Nicotine conjugate vaccine efficacy is limited by the concentration of nicotine-specific antibodies that can be reliably generated in serum. Previous studies suggest that the concurrent use of 2 structurally distinct nicotine immunogens in rats can generate additive antibody responses by stimulating distinct B cell populations. In the current study we investigated whether it is possible to identify a third immunologically distinct nicotine immunogen. The new 1′-SNic immunogen (2S)-N,N′-(disulfanediyldiethane-2,1-diyl)bis[4-(2-pyridin-3-ylpyrrolidin-1-yl)butanamide] conjugated to keyhole limpet hemocyanin (KLH) differed from the existing immunogens 3′-AmNic-rEPA and 6-CMUNic-BSA in linker position, linker composition, conjugation chemistry, and carrier protein. Vaccination of rats with 1′-SNic-KLH elicited high concentrations of high affinity nicotine-specific antibodies. The antibodies produced in response to 1′-SNic-KLH did not appreciably cross-react in ELISA with either 3′-AmNic-rEPA or 6-CMUNic-BSA or vice-versa, showing that the B cell populations activated by each of these nicotine immunogens were non-overlapping and distinct. Nicotine retention in serum was increased and nicotine distribution to brain substantially reduced in rats vaccinated with 1′-SNic-KLH compared to controls. Effects of 1′-SNic-KLH on nicotine distribution were comparable to those of 3′-AmNic-rEPA which has progressed to late stage clinical trials as an adjunct to smoking cessation. These data show that it is possible to design multiple immunogens from a small molecule such as nicotine which elicit independent immune responses. This approach could be applicable to other addiction vaccines or small molecule targets as well.

In addition to addiction, the repeated use of (+)-methamphetamine [(+)-METH], (+)amphetamine [(+)-AMP], or (±)-methylenedioxymethamphetamine [(±)-MDMA, commonly called ecstasy] canlead to life-threatening medical problems including cardiovascular injury, severe depression, and psychosis. Currently, there are no specific pharmacotherapies to treat these medical problems. In this study, we report the design and synthesis of two haptens, (S)-(+)-3-(9-carboxynonyloxy) methamphetamine [3a, (+)-METHMO10] and (S)-(+)-3-(5-carboxypentyloxy)methamphetamine [3b, (+)-METH MO6], and their use in generating high affinity (low K D value) monoclonal antibodies (mAbs) against (+)-METH, (+)-AMP, and/or (+)-MDMA. Based on results from the determination of mAb K D values and ligand specificity, the mAbs generated from hapten 3a showed the greatest promise for generating active and passive immunotherapies for treating overdose or addiction from (+)-METH-like stimulants.The abuse of (S)-(+)-methamphetamine [1a-(+)-METH] a and other amphetamine-like stimulants continues to be a major health problem worldwide. 1-3 Indeed, a study by RAND Corporation estimates the economic cost of (+)-METH use in the United States in 2005 was $23.4 billion. 4 This comprehensive estimate includes the economic burden of addiction, premature death, drug treatment, and many other aspects of the drugs impact on Americans. The 2008 National Survey on Drug Use and Health estimates that over 12 million individuals, aged 12 and older, had used (+)-METH in their lifetime, that 850,000 million had used (+)-METH during the past year, and that 314,000 individuals had used (+)-METH during the last month, which defines them as current users. 5 At present there are no specific pharmacotherapies for managing adverse (+)-METH-induced effects like acute overdose and chronic addiction. Preclinical studies in rats show that systemic administration of anti-(+)-METH monoclonal antibodies (mAbs) can rapidly remove the drug from its sites of action in critical tissues like the brain and heart suggesting that immunotherapy could provide an important new medical strategy for addressing (+)-METH-induced adverse health effects in humans, while others suggest antibody catalyzed inactivation of METH could be a possible therapeutic approach. 7 (S)-(+)-Amphetamine [1b, (+)-AMP], which is a major metabolite of (+)-METH and a drug of abuse, and (+)-methylenedioxymethamphetamine, the stimulant-inducing chemical in the racemic mixture of (±)-methylenedioxymethamphetamine [2, (±)-MDMA, commonly referred to as ecstasy] are two other widely abused and dangerous stimulants. The potency and stimulant effects of these (+)-METH-like compounds are influenced by the drug's stereochemistry, with the (+)-or (S)-isomers producing significantly more psychomimetic effects, stereotyped behavior and locomotor activity, 8 and increased production of reactive oxygen species in mice. 9 Given the medical importance of all three of these structurally related (+)-or (S)-isomers, it could be medicall...

This study reports the synthesis of the mercapto hapten (S)-N-(2-(mercaptoethyl)-6-(3-(2-(methylamino)propyl)phenoxy)hexanamide [3, (+)-METH HSMO9] and its use to prepare METH-conjugated vaccines (MCV) from maleimide activated proteins. MALDI-TOF mass spectrometry analysis of the MCV synthesized using 3 showed there was a high and controllable epitope density on two different carrier proteins. In addition, the MCV produced a substantially greater immunological response in mice than previous METH haptens, and a monoclonal antibody generated from this MCV in mice showed a very high affinity for (+)-METH (KD = 6.8 nM). The efficient covalent coupling of (+)-METH HSMO9 to the activated carrier proteins suggests this approach could be cost effective for large-scale production of MCV. In addition, the general methods described for the synthesis of (+) METH HSMO9 (3) and its use to synthesize MCV will be applicable for conjugated vaccines of small molecules and other substances of abuse such as morphine, nicotine, and cocaine.

We hypothesized that an anti-METH mAb could be used in combination with a METH-conjugate vaccine (MCV) to safely improve the overall quality and magnitude of the anti-METH immune response. The benefits would include immediate onset of action (from the mAb), timely increases in the immune responses (from the combined therapy) and duration of antibody response that could last for months (from the MCV). A novel METH-like hapten (METH-SSOO9) was synthesized and then conjugated to immunocyanin monomers of Keyhole limpet hemocyanin (ICKLH) to create the MCV, ICKLH-SOO9. The vaccine, in combination with previously discovered anti-METH mAb7F9, was then tested in rats for safety and potential efficacy. The combination antibody therapy allowed safe achievement of an early high anti-METH antibody response, which persisted throughout the study. Indeed, even after four months the METH vaccine antibodies still had the capacity to significantly reduce METH brain concentrations resulting from a 0.56 mg/kg METH dose.