Identification of novel enzyme-prodrug combinations for use in cytochrome P450-based gene therapy for cancer

Gene-directed enzyme prodrug therapy can be used to increase the therapeutic activity of anti-cancer prodrugs that undergo liver cytochrome P450 (CYP)-catalyzed prodrug to active drug conversion. The present report describes a cell-culture-based assay to identify CYP gene-CYP prodrug combinations that generate bystander cytotoxic metabolites and that may potentially be useful for CYP-based gene therapy for cancer. A panel of rat liver microsomes, comprising distinct subsets of drug-inducible hepatic CYPs, was evaluated for prodrug activation in a four-day 9L gliosarcoma cell growth inhibition assay. A strong NADPH- and liver microsome-dependent increase in 9L cytotoxicity was observed for the CYP prodrugs cyclophosphamide, ifosfamide, and methoxymorpholinyl doxorubicin (MMDX) but not with three other CYP prodrugs, procarbazine, dacarbazine, and tamoxifen. MMDX activation was potentiated approximately 250-fold by liver microsomes from dexamethasone-induced rats (IC(50) (MMDX) approximately 0.1nM), suggesting that dexamethasone-inducible CYP3A enzymes contribute to activation of this novel anthracycline anti-tumor agent. This CYP3A dependence was verified in studies using liver microsomes from uninduced male and female rats and by using the CYP3A-selective inhibitors troleandomycin and ketoconazole. These findings highlight the advantages of using cell culture assays to identify novel CYP prodrug-CYP gene combinations that are characterized by production of cell-permeable, cytotoxic metabolites and that may potentially be incorporated into CYP-based gene therapies for cancer treatment.     

Development and validation of a rapid HPLC method for simultaneous determination of tramadol, and its two main metabolites in human plasma

Tramadol, an analgesic agent, and its two main metabolites O-desmethyltramadol (M1) and N-desmethyltramadol (M2) were determined simultaneously in human plasma by a rapid and specific HPLC method. The sample preparation was a simple extraction with ethyl acetate. Chromatographic separation was achieved with a Chromolith Performance RP-18e 50 mm x 4.6 mm column, using a mixture of methanol:water (13:87, v/v) adjusted to pH 2.5 by phosphoric acid, in an isocratic mode at flow rate of 2 ml/min. Fluorescence detection (lambda(ex)=200 nm/lambda(em)=301 nm) was used. The calibration curves were linear (r(2)>0.997) in the concentration range of 2.5-500 ng/ml, 1.25-500 ng/ml and 5-500 ng/ml for tramadol, M1 and M2, respectively. The lower limit of quantification was 2.5 ng/ml for tramadol, 1.25 ng/ml for M1 and 5 ng/ml for M2. The within- and between-day precisions in the measurement of QC samples at four tested concentrations were in the range of 2.5-9.7%, 2.5-9.9% and 5.9-11.3% for tramadol, M1 and M2, respectively. The developed procedure was applied to assess the pharmacokinetics of tramadol and its two main metabolites following administration of 100mg single oral dose of tramadol to healthy volunteers.     

Use of electrospray ionization liquid chromatography-mass spectrometry to study the role of CYP2D^ in the in vitro metabolism of 5-hydroxytryptamine receptor antagonists

An electrospray ionization liquid chromatographic-mass spectrometric (ESI-LC-MS) method has been developed to study the involvement of the cytochrome P450 isoenzyme CYP2D6 in the in vitro metabolism of the indole containing 5-hydroxytryptamine (5-HT₃) receptor antagonists tropisetron, ondansetron and dolasetron in human liver microsomes. Compounds were eluted using linear gradients of acetonitrile-20 mM ammonium acetate, solvent A, (10:90, v/v) (ph 6.0) and solvent B, (60:40, v/v) (pH 6.0) and a Nucleosil C₄ column. Microsomal incubations were analysed using selected ion monitoring of the molecular ion of parent drug and the molecular ion of hydroxylated metabolites. The involvement of CYP2D6 in drug metabolism was assessed by inhibition studies using quinidine (5 μM), a specific inhibitor of human CYP2D6, as well as by incubating compounds with microsomes prepared from celss transfected with cDNA encoding human CYP2D6. Results showed that the oxidation of all three compounds involved CYP2D6, but only that of tropisetron was inhibited by over 90% in the presence of quinidine. The present method can be applied to pre-clinical compounds, at an early stage of drug discovery, to assess the involvement of CYP2D6 in their metabolism and to screen for those compounds where CYP2D6 is the only isoenzyme implicated in the formation of major metabolites.     

Advances in Molecular Modeling of Human Cytochrome P450 Polymorphism

Cytochrome P450 (CYP) is a supergene family of metabolizing enzymes involved in the phase I metabolism of drugs and endogenous compounds. CYP oxidation often leads to inactive drug metabolites or to highly toxic or carcinogenic metabolites involved in adverse drug reactions (ADR). During the last decade, the impact of CYP polymorphism in various drug responses and ADR has been demonstrated. Of the drugs involved in ADR, 56% are metabolized by polymorphic phase I metabolizing enzymes, 86% among them being CYP. Here, we review the major CYP polymorphic forms, their impact for drug response and current advances in molecular modeling of CYP polymorphism. We focus on recent studies exploring CYP polymorphism performed by the use of sequence-based and/or protein-structure-based computational approaches. The importance of understanding the molecular mechanisms related to CYP polymorphism and drug response at the atomic level is outlined.     

LC-MS/MS analysis of dextromethorphan metabolism in human saliva and urine to determine CYP2D6 phenotype and individual variability in N-demethylation and glucuronidation

In order to establish a fast screening method for the determination of the CYP2D6 metabolic phenotype a sensitive LC-MS/MS assay to quantify dextromethorphan (DEX) and its O-demethylated metabolite dextrorphan (DOR) in human saliva was developed with limits of quantitation of 1 pmol/ml. Saliva was provided by 170 medical students 2h after oral ingestion of 30 mg (81 micromol) dextromethorphan hydrobromide. Individual ratios of the concentrations DEX/DOR (metabolic ratio, MR(DEX/DOR)) varied more than 25,000-fold (0.03-780). Two groups comprising 156 'Extensive' and 14 'Poor Metabolizers' were clearly distinguished. For the investigation of individual differences in N-demethylation and glucuronidation, four additional metabolites of DEX, 3-methoxymorphinan (MOM), 3-hydroxymorphinan (HOM), and the two O-glucuronides (DORGlu and HOMGlu) were measured by LC-MS/MS analysis of 6-h urine of 24 volunteers. The N-demethylation reactions DEX-to-MOM and DOR-to-HOM defined by the respective MR were significantly correlated. The same holds for the glucuronidation pathways (MR(DOR/DORGlu) versus MR(HOM/HOMGlu)). The three poor CYP2D6 metabolizers excreted relatively high amounts of the parent compound DEX (up to 7 micromol), but only low amounts of glucuronides (DORGlu: 0.4-1.0 micromol; HOMGlu: 0.2-0.7 micromol). For the 21 'Extensive Metabolizers', the two glucuronides were the most abundant, with relatively little interindividual variation (DORGlu: 10-44 micromol; HOMGlu: 5-17 micromol). For the excretion of the glucuronides, two normal distributions provided the best fit, indicating that the determination of the glucuronides alone could allow assignment of the CYP2D6 metabolic phenotype.     

Analysis of hydroxylated and N-dealkylated metabolites of terfenadine in microsomal incubates by liquid chromatography–mass spectrometry

This report describes an assay for the H₁-receptor antagonist, terfenadine, and its two primary metabolites, terfenadine alcohol (TOH) and azacyclonol (AZ), using positive-ion, electrospray ionization–liquid chromatography–mass spectrometry. The assay was developed in support of kinetic studies of terfenadine oxidative metabolism in human liver and intestinal microsomes, which required quantification of incubate metabolites at low nanomolar concentrations. Terfenadine metabolites were extracted from basified microsomal incubates into methylene chloride. Reconstituted extracts were subject to liquid chromatographic separation on a cyano-reverse phase column. The [M+H]⁺ ions of terfenadine, terfenadine metabolites, and internal standard were monitored in the effluent by quadrupole mass spectrometry. The assay demonstrated linearity over an incubate concentration range of 5–250 and 12.5–1250 ng/ml for the metabolites and the parent drug, respectively. The respective limits of detection and quantitation for all three analytes were 1.5 and 5 ng/ml of microsomal incubate. Replicate analysis of quality control samples exhibited intra-day coefficients of variation ranging from 3.3% to 7.8% for the three analytes. The corresponding inter-day coefficients of variation ranged from 4.2% to 8.6%. The reproducibility and sensitivity of the assay, combined with the selectivity of mass spectrometric detection, should allow an accurate kinetic characterization of terfenadine oxidation mediated by the high affinity CYP3A enzymes in human liver and intestinal microsomes.     

Determination of fluoxetine and norfluoxetine in human plasma by high-performance liquid chromatography with ultraviolet detection in psychiatric patients

A rapid high-performance liquid chromatographic method is described for the simultaneous determination of the widely used antidepressant drug, fluoxetine and its principal metabolite norfluoxetine in plasma. After liquid-liquid extraction the compounds were separated in a reversed-phase column and assayed by ultraviolet absorption at 226 nm. The analytical interference from psychoactive drugs and their metabolites was also studied. The extraction recoveries were 93 and 87% for norfluoxetine and fluoxetine, respectively. The limit of quantitation under the described conditions was 14 nmol/l for both compounds. The method was found to be reproducible with coefficients of variation less than 10%. A great variability in plasma concentrations of fluoxetine and norfluoxetine as well as in fluoxetine/norfluoxetine ratios was found among the 29 patients studied. This result suggests the implication of genetically polymorphic enzymes, presumably CYP2D6, CYP2C9 and CYP2C19 in the metabolism of fluoxetine to norfluoxetine. Therapeutic drug monitoring should thus be useful in patients treated with regular doses.     

Hydrogen peroxide affects abscisic acid binding to ABAP1 in barley aleurones.

Dramatic increases in H2O2 levels have been observed following abscisic acid (ABA) treatment of plant tissues. Following ABA treatment in aleurone cells, H2O2 reached transient levels of approximately 115 micromol/L H2O2. To determine whether ABA perception was modified by such changes, the effect of H2O2 on a recently characterized ABA-binding protein (ABAP1), cloned from barley aleurone layers, was examined. ABA binding to the protein was weakened by H2O2 in a concentration-dependent manner. A concentration of 75 micromol/L H2O2 gave a 50% decline in ABA binding in a reaction following first-order kinetics, indicative of binding-site susceptibility to its microenvironment. We monitored the unfolding of ABAP1 using steady-state and time-resolved tryptophan fluorescence, while following the capacity of ABAP1 to bind ABA. ABA binding decreased by 50% following ABAP1 denaturation with 1 mol/L guanidine hydrochloride or 2 mol/L urea, while the maximum emission spectra (lambda emi) red shifted from 338 to 347 nm at 3.5 mol/L guanidine hydrochloride and 5 mol/L urea. However, only a slight blue shift of lambda emi was observed following either ABAP1 incubation with H2O2 or binding to (+)-ABA (physiologically active ABA). The equilibrium ABA dissociation rate accelerated in the presence of 250 micromol/L H2O2, with the half-time dissociation reduced to 8 min. A comparison of inactivation kinetics and conformational changes shows that inactivation of ABAP1 occurs before any noticeable conformational change. This suggests that the ABA binding site is highly responsive to its microenvironment and is situated in a region that is more flexible than the protein molecule as a whole. The results demonstrate that H2O2, generated by ABA treatment of aleurone layers, is sufficient to affect the ABA-binding capacity of ABAP1, suggesting that this may be another level of control of ABA signal transduction.     

Analysis of diclofenac and its metabolites by high-performance liquid chromatography: relevance of CYP2C9 genotypes in diclofenac urinary metabolic ratios

In humans, diclofenac is metabolised to 4'-hydroxy (OH), 3'-OH and 5-OH metabolites. The polymorphic CYP2C9 is involved in the metabolism of diclofenac to 4'-OH diclofenac and 3'-OH diclofenac. The aim of the present study was to develop a high-performance liquid chromatographic method to simultaneously measure diclofenac and its metabolites in urine, suitable for metabolic studies. After liquid-liquid extraction the compounds were separated in a reversed-phase column and measured by ultraviolet absorption at 282 nm. For all compounds intra-day and inter-day variations were less than 7%, and the limits of quantitation were 0.25 mg/l. No analytical interference with endogenous compounds was found. The relationship between diclofenac metabolic ratios among different CYP2C9 genotypes is reported. The CYP2C9*3/*3 subject had the highest diclofenac/4'-OH ratios. However no difference was found between CYP2C9*2/*2 and *1/*1 genotypes. The chromatographic method developed was sensitive and reliable for the measurement of diclofenac and its metabolites simultaneously in human urine, and is suitable for use in diclofenac metabolism studies.     

Inhibitory effect of nicotine and its metabolites on tolbutamide hydroxylation in rat liver microsomes

A simple HPLC/fluorescence method to detect hydroxytolbutamide (a major metabolite of the anti-diabetic drug tolbutamide) has been developed. The effects of nicotine and some of its metabolites on tolbutamide hydroxylation is described. An extraction procedure with diethyl ether was followed by isocratic HPLC analysis of tolbutamide hydroxylation with a binary mobile phase composed of 10 mM monobasic sodium phosphate in methanol (45:55, v/v, apparent pH 2.28). A detection limit of sub-nanogram amounts (0.353 ng) of hydroxytolbutamide was obtained with fluorescence detection at 226 nm for excitation and 318 nm for emission. Overall precision values for hydroxytolbutamide was determined with coefficients of variation of 1.4–4.6% when nanogram levels of the metabolite were analyzed. Differential inhibitory responses were demonstrated for tolbutamide hydroxylation to nicotine and its metabolites. Tolbutamide hydroxylation was apparently inhibited by cotinine and relatively less inhibited by nicotine. Nornicotine, however, caused very little inhibition of tolbutamide hydroxylation. The implication is that nornicotine may not share similar affinity for the substrate binding site for tolbutamide. The results also suggest that heavy smokers may experience reduction in tolbutamide metabolism. The assay system itself will be useful for future studies of tolbutamide, and possibly related sulfonylureas.     

In vitro metabolism of carbofuran by human, mouse, and rat cytochrome P450 and interactions with chlorpyrifos, testosterone, and estradiol

Carbofuran is a carbamate pesticide used in agricultural practice throughout the world. Its effect as a pesticide is due to its ability to inhibit acetylcholinesterase activity. Though carbofuran has a long history of use, there is little information available with respect to its metabolic fate and disposition in mammals. The present study was designed to investigate the comparative in vitro metabolism of carbofuran from human, rat, and mouse liver microsomes (HLM, RLM, MLM, respectively), and characterize the specific enzymes involved in such metabolism, with particular reference to human metabolism. Carbofuran is metabolized by cytochrome P450 (CYP) leading to the production of one major ring oxidation metabolite, 3-hydroxycarbofuran, and two minor metabolites. The affinity of carbofuran for CYP enzymes involved in the oxidation to 3-hydroxycarbofuran is significantly less in HLM (K_m = 1.950 mM) than in RLM (K_m = 0.210 mM), or MLM (K_m = 0.550 mM). Intrinsic clearance rate calculations indicate that HLM are 14-fold less efficient in the metabolism of carbofuran to 3-hydroxycarbofuran than RLM or MLM. A screen of 15 major human CYP isoforms for metabolic ability with respect to carbofuran metabolism demonstrated that CYP3A4 is the major isoform responsible for carbofuran oxidation in humans. CYP1A2 and 2C19 are much less active while other human CYP isoforms have minimal or no activity toward carbofuran. In contrast with the human isoforms, members of the CYP2C family in rats are likely to have a primary role in carbofuran metabolism. Normalization of HLM data with the average levels of each CYP in native HLM, indicates that carbofuran metabolism is primarily mediated by CYP3A4 (percent total normalized rate (% TNR) = 77.5), although CYP1A2 and 2C19 play ancillary roles (% TNR = 9.0 and 6.0, respectively). This is substantiated by the fact that ketoconazole, a specific inhibitor of CYP3A4, is an excellent inhibitor of 3-hydroxycarbofuran formation in HLM (IC₅₀: 0.31 μM). Chlorpyrifos, an irreversible non-competitive inhibitor of CYP3A4, inhibits the formation of 3-hydroxycarbofuran in HLM (IC₅₀: 39 μM). The use of phenotyped HLM demonstrated that individuals with high levels of CYP3A4 have the greatest potential to metabolize carbofuran to its major metabolite. The variation in carbofuran metabolism among 17 single-donor HLM samples is over 5-fold and the best correlation between CYP isoform activity and carbofuran metabolism was observed with CYP3A4 (r² = 0.96). The interaction of carbofuran and the endogenous CYP3A4 substrates, testosterone and estradiol, were also investigated. Testosterone metabolism was activated by carbofuran in HLM and CYP3A4, however, less activation was observed for carbofuran metabolism by testosterone in HLM and CYP3A4. No interactions between carbofuran and estradiol metabolism were observed.     

Characterization of metabolites and cytochrome P450 isoforms involved in the microsomal metabolism of aconitine

Aconitine, a major Aconitum alkaloid, is well known for its high toxicity that induces severe arrhythmias leading to death. The current study investigated the metabolism of aconitine and the effects of selective cytochrome P450 (CYP) inhibitors on the metabolism of aconitine in rat liver microsomes. The metabolites were separated and assayed by liquid chromatography-ion trap mass spectrometry (LC/MS(n)) and further identified by comparison of their mass spectra and chromatographic behaviors with reference substances. Various selective inhibitors of CYP were used to identify the isoforms of CYP, that involved in the metabolism of aconitine. A total of at least six metabolites were found and characterized in rat liver microsomal incubations. Result showed that the inhibitor of CYP 3A had an inhibitory effect on aconitine metabolism in a concentration-dependant manner, the inhibitor of CYP1A1/2 had a modest inhibitory effect, whereas inhibitors of CYP2B1/2, 2D and 2E1 had no obvious inhibitory effects on aconitine metabolism. Aconitine might be metabolized by CYP 3A and CYP1A1/2 isoforms in rat liver microsome.     

Liquid chromatographic method for the simultaneous determination of caffeine and fourteen caffeine metabolites in urine

An HPLC method has been developed for the separation and the determination of caffeine and its metabolites in urine samples using a one extraction–analysis run and UV detection. The compounds were extracted by liquid–liquid extraction using chloroform–isopropylalcohol (85:15, v/v). Chromatographic separation was accomplished on an ODS analytical column with a mobile phase containing 0.05% acetic acid/methylalcohol (92.5:7.5, v/v). Compounds were monitored at 280 nm. The method was validated for the determination of AFMU, 1X, 1U, 17X and 17U caffeine metabolites required to assess the metabolic activity of the enzymes subject to in vivo caffeine testing. The validated assay was applied to urine samples from ten healthy volunteers. The method was proved to be suitable to assess simultaneously the enzymatic activity of cytochrome P450 CYP1A2 and CYP2A6, as well as N-acetyltransferase and xanthine oxidase.     

Conversion of vitamin D3 to 1alpha,25-dihydroxyvitamin D3 by Streptomyces griseolus cytochrome P450SU-1

Streptomyces griseolus cytochrome P450SU-1 (CYP105A1) was expressed in Escherichia coli at a level of 1.0 micromol/L culture and purified with a specific content of 18.0 nmol/mg protein. Enzymatic studies revealed that CYP105A1 had 25-hydroxylation activity towards vitamin D2 and vitamin D3. Surprisingly, CYP105A1 also showed 1alpha-hydroxylation activity towards 25(OH)D3. As mammalian mitochondrial CYP27A1 catalyzes a similar two-step hydroxylation towards vitamin D3, the enzymatic properties of CYP105A1 were compared with those of human CYP27A1. The major metabolite of vitamin D2 by CYP105A1 was 25(OH)D2, while the major metabolites by CYP27A1 were both 24(OH)D2 and 27(OH)D2. These results suggest that CYP105A1 recognizes both vitamin D2 and vitamin D3 in a similar manner, while CYP27A1 does not. The Km values of CYP105A1 for vitamin D2 25-hydroxylation, vitamin D3 25-hydroxylation, and 25-hydroxyvitamin D3 1alpha-hydroxylation were 0.59, 0.54, and 0.91 microM, respectively, suggesting a high affinity of CYP105A1 for these substrates.     

Combination of docking,molecular dynamics and quantum mechanical calculations for metabolism prediction of 3,4-methylenedioxybenzoyl-2-thienylhydrazone

In modern drug discovery process, ADME/Tox properties should be determined as early as possible in the test cascade to allow a timely assessment of their property profiles. To help medicinal chemists in designing new compounds with improved pharmacokinetics, the knowledge of the soft spot position or the site of metabolism (SOM) is needed. In silico methods based on docking, molecular dynamics and quantum chemical calculations can bring us closer to understand drug metabolism and predict drug–drug interactions. We report herein on a combined methodology to explore the site of metabolism prediction of a new cardioactive drug prototype, LASSBio-294 (1), using MetaPrint2D to predict the most likely metabolites, combined with structure-based tools using docking, molecular dynamics and quantum mechanical calculations to predict the binding of the substrate to CYP2C9 enzyme, to estimate the binding free energy and to study the energy profiles for the oxidation of (1). Additionally, the computational study was correlated with a metabolic fingerprint profiling using LC-MS analysis. The results obtained using the computational methods gave valuable information about the probable metabolites of (1) (qualitatively) and also about the important interactions of this lead compound with the amino acid residues of the active site of CYP2C9. Moreover, using a combination of different levels of theory sheds light on the understanding of (1) metabolism by CYP2C9 and its mechanisms. The metabolic fingerprint profiling of (1) has shown that the metabolites founded in highest concentration in different species were metabolites M1, M2 and M3, whereas M8 was found to be a minor metabolite. Therefore, our computational study allowed a qualitative prediction for the metabolism of (1). The approach presented here has afforded new opportunities to improve metabolite identification strategies, mediated by not only CYP2C9 but also other CYP450 family enzymes.     

Chiral reversed phase high-performance liquid chromatography for determining propranolol enantiomers in transgenic Chinese hamster CHL cell lines expressing human cytochrome P450

An enantioselective assay for S-(-)- and R-(+)-propranolol in transgenic Chinese hamster CHL cell lines, expressing human cytochrome P450 (CYP), was developed. The method involves extraction of propranolol from the S(9) incubates, using S-(+)-propafenone as internal standard, chiral derivatization with 2,3,4,6-tetra-O-beta-D-glucopranosyl isothiocyanate and quantitation by reversed phase high-performance liquid chromatography system with UV detection (lambda=220 nm). A baseline separation of propranolol enantiomers was achieved on a 5-microm reverse-phase ODS column, with a mixture of methanol/water/glacial acetic acid (67:33:0.05, v/v) as mobile phase. The assay is linear from 5 to 500 microM for each enantiomer. The analytical method affords average recoveries of 99.2% and 98.8% for S-(-)- and R-(+)-propranolol, respectively. The limit of quantitation for the method is 5 microM for both S-(-)- and R-(+)-propranolol. The reproducibility of the assay is satisfactory (RSD < 10%). The method allowed study of the depletion of S-(-)- and R-(+)-propranolol in transgenic Chinese hamster CHL cell lines expressing CYP3A4, CYP2C18 and CYP2C9.     

Determination of ibuprofen in pharmaceutical formulations using time-resolved terbium-sensitized luminescence.

A sensitive and specific luminescence method for the determination of ibuprofen (IB) in pharmaceutical formulations in aqueous solution is described. The method is based on the luminescence sensitization of terbium (Tb(3+)) by formation of ternary complex with IB in the presence of tri-n-octylphosphine oxide (TOPO) and Tween-20 as surfactant. The luminescence signal for Tb-IB-TOPO is monitored at lambda(ex) = 229 nm and lambda(em) = 545 nm. Optimum conditions for the formation of the complex in aqueous system, were 16 mmol/L TRIS buffer, pH 5.7, TOPO 200 micromol/L and 15 micromol/L of Tb(3+), which allows for the determination of 9.7 x 10(-7) - 9.7 x 10(-6) mol/L IB with a detection limit of 1.2 x 10(-7) mol/L. The relative standard deviations of the method were <1.4%, indicating excellent reproducibility. The proposed method was successfully applied for the assays of IB in pharmaceutical formulations with average recoveries of 100.3-102.5%.     

Linkage between the CYP2C8 and CYP2C9 genetic polymorphisms

Cytochrome P450 (CYP) 2C8 and 2C9 are polymorphic enzymes. The CYP2C8*3 and CYP2C9*2 are the major variant alleles in Caucasian populations. The enzymes encoded by these variant alleles have impaired function for the metabolism of several drug substrates. In the present study 1468 subjects that were used as population-based controls in the Stockholm Heart Epidemiology Program (SHEEP) were genotyped by allelic discrimination using a 5'-nuclease assay for CYP2C8*1, 2C8*3, 2C9*1, 2C9*2, and 2C9*3 variant alleles in which the frequencies appeared to be 0.91, 0.095, 0.83, 0.11, and 0.066, respectively. Approximately, 96% of the subjects with CYP2C8*3 allele also carried a CYP2C9*2 and 85% of the subjects that had CYP2C9*2 variant also carried a CYP2C8*3. The number of subjects carrying both of the CYP2C8*1*3 and CYP2C9*1*2 was 4.5-fold higher than expected. This strong association may be of importance especially for the metabolism of common substrates of CYP2C8 and CYP2C9 like arachidonic acid that produces physiologically active metabolites.     

Differences in plasma homocysteine levels between Zucker fatty and Zucker diabetic fatty rats following 3 weeks oral administration of organic vanadium compounds

PURPOSE: Recently, our laboratory group has reported that rats with Type 1 diabetes have decreased plasma homocysteine and cysteine levels compared to non-diabetic controls and that organic vanadium treatment increased plasma homocysteine concentrations to non-diabetic concentrations. However, to date, no studies have been done investigating the effects of organic vanadium compounds on plasma homocysteine and its metabolites in Type 2 diabetic animal model. These studies examined the effect of organic vanadium compounds [bis(maltolato)oxovanadium(IV) and bis(ethylmaltolato)oxovanadium(IV); BMOV and BEOV] administered orally on plasma concentrations of homocysteine and its metabolites (cysteine and cysteinylglycine) in lean, Zucker fatty (ZF) and Zucker diabetic fatty (ZDF) rats. ZF rats are a model of pre-diabetic Type 2 diabetes characterized by hyperinsulinemia and normoglycemia. The ZDF rat is a model of Type 2 diabetes characterized by relative hypoinsulinemia and hyperglycemia. METHODS: Zucker lean and ZF rats received BMOV in the drinking water at a dose of 0.19 +/- 0.02 mmol/kg/day. Lean and ZDF rats received BEOV by oral gavage daily at dose of 0.1 mmol/kg. The treatment period for both studies was 21 days. At termination, animals were fasted overnight (approximately 16 h) and blood samples were collected by cardiac puncture for determination of plasma glucose, insulin and homocysteine levels. Plasma homocysteine and its metabolites levels were determined using high-pressure liquid chromatography. Plasma glucose was determined using a Glucose Analyzer 2. Plasma insulin levels were determined by radioimmunoassay. Plasma triglycerides were determined by an enzymatic assay methodology. RESULTS: ZF (n = 4) and ZDF (n = 10) rats had significantly lower plasma homocysteine as compared to their respective lean groups (ZF 0.78 +/- 0.1 micromol/L vs. Zucker lean 2.19 +/- 0.7 micromol/L; ZDF 1.71 +/- 0.2 micromol/L vs. Zucker lean 3.02 +/- 0.3 micromol/L; p < 0.05). BMOV treatment in ZF rats restored plasma homocysteine levels to those observed in lean untreated rats (ZF treated: 2.04 +/- 0.2 micromol/L; lean 2.19 +/- 0.7 micromol/L). There was a modest effect of BMOV treatment on plasma glucose levels in ZF rats. BEOV treatment significantly decreased the elevated plasma glucose levels in the ZDF rats (lean 7.9 +/- 0.1 mmol/L; lean + vanadium 7.7 +/- 0.2 mmol/L; ZDF 29.9 +/- 0.4 mmol/L; ZDF + vanadium 17.4 +/- 0.3 mmol/L, p < 0.05). Organic vanadium treatment reduced cysteine levels in both ZF and ZDF rats. No differences in total plasma cysteinylglycine concentrations were observed. CONCLUSION: Plasma homocysteine levels are significantly reduced in a pre-diabetic model of Type 2 diabetes, which was restored to lean levels upon vanadium treatment; however, this restoration of plasma homocysteine levels was not seen in ZDF Type 2 diabetic rats following vanadium treatment. In the latter case vanadium treatment may not have totally overcome the insulin resistance seen in these animals.     

Identification and characterization of a bacterial cytochrome P450 for the metabolism of diclofenac

The bacterium Actinoplanes sp. ATCC 53771 is known to perform drug metabolism of several xenobiotics similarly to humans. We identified a cytochrome P450 enzyme from this strain, CYP107E4, and expressed it in Escherichia coli using the pET101 vector. The purified enzyme showed the characteristic reduced-CO difference spectra with a peak at 450 nm, indicating the protein is produced in the active form with proper heme incorporation. The CYP107E4 enzyme was found to bind the drug diclofenac. Using redox enzymes from spinach, the reconstituted system is able to produce hydroxylated metabolites of diclofenac. Production of the human 4′-hydroxydiclofenac metabolite by CYP107E4 was confirmed, and a second hydroxylated metabolite was also produced.