Phenolic Glucuronide Conjugates Research Articles

Pharmacokinetics and metabolism of cinnamic acid derivatives and flavonoids after oral administration of Brazilian green propolis in humans.

Brazilian green propolis (BGP) has chemical compounds from botanical origin that are mainly cinnamic acid derivatives (artepillin C, baccharin, and drupanin) and flavonoids (kaempferide and 6-methoxykaempferide). These compounds are expected to play an important role in the pharmacological activities of BGP. However, there is little known about the pharmacokinetics and metabolism of these compounds after oral administration of BGP. The aim of this study is to investigate the pharmacokinetics and metabolism of BGP components in humans. Twelve volunteers received 3 capsules containing 360 mg of BGP ethanol extract powder. Plasma samples were collected before and up to 24 h after the intake of BGP capsules. The collected plasma samples with or without hydrolysis by the deconjugating enzyme were analyzed by LC/MS/MS. After enzymatic hydrolysis, the Cmax values of artepillin C and drupanin, which were detected mainly in plasma after ingestion of BGP capsules, were 1255 ± 517 and 2893 ± 711 nM, respectively, of which 89.3% and 88.2% were found to be the phenolic glucuronide conjugate. This is the first time that the pharmacokinetics of the BGP components of human metabolites have been reported. Our results could provide useful information for the design and interpretation of studies to investigate the mechanisms and pharmacological effects of BGP.

A Novel Method for the Determination of the Site of Glucuronidation by Ion Mobility Spectrometry-Mass Spectrometry

Glucuronidation not only plays a detoxifying role in living body, but it also can complicate pharmacological and toxicological profiles of new drug candidates by forming active and reactive conjugated metabolites. The opportunity to elucidate structure of conjugated metabolites has increased in drug metabolism studies at the early development stage. General methodologies for the structure elucidation of glucuronide conjugate(s) include liquid chromatography-tandem mass spectrometry (LC-MS/MS) and NMR spectroscopy. In many cases, LC-MS/MS alone cannot unequivocally identify the site(s) of conjugation in isomeric glucuronidations. In the present study, we established a new strategy for the structure elucidation of glucuronide conjugates using ion mobility spectrometry (IMS)-mass spectrometry. Linear correlation between calculated collision cross-sections (CCS) and actual drift times from IMS was found for each set of parent compound (raloxifene, losartan, telmisartan, and estradiol) and the corresponding MS/MS product ions. Thus, obtained regression lines accurately and selectively projected the actual drift times of authentic standards of glucuronide conjugate based on the theoretical CCS values. The established method was used for the accurate assignment of predominant formation of phenolic glucuronide conjugate (SCH 60663) in the isomeric (phenolic and benzylic) glucuronidations of ezetimibe in the incubated sample with cryopreserved human hepatocytes. This application demonstrates the potential to facilitate the structure identification of glucuronide conjugates at the early development stage of new drug candidates.

GLUCURONIDATION OF THE ASPIRIN METABOLITE SALICYLIC ACID BY EXPRESSED UDP-GLUCURONOSYLTRANSFERASES AND HUMAN LIVER MICROSOMES

Acetylsalicylic acid (aspirin) is a common nonsteroidal anti-inflammatory drug used for treatment of pain and arthritis. In the body, acetylsalicylic acid is rapidly deacetylated to form salicylic acid. Both compounds have been proposed as anti-inflammatory agents. Major metabolites of salicylic acid are its acyl and phenolic glucuronide conjugates. Formation of these conjugates, catalyzed by UDP-glucuronosyltransferases (UGTs), decreases the amount of pharmacologically active salicylic acid present. We aimed to identify the UGTs catalyzing the glucuronidation of salicylic acid using both heterologously expressed enzymes and pooled human liver microsomes (HLMs) and to develop a liquid chromatography-tandem mass spectrometry method to quantify glucuronidation activity of UGTs 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, and 2B17 Supersomes. All UGTs tested, except 1A4, 2B15, and 2B17, catalyzed salicylic acid phenolic and acyl glucuronidation. Ratios of salicylic acid phenolic to acyl glucuronide formation varied more than 12-fold from 0.5 for UGT1A6 to 6.1 for UGT1A1. These results suggest that all UGTs except 1A4, 2B15, and 2B17 might be involved in the glucuronidation of salicylic acid in vivo. From comparisons of apparent Km values determined in pooled HLMs and in expressed UGTs, UGT2B7 was suggested as a likely catalyst of salicylic acid acyl glucuronidation, whereas multiple UGTs were suggested as catalysts of phenolic glucuronidation. The results of this UGT screening may help target future evaluation of the effects of UGT polymorphisms on response to aspirin in clinical and population-based studies.

Identification of human UDP-glucuronosyltransferase enzyme(s) responsible for the glucuronidation of ezetimibe (Zetia).

Ezetimibe [1-(4-fluorophenyl)-3(R)-[3-(4-fluorophenyl)-3(S)-hydroxypropyl]-4(S)-(4-hydroxyphenyl)-2-azetidinone] (Zetia; Schering-Plough, Kenilworth, NJ) is the first in a new class of cholesterol-lowering agents known as cholesterol absorption inhibitors. The objective of this study was to identify the isoform(s) of human liver and intestinal UDP-glucuronosyltransferase (UGT) enzymes responsible for the glucuronidation of ezetimibe. The main circulating metabolite of this drug in human plasma is SCH 60663, the phenolic glucuronide conjugate of ezetimibe. SCH 60663 [m/z = 584 Thompsons (Th)] is also the major in vitro metabolite formed by human liver microsomes supplemented with UDP glucuronic acid (UDPGA). In contrast to the liver, human jejunum microsomes supplemented with UDPGA converted ezetimibe to two glucuronides with the same mass (m/z = 584 Th) by liquid chromatography-mass spectrometry. One corresponds to the phenolic glucuronide (1-O-[4-trans-2S,3R)-1-(4-fluorophenyl)-4-oxo-3-[3(S)-hydroxy-3-(4-fluorophenyl)propyl]-2-azetidinyl]phenyl-beta-D-glucopyranuronic acid; SCH 60663) and the other was identified as the benzylic glucuronide of ezetimibe (1-O-[1(S)-(4-fluorophenyl)-3-[1-(4-fluorophenyl)-2(S)-(4-hydroxyphenyl)-4-oxo-3(R)-azetidinyl]propyl]-beta-D-glucopyranuronic acid; SCH 488128). Recombinant human UGT1A1, UGT1A3, and UGT2B15 all exhibited catalytic activity with respect to the formation of the phenolic glucuronide. However, UGT2B7 exclusively formed SCH 488128, a trace metabolite detected in dog and human plasma samples after oral administration of ezetimibe. In conclusion, the formation of SCH 60663 is mediated via UGT1A1, UGT1A3, and UGT2B15, and the formation SCH 488128 is mediated via UGT2B7.

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 μg 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.O, 6.5 and 8.0. After 8 hr incubation, the highest concentrations ( ca. 11, μg DF equivalents/g) were produced with iso-DAG at pH 5.O, and the lowest ( ca. 2.3 μg 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 β-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 β-glucuronidases.

Effects of blockage of urine and/or bile flow on diflunisal conjugation and disposition in rats

1. The effects of surgical blockage of either or both of the urinary and biliary excretion routes on the elimination of diflunisal (DF) and its conjugates were investigated in pentobarbitone-anaesthetized rats given DF at 10 mg/kg i.v. 2. In control animals the acyl glucuronide and phenolic glucuronide conjugates were excreted predominantly in bile, whereas the sulphate conjugate was eliminated almost exclusively in urine. 3. Bilateral ureter ligation had little effect on DF elimination, except for accumulation of the sulphate conjugate in plasma. Compensatory biliary excretion did not occur. 4. Total plasma clearance of DF decreased from 1.01 to 0.68 ml/min per kg following bile duct ligation. Plasma concentrations and urinary excretion of the glucuronides were elevated. 5. In rats with blockage of both urinary and biliary excretion routes, total plasma clearance of DF decreased to 0.59 ml/min per kg. Both the sulphate and phenolic glucuronide conjugates accumulated in plasma, whereas the acyl glucuronide peaked at 30 min and then declined in parallel with DF. The latter result indicates systemic instability of DF acyl glucuronide with hydrolytic regeneration of DF as the likely major consequence.

Salicylate metabolism: Effects of age and sex in adults

The plasma concentrations and urinary excretion rates of salicylic acid (SA) and some of its metabolites (salicyluric acid [SUA] and acyl and phenolic glucuronide conjugates) were measured after an oral dose of acetylsalicylic acid to 44 healthy subjects of both sexes 20 to 78 years old. There was no change in the SA absorption rate, and plasma clearance of SA was not affected by age or sex. The volume of distribution increased with age in men but not in women. Plasma concentrations of SUA rose with age as the renal clearance of this metabolite fell. The kinetic parameters Km and Vmax for the SA-to-SUA reaction did not change with age; Vmax was significantly higher in women than in men. Urinary recovery of SA and its metabolites essentially accounted for the administered dose, and was little influenced by age or sex. We conclude that these factors play a minor role in the disposition of salicylate.

The metabolic disposition of 14C-ciramadol in humans.

Twelve subjects received single 15 mg oral doses of 14C-ciramadol. Excretion of the dose occurred almost entirely by the renal route (93.5 +/- 11.7 (S.D.)% of the dose), and only 0.7 +/- 0.6% of the dose was recovered in faeces indicating that absorption was essentially complete. More than 90% of the amount recovered in urine was excreted within 24 h after dosing. Unchanged drug accounted for 43.9 +/- 6.5% of the dose, while a phenolic glucuronide conjugate was the only major urinary metabolite accounting for a further 37.9 +/- 7.8%. A second glucuronide that was conjugated with the alicyclic ring was also identified but constituted only 2.3 +/- 0.6% of the dose. Concentrations of radioactivity in plasma reached a peak at 2 h after dosing and declined with a terminal disposition half life of 4.9 h. Only ciramadol and the aryl-O-glucuronide were detected in substantial amounts in plasma. Renal clearance of ciramadol amounted to 298 +/- 54 ml/min suggesting tubular secretion in addition to glomerular filtration.