Masri M, Rizk S, Barbari A, Stephan A, Kamel G, Rost M
Transmedical Research Institute, Beirut, Lebanon.
Transplant Proc. 2007 May;39(4):1204-6. doi: 10.1016/j.transproceed.2007.04.008.
Both tacrolimus (TAC) and sirolimus (SRL) bind to the same immunophilin FKBP12; however, their mechanisms of action are distinct. SRL inhibits mammalian target of rapamycin (TOR), which is an enzyme critical to the immune function. TOR inhibition blocks the signal that mediates T-cell proliferation by preventing cell-cycle progression from G1 to S phase. Moreover, TOR inhibition results in a decrease in antibody production by blocking B-cell proliferation and maturation into antibody producing cells. The use of SRL has resulted in a decrease in the number of rejection episodes. As with other immunosuppressive agents, SRL can cause dose-related side effects, the most notable of which are hypercholesterolemia, hyperlipidemia, anemia, and thrombocytopenia. Thus, therapeutic drug monitoring to assess efficacy and toxicity has became a necessity. SRL blood levels do not correlate with its bioactivity and are affected by the concomitant use of other immunosuppressive drugs. To determine the bioactivity of SRL we have developed an assay to determine the level of Sirolimus per lymphocyte of transplant patients. The levels were correlated with lymphocyte count.
Whole blood samples from patients on SRL were collected in Ethylene Diamine Tetra-acetic acid (EDTA) vacutainer tubes. Immediately the lymphocytes from 2 mL of blood were separated using 1.5 mL of Ficoll gradient, by centrifugation for 30 minutes at 2500 RPM. The lymphocytes were washed three times with phosphate-bufferd saline and the pellet suspended in 150 microL of Middle East research institute (MERI) drug extraction solution (Beirut, Lebanon), which was then added to 300 microL of IMx solublizing reagent. The cytoplasmic SRL concentrations in lymphocytes were measured using kits supplied from Abbott diagnostics or by high-performance liquid tomography. A corresponding whole blood sample from each patient was used to measure blood levels. To determine the level per lymphocyte, the value obtained was divided by the number of lymphocytes and expressed as Pg/cell. A pharmacokinetic profile for both blood and lymphocytes was constructed for each patient using data corresponding to predose C(0), 1 hour (C(1)) and 2 hours (C(2)) after the dose. The lymphocyte enumeration for C(0), C(1), and C(2) was performed using the FACS Calibur Flow Cytometer from Becton Dickinson. The average dose was 2.86 +/- 1.27 mg/d with a C(0) = 8.05 +/- 4.24, C(1) = 21.9 +/- 8.9 ng/mL, and C(2) = 23 +/- 0.03 ng/mL. Although there was a significant correlation (P=.0975) between the dose and C(0), there was no correlation between the dose and C(0) level on the lymphocyte count P=.897. However, there was a strong correlation between SRL lymphocyte levels (pg/cell) and the lymphocyte count (r(2)=.6.06). The higher the concentration of the drug the lower the lymphocyte counts. The assay is sensitive to within 0.45 pg/cell, reproducible with a coefficient of variance (CV) of 6.4% within assay and 7.5% for intraassay.
他克莫司（TAC）和西罗莫司（SRL）均与同一亲免素FKBP12结合；然而，它们的作用机制不同。SRL抑制雷帕霉素哺乳动物靶点（TOR），这是一种对免疫功能至关重要的酶。TOR抑制通过阻止细胞周期从G1期进展到S期来阻断介导T细胞增殖的信号。此外，TOR抑制通过阻断B细胞增殖和成熟为产生抗体的细胞导致抗体产生减少。SRL的使用导致排斥反应发作次数减少。与其他免疫抑制剂一样，SRL可引起剂量相关的副作用，其中最显著的是高胆固醇血症、高脂血症、贫血和血小板减少症。因此，进行治疗药物监测以评估疗效和毒性已成为必要。SRL血药浓度与其生物活性不相关，且受其他免疫抑制药物联合使用的影响。为了确定SRL的生物活性，我们开发了一种测定移植患者每淋巴细胞中西罗莫司水平的方法。这些水平与淋巴细胞计数相关。
用乙二胺四乙酸（EDTA）真空采血管采集接受SRL治疗患者的全血样本。立即用1.5 mL Ficoll梯度从2 mL血液中分离淋巴细胞，以2500 RPM离心30分钟。淋巴细胞用磷酸盐缓冲盐水洗涤三次，沉淀悬浮于150 μL中东研究所（MERI）药物提取溶液（黎巴嫩贝鲁特）中，然后加入300 μL IMx增溶试剂。使用雅培诊断公司提供的试剂盒或通过高效液相层析法测量淋巴细胞中的细胞质SRL浓度。使用来自每位患者的相应全血样本测量血药浓度。为了确定每淋巴细胞的水平，将获得的值除以淋巴细胞数量并表示为Pg/细胞。使用给药前C(0)、给药后1小时（C(1)）和2小时（C(2)）的数据为每位患者构建血液和淋巴细胞的药代动力学曲线。使用Becton Dickinson公司的FACS Calibur流式细胞仪进行C(0)、C(1)和C(2)的淋巴细胞计数。平均剂量为2.86±1.27 mg/d，C(0)=8.05±4.24，C(1)=21.9±8.9 ng/mL，C(2)=23±0.03 ng/mL。虽然剂量与C(0)之间存在显著相关性（P = 0.0975），但剂量与淋巴细胞计数的C(0)水平之间无相关性（P = 0.897）。然而，SRL淋巴细胞水平（pg/细胞）与淋巴细胞计数之间存在强相关性（r² = 0.606）。药物浓度越高，淋巴细胞计数越低。该测定法对0.45 pg/细胞以内的变化敏感，批内变异系数（CV）为6.4%，批间变异系数为7.5%，具有可重复性。
