Biliary excretion mechanism of CPT-11 and its metabolites in humans: involvement of primary active transporters

After administration of CTP-11, a camptothecin derivative exhibiting a wide spectrum of antitumor activity, dose-limiting gastrointestinal toxicity with great interpatient variability is observed. Because the biliary excretion is a major elimination pathway for CPT-11 and its metabolites [an active metabolite, 7-ethyl-10-hydroxy-camptothecin (SN-38), and its glucuronide, SN38-Glu], several hypotheses for the toxicity involve biliary excretion. Here, we investigated whether primary active transport is involved in the biliary excretion of anionic forms of CPT-11 and its metabolites in humans using bile canalicular membrane vesicles (cMVs). Uptake of the carboxylate form of CPT-11 and the carboxylate and lactone forms of SN38-Glu by cMVs prepared from five human liver samples was ATP dependent. The concentration dependence of the ATP-dependent uptake of the carboxylate form of CPT-11 and SN38-Glu suggests the involvement of at least two saturable transport components, both with lower affinity and higher capacity than in rats. The ATP-dependent uptake of the carboxylate form of SN-38 showed a single saturable component but was detectable only in one human cMV sample. Both carboxylate and lactone forms of SN38-Glu uptake also showed a large intersample variability, although the variability was less than that observed for the carboxylate form of SN-38. On the other hand, the carboxylate form of CPT-11 exhibited much less variability. The carboxylate forms of SN38-Glu and SN-38 almost completely inhibited the ATP-dependent uptake of leukotriene C4, a well-known substrate of canalicular multispecific organic anion transporter, whereas the inhibition by the carboxylate form of CPT-11 was not as marked. Thus, multiple primary active transport systems are responsible for the biliary excretion of CPT-11 and its metabolites, and the major transport system for CPT-11 differs from that for the other two compounds. A greater degree of inter-cMV variability in the uptake of SN-38 and SN38-Glu may imply that interindividual variability in biliary excretion of these metabolites might contribute to interpatient variability in the toxicity caused by CPT-11.     

Active efflux of CPT-11 and its metabolites in human KB-derived cell lines

To investigate the possible involvement of P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), and/or other glutathione S-conjugate export pump (GS-X pump) family members on the active efflux of irinotecan [(7-ethyl-10-[4-(1-piperidino)-1-pipertidino)-1-piperidino]carb onylox y camptothecin (CPT-11)] and its metabolites, as well as their contribution to the acquisition of resistance, we studied the uptake of CPT-11, its active metabolite SN-38, and glucuronide conjugate (SN38-Glu) using membrane vesicles from human epidermoid KB-3-1-derived cell lines. These lines included KB-C2, C-A500, and KCP-4, which overexpress P-gp, MRP, and the unidentified GS-X pump, respectively. The carboxylate form of SN-38 exhibited significant ATP-dependent transport, with a Michaelis constant of 17 microM, into membrane vesicles from C-A500 but not from other cell lines. Among these KB-derived cells, significant ATP-dependent uptake of the carboxylate form of CPT-11 was only observed in KB-C2 vesicles. In addition, the uptake of the lactone and carboxylate forms of SN38-Glu into membrane vesicles from C-A500 and KB-C2, but not KCP-4, was ATP dependent, although the transport activity in C-A500 was much higher than that in KB-C2. The 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay revealed that the resistance of KB-C2 to CPT-11 and SN-38, compared with that of KB-3-1, was 6.3- and 6.8-fold, respectively; the corresponding figures for C-A500 were 12- and 27-fold, respectively, whereas those for KCP-4 were 2.3- and 20-fold, respectively. These results suggest that MRP and P-gp are involved in the active efflux of SN-38 and CPT-11, respectively, from human KB-derived cells. In addition, a difference in substrate specificity among GS-X pump members was demonstrated.     

Plasma pharmacokinetics of 7-ethyl-10-hydroxycamptothecin (SN-38) after intravenous administration of SN-38 and irinotecan (CPT-11) to rats

We studied the plasma pharmacokinetics of the lactone form and the carboxylate form of 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of irinotecan (CPT-11), after intravenous bolus administrations of each form of SN-38 and of CPT-11 to rats. After the SN-38 lactone injection, SN-38 lactone was predominant at first, and then the lactone to the carboxylate concentration ratio (LC ratio) was maintained from 30 to 90 min after the injection. The carboxylate was predominant throughout the period after the carboxylate dosing. Model-dependent analyses showed that the SN-38 lactone had greater plasma clearance and a greater distribution volume than the carboxylate. The CPT-11 administration resulted in a predominant plasma SN-38 lactone concentration. The contribution of the SN-38 lactone AUC to the total SN-38 AUC (57%) was independent of the dose of CPT-11. These results suggest that it is possible to estimate the SN-38 lactone concentration and AUC from the total SN-38 concentration without separate determination of the lactone and carboxylate. Our results showed that both SN-38 lactone and CPT-11 administration gave the predominant SN-38 lactone in plasma; however, only CPT-11 could sustain the lactone concentration at a high level, which is necessary for antitumor activity.     

Intravenous administration of irinotecan elevates the blood beta-glucuronidase activity in rats

7-Ethyl-10-hydroxycamptothecin (SN-38) is the active metabolite of an anticancer drug, irinotecan (CPT-11). Severe late diarrhea is the dose-limiting toxic effect of CPT-11. This diarrhea has been examined regarding biliary excretion and deconjugation of SN-38 glucuronide by the enzyme beta-glucuronidase (beta-GL) in intestinal microflora. Prompted by the enzymological and structural similarity of CPT-11 to organophosphorus and carbamate insecticides, we studied the effect of CPT-11 on blood beta-GL activity in rats. The i.v. injection of CPT-11 in rats significantly elevated their plasma beta-GL activity (with phenolphthalein glucuronide as a substrate) at doses of 10 and 40 mg/kg, with peak activity observed 2-3 h after administration. SN-38 lactone and carboxylate had no effect on the plasma beta-GL level. The enhancement of the activity was also observed in serum using SN-38 glucuronide as a substrate. The serum beta-GL levels showed a close correlation between these substrates. The enhancement of plasma (serum) beta-GL activity is suggested to be a result of the release of beta-GL from liver microsomes. Serum and microsomal carboxylesterase were not significantly affected by CPT-11 administration.     

The utilities and outlooks of MR cholangiography (MRCP) as a non contrast and noninvasive technique

The objective of this study was to determine the metabolic fate and disposition of the antitumor camptothecine derivative irinotecan (CPT-11). Ten patients with histological proof of malignant solid tumor received 200 mg/m2 CPT-11 as a 90-min i.v. infusion, followed by a 1.5-h i.v. infusion of cisplatin (60 or 80 mg/m2). Plasma, urine, and feces were collected for 56 h and analyzed by a specific reversed-phase high-performance liquid chromatographic assay for the parent drug and all four metabolites positively identified to date: SN-38; its beta-glucuronide conjugate, SN-38 beta-glucoronide (SN-38G); 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]-carbonyloxycamptothecine (APC); and 7-ethyl-10-[4-N-(1-piperidino)-1-amino]-carbonyloxycamptothecine (NPC). A three-exponential decline was observed in plasma for all compounds, with a clear predominance of the parent drug [25.6+/-5.71 microM x h (CPT-11) versus 15.8+/-3.51 microM x h (total metabolites)]. Total urinary excretion was 28.1+/-10.6% of the dose, with unchanged CPT-11 and SN-38G as the main excretion products. Whereas renal clearance of SN-38 was only a minor route of drug elimination, fecal concentrations of this compound were unexpectedly high (on average, 2.45% of the dose), suggestive of intestinal hydrolysis of SN-38G by bacterial beta-glucuronidase. CPT-11 and the other metabolites could also be identified from fecal extracts, with a very minor contribution overall of the cytochrome P-450-mediated compounds 7-ethyl-10-[4-N-(1-piperidino)-1-amino]-carbonyloxycamptothecine and 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]-carbonyloxycamptothecine. Surprisingly, fecal excretion accounted for only 24.4+/-13.3% of the dose, leading to a total excretion of approximately 52%. These data indicate that half of the dose in urine and feces may constitute some further unknown nonextractable or nonfluorescent metabolites. The findings from this study should be of importance as a guide to further therapeutic evaluation of this drug.     

Functional integrity of hepatocyte canalicular membrane transport of taurocholate and bilirubin diglucuronide in Eisai hyperbilirubinuria rats

Eisai hyperbilirubinuria rats (EHBR) are characterized by conjugated hyperbilirubinemia, and impaired or defective excretion of bilirubin, reduced glutathione and other organic anions from hepatocytes. Hepatocyte canalicular membrane vesicles (CMV) from EHBR and normal SD rats were studied with regard to taurocholate (TC) transport driven by ATP or a membrane potential and bicarbonate-stimulated bilirubin diglucuronide (BDG) transport. ATP-dependent uptake or association of BDG with CMV was also studied in both strains of rats. No significant differences in the uptake of TC and BDG by CMV were observed. This indicates the functional integrity of the canalicular transporters for both organic anions in EHBR. Biliary excretion of taurolithocholic acid sulfate (TLCS) is defective in EHBR. However, TLCS inhibited ATP-dependent TC uptake by SD rat CMV competitively, which may be against the hypothesis that a common organic anion carrier is defective in canalicular membranes of jaundiced rats.     

Pharmacokinetic characteristics of the novel anticancer agent CPT-11 and its active metabolite in plasma and lung lymph fluid following intravenous administration to sheep

7-Ethyl-10-[4-(1-piperidino)-1-piperidino]-carbonyloxycamptothecin (CPT-11, 100286-90-6) is one of the most promising novel anticancer agents, especially for lung cancer. 7-Ethyl-10-hydroxycamptothecin (SN-38), an active metabolic of CPT-11, has much stronger cytotoxicity than CPT-11. The present study was designed to evaluate the distribution and behavior of CPT-11 and SN-38 in lung lymph circulation following intravenous infusion. Awake sheep with chronically instrumented lung lymph fistulas were prepared. The concentrations of CPT-11 and SN-38 in plasma and lung lymph fluid were measured after intravenous infusion of 100 mg/body of CPT-11 for 90 min. SN-38 constantly showed higher lymph to plasma concentration ratios than those of CPT-11, and the % area under the curve (AUC) ratio of SN-38/CPT-11 in lymph fluid was significantly higher than that in plasma. These data indicated that SN-38 distributed in lung tissue moved more easily into lung lymph fluid than CPT-11, and might be more rapidly metabolized in lung tissue than plasma. CPT-11 may have favorable therapeutic effects on intrathoracic malignancies such as lung cancer and lymph metastasis.     

High Helicobacter pylori eradication rate with a 1-week regimen containing ranitidine bismuth citrate

HSR-903 is a newly synthesized quinolone antibacterial agent with low toxicity. The biliary and urinary excretion of unchanged HSR-903, its R-isomer, and their glucuronides was determined after iv bolus administration (5 mg/kg) to normal Sprague-Dawley rats (SDR) and Eisai hyperbilirubinemic mutant rats (EHBR). The values for the biliary excretion clearance of HSR-903 and its glucuronide in EHBR were decreased to approximately 40 and 2% of those in SDR, respectively, whereas the values for the urinary excretion clearance of HSR-903 and its glucuronide were comparable in SDR and EHBR. The biliary excretion clearance values for the R-isomer and its glucuronide were approximately 3 times greater than those for HSR-903. These results demonstrated that the enantiomers of HSR-903 and their conjugates were excreted into bile in a stereospecific manner. The hepatic uptake of [14C]HSR-903 in vivo was evaluated by means of integration plot analysis. The results indicated that the hepatic uptake of [14C]HSR-903 was very fast and was blood flow-limited. To clarify the mechanism of excretion of HSR-903 into bile, the uptake and efflux of [14C]HSR-903 were studied using isolated hepatocytes from SDR and EHBR. The initial uptake of HSR-903 by hepatocytes was temperature-dependent, saturable, and stereospecific. Unlabeled HSR-903 (S-isomer), the R-isomer, grepafloxacin, and sparfloxacin significantly inhibited the uptake of [14C]HSR-903. The efflux of [14C]HSR-903 from hepatocytes from EHBR was significantly slower than that from hepatocytes from SDR. The addition of sodium azide or bromosulfophthalein reduced the efflux of [14C]HSR-903. These results demonstrate that HSR-903 is actively excreted into bile via the canalicular multispecific organic anion transporter, which is deficient in EHBR.     

Low-grade mucoepidermoid carcinoma on the vermilion border of the lip

BACKGROUND AND PURPOSE: CPT-11 (7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin) is anew semisynthesized derivative of camptothecin. SN-38 (7-ethyl-10-hydroxycamptothecin), a metabolite of CPT-11, plays a key role in the action of CPT-11. MATERIALS AND METHODS: To determine whether SN-38 potentiates the cytotoxic effect of radiation, we investigated the interaction of SN-38 and radiation in vitro in monolayer cultures and multicellular spheroids of HT-29 human colon adenocarcinoma cells. RESULTS: HT-29 spheroids were more resistant to both SN-38 and irradiation than monolayer cells. SN-38 at a concentration of 2.5 microg/ml, which by itself was not cytotoxic, greatly increased the lethal effects of radiation in spheroids, but not in monolayer cultures. Exposure to SN-38 following irradiation inhibited the potentially lethal damage repair (PLDR) in spheroids. It is suggested that the mechanism of the radiosensitization by SN-38 is due to the PLDR inhibition. CONCLUSIONS: These results indicate that CPT-11 may play a role as radiosensitizer and that a combination of CPT-11 and irradiation could prove to be a particularly effective strategy with which to treat human colon adenocarcinoma.     

Kinetic analysis of the primary active transport of conjugated metabolites across the bile canalicular membrane: comparative study of S-(2,4-dinitrophenyl)-glutathione and 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl)benzothiazole glucuronide

Eisai hyperbilirubinemic rat (EHBR) is a mutant strain with a hereditary defect in canalicular multispecific organic anion transporter (cMOAT). We examined the uptake and mutual inhibition of S-(2,4-dinitrophenyl)-glutathione (DNP-SG), which is a typical substrate for cMOAT, and 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole (E3040) glucuronide (E-glu) with canalicular membrane vesicles (CMV) prepared from Sprague-Dawley (SD) and EHBR rats to investigate the multiplicity of the organic anion transporter. The ATP-dependent uptake by CMV from SD rats had an apparent Km of 17.6 microM for DNP-SG and 5.7 microM for E-glu, whereas the corresponding uptake by CMV from EHBR had an apparent Km of 44.6 microM for E-glu. The effects of E-glu, 4-methylumbelliferone glucuronide (4 MUG), E3040 sulfate (E-sul) and 4-methylumbelliferone sulfate (4 MUS) on the uptake of [3H]DNP-SG were also examined. The uptake of [3H]DNP-SG was inhibited by glucuronides (E-glu and 4 MUG) in a concentration-dependent manner, although it was enhanced by the sulfate conjugates (E-sul and 4 MUS). This enhancement was shown to be caused by an increased DNP-SG affinity for the transporter. In CMV from SD rats, although ATP-dependent uptake of [3H]DNP-SG was almost completely inhibited by E-glu, that of [14C]E-glu was only reduced to about 30% of controls by DNP-SG. On the other hand, in CMV from EHBR, the ATP-dependent uptake of [14C]E-glu was not inhibited at all by DNP-SG. Kinetic analysis indicated that E-glu inhibited DNP-SG uptake competitively. In conclusion: 1) cMOAT recognizes both DNP-SG and E-glu, and another transporter present in SD rats is also involved in E-glu transport along with cMOAT; 2) the latter transporter is kinetically similar to the E-glu transporter present in EHBR; 3) E-sul enhances the uptake of DNP-SG by increasing the affinity of glucuronide for the transporter.     

Simultaneous determination of the lactone and carboxylate forms of 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of irinotecan (CPT-11), in rat plasma by high performance liquid chromatography

We established a high performance liquid chromatography (HPLC) method for the simultaneous determination of the lactone and carboxylate forms of 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of the antitumor drug irinotecan (CPT-11), in rat plasma. Plasma samples were pretreated with chilled MeOH and zinc sulfate to precipitate protein, and were then directly injected into the HPLC system. Chromatography was carried out with a Puresil C18 column (particle size 5 microns), and the mobile phase consisted of 0.1 M ammonium acetate buffer (pH 5.5) and acetonitrile (70/30, v/v) containing 20 mM of tetra-n-pentylammonium bromide. The column effluent was monitored with a spectrofluorometer (excitation wavelength 380 nm, emission wavelength 540 nm). The method was valid for SN-38 lactone (5-2500 ng/ml) and carboxylate (5-1000 ng/ml).     

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.     

Conversion of the CPT-11 metabolite APC to SN-38 by rabbit liver carboxylesterase

The anticancer drug CPT-11 (7-ethyl-[4(1-piperidino)-1-piperidino]carbonyloxycamptothecin) is a water-soluble derivative of camptothecin. We report here the conversion of APC (7-ethyl-[4-N-(5-aminopentanoic acid)-1-piperidino] carbonyloxycamptothecin), an inactive metabolite of CPT-11, to SN-38 (7-ethyl-10-hydroxycamptothecin), the active metabolite of CPT-11, by a rabbit liver carboxylesterase. This reaction is not catalyzed by any known human enzyme. The formation of SN-38 from APC was characterized by an apparent Km of 37.9 +/- 7.1 microM and a Vmax of 16.9 +/- 0.9 pmol/units/min. SN-38 was confirmed as a reaction product by high-performance liquid chromatography and mass spectrometry. A 24-h incubation of 10 microM APC with 500 units/ml of rabbit carboxylesterase produced 4 microM SN-38. The product of this reaction inhibited the growth of U373 MG human glioblastoma cells in vitro. The IC50 for a 24-h exposure of U373 MG cells to APC in the presence of 50 units/ml of rabbit carboxylesterase was 0.27 +/- 0.08 microM, whereas APC alone demonstrated no inhibition of growth at concentrations up to 1 microM. The IC50 of U373 MG cells transfected with the cDNA encoding the rabbit carboxylesterase (U373pIRESrabbit) and exposed to APC for 24 h was 0.8 +/- 0.1 microM APC, whereas the growth of cells transfected with vector control (U373pIRES) was unaffected by up to 1 microM APC. Because APC is nontoxic to human cells, we are investigating the possibility of using APC/rabbit carboxylesterase in a prodrug/enzyme therapeutic approach.     

Cyclin D1 protein is overexpressed in hyperplasia and intraductal carcinoma of the breast

Irinotecan (CPT-11) is a promising antitumor agent, recently approved for use in patients with metastatic colorectal cancer. Its active metabolite, SN-38, is glucuronidated by hepatic uridine diphosphate glucuronosyltransferases (UGTs). The major dose-limiting toxicity of irinotecan therapy is diarrhea, which is believed to be secondary to the biliary excretion of SN-38, the extent of which is determined by SN-38 glucuronidation. The purpose of this study was to identify the specific isoform of UGT involved in SN-38 glucuronidation. In vitro glucuronidation of SN-38 was screened in hepatic microsomes from normal rats (n = 4), normal humans (n = 25), Gunn rats (n = 3), and patients (n = 4) with Crigler-Najjar type I (CN-I) syndrome. A wide intersubject variability in in vitro SN-38 glucuronide formation rates was found in humans. Gunn rats and CN-I patients lacked SN-38 glucuronidating activity, indicating the role of UGT1 isoform in SN-38 glucuronidation. A significant correlation was observed between SN-38 and bilirubin glucuronidation (r = 0.89; P = 0.001), whereas there was a poor relationship between para-nitrophenol and SN-38 glucuronidation (r = 0.08; P = 0.703). Intact SN-38 glucuronidation was observed only in HK293 cells transfected with the UGT1A1 isozyme. These results demonstrate that UGT1A1 is the isoform responsible for SN-38 glucuronidation. These findings indicate a genetic predisposition to the metabolism of irinotecan, suggesting that patients with low UGT1A1 activity, such as those with Gilbert's syndrome, may be at an increased risk for irinotecan toxicity.     

Irinotecan (CPT-11) metabolites in human bile and urine

A female patient was treated with irinotecan (CPT-11) for liver metastatic colon carcinoma. She had a percutaneous biliary catheter because of extrahepatic biliary obstruction. The patient was treated with CPT-11 for three courses at doses of 350 mg/m2 for the first course and 300 mg/m2 for the remaining courses, given as a 30-min i. v. infusion. Metabolism studies in bile and urine were performed by coupling high-performance liquid chromatography to electrospray mass spectrometry. Conventional spectra [liquid chromatography/mass spectrometry (LC/MS)] allowed on-line molecular mass determination of CPT-11 and its main metabolites, whereas structural information was obtained by tandem mass spectrometry (LC/MS/MS). At least 16 metabolites were detected in bile, while 8 of them were also detected in urine. Three compounds were identified as the parent drug, the active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38), and SN-38 glucuronide. The major metabolic pathway consists in oxidations of the terminal piperidine ring of the CPT-11 side chain, which eventually results in the formation of a primary amine. Other metabolites result from oxidation of the camptothecin nucleus. Finally, decarboxylation of the carboxylate form of CPT-11 was also observed. Several metabolites result from combinations of these pathways. The structures of the identified metabolites indicate for the first time a major role of monooxygenases in the elimination of a camptothecin derivative in humans. This finding will allow better understanding of interindividual variability in pharmacokinetics and intestinal toxicity of CPT-11.     

Determinants of CPT-11 and SN-38 activities in human lung cancer cells

Irinotecan (CPT-11) is a semisynthetic camptothecin derivative with a broad spectrum of anti-tumour activity. Carboxylesterase (CE) catalyses the conversion of CPT-11 to SN-38 (7-ethyl-10-hydroxycamptothecin), the active form of CPT-11. The antiproliferative effects of CPT-11 and SN-38, CE-activity and topoisomerase I protein expression were investigated in five human small-cell lung cancer (SCLC) cell lines and four human non-small-cell lung cancer (NSCLC) cell lines. Antiproliferative activity, expressed as IC50 values, was determined using the MTT assay. CPT-11 was significantly more active in SCLC than in NSCLC cell lines (P = 0.0036), whereas no significant difference between histological types was observed with SN-38. A significant correlation (r2 = 0.52, P = 0.028) was observed between CE activity and chemosensitivity to CPT-11 but not to SN-38, and significantly higher CE activity was observed in SCLC compared with NSCLC cell lines (P = 0.025). Western blotting experiments showed topoisomerase I protein expressions within a factor of 2, and a granular nuclear staining was detectable in all cell lines by immunocytochemistry of cytospins. No correlation was observed between protein expression and sensitivity to CPT-11 or SN-38. Cellular and medium concentrations of CPT-11 and SN-38 were measured by high-performance liquid chromatography (HPLC) in one SCLC cell line with high CE activity and high sensitivity to CPT-11, and one NSCLC cell line with low sensitivity to CPT-11 and CE activity. Intracellular concentrations of CPT-11 and SN-38 were higher in the SCLC cell line, and this was associated with an increase in cellular uptake of CPT-11 compared with the medium, and an increased intracellular formation of SN-38. In conclusion, CE activity appears to be associated with higher sensitivity to CPT-11 in human lung cancer cell lines and may partly explain the difference in the in vitro sensitivity to CPT-11 between SCLC and NSCLC cells. The assessment of CE activity in clinical material of lung cancer patients undergoing treatment with CPT-11 may be warranted. However, other mechanisms may influence sensitivity to CPT-11, possibly including drug transport.     

The evaluation of eustachian tube function in patients with chronic otitis media

To assess the clinical usefulness of salivary monitoring of irinotecan (CPT-11) and its active metabolite (SN-38), we examined the clinical pharmacological profile of both drugs in 9 patients with thoracic malignancies who received 60 mg/m2 CPT-11 (21 courses). Plasma and unstimulated whole saliva were collected over a 24-h period, and concentrations of CPT-11 and SN-38 were measured by high-performance liquid chromatography. Both CPT-11 and SN-38 were detectable in saliva, and the concentration-time curves in plasma and saliva showed a very similar pattern. A good correlation was observed between the saliva concentration (C3) and the plasma concentration (Cp) for both CPT-11 and SN-38 (r = 0.732, P < 0.0001 and r = 0.611, P < 0.0001, respectively). The area under the concentration-time curve calculated for saliva (AUCs) correlated with that generated for plasma (AUCp) for both CPT-11 and SN-38 (r = 0.531, P = 0.012 and r = 0.611, P = 0.0025, respectively). These results suggest that it may be feasible to use saliva instead of plasma for pharmacokinetics/pharmacodynamics studies of CPT-11.     

Pathophysiology and therapy of irinotecan-induced delayed-onset diarrhea in patients with advanced colorectal cancer: a prospective assessment

PURPOSE: Irinotecan (CPT-11), a camptothecin derivative, has shown efficacy against colorectal cancer. Delayed-onset diarrhea is its main limiting toxicity. The aim of this study was to determine the pathophysiology of CPT-11-induced delayed-onset diarrhea and assess the efficacy of combined antidiarrheal medication in a phase II, prospective, successive-cohorts, open study. PATIENTS AND METHODS: Twenty-eight patients with advanced colorectal cancer refractory to fluorouracil (5-FU) therapy received CPT-11 350 mg/m2 every 3 weeks. The first cohort of 14 consecutive patients explored for the mechanism of diarrhea received acetorphan (a new enkephalinase inhibitor) 100 mg three times daily; the second 14-patient cohort received, in addition to acetorphan, loperamide 4 mg three times daily. Before treatment, and if late diarrhea occurred, patients underwent colon mucosal biopsies for CPT-11 and topoisomerase I levels; intestinal transit time; fecalogram; fat and protein excretion; alpha1-antitrypsin clearance; D-xylose test; blood levels for vasoactive intestinal polypeptide, glucagon, gastrin, somatostatin, prostaglandin E2, and carboxylesterase; CPT-11/SN-38 and SN-38 glucuronide pharmacokinetics; and stool cultures. RESULTS: Delayed-onset diarrhea occurred during the first three treatment cycles in 23 patients (82%). Electrolyte fecal measurements showed a negative or small osmotic gap in nine of nine patients and an increased alpha1-antitrypsin clearance in six of six patients. There were no modifications in stool cultures or hormonal dysfunction. Four of 11 patients (36%) with delayed-onset diarrhea in the first cohort responded to acetorphan, whereas nine of 10 patients (90%) responded to the combination of acetorphan and loperamide (P < .02). CONCLUSION: CPT-11-induced delayed-onset diarrhea is caused by a secretory mechanism with an exudative component. Early combined treatment with loperamide and acetorphan seems effective in controlling the diarrheal episodes.     

Recombinant human hepatocyte growth factor facilitates biliary transport after hepatocyte transplantation in Eisai hyperbilirubinemic rats

Hepatocyte transplantation may offer an attractive treatment for inborn errors of liver metabolism. However, factor(s) are required as stimuli to induce proliferation of the limited number of hepatocytes transplanted. The Eisai hyperbilirubinemic rat (EHBR) is a Sprague-Dawley (SD) mutant rat with conjugated hyperbilirubinemia. EHBRs have impaired canalicular excretory transport of organic anions, bile acid glucuronide, and sulfate. Recombinant human hepatocyte growth factor (rhHGF) (100 microg/kg) was injected intravenously at 2-hr intervals for 10 hr, immediately and 35 days following the intraportal injection of 1 x 10(7) wild-type SD rat hepatocytes. Serum bilirubin concentrations decreased significantly within 35 days and were maintained at significantly reduced levels for 120 days following transplantation. Biliary excretion was demonstrated by the biliary transport of indocyanine green and sulfobromophthalein sodium into the bile. These results indicate that hepatic transport of bile acid conjugates in EHBRs can be restored by hepatocyte transplantation combined with repeated administration of exogenous rhHGF, in conjunction with functioning of the recipient's excretory biliary system.