Supplemental oxygen administration is frequently encountered in infants and adults with pulmonary insufficiency. Hyperoxia causes lung injury in experimental animals, and contributes to acute respiratory distress syndrome (ARDS) in adults. In this investigation, we tested the hypotheses that (i) hyperoxia induces CYP1A1 via the formation of 6-Formylindolo[3,2-b]carbazole (FICZ), an endogenous ligand for the Ah receptor (AHR; and (ii) FICZ would attenuate hyperoxic lung injury in vivo. Adult (10 week old) wild type (WT) mice were maintained in room air or exposed to hyperoxia (> 95% O₂) for 6-48 h, and lungs were analyzed for the formation of FICZ. FICZ was detected in mouse lungs exposed to hyperoxia at 6 h, but not at later time points, as revealed by LC-MS/MS experiments. Further mechanistic studies showed formation of FICZ from tryptophan in the presence of Fe²⁺ and H₂O₂ in vitro, and also in H358 cells exposed to hyperoxia (80% O₂; 8 h). In a second set of experiments, WT mice were treated with FICZ (2 mg/kg), i.p., once daily for 3 days, and the animals were maintained in room air or exposed to hyperoxia (> 95% O₂) for 72 h. FICZ significantly induced CYP1A1, 1A2, and 1B1 in and We report that FICZ significantly upregulated CYP1A1 and CYP1B1 in lung and liver and CYP1A2 in liver at the mRNA, protein, and enzyme levels. Hyperoxia caused significant lung injury and inflammation as determined by increased lung weight/body weight (LW/BW) ratios and histology and neutrophil recruitment. FICZ treatment significantly suppressed lung injury mediated by hyperoxia. In a third set of experiments, WT, Cyp1a1-null, Cyp1a2-null, Cyp1b1-null, Cyp1a1/1a2-double null, Cyp1a1/1a2/1b1 triple null, and AHR-null mice were treated with FICZ (2 mg/kg), i.p, and FICZ levels were measured in lung and liver. FICZ was detected in all types of mice, but the levels were significantly higher in Cyp1a1-null, Cyp1a1/1a2-double null, Cyp1a1/1a2/1b1-triple null, and AHR-null, with FICZ being the highest in AHR-null mice. These results suggest that CYP1A1, 1A2, and 1B1 play an important, differential role in the metabolic degradation of FICZ in vivo. In conclusion, our results demonstrate t(i) novel role of FICZ in the induction of CYP1A1 by hyperoxia via the AHR; (ii) protection against hyperoxic lung injury by FICZ; and (iii) role of CYP1A1, 1A2, 1B1, and AHR-mediated mechanisms in the metabolism of FICZ in vivo. Further studies could lead to the development of novel AHR agonists in the prevention/treatment of ARDS in humans.

The CYP1A2 inhibition by a herbal formula Xin-yi-san: theophylline oxidation and risk assessment

Yune-Fang Ueng^1,2*,An-Chi Chen^1,2, Hong-Jaan Wang³, Li-Ting Kao³, Chung-Kuang Lu⁴, and Keng-Chang Tsai⁴

¹Division of Basic Chinese Medicine, National Research Institute of Chinese Medicine, Ministry of Health and Welfare, Taipei, Taiwan

²Institute of Biopharmaceutical Sciences, College of Pharmaceutical Sciences, National Yang Ming Chiao Tung University, Taipei, Taiwan

³School of Pharmacy, National Defense Medical Center, Taipei, Taiwan

⁴Division of Chinese Medicinal Chemistry, National Research Institute of Chinese Medicine, Ministry of Health and Welfare, Taipei, Taiwan

The Xin-yi-san herbal decoction (XYS) is commonly used to treat patients with allergic rhinitis. Theophylline has a narrow therapeutic index and its elimination primarily depends on CYP1A2 activity. The XYS contained inhibitors that decreased theophylline oxidation (THO) activities of rat liver microsomes and human CYP1A2. Among the herbal components of XYS decoction, Angelicae Dahuricae Radix contained potent inhibitors of THO. A furanocoumarin imperatorin was abundant in XYS and Angelicae Dahuricae Radix decoctions. In rats, oral administration of XYS and imperatorin decreased theophylline clearance. In a recombinant human CYP1A2 system, imperatorin inhibited THO activity with K_i values of 77‒84 nM, higher than those (20‒52 nM) of fluvoxamine, which clinically interacted with theophylline. A retrospective study of theophylline users showed that 2.6% of 201,093 theophylline users consumed XYS during 2017‒2018 in Taiwan. After the adjustment using inverse probability weighting, XYS users had a higher occurrence of undesired effects than non-XYS users. There was an approximately two-fold higher occurrence of headaches (odds ratio (OR), 2.14; 95% confidence interval (CI), 1.99‒2.30) and tachycardia (OR, 1.83; 95% CI, 1.21‒2.7). The incidence of irregular heartbeats increased (OR, 1.36; 95% CI, 1.07‒1.72) in the theophylline users who took a high cumulative dose (> 24 g) of XYS. However, compared with the non-XYS users, mortality in patients taking XYS and theophylline was lower (OR, 0.24; 95% CI, 0.14‒0.40). The use of XYS did not affected the occurrence of bone fracture, which was used as a negative control. In summary, CYP1A2 inhibitors were identified in XYS and undesirable effects were observed in patients receiving both theophylline and XYS. Further human studies are essential for adjusting the dosage of theophylline and exploring the reduced mortality in XYS users.

Joan Z. Zuo,

School of Pharmacy, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong SAR

The talk will provide i) an overview of enzyme mediated herb-drug interactions and related mechanisms so far with highlight on those related CYP450; ii) A case study on one of our recent findings on the genetic polymorphism based inhibition on CYP450 by Radix Puerariae lobatae (Gegen) in healthy subjects. Based on our serial preclinical findings, we hypothesized that puerarin containing Radix Puerariae lobatae could inhibit the CYP 450s and offset the activities of anticoagulation and antiplatelet drugs via alteration of the relevant biomarkers. A study was then designed aiming to evaluate the potential inhibition of CYP 450 enzymes by co-administered Gegen in healthy subjects and related mechanisms especially on the genetic polymorphism impact on such inhibition of CYP450. About fifty two healthy subjects (26 males and 26 females) were randomized into two groups (14 males & 14 females for low dose Gegen group; 12 males & 12 females for high Gegen dose group ) for investigations on the effect of Gegen at low (3.3 g/time, t.i.d) or high doses (5 g/time, t.i.d) on the cocktail probe drugs for CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6 and CYP3A4. All the healthy volunteers received a single oral dose of the six-probe cocktail as baseline (Session I) followed by a 14-day oral treatment with Gegen at the dose of 3.3g/5g per time, t.i.d. On Day 14, 2h after the Gegen dosing, each healthy volunteer received another dose of the six-probe cocktail (Session II). To further verify the pharmacogenetic impact, identification for the variant of CYP2C19 in those subjects with a reduced-function CYP2C19 genotype using PCR followed by Sanger sequencing and comparisons with reference genes from NCBI were conducted. Among the six major CYPs, orally administered Gegen could only inhibit CYP2C19 in a genotype and dose dependent manner. Considering its extensive clinical applications, genetic screening guidance on the safe use of Gegen in individuals are warranted.

The intestinal epithelium and the liver form a physical, but also a biochemical and metabolic barrier against orally ingested chemicals. The marine biotoxin okadaic acid (OA) is produced by dinoflagellates and induces diarrheic shellfish poisoning, characterized by gastrointestinal symptoms. Acute OA toxicity is based on the disruption of the physical intestinal barrier, due to the break of intestinal cell-cell contacts while, at the molecular level, the compound disturbs protein phosphorylation by acting as an inhibitor of especially protein phosphatase 2A, but also other cellular phosphatases. Aim of this study was to investigate whether sub-toxic concentrations of OA, not affecting the physical barrier function, are also able affect the metabolic barrier function of the liver.

HepaRG human liver cells were differentiated and treated with non-cytotoxic OA concentrations. Expression and activity of xenobiotic-metabolizing enzymes, especially cytochrome P450 (CYP) isoforms, and CYP-regulating nuclear receptors were analyzed. A multi-omics approach, consisting of a combination of transcriptomics with targeted and non-targeted proteomics, was used in combination with subsequent bioinformatic data mining to characterize OA-mediated alterations in cellular signaling pathways.

Non-toxic concentrations of OA were able to inhibit a broad spectrum of xenobiotic-metabolizing CYPs at the mRNA, protein and activity levels. In addition, expression and transcriptional activity of nuclear receptors was reduced. Omics data revealed characteristic patterns of altered phosphorylation-dependent signaling pathways, and by the use of specific inhibitors, it was demonstrated that activation of the transcription factor NF-κB, followed by cytokine release and subsequent activation of JAK/STAT-dependent signaling is the molecular pathway by which OA interferes with CYP expression.

CYP expression is inhibited by OA in human liver cells via activation of NF-κB and JAK/STAT. Thus, OA might not only disrupt the physical intestinal barrier at higher concentrations, but also disrupts the hepatic and intestinal metabolic, biochemical barrier against xenobiotics, already at exposure to non-cytotoxic concentrations.