Dairy consumption is associated with lower plasma dihydroceramides in women from the D.E.S.I.R. cohort

AimIn the D.E.S.I.R. cohort, higher consumption of dairy products was associated with lower incidence of hyperglycaemia, and dihydroceramide concentrations were higher in those who progressed to diabetes. Our aim here was to study the relationships between dairy consumption and concentrations of dihydroceramides and ceramides.MethodsIn the D.E.S.I.R. cohort, men and women aged 30–65 years, volunteers from West-Central France, were included in a 9-year follow-up with examinations every 3 years, including food-frequency questionnaires. Two items concerned dairy products (cheese, other dairy products except cheese). At each examination, dihydroceramides and ceramides were determined by mass spectrometry in a cohort subset; in the present study, the 105 people who did not progress to type 2 diabetes were analyzed, as the disorder per se might be a confounding factor.ResultsHigher consumption of dairy products (except cheese) was associated with total plasma dihydroceramides during the follow-up, but only in women (P = 0.01 for gender interaction). In fact, dihydroceramide levels were lower in women with high vs low consumption (P = 0.03), and were significantly increased during follow-up (P = 0.01) in low consumers only. There was also a trend for lower ceramides in women with high dairy (except cheese) intakes (P = 0.08). Cheese was associated with dihydroceramide and ceramide changes during follow-up (P = 0.04 for both), but no clear trend was evident in either low or high consumers.ConclusionThese results show that, in women, there is an inverse association between fresh dairy product consumption and predictive markers (dihydroceramides) of type 2 diabetes.     

Radiation Dose Benchmarks During Cardiac Catheterization for Congenital Heart Disease in the United States

ObjectivesThe aim of this study was to define age-stratified, procedure-specific benchmark radiation dose levels during interventional catheterization for congenital heart disease.BackgroundThere is a paucity of published literature with regard to radiation dose levels during catheterization for congenital heart disease. Obtaining benchmark radiation data is essential for assessing the impact of quality improvement initiatives for radiation safety.MethodsData were obtained retrospectively from 7 laboratories participating in the Congenital Cardiac Catheterization Project on Outcomes collaborative. Total air kerma, dose area product, and total fluoroscopy time were obtained for the following procedures: 1) patent ductus arteriosus closure; 2) atrial septal defect closure; 3) pulmonary valvuloplasty; 4) aortic valvuloplasty; 5) treatment of coarctation of aorta; and 6) transcatheter pulmonary valve placement.ResultsBetween January 2009 and July 2013, 2,713 cases were identified. Radiation dose benchmarks are presented including median, 75th percentile, and 95th percentile. Radiation doses varied widely between age groups and procedure types. Radiation exposure was lowest in patent ductus arteriosus closure and highest in transcatheter pulmonary valve placement. Total fluoroscopy time was a poor marker of radiation exposure and did not correlate well with total air kerma and dose area product.ConclusionsThis study presents age-stratified radiation dose values for 6 common congenital heart interventional catheterization procedures. Fluoroscopy time alone is not an adequate measure for monitoring radiation exposure. These values will be used as baseline for measuring the effectiveness of future quality improvement activities by the Congenital Cardiac Catheterization Project on Outcomes collaborative.     

海洋纤溶活性化合物FGFC1和FGFC2影响血小板聚集特性的研究

目的：研究新型海洋双吲哚纤溶活性化合物FGFC1对血小板聚集的影响,发现纤溶活性化合物FGFC1与血小板相关因子变化的关系,同时比较双吲哚化合物FGFC1和单吲哚化合物FGFC2作用于血小板的异同。方法：从新鲜兔血收集血小板,调整反应体系血小板密度,以乙酰水杨酸为阳性对照,采用微量比浊法分析FGFC1和FGFC2对血小板聚集率的影响,用酶联免疫法测定FGFC1和FGFC2对血小板生理因子含量的变化。结果：FGFC1对ADP和AA诱导的血小板聚集具有抑制作用,最大抑制率分别达到42.58%±1.7%和47.82%±2.18%;FGFC2对ADP、AA和胶原诱导的血小板聚集均具有抑制作用,最大抑制率分别为50.12%±1.02%、45.28%±1.09%和50.28%±2.12%。FGFC1与FGFC2均能提升血小板内环磷酸腺苷含量,且FGFC2能明显降低血小板内血栓素A2含量。结论：海洋双吲哚纤溶活性化合物显著抑制血小板聚集,FGFC1通过影响血小板环磷酸腺苷的含量实现抑制血小板聚集,FGFC2可能通过不止一种途径抑制血小板聚集。吲哚化合物抑制血小板聚集与分子结构密切相关。     

The Diffusion of Hydrogen Peroxide Into the Liquid Product During Filling Operations Inside Vaporous Hydrogen Peroxide–Sterilized Isolators Can Be Predicted by a Mechanistic Model

Isolators are commonly used in filling operations of pharmaceutical products. To ensure an aseptic inner environment, isolators are regularly sterilized with vaporized hydrogen peroxide. However, despite extensive purging with air, some residual H₂O₂ remains within the isolator atmosphere and may thus end up in the liquid pharmaceutical drug product, which subsequently may cause oxidation and impact the product’s safety and efficacy. We aimed to evaluate the extent of this phenomenon and to model it. For that purpose, we studied the diffusion of H₂O₂ into water contained in small recipients exposed to the atmosphere of a H₂O₂-sterilized small-scale test isolator. Based on the results, a mechanistic model was proposed to estimate the quantity of H₂O₂ in the product, taking into account the time, filling volume, H₂O₂ concentration, and a configuration factor. Afterward, this model was challenged by filling water at a manufacturing scale, and we observed that the diffusion model could predict the trend of increasing H₂O₂ concentration. However, a consistent difference in H₂O₂ concentration between the model and the experimental results was observed, suggesting the contribution of another parameter. Our results can be used to predict more accurately H₂O₂ concentration in a pharmaceutical product at the manufacturing level.     

Engineering of an epoxide hydrolase for efficient bioresolution of bulky pharmaco substrates

Optically pure epoxides are essential chiral precursors for the production of (S)-propranolol, (S)-alprenolol, and other β-adrenergic receptor blocking drugs. Although the enzymatic production of these bulky epoxides has proven difficult, here we report a method to effectively improve the activity of BmEH, an epoxide hydrolase from Bacillus megaterium ECU1001 toward α-naphthyl glycidyl ether, the precursor of (S)-propranolol, by eliminating the steric hindrance near the potential product-release site. Using X-ray crystallography, mass spectrum, and molecular dynamics calculations, we have identified an active tunnel for substrate access and product release of this enzyme. The crystal structures revealed that there is an independent product-release site in BmEH that was not included in other reported epoxide hydrolase structures. By alanine scanning, two mutants, F128A and M145A, targeted to expand the potential product-release site displayed 42 and 25 times higher activities toward α-naphthyl glycidyl ether than the wild-type enzyme, respectively. These results show great promise for structure-based rational design in improving the catalytic efficiency of industrial enzymes for bulky substrates.Optically pure epoxides and the corresponding vicinal diols are valuable chiral building blocks for the production of pharmaceutically active compounds and other fine chemicals (1). Existing approaches for preparing enantiopure epoxides and diols include the asymmetric epoxidation or dihydroxylation of olefin substrates and the resolution of racemic epoxides. These reactions can be accomplished with either chemical catalysts such as chiral salen cobalt complexes and porphyrin manganese adducts or biocatalysts such as monooxygenases and epoxide hydrolases (EHs) (2–4). In the past two decades, EHs have received much attention because they are cofactor-independent enzymes that are “easy to use” for catalyzing the hydrolysis of racemic epoxides to yield highly enantiopure epoxides and vicinal diols (1, 5, 6). However, application of EHs in laboratory and industry was often hindered by their narrow substrate scope, low enantioselectivity, and regioselectivity, or product inhibition (7, 8).Many protein-engineering efforts have been made to overcome these drawbacks (9, 10). For example, directed evolution by error-prone PCR or DNA shuffling has been used to enhance the activity and enantioselectivity of EHs (11–13). Structure-guided mutagenesis also generated a few EH variants with improved catalytic performance (14–16). The strategy of iterative Combinatorial Active Site-Saturation Test (CAST) combines the rational approach and directed evolution to yield high-quality and small focused mutant libraries for screening EHs with better enantioselectivity (7, 17). By mutating residues at the substrate-binding site, the substrates of EHs have been expanded to include cyclic meso-epoxides, phenyl glycidyl ether (PGE) derivatives, and other styrene oxide-like analogs (18, 19). However, the catalytic efficiency of EH is still not satisfactory for bulky epoxide substrates including precursors of (S)-propranolol, (S)-alprenolol, and other β-adrenergic receptor blocking drugs (20, 21).In this work, we select BmEH, an EH cloned from Bacillus megaterium ECU1001, to expand its substrate scope for bulky pharmaco substrate α-naphthyl glycidyl ether (NGE). This enzyme is a potential industrial biocatalyst because it has unusual (R)-enantioselectivity and resolves ortho-substituted PGEs and para-nitrostyrene oxide with excellent enantiomeric ratios (E > 200) (22). We first identified the active tunnel of BmEH by solving its crystal structure complexed with a substrate analog phenoxyacetamide (POA) and analyzing the routes of substrate entry and product release by mass spectrum analysis. Alanine scanning experiments targeted to the potential product-release site of BmEH resulted in two variants, F128A and M145A, with efficient bioresolution abilities on NGE. Further kinetic measurements and structural analysis showed that M145A has much higher activity for the transition state intermediate formation, whereas both mutants exhibited expanded product-release site. The M145A BmEH variant has been successfully applied for the preparation of (S)-propranolol on a gram scale. The engineering of the potential product-release site described herein should have great promise for structure-based rational design of better industrial enzymes.     

我国已上市治疗用提取分离类和菌体制剂类产品分析

目的：分析我国已上市治疗用提取分离类和菌体制剂类产品存在的差距,明确努力方向。方法：基于我国已上市治疗用提取分离类和菌体制剂类产品批准数据进行统计分析和对比分析。结果：对我国已上市治疗用提取分离类产品中的血液制品和多组分生化药物以及菌体制剂类产品的批准数量、企业数量进行了梳理,并与FDA批准情况进行对比分析。结论：治疗用提取分离类和菌体制剂类产品都是早期的治疗用生物药物,血液制品目前仍发挥着重要的临床价值,但重复申报情况严重。多组分生化药物和菌体制剂重复申报情况很少,但在临床上应用很少。FDA批准的两类产品数量较少。     

我国血液制品企业GMP检查缺陷分析及监管思路探索

目的：为完善我国药品GMP管理,提高企业GMP管理水平提供参考。方法：基于检查37家（次）血液制品生产企业在实施GMP中存在的381项缺陷,采用数据统计的方法进行分类评估,并提出针对性建议。结果与结论：目前,在我国血液制品GMP管理中,存在部分企业实施GMP主动意识不强、企业违反GMP成本较低等问题。建议通过落实企业主体责任、加大检查及处罚力度等办法,减少目前我国血液制品GMP监管中的问题。