Introduction
CYP3A4 (cytochrome P450) is the major
metabolic pathway taken by drugs in the human body.
Over 50% of new molecular entities (NMEs) will be metabolized
by CYP3A4. CYP3A4 is both inhibitable and inducible
by drugs, and this accounts for many important drug
interactions. The importance of CYP3A4 has been highlighted
by the recent removal of drugs from the market due to
drug interactions.
For example, terfenadine (Seldane®),
cisapride (Propulsid®), and cerivastatin (Baycol®) were
withdrawn due to toxicity produced in patients receiving
inhibitors of CYP3A4. These drugs are largely metabolized
by CYP3A4; simultaneous administration with CYP3A4 inhibitors
resulted in dangerous increases in drug blood levels.
Conversely, mibafradil (Posicor®) was withdrawn from
the market because it was a potent inhibitor of CYP3A4
and could cause toxic reactions to some drugs largely
metabolized by CYP3A4 (such as “statins”). In each case,
regulatory agencies believe that the risk management
issues presented by these drugs could have been predicted
by appropriate studies conducted during the development
programs. Regulatory agencies are therefore generally
requiring that all NDA submissions contain answers to
the following two questions:
1) Is CYP3A4 a major pathway
for metabolism of the NME? If so, concomitant administration
of drugs that are inhibitors or inducers of CYP3A4 could
result in toxicity or under treatment, respectively,
with the NME (i.e. the NME is “the victim” in the interaction).
2) Is the NME an inhibitor
or inducer of CYP3A4? If so, treatment with the NME
could result in toxicity or lack of efficacy, respectively,
from a concomitantly administered drug largely metabolized
by CYP3A4 (i.e. the NME is “the cause” of the interaction).
At most major pharmaceutical companies,
the answers to these two “key” questions are now sought
prior to lead candidate selection based on high throughput
in vitro technology.
In vitro approaches
There are three in vitro approaches now commonly
used to determine the answer to question one. One approach
is to determine if recombinant CYP3A4 is capable of
metabolizing the NME. A second approach is to determine
the effect of specific chemical or antibody inhibitors
of CYP3A4 on NME metabolism in pooled human liver microsomes.
A third approach is the determine the rates of metabolism
of the NME in a battery of human liver microsomes previously
characterized in terms of metabolism of model CYP3A4
substrates, also called “probes”. A perfect correlation
between rates of metabolism of the NME and the probe
across the entire battery of samples supports a central
role for CYP3A4.
The most common in vitro approach
used to answer question two involves measuring the concentration
dependent effect of the NME on CYP3A4 activity in liver
microsomes (or on recombinant CYP3A4) using a CYP3A4
probe.
An interesting recent discovery is that the results
obtained in these inhibition studies depends on the
CYP3A4 probe used (1).
The accepted explanation for this observation is that
CYP3A4 has at least two distinct substrate binding sites
allowing it, in effect, to behave like two different
enzymes (2).
It is therefore possible for an NME to be a potent inhibitor
of one substrate binding site but a weaker inhibitor
of another substrate binding site. As a result, one
concentration of NME may result in significant inhibition
of metabolism of some CYP3A4 substrates while having
insignificant inhibition of other CYP3A4 substrates.
For this reason, it has become customary to use at least
two structurally diverse CYP3A4 probes for in vitro
inhibition studies (3).
The ability of an NME to induce CYP3A4 can be assessed
in primary cultures of human hepatocytes. This question
is increasingly addressed by examining the ability of
the NME to activate the pregnene- X- receptor (PXR),
which is involved in transcriptional activation of the
CYP3A4 gene (4).
Limitations of in vitro testing
The above in vitro studies provide
clues to the answers to the two key questions and “go
- no go” decisions are often made based on them. However,
it is clear that data obtained in in vitro systems cannot
be automatically extrapolated to the living human. The
fallibility of the in vitro tests reflects many factors,
including an inability to predict the concentration
of NME (and potentially its metabolites) that will reach
the CYP3A4 substrate binding site, or the PXR receptor,
in vivo. Therefore, it is not yet possible to confidently
answer the two key questions based solely on in vitro
studies.
In addition, in studies performed
in vitro, it has been shown that a given compound can
be a potent inhibitor of testosterone metabolism while
being a relatively weak inhibitor of midazolam metabolism.
Conversely, some compounds potently inhibit midazolam
metabolism while having less effect on testosterone
metabolism. The accepted explanation for
this phenomenon is that CYP3A4 has at least two distinct
substrate binding domains allowing it, in effect, to
behave like two different enzymes (5 and references
therein). It is therefore becoming standard practice
to utilize substrates from both the midazolam and testosterone
subgroups in inhibition studies performed in vitro (6).
Investigators should be mindful of
this accepted in vitro practice when designing in vivo
inhibition studies. The Erythromycin Breath Test provides
an attractive alternative to using testosterone as a
CYP3A4 probe in clinical inhibition studies because
erythromycin is within the testosterone subgroup (7).
In addition, the test uses trace doses of erythromycin
so that the Erythromycin Breath Test can be added easily
to existing inhibition protocols that use substrates
within the midazolam class.
In vivo approaches
Over that last decade, techniques
have been developed that make it possible to answer
the two key questions in humans relatively early in
clinical development. These techniques follow a rationale
that is similar to that underlying in vitro approaches.
To determine the answer to question
one, two approaches are commonly taken. The most common
approach is to perform single dose pharmacokinetic studies
with the NME in a small number of humans (usually 10
or less) before and after they have been treated with
a known potent inhibitor of CYP3A4. This is directly
analogous to the use of chemical inhibitors in pooled
human liver microsomes described above. To date, ketoconazole
has generally been used as the CYP3A4 inhibitor, but
recent data suggest that the antibiotic troleandomycin,
or TAO, is a more specific and potent inhibitor (8).
Regardless of the inhibitor used, it is desirable to
document the extent of CYP3A4 inhibition that is occurring
in each subject. This can be indirectly inferred by
measuring ketoconazole blood levels (9), but blood levels
are not as helpful with TAO since a large component
of the CYP3A4 inhibition produced by TAO is mechanism-based.
It is therefore becoming standard to measure in vivo
CYP3A4 activity before and during administration of
the NME using the Erythromycin Breath Test (8-14). This
test employs less than 0.1 mg of erythromycin as a CYP3A4
probe and will not influence the disposition of other
substrates. The Erythromycin Breath Test can therefore
be administered, and CYP3A4 activity measured, at any
time during the pharmacokinetic study.
The second approach to answer question
one is to stratify the subject population based on CYP3A4
activity, and then correlate individual clearance of
the NME with CYP3A4 activity (15 -20). This approach
is directly analogous to the in vitro approach that
searches for correlations within a battery of human
liver microsomes. There is no genetic test available
that predicts CYP3A4 activity in subjects, even healthy
volunteers. It is therefore necessary
to use a probe drug to assess each subject’s CYP3A4
activity. The Erythromycin Breath Test provides a rapid
means of assessing relative CYP3A4 activity.
The advantage of the correlation approach is that it
can be incorporated into any study at any time, even
phase 1 dose escalation studies.
The use of probes can also address
question two in vivo. For inhibition studies, it has
become customary to measure apparent oral clearance
of simvastatin or lovastatin before and during steady
state dosing of the NME (at the highest dose anticipated
for clinical use). A completely negative result is generally
considered sufficient to exclude CYP3A4 inhibition.
However, most studies performed with simvastatin or
lovastatin are in fact positive (using bioequivalence
parameters). This reflects the fact that these probes
are exquisitely sensitive to CYP3A4 inhibition. For
example, consumption of grapefruit juice will increase
simvastatin AUC by up to 12- fold (21). The problem
then becomes determining the significance of the inhibition.
If a positive result is obtained with
simvastatin or lovastatin, the next step is generally
to look for inhibition using two structurally diverse
probes (similar to the practice used in vitro) (3).
The most common two probes used are midazolam and erythromycin
(using the Erythromycin Breath Test) (22-30), which
can be simultaneously administered (28,31). Importantly,
erythromycin and midazolam represent, respectively,
the two major substrate classes in terms of CYP3A4 inhibition
(1). One advantage of the Erythromycin Breath Test is
that it provides an “instantaneous” measure of CYP3A4
activity (9). It is therefore possible to correlate
the percent fall in the Erythromycin Breath Test (from
pre-dose baseline) with the total or free plasma concentration
of the NME. This allows calculation of in vivo IC50
(which approximates Ki since only trace doses of erythromycin
are involved) (9,29).
For induction studies, it is customary
to use either the Erythromycin Breath Test or midazolam
as probes(32-36). One advantage of the Erythromycin
Breath Test is that it can be repetitively administered
to determine the time course of onset and resolution
of induction (or inhibition) in relation to initiation
and completion of treatment (22,27,29).
Summary
Prior to NDA submission, it is now
expected that the sponsor will have addressed the two
key questions outlined above. In vitro studies provide
important information, but generally do not obviate
the need for focused clinical studies which measure
CYP3A4 activity using carefully selected probe drugs.
Because CYP3A4 appears to have at least two distinct
substrate binding sites, it is now recommended that
two structurally different probes should be used in
in vitro and in vivo inhibition studies. Once inhibition
or induction of CYP3A4 is detected in vivo, the time
course relative to treatment should be established.
In some cases, labeling regarding potential drug interactions
can be based on these studies alone. In other instances,
additional specific drug interaction studies may be
required for approval, but these can often be addressed
in a Phase 4 program.
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