Minipig Liver Microsomes Research Articles

2-Oxidation, 3-methyl hydroxylation, and 6-hydroxylation of skatole, a contributor to the odour of boar-tainted pork meat, mediated by porcine liver microsomal cytochromes P450 1A2, 2A19, 2E1, and 3A22

The 2-oxidation, 3-methyl hydroxylation, and 6-hydroxylation of skatole (a contributor to boar taint) mediated by minipig liver microsomes and recombinant P450 enzymes expressed in bacterial membranes were investigated. At low substrate concentrations of 10 µM, the formation rates of indole-3-carbinol, 6-hydroxyskatole, and the sum of 3-methyloxindole, indole-3-carbinol, and 6-hydroxyskatole in male minipig liver microsomes were significantly lower than those in female minipig liver microsomes. Compensatory 3-methyloxindole and indole-3-carbinol formation in minipig liver microsomes, which lack 6-hydroxyskatole formation in males, was mediated partly by liver microsomal P450 1A2 and P450 1A2/2E1, respectively. These enzymes were suppressed by typical P450 inhibitors in female minipig liver microsomes. Among the 14 pig P450 forms evaluated, P450 2A19 was the dominant form mediating 3-methyloxindole, indole-3-carbinol, and 6-hydroxyskatole formation from skatole at substrate concentrations of 100 µM. Positive cooperativity was observed in 3-methyloxindole formation from skatole mediated by male minipig liver microsomes and by pig P450 3A22 with Hill coefficients of 1.2–1.5. These results suggest high skatole 2-oxidation, 3-methyl hydroxylation, and 6-hydroxylation activities of pig P450 2A19 and compensatory skatole oxidations mediated by pig P450 1A2, 2E1, or 3A22 in male minipig liver microsomes.

Non-specific binding of compounds in in vitro metabolism assays: A comparison of microsomal and hepatocyte binding in different species and an assessment of the accuracy of prediction models.

Non-specific binding in in vitro metabolism systems leads to an underestimation of the true intrinsic metabolic clearance of compounds being studied. Therefore in vitro binding needs to be accounted for when extrapolating in vitro data to predict the in vivo metabolic clearance of a compound. While techniques exist for experimentally determining the fraction of a compound unbound in in vitro metabolism systems, early in drug discovery programmes computational approaches are often used to estimate the binding in the in vitro system. Experimental fraction unbound data (n = 60) were generated in liver microsomes (fumic) from five commonly used pre-clinical species (rat, mouse, dog, minipig, monkey) and humans. Unbound fraction in incubations with mouse, rat or human hepatocytes was determined for the same 60 compounds. These data were analysed to determine the relationship between experimentally determined binding in the different matrices and across different species. In hepatocytes there was a good correlation between fraction unbound in human and rat (r2 = 0.86) or mouse (r2 = 0.83) hepatocytes. Similar correlations were observed between binding in human liver microsomes and microsomes from rat, mouse, dog, Göttingen minipig or monkey liver microsomes (r2 of >0.89, n = 51 – 52 measurements in different species). Physicochemical parameters (logP, pKa and logD) were predicted for all evaluated compounds. In addition, logP and/or logD were measured for a subset of compounds. Binding to human hepatocytes predicted using 5 different methods was compared to the measured data for a set of 59 compounds. The best methods evaluated used measured microsomal binding in human liver microsomes to predict hepatocyte binding. The collated physicochemical data were used to predict the human fumic using four different in silico models for a set of 53 to 60 compounds. The correlation (r2) and root mean square error between predicted and observed microsomal binding was 0.69 & 0.20, 0.47 & 0.23, 0.56 & 0.21 and 0.54 & 0.26 for the Turner-Simcyp, Austin, Hallifax-Houston and Poulin models, respectively. These analyses were extended to include measured literature values for binding in human liver microsomes for a larger set of compounds (n = 697). For the larger dataset of compounds, microsomal binding was well predicted for neutral compounds (r2=0.67 - 0.70) using the Poulin, Austin, or Turner-Simcyp methods but not for acidic or basic compounds (r2 <0.5) using any of the models. While the lipophilicity-based models can be used, the in vitro binding should be measured for compounds where more certainty is needed, using appropriate calibrated assays and possibly established weak, moderate, and strong binders as reference compounds to allow comparison across databases.

Study on metabolic dynamics,metabolic enzyme phenotype and species difference of hepatic and intestinal microsome of psoralidin

This study aims to investigate metabolic activities of psoralidin in human liver microsomes( HLM) and intestinal microsomes( HIM),and to identify cytochrome P450 enzymes( CYPs) and UDP-glucuronosyl transferases( UGTs) involved in psoralidin metabolism as well as species differences in the in vitro metabolism of psoralen. First,after incubation serial of psoralidin solutions with nicotinamide adenine dinucleotide phosphate( NADPH) or uridine 5&apos;-diphosphate-glucuronic acid( UDPGA)-supplemented HLM or HIM,two oxidic products( M1 and M2) and two conjugated glucuronides( G1 and G2) were produced in HLM-mediated incubation system,while only M1 and G1 were detected in HIM-supplemented system. The CLintfor M1 in HLM and HIM were 104. 3,and57. 6 μL·min~(-1)·mg~(-1),respectively,while those for G1 were 543. 3,and 75. 9 μL·min~(-1)·mg~(-1),respectively. Furthermore,reaction phenotyping was performed to identify the main contributors to psoralidin metabolism after incubation of psoralidin with NADPH-supplemented twelve CYP isozymes( or UDPGA-supplemented twelve UGT enzymes),respectively. The results showed that CYP1 A1( 39. 5 μL·min~(-1)·mg~(-1)),CYP2 C8( 88. 0 μL·min~(-1)·mg~(-1)),CYP2 C19( 166. 7 μL·min~(-1)·mg~(-1)),and CYP2 D6( 9. 1 μL·min~(-1)·mg~(-1)) were identified as the main CYP isoforms for M1,whereas CYP2 C19( 42. 0 μL·min~(-1)·mg~(-1)) participated more in producing M2. In addition,UGT1 A1( 1 184. 4 μL·min~(-1)·mg~(-1)),UGT1 A7( 922. 8 μL·min~(-1)·mg~(-1)),UGT1 A8( 133. 0 μL·min~(-1)·mg~(-1)),UGT1 A9( 348. 6 μL·min~(-1)·mg~(-1)) and UGT2 B7( 118. 7 μL·min~(-1)·mg~(-1)) played important roles in the generation of G1,while UGT1 A9( 111. 3 μL·min~(-1)·mg~(-1)) was regarded as the key UGT isozyme for G2. Moreover,different concentrations of psoralidin were incubated with monkey liver microsomes( MkLM),rat liver microsomes( RLM),mice liver microsomes( MLM),dog liver microsomes( DLM) and mini-pig liver microsomes( MpLM),respectively. The obtained CLintwere used to evaluate the species differences.Phase Ⅰ metabolism and glucuronidation of psoralidinby liver microsomes showed significant species differences. In general,psoralidin underwent efficient hepatic and intestinal metabolisms. CYP1 A1,CYP2 C8,CYP2 C19,CYP2 D6 and UGT1 A1,UGT1 A7,UGT1 A8,UGT1 A9,UGT2 B7 were identified as the main contributors responsible for phase Ⅰ metabolism and glucuronidation,respectively. Rat and mini-pig were considered as the appropriate model animals to investigate phase Ⅰ metabolism and glucuronidation,respectively.

Interspecies metabolic diversity of artocarpin in vitro mammalian liver microsomes.

Artocarpin has shown anti-inflammation and anticancer activities. However, the metabolism differences among different species have not been reported. In this work, we used liver microsomes to explore the metabolic characteristics and possible metabolites of artocarpin among different species. The structures of six metabolites were characterized by LC-MS/MS, and hydroxylated artocarpin was the main metabolite. Enzyme kinetics and depletion studies of artocarpin among different species proved that artocarpin metabolism exhibited significant species differences; rats and monkeys showed a great metabolic ability to artocarpin, and minipigs showed the highest similarity to humans. The in vivo hepatic clearances of artocarpin in rats and humans were predicted that artocarpin was classified as a high-clearance drug in humans and rats. The glucuronidation assay of artocarpin in different liver microsomes also proved that artocarpin metabolism showed significant species difference. These findings will support further pharmacological or toxicological research on artocarpin.Abbreviations: UGT: UDP-glucuronosyltransferase; CYP: cytochrome P450; LC-MS/MS: liquid chromatography-tandem mass spectrometry; HPLC: high-performance liquid chromatography; HLMs: human liver microsomes; MLMs: monkey liver microsomes; RAMs: rabbit liver microsomes; RLMs: rat liver microsomes; DLMs: dog liver microsomes; PLMs: minipig liver microsomes; Vmax: maximum velocity; Km: Michaelis constant; CLint: intrinsic clearance; CLH: hepatic clearance; QH: hepatic blood flow.

In vitro investigation of permeability and metabolism of licoricidin

AimLicoricidin has multiple pharmacological activities. The present study was designed to investigate the permeability and pharmacokinetic behavior of licoricidin using in vitro models. Material and methodsFirst, human liver microsomes and recombinant human supersomes were used to investigate the interactions between metabolic enzymes and licoricidin. In addition, rat, minipig, rabbit, dog, monkey, and human liver microsomes were used to determine metabolic differences among species. The parallel artificial membrane permeability assay (PAMPA) was used to explore licoricidin permeability behavior. Key findingsAt 100 μM, licoricidin strongly inhibited CYP2C9, CYP2C19, CYP3A4, UGT1A3, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT2B4, UGT2B7, UGT2B15, and UGT2B17. Licoricidin metabolism exhibited considerable differences among species; dog, pig, and rat liver microsomes showed higher metabolic capacity than the other species. Seven licoricidin metabolites were identified by liquid chromatography-tandem mass spectrometry, and hydration and hydroxylation were the major metabolic pathways. The PAMPA permeability of licoricidin was moderate at both pH 4.0 and 7.4. SignificanceThe present study will support further pharmacological or toxicological research on licoricidin.

The different metabolism of morusin in various species and its potent inhibition against UDP-glucuronosyltransferase (UGT) and cytochrome p450 (CYP450) enzymes

1. The aim of this study was to investigate the inhibitory effect of morusin on Glucuronosyltransferase (UGT) isoforms and cytochrome P450 enzymes (CYP450s). We also investigated the metabolism of morusin in human, rat, dog, monkey, and minipig liver microsomes.2. 100 μM of morusin exhibited strong inhibition on all UGTs and CYP450s. The half inhibition concentration (IC50) values for CYP3A4, CYP1A2, CYP2C9, CYP2E1, UGT1A6, UGT1A7, and UGT1A8 were 2.13, 1.27, 3.18, 9.28, 4.23, 0.98, and 3.00 μM, and the inhibition kinetic parameters (Ki) were 1.34, 1.16, 2.98, 6.23, 4.09, 0.62, and 2.11 μM, respectively.3. Metabolism of morusin exhibited significant species differences. The quantities of M1 from minipig, monkey, dog, and rat were 7.8, 11.9, 2.0, and 6.3-fold of human levels. The Km values in HLMs, RLMs, MLMs, DLMs, and PLMs were 7.84, 22.77, 14.32, 9.13, and 22.83 μM, and Vmax for these species were 0.09, 1.23, 1.43, 0.15, and 0.75 nmol/min/mg, respectively. CLint (intrinsic clearance) values (Vmax/Km) for morusin obeyed the following order: monkey > rat > minipig > dog > human. CLH (hepatic clearance) values for humans, dogs, and rats were calculated to be 8.28, 17.38, and 35.12 mL/min/kg body weight, respectively.4. This study provided vital information to understand the inhibitory potential and metabolic behavior of morusin among various species.

Expression of Bama Minipig and Human CYP3A Enzymes: Comparison of the Catalytic Characteristics with Each Other and Their Liver Microsomes.

Minipigs represent a good animal model because of the physiologic and anatomic similarities they share with humans. Three cytochrome P450 (CYP) 3A isozymes, CYP3A22, CYP3A29, and CYP3A46, have recently been reported to be expressed in Bama minipigs, which have limited data relating to their metabolic characteristics. In the present study, Bama minipig CYP3A22, CYP3A29, and CYP3A46 were recombinantly expressed and their metabolic manners were compared with those of human CYP3A4 and CYP3A5 and also human and Bama minipig liver microsomes. The results indicated Bama minipigs and human CYP3A enzymes showed similar metabolic kinetics and metabolite profiles using testosterone, midazolam, and nifedipine as substrates. However, the differences in amino acid sequences change the elimination velocity and metabolic preference of CYP3A enzymes to their substrates. It was demonstrated that CYP3A29, CYP3A4, and CYP3A5 were the most active enzymes for all reactions, whereas CYP3A46 was the least active enzyme. Substrate-dependent metabolism characteristics between human and Bama minipig CYP3A isoenzymes exist.

Age-related Differences in CYP3A Abundance and Activity in the Liver of the Göttingen Minipig.

In view of paediatric drug development, regulatory authorities often request safety studies in juvenile animals, including minipigs. Unfortunately, knowledge on the ontogeny of the biotransformation processes in animal models remains scarce and impedes a correct interpretation of the toxicity findings. CYP3A4 is one of the most important drug-metabolizing enzymes in human beings and shows important similarities with CYP3A in the minipig. Therefore, the aim of this study was to assess the abundance and activity of CYP3A in liver microsomes from foetal, juvenile (days 1, 3, 7 and 28) and adult male and female Göttingen minipigs. CYP3A abundance was studied by an indirect enzyme-linked immunosorbent assay (ELISA), whereas CYP3A activity was assessed by a biotransformation assay with Luciferin-IPA. CYP3A abundance could not be detected until day 3. From day 7 onwards, a gradual increase in expression was noted, leading to the highest abundance in adult animals. CYP3A activity was not detectable in foetuses and 1-day-old animals. The CYP3A activity was detectable, but below the LLOQ in day 3 animals and increased gradually with age to reach the highest level in adults. The CYP3cide and ketoconazole inhibition, and testosterone and midazolam reduction of Luciferin-IPA metabolism in minipig liver microsomes substantiate that Luciferin-IPA is metabolized by CYP3A in minipigs. A positive correlation was found between CYP3A abundance and biotransformation of Luciferin-IPA (Pearson r = 0.863; p < 0.0001). In conclusion, both abundance and activity of CYP3A increased gradually in juvenile minipigs, but remained below the levels observed in adult animals.

Drug oxygenation activities mediated by liver microsomal flavin-containing monooxygenases 1 and 3 in humans, monkeys, rats, and minipigs

Liver microsomal flavin-containing monooxygenases (FMO, EC 1.14.13.8) 1 and 3 were functionally characterized in terms of expression levels and molecular catalytic capacities in human, cynomolgus monkey, rat, and minipig livers. Liver microsomal FMO3 in humans and monkeys and FMO1 and FMO3 in rats and minipigs could be determined immunochemically with commercially available anti-human FMO3 peptide antibodies or rat FMO1 peptide antibodies. With respect to FMO-dependent N-oxygenation of benzydamine and tozasertib and S-oxygenation of methimazole and sulindac sulfide activities, rat and minipig liver microsomes had high maximum velocity values (Vmax) and high catalytic efficiency (Vmax/Km, Michaelis constant) compared with those for human or monkey liver microsomes. Apparent Km values for recombinantly expressed rat FMO3-mediated N- and S-oxygenations were approximately 10–100-fold those of rat FMO1, although these enzymes had similar Vmax values. The mean catalytic efficiencies (Vmax/Km, 1.4 and 0.4min−1μM−1, respectively) of recombinant human and monkey FMO3 were higher than those of FMO1, whereas Vmax/Km values for rat and minipig FMO3 were low compared with those of FMO1. Minipig liver microsomal FMO1 efficiently catalyzed N- and S-oxygenation reactions; in addition, the minipig liver microsomal FMO1 concentration was higher than the levels in rats, humans, and monkeys. These results suggest that liver microsomal FMO1 could contribute to the relatively high FMO-mediated drug N- and S-oxygenation activities in rat and minipig liver microsomes and that lower expression of FMO1 in human and monkey livers could be a determinant factor for species differences in liver drug N- and S-oxygenation activities between experimental animals and humans.

CDNA Cloning and Functional Analysis of Minipig Uridine Diphosphate-Glucuronosyltransferase 1A1

Uridine diphosphate (UDP)-glucuronosyltransferase 1A1 (UGT1A1) plays important roles in the glucuronidation of various drugs and endogenous substances. Minipigs have been used as experimental animals in pharmacological and toxicological studies, because many of their physiological characteristics are similar to those of humans. In this study, the similarities and differences in enzymatic properties of UGT1A1 between humans and minipigs were precisely identified. Minipig UGT1A1 (mpUGT1A1) cDNA was firstly cloned by the rapid amplification of cDNA ends (RACE) method, and the corresponding protein as well as human UGT1A1 (hUGT1A1) enzyme was expressed in insect cells. Then the kinetics of estradiol at 3-hydroxy position (E-3OH) and 7-ethyl-10-hydroxycamptothecin (SN-38) glucuronidation by recombinant UGT1A1s as well as human and minipig liver microsomes were analyzed. The homology between mpUGT1A1 and hUGT1A1 at the amino acid level was 80.9%. E-3OH and SN-38 glucuronidation by recombinant hUGT1A1 and mpUGT1A1 showed allosteric sigmoidal kinetics. The CL value (29.1 µL/min/mg protein) for E-3OH glucuronidation of mpUGT1A1 was significantly higher (1.4-fold) than that of hUGT1A1, whereas the CL value (0.83 µL/min/mg protein) for SN-38 glucuronidation was significantly lower (27%) than that of hUGT1A1; however, the kinetic models and parameter levels for E-3OH and SN-38 glucuronidation by human and minipig liver microsomes did not parallel those in the respective species. These findings suggest that the enzymatic properties of UGT1A1 are considerably different between humans and minipigs. The information on species differences in UGT1A1 function gained in this study should help with in vivo extrapolation of xenobiotic metabolism and toxicity.

COULD VIBRATIONAL SPECTROSCOPY REVOLUTIONISE QUALITY CONTROL OF HERBAL MEDICINES?

s. PHYTOPHARM 2012 M 114 Obzory po kliniceskoj farmacologii i lekarstvennoj terapii [Reviews of clinical pharmacology and drug therapy] ТОМ 10/2012/2 COULD VIBRATIONAL SPECTROSCOPY REVOLUTIONISE QUALITY CONTROL OF HERBAL MEDICINES? © Viljoen Alvaro, Vermaak Ilze Department of Pharmaceutical Sciences, Faculty of Science, Tshwane University of Technology, Private Bag X680, Pretoria 0001, South Africa Vibrational spectroscopy includes near infrared (NiR) mid-infrared (MiR) and Raman spectroscopy. These methods in combination with multivariate data analysis have proved to be powerful analytical techniques and have been implemented in various industries such as the petrochemical, agricultural and food industries. The quality of herbal medicines is of great concern as many consumers now choose herbal medicines over Western medicines and cases of mistaken identity (substitutions) or adulteration with other species with sometimes dire health consequences or loss of consumer trust in the product due to ineffectiveness has been reported. The quality of herbal medicines is assessed both qualitatively (authentication) and quantitatively through the quantification of specific biomarkers. Traditional analytical methods used to determine quality such as high performance liquid chromatography and mass spectrometry are destructive, expensive and time-consuming. Vibrational spectroscopy methods have major advantages over these methods in that it is simple, rapid, relatively cheap and non-destructive. Various examples of quality control methods developed using vibrational spectroscopy techniques will be described for medicinally and commercially important plant species: to discriminate between closely related plant species (e. g. Agathosma betulina and A. crenulata; Pelargonium reniforme and P. sidoides), a case of mistaken identity (Eriocephalus punctulatus and E. tenuifolius) and as a quantification tool for biomarkers (e. g. pulegone, diosphenol, isomenthone etc. in A. betulina essential oils). hyperspectral imaging uses the platform of vibrational spectroscopy and multivariate data analysis but adds an extra data dimension opening up a range of novel applications in the quality control of herbal medicines such as authentication using whole dried fruits (e. g. Illicium anisatum and I. verum). from these examples it is evident, the revolution has already begun. THE METABOLISM OF LIGNANS FROM FRUCTUS SCHISANDRA © Yin Jun , Cao Yun-Feng , Yang Ling 2 School of Traditional Chinese Medicines, Shenyang Pharmaceutical University, Shenyang 110016, China Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China After metabolites from dibenzocyclooctadiene lignans (Schizandrin, deoxyschizandrin and schisantherin A) of Schisandra chinensis incubated with human liver microsomes were isolated by hPlC and their structures were identified by lC–MS, h-NMR and C-NMR, the study combined correlation analysis, chemical inhibition studies, assays with recombinant CyPs and enzyme kinetics indicated that CyP3A4 was the main hepatic isoform. Three metabolites were isolated by hPlC and their structures were identified to be 8 (R)-hydroxylschizandrin, 2-demethyl-8 (R)-hydroxyl-schizandrin, 3-demethyl-8 (R)-hydroxyl-schizandrin, Rat and minipig liver microsomes were applied to evaluate species differences, and the results showed little difference among the mentioned species. Speculated that deoxyschizandrin might undergo hydroxylation in hlMs, and schizandrin could represent the corresponding metabolite. The data also show that deoxyschizandrin metabolism to form schizandrin specially mediated by CyP3A4, is the principal route of disposition in humans. The specificity, high affinity, and high turnover make deoxyschizandrin hydroxylation an excellent probe drug for CyP3A4 activity in vitro. The metabolic pathway of schisantherin A validated the inhibitory mechanism against CyP3A4 undergoes demethylenenation in the human liver microsomes. iC 50 drift and dynamics test indicate that schisantherin A is inhibitor which shows dependency to NAdP and time, which illustrates that the methylene of schisantherin A produced active intermediate during the catalyzed reaction, thereby presents forceful inhibitory effect against CyP3A4. The lignans containing methylenedioxy abundant in fructus Schisandra probably reduce clinical value thereof. in mice liver microsomes, schisantherin A, schizandrol b and schizandrin b showed strong inhibition against metablism schizandrin and deoxschizandrin. Our research results indicate that why deoxyschizandrin presented forceful anti-multidrug resistance in vitro but reduced or vanished in vivo, and the extract of fructus Schisandra containing deoxyschizandrin presents forceful anti-multidrug resistance in vivo.

New model system for testing effects of flavonoids on doxorubicin-related formation of hydroxyl radicals

Doxorubicin belongs to anthracycline cytotoxic drugs and it is widely used as a major therapeutic agent in the treatment of various types of tumors. However,its therapeutic use is limited by the development of myelosuppression and cardiotoxicity after a specific cumulative dose is reached. The aim of this study was to investigate the effect of flavonoids, either natural or synthetic on doxorubicin-mediated formation of oxidative stress implicated in doxorubicin toxicity. Doxorubicin caused a concentration-dependent increase in the formation of hydroxyl radicals in minipig liver microsomes used as an in-vitro model system. When bacterial membranes heterologously expressing human NADPH cytochrome-P450 oxidoreductase were incubated with doxorubicin, formation of the superoxide radical under aerobic conditions and the doxorubicin–semiquinone radical under anaerobic conditions was detected. Forty different flavonoids were tested for their potency to prevent NADPH-induced or Fe2+-induced peroxidation of lipids in the microsomal system. According to the results, seven flavonoids were selected for evaluation of their potency to inhibit doxorubicin-dependent formation of hydroxyl radicals assessed by electron spin resonance. Myricetin, fisetin, and kaempferol were found to produce a significant protective effect against hydroxyl radicals in the minipig liver microsomal system. In conclusion, this study shows the use of a novel cost-effective in-vitro model system for preselection of antioxidants for testing of their protective effects against toxicity of anthracyclines and potentially other oxidative stress-inducing chemicals.

CYP3A catalyses schizandrin biotransformation in human, minipig and rat liver microsomes

Schizandrin is recognized as the major absorbed effective constituent of Fructus schisandrae, which is extensively applied in Chinese medicinal formula. The present study aimed to profile the phase I metabolites of schizandrin and identify the cytochrome P450 (CYP) isoforms involved.After schizandrin was incubated with human liver microsomes, three metabolites were isolated by high-performance liquid chromatography (HPLC) and their structures were identified to be 8(R)-hydroxyl-schizandrin, 2-demethyl-8(R)-hydroxyl-schizandrin, 3-demethyl-8(R)-hydroxyl-schizandrin, by liquid chromatography-mass spectrometry (LC-MS), 1H-nuclear magnetic resonance (NMR), and 13C-NMR, respectively. A combination of correlation analysis, chemical inhibition studies, assays with recombinant CYPs, and enzyme kinetics indicated that CYP3A4 was the main hepatic isoform that cleared schizandrin. Rat and minipig liver microsomes were included when evaluating species differences, and the results showed little difference among the species.In conclusion, CYP3A4 plays a major role in the biotransformation of schizandrin in human liver microsomes. Minipig and rat could be surrogate models for man in schizandrin pharmacokinetic studies. Better knowledge of schizandrin’s metabolic pathway could provide the vital information for understanding the pharmacokinetic behaviours of schizandrin contained in Chinese medicinal formula.

Ultra-performance liquid chromatographic–electrospray mass spectrometric determination (UPLC-ESI-MS) of O-demethylated metabolite of paeonol in vitro: Assay development, human liver microsome activities and species differences

A simple and sensitive method for determination of the O-demethylation activity of rat, dog, minipig, and human liver micrsomes toward paeonol using ultra-performance liquid chromatography with mass detection (UPLC-MS) has been developed. The method uses chemically synthesized O-demethylated metabolite of paeonol (2,4-dihydroxyacetophenone, DHA) as a standard for method validation. Validation was done with respect to specificity, linearity, detection limit, recovery, stability, precision and accuracy. The chromatographic separation was achieved on a UPLC BEH C 18 column (50 mm × 2.1 mm i.d., 1.7 μm), with phase of acetonitrile–water (ratio 30:70). Selective ion reaction (SIR) monitor was specific for paeonol, DHA and I.S. The method was specific since there were no interference peaks from the reaction matrix. The calibration curve for DHA was linear from 0.5–100 μM with r 2 = 0.9999. The newly developed method has good precision and accuracy. The method was successfully used to determine the kinetics of DHA activities toward paeonol in liver microsomes from different species. Dog liver microsomes (DLMs) were the most active in paeonol O-demethylation (709.7 pmol/min/mg protein) followed by rat liver microsomes (RLMs) (579.6 pmol/min/mg protein), HLMs (569.3 pmol/min/mg protein), and then minipig liver microsomes (PLMs) (417.3 pmol/min/mg protein). The developed method was appropriated for rapid screening paeonol O-demethylation activity in liver microsomes from different species.

Metabolic Profiling and Cytochrome P450 Reaction Phenotyping of Medroxyprogesterone Acetate

Medroxyprogesterone acetate (MPA) is one of the most frequently prescribed progestins for conception, hormone replacement therapy, and adjuvant endocrine therapy. MPA has a low oral bioavailability because of extensive metabolism; however, its metabolism was poorly documented. This study was intended to profile the phase I metabolites of MPA and the cytochrome P450 (P450) isoforms involved. After MPA was incubated with human liver microsomes and the NADPH-generating system, five main metabolites (namely M-1, M-2, M-3, M-4, and M-5) were isolated by high-performance liquid chromatography. Three major metabolites (M-2, M-4, and M-3) were tentatively identified to be 6beta-, 2beta-, and 1beta-hydroxy MPA by liquid chromatography/mass spectrometry and (1)H nuclear magnetic resonance. By consecutive metabolism of purified M-2, M-3, and M-4, M-1 and M-5 were proposed to be 2beta-, 6beta-dihydroxy MPA, and 1,2-dehydro MPA, respectively. CYP3A4 was identified to be the isoform primarily involved in the formation of M-2, M-3, and M-4 in studies with specific P450 inhibitors, recombinant P450s, and correlation analysis. Rat and minipig liver microsomes were included evaluating species differences, and the results showed little difference among the species. In human liver microsomes, the K(m) values ranged from 10.0 to 11.2 muM, and the V(m) values ranged from 194 to 437 pmol/min/mg for M-2, M-3, and M-4. In conclusion, CYP3A4 was the major P450 isoform involved in MPA hydroxylation, with 6beta, 2beta, and 1beta being the possible hydroxylation sites. Minipig and rat could be the surrogate models for man in MPA pharmacokinetic studies.

Predictive value of animal models for human cytochrome P450 (CYP)-mediated metabolism: A comparative study in vitro

One major challenge in drug development is defining of the optimal animal species to serve as a model of metabolism in man. The study compared the hepatic drug metabolism characteristics of humans and six widely used experimental animal species. Classical in vitro model enzyme assays with known human cytochrome P450 (CYP) enzyme selectivity were employed and optimized to target human hepatic CYP forms. The profile of CYP activities best resembling the human was seen in mouse followed by monkey, minipig, and dog liver microsomes, with rats displaying the most divergent. The widest interindividual variability was found in CYP3A-mediated midazolam α-hydroxylase, and omeprazole sulphoxidase activities in human and monkey liver microsomes. These data demonstrate that if hepatic xenobiotic-metabolizing characteristics were to be the sole reason for the selection of animal species for toxicity studies, then the rat might not be the most appropriate model to mimic human CYP activity patterns.

Cytochrome P450 destruction by benzene metabolites 1,4-benzoquinone and 1,4-hydroquinone and the formation of hydroxyl radicals in minipig liver microsomes

Reactive metabolites of benzene 1,4-benzoquinone and 1,4-hydroquinone exert their toxic effects through covalent and/or oxidative damage to DNA and proteins. Since minipigs have been proposed as a suitable model species in toxicological and pharmacological research, the aim of this study was to explore mechanisms by which catechol, 1,4-hydroquinone and 1,4-benzoquinone destroy cytochrome P450 (P450) and induce oxidative stress in minipig liver microsomes. Our second goal was to assess the usefulness of minipig liver microsomes as a model system for the testing of the production of oxidative stress by clinically relevant quinone-containing compounds, e.g. anthracyclines. Of the three benzene metabolites tested, the highest P450 destruction was caused by 1,4-benzoquinone. This destructive effect did not correlate with the production of hydroxyl radicals as measured by ESR spin trapping which was the highest in samples containing 1,4-hydroquinone. Our results confirm previous findings that 1,4-benzoquinone exerts its effect mainly by direct attack on macromolecules while 1,4-hydroquinone rather stimulates the production of reactive oxygen species. Doxorubicin stimulated the production of hydroxyl radicals and the destruction of P450 similarly as 1,4-hydroquinone. Minipig liver microsomes should be further tested as a possibly suitable model system for the testing of potential modulators of the toxicity of doxorubicin.

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.

MINIPIG CYTOCHROME P450 2E1: COMPARISON WITH HUMAN ENZYME

Cytochrome P450 2E1 was isolated from minipig liver microsomes. The protein has been cloned and the respective cDNA sequenced (GenBank Accession Number AY581116). Minipig CYP2E1 is two residues shorter than its human ortholog. The only difference between pig and minipig sequence is the presence of aspartic acid residue in position 346 contrary to valine in the pig enzyme. Minipig CYP2E1 was shown to be able to convert two prototypical substrates of human CYP2E1, chlorzoxazone and p-nitrophenol, to the respective metabolites. The experiments performed with both the liver microsomal fraction and reconstituted systems with human or minipig CYP2E1 confirmed the similarity of both enzymes. Inhibition with diethyldithiocarbamate gave comparable Ki values for minipig as well as for the human CYP2E1. The results indicate that the systems containing minipig CYP2E1 may be used to model the respective CYP2E1-catalyzed reactions of drug metabolism in humans.

563 The use of minipig liver microsomes in the study of interactions between cytochromes P450 and metabolites of benzene

563 The use of minipig liver microsomes in the study of interactions between cytochromes P450 and metabolites of benzene