慢性肾功能损伤的肝移植受者转换为西罗莫司治疗的疗效观察

目的 观察慢性肾功能损伤的肝移植受者转换为西罗莫司治疗的疗效.方法 应用钙调磷酸酶抑制剂(CNI)并伴有慢性肾功能损伤的肝移植受者23例(其中19例应用他克莫司,4例应用环孢素A)转换为西罗莫司(SRL)治疗.SRL的起始剂量为4mg/d,次日为2 mg/d,应用高压液相色谱法测定全血SRL浓度,当血SRL浓度达5～8 μg/L后,停用CNI类药物,同时服用吗替麦考酚酯,1 g/d.记录受者入组前的基础血清肌酐(Cr)、肌酐清除率、肾小球滤过率(GFR),并分别于用药后第1、3、6、12和24个月时监测血SRL浓度、Cr、肌酐清除率、GFR,同时监测受者体重、血压、血细胞计数、肝功能和肝脏生化指标、血脂、尿蛋白.于用药后12个月时行肝脏穿刺活检确认有无排斥反应.结果 23例平均随访29.4个月,随访期内死亡2例,另21例于用药后1、3、6、12和24月时的Cr分别为(147.40±23.36)、(152.60±20.08)、(150.20±22.64)、(137.60±18.09)、(138.30±17.04)μmol/L,与Cr的基础值[(158.91±29.13)μmol/L]相比较,1、12、24个月时的差异有统计学意义(P＜0.05).用药后1、3、6、12和24月时的肌酐清除率分别为(0.97±0.18)、(0.99±0.14)、(1.00±0.17)、(1.07±0.29)、(1.14±0.12)ml/s,与基础肌酐清除率[(0.91±0.14)ml/s]相比较,1、12、24个月时的差异有统计学意义(P＜0.05).用药后1、3、6、12和24月时的GFR分别为(0.80±0.15)、(0.78±0.11)、(0.75±0.12)、(0.84±0.10)、(0.94±0.13),与基础GFR[(0.71±0.11)ml/s]相比较,1、12、24个月时的差异有统计学意义(P＜0.05).应用SRL后第1、3、6、12和24个月时,Cr≤123μmol/L者所占的比例分别为38.1％、33.3％、28.6％、47.6％和52.4％.随访期内无受者发生排斥反应.结论 慢性肾功能损伤的肝移植受者转换为西罗莫司治疗可改善其肾功能.转换治疗未增加排斥反应的发生率.     

西罗莫司对双侧输尿管梗阻大鼠水电解质代谢紊乱的保护作用

目的 探讨西罗莫司对双侧输尿管梗阻(BUO)大鼠水电解质代谢紊乱及肾脏上皮钠通路γ(γ-ENaC)、Na+-K+-ATP酶及水通道蛋白2(AQP2)蛋白的影响.方法 Wistar大鼠48只,随机分为假手术组、BUO组和西罗莫司组,每组16只.BUO组和西罗莫司组大鼠结扎双侧输尿管制作BUO模型,假手术组仅游离双侧输尿管后缝合.BUO组及西罗莫司组双侧输尿管梗阻24 h后解除梗阻.西罗莫司组每天西罗莫司口服液(2.5 ml/次)灌胃,假手术组及UUO组用同体积生理盐水灌胃.于术后4、7d测量3组大鼠尿量并留取尿液进行生化分析;术后4、7d抽取动脉血化验,同时取出双侧肾脏,采用免疫组化及蛋白质印迹法检测肾脏γ-ENaC、Na+-K+-ATP酶及AQP2蛋白的表达.结果 BUO组大鼠输尿管梗阻解除后4、7d尿量分别为(85.31±13.15)、( 66.39±10.56)ml,显著多于假手术组( 35.36±7.74)、(33.90±8.03)ml及西罗莫司组(69.81±10.70)、(48.57±9.01)ml;尿钠浓度(42.17±7.35)、(43.63±18.39) mmol/L,显著低于假手术组(170.56±18.39)、(172.52±7.35) mmol/L及西罗莫司组(76.18±13.20)、(134.28±13.20) mmol/L,3组间比较差异均有统计学意义(P＜0.05).西罗莫司组4、7d肾脏γ-ENaC表达量分别为2.09±0.32、2.27±0.35,Na+ -K+ -ATP酶分别为2.41±0.48、2.67±0.43,AQP2分别为2.17±0.45、2.63±0.28,显著高于同时间点的BUO组(1.28±0.21、1.45±0.17,1.99±0.28、2.18±0.24,0.93±0.22、1.31±0.16),低于同时间点假手术组(2.58±0.51、2.60±0.56,2.89±0.53、2.97±0.66,3.05±0.63、3.10±0.67),3组间比较差异均有统计学意义(P＜0.05).结论 γ-ENaC、Na+-K+-ATP酶及AQP2蛋白水平降低可能是泌尿系梗阻后肾小管性钠回吸收障碍、低渗性尿量增多的主要原因之一.西罗莫司可以通过抑制肾小管γ-ENaC、Na+-K+-ATP酶及AQP2蛋白丢失,减轻术后肾脏钠回吸收障碍及低渗性尿等水电解质代谢紊乱,保护肾脏功能.     

Rapamycin retards growth and causes marked alterations in the growth plate of young rats

Rapamycin is a potent immunosuppressant with antitumoral properties widely used in the field of renal transplantation. To test the hypothesis that the antiproliferative and antiangiogenic activity of rapamycin interferes with the normal structure and function of growth plate and impairs longitudinal growth, 4-week-old male rats (n = 10/group) receiving 2 mg/kg per day of intraperitoneal rapamycin (RAPA) or vehicle (C) for 14 days were compared. Rapamycin markedly decreased bone longitudinal growth rate (94 ± 3 vs. 182 ± 3 μm/day), body weight gain (60.2 ± 1.4 vs. 113.6 ± 1.9 g), food intake (227.8 ± 2.6 vs. 287.5 ± 3.4 g), and food efficiency (0.26 ± 0.00 vs. 0.40 ± 0.01 g/g). Signs of altered cartilage formation such as reduced chondrocyte proliferation (bromodeoxiuridine-labeled cells 32.9 ± 1.4 vs. 45.2 ± 1.1%), disturbed maturation and hypertrophy (height of terminal chondrocytes 26 ± 0 vs. 29 ± 0 μm), and decreased cartilage resorption (18.7 ± 0.5 vs. 31.0 ± 0.8 tartrate-resistant phosphatase alkaline reactive cells per 100 terminal chondrocytes), together with morphological evidence of altered vascular invasion, were seen in the growth plate of RAPA animals. This study indicates that rapamycin can severely impair body growth in fast-growing rats and distort growth-plate structure and dynamics. These undesirable effects must be kept in mind when rapamycin is administered to children.     

Early experience with conversion to sirolimus in a pediatric renal transplant population

Sirolimus is an immunosuppressive agent that offers potentially significant benefits for young transplant patients facing life-long treatment. Its action of reducing cell proliferation may reduce the risk of chronic allograft nephropathy and posttransplant neoplasia. Twenty-nine children were converted from calcineurin inhibitors to sirolimus after renal transplantation and followed for a minimum of 12 months. Glomerular filtration increased transiently in those converted before 12 months after transplantation but not in those converted later, when chronic histological changes had developed. Mild acute rejection occurred after conversion in 10%, and side effects led to cessation of sirolimus in 31%. Anemia occurred in 55% of patients and responded well to darbepoetin. Most side effects (anemia, hypercholesterolemia, mouth ulcers, and myalgias) became less severe with time. The number of antihypertensive drugs required decreased significantly on sirolimus. Although side effects are frequent on sirolimus, in the majority of children, they are mild enough to allow the patient to continue taking the drug, and for these children the long-term benefits are potentially valuable.     

Multiple coronary artery aneurysm formation one year and four years after sirolimus-eluting coronary stent implantation

A 61 years old male patient having the complaint of angina pectoris underwent coronary angiography that revealed critical luminal stenosis at left anterior descending artery (LAD) and diagonal branch. Percutaneous transluminal coronary angioplasty was performed and sirolimus eluting stents (CYPHER; Johnson & Johnson – Cordis) 2.75 ×10 mm and 2.75×10 mm were deployed respectively. One year after the procedure coronary angiography revealed coronary artery aneurysm (CAA) formation in diagonal branch and mild ectasia in LAD stent area. Last coronary angiography four years and three months after initial intervention showed multiple CAA at diagonal branch and LAD. This case report is an example of late formation of CAA by sirolimus-coated coronary stent.     

Sirolimus inhibits the growth and metastatic progression of hepatocellular carcinoma

Purpose  Immunosuppressive therapy after liver transplantation for hepatocellular carcinoma (HCC) is one of the major contributory factors for HCC recurrence and metastasis. Sirolimus, a potent immunosuppressant, has been reported to be an effective inhibitor in a variety of tumors. The present study is designed to explore whether sirolimus could block the growth and metastatic progression of HCC. Methods  MHCC97H cells were used as targets to explore the effect of sirolimus on cell cycle progression, apoptosis, proliferation, and its antiangiogenic mechanism. LCI-D20, a highly metastatic model of human HCC in nude mice, was also used as the model tumor to explore the effect of sirolimus on tumor growth and metastatic progression. Results  In vitro, sirolimus induced cell cycle arrest at the G1 checkpoint and blocked proliferation of MHCC97H cells but did not induce apoptosis. In vivo, sirolimus prevented tumor growth and metastatic progression in LCI-D20. Intratumoral microvessel density and circulating levels of VEGF in tumor-bearing mice were also significantly reduced in sirolimus treatment group. Quantitative RT-PCR showed that sirolimus down-regulated the mRNA expression of VEGF and HIF-1a, but not of bFGF, and TGF-b in MHCC97H cells. Furthermore, western blot analysis confirmed that sirolimus also decreased expression of HIF-1a at protein level, in parallel with the down-regulation of the levels of VEGF protein excretion in a time-dependent manner as compared to untreated control cells following anoxia. Conclusions  The immunosuppressive macrolide sirolimus prevents the growth and metastatic progression of HCC, and suppresses VEGF synthesis and secretion by downregulating HIF-1a expression. Sirolimus may be useful for clinical application in patients who received a liver transplant for HCC. Z. Wang and J. Zhou contributed equally to this work.     

Multi-center evaluation of analytical performance of the microparticle enzyme immunoassay for sirolimus

OBJECTIVES: This study evaluated the analytical characteristics of the new Abbott microparticle enzyme immunoassay (MEIA) for sirolimus. DESIGN AND METHODS: The protocol consisted of nine sections: evaluation of antibody specificity, linearity, detection limit, quantification limit, endogenous interferents, exogenous interferents, precision, proficiency testing panel, and method comparison. RESULTS: The mean analytical detection limit was 0.68 microg/L. The sirolimus concentration corresponding to a total CV of 20% was 1.5 microg/L. Linearity of response was demonstrated across the dynamic range of the assay. Total precision (CVs) at QC control levels from 5 to 22 microg/L ranged from 5.7 to 12.6%. Assay standardization was found to be in good agreement with LC/MS/MS as compared with target values for spiked sirolimus proficiency samples from an international sirolimus proficiency testing program. Good correlations (R values) of the immunoassay were observed in comparisons to LC/MS/MS. R values tended to be lower in comparisons with LC/UV methods. Across both LC-based methods and all study sites, there was approximately 25% overall positive slope bias due to cross reactivity of the MEIA antibody to metabolites of sirolimus. The assay cross-reactivity to metabolites of sirolimus parent drug ranged from 6 to 63%. Assay interferences were minimal with the exception of hematocrit, which presented a negative relationship to measured sirolimus concentration. CONCLUSIONS: The MEIA demonstrated acceptable analytical characteristics for use for routine monitoring of sirolimus immunosuppressive therapy, and is a viable alternative to HPLC-based methods for sirolimus monitoring.     

The impact of the mTOR inhibitor sirolimus on the proliferation and function of pancreatic islets and ductal cells

Aims/hypothesis

The Edmonton Protocol for islet transplantation has provided hope for type 1 diabetic patients. However, this protocol requires lifelong immunosuppression, specifically sirolimus, a cellular antiproliferate. The effect of sirolimus on human pancreatic ductal cells (HDCs) is not known. This may be important since HDCs are believed to be islet precursors. Since neonatal porcine islets (NPIs), which contain many ductal precursor cells, could be a potential clinical source of islets, we also tested the effects of sirolimus on this tissue.

Methods

HDCs (n=4), NPIs (n=9) and human islets (n=5) were cultured with and without sirolimus (20 ng/ml) for 6 days.

Results

HDCs and NPIs cultured with sirolimus showed a 50 and 28% decrease, respectively, in cell number relative to control (p<0.05). Control cultures expanded 1.65- and 2.44-fold relative to time 0. Decreases in cell number of sirolimus-treated HDCs were not due to apoptosis as measured by TUNEL staining. No functional effects on human islets or NPIs were observed following static incubation with high glucose. Treatment of syngeneically transplanted and naïve BALC/c mice with sirolimus resulted in altered OGTT profiles with prolonged elevation of hyperglycaemia and weight gain. There was no difference in graft and organ insulin content between treatment groups.

Conclusions/interpretation

Our results indicate that sirolimus decreases ductal cell numbers in culture and alters glucose-stimulated insulin secretion in vivo. The administration of sirolimus to islet transplant recipients is likely to impair graft function as a result of decreasing ductal neogenesis and induction of insulin resistance.     

Plasma stable,pH-sensitive non-ionic surfactant vesicles simultaneously enhance antiproliferative effect and selectivity of Sirolimus

Abstract

Objective: The purpose of the present investigation was to prepare a plasma stable, pH-sensitive niosomal formulation to enhance Sirolimus efficacy and selectivity.

Materials and methods: pH-sensitive niosomal formulations bearing PEG-Poly (monomethyl itaconate)-CholC6 (PEG-PMMI-CholC6) copolymers and cholesteryl hemisuccinate (CHEMS) were prepared by a modified ethanol injection method and characterized with regard to pH-responsiveness and stability in human serum. The ability of pH-sensitive niosomes to enhance the Sirolimus cytotoxicity was evaluated in vitro using human erythromyeloblastoid leukemia cell line (K562) and compared with cytotoxicity effect on human umbilical vein endothelial cells (HUVEC).

Results and discussion: This study showed that both formulations can be rendered pH-sensitive property and were found to rapidly release their contents under mildly acidic conditions. However, the CHEMS-based niosomes lost their pH-sensitivity after incubation in plasma, whereas, PEG-PMMI-CholC6 niosomes preserved their ability to respond to pH change. Sirolimus encapsulated in pH-sensitive niosomes exhibited a higher cytotoxicity than the control conventional formulation on K562 cell line. On the other hand, both pH-sensitive niosomes showed lower antiproliferative effect on HUVEC cells.

Conclusion: Plasma stable, pH-sensitive PEG-PMMI-CholC6-based niosomes can improve the in vitro efficiency and also reduce the side effects of Sirolimus.     

雷帕霉素耐药机制及其逆转途径

雷帕霉素靶蛋白(mTOR)信号通路与肿瘤细胞的增殖、周期调控等多种病理过程密切相关.雷帕霉素通过抑制mTOR起到抗肿瘤作用,但其容易产生耐药等缺点导致临床应用受限,耐药机制主要与mTOR受抑后负反馈激活磷脂酰肌醇3-激酶-蛋白激酶B(PI3K-Akt)有关.mTOR通路相关蛋白的双重抑制剂等药物有望逆转其耐药.