WO2022033200A1 - 索拉非尼在治疗1型糖尿病中的应用 - Google Patents

索拉非尼在治疗1型糖尿病中的应用 Download PDF

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WO2022033200A1
WO2022033200A1 PCT/CN2021/102606 CN2021102606W WO2022033200A1 WO 2022033200 A1 WO2022033200 A1 WO 2022033200A1 CN 2021102606 W CN2021102606 W CN 2021102606W WO 2022033200 A1 WO2022033200 A1 WO 2022033200A1
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sorafenib
diabetes
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邓沱
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中南大学湘雅二医院
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  • Type 1 diabetes is an autoimmune disease characterized by massive damage to the insulin-producing beta cells of the pancreatic islets. It is extremely harmful and can cause a variety of complications, including cardiovascular disease, stroke, neurological disease, diabetic foot and so on. And the incidence of type 1 diabetes is increasing year by year, and the incidence rate in children worldwide is doubling every 20 years. At present, the treatment of type 1 diabetes mainly relies on insulin, but long-term insulin injection not only brings inconvenience to patients, but also causes many complications. Therefore, it is urgent to find a new treatment for type 1 diabetes.
  • T1D The destruction of ⁇ cells in T1D is mainly mediated by autoreactive T cells, so targeting autoreactive T cells brings new hope for the treatment of T1D.
  • T cell-based immunotherapies such as anti-CD3 monoclonal antibodies, CTLA4-Ig fusion proteins, and low-dose antithymocyte globulin. These immunotherapies do show some efficacy in human clinical trials, but their systemic immunosuppressive effects and transient effectiveness limit their clinical application. Facing the growing number of T1D populations, there is an urgent need to develop more effective and safer T cell-based immunotherapies.
  • antigen-presenting cells present autoantigens such as precursor proinsulin (PPI), insulinoma-associated antigen 2 (I-A2), glutamate decarboxylase (GAD) and zinc to CD4+ T cells
  • PPI precursor proinsulin
  • I-A2 insulinoma-associated antigen 2
  • GCD glutamate decarboxylase
  • IL-12 interleukin 12
  • Th1 cells produce inflammatory cytokines including IFN- ⁇ and IL-2 to activate CD8+ cytotoxic T lymphocytes and macrophages and cooperate with them to attack pancreatic ⁇ cells.
  • the IL-12/Stat4 pathway is directly mediated by two tyrosine kinases, Janus kinase 2 (Jak2) and tyrosine kinase 2 (Tyk2), and may be indirectly regulated by other tyrosine kinases.
  • sorafenib in the preparation of an oral medicine for the preventive treatment of type 1 diabetes.
  • Sorafenib for the preventive treatment of type 1 diabetes provides a new treatment approach for the majority of patients, and provides more choices for clinicians and patients.
  • sorafenib provides oral medication without the hassle of daily insulin injections.
  • sorafenib in the preparation of an oral medicine for the preventive treatment of type 1 diabetes.
  • Sorafenib for the preventive treatment of type 1 diabetes provides a new treatment approach for the majority of patients, and provides more choices for clinicians and patients.
  • sorafenib provides oral medication without the hassle of daily insulin injections.
  • Sorafenib is a small molecule compound that inhibits tumor cell proliferation, angiogenesis and increases apoptosis in a wide range of tumor models.
  • TKI oral receptor tyrosine kinase inhibitor
  • Raf serine/threonine kinase and receptor tyrosine kinase vascular endothelial growth factor body 1, 2, 3 and platelet-derived growth factor-beta, Flt-3 and c-kit.
  • Sorafenib is approved by the US FDA for the treatment of advanced inoperable hepatocellular carcinoma (HCC) and advanced renal cell carcinoma (RCC), as well as advanced radioiodine-refractory differentiated thyroid cancer (radioiodine-referectory) differentiated thyroid cancer, RRDTC).
  • HCC advanced inoperable hepatocellular carcinoma
  • RRC advanced renal cell carcinoma
  • RRDTC advanced radioiodine-refractory differentiated thyroid cancer
  • the molecular formula of Sorafenib is C 21 H 16 ClF 3 N 4 O 3 , the molecular weight is 464.82500, and the structural formula is shown in formula I.
  • the present invention first treats in vitro cultured cells with increasing concentrations of sorafenib CD4+ T cells were induced to differentiate into Th1 cells by adding IL-12, and the proportion of Th1 was detected by flow cytometry. The results showed that sorafenib inhibited IL-12-induced Th1 cell differentiation in a dose-dependent manner.
  • the present invention uses sorafenib to gavage female NOD mice prior to the onset of type 1 diabetes, and observes natural and cyclophosphamide-induced morbidity in mice. The results showed that sorafenib could prevent the occurrence of type 1 diabetes in NOD mice.
  • sorafenib can be used to protect the pancreas of NOD mice from autoreactive T cell attack.
  • Each pharmaceutical dosage form can be prepared by selecting appropriate acceptable excipients according to the actual needs of the dosage form, which belongs to the conventional dosage form preparation technology in the art. Such as capsules, tablets, injection powder and so on.
  • the invention discloses a new application of sorafenib, and those skilled in the art can learn from the content of this paper and appropriately improve the process parameters to achieve. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention.
  • the method and application of the present invention have been described through the preferred embodiments, and it is obvious that relevant persons can make changes or appropriate changes and combinations of the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and Apply the technology of the present invention.
  • the reagents involved in the examples of the present invention are all commercially available products, which can be purchased through commercial channels.
  • C57BL/6J mice were sacrificed and the spleen was removed and disrupted in PBS containing 2% fetal bovine serum (FBS). Tissue debris was removed by filtering the cell suspension through a 70 ⁇ m nylon filter. After centrifugation at 300g for 10 minutes, cells were resuspended in PBS containing 2% fetal bovine serum (FBS) to maintain a cell concentration of 1 x 10 ⁇ 8 nucleated cells/mL.
  • FBS fetal bovine serum
  • CD4+ T cells were cultured in RPMI-1640 medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Gibco).
  • IL-12 was purchased from peprotech, and sorafenib was Nexavar tablets purchased in the hospital, both dissolved in DMSO, the concentration of the stock solution was 10 mM, and it was frozen at -80°C.
  • the working solution was diluted with RPMI medium to treat the cells.
  • sorafenib On IL-12-induced Th1 cell differentiation, the above-mentioned CD4+ T cells were evenly seeded in 12-well plates, firstly adding increasing concentrations of sorafenib (concentration gradient: 0, 1, 2, 4 ⁇ g/ml), supplemented to equal volume with DMSO, and set up 3 parallel replicate groups, then An equal amount of IL-12 was added to each well, the cells were harvested after 48 hours, and the proportion of Th1 cells was detected by flow cytometry.
  • concentration gradient 0, 1, 2, 4 ⁇ g/ml
  • a is flow cytometry to detect the proportion of Th1 cells under different concentrations of sorafenib treatment
  • b is the result after quantitative analysis, it can be seen that with the increase of sorafenib concentration, the proportion of Th1 cells gradually decreases , suggesting that sorafenib can inhibit IL-12-induced Th1 differentiation in a dose-dependent manner.
  • the method is to treat with increasing concentrations of sorafenib CD4+ T cells were harvested after 48 hours, and the cells were counted. The results are shown in Figure 2.
  • Dispensing Use carboxymethylcellulose (CMC) as a solvent, sterilize CMC by autoclaving before dispensing, grind sorafenib in the tablet into powder on an ultra-clean workbench, and then weigh an appropriate amount of sorafenib. Rafenib powder is uniformly suspended in the solvent, and the prepared drug is stored at 4°.
  • CMC carboxymethylcellulose
  • Blood sugar measurement Cut off about 1mm from the tail tip of the mouse, wipe off the first drop of blood with gauze after bleeding, wait for the second drop of blood to flow out naturally, absorb and read with blood sugar test strips, two consecutive blood sugar readings > 250mg/ dl can be diagnosed as type 1 diabetes.
  • mice were divided into two groups, one group was the sorafenib administration group, the other was the solvent administration group.
  • the mice were treated by intragastric administration for 4 weeks at the age of 10 weeks, and cyclophosphamide was injected after the intragastric administration for two weeks. The onset was induced, the mice were sacrificed, the pancreas was removed, a small portion of the extracted RNA was used for RT-PCR, and the rest was mostly used for flow cytometry.
  • Figure 5 a shows the changes of CD4+ T cell subsets (Th1, Th2, Th17 and Treg) in the pancreas of the two groups of mice detected by flow cytometry.
  • the results showed that the proportion of Th1 cells in the pancreas of the sorafenib group was significantly reduced , while the proportion of other T cell subsets remains unchanged;
  • b is the gene expression level of Th1 cell marker genes (Tbet and IFN ⁇ ) and pro-inflammatory cytokines (IL-1 ⁇ , IL-6 and TNF- ⁇ ) in the pancreas detected by RT-PCR , the results showed that the gene expression levels of Th1 cell marker genes (Tbet and IFN ⁇ ) and pro-inflammatory cytokines (IL-1 ⁇ , IL-6 and TNF- ⁇ ) in the pancreas of the sorafenib group were significantly reduced; Blocks the development of type 1 diabetes by reducing the infiltration of Th1 cells in the pancreas of NOD mice.
  • mice in each group were compared with the vehicle group, and statistical analysis was performed with independent samples t-test: + means P value ⁇ 0.1, * means P value ⁇ 0.05.
  • Sorafenib inhibits IL-12-induced Th1 cell differentiation by inhibiting the phosphorylation of Stat4
  • the method is treatment with increasing concentrations of sorafenib (concentration gradient: 0, 1, 2, 4 ⁇ g/ml) CD4+ T cells were filled with DMSO to an equal volume, and 3 parallel replicate groups were set up, and then an equal amount of IL-12 was added to each well. After 48 hours, the cells were harvested, the protein was extracted, and the western blotting test (WB) was performed. The protein levels of STAT4 and p-STAT4 were detected.
  • sorafenib concentration gradient: 0, 1, 2, 4 ⁇ g/ml

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Abstract

索拉非尼或其衍生物在制备预防和/或治疗I型糖尿病的药物中的应用。

Description

索拉非尼在治疗1型糖尿病中的应用
本申请要求于2020年08月13日提交中国专利局、申请号为202010812329.3、发明名称为“索拉非尼在治疗1型糖尿病中的应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于医药技术领域，具体涉及索拉非尼的新应用，尤其是索拉非尼在预防性治疗1型糖尿病中的应用。
背景技术
1型糖尿病(T1D)是一种自身免疫性疾病，其特征在于产生胰岛素的胰岛β细胞大量受损。其危害性极大，能够引起多种并发症，包括心血管疾病，中风，神经系统病变，糖尿病足等等。并且一型糖尿病的发病率逐年增加，在全球儿童中的发病率每20年增加一倍。目前对于一型糖尿病的治疗主要还是依赖于胰岛素，但是长期注射胰岛素不仅给病人带来不便，还会引起许多并发症。因此，找到一种新的治疗1型糖尿病的方法急不可待。
T1D中β细胞的破坏主要由自身反应性T细胞介导，因此靶向自身反应性T细胞给T1D的治疗带来新的希望。目前，已经出现了多种基于T细胞的免疫疗法，例如抗CD3单克隆抗体，CTLA4-Ig融合蛋白和低剂量抗胸腺细胞球蛋白。这些免疫疗法的确在人类临床试验中显示出一些效果，但是它们的全身性免疫抑制作用和短暂的有效性限制了其临床应用。面对越来越多的T1D人群，迫切需要开发出更有效，更安全的基于T细胞的免疫疗法。
在T1D的早期阶段，抗原呈递细胞(APC)向CD4+ T细胞呈递自身抗原，例如胰岛素原前体(PPI)，胰岛素瘤相关抗原2(I-A2)，谷氨酸脱羧酶(GAD)和锌转运蛋白(ZnT8)等，与此同时，APC分泌白介素12(IL-12)诱导幼稚CD4+ T细胞分化为Th1细胞。Th1细胞产生包括IFN-γ和IL-2在内的炎性细胞因子，以激活CD8+细胞毒性T淋巴细 胞和巨噬细胞，并与它们协同攻击胰岛β细胞。通过破坏Tbet(一种Th1分化的关键转录因子)来阻止Th1细胞分化，可有效阻断NOD小鼠的胰岛炎和T1D发生。因此，Th1细胞群是T1D发病机制的关键介体。IL-12是诱导Th1细胞分化的关键细胞因子，IL-12诱导的信号转导子和转录激活因子4(Stat4)的激活是驱动Th1细胞分化的重要信号途径。越来越多的证据表明，IL-12诱导的Th1细胞分化在T1D的发病机理中起着至关重要的作用。在NOD小鼠中，每天注射IL-12可增加T1D发病率，而使用IL-12拮抗剂则可降低T1D发病率。此外，抑制Stat4的激活能够完全阻止NOD小鼠T1D的发生。在人类，IL-12b基因被认为是T1D相关基因。总之，这些研究表明IL-12诱导的Th1分化在T1D发病中扮演着关键作用。因此，靶向IL-12/Stat4轴来抑制Th1细胞分化可能是治疗T1D更特异和有效的方法。
酪氨酸激酶是一组具有催化亚基的酶，可将ATP上的磷酸基团转移至蛋白质中的一个或多个酪氨酸残基，导致蛋白质构象变化，从而影响蛋白质功能。酪氨酸激酶是信号转导的重要介体，可响应外部和内部刺激而调节细胞反应，例如细胞分裂，分化，凋亡和代谢。由于酪氨酸激酶参与多种生物过程，因此开发了酪氨酸激酶抑制剂(TKI)用于治疗多种疾病，包括癌症，传染性疾病，自身免疫和炎性疾病等。截至目前，已有39个TKI被FDA批准，还有更多的TKI正在临床试验中测试。IL-12/Stat4途径直接由两个酪氨酸激酶，Janus激酶2(Jak2)和酪氨酸激酶2(Tyk2)介导，并可能被其他酪氨酸激酶间接调控。
发现能够抑制IL-12诱导的Th1细胞分化，并且对于T1D有治疗作用的药物具有重要的现实意义。
发明内容
有鉴于此，本发明提供一种索拉非尼在制备预防性治疗1型糖尿病的口服药物中的应用。索拉非尼用于1型糖尿病的预防性治疗给广大患者提供了新的治疗途径，给临床医生及患者提供了更多选择。对于1型糖尿病患者，索拉非尼能为患者提供口服药物治疗，免于进行每日胰岛素注射 的麻烦。
为了实现上述发明目的，本发明提供以下技术方案：
有鉴于此，本发明提供一种索拉非尼在制备预防性治疗1型糖尿病的口服药物中的应用。索拉非尼用于1型糖尿病的预防性治疗给广大患者提供了新的治疗途径，给临床医生及患者提供了更多选择。对于1型糖尿病患者，索拉非尼能为患者提供口服药物治疗，免于进行每日胰岛素注射的麻烦。
为了实现上述发明目的，本发明提供以下技术方案：
索拉非尼(Sorafenib)是在广泛的肿瘤模型中抑制肿瘤细胞增殖、血管生成和增加凋亡的小分子化合物。它作为一种口服受体酪氨酸激酶抑制剂(TKI)，可抑制与肿瘤发生和肿瘤进展有关的因子，如Raf丝氨酸/苏氨酸激酶和受体酪氨酸激酶(血管内皮生长因子受体1、2、3和血小板衍生生长因子-β、Flt-3和c-kit)。索拉非尼被美国FDA批准用于治疗晚期不可手术的肝癌(hepatocellular carcinoma,HCC)和晚期肾细胞癌(renal cell carcinoma,RCC)，以及晚期放射性碘难治性分化的甲状腺癌(radioiodine-referectory differentiated thyroid cancer,RRDTC)。索拉非尼分子式为C 21H 16ClF 3N 4O 3，分子量464.82500，结构式如式I所示。
Figure PCTCN2021102606-appb-000001
在本发明中，通过细胞和小鼠模型来明确索拉非尼对1型糖尿病的预防与治疗作用。
在一些实施方案中，本发明先用递增浓度的索拉非尼处理体外培养的
Figure PCTCN2021102606-appb-000002
CD4+ T细胞，再加IL-12诱导其分化为Th1细胞，通过流式细胞术检测Th1的比例。结果显示索拉非尼以剂量依赖的方式抑制IL-12诱导的Th1细胞分化。
在一些实施方案中，本发明使用递增浓度的索拉非尼处理
Figure PCTCN2021102606-appb-000003
CD4+ T细胞两天，通过细胞计数仪进行细胞计数。结果显示，索拉非尼对T细胞没有细胞毒性。
由此可见，索拉非尼可用于抑制IL-12诱导的Th1细胞的分化。
在一些实施方案中，本发明使用索拉非尼对1型糖尿病发病前的雌性NOD小鼠进行灌胃处理，观察小鼠的自然发病率和环磷酰胺诱导的发病率。结果显示，索拉非尼能够预防NOD小鼠1型糖尿病的发生。
由此可见，索拉非尼可用于预防1型糖尿病的发生。
在一些实施方案中，本发明使用索拉非尼对发病前小鼠连续灌胃4周，将未发病的小鼠处死，取胰腺，甲醛固定，切片，HE染色，在显微镜下观察胰岛情况，比较索拉非尼组和溶剂组小鼠胰岛炎情况。结果显示，索拉非尼组胰岛炎情况比溶剂组明显改善，单核细胞浸润明显减少。
在一些实施方案中，本发明使用索拉非尼对发病前小鼠连续灌胃4周，在灌胃处理两周后给予环磷酰胺注射诱导发病，处死小鼠，取胰腺，用一小部分提RNA做RT-PCR检测胰腺中Th1细胞标志基因(Tbet和IFNγ)和促炎细胞因子(IL-1β，IL-6和TNF-α)的基因表达水平，剩下的大部分做流式细胞术检测两组小鼠胰腺中CD4+ T细胞亚群(Th1，Th2，Th17和Treg)的变化。结果显示，索拉非尼能够减少胰腺Th1细胞的浸润和炎症因子的表达。
由此可见，索拉非尼可用于保护NOD小鼠胰腺免受自身反应性T细胞攻击。
进一步的，在一些实施方案中，本发明先使用递增浓度的索拉非尼处理体外培养的
Figure PCTCN2021102606-appb-000004
CD4+ T细胞，48小时后收细胞，提蛋白，做蛋白质印 迹试验(western blot，WB)检测STAT4和p-STAT4的蛋白水平。结果显示，索拉非尼能够抑制STAT4的磷酸化，并且索拉非尼抑制STAT4激活的剂量与抑制Th1分化的剂量完全匹配。
由此可见，索拉非尼通过抑制STAT4的激活来抑制IL-12诱导的Th1细胞分化。
综上所述，本发明提供了索拉非尼在预防性治疗1型糖尿病中的应用。
其中，所述药物为索拉非尼。
进一步的，所述药物还包括药学上可接受的辅料。
所述药物可以为当前药品领域任何剂型，包括口服制剂或注射制剂。
各药物剂型可根据该剂型实际需要选取合适的可接受辅料来制备，这属于本领域常规的剂型制备技术。如制成胶囊剂、片剂、注射粉剂等。
由上述技术方案可知，本发明提供了索拉非尼在预防性治疗1型糖尿病中的应用。索拉非尼用于1型糖尿病的预防性治疗给广大患者提供了新的治疗途径，给临床医生及患者提供了更多选择。对于1型糖尿病患者，索拉非尼能为患者提供口服药物治疗，免于进行每日胰岛素注射的麻烦。索拉非尼可以化学合成，成本较生物制剂要低。且已经通过FDA及NMPA的批准上市，用于临床治疗。其副反应较少较轻，临床病人耐受性良好，且病人负担较轻。
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为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。
图1示实施例1索拉非尼以剂量依赖的方式抑制IL-12诱导的Th1细胞分化；a为流式细胞术检测不同浓度索拉非尼处理下Th1细胞的比例；b为定量分析后的结果；
图2示实施例2索拉非尼对T细胞没有细胞毒性；
图3示实施例3索拉非尼能够预防NOD小鼠1型糖尿病的发生；其中，a为索拉非尼预防NOD小鼠自发性1型糖尿病的结果图；b为索拉非尼预防NOD小鼠环磷酰胺诱导的1型糖尿病的结果图；
图4示实施例4索拉非尼能够改善NOD小鼠的胰岛炎情况；
图5示实施例5索拉非尼减少NOD小鼠胰腺Th1细胞的浸润；其中，a为流式细胞术检测两组小鼠胰腺中CD4+ T细胞亚群(Th1，Th2，Th17和Treg)的变化；b为RT-PCR检测胰腺中Th1细胞标志基因(Tbet和IFNγ)和促炎细胞因子(IL-1β，IL-6和TNF-α)的基因表达水平；
图6示实施例6索拉非尼通过抑制Stat4的磷酸化抑制IL-12诱导的Th1细胞分化；其中，a为蛋白质印迹结果，b为定量结果。
具体实施方式
本发明公开了索拉非尼的新应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
如无特殊说明，本发明实施例中所涉及的试剂均为市售产品，均可以通过商业渠道购买获得。
下面结合实施例，进一步阐述本发明：
实施例1、索拉非尼以剂量依赖的方式抑制IL-12诱导的Th1细胞分化。一、材料与方法
1、细胞
将C57BL/6J小鼠处死后取出脾脏，在含2％胎牛血清(FBS)的PBS中破坏脾脏。用70μm尼龙滤网过滤细胞悬液除去组织碎片。300g离心10分钟，然后用含2％胎牛血清(FBS)的PBS重悬细胞，保持细胞浓度为1x 10^8有核细胞/mL。用EasySep TM Mouse
Figure PCTCN2021102606-appb-000005
CD4+ T Cell Isolation Kit(STEMCELL Technologies)提取
Figure PCTCN2021102606-appb-000006
CD4+ T细胞，提出的
Figure PCTCN2021102606-appb-000007
CD4+ T细胞培养于含10％胎牛血清(Gibco)及1％青霉素/链霉素(Gibco)的RPMI-1640培养基(Gibco)中。
2、刺激因子
IL-12购于peprotech，索拉非尼为在医院购买的Nexavar片剂，均溶于DMSO，母液浓度为10mM，冻存于-80℃，工作液采用RPMI培养基稀释后处理细胞。
3.体外抑制试验
为了检测索拉非尼对IL-12诱导的Th1细胞分化的抑制作用，将上述
Figure PCTCN2021102606-appb-000008
CD4+ T细胞均匀种在12孔板，先加入递增浓度的索拉非尼(浓度梯度：0，1，2，4μg/ml)，用DMSO补齐至等体积，设3个平行重复组，然后每个孔加入等量的IL-12，48小时后收细胞，通过流式细胞术检测Th1细胞比例。
二、结果分析
图1中a图为流式细胞术检测不同浓度索拉非尼处理下Th1细胞的比例，b为定量分析后的结果，可见随着索拉非尼浓度的升高，Th1细胞的比例逐渐减少，提示索拉非尼能够抑制IL-12诱导的Th1分化，并且这种抑制作用呈剂量依赖性。
表1 图1b数据
Figure PCTCN2021102606-appb-000009
每组设3个平行孔，不同浓度组分别与0μg/ml进行比较，用独立样本t检验进行统计学分析：**代表P值<0.01，***代表P值<0.001。
实施例2、索拉非尼对T细胞没有细胞毒性
方法为使用递增浓度的索拉非尼处理
Figure PCTCN2021102606-appb-000010
CD4+ T细胞，48h后，收细胞，进行细胞计数，结果见图2。
结果显示，索拉非尼处理不会影响
Figure PCTCN2021102606-appb-000011
CD4+ T细胞的数量，对
Figure PCTCN2021102606-appb-000012
CD4+ T细胞没有细胞毒性。
表2 图2数据
Figure PCTCN2021102606-appb-000013
实施例3、索拉非尼能够预防NOD小鼠1型糖尿病的发生
一、材料与方法
1、小鼠：选择研究1型糖尿病最广泛使用的自发性1型糖尿病小鼠模型---NOD小鼠，由于雌性NOD小鼠自发性1型糖尿病发病率远高于雄性小鼠，所以本发明使用雌性NOD小鼠进行实验。本发明的雌性NOD小鼠购于上海南方模式生物科技有限公司。
2、配药：用羧甲基纤维素(carboxymethylcellulose，CMC)作为溶剂，配药前先将CMC高压灭菌，在超净工作台将片剂的索拉非尼研磨成粉末，然后称取适量的索拉非尼粉末，使其均匀的悬浮在溶剂里，配好的药物置于4°保存。
3、给药：本发明采用灌胃的形式给小鼠服药，索拉非尼给药浓度为10mg/kg体重。
4、环磷酰胺注射：给NOD小鼠注射300mg/kg体重的环磷酰胺可迅速诱导其1型糖尿病发病。
5、测血糖：剪去小鼠尾尖1mm左右，待其出血后用纱布拭去第一滴血，等第二滴血自然流出，用血糖试纸吸取并读数，连续两次血糖读数>250mg/dl即可诊断为1型糖尿病。
6、观察索拉非尼对NOD小鼠自发性1型糖尿病的预防作用：小鼠分为两组，一组为灌索拉非尼组，一组为灌CMC组。从8周龄开始，对小鼠每天灌胃一次，连续灌胃12周，观察并统计小鼠1型糖尿病自然发病率。
7、观察索拉非尼对NOD小鼠环磷酰胺诱导的1型糖尿病发生的预 防作用：小鼠分为两组，一组为灌索拉非尼组，一组为灌CMC组。从8周龄开始，对小鼠每天灌胃一次，连续灌胃7周，在灌胃4周后对每只小鼠进行环磷酰胺注射以诱导1型糖尿病的发作，观察并统计小鼠1型糖尿病发病率。
二、结果分析
图3中，a为索拉非尼对NOD小鼠自发性1型糖尿病的预防作用，观察到32周龄，索拉非尼组的小鼠1型糖尿病发病率为48％，而CMC组发病率为75％；b为索拉非尼对NOD小鼠环磷酰胺诱导的1型糖尿病发生的预防作用，环磷酰胺注射后引起NOD小鼠迅速发病，CMC组超半数发生1型糖尿病，而索拉非尼组发病率仅24％，停止灌药后索拉非尼组对1型糖尿病的预防作用仍然存在，并持续到20周；这提示索拉非尼对于1型糖尿病具有预防性治疗作用。
表3 图3a数据
Figure PCTCN2021102606-appb-000014
灌CMC(vehicle)组小鼠是20只，灌索拉非尼(sora)组小鼠是23只；在不同时间点对两组小鼠进行生存分析： +代表P值<0.1。
表4 图3b数据
Figure PCTCN2021102606-appb-000015
灌CMC(vehicle)组小鼠是14只，灌索拉非尼(sora)组小鼠是17只；在不同时间点对两组小鼠进行生存分析：*代表P值<0.05。
实施例4、索拉非尼能够改善NOD小鼠的胰岛炎情况
方法为NOD小鼠分为两组，一组为灌索拉非尼组，一组为灌CMC组。从8周龄开始，对小鼠每天灌胃一次，连续灌胃4周，将未发病的小鼠处死，取胰腺，甲醛固定，切片，HE染色，在显微镜下观察胰岛情况，拍照。
结果显示，图4索拉非尼组胰岛炎情况比CMC组明显改善，单核细胞浸润明显减少。
实施例5、索拉非尼减少NOD小鼠胰腺Th1细胞的浸润
方法为小鼠分为两组，一组为灌索拉非尼组，一组为灌溶剂组，10周龄开始灌胃处理小鼠4周，在灌胃处理两周后给予环磷酰胺注射诱导发病，处死小鼠，取胰腺，用一小部分提RNA做RT-PCR，剩下的大部分做流式细胞术。
结果：图5中a为流式细胞术检测两组小鼠胰腺中CD4+ T细胞亚群(Th1，Th2，Th17和Treg)的变化，结果显示索拉非尼组胰腺中的Th1细胞比例显著减少，而其他T细胞亚群比例不变；b为RT-PCR检测胰腺中Th1细胞标志基因(Tbet和IFNγ)和促炎细胞因子(IL-1β，IL-6和TNF-α)的基因表达水平，结果显示索拉非尼组胰腺中Th1细胞标志基因 (Tbet和IFNγ)和促炎细胞因子(IL-1β，IL-6和TNF-α)的基因表达水平显著减少；这说明索拉非尼通过减少NOD小鼠胰腺Th1细胞的浸润，来阻止1型糖尿病的发展。
表5 图5a数据
Figure PCTCN2021102606-appb-000016
每组设2或3个平行孔，sora组与vehicle组进行比较，用独立样本t检验进行统计学分析：*代表P值<0.05.
表6 图5a数据
Figure PCTCN2021102606-appb-000017
表7 图5b数据
Figure PCTCN2021102606-appb-000018
每组2或3只小鼠，sora组与vehicle组进行比较，用独立样本t检验进行统计学分析： +代表P值<0.1，*代表P值<0.05。
实施例6、索拉非尼通过抑制Stat4的磷酸化抑制IL-12诱导的Th1细胞分化
方法为使用递增浓度的索拉非尼(浓度梯度：0，1，2，4μg/ml)处理
Figure PCTCN2021102606-appb-000019
CD4+ T细胞，用DMSO补齐至等体积，设3个平行重复组，然后每个孔加入等量的IL-12，48小时后收细胞，提蛋白，做蛋白质印迹试验(western blot，WB)检测STAT4和p-STAT4的蛋白水平。
结果：图6中，a为WB结果，b为定量结果；结果显示索拉非尼能够抑制STAT4的磷酸化，并且索拉非尼抑制STAT4激活的剂量与抑制Th1分化的剂量完全匹配，这说明索拉非尼通过抑制STAT4的激活来抑制IL-12诱导的Th1细胞分化。
表8 图6b数据
Figure PCTCN2021102606-appb-000020
每组设3个平行孔，不同浓度组分别与0μg/ml进行比较，用独立样本t检验进行统计学分析： +代表P值<0.1，*代表P值<0.05，**代表P值<0.01，***代表P值<0.001。
以上对本发明所提供的索拉非尼的新应用，尤其是索拉非尼在预防性治疗1型糖尿病中的应用进行了详细介绍。本文应用了具体个例对本发明的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。

Claims (8)

  1. 索拉非尼或其衍生物在制备抑制IL-12诱导的Th1细胞分化的药物中的应用。
  2. 索拉非尼或其衍生物在制备预防和/或治疗1型糖尿病的药物中的应用。
  3. 索拉非尼或其衍生物在制备改善1型糖尿病胰岛功能的药物中的应用。
  4. 如权利要求3所述的应用，其特征在于，改善1型糖尿病胰岛功能为改善胰岛炎或减少单核细胞浸润。
  5. 索拉非尼或其衍生物在制备减少胰腺Th1细胞的浸润的药物中的应用。
  6. 索拉非尼或其衍生物在制备抑制STAT4激活的药物中的应用。
  7. 如权利要求1～6任一项所述的应用，其特征在于，所述药物还包括药学上可接受的辅料。
  8. 如权利要求1～6任一项所述的应用，其特征在于，所述药物的剂型为口服制剂或注射制剂。
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Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
"Encyclopedia of Traditional Chinese Medicine: Medical Immunology, 1st Edition", 31 December 2018, CHINA UNION MEDICAL COLLEGE PRESS, CN, ISBN: 978-7-5679-1019-5, article CAO, XUETAO ET AL.: "Th1", pages: 234 - 239, XP009534510 *
LI, RONGRONG ET AL.: "The Effects of the Changes of CD68/CD22 Positive Cells in Pancreatic Islet and the Serum IL-12 and IL-4 Levels on Type 1 Diabetes Mellitus Mice", IMMUNOLOGICAL JOURNAL, vol. 30, no. 12, 31 December 2014 (2014-12-31), pages 1048 - 1052, XP055900595, ISSN: 1000-8861 *
LIN JIUNN-CHANG, HUANG WEI-PANG, LIU CHIEN-LIANG, LEE JIE-JEN, LIU TSANG-PAI, KO WEN-CHIN, HUANG YU-CHUEN, HSU MING-LING, WU CHIH-: "Sorafenib Induces Autophagy in Human Myeloid Dendritic Cells and Prolongs Survival of Skin Allografts", TRANSPLANTATION, vol. 95, no. 6, 27 March 2013 (2013-03-27), GB , pages 791 - 800, XP055900398, ISSN: 0041-1337, DOI: 10.1097/TP.0b013e31827fac48 *

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