Oxidation of specific tryptophan residues inhibits high-affinity binding of cocaine and its metabolites to a humanized anticocaine mAb.

Cocaine addiction remains a serious problem lacking an effective pharmacological treatment. Thus, we have developed a high-affinity anti-cocaine monoclonal antibody (mAb), h2E2, for the treatment of cocaine use disorders. We show that selective tryptophan (Trp) oxidation by 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) resulted in a loss of high-affinity binding of cocaine to this mAb. The newly developed use of excess methionine (Met) to protect mAb met residues from AAPH oxidation did not substantially attenuate the effects of oxidation on cocaine binding but greatly decreased the modification of met residues in the mAb. Similar large decreases in ligand affinity (5000-10,000-fold) upon oxidation were observed using cocaine and two cocaine metabolites, cocaethylene and benzoylecgonine, which also bind with nanomolar affinity to this h2E2 mAb. The decrease in binding affinity was accompanied by a decrease of approximately 50% in Trp fluorescence, and increases in mAb 310 to 370 nm absorbance were consistent with the presence of oxidized forms of Trp. Finally, mass spectral analysis of peptides derived from control and AAPH-oxidized mAb indicated that excess free met did effectively protect mAb met residues from oxidation, and that AAPH-oxidized mAb heavy-chain Trp33 and light-chain Trp91 residues are important for cocaine binding, consistent with a recently derived h2E2 Fab fragment crystal structure containing bound benzoylecgonine. Thus, protection of the anti-cocaine h2E2 mAb from Trp oxidation prior to its clinical administration is critical for its proposed therapeutic use in the treatment of cocaine use disorders.

Cocaine augments neuro-inflammation via modulating extracellular vesicle release in HIV-1 infected immune cells.

BACKGROUND: Extracellular Vesicles (EV) recently have been implicated in the pathogenesis of HIV-1 syndromes, including neuroinflammation and HIV-1 associated neurological disorder (HAND). Cocaine, an illicit stimulant drug used worldwide is known to exacerbate these HIV-1 associated neurological syndromes. However, the effects of cocaine on EV biogenesis and roles of EVs in enhancing HIV-1 pathogenesis are not yet well defined. RESULTS: Here, we investigated the effects of cocaine on EV biogenesis and release in HIV-1 infected immune cells and explored their roles in elicitation of neuroinflammation. We found that cocaine significantly augmented the release of EVs from uninfected and HIV-1 infected T-cells, DCs and macrophages. Further analysis of the molecular components of EVs revealed enhanced expression of adhesion molecules integrin ß1 and LFA-1 in those EVs derived from cocaine treated cells. Intriguingly, in EVs derived from HIV-1 infected cells, cocaine treatment significantly increased the levels of viral genes in EVs released from macrophages and DCs, but not in T-cells. Exploring the molecular mechanism to account for this, we found that DCs and macrophages showed enhanced expression of the cocaine receptor Sigma 1-Receptor compared to T-cells. In addition, we found that cocaine significantly altered the integrity of the RNA-induced silencing complex (RISC) in HIV-1 infected macrophages and DCs compared to untreated HIV-1 infected cells. Characterizing further the molecular mechanisms involved in how cocaine increased EV release, we found that cocaine decreased the expression of the interferon-inducible protein BST-2; this resulted in altered trafficking of intracellular virus containing vesicles and EV biogenesis and release. We also observed EVs released from cocaine treated HIV-1 infected macrophages and DCs enhanced HIV-1 trans-infection to T-cells compared to those from untreated and HIV-1 infected cells. These EVs triggered release of proinflammatory cytokines in human brain microvascular endothelial cells (HBMECs) and altered monolayer integrity. CONCLUSIONS: Taken together, our results provide a novel mechanism which helps to elucidate the enhanced prevalence of neurological disorders in cocaine using HIV-1 infected individuals and offers insights into developing novel therapeutic strategies against HAND in these hosts.

Cocaine binding to the Fab fragment of a humanized anti-cocaine mAb quantitated by dye absorption and fluorescence spectroscopy.

In this work, we establish that cocaine binding to the Fab fragment of a recombinant humanized anti-cocaine mAb (h2E2) can be directly and easily quantitated using simple and inexpensive absorption and fluorescence measurements, employing dyes typically used for differential scanning fluorimetry, DASPMI and SYPRO Orange. For concentrated samples of the Fab fragment, absorbance spectroscopy employing these dyes reveals the number of cocaine sites present, using either DASPMI (by measuring the increase in dye absorbance) or SYPRO Orange (by measuring the change in dye maximal absorbance wavelength). Interestingly, we observed that cocaine binding to the Fab fragment had a much different effect on the SYPRO Orange dye absorbance than previously reported for the intact h2E2 mAb, resulting in a large decrease in the total dye absorbance for the Fab fragment, in contrast to previous results with the intact h2E2 mAb. For dilute samples of Fab fragment, a dye fluorescence emission spectroscopy assay was developed to quantitate the number of cocaine (and other high affinity cocaine metabolites) binding sites via the ligand-induced decrease in fluorescence emission of both of these extrinsic dyes. The difference in the cocaine titrations for the high affinity (Kd < 30 nM) ligands, cocaine, cocaethylene and benzoylecgonine and the low affinity (Kd > 30 µM) ligands, norcocaine, ecgonine methyl ester, and ecgonine were obvious using this assay. These simple, direct, and inexpensive techniques should prove useful for evaluation of other small molecule antigen binding Fab fragments, enabling quantitation and rapid biochemical assessments necessary for determining Fab fragment suitability for in vivo uses and other assays and experiments.

Ligand binding to a humanized anti-cocaine mAb measured by dye absorption spectroscopy.

Here we demonstrate that the antigen binding function of a humanized anti-cocaine mAb (h2E2) can be directly and easily determined using simple and inexpensive absorption spectroscopy and dyes commonly used for differential scanning fluorimetry, such as DASPMI and SYPRO Orange. Therapeutic monoclonal antibodies are commonly formulated in buffers which can interfere with necessary functional assays, containing additives and excipients such as mild detergents. Using the undiluted therapeutic product h2E2 mAb in its formulation buffer containing 0.01% polysorbate 80, the number of antigen/cocaine binding sites can be determined by the increase in absorbance (for DASPMI dye) or by the decrease in absorbance maximum wavelength (for SYPRO Orange dye), confirming proper function of the therapeutic mAb product. This ligand-induced visible dye absorption change can also be used to qualitatively evaluate the relative affinities of various metabolites of cocaine. These results are confirmed and extended by binding data obtained in the same formulation buffer using intrinsic tyrosine and tryptophan fluorescence quenching by cocaine, as well as by differential scanning fluorimetry. Interestingly, the binding of the cocaine metabolite norcocaine was demonstrated to be differentially affected by the pH 6 formulation buffer used for this mAb, presumably due to the differential ionizability of the demethylated norcocaine tropane ring nitrogen. This simple, direct, and inexpensive technique should prove useful for evaluation of other small molecule binding mAbs directly in their formulation buffers containing detergent, allowing rapid functional assessment of the produced therapeutic proteins.

Cocaine vaccine dAd5GNE protects against moderate daily and high-dose "binge" cocaine use.

The cocaine vaccine dAd5GNE is comprised of a disrupted serotype 5 adenovirus gene therapy vector covalently conjugated to the cocaine analog GNE. The vaccine evokes a high titer of circulating anti-cocaine antibodies that prevent cocaine from reaching its cognate receptors in the central nervous system. Prior studies have demonstrated the efficacy of dAd5GNE in models of occasional, moderate cocaine use. However, previous studies have not sufficiently evaluated the efficacy of dAd5GNE in models of the repetitive and high-dose "binge" use patterns common in human addicts. In the present study, we evaluated the capacity of dAd5GNE vaccination to protect against "binge" cocaine use and circumstances where vaccinated addicts attempt to override the vaccine. We modeled repetitive daily cocaine use in vaccinated Balb/c mice and African green monkeys, and evaluated high-dose "binge" scenarios in Balb/c mice. In each model of daily use the dAd5GNE vaccine prevented cocaine from reaching the central nervous system. In the high-dose "binge" model, vaccination decreased cocaine-induced hyperactivity and reduced the number of cocaine-induced seizures. Based on this data and our prior data in rodents and nonhuman primates, we have initiated a clinical trial evaluating the dAd5GNE anti-cocaine vaccine as a potential therapy for cocaine addicts who wish to stop cocaine use. If dAd5GNE vaccination is safe and produces high anti-cocaine antibody titers in the clinic, we hypothesize that the vaccine will restrict the access of cocaine to the central nervous system and inhibit cocaine-induced "highs" even in the context of moderate daily and high-dose "binge" use that might otherwise cause a drug-induced overdose.

Multi-domain unfolding of the Fab fragment of a humanized anti-cocaine mAb characterized by non-reducing SDS-PAGE.

Monoclonal antibodies and their fragments are widely used for research and therapy. Fab fragments are useful since they retain antigen binding specificity, but being smaller proteins, are better able to penetrate biological compartments and tumors, and do not induce Fc-dependent immunological system activation. Our laboratory developed an anti-cocaine mAb (named h2E2) for the treatment of cocaine use disorders, which is currently in advanced pre-clinical development. We are also interested in the Fab fragment of this mAb for potential therapy of acute cocaine overdose. Previously, we showed that this mAb and its F(ab')2 and Fab fragments undergo discrete domain unfolding, as detected by non-reducing SDS-PAGE, and that ligand-induced protein thermal stabilization can be quantitated utilizing differential scanning fluorimetry in the absence of SDS. Here, we demonstrate that multiple Fab protein gel bands observed using non-reducing SDS-PAGE in the presence and absence of cocaine and its metabolites can be explained and interpreted based on the differential stabilization of two unfolding domains in the Fab fragment. The variable domain is stabilized by ligands against SDS unfolding, while the constant domain is not. This data and its interpretation are also supported by differential scanning fluorimetry data for the Fab fragment in SDS. It is likely that these non-reducing SDS-PAGE results and the gel band domain unfolding model will be applicable to other small molecule binding antibodies. Thus, non-reducing SDS-PAGE is a widely available and simple method for assessing domain stability and multi-step unfolding of Fab fragments.

A novel differential scanning fluorimetry analysis of a humanized anti-cocaine mAb and its ligand binding characteristics.

An anti-cocaine monoclonal antibody (mAb) designated h2E2 will soon enter clinical trials for the treatment of cocaine abuse disorders. Importantly, this antibody selectively binds cocaine and its active metabolite, cocaethylene, with high affinity, while binding inactive metabolites with substantially lower affinities. Here, we used differential scanning fluorimetry (DSF) to characterize the stability and ligand binding properties of this antibody and its cocaine-binding Fab fragment. The Sypro orange dye commonly used for DSF revealed multiple overlapping thermal protein denaturation transitions for both the mAb and the Fab fragment, making quantitative analysis of ligand binding by thermal stabilization problematic. However, by using the "rotor" dye, DASPMI (4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide), which measures the rotational restriction of the fluorescent dye (as opposed to the Sypro orange dye which measures the hydrophobicity of the dye microenvironment), a simple two state thermal denaturation transition that is stabilized by ligand binding was observed for the h2E2 mAb, enabling Boltzmann fitting and quantitative thermodynamic analysis of the DASPMI DSF mAb cocaine and metabolite binding data. The computed affinities were consistent with ligand binding affinities determined using other techniques. Thus, this novel DASPMI DSF method can simply, inexpensively, and very rapidly generate ligand binding constants for the h2E2 mAb, despite the presence of multiple, overlapping, thermally unfolding protein domains characteristic of all mAbs. This approach is likely applicable to other mAbs currently in use for many research and therapeutic applications.

Structural analysis of free and liganded forms of the Fab fragment of a high-affinity anti-cocaine antibody, h2E2.

A high-affinity anti-cocaine monoclonal antibody, designated h2E2, is entering phase 1 clinical trials for cocaine abuse therapy. To gain insight into the molecular details of its structure that are important for binding cocaine and cocaine metabolites, the Fab fragment was generated and crystallized with and without ligand. Structures of the unliganded Fab and the Fab fragment bound to benzoylecgonine were determined, and were compared with each other and with other crystallized anti-cocaine antibodies. The affinity of the h2E2 antibody for cocaine is 4 nM, while that of the cocaine metabolite benzoylecgonine is 20 nM. Both are higher than the reported affinity for cocaine of the two previously crystallized anti-cocaine antibodies. Consistent with cocaine fluorescent quenching binding studies for the h2E2 mAb, four aromatic residues in the CDR regions of the Fab (TyrL32, TyrL96, TrpL91 and TrpH33) were found to be involved in ligand binding. The aromatic side chains surround and trap the tropane moiety of the ligand in the complex structure, forming significant van der Waals interactions which may account for the higher affinity observed for the h2E2 antibody. A water molecule mediates hydrogen bonding between the antibody and the carbonyl group of the benzoyl ester. The affinity of binding to h2E2 of benzoylecgonine differs only by a factor of five compared with that of cocaine; therefore, it is suggested that h2E2 would bind cocaine in the same way as observed in the Fab-benzoylecgonine complex, with minor rearrangements of some hypervariable segments of the antibody.

Disposable electrochemical immunosensor array for the multiplexed detection of the drug metabolites morphine, tetrahydrocannabinol and benzoylecgonine.

Heroin, marijuana and cocaine are widely abused drugs. Their use can be readily detected by analyzing urine for the metabolites morphine (MOR), tetrahydrocannabinol (THC) or benzoylecgonine (BZC). A multiplex immunosensor is described here for detection of MOR, THC and BZC using screen printed carbon array electrodes modified with gold nanoparticles. Antibodies against MOR, THC and BZC were immobilized on eight electrodes in a sensor array simultaneously, and a competitive assay was used for the detection. The free analytes in the sample compete with bovine serum albumin-conjugated analytes for the immobilized antibodies on the sensor surface. The array is capable of detecting the three drugs simultaneously within 20-40 min. The method has a high sensitivity, with detection limits as low as 1.2, 7.0, and 8.0 pg.mL-1 for MOR, THC and BZC, respectively. Cross reactivity testing was preformed to monitor any nonspecific binding. The results revealed good selectivity. Urine samples were spiked with the 3 drugs and tested with the multiplexed immunosensor. Recovery percentages ranged between 88 to 115%. Graphical abstract Schematic presentation of the multiplexed immunosensor for drugs of abuse,viz. tetrahydrocannabinol (THC), morphine (MOR), and benzoylecgonine (BZC)) by using an array of modified electrodes.

Unfolding of IgG domains detected by non-reducing SDS-PAGE.

Monoclonal antibodies are very important in modern therapeutics and constitute a substantial percentage of newly approved drugs. Every therapeutic monoclonal antibody must be analyzed for structural and functional integrity, and all protein heterogeneities need to be identified and quantified. The conformational stabilities of the monoclonal antibodies are also important for antibody storage and handling stabilities. One of the first and simplest of the structural analysis techniques utilized is SDS-PAGE, which can be performed both with and without prior reduction to break disulfide bonds. This permits sizing of both heavy and light chains in the reduced condition, and sizing of the intact antibody and any disulfide aggregates in the non-reduced condition. Analyzing our human anti-cocaine monoclonal antibody, we noted unexpectedly larger apparent molecular weights and apparent protein size heterogeneities using non-reducing SDS-PAGE. These apparent molecular weight heterogeneities are not consistent with other analysis techniques. Heterogeneities were noted using several heating and pre-electrophoretic sample preparation protocols, and are modified by the inclusion of small concentrations of certain alcohols such as propanol and butanol. All of these unexpected results were also observed for a commercial human IgG1 antibody, suggesting that these observations are applicable to IgGs in general. Thus, careful attention must be paid to the interpretation of non-reducing SDS-PAGE results for IgGs. It is hypothesized that differential thermal unfolding of the Fab, CH2 and CH3 domains of the IgGs in SDS give rise to the stable, discrete bands observed using different heating protocols prior to non-reducing SDS-PAGE.

Evaluation of methods to reduce background using the Python-based ELISA_QC program.

Almost all immunological approaches [immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), Western blot], that are used to quantitate specific proteins have had to address high backgrounds due to non-specific reactivity. We report here for the first time a quantitative comparison of methods for reduction of the background of commercial biotinylated antibodies using the Python-based ELISA_QC program. This is demonstrated using a recombinant humanized anti-cocaine monoclonal antibody. Several approaches, such as adjustment of the incubation time and the concentration of blocking agent, as well as the dilution of secondary antibodies, have been explored to address this issue. In this report, systematic comparisons of two different methods, contrasted with other more traditional methods to address this problem are provided. Addition of heparin (HP) at 1 µg/ml to the wash buffer prior to addition of the secondary biotinylated antibody reduced the elevated background absorbance values (from a mean of 0.313 ±â€¯0.015 to 0.137 ±â€¯0.002). A novel immunodepletion (ID) method also reduced the background (from a mean of 0.331 ±â€¯0.010 to 0.146 ±â€¯0.013). Overall, the ID method generated more similar results at each concentration of the ELISA standard curve to that using the standard lot 1 than the HP method, as analyzed by the Python-based ELISA_QC program. We conclude that the ID method, while more laborious, provides the best solution to resolve the high background seen with specific lots of biotinylated secondary antibody.

Investigations into the efficacy of multi-component cocaine vaccines.

Although cocaine addiction remains a serious health and societal problem in the United States, no FDA-approved treatment has been developed. Vaccines offer an exciting strategy for the treatment of cocaine addiction; however, vaccine formulations need to be optimized to improve efficacy. Herein, we examine the effectiveness of a tricomponent cocaine vaccine, defined as having its hapten (GNE) and adjuvant (cytosine-guanine oligodeoxynucleotide 1826, CpG ODN 1826) covalently linked via the immunogenic protein ovalbumin (OVA). The tricomponent vaccine (GNE-OVA-CpG 1826) and a vaccine of analogous, individual components (GNE-OVA+CpG ODN 1826) were found to similarly induce highly specific anticocaine antibody production in mice and block cocaine's stimulant effects in hyperlocomotor testing.

Cocaine Allergy in Drug-Dependent Patients and Allergic People.

BACKGROUND: Adverse reactions to local anesthetics (LAs), especially esters, are not uncommon, but true allergy is rarely diagnosed. To our knowledge, currently there is no reliable method of determining IgE-mediated hypersensitivity to LAs and cocaine. OBJECTIVE: To assess the clinical value of allergy tests (prick, IgE, challenges, and arrays) in people suffering hypersensitivity reactions (asthma and anaphylaxis) during local anesthesia with cocaine derivatives and drug abusers with allergic symptoms after cocaine inhalation. METHODS: We selected cocaine-dependent patients and allergic patients who suffered severe reactions during local anesthesia from a database of 23,873 patients. The diagnostic yield (sensitivity, specificity, and predictive value) of allergy tests using cocaine and coca leaf extracts in determining cocaine allergy was assessed, taking a positive challenge as the criterion standard. RESULTS: After prick tests, specific IgE, and challenge with cocaine extract, 41 of 211 patients (19.4%) were diagnosed as sensitized to cocaine. Prick tests and IgE to coca leaves (coca tea) had a good sensitivity (95.1% and 92.7%, respectively) and specificity (92.3 and 98.8%, respectively) for the diagnosis of cocaine allergy and LA-derived allergy. CONCLUSIONS: Cocaine may be an important allergen. Drug abusers and patients sensitized to local anesthesia and tobacco are at risk. Both prick tests and specific IgE against coca leaf extract detected sensitization to cocaine. The highest levels were related to severe clinical profiles.

Augmenting the efficacy of anti-cocaine catalytic antibodies through chimeric hapten design and combinatorial vaccination.

Given the need for further improvements in anti-cocaine vaccination strategies, a chimeric hapten (GNET) was developed that combines chemically-stable structural features from steady-state haptens with the hydrolytic functionality present in transition-state mimetic haptens. Additionally, as a further investigation into the generation of an improved bifunctional antibody pool, sequential vaccination with steady-state and transition-state mimetic haptens was undertaken. While GNET induced the formation of catalytically-active antibodies, it did not improve overall behavioral efficacy. In contrast, the resulting pool of antibodies from GNE/GNT co-administration demonstrated intermediate efficacy as compared to antibodies developed from either hapten alone. Overall, improved antibody catalytic efficiency appears necessary to achieve the synergistic benefits of combining cocaine hydrolysis with peripheral sequestration.

A novel Python program for implementation of quality control in the ELISA.

The use of semi-quantitative assays such as the enzyme-linked immunosorbent assay (ELISA) requires stringent quality control of the data. However, such quality control is often lacking in academic settings due to unavailability of software and knowledge. Therefore, our aim was to develop methods to easily implement Levey-Jennings quality control methods. For this purpose, we created a program written in Python (a programming language with an open-source license) and tested it using a training set of ELISA standard curves quantifying the Fab fragment of an anti-cocaine monoclonal antibody in mouse blood. A colorimetric ELISA was developed using a goat anti-human anti-Fab capture method. Mouse blood samples spiked with the Fab fragment were tested against a standard curve of known concentrations of Fab fragment in buffer over a period of 133days stored at 4°C to assess stability of the Fab fragment and to generate a test dataset to assess the program. All standard curves were analyzed using our program to batch process the data and to generate Levey-Jennings control charts and statistics regarding the datasets. The program was able to identify values outside of two standard deviations, and this identification of outliers was consistent with the results of a two-way ANOVA. This program is freely available, which will help laboratories implement quality control methods, thus improving reproducibility within and between labs. We report here successful testing of the program with our training set and development of a method for quantification of the Fab fragment in mouse blood.

Characterization of a recombinant humanized anti-cocaine monoclonal antibody produced from multiple clones for the selection of a master cell bank candidate.

We have generated a humanized anti-cocaine monoclonal antibody (mAb), which is at an advanced stage of pre-clinical development. We report here in vitro binding affinity studies, and in vivo pharmacokinetic and efficacy studies of the recombinant mAb. The overall aim was to characterize the recombinant antibody from each of the three highest producing transfected clones and to select one to establish a master cell bank. In mAb pharmacokinetic studies, after injection with h2E2 (120 mg/kg iv) blood was collected from the tail tip of mice over 28 days. Antibody concentrations were quantified using ELISA. The h2E2 concentration as a function of time was fit using a two-compartment pharmacokinetic model. To test in vivo efficacy, mice were injected with h2E2 (120 mg/kg iv), then one hour later injected with an equimolar dose of cocaine. Blood and brain were collected 5 min after cocaine administration. Cocaine concentrations were quantified using LC/MS. The affinity of the antibody for cocaine was determined using a [3H] cocaine binding assay. All three antibodies had long elimination half-lives, 2-5 nM Kd for cocaine, and prevented cocaine's entry into the brain by sequestering it in the plasma. Pharmacokinetic and radioligand binding assays supported designation of the highest producing clone 85 as the master cell bank candidate. Overall, the recombinant h2E2 showed favorable binding properties, pharmacokinetics, and in vivo efficacy.

Neutrophil extracellular traps as a potential source of autoantigen in cocaine-associated autoimmunity.

Objective: Exposure to illicit cocaine and its frequent adulterant, levamisole, is associated with ANCAs targeting neutrophil elastase (NE), neutropenia and vasculitic/thrombotic skin purpura. The mechanisms of cocaine/levamisole-associated autoimmunity (CLAA) are unknown. The aim of this study was to assess the ability of cocaine and levamisole to induce the release of neutrophil extracellular traps (NETs), a potential source of autoantigen and tissue injury. Methods: We performed quantitative and qualitative assessment of NET formation in neutrophils from healthy donors exposed to either drug in vitro . In addition, IgG from sera of individuals with CLAA (CLAA-IgG) was assessed for its ability to enhance formation of, and to bind to, drug-induced NETs. Results: Both cocaine and levamisole could induce formation of NETs enriched in NE and, potentially, inflammatory mitochondrial DNA. Both drugs could also augment simultaneous release of B cell-activating factor belonging to the TNF family (BAFF). CLAA-IgG, but not IgG from healthy individuals, could potentiate drug-induced NETosis. Furthermore, CLAA-IgG, but not ANCA + control IgG, bound to drug-induced NETs in a pattern consistent with NE targeting. Conclusion: Both cocaine and levamisole may contribute to the development of ANCAs by inducing release of potentially inflammatory NETs in association with NE autoantigen and BAFF. Enhancement of drug-induced NET release by CLAA-IgG provides a potential mechanism linking vasculitis/pupuric skin disease to acute drug exposure in patients with CLAA. Further study of this under-recognized form of autoimmunity will be likely to provide mechanistic insight into ANCA-associated vasculitis and other diseases associated with NETosis.

Efficacy of an adenovirus-based anti-cocaine vaccine to reduce cocaine self-administration and reacqusition using a choice procedure in rhesus macaques.

Immunopharmacotherapy offers an approach for treating cocaine abuse by specifically targeting the cocaine molecule and preventing its access to the CNS. dAd5GNE is a novel cocaine vaccine that attenuates the stimulant and the reinforcing effects of cocaine in rats. The goal of this study was to extend and validate dAd5GNE vaccine efficacy in non-human primates. Six experimentally naïve adult female rhesus monkeys (Macaca mulatta) were trained to self-administer 0.1mg/kg/injection intravenous (i.v.) cocaine or receive candy; then 4 monkeys were administered the vaccine and 2 monkeys were administered vehicle intramuscularly, with additional vaccine boosts throughout the study. The reinforcing effects of cocaine were measured during self-administration, extinction, and reacquisition (relapse) phases. Serum antibody titers in the vaccinated monkeys remained high throughout the study. There was no change in the preference for cocaine over candy over a 20-week period in 5 of the 6 monkeys; only one of the 4 (25%) vaccinated monkeys showed a decrease in cocaine choice. All 6 monkeys extinguished responding for cocaine during saline extinction testing; vaccinated monkeys tended to take longer to extinguish responding than control monkeys (17.5 vs. 7.0 sessions). Vaccination substantially retarded reacquisition of cocaine self-administration; control monkeys resumed cocaine self-administration within 6-41 sessions and 1 vaccinated monkey resumed cocaine self-administration in 19 sessions. The other 3 vaccinated monkeys required between 57 and 94 sessions to resume cocaine self-administration even in the context of employing several manipulations to encourage cocaine reacquisition. These data suggest that the dAdGNE vaccine may have therapeutic potential for humans who achieve cocaine abstinence as part of a relapse prevention strategy.

Cocaine Vaccine Development: Evaluation of Carrier and Adjuvant Combinations That Activate Multiple Toll-Like Receptors.

Although cocaine abuse and addiction continue to cause serious health and societal problems, an FDA-approved medication to treat cocaine addiction has yet to be developed. Employing a pharmacokinetic strategy, an anticocaine vaccine provides an attractive avenue to address these issues; however, current vaccines have shown varying degrees of efficacy, indicating that further formulation is necessary. As a means to improve vaccine efficacy, we examined the effects of varying anticocaine vaccine formulations by combining a Toll-like receptor 9 (TLR9) agonist with a TLR5 agonist in the presence of alum. The TLR9 agonist used was cytosine-guanine oligodeoxynucleotide 1826 (CpG 1826), while the TLR5 agonist was flagellin (FliC). Formulations with the TLR9 agonist elicited superior anticocaine antibody titers and blockade of hyperlocomotor effects compared to vaccines without CpG 1826. This improvement was seen regardless of whether the TLR5 agonist, FliC, or the nonadjuvanting Tetanus Toxoid (TT) was used as the carrier protein. Additional insights into the value of FliC as a carrier versus adjuvant was also investigated by generating two unique formats of the protein, wild-type and mutated flagellin (mFliC). While the mFliC conjugate retained its ability to stimulate mTLR5, it yielded reduced cocaine sequestration and functional blockade relative to FliC and TT. Overall, this work indicates that activation of TLR9 can improve the function of cocaine vaccines in the presence of TLR5 activation by FliC, with any potential additive effects limited by the inefficiency of FliC as a carrier protein as compared to TT.

The effects of a repeated dose of a recombinant humanized anti-cocaine monoclonal antibody on cocaine self-administration in rats.

BACKGROUND: Immunotherapy has shown potential as a treatment for cocaine abuse. The humanized recombinant anti-cocaine monoclonal antibody (mAb) with the preclinical designation h2E2 has been shown to decrease cocaine concentrations in the brain in rats, but its effects on cocaine self-administration behavior have never been tested. METHODS: The amount of cocaine needed to reinstate self-administration behavior (priming threshold) was calculated and the inter-injection intervals at unit doses of 0.3µmol/kg and 3µmol/kg during maintained self-administration were measured over a five-week baseline period. Rats trained to self-administer cocaine were infused with two doses of h2E2 (120mg/kg i.v.) 35days apart. Priming threshold and inter-injection intervals were measured for 35days after both injections. RESULTS: After both injections of h2E2, priming thresholds were significantly increased (3-fold) compared to expected baseline and then gradually declined over 35days. A significant decrease (15-33%) in inter-injection intervals during maintained self-administration was also observed following both h2E2 infusions at the lower dose, and after the first injection at the higher dose. No significant decreases in body weight were observed after either injection, indicating a lack of toxicity following a second injection. CONCLUSIONS: These data predict that the safety and effectiveness of h2E2 will be maintained after multiple treatments of this potential immunotherapy for cocaine abuse.