Cynomolgus Monkey Radiolabel Biodistribution Study
The cynomolgus monkey distribution study was conducted in accordance with SOPs and the protocol as approved by Eli Lilly and Company and in compliance with the requirements contained in the MPI Research (now Charles River, Mattawan, MI) Radioactive Materials License Number 21-11315-02, and all applicable regulations issued by the Nuclear Regulatory Commission (NRC). Briefly, the non-radiolabeled BsAb-1 and BsAb-2 were conjugated to diethylene triamine pentaacetic acid (DTPA) and radiolabeled with 111In at MPI Research, Inc. to target a low specific activity. Additionally, non-radiolabeled BsAb-1 and BsAb-2 were radiolabeled with 125I at MPI Research, Inc. to target the same low specific activity as the111In-DTPA. To prepare the dosing formulations, the equal amounts of 111In-DTPA and 125I radiolabeled conjugated test article were combined with the required volume of vehicle, phosphate buffered saline (PBS) and mixed to prepare the dosing formulation containing both conjugates. A dual-isotope gamma counter setting was used for all formulation measurements. The dosing formulations were prepared on the day prior to administration.
The dosing formulation was administered once via IV injection. The actual amount administered was determined by weighing the dose syringe before and after dose administration. Each dosing syringe was also counted in the dose calibrator prior to and after dosing. Doses were labeled to a target dose level of 5 mg/kg (~1 mCi/kg split equally between the two isotopes). The final dose was based on the A280 concentration, and effort was made to administer approximately the same mass and volume of dosing formulation to each group. Individual doses were based on body weights collected on Day -1. A dose wipe of the injection site was collected into a plastic container and was retained at room temperature prior to analysis by LSC to determine residual radioactivity. The radioactivity recovered was subtracted from the administered amount to give the actual radioactive dose administered.
Blood samples (~200 to 300 µL) were collected from all animals via the femoral vein into tubes containing sodium citrate. All samples were analyzed using the gamma counter to determine residual radioactivity. Blood samples were collected at 0.083, 1, 6, 24, 48, 96 and 168 hours post-dose (cohorts of three animals per group). The total weight of each blood sample was recorded and analyzed for radioactivity using the gamma counter. The %ID/g values were corrected for radioactive decay over time. Following evaluation of the samples for hematocrit values, the samples were processed to plasma, and the total weight of each packed cell fraction and plasma sample was recorded. Packed cell fraction and plasma samples were analyzed for total radioactivity via the gamma counter. A dual-isotope gamma counter setting was used for all sample measurements.
Liver biopsies were collected from all animals (cohorts of three animals per group) at 1 hour post administration for immunofluorescence analyses (described below). The adrenal gland, bladder (urinary), bone (femur), bone marrow (femur), brain, muscle (gastrocnemius, both quadriceps, and scapular region), heart, kidney, large intestine/cecum with contents, liver, lung, lymph nodes (mesenteric), pancreas, skin (ventral and upper and lower dorsal), small intestine with contents, spleen, stomach with contents, testes, thymus, thyroid, and fat pad were collected from one animal per timepoint per group at 48, 96 and 168 hours postdose. The tissues were rinsed with 0.9% saline and blotted dry as appropriate. All tissues were weighed and collected into a plastic container at room temperature and maintained under this condition until analyzed for total radioactivity via the gamma counter. Urine was collected from one animal per timepoint per group from 0 to 48, 0 to 96 and 0 to 168 hours postdose. A dual-isotope gamma counter setting was used for all sample measurements. Similar to the blood samples, the %ID/g values for the tissues and urine were corrected for radioactive decay over time.