目标:平滑(SMO),刺猬信号通路的关键组成部分,代表三阴性乳腺癌(TNBC)的治疗靶标,然而,TNBC患者的化疗反应率仅为40-50%,强调迫切需要开发新药物来有效治疗这种疾病。新化合物TPB15,一种衍生自[1,2,4]三唑并[4,3-α]吡啶的SMO抑制剂,与第一种SMO抑制剂vismodegib相比,在体外和体内均表现出优异的抗TNBC活性和较低的毒性。然而,该化合物的药代动力学特性尚不清楚。本工作旨在开发一种简单的HPLC-MS/MS方法,以描述TPB15在大鼠中的药代动力学和生物利用度,作为进一步临床研究的基础工作。
方法:在AgilentZORBAXStableBondC18柱上通过梯度洗脱,使用乙腈和0.1%甲酸作为流动相以0.3mL/min的流速进行分离。采用m/z454.2→100.0,248.1→121.1的正模式多反应监测(MRM)来确定TPB15和内标替硝唑,分别。特异性,日内和日间精度和准确性,提取回收,稳定性,基体效应,方法的稀释完整性和残留性得到验证。通过以5mg/kg的剂量静脉注射和25mg/kg的剂量口服灌胃对大鼠进行TPB15的药代动力学和生物利用度研究。使用药代动力学软件DAS2.1.1通过非隔室分析计算药代动力学参数。
结果:特异性值,日内和日间精度和准确性,提取回收,稳定性,基体效应,稀释完整性和残留满足可接受的限制。该方法的定量下限为10ng/mL,线性范围为10-2000ng/mL。然后通过灌胃(25mg/kg)和静脉注射(5mg/kg)将经过验证的方法应用于大鼠的药代动力学和生物利用度研究,TBP15在大鼠体内的口服生物利用度为16.4±3.5%。药代动力学参数计算如下:最大血浆浓度(Cmax)(PO:2787.17±279.45µg/L),达到最大血浆浓度的时间(Tmax)(PO:4.20±0.90h),浓度-时间曲线下面积0至时间(AUC0-t)(PO:17,373.03±2585.18ng/mL·h,IV:21,129.79±3360.84ng/mL·h),浓度-时间曲线下面积为0至无穷大(AUC0-∞)(PO:17,443.85±2597.63ng/mL·h,IV:17,443.85±2597.63ng/mL·h),终末消除半衰期(t1/2)(PO:7.26±2.16h,IV:4.78±1.09h)。
结论:TPB15是治疗TNBC的有希望的候选药物,已在体外和体内表现出突出的疗效和安全性。这项研究建立了一个简单的,敏感,和快速HPLC-MS/MS生物分析方法,根据FDA和EMA指南开发和验证,用于进行TPB15的药代动力学和生物利用度研究。结果表明,由于其长t1/2,药代动力学特征良好。然而,下一阶段的研究应包括配方筛选,以提高生物利用度,以及临床试验,代谢途径分析,和评估潜在的药物-药物相互作用。
OBJECTIVE: Smoothened (SMO), a key component of the hedgehog signaling pathway, represents a therapeutic target for triple negative breast cancer (TNBC), yet the chemotherapy response rate in TNBC patients is only 40-50%, underscoring the urgent need for the development of novel drugs to effectively treat this condition. The novel compound TPB15, an SMO inhibitor derived from [1,2,4] triazolo [4,3-α] pyridines, demonstrated superior anti-TNBC activity and lower toxicity compared to the first SMO inhibitor vismodegib in both in vitro and in vivo. However, the compound\'s pharmacokinetic properties remain unclear. The present work aims to develop a simple HPLC-MS/MS method to profile the pharmacokinetics and bioavailability of TPB15 in rats as a ground work for further clinical research.
METHODS: Separation was performed on an Agilent ZORBAX StableBond C18 column by gradient elution using acetonitrile and 0.1% formic acid as mobile phase at a flow rate of 0.3 mL/min. Multiple reaction monitoring(MRM) in positive mode with the transitions of m/z 454.2 → 100.0, 248.1 → 121.1 was employed to determine TPB15 and internal standard tinidazole, respectively. The specificity, intra- and inter- day precision and accuracy, extraction recovery, stability, matrix effect, dilution integrity and carryover of the method was validated. The pharmacokinetics and bioavailability study of TPB15 were carried out on rats through intravenous injection at the dose of 5 mg/kg and oral gavage at the dose of 25 mg/kg, and the pharmacokinetics parameters were calculated by the non-compartment analysis using the pharmacokinetics software DAS 2.1.1.
RESULTS: The values of specificity, intra- and inter- day precision and accuracy, extraction recovery, stability, matrix effect, dilution integrity and carryover satisfied the acceptable limits. The lower limit of quantification of this method was 10 ng/mL with a linear range of 10-2000 ng/mL. The validated method was then applied to pharmacokinetics and bioavailability studies in rat by dosing with gavage (25 mg/kg) and intravenous injection(5 mg/kg), and the oral bioavailability of TBP15 in rat was calculated as 16.4 ± 3.5%. The pharmacokinetic parameters were calculated as following: maximum of plasma concentration (Cmax) (PO: 2787.17 ± 279.45 µg/L), Time to maximum plasma concentration (Tmax) (PO: 4.20 ± 0.90 h), the area under the concentration-time curve 0 to time (AUC0-t) (PO: 17,373.03 ± 2585.18 ng/mL·h, IV: 21,129.79 ± 3360.84 ng/mL·h), the area under the concentration-time curve 0 to infinity (AUC0-∞) (PO: 17,443.85 ± 2597.63 ng/mL·h, IV: 17,443.85 ± 2597.63 ng/mL·h), terminal elimination half-life (t1/2) (PO: 7.26 ± 2.16 h, IV: 4.78 ± 1.09 h).
CONCLUSIONS: TPB15, a promising candidate for treating TNBC, has demonstrated outstanding efficacy and safety in vitro and in vivo. This study established a simple, sensitive, and rapid HPLC-MS/MS bioanalytical method, developed and validated in accordance with FDA and EMA guidelines, for conducting pharmacokinetic and bioavailability studies of TPB15. The results revealed a favorable pharmacokinetic profile owing to its long t1/2. Nevertheless, the next phase of research should include formulation screening to enhance bioavailability, as well as clinical trials, metabolism pathway analysis, and assessment of potential drug-drug interactions.