This dissertation presents an approach to optimize topotecan (TPT) intraperitoneal chemotherapy using anti-topotecan antibodies (ATAb). We hypothesize that ATAb may be used to decrease systemic unbound TPT exposure (AUCf), decrease TPT-induced systemic toxicity and enhance the efficacy of TPT intraperitoneal chemotherapy.; The limitations of 'traditional' targeting strategies, as well as different 'inverse targeting strategies' to improve the regio-selectivity of drug disposition were reviewed. A sensitive HPLC assay was developed to quantify total and unbound TPT plasma concentrations in mice, with or without the presence of anti-TPT antibodies. The assay has a working range of 1--500 ng/ml, with lower limit of quantitation of 0.02 ng. Pharmacokinetic studies suggested that TPT demonstrated "non-restrictive" elimination, as the clearance was relatively insensitive to changes in protein binding. TPT pharmacokinetics/toxicodynamics were assessed in mice following a wide range of dose and dosing protocols (i.v. bolus, i.p. bolus and i.p. infusion from 24--168 h). An integrated PK/PD model was developed to describe the relationship between TPT exposure and TPT-induced toxicity in mice. A hybridoma cell line secreting high affinity anti-TPT immunoglubin (K A = 4.8 x 108 M-1) was developed. Anti-TPT IgG and Fab fragments were produced, purified, and characterized. The effects of endogenous ATAb on TPT pharmacokinetics/toxicodynamics were investigated in mice that had been immunized with a TPT-immunoconjugate, and the therapeutic efficacy of TPT i.p. chemotherapy was evaluated in mice bearing peritoneal tumors. Results showed that immunized animals demonstrated a dramatic decrease in AUCf relative to the non-immunized animals. Endogenous ATAb protected immunized animals from severe body weight loss and allowed a 3-fold increase of the maximum tolerated dose of i.p. TPT. Additionally, immunized mice bearing peritoneal tumors showed improved survival relative to non-immunized tumor-bearing mice (100% vs. 20%). Finally, a hybrid compartmental/physiological pharmacokinetic model was developed to describe the effect of endogenous ATAb on TPT disposition. Simulations using this model predicted that the dissociation rate constant (koff) may be the most important determinant of antibody effect, and an antibody with intermediate binding affinity (KA ≈ 1 x 107 M-1) may be the most efficient to decrease systemic unbound drug exposure following intraperitoneal chemotherapy.
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机译：本文提出了一种使用抗拓扑替康抗体（ATAb）优化拓扑替康（TPT）腹膜内化疗的方法。我们假设ATAb可用于减少全身未结合的TPT暴露（AUCf），降低TPT引起的全身毒性并增强TPT腹膜内化疗的疗效。综述了“传统”靶向策略的局限性，以及不同的“逆靶向策略”以改善药物配置的区域选择性。开发了一种灵敏的HPLC分析法，可以定量检测是否存在抗TPT抗体的小鼠总TPT和未结合TPT血浆浓度。该测定的工作范围为1--500 ng / ml，定量下限为0.02 ng。药代动力学研究表明，TPT显示出“非限制性”消除，因为清除对蛋白质结合的变化相对不敏感。按照广泛的剂量和给药方案（24到168小时静脉推注，腹腔推注和腹膜内输注）评估小鼠的TPT药代动力学/毒理动力学。建立了一个完整的PK / PD模型来描述TPT暴露与TPT诱导的小鼠毒性之间的关系。开发了分泌高亲和力抗-TPT免疫球蛋白的杂交瘤细胞系（KA = 4.8 x 108 M-1）。抗-TPT IgG和Fab片段被产生，纯化和表征。在已经用TPT-免疫缀合物免疫的小鼠中研究了内源性ATAb对TPT药代动力学/毒理动力学的影响，以及TPT i.p.的治疗功效。在患有腹膜肿瘤的小鼠中评估了化学疗法。结果表明，相对于未免疫的动物，免疫的动物表现出AUCf的显着降低。内源性ATAb可保护免疫动物免于严重体重减轻，并使最大耐受i.p剂量增加3倍。 TPT。另外，相对于未免疫的荷瘤小鼠，免疫接种的腹膜肿瘤小鼠表现出更高的存活率（100％vs. 20％）。最后，建立了一个混合的区室/生理药代动力学模型来描述内源性ATAb对TPT处置的影响。使用该模型的模拟预测，解离速率常数（koff）可能是抗体效应的最重要决定因素，具有中等结合亲和力（KA＆ap; 1 x 107 M-1）的抗体可能最有效地减少系统性未结合腹膜内化疗后的药物暴露。
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