Identification and activities of human carboxylesterases for the activation of CPT-11, a clinically approved anticancer drug
Identification and activities of human carboxylesterases for the activation of CPT-11, a clinically approved anticancer drug
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DOI：
10.1021/bc0155420
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发表时间：
2001-11-01
影响因子：
4.7
通讯作者：
Wahl, AF
中科院分区：
文献类型：
--
作者：
Senter, PD;Beam, KS;Wahl, AF
CPT-11是一种临床批准的抗癌药物，用于治疗晚期结直肠癌。给药后，药物的氨基甲酸酯侧链水解，导致SN-38的释放，SN-38是一种细胞毒性活性增加约1000倍的药物。由于注射剂量的CPT-11中只有很小的百分比转化为SN-38，因此有很大的机会通过选择性激活肿瘤部位内的药物来提高其治疗功效并减少其全身毒性。我们设想用于CPT-11活化的mAb-人酶缀合物将是令人感兴趣的，特别是因为缀合物可能是最低免疫原性的，并且前药是临床批准的。为此，有必要确定能够将CPT-11转化为SN-38的最具活性的人类酶。我们从人肝微粒体中分离酶，基于它们影响转化的能力，并鉴定人羧酸酯酶2（hCE-2）具有最大的比活性。hCE-2的活性比人羧酸酯酶1高26倍，是兔肝羧酸酯酶活性的65%，兔肝羧酸酯酶是已知活性最高的CPT-11水解酶。抗p97 mAb 96.5与hCE-2连接，形成可与抗原阳性癌细胞结合并将CPT-11转化为SN-38的缀合物。细胞毒性试验证实，偶联物导致活性药物的产生，但前药活化动力学（48 pmol min（-1）mg（-1））不足以进行免疫特异性前药活化。这些结果证实了hCE-2对CPT-11活化的重要性，并强调了酶动力学对选择性前药活化的重要性。
CPT-11 is a clinically approved anticancer drug used for the treatment of advanced colorectal cancer. Upon administration, the carbamate side chain of the drug is hydrolyzed, resulting in the release of SN-38, an agent that has approximately 1000-fold increased cytotoxic activity. Since only a very small percentage of the injected dose of CPT-11 is converted to SN-38, there is a significant opportunity to improve its therapeutic efficacy and to diminish its systemic toxicity by selectively activating the drug within tumor sites. We envisioned that a mAb-human enzyme conjugate for CPT-11 activation would be of interest, particularly, since the conjugate would likely be minimally immunogenic, and the prodrug is clinically approved. Toward this end, it was necessary to identify the most active human enzyme that could convert CPT-11 to SN-38. We isolated enzymes from human liver microsomes based on their abilities to effect the conversion and identified human carboxylesterase 2 (hCE-2) as having the greatest specific activity. hCE-2 was 26-fold more active than human carboxylesterase 1 and was 65% as active as rabbit liver carboxylesterase, the most active CPT-11 hydrolyzing enzyme known. The anti-p97 mAb 96.5 was linked to hCE-2, forming a conjugate that could bind to antigen-positive cancer cells and convert CPT-11 to SN-38. Cytotoxicity assays established that the conjugate led to the generation of active drug, but the kinetics of prodrug activation (48 pmol min(-1) mg(-1)) was insufficient for immunologically specific prodrug activation. These results confirm the importance of hCE-2 for CPT-11 activation and underscore the importance of enzyme kinetics for selective prodrug activation.