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).