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Biliary excretion is the main route of disposal of bilirubin and impaired excretion results in jaundice, a well recognisable symptom of liver disease. Conjugation of bilirubin in the liver is essential for its clearance. The glucuronidation of bilirubin is catalysed by the microsomal UDP-glucuronosyltransferase UGT1A1. Patients with Crigler-Najjar syndrome type 1 and Gunn rats, mutant strain of the Wistar rats, bear an autosomal recessive disorder resulting in hyperbilirubinemia. The aim of this work is to add new data about activity of UGT1A1 during the perinatal period and adult life. The results showed that activity of UGT1A1 is detectable from day 22 of the gestation. After birth, activity of UGT1A1 gradually increases and reaches the levels of adult life. Furthermore, bilirubin azopigments have been separated and characterized by thin layer chromatography. We have found that concentration of samples by evaporation and ulterior storing at -20 degrees C seemed to be suitable for the maintenance of samples., Spanish Social Security Funds for Research, Depto. de Inmunología, Oftalmología y ORL, Fac. de Medicina, TRUE, pub

Simple Summary In this study, we clarified the evolutional features of the UGT gene family in Carnivora. We firstly analyzed the gene synteny of UGT1As, 2Bs, ana 2Es and further demonstrated the phylogenetic analysis to reveal the evolutional gene duplication and loss event in Carnivora. We found specific UGT1A duplication in Canidae, brown bear and black bear, and UGT2Bs duplication in Canidae, some Mustelidae, and Ursidae. In addition, we observed gene contraction of UGT1A7-12 in Phocidae, Otariidae, and Felidae. This study strongly suggested closely related Carnivorans also showed significant evolutional differences of UGTs, and further imply the importance of appropriate approaches to assess pharmacokinetics and toxicokinetic from experimental animals. UDP-glucuronosyltransferases (UGTs) are one of the most important enzymes for xenobiotic metabolism or detoxification. Through duplication and loss of genes, mammals evolved the species-specific variety of UGT isoforms. Among mammals, Carnivora is one of the orders that includes various carnivorous species, yet there is huge variation of food habitat. Recently, lower activity of UGT1A and 2B were shown in Felidae and pinnipeds, suggesting evolutional loss of these isoforms. However, comprehensive analysis for genetic or evolutional features are still missing. This study was conducted to reveal evolutional history of UGTs in Carnivoran species. We found specific gene expansion of UGT1As in Canidae, brown bear and black bear. We also found similar genetic duplication in UGT2Bs in Canidae, and some Mustelidae and Ursidae. In addition, we discovered contraction or complete loss of UGT1A7-12 in phocids, some otariids, felids, and some Mustelids. These studies indicate that even closely related species have completely different evolution of UGTs and further imply the difficulty of extrapolation of the pharmacokinetics and toxicokinetic result of experimental animals into wildlife carnivorans.

Simple Summary In this study, we clarified the evolutional features of the UGT gene family in Carnivora. We firstly analyzed the gene synteny of UGT1As, 2Bs, ana 2Es and further demonstrated the phylogenetic analysis to reveal the evolutional gene duplication and loss event in Carnivora. We found specific UGT1A duplication in Canidae, brown bear and black bear, and UGT2Bs duplication in Canidae, some Mustelidae, and Ursidae. In addition, we observed gene contraction of UGT1A7-12 in Phocidae, Otariidae, and Felidae. This study strongly suggested closely related Carnivorans also showed significant evolutional differences of UGTs, and further imply the importance of appropriate approaches to assess pharmacokinetics and toxicokinetic from experimental animals. UDP-glucuronosyltransferases (UGTs) are one of the most important enzymes for xenobiotic metabolism or detoxification. Through duplication and loss of genes, mammals evolved the species-specific variety of UGT isoforms. Among mammals, Carnivora is one of the orders that includes various carnivorous species, yet there is huge variation of food habitat. Recently, lower activity of UGT1A and 2B were shown in Felidae and pinnipeds, suggesting evolutional loss of these isoforms. However, comprehensive analysis for genetic or evolutional features are still missing. This study was conducted to reveal evolutional history of UGTs in Carnivoran species. We found specific gene expansion of UGT1As in Canidae, brown bear and black bear. We also found similar genetic duplication in UGT2Bs in Canidae, and some Mustelidae and Ursidae. In addition, we discovered contraction or complete loss of UGT1A7-12 in phocids, some otariids, felids, and some Mustelids. These studies indicate that even closely related species have completely different evolution of UGTs and further imply the difficulty of extrapolation of the pharmacokinetics and toxicokinetic result of experimental animals into wildlife carnivorans.

Simple Summary In this study, we clarified the evolutional features of the UGT gene family in Carnivora. We firstly analyzed the gene synteny of UGT1As, 2Bs, ana 2Es and further demonstrated the phylogenetic analysis to reveal the evolutional gene duplication and loss event in Carnivora. We found specific UGT1A duplication in Canidae, brown bear and black bear, and UGT2Bs duplication in Canidae, some Mustelidae, and Ursidae. In addition, we observed gene contraction of UGT1A7-12 in Phocidae, Otariidae, and Felidae. This study strongly suggested closely related Carnivorans also showed significant evolutional differences of UGTs, and further imply the importance of appropriate approaches to assess pharmacokinetics and toxicokinetic from experimental animals. UDP-glucuronosyltransferases (UGTs) are one of the most important enzymes for xenobiotic metabolism or detoxification. Through duplication and loss of genes, mammals evolved the species-specific variety of UGT isoforms. Among mammals, Carnivora is one of the orders that includes various carnivorous species, yet there is huge variation of food habitat. Recently, lower activity of UGT1A and 2B were shown in Felidae and pinnipeds, suggesting evolutional loss of these isoforms. However, comprehensive analysis for genetic or evolutional features are still missing. This study was conducted to reveal evolutional history of UGTs in Carnivoran species. We found specific gene expansion of UGT1As in Canidae, brown bear and black bear. We also found similar genetic duplication in UGT2Bs in Canidae, and some Mustelidae and Ursidae. In addition, we discovered contraction or complete loss of UGT1A7-12 in phocids, some otariids, felids, and some Mustelids. These studies indicate that even closely related species have completely different evolution of UGTs and further imply the difficulty of extrapolation of the pharmacokinetics and toxicokinetic result of experimental animals into wildlife carnivorans.

ObjectivesTo investigate if the nicotine metabolite ratio (NMR, the ratio of nicotine metabolites 3'-hydroxycotinine/cotinine) is a reliable phenotypic biomarker for nicotine clearance across races, and as a function of differences in the rate of nicotine, cotinine and 3'-hydroxycotinine glucuronidation and UGT genotypes.MethodsParticipants [Caucasians (Whites), African Americans (Blacks) and Asian-Americans (Asians)] received an oral solution of deuterium-labeled nicotine and its metabolite cotinine. Plasma and saliva concentrations of nicotine and cotinine were used to determine oral clearances. Rates of glucuronidation were assessed from urine glucuronide/parent ratios, and UGT2B10 and UGT2B17 genotypes from DNA.ResultsAmong the 227 participants, 96 (42%) were White, 67 (30%) Asian and 64 (28%) Black. Compared to the other two races, Whites had higher nicotine and cotinine total oral clearance, Blacks had lower nicotine and cotinine glucuronidation rates and Asians had lower 3'-hydroxycotinine glucuronidation rates. A strong positive correlation (correlations coefficients 0.77-0.84; P < 0.001) between NMR and nicotine oral clearance was found for all three races, and NMR remained a strong predictor for the nicotine oral clearance while adjusting for race, sex and age. Neither the metabolite glucuronidation ratios nor the UGT genotypes had significant effects on the ability of NMR to predict nicotine oral clearance.ConclusionNMR appears to be a reliable phenotypic biomarker for nicotine clearance across races, glucuronidation phenotypes and genotypes. Racial differences in the relationships between NMR, smoking behaviors and addiction are unlikely to be related to an inadequate estimation of nicotine clearance on the basis of NMR.

ObjectivesTo investigate if the nicotine metabolite ratio (NMR, the ratio of nicotine metabolites 3'-hydroxycotinine/cotinine) is a reliable phenotypic biomarker for nicotine clearance across races, and as a function of differences in the rate of nicotine, cotinine and 3'-hydroxycotinine glucuronidation and UGT genotypes.MethodsParticipants [Caucasians (Whites), African Americans (Blacks) and Asian-Americans (Asians)] received an oral solution of deuterium-labeled nicotine and its metabolite cotinine. Plasma and saliva concentrations of nicotine and cotinine were used to determine oral clearances. Rates of glucuronidation were assessed from urine glucuronide/parent ratios, and UGT2B10 and UGT2B17 genotypes from DNA.ResultsAmong the 227 participants, 96 (42%) were White, 67 (30%) Asian and 64 (28%) Black. Compared to the other two races, Whites had higher nicotine and cotinine total oral clearance, Blacks had lower nicotine and cotinine glucuronidation rates and Asians had lower 3'-hydroxycotinine glucuronidation rates. A strong positive correlation (correlations coefficients 0.77-0.84; P < 0.001) between NMR and nicotine oral clearance was found for all three races, and NMR remained a strong predictor for the nicotine oral clearance while adjusting for race, sex and age. Neither the metabolite glucuronidation ratios nor the UGT genotypes had significant effects on the ability of NMR to predict nicotine oral clearance.ConclusionNMR appears to be a reliable phenotypic biomarker for nicotine clearance across races, glucuronidation phenotypes and genotypes. Racial differences in the relationships between NMR, smoking behaviors and addiction are unlikely to be related to an inadequate estimation of nicotine clearance on the basis of NMR.

BackgroundVariation in CYP2A6 activity influences tobacco smoking behaviors and smoking-related health outcomes. Plasma Nicotine Metabolite Ratio (NMR) is a robust phenotypic biomarker of CYP2A6 activity and nicotine clearance. In urine, the NMR has been calculated as a ratio of free trans-3'-hydroxycotinine to free cotinine (NMRF/F), total trans-3'-hydroxycotinine to free cotinine (NMRT/F), or total trans-3'-hydroxycotinine to total cotinine (NMRT/T). We evaluated these three urinary NMR versions relative to plasma NMR and nicotine clearance and elucidated mechanisms of discrepancies among them.MethodsBaseline plasma and urine biomarker data were available from two smoking cessation clinical trials and one nicotine pharmacokinetic study (total N = 768). NMRs were compared using Pearson correlations, linear regressions and ANOVA analyses. UGT2B10 and UGT2B17 were genotyped.ResultsUrinary NMRT/F was the most highly related to plasma NMR (R2 = 0.70, P <2.2e-16) followed by NMRF/F (R2 = 0.68, P <2.2e-16), while NMRT/T was less strongly related (R2 = 0.60, P <2.2e-16); consistent across study, ethnicity, sex, heaviness of smoking, and analyte analysis. Controlling for cotinine glucuronidation, as a phenotype or UGT2B10 genotype, corrected the NMRT/T discordance with plasma NMR (Panova<0.001). Similar findings were obtained for relationships of nicotine clearance with plasma NMR > urinary NMRT/F > NMRF/F > NMRT/T (R2 = 0.41 > 0.37 > 0.35 > 0.25 respectively).ConclusionUrinary NMRT/F followed by NMRF/F are the best urinary alternatives to plasma NMR or nicotine clearance. NMRT/T has the least utility as it is influenced substantially by variation in cotinine glucuronidation.ImpactThis work highlighted the variation in urinary NMRs, and identified mechanisms for disparities among them, which facilitates their use in predicting smoking-related outcomes.

Over the last decade environmental agencies worldwide have escalated their work to phase out animal testing for the purposes of chemical regulation. Meanwhile the number of commercially available chemicals and the requirements for hazard assessments have both increased, creating a large need for substitution of traditional in vivo assays with in vitro tests. One example of this is the replacement of the OECD acute toxicity test of adult fish (test guideline [TG] 203) with zebrafish embryos (TG 236). With new insights into the toxicological properties of chemicals, the demand on these replacement tests is also changing character with a shifted focus towards mechanistic understanding of toxicity. The omics sciences encompass a group of analytical methods which have proven to be very powerful for unveiling of mechanistic information of biochemical processes. Metabolomics is one of the younger members of this family and entails the large-scale analysis of endogenous metabolites and their perturbation in living organisms. The overall objective of this thesis was to develop modifications to the TG236 OECD assay to obtain omic data suitable for use in chemical hazard assessment. To achieve this goal, we started by developing a targeted and non-targeted metabolomics workflow and evaluated the performance of the two types of analysis (Paper I). We also evaluated the efficiency of three signal drift correction approaches, which is an important step in data quality improvement for non-targeted analysis, and reported previously unlisted biochemicals present in NIST reference material. In Paper II we applied the workflow in Paper I to a newly developed, in-plate extraction method for single zebrafish embryos which were exposed to the pharmaceutical and environmental pollutant propranolol. Data processing workflows were developed to overcome challenges arising from the occurrence of the exposure compound and its biotransformation products (or in-source fragments of these) in the

Over the last decade environmental agencies worldwide have escalated their work to phase out animal testing for the purposes of chemical regulation. Meanwhile the number of commercially available chemicals and the requirements for hazard assessments have both increased, creating a large need for substitution of traditional in vivo assays with in vitro tests. One example of this is the replacement of the OECD acute toxicity test of adult fish (test guideline [TG] 203) with zebrafish embryos (TG 236). With new insights into the toxicological properties of chemicals, the demand on these replacement tests is also changing character with a shifted focus towards mechanistic understanding of toxicity. The omics sciences encompass a group of analytical methods which have proven to be very powerful for unveiling of mechanistic information of biochemical processes. Metabolomics is one of the younger members of this family and entails the large-scale analysis of endogenous metabolites and their perturbation in living organisms. The overall objective of this thesis was to develop modifications to the TG236 OECD assay to obtain omic data suitable for use in chemical hazard assessment. To achieve this goal, we started by developing a targeted and non-targeted metabolomics workflow and evaluated the performance of the two types of analysis (Paper I). We also evaluated the efficiency of three signal drift correction approaches, which is an important step in data quality improvement for non-targeted analysis, and reported previously unlisted biochemicals present in NIST reference material. In Paper II we applied the workflow in Paper I to a newly developed, in-plate extraction method for single zebrafish embryos which were exposed to the pharmaceutical and environmental pollutant propranolol. Data processing workflows were developed to overcome challenges arising from the occurrence of the exposure compound and its biotransformation products (or in-source fragments of these) in the

Over the last decade environmental agencies worldwide have escalated their work to phase out animal testing for the purposes of chemical regulation. Meanwhile the number of commercially available chemicals and the requirements for hazard assessments have both increased, creating a large need for substitution of traditional in vivo assays with in vitro tests. One example of this is the replacement of the OECD acute toxicity test of adult fish (test guideline [TG] 203) with zebrafish embryos (TG 236). With new insights into the toxicological properties of chemicals, the demand on these replacement tests is also changing character with a shifted focus towards mechanistic understanding of toxicity. The omics sciences encompass a group of analytical methods which have proven to be very powerful for unveiling of mechanistic information of biochemical processes. Metabolomics is one of the younger members of this family and entails the large-scale analysis of endogenous metabolites and their perturbation in living organisms. The overall objective of this thesis was to develop modifications to the TG236 OECD assay to obtain omic data suitable for use in chemical hazard assessment. To achieve this goal, we started by developing a targeted and non-targeted metabolomics workflow and evaluated the performance of the two types of analysis (Paper I). We also evaluated the efficiency of three signal drift correction approaches, which is an important step in data quality improvement for non-targeted analysis, and reported previously unlisted biochemicals present in NIST reference material. In Paper II we applied the workflow in Paper I to a newly developed, in-plate extraction method for single zebrafish embryos which were exposed to the pharmaceutical and environmental pollutant propranolol. Data processing workflows were developed to overcome challenges arising from the occurrence of the exposure compound and its biotransformation products (or in-source fragments of these) in the

Over the last decade environmental agencies worldwide have escalated their work to phase out animal testing for the purposes of chemical regulation. Meanwhile the number of commercially available chemicals and the requirements for hazard assessments have both increased, creating a large need for substitution of traditional in vivo assays with in vitro tests. One example of this is the replacement of the OECD acute toxicity test of adult fish (test guideline [TG] 203) with zebrafish embryos (TG 236). With new insights into the toxicological properties of chemicals, the demand on these replacement tests is also changing character with a shifted focus towards mechanistic understanding of toxicity. The omics sciences encompass a group of analytical methods which have proven to be very powerful for unveiling of mechanistic information of biochemical processes. Metabolomics is one of the younger members of this family and entails the large-scale analysis of endogenous metabolites and their perturbation in living organisms. The overall objective of this thesis was to develop modifications to the TG236 OECD assay to obtain omic data suitable for use in chemical hazard assessment. To achieve this goal, we started by developing a targeted and non-targeted metabolomics workflow and evaluated the performance of the two types of analysis (Paper I). We also evaluated the efficiency of three signal drift correction approaches, which is an important step in data quality improvement for non-targeted analysis, and reported previously unlisted biochemicals present in NIST reference material. In Paper II we applied the workflow in Paper I to a newly developed, in-plate extraction method for single zebrafish embryos which were exposed to the pharmaceutical and environmental pollutant propranolol. Data processing workflows were developed to overcome challenges arising from the occurrence of the exposure compound and its biotransformation products (or in-source fragments of these) in the

Over the last decade environmental agencies worldwide have escalated their work to phase out animal testing for the purposes of chemical regulation. Meanwhile the number of commercially available chemicals and the requirements for hazard assessments have both increased, creating a large need for substitution of traditional in vivo assays with in vitro tests. One example of this is the replacement of the OECD acute toxicity test of adult fish (test guideline [TG] 203) with zebrafish embryos (TG 236). With new insights into the toxicological properties of chemicals, the demand on these replacement tests is also changing character with a shifted focus towards mechanistic understanding of toxicity. The omics sciences encompass a group of analytical methods which have proven to be very powerful for unveiling of mechanistic information of biochemical processes. Metabolomics is one of the younger members of this family and entails the large-scale analysis of endogenous metabolites and their perturbation in living organisms. The overall objective of this thesis was to develop modifications to the TG236 OECD assay to obtain omic data suitable for use in chemical hazard assessment. To achieve this goal, we started by developing a targeted and non-targeted metabolomics workflow and evaluated the performance of the two types of analysis (Paper I). We also evaluated the efficiency of three signal drift correction approaches, which is an important step in data quality improvement for non-targeted analysis, and reported previously unlisted biochemicals present in NIST reference material. In Paper II we applied the workflow in Paper I to a newly developed, in-plate extraction method for single zebrafish embryos which were exposed to the pharmaceutical and environmental pollutant propranolol. Data processing workflows were developed to overcome challenges arising from the occurrence of the exposure compound and its biotransformation products (or in-source fragments of these) in the

Over the last decade environmental agencies worldwide have escalated their work to phase out animal testing for the purposes of chemical regulation. Meanwhile the number of commercially available chemicals and the requirements for hazard assessments have both increased, creating a large need for substitution of traditional in vivo assays with in vitro tests. One example of this is the replacement of the OECD acute toxicity test of adult fish (test guideline [TG] 203) with zebrafish embryos (TG 236). With new insights into the toxicological properties of chemicals, the demand on these replacement tests is also changing character with a shifted focus towards mechanistic understanding of toxicity. The omics sciences encompass a group of analytical methods which have proven to be very powerful for unveiling of mechanistic information of biochemical processes. Metabolomics is one of the younger members of this family and entails the large-scale analysis of endogenous metabolites and their perturbation in living organisms. The overall objective of this thesis was to develop modifications to the TG236 OECD assay to obtain omic data suitable for use in chemical hazard assessment. To achieve this goal, we started by developing a targeted and non-targeted metabolomics workflow and evaluated the performance of the two types of analysis (Paper I). We also evaluated the efficiency of three signal drift correction approaches, which is an important step in data quality improvement for non-targeted analysis, and reported previously unlisted biochemicals present in NIST reference material. In Paper II we applied the workflow in Paper I to a newly developed, in-plate extraction method for single zebrafish embryos which were exposed to the pharmaceutical and environmental pollutant propranolol. Data processing workflows were developed to overcome challenges arising from the occurrence of the exposure compound and its biotransformation products (or in-source fragments of these) in the

Objective: We report a case of accidental repeated supratherapeutic dosing of paracetamol in a neonate with hyperbilirubinaemia and prolonged paracetamol elimination. Case report: A 10-day-old, 3.5 kg, male, born at 38-weeks' gestation, was referred with an elevated paracetamol concentration. The child underwent circumcision six days after birth and was accidentally given four doses of 200 mg (56 mg/kg) of paracetamol over 24 hours (224mg/kg total). Due to misinterpretation of syringe markings, the mother dosed 200 mg (2 mL) instead of 50 mg (0.5 mL). Two days later, she realized the error when using a different syringe to administer 40mg paracetamol. Blood tests by the general practitioner 19.5 hours after this last dose revealed paracetamol concentration 381 mumol/L (57mg/L), ALT 18 IU/L (normal <40), with total bilirubin 262 mumol/L (normal <300) and gamma glutamyltransferase (GGT) 83 IU/L (normal <50). In hospital, repeat paracetamol concentration, assayed 29.5 hours postlast dose, was 236 mumol/L. Bilirubin was 284 mumol/L (16.6 mg/dL). Acetylcysteine was commenced six hours later using a 2-bag, 20-hour regimen and continued for 27 hours, when serum paracetamol was undetectable and ALT was 29 IU/L. He was discharged home clinically well. On follow up one week later, he remained well, however, serum ALT was 58 IU/L with total bilirubin 308 mumol/L. Elimination half-life calculation for paracetamol was 14.5 hours with apparent first-order elimination. Conclusion(s): Elimination half-life for paracetamol in adults after therapeutic dosing is 1.5-3 hours [1] and in healthy neonates 3.5 hours [2]. After acute overdose, neonatal elimination half-life is 5.7-9 hours. In this case, hyperbilirubinemia was unlikely to interfere with the paracetamol assay, given the concentration was eventually undetectable. We hypothesize that the prolonged halflife may have been influenced by unconjugated hyperbilirubinemia which utilises glucuronidation to form conjugated bilirubin. Th

Objective: We report a case of accidental repeated supratherapeutic dosing of paracetamol in a neonate with hyperbilirubinaemia and prolonged paracetamol elimination. Case report: A 10-day-old, 3.5 kg, male, born at 38-weeks' gestation, was referred with an elevated paracetamol concentration. The child underwent circumcision six days after birth and was accidentally given four doses of 200 mg (56 mg/kg) of paracetamol over 24 hours (224mg/kg total). Due to misinterpretation of syringe markings, the mother dosed 200 mg (2 mL) instead of 50 mg (0.5 mL). Two days later, she realized the error when using a different syringe to administer 40mg paracetamol. Blood tests by the general practitioner 19.5 hours after this last dose revealed paracetamol concentration 381 mumol/L (57mg/L), ALT 18 IU/L (normal <40), with total bilirubin 262 mumol/L (normal <300) and gamma glutamyltransferase (GGT) 83 IU/L (normal <50). In hospital, repeat paracetamol concentration, assayed 29.5 hours postlast dose, was 236 mumol/L. Bilirubin was 284 mumol/L (16.6 mg/dL). Acetylcysteine was commenced six hours later using a 2-bag, 20-hour regimen and continued for 27 hours, when serum paracetamol was undetectable and ALT was 29 IU/L. He was discharged home clinically well. On follow up one week later, he remained well, however, serum ALT was 58 IU/L with total bilirubin 308 mumol/L. Elimination half-life calculation for paracetamol was 14.5 hours with apparent first-order elimination. Conclusion(s): Elimination half-life for paracetamol in adults after therapeutic dosing is 1.5-3 hours [1] and in healthy neonates 3.5 hours [2]. After acute overdose, neonatal elimination half-life is 5.7-9 hours. In this case, hyperbilirubinemia was unlikely to interfere with the paracetamol assay, given the concentration was eventually undetectable. We hypothesize that the prolonged halflife may have been influenced by unconjugated hyperbilirubinemia which utilises glucuronidation to form conjugated bilirubin. Th

ObjectivesNicotine metabolism rates differ considerably among individuals, even after controlling for variation in the major nicotine-metabolizing enzyme, CYP2A6. In this study, the impact of genetic variation in alternative metabolic enzymes and transporters on nicotine and cotinine (COT) pharmacokinetics and smoking was investigated.MethodsWe examined the impact of UGT2B10, UGT2B17, FMO3, NAT1, and OCT2 variation on pharmacokinetics and smoking (total nicotine equivalents and topography) before and after stratifying by CYP2A6 genotype in 60 African American (AA) smokers who received a simultaneous intravenous infusion of deuterium-labeled nicotine and COT.ResultsVariants in UGT2B10 and UGT2B17 were associated with urinary glucuronidation ratios (glucuronide/free substrate). UGT2B10 rs116294140 was associated with significant alterations in COT and modest alterations in nicotine pharmacokinetics. These alterations, however, were not sufficient to change nicotine intake or topography. Neither UGT2B10 rs61750900, UGT2B17*2, FMO3 rs2266782, nor NAT1 rs13253389 altered nicotine or COT pharmacokinetics among all individuals (n=60) or among individuals with reduced CYP2A6 activity (n=23). The organic cation transporter OCT2 rs316019 significantly increased nicotine and COT Cmax (P=0.005, 0.02, respectively) and decreased nicotine clearance (P=0.05). UGT2B10 rs116294140 had no significant impact on the plasma or urinary trans-3'-hydroxycotinine/COT ratio, commonly used as a biomarker of CYP2A6 activity.ConclusionWe found that polymorphisms in genes other than CYP2A6 represent minor sources of variation in nicotine pharmacokinetics, insufficient to alter smoking in AAs. The change in COT pharmacokinetics with UGT2B10 rs116294140 highlights the UGT2B10 gene as a source of variability in COT as a biomarker of tobacco exposure among AA smokers.

Clostridium difficile causes antibiotic-associated diarrhea and is a major public health concern. Current therapies disrupt the protective intestinal flora, do not reliably prevent recurrent infections, and will be decreasingly effective should less susceptible strains emerge. CRS3123 is an oral agent that inhibits bacterial methionyl-tRNA synthetase and has potent activity against C. difficile and aerobic Gram-positive bacteria but little activity against Gram-negative bacteria, including anaerobes. This first-in-human, double-blind, placebo-controlled, dose escalation study evaluated the safety and systemic exposure of CRS3123 after a single oral dose in healthy adults. Five cohorts of eight subjects each received CRS3123 or placebo in a 3:1 ratio. Doses for the respective active arms were 100 mg, 200 mg, 400 mg, 800 mg, and 1,200 mg. Blood and urine were collected for pharmacokinetic analysis. CRS3123 concentrations were measured with validated LC-MS/MS techniques. There were no serious adverse events or immediate allergic reactions during administration of CRS3123. In the CRS3123-treated groups, the most frequent adverse events were decreased hemoglobin, headache, and abnormal urine analysis; all adverse events in the active-treatment groups were mild to moderate, and their frequency did not increase with dose. Although CRS3123 systemic exposure increased at higher doses, the increase was less than dose proportional. The absorbed drug was glucuronidated at reactive amino groups on the molecule, which precluded accurate pharmacokinetic analysis of the parent drug. Overall, CRS3123 was well tolerated over this wide range of doses. This safety profile supports further investigation of CRS3123 as a treatment for C. difficile infections. (This study has been registered at ClinicalTrials.gov under identifier NCT01551004.).

ObjectivesNicotine metabolism rates differ considerably among individuals, even after controlling for variation in the major nicotine-metabolizing enzyme, CYP2A6. In this study, the impact of genetic variation in alternative metabolic enzymes and transporters on nicotine and cotinine (COT) pharmacokinetics and smoking was investigated.MethodsWe examined the impact of UGT2B10, UGT2B17, FMO3, NAT1, and OCT2 variation on pharmacokinetics and smoking (total nicotine equivalents and topography) before and after stratifying by CYP2A6 genotype in 60 African American (AA) smokers who received a simultaneous intravenous infusion of deuterium-labeled nicotine and COT.ResultsVariants in UGT2B10 and UGT2B17 were associated with urinary glucuronidation ratios (glucuronide/free substrate). UGT2B10 rs116294140 was associated with significant alterations in COT and modest alterations in nicotine pharmacokinetics. These alterations, however, were not sufficient to change nicotine intake or topography. Neither UGT2B10 rs61750900, UGT2B17*2, FMO3 rs2266782, nor NAT1 rs13253389 altered nicotine or COT pharmacokinetics among all individuals (n=60) or among individuals with reduced CYP2A6 activity (n=23). The organic cation transporter OCT2 rs316019 significantly increased nicotine and COT Cmax (P=0.005, 0.02, respectively) and decreased nicotine clearance (P=0.05). UGT2B10 rs116294140 had no significant impact on the plasma or urinary trans-3'-hydroxycotinine/COT ratio, commonly used as a biomarker of CYP2A6 activity.ConclusionWe found that polymorphisms in genes other than CYP2A6 represent minor sources of variation in nicotine pharmacokinetics, insufficient to alter smoking in AAs. The change in COT pharmacokinetics with UGT2B10 rs116294140 highlights the UGT2B10 gene as a source of variability in COT as a biomarker of tobacco exposure among AA smokers.

Clostridium difficile causes antibiotic-associated diarrhea and is a major public health concern. Current therapies disrupt the protective intestinal flora, do not reliably prevent recurrent infections, and will be decreasingly effective should less susceptible strains emerge. CRS3123 is an oral agent that inhibits bacterial methionyl-tRNA synthetase and has potent activity against C. difficile and aerobic Gram-positive bacteria but little activity against Gram-negative bacteria, including anaerobes. This first-in-human, double-blind, placebo-controlled, dose escalation study evaluated the safety and systemic exposure of CRS3123 after a single oral dose in healthy adults. Five cohorts of eight subjects each received CRS3123 or placebo in a 3:1 ratio. Doses for the respective active arms were 100 mg, 200 mg, 400 mg, 800 mg, and 1,200 mg. Blood and urine were collected for pharmacokinetic analysis. CRS3123 concentrations were measured with validated LC-MS/MS techniques. There were no serious adverse events or immediate allergic reactions during administration of CRS3123. In the CRS3123-treated groups, the most frequent adverse events were decreased hemoglobin, headache, and abnormal urine analysis; all adverse events in the active-treatment groups were mild to moderate, and their frequency did not increase with dose. Although CRS3123 systemic exposure increased at higher doses, the increase was less than dose proportional. The absorbed drug was glucuronidated at reactive amino groups on the molecule, which precluded accurate pharmacokinetic analysis of the parent drug. Overall, CRS3123 was well tolerated over this wide range of doses. This safety profile supports further investigation of CRS3123 as a treatment for C. difficile infections. (This study has been registered at ClinicalTrials.gov under identifier NCT01551004.).

UDP-glucuronosyltransferase (UGT) superfamily are glycoproteins resident of the endoplasmic reticulum membranes that undergo post-translational modifications (PTM). UGT2B7 is of particular interest because of its action on a wide variety of drugs. Most studies currently survey common variants and are only examining a small fraction of the genetic diversity. However, rare variants (frequency <1%) might have significant effect as they are predicted to greatly outnumber common variants in the human genome. Here, we discovered a rare single nucleotide UGT2B7 variant of potential pharmacogenetic relevance that encodes a nonconservative amino acid substitution at codon 121. This low-frequency variation, found in two individuals of a population of 305 healthy volunteers, leads to the translation of an asparagine (Asn) instead of an aspartic acid (Asp) (UGT2B7 p.D121N). This amino acid change was predicted to create a putative N-glycosylation motif NX(S/T) subsequently validated upon endoglycosidase H treatment of microsomal fractions and inhibition of N-glycosylation of endogenously produced UGT2B7 with tunicamycin from HEK293 cells. The presence of an additional N-linked glycan on the UGT2B7 enzyme, likely affecting proper protein folding, resulted in a significant decrease, respectively by 49 and 40%, in the formation of zidovudine and mycophenolic acid glucuronides. A systematic survey of the dbSNP database uncovered 32 rare and naturally occurring missense variations predicted to create or disrupt N-glycosylation sequence motifs in the other UGT2B enzymes. Collectively, these variants have the potential to increase the proportion of variance explained in the UGT pathway due to changes in PTM such as N-linked glycosylation with consequences on drug metabolism.

UDP-glucuronosyltransferases (UGTs) catalyse glucuronidation reactions between glucuronic acid and drug molecules, which contain nucleophilic groups, mostly hydroxyls, amines or carboxylic acids. Glucuronidation is the most important reaction in the conjugative drug metabolism. Because these conjugates are not usually able to cross cell membranes passively, they need active efflux transport. Efflux transporters mostly belong to superfamily of ATP-binding cassette transporters (ABC). Subfamily C of ABC transporters (ABCC) are known to be involved in efflux transport of glucuronides. Especially MRP2 (ABCC2) and MRP3 (ABCC3) play key roles in the elimination of glucuronide conjugates of drugs. MRP2 is localized in the apical membranes of hepatocytes and enterocytes, whereas MRP3 is localized in the basolateral membranes of the respective cells. On the other hand, UGT1A1 and UGT2B7 are highly expressed in liver and small intestine and are the most important UGTs in drug metabolism. It is known, that UGTs and efflux transporters work together forming interplay to eliminate drugs. Therefore, studying both of them in the same in vitro system is in important focus of drug metabolism studies. The Madin Darby canine kidney cell line (MDCK) is one of the standard in vitro tools in drug metabolism studies. In this study, MDCK was chosen for a cell line to co-express UGTs (UGT1A1 or UGT2B7) and efflux transporters (MRP2 and MRP3 simultaneously. Therefore, cloning of the UGT2B7 cDNA and the ABCC3 cDNA encoding MRP3 was aimed in this study. On the other hand, the UGT1A1 cDNA was already cloned in-house and MRP2 expressing MDCK cells were established earlier. Cloning of the UGT2B7 cDNA was not successful in this study despite of several different strategies such as PCR-amplification of the cDNA fragment using kidney or liver sscDNA as template. Cloning of the ABCC3 cDNA encoding MRP3 was achieved and a mammalian expression vector containing this cDNA was constructed. In addition, t, UDP-glukuronosyylitransferaasit (UGT:t) katalysoivat glukuronidaatioreaktiota, jossa glukuronidihappo liittyy lääkeaineiden nukleofiilisiin ryhmiin kuten hydroksyyleihin, amiineihin tai karboksyylihappoihin. Glukuronidaatio on kaikkein tärkein metaboliareaktio konjugatiivisessa lääkeainemetaboliassa. Glukuronidikonjugaatit eivät pysty pääsääntöisesti läpäisemään solukalvoja passiivisesti, joten ne täytyy kuljettaa aktiivisesti ulos soluista. Efflux-transportterit vastaavat yhdisteiden aktiivisesta kuljetuksesta ulos soluista. Suurin osa efflux-transporttereista kuuluu adenosiinitrifosfaattia (ATP) sitovien efflux-transportterien perheeseen (ABC), joista etenkin C-alaperhe (ABCC) osallistuu glukuronidikonjugaattien kuljetukseen ulos soluista. MRP2 (ABCC2) ja MRP3 (ABCC3) ovat tärkeässä asemassa tässä kuljetuksessa. MRP2 on lokalisoitunut hepatosyyttien ja enterosyyttien apikaalimembraanille, kun taas MRP3 on lokalisoitunut vastaavien solujen basolateraalimembraanille. Lisäksi UGT1A1 ja UGT2B7 ekspressoituvat suuressa määrin maksassa ja ohutsuolessa, ja ovat täten kaikkein tärkeimpiä UGT-entsyymejä lääkeainemetaboliassa. Tiedetään, että UGT:t ja efflux-transportterit osallistuvat yhdessä lääkeaineiden eliminaatioon ja muodostavat täten yhteispelin. Tämän takia on tärkeää tutkia kumpaakin prosessia samassa in vitro mallissa. Madin Darby koiran munuaissolulinja (MDCK) on yksi perustyökaluista lääkeainemetaboliatutkimuksissa. Tässä tutkimuksessa MDCK-solut valittiin solulinjaksi, jossa ekspressoidaan samanaikaisesti UGT:t (UGT1A1 tai UGT2B7) sekä MRP:t (MRP2 ja MRP3). Tätä varten tämän tutkimuksen tavoite oli kloonata UGT2B7:n ja ABCC3:n, joka koodaa MRP3:a, cDNA:t. Toisaalta, UGT1A1:n cDNA oli kloonattu ja MRP2:ta ekspressoivat MDCK-solut oli muodostettu jo aikaisemmin tässä laboratoriossa. UGT2B7:n cDNA:n kloonaus ei onnistunut tässä tutkimuksessa, vaikka useita eri strategioita yritettiin, kuten cDNA fragmentin PCR-amplifikaatiota maksa- ja munuais-sscDNA:sta. Toisaal

Glucuronidation, mediated by UDP-glucuronosyltransferase enzymes (UGTs), is a major phase II biotransformation pathway and, complementary to phase I metabolism and membrane transport, one of the most important cellular defense mechanism responsible for the inactivation of therapeutic drugs, other xenobiotics and numerous endogenous molecules. Individual variability in UGT enzymatic pathways is significant and may have profound pharmacological and toxicological implications. Several genetic and genomic processes are underlying this variability and are discussed in the context of drug metabolism and diseases such as cancer.

UDP-glucuronosyltransferase (UGT) superfamily are glycoproteins resident of the endoplasmic reticulum membranes that undergo post-translational modifications (PTM). UGT2B7 is of particular interest because of its action on a wide variety of drugs. Most studies currently survey common variants and are only examining a small fraction of the genetic diversity. However, rare variants (frequency <1%) might have significant effect as they are predicted to greatly outnumber common variants in the human genome. Here, we discovered a rare single nucleotide UGT2B7 variant of potential pharmacogenetic relevance that encodes a nonconservative amino acid substitution at codon 121. This low-frequency variation, found in two individuals of a population of 305 healthy volunteers, leads to the translation of an asparagine (Asn) instead of an aspartic acid (Asp) (UGT2B7 p.D121N). This amino acid change was predicted to create a putative N-glycosylation motif NX(S/T) subsequently validated upon endoglycosidase H treatment of microsomal fractions and inhibition of N-glycosylation of endogenously produced UGT2B7 with tunicamycin from HEK293 cells. The presence of an additional N-linked glycan on the UGT2B7 enzyme, likely affecting proper protein folding, resulted in a significant decrease, respectively by 49 and 40%, in the formation of zidovudine and mycophenolic acid glucuronides. A systematic survey of the dbSNP database uncovered 32 rare and naturally occurring missense variations predicted to create or disrupt N-glycosylation sequence motifs in the other UGT2B enzymes. Collectively, these variants have the potential to increase the proportion of variance explained in the UGT pathway due to changes in PTM such as N-linked glycosylation with consequences on drug metabolism.

The use of laboratory animals for toxicity testing in chemical safety assessment meets increasing ethical, economic and legislative constraints. The development, validation and application of reliable alternatives for in vivo toxicity testing are therefore urgently needed. In order to use toxicity data obtained from in vitro assays for risk assessment, in vitro concentration-response data need to be translated into in vivo dose-response data that are needed to obtain points of departure for risk assessment, like a benchmark dose (BMD). In the present study, we translated in vitro concentration-response data of the retinoid all-trans-retinoic acid (ATRA), obtained in the differentiation assay of the embryonic stem cell test, into in vivo dose-response data using a physiologically based kinetic model for rat and human that is mainly based on kinetic model parameter values derived using in vitro techniques. The predicted in vivo dose-response data were used for BMD modeling, and the obtained BMDL10 values [lower limit of the 95 % confidence interval on the BMD at which a benchmark response equivalent to a 10 % effect size (BMR10) is reached (BMD10)] for rat were compared with BMDL10 values derived from in vivo developmental toxicity data in rats reported in the literature. The results show that the BMDL10 values from predicted dose-response data differ about sixfold from the BMDL10 values obtained from in vivo data, pointing at the feasibility of using a combined in vitro-in silico approach for defining a point of departure for toxicological risk assessment.

Hepatic biotransformation and drug transport mechanisms vary significantly between species. While these processes that determine largely the kinetic behavior of drugs have been studied abundantly in dogs, corresponding investigations in cats are hardly available, despite the increasing role of cats in veterinary practice, following the increasing popularity of cats in The Netherlands. Drug intolerance, toxic side effects or a lack of efficacy have been observed when treating feline patients with drugs licensed for other species. In this thesis, we designed a series of experimental in vitro approaches that address the major phases of hepatic drug metabolism and excretion. Main findings: Phase I - Cytochrome P450 (CYP) activities and substrate specificities differ between feline and canine liver microsomes, as expected. Also gender differences are observed. However, fluorometric assays intended for rapid analysis of CYP activity of patient-derived liver biopsies, was found to be non-realistic for a clinical routine as its sensitivity is too low and in turn the amount of liver tissue needed for quantitative results is too high to allow the application in clinical diagnostics. - Diazepam, a drug that is regularly associated with undesirable hepatic side effects in cats, is converted in feline hepatic microsomes mainly into temazepam, while in dog microsomes nordiazepam appears to be the principle metabolite. The lack of quantifiable formation of nordiazepam and oxazepam in feline liver microsomes suggests a feline CYP2B11 ortholog that significantly differs from the corresponding enzyme in dogs. Phase II - A deficient UDP-glucuronosyltransferase (UGT) 1A6 activity was confirmed in feline liver microsomes, and also functional UGT2B homologs are apparently absent in the feline liver. However a functional UGT1A1 and probably other UGT1A homologs in cats were identified, albeit with a lower capacity than dogs.

Purpose: Based on recovered metabolite ratios in urine, it has been concluded that paracetamol glucuronidation may be up-regulated upon multiple dosing. This study investigates paracetamol clearance in neonates and infants after single and multiple dosing using a population modelling approach. Methods: A population pharmacokinetic model was developed in NONMEM VI, based on paracetamol plasma concentrations from 54 preterm and term neonates and infants, and on paracetamol, paracetamol-glucuronide and paracetamol-sulphate amounts in urine from 22 of these patients. Patients received either a single intravenous propacetamol dose or up to 12 repeated doses. Results: Paracetamol and metabolite disposition was best described with one-compartment models. The formation clearance of paracetamol-sulphate was 1.46 mL/min/kg1.4, which was about 5.5 times higher than the formation clearance of the glucuronide of 0.266 mL/min/kg. The renal excretion rate constants of both meta

Pyrene (PY) is a polycyclic aromatic hydrocarbon (PAH) that is often used as a biomarker for human and wildlife exposure to PAHs. As the metabolites of PAHs, similar to their parent compounds, pose public health risks, it is necessary to study their characteristics and tissue-specific distribution. The present study was performed to experimentally characterize PY metabolites and analyze the tissuespecific distribution of the conjugated metabolites after oral administration of PY to rats. PY metabolites, such as pyrenediol-disulfate (PYdiol-diS), pyrenediol-sulfate (PYdiol-S), pyrene-1-sufate (PYOS), pyrene-1-glucuronide (PYOG) and 1-hydroxypyrene (PYOH), were detected in rat urine. Although glucuronide conjugate was the predominant metabolite, the metabolite composition varied among tissues. Interestingly, the proportion of PYOH was high in the large intestine. Furthermore, PYOH was the only PY metabolite detected in feces. KEY WORDS: conjugated metabolites, glucuronidation, rat, sulfation, tissue distribution

Pyrene (PY) is a polycyclic aromatic hydrocarbon (PAH) that is often used as a biomarker for human and wildlife exposure to PAHs. As the metabolites of PAHs, similar to their parent compounds, pose public health risks, it is necessary to study their characteristics and tissue-specific distribution. The present study was performed to experimentally characterize PY metabolites and analyze the tissuespecific distribution of the conjugated metabolites after oral administration of PY to rats. PY metabolites, such as pyrenediol-disulfate (PYdiol-diS), pyrenediol-sulfate (PYdiol-S), pyrene-1-sufate (PYOS), pyrene-1-glucuronide (PYOG) and 1-hydroxypyrene (PYOH), were detected in rat urine. Although glucuronide conjugate was the predominant metabolite, the metabolite composition varied among tissues. Interestingly, the proportion of PYOH was high in the large intestine. Furthermore, PYOH was the only PY metabolite detected in feces. KEY WORDS: conjugated metabolites, glucuronidation, rat, sulfation, tissue distribution

Glucuronidation, mediated by UDP-glucuronosyltransferase enzymes (UGTs), is a major phase II biotransformation pathway and, complementary to phase I metabolism and membrane transport, one of the most important cellular defense mechanism responsible for the inactivation of therapeutic drugs, other xenobiotics and numerous endogenous molecules. Individual variability in UGT enzymatic pathways is significant and may have profound pharmacological and toxicological implications. Several genetic and genomic processes are underlying this variability and are discussed in the context of drug metabolism and diseases such as cancer.

Estrogens are female sex hormones that have genotoxic and proliferation-enhancing effects in cells. Life-time exposure to estrogens is linked to the risk of several cancers. Estrone is only a weak agonist of estrogen receptor but it serves as a precursor for biosynthesis of 17β-estradiol, 16α-hydroxyestrone and catechol estrogens. While 16α-hydroxyestrone has relatively weak affinity for estrogen receptor, it has prolonged effect due to covalent binding to the receptor. UDP-glucuronosyltransferases (UGTs) are phase II metabolic enzymes that conjugate estrogens with glucuronic acid to render them more watersoluble. Polymorphisms in UGT genes have been linked to excretion of steroids and risk of some cancers. Generally, subfamily UGT1A enzymes conjugate the 3-hydroxyls of estrogens, while the activity of subfamily UGT2B is directed towards 16- and 17-hydroxyls. Previous results on estrone glucuronidation are incomplete and conflicting, while glucuronidation of 16α-hydroxyestrone has not been systematically studied. The aim of this study was to identify UGTs active in the glucuronidation of estrone and 16α-hydroxyestrone and to further examine the glucuronidation kinetics of the active UGTs. Also the effects of bovine serum albumin (BSA), dimethyl sulfoxide (DMSO) and mutations of UGT1A10F90 and UGT1A10F93 on glucuronidation activity were examined. Activity assays were conducted using recombinant enzymes as well as human liver and intestinal microsomes. Resulting glucuronides were analyzed using high performance liquid chromatography and quantified based on their UV absorbance. UGT1A3, UGT1A10 and UGT2A1 showed the highest activity toward estrone glucuronidation, while UGT1A10, UGT2A1 and UGT2B7 were the most efficient UGTs conjugating 16α-hydroxyestrone. UGT1A10 had the highest Vmax in the glucuronidation of both substrates, although it conjugated estrone at a higher rate than 16α-hydroxyestrone. UGT1A10F93 was shown to have a role in the different glucuronidation act, Estrogeenit ovat naissukupuolihormoneja, joilla on sekä perimää vaurioittavia että solujen jakautumista kiihdyttäviä vaikutuksia. Elämänaikainen estrogeenialtistus liittyy useiden syöpien kehittymiseen. Estroni on estrogeenireseptorin heikko agonisti, mutta se on 17β-estradiolin, 16α-hydroksiestronin ja katekoliestrogeenien biosynteesin lähtöaine. Vaikka 16α-hydroksiestronilla on heikko affiniteetti estrogeenireseptoriin, se sitoutuu reseptoriin kovalenttisesti ja vaikuttaa muita estrogeeneja pidempään. UDP-glukuronosyylitransferaasit (UGT:t) ovat faasin II metaboliaentsyymejä, jotka liittävät estrogeeneihin glukuronidihapon tehden niistä vesiliukoisempia. UGT-geenien polymorfiat liittyvät steroidien erittymiseen kehosta ja joidenkin syöpien kehitykseen. UGT1A-alaperheen entsyymit glukuronidoivat pääsääntöisesti estrogeenien 3-hydroksyyliä, kun taas UGT2B-alaperheen aktiivisuus kohdistuu 16- ja 17-hydroksyyleihin. Estronin glukuronidaatiosta julkaistut tulokset ovat epätäydellisiä ja ristiriitaisia, kun taas 16α-hydroksiestronin glukuronidaatiota ei ole aiemmin tutkittu järjestelmällisesti. Tämän tutkimuksen tarkoituksena oli tunnistaa estronia ja 16α-hydroksiestronia glukuronidoivat UGT:t ja tarkastella aktiivisten entsyymien glukuronidaatiokinetiikkaa. Lisäksi tutkittiin naudan seerumin albumiinin (BSA), dimetyylisulfoksidin (DMSO) ja aminohappojen UGT1A10F90 ja UGT1A10F93 vaikutusta glukuronidaatioaktiivisuuteen. Aktiivisuusmittauksissa käytettiin sekä rekombinanttientsyymejä että ihmisen maksa- ja suolistomikrosomeja. Syntyneet glukuronidit analysoitiin korkean suorituskyvyn nestekromatografialla ja kvantitoitiin UV-absorbanssin perusteella. UGT1A3, UGT1A10 ja UGT2A1 olivat aktiivisimpia estronin glukuronidaatiossa, ja UGT1A10, UGT2A1 ja UGT2B7 konjugoivat 16α-hydroksiestronia tehokkaimmin. UGT1A10:llä oli korkein Vmaxarvo molempien substraattien konjugaatiossa, vaikka se glukuronidoi estronia nopeammin kuin 16α-hydroksiestronia. Osoittautui, että UGT1A10F93 vai

The bioavailability of a drug can be described as the fraction of an orally administered dose that reaches the systemic circulation and is often limited by first-pass metabolism in the gut and the liver. It is important to have knowledge about these processes since the systemic blood drug concentration is tightly connected to the effect of the drug. The general aim of this project was to quantitatively examine the role of the intestine in relation to the liver in first-pass metabolism of orally administered drugs. The first-pass metabolism of verapamil and raloxifene was investigated in detail with in vivo, in vitro and simulation studies, using the pig as an experimental model. The intestine contributed to the same extent as the liver to first-pass metabolism of R/S-verapamil in vivo in pigs. The S-isomer of verapamil was found in lower plasma concentrations compared to the R-isomer after oral dosing. The in vitro metabolism of verapamil in pig and human liver showed interspecies similarity and indicated equal intrinsic clearance for R- and S-verapamil. Through physiologically based pharmacokinetic modeling the stereoselectivity was explained by a combination of several processes, including enantioselective plasma protein binding, blood-to-plasma partition, and gut and liver tissue distribution. For raloxifene the intestine was the dominating organ in first-pass glucuronidation in vivo in pigs. Furthermore, the raloxifene concentration entering the intestine or the dose administered in the gut did not influence the plasma PK of raloxifene and indicated that the intestinal metabolism was not saturable with clinical relevant doses. For both verapamil and raloxifene, a time-dependent hepatic metabolism was noted with major consequences to the pharmacokinetic of the drugs. This project has pointed out the importance of intestinal metabolism in the overall first-pass extraction of drugs and indicates that intestinal metabolism should be considered and evaluated early in

1. In this study an advanced multisampling site pig model, with simultaneous venous blood sampling pre- and post liver, was applied to quantify the role of the intestine in relation to the liver in first-pass glucuronidation of raloxifene in vivo. The pharmacokinetic of raloxifene (a BCS/BDDCS class II compound) in humans is characterized by extensive metabolism (>90%) and the major metabolite is the 4'-beta-glucuronide (R-4-G). 2. Following intra-jejunal (i.j.) single dose administration in pigs raloxifene was metabolized in the gut (E G) during first-pass to more than 70% and a high concentration (AUC(0-6 h) ratio R-4-G/raloxifene >100) of R-4-G was reached in the portal vein. The hepatic extraction (E-H) of raloxifene was similar to 50% and as in humans the bioavailability become low (similar to 7%) in pigs. Interestingly the E-H of raloxifene and R-4-G was time-dependent after i.j. administration. 3. It is clear that the gut was the dominating organ in first-pass extraction of raloxifene in vivo in pigs. The quantification in this study support earlier human data and emphasize that intestinal glucuronidation should be considered early in the pharmaceutical development.

The bioavailability of a drug can be described as the fraction of an orally administered dose that reaches the systemic circulation and is often limited by first-pass metabolism in the gut and the liver. It is important to have knowledge about these processes since the systemic blood drug concentration is tightly connected to the effect of the drug. The general aim of this project was to quantitatively examine the role of the intestine in relation to the liver in first-pass metabolism of orally administered drugs. The first-pass metabolism of verapamil and raloxifene was investigated in detail with in vivo, in vitro and simulation studies, using the pig as an experimental model. The intestine contributed to the same extent as the liver to first-pass metabolism of R/S-verapamil in vivo in pigs. The S-isomer of verapamil was found in lower plasma concentrations compared to the R-isomer after oral dosing. The in vitro metabolism of verapamil in pig and human liver showed interspecies similarity and indicated equal intrinsic clearance for R- and S-verapamil. Through physiologically based pharmacokinetic modeling the stereoselectivity was explained by a combination of several processes, including enantioselective plasma protein binding, blood-to-plasma partition, and gut and liver tissue distribution. For raloxifene the intestine was the dominating organ in first-pass glucuronidation in vivo in pigs. Furthermore, the raloxifene concentration entering the intestine or the dose administered in the gut did not influence the plasma PK of raloxifene and indicated that the intestinal metabolism was not saturable with clinical relevant doses. For both verapamil and raloxifene, a time-dependent hepatic metabolism was noted with major consequences to the pharmacokinetic of the drugs. This project has pointed out the importance of intestinal metabolism in the overall first-pass extraction of drugs and indicates that intestinal metabolism should be considered and evaluated early in

The UDP-glucuronosyltransferase (UGT) family of enzymes plays a vital role in the detoxification of carcinogens as well as clearance of anti-cancer drugs. In humans, 19 UGT family members have been identified and are expressed in a tissue specific manner throughout the body. However, the UGTs have not been previously characterized in melanocytes or melanoma. In the present study, UGT2B7, UGT2B10, and UGT2B15 were identified as being normally expressed in human melanocytes. The same three UGT family members were also expressed in the primary melanoma cell line WM115. No UGT expression was detected in another primary melanoma cell line, WM3211, or in any metastatic melanoma cell line examined. These results suggest that UGT expression is lost during melanoma progression. Treatment of WM3211 or metastatic melanoma cell lines with anti-cancer agents (including vemurafenib) induced expression of UGT2B7, UGT2B10 and UGT2B15 demonstrating that melanoma cells retain the ability to re-express these same three UGTs. The corresponding increase in glucuronidation activity in melanoma cells following anti-cancer treatment was also observed. Furthermore, knockdown of UGT2B7 in WM115 cells sensitized these cells to treatment by adriamycin and epirubicin indicating that UGT2B7 is involved in resistance to these drugs. However, knockdown of UGT2B7 had no effect on temozolomide toxicity. Taken together, these results clearly demonstrate a role for UGTs in melanoma etiology. Since the UGTs are drug metabolism enzymes, we propose that re-expression of the UGTs constitutes a previously unsuspected mechanism for intratumoral drug resistance in melanoma.

Estradioli on naisten tärkein naissukupuolihormoni, ja se metaboloituu elimistössä kahdeksi katekoliestradioliksi. 2-hydroksiestradioli (2-OHE2) on normaalisti vallitseva estradiolin katekolimetaboliitti, mutta rintasyöpäpotilailla genotoksinen 4-hydroksiestradioli (4-OHE2) kohoaa vallitsevaksi katekoliestradiolimetaboliitiksi. Molemmat katekoliestradiolit muodostavat reaktiivisia kinoneja, jotka voivat DNA:ta vaurioittamalla johtaa mutaatioihin ja lopulta syövän kehittymiseen. 4-OHE2:n karsinogeenisuuden syyt eivät ole vielä täysin selvillä, mutta 4-OHE2:sta muodostuu enemmän DNA:ta vaurioittavia kinoneja. Faasi II metaboloivat entsyymit voivat inaktivoida muodostuvia katekoliestradioleita metyloimalla (COMT) tai liittämällä substraattiin glukuronihapon (UGT:t). Nämä faasi II entsyymit voivat näin suojata rintasyövältä, sillä ainoastaan konjugoimattomat katekoliestradiolit voidaan hapettaa reaktiivisiksi kinoneiksi. Tässä työssä tutkittiin ihmisen UDP-glukuronosyylitransferaasien (UGT) katalysoimia glukuronidaatioreaktioita katekoliestradioleiden kanssa. Tätä varten 2-OHE2:n ja 4-OHE2:n glukuronideille kehitettiin omat HPLC-menetelmänsä. 2-OHE2:lla havaittiin kahta eri glukuronidia. 11 UGT-isoentsyymiä katalysoi 2-OHE2:n glukuronidaatiota, ja näistä UGT:t 1A1, 1A7 ja 1A10 muodostavat molempia glukuronideja. UGT:t 1A3, 1A8, 1A9, 2A1, 2A2, 2A3, 2B7 ja 2B15 muodostavat ainoastaan toisena retentoitunutta glukuronidia, joka oli vallitseva glukuronidi myös UGT1A1:llä, UGT1A7:llä ja UGT1A10:llä. 4-OHE2:lla muodostui kolmea eri glukuronidia, mutta ensimmäisen glukuronidin pitoisuudet jäivät alle kvantitointirajan. UGT1A10 katalysoi toisena ja UGT:t 1A7, 1A8, 1A9, 2B7 ja 2B15 katalysoivat kolmantena retentoitunutta glukuronidia. Tavoitteena oli myös selvittää mihin hydroksyyliryhmään glukuronidi kiinnittyy, mutta tämä tavoite jäi saavuttamatta, sillä sopivia standardeja ei ollut saatavilla., Estradiol is a female sex hormone which is metabolized to two different catechol estradiols. 2-hydroxyestradiol (2-OHE2) is normally the major catechol estradiol metabolite but breast cancer patients have increased amounts of genotoxic 4-hydroxyestradiol (4-OHE2) and it arises to predominant metabolite with these patients. These catechol estradiols can form reactive quinones that can bind to DNA and lead to mutations and finally cause cancer. Catechol-O-methyl transferase can add methyl groups and UDP-glucuronosyl transferase (UGT) glucuronic acid groups to catechol estradiols. These phase II enzymes play important role in the inactivation of catechol estradiols because only non-conjugated catechol estradiols can be oxidized to quinones. The aim of this study was to find out which human UGTs catalyze glucuronidation of 2-OHE2 or 4-OHE2, how many different glucuronides are formed and in which part of the substrate glucuronic acid is added. To answer these questions chromatography methods for 2-OHE2 and 4-OHE2 glucuronides were developed using HPLC. Eleven UGT-enzymes glucuronidate 2-OHE2. UGTs 1A1, 1A7 and 1A10 form two different glucuronides and UGTs 1A3, 1A8, 1A9, 2A1, 2A2, 2A3, 2B7 and 2B15 form only the second glucuronide. It was possible to detect three different glucuronides for 4-OHE2 but the amount of the first glucuronide was under quantification limit. UGT1A10 catalyzed the formation of the second glucuronide and UGTs 1A7, 1A8, 1A9, 2B7 and 2B15 catalyzed the formation of the last glucuronide. One aim of the study was to find out which part of the substrate is glucuronidated but this aim was not achieved because suitable standards were not available.

All organisms are constantly and unavoidably exposed to xenobiotics including both man–made and natural chemicals such as drugs, plant alkaloids, microorganism toxins, pollutants, pesticides, and other industrial chemicals. Formally, biotransformation of xenobiotics as well as endogenous compounds is subdivided into phase I and phase II reactions. This chapter focuses on phase II biotransformation reactions (also called ´conjugation reactions´) which generally serve as a detoxifying step in metabolism of drugs and other xenobiotics as well as endogenous substrates. On the other hand, these conjugations also play an essential role in the toxicity of many chemicals due to the metabolic formation of toxic metabolites such as reactive electrophiles. Gene polymorphism of biotransformation enzymes may often play a role in various pathophysiological processes. Conjugation reactions usually involve metabolite activation by a high–energy intermediate and have been classified into two general types: type I (e.g., glucuronidation and sulfonation), in which an activated conjugating agent combines with substrate to yield the conjugated product, and type II (e.g., amino acid conjugation), in which the substrate is activated and then combined with an amino acid to yield a conjugated product. In this chapter, we will concentrate on the most important conjugation reactions, namely glucuronide conjugation, sulfoconjugation, acetylation, amino acid conjugation, glutathione conjugation and methylation.

The UDP-glucuronosyltransferase (UGT) family of enzymes plays a vital role in the detoxification of carcinogens as well as clearance of anti-cancer drugs. In humans, 19 UGT family members have been identified and are expressed in a tissue specific manner throughout the body. However, the UGTs have not been previously characterized in melanocytes or melanoma. In the present study, UGT2B7, UGT2B10, and UGT2B15 were identified as being normally expressed in human melanocytes. The same three UGT family members were also expressed in the primary melanoma cell line WM115. No UGT expression was detected in another primary melanoma cell line, WM3211, or in any metastatic melanoma cell line examined. These results suggest that UGT expression is lost during melanoma progression. Treatment of WM3211 or metastatic melanoma cell lines with anti-cancer agents (including vemurafenib) induced expression of UGT2B7, UGT2B10 and UGT2B15 demonstrating that melanoma cells retain the ability to re-express these same three UGTs. The corresponding increase in glucuronidation activity in melanoma cells following anti-cancer treatment was also observed. Furthermore, knockdown of UGT2B7 in WM115 cells sensitized these cells to treatment by adriamycin and epirubicin indicating that UGT2B7 is involved in resistance to these drugs. However, knockdown of UGT2B7 had no effect on temozolomide toxicity. Taken together, these results clearly demonstrate a role for UGTs in melanoma etiology. Since the UGTs are drug metabolism enzymes, we propose that re-expression of the UGTs constitutes a previously unsuspected mechanism for intratumoral drug resistance in melanoma.

Carvedilol is administered orally as a racemic mixture of R(+)- and S(-)-enantiomers for treatment of angina pectoris, hypertension and chronic heart failure. We have reported that enzyme kinetic parameters for carvedilol glucuronidation by human liver microsomes (HLM) differed greatly depending on the substrate form, namely, racemic carvedilol and each enantiomer. These phenomena were thought to be caused by mutual inhibition between carvedilol enantiomers during racemate glucuronidation. The aim of this study was to clarify the mechanism of these phenomena in HLM and human intestinal microsomes (HIM) and its relevance to uridine 5'-diphosphate (UDP)-glucuronosyl transferase (UGT) 1A1, UGT2B4 and UGT2B7, which mainly metabolize carvedilol directly in phase II enzymes. HLM apparently preferred metabolizing (S)-carvedilol to (R)-carvedilol in the racemate, but true activities of HLM for both glucuronidation were approximately equal. By determination of the inhibitory effects of (S)-carvedilol on (R)-carvedilol glucuronidation and vice versa, it was shown that (R)-carvedilol glucuronidation was more easily inhibited than was (S)-carvedilol glucuronidation. UGT2B7 was responsible for (S)-carvedilol glucuronidation in HLM. Ratios of contribution to (R)-carvedilol glucuronidation were approximately equal among UGT1A1, UGT2B4 and UGT2B7. However, enzyme kinetic parameters were different between the two lots of HLM used in this study, depending on the contribution ratio of UGT2B4, in which (R)-glucuronidation was much more easily inhibited by (S)-carvedilol than was (S)-glucuronidation by (R)-carvedilol. Meanwhile, HIM preferred metabolizing (R)-carvedilol, and this tendency was not different between the kinds of substrate form.

Carvedilol is administered orally as a racemic mixture of R(+)- and S(-)-enantiomers for treatment of angina pectoris, hypertension and chronic heart failure. We have reported that enzyme kinetic parameters for carvedilol glucuronidation by human liver microsomes (HLM) differed greatly depending on the substrate form, namely, racemic carvedilol and each enantiomer. These phenomena were thought to be caused by mutual inhibition between carvedilol enantiomers during racemate glucuronidation. The aim of this study was to clarify the mechanism of these phenomena in HLM and human intestinal microsomes (HIM) and its relevance to uridine 5'-diphosphate (UDP)-glucuronosyl transferase (UGT) 1A1, UGT2B4 and UGT2B7, which mainly metabolize carvedilol directly in phase II enzymes. HLM apparently preferred metabolizing (S)-carvedilol to (R)-carvedilol in the racemate, but true activities of HLM for both glucuronidation were approximately equal. By determination of the inhibitory effects of (S)-carvedilol on (R)-carvedilol glucuronidation and vice versa, it was shown that (R)-carvedilol glucuronidation was more easily inhibited than was (S)-carvedilol glucuronidation. UGT2B7 was responsible for (S)-carvedilol glucuronidation in HLM. Ratios of contribution to (R)-carvedilol glucuronidation were approximately equal among UGT1A1, UGT2B4 and UGT2B7. However, enzyme kinetic parameters were different between the two lots of HLM used in this study, depending on the contribution ratio of UGT2B4, in which (R)-glucuronidation was much more easily inhibited by (S)-carvedilol than was (S)-glucuronidation by (R)-carvedilol. Meanwhile, HIM preferred metabolizing (R)-carvedilol, and this tendency was not different between the kinds of substrate form.

The bioavailability of a drug can be described as the fraction of an orally administered dose that reaches the systemic circulation and is often limited by first-pass metabolism in the gut and the liver. It is important to have knowledge about these processes since the systemic blood drug concentration is tightly connected to the effect of the drug. The general aim of this project was to quantitatively examine the role of the intestine in relation to the liver in first-pass metabolism of orally administered drugs. The first-pass metabolism of verapamil and raloxifene was investigated in detail with in vivo, in vitro and simulation studies, using the pig as an experimental model. The intestine contributed to the same extent as the liver to first-pass metabolism of R/S-verapamil in vivo in pigs. The S-isomer of verapamil was found in lower plasma concentrations compared to the R-isomer after oral dosing. The in vitro metabolism of verapamil in pig and human liver showed interspecies similarity and indicated equal intrinsic clearance for R- and S-verapamil. Through physiologically based pharmacokinetic modeling the stereoselectivity was explained by a combination of several processes, including enantioselective plasma protein binding, blood-to-plasma partition, and gut and liver tissue distribution. For raloxifene the intestine was the dominating organ in first-pass glucuronidation in vivo in pigs. Furthermore, the raloxifene concentration entering the intestine or the dose administered in the gut did not influence the plasma PK of raloxifene and indicated that the intestinal metabolism was not saturable with clinical relevant doses. For both verapamil and raloxifene, a time-dependent hepatic metabolism was noted with major consequences to the pharmacokinetic of the drugs. This project has pointed out the importance of intestinal metabolism in the overall first-pass extraction of drugs and indicates that intestinal metabolism should be considered and evaluated early in

Carvedilol is administered orally as a racemic mixture of R(+)- and S(-)-enantiomers for treatment of angina pectoris, hypertension and chronic heart failure. We have reported that enzyme kinetic parameters for carvedilol glucuronidation by human liver microsomes (HLM) differed greatly depending on the substrate form, namely, racemic carvedilol and each enantiomer. These phenomena were thought to be caused by mutual inhibition between carvedilol enantiomers during racemate glucuronidation. The aim of this study was to clarify the mechanism of these phenomena in HLM and human intestinal microsomes (HIM) and its relevance to uridine 5'-diphosphate (UDP)-glucuronosyl transferase (UGT) 1A1, UGT2B4 and UGT2B7, which mainly metabolize carvedilol directly in phase II enzymes. HLM apparently preferred metabolizing (S)-carvedilol to (R)-carvedilol in the racemate, but true activities of HLM for both glucuronidation were approximately equal. By determination of the inhibitory effects of (S)-carvedilol on (R)-carvedilol glucuronidation and vice versa, it was shown that (R)-carvedilol glucuronidation was more easily inhibited than was (S)-carvedilol glucuronidation. UGT2B7 was responsible for (S)-carvedilol glucuronidation in HLM. Ratios of contribution to (R)-carvedilol glucuronidation were approximately equal among UGT1A1, UGT2B4 and UGT2B7. However, enzyme kinetic parameters were different between the two lots of HLM used in this study, depending on the contribution ratio of UGT2B4, in which (R)-glucuronidation was much more easily inhibited by (S)-carvedilol than was (S)-glucuronidation by (R)-carvedilol. Meanwhile, HIM preferred metabolizing (R)-carvedilol, and this tendency was not different between the kinds of substrate form.

All organisms are constantly and unavoidably exposed to xenobiotics including both man–made and natural chemicals such as drugs, plant alkaloids, microorganism toxins, pollutants, pesticides, and other industrial chemicals. Formally, biotransformation of xenobiotics as well as endogenous compounds is subdivided into phase I and phase II reactions. This chapter focuses on phase II biotransformation reactions (also called ´conjugation reactions´) which generally serve as a detoxifying step in metabolism of drugs and other xenobiotics as well as endogenous substrates. On the other hand, these conjugations also play an essential role in the toxicity of many chemicals due to the metabolic formation of toxic metabolites such as reactive electrophiles. Gene polymorphism of biotransformation enzymes may often play a role in various pathophysiological processes. Conjugation reactions usually involve metabolite activation by a high–energy intermediate and have been classified into two general types: type I (e.g., glucuronidation and sulfonation), in which an activated conjugating agent combines with substrate to yield the conjugated product, and type II (e.g., amino acid conjugation), in which the substrate is activated and then combined with an amino acid to yield a conjugated product. In this chapter, we will concentrate on the most important conjugation reactions, namely glucuronide conjugation, sulfoconjugation, acetylation, amino acid conjugation, glutathione conjugation and methylation.

The bioavailability of a drug can be described as the fraction of an orally administered dose that reaches the systemic circulation and is often limited by first-pass metabolism in the gut and the liver. It is important to have knowledge about these processes since the systemic blood drug concentration is tightly connected to the effect of the drug. The general aim of this project was to quantitatively examine the role of the intestine in relation to the liver in first-pass metabolism of orally administered drugs. The first-pass metabolism of verapamil and raloxifene was investigated in detail with in vivo, in vitro and simulation studies, using the pig as an experimental model. The intestine contributed to the same extent as the liver to first-pass metabolism of R/S-verapamil in vivo in pigs. The S-isomer of verapamil was found in lower plasma concentrations compared to the R-isomer after oral dosing. The in vitro metabolism of verapamil in pig and human liver showed interspecies similarity and indicated equal intrinsic clearance for R- and S-verapamil. Through physiologically based pharmacokinetic modeling the stereoselectivity was explained by a combination of several processes, including enantioselective plasma protein binding, blood-to-plasma partition, and gut and liver tissue distribution. For raloxifene the intestine was the dominating organ in first-pass glucuronidation in vivo in pigs. Furthermore, the raloxifene concentration entering the intestine or the dose administered in the gut did not influence the plasma PK of raloxifene and indicated that the intestinal metabolism was not saturable with clinical relevant doses. For both verapamil and raloxifene, a time-dependent hepatic metabolism was noted with major consequences to the pharmacokinetic of the drugs. This project has pointed out the importance of intestinal metabolism in the overall first-pass extraction of drugs and indicates that intestinal metabolism should be considered and evaluated early in

The general aim of this thesis was to improve the understanding of the in vivo pharmacokinetics and, in particular, the metabolism of finasteride, a 5α-reductase inhibitor used in the treatment of enlarged prostate glands and male pattern baldness. CYP3A4 has been identified as the major enzyme involved in the sequential metabolism of finasteride to ω-OH finasteride (M1) and ω-COOH finasteride (M3). The consequences of induced and inhibited metabolism on the pharmacokinetics of finasteride and its metabolites were investigated in humans and pigs. Both studies included bile collection. The collected human and pig samples were used for the metabolite identification. As expected, induced metabolism led to reduced plasma exposure of finasteride and inhibited metabolism had the opposite effect. The interactions were investigated in detail and included examination of the biliary pharmacokinetics of finasteride and its metabolites. In pigs, the study included monitoring of the hepatic extraction over time, deconvolution and the development of a semi-physiological model for comparison of the effects on the gut wall and liver metabolism. For M3, the concentration ratios of bile to plasma and the renal clearance indicated that carrier-mediated processes are involved in the biliary and urinary excretion. This was not, however, the case for finasteride. The metabolite, M1, could not be quantified either in humans or pigs. Instead, two other OH metabolites, M1 isomers, were identified in humans. These metabolites were found to undergo glucuronide conjugation. In humans, one glucuronide was identified intact and in pigs, both glucuronides were identified intact in bile and in urine. In addition, a glucuronide of M3 was identified in human bile. In conclusion, advances have been made in the understanding of the pharmacokinetics of finasteride, in particular in relation to the metabolism. Hopefully, the findings of this comprehensive investigation can be applied to other drugs and

The general aim of this thesis was to improve the understanding of the in vivo pharmacokinetics and, in particular, the metabolism of finasteride, a 5α-reductase inhibitor used in the treatment of enlarged prostate glands and male pattern baldness. CYP3A4 has been identified as the major enzyme involved in the sequential metabolism of finasteride to ω-OH finasteride (M1) and ω-COOH finasteride (M3). The consequences of induced and inhibited metabolism on the pharmacokinetics of finasteride and its metabolites were investigated in humans and pigs. Both studies included bile collection. The collected human and pig samples were used for the metabolite identification. As expected, induced metabolism led to reduced plasma exposure of finasteride and inhibited metabolism had the opposite effect. The interactions were investigated in detail and included examination of the biliary pharmacokinetics of finasteride and its metabolites. In pigs, the study included monitoring of the hepatic extraction over time, deconvolution and the development of a semi-physiological model for comparison of the effects on the gut wall and liver metabolism. For M3, the concentration ratios of bile to plasma and the renal clearance indicated that carrier-mediated processes are involved in the biliary and urinary excretion. This was not, however, the case for finasteride. The metabolite, M1, could not be quantified either in humans or pigs. Instead, two other OH metabolites, M1 isomers, were identified in humans. These metabolites were found to undergo glucuronide conjugation. In humans, one glucuronide was identified intact and in pigs, both glucuronides were identified intact in bile and in urine. In addition, a glucuronide of M3 was identified in human bile. In conclusion, advances have been made in the understanding of the pharmacokinetics of finasteride, in particular in relation to the metabolism. Hopefully, the findings of this comprehensive investigation can be applied to other drugs and