Administration in non-human primates of escalating intravenous doses of targeted nanoparticles containing ribonucleotide reductase subunit M2 siRNA

Abstract

The results of administering escalating, i.v. doses of targeted nanoparticles containing a siRNA targeting the M2 subunit of ribonucleotide reductase to non-human primates are reported. The nanoparticles consist of a synthetic delivery system that uses a linear, cyclodextrin-containing polycation, transferrin (Tf) protein targeting ligand, and siRNA. When administered to cynomolgus monkeys at doses of 3 and 9 mg siRNA/kg, the nanoparticles are well tolerated. At 27 mg siRNA/kg, elevated levels of blood urea nitrogen and creatinine are observed that are indicative of kidney toxicity. Mild elevations in alanine amino transferase and aspartate transaminase at this dose level indicate that the liver is also affected to some extent. Analysis of complement factors does not reveal any changes that are clearly attributable to dosing with the nanoparticle formulation. Detection of increased IL-6 levels in all animals at 27 mg siRNA/kg and increased IFN-gamma in one animal indicate that this high dose level produces a mild immune response. Overall, no clinical signs of toxicity clearly attributable to treatment are observed. The multiple administrations spanning a period of 17-18 days enable assessment of antibody formation against the human Tf component of the formulation. Low titers of anti-Tf antibodies are detected, but this response is not associated with any manifestations of a hypersensitivity reaction upon readministration of the targeted nanoparticle. Taken together, the data presented show that multiple, systemic doses of targeted nanoparticles containing nonchemically modified siRNA can safely be administered to non-human primates.

Conflict of interest statement

Conflict of interest statement: M.E.D. is a consultant to and has stock in Calando Pharmaceuticals.

Figures

Fig. 1.

Schematic illustration of the delivery system (A) and its formulation with siRNA (B).

Fig. 2.

Overview of experimental protocol. Three female, non-naïve cynomolgus monkeys were treated with an siRNA-containing nanoparticle formulation. Formulations were freshly prepared on the day of treatment and had a final siRNA concentration of 1 mg/ml. A simple slow-push i.v. injection was used to administer the 3-mg/kg (with respect to siRNA) and 9-mg/kg doses. The large volume needed for the 27-mg/kg dose required administration of this dose by 2-h i.v. infusion; the 0-mg/kg dose (D5W only) was also delivered by infusion. The 0-, 3-, 9-, and 27-mg/kg doses were administered sequentially, with 2–3 days between each dose, as indicated. After the 27-mg/kg dosing, a washout period of 11–12 days was allowed before a final 3-mg/kg dose was given. Blood samples were drawn at the indicated times for analyses of complete blood counts, serum chemistry, coagulation parameters, complement factors, antibodies, cytokines, and PK.

Fig. 3.

Cytokines. At 6 h postdosing, blood was drawn from each animal, processed into plasma, and analyzed for the indicated cytokine levels via ELISA (IL-6 and IL-12) or multiplex assay (IFN-γ). Experimentally determined lower limits of quantification values were 12 ng/ml for IL-12, 11.9 pg/ml for IFN-γ, and 7.8 ng/ml for IL-6. Bars indicate averages, and error bars indicate standard deviations for each group of n = 3 measurements (one for each of the three animals).

Fig. 4.

Detection of monkey antibodies against human Tf. (A) Total antinanoparticle antibody titer was determined by ELISA on serum samples taken predosing (acclimation) and on day 18. (B) Serum samples (day 18) were incubated with human holo-Tf (1 or 100 μg/ml) or HSA (1 or 100 μg/ml) before antibody titer determination by ELISA. Incubation with HSA had a minimal effect on the ELISA signal, whereas there was a sharp dose-dependent reduction in the ELISA signal upon Tf incubation. This result supports the rationale that the antibodies generated specifically recognize the Tf moiety of the AD-PEG-Tf component of the nanoparticles.

Fig. 5.

PK of nanoparticles in monkey plasma. (A) Total plasma siRNA (ng/ml) was determined in monkey plasma as a function of time after dosing by hybridization-ligation assay. In all three animals, the t = 5 min plasma level scales with dose. Points represent the mean value for three animals, and error bars denote standard deviations. For all measurements below the lower limit of quantification (LLOQ), a value equal to half of the LLOQ (LLOQ/2) is reported. (Note: As discussed in Results and Discussion, there is reason to believe these values are underestimates of true circulating nanoparticle concentrations.) (B) Total plasma siRNA (ng/ml) concentrations at t = 5 min postdose. There is no clear difference observed between the initial (days 3 or 4) and final (day 21) 3-mg/ml dose across the three animals, suggesting that there may not be a reduction in half-life resulting from anti-Tf antibodies.