Designing a novel photoinduced electron transfer-based small-molecule fluorescent probe specific for CYP3A isozymes.

Cytochrome P450 (CYPs) are oxidoreductases distributed in various tissues in plants and animals. Among the CYP families, CYP3A is the most abundant in vivo, particularly in humans, and it is involved in the metabolism of many drugs. It is crucial to measure CYP3A activity for both pharmaceuticals and agrochemicals because inhibition or induction of this enzyme can seriously affect the occurrence of toxicity or efficacy. In the present study, a novel fluorescent probe, 6-(2,5-bis(trifluoromethyl)benzyloxy)-9-(4-methoxy-2-methylphenyl)-3H-xanthen-3-one (BMX, quantum efficiency: 21%), was designed and synthesized. The design was done by photoinduced electron transfer strategy. BMX was specifically metabolized only using CYP3A to generate 2-Me-4-MeO TokyoGreen (quantum efficiency: 85%), resulting in strong fluorescence in the presence of CYP3A isozymes. Protein assays using recombinant human, rat, and mouse CYP isozymes demonstrated the selective metabolism of BMX and production of fluorescence only by CYP3A in all species.

Combining Small-Molecule Bioconjugation and Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) to Expose Allostery: the Case of Human Cytochrome P450 3A4.

We report a novel approach to study allostery which combines the use of carefully selected bioconjugates and hydrogen-deuterium exchange mass spectrometry (HDX-MS). This strategy avoids issues related to weak substrate binding and ligand relocalization. The utility of our method is demonstrated using human cytochrome P450 3A4 (CYP3A4), the most important drug-metabolizing enzyme. Allosteric activation and inhibition of CYP3A4 by pharmaceuticals is an important mechanism of drug interactions. We performed HDX-MS analysis on several CYP3A4-effector bioconjugates, some of which mimic the allosteric effect of positive effectors, while others show activity enhancement even though the label does not occupy the allosteric pocket (agonistic) or do not show activation while still blocking the allosteric site (antagonistic). This allowed us to better define the position of the allosteric site, the protein structural dynamics associated with allosteric activation, and the presence of coexisting conformers.

Effects of alcohol-induced increase in CYP2E1 content in human liver microsomes on the activity and cooperativity of CYP3A4.

We investigate the effect of the alcohol-induced increase in the content of CYP2E1 in human liver microsomes (HLM) on the function of CYP3A4. Membrane incorporation of the purified CYP2E1 into HLM considerably increases the rate of metabolism of 7-benzyloxyquinoline (BQ) and attenuates the homotropic cooperativity observed with this CYP3A4-specific substrate. It also eliminates the activating effect of α-naphthoflavone (ANF) seen in some HLM samples. To probe the physiological relevance of these effects, we compared three pooled preparations of HLM from normal donors (HLM-N) with a pooled preparation from ten heavy alcohol consumers (HLM-A). The composition of the P450 pool in all samples was characterized by the mass-spectrometric determination of 11 cytochrome P450 species. The fractional content of CYP2E1 in HLM-A was from 2.0 to 3.4 times higher than in HLM-N. In contrast, the content of CYP3A4 in HLM-A was the lowest among all samples. Despite that, HLM-A exhibited a much higher metabolism rate and a lower homotropic cooperativity with BQ, similar to CYP2E1-enriched HLM-N. To substantiate the involvement of interactions between CYP2E1 and CYP3A4 in these effects, we probed hetero-association of these proteins in CYP3A4-containing Supersomes™ with a technique employing CYP2E1 labeled with BODIPY-618 maleimide. These experiments evinced the interactions between the two enzymes and revealed an inhibitory effect of ANF on their association. Our results demonstrate that the functional properties of CYP3A4 are fundamentally dependent on the composition of the cytochrome P450 ensemble and suggest a possible impact of chronic alcohol exposure on the pharmacokinetics of drugs metabolized by CYP3A4.

Inhibitory effects of astilbin, neoastilbin and isoastilbin on human cytochrome CYP3A4 and 2D6 activities.

Astilbin, neoastilbin and isoastilbin are three flavonoid isomers from Smilacis glabrae Roxb. (S. glabrae). Several studies have shown that consumption of flavonoids can increase the risk of food/drug-drug interaction by affecting the activities of human cytochrome CYP3A4 and 2D6. In the present study, an ultrahigh-performance liquid chromatography and triple quadrupole mass spectrometry method was developed for the determination of the interaction between three flavonoid isomers and two CYPs. Under the optimized reaction conditions, the Km values were 18.9 and 36.4 µM and the Vmax values were 0.02 and 0.20 µM/min for CYP3A4 and 2D6 in vitro, respectively. Astilbin showed the strongest inhibition on CYP3A4, followed by isoastilbin and neoastilbin with IC50 values of 2.63, 3.03 and 6.51 µM. Neoastilbin showed the strongest inhibition on CYP2D6, followed by isoastilbin and astilbin, with IC50 values of 1.48, 11.87 and 14.16 µM, respectively. The three isomers showed reversible inhibition on both enzymes. Neoastilbin and astilbin were noncompetitive type for CYP3A4 and 2D6, isoastilbin was a mixture and noncompetitive type for CYP3A4 and 2D6, respectively. Our study suggests that the three isomers may increase the risk of food/drug-drug interactions by affecting the activities of CYP3A4 and 2D6.

Development and Characterization of MDR1 (Mdr1a/b) CRISPR/Cas9 Knockout Rat Model.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) technology is widely used as a tool for gene editing in rat genome site-specific engineering. Multidrug resistance 1 [MDR1 (also known as P-glycoprotein)] is a key efflux transporter that plays an important role not only in the transport of endogenous and exogenous substances, but also in tumor MDR. In this report, a novel MDR1 (Mdr1a/b) double-knockout (KO) rat model was generated by the CRISPR/Cas9 system without any off-target effect detected. Western blot results showed that MDR1 was completely absent in the liver, small intestine, brain, and kidney of KO rats. Further pharmacokinetic studies of digoxin, a typical substrate of MDR1, confirmed the deficiency of MDR1 in vivo. To determine the possible compensatory mechanism of Mdr1a/b (-/-) rats, the mRNA levels of the CYP3A subfamily and transporter-related genes were compared in the brain, liver, kidney, and small intestine of KO and wild-type rats. In general, a new Mdr1a/b (-/-) rat model has been successfully generated and characterized. This rat model is a useful tool for studying the function of MDR1 in drug absorption, tumor MDR, and drug target validation.

Plasma extracellular nanovesicle (exosome)-derived biomarkers for drug metabolism pathways: a novel approach to characterize variability in drug exposure.

AIMS: Demonstrate the presence of cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) proteins and mRNAs in isolated human plasma exosomes and evaluate the capacity for exosome-derived biomarkers to characterize variability in CYP3A4 activity. METHODS: The presence of CYP and UGT protein and mRNA in exosomes isolated from human plasma and HepaRG cell culture medium was determined by mass spectrometry and reverse transcription-polymerase chain reaction, respectively. The concordance between exosome-derived CYP3A4 biomarkers and midazolam apparent oral clearance (CL/F) was evaluated in a small proof-of-concept study involving six genotyped (CYP3A4 *1/*1 and CYP3A5 *3/*3) Caucasian males. RESULTS: Exosomes isolated from human plasma contained peptides and mRNA originating from CYP 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2 J2, 3A4 and 3A5, UGT 1A1, 1A3, 1A4, 1A6, 1A9, 2B4, 2B7, 2B10 and 2B15, and NADPH-cytochrome P450 reductase. Mean (95% confidence interval) exosome-derived CYP3A4 protein expression pre- and post-rifampicin dosing was 0.24 (0.2-0.28) and 0.42 (0.21-0.65) ng ml-1 exosome concentrate. Mean (95% confidence interval) exosome CYP3A4 mRNA expression pre- and post-rifampicin dosing was 6.0 (1.1-32.7) and 48.3 (11.3-104) × 10-11 2-ΔΔCt , respectively. R2 values for correlations of exosome-derived CYP3A4 protein expression, CYP3A4 mRNA expression, and ex vivo CYP3A4 activity with midazolam CL/F were 0.905, 0.787 and 0.832, respectively. CONCLUSIONS: Consistent strong concordance was observed between exosome-derived CYP3A4 biomarkers and midazolam CL/F. The significance of these results is that CYP3A4 is the drug-metabolizing enzyme of greatest clinical importance and variability in CYP3A4 activity is poorly described by existing precision dosing strategies.

Sensitive and simultaneous quantitation of 6ß-hydroxycortisol and cortisol in human plasma by LC-MS/MS coupled with stable isotope dilution method.

CYP3A phenotyping provides a means for personalized drug therapy. We focused our attention on the plasma 6ß-hydroxycortisol (6ß-OHF) to cortisol ratio as an index for CYP3A phenotyping. In the present study, we developed a sensitive and reliable method for the simultaneous determination of 6ß-OHF and cortisol in human plasma using high-performance liquid chromatography/tandem mass spectrometry together with picolinylester derivatization or nonderivatization methods and 6ß-[9,11,12,12-2 H4 ]hydroxycortisol and [1,2,4,19-13 C4 ]cortisol as internal standards for in vivo CYP3A phenotyping in humans. The lower limits of quantification were 38.513 pg/mL for 6ß-OHF and 38.100 pg/mL for cortisol. The relative error and relative standard deviation of the lower limits of quantification were <5% for both methods. The intra-day and inter-day assay reproducibilities of the determined 6ß-OHF and cortisol concentrations were consistent with the actual amounts added as relative errors and relative standard deviations for both methods, which were <5.4% and <3.9%, respectively. Both methods were applied for the quantification of plasma 6ß-OHF and cortisol concentrations in healthy subjects taking oral contraceptives. The absolute concentrations and time course of 6ß-OHF and cortisol were found to be consistent when measured using the 2 methods. The ratio as an index for in vivo CYP3A activity decreased after 21 days of taking oral contraceptives for both methods. To the best of our knowledge, this is the first study reporting the detailed investigation of accuracy and precision in the simultaneous measurement of 6ß-OHF and cortisol in human plasma using liquid chromatography/tandem mass spectrometry coupled with stable isotope dilution method, which can be applied to CYP3A phenotyping.

The expression levels of CYP3A4 and CYP3A5 serve as potential prognostic biomarkers in lung adenocarcinoma.

Lung adenocarcinoma remains to be a high-mortality disease with few effective prognostic biomarkers. Novel biomarkers are urgently demanded to supplement the current prognostic biomarkers. Here, we explored the prognostic value of CYP3A4 and CYP3A5 in lung adenocarcinoma. The tissue microarray was made up of lung adenocarcinoma samples and corresponding normal lung tissues from Nanjing Medical University affiliated Cancer Hospital Tissue Bank. The expression of CYP3A4, together with CYP3A5, was detected by the chip data from Gene Expression Omnibus datasets and immunohistochemistry staining of the tissue microarray. Then, we assessed the relationships between CYP3A4 or CYP3A5 expression level and clinicopathological factors to estimate the clinical significance. Kaplan-Meier curves were applied to analyze the prognosis. Univariate and multivariate Cox analyses were subsequently applied to identify the independent prognostic factors. Immunohistochemistry staining results showed that by comparison with matched normal tissues, CYP3A4 was frequently hyper-expressed in lung adenocarcinoma tissues while CYP3A5 was hypo-expressed, which was consistent with the Gene Expression Omnibus analysis. Kaplan-Meier analysis indicated that high-CYP3A4 or low-CYP3A5 expression level predicted poor survival in lung adenocarcinoma patients. Multivariate Cox analysis found that hypo-expression of CYP3A5 was an independent prognostic factor. Further study revealed that combination of these two markers exhibited a more powerful predictor of poor prognosis, which could target to more accurate survival of lung adenocarcinoma. Our findings indicate that combination of CYP3A4 and CYP3A5 may serve as a novel prognostic biomarker in lung adenocarcinoma, which contribute to the precision of predicting the survival in lung adenocarcinoma.

Avaliação dos mecanismos envolvidos na permeabilidade de fármacos antirretrovirais por meio dos modelos ex vivo (células de Franz) e in vitro (PAMPA) / Evaluation of mechanisms involved in the permeability of antiretroviral drugs through ex vivo (Franz cells) and in vitro (PAMPA) models

Para fármacos administrados por via oral, o controle da extensão e da velocidade de absorção depende basicamente de duas importantes etapas: solubilidade do fármaco nos líquidos fisiológicos e sua permeabilidade através das membranas biológicas. Assim, o Sistema de Classificação Biofarmacêutica (SCB) foi proposto como uma ferramenta para o desenvolvimento de novos fármacos, de novas formulações e para auxiliar nos processos de bioisenção. No entanto, outro fator relacionado à biodisponibilidade e que deve ser considerado nos estudos biofarmacêuticos é o metabolismo. Desta forma, o Sistema de Classificação Biofarmacêutica de Distribuição de Fármacos (SCBDF) foi proposto com a finalidade de classificar os fármacos de acordo com suas características de solubilidade e de metabolismo de modo que seja possível avaliar e predizer o comportamento do fármaco in vivo. O metabolismo tem sido amplamente investigado, sobretudo as enzimas do citocromo P450, as quais estão presentes também nos enterócitos. Além disso, o SCBDF oferece um suporte quanto à avaliação dos mecanismos de permeabilidade envolvidos nos processos de absorção, interações fármaco-fármaco e interações fármaco-alimento. Assim, o presente trabalho teve como objetivo elucidar os mecanismos envolvidos na permeabilidade de fármacos antirretrovirais por meio dos modelos ex vivo (câmaras de difusão vertical tipo Franz) e in vitro (PAMPA, MDCK-MDR1 e microssomas) considerando os aspectos relacionados ao metabolismo intestinal e ao efluxo destes fármacos. Dada a importância da utilização de fármacos antirretrovirais na terapia medicamentosa contra a Síndrome da Imunodeficiência Adquirida (SIDA) e que estes medicamentos são normalmente administrados cronicamente, a compreensão dos mecanismos envolvidos na permeabilidade é de suma importância, uma vez que estes não estão totalmente esclarecidos e poucas informações são encontradas na literatura. Além disso, a biodisponibilidade de fármacos como estavudina, lamivudina e zidovudina indica variação na permeabilidade, necessitando de uma investigação científica mais aprofundada dos processos absortivos. Assim, segmentos de jejuno provenientes de ratos machos Wistar foram utilizados para a avaliação da permeabilidade intestinal dos referidos antirretrovirais considerando a avaliação de efluxo pela glicoproteína-P e o metabolismo intestinal pela CYP3A. De maneira complementar, estudos in vitro com o emprego de membranas artificiais paralelas (PAMPA) e culturas celulares de MDCK-MDR1 foram realizados com a finalidade de auxiliar na elucidação dos mecanismos de permeabilidade dos fármacos antirretrovirais. Além disso, a avaliação do metabolismo dos referidos fármacos foi realizada com o emprego de microssomas a fim de verificar se tais substâncias são substratos de enzimas da família CYP3A e, assim, verificar o impacto do metabolismo intestinal na absorção. Os resultados de permeabilidade obtidos em PAMPA foram: 0,74±0,11 x 10-6 cm/s para a estavudina, 0,25±0,12 x 10-6 cm/s para a lamivudina e 1,14±0,25 x 10-6 cm/s para a zidovudina. Já no modelo ex vivo com o emprego de câmaras de difusão vertical tipo Franz, os resultados foram: 1,56±0,32 x 10-5 cm/s para a estavudina, 1,26±0,27 x 10-5 cm/s para a lamivudina e 2,54±0,49 x 10-5 cm/s para a zidovudina. Portanto, com base nos resultados obtidos a partir dos dois métodos empregados, sugere-se que 30 outro mecanismo de transporte que não envolva a permeabilidade por difusão transcelular passiva possa estar relacionado à permeabilidade dos fármacos antirretrovirais. Com relação aos estudos de efluxo, os resultados obtidos a partir dos experimentos realizados em câmaras de difusão vertical tipo Franz demonstraram o aumento significativo da permeabilidade dos três antirretrovirais quando o inibidor de P-gp foi empregado, sendo: de 15,6 x 10-6 para 42,5 x 10-6 cm/s para a estavudina, de 12,6 x 10-6 para 37,5 x 10-6 cm/s para a lamivudina e de 25,4 x 10-6 para 56,6 x 10-6 cm/s para a zidovudina. Em culturas celulares MDCK-MDR1, os resultados de permeabilidade foram utilizados para a obtenção das razões entre as direções B→A e A→B. Os valores de Papp na condição inibida para os fármacos estudados apresentaram razão menor do que 1. Já a razão B→A/A→B para cada fármaco nos ensaios sem inibidor apresentou-se igual ou maior que 2, evidenciando a interação fármaco-transportador. Com base nisso, o modelo ex vivo com o emprego de segmentos intestinais em câmaras de difusão vertical tipo Franz apresentou-se adequado na avaliação do mecanismo de efluxo dos fármacos antirretrovirais, o que foi confirmado com os estudos realizados em MDCK-MDR1. Assim, os fármacos antirretrovirais estudados apresentaram interação significativa com a P-gp. Em relação aos estudos de metabolismo realizados em câmaras de difusão vertical tipo Franz, os resultados demonstraram grande variação na permeabilidade dos três antirretrovirais quando o inibidor de CYP3A foi empregado, sendo: de 15,6 x 10-6 para 23,5 x 10-6 cm/s para a estavudina, de 12,6 x 10-6 para 27,3 x 10-6 cm/s para a lamivudina e de 25,4 x 10-6 para 40,5 x 10-6 cm/s para a zidovudina. Já no modelo que emprega microssomas, os resultados de metabolização na ausência e na presença de inibidor de CYP3A foram: de 16,56% para 19,79% para a estavudina, de 14,56% para 15,55% para a lamivudina e de 17,85% para 16,48% para a zidovudina. Com base nisso, sugerese o emprego de microssomas para a determinação de metabolismo, uma vez que o método ex vivo empregado demonstrou grande variação entre os valores obtidos. Desta forma, observou-se que, para cada fármaco, não houve influência significativa no metabolismo pré-sistêmico relacionado às enzimas do complexo CYP3A, o que indica que a absorção oral das referidas substâncias não é limitada por tais enzimas. Portanto, a utilização dos diferentes métodos empregados no desenvolvimento do presente trabalho permitiu compreender os mecanismos envolvidos no transporte dos fármacos antirretrovirais, o que se torna de grande relevância nas etapas de desenvolvimento farmacêutico de novas moléculas e na compreensão de eventos clínicos ainda não esclarecidos atualmente

For orally administered drugs, control of the extent and rate of absorption depends on two important steps: solubility of the drug in physiological liquids and their permeability across biological membranes. Thus, the Biopharmaceutics Classification System (BCS) has been proposed as a tool for the development of new drugs, new formulations and aid in the biowaiver processes. However, another factor related to bioavailability that should be considered in biopharmaceutic studies is the metabolism. Thus, the Biopharmaceutics Drug Disposition Classification System (BDDCS) has been proposed for drug classification according to their solubility and metabolism characteristics, so it is possible to evaluate and predict the in vivo behavior of a compound. Metabolism has been extensively investigated, especially cytochrome P450 enzymes, which are also expressed in enterocytes. Besides, BDDCS provides support in evaluating the permeability mechanisms involved in the absorption processes, drug-drug interactions and drug-food interactions. Thus, the present study aimed to evaluate the mechanisms of permeability of antiretroviral drugs through the ex vivo (Franz cells) and in vitro (PAMPA, MDCK-MDR1 and microsomes) models considering aspects related to the intestinal metabolism and efflux of these drugs. Given the importance of the use of antiretroviral drugs in drug therapy against Acquired Immune Deficiency Syndrome (AIDS) and that these drugs are usually administered in a long-term way, understanding the mechanisms involved in the permeability is of a great importance, since they are not totally elucidated and no information is found in the literature. In addition, drugs as stavudine, lamivudine and zidovudine indicate variation in the permeability, which require further scientific investigation of absorptive processes. Thus, jejunum segments from rats were used to evaluate the intestinal permeability of these antiretroviral drugs, considering the evaluation of efflux by P-glycoprotein and intestinal metabolism by CYP3A. In a complementary manner, in vitro studies using parallel artificial membranes (PAMPA) and cell cultures MDCK-MDR1 were performed to aid in the elucidation of the permeability mechanisms of antiretroviral drugs. Also, the evaluation of the metabolism was carried out using microsomes to verify if such substances are substrates of CYP3A, and verify the impact of the intestinal metabolism in the absorption. The permeability results obtained in PAMPA were: 0.74±0.11x10-6 cm/s for stavudine, 0.25±0.12x10-6 cm/s for lamivudine and 1.14±0.25x10-6 cm/s for zidovudine. In ex vivo method using the intestinal segments in Franz cells, the results were: 1.56±0.32x10-5 cm/s for stavudine, 1.26±0.27x10-5 cm/s for lamivudine and 2.54±0.49x10-5 cm/s for zidovudine. Thus, based on the results obtained from these two methods, it is suggested that the antiretroviral drugs present other transport mechanism that is different from transcellular passive diffusion. For efflux studies, results obtained from experiments performed in Franz cells shown the increase of the permeability of the three antiretroviral drugs when the P-gp inhibitor was used: from 15.6x10-6 to 42,5x10-6 cm/s for stavudine, from 12.6x10-6 cm/s to 37.5x10-6 cm/s for lamivudine, and 25.4x10-6 to 56.6x10-6 cm/s for zidovudine. In MDCK-MDR1, the permeability results were used for obtaining ratio values between the directions B→A and A→B. The Papp values obtained with 33 inhibitor shown a ratio less than 1. For ratio B→A/A→B for each drug in experiments without inhibitor, the values obtained was equal or greater than 2, which shows the interaction between drug and transporter. Based on that, the ex vivo model using intestinal segments in Franz cells seems to be adequate for evaluation of efflux mechanism of antiretroviral drugs, which was confirmed by MDCK-MDR1 studies. Thus, the antiretroviral drugs presented interaction with P-gp. For metabolism studies in intestinal segments in Franz cells, a wide range of standard deviation was observed for the three antiretroviral drugs when the CYP3A inhibitor was used: from 15.6x10-6 cm/s to 23.5x10-6 cm/s for stavudine, from 12.6x10-6 cm/s to 27.3x10-6 cm/s for lamivudine, and from 25.4x10-6 cm/s to 40.5x10-6 cm/s for zidovudine. In experiments in microsomes, the results of metabolization in the absence and presence of CYP3A inhibitor were: from 16.56 to 19.79% for stavudine, from 14.56 to 15.55% for lamivudine and from 17.85 to 16.48% for zidovudine. Based on that, it is suggested the use of microsomes for metabolism evaluation, since the ex vivo method presented high variability between the results obtained. For each drug, no significative influence in pre-systemic metabolism related to CYP3A enzymes was observed, which indicates that the oral absorption of the drugs is not limited by these enzymes. The use of different methods in this work allowed to understand the mechanisms involved in the transport of antiretroviral drugs, which is of a great relevance in drug development and in the understanding of clinical events currently not clarified

Development of a Specific Substrate-Inhibitor Panel (Liver-on-a-Chip) for Evaluation of Cytochrome P450 Activity.

We developed a cytochrome P450 substrate-inhibitor panel for preclinical in vitro evaluation of drugs in a 3D histotypical microfluidic cell model of human liver (liver-on-a-chip technology). The concentrations of substrates and inhibitors were optimized to ensure reliable detection of the principal metabolites by HPLC-mass-spectroscopy. The selected specific substrate-inhibitor pairs, namely bupropion/2-phenyl-2-(1-piperidinyl)propane) for evaluation of CYP2B6B activity, tolbutamide/sulfaphenazole for CYP2C9, omeprazole/(+)-N-benzylnirvanol for CYP2C19, and testosterone/ketoconazole for CYP3A4, enable reliable evaluation of the drug metabolism pathway. In contrast to animal models characterized by species-specific expression profile and activity of cytochrome P450 isoforms, our in vitro model reflects the metabolism of human hepatocytes in vivo.

Identification of stable and reactive metabolite(s) of nelfinavir in human liver microsomes and rCYP3A4.

The present study was performed to detect trace level stable and reactive metabolites of nelfinavir in human liver microsomes and rCYP3A4. Initially, chromatographic and MS parameters were optimized and fragmentation pattern of the drug was delineated. The structures of metabolites were then elucidated by comparison of their MS/MS fragmentation patterns with the drug. A total of thirty nine stable metabolites were formed, of which twelve were established to be monohydroxylated, eighteen dihydroxy, two dehydrogenated, and one each a diquinone, keto, carboxylic, N-deacylated, dealkylated, oxo and dehydro monohydroxyl metabolite. Previously, a biotransformation product with hydroxylation at tert-butyl group of nelfinavir is reported as an active metabolite of the drug. In our case, ortho-diquinone and N-oxide metabolites were detected, which are known to be reactive in nature. However, these metabolites did not show any interaction with nucleophiles, possibly due to steric hindrance at the site of interface.

Flaxseed reduces the pro-carcinogenic micro-environment in the ovaries of normal hens by altering the PG and oestrogen pathways in a dose-dependent manner.

The objective of the present study was to find the optimum dose of flaxseed that would decrease PG and alter oestrogen pathway endpoints implicated in ovarian cancer. In the study, four groups of fifty 1.5-year-old chickens were fed different amounts of flaxseed (0, 5, 10 or 15% of their total diet) for 4 months and were then killed to collect blood and tissues. Levels of flaxseed lignan metabolites, Enterolactone (EL) and Enterodiol (ED) were measured in the serum, liver and ovaries by liquid chromatography-MS/MS, and n-3 and n-6 fatty acid (FA) levels were measured by GC. The effects of the varied flaxseed doses were assessed by measuring levels of PGE2 and oestrogen metabolites (16-hydroxyestrone (16-OHE1) and 2-hydroxyestrone (2-OHE1)) as well as by analysing the expression of the oestradiol metabolising enzymes CYP3A4 (cytochrome p450, family 3, subfamily A, polypeptide 4), CYP1B1 (cytochrome p450, family 1, subfamily B, polypeptide 1) and CYP1A1 (cytochrome p450, family 1, subfamily A, polypeptide 1) and that of oestrogen receptor α (ERα) in the ovaries. The ratio of n-3:n-FA increased with an increase in flaxseed supplementation and corresponded to a dose-dependent decrease in cyclo-oxygenase-2 protein and PGE2 levels. EL and ED increased in the serum, liver and ovaries with increased concentrations of flaxseed. Flaxseed decreased the expression of ERα in the ovaries. The ratio of 2-OHE1:16-OHE1 in the serum increased significantly in the 15% flaxseed diet, and there was a corresponding increase in CYP1A1 in the liver and decrease in CYP3A4 in the ovaries. CYP1B1 mRNA also decreased with flaxseed diet in the ovaries. The 15% flaxseed-supplemented diet significantly decreased inflammatory PGE2, ERα, CYP3A4, CYP1B1 and 16-OHE1, but it increased CYP1A1 and 2-OHE1, which thus reduced the inflammatory and pro-carcinogenic micro-environment of the ovaries.

Epigallocatechin-3-O-Gallate Protects Against Hepatic Damage and Testicular Toxicity in Male Mice Exposed to Di-(2-Ethylhexyl) Phthalate.

The aim of this study was to examine the effects of epigallocatechin-3-O-gallate (EGCG) on hepatic damage and testicular toxicity in male mice exposed to daily oral administration of di-(2-ethylhexyl) phthalate (DEHP). A mouse model was used to assess the effects of daily intraperitoneal EGCG injection on hepatic and testicular damage. Histological and mitochondrial membrane potential results revealed that EGCG treatment significantly arrested the progression of hepatic damage. EGCG treatment resulted in significant suppression of liver injury (i.e., reduced activities of alanine aminotransferase [ALT] and aspartate aminotransferase [AST]). The development of DEHP-induced hepatic and testicular damage altered the testosterone concentration in mouse serum, which could affect the reproductive ability of male mice. Moreover, EGCG treatment markedly attenuated testes lesions, sperm deformity, and spermatogenic cell apoptosis. At the molecular level, hepatic CYP3A4 expression was substantially reduced by EGCG treatment in mice exposed to DEHP compounds, whereas testicular aromatase expression was increased significantly in testes. Thus, these results demonstrate that EGCG administration may protect against liver damage and reproductive toxicity in males exposed to DEHP.

Short communication: Hepatic progesterone-metabolizing enzymes cytochrome P450 2C and 3A in lactating cows during thermoneutral and heat stress conditions.

Two experiments were performed to determine the effects of heat stress (HS) and insulin on hepatic mRNA abundance of enzymes responsible for metabolizing progesterone [cytochrome P450 2C and 3A (CYP2C and CYP3A)]. To distinguish the direct effects of HS from decreased dry matter intake, cohorts were pair fed (PF) in thermoneutral conditions to match the intake of the HS cows during both experiments. In the first experiment, multiparous late-lactation Holstein cows (n=12, 305±33 d in milk) housed in climate-controlled chambers were subjected to 2 experimental periods: (1) thermoneutral (TN) conditions (18°C, 20% humidity) with ad libitum intake (TN and well fed) for 9 d; and (2) either HS conditions (cyclical temperature 31-40°C, 20% humidity) fed for ad libitum intake (n=6), or TN conditions and PF to match the HS animal (n=6) for 9 d. To evaluate hepatic gene expression during experiment 1, biopsies were obtained at the end of each period. In the second experiment, multiparous mid-lactation Holstein cows (n=12, 136±8 DIM) were housed and fed in conditions similar to those described for the first experiment. Liver biopsies were obtained immediately before and after an insulin tolerance test administered on d 6 of each period. No effects of exogenous insulin were observed on any of the tested variables, nor were there interactions between environment (TN/HS or well fed/PF) and insulin administration. Heat stress decreased hepatic CYP2C expression during both experiments. The relative abundance of CYP3A was not affected by environmental conditions in the late-lactation cows (first experiment), but was reduced by HS in the mid-lactation cows (second experiment). Interestingly, during experiment 2, hepatic CYP3A expression also decreased during PF. These results suggest that HS reduces the capacity of the liver to metabolize progesterone through distinct effects on CYP2C and CYP3A, and that the effects appear to vary based upon stage of lactation. Ultimately, HS may affect reproductive outcomes by reducing the abundance of the enzymes responsible for the breakdown of progesterone. This reduction could serve as a beneficial adaptation for rescuing early embryos or may be detrimental, as it affects feedback mechanisms necessary for proper cyclicity.

A non-invasive CYP3A4 biomarker and body mass index predict cyclosporine dosage requirements in Chinese renal transplant recipients.

An endogenous CYP3A4 biomarker for in vivo metabolism of cyclosporine should be useful for optimizing individual dosage. We aimed to investigate if the combined ratio of endogenous 6ß-hydroxycortisol and 6ß-hydroxycortisone to cortisol and cortisone (HOM) in urine could be used as an endogenous probe for the prediction of cyclosporine dosage requirements in renal transplant recipients. 54 medically stable kidney transplant recipients participated in this study. Morning spot blood and urine samples were gathered. The multiple regression analysis including urinary HOM and body mass index accounted for 73.1% of variability in blood concentration/dose ratio (C/D) of cyclosporine, in which urinary HOM and body mass index contributed 64.9% and 8.2%, respectively. Based on the present approach, individual dosage regimen of CsA could be acquired without therapeutic drug monitoring and the results showed that all of the observed stable doses of CsA were within the predicted range during different post-operative periods. In summary, there is a significant relationship between endogenous CYP3A4 biomarker (assessed by urinary HOM) and in vivo metabolism of cyclosporine in renal transplant recipients. Urinary HOM and body mass index are important predictors of cyclosporine metabolism. Our findings provide clinical implications that the predictive algorithm based on a simple, safe and non-invasive CYP3A4 phenotyping can be anticipated.

Auto-induction of phase I and phase II metabolism of artemisinin in healthy Chinese subjects after oral administration of a new artemisinin-piperaquine fixed combination.

BACKGROUND: Artequick is a relatively inexpensive artemisinin (Qing-hao-su; QHS)-based combination therapy (ACT) that contains QHS and piperaquine (PQ), which has not been widely used because of the decreased concentration level of QHS after repeated oral administrations for five to seven days as a monotherapy. This study was designed to evaluate the potential auto-induction metabolism of QHS in healthy Chinese adults after a two-day oral administration of QHS-PQ. The effect of QHS-PQ on the activity of the CYP2B6 and CYP3A4 was also investigated. METHODS: Fourteen healthy Chinese subjects received two-day oral doses of QHS-PQ (Artequick). A two-drug cocktail consisting of bupropion and midazolam was used to assess the activities of CYP2B6 and CYP3A, respectively. Plasma samples were analysed for QHS and its phase I/II metabolites, probe drugs and their metabolites, using a validated liquid chromatography tandem mass spectrometric (LC-MS) method. RESULTS: Four major phase I metabolites of QHS (M1-M3 and deoxy-QHS) and two subsequent phase II metabolites (M4-M5) were detected in human plasma after oral administrations of QHS-PQ. The AUC0-t of the QHS and its phase I metabolites decreased significantly (P < 0.05) with increased oral clearance (CL/F) after two-day oral doses of QHS-PQ, whereas its phase II metabolites exhibited higher AUC (P < 0.01). The phase I metabolic capability, calculated by the AUC0-t ratio of all phase I metabolites to QHS, increased 1.5-fold after the repeated dose (P < 0.01), and the phase II metabolic capability increased 1.5-fold for M4 and 3.0-fold for M5. The enzyme activity of CYP2B6 and CYP3A4 increased 2.1-fold and 3.2-fold, respectively, after two-day oral doses of QHS-PQ. CONCLUSIONS: The auto-induction of both phase I and phase II metabolism of QHS was present in healthy Chinese subjects after a recommended two-day oral dose of QHS-PQ. The auto-induction metabolism also existed for phase I metabolites of QHS. The enzyme activity of CYP2B6 and CYP3A4 was induced after the two-day oral doses of QHS-PQ. Based on these results, the alternative common three-day regimen for QHS-PQ could probably lead to lower bioavailability of QHS and higher potential of drug-drug interaction caused by the induction of drug-metabolizing enzymes.

Phenotypic and in vivo functional characterization of immortalized human fetal liver cells.

We report the establishment and characterization of immortalized human fetal liver progenitor cells by expression of the Simian virus 40 large T (SV40 LT) antigen. Well-characterized cells at various passages were transplanted into nude mice with acute liver injury and tested for functional capacity. The SV40LT antigen-immortalized fetal liver cells showed a morphology similar to primary cells. Cultured cells demonstrated stable phenotypic expression in various passages, of hepatic markers such as albumin, CK 8, CK18, transcription factors HNF-4α and HNF-1α and CYP3A/7. The cells did not stain for any of the tested cancer-associated markers. Albumin, HNF-4α and CYP3A7 expression was confirmed by reverse transcription polymerase chain reaction (RT-PCR). Flow cytometry showed expression of some progenitor cell markers. In vivo study showed that the cells expressed both fetal and differentiated hepatocytes markers. Our study suggests new approaches to expand hepatic progenitor cells, analyze their fate in animal models aiming at cell therapy of hepatic diseases.

Vitamin D receptor agonist EB1089 is a potent regulator of prostatic "intracrine" metabolism.

BACKGROUND: A contributing factor to the emergence of castrate resistant prostate cancer (CRPC) is the ability of the tumor to circumvent low circulating levels of testosterone during androgen deprivation therapy (ADT), through the production of "intracrine" tumoral androgens from precursors including cholesterol and dehydroepiandrosterone (DHEA). As these processes promote AR signaling and prostate cancer progression their modulation is required for disease prevention and treatment. METHODS: We evaluated the involvement of the vitamin D receptor ligand EB1089 in the regulation of genes with a role in androgen metabolism using the androgen dependent cell lines LNCaP and LAPC-4. EB1089 regulation of androgen metabolism was assessed using QRT-PCR, luciferase promoter assays, western blotting, enzyme activity assays, and LC-MS analyses. RESULTS: EB1089 induced significant expression of genes involved in androgen metabolism in prostate cancer cells. Real-Time PCR analysis revealed that VDR mediated significant regulation of CYP3A4, CYP3A5, CYP3A43, AKR1C1-3, UGT2B15/17, and HSD17B2. Data revealed potent regulation of CYP3A4 at the level of mRNA, protein expression and enzymatic activity, with VDR identified as the predominant regulator. Inhibition of CYP3A activity using the specific inhibitor ritonavir resulted in alleviation of the anti-proliferative response of VDR ligands in prostate cancer cells. Mass spectrometry revealed that overexpression of CYP3A protein in prostate cancer cells resulted in a significant increase in the oxidative inactivation of testosterone and DHEA to their 6-ß-hydroxy-testosterone and 16-α-hydroxy-DHEA metabolites, respectively. CONCLUSIONS: These data highlight a potential application of VDR-based therapies for the reduction of growth-promoting androgens within the tumor micro-environment.

Investigation of the effect of the uneven distribution of CYP3A4 and P-glycoprotein in the intestine on the barrier function against xenobiotics: a simulation study.

CYP3A4 and P-glycoprotein (P-gp) have similar substrate specificities and work together to form an intestinal absorption barrier against xenobiotics. Previous reports have indicated that CYP3A4 expression decreases gradually, whereas P-gp expression increases, from the upper to lower small intestine. The physiological rationale for this uneven distribution of CYP3A4 and P-gp as a barrier against xenobiotics has not been determined. To clarify the effect of these distribution patterns on barrier function, we constructed a mathematical model that included passive membrane permeation, P-gp-mediated apical efflux, and CYP3A4-mediated metabolism, and we simulated the effects of these distribution patterns on the fraction absorbed of co-substrates without changing their overall activities. The simulation showed that the physiological distribution patterns of both CYP3A4 and P-gp result in the lowest fraction absorbed, but not for drugs with low CYP3A4 and high P-gp-mediated clearances. These results suggest that the distribution pattern of CYP3A4 is especially important for the barrier function. On the other hand, physiological distribution pattern of P-gp exerts the maximum barrier function for dual good substrates for P-gp and CYP3A4, but even distribution of P-gp mostly suppresses the intestinal absorption of good P-gp, but poor CYP3A4 substrates.

A high-speed liquid chromatography/tandem mass spectrometry platform using multiplexed multiple-injection chromatography controlled by single software and its application in discovery ADME screening.

RATIONALE: Multiplexed liquid chromatography (LC) coupled with multiple-injection-chromatogram acquisition has emerged as the method of choice for high-speed discovery bioanalysis, because it significantly reduces injection-to-injection cycle time while maintaining the chromatography quality. Historically, systems utilizing this approach had been custom built, and therefore relied on custom software tools to communicate with multiple vendor software for system control, which lacked transferability, flexibility and robustness. METHODS: In this study, we refined a multiplexed bioanalytical system previously reported, by implementing open-deck auto-sampler manifold and multiple-injection-chromatogram acquisition, all on a commercially available system with single software control. RESULTS: As a result of these improvements, the developed LC/tandem mass spectrometry (MS/MS) method on the system was nearly three times faster than the previous method, while demonstrating comparable analytical accuracy, precision and robustness. This system has been evaluated for in vitro ADME screening assays including metabolic stability, CYP inhibition and Caco-2. The biological data generated on the developed system displayed good correlation with those from the previous LC/MS/MS approaches. CONCLUSIONS: The developed platform demonstrated applicability to the in vitro screening assays evaluated and has been successfully implemented to support the high-throughput metabolic stability assay, with a significantly improved bioanalytical throughput, capacity and data turnaround.