Byung Hwa Jung

Antiretroviral drug exposure in the female genital tract: implications for oral pre- and post-exposure prophylaxis

Objectives:To describe first dose and steady state antiretroviral drug exposure in the female genital tract. Design:Non-blinded, single center, open-label pharmacokinetic study in HIV-infected women. Method:Twenty-seven women initiating combination antiretroviral therapy underwent comprehensive blood plasma and cervicovaginal fluid sampling for drug concentrations during the first dose of antiretroviral therapy and at steady-state. Drug concentrations were measured by validated HPLC/UV or HPLC-MS/MS methods. Pharmacokinetic parameters were estimated for 11 drugs by non-compartmental analysis. Descriptive statistics and 95% confidence intervals were generated using Intercooled STATA Release 8.0 (Stata Corporation, College Station, Texas, USA). Results:For all antiretroviral drugs, genital tract concentrations were detected rapidly after the first dose. Drugs were stratified according to the genital tract concentrations achieved relative to blood plasma. Median rank order of highest to lowest genital tract concentrations relative to blood plasma at steady state were: lamivudine (concentrations achieved were 411% greater than blood plasma), emtricitabine (395%), zidovudine (235%) tenofovir (75%), ritonavir (26%), didanosine (21%), atazanavir (18%), lopinavir (8%), abacavir (8%), stavudine (5%), and efavirenz (0.4%). Conclusions:This is the first study to comprehensively evaluate antiretroviral drug exposure in the female genital tract. These findings support the use of lamivudine, zidovudine, tenofovir and emtricitabine as excellent pre-exposure/post-exposure prophylaxis (PrEP/PEP) candidates. Atazanavir and lopinavir might be useful agents for these applications due to favorable therapeutic indices, despite lower genital tract concentrations. Agents such as stavudine, abacavir, and efavirenz that achieve genital tract exposures less than 10% of blood plasma are less attractive PrEP/PEP candidates.

Mass spectrometry based metabolomic approaches in urinary biomarker study of women's cancers.

BACKGROUND The metabolomic approaches for mining biomarkers of womens cancers based on gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry combined with partial least squares-discriminant analysis are described. METHODS To identify urinary potential biomarkers, the qualitative and quantitative analyses were introduced with 10 breast, 9 ovarian and 12 cervical cancer patients as well as 22 normal controls, which were considered with their ages and menopausal state. RESULTS For comprehensive metabolomic approaches, the non-targeted qualitative profiling was first achieved to get metabolic patterns of collected samples and the targeted quantitative analysis focused on hormonal metabolism was also conducted. Two known biomarkers, i.e., 5-hydroxymethyl-2-deoxyuridine and 8-hydroxy-2-deoxyguanosine, in breast cancer were also confirmed using the present methods. In addition, 3 potential biomarkers for ovarian cancer i.e. 1-methyladenosine, 3-methyluridine, and 4-androstene-3,17-dione, which were categorized in significantly increased level using one way of variance analysis (p<0.05), were identified as quantitatively targeted metabolites with pattern analysis. The cancer markers identified in this study are highly related to metabolites which are responsible for oxidative DNA damage and DNA methylation process. CONCLUSION The present metabolomic approaches are not only useful for diagnostic tools and patient stratification, but may be mapped on metabolic network to reflect disease states.

In vitro skin permeation of nicotine from proliposomes

Abstract The feasibility of proliposomes as a sustained transdermal dosage form was examined. Proliposomes containing varying amount of nicotine were prepared by a standard method using sorbitol and lecithin. The porous structure of sorbitol in the proliposomes was maintained, indicating that the majority of lecithin and nicotine is deposited within their porous matrix of the sorbitol particles. As a consequence, the flow properties of the proliposome particles was comparable to that of original sorbitol particles. Microscopic observation revealed that proliposomes are converted to liposomes almost completely within minutes following contact with water. It indicates that proliposomes may form liposomes by the sweat when they are applied on the skin under occlusive conditions in vivo. The size distribution of the reconstituted liposomes and nicotine release to pH 7.4 phosphate buffer from them were not significantly affected by the content of nicotine. The release pattern was apparently identical to the Exodus® patch, a commercially available transdermal nicotine formulation. We also studied in vitro permeation of nicotine across rat skin from proliposomes in a modified Keshary–Chien diffusion cell where the experimental set up simulates in vivo application of the proliposomes under an occlusive condition. The nicotine flux from proliposomes was initially retarded compared with that of nicotine powder. The flux from proliposomes appeared to remain constant throughout the experimental period compared with that of nicotine powder, indicating that nicotine may be delivered across the skin in a sustained manner at a constant rate from proliposomes. These results, therefore, indicate that sustained transdermal delivery of nicotine is feasible using proliposomal formulations if the formulations are topically applied under occlusive conditions.

Role of the AMPK/SREBP-1 pathway in the development of orotic acid-induced fatty liver

Orotic acid (OA), an intermediate in pyrimidine metabolism, has been used for a variety of purposes, such as dietary supplements. Although it is well documented that OA induces fatty liver in a species-specific manner, the precise molecular mechanisms remain unclear. The present study investigated the role of the adenosine monophosphate-activated protein kinase (AMPK)-sterol regulatory element-binding protein-1 (SREBP-1) pathway in the OA-induced fatty liver. Treatment with OA suppressed the phosphorylation of AMPK via proteasomal degradation of upstream kinase LKB1 and induced activation of SREBP-1 in both human hepatoma cell lines and primary rat hepatocytes. OA-induced SREBP-1 transcriptional activity was suppressed by cotreatment with aminoimidazole carboxamide ribonucleotide (AICAR) or metformin, or by overexpression of constitutively active AMPK (CA-AMPK) in the human hepatoma cell line. Importantly, in vivo data corroborated these results. Feeding 1% OA with diet decreased the phosphorylation of AMPK and increased the maturation of SREBP-1 and the expression of SREBP-responsive genes in the rat liver. OA-induced lipid accumulation was also completely inhibited by rapamycin. Mouse hepatocytes and mice were resistant to OA-induced lipogenesis because of little if any response in AMPK and downstream effectors. In conclusion, OA induces hepatic lipogenesis, mediated predominantly by the AMPK/SREBP-1 pathway in rat hepatocytes and human hepatoma cell lines.

Discovery of common urinary biomarkers for hepatotoxicity induced by carbon tetrachloride, acetaminophen and methotrexate by mass spectrometry-based metabolomics.

Liver toxicity represents an important healthcare issue because it causes significant morbidity and mortality and can be difficult to predict before symptoms appear owing to drug therapy or exposure to toxicants. Using metabolomic techniques, we discovered common biomarkers for the prediction of hepatotoxicity in rat urine using mass spectrometry. For this purpose, liver toxicity was induced by 5 days of oral administration of carbon tetrachloride (1 ml kg−1 per day), acetaminophen (1000 mg kg−1 per day) and methotrexate (50 mg kg−1 per day). Serum levels of alkaline phosphatase aspartate aminotransferase, alanine aminotransferase and histopathology in liver tissue were then checked to demonstrate liver toxicity. Global metabolic profiling with UPLC‐TOF‐MS (ultraperformance liquid chromatography–mass spectrometry), multivariate analysis (partial least square‐discriminant analysis, hierarchical analysis) and database searching were performed to discover common biomarkers for liver toxicity induced by these three compounds. Urinary concentrations of the newly discovered biomarkers were then quantified to confirm them as biomarkers of hepatotoxicity with targeted metabolic profiling using GC (gas chromatography)–MS and CE (capillary electrophoresis)–MS. In the results, steroids, amino acids and bile acids were metabolically changed between the control and drug‐treated groups in global metabolic profiling; 11β‐hydroxyandrosterone, epiandrosterone, estrone, 11‐dehydrocorticosterone, glycine, alanine, valine, leucine, dl‐ornithine, 3‐methylhistidine, cholic acid and lithocholic acid were selected as liver toxicity biomarkers after performing targeted metabolic profiling. In conclusion, we discovered metabolite biomarkers belonging to three different metabolic pathways to check for liver toxicity with mass spectrometry from a metabolomics study that could be used to evaluate hepatotoxicity induced by drugs or other toxic compounds. Copyright

Rapid and sensitive determination of sertraline in human plasma using gas chromatography-mass spectrometry.

A method for the determination of sertraline in human plasma using gas chromatography-mass spectrometry (GC-MS), with the selected ion-monitoring (SIM) mode, was described. The following was used in this study: (1) single liquid-liquid extraction at alkaline pH after deproteinization of plasma protein and (2) perfluoroacylation with HFBA, which has higher sensitivity (about 10-fold) compared with previous reported derivatization. The detection limit for the SIM of sertraline as an N-HFB derivative was 0.1 ng/ml, and its recovery was 80-85%. The linear response was obtained in the range of 0.2-10.0 ng/ml with a correlation coefficient of 0.999. The coefficient of variation (C.V.%) was less than 12.1% in the 1-30 ng/ml, and less than 18.2% at 0.2 ng/ml, and the accuracy was less than 10% at all of the concentration range. These findings indicate that this assay method has adequate precision and accuracy to determine the amount of sertraline in human plasma. After pharmacokinetics was performed with this assay method following oral administration of sertraline hydrochloride in man, moment analysis revealed that pharmacokinetic parameters for sertraline (Cmax, 10.3 ng/ml; Tmax, 8.0 h; T(1/2) 28.6 h) were similar to previously reported results. These results indicate that this simple and sensitive assay method is readily applicable to the pharmacokinetic studies of sertraline.

Increased urinary F2-isoprostanes levels in the patients with Alzheimer’s disease

Abstract Isoprostanes, novel markers of oxidative injury, are generated by the free radical-mediated peroxidation of arachidonic acid (AA). They are thought to be important in the pathogenesis of neurodegenerative diseases such as Alzheimer’s disease (AD). Using gas chromatography–mass spectrometry (GC–MS), we investigated the alteration of urinary F 2 -isoprostanes in AD patients compared to that of healthy subjects. Our results show that the levels of urinary F 2 -isoprostanes, sum of the prostaglandin F 2α isomer; prostaglandin F 2α (PGF 2α ), prostaglandin F 2β (PGF 2β ), and 8-isoprostaglandin F 2α (8-isoPGF 2α ), significantly increased in AD patients ( P 2α , one of the biomarkers of oxidative stress, was not significantly different between 34 AD patients and 20 age-matched controls ( P > 0.05). The PGF 2α , formed by endoperoxide reductase from PGH 2 , was significantly increased in AD patients, when compared with the levels of the normal controls ( P 2α , an enzymatic product of arachidonic acid, may affect the pathogenesis of AD. In addition, urinary F 2 -isoprostanes can play an important role as a biomarker in AD.

Quantitative analysis of 17 amino acids in the connective tissue of patients with pelvic organ prolapse using capillary electrophoresis-tandem mass spectrometry.

The simultaneous determination of 17 amino acids in connective tissue using capillary electrophoresis is described in this study. Separation was carried out on a fused silica capillary column (80 cm x 50 mm i.d.) with 1M formic acid as the running electrolyte. The detection was conducted on a mass spectrometer by selective reaction monitoring (SRM) mode via an electrospray ionization source. Tissue samples were prepared by reduction and acid hydrolysis to extract amino acids; over 84.3% recovery was seen for all compounds. The method allowed for sensitive, reproducible, and reliable quantification, and all 17 amino acids were separated using this method. Good linearity over the investigated concentration ranges was observed, with values of R higher than 0.993 for all the analytes. Precision and accuracy examined at three concentration levels ranged from 0.2% to 19.5% and 84.1% to 120.0%, respectively. Matrix effects were also tested and ranged from -9.1% to 15.4%. The validated method was applied to the quantitation of 17 amino acids in pelvic connective tissue of pelvic organ prolapsed patients. Methionine, glutamine, and histidine were significantly higher in the experimental patients compared to the controls. This suggests that changes in the amino acid concentrations within the connective tissue could be a factor in the genesis of pelvic organ prolapse. Therefore, this method is potentially applicable for amino acid analysis in tissue, providing a more complete understanding of pelvic organ prolapse.

Discovery of safety biomarkers for atorvastatin in rat urine using mass spectrometry based metabolomics combined with global and targeted approach

In order to develop a safety biomarker for atorvastatin, this drug was orally administrated to hyperlipidemic rats, and a metabolomic study was performed. Atorvastatin was given in doses of either 70 mg kg(-1) day(-1) or 250 mg kg(-1) day(-1) for a period of 7 days (n=4 for each group). To evaluate any abnormal effects of the drug, physiological and plasma biochemical parameters were measured and histopathological tests were carried out. Safety biomarkers were derived by comparing these parameters and using both global and targeted metabolic profiling. Global metabolic profiling was performed using liquid chromatography/time of flight/mass spectrometry (LC/TOF/MS) with multivariate data analysis. Several safety biomarker candidates that included various steroids and amino acids were discovered as a result of global metabolic profiling, and they were also confirmed by targeted metabolic profiling using gas chromatography/mass spectrometry (GC/MS) and capillary electrophoresis/mass spectrometry (CE/MS). Serum biochemical and histopathological tests were used to detect abnormal drug reactions in the liver after repeating oral administration of atorvastatin. The metabolic differences between control and the drug-treated groups were compared using PLS-DA score plots. These results were compared with the physiological and plasma biochemical parameters and the results of a histopathological test. Estrone, cortisone, proline, cystine, 3-ureidopropionic acid and histidine were proposed as potential safety biomarkers related with the liver toxicity of atorvastatin. These results indicate that the combined application of global and targeted metabolic profiling could be a useful tool for the discovery of drug safety biomarkers.

Measurement of corticoids in the patients with clinical features indicative of mineralocorticoid excess

BACKGROUND A method for the measurement of five important serum and urinary corticoids on the syndrome of mineralcorticoid excess is reported. The methodology was combined gas chromatography-mass spectrometry (GC-MS) with selected ion-monitoring mode. METHODS After extraction with a solid-phase cartridge using an Oasis HLB copolymer, the residues were derivatized with a mixture of N-methyl-N-trimethylsilyltrifluoroacetamide/ammonium iodide/dithioerythritol (1000:4:5, v/w/w), and analyzed. RESULTS The linearity as the regression coefficients were >0.979 over a range of 1-500 ng/ml, and limit of detection ranged from 1 to 3 ng/ml while their analytical recoveries varied in the range of 75.7-94.9%. The overall precision (% CV) of the method were 3.2-7.2% and 3.6-6.3% for serum and urine, respectively. The accuracy expresses as % bias ranged from -4.1 to 6.4%. This assay was used on two patients with hypokalemic hypertension, and may be useful in ruling out mineralcorticoid excess (AME) type 1 or 2. CONCLUSIONS The present GC-MS technique may be useful to differentiate between the syndrome of AME and other hypertensive diseases with clinical features suggestive of mineralcorticoid excess because of the assays reliablity and precision.