Drug Metabolism and Pharmacokinetics of Antisense Oligonucleotide Therapeutics: Typical Profiles, Evaluation Approaches, and Points to Consider Compared with Small Molecule Drugs.

Oligonucleotide therapeutics are attracting attention as a new treatment modality for a range of diseases that have been difficult to target using conventional approaches. Technical advances in chemical modification and drug delivery systems have led to the generation of compounds with excellent profiles as pharmaceuticals, and 16 oligonucleotide therapeutics have been marketed to date. There is a growing need to develop optimal and efficient approaches to evaluate drug metabolism and pharmacokinetics (DMPK) and drug-drug interactions (DDIs) of oligonucleotide therapeutics. The DMPK/DDI profiles of small molecule drugs are highly diverse depending on their structural and physicochemical characteristics, whereas oligonucleotide therapeutics share similar DMPK profiles within each chemistry type. Most importantly, the mechanisms and molecules involved in the distribution and metabolism of oligonucleotides differ from those of small molecules. In addition, there are considerations regarding experimental approaches in the evaluation of oligonucleotides, such as bioanalytical challenges, the use of radiolabeled tracers, materials for in vitro metabolism/DDI studies, and methods to study biodistribution. In this review, we attempt to summarize the DMPK characteristics of antisense oligonucleotide (ASO) therapeutics and discuss some of the issues regarding how to optimize the evaluation and prediction of the DMPK and DDI of ASOs.

2'-N-Alkylaminocarbonyl-2'-amino-LNA: Synthesis, duplex stability, nuclease resistance, and in vitro anti-microRNA activity.

2'-Amino-LNA has the potential to acquire various functions through chemical modification at the 2'-nitrogen atom. This study focused on 2'-N-alkylaminocarbonyl 2'-amino-LNA, which is a derivative of 2'-amino-LNA. We evaluated its practical usefulness as a chemical modification of anti-miRNA oligonucleotide. The synthesis of phosphoramidites of 2'-N-alkylaminocarbonyl substituted 2'-amino-LNA bearing thymine and 5-methylcytosine proceeded in good yields. Incorporating the 2'-N-alkylaminocarbonyl-2'-amino-LNA monomers into oligonucleotides improved the duplex stability for complementary RNA strands and robust nuclease resistance. Moreover, 2'-N-alkylaminocarbonyl-2'-amino-LNA is a promising scaffold that significantly increases the potency of anti-miRNA oligonucleotides.

Parallel synthesis of oligonucleotides containing N-acyl amino-LNA and their therapeutic effects as anti-microRNAs.

2'-Amino-locked nucleic acid (ALNA), maintains excellent duplex stability, and the nitrogen at the 2'-position is an attractive scaffold for functionalization. Herein, a facile and efficient method for the synthesis of various 2'-N-acyl amino-LNA derivatives by direct acylation of the 2'-amino moiety contained in the synthesized oligonucleotides and its fundamental properties are described. The introduction of the acylated amino-LNA enhances the potency of the molecules as therapeutic anti-microRNA oligonucleotides.

Altered Biodistribution and Hepatic Safety Profile of a Gapmer Antisense Oligonucleotide Bearing Guanidine-Bridged Nucleic Acids.

Guanidine-bridged nucleic acid (GuNA) is a novel 2',4'-bridged nucleic acid/locked nucleic acid (2',4'-BNA/LNA) analog containing cations that exhibit strong affinity for target RNA and superior nuclease resistance. In this study, Malat1 antisense oligonucleotide (ASO) bearing GuNA was evaluated for target knockdown (KD) activity and tolerability. The GuNA ASO did not interfere with RNase H recruitment on the target RNA/ASO heteroduplex and did show potent target KD activity in a skeletal muscle-derived cell line equivalent to that of the LNA ASO under gymnotic conditions, whereas almost no KD activity was observed in a hepatocyte-derived cell line. The GuNA ASO exhibited potent KD activity in various tissues; the KD activity in the skeletal muscle was equivalent with that of the LNA ASO, but the KD activities in the liver and kidney were clearly lower compared with the LNA ASO. In addition, despite the higher accumulation of the GuNA ASO in the liver, levels of aspartate aminotransferase and alanine aminotransferase with the GuNA ASO administration were not elevated compared with those induced by the LNA ASO. Our data indicate that the GuNA ASO is tolerable and exhibits unique altered pharmacological activities in comparison with the LNA ASO in terms of the relative effect between liver and skeletal muscle.

Prediction of human pharmacokinetics for low-clearance compounds using pharmacokinetic data from chimeric mice with humanized livers.

Development of low-clearance (CL) compounds that are slowly metabolized is a major goal in the pharmaceutical industry. However, the pursuit of low intrinsic CL (CLint ) often leads to significant challenges in evaluating the pharmacokinetics of such compounds. Although in vitro-in vivo extrapolation is widely used to predict human CL, its application has been limited for low-CLint compounds because of the low turnover of parent compounds in metabolic stability assays. To address this issue, we focused on chimeric mice with humanized livers (PXB-mice), which have been increasingly reported to accurately predict human CL in recent years. The predictive accuracy for nine low-CLint compounds with no significant turnover in a human hepatocyte assay was investigated using PXB-mouse methods, such as single-species allometric scaling (PXB-SSS) approach and a novel physiologically based scaling (PXB-PBS) approach that assumes that the CLint per hepatocyte is equal between humans and PXB-mice. The percentages of compounds with predicted CL within 2- and 3-fold ranges of the observed CL for low-CLint compounds were 89% and 100%, respectively, for both PXB-SSS and PXB-PBS approaches. Moreover, the predicted CL was mostly consistent among the methods. Conversely, the percentages of compounds with predicted CL within 2- and 3-fold ranges of the observed CL for low-CLint compounds were 50% and 63%, respectively, for multispecies allometric (MA) scaling. Overall, these PXB-mouse methods were much more accurate than conventional MA scaling approaches, suggesting that PXB-mice are useful tools for predicting the human CL of low-CLint compounds that are slowly metabolized.

Human-Induced Pluripotent Stem Cell-Derived Hepatocytes and their Culturing Methods to Maintain Liver Functions for Pharmacokinetics and Safety Evaluation of Pharmaceuticals.

Human hepatocytes are essential cell types for pharmacokinetics and the safety evaluation of pharmaceuticals. However, widely used primary hepatocytes with individual variations in liver function lose those functions rapidly in culture. Hepatic cell lines are convenient to use but have low liver functions. Human-Induced Pluripotent Stem (hiPS) cells can be expanded and potentially differentiated into any cell or tissue, including the liver. HiPS cell-derived Hepatocyte-Like Cells (hiPSHeps) are expected to be extensively used as consistent functional human hepatocytes. Many laboratories are investigating methods of using hiPS cells to differentiate hepatocytes, but the derived cells still have immature liver functions. In this paper, we describe the current uses and limitations of conventional hepatic cells, evaluating the suitability of hiPS-Heps to pharmacokinetics and the safety evaluation of pharmaceuticals, and discuss the potential future use of non-conventional non-monolayer culture methods to derive fully functional hiPS-Heps.

Precise prediction of activators for the human constitutive androstane receptor using structure-based three-dimensional quantitative structure-activity relationship methods.

The constitutive androstane receptor (CAR, NR1I3) regulates the expression of numerous drug-metabolizing enzymes and transporters. The upregulation of various enzymes, including CYP2B6, by CAR activators is a critical problem leading to clinically severe drug-drug interactions (DDIs). To date, however, few effective computational approaches for identifying CAR activators exist. In this study, we aimed to develop three-dimensional quantitative structure-activity relationship (3D-QSAR) models to predict the CAR activating potency of compounds emerging in the drug-discovery process. Models were constructed using comparative molecular field analysis (CoMFA) based on the molecular alignments of ligands binding to CAR, which were obtained from ensemble ligand-docking using 28 compounds as a training set. The CoMFA model, modified by adding a lipophilic parameter with calculated logD7.4 (S+logD7.4), demonstrated statistically good predictive ability (r2 = 0.99, q2 = 0.74). We also confirmed the excellent predictability of the 3D-QSAR model for CAR activation (r2pred = 0.71) using seven compounds as a test set for external validation. Collectively, our results indicate that the 3D-QSAR model developed in this study provides precise prediction of CAR activating potency and, thus, should be useful for selecting drug candidates with minimized DDI risk related to enzyme-induction in the early drug-discovery stage.

Involvement of hepatocyte nuclear factor 4 alpha in transcriptional regulation of the human pregnane X receptor gene in the human liver.

Pregnane X receptor (PXR; NR1I2), a key transcriptional factor that regulates genes encoding drug-metabolizing enzymes and drug transporters, is abundantly expressed in the human liver. However, studies on the molecular mechanism of human PXR gene regulation are limited. In this study, we examined the involvement of hepatocyte nuclear factor 4alpha (HNF4alpha; NR2A1) in the transcriptional regulation of the human PXR gene in the human liver. The activities of the human PXR promoter containing the direct repeat 1 (DR1) element located at -88/-76 of the promoter were significantly increased by co-expression of HNF4alpha in the human hepatocellular carcinoma cell line. In addition, introduction of mutation into the DR1 element abolished the transcriptional activation of the human PXR promoter by exogenous HNF4alpha. The results of gel mobility shift assays and chromatin immunoprecipitation assays showed that HNF4alpha was bound to the promoter region containing the DR1 element. A knock-down of HNF4alpha by siRNA significantly decreased expression levels of endogenous PXR mRNA in HepG2 cells. Furthermore, expression levels of PXR mRNA positively correlated with those of HNF4alpha mRNA in 18 human liver samples. These results suggested that HNF4alpha transactivated the human PXR gene by binding to the DR1 element located at -88/-76 of the promoter and was involved in the expression of PXR in the human liver.

Identification of HMG-CoA reductase inhibitors as activators for human, mouse and rat constitutive androstane receptor.

Constitutive active (or androstane) receptor (CAR, NR1I3), a member of the nuclear receptor family, is a major regulator for induction of cytochrome P450 2B (CYP2B) genes by phenobarbital. Phenobarbital-like inducer, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene is a potent mouse CAR ligand that has been used to study CAR target genes in mice but does not activate human CAR (hCAR) or rat CAR (rCAR). Although 6-(4-chlorophenyl) imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO) was reported to be an hCAR agonistic ligand, activation of hCAR by CITCO in cell-based reporter assay was weak. Therefore, we performed a screening of 50 drugs and chemicals using cell-based reporter assays to identify activators of hCAR. Among them, HMG-CoA reductase inhibitors (cerivastatin, simvastatin, fluvastatin, and atorvastatin) enhanced the hCAR-mediated transcriptional activation of phenobarbital-responsive enhancer module reporter gene by up to 3-fold. Similar activation by HMG-CoA reductase inhibitors was also observed with mouse and rat CARs. On the other hand, pravastatin did not activate hCAR at the concentrations tested (up to 30 microM). The extent of activation by the HMG-CoA reductase inhibitors was stronger than that by CITCO. Cerivastatin, simvastatin, fluvastatin, and atorvastatin induced CYP2B6 mRNA in stable hCAR-expressed FLC7 cells but not in original FLC7 cells. Therefore, we concluded that CAR mediates the effects of HMG-CoA reductase inhibitors on the induction of CYP2B genes, although HMG-CoA reductase inhibitors also activate pregnane X receptor. HMG-CoA reductase inhibitors such as cerivastatin would be useful to study for elucidating molecular and cellular mechanisms of hCAR.