放化疗联用在临床中被广泛进行，目的是加强抗肿瘤效果和减少彼此剂量。然而，在对放化疗联合治疗的方案，包括剂量、顺序等进行研究时，发现放疗会引起药物PK的改变，这种由放疗（电离辐射）引起的药代动力学改变被称为“RT-PK”现象。目前的“RT-PK”研究多停留在PK与代谢酶层次，对深入机制探究缺乏。我国高血压患者基数达2.45亿，且高血压是抗癌治疗最常见并发症之一，二者存在交叉。基于癌症-高血压患者的普遍性和用药特点，本研究提出一线降压药物ARBs中厄贝沙坦的潜在“RT-PK”猜想。厄贝沙坦作为CYP2C9和OATP1B1的底物，当复苏和引起这些没或转运体的表达改变时，可能改变厄贝沙坦的ADME。目前，还未有研究报道厄贝沙坦的“RT-PK”现象和基于OATP1B1的“RT-PK”机制。本研究通过体内外实验，从体内药代、代谢酶或转运体表达、分子通路多层次进行探究，旨在探究并阐明厄贝沙坦的“RT-PK”现象与机制，为临床中应对该类现象和预测并研究其他药物的“RT-PK”研究提供参考和指导。
根据研究目的，本研究首先建立了一种可以同时检测生物样品中厄贝沙坦及厄贝沙坦N-β-D-葡糖醛苷的LC-MS/MS定量分析方法，使用流动相为：A）1mM乙酸铵-0.05%甲酸-5%乙腈-水；B）纯乙腈。进样量1μl，洗脱程序为：0-0.5min, 30% B; 0.5-2.0min, 30% B升至85% B; 2.0-4.0min, 85% B；4.0-4.1min，85% B降至30% B；4.1-5.5min，30% B。随后对定量方法开展选择性与特异性、线性、准确度与精密度、提取回收率和基质效应的方法学验证，结果均符合M10生物样品定量分析指导原则的要求。
大鼠分组（对照组和辐射组），辐射组大鼠接受腹部照射（X-ray，5Gy），采集各组生物样品，前处理并使用开发的LC-MS/MS方法进行定量分析和结果统计。首先，血药浓度结果发现X-射线会导致血浆中厄贝沙坦浓度显著上升，通过计算药代动力学参数，得到455%的AUC和109%的Cmax增加，及Vd和CL显著下降。其次，组织分布结果显示，0.5h、4h、24h三个时间点中，各组织浓度均随时间下降，其中肝组织具有最高浓度的药物分布，且给药后0.5h，辐射组肝组织分布量显著低于对照组。胆汁分泌检测结果显示，厄贝沙坦N-β-D-葡糖醛苷为主要成分，且辐射组累积排泄分数（回收率）呈现下降趋势，但无显著性；尿粪累积排泄分数（回收率）结果显示，粪便是厄贝沙坦主要排泄途径，厄贝沙坦是主要存在形式，辐射组的同时间回收率显著低于对照组且更晚达到最大回收率。以上结果提示厄贝沙坦存在血液蓄积且可能为肝脏分布减少和粪中排泄减少的主要原因。
厄贝沙坦是肝脏摄取转运体OATP1B1的底物，如果辐射能够引起其表达下调，则可能导致血药蓄积。为阐明转运体和代谢酶在辐射后的变化情况并基于此对厄贝沙坦的“RT-PK”现象与分子机制进行探究。本研究首先通过对大鼠肝组织的CYP2C9、oatp4（人OATP1B1大鼠同源蛋白）、Mrp2和肠组织的Mrp2的蛋白或mRNA表达进行探究。结果显示CYP2C9在辐射后未发生显著表达改变，排除了一相代谢在厄贝沙坦“RT-PK”中的作用。同时，辐射引起oatp4的下调，提示肝脏对药物摄取的减少。肝脏Mrp2下调，可能与胆汁中厄贝沙坦N-β-D-葡糖醛苷及粪便中厄贝沙坦含量的下降有关。肠组织的Mrp2呈现上调趋势，与肝脏相反，提示组织间差异。随后，在HepG2和Caco-2细胞中进行类似的mRNA表达考察，结果与大鼠相应组织呈现不一致甚至相反趋势，需进一步研究。
核受体家族（PXR、FXR、LXR）在肝脏广泛分布并参与多种药物代谢酶与药物转运体表达调控。为探究其在调节oatp4和Mrp2中的作用和具体类型，本研究考察了其蛋白与mRNA表达情况，结果显示PXR与FXR均呈现辐射后在24-48h显著下调，LXR在24h的蛋白表达无显著下调，提示PXR和FXR是参与了oatp4和Mrp2表达调控的主要核受体类型。
炎症和氧化应激是电离辐射引起的重要生理学效应，与“RT-PK”可能存在密切关联。为进一步探究其在厄贝沙坦PK改变和转运体表达改变中的作用和更生如的分子机制，本研究首先对氧化应激和炎症水平进行检测，结果显示辐射后24-72h大鼠出现了Nrf2和下游HO-1mRNA表达上调，生化检测显示血浆中GSH与SOD在辐射后24h显著减少，而组织mRNA结果还显示IL-6在辐射后24h显著上升，但TNF-α和IL-1β显著下降，提示大鼠出现氧化应激和轻微肝组织炎症。研究进一步通过H&E切片染色探究X-射线辐射对组织影响，结果显示辐射后大鼠肝组织出现细胞破损、间隙增加、肝小叶结构改变。进一步对与炎症和氧化应激密切相关的Nf-κB及在炎症和细胞凋亡、自噬中会被激活并调控Nf-κB的PI3K/AKT通路进行研究。结果显示，辐射后AKT与Nf-κB磷酸化显著增加，提示通路激活。Nf-κB的激活能够通过负调控PXR等对靶基因的转录调控作用降低靶基因的表达。因此，我们发现了一条可能参与大鼠肝脏oatp4和Mrp2下调的分子通路，但需要进一步的实验验证。
综上，本研究发现辐射后厄贝沙坦会产生AUC与Cmax上升和Vd与CL下降，PK结果显示肝组织分布减少、粪排泄速率减缓，排泄量减少。进一步研究解释了厄贝沙坦的“RT-PK”现象及引起该现象的潜在分子机制，发现X-射线可能通过激活PI3K/AKT/Nf-κB通路负性调控PXR/FXR的转录调控作用，降低oatp4和Mrp2的表达，且PXR或FXR的表达也受到不明机制的下调，可能与细胞氧化应激激活某些通路有关。总之，本研究系统的阐释了厄贝沙坦的“RT-PK”现象和潜在的分子机制。本研究为实现系统化归纳辐射与体内酶或转运体的研究提供参考。为临床中癌症-高血压病人中使用厄贝沙坦或类似药物进行降压治疗时合理调整用药提供参考依据。

The combination of radiotherapy and chemotherapy is widely used in clinical practice, aiming to enhance the anti-tumor effect and reduce the dosage of each. However, when studying the combined treatment regimens of radiotherapy and chemotherapy, including dosage and sequence, it was found that radiotherapy can cause changes in drug PK, a phenomenon known as "RT-PK". Currently, most "RT-PK" research remains at the PK and metabolic enzyme level, lacking in-depth exploration of the mechanism. In China, the number of patients with hypertension is as high as 245 million, and hypertension is one of the most common complications of cancer treatment. There is an intersection between the two. Based on the prevalence and medication characteristics of cancer-hypertension patients, this study proposes a potential "RT-PK" hypothesis for irbesartan, a first-line antihypertensive drug in the ARBs class. As a substrate of CYP2C9 and OATP1B1, irbesartan may have its ADME altered when radiotherapy (ionizing radiation) causes changes in the expression of these enzymes or transporters. Currently, there are no studies reporting the "RT-PK" phenomenon of irbesartan or the "RT-PK" mechanism based on OATP1B1. This study explores and clarifies the "RT-PK" phenomenon and mechanism of irbesartan through in vitro and in vivo experiments, from multiple levels including in vivo PK, expression of metabolic enzymes or transporters, and molecular pathways, aiming to provide reference and guidance for clinical management of this phenomenon and for the study of "RT-PK" of other drugs.
According to the research objective, this study first established an LC-MS/MS quantitative analysis method that can simultaneously detect irbesartan and irbesartan N-β-D-glucuronide in biological samples. The mobile phase was: A) 1mM ammonium acetate - 0.05% formic acid - 5% acetonitrile - water; B) pure acetonitrile. The injection volume was 1 μl, and the elution program was: 0-0.5 min, 30% B; 0.5-2.0 min, 30% B increased to 85% B; 2.0-4.0 min, 85% B; 4.0-4.1 min, 85% B decreased to 30% B; 4.1-5.5 min, 30% B. The method was validated for selectivity and specificity, linearity, accuracy and precision, extraction recovery, and matrix effect, and all results met the requirements of the M10 guideline for quantitative analysis of biological samples.
By using X-rays to irradiate the abdomen of rats and collecting plasma samples, tissue samples, bile, feces, etc., and then using the developed LC-MS/MS method for quantitative analysis and result statistics, it was found that X-rays led to a significant increase in the plasma concentration of irbesartan. Through the calculation of pharmacokinetic parameters, a 455% increase in AUC and a 109% increase in Cmax were obtained, along with a significant decrease in Vd and CL. The tissue distribution results showed that at the three time points of 0.5 h, 4 h, and 24 h, the concentrations in each tissue decreased over time. The liver tissue had the highest drug concentration distribution, and at 0.5 h after administration, the concentration in the radiation group was significantly lower than that in the control group. The bile secretion detection results showed that irbesartan N-β-D-glucuronide was the main component, and the cumulative excretion fraction (recovery rate) in the radiation group showed a downward trend, but without significance. The cumulative excretion fraction (recovery rate) in urine and feces showed that feces was the main excretion route of irbesartan, and the recovery rate in the radiation group was significantly lower than that in the control group at the same time and reached the maximum recovery rate later. These results suggest that irbesartan accumulates in the blood and the main reasons may be the reduced distribution in the liver and the reduced excretion in feces. Irbesartan is a substrate of the liver uptake transporter OATP1B1. If radiation can cause its expression to be downregulated, it may lead to drug accumulation in the blood. To clarify the changes of transporters and metabolic enzymes after radiation and to explore the "RT-PK" phenomenon and molecular mechanism of irbesartan based on this, this study first investigated the protein or mRNA expression of CYP2C9, oatp4 (rat homolog of human OATP1B1), Mrp2 in rat liver tissue and Mrp2 in intestinal tissue. The results showed that CYP2C9 did not have significant expression changes after radiation, excluding the role of phase I metabolism in the "RT-PK" of irbesartan. Meanwhile, radiation caused the down-regulation of oatp4, suggesting a reduction in drug uptake by the liver. The down-regulation of liver Mrp2 may be related to the decrease in irbesartan N-β-D-glucuronide in bile and irbesartan content in feces. The expression of Mrp2 in intestinal tissue showed an upward trend, contrary to that in the liver, suggesting inter-organ differences. Subsequently, similar mRNA expression was investigated in HepG2 and Caco-2 cells, and the results were inconsistent or even opposite to those in the corresponding rat tissues, requiring further research.
The nuclear receptor family (PXR, FXR, LXR) is widely distributed in the liver and participates in the expression regulation of various drug-metabolizing enzymes and drug transporters. To explore their role and specific types in regulating oatp4 and Mrp2, this study examined their protein and mRNA expression. The results showed that both PXR and FXR were significantly down-regulated 24-48 hours after radiation, while LXR protein expression did not show significant down-regulation at 24 hours, suggesting that PXR and FXR are the main nuclear receptor types involved in the expression regulation of oatp4 and Mrp2.
Inflammation and oxidative stress are important physiological effects caused by ionizing radiation and may be closely related to "RT-PK". To further explore their role and underlying molecular mechanisms in the changes of irbesartan PK and transporter expression, this study first detected the levels of oxidative stress and inflammation. The results showed that Nrf2 and downstream HO-1 mRNA expression were up-regulated in rats 24-72 hours after radiation. Biochemical tests showed that GSH and SOD in plasma were significantly reduced 24 hours after radiation, while tissue mRNA results also showed that IL-6 was significantly increased 24 hours after radiation, but TNF-α and IL-1β were significantly decreased, suggesting that rats experienced oxidative stress and mild liver tissue inflammation. The study further explored the effects of X-ray radiation on tissues through H&E staining, and the results showed that liver tissue in rats had cell damage, increased gaps, and altered hepatic lobule structure after radiation. Further studies were conducted on Nf-κB, which is closely related to inflammation and oxidative stress, and the PI3K/AKT pathway, which is activated and regulates Nf-κB in inflammation and apoptosis and autophagy. The results showed that AKT and Nf-κB phosphorylation significantly increased after radiation, indicating pathway activation. The activation of Nf-κB can reduce the expression of target genes by negatively regulating the transcriptional regulation of PXR and others. Therefore, we discovered a possible molecular pathway involved in the down-regulation of oatp4 and Mrp2 in rat liver, but further experiments are needed for verification.
In conclusion, this study found that irbesartan would have an increase in AUC and Cmax and a decrease in Vd and CL after radiation, and PK results showed a reduction in liver tissue distribution and a decrease in fecal excretion rate and excretion amount. Further research explained the "RT-PK" phenomenon of irbesartan and the potential molecular mechanism causing it, and found that X-rays may negatively regulate the transcriptional regulation of PXR/FXR by activating the PI3K/AKT/Nf-κB pathway, reducing the expression of oatp4 and Mrp2, and the expression of PXR or FXR was also down-regulated by an unknown mechanism, possibly related to the activation of certain pathways by cellular oxidative stress. In conclusion, this study systematically expounded the "RT-PK" phenomenon of irbesartan and its potential molecular mechanism. This research provides a reference for the systematic induction of studies on radiation and in vivo enzymes or transporters. It also offers a basis for the rational adjustment of medication when using irbesartan or similar drugs for antihypertensive treatment in cancer patients with hypertension in clinical practice.