Evaluation of accelerator mass spectrometry in a human mass balance and pharmacokinetic study-experience with 14C-labeled (R)-6-[amino(4- chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3-chlorophenyl)-1- methyl-2(1H)-quinolinone (R115777), a farnesyl transferase inhibitor.

Accelerator mass spectrometry (AMS) has been used in a human mass balance and metabolism study to analyze samples taken from four healthy male adult subjects administered nanoCurie doses of the farnesyl transferase inhibitor 14C-labeled (R)-6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3-chlorophenyl)-1-methyl-2(1H)-quinolinone ([14C]R115777). Plasma, urine, and feces samples were collected at fixed timepoints after oral administration of 50 mg [14C]R115777 (25.4 Bq/mg or 687 pCi/mg i.e., equivalent to 76.257 x 10(3) dpm) per subject. AMS analysis showed that drug-related (14)C was present in the plasma samples with C(max) values ranging from 1.6055 to 2.9074 dpm/ml (1.0525-1.9047 microg/ml) at t(max) = 2 to 3 h. The C(max) values for acetonitrile extracts of plasma samples ranged from 0.3724 to 0.7490 dpm/ml in the four male subjects. Drug-related 14C was eliminated from the body both in the urine and the feces, with a mean total recovery of 79.8 +/- 12.9% in the feces and 13.7 +/- 6.2% in the urine. The majority of drug-related radioactivity in urine and feces was excreted within the first 48 h. High-performance liquid chromatography (HPLC)-AMS profiles were generated from radioactive parent drug plus metabolites from pooled diluted urine, plasma, and methanolic feces extracts and matched to retention times of synthetic reference substances, postulated as metabolites. All HPLC separations used no more than 5 dpm injected on-column. The radioactive metabolite profiles obtained compared well with those obtained using liquid chromatography/tandem mass spectometry. This study demonstrates the use of AMS in a human phase I study in which the administered radioactive dose was at least 1000-fold lower than that used for conventional radioactive studies.

The metabolism and excretion of galantamine in rats, dogs, and humans.

Galantamine is a competitive acetylcholine esterase inhibitor with a beneficial therapeutic effect in patients with Alzheimer's disease. The metabolism and excretion of orally administered (3)H-labeled galantamine was investigated in rats and dogs at a dose of 2.5 mg base-Eq/kg body weight and in humans at a dose of 4 mg base-Eq. Both poor and extensive metabolizers of CYP2D6 were included in the human study. Urine, feces, and plasma samples were collected for up to 96 h (rats) or 168 h (dogs and humans) after dosing. The radioactivity of the samples and the concentrations of galantamine and its major metabolites were analyzed. In all species, galantamine and its metabolites were predominantly excreted in the urine (from 60% in male rats to 93% in humans). Excretion of radioactivity was rapid and nearly complete at 96 h after dosing in all species. Major metabolic pathways were glucuronidation, O-demethylation, N-demethylation, N-oxidation, and epimerization. All metabolic pathways observed in humans occurred in at least one animal species. In extensive metabolizers for CYP2D6, urinary metabolites resulting from O-demethylation represented 33.2% of the dose compared with 5.2% in poor metabolizers, which showed correspondingly higher urinary excretion of unchanged galantamine and its N-oxide. The glucuronide of O-desmethyl-galantamine represented up to 19% of the plasma radioactivity in extensive metabolizers but could not be detected in poor metabolizers. Nonvolatile radioactivity and unchanged galantamine plasma kinetics were similar for poor and extensive metabolizers. Genetic polymorphism in the expression of CYP2D6 is not expected to affect the pharmacodynamics of galantamine.

Lack of glutathione conjugation of melphalan in the isolated in situ liver perfusion in humans.

Tumor cell resistance against melphalan (LPAM) has been associated with increased cellular reduced glutathione (GSH) levels and glutathione S-transferase activity. Therefore, GSH conjugation of LPAM has been hypothesized to be a key factor in tumor cell resistance. In the present study, we evaluated GSH conjugation of LPAM by the perfused liver in patients with colorectal cancer metastases undergoing a Phase II study of isolated liver perfusion as well as in the rat. To evaluate whether LPAM-GSH conjugates were synthesized in the rat in vivo, LPAM was infused i.v. at a rate of 2.0 micromol/kg/min. In bile samples obtained during the infusion, two major GSH conjugates were identified by mass spectrometry: mono-hydroxy-mono-GSH-LPAM and di-GSH-LPAM. The maximum biliary excretion rate of these two conjugates accounted for only 1.3% of the LPAM infusion rate. In bile or perfusate samples from patients treated for 60 min initially with 0.3 mM LPAM in the perfusion medium via isolated liver perfusion (200 mg LPAM in approximately 2 liters perfusion medium), none of the above-mentioned conjugates were detected. When comparable rat liver perfusions were performed initially with 66 microM or 0.66 mM LPAM in the perfusion medium, bile samples did contain GSH-LPAM conjugates; the cumulative biliary excretion of the two conjugates amounted to 0.4 and 0.2% of the LPAM dose, respectively. These data suggest that both in rats and humans, hepatic GSH conjugation plays a very minor (if any) role in the elimination of LPAM and, therefore, that modulation of GSH levels is unlikely to affect the rate of elimination of this drug.

Disposition of the bromosulfophthalein-glutathione conjugate in the isolated perfused rat kidney.

Renal elimination of the bromosulfophthalein-glutathione conjugate (BSP-GSH) after its i.v. administration in the rat in vivo is negligible. In our study we wanted to establish whether the high albumin-binding of BSP-GSH constitutes the major restrictive factor toward the urinary excretion of the compound. The renal disposition of BSP-GSH was studied in the isolated rat kidney during perfusions with or without albumin in the perfusate. The urinary clearance of BSP-GSH in the absence of albumin was very low (< 60 microliters/min) as compared to the inulin clearance (approximately 300 microliters/min). This indicates that albumin-binding is not the major reason for the low urinary clearance of BSP-GSH. Addition of albumin to the perfusate further decreased the urinary excretion by 60%. BSP-GSH is metabolized by the kidney into two major metabolites: the cysteinylglycine conjugate and the di-glutathione conjugate. Both metabolites appear in perfusate, which suggests that BSP-GSH undergoes tubular (re-)uptake. The di-glutathione conjugate is further metabolized to the di-cysteinylglycine conjugate. The di-glutathione conjugate and the di-cysteinylglycine conjugate are the major urinary components and the urinary elimination of BSP-GSH may depend on their formation. Inhibition of gamma-glutamyl transpeptidase activity with acivicin largely prevented the degradation to the cysteinylglycine and dicysteinylglycine conjugates of BSP. The total rate of urinary excretion, however, was only slightly lowered by acivicin. Apparently, cleavage of the gamma-glutamyl moiety is not relevant for the total urinary elimination of BSP-GSH.

Glutathione conjugation of bromosulfophthalein in relation to hepatic glutathione content in the rat in vivo and in the perfused rat liver.

The relation between the rate of glutathione (GSH) conjugation and hepatic GSH content was studied in the rat in vivo and the in situ single-pass-perfused rat liver preparation with bromosulfophthalein (BSP) as the model substrate. The biliary excretion of the BSP-GSH conjugate and the hepatic GSH content were monitored simultaneously during intravenous infusions with BSP in the rat in vivo, and during liver perfusions with BSP-containing perfusion medium. Rats were pretreated with single or multiple doses of buthionine sulfoximine, an inhibitor of the de novo synthesis of GSH. Surprisingly, the excretion of the BSP-GSH conjugate was sustained at a high rate, despite a virtually complete depletion of hepatic GSH, both in the rat in vivo as well as in the perfused rat liver. The results indicate that GSH was still available for conjugation with BSP after apparent depletion of the hepatic GSH pool, presumably because of a residual de novo synthesis of GSH in the liver. Despite the multiple pretreatment with buthionine sulfoximine, the de novo GSH synthesis was sufficient to sustain a high rate of GSH conjugation of BSP. The cosubstrate-Km for GSH conjugation of BSP in the liver was estimated to be very small (approximately 0.3 mumol/g): the excretion rate of the BSP-GSH conjugate was only impaired at minimal hepatic GSH levels.

Localization of glutathione conjugation activities toward bromosulfophthalein in perfused rat liver. Studies with the multiple indicator dilution technique.

The plasma binding and conjugation kinetics of bromosulfophthalein (BSP) with glutathione (GSH) were studied in the single-pass in situ perfused rat liver (portal vein perfusion at 10 ml/min); GSH in postmitochondrial fractions of the liver at the end of the experiment was examined as a potential rate-determining factor. BSP was highly bound to 1% albumin with at least two classes of binding sites: one of 0.17 site with an association constant of 1.9 x 10(7) M-1, and one of 7.4 equivalent sites of association constant, 1.3 x 10(5) M-1. Nonlinear binding was observed within between 5-1500 microM BSP. At varying input concentrations (0.4-250 microM) of BSP, the unbound fraction was extremely low (< 0.005) and the hepatic extraction ratio declined from 0.67 to 0.15; loss of BSP was primarily caused by GSH conjugation to form BSP-GSH, which appeared exclusively in bile. Additionally, unchanged BSP and two very minor unidentified metabolites were also excreted in bile. The formation of BSP-GSH proceeded with an apparent Vmax of 22 nmol/min/g and a KM of 0.05 microM, whereas the parameters for BSP excretion were 0.85 nmol/min/g and 0.02 microM, respectively. Within the concentration range of BSP examined, GSH availability did not appear to be rate-limiting in the formation or excretion of BSP-GSH.(ABSTRACT TRUNCATED AT 250 WORDS)

Methods for the quantitation of bromosulfophthalein and its glutathione conjugate in biological fluids.

This paper describes a solvent-gradient HPLC method which was developed for the quantitation of bromosulfophthalein (BSP) in erythrocyte and albumin containing (blood) perfusate samples, and of BSP and its glutathione conjugate (BSP-GSH) in bile samples obtained from rat liver perfusion experiments. Phenolphthalein was used as an internal standard. The cysteinyl-, N-acetylcysteinyl-, and cysteinylglycinyl-BSP conjugates did not interfere with the HPLC assay and cysteine, N-acetylcysteine, [3H]GSH, and [3H]GSSG eluted within the first 6 min postinjection onto the HPLC system. A spectrophotometric method was also developed for the rapid quantitation of BSP in blood perfusate; tracer [14C]urea was utilized as the internal standard. Although the spectrophotometric method was less sensitive than the HPLC method, good correlation was found to exist between the methods.

Glutathione conjugation and pharmacokinetics of 2-bromo-3-phenylpropionic acid in vitro and in the rat in vivo.

Glutathione (GSH) conjugation of the chiral compound 2-bromo-3-phenylpropionic acid (BPP) was studied in vitro and in the rat in vivo. GSH conjugation of BPP, catalyzed by a mixture of glutathione-S-transferases (GST's) from rat liver cytosol in vitro, was stereoselective: at a substrate concentration of 250 microM, (R)-BPP was more rapidly conjugated than (S)-BPP (R/S-ratio = 2.6). The blood elimination kinetics of the separate BPP enantiomers and the biliary excretion kinetics of the corresponding GSH conjugates were studied in the rat in vivo after administration of (R)- or (S)-BPP at a dose level of 50 mumol/kg. Elimination of (R)-BPP from blood was faster than that of (S)-BPP: half lives were 9 +/- 2 min for (R)-BPP and 13 +/- 1 min for (S)-BPP. The biliary excretion rate of the GSH conjugate of (R)-BPP declined monoexponentially, while that of the GSH conjugate of (S)-BPP displayed a biphasic profile. Half lives of excretion were 13 +/- 1 for the GSH conjugate of (R)-BPP, and 11 +/- 2 for the GSH conjugate of (S)-BPP (second phase). The first phase in the biliary excretion of the GSH conjugate of (S)-BPP could not be attributed to capacity limitation of biliary transport carriers as higher excretion rates were attained upon administration of higher doses (100 and 200 mumol/kg) of (S)-BPP). The blood elimination profiles of (R)- and (S)-BPP differed greatly from the biliary excretion profiles of the corresponding GSH conjugates. This suggests that the kinetics of BPP conjugate excretion are determined by other processes than hepatic GSH conjugation.