Effect of albumin on human liver microsomal and recombinant CYP1A2 activities: impact on in vitro-in vivo extrapolation of drug clearance.

Long-chain unsaturated fatty acids inhibit several cytochrome P450 and UDP-glucuronosyltransferase (UGT) enzymes involved in drug metabolism, including CYP2C8, CYP2C9, UGT1A9, UGT2B4, and UGT2B7. Bovine serum albumin (BSA) enhances these cytochrome P450 and UGT activities by sequestering fatty acids that are released from membranes, especially with human liver microsomes (HLM) as the enzyme source. Here, we report the effects of BSA on CYP1A2-catalyzed phenacetin (PHEN) O-deethylation and lidocaine (LID) N-deethylation using HLM and Escherichia coli-expressed recombinant human CYP1A2 (rCYP1A2) as the enzyme sources. BSA (2% w/v) reduced (p < 0.05) the K(m) values of the high-affinity components of human liver microsomal PHEN and LID deethylation by approximately 70%, without affecting V(max). The K(m) (or S(50)) values for PHEN and LID deethylation by rCYP1A2 were reduced to a similar extent. A fatty acid mixture, comprising 3 µM concentrations each of oleic acid and linoleic acid plus 1.5 µM arachidonic acid, doubled the K(m) value for PHEN O-deethylation by rCYP1A2. Inhibition was reversed by the addition of BSA. K(i) values for the individual fatty acids ranged from 4.7 to 16.7 µM. Single-point in vitro-in vivo extrapolation (IV-IVE) based on the human liver microsomal kinetic parameters obtained in the presence, but not absence, of BSA predicted in vivo hepatic clearances of PHEN O-deethylation and LID N-deethylation that were comparable to values reported in humans, although in vivo intrinsic clearances were underpredicted. Prediction of the in vivo clearances of the CYP1A2 substrates observed here represents an improvement on other experimental systems used for IV-IVE.

Characterization of niflumic acid as a selective inhibitor of human liver microsomal UDP-glucuronosyltransferase 1A9: application to the reaction phenotyping of acetaminophen glucuronidation.

Enzyme selective inhibitors represent the most valuable experimental tool for reaction phenotyping. However, only a limited number of UDP-glucuronosyltransferase (UGT) enzyme-selective inhibitors have been identified to date. This study characterized the UGT enzyme selectivity of niflumic acid (NFA). It was demonstrated that 2.5 µM NFA is a highly selective inhibitor of recombinant and human liver microsomal UGT1A9 activity. Higher NFA concentrations (50-100 µM) inhibited UGT1A1 and UGT2B15 but had little effect on the activities of UGT1A3, UGT1A4, UGT1A6, UGT2B4, UGT2B7, and UGT2B17. NFA inhibited 4-methylumbelliferone and propofol (PRO) glucuronidation by recombinant UGT1A9 and PRO glucuronidation by human liver microsomes (HLM) according to a mixed (competitive-noncompetitive) mechanism, with K(i) values ranging from 0.10 to 0.40 µM. Likewise, NFA was a mixed or noncompetitive inhibitor of recombinant and human liver microsomal UGT1A1 (K(i) range 14-18 µM), whereas competitive inhibition (K(i) 62 µM) was observed with UGT2B15. NFA was subsequently applied to the reaction phenotyping of human liver microsomal acetaminophen (APAP) glucuronidation. Consistent with previous reports, APAP was glucuronidated by recombinant UGT1A1, UGT1A6, UGT1A9, and UGT2B15. NFA concentrations in the range of 2.5 to 100 µM inhibited APAP glucuronidation by UGT1A1, UGT1A9, and UGT2B15 but not by UGT1A6. The mean V(max) for APAP glucuronidation by HLM was reduced by 20, 35, and 40%, respectively, in the presence of 2.5, 50, and 100 µM NFA. Mean K(m) values decreased in parallel with V(max), although the magnitude of the decrease was smaller. Taken together, the NFA inhibition data suggest that UGT1A6 is the major enzyme involved in APAP glucuronidation.

Spironolactone and canrenone inhibit UGT2B7-catalyzed human liver and kidney microsomal aldosterone 18beta-glucuronidation: a potential drug interaction.

Elevated plasma concentrations of aldosterone (ALDO) are observed in patients treated with spironolactone. Because ALDO is eliminated via UGT2B7-catalyzed 18beta-glucuronidation, this study aimed to determine whether spironolactone and its primary metabolites, canrenone and canrenoic acid, inhibit ALDO 18beta-glucuronidation by recombinant UGT2B7 and by human liver (HLM) and human kidney cortical (HKCM) microsomes. Initial experiments characterized the effects of all three compounds on 4-methylumbelliferone and ALDO glucuronidation by recombinant human UGT2B7. IC(50) values for spironolactone and canrenone ranged from 26 to 50 microM, whereas canrenoic acid was a weak inhibitor. Inhibitor constant (K(i)) values for spironolactone and canrenone inhibition of ALDO 18beta-glucuronidation were subsequently determined with HLM, HKCM, and UGT2B7 as the enzyme sources. Spironolactone and canrenone were competitive inhibitors of ALDO 18beta-glucuronidation by HLM, HKCM, and UGT2B7. Mean (+/-) K(i) values for spironolactone were 52 +/- 22 (HLM) and 34 +/- 4 microM (HKCM), and mean (+/-) K(i) values for canrenone were 41 +/- 19 (HLM) and 23 +/- 2 microM (HKCM). K(i) values for spironolactone and canrenone inhibition of ALDO 18beta-glucuronidation by recombinant UGT2B7 were 23 and 11 microM, respectively. "Actual" K(i) values for spironolactone and canrenone inhibition of ALDO 18beta-glucuronidation, which take into account the role of endogenous microsomal inhibitors, are predicted to be 3 to 5 and 2 to 4 microM, respectively. The data indicate that the elevated ALDO concentrations observed in patients treated with spironolactone may be due, at least in part, to a pharmacokinetic interaction, and spironolactone and canrenone should be considered to be potential inhibitors of the UGT2B7-mediated metabolic clearance of drugs in both liver and kidney.

Aldosterone glucuronidation by human liver and kidney microsomes and recombinant UDP-glucuronosyltransferases: inhibition by NSAIDs.

AIMS: To characterize: i) the kinetics of aldosterone (ALDO) 18beta-glucuronidation using human liver and human kidney microsomes and identify the human UGT enzyme(s) responsible for ALDO 18beta-glucuronidation and ii) the inhibition of ALDO 18beta-glucuronidation by non-selective NSAIDs. METHODS: Using HPLC and LC-MS methods, ALDO 18beta-glucuronidation was characterized using human liver (n= 6), human kidney microsomes (n= 5) and recombinant human UGT 1A1, 1A3, 1A4, 1A5, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B10, 2B15, 2B17 and 2B28 as the enzyme sources. Inhibition of ALDO 18beta-glucuronidation was investigated using alclofenac, cicloprofen, diclofenac, diflunisal, fenoprofen, R- and S-ibuprofen, indomethacin, ketoprofen, ketorolac, meclofenamic acid, mefenamic acid, S-naproxen, pirprofen and tiaprofenic acid. A rank order of inhibition (IC(50)) was established and the mechanism of inhibition investigated using diclofenac, S-ibuprofen, indomethacin, mefenamic acid and S-naproxen. RESULTS: ALDO 18beta-glucuronidation by hepatic and renal microsomes exhibited Michaelis-Menten kinetics. Mean (+/-SD) K(m), V(max) and CL(int) values for HLM and HKCM were 509 +/- 137 and 367 +/- 170 microm, 1075 +/- 429 and 1110 +/- 522 pmol min(-1) mg(-1), and 2.36 +/- 1.12 and 3.91 +/- 2.35 microl min(-1) mg(-1), respectively. Of the UGT proteins, only UGT1A10 and UGT2B7 converted ALDO to its 18beta-glucuronide. All NSAIDs investigated inhibited ALDO 18beta-G formation by HLM, HKCM and UGT2B7. The rank order of inhibition (IC(50)) of renal and hepatic ALDO 18beta-glucuronidation followed the general trend: fenamates > diclofenac > arylpropionates. CONCLUSION: A NSAID-ALDO interaction in vivo may result in elevated intra-renal concentrations of ALDO that may contribute to the adverse renal effects of NSAIDs and their effects on antihypertensive drug response.

Influence of N-terminal domain histidine and proline residues on the substrate selectivities of human UDP-glucuronosyltransferase 1A1, 1A6, 1A9, 2B7, and 2B10.

An N-terminal domain histidine [corresponding to position 39 of UDP-glucuronosyltransferase (UGT) 1A1] is conserved in all UGT1A and UGT2B subfamily proteins except UGT1A4 (Pro-40) and UGT2B10 (Leu-34). Unlike most UGT1A and UGT2B xenobiotic-metabolizing enzymes, UGT1A4 and UGT2B10 lack the ability to glucuronidate 4-methylumbelliferone (4MU) and 1-naphthol (1NP), both planar phenols, and naproxen (a carboxylic acid). However, only UGT1A4 glucuronidates the tertiary amines lamotrigine (LTG) and trifluoperazine (TFP). In this study, we sought to elucidate the influence of specific N-terminal histidine and proline residues on UGT enzyme substrate selectivity. The conserved N-terminal domain histidine of UGT1A1, UGT1A6, UGT1A9, and UGT2B7 was mutated to proline and leucine 34 of UGT2B10 was substituted with histidine, and the capacity of the wild-type and mutant proteins to glucuronidate 4MU, 1NP, LTG, TFP, and naproxen was characterized. Whereas UGT1A1(H39P), UGT1A6(H38P), and UGT1A9(H37P) lacked the ability to metabolize 4MU, 1NP, and naproxen, all glucuronidated LTG. K(m) values for UGT1A1(H39P) and UGT1A9(H37P) were 774 and 3812 microM, respectively, compared with 1579 microM for UGT1A4. UGT1A1(H39P) also glucuronidated TFP with a V(max)/K(m) value comparable to that of UGT1A4. In contrast to the wild-type enzyme, UGT2B10(L34H) glucuronidated 4MU and 1NP with respective K(m) values of 260 and 118 microM. UGT2B7(H35P) lacked activity toward all substrates. The data confirm a pivotal role for an N-terminal domain proline in the glucuronidation of the tertiary amines LTG and TFP by UGT1A subfamily proteins, whereas glucuronidation reactions involving proton abstraction generally, although not invariably, require a histidine at the equivalent position in both UGT1A and UGT2B enzymes.

S-Naproxen and desmethylnaproxen glucuronidation by human liver microsomes and recombinant human UDP-glucuronosyltransferases (UGT): role of UGT2B7 in the elimination of naproxen.

AIMS: To characterize the kinetics of S-naproxen ('naproxen') acyl glucuronidation and desmethylnaproxen acyl and phenolic glucuronidation by human liver microsomes and identify the human UGT isoform(s) catalysing these reactions. METHODS: Naproxen and desmethylnaproxen glucuronidation were investigated using microsomes from six and five livers, respectively. Human recombinant UGTs were screened for activity towards naproxen and desmethylnaproxen. Where significant activity was observed, kinetic parameters were determined. Naproxen and desmethylnaproxen glucuronides were measured by separate high-performance liquid chromatography methods. RESULTS: Naproxen acyl glucuronidation by human liver microsomes followed biphasic kinetics. Mean apparent K(m) values (+/-SD, with 95% confidence interval in parentheses) for the high- and low-affinity components were 29 +/- 13 microm (16, 43) and 473 +/- 108 microm (359, 587), respectively. UGT 1A1, 1A3, 1A6, 1A7, 1A8, 1A9, 1A10 and 2B7 glucuronidated naproxen. UGT2B7 exhibited an apparent K(m) (72 microm) of the same order as the high-affinity human liver microsomal activity, which was inhibited by the UGT2B7 selective 'probe' fluconazole. Although data for desmethylnaproxen phenolic glucuronidation by human liver microsomes were generally adequately fitted to either the single- or two-enzyme Michaelis-Menten equation, model fitting was inconclusive for desmethylnaproxen acyl glucuronidation. UGT 1A1, 1A7, 1A9 and 1A10 catalysed both the phenolic and acyl glucuronidation of desmethylnaproxen, while UGT 1A3, 1A6 and 2B7 formed only the acyl glucuronide. Atypical glucuronidation kinetics were variably observed for naproxen and desmethylnaproxen glucuronidation by the recombinant UGTs. CONCLUSION: UGT2B7 is responsible for human hepatic naproxen acyl glucuronidation, which is the primary elimination pathway for this drug.