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.