A recombinant humanized anti-cocaine monoclonal antibody inhibits the distribution of cocaine to the brain in rats

Abstract

The monoclonal antibody (mAb), h2E2, is a humanized version of the chimeric human/murine anti-cocaine mAb 2E2. The recombinant h2E2 protein was produced in vitro from a transfected mammalian cell line and retained high affinity (4 nM Kd) and specificity for cocaine over its inactive metabolites benzoylecgonine (BE) and ecgonine methyl ester. In rats, pharmacokinetic studies of h2E2 (120 mg/kg i.v.) showed a long terminal elimination half-life of 9.0 days and a low volume of distribution at steady state (Vdss) of 0.3 l/kg. Pretreatment with h2E2 produced a dramatic 8.8-fold increase in the area under the plasma cocaine concentration-time curve (AUC) and in brain a concomitant decrease of 68% of cocaine's AUC following an i.v. injection of an equimolar cocaine dose. Sequestration of cocaine in plasma by h2E2, shown via reduction of cocaine's Vdss, indicates potential clinical efficacy. Although the binding of cocaine to h2E2 in plasma should inhibit distribution and metabolism, the elimination of cocaine remained multicompartmental and was still rapidly eliminated from plasma despite the presence of h2E2. BE was the major cocaine metabolite, and brain BE concentrations were sixfold higher than in plasma, indicating that cocaine is normally metabolized in the brain. In the presence of h2E2, brain BE concentrations were decreased and plasma BE was increased, consistent with the observed h2E2-induced changes in cocaine disposition. The inhibition of cocaine distribution to the brain confirms the humanized mAb, h2E2, as a lead candidate for development as an immunotherapy for cocaine abuse.

Fig. 1.

The binding affinities of 2E2 and h2E2 for [³H]cocaine. Antibodies and [³H]cocaine were incubated in PBS at pH 7.0 at room temperature for 6 hours. The K_d, defined as the concentration of [³H]cocaine at which 50% of B_max occurred, was 4.4 nM for 2E2 and 3.9 nM for h2E2.

Fig. 2.

The pharmacokinetics of the anti-cocaine mAb h2E2 in rats. Animals received an i.v. infusion of 120 mg/kg h2E2. Samples of blood (10 μl) were obtained from tail veins at the indicated times after completion of the mAb infusion. Concentrations of h2E2 in blood were determined using an ELISA. Data points represent the mean ± S.E.M. from four rats. The V_dss was 0.3 l/kg. A two-compartment model with a t_1/2α of 19 hours described the distribution phase, and a t_1/2β of 9.0 days described the elimination phase, represented by the best-fit regression line.

Fig. 3.

The effect of h2E2 on the pharmacokinetics of cocaine in plasma (A) and brain (B) in rats. Rats received an i.v. infusion of 120 mg/kg h2E2. One hour later the rats received an i.v. injection of cocaine HCl (0.56 mg/kg). The animals were sacrificed at the indicated times, and blood and the brain were collected. Cocaine concentrations were determined by GC/MS. Each data point represents the mean ± S.E.M. from three rats. In the absence of h2E2 (open circles), the cocaine concentration-time profile in plasma (A) was described by a two-compartment pharmacokinetic model with a t_1/2α of 0.4 minute and a t_1/2β of 32.9 minutes. In the presence of h2E2 (closed circles), a two-compartment pharmacokinetic model indicated a t_1/2α of 2.2 minutes and a t_1/2β of 13.8 minutes. h2E2 produced a 8.8-fold increase in the area under the plasma cocaine AUC. The V_dss in the absence and presence of h2E2 was 2.6 and 0.1 l/kg, respectively. In the brain (B) in the absence of h2E2 (open circles), a two-compartment pharmacokinetic model with an AUC of 29,900 (ng/g) × minutes described the cocaine concentration-time profile. In the presence of h2E2 (closed circles), a two-compartment pharmacokinetic model with an AUC of 9460 (ng/g) × minutes described the cocaine concentration-time profile. h2E2 produced a 68% decrease in the brain cocaine AUC.

Fig. 4.

The effect of h2E2 on the formation of BE via cocaine metabolism in plasma (A) and brain (B) in rats. Concomitant to determination of cocaine concentrations using GC/MS, the concentrations of BE were determined. Each data point represents the mean ± S.E.M. from three rats. In the absence of h2E2 (open circles), BE is formed at a relatively constant rate in plasma (A), but in the brain (B) it is present in higher concentrations that decline over time. In contrast, the presence of h2E2 (closed circles) causes an increase in BE levels in plasma (A) and in the brain (B) a corresponding decrease in BE formation.