G. McKay

Bioanalytical Method Validation—A Revisit with a Decade of Progress

This report is a synthesis of (1) the earlier conference on Analytical Methods Validation−Bioavailability, Bioequivalence and Pharmacokinetic Studies (Conference held in Arlington, VA, December 3–5, 1990 and the report published in Pharmaceutical Research, 9: 588-592, 1992) and (2) the workshop on “Bioanalytical Methods Validation—A Revisit with a Decade of Progress,” (Workshop held in Arlington, VA, January 12–14, 2000), sponsored by the American Association of Pharmaceutical Scientists and the U. S. Food and Drug Administration. The bioanalytical method validation workshop of January 12–14, 2000 was directed towards small molecules. A separate workshop was held in March 1–3, 2000 to discuss validation principles for macromolecules. The purpose of this report is to represent the progress in analytical methodologies over the last decade and assessment of the major agreements and issues discussed with regard to small molecules at both the conference and the workshop. The report is also intended to provide guiding principles for validation of bioanalytical methods employed in support of bioavailability, bioequivalence, and pharmacokinetic studies in man and in animals.

Analytical methods validation: Bioavailability, bioequivalence and pharmacokinetic studies: Sponsored by the American Association of Pharmaceutical Chemists, U.S. Food and Drug Administration, Fédération Internationale Pharmaceutique, Health Protection Branch (Canada) and Association of Official Analytical Chemists

Abstract This is a summary report of the conference on ‘Analytical Methods Validation: Bioavailability, Bioequivalence and Pharmacokinetic Studies.’ The conference was held from December 3 to 5, 1990, in the Washington, DC area and was sponsored by the American Association of Pharmaceutical Scientists, U.S. Food and Drug Administration, Federation Internationale Pharmaceutique, Health Protection Branch (Canada) and Association of Official Analytical Chemists. The purpose of the report is to represent our assessment of the major agreements and issues discussed at the conference. This report is also intended to provide guiding principles for validation of analytical methods employed in bioavailability, bioequivalence and pharmacokinetic studies in man and animals. The objectives of the conference were: (1) to reach a consensus on what should be required in analytical methods validation and the procedures to establish validation; (2) to determine processes of application of the validation procedures in the bioavailability, bioequivalence and pharmacokinetic studies; and (3) to develop a report on analytical methods validation (which may be referred to in developing future formal guidelines). Acceptable standards for documenting and validating analytical methods with regard to processes, parameters or data treatments were discussed because of their importance in assessment of pharmacokinetic. bioavailability, and bioequivalence studies. Other topics which were considered essential in the conduct of pharmacokinetic studies or in establishing bioequivalency criteria, including measurement of drug metabolites and stereoselectivc determinations, were also deliberated.

Intersubject variation in the pharmacokinetics of haloperidol and reduced haloperidol.

Single oral doses (5 mg) of haloperidol were administered to 36 healthy men (26 black, 10 white) of whom 28 (22 black, 6 white) completed the study. Plasma samples harvested over 96 hours were analyzed for haloperidol and reduced haloperidol by means of a new high performance liquid chromatographic method. Reduced haloperidol was detectable in the plasma of only six of the 28 subjects (five blacks, one white). In these individuals reduced haloperidol plasma concentrations were generally much lower than those of the parent drug. This finding in the present single-dose study is in contrast to literature reports that have described levels of reduced haloperidol higher than those of the parent drug in some patients chronically medicated with haloperidol. There was wide intersubject variation in area under the plasma concentration versus time curve and apparent oral clearance values for haloperidol. The distributions of these pharmacokinetic parameters about their respective means were each leptokurtotic and skewed toward higher values. In each case the geometric mean gave a better estimate of central tendency than the arithmetic mean. Wide intersubject variation prevented the detection of significant differences in these pharmacokinetic parameters between black and white subjects or between smokers and nonsmokers.

Characterization of the human hepatic cytochromes P450 involved in the in vitro oxidation of clozapine.

It was aimed to identify the cytochrome(s) P450 (CYPs) involved in the N-demethylation and N-oxidation of clozapine (CLZ) by various approaches using human liver microsomes or microsomes from human B-lymphoblastoid cell lines. The maximum rates of formation were measured in the microsomal fraction of human livers and the Michaelis-Menten kinetics one enzyme model was found to best fit the data with mean K(M) for CLZ N-oxide and N-desmethyl-CLZ of 336 and 120 microM, respectively. Significant correlations were observed between the maximum rates of formation (Vmax) for CLZ N-oxide and N-desmethyl-CLZ with the microsomal immunoreactive contents of CYP1A2 (r = 0.92, P < 0.009 and r = 0.77, P < 0.077; respectively) and CYP3A (r = 0.89, P < 0.02 and r = 0.82, P < 0.05; respectively). Antibodies directed against CYP1A2 and CYP3A inhibited formation of CLZ N-oxide in human liver microsomes by 10.7+/-6.1%) and 37.2+/-6.9% of control, respectively, whereas CLZ N-demethylation was inhibited by 32.2+/-15.4% and 33.6+/-7.4%, respectively. Troleandomycin (CYP3A inhibitor) and furafylline (CYP1A2 inhibitor) inhibited CLZ N-oxidation in human liver microsomes by 23.2+/-12.1% and 7.8+4.3%, respectively, whereas CLZ N-demethylation was inhibited by 17.5+/-13.9% and 25.6+/-16.5%, respectively. While ketoconazole did not inhibit N-oxidation of CLZ, the N-demethylation pathway was inhibited by 34.1+/-10.0%. Formation in stable expressed enzymes indicated involvement of CYP3A and CYP1A2 in CLZ N-oxide formation and CYP2D6, CYP1A2 and CYP3A4 in CLZ N-demethylation. This apparent involvement of CYP2D6 in the N-demethylation of CLZ did not corroborate with the findings of other experiments. In conclusion, these data indicate that while both CYP isoforms readily catalyze both metabolic routes in vitro, CYP1A2 and CYP3A4 are more important in N-demethylation and N-oxidation, respectively.

Effect of quinidine on the interconversion kinetics between haloperidol and reduced haloperidol in humans: implications for the involvement of cytochrome P450IID6

SummaryHaloperidol (HAL) is a potent butyrophenone antipsychotic agent which is reversibly metabolized to reduced haloperidol (RHAL). In order to determine if this reversible metabolic pathway is linked to the debrisoquine 4-hydroxylase isozyme of cytochrome P-450 (P450IID6), HAL (5 mg) or RHAL (5 mg) was orally administered to healthy male volunteers in a randomized crossover design both with and without a prior (1 h) oral dose of quinidine (250 mg bisulfate), a potent inhibitor of this isozyme. Thirteen volunteers, 11 extensive metabolizers, 2 poor metabolizers, completed all four phases of the study. Plasma samples harvested over seven days were analysed for HAL and RHAL. An expression for the apparent fractional availability of metabolite from the parent compound given (Fappinfmsupp) was derived and was used to determine whether HAL or RHAL is the preferred metabolite, and whether quinidine co-administration alters Fapp for either compound.The AUC (0-t) for both HAL and RHAL were significantly greater following the administration of either compound with quinidine compared with AUC (0-t) values obtained in the absence of quinidine. The maximum plasma concentration (Cmax) of the administered compound was also greater following the administration of quinidine. Quinidine had no effect on the half-lives of the administered compounds. The Fapp for HAL and RHAL were not significantly affected by the administration of quinidine, indicating that the interconversion of HAL and RHAL is not linked to P450IID6. The Fapp of RHAL after administration of HAL was significantly greater than the Fapp of HAL after RHAL administration, indicating that RHAL is the preferred metabolic form. This difference was not affected by quinidine.It is concluded that: 1) RHAL is the preferred form after administration of either compound and is not affected by quinidine, 2) the interconversion of HAL and RHAL is not affected by quinidine, indicating that this reversible metabolic process is not linked to P450IID6 and 3) there is a significant increase in the AUC (0-t) and Cmax values following quinidine co-administration with either HAL or RHAL. The precise mechanism of this interaction can not be established from this study, however, the observed increases in AUC (0-t) and Cmax may be explained with a simple tissue blinding displacement mechanism.

Metabolism of methoxyphenamine in extensive and poor metabolizers of debrisoquin

Urine and plasma concentrations of methoxyphenamine (MP) and three of its metabolites were determined after a single oral 60.3 mg dose of MP hydrochloride to healthy subjects of known debrisoquin (D) phenotype. Urine was collected from five extensive (EM) and five poor (PM) metabolizers of D for 12 hours and analyzed after treatment with β‐glucuronidase/sulfatase. There were marked interphenotype differences in the total urinary excretion of O‐demethylmethoxyphenamine (ODMP) and 5‐hydroxymethoxyphenamine (5HMP), as well as in MP/ODMP and MP/5HMP ratios. In contrast, the urinary output of N‐demethylmethoxyphenamine (NDMP) or MP/NDMP ratios showed no interphenotype differences. Plasma data from two EMs and two PMs showed that the mean values for maximum concentration, t½, and total AUC for MP were two‐, three‐, and sixfold greater, respectively, in PMs than in EMs. The plasma levels of ODMP and 5HMP were higher in EMs than in PMs, whereas the converse was true for NDMP. Thus, O‐demethylation and aromatic 5‐hydroxylation of MP are defective in PMs of D, resulting in increased MP and NDMP plasma levels. The form of cytochrome P‐450 involved in the N‐demethylation of MP is different from that responsible for O‐demethylation and aromatic 5‐hydroxylation.

N+-Glucuronidation of aliphatic tertiary amines, a general phenomenon in the metabolism of H1-antihistamines in humans

1. Representative drugs of the various structural classes of H1 antihistamines were chosen for study. The drugs chosen (class name in parentheses) were chlorpheniramine maleate and pheniramine maleate (alkylamines), diphenhydramine hydrochloride and doxylamine succinate (ethanolamines), pyrilamine maleate and tripelennamine hydrochloride (ethylenediamines), promethazine hydrochloride (phenothiazine), cyclizine lactate (piperazine) and terfenadine (miscellaneous). In each case oral dose(s) were administered over no more than 6 h to two healthy volunteers and the total urine collected for 36 h. 2. Metabolites from urine were separated by h.p.l.c. and individually collected prior to mass spectrometric analysis in the fast atom bombardment mode. The structure of each metabolite identified as a quaternary ammonium-linked glucuronide metabolite was confirmed by direct comparison of its mass spectrum and chromatographic behaviour with that of a synthetic authentic compound. 3. For eight of the nine drugs studied, metabolism by the N(+)-glucuronidation pathway was observed in each of the volunteers. Terfenadine was the exception. 4. The amount of each N(+)-glucuronide in the urine was estimated by h.p.l.c. analysis. The mean proportion of dose excreted as the metabolite was 14.3%, 6.5% and 4.0% for cyclizine, tripelennamine and diphenhydramine, respectively. Promethazine was the only case where the N(+)-glucuronide accounted for less than 1.0% of the administered dose in both volunteers examined.

An ultrasensitive method for the measurement of haloperidol and reduced haloperidol in plasma by high-performance liquid chromatography with coulometric detection

A new analytical method has been developed for the simultaneous quantitation of haloperidol and reduced haloperidol in plasma. The method is based on high performance liquid chromatography (HPLC) with coulometric detection. The extraction and sample clean up procedures are simple and rapid to execute, yet yield chromatograms virtually free of interference from endogenous plasma constituents, such that the extraordinary sensitivity of the coulometric detector can be exploited fully. The detection limits for haloperidol and reduced haloperidol are 20 pg/ml plasma, and the limits of quantitation are 50 pg/ml for both drug and metabolite. Standard curves were linear down to 50 pg/ml with coefficients of variation of less than 7.0% at the limits of quantitation. The method was applied to the study of the plasma levels of haloperidol and reduced haloperidol in two healthy subjects. It was possible to monitor the plasma levels of haloperidol for at least 96 h (4 days) after the administration of a 5-mg oral dose of haloperidol. It was also possible to monitor reduced haloperidol levels over 96 h in one subject, although the metabolite was not detectable in the plasma of the other at any stage.

Fluphenazine plasma levels in patients receiving low and conventional doses of fluphenazine decanoate

Plasma fluphenazine concentrations (FLU) were measured in 45 patients with schizophrenic disorders who participated in a double-blind comparison of 5 and 25 mg fluphenazine decanoate (FD). The rise in plasma level of FLU 24 h after a “test dose” was significantly correlated with steady state FLU concentration at 12 weeks (for 5 mg patients, r=0.45, P=0.04; for 25 mg, r=0.78, P=0.005). Patients who had low FLU at baseline required nearly 6 months to reach a steady state when they received 25 mg. Patients who received 5 mg and had low FLU at baseline continued to demonstrate relatively low plasma levels for the entire 1st year. Although the mean FLU at 6 months was lower for patients who relapsed during the subsequent 18 months (0.57 ng/ml for relapsers vs 1.01 ng/ml for nonrelapsers), this difference was not statistically significant. When plasma levels from both dosage groups were combined, FLU at 12 weeks correlated significantly with factor scores for akinesia (r=0.52, P=0.002) and BPRS cluster scores for retardation (r=0.52, P=0.002). These results indicate that the measurement of fluphenazine plasma levels may be useful in determining when patients treated with FD are receiving drug doses which are likely to cause discomforting side effects.

Interconversion between haloperidol and reduced haloperidol in healthy volunteers

SummaryThe interconversion between haloperidol (HAL) and reduced haloperidol (RHAL) was examined following their separate administration in low (5 mg) single oral doses to 15 young healthy male volunteers in a crossover design. Using an ultrasensitive HPLC method plasma concentrations of HAL and RHAL were monitored over a period of one week following each administration.Except in one case, both the analytes were found in the plasma of all the volunteers following each administration, thereby indicating interconversion of the two compounds. Comparison of the AUC(0-t) ratios of RHAL/HAL and HAL/RHAL following administration of HAL and RHAL, respectively, revealed that the interconversion favours the reduction of HAL to RHAL.The disposition of HAL following administration of RHAL appears to be limited by its rate of formation and the disposition of RHAL following administration of HAL, on the other hand, is much slower than that of the parent compound.