Metabolomics investigation of cutaneous T cell lymphoma based on UHPLC-QTOF/MS.

OBJECTIVES: The identification of cutaneous T cell lymphoma (CTCL) biomarkers may serve as a predictor of disease progression and treatment response. The aim of this study was to map potential biomarkers in CTCL plasma. DESIGN AND METHODS: Plasma metabolic perturbations between CTCL cases and healthy individuals were investigated using metabolomics and ultrahigh performance liquid chromatography­quadrupole time-of-flight mass spectrometry (UHPLC-QTOF/MS). RESULTS: Principal component analysis (PCA) of the spectra showed clear metabolic changes between the two groups. Thirty six potential biomarkers associated with CTCL were found. CONCLUSIONS: Based on PCA, several biomarkers were determined and further identified by LC/MS/MS analysis. All of these could be potential early markers of CTCL. In addition, we established that heparin as anticoagulant has better pre-treatment results than EDTA with the UHPLC-QTOF/MS approach.

In vitro and in vivo evidence for amphotericin B as a P-glycoprotein substrate on the blood-brain barrier.

Amphotericin B (AMB) has been a mainstay therapy for fungal infections of the central nervous system, but its use has been limited by its poor penetration into the brain, the mechanism of which remains unclear. In this study, we aimed to investigate the role of P-glycoprotein (P-gp) in AMB crossing the blood-brain barrier (BBB). The uptake of AMB by primary brain capillary endothelial cells in vitro was significantly enhanced after inhibition of P-gp by verapamil. The impact of two model P-gp inhibitors, verapamil and itraconazole, on brain/plasma ratios of AMB was examined in both uninfected CD-1 mice and those intracerebrally infected with Cryptococcus neoformans. In uninfected mice, the brain/plasma ratios of AMB were increased 15 min (3.5 versus 2.0; P < 0.05) and 30 min (5.2 versus 2.8; P < 0.05) after administration of verapamil or 45 min (6.0 versus 3.9; P < 0.05) and 60 min (5.4 versus 3.8; P < 0.05) after itraconazole administration. The increases in brain/plasma ratios were also observed in infected mice treated with AMB and P-gp inhibitors. The brain tissue fungal CFU in infected mice were significantly lower in AMB-plus-itraconazole or verapamil groups than in the untreated group (P < 0.005), but none of the treatments protected the mice from succumbing to the infection. In conclusion, we demonstrated that P-gp inhibitors can enhance the uptake of AMB through the BBB, suggesting that AMB is a P-gp substrate.

Cochinchina momordica seed extract induces apoptosis and cell cycle arrest in human gastric cancer cells via PARP and p53 signal pathways.

Cochinchina momordica seed is the dried ripe seed of Momordica cochinchinensis (Lour.) Spreng, which is a kind of fruit and consumed for dietary as well as medicinal uses. In this study, using the human SGC7901 and MKN-28 gastric cancer cell lines, we explored the anticancer activity of the extract from cochinchina momordica seed (ECMS). ECMS inhibited significantly the survival rates of SGC7901 and MKN-28 cells in concentration- and time-dependent manners by MTT assay. The typical apoptotic morphological changes were observed by Hoechst 33258 dye assay after SGC7901 and MKN-28 cells were treated with ECMS for 48 h. Flow cytometry analysis revealed that ECMS-treatment blocked the cells at the S phase of cell cycle. Furthermore, the protein expression levels of poly (ADP-ribose) polymerase (PARP) and Bcl-2 were downregulated notably by ECMS-treatment, whereas those of Fas/Fas-associated death domain, p53, and Bax were upregulated in SGC7901 cells. ECMS dramatically enhanced the enzymatic activities of caspase-3 and caspase-9 whilst slightly increased caspase-8 activity. Taken together, this study demonstrated that ECMS exerted cytotoxic activities via PARP and p53 signal pathways in the human gastric cancer cells.

Cochinchina momordica seed extract induces G2/M arrest and apoptosis in human breast cancer MDA-MB-231 cells by modulating the PI3K/Akt pathway.

Cochinchina momordica seeds are a kind of traditional Chinese herb. In this study, anticancer activity and underlying mechanisms were investigated with an extract using human breast cancer MDA-MB-231 cells. The survival rate was reduced in a concentration- and time-dependent manner as assessed by MTT assay. After incubation for 48 h, typical apoptotic morphological changes were observed by Hoechst 33258 dye assay. Flow cytometry revealed that the treatment obviously induced G2/M arrest and apoptosis in MDA-MB-231 cells. Furthermore, western blotting demonstrated downregulation of protein expression of PI3K, Akt, NF-kB, Bcl-2, Cdk1 and cyclin B1, whereas Bax and caspase-3 were upregulated. Our results suggest that the extract induced cell cycle G2/M arrest and apoptosis in MDA-MB-231 cells by decreasing PI3K/Akt pathway. Therefore, we propose that ECMS has potential as a breast cancer chemotherapeutic agent.

[Development and identifiability analysis of parent-metabolite pharmacokinetic model for risperidone and its main active metabolite 9-hydroxyrisperidone].

To develop a parent-metabolite pharmacokinetic model for risperidone (RIP) and its major active metabolite (9-hydroxyrisperidone) and investigate their pharmacokinetics characteristics in healthy male volunteers, twenty-two healthy volunteers were orally given a single dose of 2 mg RIP. Plasma samples were collected in the period of 96 hours and concentrations of RIP and 9-hydroxyrisperidone were measured by a validated HPLC/MS method. CYP2D6 phenotypes were identified by the T1/2 of RIP and 9-hydroxyrisperidone according to the literature. Model structure identifiability analysis was performed by the similarity transformation approach to investigate whether the unknown parameters of the proposed model could be estimated from the designed experiment. Pharmacokinetics parameters were estimated using weighted least squares method, and the final pharmacokinetics model were tested and evaluated by Monte Carlo simulation. Eighteen volunteers were phenotyped as extensive metabolizers (EM) and four volunteers were identified as intermediate metabolizers (IM). The final model included central and peripheral compartment for both parent (RIP) and metabolite (9-hydroxyrisperidone) respectively. Model structure identifiability analysis indicated that the proposed model was local identifiable. However, if the ratio of RIP converted to 9-hydroxyrisperidone was assumed to be 32% in EM, and 22% in IM, the model could be globally identifiable. The predicted time-concentration curve and AUC(0-t), C(max), T(max) of RIP and 9-hydroxyrisperidone estimated by the established model were in agreement with the observations and noncompartment analysis. Rate constant of RIP conversion to 9-hydroxyrisperidone was (0.12 +/- 0.08) h(-1) and (0.014 +/- 0.007) h(-1) for EM and IM, respectively. Elimination rate constants of RIP were (0.25 +/- 0.18) and (0.05 +/- 0.23) h(-1) for EM and IM, respectively. Model validation result showed that all parameters derived from the concentration data fitted well with the theoretical value, with mean prediction error of most PK parameter within +/- 15%. The established model well defined the disposition of RIP and 9-hydroxyrisperidone simultaneously and showed large inter-individual pharmacokinetics variation in different CYP2D6 phenotype. The model also provide a useful approach to characterize pharmacokinetics of other parent-metabolite drugs.

Total and free mycophenolic acid and its 7-O-glucuronide metabolite in Chinese adult renal transplant patients: pharmacokinetics and application of limited sampling strategies.

OBJECTIVE: This study aimed to investigate the pharmacokinetic characteristics of total and free mycophnolic acid (MPA) and its 7-O-glucuronide metabolite (MPAG) in Chinese renal transplant recipients. In addition, limited sampling strategies were developed to estimate the individual area under concentration curve (AUC) of total and free MPA. METHODS: Total and free MPA and MPAG concentrations were determined by high performance liquid chromatography. Whole 12-h pharmacokinetic profiles were obtained on the 10th day after operation in 12 adult Chinese de novo renal transplant recipients administrated with mycophenolate mofetil (MMF, 750 mg bid), cyclosporine and corticosteroids. Limited sampling strategies with jackknife technique, a resampling method, and Bland-Altman analysis were employed to develop equations to estimate total and free MPA AUC. RESULTS: The pattern of total and free MPA and MPAG plasma concentration-time curves in the cohort of patients taking lower doses of MMF was consistent with previous reports of Caucasian patients taking MMF 1 g bid, except that dose-normalized exposure of total and free MPAG was much lower in the current study than in those of the Caucasians. The mean C (max) and AUC(0-12h) of total and free MPA were 9.4 +/- 3.4 mg/L, 20.2 +/- 6.5 mg x h/L and 0.4 +/- 0.4 mg/L, 0.7 +/- 0.5 mg x h/L, respectively, whereas mean C (max) and AUC(0-12h) of total and free MPAG were 97.3 +/- 32.6 mg/L, 656.0 +/- 148.0 mg.h/L and 29.9 +/- 8.5 mg/L, 222.0 +/- 58.1 mg x h/L respectively. The mean fractions of free MPA and MPAG were 3.5 +/- 2.0 and 34.6 +/- 8.0%, respectively. No determinant was identified to influence the pharmacokinetics of total and free MPA and MPAG or the free fraction of MPA and MPAG. The combinations of C (2h)-C (4h) and C (1h)-C (2h)-C (3h) were the best to estimate free and total MPA AUC(0-12h) respectively, whereas the combination of C (2h)-C (3h)-C (4h) and C (1h)-C (2h)-C (4h) was the best to estimate both simultaneously. CONCLUSION: This is the first time that the pharmacokinetics profile of total and free MPA and its main metabolite MPAG has been examined in Chinese adult renal transplant patients. The limited sampling strategies proposed to estimate individual free and total MPA AUC could be useful in optimizing patient care.

[Pharmacokinetic model for the enterohepatic circulation of mycophenolic acid].

AIM: To develop a pharmacokinetic model for the enterohepatic circulation of mycophenolic acid (MPA). METHODS: Twenty healthy volunteers were orally given a single dose of 500 mg mycophenolate mofetil. Plasma samples were collected during 48 hours and MPA concentration was measured by HPLC method. Pharmacokinetic (PK) model was established based on physiological and biopharmaceutical consideration and PK parameters were obtained using nonlinear mixed effect model. RESULTS: The proposed model included an intestinal compartment and gall bladder compartment in addition to the central compartment. The predicted time-concentration curve and AUC0-t, Cmax, Tmax estimated by the established model were in agreement with the observations. CONCLUSION: The established model was well defined for the MPA disposition and could afford a useful approach for the further clinical investigation.

Determination of quinolizidine alkaloids in Sophora tonkinensis by HPCE.

A simple, rapid and reliable high-performance capillary electrophoresis method has been developed to determine quantitatively the alkaloid content of Sophora tonkinensis, a Chinese herb commonly known as shan-dou-gen. A total of seven quinolizidine alkaloids (cytisine, sophocarpine, matrine, lehmannine, sophoranol, oxymatrine and oxysophocarpine) could be readily separated within 15 min. The running buffer was 50 mM phosphate buffer (pH 2.5) containing 1% hydroxypropyl-beta-cyclodextrin and 3.3% isopropanol in water. The applied voltage was 25 kV, the capillary temperature was 25 degrees C, the detection wavelength was 200 nm and scopolamine butylbromide was used as internal standard. The method was used to analyse the chemical constituents of two commercial alternatives to shan-dou-gen. The alkaloid constituents of authentic shan-dou-gen gave a specific HPCE electropherogram that could be used to distinguish the drug from potential substitutes. Furthermore, the content of oxymatrine and the total content of the seven quinolizidine alkaloids could be used as quantitative markers in order to assess the quality of S. tonkinensis.

Quantification of total and free mycophenolic acid in human plasma by liquid chromatography with fluorescence detection.

A simple high-performance liquid chromatographic (HPLC) method was developed for the assay of total and free mycophenolic acid (MPA) in human plasma. Prior to analysis, total mycophenolic acid was extracted by protein precipitation and free drug was isolated from plasma samples using ultrafiltration. The extracts were injected onto a Kromasil C8 column at 30 degrees C with excitation and emission wavelengths set at 342 and 425 nm, respectively. The mobile phase was consisted of acetonitrile-32 mM glycine buffer, pH 9.2 (20:80, v/v), at a flow rate of 1.0 ml/min. The method was found to be linear over the concentration range investigated, 0.05-40 mg/l for total mycophenolic acid (r>0.999) and 5-1000 microg/l (r>0.99) for free drug. The percentage error of the analytical method was below 10.9%. The intra- and inter-day reproducibility was adequate with the coefficients of variation of 8.28% or below. The run time were 4 and 6 min for free and total MPA, respectively. The method thus can be effectively applied to measure mycophenolic acid concentrations in clinical samples.