Evaluation of amidoxime derivatives as prodrug candidates of potent bis-cationic antimalarials.

Plasmodium falciparum is responsible for most of the cases of malaria and its resistance to established antimalarial drugs is a major issue. Thus, new chemotherapies are needed to fight the emerging multi-drug resistance of P. falciparum malaria, like choline analogues targeting plasmodial phospholipidic metabolism. Here we describe the synthesis of amidoxime derivatives as prodrug candidates of reverse-benzamidines and hybrid compounds able to mimic choline, as well as the design of a new series of asymmetrical bis-cationic compounds. Bioconversion studies were conducted on amidoximes in asymmetrical series and showed that amidoxime prodrug strategy could be applied on C-alkylamidine moieties, like benzamidines and that N-substituents did not alter the bioconversion of amidoximes. The antimalarial activity of the three series of compounds was evaluated in vitro against P. falciparum and in vivo against P. vinckei petteri in mice.

Characterization of a new anticancer agent, EAPB0203, and its main metabolites: nuclear magnetic resonance and liquid chromatography-mass spectrometry studies.

The present study was conducted to assess the structures of the main unknown oxygenated metabolites of EAPB0203. The first step was to assign all the (1)H and (13)C NMR of both EAPB0203 and its demethylated metabolite (EAPB0202) to the corresponding atoms in their molecular structures and to elucidate the fragmentation pathways for the [M + H](+) ions of these compounds using high-resolution mass spectrometry (MS). MS/MS spectra showed that both protonated molecules possessing an even number of electrons were unexpectedly losing radicals such as H(•), CH(3)(•), or even C(7)H(7)(•) giving stable radical cations. In vitro metabolism studies were investigated in rat and dog liver microsomes and in the filamentous fungus Cunninghamella elegans. Structural elucidation of six oxygenated metabolites was performed based on the following: (i) their fragmentation pathways in liquid chromatography-MS/MS (LC-MS/MS) analyses; (ii) comparison of their changes in their molecular masses and fragment ions with those of the parent drugs; and (iii) the results of online H/D exchange experiments that provided additional evidence in differentiating hydoxylated metabolites from N-oxides. Structures of the metabolites were elucidated by LC-MS/MS and comparison with synthetic standards; structures of these standards were confirmed using one- and two-dimensional (1)H NMR spectroscopies.

Intraperitoneal administration of novel doxorubicin loaded polymeric delivery systems against peritoneal carcinomatosis: experimental study in a murine model of ovarian cancer.

OBJECTIVE: Peritoneal spread is an adverse outcome in ovarian cancer. Despite clinical efficiency, intraperitoneal (i.p.) chemotherapy after cytoreductive surgery is associated with high systemic and local toxicity. Two polymer-drug delivery systems (P-HYD1-DOX and P-HYD2-DOX) were developed for i.p. administration by conjugating doxorubicin (DOX) to a poly(l-Lysine citramide) polymer carrier with a hydrazone-based degradable spacer. The aim of this study was to assess the antitumoral efficacy of these two conjugates in a xenograft model of human ovarian carcinomatosis. METHODS: Peritoneal carcinomatosis was generated in athymic mice by i.p. injection of SKOV3-Luc cells. Free DOX, P-HYD1-DOX and P-HYD2-DOX solutions were administered i.p. at the same dose of 10 mg/kg (DOX eq.). For each treatment, tumor load and therapeutic efficacy were compared to untreated mice and assessed by bioluminescence imaging and survival rates. Toxicity profiles in each group and biodistribution of P-HYD2-DOX after i.p. administration were also determined. RESULTS: P-HYD-1-DOX and P-HYD-2-DOX demonstrated significant antitumoral efficacy against peritoneal carcinomatosis. Compared to untreated group, P-HYD1-DOX improved median survival times from 58 to 105 days. For P-HYD2-DOX, median survival was not reached after a follow-up of 120 days. Bioluminescence showed high efficacy of P-HYD-2-DOX compared to free DOX but the difference was not significant. Biodistribution study confirmed that free and active DOX were successively released from P-HYD2-DOX in vivo. P-HYD-DOX conjugates were well tolerated by mice after i.p. injection. CONCLUSION: P-HYD-DOX conjugates demonstrated significant activity against peritoneal carcinomatosis in a xenograft model of ovarian carcinomatosis and their ability to release active DOX in i.p. deposits and tumor. These features are of clinical interest for i.p. administration in the treatment of ovarian peritoneal carcinomatosis after cytoreductive surgery.

Metabolism and pharmacokinetics of EAPB0203 and EAPB0503, two imidazoquinoxaline compounds previously shown to have antitumoral activity on melanoma and T-lymphomas.

For several years, our group has been developing quinoxalinic compounds. Two of them, N-methyl-1-(2-phenethyl)imidazo[1,2-a]quinoxalin-4-amine (EAPB0203) and 1-(3-methoxyphenyl)-N-methylimidazo[1,2-a]quinoxalin-4-amine (EAPB0503), have emerged as the most promising anticancer drugs. In the present work, we determined metabolism pathways using liver microsomes from four mammalian species including human. We identified the cytochrome P450 isoform(s) involved in the metabolism and then investigated the pharmacokinetics and metabolism of EAPB0203 and EAPB0503 in rat after intravenous and intraperitoneal administration. Biotransformation of the compounds involved demethylation and hydroxylation reactions. Rat and dog metabolized the compounds at a higher rate than mouse and human. In all species, CYP1A1/2 and CYP3A isoforms were the predominant enzymes responsible for the metabolism. From human liver microsomes, unbound intrinsic clearances were approximately 56 ml/(min · g) protein. EAPB0203 and EAPB0503 were extensively bound to human plasma proteins, mainly human serum albumin (HSA) (∼98-99.5%). Thus, HSA could act as carrier of these compounds in human plasma. Scatchard plots showed patterns in which the plots yielded upwardly convex hyperbolic curves. On the basis of the Hill coefficients, there appears to be interaction between the binding sites of HSA, suggesting positive cooperativity. The main in vitro metabolites were identified in vivo. Total clearances of EAPB0203 and EAPB0503 [3.2 and 2.2 l/(h · kg), respectively] were notably lower than the typical cardiac plasma output in rat. The large volumes of distribution of these compounds (4.3 l/kg for EAPB0203 and 2.5 l/kg for EAPB0503) were consistent with extensive tissue binding. After intraperitoneal administration, bioavailability was 22.7% for EAPB0203 and 35% for EAPB0503 and a significant hepatic first-pass effect occurred.

Quantitation of SAR97276 in mouse tissues by rapid resolution liquid chromatography-mass spectrometry.

1,12-Bis[5-(2-hydroxyethyl)-4-methyl-1,3-thiazol-3-ium]dodecane dibromide (SAR97276, T3) is a new antimalarial drug, which is currently being evaluated in clinical trials for severe malaria. Drug accumulation inside the parasite and a dual mechanism of action are a major strength of this compound, as it could help delay the development of resistance. The purpose of this article was to develop a rapid resolution LC-MS method for quantifying SAR97276 in mouse tissues. The LC system consisted of Zorbax Eclipse XDB C8 (1.8 microm, 50 x 4.6 mm, 60 degrees C) column. Elution with a gradient mobile phase consisting of ACN-trimethylamine-formate buffer (pH 3) at a flow rate of 1 mL/min yielded sharp, utmost-resolved peaks within 2 min. Tissue samples were powdered under liquid nitrogen. After protein precipitation with citric acid, SPE using WCX cartridges was used for sample preparation. There was no influence of the matrix on the detection of either SAR97276 or the IS. Assay precision was <13% and accuracy was 90-107%. The lower LOQs were 3.3 microg/kg in brain and 33 microg/kg in liver and heart. This newly developed method was used to study the tissue distribution of SAR97276 in mouse as part of the ongoing development of SAR97276.

Quantitation of imidazo[1,2-a]quinoxaline derivatives in human and rat plasma using LC/ESI-MS.

Since several years, our group developed quinoxalinic compounds. Among the synthesized compounds, in the imidazo[1,2-a]quinoxaline series, EAPB0203 has shown interesting activities both on melanoma and lymphoma. The structure of EAPB0203 has been modulated and a new compound, EAPB0503, exhibits an in vitro cytotoxic activity on melanoma cancer cell line 7-9 times higher than EAPB0203. We validated an LC/ESI-MS method to simultaneously quantify EAPB0503 and its metabolite EAPB0603 in human and rat plasma. Chromatography was performed on a C8 Zorbax eclipse XDB column with a mobile phase consisting of acetronitrile and formate buffer gradient elution. LC-MS data were acquired in SIM mode at m/z 305, 291, and 303 for EAPB0503, EAPB0603, and the internal standard, respectively. The drug/internal standard peak area ratios were linked via quadratic relationships to concentrations (low range: 5-300 microg/L, high range: 100-1000 microg/L). The method is precise (precision, < or = 14%) and accurate (recovery, 92-113%). Mean extraction efficiencies, > 72% for each analyte, were obtained. The lower LOQs were 5 microg/L. This highly specific and sensitive method was successfully used to investigate plasma concentrations of EAPB0503 and EAPB0603 in a pharmacokinetic study carried out in rat and would also be useful in clinical trials at a later stage.

Pharmacokinetics of artesunate in the domestic pig.

OBJECTIVES: The aim was to study the pharmacokinetic profile of artesunate and its metabolite dihydroartemisinin (DHA) in a pig model. METHODS: Thirteen pigs received either intravenous (iv) or intramuscular (im) artesunate (60 mg), with the alternative preparation given 24 h later in an open crossover design. Five of them also received an additional intra-arterial (ia) artesunate dose (60 mg). The plasma concentrations of artesunate and DHA were determined by high-performance liquid chromatography with electrochemical detection. Population modelling was performed with NONMEM, using a two-compartment model. RESULTS: Plasma concentration-time profiles were comparable to those observed in humans, with a rapid and biphasic decline for both artesunate and DHA. Following an iv bolus, artesunate had a median maximum plasma concentration (C(max)) of 13.8 microM [interquartile range (IQR), 10.4-22.1 microM], elimination half-life (t(1/2)) = 18 min (IQR, 16-22 min), total plasma clearance (CL) = 5.58 L/h/kg (IQR, 3.31-5.91 L/h/kg) and volume of distribution (V(d)) = 1.85 L/kg (IQR, 1.27-3.20 L/kg). The median C(max) value for DHA was 3.30 microM (IQR, 2.08-5.95 microM), t(1/2) = 26 min (IQR, 23-31 min), CL/Fm = 4.37 L/h/kg (IQR, 3.29-6.87 L/h/kg) and V(d)/Fm = 2.56 L/kg (IQR, 1.93-4.49 L/kg). Artesunate and DHA pharmacokinetic parameters were similar after ia administration. Following im dosing, median artesunate C(max) was 4.81 microM (IQR, 3.74-5.40 microM), t(1/2) = 18 min (IQR, 16-28 min), CL = 4.37 L/h/kg (IQR, 4.13-4.68 L/h/kg) and V(d) = 2.07 L/kg (IQR, 1.83-2.79 L/kg); the bioavailability was 100%. For DHA, median C(max) was 1.43 microM (IQR, 1.00-1.92 microM), t(1/2) = 27 min (IQR, 25-37 min), CL/Fm = 4.68 L/h/kg (IQR, 3.35-6.73 L/h/kg) and V(d)/Fm = 3.31 L/kg (IQR, 2.89-4.27 L/kg). CONCLUSIONS: The pharmacokinetic properties of artesunate and DHA in pigs were similar to those reported in humans, suggesting that the swine model is suitable for determining the preclinical pharmacokinetics of artemisinin derivatives.

HPLC with UV or mass spectrometric detection for quantifying endogenous uracil and dihydrouracil in human plasma.

BACKGROUND: We developed and compared 2 different methods for quantifying uracil (U) and dihydrouracil (UH(2)) in BSA and human plasma. Special attention was paid to the selectivity/specificity and the absence of a matrix effect. The UH(2)/U ratio is intended as a biomarker to identify patients with deficiency in 5-fluorouracil metabolism. METHODS: We quantified U and UH(2) with 2 liquid chromatography methods after solid-phase extraction, one with UV detection (LC-UV) and the other with mass spectrometric detection (LC-MS). We selected 2 internal standards to prevent the risk of interferences. Separation was achieved with a Waters Atlantis dC18 column (LC-MS) or a Waters SymmetryShield RP18 column connected with an Atlantis dC18 (LC-UV). Mass spectrometric data were acquired in single-ion monitoring mode. RESULTS: Assay imprecision in BSA solution was <15% (LC-UV) and <12% (LC-MS); in plasma, assay imprecision was <9.5% and <9.0%, respectively. Recoveries were 88.2%-110% (LC-UV) and 94.8%-107% (LC-MS). Extraction efficiencies were >or=89.0%. In BSA, the lower limits of quantification for U and UH(2) were 2.5 microg/L and 6.25 microg/L, respectively, for the LC-UV method and 2.5 microg/L and 3.1 microg/L for LC-MS. The corresponding values in plasma were 11.6 microg/L and 21.5 microg/L, and 4.1 microg/L and 12.1 microg/L. CONCLUSIONS: To estimate endogenous U and UH(2) concentrations and their ratio, we recommend the use of a drug-free human plasma pool in which baseline U and UH(2) concentrations have previously been measured with the standard-addition method. Our LC-MS method, which has the better test performance and is useful for measuring UH(2)/U ratios in cancer patients, is preferred when this equipment is available.

In vitro and in vivo anti-tumoral activities of imidazo[1,2-a]quinoxaline, imidazo[1,5-a]quinoxaline, and pyrazolo[1,5-a]quinoxaline derivatives.

Imidazoquinoxaline and pyrazoloquinoxaline derivatives, analogues of imiquimod, were synthesized, and their in vitro cytotoxic and pharmacodynamic activities were evaluated. In vitro cytotoxicity studies were assessed against melanoma (A375, M4Be, RPMI-7591), colon (LS174T), breast (MCF7), and lymphoma (Raji) human cancer cell lines. In vivo studies were carried out in M4Be xenografted athymic mice. EAPB0103, EAPB0201, EAPB0202, and EAPB0203 showed significant in vitro activities against A375 compared to fotemustine and imiquimod used as references. These compounds were 6-110 and 2-45 times more active than fotemustine and imiquimod, respectively. EAPB0203 bearing phenethyl as substituent at position 1 and methylamine at position 4 showed the highest activity. EAPB0203 has also a more potent cytotoxic activity than imiquimod and fotemustine in M4Be and RPMI-7591 and interesting cytotoxic activity in other tumor cell lines tested. In vivo, EAPB0203 treatment schedules caused a significant decrease in tumor size compared to vehicle control and fotemustine treatments.

A limited sampling strategy to estimate individual pharmacokinetic parameters of methotrexate in children with acute lymphoblastic leukemia.

PURPOSE: The objectives of this study were to characterize the population pharmacokinetics of MTX in patients with acute lymphoblastic leukemia (ALL) with ages ranging from 2 to 16 years and to propose a limited sampling strategy to estimate individual pharmacokinetic parameters. METHODS: Seventy-nine children were enrolled in this study; they received 1-4 courses of chemotherapy. MTX was administered at a dose of 5 g/m2. MTX population parameters were estimated from 61 patients (231 courses; age range: 2-16 years). The data were analyzed by nonlinear mixed-effect modeling with use of a two-compartment structural model. The interoccasion variability was taken into account in the model. Eighteen additional patients (70 courses) were used to evaluate the predictive performances of the Bayesian approach and to devise a limited sampling strategy. RESULTS: The following population parameters were obtained: total clearance (CL) = 8.8 l/h (inter-individual variability: 43%), initial volume of distribution (V1) = 17.3 l (48%), k12 = 0.0225 h(-1) (41%), and k21 = 0.0629 h(-1) (24%). The inter-individual variability in the initial volume of distribution was partially explained by the fact that this parameter was weight-dependent. Intercourse variability was limited, with a mean variation of 13.2%. The protocol involving two sampling times, 24 and 48 h after the beginning of infusion, allows precise and accurate determination of individual pharmacokinetic parameters and consequently, it was possible to predict the time at which the MTX concentration reached the predicted threshold (0.2 microM) below which the administration of folinic acid could be stopped. CONCLUSION: The results of this study combine the relationships between the pharmacokinetic parameters of MTX and patient covariates that may be useful for dose adjustment, with a convenient sampling procedure that may aid in optimizing pediatric patient care.

A limited sampling strategy to estimate the pharmacokinetic parameters of irinotecan and its active metabolite, SN-38, in patients with metastatic digestive cancer receiving the FOLFIRI regimen.

In this study we propose for the first time a limited sampling strategy to estimate the individual pharmacokinetic parameters of both irinotecan and SN-38 in patients treated with the irinotecan plus 5-fluorouracil (FOLFIRI) regimen. The pharmacokinetics of irinotecan and SN-38 were studied in 74 patients with advanced inoperable digestive cancer. Plasma concentrations were taken during and up to the 42 h following a 90-min infusion of irinotecan (180-225 mg/m(2)). Data splitting was used to create model-building and validation data sets, and data were analysed with the NONMEM program. The disposition of SN-38 was dependent on the disposition of irinotecan. The estimated pharmacokinetic parameters of irinotecan [terminal half-life (t(1/2)), 11.5 h; total clearance (CL), 25.0 l h(-1); area under curve (AUC), 14.9 mg x h l(-1)] and SN-38 (terminal t(1/2), 32.2 h; AUC, 0.42 mg x h l(-1)) were similar to those determined in other studies. The protocol involving two sampling times, at 1 and 24 h following the beginning of the infusion, allowed for a precise and accurate determination of the individual pharmacokinetic parameters of the two drugs. The limited sampling strategy developed in this study ought to facilitate future studies on the pharmacology and toxicity of irinotecan-based therapy.

Biodistribution of doxorubicin-alkylated poly(L-lysine citramide imide) conjugates in an experimental model of peritoneal carcinomatosis after intraperitoneal administration.

Peritoneal spread is a common manifestation in ovarian and gastrointestinal cancer. Intraperitoneal (i.p.) chemotherapy after cytoreductive surgery is associated with high systemic or local toxicity. A macromolecular drug delivery system was evaluated with the aim of improving the therapeutic index of i.p. chemotherapy. Peritoneal carcinomatosis was generated in BDIX rats (n=55) by i.p. injection of 2 million DHDK12 cells. Fourteen days later, doxorubicin (DOX) and two DOX-alkylated poly(L-lysine citramide imide) conjugates bearing 9.5% and 20.5% (w/w) chemically bound drug, respectively, were given i.p. to rats at a single 2 mg DOX/kg dose. Free and polymer-bound DOX were assessed in plasma, peritoneal fluid, abdominal tissues and heart, 15 min, 2, 6, 24, 48 and 168 h after injection. According to pharmacokinetic profiles, the peritoneal fluid areas under the concentration versus time curves (AUCs) were 2 and 2.6 times greater for the conjugates (P-DOX20 and P-DOX10, respectively) than for the free drug, respectively. Conjugates crossed the peritoneal barrier slower than the free drug. For the tumor, AUCs were, respectively, 3 and 7 times higher for the conjugates than for free DOX. The elimination half-lives of the conjugates were higher than that calculated for the free drug. Only very small concentrations were detected in peripheral organs and in the heart. In contrast, significant retention and accumulation of the conjugates were found in abdominal organs, particularly in the tumor. There was no sign of macroscopic chemical peritonitis after injection of the polymer-DOX conjugates. In conclusion, the conjugates have higher elimination half-lives than free DOX and were preferentially retained in abdominal organs and in the peritoneal carcinomatosis. This feature is of clinical interest to target tumor deposits.

Pharmacokinetics and pharmacodynamics of irinotecan and its metabolites from plasma and saliva data in patients with metastatic digestive cancer receiving Folfiri regimen.

PURPOSE: Irinotecan is extensively metabolized into at least four compounds and previous pharmacokinetic-pharmacodynamic studies have given varying results. We hypothesized that saliva, a noninvasive, safe and painless biological sampling process, could be a good predictor of the behavior of irinotecan and its metabolites. METHODS: Thirty-five patients with metastatic digestive cancer were treated with a Folfiri regimen every 2 weeks. The irinotecan-administered dose was 180 mg/m(2); 17 patients participated in a dose-escalating study. Irinotecan and its metabolites (SN-38, SN-38G, APC, NPC) were quantified in plasma and saliva by high-performance liquid chromatography with fluorescence detection. RESULTS: The mean irinotecan systemic clearance and steady-state volume of distribution values were 14.3 l/h/m(2) and 211 l/m(2), respectively. The intrapatient variability (22-28%) was far lower than the interindividual variability (33-88%). Age and weight were the two physiological parameters that influenced drug disposition. For irinotecan, SN-38, APC and NPC, similar pharmacokinetic profiles were observed from plasma and saliva data. The saliva/plasma AUC ratios averaged 1 for irinotecan, 0.3 for SN-38, 0.17 for APC and 0.27 for NPC. Neutropenia, diarrhea and nausea were the main toxicities encountered. From both plasma and saliva data, the percentage decrease in neutrophil count appeared to be related to irinotecan and SN-38 AUCs. CONCLUSIONS: All these findings provide a rationale for an individual adaptation of irinotecan dosing. In case of difficult venous access, the titration of irinotecan and of its active metabolite SN-38 in saliva may prove relevant.

Phase II study of melphalan as a single-agent infused over a 24-hour period with individual adapted dosing in patients with recurrent epithelial ovarian cancer.

The current study was performed to evaluate the efficacy and toxicity of a chemotherapy treatment using melphalan administered over a 24-h period with individual adapted dosing in advanced ovarian cancer. Melphalan was infused intravenously with an automatic infusion pump over a 24-h period. The schedule was repeated every 28 days for a maximum of 6 cycles. The initial administered dose was 25 mg/m(2). Drug adjustment was then made using a population approach, with the aim of constraining the overall area under the plasma concentration-time curve within 2.0-2.5 mg . h/l. A total of 96 courses of chemotherapy was administered. The toxicity profile did not differ greatly from that reported after a 1-h infusion and was acceptable in such a heavily pretreated patient population. Twenty-five patients were assessable for response and survival. Four (16%) partial responses were observed, which lasted 9, 10, 13 and 37 months, respectively. Three patients experienced stable disease during 8, 14 and 22 months, respectively. The 1- and 2-year survival rates were 18% and 6%, respectively. At the time of analysis, two patients remained alive with progressive disease at 22 and 37 months, respectively. In conclusion, melphalan administered over a 24-h period in platinum- and taxane-resistant ovarian cancer patients appeared to provide some clinical benefits with manageable toxicity. Based on these results, future studies comparing melphalan administered over a 24-h period and oral melphalan administrations will be scheduled.

In vitro cytotoxic effect of melphalan and pilot phase II study in hormone-refractory prostate cancer.

The objective of this study was to determine the impact of single versus sequential exposure to melphalan on the proliferation of an androgen-independent prostate cell line, PC-3, and to report the results of a pilot phase II study. For exposure to a single bolus dose, the doses were added at the start of the study and cell culture was continued for 96 h. For sequential exposure, 1/9 of the dose was added every 1.5 h over 12 h, followed by cell culture for 84 h. Cell growth inhibition was determined by the MTT assay. The clinical study was carried out on 14 patients with advanced prostate cancer. Melphalan was infused over a 24-h period. The sequential-dose schedule was more effective than the single-dose exposure, IC50 values: 0.074 versus 0.77/microg/ml. Out of the 14 patients (42 courses) enrolled into the study, two patients were removed within the first 2 weeks because of rapid disease progression. The toxicity profile did not differ greatly from that reported after a 1-h infusion. Four PR and two SD were observed. The median survival of the twelve patients was 23 weeks. Melphalan administered over a 24-h period to patients with androgen-independent prostate cancer appeared to provide some clinical benefits with manageable toxicity.

Liquid chromatography-electrospray mass spectrometry determination of free and total concentrations of ropivacaine in human plasma.

A specific and sensitive liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) method was developed for the determination of free and total ropivacaine in human plasma. The work-up procedure involved a simple precipitation of plasma proteins with methanol. Etidocaine served as the internal standard. After microscale equilibrium-dialysis, measurement of free ropivacaine levels was performed after direct injection of the dialysate into the chromatograph. The system used a Zorbax eclipse XD8 C8 analytical column packed with 5 microm diameter particles as the stationary phase. The mobile phase consisted of a 15-min gradient (mobile phase A: 0.05% (v/v) trimethylamine in acetonitrile, mobile phase B: 2mM ammonium formate buffer (pH 3)). Mass spectrometric data were acquired in single ion monitoring mode at m/z 275 for ropivacaine and m/z 277 for etidocaine. The drug/internal standard peak area ratios (plasma) or peak areas (dialysate) were linked via a quadratic relationship to concentrations. Precision ranged from 1 to 7.6% accuracy was between 92.6 and 109%. The lower limits of quantitation were 1 microg/l in plasma and 2 microg/l in the dialysate. This method was found suitable for the analysis of plasma samples collected during a clinical trial performed in 30 infants undergoing epidural anaesthesia or continuous psoas compartment block.

Liquid chromatography-electrospray mass spectrometry determination of ibogaine and noribogaine in human plasma and whole blood. Application to a poisoning involving Tabernanthe iboga root.

A liquid chromatography/electrospray ionization mass spectrometry (LC-ESI-MS) method was developed for the first time for the determination of ibogaine and noribogaine in human plasma and whole blood. The method involved solid phase extraction of the compounds and the internal standard (fluorescein) from the two matrices using OasisHLB columns. LC separation was performed on a Zorbax eclipse XD8 C8 column (5 microm) with a mobile phase of acetonitrile containing 0.02% (v/v) trimethylamine and 2mM ammonium formate buffer. MS data were acquired in single ion monitoring mode at m/z 311.2, 297.2 and 332.5 for ibogaine, noribogaine and fluorescein, respectively. The drug/internal standard peak area ratios were linked via a quadratic relationship to plasma (0.89-179 microg/l for ibogaine; 1-200 microg/l for noribogaine) and to whole blood concentrations (1.78-358 microg/kg for ibogaine; 2-400 microg/kg for noribogaine). Precision ranged from 4.5 to 13% and accuracy was 89-102%. Dilution of the samples had no influence on the performance of the method. Extraction recoveries were > or =94% in plasma and > or =57% in whole blood. The lower limits of quantitation were 0.89 microg/l for ibogaine and 1 microg/l for noribogaine in plasma, and 1.78 microg/kg for ibogaine and 2 microg/kg for noribogaine in whole blood. In frozen plasma samples, the two drugs were stable for at least 1 year. In blood, ibogaine and noribogaine were stable for 4h at 4 degrees C and 20 degrees C and 2 months at -20 degrees C. The method was successfully used for the analysis of a poisoning involving Tabernanthe iboga root.

Distribution of ibogaine and noribogaine in a man following a poisoning involving root bark of the Tabernanthe iboga shrub.

In the present paper, we report for the first time the tissue distribution of ibogaine and noribogaine, the main metabolite of ibogaine, in a 48-year-old Caucasian male, with a history of drug abuse, found dead at his home after a poisoning involving the ingestion of root bark from the shrub Tabernanthe iboga. Ibogaine and noribogaine were quantified in tissues and fluids using a fully validated liquid chromatography-electrospray mass spectrometry method. Apart from cardiac tissue, ibogaine and noribogaine were identified in all matrices investigated. The highest concentrations were found in spleen, liver, brain, and lung. The tissue/subclavian blood concentration ratios averaged 1.78, 3.75, 1.16, and 4.64 for ibogaine and 0.83, 2.43, 0.90, and 2.69 for noribogaine for spleen, liver, brain, and lung, respectively. Very low concentrations of the two drugs were found in the prostatic tissue. Both ibogaine and noribogaine are secreted in the bile and cross the blood-brain barrier. Four other compounds were detected in most of the studied matrices. One of them was identified as ibogamine. Unfortunately, we were not able to positively identify the other three compounds because of the unavailability of reference substances. Two of them could possibly be attributed to the following oxidation products: iboluteine and desmethoxyiboluteine. The third compound could be ibogaline.

Inter- and intra-individual variability in transdermal fentanyl absorption in cancer pain patients.

A transdermal therapeutic system (TTS) is recommended for use in chronic cancer pain, particularly in the advanced stages. The aim of this trial was to study intra- and interindividual variabilities in fentanyl transdermal absorption and investigate physiological and clinical parameters that can influence the absorption in patients treated using a TTS for moderate to severe cancer pain. The study group consisted of 108 patients (71 men and 37 women; mean age, 61.3 years) with chronic cancer pain. A total of 507 patches were analysed. The TTSs used to administer fentanyl were removed after a 72-h period. The amount of fentanyl remaining in the patches was determined using a high-performance liquid chromatography method with ultraviolet detection. Depending on the analgesic requirements of the patient, the dose of fentanyl administered by TTS ranged from 25 to 500 microg/h. The study period was 6 months. Large interindividual variability in the amount of remaining fentanyl in the patches occurred. For 58.1% of patches, absorption was 60 to 84%; for 33.2% of them, it was lower; and for 8.8%, it was higher than this range. The intra-individual variability ranged from 2.8 to 75.1%. The bioavailability of fentanyl was statistically different according to patient age. Patients >75 years of age absorbed 50% of the fentanyl during the selected 72-h period, whereas patients <65 years absorbed 66%. Moreover, there is a significant difference in the percentage of absorbed fentanyl according to the type of cancer. The absorption was higher in patients with breast or digestive cancer than in those with lung cancer. Hyperhidrosis, hypertrichosis and the localization of patches on the skin did not influence bioavailability. For the entire group, transdermal fentanyl treatment provided good to excellent pain relief in the majority of patients.

Quantitation of ibogaine and 12-hydroxyibogamine in human plasma by liquid chromatography with fluorimetric detection.

A high-performance liquid chromatographic (HPLC) method with fluorimetric detection was developed for the simultaneous determination of ibogaine and noribogaine in human plasma using fluorescein as internal standard. This method involved a solid phase extraction of the compounds from plasma using N-vinylpyrrolidone-divinybenzene copolymer cartridges. Separation of the three analytes was performed on a reversed-phase Supelcosil C18 analytical column (75 mm x 4.6mm i.d., 3 microm particle size). The excitation wavelength was set at 230 nm for the first 15.8min and then at 440 nm for the following 14.2 min; the emission wavelength was set at 336 nm for the first 15.8 min and then at 514 nm for the following 14.2 min. Obtained from the method validation, inter-assay precision was 6.0-12.5% and accuracy was 95.4-104%. The extraction efficiencies of the assay were higher than 94% and were constant across the calibration range. The lower limits of quantitation were 0.89 ng/ml for ibogaine and 1 ng/ml for noribogaine; at these levels, precision was < or =17% and accuracy was 95-105%. In this paper, extensive stability testing was undertaken using a wide range of storage conditions. Special attention must be paid to sample handling to avoid light degradation of the compounds.