Murthy J N, Davis D L, Yatscoff R W, Soldin S J
Department of Laboratory Medicine, Children's National Medical Center, Washington, DC 20010, USA.
Clin Biochem. 1998 Nov;31(8):613-7. doi: 10.1016/s0009-9120(98)00086-1.
Tacrolimus (FK506) is an immunosuppressive drug with great clinical promise. There is a controversy regarding the role of tacrolimus metabolites in immunosuppression and toxicity, and immunoassays and immunophilin binding assays have not been adequately tested for metabolite cross-reactivity. Methods are limited to HPLC and HPLC-MS for quantifying the parent drug. Mixed lymphocyte culture assay (MLC) is the preferred functional bioassay for the measurement of parent drug and active metabolites but it is not practical for routine laboratory use. Due to differences in assay methods and reagent specificity, the concentration of tacrolimus in a given specimen may vary among different assay kit manufacturers. The objective of this study was to evaluate the degree of cross-reactivity or interference of the three first-generation tacrolimus metabolites [13-O-demethyl (M-I), 31-O-demethyl (M-II) and 15-O-demethyl (M-III)] among two different tacrolimus immunoassays (Immunoassay: PRO-Trac II FK506, Abbott IMx tacrolimus-II); and the radioreceptor assays (RRA) using minor immunophilins (14, 37, and 52 kDa immunophilins) and tacrolimus binding protein (FKBP12).
First-generation tacrolimus metabolites (M-I, M-II, and M-III) spiked in drug-free whole blood were assayed with RRA using three minor immunophilins (14, 37, and 52 kDa) and two commercial immunoassay procedures (Incstar PRO-Trac II tacrolimus, Abbott IMx tacrolimus II). The results were compared to previously published FKBP-12 RRA data and their immunosuppressive potency.
The first generation tacrolimus metabolites (M-I, M-II, and M-III) were tested using concentrations of 10 and 20 ng/mL. The significance of the metabolite interference (% of the total interference) was calculated based on the relative concentration of each metabolite present at steady-state trough concentrations in renal transplant recipients (22). Metabolite I, which has no functional immunosuppressive activity showed minimal interference compared to M-II and M-III in all assays except the 14 kDa RRA. The Incstar PRO-Trac II tacrolimus assay showed the least M-I interference. Metabolite-II, which has a pharmacologic potency similar to the parent drug, showed a significant interference in the immunoassays and significant interference in radioreceptor assays. Metabolite III, which is pharmacologically inactive, produces 3-10% interference in the different assays if its presence in the blood is 6% of the parent drug. The total interference from these three metabolites was greater in the immunoassays than in the receptor assays. Receptor assays for tacrolimus provide results closer to the target value than do immunoassays.
他克莫司（FK506）是一种具有巨大临床应用前景的免疫抑制药物。关于他克莫司代谢产物在免疫抑制和毒性方面的作用存在争议，并且免疫测定法和免疫亲和素结合测定法尚未对代谢产物交叉反应性进行充分测试。目前方法仅限于高效液相色谱法（HPLC）和HPLC - 质谱法用于定量母体药物。混合淋巴细胞培养试验（MLC）是测量母体药物和活性代谢产物的首选功能生物测定法，但它不适合常规实验室使用。由于测定方法和试剂特异性的差异，给定标本中他克莫司的浓度在不同测定试剂盒制造商之间可能会有所不同。本研究的目的是评估两种不同的他克莫司免疫测定法（免疫测定法：PRO - Trac II FK506，雅培IMx他克莫司 - II）以及使用次要免疫亲和素（14、37和52 kDa免疫亲和素）和他克莫司结合蛋白（FKBP12）的放射受体测定法（RRA）中三种第一代他克莫司代谢产物[13 - O - 去甲基（M - I）、31 - O - 去甲基（M - II）和15 - O - 去甲基（M - III）]的交叉反应程度或干扰情况。
将添加到无药物全血中的第一代他克莫司代谢产物（M - I、M - II和M - III），使用三种次要免疫亲和素（14、37和52 kDa）以及两种商业免疫测定程序（Incstar PRO - Trac II他克莫司，雅培IMx他克莫司II）通过RRA进行测定。将结果与先前发表的FKBP - 12 RRA数据及其免疫抑制效力进行比较。
使用10和20 ng/mL的浓度对第一代他克莫司代谢产物（M - I、M - II和M - III）进行测试。基于肾移植受者稳态谷浓度下每种代谢产物的相对浓度计算代谢产物干扰的显著性（总干扰的百分比）（22）。除14 kDa RRA外，在所有测定中，无功能性免疫抑制活性的代谢产物I与M - II和M - III相比显示出最小的干扰。Incstar PRO - Trac II他克莫司测定显示M - I干扰最小。药理效力与母体药物相似的代谢产物II在免疫测定中显示出显著干扰，在放射受体测定中也有显著干扰。药理上无活性的代谢产物III，如果其在血液中的存在量为母体药物的6%，则在不同测定中产生3 - 10%的干扰。这三种代谢产物在免疫测定中的总干扰大于在受体测定中的干扰。他克莫司的受体测定提供的结果比免疫测定更接近目标值。
