Efficient Cocaine Degradation by Cocaine Esterase-Loaded Red Blood Cells

Abstract

Recombinant bacterial cocaine esterase (CocE) represents a potential protein therapeutic for cocaine use disorder treatment. Unfortunately, the native enzyme was highly unstable and the corresponding mutagenized derivatives, RBP-8000 and E196-301, although improving in vitro thermo-stability and in vivo half-life, were a partial solution to the problem. For cocaine use disorder treatment, an efficient cocaine-metabolizing enzyme with a longer residence time in circulation would be needed. We investigated in vitro the possibility of developing red blood cells (RBCs) loaded with RBP-8000 and E196-301 as a biocompatible system to metabolize cocaine for a longer period of time. RBP 8000 stability within human RBCs is limited (approximately 50% residual activity after 1 h at 37°C) and not different as for the free enzyme, while both free and encapsulated E196-301 showed a greater thermo-stability. By reducing cellular glutathione content during the loading procedure, in order to preserve the disulfide bonds opportunely created to stabilize the enzyme dimer structure, it was possible to produce an encapsulated protein maintaining 100% stability at least after 4 h at 37°C. Moreover, E196-301-loaded RBCs were efficiently able to degrade cocaine in a time- and concentration-dependent manner. The same stability results were obtained when murine RBCs were used paving the way to preclinical investigations. Thus, our in vitro data show that E196-301-loaded RBCs could act as efficient bioreactors in degrading cocaine to non-toxic metabolites to be possibly considered in substance-use disorder treatments. This approach should now be investigated in a preclinical model of cocaine use disorder to evaluate if further protein modifications are needed to further improve long term enzyme stability.

Keywords: E196-301 stability; RBP 8000 stability; cocaine degradation; cocaine esterase; cocaine use disorder; red blood cells as drug delivery system.

FIGURE 1

Crystal structure of the cocaine esterase (CocE) dimer. Ribbon representation of the homodimeric molecule showing the side chains of the soleucine and leucine residues that will be replaced with cysteine forming intersubunit disulfide bonds.

FIGURE 2

RBP 8000 stability. In vitro stability of free and red blood cell (RBC)-loaded RBP 8000 at 37°C in PBS, pH 7.2. Free enzyme was tested at 0.5 ± 0.1 and 10 ± 1 IU/ml. RBC-loaded enzyme was tested at 5 ± 1 and 15 ± 1.3 IU/ml RBCs. RBC suspensions at 10% Ht were used. All experiments were in duplicate that agree within 7.5%. Results are mean ± SD of three different experiments. Values of RBP 8000-loaded RBCs were not significantly different from control (free RBP 8000 at 10 ± 1 U/ml).

FIGURE 3

Stability comparison between RBP 8000 and E196-301. In vitro stability of RBP 8000 and 196-301 at 37°C in PBS, pH 7.2. Free enzymes were tested at 10 ± 1 and 15 ± 1 IU/ml, respectively. All experiments were in duplicate that agree within 7.5%. Results are mean ± SD of three different experiments. All values were significantly different (p < 0.005).

FIGURE 4

E196-301 stability in free and in red blood cell (RBC) encapsulated form. In vitro stability of free and RBC-loaded E196-301 at 37°C in PBS, pH 7.2. Free enzyme was tested at 15 ± 1 IU/ml, while RBC-loaded enzyme was tested at 16 ± 1 IU/ml RBCs. All experiments were in duplicate that agree within 7.5%. Results are mean ± SD of three different experiments.

FIGURE 5

Reduced glutathione (GSH) effect on E196-301 stability. Enzyme activity has been evaluated at different times (0, 1, 2, and 3 h) after incubation at 37°C in a 1 ml-final volume in the absence or in the presence of different GSH concentration in the range 0.5–3 mM. Values are expressed in percentage respect to time 0. At this time the enzyme activity was 15 IU/ml. This is representative of two different duplicate experiments that agree within 5%.

FIGURE 6

Effect of reduced glutathione (GSH) in E196-301 stability once encapsulated in red blood cells (RBCs). In vitro stability of RBC-loaded E196-301 at 37°C in PBS, pH 7.2. RBC-loaded enzyme was tested at 15 ± 2 IU/ml RBCs. All experiments were in duplicate that agree within 7.5%. Results are mean ± SD of three different experiments.

FIGURE 7

Cocaine degradation. The ability of E196-301-loaded red blood cells (RBCs) to metabolize cocaine has been evaluated at different times (range 0–2 h) after incubation at 37°C in a 500 μl-final volume with 5 μg/ml cocaine concentration. Conditions: 0.28 IU E196-301/ml RBC suspension (blue); 0.028 IU E196-301/ml RBC suspension (red); 0.0028 IU E196-301/ml RBC suspension (green); 0.00028 IU E196-301/ml RBC suspension (violet). This is representative of two different duplicate experiments that agree within 5%. For the RBC condition 1:2500 the times 0, 2, 5, and 10 min are highly significant among them (****); for the RBC 1:25000 condition each time is significantly different from the previous one up to time point 90 min (***); for the RBC condition 1:250000 a significant difference has been observed at each time (with the exception of the time points 2, 5, and 30 min) with respect to time zero (by one-way ANOVA test, ***p < 0.001, ****p < 0.0001).