Analytical method validation for biomarkers as a drug development tool: points to consider.

Biomarkers are an important drug developmental tool. Assessment of quantitative analytical methods of biomarkers is not included in any regulatory documents in Japan. Use of biomarkers in clinical evaluations and supporting the post-marketing evaluation of drug efficacy and/or adverse reactions requires assessment and full validation of analytical methods for these biomarkers. The Biomarker Analytical Method Validation Study Group is a research group in Japan comprising industry and regulatory experts. Group members discussed and prepared this 'points to consider document' covering measurements of endogenous metabolites/peptides/proteins by ligand binding assays and chromatographic methods with or without mass spectrometry. We hope this document contributes to the global harmonization of biomarker assay validation.

An iminium ion metabolite hampers the production of the pharmacologically active metabolite of a multikinase inhibitor KW-2449 in primates: irreversible inhibition of aldehyde oxidase and covalent binding with endogenous proteins.

We previously reported that KW-2449, (E)-1-{4-[2-(1H-Indazol-3-yl)vinyl]benzoyl}piperazine, a novel multikinase inhibitor developed for the treatment of leukemia patients, was oxidized to an iminium ion intermediate by monoamine oxidase B (MAO-B) and then converted to its oxo-piperazine form (M1) by aldehyde oxidase (AO). However, it was found that the significant decrease in the pharmacologically active metabolite M1 following repeated administration of KW-2449 in primates might hamper the effectiveness of the drug. The mechanism underlying this phenomenon was investigated and it was found that the AO activity was inhibited in a time-dependent manner in vitro under the co-incubation of KW-2449 and MAO-B, while neither KW-2449 nor M1 strongly inhibited MAO-B or AO activity. These results clearly suggest that MAO-B catalysed iminium ion metabolite inhibited AO, prompting us to investigate whether or not the iminium ion metabolite covalently binds to endogenous proteins, as has been reported with other reactive metabolites as a cause for idiosyncratic toxicity. The association of the radioactivity derived from 14 C-KW-2449 with endogenous proteins both in vivo and in vitro was confirmed and it was verified that this covalent binding was inhibited by the addition of sodium cyanide, an iminium ion-trapping reagent, and pargyline, a MAO-B inhibitor. These findings strongly suggest that the iminium ion metabolite of KW-2449 is highly reactive in inhibiting AO irreversibly and binding to endogenous macromolecules covalently.

Monoamine oxidase B oxidizes a novel multikinase inhibitor KW-2449 to its iminium ion and aldehyde oxidase further converts it to the oxo-piperazine form in human.

(E)-1-{4-[2-(1H-Indazol-3-yl)vinyl]benzoyl}piperazine (KW-2449) is a novel multikinase inhibitor. During our clinical study, we found that KW-2449 is mainly metabolized to its oxo-piperazine form (M1). An inhibition study suggested that monoamine oxidase-B (MAO-B) oxidizes KW-2449 to an iminium (intermediate) and aldehyde oxidase (AO) then metabolizes the intermediate to M1. The conversion of KW-2449 to the iminium (intermediate) by MAO-B was confirmed by the formation of its cyanide adduct. This cooperative metabolic pathway by MAO-B and AO was newly identified in the metabolism of piperazine. The clearance of KW-2449 by MAO-B and AO in human was estimated based on the kinetic analysis with in vitro-in vivo extrapolation. The systemic clearance in human was similar to the calculated value, indicating that the extrapolation approach was applicable to KW-2449 metabolism. Finally, we found that (E)-3-amino-1-{4-[2-(1H-Indazol-3-yl)vinyl]benzoyl}-pyrrolidine (Compound A) as a stable compound against MAO-B and AO. The total body clearance of Compound A was reduced to one tenth of KW-2449, demonstrating that preventing the metabolism of MAO and AO led to more preferable pharmacokinetic profiles. As piperazine is often introduced to drug candidates to improve lipophilicity of the compound to get more hydrophilic nature, the results of this study provide useful information for future drug development.

PARAFAC Decomposition for Ultrasonic Wave Sensing of Fiber Bragg Grating Sensors: Procedure and Evaluation.

Ultrasonic wave-sensing technology has been applied for the health monitoring of composite structures, using normal fiber Bragg grating (FBG) sensors with a high-speed wavelength interrogation system of arrayed waveguide grating (AWG) filters; however, researchers are required to average thousands of repeated measurements to distinguish significant signals. To resolve this bottleneck problem, this study established a signal-processing strategy that improves the signal-to-noise ratio for the one-time measured signal of ultrasonic waves, by application of parallel factor analysis (PARAFAC) technology that produces unique multiway decomposition without additional orthogonal or independent constraints. Through bandpass processing of the AWG filter and complex wavelet transforms, ultrasonic wave signals are preprocessed as time, phase, and frequency profiles, and then decomposed into a series of conceptual three-way atoms by PARAFAC. While an ultrasonic wave results in a Bragg wavelength shift, antiphase fluctuations can be observed at two adjacent AWG ports. Thereby, concentrating on antiphase features among the three-way atoms, a fitting atom can be chosen and then restored to three-way profiles as a final result. An experimental study has revealed that the final result is consistent with the conventional 1024-data averaging signal, and relative error evaluation has indicated that the signal-to-noise ratio of ultrasonic waves can be significantly improved.

Design and synthesis of piperidine farnesyltransferase inhibitors with reduced glucuronidation potential.

The design and synthesis of a novel piperidine series of farnesyltransferase (FTase) inhibitors with reduced potential for metabolic glucuronidation are described. The various substitution and exchange of the phenyl group at the C-2 position of the previously described 2-(4-hydroxy)phenyl-3-nitropiperidine 1a (FTase IC(50)=5.4nM) resulted in metabolically stable compounds with potent FTase inhibition (14a IC(50)=4.3nM, 20a IC(50)=3.0nM, and 50a IC(50)=16nM). Molecular modeling studies of these compounds complexed with FTase and farnesyl pyrophosphate are also described.

Determination of the anticancer drug KW-2170, a pyrazoloacridone derivative, and its metabolites in human and dog plasma by high-performance liquid chromatography using an electrochemical detector.

KW-2170, 5-(3-aminopropyl) amino-7,10-dihydroxy-2-(2-hydroxethyl)-aminoethyl-6H-pyrazolo [4,5,1-de] acridin-6-one dihydroxychloride, is a novel anticancer agent under clinical development. We have established a highly sensitive method which can simultaneously quantitate KW-2170 and its two metabolites, a carboxylic (M1) and hydroxylated (M2) derivative involving the 5-position, in human and dog plasma. KW-2170 and its metabolites were extracted from plasma using a weak cation-exchange cartridge and then determined by HPLC using an electrochemical detector (ED). Over the concentration range 0.1-50 ng/ml, precision and accuracy of intra- and inter-day assay were within 11% in human plasma. In dog plasma, they were within 17% at the lower quantitation limit and within 11% at other concentrations. These three compounds were stable during the assay procedure, freeze-thawing cycles and during long-term storage. Using this methodology, the pharmacokinetics of KW-2170 in a dog could be monitored over 24 h. This method is suitable for evaluation of the detailed pharmacokinetics of KW-2170 and its metabolites in humans and dogs.