西罗莫司与未成熟树突状细胞延长小鼠皮肤移植存活时间的协同作用

目的 研究西罗莫司（SRL）对小鼠骨髓源树突状细胞（DC）分化及成熟的影响，观察西罗莫司与未成熟树突状细胞在延长小鼠皮肤移植存活时间中的协同作用。方法 （1）在诱导C57BL／6小鼠骨髓细胞定向分化为DC时加入SRL，通过流式细胞仪检测CD11c、CD86及MHCⅡ类分子表达情况，经脂多糖（LPS）刺激后，再检测各分子表达的变化。（2）通过单向混合淋巴细胞反应（MLR）观察经SRL处理的DC刺激同种异基因小鼠T细胞增殖情况。（3）以C57BL／6小鼠为供者，BALB／c小鼠为受者建立皮肤移植模型。观察皮肤移植前7d经尾静脉注射供者未成熟DC及经胃管连续灌注SRL7d的受者移植皮片存活情况及组织学变化。结果 （1）经SRL处理的DC表面CD11c表达仅有轻度降低，但CD86和MHCⅡ类分子表达明显减少。（2）MLR显示经SRL处理的DC刺激同种异基因小鼠T细胞增殖的能力降低。（3）受者皮肤移植术前联合应用供者未成熟DC和SRL，可减轻移植皮片炎症反应并延长其存活时间。结论 SRL对DC分化的影响不明显，但可抑制DC发育成熟。受者术前应用SRL和未成熟DC可延长皮肤移植的存活时间。     

3L自动发酵罐中西罗莫司的发酵研究

采用美国NBS公司生产的BIOFLO3000型3L发酵罐进行西罗莫司发酵代谢过程的考察和代谢参数的累积,建立了较为适应的西罗莫司发酵条件,罐上发酵水平与摇瓶发酵水平相当.     

Use of Sirolimus in the Treatment of Refractory Autoimmune Hepatitis

BackgroundCorticosteroids and azathioprine are widely accepted as the initial therapy for autoimmune hepatitis. However, the disease is refractory to steroids in about 10%-20% of patients, for whom currently there is no standardized treatment. Here we describe our experience with sirolimus in treatment of steroid refractory autoimmune hepatitis.MethodsThis is a longitudinal follow-up study. Between November 2007 and January 2014, 5 subjects with steroid refractory autoimmune hepatitis were treated with sirolimus at our institution.ResultsA response, defined as a sustained >50% fall in alanine aminotransferase (ALT) levels, was achieved in 4/5 patients. A complete response, sustained normalization of ALT levels, was achieved in 2/5 patients. The need for steroids was significantly reduced in all patients (P < .05).ConclusionsIn this small series, sirolimus appears to be useful in the treatment of patients with steroid refractory autoimmune hepatitis.     

Five-year clinical outcomes of the first Korean-made sirolimus-eluting coronary stent with abluminal biodegradable polymer

This study evaluated the 5-year clinical outcomes of the Genoss DES, the first Korean-made sirolimus-eluting coronary stent with abluminal biodegradable polymer.We previously conducted the first-in-patient prospective, multicenter, randomized trial with a 1:1 ratio of patients using the Genoss DES and Promus Element stents; the angiographic and clinical outcomes of the Genoss DES stent were comparable to those of the Promus Element stent. The primary endpoint was major adverse cardiac events (MACE), which was a composite of death, myocardial infarction (MI), and target lesion revascularization (TLR) at 5 years.We enrolled 38 patients in the Genoss DES group and 39 in the Promus Element group. Thirty-eight patients (100%) from the Genoss DES group and 38 (97.4%) from the Promus Element group were followed up at 5 years. The rates of MACE (5.3% vs 12.8%, P = .431), death (5.3% vs 10.3%, P = .675), TLR (2.6% vs 2.6%, P = 1.000), and target vessel revascularization (TVR) (7.9% vs 2.6%, P = .358) at 5 years did not differ significantly between the groups. No TLR or target vessel revascularization was reported from years 1 to 5 after the index procedure, and no MI or stent thrombosis occurred in either group during 5 years.The biodegradable polymer Genoss DES and durable polymer Promus Element stents showed comparable low rates of MACE at the 5-year clinical follow-up.     

西罗莫司洗脱支架和Pixel支架治疗冠状动脉小血管病变的比较

目的对比研究西罗莫司洗脱支架(CypherTM支架)和Pixel支架治疗冠状动脉小血管病变的安全性及疗效。方法将2003年3月至2005年1月在我院住院的67例小血管病变患者随机分成A、B两组,A组34例接受CypherTM支架治疗,B组33例接受Pixel支架治疗。比较分析两组的手术成功率、并发症发生率、心脏不良事件发生率、再狭窄率及晚期管腔丢失等指标。结果两组支架置入术的成功率均为100%,无残余狭窄或残余狭窄<10%,无任何并发症。A组有30例、B组有29例患者在术后约9个月复查了定量冠状动脉造影。随访期间,A组中有2例出现支架内再狭窄导致临床心绞痛复发;B组有7例复发心绞痛,其中6例经冠状动脉造影证实为支架内再狭窄所致;两组均无一例死亡。A组的造影再狭窄率为6.7%,B组为20.7%(P<0.05);A组的晚期管腔丢失为0.19±0.58mm,B组为0.63±0.61mm(P<0.01);A组的靶血管再次血运重建率为6.7%,B组为20.7%(P<0.05)。结论CypherTM支架和Pixel支架治疗小血管病变均安全、有效,但CypherTM支架的远期疗效明显优于Pixel支架。     

顺铂联合雷帕霉素或3-甲基腺嘌呤对肺腺癌A549细胞抑制作用的研究

目的观察非小细胞肺癌(non small cell lung cancer,NSCLC)中肺腺癌A549细胞在顺铂(cisplatin,CP)联合雷帕霉素(rapamycin,RAPA)或3-甲基腺嘌呤(3-methyladenine,3-MA)作用下的结果,为提高顺铂的化疗效果提供理论依据。方法 2013年3月至2014年6月期间,通过对人肺腺癌细胞株A549(由河北医科大学第四医院科研中心提供)经四甲基偶氮唑盐3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT)实验检测顺铂、雷帕霉素和3-MA对A549细胞的增殖抑制率,计算出各药物的50%细胞抑制浓度(50%inhibitory concentration,IC50),并以此作为实验浓度。实验分为4组:对照组(无药物干预)、顺铂组(加15μmol/L的顺铂)、顺铂+雷帕霉素组(加10nmol/L的雷帕霉素1h后再加15μmol/L的顺铂)、顺铂+3-MA组(加3μmol/L的3-MA 1h后再加15μmol/L的顺铂),将对数生长期的肺癌A549细胞以每孔1.0×10^6/ml密度接种于6孔培养板中,待细胞长至孔底面积约70%~80%后分别加入稀释好的药物,分别培养24、48、72h。肺癌A549细胞的生长迁移情况用细胞划痕实验检测,mTOR、LC3-Ⅱ及Bax mRNA和蛋白的表达情况用RT-PCR(逆转录-聚合酶链反应)、Western blot(蛋白质印迹)法检测。数据处理采用SPSS 17.0统计软件进行分析。结果顺铂IC50:15μmol/L;雷帕霉素IC50:10nmol/L;3-MA IC50:3μmol/L。划痕实验显示24h顺铂+雷帕霉素组细胞迁移能力最弱,48h和72h顺铂+3-MA组细胞迁移能力最弱。RT-PCR显示顺铂+雷帕霉素组LC3-ⅡmRNA的2-△△Ct值(24h:1.686±0.069;48h:1.803±0.083;72h:1.836±0.056)与顺铂组(24h:1.489±0.031;48h:1.325±0.007;72h:1.428±0.080)相比表达量均明显上升(24hF=149.780,P<0.01;48hF=111.599,P<0.01;72hF=167.855,P<0.01);而顺铂+3-MA组的Bax mRNA的2-△△Ct值(48h:1.864±0.104;72h:1.935±0.068),与顺铂组(48h:1.346±0.080,72h:1.462±0.029)相比,表达量均明显上升(48hF=52.853,72hF=202.118;P值均<0.01)。Western blot显示顺铂+雷帕霉素组LC3-Ⅱ蛋白(48h:0.556±0.010;72h:0.571±0.009)与顺铂组(48h:0.426±0.0107;72h:0.492±0.009)相比表达量均显著上升(48hF=372.056,72hF=930.500;P值均<0.01);而顺铂+3-MA组的Bax蛋白(48h:0.897±0.022;72h:0.916±0.005),与顺铂组(48h:0.463±0.011;72h:0.581±0.007)相比,表达量均显著上升(48hF=1100.412,72hF=5715.778;P值均<0.01)。结论顺铂联合雷帕霉素或3-MA较单独使用顺铂能更好的抑制A549细胞的生长,长时间用药时顺铂联合3-MA作用最强。     

Propranolol for infantile hepatic hemangioendothelioma: Clinical evaluation of drug efficacy and safety using a single-center patient cohort

Introduction and objectivesInfantile hepatic hemangioendothelioma (IHHE) is a benign liver tumor, associated with hypothyroidism and vascular malformations along the skin, brain, digestive tract and other organs. Here, we determined a single-center patient cohort by evaluating the effectiveness and safety of propranolol and sirolimus for the treatment of IHHE.Patients and methodsWe performed a monocentric and observational study, based on clinical data obtained from 20 cases of IHHE treated with oral propranolol and sirolimus at the Shanghai Children's Medical Center (SCMC), between December 2017 and April 2019. All cases were confirmed by abdominal enhanced CT examination (18/20, 90%) and sustained decrease of alpha fetoprotein (AFP) (2/20, 10%).Propranolol treatment was standardized as once a day at 1.0 mg/kg for patients younger than 2 months, and twice a day at 1.0 mg/kg (per dose) for patients older than 2 months. Sirolimus was used to treat refractory IHHE patients after 6 months of propranolol treatment, and initial dosing was at 0.8 mg/m² body surface per dose, administered every 12 h. Upon treatment, abdominal ultrasound scanning was regularly performed to evaluate any therapeutic effects. All children were followed up for 6–22 months (mean value of 12.75 months). The clinical manifestations and therapeutic effects, including complications during drug management, were reviewed after periodic follow-up.ResultsThe effective rate of propranolol for the treatment of children with IHHE was 85% (17/20). In most cases, the AFP levels gradually decreased into the normal range. A complete response (CR) was achieved in 3 cases, partial response (PR) for 14 cases, progressive disease (PD) for 2 cases and stable disease (SD) was only detected once. Lesions decreased in two PD patients after administration of oral sirolimus. No serious adverse reactions were observed.ConclusionThis study indicates that both propranolol and sirolimus were effective drugs for the treatment of children with IHHE at SCMC.     

Targeting of the Hedgehog/GLI and mTOR pathways in advanced pancreatic cancer,a phase 1 trial of Vismodegib and Sirolimus combination

Background/ObjectivesPreclinical data indicated a functional and molecular interaction between Hedgehog (HH)/GLI and PI3K-AKT-mTOR pathways promoting pancreatic ductal adenocarcinoma (PDAC). A phase I study was conducted of Vismodegib and Sirolimus combination to evaluate maximum tolerated dose (MTD) and preliminary anti-tumor efficacy.MethodsCohort I included advanced solid tumors patients following a traditional 3 + 3 design. Vismodegib was orally administered at 150 mg daily with Sirolimus starting at 3 mg daily, increasing to 6 mg daily at dose level 2. Cohort II included only metastatic PDAC patients. Anti-tumor efficacy was evaluated every two cycles and target assessment at pre-treatment and after a single cycle.ResultsNine patient were enrolled in cohort I and 22 patients in cohort II. Twenty-eight patients were evaluated for dose-limiting toxicities (DLTs). One DLT was observed in each cohort, consisting of grade 2 mucositis and grade 3 thrombocytopenia. The MTD for Vismodegib and Sirolimus were 150 mg daily and 6 mg daily, respectively. The most common grade 3–4 toxicities were fatigue, thrombocytopenia, dehydration, and infections. A total of 6 patients had stable disease. No partial or complete responses were observed. Paired biopsy analysis before and after the first cycle in cohort II consistently demonstrated reduced GLI1 expression. Conversely, GLI and mTOR downstream targets were not significantly affected.ConclusionsThe combination of Vismodegib and Sirolimus was well tolerated. Clinical benefit was limited to stable disease in a subgroup of patients. Targeting efficacy demonstrated consistent partial decreases in HH/GLI signaling with limited impact on mTOR signaling. These findings conflict with pre-clinical models and warrant further investigations.     

Mammalian target of rapamycin inhibition in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. It is associated with a poor prognosis and has limited treatment options. Sorafenib, a multi-targeted kinase inhibitor, is the only available systemic agent for treatment of HCC that improves overall survival for patients with advanced stage disease; unfortunately, an effective second-line agent for the treatment of progressive or sorafenib-resistant HCC has yet to be identified. This review focuses on components of the mammalian target of rapamycin (mTOR) pathway, its role in HCC pathogenesis, and dual mTOR inhibition as a therapeutic option with potential efficacy in advanced HCC. There are several important upstream and downstream signals in the mTOR pathway, and alternative tumor-promoting pathways are known to exist beyond mTORC1 inhibition in HCC. This review analyzes the relationships of the upstream and downstream regulators of mTORC1 and mTORC2 signaling; it also provides a comprehensive global picture of the interaction between mTORC1 and mTORC2 which demonstrates the pre-clinical relevance of the mTOR pathway in HCC pathogenesis and progression. Finally, it provides scientific rationale for dual mTORC1 and mTORC2 inhibition in the treatment of HCC. Clinical trials utilizing mTORC1 inhibitors and dual mTOR inhibitors in HCC are discussed as well. The mTOR pathway is comprised of two main components, mTORC1 and mTORC2; each has a unique role in the pathogenesis and progression of HCC. In phase III studies, mTORC1 inhibitors demonstrate anti-tumor activity in advanced HCC, but dual mTOR (mTORC1 and mTORC2) inhibition has greater therapeutic potential in HCC treatment which warrants further clinical investigation.     

Preclinical Study of a Biodegradable Polymer-based Stent with Abluminal Sirolimus Release

Background

Bioabsorbable polymer stents with drug elution only on the abluminal surface may be safer than durable polymer drug-eluting stents.

Objective

To report the experimental findings with the Inspiron^TM stent - a bioabsorbable polymer-coated stent with sirolimus release from the abluminal surface only, recently approved for clinical use.

Methods

45 stents were implanted in the coronary arteries of 15 pigs. On day 28 after implantation, angiographic, intracoronary ultrasonographic and histomorphological data were collected. Five groups were analyzed: Group I (nine bare-metal stents); Group II (nine coated with bioabsorbable polymer on the luminal and abluminal surfaces); Group III (eight stents coated with bioabsorbable polymer on the abluminal surface); Group IV (nine stents with bioabsorbable polymer and sirolimus on the luminal and abluminal surfaces); and Group V (ten stents with bioabsorbable polymer and sirolimus only on the abluminal surface).

Results

The following results were observed for Groups I, II, III, IV and V, respectively: percentage stenosis of 29 ± 20; 36 ± 14; 33 ± 19; 22 ± 13 and 26 ± 15 (p = 0.443); late lumen loss (in mm) of 1.02 ± 0.60; 1.24 ± 0.48; 1.11 ± 0.54; 0.72 ± 0.44 and 0.78 ± 0.39 (p = 0.253); neointimal area (in mm²) of 2.60 ± 1.99; 2.74 ± 1.51; 2.74 ± 1.30; 1.30 ± 1.14 and 0.97 ± 0.84 (p = 0.001; Groups IV and V versus Groups I, II and III); and percentage neointimal area of 35 ± 25; 38 ± 18; 39 ± 19; 19 ± 18 and 15 ± 12 (p = 0.001; Groups IV and V versus Groups I, II and III). Injury and inflammation scores were low and with no differences between the groups.

Conclusion

The Inspiron^TM stent proved to be safe and was able to significantly inhibit the neointimal hyperplasia observed on day 28 after implantation in porcine coronary arteries.