Shigeji Jingu

Determination of pibutidine metabolites in human plasma by LC-MS/MS

A novel metabolite-screening procedure for pibutidine. an H2-receptor antagonist, which uses high-performance liquid chromatography/tandem mass spectrometry (LC-MS/MS), demonstrated the presence of pibutidine and its four metabolites in plasma from volunteers who received a single dose of pibutidine hydrochloride. In order to quantitatively examine the metabolism of pibutidine, an assay based on LC-MS/MS was subsequently developed for the simultaneous determination of its metabolites in human plasma. Target analytes consisted of M-5, M-7 and M-8, which were prominently detected by the screening procedure, and M-9, which has pharmacological activity as an H2-receptor antagonist. Metabolites and their deuterated internal standards were extracted from human plasma using an Oasis HLB extraction cartridge, and chromatographed on a Monitor C18M column. No isotope effects on chromatographic retention time were observed for any deuterated compounds, which were ionized using an electrospray ionization (ESI)- interface and detected by MS/MS in the selected reaction-monitoring (SRM) mode simultaneously with the corresponding metabolites. The assay was validated over the concentration range of 0.1 to 25.6 ng ml(-1) and used to determine the plasma levels of metabolites in volunteers following oral administration of a 20-mg dose of pibutidine hydrochloride.

A surrogate analyte method to determine d-serine in mouse brain using liquid chromatography–tandem mass spectrometry

A bioanalytical method for determining endogenous d-serine levels in the mouse brain using a surrogate analyte and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. [2,3,3-(2)H]D-serine and [(15)N]D-serine were used as a surrogate analyte and an internal standard, respectively. The surrogate analyte was spiked into brain homogenate to yield calibration standards and quality control (QC) samples. Both endogenous and surrogate analytes were extracted using protein precipitation followed by solid phase extraction. Enantiomeric separation was achieved on a chiral crown ether column with an analysis time of only 6 min without any derivatization. The column eluent was introduced into an electrospray interface of a triple-quadrupole mass spectrometer. The calibration range was 1.00 to 300 nmol/g, and the method showed acceptable accuracy and precision at all QC concentration levels from a validation point of view. In addition, the brain d-serine levels of normal mice determined using this method were the same as those obtained by a standard addition method, which is time-consuming but is often used for the accurate measurement of endogenous substances. Thus, this surrogate analyte method should be applicable to the measurement of d-serine levels as a potential biomarker for monitoring certain effects of drug candidates on the central nervous system.

Rapid characterization of urinary metabolites of pibutidine hydrochloride in humans by liquid chromatography/electrospray ionization tandem mass spectrometry.

The metabolic products of pibutidine hydrochloride, a new H(2)-receptor antagonist, in human urine after oral administration of 40 mg/man were characterized by high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) with electrospray ionization (ESI). Two-stage collision-induced dissociation (CID) experiments, with in-source CID by increasing the octapole offset voltage and collision-cell CID, were performed in order to develop a very rapid screening procedure that enhanced selectivity toward pibutidine-related compounds. It was possible to detect metabolites of pibutidine directly from a crude biological matrix without prior extraction, enabling confirmation of the identity of eight metabolites in urine. In addition, the linear range in ESI for pibutidine-related compounds was studied to determine the urinary excretion of pibutidine and its metabolites in humans.

A Strategy for Assessing Potential Drug-Drug Interactions of a Concomitant Agent against a Drug Absorbed via an Intestinal Transporter in Humans

A strategy for assessing potential drug-drug interactions (DDIs) based on a simulated intestinal concentration is described. The proposed prediction method was applied to the DDI assessment of luseogliflozin, a novel antidiabetic drug, against miglitol absorbed via the intestinal sodium-glucose cotransporter 1 (SGLT1). The method involves four steps: collection of physicochemical and pharmacokinetic parameters of luseogliflozin for use in a computer simulation; evaluation of the validity of these parameters by verifying the goodness of fit between simulated and observed plasma profiles; simulation of the intestinal luseogliflozin concentration-time profile using the Advanced Compartment Absorption and Transit (ACAT) model in a computer program and estimation of the time spent above a value 10-fold higher than the IC50 value (TAIC) for SGLT1; and evaluation of the DDI potential of luseogliflozin by considering the percentage of TAIC against the miglitol Tmax (time for Cmax) value (TAIC/Tmax). An initial attempt to prove the validity of this method was performed in rats. The resulting TAIC/Tmax in rats was 32%, suggesting a low DDI potential of luseogliflozin against miglitol absorption. The validity was then confirmed using an in vivo interaction study in rats. In humans, luseogliflozin was expected to have no DDI potential against miglitol absorption, since the TAIC/Tmax in humans was lower than that in rats. This prediction was proven, as expected, in a clinical interaction study. In conclusion, the present strategy based on a simulation of the intestinal concentration-time profile using dynamic modeling would be useful for assessing the clinical DDI potential of a concomitant agent against drugs absorbed via an intestinal transporter.

A sensitive and selective method for the quantitative analysis of miglitol in rat plasma using unique solid-phase extraction coupled with liquid chromatography–tandem mass spectrometry

A sensitive, selective and robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the quantification of miglitol in rat plasma. The sample preparation procedures involved protein precipitation and unique solid-phase extraction, which efficiently removed sources of ion suppression and column degradation interference present in the plasma. Chromatographic separation was achieved on an amide column using 10 mmol/L CH3 COONH4 and CH3 CN:CH3 OH (90:10, v/v) as the mobile phase under gradient conditions. Detection was performed using tandem mass spectrometry equipped with an electrospray ionization interface in positive ion mode.The selected reaction monitoring transitions for miglitol and a stable isotope-labeled internal standard were m/z 208 → m/z 146 and m/z 212 → m/z 176, respectively. The correlation coefficients of the calibration curves ranged from 0.9984 to 0.9993 over a concentration range of 0.5-100 ng/mL plasma. The quantification limit of the proposed method was more than 10 times lower than those of previously reported LC-MS/MS methods. The novel method was successfully validated and applied to a pharmacokinetic study in rats.

(2S,4S)-4-Fluoro-1-{[(2-hydroxy-1,1-dimethylethyl)amino]acetyl}-pyrrolidine-2-carbonitrile monobenzenesulfonate (TS-021) is a selective and reversible dipeptidyl peptidase IV inhibitor

The incretin hormone glucagon-like peptide-1 (GLP-1) has significant roles in the regulation of postprandial glucose metabolism, and the active form of GLP-1 is rapidly degraded by dipeptidyl peptidase (DPP)-IV. Therefore, DPP-IV inhibition is a promising approach for the treatment of type 2 diabetes. In the present study, we investigated the character of a DPP-IV inhibitor, TS-021, (2S, 4S)-4-fluoro-1-{[(2-hydroxy-1,1-dimethylethyl)amino]acetyl}-pyrrolidine-2-carbonitrile monobenzenesulfonate both in vitro and in vivo. TS-021 inhibits DPP-IV activity in human plasma with an IC(50) value of 5.34nM. In kinetics experiments, TS-021 had a relatively higher dissociation rate constant, with a k(off) value of 1.09×10(-3)s, despite exhibiting a potent human plasma DPP-IV inhibition activity with a K(i) value of 4.96nM. TS-021 exhibited significant inhibition selectivity against DPP-8 (>600 fold), DPP-9 (>1200 fold) and other peptidases examined (>15,000 fold). In normal rats, dogs and monkeys, a single oral dose of TS-021 exhibited favorable pharmacokinetic profiles. In Zucker fatty (fa/fa) rats, a rat model of obesity and impaired glucose tolerance, the oral administration of TS-021 resulted in the suppression of plasma DPP-IV activity and an increase in the active form of GLP-1. Furthermore, TS-021 exhibited a significant improvement in glucose tolerance by increasing the plasma insulin level during oral glucose tolerance tests at doses of 0.02-0.5mg/kg. These results suggest that TS-021 is a selective and reversible dipeptidyl peptidase IV inhibitor and has excellent characteristics as an oral anti-diabetic agent for postprandial hyperglycemia in patients with impaired glucose tolerance or type 2 diabetes.

Ultrasensitive determination of NE-100, a novel sigma ligand, in human plasma by liquid chromatography and electrospray ionization tandem mass spectrometry combined with a column-switching technique.

For the highly sensitive and selective determination of NE-100, a novel sigma ligand, at levels of low picogram per milliliter of human plasma, a method with excellent reliability employing liquid chromatography (LC)-electrospray ionization (ESI) tandem mass spectrometry (MS-MS) combined with a column-switching technique has been developed. The method involves the use of a stable isotope labeled compound as the internal standard (I.S.), liquid-solid extraction of a plasma specimen with a C8 cartridge, automated on-line clean-up on a short trapping column, subsequent separation on a micro-bore C18 column and detection with ESI-MS-MS using m/z 356 ([M+H]+) as a precursor ion and m/z 105 as a product ion in a selected reaction monitoring mode. The detection and the quantification limits of NE-100 in plasma were 0.5 pg/ml with a signal-to-noise ratio (S/N) of 3 and 2.3 pg/ml, respectively, with an S/N of 21. The good linearity of the calibration graph was obtained in the range of 2.3 to approximately 907.0 pg/ml with excellent reliability. The developed method was applied to the determination of NE-100 in plasma obtained from the clinical trail.

Development and validation of a liquid chromatographic-tandem mass spectrometric method for the determination of pibutidine in human urine

A liquid chromatographic-tandem mass spectrometric method for the rapid quantitative determination of pibutidine, an H2-receptor antagonist, in human urine has been developed and validated over the concentration range 0.1-25.6 microg ml(-1). Urine samples were prepared based on a simple dilution with 0.05% acetic acid, followed by reversed-phase liquid chromatographic separation. Pibutidine and its internal standard (2H10-pibutidine) were ionized using an electrospray ionization interface and detected by tandem mass spectrometry in the selected reaction-monitoring mode. Completed validation demonstrated the method to be robust, accurate, precise and specific for the direct quantification of pibutidine in human urine. This method has enabled investigation of the urinary excretion of pibutidine following oral administration of pibutidine hydrochloride to healthy subjects.

Development and validation of a liquid chromatography–tandem mass spectrometric method for the quantification of 5-thio-d-glucose in rat and human plasma

A highly selective, sensitive, and robust liquid chromatography-tandem mass spectrometric method for the determination of 5-thio-d-glucose concentrations in rat and human plasma was developed and validated. The sample preparation procedure involved protein precipitation and solid phase extraction, which efficiently removed sources of interference present in the plasma. Chromatographic separation was obtained using an NH(2)-column with distilled water and acetonitrile as the mobile phase under gradient conditions. Detection was performed using tandem mass spectrometry equipped with an electrospray ionization interface in negative ion mode. The selected reaction monitoring (SRM) transitions for 5-thio-d-glucose and an internal standard (5-thio-d-glucose-(13)C(6)) were m/z 195→m/z 105 and m/z 201→m/z 108, respectively. The correlation coefficients of the calibration curves ranged from 0.9997 to 0.9999 over a concentration range from 10 to 3000ng/mL plasma. The validated method was successfully applied to a pharmacokinetic study in rats.