UDP-glucuronosyltransferase Research Articles

Identification of genes involved in biosynthesis of paeoniflorin in Paeonia lactiflora based on transcriptome analysis

Paeoniflorin, a representative pinane monoterpene glycoside, is the main active component and quality index of Paeoniae Radix Alba and Paeoniae Radix Rubra.The possible biosynthesis of paeoniflorin is as follows: GPP is derived from mevalonate(MVA) and/or 2-C-methyl-D-erythritol 4-phosphate(MEP) pathway(s) followed by the catalysis with terpene synthase, cytochrome P450(CYP450), UDP-glucuronosyltransferase(UGT), and acyltransferase(AT), respectively.This study aims to explore the genes rela-ted to the biosynthesis of paeoniflorin.To be specific, the cDNA libraries for flowers, leaves, and roots of Paeonia lactiflora were established and sequenced.A total of 30 609 open reading frames(ORFs) were yielded.Through functional annotation and expression analysis of all CYP450 genes in the transcriptome, 11 CYP450 genes belonging to CYP71 A and CYP71 D subfamilies and showing expression trend consistent with monoterpene synthase PlPIN that may be involved in paeoniflorin biosynthesis were screened out.Subsequently, 7 UGT genes and 9 AT genes demonstrating the expression trend consistent with PlPIN which were possibly involved in paeoniflorin biosynthesis were further screened by functional annotation analysis, full-length sequence analysis, expression analysis, and phylogeny analysis.This study provided a systematic screening method with smaller number of candidate genes, thus reducing the workload of functional gene verification.The result laid a foundation for analyzing the biosynthesis pathway of paeoniflorin and the formation mechanism.

Bilirubin metabolism and UDP-glucuronosyltransferase 1A1 variants in Asians: Pathogenic implications and therapeutic response.

In the Asian general population, at least six single-nucleotide variants (SNVs) in the UDP-glucuronosyltransferase (UGT) 1A1 gene have been identified: -3279T>G, -53A(TA)6 TAA>A(TA)7 TAA, 211G>A, 686C>A, 1091C>T, and 1456T>G. Each of these six SNVs was observed in at least four ethnic groups of the 12 Asian populations studied. In East Asian populations, the descending frequency of these six SNVs was as follows: -3279G>[-53A(TA)7 TAA, 211A]>(686A, 1091T)>1456G. Because of the presence of linkage disequilibrium and the expulsion phenomenon, when the SNVs -3279G, -53A(TA)7 TAA, 211A, and 686A were simultaneously involved, 15 instead of the estimated 81 genotypes were observed. Those carrying 686AA or 1456GG developed Gilbert's syndrome or Crigler-Najjar syndrome type 2. Both -53A(TA)7 TAA/A(TA)7 TAA and 211AA are the main causes of Gilbert's syndrome in East Asian populations. In East Asian populations, the 211AA genotype is the main cause of neonatal hyperbilirubinemia, whereas -53A(TA)7 TAA/A(TA)7 TAA exerts a protective effect on hyperbilirubinemia development in neonates fed with breast milk. Both 211A and -53A(TA)7 TAA are significantly associated with adverse drug reactions induced by irinotecan (one of the most widely used anticancer agents) in Asians. However, at least three common SNVs (-3279G, -53A(TA)7 TAA, and 211A) should be comprehensively analyzed. This study investigated the clinical significance of these six SNVs and demonstrated that examining UGT1A1 variants in Asian populations is considerably challenging.

Inherited Unconjugated Hyperbilirubinemias and Radical Cancer surgery: Perioperative Concerns for Bilateral Modified Radical Mastectomy

Inherited defects of Uridine 5'-diphospho-glucuronosyltransferase (UDPGT) can cause congenital unconjugated hyperbilirubinemia. The perioperative anesthetic management of such patients poses several challenges. Here we report a case of bilateral carcinoma breast with congenital unconjugated hyperbilirubinemia posted for bilateral modified radical mastectomy. The patient was administered general anesthesia with no perioperative deterioration of liver function.

Uridine Diphosphate Glucuronosyl Transferase 2B28 (UGT2B28) Promotes Tumor Progression and Is Elevated in African American Prostate Cancer Patients.

Prostate cancer (PCa) is the second most diagnosed cancer in the United States and is associated with metabolic reprogramming and significant disparities in clinical outcomes among African American (AA) men. While the cause is likely multi-factorial, the precise reasons for this are unknown. Here, we identified a higher expression of the metabolic enzyme UGT2B28 in localized PCa and metastatic disease compared to benign adjacent tissue, in AA PCa compared to benign adjacent tissue, and in AA PCa compared to European American (EA) PCa. UGT2B28 was found to be regulated by both full-length androgen receptor (AR) and its splice variant, AR-v7. Genetic knockdown of UGT2B28 across multiple PCa cell lines (LNCaP, LAPC-4, and VCaP), both in androgen-replete and androgen-depleted states resulted in impaired 3D organoid formation and a significant delay in tumor take and growth rate of xenograft tumors, all of which were rescued by re-expression of UGT2B28. Taken together, our findings demonstrate a key role for the UGT2B28 gene in promoting prostate tumor growth.

Studies on Chemical Characterization of Ginkgo Amillaria Oral Solution and Its Drug-Drug Interaction With Piceatannol 3'-O-β-D-Glucopyranoside for Injection.

Ginkgo Amillaria oral solution (GAO) is commonly used for the treatment of cardiovascular and cerebrovascular diseases in China. Piceatannol-3′-O-β-D-glucopyranoside for injection (PGI) is mainly used for the prevention and treatment of ischemic cerebrovascular diseases. With the spread of cerebrovascular disease, the possibility of combining the two drugs has increased; however, there is no research on the drug–drug interaction (DDI) between these two medicines. In this paper, an ultrahigh-performance liquid chromatography/quadrupole–orbitrap mass spectrometry (UHPLC/Q-Orbitrap MS) method was established to characterize the chemical constituents of GAO first; 62 compounds were identified or tentatively identified based on their retention time (RT), MS, and MS/MS data. Nine main compounds were determined by ultrahigh-performance liquid chromatography/triple quadrupole mass spectrometry (UPLC-QQQ-MS). Furthermore, incubation with liver microsomes in vitro was fulfilled; the results showed that GAO had a significant inhibitory effect on UGT1A9 and UGT2B7 (p < 0.05), and PGI was mainly metabolized by UGT1A9. The identification results of in vivo metabolites of PGI showed that PGI mainly undergoes a phase II binding reaction mediated by UDP-glucuronosyltransferase (UGT) and sulfotransferase (SULT) in vivo. Therefore, pharmacokinetic studies were performed to investigate the DDI between GAO and PGI. The results showed that the AUC (p < 0.05) and T1/2 (p < 0.05) of PGI in vivo were significantly increased when administered together with GAO, whereas the CL was significantly decreased (p < 0.05). The exploration of in vitro and in vivo experiments showed that there was a DDI between GAO and PGI.

In silico prediction of UGT-mediated metabolism in drug-like molecules via graph neural network

UDP-glucuronosyltransferases (UGTs) have gained increasing attention as they play important roles in the phase II metabolism of drugs. Due to the time-consuming process and high cost of experimental approaches to identify the metabolic fate of UGT enzymes, in silico methods have been developed to predict the UGT-mediated metabolism of drug-like molecules. We developed consensus models with the combination of machine learning (ML) and graph neural network (GNN) methods to predict if a drug-like molecule is a potential UGT substrate, and then we applied the Weisfeiler-Lehman Network (WLN) model to identify the sites of metabolism (SOMs) of UGT-catalyzed substrates. For the substrate model, the accuracy of the single substrate prediction model on the test set could reach to 0.835. Compared with the single estimators, the consensus models are more stable and have better generalization ability, and the accuracy on the test set reached to 0.851. For the SOM model, the top-1 accuracy of the SOM model on the test set reached to 0.898, outperforming existing works. Thus, in this study, we proposed a computational framework, named Meta-UGT, which would provide a useful tool for the prediction and optimization of metabolic profiles and drug design.Graphical

Pediatric AIDS-Therapeutic Successes Built on a Foundation of Pediatric Clinical Pharmacology with Pharmacokinetic-Pharmacodynamic Modeling.

Pediatric AIDS-Therapeutic Successes Built on a Foundation of Pediatric Clinical Pharmacology with Pharmacokinetic-Pharmacodynamic Modeling.

Genome-Wide Evolutionary Analysis of Putative Non-Specific Herbicide Resistance Genes and Compilation of Core Promoters between Monocots and Dicots.

Herbicides are key weed-control tools, but their repeated use across large areas has favored the evolution of herbicide resistance. Although target-site has been the most prevalent and studied type of resistance, non-target-site resistance (NTSR) is increasing. However, the genetic factors involved in NTSR are widely unknown. In this study, four gene groups encoding putative NTSR enzymes, namely, cytochrome-P450, glutathione-S-transferase (GST), uridine 5′-diphospho-glucuronosyltransferase (UDPGT), and nitronate monooxygenase (NMO) were analyzed. The monocot and dicot gene sequences were downloaded from publicly available databases. Phylogenetic trees revealed that most of the CYP450 resistance-related sequences belong to CYP81 (5), and in GST, most of the resistance sequences belonged to GSTU18 (9) and GSTF6 (8) groups. In addition, the study of upstream promoter sequences of these NTSR genes revealed stress-related cis-regulatory motifs, as well as eight transcription factor binding sites (TFBS) were identified. The discovered TFBS were commonly present in both monocots and dicots, and the identified motifs are known to play key roles in countering abiotic stress. Further, we predicted the 3D structure for the resistant CYP450 and GST protein and identified the substrate recognition site through the homology approach. Our description of putative NTSR enzymes may be used to develop innovative weed control techniques to delay the evolution of NTSR.

The Protective Effect of Sulforaphane on Dextran Sulfate Sodium-Induced Colitis Depends on Gut Microbial and Nrf2-Related Mechanism.

Sulforaphane (SFN), an isothiocyanate present in cruciferous vegetables such as broccoli and brussels sprouts, has a variety of biological functions. This study was undertaken to assess the potential efficacy of SFN in ameliorating dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) in mice and to elucidate the underlying mechanisms. UC was induced in mice with administration of 2% DSS in drinking water for 7 days. Male C57BL/6 mice were treated with Mesalazine (50 and 100 mg/kg body weight) and various doses of SFN (2.5, 5, 10, and 20 mg/kg body weight). In DSS colitis mice, the hallmarks of disease observed as shortened colon lengths, increased disease activity index (DAI) scores and pathological damage, higher proinflammatory cytokines and decreased expression of tight junction proteins, were alleviated by SFN treatment. SFN also partially restored the perturbed gut microbiota composition and increased production of volatile fatty acids (especially caproic acid) induced by DSS administration. The heatmap correlation analysis indicated that Lactobacillus johnsonii, Bacteroides acidifaciens, unclassified Rikenellaceae RC9, and unclassified Bacteroides were significantly correlated with disease severity. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), Signal Transducer and Activator of Transcription 3 (STAT3), and Phase II enzyme UDP-glucuronosyltransferase (UGT) were involved in the protective effect of SFN against DSS-induced colitis. This study's findings suggest that SFN may serve as a therapeutic agent protecting against UC.

Optimisation of the HepaRG cell line model for drug toxicity studies using two different cultivation conditions: advantages and limitations.

The HepaRG cell line represents a successful model for hepatotoxicity studies. These cells are of human origin and are differentiated in vitro into mature and functional hepatocyte-like cells. The objective of this research was to compare two different culture protocols, Sison-Young et al. 2017 (hereinafter referred as Sison) and Gripon et al. 2002 (hereinafter referred as Biopredic) for HepaRG cells in order to optimise this model for drug metabolism and toxicity testing studies. HepaRG cells obtained from the same batch were cultured according to the described protocols. Using both protocols, differentiated HepaRG cells retained their drug metabolic capacity (major phase I/II enzymes) and transporters, as well as their morphological characteristics. Morphologically, HepaRG cells cultured after the Biopredic protocol formed more apical membranes and small ductular-like structures, than those cultivated using the Sison protocol. Also, the efflux activity of multidrug resistance protein 1 (MDR1) and multidrug resistance-associated protein 1 (MRP1) as well as the activity of uridine-glucuronosyltransferase (UGT) and glutathione S-transferase (GST) were significantly reduced in HepaRG cultured using the Sison protocol. Applying well-established drug cocktails to measure cytochrome P450 (CYPs) activity, we found that production of the corresponding metabolites was hampered in Sison-cultured HepaRG cells, indicating that the activity of CYP1A2, CYP2C9, CYP3A4, CYP2B6 and CYP2C19 was significantly reduced. Moreover, HepaRG sensitivity to well-known drugs, namely diclofenac, amiodarone, imipramine and paracetamol, revealed some differences between the two culture protocols. Furthermore, the HepaRG cells can be maintained with higher viability and sufficient CYPs activity and expression (i.e. CYP3A4, CYP1A2 and CYP2B6) as well as liver-specific functions, using Biopredic compared with the Sison culture protocol. These maintained liver-specific functions might be dependent on the prolongation of the culture conditions in the case of the Biopredic protocol. In conclusion, based on the metabolic activity of HepaRG cells using the standard protocol from Biopredic, we believe that this protocol is optimal for investigating drug metabolism and pharmacokinetic screening studies.

Trio-binned genomes of the woodrats Neotoma bryanti and Neotoma lepida reveal novel gene islands and rapid copy number evolution of xenobiotic metabolizing genes.

The genomic architecture underlying the origins and maintenance of biodiversity is an increasingly accessible feature of species, due in large part to third-generation sequencing and novel analytical toolsets. Applying these techniques to woodrats (Neotoma spp.) provides a unique opportunity to study how herbivores respond to environmental change. Neotoma bryanti and N. lepida independently achieved a major dietary feat in the aftermath of a natural climate change event: switching to the novel, toxic food source creosote bush (Larrea tridentata). To better understand the genetic mechanisms underlying this ability, we employed a trio binning sequencing approach with a N. bryanti×N. lepida F1 hybrid, allowing the simultaneous assembly of genomes representing each parental species. The resulting phased, chromosome-level, highly complete haploid references enabled us to explore the genomic architecture of several gene families-cytochromes P450, UDP-glucuronosyltransferases (UGTs), and ATP-binding cassette (ABC) transporters-known to play key roles in the metabolism of naturally occurring toxic dietary compounds. In addition to duplication events in the ABCG and UGT2B subfamilies, we found expansions in three P450 gene families (2A, 2B, 3A), including the evolution of multiple novel gene islands within the 2B and 3A subfamilies, which may have provided the crucial substrate for dietary adaptation. Our assemblies demonstrate that trio binning from an F1 hybrid rodent effectively recovers parental genomes from species that diverged more than a million years ago.

Contribution of genetic factors to high rates of neonatal hyperbilirubinaemia on the Thailand-Myanmar border.

Very high unconjugated bilirubin plasma concentrations in neonates (neonatal hyperbilirubinaemia; NH) may cause neurologic damage (kernicterus). Both increased red blood cell turn-over and immaturity of hepatic glucuronidation contribute to neonatal hyperbilirubinaemia. The incidence of NH requiring phototherapy during the first week of life on the Thailand-Myanmar border is high (approximately 25%). On the Thailand-Myanmar border we investigated the contribution of genetic risk factors to high bilirubin levels in the first month of life in 1596 neonates enrolled in a prospective observational birth cohort study. Lower gestational age (<38 weeks), mutations in the genes encoding glucose-6-phosphate dehydrogenase (G6PD) and uridine 5'-diphospho-glucuronosyltransferase (UGT) 1A1 were identified as the main independent risk factors for NH in the first week, and for prolonged jaundice in the first month of life. Population attributable risks (PAR%) were 61.7% for lower gestational age, 22.9% for hemi or homozygous and 9.9% for heterozygous G6PD deficiency respectively, and 6.3% for UGT1A1*6 homozygosity. In neonates with an estimated gestational age ≥ 38 weeks, G6PD mutations contributed PARs of 38.1% and 23.6% for "early" (≤ 48 hours) and "late" (49-168 hours) NH respectively. For late NH, the PAR for UGT1A1*6 homozygosity was 7.7%. Maternal excess weight was also a significant risk factor for "early" NH while maternal mutations on the beta-globin gene, prolonged rupture of membranes, large haematomas and neonatal sepsis were risk factors for "late" NH. For prolonged jaundice during the first month of life, G6PD mutations and UGT1A1*6 mutation, together with lower gestational age at birth and presence of haematoma were significant risk factors. In this population, genetic factors contribute considerably to the high risk of NH. Diagnostic tools to identify G6PD deficiency at birth would facilitate early recognition of high risk cases.

Beta-Glucuronidase Activity: Another Source of Ethyl Glucuronide.

Numerous classes of endogenous and xenobiotic compounds are conjugated to uridine-5'-diphospho (UDP)-alpha-D-glucuronic acid which is catalyzed by human UDP-Glucuronosyltransferases (UGTs). The resulting beta-D-glucuronides can be hydrolyzed to β-D-glucuronic acid and the corresponding aglycone in a configuration retaining manner by beta-glucuronidases (GUSBs), which are widely distributed in mammalians, microbiota, insects, molluscs, nematodes, fishes and plants. This study investigates GUSBs' activity in the presence of ethanol (0-70% by volume) using different β-D-glucuronides (phenolphthalein-β-D-glucuronide, 4-nitrophenol-β-D-glucuronide, morphine-3-O-β-D-glucuronide, quercetin-3-O-β-D-glucuronide and 1-/2-propyl-β-D-glucuronide) as substrates. It was found that β-D-ethyl glucuronide (EtG), which is a minor UGT-derived metabolite of ethanol in man and one of the most frequently used biomarkers of alcohol consumption today, builds up from all investigated β-D-glucuronides by means of GUSBs in the presence of ethanol. The glucuronyl transfer reaction, which was neither detected in the absence of ethanol nor in absence of GUSBs, is minor at ethanol concentrations which are commonly observed in blood and tiβues after consumption of alcoholic beverages, but predominant at higher concentrations of ethanol. In spite of in vitro characteristics, our observations point to an additional biochemical path and another source of EtG, which should be further evaluated in the context of alcohol biomarker applications. The detection of EtG in several settings independent from of human UGT-metabolism (e.g. EtG post post-collection synthesis in E.coli coli-contaminated urine samples, EtG in wine and ethanolic herbal preparations) can be explained by the described mechanism.

Abstract 5434: Discovery of SCR-7952, a novel, potent and high selective MAT2A inhibitor shows robust anti-tumor activities and better safety profile

Abstract The metabolic enzyme methionine adenosyltransferase 2A (MAT2A) was identified as a synthetic lethal target in methylthioadenosine phosphorylase (MTAP)-deleted cancers. The co-deletion of MTAP and CDKN2A was observed in approximately 15% of all cancers and predicted worse outcomes, which provides MAT2A a potential therapeutic target. AG-270, a first MAT2A inhibitor, is currently evaluated in early clinical study. Given the inhibitory activity on UGT1A1, a bilirubin uridine diphosphate glucuronosyl transferase regulating the homeostasis of bilirubin, treatment of AG-270 led to the bilirubin increase in patients, which limited the further dose escalation. Therefore, to avoid those off-targeted side effects and maximize benefit from the MAT2A targeted therapy, it is necessary to develop a new generation of MAT2A inhibitor with better activity and higher selectivity, especially, removal of UGT1A1 activity. Here, we report SCR-7952 is a novel MAT2A inhibitor with greater potency and higher selectivity. In biochemical assay, SCR-7952 exhibited potent inhibitory activity on MAT2A enzyme with IC50 of 21 nM, which was more potent than AG-270 (IC50, 68 nM). In HCT116 MTAP-/- cells, SCR-7952 strongly inhibited the cellular SAM production with IC50 of 2 nM, which was significantly more potent than AG-270 (IC50, 6 nM). And SCR-7952 potently inhibited HCT116 MTAP-/- cell growth with IC50 of 53 nM, which was almost 6-fold more potent than AG-270 tested in parallel (IC50, 300 nM). Meanwhile, SCR-7952 displayed more than 20-fold selectivity over the HCT116 parent cells on proliferation assay. However, AG-270 only demonstrated 4-fold selectivity in the same assay. Furthermore, inhibition of SCR-7952 on a panel of 47 life-dependent targets was determined at 10 μM, result showed that there is no significant inhibition observed. In addition to MTAP KO cells, SCR-7952 showed potently inhibited cell growth in multiple cell lines with natural MTAP-deficiency. SCR-7952 revealed excellent oral bioavailability and PK properties in pre-clinical species. In the HCT-116 MTAP-/- xenograft model, SCR-7952 demonstrated robust anti-tumor activity and achieved 72% tumor growth inhibition (TGI) at 1 mpk, QD, which was more potent than AG-270 (TGI 56% at 200 mpk, QD). Furthermore, SCR-7952 was well tolerant and no elevation of bilirubin was observed. In contrast, AG-270 at 200mpk resulted in significant increase of bilirubin. In conclusion, the above data demonstrate that SCR-7952 is a more potent and higher selective MAT2A, and will be filed IND in 2022. Citation Format: Feng Zhou, Feng Tang, Zhiyong Yu, Zhen Li, Wenqing Yang, Liting Xue, Ping Chen, Renhong Tang. Discovery of SCR-7952, a novel, potent and high selective MAT2A inhibitor shows robust anti-tumor activities and better safety profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5434.

Abstract 5250: Association between UGT1A1 gene polymorphism and safety and efficacy of irinotecan monotherapy as the second-line treatment for advanced gastric cancer

Abstract Objective: Uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) is a key enzyme in irinotecan metabolism. However, the relationship between UGT1A1 genotype and the safety and efficacy of irinotecan monotherapy in the treatment of Chinese advanced gastric cancer remains unclear. Methods: A total of 110 patients were enrolled. Intravenous irinotecan was administered every 3 weeks. Irinotecan dose was selected according to the polymorphism of UGT1A1 gene, which was divided into 3 groups: UGT1A1*6 wild-type (GG type): 125mg/m2, d1, d8; UGT1A1*6 mutant heterozygosity (GA type) 100mg/m2, d1, d8; UGT1A1*6 homozygosity mutation (AA type) 75mg/m2, d1, d8 or paclitaxel 125mg/m2, d1, d8. UGT1A1 genotypes were determined by direct sequencing or next-generation sequencing of genomic DNA extracted from peripheral blood. Results: Among 110 patients of the subjects, the genotypes of UGT1A1*28 were wild-type in 78 patients (70.91%), mutant heterozygosity in 28 (25.45%) and mutant homozygosity in 4 (3.64%). UGT1A1*6 were GG in 67 cases (60.91%), GA in 35 cases (31.82%), and AA in 8 cases (7.27%). There were two heterozygous mutations in 3 patients, and no homozygous mutation was detected. A total of 10 cycles of irinotecan were administered, with a median of five cycles per patient. There was no significant difference in the risk of delayed diarrhea and granulocytopenia in patients with different UGT1A1*28 genotypes (P&amp;gt;0.05), while there was significant difference in the risk of delayed diarrhea and granulocytopenia in patients with different UGT1A1*6 genotypes (P&amp;lt;0.05). The dose intensity of irinotecan was different in patients with different subtypes of UGT1A1*6 gene, and the dose intensity of heterozygous mutant patients was lower than that of wild-type patients. However, there were no significant differences in PFS and OS among patients with different subtypes after dose adjustment and program adjustment (P&amp;gt;0.05). Conclusion: UGT1A1*6 gene polymorphism was significantly associated with diarrhea and neutropenia induced by irinotecan. The safety and effectiveness of antitumor therapy might be significantly improved by individualized drug administration according to the results of UGT1A1*6 gene polymorphism. Citation Format: Huifang Lv, Yunduan He, Caiyun Nie, Beibei Chen, Jianzheng Wang, Weifeng Xu, Jing Zhao, Xiaobing Chen, Junling Zhang. Association between UGT1A1 gene polymorphism and safety and efficacy of irinotecan monotherapy as the second-line treatment for advanced gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5250.

Characterization of JNJ-2482272 [4-(4-Methyl-2-(4-(Trifluoromethyl)Phenyl)Thiazole-5-yl) Pyrimidine-2-Amine] As a Strong Aryl Hydrocarbon Receptor Activator in Rat and Human.

[4-(4-Methyl-2-(4-(trifluoromethyl)phenyl)thiazole-5-yl)pyrimidine-2-amine] (JNJ-2482272), under investigation as an anti-inflammatory agent, was orally administered to rats once daily at 60 mg/kg for 6 consecutive days. Despite high plasma exposure after single administration (Cmax of 7.1 μM), JNJ-2482272 had plasma concentrations beneath the lower limit of quantification (3 ng/ml) after 6 consecutive days of dosing. To determine if JNJ-2482272 is an autoinducer in rats, plated rat hepatocytes were treated with JNJ-2482272 for 2 days. The major hydroxylated metabolites of JNJ-2482272 were isolated and characterized by mass spectrometry and NMR analyses. Compared with the vehicle-treated cells, a concentration-dependent increase was observed in the formation of phase I- and II-mediated metabolites coinciding with greater expression of cytochrome P450s (P450s) and UDP-glucuronosyltransferases (UGTs) in rat hepatocytes. CYP1A1, CYP1A2, CYP1B1, and UGT1A6 transcripts were predominantly induced, suggesting that JNJ-2482272 is an activator of the aryl hydrocarbon receptor (AhR). In a human AhR reporter assay, JNJ-2482272 demonstrated potent AhR activation with an EC50 value of 0.768 nM, a potency more comparable to the strong AhR activator and toxin 2,3,7,8-tetrachloro-dibenzodioxin than to weaker AhR activators 3-methylcholanthrene, β-naphthoflavone, and omeprazole. In plated human hepatocytes, JNJ-2482272 induced CYP1A1 gene expression with an EC50 of 20.4 nM and increased CYP1A activity >50-fold from basal levels. In human recombinant P450s, JNJ-2482272 was exclusively metabolized by the CYP1 family of enzymes and most rapidly by CYP1A1. The summation of these in vitro findings bridges the in vivo conclusion that JNJ-2482272 is a strong autoinducer in rats and potentially in humans through potent AhR activation. SIGNIFICANCE STATEMENT: Drugs that induce their own metabolism (autoinducers) can lack sustained exposures for pharmacology and safety assessment hindering their development. JNJ-2482272 is demonstrated herein as a strong aryl hydrocarbon receptor (AhR) activator and CYP1A autoinducer, explaining its near complete loss of exposure after repeat administration in rat, which is likely translatable to human (if progressed further) considering its nanomolar potency comparable to "classical" AhR ligands like 2,3,7,8-tetrachloro-dibenzo-dioxin despite bearing a "nonclassical" drug structure.

P-113 A multicenter observational study of liposomal irinotecan and fluorouracil/leucovorin in patients with unresectable or recurrent pancreatic cancer (NAPOLEON-2): Retrospective part

Nanoliposomal irinotecan and fluorouracil with folinic acid (NAL-IRI/FU/LV) is the standard regimen after gemcitabine-based therapy for unresectable or recurrent pancreatic cancer (urPC), based on the results of a phase 3 trial, NAPOLI-1 study. However, the efficacy and toxicity of NAL-IRI/FU/LV in the clinical setting remain unclear. Therefore, we conducted this NAPOLEON-2 study to investigate the clinical features of NAL-IRI/FU/LV and explore the predictive and prognostic factors, both retrospectively and prospectively. Here we show the efficacy and toxicity of NAL-IRI/FU/LV of the retrospective part. This retrospective study collected data from urPC patients treated with NAL-IRI/FU/LV, who had received at least one previous chemotherapy, from 21 hospitals in Japan during the period from June 2020 to May 2021. Patient characteristics, treatment efficacy, and adverse events were analyzed. Overall survival (OS) and progression-free survival (PFS) were estimated by Kaplan-Meier analysis. Incidentally, OS and PFS among the therapeutic lines of NAL-IRI/FU/LV were also analyzed. NAL-IRI/FU/LV was administered to 161 patients. The median follow-up period was 5.3 months (95% confidence interval [95%CI], 4.7–6.3). All patients had previous gemcitabine-based therapy, and NAL-IRI/FU/LV was administered as 2nd/3rd/4th-line chemotherapy to 104/41/16 patients, respectively. Eighteen patients (11%) had received previous irinotecan. The median age was 67 (range, 38–85), and 88 males (55%) were included. Performance Status (PS) was 0/1/2/3 in 46/47/6/1 patients. Nineteen patients (12%) had locally advanced disease and 142 (88%) patients had metastatic disease. Eighty-nine patients (55%) had liver metastasis and 44 patients (27%) had peritoneal metastasis. The median treatment courses were 5 (range, 1–29). The median OS was 9.5 months (95%CI, 7.5–11.0). The median PFS was 3.3 months (95%CI, 2.8–4.4). The overall response rate was 5%, and the disease control rate was 52%. The relative dose intensity was 82.8% in NAL-IRI and 98.2% in FU. The initial dose of NAL-IRI was reduced in 57 patients (35%), mainly because of the uridine diphosphate glucuronosyltransferase (UGT) 1A1 examination status (8%), followed by organ function decrease (6%). Dosage reduction during the treatment (independent from the initial dosage reduction) was conducted in 63 patients (39%), mainly because of neutropenia (14%) and anorexia (10%). More frequent grade 3/4 adverse events (AEs) were neutropenia (23%), anorexia (12%), leukopenia (12%), and anemia (9%). No Grade 5 AE was observed. The median OS in NAL-IRI/FU/LV as 2nd-line therapy compared by as 3rd-line or later regimen was 9.1 vs 10.7 months (hazard ratio [HR], 0.69; 95% CI, 0.42–1.13; p=0.14). The median PFS in NAL-IRI/FU/LV as 2nd-line therapy compared to as 3rd-line or later regimen was 2.9 vs 4.2 months (HR, 0.90; 95% CI, 0.63–1.29; p=0.58). NAL-IRI/FU/LV presented appropriate efficacy compared to previous studies, and the one on OS was shown regardless of therapeutic lines. Toxicity profiles were similar to them and thought to be manageable. In conclusion, NAL-IRI/FU/LV could be a candidate for 2nd-line or later regimen in the real world.

P-95 Bevacizumab, irinotecan and biweekly trifluridine/tipiracil for pretreated metastatic colorectal cancer: MODURATE, a phase Ib study

Treatment of refractory metastatic colorectal cancer (mCRC) with trifluridine/tipiracil yields some survival benefits. Trifluridine/tipiracil combined with bevacizumab or irinotecan suggested enhanced therapeutic effects in preclinical models. A phase Ib study combining standard trifluridine/tipiracil regimens with irinotecan resulted in better antitumor activity, accompanied by a high degree of febrile neutropenia. This phase Ib study of trifluridine/tipiracil, irinotecan, and bevacizumab in previously treated mCRC was conducted at two centers, Shizuoka Cancer Center and Aichi Cancer Center in Japan, and consisted of dose escalation (3+3 design) and expansion cohorts. Key eligibility criteria included histologically confirmed colorectal adenocarcinoma, failure or intolerant to fluoropyrimidine and oxaliplatin, no prior therapy with trifluridine/tipiracil and irinotecan, age of 20–75 years, and ECOG PS of 0–1. Patients received trifluridine/tipiracil (25–35 mg/m2 twice daily on days 1–5), irinotecan (150–180 mg/m2; day 1), and bevacizumab (5 mg/kg; day 1) every 2 weeks. The recommended phase II dose (RPTD) in the dose escalation cohort was administered to at least 15 patients in both cohorts. A total of 28 patients from two centers were enrolled between August 2016 and January 2020, comprising 18 in dose escalation cohort and 10 additional patients in expansion cohort. Five dose-limiting toxicities were observed in dose escalation cohort. RPTD was defined as trifluridine/tipiracil 35 mg/m2, irinotecan 150 mg/m2, and bevacizumab 5 mg/kg. Of the 16 patients who received RPTD, patients characteristics were as follows; median age (range), 64 (38-73) years; male/female, 11/5; PS 0/1, 13/3; right-/left-sided tumor, 1/15; median number of metastatic site (range), 2 (1-5); RAS mutant/wild-type, 9/7; uridine diphosphate glucuronosyltransferase 1A1 genotype wild/single hetero/double hetero or homo, 7/8/1; prior history of bevacizumab/anti-EGFR antibody, 6/4. The most common grade 3/4 adverse events were neutropenia (86%) and leukopenia (63%) without febrile neutropenia. No treatment-related death was observed. Dose reduction, delay, and discontinuation occurred in 94%, 94%, and 6% of patients, respectively. At a median follow-up time (range) of 417 (346–611) days, the overall response rate was 19%, with 3 patients achieving a partial response. The disease control rate was 75%, with an additional 9 patients exhibiting stable disease for >4 months duration in 5 patients. The median progression-free survival and overall survival were 7.1 (95% confidence interval [CI], 3.7–not reached [NR]) months and 21.7 (95% CI, 11.3–NR) months, respectively. Biweekly administration of trifluridine/tipiracil, irinotecan, plus bevacizumab may decrease febrile neutropenia with moderate antitumor activity in previously treated mCRC patients. Further investigation of this combination therapy is required.

Firm evidence for the detoxification of senecionine-induced hepatotoxicity via N-glucuronidation in UGT1A4–humanized transgenic mice

Uridine diphosphate glucuronosyltransferase (UGT)1A4 is responsible for N-glucuronidation of tertiary amines but is a pseudogene in commonly used rodent models in toxicity and safety assessment. As a continuation of our investigation into the toxicity and safety assessment of pyrrolizidine alkaloid (PA)-containing herbs, we generated a UGT1A4–humanized (hUGT1A4) transgenic mouse model to systematically study the toxicity, metabolism network, and toxicokinetic characteristics of senecionine (a representative toxic PA) and compared with that in the wide-type controls in parallel. As results, senecionine-induced toxicity was significantly decreased as approved by mortality, pathology, and biochemistry assays in hUGT1A4 mice and cultured primary hepatocytes. More importantly N-glucuronidation adduct was exclusively identified in all the hUGT1A4 mice, liver microsomes, and cultured primary hepatocytes, yet absent in the wide-type controls. The variation in toxicokinetic characters was also observed between hUGT1A4 mice and the wide-type controls with a notably inhibition of the toxification metabolites, i.e., pyrrole-protein adducts, in hUGT1A4 mice. Conclusively, UGT1A4 plays an important role in detoxification of senecionine and the hUGT1A4 mouse model is promising for the pre-clinical evaluation of the efficacy and toxicity of tertiary amine agents in drug development and safety assessment.

Generation of HepG2 Cells with High Expression of Multiple Drug-Metabolizing Enzymes for Drug Discovery Research Using a PITCh System.

HepG2 cells are an inexpensive hepatocyte model that can be used for repeated experiments, but HepG2 cells do not express major cytochrome P450s (CYPs) and UDP glucuronosyltransferase family 1 member A1 (UGT1A1). In this study, we established CYP3A4–POR–UGT1A1–CYP1A2–CYP2C19–CYP2C9–CYP2D6 (CYPs–UGT1A1) knock-in (KI)-HepG2 cells using a PITCh system to evaluate whether they could be a new hepatocyte model for pharmaceutical studies. To evaluate whether CYPs–UGT1A1 KI-HepG2 cells express and function with CYPs and UGT1A1, gene expression levels of CYPs and UGT1A1 were analyzed by using real-time PCR, and metabolites of CYPs or UGT1A1 substrates were quantified by HPLC. The expression levels of CYPs and UGT1A1 in the CYPs–UGT1A1 KI-HepG2 cells were comparable to those in primary human hepatocytes (PHHs) cultured for 48 h. The CYPs and UGT1A1 activity levels in the CYPs–UGT1A1 KI-HepG2 cells were much higher than those in the wild-type (WT)-HepG2 cells. These results suggest that the CYPs–UGT1A1 KI-HepG2 cells expressed functional CYPs and UGT1A1. We also confirmed that the CYPs–UGT1A1 KI-HepG2 cells were more sensitive to drug-induced liver toxicity than the WT-HepG2 cells. CYPs–UGT1A1 KI-HepG2 cells could be used to predict drug metabolism and drug-induced liver toxicity, and they promise to be a helpful new hepatocyte model for drug discovery research.