The impact of pregnancy on urinary ketorolac metabolites after single intravenous bolus.

Compared to female volunteers or postpartum, ketorolac clearance is higher at delivery. To explore the alterations that explain this higher clearance, urinary ketorolac metabolites collected at delivery (n = 40) were compared to female volunteers (unpaired, n = 8) or postpartum (paired, n = 8) following intravenous administration of 30 mg ketorolac tromethamine. A mean 38 (SD 9) % of the ketorolac dose was retrieved in 8-h urine collections. This was based on mean portions of 56 (20), 10 (14) and 33 (12) % for free ketorolac, ketorolac-glucuronide and p-hydroxy-ketorolac, respectively. The mean ketorolac-glucuronide portion at delivery (5 %) was lower compared to female volunteers (21 %) or postpartum (21 %) (p = 0.003 and p = 0.002, respectively). Similarly, there was a difference in mean portion of free urinary ketorolac at delivery when compared to healthy female volunteers (60-45 %, p = 0.046). Using paired statistics, the mean portion of total urinary ketorolac was lower (62-73 %, p = 0.015) while the portion retrieved as p-hydroxy-ketorolac was significantly higher at delivery compared to postpartum (38-28 %, p = 0.031). The differences in urine metabolites suggest that the increased ketorolac clearance at delivery is in part explained by increased metabolic clearance to p-hydroxy-ketorolac, reflecting increased oxidation activity.

Determination of non-steroidal anti-inflammatory drugs in urine by combining an immobilized carboxylated carbon nanotubes minicolumn for solid-phase extraction with capillary electrophoresis-mass spectrometry.

Last years, the usefulness of the use of carbon nanotubes (CNTs) as sorbent material have been demonstrated for a wide variety of compounds. In this work, it has been demonstrated for first time that immobilized carboxylated single-walled carbon nanotubes (c-SWNTs) offer clear advantages over the use of CNTs. The higher adsorption capacity has been attributed to the special orientation of c-SWNTs molecules on the glass surface. The potential of this new sorbent was evaluated for the preconcentration of non-steroidal anti-inflammatory drugs (NSAIDs) from urine samples. Purified samples were analysed by capillary electrophoresis-mass spectrometry detection allowing the determination of 1.6 to 2.6 microg/L of NSAIDs with only 5 mL of sample. The precision of the method for the determination of real spiked urine samples ranged from 5.4 to 7.4% and the recoveries from 98.6 to 102.2%.

Identification of coenzyme A-related tolmetin metabolites in rats: relationship with reactive drug metabolites.

1. It has recently been proposed that acyl coenzyme A thioesters (acyl-CoAs) of xenobiotic carboxylic acids are electrophilic, reactive metabolites that may react with proteins. 2. The primary objective was to investigate the reactivity of the tolmetin acyl coenzyme A thioester (Tol-CoA). The second objective was to identify and quantify tolmetin (Tol) metabolites in vivo that were formed via Tol-CoA, e.g. the glycine (Tol-Gly) and taurine (Tol-Tau) conjugates. This finding would be indicative of Tol-CoA formation and thus of other acyl-CoA-related reactions that might occur, e.g. covalent binding to proteins. 3. In order to study the chemical reactivity, Tol-CoA (0.5 mM) was incubated with glutathione (5 mM) in a 0.1 M phosphate buffer (pH 7.4) at 37 degrees C. Tol-CoA reacted rapidly with glutathione in vitro to form the S-acyl glutathione conjugate at a rate of 14.9 +/- 0.7 micro M min(-1) (mean +/- SD, n = 3) from 0 to 10 min. Compared with acyl-CoAs of other xenobiotic carboxylic acids, naproxen and clofibric acid, the rate by which Tol-CoA reacted with glutathione was high. 4. Following administration of (3)H-Tol (100 mg kg(-1), 200 micro Ci kg(-1), p.o.) to male Sprague-Dawley rats, Tol-Tau and Tol-Gly were identified in urine by electrospray ionization MS-MS in both positive- and negative-ion modes. The conjugates were only formed at trace levels (< 0.5%). However, the presence of Tol-Tau and Tol-Gly showed the reactive Tol-CoA was formed in vivo.

Determination of nonsteroidal anti-inflammatory drugs in biological fluids by automatic on-line integration of solid-phase extraction and capillary electrophoresis.

A new, automatic method for the clean-up, preconcentration, separation, and quantitation of nonsteroidal anti-inflammatory drugs (NSAIDs) in biological samples (human urine and serum) using solid-phase extraction coupled on-line to capillary electrophoresis is proposed. Automatic pretreatment is carried out by using a continuous flow system operating simultaneously with the capillary electrophoresis equipment, to which it is linked via a laboratory-made mechanical arm. This integrated system is controlled by an electronic interface governed via a program developed in GWBasic. Capillary electrophoresis is conducted by using a separation buffer consisting of 20 mM NaHPO4, 20 mM beta-cyclodextrin and 50 mM SDS at pH 9.0, an applied potential of 20 kV and a temperature of 20 degrees C. The analysis time is 10 min and the detection limits were between 0.88 and 1.71 microg mL(-1). Automatic clean-up and preconcentration is accomplished by using a C-18 minicolumn and 75% methanol as eluent. The limit of detection of NSAIDs can be up to 400-fold improved when using sample clean-up. The extraction efficiency for these compounds is between 71.1 and 109.7 microg mL(-1) (RSD 2.0-7.7%) for urine samples and from 77.2 to 107.1 microg mL(-1) (RSD 3.5-7.1%) for serum samples.

Bile duct ligation promotes covalent drug-protein adduct formation in plasma but not in liver of rats given zomepirac.

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 antiinflammatory 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.

Interference by nonsteroidal anti-inflammatory drugs in EMIT and TDx assays for drugs of abuse.

Fourteen nonsteroidal anti-inflammatory drugs were evaluated for interference in EMIT and TDx assays for drugs of abuse. Only tolmetin demonstrated significant interferences in the EMIT assay. Urine samples that contained high concentrations of tolmetin (1800 mg/L) had characteristic high molar absorptivity at the wavelength used in EMIT assays (340 nm). Consequently, EMIT analysis of samples resulted in instrument error alarms on a Hitachi 704 instrument and depressed milliabsorbance values (delta A) relative to calibrators. Similar results were obtained with urine samples collected from an arthritic patient after the administration of 200 and 400 mg of tolmetin. When tolmetin samples were mixed with drugs of abuse, depressed delta A values were noted in all assays. Samples containing opiates and cannabinoids tested negative, and instrument error alarms were produced with samples that contained amphetamines. A gas chromatographic-mass spectrometric (GC-MS) assay for benzoylecgonine in the presence of tolmetin was successful, and no interferences were noted. Similar interferences by tolmetin were not observed in TDx assays, probably because of the different wavelength (525 nm) used in this assay. However, a potential for false-positive results in the TDx benzodiazepine assay was noted for urine samples containing high concentrations of fenoprofen, flurbiprofen, indomethacin, ketoprofen, and tolmetin. Generally, it was concluded that the presence of tolmetin in urine samples could lead to the production of unacceptable results by the EMIT assay for drugs of abuse. However, TDx and GC-MS assays were useful alternatives for the analysis of urine samples suspected of containing tolmetin.

Pharmacokinetic study of 4'-acetamidophenyl-2-(5'-p-toluyl-1'-methylpyrrole)acetate in the rat.

A pharmacokinetic study has been performed on a new non-steroidal anti-inflammatory drug, AU-8001 (4'-acetamidophenyl-2-(5'-p-toluyl-1'-methylpyrrole)acetate, CAS 82239-77-8) in the rat, following intravenous (10 mg/kg) and oral (50 mg/kg) administrations. The new molecule constitutes a prodrug of paracetamol and tolmetin. Compartmental and non-compartmental treatment of experimental data lead to the same conclusions. After intravenous administration, the rapid hydrolysis of the ester function of the molecule causes a short transit of the prodrug in the organism and the rapid attainment of maximum plasmatic levels of metabolites. After oral administration, the prodrug shows flip-flop behaviour in which the absorption constant rate is the limiting factor and is responsible for the slow bioconversion of the substance. The bioavailability of the prodrug is incomplete and, according to the urinary excretion data, a fraction of the dose reaches the blood stream in the form of metabolites. AU-8001, however, in spite of its limited bioavailability, is a model prodrug which could be used to prolong the activity of the original drugs.

Irreversible binding of tolmetin glucuronic acid esters to albumin in vitro.

Tolmetin glucuronide (TG), extracted and purified from human urine, was incubated with albumin in vitro. The degradation profile and irreversible binding to protein were investigated and kinetic parameters calculated. Standard conditions were as follows: TG, 30 micrograms/ml; human serum albumin (HSA), 3%; pH 7.45; 37 degrees C. Lower pH enhanced TG stability and reduced both the extent and the rate of irreversible binding. HSA also increased TG stability, compared to protein-free buffer, but the opposite was observed with bovine serum albumin (BSA). With BSA, irreversible binding was much less, but the rate of adduct formation was the same as with HSA. Essentially fatty acid free HSA behaved similarly to HSA. Preincubation of HSA with warfarin, or diazepam, or an excess of tolmetin, did not influence irreversible binding significantly. In buffer, acyl migration led predominantly to one isomer. This isomer bound irreversibly to HSA, although more slowly and to a lesser extent than the beta 1-isomer. Incubation of TG with poly-L-lysine also resulted in irreversible binding but to a lesser extent than with HSA. Our results suggest that there is more than one binding mechanism, with the preferential pathway a function of the isomers present and the experimental conditions.

Ketorolac tromethamine pharmacokinetics and metabolism after intravenous, intramuscular, and oral administration in humans and animals.

In humans, ketorolac is completely bioavailable and its kinetics are linear. It is absorbed rapidly (half-life for absorption 3.8 min) after oral (fasting) and intramuscular administration; food delays but does not reduce its absorption. The drug is highly protein bound in humans (greater than 99%). The mean plasma elimination half-life is 5-6 hours, and ketorolac is not extensively distributed outside the vascular compartment (Vd beta 15 L). Virtually all of the drug-related material circulating in plasma is in the form of ketorolac (greater than 96%), with the only metabolite the pharmacologically inactive p-hydroxyketorolac (PHK). Humans excrete about 90% of the administered dose in urine. About 60% of drug-related material recovered from urine is ketorolac, about 12% is PHK, and 28% represents polar, glucuronide conjugates of ketorolac. The animal models in which ketorolac's metabolism and kinetics are most similar to those in humans are the mouse and monkey, respectively.

Disposition and irreversible plasma protein binding of tolmetin in humans.

The pharmacokinetics and irreversible plasma protein binding of tolmetin were studied in six healthy subjects after the administration of a single, 400 mg dose of tolmetin. With HPLC analysis, tolmetin, tolmetin glucuronide, and the isomers of tolmetin glucuronide, which result from intramolecular acyl migration in vivo, were detected in the plasma up to 4 hours after administration, whereas these conjugates were present in the urine up to 24 hours. Irreversible binding of tolmetin to plasma proteins occurred in all subjects. Irreversible binding exhibited a better correlation with exposure to tolmetin glucuronide (r = 0.5618) and the isomers of tolmetin glucuronide (r = 0.8200) than with exposure to tolmetin (-0.3635). This is consistent with the hypothesis that covalent binding occurs via the acyl glucuronide.

High-performance liquid chromatographic determination of tolmetin, tolmetin glucuronide and its isomeric conjugates in plasma and urine.

A rapid and sensitive analytical procedure is described for the simultaneous measurement of tolmetin (T), tolmetin glucuronide (1 beta-TG) and the isomers of tolmetin glucuronide in plasma and urine. A reversed-phase liquid chromatographic system is used with an ion-pairing mobile phase of methanol-tetrabutylammonium hydrogensulfate buffered to pH 4.5 and kept at a constant temperature of 50 degrees C. Detection is by UV at 313 nm. Plasma (0.5 ml) and urine (0.1 ml) are collected in pre-cooled containers and immediately adjusted to pH 3.0 to minimize TG isomerization and hydrolysis. Samples are then deproteinated with acetonitrile, the supernatant is evaporated to dryness and reconstituted in an acetate buffer (pH 4.5), and 50 microliters are injected onto the system. Using zomepirac as the internal standard, the measurable, linear concentration ranges are 0.05-50 micrograms/ml for T in plasma and 0.025-50 micrograms/ml for T in urine. Chromatographic peaks representing T,1 beta-TG and three isomers of TG were identified, all with retention times less than 10 min. The need for special handling of biological samples is discussed.

Effect of probenecid on the formation and elimination of acyl glucuronides: studies with zomepirac.

When 100 mg oral zomepirac was taken with 500 mg b.i.d. oral probenecid by six healthy subjects, the disposition of zomepirac was markedly altered. Probenecid decreased total plasma clearance of zomepirac by 64%, which resulted in an increase in bioavailability from 0.55 without probenecid to 0.84 when given concurrently. The apparent metabolic clearance of zomepirac to form zomepirac acyl glucuronide was reduced 71% and zomepirac renal clearance, a minor elimination route, was reduced by 79%. When assayed by a method that prevents degradation of the labile acyl glucuronide, zomepirac glucuronide concentrations in plasma were comparable to those of zomepirac. Probenecid decreased the renal clearance of zomepirac glucuronide by 72%, which, together with the increased zomepirac levels, resulted in a 2.8-fold increase in the AUC of the conjugate. Urinary excretion of zomepirac glucuronide was reduced from 72% to 58% of the dose, but the excretion of free zomepirac was unchanged at 5% of the dose. The ratio of the total clearance/bioavailability of zomepirac in control subjects was 682 +/- 246 ml/min, which is double the value reported in previous studies of zomepirac disposition. We believe that this difference is due to degradation of the unstable zomepirac acyl glucuronide in the previous analytic methodologies used. Qualitatively, the effects of probenecid on zomepirac disposition are similar to those previously reported for other drugs of this class that are metabolized to acyl glucuronides. However, zomepirac appears unusual in that significant levels of its acyl glucuronide metabolite are found in vivo.

Continuous body fluid monitoring for zomepirac by fully automated high-performance liquid chromatography.

A sensitive, specific and rapid determination of 5-(4-chlorobenzoyl)-1,4-dimethyl-1H-pyrrole-2-acetic acid sodium salt dihydrate (zomepirac sodium, Zomax) in human plasma and urine by a continuous body fluid monitoring system based on high-performance liquid chromatography is described. Samples are directly injected into the apparatus which consists of commonly available HPLC-modules. Manual sample clean-up procedures as well as the addition of an internal standard are not needed. Using 50-microliter aliquots, the detection limit is lower than 0.05 microgram/ml and the calibration ranges from 0.5 to 100 micrograms/ml are linear for both, spiked samples and references. The recovery is 97% for plasma and 95% for urine samples at a sampling rate of about 15 samples/h.

Comparison of tolmetin kinetics in rheumatoid arthritis and matched healthy controls.

We could find no significant differences in the kinetics of tolmetin when comparing five rheumatoid arthritis patients with moderate disease activity with a carefully matched group of normal healthy volunteers. Further, there were no discernible clinically significant differences in the kinetics of tolmetin when comparing a single dose to one week of therapy. These results, although limited by the small number of subjects involved and the large interpatient variability, suggest that it may be possible to extrapolate pharmacokinetic data from normals to patients with moderately active disease.

Simultaneous determination of tolmetin and its metabolite in biological fluids by high-performance liquid chromatography.

A highly sensitive and selective high-performance liquid chromatographic assay has been developed for the separation and quantitation of tolmetin and its major metabolite in human biological fluids, viz. plasma, urine and synovial fluid. Analysis of plasma and synovial fluid required only 0.5 ml of the sample. The sample was washed with diethyl ether and extracted with diethyl ether-chloroform (2:1). The extracted compounds were injected onto a reversed-phase column (RP-2) and absorbance was measured at 313 nm. The standard curves in plasma were found to be linear for both tolmetin and the metabolite at concentrations from 0.04 to 10.0 microgram/ml. Urine samples (0.5 ml) were diluted (1:1) with methanol containing the internal standard and were directly injected onto the reversed-phase (RP-2) column. Standard curves of tolmetin and metabolite in urine were linear in the range 5-300 microgram/ml. Serum and synovial fluid concentrations of tolmetin and its metabolite in patients receiving multiple doses of tolmetin sodium were determined using the assay procedure.