Chemical and mechanistic toxicology evaluation of exon skipping phosphorodiamidate morpholino oligomers in mdx mice.

AVI-4658 is a phosphorodiamidate morpholino oligomer (PMO) designed to induce skipping of dystrophin exon 51 and restore its expression in patients with Duchenne muscular dystrophy (DMD). Preclinically, restoration of dystrophin in the dystrophic mdx mouse model requires skipping of exon 23, achieved with the mouse-specific PMO, AVI-4225. Herein, we report the potential toxicological consequences of exon skipping and dystrophin restoration in mdx mice using AVI-4225. We also evaluated the toxicological effects of AVI-4658 in both mdx and wild-type mice. In both studies, animals were dosed once weekly for 12 weeks up to the maximum feasible dose of 960 mg/kg per injection. Both AVI-4658 and AVI-4225 were well-tolerated at all doses. Findings in AVI-4225-treated animals were generally limited to mild renal tubular basophilia/vacuolation, without any significant changes in renal function and with evidence of reversing. No toxicity associated with the mechanism of action of AVI-4225 in a dystrophic animal was observed.

Repeat-dose toxicology evaluation in cynomolgus monkeys of AVI-4658, a phosphorodiamidate morpholino oligomer (PMO) drug for the treatment of duchenne muscular dystrophy.

AVI-4658 is a phosphorodiamidate morpholino oligomer (PMO) drug designed to restore dystrophin expression in a subset of patients with Duchenne muscular dystrophy (DMD). Previous reports demonstrated this clinical proof-of-principle in patients with DMD following intramuscular injection of AVI-4658. This preclinical study evaluated the toxicity and toxicokinetic profile of AVI-4658 when administered either intravenously (IV) or subcutaneously (SC) to cynomolgus monkeys once weekly over 12 weeks, at doses up to the maximum feasible dose of 320 mg/kg per injection. No drug-related effects were noted on survival, clinical observations, body weight, food consumption, opthalmoscopic or electrocardiographic evaluations, hematology, clinical chemistry, urinalysis, organ weights, and macroscopic evaluations. Drug-related microscopic renal effects were dose-dependent, apparently reversible, and included basophilic granules (minimal), basophilic tubules (minimal to moderate), and tubular vacuolation (minimal to mild). These data establish the tolerability of AVI-4658 at doses up to and including the maximum feasible dose of 320 mg/kg by IV bolus or SC injection.

Use of mixed-effect models and tolerance limits to evaluate control cynomolgus monkey body weight change and variability during preclinical toxicology studies.

Cynomolgus monkeys are an important and widely used species in preclinical toxicology studies. During the in-life phase of study, body weight effects may be indicative of toxicity; however, trends in body weight and body weight variability are often difficult to interpret due to small sample size and/or inter- and intra-animal variability. The present analysis utilizes mixed-effect modelling, which incorporates random and fixed effects into linear regression models, to evaluate control monkey body weight trends and variability relative to baseline (initial) weight and study duration. The primary aim of this analysis is to evaluate whether mixed-effect model based tolerance limits can aide in determining whether apparent test article-related changes in body weight deviate more than the predicted variability defined by the model tolerance limits. The models for this study are based on vehicle control animal body weight data from the following studies: 1-month (20 studies, 198 animals), 3-month (19 studies, 180 animals), and 9-month (17 studies, 182 animals). The analysis presented herein provides the framework for evaluating control monkey body weight change in studies with small sample size, and anticipated control monkey body weight change relative to gender and study duration.

14-day repeat-dose oral toxicity evaluation of oxazyme in rats and dogs.

Oxazyme (OC4) is an orally administered formulation that has as an active component a recombinant mutant form of Bacillus subtilis oxalate decarboxylase (OxDC) enzyme C383S, designed to degrade dietary oxalate in the stomach. Fourteen-day repeat-dose studies were conducted in rats and dogs to evaluate toxicity and determine a no observed adverse effect level (NOAEL). Animals were administered OC4 by oral gavage twice daily for 14 consecutive days. Reversibility, progression, and delayed appearance of any observed changes were evaluated in a subset of animals that underwent a recovery of 7 days following 14 days of control or test-article. There were no test-article-related adverse effects or deaths in either species. Results indicate that the NOAEL under the conditions used in the studies was 720.8 mg/kg/d in rats and 187.2 mg/kg/d in dogs, the high dose tested in each species.