COVALENT BINDING OF ACIDIC DRUGS VIA REACTIVE INTERMEDIATES - DETECTION OF BENOXAPROFEN AND FLUNOXAPROFEN PROTEIN ADDUCTS IN BIOLOGICAL-MATERIAL

The purpose of the study was the direct detection of intact protein ad ducts - resulting from in-vitro incubations of flunoxaprofen- and beno xaprofen glucuronides in biological materials or originating from in v ivo studies - by polyacrylamide gel electrophoresis (SDS-PAGE) followe d by blotting and fluorescent scan, presumably yielding better specifi city for the macromolecular binding partner and avoiding alkaline clea vage to release the aglycone. Glucuronides were isolated from urine sa mples or generated by incubation of aglycone with cofactors and rat li ver microsomes. Following dialysis against BSA solution, SDS-PAGE and subsequent electrotransblotting were performed. Apparently, albumin re presents the major binding protein for the covalent binding of these a cyl glucuronides in plasma following incubation with blank plasma. In microsomal proteins two fluorescent peaks (appr. 39 and 62 KD) were id entified for flunoxaprofen and benoxaprofen incubations. In vivo coval ent binding was detected for both flunoxaprofen and benoxaprofen in pl asma samples. For the racemically administered benoxaprofen a slight p reponderance in adduct concentrations was found for the S-enantiomer. The pharmacokinetic analysis of in vivo data obtained for R/S-benoxapr ofen (dose: 600 mg racemate) and S-flunoxaprofen (dose: 100 mg racemat e), both of which have been withdrawn from the market, (employing a st ereospecific HPLC method when analyzing volunteers' and patients' samp les collected in the last 14 years, yet not stored longer than 3-4 yea rs) demonstrated that significant amounts of glucuronides occur for bo th drugs (n = 2 for each compound; average C-max values of the glucuro nides: S-flunoxaprofen: 395 ng/ml; S-benoxaprofen: 775 ng/ml; R-benoxa profen: 563 ng/ml). Presumably because of stereoinversion in humans, a glycone and glucuronide concentrations were higher for S- than for R-b enoxaprofen. In vivo aglycone/glucuronide ratios were smaller for S- t han for R-benoxaprofen, although in vitro incubation with human liver microsomes resulted in preferential glucuronidation of the R-enantiome r of benoxaprofen. Plasma concentration-time curves of the glucuronide s paralleled those of the respective aglycones in their terminal phase . S-Benoxaprofen adduct concentrations were higher than R-benoxaprofen adduct concentrations (S: 28 ng/ml; R: 18 ng/ml covalently bound) and S-flunoxaprofen adduct concentrations with 29 ng/ml in the same range as S-benoxaprofen adducts, although for the latter the dose range as well as the respective glucuronide concentrations were higher. This in dicates a higher reactivity of S-flunoxaprofen as opposed to S-benoxap rofen glucuronides.