Species differences in small intestinal exposure-related epithelial vacuolation in rats and dogs treated with a heteroaryldihydropyrimidine molecule.

Small intestinal epithelial vacuolation induced by a heteroaryldihydropyrimidine compound (HAP-1) was observed in rats but not in dogs at termination in screening toxicity studies, despite the plasma exposure being higher in dogs. To understand the species differences, investigational studies with multiple time points following single dose (SD) and 7-day repeated dose (RD) were conducted in both species at doses resulting in comparable plasma exposures. In rats, epithelial vacuolation in the duodenum and jejunum were observed at all time points. In dogs, transient vacuolation was noted at 8 h post-SD (SD_8h) and 4 h post-RD (RD_4 h), but not at termination (RD_24 h). Special stains demonstrated lipid accumulation within enterocytes in both species and intracytoplasmic inclusion bodies in rats. Transmission electron microscopy identified these inclusion bodies as endoplasmic reticulum (ER) membranous structures. Transcriptomic analysis on jejunal mucosa at SD_8 h and RD_24 h revealed perturbations of lipid metabolism-related genes at SD_8 h in both species, but not at RD_24 h in dogs. ER stress-related gene changes at both time points were observed in rats only. Despite comparable HAP-1 plasma exposures, the duodenum and jejunum tissue concentrations of HAP-1 and acyl glucuronide metabolite were >5- and >30-fold higher in rats than in dogs, respectively. In vitro, similar cytotoxicity was observed in rat and dog duodenal organoids treated with HAP-1. In conclusion, HAP-1-induced intestinal epithelial vacuolation was related to lipid metabolism dysregulation in both species and ER-related injuries in rats only. The species differences were likely related to the difference in intestinal exposure to HAP-1 and its reactive metabolite.

Controlled and Regioselective Ring-Opening Polymerization for Poly(disulfide)s by Anion-Binding Catalysis.

Poly(disulfide)s are an emerging class of sulfur-containing polymers with applications in medicine, energy, and functional materials. However, the constituent dynamic covalent S-S bond is highly reactive in the presence of the sulfide (RS-) anion, imposing a persistent challenge to control the polymerization. Here, we report an anion-binding approach to arrest the high reactivity of the RS- chain end to control the synthesis of linear poly(disulfide)s, realizing a rapid, living ring-opening polymerization of 1,2-dithiolanes with narrow dispersity and high regioselectivity (Mw/Mn ∼ 1.1, Ps ∼ 0.85). Mechanistic studies support the formation of a thiourea-base-sulfide ternary complex as the catalytically active species during the chain propagation. Theoretical analyses reveal a synergistic catalytic model where the catalyst preorganizes the protonated base and anionic chain end to establish spatial confinement over the bound monomer, effecting the observed regioselectivity. The catalytic system is amenable to monomers with various functional groups, and semicrystalline polymers are also obtained from lipoic acid derivatives by enhancing the regioselectivity.

Discovery of a first-in-class orally available HBV cccDNA inhibitor.

BACKGROUND & AIMS: The persistence of covalently closed circular DNA (cccDNA) in infected hepatocytes is the major barrier preventing viral eradication with existing therapies in patients with chronic hepatitis B. Therapeutic agents that can eliminate cccDNA are urgently needed to achieve viral eradication and thus HBV cure. METHODS: A phenotypic assay with HBV-infected primary human hepatocytes (PHHs) was employed to screen for novel cccDNA inhibitors. A HBVcircle mouse model and a uPA-SCID (urokinase-type plasminogen activator-severe combined immunodeficiency) humanized liver mouse model were used to evaluate the anti-HBV efficacy of the discovered cccDNA inhibitors. RESULTS: Potent and dose-dependent reductions in extracellular HBV DNA, HBsAg, and HBeAg levels were achieved upon the initiation of ccc_R08 treatment two days after the HBV infection of PHHs. More importantly, the level of cccDNA was specifically reduced by ccc_R08, while it did not obviously affect mitochondrial DNA. Additionally, ccc_R08 showed no significant cytotoxicity in PHHs or in multiple proliferating cell lines. The twice daily oral administration of ccc_R08 to HBVcircle model mice, which contained surrogate cccDNA molecules, significantly decreased the serum levels of HBV DNA and antigens, and these effects were sustained during the off-treatment follow-up period. Moreover, at the end of follow-up, the levels of surrogate cccDNA molecules in the livers of ccc_R08-treated HBVcircle mice were reduced to below the lower limit of quantification. CONCLUSIONS: We have discovered a small-molecule cccDNA inhibitor that reduces HBV cccDNA levels. cccDNA inhibitors potentially represent a new approach to completely cure patients chronically infected with HBV. IMPACT AND IMPLICATIONS: Covalently closed circular DNA (cccDNA) persistence in HBV-infected hepatocytes is the root cause of chronic hepatitis B. We discovered a novel small-molecule cccDNA inhibitor that can specifically reduce cccDNA levels in HBV-infected hepatocytes. This type of molecule could offer a new approach to completely cure patients chronically infected with HBV.

Effects of Synthetic Astaxanthin on the Growth Performance, Pigmentation, Antioxidant Capacity, and Immune Response in Black Tiger Prawn (Penaeus monodon).

Synthetic astaxanthin is an effective nutritional strategy for improving shrimp body color and promoting growth. However, the optimal amount of astaxanthin in feed also varies with the synthetic technology and purity. In the present study, five diets containing different doses of synthetic astaxanthin (0% (CON), 0.02% (AX0.02), 0.04% (AX0.04), 0.08% (AX0.08), and 0.16% (AX0.16)) were administered to Penaeus monodon (initial body weight: 0.3 ± 0.03 g) for 8 weeks. With an increase in astaxanthin content in feed, weight gain and specific growth rate increased initially and subsequently decreased, with the highest value appearing at AX0.08. Dietary astaxanthin supplementation obviously improved the carapace and muscle color by enhancing astaxanthin pigmentation. Meanwhile, the fatty acid profile was altered by dietary astaxanthin, as evidenced by a decline in palmitic acid proportion, along with an increase in n-3 polyunsaturated fatty acids (n-3 PUFA) contents in muscle. In addition, dietary astaxanthin supplementation regulated prawn's antioxidant capacity. In the hemolymph, the activities of glutamic pyruvic transaminase (GPT) showed a significantly decrease trend with linear effect. The activities of glutamic oxaloacetic transaminase (GOT) and the contents of malondialdehyde (MDA) were first downregulated and then upregulated with significantly quadratic pattern. In the hepatopancreas, the activities of superoxide dismutase (SOD) and the contents of MDA were significantly downregulated with the increase of dietary astaxanthin levels. Reduced glutathione (GSH) contents and catalase (CAT) activities were also significantly decreased in group AX0.08. Correspondingly, astaxanthin decreased GSH and MDA contents under transportation stress. Moreover, the mRNA expression of immune genes (traf6, relish, and myd88) were inhibited by dietary astaxanthin supplementation. Based on the results of polynomial contrasts analysis and Duncan's test, dietary synthetic astaxanthin is a suitable feed additive to improve the growth, body color, antioxidant capacity, and nonspecific immunity of P. monodon. According to the second-order polynomial regression analysis based on the weight gain, the optimal supplementation level of dietary astaxanthin was 90 mg kg-1 in P. monodon.

MdHB1 down-regulation activates anthocyanin biosynthesis in the white-fleshed apple cultivar 'Granny Smith'.

Coloration in apple (Malus×domestica) flesh is mainly caused by the accumulation of anthocyanin. Anthocyanin is biosynthesized through the flavonoid pathway and regulated by MYB, bHLH, and WD40 transcription factors (TFs). Here, we report that the HD-Zip I TF MdHB1 was also involved in the regulation of anthocyanin accumulation. MdHB1 silencing caused the accumulation of anthocyanin in 'Granny Smith' flesh, whereas its overexpression reduced the flesh content of anthocyanin in 'Ballerina' (red-fleshed apple). Moreover, flowers of transgenic tobacco (Nicotiana tabacum 'NC89') overexpressing MdHB1 showed a remarkable reduction in pigmentation. Transient promoter activation assays and yeast one-hybrid results indicated that MdHB1 indirectly inhibited expression of the anthocyanin biosynthetic genes encoding dihydroflavonol-4-reductase (DFR) and UDP-glucose:flavonoid 3-O-glycosyltransferase (UFGT). Yeast two-hybrid and bimolecular fluorescence complementation determined that MdHB1 acted as a homodimer and could interact with MYB, bHLH, and WD40 in the cytoplasm, consistent with its cytoplasmic localization by green fluorescent protein fluorescence observations. Together, these results suggest that MdHB1 constrains MdMYB10, MdbHLH3, and MdTTG1 to the cytoplasm, and then represses the transcription of MdDFR and MdUFGT indirectly. When MdHB1 is silenced, these TFs are released to activate the expression of MdDFR and MdUFGT and also anthocyanin biosynthesis, resulting in red flesh in 'Granny Smith'.

Fast quantification of endogenous carbohydrates in plasma using hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry.

Endogenous carbohydrates in biosamples are frequently highlighted as the most differential metabolites in many metabolomics studies. A simple, fast, simultaneous quantitative method for 16 endogenous carbohydrates in plasma has been developed using hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry. In order to quantify 16 endogenous carbohydrates in plasma, various conditions, including columns, chromatographic conditions, mass spectrometry conditions, and plasma preparation methods, were investigated. Different conditions in this quantified analysis were performed and optimized. The reproducibility, precision, recovery, matrix effect, and stability of the method were verified. The results indicated that a methanol/acetonitrile (50:50, v/v) mixture could effectively and reproducibly precipitate rat plasma proteins. Cold organic solvents coupled with vortex for 1 min and incubated at -20°C for 20 min were the most optimal conditions for protein precipitation and extraction. The results, according to the linearity, recovery, precision, matrix effect, and stability, showed that the method was satisfactory in the quantification of endogenous carbohydrates in rat plasma. The quantified analysis of endogenous carbohydrates in rat plasma performed excellently in terms of sensitivity, high throughput, and simple sample preparation, which met the requirement of quantification in specific expanded metabolomic studies after the global metabolic profiling research.

The Influence of Textile Structure Characteristics on the Performance of Artificial Blood Vessels.

Cardiovascular disease is a major threat to human health worldwide, and vascular transplantation surgery is a treatment method for this disease. Often, autologous blood vessels cannot meet the needs of surgery. However, allogeneic blood vessels have limited availability or may cause rejection reactions. Therefore, the development of biocompatible artificial blood vessels is needed to solve the problem of donor shortage. Tubular fabrics prepared by textile structures have flexible compliance, which cannot be matched by other structural blood vessels. Therefore, biomedical artificial blood vessels have been widely studied in recent decades up to the present. This article focuses on reviewing four textile methods used, at present, in the manufacture of artificial blood vessels: knitting, weaving, braiding, and electrospinning. The article mainly introduces the particular effects of different structural characteristics possessed by various textile methods on the production of artificial blood vessels, such as compliance, mechanical properties, and pore size. It was concluded that woven blood vessels possess superior mechanical properties and dimensional stability, while the knitted fabrication method facilitates excellent compliance, elasticity, and porosity of blood vessels. Additionally, the study prominently showcases the ease of rebound and compression of braided tubes, as well as the significant biological benefits of electrospinning. Moreover, moderate porosity and good mechanical strength can be achieved by changing the original structural parameters; increasing the floating warp, enlarging the braiding angle, and reducing the fiber fineness and diameter can achieve greater compliance. Furthermore, physical, chemical, or biological methods can be used to further improve the biocompatibility, antibacterial, anti-inflammatory, and endothelialization of blood vessels, thereby improving their functionality. The aim is to provide some guidance for the further development of artificial blood vessels.

Chitinase-Like Protein PpCTL1 Contributes to Maintaining Fruit Firmness by Affecting Cellulose Biosynthesis during Peach Development.

The firmness of the flesh fruit is a very important feature in the eating process. Peach fruit is very hard during development, but its firmness slightly decreases in the later stages of development. While there has been extensive research on changes in cell wall polysaccharides during fruit ripening, little is known about the changes that occur during growth and development. In this study, we investigated the modifications in cell wall components throughout the development and ripening of peach fruit, as well as its impact on firmness. Our findings revealed a significant positive correlation between fruit firmness and cellulose content at development stage. However, the correlation was lost during the softening process, suggesting that cellulose might be responsible for the fruit firmness during development. Members of the chitinase-like protein (CTL) group are of interest because of their possible role in plant cell wall biosynthesis. Here, two CTL homologous genes, PpCTL1 and PpCTL2, were identified in peach. Spatial and temporal expression patterns of PpCTLs revealed that PpCTL1 exhibited high expression abundance in the fruit and followed a similar trend to cellulose during fruit growth. Furthermore, silencing PpCTL1 expression resulted in reduced cellulose content at 5 DAI (days after injection), this change that would have a negative effect on fruit firmness. Our results indicate that PpCTL1 plays an important role in cellulose biosynthesis and the maintenance of peach firmness during development.

Selection and Validation of Reliable Reference Genes for Gene Expression Studies in Different Genotypes and TRV-Infected Fruits of Peach (Prunus persica L. Batsch) during Ripening.

Real-time quantitative PCR (RT-qPCR) is a powerful tool to detect and quantify transcription abundance, and the stability of the reference gene determines its success. However, the most suitable reference gene for different genotypes and tobacco rattle virus (TRV) infected fruits was unclear in peach (Prunus persica L. Batsch). In this study, 10 reference genes were selected and gene expression was characterized by RT-qPCR across all samples, including different genotypes and TRV-infected fruits during ripening. Four statistical algorithms (geNorm, NormFinder, BestKeeper, and RefFinder) were used to calculate the stability of 10 reference genes. The geNorm analysis indicated that two suitable reference genes should be used for gene expression normalization. In general, the best combination of reference genes was CYP2 and Tua5 for TRV-infected fruits and CYP2 and Tub1 for different genotypes. In 18S, GADPH, and TEF2, there is an unacceptable variability of gene expression in all experimental conditions. Furthermore, to confirm the validity of the reference genes, the expression levels of PpACO1, PpEIN2, and PpPL were normalized at different fruit storage periods. In summary, our results provide guidelines for selecting reliable reference genes in different genotypes and TRV-infected fruits and lay the foundation for accurate evaluation of gene expression for RT-qPCR analysis in peach.

PpePL1 and PpePL15 Are the Core Members of the Pectate Lyase Gene Family Involved in Peach Fruit Ripening and Softening.

Pectin is the major component in the primary cell wall and middle lamella, maintaining the physical stability and mechanical strength of the cell wall. Pectate lyase (PL), a cell wall modification enzyme, has a major influence on the structure of pectin. However, little information and no comprehensive analysis is available on the PL gene family in peach (Prunus persica L. Batsch). In this study, 20 PpePL genes were identified in peach. We characterized their physicochemical characteristics, sequence alignments, chromosomal locations, and gene structures. The PpePL family members were classified into five groups based on their phylogenetic relationships. Among those, PpePL1, 9, 10, 15, and 18 had the higher expression abundance in ripe fruit, and PpePL1, 15, and 18 were upregulated during storage. Detailed RT-qPCR analysis revealed that PpePL1 and PpePL15 were responsive to ETH treatment (1 g L-1 ethephon) with an abundant transcript accumulation, which suggested these genes were involved in peach ripening and softening. In addition, virus-induced gene silencing (VIGS) technology was used to identify the roles of PpePL1 and PpePL15. Compared to controls, the RNAi fruit maintained greater firmness in the early storage stage, increased acid-soluble pectin (ASP), and reduced water-soluble pectin (WSP). Moreover, transmission electron microscopy (TEM) showed that cell wall degradation was reduced in the fruit of RNAi-1 and RNAi-15, which indicated that softening of the RNAi fruit has been delayed. Our results indicated that PpePL1 and PpePL15 play an important role in peach softening by depolymerizing pectin and degrading cell wall.

Discovery of Novel cccDNA Reducers toward the Cure of Hepatitis B Virus Infection.

Chronic hepatitis B virus (HBV) infection is a worldwide disease that causes thousands of deaths per year. Currently, there is no therapeutic that can completely cure already infected HBV patients due to the inability of humans to eliminate covalently closed circular DNA (cccDNA), which serves as the template to (re)initiate an infection even after prolonged viral suppression. Through phenotypic screening, we discovered xanthone series hits as novel HBV cccDNA reducers, and subsequent structure optimization led to the identification of a lead compound with improved antiviral activity and pharmacokinetic profiles. A representative compound 59 demonstrated good potency and oral bioavailability with no cellular toxicity. In an HBVcircle mouse model, compound 59 showed excellent efficacy in significantly reducing HBV antigens, DNA, and intrahepatic cccDNA levels.

Recent progress of self-powered respiration monitoring systems.

Wearable and implantable medical devices are playing more and more key roles in disease diagnosis and health management. Various biosensors and systems have been used for respiration monitoring. Among them, self-powered sensors have some special characteristics such as low-cost, easy preparation, highly designable, and diversified. The respiratory airflow can drive the self-powered sensors directly to convert mechanical energy of the airflow into electricity. One of the major goals of the self-powered sensors and systems is realizing health monitoring and diagnosis. The relationship between the output signals and the models of respiratory diseases has not been studied deeply and clearly. Therefore, how to find an accurate relationship between them is a challenging and significant research topic. This review summarized the recent progress of the self-powered respiratory sensors and systems from aspects of device principle, output property, detecting index and so on. The challenges and perspectives have also been discussed for reference to the researchers who are interested in the field of self-powered sensors.

Accelerated Skin Wound Healing by Electrical Stimulation.

When the integrity of the skin got damaged, an endogenous electric field will be generated in the wound and a series of physiological reactions will be initiated to close the wound. The existence of the endogenous electric field of the wound has a promoting effect on all stages of wound healing. For wounds that cannot heal on their own, the exogenous electric field can assist the treatment. In this review, the effects of exogenous electrical stimulation on wound healing, such as the inflammation phase, blood flow, cell proliferation and migration, and the wound scarring is overviewed. This article also reviews the new electrical stimulation methods that have emerged in recent years, such as small power supplies, nanogenerators (NGs), and other physical, chemical or biological strategies. These new electrical stimulation methods and devices are safe, low-cost, stable, and small in size. The challenge and perspective are discussed for the future trends of the electrical stimulation treatment in accelerating skin wound healing.

Pulmonary toxicity and metabolic activation of dauricine in CD-1 mice.

Dauricine is the major bioactive component isolated from the roots of Menispermum dauricum D.C. and has shown promising pharmacological activities with a great potential for clinic use. However, the adverse effects and toxicity of the alkaloid are unfortunately ignored. The objective of the current study was to evaluate the toxicity of dauricine in vitro and in vivo. Mice (CD-1) were treated intraperitoneally with dauricine at various doses, and sera and lung lavage fluids were collected after 24 h of treatment. No changes in serum aspartate aminotransferase, alanine aminotransferase, and blood urea nitrogen were noticed, whereas a dose-dependent increase in lactate dehydrogenase activity was observed in lung lavage fluids. Ethidium-based staining studies showed that remarkable cells lost membrane integrity in the lungs of the animals treated with dauricine at 150 mg/kg. Histopathological evaluation of lungs of mice showed that dauricine at the same dose caused significant alveolar edema and hemorrhage. Exposure to dauricine at 40 muM for 24 h resulted in up to 60% cell death in human lung cell lines BEAS-2B, WI-38, and A549. Ketoconazole showed protective effect on the pulmonary injury in mice given dauricine. A quinone methide metabolite of dauricine was identified in mouse lung microsomal incubations, and the presence of ketoconazole in the microsomal incubations suppressed the formation of the quinone methide metabolite. In conclusion, dauricine produced pulmonary injury in CD-1 mice. The pulmonary toxicity appears to depend on the metabolism of dauricine mediated by CYP3A. The electrophilic quinone methide metabolite probably plays an important role in the pulmonary toxicity induced by dauricine.

Detection of protein adduction derived from styrene oxide to cysteine residues by alkaline permethylation.

Styrene oxide-cysteine adduction is predominantly involved in protein covalent modification after exposure in vivo to styrene or styrene oxide. In the present study, we developed an alkaline permethylation- and GC/MS-based approach to detect styrene oxide-derived protein adduction. Permethylation of the protein adducts produced two methylthiophenylethanols, namely 2-methylthio-2-phenyl-1-ethanol and 2-methylthio-1-phenyl-1-ethanol. To improve the permethylation efficiency, reaction conditions, including temperature, time, NaOH strength, and molar ratio of CH(3)I/NaOH, were explored. Under optimized conditions, the yields of the analyte formation resulting from permethylation of authentic standard alpha- and beta-mercapturic acids, representing alpha and beta isomers of cysteine adducts, were 35% and 28%, respectively. Permethylation of styrene oxide-modified bovine serum albumin released the two methylthiophenylethanols with an alpha-/beta-adduction ratio of 1.5. A concentration-dependent increase in both alpha- and beta-adduction was observed in mouse liver microsomes incubated with styrene at various concentrations. CD-1 mice were administered intraperitoneally with styrene at doses of 0, 50, and 400mg/kg daily for 5 days. The formation of protein adducts derived from styrene oxide in whole blood in 400mg/kg group was observed with an alpha/beta ratio of 4.8, suggesting that the reaction of styrene oxide with cysteine residues took place more likely at the alpha-carbon than the beta-carbon of styrene oxide.

Transport and metabolism of flavonoids from Chinese herbal remedy Xiaochaihu- tang across human intestinal Caco-2 cell monolayers.

AIM: To investigate the limiting factors for oral bioavailability of flavonoids derived from the Chinese herbal remedy Xiaochaihu-tang. The investigational flavonoids included baicalin, wogonoside, oroxylin-A 7-O-beta-Dglucopyranosiduronide, liquiritin, liquiritin apioside, isoliquiritin, and isoliquiritin apioside, as well as their aglycones baicalein, wogonin, oroxylin-A, liquiritigenin, and isoliquiritigenin. METHODS: Caco-2 cell monolayers were used and the apparent permeability both in apical to basolateral and basolateral to apical directions was measured for each investigational compound. Meanwhile, chemoinformatics was carried out to provide insight into the mechanism governing the permeability. In addition, carrier-mediated transport with or without inhibitors, as well as the metabolism by conjugation, was also examined with Caco- 2 cell monolayers for the flavonoids. RESULTS: The investigational flavonoid aglycones exhibited favorable membrane permeability, but efficient glucuronidation and/or sulfation by the enterocytes may limit their bioavailability. For the flavonoid glycosides, their poor membrane permeability was found to be caused by high hydrogen-bonding potential. Among the glycosides, oroxylin-A 7-O-beta- D-glucopyranosiduronide, isoliquiritin, and isoliquiritin apioside were transported under the mediation of the efflux transporters multidrug resistance-associated protein and/or P-glycoprotein. CONCLUSION: The limiting factors of oral bioavailability for the flavonoids derived from Xiaochaihu-tang appeared to include poor membrane permeability, significant efflux, and efficient intestinal metabolism by conjugation.

A simultaneously quantitative method to profiling twenty endogenous nucleosides and nucleotides in cancer cells using UHPLC-MS/MS.

Endogenous nucleosides and nucleotides in biosamples are frequently highlighted as the most differential metabolites in recent metabolomics studies. We developed a rapid, sensitive, high-throughput and reliable quantitative method to simultaneously profile 20 endogenous nucleosides and nucleotides in cancer cell lines based on ultra-high performance liquid chromatography-electrospray tandem mass spectrometry (UHPLC- MS/MS) by using a porous graphitic carbon column and basic mobile phase. The results indicated that high pH value of mobile phase containing 0.12% diethylamine (DEA) and 5mM NH4OAC (pH 11.5) was the critical factor to prevent the adsorption of multi-phosphorylated species, and significantly improved peak shape and sensitivity. The optimized method was successfully validated with satisfactory linearity, sensitivity, accuracy, precision, matrix effects, recovery and stability for all analytes. The limit of quantification (LOQ) was in the range of 0.6-6nM (6-60 fmol on column). The validated method was applied to the extract of three epithelial cancer cell lines, and the significant difference in the profiling of the nucleosides and nucleotides among the cancer cell lines enables discrimination of breast cancer cell line from the colon cancer cell line and the lung cancer cell line. This quantified analytical method of 20 endogenous nucleosides and nucleotides in cancer cell lines meets the requirement of quantification in specific expanded metabolomics studies, with good selectivity and sensitivity.

Retrorsine, but not monocrotaline, is a mechanism-based inactivator of P450 3A4.

Retrorsine (RTS) and monocrotaline (MCT) cause severe toxicities via P450-mediated metabolic activation. The screening of mechanism-based inhibitors showed RTS inactivated 3A4 in the presence of NADPH. Unlike RTS, MCT failed to inhibit P450 3A4 and other enzymes tested. Further studies showed the loss of P450 3A4 activity occurred in a time- and concentration-dependent way, which was not recovered after dialysis. Dextromethorphan, a P450 3A4 substrate, protected the enzyme from the inactivation. Exogenous nucleophile glutathione (GSH) and reactive oxygen species scavengers catalase and superoxide dismutase did not protect P450 3A4 from the inactivation. GSH trapping experiments showed both P450 3A4 and 2C19 converted RTS and MCT to the corresponding electrophilic metabolites which could be trapped by GSH to form 7-GSH-DHP conjugate. We conclude that RTS and MCT are metabolically activated by P450 3A4 and 2C19, and that RTS, but not MCT, is a mechanism-based inactivator of P450 3A4.

Oral bioavailability and brain penetration of (-)-stepholidine, a tetrahydroprotoberberine agonist at dopamine D(1) and antagonist at D(2) receptors, in rats.

BACKGROUND AND PURPOSE: (-)-Stepholidine has high affinity for dopamine D(1) and D(2) receptors. The aims of the present study were to examine the oral bioavailability and brain penetration of (-)-stepholidine and to gain understanding of mechanisms governing its transport across the enterohepatic barrier and the blood-brain barrier. EXPERIMENTAL APPROACH: The pharmacokinetics of (-)-stepholidine was studied in rats and microdialysis was used to measure delivery to the brain. These studies were supported by biological measurement of unbound (-)-stepholidine. Membrane permeability was assessed using Caco-2 cell monolayers. Metabolite profiling of (-)-stepholidine in rat bile and plasma was performed. Finally, in vitro metabolic stability and metabolite profile of (-)-stepholidine were examined to compare species similarities and differences between rats and humans. KEY RESULTS: Orally administered (-)-stepholidine was rapidly absorbed from the gastrointestinal tract; two plasma concentration peaks were seen, and the second peak might result from enterohepatic circulation. Due to extensive pre-systemic metabolism, the oral bioavailability of (-)-stepholidine was poor (<2%). However, the compound was extensively transported across the blood-brain barrier, demonstrating an AUC (area under concentration-time curve) ratio of brain : plasma of approximately 0.7. (-)-Stepholidine showed good membrane permeability that was unaffected by P-glycoprotein and multidrug resistance-associated protein 2. In vitro (-)-stepholidine was metabolized predominantly by glucuronidation and sulphation in rats and humans, but oxidation of this substrate was very low. CONCLUSIONS AND IMPLICATIONS: Although (-)-stepholidine exhibits good brain penetration, future development efforts should aim at improving its oral bioavailability by protecting against pre-systemic glucuronidation or sulphation. In this regard, prodrug approaches may be useful.

Liquid chromatography/tandem mass spectrometry for the determination of fluoxetine and its main active metabolite norfluoxetine in human plasma with deuterated fluoxetine as internal standard.

Fluoxetine (F) and its N-demehylated metabolite norfluoxetine (NF) are selective inhibitors of serotonin reuptake in humans. A new sensitive rapid method for the simultaneous determination of F and NF in plasma was established and validated, and was further applied to assess the bioequivalence of two oral formulations of F in 22 healthy Chinese male volunteers who received a single oral dose of each formulation (containing 20 mg of fluoxetine hydrochloride). The new method involves using liquid chromatography/tandem mass spectrometry (LC/MS/MS) in multiple reaction monitoring mode with deuterated fluoxetine (DF) as internal standard. High levels of analytical sensitivity and specificity of MS/MS detection enabled use of a simple liquid-liquid extraction procedure. The combination of a simple sample clean-up procedure and short chromatographic run-time (5 min) considerably increased the productivity of the analytical method. The method was validated for the plasma concentration range 0.27-22 ng/mL for both of the test compounds, and the calibration curves were linear with coefficients of correlation >0.999. The limit of detection was 0.1 ng/mL for plasma F and NF. Taking the plasma sample size (200 micro L) into account the new method for determination of F and NF is more sensitive than those described previously.