Evaluation of flunixin meglumine pour-on administration on prostaglandin E2 concentration in inflammatory exudate after induction of inflammation in cattle

Accepted Manuscript Evaluation of flunixin meglumine pour-on administration on prostaglandin E concentration in inflammatory exudate after induction of inflammation in cattle

Julien Thiry, René Fournier, Olivier Roy, Mathias Catala PII: DOI: Reference:

S0034-5288(16)30676-2 doi: 10.1016/j.rvsc.2017.04.010 YRVSC 3305

To appear in:

Research in Veterinary Science

Received date: Revised date: Accepted date:

5 December 2016 24 March 2017 18 April 2017

Please cite this article as: Julien Thiry, René Fournier, Olivier Roy, Mathias Catala , Evaluation of flunixin meglumine pour-on administration on prostaglandin E concentration in inflammatory exudate after induction of inflammation in cattle. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Yrvsc(2017), doi: 10.1016/j.rvsc.2017.04.010

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ACCEPTED MANUSCRIPT Title Page:

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Type of Article: Short Communication

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Title: Evaluation of flunixin meglumine pour-on administration on Prostaglandin E

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concentration in inflammatory exudate after induction of inflammation in cattle

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Authors: Julien Thirya*, René Fournierb, Olivier Royc, Mathias Catalac Merck Animal Health, 2 Giralda Farms, Madison NJ 07940, USA

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MSD Santé Animale, 7 rue Olivier de Serres, 49071 Beaucouzé, France

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CEBIPHAR, 1 rue de la Bodinière, 37230 Fondettes, France

Corresponding author: Julien Thiry, Merck Animal Health, 2 Giralda Farms, Madison

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07940 NJ, USA, Tel: +1.973.937.5555; E-mail: [email protected]

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ACCEPTED MANUSCRIPT Abstract: The effect of flunixin transdermal pour-on solution (Finadyne® Transdermal; MSD Animal Health) on prostaglandin E2 (PGE2) synthesis in bovine inflammatory exudate was evaluated in a tissue cage model of acute inflammation. Twelve calves were

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randomly allocated to two-treatment groups over two sequences. Three weeks prior to the

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first period, sterile hollow perforated polyethylene balls were surgically embedded in the

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subcutis at four distinct sites in each animal. On the first day of each period, an aseptic inflammation was induced by injecting 0.5 mL of a 2% carrageenan solution into the

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lumen of each tissue cage. Treatment with either flunixin transdermal or negative control

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(NaCl) immediately followed. 0.5 mL of exudate was collected prior to challenge, and at 2, 4, 8, 12, 24, 36 and 48 hours after challenge. Exudate PGE2 concentrations were

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analyzed using ultra-high pressure liquid chromatography coupled with tandem mass

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spectrometry method. Mean PGE2 concentrations were consistently lower in calves treated with flunixin transdermal than those measured in calves treated with negative

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control, indicating an inhibitory activity on cyclo-oxygenase. Inhibition was the highest

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at 8 hours after treatment, and differences with the negative control were significant at +8, 24, 36 and 48 hours. The flunixin transdermal formulation was effective in reducing

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PGE2 concentrations in bovine exudate following an induced inflammation. Its antiinflammatory action started in the first hours after treatment and lasted up to 48 hours.

Keywords: Flunixin; pour-on; inflammation; prostaglandin; cattle; tissue cage model.

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ACCEPTED MANUSCRIPT Short Communication The active substance flunixin, used as the meglumine salt, is a carboxylic acid, nonsteroidal anti-inflammatory drug (NSAID) with anti-pyretic and analgesic properties. It demonstrates potent inhibition of the cyclo-oxygenase (COX-1 and COX-2), an

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enzyme in the arachidonic acid cascade which generates inflammatory mediators of the

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prostaglandin group (Lees et al., 2004). Prostaglandins, and in particular prostaglandin E (PGE), are fundamental in the inflammatory process and known to be present in large

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quantities in inflammatory exudates. In particular, PGE2 is present in higher concentrations than other prostaglandins in inflammatory processes, and is involved in all

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reactions leading to the classic signs of inflammation, i.e. pyrexia, oedema and pain

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(Dray, 1995; Ricciotti and FitzGerald, 2011).

Many of the physiological and pathological changes associated with clinical mastitis and

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naturally occurring Bovine Respiratory Disease are a result of the inflammatory response to infection (Lekeux, 2006, Pezeshki et al., 2011). It is therefore important to lessen the

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excessive inflammatory response secondary to bacterial and viral infections by using

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NSAIDs (Brentnall et al., 2012; Thiry and Brianceau, 2014). Flunixin is one of the most widely used NSAIDs in veterinary medicine, and the injectable product is used as adjunctive therapy in the treatment of respiratory diseases in cattle and piglets, as well as bovine acute mastitis (Lockwood et al., 2008; Salichs et al., 2013; Thiry et al., 2014). Flunixin exhibits a high degree of plasma protein binding (approximately 99%) (Skidmore et al., 2008). Because of preferential distribution into inflammatory exudate and slow clearance from this compartment (Espinasse et al., 1994; Landoni et al., 1995), 3

ACCEPTED MANUSCRIPT flunixin persists in inflammatory tissues and achieves high concentrations in inflammatory exudate, exceeding corresponding plasma concentrations. The half-life for flunixin in inflammatory exudate is 23 hours in cattle (Lees et al., 1996). It is clinically

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advantageous as it is the site of therapeutic action.

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A novel pour-on formulation of flunixin meglumine for transdermal absorption has been

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recently registered. Flunixin transdermal was demonstrated to be an effective adjunctive therapy in the treatment of bovine respiratory disease and bovine acute mastitis

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demonstrating anti-pyretic effects and improvement in clinical signs (Thiry and

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Brianceau, 2014; Thiry et al., 2015). The mean bioavailability of topical flunixin was 48% and the mean half-life 6.42 h. The Cmax following topical application of flunixin was

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1.17 µg/mL, after 2.14 h (Tmax) (Kleinhenz et al., 2016). However, no studies are

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available on the effect of this formulation on the synthesis of inflammatory mediators.

The tissue cage model allows the production of inflammatory exudate and measurement

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of inflammatory mediators. This model consists of subcutaneous implantation of hollow

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perforated devices and subsequent stimulation by carrageenan injection of the granulation tissue which develops within the cage. It constitutes a good interstitial tissue model, as the fluid within the chamber is comparable to interstitial fluid (Espinasse et al., 1994, Sidhu et al., 2004). The inflammation is mild, temporary and localized to the chamber, minimizing discomfort and stress to animals. Sequential sampling is easily performed, thus permitting the time course of the inflammatory process to be monitored and the use of a cross-over design. The objective of this study was to evaluate the action of flunixin, 4

ACCEPTED MANUSCRIPT when administered as a pour-on solution, on the synthesis of PGE2 in inflammatory exudate after induction of inflammation in cattle using a tissue cage model.

Three weeks before study initiation, sterile spherical perforated polypropylene tissue

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cages were surgically embedded in the subcutaneous space at four distinct sites per

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animal (one cage per site). No cage was rejected over the study period, showing the

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excellent tolerance of the implants and validating the technique used for implantation. Carrageenan solution was subsequently injected in each cage, inducing an acute aseptic

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and non-immune inflammatory reaction, which remain limited into each of the spherical

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chambers. The crossover design was used to test different treatments in the same animal and to reduce inter-animal and genetic courses of variation. Each animal received

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flunixin transdermal or negative control sequentially, with a two-week interval (wash-out

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period) between the periods. During each of the two study periods, 6 animals were administered flunixin topically on the dorsal midline at 3.3 mg/kg (Finadyne®

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Transdermal 50 mg/ml pour-on solution for cattle, 1 mL/15 kg); and 6 animals were

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administered the negative control topically on the dorsal midline at 1 mL/15 kg (0.9% NaCl). All applications of the products were made immediately after the injection of

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carrageenan solution into each cage. Exudate was collected by centesis of 3 cages per animal at 0h (prior to injection of the carrageenan solution and treatment), at 2, 4, 8, 12, 24, 36 and 48 hours after treatment. At each collection time point, all 3 samples from one animal were pooled. The PGE2 exudate concentrations were determined per animal and time-point using ultra-high pressure liquid chromatography coupled with tandem mass

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ACCEPTED MANUSCRIPT spectrometry method. The detailed experimental procedures are provided in Supplementary file 1.

Mean exudate PGE2 concentrations followed the same trend during both periods,

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however, the log ratio of concentrations are significantly different between the 2 periods

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at hours 12 and 48 (p values ≤ 0.0431). The cross-over design thus failed to produce a

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valid comparison of the effect of treatment over the two periods. Since a period effect was detected at 2 out of 7 time points, data were analysed separately for each of the two

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periods and the results of the pooled periods were not taken into consideration.

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In the negative control group, PGE2 concentrations increased sharply after challenge reaching a peak at 8 hours. This was followed by a gradual decrease without return to

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baseline level 48 hours after challenge (Table 1). In the flunixin treated group, PGE2

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concentrations remained around the pre-induction/treatment baseline level, with a peak observed at 12 hours (period 1) or at 4 hours (period 2). Mean exudate PGE2

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concentrations were almost 17 and 14 times higher in the negative control group

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compared with the flunixin treated group at the peak during the first and the second periods, respectively (Table 1). Exudate PGE2 concentrations were significantly different

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between groups at 8h and from 24 to 48 hours after treatment during the first period (twosided t-test of the least squares means of exudate PGE2 concentrations, p ≤ 0.007), and from 8 to 24 hours and at 48h during the second period (p ≤ 0.0489). By comparison to the negative control group, reduction of exudate PGE2 concentrations in the flunixin treated group was 48.1% and 44.4% as early as 4 hours after treatment during the first and the second periods, respectively (Figure 1). The maximum effect was 6

ACCEPTED MANUSCRIPT observed 8 hours after treatment, with 94.3% and 93.0% of PGE2 synthesis inhibition. The percentage of reduction of PGE2 remained at almost 90% up to 48 hours posttreatment (from 86.9% to 90.8% and from 84.8% to 91.9% during the first and the second period, respectively) (Figure 1). These results demonstrate a potent anti-inflammatory

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effect and a rapid speed of action of flunixin administered by transdermal route.

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NSAIDs act primarily to reduce the biosynthesis of prostaglandins, copious in inflammatory exudates. Inhibition of PGE2 synthesis at inflammatory sites results from

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blockade of COX-2, the enzyme isoform which is induced in the presence of acute

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inflammation. In vivo exudate PGE2 concentration is therefore used as an indicator of COX-2 activity (Brentnall et al., 2012). The reduction in PGE2 concentrations observed

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in this study after treatment with flunixin transdermal in comparison with NaCl, is a clear

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indication of the strong inhibitory activity of this product on cyclo-oxygenase. The comparison of results obtained in this study to those already published after

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administration of flunixin by the intramuscular route show similar reduction of PGE2

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concentrations in the inflammatory exudate. Indeed, a tissue cage study recently conducted demonstrated that flunixin injected intramuscularly in cattle allows decrease of

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exudate PGE2 concentrations, with the strongest effect occurring 4 hours after administration. The flunixin anti-inflammatory activity persisted up to 48 hours posttreatment (Roy et al., 2015). Other studies demonstrated that the inhibition of PGE2 concentration with flunixin injected intramuscularly ranged from 44 to 75% at +48h and reached 97% at 24 hours after treatment (Espinasse et al., 1994); or ranged from 60 to 90% for 24 hours (Landoni et al., 1995). 7

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Flunixin meglumine administered topically is rapidly absorbed and has a longer half-life compared to IV administration (Kleinhenz et al., 2016). This formulation has also the advantages of easier and safer administration for handlers, and reducing stress on cattle

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compared to other methods of administration (Thiry and Brianceau, 2014). Moreover,

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when administered once at the dose of 3.3 mg/kg, this study shows that flunixin

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transdermal was effective to reduce the PGE2 concentration in bovine exudates following an induced inflammation using a tissue cage model. Its anti-inflammatory action started

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in the first hours after treatment and lasted up to 48 hours.

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Brentnall, C., Cheng, Z., McKellar, Q.A., Lees, P., 2012. Influence of oxytetracycline

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on carprofen pharmacodynamics and pharmacokinetics in calves. J. Vet. Pharmacol.

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Espinasse, J., Thouvenot, J.P., Dalle, S., Garcia, J., Schelcher, F., Salat, O., Valarcher, J.F., Daval, S., 1994. Comparative study of the action of flunixin

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meglumine and tolfenamic acid on prostaglandin E2 synthesis in bovine inflammatory exudates. J. Vet. Pharmacol. Therap. 17, 271–274. Kleinhenz, M.D., Van Engen, N.K., Gorden, P.J., Kukanich, B., Rajewski, S.M., Walsh, P., Coetzee, J.F., 2016. The pharmacokinetics of transdermal funixin meglumine in Holstein calves. J. Vet. Pharmacol. Ther. doi: 10.1111/jvp.12314. PubMed PMID: 27121728. 8

ACCEPTED MANUSCRIPT Landoni,

M.F.,

Cunningham,

F.M.,

Lees,

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pharmacokinetics and pharmacodynamics of flunixin in calves by use of pharmacokinetic/pharmacodynamic modeling. Am. J. Vet. Res. 56, 786-794. Lees, P., Delatour, P., Foster, A., Foot, R., Baggot, D., 1996. Evaluation of carprofen

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in calves using a tissue cage model of inflammation. Brit. Vet. J. 152, 199-211.

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Ricciotti, E., FitzGerald, G.A., 2011. Prostaglandins and inflammation. Arterioscler. Thromb. Vasc. Biol. 31, 986-1000. Roy, O., Brianceau, P., Thiry, J., Catala, M., Montreuil, C., Fournier, R., 2015. Comparative efficacy of flunixin on prostaglandin E2 synthesis in a bovine tissue cage model of acute inflammation. Revue Med. Vet. 166, 185-191.

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ACCEPTED MANUSCRIPT Salichs, M., Sabaté, D., Homedes, J., 2013. Efficacy of ketoprofen administered in drinking water at a low dose for the treatment of porcine respiratory disease complex. J. Anim. Sci. 91, 4469-4475. Skidmore, A., Roder, J., Van De Ven, J., Cloet, P., Montgomery, A., 2008. Flunixin

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meglumine (Finadyne®, Banamine®) in pain management of cattle: pharmacology

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Budapest, Hungary, pp293.

Sidhu, P., Shojaee Aliabadi, F., Andrews, M., Lees, P., 2004. Tissue chamber model

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of acute inflammation in farm animal species. Res. Vet. Sci. 74, 67-77.

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Thiry, J., González-Martín, J.V., Elvira, L., Pagot, E., Voisin, F., Lequeux, G., Weingarten, A., de Haas, V., 2014. Treatment of naturally occurring bovine

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respiratory disease in juvenile calves with a single administration of a florfenicol plus

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flunixin meglumine formulation. Vet. Rec. 174, 430. Thiry, J., Brianceau, P., 2014. Flunixin meglumine transdermal pour-on solution as

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adjunct therapy in the treatment of bovine respiratory disease in calves less than 8

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weeks of age. J.J. Vet. Sci. Res. 1, 002. Thiry, J., Milon-Harnois, G., Chiquet, M., Daluzeau, L., Borchert-Stuhlträger, M.,

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Sander, B., Thomas, E., Boeckh, A., de Haas, V., Brianceau, P., 2015. Effectiveness and safety of a novel flunixin meglumine transdermal pour-on solution in the treatment of bovine mastitis. Proceedings of the European Buiatrics Forum; 2015 Oct 14-16; Rome, Italy, pp 233.

Acknowledgements 10

ACCEPTED MANUSCRIPT This work was supported by Merck Animal Health and MSD Santé Animale.

Conflict of Interest O. Roy and M. Catala are employees of CEBIPHAR. R. Fournier is employee of MSD

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Santé Animale. J. Thiry is employee of Merck Animal Health, Madison, NJ, USA.

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ACCEPTED MANUSCRIPT Table 1. Means of PGE2 concentration (pg/mL) at each period Collection time

0h

+2h

+4h

+8h

+12h

+24h

+36h

+48h

Period 1 Mean

3414.5

5138.3

5946.3

8444.7

15443.5

6168.5

3810.7

5120.3

TD

sd

1059.3

2606.5

3122.2

8006.1

14409.4

4513.6

2067.4

4670.2

Mean

2813.5a

4640.7

11455.8 147004.5 118085.3 49087.3 41534.8 40035.8

sd

3033.7

4574.3

13840.6 191055.9 153553.4 30336.4 22896.8 21869.7

Flunixin

Mean

10323.5 10648.5 12181.8 3564.0

2533.5

3045.8

3407.2

1279.2

TDb

sd

16782.1 13422.0 13529.8 3963.5

1701.9

3208.1

2160.6

1330.2

Mean

6244.7

11455.7 21900.2 51159.3

31400.8

33329.0 23951.3 8423.5

sd

5548.8

7761.3

17414.0

22754.4 20310.7 5476.1

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Flunixin

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NaCl

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Period 2

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NaCl

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14984.6 50615.6

One value below the LLOQ was replaced by 25 pg/mL(LLOQ)

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n=5 at 36h

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TD, transdermal; sd, standard deviation.

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ACCEPTED MANUSCRIPT Legend of figure Figure 1. Percent of reduction of PGE2 concentrations between the treatment groups

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(flunixin TD vs. control)

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ACCEPTED MANUSCRIPT Highlights:

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Acute inflammation in cattle was induced in a tissue cage model Flunixin transdermal was effective to reduce PGE2 concentration in bovine exudates Anti-inflammatory action started in the first hours and lengthened up to 48h

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