Inhibition of endopeptidase EC 24.11 in humans. Renal and endocrine effects.

The effects of an orally active inhibitor (UK 79300) of the neutral metalloendopeptidase EC 3.4.24.11 were investigated in six healthy male volunteers maintained on a constant diet (150 mmol sodium/day and 80 mmol potassium/day). Subjects were studied in a random order, single-blind study on two occasions, each 48 hours in length, when they were given UK 79300 (25 or 50 mg p.o.) or placebo at 12-hour intervals (each agent for 24 hours). The endopeptidase inhibitor enhanced plasma concentrations of atrial natriuretic factor in association with suppression of both plasma renin activity and aldosterone concentrations. Twenty-four-hour urinary excretion of sodium was doubled by UK 79300, and the urinary excretion rates of phosphorus, atrial natriuretic factor immunoreactivity, and cyclic guanosine monophosphate were also significantly enhanced, whereas urinary aldosterone excretion was halved. The profile of biological effects closely paralleled those previously reported with low dose infusions of atrial natriuretic factor in humans and animals. Therapeutic trials of such inhibitors are now indicated for hypertension or heart failure together with further studies to clarify the underlying mechanisms of action.

The atriopeptidase inhibitor UK 69,578 increases atrial natriuretic factor and causes a natriuresis in normal humans.

The endopeptidase EC 3.4.24.11 (atriopeptidase) degrades atrial natriuretic factor (ANF). Intravenous administration of UK 69,578 (0.025 to 10.0 mg/kg), a new specific atriopeptidase inhibitor, in 16 normal volunteers produced a two- to three-fold rise in endogenous ANF. Peak levels were reached within 2 h declining to control values by 8 h. The rise in ANF was associated with an increase in urine volume and mean urinary sodium excretion rose from 64.9 mmoles/8 h after placebo to 116.1 mmoles/8 h after 10 mg/kg UK 69,578. Despite the natriuresis, plasma active renin concentration was suppressed for up to 8 h. We conclude that inhibition of the endopeptidase EC 3.4.24.11 in humans elevates endogenous ANF and causes a natriuresis and may offer a novel therapeutic approach to the treatment of hypertension and cardiac failure.

A validation study comparing accelerator MS and liquid scintillation counting for analysis of 14C-labelled drugs in plasma, urine and faecal extracts.

A comparison has been made between accelerator mass spectrometry (AMS) analysis and liquid scintillation counting (LSC) of plasma, urine and faecal samples containing 14C-labelled drugs. In an in vitro study in which human plasma was spiked (the term spiked is used in Section 2.6) with 14C-Fluconazole (14C-FL) over a concentration range of 0.1-2.5 dpm/ml, a correlation coefficient of 0.999 was determined for AMS analysis versus extrapolated LSC data. No significant day to day (or inter-day)variation was seen (P < 0.05 by ANOVA). Coefficients of variation for these analyses ranged from 2.68 to 6.50%. In vivo studies in which rats were given a high (11.5 microCi/kg) or low (18.1 nCi/kg) radioactive dose (to model an exposure of 0.9 microSievert to man) of 14C-Fluticasone propionate(14C-FP) showed that there was also a good correspondence between AMS and LSC data. A mass balance study in a single the faeces by 96 h; less than 1% of the administered dose was excreted in the urine. The limit of reliable measurement of drug related material, above background concentrations, by AMS analysis in this study was approximately 0.1 dpm/ml for plasma, 0.01 dpm/ml for urine without any sample extraction or concentration and 0.01 dpm/ml for faecal extracts. The data reported here demonstrate that AMS is an ultrasensitive and reliable method for analysing 14C-labelled drugs in human and animal body fluids.

The metabolism and kinetics of doxazosin in man, mouse, rat and dog.

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.

The sensitive determination of abanoquil in blood by high-performance liquid chromatography/atmospheric pressure ionization mass spectrometry.

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.

Pharmacokinetics and metabolism of darifenacin in the mouse, rat, dog and man.

1. Following intravenous administration to animals at 2.5 mg/kg, darifenacin exhibited terminal plasma half-lifes < 2 h due to high plasma clearance (with respect to blood flow) and volumes of distribution greater than total body water. 2. Following oral administration to animals at doses > 4 mg/kg there was evidence of saturation of clearance since oral AUCs exceeded those expected from the high plasma clearances. In addition, terminal plasma half-lifes were greater than those estimated from intravenous administration. 3. In man, oral clearance was high with respect to liver blood flow. 4. Following oral administration of the radiolabelled drug to animals and man, unchanged darifenacin was only a minor component of the faecal radioactivity indicating that darifenacin was well absorbed from the gut. 5. Darifenacin was metabolized by three main routes in all species: monohydroxylation, oxidative dihydrobenzfuran ring opening and N-dealkylation. There were no marked species differences in the metabolism of darifenacin.

Formation and pharmacokinetics of the active drug candoxatrilat in mouse, rat, rabbit, dog and man following administration of the prodrug candoxatril.

1. Candoxatrilat, an active neutral endopeptidase inhibitor, was released rapidly from the inactive prodrug candoxatril in vivo in mouse, rat, rabbit, dog and man. 2. Oral doses of [14C]-candoxatril were cleared rapidly, mostly by ester hydrolysis to candoxatrilat, in mouse, dog and man. A complementary intravenous study in man with [14C]-candoxatrilat showed that the active drug was virtually completely renally cleared. Neither candoxatril nor candoxatrilat underwent chiral inversion in man. 3. Systemic availability of candoxatrilat from the oral prodrug was estimated to be 88, 53, 42, 17 and 32% in mouse, rat, rabbit, dog and man respectively. Plasma clearance of candoxatril was too rapid to enable pharmacokinetic parameter calculation in mouse and rabbit; for man, the apparent oral clearance was 57.9 ml/min/kg and the elimination half-life was 0.46 h. 4. For intravenous candoxatrilat, total plasma clearance values were 32, 15, 5.5, 5.8 and 1.9 ml/min/kg for mouse, rat, rabbit, dog and man respectively. Renal clearance values were 8.7, 7.2, 2.9 and 1.7 ml/min/kg for mouse, rat, dog and man and these approximate to the respective glomerular filtration rates. Allometric scaling with respect to bodyweight across the species allowed reasonable prediction of the above two clearance parameters in man.

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.