Recovery of Dopaminergic System after Cocaine Exposure and Impact of a Long-acting Cocaine Hydrolase

Abstract

Dysregulation of dopamine transporters (DAT) within the dopaminergic system is an important biomarker of cocaine exposure. Depending on cocaine amount in-taken, one-time exposure in rats could lead to most (>95% of total) of DAT translocating to plasma membrane of the dopaminergic neurons compared to normal DAT distribution (~5.7% on the plasma membrane). Without further cocaine exposure, the time course of striatal DAT distribution, in terms of intracellular and plasma membrane fractions of DAT, represents a recovery process of the dopaminergic system. In this study we demonstrated that after an acute cocaine exposure of 20 mg/kg (i.p.), the initial recovery process from days 1 to 15 in rats was relatively faster (from >95% on day 1 to ~35.4% on day 15). However, complete recovery of the striatal DAT distribution may take about 60 days. In another situation, with repeated cocaine exposures for once every other day for a total of 17 doses of 20 mg/kg cocaine (i.p.) from days 0 to 32, the complete recovery of striatal DAT distribution may take an even longer time (about 90 days), which represents a consequence of chronic cocaine use. Further, we demonstrated that a highly efficient Fc-fused cocaine hydrolase, CocH5-Fc(M6), effectively blocked cocaine-induced hyperactivity and DAT trafficking with repeated cocaine exposures by maintaining a plasma CocH5-Fc(M6) concentration ≥58.7 ± 2.9 nM in rats. The cocaine hydrolase protected dopaminergic system and helped the cocaine-altered DAT distribution to recover by preventing the dopaminergic system from further damage by cocaine.

1. INTRODUCTION

Cocaine produces its physiological effects by targeting multiple proteins in the central nervous system (CNS),¹ particularly by binding with dopamine transporter (DAT) to block normal dopamine (DA) recycling process.² This process along with the pleasure feeling would wear off shortly after abstinence of the drug. However, chronic cocaine use is associated with neuroadaptations in the dopaminergic system,^3–5 including increases in the density of DAT on plasma membrane of dopaminergic neurons.^{6, 7} Given the critical role of DAT in neurotransmission, various studies have been carried out on animal models to learn its structure, function, and distribution.⁸ Long-standing evidence supports that DAT presentation on neuron cell surface is highly plastic and is subject to regulated endocytic trafficking – it constitutively internalizes and recycles during the neurotransmission process.^{9, 10} Cocaine administration can significantly induce DAT trafficking to the plasma membrane, without significantly changing the total amount of DAT (which is a sum of the intracellular and plasma membrane DAT).^{6, 11} One-time use of cocaine increases the surface DAT expression (via DAT trafficking) for at least a month,¹² while abstinence from chronic use of cocaine would take a longer period of time for striatal DAT distribution to recover in monkey models,¹³ as normalization of dopaminergic function is usually a slow process. However, the specific role of abstinence in the DAT trafficking was difficult to determine because of the lack of measurements in groups with different abstinence periods.

Further, it is highly desired to develop a truly effective pharmacotherapy for treatment of cocaine use disorder (CUD).^14–16 However, despite of decades of effects, there is still no Food and Drug Administration (FDA)-approved pharmacotherapy specific for CUD, due to difficulty to antagonize cocaine’s physiological effects without affecting normal functions of brain. Nevertheless, through computational modelling and simulation-guided rational design,^17–20 we have developed highly efficient cocaine hydrolases (CocHs) that are rationally designed mutants of human butyrylcholinesterase (BChE),^{19, 21–24} and derived a long-acting form of it (denoted as CocH-Fc) through protein fusion with the Fc fragment (wild-type or mutant) of human immunoglobulin G1 (IgG1).^25–27 It has been demonstrated that these proteins are capable of accelerating cocaine metabolism, rapidly detoxifying cocaine and its toxic metabolites.^{22, 27–30} A single dose of the CocH-Fc can effectively block the physiological and reinforcing effects of cocaine for a long period of time (weeks).^{25, 27, 31} Specially, we have also demonstrated that CocH-Fc can also block cocaine-induced DAT trafficking in cocaine-naïve rats.¹¹ It is more interesting for us to know whether CocH-Fc could help dopaminergic system recovery after cocaine exposure and provide protection against repeated cocaine administration, allowing the dopaminergic pathway to maintain a cocaine abstinence status.

In present study, we first examined the time-dependent distribution of striatal DAT in terms of intracellular and plasma membrane fractions after different cocaine abstinence periods following acute or chronic cocaine administrations. Further, we also demonstrated that a CocH-Fc protein, known as CocH5-Fc(M6), is capable of effectively eliminating cocaine-induced hyperactivity, blocking DAT trafficking from repeated cocaine exposure, preventing further damage to DA system from the drug, and thus help the brain to recover.

2. MATERIALS AND METHODS

2.1. Animals

All in vivo experiments were conducted with male Sprague–Dawley rats (weight 200 g to 250 g at receiving), which were ordered from Harlan (Harlan, Indianapolis, IN), and housed initially as two rats per cage. All rats were kept at room temperature of around 22°C, allowing ad libitum access to food and water. Animal housing areas were maintained on a 12-hour light/12-hour dark cycle, with lights on at 8:00 AM. On all the testing days, experiments were performed in light between 9:00 AM to 4:00 PM. All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health (NIH). The animal protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Kentucky.

2.2. Drug and materials

CocH5-Fc(M6), which is a Fc fusion protein with CocH5 (the A199S/F227A/P285A/S287G/A328W/Y332G mutant of human butyrylcholinesterase) fused with the sextuple-mutated (A1V/M38Y/S40T/T42E/D142E/L144M) Fc region of human IgG1, was developed in our previous studies.^{22, 27, 32} Cocaine was provided by the National Institute on Drug Abuse (NIDA) Drug Supply Program (Bethesda, MD). Antibodies recognizing rat Na⁺/K⁺ ATPase (mouse monoclonal antibody, catalog #sc-48345), protein phosphatase 2A (PP2A, mouse monoclonal antibody, catalog #sc-13601), β-actin (Horseradish peroxidase (HRP)-conjugated mouse polyclonal antibody, catalog #sc-47778), were all ordered from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-DAT antibody (mouse monoclonal antibody, catalog #MA524796), and HRP-conjugated goat anti-mouse (catalog #G21040) antibody (polyclonal), protease and phosphatase inhibitor cocktail (100-X), Sulfo-NHS-SS-biotin (sulfosuccinimidyl-2-[biotinamido]ethyl-1,3-dithiopropionate), and monomeric avidin agarose were purchased from Thermo Fisher Scientific (Waltham, MA). All other chemicals and surgery tools were purchased from either Thermo Fisher Scientific (Waltham, MA), VWR international (Radnor, PA), or Sigma-Aldrich (St. Louis, MO).

2.3. DAT cellular distribution assay

Biotinylation and Western blotting assays were performed using a previously published method.¹¹ Briefly, rats (n = 4 for each group) were given a single or multiple intraperitoneal dose of 20 mg/kg cocaine, and then decapitated under deep anesthesia with CO₂ (40% chamber replacement rate, 3 to 4 min) to collect the brain samples at different time-points after cocaine exposure. Striatum samples were quickly isolated on ice and immediately homogenized in 1-mL ice-cold sucrose solution (0.32 M sucrose, 5 mM sodium bicarbonate, 1-X phosphatase inhibitor cocktail, and 1-X protease inhibitor cocktail, pH 7.4) with 1-min continuous homogenization of a cordless pestle motor in 1.5 mL microtubes on ice. This procedure led to the nerve terminals being torn away from their axons, the postsynaptic and glial cells to which they were connected.³³ The presynaptic membranes then resealed, enclosing the nerve terminal contents, such as cytoplasm, mitochondria, and synaptic vesicles storing DATs etc. to form a Sac-like structure (called synaptosomes), which has the functions closely mimic nerve terminals in vivo.^33–35

The homogenates were then centrifuged at 1500g for 10 min at 4°C and resulting supernatants were centrifuged at 17,000g for 25 min at 4°C. Resulting pellets were quickly washed twice with 1 mL PBS/Ca/Mg buffer (138 mM NaCl, 2.7 mM KCl, 1.5 mM KH₂PO₄, 9.6 mM Na₂HPO₄, 1 mM MgCl₂, 0.1 mM CaCl₂, 1-X phosphatase inhibitor cocktail, and 1-X protease inhibitor cocktail, pH 7.3), then resuspended in 500 μL ice-cold PBS/Ca/Mg buffer to obtain synaptosomal suspensions. Suspensions containing the total synaptosomal protein were incubated for 1.5 hour at 4°C with continual shaking in 500 μL 3 mg/mL sulfo-NHS-SS-biotin in PBS/Ca/Mg buffer. After incubation, samples were centrifuged at 17,000g for 10 min at 4°C. To remove the free biotinylation reagent, the resulting pellet was resuspended in 1 mL ice-cold 100 mM glycine in PBS/Ca/Mg buffer and centrifuged at 17,000g for 10 min at 4°C. The resuspension and centrifugation steps were repeated. Final pellets were resuspended in 1 mL ice-cold 100-mM glycine in PBS/Ca/Mg buffer and incubated with continuous shaking for 30 min at 4°C to ensure complete removal of free biotinylation agent. Subsequently, the samples were centrifuged at 17,000g for 4 min at 4°C, and the resulting pellets were resuspended in 1 mL ice-cold PBS/Ca/Mg buffer and centrifuged again. The resuspension and centrifugation steps were repeated for two more times. The final pellet was lysed by sonication for 1 to 2 min in 300 μL Triton X-100 buffer (10 mM Tris, 150 mM NaCl, 1.0% Triton X-100, 250 μM phenylmethylsulfonyl fluoride, 1-X phosphatase inhibitor cocktail, and 1-X protease inhibitor cocktail, pH 7.4) followed by incubation and continuous shaking for 30 min at room temperature (22 ± 1°C). Lysates (300 μL) were centrifuged at 17,000g for 20 min at 4°C. The pellet was discarded, and all the supernatant was incubated with continuous shaking in the presence of monomeric avidin beads (100 μL per tube, after removal of ethanol and washed with Triton X-100 buffer) for 30 min at room temperature. Samples were then centrifuged at 17,000g for 4 min at 4°C and supernatants (containing non-biotinylated, intracellular fraction proteins) were concentrated to 20 μL and stored temporarily at −20°C. The efficiency of avidin in isolating the biotinylated and non-biotinylated proteins across the protein range in the tissue extract was verified using avidin-conjugated antibody, which showed that biotinylated proteins were completely absorbed by the avidin and were not present in the supernatant. The resulting pellets containing the avidin-absorbed biotinylated (membrane) proteins were washed for at least 3 times with 1 mL 1.0% Triton X-100 buffer and centrifuged at 17,000g for 4 min at 4°C. The final pellet consisted of the biotinylated proteins adsorbed to monomeric avidin beads. The biotinylated protein (bound on avidin beads) and non-biotinylated proteins were mixed with 30 or 10 μL reducing SDS loading buffer, respectively, then went through a repeated boil-freeze cycle for 5 times (2 min/cycle), and finally boiled for 5 min to completely denature the extracted proteins. The biotinylated proteins were eluted by the reducing agent into the loading buffer. The samples were centrifuged at 6,200g for 5 min and stored temporarily at −20°C until use for immunoblotting.

To obtain the immunoreactive DAT protein in the intracellular and plasma membrane fractions, samples were thawed and subjected to gel electrophoresis and western blotting as described previously.¹¹ Briefly, proteins were separated by 8% SDS–polyacrylamide gel electrophoresis (PAGE) for 85 min at 120 V and transferred to Immobilon-P transfer membranes (0.45 μm pore size; Millipore Co., Bedford, MA, USA) in transfer buffer (50 mM Tris, 250 mM glycine, 3.5 mM SDS, 25% (v/v) methanol) using an XCell SureLock Mini-Cell (Thermo Fisher) for 1 hour at 40 V. The membranes were incubated with blocking buffer (1% BSA in TBS containing 0.5% Tween 20) for 1 hour at room temperature, followed by incubation with mouse monoclonal DAT antibody (1 : 2,000) overnight at 4°C. Transfer membranes were washed for five times with wash buffer (TBS containing 0.5% Tween 20) at room temperature and then incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (1 : 10,000 dilution in blocking buffer) for 1 hour at room temperature. Immunoreactive proteins on the transfer membranes were detected using enhanced chemiluminescence and developed on Hyperfilm (ECL-plus; Amersham Bio-sciences UK Ltd, Little Chalfont, UK). After detection and quantification of DAT protein, each blot was stripped using Tris buffer (62.5 mM Tris-HCl with 2% SDS and 100 mM β-mercaptoethanol, pH 6.8) and re-probed for detection of Na⁺/K⁺ ATPase, PP2A and β-actin. Amounts of Na⁺/K⁺ ATPase and of PP2A, that are a plasma membrane resident protein and an intracellular protein, respectively, were determined using mouse monoclonal alpha-1 Na⁺/K⁺ ATPase type-1 antibody (1 : 5,000) and mouse monoclonal demethylated protein phosphatase 2A–C antibody (1 : 500) to monitor the efficiency of the biotinylation of cell surface proteins. β-actin, a cytoskeletal protein, served as a control for loading of proteins and was determined using mouse polyclonal β-actin antibody (1: 10,000). Multiple autoradiographs were obtained using different exposure times, and immuno-reactive bands within the linear range of detection were quantified by densitometric scanning using Gel Documentation System (Bio-Rad Laboratories, Hercules, CA). Band density measurements, expressed as relative optical density, were used to calculate the levels of DAT in non-biotinylated and biotinylated fractions. Specifically, the normalized percentage of biotinylated or non-biotinylated DAT of total DAT levels were calculated based on density of DAT-immunoreactive bands in the biotinylated or non-biotinylated fraction divided by the combination of these two fractions.

2.5. Locomotor activity testing

Rats were placed in high-density, non-porous plastic chambers (50-cm L × 50-cm W × 38-cm H) in a quiet room and allowed to acclimate before testing. On the test day, following one-hour acclimation, rats were administered either saline alone i.p. (for the control group) or a pre-treatment of 1.5 mg/kg CocH5-Fc(M6) i.v., followed by a dose of 20 mg/kg cocaine i.p. at 1 hour after enzyme/saline administration, and then 20 mg/kg cocaine (i.p.) on every other day until day 32. After cocaine injection, rats were immediately returned to the test chambers. On each test day, the experiment was performed between 9:00 am to 4:00 pm. The distance traveled in every 5 minutes were tracked and recorded for two hours after cocaine administration using ANY-maze video tracking system (San Diego Instruments, San Diego, CA) in our lab. Upon completion of the tests, animals were returned to their home cages.

2.6. Determination of plasma enzyme concentration

To measure plasma enzyme concentrations, blood samples (around 75 μL) were collected from saphenous vein using heparin-treated capillary tubes at different time-points after enzyme administration. Collected blood samples were centrifuged for 5 min at a speed of 8,000g, and separated plasma was kept at 4°C before analysis. A sensitive radiometric assay as described before³⁶ was performed to measure the enzyme concentration in plasma. Briefly, 10 μL of plasma sample was mixed with 140 μL of 0.1 M phosphate buffer (pH = 7.4). 50 μL of 400 μM [³H]-(−)-cocaine was added to the mixture to initiate enzymatic reaction. The reaction was proceeded at room temperature and was stopped by adding 200 μL of 0.1 M hydrochloric acid. The radioactive product benzoic acid was extracted by toluene for scintillation counting. The enzyme concentration ([E]) was calculated based on equation [E] = V_max/k_cat, where V_max is the maximum rate the enzymatic reaction can achieve, k_cat is the turnover number of the enzyme. Enzyme kinetic parameters were obtained from our previous study.²²

2.7. Analysis of plasma cocaine and metabolites

To determine the concentrations of cocaine and its metabolites in whole blood, rat blood samples (around 75 μL) were collected from saphenous veins using heparin-treated capillary tubes at various time-points after cocaine administration and were mixed immediately with 100-μL paraoxon solution composed of 250 μM paraoxon with 10 U/mL heparin in 0.1% formic acid. Blood samples were stored at −80 °C before use. For analysis, thawed blood sample was mixed with 500 μL of 4% formic acid and 75 μL of 100 nM internal standards. The mixture was then subject to a one-step solid-phase extraction (SPE) using mixed cation exchange model solid phase extraction cartridges (Oasis MCX 1 cc Vac Cartridge, 10 mg) purchased from Waters (Milford, MA). Briefly, the loaded sample was washed twice with 1 mL methanol, followed by elution using 1 mL methanol/7.5% ammonium hydroxide (95:5, v/v).

A quantitative LC-MS/MS assay described in our previous report³⁷ was used to quantify cocaine and its metabolites in blood samples. Briefly, extracted samples were evaporated and resuspended in 0.1% formic acid before loading to a Shimadzu HPLC system (Shimadzu, Kyoto, Japan), consisting of a DGU-20A/3R degasser, LC-20 AD binary pumps, CBM-20A controller, and SIL-20A/HT auto sampler. Mobile phase A consisted of 0.1% formic acid and mobile phase B consisted of 0.1% formic acid: acetonitrile (10 : 90, v/v). Samples were loaded to an Atlantis T3 (100 Å, 3 μm, 2.1 mm X 150 mm I.D) column (Waters, Milford, MA) and were eluted by gradient. AB SCIEX tripleTOF™ 5600 (AB SCIEX, Redwood City, CA) was applied in positive ion and high sensitivity mode for analysis.

2.8. Statistics

Data are presented as mean ± SEM, and n represents the number of independent experiments for each group. Data from immunoblotting experiments were analyzed using one-way ANOVA or t-test for each of the fractions (biotinylated and non-biotinylated). The accepted level of statistical significance was p < 0.05.

3. RESULTS

3.1. DAT distribution at various time-points after cocaine exposure

Our previous study¹¹ reported that in normal rat brain, striatal DAT mainly existed in the intracellular fraction, with only a very small fraction (~5.7%) existed in the plasma membrane in rats. Notably, our finding of only ~5.7% of DAT in the plasma membrane surface in rat brain is far less than the previously reported surface DAT fraction in cultured PC12-hDAT cells (~25–40%),^38–40) but is close to the synaptosomal surface faction (~7%) of DAT in rats reported by Johnson et al.⁴¹ Cocaine at an i.p. dose of 10 mg/kg or higher, induced translocating of most DAT into the plasma membrane (with only <5% remaining in the intracellular fraction) at 24 hours after cocaine administration in rats that were naïve to cocaine. A dose of 20 mg/kg cocaine i.p. also induced very strong (a total distance of ~350 meters traveled within 1^st hour after cocaine administration) and long-period (~2 hours) hyperactivity without notably severe toxicity sign. For this reason, 20 mg/kg cocaine i.p. was used in present study to introduce cocaine exposure in rat groups with either single or multiple-time cocaine exposure.

To study striatal DAT distribution on plasma membrane and intracellular fractions at different time points after cocaine exposure, rat brain samples were collected on 15 days, 30 days, 60 days, or 90 days after a single intraperitoneal administration of 20 mg/kg cocaine.

Depicted in Figure 1A are western blots showing the striatal DAT distribution in both the plasma membrane (biotinylated) and intracellular fraction (non-biotinylated) of samples collected at various time-points after cocaine exposure. The data for the negative control group (without cocaine administration) and 24-hour group were reported previously,¹¹ and the data for all other groups in Figure 1 are reported here for the first time. The corresponding numeric data are summarized in Figure 1B. Na⁺/K⁺ ATPase α and PP2A are presented as controls for plasma membrane fraction and intracellular fraction, respectively. β-actin is a loading control. All these signals of the protein were used to normalize the DAT distribution while converting the images into numeric data.

Figure 1.

Striatal DAT immunoreactivity after acute exposure to 20 mg/kg cocaine (i.p.) (n = 4 rats per group). For the immunoblotting results of striatal DAT distribution in biotinylated membrane fraction and non-biotinylated intracellular fraction, Na⁺/K⁺ ATPase and PP2A serve as biotinylation controls, while β-actin serves as a loading control (panel A). Normalized DAT expression values (panel B) are mean ± SEM for DAT density following different time period after cocaine exposure expressed as a percentage of total DAT (biotinylated plus non-biotinylated). Statistical analysis (One-way ANOVA): *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

The data in Figure 1 shows a time course of striatal DAT distribution over 3 months after cocaine exposure. As indicated in Figure 1, the plasma membrane DAT population was upregulated from ~5.7% (negative control) to ~95.8–98.1% (24 hours) after cocaine exposure, and then was gradually recovered to 35.4 ± 2.7% (after 15 days), 26.4 ± 3.3% (30 days), 13.5 ± 0.8% (60 days), and 4.6 ± 1.6% (90 days). Our data is consistent with a known concept that cocaine can cause upregulation of plasma membrane DAT population to last for a long period of time.¹² According to the data, the initial recovery process from day 1 to day 15 was relatively faster (from ~95.8–98.1% on day 1 to ~35.4% on day 15) and then slower after day 15. The plasma membrane fraction (~26.4%) on day 30 was still significantly higher than the negative control (~5.7%). The plasma membrane fraction (~13.5%) on day 60 was non-significantly higher than the negative control (~5.7%), with p = 0.0953. So, rats may need ~60 days for the DAT distribution returning to the normal status after one-time exposure to cocaine.

3.2. DAT distribution at various time-points after cocaine exposure for multiple times

To check recovery process of the DAT distribution after cocaine exposure for multiple times, additional four groups of rats (n = 4 per group) were given an intraperitoneal dose of 20 mg/kg cocaine on every other day from day 0 (the first dose) to day 32 (the final dose) for a total of 17 doses. Brain samples were collected at 24 hours, 30 days, 60 days, or 90 days after the final dose of cocaine.

Similar to Figure 1, Figure 2A displays western blots showing the striatal DAT distribution and Figure 2B is a summary of the corresponding numeric data. Negative control results from normal rats (without cocaine exposure)¹¹ were added to Figure 2B for comparison. As shown in Figure 2, the plasma membrane fraction of DAT decreased from 97.1 ± 0.2% on day 1 (24 hours) to 40.8 ± 3.1% on day 30, 15.3 ± 1.2% on day 60, and then 12.3 ± 1.3% on day 90 after the last exposure of 20 mg/kg cocaine (i.p.). The plasma membrane fraction (~15.3%) on day 60 was still significantly higher than the negative control (~5.7%). The plasma membrane fraction (~12.3%) on day 90 was non-significantly higher than the negative control (~5.7%), with p = 0.068. So, rats may need ~90 days for the DAT distribution returning to the normal status after cocaine exposure for multiple times (a total of 17 doses of 20 mg/kg cocaine in 32 days).

Figure 2.

(A) Time course of striatal DAT immunoreactivity after multiple times of cocaine exposure (n = 4 rats per group). All groups of rats received a dose of 20 mg/kg cocaine (i.p.) every other day from days 0 to 32 days for a total of 17 doses. (B) Normalized DAT expression values are mean ± SEM for DAT density following different period after cocaine abstinence expressed as a percentage of total DAT (biotinylated plus non-biotinylated). Statistical analysis (One-way ANOVA): *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

3.3. Impact of enzyme CocH5-Fc(M6)

Once understanding the pattern of dopamine trafficking after cocaine exposure and the timing for recovery of dopaminergic system in rats, we wanted to know whether our cocaine hydrolase, such as CocH5-Fc(M6), can effectively protect the brain from repeated cocaine exposures or help cocaine-altered-dopaminergic system to recover by preventing further damage from repeated cocaine exposures. For this purpose, two groups of rats, including four rats (L1 to L4) in one group (n=4) and four rats (L5 to L8) in the other group (n=4), were administered (i.v.) with 1.5 mg/kg CocH5-Fc(M6) for three times on days 0, 10, and 20, with daily blood collection for analysis of the plasma enzyme concentrations. Specially, two days before the first enzyme administration, i.e. on day −2, only one group of rats (L1 to L4) were injected (i.p.) with 20 mg/kg cocaine. After the first enzyme administration, both groups of rats (L1 to L8) were injected (i.p.) with 20 mg/kg cocaine on every other day from day 0 to day 32. Particularly, on days 0, 10, and 20 (the days for the enzyme administration), intraperitoneal administration of 20 mg/kg cocaine occurred at 1 h after the enzyme administration. Each time, intraperitoneal administration of 20 mg/kg cocaine was followed by locomotor activity monitoring. In addition, on day 31, all the rats (L1 to L8) were also given another dose of 20 mg/kg cocaine (i.p.), followed by blood collections for analyzing plasma concentrations of cocaine and its main metabolites. On day 33, 24 hours after the final cocaine exposure on day 32, brain samples were collected from the two groups of rats (L1 to L4 and L5 to L8) for analysis of their DAT distribution.

Figure 3A shows the western blots for rats L1-L8 in two groups, that is, one group of rats (L1 to L4) with cocaine exposure on day −2 before the enzyme administration and the other group of rats (L5 to L8) without cocaine exposure before the enzyme administration. Depicted in Figure 3B are the corresponding numeric data in comparison with the DAT distribution data from the negative control group (without cocaine exposure at all) and other two positive control groups (exposed to cocaine without enzyme administration) discussed above. According to the data shown in Figure 3, the DAT distribution for the group of rats (L5-L8) is similar to that for the negative control group, indicating that the repeated cocaine exposures did not induce DAT trafficking to plasma membrane after the enzyme administration. The DAT distribution for the other group of rats (L1-L4) is similar to that for the group of rats with one-time cocaine exposure and the brain samples were collected 30 days post cocaine exposure (data also displayed in Figure 1), which further indicates that the repeated cocaine exposures after the enzyme administration did not stop recovery of the unusual DAT distribution caused by cocaine whereas the cocaine exposure on day −2 before the enzyme administration did induce DAT trafficking. All these data consistently demonstrated that the enzyme effectively blocked the effects of repeated cocaine exposures on the DAT distribution. Figure 4 shows the measured plasma concentrations of CocH5-Fc(M6), indicating that for rats (L1-L8) the plasma CocH5-Fc(M6) concentration over 32 days post the enzyme injection was always equal or higher than 58.7 ± 2.9 nM. The pharmacokinetic data in Figure 4 along with the DAT distribution data in Figure 3B indicate that enzyme CocH5-Fc(M6) at a plasma concentration of 58.7 ± 2.9 nM or higher can effectively block the effects of 20 mg/kg cocaine (i.p.) on DAT distribution.

Figure 3.

Striatal DAT immunoreactivity after repeated exposures to cocaine (L1 to L8). Rats L1 to L4 received a dose of 20 mg/kg cocaine (i.p.) pre-treatment on day −2, then all rats received a dose of 1.5 mg/kg CocH5-Fc(M6) (i.v.) on days 0, 10, and 20, followed by repeated i.p. administration of 20 mg/kg cocaine during days 0 to 32. Brain samples were collected 24 hours after the final cocaine administration. Normalized DAT expression values are mean ± SEM for DAT density following different period after cocaine abstinence expressed as a percentage of total DAT (biotinylated plus non-biotinylated). Statistical analysis (One-way ANOVA): *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Figure 4.

Time course of enzyme concentration in plasma of rats (L1 to L8, n = 8) after the first dose of CocH5-Fc(M6) administration. Rats were administered (i.v.) with 1.5 mg/kg CocH5-Fc(M6) on days 0, 10, and 20. The plasma enzyme concentrations are plotted as the mean ± SEM mg/L in plasma at each time-point.

In addition, the exogenous enzyme CocH5-Fc(M6) in plasma also effectively blocked cocaine-induced hyperactivity, as shown in Figures 5 and 6. According to the locomotor activity data in Figure 5, intraperitoneal administration of 20 mg/kg cocaine on day −2 before the enzyme administration induced hyperactivity, as expected. However, intraperitoneal administration of 20 mg/kg cocaine at 1 h after the enzyme administration on day 0 did not induce hyperactivity compared to the control group (with saline instead of cocaine). Depicted in Figure 6 are all the locomotor activity data obtained from day 0 to day 32 after the enzyme administration in comparison with the control groups, demonstrating that cocaine administration during days 0 to 32 did not induce significant hyperactivity in the two groups of the enzyme-treated rats.

Figure 5.

Cocaine-induced hyperactivity in rats (half of group, L1, L2, L3, and L4, n = 4) of pro- and post-1.5 mg/kg CocH5-Fc(M6) (i.v.). Rats were first allowed to acclimate to the test chambers for 60 min before intraperitoneal administration of cocaine or saline. These rats first received saline to obtain the baseline, then received their 1^st dose of 20 mg/kg cocaine (i.p.) 1 day later (day −2). On the 3^rd day (day 0), they received 1.5 mg/kg CocH5-Fc(M6) (i.v.), followed with another dose of 20 mg/kg cocaine (i.p.) 1 hour later. The locomotor activity data are plotted as the mean ± SEM meters travelled in 5-min bin during locomotor activity for 3 hours (including 2 hours after the cocaine administration). Statistical analysis (one-way ANOVA): *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Figure 6.

Cocaine-induced hyperactivity in rats under impact of CocH5-Fc(M6) (n = 8 for L1 to L8) in which four rats (L1 to L4) received a dose of 20 mg/kg cocaine (i.p.) 2 days (on day −2) before their 1^st dose of 1.5 mg/kg CocH5-Fc(M6) (i.v.). Rats first received saline to get the data “Saline” shown in panels A to Q (L1 to L8, n = 8), then 3 days later (on day 0) received 1.5 mg/kg CocH5-Fc(M6) (i.v.) on days 0, 10, and 20 one hour before administrated with 20 mg/kg cocaine (i.p.) on the days. After that, rats L1 to 8 received 20 mg/kg cocaine (i.p.) on every other day from days 0 to 32 (panels A to Q). Control group received only 20 mg/kg cocaine (i.p.) with the same time schedule. The locomotor activity data are plotted as the mean ± SEM meters travelled in 5-min bin during locomotor activity for 3 hours (including 2 hours after the cocaine administration). Panel R summarizes the accumulated traveled distance (meters) within the 1^st hour after cocaine administration during the entire period of locomotor activity experiment (the saline data for control group only showed in this graph). Outcomes of statistical analysis (t-test) on the statistical analysis of the accumulated traveled distances in panel R for days 0 to 32: **** (p < 0001) for rats L1 to L4 vs cocaine (20 mg/kg) control; **** (p < 0001) for rats L5 to L8 vs cocaine (20 mg/kg) control; ns (p > 0.05) for rats L1 to L4 vs saline control; ns (p > 0.05) for rats L5 to L8 vs saline control.

Further, shown in Figure 7 are time courses of the plasma concentrations of cocaine and its metabolite ecgonine methyl ester (EME) after intraperitoneal administration of 20 mg/kg cocaine on day 31 in comparison with the corresponding plasma concentrations in the control group of rats without enzyme administration. According to the cocaine concentrations shown in Figure 7A, in the presence of the enzyme (which has k_cat = 14,600 min⁻¹,²² indicating that each enzyme molecule could hydrolyze up to 14,600 cocaine molecules per minute), cocaine was quickly eliminated with a plasma concentration at 2 min after cocaine injection being more than 10-fold lower than that in the control group. The cocaine plasma concentration decreased to the detection limit of the instrument within 10 minutes after cocaine injection. The data indicated that even with a blood concentration of 97.0 ± 5.2 nM CocH5-Fc(M6) that is around one of three local minimum values in the enzyme pharmacokinetic profile (Figure 4), CocH5-Fc(M6) still powerfully protected the brain. The observed enzyme-accelerated cocaine metabolism is consistent with the considerable increase of biologically inactive metabolite EME shown in Figure 7B, because CocH5-Fc(M6)-catalyzed hydrolysis of cocaine at the benzoyl ester moiety produces EME. The data in Figure 7 suggest that CocH5-Fc(M6) at a concentration of ≥97 nM in plasma may effectively block any physiological effect of cocaine, which explains why the enzyme was able to effectively block cocaine-induced DAT trafficking and cocaine-induced hyperactivity shown previously.

Figure 7.

Time-dependent concentrations of cocaine and its metabolite EME in the blood for 3 hours on day 31 (when plasma concentration of CocH5-Fc(M6) was 97.5 ± 5.2 nM) after i.p. administration of 20 mg/kg cocaine (L1 to L8, n = 8). Control was a group of cocaine-naïve rats injected (i.p.) with a dose of 20 mg/kg cocaine.

4. DISCUSSION

We previously demonstrated¹¹ that cocaine at an intraperitoneal dose of 10 mg/kg or higher induced trafficking of most DAT to the plasma membrane (with only <5% remained in the intracellular) at 24 hour after cocaine injection in rats. In current study, we aimed to reveal how long it could take for the dopaminergic system recovering to its normal status (in terms of intracellular and plasma membrane fractions of DAT) in rats. The western blot data presented in this report showed that after an acute cocaine exposure of 20 mg/kg i.p., the initial recovery process from day 1 to day 15 was relatively faster (from ~95.8–98.1% on day 1 to ~35.4% on day 15). However, the recovery of striatal DAT distribution may take about 60 days. Further, in rats with repeated cocaine exposures for once every other day from days 0 to 32 (a total of 17 doses of 20 mg/kg cocaine, i.p.), the complete recovery of striatal DAT distribution may take an even longer time (about 90 days), which may represent a consequence of the chronic cocaine use.

Concerning a potential treatment for cocaine use disorder, our previous studies demonstrated that exogenous CocHs can powerfully rescue rats from a lethal dose of cocaine by rapidly detoxifying cocaine and its toxic metabolites.^{30, 42} Furthermore, Fc-fused CocHs can block cocaine-induced hyperactivity, cocaine discrimination, and cocaine self-administration (associated with i.v. infusion of cocaine).³¹ In particular, CocH3-Fc(M3) was proved capable of effectively limiting the brain cocaine concentration and preventing cocaine-induced hyperactivity and DAT trafficking to plasma membrane from acute cocaine exposure.¹¹ In present study, we demonstrate that CocH5-Fc(M6), which has an even higher catalytic efficiency against cocaine than CocH3-Fc(M3),²² can completely protect rats from behavioral and physiological effects, such as hyperactivity and DAT trafficking to plasma membrane induced by repeated cocaine exposures. Particularly, based on the combined striatal DAT distribution, locomotor activity, plasma cocaine concentration, and plasma enzyme concentration data in the CocH5-Fc(M6)-treated rats, enzyme CocH5-Fc(M6) effectively accelerated cocaine metabolism through cocaine hydrolysis at the benzoyl ester moiety. Through accelerating cocaine metabolism, the enzyme completely blocked both cocaine-induced hyperactivity and DAT tracking when the enzyme maintained an average plasma concentration of ≥58.7 ± 2.9 nM.

Further, as a necessary condition for effective treatment of cocaine dependence, one would like to have the DAT system recovered to normal status in terms of intracellular and plasma membrane fractions of DAT. According to our data obtained in this study, in the presence of enzyme CocH5-Fc(M6) in plasma with a concentration ≥58.7 ± 2.9 nM, further repeated cocaine exposures did not affect the recovery of the abnormal striatal DAT distribution after previous cocaine exposure. In other words, enzyme CocH5-Fc(M6) can effectively help the DAT system of rats to recover by blocking the effects of further cocaine administration.