Animals and Dosing

Twelve-week-old male Wistar rats (N = 96; average weight of ~406 g) obtained from Charles River Labs (Raleigh, NC) were fed a low-protein (2.5%) and high-phosphate (0.92%) diet (Teklad Custom Diet, TD130126, Harlan Laboratories, Indianapolis, IN) for 1 week. Thereafter, 24 rats were maintained on the same diet to serve as nonuremic controls, and 72 rats were switched to the same base diet supplemented with 0.75% adenine (TD130127) for an additional 4 weeks to induce uremia and SHPT. Orally administered adenine is metabolized to 2,8-dihydroxyadenine, which precipitates and forms tubular crystals that injure the renal tissue. These 72 rats received etelcalcetide, vehicle, or paricalcitol treatment (n = 24 per group) within 24 h of adenine introduction. Etelcalcetide was formulated in vehicle (10 mM succinic acid, 0.85% NaCl, 0.9% benzyl alcohol, pH 4.5) and injected subcutaneously (SC) at 0.3 mg/kg once a day for 4 weeks. The SC route of administration was selected because of the length of the study and challenges associated with repeated i.v. dosing in animal studies. Subcutaneous administration of etelcalcetide has been shown to effectively decrease PTH and provides good drug exposure in animal studies [22]. Paricalcitol was injected intraperitoneally (IP) at 42 ng/kg three times per week for 4 weeks. The paricalcitol dose (42 ng/kg) was selected based on its nonsignificant effects on serum calcium while reducing PTH compared to higher doses (>42 ng/kg) [15]. The goal was to avoid the contribution of hypercalcemia induced by higher doses of paricalcitol to the development of vascular calcification. Twenty-four rats on adenine diet and 24 rats on adenine-free diet received vehicle by subcutaneous injection once a day for 4 weeks. Blood samples were collected through tail vein under isoflurane anesthesia before switching to adenine diet (predose) and prior to dosing at 2 and 4 weeks after the treatment. Rats were cared for in accordance with the Guide for the Care and Use of Laboratory Animals, 8th Edition [26].