Download The Effect of an Individual`s Cytochrome CYP3A4 Activity on

Vol. 6, 1255–1258, April 2000
Clinical Cancer Research 1255
The Effect of an Individual’s Cytochrome CYP3A4 Activity on
Docetaxel Clearance1
JoAnn Hirth, Paul B. Watkins,
Myla Strawderman, Anne Schott, René Bruno,
and Laurence H. Baker2
Division of Hematology/Oncology [J. H., A. S., L. H. B.], General
Clinical Research Center [P. B. W.], and Biostatistics Care [M. S.],
University of Michigan Comprehensive Cancer Center, Ann Arbor,
Michigan 48109, and Rhône-Poulenc Rorer, Antony Cedex 92165,
France [R. B.]
ABSTRACT
Docetaxel is a chemotherapeutic agent effective in the
treatment of various solid tumors. Patients given a standard
dose of docetaxel exhibit wide interpatient variation in clearance (CL) and toxic effects. Docetaxel undergoes metabolism
by cytochrome CYP3A4. Thus, interpatient variability in
CYP3A4 activity may account in part for differences in
toxicity and CL. Twenty-one heavily pretreated patients
with metastatic sarcomas received docetaxel (100 mg/m2).
Hepatic CYP3A4 activity in each patient was measured by
the [14C-N-methyl]erythromycin breath test (ERMBT).
Blood samples were taken at selected times over the next
24 h for pharmacokinetic analysis. Phenotypic expression of
hepatic CYP3A4 activity measured by the ERMBT varied
over 20-fold (administered 14C exhaled in 1 h: mean, 2.53%;
range, 0.25–5.35%), which is similar to a normal control
population. CL of docetaxel varied nearly 6-fold (mean, 21.0
liters/h/m2; range, 5.4 –29.1 liters/h/m2). The ERMBT was
the best predictor of CL when compared with serum alanine
aminotransferase, albumin, alkaline phosphatase, or serum
␣-1-acidic glycoprotein. The natural log of ERMBT accounted for 67% of the interpatient variation in CL. Multivariate analysis showed that the natural log of ERMBT
and albumin together accounted for 72% of the interpatient
variation in CL. The greatest toxicity was seen in patients
with the lowest ERMBT. Hepatic CYP3A4 activity is the
strongest predictor of docetaxel CL and accounts for the
majority of interpatient differences in CL. Patients with low
CYP3A4 activity are at risk for having decreased CL and
may thus experience increased toxicity from docetaxel.
Those with high activity may be receiving a suboptimal dose.
By measuring CYP3A4 activity, the ERMBT may be clini-
Received 8/24/99; revised 12/1/99; accepted 12/6/99.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
1
Supported by General Clinical Research Center Grant M01 RR00042.
2
To whom requests for reprints should be addressed, at University of
Michigan Comprehensive Cancer Center, 1500 East Medical Center
Drive, 7216 CCGC, Ann Arbor, MI 48109-0948.
cally useful in tailoring doses of CYP3A4 substrates, such as
docetaxel, in certain individuals.
INTRODUCTION
Patients and healthy individuals alike display significant
differences in terms of efficacy and adverse effects after exposure to many drugs. An identical dosage of a drug can result in
widely different concentrations of the therapeutically active
compound or metabolites (1). The presence of genetic variability in the activity of enzymes involved in drug metabolism could
lead to clinically important variations in efficacy or adverse
effects for some drugs.
The cytochrome P450 system is a family of heme-containing enzymes responsible for the metabolism of nonendogenous
substances, such as drug molecules and toxins (2). Specific
isoenzymes are designated by the prefix “CYP,” and to date,
over 30 isoenzymes have been purified, cloned, sequenced, and
characterized in humans. CYP3A4 is found in the liver and
small bowel. The CYP3A4 enzyme displays large interpatient
differences in both content and catalytic activity in humans (3).
These differences exist even in the absence of medications
known to induce or inhibit the enzyme and are thus likely to
reflect genetic variability (3).
The ERMBT3 specifically measures the in vivo activity of
hepatic CYP3A4 (3, 4) and is therefore ideally suited for drugs
given i.v., such as docetaxel. This test is based on the fact that
CYP3A4 is the major enzyme responsible for erythromycin metabolism, liberating a carbon atom that is exhaled as carbon dioxide. After injecting a trace dose of radiolabeled erythromycin, the
rate of radiolabeled carbon dioxide exhaled is measured. This test
has been validated as a specific measure of CYP3A4 activity (3).
In vitro studies suggest that CYP3A4 is the major enzyme
involved in docetaxel metabolism (5). Docetaxel is excreted in
the feces with less than 8% excreted unchanged (6). It is highly
bound to plasma proteins including AAG. Pharmacokinetic data
show significant variation in CL between patients (7). Decrease
in total body CL is associated with increased frequency and
severity of side effects (8). Mulitvariate analysis demonstrates
that interpatient variability of CL is predicted by body surface
area, AAG plasma level, and elevated hepatic enzymes; however, these parameters do not account for total variability (7).
Because docetaxel binds chiefly to AAG, high levels of this
protein presumably limit the free fraction of docetaxel available
for CL in the liver. Due to lower CL of docetaxel in patients
with elevated transaminases, it is currently recommended in the
United States package insert that patients with an ALT ⬎ 1.5⫻
3
The abbreviations used are: ERMBT, erythromycin breath test; AAG,
␣-1-acidic glycoprotein; ULN, upper limit of normal; ALT, alanine
aminotransferase; ln ERMBT, natural log of ERMBT; GCRC, General
Clinical Research Center; CL, clearance.
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1256 Effect of CYP3A4 Activity on Docetaxel Clearance
Fig. 1 Drug metabolism via cytochrome CYP3A4.
the ULN concomitant with alkaline phosphatase ⬎ 2.5⫻ ULN
or bilirubin ⬎ ULN do not receive this drug. The European
Summary of Product Characteristics alternatively recommends a
dosage reduction of 25% in patients with concomitant elevations
of ALT and alkaline phosphatase.
Previous studies have shown modest activity for docetaxel
in patients with advanced sarcomas (response rates of 17% and
18%), thus we decided to try this agent in our sarcoma population (9, 10). We hypothesized that variability in CYP3A4 activity accounts for interpatient differences in docetaxel CL and
toxicity. Thus, the ERMBT was used to measure CYP3A4
activity in sarcoma patients receiving docetaxel (Fig. 1).
PATIENTS AND METHODS
Study Population. Twenty-one patients with measurable,
metastatic sarcomas of bone or soft tissue origin were entered into
our trial. Eighteen patients had soft tissue sarcomas, whereas three
had bone sarcomas. Twelve patients were men, and nine were
women (age range, 19 –72 years). Thirteen of the patients with soft
tissue sarcomas had received extensive prior chemotherapy,
whereas five patients with gastrointestinal stromal sarcomas had
not received extensive prior chemotherapy. Two of the three patients with bone sarcomas had also received extensive prior chemotherapy. All patients gave written informed consent, and the study
was approved by the Protocol Review Committee of the Cancer
Center and the Review Committee of the GCRC, as well as the
University of Michigan Institutional Review Board. All patients
had normal hematological values and liver chemistries within the
limits recommended for the use of docetaxel at the time of admission (normal total bilirubin, alkaline phosphatase ⬍ 5⫻ the ULN,
and serum transaminase levels ⬍ 1.5⫻ the ULN). Concurrent use
of drugs known to alter CYP3A4 activity such as imidazole antimycotic agents (fluconazole), macrolide antibiotics (erythromycin),
antiseizure medications (phenobarbitol and phenytoin), and rifampin were specifically avoided. Other exclusion criteria were
concurrent radiotherapy, active infection, pregnancy or lactation,
and allergy to erythromycin.
Study Schema and Procedures. Three ERMBTs were
administered to each patient at the GCRC. The test was done on
three occasions in case either the dexamethasone premedication or
the chemotherapy introduced variability. The first study served as
a baseline and was done before admission for docetaxel (range,
1–19 days before admission). The second study was done 24 h after
dexamethasone premedication was initiated and 1 h before the
infusion of docetaxel. The third study was done 3 h after the
infusion of docetaxel had been completed. A total of 3 ␮Ci (⬍0.1
mmol) of [14C-N-methyl]erythromycin obtained from Metabolic
Solutions, Inc. (Nashua, NH) was given i.v. Exhaled carbon dioxide was trapped in a solution of hyamine hydroxide, ethanol, and a
blue indicator before and 20 min after the injection. The percentage
of 14C exhaled/h ⫽ 49.496 ⫻ (percentage of 14C exhaled/min at 20
min ⫺ baseline counts) (11).
Docetaxel was administered at 100 mg/m2 i.v. over 1 h.
Each patient received the following standard premedications:
(a) dexamethasone (8 mg, p.o.) every 12 h starting the day
before infusion and continuing for 5 days; and (b) granisetron (2
mg, p.o.) 30 min before the infusion. Blood was drawn for
pharmacokinetic analysis over 24 h with specimens at optimal
sampling times: just before the end of infusion and at 0.25, 0.75,
3.00, 6.50, and 24.00 h after the end of infusion (12). Case
report forms documented actual sampling times as well as the
actual beginning and end times of the infusion. Docetaxel was
assayed from frozen plasma samples using reverse-phase highperformance liquid chromatography (13). All samples were
assayed at the same time. A Bayesian criterion was used to
calculate the docetaxel plasma concentration area under the
curve based on measured drug levels and population pharmacokinetic parameters using NONMEM software (12, 14). The
area under the curve was computed as dose/CL. CL was directly
estimated by fitting the model (12, 14). Patients had baseline
liver chemistries and serum AAG levels drawn. One patient did
not have a baseline serum ALT recorded, and the two patients
had insufficient sample to determine the AAG level. Baseline
and weekly hematological counts were checked. Granulocytecolony stimulating factor was not given during this first cycle.
Subsequent cycles of chemotherapy were given every 3
weeks if the patient was judged by us to be deriving clinical benefit.
The pharmacokinetic data were obtained only during the initial
cycle. Treatment was stopped if the disease progressed, if intolerable toxicities developed, or at the patient’s request. Radiological
evaluation of patients was done at the discretion of the treating
physician to document clinical response or progression.
Statistical Analysis. Although the distribution of the
ERMBT in a large sample of normal controls is positively skewed,
the distribution in this small sample is approximately normal. The
paired t test was used to compare the breath test values obtained at
the three time points. Pearson’s correlation coefficient (r) further
describes the strength of the linear relationship and is presented
along with the P. Nonparametric analogues to these test procedures
were conducted for confirmation but are not reported.
CL, as determined by pharmacokinetic modeling, is approximately normal. The association between CL and patient characteristics was evaluated using simple and multiple least-squares
regression models. Variables considered as potential predictors of
CL in this model included ERMBT, AAG, albumin, ALT, and
alkaline phosphatase. A natural log transformation was applied if
indicated by univariate residual plots. For instance, the relationship
between the ERMBT and CL was not linear over the entire range
of values, particularly at the lowest ERMBT values, and the low
ERMBT values had a better fit when ln ERMBT was used. The
best model for predicting CL was determined from the set of
predictors using a step-wise procedure based on the largest F
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Clinical Cancer Research 1257
Table 1 Variability in breath tests and docetaxel clearance
a
ERMBT #1
ERMBT #2
ERMBT #3
Docetaxel CLc
Mean
SD
Range
2.41
2.53
2.25b
20.42
1.08
1.17
0.93
6.30
0.125–4.86
0.41–5.35
0.53–4.37
5.4–29.1
ERMBT ⫽ percentage of 14C exhaled/h.
Significantly lower than ERMBT #2 (P ⫽ 0.0047).
c
Docetaxel CL is given in liters/h/m2.
a
b
statistic for each variable’s contribution to the model. The first
factor included using the step-wise procedure is the best single
predictor of CL. Two patients missing certain lab test results were
excluded from analyses that involved these lab tests. All two-way
interactions were examined for the final best model. All analyses
were conducted using the SAS software package.
RESULTS
The interpatient variability in ERMBTs and docetaxel CL
is evident in the range of values obtained, as shown in Table 1.
The ERMBT varied ⬎20-fold, largely because a few patients
had very low breath test results. The CL of docetaxel varied
⬎5-fold among these patients. These ERMBT data exhibit similar variability to that seen in 107 normal controls studied
previously (ERMBT mean ⫽ 2.56; SD, 1.06; range, 0.77– 6.15).
There was excellent correlation between ERMBT results on all
three occasions (r ⫽ 0.81– 0.92, P ⬍ 0.0001). Thus, there is no
evidence for a significant effect of dexamethasone premedication on the ERMBT. There is a tendency for the test value to fall
modestly after docetaxel administration (ERMBT #3 versus
ERMBT #2, P ⫽ 0.0047), likely reflecting competition between
erythromycin and docetaxel for metabolism by CYP3A4. The
ERMBT #2 was used in subsequent analyses.
Fig. 2 demonstrates the relationship between CL and the
phenotypic measurement of CYP3A4. As expected, those patients
with reduced CL had the lowest ERMBT results. As mentioned in
the statistical section, the low ERMBT values had a better fit when
ln ERMBT was used, with r2 ⫽ 0.67, P ⫽ 0.0001. Even if the
ERMBT was not log-transformed, the relationship between CL and
the ERMBT remained strong (r2 ⫽ 0.49, P ⫽ 0.001). Albumin was
also a good predictor of CL (r2 ⫽ 0.46, P ⫽ 0.001). Alkaline
phosphatase was not as strong a predictor of CL. One patient with
osteosarcoma had an exceedingly high alkaline phosphatase (5769)
from bone rather than liver origin and was therefore included in the
study. When this outlier was removed from the analysis, alkaline
phosphatase was a weak predictor of CL (r2 ⫽ 0.36). The relationships between CL and ALT or AAG were also weak (r2 ⫽ 0.23 and
0.12, respectively), but in the anticipated inverted direction. Thus,
the ERMBT proved to be the best single predictor of docetaxel CL.
Virtually all patients had National Cancer Institute grade 3
or 4 neutropenia at the nadir (83% grade 4; 6% grade 3). Blood
counts were only checked weekly, so it is possible that the nadir
was missed on a couple of patients. It is also interesting to note
that the two patients requiring hospitalization with the severest
toxicities (mucositis/sepsis/neutropenia) were those with the
lowest ERMBT and docetaxel CL results (5.4 and 10.5 liters/
h/m2). The AAG levels, which were shown previously to be a
Fig. 2 Relationship between CL and phenotypic measurement of
CYP3A4.
good predictor of CL and toxicity (8), showed no trend in
predicting this toxicity, and both patients were within the recommended range for liver chemistries.
As described in the statistical section, simple and multiple
regression analyses were conducted to model the best predictors
of CL. The best two-factor model for predicting CL was determined to be ln ERMBT and albumin (r2 ⫽ 0.72). No other
factors added significantly to this model, and no statistically
significant codependence between variables was detected. In the
two-factor model containing untransformed ERMBT and AAG,
both variables were independently predictive of CL (r2 ⫽ 0.61),
with higher AAG predicting lower CL.
DISCUSSION
Both the CL of docetaxel and CYP3A4 activity, as measured by the ERMBT, had marked interpatient variation in this
study. All patients were required to have good hepatic function
as described previously, and with the exception of dexamethasone, no patient received known potent inhibitors or inducers of
CYP3A4. Thus, it appears that the variation seen could reflect
genetic differences in the metabolism of docetaxel.
The most significant independent variable examined to predict docetaxel CL in this study was hepatic CYP3A4 activity as
measured by ERMBT. Previously, the best predictors of docetaxel
pharmacokinetics that had been identified were abnormal liver
chemistries and serum AAG level. Albumin was also a significant
predictor of CL, but its effect was of low magnitude compared with
that of AAG (9). In this study, AAG and the ERMBT results do not
appear to be correlated, and the ERMBT is a superior predictor of
CL. In fact, the ERMBT and albumin are the strongest predictors of
CL, and together they account for 72% of the interpatient variability in CL. Why is the AAG a poorer predictor in this study? The
pharmacokinetic data and AAG levels in this study were deter-
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1258 Effect of CYP3A4 Activity on Docetaxel Clearance
mined in the same manner and by the same investigator as these
prior data, so quality control should not be a factor. The small
sample size of the present study may account for this difference.
Bruno’s study (8) included 547 patients and used a statistical
approach with Bayesian estimates directed toward subpopulations
with extreme covariate values (presumably clinically at a higher
risk). This requires a large sample size. This approach is not
applicable to our study of 21 patients. The variability of AAG
levels may also be important. Docetaxel is strongly protein-bound
to AAG, thus high AAG levels should decrease docetaxel CL. In
the previous work (7), this was evident for high AAG levels;
however, the model did not improve the prediction of CL for
patients with low AAG levels (⬍0.88 g/liter). Perhaps our study
did not include as many patients with “high” AAG levels, and thus
AAG was not found to be an important predictor of docetaxel CL.
The AAG reflects the free fraction of drug available for CL in the
liver, whereas CYP3A4 activity reflects the individual’s inherent
ability to metabolize the drug. Although this is a small study, the
fact that CYP3A4 activity was a better predictor is an important
new finding and will hopefully improve our understanding of the
pharmacokinetics of docetaxel. Larger studies are under way and
will help clarify the importance of AAG and CYP3A4 activity in
relationship to CL.
Although toxicities (namely, grade 3 or 4 neutropenia) were
seen in virtually all patients at this dose level, the observation that
an ERMBT result of ⬍1 in this study was associated with the
severest toxicities is interesting. Although marked interpatient heterogeneity in the expression of CYP3A4 genes is known to exist,
the clinical implications of having a “high” or “low” CYP3A4
activity were not necessarily known. In this study “low” CYP3A4
activity (ERMBT ⬍ 1) appears to be associated with excess toxicity. It implies that consideration should be given to modifying the
dose for patients with such a low ERMBT. Similarly, one would
wonder whether patients with “high” CYP3A4 activity are at risk
of getting subtherapeutic doses of CYP3A4 substrates such as
docetaxel. This hypothesis should be studied further.
In this study, single agent docetaxel was given, but docetaxel
is now commonly included in combination regimens. Because the
additional drugs are often myelotoxic but not CYP3A4-mediated,
the ERMBT may be helpful in sorting out the specific effect
docetaxel makes with regard to the toxicity of the regimen.
Logistically, the ERMBT is easy to administer and poses a
minimal risk to patients. It proved to be highly reproducible. It was
not significantly affected by dexamethasone premedication, and
this observation is consistent with prior data (15) that showed no
substantial effect of dexamethasone premedication on docetaxel
CL. The test can therefore be done at a convenient time, days or
weeks ahead of the planned administration of docetaxel.
In conclusion, CYP3A4 activity was found to be the most
significant independent variable for predicting the CL of docetaxel. The ERMBT, perhaps together with serum albumin or
AAG, may be clinically useful in individualizing the dose of
docetaxel and possibly other drugs that are CYP3A4 substrates,
including several new antineoplastic agents. It may also be
helpful in individualizing the dose of docetaxel in patients with
markedly elevated hepatic enzymes who are currently excluded
from treatment with docetaxel.
We plan to initiate a study using tailored dosing of docetaxel based on CYP3A4 activity. We postulate that by tailor-
ing the dose, variability in patient drug exposure could be
diminished. Hopefully, modern pharmacogenetics can be put
into practical use in the clinic and allow us to treat patients with
better precision and less toxicity.
ACKNOWLEDGMENTS
We thank Mary Vestich for secretarial support, Michelle Walsh for
technical assistance, and Marlene Woo for clinical assistance. We are
also very grateful for the contributions of A. Joly and P. Baille (RhônePoulenc Rorer, Antony, France) with regard to the pharmacokinetic
analysis of docetaxel. Finally, we thank the GCRC staff, without whom
this study could not have been done.
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The Effect of an Individual's Cytochrome CYP3A4 Activity on
Docetaxel Clearance
JoAnn Hirth, Paul B. Watkins, Myla Strawderman, et al.
Clin Cancer Res 2000;6:1255-1258.
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