Potential clinical applications of small interfering RNA (siRNA) are hampered primarily by delivery issues. We have successfully addressed the delivery problems associated with off-site targeting of highly toxic chemotherapeutic agents by attaching the drugs to tumor-specific ligands that will carry the attached cargo into the desired cancer cell. Indeed, several such tumor-targeted drugs are currently undergoing human clinical trials. We now show that efficient targeting of siRNA to malignant cells and tissues can be achieved by covalent conjugation of small-molecular-weight, high-affinity ligands, such as folic acid and DUPA (2-[3-(1, 3-dicarboxy propyl)-ureido] pentanedioic acid), to siRNA. The former ligand binds a folate receptor that is overexpressed on a variety of cancers, whereas the latter ligand binds to prostate-specific membrane antigen that is overexpressed specifically on prostate cancers and the neovasculature of all solid tumors. Using these ligands, we show remarkable receptor-mediated targeting of siRNA to cancer tissues in vitro and in vivo.

Cocaine is a highly addictive drug, and despite intensive efforts, effective therapies for cocaine craving and addiction remain elusive. In recent years, we and others have reported advances in anti-cocaine immunopharmacotherapy based on specific antibodies capable of sequestering the drug before it reaches the brain. In an effort to obtain high affinity therapeutic anti-cocaine antibodies, either whole IgGs or other antibody constructs, fluorescence spectroscopic techniques could provide a means of assisting selection and engineering strategies. We report the synthesis of a series of cocaine-fluorophore conjugates (GNC-F1, GNC-F2, GNC-I) and the functional evaluation of these compounds against single-chain Fv antibodies obtained via crystallographic analysis/engineering and against commercially available anti-cocaine monoclonal antibodies with a wide range of cocaine-binding affinities. From these studies, we determined that the GNC-F2 fluorophore reproduced affinity constants obtained using [(3)H]-labeled cocaine. We anticipate that the readily synthesized and nonradioactive GNC-F2 will find use both as a tool for bioimaging and in the high-throughput selection and engineering of potential therapeutic antibodies against cocaine.

No abstract

Immunopharmacotherapy as an approach to combat drugs of abuse has become an active area of investigation. Marijuana is the most commonly used illicit drug in the U.S. The main active chemical in marijuana is delta9-tetrahydrocannabinol (delta9-THC); hence, monoclonal antibodies with high affinity and specificity for delta9-tetrahydrocannabinol could be valuable immunopharmacotherapeutic intervention and diagnostic tools. We have synthesized immunoconjugates that induce an effective immune response to delta9-THC and describe a convenient synthesis of radiolabeled delta9-THC. We demonstrate the value and use of this probe to select anti-delta9-THC antibodies that bind delta9-THC with good affinity. The synthetic route to radiolabeled delta9-THC has enabled the correct assessment of the affinity of these antibodies to their ligand and may facilitate future binding studies between delta9-THC and its analogues and the cannabinoid receptors.

One approach to treating drug abuse uses anti-drug antibodies to immunize subjects against the illicit substance rather than administering therapeutics that target the specific CNS site of action. At present, passive vaccination has recognized efficacy in treating certain gross symptoms of drug misuse, namely motor activation, self-administration, and overdose. However, the potential for antibodies to prevent drug-induced changes involving finer cognitive processes, such as benzodiazepineassociated amnesia, remains unexplored. To address this concept, a flunitrazepam hapten was synthesized and employed in the generation of a panel of high affinity monoclonal antibodies. Antiflunitrazepam mAb RCA3A3 (K d,app = 200 nM) was tested in a mouse model of passive immunization and subsequent mole-equivalent challenge with flunitrazepam. Not only was flunitrazepam-induced sedation prevented, but immunization also conferred protection to memory consolidation as assessed through contextual and cued fear conditioning paradigms. These results provide evidence that immunopharmacotherapeutic blockade of drug intoxication also preserves complex cognitive function.