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Peripheral Blockers as Treatments for Substance Abuse and Dependence

Poster Abstracts

Buddy Pouw1, Garo P. Basmadjian1, Satendra Singh1, Michael H. Shearer2, and Ronald C. Kennedy2
1Department of Medicinal Chemistry and Pharmaceutics, 2College of Pharmacy, Microbiology and Immunology, College of Medicine, University of Oklahoma Health Sciences Center


Porcine liver esterase (PLE-EC has been shown to have the highest efficiency of hydrolyzing cocaine compared to other commercially available esterases. This carboxylesterase was purified using a Concanavalin A column and complexed with a novel Transition State Analog of cocaine (TSA-III). Female BALB/cByJ mice immunized with this complex (PLE-TSA-III) produced anti-PLE-TSA-III antibodies (ELISA 1:70,000; n = 9) that hydrolyzed cocaine (1.3 fold compared to the pre-bled sera), ten days after the third sc. injection. Monoclonal antibodies were generated by hybridizing the spleen cells of the immunized mice with SP2/0-Ag14, mice myeloma cells. A purified monoclonal antibody (CD-1) hydrolyzed cocaine following Michaelis-Menten kinetics (Km: 19.4 &M, Vmax: 0.17&moles/mg/min; all wells, n = 8). This preliminary study showed that a novel and potentially fruitful approach was possible in developing a vaccine against cocaine. On the other hand, from the public health point of view, the cost-effectiveness of such preventive means can be enhanced by encapsulating the vaccine that can be delivered as a single shot immunization.

Joel Proksch
University of Arkansas for Medical Sciences


The purpose of these studies was to determine favorable therapeutic conditions for increasing phencyclidine (PCP) and anti-PCP Fab co-elimination by the kidney. In the first study, attempts to increase Fab elimination by fluid loading or systemic alkalinization did not increase the total amount of PCP or Fab in the urine compared to controls. However, urinary alkalinization did significantly increase the excretion rate of Fab during the first 3 hr following anti-PCP Fab administration. To better understand this increased excretion rate, serum and urine pharmacokinetics were determined. No differences in overall Fab pharmacokinetics were found between control and alkalinized animals. However, during the first 3 hr interval, Fab renal clearance was 50% higher in alkalinized animals, apparently due to an increase in glomerular filtration rate. Although a rapid loss of Fab within the first 3 hr accounted for the majority of Fab elimination in all rats, this elimination occurred at a faster rate in the alkalinized animals. Additionally, renal excretion of intact Fab and PCP was linear over a 30-fold range of mole-equivalent doses and averaged 60% and 28% of the dose, respectively. Without the Fab, only 2.5% of the PCP was eliminated in the urine. Furthermore, analysis of serum from selected rats showed no significant immune response against Fab. In conclusion, these data showed that Fab was safe to administer, and the serum pharmacokinetics followed first-order processes consisting of Fab losses through an initial rapid elimination phase (~60% of the dose) and a slower terminal elimination phase (~40% of the dose). (Supported by NIDA grants DA07610, RSDA KO2 DA0110 to S.M.O., and NRSA F31DA05795 to J.W.P.)

Shixian Deng, Milan N. Stojanovic, Danwen Huang, Rowena E. Almonte, Paloma De Prada, Donald W. Landry

Columbia University College of Physicians and Surgeons


Cocaine can be degraded by hydrolysis of the benzoyl ester group to yield inactive products and a catalyst for this reaction could reduce serum cocaine concentrations, deprive the cocaine abuser of the behavioral reinforcing effect of the drug, and thus favor extinction of the addiction. Catalytic antibodies are programmable artificial enzymes obtained by immunization with a transition-state analog of the substrate in the midst of the desired chemical transformation. We have synthesized several analogs of cocaine hydrolysis, immunized mice, and generated antibodies that hydrolyze cocaine. One of these enzymes has achieved the activity necessary for preclinical studies and we have demonstrated its ability to block lethal doses of cocaine in rat. We have begun a rational effort to improve on the activity of these first artificial enzymes to degrade cocaine through the synthesis of novel transition-state analogs and the mutagenesis of our most active antibody.

The strategies guiding the synthesis of novel transition-state analogs include:

Substrate-assisted enzymatic catalysis: Cyclic analogs based on phosphinate esters have been synthesized in order to elicit antibodies that would allow for the protonated bridgehead amine in cocaine to participate in intramolecular catalysis.

Heterologous immunization: C.3 2'phenylethyl analogs of cocaine have been synthesized to construct 1',2'-aminoalcohol analogs of cocaine. Consecutive immunizations with these analogs and classic phosphonate monoesters will recruit antibodies endowed with the desired orientation of functional groups.

Reactive immunization: Analogs of cocaine with the organophosphate ester functionality of nerve agents and pesticides are expected to yield abzyme mimics of choline esterases.

Anita D. Wentworth, Ph.D.

The Scripps Research Institute


Catalytic antibodies comprise a well-established class of programmable biocatalysts, the practical applications of which have been implicated in both synthetic chemistry and immunotherapies. Here we report some of the recent advances in our group which have focused on new hapten strategies to elicit catalysts for the unassisted hydrolyses of phosphodiesters and primary amides. In addition, our reactive immunization approach has now been successfully applied to yield catalysts for the homochiral production of the anti-inflammatory agent Naproxen. The unassisted hydrolyses of phosphodiester and amide bonds are considered major targets for antibody catalysis because of both the challenge posed by their inherent stability and their potential therapeutic relevance. Our preliminary success in generating an antibody phosphodiesterase using a transition-state analog approach has now been augmented with a bait-and-switch hapten design. A quaternary ammonium phosphate hapten generated an antibody, MATT.F-1, which catalyzes the hydrolysis of a p-nitrophenyl phosphate substrate
(kcat/kuncat = 3D 1.7 x 103).

A novel boronic acid hapten has been employed in a chemical selection strategy to elicit catalysts for amide bond hydrolysis. Although no catalysts were isolated using conventional hybridoma techniques, a catalytic Fab, BL-25, was highlighted following panning of a combinatorial antibody library. BL-25 catalyzes efficiently the regio- and stereoselective hydrolysis of the terminal primary amide bond in a tri peptide (kcat/kuncat = 3D 4 x 104).

The chiral resolution of (=B1)-Naproxen has been facilitated by enantioselective hydrolysis of its aryl esters using antibody catalysts generated against a reactive phosphonate diester immunogen. This approach has produced some of the most catalytically proficient antibodies yet known.

Wei Hua Xie1, C.V. Altamirano1, C.F. Bartels, R.J. Speirs2, J.R. Cashman2, and O. Lockridge1

1Eppley Institute and Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center and 2Human BioMolecular Research Institute


Life-threatening symptoms due to cocaine toxicity include grand-mal seizures, cardiac arrest, stroke, and drug-induced psychosis accompanied by elevated body temperature. Healthy young men have died after using cocaine for the first time, the most famous case being that of the University of Maryland basketball player Len Bias in 1986. It is known that the major route of cocaine detoxication is hydrolysis by butyrylcholinesterase (BChE). This enzyme occurs in several genetic variant forms. The homozygous atypical variant (D70G) occurs in 1 out of 2500 people, the J variant (E497V) in 1 out of 100,000, and the K variant (A539T) in 1 out of 100. People who have the atypical variant are unable to breathe for 2 hours after receiving a dose of succinylcholine that paralyzes most people for only 3-5 minutes. The reason for this prolonged effect is that the atypical variant has a low binding affinity for succinylcholine. We expressed these genetic variants in CHO cells, purified the BChE, and measured kcat and Km values. We found that the atypical variant had a tenfold lower binding affinity for cocaine, but a normal kcat value. The J and K variants had normal kcat and Km values. These results suggest that people with atypical BChE may experience severe or fatal cocaine intoxication after doses of cocaine that are not harmful to others. People with the K variant have a 33% decrease of BChE activity in plasma and people with the J variant have a 66% decrease, suggesting that people with these genetic variants are more susceptible to the toxic effects of cocaine, even though their binding affinities and kcat values are similar to those of wild-type.

In an attempt to improve the cocaine hydrolase activity of BChE, we mutated amino acids near the active site. The mutant A328Y had a kcat of 10.2 min-1 and Km of 9 M for a fourfold improvement in catalytic efficiency (kcat/Km) compared to wild-type BChE with its kcat of 3.9 min-1 and Km of 14 M. BChE purified from plasma of cat, horse, and chicken was tested for cocaine hydrolase activity. Compared to human BChE, horse BChE had a twofold higher kcat but a lower binding affinity, cat BChE was similar to human, while chicken BChE had only 10% of the catalytic efficiency.

Supported by the Nebraska Affiliate of the American Heart Association Grant 9707841S (to O.L.), AASERT Award DAAG55-07-1-0244 from the U.S. Army Research Office (to O.L.) NIH grants DA08531 and DA00269 (to J.R.C.), DA011707 (to O.L.), and U.S. Army Medical Research and Materiel Command Grant DAMD17-97-1-7349 (to O.L.).

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