Pharmacokinetics and metabolism of formestane in breast cancer patients.

Formestane (Lentaron(R), 4-hydroxyandrostenedione) is a steroidal aromatase inhibitor used for treatment of advanced breast cancer. Clinically, it is administered as a depot form once fortnightly by intramuscular (i.m.) injection. To investigate the pharmacokinetics, bioavailability and metabolism of the drug, seven patients received single 250 mg i.m. doses of commercial formestane on Days 0, 21, 35, 49 and 63 of this trial. On Day 63, three of the patients received an additional single intravenous (i.v.) pulse dose of 1 mg of 14C-labelled formestane. The plasma kinetics after i.m. dosing confirmed a sustained release of formestane from the site of injection. Within 24-48 h of the first dose, the circulating drug reached a C(max) of 48.0+/-20.9 nmol/l (mean+/-S.D.; N=7). At the end of the dosing interval, after 14 days, the plasma concentration was still at 2.3+/-1.8 nmol/l. The kinetic variables did not significantly change during prolonged treatment. Intramuscular doses appear to be fully bioavailable. Following i.v. injection of 14C-formestane, the unchanged drug disappeared rapidly from plasma, the terminal elimination half-life being 18+/-2 min (N=3). Plasma clearance, CL was 4.2+/-1.3 l/(h kg) and the terminal distribution volume V(z) was 1.8+/-0.5 l/kg. The drug is mainly eliminated by metabolism, renal excretion of metabolites accounting for 95% of dose. The excretory balance of 14C-compounds in urine and faeces totals up to 98.9+/-0.8% of the i.v. dose after 168 h. The 14C-compounds in plasma and urine were separated by HPLC, and three major metabolites were submitted to structural analysis by MS, NMR and UV spectroscopy. One of the metabolites is the direct 4-O-glucuronide of formestane. The other two represent 3-O-sulfates of the exocons 3beta,4beta-dihydroxy-5alpha-androstane-17-one and 3alpha,4beta-dihydroxy-5alpha-androstane-17-one, their ratio being 7:3. These exocons are formed by stereoselective 3-keto reduction, accompanied by reduction of the 4,5-enol function. The exocons do not inhibit human placental aromatase activity in vitro.

Characterization of a novel diclofenac metabolite in human urine by capillary gas chromatography-negative chemical ionization mass spectrometry.

A sensitive analytical method was developed to characterize diclofenac metabolites in small amounts of body fluids. Desalted and lyophilized urine samples were extracted with supercritical carbon dioxide directly or after acidic hydrolysis. The extracts were derivatized with N-tert.-butyldimethylsilyl-N-methyltrifluoroacetamide. The derivatives were separated by capillary gas chromatography and identified by negative chemical ionization mass spectrometry. Full mass spectra were obtained at a level of 1.10(-9) g/ml. With direct extraction, the metabolites could be analysed in one step as open-chained acids and as (cyclic) oxindoles. By acidic hydrolysis the conjugates were transformed to the oxindoles. With both methods, a new main metabolite, [2-[2,6-dichloro-4-hydroxy-3-methoxyphenyl)amino]phenyl]acetic acid, was identified The mechanism of its formation is discussed.

The disposition of [14C]-labelled benazepril HCl in normal adult volunteers after single and repeated oral dose.

1. The disposition of [14C]-labelled benazepril HCl, an ACE-inhibitor, was studied in four normal adult volunteers after a single oral dose of 20 mg and after repeated doses of 20 mg once daily for 5 days. Radioactivity was measured in plasma, urine and faeces. The prodrug ester benazepril and the pharmacologically active metabolite benazeprilat were determined quantitatively in plasma and urine by a g.c.-m.s. method. The pattern of metabolites in urine was analysed semiquantitatively by h.p.l.c.-radiometry. 2. After a single oral dose at least 37% was absorbed, as indicated by urinary recovery. The peak plasma concentration of benazepril (0.58 +/- 0.13 nmol/g (SD] was observed at 0.5h after dose, indicating rapid absorption. Peak concentrations of radioactivity (1.88 +/- 0.48 nmol/g) and of active benazeprilat (0.84 +/- 0.25 nmol/g) were observed at 1 h after dose, demonstrating rapid bioactivation. 3. The area under the plasma curve (AUC0-96 h) of total radioactivity amounted to 9.7 +/- 1.1 (nmol/g)h, 5% of which was accounted for by benazepril and about 50% by benazeprilat. 4. Over 9 days 96.8 +/- 0.5% of the dose was excreted in urine and faeces. Urinary excretion accounted for 37.0 +/- 6.0% of the dose, 80% of which was recovered in the first 8 h after dosing. 5. In urine, only 0.4% of the dose (1% of the radioactivity) was excreted as unchanged benazepril, indicating that the compound was extensively metabolized. Benazeprilat accounted for 17% of the dose (about half of the radioactivity; 0-96 h). Glucuronide conjugates of benazepril and benazeprilat constituting approximately 11% and 22% of the radioactivity (about 4% and 8% of the dose; 0-48 h) were tentatively identified. 6. Repeated oral treatment with benazepril HCl did not influence the pharmacologically relevant kinetics and disposition parameters.

Metabolic characteristics of oxcarbazepine (®trileptal) and their beneficial implications for enzyme induction and drug interactions.

Hepatic oxygenases of the cytochrome P-450 family play a major role in the clearance of various anti-epileptic drugs. These enzymes are susceptible both to induction and to inhibition. Phenytoin, carbamazepine (CBZ), primidone, and phenobarbitone, for instance, are potent enzyme inducers. Other drugs, such as chloramphenicol, propoxyphene, verapamil, and viloxazine, inhibit cytochrome P-450. Pharmacokinetic behaviour is thus often altered, especially in combined medication, so that the dosage has to be re-adjusted if an optimum therapeutic outcome is to be ensured.Oxcarbazepine (OXC) is a keto analogue of CBZ. In the human liver the keto group is readily reduced, and the resulting monohydroxy metabolite is cleared by glucuronidation. The two enzymes mediating these reactions, i.e. aldo-keto reductase and UDP-glucuronyltransferase, do not depend on cytochrome P-450. The monohydroxy metabolite is the major active substance in plasma. Its elimination is not enhanced by OXC. Moreover, OXC seems to have little effect on cytochrome P-450. Aldo-keto reductases and glucuronyltransferases are in general less sensitive to induction and inhibition than are P-450 dependent enzymes. On the whole, OXC possesses very little potential for metabolic drug interactions, and thus differs favourably from other anti-epileptic drugs.

A new metabolite of diclofenac sodium in human plasma.

1. Among the phenolic metabolites of diclofenac in human plasma, an unknown compound (metabolite VI) was detected by h.p.l.c. and g.c. methods. This was also found in baboon plasma. 2. Metabolite VI was identified as 3'-hydroxy-4'-methoxy diclofenac by mass and n.m.r. spectroscopic analysis. Comparison with synthetic reference compound confirmed its structure. 3. In plasma, metabolite VI persists much longer than do unchanged diclofenac and the other phenolic metabolites. In urine, metabolite VI and its conjugates are excreted in trace amounts only. 4. A synthetic sample of metabolite VI was shown to be virtually inactive in animal models of inflammation and pain.

Single-dose kinetics of an enteric-coated formulation of carbamazepine-10,11-epoxide, an active metabolite of carbamazepine.

The kinetics of an enteric-coated formulation of carbamazepine-10,11-epoxide (CBZ-E) were studied in healthy subjects. A single oral dose of 100 mg of CBZ-E was given to eight subjects. Four of them were also given a single oral dose of 200 mg of CBZ-E. Plasma concentrations of CBZ-E and urinary excretion of the end metabolite trans-10,11-dihydroxy-10,11-dihydro-carbamazepine (trans-CBZ-diol) were determined by high performance liquid chromatography. Plasma kinetics of CBZ-E fitted an open one-compartment model with plasma elimination half-life of 7.4 +/- 1.8 h (mean +/- SD). The clearance was 105 +/- 17 ml/kg/h and the apparent volume of distribution 1.1 +/- 0.2 L/kg assuming complete bioavailability. There was no indication of dose-dependent elimination of CBZ-E. The recovery of trans-CBZ-diol in urine collected for 3 days was 67 +/- 9% of the given dose. This enteric-coated formulation may thus in the future be used for the evaluation of the clinical effects of CBZ-E in patients.

Tricyclic drugs: potent mutagenicity of traces of a nitroarene formed in the reaction of opipramol with nitrite.

The tricyclic psychotropic drug opipramol (Insidon) reacts in vitro with sodium nitrite in acidic solution to form products including mutagens for Salmonella typhimurium TA98 and TA100. Two aspects are particularly noteworthy. The strong mutagenicity of the crude reaction mixture is almost exclusively due to a compound which is present only in trace quantities (less than 0.1%). This mutagen was identified as a nitroarene, 4-[3-(2-nitro-9-(10H)-acridinon-10-yl)propyl]-1-piperazine-ethanol . Hence, while the formation of carcinogenic N-nitroso compounds from the interaction of nitrogen-containing compounds with nitrite is well known, the present study demonstrates the formation of a highly mutagenic C-nitro compound in the presence of nitrite.

Biotransformation of oxaprotiline: isolation and identification of metabolites in urine of rat and dog.

The biotransformation of oxaprotiline has been investigated in rat and dog after oral administration of racemic 14C-labelled oxaprotiline X HCl. Rats excreted 28% dose in urine within 120 h and dogs 32% within 96 h. The metabolites were isolated by liquid chromatography and their structures elucidated by spectroscopic methods. In both species, oxaprotiline is extensively metabolized. Principal metabolic transformations are aromatic hydroxylations and formation of aromatic hydroxy-methoxy derivatives, N-demethylation, deamination and direct O-glucuronidation. Most of the primary metabolites formed by functionalization reactions occur in both free and glucuronidated form. In the rat, diastereoisomeric 3-hydroxy metabolites and the corresponding phenolic glucuronides are predominant. Products of deamination are minor, and products of direct O-glucuronidation are not detectable. In the dog, biotransformation is more complex. Major metabolites are diastereoisomeric 2- and 3-hydroxy compounds and the corresponding phenolic glucuronides. Oxidations in the side-chain and direct O-glucuronidation are minor metabolic pathways.

The metabolism of 14C-oxcarbazepine in man.

The disposition of the new anti-epileptic agent oxcarbazepine (10,11-dihydro-10-oxo-5H-dibenz[b,f]azepine-5-carboxamide) has been studied in two healthy volunteers following an oral 400 mg dose of 14C-labelled drug. The dose was excreted almost completely in the urine (94.6 and 97.1%) within six days. Faecal excretion amounted to 4.3 and 1.9% of the dose in the two subjects. In the 0-6 days urine samples the biotransformation products have been isolated and identified. 10,11-Dihydro-10-hydroxycarbamazepine (GP 47,779) and its two diastereoisomeric O-glucuronides were found as main metabolites. Taken together, they accounted for 79% of urinary 14C. Unchanged oxcarbazepine, and its sulphate and glucuronide conjugates were isolated in smaller amounts only (13%). Other minor metabolites were the trans- and cis-isomers of 10,11-dihydro-10,11-dihydroxy-carbamazepine (approximately 4%), and a phenolic derivative of GP 47,779 (less than 1%). The biotransformation of oxcarbazepine proceeds mainly by reduction to GP 47,779, and subsequent conjugation with glucuronic acid. Reduction is stereospecific, favouring the S-configuration of GP 47,779. Direct conjugation of oxcarbazepine, in the enol form, is a minor pathway. Oxidative reactions are unimportant.

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.

Isolation and stereospecific determination of the enantiomers of oxindazac by direct liquid chromatographic resolution on triacetylcellulose.

The preparation of the optically pure enantiomers of the antiphlogistic trial drug oxindazac via liquid chromatographic resolution of the corresponding tert.-butyl or benzyl ester on triacetylcellulose is described. Cleavage of the optically pure enantiomeric esters to the acids proceeds without significant racemization. The methyl ester of oxindazac is also completely resolved on the same chiral phase. Whereas oxindazac racemizes easily upon derivatization to diastereomers, no racemization is observed upon methylation to the corresponding methyl ester with diazomethane. An inverse isotope dilution method has been developed to determine both enantiomers of the drug in biological fluids after administration of 14C-labelled oxindazac. The enantiomers are converted into their methyl esters and separated on triacetylcellulose. Quantitation is performed by on-line UV detection at 290 nm and off-line radiometry. In the analysis of plasma samples, endogenous compounds do not interfere. The recoveries of [14C]oxindazac from water, rat and human plasma were 99.6 +/- 1.8% for the (+/- and 96.0 +/- 1.4% for the (-)-enantiomer. The plasma concentrations and urinary excretion of the two enantiomers were determined in a human volunteer who had received 200 mg of racemic 14C-labelled oxindazac.

Disposition and pharmacokinetics of cadralazine and individual metabolites in man.

The absorption, biotransformation and elimination of the antihypertensive drug cadralazine, 2-(3-[6-(2-hydroxypropyl)ethylamino]pyridazinyl)-ethylcarbazate , have been studied in two healthy male volunteers, following single 20 mg oral doses of the 14C-labelled preparation. Absorption was rapid and complete. In plasma total 14C-compounds reached maximum levels of 395 and 312 ng/g after 0.5 and 1.5 h in subject A and B, respectively. The levels rapidly declined to 3 and 6 ng/g after 24 h. Unchanged cadralazine constituted the major fraction, 72%, of the integrated plasma concentration (AUC, 0-24 h) of total radioactivity. As determined by an isotope dilution technique, about 1% of the AUC of plasma-14C was attributable to a pharmacologically active hydrazino-metabolite (IV) formed by decarbethoxylation and 2% to the acetylation product of the latter (V). Excretion of the radioactivity occured predominantly by the kidneys, 91 and 94% within 0-24 h in subject A and B. After 96 h 94 and 99% of the dose were found in the excreta. In the 0-48 h urine 73% of total 14C consisted of unchanged drug. The hydrazino-metabolite (IV) accounted for about 2% of urinary radioactivity, and two secondary products of the same pathway (II, V) for another 2%. Products of N-dealkylation (VI, VII) and C-oxidation (VIII) constituted together another 5%.

Indoxyl derivatives of drug metabolites.

Indoxyl derivatives were detected as minor products among the urinary metabolites of two trial drugs, a benzodiazepine (GP 55 129) and a benzophenone (CGP 11 952). Their structures were elucidated by NMR and mass spectroscopy. Presumably, metabolites containing potential aldehyde functions react spontaneously with endogenous indoxyl. Such derivatives have not hitherto been encountered in drug metabolism.

The metabolic fate of 14C-oxaprotiline X HCl in man. III. Stereospecific disposition.

The disposition of the enantiomers of oxaprotiline has been investigated after single 100 mg oral doses of racemic 14C-labelled oxaprotiline X HCl in two healthy subjects. Absorption was complete. Peak blood concentrations of total 14C were 804 and 1010 ng equiv. g-1 after 4-6 h in the two subjects. After 9 days 85 and 80 per cent of the dose were excreted in urine, and a total of 93 and 87 per cent were found in the excreta. Mean peak blood concentrations of unchanged S(+)- and R(-)-oxaprotiline amounted to 25 and 10 ng g-1 before, and 474 and 422 ng g-1 after acid hydrolysis (free plus O-glucuronide). The mean blood half-lives of the S(+) and R(-) isomers were 22 and 23 h. Direct O-glucuronidation is the major metabolic pathway and N-demethylation a minor one. The former is more marked with the S(+) isomer and the latter with the R(-) isomer. For oxaprotiline, the AUC-ration of S(+) to R(-) was 2.2 before and 1.4 after hydrolysis. For desmethyl oxaprotiline the corresponding ration was 0.8 after hydrolysis. In urine, 0.8 and 0.5 per cent of total 14C were present as unchanged S(+)- and R(-)-oxaprotiline. After acid hydrolysis of the O-glucuronides, the enantiomers account for 44.7 and 37.1 per cent. The O-glucuronides of S(+)- and R(-)-desmethyl oxaprotiline account for 4.6 and 5.7 per cent.

The metabolic fate of [14C]oxaprotiline.HCl in man. II. Isolation and identification of metabolites.

The new antidepressant agent oxaprotiline is extensively metabolized by man. Following an oral 50 mg dose of racemic [14C]oxaprotiline, most of the 14C was excreted in the urine as metabolites (greater than 98% total 14C); only 1% was excreted unchanged. Glucuronidation at the carbinol group of the molecule is the major metabolic pathway (83%). The two diastereoisomeric glucuronides were separated; the more polar O-glucuronide of S(+)-oxaprotiline predominates (44%), suggesting stereoselective disposition of the two enantiomers. Oxidative pathways are minor, and yield desmethyl oxaprotiline (10%) and 3-hydroxy R(-)-oxaprotiline (4%), both of which are conjugated with glucuronic acid. The biotransformation of oxaprotiline in man is less complex than that of other polycyclic antidepressants, which are metabolized mainly by oxidative reactions.

The metabolic fate of [14C]oxaprotiline.HCl in man. I. Disposition and preliminary pharmacokinetics.

Absorption, biotransformation and elimination of [14C]oxaprotiline.HCl have been studied after oral administration of 50 mg doses to two human subjects. Absorption was complete, and peak blood concn. of total 14C were 590 and 297 ng equiv./ml after 3-6 h in the two subjects. After 11 days, 84 and 90% dose was excreted in urine, and a total of 98% was excreted. Peak blood concn. of unchanged oxaprotiline were 16 and 19 ng/ml before, and 167 and 207 ng/ml after enzymic hydrolysis. The blood half-life in the two subjects was 23 and 29 h. The blood concn. of the desmethyl metabolite was low (2 ng equiv./ml), but also increased after hydrolysis (11-19 ng equiv./ml). Oxaprotiline was bound (83%) in vitro to serum proteins. Sixty per cent was bound to serum albumin and 20% to alpha 1-acid glycoprotein. In urine only 1% of total 14C was present as unchanged oxaprotiline, and 0.2% as the desmethyl metabolite. After enzymic hydrolysis these increased to 48 and 6%, respectively, and after acid hydrolysis to 85 and 10%.

Multiple inverse isotope dilution assay for cadralazine and four metabolites in biological fluids.

An inverse isotope dilution assay was developed for the specific determination of 14C-labelled cadralazine and four of its metabolites in biological samples. After addition of unlabelled carrier substances to the sample, metabolite IV was derivatized. The derivative and the unaltered compounds (I, II, III, V) were extracted and separated by high-performance liquid chromatography on silica gel. Quantitation was performed by on-line ultraviolet detection at 274 nm and off-line radiometry by liquid scintillation counting. Endogenous compounds and unknown metabolites did not interfere in the assay. The analysis of water, plasma and urine samples spiked with [14C]cadralazine showed mean recoveries between 98.4 and 101.3%. The lower limit of detection was 10 nmol/1 (3 ng/ml) for any of the compounds I-V. The method was used for the analysis of plasma and urine samples of rats dosed with [14C]cadralazine.

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.

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.

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

An isotope dilution assay for the determination of both oxaprotiline enantiomers in biological samples after administration of the racemic mixture has been developed. The enantiomers were reacted with synthetically prepared, optically pure N-trifluoroacetyl-S(-)-prolyl chloride, followed by high-performance liquid chromatographic separation of the diastereoisomers formed. Quantitation was performed by on-line UV detection at 260 nm and off-line radiometry by liquid scintillation counting. Endogenous compounds and metabolites do not interfere in the assay. Analysis of water and the blood and urine of rats spiked with [14C]oxaprotiline X HCl showed recoveries for S(+)-oxaprotiline X HCl (mean +/- coefficient of variation, n = 4-6) of 98.0 +/- 1.0% (water), 100.5 +/- 0.6% (blood) and 101.5 +/- 2.0% (urine), and for R(-)-oxaprotiline X HCl of 101.3 +/- 2.0% (water), 102.2 +/- 2.1% (blood) and 103.2 +/- 0.2% (urine). A pilot study to determine blood levels of the two enantiomers in two rats dosed with racemic [14C]oxaprotiline X HCl (10 mg/kg i.v.) was carried out to test the method. The results indicated stereoselective disposition of oxaprotiline enantiomers in the rat. The ratio of the areas under the blood concentration curves for R(-)-to S(+)-oxaprotiline X HCl was 1.14.