Silicone Oil-Free Polymer Syringes for the Storage of Therapeutic Proteins

Prefilled syringes are a popular choice for the delivery of biopharmaceuticals. However, glass syringes might not be the optimal primary packaging material for all biopharmaceuticals. There is evidence that the necessary lubricant silicone oil in glass syringes can interact with proteins and can be shed from the surface into the product solution. In recent years, silicone oil-free polymer syringes were developed. Despite several advantages, however, a major shortcoming of these polymer systems is their relatively high gas permeability, which might be a limitation for the storage of oxygen sensitive biopharmaceuticals. So far, no long-term protein stability studies regarding such polymer systems have been published. In this study, 2 therapeutic proteins were stored in glass syringes and in silicone oil-free polymer syringes. In addition, polymer syringes stored in nitrogen-filled aluminum pouches or covered with oxygen-tight labels were included. Similar chemical protein stability was achieved at 4°C for all syringes. However, in contrast to the polymer syringes, high particle counts were observed in the glass syringes. Polymer syringes stored in nitrogen-filled aluminum pouches presented a promising alternative for the storage of biopharmaceuticals as they do not expose patients to silicone oil and silicone oil-protein aggregates.     

盐酸文拉法辛缓释片Beagle犬体内药动学及生物等效性研究

目的 研究盐酸文拉法辛缓释片在Beagle犬体内的药动学和生物等效性。方法 8条健康Beagle犬随机分成2组,采用双周期、双交叉、单剂量分别ig盐酸文拉法辛缓释片受试制剂或参比制剂75 mg,清洗期为1周;建立血浆中盐酸文拉法辛液相色谱-质谱联用（LC-MS/MS）检测方法,进行方法精密度、准确度、提取回收率、基质效应、稳定性方法学验证;测定给药前（0 h）及给药后2、3、4、5、6、8、10、12、14、16、24、36、48、72 h血浆中盐酸文拉法辛血药浓度,运用DAS 2.1.1软件计算其药动学参数,并评价其生物等效性。结果 LC-MS/MS方法学经验证符合检测要求,受试制剂和参比制剂主要药动学参数分别如下：T_1/2分别为（7.16±2.34）和（6.95±1.57） h、Cmax分别为（522.89±201.12）和（515.22±159.29）ng/mL、T_max分别为（10.38±1.69）和（10.50±2.07）h、AUC_0-t分别为（8 398.64±3332.86）和（8 050.71±2103.15）ng·h/mL、AUC_0-∞分别为（8 701.60±3303.29）和（8 450.01±2273.45）ng·h/mL;以参比制剂为参考,受试制剂AUC_0-∞相对生物利用度为（101.0±13.1）%。结论 盐酸文拉法辛缓释片受试制剂与参比制剂在Beagle犬体内具有生物等效性。     

On the monitoring of dabigatran treatment in “real life” patients with atrial fibrillation

Introduction

The oral direct thrombin inhibitor dabigatran is increasingly used to prevent thromboembolic stroke in patients with atrial fibrillation (AF). Routine laboratory monitoring is currently not recommended, but measurements of dabigatran and/or its effect are desirable in certain situations. We studied dabigatran exposure and compared different tests for monitoring of dabigatran in a real-life cohort of AF patients.

Material and methods

Ninety AF patients (68 ± 9 years, 67% men, mean CHADS₂ score 1.5) were treated with dabigatran 150 (n = 73) or 110 mg BID (n = 17). Trough plasma concentrations of total and free dabigatran by liquid chromatography-tandem mass-spectrometry (LC-MS/MS) were compared to indirect measurements by Hemoclot thrombin inhibitors (HTI) and Ecarin clotting assay (ECA), as well as PT-INR and aPTT.

Results

Total plasma dabigatran varied 20-fold (12–237 ng/mL with 150 mg BID) and correlated well with free dabigatran (r² = 0.93). There were strong correlations between LC-MS/MS and HTI or ECA (p < 0.001) but these assays were less accurate with dabigatran below 50 ng/mL. The aPTT assay was not dependable and PT-INR not useful at all. There were weak correlations between creatinine clearance (Cockcroft-Gault) and LC-MS/MS, HTI and ECA (p < 0.001 for all). A high body weight with normal kidney function was associated with low dabigatran levels.

Conclusions

HTI and ECA reflect the intensity of dabigatran anticoagulation, but LC-MS/MS is required to quantify low levels or infer absence of dabigatran. Most real life patients with a normal creatinine clearance had low dabigatran levels suggesting a low risk of bleeding but possibly limited protection against stroke.     

LC-MS/MS法测定人血浆中法罗培南的浓度及药代动力学研究

目的：建立灵敏、快速、准确的人血浆中法罗培南浓度的HPLC-MS/MS检测方法,并将其用于测定健康受试者静脉注射法罗培南注射液后的血药浓度及药动学研究中。方法：血浆经乙腈处理后,取上清液进行HPLC-MS/MS法测定。10例健康受试者连续6天、每天2次（q12h）,进行注射用法罗培南钠的单、多次静脉输注（每次剂量400 mg）给药,分别于给药前和输注开始后0.33、0.67、1、1.17、1.5、2、2.5、3、4、5、6、8、9、12 h 采集血浆样本进行分析,测定法罗培南血药浓度并进行药代动力学参数计算。结果：血浆中法罗培南的线性范围为0.05000~50.00 μg·mL-1,最低定量限为0.05000 μg·mL-1。健康受试者的单、多次（q12h）静脉输注400 mg法罗培南后Cmax分别为（34.12±3.21） μg·mL-1和（33.44±5.67） μg·mL-1,AUC0-∞分别为（53.15±6.57） μg·h·mL-1和（49.55±7.84） μg·h·mL-1。结论：建立的方法灵敏、简便,可用于人静脉输注法罗培南注射液后血浆药物浓度的测定及药代动力学研究。     

维拉帕米和仙茅苷在大鼠体内药物相互作用研究

目的：研究维拉帕米联用仙茅苷之后,维拉帕米和仙茅苷在大鼠体内可能发生的药物相互作用。方法：24只雄性SD大鼠,随机分为单药组和联用药组,单药组灌胃仙茅苷20 mg· kg-1,联用药组灌胃维拉帕米和仙茅苷,其给药剂量分别为10 mg·kg-1和20 mg·kg-1。在不同时间点取血,采用LC-MS测定仙茅苷的的血药浓度,计算并比较仙茅苷的药代动力学参数。结果：与单药组相比,联用药组仙茅苷药代动力学参数发生明显变化,其中仙茅苷最大血药浓度从189.13 ng·mL-1增加到277.53 ng·mL-1,血浆曲线下面积从949.18 ng·h·mL-1增加到1728.08 ng·h·mL-1。结论：维拉帕米联合仙茅苷用药之后,维拉帕米能明显增强仙茅苷在大鼠体内的血药浓度。     

肠道菌群对石榴皮鞣质的分解代谢作用研究

目的考察肠道菌群对石榴皮鞣质的分解代谢作用,阐明石榴皮鞣质在肠道的代谢途径。方法将石榴皮鞣质与大鼠肠道菌液共同孵育4、6、8、12、24、48 h后,检测不同时间点溶液中的代谢成分。结果通过LC-MS/MS从石榴皮鞣质肠道菌群孵育液中共鉴别出了10个代谢产物,分别为尿石素B、没食子酸、焦性没食子酸、3-O-甲基没食子酸、尿石素A、甲基化尿石素A、尿石素C、尿石素-M6、葡萄糖醛酸化尿石素A、葡萄糖醛酸化尿石素C。结论石榴皮鞣质在肠道菌群中主要通过代谢酶的水解、还原等作用将其代谢成具有生理活性的小分子化合物,这些化合物有可能是石榴皮鞣质在机体内发挥药理活性的物质基础。     

液相色谱-质谱联用鉴定他克莫司的有关物质

采用液相色谱-质谱（LC-MS）联用技术研究他克莫司的有关物质。采用Agilent Eclipse Plus-C₁₈（150 mm × 4.6 mm,3.5 μm）色谱柱,以0.01%甲酸水溶液-乙腈-甲基叔丁基醚为流动相体系,对他克莫司及其强制降解试验样品中的有关物质进行梯度洗脱分离;电喷雾正离子化-四极杆-飞行时间串联质谱法（ESI-Q-TOF/MS）测定各有关物质的母离子及碎片离子的准确质荷比和元素组成,并解析鉴定有关物质的结构。在所建立的条件下,他克莫司与其有关物质分离良好,检测并鉴定出35个主要有关物质,其中2个分别为他克莫司的互变异构体Ⅰ和Ⅱ（他克莫司的有效成分）,3个为美国药典收载的已知杂质,其余30个为新鉴定的未知有关物质。研究结果可为他克莫司发酵生产过程的质量控制提供参考依据。     

LC-MS/MS法测定大鼠血浆中芹黄春浓度及其在药代动力学研究中的应用

目的：研究大鼠分别灌胃和静脉给药芹黄春（芹菜素-7-O-β-D吡喃葡萄糖苷）溶液后,血浆中的芹黄春药物浓度及其药代动力学性质。方法6只雌性Sprague Dawley大鼠随机分为两组,每组3只,分别经灌胃(10 mg/kg)和静脉注射(2 mg/kg)给予芹黄春溶液后,按设计的时间点从大鼠眼内眦静脉丛采集血液样品,置于肝素化的离心管中,低温离心得血浆。采用甲醇沉淀法处理血浆生物样品,利用LC-MS/MS方法对血浆生物样品进行分析。药代动力学参数由Kinetica 2000软件进行计算。结果芹黄春浓度在1~2000 ng/mL范围内线性关系良好(r=0.9974),定量下限为1 ng/mL,低(8 ng/mL)、中(100 ng/mL)、高(1200 ng/mL)3个浓度的提取回收率均大于90%,日内日间精密度和稳定性的RSD均小于12.8%,日内日间准确度在91.5%~107%,方法学考察均符合要求。大鼠经静脉注射和灌胃芹黄春后,Cmax分别为(2363±96) ng/mL和(13.3±5.8) ng/mL,AUC0~∞分别为(796±201) h· ng/mL和(28.9±9.2) h· ng/mL,大鼠经静脉注射和后t1/2为(1.20±0.17) h,芹黄春的口服生物利用度约为0.6%。结论所建立的LC-MS/MS分析方法方便、快速,灵敏度高,准确度好,可用于大鼠血浆芹黄春浓度测定及其药代动力学的研究。     

The Selection of a Pharmaceutical Salt—The Effect of the Acidity of the Counterion on Its Solubility and Potential Biopharmaceutical Performance

A roadmap for the selection of a pharmaceutical salt form for a development candidate is presented. The free base of the candidate did not have sufficient chemical stability for development. The initially selected salt form turned out to be undevelopable because it was unstable during scale-up synthesis and storage. The rationale for the new solid form screening and the criteria for selection are discussed. Before the final selection, the pH solubility profiles of the 2 new salts, a benzoate and a besylate, were compared. Atypical solubility behavior was observed for the benzoate salt in hydrochloric acid with and without normal saline. A scheme is proposed illustrating how the pKas of the counterion and active pharmaceutical ingredient, the medium composition, and final pH affect the solubility and solution equilibria of the 2 selected salt forms. This scheme also includes the equilibria between solution and solid phases in different pH ranges. The pharmaceutical importance of this research is that it sheds light on how the acidity of the counterion can affect the solubility of the selected salt form in the gastric environment. With a well-designed formulation strategy, this property potentially can be translated to optimal biopharmaceutical performance of the drug product.     

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).