Evaluation of the risk of diarrhea induced by epidermal growth factor receptor tyrosine kinase inhibitors with cultured intestinal stem cells originated from crypts in humans and monkeys.

Severe diarrhea is a common side effect of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). We aimed to evaluate the risk of EGFR-TKI-induced diarrhea using spheroids of human and monkey crypt-derived intestinal stem cells. Intestinal spheroids exhibited higher toxic susceptibility to EGFR-TKIs than Caco-2 cells. As concentration of EGFR-TKIs increased, cellular ATP first decreased relative to the control condition, followed by an increase in LDH release, in contrast with their simultaneous changes with traditional cytotoxic anticancer drugs. The toxic sensitivity of spheroids to various EGFR-TKIs corresponded to clinical diarrhea incidence. Afatinib, a second-generation EGFR-TKI, exhibited higher toxic sensitivity compared with the first-generation ones, corresponding to the clinical evidence that afatinib-induced diarrhea is almost inevitable and severe. By contrast, the third-generation osimertinib, which reduces the risk of diarrhea, showed mitigated cytotoxicity compared with afatinib. For irreversible EGFR-TKIs, the decreased ATP level persisted or its recovery was delayed even after drug removal compared with reversible ones. Furthermore, the highest drug accumulation in spheroids (TKIspheroids) and inhibition potency against EGFR (TKIspheroids/Ki) were observed for afatinib. This system would be useful for predicting the risk of EGFR-TKI-induced diarrhea; moreover, on-target cytotoxicity against intestinal stem cells might contribute to clinically observed diarrhea.

Calcium-activated chloride channel is involved in the onset of diarrhea triggered by EGFR tyrosine kinase inhibitor treatment in rats.

EGFR tyrosine kinase inhibitors (TKIs) are mainly used to treat non-small cell lung cancer; however, adverse effects such as severe diarrhea represent a major obstacle towards the continuation of EGFR-TKIs therapy. Chloride channels, which control the fluid flow in the intestinal lumen, are proposed as an important target to remediate EGFR-TKIs-induced diarrhea, but the mechanism remains unclear. The aim of this study was to clarify the mechanism underlying EGFR-TKIs-induced diarrhea with a particular focus on the role of intestinal chloride channels. Here, we show that osimertinib-treated rats exhibit diarrhea and an increase in fecal water content without showing any severe histopathological changes. This diarrhea was attenuated by intraperitoneal treatment with the calcium-activated chloride channel (CaCC) inhibitor CaCCinh-A01. These findings were confirmed in afatinib-treated rats with diarrhea. Moreover, treatment with the Japanese traditional herbal medicine, hangeshashinto (HST), decreased fecal water content and improved fecal appearance in rats treated with EGFR-TKIs. HST inhibited the ionomycin-induced CaCC activation in HEK293 cells in patch-clamp current experiments and its active ingredients were identified. In conclusion, secretory diarrhea induced by treatment with EGFR-TKIs might be partially mediated by the activation of CaCC. Therefore, blocking the CaCC could be a potential new treatment for EGFR-TKI-induced diarrhea.

Mechanism of hepatotoxicity of first-line tyrosine kinase inhibitors: Gefitinib and afatinib.

AIMS: Both gefitinib and afatinib are epidermal growth factor tyrosine kinase inhibitors (EGFR-TKI) in the treatment of non-small cell lung cancer (NSCLC). It has been reported that gefitinib and afatinib could cause hepatotoxicity during the clinic treatment, therefore it is critical to investigate their hepatotoxicity systematically. In this study, zebrafish (Danio rerio) were used as model animals to compare the hepatotoxicity and their toxic mechanism. MAIN METHODS: The zebrafish transgenic line [Tg (fabp10a: dsRed; ela3l:EGFP) was used in this study. After larvae developed at 3 days post fertilization (dpf), they were put into different concentrations of gefitinib and afatinib. At 6 dpf, the viability, liver area, fluorescence intensity, histopathology, apoptosis, transaminase reflecting liver function, the absorption of yolk sac, and the expression of relative genes were observed and analyzed respectively. KEY FINDINGS: Both gefitinib and afatinib could induce the larvae hepatotoxicity dose-dependently. Based on the liver morphology, histopathology, apoptosis and function assessments, gefitinib showed higher toxicity, causing more serious liver damage. Both gefitinib and afatinib caused abnormal expressions of genes related to endoplasmic reticulum stress (ERS) pathway and apoptosis. For example, jnk, perk, bip, chop, ire1, bid, caspase3 and caspase9 were up-regulated, while xbp1s, grp78, bcl-2/bax, and caspase8 were down-regulated. The hepatotoxicity difference of gefitinib and afatinib might be due to the different expression level of related genes.