W. Dieterle

Biotransformation and pharmacokinetics of sulfinpyrazone (Anturan®) in man

SummaryThe absorption, biotransformation and elimination of sulfinpyrazone, 1,2-diphenyl-3,5-dioxo-4-(2′-phenylsufinylethyl)-pyrazolidine, have been studied by administration of single 200 mg oral doses of a14C-labelled preparation to two male volunteers. Absorption from the gastro-intestinal tract was rapid and complete and the plasma concentration of unchanged drug reached maximum values of 22.67 and 13.04 µg/ml, respectively, after 1 – 2 hours. The elimination half-life in the two subjects, calculated from the decline between 3 and 8 hours, was 2.7 and 2.2 hours. The integrated concentration of unchanged sulfinpyrazone in plasma, estimated from the area under the concentration curves (AUC), was almost as high as that of total14C-substances, so the proportion of metabolized drug in plasma was low. In no case did the AUC of the three specifically determined metabolites, i.e. the sulphone G 31 442, the “para-hydroxy”-compound G 32 642 and the “4-hydroxy”-compound GP 52 097, exceed 4% of the sulfinpyrazone value. More than 95% of whole blood radioactivity was confined to plasma. The oral dose was rapidly and completely excreted, since within 4 days more than 95% was recovered, 85% from urine and 10% from faeces. A large proportion of the dose was excreted as unchanged drug in the two volunteers: 51 and 54% of total urinary radioactivity was present as sulfinpyrazone; 8.2 and 8.8% was present as “para-hydroxy”-metabolite, 2.7 and 3.0% as sulphone-metabolite, and 0.6 and 0.8% as “4-hydroxy”-metabolite. About 30% of urinary radioactivity consisted of highly polar metabolites. Spectroscopy of them showed that they were the C-β-glucuronides of sulfinpyrazone (28%) and the corresponding sulfone (2%). In these metabolites the C(4) of the pyrazolidine ring was directly attached to glucuronic acid, and thus they represent a new type of biosynthetic conjugate.

Biotransformation and pharmacokinetics of acenocoumarol (Sintrom) in man.

SummaryThe absorption, biotransformation and elimination of the anticoagulant acenocoumarol, 3-[α- (4′-nitrophenyl)-β-acetylethyl]-4-hydroxycoumarin, have been studied by oral administration of 12 mg of a14C-labelled preparation to two male volunteers. Absorption from the gastro-intestinal tract was rapid and the plasma concentration of unchanged drug reached a maximum of 169 and 412 ng/ml, respectively, after 3 hours. The elimination half-life in the two subjects, calculated from the decline between 6 and 24 h, was 8.7 and 8.2 hours. A constant proportion of 98.7% of the drug was bound in vitro to serum proteins over a concentration range of 0.021–8.34 µg/ml, with little interindividual variation. The major portion of the binding was to human serum albumin (97.5%) at two classes of binding sites: association constant K1=1.04×105 l/mole (n1=1) and K2=5.55×103 l/mole (n2=4). In addition to unchanged acenocoumarol, four metabolites were determined in plasma by isotope dilution techniques: the amino-, acetamido-, alcohol1- and alcohol2-metabolites. Of them, the amino-metabolite showed the highest concentration, namely 278 ng/ml, after 6 h in Subject A, and 163 ng/ml after 10 hours in Subject B. Judged from the integrated concentrations, the compounds analyzed accounted for 76 and 89%, respectively, of the total radioactivity in plasma. All the metabolites detected in plasma showed anticoagulant activity when tested in mice. The quantities of the metabolites excreted in urine from 0–120 hours were (Subject A/Subject B): acenocoumarol 0.3/0.2%, amino-metabolite 12.3/7.7%, acetamido-metabolite 19.0/11.1%, alcohol1-metabolite 4.6/9.0%, alcohol2-metabolite 1.7/4.4%, 6-hydroxy-metabolite 6.9/18.3% and 7-hydroxy-metabolite 14.0/22.2%.

Binding of chlorthalidone (Hygroton®) to blood components in man

SummaryThe binding of chlorthalidone to human blood components has been studied in vitro. The drug was preferentially taken up by red blood cells, the partition ratio between plasma and the cell fraction being dependent on the drug concentration. When the concentration of chlorthalidone in blood was less than 15–20 µg/ml, more than 98% of the compound was bound to red cells. Increasing the concentration resulted in an abrupt change of the partition ratio in favour of plasma, which indicates a saturable receptor for chlorthalidone in red cells, namely carbonic anhydrase (HCA). The association constant of the drug-enzyme complex KassHCA was 2.76×106 l/mole. For the two major isoenzymes of carbonic anhydrase, HCA-B and HCA-C, the association constants were different: KassHCA-B=2.43×106 l/mole and KassHCA-C=5.69×106 l/mole. The number of binding sites n=1 in all cases. In human serum at 37°C, over a concentration range of 0.02–7.7 µg/ml, 75.7% of chlorthalidone was bound to proteins. The major portion of the binding was to albumin (HSA), the association constant of the complex KassHSA=1.18×103 l/mole and the number of binding sites n=4. The much higher association constant of chlorthalidone with HCA than with HSA can account for selective uptake of the drug by red cells.

Pharmacokinetics of diclofenac and five metabolites after single doses in healthy volunteers and after repeated doses in patients.

1. The kinetics of diclofenac (I) and five of its metabolites (II-VI) were investigated in three healthy volunteers and in six patients. Compounds I-VI were measured by capillary column gas chromatography in plasma and urine. 2. After a single 100 mg dose of diclofenac sodium to volunteers, the drug was absorbed rapidly and showed peak plasma levels of 10-12 nmol/g. The maximum concentrations of five metabolites were comparatively low (0.36-2.94 nmol/g). The mono- and dihydroxy metabolites (II-V) had apparent terminal half-lives similar to that of I (1-3 h), but the hydroxymethoxy metabolite (VI) had a half-life of about 80 h. Renal elimination of VI within 96 h was about 1% of dose and that of I-VI was 36% (free plus conjugated). 3. Following daily treatment with 2 x 75 mg of an experimental sustained release formulation to patients for 6-10 months, steady-state trough concentrations of I-V in plasma were low (average values: 0.23-0.57 nmol/g). The mean trough concentration of VI was comparatively higher at 3.69 +/- 0.91 nmol/g presumably reflecting its accumulation. Despite this it is unlikely to contribute to the drugs therapeutic activity, since it has been shown in laboratory tests to be devoid of anti-inflammatory activity.

Preparative reversed-phase chromatography of polar and non-polar metabolites on columns packed with micronized xad-2 resin

Abstract Coarse-grade XAD-2 resin has been micronized (medium particle size 12 μm) and used as a support for preparative high-resolution liquid chromatography. By equilibration with single-phase mixtures of water, a lower aliphatic alcohol and a hydrophobic solvent a reversed-phase partition system is formed in situ . This chromatographic technique is characterized by a large capacity, a high power of resolution and a high degree of selectivity and reproducibility, and versatile application in the isolation of polar and non-polar drug metabolites or naturally occurring compounds from complex mixtures is possible. The examples given relate particularly to the separation of water-soluble, non-extractable compounds, such as glucuronides or other conjugates, and amino acids on large-diameter columns (up to 2.5 cm).

Pharmacokinetics and pharmacodynamics of the ace inhibitor benazepril hydrochloride in the elderly

SummaryThe pharmacokinetics and pharmacodynamics of a single oral dose benazepril·HCl 10 mg have been studied in 15 healthy volunteers aged 65 to 80 y. The kinetics of unchanged benazepril and its active metabolite benazeprilat did not differ significantly in males and females, so the combined kinetic data from all 15 elderly subjects were compared with a historical control group of 19–32 year-old healthy men treated in the same way.The disposition of benazepril was not affected by age. The time to maximum plasma concentration, tmax (0.5 h) and elimination half-life (0.6 h) in the elderly were the same as in young subjects. The kinetics of benazeprilat was slightly changed in the elderly; although its tmax (1.5 h) was not affected, Cmax and the AUC were 20–40% greater. The elimination half-life of benazeprilat during the first 24 h after doing in the elderly was increased by about 20% to 3.2 h. The renal plasma clearance of benazeprilat (18.1 ml·min−1) was about 20% smaller than in the young subjects. An average of 18.5% of the dose was recovered as benazeprilat in the 24 h urine from the elderly subjects, which was similar to the recovery in the young subjects. Both benazepril and benazeprilat were highly bound to serum proteins (96 and 95%, respectively).Mean systolic and diastolic blood pressures in the elderly were reduced by a maximum of 37/16 mm Hg at 6 h, in association with a small rise in pulse rate.Treatment was generally well tolerated. Three of the 15 subjects reported clinical adverse experiences judged to be possibly drug related, namely headache, abdominal pain and cold extremities.

Determination of a new angiotensin converting enzyme inhibitor and its active metabolite in plasma and urine by gas chromatography-mass spectrometry.

A specific and sensitive gas chromatographic-mass spectrometric method for the simultaneous quantification of unchanged 3-[( 1-ethoxycarbonyl-3-phenyl-(1S)-propyl]amino)-2,3,4,5-tetrahydro-2-oxo-1- 1-(3S)-benzazepine-1-acetic acid (I) and its active metabolite, the dicarboxylic acid (II), in plasma and urine has been developed and validated. 2H5-labelled analogues of I and II were used as internal standards. The compounds were isolated from plasma and urine under acidic conditions using XAD-2 resin or Extrelut 1 columns. Following derivatization with diazomethane, the samples were analysed by packed-column gas chromatography-electron-impact mass spectrometry with selected-ion monitoring. The analysis of spiked plasma and urine samples demonstrated the good accuracy and precision of the method, which is suitable for use in pharmacokinetic and bioavailability studies with the new angiotensin converting enzyme inhibitor prodrug I.HCl in humans.

Multiple inverse isotope dilution assay for oxprenolol and nine metabolites in biological fluids

An isotope dilution assay for the specific determination of 14C-labelled oxprenolol and nine of its metabolites in the same biological sample is described. After addition of unlabelled carriers to the sample, oxprenolol and the metabolites were isolated by base- and acid-specific extraction and separated by normal-phase high-performance liquid chromatography using two different mobile phases. Quantitation of the various peaks was performed by on-line ultraviolet detection at 275 nm and off-line radiometry by liquid scintillation counting. Endogenous compounds and unknown metabolites did not interfere in the assay. The analysis of rat and dog blood, plasma and urine samples spiked with [14C]oxprenolol hydrochloride, showed mean recoveries between 98.7 and 99.8%. The assay was used to investigate the metabolic fate of [14C]oxprenolol in the dog. Analyses of blood and urine demonstrated the quantitative significance of the various metabolites in the biotransformation of oxprenolol.

The disposition and metabolism of 14C-oxprenolol·HCl in man

The disposition and metabolism of oxprenolol have been investigated in two healthy male volunteers, following a single 160 mg oral dose of racemic 14C-labelled oxprenolol. Absorption was rapid and complete. Peak blood concentrations of total radioactivity were 8.83 and 8.21 nmol X g-1 after 1 and 1.5 h in the two subjects. After 4 days 93.4 and 81.9% of the dose was excreted in urine, and a total of 96.6 and 84.5% found in the excreta. Mean peak blood concentrations of unchanged R(+)- and S(-)- oxprenolol were 0.83 and 0.81 nmol X g-1. Maximal concentrations of the glucuronides of the R(+)- and S(-)- isomers were 1.98 and 3.51 nmol X g-1. The mean half-lives of both oxprenolol enantiomers were 1.8 h, those of their glucuronides were 3.2 h (R(+] and 4.6 h (S(-]. Unchanged oxprenolol and the oxprenolol glucuronides constituted 11.4 and 66.5% of the area under the blood concentration-time curve (AUC, 0-24 h) of total radioactivity. The AUC-ratio of R(+) to S(-) was 1.19 for free oxprenolol and 0.36 for the glucuronides. Free metabolites II-X represented together 4.3% of 14C-AUC, and their glucuronides 15.2%. In urine, 1.8 and 1.0% of the total radioactivity was present as unchanged R(+)- and S(-)- oxprenolol, respectively. The glucuronides of the enantiomers accounted for 24.5 and 26.5%. The percentages of free 4- and 5-hydroxy oxprenolol were 0.7 and 2.4% while those of their glucuronides were 12.3 and 7.5%. Metabolites IV-X constituted together 6.2% in free form and 5.3% in conjugated form. In conclusion, the good mass balances in blood and urine has enabled the comprehensive and quantitative description of the metabolic fate of oxprenolol in man. Oxprenolol is extensively metabolized, direct O-glucuronidation being the major metabolic pathway and oxidative reactions minor ones. The disposition of the oxprenolol enantiomers revealed no remarkable stereoselective differences.

Multiple inverse isotope dilution assay for the stereospecific determination of R(+)- and S(-)-oxprenolol in biological fluids.

An isotope dilution assay has been developed for the determination of both oxprenolol enantiomers in biological samples after administration of the racemic 14C-labelled mixture. The enantiomers were reacted with optically pure S(-)-1-phenylethyl isocyanate and the diastereoisomeric urea derivatives formed were separated by normal-phase high-performance liquid chromatography. Quantitation was performed by on-line ultraviolet detection at 275 nm and off-line radiometry. Endogenous compounds and oxprenolol metabolites did not interfere with the assay. Analysis of water and blood, plasma and urine samples of rats and dogs spiked with [14C]oxprenolol hydrochloride showed mean recoveries for R(+)-oxprenolol hydrochloride of 99.2% (water), 99.3% (blood), 99.1% (plasma) and 97.9% (urine), and for S(-)-oxprenolol hydrochloride of 99.7% (water), 98.1% (blood), 98.6% (plasma) and 96.9% (urine). In a pilot study, the presented method was used to investigate the metabolic fate of the enantiomers in two dogs dosed orally with racemic [14C]oxprenolol hydrochloride (3 mg/kg). The results show that conjugation of R(+)-oxprenolol exceeded that of S(-)-oxprenolol.