Akinboye Emmanuel S, Rogers Oliver C, Isaacs John T
Department of Oncology, Prostate Cancer Program, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, The Johns Hopkins University School of Medicine, Baltimore, Maryland.
Prostate. 2018 Jun;78(9):655-663. doi: 10.1002/pros.23494. Epub 2018 Mar 24.
The SH-group at Cys-34 of human serum albumin (HSA) is a unique and accessible functional group that can be exploited for efficient linkage of a maleimide containing cytotoxic drug derivative to albumin. The specific maleimide chemistry used for production of the maleimide-linked albumin drug (MAD) is critical, however, to minimize the plasma concentration of "free" cytotoxic drug spontaneously released from albumin carrier thus decreasing dose-limiting host toxicity while enhancing the plasma half-life from minutes to days (ie, pharmacokinetic effect) and tissue concentration of the MAD in the extracellular cellular fluid at sites of cancer (ie, EPR effect).
To accomplish this goal, a chemical synthesis was developed using 2-fluoro-5-maleimidobenzoic acid to stably link the potent cytotoxic chemically modified analogue of the naturally occurring sesquiterpene γ-lactone, thapsigargin, 8-O-(12-aminododecanoyl)-8-O-debutanoyl thapsigargin (12ADT), to Cys-34 of albumin to produce 12ADT-MAD.
Using FITC-labeling, LC/MS analysis, and in vitro growth and clonogenic survival assays on a series of 6 human prostate cancer lines (LNCaP, LAPC-4, VCap, CWR22R 1, PC3, and Du145), we documented that 12ADT-MAD is endocytosed by prostate cancer cells where it is degraded into its amino acids liberating cysteinyl-maleimide-12ADT which is both chemically stable at the acidic pH of 5.5 present in the endosome while retaining its high killing ability (IC 50 nM) via SERCA inhibition.
Based upon these positive in vitro validation results, the in vivo efficacy versus host toxicity of this 12-ADT-MAD approach is presently being evaluated against a series of patient derived androgen responsive and castration resistant human xenografts in immune-deficient mice.
人血清白蛋白（HSA）第34位半胱氨酸上的巯基是一个独特且易于接近的官能团，可用于将含马来酰亚胺的细胞毒性药物衍生物高效连接至白蛋白。然而，用于生产马来酰亚胺连接的白蛋白药物（MAD）的特定马来酰亚胺化学方法至关重要，以尽量减少从白蛋白载体自发释放的“游离”细胞毒性药物的血浆浓度，从而降低剂量限制性宿主毒性，同时将血浆半衰期从几分钟延长至数天（即药代动力学效应），并提高MAD在癌症部位细胞外液中的组织浓度（即增强渗透滞留效应）。
为实现这一目标，开发了一种化学合成方法，使用2-氟-5-马来酰亚胺基苯甲酸将天然存在的倍半萜γ-内酯毒胡萝卜素的强效细胞毒性化学修饰类似物8-O-(12-氨基十二烷酰基)-8-O-去丁酰基毒胡萝卜素（12ADT）稳定连接至白蛋白的第34位半胱氨酸，以制备12ADT-MAD。
通过对一系列6种人前列腺癌系（LNCaP、LAPC-4、VCap、CWR22R 1、PC3和Du145）进行异硫氰酸荧光素（FITC）标记、液相色谱/质谱（LC/MS）分析以及体外生长和克隆形成存活试验，我们证明12ADT-MAD被前列腺癌细胞内吞，在细胞内降解为氨基酸，释放出半胱氨酰-马来酰亚胺-12ADT，其在内涵体5.5的酸性pH条件下化学稳定，同时通过抑制肌浆网钙ATP酶（SERCA）保持其高杀伤能力（半数抑制浓度[IC50]为纳摩尔级）。
基于这些积极的体外验证结果，目前正在免疫缺陷小鼠中针对一系列患者来源的雄激素反应性和去势抵抗性人异种移植瘤评估这种12-ADT-MAD方法的体内疗效与宿主毒性。
