LC/MS分析大鼠体内氧化苦参碱及其主要代谢物LC/MS分析大鼠体内氧化苦参碱及其主要代谢物

目的研究氧化苦参碱在大鼠体内的主要代谢产物。方法以氧化苦参碱和苦参碱为对象优化液相色谱/电喷雾离子阱质谱(LC/ESI-ITMSⁿ)实验条件，分析总结其电喷雾质谱的一级电离规律和二级质谱裂解规律，作为氧化苦参碱大鼠体内代谢物结构分析的依据。健康大鼠腹腔肌注40 mg·kg^-1氧化苦参碱，收集0~24 h的尿样，尿样中的代谢物经C₁₈小柱进行富集与纯化后，在优化的LC/ESI-ITMSⁿ条件下进样分析。代谢物的结构推导主要依据代谢物的色谱保留时间及其电喷雾离子阱质谱(ESI-ITMSⁿ)电离规律。结果在大鼠尿样中有原药及其6种I相氧化及还原代谢产物，且主要代谢物为苦参碱。未发现II相代谢物。结论本法不仅操作简便、快速，而且灵敏度高、专属性强。该分析技术是研究药物代谢最有效的方法之一。     

HPLC-MSn法鉴定葫芦巴碱及其在大鼠体内的主要代谢产物

目的建立快速灵敏的LC-MSⁿ检测葫芦巴碱及其在大鼠体内代谢物的分析方法。方法以葫芦巴碱对LC-MS²色谱及质谱条件进行优化，分析其电喷雾质谱的一级电离规律和多级质谱裂解规律，以此作为葫芦巴碱大鼠体内代谢物分析鉴定的依据。健康大鼠尾静脉注射8 mg·kg^-1葫芦巴碱，收集0～48 h的尿样，经C₁₈小柱固相萃取分离纯化后，直接采用LC-MSⁿ方法对尿样进行测定。结果根据生物体内药物代谢转化规律及母体药物的色谱-质谱行为规律，在尿样中鉴定出母药及其N-去甲基、N-去甲基环氧化产物，以及母药及其N-去甲基环氧化物的甘氨酸轭合物。结论本方法灵敏、快速、选择性高、专属性好，可用于葫芦巴碱的代谢产物研究。     

四氯化碳体外与大鼠肝微粒体膜作用的研究

向大鼠肝微粒体膜悬液中加入CCl_4(0.05～1μl/ml),可致膜脂过氧化和膜脂流动性降低。二巯基苏糖醇(DTT)和普鲁卡因能拮抗上述效应。CCl_4与微粒体膜作用后,ANS荧光强度降低并出现红移。结果提示,CCl_4诱发的膜脂过氧化可致微粒体膜的物理状态发生改变。     

抗焦虑新药AF-5及其代谢物在人肝微粒体体外温孵体系中代谢研究

目的研究一类抗焦虑新药AF-5及其代谢物(I,II)在人肝微粒体体外温孵体系中代谢情况.方法自制人肝微粒体,用Lowry法测定酶活性为8.79mg·mL^-1.以此配制人肝微粒体体外温孵体系,加入药物,温孵后,提取分离,GC-MS测定.结果鉴定了AF-5在人肝微粒体体外温孵体系中的两个主要代谢物,并阐明了其体外代谢途径为AF-5的4位首先氧化为羟基,然后氧化成羰基.结论AF-5在体外人肝微粒体温孵体系中,100min后完全代谢成羟基代谢物I及羰基代谢物II,以羟基代谢物为主要代谢产物.AF-5代谢物I在人肝微粒体温孵体系中,可转化为代谢物II,而代谢物II在人肝中则不再代谢.     

普罗帕酮在中国健康受试者体内的羟基化代谢产物研究

目的:阐明普罗帕酮羟基化代谢过程的种属及种族差异。方法:选择10 名中国健康受试者单剂量po300 mg 盐酸普罗帕酮片,收集0 ～12 h 的尿样,经液 液萃取后,采用LC/ MSⁿ 技术,对羟基化代谢产物进行选择性离子监测(m/z 358) 和多级全扫描质谱分析。结果:在服药后的尿样中检测到两种羟基化代谢产物,根据质谱数据,推测这两种代谢产物分别为4′-羟基普罗帕酮和5-羟基普罗帕酮。采用微生物转化法结合半制备HPLC制备并分离了4′-羟基普罗帕酮的对照品,通过NMR 证实了其结构。结论:在10 名受试者服药后的尿样中均能检测到代谢物4′ 羟基普罗帕酮和5 羟基普罗帕酮,与文献报道的白人受试者代谢结果相比,中国受试者有较宽的羟基化代谢谱。     

注射用盐酸罂粟碱冻干曲线的研究

目的:通过研究选择一个最佳的冻干曲线用以指导注射用盐酸罂粟碱的制备.方法:通过调整冻干过程中升华干燥的时间和温度,进行冻干曲线的最终确定.结果:按照方案一生产出的成品各项指标检验合格,外观品质均好于方案二.结论:筛选出的冻干曲线生产周期短,生产运行成本低,制得成品检验合格,可用于注射用盐酸罂粟碱的工业化生产.     

9901对犬和人CYP450同工酶体内和体外作用的研究

目的9901系我国正在开发的拥有独立自主知识产权的化合物,前期的药效学结果表明9901有良好的抗寄生虫效果,尤其是对于旋毛虫有很好的杀灭效果。目前,市面上尚无高效杀灭旋毛虫的药物,因此9901的有可能开发为一个新型的抗寄生虫药物。目前对于9901安全性尚未见报道。本试验通过动物体内试验及人肝细胞体外试验,评价9901对肝细胞色素P450同工酶1A2,3A4,2A6和2D6的作用,为指导临床安全用药提供依据。方法本实验使用的犬肝脏来自于长毒试验中的犬。Beagle分为4个剂量,每日口服给予9901连续给药28d,剂量为0、7.5、30和60mg/kg。给药末期取肝脏制备微粒体,用差速离心法制备肝微粒体,分别以非那西汀、睾酮为底物,与辅酶β-NADPH共孵30min后,用HPLC测定代谢产物对氨基乙酰酚、6β-羟基睾酮的生成量以确定CYP450同工酶CYP1A2、CYP3A4的相对活力。进一步用人肝微粒体体外温孵研究9901对CYP1A2、CYP3A4、CYP2A6和CYP2D6的作用。结果试验结果显示出动物的个体差异比较大,与对照组比较经统计分析显示在本试验条件下9901对Beagle犬CYP1A2、CYP3A4无抑制或诱导作用。人肝微粒体温孵体外研究显示在浓度<200μg/ml的情况下,9901对于CYP1A2,2A6,2D6和3A4无诱导/抑制作用。结论9901对于人肝CYP450酶1A2,2A6,2D6,3A4及Beagle犬肝CYP450酶1A2,3A4没有诱导/抑制作用,提示若9901在临床上联合用药是较为安全的药物。     

63Ni-NiCl2在大鼠体内的药代动力学及其组织残留

镍是动物体必需的微量元素,参与核酸和蛋白质的代谢、激素的调节过程,具有刺激生血、促进红细胞再生的功能[1],与激素、蛋白质和脂类的代谢也密切相关.国内相关研究较少,尤其使用同位素标记法研究更少.     

优化罂粟碱注射液给药方式的研究

目的探索优化我院手外科罂粟碱注射液给药方式。方法通过对我院手外科显微外科术后的患者进行自设问卷调查,分析不良反应发生情况,制定优化罂粟碱注射液给药方式的方案,对新给药方式进行同样的问卷调查。结果显微外科术后罂粟碱注射液使用微量泵入的给药方式相比于肌肉注射可减少不良反应的发生。结论通过优化罂粟碱注射液的给药方式,使药物发挥了更好的效果,减少了不良反应,极大地提高了患者用药的依从性。     

尼莫地平在人肝微粒体内的代谢

:采用人肝微粒体在体外研究尼莫地平 (Nim)在人体内的代谢物及代谢途径 . Nim在人肝微粒体内被迅速代谢成 3个代谢物 ,分别是 Nim二氢吡啶环脱氢代谢物 M1,二氢吡啶环侧链脱甲基代谢物M2 ,二氢吡啶环脱氢及其侧链脱甲基代谢物 M3.Nim在人肝微粒体中的最初的两步代谢反应是其二氢吡啶环脱氢氧化及其侧链脱甲基反应 ,两者的代谢产物可以被进一步代谢为代谢物 M3.CYP3A的特异性抑制剂醋竹桃霉素和酮康唑可以抑制Nim的二氢吡啶环脱氢氧化及其侧链脱甲基反应 ,使 Nim的代谢速率明显下降 ,结果提示 CYP3A参与了 Nim在人肝微粒体内的代谢     

Structure elucidation of in vivo and in vitro metabolites of mangiferin

The in vivo and in vitro metabolism of mangiferin was systematically investigated. Urine, plasma, feces, contents of intestinal tract and various organs were collected after oral administration of mangiferin to healthy rats at a dose of 200mg/kg body weight. For comparison, mangiferin was also incubated in vitro with intestinal flora of rats. With the aid of a specific and sensitive liquid chromatography coupled with electrospray ionization tandem hybrid ion trap mass spectrometry (LC-ESI-IT-MS(n)), a total of thirty-three metabolites of mangiferin were detected and their structures were tentatively elucidated on the basis of the characteristics of their precursor ions, product ions and chromatographic retention times. The biotransformation pathways of mangiferin involved deglycosylation, dehydroxylation, methylation, glycosylation, glucuronidation and sulfation.     

Structural elucidation of in vivo and in vitro metabolites of anisodine by liquid chromatography-tandem mass spectrometry

Liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESIMSn) was employed to investigate the in vivo and in vitro metabolism of anisodine. Feces, urine and plasma samples were collected after ingestion of 20 mg anisodine to healthy rats. Feces and urine samples were cleaned up by liquid-liquid extraction and solid-phase extraction procedures (C18 cartridges), respectively. Methanol was added to plasma samples to precipitate plasma proteins. Anisodine was incubated with homogenized liver and intestinal flora of rats in vitro, respectively, followed by extraction with ethyl acetate. LC-MSn was used for the separation and identification of the metabolites using C18 column with mobile phase of methanol/0.01% triethylamine solution (2 mM, adjusted to pH 3.5 with formic acid) (60:40, v/v). The results revealed that five metabolites (norscopine, scopine, alpha-hydroxytropic acid, noranisodine and hydroxyanisodine) and the parent drug existed in feces. Three new metabolites (dimethoxyanisodine, tetrahydroxyanisodine and trihydroxy-methoxyanisodine) were identified in urine. Four metabolites (norscopine, scopine, hydroxyanisodine and anisodine N-oxide) and the parent drug were detected in plasma. Two hydrolyzed metabolites (scopine and alpha-hydroxytropic acid) were found in rat intestinal flora incubation mixture, and two metabolites (aponoranisodine and anisodine N-oxide) were identified in homogenized liver incubation mixture.     

Structural elucidation of in vitro and in vivo metabolites of cryptotanshinone by HPLC-DAD-ESI-MS(n)

The structural elucidation of the in vivo and in vitro metabolites of cryptotanshinone which was the major active component isolated from rhizome of Salvia miltiorrhiza Bunge and possessed significant antibacterial, anti-dermatophytic, antioxidant, anti-inflammatory and anticancer activities was described. Nineteen phase I metabolites and six phase II metabolites of cryptotanshinone were elucidated and identified by a sensitive LC-DAD-ESI-MS(n) method, and their molecular structures were proposed on the basis of the characteristics of their precursor ions, product ions, chromatographic retention time and ultraviolet spectra. The in vivo and in vitro phase I metabolites were mainly biotransformed by four main routes, which were dehydrogenation, hydroxylation, furan ring cleavage and oxidation metabolism, and among these phase I reactions, dehydrogenation was the predominant metabolic pathway. Six in vivo phase II metabolites were identified as the glucuronided and the sulfated conjugates which showed a neutral loss of 176 and 80 Da, respectively. The biotransformation pathways of cryptotanshinone were proposed on the basis of this research.     

Alkylation of RNA by vinyl bromide metabolites in vitro and in vivo

[1,2-¹⁴C]Vinyl bromide was incubated with rat liver microsomes, NADPH, and polyadenylic acid, polycytidylic acid, or RNA, respectively. Part of the adenosine moieties in RNA or in polyadenylic acid were alkylated and labelled 1,N⁶-ethenoadenosine structures were formed. Part of the cytidine moieties were converted into 3,N⁴-ethenocytidine. In addition, a further unidentified cytidine alkylation product was observed which was not seen in experiments using [1,2-¹⁴C]vinyl chloride. When rats were exposed to [1,2-¹⁴C]vinyl bromide, radioactive ethenoadenosine and ethenocytidine were present in hydrolysates of liver RNA. A further alkylation product was observed in the RNA hydrolysates which did not occur in experiments using [¹⁴C]vinyl chloride. The data show that vinyl bromide metabolites alkylate nucleic acids; although in general in this respect vinyl bromide and vinyl chloride behave similarly, some differences are observed in the alkylation behaviour of both compounds.     

罗红霉素主要代谢产物的体外抗菌活性研究

临床上广泛使用的 (E) -罗红霉素在人体内有多种代谢途径。在鉴别和制备其代谢产物的基础上 ,采用二倍稀释法 ,选用 3种生物检测实验标准菌株 ,测定了母体药物和 6种主要代谢产物的体外抗菌活性(MIC,MBC)。结果表明 ,(E) -罗红霉素经代谢转化后 ,生成的 (Z) -罗红霉素异构体的活性略下降 ,(E) -红霉素肟的活性无明显改变 ,(E) - O-单去甲罗红霉素的 MIC未改变 ,而对芽孢杆菌的 MBC有所降低 ,(E) - N-单去甲罗红霉素的活性显著降低 ,(E) -脱红霉糖罗红霉素则基本失活。被测药物及代谢物在不同菌株之间的MIC和 MBC变化趋势基本相同 ,MBC较相应的 MIC大 10 0～ 10 0 0倍左右。     

In vitro and in vivo disposition of 3H-methiothepin in brain tissues

Summary A single treatment with a large dose of methiothepin (20 mg/kg, i.p.) induced, as early as the 2nd day after injection, a significant increase (+20–35%) in the number of specific binding sites for ³H-5-HT in forebrain areas, particularly the hippocampus. Experiments with ³H-methiothepin indicated that the drug remained firmly bound to brain membranes thus maintaining a local concentration high enough to effectively block 5-HT receptors for 10–12 h after its peripheral administration. Accordingly, it can be concluded that the occupancy of central 5-HT receptor sites by methiothepin for several hours was sufficient to induce a supersensitivity phenomenon within the two following days.Although ³H-methiothepin was a useful marker for analyzing the disposition and the kinetics of the 5-HT antagonist in brain tissues, it could not be used as a specific ligand of 5-HT receptors in brain since under in vitro as well as in vivo conditions most of ³H-methiothepin bound to non-specific sites, especially to the lipid component of the membranes.D. L. Nelson is recipient of postdoctoral fellowships from the French Institut National de la Santé et de la Recherche Médicale and from the USPHS (NS 05860-01).     

高效液相色谱法测定盐酸罂栗碱注射液的含量及有关物质

目的:采用高效液相色谱法测定盐酸罂粟碱注射液的含量及有关物质。方法:采用 C_(18)色谱柱(150 mm×4.6 mm,5 μm);流动相为0.5%醋酸铵-1%三乙胺-甲醇(39:1:60);流速1 mL·min~(-1);检测波长238 nm;柱温40℃。结果:盐酸罂粟碱的线性范围为10～160 μg·mL~(-1)(r=0.9999),平均回收率为99.86%(n=9);各杂质峰与主峰达到基线分离。结论:本方法简便、快速,结果准确,重复性好。     

In vitro and in vivo alpha-blocking activity of thymoxamine and its two metabolites

The alpha-adrenoceptor potency of thymoxamine and its two metabolites deacetylthymoxamine and demethyldeacetylthymoxamine were determined on the contraction of rat vas deferens induced by noradrenaline, the blood pressure increase induced by noradrenaline given i.v. to dogs and the contraction of the nictitating membrane induced by electrical stimulation in cats. In vivo the three drugs were administered at 6.35 x 10(-6) mol kg-1 intravenously. Deacetylthymoxamine presented nearly the same alpha-blocking activity as the parent drug. This was ascribed in vivo to the rapid deacetylation of thymoxamine. Demethyldeacetylthymoxamine was less active. In vitro its pA2 was 6.20 +/- 0.09 compared with 6.75 +/- 0.20 for thymoxamine and 6.57 +/- 0.13 for deacetylthymoxamine. In vivo, it was inactive in dog and less active than the other two drugs soon after its administration in the cat. The oral LD 50 values in the mouse for the three drugs were respectively 0.81, 0.71 and 1.14 mmol kg-1 for thymoxamine, deacetylthymoxamine and demethyldeacetylthymoxamine.     

In vitro and in vivo antineoplastic activity of a novel bromopyrrole and its potential mechanism of action

Background and purpose:

Many bromopyrrole compounds have been reported to have in vitro antineoplastic activity. In a previous study, we isolated N-(4, 5-dibromo-pyrrole-2-carbonyl)-L-amino isovaleric acid methyl ester (B6) from marine sponges. Here, we investigated the in vitro and in vivo antineoplastic activity of B6 and its potential mechanism.

Experimental approach:

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to determine the in vitro antineoplastic activity of B6. Flow cytometry, western blot analysis and morphological observations were used to investigate its mechanism of action. A mouse xenograft model was used to determine its in vivo activity.

Key results:

B6 inhibited the proliferation of various human cancer cells in vitro, with highest activity on LOVO and HeLa cells. B6 also exhibited significant growth inhibitory effects in vivo in a xenograft mouse model. Acute toxicity analysis suggested that B6 has low toxicity. B6-treated cells arrested in the G1 phase of the cell cycle and had an increased fraction of sub-G1 cells. In addition, the population of Annexin V-positive/propidium iodide-negative cells increased, indicating the induction of early apoptosis. Indeed, B6-treated cells exhibited morphologies typical of cells undergoing apoptosis. Western blotting showed cleaved forms of caspase-9 and caspase-3 in cells exposed to B6. Moreover, B6-promoted Ca²⁺ release and apoptosis was associated with elevated intracellular Ca²⁺concentration.

Conclusions and implications:

B6 has significant antineoplastic activity in vitro as well as in vivo. It inhibits tumour cell proliferation by arresting the cell cycle and inducing apoptosis. With its low toxicity, B6 represents a promising antineoplastic, primary compound.     

Formation of 3,N4-ethenocytidine moieties in RNA by vinyl chloride metabolites in vitro and in vivo

Rats were exposed to [1,2-¹⁴C] vinyl chloride. Liver RNA was isolated, hydrolyzed, and the nucleosides separated on Aminex-A-6. Besides the physiological bases and 1,N⁶-ethenoadenosine, radioactivity was also incorporated into 3,N⁴-ethenocytidine. Radioactive 3,N⁴-ethenocytidine moieties were also formed on incubation of polycytidylic acid with rat liver microsomes, NADPH and [¹⁴C] vinyl chloride. These alkylation mechanisms are consistent with the mutagenic and cancerogenic properties of vinyl chloride.

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Zusammenfassung RNS wurde aus der Leber von Ratten, die einer Atmosphäre mit [1,2-¹⁴C] Vinylchlorid ausgesetzt waren, isoliert. Nach enzymatischer Hydrolyse und Trennung der Nukleoside auf Aminex-A-6 wurde außer den physiologischen Nukleosiden und dem bereits beschriebenen 1,N⁶-Äthenoadenosin auch ein Radioaktivitätseinbau in 3,N⁴-Äthenocytidin gefunden. Eine Bildung von 3,N⁴-Äthenocytidinresten wurde auch beobachtet, wenn Polycytidylsäure, Rattenlebermikrosomen, NADPH-regenerierendes System und [¹⁴C] Vinylchlorid inkubiert wurden.