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.

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.

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.

A novel method has been developed for the rapid solid phase extraction of drugs and metabolites from biological fluids, prior to further analysis. The newly designed, 96-tube micropreparation block facilitates high throughput by enabling the extraction of 96 samples simultaneously. The system is described, linked to HPLC/APCI-MS/MS, for the determination of darifenacin in human plasma. The resulting procedure, using deuterated darifenacin as internal standard, is validated over the concentration range 25-2000 pg/mliter; accuracy (0.6-4.6%) and precision (3.6-18.8%) are considered acceptable and overall recovery was determined to be approximately 50%.

In order to evaluate the contribution to the overall pharmacokinetic and pharmacological profile of amlodipine made by the side-chain ether oxygen atom and the intramolecular hydrogen bond to the DHP ring NH proton, the profile of amlodipine was compared with that of its carba and thio bioisosteres. Replacing the side-chain oxygen by carbon dramatically reduces in vitro calcium antagonist potency, an effect which may be attributed to the loss of a through-bond inductive effect on the DHP ring NH proton, while both the thio and carba analogues show lower in vitro selectivity than amlodipine for vascular over cardiac tissue. On intravenous administration to anaesthetised dogs, compounds 2 and 3 both exhibit marked depression of myocardial contractility at doses equal or close to their ED50 for reduction of coronary vascular resistance. The plasma clearances of amlodipine and analogues 3 and 4 are similar, suggesting that the conformation adopted by the 2-sidechain has little influence on this parameter although bulk and polarity are important. However, compounds 3 and 4 have markedly lower volumes of distribution (6 and 8 dm3 kg-1, respectively) than amlodipine (25 dm3 kg-1) and consequently shorter half-lives; this may be a consequence of their inability to form an intramolecular hydrogen bond.

A method is described for the determination of abanoquil in human blood. The method is based on high-performance liquid chromatography (HPLC)/atmospheric pressure positive ion chemical ionization mass spectrometry, using (2H3)abanoquil as internal standard. Multiple reaction monitoring is employed for selectivity and sensitivity, which enables quantification over the range 10-500 pg ml-1 with acceptable precision and accuracy. This assay methodology illustrates the versatility of atmospheric pressure ionization/tandem mass spectrometry, in conjunction with HPLC, for the separation and quantification of drugs in the subnanogram per millilitre range.

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.

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.

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.

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.

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.

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.

The disposition of amlodipine, a new calcium-channel blocker with a slow onset and long duration of action, has been investigated in humans and in the animal species used in the evaluation of drug efficacy and safety. Pharmacokinetic studies were conducted with nonlabeled drug using specific high-pressure liquid chromatography or gas chromatographic procedures. The metabolic fate of the drug was investigated in mice, rats, dogs, and humans using [4-14C]-amlodipine. After intravenous administration, the percentages of the dosed radioactivity recovered in urine were 62% in humans, 45% in dogs, 38% in rats, and 25% in mice. The remainder of the doses were recovered in the feces. A similar pattern of excretion was observed after oral dosing indicating complete absorption of the 14C drug. Absorbed drug is extensively metabolized because only approximately 5% of the dose was excreted unchanged in human urine. Metabolism in humans primarily involves oxidation to the pyridine derivative with subsequent oxidative deamination of the 2-aminoethyoxymethyl side chain or deesterification at the 5-methoxycarbonyl group. These metabolites were common to either the rat or dog, although some dihydropyridine derivatives were observed as metabolites in these two species. None of the metabolites identified and then synthesized was found to have any significant calcium antagonist activity relative to amlodipine. Bioavailability of unchanged drug after oral administration was high with values of 63, 88, 100, and 100% in humans, dogs, mice, and rats, respectively. Mean plasma half-life values from single-dose studies were 35 h in humans (cf nifedipine, approximately 2 h), 30 h in dogs, 3 h in rats, and 11 h in mice.(ABSTRACT TRUNCATED AT 250 WORDS)

The metabolic fate of doxazosin was investigated in man, mouse, rat and dog using 14C-labelled compound. Bioavailability and pharmacokinetic studies were also conducted with nonlabelled drug, using a specific h.p.l.c. method. Following both oral and intravenous administration, the major route of elimination of drug-related compounds was via the faeces for all species studied. Comparison of the oral and intravenous data show that doxazosin is completely absorbed in man, mouse and rat and is moderately well absorbed in dog. The drug is extensively metabolized, e.g. only about 5% of the dose was excreted unchanged in man. Metabolism in man mainly involves 6- and 7- O-demethylation and 6' and 7'-hydroxylation. These and some minor products were common to the mouse, rat or dog and man. Plasma protein binding was high in all species studied, ranging from 95.3% in the rat to 98.3% in human patients. Oral bioavailability is 60% in dog and approximately 50% in the rat, which is similar to the value of 63% reported for man at therapeutic doses. Mean plasma clearance values were 13 ml min-1 kg-1 (dogs), 30 ml min-1 kg-1 (rats) and 1.2 ml min-1 kg-1 (human subjects). Mean plasma half-life values were 5 h in dogs and 1.2 h in rats: a value of 9 h was reported for human volunteers (cf. 2.5 h for prazosin). The long plasma half-life of doxazosin provides the basis for once-daily dosing.

A series of phenylglyoxylic acids is described, many of which are able to promote carbohydrate oxidation in muscle tissue, thereby favorably altering the carbohydrate/fatty acid balance in situations where fatty acid utilization is elevated. Such situations are reported to occur in ischemic heart disease, particularly following myocardial infection. In an attempt to effectively deliver the phenylglyoxylic acids to the site of action within the cell, the L-(+)-phenylglycines were employed as prodrugs. These are known to be transaminated to phenylglyoxylic acids. L-(+)-2-(4-Hydroxyphenyl)glycine (25, oxfenicine) has been selected for clinical evaluation.

1. The major metabolite of dazoxiben in human urine was isolated by XAD-2 and silica chromatography, then identified as a glucuronide conjugate by mass spectrometry and n.m.r. spectroscopy. 2. Levels of dazoxiben and the metabolite in plasma were measured by extraction onto activated charcoal, followed by h.p.l.c. analysis. 3. The method has been used to determine the pharmacokinetics of the drug and metabolite, after intravenous and oral administration of single 100 mg doses to man.

Bioavailability of capsule and tablet formulations of tolamolol were compared by measuring plasma concentration of tolamolol and reduction in maximum exercise heart rate over a period of twelve hours in eight healthy subjects in a two-way cross-over study. Tolamol was absorbed more rapidly from capsules than from tablets; this did not result in any significant difference in the reduction in maximum exercise heart rate between the two formulations. There was no significant difference between area under curve of reduction in exercise tachycardia and area under curve of plasma concentration of tolamolol for the two formulations. Reduction in maximum exercise heart rate was related to logarithm of plasma concentration of tolamolol between two and twelve hours after both formulations.

1. [3H, 14C]Tolamolol was well absorbed after oral administration to mice, rats, guinea-pigs, rabbits and dogs. 2. The major route for excretion of radioactivity by mice, rats and guinea-pigs was the faeces; in rabbits the major route was the urine. Dogs excreted similar amounts of radioactivity by both routes. Biliary excretion of radioactivity by the rat and guinea-pig was demonstrated. 3. Tolamolol was extensively metabolized by all five species. The major metabolite in mice, rats, guinea-pigs and rabbits was the product of hydroxylation of the tolyl ring, which was excreted as such as the glucuronide and sulphate conjugates. 4. In the dog the major metabolite was the acid resulting from hydrolysis of the carbamoyl group. This acid was also excreted by the rabbit, but was only a minor metabolite in the other species studied.

Pharmacokinetic and physiological variables were measured in six healthy subjects after intravenous and oral administration of tolamolol. 2. After intravenous injection of tolamolol (20 mg), there was a biphasic decline both in plasma concentration and attenuation of maximum exercise tachycardia. First and second phase half-lives of plasma concentration were 7 min and 2.5 h respectively. WReduction of maximum exercise tachycardia declined from 32 beats/min at 2 h to 19 beats/min at 8 hours. Clearance of tolamolol from blood ranged from 0.8-1.41 min-1. 3. After the oral administration of tolamolol (100 mg), the average volume of distribution was 220.1 and plasma concentration half-life 1.8 hours. After ten eight-hourly doses of 100 mg there was no accumulation of tolamolol and the half-life of plasma clearance was unchanged. 4. Hydroxytolamolol was detected in plasma in two of six subjects after oral tolamolol. 5. There was a significant positive correlation between reduction in maximum exercise heart rate and logarithm of plasma concentration of tolamolol after both oral and intravenous administration.