Direct determination of diastereomeric carprofen glucuronides in human plasma and urine and preliminary measurements of stereoselective metabolic and renal elimination after oral administration of carprofen in man

A direct stereospecific HPLC assay for carprofen glucuronides in biologic fluids was developed that makes use of a reverse phase (C 18) gradient system, in which the mobile phase consisted of a mixture of acetonitrile and tetrabutylammonium hydroxide buffer (pH 2.5). Reference diastereomeric glucuronides of carprofen were purified from human urine after oral administration of the single enantiomers. When 0.1 ml of sample was used, the limit of detection for carprofen glucuronides was 50 ng/ml in plasma and 200 ng/ml in urine. Coefficients of variation did not exceed 12% for both intra and interday variability. This HPLC method is applicable to pharmacokinetic analysis for carprofen glucuronides in humans. After oral administration of the single enantiomers there was little indication of metabolic inversion. For the two enantiomers the apparent total and metabolic clearances were similar. The limited data available suggest that renal clearance of the (S)-glucuronide was greater than that of the (R)-glucuronide.     

Probenecid inhibits the renal clearance and renal glucuronidation of nalidixic acid

The aim of this pilot study was to demonstrate the possible inhibitory effect of probenecid on the renal glucuronidation and on the renal clearance of nalidixic acid in a human volunteer. Under acidic urine conditions, hardly any nalidixic acid is excreted unchanged (0.2%). It is excreted as acyl glucuronide (53.4%), 7-hydroxymethylnalidixic acid (10.0%), the latter's acyl glucuronide 30.9%, and 7-carboxynalidixic acid (4.2%). Under probenecid co-medication the renal glucuronidation of nalidixic acid is reduced from 53% to 16%; the renal clearance of both nalidixic acid and 7-hydroxymethylnalidixic acid are reduced (p <0.001); the intrinsict _1/2 of the metabolite 7-hydroxymethylnalidixic acid increased from 0.48 h to 4.24 h. The amount of acyl glucuronidation of 7-hydroxymethylnalidixic acid was not altered. Thein vitro protein binding of both acyl glucuronides was increased, while no effect on the unconjugated compounds was seen. Nalidixic acid had no effect on the maximal renal excretion rate of probenecid acyl glucuronide.     

Stereoselective binding of the glucuronide conjugates of carprofen enantiomers to human serum albumin

The stereoselective binding of carprofen enantiomers and carprofen glucuronide diastereomers to human serum albumin (HSA) was studied using an ultrafiltration method. Carprofen glucuronides exhibit a considerable and stereoselective affinity to HSA, although less than that seen for the parent enantiomers. The (S)-glucuronide showed a higher binding affinity to HSA than the (R)-glucuronide. The (S)-enantiomer of unmetabolized carprofen was bound to fatty acid free HSA to a much greater extent than the (R)-enantiomer. Warfarin reduced the binding of the glucuronides to a greater extent than did diazepam, but diazepam displaced the unconjugated enantiomers to a greater extent than did warfarin. These results suggest differences in binding region between the carprofen enantiomers and their glucuronides on the albumin molecule.     

Effect of probenecid on the formation and elimination kinetics of the sulphate and glucuronide conjugates of diflunisal

The effect of probenecid on the pharmacokinetics of diflunisal and its glucuronide and sulphate conjugates was studied in 8 healthy volunteers. Diflunisal 250 mg b. d. was administered p. o. for 15 days and its steady state pharmacokinetics was evaluated on Day 16 after the last dose (control phase). Probenecid 500 mg b. d. was co-administered throughout the entire study period in the treatment phase of the study.The steady state plasma concentration of diflunisal was significantly higher during the probenecid treatment phase as compared to the control phase (104.0 vs. 63.1 g·ml^–1). This was the result of a significant decrease in the plasma clearance of diflunisal from 5.8 (control) to 3.4 ml·min^–1 (probenecid co-administration). The metabolite formation clearances of both glucuronides were significantly decreased by probenecid, -45 % and -54 % for the phenolic and acyl glucuronide, respectively. The metabolite formation clearance of the sulphate conjugate was not affected by probenecid co-administration.Steady state plasma concentrations of the sulphate and glucuronide conjugates of diflunisal were 2.5- to 3.1-fold higher during probenecid co-administration, due to a significant reduction in the renal clearance of the three diflunisal conjugates. Probenecid also reduced the plasma protein binding of diflunisal, but only to a minor extent; the unbound plasma fraction of diflunisal at steady state averaged between 5 and 30 % higher during probenecid co-administration.     

Probenecid inhibits the glucuronidation of indomethacin andO-desmethylindomethacin in humans

Indomethacin is metabolized in humans byO-demethylation, and by acyl glucuronidation to the l-O-glucuronide. Indomethacin, its metabolite, and their conjugates can be measured directly by gradient high-pressure liquid chromatographic analysis without enzymic deglucuronidation. The pharrnacokinetic profile of indomethacin and some preliminary pharmacokinetic parameters of indomethacin obtained from one human volunteer are given. In plasma only the parent drug indomethacin is present, while in urine the acyl and ether glucuronides are present in high concentrations. This confirms other reports that indomethacin andO-desmethylindomethacin may be glucuronidated in the kidney. Probenecid is a known substrate for renal glucuronidation. If indomethacin is glucuronidated in the human kidney like probenecid, then this glucuronidation might be reduced or inhibited under probenecid co-medication. This pilot experiment shows that probenecid reduced the acyl glucuronidation of indomethacin by 50% and completely inhibited the formation ofO-desmethylindomethacin acyl and ether glucuronide.     

Direct high pressure liquid chromatographic analysis and preliminary pharmacokinetics of enantiomers of oxazepam and temazepam with their corresponding glucuronide conjugates

Three high pressure liquid chromatographic systems for the separation of oxazepam, temazepam and their glucuronides (system A), the separation of theirR,S glucuronide diastereomers (system B) and the chiral separation of the parent drugs (system C) are described. Preliminary pharmacokinetics ofR,S-oxazepam andR,S-temazepam in a human volunteer reveal that the protein binding of the glucuronides is lower than that of the parent drugs, but that there is no difference in protein binding between theR-oxazepam/temazepam andS-oxazepam/temazepam and their corresponding glucuronides. TheS-glucuronide is the main metabolite formed and excreted by man. The plasma ratioR/S-glucuronide is 11 for both oxazepam and temazepam. The renal clearances ofR-temazepam, andS-temazepam are similar, and those ofR-oxazepam andS-oxazepam tend to be different.     

Rectal administration of nicomorphine in patients improves biological availability of morphine and its glucuronide conjugates

The pharmacokinetics of 30 mg nicomorphine after rectal administration with a suppository are described in 8 patients under combined general and epidural anaesthesia. No nicomorphine or 6-mononicotinoylmorphine could be detected in the serum. Morphine appeared almost instantaneously with a lag-time of 8 min and had a final elimination half-life of 1.48±0.48 h. Morphine was metabolized to morphine-3-glucuronide and morphine-6-glucuronide. These glucuronide conjugates appeared after a lag-time of 12 min and the half-life of these two glucuronide conjugates was similar: about 2.8 h (P>0.8). The glucuronide conjugate of 6-mononicotinoylmorphine was not detected. In the urine only morphine and its glucuronides were found. The renal clearance value for morphine was 162 m·min^–1 and for the glucuronides 81 ml·min^–1. This study shows that administration of a suppository with 30 mg nicomorphine gives an excellent absolute bioavailability of morphine and its metabolites of 88%. The lipid-soluble prodrug nicomorphine is quickly absorbed and immediately hydrolysed to morphine.     

Glucuronidation Kinetics of R,S-Ketoprofen in Adjuvant-Induced Arthritic Rats

Purpose. A pharmacokinetic study was carried out in rats to investigate the effect of arthritis on the glucuronidation of the nonsteroidal anti-inflammatory drug ketoprofen. Methods. An iv bolus dose of R,S-ketoprofen (10 mg/kg) was administered to control (n = 6) and adjuvant-induced arthritic rats (n = 6). All experiments were carried out in bile-exteriorized animals. Concentrations of R- and S-ketoprofen in plasma, bile and urine, and of their glucuronides in bile and urine were determined by HPLC. In a separate series of experiments, the ex vivo plasma protein binding of R- and S-ketoprofen was measured in control and arthritic rats following iv administration of R,S-ketoprofen. Results. As a result of a significant decrease in plasma albumin concentrations in arthritic rats, the unbound fraction of R- and S-ketoprofen was significantly increased (approximately 2-fold) in rats with adjuvant-induced arthritis. Total (i.e., bound plus unbound) plasma clearances of R- and S-ketoprofen were not different in arthritic rats. Unbound plasma clearances of both ketoprofen enantiomers, however, were significantly reduced (by 53% and 61%, respectively). This was due to a significant impairment in the formation of the R- and S-ketoprofen glucuronides. There was no apparent effect of adjuvant-induced arthritis on the chiral inversion of R- to S-ketoprofen. Conclusions. Adjuvant-induced arthritis in the rat leads to a significant impairment in the in vivo glucuronidation of R- and S-ketoprofen.     

Interindividual variation in the capacity-limited renal glucuronidation of probenecid by humans

A dose of 1,000 mg probenecid was administered orally to 14 human volunteers in order to quantify the maximal rate of formation and excretion of probenecid acyl glucuronide in the urine. Probenecid showed dose-dependent pharmacokinetics. Plasma protein binding of probenecid was high, being somewhat higher in males (90.7±1.4%) than in females (87.9±1.4%; p=0.0019). It was shown that probenecid is metabolized by cytochrome P-450 into at least two phase I metabolites. Each of the metabolites accounted for less than 12% of the dose administered; the main metabolite probenecid acyl glucuronide, representing 42.9±13.2% of the dose, was only present in urine and not in plasma. The renal excretion rate-time profile of probenecid acyl glucuronide showed a plateau value in the presence of an acidic urine pH. This plateau value was maintained for about 10 h at the dose of 1,000 mg. The height of the plateau value depended on the individual and varied between 250 and 800g/min (15–50 mg/h). It was inferred that probenecid acyl glucuronide is formed in the kidney during blood-to-lumen passage through the tubular cells. We conclude that the plateau value in the renal excretion rate of probenecid glucuronide reflects itsV _max of formation.     

Saturable kinetics of intravenous chlorothiazide in the rhesus monkey

A range of bolus doses of ¹⁴ C-chlorothiazide and unlabeled drug (6.7–30 mg/kg) were administered to each of three unanesthetized rhesus monkeys with and without concurrent probenecid dosing. Plasma up to 4 h and urine up to 24 h were sampled frequently. Apparent terminal plasma half-lives ranged from 18 to 25 min in the absence of probenecid. No apparent trend was noted for the volume of distribution of the central compartment calculated from estimated plasma concentrations at time zero. For chlorothiazide studies, an average of 92% of the dose was recovered in urine by 24 hr. Plasma and urinary clearances at low doses were 20 to 50% higher than those found with higher doses. These dose-dependent clearances for chlorothiazide were found at doses parallel to the most often prescribed clinical doses in humans on a g chlorothiazide per kg body weight basis. Clearances in the presence of probenecid decreased two-to four-fold over those seen without probenecid. Incremental renal clearances of chlorothiazide in the studies with and without probenecid were also evaluated. Curvilinear segments characteristic of dose-dependent kinetics were demonstrated in graphs of urinary excretion rate versus plasma concentrations. Values of Michaelis-Menten constants V_max and K_m were calculated for renal excretion of chlorothiazide by active transport after intravenous doses in all three monkeys. The contribution of glomerular filtration to chlorothiazide renal clearance was found to be negligible. Values of the constant K_I (the concentration of the probenecid competitive inhibitor of chlorothiazide, which doubles the apparent K_m value of chlorothiazide) were calculated using the previously calculated V_max and K_m values.Supported in part by NIH grants GM 26691 and AM 20884. During the course of this work, Dr. Gustafson received support as an NIH Predoctoral Fellow (GM 00752) and as a Fellow of the American Foundation for Pharmaceutical Education.     

Pharmacokinetics of ketoprofen enantiomers in cholecystectomy patients: influence of probenecid

Summary Ketoprofen (KT), a 2-arylpropionic acid nonsteroidal antiinflammatory drug, is administered as a racemate. Previous reports suggest stereoselective biliary excretion of KT enantiomers. This hypothesis was tested by administering 50 mg racemic KT to five patients who required bile drainage following cholecystectomy surgery. Subsequently, to study the influence of probenecid (PB), an inhibitor of KT renal elimination, on the biliary excretion, 1000 mg PB was administered 1.5 h before KT to the same patients. The unchanged and conjugated (as glucuronides) KT enantiomers were measured in plasma, urine and bile.In general, KT enantiomers had different plasma concentration-time curves. As compared to normal subjects, these patients had comparable AUCs and shorter t_1/2s. Biliary concentrations of conjugated S-KT were greater than R-KT. Nevertheless, the total cumulative biliary excretion of conjugated KT did not exceed 2% of the dose ruling out this pathway as a significant route of KT elimination.There was a positive and significant correlation between the cumulative urinary excretion of conjugated KT enantiomers and creatinine clearance. Although PB did not influence the pattern of stereoselectivity of KT, it increased AUC and prolonged t_1/2 of the enantiomers. While reducing cumulative urinary excretion, PB increased total biliary elimination of conjugated KT enantiomers. This, however, did not totally compensate for the reduced urinary excretion.It is suggested that the impaired conjugation of KT caused by PB administration may result in the augmentation of other, otherwise minor, metabolic pathways.     

Stereoselective disposition of carprofen, flunoxaprofen, and naproxen in rats.

The stereoselective dispositions of carprofen, flunoxaprofen, and naproxen were studied in rats after i.v. administration of racemate (11 mumol/kg) or enantiomer (5.5 mumol/kg). The total clearances of the (R)-enantiomers of carprofen and flunoxaprofen were significantly greater than those of the (S)-enantiomers. The clearance of (S)-naproxen was similar to the value for (R)-naproxen. There were no marked differences in steady-state volume of distribution between (R)- and (S)-enantiomers for carprofen, flunoxaprofen, or naproxen. The (R)- to (S)-enantiomer inversion ratio for flunoxaprofen in rats was 0.54. The ratios for naproxen and carprofen were 0.02 and 0.003, respectively. Biliary excretion of (R)-carprofen and of its glucuronide were higher than those of the (S)-enantiomer and its glucuronide. In contrast, biliary excretion of the (S)-enantiomers of flunoxaprofen, naproxen, and of their glucuronides were greater than those of their antipodes. Insignificant amounts of the parent enantiomers and of the glucuronides of these three drugs were excreted in urine. These results indicate that there is a wide variation in the extent of inversion at a chiral center for these three 2-arylpropionates and in the stereoselective disposition of their acyl glucuronides.     

Studies of the renal excretion of probenecid acyl glucuronide in man

Summary The metabolism of probenecid has been investigated in both normal and gouty subjects. To carry out these studies, specific spectrophotometric methods were developed for the measurement of a major metabolite of probenecid, the acyl glucuronide. This conjugate was isolated in semi-pure state and its identity as a mono acyl glucuronide established. Our experiments indicate that about 25% of probenecid is converted to its acyl glucuronide and that only a small amount of the drug is excreted unchanged. About 80% of orally administered¹⁴C probenecid could be accounted for in urine, and about half of this was found to consist of metabolites more polar than the parent drug. The renal clearance of probenecid acyl glucuronide was shown to be about 1/3 that of creatinine clearance, whereas the clearance of probenecid was lower. The present findings in man and those of other workers in animals, raise the important question of the possible contribution of these metabolites to the overall pharmacological effects resulting from the administration of probenecid.Supported by NIH Grants A-162 and GM-14270; portions of this work were initially carried out under Grant A-4724 at the NYU Research Service, Goldwater Memorial Hospital, New York, N.Y.     

Capacity-limited renal glucuronidation of probenecid by humans A pilotVmax-finding study

Probenecid shows dose-dependent pharmacokinetics. When in one volunteer the dose is increased from 250 to 1,500 mg orally, thet _1/2 increased from 3 to 6 h. TheC _max was 14g/ml with a dosage of 250 mg, 31g/ml with 500 mg, 70g/ml with 1,000 mg and 120g/ml with 1,500 mg. Thet _max remained 1 h for all four dosages. The AUC/dose ratio increased with the dose, indicating nonlinear elimination. The total body clearance declined from 64.5 ml/min for 250 mg to 26.0 ml/min for 1,500 mg. The renal clearance of probenecid remained constant, 0.6–0.8 ml/min. Protein binding of probenecid is high (91%) and independent of the dose. The phase I metabolites show lower protein binding values (34–59%). The protein binding of probenecid glucuronidein vitro (spiked plasma) is 75%. Probenecid is metabolized by cytochrome P-450 to three phase I metabolites. Each of the metabolites accounts for less than 10% of the dose administered; the percentage recovered in the urine is independent of the dose. The main metabolite probenecid glucuronide is only present in urine and not in plasma. The renal excretion rate-time profile of probenecid glucuronide shows a plateau value of approximately 700g/min (46 mg/h) with acidic urine pH. The duration of this plateau value depends on the dose: 2 h at 500 mg, 10 h at 1,000 mg and 20 h at 1,500 mg. It is demonstrated that probenecid glucuronide must be formed in the kidney during its passage of the tubule. The plateau value in the renal excretion rate of probenecid value reflects itsV _max of formation.     

Influence of renal function on the elimination of morphine and morphine glucuronides

Summary The influence of renal function, measured by ⁵¹Cr-EDTA clearance, on morphine and morphine glucuronide kinetics has been studied in 13 patients after a single i.v. injection of morphine. Unconjugated morphine and morphine glucuronides were measured by a sensitive, specific RIA after extraction from plasma. No significant correlation was found between total body clearance of unconjugated morphine and ⁵¹Cr-EDTA clearance. However, patients with renal insufficiency had impaired elimination of morphine glucuronides, and the apparent clearance was significantly correlated with the ⁵¹Cr-EDTA clearance (r=0.94, p<0.001). A relatively long terminal elimination half-life of morphine was found in all patients (mean±SD: 9.2±2.5 h), irrespective of glomerular function.     

Competitive Inhibition of Zidovudine Clearance by Probenecid During Continuous Coadministration

The pharmacokinetics of zidovudine in the rabbit were studied during coadministration of probenecid at two infusion rates. Each animal (n = 6) served as its own control during an initial 8-hr infusion of zidovudine. In the second 8-hr infusion period, probenecid was coadministered with zidovudine. Urine samples were collected by bladder flush hourly for 19 hr. Plasma samples were taken at the midpoint of the urine collection interval and at predetermined intervals for 3 hr postinfusion. Plasma concentrations of zidovudine reached steady state during control periods but showed incomplete attainment of steady state during the infusions of probenecid at the higher rate. Total and renal clearance of zidovudine were reduced by 24.0 ± 4.0 and 20.7 ± 15%, respectively, during low-dose probenecid treatment and 48.9 ± 7.4 and 55.7 ± 3.4%, respectively, with high-dose probenecid treatment. Plasma probenecid concentrations during low-dose and high-dose infusion were 56.9 ± 12 and 248 ± 42 µg/ml. Postinfusion data showed that the zidovudine terminal half-life during high-dose probenecid treatment was longer than that with low-dose probenecid treatment (58.2 ± 4.6 vs 39.0 ± 9.1 min). The volume of distribution of zidovudine also decreased (1.76 ± 0.27 vs 1.10 ± 0.095 L/kg) as a result of probenecid coadministration. The results are consistent with competitive inhibition of renal and nonrenal clearances. A drug interaction model relating zidovudine clearances to plasma probenecid concentrations was derived. Michaelis-type constants for probenecid inhibition of zidovudine renal and nonrenal clearances were 73 and 55 µg/ml, respectively. The maximum proportion of AZT's renal clearance subject to inhibition is significantly greater (72%) than that of the nonrenal clearance (54%) and agrees closely with the fraction not filtered.     

Acute and long-term hypotensive effects and plasma concentrations of nifedipine in patients with essential hypertension

Summary A double-blind, cross-over study in 16 patients with essential hypertension was carried out, to evaluate any possible interference by indomethacin, a known prostaglandin-synthetase inhibitor, with the antihypertensive effect of oxprenolol, a non-selective beta-adrenoceptor blocking agent. Both indomethacin and oxprenolol, as well as the two drugs combined, inhibited plasma renin activity; no change was found in urinary sodium excretion or body weight. Oxprenolol alone caused a highly significant decrease in the systolic (–10.4 mmHg,p<0.001), diastolic (–7.4 mmHg,p<0.001) and mean (–7.7 mmHg,p<0.01) blood pressures, whereas indomethacin did not influence blood pressure. When the two drugs were given in combination, blood pressure decreased (systolic: –5.9 mmHg; diastolic: –4.0 mmHg; mean: –4.6 mmHg), but the changes induced in blood pressure were reduced by about 50% when compared with those in the oxprenolol alone period. The data show that indomethacin seems to interfere with the antihypertensive effect of oxprenolol, by an action which may be due to the inhibition of prostaglandin synthesis.     

Capacity-limited renal glucuronidation of probenecid by humans

Probenecid shows dose-dependent pharmacokinetics. When in one volunteer the dose is increased from 250 to 1,500 mg orally, thet _1/2 increased from 3 to 6 h. TheC _max was 14μg/ml with a dosage of 250 mg, 31μg/ml with 500 mg, 70μg/ml with 1,000 mg and 120μg/ml with 1,500 mg. Thet _max remained 1 h for all four dosages. The AUC/dose ratio increased with the dose, indicating nonlinear elimination. The total body clearance declined from 64.5 ml/min for 250 mg to 26.0 ml/min for 1,500 mg. The renal clearance of probenecid remained constant, 0.6–0.8 ml/min. Protein binding of probenecid is high (91%) and independent of the dose. The phase I metabolites show lower protein binding values (34–59%). The protein binding of probenecid glucuronidein vitro (spiked plasma) is 75%. Probenecid is metabolized by cytochrome P-450 to three phase I metabolites. Each of the metabolites accounts for less than 10% of the dose administered; the percentage recovered in the urine is independent of the dose. The main metabolite probenecid glucuronide is only present in urine and not in plasma. The renal excretion rate-time profile of probenecid glucuronide shows a plateau value of approximately 700μg/min (46 mg/h) with acidic urine pH. The duration of this plateau value depends on the dose: 2 h at 500 mg, 10 h at 1,000 mg and 20 h at 1,500 mg. It is demonstrated that probenecid glucuronide must be formed in the kidney during its passage of the tubule. The plateau value in the renal excretion rate of probenecid value reflects itsV _max of formation.     

Plasma levels of morphine and morphine glucuronides in the treatment of cancer pain: relationship to renal function and route of administration

Summary There is growing evidence that renally-impaired patients receiving morphine therapy are at greater risk of developing opiate toxicity, due to the accumulation of an active metabolite, morphine-6-glucuronide (M6G), which is usually excreted by the kidneys. This study examined the relationships between morphine dosage, renal function, and trough plasma concentrations of morphine and its glucuronide metabolites in 21 patients (aged mean: 68.5 years; 11 males) receiving either oral or subcutaneous morphine for terminal cancer pain. The median daily morphine dosages (mg · kg^–1) were: orally 1.87 (range 0.37–6.82) and subcutaneously 1.64 (range 0.22–3.60).The median plasma concentrations of morphine, morphine-3-glucuronide (M3G), and M6G (ng · ml^–1) were: 36.0, 1035.2, and 142.3, respectively. The plasma concentrations of morphine, M3G and M6G were each significantly related to the daily morphine dosage (n=21, Spearman r=0.79, 0.91, and 0.88 respectively). Accumulation of the morphine glucuronides was dependent on renal function. The plasma concentrations of M3G and M6G, when divided by the morphine concentration, were significantly related to the caluclated creatinine clearance of the patient. Patients receiving oral morphine had higher plasma concentration ratios of glucuronide/morphine than those receiving subcutaneous therapy, presumably due to first-pass glucuronidation.The results of this study confirm that accumulation of the pharmacologically active M6G is related to renal function, which probably explains the observation that morphine dosage requirements are generally reduced in patients with renal impairment.