Targeted tissue delivery of RNA therapeutics using antibody-oligonucleotide conjugates (AOCs).

Although targeting TfR1 to deliver oligonucleotides to skeletal muscle has been demonstrated in rodents, effectiveness and pharmacokinetic/pharmacodynamic (PKPD) properties remained unknown in higher species. We developed antibody-oligonucleotide conjugates (AOCs) towards mice or monkeys utilizing anti-TfR1 monoclonal antibodies (αTfR1) conjugated to various classes of oligonucleotides (siRNA, ASOs and PMOs). αTfR1 AOCs delivered oligonucleotides to muscle tissue in both species.

Early-Stage Identification and Avoidance of Antisense Oligonucleotides Causing Species-Specific Inflammatory Responses in Human Volunteer Peripheral Blood Mononuclear Cells.

A human peripheral blood mononuclear cell (PBMC)-based assay was developed to identify antisense oligonucleotide (ASO) with the potential to activate a cellular innate immune response outside of an acceptable level. The development of this assay was initiated when ISIS 353512 targeting the messenger ribonucleic acid for human C-reactive protein (CRP) was tested in a phase I clinical trial, in which healthy human volunteers unexpectedly experienced increases in interleukin-6 (IL-6) and CRP. This level of immune stimulation was not anticipated following rodent and nonhuman primate safety studies in which no evidence of exaggerated proinflammatory effects were observed.

Glucagon-receptor-antagonism-mediated β-cell regeneration as an effective anti-diabetic therapy.

Type 1 diabetes mellitus (T1D) is a chronic disease with potentially severe complications, and β-cell deficiency underlies this disease. Despite active research, no therapy to date has been able to induce β-cell regeneration in humans. Here, we discover the β-cell regenerative effects of glucagon receptor antibody (anti-GcgR).

Inhibition of Complement Factor 3 in Geographic Atrophy with NGM621: Phase 1 Dose-Escalation Study Results.

Purpose: To evaluate the safety and tolerability of single and multiple intravitreal injections of NGM621 in patients with geographic atrophy (GA) and to characterize the pharmacokinetics and immunogenic potential.

Design: Multicenter, open-label, single- and multiple-dose phase 1 study.

Methods: Fifteen patients enrolled at 4 sites in the United States.

Safety Assessment of Formulation Vehicles Following Intravitreal Administration in Rabbits.

Purpose: Evaluate 21 formulation vehicles administered to rabbits after intravitreal injection for tolerability and safety.

Methods: Forty-two Dutch Belted rabbits were anesthetized, and the eyes received a single intravitreal injection of the excipient formulation. Clinical signs and ocular irritation responses were recorded twice daily for 7 days and microscopic evaluation of the eyes, optic nerve, and eyelids was completed at 1-week post treatment.

The Distinct and Cooperative Roles of Toll-Like Receptor 9 and Receptor for Advanced Glycation End Products in Modulating In Vivo Inflammatory Responses to Select CpG and Non-CpG Oligonucleotides.

Antisense oligonucleotides (ASOs) are widely accepted therapeutic agents that suppress RNA transcription. While the majority of ASOs are well tolerated in vivo, few sequences trigger inflammatory responses in absence of conventional CpG motifs. In this study, we identified non-CpG oligodeoxy-nucleotide (ODN) capable of triggering an inflammatory response resulting in B cell and macrophage activation in a MyD88- and TLR9-dependent manner.

Assessment of the Effects of 2'-Methoxyethyl Antisense Oligonucleotides on Platelet Count in Cynomolgus Nonhuman Primates.

Decreases in platelet (PLT) counts observed in nonhuman primates (NHPs) given 2'-O-methoxyethyl modified antisense inhibitors (2'-MOE ASOs) have been reported, but the incidence and severity of the change vary considerably between sequences, studies, and animals. This article will broadly illustrate the spectrum of effects on PLT count in NHPs. From queries of an NHP safety database representing over 102 independent 2'-MOE ASOs, from 61 studies and >2200 NHPs, two patterns of PLT changes emerged.

Integrated Safety Assessment of 2'-O-Methoxyethyl Chimeric Antisense Oligonucleotides in NonHuman Primates and Healthy Human Volunteers.

The common chemical and biological properties of antisense oligonucleotides provide the opportunity to identify and characterize chemical class effects across species. The chemical class that has proven to be the most versatile and best characterized is the 2'-O-methoxyethyl chimeric antisense oligonucleotides. In this report we present an integrated safety assessment of data obtained from controlled dose-ranging studies in nonhuman primates (macaques) and healthy human volunteers for 12 unique 2'-O-methoxyethyl chimeric antisense oligonucleotides.

Hepatotoxicity of high affinity gapmer antisense oligonucleotides is mediated by RNase H1 dependent promiscuous reduction of very long pre-mRNA transcripts.

High affinity antisense oligonucleotides (ASOs) containing bicylic modifications (BNA) such as locked nucleic acid (LNA) designed to induce target RNA cleavage have been shown to have enhanced potency along with a higher propensity to cause hepatotoxicity. In order to understand the mechanism of this hepatotoxicity, transcriptional profiles were collected from the livers of mice treated with a panel of highly efficacious hepatotoxic or non-hepatotoxic LNA ASOs. We observed highly selective transcript knockdown in mice treated with non-hepatotoxic LNA ASOs, while the levels of many unintended transcripts were reduced in mice treated with hepatotoxic LNA ASOs.

Identification and characterization of modified antisense oligonucleotides targeting DMPK in mice and nonhuman primates for the treatment of myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1) is the most common form of muscular dystrophy in adults. DM1 is caused by an expanded CTG repeat in the 3'-untranslated region of DMPK, the gene encoding dystrophia myotonica protein kinase (DMPK). Antisense oligonucleotides (ASOs) containing 2',4'-constrained ethyl-modified (cEt) residues exhibit a significantly increased RNA binding affinity and in vivo potency relative to those modified with other 2'-chemistries, which we speculated could translate to enhanced activity in extrahepatic tissues, such as muscle.

Mechanistic understanding for the greater sensitivity of monkeys to antisense oligonucleotide-mediated complement activation compared with humans.

Differences in sensitivity of monkeys and humans to antisense oligonucleotide (ASO)-induced complement alternative pathway (AP) activation were evaluated in monkeys, humans, and in serum using biochemical assays. Transient AP activation was evident in monkeys at higher doses of two 2'-O-methoxyethyl (2'-MOE) ASOs (ISIS 426115 and ISIS 183750). No evidence of AP activation was observed in humans for either ASO, even with plasma ASO concentrations that reached the threshold for activation in monkeys.

Recommendations for safety pharmacology evaluations of oligonucleotide-based therapeutics.

This document was prepared by the Safety Pharmacology Subcommittee of the Oligonucleotide Safety Working Group (OSWG), a group of industry and regulatory scientists involved in the development and regulation of therapeutic oligonucleotides. The mission of the Subcommittee was to develop scientific recommendations for the industry regarding the appropriate scope and strategies for safety pharmacology evaluations of oligonucleotides (ONs). These recommendations are the consensus opinion of the Subcommittee and do not necessarily reflect the current expectations of regulatory authorities.

Utilization of causal reasoning of hepatic gene expression in rats to identify molecular pathways of idiosyncratic drug-induced liver injury.

Drug-induced liver injury (DILI) represents a leading cause of acute liver failure. Although DILI can be discovered in preclinical animal toxicology studies and/or early clinical trials, some human DILI reactions, termed idiosyncratic DILI (IDILI), are less predictable, occur in a small number of individuals, and do not follow a clear dose-response relationship. The emergence of IDILI poses a critical health challenge for patients and a financial challenge for the pharmaceutical industry.

Antisense inhibition of coagulation factor XI prolongs APTT without increased bleeding risk in cynomolgus monkeys.

A strategy to produce sufficient anticoagulant properties with reduced risk of bleeding may be possible through inhibition of factor XI (FXI), a component of the intrinsic coagulation cascade. The objective of this work was to determine the safety profile of ISIS 416858, a 2'-methoxyethoxy (2'-MOE) antisense oligonucleotide inhibitor of FXI, with focus on assessment of bleeding risk. Cynomolgus monkeys administered ISIS 416858 (4, 8, 12, and 40 mg/kg/wk, subcutaneous) for up to 13 weeks produced a dose-dependent reduction in FXI (mRNA in liver and plasma activity) and a concomitant increase in activated partial thromboplastin time (APTT).

Neutrophil-cytokine interactions in a rat model of sulindac-induced idiosyncratic liver injury.

Previous studies indicated that lipopolysaccharide (LPS) interacts with the nonsteroidal anti-inflammatory drug sulindac (SLD) to produce liver injury in rats. In the present study, the mechanism of SLD/LPS-induced liver injury was further investigated. Accumulation of polymorphonuclear neutrophils (PMNs) in the liver was greater in SLD/LPS-cotreated rats compared to those treated with SLD or LPS alone.

Effect of PF-04217329 a prodrug of a selective prostaglandin EP(2) agonist on intraocular pressure in preclinical models of glaucoma.

Better control of intraocular pressure (IOP) is the most effective way to preserve visual field function in glaucomatous patients. While prostaglandin FP analogs are leading the therapeutic intervention for glaucoma, new target classes also are being identified with new lead compounds being developed for IOP reduction. One target class currently being investigated includes the prostaglandin EP receptor agonists.

Trovafloxacin, a fluoroquinolone antibiotic with hepatotoxic potential, causes mitochondrial peroxynitrite stress in a mouse model of underlying mitochondrial dysfunction.

Trovafloxacin (TVX) is a fluoroquinolone antibiotic whose therapeutic use was severely restricted due to an unacceptable risk of idiosyncratic liver injury. Oxidative stress and mitochondrial injury have been implicated in fluoroquinolone toxicity, but the mechanisms underlying liver injury are poorly understood. Because TVX-induced hepatotoxicity cannot be modeled in normal healthy rodents, we asked whether an underlying genetic defect (heterozygous deficiency in mitochondrial superoxide dismutase, Sod2) might aggravate TVX-induced mitochondrial adverse effects.

Oxidative stress is important in the pathogenesis of liver injury induced by sulindac and lipopolysaccharide cotreatment.

Among currently prescribed nonsteroidal anti-inflammatory drugs, sulindac (SLD) is associated with the greatest incidence of idiosyncratic hepatotoxicity in humans. Previously, an animal model of SLD-induced idiosyncratic hepatotoxicity was developed by cotreating rats with a nonhepatotoxic dose of LPS. Tumor necrosis factor-alpha (TNF) was found to be critically important to the pathogenesis.

PD0325901, a mitogen-activated protein kinase kinase inhibitor, produces ocular toxicity in a rabbit animal model of retinal vein occlusion.

Objective: PD0325901, a selective inhibitor of mitogen-activated protein kinase kinase (MEK), was associated with the occurrence of ocular retinal vein occlusion (RVO) during clinical trials in patients with solid tumors. As previous animal safety studies in rats and dogs did not identify the eye as a target organ of toxicity, this work was conducted to develop a rabbit model of ocular toxicity with PD0325901.

Methods: Dutch-Belted rabbits were administered a single intravitreal injection of PD0325901 (0.

Sulindac metabolism and synergy with tumor necrosis factor-alpha in a drug-inflammation interaction model of idiosyncratic liver injury.

Sulindac (SLD) is a nonsteroidal anti-inflammatory drug (NSAID) that has been associated with a greater incidence of idiosyncratic hepatotoxicity in human patients than other NSAIDs. In previous studies, cotreatment of rats with SLD and a modestly inflammatory dose of lipopolysaccharide (LPS) led to liver injury, whereas neither SLD nor LPS alone caused liver damage. In studies presented here, further investigation of this animal model revealed that the concentration of tumor necrosis factor-alpha (TNF-alpha) in plasma was significantly increased by LPS at 1 h, and SLD enhanced this response.

L-Carnosine: multifunctional dipeptide buffer for sustained-duration topical ophthalmic formulations.

Objectives: The use of l-carnosine as an excipient in topical ophthalmic formulations containing gellan gum, a carbohydrate polymer with in-situ gelling properties upon mixing with mammalian tear fluid, was developed as a novel platform to extend precorneal duration. Specific utilisation of l-carnosine as a buffer in gellan gum carrying vehicles was characterised.

Methods: Buffer capacity was evaluated using 7.

Underlying mitochondrial dysfunction triggers flutamide-induced oxidative liver injury in a mouse model of idiosyncratic drug toxicity.

Flutamide, a widely used nonsteroidal anti-androgen, but not its bioisostere bicalutamide, has been associated with idiosyncratic drug-induced liver injury. Although the susceptibility factors are unknown, mitochondrial injury has emerged as a putative hazard of flutamide. To explore the role of mitochondrial sensitization in flutamide hepatotoxicity, we determined the effects of superimposed drug stress in a murine model of underlying mitochondrial abnormalities.

Hepatotoxic interaction of sulindac with lipopolysaccharide: role of the hemostatic system.

Sulindac (SLD) is a nonsteroidal anti-inflammatory drug (NSAID) that has been associated with a greater incidence of idiosyncratic hepatotoxicity in human patients than other NSAIDs. One hypothesis regarding idiosyncratic adverse drug reactions is that interaction of a drug with a modest inflammatory episode precipitates liver injury. In this study, we tested the hypothesis that lipopolysaccharide (LPS) interacts with SLD to cause liver injury in rats.

CpG and Non-CpG oligodeoxynucleotides induce differential proinflammatory gene expression profiles in liver and peripheral blood leukocytes in mice.

Proinflammatory effects caused by oligodeoxynucleotides (ODN) include cytokine production, splenomegaly and infiltration of mononuclear cells into tissues. Presence of one or more CpG motifs in an ODN sequence confers potency for proinflammatory properties. The objective of this research was to characterize the proinflammatory effects produced by CpG containing ODN as compared to non-CpG ODN using gene array analysis.