3.6 Effect of ningetinib on pharmacokinetics of D6-M1 in mice.
To investigate whether ningetinib could affect the pharmacokinetics of
M1 by inhibiting efflux transporters, D6-M1 was intravenously injected
to ICR mice to prevent the effect of M1 generated by ningetinib
metabolism. The results are shown in Fig. 7 and Table 4. Compared with
the control group, the blood exposure of D6-M1 in the
ningetinib-combination group was increased by approximately 75%. In
addition, the blood concentration of D6-M1 was not affected by
ningetinib within 2 h after administration. From 4 h to 24 h, the blood
exposure of D6-M1 in the ningetinib group increased nearly by 200%,
which further demonstrated that ningetinib could inhibit the elimination
of D6-M1.
Discussion
In pharmacokinetic studies, M1 was identified as the primary circulating
metabolite of ningetinib in humans. However, in clinical trials on
multi-target-based combination medication for NSCLC therapy, both the
plasma exposure and Cmax of M1 dropped dramatically and
were reduced by more than 80% when ningetinib was co-administered with
gefitinib, whereas the pharmacokinetic parameters (AUC,
Cmax and Tmax) of ningetinib were not
affected. The elimination behaviours of both ningetinib and M1 were not
obviously changed. These data suggested a DDI between ningetinib and
gefitinib, mostly likely through metabolic mechanism.
The mechanism of M1 formation was initially investigated. The enzymatic
kinetic data showed that M1 formation was mainly mediated by CYP1A1 and
to a lesser extent by CYP1B1, CYP2C9 and CYP3A4. When examining the
potency of gefitinib as an inhibitor of CYP450s in vitro , our
results were somewhat different from those reported in the literature
(Filppula, Neuvonen, & Backman, 2014;
Rahman et al., 2014). The present study
revealed that gefitinib was a strong inhibitor of CYP1A1, moderate
inhibitor of CYP1B1 and weak inhibitor of CYP2C9 but had no influence on
CYP3A4. Gefitinib significantly inhibited M1 production in HLMs, but the
concentration of the parent drug was not influenced by gefitinib, since
the amount of M1 produced in HLMs and in patients was less than 5%, andN -desmethylation was not a major metabolic pathway of ningetinib
in humans.
The high plasma exposure of M1 could be related to tissue distribution
and/or clearance. Unlike in plasma, the concentrations of M1 in tissues
were less than 30% of the parent drug, suggesting that the metabolite
tended to be retained in the plasma and its penetration into tissues was
more difficult than that of ningetinib. One possible explanation for the
findings was that M1 exhibited much higher protein binding rates both in
mouse and human plasma than the parent drug (99.9% versus 90%).
In vitro incubations showed that the liver was the primary site
of M1 formation. Thus, the hepatic clearance pathway of M1 was evaluated
subsequently. The transport studies demonstrated that M1 was a substrate
of P-gp, BCRP and MRP2. The parent drug ningetinib happened to be a
potent inhibitor of P-gp and moderate inhibitor of BCRP and MRP2.
Therefore, it was speculated that ningetinib obstructed the biliary
excretion pathway of M1 and resulted in its high plasma exposure. The
D6-M1 pharmacokinetic experiment further proved this conclusion. In
addition, the literature (Galetti et al., 2015b; Kitazaki et al., 2005)
and our results confirmed that gefitinib was also an inhibitor of P-gp
and BCRP. It might inhibit the efflux of M1 mediated by P-gp and BCRP.
However, the IC50 value of gefitinib on P-gp was
significantly higher than that of ningetinib (5.40 μM versus 0.413 μM)
and gefitinib had no inhibitory effect on MRP2. Though the
IC50 value of gefitinib on BCRP was lower than that of
ningetinib (9.09 μM versus 18.7 μM), both of them were much higher than
the peak plasma concentrations of ningetinib (1.37
μg·mL-1) and gefitinib (272 ng·mL-1)
when therapeutic doses of these drugs were administered to NSCLC
patients. The plasma Cmax of gefitinib was less than one
third of ningetinib. In this situation, it was speculated that the
efflux pathway of M1 was inhibited by ningetinib, whereas the inhibition
of efflux by gefitinib was negligible due to its higher
IC50 value and much lower in vivo concentrations.
When co-administrated with gefitinib, the pharmacokinetic alteration of
M1 showed the inhibitory effect of gefitinib on M1 formation, not on its
efflux transports. The interaction of ningetinib and gefitinib mediated
by P-gp and BCRP was also studied in the present work. The ER values of
ningetinib were decreased by not more than 50% under the effect of 50
μM of gefitinib (Table S3). Therefore, transporter-mediated effect of
gefitinib on ningetinib could be ignored.
To our knowledge, it was the first time to report that gefitinib was a
strong inhibitor of CYP1A1. CYP1A1 was the most actively studied human
pulmonary CYP enzyme involved in polycyclic aromatic hydrocarbons
metabolism, and might play a role in the development of lung cancer
(Oyama et al., 2008). It was also reported that CYP1A1 was highly
expressed in human brain, colon and bladder tumour tissues compared to
the normal tissues, as determined by mRNA level expression
(Androutsopoulos et al., 2013; Wahid, Mahjabeen, Baig, & Kayani, 2013).
In addition, CYP1A1 polymorphisms (rs4646903 and rs1048943) were
positively correlated with prostate cancer, breast cancer, lung cancer,
et,al (Lu et al., 2020). Therefore,
gefitinib’s strong inhibitory
effect on CYP1A1 may provide possibilities for its application in tumour
prevention and reversal of terminal tumour resistance. In vitroexperiments in our laboratory indicated that the amount of M1 produced
was positively correlated with the protein expression of CYP1A1 in HLMs
and HLUMs from smokers of varying degrees. The high selectivity of
ningetinib for CYP1A1 may provide its potential as an indicator of
CYP1A1.
In conclusion, this study illustrated the DDI mechanism of ningetinib
and gefitinib in patients with NSCLC. Ningetinib was metabolized by
CYP1A1 to produce a small amount of N -demethylated metabolite M1.
The low tissue affinity of M1 and the inhibitory effect of ningetinib on
M1 canalicular efflux resulted in its high plasma exposure and long
elimination half-life. When co-administrated, as a strong inhibitor of
CYP1A1, gefitinib inhibited the formation of M1 and reduced its plasma
exposure by more than 80%. However, the pharmacokinetics of the parent
drug ningetinib was not influenced because of the low metabolic yield of
M1. Due to the relatively high expression of CYP1A1 in pulmonary tumour
tissues, the concentration of the parent drug ningetinib in the target
tissue may be increased by gefitinib. As a consequence, the long-term
safety profile and efficacy of ningetinib combined with gefitinib should
be concerned in NSCLC patients. The findings also
suggested that in-depth analysis
of the pharmacokinetics of metabolites in DDI studies can help enrich
the DDI mechanism and discovery of novel inhibitors (or inducers) of
metabolic enzymes or transporters.