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.