Active Immunization with Cocaine-Protein Conjugate Attenuates Cocaine Effects

*This manuscript is published in Pharmacology, Biochemistry, and Behavior, Vol.58, No.1, pp.215-220, 1997. It is presented here solely for use by the Cocaine Research Lab at Eastern.
R.H. Ettinger * Department of Psychology Eastern Oregon University La Grande, OR 97850 email: ettinger@eosc.osshe.edu W.F. Ettinger Department of Biology Gonzaga University Spokane, WA 99258 email: ettinger@gonzaga.edu Wendy E. Harless Department of Psychology Eastern Oregon University La Grande, OR 97850 *Corresponding author

ABSTRACT

ETTINGER, R.H., W.F. ETTINGER, AND W. HARLESS. Active Immunization with Cocaine-Protein Conjugate Attenuates Cocaine Effects. PHARMACOL BIOCHEM BEHAV. Immunization with cocaine-keyhole limpet hemocyanin (KLH) conjugate elicited the formation of anti-cocaine antibody sufficient to blunt cocaine effects in rats. Cocaine was bound to KLH for immunization with the photoactivatable crosslinker N-hydroxysuccinimide-4-azidobezoate (HSAB). Immunization with the cocaine-KLH-CFA complex was effective in attenuating the analgesic and reinforcing effects of cocaine in laboratory rats. Enzyme-linked dot blot assay revealed the presence of anti-cocaine antibody in serum. Competitive binding studies suggest that the antibody was specific to cocaine. Active immunization for cocaine may provide an alternative to drug treatment and provide protection from addiction.

Key Words: Cocaine, antibodies, analgesia, reinforcement, HSAB, rats

INTRODUCTION

It is estimated that 20 to 30 million people in the United States have used cocaine and that as many as 4 million people may be addicted. Illicit cocaine use has reached epidemic proportions and there is no evidence that this trend is decreasing (9,11,21). Cocaine's powerful reinforcing effects are attributed to its ability to inhibit dopamine reuptake in the mesolimbic-cortical system. This inhibition agonizes dopamine neuraltransmission resulting in reinforcement (8,18). Although the neural mechanisms for cocaine's effects are well understood, there appears to be no way to directly prevent them pharmacologically.

Recently, several laboratories have described cocaine-specific antibodies that may effectively interfere with cocaine's ability to agonize dopamine neuraltransmission. Catalytic antibodies, which promote rapid degradation of cocaine into its metabolites, ecgonine methyl ester and benzoic acid, have been isolated (13). Whether it is possible to develop catalytic antibodies powerful enough to block the reinforcing and analgesic effects of cocaine remains to be demonstrated. Haptens such as cocaine do not normally elicit active antibody formation. However, haptens may be conjugated with immunogenic proteins by a variety of cross-linking procedures (c.f. 10). Immunization with cocaine conjugated with keyhole limpet hemocyanin (KLH) reportedly prompted the formation of anti-cocaine antibodies capable of preventing cocaine analgesia (1). This procedure required prior oxidation of cocaine ester bonds with sodium metaperiodate. The oxidized cocaine-KLH conjugate thus formed behaved as a stable immunogen. While it is easy to visualize how preliminary metaperiodate oxidation can successfully be utilized in binding haptens containing vicinal hydroxyl groups to proteins, it is difficult to understand how such a process serves in coupling cocaine, which lacks vicinal hydroxyl groups, to KLH (c.f. 7). Cocaine has also been coupled to KLH for immunization by structurally modifying the cocaine molecule (19). Immunization with this cocaine-KLH conjugate successfully elicited antibody formation sufficient to attenuate cocaine induced locomotor activity and decrease brain levels of cocaine in immunized animals. We describe and evaluate an alternative coupling reaction for active immunization against cocaine effects.

It has been reported (6) that metoclopramide (MCP) was photolytically conjugated to bovine serum albumin (BSA) using the crosslinker HSAB (N-hydroxysuccinimide-4- azidobenzoate). The bond formed was quite stable and the conjugate acted as a good immunogen. A free-radical mechanism was postulated to account for the crosslinking process. Preliminary treatment of HSAB with BSA resulted in an amide derivitative of the photoactive crosslinker by a free amino group in the BSA. Upon UV irradiation of the HSAB derivatized BSA in the presence of MCP, free radicals were generated: A nitrene radical originating from the azido group, chloride radical, and phenyl radical resulting from homolytic cleavage of the C- Cl bond in the MCP aromatic ring. Among other products, interaction between the phenyl and nitrene radicals produced a diphenylamine derivative.

It occurred to us that in addition to the free radical crosslinking postulate, another process would be expected to predominate if the UV source was of slightly longer wavelength than that used to conjugate metoclopramide to BSA (6). In such a case, photolysis of azido groups may also give rise to nitrenes by loss of N2. These reactive nitrenes have the propensity to form bonds of the electron donor-acceptor type. Since cocaine does not lack electron donating groups, we decided to follow a procedure similar to that reported utilizing HSAB to crosslink cocaine to KLH and to BSA for immunization.

We examined the effects of immunization by evaluating cocaine antinociception for thermal pain and for reinforced place preference conditioning. Cocaine's antinociceptive effects are primarily mediated by blocking sodium conductance in peripheral pain neurons (20) and by dopamine mediation of projections to the medial thalamus (22), while cocaine's reinforcing effects are mediated centrally by dopamine agonism in the mesolymbic system (8,18).

METHODS

Preparation of cocaine-KLH Immunogen

The cocaine-KLH conjugate was prepared by first dissolving HSAB (4 mg) in 100 µl of dry DMSO, 12 µl of this solution was then added to a solution of KLH (10 mg in 1 ml of 50 mM phosphate buffer, pH 7.4). The HSAB was allowed to react with the KLH at room temperature in the dark for 10 minutes, after which excess HSAB and DMSO were removed by gel filtration through a column of Sephadex-G10 (0.8 x 16 cm) equilibrated in phosphate buffer. The KLH- containing fractions (6 x 0.5 ml, determined by absorption of UV light at 280 nm) were pooled, chilled on ice, and then mixed with 68 mg cocaine HCl. Photoactivation of the HSAB-KLH conjugation in the presence of cocaine was performed in a 3 ml quartz cuvette placed in an ice bath 9 cm from a 450 Watt immersion-type UV photochemical lamp (Ace Glass). Photoactivation was allowed to proceed for 5 minutes. The cocaine-KLH conjugate was separated from nonconjugated cocaine by exhaustive dialysis against 150 mM NaCl, 50 mM phosphate buffer pH 7.4 (PBS). A cocaine-BSA conjugate and a 3[H]-cocaine-KLH conjugate were also prepared by the same method.

The amount of cocaine conjugated to KLH by HSAB was determined by performing the coupling reaction with 3[H]-cocaine (14.1 GBq/mmol). Samples of the reaction were spotted on glass fiber filters (Fisher G4) before and after photoactivation. The filters were allowed to dry and were subsequestly assayed for trichloracetic acid (TCA) precipatble radioactivity (15).

All chemicals, except for 3[H]-cocaine, were obtained from Sigma Chemical Company, St. Louis, MO. 3[H]-cocaine was obtained from New England Nuclear, Wilmington, DE.

Animals

Thirty six experimentally-naive female Long-Evans rats 9 months in age were used (Charles River, Wilmington, MA). Animals were randomly divided into three groups of 12 for immunization. All animals were housed individually in suspended stainless steel cages (17.75 x 24.5 x 17.75 cm) in a climatically-controlled room (22 0C) with 24 hour illumination. Food and water were available ad libitum.

Immunization with Cocaine-KLH conjugate

Animals in the immunization group (n=12) were injected with 0.2 ml cocaine-KLH emulsified with 0.2 ml Complete Freund's Adjuvant (CFA). A second group of 12 animals was injected with 0.2 ml of the KLH-HSAB compound photoactivated in the absence of cocaine and emulsified with 0.2 ml CFA (KLH-HSAB control). A third group of 12 animals was injected with 0.2 ml KLH in saline emulsified with 0.2 ml CFA (KLH control). Four weeks after the initial injections, and at four week intervals throughout, all animals received booster injections with the same immunogen emulsified with incomplete Freund's adjuvant. All immunizations were administered subcutaneously to the anterior dorsal area of the back.

Hot Plate Reaction Test

We first examined the effectiveness of the cocaine-KLH conjugate in blocking cocaine effects by measuring reactions of immunized animals to a hot plate following cocaine administration. Cocaine exerts antinociceptive effects both peripherally by blocking sodium conductance (20) and centrally via dopamine projections to the medial thalamus (22). A Thermolyne Hot Plate Model # HPA1915B (Barnstead/Thermolyne Corporation) was modified by the addition of a 16.8 x 11.1 x 0.4 cm steel plate for increased temperature regulation, a four-walled Plexiglas cage (16.8 x 11.1 x 30.5 cm), and a composite wood lid (25.4 x 25.4 x 0.4 cm) to confine animals to the hot plate. A remote thermometer was used to monitor the surface temperature of the hot plate.

Three separate hot plate tests were administered to the cocaine-KLH immunized and KLH control animals over a three week period. Hot plate reaction times following either saline or cocaine administered i.p. were recorded on alternate days for each test. Five weeks post- immunization (one week after booster injections) all subjects were administered 0.25 ml 9% saline solution i.p., returned to their home cage for 15 minutes, and then tested on the hot plate (54 0C). The following day all animals were injected i.p. with 25 mg/kg cocaine HCl, returned to their home cage, and tested on the hot plate 15 minutes later. The order of injections was reversed for the second hot plate test conducted six weeks post immunization, and reversed again for the third hot plate test conducted seven weeks post immunization. The hot plate was maintained at 54 0C for all subjects during all trials. Animals remained on the hot plate until paw licking, a limb withdrawal reflex occurred, or 45 seconds had elapsed. No animals were allowed to stay on the hot plate for more than 45 seconds and no animals suffered burns or severe discomfort.

Place Preference Conditioning

The place preference conditioning (PPC) method has been used to reliably evaluate the reinforcing properties of drugs including cocaine because it allows for a rapid and direct assessment of a learned association between a drug and specific environmental stimuli (16,17,23). We used cocaine reinforced PPC to evaluate the reinforcing properties of cocaine following immunization. Conditioning trials were conducted in a wooden rectangular chamber using the same 24 animals from the cocaine-KLH immunization and KLH control groups. The chamber was divided into three separate rooms separated by guillotine doors. The two end compartments measured 31 cm X 27 cm X 25 cm high. The middle compartment was 20 cm X 10 cm X 25 cm high. One end compartment was painted dark green, had a black top, and was not illuminated. The other end compartment was white, had a clear plexi-glass top, and was illuminated with a 25 watt light located 0.5 m above the chamber. Both end compartments had wire mesh floors and were suspended above cedar shavings. The center compartment was grey, had a clear plexi-glass top and a metal floor. A mirror was located above the center compartment which allowed an observer to record when animals entered or left either end compartment. White noise masked extraneous sounds.

Place preference conditioing commenced eight weeks post immunization. Phase 1 consisted of 4 consecutive days of baseline training followed by a place preference test. During training animals were placed in the center chamber of the PPC apparatus with the guillotine doors closed, after 1 minute the doors to both end compartments were opened and the animals were allowed to explore the chamber unrestricted. After 15 minutes the animals were removed and returned to their home cage. On the fifth day time spent in each compartment was recorded during a 15 minute test. An animal was considered to be in an end compartment only when its two front paws were in that compartment. Phase 2 consisted of 8 consecutive days of cocaine-reinforced PPC followed by another place preference test. During conditioning each animal was administered either 0.25 ml of 9% saline or 10 mg/kg cocaine HCL in saline i.p. on alternate days. After saline administration the animals were restricted to the dark compartment for 15 minutes, then returned to their home cage. Following cocaine administration the animals were restricted to the white, lighted compartment for 15 minutes, then returned to their home cage. On the ninth day the animals were not injected and were place in the central compartment with the doors to both end compartments open for 15 minutes. The time spent in each compartment was recorded as before. Phase 3 consisted of 8 days of conditioning in which the PPC of Phase 2 was reversed. All of the procedures in Phase 2 were followed except that animals were placed in the white lighted compartment immediately following saline injections and in the dark compartment following cocaine injections. The animals were again tested on the ninth day using the same procedure as in Phase 2.

RESULTS

Immunoassay for anti-cocaine antibody

Immunoassays for cocaine antibodies were performed on blood drawn from tail veins 13 weeks after initial immunizations (1 week after the third booster injections) from all 36 animals. Approximately 0.25 ml of serum was frozen from each animal until assays were performed over the following 2 weeks. Enzyme-linked dot blot assays for anti-cocaine antibodies were performed for each serum sample. The dot blot assays were performed by binding serial dilutions (diluted in PBS) of the cocaine-KLH immunogen, cocaine-BSA conjugate, and BSA alone to nitrocellulose. After drying, the nitrocellulose was blocked with nonfat milk-Tris buffered saline solution and then incubated with 0.25 ml serum in 25 ml nonfat milk for two hours (12). The nitrocellulose was washed extensively in nonfat milk and then incubated for another two hours with alkaline phosphatase-conjugated goat anti-Rat IgG (Sigma) diluted 1:2000 in nonfat milk. Enzyme-linked anti-Rat IgG was detected using X-phos and nitroblue tetrazolium (3). Serum from 11 of the 12 animals immunized with KLH-cocaine conjugate revealed cocaine antibodies. Anti-cocaine antibody was not detected by this method in any of the KLH control or the KLH-HSAB control animals. Representative nitrocellulose strips are presented in Figure 1. The left half of each strip shows antibody binding to dilutions of cocaine-KLH. The presence of anti-cocaine antibody is demonstrated by the anti-rat IgG binding to cocaine-BSA on the strip shown in the right half of Figure 1a. There was no evidence of anti-rat IgG binding to the strip in the right half of figure 1b which contained BSA alone. The strip shown in Figure 1c shows KLH antibody binding and no cocaine-BSA binding for KLH control animals. Animals immunized with the KLH-HSAB photoactivated in the absence of cocaine also revealed KLH antibody binding and no cocaine- BSA binding (not shown).

Fig. 1. Representative dot blot assays for anti-cocaine antibody. Two dilutions (1.0 ug and 0.1 ug) of cocaine-KLH were placed on the left side of strips A-C. Two dilutions of cocaine-BSA (1.0 ug and 0.1 ug) were placed on the right side of strips A and C. The right side of strip B contains two dilutions (1.0 ug and 0.1 ug) of BSA alone. Strips A and B were incubated with serum from a representative animal immunized with cocaine-KLH. Binding of antibodies to the strips is indicated by stain. Binding of antibody to cocaine-KLH (A & B left side), cocaine-BSA (right side of A indicated by arrows), but not to BSA alone (B right side) indicates the presence of anti-cocaine antibody in this animal. Strip C was incubated with serum from a representative animal immunized with KLH alone. Binding of antibodies to cocaine-KLH (C left) indicates the presence of anti-KLH antibody. The absence of binding to the cocaine-BSA (C right) indicates the lack of anti-BSA or anti-cocaine antibody in this animal.

To determine the specificity of the anti-cocaine antibody 0.25 ml of serum from cocaine-KLH immunized animals was incubated for 20 min in 25 ml nonfat milk in the presence of 25 mg free cocaine before incubating with nitrocellulose. None of these samples revealed anti-rat IgG binding to cocaine-BSA (not shown), indicating that the antibody present was specific to cocaine.

Hot plate reaction test

Mean differences (D in Table 1) in hot plate reaction times for the saline versus 25 mg/kg cocaine injections are compared within groups in Figure 2 for all three hot plate tests. Statistical comparisons were made with paired t-tests and are presented in Table 1. For each test there were no differences in hot plate reaction times following saline and cocaine administration for cocaine- KLH immunized animals. All three tests revealed significant differences, however, in reaction times following saline and cocaine administration for the KLH control animals. These significant increases in hot plate reaction times for the control animals suggest that cocaine attenuated thermal pain in these animals, but not in the immunized animals. These results indicate that immunization with cocaine conjugates can successfully blunt cocaine analgesia and that this immunization effect is not transient.

Fig.2. Mean hot plate reaction times for 3 separate hot plate tests. Each test (1-3) shows the mean reaction time for control (Ctl) and immunized (Imm) animals subsequently treated with saline or 25 mg/kg cocaine respectively. Cocaine significantly increased hot plate reaction times for the control animals only in all three tests. Asterisks indicate significant differences from prior condition.

Place Preference Conditioning

The results of cocaine reinforced PPC are presented in Figure 3. The figure shows mean proportions of test time spent in the white lighted area during each phase of conditioning. There were significant differences in place preference for the KLH control animals but not for the cocaine-KLH immunized animals across preference tests (F=6.47, p=.008). That is, the KLH control animals demonstrated PPC and PPC reversal using cocaine as a reinforcer. On the other hand, the cocaine-KLH immunized animals failed to demonstrate conditioning with cocaine during either Phase 2 or 3. Because the mean baseline preference for the white area was slightly higher for the cocaine-KLH animals (.55 vs .43 for the KLH control animals, t=1.05, p=.32) the failure to demonstrate PPC for the immunized animals in Phase 2 might be attributed to a ceiling effect. Therefore, we reversed PPC during Phase 3. Even though the preferences for the white area following Phase 2 were identical for both groups (.63 vs .63) only animals in the KLH control group showed a reversal in their white area preference following conditioning with cocaine to the black area in Phase 3 (t=2.85, p<.02). These results demonstrate that active immunization with cocaine conjugate blunts cocaine's reinforcing effects as measured by PPC.

Fig.3. Mean proportions of time in the white side following baseline exposure, cocaine reinforced PPC to the white area (White CS), and cocaine reinforced PPC to the black area (Black CS) for KLH control and cocaine-KLH immunized animals. Control animals show increases in preference for the white area following conditioning to white and increases in preference to the black area following conditioning to black. Immunized animals do not show changes in place preference. Asterisks indicate significant differences from prior condition.

DISCUSSION

Animals immunized with the cocaine-KLH-CFA conjugate demonstrated resistance to both cocaine's thermal analgesic and reinforcing effects. Cocaine analgesia to thermal pain was evaluated by measuring reaction times of animals placed on a standard hot plate. Cocaine reinforcement was evaluated by place preference conditioning. Because antinociception for thermal pain is primarily mediated by peripheral neurons and cocaine reinforcement by central dopamine neurons our results suggest that anti-cocaine antibody prevents cocaine effects in both peripheral and central neurons. Furthermore, the anti-cocaine antibody elicited in our animals did not appear to undergo depletion with relatively large cocaine doses (25 mg/kg in our hot plate tests and 10 mg/kg in our PPC tests) or with repetitive cocaine administration. We observered strong cocaine effects in our control animals throught testing indicating that animals were not developing tolerance to repeated cocaine exposure. These results both support and extend those previously reported (1,19). Although we have not yet determined the level of anti-cocaine antibody present in the immunized animals, the anti-cocaine antibody appeared to be specific to cocaine since competitive binding with free cocaine in serum prevented binding to our cocaine- BSA conjugate.

Cocaine is sensitive to enzymatic hydrolysis of the methyl ester resulting in the production of benzoylecgonine (5,19). It has been suggested that conjugates designed to elicit anti-cocaine antibody production, that retain the methyl ester, may become deesterfied in vivo resulting in an appreciable anti-benzoylecgonine titre which is expected to interfere with the production of optimal levels of anti-cocaine antibodies (19). The conjugate prepared in the current report should be as sensitive to methyl ester hydrolysis as free cocaine under physiological conditions. However, since the methyl ester represents only a single epitope of the entire cocaine molecule the deesterfied conjugate should still elicit an antigenic response resulting in the production of polyclonal antibodies, many of which may still recognize cocaine by its other functonal groups. The conjugation method proposed here, utilizing HSAB, likely results in several different orientations of the exposed cocaine molecule and a broad spectrum of anti-cocaine antibodies may have been elicited. Therefore, it may never be clear what proportion of the antibody population was elicited by the conjugated metabolites ecgonine methyl ester or benzoylecgonine. Nevertheless, the HSAB conjugation method is rapid, relatively efficient, and results in a product that elicits the production of anti-cocaine antibodies. An estimate of the efficiency of binding cocaine to KLH using this method was obtained by conjugating 3[H]-cocaine to KLH. This analysis revealed that 60% of the theoretical number of HSAB molecules on KLH were conjugated to cocaine. Whether other conjugation methods yield a product that more efficiently elicits the production of anti-cocaine antibodies is yet to be determined.

Further behavioral and immunological studies are currently in progress to quantify the amount of cocaine antibody present in rat serum and to explore their long term effectiveness in attenuating the drugs' reinforcing properties and cocaine-induced analgesia. Active immunization for cocaine using conjugation methods may provide an adjuvant to drug treatment and provide protection from addiction.

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TABLE CAPTION Tab.1. Statistical comparisons of the mean differences (D) in hotplate reaction times following saline and cocaine administrations for control and cocaine immunized animals. Results for each of the three hotplate tests are shown. Cocaine significantly increased hotplate reaction times in all three tests for the control animals but not for immunized animals.