Application of AKT inhibitor in preparation of medicine for inducing pancreas islet transplantation immune tolerance

Abstract

The invention belongs to the technical field of medicine application and biological medicine, and particularly relates to application of an AKT inhibitor in preparation of a medicine for inducing pancreas islet transplantation immune tolerance. The invention discovers that the AKT inhibitor MK-2206 can effectively induce immune tolerance after pancreas islet transplantation for the first time, system experiments prove that a short-term intervention scheme adopting the AKT inhibitor MK-2206 can obviously promote long-term survival of pancreas islet transplantation, and the effect of the AKT inhibitor MK-2206 is superior to that of a traditional immunosuppressor. The method belongs to secondary development of medicines with known active ingredients, and provides a potential novel 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 multiple 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 the specific and long-term unresponsive state established by the transplant recipient to the islet allograft without long-term maintenance immunosuppressive therapy, while maintaining the complete immune response ability to other antigens.

[0009] The establishment of this state is confirmed by one or more of the following aspects: Functional tolerance: the long-term survival of the graft for more than 100 days, and the stable maintenance of the blood glucose level of the recipient.

[0010] Immune regulation: significantly promoting the expansion of regulatory B cells and regulatory T cells with immune suppression function in peripheral blood and / or spleen.

[0011] Tissue and functional protection: effectively protecting the tissue structural integrity of the islet graft, significantly reducing inflammatory cell infiltration, and maintaining its normal insulin and glucagon secretion function.

[0012] 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.

[0013] 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.

[0014] 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.

[0015] 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.

[0016] 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.

[0017] Preferably, the pharmaceutical composition is formulated as a dosage form for oral administration.

[0018] In a fifth aspect, the present application provides a method for inducing immune tolerance in islet transplantation recipients, comprising administering an effective amount of MK-2206 to the islet transplantation recipient by using a short-term intervention regimen.

[0019] 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.

[0020] Preferably, the dose of each administration is 120 mg MK-2206 / kg.

[0021] 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 The significant advantage of the present application compared with the prior art is that: The present application first discovers that AKT inhibitor MK-2206 can effectively induce immune tolerance after islet transplantation, and through systematic experiments, it is verified that the short-term intervention regimen using AKT inhibitor MK-2206 can significantly promote the long-term survival of islet grafts, and the effect is better than that of traditional immunosuppressants. This belongs to the secondary development of known active ingredient drugs, and provides a potential new treatment strategy for inducing transplant immune tolerance in clinic.

[0022] 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, so as to construct 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 at home and abroad, and belongs to an important innovation of drug action mode. BRIEF DESCRIPTION OF DRAWINGS

[0023] 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.

[0024] Figure 1 The blood glucose change of C57BL / 6 diabetic mice after allogeneic islet transplantation provided by the present application.

[0025] Figure 2 The administration schematic (A) and survival curve (B) of islet grafts of the recipient mice in different treatment groups provided by the present application.

[0026] Figure 3 The flow cytometry graphs and statistical results of TIM1⁺ Bregs (A, B) and CD4⁺ Foxp3⁺ Tregs (C, D) in peripheral blood provided by the present application.

[0027] Figure 4Flow cytometry graphs and statistical results of TIM1+ Bregs (A, B) and CD4+ Foxp3+ Tregs (C, D) in the spleen provided for the present application.

[0028] Figure 5 HE staining (A) and immunofluorescence staining results (B) of kidney tissue containing a graft provided for the present application. DETAILED DESCRIPTION

[0029] The technical solutions of the present application will be described 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 skilled in the art without creative labor fall within the scope of protection of the present application.

[0030] Reference herein to "an embodiment" means that a particular feature, structure, or characteristic described in conjunction with an embodiment can be included in a variety of embodiments of the present application. Appearances of the phrase "in an embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are they necessarily mutually exclusive of one another. Those skilled in the art will understand that an embodiment described herein can be incorporated in some embodiments and not in others.

[0031] In the embodiments described herein, unless a specific technique or condition is specified, the technique or condition described in the literature in the art or according to the product manual is used. Unless the manufacturer is specified, all 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. Example 1: Establishment of a stable and repeatable islet transplantation model 1. Experimental materials and methods 1.1 Experimental animals Donor mice: 6-8 week old male Balb / C mice.

[0032] Recipient mice: 6-8 week old male C57BL / 6 mice.

[0033] 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.

[0034] 1.2 Establishment of a diabetes model and islet transplantation A diabetes model was induced in C57BL / 6 mice by intraperitoneal injection of 200 mg / kg streptozotocin, and a blood glucose level > 16.7 mmol / L for two consecutive days was considered to be a successful model.

[0035] Islets were digested by collagenase P and purified by 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 the blood glucose recovered to <11.1 mmol / L (200 mg / dL) indicated that the transplantation was successful.

[0036] 2. Experimental results 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.

[0037] Example 2: Effect of tolerance induction and verification of immune mechanism of short-range intervention scheme of MK-2206 1. Experimental materials and methods 1.1 Preparation of MK-2206 drug composition 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.

[0038] 1.2 Animal grouping and administration The successfully obtained transplanted recipient mice in Example 1 were randomly divided into three groups: solution control group (Tx), rapamycin group (RAPA) and AKT inhibitor group (MK-2206); See Figure 2 A, administered as follows: Solution control group (Tx): equal volume of 30% SBE-β-CD solution was given after transplantation.

[0039] Rapamycin group (RAPA (Sirolimus, rapamycin; Sirolimus) | mTOR inhibitor | MCE | product number HY-10219 batch 260967)): 1 mg / kg rapamycin was administered orally by gavage from the 4th day after transplantation, once a day for 10 consecutive days.

[0040] AKT inhibitor group (MK-2206): MK-2206 drug composition prepared in Example 1 was administered orally by gavage at a dose of 120 mg / kg every other day, a total of 4 times, starting from the 4th day after transplantation.

[0041] Control group: After the mice were induced to have diabetes, the left kidney was exposed by surgery, but no islets were transplanted, and the same volume of solvent was administered after the operation.

[0042] 1.3 Blood glucose monitoring and follow-up Blood glucose was monitored daily for the first 20 days after surgery, and then 2-3 times a week. Blood glucose > 11.1 mmol / L for two consecutive days was considered to have occurred immune rejection.

[0043] 1.4 Sample collection and flow cytometry analysis 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 2mM EDTA). The spleen was filtered through a 70μm nylon mesh, followed by red blood cell lysis and resuspension in staining buffer.

[0044] 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, and then stained with anti-Foxp3 antibody. The sample was collected by flow cytometry to obtain data, and FlowJo software was used for analysis.

[0045] 1.5 Statistical analysis: Statistical analysis was completed 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 evaluated by one-way ANOVA, followed by Bonferroni's multiple comparison correction test. P<0.05 was considered statistically significant.

[0046] 2. Experimental results 2.1 Graft survival and function See Figure 2In the solution control (Tx) group, the median rejection period in mice with islet allografts was 14 days. In the positive drug control rapamycin (RAPA) group, the median rejection period 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 The 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.

[0047] 2.2 Expansion of regulatory immune cells Please see FIGS. 3A and B, the flow cytometry 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. Please see Figure 3 The flow cytometry analysis in C and D for CD25⁺Foxp3⁺ Tregs shows that the proportion of such Tregs in the peripheral blood of the RAPA group increased to a certain extent, and the proportion of CD25⁺Foxp3⁺ Tregs in the MK-2206 group also increased at a more significant rate (p<0.001). The accumulation of such regulatory cells (TIM1⁺ Bregs and CD25⁺Foxp3⁺ Tregs) is highly consistent with the trend of long-term survival of the graft, suggesting that MK-2206 can inhibit the rejection reaction by remodeling 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 graft tolerance by enhancing the accumulation of immune regulatory cells such as TIM1⁺ Bregs and CD25⁺Foxp3⁺ Tregs in the peripheral blood.

[0048] Please see Figure 4A and B, Flow cytometry analysis (spleen) results showed that for TIM1⁺ Bregs, the proportion of TIM1⁺ Bregs in the MK-2206 group was significantly higher than that in the RAPA group (P<0.01); see FIG. 4C and D, the proportion of CD25⁺Foxp3⁺ Tregs in the MK-2206 group was significantly higher than that in the RAPA group (p<0.01). Highly consistent with the results of peripheral blood. This finding indicates that MK-2206 not only promotes the accumulation of immunoregulatory 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.

[0049] Through the above experiments, this embodiment proves that MK-2206 can significantly prolong the survival of transplants and induce long-term immune tolerance through a specific short-range intervention scheme, and the mechanism of action is closely related to promoting the expansion of regulatory immune cells.

[0050] Example 3: Histological protective effect of MK-2206 on transplanted islets 1. Experimental materials and methods 1.1 Collection of tissue samples At the end of the experiment, the transplanted kidney tissues of the mice in each group were taken, among which 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.

[0051] 1.2 HE staining The mouse transplanted kidney tissues were fixed in 4% paraformaldehyde for 24 hours, routinely paraffin-embedded, and the section thickness was 4 μm. After the section was deparaffinized with xylene and hydrated with gradient alcohol, it was dyed with hematoxylin for 5 minutes, washed with tap water, differentiated with hydrochloric acid alcohol for 30 seconds, washed with running water, and blue-stained with alkali solution for 1 minute; then it was re-dyed with eosin staining solution for 2 minutes, dehydrated with gradient alcohol, transparentized with xylene, and mounted with neutral resin. Finally, the histological morphology of the islet graft was observed under an optical microscope.

[0052] 1.3 Immunofluorescence staining After the paraffin section was routinely deparaffinized / hydrated, it was permeabilized with 0.3% Triton X-100 and blocked with 5% BSA for 1 h; an antibody from a different host (insulin, glucagon) was added and incubated at 4°C overnight, the next day PBS was washed, and 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 washing, and mounting with an anti-fluorescence quenching mounting agent, and the image was collected under a fluorescence microscope.

[0053] 2. Experimental results 2.1 Tissue integrity protection See Figure 5 A, HE staining results show that there is obvious inflammatory cell infiltration in the solution control group (Tx) subcapsular kidney graft, and the islet structure is severely damaged; while the islet structure of the MK-2206 treatment group is intact, and the inflammatory reaction is significantly reduced.

[0054] 2.2 Maintenance of endocrine function See Figure 5 B, immunofluorescence three-color staining results show that the expression of insulin and glucagon in the solution control group (Tx) is significantly weakened, while the insulin secretion function of the AKT inhibitor group (MK-2206) is well maintained, indicating that the function of the transplanted islets is effectively protected.

[0055] Combined with the blood glucose monitoring results of the above Example 2, 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 transplantation tolerance at the histological level, and effectively maintains the functional integrity of the transplanted islets.

[0056] The above only describes the 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. The application according to claim 1, characterized in that, The drug is used to suppress immune rejection after islet transplantation and / or prolong the survival time of islet grafts.

3. The application according to claim 1, characterized in that, The drug is used to maintain stable blood glucose levels in transplant recipients after islet transplantation.

4. The application according to claim 1, characterized in that, The drug is used to promote the amplification of TIM1⁺ Bregs and CD4⁺Foxp3⁺ Tregs in peripheral blood and / or spleen after islet transplantation.

5. The application according to claim 1, characterized in that, The drug is used to protect the tissue integrity of the islet graft after islet transplantation and / or reduce inflammatory cell infiltration of the graft.

6. The application according to claim 1, characterized in that, The drug is used to maintain insulin secretion function and glucagon expression in islet grafts after islet transplantation.

7. The application according to claim 1, characterized in that, The drug includes a pharmaceutically acceptable carrier, which includes cyclodextrin or a derivative thereof.

8. The application according to claim 7, characterized in that, The carrier is a 30% (w / v) thiobutyl ether-β-cyclodextrin solution, and the MK-2206 exists in the form of MK-2206 dihydrochloride.

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