Granulocytic differentiation of human NB4 promyelocytic leukemia cells induced by all-trans retinoic acid metabolites.

The metabolism of all-trans retinoic acid (ATRA) has been reported to be partly responsible for the in vivo resistance to ATRA seen in the treatment of human acute promyelocytic leukemia (APL). However, ATRA metabolism appears to be involved in the growth inhibition of several cancer cell lines in vitro. The purpose of this study was to evaluate the in vitro activity of the principal metabolites of ATRA [4-hydroxy-retinoic acid (4-OH-RA), 18-hydroxy-retinoic acid (18-OH-RA), 4-oxo-retinoic acid (4-oxo-RA), and 5,6-epoxy-retinoic acid (5,6-epoxy-RA)] in NB4, a human promyelocytic leukemia cell line that exhibits the APL diagnostic t(15;17) chromosomal translocation and expresses the PML-RAR alpha fusion protein. We established that the four ATRA metabolites were indeed formed by the NB4 cells in vitro. NB4 cell growth was inhibited (69-78% at 120 h) and cell cycle progression in the G1 phase (82-85% at 120 h) was blocked by ATRA and all of the metabolites at 1 microM concentration. ATRA and its metabolites could induce NB4 cells differentiation with similar activity, as evaluated by cell morphology, by the nitroblue tetrazolium reduction test (82-88% at 120 h) or by the expression of the maturation specific cell surface marker CD11c. In addition, nuclear body reorganization to macropunctated structures, as well as the degradation of PML-RAR alpha, was found to be similar for ATRA and all of its metabolites. Comparison of the relative potency of the retinoids using the nitroblue tetrazolium reduction test showed effective concentrations required to differentiate 50% of cells in 72 h as follows: ATRA, 15.8 +/- 1.7 nM; 4-oxo-RA, 38.3 +/- 1.3 nM; 18-OH-RA, 55.5 +/- 1.8 nM; 4-OH-RA, 79.8 +/- 1.8 nM; and 5,6-epoxy-RA, 99.5 +/- 1.5 nM. The ATRA metabolites were found to exert their differentiation effects via the RAR alpha nuclear receptors, because the RAR alpha-specific antagonist BMS614 blocked metabolite-induced CD11c expression in NB4 cells. These data demonstrate that the principal ATRA Phase 1 metabolites can elicit leukemia cell growth inhibition and differentiation in vitro through the RAR alpha signaling pathway, and they suggest that these metabolites may play a role in ATRA antileukemic activity in vivo.

Identification of human cytochrome P450s involved in the formation of all-trans-retinoic acid principal metabolites.

Cytochrome P450 (P450)-dependent metabolism of all-trans-retinoic acid (atRA) is important for the expression of its biological activity. Because the human P450s involved in the formation of the principal atRA metabolites have been only partially identified, the purpose of this study was to identify the human P450s involved in atRA metabolism. The use of phenotyped human liver microsomes (n = 16) allowed the identification of the following P450s: 2B6, 2C8, 3A4/5, and 2A6 were involved in the formation of 4-OH-RA and 4-oxo-RA; 2B6, 2C8, and 2A6 correlated with the formation of 18-OH-RA; and 2A6, 2B6, and 3A4/5 activities correlated with 5, 6-epoxy-RA formation (30-min incubation, 10 microM atRA, HPLC separation, UV detection 340 nm). The use of 15 cDNA-expressed human P450s from lymphoblast microsomes, showed the formation of 4-OH-RA by CYP3A7 > CYP3A5 > CYP2C18 > CYP2C8 > CYP3A4 > CYP2C9, whereas the 18-OH-RA formation involved CYPs 4A11 > 3A7 > 1A1 > 2C9 > 2C8 > 3A5 > 3A4 >2C18. Kinetic studies identified 3A7 as the most active P450 in the formation of three of the metabolites: for 4-OH-retinoic acid, 3A7 showed a V(max)/K(m) of 127.7, followed by 3A5 (V(max)/K(m) = 25.6), 2C8 (V(max)/K(m) = 24.5), 2C18 (V(max)/K(m) = 15.8), 3A4 (V(max)/K(m) = 5.7), 1A1 (V(max)/K(m) = 5.0), and 4A11 (V(max)/K(m) = 1.9); for 4-oxo-RA, 3A7 showed a V(max)/K(m) of 13.4, followed by a 10-fold lower activity for both 2C18 and 4A11 (V(max)/K(m) = 1.2); and for 18-OH-RA, 3A7 showed a V(max)/K(m) of 10.5 compared with a V(max)/K(m) of 2.1 for 4A11 and 2.0 for 2C8. 5,6-Epoxy-RA was only detected at high substrate concentrations in this system (>10 microM), and P450s 2C8, 2C9, and 1A1 were the most active in its formation. The use of embryonic kidney cells (293) stably transfected with human P450 cDNA confirmed the major involvement of P450s 3A7, 1A1, and 2C8 in the oxidation of atRA, and to a lesser extent, 1A2, 2C9, and 3A4. In conclusion, several human P450s involved in atRA metabolism have been identified, the expression of which was shown to direct atRA metabolism toward the formation of specific metabolites. The role of these human P450s in the biological and anticancer effects of atRA remains to be elucidated.

[Irinotecan pharmacokinetics]. / Pharmacocinétique de l'irinotecan.

The clinical pharmacokinetics of irinotecan (CPT11) can be described by a 2 or 3 compartment model, a mean terminal half-life of 12 hours, a volume of distribution at steady state of 168 l/m2 and a total body clearance of 15 l/m2/h. Irinotecan is 65% bound to plasma proteins. The areas under the plasma concentration-time curve (AUC) of both irinotecan and active metabolite SN38 increase proportionally to the administered dose, although interpatient variability is important. SN38 levels achieved in humans are about 100-fold lower than corresponding irinotecan levels, but these concentrations are important since SN38 is 100- to 1,000-fold more cytotoxic than the parent compound. SN38 is 95% bound to plasma proteins. SN38 plasma decay follows closely that of the parent compound. Irinotecan is extensively metabolized in the liver. The bipiperidinocarbonylxy group of irinotecan is first removed by a carboxyesterase to yield the corresponding carboxylic acid and SN38. This metabolite can be converted into SN38 glucuronide by UDP-glucuronyltransferase (1.1 isoform). A recently identified metabolite is the 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]-carbonyloxy-camptothecin (APC), which is formed by the action of cytochrome P450 3A4. Numerous other unidentified metabolites are detected in bile and urine. The mean 24 h irinotecan urinary excretion represents 17-25% of the administered dose, whereas SN38 and its glucuronide recovery in urine is minimal (0.5 and 6%, respectively). Irinotecan and SN38 pharmacokinetics are not influenced by prior exposure to the parent drug. Irinotecan and SN38 AUCs correlate significantly with leuko-neutropenia and sometimes with the intensity of diarrhea. Increased bilirubin levels appear to influence irinotecan total body clearance. The observation that most tumor responses were seen at the highest doses administered in phase I trials suggest a dose-response relationship with this drug. These pharmacokinetic-pharmacodynamic relationships may prove useful for a better clinical management of this drug aimed at a better control of toxicities and a better prediction of tumor response for the benefit of the individual patient.

Clinical pharmacokinetics of irinotecan.

This article reviews the clinical pharmacokinetics of a water-soluble analogue of camptothecin, irinotecan [CPT-11 or 7-ethyl-10-[4-(1-piperidino)-1-piperidino]-carbonyloxy-camptoth eci n]. Irinotecan, and its more potent metabolite SN-38 (7- ethyl-10-hydroxy-camptothecin), interfere with mammalian DNA topoisomerase I and cancer cell death appears to result from DNA strand breaks caused by the formation of cleavable complexes. The main clinical adverse effects of irinotecan therapy are neutropenia and diarrhoea. Irinotecan has shown activity in leukaemia, lymphoma and the following cancer sites: colorectum, lung, ovary, cervix, pancreas, stomach and breast. Following the intravenous administration of irinotecan at 100 to 350 mg/m2, mean maximum irinotecan plasma concentrations are within the 1 to 10 mg/L range. Plasma concentrations can be described using a 2- or 3-compartment model with a mean terminal half-life ranging from 5 to 27 hours. The volume of distribution at steady-state (Vss) ranges from 136 to 255 L/m2, and the total body clearance is 8 to 21 L/h/m2. Irinotecan is 65% bound to plasma proteins. The areas under the plasma concentration-time curve (AUC) of both irinotecan and SN-38 increase proportionally to the administered dose, although interpatient variability is important. SN-38 levels achieved in humans are about 100-fold lower than corresponding irinotecan concentrations, but these concentrations are potentially important as SN-38 is 100- to 1000-fold more cytotoxic than the parent compound. SN-38 is 95% bound to plasma proteins. Maximum concentrations of SN-38 are reached about 1 hour after the beginning of a short intravenous infusion. SN-38 plasma decay follows closely that of the parent compound with an apparent terminal half-life ranging from 6 to 30 hours. In human plasma at equilibrium, the irinotecan lactone form accounts for 25 to 30% of the total and SN-38 lactone for 50 to 64%. Irinotecan is extensively metabolised in the liver. The bipiperidinocarbonylxy group of irinotecan is first removed by hydrolysis to yield the corresponding carboxylic acid and SN-38 by carboxyesterase. SN-38 can be converted into SN-38 glucuronide by hepatic UDP-glucuronyltransferase. Another recently identified metabolite is 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]-carbonyloxy-camptothecin (APC). This metabolite is a weak inhibitor of KB cell growth and a poor inducer of topoisomerase I DNA-cleavable complexes (100-fold less potent than SN-38). Numerous other unidentified metabolites have been detected in bile and urine. The mean 24-hour irinotecan urinary excretion represents 17 to 25% of the administered dose. Recovery of SN-38 and its glucuronide in urine is low and represents 1 to 3% of the irinotecan dose. Cumulative biliary excretion is 25% for irinotecan, 2% for SN-38 glucuronide and about 1% for SN-38. The pharmacokinetics of irinotecan and SN-38 are not influenced by prior exposure to the parent drug. The AUC of irinotecan and SN-38 correlate significantly with leuco-neutropenia and sometimes with the intensity of diarrhoea. Certain hepatic function parameters have been correlated negatively with irinotecan total body clearance. It was noted that most tumour responses were observed at the highest doses administered in phase I trials, which indicates a dose-response relationship with this drug. In the future, these pharmacokinetic-pharmacodynamic relationships will undoubtedly prove useful in minimising the toxicity and maximise the likelihood of tumour response in patients.

Expression and inducibility of cytochrome P450 3A9 (CYP3A9) and other members of the CYP3A subfamily in rat liver.

Quantitative reverse-transcriptase polymerase chain reaction was used to determine the content of mRNA derived from four CYP3A genes (CYP3A2, CYP3A9, CYP3A18, and CYP3A23) in rat liver. CYP3A2 and CYP3A9 gene expression was age- and sex-dependent, whereas CYP3A18 and CYP3A23 mRNA were observed before and after puberty at fairly constant levels that were about 20% higher in males than in females. CYP3A9 mRNA was detected only in adult rats, with a nearly twofold higher expression in females. CYP3A9 induction was different from other CYP3A isoenzymes, since phenobarbital was a more effective inducer than dexamethasone and clotrimazole. The results presented for CYP3A23 are those anticipated for CYP3A1, which may be an allelic variant of CYP3A23 not detected in these experiments. These data show that rat CYP3A genes are variably expressed depending on age, sex, or inducer type.

Etoposide bioavailability after oral administration of the prodrug etoposide phosphate in cancer patients during a phase I study.

PURPOSE: The purpose of this study was to determine the bioavailability (F) of etoposide (E;VP-16) after oral administration of the water-soluble prodrug etoposide phosphate (EP;BMY-40481) during a phase I trial in cancer patients. PATIENTS AND METHODS: Twenty-nine patients received oral EP (capsules, 50 to 150 mg/m2/d of E equivalent) for 5 days in week 1 (course 1), followed every 3 weeks thereafter by a daily intravenous (i.v.) infusion for 5 days of E (80 mg/m2, 1-hour i.v. infusion; course 2); in three patients, the i.v. E course was given before oral EP. Plasma and urine E pharmacokinetics (high-performance liquid chromatography [HPLC]) were performed on the first day of oral EP administration and on the first day of i.v. E. RESULTS: Twenty-six of 29 patients completed two courses or more, whereas three patients received only one course due to toxicity. Myelosuppression was dose-dependent and dose-limiting, with grade 4 leukoneutropenia in four of 15 patients at 125 mg/m2 and in five of seven patients at 150 mg/m2. One patient died of meningeal hemorrhage related to grade 4 thrombocytopenia. Other toxicities were infrequent and/or manageable. No objective response was observed. The maximum-tolerated dose (MTD) is therefore 150 mg/m2, and the recommended oral dose of EP for phase II trials in this poor-risk patient population is 125 mg/m2. Twenty-six patients had pharmacokinetic data for both oral EP and i.v. E, whereas three had pharmacokinetic data on the i.v. E course only. After oral administration of EP, the pharmacokinetics of E were as follows: mean absorption rate constant (Ka), 1.7 +/- 1.7 h-1 (mean +/- SD); lag time, 0.3 +/- 0.2 hours; time of maximum concentration (t(max)), 1.6 +/- 0.8 hours; and mean half-lives (t1/2), 1.6 +/- 0.2 (first) and 10.3 +/- 5.8 hours (terminal); the increase in the area under the plasma concentration-versus-time curve (AUC) of E was proportional to the EP dose. After the 1-hour i.v. infusion of E, maximum concentration (C(max)) was 15 +/- 3 micrograms/mL; mean AUC, 88.0 +/- 22.0 micrograms.h/mL; mean total-body clearance (CL), 0.97 +/- 0.24 L/h/m2 (16.2 mL/min/m2); and mean t1/2, 0.9 +/- 0.6 (first) and 8.1 +/- 4.1 hours (terminal). The 24-hour urinary excretion of E after i.v. E was significantly higher (33%) compared with that of oral EP (17%) (P < .001). Significant correlation was observed between the neutropenia at nadir and the AUC of E after oral EP administration (r = .58, P < .01, sigmoid maximum effect [E(max)] model). The mean F of E after oral administration of EP in 26 patients was 68.0 +/- 17.9% (coefficient of variation [CV], 26.3%; F range, 35.5% to 111.8%). In this study, tumor type, as well as EP dose, did not significantly influence the F in E. There was no difference in F of E, whether oral EP was administered before or after i.v. E. Compared with literature data on oral E, the percent F in E after oral prodrug EP administration was 19% higher at either low ( < or = 100 mg/m2) or high ( > 100 mg/m2) doses. CONCLUSION: Similarly to E, the main toxicity of the prodrug EP is dose-dependent leukoneutropenia, which is dose-limiting at the oral MTD of 150 mg/m2/d for 5 days. The recommended oral dose of EP is 125 mg/m2/d for 5 days every 3 weeks in poor-risk patients. Compared with literature data, oral EP has a 19% higher F value compared with oral E either at low or high doses. This higher F in E from oral prodrug EP appears to be a pharmacologic advantage that could be of potential pharmacodynamic importance for this drug.

Experimental antitumor activity and pharmacokinetics of the camptothecin analog irinotecan (CPT-11) in mice.

Irinotecan (CPT-11) is a semi-synthetic derivative of camptothecin currently in clinical trials. In vitro, CPT-11 presented preferential cytotoxicity toward some solid tumor cells (mouse colon 38 and pancreas 03; human pancreas MIA PaCa-2) as compared to leukemia cells (L1210), whereas SN-38, a metabolite of CPT-11, was not solid tumor selective. In vivo, schedule of administration studies in P388 leukemia and mammary adenocarcinoma 16/C (MA16/C) showed that CPT-11 was not markedly schedule dependent. In order to determine its spectrum of anticancer activity, CPT-11 was evaluated against a variety of mouse and human tumors. The end points used were total log cell kill (Lck) for solid tumors and increase in life span (% ILS) for leukemia. Intravenous CPT-11 was found highly active against both early and advanced stage pancreatic ductal adenocarcinoma 03 (P03), with 60% long-term survivors and 100% complete regressions, respectively. Other responsive tumors included: colon adenocarcinomas 38 and 51 (both 1.0 Lck); MA16/C (3.4 Lck); MA13/C (1.0 Lck); human Calc18 breast adenocarcinoma (2.8 Lck); Glasgow osteogenic sarcoma (1.8 Lck); Lewis lung carcinoma (1.4 Lck); B16 melanoma (1.4 Lck); P388 leukemia (170% ILS) and L1210 leukemia (64% ILS). Of interest, CPT-11 was active against tumors with acquired resistance to vincristine (P388/Vcr), to doxorubicin (P388/Dox) and to docetaxel (Calc18/TXT). CPT-11 was also found highly active after oral administration in mice bearing P03 and MA16/C tumors. Pharmacokinetic evaluations performed i.v. at the highest non-toxic dosage in mice bearing P03 tumors revealed CPT-11 peak plasma concentrations (Cmax) of 8.9 micrograms/ml and a terminal half-life of 0.6 h. The metabolite SN-38 plasma concentrations presented a Cmax of 1.6 micrograms/ml and a terminal half-life of 7.4 h. Although the CPT-11 tumor levels were similar to the plasma concentrations for early time points, drug levels decreased more slowly in the tumor compared to plasma (half-life, 5.0 h). SN-38 tumor levels reached concentrations in the range of 0.32-0.34 micrograms/g and decayed with a half-life of 6.9 h. No significant difference in plasma or tumor pharmacokinetics of either CPT-11 or SN-38 were noted after one or five daily i.v. injections. Overall, these data show that CPT-11 has good activity in experimental models, when administered both by the i.v. and the oral routes. Compared to humans, a similar schedule of administration independence was observed and similar CPT-11 levels could be reached at efficacious dosages although metabolite SN-38 levels were found higher in mice.

In vivo induction of cytochrome P450 CYP3A expression in rat leukocytes using various inducers.

Although the induction of cytochromes P450 3A (CYP3A) is relatively well characterized in liver, its inducibility in an easily available tissue such as the peripheral leukocytes is not known. The purpose of this study was, therefore, to determine if CYP3A is inducible in vivo in peripheral leukocytes. Microsomes from rat leukocytes and liver were examined for CYP3A protein expression using Western blotting with a rabbit polyclonal antibody against rat CYP3A. Although CYP3A was not detected in control leukocytes, in vivo treatment with known CYP3A inducers (dexamethasone, clotrimazole, phenobarbital, pregnenolone-16 alpha-carbonitrile) resulted in CYP3A leukocyte levels of 0.2-0.8 pmol/mg protein. This leukocyte induction was approximately 1000-fold lower than in induced liver. Interestingly, there was an apparent linear relationship between leukocyte and liver CYP3A contents (r2 = 0.748, n = 29). These results not only demonstrate for the first time that CYP3A is inducible in rat leukocytes after in vivo treatment with various CYP3A inducers, but also suggest that peripheral leukocytes could be used to assess induction in vivo.

Phase I and pharmacology study of intoplicine (RP 60475; NSC 645008), novel topoisomerase I and II inhibitor, in cancer patients.

Intoplicine (RP 60475F; NSC 645008) is a novel 7H-benzo[e]pyrido[4,3-b]indole derivative which interacts with both topoisomerases I and II. Because of its high activity in preclinical cancer models, original mechanism of action and acceptable toxicity profile, intoplicine was further evaluated in a phase I and pharmacology study. Thirty-three (33) patients (24 men and nine women) meeting standard phase I eligibility criteria were included: median age was 56 years, performance status 0-1 in 28 patients and 2 in five patients. Tumor primary sites were head and neck (9), colon (6), lung (3) and various other sites (15). Thirty-one patients had received prior radiotherapy and/or chemotherapy. Sixty-nine coursed of intoplicine were administered as a 1 h i.v. infusion at dose levels ranging from 12 to 360 mg/m2. Dose-dependent and reproducible hepatotoxicity was dose limiting in three out of four patients at 360 mg/m2: this toxicity was reversible in two of three patients, but fatal in one. Two sudden deaths occurred in this study at 12 and 48 mg/m2, and the drug implication could not be excluded. No myelosuppression was noted. Hepatotoxicity is therefore dose limiting at 360 mg/m2, and the phase II recommended dose is 270 mg/m2 every 3 weeks with close monitoring of hepatic and cardiac functions. Intoplicine pharmacokinetics was determined in plasma (23 patients) and whole blood (18 patients) at doses ranging from 12 to 360 mg/m2. Intoplicine plasma concentration decay was either bi- or triphasic with the following pharmacokinetic values (mean +/- SEM): half-life alpha, 0.04 +/- 0.004 h; half-life beta, 0.61 +/- 0.13 h; terminal half-life, 19.4 +/- 4.0 h; mean residence-time (MRT), 11.3 +/- 2.4; total plasma clearance (CL), 74 +/- 5 l/h; volume of distribution beta (V beta), 1982 +/- 477 l: volume of distribution at steady state (Vss): 802 +/- 188 l. both the area under the plasma concentration versus time curves (AUC) and the maximum plasma concentrations (Cmax) increased linearly with the intoplicine dose, indicating linear pharmacokinetics (AUC: r = 0.937; slope = 0.01305; p < 0.001; Cmax: r = 0.847; slop = 0.01115; p < 0.001). Plasma AUC was also predicted very accurately by the Cmax values (r = 0.909; slope = 1.0701; p < 0.001). Other plasma pharmacokinetic parameter values increased significantly with dose, e.g. the terminal half-life (r = 0.748, p < 0.001) the MRT (r = 0.728, p < 0.001), the V beta (r = 0.809, p < 0.001), and the Vss (r = 0.804, p < 0.001). This was probably due to a longer detectability of the drug in plasma at higher doses. Blood pharmacokinetics was also evaluated in 18 patients since it was found that red blood cells represented a significant drug reservoir for intoplicine. Blood intoplicine disposition curves were either bi- or triphasic with the following pharmacokinetic parameter values (mean +/- SEM): half-life alpha, 0.04 +/- 0.01 h; half-life beta, 0.94 +/- 0.22 h; terminal half-life, 57.1 +/- 6.6 h; MRT, 82.2 +/- 9.9 h; CL, 18 +/- 3 l/h; V beta, 1188 +/- 147 I; Vss 1163 +/- 138 I. Blood pharmacokinetics was linear, since AUC and Cmax increased linearly with dose (AUC: r = 0.879; slop = 0.06884; p < 0.001; Cmax: r = 0.835, slop = 0.01223; p < 0.001). Blood AUC values could also be determined by the blood Cmax (r = 0.768; slop = 5.0206; p < 0.001). Other blood pharmacokinetic parameter values presented a dose dependence, e.g. the terminal half-life (r = 0.626, p = 0.005), the V beta (r = 0.682, p = 0.002) and the Vss (r = 0.555, p = 0.017). The plasma or blood intoplicine concentrations achieved in vivo in humans are potentially cytotoxic levels based on preclinical in vivo and in vitro data. In conclusion, the phase II recommended dose of intoplicine is 270 mg/m2 administered as a 1 h i.v. infusion every 3 weeks. Plasma and blood pharmacokinetics were linear within the dose range studied. Potentially cytotoxic concentrations were reached at clinically achievable doses.

Main drug-metabolizing enzyme systems in human non-Hodgkin's lymphomas sensitive or resistant to chemotherapy.

Non-Hodgkin's lymphomas (NHL) are one of the most chemosensitive human malignancies. Complete response (CR) is often achieved, but many patients relapse and a second CR is difficult to obtain because of the development of chemoresistance. In an attempt to better understand the biology and the chemosensitivity of these lymphoid tumors, we assessed the main drug-metabolizing enzyme systems in normal lymphocytes, chemosensitive NHL and chemoresistant NHL. Cytochromes P-450 (1A1/A2, 2B1/B2, 2C8-10, 2E1, 3A4), epoxide hydrolase and glutathione S-transferases (GST-alpha, -mu, -pi) were assayed by immunoblotting. UDP-glucuronosyltransferase, beta-glucuronidase, sulfotransferase, sulfatase, GST activity, and glutathione (GSH) content, were determined by spectral assays. Results showed the absence of all probed cytochromes P-450 in normal lymphocytes and NHL cells tested. GST activity was significantly lower in chemoresistant NHL compared to normal lymphocytes. GST-alpha was not detected in either normal lymphocytes or NHL cells. GST-pi was the predominant isoenzyme, and GST-mu was not detected in chemosensitive NHL. GSH content was significantly lower in chemoresistant NHL compared to other lymphoid tissues tested. The conjugating enzymes UDP-glucuronosyltransferase and sulfatase were similar in either chemoresistant NHL compared to chemosensitive NHL. The activity of the hydrolytic enzyme beta-glucuronidase was lower in chemoresistant compared to chemosensitive NHL, whereas sulfatase was higher in sensitive NHL compared to normal lymphocytes. Epoxide hydrolase was not detected in either normal or NHL cells tested. In conclusion, these studies did not show any cytochrome P-450 in human lymphoid cells tested, but pointed out noteworthy differences for other enzyme systems tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Phase I and pharmacokinetic study of irinotecan (CPT-11) administered daily for three consecutive days every three weeks in patients with advanced solid tumors.

BACKGROUND: We conducted a phase I and pharmacokinetic study to determine the maximum tolerable dose (MTD), toxicities, pharmacokinetic profile, and antitumor activity of Irinotecan (CPT-11) in patients with refractory solid malignancies. PATIENTS AND METHODS: Forty-six patients were entered in this phase I study. CPT-11 was administered intravenously over 30 minutes for 3 consecutive days every 3 weeks. Dose levels ranged from 33 mg/m2/day to 115 mg/m2/day on days 1 through 3. The pharmacokinetics of total CPT-11 and its active metabolite SN-38 were assayed by HPLC. RESULTS: The combination of leukopenia and diarrhea was dose-limiting toxicity at 115 mg/m2/day dose level, since 50% of the patients (5/10) experienced either grade 3-4 leukopenia, or diarrhea, or both. Leukopenia appeared to be a cumulative toxicity, with a global increase in its incidence and severity upon repeated administration of CPT-11. Other toxicities included nausea, vomiting, fatigue and alopecia. CPT-11 and active metabolite SN-38 pharmacokinetics were determined in 21 patients (29 courses). Both CPT-11 and SN-38 pharmacokinetics presented a high interpatient variability. CPT-11 mean maximum plasma concentrations reached 2034 ng/ml at the MTD (115 mg/m2). The terminal-phase half-life was 8.3 h and the mean residence time 10.2 h. The mean volume of distribution at steady state was 141 l/m2/h. CPT-11 rebound concentrations were observed in many courses at about 0.5 to 1 hour following the end of the i.v. infusion, which is suggestive of enterohepatic recycling. Total body clearance did not vary with increased dosage (mean = 14.3 l/h/m2), indicating linear pharmacokinetics within the dose range administered in this trial. The total area under the plasma concentration versus time curve (AUC) increased proportionally to the CPT-11 dose. Mean metabolite SN-38 peak levels reached 41 ng/ml at the MTD. A significant correlation was observed between CPT-11 area under the curve (AUC) and its corresponding metabolite SN-38 AUC (r = 0.52, p < 0.05). SN-38 rebound concentrations were observed in many courses at about 0.5 to 1 hour following the end of the i.v. infusion, which is suggestive of enterohepatic recycling. Mean 24-h urinary excretion of CPT-11 accounted for 10% of the administered dose by the third day, whereas SN-38 urinary excretion accounted for 0.18% of the CPT-11 dose. In this phase I trial, the hematological toxicity correlated with neither CPT-11 nor SN-38 AUC. Diarrhea grade correlated significantly with CPT-11 AUC. Two partial (breast adenocarcinoma and carcinoma of unknown primary) and 2 minor (hepatocarcinoma and pancreatic adenocarcinoma) responses were observed. CONCLUSION: The MTD for CPT-11 administered in a 3 consecutive-days-every-3 weeks schedule in this patient population is 115 mg/m2/day. The recommended dose for phase II studies is 100 mg/m2/day.

Population pharmacokinetics and pharmacodynamics of irinotecan (CPT-11) and active metabolite SN-38 during phase I trials.

BACKGROUND: Irinotecan (CPT-11) is a novel water-soluble camptothecin derivative selected for clinical testing based on its good in vitro and in vivo activity in various experimental systems, including pleiotropic drug-resistant tumors. Its mechanism of action appears mediated through topoisomerase I inhibition. The purpose of this study was to describe CPT-11 and active metabolite SN-38 population pharmacokinetics, examine patient characteristics that may influence pharmacokinetics, and to investigate pharmacokinetic-pharmacodynamic relationships that may prove useful in the future clinical management of this drug. PATIENTS AND METHODS: As part of 3 Phase I studies including 235 patients, pharmacokinetics of CPT-11 and metabolite SN-38 were determined in 107 patients. CPT-11 was administered as a 30-min i.v. infusion according to 3 different schedules: daily for 3 consecutive days every 3 weeks, weekly for 3 weeks, and once every 3 weeks. Patients characteristics were the following: median age 53 years; 62 men, 45 women; 105 caucasians, 2 blacks; performance status was 0-1 in 96 patients; tumor sites were predominantly colon, rectum, head and neck, lung, ovary and breast; with the exception of 6 patients, all had been previously treated with surgery, chemotherapy and/or radiotherapy. CPT-11 and metabolite SN-38 were simultaneously determined by HPLC using fluorescence detection. Pharmacokinetic parameters were determined using model-independent and model-dependent analyses. RESULTS: 168 pharmacokinetic data sets were obtained in 107 patients (97 first courses, 43 second courses, 23 third courses, 4 fourth courses, and 1 fifth course). Rebound concentrations of CPT-11 were frequently observed at about 0.5 to 1 h following the end of the i.v. infusion, which is suggestive of enterohepatic recycling of the drug. Model-independent analysis yielded the following mean population pharmacokinetic parameters for CPT-11: a terminal half-life of 10.8 h, a mean residence time (MRT) of 10.7 h, a volume of distribution at steady state (Vdss) of 150 L/m2, and a total body clearance of 14.3 L/m2/h. Model-dependent analysis disclosed a CPT-11 plasma disposition as either biphasic or triphasic with a mean terminal half-life of 12.0 h. The volume of distribution Vdss (150 L/m2) and total body clearance (14.8 L/m2/h) yielded almost identical values to the above model-independent analysis. The active metabolite SN-38 presented rebound concentrations in many courses at about 1 h following the end of the i.v. infusion which is suggestive of enterohepatic recycling. The mean time at which SN-38 maximum concentrations was reached was at 1 h since the beginning of the 0.5 h infusion (i.e., 0.5 h post i.v.). SN-38 plasma decay followed closely that of the parent compound with a mean apparent terminal half-life of 10.6 h. Mean 24 h CPT-11 urinary excretion represented 16.7% of the administered dose, whereas metabolite SN-38 recovery in urine was minimal (0.23% of the CPT-11 dose). The number of CPT-11 treatments did not influence pharmacokinetic parameters of either the parent compound or metabolite SN-38. Although CPT-11 pharmacokinetics presented an important interpatient variability, both CPT-11 maximum concentrations (Cmax) and the CPT-11 area under the plasma concentration versus time curves (AUC) increased proportionally and linearly with dosage (Cmax, r = 0.78, p < 0.001); CPT-11 AUC, r = 0.88, p < 0.001). An increase in half-life and MRT was observed at higher dosages, although this did not influence the linear increase in AUC as a function of dose. The volume of distribution at steady state (Vdss) and the total body clearance (CL) were not affected by the CPT-11 dose. Metabolite SN-38 AUC increased proportionally to the CPT-11 dose (r = 0.67, p < 0.001) and also with the parent compound AUC (r = 0.75, p < 0.001) (ABSTRACT TRUNCATED)

Phase I and pharmacologic studies of the camptothecin analog irinotecan administered every 3 weeks in cancer patients.

PURPOSE: A phase I study was undertaken to determine the maximum-tolerated dose (MTD), principal toxicities, and pharmacokinetics of the novel topoisomerase I inhibitor irinotecan (CPT-11). PATIENTS AND METHODS: Sixty-four patients meeting standard phase I eligibility criteria were included (24 women, 40 men; median age, 51 years; primary sites: colon, head and neck, lung, pleura; 60 of 64 had been previously treated). Pharmacokinetics was determined by high-performance liquid chromatography (HPLC). RESULTS: One hundred ninety CPT-11 courses were administered as a 30-minute intravenous (IV) infusion every 3 weeks (100 to 750 mg/m2). Grade 3 to 4 nonhematologic toxicities included diarrhea (16%; three hospitalizations), nausea and vomiting (9%), asthenia (14%), alopecia (53%), elevation of hepatic transaminases (8%), and one case of skin toxicity. An acute cholinergic syndrome was observed during CPT-11 administration. Diarrhea appeared dose-limiting at 350 mg/m2, but this was circumvented by using a high-dose loperamide protocol that allowed dose escalation. Dose-dependent, reversible, noncumulative granulocytopenia was the dose-limiting toxicity (nadir, days 6 to 9; median recovery time, 5 days). Grade 3 to 4 anemia was observed in 9% of patients. One patient died during the study, 8 days after CPT-11 treatment. Two complete responses (cervix, 450 mg/m2; head and neck, 750 mg/m2) and six partial responses in fluorouracil (5-FU)-refractory colon cancer were observed (260 to 600 mg/m2). Pharmacokinetics of CPT-11 and active metabolite SN-38 were performed in 60 patients (94 courses). CPT-11 plasma disposition was bi- or triphasic, with a mean terminal half-life of 14.2 +/- 0.9 hours (mean +/- SEM). The mean volume of distribution (Vdss) was 157 +/- 8 L/m2, and total-body clearance was 15 +/- 1 L/m2/h. The CPT-11 area under the plasma concentration versus time curves (AUC) and SN-38 AUC increased linearly with dose. SN-38 plasma decay had an apparent half-life of 13.8 +/- 1.4 hours. Both CPT-11 and SN-38 AUCs correlated with nadir leukopenia and granulocytopenia, with grade 2 diarrhea, and with nausea and vomiting. CONCLUSION: The MTD of CPT-11 administered as a 30-minute IV infusion every 3 weeks is 600 mg/m2, with granulocytopenia being dose-limiting. At 350 mg/m2, diarrhea appeared dose-limiting, but high-dose loperamide reduced this toxicity and allowed dose escalation. For safety reasons, the recommended dose is presently 350 mg/m2 every 3 weeks; more experience must be gained to establish the feasibility of a higher dose in large multicentric phase II studies. However, when careful monitoring of gastrointestinal toxicities is possible, a higher dose of 500 mg/m2 could be recommended in good-risk patients. The activity of this agent in 5-FU-refractory colorectal carcinoma makes it unique and mandates expedited phase II testing.

Phase I and pharmacology study of flavone acetic acid administered two or three times weekly without alkalinization.

Flavone acetic acid (FAA, NSC 347512) is a synthetic flavonoid compound with a unique form of preclinical antitumor activity, but its mechanism of action is still not known. In an attempt to exploit the remarkable preclinical activity of this compound in such a way as to allow its use as a clinically useful agent, we performed a phase I and pharmacology study with frequent administration and no hyperhydration or alkalinization. Sixteen patients (9 men, 7 women) were given FAA as 6-h i.v. infusions 2 or 3 times a week (10 and 6 patients, respectively), at doses ranging from 2.5 to 8.1 g/m2. A total of 130 doses were administered during this study. Sedation, arterial hypotension, vomiting and diarrhea were the predominant toxicities observed at the highest dose (8.1 g/m2. One patient developed severe but reversible multiple organ failure. No treatment-related deaths occurred. Pharmacokinetics was linear for the doses studied, with peak plasma levels ranging from 39 to 449 micrograms/ml and a mean terminal half-life of 3.1 h. No drug accumulation was observed with this frequent-administration schedule. No objective response was observed. Three FAA infusions per week at 8.1 g/m2 could be recommended as an optimal and tolerable schedule.

Limited sampling models for simultaneous estimation of the pharmacokinetics of irinotecan and its active metabolite SN-38.

Irinotecan (CPT-11) is a novel topoisomerase I inhibitor with clinical activity in human malignancies. The objective of this study was to develop efficient limited sampling models (LSMs) to estimate simulataneously the area under the plasma concentration versus time curves (AUC) for both CPT-11 and its active metabolite SN-38. A total of 64 pharmacokinetic sets (> or = 24-h sampling) were obtained in phase I studies at doses ranging from 50 to 750 mg/m2 (0.5-h i.v. infusion). The patients were randomly assigned to a training data set (n = 32) and a test set (n = 32). Multiple linear regression analyses were used to determine the optimal LSMs based on the correlation coefficient (r), bias (MPE%, percentage of mean prediction error), and precision (RMSE%, percentage of root mean squared prediction error). Of these LSMs, the ones including maximal concentrations of CPT-11 (0.5 h, the end of the i.v. infusion) and metabolite SN-38 (approximately 1 h) were favored along with predictive precision and clinical constraints. Several bivariate models including a 6-h time point as the last sampling time (or 7 h) were found to be highly predictive of either the CPT-11 AUC or the SN-38 AUC. The chosen sampling time points were the ones that allowed the best compromise between the accurate determination of either compound alone with the same sampling times. The simultaneously best prediction of both CPT-11 and SN-38 AUCs was obtained with sampling time points harvested at 0.5, 1, and 6 h (or 7 h). With these sampling time points a trivariate model was selected for the determination of CPT-11 AUC namely, CPT-11 AUC (ng h ml-1) = 0.820 x C0.5h + 0.402 x C1h + 15.47 x C6h + 928, and a corresponding model was selected for the determination of metabolite AUC, i.e., SN-38 AUC (ng h ml-1) = 4.05 x C0.5h -0.81 x C1h + 23.01 x C6h - 69.78, where C(t) is the concentration in nanograms per milliliter of either compound at a given time t. These models performed well with the test data sets for CPT-11 AUC (r = 0.98, MPE% = -1.4, RMSE% = 13.9) and for SN-38 AUC (r = 0.95, MPE% = -6.5, RMSE% = 37.7). In addition to the determination of AUCs (and hence clearance), these models also allow the determination of the maximal concentrations of both compounds, which might be needed for pharmacodynamics studies.(ABSTRACT TRUNCATED AT 400 WORDS)

Factors involved in clinical pharmacology variability in oncology (review).

One of the major problems in cancer pharmacology is the prediction of the outcome of treatment in terms of both toxicity and tumor response. Indeed, why is a patient presenting a high toxicity at a standard dose? Also, why is another patient presenting a response to chemotherapy, while another does not respond, even if the same doses and drugs are used? The causes of variability in the response to chemotherapy can be classified in 2 classes, these related to the host, and those related to the tumor. Concerning the host, physiopathological parameters can influence the outcome of treatment e.g. age, sex, kidney and liver functions, plasma protein binding, concomitant treatments, and pharmacogenetics. With regard to the tumor, many factors can also influence chemotherapy e.g. tumor type, localization, volume, aggressiveness, dissemination stage, prior treatments (resistance), biological marker levels, and pharmacogenetic phenotype. Although some progress has been made in the past years concerning the clinical use of some factors responsible for the variability of pharmacological response in oncology, much remains to be done in order to adapt the treatment to a particular patient. Although pharmacogenetic phenotyping has been feasible for some anticancer drugs, this area deserves more effort in the future. Indeed, pharmacogenetic phenotyping of both the patient and the tumor will probably allow to tailor chemotherapy to an individual patient, in order to optimize the patient's chances to obtain a tumor response with minimum systemic toxicity.

Principal drug-metabolizing enzyme systems in L1210 leukemia sensitive or resistant to BCNU in vivo.

1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) resistance has been mostly studied in vitro. In an attempt to better understand BCNU resistance in the in vivo situation, we compared the principal drug-metabolizing enzyme systems in two L1210 leukemia lines, one sensitive and one resistant to BCNU (L1210/BCNU), passaged in vivo in mice. The following enzymes were assayed by immunoblotting: cytochromes P-450 (1A1/1A2, 2B1/2B2, 2C8-10, 2E1, 3A), epoxide hydrolase (EH) and glutathione S-transferase (GST-alpha, -mu and -pi). The following enzymes and cofactors were assayed fluorometrically or spectrophotometrically: 1-chloro-2-4 dinitrobenzene-GST (CDNB-GST), total glutathione (GSH), UDP-glucuronosyltransferase, beta-glucuronidase, sulfatase and sulfotransferase. Results showed that cytochrome P-450 1A1/1A2 was the only isoenzyme detected in both L1210 and L1210/BCNU. CDNB-GST activity was significantly higher in L1210/BCNU compared with L1210. The isoenzyme GST-alpha was more abundant in L1210/BCNU compared with L1210, whereas GST-pi was expressed less in the BCNU-resistant leukemia line. GST-mu was not detected in either L1210 leukemia lines. GSH levels were similar in the two L1210 lines. No significant difference was observed between the two leukemia lines for the conjugative enzymes UDP-glucuronosyltransferase and sulfotransferase, whereas their corresponding hydrolytic enzymes beta-glucuronidase and sulfatase were about two-fold lower in the BCNU-resistant leukemia line. Epoxide hydrolase was 1.3-fold higher in L1210/BCNU compared with L1210 and this level was about three-fold higher than in mouse liver. In conclusion, these studies showed the presence of cytochrome P-450 1A1/1A2 in the two L1210 leukemia lines studied, and indicated noteworthy differences between the two leukemia lines for many enzyme systems such as GST, beta-glucuronidase, sulfatase and epoxide hydrolase. These data are of importance to better understand the mechanisms of drug resistance to nitrosoureas in vivo.

Phase I/II trial of continuous infusion vinorelbine for advanced breast cancer.

PURPOSE: A phase I/II trial of vinorelbine (VRL) administered by continuous infusion (CIV) was conducted in advanced breast carcinoma (ABC) patients to determine the maximum-tolerated dose (MTD) and to evaluate the toxicity pattern and antitumor activity of this alternative administration schedule to the currently recommended weekly short intravenous (IV) administration. PATIENTS AND METHODS: Between February 1990 and July 1991, 64 consecutive, eligible patients with ABC were treated; 33 had received one or two previous palliative chemotherapy combinations and 31 had not received chemotherapy for metastatic disease. VRL was administered, after an initial IV bolus of 8 mg/m2 on day 1, by a 4-day CIV at five different 24-hour dose levels (DLs) to be repeated every 21 or 28 days: DL1, 5.5 mg/m2; DL2, 7 mg/m2; DL3, 8 mg/m2, DL4, 9 mg/m2; and DL5, 10 mg/m2. RESULTS: The limiting noncumulative toxicity was neutropenia, with the MTD established at 8 mg/m2 bolus plus 10 mg/m2/d for 4 days (total dose per cycle, 48 mg/m2). At DL3 and DL4, we observed mucositis (14% of patients; five percent of cycles > grade 2), alopecia, and asthenia. By contrast, neurotoxicity was minor. The toxicity was otherwise predictable and manageable. Pharmacokinetic data obtained at DL1 and DL3 showed a mean VRL plasma concentration of 967 +/- 331 ng/mL after the initial 8 mg/m2 IV bolus dose, which declined rapidly thereafter to reach mean steady-state levels of 12 ng/mL (n = 5) for the 30-mg/m2 dose and 8 ng/mL (n = 2) for the 40-mg/m2 dose. These levels were maintained over the 96-hour CIV. The mean residence time (MRT) was 29 +/- 7 hours (terminal half-life [t1/2], 23 hours), the total-body clearance (CL) was 24 +/- 11 L/hr/m2, and the volume of distribution at steady-state (Vss) was high at 1,832 +/- 359 L/m2. Two patients achieved a complete response (CR) and 21 a partial response (PR), for an objective response rate of 36% (95% confidence interval [Cl], 23 to 49). The median duration of response was 6 months. The median survival duration was 24 months (range, 3 to 37). A relationship between given dose-intensity and objective response rate was found, with an overall response (OR) rate of 13.3% (two of 15) for 8 to 10 mg/m2/wk, 35.4% (11 of 31) for 10 to 12 mg/m2/wk, and 55.5% (10 of 18) for 12 to 14.5 mg/m2/wk. CONCLUSION: This trial, while confirming VRL activity in ABC, shows the feasability of a CIV administration schedule. A decrease of the administrated total dose per 3- to 4-week cycle to less than the weekly schedule with the same therapeutic activity suggests a better therapeutic index. The data are also suggestive of a dose-response relationship and a dose-intensity/activity correlation.