Pig Liver Microsomes Research Articles

CYP3As catalyze nifedipine oxidation in pig liver microsomes: Enzyme kinetics, inhibition and functional expression

Abstract Cytochrome P450 (CYP) 3As have received much attention because of their importance in drug metabolism. However, information on CYP3As in pigs is still largely incomplete. Herein, we focused on the involvement of CYP3As in nifedipine oxidation in pig liver microsomes. Enzyme kinetics, inhibition and immunochemical studies indicated that CYP3As might catalyze nifedipine oxidation and that ketoconazole could strongly inhibit this reaction in a noncompetitive and time-dependent manner. Furthermore, pig CYP3A46, showing the highest amino acid sequence identity to human CYP3A4, exhibited nifedipine oxidation activity. Collectively, these results provide strong evidence that CYP3As catalyze nifedipine oxidation in pig liver microsomes.

Identification of carbadox metabolites formed by liver microsomes from rats, pigs and chickens using high‐performance liquid chromatography combined with hybrid ion trap/time‐of‐flight mass spectrometry

Carbadox (methyl-3-(2-quinoxalinylmethylene)-carbazate-N(1),N(4)-dioxide) is a chemotherapeutic growth promoter added to feed for starter pigs. In this work, the metabolism of carbadox in rat, pig and chicken liver microsomes has been studied firstly. The incubation mixtures were then processed and analyzed for metabolites with a sensitive and reliable method based on high-performance liquid chromatography combined with hybrid ion trap/time-of-flight mass spectrometry (LC/MS-IT-TOF). With the help of chromatographic behavior and accurate mass measurements, it is possible to rapidly and reliably characterize the metabolites of carbadox. The structural elucidations of these metabolites were performed by comparing the changes in the accurate molecular masses and fragment ions generated from precursor ions with those of parent drug. The present results showed that the metabolism of carbadox in liver microsomes had qualitative species-difference. A total of seven metabolites were identified in rat liver microsomes. Five metabolites (Cb1-Cb3, Cb5, Cb7) were observed in pig and chicken liver microsomes. In addition, metabolite Cb6 was also detected in chicken liver microsomes. The peak areas of the metabolites in the three species are different. For the formations of Cb1, Cb2, Cb5 and Cb6, the rank order was rat>chicken>pig; Cb3; pig~chicken>rat. Cb1, Cb2 and Cb3 have been previously reported, whereas the other four metabolites were novel. The N→O group reduction and hydroxylation followed by N→O group reduction were the main metabolic pathways for carbadox in the three species.

Effects of Ketoconazole on the In Vivo Biotransformation and Hepatobiliary Transport of the Thrombin Inhibitor AZD0837 in Pigs

Ketoconazole has been shown in clinical trials to increase the plasma exposure of the oral anticoagulant prodrug AZD0837 [(2S)-N-{4- [(Z)-amino(methoxyimino)methyl]benzyl}-1-{(2R)-2-[3-chloro-5-(difluoromethoxy)phenyl]-2-hydroxyethanoyl}-azetidine-2-carboxamide] and its active metabolite, AR-H067637 [(2S)-N-{4-[amino(imino)methyl]benzyl}-1-{(2R)-2-[3-chloro-5-(difluoromethoxy)phenyl]-2-hydroxyethanoyl}-azetidine-2-carboxamide]. To investigate the biotransformation of AZD0837 and the effect of ketoconazole on this process, we used an experimental model in pigs that allows repeated sampling from three blood vessels, the bile duct, and a perfused intestinal segment. The pigs received AZD0837 (500 mg) given enterally either alone (n = 5) or together with single-dose ketoconazole (600 mg) (n = 6). The prodrug (n = 2) and its active metabolite (n = 2) were also administered intravenously to provide reference doses. The plasma data revealed considerable interindividual variation in the exposure of the prodrug, intermediate metabolite, and active metabolite. However, AR-H067637 was detected at very high concentrations in the bile with low variability (Ae(bile) = 53 ± 6% of the enteral dose), showing that the compound had indeed been formed in all of the animals and efficiently transported into the bile canaliculi. Concomitant dosing with ketoconazole increased the area under the plasma concentration-time curve for AZD0837 (by 99%) and for AR-H067637 (by 51%). The effect on the prodrug most likely arose from inhibited CYP3A-mediated metabolism. Reduced metabolism also seemed to explain the increased plasma exposure of the active compound because ketoconazole prolonged the terminal half-life with no apparent effect on the extensive biliary excretion and biliary clearance. These in vivo results were supported by in vitro depletion experiments for AR-H067637 in pig liver microsomes with and without the addition of ketoconazole.

In vitro biotransformation and investigation of metabolic enzymes possibly responsible for the metabolism of bisdesoxyolaquindox in the liver fractions of rats, chicken, and pigs

Bisdesoxyolaquindox is a reduced metabolite of olaquindox which is used as a medicinal feed additive in veterinary medicine. The relevant metabolism studies of bisdesoxyolaquindox have been carried out for the first time in rat, chicken, and pig liver subcellular fractions in order to understand the metabolic enzymes that are possibly responsible for the metabolism of olaquindox. The metabolites were characterized by high-performance liquid chromatography combined with hybrid ion trap/time-of-flight mass spectrometry. The major metabolic pathways of bisdesoxyolaquindox in the three species were the oxidation of hydroxyl to bisdesoxyolaquindox-2′-carboxyl acid (O10) and the N-dealkylation of the side chain to 3-methylquinoxaline 2-carboxamide (O12). Other metabolic pathways were also proposed which involved the direct methyl oxidation and N-oxide on the quinoxaline ring in the three species as well as N-hydroxylation only in rat. The intrinsic clearance values in the liver microsomes for O10 and O12 were ranked in the order of chicken > pig ≫ rat and rat > pig ≫ chicken, respectively. Inhibition studies indicated that 8-methoxypsoralen, 4-methylpyrazole and α-naphthoflavone could inhibit the formations of O10 and O12 in all species. Quinidine, troleandomycin, diethyldithiocarbamate, and disulfiram showed an interspecies difference in the inhibition of the formation of two metabolites. In rat and pig liver cytosol, 4-methylpyrazole, menadione and chlorpromazine strongly inhibited the formation of O10. Both diethyldithiocarbamate and disulfiram were found to inhibit O10 formation in rat cytosol but not in pig cytosol. These results indicated the following: In rat liver microsomes, CYP2A might be involved in the formation of O10, and CYP1A, CYP2A and CYP2E would be involved in the O12 formation. In pig liver microsomes, CYP1A and CYP2E might catalyze the formations of O10 and O12. In rat cytosol, alcohol dehydrogenase, aldehyde oxidase and aldehyde dehydrogenase should catalyze the O10 formation. In pig cytosol, alcohol dehydrogenase and aldehyde oxidase might be involved in the formation of O10. In chicken, it was found that various CYP isoenzymes were capable of catalyzing the two reactions; none of the inhibitors of cytosol enzymes inhibited O10 formation in chicken cytosol.

Species and Gender Differences Affect the Metabolism of Emodin via Glucuronidation

The aim of the present study was to define the mechanisms responsible for poor bioavailability of emodin by determining its metabolism using in vitro and in situ disposition models of the intestine and liver. Liver microsomes of mice, rats, guinea pigs, dogs, and humans were used along with the rat intestinal perfusion model and the rat intestinal microsomes. In the rat intestine, excretion rates of emodin-3-O-glucuronide were significantly different (p < 0.05) in four regions of the intestine and were higher in males than in females (p < 0.01). Emodin glucuronidation in liver microsomes was species-dependent, and K (m) values varied 5.7-fold (3.2-18.2 microM) in males and 2.8-fold (4.6-13.0 microM) in females. The male intrinsic clearance (CL(int)) values differed by 5-fold (27.6-138.3 mL h(-1) mg(-1) protein), and female CL(int) values differed by 4.3-fold (24.3-103.5 mL h(-1) mg(-1) protein). Since CL(int) values of emodin glucuronidation were 10-fold higher than that of isoflavones, emodin was considered rapidly glucuronidated. In contrast to the large species-dependent effects on K (m) and CL(int) values, gender had a smaller effect on these kinetic parameters (2-fold, p < 0.05). Lastly, glucuronidation rates obtained using liver microsomes from various experimental animals of the same gender correlated well with those in human liver microsomes. In conclusion, Rapid metabolism by UDP-glucuronosyltransferase is the major reason why emodin has poor bioavailability. Species and gender affected emodin metabolism to a different degree, and experimental animals are expected to be useful in predicting emodin glucuronidation in humans.

Metabolism of mequindox in liver microsomes of rats, chicken and pigs

Mequindox, 3-methyl-2-quinoxalinacetyl-1,4-dioxide, is a quinoxaline-N,N-dioxide used in veterinary medicine as a antibacterial in China. To gain an understanding of the interspecies differences in the metabolism of mequindox, comparative metabolite profiles were qualitatively and quantitatively carried out for the first time in rat, chicken and pig liver microsomes by high-performance liquid chromatography combined with hybrid ion trap/time-of-flight mass spectrometry. A total of 14 metabolites were characterized based on their accurate MS(2) spectra and known structure of mequindox. The in vitro metabolic pathways of mequindox in three species were proposed as N-->O group reduction, carbonyl reduction, N-->O group reduction followed by carbonyl reduction or methyl mono-hydroxylation. A metabolic pathway involving N-->O group reduction followed by acetyl group mono-hydroxylation in only chicken was also proposed. There was also quantitative species difference for mequindox metabolism in three species. 1-Desoxymequindox was the main metabolite in all species, but otherwise there were some qualitative interspecies differences in mequindox major metabolites. This work has revealed biotransformation characteristics of mequindox among different species, and moreover will further facilitate the explanations of the biological activities of mequindox in animals.

The metabolism and N-oxide reduction of olaquindox in liver preparations of rats, pigs and chicken

Olaquindox, N-(2-hydroxyethyl)-3-methyl-2-quinoxalinecarboxamide-1,4-di- N-oxide, is one of the quinoxaline-dioxides used widely as an antimicrobial growth promoter in pig production. Its toxicities were reported to be closely related to the formation of N-oxide reductive metabolites. The present study presents the metabolism and N-oxide reduction of olaquindox incubated with liver microsomes and liver cytosol of rats, pigs and chicken. Metabolites were identified and characterized with a novel LC/MS-ITTOF. Thirteen metabolites were found in liver microsomes of rats, three of which were identified to be novel. Seven metabolites were found in liver microsomes of pigs and chicken. The N-oxide reduction was the major metabolic pathway of olaquindox in liver microsomes of the three species. The N1-reduction of olaquindox to metabolite O2 was found in not only liver microsomes but also cytosol of the three species in the presence of NAD(P)H under hypoxic conditions. The N1-reduction could be inhibited by air and carbon monoxide, and be significantly stimulated by riboflavin under various conditions. The N1-reduction in the liver cytosol of rats and pigs could be enhanced by menadione, but the reduction in liver cytosol of chicken could not be. The N1-reduction activities in all animals were not abolished when liver microsomes and cytosol were boiled. These findings suggested that the N1-reduction of olaquindox could be mediated by non-enzymatic and enzymatic conditions. This N1-reduction of olaquindox could also be catalyzed by a quinone-dependent reducing system in liver cytosol of rats and pigs. Moreover, liver cytosol of rats and pigs had an ability of N4-reduction that catalyzed olaquindox to metabolite O1 in the presence of benzaldehyde under hypoxic conditions, but the liver cytosol of chicken did not. The N4-reduction could be inhibited markedly in the cytosol rats and pigs by menadione, chlorpromazine and promethazine. In addition, 7-hydroxycoumarin was also found to inhibit the formation of O1 in the cytosol of rats. The inhibitory results suggested that the N4-reduction might be catalyzed by aldehyde oxidase in the cytosol of pigs, and by aldehyde oxidase and xanthine oxidase in the cytosol of rats. In conclusion, the N1-reduction and N4-reduction of olaquindox are mediated by multiple mechanisms and significant species differences are involved in both reductions. This work is a contribution to the understanding of toxicities and the relativities between toxicities and metabolism of olaquindox.

Isolation of two cytochrome P450 forms, CYP2A19 and CYP1A, from pig liver microsomes

Cytochromes P450 comprise a superfamily of proteins that play a crucial role in the biotransformation of numerous chemicals. Purified CYPs can be used e.g. in studies on structure or determining the drug metabolism pathways. In this work, purification of the porcine CYP1A and CYP2A19 to electrophoretic homogeneity from the pig hepatic microsomes using octylamino Sepharose and hydroxylapatite column chromatography is reported. The proteins have been clearly recognized by commercial antibodies against rat and human CYP1A2 (porcine CYP1A) and human CYP2A6 (CYP2A19) respectively, using Western blot. Activities of both enzymes were determined by specific substrates, 7-ethoxyresorufin, 7-methoxyresorufin (CYP1A) and coumarin (CYP2A19). The isolated enzymes show kinetic parameters similar to human counterparts. Taken together, pig cytochromes can be used for the testing of veterinary drug metabolism, useful for the determination of drug residues in meat of pigs. The results obtained show that the pigs may be a suitable model for biotransformation of xenobiotics in humans.

A Professional and Personal Odyssey

As I began to write this article reflecting on my professional career in biomedical research, many memories of people, activities, situations, and experiences were swirling in my head. Because of the extraordinary support that I have received from my family, they are never far from my thoughts. I believe that sharing a few of these memories will be helpful for those who find themselves in similar situations or periods of professional development. This is not meant to be a comprehensive review of the fields to which I have had the privilege of contributing but a personal stroll down a path in which I have found extraordinary pleasure and from which I have derived a sense of accomplishment.

Oxidative and Reductive Metabolism of Tris(p-carboxyltetrathiaaryl)methyl Radicals by Liver Microsomes

Tris(p-carboxyltetrathiaaryl)methyl (TAM) radicals are particularly stable carbon-centered free radicals that are used as contrast agents in NMR imaging and as probes for in vivo oximetry by electron paramagnetic resonance (EPR) imaging. However, nothing is known so far on the metabolism of these persistent radicals in mammals. This article describes the metabolism of two TAM radicals by rat, human, and pig liver microsomes. It shows that microsomal transformation of these free radicals leads to two major metabolites resulting from an oxidation or a reduction of the present compounds. The structures of these metabolites were completely established by 1H and 13C NMR spectroscopy, mass spectrometry, and comparison with authentic compounds. Under aerobic conditions, liver microsomes catalyzed the oxidative decarboxylation of TAM radicals by NADPH and O2 with formation of the corresponding quinone-methide products. This reaction was dependent on cytochromes P450 and cytochrome P450 reductase and greatly implied the involvement of superoxide. Under anaerobic conditions, these enzymes catalyzed the reduction of TAM radicals to the corresponding triarylmethanes. This reduction was strongly inhibited by O2. These metabolic transformations should be considered when using such TAM radicals for pO2 measurement by EPR imaging, especially in tissues in which fast oxidative (inflammation sites) or reductive (hypoxic tissues) metabolism could occur.

Metabolism of cyadox in rat, chicken and pig liver microsomes and identification of metabolites by accurate mass measurements using electrospray ionization hybrid ion trap/time‐of‐flight mass spectrometry

Cyadox (CYX), (2-formylquinoxaline)-N(1),N(4)-dioxide cyanoacetylhydrazone, is a growth promoter, which is more efficient and less toxic to animals. Few studies have been performed to reveal the metabolism of CYX in animals till now. In this study, the metabolic fate of CYX in the liver microsomes of animal was investigated firstly using high-performance liquid chromatography combined with hybrid ion trap/time-of-flight mass spectrometry. CYX was incubated with rat, chicken and pig liver microsomes in the presence of a NADPH-generating system. Multiple scans of metabolites in MS and MS(2) modes and accurate mass measurements were performed simultaneously through data-dependent acquisition. Most measured mass errors were less than 10 ppm for both protonated molecules and fragment ions using external mass calibration. The structures of metabolites and their fragment ions were easily and reliably characterized based on the accurate MS(2) spectra and known structure of CYX. The relative biotransformation of CYX into characterized metabolites was estimated based on the UV absorption and the assumption that all metabolites had the same extinction coefficient as the parent compound at 305 nm. Totally, seven metabolites were identified as three reduced metabolites (cyadox 1-monoxide (Cy1), cyadox 4-monoxide (Cy2) and bisdesoxycyadox (Cy4)), three hydrolysis metabolites of the amide bond (N-decyanoacetyl cyadox (Cy5), N-decyanoacetyl cyadox 1-monoxide (Cy6) and N-decyanoacetyl bisdesoxycyadox (Cy7)) and a hydroxylation metabolite of Cy1 (Cy3). Cy1-Cy6 could be detected in rat, chicken and pig liver microsomes while metabolite Cy7 could only be observed in pig. The amounts of the metabolites in three species are different. For the formations of Cy1 and Cy3, the rank order was rat approximately chicken > pig. For Cy4 and Cy5, the order was pig > rat > chicken. Cy1 and Cy4 have been previously reported, whereas the other five metabolites were novel. The N-->O group reduction and hydroxylation were the main metabolic pathways for CYX in the three species.

Synthesis, structure characterization, and enzyme screening of clenbuterol glucuronides

Two clenbuterol O-glucuronide diastereomers were synthesized by the Koenigs–Knorr reaction. Structures and glucuronidation sites of the glucuronides were characterized by tandem mass spectrometry and nuclear magnetic resonance spectroscopy. The two diastereomers were used as standard compounds in studies of stereoselective glucuronidation of clenbuterol with liver microsomes from different species and with 15 human recombinant UDP-glucuronosyltransferases. In this study, chemical and enzymatic reactions produced only O-glucuronides of clenbuterol, although on the basis of the chemical structure of the aglycone, both O- and N-glucuronides of clenbuterol could be formed. Differences in the production of diastereomers of clenbuterol glucuronides were observed among liver microsomes from the various animals. Dog and bovine liver microsomes were significantly active, and also stereoselective, each producing only one but a different diastereomer. Liver microsomes from rabbit and rat were also rather actively glucuronidating clenbuterol, but human, pig, and moose liver microsomes produced only minor amounts of glucuronides. Human liver microsomes produced only one clenbuterol glucuronide diastereomer, and the same was true of the human UDP-glucuronosyltransferases that were active (formation of glucuronide: 1A9 > 1A10 ≫ 1A7). The marked differences in the stereoselective glucuronidation of clenbuterol show that UDP-glucuronosyltransferases in the livers of different animals do not have the same functions, activities, or distribution. This needs to be taken into account, particularly in toxicology testing.

Identification of metabolites of meisoindigo in rat, pig and human liver microsomes by UFLC–MS/MS

3-(1,2-Dihydro-2-oxo-3H-indol-3-ylidene)-1,3-dihydro-1-methyl-2H-indol-2-one, abbreviated as meisoindigo, has been a routine therapeutic agent in the clinical treatment of chronic myelogenous leukemia in China since the 1980s. To gain an understanding of the interspecies differences in the metabolism of meisoindigo, the relevant metabolism studies were carried out for the first time in rat, pig and human liver microsomes of different genders by ultra fast liquid chromatography/tandem mass spectrometry (UFLC–MS/MS). The qualitative metabolite identification was accomplished by multiple reaction monitoring (MRM) in combination with Enhanced Product Ion (EPI). The semi-quantitative metabolic stability and metabolite formation were simultaneously measured by MRM. The in vitro metabolic pathways of meisoindigo in three species were proposed as 3,3′ double bond reduction, followed by N-demethylation, and reduction followed by phenyl mono-oxidation. Two novel metabolic pathways involving direct phenyl mono-oxidation without reduction in the three species, and direct N-demethylation without reduction in only pig and human, were also proposed. It may be noted that the two metabolites formed after reduction followed by phenyl mono-oxidation at positions 4, 5, 6 or 7, as well as one metabolite formed from direct phenyl mono-oxidation at either of the two phenyl rings without reduction were found to be uniquely present in human. The in vitro t 1/2 and in vitro CL int values of meisoindigo were calculated. Statistical analysis showed there were no significant differences in the metabolic stability profiles of meisoindigo among three species, and gender effect on the metabolic stability of meisoindigo was negligible. Formation profiles of the most significant reductive metabolites were obtained in the three species.

Comparative Study on Nobiletin Metabolism with Liver Microsomes from Rats, Guinea Pigs and Hamsters and Rat Cytochrome P450

In vitro metabolism of nobiletin, a polymethoxy-flavonoid abundantly present in citrus peels, was studied using liver microsomes of rats, hamsters and guinea pigs and ten cDNA-expressed rat cytochrome P450 (P450). The effects of P450 inducers on nobiletin metabolism were also investigated. Aerobical incubation with NADPH and animal liver microsomes transformed nobiletin to five metabolites, M-1, M-2, M-3, M-4 and M-5. From LC-MS and (1)H-NMR data and a time-course study, these were assumed to be 4'-hydroxy (OH)-, 7-OH-, 6-OH-, 3',4'-diOH- and 6,7-diOH-metabolites, respectively. Pretreatment of animals with phenobarbital increased M-2 and M-3 to about 2-fold that in untreated animals. Pretreatment with 3-methylcholanthrene (MC) resulted in remarkable increases of both M-1 and M-4 (3 to 9-fold that of untreated). Males had 2-3 times higher M-2 and M-3 formation activities in rats, and for M-2 in hamsters than did females. Immunoinhibition study using antiserum against P450 revealed the involvement of hamster CYP1A2 in the formation of M-1 and M-4 in hamster liver. Of ten rat P450s, CYP2C11, CYP3A1, CYP3A2 and CYP2D1 had high activities for the formation of M-1, M-2 and M-3. Another P450s (CYP1A1, CYP2C12 and CYP1A2) also showed activity for the formation of M-1. Only CYP1A1 produced 3',4'-diOH-metabolites (M-4). However, CYP2A1, CYP2B1 and CYP2E1 had no activity for nobiletin. These results suggested that constitutive P450s such as CYP2C11, CYP2D1, CYP3A1, CYP3A2 and CYP2C12 are responsible for the demethylation at the 6-, 7-, 3'- and 4'-positions; whereas, MC-inducible P450s, CYP1A1 and CYP1A2, preferentially catalyzed demethylation at the 3'-and 4'-positions.

Determination of microsomal CYP2A6 activity by high performance liquid chromatography

Coumarin has been reported to be a reliable probe for the determination of human microsomal CYP2A6 activity. Coumarin is converted to 7-hydroxycoumarin by CYP2A6. A high pressure liquid chromatographic assay for the estimation of CYP2A6 activity in microsomes was evaluated. A RP C-18 Novapak Waters (15 cm x 3.9 mm, 5 µm) column was used for the assay. The mobile phase composition was methanol : 1% glacial acetic acid (35:65 v/v) (pH~3.1), with a flow rate of 0.6 ml/min, injection volume of 100 µl and detection at 320 nm. The retention times for coumarin and 7-hydroxycoumarin were 8.7 min and 5.3 min, respectively. The limit of detection (LOD) was 0.05 µM, and the limit of quantitation (LOQ) was 0.1 µM, for 7-hydroxycoumarin. The percent coefficient of variation associated with 7-hydroxycoumarin determination after duplicate estimation was found to be in the range 0.03 to 3.6% for buffer matrix and 0.1 to 6.5% for microsomal matrix. The mean rate of 7-hydroxycoumarin formation in guinea pig liver microsomes was 0.084 nmol/min/nmol P450. Coumarin 7-hydroxylase activity was absent in rat liver microsomes. No interference was observed from incubation mixture components.

Reduction of Nω-hydroxy-l-arginine to l-arginine by pig liver microsomes, mitochondria, and human liver microsomes

Nω-Hydroxy-l-arginine, the intermediate in nitric oxide formation from l-arginine catalyzed by NO synthase, can be released into the extracellular space. It has been suggested that it can circulate and exert paracrine effects. Since it cannot only be used as substrate by NO synthases, but can also be oxidized by cytochrome P450 and other hemoproteins in a superoxide-dependent manner, it has been proposed that it can serve as NO donor. In the present study, the in vitro reduction of Nω-hydroxy-l-arginine was examined. Pig and human liver microsomes as well as pig liver mitochondria were capable of reducing Nω-hydroxy-l-arginine to l-arginine in an oxygen-insensitive enzymatic reaction. These results demonstrate that this metabolic pathway has to be considered when suggesting Nω-hydroxy-l-arginine as NO-precursor. The reconstituted liver microsomal system of a pig liver CYP2D enzyme, the benzamidoxime reductase, was unable to replace microsomes to produce l-arginine from Nω-hydroxy-l-arginine.

On-line Formation, Separation, and Estrogen Receptor Affinity Screening of Cytochrome P450-Derived Metabolites of Selective Estrogen Receptor Modulators

We have developed a fully automated bioreactor coupled to an on-line receptor affinity detection system. This analytical system provides detailed information on pharmacologically active metabolites of selective estrogen receptor modulators (SERMs) generated by cytochromes P450 (P450s). We demonstrated this novel concept by investigating the metabolic activation of tamoxifen and raloxifene by P450-containing pig and rat liver microsomes. The high resolution screening (HRS) system is based on the coupling of a P450-bioreactor to an HPLC-based estrogen receptor alpha (ERalpha) affinity assay. P450-derived metabolites of the SERMs were generated in the bioreactor, subsequently trapped on-line with solid phase extraction, and finally separated with gradient HPLC. Upon elution, the metabolites were screened on affinity for ERalpha with an on-line HRS assay. With this HRS system, we were able to follow, in a time-dependent manner, the formation of ERalpha-binding metabolites of tamoxifen and raloxifene. By analyzing the bioaffinity chromatograms with liquid chromatography-tandem mass spectrometry, structural information of the pharmacologically active metabolites was obtained as well. For tamoxifen, 15 active and 6 nonactive metabolites were observed, of which 5 were of primary, 10 of secondary, and 6 of an as yet unknown order of metabolism. Raloxifene was biotransformed in three primary and three secondary metabolites. MS/MS analysis revealed that three of the observed active metabolites of raloxifene were not described before. The present automated on-line HRS system coupled to a P450-containing bioreactor and an ERalpha-affinity detector proved very efficient, sensitive, and selective in metabolic profiling of SERMs.

Isolation and structural elucidation of tiamulin metabolites formed in liver microsomes of pigs

Although the antimicrobial tiamulin is extensively metabolized in pigs, the metabolism is not well investigated. In this work the NADPH dependent metabolism of tiamulin in liver microsomes from pigs has been studied. The tiamulin metabolites formed in the incubations were analysed using LC–MS, and three major metabolites were isolated using solid phase extraction and preparative HPLC. The final structure elucidations were performed by tandem mass spectrometry and 1H and 13C NMR. The structures of the metabolites were found to be 2β-hydroxy-tiamulin, 8α-hydroxy-tiamulin and N-deethyl-tiamulin. In addition, the LC–MS chromatograms revealed two other minor metabolites. From their chromatography and from MS 2 analysis the structures were estimated to be 2β-hydroxy- N-deethyl-tiamulin and 8α-hydroxy- N-deethyl-tiamulin, but the structures were not confirmed by NMR. In these studies approximately 20% of tiamulin was deethylated, 10% was hydroxylated in the 2β-position and 7% was hydroxylated in the 8α-position. About 40% of tiamulin was metabolized during the incubation conditions used. The protein precipitation in the incubations was performed using perchloric acid, and the preparative purification was performed under alkaline conditions. Therefore, the stability of the metabolites under these conditions was studied. The metabolites were found to be stable in the acid solution, but under alkaline conditions, particularly at room temperature, the stability of especially 8α-hydroxy-tiamulin was considerably reduced (40% loss after 1 week).

FLUTAMIDE METABOLISM IN FOUR DIFFERENT SPECIES IN VITRO AND IDENTIFICATION OF FLUTAMIDE METABOLITES IN HUMAN PATIENT URINE BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY/TANDEM MASS SPECTROMETRY

A new metabolic scheme of flutamide is proposed in this article. Some patients treated with flutamide, a nonsteroidal antiandrogen, have developed severe hepatic dysfunction. Toxic metabolites have been proposed to be responsible for these negative effects. In this study, the qualitative aspects of the in vitro metabolism of flutamide in liver microsomes from human, dog, pig, and rat were evaluated. A direct comparison of the flutamide metabolism in liver and prostate microsomes from pig was made, and the in vivo metabolism of flutamide was investigated in urine from orally treated prostate cancer patients. Liquid chromatography/tandem mass spectrometry was used for analysis. The mass spectrometer was equipped with an electrospray interface and operated in the negative ion mode. In liver microsomes from pig, dog, and rat, extensive hydroxylation of flutamide occurred. One, two, or three hydroxy groups were attached, and isomeric forms were detected for both monohydroxylated and trihydroxylated drug. In pig liver microsomes, isomers of a third metabolite, hydroxylated 4-nitro-3-(trifluoromethyl)-aniline, were also found after incubation with either flutamide or 2-hydroxyflutamide. In human liver microsomes, the pharmacologically active 2-hydroxyflutamide was the only metabolite detected. Several phase I metabolites as well as four intact phase II metabolites could be recovered from the urine samples. For the first time in humans, glucuronic acid conjugates of hydroxylated 4-nitro-3-(trifluoromethyl)-aniline, and mono- and dihydroxylated flutamide were identified, together with hydroxylated 4-nitro-3-(trifluoromethyl)-aniline conjugated with sulfate. In addition, one mercapturic acid conjugate of hydroxylated flutamide, probably formed from flutamide via a reactive intermediate, was detected.

Metabolism of new-generation taxanes in human, pig, minipig and rat liver microsomes

The novel taxanes SB-T-1102, SB-T-1214 and SB-T-1216 are up to 1000-fold more cytotoxic for resistant tumour cells than clinically used paclitaxel and docetaxel, and the current study has examined the metabolism of these new taxanes in human, rat, pig and minipig liver microsomes. Metabolites were characterized by high-performance liquid chromatography (HPLC)/tandem mass spectrometry (MS/MS) analysis. Metabolic pathways derived from their structures were confirmed by investigating subsequent metabolism of purified metabolites. SB-T-1102, SB-T-1214 and SB-T-1216 were metabolized to 14, 10 and 11 products, respectively. In contrast to docetaxel, side-chain hydroxylation did not occur at their tert-butyl group, but on the isobutyl (SB-T-1102) or isobutenyl (SB-T-1214 and SB-T-1216) chains. Species differences in their metabolism were observed. For example, human and untreated rat microsomes hydroxylated SB-T-1216 preferentially at the side-chain, whereas pig and minipig microsomes preferentially metabolized more at the taxane core. The increased formation of secondary and tertiary metabolites in rat microsomes with high expression of CYP3A1/2 compared with uninduced rats confirmed the role of CYP3A in taxane metabolism. All major products were formed by human cDNA-expressed CYP3A4 and none by CYP1A2, 1B1, 2A6, 2C9 and 2E1, indicating the principal role of CYP3A orthologues in SB-T metabolism. The knowledge of metabolic pathways of the examined agents and of their rates of formation is important due to possible metabolic inactivation of these three novel drugs with a great potential for the therapy of taxane-resistant tumours. The relatively slow metabolism of SB-T-1102 could be favourable for its antitumour efficiency in vivo.