Rat Liver Microsomal Fraction Research Articles

LC-MS/MS identification of the principal in vitro and in vivo phase I metabolites of the novel thiosemicarbazone anti-cancer drug, Bp4eT

The iron chelator, 2-benzoylpyridine-4-ethyl-3-thiosemicarbazone (Bp4eT), was identified as a lead compound of the 2-benzoylpyridine thiosemicarbazone series, which were designed as potential anti-cancer agents. This ligand has been shown to possess potent anti-proliferative activity with a highly selective mechanism of action. However, further progress in the development of this compound requires data regarding its metabolism in mammals. The aim of this study was to identify the main in vitro and in vivo phase I metabolites of Bp4eT using liquid chromatography tandem mass spectrometry (LC-MS/MS). Two metabolites were detected after incubation of this drug with rat and human liver microsomal fractions. Based on LC-MS(n) analysis, the metabolites were demonstrated to be 2-benzoylpyridine-4-ethyl-3-semicarbazone and N (3)-ethyl-N (1)-[phenyl(pyridin-2-yl)methylene]formamidrazone, with both resulting from the oxidation of the thiocarbonyl group. The identity of these metabolites was further shown by LC-MS/MS analysis of these latter compounds which were prepared by oxidation of Bp4eT with hydrogen peroxide and their structures confirmed by nuclear magnetic resonance and infrared spectra. Both the semicarbazone and the amidrazone metabolites were detected in plasma, urine, and feces after i.v. administration of Bp4eT to rats. In addition, another metabolite that could correspond to hydroxylated amidrazone was found in vivo. Thus, oxidative pathways play a major role in the phase I metabolism of this promising anti-tumor agent. The outcomes of this study will be further utilized for: (1) the development and validation of the analytical method for the quantification of Bp4eT and its metabolites in biological materials; (2) to design pharmacokinetic experiments; and to (3) evaluate the potential contribution of the individual metabolites to the pharmacodynamics/toxico-dynamics of this novel anti-proliferative agent.

Stereoselective liquid chromatographic determination of 1′‐oxobufuralol and 1′‐hydroxybufuralol in rat liver microsomal fraction using hollow‐fiber liquid‐phase microextraction for sample preparation

A three-phase hollow-fiber liquid-phase microextraction (HF-LPME) method for the stereoselective determination of bufuralol metabolites 1'-oxobufuralol (1'-Oxo-BF) and 1'-hydroxybufuralol (1'-OH-BF) in microsomal preparations is described for the first time. The HPLC analysis was carried out using a Chiralcel OD-H column with hexane/2-propanol/methanol (97.5:2.0:0.5, v/v/v) plus 0.5% diethylamine as the mobile phase, and UV detection at 248 and 273 nm. The HF-LPME optimized conditions involved: n-octanol as the organic solvent, 0.2 mol/L acetic acid as the acceptor phase, donor phase pH adjusted to 13, sample agitation at 1500 rpm and extraction for 30 min. By using this extraction procedure, the recovery rates were in the range of 63-69%. The method was linear over the concentration range of 100-5000 ng/mL for each enantiomer of 1'-Oxo-BF (r>0.9978) and of 100-2500 ng/mL for each stereoisomer of 1'-OH-BF (r>0.9957). The quantification limits were 100 ng/mL for all analytes. The validated method was used to assess the in vitro biotransformation of bufuralol using rat liver microsomal fraction that demonstrated predominant formation of (S)-1'-Oxo-BF and (R,R)-1'-OH-BF.

The Imidazoacridinone Antitumor Drug, C-1311, Is Metabolized by Flavin Monooxygenases but Not by Cytochrome P450s

5-Diethylaminoethylamino-8-hydroxyimidazoacridinone (C-1311) is an antitumor agent that is also active against autoimmune diseases. The intention of the present studies was to elucidate the role of selected liver enzymes in metabolism of C-1311 and the less active 8-methyl derivative, 5-diethylaminoethylamino-8-methoxyimidazoacridinone (C-1330). Compounds were incubated with rat liver microsomal fraction, with a set of 16 human liver protein samples, and with human recombinant isoenzymes of cytochrome P450, flavin monooxygenases (FMO), and UDP-glucuronosyltransferase (UGT). Our results showed that C-1311 and C-1330 were metabolized with human liver microsomal enzymes but not with any tested human recombinant cytochromes P450 (P450s). Two of these, CYP1A2 and CYP3A4, were inhibited by both compounds. In addition, results of C-1311 elimination from hepatic reductase-null mice, in which liver NADPH-P450 oxidoreductase has been deleted indicated that liver P450s were slightly engaged in drug transformation. In contrast, both compounds were good substrates for human recombinant FMO1 and FMO3 but not for FMO5. The product of FMO metabolism, P(FMO), which is identified as an N-oxide derivative, was identical to P3(R) of liver microsomes. P3(R) was observed even in the presence of the P450 inhibitor, 1-aminobenzotriazole, and it disappeared after heating. Therefore, FMO enzymes could be responsible for microsomal metabolism to P3(R) = P(FMO). Glucuronidation on the 8-hydroxyl group of C-1311 was observed with liver microsomes supported by UDP-glucuronic acid and with recombinant UGT1A1, but it was not the case with UGT2B7. Summing up, we showed that, whereas liver P450 isoenzymes were involved in the metabolism of C-1311 to a limited extent, FMO plays a significant role in the microsomal transformations of this compound, which is also a specific substrate of UGT1A1.

In vitro characterization of rosiglitazone metabolites and determination of the kinetic parameters employing rat liver microsomal fraction

Rosiglitazone (RSG), a thiazolidinedione antidiabetic drug, is metabolized by CYP450 enzymes into two main metabolites: N-desmethyl rosiglitazone (N-Dm-R) and ρ-hydroxy rosiglitazone (ρ-OH-R). In humans, CYP2C8 appears to have a major role in RSG metabolism. On the other hand, the in vitro metabolism of RSG in animals has not been described in literature yet. Based on these concerns, the kinetic metabolism study of RSG using rat liver microsomal fraction is described for the first time. Maximum velocity (V (max)) values of 87.29 and 51.09 nmol/min/mg protein were observed for N-Dm-R and ρ-OH-R, respectively. Michaelis-Menten constant (K(m)) values were of 58.12 and 78.52 μM for N-Dm-R and ρ-OH-R, respectively. Therefore, these results demonstrated that this in vitro metabolism model presents the capacity of forming higher levels of N-Dm-R than of ρ-OH-R, which also happens in humans. Three other metabolites were identified employing mass spectrometry detection under positive electrospray ionization: ortho-hydroxy-rosiglitazone (ο-OH-R) and two isomers of N-desmethyl hydroxy-rosiglitazone. These metabolites have also been observed in humans. The results observed in this study indicate that rats could be a satisfactory model for RSG metabolism.

Human Multiple Inositol Polyphosphate Phosphatase as Apoptosis Biomarker

The multiple inositol polyphosphate phosphatase (MINPP) which regulates cellular levels of inositol phosphates, is located on chromosome 10q23 in human cells and is a member of histidine phosphatases family. In this study, we have determined the antigenicity of MINPP. We proposed different possible peptides and designed the experimental methodology to produce MINPP polyclonal antibodies. To further elucidate the physiological role of MINPP we have characterized the human MINPP antibodies using direct western immunoblotting of bacteria (E. coli), yeast (Saccharomyces cerevisae) expressing MINPP. SDS‐PAGE identified 58 KDa protein band using mammalian pancreatic cancer cell lysates (Panc1) and homogenate from rat liver and microsomal fraction of rat liver and kidney after immunoprecipitations with MINPP antibodies. The MINPP enzyme activity was determined by two methods. First, inositol phosphates separation by column chromatography using the microsomal fraction of rat liver and kidney, total cell lysates from rat liver in a scintillation counter using tritiated‐inositol (3H‐inositol). Second, a customized colorimetric method was developed without any radio‐active 3H ‐inositol using 96 wells plate reader. Using different apoptosis inducers, we have seen that MINPP expression increased in programmed cell death in a dose dependent manner. Supported by University of Arkansas at Little Rock.

Hollow fiber-based liquid-phase microextraction (HF-LPME) of isradipine and its main metabolite followed by chiral HPLC analysis: application to an in vitro biotransformation study

An enantioselective liquid chromatographic method using two-phase hollow fiber liquid-phase microextraction (HF-LPME-HPLC) was developed for the determination of isradipine (ISR) enantiomers and its main metabolite (pyridine derivative of isradipine, PDI) in microsomal fractions isolated from rat liver. The analytes were extracted from 1 mL of microsomal medium using a two-phase HF-LPME procedure with hexyl acetate as the acceptor phase, 30 min of extraction, and sample agitation at 1,500 rpm. For the first time, ISR enantiomers and PDI were resolved. For this separation, a Chiralpak(®) AD column with hexane/2-propanol/ethanol (94:04:02, v/v/v) as the mobile phase at a flow rate of 1.5 mL min(-1) was used. The column was kept at 23 ± 2 °C. The drug and metabolite detection was performed at 325 nm and the internal standard oxybutynin was detected at 225 nm. The recoveries were 23% for PDI and 19% for each ISR enantiomer. The method presented quantification limits (LOQ) of 50 ng mL(-1) and was linear over the concentration range of 50-5,000 and 50-2,500 ng mL(-1) for PDI and each ISR enantiomer, respectively. The validated method was employed to an in vitro biotransformation study of ISR using rat liver microsomal fraction showing that (+)-(S)-ISR is preferentially biotransformed.

Gas-phase photocatalytic degradation and detoxification of o-toluidine: Degradation mechanism and Salmonella mutagenicity assessment of mixed gaseous intermediates

The photocatalytic degradation of toluidine over titanium oxide (TiO 2) thin films under UV irradiation was investigated. The degradation efficiency of 98.7% was obtained for a toluidine concentration of about 4500 μg L −1 and illumination of 240 min. The degradation intermediates produced during photocatalytic oxidation were identified using Fourier transform-infrared spectrometry (FTIR) and gas chromatography–mass spectrometry (GC–MS). Only a small amount of intermediates, including phenol and toluene, were found in the gas phase. Many other trace amount intermediates, such as 2-hydroxybenzaldehyde, 2-nitrobenzaldehyde, 2-hydroxybenzenemethanol, 2-hydroxybenzoic acid, phenol etc., were detected on the TiO 2 surface. An Ames assay of the Salmonella typhimurium strains TA98 and TA100 was employed to evaluate the mutagenicity of toluidine and its gaseous photocatalytic degradation intermediates. With or without rat liver microsomal fraction (S9 mix) activation, neither toluidine nor its gaseous intermediates presented mutagenic activity against strains TA98 (±S9) and TA100 (−S9) at all tested doses. Toluidine, however, can induce a weak positive response to the TA100 strain with an S9 mix at doses as high as 4000 μg plate −1. An increase of revertants per plate was obtained after 30 min photocatalysis in the TA100 strain with S9 mix. As reaction time further increased, photocatalytic technology exhibited the ability to completely and efficiently detoxify toluidine. Both our chemical analysis and toxic evaluation indicate that all mutagenic intermediates in the gas can be completely eliminated within 240 min, which further suggests that photocatalytic technology is an effective approach for degrading aromatic amines.

Simultaneous determination of rosiglitazone and its metabolites in rat liver microsomal fraction using hollow‐fiber liquid‐phase microextraction for sample preparation

A three-phase hollow-fiber liquid-phase microextraction method for the analysis of rosiglitazone and its metabolites N-desmethyl rosiglitazone and ρ-hydroxy rosiglitazone in microsomal preparations is described for the first time. The drug and metabolites HPLC determination was carried out using an X-Terra RP-18 column, at 22°C. The mobile phase was composed of water, acetonitrile and acetic acid (85:15:0.5, v/v/v) and the detection was performed at 245 nm. The hollow-fiber liquid-phase microextraction procedure was optimized using multifactorial experiments and the following optimal condition was established: sample agitation at 1750 rpm, extraction for 30 min, hydrochloric acid 0.01 mol/L as acceptor phase, 1-octanol as organic phase, and donor phase pH adjustment to 8.0. The recovery rates, obtained by using 1 mL of microsomal preparation, were 47-70%. The method presented LOQs of 50 ng/mL and it was linear over the concentration range of 50-6000 ng/mL, with correlation coefficients (r) higher than 0.9960, for all analytes. The validated method was employed to study the in vitro biotransformation of rosiglitazone using rat liver microsomal fraction.

An acellular assay to assess the genotoxicity of complex mixtures of organic pollutants bound on size segregated aerosol. Part I: DNA adducts

An acellular assay consisting of calf thymus DNA with/without rat liver microsomal S9 fraction was used to study the genotoxicity of complex mixtures of organic air pollutants bound to size segregated aerosols by means of DNA adduct analysis. We compared the genotoxicity of the organic extracts (EOMs) from three size fractions of aerosol ranging from 0.17 μm to 10 μm that were collected by high volume cascade impactors in four localities of the Czech Republic differing in the extent of the environmental pollution: (1) small village in proximity of a strip mine, (2) highway, (3) city center of Prague and (4) background station. The total DNA adduct levels induced by 100 μg/ml of EOMs were analyzed by 32P-postlabelling analysis with a nuclease P1 method for adduct enrichment. The main finding of the study was most of the observed genotoxicity was connected with a fine particulate matter fraction (<1 μm). The concentrations of carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) in EOMs indicate that fine fractions (0.5–1 μm) bound the highest amount of c-PAHs in all aerosol sampling sites, which might be related to the higher specific surface of this fraction as compared with a course fraction (1–10 μm) and higher mass as compared with a condensational fraction (0.17–0.5 μm). As for aerosol mass, both fine and condensational fractions are effective carriers of c-PAHs. Similarly, the DNA adduct levels per m 3 of air were highest for the fine fraction, while the condensational fraction (strip mine site and city center) revealed the highest DNA adduct levels in cases where aerosol mass is taken into consideration. A strong correlation was found between the c-PAHs and DNA adduct levels induced by EOMs in all the localities and for various size fractions ( R 2 = 0.98, p < 0.001). It may be concluded that the analysis of total DNA adducts induced in an acellular assay with/without metabolic activation represents a relatively simple method to assess the genotoxic potential of various complex mixtures.

Comparison of the antioxidant effects of synovial fluid from equine metacarpophalangeal joints with those of hyaluronic acid and chondroitin sulfate

To evaluate the antioxidant effects of synovial fluid (SF) pooled from metacarpophalangeal joints of healthy horses and horses with various pathological conditions, and to compare then with the antioxidant effects of hyaluronic acid (HA) and chondroitin sulfate (CS). SF from 1 metacarpophalangeal joint was obtained from 42 horses immediately after humane slaughter. Samples were classified into 3 groups on the basis of origin: healthy joints or joints with chronically damaged cartilage or vascularly congested synovial membranes as detected via macroscopic evaluation. Antioxidant effects were evaluated by use if rat liver microsomal fractions treated with Fe(3+)-ascorbate as a free radical generator system leading to oxidative stress. Amounts of thiobarbituric-reactive substances and glutathione transferase (GSH-T) conjugation activity were measured. SF from healthy and chronically damaged joints inhibited microsomal lipid peroxidation, whereas SF from joints with congested synovial membranes had only a slight effect. Hyaluronic acid and CS did not inhibit microsomal lipid peroxidation. Moreover, GSH-T activity was detected in all SF samples as well as HA and CS protected rat microsomal GSH-T activity against oxidative damage. Only SF samples from joints with congested synovial membranes protected microsomal thiols against oxidation, an effect also evident with HA and CS. The antioxidant mechanisms associated with the response to metacarpophalangeal joint damage in horses appeared to act on different targets, depending on whether the damage was acute or chronic.

Assay of labile estrogen o-quinones, potent carcinogenic molecular species, by high performance liquid chromatography–electrospray ionization tandem mass spectrometry with phenazine derivatization

A sensitive and selective assay method for labile estrogen o-quinones, estrone (E 1)-2,3-quinone (Q), E 1-3,4-Q, estradiol (E 2)-2,3-Q and E 2-3,4-Q, based on the use of phenazine (Phz) derivatization with o-phenylenediamine and high performance liquid chromatography–electrospray ionization tandem mass spectrometry (HPLC–ESI-MS/MS) was described. The Phz derivatives of four estrogen o-quinones were purified by solid phase extraction and analyzed by HPLC–ESI-MS/MS. The protonated molecule was observed as a base peak for all Phz derivatives in their ESI-mass spectra (positive mode). In multiple reaction monitoring, the transition from [M+H] + to m/ z 231 was chosen for quantification. Calibration curves for the o-quinones were obtained using standard catechol estrogens after sodium metaperiodate treatment and Phz derivatization. Using this method, these four estrogen o-quinones were analyzed with the limit of quantification of 5 ng/ml in acetonitrile (MeCN)–blank matrix (1:4, v/v), respectively, on a basis of the weight of catechol estrogens. Assay accuracy and precision for four estrogen o-quinones were 89.6–113.0% and 3.1–12.6% (5, 125 and 2000 ng/ml in MeCN–blank matrix). Applications of this method enabled to determine the catalytic activities on hydroxylation and subsequent oxidation of E 1 and E 2 of Mushroom tyrosinase and rat liver microsomal fraction. It was confirmed by this method that tyrosinase exhibited 2- and 4-hydroxylation and further oxidation activities for catechols in the ring-A of estrogens. Whereas rat liver microsomal fraction possessed only 2- and 4-hydroxylation activities, and further oxidation activity for catechol estrogens was low.

In-vitro and in-vivo pharmacokinetic interactions of amprenavir, an HIV protease inhibitor, with other current HIV protease inhibitors in rats.

The drug interactions between a new human immune deficiency virus (HIV) protease inhibitor, amprenavir, and four other protease inhibitors which are presently used have been characterized by in-vitro metabolic studies using rat liver microsomal fractions and in-vivo oral administration studies. The metabolic clearance rates (Vmax/Km) of amprenavir, saquinavir, indinavir and nelfinavir in rat liver microsomes were 50.67+/- 3.77, 170.88 +/- 15.34, 73.01 +/- 2.76 and 126.06 +/- 6.23 microLmin(-1) (mg protein)(-1), respectively, and the degree of metabolicclearance was in the order of saquinavir > nelfinavir > indinavir > amprenavir > ritonavir. The inhibition constants (Ki) of ritonavir for amprenavir, indinavir, nelfinavir and saquinavir were 2.29, 0.95, 1.01 and 1.64 microM, respectively, and that of indinavir for amprenavir was 0.67, indicating that amprenavir metabolism in rat liver microsomes was strongly inhibited by indinavir. The Ki values of amprenavir for indinavir, nelfinavir and saquinavir were 7.41, 2.13 and 16.11 microM, respectively, and those of nelfinavirand saquinavirforamprenavirwere 9.15 and 34.57 microM, respectively. The area under the concentration vs time curve (AUC) of amprenavir after oral co-administration with saquinavir, indinavir, nelfinavir or ritonavir (20 mg kg(-1) for each oral dose in rats) was increased by 1.6-, 2.0-, 1.2- and 9.1-fold, respectively. The AUC values of saquinavir, indinavir and nelfinavir by co-administration with amprenavir showed about 7.3-, 1.3-, and 7.9-fold increase, respectively. These observations suggested that the oral bioavailability of amprenavir was not so affected by co-administration with saquinavir, nelfinavir or indinavir, compared with ritonavir, whereas amprenavir markedly affected the oral bioavailability of saquinavir and nelfinavir. In addition, the in-vivo effects after co-administration of two kinds of HIV protease inhibitors cannot always be predicted from in-vitro data, suggesting the presence of other interaction processes besides metabolism in the liver. However, these results provide useful information for the treatment of AIDS patients when they receive a combination therapy with two kinds of HIV protease inhibitor.

Pharmacokinetic Interactions between HIV-1 Protease Inhibitors in Rats: Study on Combinations of Two Kinds of HIV-1 Protease Inhibitors

The drug interactions between four human immune deficiency virus (HIV-1) protease inhibitors have been characterized by in-vitro metabolic studies using rat liver microsomal fractions and in-vivo oral administration. In this study, a new HPLC analytical method developed by us was used for the simultaneous determination of saquinavir and nelfinavir in rat plasma and microsomes. The metabolic clearance rates (Vmax/Km) of saquinavir, nelfinavir, and indinavir were 170.9 +/- 10.9, 126.1 +/- 4-4, and 73.0 +/- 2.0 microL min(-1) (mg protein)(-1), respectively. Ritonavir was the strongest inhibitor with inhibition constants (Ki) of 1.64 microM for saquinavir, 0.95 microM for indinavir, and 1.01 microM for nelfinavir. Nelfinavir was the second strongest inhibitor with Ki's of 2.35 microM for saquinavir and 2.14 microM for indinavir. Indinavir was the third strongest inhibitor with Ki's of 2.76 microM for nelfinavir and 3.55 microM for saquinavir. Saquinavir was the weakest inhibitor for the other three HIV- 1 protease inhibitors. After oral co-administration in combination with another HIV-1 protease inhibitor, the AUCs of saquinavir, indinavir, and nelfinavir were significantly increased compared with mono-treatment. The AUCs of saquinavir were increased about 10.1-, 3.1- and 45.9-fold in the presence of indinavir, nelfinavir and ritonavir, respectively. The AUCs of indinavir were increased about 6.8-, 5.9- and 9.4-fold in the presence of nelfinavir, saquinavir and ritonavir, respectively. The AUCs of nelfinavir were increased about 2.2-, 6.6- and 8.5-fold in the presence of indinavir, saquinavir and ritonavir, respectively. The in-vivo effects observed after co-administration of two kinds of HIV-1 protease inhibitor were not always expected from in-vitro data, suggesting the presence of other interaction processes besides metabolism in the liver. These results provide useful information for the treatment of AIDS patients receiving combination therapy with two HIV-1 protease inhibitors.

In Vitro Hepatic Biotransformation of Aspalathin and Nothofagin, Dihydrochalcones of Rooibos (Aspalathus linearis), and Assessment of Metabolite Antioxidant Activity

Aspalathin (2',3,4,4',6'-pentahydroxy-3'-C-beta-d-glucopyranosyldihydrochalcone) is the major flavonoid present in the South African herbal tea rooibos. In vitro metabolism of aspalathin and a structural analogue nothofagin, lacking the A ring catechol group, was investigated by monitoring the formation of glucuronyl and sulfate conjugates using Aroclor 1254 induced and uninduced rat liver microsomal and cytosolic subcellular fractions. Following glucuronidation of both aspalathin and nothofagin, HPLC-DAD, LC-MS, and LC-MS/MS analyses indicated the presence of two metabolites: one major and one minor. Only one aspalathin metabolite was obtained after sulfation, while no metabolites were observed for nothofagin. Two likely sites of conjugation for aspalathin are 4-OH or 3-OH on the A-ring. For nothofagin, the 4-OH (A-ring) and 6'-OH (B-ring) seem to be involved. The glucuronyl conjugates of aspalathin lack any radical scavenging properties in online postcolumn DPPH radical and ABTS radical cation assays. Deconjugation assays utilizing glucuronidase and sulfatase resulted in the disappearance of the metabolites, with the concomitant formation of the unconjugated form in the case of the glucuronidated product. The balance between conjugated and unconjugated forms of aspalathin could have important implications regarding its role in affecting oxidative status in intra- and extracellular environments in vivo.

Effects of bile acids on rat hepatic microsomal type I 11β-hydroxysteroid dehydrogenase

The aim of this study was to investigate the effect of various bile acids on hepatic type I 11β-hydroxysteroid dehydrogenase (11β-HSD1) activity in vitro. The rat liver microsome fraction was prepared and 11β-HSD1 activity was assayed using cortisol and corticosterone as substrates for the enzyme reaction. The substrate and various concentrations of bile acids were added to the assay mixture. After incubation, the products were extracted and analyzed using high-performance liquid chromatography. All bile acids tested except deoxycholic acid and 7-keto bile acids inhibited the 11β-HSD1 enzyme reaction to some degree. Ursodeoxycholic acid inhibited the activity less than cholic, chenodeoxycholic, and lithocholic acids. Deoxycholic acid and 7-keto bile acids did not inhibit, but enhanced the enzyme activity. Inhibitions of dehydrogenation by corticosterone were weaker than those by cortisol. Kinetic analysis revealed that the inhibition of 11β-HSD1 was competitive. The inhibition of 11β-HSD1 by bile acids depended on the three-dimensional structural difference in the steroid rings and the presence of the 7α-hydroxy molecule of the bile acids was important for the inhibition of rat hepatic 11β-HSD1 enzyme activity. These results suggest that bile acid administration might modulate 11β-HSD1 enzyme activity.

Chiral HPLC analysis of venlafaxine metabolites in rat liver microsomal preparations after LPME extraction and application to an in vitro biotransformation study

A three-phase LPME (liquid-phase microextraction) method for the enantioselective analysis of venlafaxine (VF) metabolites (O-desmethylvenlafaxine (ODV) and N-desmethylvenlafaxine (NDV) in microsomal preparations is described for the first time. The assay involves the chiral HPLC separation of drug and metabolites using a Chiralpak AD column under normal-phase mode of elution and detection at 230 nm. The LPME procedure was optimized using multifactorial experiments and the following optimal condition was established: sample agitation at 1,750 rpm, 20 min of extraction, acetic acid 0.1 mol/L as acceptor phase, 1-octanol as organic phase and donor phase pH adjustment to 10.0. Under these conditions, the mean recoveries were 41% and 42% for (-)-(R)-ODV and (+)-(S)-ODV, respectively, and 47% and 48% for (-)-(R)-NDV and (+)-(S)-NDV, respectively. The method presented quantification limits of 200 ng/mL and it was linear over the concentration range of 200-5,000 ng/mL for all analytes. The validated method was employed to study the in vitro biotransformation of VF using rat liver microsomal fraction. The results demonstrated the enantioselective biotransformation of VF.

Chemical synthesis of bile acid acyl-adenylates and formation by a rat liver microsomal fraction

In mammals, unconjugated bile acids formed in the intestine by bacterial deconjugation are reconjugated ( N-acylamidated) with taurine or glycine during hepatocyte transport. Activation of the carboxyl group of bile acids to form acyl-adenylates is a likely key intermediate step in bile acid N-acylamidation. To gain more insight into the process of bile acid adenylate formation, we first synthesized the adenylates of five common, natural bile acids (cholic, deoxycholic, chenodeoxycholic, ursodeoxycholic, and lithocholic acid), and confirmed their structure by proton NMR. We then investigated adenylate formation by subcellular fractions of rat liver (microsomes, mitochondria, cytosol) using a newly developed LC method for quantifying adenylate formation. The highest activity was observed in the microsomal fraction. The reaction required Mg 2+ and its optimum pH was about pH 7.0. In term of maximum velocity ( V max) and the Michaelis constant ( K m), the catalytic efficiency of the enzyme under the conditions used was highest with cholic acid of the bile acids tested. The formation of cholyl-adenylate was strongly inhibited by lithocholic and deoxycholic acid, as well as by palmitic acid; ibuprofen and valproic acid were weak inhibitors. In cholestatic disease, such adenylate formation might lead to subsequent bile acid conjugation with glutathione or proteins.

Differential Enzymatic Reductions Governing the Differential Hypoxia-Selective Cytotoxicities of Phenazine 5,10-Dioxides

Some derivatives of phenazine 5,10-dioxide are selectively toxic to hypoxic cells commonly found in solid tumors. Previous studies of the phenazine 5,10-dioxide mechanism of action indicated that a bioreduction process could be involved in its selective toxicities, maybe as result of its potential H(*)-releasing capability in hypoxia. The major unresolved aspect of the mechanism of phenazine 5,10-dioxides is the identity of the reductase(s) in the cell responsible for activating the drug to its toxic form and metabolites. We have studied the metabolism in both hypoxia and oxia of some selected 2-amino and 2-hydroxyphenazine 5,10-dioxides, 1- 5, using rat liver microsomal and cytosol fractions. Differential hypoxic/oxic metabolism was found to be correlated to a compound's cytotoxic selectivity but, in general, without metabolic differences between liver microsomal or cytosolic enzymes. Dicoumarol and ketoconazole were found to inhibit the hypoxic metabolism of the most selective phenazine 5,10-dioxide, 1, inferring a role for DT-diaphorase and cytochrome P450. The least hypoxic selective agents, 4 and 5, possess different hypoxia-metabolic profiles as compared to derivative 1, explaining the differential cytotoxic biological behavior. The nonselective derivative, 2, suffered bioreduction in both conditions and, according to the inhibition studies with dicoumarol and ketoconazole, involves both DT-diaphorase and cytochrome P450. The nontoxic derivative, 3, showed poor bioreductive behavior.

Enantioselective analysis of oxybutynin and N-desethyloxybutynin with application to an in vitro biotransformation study

An enantioselective method using liquid-phase microextraction (LPME) followed by HPLC analysis was developed for the determination of oxybutynin (OXY) and its major metabolite N-desethyloxybutynin (DEO) in rat liver microsomal fraction. The LPME procedure was optimized using multifactorial experiments. Under the optimal extraction conditions, the mean recoveries were 61 and 55% for ( R)-OXY and ( S)-OXY, respectively, and 70 and 76% for ( R)-DEO and ( S)-DEO, respectively. The validated method was employed to an in vitro biotransformation study using rat liver microsomal fraction. The results demonstrated the enantioselective biotransformation of OXY.

Oxidation of 5-methoxy- N, N-diisopropyltryptamine in rat liver microsomes and recombinant cytochrome P450 enzymes

The oxidative metabolism of 5-methoxy- N, N-diisopropyltryptamine (5-MeO-DIPT), a tryptamine-type designer drug, was studied using rat liver microsomal fractions and recombinant cytochrome P450 (CYP) enzymes. 5-MeO-DIPT was biotransformed mainly into a side-chain N-deisopropylated metabolite and partially into an aromatic ring O-demethylated metabolite in liver microsomal fractions from untreated rats of both sexes. This metabolic profile is different from our previous findings in human liver microsomal fractions, in which the aromatic ring O-demethylation was the major pathway whereas the side-chain N-deisopropylation was minor [Narimatsu S, Yonemoto R, Saito K, Takaya K, Kumamoto T, Ishikawa T, et al. Oxidative metabolism of 5-methoxy- N, N-diisopropyltryptamine (Foxy) by human liver microsomes and recombinant cytochrome P450 enzymes. Biochem Pharmacol 2006;71:1377–85]. Kinetic and inhibition studies indicated that the side-chain N-dealkylation is mediated by CYP2C11 and CYP3A2, whereas the aromatic ring O-demethylation is mediated by CYP2D2 and CYP2C6 in untreated male rats. Pretreatment of male rats with β-naphthoflavone (BNF) produced an aromatic ring 6-hydroxylated metabolite. Recombinant rat and human CYP1A1 efficiently catalyzed 5-MeO-DIPT 6-hydroxylation under the conditions used. These results provide valuable information on the metabolic fate of 5-MeO-DIPT in rats that can be used in the toxicological study of this designer drug.