A recombinant humanized anticocaine monoclonal antibody, h2E2, has shown potential in the preclinical phases for the treatment of cocaine abuse. The standard tests for cocaine usage are the detection of benzoylecgonine (BE) and cocaine in the urine. This includes workplace drug screens as well as in clinical trials for potential treatments of cocaine abuse. By sequestering cocaine into the plasma compartment, h2E2 prevents cocaine from entering the brain. Due to the altered disposition of cocaine in the presence of h2E2, we investigated the effects of h2E2 on cocaine and metabolite levels in the urine of rats to clarify the use of BE as an endpoint measurement for effectiveness in future clinical trials. The urine concentrations of cocaine and metabolites were considerably altered in the presence of h2E2. After a single injection of h2E2 (120 mg/kg) and cocaine hydrochloride (0.56 mg/kg), the concentration of cocaine and BE excreted into the urine of rats decreased by 92% and 91%, respectively, from vehicle controls. Due to the significant decrease in urinary excretion, BE is not an appropriate indicator of cocaine usage in the presence of h2E2. Another endpoint measurement must be selected for the measurement of cocaine usage in the upcoming clinical trials of h2E2. In contrast to the effects on cocaine and BE urinary excretion, there was a 3-fold increase in ecgonine methyl ester (EME) in the presence of h2E2. Therefore, we conclude that EME is a more appropriate measurement of cocaine intake in the presence of h2E2. Materials and Methods Animals. Twelve male, Sprague-Dawley cocaine-naive rats (250-325 g) were used during the course of this study (Harlan Laboratories, Indianapolis, IN). Rats were housed individually on a 14/10-hour light/dark
The recombinant monoclonal anti‐cocaine antibody, h2E2, sequesters cocaine in plasma increasing concentrations more than 10‐fold. The increased levels of cocaine in the plasma could have detrimental peripheral effects, particularly on the cardiovascular system. We investigated the duration and magnitude of the effect of cocaine on the rat heart, and if h2E2 could antagonize that effect. Echocardiography was used to evaluate cardiac function under isoflurane anesthesia, while a tail‐cuff was used to measure blood pressure. Cocaine was delivered intravenously and the rats were continuously monitored for a total of 45 min. Echocardiography measurements were recorded every 5 min and blood pressure measurements were recorded throughout the duration of the experiment using 30‐s cycles. ECG recordings were taken simultaneously with the echocardiography measurements. An increase in ejection fraction was seen after the cocaine push with the maximum change occurring at 25 min. Treatment with h2E2 1 h before the cocaine push did not have any effect on cardiac parameters. Subsequent cocaine treatment had no effect on the ejection fraction, indicating that the antibody‐bound cocaine does not affect the heart. This antagonism of cocaine's effects was greatly decreased after 1 week and entirely absent after 1 month. Cocaine in the presence of h2E2 is pharmacologically inert and h2E2 may have additional clinical utility for reversing cocaine effects on the cardiovascular system.
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 Information This work is supported by the National Institutes of Health, National Institute on Drug Abuse grant number U01DA039550 (to ABN). The pharmacokinetics of t...
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