Pharmacology of a central nervous system delivered 2'-O-methoxyethyl-modified survival of motor neuron splicing oligonucleotide in mice and nonhuman primates.

Spinal muscular atrophy (SMA) is a debilitating neuromuscular disease caused by the loss of survival of motor neuron (SMN) protein. Previously, we demonstrated that ISIS 396443, an antisense oligonucleotide (ASO) targeted to the SMN2 pre-mRNA, is a potent inducer of SMN2 exon 7 inclusion and SMN protein expression, and improves function and survival of mild and severe SMA mouse models. Here, we demonstrate that ISIS 396443 is the most potent ASO in central nervous system (CNS) tissues of adult mice, compared with several other chemically modified ASOs. We evaluated methods of ISIS 396443 delivery to the CNS and characterized its pharmacokinetics and pharmacodynamics in rodents and nonhuman primates (NHPs). Intracerebroventricular bolus injection is a more efficient method of delivering ISIS 396443 to the CNS of rodents, compared with i.c.v. infusion. For both methods of delivery, the duration of ISIS 396443-mediated SMN2 splicing correction is long lasting, with maximal effects still observed 6 months after treatment discontinuation. Administration of ISIS 396443 to the CNS of NHPs by a single intrathecal bolus injection results in widespread distribution throughout the spinal cord. Based upon these preclinical studies, we have advanced ISIS 396443 into clinical development.

Local and systemic tolerability of a 2'O-methoxyethyl antisense oligonucleotide targeting interleukin-4 receptor-α delivery by inhalation in mouse and monkey.

Antisense oligonucleotides (ASOs) bind and facilitate degradation of RNA and inhibit protein expression in pathways not easily targeted with small molecules or antibodies. Interleukin (IL)-4 and IL-13 potentiate signaling through the shared IL-4 receptor-α (IL-4Rα) subunit of their receptors. ASO targeting of IL-4Rα mRNA in a mouse model of asthma led to attenuation of airway hyperactivity, demonstrating potential benefit in asthma patients. This study focused on tolerability of inhaled IL-4Rα-targeting ASOs. Toxicity studies were performed with mouse- (ISIS 23189) and human-specific (ISIS 369645) sequences administered by inhalation. Four week (monkey) or 13 week (mouse) repeat doses at levels of up to 15 mg/kg/exposure (exp) and 50 mg/kg/exp, respectively, demonstrated dose-dependent effects limited to increases in macrophage size and number in lung and tracheobronchial lymph nodes. The changes were largely non-specific, reflecting adaptive responses that occur during active exposure and deposition of ASO and other material in the lung. Reversibility was observed at a rate consistent with the kinetics of tissue clearance of ASO. Systemic bioavailability was minimal, and no systemic toxicity was observed at exposure levels appreciably above pharmacological doses and doses proposed for clinical trials.

A Semi-Mechanistic Population Pharmacokinetic Model of Nusinersen: An Antisense Oligonucleotide for the Treatment of Spinal Muscular Atrophy.

A pharmacokinetic (PK) model was developed for nusinersen, an antisense oligonucleotide (ASO) that is the first approved treatment for spinal muscular atrophy (SMA). The model was built with data from 92 nonhuman primates (NHPs) following intrathecal doses (0.3-7 mg) and characterized the PK in cerebrospinal fluid (CSF), plasma, total spinal cord, brain, and pons. The estimated volumes were 13.6, 937, 4.5, 53.8, and 2.11 mL, respectively. Global sensitivity analysis demonstrated that the CSF-to-plasma drug distribution rate (0.09 hour-1 ) is a major determinant of the maximum nusinersen concentration in central nervous system (CNS) tissues. Physiological age-based and body weight-based allometric scaling was implemented with exponent values of -0.08 and 1 for the rate constants and the volume of distribution, respectively. Simulations of the scaled model were in agreement with clinical observations from 52 pediatric phase I PK profiles. The developed model can be used to guide the design of clinical trials with ASOs.

Control of terrestrial animal rabies in Anne Arundel County, Maryland, after oral vaccination of raccoons (1998-2007).

OBJECTIVE: To evaluate the effectiveness of an oral rabies vaccination (ORV) project conducted from 1998 through 2007 in Anne Arundel County, Md, for the control of rabies in terrestrial animals. DESIGN: Retrospective analysis of surveillance data (1997 through 2007). ANIMALS: Free-ranging raccoons (Procyon lotor) and other terrestrial mammals. PROCEDURES: Vaccinia-rabies glycoprotein recombinant virus oral rabies vaccine-bait units were distributed annually by aircraft and ground teams targeting free-ranging raccoons. Approximately 2 to 4 weeks following the vaccine-bait placement, raccoons were live trapped, sedated, processed, and then released. Serologic samples were tested for the presence of rabies virus-neutralizing antibodies (RVNAs). Bait acceptance was estimated by analysis of tetracycline biomarking of sampled teeth. Rabies incidence was determined by the passive identification of rabid terrestrial animals. RESULTS: The incidence of rabies in terrestrial animals decreased 92% between 1997 (the year prior to the start of the ORV project) and 2007. The mean RVNA prevalence across all years was 33% among trapped raccoons in areas baited with a fish meal polymer bait type, whereas the mean bait acceptance was 30%. Adult raccoons had a seropositivity rate twice that of juvenile raccoons, whereas the bait acceptance rate between adults and juveniles did not differ significantly. For areas baited with a coated sachet bait, adults and juveniles had the same seroprevalence. Juveniles had better seroprevalence when the annual campaign started in September and October, compared with August. CONCLUSIONS AND CLINICAL RELEVANCE: The ORV project contributed to a significant decrease in annual incidence of terrestrial animal rabies in Anne Arundel County, Md, during the 10-year project period. For fish meal polymer baits, juvenile raccoons accessed bait at the same rate as adult raccoons but had a significantly lower prevalence of RVNAs. For coated sachet baits, seroprevalence was the same in both age groups. The time of year the bait distribution occurred and the bait type used may be partial explanations for the difference in RVNA seroprevalence between adults and juvenile raccoons.

Renal uptake and tolerability of a 2'-O-methoxyethyl modified antisense oligonucleotide (ISIS 113715) in monkey.

The primary target organ for uptake of systemically administered phosphorothioate oligonucleotides is the kidney cortex and the proximal tubular epithelium in particular. To determine the effect of oligonucleotide uptake on renal function, a detailed renal physiology study was performed in cynomolgus monkeys treated with 10-40 mg/kg/week ISIS 113715 for 4 weeks. The concentrations of oligonucleotide in the kidney cortex ranged from 1400 to 2600 µg/g. These concentrations were associated with histologic changes in proximal tubular epithelial cells that ranged from the appearance of cytoplasmic basophilic granules to atrophic and degenerative changes at higher concentrations. However, there were no renal functional abnormalities as determined by the typical measurements of blood urea nitrogen, serum creatinine, creatinine clearance, or urine specific gravity. Nor were there changes in glomerular filtration rate, or renal blood flow. Specific urinary markers of tubular epithelial cell damage, such as N-acetyl-glucosaminidase, and α-glutathione-s-transferase were not affected. Tubular function was further evaluated by monitoring the urinary excretion of amino acids, ß(2)-microglobulin, or glucose. Renal function was challenged by administering a glucose load and by examining concentrating ability after a 4-h water deprivation. Neither challenge produced any evidence of change in renal function. The only change observed was a low incidence of increased urine protein/creatinine ratio in monkeys treated with ≥40 mg/kg/week which was rapidly reversible. Collectively, these data indicate that ISIS 113715-uptake by the proximal tubular epithelium has little or no effect on renal function at concentrations of 2600 µg/g.

An in vivo approach to structure activity relationship analysis of peptide ligands.

PURPOSE: The goals in this study were several-fold. First, to optimize the in vivo phage display methodology by incorporating phage pharmacokinetic properties, to isolate peptides that target the brain microvasculature, and then to build focused libraries to obtain structure activity relationship information in vivo to identify the optimal targeting motif. MATERIALS AND METHODS: The blood pharmacokinetics of filamentous and T7 phage were evaluated to choose the optimal platform. A randomized peptide library with a motif CX(10)C was constructed in T7 phage and used for in vivo panning. Focused peptide libraries around each structural element of the brain-specific peptide were constructed to perform kinetic structure activity relationship (kSAR) analysis in vivo. To determine potential function, sepsis was induced in mice by LPS administration and four hours later the effect of GST-peptide on adhesion of rhodamine-labelled lymphocytes or CFDA-labelled platelets to pial microvasculature was observed by intravital microscopy. RESULTS: The blood phamacokinetics of T7 was rapid (half-life of 12 min) which aids the clearance of non-specific phage. In vivo panning in brain enriched for isolates expressing the motif CAGALCY. Kinetic analysis of focused libraries built around each structural element of the peptide provided for rapid pharmacophore mapping. The computer modeling data suggested the peptide showed similarities to peptide mimetics of adhesion molecule ligands. GST-CAGALCY but not GST control protein was able to inhibit the rolling and adhesion of labeled platelets to inflamed pial vasculature. GST-CAGALCY had no effect on lymphocyte adhesion. CONCLUSIONS: Incorporating normal blood phamacokinetics of T7 phage into in vivo phage display improves the ability to recover targeting peptide motifs and allows effective lead optimization by kSAR. This approach led to the isolation of a brain-specific peptide, CAGALCY, which appears to function as an effective antagonist of platelet adhesion to activated pial microvasculature.