Metabolism of irinotecan (CPT-11) by human hepatic microsomes: participation of cytochrome P-450 3A and drug interactions.

Irinotecan (CPT-11) is a water-soluble analogue of camptothecin showing activity in colon cancer. Recently, we identified a major metabolite of CPT-11 in patients' plasma, 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino] carbonyloxycamptothecin (APC), which is produced by the oxidation of the distal piperidine ring (P. Rivory et al, Cancer Res., 56: 3689-3694, 1996). As with all active camptothecin derivatives, CPT-11 is subject to spontaneous interconversion between a lactone and a carboxylate form in aqueous media. The kinetics of biotransformation of the two forms of CPT-11 into APC was studied using pooled human liver microsomes. The formation of APC was characterized by the following parameters: Km = 18.4 +/- 1.4 and 39.7 +/- 11.6 microM; and Vmax = 26.0 +/- 0.6 and 13.4 +/- 1.7 pmol/min/mg protein for the lactone and carboxylate forms of CPT-11, respectively. This reaction was found to be catalyzed principally by cytochrome P-450 (CYP) 3A because of three key results: (a) the CYP 3A-selective inhibitors ketoconazole (1 microM) and troleandomycin (100 microM) inhibited APC formation by 98 and 100%, respectively, mostly in a competitive way; (b) using microsomes from transfected lymphoblastoid cells expressing specific CYPs, we found that only those from CYP 3A4 cDNA-transfected cells transformed CPT-11 into APC; and (c) using 15 individual preparations of human liver microsomes, we observed highly significant correlations between the activity of CPT-11 metabolism into APC and both immunoreactivity with anti-CYP 3A antibodies and testosterone 6beta hydroxylation, an activity specifically mediated by CYP 3A. The effect on this metabolism of 11 drugs used at 100 microM was studied with CPT-11 lactone at 25 microM. Amikacin, Bactrim, ciprofloxacin, rocephine, 5-fluorouracil, metoclopramide, morphine, and paracetamol had no effect, but ondansetron, loperamide, and racecadotril inhibited this pathway by 25, 50, and 50%, respectively. These concentrations exceed those expected in vivo. APC formation in patients may thus be influenced by coadministered ketoconazole therapy and may decline after administration of CPT-11 because of the lactonolysis of the latter.

Identification and properties of a major plasma metabolite of irinotecan (CPT-11) isolated from the plasma of patients.

Irinotecan [7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin (CPT-11)] is a promising water-soluble analogue of camptothecin [S. Sawada et al., Chem. & Pharm. Bull. (Tokyo), 39: 1446-1454, 1991]. We have reported previously the presence of an important polar metabolite, in addition to 7-ethyl-10-hydroxycamptothecin (SN-38) beta-glucuronide, in samples of plasma taken from patients undergoing treatment with CPT-11 (L.P. Rivory and J. Robert, Cancer Chemother. Pharmacol. 36: 176-179, 1995; L. P. Rivory and J. Robert, J. Cromatogr., 661: 133-141, 1994). Plasma samples (0.5 ml) containing comparatively large amounts of this metabolite were extracted by solid-phase columns and subjected to high-performance liquid chromatography and mass spectrometry in parallel to fluorometric detection. The metabolite yielded [M + 1] ions with a m/z of 619, representing the addition of 32 atomic mass units to CPT-11. Purified fractions were subjected to proton nuclear magnetic resonance, and the structure determined, 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]carbonyloxycampothecin (APC), was further validated following synthesis. Like CPT-11, APC was found to be only a weak inhibitor of the cell growth of KB cells in culture (IC50, 2.1 versus 5.5 micrograms/ml for CPT-11 and 0.01 microgram/ml for SN-38, the active metabolite of CPT-11) and was a poor inducer of topoisomerase I DNA-cleavable complexes (100-fold less potent than SN-38). In contrast to CPT-11, APC was not hydrolyzed to SN-38 by human liver microsomes or purified human liver carboxylesterase. Furthermore, APC did not inhibit the hydrolysis of CPT-11 in these preparations. Interestingly, APC was only a weak inhibitor of acetylcholinesterase in comparison to CPT-11 and neostigmine. It appears likely, therefore, that APC does not contribute directly to the activity and toxicity profile of CPT-11 in vivo.

Pharmacokinetic interrelationships of irinotecan (CPT-11) and its three major plasma metabolites in patients enrolled in phase I/II trials.

Irinotecan (CPT-11) is an analogue of 20(S)-camptothecin with promising activity against several tumor types. In patients, CPT-11 is metabolized to 7-ethyl-10-hydroxycamptothecin (SN-38) and to the beta-glucuronide of SN-38. Recently, we identified an additional metabolite of CPT-11, 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino] carbonyloxycamptothecin (APC; L. P. Rivory et al. , Cancer Res., 56: 3689-3694, 1996). The aim of this study was to investigate the interrelationships of all four compounds to identify factors that might be responsible for the large interpatient variability in CPT-11 and SN-38 kinetics. The plasma kinetics of CPT-11, SN-38, the beta-glucuronide of SN-38, and APC were studied in 19 patients for a total of 33 cycles (115-600 mg/m2). Although the area under the concentration curves (AUCs) of all compounds studied increased with dose, there was considerable variability. Ratios of the AUCs of the appropriate compounds were used as estimates of the major routes of metabolism (conversion of CPT-11 to SN-38, metabolism of CPT-11 to APC, and glucuronidation of SN-38). Each ratio varied more than 10-fold across the patient population, and the apparent extent of conversion of CPT-11 to SN-38 was highest at the 115 mg/m2 dose level. Interestingly, AUCSN-38 was greater in patients with both high AUCCPT-11 and AUCAPC. We conclude that the variability of the pharmacokinetics of CPT-11 and SN-38 is likely to be due to extensive interpatient differences in the pathways implicated in the metabolism of CPT-11.

Impact of different fluorouracil biochemical modulators on cellular dihydropyrimidine dehydrogenase.

In attempts to increase fluorouracil (FU) activity by pharmacomodulation, most attention has been paid to FU activation pathways without consideration of the presence and possible role of FU catabolism in the target tumoral cell itself. The first step in the catabolism of FU is hydrogenation by the enzyme dihydropyrimidine dehydrogenase (DPD). The purpose of the present study was to test the DPD-inhibitory effects of several agents whose use as FU biomodulators has been clinically established: cisplatin, hydroxyurea, dipyridamole, and allopurino. Five cancer cell lines of human origin were used. Dipyridamole and hydroxyurea were the only modulators for which an augmentation in FU cell-growth inhibition (MTT test) was clearly evident for the whole panel of cell lines investigated (P<1.10(-4) and P=0.005, respectively). With dipyridamole the efficacy of FU was multiplied by a factor of around 5. Allopurinol and cisplatin had no obvious effect on cellular DPD activity (biochemical method). For dipyridomole and hydroxyurea, DPD activity showed a more or less marked concentration-related inhibition according to the cell line tested. Only dipyridamole produced reductions in FU IC50 values (50% growth-inhibitory concentrations), i.e., potentiation of FU cytotoxicity, that were significantly related to inhibition of cellular DPD activity. This DPD-mediated interaction between dipyridamole and FU is a new finding that could be important for a better understanding of FU-dipyridamole combination chemotherapy.

The transformation of irinotecan (CPT-11) to its active metabolite SN-38 by human liver microsomes. Differential hydrolysis for the lactone and carboxylate forms.

Irinotecan (CPT-11) is a new camptothecine derivative presently in development for the treatment of several advanced malignancies. It is converted in vivo to a highly potent metabolite, SN-38, by carboxylesterases. All camptothecine derivatives undergo lactonolysis in a pH-dependent reversible manner, generating inactive carboxylate forms. We have investigated in vitro the kinetics of transformation of CPT-11 to SN-38 by human liver microsomes originating from several donors. Microsomes from seven livers were studied individually or as a pooled preparation. CPT-11, either in its lactone or its carboxylate form, was added at a range of concentrations. The SN-38 formed was measured by HPLC with fluorometric detection. In the deacylation-limited carboxylesterase reaction, the linear steady-state kinetics between 10 and 60 min were determined. At all concentrations of CPT-11, the steady-state velocity of SN-38 formation as well as the intercept concentrations of SN-38 were about 2-fold higher when the substrate was under the lactone form than under the carboxylate form. We estimated the values (+/-SD) of K'm and Vmax to be 23.3 +/- 5.3 microM and 1.43 +/- 0.15 pmol/min/mg for the lactone and 48.9 +/- 5.5 microM and 1.09 +/- 0.06 pmol/min/mg for the carboxylate form of CPT-11, respectively. We conclude that the greater rate of conversion of CPT-11 lactone may contribute to the plasma predominance of SN-38 lactone observed in vivo. The inter-individual variation of SN-38 formation was relatively high (ratio of 4 between extreme values) but no large age- or gender-related differences were seen. The effect of twelve drugs of different therapeutic classes (antibiotics, antiemetics, antineoplastics, antidiarrhoeics, analgesics), which could be administered in association with irinotecan in the clinical setting, was evaluated in this system (drug concentration: 100 microM; CPT-11 lactone concentration: 10 microM). Loperamide and ciprofloxacine where the only drugs exerting a weak inhibition of CPT-11 conversion to SN-38.

Hepatic extraction, metabolism, and biliary excretion of irinotecan in the isolated perfused rat liver.

Irinotecan (CPT-11) is a semisynthetic derivative of camptothecine that has proved activity in the treatment of colorectal carcinoma. The metabolites identified in humans include SN-38, SN-38 glucuronide, and several CYP3A-derived metabolites. We have studied the hepatic extraction, metabolism, and biliary excretion of irinotecan in the isolated perfused rat liver. After injection of a bolus dose of 5 micromol in the reservoir, irinotecan lactone disappeared from the perfusate following a two-exponential decay with half-lives of 3.5 and 120 min and a total clearance of 1.54 +/- 0.07 mL/min per gram of liver. The area under the curve (AUC) ratio lactone/total drug was 0.212 +/- 0.098 and the half-life of interconversion was 5.02 +/- 0.10 min. Bolus administrations of 2.5, 5, and 25 micromol of irinotecan gave AUCs proportional to the doses administered, indicating that no saturation occurred during dose increase. However, the relative formation of SN-38 and SN-38 glucuronide decreased at the high dose. This result was not the case for the CYP3A metabolites, which had identical metabolic ratios at all three doses. Infusions of 30 and 90 min of a dose of 5 micromol led to the same AUCs and metabolic ratios as a bolus of the same dose. Biliary elimination of irinotecan and metabolites represented 18-22% of the dose administered at 2.5 and 5 micromol but only 7-9% at 25 micromol, suggesting a saturation of this process. These data indicate that the hepatic disposition of irinotecan may vary at high dose, both at the level of biliary excretion and of activation to SN-38.

Biosynthesis of an aminopiperidino metabolite of irinotecan [7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecine] by human hepatic microsomes.

Irinotecan [7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecine++ +] is a water-soluble analogue of camptothecine used in the second-line treatment of advanced colon cancer. Recently, we identified, in the plasma of patients and in human liver microsomal incubations, the presence of a new metabolite of irinotecan, 7-ethyl-10-(4-amino-1-piperidino)carbonyloxycamptothecine (NPC), which is produced by cleavage of the distal piperidine ring of irinotecan. The kinetics of biotransformation of the lactone and carboxylate forms of irinotecan into NPC were studied using human liver microsomes. The formation of NPC was characterized by the following parameters: KM = 48.2 +/- 6.8 and 273 +/- 122 microM and Vmax = 74.1 +/- 4.9 and 78.6 +/- 27.7 pmol/min/mg of protein for the lactone and carboxylate forms of irinotecan, respectively. Interestingly, there was no formation of NPC from 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]carbonyloxycamptothecine, a major metabolite of irinotecan that has an open distal piperidine ring and could be considered a possible metabolic precursor of NPC. The transformation of irinotecan into NPC was found to be catalyzed principally by cytochrome P450 (CYP) 3A, based on three key results, as follows: 1) the CYP3A-selective inhibitors ketoconazole (1 microM) and troleandomycin (100 microM) inhibited NPC formation by 99 and 100%, respectively; 2) of a series of microsomal preparations from transfected lymphoblastoid cells expressing specific CYPs, only those from CYP3A4 cDNA-transfected cells transformed irinotecan into NPC; and 3) incubations with 15 individual preparations of human liver microsomes yielded highly significant correlations between the formation of NPC and both immunoreactivity with anti-CYP3A antibodies and testosterone 6beta-hydroxylation (an activity specifically mediated by CYP3A). The effects of 11 drugs (used at 100 microM) on this metabolism were studied with irinotecan lactone (25 microM). Although ondansetron, loperamide, and racecadotril inhibited this pathway by 75, 95, and 95%, respectively, the concentrations used may not be clinically achievable. However, significant inhibition by ketoconazole and troleandomycin indicates that NPC formation in patients may be influenced by coadministration of drugs with known anti-CYP3A activities.

Glucuronidation of SN-38, the active metabolite of irinotecan, by human hepatic microsomes.

We have investigated the glucuronidation in vitro of SN-38, the active metabolite of irinotecan, a semi-synthetic anticancer drug derived from 20(S)camptothecin. Preparations of human hepatic microsomes (final concentration : 1 mg prot./ml), were incubated for 1 hr in 0.1 M Tris buffer, pH 7.4, containing 10 mM MgCl2, in the presence of UDP-glucuronic acid (4 mM), saccharolactone (4 mM), and a detergent. Microsomes from five livers were studied individually or as a pooled preparation. SN-38, either in its lactone or its carboxylate form, was added at a range of concentrations. The SN-38 beta-glucuronide formed was measured by HPLC with fluorometric detection. The glucuronidation reaction appeared linear over 1 hr in these conditions and Brij 35 at 0.5 mg/mg prot. was the best activator. The apparent parameters of the reaction were independent of the molecular form of the substrate. The half-saturation constant was 17-20 microM and Vmax was 60-75 pmol/min./mg prot. The interindividual variation of SN-38 glucuronidation was relatively low (ratio of 1.8 between extreme values). In addition, the effect of twelve drugs currently associated with irinotecan in clinics was evaluated in this system (drug concentration: 100 microM; SN-38 concentration: 5 microM). These produced little if any interference with SN-38 glucuronidation. Therefore, major interferences of this transformation by comedications are unlikely to occur in vivo.

Identification of a new metabolite of CPT-11 (irinotecan): pharmacological properties and activation to SN-38.

Irinotecan, or CPT-11 (7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecine++ +), is a water-soluble derivative of camptothecine with promising activity against several types of malignancies. In addition to 7-ethyl-10-hydroxycamptothecine (SN-38), its active metabolite, we were able to identify several metabolites in the plasma of patients treated with this drug, especially an oxidative metabolite, 7-ethyl-10[4-N-(5-aminopentanoic acid)-1-piperidino] carbonyloxy-camptothecine. During our study of the biosynthesis of 7-ethyl-10[4-N-(5-aminopentanoic acid)-1-piperidino] carbonyloxy-camptothecine from CPT-11 by human liver microsomes, we were able to detect another quantitatively important polar metabolite, which was also present in the plasma and urine of patients treated with CPT-11. On the basis of preliminary experiments, the structure of this compound was postulated to be 7-ethyl-10-(4-amino-1-piperidino)carbonyloxycamptothecine, and this structure was synthesized by Rhône-Poulenc Rorer. Urine samples and human liver microsomal extracts were studied by high-performance liquid chromatography/atmospheric pressure chemical ionization/tandem mass spectrometry to identify its structure formally. The identification of the metabolite was supported by identical retention time, mass-to-charge ratio and tandem mass spectrometry fragmentation as a synthetic standard. Like irinotecan, 7-ethyl-10-(4-amino-1-piperidino) carbonyloxycamptothecine was a weak inhibitor of cell growth of P388 cells in culture (IC50 = 3.4 micrograms/ml vs. 2.8 micrograms/ml for irinotecan and 0.001 microgram/ml for SN-38). It was also a poor inducer of topoisomerase I-DNA cleavable complexes (100-fold less potent than SN-38). However, unlike 7-ethyl-10[4-N-(5-aminopentanoic acid)-1-piperidino] carbonyloxy-camptothecine, this new metabolite could be hydrolyzed to SN-38 by human liver microsomes and purified human liver carboxylesterase, though to a lesser extent than irinotecan. This compound can therefore contribute to the activity and toxicity profile of irinotecan in vivo.