WO2017157131A1 - 绿原酸在制备治疗以lag-3为靶点的疾病的药物中的用途 - Google Patents
绿原酸在制备治疗以lag-3为靶点的疾病的药物中的用途 Download PDFInfo
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Definitions
- the present invention belongs to the technical field of biomedicine, and more particularly to the use of chlorogenic acid for the preparation of a medicament for treating a disease targeting LAG-3.
- Chrrogenic acid CA also known as caffeic acid, is a phenolic acid formed by the condensation of caffeic acid and quinic acid.
- the chemical name is 3-o-caffeoyl quinic acid. (3-o-caffeoylquinic acid CA).
- Chlorogenic acid is a kind of phenylpropanoid synthesized by plants in the process of aerobic respiration through the pentose phosphate pathway intermediate. Chlorogenic acid has been developed into food, health care products, cosmetics and pharmaceuticals. field. As a natural small molecule monomer derived from plants, previous studies have shown that chlorogenic acid has a wide range of biological activities such as anti-oxidation, anti-inflammatory, anti-viral and anti-tumor, and exerts the above effects on which target it acts on. There are no research conclusions yet. Finding the target of chlorogenic acid, clarifying the pathway of the target and the corresponding regulatory site, is of great significance for further extensive application and precise treatment of chlorogenic acid.
- Lymphocyte-activation also known as CD233, is a cell membrane protein belonging to the immunoglobulin superfamily, an inhibitory molecule expressed on the surface of T lymphocytes. Studies have shown that LAG-3 functions on T cells. The regulation plays an important role in regulating the proliferation and activation of T lymphocytes, effector T lymphocytes, etc., and is associated with immune disorders in various disease states. The development of therapeutic drugs targeting LAG-3 may have broad application prospects.
- the present invention discloses the use of chlorogenic acid for the preparation of a medicament for inhibiting a LAG-3 target; the present invention further discloses the use of chlorogenic acid for the preparation of a medicament for treating a disease targeting LAG-3.
- chlorogenic acid can significantly act on a LAG-3 target, inhibit its content and expression, and inhibit genes, mRNAs, and proteins corresponding to LAG-3, thereby being applicable to the treatment of related diseases.
- LAG-3 is closely related to tumors. It has been shown that LAG-3 has inhibitory effects on CD4 + T and CD8 + T lymphocytes, which leads to tumor immune tolerance, promotes tumor growth and development, and inhibits LAG-3 has become a frontier research in anti-tumor immunotherapy. LAG-3 is also closely related to the virus.
- the pathogenesis of chronic hepatitis B is mainly an immunopathological reaction.
- the persistent infection of HBV is directly related to the low lymphocyte response function.
- LAG-3 is associated with the progression of chronic hepatitis B disease. Sex, LAG-3 in patients with chronic hepatitis B disease will have a positive effect on the disease.
- LAG-3 is also associated with sepsis.
- Sepsis is an inflammatory reaction and immune response that causes allergies in the body due to trauma, infection, etc., and then causes serious harm to the body.
- the mortality rate is high, and its incidence is high.
- the process is closely related to the functional inhibition of the immune system.
- Recent studies have shown that the high expression of LAG-3 on T lymphocytes in patients with sepsis is associated with a poor prognosis of this disease. Inhibition of LAG-3 may be treatment. New ideas for sepsis.
- chlorogenic acid can promote the proliferation of CD4+ T lymphocytes in experimental mice, and clear in the research and exploration of chlorogenic acid indications, chlorogenic acid pairs Many diseases have potential therapeutic value. Based on the fact that chlorogenic acid acts on CD4+ T lymphocytes and the indications corresponding to chlorogenic acid, the inventors gradually narrowed the research scope in the research process, and finally verified that chlorogenic acid directly acts on a large number of in vitro and in vivo experiments. LAG-3 target and inhibit LAG-3 The facts are expressed and chlorogenic acid is used as a drug in the treatment of related diseases targeting LAG-3.
- Chlorogenic acid is a natural substance that exists in nature.
- the inventors have applied chlorogenic acid as a drug monomer to clinical trials for the first time in the world (current clinical trials are underway, clinical trial approval number 2013L01855), green
- the original acid has a wide range of biological activities and has many pathways. It is necessary to clarify its true target of action in order to determine its possible application direction, and then screen out the population suitable for chlorogenic acid, and reversely promote some unknown pathogenesis. Progress in disease research.
- chlorogenic acid can directly act on the LAG-3 target and inhibit the expression of LAG-3 and LAG-3 proteins, thereby targeting LAG- including malignant tumors, viral infectious diseases and sepsis. 3 treats the target disease.
- chlorogenic acid can significantly act on LAG-3 target and inhibit it in cell lines with high expression of LAG-3; chlorogenic acid-containing drugs can significantly reduce peripheral blood of model mice. LAG-3 expression rate in lymphocytes.
- chlorogenic acid can significantly promote the proliferation of CD3+ T lymphocytes and CD4+ T lymphocytes, inhibit the expression of LAG-3 protein in tumors, and further have a therapeutic effect on tumors.
- chlorogenic acid can significantly reduce the alanine aminotransferase (ALT) content in the blood, thereby producing a therapeutic effect on viral infectious diseases.
- Chlorogenic acid can also treat LAG-3-associated sepsis by inhibiting the LAG-3 target.
- the drug is chlorogenic acid as an active ingredient and is added to the pharmacy.
- a preparation prepared from acceptable excipients or auxiliary ingredients.
- gastric floating tablets are mainly chlorogenic acid, hydroxypropyl cellulose as the backbone material, stearic acid as the bleaching agent, sodium bicarbonate as the foaming agent and microcrystalline cellulose and lactose. It is prepared for the filler to further improve the oral bioavailability of chlorogenic acid, and thus has a more obvious therapeutic effect on the disease targeting LAG-3.
- chlorogenic acid is prepared into a gastric floating tablet by a suitable method to further improve the oral bioavailability of chlorogenic acid.
- Fig. 1 shows the results of detecting the content of LAG-3 in each group of cells in Example 1.
- Fig. 2 shows the results of detection of the expression level of LAG-3 gene in each group of cells in Example 1.
- Figure 3 shows the expression level of LAG3 on peripheral blood lymphocytes of each group of mice in Example 2.
- Fig. 4 is a graph showing the LAG-3 expression rate (%) in peripheral blood lymphocytes of each group of mice in Example 2.
- Figure 5 shows the tumor volume after treatment of each group of glioma in situ mice in Example 3.
- FIG. 6 shows the results of immunohistochemistry of LAG-3 protein expression in brain tissue of different groups of mice in Example 3.
- Figure 7 shows the percentage of peripheral blood T lymphocytes in the saline control group and the chlorogenic acid treated group in Example 3.
- Figure 8 shows the levels of ALT in peripheral blood at different time points in each group of mice in Example 4.
- Figure 9 is a graph showing the survival rate and production time of the pharmacodynamic experiment of sepsis mice in Example 5.
- Fig. 10(a) shows the expression rate of LAG-3 in peripheral blood lymphocytes of mice in Example 5;
- Fig. 10(b) shows the ratio of CD4+T and CD8+T in peripheral blood of mice in Example 5.
- Chlorogenic acid drug substance Chlorogenic acid drug substance, cell culture dish, carbon dioxide incubator, RNAprep Pure cultured cell/bacterial total RNA extraction kit, cDNA first strand synthesis kit, fluorescence quantitative RCR instrument, human extracted from Eucommia ulmoides leaves with a purity of 99.2% Lymphocyte activation gene 3 detection kit, microplate reader, and the like.
- CLL cells and Jurkat E6-1 cells in good phase growth were inoculated into 6-well plates at a density of 5 ⁇ 105/mL, and 4 cells were set for each cell, and 3 replicate wells per group were administered.
- Three chlorogenic acid administration groups were administered with blank 1640 medium to dissolve the drug according to the required drug concentration (1 mL), and the blank control group was given the same volume of blank 1640 medium.
- the specific grouping and administration schedules are as follows. 1 shows:
- LAG-3 activity in cells of each experimental group was detected by an ELISA experiment using a human lymphocyte activating gene 3 detection kit. After the cells of each group were treated with the corresponding drugs for 48 hours, the cell culture medium of each group was collected and divided into two parts, one of which was taken, and the protein was quantified by BCA method, and the human lymphocyte activation gene 3 detection kit was used. The LAG-3 activity in each group of cells was measured, and the LAG-3 content of each group was converted.
- the total RNA in the cells was extracted using the RNA prep Pure cultured cell/bacteria total RNA extraction kit (centrifugal column type). The procedure was carried out according to the instructions. The specific extraction steps are as follows:
- step 2.2 Take another cell culture solution remaining after the equalization in step 2.2, add 200 ⁇ l of lysate RL pre-added with ⁇ -mercaptoethanol;
- the adsorption column After separately washing the adsorption column with the deproteinized solution and the rinsing liquid, the adsorption column is placed in the collection tube, and the residual liquid above is sufficiently evaporated;
- the corresponding first strand cDNA is synthesized using the cDNA first strand synthesis kit and the above extracted RNA solution.
- the specific reverse transcription process follows the instructions and is briefly described as follows:
- the tube was gently mixed and briefly centrifuged, and the PCR apparatus was incubated at 42 ° C for 50 min, followed by heating at 95 ° C for 5 min. Finally, 30 ul of RNase-Free ddH2O was added to the obtained cDNA solution and diluted to 50 ul to obtain a first strand cDNA.
- the EvaGreen fluorescent dye binds to double-stranded DNA to generate strong fluorescence, the total amount of DNA produced by the reaction can be obtained by detecting the final fluorescence intensity.
- the ⁇ -actin primer set was set to a relative value for each sample, and relative quantification was performed.
- the LAG-3 content in each group of cells was detected by ELISA.
- the results of the experiment showed that the CLL cells and Jurkat E6-1 cells in the blank group had higher LAG-3 content than the CEM-NKR cells in the control group, and the CLL and Jurkat E6-1 described in the literature were highly expressed LAG- 3 fine The cell lines were consistent.
- the content of LAG-3 in CLL cells and JurkatE6-1 cells decreased, and the degree of decline was compared with the amount of chlorogenic acid. Relevance.
- the specific experimental results are shown in Fig. 1.
- the high dose group, the middle dose group and the low dose group respectively refer to the content of LAG-3 of different cells after high, medium and low doses of chlorogenic acid.
- LAG-3 gene in each group of cells was detected by RT-PCR.
- the results of the experiment showed that the CLL cells and Jurkat E6-1 cells in the blank group had higher LAG-3 gene expression level than the CEM-NKR cells in the control group, and the CLL and Jurkat E6-1 described in the literature were highly expressed LAG. -3 cell lines were consistent.
- the expression level of LAG-3 gene in CLL cells and Jurkat E6-1 cells decreased significantly, and the degree of decline was correlated with the amount of chlorogenic acid.
- the expression level of LAG-3 gene in CHCA group (CLL cell chlorogenic acid high dose group), CMCA group (CLL cell chlorogenic acid middle dose group) and CMCA group (CLL cell chlorogenic acid low dose group)
- CB group CLL blank control group
- JHCA group Jurkat E6-1 cell chlorogenic acid high dose group
- JMHC group JurkatE6- 1 cell chlorogenic acid medium dose group
- JMHC group JurkatE6-1 cell chlorogenic acid low-dose group
- the expression level of LAG-3 gene was significantly decreased, with significant difference (*p ⁇ 0.05 ).
- the specific experimental results are as follows. As shown in Fig. 2, the high-dose group, the middle-dose group and the low-dose group respectively refer to the expression levels of LAG-3 gene in different cells after high, medium and low doses of chlorogenic acid.
- CLL cells and Jurkat E6-1 cells with high expression of LAG-3 were used as model cells, and CEM-NKR cells were used as control cells to visualize the LAG-3 of chlorogenic acid and CLL and Jurkat E6-1 cells. Expression levels were measured and expression of LAG-3 was detected from both levels of content and gene expression levels. The results showed that chlorogenic acid can significantly act on the LAG-3 target and inhibit it in the cell line with high expression of LAG-3.
- the acute T lymphocyte leukemia model mice were randomly divided into three groups, which were set as saline control group, LAG-3 antibody positive control group and injection chlorogenic acid injection group, with 6 mice in each group. According to Table 4, different groups of drugs were arranged, and the experiment was terminated after continuous intravenous injection for 15 days. The first injection was recorded as the first day, and the eyeballs of the mice were taken on the 16th day of the experiment, and the blood samples were processed and separated. Lymphocytes were stained with LAG-3PE antibody, and the expression of LAG-3 in peripheral blood cells of each group was detected by flow cytometry.
- the LAG-3 indicated by peripheral blood lymphocytes of each group of mice was detected by flow cytometry, and it was found that the LAG-3 antibody group (LAG3Ig group) and chlorogenic acid for injection were compared with the blank control group (group B).
- the group (CA group) could significantly down-regulate the expression of LAG-3 on peripheral blood lymphocytes of mice, and there was no significant difference between them (p>0.05).
- the specific experimental results are shown in Figure 3.
- the statistics are shown in Figure 4.
- LAG-3 is an inhibitory molecule present on lymphocytes.
- mice with acute T lymphocyte leukemia were used as models to investigate the effect of chlorogenic lyophilized powder injection on mice by flow cytometry.
- LAG-3 expression in peripheral blood lymphocytes of mice were used as models to investigate the effect of chlorogenic lyophilized powder injection on mice by flow cytometry.
- the results showed that the chlorogenic acid lyophilized powder injection can significantly reduce the expression rate of LAG-3 in peripheral blood lymphocytes of model mice, and the decrease of LAG-3 is close to that of LAG-3 antibody.
- Chlorogenic acid was extracted and purified from Eucommia ulmoides leaves, and the purity was about 99.52%.
- GL261 glioma cells in logarithmic growth phase were prepared into a single cell suspension of GL261 at a concentration of 5 ⁇ 105/ ⁇ L, and anesthetized with 0.75% pentobarbital sodium in an intraperitoneal injection of 6 uL/g, and the puncture site was scraped off.
- the mouse head was fixed with an ALC-H mouse brain stereotactic instrument, and the epidermis was cut longitudinally. 10 mL of GL261 single cell suspension was injected with a micro pump at a rate of 2.5 uL/min, the needle eye was closed with bone wax, and the scalp incision was carefully sutured, and a total of 35 mice were inoculated, leaving 5 normal mice.
- C57BL/6 mice inoculated with GL261 glioma cells were randomly divided into three groups: saline control group (NC group), chlorogenic acid injection treatment group (CA group) and temozolomide ( TMZ) positive control group.
- NC group saline control group
- CA group chlorogenic acid injection treatment group
- TMZ temozolomide
- the CA group received intraperitoneal injection of chlorogenic acid 40 mg/kg per day
- the NC group received intraperitoneal injection of the same volume of normal saline
- the TMZ treatment group received 20 mg/kg of temozolomide.
- the administration was continued for 7 days, and the specific animal grouping and administration schedule are shown in Table 5.
- this experiment uses a small animal nuclear magnetic imaging instrument to scan, observe the size of the tumor in situ in the mouse brain, and calculate the tumor volume.
- this experiment used immunohistochemistry to treat the mice in situ as lesions. Specimens were added to the brain tissue of normal mice as specimens to examine the expression of LAG-3 protein.
- LAG-3 is an inhibitor of T lymphocyte proliferation, which negatively regulates the proliferation of T lymphocytes, which adversely affects the prognosis of tumors and promotes the development of tumors. Detection of the proliferation of T lymphocytes can reflect the strength of LAG-3 inhibition.
- the peripheral blood of each group of mice was collected by eyeball extraction, and high-purity CD3+ T cells (>97%) were obtained by CD3 negative selection kit, and stained with LAG-3-PE to detect LAG- 3 expression.
- LAG-3 is an inhibitor of T lymphocytes and has a negative regulatory effect on the proliferation of T lymphocytes.
- the inhibition of LAG-3 should promote the proliferation of T lymphocytes.
- the percentage of CD3+ T lymphocytes and CD4+ T lymphocytes in peripheral blood lymphocytes of the two groups of mice in the saline control group and the chlorogenic acid treatment group was determined.
- the results showed that the chlorogenic acid treatment group significantly promoted the proliferation of mouse CD3+ T lymphocytes and CD4+ T lymphocytes compared with the saline control group.
- the experimental results are shown in Fig. 7.
- LAG-3 and tumor are mainly reflected in its ability to inhibit the proliferation of T lymphocytes, thereby making the tumor immune tolerance and promoting tumor development.
- GL261 in situ glioma mice were used as a model to further confirm the efficacy of chlorogenic acid injection in the treatment of glioma, further confirming the relationship between LAG-3 and tumor and peripheral blood T lymphocytes. Correlation, it was confirmed that chlorogenic acid can inhibit the expression of LAG-3 protein in glioma, and this inhibition is on the proliferation of peripheral blood lymphocytes. Can also be significantly reflected.
- mice transfected with HBV virus were randomly divided into 3 groups, 10 in each group, which were saline control group (NC group), hepatitis B immunoglobulin positive control group (HBVIG group) and green.
- NC group saline control group
- HBV group hepatitis B immunoglobulin positive control group
- CA group acid gastric floating tablets treatment group
- mice were negative control group. Specific grouping and dosing schedules are shown in Table 7.
- NC group Amount of administration Number of doses Mode of administration Saline control group (NC group) Equal volume of saline 20 Oral gavage Chlorogenic acid gastric floating tablets treatment group (CA group) 60mg/kg 20 Oral gavage Hepatitis B immunoglobulin (HBVIG group) 9IU/only 2 Intramuscular injection Normal control mice (N group) Equal volume of saline 20 Oral gavage
- ALT alanine aminotransferase
- Alanine aminotransferase is directly related to the prognosis of hepatitis. Its content can reflect the therapeutic effect of the drug. Therefore, the ALT content in peripheral blood of each group of mice at different time points after treatment was tested. The ALT was quantitatively determined by an automatic biochemical analyzer. The therapeutic effect of chlorogenic acid gastric floating tablets on hepatitis B was evaluated by comparing the ALT values between the treatment groups and the treatment group and the control group. Specifically, on the 0th, 4th, 8th, 12th, 16th, and 20th day, peripheral blood was taken from the tail vein of the mouse, and blood was used as a specimen for detection.
- the target of chlorogenic acid is LAG-3.
- the frequency of LAG-3 distribution in peripheral blood of each group of mice was detected. That is, on the second day of the experiment (ie, the 21st day of the experiment), the peripheral blood of the mice was obtained by eyeball removal, and then the expression of LAG-3 in the peripheral blood of each group of mice was detected by flow cytometry.
- mice with sepsis induced by CLP were used, and 10 normal balb/c mice.
- LAG-3 (CD223)-PE antibody CD8-PE-Cy7 antibody
- CD4-APC antibody etc.
- mice of sepsis model were randomly divided into 3 groups, 10 in each group, which were model mouse control group (NC group), chlorogenic acid gastric floating tablet treatment group (CA group) and LAG-3 antibody.
- NC group model mouse control group
- CA group chlorogenic acid gastric floating tablet treatment group
- LAG-3 antibody LAG-3 antibody
- N group another 10 normal balb/c mice were used as a negative control group (N group).
- the LAG3IG group was intramuscularly injected on the first day of the experiment and the third day of the experiment, and the CA group was intraperitoneally injected daily for a total of continuous administration.
- NC group and N group were given oral administration of equal volume of normal saline in the CA group.
- the survival rate and survival time of the mice were recorded from the beginning to the 10th day of the experiment. All mice were removed from the eyeball on the 10th day of the experiment. Whole blood is divided into two parts, spare. Specific grouping and dosing schedules are shown in Table 8.
- NC group Amount of administration Number of doses Mode of administration Saline control group (NC group) Equal volume of saline 7 Oral gavage Chlorogenic acid gastric floating tablets treatment group (CA group) 60mg/kg 7 Oral gavage LAG-3 monoclonal antibody group (LAG3IG group) 10IU/only 2 Intramuscular injection Normal control mice (N group) Equal volume of saline 20 Oral gavage
- LAG-3 is an inhibitory molecule present on T lymphocytes and has a significant effect on it.
- Add 1 ⁇ l of each antibody then add 100 ⁇ L of mixed blood to each tube, mix it with the vulgar vortex and mix it with blood for 3 s, then react at room temperature for 20 min in the dark, then add 2 mL of erythrocyte lysate, leave it at room temperature for 15 min, and wash with PBS. After 2 times, the flow cytometer was entered for detection.
- LAG-3 on peripheral blood lymphocytes of each group showed that the expression level of LAG-3 was lower in the normal control group (N group) (10a), and the corresponding amount of CD4+T and CD8+ T lymphocytes.
- N group normal
- NC group sepsis model control group
- LAG-3 expression was significantly increased (10a), and the corresponding CD4+T and CD8+ T lymphocytes were significantly decreased (10b).
- the expression of LAG-3 in septic mice was significantly lower than that in the model control group (NC group) (10a), and the corresponding CD4+T and CD8+T Lymphocytes also showed significant proliferation (10b), and the specific experimental results are shown in Figures 10a and 10b.
- LAG-3 antibody was used as a positive control, and the therapeutic effect of chlorogenic acid gastric floating tablets on septic mice was examined.
- Chlorogenic acid has a significant effect on mouse sepsis, and this effect is reflected in the effective inhibition of LAG-3 target on the peripheral blood lymphocytes of mice, corresponding CD4+T and CD8+T in peripheral blood. Lymphocytes have a significant proliferation. Further, chlorogenic acid can treat LAG-3-associated sepsis by inhibiting the LAG-3 target.
- Chlorogenic acid was extracted and purified from Eucommia ulmoides leaves, and the purity was 98.82%.
- chlorogenic acid is prepared into a gastric floating tablet by a suitable method to further improve the oral bioavailability of chlorogenic acid.
- Chlorogenic acid was extracted and purified from Eucommia ulmoides leaves, and the purity was 99.5%.
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Abstract
本发明提供绿原酸在制备抑制LAG-3靶点的药物中的用途,以及绿原酸制备治疗以LAG-3为靶点的疾病的药物中的用途。绿原酸通过抑制LAG-3可用于抗肿瘤、抗病毒和治疗脓毒症。此外,将绿原酸制成胃漂浮片剂,能提高绿原酸的口服生物利用度。
Description
本发明属于生物医药的技术领域,更具体地讲,涉及绿原酸在制备治疗以LAG-3为靶点的疾病的药物中的用途。
绿原酸(chlrogenic acid CA)又名咖啡鞣酸,是由咖啡酸(caffeic acid)和奎尼酸(quinic acid)缩合而成的缩酚酸,化学名为3-o-咖啡酰奎尼酸(3-o-caffeoylquinic acid CA)。
绿原酸是植物在进行有氧呼吸的过程中,经磷酸戊糖途径中间产物合成的一种苯丙素类物质,绿原酸已经被开发应用于食品、保健品、化妆品和药品等多个领域。作为来源于植物中的天然小分子单体物质,以往的研究显示,绿原酸具有抗氧化、抗炎、抗病毒和抗肿瘤等广泛的生物活性,对其作用于何种靶点发挥上述作用,目前尚无研究结论。找到绿原酸的作用靶点,明确该靶点所在通路和对应的调节位点,对于绿原酸的进一步广泛应用和精准治疗具有重大意义。
淋巴细胞活化基因3(lymphocyte-activation,LAG-3),又称CD233,是属于免疫球蛋白超家族的细胞膜蛋白,表达于T淋巴细胞表面的抑制性分子,研究表明LAG-3对T细胞功能的调节有重要作用,参与调节T淋巴细胞、效应T淋巴细胞等的增殖与活化,且与多种疾病状态下的免疫失调相关。以LAG-3为靶点的治疗药物的研发可能具有广阔的应用前景。
发明内容
为了解决现有技术中存在的问题,本发明的目的是提供一种绿原酸在抑制LAG-3靶点的用途。
本发明公开了绿原酸在制备抑制LAG-3靶点的药物中的用途;本发明进一步公开了绿原酸在制备治疗以LAG-3为靶点的疾病的药物中的用途。在本发明中,绿原酸能够显著地作用于LAG-3靶点,对其含量和表达产生抑制作用,并抑制LAG-3对应的基因、mRNA及蛋白,从而能够应用于相关疾病的治疗。
LAG-3与肿瘤有密切的相关性,已有研究显示LAG-3对CD4+T和CD8+T淋巴细胞均有抑制作用,从而导致肿瘤的免疫耐受性,促进肿瘤的生长和发展,抑制LAG-3已经成为抗肿瘤免疫治疗的前沿性研究。LAG-3与病毒也有密切的相关性,慢性乙型肝炎的发病机理主要是一种免疫病理反应,HBV持续感染与淋巴细胞应答功能低下有直接关系,LAG-3与慢性乙肝疾病的进展具有相关性,抑制慢性乙肝疾病患者的LAG-3将对疾病产生积极的作用。LAG-3与脓毒症同样也有相关性,脓毒症是由于创伤、感染等一些列因素引起机体过敏的炎症反应和免疫应答,继而对机体产生严重危害的疾病,死亡率很高,其发病过程与免疫***的功能抑制有密切关系,最新研究显示,LAG-3在脓毒症患者T淋巴细胞上的高表达,与这种疾病不良的预后具有相关性,抑制LAG-3,可能是治疗脓毒症的新思路。
之前的研究(包括专利内的实施例研究)都已经明确,绿原酸能够促进实验小鼠CD4+T淋巴细胞的增殖,并在绿原酸适应症的研究探索过程中明确,绿原酸对众多疾病具有潜在的治疗价值。基于绿原酸作用于CD4+T淋巴细胞的事实和绿原酸对应的适应症,发明人在研究过程中逐渐地缩小研究范围,最终通过大量的体内、外实验验证了绿原酸直接作用于LAG-3靶点并抑制LAG-3
表达的事实,并将绿原酸作为药物应用于以LAG-3为靶点的相关疾病的治疗中。
绿原酸是自然界中本身就存在的天然物质,发明人首次在全球范围内真正的将绿原酸作为药物单体应用于临床试验(目前临床试验正在进行中,临床试验批件号2013L01855),绿原酸具有广泛的生物活性且作用途径非常多,需要明确其真正的作用靶点,才能确定其可能的应用方向,进而筛选出适合使用绿原酸的人群,并且反向促进某些未知发病机理疾病的研究进展。
过往的药物开发大多在已知疾病发病机理的前提下,根据发病机理,人为地合成和机理相关的药物并使用该药物治疗相关疾病,而绿原酸作为存在于生物圈中的老物质,并不适用于以上的研发模式。发明人在不明确绿原酸作用机理和具体作用靶点的前提下,进行了大量的研究,最终确定绿原酸与LAG-3之间的密切相关性,这一关联的建立,具有非常重要的意义,其可以明显提高以绿原酸为主要有效成分的药物的有效性,从而真正地解决疾病问题。
发明人研究发现,绿原酸可以直接作用于LAG-3靶点并抑制LAG-3和LAG-3蛋白的表达,从而对包括恶性肿瘤、病毒感染性疾病和脓毒症在内的以LAG-3为靶点的疾病进行治疗。通过研究表明,在高表达LAG-3的细胞株中,绿原酸能够显著地作用于LAG-3靶点并对其产生抑制作用;含有绿原酸的药物能够显著地降低模型小鼠外周血淋巴细胞中的LAG-3表达率。并且,绿原酸可以显著地促进CD3+T淋巴细胞和CD4+T淋巴细胞的增殖,抑制LAG-3蛋白在肿瘤中的表达,进而对肿瘤产生治疗作用。此外,绿原酸可以明显地降低血液中的丙氨酸氨基转移酶(ALT)含量,进而对病毒感染性疾病产生治疗作用。绿原酸还可以通过抑制LAG-3靶点,治疗与LAG-3相关的脓毒症。
在本发明中,作为优选,所述的药物是以绿原酸为有效成分,加入药学上
可接受的辅料或辅助性成分制备而成的制剂。
具体地,本发明所述的制剂优选为胃漂浮片剂或散剂。其中,胃漂浮片剂是以绿原酸为主药、以羟丙基纤维素为骨架材料、以硬脂酸为助漂剂、以碳酸氢钠为起泡剂并以微晶纤维素和乳糖为填充剂制备而成,从而进一步提高绿原酸的口服生物利用度,进而对以LAG-3为靶点的疾病产生较为明显的治疗效果。
与现有技术相比,本发明发现了绿原酸对LAG-3靶点的抑制作用,并将其应用于抗肿瘤、抗病毒和脓毒症等以LAG-3为靶点的疾病的治疗;进一步地,采用适宜的方法将绿原酸制备成胃漂浮片剂,进一步提高绿原酸的口服生物利用度。
图1示出了实施例1中各组细胞中LAG-3的含量检测结果。
图2示出了实施例1中各组细胞中LAG-3基因表达水平的检测结果。
图3示出了实施例2中各组小鼠外周血淋巴细胞上的LAG3表达水平。
图4示出了实施例2中各组小鼠外周血淋巴细胞中的LAG-3表达率(%)。
图5示出了实施例3中各组脑胶质瘤原位小鼠治疗后的肿瘤体积大小。
图6示出了实施例3中不同组小鼠的脑组织中LAG-3蛋白表达的免疫组化结果。
图7示出了实施例3中生理盐水对照组和绿原酸治疗组的小鼠外周血T淋巴细胞百分比。
图8示出了实施例4中各组小鼠不同时间点外周血中ALT的含量。
图9示出了实施例5中脓毒症小鼠药效学实验的生存率和生产时间考察。
图10(a)示出了实施例5中小鼠外周血淋巴细胞LAG-3的表达率;图10(b)示出了实施例5中小鼠外周血中CD4+T和CD8+T的比例。
本说明书中公开的所有特征,或公开的所有方法或过程中的步骤,除了互相排斥的特征和/或步骤以外,均可以以任何方式组合。
本说明书中公开的任一特征,除非特别叙述,均可被其他等效或具有类似目的的替代特征加以替换。即,除非特别叙述,每个特征只是一系列等效或类似特征中的一个例子而已。
【实施例1】绿原酸在高表达淋巴细胞激活基因3(LAG-3)细胞系上的作用研究
1.实验材料
1.1细胞系
CLL细胞人慢性淋巴白血病细胞
JurkatE6-1人T淋巴细胞白血病细胞
CEM-NKR人淋巴瘤细胞
1.2实验药物、试剂盒及仪器
提取自杜仲叶且纯度为99.2%的绿原酸原料药、细胞培养皿、二氧化碳孵箱、RNAprep Pure培养细胞/细菌总RNA提取试剂盒、cDNA第一链合成试剂盒、荧光定量RCR仪、人淋巴细胞激活基因3检测试剂盒、酶标仪等。
2.实验方法和检测指标
2.1细胞的处理
取处于对数期生长的状态良好的CLL细胞和JurkatE6-1细胞,以5×105/mL的密度接种于6孔板中,每种细胞设置4组,每组3个复孔进行给药,
其中三个绿原酸给药组分别按照所需的药物浓度使用空白1640培养基溶解药物进行给药(1mL),空白对照组给予同等体积的空白1640培养基,具体分组和给药方案如表1所示:
表1 实施例1中的实验分组、给药量及给药时间
2.2利用人淋巴细胞激活基因3检测试剂盒检测LAG-3的活性
本实施例通过ELISA实验,利用人淋巴细胞激活基因3检测试剂盒来对各实验组细胞中的LAG-3活性进行检测。将各组细胞与对应的药物作用48h后,收集各组的细胞培养液,均分为两份,取其中一份,用BCA法进行蛋白定量后,利用人淋巴细胞激活基因3检测试剂盒对各组细胞中的LAG-3活性进行检测,换算得出各组的LAG-3含量。
2.3 RT-PCR法检测各组细胞中LAG-3基因的表达情况
(1)细胞总RNA的提取
利用RNA prep Pure培养细胞/细菌总RNA提取试剂盒(离心柱型)提取细胞内的总RNA,操作过程按说明书进行,具体提取步骤简述如下:
①取步骤2.2中均分后剩余的另一份细胞培养液,加入200μl预先加有β-巯基乙醇的裂解液RL;
②将上述液体在12,000rpm条件下离心5min,小心吸取上清液使用;
③向上清液中缓慢加入0.5倍体积的无水乙醇,混匀后转移至吸附柱CR3中于12,000rpm下离心1min,弃去废液,留吸附柱;
④加入去蛋白液RW1至吸附柱中,去除蛋白,之后离心1min,弃去废液;
⑤加入DNase I工作液,去除柱体上的DNA;
⑥用去蛋白液和漂洗液分别清洗吸附柱后,将吸附柱放于收集管中,充分挥干上面的残留液体;
⑦向吸附柱中加入60μlRNase free ddH2O,放置2min后,于12,000rpm下离心2min,即得mRNA样品;
(2)mRNA的逆转录
利用cDNA第一链合成试剂盒和上述提取得到的RNA溶液合成对应的第一链cDNA。具体逆转录过程按照说明书操作,简述如下:
取200μl的无酶离心管置于冰浴上,并于其中加入表2所示的下列溶液:
表2 实施例1中mRNA的逆转录步骤中加入的溶液及用量
RNA模板 | 5mL |
Oligo(dT)15 | 2mL |
Super Pure dNTP | 2mL |
RNase-Free ddH2O | 5.5mL |
共计 | 14.5mL |
离心后,将离心管放置于PCR仪中,在70℃下孵育5min后,简短收集液体并迅速将其转移到冰上冷却2min后补加表3所示的如下试剂:
表3 实施例1中mRNA的逆转录步骤中加入的试剂及用量
上述体系 | 14.5mL |
5×first strand buffer(含DTT) | 4mL |
RNasin | 0.5mL |
TIANScript M-MLV(200U) | 1mL |
总共 | 20mL |
将离心管轻轻混匀并简短离心后,设置PCR仪42℃下孵育50min,之后95℃加热5min。最后向得到的cDNA溶液中加入30ul RNase-Free ddH2O稀释至50ul,即得第一链cDNA。
(3)RT-PCR定量
由于EvaGreen荧光染料与双链的DNA结合会产生很强的荧光,因此通过检测最终的荧光强度可以得到反应生成DNA的总量。在试管中加入荧光染料、(1)(2)步中所合成的cDNA产物、LAG-3引物(上游引物5’-CTAGCTAGCAGCGAGCTCCTTCCAGTC-3’,下游引物5’-GACTGGAAGGAGCTCGCTGCTACGTAG-3’)并混合均匀后进行RT-PCR检测。其中,每个样品均设β-actin引物组为相对值,进行相对定量。
3.实验结果
3.1各组细胞LAG-3活性检测结果
利用ELISA法,对各组细胞中的LAG-3含量进行检测。实验结果显示,空白组的CLL细胞和JurkatE6-1细胞,与对照组的CEM-NKR细胞相比,其中的LAG-3含量较高,与文献所述的CLL和JurkatE6-1是高表达LAG-3的细
胞株相一致,在使用高、中、低剂量的绿原酸与细胞作用后,CLL细胞和JurkatE6-1细胞中的LAG-3含量均有所下降,且下降的程度与绿原酸的用量具有相关性。其中CHCA组(CLL细胞绿原酸高剂量组)和CMCA组(CLL细胞绿原酸中剂量组)中的LAG-3含量与相应的CB组(CLL空白对照组)相比,有显著性差异(*p<0.05);而在JrukatE6-1细胞中,JHCA组(JurkatE6-1细胞绿原酸高剂量组)、JMHC组(JurkatE6-1细胞绿原酸中剂量组)和JMHC组(JurkatE6-1细胞绿原酸低剂量组)与相应的JB组(JurkatE6-1空白对照组)相比,均有显著性差异(*p<0.05)。
结果显示,绿原酸能显著地抑制高表达LAG-3的CLL细胞和JurkatE6-1细胞中LAG-3的含量,且抑制作用与绿原酸的用量具有相关性,具体的实验结果如图1所示,其中,高剂量组、中剂量组和低剂量组分别是指不同的细胞在高、中、低剂量的绿原酸作用后LAG-3的含量。
3.2各组细胞中LAG-3基因的表达水平检测结果
利用RT-PCR法对各组细胞中的LAG-3基因表达情况进行检测。实验结果显示,空白组的CLL细胞和JurkatE6-1细胞与对照组的CEM-NKR细胞相比,其中的LAG-3基因表达水平较高,与文献所述的CLL和JurkatE6-1是高表达LAG-3的细胞株相一致。在使用高、中、低剂量的绿原酸与细胞作用后,CLL细胞和JurkatE6-1细胞中的LAG-3基因表达水平均显著下降,且下降的程度与绿原酸的用量具有相关性。其中CHCA组(CLL细胞绿原酸高剂量组)、CMCA组(CLL细胞绿原酸中剂量组)和CMCA组(CLL细胞绿原酸低剂量组)中的LAG-3基因表达水平与相应的CB组(CLL空白对照组)相比,有显著性差异(*p<0.05),而在JrukatE6-1细胞中,JHCA组(JurkatE6-1细胞绿原酸高剂量组)、JMHC组(JurkatE6-1细胞绿原酸中剂量组)和JMHC组
(JurkatE6-1细胞绿原酸低剂量组)与相应的JB组(JurkatE6-1空白对照组)中LAG-3基因的表达水平相比,均有显著下降,具有显著性差异(*p<0.05)。
结果显示,绿原酸能显著地抑制高表达LAG-3的CLL细胞和JurkatE6-1细胞中LAG-3基因的表达情况,且抑制作用与绿原酸的用量具有相关性,具体的实验结果如图2所示,其中,高剂量组、中剂量组和低剂量组分别是指不同的细胞在高、中、低剂量的绿原酸作用后LAG-3基因的表达水平。
本实施例采用高表达LAG-3的CLL细胞和JurkatE6-1细胞作为模型细胞,以CEM-NKR细胞作为对照细胞,直观考察了绿原酸与CLL和JurkatE6-1细胞作用后细胞LAG-3的表达水平,并从含量和基因表达水平两个层面对LAG-3的表达进行了检测。结果显示,在高表达LAG-3的细胞株中,绿原酸能够显著的作用于LAG-3靶点并对其产生抑制作用。
【实施例2】绿原酸冻干粉针剂对小鼠体内LAG-3表达水平调节的作用研究
1.实验动物及材料
急性T淋巴细胞系白血病模型小鼠、杜仲中提取的纯度为98.9%的绿原酸制备而成的注射用绿原酸冻干粉针剂、LAG-3-PE抗体、流式细胞仪等。
2.实验方法与检测指标
将急性T淋巴细胞系白血病模型小鼠随机分为3组,分别设置为生理盐水对照组、LAG-3抗体阳性对照组和注射用绿原酸针剂组,每组6只小鼠。按照表4配置不同组的药物,并连续静脉注射15天后结束实验,以实验第一次注射记为第1天,于实验第16天时摘取小鼠眼球取血,对血液样品处理后,分离淋巴细胞,采用LAG-3PE抗体进行染色后,采用流式细胞仪对各组小鼠外周血细胞中LAG-3表达情况进行检测。
表4 实施例2中的实验分组和给药方案
3.实验结果
采用流式细胞仪对各组小鼠外周血淋巴细胞表明的LAG-3进行检测后发现,与空白对照组(B组)相比,LAG-3抗体组(LAG3Ig组)和注射用绿原酸组(CA组)均能显著地下调小鼠外周血淋巴细胞上的LAG-3表达水平,且两者之间没有显著性差异(p>0.05),具体的实验结果如图3所示,具体的统计数据如图4所示。
LAG-3是存在于淋巴细胞上的抑制分子,本实施例以急性T淋巴细胞系白血病小鼠作为模型,利用流式细胞检测技术考察了注射用绿原酸冻干粉针剂作用于小鼠后,小鼠的外周血淋巴细胞中的LAG-3表达情况。结果显示,注射用绿原酸冻干粉针剂能够显著地降低模型小鼠外周血淋巴细胞中的LAG-3表达率,对LAG-3的降低作用与LAG-3抗体接近。
【实施例3】注射用绿原酸冻干粉针剂治疗小鼠恶性脑胶质瘤的药效学研究
1.实验动物及材料
1.1实验动物
6-8周龄雌雄C57BL/6小鼠40只,体重18-21g,SPF级。
1.2实验材料及仪器
流式细胞仪、OLYPMPUSPC35DX病理成像***及显微镜、酶标仪等。
2.实验方法与检测指标
2.1注射用绿原酸冻干粉针剂的制备
绿原酸由杜仲叶中提取、纯化得到,纯度约为99.52%。
取绿原酸50g、甘露醇70g、亚硫酸氢钠5g,完全溶解于注射用水,过滤并灌装冻干成含绿原酸50mg/支的冻干粉针剂。
2.2动物模型建立及给药方案
取对数生长期的GL261胶质瘤细胞,制备成浓度为5×105/μL的GL261单细胞悬液,用0.75%戊巴比妥钠按6uL/g腹腔注射麻醉后,刮除穿刺部位的毛发,用ALC-H型小鼠脑立体定向仪固定小鼠头颅,纵形切开头皮。用微量泵以2.5uL/min的速度推注10mL的GL261单细胞悬液,用骨蜡封闭针眼,仔细缝合头皮切口,共接种35只小鼠,留5只正常小鼠。
除去死亡的小鼠,将接种GL261胶质瘤细胞的C57BL/6小鼠,随机分为3组,分别为生理盐水对照组(NC组)、绿原酸注射剂治疗组(CA组)和替莫唑胺(TMZ)阳性对照组。其中CA组每天腹腔注射绿原酸40mg/kg,NC组腹腔注射等体积生理盐水,TMZ治疗组口服20mg/kg替莫唑胺进行治疗。连续给药7天,具体的动物分组及给药方案如表5所示。
表5 实施例3中小鼠脑胶质原位瘤药效学实验分组
组名 | 给药量(mg/kg) | 给药次数 | 给药方式 |
生理盐水对照组(NC组) | 等体积生理盐水 | 7 | 腹腔注射 |
绿原酸注射剂治疗组(CA组) | 40 | 7 | 腹腔注射 |
替莫唑胺治疗(TMZ组) | 20 | 7 | 口服灌胃 |
2.3考察指标
(1)肿瘤大小的药效学考察
为了最直观地衡量各组小鼠在不同治疗方式之后的肿瘤治疗情况,本实验采用小动物核磁成像仪器扫描,观察小鼠脑内原位肿瘤的大小,并计算肿瘤体积。
(2)免疫组化考察
为了考察不同组小鼠中的病灶组织中的LAG-3蛋白表达情况,确定LAG-3和疾病发展之间的相关性,本实验采用免疫组化的方式,以各组小鼠原位病灶作为标本,并加入正常小鼠的脑组织作为标本,共同考察其中的LAG-3蛋白的表达情况。
具体操作方法如下:
实验结束后,切取各组小鼠的原位病灶,切取步骤2.2所述留下的正常5只正常小鼠的脑组织,将样本均置于10%***溶液中固定24h,石蜡包埋制作蜡块,免疫组化分析SP法染色。免疫组化结果采用半定量法分析,最终统计LAG-3蛋白在不同组织样本中的表达率。
(3)T细胞增殖实验
目前的研究显示LAG-3是T淋巴细胞增殖的抑制因子,会对T淋巴细胞的增殖产生负调控,进而对肿瘤的预后产生不良影响,促进肿瘤的发展。检测T淋巴细胞的增殖情况,即可以反映出LAG-3抑制作用的强弱。实验结束后,眼球摘除法收集各组小鼠的外周血,并以CD3负选试剂盒获得高纯度CD3+T细胞(>97%),并以LAG-3-PE染色,检测其中的LAG-3表达情况。
3.实验结果
3.1肿瘤的药效学实验结果
以GL261脑胶质瘤原位小鼠为模型,考察了注射用绿原酸冻干粉针剂治疗脑胶质瘤的药效学,结果显示,与生理盐水对照组相比,绿原酸能够有效地
抑制小鼠原位病灶的生长,二者之间具有显著性差异(*P<0.05),且与阳性药替莫唑胺相比,治疗效果相当,二者之间无显著性差异。实验结果如图5所示.
3.2免疫组化实验结果
对各组小鼠的原位病灶进行免疫组化的研究。结果显示,在正常的脑组织中,未见明显的LAG-3蛋白的表达;在生理盐水模型对照组中,LAG-3蛋白高表达的平均百分率为69%;而在绿原酸治疗组中LAG-3蛋白高表达的平均百分率为23.78%;在替莫唑胺治疗组中,LAG-3蛋白高表达的百分率为44.83%。这个实验结果证明,LAG-3的高表达与肿瘤的治疗效果具有相关性,绿原酸粉针剂和替莫唑胺的肿瘤治疗效果从瘤体大小方面看,是相当的,而从LAG-3蛋白表达方面则显示,绿原酸能更大程度地抑制LAG-3蛋白的表达,是通过抑制LAG-3发挥抗肿瘤作用。实验结果如图6所示。
3.3 T细胞增殖实验结果
目前的研究显示,LAG-3是T淋巴细胞的抑制因子,对于T淋巴细胞的增殖具有负向调控作用,对LAG-3的抑制,应该会对T淋巴细胞的增殖作用产生促进作用,该实验测定了生理盐水对照组和绿原酸治疗组这两组小鼠的外周血淋巴细胞中的CD3+T淋巴细胞和CD4+T淋巴细胞的百分比。结果发现,与生理盐水对照组相比,绿原酸治疗组可以显著地促进小鼠CD3+T淋巴细胞和CD4+T淋巴细胞的增殖,实验结果如图7所示。
LAG-3与肿瘤的相关性,主要体现在其能够抑制T淋巴细胞的增殖,从而使肿瘤产生免疫耐受,促进肿瘤的发展。本实施例以GL261原位脑胶质瘤小鼠为模型,在考察绿原酸注射剂治疗脑胶质瘤的药效基础上,进一步确证了LAG-3和肿瘤以及外周血T淋巴细胞之间的相关性,确定了绿原酸可以抑制LAG-3蛋白在脑胶质瘤中的表达,且这种抑制作用在外周血淋巴细胞的增殖上
也能显著体现。
【实施例4】绿原酸胃漂浮片在慢性乙肝病毒感染小鼠体内的药效和作用机制研究
1.实验动物和材料
1.1实验动物
HBV病毒转染的慢性乙肝模型小鼠,18-22g,30只;正常balb/c小鼠,18-22g,10只。
1.2实验材料
流式细胞仪、荧光定量PCR仪等
2.实验方法
2.1绿原酸胃漂浮片的制备
按表6所示处方分别称取绿原酸原料药10g、羟丙基纤维素50g、硬脂酸30g、碳酸氢钠10g、微晶纤维素60g和乳糖40g,将原辅料分别过100目筛并采用等量递增法将原辅料初步混匀,再将原辅料过80目筛充分混匀,压制成适宜硬度的片,挤压通过20目筛制粒并用冲模压片,即得绿原酸胃漂浮片。
表6 实施例4中绿原酸胃漂浮片的处方表
成分 | 含量(质量百分比) | 作用 |
绿原酸原料药 | 5 | 主药 |
羟丙基纤维素 | 25 | 骨架剂 |
硬脂酸 | 15 | 助漂剂 |
碳酸氢钠 | 5 | 气泡剂 |
微晶纤维素 | 30 | 填充剂 |
乳糖 | 20 | 填充剂 |
2.2动物分组和给药方案
本实验将HBV病毒转染的慢性乙肝模型小鼠随机分为3组,每组10只,分别为生理盐水对照组(NC组)、乙型肝炎免疫球蛋白阳性对照组(HBVIG组)和绿原酸胃漂浮片治疗组(CA组),正常小鼠为阴性对照组。具体的分组和给药方案如表7所示。
表7 实施例4中的小鼠分组和给药方案
组名 | 给药量 | 给药次数 | 给药方式 |
生理盐水对照组(NC组) | 等体积生理盐水 | 20 | 口服灌胃 |
绿原酸胃漂浮片治疗组(CA组) | 60mg/kg | 20 | 口服灌胃 |
乙型肝炎免疫球蛋白(HBVIG组) | 9IU/只 | 2 | 肌肉注射 |
正常对照组小鼠(N组) | 等体积生理盐水 | 20 | 口服灌胃 |
2.3小鼠外周血中丙氨酸氨基转移酶(ALT)的检测
丙氨酸氨基转移酶(ALT)与肝炎的预后有直接关系,它的含量能反应药物的治疗效果,故本实验检测了各组小鼠在治疗后不同时间点外周血中的ALT含量,其中,采用全自动生化测定仪定量测定ALT,通过治疗组间及治疗组与对照组同意时间点的ALT值比较,评价绿原酸胃漂浮片对乙型肝炎的治疗作用。具体地,本实验在第0天、第4天、第8天、第12天、第16天和第20天分别从小鼠尾静脉取得外周血,以此血液为标本进行检测。
2.4小鼠外周血的LAG-3分布频率的测定
为了进一步确诊治疗效果和LAG-3之间的关系,明确绿原酸的作用靶点为LAG-3,本实验对各组小鼠治疗终点时的外周血中LAG-3分布频率进行了检测,即于实验终止第二天(即实验第21天),眼球摘除取得小鼠的外周血,随后利用流式细胞仪检测各组小鼠外周血中LAG-3表达情况。
3.实验结果
3.1绿原酸胃漂浮片的制备
3.2小鼠外周血中ALT的检测
本实验对各个小组小鼠不同时间点的外周血中的ALT含量进行了追踪检测。结果发现,与对照组小鼠(NC组)相比,HBV感染的乙型肝炎模型小鼠对照组(NC组)中,ALT含量明显增高,二者之间具有显著性差异,证明HBV感染的乙型肝炎模型小鼠具有该类疾病的典型特征,绿原酸胃漂浮片和HBIG均能明显地降低对应治疗组小鼠外周血中的ALT含量,即CA组和HBIG组与NC组相比,均具有显著性差异,证明绿原酸胃漂浮片能够显著地降低外周血中ALT的含量,实验结果如图8所示。
【实施例5】绿原酸胃漂浮片对小鼠脓毒症的治疗作用研究
1.实验方法
1.1脓毒症小鼠模型的建立
采用CLP术致脓毒症模型小鼠,共30只;正常balb/c小鼠10只。
1.2实验材料与仪器
流式细胞仪、LAG-3(CD223)-PE抗体、CD8-PE-Cy7抗体、CD4-APC抗体等
2.实验方法
2.1动物分组和给药方案
将脓毒症模型小鼠30只,随机分为3组,每组10只,分别为模型小鼠对照组(NC组)、绿原酸胃漂浮片治疗组(CA组)和LAG-3抗体治疗组(LAG3IG组),另取10只正常balb/c小鼠作为阴性对照组(N组)。其中,LAG3IG组于实验第1天和实验第3天肌肉注射,CA组每天给予腹腔注射,共连续给药
7天,NC组和N组均口服灌胃给予CA组等体积生理盐水,从开始到实验第10天,纪录小鼠的生存率及生存时间,于实验第10天将所有小鼠摘除眼球取全血,均分为两份,备用。具体的分组和给药方案如表8所示。
表8 实施例5中脓毒症小鼠药效学考察实验分组及给药方案
组名 | 给药量 | 给药次数 | 给药方式 |
生理盐水对照组(NC组) | 等体积生理盐水 | 7 | 口服灌胃 |
绿原酸胃漂浮片治疗组(CA组) | 60mg/kg | 7 | 口服灌胃 |
LAG-3单克隆抗体组(LAG3IG组) | 10IU/只 | 2 | 肌肉注射 |
正常对照组小鼠(N组) | 等体积生理盐水 | 20 | 口服灌胃 |
2.2小鼠外周血中CD4+T淋巴细胞、CD8+T淋巴细胞表面LAG-3的表达情况检测
LAG-3是存在于T淋巴细胞上的抑制分子,对其会产生明显的影响。取步骤2.1中收集的各组小鼠全血一份,在各管中加入anti-mouseCD8-PE-Cy7和anti-mouseCD4-APC和Anti-MouseLAG-3(CD223)-PE-荧光素标记单克隆抗体各1Μl,然后在各管中分别加入100μL混匀的血液,低俗漩涡混匀实际和血液3s后,室温避光反应20min,随后加入红细胞裂解液2mL,室温避光放置15min,再用PBS洗涤2次后,进入流式细胞仪进行检测。
3.实验结果
3.1小鼠生存率和生存时间的考察结果
实验期间,对各组小鼠的生存率和生存时间进行了记录。结果如下:脓毒症模型小鼠在实验结束前已全部死亡,而LAG3IG组和CA组的小鼠与脓毒症对照组相比,其生存率和生存时间有明显的改善。实验表明,绿原酸胃漂浮片(CA组)对脓毒症小鼠具有积极的治疗作用,可以提高其生存率,延长生存时
间。具体实验结果如图9所示。
3.2小鼠外周血淋巴细胞上LAG-3的表达情况
各组小鼠外周血淋巴细胞上LAG-3的表达检测结果显示,正常对照组(N组)中LAG-3表达水平较低(10a),对应的CD4+T和CD8+T淋巴细胞的量正常(10b),而在脓毒症模型对照组(NC组)小鼠中,LAG-3表达明显增高(10a),对应的CD4+T和CD8+T淋巴细胞的量显著降低(10b),LAG3IG和绿原酸胃漂浮片治疗后,脓毒症小鼠的LAG-3表达相比于模型对照组(NC组)有了明显的下降(10a),且对应的CD4+T和CD8+T淋巴细胞也有了明显的增殖(10b),具体实验结果如图10a和10b所示。
本实施例中,鉴于LAG-3靶点和脓毒症之间的相关性,以LAG-3抗体为阳性对照,考察了绿原酸胃漂浮片对脓毒症小鼠的治疗作用、结果显示,绿原酸对小鼠脓毒症具有显著的疗效,且这种作用体现在小鼠外周血淋巴细胞表面的LAG-3靶点得到有效抑制,相应的外周血中CD4+T和CD8+T淋巴细胞有了显著的增殖。进一步说明,绿原酸可以通过抑制LAG-3靶点,治疗与LAG-3相关的脓毒症。
【实施例6】绿原酸散剂的制备
绿原酸由杜仲叶中提取、纯化得到,纯度=98.82%。
取绿原酸1000g,无菌分装成含绿原酸1000mg/瓶或1000mg/袋的散剂。
综上所述,本发明发现了绿原酸对LAG-3靶点的抑制作用,并发现了其能够应用于抗肿瘤、抗病毒和脓毒症等以LAG-3为靶点的疾病的治疗;进一步地,采用适宜的方法将绿原酸制备成胃漂浮片剂,进一步提高绿原酸的口服生物利用度。
本发明并不局限于前述的具体实施方式。本发明扩展到任何在本说明书中
披露的新特征或任何新的组合,以及披露的任一新的方法或过程的步骤或任何新的组合。
【实施例7】绿原酸冻干粉针剂的制备
绿原酸由杜仲叶中提取、纯化得到,纯度=99.5%。
取绿原酸30g,甘露醇80g、亚硫酸氢钠2g,完全溶解于注射用水,过滤,灌装冻干成含绿原酸30mg/支的冻干粉针剂。
Claims (7)
- 绿原酸在制备抑制LAG-3靶点的药物中的用途。
- 绿原酸在制备治疗以LAG-3为靶点的疾病的药物中的用途。
- 根据权利要求2所述的用途,其中,所述以LAG-3为靶点的疾病包括恶性肿瘤、病毒感染性疾病和脓毒症。
- 根据权利要求1或2所述的用途,其中,所述药物以绿原酸作为LAG-3的靶点抑制剂并通过抑制LAG-3和LAG-3蛋白的表达来治疗疾病。
- 根据权利要求1或2所述的用途,其中,所述药物包括绿原酸和药学上可接受的辅料。
- 根据权利要求5所述的用途,其中,所述药物为胃漂浮片剂或散剂。
- 根据权利要求5所述的用途,其中,所述胃漂浮片剂是以绿原酸为主药、以羟丙基纤维素为骨架材料、以硬脂酸为助漂剂、以碳酸氢钠为起泡剂并以微晶纤维素和乳糖为填充剂制备而成。
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JP7391017B2 (ja) | 2017-10-06 | 2023-12-04 | カーギル インコーポレイテッド | ステビオール配糖体溶解度エンハンサー |
CN109568314B (zh) * | 2018-05-04 | 2022-04-12 | 四川九章生物科技有限公司 | 一种联合用药物及其在制备治疗术后复发标准治疗无效的高级别脑瘤的药物中的用途 |
EP3953012A1 (en) | 2019-04-06 | 2022-02-16 | Cargill, Incorporated | Methods for making botanical extract composition |
EP3952667A1 (en) | 2019-04-06 | 2022-02-16 | Cargill, Incorporated | Sensory modifiers |
CN112010972B (zh) * | 2019-05-31 | 2023-01-10 | 瑞阳(苏州)生物科技有限公司 | 与人lag-3蛋白结合的抗体及其编码基因和应用 |
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WO2009091134A2 (en) * | 2007-12-28 | 2009-07-23 | Sungkyunkwan University Foundation For Corporate Collaboration | Pharmaceutical composition for sepsis and septic shock |
CN102391119A (zh) * | 2011-11-14 | 2012-03-28 | 肖文辉 | 高纯度绿原酸制剂的制备及临床应用 |
CN105796542A (zh) * | 2016-03-15 | 2016-07-27 | 四川九章生物科技有限公司 | 绿原酸在制备治疗以lag-3为靶点的疾病的药物中的用途 |
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EP3040067A4 (en) * | 2013-08-21 | 2017-03-22 | Sichuan Jiuzhang Biological Science And Technology Co., Ltd | Chlorogenic acid powder-injection and preparation method thereof |
CN104188949B (zh) * | 2014-09-15 | 2017-08-18 | 四川九章生物科技有限公司 | 绿原酸在制备治疗少突胶质瘤的药物中的用途 |
CN104188950B (zh) * | 2014-09-15 | 2016-05-11 | 四川九章生物科技有限公司 | 绿原酸在制备治疗室管膜瘤的药物中的用途 |
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WO2009091134A2 (en) * | 2007-12-28 | 2009-07-23 | Sungkyunkwan University Foundation For Corporate Collaboration | Pharmaceutical composition for sepsis and septic shock |
CN102391119A (zh) * | 2011-11-14 | 2012-03-28 | 肖文辉 | 高纯度绿原酸制剂的制备及临床应用 |
CN105796542A (zh) * | 2016-03-15 | 2016-07-27 | 四川九章生物科技有限公司 | 绿原酸在制备治疗以lag-3为靶点的疾病的药物中的用途 |
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