Impact of microsampling on toxicological evaluation in rodent safety studies.

Recently, animal welfare has been attracting worldwide attention, and implementation of 3Rs (replacement, reduction, and refinement) is prioritized in every way possible in the drug development. Microsampling, in which small amounts of blood are collected, is attracting attention in this context. ICH S3A Q&A focused on microsampling was published in November 2017 to help accelerate the application of microsampling for toxicokinetic assessment.

No obvious toxicological influences of 50 μL microsampling from rats administered phenacetin as a drug with hematological toxicity.

According to ICH S3A Q&A focusing on microsampling, its application should be avoided in main study animals for test drugs that could exacerbate hematological parameters with frequent blood sampling. However, no study has reported the effects of microsampling on toxicity parameters of drugs known to induce hematological toxicity. Therefore, we assessed the toxicological effects of serial microsampling on rats treated with phenacetin as a model drug.

Lack of toxicological influences by microsampling (50 µL) from jugular vein of rats in a collaborative 28-day study.

Due to finalization of the ICH S3A Q&A focusing on microsampling, application of microsampling technique to regular non-clinical animal studies is expected for non-clinical safety assessment of pharmaceuticals. In Europe, microsampling from the tail vein or saphenous vein has often been used, whereas sampling from the jugular vein is thought to be more common for non-clinical studies in Japan. Therefore, we assessed the toxicological effects of serial microsampling from the jugular vein of SD rats in a common 28-day study at 4 independent organizations.

The effect of elevated α-acid glycoprotein on the pharmacokinetics of TAK-272 (SCO-272), an orally active renin inhibitor, in rats.

The pharmacokinetics of TAK-272 (SCO-272), an orally active renin inhibitor, was investigated in rats with subcutaneously injected turpentine oil, which was an inflammation animal model. Following intravenous administration of TAK-272 to the turpentine-treated rats, the systemic clearance and volume of distribution decreased with the elevated plasma α-acid glycoprotein (AGP) levels. The elevated plasma AGP levels were negatively correlated with the plasma unbound fraction of TAK-272 in the rats.

Differences in nonclinical pharmacokinetics between species and prediction of human pharmacokinetics of TAK-272 (SCO-272), a novel orally active renin inhibitor.

In the search for orally available drugs, the prediction of human pharmacokinetics (PK) is essential for successfully selecting compounds that will be clinically useful. This report describes the selection of TAK-272 (SCO-272), a novel orally active renin inhibitor, as a clinical candidate via the detailed investigation of nonclinical PK data and human PK prediction. The bioavailability (BA) of TAK-272 after oral administration to rats and monkeys was low, especially in fasted monkeys, and the systemic exposure of TAK-272 was highly variable in monkeys.

Bioanalysis of farnesyl pyrophosphate in human plasma by high-performance liquid chromatography coupled to triple quadrupole tandem mass spectrometry and hybrid quadrupole Orbitrap high-resolution mass spectrometry.

The isoprenoids farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) are pivotal intermediates for cholesterol homeostasis and cell signaling in the mevalonate pathway. We developed a sensitive and selective high-performance liquid chromatography tandem triple quadrupole mass spectrometry (LC-QQQ-MS) method for FPP in human plasma without the need for a derivatization process. We optimized the sample preparation procedure to extract FPP and C-FPP (as internal standard) from sample fluids using methanol.

A validated simultaneous quantification method for vonoprazan (TAK-438F) and its 4 metabolites in human plasma by the liquid chromatography-tandem mass spectrometry.

Vonoprazan fumarate (TAK-438) is a potassium-competitive acid blocker which was approved in Japan for a treatment of acid-related diseases. In this study a simple and validated bioanalytical method, which can simultaneously determine vonoprazan (TAK-438F) and its four metabolites (M-I, M-II, M-III and M-IV-Sul) in human plasma, was developed. The method is based on protein precipitation and subsequent ultra-high performance liquid chromatography separation followed by tandem mass spectrometry detection.

Method development for the determination of D- and L-isomers of leucine in human plasma by high-performance liquid chromatography tandem mass spectrometry and its application to animal plasma samples.

We developed a highly sensitive and specific high-performance liquid chromatography tandem mass spectrometry method with an electrospray ionization for the determination of D- and L-isomers of leucine in human plasma. Phosphate-buffered saline was used as the surrogate matrix for preparation of calibration curves and quality control samples. The extraction of D- and L-leucine in plasma samples (100 μL) was performed using cationic exchange solid-phase extraction.

Development, validation and application of the liquid chromatography tandem mass spectrometry method for simultaneous quantification of azilsartan medoxomil (TAK-491), azilsartan (TAK-536), and its 2 metabolites in human plasma.

Azilsartan medoxomil potassium salt (TAK-491) is an orally administered angiotensin II type 1 receptor blocker for the treatment of hypertension and is an ester-based prodrug that is rapidly hydrolyzed to the pharmacologically active moiety, azilsartan (TAK-536), during absorption. TAK-536 is biotransformed to the 2 metabolites M-I by decarboxylation and M-II by dealkylation. In this study, we developed and validated a LC/MS/MS method which can simultaneously determine 4 analytes, TAK-491, TAK-536, M-I and M-II.

Bioanalytical method for the simultaneous determination of D- and L-serine in human plasma by LC/MS/MS.

D-Serine is an endogenous modulator of N-methyl-D-aspartate (NMDA) receptors. Plasma concentrations of D-serine and the ratio of D-serine to total serine may be used as clinically-translatable biomarkers in NMDA receptor-related disease. We developed a highly sensitive and specific method using high performance liquid chromatography tandem mass spectrometry (LC/MS/MS) for the simultaneous determination of the D- and L-isomers of serine in human plasma.

Retrospective data analysis and proposal of a practical acceptance criterion for inter-laboratory cross-validation of bioanalytical methods using liquid chromatography/tandem mass spectrometry.

The purpose of this study is to conduct a retrospective data analysis for inter-laboratory cross-validation studies to set a reasonable and practical acceptance criterion based on a number of cross-validation results. From the results of cross-validation studies for 16 compounds and their metabolites, analytical bias and variation were evaluated. The accuracy of cross-validation samples was compared with that of quality control (QC) samples with statistical comparison of the analytical variation.

Highly sensitive liquid chromatography-tandem mass spectrometry method for quantification of TAK-448 in human plasma.

TAK-448 is a nonapeptide analogue and a novel metastin receptor agonist. The aim of this study was to develop a bioanalytical method for TAK-448F (the free base of TAK-448) in human plasma with LC/MS/MS that is sensitive and applicable for the clinical PK studies, and to evaluate the reliability and robustness of the developed method through a validation study in accordance with the regulatory guidance/guideline. The bioanalytical method developed in this study can be outlined as follows.

Species differences of organic anion transporters involved in the renal uptake of 4-amino-3-chlorophenyl hydrogen sulfate, a metabolite of resatorvid, between rats and dogs.

Previous studies on the metabolic fate of resatorvid (TAK-242) have shown that species differences in the pharmacokinetics of 4-amino-3-chlorophenyl hydrogen sulfate (M-III), a metabolite of TAK-242, between rats and dogs are mainly attributable to the urinary excretion process. In the present study, the renal uptake mechanism of M-III was investigated using kidney slices and Xenopus laevis oocytes expressing rat organic anion transporter 1 (rOat1; Slc22a6) and rOat3 (Slc22a8). The uptake of p-aminohippuric acid (PAH), a substrate for Oats, by kidney slices from rats and dogs increased at 37 °C and M-III inhibited the uptake.

Application of high-resolution ESI and MALDI mass spectrometry to metabolite profiling of small interfering RNA duplex.

We investigated the application of a high-resolution Orbitrap mass spectrometer equipped with an electrospray ionization (ESI) source and a matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometer to the metabolite profiling of a model small interfering RNA (siRNA) duplex TSR#34 and compared their functions and capabilities. TSR#34 duplex was incubated in human serum in vitro, and the duplex and its metabolites were then purified by ion exchange chromatography in order to remove the biological matrices. The fraction containing the siRNA duplex and its metabolites was collected and desalted and then subjected to high-performance liquid chromatography (HPLC) equipped with a reversed phase column.

Differences in the pharmacokinetics of 4-amino-3-chlorophenyl hydrogen sulfate, a metabolite of resatorvid, in rats and dogs.

The pharmacokinetics of 4-amino-3-chlorophenyl hydrogen sulfate, M-III of resatorvid, in rats and dogs were investigated using radiolabeled M-III ([(14)C]M-III). The elimination half-life of (14)C in the plasma of rats was approximately 1/30 of that of dogs after intravenous dosing of [(14)C]M-III at 0.5 mg/kg to rats and dogs.

Investigation of the unique metabolic fate of ethyl (6R)-6- [N- (2-chloro-4-fluorophenyl) sulfamoyl] cyclohex-1-ene-1-carboxylate (TAK-242) in rats and dogs using two types of 14C-labeled compounds having different labeled positions.

The pharmacokinetics of TAK-242 (ethyl (6R)-6- [N-(2-chloro-4-fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate, CAS 243984-11-4) and its metabolites were investigated in rats and dogs after intravenous (i.v.) dosing of TAK-242 using two types of radiolabeled TAK-242: [phenyl ring-U-14C]TAK-242 and [cyclohexene ring-U-14C]TAK-242.

Chemical reactivity of ethyl (6R)-6-[N-(2-chloro-4-fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate (TAK-242) in vitro.

Ethyl (6R)-6-[N-(2-chloro-4-fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate (TAK-242) was metabolized to cyclohexene and phenyl ring moieties in non-clinical pharmacokinetic studies and it was suggested that the cyclohexene ring moiety of TAK-242 is tightly bound to endogenous macromolecules. After incubation of TAK-242 and glutathione (GSH) in phosphate buffer (pH 7.4) at 37 °C, TAK-242 reacted with GSH to produce a glutathione conjugate of the cyclohexene ring moiety of TAK-242, which had been observed as a metabolite (M-SG) in non-clinical pharmacokinetic studies.