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Vol. 30, Issue 6, 731-733, June 2002
Institut Bergonié, BORDEAUX-cedex, France
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results and Discussion
References
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Irinotecan or CPT-11 [7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecine] is a derivative of camptothecine used in the treatment of advanced colorectal cancer. It requires activation to SN-38 (7-ethyl-10-hydroxycamptothecine) by carboxylesterase. Irinotecan and SN-38 are detoxified through two major metabolic pathways: the first one leads to oxidative degradation compounds, APC [7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]carbonyloxycamptothecine] and NPC [7-ethyl-10-(4-amino-1-piperidino)carbonyloxycamptothecine], and involves cytochrome P450 (3A4 isoform); the second one leads to SN-38 glucuronide (SN-38G) and involves UDP-glucuronosyltransferase (UGT). Using human hepatic microsomes, we studied the interactions of 15 drugs of common use in colorectal cancer patients on these metabolic pathways. Only nifedipine had a significant effect on SN-38 formation, decreasing carboxylesterase activity by 50% at 100 µM and 35% at 10 µM. Three drugs had a significant effect on SN-38G formation: clonazepam increased UGT activity by 50% at 100 µM and 30% at 10 µM, and nifedipine and vinorelbine inhibited the activity by 65 and 55% at 100 µM, respectively, with no effect at 10 µM. Five drugs exerted a significant inhibition on SN-38 formation at 100 µM: clonazepam (70%), methylprednisolone (50%), nifedipine (80%), omeprazole (85%), and vinorelbine (100%). Only omeprazole and vinorelbine still exerted a significant inhibition at 10 µM (30 and 90%, respectively), whereas only vinorelbine had a significant effect at 2 and 0.5 µM (70 and 40%, respectively). In conclusion, potential clinical interactions with the metabolism of irinotecan are likely to be important for vinorelbine, which strongly inhibits irinotecan catabolism by CYP3A4 at clinically relevant concentrations, but not for the other drugs, which exert an effect at concentrations not achievable in patients.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Irinotecan
[CPT-111;
7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecine]
is a water-soluble derivative of camptothecine (Sawada et al., 1991
)
that is currently used in the treatment of advanced colorectal cancer.
Irinotecan is a prodrug that needs to be converted to
SN-381 (7-ethyl-10-hydroxycamptothecine; Kawato
et al., 1991) through the action of carboxylesterases (Rivory et al.,
1996a; Haaz et al., 1997b). SN-38 is a very potent inhibitor of
topoisomerase I able to stabilize the cleavable complexes
DNA-topoisomerase I. Irinotecan and SN-38 are detoxified through two
major metabolic pathways. The first one independently leads to
oxidative degradation compounds, APC
[7-ethyl-10-[4-N-(5-aminopentanoic
acid)-1-piperidino]carbonyloxycamptothecine; Rivory et al., 1996b]
and NPC
[7-ethyl-10-(4-amino-1-piperidino)carbonyloxycamptothecine; Dodds et al., 1998]; in both cases, the reaction involves cytochrome P450 3A4 isoform (Haaz et al., 1998a,b). The second one leads to a
glucuronide of SN-38 (SN-38G; Rivory and Robert, 1995) and involves
UDP-glucuronosyltransferase (UGT) (isoenzyme 1A1 and other 1A isoforms)
(Haaz et al., 1997a; Iyer et al., 1998).
The availability of SN-38 to its targets is, therefore, determined by a
variety of enzyme activities, both for its formation and its
detoxification. Since these enzymes are subjected to a wide individual
variability, due to both genetic and environmental factors, there
should be a similar variability in SN-38 availability, which could
explain in turn at least part of the variability in response to
irinotecan (about 20% responders in untreated as well as in
5-fluorouracil-pretreated patients as stated by Rougier et al., 1997).
In addition, the enzyme activities responsible for irinotecan
metabolism could be affected by interactions with various drugs that
are commonly combined with irinotecan in therapeutics. Using the model
of human liver microsomes, we studied interactions of 15 drugs with the
three major pathways involving irinotecan: formation of SN-38 by
carboxylesterase, formation of NPC by CYP3A4, and formation of SN-38G
by UGT.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Chemicals and Reagents. Pure irinotecan, SN-38, and NPC were supplied by Aventis (Vitry-sur-Seine, France). 20(S)-Camptothecine was obtained from Sigma-Aldrich Chimie (Saint-Quentin-Fallavier, France). The drugs used for interaction studies were obtained from various sources: carbamazepine, clonazepam, dexamethasone, ftorafur, methylprednisolone, nifedipine, omeprazole, phenobarbital, phenytoine, ranitidine, valproic acid, and warfarin were obtained as pure chemicals from Sigma-Aldrich Chimie, as was bilirubin; capecitabine, gemcitabine, and vinorelbine were obtained as clinical formulations from Produits Roche (Neuilly-sur-Seine, France), Lilly-France (Saint-Cloud, France), and Pierre-Fabre Oncologie (Boulogne-sur-Seine, France), respectively. They were dissolved either in water or in methanol when they were not soluble in water. Solvents and reagents were of the highest grade commercially available.
Human Liver Microsomes.
Microsomes were prepared according to a standard fractionation
procedure (Berthou et al., 1989) from human liver samples obtained after approval by the relevant institutional ethical committee. We used
a pool of five individual preparations obtained from Dr C. Riché
(Faculty of Medicine, Brest, France) containing 17.4 mg of proteins per milliliter.
Incubations. Drug interactions were studied at fixed concentrations of irinotecan or SN-38 (25 or 5 µM, respectively) and of each potentially interfering drug (100 µM). When an effect was detected under these conditions, three other concentrations of the interfering drug were tested (0.5, 2, and 10 µM). Two different conditions were used: simultaneous incubation of the drug and irinotecan or SN-38 with the microsomes before the start of the reaction and preincubation of the drug with the microsomes for 20 min before the start of the reaction. Two independent experiments were performed in each condition. Control experiments were always carried out in the presence of an equivalent amount of the drug solvent (methanol or water).
For the study of the activation of irinotecan to SN-38 by carboxylesterases, microsomes were preincubated for 10 min on ice with 0.5% Triton X-100 (v/v). Aliquots of 0.32 mg of proteins were added to 0.1 M Tris buffer, pH 7.2, containing the drug tested or its solvent (final volume: 320 µl) and incubated in a shaking water bath. Irinotecan (25 µM) was then added to start the reaction. Incubation was performed for 1 h, and 37.5-µl samples were taken every 15 min for analysis. To study the glucuronidation of SN-38 by UGT, microsomes were incubated for 30 min on ice with 0.5 mg/mg of proteins Brij 35 (Sigma-Aldrich Chimie). Aliquots of 0.32 mg of microsomal proteins were added to 0.1 M Tris buffer, pH 7.4, 37°C, containing the drug tested or its solvent (final volume: 320 µl) and incubated in a water bath. SN-38 (5 µM) and saccharolactone (4 mM), an inhibitor of
-glucuronidases, were then added. The reaction was started by the
addition of uridine diphosphate -glucuronic acid (4 mM). Incubation
was performed for 1 h, and 37.5-µl samples were taken every 15 min for analysis. The effect of bilirubin was also tested on UGT
activity in the same conditions, with concentrations ranging from 1 to
100 µM.
For the study of the conversion of irinotecan to NPC by CYP3A4,
incubations were performed in 0.1 M phosphate buffer, pH 7.2, containing NADPH (1 mM), the drug tested or its solvent, and irinotecan (25 µM) (final volume: 320 µl). The reaction was started by adding the microsomes (0.32 mg of proteins). Incubation was performed for 20 min, and 37.5-µl samples were taken every 5 min for analysis.
High-Performance Liquid Chromatography Analysis. The samples of 37.5 µl were placed in 0.5-ml tubes containing 75 µl of ice-cold methanol/acetonitrile (1:1, v/v) and 50 ng of camptothecin (internal standard). The mixture was vortex-mixed and centrifuged at 8000g for 2 min. After acidification by 10 µl of 2 N HCl to 110 µl of the supernatant and vigorous stirring, samples of 100 µl were injected onto the high-performance liquid chromatography system (ThermoQuest, Saint-Quentin-en-Yvelines, France). Samples were processed via an automatic sampler (Spectra Series AS300, ThermoQuest), and the compounds were separated with a Radial-Pak NovaPak C-18 reversed-phase column (Waters Associates, Saint-Quentin-en-Yvelines, France).
Elution was performed at a constant flow rate (1.5 ml/min). For SN-38 and SN-38G, the mobile phase was isocratic and contained 78% (v/v) of 0.075 M ammonium acetate buffer, pH 5.3, 22% acetonitrile, and tetrabutylammonium phosphate (Waters) at a final concentration of 5 mM (Rivory and Robert, 1994). For NPC, elution was performed with a
programmed linear gradient over 18 min, containing 85 to 70% (v/v) of
0.075 M ammonium acetate, pH 6.0, and 15 to 30% acetonitrile, together
with tetrabutylammonium phosphate, at a final concentration of 5 mM.
Detection was achieved with a spectrofluorometer (Spectra Series F1000)
with excitation and emission wavelengths, respectively, set at 356 and
516 nm (for SN-38, 380 and 532 nm). Concentrations were calculated by
reference to standard calibration curves.
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Results and Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Drug Interactions at the Level of SN-38 Formation by
Carboxylesterases.
SN-38 formation was linear between 15 and 60 min, following an initial
burst characteristic for this enzyme (Rivory et al., 1996a; Haaz et
al., 1997b). Carboxylesterase had a mean activity in the pool of
1.15 ± 0.05 pmol/min/mg of proteins (mean ± S.E.M.). Only
one drug had a significant effect on SN-38 formation, nifedipine, which
at 100 µM altered by about 50% the carboxylesterase activity, both
with and without a 20-min preincubation of the microsomes with the
drug. No effect was detected at 0.5 and 2 µM nifedipine, whereas a
35% reduction was evidenced at 10 µM.
), no major drug interactions were observed with a panel of 11 other drugs including anticancer drugs, antiemetics, antidiarrheics,
antibiotics, analgesics, and nonsteroidal anti-inflammatory drugs. Only
loperamide and ciprofloxacine exerted a significant inhibition of ca.
50% at 100 µM. In another study, Slatter et al. (1997) identified no
relevant interactions with another group of 13 different drugs. Few
data are known about drug interactions with this enzyme activity,
except for the known inhibitors of carboxylesterases,
bis-nitrophenylphosphate and phytostigmine (Tsuji et al., 1991; Satoh
et al., 1994). Nifedipine is the only drug in our panel to contain a
carboxylester bond between two moieties of the molecule. This may
explain the inhibitory effect exerted on SN-38 formation. Nevertheless,
nifedipine probably cannot play a role in the clinical setting since,
at clinically relevant concentrations (2 µM), this effect was not
significant. Capecitabine contains an amide bond, which is cleaved by
carboxylesterases for drug (Miwa et al., 1998). Since there was no
interference of capecitabine on SN-38 formation, the enzyme responsible
for capecitabine activation is probably distinct from that responsible for irinotecan activation, which would allow the combination between these drugs in the clinical setting.
Drug Interactions at the Level of SN-38G Formation by UGT.
SN-38G formation was linear between 0 and 60 min. UGT had a mean
activity in the pool of 5.0 ± 0.6 pmol/min/mg of proteins (mean ± S.E.M.). This is about 10 times lower than expected from experiments published earlier (Haaz et al., 1997a). Such a difference can only be explained by the existence of large individual variations or by a different purification yield of the microsomal preparation. Only three drugs had a significant effect on SN-38G formation at 100 µM: clonazepam, which increased the activity about 50%, and
nifedipine and vinorelbine, which inhibited the activity by 65 and
55%, respectively. There was no additional effect of the 20-min
preincubation of the microsomes with the drug. No effects were detected
at 10 µM or below, except for clonazepam, for which a 30%
stimulation of the activity was evidenced at 10 µM. Bilirubin, a
known substrate of UGT1A1, inhibited the glucuronidation of SN-38, with
50% inhibition at 100 µM.
). It could
be expected that drug competition at the level of the glucuronidation
reaction would explain the inhibition of SN-38G formation. However,
many drugs on the total of 26 are glucuronidated in vivo, although the
isoenzyme responsible has not always been identified. In addition,
bilirubin, a known substrate of UGT1A1, has a limited effect on the
formation of SN-38G, with an IC50 of about 100 µM. This may be due to the fact that UGT isoforms other than 1A1 can
be involved in the glucuronidation of SN-38 (Ciotti et al., 1999). It
is worth noting that clonazepam exerts a stimulation of the
glucuronidation of SN-38, which is already significant at 10 µm. No
such direct stimulation of this enzyme activity has been mentioned in
the literature to our knowledge.
Drug Interactions at the Level of NPC Formation by Cytochrome P450.
We chose NPC rather than APC formation as an indication of CYP3A4
activity because this metabolite was more easily detected and
accurately quantified than APC. NPC formation was linear between 5 and
20 min with a constant y-axis intercept of about 50 nM. NPC
was formed at a mean rate of 6.34 ± 0.50 pmol/min/mg of proteins (mean ± S.E.M.), which is lower than expected from the results previously published (Haaz et al., 1998a). There again, this difference can be explained by the existence of large individual variations in
CYP3A activity. Indeed, we had observed a 40-fold difference in the
rates of formation of NPC when studying individual microsome preparations; the presence of a very active preparation in only one of
the pools would thus substantially increase the global results. Five
drugs had a significant effect on SN-38 formation at this
concentration: clonazepam, methylprednisolone, nifedipine, omeprazole,
and vinorelbine, which inhibited NPC formation by 70, 50, 80, 85, and
100%, respectively, both with and without a 20-min preincubation of
the microsomes with the drug. Only omeprazole and vinorelbine still
exerted a significant effect at 10 µM, whereas only vinorelbine also
had a significant effect at 2 and 0.5 µM. Figure
1 presents the drug interactions as a
function of their concentration. Using Dixon plots, it was possible to
evaluate by nonlinear regression the
Ki value of vinorelbine on NPC
formation, which was 1.31 ± 0.01 µM.
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,b). With the exception of methylprednisolone, all these
drugs are known substrates of CYP3A4, and drug interactions can be
explained by competition at the level of enzyme active site
(Guengerich, 1999). Nevertheless, the only interaction that can be
relevant in the clinical setting is the interaction with vinorelbine, a
concentration easily reached in the plasma of patients. As a
consequence, the combination of vinorelbine to irinotecan, which can be
proposed, for instance, in the treatment of advanced nonsmall cell lung
cancer, should be developed with caution in view of the expected
decrease in irinotecan catabolism that is likely to occur in patients.
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Footnotes |
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Received October 25, 2001; accepted February 26, 2002.
This work was supported by a grant from Aventis Oncology.
Address correspondence to: Jacques Robert, Institut Bergonié, 229 cours de l'Argonne, 33076 BORDEAUX-cedex, France. E-mail: robert{at}bergonie.org
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Abbreviations |
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Abbreviations used are: SN-38, 7-ethyl-10-hydroxycamptothecine; SN-38G, SN-38 glucuronide; APC, 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]carbonyloxy-camptothecine; NPC, 7-ethyl-10-(4-amino-1-piperidino)carbonyloxycamptothecine; UGT, UDP-glucuronosyltransferase.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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-glucuronide metabolite of SN-38 in human plasma after administration of the camptothecin derivative irinotecan.
Cancer Chemother Pharmacol
36:
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