In Vitro and Ex Vivo Inhibition of COX Isoforms by Robenacoxib in the Cat: a Comparative Study

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J. vet. Pharmacol. Therap. 33, 444–452. doi: 10.1111/j.1365-2885.2010.01166.x. In vitro and ex vivo inhibition of COX isoforms by robenacoxib in the cat: a comparative study

V. B. SCHMID* Schmid, V. B., Seewald, W., Lees, P., King, J. N. In vitro and ex vivo inhibition of W. SEEWALD COX isoforms by robenacoxib in the cat: a comparative study. J. vet. Pharmacol. Therap. 33, 444–452. P. LEESà &

J. N. KING Robenacoxib is a novel nonsteroidal anti-inﬂammatory drug (NSAID) developed for use in companion animals. Whole blood assays were used to characterize in *Novartis Centre de Recherche Sante´ the cat the pharmacodynamics of robenacoxib for inhibition of the cycloox- Animale SA, St-Aubin, Switzerland; Novartis Animal Health Inc., Basel, ygenase (COX) isoforms, COX-1 and COX-2, in comparison with other NSAIDs. Switzerland; àRoyal Veterinary College, Based on in vitro IC50COX-1:IC50COX-2 ratios, robenacoxib was COX-2 selective Hawkshead Campus, Hatﬁeld, (ratio = 32.2), diclofenac (ratio = 3.9) and meloxicam (ratio = 2.7) were only Hertfordshire, UK weakly COX-2 preferential, and ketoprofen (ratio = 0.049) was COX-1 selective. In an in vivo pharmacokinetic ex vivo pharmacodynamic study, after both p.o. (1–2 mg ⁄ kg) and subcutaneous (2 mg ⁄ kg) dosing, robenacoxib

achieved peak blood concentrations rapidly (Tmax = 1 h for both administration routes) and was cleared from blood relatively rapidly (mean residence time was 1.70 h after p.o. and 1.79 h after subcutaneous dosing). In ex vivo COX isoform inhibition assays, orally (1–2 mg ⁄ kg) or subcutaneously (2 mg ⁄ kg) administered robenacoxib signiﬁcantly inhibited COX-2, with a relatively short duration of action in the central compartment, and had no effect on COX-1. Therefore robenacoxib was COX-2 selective and spared COX-1 in vivo.In contrast, meloxicam (0.3 mg ⁄ kg via subcutaneous injection) inhibited both COX-1 and COX-2 isoforms signiﬁcantly for at least 24 h, indicating nonselec- tivity in vivo. (Paper received 15 May 2009; accepted for publication 21 December 2009) V. B. Schmid, Novartis Centre de Recherche Sante´ Animale SA, CH-1566 St-Aubin, Switzerland. E-mail: [email protected]

INTRODUCTION NSAIDs, namely deracoxib and firocoxib, have been registered for use in dogs but not in cats (Gierse et al., 2002; Millis et al., Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used 2002; McCann et al., 2004; Sessions et al., 2005; Hanson et al., in veterinary and human medicine for their analgesic, anti- 2006; Pollmeier et al., 2006). inflammatory and anti-pyretic actions. In comparison with dogs, Robenacoxib is a novel coxib NSAID which has been relatively few NSAIDs are registered for use in cats (carprofen, developed exclusively for veterinary use (King et al., 2009). It ketoprofen, meloxicam and tolfenamic acid), partially because of is an analogue of diclofenac, a preferential inhibitor of COX-2. safety issues with some of the drugs of this class in this species Recent studies have established that robenacoxib is a highly (Lascelles et al., 2007). However there is increasing recognition selective inhibitor of COX-2 in the rat, dog and cat (Giraudel of a major requirement for effective and safe analgesics for et al., 2009; King et al., 2009, 2010). The effect of robenacoxib therapeutic use in the cat (Robertson & Taylor, 2004; Taylor & on COX-1 and COX-2 in cats in vitro, compared with meloxicam Robertson, 2004; Robertson, 2005; Lascelles et al., 2007). and caprofen, was reported recently by Giraudel et al. (2009). In recent years, newer NSAIDs of the coxib class have become The aim of this study was to extend the work of Giraudel et al. available. These drugs are selective inhibitors of the COX-2 (2009) to further evaluate the effect of robenacoxib on COX-1 isoform and may offer safety advantages over nonselective and COX-2 in cats. Whole blood in vitro assays for COX-1 and inhibitors in causing less damage to the gastrointestinal tract COX-2 were used, as these are considered to be the most relevant (Watson et al., 2000; Schnitzer et al., 2004) and not interfering to NSAID actions in vivo (Patrignani et al., 1994; Pairet & with blood clotting (Masferrer et al., 1994; Warner et al., 1999). Engelhardt, 1996; Giraudel et al., 2005). In contrast to the study The safety benefits of COX-2 selective NSAIDs have been with robenacoxib by Giraudel et al. (2009), we used the established in humans (Schnitzer et al., 2004) and rats (King conventional lipopolysaccharide (LPS) stimulated production of et al., 2009), but data in cats are at present lacking. Two coxib PGE2 for the COX-2 assay. First, the effects of robenacoxib on

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COX-1 and COX-2 in vitro were compared with those of two TxB2 concentration by enzyme immunoassay using commercial other NSAIDs licensed for feline use (ketoprofen and meloxicam) kits (Amersham Pharmacia, RPN220-TxB2, Biotrak EIA, GE and with the reference COX-2 preferential drug diclofenac. Healthcare GmbH, Otelﬁngen, Switzerland). Serum samples were Second, the effect of robenacoxib, administered p.o. and subcu- assayed according to the procedures described by the manufac- taneously at the registered dosages, on COX-1 and COX-2 were turer (a separate standard curve was run for each microplate; evaluated in an in vivo pharmacokinetic ⁄ ex vivo pharmacody- standards and samples were assayed in duplicate; duplicates namic study. The effects of robenacoxib in that study were showing a CV > 15% were re-analyzed). compared to both a negative control and a positive control For the COX-2 assay, a volume of approximately 5 mL blood (meloxicam administered subcutaneously). was collected by direct venepuncture into plastic tubes contain- The hypothesis of the study was that robenacoxib would be ing citrate as anticoagulant (S-Monovette NC, Sarstedt AG, selective for COX-2 in vitro, and in consequence would inhibit Sevelen, Switzerland). Subsequent steps were performed under COX-2 and spare COX-1 when administered at the recommended aseptic conditions. Aliquots of 0.245 mL blood were pipetted into dosages to cats. microtubes containing a range of concentrations of one of the four drugs dissolved in 20 lL DMSO, followed by 5 lL LPS obtained from Sigma-Aldrich GmbH, Buchs, Switzerland (LPS, MATERIALS AND METHODS L-2630 E. coli serotype O111:B4). The LPS was dissolved in sterile phosphate buffered saline (PBS, Sigma-Aldrich GmbH, The studies were conducted under protocols approved by the Buchs, Switzerland, S-8776) at a concentration of 5.04 mg ⁄ mL, Novartis Animal Health Research Centre in St-Aubin, Switzer- divided into aliquots and frozen at )20 C. A fresh aliquot was land, and in compliance with the Swiss law for animal used in each assay. A total of 15 doubling concentrations was protection. used for each drug over the following concentration ranges: robenacoxib and diclofenac 0.0006–10 lM; ketoprofen 0.018– 300 lM; and meloxicam 0.006–100 lM. After vortexing, tubes In vitro studies were incubated for 24 h at 37 C, and then centrifuged at COX-1 and COX-2 assays 16 000 g at 4 C for 10 min. The supernatant plasma was stored

Concentration-effect relationships for inhibition of COX-1 and at )20 C for not more than 1 week before assay for PGE2 by COX-2 in feline whole blood assays were established for enzyme immunoassay using commercial kits (Amersham Phar- robenacoxib in comparison with three control drugs. Diclofenac macia, RPN222-PGE2, Biotrak EIA, GE Healthcare GmbH, was selected as a drug of similar chemical structure to robenac- Otelfingen, Switzerland). Plasma samples were processed accord- oxib; which, in previous studies in dogs, rats and humans was ing to the manufacturer’s indications (a separate standard curve nonselective or slightly preferentially selective for COX-2 (King was run for each microplate; standards and samples were assayed et al., 2009, 2010). The other comparator drugs, ketoprofen and in duplicate; duplicates showing a CV > 15% were re-analyzed). meloxicam, were chosen because both are licensed for use in cats. Each drug was supplied as a dry powder from Sigma-Aldrich Curve fitting and data analysis Chemie GmbH, Buchs, Switzerland (diclofenac, racemic ketoprofen and meloxicam) and CarboGen AG, Aarau, Switzerland All calculations were undertaken using SAS , Version 8.2 (robenacoxib). Because of the large number of samples software (SAS Institute Inc., Cary, NC, USA, 1999). for analysis, the study was conducted in two phases; robenac- For both assays the concentration-response data were fitted to oxib and diclofenac in part 1 and ketoprofen and meloxicam in a standard sigmoidal model using SAS Proc Nlin: part 2. y0 y1 Blood was collected from eight healthy cats (four male and four y ¼ y1 þ 1 þ expða þ b log xÞ female) on four occasions. For the COX-1 assay, a volume of 5 mL blood was collected by venepuncture into plastic tubes without where x = drug concentration, y = response with the following anticoagulant (S-Monovette, Sarstedt AG, Sevelen, Switzer- parameters. a = intercept, b = slope (>0), y0 = y value for land). Aliquots of 0.25 mL were pipetted into microtubes, x =0,y1 = y value for x = ¥. containing a range of concentrations of one of the four drugs IC50 is the value of x for which y =(y0 + y1) ⁄ 2, therefore under study dissolved in 20 lL dimethyl sulphoxide (DMSO; IC50 = exp (-a ⁄ b) Sigma-Aldrich Chemie GmbH, Buchs, Switzerland). A total of 15 As the model is nonlinear, the estimation of parameters doubling concentrations (a geometric sequence of two) was used involves an iterative technique. Estimates of model uncertainty for each drug over appropriate ranges as follows: robenacoxib were then based on linear approximations. It was assumed that

0.1831–3000 lM; diclofenac 0.001–30 lM; ketoprofen and me- y1 is zero. loxicam 0.018–300 lM. A control tube containing no drug was Initially, an attempt was made to fit the data for one response, used in each assay. The tubes were vortexed and then incubated one drug and one cat. However, fits were poor in all cases. for 60 min at 37 C, followed by centrifugation at 16 000 g at Therefore in the final model, the data were fitted to one response,

4 C for 10 min. The supernatant serum was collected and stored one drug and all animals with parameters a and y0 varying at )20 C for not more than 1 week prior to determination of between cats but b being the common slope for all cats.

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Geometric means were calculated for all cats and geometric tions of robenacoxib; and (ii) determine the magnitude and time mean ratios determined, as follows: IC20COX-1:IC20COX-2, course of inhibition of COX-1 and COX-2 ex vivo in whole blood

IC50COX-1:IC50COX-2, IC80COX-1:IC80COX-2 and IC20COX- assays. Samples were collected by direct puncture from a left or 1:IC80COX-2. In addition, to further characterize selectivity for right jugular or cephalic vein into S-monovette tubes (Sarstedt

COX-2, the magnitude of TxB2 inhibition was determined as a AG, Sevelen, Switzerland) containing: (i) EDTA as anticoagulant function of PGE2 inhibition. (1.2 mL) for measurement of robenacoxib concentration; (ii) sodium citrate as anticoagulant (1.4 mL) for estimation of COX- 2 activity; and (iii) without anticoagulant (1.0 mL) for determi- In vivo ⁄ ex vivo study nation of COX-1 activity. COX-1 and COX-2 activities were Animals and study design determined using the same enzyme immunoassays as described An in vivo pharmacokinetic ⁄ ex vivo pharmacodynamic study for the in vitro assays (vide supra). was undertaken under GLP compliant conditions. The main Although a total of approximately 25 mL of blood (comprising objective was to determine the time course of inhibition of an estimated 5–15% of total blood volume) was taken from the production of serum TxB2 and plasma PGE2 using whole blood cats over 24 h, the blood was not replaced. Repeated puncture of assays. A secondary objective was to establish blood concentra- different veins was preferred over catheterization to avoid the tion-time profiles and derive pharmacokinetic variables. Eight need for heparin administration or the risk of coagulation in the cats (four male, four female) of the domestic short-hair breed catheter. were used, aged 2 years 11 months to 4 years 3 months and weighing 2.5–6 kg. The cats were fed a dry pelleted feed once Robenacoxib analysis daily, when group housed. For 2 days before and 24 h after each drug administration, the cats were housed individually and fed a Blood samples were analyzed for robenacoxib using the methods mix of dry and wet food. Water was available ad libitum. described by Jung et al. (2009): an initial HPLC-UV method The cats were randomly allocated to receive each of four covering the range of approximately 500–20 000 ng ⁄ mL and, if treatments in sequence in a four-part Latin Square design with required, a subsequent analysis by LC-MS covering the range of an interval of approximately 2 weeks between phases. approximately 3–100 ng ⁄ mL. Depending on the results obtained The four treatments administered were: with the UV method, specimens were diluted in order not to exceed 100 ng ⁄ mL in the MS method. • a single tablet or fractions of a tablet of robenacoxib The analytical method was validated using as quality controls administered orally at a dose of 1–2 mg ⁄ kg using tablets (QCs) spiked matrix specimens at concentrations of 5, 20 and containing 6 mg robenacoxib (Novartis Sante´ Animale S.A.S., 50 ng ⁄ mL, run with each sequence of unknown samples and Huningue, Cedex, France). independent of calibration standards. The assay met the • robenacoxib injection administered subcutaneously at a dose requirement that two-thirds of QC samples are encompassed of 2 mg ⁄ kg (2% w ⁄ v solution for injection, Vericore Ltd., within ±15% (or ±20% for the lower limit of quantitation) of Dundee, Scotland). spiked concentrations. Samples containing robenacoxib in blood • as a positive control meloxicam injection (Metacam were shown to be stable for 5 months at )20 C. All samples 5mg⁄ mL, Labiana Life Sciences S.A., Terrassa, Spain) admin- were analyzed within 10 weeks of collection. The lower limit of istered subcutaneously at a dose of 0.3 mg ⁄ kg. quantitation of the analytical method was 3 ng ⁄ mL. • a negative control group (cats sham dosed by injection or orally). Pharmacokinetics Subcutaneous injections of robenacoxib and meloxicam were administered in the scruff of the neck using exact doses based on The following pharmacokinetic variables were determined from body weight and achieved by weighing syringes. For oral dosing the individual blood robenacoxib profiles after each administra- of robenacoxib, cats weighing 3–6 kg received one entire 6 mg tion: tablet. Cats weighing less than 3 kg were given a fraction of a tablet, to achieve a dose close to 2 mg ⁄ kg, using a tablet splitter • Area under the curve: AUC(0-t) and AUC(0-inf.) to achieve halving and quartering of tablets. Tablets or fractions • Area under the moment curve: AUMC(0-inf.) of tablets were placed on the pharynx behind the tongue, • Time of maximum blood concentration: Tmax followed by a small quantity of water to ensure ingestion. Tablets • Maximum blood concentration: Cmax or fractions of tablets were weighed prior to administration. Cats • Terminal half-life: t½ were fasted overnight and then provided with food approxi- • Mean residence time: MRT mately 2 h after dosing. The data were processed as individual time vs. blood concentration profiles using a validated pharmacokinetic evaluation Blood samples software (DEBA, Data Evaluation in BioAnalytics, release 4.1 Blood samples (3.6 mL) were collected before and at 1, 2, 4, 6, 8 XP, Copyright Dr C. N. Thumm-Kraus, Software and Consulting, and 24 h after dosing in order to: (i) measure blood concentra- 1999, Basel, Switzerland). Mean values and SDs of pharmaco-

2010 Blackwell Publishing Ltd Robenacoxib in the cat 447 kinetic variables, dose normalized (where applicable) and mean 1.3 blood concentration profiles were calculated using EXCEL. 1.2 1.1 1.0 Statistical evaluation 0.9 0.8 Data are presented as arithmetic mean, geometric mean or 0.7 median plus SD or the 95% CI. Comparisons between different 0.6 groups for PGE and TxB concentrations (as indices of COX-2 (relative units) 2 2 2 0.5 and COX-1 activity, respectively) were made statistically using 0.4 TxB repeated measures analysis of covariance (RMANCOVA) with the 0.3 following effects in the model: phase, sequence, sex, baseline 0.2 value, treatment group, time and treatment · time interaction. 0.1 In the event of overall significance, post-hoc comparisons were 0.0 made using linear contrasts. P values less than 0.05 were 0 0.3 1 3 10 30 100 300 1000 3000 considered to be statistically significant. Robenacoxib (µM) Cat 1812 2202 4070 4454 5792 6338 7665 9060

RESULTS Fig. 2. Concentration–effect relation for robenacoxib and inhibition of serum TxB2 in vitro, as an index of COX-1 activity, in eight cats. Results In vitro study are the data (symbols) and the best ﬁt (line). Baseline TxB2 concentrations (no inhibition) are represented by 1.0 relative units. Horizontal The robenacoxib concentration–response relationships for inhi- dotted lines present 100%, 80%, 50%, 20% and 0% of baseline TxB2 concentrations. bition of plasma PGE2, as an index of COX-2, and serum TxB2, as an index of COX-1, are shown in Figs 1 and 2, respectively. Geometric mean values and 95% CI for three levels of ketoprofen & meloxicam for COX-2 and ketoprofen > diclofenac > inhibition of COX-2 and COX-1; IC IC and IC , and slope 20, 50 80 meloxicam > robenacoxib for COX-1. Based on IC values, of the concentration–response relationships are presented in 50 robenacoxib was selective for COX-2, diclofenac and meloxicam Table 1. were only slightly preferential for COX-2 and ketoprofen was Slopes were relatively shallow and for each drug they were less selective for COX-1 (Table 2). steep for TxB than for PGE inhibition and this was signiﬁcant for 2 2 For diclofenac and ketoprofen, inhibition ratios were indepen- robenacoxib and meloxicam. Based on the three reported levels of dent of level of inhibition, whereas the selectivity of robenacoxib inhibition, rank order of potency was diclofenac > robenacoxib > and meloxicam increased from IC20TxB2:IC20PGE2 to IC80Tx-

B2:IC80PGE2 (Table 2). The selectivities of robenacoxib and ketoprofen for inhibition of COX-1 and COX-2, respectively, 1.4 were further conﬁrmed by the IC20TxB2:IC80PGE2 ratios of 1.3 4.233 (robenacoxib) and 0.0052 (ketoprofen). 1.2 1.1 Data in Table 3 present percentage inhibition of COX-1 for 1.0 given levels of COX-2 inhibition. 0.9 The calculated values depend both on the distance between 0.8 the COX-1 and COX-2 inhibition curves and curve slopes. Ninety 0.7 ﬁve percent inhibition of COX-2 by robenacoxib produced only

(relative units) 0.6 2 0.5 12.4% COX-1 inhibition, whereas meloxicam inhibited COX-1 by

PGE 0.4 72.7% for 95% COX-2 inhibition. In contrast, 50% COX-2 0.3 inhibition by ketoprofen was associated with 97.7% COX-1 0.2 inhibition. Figure 3 illustrates the percentage inhibition of TxB2 0.1 versus percentage inhibition of COX-1 for robenacoxib and the 0.0 three comparator drugs. 0 0.001 0.003 0.01 0.03 0.1 0.3 1 3 10 Robenacoxib (µM)

Cat 1812 2202 4070 4454 In vivo ⁄ ex vivo study 5792 6338 7665 9060 Eicosanoid inhibition Fig. 1. Concentration–effect relation for robenacoxib and inhibition of The time courses of ex vivo inhibition of plasma PGE2 and serum LPS induced production of plasma PGE in vitro, as an index of COX-2 2 TxB are illustrated in Figs 4 and 5, respectively. activity, in eight cats. Results are the data (symbols) and the best ﬁt 2 In the control group, eicosanoid concentrations showed some (line). Baseline PGE2 concentrations (no inhibition) are represented by 1.0 relative units. Horizontal dotted lines present 100%, 80%, 50%, 20% variability with time, but with no obvious increasing or and 0% of baseline PGE2 concentrations. decreasing trends.

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Table 1. Potency and slope for inhibition of plasma PGE2 and serum TxB2

Inhibition

Eicosanoid Drug n* Slope b IC20 IC50 IC80

PGE2 Robenacoxib 8 1.622 (1.396–1.849) 0.0591 (0.0462–0.0728) 0.1390 (0.1157–0.1662) 0.3267 (0.2691–0.4084) Diclofenac 8 1.417 (1.240–1.593) 0.0285 (0.0216–0.0363) 0.0758 (0.0609–0.0938) 0.2017 (0.1610–0.2591) Ketoprofen 8 1.220 (1.031–1.409) 0.1515 (0.0959–0.2223) 0.4719 (0.3293–0.6646) 1.470 (1.037–2.165) Meloxicam 8 1.479 (1.205–1.753) 0.1995 (0.1292–0.2844) 0.5093 (0.3628–0.7056) 1.300 (0.9309–1.916)

TxB2 Robenacoxib 8 1.181 (1.022–1.340) 1.383 (0.9884–1.825) 4.473 (3.551–5.551) 14.47 (11.54–18.66) Diclofenac 8 1.251 (0.8370–1.666) 0.0970 (0.0362–0.1869) 0.2937 (0.1479–0.5514) 0.8893 (0.4776–2.056) Ketoprofen 8 1.249 (1.139–1.360) 0.0077 (0.0063–0.0091) 0.0233 (0.0206–0.0261) 0.0706 (0.0626–0.0804) Meloxicam 8 0.9697 (0.8334–1.106) 0.3253 (0.2224–0.4406) 1.359 (1.085–1.668) 5.676 (4.533–7.383)

Values are geometric means with 95% CI in parentheses. *Number of cats. Slopes for inhibition of PGE2 and TxB2 were signiﬁcantly different for robenacoxib (P = 0.0016) and meloxicam (P = 0.0010).

Table 2. Potency ratios for inhibition of TxB2:PGE2, as an index of 100 selectivity for COX-2, for three levels of inhibition (20%, 50% and 80%) 90 IC20 TxB2: IC50 TxB2: IC80 TxB2: IC20 TxB2: Drug IC20 PGE2 IC50 PGE2 IC80 PGE2 IC80 PGE2 80

Robenacoxib 23.38 32.18 44.30 4.233 70 Diclofenac 3.405 3.875 4.410 0.4809

Ketoprofen 0.0506 0.0493 0.0480 0.0052 (%)

2 60 Meloxicam 1.631 2.668 4.366 0.2502 50 Values are geometric means from eight cats. 40

Inhibition TxB 30 Table 3. Percentage inhibition of serum TxB2 (as an index of COX-1) for varying percentages of inhibition of plasma PGE2 (as an index of COX-2) 20

% inhibition of TxB2 for inhibition of PGE2 of 10

Drug 50% 80% 90% 95% 0

Robenacoxib 1.6 4.4 7.6 12.4 0 102030405060708090100 Diclofenac 15.5 38.5 56.1 71.2 Inhibition PGE (%) Ketoprofen 97.7 99.4 99.8 99.9 2 Meloxicam 27.9 48.9 62.0 72.7 Robenacoxib Diclofenac Values are derived from geometric means for eight cats. Ketoprofen Meloxicam

Fig. 3. Plot of % inhibition of serum TxB2 vs. % inhibition of plasma PGE measured in in vitro whole blood assays for diclofenac, ketoprofen, Meloxicam signiﬁcantly inhibited plasma PGE2 concentration 2 relative to the control treatment at all sampling times (Fig. 5, meloxicam and robenacoxib.

Table 4). Robenacoxib also inhibited PGE2 and differences from control were marked but relatively transient; differences were signiﬁcant for 2 h after p.o. dosing and for 6 h after subcuta- after oral administration (Fig. 5, Table 4). The latter result is neous administration (Table 4). The median times to maximal judged unlikely to be a true biological effect, since robenacoxib inhibition of plasma PGE2 were: 2 h robenacoxib injection, 1 h was no longer detected in the blood after 8 h. At several robenacoxib tablets and 2 h meloxicam injection. sampling times, serum TxB2 was signiﬁcantly lower after

Meloxicam inhibited TxB2 synthesis relative to control treat- meloxicam than with either robenacoxib treatment (Table 4). ment at all times between 1 and 24 h, except for the 4 h sample when the decrease (P = 0.0583) was not significant (Fig. 5, Robenacoxib pharmacokinetics Table 4). In addition, meloxicam produced significantly greater and more persistent inhibition of serum TxB2 than the two The robenacoxib concentration-time profile in blood is presented robenacoxib groups (Table 4). in Fig. 6. Higher and more persistent concentrations were Neither oral nor parenteral dosing with robenacoxib signifi- obtained after subcutaneous dosing. Thus, arithmetic mean ± SD cantly altered serum TxB2, except for a significant effect at 24 h concentrations at 1 h were 1308 ± 481 ng ⁄ mL (subcutaneous)

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7 istration. Geometric mean values of Cmax and AUC, normalized for a dose of 1.0 mg ⁄ kg, were both lower than after subcuta- 6 neous administration. For all variables inter-animal variability 5 was greater after oral than subcutaneous dosing, as indicated by CV% values (Table 5). 4

3 DISCUSSION

concentration (ng/mL) 2 2 The results of this study confirm the findings of Giraudel et al. PGE 1 (2009) that robenacoxib is a selective inhibitor of COX-2 in vitro 0 in cats. In addition, using ex vivo assays, the recommended 01246824 dosages of robenacoxib (2 mg ⁄ kg by injection, 1–2 mg ⁄ kg by Time (h) tablets) produced significant inhibition of COX-2 while sparing Robenacoxib injection Robenacoxib tablet COX-1. Meloxicam Control The scientific literature contains many reports of in vitro studies undertaken to determine the potency of a wide range of Fig. 4. Time course of ex vivo inhibition of plasma PGE2 by four treatments: robenacoxib administered subcutaneously; robenacoxib NSAIDs as inhibitors of COX isoforms. As discussed by Warner tablet administered p.o.; meloxicam injection administered subcutane- et al. (1999) and Lees et al. (2004), assay results are markedly ously; and control (no treatment). Data are mean and SD. dependent on experimental conditions, so that isolated enzymes, broken cell preparations and intact cell assays, all in buffer solutions, and intact cell whole blood assays have yielded very disparate findings. Most investigators conducting in vitro assays 400 have preferred to use whole blood, on the ground of greatest physiological relevance; such assays use a matrix which is a 300 natural body fluid containing both platelets and leucocytes and in which the very high degree of drug binding to plasma protein, which characterises the great majority of NSAIDs, is allowed for 200 (Young et al., 1996; Pairet & Van Ryn, 1998). However, even optimized in vitro blood assays can yield for individual drugs

concentration (ng/mL) differing ratios to those obtained ex vivo and in vivo. This occurs,

2 100 for example, for those drugs such as aspirin, phenylbutazone and

TxB tepoxalin, which are converted in vivo to active metabolites (Lees 0 et al., 2004). Therefore, optimal assessment of NSAID effects on 01246824 COX-1 and COX-2 should be based on results obtained from Time (h) more than one method, as in the present investigation. The present ﬁndings conﬁrm previous results from Giraudel Robenacoxib injection Robenacoxib tablet et al. (2009), demonstrating the selectivity of robenacoxib for Meloxicam Control COX-2 compared to COX-1 inhibition in in vitro whole blood

Fig. 5. Time course of ex vivo inhibition of serum TxB2 by four assays. Further, the ex vivo data demonstrate marked inhibition treatments: robenacoxib administered subcutaneously; robenacoxib of COX-2 with no inhibition of COX-1 at the recommended tablet administered p.o.; meloxicam injection administered subcutane- dosages of robenacoxib (1–2 mg ⁄ kg orally and 2 mg ⁄ kg via ously; and control (no treatment). Data are mean and SD. subcutaneous injection). This result indicates the likely absence of metabolism of robenacoxib in vivo to active metabolite(s), in and 856 ± 639 (p.o.). Corresponding values at 4 h were relevant amounts, which possess a different pharmacodynamic 156 ± 158 and 20 ± 11 ng ⁄ mL. Mean dosages were 2.0 ± 0.0 proﬁle to robenacoxib. mg ⁄ kg (subcutaneous) and 1.45 ± 0.34 mg ⁄ kg (p.o.). It is not possible to state precisely the level and duration of After subcutaneous administration of robenacoxib the median COX-2 inhibition which should be achieved to provide appro- value of Tmax was 1 h. Geometric mean mean residence time priate levels of analgesia or inhibition of inﬂammation or fever. (MRT) was 1.79 h and terminal half-life was 0.97 h. Geometric Equally, the threshold in terms of magnitude and duration of mean values of Cmax and AUC(0-inf), normalized to a dosage of inhibition of COX-1, which should not be exceeded in order to 1mg⁄ kg, were 694 ng ⁄ mL and 1431 ng ⁄ mL.h, respectively minimise the potential for side-effects on the gastrointestinal (Table 5). tract and impairment of haemostatic mechanisms, is unknown.

After oral dosing of robenacoxib the median Tmax value was However, COX-1 inhibition should ideally be as short as possible 1.0 h, geometric mean MRT was 1.70 h and terminal half-life and not exceed 10–20% at peak concentration (Warner et al., was 1.53 h, somewhat longer than after subcutaneous admin- 1999; Lees et al., 2004). In contrast, therapeutic dosages of most

2010 Blackwell Publishing Ltd 450 V. B. Schmid et al.

Table 4. Statistical comparison (by RMANCOVA followed by linear contrasts for post-hoc comparisons) of ex vivo plasma PGE2 and serum TxB2 concentrations after four treatments

P values for pairwise comparisons*

Eicosanoid Time (h) I vs.T I vs. M T vs. M I vs. C T vs.C M vs.C

PGE2 1 0.8490 0.1573 0.1698 <.0001 I <.0001 T <.0001 M 2 0.1477 0.2265 0.8116 <.0001 I <.0001 T <.0001 M 4 0.0079 I 0.2684 0.0001 M 0.0012 I 0.3915 <.0001 M 6 0.0541 0.0481 M 0.0001 M 0.0173 I 0.5667 <.0001 M 8 0.8008 <.0001 M <.0001 M 0.2288 0.1323 <.0001 M 24 0.9544 0.0014 M 0.0008 M 0.4914 0.5043 0.0001 M

TxB2 1 0.8641 0.1613 0.1159 0.1985 0.2758 0.0087 M 2 0.1298 0.0298 M 0.0003 M 0.0506 0.6636 <.0001 M 4 0.3169 0.1978 0.0184 M 0.5640 0.6677 0.0583 6 0.6770 0.0212 M 0.0070 M 0.6981 0.9773 0.0085 M 8 0.2911 0.0016 M 0.0330 M 0.8784 0.2379 0.0012 M 24 0.0452 T 0.3673 0.3119 0.0946 0.0005 T 0.0159 M

*I = robenacoxib injection administered subcutaneously, T = robenacoxib tablet administered p.o., M = meloxicam injection administered subcutaneously, C = control (no treatment). Data from eight cats. Next to each signiﬁcant comparison (bold type), the group with the lower concentration of the eicosanoid is indicated.

inhibition for NSAIDs in animals. Based on these tentative 800 criteria, the fact that robenacoxib produced only 4.4% COX-1 inhibition at a concentration that inhibited COX-2 by 80% in the in vitro whole blood assays may be regarded as an indicator of a 600 Subcutaneous probable good safety profile in vivo. The wide safety margin of Oral robenacoxib for gastrointestinal, haemostatic and renal effects 400 has been confirmed more directly in safety studies in the rat (King et al., 2009), dog and cat [King et al., 2010 (data on file)]. In contrast, the three comparator drugs used in this study 200 produced much greater COX-1 inhibition for an 80% level of COX-2 inhibition. The findings with ketoprofen and meloxicam Blood robenacoxib (ng/mL) are of particular interest because both are licensed for clinical use 0 in the cat. The in vitro blood assays demonstrated that ketoprofen 02468is COX-1 selective in the cat; the concentration required for 50% Time (h) COX-2 inhibition produced 98% inhibition of COX-1. In the light of this observation it is of further interest to note that ketoprofen, Fig. 6. Blood robenacoxib concentration–time profiles (dose-normalized to 1 mg ⁄ kg) after single p.o. (1–2 mg ⁄ kg) and subcutaneous (2 mg ⁄ kg) when administered p.o. and intravenously to cats at recom- administration. Data are mean and SD. mended dosages, produced profound and long lasting inhibition of COX-1 ex vivo, when assessed in whole blood assays (Lees NSAIDs in humans inhibit COX-2 by more than 50% and many et al., 2003). However, ketoprofen is a chiral compound that are associated with 74–89% inhibition over the dosage interval undergoes inversion of the R to the S isomer in vivo in cats (Lees (Warner et al., 1999). Lees (2003) proposed a similar level of et al., 2003). S-ketoprofen is the eutomer, i.e. the enantiomer

Table 5. Pharmacokinetics of robenacoxib after single oral (1–2 mg ⁄ kg) and subcutaneous (2 mg ⁄ kg) administration to eight cats*

Dosing (p.o.) Dosing (subcutaneous)

Variable (units) Geometric mean Arithmetic mean SD CV (%) Geometric mean Arithmetic mean SD CV (%)

Tmax (h) 1.0 1.0 à Cmax norm. (ng ⁄ mL) 493 626 569 91 694 735 248 34 à AUC(0-t) norm. (ng ⁄ mL.h) 694 858 722 84 1421 1584 673 43 à AUC(0-inf) norm. (ng ⁄ mL.h) 719 878 716 82 1431 1597 692 43 MRT (h) 1.70 1.87 0.98 52 1.79 1.82 0.34 19

Terminal t½ (h) 1.53 1.95 1.56 80 0.97 0.98 0.17 18

*Pharmacokinetic calculations performed with the software DEBA. AUC was calculated by linear trapezoidal integration. For Tmax there was no Gaussian distribution; therefore median values are presented. àNormalized to a dosage of 1 mg ⁄ kg for all animals.

2010 Blackwell Publishing Ltd Robenacoxib in the cat 451 with greatest potency. Therefore in vitro and in vivo effects of duration in the central compartment. However, robenacoxib ketoprofen on COX-1 and COX-2 may be expected to differ. Also belongs to the class of acidic and highly protein bound NSAIDs to be noted is the possibility of species variation in the selectivity which accumulate preferentially at and are slowly cleared from of ketoprofen for COX isoforms. Using the same in vitro whole inﬂammed sites (Lees et al., 2004; Brune & Furst, 2007) and blood assays as those used in the present investigation, King therefore its pharmacodynamic duration of action is longer than et al. (2010) reported for ketoprofen in the dog an IC50COX- predictable from its short residence time in the blood (King et al.,

1:IC50COX-2 ratio of 0.88, suggesting that it is a nonselective 2009). Further studies are required to evaluate the effect of COX inhibitor in this species. robenacoxib on COX-1 and COX-2 in inflammatory exudates in The present results confirm previous work that meloxicam has cats. low selectivity for COX-2 in vitro in cats (Giraudel et al., 2009). The low COX-2 selectivity observed in vitro translated into a lack of selectivity in vivo, with significant inhibition of both COX-1 ACKNOWLEDGMENTS and COX-2 with the 0.3 mg ⁄ kg dosage with subcutaneous injection. The authors thank Christophe Chardonnens and Martin Jung for In view of possible inter-species differences and differences their technical assistance in the analytical part of these studies. attributable to experimental technique, even when using whole The authors also wish to thank especially the late Catherine blood as the matrix, it is relevant to compare for robenacoxib Rudaz for her commitment and friendship, as well as for her COX inhibition potencies and potency ratios in several species, invaluable technical expertise. including those of intended clinical use, the cat and dog. For the dog (King et al., 2010) and the cat (this study), IC50 values for COX-1 inhibition, respectively, were 10.77 and 4.98 lM. Corre- REFERENCES sponding values for COX-2 inhibition were 0.079 and 0.111 lM and IC50COX-1:IC50COX-2 inhibition ratios were 129:1 and Brune, K. & Furst, D.E. (2007) Combining enzyme specificity and tissue 32:1. 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