Abstract
The ability of active immunization to alter nicotine distribution was studied in rats. Animals were immunized with 6-(carboxymethylureido)-(±)-nicotine (CMUNic) linked to keyhole limpet hemocyanin (KLH). Antibody titers determined by ELISA, using CMUNic coupled to albumin as the coating antigen, were greater than 1:10,000. Antibody binding was inhibited by neither of the nicotine metabolites cotinine and nicotine-N-oxide but was inhibited to a greater extent by CMUNic than by nicotine; this suggests the presence of antibodies to the linker structure as well as antibodies to nicotine. Antibody affinity for nicotine measured by soluble radioimmunoassay was 2.4 ± 1.6 × 107 M−1, and binding capacity was 1.3 ± 0.7 × 10−6 M, which corresponds to 0.1 ± 0.05 mg/ml of nicotine-specific IgG per milliliter of serum. One week after their second boost, groups of eight anesthetized rats immunized with either CMUNic-KLH or KLH alone received nicotine 0.03 mg/kg (equivalent to two cigarettes in a human) via the jugular vein over 10 sec. This dosing regimen was shown to mimic the arterio-venous nicotine concentration gradient typical of nicotine delivered by cigarette smoking in humans. Plasma nicotine concentrations at 10 to 40 min were 4 to 6-fold higher in the CMUNic-KLH rats than in controls (P < .001). Nicotine binding in plasma determined by equilibrium dialysis was markedly increased in the CMUNic-KLH group (83.4 ± 6.8% vs. 16.4 ± 14.2%), but brain nicotine concentrations at 40 min did not differ (37.9 ± 4.5 vs. 44.0 ± 8.4 ng/g, CMUNic-KLHvs. KLH, P = .1). The amount of nicotine bound to antibody in plasma, estimated from the in vivo data, was 9% of the administered dose. These data demonstrate that active immunization can bind a significant fraction of a clinically relevant nicotine dose in plasma. Observing this effect with antibodies of modest affinity and titer is encouraging, but better immunogens may be needed to alter nicotine distribution to brain and modify nicotine’s behavioral effects.
Cigarette smoking is the leading cause of preventable death in the United States (McGinnis and Foege, 1993). Recent advances in behavioral and pharmacologic treatment approaches have improved a smoker’s chances of quitting (Fiore et al., 1994; Henningfield, 1995; Hjalmarsonet al., 1994). Nevertheless, the vast majority of those who try to quit will fail.
A pivotal role for nicotine in maintaining cigarette smoking is well established. Smokers tightly regulate their nicotine intake. Subjects who are switched from their usual brand to lower-nicotine cigarettes alter their smoking behavior to increase their nicotine yield per cigarette (Benowitz et al., 1986). Conversely, supplementation of nicotine intake by i.v. infusion reduces cigarette smoking (Lucchesi et al., 1967). Cessation of nicotine intake produces a withdrawal syndrome, and replacement of nicotine reduces its severity (Hughes et al., 1984). Thus an intervention aimed at reducing nicotine distribution to target tissues such as brain might provide a strategy for promoting cessation of smoking.
The possibility of using drug-specific antibodies to reduce the effects of drugs of abuse was studied more than 20 years ago by Bonese et al. (1974), who actively immunized one monkey against heroin after it had been trained to self-administer both heroin and cocaine. Immunization reduced the self-administration of heroin but not of cocaine, a result that demonstrated both its efficacy and its specificity. Similar efficacy was produced by transfusing two nonimmunized monkeys with morphine-specific antiserum from immunized monkeys (Killian et al., 1978). Altered patterns of heroin self-administration lasted more than 3 weeks, the effect of immunization waning in parallel with antibody titers.
More recently, it has been shown that the behavioral effects of cocaine can be modified by active immunization. Immunized rats challenged with a substantial dose of cocaine (15 mg/kg i.p.) showed significantly reduced locomotor activity and lower brain cocaine concentrations than controls (Carrera et al., 1995). In a separate study, self-administration of i.v. cocaine by rats at clinically relevant doses was rapidly suppressed by the passive administration of cocaine-specific antibodies (Fox et al., 1996). This result is particularly interesting because the cocaine dose delivered by each infusion (1 mg/kg = 1 μmol per 300-g rat) greatly exceeded the binding capacity of drug-specific antibody administered (4 mg = 0.05 μmol of binding sites). Presumably, the observed blunting of cocaine distribution to brain was sufficient to alter its behavioral effects.
The general concept of immunization as a means of blocking the effects of drugs of abuse is attractive because of its specificity; as a pharmacokinetic intervention that acts outside the CNS and does not affect neurotransmitters or receptors, it should not have many of the adverse effects associated with other treatments. Nicotine is an even better candidate for this approach than heroin or cocaine, because the dose administered is much lower. The dose of nicotine (molecular weight 162 D) delivered by one cigarette is approximately 1 mg, whereas that of a single psychoactive dose of cocaine (molecular weight 303 D) is about 35 mg, a molar ratio of 1:18 (Benowitz and Jacob, 1984; Pentelet al., 1994). The molar ratio of total daily doses (nicotine, 37 mg; cocaine, 1 g) is about the same. Thus the efficacy of immunization in modifying the behavioral effects of cocaine suggests that it could be even more effective in altering the behavioral effects of nicotine. Nicotine is also a good candidate for this approach because many smokers who are trying to quit are highly motivated and thus are unlikely to try to overcome the effect of antibody by purposely increasing their nicotine intake.
As a first step in evaluating this question, the current study examined the effects of active immunization on the distribution kinetics of nicotine in rats. We hypothesized that immunization would alter nicotine distribution by increasing nicotine binding in plasma and reducing the nicotine concentration in brain. A pharmacokinetically relevant model that mimics the marked arterio-venous nicotine concentration gradient produced by cigarette smoking was used, along with clinically relevant doses of nicotine.
Materials and Methods
Drugs and reagents.
(±)-Nicotine sulfate, (−)-cotinine, (−)-[methyl-3H]-nicotine 70 Ci/mmol, and goat anti-IgG-peroxidase conjugate were obtained from Sigma Chemical Co. (St. Louis, MO). (±)-Nicotine-N-oxide was a gift from Professor John Gorrod. Internal standards for the nicotine/cotinine assay (5-methyl nicotine bis-oxalate and 5-methyl cotinine perchlorate) were a gift from Dr. Peyton Jacob.
Synthesis of immunogen.
(fig.1) Production of CMUNic was accomplished by the synthesis of 6-amino-(±)-nicotine, reaction with ethyl isocyanatoacetate to form 6-(carboxyethylureido)-(±)-nicotine and hydrolysis by lithium hydroxide to CMUNic. All structures were confirmed by nuclear magnetic resonance (360 or 90 MHz), infrared spectra and elemental analysis.
6-Amino nicotine was synthesized by the method of Tschitschibabin and Kirssanow (1924) and separated from 2-amino nicotine by silica gel chromatography with methanol solvent (Rf = 0.70). HCl generated by the action of concentrated sulfuric acid on NaCl was bubbled into an ethereal solution of 6-amino nicotine, and the resultant yellow-brown solid was filtered and recrystallized from ethanol to yield 6-amino nicotine HCl.
6-Amino nicotine (4.58 g, 25.8 mmol) and an excess (3.5 ml, 3.53 g, 27 mmol) of ethyl isocyanatoacetate were stirred in 25 ml of anhydrous toluene for 2 days, and the product 6-(carboxyethylureido)-(±)-nicotine was recrystallized from ethanol and purified by silica gel column chromatography. Lithium hydroxide (3 eq) and 0.5 g (1.63 mmol) of 6-(carboxyethylureido)-(±)-nicotine were stirred in 3 ml water/3 ml tetrahydrofuran for 2 days. The solvent was evaporated, and the product CMUNic was purified by silica gel column chromatography (Rf = 0.58) using chloroform/methanol/ammonia (6:3:1) as the solvent; this yielded an off-white solid.
Immunization of rats.
Male Holtzman rats weighing 175 to 200 g were immunized with 25 μg of CMUNic-KLH or KLH alone in 0.4 ml of complete Freund’s adjuvant injected i.p. At 21 and 35 days, animals were boosted with 25 μg of CMUNic-KLH or KLH alone in 0.4 ml of incomplete Freund’s adjuvant injected i.p. Experiments were performed 4 to 12 days after the second booster injection. Nicotine-specific IgG titers were stable over this period of time. At the time of the experiment, animals weighed 285 to 400 g. IgG titers were measured from plasma obtained at the beginning of each experiment.
Characterization of antibodies.
We measured serum IgG titers and specificity by ELISA (Keyler et al., 1994), using CMUNic-albumin as the coating antigen to avoid detecting antibodies directed at KLH and using goat anti-rat IgG coupled to peroxidase as the detecting antibody. Specificity was determined by competition with CMUNic, nicotine, acetylcholine (ACh) and the two major nicotine metabolites in the rat, cotinine and nicotine-N-oxide.
Because IgG binding measured by ELISA could reflect binding to linker determinants, antibody affinity and binding capacity were determined using a soluble radioimmunoassay. The method of Muller was used because knowledge of the antibody concentration is not required for the calculation of affinity and binding capacity (Pentel et al., 1987; Muller, 1983). In brief, the dilution of serum that produced 50% precipitation of 3H(−)-nicotine by ammonium sulfate was determined, and affinity was measured at this plasma dilution by competition with unlabeled nicotine.
Protocol 1: Arterio-venous nicotine concentrations.
For studies of arterio-venous nicotine concentrations, eight male Holtzman rats weighing 210 to 270 g were anesthetized with Innovar Vet (droperidol 4 mg/ml and fentanyl 0.08 mg/ml) 1 ml/kg i.m., and PE50 catheters were placed in the left femoral artery and vein and in the right jugular vein. 3H(−)-nicotine (3 × 106 dpm) in 0.25 ml of 0.9% saline plus unlabeled nicotine 0.03 mg/kg was infused via the jugular vein over 10 sec. Heparinized blood samples of 0.3 ml were removed from the femoral arterial and venous catheters over 15-sec intervals (0–15, 16–30, 31–45, 46–60, 61–75, 76–90 sec), centrifuged immediately and assayed by scintillation counting. Terminal (90-sec) plasma samples from six additional animals were assayed by gas chromatography for cotinine to determine whether any measurable metabolism of nicotine had taken place over this period of time.
Protocol 2: Effects of immunization.
For studies of the effects of immunization on nicotine distribution, groups of eight animals immunized with CMUNic-KLH or KLH alone were anesthetized with Innovar Vet, and PE50 catheters were placed in the left femoral and right jugular veins. Nicotine 0.03 mg/kg was administered over 10 secvia the jugular vein catheter, 1.0-ml blood samples were obtained at 10, 20 and 40 min from the femoral vein catheter and plasma was stored at −20°C for measurement of plasma nicotine concentrations. At 40 min, an additional 4 to 5 ml of blood was obtained for measurement of protein binding, animals were sacrificed with KCl and organs were perfused with normal saline by injecting 30 ml into the left ventricle of the heart over 3 min. The brain was rapidly removed and the cortex and cerebellum stored at −20°C for assay of nicotine concentrations.
Protein binding.
Protein binding was measured by equilibrium dialysis for 4 h at 37°C using 0.7 ml of plasma, Teflon semi-micro cells, Spectrapor 2 membranes with a molecular weight cutoff of 12 to 14 kD and Sorenson’s buffer (0.13 M phosphate, pH 7.4) (Pentel and Keyler, 1987). Plasma pH was measured at the end of each run, and samples were used only if the final pH was 7.30 to 7.45.
Nicotine assay.
Nicotine and cotinine concentrations in plasma were measured by gas chromatography with nitrogen-phosphorus detection (Jacob et al., 1981). Sensitivity of the assay is 1 ng/ml nicotine and 5 ng/ml cotinine. Brain samples were digested in 5 volumes of NaOH overnight and homogenized, and internal standards were added. The pH was adjusted to less than 4.0 with 1 M sulfuric acid, and 3 ml of Toluene/1-butanol (70:30) was added. The suspension was mixed and centrifuged and the organic layer removed. The aqueous layer was decanted from the remaining tissue and extracted in the same manner as plasma samples. Nicotine and cotinine recovery from brain extracted in this manner is greater than 90%.
Calculation of nicotine binding capacity in plasma.
Nicotine binding capacity in plasma of immunized rats was calculated from thein vivo data as the difference in mean plasma nicotine concentration between CMUNic-KLH and KLH groups at 10 min multiplied by the estimated plasma volume of the rat, 40 ml/kg (Cocchetto and Bjornsson, 1983). Nicotine binding capacity in serum was also calculated from the serum radioimmunoassay data using the method ofMuller (1983). The molecular weight of IgG was assumed to be 150 kD.
Statistical methods.
Plasma radiolabel and nicotine concentrations were compared between groups by repeated-measures ANOVA and Scheffe’s contrast. When group differences were significant, individual time-points were compared using one-tailed unpairedt tests. Differences in brain nicotine concentrations, protein binding of nicotine in plasma and the brain/plasma nicotine concentration ratio between groups were compared using two-tailed unpaired t tests. One animal in the CMUNic group, whose plasma showed no binding activity for nicotine by ELISA (determined after the experiments were run), was excluded from the analysis of the effects of immunization on nicotine distribution.
Results
Antibody characteristics.
Among animals immunized with CMUNic-KLH, IgG titers measured by ELISA were all greater than 1:10,000, with the exception of one animal with a titer of 1:2500 that showed inhibition of binding by CMUNic but not by nicotine (this animal was excluded from further analysis). Titers were generally the same after one or two immunogen boosts. ELISA inhibition curves showed that CMUNic had a lower IC50 than nicotine (1.5 × 10−6vs. 5.8 × 10−4, fig.2). The nicotine metabolites cotinine and nicotine-N-oxide produced very little inhibition at concentrations up to 10−2 M and no more than the irrelevant control propranolol. ACh also produced no more inhibition than propranolol (data not shown). All animals immunized with KLH alone had titers of less than 1:100.
Antibody titers measured by radioimmunoassay were 1:5 to 1:10. Affinity for nicotine was 2.4 ± 1.6 × 107M−1 (mean ± S.D.). Nicotine binding capacity calculated from these data was 1.3 ± 0.7 × 10−6 M, which is equivalent to 0.1 ± 0.05 mg/ml of nicotine-specific IgG. Assuming that the concentration of total IgG in serum is 10 mg/ml (Harlow and Lane, 1988), nicotine-specific IgG represented approximately 1% of the total. Assuming a molecular weight for nicotine of 162 D and two binding sites per IgG, this nicotine-specific IgG concentration in serum corresponds to a nicotine binding capacity in serum of 210 ± 110 ng/ml.
Arterio-venous nicotine concentrations.
Assay of terminal (90-sec) plasma samples by gas chromatography detected no cotinine, so radiolabel concentrations may be taken as representative of the parent compound, nicotine. Arterial radiolabel concentrations exceeded venous concentrations (P < .001), the difference being greatest initially and diminishing rapidly thereafter (fig.3).
Effects of immunization on nicotine distribution.
Mean plasma nicotine concentrations were 4 to 6-fold greater in rats immunized with CMUNic-KLH than in controls immunized with KLH alone (P < .001, fig. 4). The higher nicotine concentration in the CMUNic-KLH group was apparent at the first sampling time (10 min). Plasma nicotine concentrations decreased modestly over the next 30 min in both groups, but the difference between groups was sustained. The mean brain nicotine concentration was 13.8% lower in the CMUNic-KLH group than in the KLH group (37.9 ± 4.5 vs. 44.0 ± 8.4 ng/g), but this difference was not significant (P = .1, fig. 5). Brain/plasma ratios were significantly different between the two groups (0.8 ± 0.6 vs. 3.8 ± 0.5, P < .001), reflecting primarily the increase in the plasma nicotine concentration in the CMUNic-KLH group. Protein binding of nicotine in plasma determined by equilibrium dialysis was significantly higher in the CMUNic-KLH group (83.4 ± 6.8% vs. 16.4 ± 14.2%, P < .001). Unbound nicotine concentrations did not differ (table 1).
Discussion
Previous studies have shown that immunization can modify the behavioral effects of heroin (Bonese et al., 1974; Killianet al., 1978) and cocaine (Carrera et al., 1995;Fox et al., 1996), presumably by binding drug in serum (and perhaps elsewhere) and preventing its distribution to brain. However, limited information is available on the quantitative requirements for immunization to alter drug distribution, such as the antibody affinity, binding capacity and extent of drug binding necessary to reduce drug concentrations in brain. The current study provides preliminary information relevant to these considerations for the use of immunization to alter nicotine distribution. These data should be useful in the design and screening of immunogens and immunization regimens intended to alter the behavioral effects of nicotine.
The rat was chosen for this study because of the ease of immunization (Carrera et al., 1995) and because nicotine pharmacokinetics in rats is quite similar to that in humans. Both are characterized by a high systemic clearance (human 91 vs. rat 152 l/min · kg), short elimination half-life (human 119 vs. rat 66 min), relatively large volume of distribution (human 2.6 vs. rat 4.1 l/kg), low protein binding in serum (human 4.9 vs. rat 9.5%) and limited renal excretion of unchanged drug (human 16vs. rat 17%) (Benowitz et al., 1982; Benowitzet al., 1989; Plowchalk et al., 1992; Milleret al., 1977). The fraction of a dose converted to cotinine is lower in rats than in humans (27 vs. 70%), and the fraction converted to nicotine-N-oxide is probably greater (Jacobet al., 1981; Shulgin et al., 1987), but this should be of little consequence for this model so long as the antibodies elicited do not appreciably bind metabolites.
The nicotine dosing regimen used in this study was designed to simulate several critical features of nicotine distribution observed with cigarette smoking in humans. The nicotine dose was equivalent to that delivered by two cigarettes (Benowitz and Jacob, 1984), and it produced venous plasma nicotine concentrations in the 10 to 40-ng/ml range observed in regular smokers (Benowitz et al., 1983). The administration of nicotine by rapid i.v. injection was intended to simulate the rapid absorption of nicotine from the lungs associated with smoking. In humans, this results in arterial nicotine concentrations than are up to 10-fold higher than venous nicotine concentrations 1 min after a single cigarette (Henningfield et al., 1993) because of rapid delivery of the nicotine bolus from the pulmonary circulation to the heart. This results in the rapid delivery of these very high arterial concentrations of nicotine to the brain. This aterio-venous concentration difference, although not identical in time course, was also observed in our model. Because immunotherapy to reduce the behavioral effects of nicotine must overcome this rapid delivery of nicotine to the brain, it is a critical feature of a model for this intervention.
The immunogen used in this study was originally designed to produce antibodies for a radioimmunoassay to measure nicotine concentrations. Derivatization at the 6-position of the pyridine ring was chosen because it is remote from the major sites of nicotine metabolism (to cotinine and nicotine-N-oxide) on the N-methyl pyrrolidine ring, an approach that has also been used by others to produce antibodies specific to parent drug (Castro et al., 1980). This strategy appeared to be successful, in that cross-reactivity with these metabolites in the serum of immunized rats was negligible. However, the ELISA binding activity of serum was inhibited more readily by the complete immunogen CMUNic than by nicotine, which indicates that serum antibodies recognized the carboxymethylureido linker. These data suggest that derivatization of nicotine at the 6-position produces antibodies that recognize parent compound and not metabolites, but that a linker other than CMUNic would be preferable to improve antibody specificity further. The use of a chiral immunogen to target the naturally occurring and active (−)-nicotine isomer, rather than the racemic immunogen used in this study, might also improve antibody specificity.
The affinity of serum antibodies for nicotine, measured using a soluble radioimmunoassay, was modest (2.4 ± 1.6 × 107M−1). The affinity of drug-specific antibodies used by Foxet al. (1996) to alter cocaine self-administration was reported to be higher, with a range of 4 × 107 to 2 × 109 M−1. Additional data on the required affinity are available from the literature on the use of passive immunization as a means of treating drug overdose. In this setting, heterologous drug-specific antibodies are administered rapidly (typically over minutes) to animals or patients with acute drug toxicity. The drug-specific antibodies bind drug in serum, reduce the unbound drug concentration in serum and provide a concentration gradient for drug to move out of target organs. If a sufficient dose of antibody is administered, toxicity can be reduced or completely reversed. Digoxin (Smith et al., 1982) and colchicine (Baudet al., 1995) toxicity have been treated in patients in this manner, and phencyclidine (Valentine et al., 1996), desipramine (Brunn et al., 1992) paraquat (Chen et al., 1994) and ricin (Houston, 1982) toxicity have been reversed in animals. Most antibodies used to treat drug overdose have had affinities for drug of 108 M−1 or higher, (1010 M−1 for digoxin, 2 × 1010 M−1 for colchicine, 5.6 × 108 M−1 for phencyclidine, 3 × 108 M−1 for desipramine), although a direct comparison of antibodies with differing affinities is not available to address the question of what affinity is actually required (Smithet al., 1982; Baud et al., 1995; Valentine and Owens, 1996).
Despite the modest affinity of the elicited nicotine-specific antibodies, immunization in the current study had a substantial effect to nicotine distribution. Nicotine binding in plasma was increased from 16.4% in control rats to 83.4% in the CMUNic group. As a result, total nicotine concentrations in plasma were 4 to 6-fold higher in the CMUNic group. Using the plasma nicotine concentration difference between the CMUNic and control groups, and the estimated plasma volume of the rat, a calculated 9% of the administered nicotine dose was bound by drug-specific antibodies in serum. Although serum represents the largest reservoir for IgG (Harlow and Lane, 1988), this figure could be higher if some binding takes place outside of serum. The nicotine binding capacity of plasma calculated from radioimmunoassay data (210 ng/ml) was 3-fold higher than that which was measuredin vivo (67 ng/ml). This probably reflects the modest affinity of the serum antibodies for nicotine resulting in incomplete occupancy of antibody by drug.
The nicotine-specific IgG concentration achieved in immunized rats was 0.1 mg/ml, or about 1% of total IgG. This is comparable to the 0.08 mg/ml produced by passive immunization with cocaine-specific antibodies and found to be effective in abolishing cocaine self-administration (Fox et al., 1996). Although they are typically lower, antigen-specific IgG concentrations in this range have been reported in humans (Weiss et al., 1995; Janoff et al., 1991), which supports the clinical relevance of this rat model.
The brain/plasma nicotine ratio in control animals of 3.8 was similar to the value of 3.3 reported previously in rats 60 min after nicotine dosing (Chowdhury et al., 1993). The brain/plasma nicotine ratio was significantly lower in the CMUNic group (0.8), but this was primarily due to the increase in the plasma nicotine concentration. Brain nicotine concentrations in the two groups were not significantly different. The brain nicotine concentration in the CMUNic group was, however, 13.8% lower than in controls, a result similar to the estimate of the amount of nicotine bound in plasma. It is possible that the brain nicotine concentration was in fact somewhat lower in the CMUNic animals but that the limited power of this small study was insufficient to demonstrate this effect. It is not clear whether such a small reduction in brain nicotine concentration would be sufficient to produce behavioral effects. Although this possibility should be studied, better immunogens that produce higher-affinity antibodies would clearly be desirable. The more pronounced (approximately 50%) reductions in brain cocaine concentrations reported by others to result from immunization (Carrera et al., 1995; Fox et al., 1996) may reflect higher affinities or serum titers of these drug-specific antibodies, differences in drug doses or differences in sampling times. In particular, measuring the brain nicotine concentration earlier than 40 min would be of interest.
In summary, the effects of active immunization on nicotine distribution were studied in a rat model that simulated the arterio-venous difference in nicotine levels produced by cigarette smoking and resulted in clinically relevant plasma nicotine concentrations. Immunization markedly increased the binding of nicotine in plasma, an estimated 9% of the administered dose being bound by antibody. These effects that were observed despite the use of an immunogen that produced antibodies of only modest affinity suggests that immunotherapy, particularly with improved immunogens, has the potential to alter substantially the distribution of nicotine and perhaps alter its behavioral effects.
Acknowledgments
We wish to thank Dr. Peyton Jacob (University of California, San Francisco) for the tissue nicotine extraction method and for supplying internal standards, and Professor John Gorrod (King’s College, London) for supplying nicotine-N-oxide.
Footnotes
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Send reprint requests to: Paul Pentel, M.D., Department of Medicine, Hennepin County Medical Center, 701 Park Ave S., Minneapolis, MN 55415.
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↵1 Supported by NIDA grants #P50-DA09259 and DA10714.
- Abbreviations:
- CMUNic
- 6-(carboxymethylureido)-(±)-nicotine
- KLH
- keyhole limpet hemocyanin
- BSA
- bovine serum albumin
- Received May 14, 1997.
- Accepted August 8, 1997.
- The American Society for Pharmacology and Experimental Therapeutics