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A sensitive and simple detection method coupling ultra-performance liquid chromatography with tandem mass spectrometry was developed and validated to analyze sumatriptan levels in human plasma. The plasma sample preparations for the analysis were based on liquid–liquid extraction with ethyl acetate, evaporation, and reconstitution. MS/MS detection was performed on a triple-quadrupole tandem mass spectrometer by monitoring the protonated parent → daughter ion pairs at m/z 296 → 58 and m/z 388 → 71 for sumatriptan and terazosin (internal standard), respectively. The method was validated with respect to its specificity, linearity, sensitivity, accuracy, precision, recovery, and stability. The calibration curve was linear from 0.5 to 50 ng/mL (r > 0.999). The mean extraction recovery for sumatriptan was higher than 62.3%. The method accuracy was within 97.4%, and the relative standard deviation of the intra- and inter-day precision values was within 11.7% at all quality control levels. Plasma samples that contained sumatriptan were stable under three freeze–thaw cycles, short- and long-term storage, and autosampler conditions. This method was successfully applied to a pharmacokinetic study conducted with 10 healthy volunteers. After oral administration of 50-mg sumatriptan and serial blood sampling over 12 h, the mean area under the plasma concentration–time curve from time 0 to 12 h and the maximum plasma concentration were 116.2 ng h/mL and 33.2 ng/mL, respectively.

The aim of this study was to investigate the pharmacokinetics and dose proportionality of a single, intravenous dose of pazufloxacin mesilate, an injectable fluoroquinolone antibiotic, in healthy Korean male volunteers.In this open-label, four-dose, parallel study, subjects were randomized to receive a single dose of pazufloxacin mesilate 300, 500, 600, and 1,000 mg (n = 6, 20, 6, and 8, respectively) administered as a 1-h intravenous infusion. Blood and urine samples were collected serially from 0 to 24 h after drug administration and analyzed using a validated HPLC method. Tolerability was assessed by monitoring clinical laboratory parameters and adverse events.After single-dose intravenous administration of pazufloxacin mesilate, the mean C(max) for groups treated with 300, 500, 600, and 1,000 mg doses ranged from 5.11 to 18.06 μg/mL; the mean AUC(0-t) ranged from 13.70 to 58.60 μg × h/mL. Pazufloxacin exhibits Lack of dose proportionality was concluded over the dose range of 300 - 1,000 mg, based on linear regression model and power model . At all four dosages studied, pazufloxacin mesilate was well tolerated.Our data suggest that all regimens of pazufloxacin administration were well tolerated. Pazufloxacin exhibits lack of dose proportionality over the dose range of 300 - 1,000 mg.

Levofloxacin and cyclosporine show different pharmacokinetic properties, but are known to be dose proportional within the therapeutic range. The authors evaluated the pharmacokinetic proportionality of levofloxacin and cyclosporine over a 100-fold dose range in healthy human volunteers, by liquid chromatography-tandem mass spectrometry (LC-MS/MS).Two independent, randomized, crossover studies were performed. For levofloxacin, eight volunteers were randomly assigned in a 1:1 ratio to receive a low dose (7.5 mg) orally or intravenously, followed by a 1-week washout period and administration via the alternate route. After another 1-week washout period, a therapeutic dose (750 mg) was administered to all eight subjects. For cyclosporine, another eight volunteers received a low dose (2 mg) or a therapeutic dose (200 mg) orally with a 1-week washout period. Drug concentrations were determined by LC-MS/MS.For levofloxacin, the mean values for dose-normalized C(max) and AUC(last) with the two doses were as follows: therapeutic dose, 15.2 ± 4.6 ng/ml/mg and 103.6 ± 15.5 ng·h/ml/mg, respectively; low dose, 17.1 ± 6.5 ng/ml/mg and 72.6 ± 8.7 ng·h/ml/mg, respectively. For cyclosporine, the mean values for dose-normalized C(max) and AUC(last) were as follows: therapeutic dose, 4.9 ± 1.5 ng/ml/mg and 15.4 ± 4.9 ng·h/ml/mg, respectively; low dose, 1.6 ± 0.6 ng/ml/mg and 9.3 ± 7.3 ng·h/ml/mg, respectively.In this study levofloxacin, which is completely absorbed and primarily eliminated renally without modification, showed better pharmacokinetic proportionality than cyclosporine, which is poorly absorbed and extensively metabolized.

An enteric-coated formulation of triflusal (triflusal EC), an antiplatelet agent, was developed to reduce the high incidence of gastrointestinal adverse events (AEs). The aim of this study is to compare the pharmacokinetics, pharmacodynamics and safety of triflusal EC with triflusal in healthy Korean male subjects to determine bioequivalence and non-inferiority for the purposes of marketing approval.A randomized, open-label, two-period, crossover study was conducted in 38 subjects. Either triflusal EC or triflusal was administered orally as a single 900 mg loading dose (day 1) followed by eight 600 mg/day maintenance doses on days 2 - 9, with a 13-day washout period. The plasma concentrations of 2-hydroxy-4-trifluoromethyl benzoic acid (HTB), the predominant active metabolite of triflusal, were assessed after administration of the loading dose, using HPLC/MS/MS. The platelet aggregation response to arachidonic acid was determined using turbidimetric aggregometry.The 90% CIs, for the geometric mean ratios of the log-transformed AUC(τ) and C(max) of HTB were seen to be within the predetermined range of 0.8 - 1.25. Triflusal EC was also shown to be non-inferior in its anti-aggregatory effect. No serious AEs were reported during this study.The pharmacokinetic and pharmacodynamic profiles of the two triflusal formulations met the requirements for bioequivalence and non-inferiority, respectively. Both formulations were well tolerated.

The aim of this study was to compare the pharmacokinetics (PK) and safety of fimasartan (BR-A-657), an angiotensin II receptor antagonist, between healthy young (19 - 45 years) and older (≥ 65 years) male subjects.To assess the effect of age on PK and safety, fimasartan was administered as a single 240 mg tablet to 12 young and 10 older male subjects, followed by serial blood sampling over 48 h. Plasma concentrations of fimasartan were analyzed using validated HPLC-MS/MS. Clinical and laboratory adverse events were assessed.After oral administration of 240 mg fimasartan, the mean area under the plasma concentration-time curve from time zero to infinity (AUC(0→∞)) was 2899.0 ng/ml/h in the older, which was significantly greater than in young subjects (1767.4 ng/ml/h; p = 0.03). The geometric mean AUC(0→∞) was 69.4% higher in older than in young subjects. The maximum plasma concentration (C(max)), time to reach C(max) and elimination half-life for fimasartan did not differ significantly between the older and young groups. Importantly, fimasartan was well tolerated during this study.While some PK parameters were statistically different between the two groups, the effect of age on the PK was modest (e.g., AUC increasetwofold in older subjects).

An improved method for determining levels of levosulpiride in human plasma using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed and validated. The protein precipitation method was used for plasma sample preparation. Levosulpiride and an internal standard (IS) were isocratically separated on a UPLC BEH C(18) column with a mobile phase of ammonium formate buffer (1mM, adjusted to pH 3 with formic acid) and acetonitrile (60:40, v/v). MS/MS detection was performed by monitoring the parent-->daughter pair of levosulpiride and the IS at m/z 342-->112 and 329-->256, respectively. The method was linear from 2.5 to 200ng/mL and exhibited acceptable precision and percent recovery. The method was successfully demonstrated in pharmacokinetic and bioequivalence studies of two levosulpiride oral formulations administered to healthy volunteers. When compared to the previous LC-MS methods, the proposed method is faster, well-validated, and uses lesser plasma volume and a similar sensitivity. The use of UPLC allowed rapid and sensitive quantification of levosulpiride, making this method suitable for high-throughput clinical applications.

A rapid, specific, and sensitive method utilizing reversed-phase ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) was developed and validated to determine finasteride levels in human plasma. The plasma samples were prepared by liquid–liquid extraction with ethyl acetate, evaporation, and reconstitution. MS/MS analyses were performed on a triple–quadrupole tandem mass spectrometer by monitoring protonated parent → daughter ion pairs at m/z 373 → 305 for finasteride and m/z 237 → 194 for carbamazepine (internal standard, IS). The method was validated with respect to linearity, recovery, specificity, accuracy, precision, and stability. The method exhibited a linear response from 0.1 to 30 ng/mL ( r 2 > 0.998). The limit of quantitation for finasteride in plasma was 0.1 ng/mL. The relative standard deviation (RSD) of intra- and inter-day measurements was less than 15% and the method was accurate within −6.0% to 2.31% at all quality-control levels. The mean extraction recovery was higher than 83% for finasteride and 84% for the IS. Plasma samples containing finasteride were stable under the three sets of conditions tested and the processed samples were stable up to 29 h in an autosampler at 5 °C. Detection and quantitation of both analytes within 3 min make this method suitable for high-throughput analyses. The method was successfully applied to a pharmacokinetic study of finasteride in healthy volunteers following oral administration.

The c-Myc oncoprotein functions as a transcription factor that can transform normal cells into tumor cells, as well as playing a direct role in normal cell proliferation. The c-Myc protein transactivates cellular promoters by recruiting nuclear cofactors to chromosomal sites through an N-terminal transactivation domain. We have previously reported the identification and functional characterization of four different c-Myc cofactors: TRRAP, hGCN5, TIP49, and TIP48. Here we present the identification and characterization of the actin-related protein BAF53 as a c-Myc-interacting nuclear cofactor that forms distinct nuclear complexes. In addition to the human SWI/SNF-related BAF complex, BAF53 forms a complex with TIP49 and TIP48 and a separate biochemically distinct complex containing TRRAP and a histone acetyltransferase which does not contain TIP60. Using deletion mutants of BAF53, we show that BAF53 is critical for c-Myc oncogenic activity. Our results indicate that BAF53 plays a functional role in c-Myc-interacting nuclear complexes.

The ATM-related TRRAP protein is a component of several different histone acetyltransferase (HAT) complexes but lacks the kinase activity characteristic of other ATM family members. We identified a novel function for this evolutionarily conserved domain in its requirement for the assembly of a functional HAT complex. Ectopic expression of TRRAP protein with a mutation in the ATM-related domain inhibits Myc-mediated oncogenic transformation. The Myc-binding region of TRRAP maps to a separable domain, and ectopic expression of this domain inhibits cell growth. These findings demonstrate that the ATM-related domain of TRRAP forms a structural core for the assembly and recruitment of HAT complexes by transcriptional activators.

Cyclosporine is an immunosuppressant drug used in organ transplants or for the treatment of autoimmune diseases. We developed and validated a simple, sensitive, and specific method using UPLC-MS/MS to determine cyclosporine levels in human whole blood. MS/MS detection was performed in the positive electrospray ionization mode with multiple reaction monitoring. Cyclosporine was extracted from whole-blood samples using ascomycin as an internal standard. The mass transitions m/z 1203.49 → 1185.53 and m/z 814.71 → 796.67 were used to assay the analyte and IS. This method was validated with respect to linearity, specificity, accuracy, precision, recovery, and stability. The method exhibited a linear response from 10 to 1000 ng mL(-1) with correlation coefficient values >0.99. The precision and the accuracy values were within 15%, except at the lower limit of quatification (LLOQ). Cyclosporine was stable in whole blood with no evidence of degradation. This method was successfully applied to a pharmacokinetic study of cyclosporine in healthy volunteers following oral administration.

Bo-yang-hwan-o-tang (BHT) is an oriental herbal medicine for treating brain disorders such as cerebral ischemia. The objective of this study was to develop an economically feasible and time-saving high-throughput screening method to monitor the potential inhibitory effects of BHT on human cytochrome P450 (CYP) enzymes in vitro. Two cocktail sets were used for incubation of human liver microsomes: Cocktail A: 6 probe substrates for CYP1A2, CYP2A6, CYP2C8, CYP2C19, CYP2D6, CYP3A4; Cocktail B: 3 for CYP2B6, CYP2C9, CYP2E1. The concentrations of the substrate metabolites were simultaneously analyzed using UPLC/MS/MS. The BHT extract had almost negligible inhibitory effects on the nine human CYP isoforms tested, with the half-maximal inhibitory concentration value ranged from 3624.99 to 45412.44 μg/ml. The results suggest that BHT extract has no inhibitory effects on CYP isoforms within the clinically recommended dosage range. We conclude that BHT might be free of drug-herb interactions when co-administered with other medicines. However, more in vivo human studies are needed to confirm these results. The high-throughput screening method can be a useful tool for drug discovery and for understanding drug interactions.

In metabolomic research, it is important to reduce systematic error in experimental conditions. To ensure that metabolomic data from different studies are comparable, it is necessary to remove unwanted systematic factors by data normalization. Several normalization methods are used for metabolomic data, but the best method has not yet been identified. In this study, to reduce variation from non-biological systematic errors, we applied 1-norm, 2-norm, and quantile normalization methods to liquid chromatography-mass spectrometry (LC-MS)-based metabolomic data from human urine samples after oral administration of cyclosporine (high- and low-dose) in healthy volunteers and compared the effectiveness of the three methods. The principal component analysis (PCA) score plot showed more obvious groupings according to the cyclosporine dose after quantile normalization than after the other two methods and prior to normalization. Quantile normalization is a simple and effective method to reduce non-biological systematic variation from human LC-MS-based metabolomic data, revealing the biological variance.

HMC05, a formulation containing eight different herbal extracts, has been used widely for several thousand years in China, Japan, and Korea as a remedy for hypertension and headache. Although its anti-inflammatory effects in mouse monocytic cell lines and anti-atherosclerotic effects in apoE-knockout mice have been reported, the pharmacodynamic effects of HMC05 in human subjects have not yet been investigated. We evaluated the efficacy and tolerability of this drug in 14 healthy male Korean subjects with normal or high-normal blood pressure (BP) in a randomized, single-blind, crossover study with a 2-week washout period. Four 500-mg tablets of HMC05 or placebo were orally administered three times daily to nine subjects with normal BP and five subjects with high-normal BP for 4 weeks. To assess the pharmacodynamic effects of HMC05, levels of high-sensitivity C-reactive protein and homocysteine, BP, and flow-mediated vasodilation were measured before and after the 4-week medication period with evaluation of tolerability. All 14 subjects completed the study, and HMC05 was well tolerated with no significant adverse events. HMC05 did not exhibit a significant BP-lowering effect in either BP group, and there were no significant differences in other pharmacodynamic values after HMC05 or placebo administration in the two groups. Further study is needed to evaluate the efficacy and tolerability of HMC05 in an adequate number of patients with hypertension.

We demonstrate that transformation-transactivation domain-associated protein (TRRAP) binding and the recruitment of histone H3 and H4 acetyltransferase activities are required for the transactivation of a silent telomerase reverse transcriptase (TERT) gene in exponentially growing human fibroblasts by c-Myc or N-Myc protein. However, recruitment of TRRAP by c- or N-Myc is dispensable for the partial induction of several basally expressed genes in exponentially growing primary and immortalized fibroblasts. Furthermore, recruitment of TRRAP is required for c-Myc- or N-Myc-mediated oncogenic transformation but not for the partial restoration of the growth defect in myc-null fibroblasts. A segment of the adenovirus E1A protein fused to a transformation-defective N-Myc protein carrying a small deletion in the transactivation domain specifically restores interaction with TRRAP, activates the silent TERT gene, induces acetylation of histones H3 and H4 at the TERT promoter, and transforms primary cells. Accordingly, wild-type L-Myc is much less efficient in TRRAP binding, activation of the silent TERT gene, and transformation of primary fibroblasts. Nevertheless, L-Myc is a potent activator of several basally expressed genes and can fully restore the growth defect of myc-null cells. These results suggest a differential requirement for TRRAP for several Myc-mediated activities.