Inhibitory Effects of Tricyclic Antidepressants on Human Liver Microsomal Morphine Glucuronidation: Application of IVIVE to Predict Potential Drug-Drug Interactions in Humans.

BACKGROUND: Tricyclic antidepressants (TCAs) are commonly co-administered with morphine as an adjuvant analgesic. Nevertheless, there remains a lack of information concerning metabolic drug-drug interactions (DDIs) resulting from TCA inhibition on morphine glucuronidation. OBJECTIVE: This study aimed to (i) examine the inhibitory effects of TCAs (viz., amitriptyline, clomipramine, imipramine, and nortriptyline) on human liver microsomal morphine 3- and 6-glucuronidation and (ii) evaluate the potential of DDI in humans by employing in vitro-in vivo extrapolation (IVIVE) approaches. METHOD: The inhibition parameters for TCA inhibition on morphine glucuronidation were derived from the in vitro system containing 2% BSA. The Ki values were employed to predict the DDI magnitude in vivo by using static and dynamic mechanistic PBPK approaches Results: TCAs moderately inhibited human liver microsomal morphine glucuronidation, with clomipramine exhibiting the most potent inhibition potency. Amitriptyline, clomipramine, imipramine, and nortriptyline competitively inhibited morphine 3- and 6-glucuronide formation with the respective Ki values of 91 ± 7.5 and 82 ± 11 µM, 23 ± 1.3 and 14 ± 0.7 µM, 103 ± 5 and 90 ± 7 µM, and 115 ± 5 and 110 ± 3 µM. Employing the static mechanistic IVIVE, a prediction showed an estimated 20% elevation in the morphine AUC when co-administered with either clomipramine or imipramine, whereas the predicted increase was <5% for amitriptyline or nortriptyline. PBPK modelling predicted an increase of less than 10% in the morphine AUC due to the inhibition of clomipramine and imipramine in both virtual healthy and cirrhotic populations. CONCLUSION: The results suggest that the likelihood of potential clinical DDIs arising from tricyclic antidepressant inhibition on morphine glucuronidation is low.

Inhibitory effects of non-steroidal anti-inflammatory drugs on human liver microsomal morphine glucuronidation: Implications for drug-drug interaction liability.

The inhibitory effects of fifteen NSAIDs from six structurally distinct classes on human liver microsomal morphine glucuronidation were investigated. Ki values of selected NSAIDs were generated and employed to assess DDI liability in vivo. Potent inhibition was observed for mefenamic acid and tolfenamic acid; respective IC50 values for morphine 3- and 6-glucuronidation were 9.2 and 13.5 µM, and 5.3 and 8.3 µM. Diclofenac and celecoxib showed moderate inhibition with IC50 values of 78 and 52 µM, and 83 and 214 µM, respectively. Estimated IC50 values for the other NSAIDs screened were >100 µM. Mefenamic acid, diclofenac, and S-naproxen competitively inhibited morphine 3- and 6-glucuronidation, with the Ki values of 11 and 12 µM, 110 and 76 µM, and 319 and 650 µM, respectively. Using the static mechanistic IVIVE approach, an approximate 40% increase in the AUC of morphine was predicted when co-administered with mefenamic acid, whereas the increase was <10% with diclofenac and S-naproxen. PBPK modeling predicted <15% increases in the morphine AUC from diclofenac and S-naproxen inhibition in virtual healthy and cirrhotic subjects. The data suggest that potential clinically significant DDIs arising from NSAID inhibition of morphine glucuronidation is unlikely, with the possible exception of some fenamates.

In vitro inhibitory effects of major bioactive constituents of Andrographis paniculata, Curcuma longa and Silybum marianum on human liver microsomal morphine glucuronidation: A prediction of potential herb-drug interactions arising from andrographolide, curcumin and silybin inhibition in humans.

This study aimed to investigate the liver microsomal inhibitory effects of silybin, silychristin, andrographolide, and curcumin by using morphine as an in vitro UGT2B7 probe substrate, and predict the magnitude of the herb-drug interaction arising from these herbal constituents' inhibition in vivo. Studies were performed in the incubation with and without bovine serum albumin (BSA). Andrographolide and curcumin showed a marked inhibition on morphine 3- and 6-glucuronidation with IC50 of 50&87 and 96&111 µM, respectively. In the presence of 2%BSA, andrographolide also showed a strong inhibition on morphine 3- and 6-glucuronidation (IC50 4.4&21.6 µM) whereas curcumin showed moderate inhibition (IC50 338&333 µM). In the absence and presence of 2%BSA, morphine 3- and 6-glucuronidation was moderately inhibited by silybin (IC50 583&862 and 1252&1421 µM, respectively), however was weakly inhibited by silychristin (IC50 3527&3504 and 1124&1530 µM, respectively). The Ki of andrographolide, curcumin and silybin on morphine 3- and 6-glucuronidation were 7.1&9.5, 72.7&65.2, and 224.5&159.7 µM, respectively, while the respective values generated from the system containing 2%BSA were 2.4&3.1, 96.4&108.8, and 366.3&394.5 µM. Using the in vitro and in vivo extrapolation approach, andrographolide was herbal component that may have had a potential interaction in vivo when it was co-administered with morphine.

Prevalence of potential drug interactions in Thai patients receiving simvastatin: The causality assessment of musculoskeletal adverse events induced by statin interaction.

Drug-drug interactions are one of the major risk factors associated with statin-induced myopathy. Although simvastatin is widely used in Thailand, studies investigating the prevalence of potential simvastatin-drug interactions (SDIs) and its clinical relevance in Thai population are still limited. We aimed to investigate the prevalence of potential SDIs (phase 1 study) and musculoskeletal adverse effects (AEs) associated with those interactions (phase 2 study). A phase 1 study was retrospectively conducted with outpatients at a 60-bed hospital who received simvastatin between July 1, 2012 and June 30, 2013. In phase 2, study was cross-sectionally conducted in outpatients whose prescriptions contain potential SDIs. Musculoskeletal AEs were evaluated by using symptom checklist questionnaires and measuring plasma creatinine kinase (CK). The causal relationship between the AEs and the potential SDIs was assessed using a Drug Interaction Probability Scale. Out of 3447 simvastatin users, potential SDIs were found in 314 patients (9.1%). The prevalence of prescriptions containing potential SDIs was in the range of 4.7-6.0%. Two-thirds of the potential SDIs were rated to be highly significant while more than 70% were in contraindication list. The most common precipitant drugs were gemfibrozil (382 prescriptions), colchicine (171 prescriptions) and amlodipine (152 prescriptions). Of 49 patients recruited into phase 2 study, we found that 31 patients (63.3%) had myopathy. Myalgia was the most frequently identified AEs (n = 18, 58.1%), followed by asymptomatic rising CK (n = 8, 25.8%), and myositis (n = 5, 16.1%). Musculoskeletal AEs associated with SDIs were found in 16 patients (51.6%). Of these, we found 50.0%, 31.3% and 18.8% had asymptomatic rising CK, myalgia, and myositis, respectively. Precipitant drugs associated with myopathy were amlodipine (2 possible cases), colchicine (3 possible cases), gemfibrozil (8 possible and 1 probable cases), nevirapine (1 possible case), and nicotinic acid (1 possible case). Potential SDIs have been found in the Thai population with a prevalence that is consistent with previous reports. Half of the musculoskeletal AEs identified were associated with SDIs. Systematic screening and management with interdisciplinary co-operation are needed to increase awareness of potential SDIs.

Initial development and testing of an instrument for patient self-assessment of adverse drug reactions.

PURPOSE: To develop and conduct preliminary testing of a causality assessment tool for patients, for potential use in encouraging both discussions with clinicians about suspected adverse drug reactions (ADRs) and reporting to authorities. METHODS: Ten causality statements, developed from qualitative studies involving patients, with a scoring system allowing categorization, were embedded in a questionnaire which also included a symptom checklist and additional details about one suspected ADR and medicine, selected for causality assessment. Patients with experiences of suspected ADRs were involved in cognitive interviews (15), piloting (20) and psychometric testing (120). Test-retest reliability, construct validity and criterion-related validity were evaluated, through repeated causality assessment, comparison with a visual analogue scale assessing certainty of causality and comparison with causality assessment using World Health Organization-Uppsala Monitoring Centre (WHO-UMC) criteria, respectively. The study involved outpatients at a university hospital in northeast Thailand. RESULTS: Ninety-eight patients completed causality assessment twice: both causality scores (Spearman rs = 0.715; p < 0.001) and causality classification [percentage of positive agreement (PPA) = 68.4; κ = 0.419; p < 0.001] showed satisfactory reliability. Causality scores were positively correlated with certainty of causality (Spearman rs = 0.556; p < 0.01). There was moderate agreement against WHO-UMC criteria [PPA = 70.4; κ = 0.440; p < 0.001]. Of the 91 completing an evaluation, 88% agreed that the tool should be used routinely, 78% agreed that it gave them useful results and 80% agreed that it was easy to use. CONCLUSIONS: This novel instrument has satisfactory psychometric properties and was acceptable to Thai patients, but it requires further testing. It has potential for use in supporting patients with suspected ADRs to discuss these with health professionals, and perhaps to report directly.

A qualitative study to explore how patients identify and assess symptoms as adverse drug reactions.

PURPOSE: To explore how Thai patients assess symptoms as adverse drug reactions (ADRs). METHODS: Out-patients at two hospitals in Thailand previously reporting suspected ADRs to statins were purposively selected to cover factors relevant to the accuracy of ADR reports. Semi-structured interviews explored the mechanisms participants used to work out whether their symptoms were related to their statin. All interviews were audio-recorded, transcribed and independently thematically analyzed by two researchers. RESULTS: One hundred interviews were suitable for analysis; 52 were male, age range was 36 to 77 years (mean ± S.D.: 59.83 ± 9.14) and most (92) were taking other medicines in addition to statins. Patient assessment of symptoms as ADRs fell into two major themes: medicine-related factors and external factors. Timing relationships were mentioned most frequently (74), followed by information received (55), seeing similar symptoms in others (7) and diagnosis through blood tests (4). Use of multiple medicines, consideration of the medicine versus diseases, symptoms occurring with more than one medicine or relieved through treatment reduced confidence in ADR attribution. Many participants proposed alternative explanations for symptoms, including old age. Lack of information and knowledge were obstacles to the assessment process. CONCLUSIONS: Patients assessed possible ADRs most often by considering timing relationships. While they also used medicine information, Thai patients received inadequate information to help them assess their symptoms. Patients expressed uncertainty and difficulties in deciding attribution when concomitant medicines and diseases were involved. The findings could support the development of a patient-friendly systematic tool for identifying and assessing possible ADRs.

The glucuronidation of R- and S-lorazepam: human liver microsomal kinetics, UDP-glucuronosyltransferase enzyme selectivity, and inhibition by drugs.

The widely used hypnosedative-anxiolytic agent R,S-lorazepam is cleared predominantly by conjugation with glucuronic acid in humans, but the enantioselective glucuronidation of lorazepam has received little attention. The present study characterized the kinetics of the separate R and S enantiomers of lorazepam by human liver microsomes (HLMs) and by a panel of recombinant human UDP-glucuronosyltransferase (UGT) enzymes. Respective mean K(m) and V(max) values for R- and S-lorazepam glucuronidation by HLM were 29 ± 8.9 and 36 ± 10 µM, and 7.4 ± 1.9 and 10 ± 3.8 pmol/min ⋅ mg. Microsomal intrinsic clearances were not significantly different, suggesting the in vivo clearances of R- and S-lorazepam are likely to be similar. Both R- and S-lorazepam were glucuronidated by UGT2B4, 2B7, and 2B15, whereas R-lorazepam was additionally metabolized by the extrahepatic enzymes UGT1A7 and 1A10. Based on in vitro clearances and consideration of available in vivo and in vitro data, UGT2B15 is likely to play an important role in the glucuronidation of R- and S-lorazepam. However, the possible contribution of other enzymes and the low activities observed in vitro indicate that the lorazepam enantiomers are of limited use as substrate probes for UGT2B15. To identify potential drug-drug interactions, codeine, fluconazole, ketamine, ketoconazole, methadone, morphine, valproic acid, and zidovudine were screened as inhibitors of R- and S-lorazepam glucuronidation by HLM. In vitro-in vivo extrapolation suggested that, of these drugs, only ketoconazole had the potential to inhibit lorazepam clearance to a clinically significant extent.

Patient reporting of suspected adverse drug reactions to antiepileptic drugs: factors affecting attribution accuracy.

This study was aimed to assess the frequency and number of suspected ADRs reported by patients taking antiepileptic drugs (AEDs) and to explore the factors that may affect patients' symptom attribution accuracy. A validated questionnaire containing an extensively checklist of symptoms was distributed to outpatients prescribed one or more AEDs. Data on concomitant drugs and diseases were obtained from outpatient records. All symptoms identified were assessed for causality. Of 1388 questionnaires distributed to 1214 patients, 830 completed questionnaires were returned (59.8%) from 727 patients. In total, 7815 symptoms were identified on 757 questionnaires (91.2%). Symptom severity ratings were positively related to the number of symptoms reported (p=0.003). Causality assessment found that 71.9% of the symptoms were 'true' ADRs and 28.1% were 'false' ADRs. Attribution accuracy was primarily influenced by the number of symptoms identified and indication for AED therapy, fewer symptoms and use for non-epilepsy indications being associated with greater attribution accuracy.

Effects of ketamine on human UDP-glucuronosyltransferases in vitro predict potential drug-drug interactions arising from ketamine inhibition of codeine and morphine glucuronidation.

In this study, the selectivity of UDP-glucuronosyltransferase (UGT) enzyme inhibition by ketamine (KTM) and the kinetics of KTM inhibition of human liver microsomal morphine (MOR) and codeine (COD) glucuronidation were characterized to explore a pharmacokinetic basis for the KTM-opioid interaction. With the exception of UGT1A4, KTM inhibited the activities of recombinant human UGT enzymes in a concentration-dependent manner. However, IC(50) values were <100 µM only for UGT2B4, UGT2B7, and UGT2B15. UGT2B7 catalyzes MOR 3- and 6-glucuronidation and the 6-glucuronidation of COD, with an additional substantial contribution of UGT2B4 to the latter reaction. Consistent with the effects of KTM on the activities of recombinant UGT2B enzyme activities, KTM competitively inhibited human liver microsomal MOR and COD glucuronidation. K(i) values for KTM inhibition of MOR 3- and 6-glucuronidation and COD 6-glucuronidation by human liver microsomes supplemented with 2% bovine serum albumin were 5.8 ± 0.1, 4.6 ± 0.2, and 3.5 ± 0.1 µM, respectively. Based on the derived inhibitor constants, in vitro-in vivo extrapolation was used to predict the effects of anesthetic and analgesic doses of KTM on MOR and COD clearances. Potentially clinically significant interactions (>50% increases in the in vivo area under the curve ratios) with MOR and COD were predicted for anesthetic doses of KTM and for a subanesthetic dose of KTM on COD glucuronidation.

In vitro-in vivo extrapolation of CYP2C8-catalyzed paclitaxel 6α-hydroxylation: effects of albumin on in vitro kinetic parameters and assessment of interindividual variability in predicted clearance.

OBJECTIVES: This study aimed to characterize the effects of bovine serum albumin (BSA) on the kinetics of CYP2C8-catalyzed paclitaxel 6α-hydroxylation in vitro; determine whether the addition of BSA to incubations improves the prediction of paclitaxel hepatic clearance via this pathway in vivo; and assess interindividual variability in predicted clearance. METHODS: The kinetics of paclitaxel 6α-hydroxlation by human liver microsomes (HLM) and recombinant CYP2C8 were characterized in incubations performed with and without BSA (2% w/v) supplementation, and the in vitro kinetic data were extrapolated to provide estimates of in vivo clearances. The Simcyp population-based ADME simulator was used to determine interindividual variability in the predicted clearances. RESULTS: Supplementation of incubations of HLM with BSA resulted in a 3.6-fold increase in the microsomal intrinsic clearance for paclitaxel 6α-hydroxylation, due mainly to a reduction in K(m) (7.08 ± 2.50 to 2.26 ± 0.39 µM), while addition of BSA to incubations of recombinant CYP2C8 resulted in an approximate doubling of intrinsic clearance. Mean values of predicted in vivo hepatic clearance were in good agreement with clinical data when in vitro data obtained in the presence of BSA were used for IV-IVE. Simcyp predicted 20- to 30-fold interindividual variability in in vivo paclitaxel hepatic clearance via the 6α-hydroxylation pathway. CONCLUSIONS: Human liver microsomal K(m) and intrinsic clearance values are over- and underpredicted, respectively, when incubations of the CYP2C8 substrate paclitaxel are performed without BSA supplementation. IV-IVE based on kinetic parameters generated in the presence of BSA improves the accuracy of predicted paclitaxel hepatic clearance.

Formulation development of morphine sulfate sustained-release tablets and its bioequivalence study in healthy Thai volunteers.

The objectives of this study were to develop morphine sulfate sustained-release tablet formulations and to evaluate the bioequivalence compared with a commercial brand. The physicochemical properties of the formulated and commercial tablets were determined and compared. The bioequivalence investigation was carried out in 15 healthy male volunteers who received a single dose in a randomized two-way crossover design. After dosing, serial blood samples were collected for a period of 24 h. Morphine concentration was assayed by high-performance liquid chromatography with electrochemical detector. The log-transformed C(max) and AUC(s) were statistically compared by analysis of variance, and the 90% confidence intervals (CIs) of the ratio of the log-transformed C(max) and AUC(s) between the most promising developed formulation and the commercial product were determined. It was found that the dissolution rate profile of a developed formulation was similar to the commercial brand. Their similarity and difference factors were well within limits. In the bioequivalence study, the AUC(last) and AUC(inf) between the test and the reference products were not statistically different (p = 0.227 and p = 0.468, respectively), with the 90% CIs of 83.4-102.6% and 87.7-139.4%, respectively. However, the C(max) of the two formulations was significantly different (p = 0.019). The 90% CI of the developed formulation was 72.0-93.0% compared to the commercial product. In vitro dissolution of locally prepared morphine sulfate sustained-release tablets was comparable to commercial brand. However, the results justified the conclusion of lack of bioequivalence of the developed product to the commercial one.

Effects of Andrographis paniculata and Orthosiphon stamineus extracts on the glucuronidation of 4-methylumbelliferone in human UGT isoforms.

The effects of Andrographis paniculata and Orthosiphon stamineus extracts on the in vitro glucuronidation of 4-methylumbelliferone (4MU) by recombinant human UGTs, UGT1A1, UGT1A3, UGT1A6, UGT1A7, UGT1A8, UGT1A10, UGT2B7 and UGT2B15 were determined. The potential inhibitory effects of both of the extracts on the activity of each of the UGT isoforms were investigated using 4MU as the substrate. Incubations contained UDP-glucuronic acid (UDPGA) as the cofactor, MgCl(2), cell lysate of respective isoform, and 4MU at the approximate apparent K(m) or S(50) value of each isoform. Final concentrations of Andrographis paniculata and Orthosiphon stamineus extracts used were 0.025, 0.25, 2.5, 25 and 50 microg/mL and 0.01, 0.10, 1.0, 10 and 50 microg/mL respectively. Both extracts variably inhibited the activity of most of the isoforms in a concentration dependent manner. Andrographis paniculata extract was the better inhibitor of all the isoforms studied (IC(50) 1.70 microg/mL for UGT1A3, 2.57 microg/mL for UGT1A8, 2.82 microg/mL for UGT2B7, 5.00 micorg/mL for UGT1A1, 5.66 microg/mL for UGT1A6, 9.88 microg/mL for UGT1A7 and 15.66 microg/mL for UGT1A10). Both extracts showed less than 70% inhibition of UGT2B15, so the IC(50) values were >50 microg/mL. The inhibition of human UGTs by Andrographis paniculata and Orthosiphon stamineus extracts in vitro suggests a potential for drug-herbal extract interactions in the therapeutic setting.

In vitro-in vivo extrapolation predicts drug-drug interactions arising from inhibition of codeine glucuronidation by dextropropoxyphene, fluconazole, ketoconazole, and methadone in humans.

Because codeine (COD) is eliminated primarily via glucuronidation, factors that alter COD glucuronide formation potentially affect the proportion of the dose converted to the pharmacologically active metabolite morphine. Thus, in vitro-in vivo extrapolation approaches were used to identify potential drug-drug interactions arising from inhibition of COD glucuronidation in humans. Initial studies characterized the kinetics of COD-6-glucuronide (C6G) formation by human liver microsomes (HLM) and demonstrated an 88% reduction in the Michaelis constant (K(m)) (0.29 versus 2.32 mM) for incubations performed in the presence of 2% bovine serum albumin (BSA). Of 13 recombinant UDP-glucuronosyltransferase (UGT) enzymes screened for COD glucuronidation activity, only UGT2B4 and UGT2B7 exhibited activity. The respective S(50) values (0.32 and 0.27 mM) generated in the presence of BSA were comparable with the mean K(m) observed in HLM. Known inhibitors of UGT2B7 activity in vitro or in vivo and drugs marketed as compound formulations with COD were investigated for inhibition of C6G formation by HLM. Inhibition screening identified potential interactions with dextropropoxyphene, fluconazole, ketoconazole, and methadone. Inhibitor constant values generated for dextropropoxyphene (3.5 microM), fluconazole (202 microM), ketoconazole (0.66 microM), and methadone (0.32 microM) predicted 1.60- to 3.66-fold increases in the area under the drug plasma concentration-time curve ratio for COD in vivo. Whereas fluconazole and ketoconazole inhibited UGT2B4- and UGT2B7-catalyzed COD glucuronidation to a similar extent, inhibition by dextropropoxyphene and methadone resulted largely from an effect on UGT2B4. Interactions with dextropropoxyphene, fluconazole, ketoconazole, and methadone potentially affect the intensity and duration of COD analgesia.

The smoothness of blood pressure control of ramipril in essential hypertensive Thai patients evaluation by 24-hour ambulatory blood pressure monitoring.

OBJECTIVE: Evaluate the efficacy of ramipril 2.5 and 5 mg once daily on the degree and homogeneity of 24-hour blood pressure reduction in essential hypertensive Thai patients. MATERIAL AND METHOD: Nineteen male subjects, aged 30 to 60 years, with newly diagnosed essential hypertension were evaluated using the 24-hour ambulatory blood pressure (24-h ABP) measurement. RESULTS: Twelve subjects responded and/or normalized with ramipril once daily, where the office and 24-h ABP were decreased significantly from baseline (p < 0.01). The percentage and magnitude of 24-h SBP/DBP loads after treatment were significantly decreased from 92 +/- 9.7/91 +/- 15.9 to 67 +/- 23.8/65 +/- 27.6 (p < 0.01) and from 23 +/- 10.6/16 +/- 5.3 mmHg to 17 +/- 10.3/10 +/- 4.8 mmHg ( p < 0.05). Trough to peak ratio for SBP/DBP was 0.59/0.52 (overall estimated) and 0.68 +/- 0.23/0.52 +/- 0.22 (individual estimated), while the smoothness index was 0.89/1.03. CONCLUSION: Ramipril 2.5 and 5 mg once daily exerted the smooth 24-hour blood pressure reduction in essential hypertensive Thai patients.

Kinetic modeling of the interactions between 4-methylumbelliferone, 1-naphthol, and zidovudine glucuronidation by udp-glucuronosyltransferase 2B7 (UGT2B7) provides evidence for multiple substrate binding and effector sites.

Interactions between the UGT2B7-catalyzed glucuronidation of zidovudine (AZT), 4-methylumbelliferone (4MU), and 1-naphthol (1NP) were analyzed using multisite and empirical kinetic models to explore the existence of multiple substrate and effector binding sites within this important drug metabolizing enzyme. 4MU and 1NP glucuronidation by UGT2B7 exhibit sigmoidal kinetics characteristic of homotropic cooperativity (autoactivation), which may be modeled assuming the existence of two equivalent, interacting substrate binding sites. In contrast, UGT2B7-catalyzed AZT glucuronidation follows hyperbolic (Michaelis-Menten) kinetics. Although 4MU and 1NP decreased the binding affinity of AZT, the kinetics of AZT glucuronidation changed from hyperbolic to sigmoidal in the presence of both modifiers. Data were well described by a generic two-substrate binding site model in which there is no interaction between the sites in the absence of 4MU or 1NP, but heterotropic cooperativity results from the binding of modifier. Inhibition of 4MU and 1NP glucuronidation by AZT and interactions between 4MU and 1NP required more complex three-site models, where the modifier acts via a distinct effector site to alter either substrate binding affinity or Vmax without affecting the homotropic cooperativity characteristic of 4MU and 1NP glucuronidation. It is noteworthy that 1NP inhibited 4MU glucuronidation, whereas 4MU activated 1NP glucuronidation. The results are consistent with the existence of two "catalytic" sites for each substrate within the UGT2B7 active site, along with multiple effector sites. The multiplicity of binding and effector sites results in complex kinetic interactions between UGT2B7 substrates, which potentially complicates inhibition screening studies.

Relationship between hyperbilirubinaemia and UDP-glucuronosyltransferase 1A1 (UGT1A1) polymorphism in adult HIV-infected Thai patients treated with indinavir.

OBJECTIVES: To investigate the association between the UGT1A1*6 (G71R) and UGT1A1*28 (promoter (TA)7-repeat) genotypes and hyperbilirubinaemia in Thai patients treated with indinavir, and characterize the inhibition of human UGTs by indinavir in vitro. METHODS: Ninety-six Thai HIV patients receiving indinavir, 800 mg t.i.d. or 800 mg b.i.d. "boosted" with ritonavir (100 mg b.i.d.), had serum bilirubin levels measured to 24 weeks post-treatment and were genotyped for UGT1A1*6 and UGT1A1*28. The inhibition selectivity and kinetics of indinavir were determined using a panel of recombinant human UGTs. RESULTS: UGT1A1*6 and UGT1A1*28 frequencies in the Thai patients were 10.4% and 15.6%, respectively. Total, conjugated (direct) and unconjugated (indirect) serum bilirubin concentrations increased significantly at 24 weeks of indinavir treatment for all four genotypes, with a trend towards higher levels depending on the number of UGT1A1 mutant alleles; *6/*28 > *6 > *28 > reference. The hazards ratio (HR) for serious hyperbilirubinaemia (total bilirubin > 2.5 mg/dl) at week 24 was statistically significant only in those patients carrying the UGT1A1*6 (HR 2.87) and UGT1A1*6/*28 (HR 11.42) genotypes. The Ki values for indinavir inhibition of UGT1A1 and UGT1A1*6 were 4.1 and 10.7 mumol/l respectively. However, indinavir was also shown to inhibit other human UGTs, notably UGT1A3 and UGT1A7. CONCLUSIONS: In contrast to Caucasian HIV-infected patients treated with indinavir, the promoter polymorphism (UGT1A1*28) is of less significance than the coding region (UGT1A1*6) mutation as a risk factor for hyperbilirubinaemia. The Ki values determined for indinavir inhibition of UGT1A1 are consistent with an interaction in vivo, with an additive effect in patients with already impaired bilirubin glucuronidation activity.

Quantitative prediction of in vivo inhibitory interactions involving glucuronidated drugs from in vitro data: the effect of fluconazole on zidovudine glucuronidation.

AIMS: Using the fluconazole-zidovudine (AZT) interaction as a model, to determine whether inhibition of UDP-glucuronosyltransferase (UGT) catalysed drug metabolism in vivo could be predicted quantitatively from in vitro kinetic data generated in the presence and absence bovine serum albumin (BSA). METHODS: Kinetic constants for AZT glucuronidation were generated using human liver microsomes (HLM) and recombinant UGT2B7, the principal enzyme responsible for AZT glucuronidation, as the enzyme sources with and without fluconazole. K(i) values were used to estimate the decrease in AZT clearance in vivo. RESULTS: Addition of BSA (2%) to incubations decreased the K(m) values for AZT glucuronidation by 85-90% for the HLM (923 +/- 357 to 91 +/- 9 microm) and UGT2B7 (478-70 microm) catalysed reactions, with little effect on V(max). Fluconazole, which was shown to be a selective inhibitor of UGT2B7, competitively inhibited AZT glucuronidation by HLM and UGT2B7. Like the K(m), BSA caused an 87% reduction in the K(i) for fluconazole inhibition of AZT glucuronidation by HLM (1133 +/- 403 to 145 +/- 36 microm) and UGT2B7 (529 to 73 microm). K(i) values determined for fluconazole using HLM and UGT2B7 in the presence (but not absence) of BSA predicted an interaction in vivo. The predicted magnitude of the interaction ranged from 41% to 217% of the reported AUC increase in patients, depending on the value of the in vivo fluconazole concentration employed in calculations. CONCLUSIONS: K(i) values determined under certain experimental conditions may quantitatively predict inhibition of UGT catalysed drug glucuronidation in vivo.

Selectivity of substrate (trifluoperazine) and inhibitor (amitriptyline, androsterone, canrenoic acid, hecogenin, phenylbutazone, quinidine, quinine, and sulfinpyrazone) "probes" for human udp-glucuronosyltransferases.

Relatively few selective substrate and inhibitor probes have been identified for human UDP-glucuronosyltransferases (UGTs). This work investigated the selectivity of trifluoperazine (TFP), as a substrate, and amitriptyline, androsterone, canrenoic acid, hecogenin, phenylbutazone, quinidine, quinine, and sulfinpyrazone, as inhibitors, for human UGTs. Selectivity was assessed using UGTs 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B7, and 2B15 expressed in HEK293 cells. TFP was confirmed as a highly selective substrate for UGT1A4. However, TFP bound extensively to both HEK293 lysate and human liver microsomes in a concentration-dependent manner (fuinc 0.20-0.59). When corrected for nonspecific binding, Km values for TFP glucuronidation were similar for both UGT1A4 (4.1 microM) and human liver microsomes (6.1+/-1.2 microM) as the enzyme sources. Of the compounds screened as inhibitors, hecogenin, alone, was selective; significant inhibition was observed only for UGT1A4 (IC50 1.5 microM). Using phenylbutazone and quinine as "models," inhibition kinetics were variously described by competitive and noncompetitive mechanisms. Inhibition of UGT2B7 by quinidine was also investigated further, because the effects of this compound on morphine pharmacokinetics (a known UGT2B7 substrate) have been ascribed to inhibition of P-glycoprotein. Quinidine inhibited human liver microsomal and recombinant UGT2B7, with respective Ki values of 335+/-128 microM and 186 microM. In conclusion, TFP and hecogenin represent selective substrate and inhibitor probes for UGT1A4, although the extensive nonselective binding of the former should be taken into account in kinetic studies. Amitriptyline, androsterone, canrenoic acid, hecogenin, phenylbutazone, quinidine, quinine, and sulfinpyrazone are nonselective UGT inhibitors.

Human udp-glucuronosyltransferases: isoform selectivity and kinetics of 4-methylumbelliferone and 1-naphthol glucuronidation, effects of organic solvents, and inhibition by diclofenac and probenecid.

The glucuronidation kinetics of the prototypic substrates 4-methylumbelliferone (4MU) and 1-naphthol (1NP) by human UDP-glucuronosyltransferases (UGT) 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B7, 2B15, and 2B17 were investigated. Where activity was demonstrated, inhibitory effects of diclofenac, probenecid, and the solvents acetone, acetonitrile, dimethyl sulfoxide, ethanol, and methanol were characterized. All isoforms except UGT1A4 glucuronidated 4MU, whereas all but UGT 1A4, 2B15, and 2B17 metabolized 1NP. However, kinetic models varied with substrate (for the same isoform) and from isoform to isoform (with the same substrate). Hyperbolic (Michaelis-Menten), substrate inhibition, and sigmoidal kinetics were variably observed for both 4MU and 1NP glucuronidation by the various UGTs. K(m) or S(50) (sigmoidal kinetics) and V(max) values varied 525- (8-4204 microM) and 1386-fold, respectively, for 4MU glucuronidation, and 1360- (1.3-1768 microM) and 37-fold, respectively, for 1NP glucuronidation. The use of a two-site model proved useful for those reactions exhibiting non-Michaelis-Menten glucuronidation kinetics. The organic solvents generally had a relatively minor effect on UGT isoform activity. UGT 2B15 and 2B17 were most susceptible to the presence of solvent, although solvent-selective inhibition was occasionally observed with other isoforms. Diclofenac and probenecid inhibited all isoforms, precluding the use of these compounds for the reaction phenotyping of xenobiotic glucuronidation pathways in human tissues. Diclofenac and probenecid K(i) values, determined for selected isoforms, ranged from 11 to 52 microM and 96 to 2452 microM, respectively. Overall, the results emphasize the need for the careful design and interpretation of kinetic and inhibition studies with human UGTs.