Recent advances in preclinical in vitro approaches towards quantitative prediction of hepatic clearance and drug-drug interactions involving organic anion transporting polypeptide (OATP) 1B transporters

Hepatic uptake mediated by organic anion transporting polypeptide (OATP) 1B1 and 1B3 can serve as a major elimination pathway for various anionic drugs and as a site of drug-drug interactions (DDIs). This article provides an overview of the in vitro approaches used to predict human hepatic clearance (CL_h) and the risk of DDIs involving OATP1Bs. On the basis of the so-called extended clearance concept, in vitro–in vivo extrapolation methods using human hepatocytes as in vitro systems have been used to predict the CL_h involving OATP1B-mediated hepatic uptake. CL_h can be quantitatively predicted using human donor lots possessing adequate OATP1B activities. The contribution of OATP1Bs to hepatic uptake can be estimated by the relative activity factor, the relative expression factor, or selective inhibitor approaches, which offer generally consistent outcomes. In OATP1B1 inhibition assays, substantial substrate dependency was observed. The time-dependent inhibition of OATP1B1 was also noted and may be a mechanism underlying the in vitro–in vivo differences in the inhibition constant of cyclosporine A. Although it is still challenging to quantitatively predict CL_h and DDIs involving OATP1Bs from only preclinical data, understanding the utility and limitation of the current in vitro methods will pave the way for better prediction.     

Restriction endonuclease fingerprinting enhanced conformation sensitive gel electrophoresis (REF-CSGE) in the analysis of BRCA1 exon 11 mutations in a high-risk breast cancer cohort

Efficient genetic analysis of large exonic regions containing heterozygous mutations and common polymorphisms can be difficult. We have analyzed 30 patients for inherited susceptibility mutations (ISM) within exon 11 of the BRCA1 gene as part of an ongoing genetic epidemiological study of high-risk breast cancer (HRBC). A novel combination of restriction endonuclease fingerprinting (REF) and conformation sensitive gel electrophoresis (CSGE) was developed for rapid and efficient screening of mutations. This method (REF-CSGE) was compared side-by-side with standard CSGE and evaluated for both efficiency and sensitivity of detection. REF-CSGE detected 100% of the alterations found by CSGE. However, one variant was only detectable by REF-CSGE. All samples with variant bands were sequenced to confirm the nature of the alteration. In total, two small deletions (frameshifts) and 62 point mutations (60 known polymorphisms and two variants of unknown significance) were found in our cohort. The majority of the exon 11 polymorphisms detected are inherited as a linked haplotype. Point mutations that comprise these haplotypes could be simultaneously detected on a single gel by REF-CSGE, thereby decreasing the number of sequencing reactions necessary to elucidate heteroduplex patterns seen on CSGE gels. An analysis of the overall efficiency of both techniques revealed that REF-CSGE required 67% fewer confirmatory sequencing reactions, resulting in savings in both reagents and technician time.     

Estimating the decline in excess risk of chronic obstructive pulmonary disease following quitting smoking – A systematic review based on the negative exponential model

We quantified the decline in COPD risk following quitting using the negative exponential model, as previously carried out for other smoking-related diseases. We identified 14 blocks of RRs (from 11 studies) comparing current smokers, former smokers (by time quit) and never smokers, some studies providing sex-specific blocks. Corresponding pseudo-numbers of cases and controls/at risk formed the data for model-fitting. We estimated the half-life (H, time since quit when the excess risk becomes half that for a continuing smoker) for each block, except for one where no decline with quitting was evident, and H was not estimable. For the remaining 13 blocks, goodness-of-fit to the model was generally adequate, the combined estimate of H being 13.32 (95% CI 11.86–14.96) years. There was no heterogeneity in H, overall or by various studied sources. Sensitivity analyses allowing for reverse causation or different assumed times for the final quitting period little affected the results. The model summarizes quitting data well. The estimate of 13.32 years is substantially larger than recent estimates of 4.40 years for ischaemic heart disease and 4.78 years for stroke, and also larger than the 9.93 years for lung cancer. Heterogeneity was unimportant for COPD, unlike for the other three diseases.     

Differential roles of hypothalamic serotonin receptor subtypes in the regulation of prolactin secretion in the turkey hen

In the turkey, exogenous serotonin (5-hydroxytryptamine, 5-HT) increases prolactin (PRL) secretion by acting through the dopaminergic (DAergic) system. In the present study, infusion of the 5-HT_2C receptor agonist, (R)(−)-DOI hydrochloride (DOI), into the third ventricle stimulates PRL secretion, whereas the 5-HT_1A receptor agonist, (+/−)-8-OH-DPAT hydrobromide (DPAT), inhibits PRL secretion. Using the immediate-early gene, c-fos, as an indicator of neuronal activity, in situ hybridization histochemistry showed preferential c-fos co-localization within tyrosine hydroxylase immunoreactive neurons (the rate limiting enzyme in DA synthesis) in the areas of the nucleus preopticus medialis (POM) and the nucleus premammillaris (PMM), in response to DPAT and DOI, respectively. To clarify the involvement of 5-HT_1A and 5-HT_2C receptors in PRL regulation, their mRNA expression was determined on hypothalamic tissue sections from birds in different reproductive stages. A significant difference in 5-HT_1A receptor was observed, with the POM of hypoprolactinemic short day and photorefractory birds showing the highest expression. 5-HT_2C receptors mRNA did not change during the reproductive cycle. The data presented support the notion that DA neurons in the PMM and POM mediate the stimulatory and inhibitory effects of 5-HT, respectively, on PRL secretion and the 5-HTergic system can both stimulate and inhibit PRL secretion.     

The effects of energy beverages on cultured cells

The popularity and prevalence of energy beverages makes it essential to examine the interactions between the ingredients and their effects on the safety of these beverages. In this study, we used in vitro assays to examine the effects of two energy beverages on mesenchymal, epithelial and neuronal cells. Our results showed that treatment of epithelial and mesenchymal cells with either energy beverage resulted in a dose dependent delay in wound closure, in a scratch wound healing assay. In rat embryonic fibroblasts, treatment with the energy beverages led to decreased lamellipodia formation and decreased proliferation/viability; whereas in MDCK cells, energy beverage treatment resulted in actin disorganization without any effects on cell proliferation. This suggests that the mechanisms underlying delayed wound healing might be different in the two cell types. Interestingly, the delays in both cell types could not be mimicked by treatment of caffeine, taurine and glucose alone or in combinations. Furthermore, treatment of chick forebrain neuronal cultures with energy beverages resulted in a dose dependent inhibition of neurite outgrowth. The cellular assays used in this study provide a consistent, qualitative and quantitative system for examining the combinatorial effects of the various ingredients used in energy beverages.     

Highly sensitive mutation screening by REF with low concentrations of urea: A blinded analysis of a 2‐kb region of the p53 gene reveals two common haplotypes

Restriction endonuclease fingerprinting (REF), a hybrid modification of single‐strand conformation polymorphism (SSCP) and restriction endonuclease digestion, has been used previously to detect mutations in 1‐ to 2‐kb segments of DNA. This paper demonstrates that fragment resolution, and thus sensitivity of REF, can be markedly improved by electrophoresis under partially denaturing, rather than nondenaturing, conditions, for genes with a high G+C content. A 2.1‐kb segment of the p53 tumor suppressor gene (54.5% G+C) containing exons 5–9, including the intervening introns, was screened in a blinded analysis of 48 samples from human breast tumors containing known wild‐type or mutant p53 genes. In gels containing 0.5 M urea, 97% of the mutant samples were detected correctly, and more than 80% of the mutations were localized within a 200‐bp region. In the process of this methodological analysis, it was discovered that: (1) there are two common and four uncommon haplotypes; (2) the two common haplotypes occurred in the three races examined, suggesting an ancient origin; and (3) haplotype II is of substantially higher frequency in the Chinese relative to Japanese (P = 0.023) and Caucasians (P = 0.005). Two other improvements in the REF procedure included (1) the selection of an optimal set of restriction endonucleases by new software (REF Select) developed recently in our laboratory; and (2) the addition of an oligonucleotide “tail,” containing two recognition sequences for restriction endonucleases, to the PCR primers to prevent coterminal fragments at the end of amplified products. These modifications facilitate the use of REF for efficient and sensitive mutation screening in p53 and other genes with a high G+C content. Hum Mutat 14:175–180, 1999. © 1999 Wiley‐Liss, Inc.     

Estimating the decline in excess risk of cerebrovascular disease following quitting smoking – A systematic review based on the negative exponential model

We attempted to quantify the decline in stroke risk following quitting using the negative exponential model, with methodology previously employed for IHD. We identified 22 blocks of RRs (from 13 studies) comparing current smokers, former smokers (by time quit) and never smokers. Corresponding pseudo-numbers of cases and controls/at risk formed the data for model-fitting. We tried to estimate the half-life (H, time since quit when the excess risk becomes half that for a continuing smoker) for each block. The method failed to converge or produced very variable estimates of H in nine blocks with a current smoker RR <1.40. Rejecting these, and combining blocks by amount smoked in one study where problems arose in model-fitting, the final analyses used 11 blocks. Goodness-of-fit was adequate for each block, the combined estimate of H being 4.78(95%CI 2.17–10.50) years. However, considerable heterogeneity existed, unexplained by any factor studied, with the random-effects estimate 3.08(1.32–7.16). Sensitivity analyses allowing for reverse causation or differing assumed times for the final quitting period gave similar results. The estimates of H are similar for stroke and IHD, and the individual estimates similarly heterogeneous. Fitting the model is harder for stroke, due to its weaker association with smoking.     

Prognostic Impact of Low-Flow Severe Aortic Stenosis in Small-Body Patients Undergoing TAVR: The OCEAN-TAVI Registry

Objectives

This study aimed to analyze the prognostic impact of low-flow (LF) severe aortic stenosis in small-body patients undergoing transcatheter aortic valve replacement (TAVR).

桦木尘提取液对小鼠肺组织脂质过氧化及REF-1基因表达的影响

目的：观察桦木尘对小鼠肺组织脂质过氧化和氧化还原相关基因REF－I表达的影响。方法：采用桦木尘水和有机提取液，建立小鼠肺肿瘤短期诱发实验二阶段模型，将染毒小鼠处死后分为染毒正常肺组织组，肺乳头状腺瘤组织组，肺腺瘤组织组和肺混合型腺瘤组织组，另将溶剂组设为对照组，TBA法检测组织匀浆脂质过氧化物。免疫组织化学染色法检测REF－I基因的表达。结果：所有染毒组肺组织匀浆TBARS显著高于溶剂对照组，其中，肺乳头状腺瘤组，肺腺瘤组和肺混合型腺瘤组P＜0．01，染毒正常肺组织组P＜0．05，与染毒正常肺组织组相比，肺乳头状腺瘤组织和肺腺瘤组织组TBARS升高，P＜0．05，溶剂对照组和染毒正常肺组织REF－1蛋白免疫组织化学染色反应为阴性。肺乳头状腺瘤表现为强烈的核着色，肺腺瘤和肺混合腺瘤则表现为核和胞浆着色，结论：桦木尘的毒作用机制可能与其改变REF－1蛋白的氧化还原调控功能，扰乱机体的氧化与抗氧化平衡状态有关。