Drugs, other xenobiotics and endogenous compounds often have the ubiquitous carboxylic acid functional group. A common metabolic pathway for such chemicals in humans is conjugation with glucuronic acid to yield an acyl (ester) glucuronide. Recent studies have shown that acyl glucuronides are unstable and reactive both in vitro and in vivo. Acyl glucuronides can undergo intramoleculuar rearrangement to isomeric conjugates which often confound metabolism studies of acidic drugs. More importantly, these reactive metabolites form covalenty adducts with plasma proteins in humans and animals. Previous scientific evidence has documented that covalent adducts of xenobiotics with macromolecules are implicated with immunological reactions, tissue damage and carcinogenesis. Therefore, protein adducts from acyl glucuronides may have significant pharmacological and toxicological consequences. Drugs metabolized in this manner are of serious health concern to the public as evidenced by congressional hearings in the past five years for drugs withdrawn from the market due to toxicity - benoxaprofen, zomepirac and suprofen. A wide range of drugs are metabolized to acyl glucuronides and are often used chronically by millions of patients in the U.S., e.g. nonsteroidal nati-inflammatory drugs for arthritis. Therefore, a better understanding of the disposition and covalent binding of reactive metabolites of this type is warranted and has led to the proposed study to address the following: 1. Is covalent binding to proteins a general phenomenon for compounds that have acyl glucuronide metabolites present in vivo? 2. What is the chemical mechanism for this covalent binding to proteins? 3. Does formation of these covalent adducts alter the function of proteins? Studies will be conducted in vitro and in animals using purified acyl glucuronides of model compounds (e.g. zomepirac, suprofen, clofibric acid) to determine whether they undergo cyl migration or form covalent adducts with proteins as albumin and hemoglobin. The effect of esterase inhibition on the stagility of the acyl glucuronides will be evaluated first in vitro with various tissues from several animal species. The factors that may affect the exposure to acyl glucuronides in vivo, as esterase activity, biliary excretion, and UDP-GT activity, will be modified in animals and their effect on covalent binding will be determined. The disposition of protein adducts in animals will be examined after dosing zomepirac or adduct to autologous albumin, utilizing methods that distinguish the aglycone from the glucuronic acid portion of the adduct. The mechanism of covalent binding will be studied for several compounds to clarify whether """"""""nucleophilic displacement"""""""" or """"""""immune formation"""""""" with Amadori rearrangement is the dominant process. Acyl migration and reversible protein binding of the acyl glucuronides will be studied empirically and theoretically with regard to their role in covalent binding. Finally, the effect of covalent adduct formation on the function of proteins will be evaluated in vitro with albumin, hemoglobin and eye lense crystallin.
