Cyclooxygenase selectivity of nonsteroidal anti-inflammatory drugs in canine blood

OBJECTIVE: To evaluate cyclooxygenase (COX) selectivity of several nonsteroidal anti-inflammatory drugs (NSAID) in canine blood in vitro. ANIMALS: 11 healthy adult male hound crosses. PROCEDURE: 9 NSAID were studied at 5 concentrations. Thromboxane B2 (TxB2) was assayed as a measure of COX-1 activity in clotted blood. Prostaglandin E2 (PGE2) was assayed as a measure of COX-2 activity in heparinized, lipopolysaccharide (LPS)-stimulated blood. All assays were competitive ELISA tests. Cyclooxygenase selectivity was expressed as a ratio of the concentration of an NSAID that inhibited 50% of the activity (IC50) of COX-1 to the IC50 of COX-2. A separate ratio of the concentration that inhibited 80% of COX activity (IC80) was also determined. A ratio of < 1.0 indicated selectivity for COX-1, whereas a ratio of > 1.0 indicated COX-2 selectivity. RESULTS: Ketoprofen, aspirin, and etodolac were COX-1 selective. Piroxicam, meloxicam, and carprofen had COX-2 selectivity. The IC50 and IC80 values were similar for most NSAID. CONCLUSIONS: This methodology provides repeatable data from individual dogs and is comparable to results of previous in vitro and ex vivo models. Findings are also consistent with those of canine studies performed in vivo, suggesting that this is a viable in vitro assessment of the COX selectivity of NSAID in dogs.     

Cloning,expression, and selective inhibition of canine cyclooxygenase-1 and cyclooxygenase-2

Cyclooxygenase (COX) performs the critical initial reaction in the arachidonic metabolic cascade, leading to formation of proinflammatory prostaglandins, thromboxanes, and prostacyclins. The discovery of a second COX isoform (COX-2) associated with inflammation led to agents that selectively inhibit COX-2. Cyclooxygenase-2 inhibitors are also being developed for canine applications. To assess the compound potency on canine enzymes, canine COX-1 and COX-2 were cloned, expressed, and purified. Cyclooxygenase-1 was cloned from a canine kidney complementary DNA (cDNA) library, with 96 % sequence homology to human COX-1. Cyclooxygenase-2 was cloned from canine kidney and lipopolysaccharide-stimulated macrophage cDNA libraries, with a 93 % sequence homology to human COX-2. The arachidonic acid Michaelis constants for canine COX-1 and COX-2 were 4.8 and 6.6 micrometer, respectively, compared with 9.6 and 10.2 micrometer for ovine. Inhibition results indicated that, for all compounds tested, there was no significant difference between potencies determined for canine enzymes and those for human enzymes.     

Evaluation of cyclooxygenase-1 and cyclooxygenase-2 expression and the effect of cyclooxygenase inhibitors in canine prostatic carcinoma

Prostatic carcinoma occurs primarily in older castrated male dogs and is typically a fatal disease (most dogs die within few months after the initial diagnosis). Surgery, i.e., total prostatectomy, or radiation therapy is often not pursued due to risks of complications and a high rate of distant metastasis. Cyclooxygenase‐2 (Cox‐2) expression has been documented in several malignancies, including canine prostatic carcinoma. Cox‐2 inhibition has been reported to have preventative effects on several human malignancies and has therapeutic effects on both laboratory and spontaneous tumour models. The purpose of this retrospective study was to evaluate Cox expression and the effects of Cox inhibitors on survival in dogs with prostatic carcinoma. 94.1 and 88.2% of the tumours expressed Cox‐1 and Cox‐2, respectively. Furthermore, dogs treated with Cox inhibitors (piroxicam or carprofen) lived significantly longer than untreated dogs, 6.9 versus 0.7 months (P < 0.0001), suggesting that Cox inhibitors may have an important role in canine prostate cancer therapy.     

Clinical pharmacology of nonsteroidal anti-inflammatory drugs in dogs

OBJECTIVES: To discuss the clinical pharmacology of currently licensed veterinary NSAIDs and to review gastrointestinal and renal adverse effects as well as drug-drug interactions that have been reported with these drugs. To review the use of NSAIDs in the peri-operative setting and their use in patients with osteoarthritis. To further review the reported effects of NSAIDs on canine articular cartilage and liver as well as the clinical relevance of a washout period. DATABASES USED: PubMed, CAB abstracts and Google Scholar using dog, dogs, nonsteroidal anti-inflammatory drugs and NSAID(s) as keywords. CONCLUSIONS: A good understanding of the mechanisms by which NSAIDs elicit their analgesic effect is essential in order to minimize adverse effects and drug-drug interactions. Cyclooxygenase (COX) is present in at least two active isoforms in the body and is the primary pharmacologic target of NSAIDs. Inhibition of COX is associated with the analgesic effects of NSAIDs. COX is present in the gastrointestinal tract and kidneys, along with other areas of the body, and is also the likely reason for many adverse effects including gastrointestinal and renal adverse effects. The newer veterinary approved NSAIDs have a lower frequency of gastrointestinal adverse effects in dogs compared to drugs such as aspirin, ketoprofen and flunixin, which may be due to differential effects on the COX isoforms. There are currently no published reports demonstrating that the newer NSAIDs are associated with fewer renal or hepatic adverse effects in dogs. NSAIDs remain the cornerstone of oral therapy for osteoarthritis unless contraindicated by intolerance, concurrent therapies or underlying medical conditions. NSAIDs are also effective and frequently used for the management of post-operative pain.     

Molecular characterisation of canine nonsteroidal anti-inflammatory drug-activated gene (NAG-1)

Nonsteroidal anti-inflammatory drug (NSAID)-activated gene (NAG-1), a divergent member of the transforming growth factor beta superfamily, was previously identified as a gene induced by several anti-tumorigenic compounds, including NSAIDs and peroxisome proliferator-activated receptor gamma (PPARgamma) ligands in humans. In this study, canine NAG-1 was characterised from a canine genomic database. Gene induction by some NSAIDs and PPARgamma ligands was demonstrated in canine osteosarcoma cell lines. Phylogenetic analysis indicates that canine NAG-1 is more homologous with the corresponding mouse and rat genes than with human NAG-1. Expression of canine NAG-1 was increased by treatment with piroxicam and SC-560 (NSAIDs) and the PPARgamma ligand rosiglitazone. This study demonstrates that canine NAG-1 is up-regulated by some anti-tumorigenic compounds in osteosarcoma cell lines and may provide an important target of chemotherapy in canine cancer.     

Immunohistochemical expression of cyclooxygenase-2 in canine ovarian carcinomas

Ovarian tumours among domestic animals are frequently encountered in bitch. Cyclooxygenase-2 (COX-2) expression has been evaluated in different kind of canine primary epithelial neoplasms. Eleven canine ovarian carcinomas and two normal samples were evaluated immunohistochemically for COX-2 expression. Nine of 11 carcinoma samples (81%) expressed COX-2 enzyme isoform. The immunoreactivity was intracytoplasmically recorded and the intensity ranged from faint to strong. Our results show that COX-2 is expressed in canine ovarian carcinoma, suggesting a potential role of COX-2 in canine ovarian carcinogenesis.     

Use of nonsteroidal anti-inflammatory drugs in food animal practice.

We attempted to determine the extent to which nonsteroidal anti-inflammatory drugs (NSAID) are used in the treatment of food animals, and whether withdrawal times for milk and slaughter are recommended to clients. A survey questionnaire was mailed to a stratified random sample of 2,000 veterinarians whose practices were at least half food animals. A cross-sectional study was used to examine the responses to determine whether differences existed on the basis of a respondent's geographic location, number of years since graduation from veterinary college, and percentage of practice devoted to beef and dairy cattle. The response rate was 71% (1,424/2,000). Of those practitioners responding, 93% (1,325/1,424) reported using NSAID, with approximately 57 (751/1,322), 24 (327/1,322), and 18% (244/1,322) of respondents reporting use more than once a week, once a week, and 1 to 2 times per month, respectively. Dairy practitioners reported more frequent use than did beef practitioners. Use of flunixin meglumine was reported more frequently than the use of aspirin, phenylbutazone, or dipyrone. Approximately 88% (1,146/1,306) of respondents that used NSAID did so in combination with antibiotics. Withdrawal times for milk and meat were made on the basis of guidelines for the antibiotic. When using NSAID alone, recommendations for withdrawal times for milk and meat varied extensively. Overall, practitioners indicated that NSAID were useful and necessary for the treatment of food-producing animals.     

Cyclooxygenase expression and prostanoid production in pyloric and duodenal mucosae in dogs after administration of nonsteroidal anti-inflammatory drugs

OBJECTIVE: To assess cyclooxygenase (COX) expression and prostanoid concentrations in pyloric and duodenal mucosae of dogs after administration of nonsteroidal anti-inflammatory drugs (NSAIDs). ANIMALS: 8 healthy dogs. PROCEDURES: Each dog received carprofen (4.4 mg/kg, q 24 h), deracoxib (2 mg/kg, q 24 h), aspirin (10 mg/kg, q 12 h), and placebo (1 dog treat, q 24 h) orally for 3 days (4-week interval between treatments). Before study commencement (baseline) and on day 3 of each treatment, pyloric and duodenal mucosal appearance was assessed endoscopically and biopsy specimens were obtained for histologic examination. Cyclooxygenase-1 and COX-2 protein expressions were assessed via western blotting, and prostanoid concentrations were measured via ELISAs. An ANOVA was used to analyze data. RESULTS: Treatments had no effect on mucosal appearance and ulceration was not evident histologically. In pyloric and duodenal mucosae, COX-1 expression was unaffected by treatments. Cyclooxygenase-2 expression remained unchanged in pyloric mucosa; in duodenal mucosa, aspirin significantly increased COX-2 expression, compared with effects of deracoxib and carprofen. At baseline, total prostaglandin and thromboxane B2 concentrations in pyloric mucosa were significantly greater than those in duodenal mucosa. Aspirin significantly decreased both prostanoid concentrations in both mucosal tissues, compared with other treatments. In pyloric mucosa, carprofen administration significantly decreased total prostaglandin and thromboxane B2 concentrations, compared with deracoxib administration. CONCLUSIONS AND CLINICAL RELEVANCE: In dogs, prostanoid synthesis was greater in pyloric mucosa than it was in duodenal mucosa. Nonselective NSAIDs significantly decreased prostanoid concentrations in these mucosae, compared with the effects of a selective COX-2 NSAID.     

Pharmacodynamics and pharmacokinetics of nonsteroidal anti-inflammatory drugs in species of veterinary interest

This review summarises selected aspects of the pharmacokinetics (PK) and pharmacodynamics (PD) of nonsteroidal anti-inflammatory drugs (NSAIDs). It is not intended to be comprehensive, in that it covers neither minor species nor several important aspects of NSAID PD. The limited objective of the review is to summarise those aspects of NSAID PK and PD, which are important to an understanding of PK-PD integration and PK-PD modelling (the subject of the next review in this issue). The general features of NSAID PK are: usually good bioavailability from oral, intramuscular and subcutaneous administration routes (but with delayed absorption in horses and ruminants after oral dosing), a high degree of binding to plasma protein, low volumes of distribution, limited excretion of administered dose as parent drug in urine, marked inter-species differences in clearance and elimination half-life and ready penetration into and slow clearance from acute inflammatory exudate. The therapeutic effects of NSAIDs are exerted both locally (at peripheral inflammatory sites) and centrally. There is widespread acceptance that the principal mechanism of action (both PD and toxicodynamics) of NSAIDs at the molecular level comprises inhibition of cyclooxygenase (COX), an enzyme in the arachidonic acid cascade, which generates inflammatory mediators of the prostaglandin group. However, NSAIDs possess also many other actions at the molecular level. Two isoforms of COX have been identified. Inhibition of COX-1 is likely to account for most of the side-effects of NSAIDs (gastrointestinal irritation, renotoxicity and inhibition of blood clotting) but a minor contribution also to some of the therapeutic effects (analgesic and anti-inflammatory actions) cannot be excluded. Inhibition of COX-2 accounts for most and possibly all of the therapeutic effects of NSAIDs. Consequently, there has been an intensive search to identify and develop drugs with selectivity for inhibition of COX-2. Whole blood in vitro assays are used to investigate quantitatively the three key PD parameters (efficacy, potency and sensitivity) for NSAID inhibition of COX isoforms, providing data on COX-1:COX-2 inhibition ratios. Limited published data point to species differences in NSAID-induced COX inhibition, for both potency and potency ratios. Members of the 2-arylpropionate sub-groups of NSAIDs exist in two enantiomeric forms [R-(-) and S-(+)] and are licensed as racemic mixtures. For these drugs there are marked enantiomeric differences in PK and PD properties of individual drugs in a given species, as well as important species differences in both PK and PD properties.     

非甾体类抗炎药前处理及检测方法研究进展

非甾体类抗炎药是目前兽医临床中使用最为广泛的一类药物,但此类药物在动物源性食品中的残留对人类的健康造成严重威胁。通过综述国内外非甾体类抗炎药在动物源性食品中的前处理方法和检测方法的研究现状,总结不同前处理方法的优缺点以及多种检测方法的灵敏度和准确性,对非甾体类药物的残留检测发展趋势进行展望,旨在为今后的检测方法开发提供参考。     

Gene expressions of canine angiopoietin-1 and -2 in normal tissues and spontaneous tumours

The angiopoietin (Ang) family of proteins are central to the regulation of angiogenesis. The purposes of this study were to determine cDNA sequences of canine Ang-1 and Ang-2 and investigate their expressions in normal tissues and spontaneous tumours. The cDNA sequences of canine Ang-1 and Ang-2 were 1,494 and 1,488 bp, and the deduced amino acid sequences were 497 and 495 residues, respectively. The cDNA sequences of canine Ang-1 and Ang-2 showed high homology with those of the other mammalian species. Canine Ang-1 and Ang-2 mRNA were detectable in all 22 normal tissues and spontaneous tumours. Higher mRNA expression level of canine Ang-2 was demonstrated in mammary simple carcinomas, haemangiosarcoma and hepatocellular carcinoma in comparison with normal tissues.     

Expression of cyclooxygenase-2 in canine nasal carcinomas

Cyclooxygenase(COX)-2 expression was evaluated in 24 paraffin-embedded canine nasal carcinoma tissue samples by immunohistochemistry. Several different tumor types were represented, including carcinomas, adenocarcinomas and squamous cell carcinomas. COX-2 expression was identified in 17/24 cases (71%). The proportion of positive cells expressing COX-2 ranged from 10 to 95% and COX-2 expression was predominantly localized in the cytoplasm. Treatment with a COX-2 inhibitor should be investigated, along with the utilization of COX-2 expression as a prognostic marker.     

Molecular cloning of canine nm23 cDNAs and their expression in normal and tumor tissues

Two canine nm-23 cDNAs, designated as nm23-C1 and -C2, were isolated and characterized. Both have a putative open reading frame consisting of 459-bp encoding 152 amino acids and are highly similar to human, mouse and rat homologues. To understand the potential role of nm23-C1 and -C2 in the development of mammary gland tumors (MGT), we analyzed the mRNA expression in 14 MGT samples by RT-PCR. The samples were divided into categories according to their histopathology (benign/malignant) and metastasis. No significant difference in the mRNA expression levels of either nm23-C1 or -C2 were observed between the benign and malignant groups or the metastatic and non-metastatic groups. These results suggest that nm23-C1 and -C2 are not related to the establishment of malignancy and metastatic lesions in canine MGT cases.     

PK-PD integration and PK-PD modelling of nonsteroidal anti-inflammatory drugs: principles and applications in veterinary pharmacology

Much useful information relevant to elucidation of mechanism of action of nonsteroidal anti-inflammatory drugs (NSAIDs) at the molecular level can be obtained from integrating pharmacokinetic (PK) and pharmacodynamic (PD) data, such data being obtained usually, although not necessarily, in separate studies. Integrating PK and PD data can also provide a basis for selecting clinically relevant dosing schedules for subsequent evaluation in disease models and clinical trials. The principles underlying and uses of PK-PD integration are illustrated in this review for phenylbutazone in the horse and cow, carprofen and meloxicam in the horse, carprofen and meloxicam in the cat and nimesulide in the dog. In the PK-PD modelling approach for NSAIDs, the PK and PD data are generated (usually though not necessarily) in vivo in the same investigation and then modelled in silico, usually using the integrated effect compartment or indirect response models. Drug effect is classically modelled with the sigmoidal E(max) (Hill) equation to derive PD parameters which define efficacy, potency and sensitivity. The PK-PD modelling approach for NSAIDs can be undertaken at the molecular level using surrogates of inhibition of cyclooxygenase (COX) isoforms (or indeed other enzymes e.g. 5-lipoxygenase). Examples are provided of the generation of PD parameters for several NSAIDs (carprofen, ketoprofen, vedaprofen, flunixin and tolfenamic acid) in species of veterinary interest (horse, calf, sheep and goat), which indicate that all drugs investigated except vedaprofen were non-selective for COX-1 and COX-2 in the four species investigated under the experimental conditions used, vedaprofen being a COX-1 selective NSAID. In these studies, plasma concentration was linked to COX inhibitory action in the biophase using an effect compartment model. Data for S-(+)-ketoprofen have been additionally subjected to inter-species modelling and allometric scaling of both PK and PD parameters. For several species values of four PK parameters were highly correlated with body weight, whilst values for PD parameters based on COX inhibition lacked allometric relationship with body weight. PK-PD modelling of NSAIDs has also been undertaken using clinical end-points and surrogates for clinical end-points in disease models. By measurement of clinically relevant indices in clinically relevant models, data generated for PD parameters have been used to set dosages and dose intervals for evaluation and confirmation in clinical trials. PK-PD modelling of NSAIDs is likely to prove superior to conventional dose titration studies for dosage schedule determination, as it sweeps the whole of the concentration-effect relationship for all animals and therefore permits determination of genuine PD parameters. It also introduces time as a second independent variable thus allowing prediction of dosage interval. Using indirect response models and clinically relevant indices, PD data have been determined for flunixin, phenylbutazone and meloxicam in the horse, nimesulide in the dog and meloxicam in the cat.     

Expression of cyclooxygenase-2 in canine renal cell carcinoma

Cyclooxygenase-2 (COX-2) has been shown to be the primary enzyme responsible for prostaglandin production during inflammation but is absent in most tissues under normal physiological states. High levels of COX-2 expression have been observed in the macula densa and thick ascending limbs of fetal kidneys; this expression declines to minimal levels during renal maturation. We hypothesized that the neoplastic cells of renal cell carcinoma (RCC) may revert to high expression of COX-2, and we evaluated its expression in three spontaneous cases of canine RCC by using immunohistochemical methods. The neoplastic cells of two of the three cases exhibited moderate to marked COX-2 immunoreactivity. These results suggest that some canine renal cell carcinomas express high levels of COX-2, which may play a role in the modulation of neoplastic cell growth.     

Changes in platelet function, hemostasis, and prostaglandin expression after treatment with nonsteroidal anti-inflammatory drugs with various cyclooxygenase selectivities in dogs

OBJECTIVE: To determine the effects of nonsteroidal anti-inflammatory drugs of various cyclooxygenase selectivities on hemostasis and prostaglandin expression in dogs. ANIMALS: 8 client-owned dogs with clinical signs of osteoarthritis. PROCEDURES: Dogs received aspirin (5 mg/kg, PO, q 12 h), carprofen (4 mg/kg, PO, q 24 h), deracoxib (2 mg/kg, PO, q 24 h), and meloxicam (0.1 mg/kg, PO, q 24 h) for 10 days each, with an interval of at least 14 days between treatments. On days 0 and 10, blood was collected for platelet aggregation assays, thrombelastography, and measurement of lipopolysaccharide-stimulated prostaglandin E(2), platelet thromboxane B(2) (TXB(2)), and free serum TXB(2) and 6-keto-prostaglandin F (PGF)-1alpha concentrations. RESULTS: Platelet aggregation decreased after treatment with aspirin and carprofen, whereas significant changes from baseline were not detected for the other drugs tested. Thrombelastograms obtained after treatment with carprofen revealed decreased maximum amplitude and alpha-angle, suggesting hypocoagulability. Maximum amplitude and coagulation index increased after treatment with deracoxib. Plasma concentrations of prostaglandin E(2) decreased after treatment with carprofen or deracoxib, and platelet TXB(2) production increased after treatment with aspirin. Serum concentrations of the prostacyclin metabolite 6-keto-PGF-1alpha did not change significantly after treatment with any of the drugs, although the ratio of free TXB(2) to 6-keto-PGF-1alpha decreased slightly after treatment with carprofen and increased slightly after treatment with deracoxib. CONCLUSIONS AND CLINICAL RELEVANCE: At the dosages tested, treatment with meloxicam affected platelet function minimally in dogs with osteoarthritis. Treatment with carprofen decreased clot strength and platelet aggregation. Clot strength was increased after treatment with deracoxib.