Preclinical Mammalian Safety Studies of EPHARNA (DOPC Nanoliposomal EphA2-Targeted siRNA)

Abstract

To address the need for efficient and biocompatible delivery systems for systemic siRNA delivery, we developed 1,2-Dioleoyl-sn-Glycero-3-Phosphatidylcholine (DOPC) nanoliposomal EphA2-targeted therapeutic (EPHARNA). Here, we performed safety studies of EPHARNA in murine and primate models. Single dosing of EPHARNA was tested at 5 concentrations in mice (N = 15 per group) and groups were sacrificed on days 1, 14, and 28 for evaluation of clinical pathology and organ toxicity. Multiple dosing of EPHARNA was tested in mice and Rhesus macaques twice weekly at two dose levels in each model. Possible effects on hematologic parameters, serum chemistry, coagulation, and organ toxicity were assessed. Following single-dose EPHARNA administration to mice, no gross pathologic or dose-related microscopic findings were observed in either the acute (24 hours) or recovery (14 and 28 days) phases. The no-observed-adverse-effect level (NOAEL) for EPHARNA is considered >225 μg/kg when administered as a single injection intravenously in CD-1 mice. With twice weekly injection, EPHARNA appeared to stimulate a mild to moderate inflammatory response in a dose-related fashion. There appeared to be a mild hemolytic reaction in the female mice. In Rhesus macaques, minimal to moderate infiltration of mononuclear cells was found in some organs including the gastrointestinal tract, heart, and kidney. No differences attributed to EPHARNA were observed. These results demonstrate that EPHARNA is well tolerated at all doses tested. These data, combined with previously published in vivo validation studies, have led to an ongoing first-in-human phase I clinical trial (NCT01591356). Mol Cancer Ther; 16(6); 1114-23. ©2017 AACR.

Conflict of interest statement

Conflicts of Interest: Anil K. Sood and Gabriel Lopez-Berestein are co-inventors on a patent related to this work. Kirstin Barnhart is currently an employee at Abbvie Inc. The other authors declare no potential conflicts of interest.

©2017 American Association for Cancer Research.

Figures

Figure 1. Schematic of Dosing Experiments

Single dose effects were assessed by administering a dose of EPHARNA and groups of mice underwent necropsy at days +1, +14, or day +28 from EPHARNA dosing. To assess continuous dosing of EPHARNA, groups of mice were given EPHARNA twice weekly and underwent necropsy after 4 weeks of treatment. Non-human primates were given EPHARNA twice weekly for 9 administrations and underwent necropsy 43–45 days after the initial dosing.

Figure 2. In situ hybridization for anti-EphA2 siRNA in mouse spleen and brain

(A) Spleen, EPHARNA treated mouse, control probe; (B) Spleen, EPHARNA treated mouse, anti-siRNA probe; (C) CNS, EPHARNA treated mouse, control probe; (D) CNS, EPHARNA treated mouse, anti-siRNA probe; (E) Spleen, Control DOPC treated mouse, control probe (F) Spleen, control DOPC treated mouse, anti-siRNA probe; (G) CNS, control DOPC treated mouse, control probe; (H) CNS, control DOPC treated mouse, anti-siRNA probe

Figure 3

Immunologic parameters in non-human primates did not appear affected by EPHARNA administration.