Cynomolgus monkey pharmacokinetic study, bioanalytical assays and
pharmacokinetic data analysis
A cynomolgus monkey pharmacokinetic study for the BsAbs was conducted in
accordance with Standard Operating Procedures (SOPs) and protocols as
approved by Eli Lilly and Company and in compliance with the
requirements of Covance Laboratories (Madison, WI).
The cynomolgus monkey pharmacokinetic study was performed with male
cynomolgus monkeys (2.3-3.2 kg). Three monkeys were assigned to each
study group and all animals received a single intravenous (IV) bolus
dose of either BsAb-1 or BsAb-2 dissolved in PBS (pH
~7.4) at 5.0 mg/kg. Each animal had blood samples
collected via a femoral vein. Blood samples were collected at predose,
1, 6, 12, 24, 48, 72, 96, 168 and 240 hours after administration of the
dose. The blood samples were collected into tubes containing sodium
citrate anticoagulant maintained in chilled cyroracks and centrifuged to
obtain plasma.
Concentrations of BsAb-1 or BsAb-2 in cynomolgus monkey plasma were
determined using anti-human IgG ELISAs for each of the molecules. In
brief, each well of an Immulon® 4HBX microtiter plate (Thermo
Scientific™, Waltham, MA) was coated with either goat anti-human IgG
(Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) at 4°C
overnight. After washing and blocking, all other standards, control
samples, and study samples were added to the plates then incubated for
one hour at room temperature. After washing, the bound molecules were
detected with an HRP-conjugated mouse anti-human IgG (Fc) antibody
(Southern Biotechnology Associates, Birmingham, AL) by TMB Microwell
Peroxidase Substrate System (KPL, Gaithersburg, MD) for a colorimetric
response. Plates were read at 450-493 nm with a reference of 630nm.
Concentrations from plasma samples were determined from a standard curve
prepared with known amounts of BsAb-1 or BsAb-2 in the appropriate
cynomolgus monkey matrix using a 4/5-parameter algorithm. The standard
curve range for BsAb-1 or BsAb-2 was from 3.91 to 500ng/mL, and the
lower limit of quantitation (LLOQ) was defined as 25 ng/mL. LC/MS was
used to determine the intact molecule.
Plasma concentration-time data following IV administration was described
using a model-independent method according to the statistical moment
theory using the either WinNonlin® Professional 6.3 or Phoenix®
WinNonlin® software package (Pharsight, A Certara™ Company, St. Louis,
MO). The parameters calculated included the maximum serum concentration
(Cmax), area under the curve (AUC0-∞),
clearance (CL), and elimination half-life (t1/2).