HUMAN GENE THERAPY CLINICAL DEVELOPMENT 25:40–49 (March 2014) ª Mary Ann Liebert, Inc. 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 and Ronald G. Crystal1

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 vaccinationmediated 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.

gers 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

Introduction

C

ocaine 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 trig1

Department of Genetic Medicine, Weill Cornell Medical College, New York, NY 10065. Division on Substance Abuse, Department of Psychiatry, New York State Psychiatric Institute, New York, NY 10032. 3 College of Physician and Surgeons, Columbia University, New York, NY 10032. 4 Center for Human Toxicology, University of Utah, Salt Lake City, UT 84112. 5 Committee on the Neurobiology of Addictive Disorders, The Scripps Research Institute, La Jolla, CA 92037. 6 The Departments of Chemistry and Immunology, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037. 7 Worm Institute for Research and Medicine, The Scripps Research Institute, La Jolla, CA 92037. 8 Center of Comparative Medicine & Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY 10065. 2

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COCAINE BIODISTRIBUTION

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 Ad5bgal, a recombinant E1a - , partial E1b - , and the E3 - serotype 5 Ad vector with bgalactosidase in the expression cassette (Hicks et al., 2011; Wee et al., 2012). The Ad5bgal vector was disrupted in 0.5% sodium dodecyl sulfate at 56C for 45 sec. The cocaine hapten GNE (0.3 mg) was activated overnight at 4C after the addition of 7.2 ll charging solution (2.4 mg of 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride and 2 mg of N-hydroxysulfosuccinimide in 4 ll H2O and 40 ll dimethylformamide) (Carrera et al., 1995; Hicks et al., 2011; Wee et al., 2012; Maoz et al., 2013). The conjugation of the disrupted Ad5bgal vector (200 lg) with the charged GNE (67 lg; 300:1 GNE to Ad capsomere molar ratio) was carried out by overnight incubation at 4C in phosphatebuffered saline (PBS; pH 7.4). The amount of Ad vector proteins was quantified using the bicinchoninic acid assay (Pierce Biotechnology, Rockford, IL).

41 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 lg, 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, 23C), and centrifuged at 3,000 · g for 15 min. The isolated serum was stored at -20C. 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 lg/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 - 80C 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 ll 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 4C. The plates were washed with 0.05% Tween 20 in PBS (PBSTween) and blocked with 5% dry milk in PBS for 30 min at 23C. Two-fold serial dilutions of serum were added to each well and incubated for 90 min at 23C. The plates were washed 4 times with PBS-Tween. Diluted horseradish peroxidase-conjugated goat antimonkey IgG (Santa Cruz Biotechnology, Santa Cruz, CA), 100 ll of 1:2,000 in 1%

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dry milk in PBS, was added to each well, and incubated for 90 min at 23C. The plates were washed four times. Peroxidase substrate (100 ll/well; Bio-Rad, Hercules, CA) was added and incubated for 15 min at 23C. The peroxidase reaction was stopped with addition of 2% oxalic acid (100 ll/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

HICKS ET AL.

specimens was performed using liquid chromatography– electrospray ionization–tandem mass spectrometry (LCESI-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

Fate of systemically administered cocaine in nonhuman primates treated with the dAd5GNE anticocaine vaccine.

Cocaine use disorders are mediated by the cocaine blockade of the dopamine transporter in the central nervous system (CNS). On the basis of the concep...
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