Population pharmacokinetic and dynamic analysis of the topoisomerase I inhibitor lurtotecan in phase II studies.

Population pharmacokinetic-dynamic analysis was prospectively integrated in a broad phase II program of lurtotecan (GI147211), a novel camptothecin derived topoisomerase I inhibitor, to determine the population pharmacokinetic profile in a larger population, to estimate individual pharmacokinetic parameters and to investigate relationships with clinical outcome. A sparse sampling method was applied during course one, which involved two sampling time-points. A Bayesian algorithm was used to estimate individual pharmacokinetic parameters, in particular total plasma clearance (CL) and volume of distribution. In total, samples were collected of 109 (63%) of 173 patients. Pharmacokinetic-dynamic evaluation could be carried out successfully in 85 (78%) of the sampled patients. CL of lurtotecan showed substantial variability (mean 87 +/- 28 L/h) and was of the same magnitude as in the phase I studies where full pharmacokinetic curves were used. Residual variability in the population estimate of CL was 9.9%. No significant relationships were observed between exposure parameters and toxicity nor likelihood of tumor response, however the latter relationship may well have been obscured by the heterogeneity of the studied population. Prospective implementation of large scale population pharmacokinetic-dynamic analysis is feasible and important to establish whether interpatient variability in drug exposure is a major determinant of toxicity or activity.

Phase I/pharmacokinetic study of the topoisomerase I inhibitor GG211 administered as a 21-day continuous infusion.

BACKGROUND: Preclinical results support a prolonged schedule of administration for topoisomerase I inhibitors, and we have previously demonstrated the safety and activity of the novel water-soluble topoisomerase I inhibitor GG211 when given as a 72-hour continuous infusion to cancer patients. PATIENTS AND METHODS: In a three-center international phase I trial, 38 patients received GG211 doses from 0.3 to 0.5 mg/m2/day by continuous intravenous infusions for seven, 14, and 21 days. Patients' median performance status was 1; nearly half had colorectal cancer, and 35 patients had prior chemotherapy. RESULTS: The first patient cohort received 0.3 mg/m2/day for seven days with no significant toxicities. Subsequent cohorts received continuous infusions for 14 and 21 days at this dose level with only mild myelosuppression noted. Dose-escalation on the 21-day schedule was then performed. No dose-limiting toxicity occurred at the 0.4 mg/m2/day dose level. Thrombocytopenia was dose-limiting with 0.5 mg/m2/day dosing but was not cumulative. Other grade 3 4 toxicities included neutropenia, nausea, vomiting, diarrhea, and fatigue. Partial responses occurred with 21-day infusion in two patients with breast and ovarian cancer at the 0.3 and 0.4 mg/m2/day dose levels, respectively. Mean GG211 lactone Css ranged from 0.17 to 0.64 ng/ml. CONCLUSION: The maximum tolerated dose of GG211 administered as a 21-day continuous infusion is 0.4 mg/m2/day with antitumor activity noted at tolerable doses.

Determination of remifentanil in human heparinised whole blood by tandem mass spectrometry with short-column separation.

For the use in pharmacokinetic studies, a fast and sensitive assay method was developed for the determination of remifentanil in human heparinised whole blood samples of 0.5 ml. The assay method is based on tandem mass spectrometry detection (LC-MS/MS). The limit of quantification is 0.1 ng/ml and linear up to 50 ng/ml. The precision, accuracy, recovery and applicability were found to be adequate for pharmacokinetic studies.

Pharmacokinetics of amiloride after inhalation and oral administration in adolescents and adults with cystic fibrosis.

STUDY OBJECTIVE: To compare the pharmacokinetics and systemic exposure of nebulized and oral amiloride in adolescents and adults with mild to moderate cystic fibrosis (CF). DESIGN: Open-label, randomized, two-way crossover, single-dose pharmacokinetic study. SETTING: University hospital clinical research unit. PATIENTS: Nine adolescents and 10 adults with mild to moderate CF (forced expiratory volume in 1 sec > or = 50% predicted, Brasfield score > or = 15). INTERVENTIONS: Patients received amiloride solution orally (10 mg of amiloride 1-mg/ml solution) and by inhalation [4.5 ml amiloride of 1-mg/ml solution in 12% saline (approximately 3.8 mmol/L) by DeVilbiss 646 nebulizer] during two study phases separated by a 7- to 28-day washout period. Serial blood and urine samples were collected for 48 and 72 hours, respectively. MEASUREMENTS AND MAIN RESULTS: After oral dosing, the mean +/- SD maximum peak concentration (Cmax) was 20.6 +/- 10.0 ng/ml at 3.2 +/- 1.2 hours in adults and 21.7 +/- 4.88 at 2.9 +/- 0.6 hours in the adolescents. Mean area under the concentration-time curve (AUC) from time zero to infinity hours was 275 +/- 115 and 254 +/- 60 ng.hr/ml in the adult and adolescent groups; half-life was 16.0 +/- 0.7 and 13.4 +/- 1.4 hours, respectively. After nebulization, 14 of 19 subjects exhibited two concentration peaks (Cmax1 and Cmax2) with mean values of 1.57 +/- 1.67 ng/ml at 0.5 +/- 0.2 hours and 1.37 +/- 1.21 ng/ml at 4.0 +/- 1.0 hours for adults, and 1.49 +/- 0.99 ng/ml at 0.5 +/- 0.1 hours and 1.52 +/- 0.81 ng/ml at 3.3 +/- 0.5 hours for adolescents. Estimated mean +/- SD dose nebulized was 1.91 +/- 0.66 and 2.28 +/- 0.30 mg in the adult and adolescent groups, respectively. Mean +/- SD AUC from time zero to the last measurable plasma amiloride concentration after inhalation was 14.4 +/- 17.6 and 15.4 +/- 10.1 ng.hr/ml in the adults and adolescents. No significant adverse events occurred during the study. Pharmacokinetic parameters were not statistically different between the adolescent and adult groups by route of administration. However significant differences in peak amiloride concentration, AUC, and urinary amiloride excretion were evident when comparing oral versus inhalation administration within each group. CONCLUSIONS: Mean amiloride plasma concentration peaks and AUC after inhalation were significantly lower than after oral dosing. In addition, the second amiloride plasma concentration peak may be due to oral ingestion of the nebulized amiloride, whereas the earlier Cmax1 after inhalation may be due to pulmonary absorption of amiloride. These results suggest that single-dose amiloride inhalation in patients with mild to moderate CF results in minimal systemic exposure compared with oral dosing, and that drug disposition is similar in adolescents and adults with CF.

Determination of remifentanil, an ultra-short-acting opioid anesthetic, in rat blood by high performance liquid chromatography with ultraviolet detection.

Remifentanil is an ultra-short-acting opioid under evaluation for use during surgical procedures which require opioid analgesia or anesthesia. It has an N-substituted methyl propanoate ester group which is highly susceptible to clevage by blood and tissue esterases as well as to chemical hydrolysis. A selective and specific high performance liquid chromatography method was developed to quantitate remifentanil in rat blood. A liquid-liquid extraction method using n-butyl chloride was used to separate interfering endogenous products from the compound of interest. Reverse phase chromatography with ultraviolet (lambda 210 nm) detection was used to quantitate the eluate. The calibration curves were found to be linear in the range 2.5-250 ng ml-1. Intra-day assay variability was 15% or less for all standards evaluated. The method was applied to blood samples collected from a short-term infusion study in rats. Good recovery, linearity, accuracy and precision were achieved with the assay.

Phase I and pharmacological study of the new topoisomerase I inhibitor GI147211, using a daily x 5 intravenous administration.

Topoisomerase I inhibitors are interesting anti-cancer agents with a novel mechanism of action. We performed a phase I study with intravenous GI147211, a new semisynthetic camptothecin analogue, using a daily x 5 schedule administered every 3 weeks, to evaluate the side-effects and pharmacokinetics of the agent. Patients with a histologically confirmed diagnosis of a solid tumour refractory to standard froms of therapy were eligible for the study. GI147211 was given as a 30 min intravenous infusion daily for 5 consecutive days, repeated every 3 weeks. In subsequent patient cohorts the dose was escalated from 0.3 to 1.5 mg m-2 day-1. Pharmacokinetics analysis was performed on days 1 and 4 of the first course using a validated high-performance liquid chromatographic assay and non-compartmental methods. A total of 19 patients were entered into the study, one patient was not evaluable for toxicity because only one drug administration was given. Eighteen patients received a total of 67 courses through four dose levels. The dose-limiting toxicities were neutropenia and thrombocytopenia at the dose of 1.5 mg m-2 day-1. Nadirs occurred on day 15 and day 15 respectively. Other toxicities were mild and infrequent and included nausea/vomiting, headache and alopecia. The maximal tolerated dose was 1.2 mg m-2 day-1. One partial response was observed in a patient with colorectal cancer. The total plasma clearance was 999+/-184 ml min-1 (range 640-1329). The volume of distribution was 190+/-461 m-2 and the terminal half-life was 3.7+/-1.2 h. The AUC increased linearly with the administered dose. A steep and significant sigmoid relationship was established between the AUC and the percent decrease of ANC. GI147211 is a new topoisomerase I inhibitor that induced dose-limiting neutropenia and thrombocytopenia in this phase I study. The recommended dose for phase II studies with this schedule is 1.2 mg m-2 x 5 every 3 weeks.

Determination of the novel sialic acid analog GG167 (GR121167X) in human urine by liquid chromatography: direct injection with column switching.

GG167 is a novel compound which selectively inhibits viral neuraminidase and has demonstrated activity against influenza A and B. A liquid chromatography (LC) method for the determination of GG167 in human urine has been developed and validated. The method allows direct injection of urine (7 microliters) using LC column switching followed by UV detection. Initial chromatography is performed using a Nucleosil-Diol column (7 microns, 250 mm x 4.6 mm), eluted with 20 mM phosphate buffer (pH 2.5):acetonitrile (18:82, v/v) at 2.0 ml min-1. GG167 is "heart-cut" to a Spherisorb-SCX column (5 microns, 100 mm x 4.6 mm) and eluted with 35 mM phosphate buffer (pH 2.5):acetonitrile (50:50, v/v) at 1.5 ml min-1 for final separation. GG167 is detected by UV absorbance at lambda = 238 nm. UV detection and peak shape are enhanced at pH < 2.5. The quantitation range of the assay is 0.3-100 micrograms ml-1. The method has demonstrated sufficient ruggedness to be used in support of GG167 clinical trials.

Inspection by variables as an acceptance criterion in bioanalysis--a proposal.

Inspection by variables is proposed as an acceptance criterion for use in bioanalysis. The criteria currently used are deficient either by ignoring the issues of precision (fixed range) and/or accuracy (99% confidence interval), not being able to provide immediate answers (quality charts), or even not being scientifically justified (fixed range). Inspection (sampling) by a variables procedure was originally developed to drive the quality of military supplies (MIL-STD-414) and was consequently incorporated in ISO 3951 as a part of industrial quality control. It is based on the concept of acceptable quality level (AQL), which is the maximum per cent defective (number of results outside of specification per 100 results) that can be considered satisfactory as a process average. It also correlates sample size with batch size. An AQL of 6.50% is proposed as a standard with specification set at +/- 20% or +/- 15%, which should result in coefficients of variation of approximately 15% and 12%, respectively. This concept has been applied post factum to 14 authentic data sets, thus proving its utility and validity.

Determination of GI147211 in human blood by HPLC with fluorescence detection.

GI147211 (GG211) is a camptothecin analogue, which exhibits antileukemic and antitumor activity by blocking DNA synthesis. The drug stability considerations and specimen handling were important aspects in method development and validation. This method involves collection of blood at the clinical site, immediate freezing, and storage at -70 degrees C. The lactone form is extracted from blood at physiological pH with a mixture of n-butyl chloride and acetonitrile (4:1); the carboxylate is not extracted under these conditions. After evaporation the extract is injected into an HPLC system with a fluorescence detector set at 378/420 nm. The internal standard used is 6,7-dimethoxy-4-methylcoumarin. The main advantages of the procedure are the separation of lactone and carboxylate by means of extraction, simplified specimen collection at clinical sites and the ability to inject almost all of the extracted material (extraction recovery, 60%) into an HPLC system. The method has been validated over the range 0.15-100 ng ml-1 with sufficient precision and accuracy (coefficient of variation below 10%) to support pharmacokinetic studies. Under the conditions of this procedure, the drug is stable in human blood at -70 degrees C for at least 93 days, as well as through two additional freeze-thaw cycles.

Measurement of amiloride in airway surface liquid utilizing HPLC and fluorescence detection.

A high-performance liquid chromatographic method for the determination of amiloride in airway surface liquid is described. It involves extraction of the drug with methanol from filter paper on which the sample is absorbed and chromatography on a Zorbax Rx column; the mobile phase is 25% acetonitrile in 0.05 M phosphate buffer; detection by fluorescence at 360/420 nm. Triamterene is used as an internal standard. The range of the assay is 2.0-2033.4 ng/sample, with adequate precision and accuracy. A power curve y = axb best describes the relationship between the peak-height ratio and concentration. Recovery of amiloride is non-linear, probably due to an adsorption process. Accuracy of the assay at lower amounts may be affected by the choice of filter paper a well as by presence of endogenous plasma components. The assay was used to measure amiloride amount in airway surface liquid after administration of 2.5 and 4.5 mg of nebulized amiloride.

Analysis of enloplatin by liquid chromatography and of platinum by atomic absorption spectrometry in various biological fluids.

Analytical methods were developed and validated for the determination of enloplatin (an anticancer agent) in plasma by reversed-phase LC and for platinum (an elemental component of enloplatin) in plasma, plasma ultrafiltrate (PUF) and whole blood by flameless atomic absorption spectrometry (FAAS). The LC procedure involved protein precipitation with dilute perchloric acid. The supernatant was mixed with sodium phosphate buffer and injected into the LC system. A C18 or a cyano column was used, depending on sample matrix, with UV detection at 230 nm. The LC method was linear from 0.50 to 50.0 micrograms ml-1. Inter-day and intra-day precision (RSD%) and accuracy (relative error%) were < +/- 14%. The FAAS procedure utilized a graphite furnace, a hollow cathode platinum (Pt) lamp, and Zeeman background correction. An aliquot of plasma, PUF, or whole blood was mixed with a solution of Triton X-100 and Antifoam-B and injected into the FAAS system. The FAAS method showed goodness of fit from 0.05 to 10.0 micrograms Pt/ml. Inter-day and intra-day precision and accuracy were < +/- 15%. The methods were developed to support pharmacokinetic studies in humans, dogs and rats.

Determination of remifentanil in human and dog blood by HPLC with UV detection.

Remifentanil (GI87084) is a phenylaminopiperidine derivative of the fentanyl type with a potent analgesic activity and ultra-short half-life. The N-substituted labile methyl propanoate ester group is highly susceptible to cleavage by endogenous esterases and by chemical hydrolysis. The hydrolysis is stopped by addition of 20 microliters of 50% citric acid per 1 ml of blood. The method involves a liquid extraction of chilled blood at pH 7.4 with butyl chloride and back-extraction into 0.01 M HCl. The chromatographic conditions are: column--Zorbax SB-CN 4.6 x 250 mm; mobile phase--7% acetonitrile--14% methanol in phosphate buffer (0.03 M; pH 3.0); detection--UV at 210 nm. The internal standard used was GI97559--an ethyl analogue of the drug. The method has been validated in human blood over the range of 1-200 ng ml-1 and in dog over the range 10-60, 135 ng ml-1 with the latter assay being used in a toxicological support study. Additionally, it was used to characterize the hydrolysis of the drug, the enzymes involved in the process, and ex vivo drug interactions.

Determination of 2'-deoxy-3'-thiacytidine (3TC) in human urine by liquid chromatography: direct injection with column switching.

3TC (GR109714X) is a cytidine dideoxynucleoside analogue which has been shown to have in vitro activity against a variety of strains of HIV-1 and is currently being investigated in clinical trials as a treatment for HIV infection. An HPLC method for the determination of 3TC in human urine has been developed and validated. The method allows direct injection of urine (10 microliters) using HPLC column switching followed by UV detection. On-line extraction is performed using a Spherisorb-SCX (5 microns, 20 x 4.0 mm) eluted with deionized water at 1 ml min-1. 3TC is retained while the bulk of urine constituents are eluted to waste. The SCX column is then backflushed to a BDS-Hypersil-C18 (5 microns, 250 x 4.6 mm) and eluted with 100 mM acetate pH 4.5-methanol (95:5, v/v) for final separation. 3TC is detected by UV absorbance at lambda = 285. The quantitation range of the assay was 0.5-500 micrograms ml-1. The method has demonstrated sufficient ruggedness to be used in support of 3TC clinical trials. Application to other cytidine analogues including DDC has been demonstrated.

A liquid chromatographic method for the determination of danazol in human serum.

A liquid chromatographic method for the determination of danazol in human serum has been developed. Reversed-phase C8 and C18 columns were used with a column-switching valve, isocratic elution and UV detection. Sample pretreatment involved extraction of the drug with pentane-methylene chloride. The method enabled the measurement of the drug at a concentration as low as 1 ng ml-1, with precision of 15.0% and accuracy of 8.3%. The method was used to run a two way replicated pharmacokinetic study of danazol. The main pharmacokinetic parameters were (mean of two periods): AUCinf = 480.94 ng x h ml-1, Cmax = 53.2 ng ml-1, tmax = 2.5 h, t0.5 = 18.00 h.

Simultaneous determination of flurazepam and its metabolites in human plasma by high-performance liquid chromatography.

A sensitive isocratic high-performance liquid chromatographic method is described, which allows the precise and accurate quantification of flurazepam and four metabolites with a single determination. A pharmacokinetic study was performed on nine volunteers and the main pharmacokinetic data are reported. The method was used to demonstrate that monodesethylflurazepam and didesethylflurazepam are major metabolites in men. One more unidentified flurazepam metabolite was detected.

Determination of encainide and its metabolites by high-performance liquid chromatography.

Encainide (ENC) and its metabolites O-demethylencainide (ODE), 3-methoxy-O-demethylencainide (MODE), N-demethylencainide (NDE) and bis-N,O-demethylencainide (NODE) have been measured by two HPLC procedures. The method of Mayol using a mu Porasil column with ethanol-water-methanesulphonic acid as mobile phase was not able to separately measure NODE and NDE in plasma. A new method is described using a mu Bondapak Phenyl column with acetonitrile-phosphate buffer (0.05 M, pH 7.5) that yields satisfactory separation of ENC and its metabolites. NODE was not identified as a metabolite in 23 patients analysed.