Clinical analysis of sampatrilat, a combined renal endopeptidase and angiotensin-converting enzyme inhibitor I: assay in plasma of human volunteers by atmospheric-pressure ionisation mass-spectrometry following derivatisation with BF3-methanol.

Sampatrilat is a dual inhibitor of angiotensin converting enzyme (ACE) and neutral endopeptidase (NEP) under development for the treatment of hypertension and congestive heart failure. In order to support the early clinical development (with oral administration and an expected low bioavailability), a sensitive and selective assay was required. A method for plasma was developed and validated employing HPLC APCI MS-MS. The plasma samples were extracted on solid-phase extraction cartridges, derivatised with BF3-methanol, diluted, extracted again and then subjected to HPLC APCI-MS-MS. Derivatisation was necessary because the two carboxyl group in the molecule prevented efficient ionisation in the heated nebuliser source. The calibration range was from 0.5 to 20 ng ml(-1) and the lower limit of quantification was 0.5 ng ml(-1). Imprecision and inaccuracy were determined on three separate occasions at three concentrations (0.5, 5 and 20 ng ml[-1]) and shown to be lower than 10% in every case.

Clinical analysis of sampatrilat, a combined renal endopeptidase and angiotensin-converting enzyme inhibitor II: assay in the plasma and urine of human volunteers by dissociation enhanced lanthanide fluorescence immunoassay (DELFIA).

Sampatrilat is a dual inhibitor of angiotensin converting enzyme (ACE) and neutral endopeptidase (NEP) under development for the treatment of hypertension and congestive heart failure. In order to support the early clinical development (with oral administration and an expected low bioavailability), a sensitive and selective assay was required. An HPLC-atmospheric-pressure chemical ionisation mass-spectrometric (HPLC-APCI-MS-MS) assay had been already validated (R.F. Venn et al., J. Pharm. Biomed. Anal., in press), but due to its low throughput an alternative method was sought. As the molecule is peptide-like and not metabolised, we believed the immunoassay approach was appropriate. Thus we developed an immunoassay for the compound using time-resolved fluorescence as an end-point (DELFIA) with lower limits of quantification of 0.2 ng ml(-1) for the plasma assay and 5 ng ml(-1) for the assay in urine. This assay is a 96-well plate based competitive immunoassay; the end-point is the determination of a (non-radioactive) europium label by time-resolved fluorimetry. Sampatrilat is labelled with chelated europium via isothiocyanate chemistry. The advantage of this assay is its extremely high throughput, allowing rapid analysis of many thousands of samples. The DELFIA method was successfully cross-validated with the HPLC-APCI-MS-MS method.

Rapid, solid phase extraction technique for the high-throughput assay of darifenacin in human plasma.

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%.

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.

Microbore liquid chromatography coupled to a flow fast atom bombardment probe for the on-line detection of the Tyr-Pro cleavage of a nonapeptide by recombinant HIV-1 protease.

The nonapeptide Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln has been reported as a model substrate for an aspartyl protease produced by the human immunodeficiency virus (HIV-1). Cleavage of this peptide at the Tyr-Pro linkage to produce tetra- and pentapeptide fragments is the basis of high-performance liquid chromatographic assays to detect HIV-1 protease activity. Confirmation of the cleavage site has been proved by using microbore liquid chromatography coupled to a dynamic fast atom bombardment interface. Comparison with fortified control incubates indicates that an approximate stoichiometric amount of the tetrapeptide was formed from the nonapeptide, confirming that the cleavage of the substrate by HIV-1 protease is both specific and quantitative.

Fluconazole levels in plasma and vaginal secretions of patients after a 150-milligram single oral dose and rate of eradication of infection in vaginal candidiasis.

Mean peak concentrations of fluconazole in plasma and vaginal secretions of females after a 150-mg single oral dose were shown to be 2.82 micrograms/ml and 2.43 micrograms/g, respectively. Our results indicate that clinically efficacious concentrations of fluconazole in vaginal secretions are easily achieved after this single oral dose.

Biotransformation of amlodipine. Identification and synthesis of metabolites found in rat, dog and human urine/confirmation of structures by gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry.

Metabolism of the dihydropyridine calcium antagonist (R,S)-2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-3-ethoxycarbony l-5- methoxycarbonyl- 6 -methyl- 1,4-dihydropyridine (amlodipine) has been studied in animals and man using 14C-labelled drug. The metabolite patterns are complex; 18 metabolites have been isolated from rat, dog and human urine. Based on chromatographic and mass-spectral evidence, structures have been proposed for the main metabolites and confirmed by synthesis of unambiguous reference compounds. Comparison of all reference compounds and isolated metabolites was made by gas chromatography-mass spectrometry pressure liquid chromatography on-line thermospray-mass spectrometry of underivatised compounds directly in urine. The metabolites are largely pyridine derivatives. The methods used in structure designation are presented, along with the proposed route of metabolism, which indicates that the metabolic pattern for amlodipine in man has features in common with those of both rat and dog.

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.

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.

The metabolism and pharmacokinetics of amlodipine in humans and animals.

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)