Injectable Biodegradable Silica Depot for Controlled Subcutaneous Delivery of Antisense Oligonucleotides with beyond Monthly Administration.

Single-stranded antisense oligonucleotides (ASOs) are typically administered subcutaneously once per week or monthly. Less frequent dosing would have strong potential to improve patient convenience and increase adherence and thereby for some diseases result in more optimal therapeutic outcomes. Several technologies are available to provide sustained drug release via subcutaneous (SC) administration. ASOs have a high aqueous solubility and require relatively high doses, which limits the options available substantially. In the present work, we show that an innovative biodegradable, nonporous silica-based matrix provides zero-order release in vivo (rats) for at least 4 weeks for compositions with ASO loads of up to about 100 mg/mL (0.5 mL injection) without any sign of initial burst. This implies that administration beyond once monthly can be feasible. For higher drug loads, substantial burst release was observed during the first week. The concentrations of unconjugated ASO levels in the liver were found to be comparable to corresponding bolus doses. Additionally, infusion using a minipump shows a higher liver exposure than SC bolus administration at the same dose level and, in addition, clear mRNA knockdown and circulating protein reduction comparable to SC bolus dosing, hence suggesting productive liver uptake for a slow-release administration.

In Vivo Metabolite Profiles of an N-Acetylgalactosamine-Conjugated Antisense Oligonucleotide AZD8233 Using Liquid Chromatography High-Resolution Mass Spectrometry: A Cross-Species Comparison in Animals and Humans.

AZD8233, a liver-targeting antisense oligonucleotide (ASO), inhibits subtilisin/kexin type 9 protein synthesis. It is a phosphorothioated 3-10-3 gapmer with a central DNA sequence flanked by constrained 2'-O-ethyl 2',4'-bridged nucleic acid (cEt-BNA) wings and conjugated to a triantennary N-acetylgalactosamine (GalNAc) ligand at the 5'-end. Herein we report the biotransformation of AZD8233, as given by liver, kidney, plasma and urine samples, after repeated subcutaneous administration to humans, mice, rats, rabbits, and monkeys. Metabolite profiles were characterized using liquid chromatography high-resolution mass spectrometry. Metabolite formation was consistent across species, mainly comprising hydrolysis of GalNAc sugars, phosphodiester-linker hydrolysis releasing the full-length ASO, and endonuclease-mediated hydrolysis within the central DNA gap followed by exonuclease-mediated 5'- or 3'-degradation. All metabolites contained the 5'- or 3'-cEt-BNA terminus. Most shortmer metabolites had the free terminal alcohol at 5'- and 3'-positions of ribose, although six were found retaining the terminal 5'-phosphorothioate group. GalNAc conjugated shortmer metabolites were also observed in urine. Synthesized metabolite standards were applied for (semi)quantitative metabolite assessment. Intact AZD8233 was the major component in plasma, whereas the unconjugated full-length ASO was predominant in tissues. In plasma, most metabolites were shortmers retaining the 3'-cEt-BNA terminus, whereas metabolites containing the 5'- or 3'-cEt-BNA terminus were detected in both tissues and urine. All metabolites in human plasma were also detected in all nonclinical species, and all human urine metabolites were detected in monkey urine. In general, metabolite profiles in animal species were qualitatively similar and quantitatively exceeded the exposures of the circulating metabolites in humans at the doses studied. SIGNIFICANCE STATEMENT: This study presents metabolite identification and profiling of AZD8233, an N-acetylgalactosamine-conjugated antisense oligonucleotide (ASO), across species. A biotransformation strategy for ASOs was established by utilizing biologic samples collected from toxicology and/or clinical studies and liquid chromatography high-resolution mass spectrometry analysis without conducting bespoke radiolabeled absorption, distribution, metabolism, and excretion studies. The generated biotransformation package was considered adequate by health authorities to progress AZD8233 into a phase 3 program, proving its applicability to future metabolism studies of ASOs in drug development.

Multiple compound-related adverse properties contribute to liver injury caused by endothelin receptor antagonists.

Drug-induced liver injury has been observed in patients treated with the endothelin receptor antagonists sitaxentan and bosentan, but not following treatment with ambrisentan. The aim of our studies was to assess the possible role of multiple contributory mechanisms in this clinically relevant toxicity. Inhibition of the bile salt export pump (BSEP) and multidrug resistance-associated protein 2 was quantified using membrane vesicle assays. Inhibition of mitochondrial respiration in human liver-derived HuH-7 cells was determined using a Seahorse XF(e96) analyzer. Cytochrome P450 (P450)-independent and P450-mediated cell toxicity was assessed using transfected SV40-T-antigen-immortalized human liver epithelial (THLE) cell lines. Exposure-adjusted assay ratios were calculated by dividing the maximum human drug plasma concentrations by the IC50 or EC50 values obtained in vitro. Covalent binding (CVB) of radiolabeled drugs to human hepatocytes was quantified, and CVB body burdens were calculated by adjusting CVB values for fractional drug turnover in vitro and daily therapeutic dose. Sitaxentan exhibited positive exposure-adjusted signals in all five in vitro assays and a high CVB body burden. Bosentan exhibited a positive exposure-adjusted signal in one assay (BSEP inhibition) and a moderate CVB body burden. Ambrisentan exhibited no positive exposure-adjusted assay signals and a low CVB body burden. These data indicate that multiple mechanisms contribute to the rare, but potentially severe liver injury caused by sitaxentan in humans; provide a plausible rationale for the markedly lower propensity of bosentan to cause liver injury; and highlight the relative safety of ambrisentan.

Understanding Statin-Roxadustat Drug-Drug-Disease Interaction Using Physiologically-Based Pharmacokinetic Modeling.

A different drug-drug interaction (DDI) scenario may exist in patients with chronic kidney disease (CKD) compared with healthy volunteers (HVs), depending on the interplay between drug-drug and disease (drug-drug-disease interaction (DDDI)). Physiologically-based pharmacokinetic (PBPK) modeling, in lieu of a clinical trial, is a promising tool for evaluating these complex DDDIs in patients. However, the prediction confidence of PBPK modeling in the severe CKD population is still low when nonrenal pathways are involved. More mechanistic virtual disease population and robust validation cases are needed. To this end, we aimed to: (i) understand the implications of severe CKD on statins (atorvastatin, simvastatin, and rosuvastatin) pharmacokinetics (PK) and DDI; and (ii) predict untested clinical scenarios of statin-roxadustat DDI risks in patients to guide suitable dose regimens. A novel virtual severe CKD population was developed incorporating the disease effect on both renal and nonrenal pathways. Drug and disease PBPK models underwent a four-way validation. The verified PBPK models successfully predicted the altered PKs in patients for substrates and inhibitors and recovered the observed statin-rifampicin DDIs in patients and the statin-roxadustat DDIs in HVs within 1.25- and 2-fold error. Further sensitivity analysis revealed that the severe CKD effect on statins PK is mainly mediated by hepatic BCRP for rosuvastatin and OATP1B1/3 for atorvastatin. The magnitude of statin-roxadustat DDI in patients with severe CKD was predicted to be similar to that in HVs. PBPK-guided suitable dose regimens were identified to minimize the risk of side effects or therapeutic failure of statins when co-administered with roxadustat.

Distribution and biotransformation of therapeutic antisense oligonucleotides and conjugates.

Drug properties of antisense oligonucleotides (ASOs) differ significantly from those of traditional small-molecule therapeutics. In this review, we focus on ASO disposition, mainly as characterized by distribution and biotransformation, of nonconjugated and conjugated ASOs. We introduce ASO chemistry to allow the following in-depth discussion on bioanalytical methods and determination of distribution and elimination kinetics at low concentrations over extended periods of time. The resulting quantitative data on the parent oligonucleotide, and the identification and quantification of formed metabolites define the disposition. Proper quantitative understanding of disposition is pivotal for nonclinical to clinical predictions, supports communication with health agencies, and increases the probability of delivering optimal ASO therapy to patients.

An oral antisense oligonucleotide for PCSK9 inhibition.

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.

Drug metabolism and pharmacokinetic strategies for oligonucleotide- and mRNA-based drug development.

Oligonucleotide and modified mRNA therapeutics have great potential to treat diseases that are currently challenging to cure and are expanding into global and chronic disease areas such as cancer and various cardiovascular diseases. Advanced drug delivery systems or ligand-drug conjugates are utilized to achieve 'the right dose to the right target' to benefit efficacy and safety in patients. Chemistry and ADME characteristics distinguish these therapeutics from small molecules. Understanding the scalability and translatability between species and compound properties is crucial for robust nonclinical PKPD predictions to support clinical study design. Although the field has been developing for three decades, there is still room for innovation but also a need for nonclinical regulatory guidance to address these new modalities.

Tesaglitazar, a PPARalpha/gamma agonist, induces interstitial mesenchymal cell DNA synthesis and fibrosarcomas in subcutaneous tissues in rats.

The development of the dual peroxisome proliferator-activated receptor (PPAR) alpha/gamma agonist tesaglitazar as an oral antidiabetic was recently discontinued. Here we present tumor data from a 2-year carcinogenicity study in rats given 0.3, 1, 3, and 10 micromol/kg tesaglitazar is presented with focus on the findings of subcutaneous fibrosarcomas. To investigate the mechanism for induction of fibrosarcomas, replicative DNA synthesis (immunohistochemical detection of BrdU-labeled cells) and expression of PPARgamma (immunohistochemistry and reverse transcription-polymerase chain reaction) in subcutaneous adipose tissues was assessed in rats administered 1 or 10 micromol/kg for 2 weeks or 3 months. Poorly differentiated subcutaneous mesenchymal sarcomas with a predominant spindle cell appearance occurred at the highest dose level of 10 micromol/kg in both sexes, and these tumors were diagnosed as fibrosarcomas. The 10-micromol/kg dose was at or above the maximum tolerated dose and caused considerable cardiovascular mortality. Tesaglitazar stimulated DNA synthesis mainly in subcutaneous interstitial mesenchymal cells. The percentage of BrdU-labeled interstitial cells was increased at 1 and 10 micromol/kg after 2 weeks. The increase in DNA synthesis was still significant at the end of the 12-week treatment at 10 mumol/kg, the dose producing fibrosarcoma. However, at 1 micromol/kg, a dose below the no-observed-effect level for fibrosarcoma, the level of DNA synthesis was similar to control levels at 12 weeks. Immunohistochemical analyses showed no detectable PPARgamma protein in the majority of BrdU-labeled interstitial mesenchymal cells in white and brown fat. This indicates that stimulation of DNA synthesis is not mediated via direct activation of PPARgamma in these cells. The results suggest that the induction of rat fibrosarcoma by tesaglitazar, at exposures 100-fold above the human therapeutic exposure, may involve proliferation of undifferentiated mesenchymal cells in subcutaneous tissues.

Effect of broad-spectrum matrix metalloproteinase inhibition on atherosclerotic plaque stability.

OBJECTIVE: Matrix metalloproteinases (MMPs) form a large family of enzymes that collectively can degrade all components of the extracellular matrix, and there is widespread interest in developing MMP inhibitors for the prevention of atherosclerotic plaque rupture. We have therefore investigated the effects of a broad-spectrum MMP inhibitor, RS-130830, on plaque development and stability. This compound inhibits a wide range of MMPs at concentrations below 20 nmol/L. METHODS: Apolipoprotein E knockout mice were fed a Western diet. Dietary administration of RS-130830 commenced at the same time as fat-feeding and continued for 8, 12, 26 or 36 weeks. To investigate the effect of RS-130830 on established plaques, mice were fed high-fat diet for 16 weeks before initiation of drug treatment and were terminated 20 weeks after this. RESULTS: Broad-spectrum MMP inhibition was associated with a significant increase in plaque area, but there was no change in the incidence of plaque rupture. There were unfavourable changes in phenotypic characteristics associated with plaque instability, such as an increased lipid content and decreased collagen content. CONCLUSIONS: These data suggest that broad-spectrum MMP inhibition RS-130830 does not have a beneficial effect on atherosclerosis in the apolipoprotein E knockout mouse model, and indicate that more selective compounds would be preferable.

Effects of Tenapanor on Cytochrome P450-Mediated Drug-Drug Interactions.

Tenapanor (RDX5791, AZD1722) is an inhibitor of sodium/hydrogen exchanger isoform 3 in development for the treatment of constipation-predominant irritable bowel syndrome and the treatment of hyperphosphatemia in patients with chronic kidney disease on dialysis. We aimed to investigate whether tenapanor inhibits or induces cytochrome P450s (CYPs). In vitro experiments assessing the potential of tenapanor to affect various CYPs indicated that it could inhibit CYP3A4/5 (IC50 0.4-0.7 µM). An open-label, phase 1 clinical study (NCT02140268) evaluated the pharmacokinetics of the CYP3A4 substrate midazolam when administered with and without tenapanor. Healthy volunteers received a single oral dose of midazolam 7.5 mg on day 1 followed by tenapanor 15 mg twice daily on days 2 to 15, with an additional single 7.5-mg midazolam dose coadministered on day 15. Midazolam exposure was similar whether it was administered alone or with tenapanor (geometric least-squares mean ratio [90%CI] for [midazolam + tenapanor]/midazolam: area under the concentration-time curve, 107% [101% to 113%]; Cmax 104% [89.6% to 122%]). Findings were similar for metabolites of midazolam. These results indicate that tenapanor 15 mg twice daily does not have a clinically relevant impact on CYP3A4 in humans and suggest that tenapanor can be coadministered with CYP3A4-metabolized drugs without affecting their exposure.