Fumihiro Jinno

Phase I first-in-human study of TAK-285, a novel investigational dual HER2/EGFR inhibitor, in cancer patients

Background:This phase I first-in-human study was conducted in Japanese patients to investigate the safety, pharmacokinetics (PKs), and determine the maximum tolerated dose (MTD) of oral TAK-285, a novel dual erbB protein kinase inhibitor that specifically targets human epidermal growth factor receptor (EGFR) and HER2.Methods:The TAK-285 dose was escalated until MTD was determined. A second patient cohort received TAK-285 at the MTD for at least 4 weeks.Results:In all, 26 patients received TAK-285 at doses ranging from 50 to 400 mg once daily (q.d.) or twice daily (b.i.d.); 20 patients made up the dose escalation cohort and the remaining 6 patients were the repeated administration cohort. TAK-285 was well tolerated. Dose-limiting toxicities noted in two patients who received 400 mg b.i.d. were grade 3 increases in aminotransferases and grade 3 decreased appetite. Consequently, the MTD was determined to be 300 mg b.i.d. Absorption of TAK-285 was rapid after oral dosing, and plasma exposure at steady-state increased in a dose-proportional fashion for doses ranging from 50 to 300 mg b.i.d. A partial response was observed for one patient with parotid cancer who received 300 mg b.i.d.Conclusion:The toxicity profile and PK properties of oral TAK-285 warrant further evaluation.

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. Formation of M‐SG was time dependent with a first order reaction and M‐I, a metabolite from the phenyl ring moiety of TAK‐242, was also produced in parallel. The formation of M‐SG was accelerated with increasing pH, therefore it was indicated that TAK‐242 reacted with GSH by a nucleophilic substitution reaction. Because glutathione transferase (GST) enhanced M‐SG formation in vitro, it is expected that the conjugation of TAK‐242 with GSH is also facilitated by GST in vivo in addition to a spontaneous chemical reaction. When radio‐labeled TAK‐242 ([cyclohexene ring‐U‐14 C]TAK‐242) was incubated with rat serum albumin (RSA) or human serum albumin (HSA) in vitro, the radioactive material was covalently bound to RSA and HSA, and M‐I was generated simultaneously in the reaction mixture. The chemical structure of the TAK‐242 adduct covalently bound to HSA was characterized by the accurate mass spectra that cyclohexene ring moiety of TAK‐242 was covalently bound to the lysine residue in HSA. The adduct was also detected in the plasma of rats and humans after single i.v. dosing of TAK‐242 (in vivo). Copyright

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. Since D- and L-serine are endogenous components, phosphate buffered saline was used as the surrogate matrix. D- and L-serine in human plasma and PBS were treated by cationic exchange solid phase extraction. D-Serine (m/z 106.1 > 60.0), L-serine (m/z 106.1 > 60.1) and DL-serine-d3 (m/z 109.1 > 63.0) were detected using a multiple reaction monitoring. The enantiomer separation of D- and L-serine was successfully achieved without any derivatization step using tandemly-arranged and ice-cold CROWNPAK CR-I(+) columns with an isocratic mobile phase comprised of 0.3% trifluoroacetic acid in 10% acetonitrile. The standard curves were linear throughout the calibration range with 0.01-10 μg/mL (D-serine) and 0.1-100 μg/mL (L-serine), respectively. Intra-day and inter-day precision and accuracy of the quality control samples were within relative standard deviations of less than 15%. The endogenous concentrations of D- and L-serine in human plasma were 0.124-0.199 and 7.97-13.1 μg/mL, respectively.

Method development for the determination of 24S‐hydroxycholesterol in human plasma without derivatization by high‐performance liquid chromatography with tandem mass spectrometry in atmospheric pressure chemical ionization mode

We developed a highly sensitive and specific high-performance liquid chromatography with tandem mass spectrometry method with an atmospheric pressure chemical ionization interface to determine 24S-hydroxycholesterol, a major metabolite of cholesterol formed by cytochrome P450 family 46A1, in human plasma without any derivatization step. Phosphate buffered saline including 1% Tween 80 was used as the surrogate matrix for preparation of calibration curves and quality control samples. The saponification process to convert esterified 24S-hydroxycholesterol to free sterols was optimized, followed by liquid-liquid extraction using hexane. Chromatographic separation of 24S-hydroxycholesterol from other isobaric endogenous oxysterols was successfully achieved with gradient mobile phase comprised of 0.1% propionic acid and acetonitrile using L-column2 ODS (2 μm, 2.1 mm id × 150 mm). This assay was capable of determining 24S-hydroxycholesterol in human plasma (200 μL) ranging from 1 to 100 ng/mL with acceptable intra- and inter-day precision and accuracy. The potential risk of in vitro formation of 24S-hydroxycholesterol by oxidation from endogenous cholesterol in human plasma was found to be negligible. The stability of 24S-hydroxycholesterol in relevant solvents and human plasma was confirmed. This method was successfully applied to quantify the plasma concentrations of 24S-hydroxycholesterol in male and female volunteers.

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. The mass spectrometric parameters for detection of TAK-438F, M-I, M-III and M-IV-Sul were modified from their optimum values in order to achieve a simultaneous quantification while retaining enough sensitivity and wide dynamic ranges for all the target analytes. The validity and robustness of the method was verified through a validation study as per the regulatory guidance on bioanalytical method validation. The calibration ranges are 0.1-100 ng/mL for TAK-438F and M-III, and 1-1000 ng/mL for M-I, M-II and M-IV-Sul using the 100 μL of human plasma. The total run time per sample is 5 min. The working solution for M-III was recommended to be prepared separately, especially for the long-term use, in order to avoid the instability of M-III in the mixed working solutions, which could cause the high consumption of reference standards. The established method was applied to clinical pharmacokinetic studies and concentrations of all the analytes in human plasma were successfully determined with high reproducibility ensured by incurred sample reanalysis, indicating the suitableness of the established method.

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 ([14C]M-III). The elimination half-life of 14C in the plasma of rats was approximately 1/30 of that of dogs after intravenous dosing of [14C]M-III at 0.5 mg/kg to rats and dogs. The in vitro and in vivo plasma protein binding ratios of M-III were relatively high and were the same in both species. The intrinsic clearance (CLint) of M-III in rats was much higher than the glomerular filtration rate in rats. Furthermore, the concentration of [14C]M-III in the kidney of rats was much higher than that in the plasma. On the contrary, in dogs, the concentration of [14C]M-III in the kidney was very much lower than that in the plasma. These results indicated that M-III was effectively taken up into the kidney and was excreted into the urine in rats; however, in dogs, ineffective renal uptake of M-III was presumed. When [14C]M-III and probenecid were simultaneously and continually infused intravenously to rats, the CLint of M-III decreased with increasing plasma concentrations of probenecid, indicating that kidney uptake of M-III in rats was inhibited by probenecid. It was also thought that uptake by the organic anion transport system(s) in the basolateral membrane is involved in the renal uptake of M-III in rats. The pharmacokinetic differences of M-III between rats and dogs are considered to be mainly caused by the difference in the urinary excretion via the renal distribution processes.

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

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. The results of mass balance studies in animals suggested that the absorbed TAK‐272 was largely eliminated by metabolism. In vitro studies revealed that TAK‐272 was mainly metabolized by CYP3A4/5 in humans, and it was a P‐glycoprotein substrate. PK analysis suggested that the factors responsible for the low BA were different in rats and monkeys. First‐pass hepatic extraction was high in rats, while the fraction absorbed from the gastrointestinal tract (Fa * Fg) was low in monkeys. It was predicted that humans would have a higher BA and a longer half‐life in the plasma compared with the animals by a simple calculation using intrinsic hepatic clearance in monkeys, which correlates well with human values for CYP3A4 substrates, and Fa * Fg in rats, which correlates relatively well with human values. TAK‐272 was finally selected as a clinical candidate based on the result of human PK prediction. The actual human PK after oral administration of TAK‐272 was comparable to the predicted profile and was preferable for clinical usage.

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. The bioanalytical method can be outlined as follows: two structural analogues are used as the internal standards. The analytes and the IS are extracted from human plasma using solid phase extraction. After evaporating, the residue is reconstituted and injected into a LC/MS/MS system with an ESI probe and analyzed in the positive ion mode. Separation is performed through a conventional reversed-phase column with a mobile phase of water/acetonitrile/acetic acid (40:60:0.05, v/v/v) mixture at a flow rate of 0.2mL/min. The total run time is 8.5min. The calibration range is 1-2500ng/mL in human plasma for all the analytes. Instability issues of the prodrug, TAK-491, were overcome and all the validation results met the acceptance criteria in accordance with the regulatory guideline/guidance. As a result of the clinical study, the human PK profiles of TAK-536, M-I and M-II were successfully obtained and also it was confirmed that TAK-491 was below the LLOQ (1ng/mL) in the human plasma samples.

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. An acceptance criterion was derived with a confidential interval approach. As the results, while a larger bias was observed for the cross-validation samples, the bias was not fully caused by analytical variation or bias attributable to the analytical methods. The direction of the deviation between the cross-validation samples and QC samples was random and not concentration-dependent, suggesting that inter-laboratory variability such as preparation errors could be a source of bias. A derived acceptance criterion corresponds to one prescribed in the Guideline on bioanalytical method validation from the Ministry of Health, Labour and Welfare in Japan and is a little wider than one in the European Medical Agency. In conclusion, thorough retrospective data analysis revealed potential causes of larger analytical bias in inter-laboratory cross-validation studies. A derived acceptance criterion would be practical and reasonable for the inter-laboratory cross-validation study.

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. The structural analogue, TAK-683, was used as the internal standard (IS). TAK-448F and the IS were extracted from human plasma using solid phase extraction (SPE) with a polymer-based weak cationic exchanger. After evaporating, the residue was reconstituted and injected into a LC-MS/MS system with ESI probe and analyzed by the selected reaction monitoring (SRM) in the positive ion mode. Separation was performed through an UPLC BEH Phenyl column with the mobile phase of water/methanol/formic acid mixture at a flow rate of 0.2 mL/min. The total run time was 10 minutes. The LLOQ was achieved to be 5 pg/mL with 0.5 mL of human plasma sample. All the validation results met the acceptance criteria in accordance with the regulatory guidance/guideline proving its reliability and robustness. As a result of the clinical study, the human PK profiles of TAK-448F were successfully obtained with this method.