Characterization of the highly variable bioavailability of tiludronate in normal volunteers using population pharmacokinetic methodologies.

Currently, the use of classical bioequivalence criteria is being called into question for certain classes of drugs such as bisphosphonates. These compounds typically possess a wide therapeutic index but may be characterized by low and variable absorption. The purpose of this communication was to characterize the highly variable bioavailability of tiludronate using a population pharmacokinetic method (NONMEM program) and compare the results to a standard 2 way cross-over bioequivalence trial in healthy subjects. Over 3500 plasma samples from 153 healthy subjects, representing 12 different clinical trials were pooled for mixed effect modeling purposes (complete data set). These studies, conducted under single and multiple dose conditions, contained all the directly comparable data available in healthy subjects administered a 400 mg dose of tiludronate. A two compartment model with first order absorption was fit to the plasma concentration-time data and a term for relative bioavailability (BA) was included. Intersubject and residual variability were modeled using a constant coefficient of variation (CCV) model. A pilot model development data set was obtained from a 24 subject cross-over bioequivalence study. Population estimates of BA and its associated 90% confidence interval of 1.12 and 0.89-1.35 compared favorably to standard bioequivalence methodology (1.15 and 0.93-1.42, respectively). Since a good fit of predicted and observed plasma concentrations as well as estimates of BA were obtained, a two compartment model with a term for BA was then applied to the complete data set. Under these conditions, BA and its 90% confidence interval were found to be 1.17 and 0.98-1.36. Intersubject variability of 31%, compared with 38% in the pilot model development data set and residual variability of 38% were seen. No differences in absorption characteristics as measured by Ka were found. Good agreement between the population pharmacokinetic parameters were observed when the pilot data set was compared with the full data set. The proposed model was confirmed by creating 10 additional smaller data sets that were matched for the number of subjects given both formulations under single and multiple dose conditions. No change in the estimate of BA was observed under these study conditions. This study demonstrated that population pharmacokinetic methodology can be applied successfully to problematical bioequivalence issues that may occur during the development process. Increasing the number of subjects in the overall analysis did not alter the estimate of BA or its 90% confidence interval, when compared to the original cross-over bioequivalence study. Bayesian approaches can be of value in large clinical trials where typically relatively few plasma samples are obtained from individual subjects.

An approach for widening the bioequivalence acceptance limits in the case of highly variable drugs.

PURPOSE: Highly variable drugs pose a problem in bioequivalence assessment because they often fail to meet current regulatory acceptance criteria for average bioequivalence (80-125%). This paper examines alternative approaches to establishing bioequivalence. METHODS: Suggested solutions have included alternate study designs, e.g., replicate and multiple dose studies, reducing the level of the confidence interval, and widening the acceptance limits. We focus on the latter approach. RESULTS: A rationale is presented for defining wider acceptance limits for highly variable drugs. Two previously described methods are evaluated, and a new method having more desirable properties is proposed. CONCLUSIONS: We challenge the "one size fits all" current definition of bioequivalence acceptance limits for highly variable drugs, proposing alternative limits or "goal posts" which vary in accordance with the intrasubject variability of the reference product.

Involvement of cytochrome P-450IIIA in metabolism of potassium canrenoate to an epoxide: mechanism of inhibition of the epoxide formation by spironolactone and its sulfur-containing metabolite.

In vitro metabolism studies of potassium canrenoate (PC) were conducted to examine whether spironolactone (SP) and/or its sulfur-containing metabolites inhibit the PC metabolic pathways to mutagenic metabolites and to elucidate the mechanism for any observed inhibitory effect. The mechanistic study was conducted using liver microsomes prepared from male and female rats with and without pretreatment of a cytochrome (Cyt) P-450IIIA inducer [pregnenolone-16 alpha-carbonitrile (PCN) or dexamethasone (DEX)] and with and without a Cyt P-450IIIA inhibitor, triacetyloleandomycin (TAO). The present study demonstrates that SP and its sulfur-containing metabolite 7 alpha-thio-spirolactone substantially inhibited the formation of promutagen 6 beta, 7 beta-epoxycanrenone (6 beta, 7 beta-epoxy-CAN) from PC. The sulfur-containing metabolite of SP that inhibit promutagen formation were not formed from PC, although a glutathione conjugate of PC was formed. The formation rate of 6 beta, 7 beta-epoxy-CAN was greater in liver microsomes prepared from rats pretreated with a Cyt P-450IIIA inducer (PCN or DEX) than in liver microsomes prepared from the untreated rats. The formation rate of the epoxide metabolite was lower after in vitro addition of TAO. Pretreatment of animals with TAO 4 hr before sacrifice produced similar results. Erythromycin, which is N-demethylated by Cyt P-450IIIA, also reduced the formation rate of 6 beta, 7 beta-epoxy-CAN. Inhibition of PC metabolism to 6 beta, 7 beta-epoxy-CAN by TAO and erythromycin, and its induction by DEX and PCN, suggest involvement of Cyt P-450IIIA, which is in turn inhibited by SP and 7 alpha-thio-spirolactone.

Identification of N-beta-L-aspartyl-L-phenylalanine as a normal constituent of human plasma and urine.

A new beta-aspartyl dipeptide, N-beta-L-aspartyl-L-phenylalanine (beta-AP), has been isolated and identified in urine and plasma from normal human volunteers. beta-AP was isolated from urine samples by high performance liquid chromatography (HPLC). Its identity and stereochemistry were demonstrated by HPLC and gas chromatography/mass spectrometry (GC-MS). The mean urinary beta-AP concentration in the subjects was 0.63 +/- 0.14 microgram/mg creatinine when averaged over two consecutive days of urine collection. Daily beta-AP excretion, determined from two 24-h urine samples collected from five individuals, was 801 +/- 117 micrograms/d (2.7 mumol/d). No diurnal rhythm was evident within the 24-h collection periods. beta-AP was also isolated from human plasma by HPLC and identified by GC-MS. Plasma from subjects contained approximately 5 ng beta-AP/ml. Furthermore, beta-AP was formed when asparagine and phenylalanine were incubated with an enzyme extract from human kidney. Thus, at least some of the beta-AP present in humans, and presumably other beta-aspartyl dipeptides as well, appears to be synthesized endogenously.

Absorption by the rhesus monkey of phenylalanine methyl ester and species differences in its metabolism by blood, plasma and intestinal mucosa.

Phenylalanine methyl ester (PM) is a decomposition product of the sweetening agent aspartame. The potential for absorption of PM was examined following intragastric and intraduodenal administration of 20-mg doses of [14C]PM to rhesus monkeys implanted with hepatic portal vein cannulae. Small amounts of unchanged PM (less than 0.1 micrograms/ml) were detected in portal and peripheral blood samples during the first 1-2 hours after administration, but none was detectable (less than 0.001 micrograms/ml) at later times. Comparison of the areas under the PM and total 14C blood concentration-time curves indicated that only 0.2% of the administered PM reached the portal blood unchanged, and 0.1% or less reached the peripheral blood unchanged. Blood and plasma from monkeys and humans hydrolyzed PM in vitro at very similar rates, but plasma PMase activity was much higher in the dog and rat than in the monkey or human. Hydrolysis of PM by intestinal mucosa homogenates was also faster for the rat and dog than for the monkey. The in vitro results suggest that absorption of intact PM in the human would be no greater than that found in the monkey.

A review of the metabolism of the aspartyl moiety of aspartame in experimental animals and man.

Aspartame (3-amino-N-(alpha-carboxyphenethyl) succinamic acid, methyl ester; the methyl ester of aspartylphenylalanine, SC-18862) is hydrolyzed in the gut to yield aspartic acid, phenylalanine, and methanol. This review of the literature describes the metabolic paths followed by aspartate in its conversion to CO2 or its incorporation into body constituents. About 70 percent of 14C from [asp-14C]-aspartame is converted in the monkey to 14CO2. Some of the aspartate is converted at the intestinal mucosal level to alanine by decarboxylation. This amino acid may be oxidized to CO2 by entering the tricarboxylic acid cycle via pyruvate and acetyl CoA. In addition, transamination of aspartate to oxaloacetate permits this product also to enter the tricarboxylic acid cycle. Aspartate may also be incorporated into body constitutents such as other amino acids, proteins, pyrimidines, asparagine, and N-acetylaspartic acid. It is concluded that the aspartate moiety of aspartame is metabolized in a manner similar to that of dietary aspartic acid.

Comparative metabolism of aspartame in experimental animals and humans.

Aspartame [SC-18862; 3-amino-N-(alpha-carboxyphenethyl) succinamic acid, methyl ester, the methyl ester of aspartylphenylalanine] is a sweetening agent that organoleptically has about 180 times the sweetness of sugar. The metabolism of aspartame has been studied in mice, rats, rabbits, dogs, monkeys, and humans. The compound was digested in all species in the same way as are natural constituents of the diet. Hydrolysis of the methyl group by intestinal esterases yielded methanol, which was oxidized in the one-carbon metabolic pool to CO2. The resultant dipeptide was split at the mucosal surface by dipeptidases and the free amino acids were absorbed. The aspartic acid moiety was transformed in large part to CO2 through its entry into the tricarboxylic acid cycle. Phenylalanine was primarily incorporated into body protein either unchanged or as its major metabolite, tyrosine.