Searching the Cytochrome P450 Enzymes for the Metabolism of Meranzin Hydrate: A Prospective Antidepressant Originating from Chaihu-Shugan-San

Meranzin hydrate (MH), an absorbed bioactive compound from the Traditional Chinese Medicine (TCM) Chaihu-Shugan-San (CSS), was first isolated in our laboratory and was found to possess anti-depression activity. However, the role of cytochrome P450s (CYPs) in the metabolism of MH was unclear. In this study, we screened the CYPs for the metabolism of MH in vitro by human liver microsomes (HLMs) or human recombinant CYPs. MH inhibited the enzyme activities of CYP1A2 and CYP2C19 in a concentration-dependent manner in the HLMs. The K_m and V_max values of MH were 10.3±1.3 µM and 99.1±3.3 nmol/mg protein/min, respectively, for the HLMs; 8.0±1.6 µM and 112.4±5.7 nmol/nmol P450/min, respectively, for CYP1A2; and 25.9±6.6 µM and 134.3±12.4 nmol/nmol P450/min, respectively, for CYP2C19. Other human CYP isoforms including CYP2A6, CYP2C9, CYP2D6, CYP2E1 and CYP3A4 showed minimal or no effect on MH metabolism. The results suggested that MH was simultaneously a substrate and an inhibitor of CYP1A2 and CYP2C9, and MH had the potential to perpetrate drug-drug interactions with other CYP1A2 and CYP2C19 substrates.     

Screening of Drug Metabolizing Enzymes for the Ginsenoside Compound K In Vitro: An Efficient Anti-Cancer Substance Originating from Panax Ginseng

Ginsenoside compound K (CK), a rare ginsenoside originating from Panax Ginseng, has been found to possess unique pharmacological activities specifically as anti-cancers. However, the role of cytochrome P450s (CYPs) in the metabolism of CK is unclear. In this study, we screened the CYPs for the metabolism of CK in vitro using human liver microsomes (HLMs) or human recombinant CYPs. The results showed that CK inhibited the enzyme activities of CYP2C9 and CYP3A4 in the HLMs. The K_m and V_max values of CK were 84.20±21.92 μM and 0.28±0.04 nmol/mg protein/min, respectively, for the HLMs; 34.63±10.48 μM and 0.45±0.05 nmol/nmol P450/min, respectively, for CYP2C9; and 27.03±5.04 μM and 0.68±0.04 nmol/nmol P450/min, respectively, for CYP3A4. The IC₅₀ values were 16.00 μM and 9.83 μM, and K_i values were 14.92 μM and 11.42μM for CYP2C9 and CYP3A4, respectively. Other human CYP isoforms, including CYP1A2, CYP2A6, CYP2D6, CYP2E1, and CYP2C19, showed minimal or no effect on CK metabolism. The results suggested that CK was a substrate and also inhibitors for both CYP2C9 and CYP3A4. Patients using CK in combination with therapeutic drugs that are substrates of CYP2C9 and CYP3A4 for different reasons should be careful, although the inhibiting potency of CK is much poorer than that of enzyme-specific inhibitors.     

Identification of human cytochrome P450 isoforms involved in the 7-hydroxylation of chlorpromazine by human liver microsomes

Studies to identify the cytochrome P450 (CYP) isoform(s) involved in chlorpromazine 7-hydroxylation were performed using human liver microsomes and cDNA-expressed human CYPs. The kinetics of chlorpromazine 7-hydroxylation in human liver microsomes showed a simple Michaelis-Menten behavior. The apparent Km and Vmax values were 3.4+/-1.0 microM and 200.5+/-83.7 pmol/min/mg, respectively. The chlorpromazine 7-hydroxylase activity in human liver microsomes showed good correlations with desipramine 2-hydroxylase activity (r = 0.763, p < 0.05), a marker activity for CYP2D6, and phenacetin O-deethylase activity (r = 0.638, p < 0.05), a marker activity for CYP1A2. Quinidine (an inhibitor of CYP2D6) completely inhibited while alpha-naphthoflavone (an inhibitor of CYP1A2) marginally inhibited the chlorpromazine 7-hydroxylase activity in a human liver microsomal sample showing high CYP2D6 activity. On the other hand, alpha-naphthoflavone inhibited the chlorpromazine 7-hydroxylase activity to 55-65% of control in a human liver microsomal sample showing low CYP2D6 activity. Among eleven cDNA-expressed CYPs studied, CYP2D6 and CYP1A2 exhibited significant activity for the chlorpromazine 7-hydroxylation. The Km values for the chlorpromazine 7-hydroxylation of both cDNA-expressed CYP2D6 and CYP1A2 were in agreement with the Km values of human liver microsomes. These results suggest that chlorpromazine 7-hydroxylation is catalyzed mainly by CYP2D6 and partially by CYP1A2.     

Pulmonary cytochrome P450 enzymes belonging to the CYP4B subfamily from an Australian marsupial,the tammar wallaby (Macropus eugenii)

Cytochromes P450 (CYPs) are critically important in the oxidative metabolism of a diverse array of xenobiotics and endogenous substrates. We have previously reported the cloning and characterisation of the koala CYP4A15, the first reported member of the CYP4 family from marsupials, and have demonstrated important species differences in CYP4A activity and tissue expression. In the present study, the cloning of CYP4B1 in the wallaby (Macropus eugenii) and their expression across marsupials is described. Rabbit anti-mouse CYP4B1 antibody detected immunoreactive proteins in lung and liver microsomes from all test marsupials, with relative weak signal detected from the koala, suggesting a species-specific expression. Microsomal 2-aminofluorene bio-activation (a CYP4B1 marker) in wallaby lung was comparable to that of rabbit, with significant higher activities detected in wallaby liver and kidneys compared to rabbit. A 1548 bp wallaby lung CYP4B complete cDNA, designated CYP4B1, which encodes a protein of 510 amino acids and shares 72% nucleotide and 69% amino acid sequence identity to human CYP4B1, was cloned by polymerase chain reaction approaches. The results demonstrate the presence of wallaby CYP4B1 that shares several common features with other published CYP4Bs; however the wallaby CYP4B1 cDNA contains four extra amino acid residues at the NH₂-terminal, a fundamentally conserved transmembrane anchor of all eukaryote CYPs.     

Investigation of cytochrome P450 inhibitory properties of maslinic acid,a bioactive compound from Olea europaea L., and its structure–activity relationship

Maslinic acid (MA), the main pentacyclic triterpene of Olea europaea L. fruit, possesses a variety of pharmacological actions, including hypoglycemic, antioxidant, cardioprotective and antitumoral activities. Despite its importance, little is known about its effects on the cytochrome P450 (CYP) activity in both humans and animals. Therefore, the aim of this study was to investigate the effects of MA on the CYP 1A2, 2C9/11, 2D1/6, 2E1 and 3A2/4 activities by human and rat liver microsomes and specific CYP isoforms. In humans, MA only weakly inhibited CYP3A4 activity in human liver microsomes and specific CYP3A4 isoform with IC₅₀ value at 46.1 and 62.3 µM, respectively. In rats, MA also exhibited weak inhibition on CYP2C11, CYP2E1 and CYP3A2 activities with IC₅₀ values more than 100 µM. Enzyme kinetic studies showed that the MA was not only a competitive inhibitor of CYP3A4 in humans, but also a competitive inhibitor of CYP2C11 and 3A2 in rats, with K_i of 18.4, 98.7 and 66.3 µM, respectively. Moreover, the presence of hydroxyl group at C-2 position of triterpenic acid in MA compared with oleanolic acid could magnify its competitive inhibition on human CYP3A4 activity. The relatively high K_i values of MA would have a low potential to cause the possible toxicity and drug interactions involving CYP enzymes, thus suggesting a sufficient safety for its putative use as a nutraceutical taken together with drugs.     

Inhibition of human cytochromes P450 2A6 and 2A13 by flavonoids,acetylenic thiophenes and sesquiterpene lactones from Pluchea indica and Vernonia cinerea

The human liver cytochrome P450 (CYP) 2A6 and the respiratory CYP2A13 enzymes play role in nicotine metabolism and activation of tobacco-specific nitrosamine carcinogens. Inhibition of both enzymes could offer a strategy for smoking abstinence and decreased risks of respiratory diseases and lung cancer. In this study, activity-guided isolation identified four flavonoids 1–4 (apigenin, luteolin, chrysoeriol, quercetin) from Vernonia cinerea and Pluchea indica, four hirsutinolide-type sesquiterpene lactones 5–8 from V. cinerea, and acetylenic thiophenes 9–11 from P. indica that inhibited CYP2A6- and CYP2A13-mediated coumarin 7-hydroxylation. Flavonoids were most effective in inhibition against CYP2A6 and CYP2A13, followed by thiophenes, and hirsutinolides. Hirsutinolides and thiophenes exhibited mechanism-based inhibition and in irreversible mode against both enzymes. The inactivation kinetic K_I values of hirsutinolides against CYP2A6 and CYP2A13 were 5.32–15.4 and 0.92–8.67 µM, respectively, while those of thiophenes were 0.11–1.01 and 0.67–0.97 µM, respectively.     

Metabolism of BYZX in Human Liver Microsomes and Cytosol: Identification of the Metabolites and Metabolic Pathways of BYZX

BYZX, [(E)-2-(4-((diethylamino)methyl)benzylidene)-5,6-dimethoxy-2,3-dihydroinden-one], belongs to a series of novel acetylcholinesterase inhibitors and has been synthesized as a new chemical entity for the treatment of Alzheimer’s disease symptoms. When incubated with human liver microsomes (HLMs), BYZX was rapidly transformed into its metabolites M1, M2, and M3. The chemical structures of these metabolites were identified using liquid chromatography tandem mass spectrometry and nuclear magnetic resonance, which indicated that M1 was an N-desethylated and C = C hydrogenation metabolite of BYZX. M2 and M3 were 2 precursor metabolites, which resulted from the hydrogenation and desethylation of BYZX, respectively. Further studies with chemical inhibitors and human recombinant cytochrome P450s (CYPs), and correlation studies were performed. The results indicated that the N-desethylation of BYZX and M2 was mediated by CYP3A4 and CYP2C8. The reduced form of β-nicotinamide adenine dinucleotide 2′-phosphate was involved in the hydrogenation of BYZX and M3, and this reaction occurred in the HLMs and in the human liver cytosol. The hydrogenation reaction was not inhibited by any chemical inhibitors of CYPs, but it was significantly inhibited by some substrates of α,β-ketoalkene C = C reductases and their inhibitors such as benzylideneacetone, dicoumarol, and indomethacin. Our results suggest that α,β-ketoalkene C = C reductases may play a role in the hydrogenation reaction, but this issue requires further clarification.     

浙江省红楠生境地群落数量分类和环境解析

为探讨群落结构与环境因子对红楠Machilus thunbergii在浙江省分布及发展的影响,采用双向指示种分类(TWINSPAN)、典范对应分析(CCA)并结合种间联结分析,对浙江省172个红楠生境地群落进行数量分类排序与环境解析。结果表明:(1)TWINSPAN将172个样地群落分成8个群落类型,红楠在群落中为优势种或主要伴生种。群落类型的不同是影响红楠生长及更新的重要原因。(2)海拔高度及坡度是决定红楠分布及发展的主导环境因子。红楠作为优势种的生境地主要特征为海拔900m以下、坡度35°以下。(3)甜槠(Castanopsis eyrei)、青冈(Cyclobalanopsis glauca)、木荷(Schima superba)为群落中的稳定优势种,种间联结结果显示与红楠均为显著正关联。     

Lignans from machilus thunbergii

Five new lignans, machilin A[(2S,3R)-2,3-dimethyl-1,4-dipiperonyl-butane], machilin B [(2S,3S)-2,3-dihydro-7-methoxy-3-methyl-2-piperon threo-2-(2-methoxy-4-trans-propenylphenoxy)-1-(4-hydroxy-3-methoxyphenyl)propan-1-ol], machilin E (erythro-1-acetoxy-2-(2-methoxy-4-trans-(3-hydroxy-1-propenyl)phenoxy]-l-piperonylpropane) were isolated from the bark of Machilus thunbergii and their structures were characterized.     

Expression and role of CYP505A1 in pathogenicity of Fusarium oxysporum f. sp. lactucae

BackgroundCytochrome P450 enzymes (CYPs) are monooxygenases present in every domain of life. In fungi CYPs are involved in virulence. Fusarium wilt of lettuce, caused by F. oxysporum f. sp. lactucae, is the most serious disease of lettuce. F. oxysporum f.sp. lactucae MSA35 is an antagonistic fungus. Pathogenic formae specialis of F. oxysporum possess a CYP belonging to the new family CYP505. This enzyme hydroxylates saturated fatty acids that play a role in plant defence.MethodsMolecular tools were adopted to search for cyp505 gene in MSA35 genome. cyp505 gene expression analysis in pathogenic and antagonistic Fusarium was performed. The enzyme was expressed in its recombinant form and used for catalytic reactions with fatty acids, the products of which were characterized by mass spectrometry analysis.ResultsA novel MSA35 self-sufficient CYP505 is differentially expressed in antagonistic and pathogenic F. oxysporum. Its expression is induced by the host plant lettuce in both pathogenesis and antagonism during the early phase of the interaction, while it is silenced during the late phase only in antagonistic Fusarium. Mass-spectrometry investigations proved that CYP505A1 mono-hydroxylates lauric, palmitic and stearic acids.ConclusionsThe ability of CYP505A1 to oxidize fatty acids present in the cortical cell membranes together with its differential expression in its Fusarium antagonistic form point out to the possibility that this enzyme is associated with Fusarium pathogenicity in lettuce.General significanceThe CYP505 clan is present in pathogenic fungal phyla, making CYP505A1 enzyme a putative candidate as a new target for the development of novel antifungal molecules.     

Antitumor drug ellipticine inhibits the activities of rat hepatic cytochromes P450

Ellipticine is a potent antineoplastic agent, whose mode of action is considered to be based mainly on DNA intercalation and/or inhibition of topoisomerase II. Recently, we found that ellipticine also forms the cytochrome P450 (CYP)-mediated covalent DNA adducts. Here, we study the effect of ellipticine on CYP enzymes in rat hepatic microsomes, studying its binding to the enzymes and its potential to inhibit the CYP activities measured with their selective substrates. Although ellipticine was reported to be a selective and strong inhibitor of CYP1A1/2, we found that its inhibitory potential is non-specific. Ellipticine is the most potent inhibitor for CYP3A-dependent 6beta-hydroxylation of progesterone, followed by CYP1A1/2-dependent ethoxyresorufin O-deethylation and CYP2B-mediated pentoxyresorufin O-depentylation. Lower inhibition was detected for 1'-hydroxylation of bufurarol, 21-hydroxylation of progesterone and 6-hydroxylation of chlorzoxazone catalyzed by CYP2D, CYP2C and CYP2E1, respectively. Ellipticine binds to several CYPs of rat hepatic microsomes. The binding titration of ellipticine typically give reverse type I spectrum with CYPs in rat hepatic microsomes. The results indicate that inhibition of CYPs by ellipticine cannot be explained only by its differential potency to bind to individual CYPs.     

The Cytochrome P450 Enzyme Responsible for the Production of (Z)‐Norendoxifen in vitro

Norendoxifen, an active metabolite of tamoxifen, is a potent aromatase inhibitor. Little information is available regarding production of norendoxifen in vitro. Here, we conducted a series of kinetic and inhibition studies in human liver microsomes (HLMs) and expressed P450s to study the metabolic disposition of norendoxifen. To validate that norendoxifen was the metabolite of endoxifen, metabolites in HLMs incubates of endoxifen were measured using a HPLC/MS/MS method. To further probe the specific isoforms involved in the metabolic route, endoxifen was incubated with recombinant P450s (CYP 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4, 3A5 and CYP4A11). Formation rates of norendoxifen were evaluated in the absence and presence of P450 isoform specific inhibitors using HLMs. The peak of norendoxifen was found in the incubations consisting of endoxifen, HLMs, and cofactors. The retention times of norendoxifen, endoxifen, and the internal standard (diphenhydramine) were 7.81, 7.97, and 5.86 min, respectively. The K_m (app) and V_max (app) values of norendoxifen formation from endoxifen in HLM was 47.8 μm and 35.39 pmol min⁻¹ mg⁻¹. The apparent hepatic intrinsic clearances of norendoxifen formation were 0.74 μl mg⁻¹ min. CYP3A5 and CYP2D6 were the major enzymes capable of norendoxifen formation from endoxifen with the rates of 0.26 and 0.86 pmol pmol⁻¹ P450 × min. CYP1A2, 3A2, 2C9, and 2C19 also contributed to norendoxifen formation, but the contributions were at least 6‐fold lower. One micromolar ketoconazole (CYP3A inhibitor) showed an inhibitory effect on the rates of norendoxifen formation by 45%, but 1 μm quinidine (CYP2D6 inhibitor) does not show any inhibitory effect. Norendoxifen, metabolism from endoxifen by multiple P450s that including CYP3A5.     

Herb-drug interaction between Styrax and warfarin: Molecular basis and mechanism

BackgroundStyrax, one of the most famous folk medicines, has been frequently used for the treatment of cardiovascular diseases and skin problems in Asia and Africa. It is unclear whether Styrax or Styrax-related herbal medicines may trigger clinically relevant herb-drug interactions.PurposeThis study was carried out to investigate the inhibitory effects of Styrax on human cytochrome P450 enzymes (CYPs) and to clarify whether this herb may modulate the pharmacokinetic behavior of the CYP-substrate drug warfarin when co-administered.Study DesignThe inhibitory effects of Styrax on CYPs were assayed in human liver microsomes (HLM), while the pharmacokinetic interactions between Styrax and warfarin were investigated in rats. The bioactive constituents in Styrax with strong CYP3A inhibitory activity were identified and their inhibitory mechanisms were carefully investigated.MethodsThe inhibitory effects of Styrax on human CYPs were assayed in vitro, while the pharmacokinetic interactions between Styrax and warfarin were studied in rats. Fingerprinting analysis of Styrax coupled with LC-TOF-MS/MS profiling and CYP inhibition assays were used to identify the constituents with strong CYP3A inhibitory activity. The inhibitory mechanism of oleanonic acid (the most potent CYP3A inhibitor occurring in Styrax) against CYP3A4 was investigated by a panel of inhibition kinetics analyses and in silico analysis.ResultsIn vitro assays demonstrated that Styrax extract strongly inhibited human CYP3A and moderately inhibited six other tested human CYPs, as well as potently inhibited warfarin 10-hydroxylation in liver microsomes from both humans and rats. In vivo assays demonstrated that compared with warfarin given individually in rats, Styrax (100 mg/kg) significantly prolonged the plasma half-life of warfarin by 2.3-fold and increased the AUC_(0-inf) of warfarin by 2.7-fold when this herb was co-administrated with warfarin (2 mg/kg) in rats. Two LC fractions were found with strong CYP3A inhibitory activity and the major constituents in these fractions were characterized by LC-TOF-MS/MS. Five pentacyclic triterpenoid acids (including epibetulinic acid, betulinic acid, betulonic acid, oleanonic acid and maslinic acid) present in Styrax were potent CYP3A inhibitors, and oleanonic acid was a competitive inhibitor against CYP3A-mediated testosterone 6β-hydroxylation.ConclusionStyrax and the pentacyclic triterpenoid acids occurring in this herb strongly modulate the pharmacokinetic behavior of warfarin via inhibition of CYP3A.     

SDS‐PAGE‐free protocol for comprehensive identification of cytochrome P450 enzymes and uridine diphosphoglucuronosyl transferases in human liver microsomes

Comprehensive identification of cytochrome P450 enzymes (CYPs) and uridine diphosphoglucuronosyl transferases (UGTs) in human liver microsomes (HLMs) was performed with an SDS‐PAGE‐free protocol. HLMs were solubilized with 5% v/v ionic liquid, 1‐butyl‐3‐methyl imidazolium tetrafluoroborate, followed by tryptic digestion, and 2D‐SCX‐RPLC‐ESI‐MS/MS (LTQ XL) analysis in triplicate. In total, 27 CYPs and 12 UGTs were confidently identified with average sequence coverage as 30.99 and 25.07%, average peptide number as 14 and 13, and average unique peptide number as 7 and 4, respectively. The highly similar isoforms of CYP3A, CYP2C, and CYP4F subfamilies could be unambiguously differentiated from each other, despite the fact that the sequence similarity of CYP2C9 and CYP2C19 is 91%. In addition, protein spectral count was used to approximately evaluate the relative abundance of identified CYPs and UGTs, and the results agreed with previous immunochemistry reports.     

Andrographolide and 14-deoxy-11, 12-didehydroandrographolide inhibit cytochrome P450s in HepG2 hepatoma cells

AimsCytochrome P450 (CYP) enzymes have been implicated in a large number of preventable drug–herb interactions. Andrographis paniculata Nees, a tropical herb widely used for various health conditions contains two major diterpenoids, andrographolide and 14-Deoxy-11, 12-Didehydroandrographolide. These compounds were evaluated systematically for their effects on CYP1A2, CYP2D6 and CYP3A4 expressions in HepG2 cells.Main methodsQuantitative RT-PCR and Western blot analysis were used to assess the mRNA and protein expression of the three CYPs. CYP3A4 enzyme activity was evaluated using P450-Glo? Assays. The LanthaScreen® TR-FRET PXR (SXR) Competitive Binding Assay was used to determine if the compounds are potential PXR-ligands.Key findingsBoth diterpenoids inhibited the mRNA and protein expressions of CYP1A2, CYP2D6, and CYP3A4. Interestingly, the lowest concentration of both diterpenoids produced a more than 50% reduction in the mRNA and protein expression of CYP3A4 and this reduction was consistent with the enzyme activity. Further experiments revealed that both diterpenoids were also capable of attenuating the ability of dexamethasone to induce CYP3A4 expression, and 14-Deoxy-11, 12-Didehydroandrographolide tended to bind to the PXR-LBD site in a concentration-dependent manner.SignificanceThese diterpenoids are potential CYP3A4 inhibitors and the effects on CYP3A4 may be clinically significant. 14-Deoxy-11, 12-Didehydroandrographolide inhibits CYP3A4 by binding and antagonizing PXR function.     

Homology modeling of the three membrane proteins of the dhurrin metabolon: catalytic sites, membrane surface association and protein-protein interactions

Formation of metabolons (macromolecular enzyme complexes) facilitates the channelling of substrates in biosynthetic pathways. Metabolon formation is a dynamic process in which transient structures mediated by weak protein-protein interactions are formed. In Sorghum, the cyanogenic glucoside dhurrin is derived from l-tyrosine in a pathway involving the two cytochromes P450 (CYPs) CYP79A1 and CYP71E1, a glucosyltransferase (UGT85B1), and the redox partner NADPH-dependent cytochrome P450 reductase (CPR). Experimental evidence suggests that the enzymes of this pathway form a metabolon. Homology modeling of the three membrane bound proteins was carried out using the Sybyl software and available relevant crystal structures. Residues involved in tight positioning of the substrates and intermediates in the active sites of CYP79A1 and CYP71E1 were identified. In both CYPs, hydrophobic surface domains close to the N-terminal trans-membrane anchor and between the F′ and G helices were identified as involved in membrane anchoring. The proximal surface of both CYPs showed positively charged patches complementary to a negatively charged bulge on CPR carrying the FMN domain. A patch of surface exposed, positively charged amino acid residues positioned on the opposite face of the membrane anchor was identified in CYP71E1 and might be involved in binding UGT85B1 via a hypervariable negatively charged loop in this protein.