Cloning and tissue expression of cytochrome P450 2S1, 4V2, 7A1, 7B1, 8B1, 24A1, 26A1, 26C1, 27A1, 39A1, and 51A1 in marmosets

Common marmoset (Callithrix jacchus) is an attractive animal model primate species for potential use in drug metabolism and pharmacokinetic studies. In this study, marmoset cytochrome P450 (P450) 2S1, 4V2, 7A1, 7B1, 8B1, 24A1, 26A1, 26C1, 27A1, 39A1, and 51A1 cDNAs were isolated from marmoset tissues (brains, lungs, livers, kidneys, and jejunums). Deduced amino acid sequences (89–98% homologous) of the marmoset P450 gene suggested similarity of molecular characteristics of marmoset P450s to human counterparts, compared with those of pig, rabbit, and rodents. Phylogenetic analysis using amino acid sequences indicated 11 marmoset P450 forms clustered with those of human and other primate counterparts, suggesting marmoset P450s have an evolutionary close relationship to human and other primate counterparts. Tissue expression patterns of these P450 mRNAs except for P450 7B1 mRNA were generally similar to those of human P450s in the five tissue types analyzed. These results suggest similarity of molecular characteristics for P450 2S1, 4V2, 7A1, 7B1, 8B1, 24A1, 26A1, 26C1, 27A1, 39A1, and 51A1 between marmosets and humans, in addition to the orthologs of human P450 1, 2, 3, and 4 families previously identified and characterized in marmosets.     

Solubility of lovastatin in a family of six alcohols: Ethanol, 1-propanol, 1-butanol, 1-pentanol, 1-hexanol, and 1-octanol

Accurate experimental determination of solubility of active pharmaceutical ingredients (APIs) in solvents and its correlation, for solubility prediction, is essential for rapid design and optimization of isolation, purification, and formulation processes in the pharmaceutical industry. An efficient material-conserving analytical method, with in-line reversed HPLC separation protocol, has been developed to measure equilibrium solubility of lovastatin in ethanol, 1-propanol, 1-butanol, 1-pentanol, 1-hexanol, and 1-octanol between 279 and 313K. Fusion enthalpy DeltaH(fus), melting point temperature, Tm, and the differential molar heat capacity, DeltaC(P), were determined by differential scanning calorimetry (DSC) to be 43,136J/mol, 445.5K, and 255J/(molK), respectively. In order to use the regular solution equation, simplified assumptions have been made concerning DeltaC(P), specifically, DeltaC(P)=0, or DeltaC(P)=DeltaS. In this study, we examined the extent to which these assumptions influence the magnitude of the ideal solubility of lovastatin, and determined that both assumptions underestimate the ideal solubility of lovastatin. The solubility data was used with the calculated ideal solubility to obtain activity coefficients, which were then fitted to the van't Hoff-like regular solution equation. Examination of the plots indicated that both assumptions give erroneous excess enthalpy of solution, H(infinity), and hence thermodynamically inconsistent activity coefficients. The order of increasing ideality, or solubility of lovastatin was butanol>1-propanol>1-pentanol>1-hexanol>1-octanol.     

人参皂苷CK水合物的制备及其稳定性研究

摘要：目的 研究人参皂苷CK的水合物及其稳定性差异。方法 采用X-射线粉末衍射、差示扫描量热仪和热重分析仪 对3种人参皂苷CK水合物进行表征;通过高温、高湿、光照、研磨及悬浮转晶等实验对3种水合物的稳定性进行对比研究。结 果 高温条件下,人参皂苷CK一水合物和半水合物稳定而二水合物会失水变化,而在高湿、光照、研磨条件下,3种水合物的 稳定性均良好;一水合物在乙腈/水(3:1)的混合溶剂中稳定,半水合物在丙酮溶剂中稳定,二水合物在甲醇溶剂中稳定,而在水 中3种水合物均不会发生晶型转变。结论 本研究确定人参皂苷CK一水合物更适合作为药用晶型,并为其结晶工艺和制剂工艺 的开发提供了数据基础。     

1-甲基-1,2,3,4-四氢异喹啉的简易合成

目的制备1-甲基-1,2,3,4-四氢异喹啉。方法以苯乙胺为原料,经酰化反应得乙酰苯乙胺,在多聚磷酸的作用下环合得1-甲基-3,4-二氢异喹啉,经硼氢化钠还原得1-甲基-1,2,3,4-四氢异喹啉。结果反应总收率80%,比文献收率提高了10%,产物结构由1H-NMR光谱确证。结论经酰化、环合、还原三步反应制备1-甲基-1,2,3,4-四氢异喹啉的方法简单,原料便宜,处理容易,副产物较少。     

黄精降低活性氧水平促进衰老内皮祖细胞功能的研究

目的观察黄精在老年大鼠内皮祖细胞(endothelial progenitor cells,EPCs)传代培养衰老进程中对细胞功能及活性氧(reactive oxygen species,ROS)水平的影响。方法分离培养大鼠骨髓EPCs并鉴定,将培养至第2代的EPCs分为4组,分别用黄精含药血清和空白对照血清继续培养至第4、6、8代。β-半乳糖苷酶染色法检测细胞衰老程度;MTT法检测细胞增殖功能;Transwell小室检测迁移功能;体外成血管试剂盒检测成血管功能;流式细胞仪检测ROS水平。结果 EPCs在体外传代培养中,其衰老程度逐渐加重,并伴有增殖、迁移和成血管功能的明显减退,ROS水平急剧升高(P<0.05);经黄精干预后,EPCs衰老程度有所减轻,其功能有所增强,ROS水平明显降低(P<0.05)。结论黄精可能通过降低ROS水平来延缓传代培养EPCs的衰老进程,保护EPCs的功能。     

1,1,2-三甲基-1H-苯并[e]吲哚的制备

目的合成1,1,2-三甲基-1H-苯并[e]吲哚。方法由2-萘胺经重氮化反应、还原反应制得2-萘肼,再与甲基异丙基酮经费歇尔吲哚合成反应制得目标化合物。结果与结论经熔点测定及1H-NMR分析确证目标化合物结构,总收率为26.4%。     

CYP1A1 and CYP1B1 genotypes, haplotypes, and TCDD-induced gene expression in subjects from Seveso, Italy

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is highly toxic in experimental animals, and is known to induce cytochrome P450 (CYP) gene expression. We investigated the effect of CYP1A1 and CYP1B1 variant genotypes and haplotypes on CYP1A1 and CYP1B1 mRNA expression and ethoxyresorufin-O-deethylase (EROD) activity in lymphocytes from 121 subjects from the Seveso population, Italy, accidentally exposed to TCDD in 1976. The 3'UTR 3801T>C and I462V variants of CYP1A1 were present in 16% and 6% of the subjects, respectively. The frequency of CYP1B1 variants was 85.2% for L432V, 49.6% for R48G and A119S, and 28.7% for N453S. There was complete linkage disequilibrium (LD) among the CYP1B1 variant loci (D'=-1) and high LD among the CYP1A1 loci (D'=0.86). Gene expression measured by RT-PCR did not vary by CYP1B1 genotype in uncultured lymphocytes. However, when lymphocytes were treated in vitro with 10 nM TCDD, CYP1B1 and CYP1A1 mRNA expression was strongly induced and modified by CYP variant alleles. Specifically, the CYP1B1*3 haplotype (L432V) was associated with increased CYP1B1 mRNA expression (P=0.03), following an additive model; the CYP1A1 I462V polymorphism was positively, although not significantly, associated with CYP1A1 expression. The CYP1B1*3 variant may have affected CYP1B1 expression in subjects highly and acutely exposed to dioxin at the time of the accident. Although based on small number of subjects, a slight increase in eczema (P=0.05, n=8) and urticaria (P=0.02, n=2) was observed 20 years after the accident in subjects carrying the CYP1B1*3 allele. Genetic variation in cytochrome P450 induction may identify subjects with variable responsiveness to TCDD and potentially increased risk of disease.     

Role of glutathione S-transferases A1-1, M1-1, and P1-1 in the detoxification of 2-phenylpropenal, a reactive felbamate metabolite

Felbamate has proven to be an effective therapy for treating refractory epilepsy. However, felbamate therapy has been limited due to the associated reports of hepatotoxicity and aplastic anemia. Previous research from our laboratory has proposed 2-phenylpropenal as the reactive metabolite in felbamate bioactivation and identified its mercapturates in the urine of rats and patients undergoing felbamate therapy. While the reaction between 2-phenylpropenal and GSH has been shown to occur spontaneously under physiological conditions, the potential catalysis by glutathione transferases (GST) has remained unknown. The work presented here demonstrates a role for GST in the detoxification of 2-phenylpropenal. The kinetic data show that 2-phenylpropenal is a substrate for all three isoforms tested, with a k(cat)/K(m) of 0.275 +/- 0.035 microM(-1) s(-1) for GSTM1-1, 0.164 +/- 0.005 microM(-1) s(-1) for GSTP1-1, and 0.042 +/- 0.005 microM(-1) s(-1) for GSTA1-1. Given that electrophilic substrates such as 2-propenal have been shown to inhibit GSTs, we also examined the inhibition of GSTM1-1, GSTP1-1 and GSTA1-1 by 2-phenylpropenal. The enzyme inhibition studies demonstrate that 2-phenylpropenal inhibits GSTP1-1 and GSTM1-1. The inhibition of GSTP1-1 was completely reversible upon filtration and reconstitution in buffer containing 10 mM GSH. However, 2-phenylpropenal inhibition of GSTM1-1 was irreversible under the same conditions. The irreversible inhibition of GSTM1-1 may be important in understanding the toxicities associated with felbamate. Given that 2-phenylpropenal is both a substrate and irreversible inhibitor for GSTM1-1, GSTM1-1 represents a potential target for 2-phenylpropenal haptenization in vivo, which may in turn mediate the observed idiosyncratic reactions.     

NQO1, MPO, CYP2E1, GSTT1 and GSTM1 polymorphisms and biological effects of benzene exposure--a literature review

BACKGROUND: Benzene is a ubiquitous toxic environmental pollutant. Biological effects have been detected as a result of low-level environmental exposures, suggesting that a large proportion of the population may potentially suffer ill health effects. Polymorphisms in genes involved in benzene metabolism are thought to influence individual susceptibility to various levels of benzene exposure. METHODS: Medline literature database search for articles relating to benzene exposure and polymorphisms in genes known to be involved in benzene metabolism (NQO1, CYP2E1, GSTT1, GSTM1 and MPO). Twenty-two reports were included in this review. RESULTS: A modest effect of the studied gene polymorphisms on the analyzed biomarkers was observed. GSTM1 and GSTT1 showed some consistent associations with both biomarkers of exposure and effect. CONCLUSION: Genetic polymorphisms on the benzene metabolism pathway should be taken into account when studying the biological effects of benzene exposure. Unique combinations of genetic polymorphisms may increase susceptibility of individuals and/or population subgroups. However, gene-gene interactions, and the biological effects of long-term and low-level exposure to benzene are not yet analyzed with well-designed studies that incorporate multiple biological end-points and multiple genes.     

基于电脑分析的药材微杂质快速鉴定法

本文建立了一种可用于快速鉴别细小药材中掺杂质的方法。以微性状观察拍摄结合计算机图像分析对药材中细微杂质进行初步的定性和定量分析。实验表明,该方法简单快速,可以大大地减轻检验工作量,可成为快检的重要手段。     

Structure-function relationships of inhibition of human cytochromes P450 1A1, 1A2, 1B1, 2C9, and 3A4 by 33 flavonoid derivatives

Structure-function relationships for the inhibition of human cytochrome P450s (P450s) 1A1, 1A2, 1B1, 2C9, and 3A4 by 33 flavonoid derivatives were studied. Thirty-two of the 33 flavonoids tested produced reverse type I binding spectra with P450 1B1, and the potencies of binding were correlated with the abilities to inhibit 7-ethoxyresorufin O-deethylation activity. The presence of a hydroxyl group in flavones, for example, 3-, 5-, and 7-monohydroxy- and 5,7-dihydroxyflavone, decreased the 50% inhibition concentration (IC50) of P450 1B1 from 0.6 μM to 0.09, 0.21, 0.25, and 0.27 μM, respectively, and 3,5,7-trihydroxyflavone (galangin) was the most potent, with an IC50 of 0.003 μM. The introduction of a 4'-methoxy- or 3',4'-dimethoxy group into 5,7-dihydroxyflavone yielded other active inhibitors of P450 1B1 with IC50 values of 0.014 and 0.019 μM, respectively. The above hydroxyl and/or methoxy groups in flavone molecules also increased the inhibition activity with P450 1A1 but not always toward P450 1A2, where 3-, 5-, or 7-hydroxyflavone and 4'-methoxy-5,7-dihydroxyflavone were less inhibitory than flavone itself. P450 2C9 was more inhibited by 7-hydroxy-, 5,7-dihydroxy-, and 3,5,7-trihydroxyflavones than by flavone but was weakly inhibited by 3- and 5-hydroxyflavone. Flavone and several other flavonoids produced type I binding spectra with P450 3A4, but such binding was not always related to the inhibitiory activities toward P450 3A4. These results indicate that there are different mechanisms of inhibition for P450s 1A1, 1A2, 1B1, 2C9, and 3A4 by various flavonoid derivatives and that the number and position of hydroxyl and/or methoxy groups highly influence the inhibitory actions of flavonoids toward these enzymes. Molecular docking studies suggest that there are different mechanisms involved in the interaction of various flavonoids with the active site of P450s, thus causing differences in inhibition of these P450 catalytic activities by flavonoids.     

1 H-1,2,3-三唑的合成

目的：研究适宜大量制备1H-1,2,3-三唑的合成方法。方法：以邻苯二胺为原料,经重氮化、环合、氧化、脱羧,制得1H-1,2,3-三唑。结果：所得产物的结构经^1HNMR,^13CNMR,MS-EI确证,并且产物熔点与文献报道一致,总收率42.0%。结论：该方法操作简便,反应条件温和,适宜工业规模制备1H-1,2,3-三唑。