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. 2014 Feb 14;25(1):40–49. doi: 10.1089/humc.2013.231

Fate of Systemically Administered Cocaine in Nonhuman Primates Treated with the dAd5GNE Anticocaine Vaccine

Martin J Hicks 1, Stephen M Kaminsky 1, Bishnu P De 1, Jonathan B Rosenberg 1, Suzette M Evans 2,,3, Richard W Foltin 2,,3, David M Andrenyak 4, David E Moody 4, George F Koob 5, Kim D Janda 6,,7, Rodolfo J Ricart Arbona 8, Michelle L Lepherd 8, Ronald G Crystal 1,✉
PMCID: PMC4047994  PMID: 24649839

Abstract

Cocaine use disorders are mediated by the cocaine blockade of the dopamine transporter in the central nervous system (CNS). On the basis of the concept that these effects could be obviated if cocaine were prevented from reaching its cognate receptors in the CNS, we have developed an anticocaine vaccine, dAd5GNE, based on a cocaine analog covalently linked to capsid proteins of an E1−E3− serotype 5 adenovirus. While the vaccine effectively blocks systemically administered cocaine from reaching the brain by mediating sequestration of the cocaine in the blood, the fact that cocaine also has significant peripheral effects raises concerns that vaccination-mediated redistribution could lead to adverse effects in the visceral organs. The distribution of systemically administered cocaine at a weight-adjusted typical human dose was evaluated along with cocaine metabolites in both dAd5GNE-vaccinated and control nonhuman primates. dAd5GNE sequestration of cocaine to the blood not only prevented cocaine access to the CNS, but also limited access of both the drug and its metabolites to other cocaine-sensitive organs. The levels of cocaine in the blood of vaccinated animals rapidly decreased, suggesting that while the antibody limits access of the drug and its active metabolites to the brain and sensitive organs of the periphery, it does not prolong drug levels in the blood compartment. Gross and histopathology of major organs found no vaccine-mediated untoward effects. These results build on our earlier measures of efficacy and demonstrate that the dAd5GNE vaccine-mediated redistribution of administered cocaine is not likely to impact the vaccine safety profile.

Introduction

Cocaine is a highly addictive small-molecule drug of abuse with 1.6 million users in the United States (Goldstein et al., 2009; Koob and Volkow, 2010; Substance Abuse and Mental Health Services Administration, 2012). Cocaine inhibits neuronal monoamine transporters, primarily the dopamine reuptake transporter (DAT), leading to accumulation of dopamine in the ventral striatum, caudate, and putamen of the basal ganglia, with consequent increased dopaminergic neurotransmission (Di and Imperato, 1988; Benowitz, 1993; Koob and Volkow, 2010). Repeat use triggers adaptive changes in neuronal circuits underlying reinforcement, reward, and sensitization, associated with addictive behavior (Koob and Volkow, 2010; Kreek et al., 2012).

On the basis of the concept that the effects of cocaine on the central nervous system (CNS) could be obviated if cocaine could be prevented from reaching its cognate receptors in the CNS, we have developed a vaccine (dAd5GNE) consisting of a disrupted serotype 5 E1−E3− human adenovirus to which the cocaine analog GNE has been covalently linked (Hicks et al., 2011; Wee et al., 2012; Maoz et al., 2013; Cai et al., 2013). Studies in mice, rats, and nonhuman primates have demonstrated that dAd5GNE induces high levels of high-affinity anticocaine antibodies that sequester systemically administered cocaine in the blood, preventing cocaine-induced hyperlocomotor activity and sensitization (Hicks et al., 2011; Wee et al., 2012; Maoz et al., 2013).

While the dAd5GNE vaccine prevents access of cocaine to the CNS, cocaine can affect organ systems outside of the CNS. Cocaine disruption of monoamine homeostasis occurs both in the CNS and systemically (Muscholl, 1961; Calligaro and Eldefrawi, 1987; Brody et al., 1990; Lipton et al., 2000; Fowler et al., 2007; Goldstein et al., 2009; Koob and Volkow, 2010). All subtypes of dopamine receptors are expressed in varying proportions in the kidney, adrenal glands, sympathetic ganglia, gastrointestinal tract, blood vessels, and heart (Lackovic and Neff, 1983; Volkow et al., 1992; Benowitz, 1993; Boschetti et al., 2010). Functions mediated by dopamine receptors that are localized outside the CNS include olfaction, vision, and hormone regulation, including adrenocorticotropic hormone, corticosterone, epinephrine, norepinephrine, prolactin, renin, and aldosterone and regulation of sympathetic tone, renal function, blood pressure, and gastrointestinal motility (Mueller et al., 1990; Volkow et al., 1992; Benowitz, 1993; Gray, 1993; Mendelson et al., 2003). Cocaine also alters the homeostasis of other monoamines in peripheral organs, including the serotonin transporter in the gastrointestinal-digestive tract and heart, and norepinephrine transporters in the adrenal glands, liver, and heart (Nayak et al., 1976; Benowitz, 1993; Lipton et al., 2000; Ding et al., 2003).

In the context that cocaine has widespread systemic effects, one potential adverse consequence of an effective anticocaine vaccine could be the altered distribution of cocaine in the viscera, driven by antibody–cocaine complexes, resulting in high levels of local organ cocaine accumulation and subsequent toxicity (Narvaez et al., 2013). To assess this issue, we evaluated the biodistribution of cocaine and its metabolites in blood and relevant organs and examined all major organs for evidence of toxicology after systemic administration of cocaine to naive and dAd5GNE-vaccinated nonhuman primates.

Methods

dAd5GNE vaccine

The source of the adenovirus capsid proteins for the dAd5GNE vaccine was Ad5βgal, a recombinant E1a−, partial E1b−, and the E3− serotype 5 Ad vector with β-galactosidase in the expression cassette (Hicks et al., 2011; Wee et al., 2012). The Ad5βgal vector was disrupted in 0.5% sodium dodecyl sulfate at 56°C for 45 sec. The cocaine hapten GNE (0.3 mg) was activated overnight at 4°C after the addition of 7.2 μl charging solution (2.4 mg of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and 2 mg of N-hydroxysulfosuccinimide in 4 μl H2O and 40 μl dimethylformamide) (Carrera et al., 1995; Hicks et al., 2011; Wee et al., 2012; Maoz et al., 2013). The conjugation of the disrupted Ad5βgal vector (200 μg) with the charged GNE (67 μg; 300:1 GNE to Ad capsomere molar ratio) was carried out by overnight incubation at 4°C in phosphate-buffered saline (PBS; pH 7.4). The amount of Ad vector proteins was quantified using the bicinchoninic acid assay (Pierce Biotechnology, Rockford, IL).

Study design

Six female Rhesus macaques (Macaca mulatta) received the vaccine or a control: four were vaccinated with the dAd5GNE vaccine (animals V1, V2, V3, and V4) formulated in 20% Adjuplex™ (Advanced BioAdjuvants LLC, Omaha, NE) and two controls received placebo (animals C1 and C2). The vaccinated animals received dAd5GNE (100 μg, 0.5 ml) by intramuscular injection to the quadriceps as needed to maintain high anticocaine titers. The vaccine was administered an average 7.2±0.7 times over 12 months and the animals were subjected to cocaine challenges of either 1 mg/kg once every 2 months or on a near-daily basis over a 15-month period. To determine specificity of elicited antibodies for cocaine, venous blood was collected at 8–9 weeks after the initial vaccination and the mean % inhibition by cocaine or cocaine metabolite of anticocaine antibody binding was assayed by competitive ELISA. To assess anticocaine antibody titer at the time of the cocaine biodistribution study, venous blood was collected the same day (V1, V2, V3, C1, and C2) or 1 week before (V4). After collection, the blood was allowed to clot (30 min, 23°C), and centrifuged at 3,000×g for 15 min. The isolated serum was stored at −20°C. The weight of the animals at the time of necropsy was 6.6±0.5 kg.

Monkeys were anesthetized with ketamine (7.3±0.9 mg/kg) and dexmedetomidine (14±2 μg/kg). Cocaine (1 mg/kg) was delivered intravenously. Blood samples were collected (0, 2.5, 15, and 60 min) in tubes containing sodium fluoride and potassium oxalate, stored on ice, centrifuged at 3,000×g for 15 min, and serum was collected. After 60 min, the monkey was euthanized with an intravenous administration of pentobarbital (82±9 mg/kg) and phenytoin (9.8±1.2 mg/kg). The animals were then perfused with 8 liters cold PBS, and the brain and organs were immediately collected. Each organ was harvested separately and evaluated for gross lesions and histopathology by a board-certified veterinary pathologist. Samples of each organ were collected in 0.1–2 g aliquots. Upon collection, serum and organ samples were either immediately flash-frozen in liquid nitrogen, transferred to dry ice for transport, and stored at −80°C or fixed in 10% neutral buffered formalin for histopathology (Supplementary Table S1; Supplementary Data are available online at www.liebertpub.com/humc). The fixed tissues were embedded in paraffin, sectioned, and stained with hematoxylin and eosin for microscopic examination.

Anticocaine antibody titers

To quantify anticocaine titers after vaccination, wells of flat-bottomed 96-well EIA/RIA plates (Corning, New York, NY) were coated with 100 μl of 1 mg/ml of the cocaine hapten GNE (Hicks et al., 2011; Wee et al., 2012; Cai et al., 2013) conjugated to bovine serum albumin at a molar ratio of 66:1 in bicarbonate buffer, pH 9.4, overnight at 4°C. The plates were washed with 0.05% Tween 20 in PBS (PBS-Tween) and blocked with 5% dry milk in PBS for 30 min at 23°C. Two-fold serial dilutions of serum were added to each well and incubated for 90 min at 23°C. The plates were washed 4 times with PBS-Tween. Diluted horseradish peroxidase-conjugated goat antimonkey IgG (Santa Cruz Biotechnology, Santa Cruz, CA), 100 μl of 1:2,000 in 1% dry milk in PBS, was added to each well, and incubated for 90 min at 23°C. The plates were washed four times. Peroxidase substrate (100 μl/well; Bio-Rad, Hercules, CA) was added and incubated for 15 min at 23°C. The peroxidase reaction was stopped with addition of 2% oxalic acid (100 μl/well). Absorbance was measured at 415 nm. Anticocaine antibody titers were calculated by interpolation of the log(OD)–log(dilution) with a cutoff value equal to twofold the absorbance of background. To assess dAd5GNE-evoked antibody specificity for cocaine and its metabolites, inhibition of dAd5GNE sera binding to anticocaine ELISA plate was performed in the presence of increasing concentrations (10−9 to 10−3 M) of cocaine, cocaethylene, benzoylecgonine, norcocaine, ecgonine methyl ester, and procaine.

Cocaine and metabolite analysis

The analysis of cocaine and cocaine metabolites was carried out at the Center for Human Toxicology at the University of Utah. The analysis of the serum and organ specimens was performed using liquid chromatography–electrospray ionization–tandem mass spectrometry (LC-ESI-MS/MS). The basis of the method as developed for cocaine and benzoylecgonine was as described by Lin et al. (2001) and methods for assessment of ecgonine methyl ester, and norcocaine as described by Lin et al. (2003).

The tissues were first weighed and then homogenized with 9 parts of buffer (0.1 M sodium phosphate pH 6.0 with 1% sodium fluoride). The tissue analysis also included an additional set of positive controls prepared from homogenates of brains from untreated rats (no cocaine administration) that were fortified to 50 ng/ml with cocaine and metabolites. The assay has an analytical range of 2.5–750 ng/ml with an undiluted 1.0 ml aliquot. With dilutions because of homogenization and/or <1.0 ml aliquot size, the lower limits of detection were as follows: serum, 10–25 ng/ml; organs (spleen, lung, liver, and heart), 25 mg/g; and the putamen and adrenal gland, 100 ng/g.

Deuterated cocaine, benzoylecgonine, and ecgonine methyl ester or norcocaine were added to plasma (1.0 ml) as the internal standards. The pH of the plasma was made acidic (pH 4.0) by the addition of acetate buffer and the mixture extracted using mixed-mode octyl and benzoyl sulfonate solid-phase extraction. The eluant was evaporated and reconstituted with methanol/0.1% formic acid in water mixture (1:9) and analyzed by LC-ESI-MS/MS. The mass spectrometer was operated in the selected reaction-monitoring mode. Quadrupole Q1 was set to pass only the MH+ions that are caused to undergo collision-induced dissociation in quadrupole Q2 to abundant product ions as follows: cocaine and cocaine-d3 at m/z 304–182, and 307–185, respectively; benzoylecgonine and benzoylecgonine-d3 at m/z 290–168 and 293–171, respectively; and ecgonine methyl ester and ecgonine methyl ester-d3 at m/z 200–182 and 203–185, respectively. The product ions were selectively filtered by quadrupole Q3 for analysis. The concentration of the analytes was determined from the ratio of the analyte peak area divided by the peak area of the spiked analyte internal standard; the equations from standard curves enabled the conversion to concentration for each cocaine metabolite and cocaine in the human plasma.

The tissues were first weighed and then homogenized with 9 parts of buffer (0.1 M sodium phosphate pH 6.0 with 1% sodium fluoride). From this, a 1 ml aliquot was taken. This resulted in a 1:10 dilution and an analytical range of approximately 10–25 up to 7,500 ng/ml per sample. The tissue analysis also included an additional set of negative controls from the homogenate of the brain from an untreated (no cocaine administration) rat that was established in previous experiments. The lower limits of detection for samples were as follows: serum, 10–25 ng/ml; organs (spleen, lung, liver, and heart), 25 ng/ml; and the putamen and adrenal gland, 100 ng/ml.

Results

Cocaine is metabolized into active, norcocaine, and cocaethylene and inactive, benzoylecgonine, and ecgonine methyl ester that do or do not block the dopamine uptake transporter (Fig. 1A). Vaccination with the dAd5GNE vaccine evoked antibodies with high specificity for cocaine and the active metabolite, cocaethylene (physiological by-product from the covalent interaction of cocaine and alcohol), and with lower specificity for the active metabolite, norcocaine (Fig. 1B) (Dean et al., 1992; Brzezinski et al., 1997; Allen, 2011). The anticocaine antibodies showed lower specificity for the nonactive metabolites, benzoylecgonine and ecgonine methyl ester, and much lower binding affinity for procaine, a low affinity control.

FIG. 1.

FIG. 1.

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dAd5GNE generated anticocaine antibody specificity for cocaine compared with cocaine metabolites. (A) Cocaine metabolic pathway (active and nonactive metabolites). (B) dAd5GNE evoked anticocaine antibody specificity for cocaine and active metabolites (cocaethylene and norcocaine) and nonactive metabolites (benzoylecgonine and ecgonine methyl ester). Procaine was used as a low-affinity control. Specificity is displayed as a function of serum anticocaine antibody binding (% inhibition) according to molar concentration of cocaine or cocaine metabolite competitor (M). Each value is % inhibition (average of 3 vaccinated animals±SEM).

Administration of the dAd5GNE vaccine evoked high titers of anticocaine antibody in the animals (n=4), whereas nonvaccinated controls (n=2) showed no anticocaine antibody titers (Fig. 2). After intravenous administration of cocaine, examination of the blood compartment revealed that cocaine and metabolites were at the expected levels in control animals, and at higher levels in the blood of vaccinated animals (Fig. 3). Cocaine serum concentration rapidly increased in the first 2.5 min, with vaccinated animals having a threefold or greater increase in cocaine serum levels (mean of 3,354±456 ng/ml) compared with controls, demonstrating that the anticocaine antibody retained the cocaine molecule in the blood compartment (Fig. 3A and B). The serum levels of cocaine of vaccinated animals remained high during the course of the 60 min study and dropped below the level of detection in the controls by 15 min, suggesting that the cocaine was metabolized and/or sequestered into other compartments.

FIG. 2.

FIG. 2.

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Anticocaine antibody titers of dAd5GNE immunized nonhuman primates (V1, V2, V3, and V4) compared with control (C1 and C2) animals. Displayed is the dAd5GNE-evoked anticocaine antibody titer−1×105 in vaccinated (immunized) animals as assessed by ELISA.

FIG. 3.

FIG. 3.

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Cocaine and metabolites (ng/ml serum) in the blood over 60 min time course after the administration of cocaine (1 mg/kg, intravenous). (A) Cocaine, controls. (B) Cocaine, immunized. (C) Benzoylecgonine, control. (D) Benzoylecgonine, immunized. (E) Ecgonine methyl ester, controls. (F) Ecgonine methyl ester, immunized. (G) Norcocaine, controls. (H) Norcocaine, immunized. Concentrations are displayed on y-ordinate as ng of cocaine or cocaine metabolite per ml of serum at time points indicated (0, 2.5, 15, and 60 min after cocaine administration). Dashed lines show limit of detection (25 ng/ml) of cocaine or metabolite in the serum compartment. Note change in scale on y-ordinate: (A, B) 0–5,000 ng/ml; (C, D) 0–350 ng/ml; (E–H) 0–150 ng/ml.

The serum levels of benzoylecgonine and ecgonine methyl ester were similar in vaccinated and control animals at early time points (2.5 and 15 min). At the later time point (60 min), the metabolites (benzoylecgonine, ecgonine methyl ester, and norcocaine) were more abundant in the vaccinated animals, indicating that the cocaine molecule was retained in the blood compartment of vaccinated animals and metabolic breakdown into benzoylecgonine, ecgonine methyl ester, and norcocaine was not slowed (Fig. 3A–H).

Evaluation of cocaine content in the blood, the brain (putamen), and peripheral organs (adrenal gland, spleen, lung, heart, and liver) at 60 min postdrug administration revealed striking differences between controls and vaccinated animals (Fig. 4). At the 60 min time point, both control animals showed no cocaine in the blood compartment but high levels of cocaine in the putamen (Fig. 4A), critical to the addiction pathway and abundant in the high-affinity receptors for cocaine and DAT. Analysis of the adrenal gland confirmed significantly high levels typical for the cocaine-stimulated hypothalamo-pituitary-adrenal axis (Fig. 4A). In control animals, cocaine levels in the heart, kidney, and liver were detectable, with levels in the spleen and lung less consistent but easily detectable (Fig. 4A). In contrast, the vaccinated animals showed high levels of cocaine retained in the blood (Fig. 4B), but there were no detectable levels of cocaine in the putamen or adrenal gland and cocaine levels in the vaccinated animals were lower in the spleen, lung, heart, and kidney relative to control animals. The liver showed similar distribution of cocaine in the vaccinated as compared with the control animals. These findings indicate that vaccination not only blocked cocaine from crossing the blood–brain barrier but also prevented cocaine access to cocaine-sensitive organs in the chest and abdomen (Fig. 4B vs. 4A).

FIG. 4.

FIG. 4.

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Distribution of cocaine in the blood, brain (putamen), and peripheral organs 1 hr after administration of cocaine (1 mg/kg, intravenous). (A) Control. (B) Immunized. Blood was collected at 60 min, animals were immediately euthanized, and brain and organs were collected. The y-axis shows the drug concentration (ng cocaine/ml serum or g tissue). The x-axis indicates the distribution according to blood compartment (serum), brain (putamen), and peripheral organs (adrenal, spleen, lung, heart, kidney, and liver). Black dashed line shows limit of detection in the putamen and adrenal (100 ng/g). Gray dashed line shows limit of detection (25 ng/ml or ng/g in blood compartment [serum] and specific organs [spleen, lung, heart, kidney, and liver]). The scale on y-ordinate is same for both panels, 0–1,600 ng/ml or ng/g.

At 60 min after cocaine administration, the major metabolite of cocaine, benzoylecgonine, was in low abundance in the serum in control animals, with no detectable levels in the putamen, adrenal gland, or spleen (Fig. 5). Benzoylecgonine was detectable in the lung of one control and detectable levels were also found in the heart, kidney, and liver (Fig. 5A). The vaccinated animals showed abundant levels of benzoylecgonine in the serum, suggesting that antibody binding to cocaine did not prevent metabolism to the major nonactive metabolite (Fig. 5A and B). Benzoylecgonine levels were below the limit of detection in the putamen, adrenal gland, and spleen in all vaccinated animals and in liver of three of four vaccinated animals. Only one animal showed minimal levels in the lung and heart (Fig. 5B). These results suggest that vaccination protects not only the brain, but also peripheral organs from cocaine toxicity and also from cocaine metabolite benzoylecgonine.

FIG. 5.

FIG. 5.

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Distribution of cocaine metabolites (benzoylecgonine, ecgonine methyl ester, and norcocaine) in the blood, brain (putamen), and peripheral organs (adrenal, spleen, lung, heart, kidney, and liver) 1 hr after administration of cocaine (1 mg/kg, intravenous). (A) Benzoylecgonine, control. (B) Benzoylecgonine, immunized. (C) Ecgonine methyl ester, controls. (D) Ecgonine methyl ester, immunized. (E) Norcocaine, controls. (F) Norcocaine, immunized. Blood was collected at 60 min, animals were immediately euthanized, and brain and organs were collected. The y-axis shows the concentration of drug metabolite (ng of cocaine metabolite per ml of serum or g of tissue). The x-axis indicates the distribution according to blood compartment (serum), brain (putamen), and peripheral organs (adrenal, spleen, lung, heart, kidney, and liver). Black dashed line shows limit of detection in the putamen and adrenal (100 ng/g). Gray dashed line shows limit of detection (25 ng/ml or ng/g) in blood compartment (serum) and specific organs (spleen, lung, heart, kidney, and liver). Note that scale on y-ordinate is same for all panels, 0–350 ng/ml or ng/g.

The second major metabolite in the breakdown of cocaine, ecgonine methyl ester, was just above the limit of detection in the serum of the two control animals (Fig. 5C) and detectable levels were observed in the brain, as well as all peripheral organs of the two controls. In comparison, in the vaccinated animals, ecgonine methyl ester was below detectable levels in the brain, adrenal gland, and heart (Fig. 5D), but detectable in the spleen and kidney, with minimal levels in the lung and liver of two vaccinated animals.

Cocaine is also demethylated, mostly in the liver, to form the minor active metabolite, norcocaine, and therefore its detection only in that organ is not unexpected (Fig. 5E). Vaccinated animals showed similar levels of norcocaine in the liver relative to controls (Fig. 5E and F). Higher levels of norcocaine were found in the kidney and to a lesser degree in the lung and spleen of the vaccinated versus control animals, but there were no detectable levels of norcocaine in the brain, heart, or adrenal gland.

Gross and microscopic examination found that one vaccinated animal had tissue necrosis around the site of injection, which may have been caused by nearly concurrent intramuscular injection of sedatives. One vaccinated monkey had a focal area of gliosis in olivary complex in the brain, with inflammatory cell infiltration. The significance of this lesion is unknown. All other findings were deemed typical of Rhesus macaques and none of these findings were considered vaccine related (Supplementary Tables S2 and S3).

In this study, we did not assess the affinity of the antibodies evoked by dAd5GNE for anhydroecgonine methyl ester, a cocaine pyrolysis product of crack cocaine, and thus the distribution of this component in crack cocaine users is unknown.

Discussion

Cocaine is a highly addictive small-molecule drug abused worldwide (Volkow, 2010; Substance Abuse and Mental Health Services Administration, 2012). The allure of cocaine is the rapid euphoria experienced by the user, resulting from rapid access of the molecule to the CNS and blockage of the dopamine transporter resulting in accumulation of dopamine in the synaptic cleft of mesolimbic neurons (Di and Imperato, 1988; Koob and Volkow, 2010). To attenuate the positive subjective effects and the direct reinforcing effects of cocaine, we have developed dAd5GNE, an anticocaine vaccine designed to prevent administered cocaine from reaching the brain (Hicks et al., 2011; Wee et al., 2012; Maoz et al., 2013). dAd5GNE is comprised of a disrupted serotype 5 adenovirus E1−E3− gene transfer vector to which GNE (Cai et al., 2013), a cocaine analog, is covalently attached. The vaccine is designed to leverage the strong antiadenovirus immune response in humans to produce high-avidity antibodies against cocaine (Harvey et al., 1999; Chirmule et al., 1999; Hackett et al., 2000; Hicks et al., 2011; Wee et al., 2012; Maoz et al., 2013; Cai et al., 2013). High levels of high-avidity anticocaine antibodies prevent systemically administered cocaine from crossing the blood–brain barrier and entering the CNS and reaching the cognate receptors in the brain (Maoz et al., 2013; Cai et al., 2013). With the goal of assessing the fate of systemically administered cocaine in the context of vaccination with dAd5GNE, we have evaluated the levels of cocaine and its metabolites in the brain, blood, and peripheral organs after intravenous administration of cocaine to nonhuman primates vaccinated with dAd5GNE after chronic exposure to cocaine. The results demonstrate that dAd5GNE vaccination significantly shielded both the CNS and peripheral organs, including the adrenal gland, spleen, lung, kidney, and liver from cocaine and the metabolites benzoylecgonine, ecgonine methyl ester. No findings in peripheral organs suggested vaccine-mediated toxicity.

Cocaine metabolism

To be effective, the anticocaine antibodies evoked by the dAd5GNE vaccine must block not only cocaine itself but also its active cocaine metabolites from access to the CNS. Cocaine is extensively metabolized, with less than 10% excreted in the urine (Barnett et al., 1981; Jeffcoat et al., 1989; Benowitz, 1993; Scheidweiler et al., 2010; Bystrowska et al., 2012). Enzymatic and nonenzymatic hydrolysis to benzoylecgonine accounts for an average of 45% of cocaine, while enzymatic hydrolysis to ecgonine methyl ester accounts for about 40% of cocaine metabolism, and microsomal oxidative metabolism of cocaine to norcocaine only accounts for a small percentage (1–3%) of the metabolized drug (Jeffcoat et al., 1989; Benowitz, 1993; Gorelick, 1997; Scheidweiler et al., 2010; Yao et al., 2013). These cocaine metabolites and cocaine per se are potentially toxic to not only the CNS but also the heart, liver, and other organs (Kloss et al., 1983; Silva et al., 1991; Benowitz, 1993; Hollander, 1995; Restrepo et al., 2007). Therefore, vaccine-mediated redistribution of administered cocaine and/or its metabolites represents a potential safety concern.

Distribution of cocaine and cocaine metabolites in the context of dAd5GNE immunization

Cocaine rapidly distributes to body tissues with a moderate affinity (Jeffcoat et al., 1989; Benveniste et al., 2005). The heart and cardiovascular systems are particularly sensitive to cocaine accumulation (Benowitz, 1993; Hollander, 1995; Connors and Hoffman, 2013; Pilgrim et al., 2013; Ibrahim et al., 2013). In the heart, cocaine inhibits membrane permeability to sodium during depolarization of cell membranes, thereby blocking the transmission of fast sodium currents and the electrical impulse of sensory neurons. This sedative effect on heart tissue can contribute to the arrhythmic potential of cocaine. It also activates α-adrenoreceptors in the heart causing vasoconstriction in coronary arteries, increases the concentration of calcium in cardiac myocytes, and, at high dose, inhibits norepinephrine reuptake that may affect cardiac action potentials (Muscholl, 1961; Derlet and Albertson, 1989; Brody et al., 1990; Rump et al., 1995; Lipton et al., 2000). Autonomic and neuromuscular effects include tachycardia, hypertension, hyperthermia, respiratory arrest, and cardiovascular collapse (Derlet and Albertson, 1989; Lipton et al., 2000; Lange and Hillis, 2001). In addition to its profound effects on the brain and the heart, cocaine also has significant effects on peripheral organs, altering metabolism in the blood vessels, adrenal glands, and kidneys within minutes of cocaine administration (Benowitz, 1993; Blake et al., 1994; Mendelson et al., 2003). In these organs, butylcholinesterase and hydrolases enzymatically degrade the cocaine molecule (Barnett et al., 1981; Jeffcoat et al., 1989; Dean et al., 1992; Brzezinski et al., 1997; Gorelick, 1997), and these cocaine degradation products are potentially toxic in the organs in which they accumulate (Kloss et al., 1983; Silva et al., 1991; Lipton et al., 2000).

The data in the present study demonstrate that dAd5GNE-evoked antibodies prevent cocaine from reaching the brain and other critical peripheral organs without delaying metabolic breakdown of the cocaine. In the serum of vaccinated animals, the levels of cocaine were reduced by threefold in 1 hr, where it is degraded into its major metabolite, benzoylecgonine, and to a lesser degree ecgonine methyl ester and norcocaine. At the same time, in vaccinated animals, cocaine did not accumulate in the brain or peripheral organs. dAd5GNE vaccination also reduced the accumulation of cocaine and its metabolites in peripheral tissues. The heart is not the only organ sensitive to cocaine-induced α-adrenoreceptor activation and the release of catelcholamines, norepinephrine, and epinephrine (Virmani et al., 1988; Derlet and Albertson, 1989; Brody et al., 1990; Lipton et al., 2000). Cocaine-associated ischemic injury has also been documented in many peripheral organs, including the muscle, kidney, placenta, large intestine, and liver (Muscholl, 1961; Benowitz, 1993; Gray, 1993; Lange and Hillis, 2001; Restrepo et al., 2007). dAd5GNE immunization reduced cocaine, benzoylecgoine, and ecgonine methyl ester distribution in the brain, adrenal glands, spleen, lung, heart, and kidney. The liver of dAd5GNE-vaccinated animals did not show increased levels of cocaine and its metabolite, norcocaine, and lower levels of benzoylecgoine and ecgonine methyl ester were observed. It is likely, therefore, that dAd5GNE would protect the brain, heart, lungs, and spleen from cocaine-induced vasoconstriction and not result in adverse effects on the liver or kidney.

In previous studies, we have shown that dAd5GNE vaccination results in high-titer, high-affinity anticocaine antibodies that can abrogate cocaine-mediated locomotor behaviors in mice and rats, alter drug self-administration in rats, and bind and sequester cocaine in the blood compartment in nonhuman primates such that cocaine binding in the CNS remains below the threshold of reinforcement (Volkow et al., 1997; Wee et al., 2012; Maoz et al., 2013; De et al., 2013). In the present study, we demonstrate that the vaccine protects not only the brain but also the relevant peripheral organs from cocaine and its active metabolites without toxicity. If these findings are replicated in human clinical trials, dAd5GNE may prove to be a therapeutic option for cocaine addiction.

Supplementary Material

Supplemental data
Supp_Table1.pdf (19.1KB, pdf)
Supplemental data
Supp_Table2.pdf (22.9KB, pdf)
Supplemental data
Supp_Table3.pdf (28.8KB, pdf)

Acknowledgments

We thank Jeannine C. Rodgers and the veterinarian staff of the Research Animal Resource Center Memorial Sloan-Kettering Cancer Center, New York, NY, for help with the study, and D.N. McCarthy and N. Mohamed, Weill Cornell Medical College, New York, NY, for help in preparing this article. These studies were supported, in part, by the National Institute on Drug Abuse (NIDA) RC2DA028847, R01DA032702, N01DA-9-7767, DA08590 (K.D.J.), and DA031749. We also thank the NIDA drug supply program for the cocaine used in this study and Advanced BioAdjuvants LLC (Omaha, NE) for the Adjuplex™ used in this study. M.J.H. was supported, in part, by T32HL094284.

Author Disclosure Statement

No conflict of interest declared by any of the authors.

References

  1. Allen K.R. (2011). Screening for drugs of abuse: which matrix, oral fluid or urine? Ann. Clin. Biochem. 48, 531–541 [DOI] [PubMed] [Google Scholar]
  2. Barnett G., Hawks R., and Resnick R. (1981). Cocaine pharmacokinetics in humans. J. Ethnopharmacol. 3, 353–366 [DOI] [PubMed] [Google Scholar]
  3. Benowitz N.L. (1993). Clinical pharmacology and toxicology of cocaine. Pharmacol. Toxicol. 72, 3–12 [DOI] [PubMed] [Google Scholar]
  4. Benveniste H., Fowler J.S., Rooney W., et al. (2005). Maternal and fetal 11C-cocaine uptake and kinetics measured in vivo by combined PET and MRI in pregnant nonhuman primates. J. Nucl. Med. 46, 312–320 [PubMed] [Google Scholar]
  5. Blake M.J., Buckley A.R., Buckley D.J., et al. (1994). Neural and endocrine mechanisms of cocaine-induced 70-kDa heat shock protein expression in aorta and adrenal gland. J. Pharmacol. Exp. Ther. 268, 522–529 [PubMed] [Google Scholar]
  6. Boschetti M., Gatto F., Arvigo M., et al. (2010). Role of dopamine receptors in normal and tumoral pituitary corticotropic cells and adrenal cells. Neuroendocrinology 92Suppl 1, 17–22 [DOI] [PubMed] [Google Scholar]
  7. Brody S.L., Slovis C.M., and Wrenn K.D. (1990). Cocaine-related medical problems: consecutive series of 233 patients. Am. J. Med. 88, 325–331 [DOI] [PubMed] [Google Scholar]
  8. Brzezinski M.R., Spink B.J., Dean R.A., et al. (1997). Human liver carboxylesterase hCE-1: binding specificity for cocaine, heroin, and their metabolites and analogs. Drug Metab. Dispos. 25, 1089–1096 [PubMed] [Google Scholar]
  9. Bystrowska B., Adamczyk P., Moniczewski A., et al. (2012). LC/MS/MS evaluation of cocaine and its metabolites in different brain areas, peripheral organs and plasma in cocaine self-administering rats. Pharmacol. Rep. 64, 1337–1349 [DOI] [PubMed] [Google Scholar]
  10. Cai X., Whitfield T., Hixon M.S., et al. (2013). Probing active cocaine vaccination performance through catalytic and noncatalytic hapten design. J. Med. Chem. 56, 3701–3709 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Calligaro D.O., and Eldefrawi M.E. (1987). Central and peripheral cocaine receptors. J. Pharmacol. Exp. Ther. 243, 61–68 [PubMed] [Google Scholar]
  12. Carrera M.R., Ashley J.A., Parsons L.H., et al. (1995). Suppression of psychoactive effects of cocaine by active immunization. Nature 378, 727–730 [DOI] [PubMed] [Google Scholar]
  13. Chirmule N., Propert K., Magosin S., et al. (1999). Immune responses to adenovirus and adeno-associated virus in humans. Gene Ther. 6, 1574–1583 [DOI] [PubMed] [Google Scholar]
  14. Connors N.J., and Hoffman R.S. (2013). Experimental treatments for cocaine toxicity: a difficult transition to the bedside. J. Pharmacol. Exp. Ther. 347, 251–257 [DOI] [PubMed] [Google Scholar]
  15. De B.P., Pagovich O.E., Hicks M.J., et al. (2013). Disrupted adenovirus-based vaccines against small addictive molecules circumvent anti-adenovirus immunity. Hum. Gene Ther. 24, 58–66 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Dean R.A., Harper E.T., Dumaual N., et al. (1992). Effects of ethanol on cocaine metabolism: formation of cocaethylene and norcocaethylene. Toxicol. Appl. Pharmacol. 117, 1–8 [DOI] [PubMed] [Google Scholar]
  17. Derlet R.W., and Albertson T.E. (1989). Emergency department presentation of cocaine intoxication. Ann. Emerg. Med. 18, 182–186 [DOI] [PubMed] [Google Scholar]
  18. Di C.G., and Imperato A. (1988). Drugs abused by humans preferentially increase synaptic dopamine concentrations in the mesolimbic system of freely moving rats. Proc. Natl. Acad. Sci. USA 85, 5274–5278 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ding Y.S., Lin K.S., Garza V., et al. (2003). Evaluation of a new norepinephrine transporter PET ligand in baboons, both in brain and peripheral organs. Synapse 50, 345–352 [DOI] [PubMed] [Google Scholar]
  20. Fowler J.S., Kroll C., Ferrieri R., et al. (2007). PET studies of d-methamphetamine pharmacokinetics in primates: comparison with l-methamphetamine and (-)-cocaine. J. Nucl. Med. 48, 1724–1732 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Goldstein R.A., DesLauriers C., Burda A., et al. (2009). Cocaine: history, social implications, and toxicity: a review. Semin. Diagn. Pathol. 26, 10–17 [DOI] [PubMed] [Google Scholar]
  22. Gorelick D.A. (1997). Enhancing cocaine metabolism with butyrylcholinesterase as a treatment strategy. Drug Alcohol Depend. 48, 159–165 [DOI] [PubMed] [Google Scholar]
  23. Gray J.D. (1993). Medical consequences of cocaine. Can. Fam. Physician 39, 1975–1981 [PMC free article] [PubMed] [Google Scholar]
  24. Hackett N.R., Kaminsky S.M., Sondhi D., et al. (2000). Antivector and antitransgene host responses in gene therapy. Curr. Opin. Mol. Ther. 2, 376–382 [PubMed] [Google Scholar]
  25. Harvey B.G., Hackett N.R., El-Sawy T., et al. (1999). Variability of human systemic humoral immune responses to adenovirus gene transfer vectors administered to different organs. J. Virol. 73, 6729–6742 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Hicks M.J., De B.P., Rosenberg J.B., et al. (2011). Cocaine analog coupled to disrupted adenovirus: a vaccine strategy to evoke high-titer immunity against addictive drugs. Mol. Ther. 19, 612–619 [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hollander J.E. (1995). The management of cocaine-associated myocardial ischemia. N. Engl. J. Med. 333, 1267–1272 [DOI] [PubMed] [Google Scholar]
  28. Ibrahim M., Maselli D.J., Hasan R., et al. (2013). Safety of beta-blockers in the acute management of cocaine-associated chest pain. Am. J. Emerg. Med. 31, 613–616 [DOI] [PubMed] [Google Scholar]
  29. Jeffcoat A.R., Perez-Reyes M., Hill J.M., et al. (1989). Cocaine disposition in humans after intravenous injection, nasal insufflation (snorting), or smoking. Drug Metab. Dispos. 17, 153–159 [PubMed] [Google Scholar]
  30. Kloss M.W., Rosen G.M., and Rauckman E.J. (1983). N-demethylation of cocaine to norcocaine. Evidence for participation by cytochrome P-450 and FAD-containing monooxygenase. Mol. Pharmacol. 23, 482–485 [PubMed] [Google Scholar]
  31. Koob G.F., and Volkow N.D. (2010). Neurocircuitry of addiction. Neuropsychopharmacology 35, 217–238 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kreek M.J., Levran O., Reed B., et al. (2012). Opiate addiction and cocaine addiction: underlying molecular neurobiology and genetics. J. Clin. Invest. 122, 3387–3393 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lackovic Z., and Neff N.H. (1983). Minireview. Evidence that dopamine is a neurotransmitter in peripheral tissues. Life Sci. 32, 1665–1674 [DOI] [PubMed] [Google Scholar]
  34. Lange R.A., and Hillis L.D. (2001). Cardiovascular complications of cocaine use. N. Engl. J. Med. 345, 351–358 [DOI] [PubMed] [Google Scholar]
  35. Lin S.N., Moody D.E., Bigelow G.E., et al. (2001). A validated liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry method for quantitation of cocaine and benzoylecgonine in human plasma. J. Anal. Toxicol. 25, 497–503 [DOI] [PubMed] [Google Scholar]
  36. Lin S.N., Walsh S.L., Moody D.E., et al. (2003). Detection and time course of cocaine N-oxide and other cocaine metabolites in human plasma by liquid chromatography/tandem mass spectrometry. Anal. Chem. 75, 4335–4340 [DOI] [PubMed] [Google Scholar]
  37. Lipton J.W., Mangan K.P., and Silvestri J.M. (2000). Acute cocaine toxicity: pharmacology and clinical presentations in adult and pediatric populations. J. Pharm. Practice 13, 159–169 [Google Scholar]
  38. Maoz A., Hicks M.J., Vallabhjosula S., et al. (2013). Adenovirus capsid-based anti-cocaine vaccine prevents cocaine from binding to the nonhuman primate CNS dopamine transporter. Neuropsychopharmacology 38, 2170–2178 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Mendelson J.H., Sholar M.B., Mutschler N.H., et al. (2003). Effects of intravenous cocaine and cigarette smoking on luteinizing hormone, testosterone, and prolactin in men. J. Pharmacol. Exp. Ther. 307, 339–348 [DOI] [PubMed] [Google Scholar]
  40. Mueller P.D., Benowitz N.L., and Olson K.R. (1990). Cocaine. Emerg. Med. Clin. North Am. 8, 481–493 [PubMed] [Google Scholar]
  41. Muscholl E. (1961). Effect of cocaine and related drugs on the uptake of noradrenaline by heart and spleen. Br. J. Pharmacol. Chemother. 16, 352–359 [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Narvaez J.C., Magalhaes P.V., Fries G.R., et al. (2013). Peripheral toxicity in crack cocaine use disorders. Neurosci. Lett. 544, 80–84 [DOI] [PubMed] [Google Scholar]
  43. Nayak P.K., Misra A.L., and Mule S.J. (1976). Physiological disposition and biotransformation of (3H) cocaine in acutely and chronically treated rats. J. Pharmacol. Exp. Ther. 196, 556–569 [PubMed] [Google Scholar]
  44. Pilgrim J.L., Woodford N., and Drummer O.H. (2013). Cocaine in sudden and unexpected death: a review of 49 post-mortem cases. Forensic Sci. Int. 227, 52–59 [DOI] [PubMed] [Google Scholar]
  45. Restrepo C.S., Carrillo J.A., Martinez S., et al. (2007). Pulmonary complications from cocaine and cocaine-based substances: imaging manifestations. Radiographics 27, 941–956 [DOI] [PubMed] [Google Scholar]
  46. Rump A.F., Theisohn M., and Klaus W. (1995). The pathophysiology of cocaine cardiotoxicity. Forensic Sci. Int. 71, 103–115 [DOI] [PubMed] [Google Scholar]
  47. Scheidweiler K.B., Spargo E.A., Kelly T.L., et al. (2010). Pharmacokinetics of cocaine and metabolites in human oral fluid and correlation with plasma concentrations after controlled administration. Ther. Drug Monit. 32, 628–637 [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Silva M.O., Roth D., Reddy K.R., et al. (1991). Hepatic dysfunction accompanying acute cocaine intoxication. J. Hepatol. 12, 312–315 [DOI] [PubMed] [Google Scholar]
  49. Substance Abuse and Mental Health Services Administration. (2012). Results from the 2011 National Survey on Drug Use and Health: Mental Health Findings. NSDUH Series H-45, HHS Publication NO. (SMA) 12–4725, Rockville, MD [Google Scholar]
  50. Virmani R., Robinowitz M., Smialek J.E., et al. (1988). Cardiovascular effects of cocaine: an autopsy study of 40 patients. Am. Heart J. 115, 1068–1076 [DOI] [PubMed] [Google Scholar]
  51. Volkow N.D. (2010). Cocaine: Abuse and Addiction. NIH Publication 10-4166, 1-8 National Institutes of Health National Institute on Drug Abuse, Bethesda, MD [Google Scholar]
  52. Volkow N.D., Fowler J.S., Wolf A.P., et al. (1992). Distribution and kinetics of carbon-11-cocaine in the human body measured with PET. J. Nucl. Med 33, 521–525 [PubMed] [Google Scholar]
  53. Volkow N.D., Wang G.J., Fischman M.W., et al. (1997). Relationship between subjective effects of cocaine and dopamine transporter occupancy. Nature 386, 827–830 [DOI] [PubMed] [Google Scholar]
  54. Wee S., Hicks M.J., De B.P., et al. (2012). Novel cocaine vaccine linked to a disrupted adenovirus gene transfer vector blocks cocaine psychostimulant and reinforcing effects. Neuropsychopharmacology 37, 1083–1091 [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Yao D., Shi X., Wang L., et al. (2013). Characterization of differential cocaine metabolism in mouse and rat through metabolomics-guided metabolite profiling. Drug Metab. Dispos. 41, 79–88 [DOI] [PMC free article] [PubMed] [Google Scholar]

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