P450s 3A4 Research Articles

Inactivation of CYP2A6 by the Dietary Phenylpropanoid trans-Cinnamic Aldehyde (Cinnamaldehyde) and Estimation of Interactions with Nicotine and Letrozole.

Human exposure to trans-cinnamic aldehyde [t-CA; cinnamaldehyde; cinnamal; (E)-3-phenylprop-2-enal] is common through diet and through the use of cinnamon powder for diabetes and to provide flavor and scent in commercial products. We evaluated the likelihood of t-CA to influence metabolism by inhibition of P450 enzymes. IC50 values from recombinant enzymes indicated that an interaction is most probable for CYP2A6 (IC50 = 6.1 µM). t-CA was 10.5-fold more selective for human CYP2A6 than for CYP2E1; IC50 values for P450s 1A2, 2B6, 2C9, 2C19, 2D6, and 3A4 were 15.8-fold higher or more. t-CA is a type I ligand for CYP2A6 (KS = 14.9 µM). Inhibition of CYP2A6 by t-CA was metabolism-dependent; inhibition required NADPH and increased with time. Glutathione lessened the extent of inhibition modestly and statistically significantly. The carbon monoxide binding spectrum was dramatically diminished after exposure to NADPH and t-CA, suggesting degradation of the heme or CYP2A6 apoprotein. Using a static model and mechanism-based inhibition parameters (K(I) = 18.0 µM; k(inact) = 0.056 minute(-1)), changes in the area under the concentration-time curve (AUC) for nicotine and letrozole were predicted in the presence of t-CA (0.1 and 1 µM). The AUC fold-change ranged from 1.1 to 3.6. In summary, t-CA is a potential source of pharmacokinetic variability for CYP2A6 substrates due to metabolism-dependent inhibition, especially in scenarios when exposure to t-CA is elevated due to high dietary exposure, or when cinnamon is used as a treatment of specific disease states (e.g., diabetes).

Risks of Adverse Events Following Coprescription of Statins and Calcium Channel Blockers: A Nationwide Population-Based Study.

Some statins (simvastatin, lovastatin, and atorvastatin) are metabolized by cytochrome P450s 3A4 (CYP3A4). Inhibitors of CYP3A4 including some calcium channel blockers (CCBs) might increase statin blood concentration, owing to drug–drug interactions. Risk of adverse events such as acute kidney injury might occur following the coprescription of CYP3A4-metabolized statins and CCBs that inhibit CYP3A4.This was a population-based cohort study. The study analyzed data of patients treated between 1997 and 2011, retrieved from Taiwan's National Health Insurance database. We enrolled 32,801 patients who received coprescription of statins and CCBs that inhibit CYP3A4 (amlodipine, diltiazem, felodipine nicardipine, nifedipine, and verapamil). These patients were divided into 2 groups, according to whether they had received CYP3A4-metabolized statins (lovastatin, simvastatin, and atorvastatin) or non-CYP3A4-metabolized statins (fluvastatin, rosuvastatin, and pitavastatin). These 2 groups were 1:1 matched by age, gender, and Carlson comorbidity index. All outcomes were assessed within 90 days following drug coprescription.In this study, 5857 patients received coprescription of CYP3A4-metabolized statins and CCBs that inhibit CYP3A4. There were no differences in comorbidity or use of antihypertensive drugs between patients who received CYP3A4-metabolized statins and those who received non-CYP3A4-metabolized statins. Patients who received CYP3A4-metabolized statins had significantly higher risk of acute kidney injury (adjusted odds ratio [OR] = 2.12; 95% CI = 1.35–3.35), hyperkalemia (adjusted OR = 2.94; 95% CI = 1.36–6.35), acute myocardial infarction (adjusted OR = 1.55; 95% CI = 1.16–2.07), and acute ischemic stroke (adjusted OR = 1.35; 95% CI = 1.08–1.68) than those who received non-CYP3A4-metabolized statins.This nationwide cohort study demonstrated the increased risk of adverse events following the coprescription of CYP3A4-metabolized statins and CCBs that inhibit CYP3A4. Therefore, it is important to take into account the potential adverse events while coprescribing CYP3A4-metabolized statins and CCBs that inhibit CYP3A4.

Development of HepG2-derived cells expressing cytochrome P450s for assessing metabolism-associated drug-induced liver toxicity

The generation of reactive metabolites from therapeutic agents is one of the major mechanisms of drug-induced liver injury (DILI). In order to evaluate metabolism-related toxicity and improve drug efficacy and safety, we generated a battery of HepG2-derived cell lines that express 14 cytochrome P450s (CYPs) (1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5 and 3A7) individually using a lentiviral expression system. The expression/production of a specific CYP in each cell line was confirmed by an increased abundance of the CYP at both mRNA and protein levels. Moreover, the enzymatic activities of representative CYPs in the corresponding cell lines were also measured. Using our CYP-expressed HepG2 cells, the toxicity of three drugs that could induce DILI (amiodarone, chlorpromazine and primaquine) was assessed, and all of them showed altered (increased or decreased) toxicity compared to the toxicity in drug-treated wild-type HepG2 cells. CYP-mediated drug toxicity examined in our cell system is consistent with previous reports, demonstrating the potential of these cells for assessing metabolism-related drug toxicity. This cell system provides a practical in vitro approach for drug metabolism screening and for early detection of drug toxicity. It is also a surrogate enzyme source for the enzymatic characterization of a particular CYP that contributes to drug-induced liver toxicity.

A Ligand-Based Drug Design. Discovery of 4-Trifluoromethyl-7,8-pyranocoumarin as a Selective Inhibitor of Human Cytochrome P450 1A2.

In humans, cytochrome P450 1A2 is the major enzyme metabolizing environmental arylamines or heterocyclic amines into carcinogens. Since evidence shows that planar triangle-shaped molecules are capable of selectively inhibiting P450 1A2, 16 triangular flavone, and coumarin derivatives were designed and synthesized for these studies. Among these compounds, 7,8-furanoflavone time-dependently inhibits P450 1A2 with a K(I) value of 0.44 μM. With a 5 min preincubation in the presence of NADPH, 0.01 μM 7,8-furanoflavone completely inactivates P450 1A2 but does not influence the activities of P450s 1A1 and 1B1. Another target compound, 7,8-pyrano-4-trifluoromethylcoumarin, is found to be a competitive inhibitor, showing high selectivity for the inhibition of P450 1A2 with a K(i) of 0.39 μM, 155- and 52-fold lower than its K(i) values against P450s 1A1 and 1B1, respectively. In yeast AhR activation assays, 7,8-pyrano-4-trifluoromethylcoumarin does not activate aryl hydrocarbon receptor when the concentration is lower than 1 μM, suggesting that this compound would not up-regulate AhR-caused P450 enzyme expression. In-cell P450 1A2 inhibition assays show that 7,8-pyrano-4-trifluoromethylcoumarin decreases the MROD activity in HepG2 cells at concentrations higher than 1 μM. Thus, using 7,8-pyrano-4-trifluoromethylcoumarin, a selective and specific P450 1A2 action suppression could be achieved, indicating the potential for the development of P450 1A2-targeting cancer preventive agents.

Cytochrome P450 3A Enzymes Catalyze the O6-Demethylation of Thebaine, a Key Step in Endogenous Mammalian Morphine Biosynthesis

Morphine, first characterized in opium from the poppy Papaver somniferum, is one of the strongest known analgesics. Endogenous morphine has been identified in several mammalian cells and tissues. The synthetic pathway of morphine in the opium poppy has been elucidated. The presence of common intermediates in plants and mammals suggests that biosynthesis occurs through similar pathways (beginning with the amino acid L-tyrosine), and the pathway has been completely delineated in plants. Some of the enzymes in the mammalian pathway have been identified and characterized. Two of the latter steps in the morphine biosynthesis pathway are demethylation of thebaine at the O(3)- and the O(6)-positions, the latter of which has been difficult to demonstrate. The plant enzymes responsible for both the O(3)-demethylation and the O(6)-demethylation are members of the Fe(II)/α-ketoglutarate-dependent dioxygenase family. Previous studies showed that human cytochrome P450 (P450) 2D6 can catalyze thebaine O(3)-demethylation. We report that demethylation of thebaine at the O(6)-position is selectively catalyzed by human P450s 3A4 and 3A5, with the latter being more efficient, and rat P450 3A2. Our results do not support O(6)-demethylation of thebaine by an Fe(II)/α-ketoglutarate-dependent dioxygenase. In rat brain microsomes, O(6)-demethylation was inhibited by ketoconazole, but not sulfaphenazole, suggesting that P450 3A enzymes are responsible for this activity in the brain. An alternate pathway to morphine, oripavine O(6)-demethylation, was not detected. The major enzymatic steps in mammalian morphine synthesis have now been identified.

Oxidation of Salicylic Acid and Aspirin by Human Cytochromes P450

Aspirin (acetylsalicylic acid) is a commonly used analgesic. It is rapidly deacetylated both enzymatically and non‐enzymatically to salicylic acid. Salicylic acid can then be further metabolized by conjugation with glycine or glucuronides or by hydroxylation to form 2,3‐dihydroxybenzoic acid (2,3‐DHBA) and 2,5‐dihydroxybenzoic acid (2,5‐DHBA). This oxidation has been suspected to occur from oxygen radicals via Fenton reactions as well as by cytochrome P450 enzymes. We analyzed the formation of 2,3‐DHBA and 2,5‐DHBA in the presence of human liver microsomes by HPLC with fluorescence detection. We were able to determine that microsomal oxidation was much faster for salicylic acid than aspirin. 2,3‐DHBA was found to be the major oxidative product formed from salicylic acid, whereas aspirin could detect only 2,5‐DHBA. Recombinant human P450s 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 all catalyzed the 5‐hydroxylation of salicylic acid. Inhibitor studies were able to confirm that all six of these enzymes played a role in the formation of 2,3‐ and 2,5‐DHBA, as well as found that 2A6 and 2B6 played a role in the hydroxylation to 2,5‐DHBA. It was also shown in the inhibitor studies that P450 2E1 was the major enzyme contributing to the in both 3‐ and 5‐hydroxylations.This work was supported in part by National Institutes of Health grant R37 CA090426.

Aromatic hydroxylation of salicylic acid and aspirin by human cytochromes P450

Aspirin (acetylsalicylic acid) is a well-known and widely-used analgesic. It is rapidly deacetylated to salicylic acid, which forms two hippuric acids—salicyluric acid and gentisuric acid—and two glucuronides. The oxidation of aspirin and salicylic acid has been reported with human liver microsomes, but data on individual cytochromes P450 involved in oxidation is lacking. In this study we monitored oxidation of these compounds by human liver microsomes and cytochrome P450 (P450) using UPLC with fluorescence detection. Microsomal oxidation of salicylic acid was much faster than aspirin. The two oxidation products were 2,5-dihydroxybenzoic acid (gentisic acid, documented by its UV and mass spectrum) and 2,3-dihydroxybenzoic acid. Formation of neither product was inhibited by desferrioxamine, suggesting a lack of contribution of oxygen radicals under these conditions. Although more liphophilic, aspirin was oxidized less efficiently, primarily to the 2,5-dihydroxy product. Recombinant human P450s 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 all catalyzed the 5-hydroxylation of salicylic acid. Inhibitor studies with human liver microsomes indicated that all six of the previously mentioned P450s could contribute to both the 5- and 3-hydroxylation of salicylic acid and that P450s 2A6 and 2B6 have contributions to 5-hydroxylation. Inhibitor studies indicated that the major human P450 involved in both 3- and 5-hydroxylation of salicylic acid is P450 2E1.

Characterization of human cytochrome P450s involved in the bioactivation of tri-ortho-cresyl phosphate (ToCP).

Tri-ortho-cresyl phosphate (ToCP) is a multipurpose organophosphorus compound that is neurotoxic and suspected to be involved in aerotoxic syndrome in humans. It has been reported that not ToCP itself but a metabolite of ToCP, namely, 2-(ortho-cresyl)-4H-1,2,3-benzodioxaphosphoran-2-one (CBDP), may be responsible for this effect as it can irreversibly bind to human butyrylcholinesterase (BuChE) and human acetylcholinesterase (AChE). The bioactivation of ToCP into CBDP involves Cytochrome P450s (P450s). However, the individual human P450s responsible for this bioactivation have not been identified yet. In the present study, we aimed to investigate the metabolism of ToCP by different P450s and to determine the inhibitory effect of the in vitro generated ToCP-metabolites on human BuChE and AChE. Human liver microsomes, rat liver microsomes, and recombinant human P450s were used for that purpose. The recombinant P450s 2B6, 2C18, 2D6, 3A4 and 3A5 showed highest activity of ToCP-bioactivation to BuChE-inhibitory metabolites. Inhibition experiments using pooled human liver microsomes indicated that P450 3A4 and 3A5 were mainly involved in human hepatic bioactivation of ToCP. In addition, these experiments indicated a minor role for P450 1A2. Formation of CBDP by in-house expressed recombinant human P450s 1A2 and 3A4 was proven by both LC-MS and GC-MS analysis. When ToCP was incubated with P450 1A2 and 3A4 in the presence of human BuChE, CBDP-BuChE-adducts were detected by LC-MS/MS which were not present in the corresponding control incubations. These results confirmed the role of human P450s 1A2 and 3A4 in ToCP metabolism and demonstrated that CBDP is the metabolite responsible for the BuChE inactivation. Interindividual differences at the level of P450 1A2 and 3A4 might play an important role in the susceptibility of humans in developing neurotoxic effects, such as aerotoxic syndrome, after exposure to ToCP.

Oxidation of Acenaphthene and Acenaphthylene by Human Cytochrome P450 Enzymes

Acenaphthene and acenaphthylene, two known environmental polycyclic aromatic hydrocarbon (PAH)pollutants, were incubated at 50 μM concentrations in a standard reaction mixture with human P450s 2A6, 2A13, 1B1,1A2, 2C9, and 3A4, and the oxidation products were determined using HPLC and LC-MS. HPLC analysis showed that P450 2A6 converted acenaphthene and acenaphthylene to several mono- and dioxygenated products. LC-MS analysis of acenaphthene oxidation by P450s indicated the formation of1-acenaphthenol as a major product, with turnover rates of 6.7,4.5, and 3.6 nmol product formed/min/nmol P450 for P4502A6, 2A13, and 1B1, respectively. Acenaphthylene oxidation by P450 2A6 showed the formation of 1,2-epoxyacenaphthene as a major product (4.4 nmol epoxide formed/min/nmol P450) and also several mono- and dioxygenated products.P450 2A13, 1B1, 1A2, 2C9, and 3A4 formed 1,2-epoxyacenaphthene at rates of 0.18, 5.3 2.4, 0.16, and 3.8 nmol/min/nmol P450, respectively. 1-Acenaphthenol, which induced Type I binding spectra with P450 2A13, was further oxidized by P450 2A13 but not P450 2A6. 1,2-Epoxyacenaphthene induced Type I binding spectra with P450 2A6 and 2A13 (K(s) 1.8 and 0.16 μM,respectively) and was also oxidized to several oxidation products by these P450s. Molecular docking analysis suggested different orientations of acenaphthene, acenaphthylene, 1-acenaphthenol, and 1,2-epoxyacenaphthene in their interactions with P450 2A6a nd 2A13. Neither of these four PAHs induced umu gene expression in a Salmonella typhimurium NM tester strain. These results suggest, for the first time, that acenaphthene and acenaphthylene are oxidized by human P450s 2A6 and 2A13 and other P450s to form several mono- and dioxygenated products. The results are of use in considering the biological and toxicological significance of these environmental PAHs in humans.

Ethynylflavones, highly potent, and selective inhibitors of cytochrome P450 1A1.

The flavone backbone is a well-known pharmacophore present in a number of substrates and inhibitors of various P450 enzymes. In order to find highly potent and novel P450 family I enzyme inhibitors, an acetylene group was incorporated into six different positions of flavone. The introduction of an acetylene group at certain locations of the flavone backbone lead to time-dependent inhibitors of P450 1A1. 3′-Ethynylflavone, 4′-ethynylflavone, 6-ethynylflavone, and 7-ethynylflavone (KI values of 0.035–0.056 μM) show strong time-dependent inhibition of P450 1A1, while 5-ethynylflavone (KI value of 0.51 μM) is a moderate time-dependent inhibitor of this enzyme. Meanwhile, 4′-ethynylflavone and 6-ethynylflavone are highly selective inhibitors toward this enzyme. Especially, 6-ethynylflavone possesses a Ki value of 0.035 μM for P450 1A1 177- and 15-fold lower than those for P450s 1A2 and 1B1, respectively. The docking postures observed in the computational simulations show that the orientation of the acetylene group determines its capability to react with P450s 1A1 and 1A2. Meanwhile, conformational analysis indicates that the shape of an inhibitor determines its inhibitory selectivity toward these enzymes.

Affinity purification of recombinant human cytochrome P450s 3A4 and 1A2 using mixed micelle systems

Recombinant cytochrome P450 (CYP or P450) enzymes are useful for drug metabolism research and thereby many expression and purification systems have been developed. Here, we provide a method for the purification of human P450s 3A4 and 1A2 expressed in Escherichia coli using mixed micelles containing anionic phospholipids. This method does not require any protein-tagging system for protein isolation and has a further advantage that the purification is concomitantly conducted with reconstitution of the enzymes into a phospholipid environment, which is crucial for the catalytic activity assay of P450 enzyme. This method may also be applied to high-throughput catalytic assays of the enzymes because the purification procedures can be undertaken in a 96-well plate.

The effect of CYP1A1 and CYP1A2 polymorphisms on gastric cancer risk among different ethnicities: a systematic review and meta-analysis

Potential Cytochrome P450s (CYPs) 1A1 MspI, 1A1 Ile462Val, 1A2*1F, and/or 1A2*1C polymorphisms have been implicated in gastric cancer risk among different ethnicities. We aimed to explore the effect of CYP 1A1 MspI, 1A1 Ile462Val, 1A2*1F, and/or 1A2*1C polymorphisms on the susceptibility to gastric cancer among different ethnicities through a systematic review and meta-analysis. Each initially included article was scored for quality appraisal. Desirable data were extracted and registered into databases. A number of 11 studies were ultimately eligible for the meta-analysis of CYP1A1 MspI polymorphism, eight studies for the meta-analysis of 1A1 Ile462Val polymorphism, and two studies for the meta-analysis of 1A2*1F polymorphism. None of genetic model was evidently suggested, and thus all the genetic models were presented. Potential sources of heterogeneity were sought out via subgroup and sensitivity analyses, and publication biases were estimated. In our meta-analysis, significant results could be found in mutational heterozygous CT genotype, compared with wild TT genotype, among large sample size subgroup for CYP1A1 MspI polymorphism. Regarding CYP1A1 Ile462Val polymorphism, no statistically significant results could be found. For CYP1A2*1F polymorphism, mutational heterozygous AC genotype, compared with wild-type AA, has deleterious effects, whereas mutational homozygous CC genotype, compared with mutational heterozygous type AC, has protective effects but lacks statistically significant difference despite its a proximity to 0.05. Combined mutational homozygous CC genotype and wild-type homozygous AA, compared with mutational heterozygous AC genotype, has protective effects. Our meta-analysis suggests no associations between CYP1A1 Ile462Val polymorphism and gastric cancer, but possible associations between CYP1A1 MspI and CYP1A2*1F polymorphisms and gastric cancer, which needs to be further reinforced or refuted among different ethnicities in well-designed large-scale high-quality studies.

Molecular cloning and cDNA characterization of Camelus dromedarius putative cytochrome P450s 1A1, 2C, and 3A

The domesticated one-humped Arabian camel, Camelus dromedarius, is one of the most important animals in the Arabian Peninsula. Most of its life, this animal is exposed to both intrinsic and extrinsic genotoxic factors that are known to cause gross metabolic alterations in many organisms. This study determined the full length coding sequence of 3 cytochrome P450s cDNAs; namely, CYP450 1A1, CYP450 2C and CYP450 3A using reverse transcription polymerase chain reaction. The C. dromedarius CYP450s 1A1, 2C, and 3A have open reading frames of 1563, 1473, and 1566 bp and cDNAs that encode proteins of 520, 490, and 521 amino acid residues, respectively. The molecular weights calculated for CYP1A1, 2C, and 3A were found to be 58.651, 56.03, and 58.594 kDa, while the predicted calculated isoelectric points using a computer algorithm were 7.315, 6.579, and 9.46. The deduced amino acid sequences of these CYPs showed the membrane anchored signal peptide, the conserved proline-rich amino terminus and the characteristic heme-binding signature localized near the carboxy terminus of the protein.

Human Cytochrome P450 Oxidation of 5-Hydroxythalidomide and Pomalidomide, an Amino Analogue of Thalidomide

The sedative and antiemetic drug thalidomide [α-(N-phthalimido)glutarimide] was withdrawn in the early 1960s because of its potent teratogenic effects but was approved for the treatment of lesions associated with leprosy in 1998 and multiple myeloma in 2006. The mechanism of teratogenicity of thalidomide still remains unclear, but it is well-established that metabolism of thalidomide is important for both teratogenicity and cancer treatment outcome. Thalidomide is oxidized by various cytochrome P450 (P450) enzymes, the major one being P450 2C19, to 5-hydroxy-, 5'-hydroxy-, and dihydroxythalidomide. We previously reported that P450 3A4 oxidizes thalidomide to the 5-hydroxy and dihydroxy metabolites, with the second oxidation step involving a reactive intermediate, possibly an arene oxide, that can be trapped by glutathione (GSH) to GSH adducts. We now show that the dihydroxythalidomide metabolite can be further oxidized to a quinone intermediate. Human P450s 2J2, 2C18, and 4A11 were also found to oxidize 5-hydroxythalidomide to dihydroxy products. Unlike P450s 2C19 and 3A4, neither P450 2J2, 2C18, nor 4A11 oxidized thalidomide itself. A recently approved amino analogue of thalidomide, pomalidomide (CC-4047, Actimid), was also oxidized by human liver microsomes and P450s 2C19, 3A4, and 2J2 to the corresponding phthalimide ring-hydroxylated product.

Cytochrome P450 Family 1 Inhibitors and Structure-Activity Relationships

With the widespread use of O-alkoxyresorufin dealkylation assays since the 1990s, thousands of inhibitors of cytochrome P450 family 1 enzymes (P450s 1A1, 1A2, and 1B1) have been identified and studied. Generally, planar polycyclic molecules such as polycyclic aromatic hydrocarbons, stilbenoids, and flavonoids are considered to potentially be effective inhibitors of these enzymes, however, the details of the structure-activity relationships and selectivity of these inhibitors are still ambiguous. In this review, we thoroughly discuss the selectivity of many representative P450 family 1 inhibitors reported in the past 20 years through a meta-analysis.

Covalent Thiol Adducts Arising from Reactive Intermediates of Cocaine Biotransformation

Exposure to cocaine results in the depletion of hepatocellular glutathione and macromolecular protein binding in humans. Such cocaine-induced responses have generally been attributed to oxidative stress and reactive metabolites resulting from oxidative activation of the cocaine tropane nitrogen. However, little conclusive data exists on the mechanistic pathways leading to protein modification or the structure and specificity of cocaine-derived adduction products. We now report a previously uncharacterized route of cocaine bioactivation leading to the covalent adduction of biological thiols, including cysteine and glutathione. Incubation of cocaine with biological nucleophiles in an in vitro biotransformation system containing human liver microsomes identified a monooxygenase-mediated event leading to the oxidation of, and subsequent sulfhydryl addition to, the cocaine aryl moiety. Adduct structures were confirmed using ultra-high performance liquid chromatography coupled to high resolution, high mass accuracy mass spectrometry. Examination of assays containing transgenic bactosomes expressing single human cytochrome P450 isoforms determined the role of P450s 1A2, 2C19, and 2D6 in the oxidation process resulting in adduct formation. P450-catalyzed aryl epoxide formation and subsequent attack by free nucleophilic moieties is consistent with the resulting adduct structures, mechanisms of formation, and the empirical observation of multiple structural and stereo isomers. Analogous adduction mechanisms were maintained across all sulfhydryl-containing nucleophile models examined; N-acetylcysteine, glutathione, and a synthetic cysteine-containing hexapeptide. Predictive in silico calculations of molecular reactivity and electrophilicity/nucleophilicity were compared to the results of in vitro assay incubations in order to better understand the adduction process using the principles of hard and soft acid and base (HSAB) theory. This study elucidated a novel metabolic pathway that may be of particular significance to the clinical and forensic toxicology of cocaine and provides analytical tools and methods that can be applied to the determination of these conjugates in humans, opening a new area of research on cocaine biotransformation and toxicology.

In vitro inhibition of metabolism but not transport of gliclazide and repaglinide by Cree medicinal plant extracts

Ethnopharmacological relevanceInteractions between conventional drug and traditional medicine therapies may potentially affect drug efficacy and increase the potential for adverse reactions. Cree traditional healing is holistic and patients may use medicinal plants simultaneously with the conventional drugs. However, there is limited information that these medicinal plants may interact with drugs and additional mechanistic information is required. In this study, extracts from traditionally used Cree botanicals were assessed for their potential interaction that could alter the disposition of two blood glucose lowering drugs, gliclazide (Diamicron) and repaglinide (Gluconorm) though inhibition of either metabolism or transport across cell membranes. Materials and methodsThe effect of 17 extracts on metabolism was examined in a human liver microsome assay by HPLC and individual cytochrome P450s 2C9, 2C19, 2C8 and 3A4 in a microplate fluorometric assay. Gliclazide, rhaponticin and its aglycone derivative, rhapontigenin were also examined in the fluorometric assay. The effect on transport was examined with 11 extracts using the intestinal epithelial Caco-2 differentiated cell monolayer model at times up to 180min. ResultsBoth blood glucose lowering medications, gliclazide and repaglinide traversed the Caco-2 monolayer in a time-dependent manner that was not affected by the Cree plant extracts. Incubation of the Cree plant extracts inhibited CYP2C9, 2C19, 2C8 and 3A4-mediated metabolism, and the formation of four repaglinide metabolites: M4, m/z 451-A, m/z 451-B and the glucuronide of repaglinide in the human liver microsome assay. Gliclazide caused no significant inhibition. Likewise, rhaponticin had little effect on the enzymes causing changes of less than 10% with an exception of 17% inhibition of CYP2C19. By contrast, the aglycone rhapontigenin showed the greatest effects on all CYP-mediated metabolism. Its inhibition ranged from a mean of 58% CYP3A4 inhibition to 89% inhibition of CYP2C9. While rhaponticin and the aglycone did not show significant effects on repaglinide metabolism, they demonstrated inhibition of gliclazide metabolism. The aglycone significantly affected levels of gliclazide and its metabolites. ConclusionThese studies demonstrate that the Cree plant extracts examined have the potential in vitro to cause drug interactions through effects on key metabolic enzymes.

A Correlation Between the In Vitro Drug Toxicity of Drugs to Cell Lines That Express Human P450s and Their Propensity to Cause Liver Injury in Humans

Drug toxicity to T-antigen-immortalized human liver epithelial (THLE) cells stably transfected with plasmid vectors that encoded human cytochrome P450s 1A2, 2C9, 2C19, 2D6, or 3A4, or an empty plasmid vector (THLE-Null), was investigated. An automated screening platform, which included 1% dimethyl sulfoxide (DMSO) vehicle, 2.7% bovine serum in the culture medium, and assessed 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium reduction, was used to evaluate the cytotoxicity of 103 drugs after 24h. Twenty-two drugs caused cytotoxicity to THLE-Null cells, with EC₅₀ ≤ 200 μM; 21 of these drugs (95%) have been reported to cause human liver injury. Eleven drugs exhibited lower EC₅₀ values in cells transfected with CYP3A4 (THLE-3A4 cells) than in THLE-Null cells; 10 of these drugs (91%) caused human liver injury. An additional 8 drugs, all of which caused human liver injury, exhibited potentiated THLE-3A4 cell toxicity when evaluated using a manual protocol that included 0.2% or 1% DMSO, but not bovine serum. Fourteen of the drugs that exhibited potentiated THLE-3A4 cell toxicity are known to be metabolized by P450s to reactive intermediates. These drugs included troglitazone, which was shown to undergo metabolic bioactivation and covalent binding to proteins in THLE-3A4 cells. A single drug (rimonabant) exhibited marked THLE cell toxicity but did not cause human liver injury; this drug had very low reported plasma exposure. These results indicate that evaluation of toxicity to THLE-Null and THLE-3A4 cell lines during drug discovery may aid selection of drugs with reduced propensity to cause drug-induced liver injury and that consideration of human exposure is required to enhance data interpretation.

Genotoxicity-related chemistry of human metabolites of benzo[ghi]perylene (B[ghi]P) investigated using electro-optical arrays and DNA/microsome biocolloid reactors with LC-MS/MS.

There is limited and sometimes contradictory information about the genotoxicity of the polycyclic aromatic hydrocarbon benzo[ghi]perylene (B[ghi]P). Using recently developed metabolic toxicity screening arrays and a biocolloid reactor-LC-MS/MS approach, both featuring films of DNA and human metabolic enzymes, we demonstrated the relatively low reactivity of metabolically activated B[ghi]P toward DNA. Electro-optical toxicity screening arrays showed that B[ghi]P metabolites damage DNA at a 3-fold lower rate than benzo[a]pyrene (B[a]P), whose metabolites have a strong and well-understood propensity for DNA damage. Metabolic studies using magnetic bead biocolloid reactors coated with microsomal enzymes in 96-well plates showed that cyt P450s 1A1 and 1B1 provide high activity for B[ghi]P and B[a]P conversion. Consistent with published results, the major metabolism of B[ghi]P involved oxidations at 3,4 and 11,12 positions, leading to the formation of B[ghi]P 3,4-oxide and B[ghi]P 3,4,11,12-bisoxide. B[ghi]P 3,4-oxide was synthesized and reacted with deoxyadenosine at N6 and N7 positions and with deoxyguanosine at the N2 position. B[ghi]P 3,4-oxide is hydrolytically unstable and transforms into the 3,4-diol or converts to 3- or 4-hydroxy B[ghi]P. LC-MS/MS of reaction products from the magnetic biocolloid reactor particles coated with DNA and human enzymes revealed for the first time that a major DNA adduct results from the reaction between B[ghi]P 3,4,11,12-bisoxide and deoxyguanosine. Results also demonstrated 5-fold lower formation rates of the major DNA adduct for B[ghi]P metabolites compared to B[a]P. Overall, results from both the electro-optical array and biocolloid reactor-LC-MS/MS consistently suggest a lower human genotoxicity profile of B[ghi]P than B[a]P.

Development of Flavone Propargyl Ethers as Potent and Selective Inhibitors of Cytochrome P450 Enzymes 1A1 and 1A2

Naturally occurring flavonoids are known to be metabolized by several cytochrome P450 enzymes including P450s 1A1, 1A2, 1B1, 2C9, 3A4, and 3A5. In general flavonoids can act as substrates, inducers, and/or inhibitors of P450 enzymes. The position of the substituents on the flavone backbone has been shown to impact the biological activity against P450 enzymes. To explore the effect of a propargyl ether substitution on flavones and flavanones, 2´-flavone propargyl ether (2´-PF), 3´-flavone propargyl ether (3´-PF), 4´-flavone propargyl ether (4´-PF), 5-flavone propargyl ether (5-PF), 6-flavone propargyl ether (6-PF), 7-flavone propargyl ether (7-PF), 6-flavanone propargyl ether (6-PFN), and 7- flavanone propargyl ether (7-PFN) were synthesized. All of the newly synthesized compounds and the parent hydroxy flavones were tested for both direct inhibition and mechanism-based inhibition of cytochrome P450 enzymes 1A1, 1A2, 2A6, and 2B1. The flavone propargyl ether derivatives were found to be more potent inhibitors of P450s 1A1 and 1A2. None of the flavones and flavanones in our study showed any inhibition of P450 2A6. Only 2´-PF and 6-PFN inhibited P450 2B1. 3´-PF showed direct inhibition of P450 1A1 with the highest observed potency of 0.02 µM, in addition to its ability to cause mechanism-based inhibition with KI and kinactivation values of 0.24 µM and 0.09 min-1 for this enzyme. 7- Hydroxy flavone also exhibited mechanism-based inhibition of P450 1A1 with KI and kinactivation values of 2.43 µM and 0.115 min-1. Docking studies and QSAR studies on P450 enzymes 1A1 and 1A2 were performed which revealed important insights into the nature of binding of these molecules and provided us with good QSAR models that can be used to design new flavone derivatives.