Glycine‐modified growth hormone secretagogues identified in seized doping material

A number of unknown pharmaceutical preparations seized by Danish customs authorities were submitted for liquid chromatography–high resolution mass spectrometry (LC–HRMS) analysis. Comparison with reference standards unequivocally identified the content of the powders as analogs of the growth hormone secretagogues GHRP‐2 (Pralmorelin), GHRP‐6, Ipamorelin, and modified growth hormone releasing factor (modified GRF 1–29), which can be used as performance‐enhancing substances in sports. In all cases, the detected modification involved the addition of an extra glycine amino acid at the N‐terminus, and analytical methods targeting growth hormone secretagogues should hence be updated accordingly.     

注射用青藤碱脂质体温敏凝胶的制备及其药动学研究

目的通过脂质体与温敏凝胶相结合,制备新型注射用关节腔给药系统,并研究其药物代谢动力学(简称药动学)。方法采用逆向蒸发法制备青藤碱脂质体,通过单因素考察、星点设计-效应面法优化处方。选取壳聚糖/β-甘油磷酸(CS/β-GP)为温敏凝胶制备体系,并采用透析袋法对青藤碱脂质体温敏凝胶的体外释放度进行测定。建立家兔关节腔给药模型,应用DAS 3.1.0软件进行数据处理进而研究其药动学。结果制备青藤碱脂质体最优处方为药脂比1∶6,大豆磷脂与胆固醇比6∶1,油水比1∶6。体外释放结果显示,青藤碱脂质体温敏凝胶缓释效果明显,至114 h还未释放完全。家兔体内药动学研究表明,青藤碱脂质体温敏凝胶的生物利用度约为青藤碱注射液的2.0倍(P0.01),达峰时间显著延长,约为青藤碱注射液的3.0倍(P0.01),血药峰浓度降低(P0.05),缓释效果优于青藤碱注射剂。结论青藤碱脂质体温敏凝胶性状稳定,缓释效果明显,注射于家兔关节腔后,经体液侵蚀后自行消解,可用于关节腔注射给药。     

Antimicrobial Activity and Biocompatibility of Antibiotic-Loaded Chitosan Hydrogels as a Potential Scaffold in Regenerative Endodontic Treatment

IntroductionThe objective of this study was to determine the effectiveness of several antibiotic-loaded hydrogel scaffolds against Enterococcus faecalis, as well as their ability to stimulate proliferation and mineralization of dental pulp stem cells.MethodsFibrin (Fg) or chitosan-fibrin hydrogels (Ch) were prepared using 12.5 mg/mL fibrinogen and 0.4% (w/v) chitosan. Triple antibiotics, clindamycin-modified triple antibiotic paste, or double antibiotics were loaded in gels (1 mg/mL). Antibacterial effect against E. faecalis biofilm was determined by using colony-forming units (CFUs) and confocal laser scanning microscope (CLSM). Cell viability and morphology were determined by loading cells into different gels at 7 and 14 days using the water-soluble tetrazolium salt-1 cell viability assay and Live & Dead cell analysis. Mineralization was detected by using alkaline phosphatase and alizarin red staining activity.ResultsAntibiotic-loaded Fg gel and Ch gel alone without antibiotics resulted in a significant reduction in CFUs compared with the positive control (P < .05). When antibiotics were loaded in Ch gel, there were no CFUs detected in any groups (P < .05). CLSM images showed dense red areas with mostly dead bacteria on the dentin surface in antibiotic-loaded Ch groups, which showed significantly less live bacteria compared with the other groups (P < .05). Triple antibiotic-loaded Fg and Ch gels resulted in a dramatic decrease in the mineralized nodule formation compared with all other gel groups (P < .05). Ch hydrogels resulted in round cell morphology up to 7 days. Ch alone or with double antibiotic paste showed more cell spreading with spindle-shaped morphology at 14 days and higher alkaline phosphatase activity compared with other antibiotic-loaded Ch groups (P > .05).ConclusionsDouble antibiotic-loaded Ch gel appears to enhance the antibacterial properties while maintaining higher cell viability, cell spreading, and mineralization activity, compared with all the other scaffolds investigated.     

白杨素固体脂质纳米粒水凝胶骨架缓释片的制备

目的制备白杨素固体脂质纳米粒水凝胶骨架缓释片。方法乳化超声-低温固化法制备固体脂质纳米粒后进一步制成冻干粉,再以HPMC 15KM为缓释材料制备水凝胶骨架缓释片。在单因素试验基础上,以HPMC 15KM用量、PEG400与PEG4000比例、PEG用量、硬脂酸镁用量为影响因素,累积释放度为评价指标,正交试验优化处方,再进行释药模型拟合。结果最优处方为HPMC K15 M用量50 mg,PEG 400与PEG 4000比例2∶1,PEG用量30 mg,硬脂酸镁用量0.5%,12 h内累积释放度为93.19%。水凝胶骨架缓释片体外释放符合一级方程(r=0.994 1),释药机制为骨架溶蚀与扩散并存。结论该方法简便可靠,可用于制备具有明显体外缓释特征的白杨素固体脂质纳米粒水凝胶骨架缓释片。     

多肽药物专家共识

中国生化制药工业协会多肽分会日前召开多肽药物专家共识研讨会,专家们就中国多肽药物发展现状,发展重点方向及对相关部门的建议达成共识。     

Synthetic conjugate peptide Fba-Met6 (MP12) induces complement-mediated resistance against disseminated Candida albicans

The fungal genus Candida includes common commensals of the human mucosal membranes, and the most prevalently isolated species, C. albicans, poses a threat of candidemia and disseminated infection associated with an unacceptably high mortality rate and an immense $4 billion burden (US) yearly. Nevertheless, the demand for a vaccine remains wholly unfulfilled and increasingly pressing. We developed a double-peptide construct that is feasible for use in humans with the intention of preventing morbid infection by targeting epitopes derived from fructose bisphosphate aldolase (Fba) and methionine synthase (Met6) which are expressed on the C. albicans cell surface. To test the applicability of the design, we vaccinated mice via the intramuscular (IM) route with the conjugate denoted Fba-Met6 MP12 and showed that the vaccine enhanced survival against a lethal challenge. Because overall endpoint IgG1 and IgG2a antibody titers were robust and these mouse subclasses are associated with protective functionality, we investigated the potential of Fba and Met6 specific antibodies to facilitate the well-defined anti-Candida response by complement, which opsonizes fungi for degradation by primary effectors. Notably, reductions in the fungal burdens and enhanced survival were both abrogated in MP12-vaccinated mice that were pre-challenge dosed with cobra venom factor (CVF), a complement depleting factor. Altogether, we demonstrated that complement is relevant to MP12-based protection against disseminated C. albicans, delineating that a novel, multivalent targeted vaccine against proteins on the surface of C. albicans can enhance the natural response to infection.     

Preclinical evaluation of injectable bone substitute materials

Injectable bone substitutes (IBSs) represent an attractive class of ready‐to‐use biomaterials, both ceramic‐ and polymer‐based, as they offer the potential benefit of minimally invasive surgery and optimal defect filling. Although in vitro assessments are the first step in the process of development, the safety and efficacy of an IBS strongly depend on validated preclinical research prior to clinical trials. However, the selection of a suitable preclinical model for performance evaluation of an IBS remains a challenge, as no gold standard exists to define the best animal model. In order to succeed in this attempt, we identified three stages of development, including (a) proof‐of‐principle, (b) predictive validity and (c) general scientific legitimacy, and the respective criteria that should be applied for such selection. The second part of this review provides an overview of commonly used animals for IBSs. Specifically, scientific papers published between January 1996 and March 2012 were retrieved that report the use of preclinical models for the evaluation of IBSs in situations requiring bone healing and bone augmentation. This review is meant not only to describe the currently available preclinical models for IBS application, but also to address critical considerations of such multi‐factorial evaluation models (including animal species, strain, age, anatomical site, defect size and type of bone), which can be indicative but in most cases edge away from the human reality. Consequently, the ultimate goal is to guide researchers toward a more careful and meaningful interpretation of the results of experiments using animal models and their clinical applications. Copyright © 2012 John Wiley & Sons, Ltd.     

The effects of dynamic compressive loading on human mesenchymal stem cell osteogenesis in the stiff layer of a bilayer hydrogel

Bilayer hydrogels with a soft cartilage‐like layer and a stiff bone‐like layer embedded with human mesenchymal stem cells (hMSCs) are promising for osteochondral tissue engineering. The goals of this work were to evaluate the effects of dynamic compressive loading (2.5% applied strain, 1 Hz) on osteogenesis in the stiff layer and spatially map local mechanical responses (strain, stress, hydrostatic pressure, and fluid velocity). A bilayer hydrogel was fabricated from soft (24 kPa) and stiff (124 kPa) poly (ethylene glycol) hydrogels. With hMSCs embedded in the stiff layer, osteogenesis was delayed under loading evident by lower OSX and OPN expressions, alkaline phosphatase activity, and collagen content. At Day 28, mineral deposits were present throughout the stiff layer without loading but localized centrally and near the interface under loading. Local strains mapped by particle tracking showed substantial equivalent strain (~1.5%) transferring to the stiff layer. When hMSCs were cultured in stiff single‐layer hydrogels subjected to similar strains, mineralization was inhibited. Finite element analysis revealed that hydrostatic pressures ≥~600 Pa correlated to regions lacking mineralization in both hydrogels. Fluid velocities were low (~1–10 nm/s) in the hydrogels with no apparent correlation to mineralization. Mineralization was recovered by inhibiting ERK1/2, indicating cell‐mediated inhibition. These findings suggest that high strains (~1.5%) combined with higher hydrostatic pressures negatively impact osteogenesis, but in a manner that depends on the magnitude of each mechanical response. This work highlights the importance of local mechanical responses in mediating osteogenesis of hMSCs in bilayer hydrogels being studied for osteochondral tissue engineering.     

The emerging role of ultrasound in detecting interstitial lung disease in patients with rheumatoid arthritis

ObjectiveTo investigate the potential role of US in the detection of ILD in a cohort of patients with RA.MethodsPatients with diagnosis of RA were consecutively enrolled. All patients underwent pulmonary examination, laboratory data, DLCO measure, chest HRCT and radiographs, and US examination. A healthy group was included as control group. US was performed according the 14-intercostal space scanning protocol using the following semiquantitative scale [0 = normal (≤ 5 B-lines); 1 = slight (≥ 6 and ≤ 15 B-lines); 2 = moderate, (≤ 16 and ≥ 30 B-lines); 3 = severe (≥ 30 B-lines)].ResultsA total of 74 RA patients and 74 healthy controls were included. Thirty of 74 patients (40.5%) showed US signs of ILD with respect to the healthy controls (3 subjects, 4.1%) (P < 0.001); whereas HRCT showed ILD in 27 (36.4%) of 74 patients. Among the 30 patients that showed US findings of ILD, 17 (56.6%) were asymptomatic from respiratory view-point. The sensitivity and specificity of US were 92% and 89% respectively. A positive correlation between US and HRCT findings were found (P < 0.001) whereas no correlation was found with chest radiographs and DLCO findings. Positive association between US findings and DAS28-ESR, anti-CCP and RF (P < 0.01 for each respectively) was found. Feasibility, represented by the mean time spent to perform the pulmonary US assessment was 7.8 minutes (± SD 1.2, range 6 to 10 minutes).ConclusionsOur results support the potential of US in detect accurately ILD in patients with RA and provide a rationale to consider it as a friendly screening tool to be implemented in early phases of the disease.     

Methodological advances in the design of peptide-based vaccines

The tremendous advances in genomics, recombinant DNA technology, bioengineering and nanotechnology, in conjunction with the development of high-end computations, have been instrumental in the process of rational design of peptide-based vaccines. The use of peptide vaccines was limited owing to their inherent instability when systemically administered; however, advanced formulation techniques have been developed for their systemic delivery, thereby overcoming their degradation, clearance, cellular uptake and off-target effects. With the rise of sophisticated immunological predictors and experimental techniques, several methodological advances have occurred in this field. This review examines contemporary methods to identify and optimize epitopes, engineer their immunogenic properties and develop their safe and efficient delivery into the host.