Context Cocaine dependence, which affects 2.5 million Americans annually, has no FDA approved pharmacotherapy. Objective To evaluate the immunogenicity, safety, and efficacy of a novel cocaine vaccine to treat cocaine dependence. Design 24 week Phase IIb randomized double-blind placebo-controlled trial with efficacy assessed during weeks 8 to 20 and follow-up to week 24. Setting Cocaine and opioid dependent persons recruited from 2003–2005 from greater New Haven, CT. Participants 115 methadone maintained subjects (67% male, 87% Caucasian, aged 18–46) were randomized to vaccine or placebo and 82% completed the trial. Most smoked crack cocaine along with using marijuana (18%), alcohol (10%), and non-prescription opioids (44%). Intervention Over 12 weeks 109/115 subjects received five vaccinations of placebo or succinylnorcocaine linked to cholera B protein. Main Outcome Measure Semi-quantitative urinary cocaine metabolite levels measured thrice weekly with positive cutoff of 300 ng/ml. Results The 38% of vaccinated subjects who attained serum IgG anti-cocaine levels ≥ 43 µg/mL (high IgG) had significantly more cocaine-free urines than those with < 43 µg/mL (low IgG) and the placebo subjects during weeks 9 to 16 (45% vs 35%). The proportion of subjects having a 50% reduction in cocaine use was significantly greater in the high IgG than low IgG subjects (0.53 vs. 0.23) (P<0.04). The most common side effects were injection site induration and tenderness. There were no treatment related serious adverse events, withdrawals, or deaths. Conclusions Attaining high (≥ 43 µg/mL) IgG anti-cocaine antibody levels was associated with significantly reduced cocaine use, but only 38% of the vaccinated attained these IgG levels and they had only 2 months of adequate cocaine blockade. Thus, we need improved vaccines and boosters.

ABSTRACT

Aerosol delivery of plasmid DNA to the lungs offers the possibility of direct application of gene preparations to pulmonary surfaces as a means of treating a variety of genetic pulmonary disorders. However, the process of jet nebulization rapidly degrades naked DNA, viral vectors, and many lipid-based formulations. While complexing DNA with cationic lipids has been shown to significantly stabilize plasmid DNA, losses of biological activity often occur during nebulization, severely limiting the efficiency of aerosol delivery of many such complexes. In conjunction with the design of aerosol delivery systems appropriate for DNA delivery, we have developed formulations using polyethyleneimine (PEI, a polycationic polymer) and DNA that result in a high level of pulmonary transfection (10- to 100-fold greater than many cationic lipids) and are stable during nebulization. In addition, these PEI-based formulations exhibit a high degree of specificity for the lungs. The properties of PEI-based formulations that make them resistant to nebulization and efficient as DNA delivery vectors for pulmonary sites have been investigated. Potential applications of this technology, including the use of aerosolized PEI-DNA for genetic immunization, are discussed.

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.

DNA degradation is a fundamental problem for any gene therapy or genetic immunization approach, since destruction of incoming genes translates into loss of gene expression. To characterize the biology of DNA degradation after naked DNA injection, the location and levels of tissue nucleases were assessed. Extracts from the serum, kidney, and liver of mice had high levels of calcium-dependent endonuclease activity. High levels of acidic endonuclease activity were identified in the spleen, liver, kidney, and skin with little activity in skeletal or cardiac muscle. Relatively little exonuclease activity was observed in any tissue. The presence of endonucleases in the skin and muscle mediated degradation of 99% of naked DNA within 90 min of injection. This degradation most likely occurred in the extracellular space upstream of other cellular events. Despite this massive destruction, gross tissue nuclease levels did not determine skin-to-muscle transfection efficiency, or site-to-site transfection efficiency in the skin. While gross tissue nuclease levels do not appear to determine differences in transfection efficiency, the presence of robust tissue nuclease activity still necessitates that massive amounts of DNA be used to overcome the loss of 99% of expressible DNA. In addition to destroying genes, the nucleases may play a second role in genetic immunization by converting large plasmids into small oligonucleotides that can be taken up more easily by immune cells to stimulate CpG-dependent Th1 immune responses. For genetic immunization, vaccine outcome may depend on striking the right balance of nuclease effects to allow survival of sufficient DNA to express the antigen, while concomitantly generating sufficient amounts of immunostimulatory DNA fragments to drive Th1 booster effects. For gene therapy, all nuclease effects would appear to be negative, since these enzymes destroy gene expression while also stimulating cellular immune responses against transgene-modified host cells.

Current medications for drug abuse have had only limited success. Anti-addiction vaccines to elicit antibodies that block the pharmacological effects of drugs have great potential for treating drug abuse. We review the status for two vaccines that are undergoing clinical trials (cocaine and nicotine) and two that are still in pre-clinical development (methamphetamine and heroin). We also outline the challenges and ethical concerns for anti-addiction vaccine development and their use as future therapeutics.

Conventional substance abuse treatments have only had limited success for drugs such as cocaine, nicotine, methamphetamine, and phencyclidine. New approaches, including vaccination to block the effects of these drugs on the brain, are in advanced stages of development. Although several potential mechanisms for the effects of anti-drug vaccines have been suggested, the most straightforward and intuitive mechanism involves binding of the drug by antibodies in the bloodstream, thereby blocking entry and/or reducing the rate of entry of the drug into the central nervous system. The benefits of such antibodies on drug pharmacodynamics will be influenced by both the quantitative and the qualitative properties of the antibodies. The sum of these effects will determine the success of the clinical applications of anti-drug vaccines in addiction medicine. This review will discuss these issues and present the current status of vaccine development for nicotine, cocaine, methamphetamine, phencyclidine, and morphine.

Background: Ara h 2 is a major peanut allergen recognized by IgE in more than 90% of patients. After electrophoretic separation the purified protein exists as a doublet, and sequences of one incomplete cDNA and one genomic clone for this allergen have been reported. Methods: Ara h 2 isoforms were purified and analyzed by mass spectroscopy, and PCR amplification products of Ara h 2 were cloned and sequenced. Results: Mass spectroscopy of purified Ara h 2 clearly identified a molecular doublet of 16,670 and 18,050 Daltons. Amplification of a peanut cDNA library using PCR primer pairs located at the amino- and carboxy-terminus revealed 2 bands separated by 50 base pairs, which we cloned and sequenced. Two types of complete cDNA clones were obtained, Ara h 2.01 and Ara h 2.02. Compared to Ara h 2.01 and the previously reported cDNA sequences, Ara h 2.02 is characterized by a 12 amino acid insertion starting at position 75 that contains a third repeat of the major IgE binding epitope DPYSPS. Conclusion: We demonstrated the molecular and genetic characteristics of two Ara h 2 isoforms, revealing that one, Ara h 2.02, might be the more potent allergen.