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PubMed2006

N-Glucuronidation of the antiepileptic drug retigabine: results from studies with human volunteers, heterologously expressed human UGTs, human liver, kidney, and liver microsomal membranes of Crigler-Najjar type II.

Retigabine (D-23129), an N-2-amino-4-(4-fluorobenzylamino)phenylcarbamine acid ethyl ester, is a novel antiepileptic drug which is currently in phase II clinical development. This drug undergoes N-glucuronidation. We aimed to identify the principal enzymes involved in the N-glucuronidation pathway of retigabine and compared our findings with those obtained from human liver (a pool of 30 donors) and kidney microsomes (a pool of 3 donors) and with results from a human absorption, distribution, metabolism, and excretion study upon administration of 200 microCi of [(14)C]-D-23129. Essentially, microsomal assays with UGT1A1 produced only one of the 2 N-glucuronides, whereas UGT1A9 is capable of forming both N-glucuronides. The rates of metabolism for UGT1A9, human liver microsomes, and UGT1A1 were 200, 100, and 100 pmol N-glucuronide per minute per milligram of protein, respectively. At the 50 micromol/L uridine diphosphate glucoronic acid (UDPGA) concentration, UGT1A4 also catalyzed the N-glucuronidation of retigabine, the rates being approximately 5 and 6 pmol/(min.mg protein). With UGT1A9, the production of metabolites 1 and 2 proceeded at a K(m) of 38+/-25 and 45+/-15 micromol/L, whereas the K(m) for retigabine N-glucuronidation by human liver microsomal fractions was 145+/-39 micromol/L. Furthermore, a V(max) of 1.2+/-0.3 (nmol/[min.mg protein]) was estimated for human liver microsomes (4 individual donors). We investigated the potential for drug-drug interaction using the antiepileptic drugs valproic acid, lamotrigine, the tricyclic antidepressant imipramine, and the anesthetic propofol. These are commonly used medications and are extensively glucuronidated. No potential for drug-drug interactions was found at clinically relevant concentrations (when assayed with human liver microsomes or UGT1A9 enzyme preparations). Notably, the biosynthesis of retigabine-N-glucuronides was not inhibited in human liver microsomal assays in the presence of 330 micromol/L bilirubin, and glucuronidation of retigabine was also observed with microsomal preparations from human kidney and Crigler-Najjar type II liver. This suggests that lack of a particular UDP-glucuronosyltransferase (UGT) isoform (eg, UGT1A1 in kidney) or functional loss of an entire UGT1A gene does not completely abolish disposal of the drug. Finally, chromatographic separations of extracts from microsomal assays and human urine of volunteers receiving a single dose of (14)C-retigabine provided clear evidence for the presence of the 2 N-glucuronides known to be produced by UGT1A9. We therefore suggest N-glucuronidation of retigabine to be of importance in the metabolic clearance of this drug.

Anticonvulsants↗
PubMed2000

Stability and enzymatic hydrolysis of quaternary ammonium-linked glucuronide metabolites of drugs with an aliphatic tertiary amine-implications for analysis.

Quaternary ammonium-linked glucuronide (N+-glucuronide) metabolites formed at aliphatic tertiary amine functional groups of xenobiotics have not been previously systematically studied with respect to their stability over a wide pH range and the ease of enzymatic hydrolysis by beta-glucuronidase from various sources. Three and four N+-glucuronide metabolites were respectively studied regarding their non-enzymatic and enzymatic stabilities where the metabolites were quantified by HPLC procedures. The N+-glucuronide metabolites of clozapine, cyclizine, and doxepin were stored at 18-22 degrees C in buffers at each nominal pH value over the 1-11 pH range. All three metabolites were stable for 3 months over the 4-10 pH range, while two metabolites slowly degraded (k in the range 0.002-0.01 days(-1)) at each of the other extreme pH values. In the initial enzymatic study the N+-glucuronide metabolites of chlorpromazine, clozapine, cyclizine, and doxepin were each treated in pH 5.0 and 7.4 buffers at 37 degrees C with beta-glucuronidase from three different sources, namely commercial brands from bovine liver, mollusks (Helix pomatia), and bacteria (Escherichia coli). Clozapine N+-glucuronide and the standard phenolphthalein O-glucuronide were susceptible to hydrolysis by the enzyme from all three sources. In contrast, the other three N+-glucuronide metabolites were resistant to hydrolysis, except for the E. coli source of beta-glucuronidase at pH 7.4. Also when examined at 50-fold increase in concentration of the enzyme sources from bovine liver and H. pomatia cyclizine N+-glucuronide was still resistant to hydrolysis by the former enzyme preparation. The optimum pH for the hydrolysis of each of the four N+-glucuronide metabolites from the E. coli enzyme source was investigated and was found to be in the pH range 6.5-7.4. These data have important implications with respect to the analysis of N+-glucuronide metabolites formed at an aliphatic tertiary amine: in general, their non-enzymatic stability will not be an important factor in the development of an analytical procedure, and when developing an indirect approach to the analysis of N+-glucuronide metabolites that involves beta-glucuronidase hydrolysis to the aglycone preliminary work should involve determining the appropriate enzyme source, buffer pH, and length of time of incubation.

Animals↗
PubMedDate not supplied

Biosynthesis of drug glucuronides for use as authentic standards.

INTRODUCTION: Glucuronidation by the uridine diphosphate glucuronosyltransferases (UGTs) plays a pivotal role in the clearance mechanism of both xenobiotics and endobiotics. The detection of glucuronides at low micromolar concentrations is required to accurately model in vitro enzyme kinetics and in vivo pharmacokinetics. However, relatively few glucuronides are currently available as standards for developing liquid chromatography and mass spectroscopy (LC/MS) bioanalytical methods. METHODS: The glucuronidation capacity of hepatic microsomes prepared from rat (RLM), dog (DLM), monkey (MLM), and human (HLM) was examined for five xenobiotic substrates. In each case, glucuronide standards were produced using the enzyme source most efficient for the production of that specific glucuronide. RESULTS: Dog hepatic microsomes were used to produce glucuronides for anthraflavic acid (yield: 14 mg), buprenorphine (yield: 14 mg), and octylgallate (total yield: 13 mg), whereas propofol glucuronide (yield: 20 mg), and ethinylestradiol glucuronide (yield: 8 mg) were prepared using HLM. All glucuronides were characterized by LC/MS/MS and nuclear magnetic resonance (NMR) spectroscopy. DISCUSSION: The multimilligram quantities of glucuronide standards produced by this method have many applications throughout drug discovery and toxicology. In addition to allowing the quantification of glucuronide formation from in vitro and in vivo studies, the authentic standards produced could also be used to assess potential pharmacological or toxicological effects of metabolites.

Animals↗
PubMed2004

Human UDP-glucuronosyltransferase 1A1 is the primary enzyme responsible for the N-glucuronidation of N-hydroxy-PhIP in vitro.

UDP-glucuronosyltransferase 1A proteins (UGT1A) catalyze the glucuronidation of many endogenous and xenobiotic compounds including heterocyclic amines and their hydroxylated metabolites. Studies have shown that in humans UGT1A-mediated glucuronidation is an important pathway in the detoxification of food-borne carcinogenic heterocyclic amines. The biotransformation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most mass abundant heterocyclic amine found in cooked meats, is highly dependent on cytochrome P4501A2 hydroxylation followed by UGT-catalyzed glucuronidation of the N-hydroxy-PhIP reactive intermediate. To determine which UGT1A proteins are involved in the glucuronidation of N-hydroxy-PhIP, microsomal preparations from baculovirus-infected insect cells that express all of the known functional human UGT1A isozymes (UGT1A1, -1A3, -1A4, -1A6, -1A7, -1A8, -1A9, and -1A10) were exposed to N-hydroxy-PhIP and the reaction products were isolated by HPLC. All UGT1A proteins except UGT1A6 showed some degree of activity toward N-hydroxy-PhIP. The formation of both N-hydroxy-PhIP-N(2)-glucuronide and N-hydroxy-PhIP-N3-glucuronide was both time- and substrate concentration-dependent. UGT1A1 was the most efficient in converting N-hydroxy-PhIP to both conjugates producing five times more of the N(2)-conjugate than UGT1A4, the next most active UGT, and 286 times more than UGT1A7, the least active UGT. With an apparent K(m) of 52 microM and a K(cat) of 114 min(-)(1), UGT1A1 was also the most catalytically efficient in forming N-hydroxy-PhIP-N(2)-glucuronide. The catalytic efficiency for N-hydroxy-PhIP-N3-glucuronide formation was 8, 10, and 6 times lower for UGT1A1, -1A4, and -1A8, respectively, when compared to the K(cat) values for N-hydroxy-PhIP-N(2)-glucuronide formation. These results clearly show that UGT1A1 has the highest specificity for glucuronidating N-hydroxy-PhIP. Polymorphic expression resulting in decreased UGT1A1 activity in humans can cause reduced rates of glucuronidation, which can change the metabolic ratio between bioactivation and detoxification to favor bioactivation. This change will increase the susceptibility to the deleterious effects from PhIP exposure because the capacity to form nontoxic N-hydroxy-PhIP glucuronide conjugates will be diminished.

Biotransformation↗
PubMed2006

The glucuronidation of Delta4-3-Keto C19- and C21-hydroxysteroids by human liver microsomal and recombinant UDP-glucuronosyltransferases (UGTs): 6alpha- and 21-hydroxyprogesterone are selective substrates for UGT2B7.

The stereo- and regioselective glucuronidation of 10 Delta(4)-3-keto monohydroxylated androgens and pregnanes was investigated to identify UDP-glucuronosyltransferase (UGT) enzyme-selective substrates. Kinetic studies were performed using human liver microsomes (HLMs) and a panel of 12 recombinant human UGTs as the enzyme sources. Five of the steroids, which were hydroxylated in the 6beta-, 7alpha-, 11beta- or 17alpha-positions, were not glucuronidated by HLMs. Of the remaining compounds, comparative kinetic and inhibition studies indicated that 6alpha- and 21-hydroxyprogesterone (OHP) were glucuronidated selectively by human liver microsomal UGT2B7. 6alpha-OHP glucuronidation by HLMs and UGT2B7 followed Michaelis-Menten kinetics, whereas 21-OHP glucuronidation by these enzyme sources exhibited positive cooperativity. UGT2B7 was also identified as the enzyme responsible for the high-affinity component of human liver microsomal 11alpha-OHP glucuronidation. In contrast, UGT2B15 and UGT2B17 were the major forms involved in human liver microsomal testosterone 17beta-glucuronidation and the high-affinity component of 16alpha-OHP glucuronidation. Activity of UGT1A subfamily enzymes toward the hepatically glucuronidated substrates was generally low, although UGT1A4 and UGT1A9 contribute to the low-affinity components of microsomal 16alpha- and 11alpha-OHP glucuronidation, respectively. Interestingly, UGT1A10, which is expressed only in the gastrointestinal tract, exhibited activity toward most of the glucuronidated substrates. The results indicate that 6alpha- and 21-OHP may be used as selective "probes" for human liver microsomal UGT2B7 activity and, taken together, provide insights into the regio- and stereoselectivity of hydroxysteroid glucuronidation by human UGTs.

Cell Line↗
PubMed2002

Differential modulation of UDP-glucuronosyltransferase 1A1 (UGT1A1)-catalyzed estradiol-3-glucuronidation by the addition of UGT1A1 substrates and other compounds to human liver microsomes.

Previous results demonstrating homotropic activation of human UDP-glucuronosyltransferase (UGT) 1A1-catalyzed estradiol-3-glucuronidation led us to investigate the effects of 16 compounds on estradiol glucuronidation by human liver microsomes (HLM). In confirmation of previous work using alamethicin-treated HLM pooled from four livers, UGT1A1-catalyzed estradiol-3-glucuronidation demonstrated homotropic activation kinetics (S(50) = 22 microM, Hill coefficient, n = 1.9) whereas estradiol-17-glucuronidation (catalyzed by other UGT enzymes) followed Michaelis-Menten kinetics (K(m) = 7 microM). Modulatory effects of the following compounds were investigated: bilirubin, eight flavonoids, 17alpha-ethynylestradiol (17alpha-EE), estriol, 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP), anthraflavic acid, retinoic acid, morphine, and ibuprofen. Although the classic UGT1A1 substrate bilirubin was a weak competitive inhibitor of estradiol-3-glucuronidation, the estrogens and anthraflavic acid activated or inhibited estradiol-3-glucuronidation dependent on substrate and effector concentrations. For example, at substrate concentrations of 5 and 10 microM, estradiol-3-glucuronidation activity was stimulated by as much as 80% by low concentrations of 17alpha-EE but was unaltered by flavanone. However, at higher substrate concentrations (25-100 microM) estradiol-3-glucuronidation was inhibited by about 55% by both compounds. Anthraflavic acid and PhIP were also stimulators of estradiol 3-glucuronidation at low substrate concentrations. The most potent inhibitor of estradiol 3-glucuronidation was the flavonoid tangeretin. The UGT2B7 substrates morphine and ibuprofen had no effect on estradiol 3-glucuronidation, whereas retinoic acid was slightly inhibitory. Estradiol-17-glucuronidation was inhibited by 17alpha-EE, estriol, and naringenin but was not activated by any compound. This study demonstrates that the interactions of substrates and inhibitors at the active site of UGT1A1 are complex, yielding both activation and competitive inhibition kinetics.

Algorithms↗
PubMed2002

Conjugation of desmethylnaproxen in the rat--a novel acyl glucuronide-sulfate diconjugate as a major biliary metabolite.

The nonsteroidal anti-inflammatory drug naproxen is primarily metabolized in humans by acyl glucuronidation to form naproxen acyl glucuronide and by O-dealkylation to form 6-O-desmethylnaproxen (DMN). DMN contains both carboxy and phenolic groups and has been shown to form acyl glucuronide and sulfate conjugates. This project aimed to investigate whether DMN formed a phenolic glucuronide and diglucuronide(s) (with both the carboxy and phenolic groups glucuronidated). Male Sprague-Dawley rats (300-350 g) with exteriorized bile flow were dosed i.v. with DMN at 50 mg/kg. Four major DMN-related peaks were detected in bile by high-performance liquid chromatography (HPLC) analysis at 225 nm, including the known acyl glucuronide and sulfate conjugates. Selective hydrolyses using acidic and alkaline conditions and digestion with beta-glucuronidase allowed tentative identification of the two unknown peaks as the phenolic glucuronide of DMN and a novel acyl glucuronide-sulfate diconjugate of DMN (i.e., formed by sulfonation of the phenolic group and glucuronidation of the carboxy group). The identities were confirmed by liquid chromatography-tandem mass spectrometry analysis of individual HPLC fractions. Total recovery of the DMN dose was approximately 80%, with the sulfate conjugate (50%) and unchanged DMN (10%) being excreted predominantly in urine and the acyl glucuronide (10%), phenolic glucuronide (6%), and acyl glucuronide-sulfate diconjugate (4%) being excreted predominantly or exclusively in bile. No evidence for a diglucuronide metabolite of DMN was found in either bile or urine of the DMN-dosed rats.

Animals↗
PubMed2002

Inhibition of mycophenolic acid glucuronidation by niflumic acid in human liver microsomes.

OBJECTIVES: The first aim of this investigation was to study the variability of mycophenolic acid (MPA) glucuronidation rate in human liver. The second aim was to study the inhibition type of niflumic acid (NA) for MPA glucuronidation in human liver. The third aim was to study the variability of the IC(50) value of NA for MPA glucuronidation in human liver. METHODS: The rate of MPA glucuronidation was measured by employing an assay based on uridine 5'-diphosphate-[U-(14)C]-glucuronic acid (UDPGA), and MPA glucuronide was isolated by means of thin-layer chromatography. The necessary concentration for UDPGA and MPA was 1 mM. The rate of MPA glucuronidation was measured in 50 human liver samples. The inhibition type of NA for MPA glucuronidation was studied in 5 human liver samples. The NA IC(50) value was measured in 27 human liver samples using six concentrations of NA ranging from 1.05 microM to 34 microM. RESULTS: MPA glucuronidation rate was positively skewed, was not gender regulated and did not correlate with the liver donor's age. The rate of MPA glucuronidation varied 4.8-fold within the 5th and 95th percentiles, with a mean+/-SD and a median of 2.8+/-1.0 nmol/min/mg and 2.5 nmol/min/mg, respectively. The inhibition type of NA for MPA glucuronidation was mixed non-competitive. The Ki value of NA (mean+/-SD) was 15+/-10 microM and, in non-inhibited samples, the K(m) value for MPA was 0.41+/-0.06 mM. The distribution of NA IC(50) value varied 3.3-fold within the 5th and 95th percentiles with a mean+/-SD and a median of 5.6+/-2.1 microM and 5.2 microM, respectively. The distribution of NA IC(50) value did not deviate significantly from normality. CONCLUSION: The range of hepatic rate of MPA glucuronidation is narrow relative to those of ethinyloestradiol, testosterone and zidovudine glucuronidation obtained from literature. The Ki value of NA is one order of magnitude lower than the K(m) for MPA in non-inhibited samples. This indicates that the inhibitor affinity for glucuronosyl transferase is greater than that of the substrate. The range of variation of NA IC(50) values is narrow.

Adult↗
PubMed1989

Androgen sulfate and glucuronide conjugates in nonhirsute and hirsute women with polycystic ovarian syndrome.

Peripheral androgen action largely determines the occurrence of hirsutism in women. Although serum 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol) glucuronide signifies skin 5 alpha-reductase activity and has been used as a marker of hirsutism and peripheral androgen metabolism, other C19 androgen conjugates have recently been measured and may also be useful markers of hirsutism in women. In addition to normal controls we studied both hirsute and nonhirsute patients with polycystic ovarian syndrome who had similar levels of circulating androgen precursors. In these three groups we measured various C19 sulfates and glucuronides including serum 3 alpha-diol glucuronide. Serum androgen precursors were elevated, but were equal in the hirsute and nonhirsute patients. Serum androsterone sulfate and glucuronide, and 3 alpha-diol sulfate and glucuronide clearly differentiated the hirsute from the nonhirsute group. Among the conjugates, androsterone glucuronide was most reflective of the difference between the two groups (100.3 +/- 28.0 versus 42.9 +/- 4.0 ng/ml, p less than 0.05). In hirsute compared with nonhirsute patients with polycystic ovarian syndrome, serum 3 alpha-diol glucuronide was increased by the smallest amount (32%), followed by androsterone sulfate (38%), 3 alpha-diol sulfate (59%), and androsterone glucuronide with the largest increase (134%). Serum androsterone glucuronide and 3 alpha-diol glucuronide both correlated with androstenedione and dehydroepiandrosterone sulfate in hirsute women but not in nonhirsute women. These data suggest that besides serum 3 alpha-diol glucuronide, other C19 sulfate and glucuronide conjugates may reflect peripheral androgen action.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1992

Stereoselective interactions of ketoprofen glucuronides with human plasma protein and serum albumin.

A clearance pathway common to many aryl alkanoic acids is the generation of renally eliminated ester glucuronides. These metabolites are susceptible to systemic hydrolysis which generates the parent aglycone. We have conducted in vitro studies with biosynthetic R- and S-ketoprofen glucuronides to elucidate the mechanism of this phenomenon. These conjugates were incubated in human plasma, various concentrations of human serum albumin (HSA) and protein-free buffer. It was apparent that albumin, rather than plasma esterases, catalysed the hydrolysis of the glucuronides. The albumin-catalysed hydrolysis of ketoprofen glucuronides was highly stereoselective. The mean (+/- SD) hydrolysis half-life of R-ketoprofen glucuronide in plasma (N = 4) at physiological pH and temperature was 1.37 (+/- 0.30) hr. The corresponding value for S-ketoprofen glucuronide, 3.46 (+/- 0.84) hr, was significantly different (P less than 0.005). In contrast, synthetic ethyl esters of R- and S-ketoprofen were hydrolysed by plasma esterases, but not by HSA, and with little stereoselectivity. The reversible protein binding of ketoprofen glucuronides was determined at physiological pH and temperature by a rapid ultra-filtration method. The binding of R- and S-ketoprofen glucuronide to human plasma protein was independent of concentration (P greater than 0.05) over the range of 1-20 micrograms/mL. The mean (+/- SD) percentage unbound in plasma (N = 4) of R-ketoprofen glucuronide was 12.6 (+/- 1.4)%. The corresponding value for S-ketoprofen glucuronide, 9.12 (+/- 0.54)%, was significantly different (P less than 0.005). S-Ketoprofen glucuronide was also more avidly protein bound in physiological concentrations of HSA. However, this stereoselectivity decreased in more dilute HSA solutions. Based on the hydrolysis and protein binding data for ketoprofen glucuronides, we propose the existence of separate binding and catalytic sites on the albumin molecule for these metabolites.

Adult↗
PubMed1996

Analysis of the glucuronidation of 7-hydroxycoumarin by HPLC.

The in-vitro metabolism of 7-hydroxycoumarin to 7-hydroxycoumarin-glucuronide was investigated in bovine liver homogenate. A metabolic reaction mixture was prepared that included a crude preparation of uridine diphosphate (UDP) glucuronyl transferase, 7-hydroxycoumarin and UDP-glucuronic acid. A HPLC method was developed to separate coumarin, 7-hydroxycoumarin, 7-hydroxycoumarin-glucuronide and an internal standard, 4-hydroxycoumarin. Samples were separated by reverse-phase HPLC, on a C18 column, with a 1 ml min-1 gradient elution with UV detection at 320 nm. The limit of quantification of the method, for 7-hydroxycoumarin-glucuronide, was 1.47 microM, and the linear range was from 0-295.7 microM. Concentrations of 7-hydroxycoumarin-glucuronide produced were calculated from a plot of 7-hydroxycoumarin-glucuronide concentration versus the mean absorbance ratio (n = 4) (7-hydroxycoumarin-glucuronide absorbance/4-hydroxycoumarin absorbance). It was possible to monitor the decrease in the 7-hydroxycoumarin content as it was metabolised as well as the increase in 7-hydroxycoumarin-glucuronide as it was produced enzymatically. The identity of the compound produced was confirmed by photodiode array spectral analysis. A plot of time versus 7-hydroxycoumarin-glucuronide produced indicates that the metabolism is linear for the first 90 min and reached a plateau at 150 min. The rate of reaction in the first 90 min was 2.96 +/- 0.06 (RSD 1.7%, n = 3) nmol of 7-hydroxycoumarin-glucuronide produced per minute per milligram of protein. After 150 min 0.34 +/- 0.005 mM (RSD 1.4%) 7-hydroxycoumarin-glucuronide was produced, from 0.77 mM 7-hydroxycoumarin introduced into the reaction mixture and 58.0% +/- 5.3% (or 0.44 +/- 0.02 mM) of the 7-hydroxycoumarin remained. These results show that it is possible to monitor the production of the phase II metabolite of coumarin with minimal sample clean-up and without the need for deconjugation of the glucuronide moiety. The method was very reliable and applicable for the direct determination of 7-hydroxycoumarin-glucuronide in an in-vitro metabolic assay.

Animals↗
PubMed1995

Effect of probenecid on the enantioselective pharmacokinetics of oxprenolol and its glucuronides in the rabbit.

PURPOSE: To study the effect of probenecid on the stereoselective pharmacokinetics of oxprenolol and its glucuronides in the rabbit. METHODS: An oral dose of 50 mg/kg racemic oxprenolol was given to nine rabbits twice, in random sequence with and without the concurrent administration of probenecid. Oxprenolol enantiomers were determined in plasma and urine by an enantioselective HPLC method. Oxprenolol glucuronides were measured in plasma and urine after enzymatic hydrolysis. RESULTS: The disposition of the oxprenolol enantiomers in rabbits is stereoselective, mainly due to a difference in metabolism. Renal excretion is only a minor elimination route for unchanged oxprenolol, and the renal clearances of the enantiomers are similar. Pretreatment with probenecid did not affect the plasma concentrations of the oxprenolol enantiomers, but there was a slight decrease in their urinary excretion. The plasma concentrations of the oxprenolol glucuronides are much higher than those of the parent enantiomers, and those of (S)-glucuronide are about twice those of its antipode. About 10% of the oxprenolol dose is excreted in the urine as glucuronides. The renal clearances of both glucuronides are similar, and markedly higher than the creatinine clearance. After probenecid, the mean glucuronide plasma levels were markedly higher, with for both glucuronides a more than twofold increase in mean AUC. Probenecid decreased the renal clearance of both glucuronides to about 30%. Moreover, it decreased slightly the formation clearance of (S)-glucuronide, while the formation clearance of (R)-glucuronide was not significantly influenced. CONCLUSIONS: Our results show that in the rabbit, both oxprenolol glucuronide diastereomers are actively secreted by the kidney, and that this process is inhibited by probenecid.

Animals↗
PubMed1992

The analgesic activity of morphine-6-glucuronide.

1. The pharmacokinetics, cardio-respiratory effects and analgesic effects of intravenous morphine-6-glucuronide were studied in 20 cancer patients with pain. Four different dose levels (0.5, 1, 2, and 4 mg 70 kg-1) were studied. Plasma concentrations of morphine-6-glucuronide were measured for 12 h after dosing. Pulse rate, respiratory rate and blood pressure were monitored, and pain relief was measured using two rating scales and a visual analogue scale. 2. The mean elimination half-life (+/- s.d.) of morphine-6-glucuronide was 3.2 +/- 1.6 h. The mean AUC standardised to a dose of 1 mg 70 kg-1 was 390 +/- 263 nmol l-1 h. Mean morphine-6-glucuronide clearance was 96 +/- 38 ml min-1. There was a direct relationship between morphine-6-glucuronide plasma clearance and calculated creatinine clearance (r = 0.81, P less than 0.001). 38 +/- 22% of the dose of morphine-6-glucuronide was recovered unchanged in the urine in 24 h. No morphine or morphine-3-glucuronide was detected in the plasma or urine of any patient after morphine-6-glucuronide treatment. 3. Morphine-6-glucuronide exerted a useful analgesic effect in 17/19 assessable patients for periods ranging between 2 and 24 h. No correlation was observed between dose or plasma morphine-6-glucuronide concentrations, and duration or degree of analgesia. No clinically significant changes in cardio-respiratory parameters were observed. No patients reported sedation or euphoria. Nausea and vomiting were notably absent in all cases. 4. Morphine-6-glucuronide is an effective and well-tolerated analgesic. It is likely that the majority of the therapeutic benefit of morphine is mediated by morphine-6-glucuronide.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

Pharmacology of morphine and morphine-3-glucuronide at opioid, excitatory amino acid, GABA and glycine binding sites.

Morphine in high doses and its major metabolite, morphine-3-glucuronide, cause CNS excitation following intrathecal and intracerebroventricular administration by an unknown mechanism. This study investigated whether morphine and morphine-3-glucuronide interact at major excitatory (glutamate), major inhibitory (GABA or glycine), or opioid binding sites. Homogenate binding assays were performed using specific radioligands. At opioid receptors, morphine-3-glucuronide and morphine caused an equipotent sodium shift, consistent with morphine-3-glucuronide behaving as an agonist. This suggests that morphine-3-glucuronide-mediated excitation is not caused by an interaction at opioid receptors. Morphine-3-glucuronide and morphine caused a weak inhibition of the binding of 3H-MK801 (non-competitive antagonist) and 125I-ifenprodil (polyamine site antagonist), but at unphysiologically high concentrations. This suggests that CNS excitation would not result from an interaction of morphine-3-glucuronide and high-dose morphine with these sites on the NMDA receptor. Morphine-3-glucuronide and morphine inhibited the binding of 3H-muscimol (GABA receptor agonist). 3H-diazepam and 3H-flunitrazepam (benzodiazepine agonists) binding very weakly, suggesting the excitatory effects of morphine-3-glucuronide and high-dose morphine are not elicited through GABAA receptors. Morphine-3-glucuronide and high-dose morphine did not prevent re-uptake of glutamate into presynaptic nerve terminals. In addition, morphine-3-glucuronide and morphine did not inhibit the binding of 3H-strychnine (glycine receptor antagonist) to synaptic membranes prepared from bovine spinal cord. It is concluded that excitation caused by high-dose morphine and morphine-3-glucuronide is not mediated by an interaction with postsynaptic amino acid receptors.

Animals↗
PubMed1988

Effect of butylated hydroxyanisole on hepatic glucuronidation and biliary excretion of drugs in mice.

Inhibition of glucuronidation by depletion of UDP-glucuronic acid from liver impairs the hepatobiliary transport of glucuronidated xenobiotics. However, it is not known if enhancement of hepatic glucuronidation increases the biliary excretion of these compounds. Therefore, the effect of treatment with butylated hydroxyanisole (BHA), which increases hepatic glucuronidation capacity, on the biliary excretion of compounds undergoing glucuronidation was studied in mice. BHA-feeding (1% for 10 days) increased hepatic UDP-glucuronic acid content by 240% and enhanced hepatic UDP-glucuronosyltransferase activities (expressed per kg body weight) toward valproic acid, phenolphthalein, iopanoic acid and bilirubin 220, 180, 120 and 60%, respectively. BHA treatment did not influence the biliary excretion of unmetabolized cholephils, phenol-3,6-dibromphthalein disulphonate and phenolphthalein glucuronide, but enhanced that of phenolphthalein (+108%), iopanoic acid (+63%) and bilirubin (+33%) as glucuronides. However, these increases were apparent only in the initial phase of excretion. In contrast, BHA markedly decreased (-43%) the biliary excretion of valproic acid glucuronides. Simultaneously, BHA increased the urinary excretion of the glucuronides of phenolphthalein (+48%), iopanoic acid (+450%) and valproic acid (+150%). A shift in the distribution of iopanoic acid and valproic acid and metabolites from liver to kidney was also apparent in BHA-fed mice. Thus, enhanced glucuronidation does not facilitate the biliary excretion of all glucuronidated compounds and only transiently increases others. It is likely that this phenomenon is the result of the glucuronides readily entering the plasma and being excreted by the kidney.

Animals↗
PubMed1992

In vitro N-glucuronidation of a novel antiepileptic drug, lamotrigine, by human liver microsomes.

The metabolism of the antiepileptic drug lamotrigine was characterized in human liver microsomes. For that purpose a high performance liquid chromatography method allowing the separation of lamotrigine glucuronide from the parent compound, and the quantitation of the glucuronide, was developed. The drug undergoes glucuronidation on the 2-nitrogen atom of the triazine ring, leading to a quaternary ammonium-linked glucuronide. This metabolite was positively identified from its hydrolysis by beta-glucuronidase and its associated radioactivity when UDP-[U-14C] glucuronic acid was used as the cosubstrate. Structural confirmation of the glucuronide was finally obtained by high performance liquid chromatography-mass spectrometry, by using a thermospray interface. The reaction proceeded with an apparent Vmax of 0.65 nmol/min/mg and Km of 2.56 mM. The average value of lamotrigine glucuronidation in four human samples of transplantable liver was 0.43 +/- 0.14 nmol/min/mg, thus indicating a large interindividual variation. An interspecies comparison of hepatic lamotrigine glucuronidation (human, rabbit, rat, monkey) revealed that the rate of glucuronidation was low. Of all the species considered, humans glucuronidated the drug to the greatest extent, with a specific activity 2-fold higher than that observed in rabbit liver microsomes. In contrast, the activity was greater than 20 times lower in monkey (0.019 nmol/min/mg) and at the limit of detection in rat liver microsomes. However, in this species, phenobarbital treatment enhanced lamotrigine glucuronidation slightly (0.017 nmol/min/mg). Among the drugs that undergo quaternary ammonium-linked glucuronidation, chlorpromazine, but not imipramine, amitriptyline and cyproheptadine, inhibited the glucuronidation of lamotrigine in vitro (IC50 of 5.0 x 10(-4) M).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Improvement of estradiol 17 beta-D-glucuronide cholestasis by intravenous administration of dimethylethanolamine in the rat.

The intravenous administration of dimethylethanolamine in the rat promotes a selective enrichment of liver membranes with polyunsaturated phosphatidylcholines. The effect of dimethylethanolamine pretreatment on cholestasis induced by estradiol 17 beta-D-glucuronide, a potent cholestatic agent, was assessed in this study. Dimethylethanolamine, dissolved in sodium-taurocholate was infused intravenously (0.3 mg/kg/min) for 15 hr. One group of control rats (estradiol 17 beta-D-glucuronide controls) received the bile salt only. An estradiol 17 beta-D-glucuronide bolus was then injected intravenously (10.4 mg/kg) into dimethylethanolamine-pretreated and estradiol 17 beta-D-control rats, and its effect on bile flow and biliary lipid secretion was compared for 3 hr. The estradiol 17 beta-D-glucuronide inhibitory effect on bile flow and biliary lipid secretion was significantly antagonized by dimethylethanolamine pretreatment. The maximum inhibition of bile flow was found 30 min after estradiol 17 beta-D-glucuronide administration, when it decreased from 3.5 +/- 0.4 microliters/min/100 gm (basal) to 0.9 +/- 0.3 microliters/min/100 gm in estradiol 17 beta-D-glucuronide controls, whereas in dimethylethanolamine-pretreated rats this decreased only from 3.2 +/- 0.4 (basal) to 2.3 +/- 0.4 microliters/min/100 gm. Bile flow and the biliary secretion of cholesterol, phosphatidylcholine and bile salts were significantly higher in the dimethylethanolamine-pretreated rats than in estradiol 17 beta-D-glucuronide controls (p less than 0.02) during the cholestatic phase. The inhibitory effect of estradiol 17 beta-D-glucuronide on bile flow was associated with a marked decrease of membrane fluidity (p less than 0.001) assessed by 1,6-diphenyl-1,3,5-hexatriene fluorescence anisotropy and with a cholesterol enrichment of microsomes, sinusoidal and canalicular liver plasma membranes and inhibition of sinusoidal Na+,K(+)-ATPase activity (p less than 0.05). These membrane alterations persisted 180 min after estradiol 17 beta-D-glucuronide administration despite complete normalization of bile flow. Dimethylethanolamine pretreatment significantly counteracted the reduction of membrane fluidity (p less than 0.001), the cholesterol enrichment and the inhibition of Na+,K(+)-ATPase (p less than 0.05) promoted by estradiol 17 beta-D-glucuronide administration in all membrane subfractions 30 and 180 min after administration. In addition, dimethylethanolamine-pretreated rats had more polyunsaturated fatty acids in membrane phosphatidylcholine with respect to the control groups. Dilatation of canaliculi and loss of microvilli were evident in estradiol 17 beta-D-glucuronide controls 180 min after estradiol 17 beta-D-glucuronide administration.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

Effect of renal dysfunction on the individual components of the acyl-glucuronide futile cycle.

Drugs which are cleared predominantly by forming acyl-glucuronides undergo a futile cycle in which plasma drug clearance is a function of the formation, hydrolysis and renal clearance of the glucuronide conjugate. The effect of impaired renal function, induced by uranyl nitrate administration, on each component of this process was studied in rabbits using diphenylacetic acid. Uranyl nitrate administration produced a decrease in creatinine clearance of approximately 70% and diphenylacetic acid plasma clearance of approximately 35%. In healthy rabbits the primary determinant of diphenylacetic acid net clearance was the very large component of glucuronide renal clearance (14.10 ml/min/kg) compared with glucuronide formation (4.79 ml/min/kg) or glucuronide hydrolysis (2.56 ml/min/kg). Uranyl nitrate treatment reduced both creatinine clearance and the glucuronide renal clearance by approximately 70%. Renal dysfunction had little effect on the hydrolysis clearance of the glucuronide, but reduced its formation clearance by 22.8%. The reduction in plasma clearance of diphenylacetic acid was a function of both the decrease in glucuronidation (60% contribution) and the decrease in renal clearance of the glucuronide (40% contribution). This study supports the futile cycle mechanism of reversible glucuronide conjugation for acyl-glucuronides in general, and provides a mechanism for the impairment of the plasma clearance in renal failure of drugs forming acyl-glucuronides.

Animals↗