Application of an alkaloid in preparing T cell immunosuppressant

Abstract

The present invention relates to an application of an alkaloid in preparing a T cell immunosuppressant, belonging to the technical field of natural product applications. The present invention discloses an application of an alkaloid, a natural product of Allium sibiricum, in preparing a T cell immunosuppressant, especially a drug for acute rejection in allogeneic transplantation, more especially a drug for acute rejection in allogeneic heart transplantation. The alkaloid in the present invention is used to inhibit the proliferation of T lymphocytes and to reduce the secretion of inflammatory factors: wherein the T lymphocytes are CD3 + T lymphocytes; inflammatory factors are Th1 type inflammatory factors, Th2 type inflammatory factors and / or Th17 type inflammatory factors, Th1 type inflammatory factors are IL-2, TNF-α and IFN-γ; Th2 type inflammatory factors are IL-4, IL-6 and IL-10; Th17 type inflammatory factors are IL-17.

Description

Technical Field

[0001] The present invention belongs to the technical field of natural biomaterial application, and more specifically, relates to the application of an alkaloid in preparing a T cell immunosuppressant, and especially relates to the application of an alkaloid in preparing an acute rejection drug in allogeneic transplantation. Background Art

[0002] Heart transplantation is the only effective treatment for end-stage heart failure, but rejection remains a major factor affecting the proper function of the transplanted organ and the long-term survival of the recipient, severely restricting its development. Suppressing acute rejection can significantly prolong the survival of the transplanted organ. Therefore, mitigating or preventing rejection is a major clinical issue that urgently needs to be addressed. Immunosuppressants are a class of drugs that suppress the body's immune function. Their primary function is to inhibit abnormal immune responses, thereby achieving the goal of treating organ transplant rejection. The most commonly used immunosuppressants for long-term maintenance therapy in clinical practice can be divided into the following categories: 1. Calcineurin inhibitors, represented by cyclosporine and tacrolimus, are the core drugs in current immunosuppressant regimens. Their main mechanism of action is to inhibit calcineurin, thereby inhibiting T cell activation and producing an immunosuppressive effect. 2. Rapamycin inhibitors, represented by sirolimus and everolimus, form a complex with tacrolimus binding protein-12 (FKBP-12) and then bind to the mammalian target of rapamycin (mTOR), blocking IL-2-induced immune cell protein and DNA synthesis, inhibiting immune cell proliferation and thus exerting an anti-immune rejection effect. 3. Antimetabolites, represented by mycophenolic acid drugs, mainly inhibit lymphocyte proliferation and differentiation by reversibly inhibiting non-competitive inosine mononucleotide dehydrogenase. Because T cell-mediated immune responses play a key role in acute immune rejection, the first-line maintenance immunosuppressants currently used in clinical practice primarily target T cell activation.

[0003] However, all of the aforementioned anti-rejection drugs suffer from numerous drawbacks, including inadequate efficacy in some patients, high drug costs, and the potential for drug resistance. Furthermore, these immunosuppressants also present with significant adverse reactions. Tacrolimus can cause hypertension, angina, palpitations, pericardial effusion, and pleural effusion; cyclosporine has strong hepatotoxicity, allergic reactions, hirsutism, and hyperbilirubinemia; rapamycin can cause headaches, nausea, dizziness, epistaxis, joint pain, thrombocytopenia, leukopenia, hypertriglyceridemia, hypercholesterolemia, hyperglycemia, elevated lactate dehydrogenase, and hypomagnesemia; and mycophenolate mofetil not only causes significant gastrointestinal side effects but can also induce tumors. Therefore, the development of new, highly effective, and less toxic T-cell immunosuppressants is imperative.

[0004] Natural products are the most common source of pharmaceuticals. Statistics show that from the 1980s to the present, 30–40% of marketed drugs each year are directly or indirectly derived from natural products. In 2010, the proportion of drugs derived from natural products reached 50%. Plant secondary metabolites are extremely diverse, and with increasing research in recent years, a growing number of highly active compounds have been discovered, laying a foundation for the development of new drugs. The whole herb, including the bulb, of Zephyranthes candida is a folk herbal remedy with liver-calming, heart-soothing, and sedative properties. It is primarily used to treat infantile convulsions and epilepsy. (+)-trans-dihydronarciclasine (TDN) is the main component of Zephyranthes candida, but its biological activity is relatively limited. Only a few studies have shown antitumor activity, and no research has been conducted on its use as a T cell immunosuppressant. In addition, there are no reports of (+)-trans-dihydronarciclasine and its analogs being marketed as immunosuppressants either domestically or internationally. To address this issue, the inventors conducted in vivo experiments using an allogeneic heart transplant acute rejection model mediated by alloreactive T cells. They also explored the immunosuppressive effects of TDN on T cells using an in vitro model of T cell receptor-specific stimulation and mixed culture of allogeneic lymphocytes. These results indicate that TDN can be used to suppress acute rejection reactions in allogeneic organ transplants and also has potential in the treatment of other T cell autoimmune diseases. Summary of the Invention

[0005] The present invention solves the technical problems in the prior art of T cell immunosuppressants, such as insufficient efficacy, high drug prices, easy drug resistance, and easy adverse reactions. The purpose of the present invention is to provide an application of alkaloids in the preparation of T cell immunosuppressants.

[0006] According to a first aspect of the present invention, there is provided a use of an alkaloid for preparing a T cell immunosuppressant, wherein the structural formula of the alkaloid is as follows:

[0007]

[0008] Preferably, the T cell immunosuppressant is a drug for acute rejection in allogeneic transplantation.

[0009] Preferably, the acute rejection drug in allogeneic transplantation is an acute rejection drug in allogeneic heart transplantation.

[0010] Preferably, the alkaloid is used to inhibit the proliferation of T lymphocytes.

[0011] Preferably, the T lymphocytes are CD3 + T lymphocytes.

[0012] Preferably, the alkaloid is used to reduce the secretion of inflammatory factors.

[0013] Preferably, the inflammatory factors are Th1 inflammatory factors, Th2 inflammatory factors and / or Th17 inflammatory factors.

[0014] Preferably, the Th1 type inflammatory factors are IL-2, TNF-α and IFN-γ.

[0015] Preferably, the Th2 inflammatory factors are IL-4, IL-6 and IL-10.

[0016] Preferably, the Th17 inflammatory factor is IL-17.

[0017] In general, the above technical solutions conceived by the present invention have the following technical advantages compared with the existing technology:

[0018] (1) The alkaloid TDN in the present invention has a good inhibitory effect on T cell-mediated acute rejection of organ transplantation and can effectively prolong the survival time of the transplant. Its mechanism is that the compound can inhibit the proliferation of T lymphocytes and reduce the secretion of inflammatory factors, which is of great significance for the research and development of immunosuppressants.

[0019] (2) The alkaloid TDN in the present invention is a novel alkaloid compound used to treat T cell-related autoimmune diseases such as organ transplant rejection, providing a candidate drug molecule for the treatment of related diseases and the development of new drugs.

[0020] (3) The compound TDN of the present invention is administered once every three days, while existing commonly used immunosuppressants in clinical practice, such as tacrolimus, cyclosporine, and rapamycin, require once a day. The administration frequency of compound TDN is lower;

[0021] (4) No obvious drug toxicity or side effects were observed in the TDN treatment group during the experiment. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the high-resolution mass spectrum of TDN.

[0023] Figure 2 For TDN 13 C-NMR spectrum.

[0024] Figure 3 The measured ECD diagram of TDN and the calculated ECD diagrams of (+)-trans-dihydronarciclasine and (-)-trans-dihydronarciclasine are shown.

[0025] Figure 4 To inhibit the acute rejection reaction of the transplanted heart (transplanted heart beating time).

[0026] Figure 5 The effect of TDN on the acute rejection of transplanted heart (immunohistochemical staining and CD3 + T cell infiltration).

[0027] Figure 6 TDN inhibits the proliferation and cytokine secretion of mouse spleen lymphocytes stimulated by anti-CD3 / anti-CD28 antibodies: (A) Proliferation; (B) Cytokine secretion. DETAILED DESCRIPTION

[0028] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely for the purpose of explaining the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

[0029] The alkaloid compound of the present invention has the structural formula as shown in Formula 1:

[0030]

[0031] Compound 1: (+)-trans-dihydronarciclasine (TDN), extracted from Zephyranthes candida with a purity of >99.0% (Formula 1). The main extraction steps are: mince the Zephyranthes candida bulb, dry it, and grind it to obtain Zephyranthes candida bulb powder. After grinding, extract it with 95% ethanol in a multifunctional extraction tank at 40°C for four times. The crude Zephyranthes candida extract is obtained after vacuum concentration. The total extract is suspended in warm water, adjusted to pH 2 with dilute hydrochloric acid, and extracted with chloroform to obtain a chloroform fraction with a pH of 2. The acidic aqueous layer is adjusted to pH 10 with aqueous ammonia and extracted with chloroform to obtain a chloroform fraction with a pH of 10. The chloroform fraction extract is then acidified and applied to a molecular sieve chromatography column to remove sugars. The column is then eluted with an ethanol solution containing aqueous ammonia. The eluate is collected and concentrated to obtain TDN.

[0032] The high-resolution mass spectrometry ( Figure 1 ), and its molecular formula is speculated to be C 14 H 14 NO7 (measured value 308.07770; calculated value [M–H] 308.07770, the calculated value is completely consistent with the measured value); test its NMR data ( Figure 2 ), and compared with known compounds reported in the literature, it was found that TDN 13 The C-NMR data were almost completely consistent with those of the known compound trans-dihydronarciclasine (Chemistry Select, 2016, 1, 5895–5899; Tetrahedron, 2018, 74, 5752–5757; Journal of Natural Products, 2017, 80, 1909–1917), demonstrating that the planar structure and relative configuration of TDN were consistent with those of trans-dihydronarciclasine. Since trans-dihydronarciclasine has two enantiomers, (+)-trans-dihydronarciclasine and (–)-trans-dihydronarciclasine, and ECD calculation is the authoritative method to determine the stereo configuration, we tested the ECD data of TDN and calculated the two configurations of (+)-trans-dihydronarciclasine and (–)-trans-dihydronarciclasine. The results showed that the measured ECD data of TDN was consistent with that of (+)-trans-dihydronarciclasine ( Figure 3). In summary, TDN was finally identified as (+)-trans-dihydronarciclasine.

[0033] The inventors evaluated Compound 1 in an in vivo model for the treatment of acute rejection in cardiac transplants and found that it can effectively prolong graft survival and inhibit the infiltration of alloreactive T cells. Furthermore, using an in vitro model of T cell receptor-specific stimulation and mixed allogeneic lymphocyte culture, the inventors found that the compound can effectively inhibit T cell proliferation, activation, and cytokine secretion. This compound could be directly used as a drug or lead compound for the development of treatments for acute rejection in allogeneic organ transplants.

[0034] Example 1

[0035] The specific implementation is as follows: TDN inhibits the acute rejection of heart transplantation.

[0036] In this experiment, a mouse allogeneic acute rejection model was established using 6-8 week-old C57BL / 6 and BALB / c mice. The specific method was as follows: BALB / c mice were anesthetized with 100 mg / kg of 1% sodium pentobarbital intraperitoneally and used as donors. A midline abdominal incision was performed to expose the inferior vena cava and inject heparinized saline. The ribs were cut open to open the chest, and the aortic arch and pulmonary artery were exposed. The superior vena cava and part of the right atrial appendage were ligated and then distally divided. The ascending aorta and pulmonary artery were divided proximal to the aortic arch and pulmonary artery bifurcation. The pulmonary portal vein and inferior vena cava were ligated, and the donor heart was removed. Anesthetized C57BL / 6 mice were used as recipients. A midline abdominal incision was performed to expose the abdominal aorta and inferior vena cava. The abdominal aorta and inferior vena cava were occluded below the renal vascular openings. A longitudinal incision was made in the anterior abdominal aorta. The recipient abdominal aorta was anastomosed end-to-end to the ascending aorta of the donor heart. The recipient inferior vena cava and the donor heart and pulmonary arteries were then anastomosed end-to-end. After removing the clamp, observe whether the atria and coronary arteries are filled with blood and whether the donor heart has resumed beating. Suture the abdominal incision. Palpate the beating of the donor heart daily after surgery and compare the survival time of the donor hearts in each group. Alternatively, 7 days after surgery, the transplanted hearts of the recipient mice were harvested and pathological changes were observed by hematoxylin-eosin (H&E) staining and CD3 expression by immunofluorescence. + Infiltration of T lymphocytes.

[0037] This experiment was divided into a blank group (control group), a model group (model group), and a TDN intervention group (3.2 mg / kg TDN, once every three days). The blank group used BALB / c mice as donors and recipients, and the other groups used BALB / c mice as donors and C57BL / 6 mice as recipients. The TDN intervention group started to receive medication from the day before transplantation. Figure 4As shown in the figure, on the 7th day after transplantation, the transplanted hearts of all mice in the control group could be palpated completely. The survival time of the transplanted hearts in the TDN-treated group was significantly prolonged. The first transplanted heart began to stop beating on the 20th day. After 50 days after transplantation, 70% of the mice still had beating transplanted hearts. After 86 days after transplantation, the last transplanted heart stopped beating. Figure 5 As shown, on the 7th day after transplantation, the transplanted hearts of the control group and the TDN treatment group were taken for examination at the same time. It was found that the transplanted hearts of the blank group were red, with no obvious adhesion to the surrounding tissues; the transplanted hearts of the model group were swollen, dark red, and obviously adhered to the surrounding tissues; the transplanted hearts of the TDN intervention group were slightly swollen, slightly red, and not obviously adhered to the surrounding tissues. H&E staining showed that: there was no lymphocyte infiltration in the blank group, the myocardial structure was intact, there was no myocardial cell necrosis and myocardial interstitial edema, and the vascular endothelium was basically undamaged; diffuse lymphocyte infiltration, myocardial interstitial edema, myocardial cell hemorrhage and necrosis, and vascular endothelial damage were observed in the model group; there was a small amount of lymphocyte infiltration in the TDN intervention group, slight myocardial interstitial edema, and mild vascular endothelial damage. Immunofluorescence results showed that there was no or very little CD3 in the blank group. + T lymphocyte infiltration; a large number of CD3 + T lymphocytes; the infiltrating T lymphocytes in the TDN intervention group were less.

[0038] like Figure 6 As shown in Figure A, in vitro experiments showed that TDN inhibited the proliferation and cytokine secretion of activated T lymphocytes, and this inhibitory effect was dose-dependent. Figure 6 As shown in Figure B, stimulation of mouse spleen lymphocytes with anti-CD3 / anti-CD28 antibodies can induce strong cell proliferation and release of a large amount of cytokines. The addition of TDN to the stimulation system can produce a significant inhibitory effect on proliferation at a concentration of 3.9 to 125 nM, and can inhibit the secretion of Th1 (IL-2, TNF-α and IFN-γ), Th2 (IL-4, IL-6 and IL-10) and Th17 (IL-17) cytokines after stimulation at a concentration of 3.9 to 62.5 nM.

[0039] Conclusion: TDN has a good inhibitory effect on T cell-mediated acute organ transplant rejection and can effectively prolong graft survival. Its mechanism is that the compound can inhibit the proliferation of T lymphocytes and reduce the secretion of inflammatory factors, which is of great significance for the research and development of immunosuppressants.

[0040] It will be easily understood by those skilled in the art that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An alkaloid for use in the preparation of a T cell immunosuppressant, wherein the T cell immunosuppressant is a drug for acute rejection in allogeneic heart transplantation, characterized in that: The structural formula of the alkaloid is shown below:

2. The use according to claim 1, characterized in that The alkaloids are used to inhibit the proliferation of T lymphocytes.

3. The use according to claim 2, characterized in that The T lymphocytes are CD3 + T lymphocytes.

4. The use according to claim 1, wherein The alkaloids are used to reduce the secretion of inflammatory factors.

5. The use according to claim 4, characterized in that The inflammatory factors are Th1 inflammatory factors, Th2 inflammatory factors and / or Th17 inflammatory factors.

6. The use according to claim 5, characterized in that The Th1 type inflammatory factors are IL-2, TNF-α and IFN-γ.

7. The use according to claim 5, characterized in that The Th2 type inflammatory factors are IL-4, IL-6 and IL-10.

8. The use according to claim 5, characterized in that The Th17 type inflammatory factor is IL-17.

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