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Kinase inhibitors (KIs) are a rapidly expanding class of drugs used primarily for the treatment of cancer. Data relating to the inhibition of UDP-glucuronosyltransferase (UGT) enzymes by KIs is sparse. However, lapatinib (LAP), pazopanib (PAZ), regorafenib (REG) and sorafenib (SOR) have been implicated in the development of hyperbilirubinemia in patients. This study aimed to characterise the role of UGT1A1 inhibition in hyperbilirubinemia and assess the broader potential of these drugs to perpetrate drug-drug interactions arising from UGT enzyme inhibition. Twelve recombinant human UGTs from subfamilies 1A and 2B were screened for inhibition by LAP, PAZ, REG and SOR. IC50 values for the inhibition of all UGT1A enzymes, except UGT1A3 and UGT1A4, by the four KIs were

Aim To determine the scaling factors required for inclusion of renal drug glucuronidation clearance in the prediction of total clearance via glucuronidation (CLUGT ). Methods Microsomal protein per gram of kidney (MPPGK) was determined for human 'mixed' kidney (n = 5) microsomes (MKM). The glucuronidation activities of deferiprone (DEF), propofol (PRO) and zidovudine (AZT) by MKM and paired cortical (KCM) and medullary (KMM) microsomes were measured, along with the UGT 1A6, 1A9 and 2B7 protein contents of each enzyme source. Unbound intrinsic clearances (CLint,u,UGT ) for PRO and morphine (MOR; 3- and 6-) glucuronidation by MKM, human liver microsomes (HLM) and recombinant UGT1A9 and 2B7 were additionally determined. Data were scaled using in vitro-in vivo extrapolation (IV-IVE) approaches to assess the influence of renal CLint,u,UGT on the prediction accuracy of the calculated CLUGT values of PRO and MOR. Results MPPGK was 9.3 ± 2.0 mg g(-1) (mean ± SD). The respective rates of DEF (UGT1A6), PRO (UGT1A9) and AZT (UGT2B7) glucuronidation by KCM were 1.4-, 5.2- and 10.5-fold higher than those for KMM. UGT 1A6, 1A9 and 2B7 were the only enzymes expressed in kidney. Consistent with the activity data, the abundance of each of these enzymes was greater in KCM than in KMM. The abundance of UGT1A9 in MKM (61.3 pmol mg(-1) ) was 2.7 fold higher than that reported for HLM. Conclusions Scaled renal PRO glucuronidation CLint,u,UGT was double that of liver. Renal CLint,u,UGT should be accounted for in the IV-IVE of UGT1A9 and considered for UGT1A6 and 2B7 substrates.

1. This study compared the extent, affinity, and kinetics of drug binding to human serum albumin (HSA) and liver fatty acid binding protein (LFABP) using ultrafiltration and surface plasmon resonance (SPR). 2. Binding of basic and neutral drugs to both HSA and LFABP was typically negligible. Binding of acidic drugs ranged from minor (fu 0.8) to extensive (fu 0.1). Of the compounds screened, the highest binding to both HSA and LFABP was observed for the acidic drugs torsemide and sulfinpyrazone, and for β-estradiol (a polar, neutral compound). 3. The extent of binding of acidic drugs to HSA was up to 40% greater than binding to LFABP. SPR experiments demonstrated comparable kinetics and affinity for the binding of representative acidic drugs (naproxen, sulfinpyrazone, and torsemide) to HSA and LFABP. 4. Simulations based on in vitro kinetic constants derived from SPR experiments and a rapid equilibrium model were undertaken to examine the impact of binding characteristics on compartmental drug distribution. Simulations provided mechanistic confirmation that equilibration of intracellular unbound drug with the extracellular unbound drug is attained rapidly in the absence of active transport mechanisms for drugs bound moderately or extensively to HSA and LFABP.

Recent advances in the identification and characterization of renal drug transporters and drug-metabolizing enzymes has led to greater understanding of their roles in drug and chemical elimination and in modulation of the intrarenal exposure and response to drugs, nephrotoxic compounds, and physiological mediators. Furthermore, there is increasing awareness of the potential importance of drug-drug interactions (DDIs) arising from inhibition of renal transporters, and regulatory agencies now provide recommendations for the evaluation of transporter-mediated DDIs. Apart from the well-recognized effects of kidney disease on renal drug clearance, there is a growing body of evidence demonstrating that the nonrenal clearances of drugs eliminated by certain transporters and drug-metabolizing enzymes are decreased in patients with chronic kidney disease (CKD). Based on these observations, renal impairment guidance documents of regulatory agencies recommend pharmacokinetic characterization of both renally cleared and nonrenally cleared drugs in CKD patients to inform possible dosage adjustment.

Knowledge of the metabolic stability of newly discovered drug candidates eliminated by metabolism is essential for predicting the pharmacokinetic (PK) parameters that underpin dosing and dosage frequency. Further, characterization of the enzyme(s) responsible for metabolism (reaction phenotyping) allows prediction, at least at the qualitative level, of factors (including metabolic drug-drug interactions) likely to alter the clearance of both new chemical entities (NCEs) and established drugs. Microsomes are typically used as the enzyme source for the measurement of metabolic stability and for reaction phenotyping because they express the major drug-metabolizing enzymes cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT), along with others that contribute to drug metabolism. Described in this unit are methods for microsome isolation, as well as for the determination of metabolic stability and metabolite formation (including kinetics). © 2016 by John Wiley & Sons, Inc.

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.

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.

The evidence linking the use of non-steroidal anti-inflammatory drugs (NSAIDs) with increased atherothrombotic risk is controversial, particularly in older patients. This population is consistently underrepresented in epidemiological studies. Moreover, several confounding factors such as co-morbidities, polypharmacy, and institutionalisation might affect the interpretation of studies on the real association between NSAID use and cardiovascular risk. These issues are herewith discussed together with a proposed mechanism to explain the results of recent studies demonstrating a relatively low atherothrombotic risk associated with NSAIDs in older patients. Suggestions for future research directions are also provided.

Major advances in the characterization of uridine diphosphate (UDP)-glucuronosyltransferase (UGT) enzyme substrate and inhibitor selectivities and the development of experimental paradigms to investigate xenobiotic glucuronidation in vitro now permit the prediction of a range of drug-glucuronidation parameters in humans. In particular, the availability of substrate and inhibitor "probes" for the major hepatic drug metabolizing UGTs together with batteries of recombinant enzymes allow the reaction phenotyping of drug glucuronidation reactions. Additionally, in vitro experimental approaches and scaling strategies have been successfully applied to the quantitative prediction of in vivo clearance via glucuronidation and drug-drug interaction potential.

This work characterized for the first time the binding of acidic, neutral, and basic drugs to human intestinal fatty acid binding protein (IFABP) and, for comparison, to bovine serum albumin (BSA). In addition, the study investigated whether IFABP can substitute for BSA as a constituent in incubations of human liver microsomes (HLMs) in in vitro-in vivo extrapolation (IV-IVE) studies. Each molecule of purified IFABP bound a single molecule of the fluorescent probe 1-anilino-8-naphthalene sulfonate or arachidonic acid with K d values similar to those reported for rat IFABP. Basic drugs bound negligibly to IFABP. Based on fraction unbound (f u ) at a protein concentration of 0.5% (w/v), binding of acidic and neutral drugs ranged from minor (f u > 0.8) to moderate (f u 0.5–0.8). Of the compounds screened, highest binding to IFABP was observed for sulfinpyrazone (an acid) and β-estradiol (a neutral compound). However, binding to IFABP was lower than to BSA for all the drugs investigated. To determine the potential suitability of IFABP as an alternative to BSA for enhancing the prediction accuracy of IV-IVE based on human liver microsomal kinetic data, the kinetics of zidovudine (AZT) glucuronidation by HLM were characterized in the absence and presence of BSA and IFABP (0.5–2.5%, w/v). Each protein reduced the K m for AZT glucuronidation in a concentration-dependent manner, although a higher content of IFABP in incubations (2.5 versus 1–1.5% for BSA) was necessary for a 10-fold reduction in this parameter. The results indicate that IFABP is likely to have advantages over BSA in microsomal kinetic studies with drugs that bind extensively to albumin.

Studies were performed to elucidate the mechanism responsible for the reduction in Km values of UDP-glucuronosyltransferase 2B7 (UGT2B7) substrates observed for incubations conducted in the presence of albumin. Addition of bovine serum albumin (BSA) and fatty acid-free human serum albumin (HSA-FAF), but not "crude" HSA, resulted in an approximate 90% reduction in the Km values for the glucuronidation of zidovudine (AZT) by human liver microsomes (HLM) and UGT2B7 and a 50 to 75% reduction in the S50 for 4-methylumbelliferone (4MU) glucuronidation by UGT2B7, without affecting Vmax. Oleic, linoleic, and arachidonic acids were shown to be the most abundant unsaturated long-chain fatty acids present in crude HSA and in the membranes of HLM and human embryonic kidney (HEK)293 cells, and it was demonstrated that these and other unsaturated long-chain fatty acids were UGT2B7 substrates. Glucuronides with Rf (retention factor) values corresponding to the glucuronides of linoleic and arachidonic acid were detected when HLM and HEK293 cell lysates were incubated with radiolabeled cofactor, and the intensity of the bands was modulated by the presence of crude HSA (increased) and BSA or HSA-FAF (decreased). Oleic, linoleic, and arachidonic acid inhibited AZT and 4MU glucuronidation by HLM and/or UGT2B7, due to an increase in Km/S50 without a change in Vmax. Addition of BSA and HSA-FAF reversed the inhibition. Likewise, coexpression of UGT2B7 and HSA in HEK293 cells reduced the Km/S50 values of these substrates. It is postulated that BSA and HSA-FAF sequester inhibitory fatty acids released during incubations, and the apparent high Km values observed for UGT2B7 substrates arise from the presence of these endogenous inhibitors.

To determine the scaling factors required for inclusion of renal drug glucuronidation clearance in the prediction of total clearance via glucuronidation (CLUGT ).Microsomal protein per gram of kidney (MPPGK) was determined for human 'mixed' kidney (n = 5) microsomes (MKM). The glucuronidation activities of deferiprone (DEF), propofol (PRO) and zidovudine (AZT) by MKM and paired cortical (KCM) and medullary (KMM) microsomes were measured, along with the UGT 1A6, 1A9 and 2B7 protein contents of each enzyme source. Unbound intrinsic clearances (CLint,u,UGT ) for PRO and morphine (MOR; 3- and 6-) glucuronidation by MKM, human liver microsomes (HLM) and recombinant UGT1A9 and 2B7 were additionally determined. Data were scaled using in vitro-in vivo extrapolation (IV-IVE) approaches to assess the influence of renal CLint,u,UGT on the prediction accuracy of the calculated CLUGT values of PRO and MOR.MPPGK was 9.3 ± 2.0 mg g(-1) (mean ± SD). The respective rates of DEF (UGT1A6), PRO (UGT1A9) and AZT (UGT2B7) glucuronidation by KCM were 1.4-, 5.2- and 10.5-fold higher than those for KMM. UGT 1A6, 1A9 and 2B7 were the only enzymes expressed in kidney. Consistent with the activity data, the abundance of each of these enzymes was greater in KCM than in KMM. The abundance of UGT1A9 in MKM (61.3 pmol mg(-1) ) was 2.7 fold higher than that reported for HLM.Scaled renal PRO glucuronidation CLint,u,UGT was double that of liver. Renal CLint,u,UGT should be accounted for in the IV-IVE of UGT1A9 and considered for UGT1A6 and 2B7 substrates.

Drugs and other chemicals frequently bind nonspecifically to the constituents of an in vitro incubation mixture, particularly the enzyme source [e.g., human liver microsomes (HLM)]. Correction for nonspecific binding (NSB) is essential for the accurate calculation of the kinetic parameters Km, Clint, and Ki. Many tyrosine kinase inhibitors (TKIs) are lipophilic organic bases that are nonionized at physiologic pH. Attempts to measure the NSB of several TKIs to HLM by equilibrium dialysis proved unsuccessful, presumably due to the limited aqueous solubility of these compounds. Thus, the addition of detergents to equilibrium dialysis samples was investigated as an approach to measure the NSB of TKIs. The binding of six validation set nonionized lipophilic bases (felodipine, isradipine, loratidine, midazolam, nifedipine, and pazopanib) to HLM (0.25 mg/ml) was shown to be unaffected by the addition of CHAPS (6 mM) to the dialysis medium. This approach was subsequently applied to measurement of the binding of axitinib, dabrafenib, erlotinib, gefitinib, ibrutinib, lapatinib, nilotinib, nintedanib, regorafenib, sorafenib, and trametinib to HLM (0.25 mg/ml). As with the validation set drugs, attainment of equilibrium was demonstrated in HLM-HLM and buffer-buffer control dialysis experiments. Values of the fraction unbound to HLM ranged from 0.14 (regorafenib and sorafenib) to 0.93 (nintedanib), and were generally consistent with the known physicochemical determinants of drug NSB. The extensive NSB of many TKIs to HLM underscores the importance of correction for TKI binding to HLM and, presumably, other enzyme sources present in in vitro incubation mixtures.

Enzymes of the UDP-glucuronosyltransferase (UGT) superfamily are responsible for the metabolism of many drugs, environmental chemicals and endogenous compounds. Identification of the UGT(s) involved in the metabolism of a given compound ('reaction phenotyping') currently relies on multiple confirmatory approaches, which may be confounded by the dependence of UGT activity on enzyme source, incubation conditions, and the occurrence of atypical glucuronidation kinetics. However, the increasing availability of substrate and inhibitor 'probes' for the individual UGTs provides the prospect for reliable phenotyping of glucuronidation reactions using human liver microsomes or hepatocytes, thereby providing data directly relevant to drug metabolism in humans. While the feasibility of computational prediction of UGT substrate selectivity has been demonstrated, the development of easily interpretable and generalisable models requires further improvement in the datasets available for analysis. Quantitative prediction of the hepatic clearance of glucuronidated drugs and the magnitude of inhibitory interactions based on in vitro kinetic data is more problematic. Intrinsic clearance (CL(int)) values generated using human liver microsomes under-predict in vivo hepatic clearance, typically by an order of magnitude. In vivo clearances of glucuronidated drugs are also generally under-predicted by CL(int) values from human hepatocytes, but to a lesser extent than observed with the microsomal model. While it is anticipated that systematic analysis of the potential causes of under-prediction may provide more reliable in vitro-in vivo scaling strategies, mechanistic interpretation of in vitro-in vivo correlation more broadly awaits further advances in our understanding of the structural and cellular determinants of UGT activity.

Non-selective nonsteroidal anti-inflammatory drugs (NSAIDs) have long been known to cause gastrointestinal and renal toxicity. More recently, adverse cardiovascular effects have been associated with the selective COX-2 inhibitors. However, current studies that show an increased cardiovascular risk with non-selective NSAIDs raise the question of the exclusive contribution of COX-2 to this type of toxicity. Aldosterone, a key cardiovascular hormone, can induce deleterious effects, such as myocardial fibrosis and vascular stiffening. Non-selective NSAIDs inhibit the metabolism of aldosterone in vitro by human renal tissue, predicating an increased plasma aldosterone concentration in vivo. The question remains whether inhibition of aldosterone metabolism by non-selective NSAIDs is a casual or causal factor in NSAID-induced cardiovascular toxicity.

Adverse cardiovascular (CV) effects of non-steroidal anti-inflammatory drugs (NSAIDs) are largely independent of their cyclooxygenase (COX) enzyme selectivity, but could be a consequence of aldosterone 18ß-glucuronidation inhibition (AGI), which varies between NSAIDS. This study assesses the chronic effects of celecoxib (selective COX-2 inhibitor) versus diclofenac (non-selective NSAID) therapy on arterial dysfunction in patients with rheumatoid arthritis (RA).AGI was assessed in vitro using human kidney cortical microsomes. Arterial function was measured clinically as the extent (augmentation index, AIX%) and timing (reflected wave transit time, RWTT, msec) of arterial wave reflection using radial applanation pulse wave analysis (SphygmoCor PWA device) in 39 RA patients without overt CV disease aged 40-65. A higher AIX% (and lower RWTT) indicates arterial dysfunction. Clinical assessment on a single occasion included a fasting blood sample, patient questionnaire and medical record review. Multivariable analysis was used to adjust for sex, mean blood pressure, arthritis duration, cumulative ESR-years and current DMARD therapy.The inhibition constant (Ki) for celecoxib was lower than that of diclofenac (Ki, 3.5 vs. 8.4 μM). Chronic celecoxib use was associated with a higher AIX% (34.8 vs. 32.3) and lower RWTT (130.1 vs. 132.7 msec) compared with diclofenac. Adjusted mean differences were AIX% 4.7 (95%CI 0.6 to 8.9; p=0.03) and RWTT -3.6 (95%CI -10.0 to 2.7; p=0.26).Celecoxib has a greater potency for AGI than diclofenac and its use is associated with a significantly higher AIX%. Our findings support AGI as a plausible mechanism for the CV toxicity of NSAIDs.

Although knowledge of human renal cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes and their role in xenobiotic and endobiotic metabolism is limited compared with hepatic drug and chemical metabolism, accumulating evidence indicates that human kidney has significant metabolic capacity. Of the drug metabolizing P450s in families 1 to 3, there is definitive evidence for only CYP 2B6 and 3A5 expression in human kidney. CYP 1A1, 1A2, 1B1, 2A6, 2C19, 2D6 and 2E1 are not expressed in human kidney, while data for CYP 2C8, 2C9 and 3A4 expression are equivocal. It is further known that several P450 enzymes involved in the metabolism of arachidonic acid and eicosanoids are expressed in human kidney, CYP 4A11, 4F2, 4F8, 4F11 and 4F12. With the current limited evidence of drug substrates for human renal P450s drug–endobiotic interactions arising from inhibition of renal P450s, particularly effects on arachidonic acid metabolism, appear unlikely. With respect to the UGTs, 1A5, 1A6, 1A7, 1A9, 2B4, 2B7 and 2B17 are expressed in human kidney, whereas UGT 1A1, 1A3, 1A4, 1A8, 1A10, 2B10, 2B11 and 2B15 are not. The most abundantly expressed renal UGTs are 1A9 and 2B7, which play a significant role in the glucuronidation of drugs, arachidonic acid, prostaglandins, leukotrienes and P450 derived arachidonic acid metabolites. Modulation by drug substrates (e.g. NSAIDs) of the intrarenal activity of UGT1A9 and UGT2B7 has the potential to perturb the metabolism of renal mediators including aldosterone, prostaglandins and 20-hydroxyeicosatetraenoic acid, thus disrupting renal homeostasis.

1. Activity of rat hepatic microsomal and peroxisomal long-chain (palmitoyl) and nafenopin-CoA ligases were studied following administration of either clofibric acid, di-(2-ethylhexyl)phthalate (DEHP) or phenobarbitone. 2. Clofibric acid significantly induced the peroxisomal palmitoyl and nafenopin-CoA ligases, whilst no induction of the equivalent enzymes was observed in the microsomal fraction. 3. DEHP induced only palmitoyl-CoA formation in peroxisomes, whilst all enzymes were refractory to phenobarbitone treatment. 4. The enzyme-specific patterns of inductions both intra- and inter-organelle suggest that the palmitoyl and nafenopin-CoA ligases are under different regulatory control. 5. Modulation of both the rate and extent of nafenopin- and palmitoyl-CoA formation was both agent and organelle specific. 6. This study highlights the difficulty in delineating the individual roles of both fatty acyl-CoAs and xenobiotic-CoAs in peroxisome proliferation.

Amino acid conjugation is a novel biotransformation pathway in man for a limited number of xenobiotic carboxylic acids. It involves two mitochondrial enzymatic processes: formation of a reactive xenobiotic-CoA thioester intermediate, catalyzed by an ATP-dependent acid:CoA synthetase (ACSM), and linkage of the activated acyl group via an acyl-CoA:amino acid N-acyltransferase (GLYAT) to the amino group of the acceptor amino acid. However, knowledge of amino acid conjugation in terms of enzyme multiplicity, protein structure, and xenobiotic substrate selectivity is not well advanced. Keywords: amino acid conjugation; medium chain acyl-CoA synthetase; acyl-CoA:amino acid N-acyltransferase; salicylic acid; benzoic acid; hippuric acid; salicylurate; glycine; xenobiotic-CoA thioesters

The hypothesis that the anti-inflammatory activity of NSAIDs derives from COX inhibition is well established. It also underpins the accepted mechanism of the gastrointestinal and renal toxicity of NSAIDs. However, in terms of NSAID-induced cardiovascular toxicity, is COX inhibition then guilty by association? Multiple experimental models of COX-1/COX-2 inhibition have enabled ranking of the relative inhibitory activity of NSAIDs. Inhibition is expressed as an IC(50) value and the index of COX selectivity as the ratio of the IC(50) value for COX-2 and COX-1. These data informed the 'imbalance hypothesis' that the cardiovascular risk of NSAIDs results from an imbalance in the detrimental actions of COX-1-derived thromboxane A(2) and the beneficial actions of COX-2-derived prostacyclin (PGI(2)). Data derived from in vitro models used to generate NSAID IC(50) values are discussed in the context of the difficulties in defining COX selectivity and hence understanding the toxicity of NSAIDs in current clinical use.

Recent professional statements from established bodies, i.e. American Heart Association, European Medicines Agency, and Medicines and Healthcare products Regulatory Agency, have cautioned on the use of non-steroidal anti-inflammatory drugs (NSAIDs) given the potential increase in atherothrombotic risk associated with their long-term use. However, pharmacoepidemiological studies on the association between NSAID use and the risk of stroke, one of the leading causes of death, disability, and institutionalisation in old age, have shown contrasting results. Notably, very few such studies have addressed the risk in the older population, in particular patients75-80 years, perhaps the biggest consumer group of these drugs. This article reviews the current evidence on the association between NSAIDs and risk of stroke in the older population. It also discusses the potential clinical, demographic, and pathophysiological factors potentially accounting for the discrepancies in the results of the pharmacoepidemiological studies and provides suggestions for future research directions.

Objectives This study was designed to investigate the renal disposition of 4-methylumbelliferone (4MU) and 4-methylumbelliferyl glucuronide (4MUG) to characterise the contribution of excretion and metabolic clearance to total clearance in the kidney. Methods The isolated perfused kidney (IPK) from the male Sprague–Dawley rat was used in filtering and non-filtering mode to study the renal disposition of 4MU, renally generated 4MUG and preformed 4MUG. Perfusate and urine (filtering IPK only) was collected for up to 120 min and 4MU and 4MUG in perfusate and urine were determined by HPLC. Analytes were also measured in kidney tissue collected at 120 min. Non-compartmental analysis was used to derive pharmacokinetic parameters. Key findings The concentration of 4MU in perfusate declined with a terminal half-life of approximately 120 min following administration to the filtering IPK and nonfiltering IPK. There was a corresponding increase in the concentration of 4MUG. Metabolic clearance of 4MU accounted for 92% of total renal clearance. After bolus dosing of preformed 4MUG in the perfusion reservoir of the filtering IPK, the perfusate concentration declined with the terminal half-life of approximately 260 min. The renal excretory clearance of preformed 4MUG accounted for 96% of total renal clearance. 4MU was extensively metabolized by glucuronidation in the filtering and nonfiltering IPK, and the total renal clearance of 4MU was far greater than its renal excretory clearance. This indicated that glucuronidation was the major elimination pathway for 4MU in the kidney. Conclusions The data confirmed an important role for the kidney in the metabolic clearance of xenobiotics via glucuronidation and signalled the lack of impact of impaired glomerular filtration on renal drug metabolism.

We studied the association between either non-selective NSAIDs (ns-NSAIDs), selective COX-2 inhibitors, or any NSAID and risk of incident myocardial infarction (MI) and heart failure (HF), and all-cause mortality in elderly subjects.We conducted a retrospective nested case-control study on Australian veterans using nationwide hospital admission and pharmacy dispensing data. We estimated adjusted odds ratios (OR) with 95% confidence intervals (CI) for the risk of events for three different measures of prescription supply exposure over the last 2 years: (i) supplied at least once, (ii) supply frequency: supplied more than twice within the last 30 days, once or twice within the last 30 days, and once or more 30 days to 2 years and (iii) total supplies.We identified 83 623 cases and 1 662 099 matched controls (1:20) contributing 3 862 931 persons-years of observation. NSAID use at least once within the last 2 years did not significantly affect the risk of MI (OR 1.00, 95% CI 0.96, 1.04) but was associated with a mildly reduced risk of HF (OR 0.95, 95% CI 0.92, 0.98). There was a reduced all-cause mortality with at least one supply of either ns-NSAIDs (OR 0.94, 95% CI 0.90, 0.97), selective COX-2 inhibitors (OR 0.90, 95% CI 0.88, 0.93), or any NSAID (OR 0.87, 95% CI 0.85, 0.90). Risk of death was also inversely associated with the number of prescription supplies.NSAID use is not associated with an increased risk of incident MI and HF but is associated with a reduction in all-cause mortality in Australian veterans.

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.

Purpose Studies on the risk of stroke in users of non-steroidal anti-inflammatory drugs (NSAIDs) have provided conflicting results. We studied the association between the use of non-selective ns-NSAIDs, selective COX-2 inhibitors, or either of these NSAIDs, and the incidence of stroke-related hospitalization in elderly subjects. Methods We conducted a retrospective nested case-control study on Australian veterans using nationwide hospital admission and pharmacy dispensing data. Conditional logistic regression analysis was used to estimate both crude and adjusted odds ratios (OR) and 95% confidence intervals (CI) for the risk of events for three different measures of prescription supply exposure over the last 2 years; (1) whether supplied at least once; (2) supply frequency: supplied more than twice within the last 30 days, once or twice within the last 30 days, or once or more within 30 days to 2 years; and (3) total supplies. Results There was a trend toward a reduced risk of ischemic stroke with any NSAID (OR 0.95, 95%CI 0.89–1.00) if supplied at least once within the last 2 years and a mildly reduced risk in those supplied any NSAID once or twice within the last 30 days (OR 0.89, 95%CI 0.81–0.98). Use of either ns-NSAIDs or selective COX-2 inhibitors were not associated with a significant change in risk. Conclusions The use of any NSAIDs is not associated with an increase in the risk of ischemic stroke in Australian veterans. Copyright © 2010 John Wiley & Sons, Ltd.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT • Current evidence suggests the use of non-steroidal anti-inflammatory drugs (NSAIDs) increases the risk of myocardial infarction and heart failure. However, some studies have failed to demonstrate a significant relationship. • Differences in patient demographics, study design, and the varying types and classes of NSAIDs studied might either confound or modify the association between NSAID use and risk of myocardial infarction and heart failure, and prevent accurate data interpretation. WHAT THIS STUDY ADDS • NSAID use is not associated with an increased risk of either incident myocardial infarction or heart failure in elderly patients. • NSAID use is associated with a reduction in all-cause mortality in this cohort. AIMS We studied the association between either non-selective NSAIDs (ns-NSAIDs), selective COX-2 inhibitors, or any NSAID and risk of incident myocardial infarction (MI) and heart failure (HF), and all-cause mortality in elderly subjects. METHODS We conducted a retrospective nested case-control study on Australian veterans using nationwide hospital admission and pharmacy dispensing data. We estimated adjusted odds ratios (OR) with 95% confidence intervals (CI) for the risk of events for three different measures of prescription supply exposure over the last 2 years: (i) supplied at least once, (ii) supply frequency: supplied more than twice within the last 30 days, once or twice within the last 30 days, and once or more 30 days to 2 years and (iii) total supplies. RESULTS We identified 83 623 cases and 1 662 099 matched controls (1:20) contributing 3 862 931 persons-years of observation. NSAID use at least once within the last 2 years did not significantly affect the risk of MI (OR 1.00, 95% CI 0.96, 1.04) but was associated with a mildly reduced risk of HF (OR 0.95, 95% CI 0.92, 0.98). There was a reduced all-cause mortality with at least one supply of either ns-NSAIDs (OR 0.94, 95% CI 0.90, 0.97), selective COX-2 inhibitors (OR 0.90, 95% CI 0.88, 0.93), or any NSAID (OR 0.87, 95% CI 0.85, 0.90). Risk of death was also inversely associated with the number of prescription supplies. CONCLUSIONS NSAID use is not associated with an increased risk of incident MI and HF but is associated with a reduction in all-cause mortality in Australian veterans.

Amino acid conjugation is a route of metabolism for various xenobiotic carboxylic acids and bile acids; however, knowledge of the enzymology (e.g., multiplicity of enzymes, substrate selectivity, catalytic mechanism, and protein structure), polymorphisms, and gene regulation is not as advanced as the cytochromes P450 and uridine 5′-diphosphate (UDP)-glucuronosyltransferases. Mechanistically, amino acid conjugation is a coupled enzyme system involving a mitochondrial adenosine triphosphate (ATP)-dependent acid medium-chain acyl-CoA synthetase (ACSM), which catalyzes the formation of an acyl-CoA thioester, and an acyl-CoA:glycine N -acyltransferase (GLYAT) that links the acyl group to glycine. Conjugation of bile acids is extramitochondrial involving initially enzymes located in the endoplasmic reticulum, while conjugation of bile acid-CoAs involves a peroxisomal bile acid-CoA:amino acid N -acyltransferase (BAAT). Within mitochondria, amino acid conjugation terminates the biological reactivity of xenobiotic acyl-CoAs, whereas conjugation of bile acids increases aqueous solubility and promotes bile acid flow and intestinal absorption. For both pathways, the amino acid utilized is species dependent. In mammals, excretion is predominantly as either glycine or taurine conjugates. Amino acid conjugation is important in both health and disease. Manipulation of mitochondrial amino acid conjugation is used in the treatment of nonketotic hyperglycinemia and hyperammonemia, and failure to conjugate bile acids can lead to cholestasis and loss of absorption of fat and fat-soluble vitamins. This chapter reviews the enzymology, substrate and inhibitor profile, and factors that regulate the activity of ACSM, GLYAT, and BAAT.

ATP-dependent coenzyme A (CoA) ligases catalyse the formation of the acyl-CoA thioesters of xenobiotic carboxylic acids and the formation of xenobiotic-CoAs has been implicated as being a causative factor in peroxisomal proliferation. In this study we have demonstrated using rat liver peroxisomes that the formation of palmitoyl-CoA is inhibited by a variety of xenobiotic carboxylic acids. Palmitoyl-CoA formation exhibited biphasic kinetics indicative of two isoforms, a high affinity (Km1 2.3 microM) low capacity form and a low affinity (Km2 831 microM) high capacity form. These forms were differentially inhibited by a range of xenobiotics. However, it would appear that the low affinity component may not contribute to any major extent to the formation of xenobiotic-CoAs in vivo. At a concentration of 1 mM, greater than 20% inhibition of the high affinity form was observed with the 2-arylpropionates, ibuprofen, naproxen, benoxaprofen, fenoprofen, indoprofen, ketoprofen, tiaprofenic acid and cicloprofen, the hypolipidaemics, nafenopin and ciprofibrate, and the herbicides, silvex and 2,4,5-trichlorophenoxyacetate. Valproic acid, clofibric acid, salicylic acid and 2,4-dichlorophenoxy-acetate were non-inhibitory at all concentrations studied (0.1-2.5 mM). Analysis of the type of inhibition established that only nafenopin (Ki 430 microM) and ciprofibrate (Ki 97 microM) were competitive inhibitors of palmitoyl-CoA formation suggesting that they bind at the active site and thus potentially function as alternative substrates for the peroxisomal ligase. Notably, clofibric acid which has previously been shown to form clofibroyl-CoA in peroxisomes did not interact with the palmitoyl-CoA ligase thereby suggesting that activation is mediated via an alternative peroxisomal CoA ligase. In addition, the xenobiotic inhibitors of the peroxisomal palmitoyl-CoA ligase differed from those previously reported for the equivalent microsomal enzyme suggesting that the organellar forms may be functionally distinct. This study establishes that numerous xenobiotic carboxylic acids interact with the peroxisomal palmitoyl-CoA ligase; however, it would appear that relatively few function as alternative substrates. The toxicological ramifications of peroxisomally mediated xenobiotic-CoA formation and the identification of other peroxisomal xenobiotic-CoA ligase(s) remain to be elucidated.

Inhibition of UDP-glucuronosyltransferase (UGT) activity gives rise to both drug–drug (DDIs) and drug–endobiotic interactions in vivo. Furthermore, several glucuronides have been shown to reduce the metabolic clearances of cytochrome P4502C8 substrates. Experimental paradigms, based on the use of human liver microsomes (HLM), hepatocytes, and recombinant UGTs as the enzyme sources, are now available for the investigation of drug glucuronidation in vitro including the prediction and characterization of DDIs. The reaction phenotyping of drug glucuronidation is becoming increasingly feasible with the availability of ‘batteries’ of recombinant enzymes along with substrate and inhibitor probes for the major hepatic drug metabolizing UGTs. However, the occurrence of homo- and heterotropic activation potentially complicates screening for DDIs in vitro. Recent advances in knowledge of factors that influence UGT activity in vitro have also led to the development of experimental approaches that accurately predict the magnitude of known DDIs involving glucuronidated drugs, but further work in this area is required to demonstrate the generalizability of these models.

The role of the kidney in drug and chemical disposition has traditionally focused on the excretion of polar xenobiotics and metabolites. However, there is increasing evidence demonstrating that renal UGTs are integral to the “local” intrarenal, and, possibly, systemic, metabolic clearance of numerous drugs and nondrug xenobiotics, as well as to the maintenance of renal homeostasis through limiting the biological activity of endogenous renal mediators that control electrolyte balance and renal blood flow. The common involvement of UGT1A9 and UGT2B7 in the metabolism of both endogenous and exogenous compounds in kidney predicates significant renal drug-endobiotic interactions that may explain, in part, the adverse renal effects of some drugs.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT • Carboxylic acid NSAIDs are extensively glucuronidated as either the parent drug or hydroxylated metabolites and UGT2B7 is ranked highest in terms of NSAID-glucuronidation activity. • NSAIDs cause adverse renal effects including sodium and water retention and hyperkalaemia. • In human kidney the mineralocorticoid aldosterone is glucuronidated directly to form aldosterone 18β-glucuronide. WHAT THIS STUDY ADDS • Human liver and kidney microsomes and UGT1A10 and UGT2B7 catalyze aldosterone18β-glucuronidation. • Non-selective NSAIDs inhibit renal and hepatic aldosterone18β-glucuronidation and in vivo this may lead to elevated intra-renal concentrations of this hormone. • Common involvement of UGT2B7 in NSAID and aldosterone glucuronidation predicates an intra-renal NSAID-aldosterone interaction that may explain in part the clinical observations of variable effects of NSAIDs on electrolytes, fluid retention and blood pressure. AIMS To characterize: i) the kinetics of aldosterone (ALDO) 18β-glucuronidation using human liver and human kidney microsomes and identify the human UGT enzyme(s) responsible for ALDO 18β-glucuronidation and ii) the inhibition of ALDO 18β-glucuronidation by non-selective NSAIDs. METHODS Using HPLC and LC-MS methods, ALDO 18β-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 18β-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 (IC50) was established and the mechanism of inhibition investigated using diclofenac, S-ibuprofen, indomethacin, mefenamic acid and S-naproxen. RESULTS ALDO 18β-glucuronidation by hepatic and renal microsomes exhibited Michaelis-Menten kinetics. Mean (±SD) Km, Vmax and CLint values for HLM and HKCM were 509 ± 137 and 367 ± 170 µm, 1075 ± 429 and 1110 ± 522 pmol min−1 mg−1, and 2.36 ± 1.12 and 3.91 ± 2.35 µl min−1 mg−1, respectively. Of the UGT proteins, only UGT1A10 and UGT2B7 converted ALDO to its 18β-glucuronide. All NSAIDs investigated inhibited ALDO 18β-G formation by HLM, HKCM and UGT2B7. The rank order of inhibition (IC50) of renal and hepatic ALDO 18β-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.

In order to gain insights into the renal and hepatic glucuronidation of frusemide (FSM), this study: (i) characterised the kinetics of FSM glucuronidation by human liver microsomes (HLM) and human kidney cortical- (HKCM) and medullary- (HKMM) microsomes, and (ii) identified the human UDP-glucuronosyltransferase enzyme(s) involved in this pathway. HLM, HKCM and HLMM efficiently glucuronidated FSM. FSM glucuronide (FSMG) formation followed Michaelis-Menten kinetics in all tissues. While the mean K(m) for FSMG formation by HKMM (386 +/- 68 microM) was lower than the K(m) values for HLM (988 +/- 271 microM) and HKCM (704 +/- 278 microM), mean V(max)/K(m) values were comparable for the three tissues. A panel of recombinant UGT enzymes was screened for the capacity to glucuronidate FSM. UGT 1A1, 1A3, 1A6, 1A7, 1A9, 1A10 and 2B7 metabolised FSM. Of the renally and hepatically expressed enzymes, comparison of kinetic parameters suggests a predominant role of UGT1A9 in FSM glucuronidation, although UGT1A1 may also contribute to FSMG formation by HLM. Consistent with these observations, the UGT1A selective inhibitors phenylbutazone and sulfinpyrazone decreased FSMG formation by HLM, HKCM and HKMM by 60-80%, whereas the UGT2B7 selective inhibitor fluconazole reduced FSM glucuronidation byor =20%. The ability of HKCM and HKMM to form FSMG supports the proposition that the kidney is the main organ involved in FSM glucuronidation in vivo, although a role for hepatic metabolism remains a possibility in renal dysfunction. The data further demonstrate the potential importance of both the medulla and cortex in renal drug metabolism and detoxification.

This study characterized the mechanism by which bovine serum albumin (BSA) reduces the K(m) for phenytoin (PHY) hydroxylation and the implications of the "albumin effect" for in vitro-in vivo extrapolation of kinetic data for CYP2C9 substrates. BSA and essentially fatty acid-free human serum albumin (HSA-FAF) reduced the K(m) values for PHY hydroxylation (based on unbound substrate concentration) by human liver microsomes (HLMs) and recombinant CYP2C9 by approximately 75%, with only a minor effect on V(max). In contrast, crude human serum albumin increased the K(m) with both enzyme sources. Mass spectrometric analysis of incubations containing HLMs was consistent with the hypothesis that BSA sequesters long-chain unsaturated acids (arachidonic, linoleic, oleic) released from membranes. A mixture of arachidonic, linoleic and oleic acids, at a concentration corresponding to 1/20 of the content of HLMs, doubled the K(m) for PHY hydroxylation by CYP2C9, without affecting V(max). This effect was reversed by addition of BSA to incubations. K(i) values for arachidonic acid inhibition of human liver microsomal- and CYP2C9-catalyzed PHY hydroxylation were 3.8 and 1.6 microM, respectively. Similar effects were observed with heptadecanoic acid, the most abundant long-chain unsaturated acid present in Escherichia coli membranes. Extrapolation of intrinsic clearance (CL(int)) values for each enzyme source determined in the presence of BSA and HSA-FAF accurately predicted the known CL(int) for PHY hydroxylation in vivo. The results indicate that previously determined in vitro K(m) values for CYP2C9 substrates are almost certainly overestimates, and accurate in vitro-in vivo extrapolation of kinetic data for CYP2C9 substrates is achievable.

There is currently little information regarding the localization of UDP-glucuronosyltransferases (UGTs) in human renal cortex and medulla, and the functional contribution of renal UGTs to drug glucuronidation remains poorly defined. Using human kidney sections and human kidney cortical microsomes (HKCM) and human kidney medullary microsomes (HKMM), we combined immunohistochemistry to investigate UGT1A and UGT2B7 expression with in vitro microsomal studies to determine the kinetics of S-naproxen acyl glucuronidation. With the exception of the glomerulus, Bowman's capsule, and renal vasculature, UGT1A proteins and UGT2B7 were expressed throughout the proximal and distal convoluted tubules, the loops of Henle, and the collecting ducts. Additionally, UGT1A and UGT2B7 expression was demonstrated in the macula densa, supporting a potential role of UGTs in regulating aldosterone. Consistent with the immunohistochemical data, S-naproxen acyl glucuronidation was catalyzed by HKCM and HKMM. Kinetic data were well described by the two-enzyme Michaelis-Menten equation. K(m) values for the high-affinity components were 34 +/- 14 microM (HKCM) and 45 +/- 14 microM (HKMM). Fluconazole inhibited the high-affinity component establishing UGT2B7 as the enzyme responsible for S-naproxen glucuronidation in cortex and medulla. The low-affinity component was relatively unaffected by fluconazole (

We noted with interest the comments of Dr Sutton [1] that ‘a Google search on aldosterone, stress, mood produced ‘52 000 English or French pages’ in 0.33 s' and that our paper [2] ‘made no mention of stress as a possible cause of increases in aldosterone during NSAID treatment’. Our research [2] was conducted in vitro using human kidney (and liver) microsomes and demonstrated that nonselective NSAIDs inhibit the glucuronidation of aldosterone, predicating an increase in plasma and tissue (renal) aldosterone concentration in vivo. To suggest that stress is an explantion for our in vitro data indicates that Dr Sutton either did not read or understand the article and its conclusions. Although aldosterone synthesis and secretion is regulated principally by angiotensin II, extracellular potassium and ACTH we concur with Dr Sutton that other factors can stimulate or inhibit aldosterone production in vivo including catecholamines, 5-hydroxytryptamine, heparin and vasoactive intestinal polypeptide [3]. Undoubtedly aldosterone is a key cardiovascular hormone contributing to the pathophysiology of heart failure [3] and the development of hypertension [4]. The potential for a synergistic effect of NSAIDs and ‘stress’ on aldosterone concentration in vivo might lead us to conclude that in stressful situations the adage ‘take an aspirin and lie down’ should be revised simply to ‘lie down’!

The exact mechanism(s) of NSAID-induced nephrotoxicity remains unclear, but most theories centre on the initial inhibition of COX and the subsequent perturbation of the numerous actions of COX in the kidney. Since the nineteenth century no NSAIDs have been developed that are devoid of renal adverse effects, including the COX-2 selective inhibitors. Formation of renal eicosanoids from arachidonic acid is significantly increased in the presence of various stimuli, and metabolic degradation of arachidonic acid and its biologically active metabolites is crucial to the maintenance of renal homeostatic mechanisms. An important family of enzymes that function in this capacity are the uridine 5'-diphosphate glucuronosyltransferases (UGTs) that variously metabolise arachidonic acid and its metabolites. This review focuses on arachidonic acid and its biologically active metabolites and their respective fates subsequent to COX inhibition by NSAIDs. The common involvement of UGT in the metabolism of arachidonic acid, eicosanoids and NSAIDs is discussed in the context of novel mechanisms of NSAID-induced nephrotoxicity.

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 Km values (±SD, with 95% confidence interval in parentheses) for the high- and low-affinity components were 29 ± 13 µm (16, 43) and 473 ± 108 µm (359, 587), respectively. UGT 1A1, 1A3, 1A6, 1A7, 1A8, 1A9, 1A10 and 2B7 glucuronidated naproxen. UGT2B7 exhibited an apparent Km (72 µm) 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.

This review focuses primarily on the mammalian medium and long-chain fatty acid coenzyme A ligases that have been implicated in the metabolism of xenobiotic carboxylic acids such as pesticides, arylpropionate non steroidal anti-inflammatory drugs and the hypolipidaemic clofibrate and its congeners. Evidence of multiplicity of mitochondrial and microsomal enzymes and their respective substrate/inhibitor profiles are discussed. For completeness, where appropriate, details of non-substrate inhibitors have also been included. Although knowledge is limited at present with respect to the medium-chain enzymes, aspects of regulation particularly the in vivo, in vitro role of peroxisome proliferators and current knowledge of the molecular biology of the long-chain fatty acid CoA ligase superfamily are documented. Additionally, alignment of thirteen cloned mammalian fatty acid CoA ligases using criteria established for the CYP and UGT superfamilies has enabled construction of a phylogenetic tree that clearly defines three families. Catalytic data are still limited and the xenobiotic substrate/inhibitor profiles of the recombinant proteins are incomplete. Finally, with increasing recognition of the importance of fatty acyl-CoA esters as physiological regulators of cell function including gene expression, the review concludes with a discussion of the metabolic fate and toxicity of xenobiotic acyl-CoA esters.

Endogenous fatty acyl-CoAs play an important role in the acylation of proteins. A number of xenobiotic carboxylic acids are able to mimic fatty acids, forming CoA conjugates and acting as substrates in pathways of lipid metabolism. In this study nafenopin, a substrate for human hepatic fatty acid-CoA ligases, was chosen as a model compound to study xenobiotic acylation of human liver proteins. (3)H-nafenopin (+/- unlabeled palmitate) or (14)C-palmitate (+/- unlabeled nafenopin) were incubated for up to 120 min at 37 degrees C with ATP, CoA, and homogenate protein (1 mg/ml) from four individual human livers. Nafenopin covalently bound to proteins was detectable in all human livers and increased with time. Nafenopin adduct formation was directly proportional to nafenopin-CoA formation (r = 0.985, p0.05). Attachment of nafenopin to proteins involved both thioester and amide linkages with 76 and 24% of adducts formed with proteins100 and 50-100 kDa, respectively. Protein acylation by palmitate was also demonstrated. Palmitate significantly inhibited nafenopin-CoA formation by 29% but had no effect on nafenopin-CoA-mediated protein acylation. In contrast, nafenopin significantly inhibited protein palmitoylation by palmitoyl-CoA. This is the first study to demonstrate a direct relationship between xenobiotic-CoA formation, acylation of human liver proteins, and inhibition of endogenous palmitoylation. The ability of xenobiotics to acylate tissue proteins may have important biological consequences including perturbation of endogenous regulation of protein localization and function.

SUMMARY 1. Formation of acyl-coenzymes (Co)A occurs as an obligatory step in the metabolism of a variety of endogenous substrates, including fatty acids. The reaction is catalysed by ATP-dependent acid:CoA ligases (EC 6.2.1.1-2.1.3; AMP forming), classified on the basis of their ability to conjugate saturated fatty acids of differing chain lengths, short (C2-C4), medium (C4-C12) and long (C10-C22). The enzymes are located in various cell compartments (cytosol, smooth endoplasmic reticulum, mitochondria and peroxisomes) and exhibit wide tissue distribution, with highest activity associated with liver and adipose tissue. 2. Formation of acyl-CoA is not unique to endogenous substrates, but also occurs as an obligatory step in the metabolism of some xenobiotic carboxylic acids. The mitochondrial medium-chain CoA ligase is principally associated with metabolism via amino acid conjugation and activates substrates such as benzoic and salicylic acids. Although amino acid conjugation was previously considered an a priori route of metabolism for xenobiotic-CoA, it is now recognized that these highly reactive and potentially toxic intermediates function as alternative substrates in pathways of intermediary metabolism, particularly those associated with lipid biosyntheses. 3. In addition to a role in fatty acid metabolism, the hepatic microsomal and peroxisomal long-chain-CoA-ligases have been implicated in the formation of the acyl-CoA thioesters of a variety of hypolipidaemic and peroxisome proliferating agents (e.g. cloflbric acid) and of the r(-)-enantiomers of the commonly used 2-arylpropionic acid non-steroidal anti-inflammatory drugs (e.g. ibuprofen). In vitro kinetic studies using rat hepatic microsomes and peroxisomes have alluded to the possibility of xenobiotic-CoA ligase multiplicity. Although cDNA encoding a long-chain ligase have been isolated from rat and human liver, there is currently no molecular evidence of multiple isoforms. The gene has been localized to chromosome 4 and homology searches have revealed a significant similarity with enzymes of the luciferase family. 4. Increasing recognition that formation of a CoA conjugate increases chemical reactivity of xenobiotic carboxylic acids has led to an awareness that the relative activity, substrate specificity and intracellular location of the xenobiotic-CoA ligases may explain differences in toxicity. 5. Continued characterization of the human xenobiotic-CoA ligases in terms of substrate/inhibitor profiles and regulation, will allow a greater understanding of the role of these enzymes in the metabolism of carboxylic acids.

The effects of gender and oral contraceptive steroids on the pharmacokinetics of (R)-ibuprofen were studied in groups of healthy adult males, females and oral contraceptive steroid (OCS) using females. The values of AUC, CLpo, t1/2 and Vss, app did not differ significantly between the groups. Similarly, the percentage unbound of (R)-ibuprofen in pooled plasma from the three groups was not statistically different. Since chiral inversion is the major determinant of (R)-ibuprofen clearance in humans, it may be inferred from these data that gender and OCS have little or no effect on conversion of (R)-ibuprofen to the pharmacologically active S-enantiomer. Moreover, it is unlikely that hormonal factors influence the activity of the human hepatic long-chain fatty-acid:CoA ligase, the enzyme mediating the rate limiting step of (R)-ibuprofen inversion.In Australia, clinical researchers studied the effects of gender and oral contraceptive (OC) steroids on the pharmacokinetics of (R)-ibuprofen in 8 healthy adult males (mean age = 21 years), adult females (24 years), and OC users (22 years). There were no statistically significant differences between males, females, and OC users for any pharmacokinetic parameter for (R)-ibuprofen. These parameters included areas under the plasma total concentration-time curve (AUC), maximal plasma concentration, time to maximal plasma concentration, half-life, CLpo, and apparent steady-state volumes of distribution. The AUC to the last data point observed for (S)-ibuprofen (derived from (R)-ibuprofen) was similar, suggesting that hormonal factors do not affect plasma clearance of (S)-ibuprofen. The average percentages unbound of (R)- and (S)-ibuprofen across the concentration was not statistically different between the groups: 1.82% and 2.84% for males, 1.83% and 3.01% for females, and 2.1% and 2.97% for OC users, respectively. The mean fraction unbound of (S)-ibuprofen was 53.6% greater than that of (R)-ibuprofen. Since chiral inversion may explain 62-92% of (R)-ibuprofen clearance in humans, these data may suggest that gender and OCs do not effect or have only a limited effect on the conversion of (R)-ibuprofen to the pharmacologically active S-enantiomer. These findings indicate that hormonal factors probably do not affect the activity of the human hepatic long-chain fatty-acid:CoA ligase, the enzyme mediating the rate limiting step of (R)-ibuprofen inversion.

In this study we have demonstrated that rat hepatic peroxisomes catalyse the formation of nafenopin-CoA. The process is mediated by apparent high affinity (Km 6.7 microM), low capacity (Vmax 0.31 nmol/mg/min) and low affinity, high capacity isoforms. Palmitic acid (Ki 1.1 microM), R(-) ibuprofen (Ki 7.9 microM), ciprofibrate (Ki 60.2 microM) and clofibric acid (Ki 86.8 microM) competitively inhibited nafenopin-CoA formation catalysed by the apparent high affinity isoform. An antibody raised against the microsomal palmitoyl-CoA ligase inhibited the equivalent peroxisomal enzyme significantly (P0.001) but did not inhibit peroxisomal nafenopin-CoA ligase activity. These data suggest that nafenopin-CoA formation is catalysed by a peroxisomal CoA ligase which differs from the peroxisomal long chain fatty acid-CoA ligase in relation to its xenobiotic/antibody inhibitor profile and kinetic characteristics.

The first conjugation reaction, the conversion of benzoic acid to hippuric acid, was reported in 1842. Subsequent research lead to the discovery of major pathways involving conjugation with glucuronic acid, sulfate, glutathione, methyl groups and coenzyme A, which are mediated by families of enzymes in the cytoplasm and in intracellular membranes. Conjugation often produces less biologically active products that are readily excreted. However, in some cases, the conjugate may be more active or may be further metabolized to products that are toxic to the cell. The potential for predicting individuals that may be at risk for drug- or xenobiotic-induced toxicities has been enhanced by the characterization of the cDNAs and genes encoding conjugating enzymes and the discovery of mutations and polymorphisms in these genes.

Multiplicity of hepatic microsomal coenzyme A ligases catalyzing acyl-CoA thioester formation is an important factor for consideration in relation to the metabolism of xenobiotic carboxylic acids. In this study the kinetic characteristics of rat hepatic microsomal nafenopin-CoA ligase were studied and compared with those of long-chain fatty acid (palmitoyl) CoA ligase. The high affinity component of palmitoyl-CoA formation was inhibited by nafenopin ( K i 53 μ M) and ciprofibrate ( K i 1000 μ M). Analagous to palmitoyl-CoA, nafenopin-CoA formation was catalyzed by an apparent high affinity low capacity isoform ( K m 6 ± 2.5 μ M, (V max 0.33 ± 0.12 nmol/mg per min) which was inhibited competitively by palmitic acid (mean K i 1.7 μ M, n = 5) and R -ibuprofen (mean K i 10.8 μ M, n = 5) whilst ciprofibrate and clofibric acid were ineffective as inhibitors. The intrinsic metabolic clearance of nafenopin to nafenopin-CoA (V max / K m 0.057 ± 0.011 nmol/mg/min ± M) was similar to that reported recently for the formation of ibuprofenyl-CoA by rat liver microsomes. Evidence of both a substantial difference between the K m and K i for nafenopin and lack of commonality with regard to xenobiotic inhibitors suggests that the high affinity microsomal nafenopin-CoA and long-chain fatty acid-CoA ligases are kinetically distinct. Thus until the current ‘long-chain like’ xenobiotic-CoA ligases are fully characterised in terms of substrate specificity, inhibitor profile, etc, it will be impossible to rationalize (and possibly predict) the metabolism and hence toxicity of xenobiotic carboxylic acids forming acyl-CoA thioester intermediates.

In vivo and in vitro (-)R-ibuprofen is inverted to the (+)S antipode via stereoselective formation of an R-ibuprofenyl-CoA intermediate. In this study the effects of (-)R- and (+)S-ibuprofen on metabolism and respiration were studied using isolated rat hepatocytes and mitochondria. R-Ibuprofen significantly increased the lactate to pyruvate ratio, perturbed mitochondrial ketogenesis as evidenced by alterations in the beta-hydroxybutyrate to acetoacetate ratio and uncoupled mitochondrial oxidative phosphorylation. In addition, substantial dose- and time-dependent sequestration of reduced CoA (CoASH) occurred in the presence of the R enantiomer. Similarly, S-ibuprofen altered both the cytosolic and mitochondrial redox states although the magnitude of the effect was substantially less than that observed with the R enantiomer. In contrast to R-ibuprofen, S-ibuprofen did not uncouple oxidative phosphorylation or sequester hepatocyte CoASH. It is proposed that the perturbations observed in hepatocyte intermediary metabolism and mitochondrial function are attributable to a combination of the direct effects of R-ibuprofen per se and the sequestration of CoASH as R-ibuprofenyl-CoA during the process of chiral inversion. On the basis of these results, R-ibuprofen should be considered more in terms of metabolism to a reactive acyl-CoA intermediate rather than as a pro-drug for the pharmacologically active S-enantiomer.

Microsomal long chain fatty acid CoA ligase (EC 6.2.1.3) has been implicated in the formation of CoA thioesters of xenobiotics containing a carboxylic acid moiety. In this study we have demonstrated that the microsomal enzyme from rat liver exhibits biphasic kinetics for the formation of palmitoyl-CoA, i.e. there are high affinity low capacity Kmhigh, 1.6 microM, Vmaxhigh, 12.9 nmol/mg/min) and low affinity high capacity (Kmlow, 506 microM, Vmaxlow, 58.3 nmol/mg/min) components. Inhibition of the high affinity isoform was studied using the R and S enantiomers of ibuprofen, fenoprofen, ketoprofen and naproxen. The high affinity component of palmitoyl-CoA formation was competitively inhibited by R-fenoprofen (Ki 15.4 microM) while R-ibuprofen exhibited mixed inhibition kinetics. In contrast the R and S enantiomers of ketoprofen and naproxen were non-competitive inhibitors. This diversity of inhibition kinetics observed argues in favour of a binding site in addition to the catalytic site. A competitive interaction with the high affinity form correlated with literature evidence of enantiospecific chiral inversion and "hybrid" triglyceride formation for the R enantiomers of fenoprofen and ibuprofen. Paradoxically, R-ketoprofen which is extensively inverted in rats was a non-competitive inhibitor of palmitoyl-CoA formation by the high affinity isoform suggesting that it may not act as an alternate substrate. The results of this study clearly indicate that formation of R-2-arylpropionate-CoAs is not fully explained by interaction with the high affinity isoform of a microsomal long chain (palmitoyl) CoA ligase and therefore the involvement of other isoforms cannot be discounted.