Pharmacokinetics, biodistribution, stability and toxicity of a cell-penetrating peptide-morpholino oligomer conjugate
Pharmacokinetics, biodistribution, stability and toxicity of a cell-penetrating peptide-morpholino oligomer conjugate
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DOI：
10.1021/bc070060v
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发表时间：
2007-07-01
影响因子：
4.7
通讯作者：
Iversen, Patrick L.
中科院分区：
文献类型：
--
作者：
Amantana, Adams;Moulton, Hong M.;Iversen, Patrick L.
目的：将富含精氨酸的细胞穿透肽(CPP)偶联到磷酸二氢吗啉齐聚物(PMO)上，可促进PMO的胞浆和胞核转运。然而，CPP-PMO的体内分布在很大程度上是未知的。在这项研究中，我们研究了一种与CPP(RXR)(4)(X=6-氨基己酸)偶联的抗c-myc PMO在大鼠体内的药代动力学、组织分布、稳定性和安全性。方法：大鼠静脉注射PMO和CPP-PMO。用高效液相色谱法测定血浆和组织中PMO和CPP-PMO的浓度。用质谱法测定CPP-PMO结合物中CPP部分在大鼠血浆和组织裂解物中的稳定性。通过体重变化、血清化学和动物行为来评估CPP-PMO的安全性。结果：CPP偶联改善了PMO的动力学行为，估计消除半衰期增加2倍，分布体积增加4倍，血药浓度-时间曲线下面积增加。与改进的药代动力学曲线一致的是，与CPP的结合增加了PMO在除脑外的所有组织中的摄取，不同器官类型的摄取有所不同，肝、脾和肺的摄取增加更大。CPP-PMO结合物比相应的PMO具有更大的组织保留力。质谱学数据表明PMO部分没有明显的降解，而CPP部分有可识别的降解。CPP在血浆和组织裂解物中的降解具有时间依赖性，且在血浆中的降解速度更快。血浆和裂解物中降解产物的模式不同。安全性评价数据表明，CPP-PMO在15 mg/kg剂量下耐受性良好，没有明显的毒性迹象。相反，在剂量为150 mg/kg时，记录了一些不良事件，如嗜睡、体重减轻、血尿素氮(P<；0.01)和血清肌酐(P<；0.001)水平升高。在毒性剂量下，自由补充L精氨酸可提高血清肌酐(P<；0.0 5)和尿素氮(P<；0.0 1)的清除率，提示CPP具有肾脏毒性。结论：本研究表明，CPP与PMO的偶联可增强PMO的药代动力学、组织摄取和随后的滞留。因此，当剂量为
Objective: Conjugation of arginine-rich cell-penetrating peptide (CPP) to phosphorodiamidate morpholino oligomers (PMO) has been shown to enhance cytosolic and nuclear delivery of PMO. However, the in vivo disposition of CPP-PMO is largely unknown. In this study, we investigated the pharmacokinetics, tissue distribution, stability, and safety profile of an anti-c-myc PMO conjugated to the CPP, (RXR)(4) (X = 6-aminohexanoic acid) in rats. Methods: The PMO and CPP-PMO were administrated intravenously into rats. The concentrations of the PMO and the CPP-PMO in plasma and tissues were monitored by HPLC. The stability of the CPP portion of the CPP-PMO conjugate in rat plasma and tissue lysates was determined by mass spectrometry. The safety profile of the CPP-PMO was assessed by body weight changes, serum chemistry, and animal behavior. Results: CPP conjugation improved the kinetic behavior of PMO with a 2-fold increase in the estimated elimination half-life, a 4-fold increase in volume of distribution, and increased area under the plasma concentration vs time curve. Consistent with the improved pharmacokinetic profile, conjugation to CPP increased the uptake of PMO in all tissues except brain, varied between organ type with greater uptake enhancement occurring in liver, spleen, and lungs. The CPP-PMO conjugate had greater tissue retention than the corresponding PMO. Mass spectrometry data indicated no observable degradation of the PMO portion, while there was identifiable degradation of the CPP portion. Time-dependent CPP degradation was observed in plasma and tissue lysates, with the degradation in plasma being more rapid. The pattern of degraded products differed between the plasma and lysates. Safety evaluation data showed that the CPP-PMO was well-tolerated at the dose of 15 mg/kg with no apparent signs of toxicity. In contrast, at the dose of 150 mg/kg, adverse events such as lethargy, weight loss, and elevated BUN (p < 0.01) and serum creatinine (p < 0.001) levels were recorded. Supplementation with free L-arginine ad libitum showed improved clearance of serum creatinine (p < 0.05) and BUN (p < 0.01) at the toxicological dose, suggesting that the CPP caused toxicity in kidney. Conclusion: This study demonstrates that conjugation of CPP to PMO enhances the PMO pharmacokinetic profile, tissue uptake, and subsequent retention. Therefore, when dosed at