Discussion
There have been
many
reports verifying the involvement of CYP3A4 and CYP2J2 in the metabolism
of rivaroxaban; however, the respective metabolic contributions of CYP
isoforms were unknown. In this study, the contributions of CYP3A4 and
CYP2J2 were systematically evaluated and compared. Firstly,
CYP-dependent M1 formation analysis indicated that CYP2J2 showed a
strongest catalytic activity. Moreover, in CYP3A subfamily, except
CYP3A4, CYPs 3A5 and 3A7 also participated in the rivaroxaban
hydroxylation (M1 area, CYP3A4: CYP3A5: CYP3A7 = 25.0: 8.6: 6.3).
Secondly, kinetic studies further verified the highest catalytic
efficiency of CYP2J2, approximately 39-fold to that of CYP3A4. Finally,
CYP-specific inhibition experiments were used to access the
contributions of CYPs. Ketoconazole (CYP3A-specific inhibitor) and
danazol (CYP2J2-specific inhibitor) inhibited 43.3% and 41.1%
rivaroxaban metabolism in HLM. Based on the ratio of M1 area by CYP3A
subfamily in CYP screen (M1 area, CYP3A4: CYP3A5: CYP3A7 = 25.0: 8.6:
6.3), the contributions of CYPs 3A4, 3A5 and 3A7 were 27.3%, 9.4% and
6.9% respectively. Therefore, our studies identified the predominated
role of CYP2J2 in the rivaroxaban hydroxylation with a contribution of
41.1%, which were much higher than the contribution of 27.3% by
CYP3A4.
In the analysis of M1 formation by CYPs, CYP2J2 produced the highest
catalytic activity which was nearly 16 times higher than that of CYP3A4
(Figure 3B). Kinetic studies further demonstrated that the intrinsic
clearance value of CYP2J2 was far higher than that of CYP3A4,
approximately 39-fold (Table 1). Finally, the respective contributions
of CYP2J2 and CYP3A4 in HLM were determined by the CYP-specific chemical
inhibition study. Even though the inhibitory effect of CYP2J2-specific
inhibitor danazol and CYP3A-specific inhibitor ketoconazole were
comparable, being 41.1% and 43.3% respectively, there were dramatic
differences in the content of each isoenzyme in the HLM. CYP3A subfamily
is responsible for the metabolism of approximately 30.2% of clinical
drugs, and CYP3A4 as the major isoform of the CYP3A subfamily represents
about 14–24% of the microsomal P450 pool, on average [20-23]. In
contrast, CYP2J2 is the least abundant P450 isoform of those involved in
clinical drug metabolism [23]. CYP3A was the most
abundantly-expressed subfamily in the liver, at ~28.8%
of the total hepatic CYPs, whereas CYP2J2 abundance was less than 1%
[23 24]. Moreover, proteomic analysis demonstrated the protein
concentration of CYP3A4 to be about 50-fold that of CYP2J2 in HLM
[25]. Therefore, we inferred that the results of inhibitory
experiments in HLM, CYP2J2 and CYP3A4-specific inhibitors produced
comparable inhibition ratio, were most likely caused by the dramatic
differences in content for two isoforms in HLM. With such a low content
in HLM, CYP2J2 produced a comparable contribution with that by CYP3A,
the most abundant subfamily in liver, it suggested the higher catalytic
efficiency of CYP2J2 than that of CYP3A4 in rivaroxaban hydroxylation.
In the inhibition study, the total inhibitory activity in HLMs was over
100% at 130.27%, which may have been due to the poor selectivity of
the high-concentration inhibitors. Additionally, the concentration of
specific inhibitors had considerable impact on their inhibition
selectivity. For example, although ketoconazole is known as a specific
inhibitor of the CYP3A subfamily, it has also shown inhibitory activity
in the CYP1A1-mediated metabolism of 7-ethoxycoumarin, with anIC 50 value of 0.33 ± 0.03 μM [26]. In the
present study, the concentration of ketoconazole used for inhibiting
CYP3A was set as 1 μM―which was about 10 x Ki ―to
ensure absolute inhibition. At this concentration, ketoconazole may also
inhibit CYP1A1, which was also found to be involved in the metabolism of
rivaroxaban (Figure 3); this could explain why high concentrations of
ketoconazole affected high total inhibition activity. A similar outcome
was observed with quinidine, which was used to specifically inhibit the
activity of CYP2D6. Quinidine displayed significant inhibition of
CYP1A1-mediated 7-ethoxyresorufin O-deethylation, with anIC 50 value of 1.1 μM [27]. However, the
concentration of quinidine in the present study was set at 10 μM, which
probably also inhibited the CYP1A1-mediated metabolism of rivaroxaban
and led to a much higher total inhibition activity. Hence, poorly
selective inhibitory effects of high-concentration inhibitors resulted
in a more than 100% inhibition ratio in the present
study.
In addition to poor selectivity of some inhibitors, the higher
expression level of CYP2C9 in the liver may also partly account for its
high inhibition. In the CYP-specific inhibition study, the inhibition
ratio of CYP2C9 was the third highest after CYP2J2 and CYP3A4, while the
M1 peak area produced by CYP2C9 in the CYP screen was less than 10, and
approximately 0.38% of that produced by CYP2J2. The abundant content of
CYP2C9 in the liver played an important role in this difference between
the two results. First, CYP2C9 is one of the most highly expressed
members of all P450 isoforms, with a similar or lower protein level to
CYP3A4 [22 28]. More importantly, the CYP2C9 protein level was
higher than that of CYP3A4 in HLM, and much higher than that of CYP2J2
[25].
Based
on this rationale, the inhibition ratio achieved with CYP2C9-specific
sulfaphenazole was much higher than its actual efficacy.
The difference in the protein contents of CYP2J2 and CYP3A4 in the liver
resulted in their different status in clinical drug-drug interaction
research. The drug-drug interactions of rivaroxaban have been
extensively assessed in combination with many drugs, including CYP3A4 or
P-gp substrates, inhibitors and
inducers.
However,
results have demonstrated that the combination of rivaroxaban with
agents that are strong inhibitors of both CYP3A4 and P-gp can increase
rivaroxaban plasma concentrations in vivo, prompting caution regarding
its co-administration [15]. In contrast, owing to its low expression
levels in liver microsomes, CYP2J2 is usually not considered in routine
drug-drug interactions, thus, the potential for CYP2J2 regulators to
change rivaroxaban clearance has not been widely evaluated to
date.
However, it is expressed at an extremely high level in the
cardiovascular system: an evaluation of the mRNA levels of P450 isoforms
in the heart showed CYP2J2 mRNA levels largely exceeded those of other
detected isozymes by 3 million to 62 times [29 30]. And, in the
aorta and coronary artery, the expression of CYP2J2 ranks second only to
CYP2C9 [31]. These distribution characteristics of CYP2J2 are
consistent with the function of transforming arachidonic acid into
epoxyeicosatrienoic acids, which plays a vital role in cardiovascular
homeostasis and regulating vascular tone [32 33]. Importantly, a few
rivaroxaban drug-drug interaction studies indicated that it may interact
with other cardiovascular drugs that target CYP2J2, such as amiodarone
and dronedarone [34]. Therefore, the possibility of the interaction
between rivaroxaban and other drugs, especially with drugs to treat
cardiovascular diseases, can be more exactly evaluated if the heart is
set as the target organ and the content of CYP2J2 is considered.
In vitro drug-drug interaction (DDI) studies have been one of the major
methods for evaluating the efficiency and safety of drugs. For
rivaroxaban, the evaluations targeting CYP3A4 and P-gp are the main DDI
research direction so far, which was due to the rich content of CYP3A4
in the liver and the key role of P-gp in transporter-mediated DDIs. Our
results, showing the dominant role of CYP2J2 in the metabolism of
rivaroxaban, fill a gap in the basic metabolism studies of rivaroxaban
and also give a new insight into DDI studies involving rivaroxaban.
In summary, multiple CYP isoforms
were found to be involved in the hydroxylation of rivaroxaban, with
CYP2J2 identified as the predominant isoenzyme involved.