Simultaneous determination of six coptis alkaloids in urine and feces by LC-MS/MS and its application to excretion kinetics and the compatibility mechanism of Jiao-Tai-Wan in insomniac rats.

Jiao-Tai-Wan (JTW) is a well-known traditional Chinese medicine prescription composed of Rhizoma Coptidis (RC) and Cortex Cinnamon (10:1, g/g). It has been used to treat insomnia in China for centuries. This study investigates the excretion properties of coptis alkaloids from RC and JTW in normal and insomniac rats, and it examines the compatibility mechanism for this prescription. A new liquid chromatography-tandem mass spectrometry method was developed for the simultaneous determination of six alkaloids - berberine, epiberberine, coptisine, jatrorrhizine, palmatine and magnoflorine - in rat urine and feces. The normal and model rats were orally treated with RC and JTW powder at a dosage containing 3.0 g kg-1  day-1 RC once per day for 7 days. Briefly, the results showed that the cumulative amounts of urinary and fecal excretion of the six alkaloids were significantly different in the pathological condition, as well as in compatibility. In normal rats, the urinary and fecal excretion of coptis alkaloids, especially berberine, coptisine and palmatine, increased significantly in the JTW group compared with the RC group, while the urinary and fecal excretion of six alkaloids decreased in insomniac rats. These data suggested that pathological conditions might have a notable influence on the excretion of alkaloids in rats, and demonstrated that the compatibility could promote better therapeutic effects through the accumulation of alkaloids in the body. These results might explain the compatibility of JTW.

[Study on mechanism of hepatotoxicity of Ploygoni Multiflori Caulis based on function inhibition of bilirubin-associated transporters in idiosyncratic rat].

To explore the possible mechanism of liver injury, the effects of Ploygoni Multiflori Caulis and its extractive on the function of bilirubin-associated transporters were investigated in normal (N) and idiosyncratic (LPS) rats (M). The normal and LPS rats were respectively administrated powder of Ploygoni Multiflori Caulis, its extractive and same volume of 0.5% CMC-Na solution for 7 d. BSP, a substrate of the transporters of Oatp1a1 and Oatp1b2 was selected, and its pharmacokinetic parameters of intravenous injection were determined to examined the activity these transporters. Meanwhile the mRNA expressions of transporters were detected. Compared with N-blank control group, besides M-powder group, the Cmax has no significantly different from other groups, t1/2, AUC0-t and AUC0-∞ were significantly increased, and CL were significantly decreased. However, compared with N- blank control group, AST and ALT decreased significantly. The expression of Oatp1a1, Oatp1b2 and MRP2 mRNA was significantly decreased (P<0.05), but there was no act synergistically when Ploygoni Multiflori Caulis and extractive were combined with LPS. The function of Oatp1a1, Oatp1b2 and MRP2 in rats were significantly inhibited by Ploygoni Multiflori Caulis and extractive, which may be an important cause of hepatotoxicity.

[In vitro effects of Genkwa Flos chloroform extract on activity of human liver microsomes UGTs and UGT1A1].

To predict the mechanism of liver injury induced by Genkwa Flos, we investigated the effect of chloroform extract on UGTs and UGT1A1 activities of the liver microsomes in rat and human. In the present study, 4-nitrophenol(4-NP) and ß-estradiol were elected as substrates to determine activities of UGTs and UGT1A1 by UV and HPLC. The results showed that there were 1.00% of apigenin, 6.40% of hydroxygenkwanin and 18.38% of genkwanin in chloroform extract; and total diterpene mass fraction was 31.40%. Compared with the control group, chloroform extract could significantly inhibit the activity of UGTs in rat liver microsomes(RLM) system, while the inhibitory effect was not obvious in human liver microsomes(HLM) system. UGT1A1 activity was inhibited by chloroform extract in rat liver microsomes and human liver microsomes (based on genkwanin, IC50=8.76, 10.36 µmol•L⁻¹). The inhibition types were non-competitive inhibition(RLM) and uncompetitive inhibition(HLM). In conclusion, the results indicated that chloroform extract showed different inhibitory effects on UGTs and UGT1A1 activity, which may be one of the mechanisms of liver injury induced by Genkwa Flos.

Enhanced anti-hepatocarcinoma efficacy by GLUT1 targeting and cellular microenvironment-responsive PAMAM-camptothecin conjugate.

The efficient targeting of drugs to tumor cell and subsequent rapid drug release remain primary challenges in the development of nanomedicines for cancer therapy. Here, we constructed a glucose transporter 1 (GLUT1)-targeting and tumor cell microenvironment-sensitive drug release Glucose-PEG-PAMAM-s-s-Camptothecin-Cy7 (GPCC) conjugate to tackle the dilemma. The conjugate was characterized by a small particle size, spherical shape, and glutathione (GSH)-sensitive drug release. In vitro tumor targeting was explored in monolayer (2D) and multilayer tumor spheroid (3D) HepG2 cancer cell models (GLUT1+). The cellular uptake of GPCC was higher than that in the control groups and that in normal L02 cells (GLUT1-), likely due to the conjugated glucose moiety. Moreover, the GPCC conjugate exhibited stronger cytotoxicity, higher S arrest and enhanced apoptosis and necrosis rate in HepG2 cells than control groups but not L02 cells. However, the cytotoxicity of GPCC was lower than that of free CPT, which could be explained by the slower release of CPT from the GPCC compared with free CPT. Additional in vivo tumor targeting experiments demonstrated the superior tumor-targeting ability of the GPCC conjugate, which significantly accumulated in tumor meanwhile minimize in normal tissues compared with control groups. The GPCC conjugate showed better pharmacokinetic properties, enabling a prolonged circulation time and increased camptothecin area under the curve (AUC). These features contributed to better therapeutic efficacy and lower toxicity in H22 hepatocarcinoma tumor-bearing mice. The GLUT1-targeting, GSH-sensitive GPCC conjugate provides an efficient, safe and economic approach for tumor cell targeted drug delivery.

The oral bioavailability, excretion and cytochrome P450 inhibition properties of epiberberine: an in vivo and in vitro evaluation.

Epiberberine (EPI) is a novel and potentially effective therapeutic and preventive agent for diabetes and cardiovascular disease. To evaluate its potential value for drug development, a specific, sensitive and robust high-performance liquid chromatography-tandem mass spectrometry assay for the determination of EPI in rat biological samples was established. This assay was used to study the pharmacokinetics, bioavailability and excretion of EPI in rats after oral administration. In addition, a cocktail method was used to compare the inhibition characteristics of EPI on cytochrome P450 (CYP450) isoforms in human liver microsomes (HLMs) and rat liver microsomes (RLMs). The results demonstrated that EPI was rapidly absorbed and metabolized after oral administration (10, 54 or 81 mg/kg) in rats, with Tmax of 0.37-0.42 h and T1/2 of 0.49-2.73 h. The Cmax and area under the curve values for EPI increased proportionally with the dose, and the oral absolute bioavailability was 14.46%. EPI was excreted mainly in bile and feces, and after its oral administration to rats, EPI was eliminated predominantly by the kidneys. A comparison of the current half-maximal inhibitory concentration and Ki values revealed that EPI demonstrated an obvious inhibitory effect on CYP2C9 and CYP2D6. Furthermore, its effect was stronger in HLM than in RLM, more likely to be a result of noncompetitive inhibition.

Overcoming Multidrug Resistance through the GLUT1-Mediated and Enzyme-Triggered Mitochondrial Targeting Conjugate with Redox-Sensitive Paclitaxel Release.

Multidrug resistance (MDR) is thought to be the major obstacle leading to the failure of paclitaxel (PTX) chemotherapy. To solve this problem, a glucose transporter-mediated and matrix metalloproteinase 2 (MMP2)-triggered mitochondrion-targeting conjugate [glucose-polyethylene glycol (PEG)-peptide-triphenylphosponium-polyamidoamine (PAMAM)-PTX] composed of a PAMAM dendrimer and enzymatic detachable glucose-PEG was constructed for mitochondrial delivery of PTX. The conjugate was characterized by a 30 nm sphere particle, MMP2-sensitive PEG outer layer detachment from PAMAM, and glutathione (GSH)-sensitive PTX release. It showed higher cellular uptake both in glucose transporter 1 (GLUT1) overexpressing MCF-7/MDR monolayer cell (2D) and multicellular tumor spheroids (3D). The subcellular location study showed that it could specifically accumulate in the mitochondria. Moreover, it exhibited higher cytotoxicity against MCF-7/MDR cells, which significantly reverse the MDR of MCF-7/MDR cells. The MDR reverse might be caused by reducing the ATP content through destroying the mitochondrial membrane as well as by down-regulating P-gp expression. In vivo imaging and tissue distribution indicated more conjugate accumulated in the tumor of the tumor-bearing mice model. Consequently, the conjugate showed better tumor inhibition rate and lower body weight loss, which demonstrated that it possessed high efficiency and low toxicity. This study provides glucose-mediated GLUT targeting, MMP2-responsive PEG detachment, triphenylphosponium-mediated mitochondria targeting, and a GSH-sensitive intracellular drug release conjugate that has the potential to be exploited for overcoming MDR of PTX.