Metabolic Stability and Metabolite Identification of CYP450 Probe Substrates in Ferret Hepatocytes.

BACKGROUND: Ferrets exhibit similar lung physiology to humans and display similar clinical signs following influenza infection, making them a valuable model for studying high susceptibility and infection patterns. However, the metabolic fate of several common human CYP450 probe substrates in ferrets is still unknown and has not been studied. OBJECTIVE: The purpose of this study was to investigate the metabolism of nine human CYP450 probe substrates in ferret hepatocytes and explore their metabolic rate differences between ferrets and other species. METHOD: Nine substrates were individually incubated in ferret hepatocytes for up to 120 min. At each time point, 30 µL mixtures were extracted for stability analysis using LC-MS/MS methods. After a 120-minute incubation period, 400 µL of the mixtures were extracted for metabolite identification using UHPLC-QExactive Plus. RESULTS: The metabolic clearance was determined as follows: diclofenac > taxol > chlorzoxazone > dextromethorphan > midazolam > omeprazole > bupropion > phenacetin > testosterone. Seven metabolites were identified from phenacetin. Deethylation was found to be the major pathway, and the major metabolite was matched with acetaminophen as probed with the CYP1A2 enzyme. Six metabolites were identified from diclofenac. Glucuronidation was the primary pathway, and a metabolite was found to match 4-OH-diclofenac as probed with the CYP2C9 enzyme. Twenty-two metabolites were identified from omeprazole. The major metabolic pathways included mono-oxygenation and sulfoxide to thioether conversion. No metabolite was found to match with the 5-OH-omeprazole as probed with the CYP2C19 enzyme. Twenty-two metabolites were identified from dextromethorphan. Demethylation was found to be the major metabolic pathway, and one demethylation metabolite was matched with dextrorphan as probed with CYP2D6. Fourteen metabolites were identified from midazolam. Mono-oxygenation was found to be the primary metabolic pathway, and one of the mono-oxygenation metabolites was matched with 1-OH-midazolam as probed with the CYP3A4 enzyme. Eight metabolites were identified from testosterone. Mono-oxygenation and glucuronidation were identified as the major metabolic pathways. One mono-oxygenation was matched with 6-ß-testosterone as probed with CYP3A4 enzyme. Six metabolites were identified from taxol. Hydrolysis and mono-oxygenation were the top two metabolic pathways. No metabolite was matched with 6-α-OH-taxol as probed with the CYP2C8 enzyme. Ten metabolites were identified from bupropion. Mono-oxygenation and hydrogenation were identified as the top two metabolic pathways. No mono-oxygenation metabolite was matched with hydroxy-bupropion as probed with the CYP2B6 enzyme. Nine metabolites were identified from chlorzoxazone. Monooxygenation and sulfation were the top two metabolic pathways. One mono-oxygenation metabolite was matched with 6-OH-chlorzoxazone as probed with the CYP2E1 enzyme. CONCLUSION: Nine human CYP probe substrates were clearly metabolized in ferret hepatocytes, demonstrating substrate-dependent metabolic rates in ferret hepatocytes and species-dependent metabolic rates in mouse, rat, dog, monkey, and human hepatocytes. Except for 6-a-5-OH-omeprazole, 6-α-OH-taxol, and hydroxy-bupropion, specific metabolites of other six probe substrates in ferret hepatocytes were detected and identified as probed with six human CYP enzymes, respectively.

Multiple dyes applications for fluorescent convertible polymer capsules as macrophages tracking labels.

Tracing individual cell pathways among the whole population is crucial for understanding their behavior, cell communication, migration dynamics, and fate. Optical labeling is one approach for tracing individual cells, but it typically requires genetic modification to induce the generation of photoconvertible proteins. Nevertheless, this approach has limitations and is not applicable to certain cell types. For instance, genetic modification often leads to the death of macrophages. This study aims to develop an alternative method for labeling macrophages by utilizing photoconvertible micron-sized capsules capable of easy internalization and prolonged retention within cells. Thermal treatment in a polyvinyl alcohol gel medium is employed for the scalable synthesis of capsules with a wide range of fluorescent dyes, including rhodamine 6G, pyronin B, fluorescein, acridine yellow, acridine orange, thiazine red, and previously reported rhodamine B. The fluorescence brightness, photostability, and photoconversion ability of the capsules are evaluated using confocal laser scanning microscopy. Viability, uptake, mobility, and photoconversion studies are conducted on RAW 264.7 and bone marrow-derived macrophages, serving as model cell lines. The production yield of the capsules is increased due to the use of polyvinyl alcohol gel, eliminating the need for conventional filtration steps. Capsules entrapping rhodamine B and rhodamine 6G meet all requirements for intracellular use in individual cell tracking. Mass spectrometry analysis reveals a sequence of deethylation steps that result in blue shifts in the dye spectra upon irradiation. Cellular studies on macrophages demonstrate robust uptake of the capsules. The capsules exhibit minimal cytotoxicity and have a negligible impact on cell motility. The successful photoconversion of RhB-containing capsules within cells highlights their potential as alternatives to photoconvertible proteins for individual cell labeling, with promising applications in personalized medicine.

In vitro and in vivo studies on the metabolism and pharmacokinetics of the selective gut microbial ß-glucuronidase targeting compound Inh 1.

In vitro studies using rat, mouse, and human microsomes and hepatocytes on the bacterial ß-glucuronidase inhibitor 1-((6,8-dimethyl-2-oxo-1,2-dihydroquinolin-3-yl)methyl)-3-(4-ethoxyphenyl)-1-(2-hydroxyethyl)thiourea) (Inh 1) revealed extensive metabolism in all species.The intrinsic clearances of Inh 1 in human, mouse, and rat hepatic microsomes were 30.9, 67.8, and 201 µL/min/mg, respectively. For intact hepatocytes intrinsic clearances of 21.6, 96.0, and 129 µL/min/106 cells were seen for human, mouse and rat, respectively.The metabolism of Inh 1 involved an uncommon desulphurisation reaction in addition to oxidation, deethylation, and conjugation reactions at multiple sites. Six metabolites were detected in microsomal incubations in human and rat, and seven for the mouse. With hepatocytes, 18 metabolites were characterised, 9 for human, and 11 for mouse and rat.Following IV administration to mice (3 mg/kg), plasma concentrations of Inh 1 exhibited a monophasic decline with a terminal elimination half-life of 0.91 h and low systemic clearance (11.8% of liver blood flow). After PO dosing to mice (3 mg/kg), peak observed Inh 1 concentrations of 495 ng/mL were measured 0.5 h post dose, declining to under 10 ng/mL at 8 h post dose. The absolute oral bioavailability of Inh 1 in the mouse was ca. 26%.

The in vitro metabolism and in vivo pharmacokinetics of the bacterial ß-glucuronidase inhibitor UNC10201652.

In vitro incubation of the bacterial ß-glucuronidase inhibitor UNC10201652 (4-(8-(piperazin-1-yl)-1,2,3,4-tetrahydro-[1,2,3]triazino[4',5':4,5]thieno[2,3-c]isoquinolin-5-yl)morpholine) with mouse, rat, and human liver microsomes and hepatocytes generated metabolites at multiple sites via deethylations, oxidations and glucuronidation.Two UNC10201652 metabolites were detected in human, and four in mouse and rat liver microsomal incubations. Intrinsic clearances of UNC10201652 in human, mouse, and rat liver microsomes were 48.1, 115, and 194 µL/min/mg respectively.Intrinsic clearances for human, mouse, and rat hepatocytes were 20.9, 116, and 140 µL/min/106 cells respectively and 24 metabolites were characterised: 9 for human and 11 for both rodent species.Plasma clearance was 324.8 mL/min/kg with an elimination half-life of 0.66 h following IV administration of UNC10201652 to Swiss Albino mice (3 mg/kg). Pre-treatment with 1-aminobenzotriazole (ABT) decreased clearance to 127.43 mL/min/kg, increasing the t1/2 to 3.66 h.Comparison of profiles after oral administration of UNC10201652 to control and pre-treated mice demonstrated a large increase in Cmax (from 15.2 ng/mL to 184.0 ng/mL), a delay in Tmax from 0.25 to 1 h and increased AUC from 20.1 to 253 h ng/ml. ABT pre-treatment increased oral bioavailability from 15% to >100% suggesting that CYP450's contributed significantly to UNC10201652 clearance in mice.

Inhibitory and inductive effects of 4- or 5-methyl-2-mercaptobenzimidazole, thyrotoxic and hepatotoxic rubber antioxidants, on several forms of cytochrome P450 in primary cultured rat and human hepatocytes.

Effects of 4-methyl-2-mercaptobenzimidazole (4-MeMBI) and 5-methyl-2- mercaptobenzimidazole (5-MeMBI) on cytochrome P450 (CYP) activity were examined in primary cultured rat hepatocytes. Hepatocytes from male Wistar rats were cultured in the presence of 4-MeMBI or 5-MeMBI (0-400 µM), and the activity of CYPs 3A2/4 (48 and 96 h) and 1A1/2 (48 h) was determined by measuring the activity of testosterone 6ß-hydroxylation and 7-ethoxyresorufin O-deethylation, respectively. As a result, 4-MeMBI and 5-MeMBI (≥12.5 µM) inhibited CYP3A2 activity. On the other hand, 4-MeMBI (≥25 µM) and 5-MeMBI (≥100 µM) induced CYP1A1/2 activity, being consistent with the previous in vivo results. In a comparative metabolism study using primary cultured human hepatocytes from two Caucasian donors, 4-MeMBI and 5-MeMBI induced the activity of CYPs 3A4 and 1A1/2 with individual variability. It was concluded from these results that 4-MeMBI, 5-MeMBI and MBI caused inhibition of CYP3A2 activity in primary cultured rat hepatocytes, suggesting their potential for metabolic drug-drug interactions. Primary cultured rat and human hepatocytes were considered to be useful for the evaluation of effects of the benzimidazole compounds on their inducibility and inhibitory activities of cytochrome P450 forms.

Enhancement of photocatalytic efficiency by in situ fabrication of BiOBr/BiVO4 surface junctions.

Surface junctions between BiOBr and BiVO4 were synthesized. The BiOBr/BiVO4 with 1wt.% of BiOBr exhibited the highest photocatalytic activity in the degradation of RhB under visible-light irradiation. It was found that the highly efficient adsorption of RhB molecules via the electrostatic attraction between Br- and cationic N(Et)2 group played a key role for the high photocatalytic activities of BiOBr/BiVO4. This efficient adsorption promoted the N-deethylation of RhB and thus accelerated the photocatalytic degradation of RhB. Moreover, the metal-to-metal charge transfer (MMCT) mechanism was proposed, which revealed the concrete path paved with Bi-O-Bi chains for the carrier migration in BiOBr/BiVO4. The interaction between photoexcited RhB* and the Bi3+ in BiVO4 provided the driving force for the migration of photo-generated carriers along the Bi-O-Bi chains. This work has not only demonstrated the important role of efficient adsorption in the photocatalytic degradation of organic contaminants, but also developed a facile strategy to improve the efficiency of photocatalysts.

Effects of inducers of cytochrome P450s on enrofloxacin N-deethylation in crucian carp Carassius auratus gibelio.

In this study with crucian carp (Carassius auratus gibelio), the effect on enrofloxacin (EF) and its metabolite ciprofloxacin (CF) and on the activity of cytochrome P450 1A (CYP1A) and cytochrome P450 3A (CYP3A) was estimated following the oral administration of rifampicin (RIF) (12mg/kg) and ß-naphthoflavone (BNF) (12mg/kg), respectively. First, reversed-phase high-performance liquid chromatography (RP-HPLC) was used to detect the pharmacokinetics of EF with continual blood sampling. In RIF-treated, BNF-treated and control groups, the value of the CmaxCF/CmaxEF ratio was 4.41, 0.81 and 0.95, and the corresponding value of the AUC0-t-CF/AUC0-t-EF ratio was 3.69, 1.84 and 1.76, respectively. In the RIF-treated, BNF-treated and control groups, the MRT values of EF were 26.57, 27.45 and 30.88h, and the corresponding values for CF were 5.79, 35.18 and 38.11h, respectively. Based on these results for crucian carp, the accumulation and elimination of EF and CF in the RIF-treated group were more rapid than in BNF-treated and control groups. Second, liver microsomes were pretreated with the inducer of CYP1A for BNF and that of CYP3A for RIF, and then the enzymatic activities of CYP1A and CYP3A were measured, respectively. The activities of ethoxyresorufin-O-deethylation (EROD) and erythromycin-N-demethylation (ERND) increased significantly (P<0.05) for CYP1A and CYP3A, respectively. However, in further experiments on the formation of CF, the level of EF N-deethylation was significantly induced by RIF and inhibited by ketoconazole (KTZ) for CYP3A but had no influence for CYP1A, BNF and berberine chloride (BER). We concluded that CYP3A might be responsible for the N-deethylation of EF and because of this activity, could also serve as a toxicity biomarker in crucian carp.

The relationship between DNA adduct formation by benzo[a]pyrene and expression of its activation enzyme cytochrome P450 1A1 in rat.

Benzo[a]pyrene (BaP) is a human carcinogen requiring metabolic activation prior to reaction with DNA. Cytochrome P450 (CYP) 1A1 is the most important hepatic and intestinal enzyme in both BaP activation and detoxification. CYP1A2 is also capable of oxidizing BaP, but to a lesser extent. The induction of CYP1A1/2 by BaP and/or ß-naphthoflavone in liver and small intestine of rats was investigated. Both BaP and ß-naphthoflavone induced CYP1A expression and increased enzyme activities in both organs. Moreover, the induction of CYP1A enzyme activities resulted in an increase in formation of BaP-DNA adducts detected by (32)P-postlabeling in rat liver and in the distal part of small intestine in vivo. The increases in CYP1A enzyme activity were also associated with bioactivation of BaP and elevated BaP-DNA adduct levels in ex vivo incubations of microsomes of both organs with DNA and BaP. These findings indicate a stimulating effect of both compounds on BaP-induced carcinogenesis.

Inhibition of CYP1 by berberine, palmatine, and jatrorrhizine: selectivity, kinetic characterization, and molecular modeling.

Cytochrome P450 (P450, CYP) 1 family plays a primary role in the detoxification and bioactivation of polycyclic aromatic hydrocarbons. Human CYP1A1, CYP1A2, and CYP1B1 exhibit differential substrate specificity and tissue distribution. Berberine, palmatine, and jatrorrhizine are protoberberine alkaloids present in several medicinal herbs, such as Coptis chinensis (Huang-Lian) and goldenseal. These protoberberines inhibited CYP1A1.1- and CYP1B1.1-catalyzed 7-ethoxyresorufin O-deethylation (EROD) activities, whereas CYP1A2.1 activity was barely affected. Kinetic analysis revealed that berberine noncompetitively inhibited EROD activities of CYP1A1.1 and CYP1B1.1, whereas palmatine and jatrorrhizine caused either competitive or mixed type of inhibition. Among protoberberines, berberine caused the most potent and selective inhibitory effect on CYP1B1.1 with the least Ki value of 44±16 nM. Berberine also potently inhibited CYP1B1.1 activities toward 7-ethoxycoumarin and 7-methoxyresorufin, whereas the inhibition of benzo(a)pyrene hydroxylation activity was less pronounced. Berberine inhibited the polymorphic variants, CYP1B1.3 (V432L) and CYP1B1.4 (N453S), with IC50 values comparable to that for CYP1B1.1 inhibition. Berberine-mediated inhibition was abolished by a mutation of Asn228 to Thr in CYP1B1.1, whereas the inhibition was enhanced by a reversal mutation of Thr223 to Asn in CYP1A2.1. This result in conjugation with the molecular modeling revealed the crucial role of hydrogen-bonding interaction of Asn228 on CYP1B1.1 with the methoxy moiety of berberine. These findings demonstrate that berberine causes a selective CYP1B1-inhibition, in which Asn228 appears to be crucial. The inhibitory effects of berberine on CYP1B1 activities toward structurally diverse substrates can be different.

Catalytic Assays for Human Cytochrome P450 : An Introduction.

Cytochrome P450 (P450) is a superfamily of individual monooxygenase enzymes that metabolize structurally diverse xenochemicals, including many clinically useful drugs and foreign chemicals widespread in the environment. P450 substrates that can be used to selectively monitor individual P450 enzymes or P450 subfamilies have been identified through studies using P450 enzyme-selective inhibitory antibodies and chemical inhibitors in conjunction with experiments utilizing individual cDNA-expressed P450 enzymes. This chapter describes P450 form-selective substrates that can be used to monitor the activities of human P450 enzymes CYP1A, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A, CYP4A11, and CYP7A1. Cautions that need to be exercised when using these substrates to probe for individual P450 activities in human liver and other tissues are discussed.