Use of 12-ketolithocholic acid for the preparation of a medicament for the prevention and treatment of acute graft-versus-host disease

Abstract

The application relates to the technical field of immunoregulation drugs, in particular to application of 12-ketolithocholic acid or a pharmaceutically acceptable salt thereof in preparation of a drug for preventing and / or treating acute graft-versus-host disease. The application discloses, for the first time, the application of 12-ketolithocholic acid or a pharmaceutically acceptable salt thereof in the direction of preventing and / or treating acute graft-versus-host disease, 12-ketolithocholic acid can effectively improve the disease process of acute graft-versus-host disease induced by allogeneic hematopoietic stem cell transplantation, reduce the disease severity and mortality, and provide a highly selective, low-toxicity endogenous small-molecule drug candidate for prevention and / or treatment of acute graft-versus-host disease. 12-ketolithocholic acid can also be used as an auxiliary drug for existing treatment schemes such as glucocorticoids and JAK inhibitors, and provides a new technical scheme for prevention and / or treatment of acute graft-versus-host disease.

Description

Technical Field

[0001] This invention relates to the field of immunomodulatory drug technology, specifically to the application of 12-ketodeoxycholic acid (12-ketoLCA, also known as 12-keto-type bile acid) in the preparation of drugs for the prevention and treatment of acute graft-versus-Host disease (aGVHD). Background Technology

[0002] Acute graft-versus-host disease (APH) is one of the most serious and fatal complications following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Its pathogenic mechanism primarily stems from the abnormal activation and widespread proliferation of donor-derived T cells in the recipient, releasing large amounts of pro-inflammatory cytokines that attack target organs such as the intestines, liver, skin, and lungs, leading to severe tissue inflammation and even recipient death. In the development of APH, helper T cells (Th1) are one of the key effector T cell subsets mediating target organ immune damage. Th1 cells can produce inflammatory cytokines such as interferon-γ (IFN-γ), promoting the inflammatory cascade and participating in the APH-related tissue damage process. Therefore, regulating T cell differentiation and inhibiting abnormal Th1 immune responses are important therapeutic directions for alleviating APH.

[0003] Currently, clinical treatment of acute graft-versus-host disease (APHD) typically relies heavily on glucocorticoids and broad-spectrum immunosuppressants. However, this type of therapy has significant limitations, including the tendency to induce hormone resistance and the indiscriminate suppression of the immune system leading to a surge in infection risk.

[0004] To overcome the current bottlenecks in the treatment of acute graft-versus-host disease (APH), existing literature and patents have disclosed technical solutions using small molecule drugs to alleviate or treat APH. For example, patent application number 202310522782.4 utilizes itaconic acid or its derivatives to alleviate or treat APH, and patent application number 202310479412.7 uses betulinol to alleviate or treat APH. These technical solutions have demonstrated that APH symptoms can be alleviated through antioxidant activity, inhibition of abnormal expression of inflammatory factors, or reduction of Th1 / Tc1 cell numbers. However, most of these small molecule drugs exhibit broad-spectrum T cell subset suppression activity. While inhibiting harmful effector T cells, they tend to indiscriminately inhibit regulatory T cells, which are crucial for maintaining post-transplant immune tolerance, making it difficult to achieve selective and precise regulation of T cell subsets.

[0005] Recent immunological and metabolic studies have revealed that bile acids, as endogenous metabolites in the enterohepatic circulation, possess good biocompatibility and immunomodulatory activity. Furthermore, bile acids and their metabolites with different structures exhibit high structure-activity specificity in regulating T cell subset differentiation. However, no studies have yet elucidated the role of bile acids in the prevention and treatment of acute graft-versus-host disease (APHD) and in regulating T cell differentiation. Therefore, there is a need to develop a highly selective, low-toxicity small molecule endogenous bile acid that can specifically inhibit Th1 cell differentiation, in order to overcome the current bottleneck in the treatment of APHD and improve the prognosis of hematopoietic stem cell transplant patients. Summary of the Invention

[0006] The purpose of this invention is to provide the application of 12-ketolithocholic acid in the preparation of drugs for the prevention and treatment of acute graft-versus-host disease. 12-ketolithocholic acid reduces the inflammatory response associated with acute graft-versus-host disease by regulating abnormally activated T cell immune responses, thereby exerting preventive and / or therapeutic effects.

[0007] To achieve the above objectives, the present invention provides the following technical solution: the use of 12-ketolithocholic acid or a pharmaceutically acceptable salt thereof in the preparation of drugs for the prevention and / or treatment of acute graft-versus-host disease.

[0008] Furthermore, the 12-ketolithocholic acid or a pharmaceutically acceptable salt thereof exerts preventive and / or therapeutic effects by modulating abnormally activated T-cell immune responses and reducing acute graft-versus-host disease-related inflammatory responses.

[0009] Furthermore, the 12-ketolithocholic acid or a pharmaceutically acceptable salt thereof selectively inhibits CD4. + T cells play a role in differentiating into Th1 cells.

[0010] Furthermore, the 12-ketolithocholic acid or a pharmaceutically acceptable salt thereof, while inhibiting Th1 cell differentiation, does not significantly affect CD4. + T cells differentiate into Th17 cells and Treg cells.

[0011] Furthermore, the acute graft-versus-host disease is acute graft-versus-host disease induced by allogeneic hematopoietic stem cell transplantation.

[0012] Furthermore, the 12-ketolithocholic acid or a pharmaceutically acceptable salt thereof is used to alleviate inflammatory damage to target organs associated with acute graft-versus-host disease; The target organs include one or more of the intestines, liver, and lungs.

[0013] Furthermore, the dosage of the 12-ketolithocholic acid or a pharmaceutically acceptable salt thereof is 80 mg / kg / day to 120 mg / kg / day.

[0014] This application also provides a pharmaceutical composition for the prevention and / or treatment of acute graft-versus-host disease. The pharmaceutical composition comprises an effective amount of 12-ketolithocholic acid or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent, or excipient, for reducing Th1-type immune responses during acute graft-versus-host disease and alleviating inflammatory damage to tissues associated with acute graft-versus-host disease.

[0015] Furthermore, the pharmaceutical composition further includes one or more adjuvant therapeutic agents; The adjuvant therapy drugs are selected from glucocorticoids or JAK inhibitors.

[0016] The beneficial effects of this invention are as follows: This application discloses for the first time the application of 12-ketolithocholic acid or a pharmaceutically acceptable salt thereof in the prevention and / or treatment of acute graft-versus-host disease. 12-ketolithocholic acid can effectively improve the disease course of acute graft-versus-host disease induced by allogeneic hematopoietic stem cell transplantation, reduce disease severity and mortality, and improve inflammatory pathological damage to related target organs, providing a highly selective and low-toxicity endogenous small molecule drug candidate for the prevention and / or treatment of acute graft-versus-host disease.

[0017] 12-Ketolithocholic acid exhibits excellent selective regulation of T cell subsets. While precisely blocking pathogenic Th1 immune responses, it can maximally preserve cell subsets that maintain immune homeostasis, thus facilitating the remodeling of immune tolerance and balance after transplantation. 12-Ketolithocholic acid directly and specifically inhibits the differentiation of CD4+ T cells into pathogenic Th1 subsets and significantly downregulates T-bet expression; meanwhile, it has no significant effect on the differentiation of Th17 cells and Treg cells.

[0018] 12-Ketolithocholic acid can significantly reduce the infiltration of inflammatory cells in the core target organs such as the intestine, liver and lungs of the recipient by blocking the Th1-mediated systemic inflammatory cascade, effectively barrier and repair tissue structural damage, and significantly alleviate the systemic multi-organ pathological changes caused by acute graft-versus-host disease.

[0019] 12-Ketolithocholic acid can also be used as an adjunct to existing treatment regimens such as glucocorticoids and JAK inhibitors, providing a new technical solution for the prevention and / or treatment of acute graft-versus-host disease.

[0020] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Attached Figure Description

[0021] Figure 1 The figure shows the phenotypic improvement effect of 12-ketolithocholic acid on an allogeneic hematopoietic stem cell transplantation-induced acute graft-versus-host disease mouse model as shown in this invention. Figure 2 Comparison of hematoxylin-eosin (HE) stained sections showing the effect of 12-ketolithocholic acid on improving the pathological damage of major target organs in recipient mice as shown in this invention. Figure 3 This is a flow cytometry-based bar chart showing the changes in the proportion of T cell subsets in different target organs of recipient mice after treatment with 12-ketolithocholic acid, as described in this invention. Figure 4 The present invention demonstrates the effect of 12-ketolithocholic acid on CD4 in an in vitro directed differentiation model. + Flow cytometry scatter plot and statistical bar chart showing the influence of T cell differentiation into Th1, Th17 and Treg subsets. Detailed Implementation

[0022] The technical solutions of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0023] It should be noted that the bile acids, such as 12-ketolithocholic acid, and their metabolites used in this application are all commercially available finished compounds. Any 12-ketolithocholic acid having the same chemical structure and capable of achieving the preventive and / or therapeutic effects of acute graft-versus-host disease as described in this invention can be used in this invention.

[0024] Through large-scale high-throughput sequencing and metabolomics comparative analysis of disease-onset and non-onset models, a class of endogenous bile acids and their metabolites with potential immunoprotective effects were screened, including 12-ketolithocholic acid, 3-ketolithocholic acid (3-ketoLCA), 3α,12α-dihydroxycholanic acid, etc. Furthermore, in vitro and in vivo animal experiments confirmed that these bile acids and their metabolites can effectively intervene in disease progression. A preferred embodiment of this application provides the application of 12-ketolithocholic acid or its pharmaceutically acceptable salts in the preparation of drugs for the prevention and / or treatment of acute graft-versus-host disease. This embodiment opens up a new use for 12-ketolithocholic acid or its pharmaceutically acceptable salts in intervening in abnormal immune responses after transplantation, which can significantly inhibit the progression of acute graft-versus-host disease and effectively improve the clinical symptoms and prognosis of transplant recipients. As a highly selective and low-toxicity endogenous small molecule, 12-ketolithocholic acid or its pharmaceutically acceptable salts have shown unique advantages in the preparation of drugs for the prevention and / or treatment of acute graft-versus-host disease. They can overcome the clinical bottlenecks of limited efficacy and large toxic side effects of existing glucocorticoids and traditional broad-spectrum immunosuppressants, providing a new drug intervention strategy for the clinical prevention and treatment of acute graft-versus-host disease.

[0025] In one embodiment, 12-ketolithocholic acid or a pharmaceutically acceptable salt thereof exerts a preventive and / or therapeutic effect by modulating abnormally activated T-cell immune responses and reducing inflammatory responses associated with acute graft-versus-host disease (APVD). 12-ketolithocholic acid can target key pathological processes in the development of APVD by modulating abnormally activated T-cell immune responses, significantly reducing APVD-related inflammatory damage. Compared to traditional broad-spectrum immunosuppressive therapy, this approach effectively curbs pathological immune overactivation while greatly reducing the risk of serious infections caused by excessive immunosuppression, thereby significantly improving the targeting and safety of clinical treatment for APVD.

[0026] In one embodiment, 12-ketolithocholic acid or a pharmaceutically acceptable salt thereof selectively inhibits CD4. +T cells differentiate into Th1 cells. In this embodiment or other embodiments, 12-ketolithocholic acid or a pharmaceutically acceptable salt thereof can downregulate the expression level of the transcription factor T-bet, thereby blocking the differentiation and development of Th1 cells, reducing the generation of pathogenic Th1 effector cells during acute graft-versus-host disease (APHD) at its source, and reducing Th1-mediated inflammatory damage. This mechanism of action differs from non-specific traditional immunosuppression strategies, enabling targeted regulation of key pathogenic T cell subsets and significantly alleviating post-transplant immune damage. In this embodiment or other embodiments, 12-ketolithocholic acid or a pharmaceutically acceptable salt thereof works by reducing the generation of Th1 effector cells and decreasing the expression or release of Th1 cell-related inflammatory factors, thereby further blocking the Th1-mediated inflammatory cascade response, reducing damage to target organs such as the intestine, liver, and lungs by inflammatory factors, and thus improving tissue inflammation and pathological changes related to AHD. Th1 cell-related inflammatory factors include interferon-γ (IFN-γ).

[0027] In one embodiment, 12-ketolithocholic acid or a pharmaceutically acceptable salt thereof inhibits Th1 cell differentiation without significantly affecting CD4. + T cells differentiate into Th17 cells and regulatory T cells (Tregs). This selective inhibition avoids non-specific interference from 12-ketolithocholic acid or its pharmaceutically acceptable salts on cell subsets that maintain immune homeostasis, precisely blocking pathogenic Th1 immune responses while maximally preserving post-transplant immune balance, thereby significantly improving drug safety and therapeutic window.

[0028] In one embodiment, acute graft-versus-host disease (APH) is acute APH induced by allogeneic hematopoietic stem cell transplantation (allo-HSCT). By clarifying this application scenario, the intervention target of 12-ketolithocholic acid or its pharmaceutically acceptable salt precisely matches the pathogenesis of major clinical acute APH, effectively curbing tissue immune damage caused by abnormal activation of donor T cells after allogeneic transplantation, and significantly improving the applicability and translational value of this technical solution.

[0029] In one embodiment, 12-ketolithocholic acid or a pharmaceutically acceptable salt thereof is used to reduce inflammatory damage in target organs associated with acute graft-versus-host disease (APV). By using 12-ketolithocholic acid or a pharmaceutically acceptable salt thereof to reduce inflammatory damage in target organs associated with APV, particularly improving inflammatory infiltration and structural damage in tissues such as the intestine, liver, and lungs, the degree of multi-organ damage caused by APV can be reduced, thereby improving the overall disease state of the recipient and prolonging survival.

[0030] In one embodiment, the dosage of 12-ketolithocholic acid or a pharmaceutically acceptable salt thereof is 80 mg / kg / day to 120 mg / kg / day. Limiting the dosage to this specific range allows the drug to fully exert its immunomodulatory efficacy in treating acute graft-versus-host disease while avoiding potential toxic side effects due to overdose. This dosage range not only establishes a therapeutic window for 12-ketolithocholic acid that combines efficacy and safety but also significantly improves its operability and safety in clinical application.

[0031] One embodiment provides a pharmaceutical composition for the prevention and / or treatment of acute graft-versus-host disease (APH), comprising an effective amount of 12-ketolithocholic acid or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent, or excipient, for inhibiting the Th1-type immune response during APH and reducing APH-related tissue inflammatory damage. By constructing a pharmaceutical composition comprising 12-ketolithocholic acid or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent, or excipient, 12-ketolithocholic acid can exert its anti-APH effect in a form suitable for clinical administration. This pharmaceutical composition can reduce inflammatory damage and improve therapeutic efficacy by inhibiting the Th1-type immune response during APH, and provides a basis for subsequent pharmaceutical formulation development and clinical application.

[0032] In one embodiment, the pharmaceutical composition may further include one or more adjuvant therapeutic agents. The adjuvant therapeutic agents may be selected from glucocorticoids or JAK inhibitors. By combining the 12-ketolithocholic acid pharmaceutical composition with adjuvant therapeutic agents such as glucocorticoids or JAK inhibitors, synergistic regulation between different mechanisms of action can be achieved. The highly selective inhibitory effect of 12-ketolithocholic acid on Th1-type immune responses precisely reduces tissue inflammatory damage, while the combination with adjuvant agents rapidly inhibits abnormal systemic immune activation. This combined regimen can significantly improve the overall therapeutic effect of acute graft-versus-host disease and provides a feasible strategy for reducing the dosage of single drugs, reducing toxic side effects, and improving drug safety.

[0033] This application utilizes commercially available pure 12-ketolithocholic acid as experimental material, the chemical structure of which is confirmed through product information or routine chemical analysis. Based on the dosage or treatment concentrations used in animal and cell experiments, 12-ketolithocholic acid is dissolved in a suitable solvent system to prepare a stock solution, which is further diluted to the appropriate concentration of working solution as needed. The solvent system can be selected based on the solubility characteristics of 12-ketolithocholic acid, for example, phosphate-buffered saline (PBS) containing an appropriate amount of dimethyl sulfoxide (DMSO) or other suitable pharmaceutically acceptable solvent systems. In animal experiments, the control group uses the same dosage volume and solvent system composition as the 12-ketolithocholic acid treatment group as a solvent control to eliminate the influence of solvent factors on the experimental results. In cell experiments, the control group is supplemented with the same volume and final concentration of DMSO as the 12-ketolithocholic acid treatment group as a solvent control. This invention does not limit the specific commercial source of 12-ketolithocholic acid. Any compound used that has the same chemical structure as 12-ketolithocholic acid and can achieve the preventive and / or therapeutic effects of acute graft-versus-host disease as described in this invention can be used in this invention.

[0034] I. 12-Ketolithocholic Acid Reduces the Severity of Acute Graft-versus-Host Disease To evaluate the therapeutic effect of 12-ketolithocholic acid on acute graft-versus-host disease (GVHD), a mouse model of GVHD induced by allogeneic hematopoietic stem cell transplantation was established. Figure 1 As shown in Figure A, 6- to 8-week-old female C57BL / 6 (H-2b) mice were used as donors, and female BALB / c (H-2d) mice were used as recipients. First, BALB / c recipient mice underwent a single 6.25 Gy whole-body X / γ-ray irradiation (TBI). Subsequently, they were randomly divided into a control group (aGVHD model group) and a 12-ketolithocholic acid treatment group (12-ketoLCA group), with 15 mice in each group. On the day of transplantation (day 0), the C57BL / 6 donor mice were sacrificed, and the spleen, femur, and tibia were harvested under aseptic conditions. The spleen was thoroughly ground until no obvious tissue fragments remained, and then filtered through a 200-mesh sieve to prepare a spleen single-cell suspension. Simultaneously, the medullary cavities of the femur and tibia were flushed to obtain bone marrow cells, which were then filtered through a 200-mesh sieve to obtain a bone marrow cell suspension. Ammonium chloride erythrocyte lysis buffer was added to both the single-cell suspension and the bone marrow cell suspension for erythrocyte lysis. After lysis, the cells were washed, resuspended, and counted to obtain donor spleen cells and donor bone marrow cells, respectively.

[0035] Donor spleen cells and donor bone marrow cells were mixed in predetermined quantities to prepare a donor cell suspension. This donor cell suspension was administered via tail vein injection to each recipient mouse in the 12-ketolithocholic acid treatment group and the control group. Each recipient mouse received 0.3 mL of the donor cell suspension, which contained 1 × 10⁶ spleen cells from C57BL / 6 donor mice. 6 6 × 10 bone marrow cells 6 indivual.

[0036] From the day of transplantation (day 0 post-transplantation), recipient mice in the 12-ketolithocholic acid treatment group were administered 12-ketolithocholic acid working solution daily by gavage at a dose of 100 mg / kg / day for 14 consecutive days; the control group received an equal volume of solvent-based control. General clinical manifestations, survival status, and weight changes of mice in each group were continuously monitored for 40 days post-transplantation, and the mice were scored according to the clinical scoring criteria for acute graft-versus-host disease commonly used in this field. Scoring indicators included weight change, activity level, hair condition (erect / shedding), posture (arched back posture), and skin condition. To reduce bias caused by subjective evaluation, disease scoring was performed by the researchers without knowledge of the group assignments. Results are as follows: Figure 1 As shown.

[0037] Depend on Figure 1 As shown in Figure B, the overall survival of recipient mice in the control group was 0 at approximately 30 days. Compared with the control group, the overall survival of recipient mice in the 12-ketolithocholic acid treatment group was improved, the survival time was prolonged, and the survival rate at 30 days post-transplantation was increased; the difference between the groups was statistically significant (**P<0.01). Figure 1 As shown in Figure C, both the 12-ketolithocholic acid-treated group and the control group experienced a significant, phased decrease in body weight after transplantation, followed by gradual recovery. Compared to the control group, the 12-ketolithocholic acid-treated group showed a significantly smaller rate of weight loss after transplantation and demonstrated better weight maintenance and recovery during the subsequent observation period. Figure 1 The results showed that, compared with the control group, the clinical disease scores of mice treated with 12-ketolithocholic acid remained at a lower level overall; especially during the peak of disease onset around day 7 post-transplantation, 12-ketolithocholic acid intervention significantly reduced the peak of disease severity. This result indicates that 12-ketolithocholic acid can effectively improve the clinical symptoms of acute graft-versus-host disease induced by allogeneic hematopoietic stem cell transplantation, reduce histopathological damage, and decrease mortality, demonstrating good therapeutic potential against acute graft-versus-host disease.

[0038] II. 12-Ketolithocholic Acid Reduces Pathological Damage to Target Organs Related to Acute Graft-versus-Host Disease To further evaluate the effect of 12-ketolithocholic acid on the pathological damage of target organs associated with acute graft-versus-host disease, some recipient mice from the control group and the 12-ketolithocholic acid treatment group were sacrificed on day 14 post-transplantation, and their liver, lung, small intestine, and large intestine tissues were collected for histopathological examination. The collected target organ tissues were fixed in 4% polyformaldehyde (PFA) for 24–48 h, then dehydrated with graded ethanol, cleared in xylene, embedded in paraffin, and sectioned into 4 μm–5 μm thick sections using an embedding microtome. Subsequently, the sections corresponding to each target organ were dewaxed in xylene, rehydrated with graded ethanol, and then stained with hematoxylin and eosin. Specifically, the sections were stained in hematoxylin for 3–5 min, rinsed with running water, differentiated with hydrochloric acid alcohol, and blued with ammonia, and then stained in eosin for 1–3 min. After staining, the sections were dehydrated with graded ethanol, cleared in xylene, and mounted with neutral resin. Pathological observation was performed on sections corresponding to each group of target organs using a microscopic optical imaging system. Results are as follows: Figure 2 As shown.

[0039] Depend on Figure 2 It was found that the liver, lungs, small intestine, and large intestine of the recipient mice in the control group all showed varying degrees of pathological damage, exhibiting typical pathological damage of acute graft-versus-host disease. Specifically, the liver showed pathological changes such as inflammatory cell infiltration; the lung tissue showed significant thickening of the alveolar septa, accompanied by inflammatory cell exudation and structural disorder; the mucosa and submucosa of the small and large intestine showed extensive inflammatory cell infiltration, epithelial cell necrosis and shedding, severe damage, atrophy, and fusion of intestinal villi, and loss of crypt structures. Compared with the control group, the 12-ketolithocholic acid treatment group showed reduced inflammatory cell infiltration in the liver and lung tissues of the recipient mice, with relatively intact alveolar structures; the mucosal structures of the small and large intestines were well maintained, with significantly reduced damage, neatly arranged intestinal villi, and a substantial reduction in crypt damage and villi shedding. This result indicates that 12-ketolithocholic acid can significantly reduce inflammatory cell infiltration in target organs such as the liver, lungs, small intestine, and large intestine of recipient mice during acute graft-versus-host disease (APHD), effectively protect the structural integrity of target organs, and significantly reduce pathological damage to related target organ tissues such as the liver, lungs, small intestine, and large intestine during AHD induced by allogeneic hematopoietic stem cell transplantation.

[0040] III. Effects of 12-ketolithocholic acid on reducing the proportions of Th1, Th17, and Treg cells in different tissues of mice with acute graft-versus-host disease. To further investigate the immunological effects of 12-ketolithocholic acid in improving acute graft-versus-host disease, some recipient mice from the control group and the 12-ketolithocholic acid-treated group were sacrificed on day 14 post-transplantation, and their spleen, intestine, and liver tissues were collected intact. Flow cytometry was used to detect CD4 levels in different tissues. +The proportions of T cell subsets. Spleen tissue was ground and filtered through a 200-mesh sieve, followed by erythrocyte lysis to obtain a single-cell suspension. Intestinal tissue was processed appropriately to obtain a single-cell suspension of the intestinal lamina propria. Liver tissue was ground, filtered through a 200-mesh sieve, and separated into liver lymphocytes using Percoll density gradient centrifugation. Cells obtained from each tissue were washed and resuspended for flow cytometry analysis. Th1 cells, with CD4+ as the dominant cytokine, were analyzed. + T-bet + Cell characterization; Th17 cells were characterized using CD4 + Roryt + Cell characterization; Treg cells use CD4+ + FoxpIII + Cell characterization was performed. Specific analytical results are as follows: Figure 3 As shown.

[0041] Depend on Figure 3 As shown in Figure A, compared with the control group, the proportion of Th1 cells in the spleen of recipient mice treated with 12-ketolithocholic acid was significantly reduced (**P<0.01); while the proportions of Th17 cells and Treg cells were not significantly different from those in the control group (ns). Figure 3 As shown in Figure B, compared with the control group, the proportion of Th1 cells in the intestinal lamina propria of the 12-ketolithocholic acid-treated mice was significantly reduced (***P<0.001); while the proportion of Th17 cells and Treg cells remained stable and was not significantly affected (ns). Figure 3 As shown in Figure c, compared with the control group, the proportion of Th1 cells in the liver of recipient mice treated with 12-ketolithocholic acid was significantly reduced (**P<0.01), while the proportion of Treg cells did not change significantly (ns). Although the proportion of Th17 cells in the liver decreased to some extent (*P<0.05), the decrease was much smaller than that of Th1 cells. This result indicates that 12-ketolithocholic acid can significantly and specifically inhibit the generation and accumulation of pathogenic Th1 cells in major organs such as the spleen, intestine, and liver in vivo, block the Th1-type immune-mediated cascade inflammatory response, and maximally maintain the homeostasis of regulatory cell subsets such as Treg cells and Th17 cells, demonstrating excellent T cell subset-selective immunomodulatory activity.

[0042] IV. 12-Ketolithocholic Acid In Vitro Inhibition of CD4 + T cells differentiate into Th1 cells To further verify the direct regulatory effect of 12-ketolithocholic acid on Th1 cell differentiation, an initial CD4 group was established. +A T-cell directed differentiation model was established in vitro. Specifically, spleen and peripheral lymph node tissues from 6- to 8-week-old C57BL / 6 mice were collected, ground under sterile conditions, and filtered through a 200-mesh cell sieve to prepare single-cell suspensions. Red blood cell lysis buffer was added to the single-cell suspensions for erythrocyte lysis. After erythrocyte lysis, naïve CD4+ T cells were enriched using magnetic bead sorting. These naïve CD4+ T cells... + T cells can be identified or enriched based on surface markers such as CD4 and CD62L.

[0043] The culture plates were pre-coated with anti-CD3 antibody and anti-CD28 antibody at concentrations of 1 μg / mL to 5 μg / mL and 1 μg / mL to 2 μg / mL, respectively. The initial CD44 cells obtained from sorting were then... + T cells are produced at a rate of approximately 1 × 10 6 Cells were seeded at a density of [number] cells / mL on coated culture plates and cultured. Subsequently, in vitro directed differentiation systems for Th1, Th17, and Treg cells were established. The Th1 cell induction system was supplemented with recombinant mouse IL-12, anti-IL-4 neutralizing antibody, and IL-2; the Th17 cell induction system was supplemented with recombinant mouse TGF-β1, IL-6, anti-IFN-γ neutralizing antibody, and anti-IL-4 neutralizing antibody; and the Treg cell induction system was supplemented with recombinant mouse TGF-β1 and IL-2. Each cell differentiation system was divided into two groups: a control group and a 12-ketolithocholic acid treatment group. The 12-ketolithocholic acid treatment group was supplemented with a final concentration of 20 μM 12-ketolithocholic acid solution, while the control group received an equal volume of solvent control (DMSO). The cells were then incubated at 37°C in a 5% CO2 incubator for 72 hours. After culture, cells were collected, fixed, permeabilized, and stained with intracellular / nuclear fluorescent antibodies. Flow cytometry was then used to detect the differentiation ratios of Th1, Th17, and Treg cells. Results are as follows: Figure 4 As shown. Among them, Th1 cells use CD4 + T-bet + Cell characterization; Th17 cells were characterized using CD4 + Roryt + Cell characterization; Treg cells use CD4+ + FoxpIII + Cells were characterized.

[0044] Depend on Figure 4 It can be seen that in the Th17 cell directed differentiation system, compared with the control group, CD4 cells treated with 20 μM 12-ketolithocholic acid showed increased activity. + T-bet +The differentiation rate of cells was significantly reduced (P<0.001); while under the same intervention conditions, in the Th17 cell directed differentiation system, the CD4 group treated with 12-ketolithocholic acid showed a significantly lower differentiation rate (P<0.001); + Roryt + No significant difference was observed in cell proportion (ns); in the Treg cell directed differentiation system, the CD4 count in the 12-ketolithocholic acid treatment group was significantly higher. + FoxpIII + No significant difference was observed in cell proportion (ns). This result indicates that, under the established in vitro directed differentiation conditions, 12-ketolithocholic acid can directly inhibit naïve CD4 cells. + T cells differentiated into Th1 cells, but no significant effect was observed on Th17 and Treg cell differentiation. Combined with the above in vivo experimental results, it can be concluded that 12-ketolithocholic acid has a relatively significant selective regulatory effect on the Th1-type immune response associated with acute graft-versus-host disease (APVD). This effect may be one of the important mechanisms by which it improves the severity of APVD and reduces pathological damage to related target organs.

[0045] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0046] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. 12-Ketolithocholic acid or a pharmaceutically acceptable salt thereof in the preparation of drugs for the prevention and / or treatment of acute graft-versus-host disease.

2. The application as described in claim 1, characterized in that, The 12-ketolithocholic acid or its pharmaceutically acceptable salt exerts preventive and / or therapeutic effects by modulating abnormally activated T-cell immune responses and reducing acute graft-versus-host disease-related inflammatory responses.

3. The application as described in claim 2, characterized in that, The 12-ketolithocholic acid or its pharmaceutically acceptable salt selectively inhibits CD4. + T cells play a role in differentiating into Th1 cells.

4. The application as described in claim 3, characterized in that, The 12-ketolithocholic acid or its pharmaceutically acceptable salts inhibit Th1 cell differentiation without significantly affecting CD4. + T cells differentiate into Th17 cells and Treg cells.

5. The application as described in claim 1, characterized in that, The acute graft-versus-host disease mentioned is an acute graft-versus-host disease induced by allogeneic hematopoietic stem cell transplantation.

6. The application as described in claim 5, characterized in that, The 12-ketolithocholic acid or a pharmaceutically acceptable salt thereof is used to reduce inflammatory damage to target organs associated with acute graft-versus-host disease; The target organs include one or more of the intestines, liver, and lungs.

7. The application as described in any one of claims 1-6, characterized in that, The dosage of the 12-ketolithocholic acid or a pharmaceutically acceptable salt thereof is 80 mg / kg / day to 120 mg / kg / day.

8. A pharmaceutical composition for the prevention and / or treatment of acute graft-versus-host disease, characterized in that, Including an effective amount of 12-ketolithocholic acid or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent or excipient, for reducing Th1-type immune responses during acute graft-versus-host disease and alleviating inflammatory damage to tissues associated with acute graft-versus-host disease.

9. The pharmaceutical composition according to claim 8, characterized in that, The pharmaceutical composition further includes one or more adjuvant therapeutic agents; The adjuvant therapy drugs are selected from glucocorticoids or JAK inhibitors.

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