Reactivity of diflunisal acyl glucuronide in human and rat plasma and albumin solutions

doi:10.1016/0006-2952(90)90286-T

Biochemical Pharmacology

Volume 39, Issue 6, 15 March 1990, Pages 1067-1075

https://doi.org/10.1016/0006-2952(90)90286-T Get rights and content

Abstract

Diflunisal acyl glucuronide (DAG) is a major metabolite of diflunisal (DF) in rats and humans. We have investigated the reactivity of DAG, in purified albumin solutions and plasma from both rat and human sources, along three interrelated pathways: rearrangement via acyl migration to yield positional isomers of DAG, hydrolysis of DAG and/or its isomers to liberate DF, and formation of covalent adducts of DF (via DAG and/or its isomers) with plasma protein. Two initial concentrations of DAG (ca. 50 and 10 μg DF equivalents/mL) were used throughout. In all incubations, the order of quantitative importance of the reactions was: rearrangement > hydrolysis >covalent binding. At pH 7.4 and 37°, degradation of DAG in albumin solutions (e.g. half-life ca. 95 min in fatty acid-free human serum albumin) was retarded in comparison to that found in buffer alone (half-life ca. 35 min). Degradation in unbuffered rat and human plasma containing heparin was comparable to that found in buffer. Maximal covalent binding to protein was achieved after 4–8 hr incubation, and was greatest for fatty acid-free human serum albumin (165ng DF/mg albumin). Thereafter, slow degradation of the adducts was observed. Formation of DF-plasma protein adducts in vivo was also found in rats and humans dosed with DF.

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In addition to the transacylation of 1-O-β-AG, ring-opened AGs (aldose), generated from positional isomers, also form covalent protein adducts, known as Schiff bases, followed by an Amadori rearrangement to form glycated proteins [52]. 1-O-β-AGs and their positional isomers of various carboxylic acid-containing drugs, such as zomepirac, diflunisal, etodolac, ketoprofen, and fibrates, covalently bind to proteins in human plasma and/or human serum albumin (HSA) [51,53–56]. The degradation rate constants of nine kinds of AGs in an incubation mixture containing HSA were strongly correlated with the levels of covalent binding to HSA (r2 = 0.995) [57].
Idiosyncratic drug toxicity (IDT) is a serious problem in drug development. Reactive metabolites are postulated to be one of the causes for IDT. Conjugated metabolites are generally non-reactive except for acyl glucuronides (AGs), which are sufficiently reactive to covalently bind to endogenous proteins. Thus, it has been suggested that AGs would contribute to IDT caused by carboxylic acid-containing drugs. Glucuronidation of a carboxylate residue is catalyzed by UDP-glucuronosyltransferase 1A and 2B isoforms. Unstable AGs undergo intramolecular rearrangements as well as non-enzymatic and enzymatic hydrolysis. The instability and reactivity toward proteins have been well studied for a large number of AGs. Moreover, the half-life of AGs in neutral buffer is becoming a common marker for the prediction of toxicity caused by carboxylic acid-containing drugs in the screening of new chemical entities; however, the underlying mechanisms of the toxicity are not elucidated. Recently, an immunostimulation assay has been proposed for the assessment of the toxicological potential of AGs, which may have a better predictability compared with half-life and peptide adduct assays. In addition to in vitro studies, studies in model animals indicate the in vivo toxicological potential of AGs and help understand the mechanisms of the AG toxicity.
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Acyl glucuronides can be reactive metabolites capable of undergoing hydrolysis, intramolecular acyl migration, and covalent binding to proteins, both in vitro and in vivo. Glucuronidation occurs primarily in the liver and intestines, and is the major route of elimination for many xenobiotic and endogenous compounds containing a carboxylic acid function. Although glucuronidation is recognized as a detoxification process, acyl-linked glucuronides are considered chemically reactive electrophiles capable of covalent modification with nucleophilic sites on proteins. The extent of covalent binding with lysine amino groups of proteins correlates well with acyl glucuronide degradation rates (acyl migration and hydrolysis) and occurs either through a direct transacylation reaction or by a Schiff base mechanism via the open-chain aldehyde form of the acyl-migrated isomer. Subsequent formation of drug protein adducts has been proposed to elicit a direct toxicity or an immune-based reaction, both of which are consistent with the idiosyncratic toxicity commonly associated with carboxylic acid-containing drugs.
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Inhibition of proliferation of HT-29 colon adenocarcinoma cells by carboxylate NSAIDs and their acyl glucuronides
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Many nonsteroidal anti-inflammatory drugs (NSAIDs) which have antiproliferative activity in colon cancer cells are carboxylate compounds forming acyl glucuronide metabolites. Acyl glucuronides are potentially reactive, able to hydrolyse, rearrange into isomers, and covalently modify proteins under physiological conditions. This study investigated whether the acyl glucuronides (and isomers) of the carboxylate NSAIDs diflunisal, zomepirac and diclofenac had antiproliferative activity on human adenocarcinoma HT-29 cells in culture. Included as controls were the carboxylate NSAIDs themselves, the non-carboxylate NSAID piroxicam, and the carboxylate non-NSAID valproate, as well as its acyl glucuronide and isomers. The compounds were incubated at 1–3000 μM with HT-29 cells for 24 hr, with [³H]-thymidine added for an additional 2 hr incubation. IC₅₀ values were calculated from the concentration-inhibition response curves for thymidine uptake. The four NSAIDs inhibited thymidine uptake, with IC₅₀ values about 200–500 μM. All of the NSAID acyl glucuronides (and isomers, tested in the case of diflunisal) showed antiproliferative activity broadly comparable to the parent drugs. This activity may stem from direct uptake of intact glucuronide/isomers followed by covalent modification of proteins critical in the cell replication process. However, hydrolysis during incubation and cellular uptake of liberated parent NSAID will play a role. In HT-29 cells incubated with zomepirac, covalently modified proteins in cytosol were detected by immunoblotting with a zomepirac antibody, suggesting that HT-29 cells do have the capacity to glucuronidate zomepirac. The anti-epileptic drug valproate had no effect on inhibition of thymidine uptake, though, surprisingly, its acyl glucuronide and isomers were active. The reasons for this are unclear at present.
Bile duct ligation promotes covalent drug-protein adduct formation in plasma but not in liver of rats given zomepirac
2000, Life Sciences
Acyl glucuronides are reactive electrophilic metabolites of carboxylate drugs, capable of undergoing hydrolysis, rearrangement and covalent binding reactions with proteins in vivo. Such covalent drug-protein adducts may be prerequisites for certain idiosyncratic immune and toxic responses in susceptible individuals. The present study examined the effect of experimental cholestasis on the extent and pattern of formation of protein adducts in plasma and liver of rats given the non-steroidal anti-inflammatory drug (NSAID) zomepirac (ZP). Groups of intact, bile-exteriorized and bile duct-ligated rats given a 50 mg/kg i.v. dose of ZP were studied for 24 hr. In intact rats, only 1.4% of the dose was recovered as the sum of ZP, ZP acyl glucuronide (ZAG) and its rearrangement isomers (iso-ZAG) in urine in 24 hr. In bile-exteriorized animals, 0.5% of the dose was recovered in urine in 24 hr, with 31.6% of the dose being recovered in bile (2.7% as ZP, 20.0% as ZAG and 8.9% as iso-ZAG). In the bile duct-ligated group, recovery of dose in 24 hr urine totalled 17.5% (1.7% as ZP, 6.7% as ZAG and 9.1% as iso-ZAG). ZAG and iso-ZAG were measurable in plasma only in the bile duct-ligated group, and covalent binding of ZP to plasma proteins was much higher (5–6 fold) than in intact or bile-exteriorized rats. Total adduct concentrations in liver were not significantly different among the three groups. Immunoblotting using a polyclonal ZP antiserum confirmed that serum albumin was a major target protein in plasma. The major ZP-modified bands in the livers of intact and bile-exteriorized rats were at about 110, 140 and 200 kDa. However, the bands at 110 and 140 kDa were much lower in the livers of bile duct-ligated rats. The results show that about 30% of ZP doses are normally excreted as ZAG and its isomers in bile, with only minor excretion in urine. Bile duct ligation shunts the glucuronide into blood (and urine), strongly promoting adduct formation with plasma proteins, and alters the pattern but not the total quantity of drug-modified proteins formed in the liver.
Hepatobiliary transport of diflunisal conjugates and taurocholate by the perfused rat liver: The effect of chronic exposure of rats to diflunisal
1998, Life Sciences
Acyl glucuronides are reactive electrophilic metabolites of carboxylate drugs which can form covalent adducts with endogenous macromolecules such as serum albumin and hepatic proteins. Such adducts have been suggested as initiating factors in certain immune and toxic responses to acidic drugs. In the present study, pretreatment of rats with high daily doses (50 mg/kg orally) of the non-steroidal anti-inflammatory drug (NSAID) diflunisal (DF) for 35 days, followed by perfusion of the isolated liver with 3 mg DF for 3 hr, resulted in appreciable concentrations of covalent adducts of DF with hepatic tissue (3.68 μg DF/g liver). Immunoblotting using a rabbit polyclonal DF antiserum showed the major DF-modified bands at about 110, 140 and 200 kDa. A vehicle-pretreated control group achieved adduct concentrations of only 0.37 μg DF/g liver, with the 200 kDa band not detectable in immunoblots. Elimination of DF from perfusate of the isolated perfused rat liver (IPRL) preparation was the same ( $t_{12}$ about 3.4 hr) in both DF- and vehicle-pretreated groups. Appearance of the sulfate (DS) conjugate, the major metabolite in perfusate, was also similar. However, higher concentrations of the acyl glucuronide (DAG) and phenolic glucuronide (DPG) conjugates were found in perfusate at later times, though a statistically significant difference in area under the concentration-time curve was found only in the case of DAG. At 3 hr, recoveries of dose as DAG and DPG were significantly higher in perfusate, but not in bile. No significant differences in uptake and biliary excretion of taurocholate were found between the two groups. The finding of higher perfusate concentrations of DAG and DPG could signal a minor compromise to biliary excretion processes for the glucuronides, though whether such a result is simply coincident with or attributable to DAG-derived covalent DF-protein adducts in liver remains indeterminate.

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Reactivity of diflunisal acyl glucuronide in human and rat plasma and albumin solutions

Abstract

J Chromatogr

J Pharmaceut Biomed Anal

Biochem Pharmacol

J Chromatogr

Physiological disposition and metabolism of 5-(2',4'-dinuoropnenyl)salicylic acid. a new salicylate

Drug Metab Dispos

Effect of enterohepatic circulation on thé pharmacokinetics of diflunisal in rats

Drug Metab Dispos

Isolation and identification of a new major metabolite of diflunisal in man. The sulfate conjugate

Drug Metab Dispos

Reactivity considerations in the analysis of glucuronide and sulfate conjugates of diflunisal

Ther Drug Monit

Assay methodology for quantification of the ester and ether glucuronide conjugates of diflunisal in human urine

J Chromatogr

Glucuronic acid conjugates of bilirubin-IXα in normal bile compared with post-obstructive bile. Transformation of the 1-O-acylglucuronides into 2-, 3-, and 4-O-acylglucuronides

Biochem J

Separation of two conjugates of clofibric acid (CPIB) found in the urine of subjects taking clofibrate

Clin Exp Pharmacol Physiol

pH dependent formation of β-glucuronidase resistant conjugates from the biosynthetic ester glucuronide of isoxepac

Biochem Pharmacol

Apparent intramolecular acyl migration of zomepirac glucuronide

Drug Metab Dispos

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

Drug Metab Dispos

pH-Dependent rearrangement of the biosynthetic ester glucuronide of valproic acid to β-glucuronidase-resistant forms

Drug Metab Dispos

Furosemide 1-O-acyl glucuronide. In vitro biosyn-thesis and pH-dependent isomerization to β-glucuronidase-resistant forms

Drug Metab Dispos

Acylation of amino acids and other endobiotics by xenobiotic carboxylic acids

Effect of pH on acyl migration and hydrolysis of tolmetin glucuronide

Drug Metab Dispos

Properties of acyi glucuronides: implications for studies of the pharmacokinetics and metabolism of acidic drugs

Drug Metab Rev