The pharmacokinetics of the Fab fragment of a humanized anti‐cocaine monoclonal antibody and its effects on cocaine’s distribution in mice

Abstract

There is currently no FDA approved treatment for cocaine abuse. Our laboratory has created a humanized anti‐cocaine monoclonal antibody (mAb), h2E2, that has high affinity and specificity for cocaine and is cleared slowly in rodents. The mAb h2E2 sequesters cocaine in the plasma and prevents cocaine entry into the brain in rodents. The antigen binding portion, the Fab fragment, of h2E2 has the same high affinity and selectivity as its whole mAb counterpart. The Fab fragment should also sequester cocaine into the plasma compartment but is expected to have a more rapid clearance than the intact mAb. This study elucidated the pharmacokinetics of the Fab fragment and its effects on cocaine’s pharmacokinetics in mice. Catheterized mice were injected with Fab (80 mg/kg, i.v., pH=7.4, PBS) (n=8) and blood samples collected from the tip of the tail at designated time points over three days. Fab concentrations in blood samples were quantified using an ELISA assay. Two groups of catheterized mice were injected under mild restraint with Fab (82 mg/kg, i.v. pH= 7.4, PBS, generated by enzymatic digestion of whole mAb h2E2) or vehicle (PBS, pH=7.4). One hour later, mice were injected with an equimolar dose of cocaine HCl (0.56 mg/kg, i.v.). At 8 time points between 0.75 and 60 min, mice were decapitated, and trunk blood was collected. Sodium pentobarbital (50 mg/kg, i.p.) was administered 3 minutes prior to decapitation. For the 0.75 and 1.5‐minute time points, cocaine was injected into anesthetized rats. Blood (typically 0.5–2 mL) was placed into 2‐mL polypropylene tubes and then centrifuged at 5000 g for 3 minutes. Plasma was pipetted into polypropylene microcentrifuge tubes and rapidly frozen on dry ice and stored at −80°C until LC‐MS/MS analysis. This experiment was performed three times. Total cocaine concentrations were quantified using LC‐MS/MS. The pharmacokinetic profile of the Fab fragment in mice was described by a 2‐compartment model (Figure 1). The Fab fragment has a mean distribution half‐life (t1/2α) of 16.7 minutes and a terminal elimination half‐life (t1/2β) of 7.1 hours (Figure 1). In the presence of the Fab fragment, cocaine concentrations in the plasma increased by 4.5‐fold compared to vehicle animals (Figure 2). The initial volume of distribution of cocaine decreased approximately 4.5‐fold in the presence of Fab. The Fab fragment increases the concentration of cocaine in the plasma by approximately 4.5‐fold compared to vehicle controls. Although the data is not shown, it is hypothesized that the Fab fragment, like its mAb counterpart, prevents cocaine from crossing the blood‐brain barrier and entering the brain. With a 24‐fold decrease in elimination half‐life (7.1 hours) compared to the intact h2E2 (7.8 days) and predicted rapid urinary excretion, the Fab fragment may be useful for the treatment of cocaine overdoses.Support or Funding InformationThis work is supported by the National Institutes of Health, National Institute on Drug Abuse grant number U01DA039550 (to ABN).The pharmacokinetics of the Fab fragment in male miceFigure 1The pharmacokinetics of cocaine in the presence of the Fab fragmentFigure 2