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Clinical Trials |
Departments of Pharmaceutical Sciences [M. K. M., W. C. Z., K. M. R., S. K. H., A. K. S., C. F. S.], Hematology and Oncology [W. L. F., V. M. S.], and Molecular Pharmacology [P. J. H.], St. Jude Children’s Research Hospital, Memphis, Tennessee 38105-2794, and Department of Pharmacology [P. J. H.] and The Center for Pediatric Pharmacokinetics and Therapeutics [P. J. H., C. F. S.], University of Tennessee, Memphis, Tennessee 38163
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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6) was 90.9 ± 96.4, 103.7 ± 62.4,
and 95.3 ± 63.9 at IRN doses of 20, 24, and 29 mg/m2,
respectively. The relative extent of IRN conversion to SN-38 and
metabolism to APC measured after dose 1 were 0.49 ± 0.33 and
0.29 ± 0.17 (mean ± SD). No statistically significant
intrapatient difference was noted for SN-38 area under the
concentration-time curve. Large interpatient variability in IRN and
metabolite disposition was observed. The relative extent of conversion
and the SN-38 systemic exposure achieved with this protracted schedule
of administration were much greater than reported in adults or children
receiving larger intermittent doses. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, IRN undergoes deesterification by
carboxylesterase to an active metabolite, SN-38
(7-ethyl-10-hydroxycamptothecin). SN-38 subsequently undergoes
conjugation to form SN-38G. Recently, two oxidative metabolites, APC
(7-ethyl-10-[4-N-(5-aminopentanoic
acid)-1-piperidino]-carbonyloxycamptothecin) and NPC
[7-ethyl-10-(4-amino-1-piperidino)-carbonyloxycamptothecin], have
been identified in an adult clinical trial (5
, 6)
. The
biotransformation of IRN to either APC or NPC was catalyzed mainly by
cytochrome P450 3A4 (6
, 7)
.
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Although much was known regarding the pharmacokinetics and pharmacodynamics of IRN in adults (9, 10, 11) , the disposition of IRN and metabolites in children has not been reported. Before initiating a pediatric clinical trial in which the IRN dose is adjusted to attain the desired SN-38 plasma systemic exposure, the inter- and intrapatient variability in drug disposition must be defined. Thus, we conducted this study to describe the pharmacokinetics and pharmacodynamics of IRN and its metabolites in children with recurrent solid tumors.
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PATIENTS AND METHODS |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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3 x normal for age),
adequate hepatic function (bilirubin and alanine aminotransferase 3 x normal), and normal metabolic parameters (serum
electrolytes, blood sugar, calcium, and phosphorous). Before study
entry, written informed consent was obtained from patients, parents, or
guardians according to institutional guidelines.
Treatment Protocol.
IRN (Camptosar) was diluted with 50 ml of 5% dextrose injection (D5W),
USP, and administered by 1-h i.v. infusion once daily for 5 consecutive
days followed by a 2-day rest and then an additional 5 consecutive days
of treatment [(d x 5 x 2)]. The dosages studied in this
pharmacokinetic study included 20, 24, and 29
mg/m2/day.
Pharmacokinetic Studies.
Pharmacokinetic studies were performed after doses 1 and 10 of the
first course. Blood samples (3 ml) were collected in heparinized tubes
immediately before IRN infusion and at 0.25, 0.5, 1, 2, 4, and 6 h
after the end of the infusion. All blood samples were obtained from a
site contralateral to the infusion site. All blood samples were
immediately centrifuged at 10,000 rpm for 2 min on a table-top
centrifuge. Plasma was separated, and proteins were precipitated by the
addition of 200 µl of plasma to 800 µl of cold methanol (-30°C),
followed by vigorous agitation with a vortex mixer and repeat
centrifugation at 10,000 rpm for 2 min. The supernatant was decanted
and stored at -70°C until analysis (3
, 4)
.
Quantification of IRN, SN-38, and APC.
IRN, SN-38, and APC lactone plasma concentrations were determined using
an isocratic high-performance liquid chromatography assay with
fluorescence detection as described previously in detail
(12)
. Excitation and emission wavelengths were 370 and 520
nm. The lower level of quantitation for this assay was 1 ng/ml for
CPT-11, SN-38, and APC (2)
. All calibrators and controls
were prepared using single-donor plasma.
Pharmacokinetic Analysis.
The disposition of IRN and metabolites was evaluated using a
four-compartment model with linear distribution and elimination. As
depicted in Fig. 2
, the four-compartment
pharmacokinetic model consisted of an IRN central compartment, an IRN
peripheral tissue compartment, and SN-38 and APC plasma compartments. A
simplifying assumption was made that the apparent volumes of
distribution for IRN, SN-38, and APC were identical. Pharmacokinetic
parameters for each set of data were initially fit by ML estimation as
implemented in ADAPT II, and the mean and SD for each parameter were
calculated (13)
. These mean parameter estimates were then
used as the revised initial estimates for another ML estimation. The
mean and SD for each parameter were updated until the parameter
estimates for all parameters were stable (defined as no net change in
the third significant digit). The refined mean and SD from the final ML
run were used to construct a diagonal covariance matrix for use in a
Bayesian algorithm. All sets of data were modeled using a
MAP-Bayesian approach to further refine estimates and update the
covariance matrix. This was repeated until stable estimates of the
model parameters were obtained. Each observation was assessed for the
goodness of fit by an estimate of the variance for the predicted
values.
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Although the ratio of metabolite:parent compound is not a direct measure of conversion or metabolism, the ratio is useful to characterize the relative conversion from the parent compound to metabolites among patients (14, 15, 16) . The calculation of the REC is defined as the ratio of SN-38 AUC:IRN AUC (14) . The same principle was applied to the REM, defined as the ratio of APC AUC:IRN AUC.
SN-38 Protein Binding.
In patients with adequate plasma samples, SN-38 lactone protein binding
studies were conducted as described previously (17)
.
Briefly, a plasma sample was obtained from a patient before IRN
administration and spiked with SN-38 lactone to attain a final
concentration of 1.2 µM. The plasma was placed in a
Micropartition System (Amicon Corp., Danvers, MA), and the plasma
ultrafiltrate was collected. In preliminary experiments with human
plasma, no significant adsorption of SN-38 to the filter membrane was
noted, so preconditioning of the membrane was not necessary. The
percentage of SN-38 lactone unbound (% unbound) was calculated from
the concentration of SN-38 lactone in the ultrafiltrate in relation to
the SN-38 lactone plasma concentration before ultrafiltration (protein
bound and unbound). The BR is the ratio of molar concentration of drug
bound:drug free (Fu) and is determined from the
following equation: BR = (1/Fu) - 1.
Pharmacodynamic Analysis.
Each patient was assessed for myelosuppression using the NCI Common
Toxicity Criteria and responses coded by standard criteria. The
relationship between myelosuppression and SN-38 systemic exposure was
correlated using the percentage change in ANC, which was defined as a
ratio of the differential between pretreatment ANC (pre) and the nadir
to the pretreatment ANC.
 | (1) |
The patient or the patient’s family reported the onset and frequency of diarrhea, and the grade of diarrhea was determined using the NCI Common Toxicity Criteria. Pharmacodynamic relationships were investigated using multivariate regression analysis between grade of diarrhea and SN-38 systemic clearance, cumulative SN-38 systemic exposure up to the onset of diarrhea in all patients, or cumulative SN-38 systemic exposure in a subset of patients who experienced delayed onset of diarrhea.
Statistical Analysis.
Statistical analysis was performed with the Statistica software package
(release 6.0). Data are reported as the mean ± SD and median with
range. The relationship between duration and severity of diarrhea was
assessed using the Spearman correlation. An exact Wilcoxon test was
used to determine statistical significance between SN-38 systemic
exposure measured after doses 1 and 10. All comparisons were considered
significant at a type I error rate of 
= 0.05.
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RESULTS |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Of the 24 patients evaluated, 19 had
pharmacokinetic studies after doses 1 and 10. Four patients had studies
completed only after the first dose, and one patient was studied at
dose 10 only.
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. IRN, SN-38, and APC lactone
plasma concentration-time data from a representative patient given IRN
at a dose of 20 mg/m2 are depicted in Fig. 3
. IRN exhibited biphasic plasma
elimination with a rapid decrease after the end of the 60-min infusion.
SN-38 plasma concentrations displayed monoexponential first-order
elimination. In two patients, SN-38 plasma concentrations revealed a
bimodal distribution, suggesting enterohepatic recycling. APC terminal
plasma concentrations displayed first-order decay.
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. The mean ± SD ng/ml·h
single-day lactone SN-38 AUC06 was 90.9 ± 96.4, 103.7 ± 62.4, and 95.3 ± 63.9 at IRN doses of 20,
24, and 29 mg/m2, respectively. The IRN
AUC06 accounted for greater than 70% of the
IRN AUC0
in all of the 43 pharmacokinetic
studies. Likewise, the SN-38 AUC06 accounted
for greater than 70% of the SN-38 AUC 0- in
38 of 43 pharmacokinetic studies. Thus, we chose
AUC06 to report the systemic exposure.
As shown in Table 3
, large interpatient variability was observed in
IRN, SN-38, and APC systemic exposure or AUC06
after the first IRN dose. A 5-fold variation in IRN systemic exposures
among patients was observed during course 1 over three dosage levels,
and more than 30- and 10-fold variations in SN-38 and APC
systemic exposures were observed, respectively. In the 19 patients who
had pharmacokinetic studies after doses 1 and 10, we observed no
statistically significant difference in SN-38 AUC (P =
0.10, exact Wilcoxon test).
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Adequate sample volume was available to determine the extent of SN-38 protein binding in 13 patients. The serum albumin levels were normal in these patients, ranging from 3.0–4.4 g/dl. Median SN-38 percentage unbound was 3.3 (range, 0.7–6.5) over all dose levels. No relation was noted between serum albumin and SN-38 percentage unbound or BR.
Myelosuppression was minimal and was not dose limiting in any child during the first course of therapy, except for one patient that developed a grade 3 neutropenia lasting for 11 days after starting an IRN dose of 24 mg/m2. We were unable to identify any pharmacodynamic relationship between SN-38 systemic exposure and myelosuppression.
Diarrhea was seen at all three dose levels. Among six patients who
developed grade 3 diarrhea, two patients had positive cultures for
Clostridium difficile. Acute onset of diarrhea within 8 h of the initial IRN dose occurred in two patients. The median onset of
diarrhea occurred on day 9 (range, 1–16 days) after the first dose of
the first course of IRN. All patients took loperamide at the first
onset of loose stools. Diarrhea was resolved after a median of 4 days
(range, 0–16 days) after the last dose of IRN. We observed no
significant correlation between the duration or severity of diarrhea
and pharmacokinetic measures of drug exposure, including IRN, SN-38,
and APC AUC06.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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IRN displayed a biexponential elimination, whereas SN-38 exhibited a monophasic decline, perhaps because of the low measured SN-38 plasma concentrations. In addition, we measured APC plasma concentrations as an indicator of the extent of oxidative metabolism of IRN. We incorporated APC plasma concentration data into the model and reported the first APC pharmacokinetic parameters in children. The mean terminal half-lives of IRN and SN-38 were shorter than those reported in adults (22) . The mean IRN systemic clearance in our population was comparable to that observed in adults; however, a greater interpatient variability was observed in this pediatric population than in adults (22) . IRN systemic clearance appeared to be dose independent, although the dosage range evaluated was narrow (20–29 mg/m2), and the sample size, especially at the 29 mg/m2 dosage level, was small.
The range of SN-38 protein binding observed in our study was consistent with that reported from an in vitro SN-38 protein binding study (23) . We were unable to find a relation between serum albumin and SN-38 protein binding. However, the 10-fold interpatient variability in SN-38 percentage unbound is likely to be clinically relevant.
To compare the SN-38 systemic exposures observed in our patients with those reported in adults, we calculated the relative extent of IRN conversion to SN-38 (REC). The median REC at each IRN dose level studied was 0.37, 0.51, and 0.21 at IRN dosages of 20, 24, and 29 mg/m2/day, respectively. This was greater than the REC reported in another group of children (15) and also in a group of adults (14) . However, our daily IRN dose was approximately one-tenth of those given in these two studies. We therefore compared the absolute SN-38 production in our study with that reported in adult trials using the Food and Drug Administration-approved IRN schedule and dosage (20 , 22 , 24) . At each dose level, our protracted administration schedule was associated with as much as a 4-fold greater cumulative SN-38 systemic exposure per course than that obtained in the adult trials where cumulative IRN doses were two to three times less. However, a recent report of a low-dose (10 mg/m2), 14-day continuous infusion of IRN in adults showed that the mean REC was 0.16 compared with 0.03–0.05 after short infusions of therapeutic dosages. The investigators also noted two partial responses in this heavily pretreated Phase I patient population. Thus, it is conceivable that lower IRN doses, given over protracted intervals, may be more effective in adults because of a greater production of the active SN-38 metabolite (25) .
The median REM at an IRN dose of 20 mg/m2 was 0.29 (range, 0.10–0.57), which was similar to the finding in another pediatric study in which a 10-fold higher IRN dose of 200–420 mg/m2 every 3 weeks was administered (15) . Vassal et al. (15) reported a median REM of 0.12 (range, 0.04–0.25) in a group of children whose age ranged from 10 months to 17.5 years. Estimated REM measurements in both groups of children were much lower than that reported previously in adults (median, 2.2; range, 0.65–3.9) receiving IRN at 115 mg/m2 over a 90-min period every 3 weeks (14) . This illustrates the differences in metabolite formation between children and adults. The exact mechanisms by which APC metabolism is mediated in children and adults are still unknown.
In a recent study of adult glioma patients, Friedman and colleagues
(26
, 27) suggested that the concurrent administration of
anticonvulsants and corticosteroids with IRN altered the disposition of
IRN and metabolites. Their results showed that the mean IRN and SN-38
AUC was approximately 39% and 22%, respectively, of that measured in
a group of gastrointestinal cancer patients treated with IRN on a
similar schedule and dose, but without concurrent anticonvulsants and
corticosteroids. They did not report the APC data from their patients.
Although the present study was not designed to evaluate the interaction
of corticosteroids and anticonvulsants with IRN, two patients were
treated at 24 mg/m2 and received concomitant
dexamethasone and anticonvulsants (e.g., valproic acid,
phenobarbital, and carbamazepine). The REC value for each patient was
below the median determined for all patients after dose 1 (see Table 4
). Similarly, these two patients
receiving dexamethasone and anticonvulsants had REM values 2-fold that
of the median value determined for all patients after dose 1 (see Table 4
). These preliminary observations warrant further investigation into
the mechanisms for possible drug interactions.
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We were unable to identify a pharmacodynamic relationship between SN-38 or IRN systemic exposure and gastrointestinal toxicity. Although we used the NCI Common Toxicity Criteria for classifying the severity of diarrhea, these results remain subjective. Most end point measurements of diarrhea are based largely on patient or family self-reports. We conclude that a more systematic quantification of diarrhea in this patient population will be essential to additional studies of the mechanism of this toxicity. In single-dose studies of IRN in adults, delayed onset of diarrhea occurred at least 2 days after dosing (29) . However, in our pediatric patients who received this protracted schedule, it was difficult to distinguish whether diarrhea occurring late in the schedule was attributed to cumulative systemic exposure or to a delayed onset.
The major elimination pathway of SN-38 is conjugation to glucuronic acid. SN-38G has been shown to be secreted in bile and deconjugated by intestinal flora to form SN-38 (30) . This local accumulation of SN-38 is believed to contribute to the delayed gastrointestinal toxicity observed in patients receiving IRN. Gupta et al. (31) described a linear relationship between biliary index and grade of gastrointestinal toxicity in adults. Our plasma sampling schedule was not designed to identify enterohepatic recycling in these pediatric patients; however, the SN-38 plasma profiles displayed a secondary peak suggestive of enterohepatic recycling in two patients. We were unable to measure plasma SN-38G concentrations in the current study because of a limited sample volume. A follow-up study in children that will include measurements of SN-38G is ongoing.
In summary, the description of the disposition of IRN, SN-38, and APC with one pharmacokinetic model may have important clinical implications. Specifically, this model could be used to prospectively adjust the IRN dose in clinical trials to attain putative cytotoxic systemic exposures as defined in the xenograft model. However, because of the wide interpatient variability noted in IRN and metabolite disposition, additional pharmacokinetic studies are needed to refine population priors for use in a Bayesian approach to target cytotoxic systemic exposures. Moreover, additional studies of the genetic polymorphisms in IRN-metabolizing enzymes (e.g., UGT1A1, CYP3A4) or plasma protein binding may explain some of the interpatient variability of IRN and metabolite disposition in children.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by USPHS Grant CA 23099,
Cancer Center Support Grant CA 21765, and by American Lebanese Syrian
Associated Charities. 
2 To whom requests for reprints should be
addressed, at St. Jude Children’s Research Hospital, 332 North
Lauderdale, Memphis, TN 38105-2794. Phone: (901) 495-3665; Fax: (901)
525-6869; E-mail: clinton.stewart{at}stjude.org
3 The abbreviations used are: IRN, irinotecan
[7-ethyl-10-(4-[1-piperidino]-1-piperidino)-carbonyloxycamptothecin];
SN-38G, SN-38 glucuronide; AUC, area under the plasma-concentration
versus time curve; ML, maximum likelihood; REC, relative
extent of conversion; REM, relative extent of metabolism; BR, binding
ratio; NCI, National Cancer Institute; ANC, absolute neutrophil
count.
Received 8/30/99; revised 11/16/99; accepted 11/19/99.
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REFERENCES |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), and APC (
) plasma
concentration versus time plot in a representative
patient (patient 2, dose 1) who received IRN at 20 mg/m2
over a 1-h period. Symbols represent measured plasma
concentrations, and solid lines represent the best fit
line from the Bayesian estimation.