Application of alkaloid in preparation of T cell immunosuppressor

Abstract

The invention relates to application of alkaloid in preparation of a T cell immunosuppressor, and belongs to the technical field of application of natural products. The invention discloses an application of a natural product alkaloid of zephyranthes candida in preparation of a T cell immunosuppressor, especially a medicine for acute rejection in allogeneic transplantation, and more particularly a medicine for acute rejection in allogeneic heart transplantation. The alkaloid is used for inhibiting proliferation of T lymphocytes and reducing secretion of inflammatory factors, wherein the T lymphocytes are CD3 + T lymphocytes; the inflammatory factors are a Th1 type inflammatory factor, a Th2 type inflammatory factor and / or a Th17 type inflammatory factor, and the Th1 type inflammatory factor is IL-2, TNF-alpha and IFN-gamma; the Th2 type inflammatory factors are IL-4 (Interleukin-4), IL-6 (Interleukin-6) and IL-10; the Th17 type inflammatory factor is IL-17 (Interleukin-17).

Description

Technical Field

[0001] The present invention belongs to the technical field of application of natural biomaterials, and more specifically, relates to the application of an alkaloid in preparing a T cell immunosuppressant, and in particular 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 is still one of the main factors that affect the normal function of the transplant and the long-term survival of the recipient after heart transplantation, which seriously restricts the development of heart transplantation. By inhibiting acute rejection, the survival of the transplant can be better prolonged. Therefore, how to reduce or avoid rejection is a major problem that urgently needs to be solved in clinical practice. Immunosuppressants are a class of drugs that have an inhibitory effect on the body's immune function. Their main function is to inhibit the body's abnormal immune response, thereby achieving the purpose 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 the current immunosuppressant regimen. Their main mechanism of action is to inhibit calcineurin, thereby inhibiting the activation of T cells and producing immunosuppressive effects; 2. Rapamycin inhibitors represented by sirolimus and everolimus, which can block the synthesis of immune cell protein DNA induced by IL-2 and inhibit the proliferation of immune cells by binding to tacrolimus binding protein-12 (FKBP-12) and then binding to mammalian target of rapamycin (mTOR), thereby exerting an anti-immune rejection effect; 3. Antimetabolites represented by mycophenolic acid drugs mainly inhibit the proliferation and differentiation of lymphocytes by reversibly inhibiting non-competitive hypoxanthine mononucleotide dehydrogenase. Since T cell-mediated immune response plays an important role in acute immune rejection, the first-line maintenance therapy immunosuppressants currently used in clinical practice are mainly aimed at T cell activation.

[0003] However, the above-mentioned anti-rejection drugs all have many shortcomings, such as insufficient efficacy in some patients, high drug prices, and easy drug resistance. In addition, the above-mentioned immunosuppressants also have relatively serious adverse drug reactions. Among them, tacrolimus can cause hypertension, angina pectoris, palpitations, pericardial effusion, and pleural effusion; cyclosporine has strong liver and kidney toxicity, allergic reactions, hirsutism, hyperbilirubinemia and other adverse reactions; rapamycin can cause headaches, nausea, dizziness, nosebleeds, joint pain, thrombocytopenia, leukopenia, hypertriglyceridemia, hypercholesterolemia, hyperglycemia, elevated lactate dehydrogenase and hypomagnesemia; and mycophenolate mofetil not only has a large gastrointestinal reaction, but may also induce tumors. Therefore, it is imperative to develop new, efficient and low-toxic T cell immunosuppressants.

[0004] Natural products are the most common source of drugs. According to statistics, from the 1980s to the present, 30-40% of the drugs marketed each year are directly or indirectly derived from natural products. In 2010, the proportion of drugs from natural product-related sources reached 50%. The types of plant secondary metabolites are extremely rich. With the increasing research on them in recent years, more and more active compounds have been discovered, laying a certain foundation for the development of new drugs. The whole herb with bulbs of Zephyranthes candida is a folk herbal medicine that has the effects of calming the liver, calming the heart, extinguishing wind and calming down. It is mainly used to treat infantile convulsions and epilepsy. (+)-trans-dihydronarciclasine (hereinafter referred to as TDN) is the main component of Zephyranthes candida, but there are few studies on its biological activity. Only a small number of literatures show that the compound has anti-tumor activity, and no research has been carried out on its T cell immunosuppressant. In addition, there are no reports of (+)-trans-dihydronarciclasine and its analogs being marketed as immunosuppressants at home and abroad. To address this issue, the inventors conducted in vivo experiments using an acute rejection model of allogeneic heart transplantation mediated by alloreactive T cells, and explored the immunosuppressive effect of TDN on T cells through an in vitro model of T cell receptor-specific stimulation and mixed culture of allogeneic lymphocytes, indicating that TDN can be used to suppress acute rejection reactions in allogeneic organ transplantation 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 that T cell immunosuppressants have insufficient efficacy, high drug prices, are prone to drug resistance, and are prone to 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 an application 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-type inflammatory factors, Th2-type inflammatory factors and / or Th17-type inflammatory factors.

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

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

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

[0017] In general, the above technical solution conceived by the present invention has the following technical advantages compared with the prior art:

[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. The 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, and provides candidate drug molecules for the treatment of related diseases and the development of new drugs.

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

[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.

[0025] Figure 4 To inhibit the acute rejection of 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 purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain 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 can be combined with each other as long as they do not conflict with each other.

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

[0030]

[0031] Compound 1: (+)-trans-dihydronarciclasine (abbreviated as TDN), extracted from Zephyranthes candida, with a purity of >99.0% (Formula 1), the main extraction steps are: chop the Zephyranthes candida bulb, dry it, and crush it to obtain Zephyranthes candida bulb powder, and then extract it with 95% ethanol in a multifunctional extraction tank at 40°C for 4 times, and then concentrate it under reduced pressure to obtain a crude Zephyranthes candida extract. After the total extract is suspended in warm water, dilute hydrochloric acid is added to adjust the pH to 2, and then it is extracted with chloroform to obtain a chloroform part with a pH of 2. After the acidic water layer is adjusted to pH 10 with ammonia water, it is extracted with chloroform to obtain a chloroform part with a pH of 10. The pH value of the chloroform part extract is adjusted to acidic and then applied to a molecular sieve chromatography column; sugars in the molecular sieve chromatography column are removed; the molecular sieve chromatography column is eluted with an ethanol solution containing ammonia water, and the eluate is collected and concentrated to obtain TDN.

[0032] The high-resolution mass spectrometry ( Figure 1 ), and its molecular formula is estimated 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 are almost completely consistent with the known compound trans-dihydronarciclasine (Chemistry Select, 2016, 1, 5895–5899; Tetrahedron, 2018, 74, 5752–5757; Journal of Natural Products, 2017, 80, 1909–1917), proving that the planar structure and relative configuration of TDN are consistent with trans-dihydronarciclasine. Since trans-dihydronarciclasine has two enantiomers, (+)-trans-dihydronarciclasine and (–)-trans-dihydronarciclasine, and calculating ECD 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 the effect of compound 1 on acute rejection of cardiac transplantation in an in vivo model and found that the compound can effectively prolong the survival time of the transplant and inhibit the infiltration of alloreactive T cells; through an in vitro model of T cell receptor-specific stimulation and mixed culture of allogeneic lymphocytes, the inventors found that the compound can effectively inhibit the proliferation, activation and cytokine secretion of T cells. The compound can be directly used as a drug or lead compound for the development of drugs for acute rejection in allogeneic organ transplantation.

[0034] Example 1

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

[0036] In this experiment, 6-8 week old C57BL / 6 and BALB / c mice were used to establish a mouse allogeneic acute rejection model. The specific method is as follows: BALB / c mice were anesthetized by intraperitoneal injection of 100 mg / kg of 1% sodium pentobarbital and used as donors. A midline incision was made in the abdomen to expose the inferior vena cava and inject heparin saline. The ribs were cut open to open the chest to expose the aortic arch and pulmonary artery. The superior vena cava and part of the right atrial appendage were then ligated and cut off at the distal end. The ascending aorta and pulmonary artery were cut off at the proximal end of the aortic arch and the pulmonary artery bifurcation. The pulmonary portal vein and inferior vena cava were ligated and the donor heart was removed. C57BL / 6 mice were used as recipients. After anesthesia, a midline incision was made in the abdomen to expose the abdominal aorta and inferior vena cava. The abdominal aorta and inferior vena cava below the opening of the renal vessels were blocked. A longitudinal incision was made on the anterior wall of the abdominal aorta. The recipient abdominal aorta was anastomosed end to end with the ascending aorta of the donor heart, and then the recipient inferior vena cava and the donor heart and pulmonary artery were anastomosed end to end. After removing the vascular clamp, observe whether the atria and coronary arteries are filled with blood and whether the donor heart has resumed beating. Suture the abdominal incision. Touch the beating of the donor heart every day after surgery and compare the survival time of the donor hearts in each group. Or 7 days after surgery, the transplanted hearts of the recipient mice were obtained and the pathological changes were observed by hematoxylin-eosin (H&E) staining and CD3 by immunofluorescence. + Infiltration of T lymphocytes.

[0037] This experiment was divided into a control group, a model group, and a TDN intervention group (3.2 mg / kg TDN, once every three days). The control 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 was given medication starting 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 touched to have completely stopped beating. The survival time of the transplanted hearts in the TDN-treated group was significantly prolonged. The first transplanted heart stopped beating on the 20th day. After 50 days after transplantation, 70% of the mice still had transplanted hearts beating. 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 examined 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 enlarged, dark red, and obviously adhered to the surrounding tissues; the transplanted hearts of the TDN intervention group were slightly enlarged, 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 B, stimulating mouse spleen lymphocytes with anti-CD3 / anti-CD28 antibodies can induce strong cell proliferation and release of a large amount of cytokines. Adding 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 rejection of organ transplantation 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 only 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 protection scope of the present invention.

Claims

1. An application of an alkaloid in preparing a T cell immunosuppressant, characterized in that: The structural formula of the alkaloid is shown below:

2. The use according to claim 1, characterized in that The T cell immunosuppressant is a drug for acute rejection in allogeneic transplantation.

3. The use according to claim 2, characterized in that The acute rejection drug in allogeneic transplantation is an acute rejection drug in allogeneic heart transplantation.

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

5. The use according to claim 4, characterized in that The T lymphocytes are CD3 + T lymphocytes.

6. The use according to claim 1, characterized in that The alkaloids are used to reduce the secretion of inflammatory factors.

7. The use according to claim 6, characterized in that The inflammatory factors are Th1 type inflammatory factors, Th2 type inflammatory factors and / or Th17 type inflammatory factors.

8. The use according to claim 7, characterized in that The Th1 type inflammatory factors are IL-2, TNF-α and IFN-γ.

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

10. The use according to claim 7, characterized in that The Th17 type inflammatory factor is IL-17.

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