Exosome-like nanoplatform modified with targeting ligand improves anti-cancer and anti-inflammation effects of imperialine.

Currently, most anti-cancer therapies are still haunted by serious and deleterious adverse effects. Here, we report a highly biocompatible tumor cell-targeting delivery systems utilizing exosome-like vesicles (ELVs) that delivers a low-toxicity anti-cancer agent imperialine against non-small cell lung cancer (NSCLC). First, we introduced a novel micelle-aided method to efficiently load imperialine into intact ELVs. Then, integrin α3ß1-binding octapeptide cNGQGEQc was modified onto ELV platform for tumor targeting as integrin α3ß1 is overexpressed on NSCLC cells. This system not only significantly improved imperialine tumor accumulation and retention, but also had extremely low systemic toxicity both in vitro and in vivo. Our discoveries offer new ways to utilize ELV more efficiently for both drug loading and targeting. The solid pharmacokinetics improvement and extraordinary safety of this system also highlight possibilities of alternative long course cancer therapies using similar strategies.

Simultaneous Determination and Pharmacokinetics of Peimine and Peiminine in Beagle Dog Plasma by UPLC-MS/MS after the Oral Administration of Fritillariae ussuriensis Maxim and Fritillariae thunbergii Miq Powder.

A simple and high sensitive ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the simultaneous determination of peimine and peiminine in beagle dog plasma after the oral administration of Fritillariae ussuriensis Maxim and Fritillariae thunbergii Miq powder. Chromatographic separation was achieved on an ACQUIT UPLC® BEH C18 column (1.7 µm, 2.1 × 100 mm) in a gradient elution way with a mobile phase consisting of acetonitrile and water containing 0.1% formic acid at a flow rate of 0.4 mL/min. The plasma samples were prepared by a liquid⁻liquid extraction (LLE) method with ethyl acetate. The analytes were detected with a triple quadrupole tandem mass spectrometry (MS) in multiple reaction monitoring (MRM) mode and a positive ion electrospray ionization (ESI) of the transitions at m/z 432.4→414.4 for peimine and m/z 430.3→412.3 for peiminine. The method was linear for two analytes over the investigated range with all determined correlation coefficients exceeding 0.9900. The lower limit of quantification (LLOQ) was 0.988 ng/mL for peimine and 0.980 ng/mL for peiminine. The mean extraction recoveries of peimine and peiminine at three quality control samples (QC) levels were ranged from 82.56 to 88.71%, and matrix effects ranged from 92.06 to 101.2%. The intra-day and inter-day precision and accuracy were within the acceptable limits at LLOQ and QC levels. The method was effectively and successfully applied to the pharmacokinetics of peimine and peiminine after oral administration of powder to beagle dogs. The obtained results may be help to guide the clinical application of Fritillaria ussuriensis Maxim and Fritillaria thunbergii Miq.

A (polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer)-dispersed sustained-release tablet for imperialine to simultaneously prolong the drug release and improve the oral bioavailability.

Imperialine, extracted from Bulbus Fritillariae Cirrhosae, is an efficient antitussive and expectorant medicine. However, its short half-life and stomach degradation limited imperialine from further clinical use. The current study was conducted to develop a sustained-release tablet for imperialine both to prolong absorption time and to improve the oral bioavailability of the drug. The tablets were prepared by a direct compression method formulated on optimized solid dispersion (SD) for imperialine based on polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (Soluplus(®)) with imperialine/Soluplus(®) ratio of 1:8 (w/w). In order to obtain the optimized formulation, factors that affected the drug release were investigated by in vitro dissolution studies in the media of pH1.2, 5.8, 7.0 and 7.4. Powder X-ray diffraction and scanning electron microscope confirmed that the imperialine in SD was amorphous instead of crystalline, and still stayed amorphous even after the direct compression. And besides, pharmacokinetic study in Beagle dogs was performed to inspect the in vivo sustained release. Plasma concentration-time curves and pharmacokinetic parameters were gained. As a result, the Cmax of imperialine was one-fold reduced and Tmax was two-fold prolonged, and the mean AUC0-24 was expressed as 89.581±21.243µgh/L, which showed that the oral bioavailability of imperialine was 2.46-fold improved. Moreover, the in vitro-in vivo correlation was recommended to carry out, demonstrating the percentages of drug release in vitro were well-correlated with the absorptive fraction in vivo with the correlation coefficients above 0.9900. By mathematically modeling and moment imaging of the drug release, Peppas equation was selected as the most fitted model for the sustained-release tablets with the diffusional coefficient in the range of 0.59-0.62, indicating the release of imperialine from the sustained-release tablets was an anomalous process involving polymer swelling, drug diffusion and matrix erosion.

Pharmacokinetics, tissue distribution and excretion of verticinone from F. hupehensis in rats.

Verticinone, the main active component in F. hupehensis, exhibits potent antitussive and expectorant effects. Here, a LC-MS method was developed and applied to study the pharmacokinetics, tissue distribution and excretion of verticinone in rats, and its plasma protein binding in vitro. A significant gender difference in the pharmacokinetics of verticinone in rats was observed, as its absolute oral bioavailability in male and female rats was 45.8% and 2.74%, respectively. The relative bioavailability of verticinone was significantly lower in female rats as compared to male, following intragastrical (i.g.) and intravenous (i.v.) administration. After successive i.g. administration of verticinone, accumulation was observed in female rats but not in the male ones. The tissue distribution study showed that verticinone had a good tissue penetrability and a high tissue affinity in most studied tissues, except brain. After a 2 mg/kg oral dose, less than 4% of the dose was excreted as unchanged parent compound in male rats, and less than 1% in female rats, which indicated that verticinone was metabolized more extensively in female rats than in male rats.

Novel LC-MS/MS method for analyzing imperialine in rat plasma: development, validation, and application to pharmacokinetics.

A sensitive, selective, rapid liquid chromatography-electrospray ionization-tandem mass spectrometric method was developed and validated in rat plasma to quantify imperialine, a major active constituent extracted from Bulbus Fritillariae Cirrhosae. Before analysis, plasma samples were pre-treated using cost-effective protein precipitation in order to extract imperialine and the internal standard, carbamzepine. The two analytes were then separated on a Diamonsil ODS chromatography column using gradient elution with a mobile phase of 0.1% aqueous formic acid and acetonitrile. Mass spectrometry was carried out in multiple reaction monitoring mode using a positive electrospray ionization interface. The calibration curve was linear (r(2)=0.9998) over the concentration range 2-1000ng/mL, while the validated limit of determination (LOD) was 0.5ng/mL. Precision varied from 0.1% to 7.1%, and the accuracy varied within ±2%. The method proved robust to sample freezing and thawing, as well as short- and long-term sample storage. The developed method was successfully applied to the pharmacokinetic study of imperialine in rats. Different amounts of imperialine were administered in single doses orally or through the caudal vena cava, and pharmacokinetic parameters were evaluated. Oral bioavailability with a dose of 1mg/kg was 31.2%; 5mg/kg, 53.6%; and 10mg/kg, 47.4%.

A simple and sensitive LC-MS/MS method for quantitation of peimine and its pharmacokinetic in beagle dogs.

A simple LC-MS/MS method was developed for determination and pharmacokinetic study of peimine in beagle dogs. The chromatographic separation was carried out on a Agilent Zobax SB C18 column with a mobile phase consisting of acetonitrile-10 mmol/L ammonium formate (35:65, v/v) at a flow rate of 0.3 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by MRM via electro spray ionization source with positive mode. The standard curve for peimine was linear (r>0.999) over the concentration range of 1-200 ng/mL with a lower limit of quantification of 1 ng/mL. The intra- and inter-day precision (relative standard deviation) values were not higher than 15% and the accuracy (relative error) was <5% at 3 quality control levels. This simple, fast and highly sensitive method was fully validated and successfully applied to a preclinical pharmacokinetic study of peimine in dogs after oral and intravenous administration.

[Rat intestinal absorption trait of peimine and peiminine in Thunberg fritillary bulb extract].

To study the in situ intestinal absorption kinetics and compatibility influence of peimine and peiminine in rats, the absorption of peimine and peiminine in small intestine (duodenum, jejunum and ileum) and colon of rats was investigated using in situ single-pass perfusion method and the drug content was measured by HPLC-ELSD. Perfusion rate, pH, concentration of drug, gender and bile duct ligation can significantly affect the absorption of peimine and peiminine, the Ka, and Papp values in the condition of pH 6.8 and pH 7.4 had significant difference (P<0.01), as drug concentration irlcreased, the absorption parameters of peimine and peiminine decreased, Ka and Papp between low concentrations and middle concentrations was significant difference (P<0.01). Verapamil can not affect Ka and Papp of peimine and peiminine which are in the extract (P> 0.05). Bitter almonds and licorice can significantly reduce the absorption of peimine and peiminine with the usual dose (P<0.01), extracted separately and together had no significant difference on Ka and Papp (P> 0.05). Experimental results show that the absorption features of peimine and peiminine are basically the same, both of them could be absorbed at all segments of the intestine in rats and had no special absorption window, and with significant differences between male and female individuals. The absorption of peimine and peiminine complies with the active transport and facilitated diffusion in the general intestinal segments. Bitter almond and licorice can reduce the intestinal absorption rate ofpeimine and peiminine.

Sex dependent pharmacokinetics, tissue distribution and excretion of peimine and peiminine in rats assessed by liquid chromatography-tandem mass spectrometry.

ETHNOPHARMACOLOGICAL RELEVANCE: Fritillaria thunbergii Miq. has been traditionally used in China as antitussive and expectorant herbs, and newly used in the clinical treatment of leukemia in recent years. AIM: To investigate whether gender exerted a significant influence on the pharmacokinetics, tissue distribution and excretion of peimine and peiminine in Sprague-Dawley rats who were given a single oral administration of 4.25 g/kg Fritillaria thunbergii Miq. extract. MATERIALS AND METHODS: Sprague-Dawley rats were assigned into two groups based on the gender and orally administered 4.25 g/kg Fritillaria thunbergii Miq. extract for each individual pharmacokinetics, tissue distribution and excretion study. RESULTS: Compared with female rats, peimine and peiminine were eliminated slowly from male rat plasma, and significant gender-related differences were observed in the pharmacokinetic parameters. Drug blood and tissue levels in male rats were significantly higher than the female counterparts except for several tissues, such as fat, muscle and skin. Gender also exerted a significant influence on the urine excretion but such effect was not observed in the feces excretion study. CONCLUSIONS: Gender exerted a significant influence on the pharmacokinetics, tissue distribution and urine excretion of peimine and peiminine. It is assumed that the sex-associated differences of peimine and peiminine in rats might be mainly result from sex-dependent expression and activity of drug metabolism enzymes and P-glycoprotein.

Comparative pharmacokinetic studies of peimine and peiminine in rat plasma by LC-MS-MS after oral administration of Fritillaria thunbergii Miq. and Fritillaria thunbergii Miq. - Glycyrrhiza uralensis Fisch. couple extract.

A sensitive LC-MS-MS method has been successfully applied to pharmacokinetic study of peimine and peiminine in rat plasma after oral administration of Fritillaria thunbergii Miq. exact and Fritillaria thunbergii Miq. - Glycyrrhiza uralensis Fisch. couple extract. The results indicated that plasma profiles of peimine and peiminine confirmed to two-compartment open model with weighting function of 1/C2 for data fitting and parameter estimation and the utilization with Glycyrrhiza uralensis Fisch. could decrease C(max) and prolong MRT(0-infinity) and t1/2 of peimine remarkly with the bioavailability of peimine remained practically unchanged. Meanwhile, the concentration of peimine in rat plasma was more stable. Nevertheless, there were no significant differences among all calculated parameters of peiminine.

[Simultaneous determination of peimine and peiminine in rat plasma by LC-MS/MS and its application in the pharmacokinetic study].

To establish an LC-MS/MS method for simultaneous determination of peimine and peiminine in rat plasma after oral and intravenous administration of Fritillaria thunbergii Miq. extract, the pharmacokinetic parameters were calculated as well. Peimine, peiminine and internal standard carbamazepine were extracted from plasma with liquid-liquid extraction by ethyl acetate, then separated on a Luna C18 column by using acetonitrile-water containing 10 mmol x L(-1) ammonium formate (35:65), as mobile phase. The electrospray ionization (ESI) source was applied and operated in positive ion mode. Peimine was detected at m/z 432.4 --> 414.4, peiminine at m/z 430.4 --> 412.4 and carbamazepine (IS) at 237.1 --> 194.2. The linear calibration curves were obtained at the concentration range of 0.8-800 ng x mL(-1) for peimine and peiminine. The extraction recoveries were 94.1%-105.3% and 85.8%-98.6%, respectively. The precisions, accuracy and stability of the analytes meet the requirements. The method was shown to be effective, convenient, and suitable for simultaneous pharmacokinetic study of peimine and peiminine in rat.

Development and validation of a liquid chromatography-mass spectrometry method for the determination of verticinone in rat plasma and its application to pharmacokinetic study.

We have developed and validated a sensitive liquid chromatography-electrospray ionization-mass spectrometric (LC-ESI-MS) method for the quantification of verticinone, a major active constituent from Fritillaria hupehensis Hsiao et KC Hsia., in rat plasma. Verticinone and the internal standard (IS), hupehenine, were extracted from plasma samples by a simple liquid-liquid extraction with ethyl acetate after being alkalified by 1M ammonia hydroxide. Chromatographic separation was achieved on a C(18) column using a gradient elution program with methanol and water as the mobile phase. The detection was performed by selected ion monitoring (SIM) mode via positive electrospray ionization (ESI) interface. The lower limit of quantification (LLOQ) was 0.1 ng/mL. The calibration curves were linear (r(2)>0.998) over the concentration range of 0.1-200 ng/mL. Within- and between-run precision was less than 6.5% and accuracy was within +/-10.7%. The validated method was applied to the pharmacokinetic study of verticinone in rats after a single oral administration of 1 mg/kg.

Turbulent-flow chromatography coupled on-line to fast high-performance liquid chromatography and mass spectrometry for simultaneous determination of verticine, verticinone and isoverticine in rat plasma.

A method based on the on-line turbulent-flow chromatography and fast high-performance liquid chromatography/mass spectrometry (TFC-LC/MS) was developed for sensitive and high throughput pharmacokinetic study of traditional Chinese medicines (TCMs). In this method, an on-line extraction column (Waters Oasis HLB) and a fast HPLC column with sub-2 microm particle size (Agilent Zorbax StableBond-C(18), 4.6 mm x 50 mm, 1.8 microm) in a column-switching set-up were utilized. HLB is a reversed-phase extraction column with hydrophilic-lipophilic balanced copolymer (2.1 mm x 20 mm, 25 microm particle size), which will exhibit some turbulent-flow properties at a high-flow rate. The method combines the speed and robustness of turbulent-flow extraction and the sensitivity and separation efficiency of fast HPLC-MS to analyze multiple and trace constituents of TCMs in plasma matrix. This method was successfully applied for pharmacokinetic study of verticine, verticinone and isoverticine, the chemical markers of Fritillaria thunbergii, after oral administration of total steroidal alkaloids extract of F. thunbergii to rats. Each plasma sample was analyzed within 7 min. The method demonstrated good linearity (R>0.999) ranged from 0.505 to 96.0 ng/mL with satisfactory accuracy and precision, and the lower limit of quantifications of verticine, verticinone and isoverticine were estimated to be 0.120, 0.595 and 0.505 ng/mL, respectively. These results indicate that the proposed method is fast, sensitive, and feasible for pharmacokinetic study of TCMs.

Simultaneous determination of peimine and peiminine in rat plasma by LC-ESI-MS employing solid-phase extraction.

A simple and reliable LC-ESI-MS method for the determination of peimine and peiminine in rat plasma was developed for the first time. The method was proven to be specific and sensitive by carrying out validation. The analytes were extracted from rat plasma via solid-phase extraction on Waters Oasis MCX cartridges. Chromatography separation was achieved on a C(18) column using 10 mM ammonium acetate (adjusted to pH 3.0 with glacial acetic acid)-acetonitrile (85:15, v/v) as mobile phase. The linear range was 1-100 ng/mL for peimine and peiminine. Intra- and inter-day precisiond were less than 10%. Accuracies were within 85-115% of their nominal concentrations. The limit of quantification was 1 ng/mL for both analytes. The developed assay was successfully applied to pharmacokinetic study of peimine and peiminine in rats orally administered the alkaloids extracts from Bulbus Fritillariae, demonstrating a possible broader spectrum of applications of this method.

3-alpha-Chlorimperialine: an M2-selective muscarinic receptor antagonist that penetrates into brain.

Muscarinic M2 receptors have been found to be severely depleted in post-mortem brains of Alzheimer's patients. This loss of receptor may represent a useful diagnostic marker, if it could be quantitatively imaged with single-photon emission computed tomography (SPECT) or positron emission tomography (PET) imaging. In order to develop a radioligand with selectivity for muscarinic M2 receptors, we now report that 3-alpha-chlorimperialine is a potent M2 receptor antagonist with a Ki of 0.32 nM at M2 receptors, a 12-fold selectivity for M2 over M1 receptors, and a 5-fold selectivity for M2 over M4 receptors. Furthermore, 2% of the injected dose of 3-alpha-chlorimperialine per gram tissue penetrates into brain within 30 min, then washes out gradually. Taken together, these studies demonstrate that 3-alpha-chlorimperialine is a potent M2-selective muscarinic antagonist that penetrates into brain and may be a useful substrate for radioiodination and subsequent imaging of brain muscarinic M2 receptors.