Chun-Yu Liu

An LC–MS/MS method for the simultaneous determination of chlorogenic acid, forsythiaside A and baicalin in rat plasma and its application to pharmacokinetic study of Shuang-huang-lian in rats

An LC-MS/MS method was developed for the simultaneous determination of chlorogenic acid, forsythiaside A and baicalin, three major ingredients in Shuang-huang-lian preparations, in rat plasma. Following extraction by methanol-ethyl acetate-trifluoroacetic acid (49:49:2, v/v/v), the extracted analytes were separated on a reverse phase C12 column using a gradient mobile phase system of acetonitrile-water containing 0.1% formic acid. The limits of quantification were between 1.0 and 2.1ng/mL, the precision was <7% and the accuracy was between 94% and 107%. The validated method was applied to a comparative pharmacokinetic study in rats after administration of Shuang-huang-lian solutions via intravenous, peroral or intratracheal routes. The results showed that the three chemical markers were more rapidly and thoroughly absorbed following pulmonary delivery as compared with peroral administration.

Involvement of the Inhibition of Intestinal Glucuronidation in Enhancing the Oral Bioavailability of Resveratrol by Labrasol Containing Nanoemulsions

Nanoemulsions have been developed for the oral delivery of poorly bioavailable phenolic compounds that are sensitive to intestinal glucuronidation. However, little is known about the contribution of UDP-glucuronosyltransferase (UGT) inhibitory excipients in nanoemulsions toward the inhibition of intestinal glucuronidation and the consequent enhanced bioavailability. In this study, Labrasol but not poloxamer 188 (F68) was found to inhibit the glucuronidation of resveratrol (RES), a model phenolic compound, in an inhibition assay with rat microsomes. Subsequently, two nanoemulsions, Lab-N and F68-N, were prepared with similar particle size distribution, zeta potentials, and entrapment efficiency by coemulsifying with Labrasol or F68, respectively. Although Lab-N exhibited inferior or comparable profiles of in vitro release, cellular uptake in Caco-2 cells, and lymphatic transport in rats to F68-N, the in vitro absorption study with everted sacs suggested that Labrasol containing formulations significantly and dose-dependently increased the transport of RES relative to free RES or F68-N by decreasing the amount of permeated metabolite, RES-3-glucuronide (RES-G). The in vivo pharmacokinetic experiments indicated that Lab-N exhibited increments in the maximum plasma concentration and the bioavailability of RES by 1098% and 560%, respectively, and significant decreases in those of RES-G, compared to F68-N. The overall results demonstrated that the improved oral bioavailability of RES by Lab-N was mainly attributable to the inhibition of intestinal glucuronidation by the presence of UGT inhibitory excipient.

Pulmonary delivery of scutellarin solution and mucoadhesive particles in rats.

The objectives of this study were to investigate the effects of mucoadhesive excipients on systemic bioavailability of an inhaled drug and to evaluate the feasibility of using the pulmonary route for non-invasive systemic delivery of scutellarin, a poorly orally absorbed flavonoid glucuronide. Following intratracheal spray of the scutellarin solution, the bioavailability was found to be approximately 77% in rats, which was >30-fold higher than that via the peroral route. In addition, the pulmonary absorption of scutellarin appeared to avoid the intestinal first-pass metabolism accompanied by peroral administration. Spray-dried scutellarin particles with the presence of mucoadhesive excipients were found to affect the corresponding mucociliary transport rate (MTR) as evaluated by a frog palate model. The pharmacokinetic results indicated that the magnitude of AUC(0-480) of intrapulmonary delivered drug particles was not correlated to the fine particle fraction (FPF) but inversely related to the MTR. Incorporating mucoadhesive polymeric mixtures into the scutellarin particles, the MTR decreased by sixfold, and the absolute bioavailability of the drug was found to increase from 70.1% to 97.9% despite a decrease in the FPF. Moreover, in vitro results evaluated using Calu-3 and A549 cell lines showed that scutellarin and spray-dried particles with or without the presence of mucoadhesives exhibited no local cell cytotoxic effects in the tested concentration range. In conclusion, the conducting airway is well permeable to scutellarin, and scutellarin may be effectively delivered systemically through inhalation of respirable droplets or particles.

Oily nanosuspension for long-acting intramuscular delivery of curcumin didecanoate prodrug: Preparation, characterization and in vivo evaluation

The objective of this study was to prepare the nanocrystals of curcumin didecanoate (CurDD) by wet ball milling and to investigate the comparative pharmacokinetics of oily nano- and micro-suspensions after intramuscular (i.m.) administration to rats. Upon optimizing the wet ball milling parameters, CurDD nanocrystals were produced with median particle size of ~500 nm and the freeze-dried nanocrystals were readily dispersed in peanut oil to form stable nanosuspensions. Although the nanosuspension appeared to exhibit slower clearance from the injection site after i.m. injection, compared to microsuspension (~5 μm), a significantly higher maximum plasma curcumin concentration (69.0 ng/ml) was observed for the former than that for the latter (18.5 ng/ml). In addition, the nanosuspension provided significant higher plasma curcumin concentrations and brain CurDD contents for at least 15 days than the microsuspension, except for the initial times. A single i.m. injection of nanosuspension appeared to achieve reversal effect on reserpine-induced hypothermia for at least 13 days. This study demonstrates that CurDD nanosuspension may act as a long-acting i.m. injectable for sustained delivery of curcumin, potentially applicable to elicit a long-lasting antidepressant effect.

Stabilization and sustained release of zeylenone, a soft cytotoxic drug, within polymeric micelles for local antitumor drug delivery.

Use of soft drugs has resulted in mixed success with the applicability to chemotherapeutics yet being confirmed. We hypothesize that incorporation of a soft cytotoxic agent into polymeric micelles, which confer to stabilizing and sustained release effect, will improve and prolong the local antitumor efficacy, thus achieving the therapeutic potential of soft cytotoxic agents. We incorporated a model soft cytotoxic agent, zeylenone, into mPEG-PLGA micelles by solvent evaporation method. The drug loaded micelles were characterized in terms of drug encapsulation, dynamic size, zeta potential, drug stability and in vitro and in vivo release. The in vivo antitumor efficacy was evaluated in A549 tumor-bearing mice. Zeylenone-loaded micelles exhibited core-shell morphology with dynamic size of about 36 nm and offered efficient solubilizing and stabilizing effects. In vitro release and in vivo pharmacokinetic results indicated sustained release of zeylenone in micelles. In addition, local delivered zeylenone-loaded micelles showed improved and sustained antitumor effect in vivo, compared with intravenous administration or local delivery of free drug solution. This study demonstrates the feasibility of soft cytotoxic agent to achieve local antitumor efficacy after the drug was stabilized and sustained the release within polymeric micelles.

Evaluating pulmonary toxicity of Shuang–Huang–Lian in vitro and in vivo

UNLABELLED ETHNOPHARMAOCOLOGICAL RELEVANCE: Shuang-Huang-Lian (SHL) is a traditional Chinese formula and has been used for the treatment of respiratory tract infections by inhalation. However, the pulmonary toxicity via inhalation is largely uninvestigated. AIM OF STUDY To evaluate the pulmonary toxicity of SHL following in vivo intratracheal spray to rats and in vitro exposures to A549 and Calu-3 cells. METHODS Calu-3 and A549 cells were exposed to SHL, chlorogenic acid, baicalin and forsythin solutions and in vitro cytotoxicity was evaluated using an MTT assay, whilst rats were subjected to intratracheal administration of SHL solutions and in vivo toxicity was indicated by assaying the LDH activity and total protein content in bronchoalveolar lavage fluid (BALF) and observing the histopathologic changes of the lungs. Secretion of inflammatory mediators, including IL-6, IL-8 and TNF-α, in cell culture media and BALF was quantified by ELISA. RESULTS The MTT cell viability data revealed the presence of minor toxicity to Calu-3 or A549 cells following exposure to SHL and its major ingredients for 24h or 48 h. However, the cell cultural media showed no sign of inflammatory responses. The in vivo results showed that exposures to SHL at doses of up to 50mg/kg did not significantly increase the total protein content, the LDH activity and the concentrations of IL-6, IL-8 and TNF-α in BALF. However, although intratracheal sprayed SHL at doses of up to 6 mg/kg for histopathologic study and up to 25mg/kg for cell counts showed no sign of adverse effects, inhaled SHL at elevated doses appeared to induce alveolar fusion in the lung and significant increases in the cell number of monocytes and granulocytes in the BALF. CONCLUSION The results demonstrated that the pulmonary safety of inhaled SHL was dependent on the administered dose. Inhalation therapy of SHL may be safely used when the inhaled dose was properly controlled.

Solid lipid nanoparticles for sustained pulmonary delivery of Yuxingcao essential oil: Preparation, characterization and in vivo evaluation.

The objective of this study was to prepare solid lipid nanoparticles (SLNs) for sustained pulmonary delivery of Yuxingcao essential oil (YEO). Three YEO loaded SLNs (SLN-200, SLN-400 and SLN-800) with different particle size were prepared and separated following a high-shear homogenization technique using Compritol 888 ATO as lipid and polyvinyl alcohol as an emulsifier. The particle size, zeta potential, drug encapsulation efficiency and drug loading of the SLNs were determined to be between 171 and 812nm, -17.1 and -19.3mV, between 76.6 and 90.2% and between 2.34 and 3.12%, respectively whereas the in vitro release data showed that the SLNs led to sustained drug release up to 48h. In addition, the SLN suspensions after nebulization conferred the fine particle fractions (<5.4μm) of 67.4-75.8%. Following intratracheal administration to rats, YEO loaded SLNs not only prolonged pulmonary retention up to 24h, but also increased AUC values (15.4, 18.2 and 26.3μg/gh for SLN-200, SLN-400 and SLN-800, respectively) by 4.5-7.7 folds compared to the intratracheally dosed YEO solution and by 257-438 folds to the intravenously dosed YEO solution, respectively. The present results were the first to show that YEO loaded SLNs may sustain YEO inhalation delivery and improve local bioavailability, representing a promising inhalable carrier to attain once daily application.

In vitro uptake and transport studies of PEG-PLGA polymeric micelles in respiratory epithelial cells

&NA; Polymeric micelles are considered promising carriers for pulmonary drug delivery. Their interaction with the respiratory epithelium, however, is mostly unknown. In the present study, methoxypoly (ethylene glycol) (mPEG)‐poly (lactic‐co‐glycolic acid) (PLGA) micelles containing curcumin acetate (CA) or a mixture of CA and Nile Red (NR) were prepared using the solvent evaporation method. Calu‐3 and NCI‐H441 human respiratory epithelial cell monolayers were used as in vitro models of upper and lower respiratory tract epithelium barrier, respectively, to study the cellular uptake and transport of the vesicles. The results show that Calu‐3 and NCI‐H441 cells internalized micellar particles and that micelles were able to translocate across the cell monolayers. Micelles were more readily internalized into and permeated across Calu‐3 cell monolayers when compared to NCI‐H441 cells. Furthermore, the presence of inhibitors of endocytic processes, such as methyl‐&bgr;‐cyclodextrin, NaN3 and hypertonic sucrose attenuated the cellular uptake and trafficking of micelles. In conclusion, this study demonstrated that mPEG‐PLGA micelles translocate human respiratory epithelium in vitro through clathrin‐, energy‐ and cholesterol‐mediated endocytosis. Graphical abstract Figure. No caption available.

Stabilization of zeylenone in rat plasma by the presence of esterase inhibitors and its LC-MS/MS assay for pharmacokinetic study.

Six esterase inhibitors, namely EDTA·2Na(+), NaF, phenylmethanesulfonyl fluoride, dichlorvos, bis-nitrophenyl phosphate (BNPP) and thenoyltrifluoroacetone, and the mixture of NaF and BNPP, were evaluated for the stabilization of labile benzoate containing zeylenone in rat plasma. The mixture appeared to exhibit the most effectively stabilizing effect with the degraded content of zeylenone decreasing from >60% (in the absence of inhibitors) to <6%. Following the stabilization by the addition of NaF (5 mM) and BNPP (5 mM), the analytes in rat plasma were acidified by formic acid and extracted into ethyl acetate at 0°C. After chromatographic separation, the detection of zeylenone was performed on a 3200 Q-Trap with positive ion electrospray mode, monitoring the ion transition m/z 383.2 → 105.0. The method was validated over the range from 2.68 to 1340 ng/mL with inter- and intra-run precision for the quality control samples being less than 6.8%. The assay accuracy was within 100 ± 7.0%. The validated method was successfully applied to a pharmacokinetic study in rats after the intratracheal administration of zeylenone in free drug or polymeric micellar solutions. The results showed that the pulmonary absorption of zeylenone loaded in micelles was significantly retarded compared with that of free drug solutions.

Preparation and evaluation of self-microemulsions for improved bioavailability of ginsenoside-Rh1 and Rh2

Due to intestinal cytochrome P450 (CYP450)-mediated metabolism and P-glycoprotein (P-gp) efflux, poor oral bioavailability hinders ginsenoside-Rh1 (Rh1) and ginsenoside-Rh2 (Rh2) from clinical application. In this study, Rh1 and Rh2 were incorporated into two self-microemulsions (SME-1 and SME-2) to improve oral bioavailability. SME-1 contained both CYP450 and P-gp inhibitory excipients while SME-2 only consisted of P-gp inhibitory excipients. Results for release, cellular uptake, transport, and lymph node distribution demonstrated no significant difference between either self-microemulsions in vivo, but were elevated significantly in comparison to the free drug. The pharmaceutical profiles in vivo showed that the bioavailability of Rh1 in SME-1 (33.25%) was significantly higher than that in either SME-2 (21.28%) or free drug (12.92%). There was no significant difference in bioavailability for Rh2 between SME-1 (48.69%) or SME-2 (41.73%), although they both had remarkable increase in comparison to free drug (15.02%). We confirmed that SME containing CYP450 and P-gp inhibitory excipient could distinctively improve the oral availabilities of Rh1 compared to free drug or SME containing P-gp inhibitory excipient. No notable increase was observed between either SME for Rh2, suggesting that Rh2 undergoes P-gp-mediated efflux, but may not undergo distinct CYP450-mediated metabolism.