Trace analysis of rapamycin in human blood by micellar electrokinetic chromatography

A capillary electrophoretic method with UV detection at 278 nm has been developed for analysis of the immunosuppressant rapamycin (sirolimus) in human blood at low microgram per liter levels. Separation has been achieved in an acidic carrier electrolyte containing sodium dodecylsulfate and 30% (v/v) acetonitrile. For sample clean-up and preconcentration, an off-line solid-phase extraction step using a silica-based reversed-phase material and an on-capillary focussing technique were employed. The latter allows the injection of increased sample volumes without excessive band broadening. Although this new method is less sensitive than existing liquid chromatographic procedures combined with mass spectrometry, it is fully suited to routine analysis of rapamycin in blood from patients treated with this drug. Last but not least the low costs make it an attractive alternative to established methods.     

诱导血红素氧合酶-1及雷帕霉素对PDGF诱导的大鼠平滑肌细胞表型转化的抑制作用

目的观察血红素氧合酶-1(HO-1)与雷帕霉素对血小板源性生长因子-BB(PDGF-BB)诱导的大鼠血管平滑肌细胞(VSMCs)表型转化的作用。方法原代大鼠VSMCs给予PDGF-BB诱导表型转化。同时给予不同剂量的血红素氧合酶诱导剂氯血红素诱导HO-1的表达,和雷帕霉素共培养24h。结果PDGF-BB20nmol/ml作用原代VSMCs24h后,SM-α-actin与PCNA表达减弱,PCNA表达增强。氯血红素诱导HO-1后可以抑制PDGF-BB诱导的VSMCs表型改变,并且随剂量的增加,抑制强度增加。较低剂量的雷帕霉素可以抑制PDGF诱导的VSMCs表型转化。结论PDGF-BB亚型可以促进原代培养的大鼠VSMCs由收缩表型向合成表型转化,给予氯血红素诱导HO-1表达可以对抗PDGF-BB诱导的大鼠VSMCs表型转化,并呈剂量相关。     

Proactively Reducing Anti‐Drug Antibodies via Immunomodulatory Bioconjugation

Although PEGylation reduces the immunogenicity of protein drugs to some extent, its limitations for highly immunogenic biotherapeutics have been demonstrated. Herein, a proactive strategy to alleviate the development of anti‐drug antibodies (ADAs) against protein drugs by immunomodulatory bioconjugation is reported. Rapamycin was conjugated to a PEGylated protein therapeutic via a cleavable disulfide linker. The conjugated rapamycin can be released from the bioconjugate and prevent immune responses once the bioconjugate is uptaken by antigen‐presenting cells. The immunomodulatory bioconjugate significantly reduced the titers of ADAs compared with a PEGylated protein. The inhibition of immune responses was specific to the conjugated antigen, avoiding systemic immune suppression and the risk of increased susceptibility to infections. The reported approach breaks the limitations of PEGylation by the proactive prevention of ADAs.     

A novel technique for on-capillary preconcentration of anionic compounds applied to the trace analysis of rapamycin in human blood by capillary electrophoresis

A capillary electrophoretic (CE) method with UV detection at 278 nm has been developed for analysis of the immunosuppressant rapamycin (sirolimus) in human blood at low microg.L(-1) levels. Separation has been achieved in an acidic carrier electrolyte containing sodium dodecyl sulfate and 20% v/v methanol. For sample cleanup and preconcentration, both an off-line solid-phase extraction step using a silica-based reversed-phase material and a newly developed on-capillary focusing technique have been employed. The subsequent treatment of rapamycin under alkaline conditions leads to a cleavage of the lacton bond of the molecule, generating a negatively charged carboxylic group which allows electrokinetic injection into the CE instrument. During the injection process, the negatively charged analyte migrates into an acidic carrier electrolyte, so that it becomes neutral due to protonation and is focused at the capillary inlet. Injection times of 300 s at -7.5 kV could be applied without band-broadening. Results for real samples indicated that the method is fully suited for routine applications and may be an attractive alternative to established liquid chromatographic techniques.     

Synthesis, isolation, and characterization of ABT-578 equilibrium isomers

ABT-578, an anti-restenosis agent exists as two isomers, a major pyran form and a minor oxepane form. The existence of the two isomeric forms was established by isolation and equilibration studies under buffered and physiological conditions. Finally their structures were confirmed by converting the major pyran form to the oxepane form by synthesis, isolation, and characterization.     

Preparation and in vitro release profiles of drug-eluting controlled biodegradable polymer coating stents

In the present study, the different drug-eluting controlled biodegradable polymer coatings were fabricated on stainless steel stents. The coatings were not only uniform and smooth but also had excellent mechanical property. The drug release profiles of drug-eluting stents were studied in detail in this study. Depending on the drug type, different drug-eluting stents exhibited different drug release profile. There were two basic release profiles for different drug-eluting stents, i.e., two-phase release profile with burst release or linear release profile without burst release. Incorporating heparin in the rapamycin or curcumin eluting stents can improve the average drug release rate of both and the burst release of rapamycin. The average drug release rate increased with the increase of drug loading but was not proportional to increase of the ratio of drug/polymer. Fabricating the control release layer on rapamycin-eluting stent surface can prevent the burst release of rapamycin and prolong the release period of rapamycin. All results showed that the drug release profile of drug-eluting stents depends on many parameters including drug type, ratio of drug/polymer, and drug carrier properties.     

A simple and efficient route to the FKBP-binding domain from rapamycin

A simple and highly efficient route to the FKBP-binding domain (FKBD) from the natural product rapamycin has been developed, which entails a sequence of ozonolysis/Baeyer-Villiger/Wittig reactions. The newly synthesized FKBD may serve as a core to assemble hybrid macrocyclic libraries for the discovery of novel probes of protein function and to synthesize new ligands for the FKBP family of proteins.     

REDV/雷帕霉素复合涂层构建内皮细胞选择性功能界面

运用复合涂层的概念构建了兼具药物洗脱和内皮促进作用的载药涂层. 以载雷帕霉素(Rapamycin, RAP)的聚乙二醇甲基丙烯酸酯(PEGMA)-甲基丙烯酸丁酯(BMA)(PEGMA-BMA, PEGB)为内层, Arg-Glu-Asp-Val(REDV)多肽修饰的PEGBN为外层包裹载药涂层. 体外药物释放结果表明, 雷帕霉素可以维持缓慢稳定的长效释放, 释放过程中没有出现暴释现象. 表面细胞生长行为表明, 雷帕霉素可以有效地阻抗内皮细胞和平滑肌细胞的黏附, 抑制细胞活性; 随着药物释放的进行, 雷帕霉素浓度逐渐减低, 但涂层依然维持对平滑肌细胞的非特异性阻抗; 而REDV修饰的外涂层开始呈现内皮细胞的选择性黏附, 随着释放时间延长, 内皮细胞特异选择性也逐渐加强. 雷帕霉素和REDV多肽协同构建的复合涂层能够有效抑制平滑肌细胞的增殖, 获得内皮细胞选择性黏附.