Inhibitors of proprotein convertase subtilisin/kexin type 9 (PCSK9) reduce low-density lipoprotein (LDL) cholesterol and are used for treatment of dyslipidemia. Current PCSK9 inhibitors are administered via subcutaneous injection. We present a highly potent, chemically modified PCSK9 antisense oligonucleotide (ASO) with potential for oral delivery. Past attempts at oral delivery using earlier-generation ASO chemistries and transient permeation enhancers provided encouraging data, suggesting that improving potency of the ASO could make oral delivery a reality. The constrained ethyl chemistry and liver targeting enabled by N-acetylgalactosamine conjugation make this ASO highly potent. A single subcutaneous dose of 90 mg reduced PCSK9 by >90% in humans with elevated LDL cholesterol and a monthly subcutaneous dose of around 25 mg is predicted to reduce PCSK9 by 80% at steady state. To investigate the feasibility of oral administration, the ASO was coformulated in a tablet with sodium caprate as permeation enhancer. Repeated oral daily dosing in dogs resulted in a bioavailability of 7% in the liver (target organ), about fivefold greater than the plasma bioavailability. Target engagement after oral administration was confirmed by intrajejunal administration of a rat-specific surrogate ASO in solution with the enhancer to rats and by plasma PCSK9 and LDL cholesterol lowering in cynomolgus monkey after tablet administration. On the basis of an assumption of 5% liver bioavailability after oral administration in humans, a daily dose of 15 mg is predicted to reduce circulating PCSK9 by 80% at steady state, supporting the development of the compound for oral administration to treat dyslipidemia.

Pharmacokinetics (PK) of antisense oligonucleotides (ASOs) is characterized by rapid distribution from plasma to tissue and slow terminal plasma elimination driven by re-distribution from tissue. Quantitative understanding of tissue PK and RNA knockdown for various ASO chemistries, conjugations, and administration routes is critical for successful drug discovery. Here, we report concentration-time and RNA knockdown profiles for a gapmer ASO with locked nucleic acid ribose chemistry in mouse liver, kidney, heart, and lung after subcutaneous and intratracheal administration. Additionally, the same ASO with liver targeting conjugation (galactosamine-N-acetyl) is evaluated for subcutaneous administration. Data indicate that exposure and knockdown differ between tissues and strongly depend on administration route and conjugation. In a second study, we show that tissue PK is similar between the three different ribose chemistries locked nucleic acid, constrained ethyl and 2'-O-methoxyethyl, both after subcutaneous and intratracheal administration. Further, we show that the half-life in mouse liver may vary with ASO sequence. Finally, we report less than dose-proportional increase in liver concentration in the dose range of 3-30 μmol/kg. Overall, our studies contribute pivotal data to support design and interpretation of ASO in vivo studies, thereby increasing the probability of delivering novel ASO therapies to patients.

Brown adipose tissue (BAT) is considered an interesting target organ for the treatment of metabolic disease due to its high metabolic capacity. Non-shivering thermogenesis, once activated, can lead to enhanced partitioning and oxidation of fuels in adipose tissues, and reduce the burden of glucose and lipids on other metabolic organs such as liver, pancreas and skeletal muscle. Sustained long-term activation of BAT may also lead to meaningful bodyweight loss. In this review, we discuss three different drug classes (the thiazolidinedione (TZD) class of PPAR agonists, 3 adrenergic receptor agonists and fibroblast growth factor 21 (FGF21) analogues) that have been proposed to regulate BAT and beige recruitment or activation, or both, and which have been tested in both rodent and human. The learnings from these classes suggest that restoration of functional BAT and beige mass as well as improved activation might be required to fully realize the metabolic potential of these tissues. Whether this can be achieved without the undesired cardiovascular side-effects exhibited by the TZD PPAR agonists and 3 adrenergic receptor agonists remains to be resolved.

Today, there is great interest in diets proposing new combinations of macronutrient compositions and fasting schedules. Unfortunately, there is little consensus regarding the impact of these different diets, since available studies measure different sets of variables in different populations, thus only providing partial, non-connected insights. We lack an approach for integrating all such partial insights into a useful and interconnected big picture. Herein, we present such an integrating tool. The tool uses a novel mathematical model that describes mechanisms regulating diet response and fasting metabolic fluxes, both for organ-organ crosstalk, and inside the liver. The tool can mechanistically explain and integrate data from several clinical studies, and correctly predict new independent data, including data from a new study. Using this model, we can predict non-measured variables,e.g. hepatic glycogen and gluconeogenesis, in response to fasting and different diets. Furthermore, we exemplify how such metabolic responses can be successfully adapted to a specific individual’s sex, weight, height, as well as to the individual’s historical data on metabolite dynamics. This tool enables an offline digital twin technology.

Current research on metabolic disorders and diabetes relies on animal models because multi-organ diseases cannot be well studied with standard in vitro assays. Here, we have connected cell models of key metabolic organs, the pancreas and liver, on a microfluidic chip to enable diabetes research in a human-based in vitro system. Aided by mechanistic mathematical modeling, we demonstrate that hyperglycemia and high cortisone concentration induce glucose dysregulation in the pancreas-liver microphysiological system (MPS), mimicking a diabetic phenotype seen in patients with glucocorticoid-induced diabetes. In this diseased condition, the pancreas-liver MPS displays beta-cell dysfunction, steatosis, elevated ketone-body secretion, increased glycogen storage, and upregulated gluconeogenic gene expression. Conversely, a physiological culture condition maintains glucose tolerance and beta-cell function. This method was reproducible in two laboratories and was effective in multiple pancreatic islet donors. The model also provides a platform to identify new therapeutic proteins, as demonstrated with a combined transcriptome and proteome analysis.