Use of open-cage borane compounds for the preparation of immunosuppressants

Abstract

The present application relates to the application of open-cage borane compounds in the preparation of immunosuppressants, belonging to the technical field of biological medicine. The open-cage borane compounds of the present application include nest borane compounds and / or spider web borane compounds, and the pharmaceutically acceptable solvates or pharmaceutically acceptable complexes of the nest borane compounds and the spider web borane compounds. The open-cage boron clusters of the present application have dehydrogenase-like activity, can catalyze the dehydrogenation reaction of hydroxyl-containing organic substrates in a biological system, promote the conversion of NAD(P) + to NAD(P)H, further improve the intracellular oxidation-reduction ratio, induce cell redox stress, and further reshape the metabolic pattern of HSCs from the source, promote the differentiation of immune cells to immunosuppressive phenotype, and play an immunosuppressive role.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of open-cage borane compounds in the preparation of immunosuppressants. Background Technology

[0002] Hematopoietic stem cell transplantation (HSCT) is currently a core clinical approach for the radical treatment of various hematological diseases, widely used in dozens of benign and malignant hematological and metabolic disorders, including acute myeloid leukemia, acute lymphoblastic leukemia, myelodysplastic syndrome (MDS), aplastic anemia, and multiple myeloma. With continuous advancements in human leukocyte antigen (HLA) matching technology, optimization of pretreatment protocols, and overall improvement in transplant supportive care, the clinical application of allogeneic hematopoietic stem cell transplantation (allo-HSCT) continues to expand.

[0003] However, despite the increasing sophistication of transplantation techniques, graft-versus-host disease (GVHD) remains a critical obstacle severely impacting the efficacy of allo-HSCT and the long-term survival of patients. Therefore, effectively preventing and controlling the occurrence and development of aGVHD remains a significant challenge and a key bottleneck in the field of hematopoietic stem cell transplantation.

[0004] Currently, clinical immunosuppressive therapy for aGVHD has evolved from non-specific broad-spectrum inhibition to targeted intervention. This mainly includes the following three aspects: (1) Traditional immunosuppressants: In terms of non-specific broad-spectrum inhibition, the traditional regimen, represented by glucocorticoids combined with calcineurin inhibitors (such as cyclosporine A (CsA) and tacrolimus (FK506)), remains the core cornerstone of first-line prevention and treatment by non-selectively inhibiting T cell activation and proliferation. However, the clinical efficacy of this strategy is limited by two inherent defects: i) the mechanism of action lacks selectivity, which interferes with the homeostasis of regulatory immune cells while inhibiting pathogenic T cells, leading to significant adverse reactions such as opportunistic infections and organ toxicity; ii) many patients do not respond to hormone therapy or only achieve partial remission, progressing to hormone-refractory aGVHD.

[0005] (2) Targeted Immunosuppressants: In recent years, significant breakthroughs have been made in inhibitors targeting the JAK-STAT signaling pathway. Based on the positive results of two pivotal Phase III clinical trials, REACH2 and REACH3, the JAK1 / 2 inhibitor ruxolitinib has been approved for the treatment of hormone-refractory aGVHD and chronic GVHD, becoming the most important treatment advancement in this field in the past decade. Compared with ruxolitinib, baricitinib has shown superior regulatory T cell (Treg) protection and epithelial repair promotion effects in preclinical models, and theoretically has a lower risk of myelosuppression; as a dual-target inhibitor of JAK2 / FLT3, baricitinib can maintain T cell homeostasis by preserving the IL-2 signaling pathway, and has also achieved a good balance between efficacy and safety in animal models. Although significant progress has been made in the treatment of aGVHD, existing immunosuppressive strategies still face multiple challenges such as limited efficacy, significant toxicity, and large individual variability.

[0006] (3) Biological immunomodulators: In recent years, various improvement strategies have been explored for biological immunomodulators, including in vitro expansion and reinfusion of regulatory T cells (Tregs), infusion of mesenchymal stem cells (MSCs), intervention with biological agents targeting inflammatory factors such as IL-6R and TNF-α, and personalized medicine based on single-cell sequencing.

[0007] However, current immunosuppressive therapy for aGVHD has the following drawbacks: 1) Efficacy bottleneck: Regardless of traditional drugs or new targeted drugs, there is a considerable proportion of non-responding patients, and the long-term survival rate of hormone-refractory aGVHD remains low. 2) Toxicity issues: Non-selective immunosuppression leads to safety issues such as infection and organ toxicity, which limits the therapeutic window; 3) Significant individual differences: The lack of biomarkers to predict treatment efficacy makes it difficult to achieve precise stratification and personalized medication. 4) Transformational barriers in cell therapy: Although novel strategies such as regulatory T cells and mesenchymal stem cells have potential, they are complex to prepare, costly, difficult to control in terms of quality, and have poor clinical accessibility. 5) Limitations of intervention strategies: Existing strategies all focus on end-effect cells or downstream inflammatory mediators, which are "passive response" regulation. Although they can relieve symptoms in the short term, they cannot reshape the abnormal immune cell differentiation pattern from the source, making it difficult to achieve a fundamental therapeutic breakthrough.

[0008] Therefore, there is an urgent need to provide new aGVHD immunosuppressive drugs with better efficacy and safety. Summary of the Invention

[0009] To address the aforementioned technical problems, this invention provides the application of borane compounds in the preparation of anti-aGVHD immunosuppressive drugs. The open-cage boron clusters of this invention include nested and spiderweb-like borane compounds (a schematic diagram illustrating the process by which borane compounds exert immunosuppressive activity is shown in the figure). Figure 1 As shown in the figure, it possesses dehydrogenase-like activity, capable of catalyzing the dehydrogenation reaction of hydroxyl-containing organic substrates in biological systems, and promoting NAD(P) dehydrogenation. + It converts to NAD(P)H and further increases the intracellular NADPH / NADP ratio. + NADH / NAD + The invention also utilizes redox ratios such as GSH / GSSG to induce cellular reductive stress, thereby fundamentally reshaping the metabolic patterns of HSCs and promoting the differentiation of immune cells into immunosuppressive phenotypes, thus exerting an immunosuppressive effect. In summary, this invention develops a non-enzyme-dependent open-cage borane compound to catalyze NAD(P)H regeneration, targeting key metabolic nodes in hematopoietic stem cells, reshaping the immune cell lineage bias at the differentiation source level, and fundamentally reducing the generation of pathogenic effector T cells.

[0010] This invention is achieved through the following technical solution:

[0011] The purpose of this invention is to provide the use of open-cage borane compounds in the preparation of immunosuppressants, characterized in that the open-cage borane compounds include nested borane compounds and / or cobweb borane compounds, as well as pharmaceutically acceptable solvates or pharmaceutically acceptable complexes of the nested borane compounds and cobweb borane compounds, respectively. The open-cage borane compounds are non-closed-cage borane compounds relative to closed-cage structures.

[0012] In one embodiment of the present invention, the general formula of the open-cage borane compound is B. n H n+q-m R m Where n is an integer from 5 to 12, q is an integer from 4 to 6, m is an integer from 0 to 6, and m ≤ q; R are each independently selected from hydroxyl, carboxyl, amino, mercapto, halogen, phenyl, tolyl, nitrile Lewis base ligands or thioether Lewis base ligands; the open-cage borane compound contains at least one BHB bridging hydrogen structural unit.

[0013] In one embodiment of the present invention, the open-cage borane compound is B. 10 H 12 (CNCH3)2、B 10 H 12 (SEt2)2、[Et3NH][B 11 H 14 ]、B 10 H 14 and B11 H 15 One or more of them.

[0014] In one embodiment of the present invention, the concentration of the open-cage borane compound in the immunosuppressant is 0.01 mg / kg to 10 mg / kg.

[0015] In one embodiment of the present invention, the immunosuppressant is administered once every 6 h to 48 h for 7 to 28 consecutive days.

[0016] In one embodiment of the present invention, the immunosuppressant is a drug used to treat or prevent acute graft-versus-host disease, chronic graft-versus-host disease, inflammatory bowel disease, vasculitis, or rheumatoid arthritis.

[0017] In one embodiment of the present invention, the dosage form of the immunosuppressant is selected from tablets, capsules, granules, oral liquids, emulsions, dry suspensions, dry extracts, or injections.

[0018] In one embodiment of the present invention, the immunosuppressant further includes a pharmaceutically or pharmacologically acceptable carrier, an inorganic acid salt, and / or an organic acid salt.

[0019] In one embodiment of the present invention, the carrier is selected from one or more of the following: disintegrant, diluent, lubricant, adhesive, humectant, flavoring agent, filler, suspending agent, surfactant, and preservative.

[0020] In one embodiment of the invention, the filler is selected from one or more of starch, sucrose, and lactose; the wetting agent includes glycerin; and the surfactant includes hexadecyl alcohol.

[0021] In one embodiment of the present invention, the adhesive is selected from one or more of cellulose derivatives, alginates, gelatin and polyvinylpyrrolidone.

[0022] In one embodiment of the present invention, the disintegrant is selected from one or more of agar, calcium carbonate, and sodium bicarbonate.

[0023] In one embodiment of the present invention, the organic acid salt is selected from alkyl sulfonates and / or aryl sulfonates.

[0024] The open-cage borane compounds of the present invention possess catalytic activity, including dehydrogenase-like activity, and can catalyze the conversion of NAD(P). + NAD(P)H is generated, promoting an increase in GSH and inducing cellular reducing stress. The NADPH / NADP ratio in cells is used as an indicator of reducing stress. + NADH / NAD +The ratios of the three classic redox pairs, GSH / GSSG, were significantly higher than those in the blank control group.

[0025] The open-cage borane compounds of this invention induce reducing stress, further promoting cell differentiation into an immunosuppressive phenotype, and promoting the secretion of immunosuppressive factors while inhibiting the release of immune-activating factors, ultimately exerting an immunosuppressive effect. The cells include stem cells, macrophages, T cells, and B cells. The immunosuppressive phenotype includes Treg cells, M2 macrophages, and Breg cells. The immunosuppressive and immune-activating factors include, among others, IL-10, IL-4, and TGF-β; and IL-1β, IL-17, and TNF-α.

[0026] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0027] This invention provides the application of open-cage borane compounds in the preparation of immunosuppressants. The immunosuppressants of this invention address the root cause of aGVHD prevention and treatment, and have the following five important functions:

[0028] (1) This invention is the first to discover that open-cage borane compounds have dehydrogenase-like catalytic activity, which can efficiently catalyze the dehydrogenation reaction of metabolic substrate molecules and generate NAD(P)H.

[0029] (2) The catalytic activity of open-cage borane compounds is usually closely related to their electron-deficient boron cluster skeleton and open structure: i) The B–H–B bridging hydrogen (three-center two-electron bond) widely present in the open-cage structure endows the boron cluster with strong electron delocalization characteristics and hydrogen transfer ability, which is beneficial to the activation of hydroxyl-containing substrates and dehydrogenation-related reactions; ii) Compared with closed-cage boranes, open-cage boron clusters have a more open surface structure, higher skeleton electronic flexibility and more exposed boron active sites, thus making them more likely to react with hydroxyl-containing substrates and NAD(P). + This creates a favorable reaction microenvironment, promoting electron transfer and catalytic processes. In contrast, closed-cage boranes typically possess highly closed and electron-delocalized polyhedral structures, limiting the exposure of their surface active sites and their electronic controllability. Therefore, open-cage boranes generally exhibit structural features more favorable for catalytic reactions in terms of spatial accessibility and dynamic electronic activity, while closed-cage boranes lack these catalytic activities.

[0030] (3) In order to break through the limitations of the existing strategy of "downstream intervention and passive response", this invention moves the prevention and treatment checkpoint to the source of immune reconstitution, the donor hematopoietic stem cells, and focuses on the key node of metabolism-immune coupling, NAD(P)H redox homeostasis. It explains the pathogenesis of aGVHD from the perspective of energy metabolism and redox signal coupling, and provides a theoretical framework for deepening the understanding of the law of immune reconstitution after transplantation.

[0031] (4) This invention introduces the catalytic regeneration strategy into the field of stem cell metabolic regulation for the first time, and uses non-enzyme-dependent catalytic small molecules to achieve efficient recycling and regeneration of NAD(P)H, opening up a new technical path for cell metabolic intervention that is both efficient and precise.

[0032] (5) This invention addresses the clinical dilemma of high mortality rate in hormone-refractory aGVHD and the limitations of existing targeted drugs in bone marrow suppression and drug resistance. By reshaping the differentiation fate of HSCs, it fundamentally reduces the generation of pathogenic effector T cells, providing a safer and more durable new source treatment strategy for clinical practice. Attached Figure Description

[0033] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0034] Figure 1 This is the present invention. Figure 1 A schematic diagram illustrating the process by which borane compounds exert their immunosuppressive activity;

[0035] Figure 2 This invention compares the dehydrogenase-like activities of different cage-type borane compounds; wherein, A represents 5 borane compounds; B represents the catalytic activity of borane compounds detected by colorimetric method (n=3); C represents the NADH catalytic product detected by HPLC; and D represents the NADPH catalytic product detected by HPLC.

[0036] Figure 3 This is the Michaelis-Menten equation for the borane compounds of this invention; wherein, A represents the catalytic kinetics of the borane compounds that conform to the Michaelis-Menten equation; and B represents the catalytic kinetics of the lactate dehydrogenase LDH that conform to the Michaelis-Menten equation.

[0037] Figure 4 This invention relates to the reducing stress induced in cells by borane compounds; wherein, A is the intracellular NADPH / NADP ratio quantified by LC-MS / MS. + NADH / NAD + Redox contrast ratio changes (n=3); B shows the changes in GSH content in cells after GSHtracer staining observed by fluorescence confocal microscopy (scale bar: 10 μm).

[0038] Figure 5The present invention relates to the immunosuppressive effect induced in T cells by the borane compounds;

[0039] Figure 6 This invention describes the immunosuppression induced by the borane compound in the spleen of mice; wherein, A is a schematic diagram of the C57BL / 6→BALB / cMHC completely mismatched allogeneic bone marrow transplantation model; B is the absolute number of donor-derived T cells in the spleen of recipient mice +14 days after transplantation; C is the number of donor-derived CD8 cells in the spleen of recipient mice. + T cell ratio; D represents donor-derived CD4+ in the spleen of the recipient mouse. + T cell proportion; E represents the proportion of donor-derived Treg cells in the spleen of recipient mice; F represents the proportion of donor-derived Th1 cells in the spleen of recipient mice; G represents the cytokine expression level in mouse serum; H represents the image and score under HE staining. Detailed Implementation

[0040] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0042] BEAS-2B lung epithelial cells were purchased from ATCC, CRL-3588;

[0043] 4T1 breast cancer cells were purchased from ATCC, CRL-2539;

[0044] Lung cancer cells A549 were purchased from ATCC, CCL-185.

[0045] HepG2 liver cancer cells were purchased from ATCC, HB-8065;

[0046] Lactate dehydrogenase (LDH) was purchased from Aladdin, 9028-36-8.

[0047] Example 1: Characterization of the dehydrogenation catalytic activity of borane compounds.

[0048] This example collects five typical borane compounds, including closed-cage, cobweb, and nested types. Figure 2 (A) To investigate the dehydrogenation activity of borane compounds, lactic acid (LA) was used as the dehydrogenation substrate, 3-(4,5-dimethylthiazol-2-yl)-5-(triazol-2-yl)-2,5-diaminothiazolium was used as the chromogenic substrate, and NAD+ was used. +Colorimetric method as a hydrogen acceptor. The specific procedure is as follows: First, prepare NADH series solutions with concentrations ranging from 0-1 mM, and mix them with MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazole) to a final concentration of 0.4 mg / mL. After incubation at 37°C in the dark for 10 min, measure the absorbance at 490 nm and plot a standard curve. Then, mix a borane compound with a final concentration of 50 μg / mL and a NADH solution with a final concentration of 1 mM. + The experimental group consisted of a mixture of 3 mM LA (pH adjusted to 7 with 0.1 M NaOH) and 0.4 mg / mL MTS; the final concentration was 1 mM NAD. + A mixture of 3 mM LA (pH adjusted to 7 with 0.1 M NaOH) and 0.4 mg / mL MTS was used as a control group. The reaction was carried out at 37℃ and 200 rpm for 30 min with shaking. After the reaction, the reaction solution was transferred to a 96-well plate with a clear bottom and the absorbance was measured at 490 nm. The concentration of NADH generated in the reaction system was calculated according to the standard curve. Finally, the lactate dehydrogenase activity (LDH-like activity) was calculated by substituting the following formula:

[0049] ;

[0050] Where B is the amount of NADH generated during the reaction (nmol), V (mL) is the volume of the reaction solution, and T (min) is the reaction time. The results show that the closed-cage type B... 12 H 12 Na2(1) and C2B 10 H 12 (2) After incubation with MTS and substrate for 30 min, it showed a bright yellow color, similar to the control group; while the cobweb-like B... 10 H 12 (SEt2)2(3)and B 10 H 12 (CNCH3)2 (4) and nested B 11 H 14 The (NH4)2 (5) group showed a brownish color, and the calculated lactate dehydrogenase activities were 20.8 mU / mL, 23.2 mU / mL, and 22.4 mU / mL, respectively, indicating that the cobweb-like B 10 H 12 (SEt2)2、B 10 H 12 (CNCH3)2 and nested B 11 H 14The three compounds (NH4)2 may possess dehydrogenase-like activities. Figure 2 (B in the middle).

[0051] To further confirm the dehydrogenation catalytic reaction, compound B was selected. 10 H 12 (CNCH3)2 (abbreviated as aB) 10 The dehydrogenation catalytic product NAD(P)H of this borane compound was identified by high-performance liquid chromatography (HPLC). The results showed that NADH and NAD... + The standards showed peak elution times of 7.6 min and 4.5 min on HPLC. Comparison of the reaction products revealed that the borane compound catalyzed the reaction of NAD... + The peak decreased significantly, while the NADH peak of the product increased significantly. Figure 2 (C in the text). Similarly, with G6P and NADP... + Using this borane compound as a substrate, catalytic reactions were conducted, and the results showed that this borane compound could also catalyze NADP. + Generate NADPH ( Figure 2 (D in the text). The above results indicate that the cage-like structure of boron clusters is closely related to their reactivity, and that spiderweb and nested borane molecular structures with open faces or missing tops can catalyze NAD(P). + NAD(P)H is generated.

[0052] Example 2: Catalytic kinetic characterization of boronane compounds.

[0053] To further investigate the dehydrogenation activity of borane compounds, lactic acid was used as the dehydrogenation substrate, 3-(4,5-dimethylthiazol-2-yl)-5-(triazol-2-yl)-2,5-diaminothiazolium was used as the chromogenic substrate, and NAD+ was used. + Colorimetric method as a hydrogen acceptor, to investigate aB 10 The catalytic reaction kinetics curve of the molecule was obtained, with lactate dehydrogenase (LDH) as a positive control. The specific procedure was as follows: 50 μg / mL of aB... 10 With NAD+ in a final concentration range of 0-20 mM + The LA solution (pH adjusted to 7 with 0.1 M NaOH) was mixed in a 96-well plate, and finally, MTS was added to a final concentration of 0.4 mg / mL. The kinetic reaction program was set in a microplate reader: 37℃, continuous shaking, and absorbance measured at 490 nm every 1 min. Finally, the Michaelis-Menten equation was used for fitting analysis based on the reaction rates measured at different substrate concentrations. The results showed that aB 10 The catalytic kinetics of the compound conforms to the Michaelis-Menten equation, and its maximum reaction rate (Vt) is... maxThe reaction rate was 27.6 μM / min, close to the maximum reaction rate of lactate dehydrogenase; while the Michaelis constant Km was 1.14 mM. Figure 3 This result further demonstrates that the borane possesses dehydrogenation catalytic activity.

[0054] Example 3

[0055] Borane compounds induce the accumulation of cellular reducing equivalents. (The text abruptly shifts to a seemingly unrelated topic about aB protein.) 10 Representative open-cage type B 10 Taking borane compounds as an example, the reduction equivalent accumulation induced by them was investigated in BEAS-2B lung epithelial cells. The specific procedure was as follows: BEAS-2B lung epithelial cells in logarithmic growth phase and in good condition were taken, washed with PBS, and digested with trypsin. Digestion was stopped when the cells became round and bright, and the cells were pipetted into a single-cell suspension. After centrifugation at 1000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in PBS and counted. The NAD(P)H / NAD(P) ratio was then calculated. + Each assay kit requires approximately 3 million cells. Prepare a cell suspension of appropriate concentration and seed it in culture flasks for overnight incubation. After aspirating the supernatant, add 5 mL of aB to each flask to a final concentration of 5 μg / mL. 10 (Diluted in BEGM medium), cultured in a cell culture incubator at 37 ℃ for 12 h. At the endpoint, discard the supernatant, wash twice with PBS, collect cells using a cell scraper, centrifuge at 1000 rpm for 5 min, discard the supernatant, add 100 μL of lysis buffer to each cell sample, sonicate in an ice-water bath, and centrifuge at 18000 rpm for 5 min to collect the supernatant. Simultaneously prepare NAD(P)H standard solution and freshly prepared NAD(P)H / NAD(P) solution. + Detection working solution. Samples were prepared separately according to the NAD(P)H group and NAD(P) group. + The appropriate treatment solution was added to each group, and the mixture was incubated at 37 °C in the dark for 15 min, then neutralized. The precipitate was removed by centrifugation at 18000 rpm for 10 min. Subsequently, 75 μL of detection working solution was added to each well, making the total volume 150 μL. The reaction was carried out at room temperature in the dark for 2 h. Fluorescence intensity was detected at Ex / Em = 540 / 590 nm, and intracellular NAD(P)H and NAD(P) were calculated based on the standard curve. + The content was determined, and the NAD(P)H / NAD(P) ratio was obtained. + The ratio. Results showed that, compared to the control group PBS, aB... 10 NADPH / NADP in the treated cells + NADH / NAD + The redox ratio increased by 1.3-1.5 times. Figure 4(A) This demonstrates that the borane compound can induce cellular reduction equivalent accumulation, resulting in reduction stress. To further demonstrate the universality of the material-induced reduction stress, the cell types were expanded to include breast cancer cells 4T1, lung cancer cells A549, and liver cancer cells HepG2. Reduction stress was detected by staining intracellular GSH with the ratiometric fluorescent probe GSHtracer. The results showed that aB 10 The above three cell types can induce an increase in the GSH / GSSG ratio ( Figure 4 (B in the text). This demonstrates that the borane compound can accelerate cellular dehydrogenation catalysis, induce the accumulation of reducing equivalents, and generate reducing stress in various cell types.

[0056] Example 4

[0057] The immunosuppressive effects of borane compounds were investigated at the cellular level. (Using aB) 10 Taking borane compounds as an example, we investigated their immunosuppressive effects at the cellular level. First, following the methods described on the STEMCELL Technologies website, we extracted and isolated naïve CD4 cells from mouse spleens. + T cells (naïve CD4) + T). Add 6.25 μg / mL aB. 10 After 30 min of treatment, the cells were washed off, and then cytokines (20 ng / mL IL-2 and 2.5 ng / mL TGF-β) were added to induce differentiation. The cells were incubated at 37 °C for 72 h, and then collected. Flow cytometry staining (anti-CD4, anti-CD25, and anti-FOXP3 antibodies) was used to evaluate cell differentiation. The results showed that, compared to the control group induced by culture medium containing only cytokines, aB... 10 Small molecules can increase the proportion of induced regulatory T cells (Tregs) by 4 times. The core function of Tregs is to suppress the immune response and prevent excessive inflammation and autoimmunity. This demonstrates aB 10 It induces a significant immunosuppressive effect at the cellular level. Figure 5 ).

[0058] Example 5: Investigation of the immunosuppressive effect of borane compounds at the animal level.

[0059] With aB 10 To investigate its immunosuppressive effects at the animal level, a classic C57BL / 6→BALB / c(H-2b→H-2d) MHC fully mismatched allogeneic bone marrow transplantation model was used. The transplantation was performed using 6.25 μg / mL aB... 10After co-incubation with T-cell-depleted C57BL / 6 bone marrow cells for 30 min, the cells were eluted. The processed bone marrow T-cells and spleen T cells sorted by magnetic beads were injected into BALB / c cells via the tail vein, followed by oral administration via drinking water. 10 BALB / c mice were administered a dose of 1 mg / kg for 14 consecutive days. Figure 6 (A) After drug administration, mouse spleen (SP) cells were isolated, and flow cytometry was used to analyze the composition and functional subset differentiation of donor T cells in the spleen. The results showed that aB 10 There was no significant difference in the absolute number of T cells after drug administration. Figure 6 (B in the text); regarding the composition of T cell subsets, aB 10 Treatment significantly reduced the proportion of cytotoxic T cells with cytotoxic effects, namely CD8+. + The proportion of T cells was significantly reduced (aB) 10 Group 13.6% ( Figure 6 In the control group (C), 28.9% (p < 0.0001), while helper T cells (CD4+) were present. + The proportion of T cells was relatively preserved (aB) 10 Group 9.5%, control group 14.7%, p > 0.05) Figure 6 (D) in CD4. + Among T cell subsets, CD25, which has immunomodulatory functions + FoxP3 + The proportion of regulatory T cells (Tregs) increased significantly (aB 10 Group 5.72%, control group 3.0%, p < 0.0001) Figure 6 (E in the middle), on the contrary, CD4 promotes inflammation. + IFN-γ + Th1(aB) 10 The percentage of cells in the control group was 8.5%, while that in the control group was 13.4% (p < 0.0001), indicating a decrease in cell proportion. Figure 6 (F in the original text). In summary, B 10 H 12 In in vivo experiments, (CNCH3)2 does not cause immunosuppression by killing T cells, but rather by altering the composition of immune cells and reshaping the immune microenvironment to achieve the effect of immunosuppression.

[0060] Simultaneously, mouse serum was collected on day 14, and cytokines in the mouse serum were quantitatively detected using LEGENDplex™ multifactor detection technology. The results showed that anti-inflammatory factors IL-10 and TGF-β were significantly upregulated, while the expression levels of pro-inflammatory factors TNF-α and IL-17 were significantly downregulated, consistent with the results of immune cell differentiation phenotype analysis. Figure 6(G in the text). Pathological sections were stained with H&E, and the results showed that the liver, intestines, skin, and other target organs of mice in the untreated group exhibited significant inflammatory recruitment, while aB... 10 The treatment group showed a significant reduction in inflammation of target organs such as the liver, intestines, and skin. Figure 6 (H in the example). The above embodiments show that aB 10 Oral administration can induce immunosuppression in mice and significantly alleviate GVHD.

[0061] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. The application of open-cage borane compounds in the preparation of immunosuppressants, characterized in that, The open-cage type borane compound is B. 10 H 12 (CNCH3)2、B 10 H 12 (SEt2)2 or [Et3NH][B] 11 H 14 The structure of the open-cage type borane compound is as follows: , or ; The immunosuppressant is a drug used to treat or prevent acute graft-versus-host disease and chronic graft-versus-host disease.

2. The application according to claim 1, characterized in that, The concentration of the open-cage borane compound in the immunosuppressant is 0.01 mg / kg to 10 mg / kg.

3. The application according to claim 1, characterized in that, The immunosuppressant is administered every 6-48 hours for 7-28 consecutive days.

4. The application according to claim 1, characterized in that, The dosage form of the immunosuppressant is selected from tablets, capsules, granules, oral liquids, emulsions, dry suspensions, dry extracts, or injections.

5. The application according to claim 1, characterized in that, The immunosuppressants also include pharmaceutically acceptable carriers, inorganic acid salts, and / or organic acid salts.

6. The application according to claim 5, characterized in that, The carrier is selected from one or more of the following: disintegrant, diluent, lubricant, adhesive, humectant, flavoring agent, filler, suspending agent, surfactant, and preservative.

7. The application according to claim 5, characterized in that, The organic acid salt is selected from alkyl sulfonates and / or aryl sulfonates.

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