Nicole L. Hatch and Dr. Daniel L. Simmons, Chemistry and Biochemistry
Non-steroidal anti-inflammatory drugs (NSAIDS) are the basis for a multi-billion dollar pharmaceutical market. Their target, Cyclooxygenase (COX), might from an economic standpoint be considered the most important enzyme known to man (1). NSAIDS inhibit COX from producing arachdonic acid, a precursor to prostaglandins (2). Prostaglandins have been indicated in platelet aggregation, cancer, the processing of pain information in the spinal cord, septic shock, smooth muscle contraction during ovulation, cell proliferation, rheumatoid arthritis and inflammation.
Two forms of COX have been isolated. A constitutive form, normally present in most cells, was cloned in sheep in 1988 (3). A second isoform that is inducible was cloned in 1991 (4). The isoforms were named COX-1 and COX-2 respectively. Although very similar in structure and catalytic activity, there are sine very important differences between COX-1 and COX-2. One of the most important distinctions, and a major reason for this study, is that COX-1 produces prostaglandins that protect the stomach and digestive tract. When NSAIDs specifically inhibit COX-1, the gastro-protective function is blocked and gastric and intestinal ulcers often result. In the United States all prescription COX-inhibition NSAIDs carry a warning about life threatening gastrointestinal ulcers (5). By specifically targeting COX-2 with NSAIDs, the gastric side effects of COX-1 inhibition may be eliminated.
Although studies on the selectivity of NSAIDs in humans are well underway, little is known about the pharmacological properties of NSAIDs in canine, particularly with regard to their COX-1 and COX-2 inhibition selectivity. Yet, the treatment of dogs for inflammation and pain is the largest potential veterinary market for NSAIDs. To develop canine COX-2 selective drugs for the treatment of arthritis and pain in dogs, it is necessary to develop accurate models for evaluating COX-1 and COX-2 inhibition. The serum albumin in dog’s blood is well known to bind NSAIDs with much less efficiency then in humans, therefore dogs must be examined separately from other species.
Seven cell lines were screened for COX-2 induction using radioactive immuno assay. CCl-34 Madin-Darby canine kidney cells showed the highest induced COX-2 levels and used for the remainder of the experiments. Twelve flasks of CCl-34s were exposed to phorbol ester for six hours in order to stimulate the production of COX-2. Total RNA was then isolated through the guanidinium isothiocynate method (6). The isolated RNA was Poly-A selected to isolate mRNA from total RNA. First strand synthesis using four primers was followed by PCR amplification (7). (40 cycles, 1 minute denaturation at 93°C, 1 minute annealing at 54°C, 2 minutes elongation at 72°C, and 7 minutes final elongation.) The products were electrophoresed in a 2% agarose gel at 125mV and gave a band in the 200 kbp region as expected. Following ligation and transformation into supercompetent cells, the plasmid was mini preped for sequencing. The clones of canine COX-2 PCR fragments were sent to BYU’s sequencing center where the sequence was also compared to all known sequences of COX-2 through a NCBI Blast search.
Work has begun to isolate and sequence canine COX-1, but has been slow going. Once COX-1 is sequenced several steps still remain before a well-characterized whole blood assay is finished. First canine anti-sera recombinant against both the COX-1 and COX-2 proteins must be made following the elucidation of both sequences. Using the anti-sera the blood assay can then be characterized. The inhibition specificity of around a dozen NSAIDs will then be measured using the whole blood assay.
References
- Smith, W. l., L. J. Marnett. 1991 Review: prostaglandin endoperoxide synthase: structure and catalysis. Biochem. Et Biophys. Acta. 1083, 1-17.
- Vane, J. 1994. Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nat. New Biol.231, 232-235.
- DeWitt, D.L., W.L. Smith. 1988 Primary structure of prostaglandin G/H synthase from sheep vesicular gland determined from the complementary DNA sequence. Proc. Natl. Acad. Sci. 85, 1412-1416.
- Xie, W., J.G. Chipman, D.L. Robertson, R.L. Erikson, and D.L. Simmons. 1991. Expression of mitogen-responsive gene encoding prostaglandin synthase is regulated by mRNA splicing. Proc. Natl. Acad. Sci. 88, 2692-2696.
- Cryer, B., M. Feldman. 1998 Cyclooxygenase-1 and Cyclooxygenase-2 selectivity of widely used non-steroidal anti-inflammatory drugs. Am. J. Med. 104, 413-421.
- Chirgwin. 1979. Biochem 18:5294
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