Interactions between oxaprozin glucuronide and human serum albumin.

1. The first step in the interaction between oxaprozin glucuronide and human serum albumin (HSA) is formation of a reversible complex which then leads to the following reactions; (a) acyl migration of the aglycone from position 1 to positions 2, 3 and 4 of the glucuronic acid moiety; (b) hydrolysis of the glycosidic bond; and (c) covalent binding of oxaprozin to the HSA molecule. The isomers of oxaprozin glucuronide formed in (a) and the covalently bonded drug in (c) are also hydrolyzed to oxaprozin. 2. Oxaprozin and ligands known to bind at Site II as classified by Sudlow et al. (1976), also called the benzodiazepine binding site (Müller and Wollert 1975), inhibit these reactions with oxaprozin glucuronide, while ligands which are known to bind at other sites on HSA do not. 3. Modification of a single tyrosine residue, located within Site II, with tetranitromethane, diisopropylfluorophosphate, and p-nitrophenylacetate causes significant reduction of the covalent binding of oxaprozin to HSA. 4. Tetranitromethane modification of HSA decreases all three reactions, while not inhibiting the formation of the reversible complex, indicating that the tyrosine located in Site II (tyr-411)acts as the nucleophile in these reactions. 5. Chemical modification of lysine residues has only a small effect on the reactions while modification of the lone free sulphhydryl (cys) in HSA has no effect.

Species-dependent enantioselective glucuronidation of three 2-arylpropionic acids. Naproxen, ibuprofen, and benoxaprofen.

The enantioselective glucuronidation of several racemic 2-arylproprionic acids (naproxen, ibuprofen, and benoxaprofen) was investigated in vitro with immobilized microsomal protein from human, rhesus monkey, and rabbit liver as the source of UDP-glucuronyl-transferases. Human microsomes, solubilized microsomal protein, and immobilized protein all gave comparable enantioselectivity. The diastereomeric glucuronides were separated and quantitated by HPLC and characterized stereochemically by co-elution with glucuronides formed from authentic resolved enantiomers. Conjugation of the carboxylic acid moieties occurred stereoselectively with all three substrates. However, enantioselectivity varied qualitatively and quantitatively with substrate as well as with species. The glucuronidation of (S)-naproxen by human liver enzymes was inhibited in the presence of (R)-naproxen and vice versa. The ratio of the glucuronides of (S)-benoxaprofen to that of (R)-benoxaprofen in rhesus monkey urine varied between individual animals and appeared to change through time as dosing continued. Hydrolysis of the diasteriomeric glucuronides of (R)- and (S)-naproxen was differentially inhibited by addition of 1,4-saccharolactone.

Extrapolation from animals to man: predictions, pitfalls and perspectives.

Comparative drug disposition studies can be useful in extrapolating from animals to man provided that the criteria indicating interspecies similarity in disposition reflect similar exposure to the foreign compound. Interspecies variability, on the other hand, can often be related to physiological or biochemical differences, thereby providing a rationale for the unsuitability or limitations of a species as a model for human metabolism. Retrospective evaluation of the following examples illustrates the relevance of the indicated disposition characteristics to risk and efficacy assessment: (a) oxaprozin (route of excretion, enterohepatic circulation and exposure; plasma concentrations and efficacy prediction); (b) ciramadol (species differences in presystemic elimination and major metabolic pathway); (c) acebutolol (pharmacologically active human metabolite absent in one of the toxicology species); (d) esmolol (duration of pharmacologic effect controlled by species dependent nature of blood esterases). Stereochemical preferences in the disposition of racemic drugs often differ among species. Extrapolations from one species to another cannot be made in this situation. Pharmacokinetic parameters based on measurements of the sum of the isomers are meaningless and potentially misleading. Future improvements can come from: computer assisted predictions of metabolic pathways; increased use of human tissues; and use of animal species physiologically similar to humans, e.g. the miniature swine.

Inhibition of ciramadol glucuronidation by benzodiazepines.

Studies on the inhibition of ciramadol glucuronidation by benzodiazepines were performed in vitro and in vivo. Ciramadol glucuronidation was slower (Vmax, 1.56 vs. 5.40 nmol/min/mg of microsomal protein) in human than in dog liver microsomes. Inhibition constants (Ki) for lorazepam and oxazepam were 3 to 4 times higher than that calculated for diazepam. Rates of morphine glucuronidation in human liver microsomes were assessed for comparative purposes and agreed with literature values. Each benzodiazepine appeared to be a competitive inhibitor of ciramadol and morphine UDP-glucuronyltransferase activity. The in vivo disposition of ciramadol was unchanged in dogs pretreated with lorazepam. After diazepam treatment no change in the Vdss of ciramadol occurred, but plasma clearance was significantly reduced, resulting in a prolongation of t1/2. Diazepam caused a significant reduction in the oral clearance of ciramadol, whereas no change occurred in systemic availability. Thus, diazepam may have had a secondary effect on hepatic blood flow (QH) and produced offsetting alterations in both intrinsic clearance (Cl int) and QH. A decrease in the area under the plasma concentration time curves of ciramadol aryl O-glucuronide following iv treatment with diazepam coupled with the in vitro data indicate that the mechanism for the decrease in the clearance of ciramadol is inhibition of its glucuronidation by diazepam. Since glucuronidation plays a major role in the elimination of ciramadol in man and dog, these experiments suggest that the disposition of ciramadol in man would not be affected by coadministration of lorazepam, whereas the potential for a diazepam/ciramadol drug interaction in humans exists.

Reactions of oxaprozin-1-O-acyl glucuronide in solutions of human plasma and albumin.

Hydrolysis and rearrangement (isomerization by acyl migration) of oxaprozin glucuronide are greatly accelerated by plasma and human serum albumin. Albumin accounts for all the hydrolytic activity in plasma and no esterase is involved. The isomeric esters formed by rearrangement are also good substrates for the hydrolysis reaction. Another reaction between oxaprozin glucuronide and albumin leads to covalent binding of the aglycone. Similar reactions leading to covalent binding have been described for other acyl glucuronides by several investigators. In the case of oxaprozin, there is little or no potential for biological significance of covalent binding because the reaction is almost entirely inhibited by low concentrations of the drug. All three reactions are pH dependent but not to the same extent. They can be considered to be transacylations to the hydroxyl ion (hydrolysis), to a different OH-group of the glucuronic acid moiety (rearrangement) or to a nucleophilic group on the albumin molecule (covalent binding). All three reactions are greatly inhibited by the same compounds suggesting a common reaction site. This site has certain features in common with the indole or benzodiazepine binding site of human serum albumin. A scheme is proposed in which the first step is reversible binding of the acyl glucuronide to this site in analogy to the known reversible binding of reactive esters (such as p-nitrophenyl acetate) to the same site. All three reactions are inhibited by compounds such as naproxen and decanoic acid which are known to also inhibit the acylation of albumin by reactive esters and the reversible binding of benzodiazepines.

Protein binding of oxazepam and its glucuronide conjugates to human albumin.

The binding of oxazepam and its glucuronide conjugates to human serum albumin (HSA), as well as the binding interactions of the drug and its metabolites, were examined by equilibrium dialysis and kinetic probe studies. Oxazepam and its S(+) glucuronide are bound to the HSA molecule with affinity constants of 3.5 X 10(5) M-1 and 5.5 X 10(4) M-1, respectively, which were independent of protein concentration over a range of 0.1 to 5.0 g/dl. The R(-) glucuronide bound weakly to albumin, with the binding parameter, N X K, increasing at lower albumin concentrations. Pre-acetylation of fatty acid free-HSA resulted in decreased binding of all three compounds, probably by altering the conformation of the binding sites. Kinetic probe studies with p-nitrophenyl acetate indicate that oxazepam and its S(+) glucuronide shared a common binding site on HSA, but that the R(-) glucuronide bound at another site. Oxazepam binding was unaffected by the presence of its glucuronide conjugates but was inhibited by fatty acids. The percentage of oxazepam bound to plasma proteins in patients with renal impairment (94%) was lower than in normal volunteers (97%). This lower binding can neither be attributed to lower albumin concentrations because of the large binding capacity of the protein and linearity of N X K nor to displacement by elevated concentrations of glucuronide conjugates, but it may be ascribed partly to increased plasma fatty acids.

Effects of food on oxaprozin bioavailability.

Twelve healthy volunteers received single 1200-mg oral doses of oxaprozin while fasting and immediately after a standard breakfast in a two-period crossover design with three weeks between administrations. Oxaprozin plasma concentrations were monitored during a 10-day period after each dose. No statistically significant differences were noted between kinetic parameters obtained in the fasting and post-prandial states for mean peak plasma concentrations (Cmax, 103 vs. 109 micrograms/ml), absorption rate constants (ka, 1.1 vs. 0.8 h-1), or total AUC (7042 vs. 7066 micrograms/ml X hr). Compared with doses administered during fasting, postprandial doses led to a delay in the onset of absorption in the gastrointestinal tract (lag time t0, 24 vs. 9 min), but not in the peak time (tmax approximately 5 hours). Oxaprozin's mean residence time t was slightly shorter for subjects in the postprandial state (72 hours) than for those in fasting state (73 hours), probably because of the intrasubject variability in half-life (48 vs. 50 hours). The results of this study indicate that the ingestion of food has no effect on the bioavailability of oxaprozin.

Determination of ciramadol in plasma by gas-liquid chromatography.

An analytical method for determining ciramadol concentrations in plasma was developed and evaluated for its specificity, precision, linearity, and sensitivity. GLC-electron capture detection of a dipentafluorobenzoyl derivative of the drug was used for quantitation. An isomer of the drug served as an internal standard. Resulting mean ratios of the peak height of derivatized drug to that of derivatized internal standard varied with a coefficient of variation that ranged from 3.8 to 11.1%. The mean ratio was linearly related to ciramadol content (8.75-175 ng) with a correlation coefficient greater than to 0.999. The minimum quantifiable concentration was 4 ng/ml with a 2-ml specimen. An application of this method is presented.

Metabolic formation of N- and O-glucuronides of 3-(p-chlorophenyl)thiazolo[3,2-a]benzimidazole-2-acetic acid. Rearrangement of the 1-o-acyl glucuronide.

Excretion of 3-(p-chlorophenyl)thiazolo[3,2-a]benzimidazole-2-acetic acid (I) and its metabolites was studied in rats, beagle dogs, and rhesus monkeys given 20-mg/kg doses of 14C-labeled drug. The urine of rhesus monkeys contained two metabolites in addition to unchanged drug. Both metabolites were hydrolyzed to I by beta-glucuronidase and the hydrolysis was inhibited by 1,4-saccharolactone, indicating that they were glucuronides of I. One of the metabolites (III) was not hydrolyzed by dilute alkali. Its NMR spectrum indicated that the site of conjugation was one of the nitrogen atoms, i.e., it was a quaternary N-glucuronide. The FAB mass spectrum was in conformity with this assignment. This metabolite was not present in the urine of dogs or rats given labeled drug. The other metabolite (II) was excreted in the urine of all three species as well as in the bile of the rat. It was readily hydrolyzed by dilute alkali (pH 11 for 0.5 hr at 37 degrees C), indicating that this metabolite was an acyl glucuronide. The metabolite was stable at pH 4.5 but it was readily converted to three isomers at 37 degrees C within 1 hr at pH 6.5 and above. The mass spectra of the derivatized isomers and metabolite were similar. The isomers were hydrolyzed to I by dilute alkali but not by beta-glucuronidase. They exhibited reducing properties (whereas metabolite II did not), suggesting that they were formed by acyl migration of the aglycone to the second, third, and fourth carbon atoms of the glucuronic acid moiety. Acyl migration probably plays a role in the disposition of I as well as other drugs that form labile glucuronides.

Species-related differences in the stereoselective glucuronidation of oxazepam.

The concentrations of (R)-(-)- and (S)-(+)-oxazepam glucuronides in plasma and urine of several species have been measured. The relative amounts of these diastereoisomers vary among species. Thus, in the plasma and urine of rhesus monkeys the concentrations of the R-isomer are higher, whereas in man and dog more of the S-isomer is present. In plasma and urine of miniature swine the amounts of the two diastereoisomers are about equal. In the urine of rabbits the S-isomer prevails. Similar species-related differences are observed in the in vitro formation of the isomeric oxazepam glucuronides. Homogenates of dog, miniature swine, rabbit, and rat liver produce more of the S-isomer, whereas with monkey liver the formation of (R)-oxazepam glucuronide is favored. The agreement between in vivo and in vitro data is fairly good for rhesus monkey, miniature swine, and rabbit. However, for the dog the ratio of S- to R-isomers in the liver homogenate is much higher than in plasma and urine. This species-dependent stereoselective glucuronidation of oxazepam is not related to the phylogenetic or dietary grouping of these species.

Pharmacokinetics of parenteral dezocine in rhesus monkeys and dogs.

Pharmacokinetic parameters of the analgesic, dezocine, were determined after intravenous and intramuscular injection (1 mg/kg) to rhesus monkeys and dogs. In both species, the drug was rapidly distributed after intravenous administration and then eliminated with a mean half-life of 2.4 hr. Systemic clearance was 54.8 +/- 8.6(SD) and 65.8 +/- 14.0(SD) ml/min/kg in the rhesus monkey and dog, respectively. Glucuronidation was recognized as a major metabolic pathway in both species, and sulfate conjugation was indicated in the dog. Renal elimination of dezocine was minimal. Less than 4% of the dose was eliminated as unchanged dezocine in urine of rhesus monkeys and 1% of the dose in dog urine. After im administration, release from the injection site was rapid and no metabolism at the injection site was indicated. Multiple-dose experiments in dogs did not reveal accumulation. The acquisition of data was made possible by the development of a sensitive, specific assay, which depends on gas-liquid (electron capture) chromatography of a pentafluorobenzoylated derivative of dezocine.

Gas chromatographic determination of guanabenz in biological fluids by electron-capture detection.

A sensitive gas chromatographic method for the determination of guanabenz[(2,6-dichlorobenzylidene)amino]guanidine in urine and plasma was developed. The method depends upon the acid hydrolysis of guanabenz to 2,6-dichlorobenzaldehyde, which has strong electron capturing properties and is volatile enough to be eluted from a gas chromatographic column. Concentrations as low as 0.1 ng of guana-benz/ml can be determined and recovery of the drug from urine and plasma samples is 81.8+/- 5.5% (SD). No interferences arising from plasma, urine, or reagents were encountered. Examples of the application of the method are given.

Protein binding and clearance of oxaprozin, a highly bound anti-inflammatory agent.

Conventional dialysis cells were used in initial attempts to determine the binding characteristics of oxaprozin (4,5-diphenyl-2-oxazolepropionic acid, Wy-21,743). Equilibration required dialysis times up to 22 hours at 37 degrees C resulting in deterioration of plasma proteins, which in turn leads to highly variable binding values. In contrast, dialysis with Dianorm cells requires less than 4 hours to reach equilibrium. The configuration of the cell optimizes the contact between the solutes and the membrane and allows for a more efficient mixing and exchanging of the solute. The percentage of unbound drug was linearly related to total drug in human plasma samples to which oxaprozin in clinically relevant concentrations (55-405 micrograms/ml) had been added. Likewise, a linear relationship between total drug concentration and the percentage unbound was observed in specimens from a pharmacokinetic study in healthy volunteers. Clearance of total oxaprozin from plasma correlated with the percentage unbound drug. Thus the higher clearance observed under steady-state conditions (where concentrations are higher than following single dose administration) was caused by a larger unbound fraction available to the elimination sites.

Oxaprozin disposition in renal disease.

Effects of renal disease on the disposition kinetics of oxaprozin, a nonsteroidal antiinflammatory analgesic, were assessed in 15 subjects who were normal, renally impaired, or who had been undergoing hemodialysis. Oral dose clearance (Cloral), volume of distribution at steady-state (V88d), and elimination half-life (t 1/2) did not substantially differ among the three groups. Mean fraction unbound oxaprozin in plasma (fup) increased from 0.08% in the normal group to 0.18% and 0.28% in the two azotemic groups. Consequently, unbound drug kinetic parameters, including intrinsic clearance (Clint) and V88du of unbound drug were reduced from 2.9 l/hr/kg and 193 l/kg in normal subjects to approximately 1.6 l/hr/kg and 91 l/kg in azotemic patients. The smaller volume of distribution is consistent with a decrease in oxaprozin tissue binding in azotemia. The decreased plasma and tissue binding and lower Clint suggest that, in the treatment of azotemic patients with rheumatoid arthritis, the dose of oxaprozin should begin at 600 mg once a day.

Metabolic disposition of ciramadol in rhesus monkeys and rats.

Studies on the metabolic disposition of ciramadol, a new orally effective analgesic, have been conducted in the rhesus monkey and male rat. The drug is well absorbed but undergoes extensive presystemic metabolism when given by the intragastric route (1 mg/kg) to rhesus monkeys. Maximum concentrations in plasma do not exceed 4 ng/ml. The major transformation occurs by glucuronidation and leads to two metabolites, and aryl O- and an alicyclic O-glucuronide. Phase I transformations are apparently minor and include N-demethylation, formation of benzoxazinyl metabolites from the reaction of endogenous acetaldehyde and formaldehyde with N-desmethylciramadol, and oxidation of the alicyclic and aromatic rings. Mass-spectrometric analyses of the isolated glucuronides and the minor phase I metabolites have been employed for structural assignments. Excretion of the intragastric dose occurs predominantly by the renal route. When given intramuscularly (1 mg/kg), concentrations of ciramadol peak at 1150 ng/ml by 0.5 hr after medication and decline rapidly to less than 7 ng/ml by 8 hr. Approximately 10% of the dose is excreted as unchanged ciramadol after im dosing. In male rats, the drug is absorbed rapidly after single 1-mg/kg intragastric doses and is taken up notably by liver, lung, kidney and spleen. Unlike its disposition in rhesus monkeys, ciramadol is present in plasma and excreted into urine mainly in the unconjugated form. Concentrations of drug in rat plasma are higher than those in rhesus monkey plasma, suggesting a smaller first-pass effect in the rat.

Pharmacokinetic disposition of guanabenz in the rhesus monkey.

The pharmacokinetics of guanabenz (E-2,6-dichlorobenzylidene aminoguanidine acetate, Wy-8678) in rhesus monkeys given 14C-labeled and unlabeled drug were investigated. The radioactive dose was well absorbed after intragastric (ig) administration of 1 mg of the labeled drug per kg, as indicated by tissue and urinary recovery of the label. Excretion into urine accounted for 57 +/- 3 (SE)% of the radioactive ig dose. Recovery of radioactivity in urine after iv administration of 0.2 mg/kg was 79 +/- 0.6% of the radioactive dose. Less than 1% of the dose was recovered in urine as unchanged drug after either route of administration. Plasma concentration/time profiles after 1-mg/kg iv and ig doses were fitted by polyexponential equations with a terminal elimination half-life of 12.0 +/- 1.1 hr. A large volume of distribution (VSSD = 10.3 +/- 0.7 liters/kg) indicated extensive extravascular distribution of the drug, which was confirmed by 14C-distribution studies. The systemic clearance was 27.5 +/- 1.4 ml/min/kg with hepatic clearance appearing to be the major determinant in guanabenz elimination. Dose proportionality was evident from a comparison of areas under the plasma concentration-time curves (AUC) of 1- and 5-mg/kg ig doses. The low systemic availability of 0.19-0.31 reflects the extensive presystemic extraction (first-pass effect) of the drug. Similarities in the pharmacokinetics of guanabenz in man and the rhesus monkey indicate that the latter species may serve as a satisfactory model for man in disposition studies.

Relationship of guanabenz concentrations in brain and plasma to antihypertensive effect in the spontaneously hypertensive rat.

The antihypertensive agent guanabenz (E-2,6-dichlorobenzylidene aminoguanidine acetate. Wy-8678) was administered i.v. in single doses of 10, 32 and 100 microgram/kg to groups of spontaneously hypertensive rats. Mean arterial blood pressure and heart rate were monitored continuously. Animals were decapitated at predetermined time intervals and concentrations of the drug in whole brain and plasma were measured by a specific gas chromatographic method. After an initial transient increase in blood pressure, significant dose-dependent maximal decreases in pressure of 15 +/- 8, 46 +/- 20 and 59 +/- 15 mm Hg (mean +/- S.D.) were observed 15 to 30 min after injection of the above respective doses. At the line of the maximal decreases in pressure (T delta max), concentrations of guanabenz in brain were 10 +/- 2, 29 +/- 8 and 89 +/- 21 ng/g and correlated significantly with dose and the magnitude of blood pressure change. The correlation between concentration in brain and change in blood pressure was also significant at subsequent sacrifice times. Concentrations of guanabenz in plasma were below the limit of detection after the 10 microgram/kg dose. At the two higher doses, concentrations in plasma were dose related but did not correlate with decreases in blood pressure. The correlation between concentration in brain and change in blood pressure is in agreement with the predominantly central mechanism of action of guanabenz.

Metabolism and kinetics of oxaprozin in normal subjects.

Absorption, biotransformation, excretion, and kinetics of oxaprozin (4,5-diphenyl-2-oxazolepropionic acid) were examined in subjects after an oral dose of 14C-oxaprozin alone as well as before, during, and after long-term administration of unlabeled drug. A single dose of 14C-oxaprozin was rapidly absorbed and the unchanged drug was essentially the only labeled substance in plasma. Recovery of radioactivity in excreta, mostly in urine, exceeded 90%. Major biotransformation routes were glucuronidation of the carboxyl group and hydroxylation of the phenyl rings followed by glucuronidation. Administration of unlabeled oxaprozin did not affect the absorption, qualitative, or quantitative metabolite profile, or recovery of 14C-oxaprozin. Following a single dose, the kinetic parameters for 14C and unchanged drug in plasma were nearly the same. A2-compartment model with first-order elimination adequately describes kinetic disposition. The slow clearance (Clp), 0.08 to 0.12 1/hr, was almost entirely due to biotransformation and the plasma half-lifes, which ranged from 49 to 69 hr, reflected the small Clp. The small volume of distribution (VD beta = 8 to 9 1) indicates limited extravascular distribution. Multiple doses of unlabeled drug, especially when given concurrently, increased the Clp of 14C-oxaprozin. This effect is apparently related to decreased binding of high concentrations of oxaprozin to plasma protein. As a result of increased Clp, steady-state levels are only 40% of levels predicted from the single-dose study.