Structural determinants of steroids for cytochrome P450 3A4-mediated metabolism

Cytochrome P450 3A4 (CYP3A4), a major member of cytochrome P450 isoenzymes, metabolizes the majority of steroids in 6β-position. For the purpose of determining requisite structural features of a series of structurally related steroids for CYP3A4-mediated metabolism, three-dimensional pharmacophore modeling as well as electrotopological state map were conducted for 15 steroids. Though prior studies speculated that the chemical reactivity of the allylic 6β-position might have a greater influence on CYP3A4 selective 6-hydroxylation than steric constraints in the enzyme, our results reveal that for CYP3A4 steroidal substrates, it is not the chemical reactivity of atoms at 6β-site, but the pharmacophoric features, i.e. the two hydrophobic rings together with two H-bond donors, that act as the key factors responsible for determining the CYP3A4 selective 6-hydroxylation of steroids.     

以睾酮为探针采用高效液相色谱法测定细胞色素CYP450 3A4的酶活性

建立了一种快速、高效的以睾酮作为探针药物评价细胞色素P450 3A4(CYP3A4)酶活性的高效液相色谱-紫外检测方法。采用的色谱柱为Phenomenex C18柱(4.6 mm×150 mm,5 μm),梯度洗脱,流速1.0 mL/min,紫外检测波长245 nm,柱温30 ℃。睾酮与大鼠肝微粒体温孵后,过已活化好的C18固相萃取小柱,收集甲醇洗脱液,于37 ℃水浴中通N2吹干,用50%甲醇复溶后进样分析测定。研究结果表明,6β-羟基睾酮的 保留时间为11.60 min,线性范围为0.5～32 μg/mL,最低检出质量浓度为0.02 μg/mL,提取率为88.41%～92.73%,方法的回收率为99.07%～101.30%;睾酮的保留时间为19.27 min,线性范围为0.5～40 μg/mL,最低检出质量浓度为0.01 μg/mL,提取率为89.59%～92.66%,方法的回收率为96.50%～98.03%。两者的日内、日间相对标准偏差均小于10%,温孵体系中的其他内源性物质不干扰测定。该方法快速、稳定、灵敏度高,适合体外睾酮及其代谢物6β-羟基睾酮的测定,可应用于体外CYP3A4酶活性的评价及酶动力学的研究。     

Enantioselective capillary electrophoresis for identification and characterization of human cytochrome P450 enzymes which metabolize ketamine and norketamine in vitro

Ketamine, a phencyclidine derivative, is used for induction of anesthesia, as an anesthetic drug for short term surgical interventions and in subanesthetic doses for postoperative pain relief. Ketamine undergoes extensive hepatic first-pass metabolism. Enantioselective capillary electrophoresis with multiple isomer sulfated β-cyclodextrin as chiral selector was used to identify cytochrome P450 enzymes involved in hepatic ketamine and norketamine biotransformation in vitro. The N-demethylation of ketamine to norketamine and subsequently the biotransformation of norketamine to other metabolites were studied via analysis of alkaline extracts of in vitro incubations of racemic ketamine and racemic norketamine with nine recombinantly expressed human cytochrome P450 enzymes and human liver microsomes. Norketamine was formed by CYP3A4, CYP2C19, CYP2B6, CYP2A6, CYP2D6 and CYP2C9, whereas CYP2B6 and CYP2A6 were identified to be the only enzymes which enable the hydroxylation of norketamine. The latter two enzymes produced metabolic patterns similar to those found in incubations with human liver microsomes. The kinetic data of ketamine N-demethylation with CYP3A4 and CYP2B6 were best described with the Michaelis–Menten model and the Hill equation, respectively. This is the first study elucidating the individual enzymes responsible for hydroxylation of norketamine. The obtained data suggest that in vitro biotransformation of ketamine and norketamine is stereoselective.     

Kinetic study of paracetamol on prolidase activity in erythrocytes by capillary electrophoresis with Ru(bpy)(3) (2+) electrochemiluminescence detection

We explored the CE with (bpy) (2+) (3) electrochemiluminescence detection for the kinetic study of drug-enzyme interaction. Effects of four nonsteroidal anti-inflammatory drugs including aspirin, paracetamol, sodium salicylate and phenacetin on prolidase (PLD) activity in erythrocytes were investigated. Aspirin enhanced PLD activity whereas the other three had inhibiting effects. This may reveal their different effects on the collagen biosynthesis and catabolism that influence tumor invasiveness. Kinetic study of paracetamol on PLD showed that the value of Michaelis constant K(m) for PLD was 1.23 mM. The mechanism of PLD inhibition by paracetamol is noncompetitive inhibition, and the inhibitor constant K(i) value obtained in our research was 9.73 x 10(3) microg/L.     

基于3D-药效团的P糖蛋白和细胞色素P4503A4的协同作用机理研究

P糖蛋白(P-glycoprotein,Pgp)和细胞色素P4503A4(CYP3A4)是决定药物ADME性质的两个重要蛋白,目前还无法通过实验方法,从分子水平清晰阐明这两个蛋白采取怎样的互补作用机理来降低外来药物的生物利用度.通过3D-药效团模建方法,提取Pgp和CYP3A4的共同底物的特征阐明这两个蛋白可能的协同作用模式.所得的药效团有助于理解药物分子同这两个蛋白的作用模式,同时该模型可以指导新药设计和改造,从而提高药物的生物利用度.     

Molecular modeling of cytochrome P450 3A4

The three-dimensional structure of human cytochrome P450 3A4 was modeled based on crystallographic coordinates of four bacterial P450s: P450 BM-3, P450cam, P450terp, and P450eryF. The P450 3A4 sequence was aligned to those of the known proteins using a structure-based alignment of P450 BM-3, P450cam, P450terp, and P450eryF. The coordinates of the model were then calculated using a consensus strategy, and the final structure was optimized in the presence of water. The P450 3A4 model resembles P450 BM-3 the most, but the B helix is similar to that of P450eryF, which leads to an enlarged active site when compared with P450 BM-3, P450cam, and P450terp. The 3A4 residues equivalent to known substrate contact residues of the bacterial proteins and key residues of rat P450 2B1 are located in the active site or the substrate access channel. Docking of progesterone into the P450 3A4 model demonstrated that the substrate bound in a 6-orientation can interact with a number of active site residues, such as 114, 119, 301, 304, 305, 309, 370, 373, and 479, through hydrophobic interactions. The active site of the enzyme can also accommodate erythromycin, which, in addition to the residues listed for progesterone, also contacts residues 101, 104, 105, 214, 215, 217, 218, 374, and 478. The majority of 3A4 residues which interact with progesterone and/or erythromycin possess their equivalents in key residues of P450 2B enzymes, except for residues 297, 480 and 482, which do not contact either substrate in P450 3A4. The results from docking of progesterone and erythromycin into the enzyme model make it possible to pinpoint residues which may be important for 3A4 function and to target them for site-directed mutagenesis.     

Effect of CYP3A4 on liver injury induced by triptolide

Triptolide (TP), one of the main bioactive diterpenes of the herbal medicine Tripterygium wilfordii Hook F, is used for the treatment of autoimmune diseases in the clinic and is accompanied by severe hepatotoxicity. CYP3A4 has been reported to be responsible for TP metabolism, but the mechanism remains unclear. The present study applied a UPLC–QTOF–MS-based metabolomics analysis to characterize the effect of CYP3A4 on TP-induced hepatotoxicity. The metabolites carnitines, lysophosphatidylcholines (LPCs) and a serious of amino acids were found to be closely related to liver damage indexes in TP-treated female mice. Metabolomics analysis further revealed that the CYP3A4 inducer dexamethasone improved the level of LPCs and amino acids, and defended against oxidative stress. On the contrary, pretreatment with the CYP3A4 inhibitor ketoconazole increased liver damage with most metabolites being markedly altered, especially carnitines. Among these metabolites, except for LPC18:2, LPC20:1 and arginine, dexamethasone and ketoconazole both affected oxidative stress induced by TP. The current study provides new mechanistic insights into the metabolic alterations, leading to understanding of the role of CYP3A4 in hepatotoxicity induced by TP.     

An NH2-terminal truncated cytochrome P450 CYP3A4 showing catalytic activity is present in the cytoplasm of human liver cells

Cytochrome P450 3A4 (CYP3A4), is the dominant human liver hemoprotein enzyme localized in the endoplasmic reticulum (ER), and is responsible for the metabolism of more than 50% of clinically relevant drugs. While we were studying CYP3A4 expression and activity in human liver, we found that anti-CYP3A4 antibody cross-reacted with a lower band in liver cytoplasmic fraction. We assessed the activities of CYP3A4 and its truncated form in the microsomal and cytoplasmic fraction, respectively. In the cytoplasmic fraction, truncated CYP3A4 showed catalytic activity when reconstituted with NADPH-cytochrome P-450 reductase and cytochrome b5. In order to determine which site was deleted in the truncated form in vitro, we transfected cells with N-terminal tagged or C-terminal tagged human CYP3A4 cDNA. The truncated CYP3A4 is the N-terminal deleted form and was present in the soluble cytoplasmic fraction. Our result shows, for the first time, that N-terminal truncated, catalytically active CYP3A4 is present principally in the cytoplasm of human liver cells.     

The stereochemistry of fatty acid hydroxylation by cytochrome P450BM3

The stereochemical preference for the cytochrome P450_BM3-catalysed hydroxylation of tetradecanoic and pentadecanoic acids has been determined via comparison with authentic non-racemic standards utilising enantioselective HPLC. The sub-terminal hydroxylation of these fatty acids by P450_BM3 is highly selective for the formation of the R-alcohols. This is the same enantioselectivity as is seen for hexadecanoic acid oxidation but contrasts with a previous report of S-hydroxylation of pentadecanoic acid by P450_BM3.     

Chiral capillary electrophoretic analysis of verapamil metabolism by cytochrome P450 3A4

Cytochrome P450 (CYP), which is one of the most important enzymes in human liver, is responsible for a large portion of the first-pass metabolism of drugs. Many studies have focused on the determination of CYP activity by substrate assays. Most of them used liquid chromatography (LC) as analytical technique, while only a few studies used capillary electrophoresis (CE) for the separation and quantitation of reaction components. In this study, the feasibility of using CE in an in vitro metabolism study with CYP was tested. Verapamil was chosen as the substrate for CYP 3A4 isozyme (Supersome). A chiral capillary electrophoretic method was developed and validated for the simultaneous determination of R,S-verapamil (VER) and their major metabolites, R,S-norverapamil (NOR). A method for CYP 3A4 activity assay was proposed with VER as a probe. At the same time, the enantioselective metabolism of VER was studied. Michaelis-Menten constants of R- and S-VER were determined. S-VER was metabolised faster and more extensively than R-VER, with K(m)=167+/-23 microM, V(max)=3,418+/-234 pmol/min/mg for S-VER, and K(m)=168+/-35 microM, V(max)=2,502+/-275 pmol/min/mg for R-VER.     

Separation and determination of carnosine-related peptides using capillary electrophoresis with laser-induced fluorescence detection

A capillary electrophoresis (CE) method with laser-induced fluorescence (LIF) detection was developed for the separation and detection of carnosine-related peptides (carnosine, anserine, and homocarnosine). A sensitive and fluorogenic regent, 3-(4-carboxybenzoyl) quinoline-2-carboxaldehyde (CBQCA) was selected as a precapillary labeling reagent for imidazole dipeptides to form isoindole derivatives. The optimized molar ratio between CBQCA and peptide was found to be 75:1, and 50 mmol/L borate buffer (pH 9.2) was used for the derivatization in order to achieve good efficiency. Three imidazole dipeptides were baseline-separated within 20 min by using 112 mmol/L sodium borate (pH 10.4-10.8) as running buffer. Concentration detection limits (signal-to-noise ratios) for carnosine, anserine, and homocarnosine were 4.73, 4.37, and 3.94 nmol/L, respectively. This method has been applied to the analysis of human cerebrospinal fluid (CSF) and meat dry powder of pig and sheep. Recoveries were in the range of 82.9-104.8% for homocarnosine in CSF. For carnosine and anserine, the recoveries are 98.3% and 80.2% in meat dry powder of pig and 111.2% and 112.8% in meat dry powder of sheep, respectively.     

Nonaqueous capillary electrophoresis with laser-induced fluorescence detection: a case study of comparison with aqueous media

A novel method based on separation by nonaqueous capillary electrophoresis (NACE) combined with laser-induced fluorescence (LIF) detection was developed and compared with classic aqueous modes of electrophoresis in terms of resolution of solutes of interest and sensitivity of the fluorescence detection. Catecholamines derivatized with 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) were chosen as test analytes for their subtle fluorescence properties. In aqueous systems, capillary zone electrophoresis (CZE) was not suitable for the analysis of test analytes due to complete fluorescence quenching of NBD-labeled catecholamines in neat aqueous buffer. The addition of micelles or microemulsion droplets into aqueous running buffer can dramatically improve the fluorescence response, and the enhancement seems to be comparable for micellar electrokinetic chromatography (MEKC) and microemulsion electrokinetic chromatography (MEEKC). As another alternative, NACE separation was advantageous when performing the analysis under the optimum separation condition of 20 mM sodium tetraborate, 20 mM sodium dodecyl sulfate (SDS), 0.1% (v/v) glacial acetic acid, 20% (v/v) acetonitrile (ACN) in methanol medium after derivatization in ACN/dimethyl sulfoxide (DMSO) (3:2, v/v) mixed aprotic solvents containing 20 mM ammonium acetate. Compared with derivatization and separation in aqueous media, NACE-LIF procedure was proved to be superior, providing high sensitivity and short migration time. Under respective optimum conditions, the NACE procedure offered the best fluorescence response with 5-24 folds enhancement for catecholamines compared to aqueous procedures. In addition, the mechanisms of derivatization and separation in nonaqueous media were elucidated in detail.     

Allosteric effects on substrate dissociation from cytochrome P450 3A4 in nanodiscs observed by ensemble and single-molecule fluorescence spectroscopy

Cytochrome P450 (CYP) 3A4 is a major human drug-metabolizing enzyme and displays pharmacologically relevant allosteric kinetics caused by multiple substrate and/or effector binding. Here, in the first single-molecule (SM) fluorescence studies of CYPs, we use total internal reflection fluorescence microscopy to measure residence times of the fluorescent dye Nile Red in CYP3A4 incorporated in surface-immobilized lipid Nanodiscs, with and without the effector alpha-naphthoflavone. We find direct evidence that CYP3A4 effectors can decrease substrate off-rates, providing a possible mechanism for effector-mediated enhancement of substrate metabolism. These interesting results highlight the potential of SM methods in studies of CYP allosteric mechanisms.     

Molecular Docking of 3-Methylindole-containing Drugs Binding into CYP3A4

Drugs SPD-304(6,7-dimethyl-3-{[methyl-(2-{methyl-[1-(3-trifluoromethyl-phenyl)-1H-indol-3-ylmethyl]- amino}-ethyl)-amino]-methyl}-chromen-4-one) and zafirlukast contain a common structural element of 3-substituted indole moiety which closely relates to a dehydrogenated reaction catalyzed by cytochrome P450s(CYPs). It was reported that the dehydrogenation can produce a reactive electrophilic intermediate which cause toxicities and inactivate CYPs. Drug L-745,870(3-{[4-(4-chlorophenyl)piperazin-1-yl]-methyl}-1H-pyrrolo- 2,3-β-pyridine) might have similar effect since it contains the same structural element. We used molecular docking approach combined with molecular dynamics(MD) simulation to model three-dimensional(3D) complex structures of SPD-304, zafirlukast and L-745,870 into CYP3A4, respectively. The results show that these three drugs can stably bind into the active site and the 3-methylene carbons of the drugs keep a reasonable reactive distance from the heme iron. The complex structure of SPD-304-CYP3A4 is in agreement with experimental data. For zafirlukast, the calculation results indicate that 3-methylene carbon might be the dehydrogenation reaction site. Docking model of L-745,870-CYP3A4 shows a potential possibility of L-745,870 dehydrogenated by CYP3A4 at 3-methylene carbon which is in agreement with experiment in vivo. In addition, residues in the phenylalanine cluster as well as S119 and R212 play a critical role in the ligands binding based on our calculations. The docking models could provide some clues to understand the metabolic mechanism of the drugs by CYP3A4.     

Kinetic study of all-or-none hemolysis induced by cationic amphiphilic polymethacrylates with antimicrobial activity

To gain an understanding of the toxicity of antimicrobial polymers to human cells, their hemolytic action was investigated using human red blood cells (RBCs). We examined the hemolysis induced by cationic amphiphilicmethacrylate random copolymers, which have amino ethyl sidechains as cationic units and either butyl or methyl methacrylate as hydrophobic units. The polymer with 30 mol% butyl sidechains (B30) displayed higher hemolytic toxicity than the polymer with 59 mol% methyl sidechains (M59). B30 also induced faster release of hemoglobin from RBCs than M59. A new theoretical model is proposed based on two consecutive steps to form active polymer species on the RBC membranes, which are associated to RBC lysis. This model takes the all-or-none release of hemoglobin by the rupture of RBCs into account, providing new insight into the polymer-induced hemolysis regarding how individual or collective cells respond to the polymers.     

Analysis of cerebrospinal fluid γ‐aminobutyric acid by capillary electrophoresis with laser‐induced fluorescence detection

The measurement of γ‐aminobutyric acid (GABA) is suitable for investigating various neurological disorders. In this study, a sensitive and selective method for free GABA quantification in cerebrospinal fluid (CSF) has been standardised. This method is based on CE with LIF detection using 4‐fluoro‐7‐nitrobenzo‐2‐oxa‐1,3‐diazole (NBD‐F) as a derivatisating agent. The reaction conditions (NBD‐F concentration, pH, temperature and reaction time) and the electrophoretic parameters (run buffer composition and pH and separation voltage) were optimised to obtain the maximum derivatisation efficiency and electrophoretic resolution. The best resolution was obtained using 200 mM sodium borate, 10 mM SDS, 8.5 mM β‐CD, pH 10 and 20 kV voltage. The method was linear in the concentration range of 2.5–1000 nM with good inter‐ and intra‐assay precision values. The effects of CSF handling on free GABA concentrations were also evaluated. Our results show that the time delay between CSF collection and freezing strongly increases the CSF GABA values. Age‐related reference values were established in 55 paediatric controls. The influence of antiepileptic therapy on free CSF GABA was studied in 38 neuropaediatric patients. Significantly, higher GABA values were obtained in patients taking valproic acid or vigabatrin therapy, which are antiepileptic drugs that modulate GABA metabolism.     

Immunoassay of P-glycoprotein on single cell by capillary electrophoresis with laser induced fluorescence detection

The cellular mechanism based on P-glycoprotein (PGP) for its drug pump function has become very important in multidrug resistance (MDR) research. A method has been established to characterize PGP on single K562 cell by coupling capillary electrophoresis with laser induced fluorescence detection. A permeable intact cell after the immunoassay binding with fluorescence labeling antibody was injected into the capillary and directly separated without lysis. It was found that once 5-10 optional cells were detected in batch, the PGP amount on this cell line could be outlined and calculated clearly. The PGP amount on K562 MDR cell line is 3.88 times higher than that on K562 sensitive cell line. These two cell lines with immunoassay binding were also analyzed by injection of multi-cells in order to improve the throughput. A resistance factor so called multidrug resistance multiple (MRM) was introduced to evaluate the MDR difference between cell lines. The MRM values of the cell line K562 measured by single cell analysis are well correlated with those by flow cytometry, which also prove the validity of our method in single cell analysis for the possibility of cancer diagnosis, pharmacokinetics and drug screening in future.     

毛细管电泳高频电导法测定尿中MDMA及其代谢物

建立了尿样中MDMA及其代谢物MDA的毛细管电泳高频电导法检测分析方法。对电泳分离缓冲介质的种类、浓度、pH值以及分离操作电压和进样时间等实验条件进行了优化。缓冲液为2．5mmol／L NH4 Ac 0．5mmol／L HAc 2mmol／L SDS 10mmol／L β-CD,分离电压为15kV时,可实现较好的分离与检测。该法在2．5～80μg/mL范围内。MDMA和MDA线性相关系数r分别为0．998和0．999,检出限均为1．0μg/mL(S／N=3)。不同添加浓度水平尿样,日间和日内RSD均小于5％,回收率均在90％以上,适于法医毒物分析和临床药物监测的需要。     

Determination of 1-aminocyclopropane-1-carboxylic acid in apple extracts by capillary electrophoresis with laser-induced fluorescence detection

A rapid and sensitive method for the determination of 1-aminocyclopropane-1-carboxylic acid (ACC) in apple tissues has been described. This method is based on the derivatization of ACC with 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ), and separation and quantification of the resulting FQ-ACC derivative by capillary electrophoresis coupled to laser-induced fluorescence detection (CE-LIF). Our results indicated that ACC derivatized with FQ could be well separated from other interfering amino acids using 20 mM borate buffer (pH 9.35) containing 40 mM sodium dodecyl sulfate and 10 mM Brij 35. The linearity of ACC was determined in the range from 0.05 to 5 microM with a correlation of 0.9967. The concentration detection limit for ACC was 10 nM (signal-to-noise = 3). The sensitivity and selectivity of this described method allows the analysis of ACC in crude apple extracts without extra purification and enrichment procedure.