P450 Isoforms Research Articles

Evaluation of the impact of vindoline, an active components of Catharanthus roseus, on rat hepatic cytochrome P450 enzymes by using a cocktail of probe drugs.

The objection of this study was to investigate the effects of vindoline(VDL) on the cytochrome P450 (CYP 450) isoforms (CYP1A2, 2B, 2C11, 2D1 and 3A) in rats. Firstly, the rats were randomly divided into VDL pretreatment group and blank group, each group had six rats. VDL pretreatment group was administrated VDL (20 mg·kg-1) by oral gavage for fifteen days consecutively, and the equivalent CMC-Na solution without VDL was given to the blank group by gavage. Secondly, a cocktail of caffeine, bupropion, diclofenac, dextromethorphan and midazolam was then administered on the sixteenth day. Finally, blood samples were collected at the specified time point, and the plasma concentration of the probe drug was determined by UHPLC-QTOF-MS/MS. The effects of VDL on the activity of these CYP enzymes in rats were evaluated by pharmacokinetic parameters. VDL pretreatment group compared with the blank group, accelerated the metabolism of diclofenac, and weakened the metabolism of caffeine. These results suggested that VDL could induce the activity of CYP2C11, and inhibits the activity of CYP1A2, but had no significant effects on CYP2B, CYP2D1 and CYP3A. The results in this study can provide beneficial information for the later clinical application of VDL.

Taming Food-Drug Interaction Risk: Potential Inhibitory Effects of Citrus Juices on Cytochrome Liver Enzymes Can Safeguard the Liver from Overdose Paracetamol-Induced Hepatotoxicity.

Paracetamol overdose is the leading cause of drug-induced hepatotoxicity worldwide. Because of N-acetyl cysteine's limited therapeutic efficacy and safety, searching for alternative therapeutic substitutes is necessary. This study investigated four citrus juices: Citrus sinensis L. Osbeck var. Pineapple (pineapple sweet orange), Citrus reticulata Blanco × Citrus sinensis L. Osbeck (Murcott mandarin), Citrus paradisi Macfadyen var. Ruby Red (red grapefruit), and Fortunella margarita Swingle (oval kumquat) to improve the herbal therapy against paracetamol-induced liver toxicity. UHPLC-QTOF-MS/MS profiling of the investigated samples resulted in the identification of about 40 metabolites belonging to different phytochemical classes. Phenolic compounds were the most abundant, with the total content ranked from 609.18 to 1093.26 μg gallic acid equivalent (GAE)/mL juice. The multivariate data analysis revealed that phloretin 3',5'-di-C-glucoside, narirutin, naringin, hesperidin, 2-O-rhamnosyl-swertisin, fortunellin (acacetin-7-O-neohesperidoside), sinensetin, nobiletin, and tangeretin represented the crucial discriminatory metabolites that segregated the analyzed samples. Nevertheless, the antioxidant activity of the samples was 1135.91-2913.92 μM Trolox eq/mL juice, 718.95-3749.47 μM Trolox eq/mL juice, and 2304.74-4390.32 μM Trolox eq/mL juice, as revealed from 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid, ferric-reducing antioxidant power, and oxygen radical absorbance capacity, respectively. The in vivo paracetamol-induced hepatotoxicity model in rats was established and assessed by measuring the levels of hepatic enzymes and antioxidant biomarkers. Interestingly, the concomitant administration of citrus juices with a toxic dose of paracetamol effectively recovered the liver injury, as confirmed by normal sections of hepatocytes. This action could be due to the interactions between the major identified metabolites (hesperidin, hesperetin, phloretin 3',5'-di-C-glucoside, fortunellin, poncirin, nobiletin, apigenin-6,8-digalactoside, 6',7'-dihydroxybergamottin, naringenin, and naringin) and cytochrome P450 isoforms (CYP3A4, CYP2E1, and CYP1A2), as revealed from the molecular docking study. The most promising compounds in the three docking processes were hesperidin, fortunellin, poncirin, and naringin. Finally, a desirable food-drug interaction was achieved in our research to overcome paracetamol overdose-induced hepatotoxicity.

A 3D-printed micro-perfused culture device with embedded 3D fibrous scaffold for enhanced biomimicry

Additive manufacturing has rapidly revolutionized the medical sectors since it is a versatile, cost-effective, assembly free technique with the ability to replicate geometrically complicated features. Some of the widely reported applications include the printing of scaffolds, implants, or microfluidic devices. In this study, a 3D-printed micro-perfused culture (MPC) device embedded with a nanofibrous scaffold was designed to create an integrated micro-perfused 3D cell culture environment for living cells. The addition of 3D fibrous scaffold onto the microfluidic chip was to provide a more physiologically relevant microenvironment for cell culture studies. Stereolithography was adopted in this study as this technique obviates excessive preassembly and bonding steps, which would otherwise be needed in conventional microfluidic fabrication. Huh7.5 hepatocellular carcinoma cells were used as model cells for this platform since liver cells experience similar perfused microenvironment. Preliminary cell studies revealed that gene expressions of albumin (ALB) and cytochrome P450 isoform (CYP3A7) were found to be significantly upregulated on the 3D-printed MPC device as compared to the static counterpart. Taken together, the 3D-printed MPC device is shown to be a physiologically relevant platform for the maintenance of liver cells. The device and printing technique developed in this study is highly versatile and tailorable to mimic local in vivo microenvironment needs of various tissues, which could be studied in future.  

Application to Butterbur Products of a Suggested Daily Intake-Based Safety Evaluation of Individual Herbal Supplements with Cytochrome P450 Expression as a Major Index.

The present paper first proposes a method for ensuring the safety of commercial herbal supplements, termed the suggested daily intake-based safety evaluation (SDI-based safety evaluation). This new method was inspired as a backward analog of the acceptable daily intake (ADI) derivation from the no observed adverse effect level (NOAEL), the basis of food additive risk analysis; namely, rats are dosed with individual herbal supplement products at the SDI for human use multiplied by 100 (the usual uncertainty factor value) per body weight for 8 d. The primary endpoint is the sign of adverse effects on liver, especially gene expression of cytochrome P450 (CYP) isoforms. The proposed method was then applied to three butterbur (Petasites hybridus) products without pyrrolizidine alkaloids but lacking clear safety information. Results showed that two oily products markedly enhanced the mRNA expression of CYP2B (>10-fold) and moderately enhanced that of CYP3A1 (<4-fold) with liver enlargement. These products also caused the renal accumulation of alpha 2-microglobulin. One powdery product showed no significant effect on liver and kidney. The large difference in effects of products was due to the difference in chemical composition revealed by liquid chromatography-mass spectroscopy. The oily and the powdery products required attention in terms of safety and effectiveness, respectively. Finally, the results from the SDI-based safety evaluation of butterbur and other herbal supplement products were grouped into four categories and cautionary notes were discussed. The SDI-based safety evaluation of their products by herbal supplement operators would contribute to safe and secure use by consumers.

Resistance to Resveratrol Treatment in Experimental PTSD Is Associated with Abnormalities in Hepatic Metabolism of Glucocorticoids.

Glucocorticoids are metabolized by the CYP3A isoform of cytochrome P450 and by 11-β-hydroxysteroid dehydrogenase type 1 (11β-HSD-1). Experimental data suggest that post-traumatic stress disorder (PTSD) is associated with an increase in hepatic 11β-HSD-1 activity and a concomitant decrease in hepatic CYP3A activity. Trans-resveratrol, a natural polyphenol, has been extensively studied for its antipsychiatric properties. Recently, protective effects of trans-resveratrol were found in relation to PTSD. Treatment of PTSD rats with trans-resveratrol allowed the rats to be divided into two phenotypes. The first phenotype is treatment-sensitive rats (TSR), and the second phenotype is treatment-resistant rats (TRRs). In TSR rats, trans-resveratrol ameliorated anxiety-like behavior and reversed plasma corticosterone concentration abnormalities. In contrast, in TRR rats, trans-resveratrol aggravated anxiety-like behavior and decreased plasma corticosterone concentration. In TSR rats, hepatic 11β-HSD-1 activity was suppressed, with a concomitant increase in CYP3A activity. In TRR rats, the activities of both enzymes were suppressed. Thus, the resistance of PTSD rats to trans-resveratrol treatment is associated with abnormalities in hepatic metabolism of glucocorticoids. The free energy of binding of resveratrol, cortisol, and corticosterone to the human CYP3A protein was determined using the molecular mechanics Poisson-Boltzmann surface area approach, indicating that resveratrol could affect CYP3A activity.

Development and validation of a proteomic correlation profiling technique to detect and identify enzymes involved in metabolism of drugs of concern.

It is important to be able to detect previously unknown and unsuspected enzymes involved in drug metabolism, in order to predict the variation of pharmacological or toxicological effect caused by pharmacokinetic variance. We investigated the use of proteomic correlation profiling (PCP) as a technique to identify the enzymes involved in metabolism of drugs of concern. By evaluating the metabolic activities of each enzyme (including isoforms of cytochrome P450, uridine 5' diphospho‑glucuronosyltransferase, and hydrolases, plus aldehyde oxidase and carbonyl reductase) on their typical substrates using a panel of human liver samples, we were able to show the validity of PCP for this purpose. Pearson or Spearman's rank correlation coefficient and P values were calculated for the association between the protein abundance profile of each protein and the metabolic rate profile of each typical substrate. For the 18 enzymatic activities examined, 13 of the enzymes reported to be responsible for the reactions had correlation coefficients higher than 0.7, and were ranked first to third. For the remaining five activities, the responsible enzymes had correlation coefficients lower than 0.7 and lower rankings. The reasons for this were diverse, including confounding resulting from low protein abundance ratios, artificially high correlations of other enzymes due to limited sample numbers, the presence of inactive enzyme forms, and genetic polymorphisms. Overall, PCP was able to identify the majority of responsible drug-metabolizing enzymes across several enzyme classes (oxidoreductase, transferase, hydrolase); use of this methodology could allow more timely and accurate identification of unknown drug-metabolizing enzymes. Significance Statement Proteomic correlation profiling using samples from individual human donors was proven to be a useful methodology for the identification of enzymes responsible for drug-metabolism. This methodology could accelerate the identification of unknown drug-metabolizing enzymes in the future.

Impact of tectoridin on the pharmacokinetics of florfenicol via targeting cytochrome P450 and P-glycoprotein of rats

Belamcanda chinensis (L.) DC, commonly used with florfenicol in Chinese veterinary clinics for respiratory tract infections, contains the major effective isoflavone, tectoridin (TEC). This study aimed to investigate the impact of TEC co-administration on the pharmacokinetics of florfenicol in vivo. Male rats received oral TEC (50 mg/kg BW) or sterile water for seven days, followed by a single oral dose of florfenicol (25 mg/kg BW) on the 8th day. Non-compartmental methods analysed the pharmacokinetics of florfenicol, while real-time reverse transcription polymerase chain reaction (RT-PCR), Western blot, and immunohistochemical analyses measured expression levels of cytochrome P450 (CYP) isoforms in the liver and P-glycoprotein (P-gp) in the jejunum. TEC significantly decreased florfenicol’s AUC(0–∞), MRT(0–∞), t 1/2z, Vz/F, and C max by 24.75%, 18.43%, 55.47%, 43.05%, and 19.48%, while increasing CLz/F by 33.33%. TEC also up-regulated hepatic CYP1A2 and CYP3A1 mRNA expression, as well as intestinal MDR1, by 1.39-fold, 1.85-fold, and 1.65-fold. This coincided with a respective increase in protein expression by 1.37-fold, 1.39-fold, and 1.43-fold. These findings suggest that TEC-induced alterations in the pharmacokinetics of florfenicol may be attributed to increased CYP and P-gp expression. Further investigations are warranted to understand the implications of these findings on the clinical effectiveness of florfenicol in veterinary practice.

Abstract 5338: A novel in vitro assay to evaluate the roles of hepatic metabolism on anticancer drug safety and efficacy

Abstract It has been well-established that hepatic drug metabolism may have significant impact on the efficacy and off-target toxicity of anticancer drugs. We present here an in vitro assay, the metabolism-dependent cytotoxicity assay (MDCA), for the evaluation of the roles of specific pathways on the efficacy of anticancer drugs. The assay employs a novel in vitro hepatic metabolic system, the permeabilized cofactor-supplemented human hepatocytes (MetMax Human Hepatocytes, MMHH), as an exogenous metabolic activating system for the evaluation of the in vitro cytotoxicity of anticancer agents towards a designated tumor and nontumor cells. MMHH are derived from cryopreserved human hepatocytes isolated from human livers procured for but not used for transplantation provided to our laboratory from the International Institute for the Advancement of Medicine. We have previously demonstrated that MMHH retained all drug metabolizing enzyme pathways present in human hepatocytes. The advantages of MMHH include the following: 1. Drug metabolizing enzyme activities are not affected by cytotoxicity of the drug substrate, a major limitation of intact human hepatocytes; and 2. Drug metabolism pathways can be selected by cofactor specification (e.g. NADPH/NAD+ for phase 1 oxidation; Uridine 5′-diphosphoglucuronic acid (UDPGA) for glucuronidation; 3'-phosphoadenosine 5'-phosphosulfate (PAPS) for sulfation; N-acetyl coenzyme A for N-acetylation). We present here a proof-of-concept study to evaluate the role of various hepatic metabolic pathways towards the cytotoxicity of cyclophosphamide, a commonly used drug for the treatment of renal carcinoma, towards a renal cancer cell line, HEK293 cells. We observed that NADPH enhanced the cytotoxicity, consistent with the known metabolic activation of cyclophosphamide by P450 isoforms (e.g. CYP2B6; CYP3A4) to the known metabolic activation of cyclophosphamide to 4-hydroxycloophosphamide, followed by the formation of the ultimate cytotoxic metabolites phosphoramide mustard and acrolein, which are responsible for its anticancer properties. In addition, we found that the addition of L-glutathione attenuated cyclophosphamide cytotoxicity towards the HEK293 cells, an observation consistent with the roles of this cellular detoxification system in the resistance of tumor cells to chemotherapeutic agents. Minimal effects were observed for UDPGA and PAPS, consistent with the known noninvolvement of these phase 2 conjugating pathways in cyclophosphamide metabolism. MDCA represents an in vitro experimental system that can be routinely applied towards the discovery and development of novel anticancer drugs to evaluate the roles of drug metabolism in efficacy and safety, with various cancer cells (e.g. prostate carcinoma, neuroblastoma) and cells from normal tissues (e.g. cardiomyocytes, intestinal mucosal cells), respectively, as target cells. Citation Format: Albert P. Li, Hong Wei. A novel in vitro assay to evaluate the roles of hepatic metabolism on anticancer drug safety and efficacy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5338.

Coptisine modulates the pharmacokinetics of florfenicol by targeting CYP1A2, CYP2C11 and CYP3A1 in the liver and P-gp in the jejunum of rats: a pilot study

Coptisine (COP) is the main active ingredient of Coptis chinensis. In Chinese veterinary clinics, Coptis chinensis is commonly used alongside florfenicol to treat intestinal infections. The goal of this study was to investigate the impact of COP co-administration on the pharmacokinetics of florfenicol in rats. Male Sprague-Dawley rats were orally administered COP (50 mg/kg BW) or sterile water for 7 consecutive days, followed by a single oral dose of florfenicol (25 mg/kg BW) on the 8th day. Pharmacokinetics of florfenicol were analysed using non-compartmental methods, while expression levels of cytochrome P450 (CYP) isoforms in the liver and P-glycoprotein (P-gp) in the jejunum were measured using real-time RT-PCR, Western blot and immunohistochemical analyses. Co-administration of COP and florfenicol significantly increased AUC(0-∞), MRT(0-∞), and Cmax of florfenicol, while CLz/F was significantly decreased. COP down-regulated the expression of CYP1A2, CYP2C11, and CYP3A1 in the liver, as well as P-gp in the jejunum. These findings suggest that co-administration of COP with florfenicol alters the pharmacokinetics of florfenicol in rats. The down-regulation of CYP and P-gp expression may contribute to this effect. Therefore, the co-administration of COP with florfenicol may enhance the prophylactic or therapeutic efficacy of florfenicol in veterinary practice.

In vitro metabolic characterization of the SARS-CoV-2 papain-like protease inhibitors GRL0617 and HY-17542.

The SARS-CoV-2 pandemic requires a new therapeutic target for viral infection, and papain-like protease (Plpro) has been suggested as a druggable target. This in-vitro study was conducted to examine the drug metabolism of the GRL0617 and HY-17542, Plpro inhibitors. Metabolism of these inhibitors was studied to predict the pharmacokinetics in human liver microsomes. The hepatic cytochrome P450 (CYP) isoforms responsible for their metabolism were identified using recombinant enzymes. The drug-drug interaction potential mediated by cytochrome P450 inhibition was estimated. In human liver microsomes, the Plpro inhibitors had phase I and phase I + II metabolism with half-lives of 26.35 and 29.53min, respectively. Hydroxylation (M1) and desaturation (-H2, M3) of the para-amino toluene side chain were the predominant reactions mediated with CYP3A4 and CYP3A5. CYP2D6 is responsible for the hydroxylation of the naphthalene side ring. GRL0617 inhibits major drug-metabolizing enzymes, including CYP2C9 and CYP3A4. HY-17542 is structural analog of GRL0617 and it is metabolized to GRL0617 through non-cytochrome P450 reactions in human liver microsomes without NADPH. Like GRL0617 and HY-17542 undergoes additional hepatic metabolism. The in-vitro hepatic metabolism of the Plpro inhibitors featured short half-lives; preclinical metabolism studies are needed to determine therapeutic doses for these inhibitors.

MD Investigation on the Interaction between Carbamazepine and Two CYP Isoforms, CYP3A4 and CYP3A5.

Carbamazepine (CBZ), a commonly prescribed antiepileptic drug, in human liver, is mainly metabolized by two isoforms of cytochrome P450 (CYP), CYP3A4 and CYP3A5. Therefore, the binding of CBZ with these two enzymes plays crucial role in the biotransformation of the drug into its active metabolite. In the present work, classical molecular dynamics (MD) simulation was used to investigate the detailed interaction mechanism between CBZ and these two CYP isoforms at the atomic level. The results revealed that although CBZ can bind with the two proteins, all kinds of the interactions, including hydrogen bonds, salt bridges, hydrophobic interaction, and π-π interaction, are isoform specific. The specificity directly leads to a binding environment difference at the active sites of the two isoforms, as represented by the electrostatic surface potential maps, which further results in the varied dynamic behavior of CBZ in the two isoforms. Our research will help to deepen the understanding of the physiological functions of CYP isoforms and opens the door for the rational design and development of isoform-specific inhibitors.

Methoxol's Impact on the Hepatic Microsomal Mixed Function Oxidase System Induced by Phenobarbital and Pyrene in Rats

Insecticides like methomyl (Lannate) are commonly used to keep pests at bay in vineyards. The mixed-function oxidase system undergoes dramatic change after being exposed to methomyl. This research set out to determine if methomyl inhibits phenobarbital-induced CYP 2B1, 2B2, and 3A, as well as benzo[a]pyrene-induced CYP 2D6 (CYP 1A1). There were a total of eight groups, each including six adult male rats. Calcium precipitation was used to separate the microsomes. Extinction coefficients were used to quantify the amounts of the electron transport components cytochrome P450, cytochrome B5, and cytochrome c-reductase. Drug metabolizing enzymes were tested for their efficacy. Mixed function oxidase was significantly elevated in rats when exposed to inducers like phenobarbital and benzo[a]pyrene. Inducer-(Phenobarbital, benzop [a] pyrene) pretreated rats given 4mg/kg methomyl had significantly lower levels of electron transport components and drug-metabolizing enzyme activity compared to inducer treatment alone. The enzymes involved in mixed-function oxidation were likewise affected by methomyl's influence on the induction of phenobarbital. The levels of electron transport components and drug metabolizing enzymes in methomyl-pretreated rats treated with benzo[a]pyrene were much lower than in rats treated with benzo[a]pyrene alone. These findings show that phenobarbital and benzo[a]pyrene increased cytochrome P450 isoform susceptibility to methomyl (CYP 2B1, 2B2, 3A; and CYP 1A1) and the induction pattern of some inducers with respect to CYP 450 isoforms are both modified by methomyl

Effects of Medicinal Plants on Carbon Tetrachloride-Induced Liver Injury: A Review

The liver is the primary organ for metabolizing and eliminating foreign chemicals. When liver cells are exposed to large concentrations of dangerous substances, hepatocyte malfunction, membrane damage, and degradation may occur. Another chemical that can harm the liver is carbon tetrachloride (CCl4). CCl4 is converted into trichloromethyl radical [CCl3]- by several cytochrome P450 isoforms. [CCl3]- subsequently reacts with oxygen to generate trichloromethyl peroxyl radical [CCl3OO]- and induce lipid peroxidation in cell membranes. In this study, the mechanism by which a variety of recently identified medicinal plants display hepatoprotective activity against CCl4-induced liver injury is investigated. We find that animal models have been used in investigations of plant extracts. Herbal plants largely protect against CCl4-induced hepatotoxicity because their phytochemicals have an inhibitory nature. The antioxidant properties of phytochemicals can halt lipid oxidation and restrict the production of free radicals by inhibiting microsomal enzymes. They can also help liver cells in their fight against CCl4-induced inflammation by strengthening and fortifying them. The effectiveness of various herbs that are hepatoprotective and are thus plausible candidates for use in medicine must be confirmed. Experiments using entire plant extracts should be replaced with tests that pinpoint the active ingredients and assess the extracts’ effect on a variety of liver cell lines.

Design, synthesis and biological evaluation of pyrazolo[3,4-b]pyridine derivatives as TRK inhibitors.

Tropomyosin receptor kinases (TRKs) are associated with the proliferation and differentiation of cells, and thus their continuous activation and overexpression cause cancer. Herein, based on scaffold hopping and computer-aid drug design, 38 pyrazolo[3,4-b]pyridine derivatives were synthesised. Further, we evaluated their activities to inhibit TRKA. Among them, compound C03 showed acceptable activity with an IC50 value of 56 nM and it inhibited the proliferation of the Km-12 cell line with an IC50 value of 0.304 μM together with obvious selectivity for the MCF-7 cell line and HUVEC cell line. Furthermore, compound C03 possessed good plasma stability and low inhibitory activity to a panel of cytochrome P450 isoforms except CYP2C9. Overall, C03 has potential for further exploration.

The relationship of cytochrome P450 isoforms with the efficacy and safety of antipsychotic and antidepressant therapy

Cytochrome P450 (CYP450) is the leading enzyme in the biotransformation of most psychotropic drugs. CYP450 gene polymorphisms determine a patient's endophenotype with respect to the activity of enzymes of the family and affect the metabolism of prescribed antipsychotics and antidepressants. Categorizing patients by endophenotype during genotyping is likely to help simplify the selection of therapy in clinical practice. Co-prescribing drugs that may be inhibitors or inducers of CYP450 isoforms, in turn, may lead to adverse reactions or no effect of therapy. The article presents a compilation of known pharmacogenetic recommendations regarding the four major endophenotypes of metabolizers.

Probing the Role of CYP2 Enzymes in the Atropselective Metabolism of Polychlorinated Biphenyls Using Liver Microsomes from Transgenic Mouse Models.

Chiral polychlorinated biphenyls (PCB) are environmentally relevant developmental neurotoxicants. Because their hydroxylated metabolites (OH-PCBs) are also neurotoxic, it is necessary to determine how PCB metabolism affects the developing brain, for example, in mouse models. Because the cytochrome P450 isoforms involved in the metabolism of chiral PCBs remain unexplored, we investigated the metabolism of PCB 91 (2,2',3,4',6-pentachlorobiphenyl), PCB 95 (2,2',3,5',6-pentachlorobiphenyl), PCB 132 (2,2',3,3',4,6'-hexachlorobiphenyl), and PCB 136 (2,2',3,3',6,6'-hexachlorobiphenyl) using liver microsomes from male and female Cyp2a(4/5)bgs-null, Cyp2f2-null, and wild-type mice. Microsomes, pooled by sex, were incubated with 50 μM PCB for 30 min, and the levels and enantiomeric fractions of the OH-PCBs were determined gas chromatographically. All four PCB congeners appear to be atropselectively metabolized by CYP2A(4/5)BGS and CYP2F2 enzymes in a congener- and sex-dependent manner. The OH-PCB metabolite profiles of PCB 91 and PCB 132, PCB congeners with one para-chlorine substituent, differed between null and wild-type mice. No differences in the metabolite profiles were observed for PCB 95 and PCB 136, PCB congeners without a para-chlorine group. These findings suggest that Cyp2a(4/5)bgs-null and Cyp2f2-null mice can be used to study how a loss of a specific metabolic function (e.g., deletion of Cyp2a(4/5)bgs or Cyp2f2) affects the toxicity of chiral PCB congeners.

Metabolism of β-naphthoflavone by cytochrome P450 isoforms in three air breathing fish, Channa punctatus, Heteropneustes fossilis and Clarias batrachus

The crucial phase I xenobiotic metabolising enzyme cytochrome P450 (CYP 450) has a variety of isoforms and a wide range of substrate specificities. While the roles of mammalian CYP 450 are well established, those of non-mammalian species are much less defined. These roles are frequently extrapolated from mammalian data and assumed to be similar across vertebrate species. In the present study, three air breathing fish species, Channa punctatus, Heteropneustes fossilis and Clarias batrachus were injected intraperitoneally (IP) with 50 mg/kg β-naphthoflavone (β-NF) and its effects on hepatic CYP 450 system were evaluated. β-NF treatment resulted in significant increase (p&lt;0.01) in CYP 450 content and CYP1A mediated EROD (Ethoxyresorufin O-deethylase) activity in all three treated fish groups compared to control. CYP2B mediated N,N-dimethylaniline demethylase activity and CYP2E1 mediated aniline hydroxylase activity showed no response suggesting insensitivity to β-NF treatment. All three fish species showed an increase in CYP3A4 mediated erythromycin N-demethylase activity, but only H. fossilis displayed a significant difference (p&lt;0.05). Of all the activities investigated, EROD activity mediated by CYP1A responded towards β-NF treatment the best and could serve as a sensitive biomarker to gauge the effects of chemical pollutants in the aquatic environment.

In vitro effects and in silico analysis of newly synthetized pyrrole derivatives on the activity of different isoforms of Cytochrome P450: CYP1A2, CYP2D6 and CYP3A4

Four pyrrole based hydazide-hydrazones with established low hepatotoxicity and promising antiproliferative activity were evaluated (at 1µM concentration) for possible inhibitory activity on human isoforms of Cytochrome P450 CYP1A2, CYP3A4 and CYP2D6. The compounds didn’t exert any statistically significant inhibitory effects on CYP1A2 and CYP2D6. However on CYP3A4 only 12 resulted in low statistically significant inhibitory effect decreasing the enzyme activity by 20%, compared to the control (pure CYP3A4). In addition the potential interactions of 12 and the evaluated CYP isoforms were displayed after molecular docking with Glide (Schrödinger). Induced-fit simulations and binding free energy (MM/GBSA) calculations were applied to elucidate the accessibility in each CYP isoform. The most active CYP3A4 inhibitor 12 demonstrated good binding affinity and was in close vicinity to the het Fe ion (2.88 Å). Overall, good correlation between the in vitro results and the free binding MM/GBSA recalculations were observed.

Design, synthesis, and biological evaluation of aminopyridine derivatives as novel tropomyosin receptor kinase inhibitors.

Tropomyosin receptor kinase (TRK) is a successful target for the treatment of various cancers caused by NTRK gene fusions. Herein, based on a rational drug design strategy, we designed and synthesized 35 aminopyrimidine derivatives that were shown to be TRKA inhibitors in the enzyme assay, among which compounds C3, C4, and C6 showed potent inhibitory activities against TRKA with IC50 values of 6.5, 5.0, and 7.0 nM, respectively. In vitro antiproliferative activity study showed that compound C3 significantly inhibited the proliferation of KM-12 cellsbut had weak inhibitory effect on MCF-7 cells and HUVEC cells. The preliminary druggability evaluation showed that compound C3 exhibited favorable liver microsomal and plasma stabilitiesand had weak or no inhibitory activity against cytochrome P450 isoforms at 10 µM. Compounds C3, C4, andC6 were also selected for ADME (absorption, distribution, metabolism, and elimination) properties prediction and molecular docking studies. Inhibition experiments showed that compound C3 was not selective for TRK subtypes. All results indicated that compound C3 was a useful candidate for the development of TRK inhibitors.

In vitro and in silico inhibition performance of choline against CYP1A2, CYP2D6 and CYP3A4

Choline is an essential nutrient involved in the synthesis of acetylcholine in the cholinergic neurons. The pharmacokinetic properties of choline are well described; however, there is a lack of data about its activity toward the CYP450 superfamily of enzymes. Therefore, the aim of this study was to conduct in silico and in vitro activity assessments of choline against three major CYP450 isoforms—CYP1A2, CYP2D6, and CYP3A4. Preliminary in silico modeling was performed on a specialized DL-CYP Prediction Server to evaluate the affinity of choline toward the enzymes. The in vitro study contained specific cytochrome P450 isoform inhibitors and substrates (for CYP1A2, CYP2D6, and CYP3A4) to determine the inhibition performance of choline at five different concentrations (0.150 − 1 µmol/L). The potential interactions of choline and CYPs were displayed after molecular docking with Glide (Schrödinger). In addition, induced-fit simulations and binding free energy calculations MM/GBSA (Molecular Mechanics-Generalized Born Surface Area) were applied to predict the accessibility in each CYP isoform. The initial in silico simulations revealed that choline lacks inhibition potency against the aforementioned enzymes. The in vitro evaluations confirmed that choline possessed no effect against CYP1A2; however, at 1 µmol/L choline exerted 22% and 27% blocking capacity against CYP2D6 and CYP3A4, respectively. Furthermore, there was good correlation between the in vitro results and the complexes’ free binding energy recalculations. Overall, the assessments indicated that choline is a weak CYP2D6 and CYP3A4 inhibitor. The latter results should be considered as a source of future unwanted drug–drug interactions.