Andrew R. King

Studies on the reactivity of acyl glucuronides—II: Interaction of diflunisal acyl glucuronide and its isomers with human serum albumin in vitro

A major metabolite of diflunisal (DF) is its reactive acyl glucuronide conjugate (DAG) which can undergo hydrolysis (regeneration of DF), intramolecular rearrangement (isomerization via acyl migration) and intermolecular reactions with nucleophiles. We have compared the fate of DAG and its individual 2-, 3- and 4-O-acyl positional isomers (at ca. 55 micrograms DF equivalents/mL) after incubation with human serum albumin (HSA, 40 mg/mL) at pH 7.4 and 37 degrees. Initial half-lives (T1/2) for DAG and its 2-, 3- and 4-isomers were 53, 75, 61 and 26 min, respectively. DAG was more labile to hydrolysis than any of its isomers but the latter, in particular the 4-isomer, were much better substrates for formation of covalent DF-HSA adducts. After a 2-hr incubation, 2.4, 8.2, 13.7 and 36.6% of substrate DAG and its 2-, 3- and 4-isomers (respectively) were present as DF-HSA adducts. With long term incubation, the concentrations of adducts so generated in situ declined in a biphasic manner, with apparent terminal T1/2 values of ca. 28 days. DAG was much more labile to transacylation with methanol (i.e. formation of DF methyl ester) than an equimolar mixture of its isomers after incubation in a 1:1 methanol:pH 7.4 buffer solution at 37 degrees (T1/2 values of 5 and 70 min, respectively). The data do not support direct transacylation with nucleophilic groups on protein as the predominant mechanism of formation of covalent DF-HSA adducts in vitro.

Studies on the reactivity of acyl glucuronides-IV. Covalent binding of diflunisal to tissues of the rat

Acyl glucuronides have been shown to be reactive electrophilic metabolites capable of undergoing hydrolysis, rearrangement (isomerization via acyl migration) and covalent binding reactions to plasma protein. The present study was undertaken to explore the occurrence and extent of in vivo formation of covalent adducts of diflunisal (DF), a salicylate derivative which forms a reactive acyl glucuronide, with tissues and plasma protein of rats. Groups of rats were given 50 mg DF/kg i.v. twice daily for periods of up to 7 days. Steady state plasma concentrations of reversibly bound DF and its conjugates (as measured 6 hr after a dose) were achieved by the third day of dosing. T 1/2 values after cessation of dosing were about 5-10 hr. By contrast, covalent DF-tissue adducts steadily accumulated over the 7-day dosing period. Maximum concentrations, measured 6 hr after the last dose, were 4.8 (liver), 1.0 (kidney), 0.74 (plasma), 0.26 (small intestine minus contents), 0.27 (large intestine minus contents) and 0.20 (skeletal muscle) microgram DF/g tissue or/mL plasma. T 1/2 values of about 50, 67, 18, 38 and 43 hr were obtained for liver, kidney, plasma and small and large intestine (respectively) after cessation of dosing. Thus, the study of acyl glucuronide reactivity and the question of any derived toxicity or immune responses should consider the formation of long-lived adducts in tissues as well as in plasma.

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

Reactivity of glucuronide and sulfate conjugates was taken into account in development of a simple isocratic HPLC method for direct assay of diflunisal (DF) and its acyl glucuronide (DAG), phenolic glucuronide (DPG), and sulfate (DS) conjugates. Whereas DPG was stable over the pH range 0–9, DAG was highly labile at neutral to slightly alkaline pH, undergoing rearrangement (isomerisation via acyl migration), hydrolysis, and, in the presence of methanol, transesterification to DF methyl ester. The 2-, 3-, and 4-O-acyl positional isomers of DAG appeared as three pairs of peaks. Interconversion between partners of each pair occurred even under acidic conditions inhibitory to acyl migration, implicating mutarotation. DS was stable at neutral to slightly alkaline pH, but underwent hydrolysis under relatively strongly acidic conditions. However, this hydrolysis was remarkably catalyzed (e.g., by 1,000-fold) in the presence of solvents (i.e., solvolysis) such as diethyl ether and ethyl acetate. DS (an acid) could not be extracted from aqueous solution because of this acidic solvolysis. Suitable conditions for simultaneous direct analysis (nonextractive, nonconcentrative) of DF and its reactive (DAG and DS) and unreactive (DPG) conjugates were achieved by working at pH of approximately 4.5. The procedure thus developed is suitable for plasma, urine, and bile samples, and has revealed the presence of new, as yet unidentified, metabolites of DF.

CONTRASTING SYSTEMIC STABILITIES OF THE ACYL AND PHENOLIC GLUCURONIDES OF DIFLUNISAL IN THE RAT

1. Diflunisal (DF) is metabolized in humans and rats primarily to its acyl glucuronide, phenolic glucuronide and sulphate conjugates. 2. After i.v. administration of DF acyl glucuronide to pentobarbitone-anaesthetized rats, DF and its phenolic glucuronide and sulphate conjugates appeared rapidly in plasma, indicating ready systemic hydrolysis of the acyl glucuronide and subsequent biotransformation of liberated DF. 3. Approximately 72% of the acyl glucuronide dose was recovered in bile and urine over 6 h: 52% as acyl glucuronide, 6% as phenolic glucuronide, 5% as sulphate, and 8% as isomers of the acyl glucuronide arising from intramolecular acyl migration. 4. Blockage of excretion routes by ligation of the ureters, bile duct, and both ureters and bile duct, decreased plasma clearance of the acyl glucuronide from 7.8 ml/min per kg to 6.0, 3.2 and 2.2 ml/min per kg respectively, and increased the apparent terminal plasma half-life of DF from 2.1 h to 2.6, 3.4 and 6.3 h, respectively. 5. By contrast, DF phenolic glucuronide was quite stable after i.v. administration at the same dose. 6. This study shows that systemic cycling between DF and its acyl glucuronide exists in the rat in vivo, with portions of each cycle of unstable acyl glucuronide through DF yielding stable phenolic glucuronide and (presumptively stable) sulphate conjugate.

A-1048400 is a novel, orally active, state-dependent neuronal calcium channel blocker that produces dose-dependent antinociception without altering hemodynamic function in rats.

Blockade of voltage-gated Ca²⁺ channels on sensory nerves attenuates neurotransmitter release and membrane hyperexcitability associated with chronic pain states. Identification of small molecule Ca²⁺ channel blockers that produce significant antinociception in the absence of deleterious hemodynamic effects has been challenging. In this report, two novel structurally related compounds, A-686085 and A-1048400, were identified that potently block N-type (IC₅₀=0.8 μM and 1.4 μM, respectively) and T-type (IC₅₀=4.6 μM and 1.2 μM, respectively) Ca²⁺ channels in FLIPR based Ca²⁺ flux assays. A-686085 also potently blocked L-type Ca²⁺ channels (EC₅₀=0.6 μM), however, A-1048400 was much less active in blocking this channel (EC₅₀=28 μM). Both compounds dose-dependently reversed tactile allodynia in a model of capsaicin-induced secondary hypersensitivity with similar potencies (EC₅₀=300-365 ng/ml). However, A-686085 produced dose-related decreases in mean arterial pressure at antinociceptive plasma concentrations in the rat, while A-1048400 did not significantly alter hemodynamic function at supra-efficacious plasma concentrations. Electrophysiological studies demonstrated that A-1048400 blocks native N- and T-type Ca²⁺ currents in rat dorsal root ganglion neurons (IC₅₀=3.0 μM and 1.6 μM, respectively) in a voltage-dependent fashion. In other experimental pain models, A-1048400 dose-dependently attenuated nociceptive, neuropathic and inflammatory pain at doses that did not alter psychomotor or hemodynamic function. The identification of A-1048400 provides further evidence that voltage-dependent inhibition of neuronal Ca²⁺ channels coupled with pharmacological selectivity vs. L-type Ca²⁺ channels can provide robust antinociception in the absence of deleterious effects on hemodynamic or psychomotor function.

Studies on the reactivity of acyl glucuronides—VII: Salicyl acyl glucuronide reactivity in vitro and covalent binding of salicylic acid to plasma protein of humans taking aspirin☆

Salicyl acyl glucuronide (SAG) is a significant metabolite of salicylic acid (SA) and aspirin. We have shown that, under physiological conditions in vitro, SAG undergoes rearrangement in a manner consistent with acyl migration to its 2-, 3- and 4-O-acyl positional isomers as the predominant pathway (T1/2 values were 1.4-1.7 hr in buffer at pH 7.4 and 37 degrees). Incubation of SAG or a mixture of its rearrangement isomers (iso-SAG) (each at approximately 50 micrograms SA equivalents/mL) with human serum albumin (HSA, at approximately 40 mg/mL) revealed the formation of covalent adducts with the protein, with peak concentrations of 1-2 micrograms SA equivalents/mL. The data support a role for the rearrangement/glycation mechanism of adduct formation. Covalent adducts of SA were also detected in the plasma of humans taking aspirin (at > or = 1200 mg/day), but the concentrations were low (<< 100 ng SA equivalents/mL). Reactivity of SAG thus provides a mechanism (though of uncertain quantitative importance) of covalent attachment of the salicyl moiety of aspirin to tissue macromolecules, which is in addition to its well-known acetylating capacity.

Fallacious results from measuring salivary carbamazepine concentrations

Summary During a carbamazepine (CBZ) relative bioavailability study involving tablets and a syrup preparation, salivary drug concentrations appeared disproportionately high relative to simultaneous plasma drug concentrations in the first 2–3 h after oral drug intake. This raised the suspicion of contamination of saliva by retention of drug in the mouth. In a separate study CBZ was retained in the mouth in tablet form (whole or crushed) or in syrup, for only 5 s before being spat out, and the mouth was carefully rinsed. Despite this, measurable salivary concentrations, sufficient to cause substantial error if extrapolated to simultaneous plasma drug concentrations, were present for at least 2 h after drug administration. CBZ in these studies disappeared from saliva with an apparent mean half-life of 21.0 ± 4.8 min. This experience suggests that, in therapeutic drug monitoring, salivary CBZ concentrations for at least 2 h after dosage may lead to invalid conclusions about simultaneous plasma CBZ concentrations.

Studies on the reactivity of acyl glucuronides—V. Glucuronide-derived covalent binding of diflunisal to bladder tissue of rats and its modulation by urinary pH and β-glucuronidase☆

Acyl glucuronide conjugates of acidic drugs have been shown to be reactive metabolites capable of undergoing non-enzymic hydrolysis, rearrangement (isomerization via acyl migration) and covalent binding reactions with plasma protein. In an earlier study (King and Dickinson, Biochem Pharmacol 45: 1043-1047, 1993), we documented formation of covalent adducts of diflunisal (DF), a salicylate derivative which is metabolized in part to a reactive acyl glucuronide (DAG), with liver, kidney, skeletal muscle and small and large intestine (in addition to plasma protein) of rats given the drug i.v. twice daily at 50 mg DF/kg for 7 days. The present study shows that covalent adducts of DF were also formed with urinary bladder tissue of these rats, achieving concentrations (ca. 5 micrograms DF equivalents/g tissue) higher than those found in the other tissues noted above. After cessation of dosing, the adduct concentrations declined with an apparent T 1/2 value of ca. 20 hr. Adducts were also formed ex vivo in excised rat bladders in which DAG or a prepared mixture of its acyl migration isomers (iso-DAG) were incubated at pH 5.0, 6.5 and 8.0. After 8 hr incubation, the highest concentrations (ca. 11 micrograms DF equivalents/g) were produced with iso-DAG at pH 5.0, and the lowest (ca. 2.3 micrograms DF equivalents/g) with DAG at pH 5.0. However, a major competing reaction for DAG (at least at pH 5.0) was hydrolysis by beta-glucuronidases originating from bladder tissue. By contrast, iso-DAG was quite resistant to such hydrolysis. The phenolic glucuronide conjugate, another important metabolite of DF, was hydrolysed only slowly. Similar results were obtained in fresh rat urine adjusted to pH 5.0. The results support covalent DF adduct formation in rat bladder originating from both DAG and iso-DAG as ultimate reactants, though the extent of binding is modulated by both urinary pH and beta-glucuronidases.

ELIMINATION OF DIFLUNISAL AS ITS ACYL GLUCURONIDE, PHENOLIC GLUCURONIDE AND SULFATE CONJUGATES IN BILE‐EXTERIORIZED AND INTACT RATS

1. The disposition of diflunisal (DF) was investigated in both bile‐exteriorized and intact rats given 10 and 100 mg/kg doses intravenously (i.v.).

Simultaneous plasma carbamazepine and carbamazepine epoxide concentrations in pharmacokinetic and bioavailability studies

Summary A comparative bioavailability study of carbamazepine (CBZ) in tablets and a syrup preparation was carried out in six volunteers, using a crossover design. Plasma and saliva samples were collected at appropriate times, and the plasma specimens were analyzed by high performance liquid chromatography for concentrations of CBZ and its epoxide metabolite (CBZ-EP). Analysis of the data showed that the preparations were equally bioavailable, although absorption was faster from the syrup and gave higher maximum plasma levels of CBZ. In other respects the preparations were pharmacokinetically equivalent. Analysis of simultaneous plasma CBZ and CBZ-EP concentration-time data by iterative curve fitting to a one-compartment linear model permitted the calculation of elimination kinetic parameters of the CBZ-EP; the mean elimination half-life was 6.9 ± 2.7 h. Failure of the salivary CBZ concentrations soon after drug intake to correlate with simultaneous plasma CBZ levels rendered the salivary data useless for bioavailability comparison, but prompted a further study of salivary CBZ levels.