Cytochrome P450 Activity Research Articles

Safer alternatives? Bisphenol F and Bisphenol S induce oxidative stress in Drosophila melanogaster larvae and trigger developmental damage.

Bisphenol F (BPF) and Bisphenol S (BPS) are safe alternatives substances? Here Drosophila melanogaster were exposed during development (larval stage) to BPF and BPS (0.25, 0.5 and 1mM). Upon reaching the last larval stage (3rd stage), markers of oxidative stress and metabolism of both substances were evaluated, along with investigation of mitochondrial and cell viability. This study is attributed to an unprecedented fact: BPF and BPS exposed larvae, both at concentrations of 0.5 and 1mM, showed higher cytochrome P-450 (CYP450) activity. The GST activity increased in all BPF and BPS concentrations, and reactive species, lipid peroxidation, superoxide dismutase, and catalase activity increased in larvae (BPF and BPS; 0.5, and 1mM); nonetheless, mitochondrial and cell viability decreased with 1mM of BPF and BPS. In addition, the reduced number of pupae formed in the 1mM BPF and BPS groups and melanotic mass formation may be attributed to oxidative stress. From the pupae formed, the hatching rate reduced in the 0.5 and 1mM BPF and BPS groups. Thus, the possible presence of toxic metabolites may be related to the larval oxidative stress condition, which is detrimental to the complete development of Drosophila melanogaster.

A linked physiologically based pharmacokinetic model for hydroxychloroquine and metabolite desethylhydroxychloroquine in SARS-CoV-2(-)/(+) populations.

Hydroxychloroquine (HCQ) is Food and Drug Administration (FDA)-approved for malaria, systemic and chronic discoid lupus erythematosus, and rheumatoid arthritis. Because HCQ has a proposed multimodal mechanism of action and a well-established safety profile, it is often investigated as a repurposed therapeutic for a range of indications. There is a large degree of uncertainty in HCQ pharmacokinetic (PK) parameters which complicates dose selection when investigating its use in new disease states. Complications with HCQ dose selection emerged as multiple clinical trials investigated HCQ as a potential therapeutic in the early stages of the COVID-19 pandemic. In addition to uncertainty in baseline HCQ PK parameters, it was not clear if disease-related consequences of SARS-CoV-2 infection/COVID-19 would be expected to impact the PK of HCQ and its primary metabolite desethylhydroxychloroquine (DHCQ). To address the question whether SARS-CoV-2 infection/COVID-19 impacted HCQ and DHCQ PK, dried blood spot samples were collected from SARS-CoV-2(-)/(+) participants administered HCQ. When a previously published physiologically based pharmacokinetic (PBPK) model was used to fit the data, the variability in exposure of HCQ and DHCQ was not adequately captured and DHCQ concentrations were overestimated. Improvements to the previous PBPK model were made by incorporating the known range of blood to plasma concentration ratios (B/P) for each compound, adjusting HCQ and DHCQ distribution settings, and optimizing DHCQ clearance. The final PBPK model adequately captured the HCQ and DHCQ concentrations observed in SARS-CoV-2(-)/(+)participants, and incorporating COVID-19-associated changes in cytochrome P450 activity did not further improve model performance for the SARS-CoV-2(+) population.

Target and nontarget mechanisms of AHAS inhibitor cross-resistance patterns in Cyperus difformis

Weed resistance to acetohydroxyacid synthase (AHAS) inhibiting herbicides has been a critical issue for rice growers worldwide since the early 1990's. In California, resistance to bensulfuron-methyl was first detected in Cyperus difformis in 1993. Since then, populations of most major weeds of rice in California have been reported to show resistance to at least one AHAS inhibitor. We sought to describe the magnitude and mechanisms of AHAS inhibitor cross-resistance in California populations of C. difformis. Sixty-two populations were collected and screened for cross-resistance to bensulfuron-methyl (BEN), halosulfuron-methyl (HAL), bispyribac‑sodium (BIS), and penoxsulam (PEN), revealing six major patterns of cross-resistance. Representative C. difformis populations from each cross-resistance pattern were then subjected to dose-response, cytochrome P450 inhibition, AHAS gene sequencing, and metabolic studies with the same herbicides as in the screening. Dose-response confirmed the detected resistances in the representative populations, and suggested that the majority of observed resistance was dose-dependent. Cytochrome P450 inhibition via malathion revealed evidence of increased metabolic activity in resistant populations to BEN, BIS, and PEN. AHAS gene sequencing revealed amino acid substitutions in five of six populations: R3 (Pro197-Ser), R4 (Pro97-His), R10 (Asp376), R41 (Ala122-Asn), and R18 (Trp574-Leu). Metabolic studies confirmed evidence of increased activity of cytochrome P450s in all populations. Metabolic BEN and HAL analysis did not yield similar results to malathion inhibition, suggesting different P450's or other pathways. Taken together, the results of the studies confirm the complexity of AHAS inhibitor cross-resistance in C. difformis, and the presence of both target-site and metabolic resistance in most of the representative populations underscores the importance of proper herbicide selection, rotation, and scouting in fields.

New in vitro screening system to detect drug-induced liver injury using a culture plate with low drug sorption and high oxygen permeability

Drug-induced liver injury (DILI) is a major factor underlying drug withdrawal from the market. Therefore, it is important to predict DILI during the early phase of drug discovery. Metabolic activation and mitochondrial toxicity are good indicators of the potential for DILI. However, hepatocyte function, including drug-metabolizing enzyme activity and mitochondrial function, reportedly decreases under conventional culture conditions; therefore, these conditions fail to precisely detect metabolic activation and mitochondrial toxicity-induced cell death. To resolve this issue, we employed a newly developed cell culture plate with high oxygen permeability and low drug sorption (4-polymethyl-1-pentene [PMP] plate). Under PMP plate conditions, cytochrome P450 (CYP) activity and mitochondrial function were increased in primary rat hepatocytes. Following l-buthionine-sulfoximine-induced glutathione depletion, acetaminophen-induced cell death significantly increased under PMP plate conditions. Additionally, 1-aminobenzotriazole reduced cell death. Moreover, mitochondrial toxicity due to mitochondrial complex inhibitors (ketoconazole, metformin, and phenformin) increased under PMP plate conditions. In summary, PMP plate conditions could improve CYP activity and mitochondrial function in primary rat hepatocytes and potentially detect metabolic activation and mitochondrial toxicity.

5-ALA treatment increases intracellular heme levels and enhances CYP3A4 activity in genome-edited Caco-2 cells

In nonclinical studies, models that can predict the metabolism of drug candidates by cytochrome P450 (CYP), including Cytochrome P450 family 3 subfamily A member 4 (CYP3A4) are helpful. CYP3A4-overexpressing human cells have been used universally to evaluate whether CYP3A4 metabolizes drug-candidate compounds. However, CYP3A4-overexpressing human cell lines are problematic because their activity levels are lower than that of in vivo human CYP3A4. Heme plays a paramount role in CYP activity. The rate-limiting step in heme biosynthesis is the generation of 5-aminolevulinic acid (5-ALA). In this study, we examined whether treatment with 5-ALA to CYP3A4-POR-UGT1A1-CES2 knockin and CES1 knockout (genome-edited) Caco-2 cells enhances CYP3A4 activity. A 7-day 5-ALA treatment increased intracellular heme levels in genome-edited Caco-2 cells without cytotoxicity. Moreover, consistent with the increase in intracellular heme content, 5-ALA treatment increased CYP3A4 activity in genome-edited Caco-2 cells. The results of this research are expected to be applied to pharmacokinetic studies using CYP-overexpressing human cells containing CYP3A4.

Role of Cytochrome P450 2C9 in COVID-19 Treatment: Current Status and Future Directions.

The major human liver drug metabolising cytochrome P450 (CYP) enzymes are downregulated during inflammation and infectious disease state, especially during coronavirus disease 2019 (COVID-19) infection. The influx of proinflammatory cytokines, known as a 'cytokine storm', during severe COVID-19 leads to the downregulation of CYPs and triggers new cytokine release, which further dampens CYP expression. Impaired drug metabolism, along with the inevitable co-administration of drugs or 'combination therapy' in patients with COVID-19 with various comorbidities, could cause drug-drug interactions, thus worsening the disease condition. Genetic variability or polymorphism in CYP2C9 across different ethnicities could contribute to COVID-19 susceptibility. A number of drugs used in patients with COVID-19 are inducers or inhibitors of, or are metabolised by, CYP2C9, and co-administration might cause pharmacokinetic and pharmacodynamic interactions. It is also worth mentioning that some of the COVID-19 drug interactions are due to altered activity of other CYPs including CYP3A4. Isoniazid/rifampin for COVID-19 and tuberculosis co-infection; lopinavir/ritonavir and cobicistat/remdesivir combination therapy; or multi-drug therapy including ivermectin, azithromycin, montelukast and acetylsalicylic acid, known as TNR4 therapy, all improved recovery in patients with COVID-19. However, a combination of CYP2C9 inducers, inhibitors or both, and plausibly different CYP isoforms could lead to treatment failure, hepatotoxicity or serious side effects including thromboembolism or bleeding, as observed in the combined use of azithromycin/warfarin. Further, herbs that are CYP2C9 inducers and inhibitors, showed anti-COVID-19 properties, and in silico predictions postulated that phytochemical compounds could inhibit SARS-CoV-2 virus particles. COVID-19 vaccines elicit immune responses that activate cytokine release, which in turn suppresses CYP expression that could be the source of compromised CYP2C9 drug metabolism and the subsequent drug-drug interaction. Future studies are recommended to determine CYP regulation in COVID-19, while recognising the involvement of CYP2C9 and possibly utilising CYP2C9 as a target gene to tackle the ever-mutating SARS-CoV-2.

Metabolic Exploration for Cyhalofop-Butyl Antagonism in Barnyardgrass [Echinochloa crus-galli (L.) P. Beauv.] Following Pretreatment of Malathion.

The present study investigated the effects of broad-spectrum metabolic inhibitors malathion (cytochrome P450 inhibitor) and/or 4-chloro-7-nitrobenzofurazan (NBD-Cl; glutathione S-transferase inhibitor) on the metabolism of cyhalofop-butyl (CyB) in barnyardgrass [Echinochloa crus-galli (L.) P. Beauv.] biotypes confirmed previously with multiple resistance to two herbicides CyB and florpyrauxifen-benzyl. The metabolic inhibitors were not effective at recovering the sensitivity of resistant barnyardgrass biotypes to CyB treated at the labeled rate (313 g ai ha-1). Rather, treatment with malathion followed by CyB caused antagonism, reducing the efficacy of CyB and promoting the growth of resistant biotypes. Pretreatment with malathion did not influence absorption/translocation of the applied form CyB and its conversion to the active herbicide form cyhalofop-acid (CyA), in both susceptible and resistant biotypes. In contrast, metabolism of the applied form (CyB) decreased 1.5 to 10.5 times by the malathion pretreatment. Taken together, the maintained CyA production against the reduced CyB metabolism could be the mechanism to account for the cause of CyB antagonism observed in barnyardgrass following malathion pretreatment. Additionally, the evolution of CyB resistance in barnyardgrass might be associated with reduced production of CyA in resistant biotypes, independent of activities of cytochrome P450 or GST enzymes.

Molecular Basis of Resistance to Bensulfuron-Methyl in a Smallflower Umbrella Sedge (Cyperus difformis L.) Population from China

Smallflower umbrella sedge (Cyperus difformis L.) is an invasive weed, and infestations of C. difformis are increasing in rice (Oryza sativa L.) fields in China. Bensulfuron-methyl is a widely used sulfonylurea herbicide that inhibits the acetolactate synthase (ALS) enzyme and has been used in recent years for effectively controlling annual weeds in the Cyperaceae family. In this study, a suspected resistant population of C. difformis (BBHY1) was collected from a rice field in Huaiyuan County, Anhui Province, China, that survived treatment with bensulfuron-methyl at the field-recommended rate (FRR). Single-dose tests and whole-plant bioassays confirmed that the BBHY1 population was resistant to bensulfuron-methyl and had evolved a high level of resistance, with a resistance index (RI) of 12.87. Sequencing of the ALS gene revealed a CCT to CAT point mutation at codon 197, which caused a P-to-H substitution in the resistant plants. Analysis of the relative expression of ALS revealed no significant differences between the resistant and susceptible populations. Inhibiting the activity of cytochrome P450s (P450s) or glutathione S-transferases (GSTs) had no significant effect on bensulfuron-methyl resistance. The BBHY1 population exhibited cross-resistance to pyrazosulfuron-ethyl, penoxsulam, and bispyribac-sodium, with RIs ranging from 5.48 to 20.63, but remained susceptible to MCPA sodium, florpyrauxifen-benzyl, and bentazon, with RIs of <1.00. These herbicides could be potentially used as alternatives for controlling resistant populations and managing herbicide resistance in other aggressive weeds in rice fields.

Oxidative stress and inflammation: the root causes of aging

Oxygen free radicals [reactive oxygen species (ROS)] and nitrogen free radicals [reactive nitrogen species (RNS)] are generated by mitochondria during adenosine triphosphate synthesis, and catalytic activities of cytochrome P450, nicotinamide adenine dinucleotide phosphate oxidases (NOXs), cyclooxygenases, and nitric oxide synthases during drug catabolism, phagocytosis, and acute inflammation. Under normal circumstances, low levels of ROS and RNS provide redox signalings that control many essential physiological processes. As age progresses ROS and RNS increase excessively due to dysfunctional mitochondria, dysregulated NOX, and other free-radical generating sources, leading to oxidative stress, which causes oxidation and denaturation of key cellular components including DNA, proteins, and lipids, which become abnormal, constituting damage-associated molecular pattern (DAMP), recognized as ‘non-self’ by immune cells, leading to inflammation which is mediated by nuclear factor kappa B-inflammasome, p38-c-Jun N-terminal kinase and Janus kinase-signal transducer and activator of transcription pathways. DAMPs are continuously released from damaged and senescent cells, causing an otherwise normally transient inflammation turning into systemic chronic inflammation, the root cause of aging and age-associated diseases (AADs). Cells restore redox balance by activating the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway that induces the synthesis and release of antioxidation molecules and enzymes including haem oxygenase-1, which also inhibits the three inflammatory pathways. Furthermore, upregulation of autophagy (AP) can get rid of abnormal molecules, prevent the generation of DAMPs, and attenuate inflammation. Both AP and Nrf2 signalings decrease with age. The upregulations of Nrf2, AP, and downregulation of inflammation are controlled by sensors of energy and stress levels, i.e., adenosine monophosphate-activated protein kinase, silent information regulator 1, and Sestrins, as well as the extracellular matrix, while mammalian targets for rapamycin complex 1, a nutrient sensor, act in the opposite direction. If the balance of these sensor systems becomes dysregulated, aging process accelerates, and the risk of AADs increases.

A cocktail probe approach to evaluate the effect of hormones on the expression and activity of CYP enzymes in human hepatocytes with conditions simulating late stage of pregnancy.

Pregnancy-mediated physiological and biochemical changes contribute to alterations in the pharmacokinetics of certain drugs. There is a paucity of data on the systematic evaluation of the underlying mechanisms. The objective of the current study was to examine the impact of changes in circulating and tissue hormonal concentrationduring the late stage of pregnancy on the activity and expression of hepatic cytochrome P450 (CYP) enzymes using a cocktail probe approach. Freshly isolated primary human hepatocytes were incubated with third trimester physiologic (plasma) and projected liver (ten-fold higher) concentrations of female hormones: progesterone (2µM), estradiol (0.3µM), estriol (0.8µM), estrone (0.2µM), 17α-hydroxyprogesterone (0.1µM), and human growth hormone (0.005µM). The metabolic activity of the hepatocytes was assessed using a cocktail of isozyme-specific P450 probe substrates (CYP1A2 (phenacetin), CYP2C9 (diclofenac), CYP2C19 (S-mephenytoin), CYP2D6 (dextromethorphan), and CYP3A4 (testosterone)). A validated LC-MS/MS assay was used to measure the corresponding metabolite concentrations. CYP450 protein and mRNA levels were measured using western blot and qRT-PCR, respectively. Female hormones at projected third-semester hepatic concentrations significantly enhanced mRNA and protein expression and increased the metabolic activity of CYP3A4. The expression and activity of other CYP450 enzymes studied were not affected by mixtures of female hormones at concentrations used. The increased activity of CYP3A4 is consistent with the clinically observed increase in clearance of CYP3A4 substrates during pregnancy. Overall expression and activity of CYP450 isozymes are differentially regulated during pregnancy.

The Disconnect in Intrinsic Clearance Determined in Human Hepatocytes and Liver Microsomes Results from Divergent Cytochrome P450 Activities.

Candidate drugs may exhibit higher unbound intrinsic clearances (CLint,u) in human liver microsomes (HLMs) relative to human hepatocytes (HHs), posing a challenge as to which value is more predictive of in vivo clearance (CL). This work was aimed at better understanding the mechanism(s) underlying this 'HLM:HH disconnect' via examination of previous explanations, including passive permeability limited CL or cofactor exhaustion in hepatocytes. A series of structurally related, passively permeable (Papps > 5 × 10-6 cm/s), 5-azaquinazolines were studied in different liver fractions, and metabolic rates and routes were determined. A subset of these compounds demonstrated a significant HLM:HH (CLint,u ratio 2-26) disconnect. Compounds were metabolized via combinations of liver cytosol aldehyde oxidase (AO), microsomal cytochrome P450 (CYP) and flavin monooxygenase (FMO). For this series, the lack of concordance between CLint,u determined in HLM and HH contrasted with an excellent correlation of AO dependent CLint,u determined in human liver cytosol[Formula: see text], r2 = 0.95, P < 0.0001). The HLM:HH disconnect for both 5-azaquinazolines and midazolam was as a result of significantly higher CYP activity in HLM and lysed HH fortified with exogenous NADPH relative to intact HH. Moreover, for the 5-azaquinazolines, the maintenance of cytosolic AO and NADPH-dependent FMO activity in HH, relative to CYP, supports the conclusion that neither substrate permeability nor intracellular NADPH for hepatocytes were limiting CLint,u Further studies are required to identify the underlying cause of the lower CYP activities in HH relative to HLM and lysed hepatocytes in the presence of exogenous NADPH. SIGNIFICANCE STATEMENT: Candidate drugs may exhibit higher intrinsic clearance in human liver microsomes relative to human hepatocytes, posing a challenge as to which value is predictive of in vivo clearance. This work demonstrates that the difference in activity determined in liver fractions results from divergent cytochrome P450 but not aldehyde oxidase or flavin monooxygenase activity. This is inconsistent with explanations including substrate permeability limitations or cofactor exhaustion and should inform the focus of further studies to understand this cytochrome P450 specific disconnect phenomenon.

DEEPCYPs: A deep learning platform for enhanced cytochrome P450 activity prediction.

Cytochrome P450 (CYP) is a superfamily of heme-containing oxidizing enzymes involved in the metabolism of a wide range of medicines, xenobiotics, and endogenous compounds. Five of the CYPs (1A2, 2C9, 2C19, 2D6, and 3A4) are responsible for metabolizing the vast majority of approved drugs. Adverse drug-drug interactions, many of which are mediated by CYPs, are one of the important causes for the premature termination of drug development and drug withdrawal from the market. In this work, we reported in silicon classification models to predict the inhibitory activity of molecules against these five CYP isoforms using our recently developed FP-GNN deep learning method. The evaluation results showed that, to the best of our knowledge, the multi-task FP-GNN model achieved the best predictive performance with the highest average AUC (0.905), F1 (0.779), BA (0.819), and MCC (0.647) values for the test sets, even compared to advanced machine learning, deep learning, and existing models. Y-scrambling testing confirmed that the results of the multi-task FP-GNN model were not attributed to chance correlation. Furthermore, the interpretability of the multi-task FP-GNN model enables the discovery of critical structural fragments associated with CYPs inhibition. Finally, an online webserver called DEEPCYPs and its local version software were created based on the optimal multi-task FP-GNN model to detect whether compounds bear potential inhibitory activity against CYPs, thereby promoting the prediction of drug-drug interactions in clinical practice and could be used to rule out inappropriate compounds in the early stages of drug discovery and/or identify new CYPs inhibitors.

Optimization of Campesterol-Producing Yeast Strains as a Feasible Platform for the Functional Reconstitution of Plant Membrane-Bound Enzymes.

Campesterol is a major phytosterol that plays important roles in regulating membrane properties and serves as the precursor to multiple specialized metabolites, such as the phytohormone brassinosteroids. Recently, we established a campesterol-producing yeast strain and extended the bioproduction to 22-hydroxycampesterol and 22-hydroxycampest-4-en-3-one, the precursors to brassinolide. However, there is a trade-off in growth due to the disrupted sterol metabolism. In this study, we enhanced the growth of the campesterol-producing yeast by partially restoring the activity of the sterol acyltransferase and engineering upstream FPP supply. Furthermore, genome sequencing analysis also revealed a pool of genes possibly associated with the altered sterol metabolism. Retro engineering implies an essential role of ASG1, especially the C-terminal asparagine-rich domain of ASG1, in the sterol metabolism of yeast especially under stress. The performance of the campesterol-producing yeast strain was enhanced with the titer of campesterol to 18.4 mg/L, and the stationary OD600 was improved by ∼33% compared to the unoptimized strain. In addition, we examined the activity of a plant cytochrome P450 in the engineered strain, which exhibits more than 9-fold higher activity than when expressed in the wild-type yeast strain. Therefore, the engineered campesterol-producing yeast strain also serves as a robust host for the functional expression of plant membrane proteins.

Insecticide resistance monitoring and diagnostics of resistance mechanisms in cotton‐melon aphid, Aphis gossypii Glover in Central China

AbstractAphis gossypii Glover is one of the most serious pests in China, and the management of this pest mainly relies on insecticides. However, the resistance status of A. gossypii in Central China is still unknown. Therefore, in this study, we firstly determined the susceptibility of 15 A. gossypii field populations from Central China to six chemical insecticides using leaf‐dipping method during 2020 and 2021 and explored the potential resistance mechanisms of these populations. Most of the field populations of A. gossypii have evolved low to high levels of resistance to imidacloprid (RR = 5.98–2352.96‐fold), thiamethoxam (RR = 5.98–2194.96‐fold), flonicamid (RR = 7.18–306.07‐fold), and pirimicarb (RR = 6.90–283.00‐fold); however, 13 tested field populations remained susceptible to chlorpyrifos. For λ‐cyhalothrin, most populations have evolved low to moderate levels of resistance (RR = 5.72–47.38‐fold). The results of correlation analysis indicated that there was a prominent positive correlation between the enzyme activity of cytochrome P450 and the logarithm of LC50 to thiamethoxam and pirimicarb. Carboxylesterase (CarE) was positively correlated with λ‐cyhalothrin, while glutathione S‐transferase (GST) was negatively correlated with λ‐cyhalothrin. Furthermore, we found that multiple resistance associated target site mutations (V62I, R81T, A302S, S431F, M918L) can be detected in the field populations of A. gossypii. Aphis gossypii in Central China have developed different degrees of resistance to tested insecticides and enhanced detoxification of cytochrome P450 and carboxylesterase and co‐existed resistance mutations contribute to the multiple resistance of these field populations. Our results provide valuable information about the status of insecticide resistance in China and are helpful for designing sustainable management strategies of A. gossypii.

Risk Assessment of Fluxametamide Resistance and Fitness Costs in Fall Armyworm (Spodoptera frugiperda).

The fall armyworm (FAW), Spodoptera frugiperda, is one of the most devastating invasive polyphagous pests, which has attracted recent global attention by developing resistance to various insecticidal active ingredients with independent mode of action. Fluxametamide, a newly commercialized isoxazoline insecticide, is exceptionally selective towards several lepidopteran pests. The present study aimed to evaluate resistance risk in FAW to fluxametamide and the fitness costs associated with fluxametamide resistance. A field-collected and genetically mixed population of FAW was artificially selected through continuous exposure to fluxametamide. After successive selection of 10 generations, there was no obvious increase in the LC50 (RF: 2.63-fold). The realized heritability (h2) of fluxametamide resistance was estimated as h2 = 0.084 using a quantitative genetic approach. Compared with the susceptible F0 strain, the Flux-SEL (F10) strain of FAW displayed no significant cross-resistance to broflanilide, chlorantraniliprole, fipronil, indoxacarb, lambda cyhalothrin, spinetoram, and tetraniliprole, except emamectin benzoate (RF: 2.08-fold). Increased activity of glutathione S-transferase (ratio 1.94) was observed in the Flux-SEL (F10) strain of FAW, while the cytochrome P450 and carboxylesterase activities were not altered. The fluxametamide-selection significantly affected the development and reproductive traits of FAW with a lower R0, T and relative fitness (Rf = 0.353). The results alluded that the risk of fluxametamide resistance evolution in FAW is relatively lower; however, proactive implementation of resistance management approaches should be done to maintain the field efficacy of fluxametamide against FAW.

Decreased plasma exposure of clopidogrel active metabolite in rats after long-term treatment with clopidogrel.

Clopidogrel (Clop) is oxidized by cytochrome P450s (CYPs) to an active thiol metabolite, Clop-AM, to inhibit platelet activation and aggregation. As an irreversible inhibitor of CYP2B6 and CYP2C19, clopidogrel may inhibit its own metabolism after long-term administration. The study compared the pharmacokinetic profiles of clopidogrel and its metabolites in rats receiving a single or a 2 week administration of Clop. The mRNA and protein levels of hepatic clopidogrel-metabolizing enzymes and their enzymatic activities were analyzed to explore their contribution to any altered plasma exposure of Clop and its metabolites. The results showed that long-term treatment with clopidogrel significantly decreased the AUC(0-t) and Cmax values of Clop-AM in rats, accompanied with markedly impaired catalytic activities of Clop-metabolizing CYPs including CYP1A2, CYP2B6, CYP2C9, CYP2C19, and CYP3A4. It suggests that consecutive administration of Clop to rats decreases hepatic CYPs activities, which may, in turn, inhibit clopidogrel metabolism and then reduce Clop-AM plasma exposure. Therefore, long-term treatment with clopidogrel has the potential to reduce its anti-platelet activity and to increase the risk of drug-drug interaction.

Effect of CdSTe QDs’ Crystal Size on Viability and Cytochrome P450 Activity of CHO-K1 and HEP-G2 Cells

In the last few years, quantum dots (QDs) have attracted research interest in different fields of science and technology. Despite their applications, it is essential to understand how nanomaterials (with different crystal sizes) are metabolized inside organisms. Thus, the focus of this study was on an evaluation of how crystal sizes of CdSTe QDs affect the viability and response of the cytochrome P450 system in CHO-K1 and HEP-G2 cells. CdSTe QDs were synthesized using a microwave-assisted system at different reaction temperatures (60, 120, 150, and 180 °C) to obtain different crystal sizes. The optical and structural characterization confirmed four crystal sizes from 3 to 8 nm. Fluorescence microscopy confirmed that CdSTe QDs are incorporated into both cell lines. Viability studies suggested that CHO-K1 cells are more sensitive than HEP-G2 cells to CdSTe QDs and Cd+2 ions. The responsible mechanisms for the toxicity of QDs and Cd+2 are apoptosis followed by necrosis. The activity of CYP 1A1, 1A2, and 3A4 isoenzymes suggests that the smallest CdSTe crystals are recognized in a manner similar to that of Cd+2. Furthermore, the largest CdSTe crystals can have different metabolic routes than Cd+2.

Pharmacogenetics to Personalize Dosing of Tricyclic Antidepressants in Major Depressive Disorder: A Randomized Clinical Trial (PITA study)

IntroductionTricyclic antidepressants (TCAs) have an important role in pharmacotherapy of Major Depressive Disorder (MDD). TCA dosing is aimed at achieving a concentration in the therapeutic window. Due to inter-individual differences in activity of Cytochrome P450 (CYP) 2D6 and 2C19, (enzymes involved in TCA metabolism) patients require an individualized dose. Currently, finding of a suitable dosage is a process of trial-and-error, including multiple drug concentration measurements and dose adjustments. A starting dose based on a patient CYP2D6 and CYP2C19 metabolizer phenotype may contribute to achieving a therapeutic concentration more quickly, possibly resulting in higher treatment efficacy and the occurrence of fewer adverse effects.ObjectivesWe aimed to study whether genotype-informed dosing in TCAs leads to faster attainment of therapeutic plasma concentrations, a higher reduction of depression severity symptoms and less adverse effects.MethodsWe conducted a double-blind randomized clinical trial, in which patients (18-65 years) diagnosed with severe MDD and eligible for treatment with a TCA were randomized in two treatment arms: The intervention arm (Pharmacogenetics Informed Treatment; PIT) and standard treatment (Treatment As Usual; TAU). Patients were treated with nortriptyline, clomipramine or imipramine for seven weeks and depressive symptom severity and adverse effects were monitored weekly. The primary outcome measure, time needed to attain a therapeutic drug concentration, was analyzed using Kaplan-Meier analyses. The secondary outcome measures, reduction in depressive symptoms and adverse effects, were analyzed with mixed model linear regression.ResultsIn total, we randomized 111 patients. The PIT group (n=56) reached a therapeutic plasma concentration significantly faster (Kaplan Meier, X2 (1) = 4.3, p=0.039) compared to TAU (n=55), especially for patients treated with nortriptyline. On average depressive symptoms decreased relatively more in PIT than in TAU, although this was not significantly different: F(6) = 0.45, p = 0.84. The severity of adverse events during the study period differed significantly between PIT and TAU (F(6)=3.10, p=0.008). PIT experienced fewer adverse effects than TAU, especially in the last two weeks of the study.ConclusionsGenotype-informed dosing leads to faster attainment of therapeutic TCA concentrations and does not lead to more adverse effects. Although we were not able to demonstrate that genotype-informed dosing leads to a significantly better response, we conclude that pharmacogenetics can contribute to the optimization of pharmacological treatment. Future research may focus on subgroups for which pharmacogenetics may be of great value, such as specific antidepressants or patients with an abnormal pharmacogenetic profile.Disclosure of InterestNone Declared

Oxidative stress, inflammation, dysfunctional redox homeostasis and autophagy cause age-associated diseases

Aging and age-associated diseases (AADs) are growing risk factors in societies worldwide. During aging, there is an accumulation of excessive oxygen free radicals [reactive oxygen species (ROS)] and nitrogen free radicals [reactive nitrogen species (RNS)] due to dysfunctional mitochondria, dysregulated catalytic activities of cytochrome P450 (CYP), nicotinamide adenine dinucleotide (NAD) phosphate [NADP(H)] oxidase (NOX), cyclooxygenases, and nitric oxide synthases (NOS) over the threshold of physiological levels, creating oxidative stress (OS). Excessive ROS and RNS oxidize, break, denature, and sometimes cause aggregations of key cellular components including DNA, proteins, and lipids. Normally, these denatured molecules and their aggregates are eliminated by autophagy (AP) and ubiquitin-proteosome system (UPS). However, these two proteostatic mechanisms are impaired as age progresses. As a result, these abnormal molecules turn into damage-associated molecular patterns (DAMPs), recognized as non-self by immune cells, leading to systemic chronic inflammation (SCI), which together with OS are the major causes of aging and AADs. Therefore, instead of trying to prevent and cure AADs individually, the logical approach should be the restoration of redox homeostasis by the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway which can prevent the damaging effect of OS and the upregulation of AP and UPS to eliminate DAMPs, and together they attenuate SCI to lessen the effect of inflammation. The central regulators, adenosine monophosphate-activated protein kinase (AMPK), sirtuin 1 (SIRT1), and Sestrins, synergistically control the restoration of the redox homeostasis by activating Nrf2, the upregulation of AP-UPS, and the inhibition of SCI. The activation of these central regulators can be achieved by exercise, caloric restriction (CR), and intakes of certain CR mimetic natural compounds. Consequently, the activations of these regulators may lead to the prevention and/or attenuation of the AADs.

Practice of CYP450 genotyping and phenotyping in children in a real-life setting.

Pharmacokinetics varies widely between children. Many factors play an important role in this variability, such as ontogeny, pharmacogenetics, gender, comorbidities, and drug-drug interactions. Significant work has already been done in adults to understand the impact of genetic polymorphisms on drug-metabolizing enzyme activity and drug response. Data remain poor in children due to ontogeny that impacts genotyping-phenotyping correlation and the difficulty enrolling children in prospective studies. Our study aimed to describe the use of cytochromes P450 (CYP) phenotyping and/or genotyping tests in children in a real-life setting and assess the correlation between the genotype and the phenotype. We reviewed the results of tests performed between January 2005 and December 2020. Fifty-two children were genotyped and/or phenotyped. Four patients were excluded from the present analysis as they only underwent ABCB1 genotyping, without CYP testing. Of the remainder, 18 underwent simultaneous CYP genotyping and phenotyping, while 17 underwent CYP genotyping only, and 13 underwent CYP phenotyping only. In all cases, investigations were performed after the following situations: insufficient clinical response to treatment, low plasma concentrations, and adverse drug reactions (ADR). The vast majority of cases were related to immunosuppressive or antipsychotic therapy. Genotyping and/or phenotyping explained or contributed to the aforementioned clinical events in 56% of cases. The correlation between the genotype and the phenotype showed variability depending on the assessed cytochrome. In several cases, the phenotype did not correspond to the genotype because of comedications. In conclusion, there is clearly value in guiding drug based on CYP activity in children.