This project is a series of translational studies evaluating novel analgesic strategies and therapeutic agents with potential for enhanced efficacy and safety in comparison to standard treatments. Specific objectives of this project are: 1) to evaluate the analgesic potential of investigational analgesics which alter either the synthesis or receptor activation of inflammatory and neuroendocrine mediators; 2) to develop novel analgesic strategies for preventing pain and optimizing analgesia while minimizing side effect potential for acute pain in ambulatory patients; and 3) to characterize the molecular-genetic responses to surgical stress, acute pain, and inflammation. Subjects in these investigations undergo oral surgery performed with local anesthesia with resultant neuroendocrine and inflammatory responses to pain and surgical stress during the perioperative period as well as acute pain for several days. Samples are collected from blood to evaluate systemic responses, by microdialysis at the surgical site to measure local release of inflammatory mediators such as prostaglandins, and by tissue biopsy to measure changes in gene expression due to inflammation. DNA is extacted from blood for selected studies to evaluate single nucleotide polymorphisms (SNPs) and their haplotypes that may be associated with inter-individual variations in response to pain and analgesia. Subjects remain at the clinic up to seven hours postoperatively and may return at 24 and 48 hours for additional observations and collection of biopsy samples A variety of cultural, psychological and physiological factors contribute to variability in both clinical and experimental contexts. The role of genetic factors in human pain sensitivity is also recognized as an important element. A study evaluating the genetic influences on variability in human pain sensitivity associated with gender, ethnicity, and temperament measured responses to experimental painful thermal and cold stimuli with visual analog scale ratings; temperament dimensions of personality were also evaluated. Loci in the vanilloid receptor subtype 1 gene (TRPV1), delta receptor opioid subtype 1 gene (OPRD) and catechol O-methyltransferase gene (COMT) were genotyped. Female European Americans with a TRPV1 allele and males with low harm avoidance showed longer cold withdrawal times based on classification and regression tree analysis. Gender, an OPRD1 polymorphism and temperament dimensions of personality were primary determinants of heat pain sensitivity. These observations demonstrate that gender, ethnicity and temperament contribute to individual variations in thermal and cold sensitivity through interactions with TRPV1 and OPRD1 SNPs. We further evaluated the pain sensitivity of normal subjects to experimentally induced pain to delineate individual response patterns and pain phenotypes; a subset of subjects also underwent standardized oral surgery and the responses to clinically induced inflammatory pain were evaluated. The latency for withdrawal in the cold pressor test exhibited a dichotomous distribution of short and long times. Women exhibited higher responses to cold and thermal stimuli than men. Ethnicity affected pain responses to thermal stimuli and cold stimuli. Neither gender nor ethnicity affected pain responses to clinical pain. Cross-modality comparisons of responses within experimental pain stimuli showed strong correlations but weaker correlations to clinical pain. These findings suggest that background factors and characteristics of experimental pain responses differ from those of clinical pain; therefore experimental pain ratings alone are not sufficient to predict responses to acute clinical acute pain. Given the prominent role of cyclooxygenase (COX) in the inflammatory responses to tissue injury, we investigated expression patterns of COX genes encoding enzymes related to prostaglandin production in a clinical model off tissue injury and their relationship with genetic polymorphisms. Pre- and post-surgical biopsies were taken from patients undergoing third molar removal; the second biopsies were done within the first 4 hours or at 48 hours following surgery. The effects of genetic variation were investigated with haplotypes based on SNPs in the COX gene. COX-2 expression at early time points was significantly increased but returned to pre-surgical levels with wide variance by 48 hours. COX-1 expression was decrease slightly at early time points but little detectable change was seen at 48 hours. Haploltypes and individual SNPs in COX-1 and COX-2 did not affect expression of either gene. Transcriptional activity of sPLA2IIA, dPGES, and mPGES was co-activated with COX-2. While these observations support the role of COX-2 in acute inflammation and pain as a target for analgesia, they also suggest that wide inter-individual variation in COX-2 expression may influence the analgesic response to selective COX-2 inhibitors. These observations also suggest sPLA2IIA, cPGES and mPGES as targets for anti-inflammatory drug development. Consistent with these findings are the results of a subsequent study evaluating the effects of a dual COX-1/-2 inhibitor (ibuprofen) and a selective COX-2 ihibitor (rofecoxib) on expression patterns of pro-inflammatory genes related to prostaglandin formation. COX-2 expression at 48 hours varied widely with drug administration: no change in the placebo group but increased COX-2 mRNA from ibuprofen and rofecoxib treatment. Three other enzymes related to PG production were significantly increased and 15-PGDH (related to PG degradation) was significantly decreased in the drug groups This demonstrates that a dual COX-1/COX-2 inhibitor and a selective COX-2 inhibitor change expression patterns of pro-inflammatory genes following tissue injury to stimulate the arachidonic acid cascade and may attenuate the inhibitory actions of these drugs by increased production of PG. The influence of common genetic polymorphisms of COX-1 and COX-2 genes on the modulation of responses to clinically induced pain in humans was examined in patients (N=200) undergoing third molar removal. COX-2 gene polymorphisms showed significant association for the time to onset of postoperative pain. Slight but significant variation in postoperative pain measures can be attributed to these SNPS, suggesting that COX-2 gene polymorphisms contribute to pain following tissue injury and may help to explain inter-individual variation in COX-2 mediated inflammatory pain. The combinations of COX gene polymorphisms in 4 major ethnic populations were generated by Haploview analysis to identify COX gene haploblocks. This information provides basic ethnic background for analysis of genetic effects of COX genes on individual variance in pain sensitivity and responses to drugs in future studies. We further evaluated gene expression in the arachidonic acid pathway due to tissue injury and inflammation, an NSAID and a coxib in the oral surgery model by use of Affymetric microarray chips. In post-surgery samples in the placebo group, inflammation related genes were up-regulate from baseline samples whereas these genes were down-regulated or unchanged in the ibuprofen and rofecoxib groups. Genes involved in inhibition of PLA2 were upregulated in the ibuprofen and rofecoxib groups; several genes related to PG activity were also upregulated by rofecoxib. LOX3, a gene in the lipoxygenase pathway was down-regulated by ibuprofen but unaffected by rofecoxib. Differential drug effects were also observed for genes related to coagulation. These results suggest that coxibs and NSAIDs result in differential effects on AA cascade gene expression following tissue injury that reflects the inhibition of COX by these drugs.
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