Hepatic Microsomes Research Articles

Characterization of Neonicotinoid Metabolites by Cytochrome P450-Mediated Metabolism in Poultry.

Neonicotinoids, a neuro-effective class of insecticides, are heavily applied in agricultural activities worldwide. Poultry can be exposed to neonicotinoids by several routes, but the knowledge of neonicotinoid's metabolism in poultry and its associated interspecies differences is highly limited. Hence, this study aims to investigate the species differences in metabolite formations, as well as cytochrome P450 (CYP)-dependent metabolism of four major neonicotinoid compounds, acetamiprid, imidacloprid, clothianidin, and thiamethoxam, in poultry. In vitro biotransformation assays using hepatic microsomes of chicken, ducks, geese, quails, and rats were conducted. Metabolites of neonicotinoids were then screened by LC/Q-TOF and quantified by LC/MS/MS. The results revealed an existence of interspecies differences in the formations of N-[(6-chloro-3-pyridyl) methyl] -N-methyl acetamidine (IM-1-5) of acetamiprid and dm-clothianidin of clothianidin between chicken and other species. In addition, the greatest CYP activities in the metabolism of most neonicotinoid substrates, such as acetamiprid to dm-acetamiprid, imidacloprid to hydroxylated-imidacloprid and imidacloprid-olefin, clothianidin to dm-clothianidin, and thiamethoxam to clothianidin, were found in chicken. These results suggested that the CYPs in chicken may have a greater capacity for metabolism of neonicotinoids compared to other poultry. This study further revealed that the maximum intrinsic clearance of dn-imidacloprid and dn-clothianidin in ducks may be superintended by CYP-mediated nitro-reductions of imidacloprid and clothianidin. Further studies employing CYP recombinant enzymes may be required to elucidate the specific CYP isoforms that may be involved in neonicotinoid metabolism in avian species.

Sex-Linked Changes in Biotransformation of Phenol in Brook Trout (Salvelinus fontinalis) over an Annual Reproductive Cycle

The microsomal metabolism of phenol (11 °C) over an annual reproductive cycle from June to December was studied using fall spawning adult brook trout (Salvelinus fontinalis). Hepatic microsomes were isolated from three male and three female fish each month. Incubations were optimized for time, cofactor concentration, pH, and microsomal protein concentration. The formation of phase I ring-hydroxylation metabolites, i.e., hydroquinone (HQ) and catechol (CAT), was quantified by HPLC with dual-channel electrochemical detection. Sample preparation and chromatographic conditions were optimized to achieve the separation and sensitivity required for the analysis of these labile products. Biotransformation of phenol over a range of substrate concentrations (1 to 150 mM) was quantified for the calculation of Michaelis–Menten constants (Km and Vmax) for each month. Results indicate a nearly equal production of HQ and CAT among males and females in late June. At the peak of maturity in October, there was an approximate ten-fold greater production of ring-hydroxylation metabolites noted in females in comparison with males on a total liver basis. In vitro phase II biotransformation of phenol glucuronidation was assessed by determining the Michaelis–Menten constants (Km, Vmax) using brook trout hepatic microsomes over a range of substrate concentrations (1 to 60 mM). Initially, there were no significant differences in the glucuronide rate of formation (pmol/min/mg protein) or total capacity (nmol/min/liver) between females and males. At the peak of maturation, the maximum rate of glucuronide formation was 4-fold less in females; however, the total capacity was 2-fold less in females due to the increased liver size in the females. The alterations in biotransformation coincided with increases in the hepatic and gonadal somatic indices and with changes in plasma hormone concentrations. These experiments provide insight into the metabolic deactivation of xenobiotics and to provide data for the prediction of altered hepatic biotransformation rates and pathways during the reproductive cycle.

Characterization of antimalarial activity of artemisinin-based hybrid drugs.

In response to the spread of artemisinin (ART) resistance, ART-based hybrid drugs were developed, and their activity profile was characterized against drug-sensitive and drug-resistant Plasmodium falciparum parasites. Two hybrids were found to display parasite growth reduction, stage-specificity, speed of activity, additivity of activity in drug combinations, and stability in hepatic microsomes of similar levels to those displayed by dihydroartemisinin (DHA). Conversely, the rate of chemical homolysis of the peroxide bonds is slower in hybrids than in DHA. From a mechanistic perspective, heme plays a central role in the chemical homolysis of peroxide, inhibiting heme detoxification and disrupting parasite heme redox homeostasis. The hybrid exhibiting slow homolysis of peroxide bonds was more potent in reducing the viability of ART-resistant parasites in a ring-stage survival assay than the hybrid exhibiting fast homolysis. However, both hybrids showed limited activity against ART-induced quiescent parasites in the quiescent-stage survival assay. Our findings are consistent with previous results showing that slow homolysis of peroxide-containing drugs may retain activity against proliferating ART-resistant parasites. However, our data suggest that this property does not overcome the limited activity of peroxides in killing non-proliferating parasites in a quiescent state.

Steroid sulfatase in mouse liver and testis: Characterization, ontogeny and localization

Steroid hormones often circulate in the plasma as inactive sulfated forms, such as estrone sulfate and dehydroepiandrosterone sulfate. The enzyme steroid sulfatase (STS) converts these steroids into active forms, mainly estrogens, in peripheral tissues. STS is present in most tissues, but it occurs at higher levels in certain organs, notably liver and placenta. In this study, we examined the tissue distribution of STS in a prominent laboratory model, the house mouse (Mus musculus). Tissues included were heart, liver, small intestine, skeletal muscle, and gonads of both sexes. An 3H-estrone-sulfate conversion assay was used to measure STS activity in tissue homogenates and extracts. STS activities were high for hepatic tissue homogenates of both genders. Testicular STS levels were similar to those of liver, while STS activities of ovary, small intestine, heart, and muscle were considerably lower. The specific STS inhibitors, EMATE and STX-64 virtually eliminated STS activity in hepatic microsomes and cytosols, verifying that the observed enzyme activity was due to STS. Enzyme kinetic assays showed Km values of 8.6 µM for liver and 9.1 µM for testis, using E1S as substrate. Hepatic and testicular STS activities, measured in CHAPS-extracted microsome, were low up to 5 weeks of age and were higher through 56 weeks. Western blotting, with a specific STS antibody, confirmed the presence of STS protein (65 Da) in both liver and testis. Immunofluorescence of tissue sections detected the presence of STS protein in hepatocytes, in testicular Leydig cells and in seminiferous tubules (Leydig cells and developing germ cells). These results suggest that STS may have a significant role in testicular function.

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

In vitro studies using rat, mouse, and human microsomes and hepatocytes on the bacterial β-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%.

Specific human CYP enzymes-dependent mutagenicity of tris(2-butoxyethyl) phosphate (an organophosphorus flame retardant) in human and hamster cell lines

Tris(2-butoxyethyl) phosphate (TBOEP) is an organophosphorus flame retardant ubiquitously present in the environment and even the human body. TBOEP is toxic in multiple tissues, which forms dealkylated and hydroxylated metabolites under incubation with human hepatic microsomes; however, the impact of TBOEP metabolism on its toxicity, particularly mutagenicity (typically requiring metabolic activation), is left unidentified. In this study, the mutagenicity of TBOEP in human hepatoma cell lines (HepG2 and C3A) and the role of specific CYPs were studied. Through molecular docking, TBOEP bound to human CYP1A1, 1B1, 2B6 and 3A4 with energies and conformations favorable for catalyzing reactions, while the conformations of its binding with human CYP1A2 and 2E1 appeared unfavorable. In C3A cells (endogenous CYPs being substantial), TBOEP exposing for 72 h (2-cell cycle) at low micromolar levels induced micronucleus, which was abolished by 1-aminobenzotriazole (inhibitor of CYPs); in HepG2 cells (CYPs being insufficient) TBOEP did not induce micronucleus, whose effect was however potentiated by pretreating the cells with PCB126 (CYP1A1 inducer) or rifampicin (CYP3A4 inducer). TBOEP induced micronucleus in Chinese hamster V79-derived cell lines genetically engineered for stably expressing human CYP1A1 and 3A4, but not in cells expressing the other CYPs. In C3A cells, TBOEP selectively induced centromere protein B-free micronucleus (visualized by immunofluorescence) and PIG-A gene mutations, and elevated γ-H2AX rather than p-H3 (by Western blot) which indicated specific double-strand DNA breaks. Therefore, this study suggests that TBOEP may induce DNA/chromosome breaks and gene mutations in human cells, which requires metabolic activation by CYPs, primarily CYP1A1 and 3A4.

Activity-Based Protein Profiling to Probe Relationships between Cytochrome P450 Enzymes and Early-Age Metabolism of Two Polycyclic Aromatic Hydrocarbons (PAHs): Phenanthrene and Retene.

A growing body of literature has linked early-life exposures to polycyclic aromatic hydrocarbons (PAH) with adverse neurodevelopmental effects. Once in the body, metabolism serves as a powerful mediator of PAH toxicity by bioactivating and detoxifying PAH metabolites. Since enzyme expression and activity vary considerably throughout human development, we evaluated infant metabolism of PAHs as a potential contributing factor to PAH susceptibility. We measured and compared rates of phenanthrene and retene (two primary PAH constituents of woodsmoke) metabolism in human hepatic microsomes from individuals ≤21 months of age to a pooled sample (n = 200) consisting primarily of adults. We used activity-based protein profiling (ABPP) to characterize cytochrome P450 enzymes (CYPs) in the same hepatic microsome samples. Once incubated in microsomes, phenanthrene demonstrated rapid depletion. Best-fit models for phenanthrene metabolism demonstrated either 1 or 2 phases, depending on the sample, indicating that multiple enzymes could metabolize phenanthrene. We observed no statistically significant differences in phenanthrene metabolism as a function of age, although samples from the youngest individuals had the slowest phenanthrene metabolism rates. We observed slower rates of retene metabolism compared with phenanthrene also in multiple phases. Rates of retene metabolism increased in an age-dependent manner until adult (pooled) metabolism rates were achieved at ∼12 months. ABPP identified 28 unique CYPs among all samples, and we observed lower amounts of active CYPs in individuals ≤21 months of age compared to the pooled sample. Phenanthrene metabolism correlated to CYPs 1A1, 1A2, 2C8, 4A22, 3A4, and 3A43 and retene metabolism correlated to CYPs 1A1, 1A2, and 2C8 measured by ABPP and vendor-supplied substrate marker activities. These results will aid efforts to determine human health risk and susceptibility to PAHs exposure during early life.

In Vitro Evaluation of Drug-Drug Interaction Potential of Epetraborole, a Novel Bacterial Leucyl-tRNA Synthetase Inhibitor.

Epetraborole (EBO) is a boron-containing inhibitor of bacterial leucyl-tRNA synthetase, with potent activity against nontuberculous mycobacteria (NTM) and Gram-negative bacteria, including Burkholderia pseudomallei. EBO is being developed for the treatment of NTM lung disease and melioidosis, administered in combination with other therapeutic agents in both diseases. Therefore, EBO and its major circulating metabolite M3 were evaluated in comprehensive drug-drug interaction (DDI) in vitro studies. The CYP inhibitory and substrate potential of EBO and M3 were assessed using hepatic microsomes. Stably transfected cells that expressed individual efflux or uptake transporters were used to determine whether EBO or M3 were substrates or inhibitors for these receptors. Stability studies indicated that EBO is a poor substrate for major CYP enzymes. Neither EBO nor M3 was a potent reversible or time-dependent inhibitor of major CYP enzymes. EBO was not an inducer of CYP1A2 mRNA, while it was a weak inducer of CYP2B6 and CYP3A4. EBO was a substrate only for OCT2. At clinically relevant concentrations, neither EBO nor M3 inhibited major human efflux or uptake transporters. Based on these data, at clinically relevant concentrations of EBO and M3, there is a low risk of victim or perpetrator DDI.

Chronic Exposure to E-Cigarettes Elevates CYP2A5 Activity, Protein Expression, and Cotinine-Induced Production of Reactive Oxygen Species in Mice.

Coumarin 7'-hydroxylase activity, a specific marker of CYP2A5 activity, and the protein level were measured in liver microsomes of male mice after chronic exposure to e-cigarettes (e-cigs) (2.4% nicotine). After exposure for 240 minutes per day for 5 days, the activity and the protein level in preproenkephalin (ppENK)-heterozygous [ppENK (+/-)] mice were significantly elevated (P <0.05) compared with the untreated control. This elevation was not due to deletion of the ppENK gene because the activity did not differ among untreated ppENK (+/-), ppENK (-/-), and wild-type ppENK (+/+) controls. Hence, the elevation can reasonably be attributed to nicotine exposure. The production of reactive oxygen species (ROS) upon incubation of the hepatic microsomes of these mice with cotinine was higher in microsomes from the e-cig-treated mice compared with the untreated controls (P < 0.01). Liquid chromatography mass spectrometry assay showed three oxidation products of cotinine, viz trans 3'-hydroxycotinine (3'-HC), 5'-hydroxycotinine (5'-HC), and cotinine N-oxide (CNO) in the plasma of these mice. The result identifies these three oxidation reactions as the source of the observed ROS and also shows that, in nicotine-treated mice, the appropriate "nicotine metabolite ratio" is (3'-HC + 5'-HC + CNO)/cotinine. The results suggest intriguing possibilities that 1) this metabolite ratio may correlate with plasma nicotine clearance and hence impact nicotine's psychoactive effects and 2) chronic e-cig treatment causes ROS-induced oxidative stress, which may play a major role in the regulation of CYP2A5 expression. Our present results clearly show that both the activity and the protein level of CYP2A5 are elevated by repeated exposure to nicotine. SIGNIFICANCE STATEMENT: Nicotine, the psychoactive ingredient of tobacco, is eliminated as the oxidation products of cotinine in reactions catalyzed by the enzymes CYP2A5 in mice and CYP2A6 in humans. This study shows that repeated exposure to e-cigarettes elevates the level of CYP2A5 and the formation of reactive oxygen species. The results suggest an intriguing possibility that CYP2A5 may be upregulated by chronic nicotine exposure due to oxidative stress caused by the oxidation of cotinine in this preclinical model of human smokers.

Sigma receptor and aquaporin modulators: chiral resolution, configurational assignment, and preliminary biological profile of RC752 enantiomers

The key role of chiral small molecules in drug discovery programs has been deeply investigated throughout last decades. In this context, our previous studies highlighted the influence of the absolute configuration of different stereocenters on the pharmacokinetic, pharmacodynamic and functional properties of promising Sigma receptor (SR) modulators. Thus, starting from the racemic SR ligand RC752, we report herein the isolation of the enantiomers via enantioselective separation with both HPLC and SFC. After optimization of the eco-sustainable chiral SFC method, both enantiomers were obtained in sufficient amount (tens of mg) and purity (ee up to 95%) to allow their characterization and initial biological investigation. Both enantiomers a) displayed a high affinity for the S1R subtype (Ki = 15.0 ± 1.7 and 6.0 ± 1.2 nM for the (S)- and (R)-enantiomer, respectively), but only negligible affinity toward the S2R (> 350 nM), and b) were rapidly metabolized when incubated with mouse and human hepatic microsomes. Furthermore, the activity on AQP-mediated water permeability indicated a different functional profile for the enantiomers in terms of modulatory effect on the peroxiporins gating.

The variability in CYP3A4 activity determines the metabolic kinetic characteristics of ketamine

Ketamine is a psychotropic drug that can cause significant neurological symptoms and is closely linked to the activity of the CYP3A4 enzyme. This study aimed to examine the diversity of CYP3A4 activity affects the metabolism of ketamine, focusing on genetic variation and drug-induced inhibition. We used a baculovirus-insect cell expression system to prepare recombinant human CYP3A4 microsomes. Then, in vitro enzyme incubation systems were established and used UPLC-MS/MS to detect ketamine metabolite. In rats, we investigated the metabolism of ketamine and its metabolite in the presence of the CYP3A4 inhibitor voriconazole. Molecular docking was used to explore the molecular mechanism of inhibition. The results showed that the catalytic activity of CYP3A4.5, .17, .23, .28, and .29 significantly decreased compared to CYP3A4.1, with a minimum decrease of 3.13%. Meanwhile, the clearance rate of CYP3A4.2, .32, and .34 enhanced remarkably, ranging from 40.63% to 87.50%. Additionally, hepatic microsome incubation experiments revealed that the half-maximal inhibitory concentration (IC50) of voriconazole for ketamine in rat and human liver microsomes were 18.01 ± 1.20 µM and 14.34 ± 1.70 µM, respectively. When voriconazole and ketamine were co-administered, the blood exposure of ketamine and norketamine significantly increased in rats, as indicated by the area under the concentration-time curve (AUC) and maximum concentration (Cmax). The elimination half-life (t1/2Z) of these substances was also prolonged. Moreover, the clearance (CLz/F) of ketamine decreased, while the apparent volume of distribution (Vz/F) increased significantly. This might be attributed to the competition between voriconazole and ketamine for binding sites on the CYP3A4 enzyme. In conclusion, variations in CYP3A4 activity would result in the stratification of ketamine blood exposure.

Probing a distinct druggable tubulin binding site with gatorbulins 1–7, their metabolic and physicochemical properties, and pharmacological consequences

Microtubules, consisting of α/β-tubulin heterodimers, are prime targets for anticancer drug discovery. Gatorbulin-1 (GB1, 1a) is a recently described marine natural product that targets tubulin at a new, seventh pharmacological site at the tubulin intradimer interface. Using our previously developed robust route towards GB1 (1a), we synthesized simplified, first-generation gatorbulins, GB2–7 (1b–1g) of this highly modified cyclodepsipeptide (GB1) that does not contain any proteinogenic amino acid. We systematically investigated the structure-activity relationship at the biochemical and cellular level using GB1-susceptible ovarian and cervical cancer cells. We validated that the hydroxamate moiety in the N-methyl-alanine residue is critical for activity. All other structural modifications present in GB1, including C-hydroxylation of asparagine, methylation at C-4 of proline, and sp2 hybridization in dehydro-alanine, were proven to be functionally relevant. Replacement of the primary amide with a methyl ester also resulted in reduced activity, indicating the intricate scaffold optimization by the GB1-producing cyanobacterium. Inhibition of tubulin polymerization in vitro and binding affinities correlated very well, translating into differentials in cellular efficacy. We used docking and molecular dynamics to evaluate the effects of the chemical simplification at the structural level, indicating that each modification resulted in loss of target interactions, although energetically modest. Similar to cevipabulin that targets two different sites on the tubulin dimer, GB1 promotes proteasome-mediated tubulin degradation but by an unknown mechanism, presumably distinct from that of cevipabulin. Comparison with cevipabulin indicated that this compound binds to the same tubulin region as GB1 (1a), although the binding mode is distinct. Cevipabulin almost exclusively interacts with α-tubulin, including nonexchangeable GTP. In contrast, GB1 (1a) makes extensive contact and hydrogen bonds with both α- and β-chains of tubulin. GB1-7 showed excellent solubility and much higher than that of paclitaxel. Hepatic microsome stability was excellent, human cytochrome P450s were not inhibited and plasma binding was minimal with high free fractions. Passive permeability was predicted to be high based on PAMPA. Parent compound GB1 (1a) was further evaluated using a cellular model with MDCK cells stably transduced with the human efflux transporter MDR1/P-gp, showing similar permeability with and against transporter gradient, indicating that GB1 (1a) is a poor P-gp substrate.

Identification and cross-species comparison of in vitro phase I brevetoxin (BTX-2) metabolites in northern Gulf of Mexico fish and human liver microsomes by UHPLC-HRMS(/MS).

Brevetoxins (BTX) are a group of marine neurotoxins produced by the harmful alga Karenia brevis. Numerous studies have shown that BTX are rapidly accumulated and metabolized in shellfish and mammals. However, there are only limited data on BTX metabolism in fish, despite growing evidence that fish serve as vectors for BTX transfer in marine food webs. In this study, we aimed to investigate the in vitro biotransformation of BTX-2, the major constituent of BTX profiles in K. brevis, in several species of northern Gulf of Mexico fish. Metabolism assays were performed using hepatic microsomes prepared in-house as well as commercially available human microsomes for comparison, focusing on phase I reactions mediated by cytochrome P450 monooxygenase (CYP) enzymes. Samples were analyzed by UHPLC-HRMS(/MS) to monitor BTX-2 depletion and characterize BTX metabolites based on MS/MS fragmentation pathways. Our results showed that both fish and human liver microsomes rapidly depleted BTX-2, resulting in a 72-99% reduction within 1h of incubation. We observed the simultaneous production of 22 metabolites functionalized by reductions, oxidations, and other phase I reactions. We were able to identify the previously described congeners BTX-3 and BTX-B5, and tentatively identified BTX-9, 41,43-dihydro-BTX-2, several A-ring hydrolysis products, as well as several novel metabolites. Our results confirmed that fish are capable of similar BTX biotransformation reactions as reported for shellfish and mammals, but comparison of metabolite formation across the tested species suggested considerable interspecific variation in BTX-2 metabolism potentially leading to divergent BTX profiles. We additionally observed non-enzymatic formation of BTX-2 and BTX-3 glutathione conjugates. Collectively, these findings have important implications for determining the ecotoxicological fate of BTX in marine food webs.

Liquid chromatography-tandem mass spectrometry for pharmacokinetics evaluation of AZD5305, a selective PARP1 inhibitor, in mice

Poly (ADP-ribose) polymerase (PARP) inhibitors that are effective against ovarian and breast cancers with breast cancer susceptibility gene (BRCA) mutations have undesirable side effects, such as hematological toxicity. AZD5305, a selective PARP1 inhibitor currently in Phase 1/2 clinical trials, may avoid the side effects caused by PARP2. However, the in vivo pharmacokinetic characteristics of AZD5305 and its bioanalytical methods are unknown. Therefore, a method based on liquid chromatography with tandem mass spectroscopy (LC–MS/MS) was developed and validated to quantify AZD5305 in plasma of mice. Optimal chromatographic separation in terms of peak intensity and symmetry was acquired using a 4-µm Polar-RP 80 Å (2.0 × 150 mm) column with ammonium acetate (5 mM) in distilled water–acetonitrile (50:50, v/v). The retention times of AZD5305 and internal standard (IS; olaparib) were 1.82 min and 1.99 min, respectively. Detection was carried out via triple quadrupole mass spectrometry in positive ion mode employing multiple reaction monitoring transitions at m/z 407.0 → 376.0 for AZD5305 and m/z 435.0 → 281.2 for the IS. The LC–MS/MS method was linear in the range 1–1000 ng/mL with a correlation coefficient ≥ 0.990 and showed acceptable values of major parameters including accuracy, precision, and recovery. Additionally, AZD5305 showed high stability under various conditions. The in vivo and in vitro pharmacokinetics of AZD5305 were successfully characterized by employing the validated LC–MS/MS method. A high level of drug exposure and linear pharmacokinetics were observed after intravenous (IV) bolus and oral administration (PO) of AZD5305 at 0.1–1 mg/kg and 0.1–3 mg/kg, respectively. The bioavailability was close to 100%, and the metabolic stability of AZD5305 in hepatic microsomes of mice and humans was very high. These results may contribute to the improvement of PARP inhibitors that are used to treat malignancies originating from BRCA mutations.

Dietary phenolics and their microbial metabolites are poor inhibitors of trimethylamine oxidation to trimethylamine N-oxide by hepatic flavin monooxygenase 3

High circulating levels of trimethylamine N-oxide (TMAO) have been associated with cardiovascular disease risk. TMAO is formed through a microbiome-host pathway utilizing primarily dietary choline as a substrate. Specific gut microbiota transform choline into trimethylamine (TMA), and, when absorbed, host hepatic flavin-containing monooxygenase 3 (FMO3) oxidizes TMA into TMAO. Chlorogenic acid and its metabolites reduce microbial TMA production in vitro. However, little is known regarding the potential for chlorogenic acid and its bioavailable metabolites to inhibit the last step: hepatic conversion of TMA to TMAO. We developed a screening methodology to study FMO3-catalyzed production of TMAO from TMA. HepG2 cells were unable to oxidize TMA into TMAO due to their lack of FMO3 expression. Although Hepa-1 cells did express FMO3 when pretreated with TMA and NADPH, they lacked enzymatic activity to produce TMAO. Rat hepatic microsomes contained active FMO3. Optimal reaction conditions were: 50 µM TMA, 0.2 mM NADPH, and 33 µL microsomes/mL reaction. Methimazole (a known FMO3 competitive substrate) at 200 µM effectively reduced FMO3-catalyzed conversion of TMA to TMAO. However, bioavailable chlorogenic acid metabolites did not generally inhibit FMO3 at physiological (1 µM) nor supra-physiological (50 µM) doses. Thus, the effects of chlorogenic acid in regulating TMAO levels in vivo are unlikely to occur through direct FMO3 enzyme inhibition. Potential effects on FMO3 expression remain unknown. Intestinal inhibition of TMA production and/or absorption are thus likely their primary mechanisms of action.

Danazol increases the oral bioavailability of midazolam by inactivation of hepatic and intestinal CYP3A in rats

Danazol (DNZ) is a synthetic androgen derivative used for the treatment of intractable hematological disorders. In this study, we investigated the effects of DNZ on CYP3A activity in hepatic and small intestinal microsomes and the pharmacokinetics of midazolam (MDZ), a typical substrate for CYP3A, in rats. MDZ 4-hydroxylation activities in hepatic and small intestinal microsomes significantly decreased 24 h after DNZ (100 mg/kg, i.p.) treatment. Time-dependent inactivation of MDZ 4-hydroxylation activities was noted when microsomes were pre-incubated with DNZ in the presence of a NADPH-generating system. The Western blot analysis indicated that the decrease observed in enzyme activity was not due to changes in the protein expression of CYP3A. In contrast to the intravenous administration, serum MDZ concentrations in DNZ-treated rats were markedly higher than those in control rats when administered orally. DNZ treatment increased MDZ oral bioavailability by approximately 2.5-folds. We herein demonstrated that DNZ increased the bioavailability of orally administered MDZ through irreversible inactivation of hepatic and intestinal CYP3A in rats.

1716. In Vitro Drug-Drug Interaction Evaluation of Epetraborole, a Novel Bacterial Leucyl-tRNA Synthetase Inhibitor

Abstract Background Epetraborole (EBO) is a boron-containing oral inhibitor of bacterial leucyl-tRNA synthetase, an essential enzyme in protein synthesis; EBO demonstrates potent activity against nontuberculous mycobacteria. EBO is being developed for the treatment of Mycobacterium avium complex lung disease patients and will be used in combination with other drugs. Therefore, EBO and its major circulating metabolite M3 were evaluated in a comprehensive drug-drug interactions (DDI) risk assessment. Methods Stability of EBO was evaluated in human liver microsomes, hepatocytes and recombinant CYP enzymes. The inhibitory potential of EBO (0.03-100 μM) and M3 (1-1000 μM) on cytochrome P450 (CYP) activities was assessed using human hepatic microsomes. The CYP induction potential of EBO (25-200 μM), M3 was evaluated and compared to prototypical inducers in human hepatocytes (three donors) and mRNA. Fold increase in mRNA expression was utilized to investigate the CYP induction potential of EBO (0.3-100 µM) and M3 (1-250 µM). Stably transfected cell lines that expressed individual transporters were used to determine whether EBO or M3 were substrates or inhibitors for these proteins. Results In vitro studies with microsomes, hepatocytes and recombinant cytochrome P450 (CYP) enzymes indicated that EBO was a poor substrate for major CYP enzymes; drug interactions with epetraborole as victim are considered unlikely. Neither EBO nor its major metabolite M3 was a potent reversible or time-dependent inhibitor of major CYP enzymes. Half maximal inhibitory concentration (IC50) values for CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4 were &amp;gt;100 µM. EBO was not an inducer of CYP1A2 mRNA in human hepatocytes from three donors, while it was a weak inducer of CYP2B6 and CYP3A4. No induction of mRNA was observed in human hepatocytes at concentrations relevant to planned clinical doses. EBO is unlikely to be a substrate for P-gp, BCRP, OATP1B1, OATP1B3, OAT1, OAT3, MATE1 and MATE2K. EBO was an in vitro substrate for OCT2, a transporter involved in active renal secretion. At clinically relevant concentrations, neither EBO nor M3 inhibited major human efflux or uptake transporters. Conclusion At clinically relevant concentrations of EBO and its major circulating metabolite M3, there is a low risk of victim or perpetrator DDI. Disclosures Afshin Shafiee, PhD, AN2 Therapeutics: Stocks/Bonds Bradley K. Wong, PhD, AN2 Therapeutics: Advisor/Consultant Sanjay Chanda, PhD, AN2 Therapeutics: Stocks/Bonds.

Cold Exposure Induces Vaso-Occlusion and Hyperalgesia in Mice with Sickle Cell Disease That Is Dependent on Complement Activation

BACKROUND AND AIM: Vaso-occlusive crises (VOC) cause severe pain in patients with sickle cell disease (SCD). To date, epidemiological studies have shown seasonably colder temperatures exacerbate SCD-related pain. Although literature and clinical observations suggest a connection between cold and triggering of acute VOC pain, the biologic mechanisms underlying VOC-evoked pain in SCD patients are poorly understood. Repeated cold or painful events results in vasoconstriction and decreased perfusion, which increases red cell transit time, deoxygenation, and hemoglobin S polymerization [Veluswamy et al. Blood. 2020;136:1191]. SCD mice exhibit ischemia-reperfusion (I/R) pathophysiology in response to hypoxia-reoxygenation with a concomitant increase in hyperalgesia, inflammation, and complement activation. Because complement activation plays a crucial role in animal pain models, we used a new model of cold-evoked VOC and I/R in HbSS mice to examine complement C5a/C5aR signaling as a mechanistic link between vaso-occlusion and acute pain in SCD. MATERIALS AND METHODS: We used a dorsal skinfold chamber model to measure microvascular stasis (vaso-occlusion) and von Frey filaments applied to the plantar surface of the hind paws to assess mechanical hyperalgesia (pain) in Townes HbSS and control HbAA mice. Non-hyperalgesic mice were exposed to cold (10oC/50oF) for 1 hour (h) and then returned to room temperature. Measurements of VOC and paw withdrawal threshold were collected at baseline before cold and at 1-4 h after cold exposure. After the 4 h stasis measurement, mice were sacrificed, and livers removed and frozen. Hepatic microsomes and nuclear extracts were isolated by differential centrifugation and liver inflammation markers were assessed on Western blots. Hepatic nuclear extracts were blotted with antibodies to NF-ĸB phospho-p65 and total p65. Hepatic microsomes were blotted with antibodies to VCAM-1 and ICAM-1. Plasma samples were blotted with antibodies to alternative pathway factor B activation fragment Bb and complement anaphylatoxin C5a. To examine the role of complement activation in stasis and hyperalgesia produced by cold-evoked VOC, HbSS mice were infused 30 minutes before cold exposure with an inhibitory monoclonal antibody (mAb) to murine C5 to prevent C5 cleavage or C5aR to prevent C5a binding to C5aR and subsequent C5a/C5aR signaling, or a control mAb (1.2 mg/kg body weight). RESULTS: Cold exposure induced more microvascular stasis (vaso-occlusion), 1, 2, 3, and 4 h after cold exposure in subcutaneous venules of HbSS mice as compared to HbAA mice exposed to cold and HbSS mice left at room temperature (p<0.001). Cold exposure also evoked mechanical hyperalgesia in HbSS mice 1, 2, and 24 h after cold as compared to baseline prior to cold (p<0.01) and cold exposed HbAA mice (p<0.05). Vaso-occlusion and mechanical hyperalgesia were accompanied by an increase in Bb and C5a levels in the plasma of HbSS, but not HbAA mice 4 h after cold exposure (p<0.01). In addition, there was an increase in NF-κB activation and VCAM-1 and ICAM-1 adhesion molecule expression in the livers of HbSS mice after cold as compared to HbAA mice exposed to cold and HbSS mice unexposed to cold (p<0.01). Importantly, pretreatment of HbSS mice before cold exposure with a blocking mAb to C5 or C5aR inhibited vaso-occlusion (Figure 1A), mechanical hyperalgesia (Figure 1B), and complement activation (Figure 1C) as compared to cold-exposed HbSS mice treated with a control mAb. CONCLUSION: This mouse model of cold-evoked VOC provides an opportunity to examine potential mediators between vaso-occlusion and pain in SCD. Our results demonstrate that in response to cold temperatures, complement activation contributes to vaso-occlusion and acute hyperalgesia, suggesting a linkage between complement activation, vaso-occlusion, and pain during cold-induced VOC. These studies provide a rationale for clinical use of complement inhibitors to prevent and treat pain in SCD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Carboxylesterase-2 plays a critical role in dabigatran etexilate active metabolite formation

Dabigatran etexilate (DABE), an oral anticoagulant prodrug, is nearly completely metabolized to the dabigatran (DAB) active metabolite by carboxylesterase-1 (CES1) and carboxylesterase-2 (CES2). The high interpatient variation in DAB plasma concentrations, coupled with its low therapeutic index, emphasizes the need to understand how CES1 and CES2 impact active metabolite formation. Previous work focused on CES1 enzyme activity but the contributions of CES2 remain unclear. The purpose of this study was to determine how CES2 activity influences DAB active metabolite formation. We compared the efficiency of DAB formation from DABE when exposed sequentially to human intestinal and then human hepatic microsomes (mimicking the normal metabolic sequence) with the reverse metabolic sequence in which DABE is exposed to hepatic and then intestinal microsomes. The poor efficiency of DAB formation with reverse sequential hydrolysis indicates that CES2 activity is crucial for active metabolite formation. Thus, the decrease in DAB formation with normal sequential hydrolysis was more sensitive to CES2 inhibition by verapamil (CES2 IC50 = 3.4 μM) than CES1 inhibition by diltiazem (CES2 IC50 = 9.1 μM). These results show CES2 activity plays a crucial role in DAB formation and that variability in its activity is an important determinant of therapeutic response.

Identification of 5-Hydroxycotinine in the Plasma of Nicotine-Treated Mice: Implications for Cotinine Metabolism and Disposition in Vivo.

Two oxidation products of cotinine, 5-hydroxycotinine (5-HC) and cotinine N-oxide (CNO), were identified for the first time in vivo in the plasma of C57BL/6 mice after injection of nicotine (1 mg/kg) or exposure to an e-cigarette (e-cig) containing 2.4% nicotine. Liquid chromatography-mass spectrometry was used to separate 3-hydroxycotinine (3-HC), 5-HC, and CNO and to quantify each by the sensitive direct detection of their parent ion with m/z of 193.097. In nicotine-injected mice, 5-HC was as abundant as 3-HC 15 minutes postinjection, and CNO was readily detectable. In e-cig-exposed mice with plasma nicotine levels resembling that of human smokers, plasma 5-HC and CNO, as well as 3-HC, were readily quantifiable at the end of the 4-hour exposure time. In nicotine-injected mice, the combined concentration of 3-HC plus 5-HC plus CNO, all formed from cotinine by CYP2A5, was higher (P < 0.01) in females than in males, although the male-female difference in cotinine plasma level did not reach statistical significance. The result highlights the importance of considering these three oxidation products of cotinine in examining cotinine metabolism and disposition. Coumarin 7-hydroxylase activity, a specific marker of CYP2A5, measured in the hepatic microsomes of untreated mice showed that females have higher activity (P < 0.001) than males (N = 8 per sex). The abundance of plasma 5-hydroxycotinine in nicotine-treated mice raises intriguing questions about the site of its origin (hepatic or possibly kidney CYP2A5) and the routes of its disposition because urinary excretion of 5-HC has not been detected by liquid chromatography with tandem mass spectrometry in mice and is controversial in human smokers. SIGNIFICANCE STATEMENT: Nicotine is the active ingredient of tobacco, but its elimination route through its biomarker cotinine is not fully understood. By liquid chromatography-mass spectrometry, this study has identified and quantified for the first time 5-hydroxycotinine and cotinine N-oxide, which are oxidation products of cotinine, in the plasma of mice treated with nicotine or exposed to e-cigarettes. The results raise intriguing questions about nicotine disposition in vivo in this well established preclinical model of human smokers.