Near-infrared light-responsive nanoparticles for improved anticancer efficacy through synergistic chemo-photothermal therapy.

Combined treatment is more effective than single treatment against most forms of cancer. The synergistic chemo-thermotherapy mediated by nanoparticles has superior advantages of lesser adverse effects as a promising cancer therapy modality. In this study, we report a theranostic carrier system co-encapsulating Doxorubicin (DOX) and Indocyanine green (ICG) into the D-α-Tocopheryl polyethylene glycol 1000 succinate (TPGS). Full physicochemical characterization studies of the DOX/ICG-loaded TPGS nanoparticles (TNPs) are performed. TNPs have a mean size around 60 nm with superior photostability, and entrapment efficiency of drugs in TNPs was 75.0% for ICG and 68.3% for DOX. TNPs also exhibit a longer sustained release with around 63% of the entrapped drug in 24 h. In vitro studies, TNPs could effectively enhance cellular uptake of DOX and ICG, which permitted high therapeutic efficacy against cancer cells. Further, we investigate antitumor efficacy of TNPs along with its impact on major organs in vivo, TNPs also exhibit a complete inhibition of tumor growth and minimal side effects after irradiation. Collectively, these results suggest that near-infrared light-responsive TNPs can further enhance antitumor effects by synergistic chemo-photothermal therapy.

Ganoderic Acid A Metabolites and Their Metabolic Kinetics.

Ganoderic acid A (GAA), a representative active triterpenoid from Ganoderma lucidum, has been reported to exhibit antinociceptive, antioxidative, cytotoxic, hepatoprotective and anticancer activities. The present study aims (1) to identify GAA metabolites, in vivo by analyzing the bile, plasma and urine after intravenous administration to rats (20 mg/kg), and in vitro by incubating with rat liver microsomes (RLMs) and human liver microsomes (HLMs); (2) to investigate the metabolic kinetics of main GAA metabolites. Using HPLC-DAD-MS/MS techniques, a total of 37 metabolites were tentatively characterized from in vivo samples based on their fragmentation behaviors. The metabolites detected in in vitro samples were similar to those found in vivo. GAA underwent extensive phase I and II metabolism. The main metabolic soft spots of GAA were 3, 7, 11, 15, 23-carbonyl groups (or hydroxyl groups) and 12, 20, 28 (29)-carbon atoms. Ganoderic acid C2 (GAC2) and 7ß,15-dihydroxy-3,11,23-trioxo-lanost-26-oic acid were two main reduction metabolites of GAA, and their kinetics followed classical hyperbolic kinetics. The specific isoenzyme responsible for the biotransformation of the two metabolites in RLMs and HLMs was CYP3A. This is the first report on the comprehensive metabolism of GAA, as well as the metabolic kinetics of its main metabolites.

Plasma and brain pharmacokinetics of ganoderic acid A in rats determined by a developed UFLC-MS/MS method.

Ganoderic acid A (GAA), an active triterpenoid of the traditional Chinese herbal medicine Lingzhi, has been reported to exhibit antinociceptive, antioxidative, and anti-cancer activities. The present study aims to establish a sensitive and rapid UPLC-MS/MS method for studying the plasma and brain pharmacokinetics of GAA in rats. The analytes were separated on a C18 column eluted with a gradient mobile phase consisting of acetonitrile and 0.1% aqueous formic acid at 0.3mL/min. The eluate was monitored by a mass detector using an MRM (m/z, 515.3-285.1) model in negative electrospray ionization. The calibration curve showed good linearity (r2>0.99), with limits of detection and quantification of 0.25 and 2.00 nmol/L, respectively. The intra- and inter-day precision and accuracy were less than 9.99% and ranged from 97.45% to 114.62%, respectively. The extraction recovery from plasma was between 92.89% and 98.87%. GAA was found to be stable in treated samples at room temperature (22°C) for 12h and in plasma at -20°C for 7d. The developed method was successfully applied to a pharmacokinetic study of GAA in rats. GAA could be rapidly absorbed into the circulation (Tmax, 0.15h) and eliminated relatively slowly (t1/2, 2.46h) after orally dosing, and could also be detected in the brain lateral ventricle (Tmax, 0.25h and t1/2, 1.40h) after intravenously dosing. The absolute oral bioavailability and brain permeability of GAA were estimated to be 8.68% and 2.96%, respectively.

Pharmacokinetic profiles of the five isoflavonoids from Pueraria lobata roots in the CSF and plasma of rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicine Radix Puerariae, the roots of Pueraria lobata (Wild.) Ohwi., has been widely used for the treatment of cardiovascular and cerebrovascular diseases in China for centuries. Isoflavonoids are believed the active components of this herb. AIM OF THIS STUDY: The present study aims to investigate the brain penetration and pharmacokinetics of five active isoflavonoids in the ventricular CSF and plasma of rats after intravenous administration of a Pueraria isoflavonoids (PIF) extract, to better understand the active components of this herb for neuro-activities. MATERIAL AND METHODS: Under anesthesia condition, SD rats (n=6) were successively suffered two surgeries for implanting cannulas at lateral ventricle and right jugular vein for brain microdialysis and blood collection, respectively. After recovery, the rats received intravenous dose of PIF at 80mg/kg and the concentrations of puerarin (PU), 3'-methoxypuerarin (MPU), 3'-hydroxypuerarin (HPU), daidzein (DA) and daidzein-8-C-apiosyl-(1-6)-glycoside (DAC) in the ventricular dialysate and plasma samples were determined using a ultra-fast liquid chromatography tandem mass spectrometry method. RESULTS: Complete concentration versus time profiles of the five components in plasma and four components except for HPU in ventricular CSF were obtained. After dosing, the average C0 values of PU, MPU, DA, DAC and HPU in plasma were estimated 6.53, 13.72, 1.54, 15.84 and 86.07µg/mL, and PU, MPU, DA and DAC were rapidly penetrated to the brain and reached to their Cmax of 521.52, 415.00, 74.34 and 380.03ng/mL in CSF at about 0.5-0.8h, respectively. The elimination t1/2 of PU, DA and DAC in CSF and plasma were no significant difference, while the t1/2 of MPU in ventricular CSF was longer than that in plasma which may attributable to the different physiological environment of central and peripheral compartments. The brain penetration index (AUCCSF/AUCplasma) was found to be about 9.29, 7.25, 11.96, and 4.21% for PU, MPU, DA, and DAC respectively. CONCLUSION: PU, MPU, DA, DAC can quickly penetrate to the brain through the blood brain barrier (BBB) and might be responsible for the neuro-pharmacological activities of P. lobata.

Metabolism profiles of nuciferine in rats using ultrafast liquid chromatography with tandem mass spectrometry.

Nuciferine (NF) is one of the main aporphine alkaloids existing in the traditional Chinese medicine Folium Nelumbinis (lotus leaves). Modern pharmacological studies have demonstrated that NF has a broad spectrum of bioactivities, such as anti-HIV and anti-hyperlipidemic effects, and has been recommended as a leading compound for new drug development. However, the metabolites and biotransformation pathway of NF in vivo have not yet been comprehensively investigated. The present study was performed to identify the metabolites of NF for exploring in vivo fates. Rat plasma and urine samples were collected after oral administration and prepared by liquid-liquid extraction with ethyl acetate. A method based on ultrafast liquid chromatography with tandem mass spectrometry was applied to identify the metabolites. Q1 (first quadrupole) full scan combined with a multiple reaction monitoring (MRM) survey scan were used for the detection of metabolites. MRM-information-dependent acquisition of enhanced product ions was used for the structural identification of detected metabolites. A total of 10 metabolites were identified, including phase I (demethylation, oxidation and dehydrogenation) and phase II (glucuronidation, sulfation and glutathione) biotransformation products. Demethylation is the main metabolic pathway of NF in the body. These results can help in improving understanding of the disposition and pharmacological mechanism of NF in the body. Copyright © 2016 John Wiley & Sons, Ltd.

Lotus Leaf Alkaloid Extract Displays Sedative-Hypnotic and Anxiolytic Effects through GABAA Receptor.

Lotus leaves have been used traditionally as both food and herbal medicine in Asia. Open-field, sodium pentobarbital-induced sleeping and light/dark box tests were used to evaluate sedative-hypnotic and anxiolytic effects of the total alkaloids (TA) extracted from the herb, and the neurotransmitter levels in the brain were determined by ultrafast liquid chromatography-tandem mass spectrometry. The effects of picrotoxin, flumazenil, and bicuculline on the hypnotic activity of TA, as well as the influence of TA on Cl(-) influx in cerebellar granule cells, were also investigated. TA showed a sedative-hypnotic effect by increasing the brain level of γ-aminobutyric acid (GABA), and the hypnotic effect could be blocked by picrotoxin and bicuculline, but could not be antagonized by flumazenil. Additionally, TA could increase Cl(-) influx in cerebellar granule cells. TA at 20 mg/kg induced anxiolytic-like effects and significantly increased the concentrations of serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), and dopamine (DA). These data demonstrated that TA exerts sedative-hypnotic and anxiolytic effects via binding to the GABAA receptor and activating the monoaminergic system.

An LC-MS/MS method for simultaneous determination of three Polygala saponin hydrolysates in rat plasma and its application to a pharmacokinetic study.

ETHNOPHARMACOLOGICAL RELEVANCE: Radix Polygala has a long history of use as a sedative in traditional Chinese medicine and its major ingredients are saponins, which are recognized effective in memory improvement but highly toxic to gastricintestinal mucosa. Polygala saponin hydrolysates (PSH), an alkaline hydrolysis product and also the intestinal metabolites of the saponins, exhibited stronger effects in improving memory of mice and had less toxicity than its original saponins. The present study aims to develop a sensitive LC-MS/MS method for simultaneously determining PSH three major active components, 3,4,5-trimethoxycinnamylic acid (TMCA), p-methoxycinnamylic acid (PMCA) and tenuifolin (TF), in rat plasma and apply the method to a pharmacokinetic study. MATERIALS AND METHODS: The acidic plasma (100µl) was treated by liquid-liquid extraction with ethyl acetate and reconstituted sample was analyzed on a C18 column eluted with acetonitrile-water (50:50) containing 0.2% formic acid at 0.4ml/min. The mass detection in negative electrospray ionization was used. The ion pairs for multiple reaction monitoring were set at m/z 237.0/103.0, 177.0/116.6 and 679.5/425.3 for TMCA, PMCA and TF, respectively. Their pharmacokinetic profiles were studied in rats after intravenous and oral dose of PSH at 20 and 100mg/kg, respectively. RESULTS: The calibration curves had good linearity (r(2)>0.99) for TMCA, PMCA and TF within the tested concentration ranges. The limits of detection and quantification were 1, 10, 0.5ng/ml and 10.0, 20.0, 1.0ng/ml, respectively. The intra-day and inter-day precisions were less than 18.9% and accuracies between 93.2% and 113.3%, and the extraction recovery ranged from 91.2% to 112.1% for all analytes. The pharmacokinetic study showed that TMCA, PMCA and TF could be rapidly absorbed into the circulation and reached their peak concentrations at about 9.1, 9.0 and 24.0min, respectively. TF had a lower oral bioavailability (2.0%) than TMCA (90.1%) and PMCA (96.5%), but it remained in the body much longer (t1/2, λz, 4.8h, oral dose) than TMCA (0.6h) and PMCA (0.9h). CONCLUSIONS: A sensitive LC-MS/MS method was developed and applied to a pharmacokinetic study of TMCA, PMCA and TF of PSH in rats. The three components are proved to be bio-available active components of PSH and might display their in vivo pharmacological activities at different levels and different time periods after oral administration.

Identification and characterization of potent CYP2D6 inhibitors in lotus leaves.

ETHNOPHARMACOLOGICAL RELEVANCE: The herb of lotus (Nelumbo nucifera) leaves is a commonly used traditional Chinese herbal medicine that is utilized for the treatment of sunstroke, to assuage thirst, and to cure both diarrhea and fever in China. Modern pharmacological studies have demonstrated that the herb exhibits various pharmacological effects, such as anti-hyperlipidemia, anti-obesity, anti-oxidant, anti-HIV, anti-microbial, and anti-hypoglycemic activities. Currently, the herb is becoming more popular in China as a "tea drink" or as a main ingredient of some herbal formulations, which implies that the herb and/or its products are now more likely to be concurrently administered with conventional medicines for losing body weight and reducing blood lipids. However, its potential inhibitory effect on human cytochrome P450 (CYP) has not been systemically investigated to date. The present study was performed to assess the potential inhibitory effects of lotus leaf alcoholic extract (LAE), its major fractions, and its main compounds on five CYP isoenzymes (CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4) in vitro. MATERIAL AND METHODS: Five probe substrates were incubated with human liver microsomes in the presence or absence of the LAE, the alkaloid fraction (AF), the flavonoid fraction (FF), or the individual aporphine alkaloids, namely, nuciferine (NF), N-nornuciferine (N-NF), and 2-hydroxy-1-methoxyaporphine (HMA). After the incubation, the relative metabolites of the substrates were analyzed using LC-MS/MS. RESULTS: The results showed that the LAE strongly inhibited CYP2D6 with an IC50 value of 12.05µg/mL and weakly inhibited other isoenzymes. In addition, FF was found to weakly inhibit CYP2D6, whereas AF exerted a markedly higher inhibitory effect on CYP2D6 activity with an IC50 value of 0.96µg/mL. The three aporphine alkaloids isolated from the AF (NF, N-NF, and HMA) significantly inhibited CYP2D6 with IC50 values of 3.78, 3.76, and 3.15µM, respectively. Their Lineweaver-Burk plots and Dixon plots showed that NF, N-NF, and HMA competitively inhibited CYP2D6 activity with Ki values of 1.88, 2.34, and 1.56µM, respectively. CONCLUSION: The study revealed that the alkaloid compounds in lotus leaves exert a potent inhibitory effect on CYP2D6 isoenzyme. The possible drug interactions of the leaves and their preparations with conventional medicines should thus be taken into account.

Pharmacokinetic mechanism of enhancement by Radix Pueraria flavonoids on the hyperglycemic effects of Cortex Mori extract in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Diabetes mellitus, characterized by abnormal blood glucose evaluation, is a serious chronic disease. In the treatment of the disease, α-glycosidase inhibitors play an important role for controlling the postprandial blood glucose level. Cortex Mori, a traditional Chinese herbal medicine, has a long history of use for the treatment of headaches, cough, edema and diabetes. Modern pharmacological studies have shown that the herb has beneficial effects on the suppression of postprandial blood glucose levels by inhibiting α-glycosidase activity in the small intestine. 1-Deoxynojirimycin (DNJ), the main active ingredient of this herb, is recognized as a potent α-glycosidase inhibitor. Our previous studies have shown that the hypoglycemic effect of Cortex Mori extract (CME) was significantly improved when giving CME in combination with Radix Pueraria flavonoids (RPF). In the present study, the pharmacokinetics and intestinal permeability of DNJ were comparatively investigated in rats after being given orally or by intestinal perfusion with CME alone or in CME-RPF pairs, to explore the mechanism of this synergistic effect. MATERIALS AND METHODS: The role of RPF on the plasma and urine concentrations of DNJ from CME orally administered was investigated. Four groups of rats received a single oral dose of either CME or CME-RPF, at DNJ equivalent doses of 20 and 40mg/kg, respectively. After dosing, plasma and urine were collected and assayed by LC/MS/MS. In addition, another two groups of rats were used for small intestinal perfusion with CME or CME-RPF at DNJ concentration of 10µM. RESULTS: Compared to the data when dosing with CME alone, the Cmax of DNJ were decreased from 5.78 to 2.94µg/ml (p<0.05) and 10.66 to 5.35µg/ml (p<0.01); Tmax were delayed from 0.40 to 0.55h and 0.35 to 0.50h (p<0.05); and MRT were significantly prolonged from 1.14 to 1.72h (p<0.05) and 0.95 to 1.62h (p<0.01), after dosing with CME-RPF at DNJ doses of 20 and 40mg/kg, respectively. In addition, the urinary recovery of DNJ over the first 4h after dosing significantly decreased from 48.76% to 33.86%. Effective permeability (Peff) of DNJ was decreased from 7.53×10(-3) to 3.09×10(-3)cm/s (p<0.05) when RPF was added to CME, when it was evaluated using the rat intestinal perfusion model. CONCLUSIONS: All the above results demonstrate that RPF was able to suspend and delay the absorption of DNJ, but did not affect the total amount of DNJ in the body. The resulting higher concentration of DNJ in the small intestine produced a relatively stronger effect of depressing the elevation of the postprandial blood glucose level. These findings support the important role of RPF in the application of CME on blood glucose control.

Different pharmacokinetics of the two structurally similar dammarane sapogenins, protopanaxatriol and protopanaxadiol, in rats.

Dammarane Sapogenins (DS), with main ingredients of protopanaxatriol (PPT, 33%) and protopanaxadiol (PPD, 16%), is an alkaline hydrolyzed product of ginsenosides and had significant activities in improving learning and memory and decreasing chemotherapy-induced myelosuppression. In the present study, the pharmacokinetics and oral bioavailabilities of PPT and PPD were investigated when a single dose of DS was administrated orally (75 mg/kg) and intravenously (i.v., 30 mg/kg) to rats. Their in vitro stabilities in the GI tract were also investigated. PPT and PPD concentrations were measured by LC-MS. The results showed that PPT was eliminated rapidly from the body with an average t1/2, λz value of 0.80 h and CL of 4.27 l/h/kg after i.v. administration, while PPD was eliminated relatively slowly with a t1/2, λz of 6.25 h and CL of 0.98l/h/kg. After oral administration, both PPD and PPT could be absorbed into the body, but their systemic exposures were quite different. PPT was absorbed into the body quickly, with a Tmax of 0.58 h and a Cmax of 0.13 µg/ml, while PPD was absorbed relatively slowly with a Tmax of 1.82 h and a Cmax of 1.04 µg/ml. The absolute bioavailabilities of PPT and PPD were estimated as 3.69% and 48.12%, respectively. The stability test found that PPT was instable in the stomach with 40% degradation after 4h incubation at 37°C, both in pH1.2 buffer and in the stomach content solution. The instability in the stomach might be one of the reasons for PPT's poor bioavailability.