flow cytometry was used to analyze the proportion of donor-derived nucleated cells in the peripheral blood of recipient mice. (For other experimental details, see Example 3 for experimental methods.)

[0226] 3. Experimental Results:

[0227] The experimental results are shown in Figure 11 .

[0228] Conclusion: In mixed chimeric mice, administration of agonist 2 after transplantation and detection of peripheral blood cell chimerism rate 4 weeks post-transplantation resulted in different experimental results depending on the timing of administration. Compared with other administration times, the optimal time was to start administration on day 8, with a significantly higher mean donor chimerism rate than the control group, showing a statistically significant difference (P = 0.0160).

[0229] Mean ± standard error of peripheral blood chimerism rate (%) in each group of mice: Control: 56.70 ± 6.634; Activator2-d8: 76.31 ± 3.045; Activator2-d10: 68.65 ± 3.809; Activator2-d12: 63.69 ± 5.763.

[0230] Example 12:

[0231] The model mice were administered the drug on days 8, 10, 12 to 28 post-transplantation, and the peripheral blood chimerism rate of each group of mice was measured on day 56 post-transplantation.

[0232] 1. Experimental materials:

[0233] (1) Mice: The donor mice were inbred SPF grade C57BL / 6J(H-2K) mice. b Male, 6-8 weeks old; recipient mice were SPF grade Balb / c (H-2K) mice. d Female, 6-8 weeks old, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0234] (2) Integrin α4β1 agonist: Activator-2.

[0235] (3) Main reagents: Phosphate-buffered saline (PBS) (Gibco, USA), fetal bovine serum (Gibco, USA), RPMI 1640 medium (Gibco, USA), FACS buffer (StemCell, Canada), serum-free SFEM medium (StemCell, Canada), lymphocyte separation medium (StemCell, Canada), PE-H-2K d Monoclonal antibody (BD Pharmingen, USA), APC-H-2Kb Monoclonal antibody (BD Pharmingen, USA), cyclophosphamide (Sigma-Aldrich, USA), EDTA anticoagulant (Gibco, USA), red blood cell lysis buffer (China Sangon Biotech), DMSO (Sigma-Aldrich, USA).

[0236] (4) Instruments: Small animal irradiation instrument (Kubtec, USA), centrifuge (Eppendorf, Germany), flow cytometer (BD Pharmingen, USA).

[0237] 2. Experimental Methods:

[0238] (1) Based on Example 11: A hybrid chimeric mouse model was established, and agonist No. 2 was injected intraperitoneally daily from day 8 to day 28, day 10 to day 28, and day 12 to day 28 after transplantation (for specific experimental details, see the experimental methods in Example 3 and Example 11).

[0239] (2) Peripheral blood chimerism level was detected on day 56 post-transplantation: Peripheral blood cell suspension from recipient mice was taken and PE-H-2K was added. d and APC-H-2K b Monoclonal antibodies were used to distinguish between donors and recipients, and flow cytometry was used to analyze the proportion of donor-derived nucleated cells in the peripheral blood of recipient mice.

[0240] 3. Experimental Results:

[0241] The experimental results are shown in Figure 12 .

[0242] Conclusion: In mixed chimeric mice, administration of agonist 2 after transplantation and detection of peripheral blood cell chimerism rate at 8 weeks post-transplantation showed that day 8 was the optimal time for administration compared to other administration points. The mean donor chimerism rate was significantly higher in this group than in the control group. In the control group, some mice had completely expelled donor cells, while in the administration group, some mice had completely implanted donor cells. The difference between the two groups was statistically significant (P = 0.0010).

[0243] Mean ± standard error of peripheral blood chimerism rate (%) in each group of mice: Control: 36.44 ± 9.958; Activator2-d8: 69.44 ± 4.042; Activator2-d10: 44.91 ± 10.44; Activator2-d12: 34.93 ± 11.84.

[0244] Example 13:

[0245] The model mice were administered the drug between days 8 and 28 post-transplantation, and the donor chimerism rate of each cell line in the peripheral blood of the mice was detected on day 56 post-transplantation.

[0246] 1. Experimental materials:

[0247] (1) Mice: The donor mice were inbred SPF grade C57BL / 6J(H-2K) mice. b Male, 6-8 weeks old; recipient mice were SPF grade Balb / c (H-2K) mice. d Female, 6-8 weeks old, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0248] (2) Integrin α4β1 agonist: Activator-2.

[0249] (3) Main reagents: Phosphate-buffered saline (PBS) (Gibco, USA), fetal bovine serum (Gibco, USA), RPMI 1640 medium (Gibco, USA), FACS buffer (StemCell, Canada), serum-free SFEM medium (StemCell, Canada), lymphocyte separation medium (StemCell, Canada), cyclophosphamide (Sigma-Aldrich, USA), EDTA anticoagulant (Gibco, USA), erythrocyte lysis buffer (China Sangon Biotech Co., Ltd.), DMSO (Sigma-Aldrich, USA), PE-H-2K d APC-H-2K b Ter119-FITC, B220-PerCp-Cyanine, Gr-1-APC-Cyanine7, CD71-PE-Cyanine7, CD3-BV421, CD11b-BV510, F4 / 80-BV605 flow cytometry antibody, DAPI (BD Pharmaceuticals, USA).

[0250] (4) Instruments: Small animal irradiation instrument (Kubtec, USA), centrifuge (Eppendorf, Germany), flow cytometer (BD Pharmingen, USA).

[0251] 2. Experimental Methods:

[0252] (1) Based on Example 11: A hybrid chimeric mouse model was established, and agonist No. 2 was injected intraperitoneally daily from day 8 to day 28 after transplantation (for specific experimental details, see the experimental methods in Example 3 and Example 11).

[0253] (2) On day 56 post-transplantation, the chimerism level of peripheral blood cells was detected: peripheral blood cell suspension from recipient mice was taken and PE-H-2K was added. d and APC-H-2K bMonoclonal antibodies were used to distinguish between donors and recipients, and flow cytometry was used to analyze the proportion of donor-derived nucleated cells in the peripheral blood of recipient mice.

[0254] (3) Staining protocol for surface markers of peripheral blood cell lineages: Peripheral blood cell suspension was stained with the corresponding fluorescent antibodies: Ter119-FITC, B220-PerCp-Cyanine, Gr-1-APC-Cyanine7, CD71-PE-Cyanine7, CD3-BV421, CD11b-BV510, F4 / 80-BV605. After incubation at 4°C in the dark for 20 min, FACS buffer was added and washed twice. Finally, the suspension was resuspended in 300 μL of FACS buffer with DAPI and analyzed within 4 hours.

[0255] 3. Experimental Results:

[0256] The experimental results are shown in Figure 13 .

[0257] Conclusion: In mixed chimeric mice, administration of agonist 2 after transplantation and subsequent detection of peripheral blood cell chimerism rates at 8 weeks post-transplantation showed that the donor chimerism rates of myeloid (Mye) cells, B cells, T cells, and erythroid (Ery) cells were significantly higher than those in the control group (P values ​​were 0.0414, 0.0400, 0.0074, and 0.0238, respectively).

[0258] Mean ± standard error of peripheral blood chimerism rate (%) in each group of mice: Mye strain: Control: 33.89±10.840, Activator-2: 58.71±6.263; B strain: Control: 57.71±12.492, Activator-2: 88.79±2.383; T strain: Control: 21.79±8.599, Activator-2: 51.72±5.329; Ery strain: Control: 36.86±9.840, Activator-2: 68.77±3.995.

[0259] Example 14:

[0260] The model mice were administered the drug between days 8 and 28 post-transplantation, and the donor chimerism rate of various hematopoietic stem / progenitor cells in the bone marrow of the mice was detected on day 56 post-transplantation.

[0261] 1. Experimental materials:

[0262] (1) Mice: The donor mice were inbred SPF grade C57BL / 6J(H-2K) mice. b Male, 6-8 weeks old; recipient mice were SPF grade Balb / c (H-2K) mice. dFemale, 6-8 weeks old, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0263] (2) Integrin α4β1 agonist: Activator-2.

[0264] (3) Main reagents: Phosphate-buffered saline (PBS) (Gibco, USA), fetal bovine serum (Gibco, USA), RPMI 1640 medium (Gibco, USA), FACS buffer (StemCell, Canada), serum-free SFEM medium (StemCell, Canada), lymphocyte separation medium (StemCell, Canada), cyclophosphamide (Sigma-Aldrich, USA), EDTA anticoagulant (Gibco, USA), erythrocyte lysis buffer (China Sangon Biotech Co., Ltd.), DMSO (Sigma-Aldrich, USA), PE-H-2K d APC-H-2K b Biotin-conjugated Lineage (CD3e, B220, Ter119, CD41, and Gr1) antibodies, Streptavidin FITC, Sca1-PerCp-Cyanine 5.5, c-Kit-APC-Cyanine 7, CD48-BV510, CD150-BV421 flow cytometry antibodies, and DAPI (BD Pharmaceuticals, USA).

[0265] (4) Instruments: Small animal irradiation instrument (Kubtec, USA), centrifuge (Eppendorf, Germany), flow cytometer (BD Pharmingen, USA).

[0266] 2. Experimental Methods:

[0267] (1) Based on Example 11: A hybrid chimeric mouse model was established, and agonist No. 2 was injected intraperitoneally daily from day 8 to day 28 after transplantation (for specific experimental details, see the experimental methods in Example 3 and Example 11).

[0268] (2) Obtaining mouse bone marrow single-cell suspension: After euthanasia, the femurs of both sides of the mice were placed in FACS buffer. Holes were cut at the edges of the intact bones with scissors. Then, bone marrow cells were flushed out into a 15mL centrifuge tube with FACS buffer using a 1mL syringe. The tube was centrifuged at 300g and 4℃ for 5min. The supernatant was discarded. 1mL of red blood cell lysis buffer was added and mixed by pipetting. The cells were lysed at room temperature for 5min. 5mL of FACS buffer was added. The tube was centrifuged at 300g and 4℃ for 5min. The supernatant was discarded. The cells were washed once with FACS buffer. The cells were then resuspended in 1mL of FACS buffer and filtered through a 40μm filter membrane to obtain a single-cell suspension.

[0269] (3) On day 56 post-transplantation, the chimerism level of bone marrow cells was detected: bone marrow cell suspension from recipient mice was taken and PE-H-2K was added. d and APC-H-2K b Monoclonal antibodies were used to distinguish between donors and recipients, and flow cytometry was used to analyze the proportion of donor-derived nucleated cells in the bone marrow of recipient mice.

[0270] (4) Staining protocol for hematopoietic stem cell surface markers: Bone marrow cell suspension was stained with biotin-conjugated Lineage (CD3e, B220, Ter119, CD41, and Gr1) antibodies, incubated at 4°C for 20 min, washed once with FACS buffer, and then stained with Streptavidin FITC, Sca1-PerCp-Cyanine 5.5, c-Kit-APC-Cyanine 7, CD48-BV510, CD150-BV421 and donor-recipient typing fluorescent antibodies. After incubation at 4°C in the dark for 20 min, FACS buffer was added and washed twice. Finally, the suspension was resuspended in 400 μL of FACS buffer with DAPI and analyzed within 4 hours. (Note: Hematopoietic progenitor cells (HPCs) are labeled with Lin-Sca1+c-kit+CD48+ by flow cytometry; multipotent progenitor cells (MPPs) are labeled with Lin-Sca1+c-kit+CD48-CD150- by flow cytometry; LSK cells are labeled with Lin-Sca1+c-kit+ by flow cytometry; and hematopoietic stem cells (HSCs) are labeled with Lin-Sca1+c-kit+CD48-CD150+ by flow cytometry.)

[0271] 3. Experimental Results:

[0272] The experimental results are shown in Figure 14 .

[0273] Conclusion: In mixed chimeric mice, administration of agonist 2 after transplantation and detection of donor chimerism rates of hematopoietic stem / progenitor cells in bone marrow at 8 weeks post-transplantation showed that the donor chimerism rates of hematopoietic progenitor cells (HPC), multifunctional progenitor cells (MPP), LSK cells, and hematopoietic stem cells (HSC) were significantly higher than those in the control group, with statistically significant differences (P values ​​were 0.0116, 0.0019, 0.0023, and 0.031, respectively).

[0274] Mean ± standard error of peripheral blood chimerism rate (%) in each group of mice: HPC line: Control: 38.46±11.608, Activator-2: 72.60±4.154; MPP line: Control: 58.82±11.510, Activator-2: 94.32±1.488; LSK line: Control: 40.95±11.868, Activator-2: 75.73±3.748; HSC line: Control: 60.15±13.843, Activator-2: 84.84±2.763.

[0275] Example 15:

[0276] The model mice were administered the drug between days 8 and 28 post-transplantation, and the results of mouse bone marrow cell clonogenesis were detected on day 56 post-transplantation.

[0277] 1. Experimental materials:

[0278] (1) Mice: The donor mice were inbred SPF grade C57BL / 6J(H-2K) mice. b Male, 6-8 weeks old; recipient mice were SPF grade Balb / c (H-2K) mice. d Female, 6-8 weeks old, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0279] (2) Integrin α4β1 agonist: Activator-2.

[0280] (3) Main reagents: phosphate buffered saline (PBS) (Gibco, USA), fetal bovine serum (Gibco, USA), RPMI 1640 medium, IMDM medium (Gibco, USA), GF M3434 medium (StemCell, Canada), lymphocyte separation medium (StemCell, Canada), cyclophosphamide (Sigma-Aldrich, USA), EDTA anticoagulant (Gibco, USA), erythrocyte lysis buffer (Sangon Biotech, Inc.), DMSO (Sigma-Aldrich, USA).

[0281] (4) Instruments: Small animal irradiator (Kubtec, USA), centrifuge (Eppendorf, Germany), cell culture incubator.

[0282] 2. Experimental Methods:

[0283] (1) Based on Example 11: A hybrid chimeric mouse model was established, and agonist No. 2 was injected intraperitoneally daily from day 8 to day 28 after transplantation (for specific experimental details, see the experimental methods in Example 3 and Example 11).

[0284] (2) Obtaining mouse bone marrow single-cell suspension: After euthanasia, the femurs of both sides of the mice were placed in FACS buffer. Holes were cut at the edges of the intact bones with scissors. Then, bone marrow cells were flushed out into a 15mL centrifuge tube with FACS buffer using a 1mL syringe. The tube was centrifuged at 300g and 4℃ for 5min. The supernatant was discarded. 1mL of red blood cell lysis buffer was added and mixed by pipetting. The cells were lysed at room temperature for 5min. 5mL of FACS buffer was added. The tube was centrifuged at 300g and 4℃ for 5min. The supernatant was discarded. The cells were washed once with FACS buffer. The cells were then resuspended in 1mL of FACS buffer and filtered through a 40μm filter membrane to obtain a single-cell suspension.

[0285] (3) CFU (Clonogenesis) assay: Bone marrow cells were collected in IMDM medium + 2% FBS, and the cell concentration was adjusted to 1×10⁻⁶. 5 / mL, take 300μL of cell suspension into 3mL of M3434 medium, vortex to mix, let stand at 4℃ for 10 minutes, then take 1.1mL and put it into a 35mm culture dish, for a total of three replicates, and then culture in a 37℃, 5% CO2 cell culture incubator for 7-12 days. Observe the morphology of multipotent cell colonies (CFU-GEMM) clones under a microscope and count and analyze them.

[0286] 3. Experimental Results:

[0287] The experimental results are shown in Figure 15 .

[0288] Conclusion: In mixed chimeric mice, administration of agonist 2 after transplantation resulted in significantly higher levels of multipotent cell colonies (CFU-GEMM) in bone marrow cells at 8 weeks post-transplantation compared to the control group, with a statistically significant difference (P = 0.0248).

[0289] Mean ± standard error of clone counts for the two groups: Control: 3.43 ± 0.582; Activator-2: 6.00 ± 0.822.

[0290] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make various improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any modifications, alterations, and equivalent changes made by those skilled in the art based on the above-disclosed technical content without departing from the spirit and scope of the present invention are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. Use of an Integrin α4β1 agonist in the preparation of a product that improves the success rate of hematopoietic stem cell engraftment, wherein the Integrin α4β1 agonist is selected from compounds of formula (I) or pharmaceutically acceptable salts thereof, or compounds of formula (II) or pharmaceutically acceptable salts thereof; 2. The use according to claim 1, characterized in that, Integrinα4β1 agonists improve the success rate of hematopoietic stem cell engraftment by increasing the expression or activity of Integrinα4β1 on the surface of hematopoietic stem cells.

3. The use according to claim 1 or 2, characterized in that, Integrin α4β1 agonists enhance hematopoietic stem cell engraftment by increasing the expression or activity of Integrin α4β1 on the surface of hematopoietic stem cells, thereby promoting stem cell engraftment and increasing the success rate of hematopoietic stem cell engraftment.

4. The use according to claim 1, characterized in that, The pharmaceutically acceptable salts of compounds of formula (I) or formula (II) are selected from pharmaceutically acceptable inorganic or organic acid salts.

5. The use according to claim 1, characterized in that, The hematopoietic stem cells mentioned are allogeneic hematopoietic stem cells.

Images