The fate of bacterial cocaine esterase (CocE): an in vivo study of CocE-mediated cocaine hydrolysis, CocE pharmacokinetics, and CocE elimination

Abstract

Cocaine abuse and toxicity remain widespread problems in the United States. Currently cocaine toxicity is treated only symptomatically, because there is no Food and Drug Administration-approved pharmacotherapy for this indication. To address the unmet need, a stabilized mutant of bacterial cocaine esterase [T172R/G173Q-CocE (DM-CocE)], which hydrolyzes cocaine into inactive metabolites and has low immunogenic potential, has been developed and previously tested in animal models of cocaine toxicity. Here, we document the rapid cocaine hydrolysis by low doses of DM-CocE in vitro and in vivo, as well as the pharmacokinetics and distribution of the DM-CocE protein in rats. DM-CocE at 50.5 μg/kg effectively eliminated 4 mg/kg cocaine within 2 min in both male and female rats as measured by mass spectrometry. We expanded on these findings by using a pharmacologically relevant dose of DM-CocE (0.32 mg/kg) in rats and monkeys to hydrolyze convulsant doses of cocaine. DM-CocE reduced cocaine to below detection limits rapidly after injection; however, elimination of DM-CocE resulted in peripheral cocaine redistribution by 30 to 60 min. Elimination of DM-CocE was quantified by using [³⁵S] labeling of the enzyme and was found to have a half-life of 2.1 h in rats. Minor urinary output of DM-CocE was also observed. Immunohistochemistry, Western blotting, and radiography all were used to elucidate the mechanism of DM-CocE elimination, rapid proteolysis, and recycling of amino acids into all tissues. This rapid elimination of DM-CocE is a desirable property of a therapeutic for cocaine toxicity and should reduce the likelihood of immunogenic or adverse reactions as DM-CocE moves toward clinical use.

Fig. 1.

In vitro and in vivo cocaine hydrolysis by DM-CocE. A, in vitro DM-CocE dose response analysis. Human samples were spiked with 8 μM cocaine at t = 0. After 5 min at 37°C, DM-CocE was added to plasma at the given concentrations, aliquots were taken, and all hydrolysis was stopped at the given points. Aliquots were assessed for the remaining cocaine concentration by LC/MS analysis. B, in vivo assessment of cocaine hydrolysis by DM-CocE. Sprague-Dawley rats were intravenously administered 0, 13.6, or 50.5 μg/kg DM-CocE followed 2 min later by an intravenous administration of 4 mg/kg cocaine. Blood samples were taken at the times after cocaine injection shown, hydrolysis activity was stopped, and samples were evaluated by LC/MS for cocaine concentration. Significance was assessed by two-way analysis of variance with Bonferroni posttests (F (6, 60) = 7.37). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Fig. 2.

DM-CocE hydrolysis of high-dose cocaine across species. A, figure adapted from Brim et al. (2011). Three male Sprague-Dawley rats were administered 5.6 mg/kg cocaine (a physiologically equivalent dose to 3.2 mg/kg in the rhesus monkey) at time 0 and DM-CocE or PBS at time 10 min. PBS data are the average data from all three rats. DM-CocE data are separated by individual rats. DM-CocE rapidly removed cocaine to below the limit of quantification within 45 s. B, a female rhesus monkey was administered 3.2 mg/kg cocaine intravenously at time 0 and DM-CocE or PBS at time 10 min. Plasma cocaine concentrations were assessed at the given time points by mass spectrometry. Administration regimen was performed once every two weeks for 6 weeks. After DM-CocE administration, cocaine levels fell below the 30-nM limit of quantification, At times later than 30 min, low concentrations of cocaine appeared in the plasma, suggesting that the elimination of DM-CocE from the serum allowed very low amounts of cocaine to diffuse back into the blood from other body compartments. Monkey F exhibited the same pattern of cocaine elimination as the rat, suggesting no effect of species or repeat dosing of DM-CocE in the nonhuman primate. C, a male rhesus monkey was administered 3.2 mg/kg cocaine intravenously at time 0 and DM-CocE or PBS at time 1 min. Dosing regimen and sample analysis were conducted as described above for the female monkey. Like the rat and the female, cocaine reappeared at higher concentrations by 40 min after cocaine injection.

Fig. 3.

Elimination of [³⁵S]DM-CocE from the blood. A, concentration of DM-CocE in the serum of rats (n = 3) as measured by [³⁵S]methionine labeling over time. [³⁵S]DM-CocE (8 mg/kg; 18 μCi/kg) was administered intravenously through an indwelling jugular catheter at time 0. Blood samples (3 μl) were taken and assessed for radioactive content by scintillation counting. The concentration of DM-CocE was calculated by using the specific activity of each radioactive dose and plotted against time. DM-CocE was eliminated rapidly over the first 4 h after injection, until reaching a plateau state (inset). Both normal and unilaterally nephrectomized animals are shown. B, representative autoradiograph of [³⁵S] in serum over time from rats administered 8 mg/kg [³⁵S]DM-CocE. Male Sprague-Dawley rats were administered 8 mg/kg [³⁵S]DM-CocE at time 0. At 0.5,1, 2, 4, 6, 8, 12, and 18 h after DM-CocE injection, serum samples were taken. Total serum protein (25 μg) from each time point was loaded onto a 10% SDS/PAGE gel. Gel was transferred onto a PVDF membrane, and membrane was exposed to film for 2 weeks. C, representative Western blot of DM-CocE in serum from rats administered 8 mg/kg DM-CocE. Serum protein was handled as described above, but membrane was subjected to Western blotting with an anti-CocE antibody. The immunolabeled protein was visualized with chemiluminescence and film. D, comparison of the 65-kDa band of both the Western blots (n = 3) and autoradiograph (n = 3). Both the chemiluminescent and radiographic data were assessed by using densitrometry of the 65-kDa band. Raw values are plotted against time.

Fig. 4.

Appearance of [³⁵S]DM-CocE in the urine. A, cumulative urine accumulation of [³⁵S] radioactivity. After [³⁵S]DM-CocE administration to rats (n = 3), urine was collected and assessed for radioactive content by scintillation counting. Cumulative counts per minute per kilogram are shown against time and fit to a one-phase association model [Y = Y0 + (plateau − Y0) × (1 − exp(−K × x))]. Data plotted are S.E.M. B, Western blot analysis of the presence of DM-CocE in urine. Samples were added to SDS and ß-mercaptoethanol loading buffer and immediately loaded onto 10% SDS/PAGE gels. DM-CocE (40 ng) was loaded as a positive control, desalted urine alone was a negative control, and 40 ng DM-CocE spiked into urine was to control for protein recovery from urine desalting. Urine was collected at the times shown after CocE injection and was loaded in a time-dependent order.

Fig. 5.

Immunohistochemical analysis of DM-CocE distribution in perfused organs from Sprague-Dawley rats. Sprague-Dawley rats received intravenous administration of the DM-CocE (8 mg/kg) or vehicle once daily for 14 days (n = 3 each) or DM-CocE (24 mg/kg) once every 4 days for 14 days (n = 3). Six hours after the final dose of DM-CocE, rats were sacrificed and perfused, with organs fixed and embedded in paraffin for immunohistochemical analysis. Sections were counterstained with hematoxylin (blue), and DM-CocE reactivity was indicated by brown precipitate formed by diaminobenzadine. Positive DM-CocE reactivity is dose-dependently seen at the tip of the renal papilla. Images are from one representative animal from each group.

Fig. 6.

Immunohistochemical analysis of DM-CocE accumulation the renal papilla. A, Sprague-Dawley rats (n = 3) were administered 8 mg/kg DM-CocE intravenously. At the times shown after injection, rats were sacrificed and perfused with saline, and kidneys were fixed and embedded in paraffin. Immunohistochemistry was performed on kidney sections. Representative images from each time point are shown. Each differential interference contrast (DIC) image shows the area of the papilla that the fluorescence image (TXR) highlights. The overall area of the papilla that the images are taken from is exemplified by the saline sample. B, analysis of kidneys from three animals at each time point reveals peak DM-CocE accumulation at 2 h, after which there is a rapid decline. At 24 h, DM-CocE reactivity is no longer seen. *, one-way analysis of variance; F_{7, 16} = 2.47; Dunnetts Multiple Comparison Test, p < 0.05. Data are plotted as S.E.M. n/s, not significant.

Fig. 7.

Radiography and immunohistochemistry of organs 24 h post-DM-CocE injection. Rats administered either DM-CocE or [³⁵S]DM-CocE (n = 3) were sacrificed and perfused with saline 29 or 24 h after administration, respectively. Organ slices were subjected to immunohistochemistry (A) or radiography (B), and representative images are shown. No DM-CocE reactivity was detected with the anti-CocE antibody; however, evenly distributed and very dense radioactivity was detected in all organ slices.