Proinflammatory Gene Expression and Prostanoid Levels in a Clinical Model of Tissue Injury

Two cyclooxygenase isozymes, COX-1 and -2, are known to catalyze the rate-limiting step of prostaglandin synthesis and are the targets of nonsteroidal anti-inflammatory drugs. Recently, the presence of a variant of COX-1, named COX-3, has been demonstrated that is especially sensitive to acetaminophen and strongly expressed in brain tissue. The proposed study will examine prostanoid suppression in the periphery by inhibition of COX-1, -2, and -3, and evaluate the time course of expression of COX isoenzymes in terms of level of mRNA and protein itself. Subjects (N equals 88) will be healthy volunteers scheduled for the surgical removal of impacted third molars. Using a double-blinded, randomized, parallel study design, subjects will be allocated to one of four treatment groups and will be administered a dose of blinded medication or placebo. One hour prior to oral surgery, rofecoxib 50 mg, acetaminophen 1000 mg or placebo in two groups will be administered orally. Half hour prior to surgery, ketorolac 30 mg or placebo, will be administered intravenously. Microdialysis will be performed with sample collection concurrent with pain report over the immediate postoperative period of three hours, and the collected transudate will be analyzed by ELISA to evaluate prostanoid (prostaglandin E2, thromboxane B2) production. Subjects within each group will be further randomized to have a biopsy collected at baseline prior to surgery and either at the time when a subject asks for rescue drug within 3 hours or at 24 hours following surgery. The biopsies will be frozen in liquid nitrogen and assayed later by RT-PCR for levels of COX-1, COX-2, or COX-3 mRNA, and by microarray for evaluation of change in overall mRNA expression. A portion of the biopsy samples will be immunostained to reveal the tissue-specific pattern of each COX isoenzymes expression within the inflammatory tissue, and another part of the biopsy samples will be analyzed by Western blot to quantify the amount of protein. Fifty ml of blood will be collected from all subjects, and the single nucleotide polymorphisms (SNPs) in the genes regulated by inflammatory responses such as COX-1, COX-2, PGE receptors (EPs), and microsomal PGE synthase (mPGES) genes, will be analyzed to investigate the role of genetic factors in individual differences of drug responses. The analgesic effect of the drugs will be assessed in the clinic every 20 minutes for the first three hours after extractions, and on the next morning before taking any medication using two pain intensity assessment instruments: a category scale, and a visual analog scale (VAS). We anticipate that non-selective COX inhibitor, the selective COX-2 inhibitor, and the selective COX-3 inhibitor will differentially alter prostanoid production over the time course evaluated, and that primary sensory neurons innervating in the inflammatory tissues may differentially express COX isoenzymes that differ in sensitivity to prototypic NSAIDs, coxibs, and acetaminophen. We also anticipate that several SNPs will contribute to the individual differences in COX expression and drug responses, which has implications for individual variation in pain, analgesic responses, and neuronal plasticity.