these methods do not directly assess the potential of a compound to induce
CYP3A4 in humans under clinical conditions. The prediction of CYP3A4
induction based on results obtained indirectly could be inaccurate. The direct
measurement of CYP3A4 activity in humans exposed to drugs or test
compounds will, therefore, provide the most directly useful assessment of
CYP3A4 induction.
In the late 1980s, Watkins et al. developed the erythromycin breath test
(Watkins et al., 1989). Erythromycin is metabolized by human CYP3A4 via
N-demethylation. Metabolism of erythromycin that is isotopically labeled at the
N-methyl group results in the elimination in expired air of [^14 C]CO 2 , while the
N-desmethyl erythromycin is excreted into bile. In the standard assay, patients
receive 4mCi of [^14 CN-methyl] erythromycin (equivalent to 0.074mmol),
dissolved in 2 mL of 5% dextrose immediately before intravenous administra-
tion. The breath of patients is collected at timed intervals thereafter through a
tube containing 4 mL of hyamine hydroxide and ethanol (1 : 1, v/v), and a trace
amount of phenolphthalein. The amount of trapped [^14 C]CO 2 is then
determined using scintillation counting. Hepatic CYP3A4 activity, represented
by the production of [^14 C]CO 2 in the breath (expressed as percentage of total
administrated radiolabel eliminated in breath per minute), was significantly
increased in patients treated with the known CYP3A4 inducers dexamethasone
or hydrocortisone for 2 days or longer, or with rifampicin (600 mg) for 4 days.
CYP3A4-dependent generation of [^14 C]CO 2 was decreased by 80% in patients
who received a single oral dose of the CYP3A4-specific inhibitor troleando-
mycin (500 mg). This method is now widely accepted and utilized. For example,
the erythromycin breath test has been used to demonstrate CYP3A4 induction
by many xenobiotics, including St. John’s wort (Durr et al., 2000), efavirenz
(Mouly et al., 2002), progestins (Tsunoda et al., 1998), dexamethasone
(McCune et al., 2000), and rifampicin (Floyd et al., 2003).
In the format originally devised, the erythromycin breath test requires
intravenous injection of a compound that is radiolabeled, and therefore
potentially hazardous. Several nonradiolabeled CYP3A4-specific substrates
have been identified that make it possible to more safely assess CYP3A4
induction directly in test subjects. These CYP3A4 selective substrates include
some clinically important drugs such as midazolam, buspirone, felodipine,
simvastatin, lovastatin, as well as testosterone (Bjornsson et al., 2003; Tucker
et al., 2001). For example, the induction by rifampicin of the CYP3A4-
dependent systemic clearance of midazolam was measured in 57 healthy
subjects (Floyd et al., 2003). The subjects received rifampicin (600 mg PO, once
daily) for 16 consecutive days. The systemic clearance of midazolam was
measured on day 1, and again on day 15. Five milliliter blood samples were
collected into EDTA-containing tubes via an intravenous catheter, before and
5, 15, 30 min, and 1, 2, 3, 4, 5, 6, and 8 h after intravenous administration of
1 mg of midazolam. Plasma concentrations of midazolam and its major
metabolite, 1^0 -OH midazolam, were determined using high-performance liquid
chromatography-tandem mass spectrometry (Wandel et al., 2000). The study
ASSESSMENTS 563