An LC/MS quantitative and microdialysis method for cyclovirobuxine D pharmacokinetics in rat plasma and brain: The pharmacokinetic comparison of three different drug delivery routes.

To explore the brain-targeting of cyclovirobuxine D(CVB-D) after administered intranasally, the pharmacokinetics of CVB-D via three different drug delivery routes: intragastric (i.g.), intranasal (i.n.), and intravenous (i.v.) in rat brain and blood was compared. Firstly, an in vivo microdialysis method for sampling CVB-D in both plasma and brain of the rat was established. Secondly, a liquid chromatography-tandem mass spectrometry method has been developed and validated for determination of CVB-D in microdialysis samples. For plasma and brain microdialysis samples, liquid-liquid extraction was used and donepezil was chosen as internal standard. Both were followed by HPLC separation and positive electrospray ionization tandem mass spectrometry detection (ESI-MS/MS). Chromatographic separation was achieved on a agilent C18 column with a mobile phase of methanol-water (50:50, v/v) (pH 3.2) containing 0.1% formic acid and 5mM ammonium acetate. Mass spectrometric detection in the positive ion mode was carried out by selected reaction monitoring (MRM) of the transitions at m/z 403.4→372.3 for CVB-D and m/z 380.2→243.1 for donepezil (IS). Good linearities were obtained in the range of 10-4000ng/mL in rat microdialysates for CVB-D. The lowest limit of quantitation was 5ng/mL, with an extraction recovery >75%, and no significant matrix effects. Intra- and inter-day precisions were all <15% with accuracies of 97.26-116.20%. All of which proved that the established method was successfully applied to the pharmacokinetic study of CVB-D. Simultaneously, brain uptake and pharmacokinetic studies were performed by determination of CVB-D concentration in blood and brain respectively for CVB-D i.g., i.n. and i.v.. Results showed that the intranasal CVB-D could improve brain targeting and had advantages for direct nose to brain transport of CVB-D when compared with injection and oral delivery routes, which indicates that intranasal administration of CVB-D could be a promising approach for the treatment of cerebrovascular disease.

Controllable drug uptake and nongenomic response through estrogen-anchored cyclodextrin drug complex.

Breast cancer is a leading killer of women worldwide. Cyclodextrin-based estrogen receptor-targeting drug-delivery systems represent a promising direction in cancer therapy but have rarely been investigated. To seek new targeting therapies for membrane estrogen receptor-positive breast cancer, an estrogen-anchored cyclodextrin encapsulating a doxorubicin derivative Ada-DOX (CDE1-Ada-DOX) has been synthesized and evaluated in human breast cancer MCF-7 cells. First, we synthesized estrone-conjugated cyclodextrin (CDE1), which formed the complex CDE1-Ada-DOX via molecular recognition with the derivative adamantane-doxorubicin (Ada-DOX) (Kd =1,617 M(-1)). The structure of the targeting vector CDE1 was fully characterized using (1)H- and (13)C-nuclear magnetic resonance, mass spectrometry, and electron microscopy. CDE1-Ada-DOX showed two-phase drug-release kinetics with much slower release than Ada-DOX. The fluorescence polarization analysis reveals that CDE1-Ada-DOX binds to recombinant human estrogen receptor α fragments with a Kd of 0.027 µM. Competition assay of the drug complex with estrogen ligands demonstrated that estrone and tamoxifen competed with CDE1-Ada-DOX for membrane estrogen receptor binding in MCF-7 cells. Intermolecular self-assembly of CDE1 molecules were observed, showing tail-in-bucket and wire-like structures confirmed by transmission electronic microscopy. CDE1-Ada-DOX had an unexpected lower drug uptake (when the host-guest ratio was >1) than non-targeting drugs in MCF-7 cells due to ensconced ligands in cyclodextrins cavities resulting from the intermolecular self-assembly. The uptake of CDE1-Ada-DOX was significantly increased when the host-guest ratio was adjusted to be less than half at the concentration of CDE1 over 5 µM due to the release of the estrone residues. CDE1 elicited rapid activation of mitogen-activated protein kinases (p44/42 MAPK, Erk1/2) in minutes through phosphorylation of Thr202/Tyr204 in MCF-7 cells. These results demonstrate a targeted therapeutics delivery of CDE1-Ada-DOX to breast cancer cells in a controlled manner and that the drug vector CDE1 can potentially be employed as a molecular tool to differentiate nongenomic from genomic mechanism.

Synthesis and biological evaluation of novel folic acid receptor-targeted, ß-cyclodextrin-based drug complexes for cancer treatment.

Drug targeting is an active area of research and nano-scaled drug delivery systems hold tremendous potential for the treatment of neoplasms. In this study, a novel cyclodextrin (CD)-based nanoparticle drug delivery system has been assembled and characterized for the therapy of folate receptor-positive [FR(+)] cancer. Water-soluble folic acid (FA)-conjugated CD carriers (FACDs) were successfully synthesized and their structures were confirmed by 1D/2D nuclear magnetic resonance (NMR), matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS), high performance liquid chromatography (HPLC), Fourier transform infrared spectroscopy (FTIR), and circular dichroism. Drug complexes of adamatane (Ada) and cytotoxic doxorubicin (Dox) with FACD were readily obtained by mixed solvent precipitation. The average size of FACD-Ada-Dox was 1.5-2.5 nm. The host-guest association constant K a was 1,639 M(-1) as determined by induced circular dichroism and the hydrophilicity of the FACDs was greatly enhanced compared to unmodified CD. Cellular uptake and FR binding competitive experiments demonstrated an efficient and preferentially targeted delivery of Dox into FR-positive tumor cells and a sustained drug release profile was seen in vitro. The delivery of Dox into FR(+) cancer cells via endocytosis was observed by confocal microscopy and drug uptake of the targeted nanoparticles was 8-fold greater than that of non-targeted drug complexes. Our docking results suggest that FA, FACD and FACD-Ada-Dox could bind human hedgehog interacting protein that contains a FR domain. Mouse cardiomyocytes as well as fibroblast treated with FACD-Ada-Dox had significantly lower levels of reactive oxygen species, with increased content of glutathione and glutathione peroxidase activity, indicating a reduced potential for Dox-induced cardiotoxicity. These results indicate that the targeted drug complex possesses high drug association and sustained drug release properties with good biocompatibility and physiological stability. The novel FA-conjugated ß-CD based drug complex might be promising as an anti-tumor treatment for FR(+) cancer.