Vaccination against nicotine during continued nicotine administration in rats: immunogenicity of the vaccine and effects on nicotine distribution to brain

Vaccination against nicotine during continued nicotine administration in rats: immunogenicity of the vaccine and effects on nicotine distribution to brain

International Journal of Immunopharmacology 22 (2000) 809±819 www.elsevier.com/locate/ijimmpharm Vaccination against nicotine during continued nicoti...

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International Journal of Immunopharmacology 22 (2000) 809±819 www.elsevier.com/locate/ijimmpharm

Vaccination against nicotine during continued nicotine administration in rats: immunogenicity of the vaccine and e€ects on nicotine distribution to brain Y. Hieda a, D.E. Keyler a,c, S. Ennifar b, A. Fattom b, P.R. Pentel a,c,* a

Minneapolis Medical Research Foundation, Minneapolis, MN, USA b Nabi, Rockville, MD, USA c Department of Medicine, Hennepin County Medical Center, Minneapolis, MN, USA Received 8 February 2000; accepted 26 May 2000

Abstract Vaccination against nicotine has been proposed as a potential treatment for nicotine dependence. Because vaccination may take months to elicit satisfactory antibody levels, the clinical usefulness of this approach will be enhanced if vaccination can be accomplished during continued nicotine intake (e.g., before a smoker quits). The current study examined the immunogenicity of a nicotine conjugate vaccine during continued nicotine dosing in rats, and its e€ects on nicotine distribution to brain. In the ®rst experiment, nicotine was administered over 11 weeks as 20 intra venous (i.v.) bolus injections per day during the rat's active cycle to simulate the usual pattern of nicotine intake from cigarette smoking. In the second experiment, rats received a continuous s.c. infusion of nicotine by osmotic pump for 11 weeks to provide serum nicotine concentrations equivalent to those of a heavy smoker and 24 h/day nicotine exposure. Nicotine-speci®c antibody titers after the third booster dose were not compromised by either regimen of concurrent nicotine administration compared to those of rats receiving saline. A single additional i.v. nicotine dose was administered at the end of each experiment. The distribution of this single nicotine dose to brain was reduced by 40±60% in vaccinated rats compared to controls. Vaccine ecacy in reducing nicotine distribution to brain was not compromised by concurrent nicotine administration. These data suggest that vaccination during concurrent nicotine administration is feasible, and that the ability of vaccination to reduce nicotine distribution to brain is preserved even after months of nicotine dosing at rates approximating cigarette smoking. 7 2000 International Society for Immunopharmacology. Published by Elsevier Science Ltd. All rights reserved. Keywords: Nicotine; Vaccine; Immunization; Antibody; Pharmacokinetics; Dependence

* Corresponding author at Department of Medicine, Hennepin County Medical Center, 701 Park Avenue S., Minneapolis, MN 55415, USA. Tel.: +1-612-347-6426; fax: +1-612-904-4366. E-mail address: [email protected] (P.R. Pentel). 0192-0561/00/$20.00 7 2000 International Society for Immunopharmacology. Published by Elsevier Science Ltd. All rights reserved. PII: S 0 1 9 2 - 0 5 6 1 ( 0 0 ) 0 0 0 4 2 - 4

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1. Introduction Use of tobacco is a leading preventable cause of death worldwide, responsible for an estimated 4,000,000 deaths in 1998 [1]. Despite recent advances in behavioral and pharmacologic treatments, the vast majority of cigarette smokers who try to quit will fail [2]. New approaches to smoking cessation and prevention are clearly needed. Vaccination has been suggested as a general approach to the treatment of drug abuse. Drug-speci®c antibodies elicited by a suitable vaccine can bind drug in blood, reduce its distribution to brain, and thereby reduce its addictive e€ects [3±5]. A cocaine vaccine has been shown to reduce cocaine self-administration in rats, and is currently undergoing clinical evaluation (personal communication, M Kasaian). Nicotine is the principal addictive component of tobacco [6]. Vaccination against nicotine is therefore of interest as a potential means of treating or preventing tobacco dependence. A nicotine conjugate vaccine has been developed consisting of nicotine linked to a carrier protein, recombinant Pseudomonas aeruginosa exoprotein A. When administered to rats, this vaccine elicits high titers of nicotine-speci®c antibodies, reduces nicotine distribution to brain, and reduces some of the physiologic and behavioral e€ects of nicotine [7]. In actively immunized (vaccinated) rats, the distribution to brain of a single nicotine dose of 0.03 mg/kg (equivalent to the nicotine intake from two cigarettes in a human) was reduced by 64%. In passively immunized rats (treated with infusions of nicotine-speci®c IgG from rabbits), the pressor e€ect of a single nicotine dose was substantially reduced, and nicotine-induced locomotor activation was completely prevented. These e€ects were observed at clinically relevant nicotine doses and with serum antibody concentrations that are potentially achievable in humans [8,9]. If a nicotine vaccine is to be used for an application such as smoking cessation, it would be desirable for the patient to have therapeutic antibody titers at the time that he or she attempts to quit smoking. To accomplish this, patients will need to be vaccinated while they are still smoking

because immunization with a conjugate vaccine typically requires several weeks to months to elicit maximal antibody levels [7,10]. This requirement raises two questions; will concurrent exposure to nicotine (free hapten) compromise the vaccine's immunogenicity, and will it compromise the ability of nicotine-speci®c antibodies to reduce nicotine distribution to brain. Successful immunization against cocaine during daily cocaine administration has been reported, although the cocaine dosing schedule was not described [5], and vaccination against methamphetamine has been observed to be e€ective during repeated methamphetamine dosing (personal communication, M. Owens). However, exposure to nicotine (which typically occurs over at least 12 h/day for a smoker) is more regular and sustained than for either cocaine or methamphetamine. Whether immunization against nicotine can be achieved during chronic nicotine dosing at rates that approximate cigarette smoking is not known. The possible e€ect of chronic nicotine administration on the ability of vaccination to reduce nicotine distribution to brain is critical to its potential as a treatment for tobacco dependence. Chronic exposure to nicotine could saturate the available nicotine-speci®c antibodies, reduce their ability to bind nicotine in serum and extracellular ¯uid, and reduce their ability to block nicotine entry into brain. It is, therefore, important to determine the extent to which chronic exposure to nicotine, at doses that approximate nicotine delivery from cigarette smoking, reduces the pharmacokinetic ecacy of vaccination. The current study examined the immunogenicity of a nicotine conjugate vaccine in rats during two regimens of nicotine exposure extending over 11 weeks. The ®rst regimen provided nicotine as repeated intra venous (i.v.) bolus doses delivered over 12 h/day. Each individual nicotine dose was equivalent on a mg/kg basis to the nicotine absorbed by a smoker from one cigarette, and the total daily nicotine exposure was equivalent to one pack of cigarettes. The second regimen provided nicotine as a continuous s.c. infusion at a higher total daily dose, equivalent to the nicotine absorbed from 3.4 packs of cigarettes daily,

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to provide the more rigorous challenge of higher serum nicotine concentrations and 24 h/day exposure. In addition, a single nicotine dose was administered to rats in both protocols just prior to terminating the experiment to assess the e€ects of chronic nicotine dosing on the ability of immunization to reduce the distribution of a single nicotine dose to brain. 2. Methods 2.1. Materials (ÿ)-Nicotine bitartrate, (ÿ)-cotinine, (ÿ)[methyl-3H]-nicotine 81 Ci/mmol, and goat antiIgG-peroxidase conjugate were obtained from Sigma Chemical (St. Louis, MO). Internal standards for the nicotine/cotinine assay were a gift from Dr. Peyton Jacob. Nicotine was administered to rats as nicotine bitartrate but all doses and measured concentrations are expressed as the base (mw 162 D). 2.2. Nicotine vaccine The hapten trans-3 '-aminomethylnicotine was prepared as previously described [7] and conjugated at the 3' position to the carrier protein recombinant Pseudomonas aeruginosa exoprotein A through a succinic acid linker to form the complete immunogen [11].

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2.4. Immunologic assays Serum nicotine-speci®c IgG titers were measured by ELISA using a polyglutamate±hapten conjugate as the coating antigen [12]. Titers were calculated from the plot of optical density versus log dilution, as the dilution corresponding to an optical density reading of 50% of the maximal value. Cross-reactivity of immune serum with the major nicotine metabolites and the nicotine receptor ligand acetylcholine has been previously shown to be low (2.7% with cotinine, <1% with nicotine-N-oxide or acetylcholine) [7]. Because assay of antibody titers in the current study was done in the presence of nicotine (owing to the concurrent administration of nicotine and immunization), the ELISA was ®rst validated by running it with nicotine 1000 ng/ml added. This serum nicotine concentration was chosen to exceed the maximum value expected in immunized rats. Antibody anity for nicotine and binding capacity in serum were measured by soluble radioimmunoassay (RIA) [12]. In contrast to the ELISA assay, the RIA was found in pilot experiments to be sensitive to nicotine concentrations in the range found in experimental animals. RIA was, therefore, performed only on sera containing no nicotine; blood samples from the nicotine vaccine groups receiving chronic saline, taken before administration of the single nicotine dose just prior to sacri®ce.

2.3. Vaccination of rats Rats were vaccinated with nicotine immunogen 25 mg i.p. in complete Freund's adjuvant on day 0, and boosted with 25 mg of immunogen in incomplete Freund's adjuvant on days 21, 35 and 63. Experiments were performed between days 70 and 76, when antibody titers were expected to be highest [7]. Control animals were similarly immunized with carrier protein alone (no hapten). Serum antibody titers were measured by ELISA 1 week after each booster dose. Only rats with serum antibody titers of >1:10,000 after the third booster dose were included in the study. Assignment to treatment groups was randomized.

2.5. Nicotine assay Concentrations of nicotine in serum and brain were measured by gas chromatography with nitrogen±phosphorus detection [13]. All nicotine concentrations are expressed as the base. Brain nicotine concentration is expressed as ng/g wet weight, corrected for brain blood content [10]. Limits of quantitation are 2 ng/ml nicotine and 10 ng/ml cotinine. 3H-nicotine was measured by adding 0.1 ml of serum or brain homogenate to scintillation ¯uid.

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Table 1 Treatment group (Chronic bolus dose protocol)a Group

Nb

Immunizationc

Chronic bolus drug

1 2 3 4

8 4 4 5

Control vaccine Nicotine vaccine Control vaccine Nicotine vaccine

Saline Saline Nicotine Nicotine

a Rats were immunized over a period of 11 weeks with either nicotine vaccine or a control vaccine (consisting of carrier protein alone), while also receiving 20 bolus injections per day of either nicotine (0.015 mg/kg per injection) or saline. b A number of animals did not complete the protocol because of catheter or computer problems. c Rats were immunized on days, 0, 21, 35 and 63.

2.6. Chronic nicotine bolus dosing protocol (Table 1). Two methods of chronic nicotine dosing were used. For bolus dosing, male Sprague-Dawley rats weighing 310±435 g and accommodated to a reversed 12/12 h light/dark cycle were anesthetized with droperidol/fentanyl i.m. and silastic catheters were placed in the right jugular vein [14]. Animals were then placed in cages with their catheters attached to a computer controlled infusion pump via a swivel tether. Two groups of eight rats received nicotine bitartrate as 20 i.v. bolus injections spaced evenly throughout their waking (dark) cycle at intervals of 36 min. Individual dose size was 0.015 mg/kg nicotine delivered over 1 s in a volume of 50 ml, for a total daily dose of 0.3 mg/kg. This dose was chosen because it is equivalent, on a mg/kg

basis, to the usual daily intake of nicotine by a 1 pack/day cigarette smoker, divided into individual doses the size of a single cigarette [15]. Nicotine dosing was started 3 days after catheter implantation to allow time for recovery from surgery. Two additional groups of eight rats received saline infusions on the same schedule. Immunization was started 2 days later (5 days after catheter implantation) to assure that steady state nicotine concentrations were already present. Two groups received nicotine vaccine and two groups received control vaccine (Table 1). One week after the ®rst and second booster doses of vaccine, rats were lightly anesthetized with droperidol/fentanyl and 0.3 ml blood was obtained by tail vein bleeding to measure nicotine-speci®c antibody titers (Table 2). Approximately 1 week after the third booster dose, rats were anesthetized for administration of the ®nal nicotine dose via the jugular catheter. Two ml of blood was removed as a baseline sample prior to the ®nal nicotine dose by tail vein bleeding. This procedure was timed so that the baseline blood sample was removed 30 min after the sixth nicotine dose of the day. Rats then received a ®nal dose of nicotine bitartrate 0.03 mg/kg (of the base) equivalent to the nicotine dose absorbed from two cigarettes in a human, with 3 mCi 3Hnicotine added, infused over 1 s [15]. This nicotine dose was used because it produces peak serum nicotine concentrations in the 10±40 ng/ml range observed in regular cigarette smokers, and because it represents a behaviorally active nic-

Table 2 Chronic bolus dose protocol Ð timeline Day

Procedure

ÿ5 ÿ3 0 21 28 35 42 63 70

Catheter implantation Begin chronic bolus dosing of nicotine or saline and continue throughout Primary vaccination First booster dose of vaccine Obtain blood for antibody titer measurement Second booster dose of vaccine Obtain blood for antibody titer measurement Third booster dose of vaccine Final 3H-nicotine dose, sacri®ce

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otine dose in both humans and rats [15±17]. Three minutes after the ®nal nicotine dose, rats were decapitated, trunk blood was collected, and brain was removed and stored at ÿ208C for analysis. Trunk blood can be used for this purpose because di€erences in nicotine concentrations between arterial and venous blood in rats 3 min after nicotine infusion are negligible [10]. 2.7. Chronic nicotine continuous infusion protocol The design of this protocol was parallel to that of the bolus dosing protocol except that nicotine or saline was delivered by s.c. osmotic pump. Rats were anesthetized and Alzet 2ML4 pumps placed in the interscapular area. Pumps delivered 60 ml/day of nicotine bitartrate for a total daily dose of 1 mg/kg. This dose was chosen to produce the maximal serum nicotine concentration of a moderate to heavy smoker, or about 10±40 ng/ml [16]. Immunization was started 3 days after pump implantation. Pumps were replaced at 4 and 8 weeks to provide drug delivery over the entire period of immunization. A single additional dose of nicotine was administered just prior to sacri®ce on day 70, as described for the bolus dosing protocol. Treatments are summarized in Table 3.

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nicotine) were compared by repeated measures ANOVA. Because di€erences in antibody titers could have emerged for the ®rst time after the third booster dose, ELISA titers at the ®nal sampling time were also compared using a twotailed t-test. Di€erences among groups in serum or brain nicotine concentrations were analyzed by one way ANOVA. If ANOVA showed an overall signi®cant di€erence among groups, di€erences between individual groups were analyzed with Fisher's PLSD test. 3. Results 3.1. ELISA assay validity Fig. 1 shows that the performance of the ELISA assay was una€ected by a concentration of nicotine exceeding those measured in the in

2.8. Statistical analysis Antibody ELISA titers over time in the two nicotine vaccine groups (with or without chronic Table 3 Treatment group (Chronic infusion protocol)a Group

N

Immunizationb

Chronic infusion drug

1 2 3 4

8 8 8 8

Control vaccine Nicotine vaccine Control vaccine Nicotine vaccine

Saline Saline Nicotine Nicotine

a

Rats were immunized over a period of 11 weeks with either nicotine vaccine or control vaccine, while also receiving a continuous infusion of either nicotine 1 mg/kg/day or saline via s.c. osmotic pump. b Rats were immunized on days, 0, 21, 35 and 63.

Fig. 1. Top panel: ELISA nicotine-speci®c antibody titers with or without nicotine 1000 ng/ml added. Bottom panel: inhibition of ELISA absorbance by addition of nicotine. Both assays show that ELISA performance was not altered by the added nicotine, validating its use even in animals that had been treated with nicotine.

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vivo experiments which follow. This assay could, therefore, be used to follow the ecacy of immunization regardless of whether animals were receiving concurrent nicotine. 3.2. RIA Serum antibody anity for nicotine, measured from the actively immunized group pretreated with chronic saline infusion was 5:421:6  107 Mÿ1 …mean2SE). The corresponding nicotine binding capacity in serum was 1:220:2  10 ÿ6 M. Assuming a serum volume of 40 ml/kg for the rat [18] and that 3/4 of the antibody exists outside of serum [19], one can estimate that the

total nicotine binding capacity per rat was 1:9  10 ÿ7 mol/kg. By comparison, the total daily dose of nicotine administered in the chronic infusion experiment was 6:2  10 ÿ6 mol/kg. Thus, daily nicotine doses exceeded the estimated nicotine binding capacity by approximately 33-fold. 3.3. Antibody titers with and without concurrent nicotine (Fig. 2). All vaccinated animals achieved serum antibody titers of >1:10,000 (range 1:28,000± 1:278,000), and none were excluded from the study because of inadequate titers. Several animals in three of the four groups of the bolus dose protocol did not complete the study because of catheter or computer problems, and were not included in any analyses (Table 1). Antibody titers in rats receiving bolus dose nicotine during the period of immunization did not di€er from those of animals receiving bolus dose saline instead (repeated measures ANOVA, p ˆ 0:72, ttest of titers after the third booster dose, p ˆ 0:58). Antibody titers in rats receiving s.c. nicotine infusion during the period of immunization did not di€er from those of animals receiving s.c. saline infusion, as analyzed by repeated measures ANOVA …p ˆ 0:79). Titers after the third booster dose were higher in rats receiving s.c. nicotine infusion when analyzed by t-test …1:161,000249,000 versus 1:53,000219,000, mean2SE, p ˆ 0:014). 3.4. Serum nicotine concentrations

Fig. 2. E€ects of concurrent nicotine administration on the development of nicotine-speci®c antibody titers in serum. Serum nicotine-speci®c antibody titers were measured over the 11-week course of immunization in rats receiving 20 daily injections of nicotine or saline (top panel) and in rats receiving chronic s.c. infusions of either nicotine or saline (bottom panel). Titers did not di€er as indicated by repeated measures ANOVA …p ˆ 0:72 for chronic bolus dosing, p ˆ 0:79 for chronic infusion). t-tests of just the titers measured after the third booster dose did not di€er in the chronic bolus dose experiment …p ˆ 0:58), but were modestly higher in the nicotineexposed animals in the chronic infusion experiment …p ˆ 0:014).

3.4.1. Bolus dose protocol (Fig. 3, top) Serum unlabeled nicotine concentrations just prior to the ®nal nicotine dose (representing the contribution of chronic nicotine dosing alone) were 6 ng/ml in the group receiving chronic nicotine with control vaccine, and 193 ng/ml in the group receiving chronic nicotine with nicotine vaccine. This demonstrates that there was considerable nicotine retention in serum during the period of chronic nicotine dosing in the actively immunized group. Serum unlabeled nicotine concentrations at the time of sacri®ce (representing total nicotine con-

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control vaccine group …p < 0:001 for ANOVA and p < 0:01 for individual comparisons). 3.4.2. Continuous s.c. infusion protocol (Fig. 3, bottom) Results of this protocol generally paralleled those of the bolus dose protocol. Serum unlabeled nicotine prior to the ®nal nicotine dose was 30 ng/ml in the control vaccine group and 525 ng/ml in the nicotine vaccine group. These levels were higher than the comparable levels from the bolus dose experiment, con®rming that the s.c. infusion regimen provided the desired higher serum nicotine concentrations. Serum unlabeled nicotine concentrations after the ®nal nicotine dose were signi®cantly di€erent by ANOVA …p < 0:001), and signi®cantly higher in each of the nicotine vaccine groups compared to the control vaccine groups …p < 0:01, Fig. 3, bottom). 3H-nicotine concentrations were similarly higher in the nicotine vaccine groups. 3.5. Brain nicotine concentrations

Fig. 3. Serum nicotine concentrations …mean2SE). ``Pre'' refers to the blood sample obtained just prior to the ®nal 3Hnicotine dose of 0.03 mg/kg. The remaining samples were obtained 3 min after infusion of that dose. ``Nicotine'' refers to unlabeled nicotine measured by gas chromatography, and representing total nicotine from both chronic dosing and the ®nal 3H-nicotine dose. 3H-nicotine represents only that nicotine administered with the ®nal dose. 3H-nicotine concentrations (dpm/ml) were converted to nicotine concentrations (ng/ml) by correcting for the speci®c activity of the administered dose. Top panel: chronic bolus dosing protocol. Bottom panel: chronic infusion protocol. Serum unlabeled and 3H-nicotine concentrations were higher in each of the nicotine vaccine groups compared to the corresponding control vaccine groups in both protocols. p < 0:05, p < 0:01, p < 0:001:

tributed by both chronic dosing and the ®nal dose administered just prior to sacri®ce) were signi®cantly di€erent by ANOVA …p < 0:01), and were markedly higher in each of the nicotine vaccine groups compared to the control vaccine groups …p < 0:05† Like the unlabeled nicotine concentrations, serum 3H-nicotine concentrations (representing only nicotine contributed by the ®nal nicotine dose just prior to sacri®ce) were higher in both nicotine vaccine groups than in either

3.5.1. Bolus dose protocol (Fig. 4, top) Brain unlabeled nicotine concentrations were signi®cantly di€erent by ANOVA …p < 0:001† and were lower in each nicotine vaccine group compared to its control vaccine counterpart. The extent of reduction was 75% in the groups receiving chronic saline and 60% in the groups receiving chronic nicotine. Reductions in brain 3H-nicotine concentrations were similar to those of unlabeled nicotine. The extent of reduction was 66% in the groups receiving chronic saline and 61% in the groups receiving chronic nicotine. Thus, chronic nicotine bolus dosing did not compromise the ability of immunization to reduce the brain 3H-nicotine concentration. 3.5.2. Continuous s.c. infusion protocol (Fig. 4, bottom) Brain unlabeled nicotine concentrations were lower in each nicotine vaccine group compared to its control vaccine counterpart. The extent of reduction was 42% in the group receiving chronic saline infusion and 29% in the group receiving chronic nicotine infusion. Reductions in brain 3H-nicotine concentrations

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4. Discussion

Fig. 4. Brain nicotine concentrations …mean2SE). ``Nicotine'' refers to unlabeled nicotine measured by gas chromatography, and representing total nicotine from both chronic dosing and the ®nal nicotine dose. 3H-nicotine concentrations (dpm/ml) were converted to nicotine concentrations (ng/g) by correcting for the speci®c activity of the administered dose. Top panel: chronic bolus dosing protocol. Bottom panel: chronic infusion protocol. Brain unlabeled nicotine and 3H-nicotine concentrations were reduced in each of the nicotine vaccine groups compared to the corresponding control vaccine groups in both protocols. p < 0:05, p < 0:01, p < 0:001:

were similarly lower in each nicotine vaccine group compared to its control vaccine counterpart …p < 0:001). The extent of reduction was 30% in the group receiving chronic saline infusion and 40% in the group receiving chronic nicotine infusion. In summary, immunization with nicotine vaccine substantially reduced the brain nicotine concentration as measured by either unlabeled nicotine, re¯ecting cumulative nicotine dosing, or 3 H-nicotine, re¯ecting just the ®nal nicotine dose. The e€ect of immunization on the distribution of the ®nal nicotine dose to brain was not in¯uenced by prior chronic nicotine dosing.

The major ®ndings of this study are that the chronic administration of nicotine to rats at clinically relevant doses did not impair the immunogenicity of a nicotine conjugate vaccine, nor did it prevent vaccination from reducing the distribution of nicotine to brain. These observations comment on both the mechanism of action of vaccination in this setting, and on its potential clinical applications. There is no completely satisfactory rat model of nicotine absorption from cigarette smoking. We, therefore, used two protocols of chronic nicotine dosing to mimic various aspects of nicotine exposure in humans. The ®rst protocol delivered nicotine to rats as 20 i.v. boluses throughout the ``day'' (waking period), with each bolus of 0.015 mg/kg equivalent (on a mg/kg basis) to the nicotine absorbed from one cigarette by a human [15]. The total daily dose was equivalent to 20 cigarettes or one pack. Aside from delivering nicotine intermittently, as is characteristic of cigarette smoking, this method of delivery has been shown to mimic the rapid absorption of nicotine into arterial blood and rapid delivery of nicotine to brain which is characteristic of cigarette smoking [20,21]. One limitation of this dosing method was that it produced only modest venous serum nicotine concentrations (mean of 6 ng/ml measured 30 min after a nicotine dose) compared to those of a regular smoker (10±40 ng/ml) [16]. A second potential limitation was that the elimination half-life of nicotine in the rat is shorter (0.8 h) than that of humans (2 h) so that serum nicotine concentrations after 12 h of abstinence were probably quite low [22,23]. To address these limitations, a second protocol of continuous nicotine infusion by subcutaneous pump was used, with an infusion rate that produced serum nicotine concentrations (mean of 30 ng/ ml) more typical of heavy smokers, and continuous 24 h/day exposure to these levels. Together, these protocols should have provided a rigorous test of the impact of chronic nicotine exposure on vaccine immunogenicity and e€ects. Vaccine immunogenicity, as measured by ELISA titers, was not impaired by either mode

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of chronic nicotine exposure. Antibody titers were in fact higher in the nicotine-exposed group than in the chronic infusion experiment, but the di€erence was small (one tube dilution) and likely of little importance. These data suggest that vaccination could potentially be initiated before a smoker quits, so that maximal antibody titers are present at the time of quitting. This is important because relapse to smoking after a quit attempt most often occurs within the ®rst few weeks. Vaccination that is ®rst initiated at the time of quitting would be unlikely to have a prompt enough e€ect to prove useful. Whether the presence of nicotine would be expected to interfere with vaccine immunogenicity is unclear, as few data are available which directly address this question. Some interference from free hapten (nicotine) might be anticipated, as it could potentially occupy recognition sites on B cells and prevent them from binding processed immunogen. Apparent success in immunizing animals against cocaine [5] or methamphetamine (personal communication, M. Owens), despite continued drug administration, may have been in¯uenced by intermittent drug dosing which would allow intervals during which little or no drug was present. In the current study, exposure to nicotine during immunization was provided as either regular daytime exposure simulating usual smoking patterns, or continuous exposure; yet neither regimen interfered with vaccine immunogenicity. Perhaps the concentrations of nicotine achieved, which spanned the range of those observed in regular smokers, was still too low to substantially interfere with the binding of immunogen to B cells. Vaccination has been previously shown to reduce the distribution to brain of a single dose of nicotine (equivalent on a mg/kg basis to two cigarettes in a human) by 64%. A smaller 29% reduction was found after ®ve repeated doses of nicotine (equivalent to 10 cigarettes) delivered over 80 min to simulate a period of heavy smoking [23]. It is, therefore, possible that repeated or chronic nicotine delivery could saturate the available antibody and reduce its ability to bind additional nicotine and block its distribution to brain. The current study tested the ability of vac-

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cination to reduce nicotine distribution to brain in rats receiving chronic nicotine at doses comparable to those associated with regular cigarette smoking. With both protocols, the distribution of a single nicotine dose (the ®nal bolus dose of 3Hnicotine) under these conditions was substantially reduced (61% with the intermittent dosing protocol, 40% with the continuous infusion). The magnitude of reduction was comparable to that of the previous studies cited above, and was not diminished by chronic nicotine infusion. These data suggest that chronic nicotine exposure does not prevent vaccination from reducing nicotine distribution to brain. The unlabeled nicotine concentration in brain (re¯ecting both chronically administered nicotine and the ®nal nicotine dose) was also reduced in the nicotine vaccine groups. In contrast to the 3 H-nicotine concentration, the unlabeled nicotine concentration in brain was reduced less in rats receiving chronic nicotine than in those receiving chronic saline (60% versus 75% in the bolus dose protocol, 29% versus 42% in the chronic infusion protocol). Thus, chronic nicotine administration may have interfered with the ability of vaccination to reduce the accumulation of nicotine in brain over several months, whereas, it did not interfere with the ability of vaccination to reduce the distribution of a single nicotine dose to brain. Vaccination could have a€ected these processes di€erently because the 3H-nicotine concentration in brain after the single nicotine dose was measured just 3 min after the dose, thus representing its early distribution, whereas the unlabeled nicotine concentration represented e€ects on the accumulation of nicotine in brain under steady state conditions. Because the subjective e€ects of nicotine in humans peak within minutes after a nicotine dose [24], vaccination e€ects on the initial distribution of each single dose may be more important than its e€ects on chronic nicotine accumulation. The reduction in the distribution of the ®nal nicotine dose to brain due to vaccination, among groups receiving chronic nicotine, was greater in the chronic nicotine bolus protocol (61%) than in the chronic nicotine infusion protocol (40%). This di€erence between protocols was not due to

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the manner in which chronic nicotine was administered, as a similar di€erence was observed in the reduction due to vaccination in the chronic saline groups (66% versus 30%). The reason for this discrepancy between protocols is not clear. Regardless of the reason, both experiments were in agreement in indicating that chronic nicotine exposure did not prevent vaccination from reducing nicotine distribution to brain. The measured serum nicotine concentrations support this conclusion. Nicotine vaccine groups in both the bolus and infusion protocols had substantially higher serum 3H-nicotine concentrations than the control vaccine groups, demonstrating retention of the ®nal dose of nicotine in serum by antibody. Chronic nicotine administration did not substantially impair the retention of 3H-nicotine in serum. Together with the brain concentrations, these data provide the ®rst indication that vaccination may remain pharmacokinetically e€ective even in the face of chronic nicotine exposure at substantial doses. The pharmacokinetic ecacy of vaccination, despite chronic nicotine dosing, is curious in that the total daily doses of nicotine received by animals in this study exceeded the estimated nicotine binding capacity of antibodies 33-fold. We have previously shown that the binding of nicotine by nicotine-speci®c antibody prolongs the nicotine elimination half-life in rats from 0.8 h to approximately 2±7 h [23]. Possibly, even this slowed rate of nicotine elimination is sucient to prevent saturation of antibody and excessive loss of nicotine binding capacity. Whether a 40±61% reduction in distribution of nicotine to brain is sucient to alter smoking behavior was not addressed in this study. However, a similar reduction in nicotine distribution to brain in another study was associated with complete blockade of the ability of nicotine to alleviate nicotine withdrawal in vaccinated rats (personal communication, D. Malin). In summary, chronic exposure of rats to nicotine at clinically relevant doses did not impair the immunogenicity of a nicotine conjugate vaccine, nor did it prevent it from reducing nicotine distribution to brain. These data support a potential role for vaccination as an adjunct to

smoking cessation interventions, and speci®cally suggest that vaccination can be successfully accomplished even during ongoing nicotine exposure.

Acknowledgements Supported by NIDA grant DA10714 and a grant from Nabi.

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