Rate Of Glucuronidation Research Articles

Species Differences in Ezetimibe Glucuronidation

Background: Peclinical and clinical studies have revealed that ezetimibe, an approved cholesterol-absorption inhibitor, is rapidly and extensively metabolized to a more potent metabolite, ezetimibe glucuronide. Since different species are commonly used in the pharmacokinetic and pharmacodynamic studies of ezetimibe, it is essential to determine the species difference in glucuronidation of ezetimibe in order to accurately evaluate ezetimibe’s pharmacokinetics and pharmacodynamics. The purpose of the study was to compare species differences in ezetimibe glucuronidation rates using intestinal microsomes from humans, rats, mice, monkeys, and dogs. Method: Intestinal microsomes from different species were used to assess the ezetimibe glucuronidation rates. Multiple substrate concentrations at 0.5, 2, 5, 10, 20, 30, 40, and 50 µM were tested and fitted into the Michaelis–Menten model to determine the enzyme kinetic parameters. Results: The results showed that the glucuronidation rates with these tested species were significantly different. Kinetic studies revealed that the maximum metabolic rate (Vmax) was higher in monkeys (3.87 ± 0.22 nmol/mg/min) than that in rat (2.40 ± 0.148 nmol/mg/min) and mouse (2.23 ± 0.10 nmol/mg/min), and then human (1.90 ± 0.08 nmol/mg/min) and dog (1.19 ± 0.06 nmol/mg/min). The CLint was an 8.17-fold difference among these species, following the order of mouse > dog > human > rat = monkey. Conclusions: The study revealed that the rate of ezetimibe glucuronidation in the intestine was different in different species and has an impact on ezetimibe glucuronidation in the intestine. When analyzing the pharmacodynamics, pharmacokinetics, or toxicology of ezetimibe using different models, these species differences must be taken into consideration.

Absorption, distribution, metabolism and excretion of linaprazan glurate in rats

Linaprazan (AZD0865, TX07) is one of potassium-competitive acid blockers. However, linaprazan is rapidly excreted from the body, shortening its acid inhibition property. Linaprazan glurate (X842) is a prodrug of linaprazan with a prolonged inhibitory effect on gastric acid secretion. Linaprazan glurate has entered clinical trials, but few studies have reported its metabolism in non-clinical and clinical settings. In this study, we studied the pharmacokinetics, tissue distribution, mass balance, and metabolism of linaprazan glurate in rats after a single oral dose of 2.4 mg/kg (100 µCi/kg) [14C]linaprazan glurate. The results demonstrated that linaprazan glurate was mainly excreted via feces in rats with 70.48% of the dose over 168 h. The plasma AUC0–∞ of linaprazan glurate in female rats was 2 times higher than that in male rats. Drug-related substances were mainly concentrated in the stomach, eyes, liver, small intestine, and large intestine after administration. In blood, drug-related substances were mostly distributed into plasma instead of hemocytes. In total, 13 metabolites were detected in rat plasma, urine, feces, and bile. M150 (2,6-dimethylbenzoic acid) was the predominant metabolite in plasma, accounting for 80.65% and 67.65% of AUC0–24h in male and female rats, respectively. Based on the structures, linaprazan glurate was mainly hydrolyzed into linaprazan, followed by a series of oxidation, dehydrogenation, and glucuronidation in rats. Besides, CES2 is the main metabolic enzyme involved in the hydrolysis of linaprazan glurate to linaprazan.

Influence of polymorphism of enzymes of the UDP family-glucuronyl transferases on the biotransformation of tamoxifen in the therapy of luminal forms of breast cancer

Annotation. Tamoxifen (TAM) (1-[4-(2-dimethylaminoethoxy)-phenyl]-1,2-diphenylbut-1(Z)-ene) is a non-steroidal selective estrogen receptor modulator (SERM), which is recognized as the "gold standard" of hormone therapy for estrogen-dependent breast cancer (BC). It is known that adjuvant treatment with TAM increases recurrence-free survival and overall survival in patients with hormone-receptor-positive breast cancer. Also, tamoxifen manifests itself as a partial estrogen agonist, which can be associated with the development of complications such as endometrial cancer, venous thromboembolism, etc. The presence of resistance and relapses during TAM therapy, which reach up to 30%, remains an actual problem. Therefore, studying the mechanisms underlying the individualization of both therapeutic effect and toxicity associated with TAM remains an important challenge. In the detoxification of both TAM and its active metabolites, glucuronidation processes, which belong to the second phase of biotransformation of xenobiotics and actively take place in the liver as well as in the mammary gland, play an important role, and therefore the study of this process can contribute to the understanding of the interindividual variability of the therapeutic effect and toxicity of TAM. The aim – to analyze the data of the scientific literature on the study of the influence of glucuronyltransferase (UGT) enzymes and their polymorphic forms on the biotransformation of TAM and its active metabolites in the treatment of hormone-receptor-positive breast cancer. A retrospective analysis of the literature of scientific databases Scopus, Web of Science, PubMed., MedLines for 2013-2023 was carried out. It is possible to draw the following conclusions that UGT isozymes are responsible for the conjugation and detoxification of tamoxifen and its metabolites in the form of glucuronides 4-OH-tamoxifen-N-glucuronide, 4-OH-tamoxifen-O-glucuronide and endoxifen-O-glucuronide. UGT1A8, UGT1A10, UGT2B7, UGT2B15 and UGT2B17 isoforms played the greatest role in glucuronidation of tamoxifen and its active metabolites, but UGT1A4 was recognized as the main one. Depending on the content of active TAM metabolites and their glucuronides in the blood plasma, it can be stated that carriers of the UGT2B15 Lys523Thr and UGT2B17del alleles demonstrated increased enzyme activity, and individuals with one variant UGT2B15 523Thr allele can even be considered superactive metabolizers of 4-OH-tamoxifen-O- glucuronide and endoxifen-glucuronide. Also, high levels of 4-OH-tamoxifen-N-glucuronide were observed in carriers of the allele of the UGT2B17del genotype. Carriers of the above alleles have high activity of glucuronidation processes and low levels of active metabolites of TAM, which calls into question the rationality of prescribing TAM as hormone therapy. In contrast, patients with UGT1A4 48Val, UGT2B7 268Tyr alleles, or with wild-type genotypes for UGT2B17 nodel and UGT2B15 523Lys, will have high levels of active metabolites and are the group of choice for tamoxifen therapy in estrogen-receptor-positive breast cancer because they will have a low rate of glucuronidation and detoxification. However, in order to create a system of clinical algorithms for the formation of tamoxifen-sensitive groups of patients, further detailed study of other possibilities of the biotransformation system in the metabolism of tamoxifen is required.

Urolithin A conjugation with NSAIDs inhibits its glucuronidation and maintains improvement of Caco-2 monolayers' barrier function

Urolithin A (UA) is an ellagitannin-derived postbiotic metabolite which emerged as a promising health-boosting agent, promoting mitophagy, improving skeletal muscle function, and suppressing the inflammatory response. However, phase II intestinal metabolism severely limits its biopotency, leading to the formation of nonactive glucuronides. To address this constraint, a set of new UA derivatives (UADs), conjugated with nonsteroidal anti-inflammatory drugs (NSAIDs), was synthesized. The bioavailability and inhibitory activity of UADs against UA-glucuronidation were evaluated using differentiated Caco-2 cell monolayers. Parallelly, after the administration of tested substances, the transepithelial electrical resistance (TEER) of the cell monolayers was continuously monitored using the CellZscope device. Though investigated UADs did not penetrate Caco-2 monolayers, all of them significantly suppressed the glucuronidation rate of UA, while conjugates with diclofenac increased the concentration of free molecule on the basolateral side. Moreover, esters of UA with diclofenac (DicloUA) and aspirin (AspUA) positively influenced cell membrane integrity. Western blot analysis revealed that some UADs, including DicloUA, increased the expression of pore-sealing tight junction proteins and decreased the level of pore-forming claudin-2, which may contribute to their beneficial activity towards the barrier function. To provide comprehensive insight into the mechanism of action of DicloUA, Caco-2 cells were subjected to transcriptomic analysis. Next-generation sequencing (NGS) uncovered substantial changes in the expression of genes involved, for instance, in multivesicular body organization and zinc ion homeostasis. The results presented in this study offer new perspectives on the beneficial effects of modifying UA's structure on its intestinal metabolism and bioactivity in vitro.

Physiologically-based pharmacokinetic modeling of the postbiotic supplement urolithin A predicts its bioavailability is orders of magnitudes lower than concentrations that induce toxicity, but also neuroprotective effects.

A range of health benefits are attributed to consuming urolithin A (UA), such as improved muscle health, anti-aging activity and neuroprotection, whereas few studies raise possible adverse effects at high doses, including genotoxicity and estrogenic effects. Therefore, understanding UA bioactivity and safety depends on its pharmacokinetics. However, there is no physiologically-based pharmacokinetic (PBPK) model available for UA, thus limiting reliable assessment of effects observed from in vitro experimentation. We characterized glucuronidation rates of UA by human S9 fractions. Partitioning and other physicochemical parameters were predicted using quantitative structure-activity relationship tools. Solubility and dissolution kinetics were determined experimentally. These parameters were used to construct a PBPK model, and results were compared with data from human intervention studies. We evaluated how different supplementation scenarios may influence UA plasma and tissue concentrations. Concentrations at which either toxic or beneficial effects were previously observed in vitro appear unlikely to be achieved in vivo. We established a first PBPK model for UA. It enables prediction of systemic UA concentrations and is critical for extrapolating in vitro results to in vivo uses. Results support the safety of UA, but also challenge the potential for readily achieving beneficial effects by postbiotic supplementation. This article is protected by copyright. All rights reserved.

Discovery of novel pyridinones as MGAT2 inhibitors for the treatment of metabolic disorders

Inhibition of monoacylglycerol transferase 2 (MGAT2) has recently emerged as a potential therapeutic strategy for the treatment of metabolic diseases such as obesity, diabetes and non-alcoholic steatohepatitis (NASH). Metabolism studies with our clinical lead (1) suggested variability in in vitro glucuronidation rates in liver microsomes across species, which made projection of human doses challenging. In addition, the observation of deconjugation of the C3-C4 double bond in the dihydropyridinone ring of 1 in solution had the potential to complicate its clinical development. This report describes our lead optimization efforts in a novel pyridinone series, exemplified by compound 33, which successfully addressed both of these potential issues.

Evaluation of metabolism and cytochrome P450 mediated interaction liabilities of naringenin

Naringenin is one of the major components of grapefruit juice. It has a broad spectrum of pharmacological activities, and many studies report that grapefruit juice inhibits cytochrome P450 (CYP) 3A4 leading to drug interactions. Naringenin was profiled through various in vitro studies like metabolic stability and glucuronidation in rat and human liver microsomes while, CYP inhibition using human liver microsomes. In addition, pharmacokinetic profiling was conducted upon intravenous (i.v.) and oral administration in rats. Naringenin undergoes both phase I and phase II metabolism in rat liver microsomes, and in human liver microsomes, it is predominantly metabolized by phase II. Glucuronidation which is addition (conjugation) of glucuronic acid to various functional groups is one of the major metabolic pathways of Naringenin. Naringenin, at 1.0 μM and 10.0 μM, did not elicit any appreciable inhibition of the 5 major CYP isoforms (CYP1A2, CYP3A4, CYP2C9, CYP2C19, and CYP2D6). Oral pharmacokinetic studies at 100, 300,and 1000 mg/kg dose and intravenous pharmacokinetic studies at 1 mg/kg dose were performed in male SD rats. Naringenin exhibited very short half-life (0.27 h) and rapid elimination (Clearance=110.65 mL/min/kg) after i.v. administration. There was saturation in Cmax and exposure beyond 100 mg/kg, and the absolute bioavailability was found to be ≤ 5% at the tested oral doses. This present experiment suggests that naringenin does not substantially inhibit CYP3A4 (or any of the tested five isoforms) isoforms per se. Given the minimal involvement of CYP enzymes in the metabolism of naringenin and minimal inhibition of CYP enzymes (IC50> 10 μM), the potential for drug-drug interactions involving CYP substrates and inhibitors is very minimal in humans.

Characterization of Enzymes Involved in Nintedanib Metabolism in Humans.

Nintedanib, which is used to treat idiopathic pulmonary fibrosis and non-small cell lung cancer, is metabolized to a pharmacologically inactive carboxylate derivative, BIBF1202, via hydrolysis and subsequently by glucuronidation to BIBF1202 acyl-glucuronide (BIBF1202-G). Since BIBF1202-G contains an ester bond, it can be hydrolytically cleaved to BIBF1202. In this study, we sought to characterize these metabolic reactions in the human liver and intestine. Nintedanib hydrolysis was detected in human liver microsomes (HLMs) (Clearance [CL int]: 102.8 ± 18.9 µL/min per mg protein) but not in small intestinal preparations. CES1 was suggested to be responsible for nintedanib hydrolysis according to experiments using recombinant hydrolases and hydrolase inhibitors as well as proteomic correlation analysis using 25 individual HLM. BIBF1202 glucuronidation in HLM (3.6 ± 0.3 µL/min per mg protein) was higher than that in human intestinal microsomes (1.5 ± 0.06 µL/min per mg protein). UGT1A1 and gastrointestinal UGT1A7, UGT1A8, and UGT1A10 were able to mediate BIBF1202 glucuronidation. The impact of UGT1A1 on glucuronidation was supported by the finding that liver microsomes from subjects homozygous for the UGT1A1*28 allele showed significantly lower activity than those from subjects carrying the wild-type UGT1A1 allele. Interestingly, BIBF1202-G was converted to BIBF1202 in HLS9 at 70-fold higher rates than the rates of BIBF1202 glucuronidation. An inhibition study and proteomic correlation analysis suggested that β-glucuronidase is responsible for hepatic BIBF1202-G deglucuronidation. In conclusion, the major metabolic reactions of nintedanib in the human liver and intestine were quantitatively and thoroughly elucidated. This information could be helpful to understand the inter- and intraindividual variability in the efficacy of nintedanib. SIGNIFICANCE STATEMENT: To our knowledge, this is the first study to characterize the enzymes responsible for each step of nintedanib metabolism in the human body. This study found that β-glucuronidase may contribute to BIBF1202-G deglucuronidation.

Abstract P1-04-18: Identification of UGT2B family genes as potential biomarkers of response to neoadjuvant therapy in HER2+ breast cancer

Abstract INTRODUCTION Human Epidermal Growth Factor Receptor (HER2) is overexpressed and/or amplified in 15-20% of breast cancer at time of first diagnosis and is associated with an aggressive clinical progression disease. Management of HER2+ early breast cancer, include a combination of sequential chemotherapy and HER2-targeted therapy, which is currently the gold standard of care. Achieving pathological complete response (pCR) to after neoadjuvant therapy has been proposed as a prognostic marker for the prediction of disease-free survival (DFS). Although there are different criteria to define pCR, in general pathological complete response is the absence of residual disease in breast and lymph nodes in the pathology analysis of the surgical piece resection (ypTo/is ypN0). However, there is a compelling need to identify molecular biomarkers that will help determine which patients will benefit from neoadjuvant therapy. Considering that the UGT2B family plays an important role in drug metabolism, our aim is to analyze its role as potential predictive biomarkers of pCR to neoadjuvant therapy in breast cancer. METHODS We included 18 female patients diagnosed as early breast cancer HER2+ at the time of neoadjuvant treatment. Tissue samples were obtained from the surgical resection performed at Virgen del Rocio Hospital (Seville, Spain) and were formalin-fixed paraffin-embedded (FFPE) to subsequently carry out the pertinent analyses of the study. All patients were treated with chemotherapy plus anti-HER2-targeted therapy and were stratified according to response to neoadjuvant treatment. We defined two groups: responder (R; RCB-0; n=14) and non-responder (NR; RCB-II/III; n=4). Total RNA from FFPE tissue was extracted using the RecoverAll Total Nucleic Acid Isolation commercial kit (Ambion, Austin, TX, USA) following the manufacturer’s instructions. RNA concentration was measured using the NanoDrop ND-1000 spectrophotometer (Nanodrop Tech, DE, USA). A total of 18 samples were labeled and hybridized with a Clariom D pico Array microarray (Affymetrix, Santa Clara, CA, USA) following the manufacturer’s instructions. Differences in expressed genes according to response were identified using the Mann–Whitney U test where p -values. < 0.05 were considered significant and ROC curve was used to determine predictor value of complete response for each member of UGT2 family. Differential expression between the two groups was analyzed using the statistical packages SPSS version 28 and R 4.1.1.1. Statistical significance was established for p-values ​​< 0.05 and fold change ≥ 2 RESULTS We found 954 differentially expressed transcripts: 311 downregulated transcripts (lower expression in R) and 643 upregulated transcripts (higher expression in R). Our data showed a significant upregulation in 5 genes of the UGT2B family in non-responder patients (p-value< 0.05). 3 genes of the UGT2b family showed an acceptable ability to discriminate responders and non-responders to neoadjuvant treatment of HER2-positive in the ROC (receiver operating characteristic curve) analysis. CONCLUSIONS We identified a set of transcript differentially expressed (FC>2; adjusted p-value< 0.05) in patients with residual disease (nR) when compared with tissue samples in patients that achieve pCR (R). UGT2B family genes are consistently upregulated in non-responder patients. UGT2B genes encode enzymes involved in glucuronidation. They are phase II drug-metabolizing enzymes with steroids removal capabilities in the liver and various steroid target tissues. An increased rate of glucuronidation is been associated with a loss of potency for the target drugs. These genes would be used as predictive biomarkers of pCR to neoadjuvant therapy in breast cancer. Citation Format: Ana Gil-Torralvo, Marta Benavent, Maria A Dominguez-Cejudo, Alejandro Falcon, Mónica Cejuela, Begoña Vieites, Sonia Molina-Pinelo, Manuel Ruiz Borrego, Juan de la Haba-Rodríguez, Maria I Queipo, Francisco Javier Salvador Bofill. Identification of UGT2B family genes as potential biomarkers of response to neoadjuvant therapy in HER2+ breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-04-18.

Contribution of UGT Enzymes to Human Drug Metabolism Stereoselectivity: A Case Study of Medetomidine, RO5263397, Propranolol, and Testosterone.

The enantiomeric forms of chiral compounds have identical physical properties but may vary greatly in their metabolism by individual enzymes. Enantioselectivity in UDP-glucuronosyl transferase (UGT) metabolism has been reported for a number of compounds and with different UGT isoforms involved. However, the impact of such individual enzyme results on overall clearance stereoselectivity is often not clear. The enantiomers of medetomidine, RO5263397, and propranolol and the epimers testosterone and epitestosterone exhibit more than a 10-fold difference in glucuronidation rates by individual UGT enzymes. In this study, we examined the translation of human UGT stereoselectivity to hepatic drug clearance considering the combination of multiple UGTs to overall glucuronidation, the contribution of other metabolic enzymes such as cytochrome P450s (P450s), and the potential for differences in protein binding and blood/plasma partitioning. For medetomidine and RO5263397, the high individual enzyme (UGT2B10) enantioselectivity translated into ∼3- to >10-fold differences in predicted human hepatic in vivo clearance. For propranolol, the UGT enantioselectivity was irrelevant in the context of high P450 metabolism. For testosterone, a complex picture emerged due to differential epimeric selectivity of various contributing enzymes and potential for extrahepatic metabolism. Quite different patterns of P450- and UGT-mediated metabolism were observed across species, as well as differences in stereoselectivity, indicating that extrapolation from human enzyme and tissue data are essential when predicting human clearance enantioselectivity. SIGNIFICANCE STATEMENT: Individual enzyme stereoselectivity illustrates the importance of three-dimensional drug-metabolizing enzyme-substrate interactions and is essential when considering the clearance of racemic drugs. However, translation from in vitro to in vivo can be challenging as contributions from multiple enzymes and enzyme classes must be combined with protein binding and blood/plasma partitioning data to estimate the net intrinsic clearance for each enantiomer. Preclinical species may be misleading as enzyme involvement and metabolism stereoselectivity can differ substantially.

Dimethandrolone, a Potential Male Contraceptive Pill, is Primarily Metabolized by the Highly Polymorphic UDP-Glucuronosyltransferase 2B17 Enzyme in Human Intestine and Liver.

Dimethandrolone undecanoate (DMAU), an oral investigational male hormonal contraceptive, is a prodrug that is rapidly converted to its active metabolite, dimethandrolone (DMA). Poor and variable oral bioavailability of DMA after DMAU dosing is a critical challenge to develop it as an oral drug. The objective of our study was to elucidate the mechanisms of variable pharmacokinetics of DMA. We first identified DMA metabolites formed in vitro and in vivo in human hepatocyte incubation and serum samples following oral DMAU administration in men, respectively. The metabolite identification study revealed two metabolites, DMA-glucuronide (DMA-G; major) and the androstenedione analog of DMA (minor), in the hepatocyte incubations. After oral DMAU administration, only DMA-G was detected in serum, which was >100-fold compared with DMA levels, supporting glucuronidation as the major elimination mechanism for DMA. Next, 13 clinically relevant UDP-glucuronosyltransferase (UGT) enzymes were tested for their involvement in DMA-G formation, which revealed a major role of UDP-glucuronosyltransferase 2B17 (UGT2B17) isoform with a smaller contribution of UGT1A9 in DMA-G formation. These data were confirmed by dramatically higher DMA glucuronidation rates (>200- and sevenfold) in the high versus the null UGT2B17-expressing human intestinal and liver microsomes, respectively. Since human UGT2B17 is a highly variable enzyme with a 20%-80% gene deletion frequency, the in vitro data suggest a major role of UGT2B17 polymorphism on the first-pass metabolism of DMA. Further, considering DMA is a selective and sensitive UGT2B17 substrate, it could be used as a clinical probe of UGT2B17 activity. SIGNIFICANCE STATEMENT: Dimethandrolone (DMA) is an active metabolite of dimethandrolone undecanoate (DMAU), an investigational male hormonal contraceptive. Previous studies have indicated poor and inconsistent bioavailability of DMAU following oral administration. This study found that UDP-glucuronosyltransferase 2B17-mediated high intestinal first-pass metabolism is the key mechanism of variable DMA bioavailability.

Simulation of the crosstalk between glucose and acetaminophen metabolism in a liver zonation model.

The liver metabolizes a variety of substances that sometimes interact and regulate each other. The modeling of a single cell or a single metabolic pathway does not represent the complexity of the organ, including metabolic zonation (heterogeneity of functions) along with liver sinusoids. Here, we integrated multiple metabolic pathways into a single numerical liver zonation model, including drug and glucose metabolism. The model simulated the time-course of metabolite concentrations by the combination of dynamic simulation and metabolic flux analysis and successfully reproduced metabolic zonation and localized hepatotoxicity induced by acetaminophen (APAP). Drug metabolism was affected by nutritional status as the glucuronidation reaction rate changed. Moreover, sensitivity analysis suggested that the reported metabolic characteristics of obese adults and healthy infants in glucose metabolism could be associated with the metabolic features of those in drug metabolism. High activities of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphate phosphatase in obese adults led to increased APAP oxidation by cytochrome P450 2E1. In contrast, the high activity of glycogen synthase and low activities of PEPCK and glycogen phosphorylase in healthy infants led to low glucuronidation and high sulfation rates of APAP. In summary, this model showed the effects of glucose metabolism on drug metabolism by integrating multiple pathways into a single liver metabolic zonation model.

Abstract P2-07-08: Identification of UGT2B15 as a potential biomarker in response to neoadjuvant therapy in HER2+ breast cancer

Abstract INTRODUCTION. In response to neoadjuvant therapy, pathological complete response (pCR; lack of residual disease in breast and lymph nodes), has been proposed as a prognostic marker for long-term clinical outcomes, such as disease-free (DFS) and overall survival (OS), in human epidermal growth factor receptor 2 (HER2)-positive and triple-negative breast cancer. In clinical practice, recognizing those patients likely to achieve such responses proves challenging to the oncologist, making the identification of new useful biomarkers vital. Here, we searched for potential biomarkers that anticipate pCR to neoadjuvant therapy in HER2-positive, hormone receptor-negative breast cancer tissue samples. METHODS. Patients with early and locally advanced breast cancer, diagnose as HER2-positive, hormone receptor-negative by immunohistochemistry (IHC) at the time of neoadjuvant treatment were included. All samples were collected from biobank at Hospital Universitario Virgen del Rocio. We defined two groups: responder (R) and non-responder (nR) and analyzed 18 samples in the discovery cohort (10 R vs 8 nR) and 12 samples in the validation cohort (6 R vs 6 nR).The RNA for the study was extracted from tissue fixed in formaldehyde and paraffin embedded. The extraction of the RNA was carried out using the commercial kit RecoverAll Total Nucleic Acid Isolation Kit from Ambion (Applied Biosystems). RNA was quantified by Qubit RNA HS Assay Kit (Molecular Probes). Before hybridization, RNA for the discovery cohort was amplified using GeneChip WT Pico Kit (Applied Biosystems).We analyzed transcript expression using ClariomD array. Differential expression between the two groups was analyzed using in-house R scripts (version 3.5.1). Data were corrected and normalized using Robust Multi-array Average method. Expression was summarized at gene level using the corresponding annotation for ClariomD BrainArray. Gene validation was performed by qPCR using TaqMan Gene Expression Assay (Applied Biosystem). RESULTS. Considering a fold change ≥ 2 and an adjusted p-value <0.05 as statistically significant, we found 53 differentially expressed transcripts: 51 downregulated transcripts (lower expression in R) and 2 upregulated transcripts (higher expression in R). The RNA was annotated as non-coding RNAs in over 25% of the cases. The distribution of such molecules was as followed: 8 long non-coding RNAs (56%); 3 non-coding RNAs (22%) and 3 pseudo-genes (22%). Regarding protein coding transcripts, gene ontology analysis revealed an enrichment of terms associated to metabolic processes and response to toxic substance.As expected, ERBB2, which encodes for HER2, appeared at the top of the list with a patent upregulation of expression in the responder group when compared with non-responder patients. To validate the data, we used qPCR and ERBB2 expression as a positive control of the results (p-value=0.0380). Our data showed a significant downregulation of UDP-glucuronosyltransferase 2B15 (UGT2B15; p-value= 0.0173), which encodes a glycosyltransferase, involved in the metabolism and elimination of toxic compounds. CONCLUSIONS. The ability to predict which patients will benefit from neoadjuvant therapy and achieve a pCR would allow for improved patient stratification and more personalized medicine. Here, we identified a set of transcript differentially expressed (FC>2; adjusted p-value<0.05) in patients that achieve pCR (R) when compared with tissue samples with residual disease (nR). We usedERBB2 expression as a positive control to validate the data and show that UGT2B15 is consistently upregulated in non-responder patients. Further work is needed to elucidate UGT2B15 role, but it is worth mentioning that an increased rate of glucuronidation has been associated with a loss of potency for the target drugs. Citation Format: Ana Gil-Torralvo, Marta Benavent, Maria A Dominguez-Cejudo, Alejandro Falcon, Begoña Vieites, Sonia Molina-Pinela, Manuel Ruiz, Álvaro Montaño, Rosario Gónzalez, Julia Martínez, Juan de la Haba, Antonio Rodríguez, Maria I Queipo, Begoña Jímenez, Javier Salvador-Bofill. Identification of UGT2B15 as a potential biomarker in response to neoadjuvant therapy in HER2+ breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-07-08.

Species-dependent hepatic and intestinal metabolism of selective estrogen receptor degrader LSZ102 by sulfation and glucuronidation

1. LSZ102 is an orally bioavailable selective estrogen receptor degrader in clinical development for the treatment of breast cancer. Preclinical studies showed efficacy in xenograft models on oral dosing. However, oral bioavailability was relatively low in several preclinical species (7-33%), and was associated with first-pass metabolism, particularly intestinal first-pass. 2. To investigate metabolism and first-pass effects, metabolites were analysed in human plasma samples after oral dosing of LSZ102 to patients, rat plasma samples after oral dosing of [14C]LSZ102, and in vitro incubations of [14C]LSZ102 with human and rat hepatocytes and intestinal S9 fractions. The kinetics of human sulfotransferase enzymes potentially involved in metabolism of LSZ102 were characterized. 3. Sulfate metabolites were found to be the major components in human plasma, as well as in human hepatocytes and intestinal S9 fractions. Contrastingly, glucuronidation was predominant in rat plasma, hepatocytes and intestinal S9. LSZ102 was found to be metabolized by several human sulfotransferases expressed in liver and intestine. The combined metabolism data in rat and human provide supporting evidence for an extensive intestinal first-pass metabolism effect via sulfation in human but glucuronidation in rat. 4. As LSZ102 is metabolized by a number of different sulfotransferases, drug-drug interactions resulting from the inhibition of one sulfotransferase are unlikely. 5. Despite the observed species difference in metabolism, the major human metabolites of LSZ102, sulfate M5, glucuronide M4, and secondary glucuronide/sulfate metabolite M12, have no or weak pharmacological activity and are not considered a toxicity risk as they are phase II conjugative metabolites.

Comparative In vitro Metabolism of Enflicoxib in Dogs, Rats, and Humans: Main Metabolites and Proposed Metabolic Pathways.

BackgroundEnflicoxib is a non-steroidal anti-inflammatory drug of the coxib family characterized by a long-lasting pharmacological activity that has been attributed to its active metabolite E-6132.ObjectivesThe aim of this work was to explore enflicoxib biotransformation in vitro in humans, rats and dogs, and to determine its metabolic pathways.MethodsDifferent in vitro test systems were used, including hepatocytes and liver and non-hepatic microsomes. The samples were incubated with enflicoxib and/or any of its metabolites at 37°C for different times depending on the test system. The analyses were performed by liquid chromatography coupled with either radioactivity detection or high-resolution mass spectrometry.ResultsEnflicoxib was efficiently metabolized by cytochrome P-450 into three main phase I metabolites: M8, E-6132, and M7. The non-active hydroxy-pyrazoline metabolite M8 accounted for most of the fraction metabolized in all the three species. The active pyrazol metabolite E-6132 showed a slow formation rate, especially in dogs, whereas metabolite M7 was a secondary metabolite formed by oxidation of M8. In hepatocytes, diverse phase II metabolite conjugates were formed, including enflicoxib glucuronide, M8 glucuronide, E-6132 glucuronide, M7 glucuronide, and M7 sulfate. Metabolite E-6132 was most probably eliminated by a unique glucuronidation reaction at a very low rate.ConclusionThe phase I metabolism of enflicoxib was qualitatively very similar among rats, humans and dogs. The low formation and glucuronidation rates of the active enflicoxib metabolite E-6132 in dogs are postulated as key factors underlying the mechanism of its long-lasting pharmacokinetics and enflicoxib's overall sustained efficacy.

Quantitative Investigation of Irinotecan Metabolism, Transport, and Gut Microbiome Activation.

The anticancer drug irinotecan shows serious dose-limiting gastrointestinal toxicity regardless of intravenous dosing. Although enzymes and transporters involved in irinotecan disposition are known, quantitative contributions of these mechanisms in complex in vivo disposition of irinotecan are poorly understood. We explained intestinal disposition and toxicity of irinotecan by integrating 1) in vitro metabolism and transport data of irinotecan and its metabolites, 2) ex vivo gut microbial activation of the toxic metabolite SN-38, and 3) the tissue protein abundance data of enzymes and transporters relevant to irinotecan and its metabolites. Integration of in vitro kinetics data with the tissue enzyme and transporter abundance predicted that carboxylesterase (CES)-mediated hydrolysis of irinotecan is the rate-limiting process in the liver, where the toxic metabolite formed is rapidly deactivated by glucuronidation. In contrast, the poor SN-38 glucuronidation rate as compared with its efficient formation by CES2 in the enterocytes is the key mechanism of the intestinal accumulation of the toxic metabolite. The biliary efflux and organic anion transporting polypeptide-2B1-mediated enterocyte uptake can also synergize buildup of SN-38 in the enterocytes, whereas intestinal P-glycoprotein likely facilitates SN-38 detoxification in the enterocytes. The higher SN-38 concentration in the intestine can be further nourished by β-d-glucuronidases. Understanding the quantitative significance of the key metabolism and transport processes of irinotecan and its metabolites can be leveraged to alleviate its intestinal side effects. Further, the proteomics-informed quantitative approach to determine intracellular disposition can be extended to determine susceptibility of cancer cells over normal cells for precision irinotecan therapy. SIGNIFICANCE STATEMENT: This work provides a deeper insight into the quantitative relevance of irinotecan hydrolysis (activation), conjugation (deactivation), and deconjugation (reactivation) by human or gut microbial enzymes or transporters. The results of this study explain the characteristic intestinal exposure and toxicity of irinotecan. The quantitative tissue-specific in vitro to in vivo extrapolation approach presented in this study can be extended to cancer cells.

Glucuronidation Of Hydroxylated Bromodiphenyl Ethers In Liver Microsomes Of A Marine And A Freshwater Fish

There are two sources of hydroxylated bromodiphenyl ethers in aquatic environments, biotransformation products of bromodiphenyl ether (BDE) flame retardants, and natural products biosynthesized by marine bacteria. Regardless of source, BDEs and their hydroxylated metabolites (OH-BDEs) are lipophilic molecules that can bioaccumulate in animals. OH-BDEs are potential substrates for glucuronidation and sulfonation, as demonstrated with human liver samples (Cisneros et. al. Chemosphere, 226, 132-139, 2019). The objective of the present study was to determine how well fish can eliminate selected OH-BDEs by glucuronidation. The four OH-BDEs studied here were natural products, 4-OH-BDE47, 2’-OH-BDE68, 6-OH-2’MeO-BDE68 or closely related to natural products, 4’-OH-BDE68. Liver microsomes were prepared from laboratory-maintained adult Channel Catfish, Ictalurus punctatus, two females and one male, held in freshwater tanks, and from wild-caught adult Red Snapper, Lutjanus campechanus, from the Gulf of Mexico, two females and two males. To determine rates of glucuronidation, uridine-diphosphate-14C-glucuronic acid (UDPGA), 1 mM, was incubated with liver microsomes, 0.05 mg, in the presence of 0.1M Tris-Cl buffer, pH 7.6, magnesium chloride, 5 mM, and varying concentrations of OH-BDE, 2.5 to 50 µM for 5 min. The amount of protein and incubation time were determined from preliminary studies to show linear formation of the glucuronide products. After 5 min, reactions were stopped by addition of tetrabutylammonium dihydrogen phosphate and acetic acid, and the ion-pair of the glucuronide was extracted into ethyl acetate for determination of 14C, following a previously published procedure. In both fish species, the rates of glucuronidation with increasing substrate concentration followed Michaelis-Menten kinetics. The Km values for the OH-BDEs ranged from 3 to 35 µM and were generally somewhat lower in the catfish than the red snapper, though this only reached statistical significance, p<0.05, for 2’-OH-BDE68. Vmax values ranged from 0.9 to 5.4 nmol glucuronide per min per mg protein and for each substrate were variable between individual fish. Mean glucuronidation enzyme efficiency was highest for 2’-OH-BDE68 in the channel catfish, 715 ± 119 µL.min-1.mg-1 (mean ± S.D., n = 3), significantly higher than for red snapper, 239 ± 74 µL.min-1.mg-1 (n=4). The lowest glucuronidation efficiencies were found for 6-OH-BDE47, 145 ± 57 µL.min-1.mg-1, n=3 in Channel Catfish and 68 ± 37 µL.min-1.mg-1, n=4, in Red Snapper. Because of the variability, the efficiencies of 6-OH-BDE47 glucuronidation were similar between the two species. The glucuronidation efficiencies reported here for two fish species are of similar orders of magnitude to those observed with human liver microsomes. Both fish species were able to efficiently catalyze glucuronidation of the studied OH-BDEs, suggesting these compounds should be readily excreted from exposed Channel Catfish or Red Snapper.

Proteomics‐Informed Scaling of Irinotecan Metabolism, Transport, and Gut Microbial Activation Explain its Intestinal Toxicity after Intravenous Dose

Objective Anticancer drug, irinotecan causes dose-limiting intestinal side effects after an intravenous dose. We hypothesized that differential tissue abundance of irinotecan metabolizing enzymes, transporters, and gut microbial activation can explain intestinal accumulation of the toxic metabolite, SN-38, and its toxicity. We integrated i) in vitro metabolism and transport data of irinotecan and its metabolites, ii) ex vivo microbiome data of SN-38 activation, and iii) tissue quantitative proteomics data of the relevant enzymes and transporters. Methods We first characterized the metabolism and transport kinetics of irinotecan using in vitro systems (recombinant proteins, cells, or membrane vesicles) expressing carboxylesterases (CESs), uridine glucuronosyltransferases (UGTs) and organic anion transporting polypeptides (OATPs), P-glycoprotein (P-gp), breast cancer resistant protein (BCRP), and multidrug resistance-associated proteins (MRPs). Then, using tissue proteomics data, we estimated the tissue-specific fractional contributions of individual enzymes and transporters. The tissue-specific metabolism was verified by performing incubations with tissue S9 fractions and hepatocytes. Finally, we estimated SN-38 reactivation by gut microbial β-glucuronidases in human fecal homogenates. Results Irinotecan and its major metabolites, SN-38, and SN-38-glucuronide (SN-38-G) were substrates of multiple uptake and efflux transporters, with OATP1B1, P-gp, and BCRP being the predominant contributors. Integration of the in vitro kinetics data with the tissue enzyme and transporter proteomics predicted that CES mediated hydrolysis of irinotecan is the rate-limiting process in the liver, whereas the toxic metabolite, SN-38 is rapidly neutralized by its hepatic glucuronidation. In contrast, the poor glucuronidation rate as compared to the efficient formation by CES2 in the enterocytes is the key mechanism of SN-38 accumulation in the intestine (Figure 1). The biliary efflux and OATP2B1 mediated enterocyte uptake are the potential mechanisms that synergize buildup of SN-38 into the enterocytes, whereas intestinal P-gp likely facilitate detoxification of the enterocytes. The higher SN-38 concentration in the intestine can be further nourished by β-d-glucuronidases leading to the higher exposure of the toxic metabolite. Conclusions: CES2, UGT1As, OATP2B1, P-gp, BCRP, and gut microbiome play crucial roles in regulating intestinal exposure and tissue-specific intestinal toxicity of irinotecan. Understanding the quantitative significance of the key disposition processes of irinotecan can be leveraged to alleviate the intestinal side effects. The proteomics-informed translational approach used here can be applied to other drugs involving complex disposition mechanisms.

Effect of race and glucuronidation rates on the relationship between nicotine metabolite ratio and nicotine clearance.

To 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. Participants [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. Among 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. NMR 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.

A fluorescence-based microplate assay for high-throughput screening and evaluation of human UGT inhibitors

Human UDP-glucuronosyltransferase enzymes (hUGTs), one of the most important classes of conjugative enzymes, are responsible for the glucuronidation and detoxification of a variety of endogenous substances and xenobiotics. Inhibition of hUGTs may cause undesirable effects or adverse drug-drug interactions (DDI) via modulating the glucuronidation rates of endogenous toxins or the drugs that are primarily conjugated by the inhibited hUGTs. Herein, to screen hUGTs inhibitors in a more efficient way, a novel fluorescence-based microplate assay has been developed by utilizing a fluorogenic substrate. Following screening of series of 4-hydroxy-1,8-naphthalimide derivatives, we found that 4-HN-335 is a particularly good substrate for a panel of hUGTs. Under physiological conditions, 4-HN-335 can be readily O-glucuronidated by ten hUGTs, such reactions generate a single O-glucuronide with a high quantum yield (Ф = 0.79) and bring remarkable changes in fluorescence emission. Subsequently, a fluorescence-based microplate assay is developed to simultaneously measure the inhibitory effects of selected compound(s) on ten hUGTs. The newly developed fluorescence-based microplate assay is time- and cost-saving, easy to manage and can be adapted for 96-well microplate format with the Z-factor of 0.92. We further demonstrate the utility of the fluorescence-based assay for high-throughput screening of two compound libraries, resulting in the identification of several potent UGT inhibitors, including natural products and FDA-approved drugs. Collectively, this study reports a novel fluorescence-based microplate assay for simultaneously sensing the residual activities of ten hUGTs, which strongly facilitates the identification and characterization of UGT inhibitors from drugs or herbal constituents and the investigations on UGT-mediated DDI.