Covalent binding of radioactivity from [14C]rofecoxib, but not [14C]celecoxib or [14C]CS-706, to the arterial elastin of rats.

Rofecoxib is a cyclooxygenase-2 (COX-2) inhibitor that has been withdrawn from the market because of an increased risk of cardiovascular (CV) events. With a special focus on the arteries, the distribution profiles of radioactivity in rats orally administered [14C]rofecoxib were investigated in comparison with two other COX-2 inhibitors, [14C]celecoxib and [14C]CS-706 (2-(4-ethoxyphenyl)-4-methyl 1-(4-sulfamoylphenyl)-1H-pyrrole), a novel selective COX-2 inhibitor. Whole-body autoradioluminography and quantitative determination of the tissue concentrations showed that considerable radioactivity is retained by and accumulated in the thoracic aorta of rats after oral administration of [14C]rofecoxib, but not [14C]celecoxib or [14C]CS-706. Acid, organic solvent, and proteolytic enzyme treatments of aorta retaining high levels of radioactivity from [14C]rofecoxib demonstrated that most of the radioactivity is covalently bound to elastin. In agreement with this result, the radioactivity was found to be highly localized on the elastic fibers in the aorta by microautoradiography. The retention of radioactivity on the elastic fibers was also observed in the aortic arch and the coronary artery. These findings indicate that [14C]rofecoxib and/or its metabolite(s) are covalently bound to elastin in the arteries. These data are consistent with the suggestion of modified arterial elasticity leading to an increased risk of CV events after long-term treatment with rofecoxib.     

Protein covalent binding of maxipost through a cytochrome P450-mediated ortho-quinone methide intermediate in rats.

(3S)-(+)-(5-Chloro-2-methoxyphenyl)-1,3-dihydro-3-fluoro-6-(trifluoromethyl)-2H-indole-2-one) (MaxiPost, BMS-204352) is a potent and specific opener for maxi-K channels and has potential to prevent and treat ischemic stroke. Following single intravenous doses of [14C]BMS-204352 to rats, only 10 to 12% of radioactivity was extractable from plasma with organic solvents. The unextractable radioactivity remained associated with the proteins (mostly albumin) after SDS-polyacrylamide gel electrophoresis or dialysis. Following acid hydrolysis in 6 M HCl for 24 h at 110 degrees C from plasma proteins collected from nine rats dosed with [14C]BMS-204352, one major radioactive product was isolated and identified as a lysine-adduct of des-fluoro des-O-methyl BMS-204352 by liquid chromatography/mass spectrometry and NMR analyses as well as by comparison with the synthetic analog, lysine-adduct of des-fluoro BMS-204352 (BMS-349821). The covalent binding of BMS-204352 results from the displacement of the ring-fluorine atom of des-O-methyl BMS-204352 with the epsilon-amino group of a lysine residue. Microsomal incubations of [14C]BMS-204352 resulted in low levels of covalent binding of radioactivity to proteins. This in vitro covalent binding required cytochrome P450-reductase cofactor NADPH and was attenuated by glutathione. P4503A inhibitors ketoconazole and troleadomycin selectively prevented the covalent binding in vitro. Based on these observations, a two-step bioactivation process for the protein covalent binding of BMS-204352 was postulated: 1) P4503A-mediated O-demethylation leading to spontaneous release of HF and the formation of an ortho-quinone methide reactive metabolite and 2) nucleophilic addition of the epsilon-amino group of protein lysine residue(s) in protein to form des-fluoro des-O-methyl BMS-204352 lysine adduct.     

The disposition and metabolism of rofecoxib, a potent and selective cyclooxygenase-2 inhibitor, in human subjects.

The disposition and metabolism of rofecoxib, a selective cyclooxygenase-2 inhibitor, were examined in healthy human subjects and in cholecystectomy patients. After oral administration of [(14)C]rofecoxib (125 mg, 100 micro Ci) to healthy subjects, the mean concentrations of total radioactivity and rofecoxib in plasma as a function of time indicated that the t(max) was achieved at 9 h postdose. After t(max), levels of both radioactivity and rofecoxib decreased in a parallel, exponential fashion (effective t(1/2) approximately equal 17 h). A similar result was obtained after oral administration of [(14)C]rofecoxib (142 mg, 100 micro Ci) to cholecystectomy patients equipped with an L-tube. In healthy subjects, radioactivity was recovered predominantly from the urine (71.5% of dose), with a small amount excreted in feces (14.2%). In patients with an L-tube, half the radioactive dose was recovered in feces, with a lesser amount excreted in urine (28.8%) and a negligible fraction in bile (1.8%). Rofecoxib underwent extensive metabolism in humans, and very little parent drug was recovered unchanged in urine (<1%). Products resulting from both oxidative and reductive pathways were identified by a combination of (1)H NMR and liquid chromatography-tandem mass spectrometry analyses, and included rofecoxib-3',4'-trans-dihydrodiol, 4'-hydroxyrofecoxib-O-beta-D-glucuronide, diastereomeric 5-hydroxyrofecoxib-O-beta-D-glucuronide conjugates, 5-hydroxyrofecoxib, rofecoxib-erythro-3,4-dihydrohydroxy acid, and rofecoxib-threo-3,4-dihydrohydroxy acid. Interconversion of rofecoxib and 5-hydroxyrofecoxib appeared not to be a quantitatively important pathway of rofecoxib disposition in human subjects, in contrast to previous findings in rats.     

Covalent interaction of 5-nitroimidazoles with DNA and protein in vitro: mechanism of reductive activation

Human hepatic microsomal enzymes catalyzed the NADPH-dependent anaerobic reductive activation of [1-14C]metronidazole [1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole] and [4,5-14C]ronidazole [(1-methyl-5-nitroimidazole-2-yl)methyl carbamate] to species that became covalently bound to proteins. Due to the low efficiency of the enzyme-catalyzed covalent binding of metronidazole, the stoichiometry of anaerobic reductive activation was studied with dithionite as the reductant. Two moles of dithionite was consumed per mole of [1-14C]metronidazole for maximal covalent binding to either DNA or immobilized sulfhydryl groups, demonstrating that four electrons are required for the reductive activation of metronidazole. These data implicate the involvement of a hydroxylamine in covalent binding. Maximal covalent binding of [4,5-14C]ronidazole to DNA also required four-electron reduction, consistent with previous studies of the covalent binding of this agent to immobilized sulfhydryl groups [Kedderis et al. (1988) Arch. Biochem. Biophys. 262, 40-48]. Studies of the covalent binding of variously radiolabeled ronidazole molecules to DNA suggested that the imidazole ring was intact while greater than 80% of the 2-carbamoyl group and the C4 proton were not present in the DNA adduct. Studies of both the chemical and human hepatic microsomal reduction of [4-3H]metronidazole demonstrated that covalent binding occurred with the stoichiometric loss of this label, implicating binding at the C4 position. These results suggest that the reductive activation of 5-nitroimidazoles generally proceeds via four-electron reduction to form hydroxylamines followed by nucleophilic attack at C4.     

The biochemical selectivity of novel COX-2 inhibitors in whole blood assays of COX-isozyme activity

We have evaluated the biochemical selectivity of novel cyclo-oxygenase (COX)-2 inhibitors, etoricoxib, valdecoxib, DFU and DFP, vs rofecoxib and celecoxib, using the human whole blood assays of COX-isozyme activity, in vitro. Compounds were incubated with human whole blood samples, allowed to clot for 1 h at 37 degrees C, or stimulated with lipopolysaccharide (10 microg/ml) for 24 h at 37 degrees C. Serum thromboxane (TX) B2 and plasma prostaglandin (PG) E2 levels were measured by specific radioimmunoassays as indices of platelet COX-1 and monocyte COX-2 activity, respectively. Valdecoxib, etoricoxib, DFU and DFP inhibited platelet COX-1 and monocyte COX-2 with the following COX-1/COX-2 IC50 ratios: 61.5, 344, 660 and 1918, respectively. The reference compounds, celecoxib and rofecoxib had corresponding values of 29.6 and 272. In conclusion, a second wave of COX-2 inhibitors with higher biochemical selectivity than the existing coxibs has been developed. Whether their administration will be associated with improved clinical efficacy and/or safety vis-à-vis celecoxib and rofecoxib remains to be established.     

The absorption, distribution, metabolism and excretion of rofecoxib, a potent and selective cyclooxygenase-2 inhibitor, in rats and dogs.

Absorption, distribution, metabolism, and excretion studies were conducted in rats and dogs with rofecoxib (VIOXX, MK-0966), a potent and highly selective inhibitor of cyclooxygenase-2 (COX-2). In rats, the nonexponential decay during the terminal phase (4- to 10-h time interval) of rofecoxib plasma concentration versus time curves after i.v. or oral administration of [(14)C]rofecoxib precluded accurate determinations of half-life, AUC(0-infinity) (area under the plasma concentration versus time curve extrapolated to infinity), and hence, bioavailability. After i.v. administration of [(14)C]rofecoxib to dogs, plasma clearance, volume of distribution at steady state, and elimination half-life values of rofecoxib were 3.6 ml/min/kg, 1.0 l/kg, and 2.6 h, respectively. Oral absorption (5 mg/kg) was rapid in both species with C(max) occurring by 0.5 h (rats) and 1.5 h (dogs). Bioavailability in dogs was 26%. Systemic exposure increased with increasing dosage in rats and dogs after i.v. (1, 2, and 4 mg/kg), or oral (2, 5, and 10 mg/kg) administration, except in rats where no additional increase was observed between the 5 and 10 mg/kg doses. Radioactivity distributed rapidly to tissues, with the highest concentrations of the i.v. dose observed in most tissues by 5 min and by 30 min in liver, skin, fat, prostate, and bladder. Excretion occurred primarily by the biliary route in rats and dogs, except after i.v. administration of [(14)C]rofecoxib to dogs, where excretion was divided between biliary and renal routes. Metabolism of rofecoxib was extensive. 5-Hydroxyrofecoxib-O-beta-D-glucuronide was the major metabolite excreted by rats in urine and bile. 5-Hydroxyrofecoxib, rofecoxib-3',4'-dihydrodiol, and 4'-hydroxyrofecoxib sulfate were less abundant, whereas cis- and trans-3,4-dihydro-rofecoxib were minor. Major metabolites in dog were 5-hydroxyrofecoxib-O-beta-D-glucuronide (urine), trans-3, 4-dihydro-rofecoxib (urine), and 5-hydroxyrofecoxib (bile).     

Non-enzymatic covalent binding of radioactivity from [14C]thiourea to rat lung protein

In vitro covalent binding of radioactivity from [14C]thiourea (TU) to rat lung protein occurs in the presence of 100% CO2. Prior boiling of lung slices results in a subsequent increase in covalent binding. Covalently bound radioactivity was released from rat lung homogenates and acid-insoluble protein obtained from animals treated with [14C]TU. The release was achieved with ammonium ion. One possibility suggested by these results is that the bound species may involve binding of isothiocyanic acid via a thiocarbamylation reaction.     

In vitro biotransformation and genotoxicity of the drinking water disinfection byproduct bromodichloromethane: DNA binding mediated by glutathione transferase theta 1-1

The drinking water disinfection byproduct bromodichloromethane (CHBrCl(2)) was previously shown to be mutagenic in Salmonella typhimurium that overexpress rat glutathione transferase theta 1-1 (GSTT1-1). Several experimental approaches were undertaken in this study to investigate the DNA covalent binding potential of reactive intermediates generated by GSTT1-1-mediated metabolism of CHBrCl(2). First, rodent hepatic cytosol incubations containing [(14)C]CHBrCl(2), supplemented glutathione (GSH), and calf thymus DNA resulted in approximately 3-fold (rat liver cytosol) and 7-fold (mouse liver cytosol) greater amounts of total radioactivity (RAD) associated with the purified DNA as compared to a control (absence of rodent cytosol) following liquid scintillation counting (LSC) of isolated DNA. The relative increase in DNA labeling is consistent with the conjugation activity of these rodent cytosols toward CHBrCl(2). Second, exposure of GSTT1-1-expressing S. typhimurium to [(14)C]CHBrCl(2) resulted in a concentration-dependent increase of bacterial DNA-associated total radioactivity. Characterization of DNA-associated radioactivity could not be assigned to a specific deoxynucleoside adduct(s) following enzymatic hydrolysis of DNA and subsequent HPLC analysis. A possible explanation for this observation was formation of a 'transient' adduct that was unstable in the DNA isolation and hydrolysis procedures employed. To circumvent problems of adduct instability, reactions of [(14)C]CHBrCl(2) with GSH catalyzed by recombinant rat GSTT1-1 were performed in the presence of calf thymus DNA or, alternatively, the model nucleophile deoxyguanosine. Hydroxyapatite chromatography of [(14)C]-labeled DNA or HPLC chromatography of [(14)C]-labeled deoxyguanosine derivatives demonstrated the covalent binding of [(14)C]CHBrCl(2)-derived metabolites to DNA and deoxyguanosine in low yield (approximately 0.02% of [(14)C]CHBrCl(2) biotransformed by GSTT1-1 resulted in DNA adducts). Cytochrome P450 (CYP)- and GST-catalyzed biotransformation of CHBrCl(2) in rat tissues (kidney and large intestine) that develop tumors following chronic CHBrCl(2) exposure were compared with rat liver (a nontarget tissue). Rat liver had a significant capacity to detoxify CHBrCl(2) (to carbon dioxide) compared with kidney and large intestine as a result of CYP-catalyzed oxidation, liver was approximately 16-fold more efficient than kidney and large intestine when intrinsic clearance values (V(max)/K(m)) were compared. In contrast, the efficiency of GST-mediated GSH conjugation of CHBrCl(2) in kidney and large intestine was only slightly lower than liver (approximately 2- to 4-fold lower), thus, the relative amounts of reactive intermediates that are produced with the capacity to covalently modify DNA may be enhanced in these extrahepatic tissues. The significance of these findings is that conjugation of CHBrCl(2) with GSH can result in the covalent modification of DNA and that cancer target tissues in rats have a much reduced detoxification capacity, but only a modest decrease in bioactivation capacity, as compared to the liver (a nontarget tissue in rats).     

Pharmacokinetics and tissue distribution of a novel PDE5 inhibitor, SK-3530, in rats

Aim： To investigate the pharmacokinetic profile and tissue distribution of a novel phosphodiesterase type 5 inhibitor, 5-ethyl-2-{5-[4-（2-hydroxy-ethyl）-piperazine- 1-sulfonyl]-2-propoxy-phenyl }-7-propyl-3,5-dihydro-pyrrolo（3,2-d）pyrimidin-4- one （SK-3530）, in rats after administration of the ^14C-labeled compound. Methods： The pharmacokinetic parameters of SK-3530 were measured based on the total radioactivity and parent SK-3530 concentration in rat plasma after intravenous and oral administration. The tissue distribution of total radioactivity after a single oral administration of [^14C]SK-3530 at a dose of 40 mg/kg was assayed. The plasma protein binding rates of SK-3530 were assessed by in vitro and ex vivo assay. Results： The total radioactivity profiles showed linear pharmacokinetics. The maximum plasma concentration and area under the curve of the parent SK3530 were 10%-20% compared to those of the total radioactivity. After the oral administration of [^14C]SK-3530, the radioactivity was widely distributed in all tissues, and the tissue/plasma ratio of the radioactivity 1 h after administration was calculated as 0.5-2.6 with the exception of excretory organs. A relatively high penetration was shown in the adrenal glands, liver, and lung. In vitro and ex vivo plasma protein binding assay by ultrafiltration showed a considerably high binding rate of more than 97%. Condusion： SK-3530 was relatively well absorbed in the gastrointestinal tract and showed linear pharmacokinetics over the investigated dose range. SK-3530 had low oral bioavailability due to a high, first-pass metabolism.     

Synthesis and in vivo evaluation of halogenated N,N-dimethyl-2-(2'-amino-4'-hydroxymethylphenylthio)benzylamine derivatives as PET serotonin transporter ligands

N, N-dimethyl-2-(2'-amino-4'-hydroxymethylphenylthio)benzylamine (38), substituted on ring A, was reported to display high binding affinity and selectivity to the human brain serotonin transporter (SERT). In an attempt to explore the potential of compounds substituted on ring B of the phenylthiophenyl core structure, three derivatives of 38 were synthesized: N, N-dimethyl-2-(2'-amino-4'-hydroxymethyl-phenylthio)-5-fluorobenzylamine (35), N, N-dimethyl-2-(2'-amino-4'-hydroxymethyl-phenylthio)-5-bromobenzylamine (36), and N, N-dimethyl-2-(2'-amino-4'-hydroxymethyl-phenylthio)-5-iodobenzylamine (37). The in vitro binding studies in cells transfected with human SERT, norepinephrine transporter (NET), and dopamine transporter (DAT) showed that 35, 36, and 37 exhibited high SERT affinity with K is (SERT) = 1.26, 0.29, and 0.31 nM (vs [(3)H]citalopram), respectively. [(11)C]-(35), [(11)C]-(36), and [(11)C]-( 37) were prepared by methylation of their monomethyl precursors 16, 17, and 18, with [(11)C]iodomethane in 28, 11, and 14% radiochemical yields, respectively. The microPET images of [(11)C]-(35), [(11)C]-(36), and [(11)C]-(37) showed high uptake in the monkey brain regions rich in SERT with peak midbrain to cerebellum ratios of 3.41, 3.24, and 3.00 at 85 min post-injection, respectively. In vivo bindings of [(11)C]-(35), [(11)C]-(36), and [(11)C]-(37) were shown to be specific to the SERT as displacement with citalopram (a potent SERT ligand) reduced radioactivity in SERT-rich regions to the cerebellum level. These results suggest that [(11)C]-(35), [(11)C]-(36), and [(11)C]-(37) could be potential agents for mapping human SERT by PET and radiolabeling 37 with iodine-123, which could afford the first SPECT SERT imaging agent exhibiting fast kinetics.     

Metabolism, pharmacokinetics, and protein covalent binding of radiolabeled MaxiPost (BMS-204352) in humans.

MaxiPost [(3S)-(+)-(5-chloro-2-methoxyphenyl)-1,3-dihydro-3-fluoro-6-(trifluoromethyl)-2H-indole-2-one); BMS-204352] is an investigational maxi-K channel opener to treat ischemic stroke. This study reports the disposition, metabolism, pharmacokinetics, and protein covalent binding of (14)C-labeled MaxiPost in healthy male volunteers as well as in dogs and rats. After each human subject received a single dose of 10 mg (14)C-labeled BMS-204352 (50 microCi) as a 5-ml intravenous infusion lasting 5 min, the plasma radioactivity concentrations showed a unique profile, wherein the concentration appeared to increase initially, followed by a terminal decline. The mean terminal t(1/2) of plasma radioactivity (259 h) was prolonged compared with that of unchanged parent (37 h). Furthermore, the extractability of radioactivity in plasma decreased over time, reaching approximately 20% at 4 h after dosing. The unextractable radioactivity was covalently bound to plasma proteins through a des-fluoro-des-methyl BMS-204352 lysine adduct. Unchanged BMS-204352 and minor metabolites were identified in plasma extract following protein precipitation. The recovery of the radioactive dose in urine and feces was nearly complete in 14-day collections (approximately 37% in urine and 60% in feces). The N-glucuronide of the parent was the prominent metabolite in urine (16.5% of dose), whereas the parent was a major drug-related component in feces (11% of dose). Similar disposition, metabolism, pharmacokinetic, and protein covalent binding properties of (14)C-labeled BMS-204352 were observed in humans, dogs, and rats.     

Characterization of celecoxib and valdecoxib binding to cyclooxygenase

Two compounds (celecoxib and valdecoxib) from the diarylheterocycle class of cyclooxygenase inhibitors were radiolabeled and used to characterize their binding to cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), several single-point variants of COX-2 (Val523Ile, Tyr355Ala, Arg120Ala, Arg120Gln, Arg120Asn) and one triple-point variant of COX-2 [Val523Ile, Arg513His, Val434Ile (IHI)]. We demonstrate highly specific and saturable binding of these inhibitors to COX-2. Under the same assay conditions, little or no specific binding to COX-1 could be detected. The affinity of [(3)H]celecoxib for COX-2 (K(D) = 2.3 nM) was similar to the affinity of [(3)H]valdecoxib (K(D) = 3.2 nM). The binding to COX-2 seems to be both rapid and slowly reversible with association rates of 5.8 x 10(6)/M/min and 4.5 x 10(6)/M/min and dissociation rates of 14 x 10(-3)/min (t(1/2) = 50 min) and 7.0 x 10(-3)/min (t(1/2) = 98 min) for [(3)H]celecoxib and [(3)H]valdecoxib, respectively. These association rates increased (4- to 11-fold) when the charged arginine residue located at the entrance to the main hydrophobic channel was mutated to smaller uncharged amino acids (Arg120Ala, Arg120Gln, and Arg120Asn). Mutation of residues located within the active site of COX-2 that define a 'side pocket' (Tyr355Ala, Val523Ile, IHI) of the main channel had a greater effect on the dissociation rate than the association rate. These mutations, which modified the shape of and access to the 'side pocket', affected the binding affinity of [(3)H]valdecoxib more than that of [(3)H]celecoxib. These binding studies provide direct insight into the properties and binding constants of celecoxib and valdecoxib to COX-2.     

Species differences in metabolism of tiaramide hydrochloride, a new anti-inflammatory agent.

1. Urinary excretion of the radioactivity in 24 h after oral administration of [14C]tiaramide hydrochloride was 67% of the dose in mice, 59% in rats, 41% in dogs and 74% in monkeys. 2. The serum half-lives of tiaramide after intravenous administration were approximately 0-2 h in mice, 0-8 h in rats and 0-5 h in dogs. 3. Marked species variations were noted in the composition of metabolites in the serum and urinary radioactivity. The major metabolites found were 1-[(5-chloro-2-oxo-3(2H)-benzothiazolyl)acetyl]-piperazine (DETR) and 4-[(5-chloro-2-oxo-3(2H)-benzothiazolyl)acetyl]-1-piperazineacetic acid (TRAA) in mice, TRAA and 4-[(5-chloro-2-oxo-3(2H)-benzothiazolyl)acetyl]-1-piperazineethanol 1-oxide (TRNO) in rats, TRNO and tiaramide-O-glucuronide (TR-O-Glu) in dogs, and TRAA and TR-O-Glu in monkeys. 4. The binding of tiaramide to plasma protein of the various species of animals and human was about 24-34% and the extent of the binding of tiaramide to human plasma protein was independent of drug concentration within the range of 1-100 micron.     

Pharmacological separation between peripheral and central functions of cyclooxygenase-2 with CIAA, a novel cyclooxygenase-2 inhibitor

There are many reports concerning the physiological and pathological involvement of cyclooxygenase (COX)-2 in the central nervous system and peripheral tissue cells. Selective COX-2 inhibitors that mainly distribute peripherally have not been reported thus far. Therefore central and peripheral roles of COX-2 remain classified pharmacologically. In this study, in vivo pharmacological profiles of CIAA ([6-chloro-2-(4-chlorobenzoyl)-1H-indol-3-yl]acetic acid), a novel selective COX-2 inhibitor which distributes at higher concentrations in plasma than in brain, were compared with those of well-known selective COX-2 inhibitors, celecoxib and rofecoxib. Additionally, the possibility of pharmacological separation between peripheral and central actions of COX-2 with the inhibitors was investigated. CIAA selectively inhibited COX-2 activity compared with COX-1 in in vitro assays with rat whole blood. The compound exhibited lower brain penetration and higher plasma concentration (the brain/plasma concentration ratio was approximately 0.02) than celecoxib and rofecoxib after oral administration. Therefore, CIAA is mainly expected to act peripherally. Edema and prostaglandin E2 (PGE2) production in Carrageenan-injected rat paws, and pyrexia and PGE2 production in the brain in lipopolysaccharide (LPS)-injected rats were measured in in vivo experiments. CIAA exhibited lower ratios of anti-pyretic/anti-edematous activities and of inhibitory activities of PGE2 production in brain/paw than those of celecoxib and rofecoxib, and these ratios well-reflected brain/plasma concentration ratios. In conclusion, we discovered a novel selective COX-2 inhibitor, CIAA, which distributes at higher concentrations in plasma than in brain, which would make possible the pharmacological separation of the peripheral and central functions of COX-2.     

Pharmacokinetics, COX-2 specificity, and tolerability of supratherapeutic doses of rofecoxib in humans

Objective: Prostaglandin synthesis is catalyzed by a constitutive cyclo-oxygenase isoform (COX-1) and an inducible isoform (COX-2). It is hypothesized that the analgesic and anti-inflammatory effects of nonsteroidal anti-inflammatory drugs (nonspecific COX-1/COX-2 inhibitors) such as ibuprofen principally derive from COX-2 inhibition. The purpose of this study was to evaluate steady-state pharmacokinetics, biochemical selectivity and tolerability of rofecoxib (Vioxx^TM), characterized in vitro as a COX-2 inhibitor. Methods: Four panels of healthy men (n=8 per panel) were administered rofecoxib (n=6) (25, 100, 250, 375 mg) or placebo (n=2) once daily on day 1 and days 3–14. Blood samples for assays of rofecoxib plasma concentration and COX isoform activity were obtained pre-dose and at specified time points post-dose. Results: Rofecoxib pharmacokinetics were found to be complex and nonlinear. Elimination half-life ranged from 9.9 h to 17.5 h after multiple dosing with an accumulation ratio close to 2 for all doses. COX-2 inhibitory activity as assessed by average inhibition of whole blood lipopolysaccharide-stimulated prostaglandin E₂ over the 8-h post-dose period on day 14 was 0.3, 67, 96, 92 and 96% for the placebo and the 25-, 100-, 250- and 375-mg treatment groups, respectively. No treatment group showed significant inhibition of COX-1 as assessed by thromboxane B₂ generation in clotting whole blood. Side effects were mild and transient. Conclusion: The results indicate that rofecoxib is a potent and specific inhibitor of COX-2 in humans even at doses more than tenfold higher than those associated with efficacy in patients with osteoarthritis. Received: 26 July 1999 / Accepted in revised form: 22 December 1999     

Absorption, metabolism and distribution of [14C]-O-methyldopa and [14C]-L-dopa after oral administration to rats

1 The absorption, tissue distribution, and metabolism of [(14)C]-O-methyldopa were compared with those of [(14)C]-L-DOPA after oral administration to rats.2 Total radioactivity in the plasma and brain of rats treated with [(14)C]-O-methyldopa was significantly higher (2 fold and 30-50 fold, respectively) than that of rats treated with [(14)C]-L-DOPA.3 Total radioactivity in the gut washings and intestinal tissue 2 h after oral administration was significantly higher in rats treated with [(14)C]-L-DOPA than in rats treated with [(14)C]-O-methyldopa. The reverse was observed in the stomach tissues.4 Peripheral metabolism of [(14)C]-O-methyldopa was much lower than that of [(14)C]-L-DOPA; the major metabolite of [(14)C]-O-methyldopa in the plasma is L-DOPA, whereas L-DOPA is mainly metabolized to phenylcarboxylic acids.     

Hypouricemic effects of novel concentrative nucleoside transporter 2 inhibitors through suppressing intestinal absorption of purine nucleosides

We have developed concentrative nucleoside transporter 2 (CNT2) inhibitors as a novel pharmacological approach for improving hyperuricemia by inhibiting intestinal absorption of purines. Dietary purine nucleosides are absorbed in the small intestines by CNTs expressed in the apical membrane. In humans, the absorbed purine nucleosides are rapidly degraded to their final end product, uric acid, by xanthine oxidase. Based on the expression profile of human CNTs in digestive tract tissues, we established a working hypothesis that mainly CNT2 contributes to the intestinal absorption of purine nucleosides. In order to confirm this possibility, we developed CNT2 inhibitors and found that (2R,3R,4S,5R)-2-(6-amino-8-{[3'-(3-aminopropoxy)-biphenyl-4-ylmethyl]-amino}-9H-purin-9-yl)-5-hydroxymethyl-tetrahydrofuran-3,4-diol (KGO-2142) and 1-[3-(5-{[1-((2R,3R,4S,5R)-3,4-dihydroxy-5-hydroxymethyl-tetrahydrofuran-2-yl)-1H-benzimidazol-2-ylamino]-methyl}-2-ethoxyphenoxy)-propyl]-piperidine-4-carboxylic acid amide (KGO-2173) were inhibitory. These CNT2 inhibitors had potent inhibitory activity against inosine uptake via human CNT2, but they did not potently interfere with nucleoside uptake via human CNT1, CNT3 or equilibrative nucleoside transporters (ENTs) in vitro. After oral administration of KGO-2173 along with [(14)C]-inosine, KGO-2173 significantly decreased the urinary excretion of radioactivity at 6 and 24h in rats. Since dietary purine nucleosides are not utilized in the body and are excreted into the urine rapidly, this decrease in radioactivity in the urine represented the inhibitory activity of KGO-2173 toward the absorption of [(14)C]-inosine in the small intestines. KGO-2142 almost completely inhibited dietary RNA-induced hyperuricemia and the increase in urinary excretion of uric acid in cebus monkeys. These novel CNT2 inhibitors, KGO-2142 and KGO-2173, could be useful therapeutic options for the treatment of hyperuricemia.     

Evidence for oxidative activation of mitoxantrone in human, pig, and rat.

A new metabolite of mitoxantrone in human, rat, and pig urine has been discovered by means of HPLC. The metabolite has been isolated by preparative HPLC from patient urine and is characterized by tandem mass spectrometry and UV-visible spectroscopy as 8,11-dihydroxy-4-(2-hydroxyethyl)-6-[[2-[(2-hydroxyethyl)amino]ethyl] amino]-1,2,3,4,7,12-hexahydronaphtho-[2,3-f]-chinoxaline-7,1 2-dione. Final structural proof has been obtained by independent synthesis. The new metabolite is a product of the enzymatic oxidation of the phenylenediamine substructure of mitoxantrone. An important biological consequence of the oxidative biotransformation is the possibility of covalent binding to intracellular targets via a highly electrophilic intermediate. Thus, alkylation may be an important mode of action of mitoxantrone. Incubation of mitoxantrone with horseradish peroxidase/hydrogen peroxide in the presence of glutathione led to the formation of two glutathione conjugates of mitoxantrone. Their structures have been elucidated by combination of IonSpray (Sciex, Canada) ionization and tandem mass spectrometry. Radioactive mitoxantrone, synthesized from sodium [14C]cyanide, was used to determine interspecies variations between human and rat. The collected rat urine was analyzed by HPLC using a radioactivity monitoring detector and revealed significant differences in the biotransformation of mitoxantrone in rat compared to human. The main metabolites thus far described in human urine are not observed in rat urine.     

Role of mixed-function oxidases and non-protein sulfhydryl content in [14C]-2-chloro-4-acetotoluidide binding to liver and kidney in starlings

The compound 2-chloro-4-acetotoluidide (CAT) is highly toxic to many avian species, including the starling. In our earlier work, we demonstrated the covalent binding of radioactivity from [14C]-CAT to liver and kidneys of the starling. In the present study, the effects of inducers of mixed-function oxidase (MFO) and non-protein sulfhydryl (NPSH) depletor on the total and covalent binding of [14C]-CAT radioactivity to liver and kidney of the starling were examined. The total and covalently bound radioactivity from [14C]-CAT to liver and kidney were decreased significantly in the starling pretreated with the MFO inducer, 3-methylcholanthrene. However, pretreatment with phenobarbital, another inducer of MFO, had no effect. Pretreatment with the inhibitor of MFO, SKF 525-A, reduced the covalent binding of [14C]-CAT radioactivity to liver but not to kidney. There was no reduction in the NPSH content of liver or kidney following intravenous administration of CAT (3.5 mg kg-1). Reduction of NPSH levels in the liver or kidney following treatment with diethyl maleate caused a significant increase in the covalent binding of [14C]-CAT to kidney but not to liver.     

The Biochemical Selectivity of Novel COX-2 Inhibitors in Whole Blood Assays of COX-isozyme Activity

Summary

We have evaluated the biochemical selectivity of novel cyclo-oxygenase (COX)-2 inhibitors, etoricoxib, valdecoxib, DFU and DFP, vs rofecoxib and celecoxib, using the human whole blood assays of COX-isozyme activity, in vitro. Compounds were incubated with human whole blood samples, allowed to clot for 1?h at 37°C, or stimulated with lipopolysaccharide (10|ig/ml) for 24?h at 37°C. Serum thromboxane (TX) B2 and plasma prostaglandin (PG) E2 levels were measured by specific radioimmunoassays as indices of platelet COX-1 and monocyte COX-2 activity, respectively. Valdecoxib, etoricoxib, DFU

and DFP inhibited platelet COX-1 and monocyte COX-2 with the following COX-1/COX-2 IC50 ratios: 61.5, 344, 660 and 1918, respectively. The reference compounds, celecoxib and rofecoxib had corresponding values of 29.6 and 272. In conclusion, a second wave of COX-2 inhibitors with higher biochemical selectivity than the existing coxibs has been developed. Whether their administration will be associated with improved clinical efficacy and/or safety visà-vis celecoxib and rofecoxib remains to be established.