In vitro co-culture systems of hepatic and intestinal cells for cellular pharmacokinetic and pharmacodynamic studies of capecitabine against colorectal cancer

Abstract

Background As a prodrug of 5-fluorouracil (5-FU), orally administrated capecitabine (CAP) undergoes preliminary conversion into active metabolites in the liver and then releases 5-FU in the gut to exert the anti-tumor activity. Since metabolic changes of CAP play a key role in its activation, a single kind of intestinal or hepatic cell can never be used in vitro to evaluate the pharmacokinetics (PK) and pharmacodynamics (PD) nature. Hence, we aimed to establish a novel in vitro system to effectively assess the PK and PD of these kinds of prodrugs. Methods Co-culture cellular models were established by simultaneously using colorectal cancer (CRC) and hepatocarcinoma cell lines in one system. Cell Counting Kit-8 (CCK-8) and flow cytometric analysis were used to evaluate cell viability and apoptosis, respectively. Apoptosis-related protein expression levels were measured using western blot analysis. A selective liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed for cellular PK in co-culture models. Results CAP had little anti-proliferative effect on the five monolayer CRC cell lines (SW480, LoVo, HCT-8, HCT-116 and SW620) or the hepatocarcinoma cell line (HepG2). However, CAP exerted marked anti-tumor activities on each of the CRC cell lines in the co-culture models containing both CRC and hepatocarcinoma cell lines, although its effect on the five CRC cell lines varied. Moreover, after pre-incubation of CAP with HepG2 cells, the culture media containing the active metabolites of CAP also showed an anti-tumor effect on the five CRC cell lines, indicating the crucial role of hepatic cells in the activation of CAP. Conclusion The simple and cost‑effective co-culture models with both CRC and hepatocarcinoma cells could mimic the in vivo process of a prodrug dependent on metabolic conversion to active metabolites in the liver, providing a valuable strategy for evaluating the PK and PD characteristics of CAP-like prodrugs in vitro at the early stage of drug development.

Advanced three‐dimensional in vitro liver models to study the activity of anticancer drugs

“…This is clear evidence that culture in hydrogels allows 3D liver models to be adequately nourished as was presented by others. [45] In this study, we evaluated the effects of the cellular composition of 3D models, the culture environment and time on 3D liver aggregate ability to biotransformation two selected anticancer drugs in free form (5FU) and as a prodrug (CAP). Both drugs at a concentration of 100 µM were incubated for 24 h with liver models, and then the culture supernatants were transferred to breast cancer (MDA-MB 231) cells seeded in multiwell plates.…”

“…This is clear evidence that culture in hydrogels allows 3D liver models to be adequately nourished as was presented by others. [ 45 ]…”

“…Similar results were presented in other study, where it was shown that incubation with liver cells allows CAP activation. [ 45,47,48 ] However, triculture models had the highest ability to transform CAP, which is evidence that the presence of parenchymal cells in in vitro liver models specifically affects their properties for drug biotransformation. No significant effect of the culture environment on the biotransformation abilities of 3D liver models was noted.…”

Advanced prodrug strategies in nucleoside analogues targeting the treatment of gastrointestinal malignancies

“…To circumvent the unacceptable gastrointestinal toxicity of doxifluridine, researchers at Roche continually developed a series of N 4 -substituted 5′-deoxy-5-fluorocytidine derivatives, which could be hydrolyzed to doxifluridine and 5-FU. As a result, N 4 -pentyloxycarbonyl-5′-deoxy-5-fluorocytidine (capecitabine, CAP, Table 1 ) stood out by such prodrug strategy as an orally chemotherapeutic drug that was FDA-approved for the treatment of colorectal cancer, pancreatic adenocarcinoma, stomach cancer, esophageal cancer, or gastroesophageal junction cancer ( Siddiqui et al, 2019 ; Pouya et al, 2021 ; Ge et al, 2023 ). As mentioned above, the gastrointestinal toxicity of doxifluridine is attributed to the liberation of 5-FU in intestine tract under the action of thymidine phosphorylase, capecitabine was thus designed as a prodrug of doxifluridine that could not be the substrate for thymidine phosphorylase in the intestine with an oral bioavailability of about 100%, C max of 3.9 mg/L, t max of 1.5–2 h, and AUC of 5.96 mg·h/L ( Walko and Lindley, 2005 ; Rehman et al, 2022 ).…”

Determination of Capecitabine and Its Metabolites in Plasma of Egyptian Colorectal Cancer Patients

“…At the dose of 1250 mg/m 2 on Day 14, the time taken for the concentration to peak in plasma (t max in hours) was 1.50, 2.00, 2.00 and 2.00 for CCB, DFCR, DFUR and FU, respectively. The AUC values (µg•h/mL) were 7.75, 7.24, 24.6 and 2.03 [1, 3,4]. Figure 1 shows the chemical structure of CCB and its major metabolites DFCR, DFUR, FU and DHFU.…”