Development and validation of qPCR methods for nucleic acid biomarkers as a drug development tool: points to consider.

Nucleic acid (NA) biomarkers play critical roles in drug development. However, the global regulatory guidelines for assessing quantification methods specific to NA biomarkers are limited. The validation of analytical methods is crucial for the use of biomarkers in clinical and post-marketing evaluations of drug efficacy and adverse reactions. Given that quantitative polymerase chain reaction (qPCR) and reverse transcription qPCR (RT-qPCR) methods are the gold standards for the quantification of NA biomarkers, the Biomarker Analytical Method Validation Study Group in Japan has discussed considerations and made recommendations for the development and validation of qPCR- and RT-qPCR-based analytical methods for endogenous NA biomarkers as drug development tools. This white paper aims to contribute to the global harmonization of NA biomarker assay validation.

Involvement of concentrative nucleoside transporter 1 in intestinal absorption of trifluorothymidine, a novel antitumor nucleoside, in rats.

ααα-Trifluorothymidine (TFT), an anticancer nucleoside analog, is a potent thymidylate synthase inhibitor. TFT exerts its antitumor activity primarily by inducing DNA fragmentation after incorporation of the triphosphate form of TFT into the DNA. Although an oral combination of TFT and a thymidine phosphorylase inhibitor has been clinically developed, there is little information regarding TFT absorption. Therefore, we investigated TFT absorption in the rat small intestine. After oral administration of TFT in rats, more than 75% of the TFT was absorbed. To identify the uptake transport system, uptake studies were conducted by using everted sacs prepared from rat small intestines. TFT uptake was saturable, significantly reduced under Na(+)-free conditions, and strongly inhibited by the addition of an endogenous pyrimidine nucleoside. From these results, we suggested the involvement of concentrative nucleoside transporters (CNTs) in TFT absorption into rat small intestine. In rat small intestines, the mRNAs coding for rat CNT1 (rCNT1) and rCNT2, but not for rCNT3, were predominantly expressed. To investigate the roles of rCNT1 and rCNT2 in TFT uptake, we conducted uptake assays by using Xenopus laevis oocytes injected with rCNT1 complementary RNA (cRNA) and rCNT2 cRNA. TFT uptake by X. laevis oocytes injected with rCNT1 cRNA, and not rCNT2 cRNA, was significantly greater than that by water-injected oocytes. In addition, in situ single-pass perfusion experiments performed using rat jejunum regions showed that thymidine, a substrate for CNT1, strongly inhibited TFT uptake. In conclusion, TFT is absorbed via rCNT1 in the intestinal lumen in rats.

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.

Safety, Tolerability, Pharmacokinetics, and Food Effects on TAC-302 in Healthy Participants: Randomized, Double-Blind, Placebo-Controlled, Single-Dose and Multiple-Dose Studies.

TAC-302 stimulates neurite outgrowth activity and is expected to restore urinary function in patients with lower urinary tract dysfunction. We conducted 2 phase 1, randomized, placebo-controlled studies to confirm the safety and pharmacokinetics (PK) of TAC-302 in healthy adult Japanese male volunteers. In the first-in-human single-dose study (n = 60), TAC-302 was administered at doses from 100 to 1200 mg after an overnight fast. The effects of a meal on the PK of TAC-302 400 mg were also examined. A multiple-dose study (n = 36) evaluated the effects of meal fat content on the PK of single doses of TAC-302 (100, 200, or 400 mg) and multiple doses of TAC-302 administered for 5 days (100, 200, and 400 mg twice daily). TAC-302 showed linear PK up to doses of 1200 mg in the fasting state, and across the dose range of 100-400 mg in the fed state. No accumulation of TAC-302 was observed. Food, particularly with high fat content, increased TAC-302 plasma concentrations. No differences were observed in the adverse event incidence between the TAC-302 and placebo groups in either study. TAC-302 showed a wide safety margin.

TAS-121, A Selective Mutant EGFR Inhibitor, Shows Activity Against Tumors Expressing Various EGFR Mutations Including T790M and Uncommon Mutations G719X.

TAS-121 is a novel orally active selective covalent inhibitor of the mutant EGFR. We performed preclinical characterization of TAS-121 and compared its efficacy and selectivity for common EGFR mutations (Ex19del and L858R), first- and second- generation EGFR-tyrosine kinase inhibitor (EGFR-TKI) resistance mutation (T790M), and uncommon mutations (G719X and L861Q) with those of other EGFR-TKIs. We also commenced investigation of the clinical benefits of TAS-121. The IC50 for intracellular EGFR phosphorylation was determined by using Jump-In GripTite HEK293 cells transiently transfected with EGFR expression vectors. Mouse xenograft models were used to evaluate the antitumor activity of TAS-121. TAS-121 potently inhibited common activating and resistance EGFR mutations to the same extent as another third-generation EGFR-TKI (osimertinib). In addition, TAS-121 showed equivalent inhibitory activity against some uncommon mutations such as G719X and L861Q. Furthermore, TAS-121 demonstrated greater selectivity for mutant EGFRs versus the wild-type EGFR compared with other EGFR-TKIs. Moreover, TAS-121 displayed antitumor activity in SW48 (EGFR G719S) and NCI-H1975 (EGFR L858R/T790M) xenograft models, and achieved an objective response in patients with NSCLC with EGFR mutations including G719A mutation. In conclusion, TAS-121 is a novel third-generation EGFR-TKI and demonstrates antitumor activities in patients with NSCLC expressing either common or uncommon EGFR mutations.

TAS6417, A Novel EGFR Inhibitor Targeting Exon 20 Insertion Mutations.

Activating mutations in the EGFR gene are important targets in cancer therapy because they are key drivers of non-small cell lung cancer (NSCLC). Although almost all common EGFR mutations, such as exon 19 deletions and the L858R point mutation in exon 21, are sensitive to EGFR-tyrosine kinase inhibitor (TKI) therapies, NSCLC driven by EGFR exon 20 insertion mutations is associated with poor clinical outcomes due to dose-limiting toxicity, demonstrating the need for a novel therapy. TAS6417 is a novel EGFR inhibitor that targets EGFR exon 20 insertion mutations while sparing wild-type (WT) EGFR. In cell viability assays using Ba/F3 cells engineered to express human EGFR, TAS6417 inhibited EGFR with various exon 20 insertion mutations more potently than it inhibited the WT. Western blot analysis revealed that TAS6417 inhibited EGFR phosphorylation and downstream molecules in NSCLC cell lines expressing EGFR exon 20 insertions, resulting in caspase activation. These characteristics led to marked tumor regression in vivo in both a genetically engineered model and in a patient-derived xenograft model. Furthermore, TAS6417 provided a survival benefit with good tolerability in a lung orthotopic implantation mouse model. These findings support the clinical evaluation of TAS6417 as an efficacious drug candidate for patients with NSCLC harboring EGFR exon 20 insertion mutations. Mol Cancer Ther; 17(8); 1648-58. ©2018 AACR.

Comparative study of aryl hydrocarbon receptor ligand activities of six chemicals in vitro and in vivo.

The aryl hydrocarbon receptor (AhR) ligand activities of six known AhR ligands were compared in vivo and in vitro. The in vivo ligand activity was estimated in terms of induction of cytochrome P450 1A1/2 activities, i.e., ethoxyresorufin-O-dealkylase (EROD) and methoxyresorufin-O-dealkylase (MROD) activities, and in vitro ligand activity was evaluated with a recombinant yeast reporter gene assay. The test chemicals were 3-methylcholanthrene (MC), beta-naphthoflavone (beta-NF), indirubin, indigo, 3,3'-diindolylmethane (DIM) and diphenyl-p-phenylenediamine (DPPD). The first four showed potent AhR ligand activity in vitro, comparable with that of 2,3,7,8-tetrachlorodibenzo-p-dioxin, while DIM and DPPD showed weaker activity. Administration of MC and beta-NF to mice caused significant induction of EROD and MROD activities, while indirubin, indigo and DIM also induced these activities, but less potently. DPPD also induced the activities, but was toxic at higher doses. These enhancing effects were lost or greatly reduced in Ahr-null mice (Ahr (-/-)). Our results suggest that EROD and MROD activity assays are useful for evaluating the AhR ligand activity of chemicals in vivo, where the biodynamics of the chemicals plays an important role.

Aryl hydrocarbon receptor-mediated induction of microsomal drug-metabolizing enzyme activity by indirubin and indigo.

Indirubin and indigo, which are thought to be natural ligands for aryl hydrocarbon receptor (AhR), showed marked AhR ligand activities in a reporter gene assay using recombinant yeast. Their activities were comparable with or more potent than that of 2,3,7,8-tetrachlorodibenzo-p-dioxin. When indirubin and indigo were administered to mice, ethoxyresorufin-O-dealkylase and methoxyresorufin-O-dealkylase activities in the liver were increased, but subsequently decreased within 2 days. Indirubin was more potent than indigo. Levels of cytochrome P450 1A1/2 proteins and mRNAs in the liver of mice dosed with indirubin were also enhanced. These enhancing effects of indirubin and indigo were not observed in AhR knock-out mice. Ethoxyresorufin-O-dealkylase and methoxyresorufin-O-dealkylase activities in rat hepatocytes and HepG2 cells were enhanced by the addition of indirubin or indigo, but less potently than by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Indigocarmine, a sulfate derivative of indigo, which is used as food additive, did not show these inducing effects on drug-metabolizing enzymes. Our results suggest that indirubin and indigo act as inducers for cytochrome P450 1A1/2 mediated by AhR in mammals in vivo.