Use of an akt inhibitor in the manufacture of a medicament for inducing immune tolerance of a pancreatic islet transplant

Abstract

The application belongs to the field of pharmaceutical application and biological medicine technology, and particularly relates to application of AKT inhibitor in preparation of drugs for inducing immune tolerance of islet transplantation. It is found for the first time that AKT inhibitor MK-2206 can effectively induce immune tolerance after islet transplantation, and it is proved through systematic experiments that a short-term intervention scheme of AKT inhibitor MK-2206 can significantly promote long-term survival of islet grafts, and the effect is better than that of traditional immunosuppressants. This belongs to secondary development of known active ingredient drugs, and provides a potential new treatment strategy for clinical induction of transplantation immune tolerance.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical applications and biological medicine technology, in particular, to the application of AKT inhibitor in the preparation of drugs for inducing immune tolerance of islet transplantation. BACKGROUND

[0002] Islet transplantation is a potential curative method for the treatment of type 1 diabetes, which restores insulin secretion function by transplanting donor islets, thereby achieving blood glucose control. However, its clinical promotion is severely limited by immune rejection. Although existing immunosuppressive agents (such as cyclosporine, tacrolimus, rapamycin, etc.) can delay rejection, their mechanism relies on long-term and broad-spectrum inhibition of T cell activation, which cannot induce specific immune tolerance to the graft, and must be taken for a long time or even for life. Once the drug is stopped, the graft rejection occurs rapidly. At the same time, long-term systemic immunosuppression brings serious side effects, including infection, nephrotoxicity and tumor risk, which greatly limits its application.

[0003] The expansion and functional stability of regulatory T cells (Tregs) and regulatory B cells (Bregs) play a core role in maintaining immune tolerance of transplantation. Although existing immunosuppressive agents can temporarily suppress the effector immune response, they are difficult to induce specific immune tolerance to the graft, and also fail to effectively promote the sustained accumulation and function maintenance of regulatory immune cells. Therefore, there is an urgent need in the art for new intervention strategies to achieve long-term islet transplantation tolerance.

[0004] The AKT signaling pathway is a key node of metabolism and immunity, and excessive activation can promote effector T cell response and disrupt immune balance; moderate inhibition of AKT signaling may promote the expansion and function maintenance of regulatory cells. MK-2206 is a highly selective, orally available AKT inhibitor, which has been verified for safety in various tumor clinical trials, but there is no report on its application in the induction of islet transplantation tolerance. Therefore, it is of great significance to explore the immune regulation of MK-2206 in islet transplantation. SUMMARY

[0005] In view of this, in order to overcome the shortcomings of the prior art, the present application aims to provide a new use of AKT inhibitor in the preparation of drugs for inducing immune tolerance of islet transplantation.

[0006] The present application discloses for the first time that AKT inhibitor MK-2206 can effectively induce immune tolerance after islet transplantation, and exhibits excellent efficacy under a specific dosing regimen.

[0007] In a first aspect, the present application provides the use of AKT inhibitor in the preparation of drugs for inducing immune tolerance of islet transplantation.

[0008] In the present application, the "immune tolerance" refers to a specific, long-term unresponsive state established by the transplant recipient to the islet allograft without long-term maintenance immunosuppressive therapy, while maintaining the full immune response ability to other antigens.

[0009] The establishment of this state is confirmed by one or more of the following aspects:

[0010] Functional tolerance: the graft survives for more than 100 days, and the blood glucose level of the recipient is stable.

[0011] Immune regulation: significantly promotes the expansion of regulatory B cells and regulatory T cells with immune suppression function in peripheral blood and / or spleen.

[0012] Tissue and functional protection: effectively protects the tissue structure integrity of the islet graft, significantly reduces the inflammatory cell infiltration, and maintains its normal insulin and glucagon secretion function.

[0013] In a second aspect, the present application provides the use of AKT inhibitor MK-2206 in the preparation of a medicament for inhibiting the immune rejection after islet transplantation.

[0014] In a third aspect, the present application provides the use of AKT inhibitor MK-2206 in the preparation of a medicament for prolonging the survival time of islet grafts.

[0015] In a fourth aspect, the present application provides a pharmaceutical composition for inducing immune tolerance of islet transplantation. The pharmaceutical composition comprises AKT inhibitor MK-2206 as an active ingredient, and a pharmaceutically acceptable carrier.

[0016] Preferably, the AKT inhibitor is MK-2206 dihydrochloride, which can be obtained commercially or can be prepared according to the known methods in the art.

[0017] Preferably, the pharmaceutically acceptable carrier comprises cyclodextrin or its derivative; more preferably, it is a 30% (w / v) aqueous solution of sulfobutyl ether-β-cyclodextrin, which can significantly improve the oral bioavailability and preparation stability of MK-2206 dihydrochloride.

[0018] Preferably, the pharmaceutical composition is formulated as a dosage form for oral administration.

[0019] In a fifth aspect, the present application provides a method for inducing immune tolerance in an islet transplantation recipient, comprising administering an effective amount of MK-2206 to the islet transplantation recipient by using a short-term intervention regimen.

[0020] Preferably, the short-term intervention regimen is specifically: starting on the 4th day after islet transplantation, oral administration once every two days, a total of 4 times.

[0021] Preferably, the dose for each administration is 120 mg MK-2206 / kg.

[0022] Surprisingly, this short-term intervention regimen is sufficient to initiate and establish a stable immune tolerance program, which can maintain long-term survival (>100 days) and functional stability of the graft after drug withdrawal

[0023] The significant advantage of the present application compared to the prior art is that:

[0024] The present application first discovered that AKT inhibitor MK-2206 can effectively induce immune tolerance after islet transplantation, and confirmed by systematic experiments that the short-term intervention regimen using AKT inhibitor MK-2206 can significantly promote the long-term survival of islet grafts, which is better than the traditional immunosuppressive agents. This belongs to the secondary development of known active ingredient drugs, and provides a potential new treatment strategy for inducing immune tolerance in clinical transplantation.

[0025] The experimental results show that, at the immune cell level, MK-2206 can specifically expand TIM1⁺ regulatory B cells and CD4⁺ Foxp3⁺ regulatory T cells in peripheral blood and spleen, thereby constructing an immune microenvironment conducive to tolerance; at the tissue function level, it can effectively protect the structural integrity of islets, reduce inflammatory infiltration, and long-term maintain normal insulin and glucagon secretion function. This mechanism has not been reported in the field of islet transplantation immune tolerance research at home and abroad, and is an important innovation in drug action mode. BRIEF DESCRIPTION OF DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the specific embodiments or prior art of the present application, the drawings needed in the specific embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0027] Figure 1 The blood glucose change of C57BL / 6 diabetic mice after allogeneic islet transplantation is provided for the present application.

[0028] Figure 2 The administration schematic (A) and survival curve (B) of islet grafts of the different treatment groups of recipient mice provided for the present application.

[0029] Figure 3 The flow cytometry graphs and statistical results of TIM1⁺ Bregs (A, B) and CD4⁺ Foxp3⁺ Tregs (C, D) in peripheral blood provided for the present application.

[0030] Figure 4 Flow cytometry graphs and statistical results of TIM1+ Bregs (A, B) and CD4+ Foxp3+ Tregs (C, D) in the spleen provided for the present application.

[0031] Figure 5 HE staining (A) and immunofluorescence staining results (B) of kidney tissue containing a graft provided for the present application. DETAILED DESCRIPTION

[0032] The technical solutions of the present application will be described clearly and completely below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0033] Reference to "an embodiment" herein means that a particular feature, structure, or characteristic described in connection with the embodiment can be included in a variety of embodiments of the present application. The appearance of the phrase in various places in the specification does not necessarily all refer to the same embodiment, nor is it necessarily mutually exclusive of other embodiments. It is explicitly and implicitly understood that the embodiments described herein are capable of combination with other embodiments.

[0034] Unless otherwise specified in the embodiments herein, the techniques or conditions described in the literature in the art, or according to the product instructions are used. Unless otherwise specified, the reagents or instruments used are conventional products that can be purchased through regular channels. The experimental methods in the following embodiments are conventional methods unless otherwise specified. The test materials used in the following embodiments are conventional commercially available products unless otherwise specified

[0035] Example 1: Establishment of a stable and repeatable islet transplantation model

[0036] 1. Experimental materials and methods

[0037] 1.1 Experimental animals

[0038] Donor mice: 6-8 week old male Balb / C mice.

[0039] Recipient mice: 6-8 week old male C57BL / 6 mice.

[0040] All mice were purchased from Jiangsu Jizhuangkang Biotechnology Co., Ltd. and were raised in a SPF level environment with free access to food and water.

[0041] 1.2 Establishment of a diabetic model and islet transplantation

[0042] C57BL / 6 mice were induced to diabetes model by intraperitoneal injection of 200 mg / kg streptozotocin, and blood glucose > 16.7 mmol / L for two consecutive days was considered to be successful modeling.

[0043] Islets were purified by collagenase P digestion and discontinuous density gradient centrifugation, and islet cells were manually picked. 300-500 islet cells were transplanted into the left kidney capsule of C57BL / 6 recipient mice, and blood glucose recovery < 11.1 mmol / L (200 mg / dL) indicated successful transplantation.

[0044] 2. Experimental results

[0045] See Figure 1 , 1-2 days after operation, the blood glucose of many recipient mice recovered to below 11.1 mmol / L, indicating that the transplanted islets had recovered function. Through continuous blood glucose monitoring, the recovery speed of islet function, rejection time and blood glucose fluctuation range of many recipient mice in this model were consistent. This embodiment successfully established a stable and repeatable allogeneic islet transplantation model.

[0046] Example 2: Effect of tolerance induction and verification of immune mechanism of short-range intervention scheme of MK-2206

[0047] 1. Experimental materials and methods

[0048] 1.1 Preparation of MK-2206 drug composition

[0049] MK-2206 dihydrochloride (MK-2206 2HCl | Akt inhibitor | TargetMol product number T1952 | batch 159551) was dissolved in freshly prepared and sterile filtered 30% sulfobutyl ether-β-cyclodextrin (SBE-β-CD) aqueous solution to prepare a uniformly dispersed solution for subsequent oral gavage administration.

[0050] 1.2 Animal grouping and administration

[0051] The successfully obtained recipient mice in Example 1 were randomly divided into three groups:

[0052] Solution control group (Tx), rapamycin group (RAPA) and AKT inhibitor group (MK-2206);

[0053] See Figure 2 A, administered as follows:

[0054] Solution control group (Tx): equal volume of 30% SBE-β-CD solution was given after transplantation.

[0055] Rapamycin group (RAPA (Sirolimus, Rapamycin; Sirolimus) | mTOR inhibitor | MCE | Product No. | HY-10219 Batch: 260967): 1 mg / kg rapamycin was orally gavaged from the 4th day after transplantation, once a day, for 10 consecutive days.

[0056] AKT inhibitor group (MK-2206): MK-2206 drug composition prepared in Example 1 was orally gavaged every two days from the 4th day after transplantation, with a dosage of 120 mg / kg, for a total of 4 times.

[0057] Blank control group was set again: after the mice were induced to have diabetes, the left kidney was surgically exposed, but no islets were transplanted, and the same volume of solvent was administered after the operation.

[0058] 1.3 Blood glucose monitoring and follow-up

[0059] Blood glucose was monitored daily for the first 20 days after the operation, and then 2-3 times a week. Blood glucose > 11.1 mmol / L for two consecutive days was considered to have occurred immune rejection.

[0060] 1.4 Sample collection and flow cytometry analysis

[0061] At the end of the experiment, the mice were anesthetized with isoflurane, and blood and spleen were collected. The blood sample was treated with 0.8% NH4Cl red blood cell lysis buffer twice (5 minutes each time) to remove red blood cells, and the resulting white blood cells were resuspended in staining buffer (PBS containing 2% fetal bovine serum and 2 mM EDTA). The spleen was filtered through a 70 μm nylon mesh, followed by red blood cell lysis and resuspension in staining buffer.

[0062] The single cell suspension was incubated with anti-CD16 / 32 antibody for 10 minutes for Fc receptor blocking, and then surface staining was performed with fluorescein-labeled anti-CD4, CD19, CD45, TIM-1, and CD25 antibodies for 30 minutes at 4°C in the dark. For intracellular staining, Foxp3 / transcription factor staining buffer kit was used to fix and permeabilize the cells, followed by staining with anti-Foxp3 antibody. The sample was collected by flow cytometry to obtain data, and FlowJo software was used for analysis.

[0063] 1.5 Statistical analysis:

[0064] Statistical analysis was performed using GraphPad Prism software. Data were expressed as mean ± standard error (SEM). Transplant survival was analyzed using Kaplan-Meier survival curves and compared by log-rank (Mantel-Cox) test. Differences between groups were assessed using one-way ANOVA followed by Bonferroni's multiple comparison correction test. P values < 0.05 were considered statistically significant.

[0065] 2. Experimental results

[0066] 2.1 Graft survival and function

[0067] See Figure 2 In the solution control (Tx) group, the median rejection period in mice with allogeneic islet transplantation was 14 days. The median rejection period in the rapamycin (RAPA) group, a positive drug control, was extended to about 27 days, indicating that traditional immunosuppressants can only temporarily delay the rejection reaction. In the AKT inhibitor (MK-2206) group, about 75% of the mice had stable blood glucose (<11.1 mmol / L) in more than 100 days of follow-up, showing a significant effect of long-term tolerance. Figure 2 Kaplan-Meier survival curve analysis in B shows that the graft survival rate in the MK-2206 group was significantly higher than that in the control group (P < 0.001), and the difference compared with the rapamycin (RAPA) group was also statistically significant. Therefore, MK-2206 can achieve long-term graft tolerance through short-term intervention, and its effect is better than that of traditional immunosuppressants.

[0068] 2.2 Regulatory immune cell expansion

[0069] See Figures 3A and B, flow cytometry analysis results show that the proportion of TIM1⁺CD19⁺ Bregs in the peripheral blood of the rapamycin (RAPA) group increased compared with the Tx group; in contrast, the proportion of TIM1⁺ Bregs in the MK-2206 group increased significantly (p < 0.001), and the expansion effect was more prominent. See Figure 3Flow cytometric analysis of C and D for CD25+Foxp3+Tregs showed that the proportion of such Tregs in the peripheral blood of the RAPA group was increased to a certain extent, while the proportion of CD25+Foxp3+Tregs in the MK-2206 group was also increased by a more significant margin (p<0.001). The accumulation of such regulatory cells (TIM1+Bregs and CD25+Foxp3+Tregs) was highly consistent with the trend of long-term survival of the graft, suggesting that MK-2206 can inhibit rejection by reshaping the immune microenvironment, and its tolerance induction mechanism is more focused on optimizing the proportion of regulatory cells. In summary, MK-2206 helps to build an immune environment conducive to transplantation tolerance by enhancing the accumulation of immune regulatory cells such as TIM1+Bregs and CD25+Foxp3+Tregs in the peripheral blood.

[0070] See Figure 4 A and B, flow cytometric analysis (spleen) results show that for TIM1+Bregs, the proportion of TIM1+Bregs in the MK-2206 group is significantly higher than that in the RAPA group (P<0.01); see FIGS. 4C and D, the proportion of CD25+Foxp3+Tregs in the MK-2206 group is significantly higher than that in the RAPA group (p<0.01). Highly consistent with the results of the peripheral blood. This finding shows that MK-2206 not only promotes the accumulation of immune regulatory cells in the peripheral circulation, but also enhances the expansion and maintenance of tolerance-related cells in the main immune organs. In summary, MK-2206 also significantly increases the proportion of regulatory immune cells in the spleen, providing a solid immunological basis for the establishment of long-term transplantation tolerance.

[0071] Through the above experiments, this embodiment proves that MK-2206 can significantly prolong the survival of the graft and induce long-term immune tolerance through a specific short-range intervention scheme, and its mechanism of action is closely related to promoting the expansion of regulatory immune cells.

[0072] Example 3: Histological protective effect of MK-2206 on transplanted islets

[0073] 1. Experimental materials and methods

[0074] 1.1 Collection of tissue samples

[0075] At the end of the experiment, the transplanted kidney tissues of the mice in each group were collected, wherein the solution control group (Tx) was collected at 14 days after transplantation (i.e., the median rejection period); the RAPA group collected tissues at 27 days after the median rejection period; the MK-2206 treatment group collected tissues at 100 days after transplantation.

[0076] 1.2 HE staining

[0077] Mouse transplanted kidney tissues were fixed in 4% paraformaldehyde for 24 hours, routinely paraffin-embedded, and sectioned at a thickness of 4 μm. After dewaxing with xylene and hydration with gradient alcohol, the sections were stained with hematoxylin for 5 minutes, rinsed with tap water, differentiated with hydrochloric alcohol for 30 seconds, rinsed with running water, and blue-reverted with alkali solution for 1 minute. Then, the sections were re-stained with eosin for 2 minutes, dehydrated with gradient alcohol, cleared with xylene, and mounted with neutral balsam. Finally, the histological morphology of the islet grafts was observed under an optical microscope.

[0078] 1.3 Immunofluorescence staining

[0079] After routine dewaxing / hydration, the paraffin sections were permeabilized with 0.3% Triton X-100 and blocked with 5% BSA for 1 h; an antibody (insulin, glucagon) from a different host was added and incubated at 4°C overnight, and the next day, after PBS washing, a corresponding fluorescently labeled secondary antibody (Alexa Fluor 488 / Alexa Fluor 594) was added and incubated at room temperature for 1 h in the dark, followed by DAPI nuclear staining, PBS rinsing, and mounting with an anti-fluorescence quenching mounting agent, and images were collected under a fluorescence microscope.

[0080] 2. Experimental results

[0081] 2.1 Tissue integrity protection

[0082] See Figure 5 A, the HE staining results show that there is obvious inflammatory cell infiltration in the solution control group (Tx) graft under the kidney capsule, and the islet structure is severely damaged; while in the MK-2206 treatment group, the islet structure of the graft is intact, and the inflammatory reaction is significantly reduced.

[0083] 2.2 Maintenance of endocrine function

[0084] See Figure 5 B, the results of triple immunofluorescence staining show that the expression of insulin and glucagon is significantly weakened in the solution control group (Tx), while the insulin secretion function is well maintained in the AKT inhibitor group (MK-2206), indicating that the function of the transplanted islets is effectively protected.

[0085] In combination with the blood glucose monitoring results of Example 2 above, MK-2206 not only prolongs the survival time of the graft, but also maintains its endocrine function and tissue integrity. In summary, MK-2206 verifies its effect of promoting long-term graft tolerance at the histological level, and effectively maintains the functional integrity of the transplanted islets.

[0086] The above merely describes preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. The application of an AKT inhibitor in the preparation of drugs for inducing immune tolerance in islet transplantation, characterized in that, The AKT inhibitor is MK-2206.

2. Use according to claim 1, characterized in that, The drug includes a pharmaceutically acceptable carrier, which includes a cyclodextrin.

3. Use according to claim 2, characterized in that, The carrier is a 30% (w / v) solution of sulfobutyl ether-beta-cyclodextrin, and the MK-2206 is in the form of MK-2206 dihydrochloride.

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