D. A. Stopher

A rapid HPLC assay for voriconazole in human plasma

This report describes a simple, rapid and reproducible method with a calibration range of 0.2-10 micrograms ml-1 voriconazole in human plasma which is more appropriate for routine clinical use than the authors previously published method. The method utilises protein precipitation with acetonitrile as the only sample preparation involved prior to reverse phase HPLC. No internal standard was required.

Determination of a new antifungal agent, voriconazole, by multidimensional high-performance liquid chromatography with direct plasma injection onto a size-exclusion column.

A fully automated method has been developed for the analysis of a new antifungal agent, voriconazole, in human plasma. Multidimensional chromatography was used with size-exclusion chromatography as the first step to separate plasma protein from the drug and internal standard which were then trapped on a precolumn of pellicular ODS. A reversed-phase column, Spherisorb ODS2, then separated drug and internal standard from one another and from remaining plasma components. With an injection of 0.56 ml plasma the limit of quantitation of the method was 5 ng/ml.

Metabolism and kinetics of amlodipine in man

1. The disposition of amlodipine, R,S,2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-3-ethoxycarbonyl- 5- methoxycarbonyl-6-methyl-1,4-dihydropyridine has been studied in two human volunteers using single oral and intravenous doses of 14C-amlodipine. The drug was well absorbed by the oral route while the mean oral bioavailability for unchanged drug was 62.5%. 2. Renal elimination was the major route of excretion with about 60% of the dosed radioactivity recovered in urine. Mean total recovered radioactivity in urine and faeces amounted to 84% for both the oral and intravenous routes. 3. Apart from a small amount of unchanged amlodipine (10% of urine 14C), only pyridine metabolites of amlodipine were excreted in urine. The majority (greater than 95%) of the metabolites excreted in the 0-72 h post-dose period were identified; the major metabolite was 2-([4-(2-chlorophenyl)-3-ethoxycarbonyl-5-methoxycarbonyl-6-methyl- 2-pyridyl]methoxy) acetic acid and this represented 33% of urinary radioactivity. The data indicate that oxidation of amlodipine to its pyridine analogue is the principal route of metabolism with subsequent metabolism by oxidative deamination, de-esterification and aliphatic hydroxylation. 4. For the two volunteers, amlodipine concentrations in plasma declined with a mean half-life of 33 h, while slower elimination of total drug-related material from plasma was observed, consistent with prolonged excretion (up to 12 days) of metabolites in urine and faeces. Only amlodipine and pyridine metabolites were found in the circulation. As these pyridine derivatives have minimal calcium antagonist activity the efficacy of amlodipine in man can most probably be attributed to the parent drug.

Pharmacokinetics and metabolism of dofetilide in mouse, rat, dog and man

1. Pharmacokinetics of dofetilide were studied in man, dog, rat and mouse after single i.v. and oral doses of dofetilide or 14C-dofetilide. 2. Dofetilide was absorbed completely in all species. Low metabolic clearance in man resulted in complete bioavailability following oral administration. Higher metabolic clearance in rodents, and to a lesser extent dogs, resulted in decreased bioavailability because of first-pass metabolism. 3. Following i.v. administration, the volume of distribution showed only moderate variation in all species (2.8-6.3 l/kg). High plasma clearance in rodents resulted in short half-life values (mouse 0.32, male rat 0.5 and female rat 1.2 h), whilst lower clearance in dog and man gave longer terminal elimination half-lives (4.6 and 7.6 h respectively). 4. After single i.v. doses of 14C-dofetilide, unchanged drug was the major component excreted in urine of all species with several metabolites also present. 5. Metabolites identified in urine from all species were formed by N-oxidation or N-dealkylation of the tertiary nitrogen atom of dofetilide. 6. After oral and i.v. administration of 14C-dofetilide to man, parent compound was the only detectable component present in plasma and represented 75% of plasma radioactivity. No single metabolite accounted for greater than 5% of plasma radioactivity.

Metabolism of amlodipine in the rat and the dog: A species difference

1. Following oral and i.v. doses of 14C-amlodipine to rat and dog, 40-50% of the dose was excreted in the urine indicating that the oral dose was well absorbed. Urinary and faecal excretion in rat was essentially complete within 48 h but was prolonged during 168 h in dog. 2. Metabolite patterns were dissimilar for rat and dog for both urine and faeces. The majority (about 95%) of the urinary metabolites were identified for both species; unchanged drug accounted for 10% and 2% of the urinary radioactivity in rat and dog respectively. 3. In rat, the principal route of metabolism involved cleavage of the 5-methoxy-carbonyl group of both the parent dihydropyridine and its pyridine analogue. In contrast, metabolism in dog involved oxidative deamination of the 2-aminoethoxy-methyl side-chain. 4. Secondary metabolism in both rat and dog was similar to that of other calcium channel blockers of the dihydropyridine class, with oxidation to the pyridine form being followed by aliphatic hydroxylation in the 6-position or O-dealkylation in the 2-position and lactonization.

Measurement of the class III antidysrhythmic drug, UK-68,798, in plasma by radioimmunoassay.

A sensitive radioimmunoassay (RIA) for the specific determination of 1-(4-methanesulphonamidophenoxy)-2-[N-(4-methanesulphonamido -phenethyl)-N- methylamino]ethane (UK-68,798), a novel class III antidysrhythmic agent, in human plasma is described. Specific antisera were raised in sheep using desmesyl-UK-68,798-succinate-ovalbumin conjugate as the antigenic hapten carrier protein. The antisera produced exhibited high specificity for UK-68,798 compared with known metabolites from animals, other antidysrhythmic agents and co-administered drugs. Good correlation was found in a comparison of the RIA method with a high-performance liquid chromatography (HPLC) method (r = 0.997) and a 10-fold lower limit of determination was observed for the RIA method compared with the HPLC method (0.05 and 0.5 ng ml-1, respectively). The RIA method was applied to the analysis of UK-68,798 in plasma obtained from human volunteers receiving the compound.

Bioanalytical data in decision making : discovery and development

1. Bioanalysis is traditionally associated with the development phase of drugs; its use in discovery programmes is often ignored but can have a major impact. 2. Pharmacokinetic studies conducted in conjunction with pharmacology screening can provide additional information to that considered in conventional structure activity relationships. Such factors as half-life and bioavailability can be critical in designing improved drugs. 3. Analytical methods in discovery programmes may differ from those used in later development work: for instance bioassay allows a common assay system for a large number of project compounds. Moreover its use, when combined with conventional methods, such as h.p.l.c., allows active metabolites to be readily detected. 4. Bioanalytical data generated in discovery and pre-clinical programmes are a valuable guide to early clinical programmes. Plasma concentration-response data from these programmes can be compared with those obtained in man. Such comparisons are particularly valuable during the phase one-initial dose escalation study. To maximize this it is our practice to generate pharmacokinetic data between each dose increase.

Pharmacokinetics of a series of bis(methanesulphonamido-arylalkyl)amines in the beagle dog

1. The pharmacokinetics of three closely related analogues of dofetilide have been investigated in the beagle dog. These have been compared with those of dofetilide and related to physicochemical properties and structural features of the molecules. 2. Following intravenous administration, the four compounds exhibit elimination half-lives ranging from 4.6 to 19 h. This range is due to changes in both volume of distribution and plasma clearance across the series. 3. In vitro plasma protein shows a relationship to lipophilicity within this series. Protein binding increasing from 54% for dofetilide, the least lipophilic compound (log D7.4 = 0.73) to 92% for the most lipophilic analogue (log D7.4 = 2.07). There is a trend for a decrease in the volume of distribution with increased plasma protein binding. 4. Plasma clearance values range from 2.4 to 10.2 ml/min/kg and are comprised of renal and non-renal components. Renal clearance ranges fro 0.11 to 2.9 ml/min/kg and shows an inverse correlation with and lipophilicity of the compounds. Values for the renal clearance of unbound drug suggest that only the most lipophilic derivative (III), has sufficient membrane affinity to undergo tubular reabsorption. 5. Non-renal clearance of either total or free drug shows no relationship with lipophilicity. Highest values are observed for the two compounds with a methyl substituent on the tertiary amine and lowest values for the two compounds in which the tertiary amine is incorporated into a 7-membered ring. In vitro metabolism in dog liver microsomes also shows increased lability for the two N-methyl compounds. The N-desmethyl metabolite is the major product in both cases.

Liquid—liquid extraction and high-performance liquid chromatography for the determination of a novel antidysrhythmic agent (UK-68,798) in human urine

A routine method for the determination of a novel class III antidysrhythmic agent, 1-(4-methanesulphonamidophenoxy)-2-[N-(4-methanesulponamidophen ethyl)- N-methylamino]ethane, in human urine has been developed. The method involves solvent extraction followed by high-performance liquid chromatography on an unmodified silica column with ultraviolet detection. Despite a low recovery of drug through the three-stage extraction procedure a reliable assay with high precision (coefficient of variation less than 6%) and a limit of determination of 2.5 ng/ml was achieved. The method has been applied to the analysis of samples following single oral and intravenous doses of 1-12.5 micrograms/kg of the drug to human volunteers.