3.2 Estimation of the extraction ratio in the liver part by the drug efficacy test using the MO–MPS
The density ratios of A549 cells exposed to CPT-11 for 72 h on the MO–MPS with and without bypass channels decreased significantly to 33.2% and 25.6%, respectively, as compared to that of the control, i.e., not exposed to CPT-11 (Fig. 3A) with a significant difference between the MO–MPS with and without bypass channels.
Prodrugs are primarily metabolized by metabolic enzymes in the liver, following which, the metabolites distributed by blood flow, show the drug effects and side effects on tissues and organs in vivo . CPT-11 is metabolized mainly by CES2 and CYP3A4, which are metabolic enzymes in hepatocytes (Fig. 3C).(Ma et al., (2000); Mullangi et al., (2010)) CPT-11 is metabolized to inactive metabolites (i.e., APC and NPC), which do not have anticancer effects, by CYP3A4, whereas it is metabolized to SN-38, which has approximately 1,000 times the anticancer effect of CPT-11, by CES2.(Uchida et al., (2013)) SN-38 exhibits strong anticancer effect by inhibiting the activity of Type-1 topoisomerase, which is necessary for cell proliferation.(Kurita and Kaneda, (1999)) Moreover, SN-38 is conjugated by UDP-glucuronosyltransferase 1A1 (UGT1A1) to yield SN-38G, which is inert, and thereby has no anticancer activity.(ref) The significant decrease in the density ratio of the A549 cells exposed to CPT-11 indicates that CPT-11 was metabolized by the HepG2 cells, and Type-1 topoisomerase were subsequently inhibited by SN-38. The cell density ratio of the MO–MPS with the bypass channel was significantly higher than that without the bypass channel because the AUC of SN-38 on the MO–MPS with bypass channel did not increase due to the decline in clearance, which indicates the ability to remove drugs by metabolism, in the liver part with lower flow rate.
The AUC of SN-38 were obtained from the cell density ratio in the drug efficacy test using the PD model (eq. 7). Then, the extraction ratios of both CPT-11 and SN-38 in the liver part were estimated by AUC of SN-38 and the PK model (eq. 6). The expression level of CYP3A4 in HepG2 is extremely low, (Levy et al., (2015); Wilkening et al., (2003)) and CPT-11 is only metabolized to SN-38 by CES2. Therefore, thefmCES2 , which represents the fraction of metabolism of CPT-11 by CES2, was set to 1.0. We have assumed that the metabolism of SN-38 to SN-38G is nonreversible in the PK–PD model because SN-38G does not convert to SN-38 in the MO–MPS. SN-38G normally changes by conjugation with β-glucuronidase on intestinal bacteriain vivo . Vd was defined as the volume of the microchannel on the MO–MPS because the compound does not effuse outside. The AUCs of SN-38 were calculated to be 8.15 \(h\bullet ng/uL\) and 19.63\(h\bullet ng/uL\) on the MO–MOP with and without the bypass channel, respectively (Fig. 3B). The extraction ratios of CPT-11 and SN-38 in the liver part were estimated to be 0.4% and 8.4%, respectively. When human liver microsomes were exposed to CPT-11, SN-38G was generated approximately 17 times more than the amount of SN-38,(Van Der Bol et al., (2011)) suggesting that the fraction of SN-38 metabolized to SN-38G per unit time is larger than the fraction of CPT-11 metabolized to SN-38 per unit time. The same observation was made in HepG2 model cells.