Date of Graduation


Document Type


Degree Type



School of Medicine


Physiology, Pharmacology & Neuroscience

Committee Chair

Patrick S. Callery.


Free-base ("crack") cocaine smoking continues to be a popular method of drug abuse in the United States. Pyrolysis of free-base cocaine readily occurs, forming primarily benzoic acid and anhydroecgonine methyl ester (AEME), which structurally contains an alpha,beta-unsaturated carbonyl functional group. This electrophilic structure is suggestive of chemical reactivity with ubiquitously occurring cellular nucleophiles, such as glutathione. Glutathione adducts formed in vivo are metabolized to N-acetylcysteine derivatives by the enzymatically driven mercapturic acid pathway. AEME and its transesterification product in the presence of ethanol, anhydroecgonine ethyl ester (AEEE), were synthesized from cocaine hydrochloride. Glutathione (GSH) and mercapturic acid (NAC) conjugates of AEME were synthesized, and the stereochemistry of the AEME-NAC conjugate was structurally elucidated by NMR and mass spectrometry. Using Ellman's method to monitor glutathione depletion over time, AEME reacted with GSH at a slower rate (6.8 x 10 -3 mM-1min-1) than equimolar concentrations of arecoline or ethacrynic acid (2.2 x 10-1 mM-1min -1 and 4.1 x 10-1 mM-1min-1, respectively). AEME also reduced the chemical formation of the DCNB-SG conjugate. In pooled human liver cytosol, the incubation of AEME with GSH at 37°C for 60 minutes at different pH levels was analyzed by LC-MS. We found unequivocal data suggesting enzymatic catalysis of AEME to AEME-SG in the presence of reduced GSH. However, AEME (10 mM) significantly reduced GST activity (p < 0.005) following a 20 hour incubation with HLC (1 mg). Additionally, AEME exhibited mixed-linear inhibition (Ki = 334 muM) towards cytosolic GST activity. A solid phase extraction (SPE) method was developed to extract cocaine and cocaine metabolites from urine. We identified approximately 50 ng/mL of AEEE by LC-MS in the urine of a known freebase cocaine/ethanol abuser, and additionally confirmed the structure of the metabolite by LC-MS/MS. This SPE method is useful in detecting AEME conjugates in biological fluids. We studied the cytotoxicity of pyrolysis products on A549 lung fibroblasts using trypan blue exclusion and flow cytometry. To summarize, our work suggests that AEME plays a potential role in the sequelae of abused cocaine related toxicities.