PK sampling
PK parameters of INM004 levels were determined in all subjects receiving
at least one dose of the medication. In stage I the participants were
hospitalized for 24 hours after the infusion and PK samples were
obtained at the following times: 0, 15, 30, 45, 60, 90, 120, 150, 180,
210, 240, 270, 300, 360, and 480 min, and at 12, 24, 48, and 72 hours
after infusion. A baseline and final laboratory tests for immunogenicity
were obtained at day 0 and 30, respectively. Ambulatory PK samples,
clinical visits, and safety laboratories at days 3, 4, 5, and 30 after
infusion were scheduled. During stage II, the participants received
three repeated doses of 4 mg.kg-1 every 24 hours during 3 days. The
volunteers stayed hospitalized for 24 hours after the last infusion. PK
samples were obtained at the following times: 0, 30, 60, 120, 180, 240,
360, and 480 min, and at 12 and 24 hours after first infusion; at 60,
120, 360 min, and 12 and 24 hours after the second infusion; and at 60,
120, 360 min, and 12, 24, 48, 72, 96 and 120 hours, and at day 30 after
the third infusion. Ambulatory visits and safety laboratory tests were
identical to those scheduled in stage I.
Quantification of the F(ab´)2 equine polyclonal antibody
concentrations in plasma was performed by Enzyme-Linked Immunosorbent
Assay (ELISA) developed for preclinical studies of INM004 and validated
for human plasma samples11. Blood samples (5 ml) for
PK analyses were collected by venipuncture, and the results analyzed by
non-compartmental (NCA) PK methods. The fixed main effects treatment,
period and sequence were analyzed for the PK parameters of interest
[Cmax, AUC (0-t) and AUC (0-Inf)]. The AUC was
obtained from the graph of the drug plasma levels over time for each
subject. The 90% confidence interval limits for PK were calculated.
Cmax and Tmax were estimated directly
from the curve with interpolation. AUC (0-inf) and AUC (0-t) were
calculated using the trapezoidal rule based on the individual
concentrations observed. The elimination half-time (t½) was calculated
as Ln (2) / λz, where λz was estimated from a log-linear regression of
the last 3 to 5 points of the curve, after reaching plateau.
Coefficients of variation were calculated for Cmax and AUC(0‑inf).