Targeting the PI3K/AKT/mTOR pathway in biliary tract cancers: A review of current evidences and future perspectives

Biliary tract cancers (BTCs) are a group of invasive neoplasms, with increasing incidence and dismal prognosis. In advanced disease, the standard of care is represented by first-line chemotherapy with cisplatin and gemcitabine. In subsequent lines, no clear recommendations are currently available, highlighting the need for novel therapeutic approaches.The PI3K/AKT/mTOR pathway is a core regulator of cell metabolism, growth and survival, and is involved in BTCs carcinogenesis and progression. Mutations, gene copy number alterations and aberrant protein phosphorylation of PI3K, AKT, mTOR and PTEN have been thoroughly described in BTCs and correlate with poor survival outcomes.Several pre-clinical evidences state the efficacy of PI3K/AKT/mTOR pathway inhibitors in BTCs, both in vitro and in vivo. In the clinical setting, initial studies with rapamycin analogs have shown interesting activity with an acceptable toxicity profile. Novel strategies evaluating AKT and PI3K inhibitors have risen serious safety concerns, pointing out the need for improved patient selection and increased target specificity for the clinical development of these agents, both alone and in combination with chemotherapy.This review extensively describes the role of the PI3K/AKT/mTOR pathway in BTCs and examines the rationale of its targeting in these tumors, with particular focus on clinical activity, toxicities and perspectives on further development of PI3K/AKT/mTOR pathway inhibitors.     

月华胶囊对耐多药结核分支杆菌感染自噬的影响

目的:以月华胶囊含药血清对耐多药结核分支杆菌感染小鼠单核巨噬细胞(RAW264.7)进行干预,探讨其对耐多药结核分支杆菌感染巨噬细胞自噬的作用及其机制。方法:低温冷冻干燥法制备月华胶囊,大鼠按3.02 g·kg^-1灌胃给药7 d,制备月华胶囊含药血清。体外培养RAW264.7,耐多药结核分子杆菌。以细胞增殖毒性检测试剂盒(CCK-8)检测含药血清对RAW264.7的增殖能力并选定其有效浓度。细胞分为模型组(10%胎牛血清);月华胶囊含药血清组(10%月华胶囊);自噬抑制剂3-甲基腺嘌呤(3-MA)+月华胶囊含药血清组(5 mg·L^-1的3-MA+10%月华胶囊含药血清);雷帕霉素(Rap)组(200 mg·L^-1的Rap+10%胎牛血清);正常组(10%胎牛血清)。除正常组外,各组细胞培养24 h后,按细胞-细菌1∶10感染4 h。透射电镜观察细胞自噬体的出现和形成;蛋白免疫印迹法(Western blot)检测细胞中I型微管相关蛋白轻链3(LC-3Ⅰ),Ⅱ型微管相关蛋白轻链3/I型微管相关蛋白1轻链3(LC-3Ⅱ/LC-3Ⅰ),Beclin-1蛋白的表达;间接免疫荧光染色法观察细胞中LC3B荧光颗粒、斑点及亮度;实时荧光定量聚合酶链式反应(Real-time PCR)检测细胞中LC3,Beclin-1 mRNA的表达。结果:与正常组比较,模型组细胞未见自噬体,细胞中LC-3Ⅱ,LC-3Ⅱ/LC-3Ⅰ,Beclin-1蛋白及LC3,Beclin-1 mRNA的相对表达量均无显著变化,细胞无荧光颗粒、斑点,无荧光亮度。与模型组比较,月华胶囊含药血清组及Rap组细胞,均可观察到自噬体的形成,细胞LC-3Ⅱ,LC-3Ⅱ/LC-3Ⅰ,Beclin-1蛋白及LC3,Beclin-1 mRNA的相对表达量值均明显升高(P<0.05),月华胶囊含药血清组细胞荧光颗粒和荧光斑点明显增多,Rap组细胞荧光颗粒和荧光斑点增多非常明显,荧光闪亮,可判定细胞荧光呈现为阳性;自噬抑制剂3-MA+月华胶囊含药血清组细胞未见自噬体,细胞中LC-3Ⅱ,LC-3Ⅱ/LC-3Ⅰ,Beclin-1蛋白及LC3,Beclin-1 mRNA的相对表达量值均无明显升高。结论:月华胶囊含药血清能使耐多药结核分支杆菌感染细胞产生自噬而发挥抗结核的免疫效应,其机制是通过调控自噬相关蛋白LC3,Beclin-1蛋白及其mRNA的表达水平,促使LC3-I趋向LC3-Ⅱ的转化加快而实现的。     

青娥丸对绝经后骨质疏松症患者骨密度、骨代谢指标和骨硬化蛋白的影响

目的观察青娥丸(QEW)对绝经后骨质疏松症患者骨密度、骨代谢指标和骨硬化蛋白的影响。方法 2016年1月至6月期间,我院收治的120例门诊和住院PMOP患者随机分为QEW组(给予钙片和青娥丸),ALF组(给予钙片和阿法骨化醇)和对照组(给予钙片)(每组n=40),随访期为1年。测量基线和治疗1年后血清骨硬化蛋白、25羟维生素D和骨转换标志物(β-CTX,N-MID和T-PINP)的水平。结果 QEW组和ALF组治疗后1年血清骨硬化素水平显著高于对照组(P0. 05),但QEW组和ALF组比较差异无统计学意义(P0. 05)。治疗后1年QEW组和ALF组血清β-CTX,N-MID和T-PINP水平均降低,而两组间差异无统计学意义(P0. 05)。但QEW组和ALF组血清β-CTX,N-MID和T-PINP水平显著低于对照组(P0. 05)。结论 QEW调节绝经后骨质疏松症患者骨代谢的机制可能与QEW增加硬化蛋白表达的作用有关。     

A preliminary report of a randomized controlled phase 2 trial of the safety and immunogenicity of mRNA-1273 SARS-CoV-2 vaccine

BackgroundVaccines are urgently needed to prevent the global spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We assessed the safety and immunogenicity of vaccine candidate mRNA-1273, encoding the prefusion-stabilized spike protein of SARS-CoV-2.MethodsThis phase 2, randomized, observer-blind, placebo-controlled trial was conducted at 8 sites in the USA, in healthy adults aged ≥18 years with no known history or risk of SARS-CoV-2 infection, and had not previously received an investigational CoV vaccine or treatment. Participants were stratified into two age cohorts (≥18-<55 and ≥55) and were randomly assigned (1:1:1) to either 50 or 100 µg of mRNA-1273, or placebo administered as two intramuscular injections 28 days apart. The primary outcomes were safety, reactogenicity, and immunogenicity assessed by anti-SARS-CoV-2-spike binding antibody level (bAb). Secondary outcome was immunogenicity assessed by SARS-CoV-2 neutralizing antibody (nAb) response.ResultsBetween 29 May and 8 July 2020, 600 participants were randomized, 300 per age cohort. The most common solicited adverse reactions were pain at injection site, headache, and fatigue following each vaccination in both age cohorts. One serious adverse event deemed unrelated by the site investigator occurred 33 days post-vaccination one. mRNA-1273 induced bAb and nAb by 28 days post-vaccination one that were higher at the 100 µg dose relative to the 50 µg dose; this difference was less apparent post-vaccination two. Binding antibodies and nAb increased substantially by 14 days following the second vaccination (day 43) to levels exceeding those of convalescent sera and remained elevated through day 57.ConclusionsVaccination with mRNA-1273 resulted in significant immune responses to SARS-CoV-2 in participants 18 years and older, with an acceptable safety profile, confirming the safety and immunogenicity of 50 and 100 µg mRNA-1273 given as a 2 dose-regimen.ClinicalTrials.gov; NCT04405076.     

2015—2019年某焦化厂下风向居民血清免疫球蛋白分析

目的： 比较2015—2019年某焦化厂下风向居民不同人群血清免疫球蛋白的含量并分析其变化趋势。方法： 2015—2019年夏季，选取中国北部某焦化厂下风向（1~2 km）两个村庄的常住居民为研究群体，按照一定的纳入排除标准确定研究对象，采集其血液及尿液通过全自动生化分析仪分别检测血清免疫球蛋白（IgG、IgA和IgM）含量及尿肌酐（Cr），并对血清免疫球蛋白含量异常率进行统计分析。尿液中多环芳烃代谢物1-羟基芘的含量采用高效液相色谱-荧光法检测。结果： 2015—2019年某焦化厂下风向居民中，女性血清IgG含量均值在各年份间均高于男性（P<0.01），IgM含量均值在2015年和2016年高于男性（P<0.05）；成人血清IgG和IgA含量均值在各年份间均高于未成年人（P<0.01），IgM含量均值在2016年低于未成年人（P<0.01）。2015—2019年某焦化厂下风向居民血清免疫球蛋白含量异常率总体呈先上升后下降的趋势，血清IgA和IgM在不同年份间的差异具有统计学意义（P<0.01），且2016年成人血清IgG、IgA和IgM含量异常率均高于未成年人（P<0.05），2015年和2016年女性血清IgG含量异常率高于男性（P<0.05）。2015—2019年，该地区居民尿液中多环芳烃代谢产物1-羟基芘的检出水平（用尿肌酐含量校正）中位数依次为0.468、0.797、0.678、0.169和0.021 μg/g。结论： 2015—2019年焦化厂下风向居民血清免疫球蛋白（IgG、IgA和IgM）含量具有一定的性别差异和年龄差异，其异常率总体呈现先上升后下降的趋势。     

Metabonomics analysis of serum from rats given long-term and low-level cadmium by ultra-performance liquid chromatography–mass spectrometry

1. This study evaluated the toxicity of chronic exposure to low-level cadmium (Cd) in rats using ultra-performance liquid chromatography–mass spectrometry (UPLC–MS). Forty male Sprague–Dawley rats were randomly assigned to four groups, namely, the control group, low-dose group (0.13?mg/kg·bw), middle-dose group (0.8?mg/kg·bw) and high-dose group (4.89?mg/kg·bw). The rats continuously received CdCl₂ via drinking water for 24?weeks. Serum samples were collected for metabonomics analysis. The data generated from the UPLC–MS was analysed using principal components analysis (PCA) and partial least-squares discriminant analysis (PLS-DA). PLS-DA model with satisfactory explanatory and predictive ability is capable of discriminating the treatment groups from the control group.

2. Finally, the 10 metabolites were identified and showed significant changes in some treatment groups compared with that in the control group (p?p?serum.

3. Results suggest that exposure to Cd can cause disturbances in the lipid metabolism, amino acid metabolism, nervous system, antioxidant defence system, liver and kidney function.     

Evaluation of Transdermal Drug Permeation as Modulated by Lipoderm and Pluronic Lecithin Organogel

The transdermal delivery of 2 fluorescent probes with similar molecular weight but different lipophilicity, into and through the skin from 2 commercially available transdermal bases, pluronic lecithin organogel, and Lipoderm^® has been evaluated. First, in vitro penetration of fluorescein sodium and fluorescein (free acid) through porcine skin was evaluated. Retention and depth distribution profiles in skin were obtained by tape stripping and then followed by optical sectioning using multiphoton microscopy. The results showed that Lipoderm^® led to an enhanced penetration of the hydrophilic compound, fluorescein sodium. For the lipophilic compound fluorescein (free acid), Lipoderm^® performed similar to pluronic lecithin organogel base, where minimal drug was detected in either receptor phase. The skin retention and depth distribution results also showed that the hydrophilic fluorescein sodium had high skin retention with Lipoderm^®, whereas fluorescein (free acid) had very low penetration and retention with increasing skin depth. Moreover, optical sectioning by multiphoton microscopy revealed an uneven distribution of probes across the skin in the x-y plane for both transdermal bases. This work showed that a hydrophilic compound has significantly increased skin penetration and retention when formulated with Lipoderm^®, and the skin retention of the probe was the main determinant of its skin flux.     

Development of Stabilizing Formulations of a Trivalent Inactivated Poliovirus Vaccine in a Dried State for Delivery in the Nanopatch™ Microprojection Array

The worldwide switch to inactivated polio vaccines (IPVs) is a key component of the overall strategy to achieve and maintain global polio eradication. To this end, new IPV vaccine delivery systems may enhance patient convenience and compliance. In this work, we examine Nanopatch? (a solid, polymer microprojection array) which offers potential advantages over standard needle/syringe administration including intradermal delivery and reduced antigen doses. Using trivalent IPV (tIPV) and a purpose-built evaporative dry-down system, candidate tIPV formulations were developed to stabilize tIPV during the drying process and on storage. Identifying conditions to minimize tIPV potency losses during rehydration and potency testing was a critical first step. Various classes and types of pharmaceutical excipients (～50 total) were then evaluated to mitigate potency losses (measured through D-antigen ELISAs for IPV1, IPV2, and IPV3) during drying and storage. Various concentrations and combinations of stabilizing additives were optimized in terms of tIPV potency retention, and 2 candidate tIPV formulations containing cyclodextrin and a reducing agent (e.g., glutathione), maintained ≥80% D-antigen potency during drying and subsequent storage for 4 weeks at 4°C, and ≥60% potency for 3 weeks at room temperature with the majority of losses occurring within the first day of storage.     

Inhibition of Organic Anion Transporting Polypeptide 1B1 and 1B3 by Betulinic Acid: Effects of Preincubation and Albumin in the Media

Betulinic acid (BA), a plant-derived pentacyclic triterpenoid, may interact with the members of the organic anion transporting polypeptide 1B subfamily. Here, we investigated the interactions of BA and its analogs with OATP1B1/3 and rat Oatp1b2 in vitro and in vivo. BA inhibited the activity of OATP1B1/3 and rat Oatp1b2 in vitro. Systemic exposure of atorvastatin was substantially altered with the intravenous co-administration of BA (20 mg/kg). Preincubation (incubation with inhibitors, followed by washout) with BA led to a sustained inhibition of OATP1B3, which recovered rapidly in the media containing 10% fetal bovine serum. The addition of albumin to the media decreased intracellular concentrations of BA and expedited the recovery of OATP1B3 activity following preincubation. For asunaprevir and cyclosporin A (previously known to inhibit OATP1B3 upon preincubation), the addition of albumin to the media shortened recovery time with asunaprevir, but not with cyclosporin A. Overall, our results showed that BA inhibits OATP1B transporters in vitro and may incur hepatic transporter-mediated drug interactions in vivo. Our results identify BA as another OATP1B3 inhibitor with preincubation effect and suggest that the preincubation effect and its duration is impacted by altered equilibrium of inhibitors between intracellular and extracellular space (e.g., albumin in the media).