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  • Author or Editor: Sally J. Benn x
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Abstract

Objective—To establish an in vitro assay and determine the differential suppressive activity of non steroidal anti-inflammatory drugs (NSAID) on cyclooxygenase (COX)-1 and COX-2 isoenzymes in dogs.

Procedure—COX activity was evaluated in the presence and absence of 4 NSAID (meloxicam, tolfenamic acid, carprofen, and ketoprofen), using a canine monocyte/macrophage cell line that constitutively expresses COX-1, but can be induced to express COX-2 when incubated with lipopolysaccharide. Inhibition of prostaglandin E2 (PGE2) synthesis by each NSAID was measured by enzyme immunoassay and attributed to specific COX-1 or COX-2 activity through assessment of COX messenger RNA expression by use of northern blot analysis and reverse transcription- polymerase chain reaction (RT-PCR). The COX selectivity of each drug was evaluated from dose-response curves by calculating a ratio (COX- 1:COX-2) of inhibitory concentration values on the basis of concentrations that reduced PGE2 by 50% in each COX model.

Results—Meloxicam and tolfenamic acid preferentially inhibited COX-2, with meloxicam inhibiting COX-2 activity 12 times more effectively than COX-1 activity. Carprofen was only 1.75 times more selective for COX-2 than for COX-1, and ketoprofen was slightly more selective for COX-1.

Conclusions—COX-1 and COX-2 were differentially sensitive to inhibition in vitro by NSAID. Meloxicam and tolfenamic acid were selective for COX-2. Effects of carprofen and ketoprofen approached equipotency against both isoenzymes. Selective COX-2 inhibitors are a new class of drugs with anti-inflammatory effects similar to conventional NSAID but with fewer adverse effects. Development of these agents for veterinary use would be facilitated by the convenience of using a canine cell line as a model system to screen COX- 1 and COX-2 inhibitor activities in vitro. (Am J Vet Res 2000;61:802–810)

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in American Journal of Veterinary Research

Abstract

Objective—To examine cyclooxygenase (COX) expression in canine platelets and Madin-Darby canine kidney (MDCK) cells in culture.

Sample Population—Canine platelets and MDCK cells.

Procedure—Total RNA was recovered from isolated canine platelets and MDCK cells. Northern blot analysis and reverse transcription-polymerase chain reaction (RTPCR), using complementary DNA probes and primers designed from the human COX sequences, were used to determine COX-1 and -2 (cyclooxygenase isoforms 1 and 2) messenger RNA (mRNA) expression.

Results—Following northern blot analysis, canine platelets were found to express only the 2.8-kb COX- 1 transcript; COX-2 was not detected. Canine MDCK cells expressed the 4.5-kb COX-2 transcript, in addition to the 2.8-kb COX-1 transcript. A single DNA band of 270 base pairs was identified following gel electrophoresis of the product obtained from RT-PCR of mRNA from canine platelets. Sequencing revealed that this PCR product was 90% homologous to a portion of the human COX-1 gene (Genbank M59979).

Conclusions and Clinical Relevance—Detection of COX-1 by RT-PCR of RNA obtained from canine platelets is a novel finding. The 90% homology of the PCR product with the human sequence suggests strong conservation between the canine and human COX-1 gene. Cloning and sequencing of the canine gene will be required to fully characterize homologous regions. Because of the importance of COX in the inflammatory process and as a potential target of currently available nonsteroidal anti-inflammatory drugs (NSAID), a better understanding of canine COX may improve our ability to use NSAID appropriately, achieve efficacy, and avoid potential adverse drug effects in dogs. (Am J Vet Res 2000;61:1512–1516)

Full access
in American Journal of Veterinary Research