利多卡因在人肝微粒体中的体外生物转化

目的 建立人肝微粒体体外生物转化利多卡因的方法。方法分别改变反应体系中人肝微粒体的浓度、生物转化时间、利多卡因的浓度，以高效液相色谱(HPLC)法测定利多卡因及其代谢产物单乙基甘氨二甲基苯酰胺(MEGX)和甘氨二甲基苯酰胺(GX)的含量。结果最佳的生物转化条件为：2.0 mg·L^-1利多卡因在1.0 g·L^-1微粒体中，生物转化60 min。结论该方法快速有效，可用于利多卡因在人肝微粒体体外代谢的研究。     

A Possible Mechanism of Naproxen‐Induced Lipid Peroxidation in Rat Liver Microsomes

Abstract: Previous papers from our laboratory report that naproxen and salicylic acid induced lipid peroxidation in rat liver microsomes, however, the mechanism is still unclear. In the present paper, ferrous iron release, nicotinamide‐adenine dinucleotide phosphate reduced form (NADPH) oxidation and hydrogen peroxide (H₂O₂) formation have been measured to find out which mechanisms are involved in naproxen‐ and salicylic acid‐induced lipid peroxidation. While the increase of ferrous iron release was observed with high concentrations of naproxen, salicylic acid did not stimulate ferrous iron release. Neither of these drugs stimulated NADPH oxidation and H₂O₂ formation. However hexobarbital and perfluorohexane, known as uncouplers of cytochrome P450, stimulated microsomal NADPH oxidation, O₂ consumption, H₂O₂ formation and water (H₂O) formation involving four‐electron oxidase reaction. These results suggest that ferrous iron release contributes to naproxen‐induced microsomal lipid peroxidation and that naproxen and salicylic acid are not uncouplers of cytochrome P450. Apparently H₂O₂ does not play an important role in naproxen‐ and salicylic acid‐induced microsomal lipid peroxidation.     

Effects of Antioxidants on Oxygen Toxicity in Vivo and Lipid Peroxidation in Vitro

Abstract: Convulsions and pulmonary damage result when animals are exposed to hyperbaric oxygen at pressures above about 300 kPa. Several hydroxyl radical scavengers (namely dimethylsulphoxide, dimethylthiourea and mannitol), the iron chelator desferoxamine and the lipid antioxidant butylated hydroxytoluene were tested for possible protection against such hyperbaric oxygen toxicity. Dimethylthiourea and dimethylsulphoxide prolonged the latency to the first convulsion, but, surprisingly, dimethylthiourea very significantly increased pulmonary damage at both pressures used (515 and 585 kPa). Desferoxamine also slightly increased lung damage at 585 kPa. Other antioxidants did not alter neurotoxicity or pulmonary toxicity induced by hyperbaric oxygen at 515 or 585 kPa. The antioxidants were also tested for their ability to inhibit lipid peroxidation (TBARS formation) in vitro. Desferoxamine (5 and 50 μM), and butylated hydroxytoluene (0.1 mM and 1 mM) greatly inhibited TBARS formation in brain and lung homogenates incubated at 37°. None of the hydroxyl radical scavengers affected TBARS levels in homogenates. There was no correlation between in vitro inhibition of lipid peroxidation and in vivo protection against oxygen toxicity.     

Biotransformation of Alprenolol in Dog,Guinea-pig and Rat Liver Microsomes

1. Metabolites of alprenolol were isolated and identified in dog, guinea-pig and rat liver microsomes by means of g.l.c-mass spectrometry and comparison with synthetic reference compounds.

2. The compounds were chromatographed as n-butylboronate derivatives, giving a series of diagnostic ions in the mass spectral fragmentation, which was elucidated by using stable isotopes.

3. Alprenolol was metabolized by aromatic ring hydroxylation, oxidation of the allylic function, and degradation of the isopropylaminopropanol side-chain. Alprenolol and four metabolites were quantified by h.p.l.c. and batch extraction techniques based on radioactivity measurements.

4. Five metabolites were detected in rat and guinea-pig liver microsomes and four in the dog. A species variation in the biotransformation of the allyl function in alprenolol was observed. The metabolite formed by oxidation of the allyl double bond was detected in significant amounts in the guinea-pig, and was also formed in the rat but could not be detected in dog liver microsomes.     

Inhibition of Cumene Hydroperoxide-Induced Lipid Peroxidation by a Novel Pyridoindole Antioxidant in Rat Liver Microsomes

Abstract: The ability of stobadine, a novel pyridoindole antioxidant, to inhibit lipid peroxidation induced by cumene hydroperoxide was investigated in rat liver microsomes. In the micromolar range stobadine effectively inhibited lipid peroxidation as measured by the formation of thiobarbituric acid reactive products. The peroxidation-related degradation of microsomal cytochrome P-450 was prevented by stobadine in the same pattern. Another line of evidence in support of the antioxidant action of stobadine was given by its inhibition of cumene hydroperoxide-induced oxygen consumption in microsomal incubations. Inhibition of lipid peroxidation was not a function of decreased bioactivation of cumene hydroperoxide, as stobadine did not affect the rate of cytochrome P-450 dependent cleavage of cumene hydroperoxide. Neither had stobadine any effect on cytochrome P-450 peroxidase function characterized by the rate of cumene hydroperox-ide-dependent oxidation of TMPD, and no direct spectral interaction with microsomal cytochrome P-450 was observed in the micromolar region. We suggest that it is the ability of stobadine to scavenge alkoxyl and peroxyl radicals that is predominantly responsible for the observed antioxidant effect.     

In Vitro Glucuronidation of Ochratoxin A by Rat Liver Microsomes

Ochratoxin A (OTA), one of the most toxic mycotoxins, can contaminate a wide range of food and feedstuff. To date, the data on its conjugates via glucuronidation request clarification and consolidation. In the present study, the combined approaches of ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), UHPLC-Orbitrap-high resolution mass spectrometry (HRMS) and liquid chromatography-multiple stage mass spectrometry (LC-MSⁿ) were utilized to investigate the metabolic profile of OTA in rat liver microsomes. Three conjugated products of OTA corresponding to amino-, phenol- and acyl-glucuronides were identified, and the related structures were confirmed by hydrolysis with β-glucuronidase. Moreover, OTA methyl ester, OTα and OTα-glucuronide were also found in the reaction solution. Based on these results, an in vitro metabolic pathway of OTA has been proposed for the first time.     

Stabilized Lipid Coated Lipoplexes for the Delivery of Antisense Oligonucleotides to Liver Endothelial Cells In Vitro and In Vivo

We report on the preparation and in vivo/in vitro disposition of antisense ODN encapsulating coated cationic lipoplexes (CCLs), prepared by a procedure essentially developed by Stuart and Allen (Stuart, D.D. and Allen, T.M. (2000) “A new liposomal formulation for antisense oligodeoxynucleotides with small size, high incorporation efficiency and good stability”, Biochim. Biophys. Acta 1463, pp. 219–229). The behavior of untargeted CCLs was compared with CCLs that were targeted to scavenger receptors on liver endothelial cells by covalent coupling of the poly-anion aconitylated human serum albumin (Aco-HSA) to the particle surface.

By means of cryo transmission electron microscopy (cryo-TEM) particles of high electron density could be distinguished from electron-translucent particles, representing high and low ODN encapsulation, respectively. The two populations were separated by sucrose density gradient centrifugation.

Upon injection into rats, the untargeted particles showed long circulating properties with a half-life of >10 h. These untargeted CCLs barely bound to liver endothelial cells in vitro while Aco-HSA CCLs massively and specifically interacted with scavenger receptors on these cells.

With J774 cells, a macrophage cell line expressing scavenger receptors, downregulation of ICAM-1 mRNA levels was achieved when the ODN was specifically delivered by Aco-HSA targeted CCLs.     

Prediction of In Vivo Interaction Between Triazolam and Erythromycin Based on In Vitro Studies Using Human Liver Microsomes and Recombinant Human CYP3A4

Purpose. To quantitatively predict the in vivo interaction betweentriazolam and erythromycin, which involves mechanism-basedinhibition of CYP3A4, from in vitro studies using human liver microsomes(HLM) and recombinant human CYP3A4 (REC). Methods. HLM or REC was preincubated with erythromycin in thepresence of NADPH and then triazolam was added. - and 4-hydroxy(OH) triazolam were quantified after a 3 min incubation and the kineticparameters for enzyme inactivation (k_inact and K _app) were obtained.Drug-drug interaction in vivo was predicted based on aphysiologically-based pharmacokinetic (PBPK) model, using triazolam anderythromycin pharmacokinetic parameters obtained from the literature and kineticparameters for the enzyme inactivation obtained in the in vitro studies. Results. Whichever enzyme was used, triazolam metabolism was notinhibited without preincubation, even if the erythromycin concentrationwas increased. The degree of inhibition depended on preincubationtime and erythromycin concentration. The values obtained for k_inactand K _app were 0.062 min^–1 and 15.9 M (-OH, HLM), 0.055 min^–1and 17.4 M (4-OH, HLM), 0.173 min^–1 and 19.1 M (-OH, REC),and 0.097 min^–1 and 18.9 M (4-OH, REC). Based on the kineticparameters obtained using HLM and REC, the AUCpo of triazolamwas predicted to increase 2.0- and 2.6-fold, respectively, followingoral administration of erythromycin (333 mg t.i.d. for 3 days), whichagreed well with the reported data. Conclusions. In vivo interaction between triazolam and erythromycinwas successfully predicted from in vitro data based on a PBPK modelinvolving a mechanism-based inhibition of CYP3A4.     

Optimization of Lipid Composition in Cationic Emulsion as In Vitro and In Vivo Transfection Agents

Purpose. To enhance in vitro and in vivo transfection activity by optimizing lipid composition of cationic lipid emulsions.Methods. Various emulsion formulations having different cationic lipids as emulsifiers, and additional helper lipids as co-emulsifiers, were prepared. The stability of the emulsion and its complex with DNA was investigated by measuring the particle size change in phosphate buffer saline (PBS) over a period of 20 days. The activity of the emulsions in transfecting pCMV-beta into COS-1 cells in the presence or absence of 80% serum was evaluated. We also evaluated in vivo transfection activity using intravenously administered pCMV-Luc+ as a reporter gene.Results. Among the cationic emulsifiers, 1,2-dioleoyl-sn-glycero-3-trimethylammonium-propane (DOTAP) formed the most stable and efficient emulsion gene carrier. Addition of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) increased in vitro transfection activity, but slightly compromised the stability of the emulsion. The loss was compensated for by including small amounts of Tween 80 in the emulsion. The in vitro and in vivo transfection activities were also increased by adding Tween 80. Even though in vitro transfection activity of liposomes was high in the absence of serum, the transfection activity of emulsions was far greater than that of liposomes in the presence of serum and for in vivo applications.Conclusions. By including DOPE as an endosomolytic agent and Tween 80 as a stabilization agent, the cationic emulsion becomes a more potent gene carrier for in vitro and in vivo applications, especially in the presence of serum.     

In Vitro and In Vivo Study of Solid Lipid Nanoparticles Loaded with Superparamagnetic Iron Oxide

Solid Lipid Nanoparticles (SLN) are already under investigation as a pharmaceutical tool able to change the pharmacokinetic and biodistribution of carried molecules. SLN are able to target drugs to lymph after duodenal administration and to overcome the Blood Brain Barrier (BBB). In this study, superparamagnetic SLN have been prepared, have colloidal size, in vitro analysis showed relaxometric properties similar to Endorem_®. In vivo Magnetic Resonance Imaging (MRI) of the central nervous system (CNS) with both SLN and Endorem_® showed that superparamagnetic SLN have slower blood clearance than Endorem_®. MRI data are consistent with CNS uptake of SLN lasting up to the end of the experiment (135 min). These findings confirm the ability of SLN to overcome the BBB; SLN might be used as a CNS MRI contrast agent.     

Inhibition of ATP-Induced Contraction in the Guinea-pig Urinary Bladder in Vitro and in Vivo

Abstract Contractions were elicited by adenosine 5′-triphosphate (ATP) in the guinea-pig urinary bladder in vitro and in vivo. In isolated detrusor strips, tetrodotoxin (3.1 × 10^-6-4.4 × 10^-5M) did not affect contractions induced by a submaximum concentration (10^-3M) of ATP, nor did atropine (1.7 × 10^-6 - 2.1 × 10^-4M), or the anticholinergic agent PR 197 within the concentration range 2.6 × 10^-8 - 2.6 × 10^-5M. In higher concentrations (5.2 × 10^-5-2.6 × 10^-4M), PR 197 inhibited the ATP-response by 60-70% in a way that was not clearly concentration-related. Isoprenaline (10^-7-2.0 × 10^-5M) and noradrenaline (2.5 × 10^-6-10^-4M) reduced the ATP-induced contractions by up to 79%. The effects of the amines were abolished by propranolol (5.2 × 10^-6-3.8 × 10^-5M). Adenosine, 1.0-2.0 × 10^-2M, reduced the ATP-response by about 50%; in lower concentrations, it had no effect. Nifedipine, 7.8 × 10^-7-1.2 × 10^-5M, reduced the responses by 15-795%. Indomethacin (≤2.0 × 10^-4M), and theophylline (2.0 × 10^-4M) had no consistent effects on ATP-induced contractions. Exposure of the preparations to a calcium-free medium reduced and abolished the ATP-response within 60 min. Intravenous injection of ATP (1 -20 mg/kg) caused a rapid and transient increase in intravesical pressure in the anaesthetized guinea-pig. The effect of ATP (3mg/kg) was reduced by atropine(5-10 mg/kg) by approximately 35%. PR 197 (2.5-5 mg/kg) abolished the ATP-response. Isoprenaline (5-100 pg/kg) caused a 53-95% inhibition that could be blocked by propranolol (1 mg/kg). The inhibiting effect of noradrenaline (10-100,μg/kg) could not be blocked by propranolol (1 mg/kg). Adenosine (1.5-3.0 mg/kg) given immediately before ATP completely inhibited the ATP-response. Nifedipine, 0.1-0.2 mg/kg, reduced the ATP-induced contraction by 34 to 100%. It is concluded that the ATP-induced contraction is elicited by a direct effect on the smooth muscle cell. It can be inhibited non-specifically by drugs with different modes of action.     

Docetaxel-Loaded Solid Lipid Nanoparticles: Preparation,Characterization, In Vitro,and In Vivo Evaluations

The aim of the present study was to prepare, characterize, and evaluate solid lipid nanoparticles (SLNs) containing docetaxel (DTX) to improve the efficacy of this chemotherapeutic agent. SLNs containing DTX (SLN-DTX) were prepared by microemulsion and probe sonication techniques. In vitro cytotoxicity of SLN-DTX compared with Taxotere® (TXT) was evaluated on colorectal (C-26) and malignant melanoma (A-375) cell lines. Cellular uptake experiment was also carried out on C-26 cells. In in vivo tests, tumor inhibitory efficacy and survival were compared with TXT on C-26-implanted BALB/c mice. SLN-DTX particle size was 180 nm and PDI of 0.2 with spherical shape. Encapsulation efficacy was more than 98%. SLN-DTX at concentration of 100 μM caused 100% and 99.9% viability reduction in C-26 and A-375 after 48 and 72 h, respectively. The half maximal inhibitory concentration (IC₅₀) of SLN-DTX on C-26 and A-375 was respectively 0.769 and 28.132 μM after 24 h. DTX cell uptake from SLN-DTX was remarkably higher than TXT. SLN-DTX showed better tumor inhibitory efficacy and survival at a dose of 10 mg/kg versus 10 and 20 mg/kg TXT. In conclusion, the results of the present study showed that the efficacy of SLN-DTX was better than TXT in cell-uptake and in vivo experiments.     

Chrysotile Inhibits Glutathione-Dependent Protection against the Onset of Lipid Peroxidation in Rat Lung Microsomes

Abstract: The glutathione and vitamin E-dependent protection of lipid peroxidation in an NADPH (0.4 mM) and chrysotile (500 μg/ml) containing system were investigated in vitro in rat lung microsomes. Addition of 1 mM glutathione to the above reaction system containing microsomes supplemented with vitamin E (1 nmol/mg protein) reduced lipid peroxidation. Similar protection by glutathione could be observed in normal unsupplemented microsomes though the degree of protection was less pronounced. Addition of free radical scavengers such as, superoxide dismutase (100 units/ml), catalase (150 units/ml), mannitol (1 mM) and β-carotene (0.5 mM) to the reaction system showed an insignificant effect on lipid peroxidation. When the reaction was carried out in absence of glutathione, vitamin E content of peroxidizing microsomes decreased rapidly. In this system a concomitant increase in the activity of microsomal glutathione-S-transferase was observed which may serve as an alternative pathway to detoxify lipid peroxides. Addition of glutathione alone to the reaction system prevented both against the loss in vitamin E content and increase in the activity of glutathione-S-transferase. Supplementation of both vitamin E and glutathione was found to be effective in lowering glutathione-S-transferase activity to that of normal basal level. Our results suggest that chrysotile-mediated stimulation of NADPH-dependent lipid peroxidation may be due to hampering of glutathione-dependent protection which may ultimately exhaust membrane bound vitamin E. Our data further suggest that the lung tissue may have an inbuilt mechanism whereby glutathione-S-transferase may be triggered to cope with the excessive production of lipid peroxides.     

虫草菌丝对肝纤维化小鼠肝脏脂质过氧化的影响

目的:明确虫草菌丝对四氯化碳小鼠肝纤维化的作用及肝脏脂质过氧化的影响.方法:四氯化(碳)皮下注射复制BALB/c小鼠肝纤维化模型,虫草组在造模同时予以虫草菌丝煎剂,直至造模结束.HE染色观察肝组织炎症天狼猩红染色观察肝脏胶原沉积,生化法测定肝组织羟脯氨酸(HyP)、超氧化物歧化酶(SOD)含量,Nothern Blot测定Ⅰ型前(人)原mRNA.结果:与正常小鼠比较,肝纤维化小鼠肝脏肝静脉与汇管区周围肝细胞明显脂肪变性,肝脏胶原沉积,可见纤维(I)隔形成,肝脏Hyp含量及Ⅰ型前胶原基因表达均显著增加,肝脏SOD减少;与模型组比较,虫草组肝脏炎症与胶原沉积减(少)(P＜0.01),Hyp含量及Ⅰ型前胶原mRNA表达明显降低(P＜0.01),SOD活性明显增加(P＜0.01).结论:虫草菌丝制(剂)良好的抗肝纤维化作用,其作用机制可能提高肝脏SOD水平、降低Ⅰ型前胶原mRNA的表达有关.     

In Vitro Covalent Binding of New Brain Tracer,Para 125 I-Amphetamine,to Rat Liver and Lung Microsomes

ABSTRACT

Butachlor action on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities in central nervous tissue of the snail Pita globosa was assayed following the method of ELLMAN et al¹, in vitro by adding butachlor directly (10–100 μ moles), to tissue homogenates and in in vivo by exposing the snails to sub-lethal concentration (26.6 ppm) and taking out the tissue for experimentation at different intervals (3, 6,12, 24 and 48 h) of exposure. The enzyme activities decreased in a dose-dependent manner in vitro, and up to 12–24 h in vivo after which they showed recovery towards the control. The inhibition of cholinesterases by butachlor in vitro indicates a direct action of the herbicide on these enzymes. Presumably butachlor exercises its neurotoxic effects through cholinergic impairment in a way similar to that of organophosphates and carbamates.     

丁基苯酞对缺血脑组织中MDA形成及超氧阴离子自由基产生的抑制作用(英)

局灶性脑缺血6小时再灌注1小时后,缺血脑组织中脂质过氧化产物雨二醛（MDA）的含量明显增加,表明再灌注导致了脂质过氧化损伤。丁基苯酞（dl－NBP,20mg·kg-1,ip）可显著降低缺血皮层组织中MDA的含量。进一步研究表明,dl－NBP,d－NBP和l－NBP皆可抑制黄嘌呤-黄嘌呤氧化酶反应系统中超氧阴离子自由基的形成,此作用可能与其抑制了黄嘌呤氧化酶活性有关。     

丁基苯酞对缺血脑组织中MDA形成及超氧阴离子自由基产生的抑制作用

局灶性脑缺血6小时再灌注1小时后,缺血脑组织中脂质过氧化产物丙二醛（MDA）的含量明显增加,表明再灌注导致了脂质过氧化损伤,丁基苯酞（dl-NBP,20mg.kg^-1.ip)可显著降低缺血皮层组织在MDA的含量。进一步研究表明,dl-NPB,d-NBP和l-NBP皆可抑制黄嘌呤－黄嘌呤氧化酶反应系统中超氧阴离子自由基的形成,此作用可能与其抑制了黄嘌呤氧化酶活性有关。