Hideo Takakusa

To meet unmet medical needs, middle-to-large molecules, including peptides and oligonucleotides, have emerged as new therapeutic modalities. Owing to their middle-to-large molecular sizes, middle-to-large molecules are not suitable for oral absorption, but there are high expectations around orally bioavailable macromolecular drugs, since oral administration is the most convenient dosing route. Therefore, extensive efforts have been made to create bioavailable middle-to-large molecules or develop absorption enhancement technology, from which some successes have recently been reported. For example, Rybelsus^® tablets and Mycapssa^® capsules, both of which contain absorption enhancers, were approved as oral medications for type 2 diabetes and acromegaly, respectively. The oral administration of Rybelsus and Mycapssa exposes their pharmacologically active peptides with molecular weights greater than 1000, namely, semaglutide and octreotide, respectively, into systemic circulation. Although these two medications represent major achievements in the development of orally absorbable peptide formulations, the oral bioavailability of peptides after taking Rybelsus and Mycapssa is still only around 1%. In this article, we review the approaches and recent advances of orally bioavailable middle-to-large molecules and discuss challenges for improving their oral absorption.

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.

Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease caused by out-of-frame or nonsense mutation in the dystrophin gene. It begins with a loss of ambulation between 9 and 14 years of age, followed by various other symptoms including cardiac dysfunction. Exon skipping of patients’ DMD pre-mRNA induced by antisense oligonucleotides (AOs) is expected to produce shorter but partly functional dystrophin proteins, such as those possessed by patients with the less severe Becker muscular dystrophy. We are working on developing modified nucleotides, such as 2′-O,4′-C-ethylene-bridged nucleic acids (ENAs), possessing high nuclease resistance and high affinity for complementary RNA strands. Here, we demonstrate the preclinical characteristics (exon-skipping activity in vivo, stability in blood, pharmacokinetics, and tissue distribution) of renadirsen, a novel AO modified with 2′-O-methyl RNA/ENA chimera phosphorothioate designed for dystrophin exon 45 skipping and currently under clinical trials. Notably, systemic delivery of renadirsen sodium promoted dystrophin exon skipping in cardiac muscle, skeletal muscle, and diaphragm, compared with AOs with the same sequence as renadirsen but conventionally modified by PMO and 2′OMePS. These findings suggest the promise of renadirsen sodium as a therapeutic agent that improves not only skeletal muscle symptoms but also other symptoms in DMD patients, such as cardiac dysfunction.

The metabolic bioactivation of a drug to a reactive metabolite (RM) and its covalent binding to cellular macromolecules is believed to be involved in clinical adverse events, including idiosyncratic drug toxicities. Therefore, it is important to assess the potential of drug candidates to generate RMs and form drug-protein covalent adducts during lead optimization processes. In this study, the RM formation of some marketed drugs were quantitatively assessed by means of a sensitive and robust detection method that we have established using (35)S-glutathione ((35)S-GSH) as a trapping agent. Problematic drugs well-known to generate RMs exhibited a relatively high rate of (35)S-GS-adducts to RM (RM-GS) formation, which contrasted with safe drugs. For practical use in lead optimization processes, a series of new chemical entities were tested and hints on the structural modifications needed in order to minimize their RM formation were provided. Furthermore, the RM-GS formation rates of a number of compounds were compared using their in vitro covalent binding yields to liver proteins determined with (14)C-labeled compounds, demonstrating that the RM-GS formation rate could be a substitute for the covalent binding yield within the same series of compounds.

Bioactivation of a drug to a reactive metabolite and its covalent binding to cellular macromolecules is believed to be involved in clinical adverse events, including idiosyncratic drug toxicities (IDTs). For the interpretation of the covalent binding data in terms of risk assessment, the in vitro and in vivo covalent binding data of a variety of drugs associated with IDTs or not were determined. Most of the “problematic” drugs, including “withdrawn” and “warning” drugs, exhibit higher human liver microsome (HLM) in vitro covalent binding yields than the “safe” drugs. Although some of the problematic drugs that are known to undergo bioactivation other than cytochrome P450-mediated oxidation exhibited only trace levels of HLM covalent binding like safe drugs, a rat in vivo covalent binding study could assess the bioactivation of such drugs. Furthermore, the tissue distribution/retention of the drugs was also examined by rat autoradiography (ARG). The residual radioactivity in the liver observed at 72 or 168 h postdose was found to be well correlated with the rat in vivo covalent binding to liver proteins; thus, the in vivo covalent binding yields of the drugs could be extrapolated from the retention profiles observed by means of ARG. Long-term retention of radioactivity in the bone marrow was observed with some drugs associated with severe agranulocytosis, suggesting a spatial relationship between the toxicity profile and drug distribution/retention. Taken together, the covalent binding and tissue distribution/retention data of the various marketed drugs obtained in the present study should be quite informative for the interpretation of data in terms of risk assessment.

The metabolic activation of a drug to an electrophilic reactive metabolite and its covalent binding to cellular macromolecules is considered to be involved in the occurrence of idiosyncratic drug toxicity (IDT). As a cellular defense system against oxidative and electrophilic stress, phase II enzymes are known to be induced through a Kelch-like ECH-associated protein 1/nuclear factor E2-related factor 2/antioxidant response element system. We presumed that it is important for the risk assessment of drug-induced hepatotoxicity and IDTs to observe the biological responses evoked by exposure to reactive metabolites, and then investigated the mRNA induction profiles of phase II enzymes in human hepatocytes after exposure to problematic drugs associated with IDTs, such as ticlopidine, diclofenac, clozapine, and tienilic acid, as well as safe drugs such as levofloxacin and caffeine. According to the results, the problematic drugs exhibited inductive effects on heme oxygenase 1 (HO-1), which contrasted with the safe drugs; therefore, the induction of HO-1 mRNA seems to be correlated with the occurrence of drug toxicity, including IDT caused by electrophilic reactive metabolites. Moreover, glutathione-depletion and cytochrome P450 (P450)-inhibition experiments have shown that the observed HO-1 induction was triggered by the electrophilic reactive metabolites produced from the problematic drugs through P450-mediated metabolic bioactivation. Taken together with our present study, this suggests that HO-1 induction in human hepatocytes would be a good marker of the occurrence of metabolism-based drug-induced hepatotoxicity and IDT caused by the formation of electrophilic reactive metabolites.