Clinical pharmacogenetics of irinotecan (CPT-11)

Irinotecan (CPT-11) is now widely used, especially for colorectal and lung cancers, whereas the drug causes severe adverse drug reactions (ADR), such as leukopenia/neutropenia or diarrhea. Irinotecan undergoes drug metabolism to form an active SN-38, which is further converted to its beta-glucuronide by UDP-glucuronosyltransferase (UGT) 1A1. A variant in the promoter of UGT1A1 gene, UGT1A1*28 allele, has been extensively studied, and pharmacogenetic relationships between the variant and ADR to irinotecan have been reported. A case-control study of Japanese cancer patients demonstrated that the patients having UGT1A1*28 were at significantly increased risk of severe ADR to irinotecan. To date, genetic variations of the UGT1A1 gene is the most important hereditary factor to predict severe ADR to irinotecan. The UGT1A1*28 is the only one variant that has multiple lines of clinical evidence in multiple races, whereas genetic variations of other UGT isoforms, drug-metabolizing enzymes and drug transporters need more confirmations of its clinical significance in multiple patient groups. At present, irinotecan chemotherapy based on a patient's UGT1A1 genetic status is scientifically reasonable.     

Toxicity of irinotecan (CPT-11) and hepato-renal dysfunction

Various clinical and laboratory parameters have been investigated for their ability to predict toxicity arising from the use of the anticancer drug, irinotecan (CPT-11). In particular, patients deficient in the conjugation of SN-38, a metabolite of CPT-11, are known to be at greater risk. We describe one case of a patient with metastatic colorectal cancer treated with a single dose of CPT-11 at 125 mg/m(2). Although this patient lacked any known predictive factors for toxicity, he experienced severe side-effects several days later. We hypothesized that the toxicity in this patient was due to compromised SN-38 conjugation. Plasma samples were analyzed by reversed-phase high-performance liquid chromatography assay for CPT-11 and its metabolites at 96, 144, 168, 192 and 288 h post-administration. We observed that the concentrations of both the parent drug and its metabolites were markedly raised (11- to 60-fold expected). Additionally the estimated terminal half-lives were 1.5-7 times those expected (29.5, 101, 39.6 and 41.8 h for CPT-11, APC, SN-38G and SN-38, respectively). We conclude that the toxicity in this patient was not caused by deficient SN-38 conjugation, but by decreased drug excretion through both hepatic and renal routes.     

The in vitro metabolism of irinotecan (CPT-11) by carboxylesterase and beta-glucuronidase in human colorectal tumours

AIMS: Irinotecan (CPT-11) is a prodrug that is used to treat metastatic colorectal cancer. It is activated to the topoisomerase poison SN-38 by carboxylesterases. SN-38 is metabolized to its inactive glucuronide, SN-38 glucuronide. The aim of this study was to determine, the reactivation of SN-38 from SN-38 glucuronide by beta-glucuronidase may represent a significant pathway of SN-38 formation. METHODS: The production of SN-38 from irinotecan and SN-38 glucuronide (2.4, 9.6 and 19.2 microm) was measured in homogenates of human colorectal tumour, and matched normal colon mucosa from 21 patients). RESULTS: The rate of conversion of irinotecan (9.6 microm) was lower in tumour tissue than matched normal colon mucosa samples (0.30+/-0.14 pmol min-1 mg-1 protein and 0.77+/-0.59 pmol min-1 mg-1 protein, respectively; P<0.005). In contrast, no significant difference was observed in beta-glucuronidase activity between tumour and matched normal colon samples (4.56+/-6.9 pmol min-1 mg-1 protein and 3.62+/-2.95 pmol min-1 mg-1 protein, respectively, using 9.6 microm SN-38 glucuronide; P>0.05). beta-Glucuronidase activity in tumour correlated to that observed in matched normal tissue (r2>0.23, P<0.05), whereas this was not the case for carboxylesterase activity. At equal concentrations of irinotecan and SN-38 glucuronide, the rate of beta-glucuronidase-mediated SN-38 production was higher than that formed from irinotecan in both tumour and normal tissue (P<0.05). However, at concentrations that reflect the relative plasma concentrations observed in patients, the rate of SN-38 production via these two pathways was comparable. CONCLUSIONS: Tumour beta-glucuronidase may play a significant role in the exposure of tumours to SN-38 in vivo.     

Docetaxel and irinotecan (CPT-11) in the treatment of malignant pleural mesothelioma--a feasibility study

We chose to treat malignant pleural mesothelioma with a combination of docetaxel and irinotecan (CPT-11), because there have been preliminary reports that CPT-11 is active against mesothelioma, and docetaxel and CPT-11 were the most active agents in our in vitro experiments in human mesothelioma cell lines. Fifteen previously untreated patients with pleural mesothelioma (IMIG Stage III-IV) were given docetaxel 60 mg/m2 followed by CPT-11 190 mg/m2 on day 1, repeated every 3 weeks. All the patients were evaluable for toxicity and 13 patients were evaluated for response. No objective responses (complete or partial) were achieved, but there were two minor responses (overall response rate 15%) each of a duration of 4 months. Three patients had stable disease (23%); median time to progression was 7 months. Median survival in all the patients was 8.5 months from the first chemotherapy cycle and 11 months from diagnosis. Toxicity was severe with seven of 15 patients suffering neutropenic fever and six of 15 patients grade 3-4 diarrhea. The trial was discontinued because of toxicity and lack of activity. We do not recommend the combination of docetaxel and CPT-11 using the schedule presented here for further investigation in malignant mesothelioma. However, CPT-11 and docetaxel, individually, still warrant further study in this disease, especially in combination with cisplatin.     

Loperamide inhibits the biliary excretion of irinotecan (CPT-11) in the rat isolated perfused liver

Patients treated with irinotecan (CPT-11) occasionally suffer from severe diarrhoea and aggressive treatment with loperamide at the first signs of loose stools is recommended. We have examined the effect of loperamide on the hepatic metabolism and biliary excretion of CPT-11 in the isolated perfused rat liver (IPRL). CPT-11 (0.5 mumol) was injected as a bolus into the IPRL reservoir, and perfusate and bile samples were collected over 3 h. Experiments were conducted using loperamide-free perfusate (n = 5) or perfusate containing 10 muM loperamide (n = 6). Perfusate and bile concentrations of total CPT-11 and the major metabolites SN-38 (7-ethyl-10-hydroxy-camptothecin), SN-38G (7-ethyl-10-hydroxy-camptothecin glucuronide) and APC (7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidine] carbonyloxycamptothecin) were determined by HPLC. The unchanged parent drug was the predominant species in bile, with approximately 4% of the dose recovered over 180 min as compared with only 1% for the metabolites. Loperamide significantly reduced the biliary excretion of CPT-11 by approximately 50% (2.0 +/- 0.9% dose compared with 3.8 +/- 1.0% in the control group, P = 0.019) over the same period. In contrast, the biliary excretion of SN-38, SN-38G and APC was not significantly affected by loperamide (P > 0.05). Furthermore, bile flow rate was not affected by loperamide. Loperamide appeared to selectively inhibit the biliary excretion of CPT-11, although the extent to which loperamide altered the disposition of CPT-11 in the clinical setting remains to be determined.     

Active transepithelial transport of irinotecan (CPT-11) and its metabolites by human intestinal Caco-2 cells

Irinotecan (CPT-11) is a camptothecin analog with low (about 10--20%) and variable oral bioavailability in animal models. Here, Caco-2 cells were used to evaluate the transepithelial transport of CPT-11 and its metabolites. Caco-2 cells demonstrated significant expression of P-glycoprotein (P-gp), multidrug resistance-associated protein and canalicular multispecific organic anion transporter. Both the lactone and carboxylate forms of CPT-11 and SN-38 were actively transported across the cell monolayers, mainly by the apical-localized P-gp pump. Cellular permeability of CPT-11 at a concentration of 17 microM converted from active to passive-diffusional transport between the 2 and 6 h exposure time points. Antiproliferative effects of CPT-11 were related to permeability of the lactone form, whereas for SN-38 efficacy was dependent on lactone accumulation. Exposure of CPT-11 with cyclosporin A significantly enhanced its efficacy, whereas this was not observed with verapamil and R101933. In contrast, SN-38 efficacy decreased in the presence of P-gp inhibitors due to active transport toward the basolateral side, thereby reducing drug accumulation. Hence, multiple-active transport systems could be demonstrated to be responsible for not only accumulation profiles but also cytotoxic efficacy of CPT-11 and SN-38 in the intestinal Caco-2 cells. It is suggested that CPT-11 might act in a time-dependent manner and that SN-38-mediated cytotoxicity relates to (dose-dependent) lactone kinetics. The results detailed in this report could contribute toward the development of a clinically useful oral formulation of CPT-11 with improved absorption characteristics and suggest that cyclosporin A is a suitable agent for further research of this concept.     

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 60min 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 V _max to be 23.3±5.3μM and 1.43±0.15pmol/min/mg for the lactone and 48.9±5.5μM and 1.09±0.06pmol/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: 100μM; CPT-11 lactone concentration: 10μM). Loperamide and ciprofloxacine where the only drugs exerting a weak inhibition of CPT-11 conversion to SN-38. Received: 30 December 1996 /Accepted: 30 April 1997     

Pharmacokinetics, metabolism, and excretion of irinotecan (CPT-11) following I.V. infusion of [(14)C]CPT-11 in cancer patients.

This study determined the disposition of irinotecan hydrochloride trihydrate (CPT-11) after i.v. infusion of 125 mg/m(2) (100 microCi) [(14)C]CPT-11 in eight patients with solid tumors. Mean +/- S.D. recovery of radioactivity in urine and feces was 95.8 +/- 2.7% (range 92.2-100.3%, n = 7) of dose. Radioactivity in blood, plasma, urine, and feces was determined for at least 168 h after dosing. Fecal excretion accounted for 63.7 +/- 6.8 (range 54.2-74.9%, n = 7) of dose, whereas urinary excretion accounted for 32.1 +/- 6.9% (range 21.7-43.8%; n = 7) of dose. One patient with a biliary T-tube excreted 30.1% of dose in bile, 14.2% in feces, and 48.2% in urine. Quantitative radiometric HPLC revealed that CPT-11 was the major excretion product in urine, bile, and feces. Aminopentane carboxylic acid (APC) and SN-38 glucuronide (SN-38G) were the most significant metabolites in urine and bile, whereas SN-38 and NPC, a primary amine metabolite, were relatively minor excretion products. SN-38 and APC were the most significant metabolites in feces. The relatively higher amount of SN-38 in feces compared with bile is presumably due to hydrolysis of SN-38G to SN-38 by enteric bacterial beta-glucuronidases. There was close correspondence between quantitative fluorescence HPLC and mass balance findings. CPT-11 was the major circulating component in plasma (55% of the mean radiochemical area under the curve), and CPT-11, SN-38, SN-38G, and APC accounted for 93% of the mean radiochemical AUC. These results show that the parent drug and its three major metabolites account for virtually all CPT-11 disposition, with fecal excretion representing the major elimination pathway.     

Population pharmacokinetics of CPT-11 (irinotecan) in gastric cancer patients with peritoneal seeding after its intraperitoneal administration

Purpose  

It is well known that CPT-11 (irinotecan) is biotransformed to its active metabolite, SN-38, by carboxylesterase in the liver and other tissues. However, little is known about its pharmacokinetics (PK) when administered intraperitoneally. The aim of our study was to develop a population pharmacokinetic model for CPT-11 and SN-38 following the intraperitoneal (IP) administration of CPT-11.     

Clinical Pharmacogenetics of Irinotecan (CPT-11)

Irinotecan (CPT-11) is now widely used, especially for colorectal and lung cancers, whereas the drug causes severe adverse drug reactions (ADR), such as leukopenia/neutropenia or diarrhea. Irinotecan undergoes drug metabolism to form an active SN-38, which is further converted to its β-glucuronide by UDP-glucuronosyltransferase (UGT) 1A1. A variant in the promoter of UGT1A1 gene, UGT1A1*28 allele, has been extensively studied, and pharmacogenetic relationships between the variant and ADR to irinotecan have been reported. A case-control study of Japanese cancer patients demonstrated that the patients having UGT1A1*28 were at significantly increased risk of severe ADR to irinotecan. To date, genetic variations of the UGT1A1 gene is the most important hereditary factor to predict severe ADR to irinotecan. The UGT1A1*28 is the only one variant that has multiple lines of clinical evidence in multiple races, whereas genetic variations of other UGT isoforms, drug-metabolizing enzymes and drug transporters need more confirmations of its clinical significance in multiple patient groups. At present, irinotecan chemotherapy based on a patient's UGT1A1 genetic status is scientifically reasonable.     

Phase I/II study of irinotecan (CPT-11) and S-1 in the treatment of advanced gastric cancer

A phase I/II study to determine the recommended dose for combination therapy with CPT-11 (irinotecan hydrochloride) and S-1 (tegafur, gimestat and otastat potassium) for advanced or recurrent gastric cancer, and to assess the safety and efficacy of this therapy. In the phase I portion of the study, S-1 was administered from day 1 to 14 at a fixed dose approved in Japan (80 mg/m2/day), and CPT-11 was administered on days 1 and 8, with its dose being escalated to 100 from 80 mg/m2. This regimen was repeated at 3-week intervals. The phase II portion of the study assessed the efficacy and safety of this regimen at the recommended dose determined in the phase I portion of the study. Seven patients were enrolled in the phase I portion of the study. The dose-limiting toxicity was the delay of administration owing to adverse reactions (leucopenia and diarrhea). The maximum tolerated dose of CPT-11 was 100 mg/m2 and the recommended dose was determined to be 80 mg/m2. In the phase II portion of the study, 10 patients with no prior chemotherapy regimen were enrolled. The median number of treatment cycles given was 4.5, the response rate was 20.0% (2/10) in all patients, the tumor control rate stable disease or better response was 60% (6/10) and the mean survival time was 311 days. Major adverse reactions included a decreased hemoglobin level, diarrhea, nausea and anorexia of grade 3 or worse (each occurred in 10% of the patients). Other adverse reactions were slight and well tolerated. The present combination therapy with CPT-11 and S-1 produced a low response rate but a high tumor control rate (stable disease or better response) and slight prolongation of survival time. This is a well-tolerated ambulatory regimen for advanced gastric cancer.     

In vitro partition of irinotecan (CPT-11) in human volunteer blood: the influence of concentration, gender and smoking

We have performed in vitro incubations of blood from male and female volunteers, smokers and non-smokers, with irinotecan at a gradient of different concentrations in order to investigate changes of partition between red blood cells (RBCs), total plasma and the free fraction. Since irinotecan (CPT-11) is not metabolized in vitro, there is no data available on its active metabolite SN-38. After extraction and sample pre-treatment, a validated high-performance liquid chromatography method followed by fluorescence detection was used to determine the concentration of the drug in the different blood constituents. The partition ratio [the concentration in the erythrocytes divided by the concentration in plasma (E/P)] was calculated. The partition ratio of CPT-11 varied from 0.7 to 2.8, reflecting its relatively high affinity for the erythrocyte, probably because of its only moderate plasma protein binding (65%). The partition ratios increased significantly with higher whole-blood concentrations, favoring uptake in the erythrocytes when plasma protein binding is saturated. No gender difference was detected, but we found relatively more CPT-11 in the erythrocytes of non-smokers compared to smokers. The incorporation of drugs into the RBC pool may be important for transportation to tumor tissue and efficacy. Smoking can have a significant influence on drug partition in the blood.     

High-performance liquid chromatographic analysis of the anticancer drug irinotecan (CPT-11) and its active metabolite SN-38 in human plasma

A rapid, sensitive, and specific high-performance liquid chromatography (HPLC) method for the simultaneous determination of irinotecan (CPT-11) and its active metabolite SN-38 in human plasma is described. The analytes are quantified as the totals of their carboxylate and lactone form. The sample pretreatment consisted of a simple protein precipitation with acetonitrile-methanol (1:1, v/v), after which CPT-11 and SN-38 were quantitatively converted to their carboxylate form by adding 0.01 mol/L sodium tetraborate (pH, 9). Chromatography was carried out on a Zorbax SB-C18 column with fluorescence detection. The method has been validated, and stability tests under various clinically relevant conditions have been performed. The lower limit of quantification (LLOQ) was 5.0 ng/mL for CPT-11 and 0.5 ng/mL for SN-38. Standard concentration ranges were linear between 5 and 1,500 ng/mL for CPT-11 and between 0.5 and 100 ng/mL for SN-38. This assay is simple, rapid, and very useful for therapeutic monitoring of CPT-11 and SN-38.     

ATP-Dependent efflux of CPT-11 and SN-38 by the multidrug resistance protein (MRP) and its inhibition by PAK-104P

Non-P-glycoprotein-mediated multidrug-resistant C-A120 cells that overexpressed multidrug resistance protein (MRP) were 10.8- and 29. 6-fold more resistant to 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin (CPT-11) and SN-38, respectively, than parental KB-3-1 cells. To see whether MRP is involved in CPT-11 and SN-38 resistance, MRP cDNA was transfected into KB-3-1 cells. The transfectant, KB/MRP, which overexpressed MRP, was resistant to both CPT-11 and SN-38. 2-[4-Diphenylmethyl)-1-piperazinyl]ethyl-5-(trans-4,6-dimethyl-1,3 , 2-dioxaphosphorinan-2-yl)-2, 6-dimethyl-4-(3-nitrophenyl)-3-pyridinecarboxylate P-oxide (PAK-104P) and MK571, which reversed drug resistance in MRP overexpressing multidrug-resistant cells, significantly increased the sensitivity of C-A120 and KB/MRP cells, but not of KB-3-1 cells, to CPT-11 and SN-38. The accumulation of both CPT-11 and SN-38 in C-A120 and KB/MRP cells was lower than that in KB-3-1 cells. The treatment with 10 microM PAK-104P increased the accumulation of CPT-11 and SN-38 in C-A120 and KB/MRP cells to a level similar to that found in KB-3-1 cells. The ATP-dependent efflux of CPT-11 and SN-38 from C-A120 and KB/MRP cells was inhibited by PAK-104P. DNA topoisomerase I expression, activity, and sensitivity to SN-38 were similar in the three cell lines. Furthermore, the conversion of CPT-11 to SN-38 in KB-3-1 and C-A120 cell lines was similar. These findings suggest that MRP transports CPT-11 and SN-38 and is involved in resistance to CPT-11 and SN-38 and that PAK-104P reverses the resistance to CPT-11 and SN-38 in tumors that overexpress MRP.     

Activity of weekly irinotecan (CPT-11) in patients with advanced non-small cell lung cancer pretreated with platinum and taxanes

Irinotecan (CPT-11), a semisynthetic derivative of camptothecin, is active in the treatment of non-small cell lung cancer (NSCLC). In this report we describe our experience with this drug when used as a single agent in patients with advanced NSCLC refractory to chemotherapy with platinum and taxanes. Nineteen NSCLC patients (thirteen males and six females; 53% adenocarcinoma and 26% squamous cell carcinoma) with a median age of 52 years (range 34–71) and a Karnofsky performance status of 60% (60–80%) were included in the study. At baseline, the patients had a median of two disease sites and had been treated with a median of two prior regimens. Irinotecan was given at a dose of 100mg/m² i.v.) weekly for 4 weeks followed by 1 week of rest. A total of 123 weekly infusions were administered, and each patient received a median of 4 weeks of treatment (range 1–32). All patients were evaluated by intention-to-treat analysis for efficacy and safety. Main toxicities reported were grade 3 neutropenia (10% of patients), diarrhea (10% of patients), and grade 4 thrombocytopenia (5% of patients). The overall clinical response rate was 16% (95% CI: 8–24) with three partial responses and 9 (47%) patients with stable disease. The median time to progression and the median survival time were 7 and 15 weeks, respectively. In conclusion, weekly irinotecan showed antitumoral activity and minimum toxicity in NSCLC patients refractory to platinum and taxanes.     

Interaction of irinotecan (CPT-11) and its active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38) with human cytochrome P450 enzymes.

The inhibition and mechanism-based inactivation potencies of irinotecan (7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin; CPT-11) and its active metabolite (7-ethyl-10-hydroxycamptothecin; SN-38) for human cytochrome P450 (P450) enzymes were investigated to evaluate the potential for drug interactions involving CPT-11 using microsomes from insect cells expressing specific human P450 isoforms. The mechanism and potential for interaction were examined by Lineweaver-Burk analysis, and NADPH-, time- and concentration-dependent effects were observed. CPT-11 and SN-38 competitively inhibited CYP3A4 (testosterone 6 beta-hydroxylation) activity with K(i) values of 129 and 121 microM, respectively. CYP2A6 (coumarin 7-hydroxylation) and CYP2C9 (diclofenac 4'-hydroxylation) activities exhibited a mixed type of inhibition comprising competitive and noncompetitive components in response to SN-38, the K(i) values being 181 and 156 microM, respectively. On the other hand, CYP1A2 (phenacetin O-deethylation), CYP2B6 (7-ethoxycoumarin O-deethylation), CYP2C8 (paclitaxel 6 alpha-hydroxylation), CYP2C19 (S-mephenytoin 4'-hydroxylation), CYP2D6 (bufuralol 1'-hydroxylation), and CYP2E1 (chlorzoxazone 6-hydroxylation) were hardly affected by either compound. Furthermore, CPT-11 and SN-38 were suggested to be mechanism-based inactivators of CYP3A4. The k(inact) and K(I) values of CPT-11 and SN-38 were 0.06 min(-1) and 24 microM and 0.10 min(-1) and 26 microM, respectively. However, no inactivation of CYP2A6 and CYP2C9 by SN-38 was observed. These results mean that CPT-11 and SN-38 interact with human P450 isoforms, such as CYP2A6, CYP2C9, and CYP3A4, in vitro and imply that the significant drug interactions involving CPT-11 may be caused by a mechanism-based inactivation of CYP3A4 by SN-38 as an active metabolite of CPT-11 rather than competitive inhibition.     

盐酸伊立替康脂质体的制备

目的结合脂质体这种新型给药载体的特征将盐酸伊利替康(Irinotecan,OPT-11)制备成脂质体以解决喜树碱类药物在生理条件下其结果中的内酯环易发生水解反应转变为羟酸盐形式,从而失去活性这个难问题.方法以包封率为主要评价指标,比较了传统的脂质体制备方法(薄膜分散法、乙醇注入法、反向蒸发法等)与主动载药方法-硫酸铵梯度法对伊立替康脂质体制备的影响.结果采用硫酸铵梯度法制备伊立替康脂质体可以获得较高的包封率、较大的药酯比.结论硫酸铵溶液的浓度、孵育时间和温度空白脂质体的粒径大小等是影响伊立替康脂质体包封率的主要因素.