Preclinical efficacy of an anti-methamphetamine vaccine using E6020 adjuvant.

BACKGROUND AND OBJECTIVE: Methamphetamine (MA) substance use disorder (SUD) does not have an efficacious pharmacotherapy. We developed a MA vaccine and investigated its potential to attenuate MA induced responses. METHODS: We examined a novel adjuvant, E6020, a Toll-like receptor-4 (TLR-4) agonist combined with tetanus-toxoid conjugated to succinyl-methamphetamine (TT-SMA) adsorbed on aluminum hydroxide (alum). Adult BALB/c female mice received the vaccine and booster injections at weeks 0, 3, and 6. The efficacy of the vaccine was assessed by the level and affinity of anti-MA antibodies elicited, its ability to attenuate MA induced locomotor activation and its reduction in the amount of MA entering the brains of vaccinated mice. RESULTS: The TT-SMA vaccine containing alum and E6020 adjuvant produced anti-MA antibodies with nanomolar affinities and showed threefold greater peak titer levels than without E6020 (700 vs 250 µg/ml). These antibodies significantly decreased MA-induced locomotor activation (p < .05), and reduced the brain (p < .005) MA levels following MA administration in actively immunized mice. CONCLUSIONS: Thus, this anti-MA vaccine formulated with E6020 demonstrated effective functional protection against behavioral disruptions induced by MA. SCIENTIFIC SIGNIFICANCE: Together, anti-MA vaccine showing a promising improvement in the efficacy of the vaccine that could be an effective candidate vaccine for methamphetamine use disorder (MUD). Furthermore, combinations of adjuvants may be a tool to design vaccines for MA dependence in humans. (Am J Addict 2019;XX:1-8).

Altered methamphetamine place conditioning in mice vaccinated with a succinyl-methamphetamine-tetanus-toxoid vaccine.

BACKGROUND AND OBJECTIVES: We previously reported that an anti-methamphetamine (MA) vaccine attenuated drug-conditioned effects in mice, but it used a carrier protein and adjuvant not available for clinical use. Here we produced a vaccine with the same hapten (succinyl-methamphetamine, SMA) but attached to tetanus toxoid (SMA-TT) and adsorbed to aluminum hydroxide, components approved for use in humans. We then assessed the vaccine's ability to generate anti-MA antibodies, alter acquisition and reinstatement of MA place conditioning, and prevent MA brain penetration. METHODS: Mice were administered SMA-TT at weeks 0 and 3 and non-vaccinated mice received saline. Anti-MA antibody concentrations were determined at 8 and 12 weeks. Place conditioning began during week 9 in which vaccinated and non-vaccinated mice were divided into groups and conditioned with .5, or 2.0 mg/kg MA. Following acquisition training, mice were extinguished and then a reinstatement test was performed in which mice were administered their original training dose of MA. Separate groups of non-vaccinated and vaccinated mice were administered .5 and 2.0 mg/kg MA and brain MA levels determined. RESULTS AND CONCLUSIONS: Anti-MA antibody levels were elevated at week 8 and remained so through week 12. The SMA-TT vaccine attenuated acquisition and reinstatement of MA place conditioning. Significantly greater proportions of vaccinated mice during acquisition and reinstatement tests showed conditioned place aversion. Moreover, MA brain levels were decreased in vaccinated mice following administration of both doses of MA. SCIENTIFIC SIGNIFICANCE: Results support further development of anti-MA vaccines using components approved for use in humans.

Vaccines against stimulants: cocaine and MA.

While the worldwide prevalence of cocaine use remains significant, medications, or small molecule approaches, to treat drug addictions have met with limited success. Anti-addiction vaccines, on the other hand, have demonstrated great potential for treating drug abuse using a distinctly different mechanism of eliciting an antibody response that blocks the pharmacological effects of drugs. We provide a review of vaccine-based approaches to treating stimulant addictions; specifically and cocaine addictions. This selective review article focuses on the one cocaine vaccine that has been into clinical trials and presents new data related to pre-clinical development of a methamphetamine (MA) vaccine. We also review the mechanism of action for vaccine induced antibodies to abused drugs, which involves kinetic slowing of brain entry as well as simple blocking properties. We present pre-clinical innovations for MA vaccines including hapten design, linkage to carrier proteins and new adjuvants beyond alum. We provide some new information on hapten structures and linkers and variations in protein carriers. We consider a carrier, outer membrance polysaccharide coat protein (OMPC), that provides some self-adjuvant through lipopolysaccharide components and provide new results with a monophosopholipid adjuvant for the more standard carrier proteins with cocaine and MA. The review then covers the clinical trials with the cocaine vaccine TA-CD. The clinical prospects for advances in this field over the next few years include a multi-site cocaine vaccine clinical trial to be reported in 2013 and phase 1 clinical trials of a MA vaccine in 2014.

Attenuation of cocaine-induced locomotor activity in male and female mice by active immunization.

BACKGROUND AND OBJECTIVES: Immunotherapy for drug addiction is being investigated in several laboratories but most studies are conducted in animals of one sex. Yet, women show heightened immune responses and are more likely to develop autoimmune diseases than men. The purpose of this study was to compare the effects of an active anti-cocaine vaccine, succinyl-norcocaine conjugated to keyhole limpet hemocyanin, for its ability to elicit antibodies and alter cocaine-induced ambulatory activity in male versus female mice. METHODS: Male and female BALB/c mice were vaccinated (n = 44) or served as non-vaccinated controls (n = 34). Three weeks after initial vaccination, a booster was given. Ambulatory activity induced by cocaine (20 mg/kg) was assessed at 7 weeks and plasma obtained at 8 weeks to assess antibody levels. RESULTS: High antibody titers were produced in mice of both sexes. The vaccine reduced ambulatory activity cocaine-induced but this effect was greater in female compared to male mice. DISCUSSION AND CONCLUSIONS: The efficacy of this anti-cocaine vaccine is demonstrated in mice of both sexes but its functional consequences are greater in females than males. SCIENTIFIC SIGNIFICANCE: Results point to the importance of testing animals of both sexes in studies of immunotherapies for addiction.

Spontaneous development of IgM anti-cocaine antibodies in habitual cocaine users: effect on IgG antibody responses to a cocaine cholera toxin B conjugate vaccine.

BACKGROUND AND OBJECTIVES: In cocaine vaccine studies, only a minority of subjects made strong antibody responses. To investigate this issue, IgG and IgM antibody responses to cocaine and to cholera toxin B (CTB-the carrier protein used to enhance immune responses to cocaine) were measured in sera from the 55 actively vaccinated subjects in a Phase IIb randomized double-blind placebo-controlled trial (TA-CD 109). METHODS: Isotype specific ELISAs were used to measure IgG and IgM anti-cocaine and anti-CTB antibody in serial samples collected prior to and at intervals after immunization. We assessed IgG anti-cocaine responses of patients with pre-vaccination IgM anti-cocaine antibodies. Competitive inhibition ELISA was used to evaluate antibody specificity. RESULTS AND CONCLUSIONS: Before immunization, 36/55 subjects had detectable IgM antibodies to cocaine, and 9 had IgM levels above the 95% confidence limit of 11 µg/ml. These nine had significantly reduced peak IgG anti-cocaine responses at 16 weeks, and all were below the concentration (40 µg/ml) considered necessary to discourage recreational cocaine use. The IgG anti-CTB responses of these same subjects were also reduced. SCIENTIFIC SIGNIFICANCE: Subjects who develop an IgM antibody response to cocaine in the course of repeated recreational exposure to this drug are significantly less likely to produce high levels of IgG antibodies from the cocaine conjugate vaccine. The failure may be due to recreational cocaine exposure induction of a type 2 T-cell independent immune response. Such individuals will require improved vaccines and are poor candidates for the currently available vaccine.

Active and Passive Immunization with an Anti-Methamphetamine Vaccine Attenuates the Behavioral and Cardiovascular Effects of Methamphetamine.

BACKGROUND: Methamphetamine use disorder (MUD) is a growing health concern with no FDA-approved treatment. The present series of studies build upon our previous work developing an anti-methamphetamine (MA) vaccine for MUD. We determined the effects of a formulation that included tetanus-toxoid (TT) conjugated to succinyl-methamphetamine (TT-SMA) adsorbed onto aluminum hydroxide (alum) in combination with the novel Toll-Like Receptor-5 agonist, entolimod. METHODS: Mice were vaccinated (0, 3, 6 weeks) with TT-SMA+alum and various doses of entolimod to determine an optimal dose for enhancing immunogenicity against MA. Functional effects were then assessed using MA-induced locomotor activation in mice. Experiments using passive immunization of antibodies generated by the vaccine tested its ability to attenuate MA-induced cardiovascular effects and alter the reinforcing effects of MA in an MA-induced reinstatement of a drug seeking model of relapse in male and female rats. RESULTS: Antibody levels peaked at 10 weeks following vaccination with TT-SMA+alum combined with entolimod (1, 3 and 10 µg). MA-induced locomotor activation was significantly attenuated in vaccinated vs. unvaccinated mice and antibody levels significantly correlated with ambulation levels. Passive immunization decreased mean arterial pressure following MA dosing in rats of both sexes but did not alter heart rate. Passive immunization also attenuated the ability of MA to reinstate extinguished drug-seeking behavior in male and female rats. Results support further development of this vaccine for relapse prevention for individuals with MUD.

Active immunotherapy for the Treatment of Cocaine Dependence.

Although cocaine is illegal in most countries of the world, addiction is common and increasing in many populations, and the effectiveness of current treatment options for those afflicted has been very limited. The availability of an anti-cocaine vaccine could offer help to those who wish to quit their addiction. A number of vaccines differing in their chemical nature have been developed, and one has advanced into clinical trials. This review will discuss the successes and limitations of the various vaccines and the results of clinical trials of the vaccine using succinyl norcocaine conjugated to cholera toxin B. This latter vaccine shows considerable promise for those individuals whose antibody response is adequate..

Vaccines for cocaine abuse.

Treatments for cocaine abuse have been disappointingly ineffective, especially in comparison with those for some other abused substances. A new approach, using vaccination to elicit specific antibodies to block the access of cocaine to the brain, has shown considerable promise in animal models, and more recently in human trials. The mechanism of action for the antibody effect on cocaine is very likely to be the straightforward and intuitive result of the binding of the drug in circulation by antibodies, thereby reducing its entry into the central nervous system and thus its pharmacological effects. The effectiveness of such antibodies on drug pharmacodynamics is a function of both the quantitative and the qualitative properties of the antibodies, and this combination will determine the success of the clinical applications of anti-cocaine vaccines in helping addicts discontinue cocaine abuse. This review will discuss these issues and present the current developmental status of cocaine conjugate vaccines.

Non-viral gene delivery to the lungs.

The lung represents an important target for gene therapy: for correction of genetic abnormalities such as cystic fibrosis, for lung cancer therapy, and for vaccination. Genes in the form of expression plasmids can be delivered both by the intravenous route and via the airways. So-called "naked" DNA can be delivered by both of these methods, but gene expression is low. Successful delivery is usually accomplished by complexing the DNA with cationic lipids or with polycations. This review will discuss the efficacy of delivery for particular purposes by various methods and complexing agents, as well as issues of biodistribution, inflammatory reactions, and improvements in formulations. Non-viral gene delivery to the lung has a long history of development, and it is now poised to represent a significant addition to the medical arsenal.

Intravenous immunoglobulin therapy suppresses manifestations of the angioedema with hypereosinophilia syndrome.

Angioedema with hypereosinophilia syndrome has a dramatic clinical presentation that may result from T-cell dysregulation and/or eosinophil dysfunction. Symptoms may be either episodic or persistent and are usually responsive to systemic glucocorticosteroid therapy. This diabetic patient had a dramatically severe presentation, responsive to high-dose steroids but relapsing when prednisone was tapered. To decrease his risks from long-term steroids, a therapeutic trial of intravenous immunoglobulin (IVIG) was tried, and a slow taper off of glucocorticosteroids achieved a prolonged remission. A brief recurrence and subsequent remission of eosinophilia and symptoms were associated with changes in the IVIG preparation lot and source. We conclude that some cases of angioedema with hypereosinophilia syndrome may be highly responsive to IVIG therapy and, furthermore, that specific sources and/or lots of IVIG may have significantly different immunoregulatory properties.

Hapten optimization for cocaine vaccine with improved cocaine recognition.

In the absence of any effective pharmacotherapy for cocaine addiction, immunotherapy is being actively pursued as a therapeutic intervention. While several different cocaine haptens have been explored to develop anticocaine antibodies, none of the hapten was successfully designed, which had a protonated tropane nitrogen as is found in native cocaine under physiological conditions, including the succinyl norcocaine (SNC) hapten that has been tested in phase II clinical trials. Herein, we discuss three different cocaine haptens: hexyl norcocaine (HNC), bromoacetamido butyl norcocaine (BNC), and succinyl butyl norcocaine (SBNC), each with a tertiary nitrogen structure mimicking that of native cocaine which could optimize the specificity of anticocaine antibodies for better cocaine recognition. Mice immunized with these haptens conjugated to immunogenic proteins produced high titre anticocaine antibodies. However, during chemical conjugation of HNC and BNC haptens to carrier proteins, the 2ß methyl ester group is hydrolyzed, and immunizing mice with these conjugate vaccines in mice produced antibodies that bound both cocaine and the inactive benzoylecgonine metabolite. While in the case of the SBNC conjugate, vaccine hydrolysis of the methyl ester did not appear to occur, leading to antibodies with high specificity to cocaine over BE. Although we observed similar specificity with a SNC hapten, the striking difference is that SBNC carries a positive charge on the tropane nitrogen atom, and therefore, it is expected to have better binding of cocaine. The 50% cocaine inhibitory concentration (IC50 ) value for SBNC antibodies (2.8 µm) was significantly better than the SNC antibodies (9.4 µm) when respective hapten-BSA was used as a substrate. In addition, antibodies from both sera had no inhibitory effect from BE. In contrast to BNC and HNC, the SBNC conjugate was also found to be highly stable without any noticeable hydrolysis for several months at 4 °C and 2-3 days in pH 10 buffer at 37 °C.

The future potential for cocaine vaccines.

INTRODUCTION: Addiction to cocaine is a major problem around the world, but especially in developed countries where the combination of wealth and user demand has created terrible social problems. Although only some users become truly addicted, those who are often succumb to a downward spiral in their lives from which it is very difficult to escape. From the medical perspective, the lack of effective and safe, non-addictive therapeutics has instigated efforts to develop alternative approaches for treatment, including anticocaine vaccines designed to block cocaine's pharmacodynamic effects. AREAS COVERED: This paper discusses the implications of cocaine pharmacokinetics for robust vaccine antibody responses, the results of human vaccine clinical trials, new developments in animal models for vaccine evaluation, alternative vaccine formulations and complementary therapy to enhance anticocaine effectiveness. EXPERT OPINION: Robust anti-cocaine antibody responses are required for benefit to cocaine abusers, but since any reasonably achievable antibody level can be overcome with higher drug doses, sufficient motivation to discontinue use is also essential so that the relative barrier to cocaine effects will be appropriate for each individual. Combining a vaccine with achievable levels of an enzyme to hydrolyze cocaine to inactive metabolites, however, may substantially increase the blockade and improve treatment outcomes.

Vaccine for cocaine dependence: a randomized double-blind placebo-controlled efficacy trial.

AIMS: We evaluated the immunogenicity, efficacy, and safety of succinylnorcocaine conjugated to cholera toxin B protein as a vaccine for cocaine dependence. METHODS: This 6-site, 24 week Phase III randomized double-blind placebo-controlled trial assessed efficacy during weeks 8 to 16. We measured urine cocaine metabolites thrice weekly as the main outcome. RESULTS: The 300 subjects (76% male, 72% African-American, mean age 46 years) had smoked cocaine on average for 13 days monthly at baseline. We hypothesized that retention might be better and positive urines lower for subjects with anti-cocaine IgG levels of ≥42 µg/mL (high IgG), which was attained by 67% of the 130 vaccine subjects receiving five vaccinations. Almost 3-times fewer high than low IgG subjects dropped out (7% vs 20%). Although for the full 16 weeks cocaine positive urine rates showed no significant difference between the three groups (placebo, high, low IgG), after week 8, more vaccinated than placebo subjects attained abstinence for at least two weeks of the trial (24% vs 18%), and the high IgG group had the most cocaine-free urines for the last 2 weeks of treatment (OR=3.02), but neither were significant. Injection site reactions of induration and tenderness differed between placebo and active vaccine, and the 29 serious adverse events did not lead to treatment related withdrawals, or deaths. CONCLUSIONS: The vaccine was safe, but it only partially replicated the efficacy found in the previous study based on retention and attaining abstinence.

Prospects, promise and problems on the road to effective vaccines and related therapies for substance abuse.

This review addresses potential new treatments for stimulant drugs of abuse, especially cocaine. Clinical trials of vaccines against cocaine and nicotine have been completed with the generally encouraging result that subjects showing high titers of antidrug antibody experience a reduction in drug reward, which may aid in cessation. New vaccine technologies, including gene transfer of highly optimized monoclonal antibodies, are likely to improve such outcomes further. In the special case of cocaine abuse, a metabolic enzyme is emerging as an alternative or added therapeutic intervention, which would also involve gene transfer. Such approaches still require extensive studies of safety and efficacy, but they may eventually contribute to a robust form of in vivo drug interception that greatly reduces the risks of addiction relapse.

Effects of anti-cocaine vaccine and viral gene transfer of cocaine hydrolase in mice on cocaine toxicity including motor strength and liver damage.

In developing an vivo drug-interception therapy to treat cocaine abuse and hinder relapse into drug seeking provoked by re-encounter with cocaine, two promising agents are: (1) a cocaine hydrolase enzyme (CocH) derived from human butyrylcholinesterase and delivered by gene transfer; (2) an anti-cocaine antibody elicited by vaccination. Recent behavioral experiments showed that antibody and enzyme work in a complementary fashion to reduce cocaine-stimulated locomotor activity in rats and mice. Our present goal was to test protection against liver damage and muscle weakness in mice challenged with massive doses of cocaine at or near the LD50 level (100-120 mg/kg, i.p.). We found that, when the interceptor proteins were combined at doses that were only modestly protective in isolation (enzyme, 1mg/kg; antibody, 8 mg/kg), they provided complete protection of liver tissue and motor function. When the enzyme levels were ~400-fold higher, after in vivo transduction by adeno-associated viral vector, similar protection was observed from CocH alone.

A vaccine against methamphetamine attenuates its behavioral effects in mice.

BACKGROUND: Vaccines have treatment potential for methamphetamine (MA) addiction. We tested whether a conjugate vaccine against MA (succinyl-methamphetamine-keyhole limpet hemocyanin carrier protein; SMA-KLH) would generate MA antibodies and alter MA-induced behaviors. METHODS: Mice were injected with SMA-KLH and received booster administrations 3 and 20 weeks later. Serum antibody titers reached peak levels by 4-6 weeks, remained at a modest level through 18 weeks, peaked again at 22 weeks after the second boost, and were still elevated at 35 weeks. At 7 weeks, groups of vaccinated and non-vaccinated mice were administered one of three MA doses (1, 2 or 3 mg/kg) to assess locomotor activity. RESULTS: Non-vaccinated mice showed dose-dependent effects of MA with hypolocomotion at the lowest dose and elevated activity levels at the highest dose. Both dose effects were reduced in SMA-KLH groups, particularly low dose-induced hypolocomotion at later times post MA administration. Separate groups of vaccinated and non-vaccinated mice were trained in MA place conditioning at 30 weeks with either 0 (vehicle) or 0.5mg/kg MA. Although times spent in the MA-paired side did not differ between groups on test vs. baseline sessions, SMA-KLH mice conditioned with MA showed reduced conditioned approach behaviors and decreased conditioned activity levels compared to control groups. CONCLUSION: These data suggest SMA-KLH attenuates the ability of MA to support place conditioning and reduces or delays its locomotor effects. Overall, results support SMA-KLH as a candidate MA vaccine.

Anti-cocaine antibody and butyrylcholinesterase-derived cocaine hydrolase exert cooperative effects on cocaine pharmacokinetics and cocaine-induced locomotor activity in mice.

We are investigating treatments for cocaine abuse based on viral gene transfer of a cocaine hydrolase (CocH) derived from human butyrylcholinesterase, which can reduce cocaine-stimulated locomotion and cocaine-primed reinstatement of drug-seeking behavior in rats for many months. Here, in mice, we explored the possibility that anti-cocaine antibodies can complement the actions of CocH to reduce cocaine uptake in brain and block centrally-evoked locomotor stimulation. Direct injections of test proteins showed that CocH (0.3 or 1mg/kg) was effective by itself in reducing drug levels in plasma and brain of mice given cocaine (10mg/kg, s.c., or 20mg/kg, i.p). Administration of cocaine antibody per se at a low dose (8 mg/kg, i.p.) exerted little effect on cocaine distribution. However, a higher dose of antibody (12 mg/kg) caused peripheral trapping (increased plasma drug levels), which led to increased cocaine metabolism by CocH, as evidenced by a 6-fold rise in plasma benzoic acid. Behavioral tests with small doses of CocH and antibody (1 and 8 mg/kg, respectively) showed that neither agent alone reduced mouse locomotor activity triggered by a very large cocaine dose (100mg/kg, i.p.). However, dual treatment completely suppressed the locomotor stimulation. Altogether, we found cooperative and possibly synergistic actions that warrant further exploration of dual therapies for treatment of cocaine abuse.

A morphine conjugate vaccine attenuates the behavioral effects of morphine in rats.

Vaccines for opioid dependence may provide a treatment that would reduce or slow the distribution of the drug to brain, thus reducing the drug's reinforcing effects. We tested whether a conjugate vaccine against morphine (keyhole limpet hemocyanin-6-succinylmorphine; KLH-6-SM) administered to rats would produce antibodies and show specificity for morphine or other heroin metabolites. The functional effects of the vaccine were tested with antinociceptive and conditioned place preference (CPP) tests. Rats were either vaccinated with KLH-6-SM and received two boosts 3 and 16 weeks later or served as controls and received KLH alone. Anti-morphine antibodies were produced in vaccinated rats; levels increased and were sustained at moderate levels through 24 weeks. Antibody binding was inhibited by free morphine and other heroin metabolites as demonstrated by competitive inhibition ELISA. Vaccinated rats showed reduced morphine CPP, tested during weeks 4 to 6, and decreased antinociceptive responses to morphine, tested at week 7. Brain morphine levels, assessed using gas-chromatography coupled to mass spectrometry (GC-MS) on samples obtained at 26 weeks, were significantly lower in vaccinated rats. This suggests that morphine entry into the brain was reduced or slowed. These results provide support for KLH-6-SM as a candidate vaccine for opioid dependence.

Dose-dependent changes in the locomotor responses to methamphetamine in BALB/c mice: low doses induce hypolocomotion.

The overall goal of the present study was to determine the effects of different doses of (+)-methamphetamine (meth) on locomotor activity of Balb/C mice. Four experiments were designed to test a wide range of meth doses in BALB/c female mice. In Experiment 1, we examined locomotor activity induced by an acute administration of low doses of meth (0.01 and 0.03mg/kg) in a 90-min session. Experiment 2 was conducted to test higher meth doses (0.3-10mg/kg). In Experiment 3, separate sets of mice were pre-treated with various meth doses once or twice (one injection/week) prior to a locomotor challenge with a low meth dose. Finally, in Experiment 4, we tested whether locomotor activation would be affected by pretreatment with a low or moderate dose of meth one month prior to the low meth dose challenge. Results show that low doses of meth induce hypolocomotion whereas moderate to high doses induce hyperlocomotion. Prior exposure to either one moderate or high dose of meth or to two, low doses of meth attenuated the hypolocomotor effect of a low meth dose one week later. This effect was also attenuated in mice tested one month after administration of a moderate meth dose. These results show that low and high doses of meth can have opposing effects on locomotor activity. Further, prior exposure to the drug leads to tolerance, rather than sensitization, of the hypolocomotor response to low meth doses.

Probing cocaine-antibody interactions in buffer and human serum.

BACKGROUND: Despite progress in cocaine immunotherapy, the kinetic and thermodynamic properties of antibodies which bind to cocaine and its metabolites are not well understood. It is also not clear how the interactions between them differ in a complex matrix such as the serum present in the human body. In the present study, we have used microscale thermophoresis (MST), isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR) we have evaluated the affinity properties of a representative mouse monoclonal (mAb08) as well as those of polyclonal antibodies purified from vaccinated mouse and human patient serum. RESULTS: MST analysis of fluorescently tagged mAb08 binding to cocaine reveals an approximately 15 fold decrease in its equilibrium dissociation constant in 20-50% human serum compared with that in saline buffer. A similar trend was also found using enriched polyclonal antibodies purified from vaccinated mice and patient serum, for which we have used fluorescently tagged bovine serum albumin conjugated to succinyl norcocaine (BSA-SNC). This conjugate closely mimics both cocaine and the hapten used to raise these antibodies. The ITC data also revealed that cocaine has a moderate affinity of about 2 µM to 20% human serum and very little interaction with human serum albumin or nonspecific human IgG at that concentration range. In a SPR inhibition experiment, the binding of mAb08 to immobilized BSA-SNC was inhibited by cocaine and benzoylecgonine in a highly competitive manner, whereas the purified polyclonal antibodies from vaccinated humans and mice, revealed preferential selectivity to pharmacologically active cocaine but not to the inactive metabolite benzoylecgonine. We have also developed a simple binding model to simulate the challenges associated with cocaine immunotherapy using the variable quantitative and kinetic properties of the antibodies. CONCLUSIONS: High sensitivity calorimetric determination of antibody binding to cocaine and its metabolites provide valuable information for characterization of their interactions and thermodynamic properties. In addition MST measurements of antibody affinity in the presence of biological fluids will provide a better opportunity to make reliable decisions and facilitate the design of cocaine vaccines and immunization conditions. The methods should be more widely adopted in characterization of antibody complexes.