Table_1_In vitro Cytotoxicity and Pharmacokinetic Evaluation of Pharmacological Ascorbate in Dogs.DOCX

Background: High-dose, pharmacological ascorbate (P-AscH−) is preferentially cytotoxic to human cancer cells in vitro. Investigations on the efficacy of P-AscH− as an adjuvant treatment for multiple human cancers are on-going, but has yet to be formally investigated in dogs. The primary objectives of this study were to determine the pharmacokinetic (PK) profile of P-AscH− in healthy Beagle dogs and the effects of P-AscH− on canine osteosarcoma cells in vitro.
Methods: Eight purpose-bred, healthy, spayed female Beagle dogs, between 20 and 21 months old, and 8–10 kg were administered two doses of P-AscH− (550 or 2,200 mg/kg) via intravenous infusion over 6 h, on separate days. Plasma ascorbate concentrations were measured at 12 time points during and after infusion for PK analysis using nonlinear mixed-effects (NLME) and non-compartmental analysis (NCA). Clonogenic assays were performed on 2 canine osteosarcoma cell lines (D-17 and OSCA-8) and canine primary dermal fibroblasts after exposure to high concentrations of ascorbate (75 pmoles/cell).
Results: Plasma ascorbate levels in the dogs peaked at approximately 10 mM following 2,200 mg/kg and returned to baseline 6–8 h after dosing. Minor adverse effects were seen in two dogs. Ascorbate (75 pmoles/cell) significantly decreased survival in both the osteosarcoma cell lines (D-17 63% SD 0.010, P = 0.005; OSCA-8 50% SD 0.086, P = 0.026), compared to normal fibroblasts (27% SD 0.056).
Conclusions: Pharmacological ascorbate is preferentially cytotoxic to canine-derived cancer cells. High levels of ascorbate can be safely administered to dogs. Further studies are needed to determine the effects of P-AscH− on canine patients.

背景：
高剂量药物性抗坏血酸（P-AscH−）在体外对人类癌细胞具有选择性细胞毒性。目前针对P-AscH−作为多种人类癌症辅助治疗手段的疗效研究正在进行中，但尚未在犬类中开展正式研究。本研究的首要目标是确定健康比格犬体内P-AscH−的药代动力学（PK）特征，以及P-AscH−在体外对犬骨肉瘤细胞的作用效果。

方法：
本研究选取8只培育用于实验的健康绝育雌性比格犬，年龄为20~21月龄，体重8~10 kg，在不同日期通过6小时静脉输注给予两次P-AscH−给药（剂量分别为550或2200 mg/kg）。在输注过程中及输注后的12个时间点采集血浆抗坏血酸浓度，采用非线性混合效应（NLME）和非房室分析（NCA）进行药代动力学分析。将两种犬骨肉瘤细胞系（D-17和OSCA-8）以及犬原代皮肤成纤维细胞置于高浓度抗坏血酸（75 pmoles/细胞）环境中培养后，开展集落形成实验。

结果：
给予2200 mg/kg剂量后，犬的血浆抗坏血酸浓度峰值约为10 mM，给药后6~8小时恢复至基线水平。两只犬出现轻微不良反应。与正常成纤维细胞（存活率27%，标准差0.056）相比，75 pmoles/细胞浓度的抗坏血酸可显著降低两种骨肉瘤细胞系的存活率：D-17细胞存活率为63%，标准差0.010，P=0.005；OSCA-8细胞存活率为50%，标准差0.086，P=0.026。

结论：
药物性抗坏血酸对犬源癌细胞具有选择性细胞毒性。高剂量抗坏血酸可安全地给予犬类。仍需开展进一步研究以明确P-AscH−对犬类患者的作用效果。