Clinical Chemistry
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Clinical Chemistry 49: 1900-1908, 2003; 10.1373/clinchem.2003.023481
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (10)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Poujol, S.
Right arrow Articles by Bressolle, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Poujol, S.
Right arrow Articles by Bressolle, F.
Related Collections
Right arrow Laboratory Management
Right arrow Clinical Immunology
Right arrow Drug Monitoring and Toxicology
(Clinical Chemistry. 2003;49:1900-1908.)
© 2003 American Association for Clinical Chemistry, Inc.

Automation and Analytical Techniques

Sensitive HPLC-Fluorescence Method for Irinotecan and Four Major Metabolites in Human Plasma and Saliva: Application to Pharmacokinetic Studies

Sylvain Poujol1, Frédéric Pinguet1, Françoise Malosse1, Cécile Astre1, Marc Ychou2, Stéphane Culine2 and Françoise Bressolle3,a

1 Oncopharmacology Department, Pharmacy Service, and
2 Department of Medicine, Val d’Aurelle Anticancer Centre, Parc Euromédecine, 34298 Montpellier, Cedex 5 France.

3 Clinical Pharmacokinetic Laboratory, Faculty of Pharmacy, 15 Avenue Ch. Flahault, University Montpellier I, 34093 Montpellier Cedex 5, France.

aAddress correspondence to this author at: Laboratoire de Pharmacocinétique Clinique, Faculté de Pharmacie, BP 14491, 34093 Montpellier Cedex 5, France. Fax 33-4-6754-8075; e-mail Fbressolle{at}aol.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Background: We developed gradient HPLC methods for quantification of the antimitotic drug irinotecan (CPT-11) and its four metabolites, SN-38, SN-38 G, 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]-carbonyloxycamptothecin (APC), and 7-ethyl-10-[4amino-1-piperidino]-carbonyloxycamptothecin (NPC), as the sum of the lactone and carboxylate forms, in human plasma and saliva. Camptothecin was used as internal standard.

Methods: The sample pretreatment involved protein precipitation with methanol–acetonitrile (50:50 by volume) followed by acidification with hydrochloric acid to convert the lactone ring-opened form into its lactone form, quantitatively. HPLC separation was performed on a Xterra RP18 column. The excitation wavelength was 370 nm, and the emission wavelength was set at 470 nm for the first 24 min and then at 534 nm for the next 4 min. The stabilities of irinotecan and its four metabolites in plasma, saliva, and acidic extracts were also investigated under various conditions.

Results: Assays were linear in the tested range of 0.5–1000 µg/L. For the five analytes, limits of quantification were 0.5 µg/L in both matrices. The interassay imprecision (as relative standard deviation) was 3.2–14% in plasma and 2.6–5.6% in saliva. Assay recoveries ranged from 92.8% to 111.2% for plasma and 100.1% to 104.1% for saliva. Mean extraction recovery from plasma or saliva was 90%.

Conclusion: The developed assay can be used to determine pharmacokinetic parameters for CPT-11, SN-38, SN-38 G, APC, and NPC in plasma and saliva from patients with metastatic colorectal cancer.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
The antineoplastic agent irinotecan hydrochloride (7-ethyl-10-[4-(1-piperidino)-1-piperidino]-carbonyloxy-camptothecin; CPT-11; Fig. 1 ) is a semisynthetic derivative of the natural product camptothecin (1)(2). This drug has demonstrated good antitumor activity both in vitro and in vivo against various experimental tumor models (1)(3), including multidrug-resistant lines (4). CPT-11 has shown anticancer activity against a variety of solid tumors in clinical trials, including colorectal cancer, gynecologic cancers, non-small cell and small cell lung cancers, and refractory cervical cancer (5)(6)(7)(8)(9)(10)(11). Like camptothecin, CPT-11 acts by inhibiting mammalian DNA topoisomerase I (12)(13). Cell death results from stabilization of cleavable complexes formed between topoisomerase I and DNA during DNA replication, transcription, and repair (14)(15)(16)(17).



View larger version (17K):
[in this window]
[in a new window]
  		Figure 1. Chemical structures of irinotecan and its major metabolites.

UDP-GT, UDP-glucuronosyltransferase.

CPT-11 is extensively metabolized in the liver into various metabolites. It is enzymatically cleaved by carboxylesterases to form 7-ethyl-10-hydroxycamptothecin (SN-38), which has a cytotoxic activity 100 to 1000 times greater than that of the parent drug (18)(19)(20). SN-38 is subsequently conjugated by uridine diphosphate glucuronosyltransferases to an inactive ß-glucuronide derivative (SN-38G) (20)(21)(22). Other known human metabolites of CPT-11 include 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]-carbonyloxycamptothecin (APC) 1 and a primary amine metabolite, 7-ethyl-10-[4-amino-1-piperidino]-carbonyloxycamptothecin (NPC), which results from ring-opening oxidation of the terminal piperidine ring of CPT-11 mediated by cytochrome P450 3A4 enzymes (23)(24). APC and NPC have weak inhibitory activity against cell growth in vivo. The structures of these metabolites are presented in Fig. 1Up .

CPT-11 and its metabolites contain an {alpha}-hydroxy-{delta}-lactone ring, which is chemically unstable and undergoes pH-dependent reversible hydrolysis to a hydroxyl-carboxylate form. Only the closed lactone forms of the drug inhibit topoisomerase I. Rivory et al. (25) found low variability of this interconversion despite the fact that the patients received different doses and were at different stages of treatment.

Several HPLC methods have been developed to quantify irinotecan and its major metabolites in human plasma (26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37). These methods involve the simultaneous quantification of the lactone and carboxylate forms (27)(29)(32)(37) or quantification of the total forms, i.e., lactone plus carboxylate (26)(28)(30)(31)(33)(34)(35)(36). In most of these methods, both CPT-11 and SN-38 were quantified and the related compound camptothecin was used as the internal standard. Only Sparreboom et al. (30), Sai et al.(35), and Owens et al. (37) developed a method for the determination of CPT-11 and the three metabolites SN-38, SN-38G, and APC in biological matrices. In most of these published methods, chromatography was carried out with fluorescence detection. Liquid chromatography–mass spectrometry methods have also been described (33)(35), but despite the latter methods providing good results, the instrumentation involved is expensive and not always readily available for routine drug monitoring.

Takahashi et al. (38) found that both CPT-11 and SN-38 were detectable in saliva and that the patterns of their concentration–time curves in plasma and saliva were very similar. To date, however, no bioanalytical assay has been validated for the determination of irinotecan and its metabolites in saliva.

This report describes rapid, specific, reliable, and sensitive analytical methods to simultaneously quantify irinotecan and four metabolites (SN-38, SN-38G, APC, and NPC) in human plasma and saliva. These methods, involving the use of an internal standard, were validated according to validation procedures, parameters, and acceptance criteria based on US Pharmacopoeia XXIII guidelines and Food and Drug Administration guidance (39)(40)(41)(42). We also evaluated the stability of these analytes under various conditions. The suitability of the developed methods for clinical use was demonstrated by the determination of irinotecan and its four metabolites in plasma and saliva obtained from patients with metastatic colorectal cancer.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
materials and reagents
Irinotecan (hydrochloride salt; CPT-11), SN-38, SN-38 G, APC, and NPC were kindly donated by Aventis Pharma Laboratories (Vitry-sur-Seine, France). The internal standard, camptothecin, was obtained from Sigma. Stock solutions of each compound were prepared in dimethyl sulfoxide (Carlo Erba) at the concentration of 1 g/L each and stored at -80 °C. Methanol, dimethyl sulfoxide, acetone, acetonitrile, 5 mol/L HCl, monopotassium phosphate, orthophosphoric acid (all from Merck), and 1-heptanesulfonic acid (Sigma) were all of analytical-reagent grade. The 5 mol/L HCl was diluted fivefold in sterile water (Fresenius, France Pharma). The phosphate buffer consisted of 13.609 g of monopotassium phosphate and 0.607 g of 1-heptanesulfonic acid in 1 L of sterile water adjusted to pH 4.0 with orthophosphoric acid.

For method validation, human plasma (Etablissement Français du sang) and parotid saliva were obtained from pooled samples collected from healthy volunteers. Saliva and plasma were stored at -20 °C before use.

instrumentation
The chromatographic system consisted of two Shimadzu LC-10AT pumps and a mixing chamber, a Shimadzu Model RF-10Axl fluorescence detector, an automatic sample injection system (Model 717; Waters) thermostated at 10 °C, and a Shimadzu data acquisition station (Class VP 4.2). The excitation wavelength was 370 nm; the emission wavelength was set at 470 nm for the first 24 min (SN-38G, NPC, APC, CPT-11, and camptothecin) and then at 534 nm (SN-38) for the next 4 min. HPLC separation was performed in a stainless-steel Xterra RP18 column [250 x 4.6 mm (i.d.)], packed with 5-µm particles (Waters). A guard Xterra RP18 column [20 x 3.9 mm (i.d.); 5-µm particle size; Waters] was placed just before the inlet of the analytical column to reduce contamination.

chromatographic conditions
The eluent was pumped through the column at a flow rate of 1 mL/min and consisted of a mixture of acetonitrile–sterile water (75:25 by volume; solvent A) and phosphate buffer, pH 4 (solvent B). The mobile phases were filtered through a 0.45 µm HV filter (Millipore), then degassed ultrasonically before use. Table 1 shows the various proportions of solvents A and B used for the methods. Chromatography was performed at ambient temperature (23 °C).


View this table:
[in this window]
[in a new window]
  		Table 1. Proportions of solvent A and solvent B for the HPLC method.

sample preparation
To a polypropylene microtube containing 100 µL of an internal standard solution (100 µg/L in acetone), previously dried under nitrogen stream, we added 0.5 mL of plasma (or 0.1 mL of saliva) and a mixture of methanol–acetonitrile (50:50 by volume; 1 mL for plasma or 0.2 mL for saliva). Proteins were denatured by full-speed vortex-mixing for 15 s; samples were then centrifuged at 9500g for 4 min. The clear supernatant (550 µL for plasma or 220 µL for saliva) was aspirated and placed in another microtube to which 250 µL (or 100 µL) of 1 mol/L HCl was added. Samples were vortex-mixed for 15 s, and an aliquot of this solution (10 µL at high concentrations or 100 µL at low concentrations) was injected on the column.

Calibrators (0.5, 1, 2.5, 5, 10, 25, 50, 100, 250, 500, and 1000 µg/L) were prepared by mixing drug-free plasma or saliva with appropriate volumes of working solutions in acetonitrile–purified water (50:50 by volume). The sample pretreatment procedure was identical to that described above.

data analysis
For each calibration curve (0.5–10, 10–250, and 250-1000 µg/L), we applied an unweighted least-squares linear regression of the responses (peak-area ratios of compound to internal standard) as a function of the nominal concentrations. The detector was set at a sensitivity of 2 and a gain of 2. The parameters of each calibration curve were used to compute back-calculated concentrations and to obtain concentration values for that day’s quality-control (QC) samples and unknown samples.

The "lack-of-fit" test was used to confirm the linearity of the method. In addition, the back-calculated concentrations were compared with the nominal concentrations, and the mean differences (or mean predictor error) with the 95% confidence interval were computed.

selectivity
We investigated the specificity of the method by analyzing 10 different batches of blank human plasma and saliva samples from healthy volunteers to determine whether endogenous constituents coeluted with the different analytes. The retention times (tr) of endogenous compounds in the matrix were compared with those of the compounds of interest.

Plasma and saliva samples from patients receiving other drugs were analyzed for interference. The following drugs were checked: granisetron, ondansetron, tropisetron, dolasetron, alizapride, zolpidem, tianeptine, methylprednisolone, fluoxetine, dextropropoxyphene, acetaminophen, enoxaparin, nifedipine, bromazepam, lorazepam, chlorazepate, levothyroxine, zopiclone, tinzaparin, cetirizine, simvastatin, famotidine, glibenclamide, and omeprazole.

imprecision and recovery
We assessed the within- and between-day imprecision and the recoveries by performing replicate analyses of QC samples (0.75, 75, and 750 µg/L) in plasma or saliva and comparing them against the calibration curves. The procedure was repeated on different days with the same calibrators to determine between-day values. Intraday values were determined by treating samples to which calibrators had been added in replicate on the same day.

The mean recovery was calculated as (mean measured concentration/theoretical concentration) x 100. Imprecision is given as the relative standard deviation (RSD).

extraction efficiency
We determined the recoveries of CPT-11, SN-38, SN-38 G, APC, and NPC from plasma and saliva twice at three concentrations (0.75, 75 and 750 µg/L) by calculating the percentage difference between the peak areas of extracted calibrators and those of the authentic (unextracted) calibrators in the relevant concentration ranges prepared in acetonitrile–water–1 mol/L HCl (183:367:250 by volume). The extraction recovery was also computed for the internal standard.

determination of the lower limit of quantification
The lower limit of quantification, estimated from QC samples, was defined as the lowest drug concentration that could be determined with a RSD <=20% and a recovery of 100 (20)% on a day-to-day basis (39)(40)(41)(42).

stability study
The stability of the lactone plus carboxylate forms of irinotecan and its four metabolites in plasma and saliva was determined by use of QC samples at concentrations of 0.75, 75, and 750 µg/L as follows:

QC samples were analyzed immediately after preparation (reference values) and at selected time intervals after storage over the study period. Three replicates were analyzed at each time point. Stability was defined as <10% loss of initial drug concentration.

pharmacokinetic study
The pharmacokinetic study was carried out in patients with metastatic colorectal cancer. It was conducted according to the current revision of the Helsinki Declaration, and all patients gave informed consent. Irinotecan was administered in a 90-min infusion every 2 weeks (180 mg/m2 for the first course, 220 mg/m2 for the second course, and 260 mg/m2 for the following courses). The patients had satisfactory WHO Cooperative Oncology Group scores (0 or 2) and liver and kidney functions. Special attention was paid to sample handling. Blood collected in heparin-containing tubes and unstimulated saliva samples were drawn before drug administration (t0) and at 30, 60, and 90 min and 4, 8, 12, 24, and 42 h after the start of infusion. Blood samples were centrifuged (1500g for 10 min) at 4 °C. A 1-mL aliquot of plasma was transferred to another tube. Saliva samples were centrifuged at 9500g for 4 min before storage. Plasma and saliva samples were stored at -80 °C until assay. To quantify the different compounds in plasma and saliva samples, we ran calibration curves and three QC samples with every set of 20 unknown samples. For CPT-11 concentrations higher than the highest calibration point, we diluted samples with drug-free plasma to bring the concentrations within the range of calibration curve. Pharmacokinetic parameters were calculated using Pk-fit software (43). The areas under the plasma or saliva concentration–time curves (AUC) from time zero to infinity were obtained by linear trapezoidal approximation with correction to time infinity by dividing the last observed data point by the elimination rate constant. Elimination half-life was determined from the slope of the linear part of the semilogarithmic curves.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
retention times and specificity
Representative chromatograms of drug-free human plasma and saliva; plasma and saliva with added CPT-11, SN-38, SN-38G, APC, NPC, and internal standard; and postdose human plasma and saliva samples collected from the clinical study are shown in Figs. 2 and 3 , respectively. Under the chromatographic conditions used, the number of theoretical plates (computed on the peak of internal standard) was ~12 531. The precolumn was exchanged every 200 sample runs, and the column was replaced when the number of theoretical plates had decreased to <7604. SN-38G [mean (SD) tr = 5.54 (0.15) min; n = 40], NPC [tr = 13.9 (0.23) min; n = 40], APC [tr = 16.0 (0.36) min; n = 40], CPT-11 [tr = 19.6 (0.56) min; n = 40], internal standard [tr = 22.5 (0.54) min; n = 40], and SN-38 [tr = 28.2 (0.83) min; n = 40] exhibited well-separated peaks ({alpha}1,2 = 1.4, {alpha}2,3 = 1.3, {alpha}3,4 = 1.4, {alpha}4,5 = 1.3, {alpha}5,6 = 1.5) under the chromatographic conditions described. The k' values were 0.89, 3.56, 4.27, 5.43, 6.36, and 8.3 for the six analytes, respectively. We evaluated peak skew using the asymmetry coefficient As = b/a, where b is the distance after the peak maximum and a is the distance before the peak maximum, both a and b being measured at 10% of the total peak height. The asymmetry coefficients were 1.09 for SN-38G, 1.03 for NPC, 1.23 for APC, 1.27 for CPT-11, and 0.9 for SN-38. No endogenous interfering peaks were visible at the retention times of the different analytes (Figs. 2A and 3A ). No interference was found with all tested drugs that could be coadministered.



View larger version (16K):
[in this window]
[in a new window]
  		Figure 2. Chromatograms of blank plasma (A), plasma to which 2.5 (B) and 250 µg/L (C) of each analyte had been added, and plasma from a patient receiving 180 mg/m2 of irinotecan (D).

Concentrations 90 min after the start of the infusion: SN-38G, 84.6 µg/L; NPC, 9.4 µg/L; APC, 106.7 µg/L; CPT-11, 2516.2 µg/L; and SN-38, 28.7 µg/L. Peaks: 1, SN-38G; 2, NPC; 3, APC; 4, CPT-11; 5, internal standard; 6, SN-38. For chromatographic conditions, see the text.



View larger version (15K):
[in this window]
[in a new window]
  		Figure 3. Chromatograms of blank saliva (A), saliva with 2.5 (B) and 250 µg/L (C) of each analyte added, and saliva from a patient receiving 260 mg/m2 of irinotecan (D).

Concentrations 4 h after the start of the infusion: NPC, 0.83 µg/L; APC, 8.57 µg/L; CPT-11, 390.5 µg/L; and SN-38, 2.99 µg/L. Peaks: 1, SN-38G; 2, NPC; 3, APC; 4, CPT-11; 5, internal standard; 6, SN-38. For chromatographic conditions, see the text.

linearity
The calculated peak-area ratios and the added concentrations of each analyte displayed linear relationships over the selected concentration ranges with consistent slopes and coefficients of determination (r2) >0.99 throughout the validation runs. The RSD for the slopes of calibration curves prepared on the same day or on different days was <13% in plasma and <9.5% in saliva. The lack-of-fit test showed no significant deviation from linearity. For each calibration point, the concentrations were back-calculated from the equation of the linear regression curves. Linear regression of the back-calculated concentrations vs the nominal concentrations provided a unit slope and an intercept equal to 0 (Student t-test). The distribution of the residuals (difference between nominal and back-calculated concentrations) showed random variations, the number of positive and negative values being approximately equal. Moreover, they were normally distributed and centered around zero. The mean value of residuals was not statistically different from 0 (Student t-test), and the 95% confidence interval included the 0 value.

imprecision and recovery
The recovery and imprecision results are shown in Table 2 . The mean (SD) extraction efficiencies for CPT-11, SN-38, SN-38G, APC, and NPC from human plasma were 95.5 (5.4)%, 97.2 (5.9)%, 93.4 (5.0)%, 95.5 (5.8)%, and 99.2 (4.69)%, respectively (n = 6). In saliva, they were 97.5 (4.2)%, 96.8 (9.5)%, 94.6 (8.2)%, 103.9 (6.3)%, and 100.8 (7.4)%, respectively (n = 6). Recoveries for the internal standard were 99.5 (3.0)% (n = 3) in plasma and 99.2 (5.0)% (n = 3) in saliva.


View this table:
[in this window]
[in a new window]
  		Table 2. Recovery and imprecision of the method.

The extraction efficiency was independent of concentration over the range studied.

lower limit of quantification
In plasma and saliva, the lower limit of quantification was 0.5 µg/L for irinotecan and its four metabolites. For QC samples, the RSD did not exceed 20%, and recoveries were 83–108%. Compared with the published methods for most of the analytes, this limit of quantification was better than those published in the literature (26)(27)(28)(29)(30)(33)(34)(35)(36)(37).

stability
Frozen QC samples tested over a 4-month period showed no sign of either degradation or loss in both plasma and saliva (P >0.05). We performed stability assays on frozen plasma samples over a 6-month period. We observed significant degradation, averaging 37% for SN-38G at 0.75 µg/L, but observed no degradation for SN-38G at the other concentrations of for any of the other compounds at any of the concentrations studied.

Plasma and saliva QC samples with added CPT-11, SN-38, SN-38G, APC, and NPC allowed to stand at room temperature or at 4 °C for 24 h showed no sign of decrease in the nominal starting concentration.

In acidic extracts, APC showed a substantial decrease after 24 h of storage at ambient temperature; mean (SD) losses were 49.1 (2.1)% at 0.75 µg/L, 45.8 (2.5)% at 75 µg/L, and 38.9 (1.9)% at 750 µg/L. For this compound, the decrease in drug concentration followed a monoexponential pattern; the corresponding half-life value was 21 h. Fig. 4 shows the stability at ambient temperature of the different analytes (concentration, 75 µg/L) in acidic extracts for the first 16 h. At 10 °C, losses were <10% after 8 h of storage, whereas after 24 h, mean losses were 9.1 (5.2)%, 21.7 (4.1)%, and 17.6 (2.8)% at the three concentrations, respectively. Losses were lower at 4 °C [7.8 (0.5)%, 18.6 (1.0)%, and 11.1 (1.2)%, respectively, after 72 h of storage]. NPC concentrations increased in proportion to the decrease in APC (30–44% increase after 24 h at ambient temperature), suggesting a partial conversion of APC into NPC in acidic extracts. The stability of SN-38G in acidic extracts indicated that no statistically significant degradation occurred over a span of 48 h at ambient temperature at 0.75 µg/L [recovery, 106 (8.1)%]. For the two other concentrations tested, we observed a significant decrease after 24 h [mean recovery, 80.3 (1.4)% at 75 µg/L and 81.8 (4.0)% at 750 µg/L]. At 4 and 10 °C, SN-38G was stable for 72 h. CPT-11, SN-38, and the internal standard were stable in the different tested conditions.



View larger version (19K):
[in this window]
[in a new window]
  		Figure 4. Stability of SN-38G ({triangleup}), NPC ({blacksquare}), APC ({square}), CPT-11 ({circ}), SN-38 ({blacktriangleup}), and internal standard (•) in acidic extracts at ambient temperature.

Concentration of each analyte, 75 µg/L. Error bars, SD.

Interestingly, both plasma and saliva samples showed no sign of degradation after the freeze–thaw procedure.

These stability results are of great interest for the routine analysis of a large number of samples during pharmacokinetic studies. Thus, acidic extracts from biological samples must be kept at low temperatures before and during analysis to avoid rapid degradation.

pharmacokinetic study
The plasma and saliva concentration–time profiles of CPT-11 and the observed metabolites determined by the method described above are presented in Fig. 5 . These results were obtained from the first three patients included in the study (administered dose, 180 mg/m2). For the parent drug and its metabolites, similar pharmacokinetic profiles were observed based on plasma and salivary data. These data could be described by a biexponential pattern. The main pharmacokinetic parameters are given in Table 3 . From the plasma data, the CPT-11 total clearance was 15.6 (4.0) L · h-1 · m-2. Rapid diffusion of irinotecan in saliva occurred; its concentrations were of the same order of magnitude in saliva and plasma samples, and the saliva/plasma AUC ratio averaged 0.98. For the active metabolite SN-38, concentrations in saliva were approximately twofold lower than in plasma; the saliva/plasma AUC ratio was 0.40. For the two metabolites APC and NPC, diffusion in saliva was low, whereas SN-38G concentrations in saliva were lower than the lower limit of quantification of the method. The low diffusion of these three metabolites could be explained by their high hydrophilic properties. Considering the high binding rates of CPT-11 and SN-38 to plasma proteins, ~63% and 95%, respectively (Aventis Pharma Laboratories data), the secretion of these two compounds in saliva remains important. The carboxylate is the predominant form of irinotecan in plasma soon after the end of infusion (25); because this form is unlikely to cross biological barriers, the secretory mechanisms of the irinotecan lactone in saliva are not attributable to simple passive diffusion but rather to active processes, as reported recently for topotecan (44). Thus, the saliva concentration could be a good predictor of the behavior of irinotecan in the body.



View larger version (19K):
[in this window]
[in a new window]
  		Figure 5. Mean (SD; error bars) plasma (A) and saliva (B) concentration–time profiles for irinotecan and its four metabolites from the first three patients included in the pharmacokinetic study (administered dose, 180 mg/m2).

x, SN-38G; {diamondsuit} and {diamond}, NPC; {blacktriangleup} and {triangleup}, APC; • and {circ}, CPT-11; {blacksquare} and {square}, SN-38.


View this table:
[in this window]
[in a new window]
  		Table 3. Main pharmacokinetic parameters [mean (SD)] for irinotecan and its metabolites computed from results for the first three patients included in the study.

In conclusion, this fully validated method permits simple and rapid quantification of irinotecan and its four main metabolites in plasma and saliva samples. In many clinical situations, including patients with difficult venous access, the determination of irinotecan and its active metabolite SN-38 in saliva may be relevant.


   Acknowledgments
 
We gratefully acknowledge support of this work by the "Ligue Nationale de Lutte contre le Cancer" (Montpellier, France). We give special thanks to B. Hawkins, Anticancer Centre, Montpellier, for assistance in the preparation of this manuscript.


   Footnotes
 
1 Nonstandard abbreviations: APC, 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]-carbonyloxycamptothecin; NPC, 7-ethyl-10-[4-amino-1-piperidino]-carbonyloxycamptothecin; QC, quality-control; tr, retention time; RSD, relative standard deviation; and AUC, area under the curve.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

  1. Kunimoto T, Nitta K, Tanaka T, Uehara N, Baba H, Takeuchi M, et al. Antitumor activity of 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxy-camptothecin, a novel water-soluble derivative of camptothecin, against murine tumors. Cancer Res 1987;47:5944-5947.[Abstract/Free Full Text]
  2. Sawada S, Okajima S, Aiyama R, Nokata K, Furuta T, Yokokura T, et al. Synthesis and antitumor activity of 20(S)-camptothecin derivatives: carbamate-linked, water-soluble derivatives of 7-ethyl-10-hydroxycamptothecin. Chem Pharm Bull 1991;39:1446-1450.
  3. Bissery MC, Mathieu-Boué A, Lavelle F. Preclinical evaluation of CPT-11, a camptothecin derivative. Proc Am Assoc Cancer Res 1991;32:402.
  4. Tsuruo T, Matsuzaki T, Matsushita M, Saito H, Yokokura T. Antitumor effect of CPT-11, a new derivative of camptothecin, against pleiotropic drug-resistant tumors in vitro and in vivo. Cancer Chemother Pharmacol 1988;21:71-74.[Web of Science][Medline] [Order article via Infotrieve]
  5. Shimada Y, Yoshino M, Wakui A, Nakao I, Futatsuki K, Sakata Y, et al. Phase II study of CPT-11, a new camptothecin derivative, in metastatic colorectal cancer. CPT-11 Gastrointestinal Cancer Study Group. J Clin Oncol 1993;11:909-913.[Abstract/Free Full Text]
  6. Rothenberg ML, Eckardt JR, Kuhn JG, Burris HA, 3rd, Nelson J, Hilsenbeck SG, et al. Phase II trial of irinotecan in patients with progressive or rapidly recurrent colorectal cancer. J Clin Oncol 1996;14:1128-1135.[Abstract/Free Full Text]
  7. Conti JA, Kemeny NE, Saltz LB, Huang Y, Tong WP, Chou TC, et al. Irinotecan is an active agent in untreated patients with metastatic colorectal cancer. J Clin Oncol 1996;14:709-715.[Abstract/Free Full Text]
  8. Fukuoka M, Niitani H, Suzuki A, Motomiya M, Hasegawa K, Nishiwaki Y, et al. A phase II study of CPT-11, a new derivative of camptothecin, for previously untreated non-small-cell lung cancer. J Clin Oncol 1992;10:16-20.[Abstract]
  9. Masuda N, Fukuoka M, Kusunoki Y, Matsui K, Takifuji N, Kudoh S, et al. CPT-11: a new derivative of camptothecin for the treatment of refractory or relapsed small-cell lung cancer. J Clin Oncol 1992;10:1225-1229.[Abstract/Free Full Text]
  10. Creemers GJ, Lund B, Verweij J. Topoisomerase I inhibitors: topotecan and irinotecan. Cancer Treat Rev 1994;20:73-96.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  11. Gerrits CJ, de Jonge MJ, Schellens JH, Stoter G, Verweij J. Topoisomerase I inhibitors: the relevance of prolonged exposure for present clinical development. Br J Cancer 1997;76:952-962.[Web of Science][Medline] [Order article via Infotrieve]
  12. Hsiang YH, Hertzberg R, Hecht S, Liu LF. Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. J Biol Chem 1985;260:14873-14878.[Abstract/Free Full Text]
  13. Hsiang YH, Liu LF. Identification of mammalian DNA topoisomerase I as an intracellular target of the anticancer drug camptothecin. Cancer Res 1988;48:1722-1726.[Abstract/Free Full Text]
  14. Kessel D, Bosmann HB, Lohr K. Camptothecin effects on DNA synthesis in murine leukemia cells. Biochem Biophys Acta 1972;269:210-216.[Medline] [Order article via Infotrieve]
  15. Wang JC. DNA topoisomerase. Ann Rev Biochem 1985;54:665-697.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  16. Hsiang YH, Liu LF, Wall ME, Wani MC, Nicholas AW, Manikumar G, et al. DNA topoisomerase I-mediated DNA cleavage and cytotoxicity of camptothecin analogues. Cancer Res 1989;49:4385-4389.[Abstract/Free Full Text]
  17. Liu LF. DNA topoisomerase poisons as antitumor drugs. Annu Rev Biochem 1989;58:351-375.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  18. Mathijssen RH, van Alphen RJ, Verweij J, Loos WJ, Nooter K, Stoter G, et al. Clinical pharmacokinetics and metabolism of irinotecan (CPT-11). Clin Cancer Res 2001;7:2182-2194.[Abstract/Free Full Text]
  19. Kawato Y, Aonuma M, Hirota Y, Kuga H, Sato K. Intracellular roles of SN-38, a metabolite of the camptothecin derivative CPT-11, in the antitumor effect of CPT-11. Cancer Res 1991;51:4187-4191.[Abstract/Free Full Text]
  20. Canal P, Gay C, Dezeuze A, Douillard JY, Bugat R, Brunet R, et al. Pharmacokinetics and pharmacodynamics of irinotecan during a phase II clinical trial in colorectal cancer. Pharmacology and Molecular Mechanisms Group of the European Organization for Research and Treatment of Cancer. J Clin Oncol 1996;14:2688-2695.[Abstract/Free Full Text]
  21. Rivory LP, Robert J. Identification and kinetics of a ß-glucuronide metabolite of SN-38 in human plasma after administration of the camptothecin derivative irinotecan. Cancer Chemother Pharmacol 1995;36:176-179.[Web of Science][Medline] [Order article via Infotrieve]
  22. Haaz MC, Rivory L, Jantet S, Ratanasavanh D, Robert J. Glucuronidation of SN-38, the active metabolite of irinotecan, by human hepatic microsomes. Pharmacol Toxicol 1997;80:91-96.[Web of Science][Medline] [Order article via Infotrieve]
  23. Rivory LP, Riou JF, Haaz MC, Sable S, Vuilhorgne M, Commercon A, et al. Identification and properties of a major plasma metabolite of irinotecan (CPT-11) isolated from the plasma of patients. Cancer Res 1996;56:3689-3694.[Abstract/Free Full Text]
  24. Dodds HM, Haaz MC, Riou JF, Robert J, Rivory LP. Identification of a new metabolite of CPT-11 (irinotecan): pharmacological properties and activation to SN-38. J Pharmacol Exp Ther 1998;286:578-583.[Abstract/Free Full Text]
  25. Rivory LP, Chatelut E, Canal P, Mathieu-Boué A, Robert J. Kinetics of the in vivo interconversion of the carboxylate and lactone forms of irinotecan (CPT-11) and its metabolite SN-38 in patients. Cancer Res 1994;54:6330-6333.[Abstract/Free Full Text]
  26. Barilero I, Gandia D, Armand JP, Mathieu-Boue A, Re M, Gouyette A, et al. Simultaneous determination of the camptothecin analogue CPT-11 and its active metabolite SN-38 by high-performance liquid chromatography: application to plasma pharmacokinetic studies in cancer patients. J Chromatogr 1992;575:275-280.[Web of Science][Medline] [Order article via Infotrieve]
  27. Rivory LP, Robert J. Reversed-phase high-performance liquid chromatographic method for the simultaneous quantitation of the carboxylate and lactone forms of the camptothecin derivative irinotecan, CPT-11, and its metabolite SN-38 in plasma. J Chromatogr B Biomed Appl 1994;661:133-141.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  28. Sumiyoshi H, Fujiwara Y, Ohune T, Yamaoka N, Tamura K, Yamakido M. High-performance liquid chromatographic determination of irinotecan (CPT-11) and its active metabolite (SN-38) in human plasma. J Chromatogr B Biomed Appl 1995;670:309-316.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  29. de Bruijn P, Verweij J, Loos WJ, Nooter K, Stoter G, Sparreboom A. Determination of irinotecan (CPT-11) and its active metabolite SN-38 in human plasma by reversed-phase high-performance liquid chromatography with fluorescence detection. J Chromatogr B Biomed Sci Appl 1997;698:277-285.[CrossRef][Medline] [Order article via Infotrieve]
  30. Sparreboom A, de Bruijn P, de Jonge MJ, Loos WJ, Stoter G, Verweij J, et al. Liquid chromatographic determination of irinotecan and three major metabolites in human plasma, urine and feces. J Chromatogr B Biomed Sci Appl 1998;712:225-235.[CrossRef][Medline] [Order article via Infotrieve]
  31. Rivory LP, Findlay M, Clarke S, Bishop J. Trace analysis of SN-38 in human plasma by high-performance liquid chromatography. J Chromatogr B Biomed Sci Appl 1998;714:355-359.[CrossRef][Medline] [Order article via Infotrieve]
  32. Chollet DF, Goumaz L, Renard A, Montay G, Vernillet L, Arnera V, et al. Simultaneous determination of the lactone and carboxylate forms of the camptothecin derivative CPT-11 and its metabolite SN-38 in plasma by high-performance liquid chromatography. J Chromatogr B Biomed Sci Appl 1998;718:163-175.[CrossRef][Medline] [Order article via Infotrieve]
  33. Ragot S, Marquet P, Lachatre F, Rousseau A, Lacassie E, Gaulier JM, et al. Sensitive determination of irinotecan (CPT-11) and its active metabolite SN-38 in human serum using liquid chromatography-electrospray mass spectrometry. J Chromatogr B Biomed Sci Appl 1999;736:175-184.[CrossRef][Medline] [Order article via Infotrieve]
  34. Escoriaza J, Aldaz A, Castellanos C, Calvo E, Giraldez J. Simple and rapid determination of irinotecan and its metabolite SN-38 in plasma by high-performance liquid-chromatography: application to clinical pharmacokinetic studies. J Chromatogr B Biomed Sci Appl 2000;740:159-168.[CrossRef][Medline] [Order article via Infotrieve]
  35. Sai K, Kaniwa N, Ozawa S, Sawada J. An analytical method for irinotecan (CPT-11) and its metabolites using a high-performance liquid chromatography: parallel detection with fluorescence and mass spectrometry. Biomed Chromatogr 2002;16:209-218.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  36. Schoemaker NE, Rosing H, Jansen S, Schellens JH, Beijnen JH. High-performance liquid chromatographic analysis of the anticancer drug irinotecan (CPT-11) and its active metabolite SN-38 in human plasma. Ther Drug Monit 2003;25:120-124.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  37. Owens TS, Dodds H, Fricke K, Hanna SK, Crews KR. High-performance liquid chromatographic assay with fluorescence detection for the simultaneous measurement of carboxylate and lactone forms of irinotecan and three metabolites in human plasma. J Chromatogr B Biomed Sci Appl 2003;788:66-74.
  38. Takahashi T, Fujiwara Y, Sumiyoshi H, Isobe T, Yamaokab N, Yamakido M. Salivary drug monitoring of irinotecan and its active metabolite in cancer patients. Cancer Chemother Pharmacol 1997;40:449-452.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  39. US Food and Drug Administration. Guidance for industry. Bioanalytical method validation. May 2001. http://www.fda.gov/cder/guidance/index.htm (Accessed December 2001)..
  40. Validation of compendia methods. United States Pharmacopoeia XXXIII 2003:2439 The United States Pharmacopeia Convention Rockville, MD. .
  41. Shah VP, Midha KK, Dighe S, McGilveray IJ, Skelly JP, Yacobi A, et al. Analytical methods validation: bioavailability, bioequivalence, and pharmacokinetic studies. J Pharm Sci 1992;81:309-312.[CrossRef][Web of Science]
  42. Bressolle F, Bromet-Petit M, Audran M. Validation of liquid chromatographic and gas chromatographic methods. Applications in pharmacokinetics. J. Chromatogr. B 1996;686:3-10.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  43. . RDPP. Pk-fit computer program, Ver. 2.1 1999 RDPP Montpellier, France. .
  44. Boucaud M, Pinguet F, Poujol S, Romieu G, Cupissol D, Astre C, et al. Salivary and plasma pharmacokinetics of topotecan in patients with metastatic epithelial ovarian cancer. Eur J Cancer 2001;37:2357-2364.



The following articles in journals at HighWire Press have cited this article:


Home page
 Molecular Cancer TherapeuticsHome page
Z. M. Prijovich, K.-C. Chen, and S. R. Roffler
Local enzymatic hydrolysis of an endogenously generated metabolite can enhance CPT-11 anticancer efficacy
Mol. Cancer Ther., April 1, 2009; 8(4): 940 - 946.
[Abstract] [Full Text] [PDF]

Home page
 The Annals of PharmacotherapyHome page
D. Czock, F. M. Rasche, B. Boesler, M. Shipkova, and F. Keller
Irinotecan in Cancer Patients with End-Stage Renal Failure
Ann. Pharmacother., February 1, 2009; 43(2): 363 - 369.
[Abstract] [Full Text] [PDF]

Home page
 JCOHome page
G. Toffoli, E. Cecchin, G. Corona, A. Russo, A. Buonadonna, M. D'Andrea, L. M. Pasetto, S. Pessa, D. Errante, V. De Pangher, et al.
The Role of UGT1A1*28 Polymorphism in the Pharmacodynamics and Pharmacokinetics of Irinotecan in Patients With Metastatic Colorectal Cancer
J. Clin. Oncol., July 1, 2006; 24(19): 3061 - 3068.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
E. Cecchin, G. Corona, S. Masier, P. Biason, G. Cattarossi, S. Frustaci, A. Buonadonna, A. Colussi, and G. Toffoli
Carboxylesterase Isoform 2 mRNA Expression in Peripheral Blood Mononuclear Cells Is a Predictive Marker of the Irinotecan to SN38 Activation Step in Colorectal Cancer Patients
Clin. Cancer Res., October 1, 2005; 11(19): 6901 - 6907.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (10)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Poujol, S.
Right arrow Articles by Bressolle, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Poujol, S.
Right arrow Articles by Bressolle, F.
Related Collections
Right arrow Laboratory Management
Right arrow Clinical Immunology
Right arrow Drug Monitoring and Toxicology

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS