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Open Veterinary Journal, (2017), Vol. 7(3): 254-260 ISSN: 2226-4485 (Print) Original Article ISSN: 2218-6050 (Online) DOI: http://dx.doi.org/10.4314/ovj.v7i3.9

______Submitted: 09/10/2016 Accepted: 01/08/2017 Published: 24/08/2017

Effects of topical flurbiprofen sodium, sodium, tromethamine and on corneal sensitivity in normal dogs

Raquel de Araújo Cantarella1, Juliana Kravetz de Oliveira1, Daniel M. Dorbandt2,3 and Fabiano Montiani-Ferreira1,*

1Universidade Federal do Paraná (UFPR), Departamento de Medicina Veterinária, Rua dos Funcionários, 1540, Bairro Juvevê, 80035-050, Curitiba – PR, Brazil 2Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois at Urbana- Champaign, 1008, West Hazelwood Drive, Urbana, Illinois 61802, USA 3Central Hospital for Veterinary Medicine, North Haven, Connecticut 06473, USA ______Abstract To evaluate corneal sensitivity by using the Cochet-Bonnet® esthesiometer in normal canine eyes at different time points following instillation of three different topical non-steroidal anti-inflammatory drugs (flurbiprofen sodium 0.03%, diclofenac sodium 0.1% and ketorolac tromethamine 0.5%) and benzalkonium chloride 0.01%. Six healthy mixed breed dogs from the same litter were used in two different stages. First, one drop of flurbiprofen sodium 0.03% and diclofenac sodium 0.1% in each eye; second, one drop of ketorolac tromethamine 0.5% and benzalkonium chloride 0.01% in each eye. Baseline esthesiometry was obtained before application and every 15 minutes thereafter until a total of 105 minutes of evaluation time. A one-week interval was allowed between the two treatment phases. Statistical analysis was used to compare means according to time of evaluation and drug used. Diclofenac sodium 0.1% decreased corneal sensitivity at 75 and 90 minutes (P > 0.015) with possible interference on neuronal nociceptive activity and effect while ketorolac tromethamine 0.5% did not show any variation for esthesiometry means along the evaluation. Flurbiprofen sodium 0.03% resulted in increased esthesiometry values 30 minutes after instillation (P > 0.013), increasing corneal sensitivity and possibly producing a greater irritant corneal effect over its analgesic properties. Benzalkonium chloride 0.01% significantly increased corneal sensitivity at 15 minutes of evaluation (P > 0.001), most likely resulting from its irritating effect. Esthesiometry did not allow a definite conclusion over the analgesic effect of the NSAIDs tested; however it was effective in detecting fluctuations in corneal sensitivity. Keywords: Cochet-Bonnet, Cornea, Nociceptors, NSAID. ______

Introduction canine eye (Maggs, 2008). After biosynthesis, Corneal and bulbar conjunctival innervation is supplied bind to receptors called pain facilitators by a relatively small number of primary sensorial (Tranquilli and Thurman, 1999; Marfurt et al. 2001), nerves originating from the ipsilateral trigeminal which promote variations in the resting potential of ganglion (about 1.5% of the neurons from ganglion´s neuronal membranes. These variations in membrane total number) (De Felipe et al., 1999). However, the resting potentials result in pain amplification (Bloom, small dimension of the corneal surface with the vast 1996; Tasaka, 1999; Acosta et al., 2005; Tranquilli and branching of peripheral nerve axons results in the Thurman, 1999). cornea being the most densely innerved structure of the NSAIDS are commonly used in ophthalmology to body (De Felipe et al., 1999). Additionally, 70% of control pain caused by tissue injury and reduce these sensorial fibers are classified as polymodal localized inflammation and after surgical procedures nociceptors (Belmonte et al., 2004). (Chen et al., 1997; Aragona et al., 2000; Weaver and Non-steroidal anti-inflammatory drugs (NSAIDS) are a Terrell, 2003; Giuliano, 2004; Acosta et al., 2007; Kim group comprised of several drugs that are able control et al., 2015). Topical NSAIDs such as flurbiprofen, inflammation, promote analgesia and reduce diclofenac and ketorolac tromethamine are commonly hyperthermia by inhibiting activity, prescribed in human and veterinary medicine due to thereby reducing eicosanoid synthesis created through their availability and relatively low cost. Topical the arachinodic acid cascade (Bloom, 1996; Insel, NSAIDS are primarily used for inflammatory ocular 1996; Aragona et al., 2000). Prostaglandins are one of conditions such as the suppression of uveitis that may the primary eicosanoids released upon insult to the be present before and after intraocular surgery

______*Corresponding Author: Fabiano Montiani-Ferreira. Universidade Federal do Paraná (UFPR), Departamento de Medicina 254 Veterinária, Rua dos Funcionários, 1540, Bairro Juvevê, 80035-050, Curitiba – PR, Brazil. Email: [email protected] http://www.openveterinaryjournal.com R. Cantarella et al. Open Veterinary Journal, (2017), Vol. 7(3): 254-260 ______(Giuliano, 2004; Sigle and Nasisse, 2006; Kim et al., Corneal sensitivity data were acquired using a Cochet- 2010; Maca et al., 2010; Klein et al., 2011). Bonnet esthesiometer (Luneau Ophtalmologie, Chen et al. (1997) showed that the application of Chartres Cedex, France) following a previously topical NSAID ophthalmic solutions effectively described protocol (Barrett et al., 1991; Good et al., reduced corneal sensitivity as well as the sensitivity of 2003). Data collection began approximately two weeks polymodal nociceptors. These findings support that after the screening examination. The handheld analgesia and ocular comfort reported by human beings esthesiometer possesses a thin, retractable, nylon may, in part, be due to decreased PGs excitatory monofilament that extends up to 6 cm in length. Since activity on nervous fibers (Martini et al., 1984; Yamada normal baseline corneal sensitivity threshold range of et al., 2002). the normal dog varies from 1.0 to 3.5 cm (Stiles et al. The aim of this study is to evaluate variations in the 2001; Blocker et al., 2007; Kobashigawa et al., 2015; corneal sensitivity of dogs after topical application of Venturi et al., 2016), measurements started with a three different NSAIDS and one preservative agent nylon monofilament length of 4 cm, which is a usual using a Cochet-Bonnet esthesiometer, which measures initial set point for corneal sensitivity evaluation in the corneal surface sensitivity threshold. In dogs, the dogs (Venturi et al., 2016). accepted criteria for measurement of corneal sensitivity The esthesiometer was gently advanced threshold is the degree of corneal stimulation that is perpendicularly toward the center of corneal surface required to elicit a blink reflex (Barrett et al., 1991; until a slight deflection of the monofilament was noted Stiles et al., 2001; Good et al., 2003; Blocker et al., after contact. If a blink reflex was not observed on at 2007; Venturi et al., 2016; Dorbandt et al., 2017). least three attempts, the length of the monofilament was Materials and Methods decreased 0.25 cm, and the procedure was then This study followed ARVO´s Statement for the Use of repeated. The length of the filament is directly Animals in Ophthalmic and Vision Research and was proportional to corneal sensitivity such that a longer approved by the Ethics Committee of the Agrarian nylon monofilament length required to stimulate a Sciences Sector, Federal University of Parana. blinking reflex equates to a more sensitive cornea. The Six nine-month-old nonbrachycephalic mixed breed experiment was conducted in two different stages, and dogs, all littermates, were used in the study. Three for each stage, a repetition was made in the same group males and three females were used, and all dogs were of animals. previously evaluated, vaccinated and free from signs of The drug bottles were labeled as ‘A’, ‘B,’ ‘C’, ‘D’, and systemic or ophthalmic diseases. Prior to enrollment in the investigators responsible for application of the the study, a complete ophthalmic examination was study drug and esthesiometry measurements were performed including a Schirmer tear test I (STT-I) masked to the drug being used. The key was maintained (Drogavet, Curitiba, Brazil), fluorescein stain by the principal investigator and revealed to the study (fluorescein sodium; Drogavet, Curitiba, Brazil), investigators at the conclusion of the study. In the first tonometry (Tono-Vet; Icare Finland, Espoo, Finland), stage, one drop of flurbiprofen sodium (drug A) was slit-lamp biomicroscopy (Hawk Eye; Dioptrix, administered onto the right eye, and one drop of L’Union, France), and indirect ophthalmoscopy (Heine diclofenac sodium (drug B) was administered onto the Omega 180 Headworn Binocular Indirect left eye of each animal. In the second stage, after a Ophthalmoscope, Dove, USA). Additionally, a washout period of 7 days, one drop of ketorolac complete blood count, serum biochemical analysis, and tromethamine (drug C) was administered onto the right urinalysis were obtained. eye and one drop of benzalkonium chloride was A masked prospective study design was used to administered onto the left eye (drug D). determine the immediate effects of three commonly For each stage, the length of the nylon monofilament available ophthalmic NSAIDS and one common able to stimulate the dogs’ blink reflex was recorded. ophthalmic preservative on corneal sensitivity in Baseline esthesiometry (before the administration of normal canine eyes. The ophthalmic NSAIDS any drug) was represented as time 0. Once each eye evaluated were flurbiprofen sodium 0.03% drop was applied onto the ocular surface, subsequent (ALLERGAN; Syntex Inc., USA), diclofenac sodium esthesiometry data were collected every 15 minutes 0.1% (ALLERGAN; Syntex Inc., USA) and ketorolac after initial administration until one hour and 45 tromethamine 0.5% (ALLERGAN; Syntex Inc., USA). minutes after the last drop (105 minutes). The ophthalmic preservative evaluated was Each stage was performed with same room conditions, benzalkonium chloride 0.01%, which is the controlling external variables such as weather, noise, preservative agent found in ketorolac tromethamine. and people and animal traffic inside and outside the Benzalkonium chloride eye drops were specially room. Only two study investigators were present during prepared in a compounding pharmacy the data collection, one responsible for esthesionometry (OPHTHALMOS, São Paulo, Brazil). and the other responsible for animal restraint. The same

255 http://www.openveterinaryjournal.com R. Cantarella et al. Open Veterinary Journal, (2017), Vol. 7(3): 254-260 ______study investigator was responsible for each function significant differences between esthesiometry data throughout the experiment. Temperature and humidity means and baseline means at 15 minutes (P = 0.1), 30 were evaluated during each stage of the investigation minutes (P = 0.8) or at any other time point after topical using a wireless thermos hygrometer (TM005X-M administration. After topical application of Meade Instruments, Irvine, CA). benzalkonium chloride 0.01%, a significant increase (P Statistical analyses were performed to compare the = 0.001) in corneal sensitivity values compared with esthesiometry data obtained in relation to time (in baseline values was observed at 15 minutes. minutes) and each drug used. To check whether or not Discussion the distribution of the data errors followed a Gaussian It is well known that corneal sensitivity exhibits a great distribution, a Shapiro-Wilk test was applied. The test variability and it is influenced by several factors. For revealed that the data did not follow a non-Gaussian instance, corneal sensitivity is significantly reduced in distribution. Therefore the non-parametric Friedman´s brachycephalic and corneal region evaluated, with the test (similar to the parametric repeated measures central part being more sensitive that the peripheral ANOVA) was used to detect differences in treatments cornea in dogs (Barrett et al., 1991) and cats (Blocker across multiple test attempts on the same group of and Van Der Woerdt, 2001). Humans over the age of animals (paired data). Descriptive and inferential 40 have a significantly reduced corneal sensitivity statistical analysis were performed using the JMP v7 (Millodot, 1972). Interestingly, the influence of age (SAS Institute, Cary, NC, EUA), statistical package for was not observed in dogs (Barrett et al., 1991) or cats computers. (Blocker and Van Der Woerdt, 2001). These variables Results were controlled in the present study since a repeated In the first stage, the mean environmental conditions measure statistical design was used with the same test- during the time in which the experiment was conducted subjects (paired data), including the initial control. Data was 21°C and 71% humidity. During the second stage, were collected in a longitudinal fashion in which temperature was 18ºC with 77% humidity. change over time was assessed and time 0 is the control Table 1 shows the esthesiometry results (medians) by of each subject. This method reduces the variance of each evaluation time for each drug tested. No estimates of treatment-effects, allowing statistical significant difference was found comparing baseline inference to be made with fewer subjects (Gueorguieva results (time 0). An overall (considering all drugs and Krystal, 2004). Additionally, to reduce the together) significant difference for higher corneal influence of different examiners on data variability, the sensitivity values within the initial 15 (P = 0.001) and same study investigator was responsible for all corneal 30 minutes (P = 0.03) after topical administration of all sensitivity evaluations. eye drops tested was observed compared to baseline Topical application of diclofenac sodium 0.1% was the values. This initial increase in corneal sensitivity was only study drug that resulted in a decrease in corneal followed by a gradual and significant decrease in mean sensitivity, and this occurred at 75 and 90 minutes post- values (P≤ 0.025) compared to baseline values. application. Paradoxically, topical application of Considering the results for the individual drugs, after flurbiprofen sodium 0.03% demonstrated a significant the topical use of flurbiprofen sodium 0.03%, an increase in corneal sensitivity 30 minutes post- increase in corneal sensitivity at 30 minutes was application while a nonsignificant trend for an increase observed (P = 0.013) when compared with baseline in corneal sensitivity occurred for all other study drugs values. Subsequently, a tendency towards a decrease in during the initial 15 to 30 minutes. For all treatment mean corneal sensitivity values was observed until the protocols, this initial increase in corneal sensitivity was last evaluation time. However these lower mean followed by a steady decrease (Table 1).The magnitude corneal sensitivity values were not significantly and significance of the initial increase and subsequent different than baseline values. A slight increase of decrease is different between drugs, but this trend is esthesiometry means also was observed 30 minutes consistently observed for each specific anti- after topical administration of diclofenac sodium 0.1%; inflammatory drug evaluated.The control of corneal however, this slight increase was not statistically pain has limited options, as topical sodium channel significant (P=0.75). blockers, such as proparacaine and tetracaine, are not Subsequently, however, significant decreases (P ≤ acceptable due to their limited duration of activity and 0.034) in corneal sensitivity were observed between 75 epitheliotoxic effects (Grant and Acosta, 1994; Herring and 90 minutes post-application of diclofenac sodium et al., 2005; Venturi et al., 2016). Topical has 0.1% when compared to baseline values. Nonetheless, been shown to provide acceptable analgesia without a at 105 minutes after initial topical administration of delay of corneal epithelialization (Peyman et al., 1994; diclofenac, the difference was no longer significant. Stiles et al., 2003; Clark et al., 2011); however, the Corneal sensitivity data obtained after topical corneal analgesic effectiveness in dogs has recently application of ketorolac tromethamine 0.5% showed no come into question (Thomson et al., 2013).

256 http://www.openveterinaryjournal.com R. Cantarella et al. Open Veterinary Journal, (2017), Vol. 7(3): 254-260 ______Table 1. Esthesiometry values (median in cm) in relation with each time point evaluated after topical use of sodium flurbiprofen 0.03%, sodium diclofenac 0.01%, ketorolac tromethamine 0.5% and benzalkonium chloride 0.01% on corneal surface in dogs.

Time (minutes) Drugs 0 15 30 45 60 75 90 105 Flurbiprofen Sodium 0.03% 0.875 1.125 1.375* 0.875 1.000 1.000 0.875 0.875 Diclofenac Sodium 0.1% 1.250 1.125 1.250 1.000 1.00 0.750* 0.750* 0.875 Ketorolac Tromethamine 0.5% 1.250 1.375 1.250 1.250 1.250 1.000 1.000 1.000 Benzalkonium Chloride 0.01% 1.000 1.5* 1.375 1.125 1.000 1.000 1.250 1.000

*Means that presented significant differences compared with the baseline esthesiometry, P < 0.05.

Therefore, it is paramount to identify alternative potentially higher corneal irritant effect. This finding options for corneal analgesia. In humans, the use of was interesting, as a change in corneal sensitivity was topical NSAIDS has been shown to decrease corneal not expected because benzalkonium chloride is a pain (Weaver and Terrell, 2003; Kim et al., 2015). To preservative devoid of anti-inflammatory properties the authors’ knowledge, there are no studies evaluating and is instead used in ophthalmic solutions to increase the antinociceptive effect of topical NSAIDS on dogs drug penetration through the lipid cell membrane with corneal ulcers. The current study provides (Madhu et al., 1996; Malhotra and Majumdar, 2006). It evidence that diclofenac decreases corneal sensitivity, is also an ingredient present in ketorolac ophthalmic and this may correlate with improved comfort when solution. Importantly, benzalkonium chloride has also used in the presence of a corneal ulceration; however, been shown to promote eicosanoid synthesis and, controlled studies should be performed to evaluate its consequently, ocular irritation suggests that topical efficacy. This decrease in corneal sensitivity after application of this preservative, even in concentrations topical application of diclofenac has also been reported as low as 0.0001%, decreases proliferative cellular in humans (Szerenyi et al., 1994; Seitz et al., 1996). activity and promotes apoptosis (Madhu et al., 1996; In contrast to the effect of diclofenac, topical De Saint Jean et al., 1999). application of flurbiprofen demonstrated a possible Although the present study demonstrated an increase in increase of nociceptor excitatory activity after 30 corneal sensitivity after application of benzalkoniun minutes. However, between 45 to 105 minutes after chloride, the same effect was not observed with application, corneal sensitivity decreased, and ketorolac, despite containing benzalkonium chloride as nociceptor activity conceivably started to decline as a preservative. It is possible that the anti-inflammatory well. During this period of decreasing sensitivity, mean action of ketorolac may reduce the irritant effect of corneal sensitivity values still remained higher than benzalkonium chloride on corneal nociceptor fibers. baseline. Our data support that flurbiprofen may result The results of flurbiprofen and diclofenac identified in in a more evident irritant action than an analgesic one. the current study are somewhat different than the The authors hypothesize that the initial increase reflects results obtained in a recent study (Dorbandt et al., a common time pattern for the irritant action on the 2017), and this may be explained, in part, by variations canine corneal surface after topical administration of all in study design as well as differences in ambient drugs tested. humidity between the two studies. The current study The authors believe that blocking action evaluated corneal sensitivity with decreasing in peripheral nociceptors is able to promote a decrease increments of 0.25 cm, and this may have allowed the of neuronal excitatory activity even in non-inflamed investigators to assess for smaller changes in corneal eyes, which is supported by previous studies (Chen, et sensitivity. The current study also evaluated corneal al., 1997; Aragona et al., 2000). Esthesiometry data sensitivity up to 105 minutes after application of the obtained after topical application of diclofenac in the specified study drug. Specifically, for diclofenac, a current study suggests the peak analgesic action of this decrease in corneal sensitivity was observed at 75 and drug to occur 75 to 90 minutes after topical application. 90 minutes after application (Table 1). One of the most After this period, however, corneal sensitivity returned important differences between the two studies is the to baseline values. Topical application of ketorolac did ambient humidity. In the current study, ambient not produce significant changes in corneal sensitivity. humidity values during data collection were 71% to Data obtained after topical application of benzalkonium 77%, and it has been shown that humidity may have chloride demonstrates a significant increase in corneal profound effects on corneal esthesiometry sensitivity at 15 minutes compared with baseline measurements (Dorbandt et al., 2017). In the recent values. Additionally, higher mean sensitivity values comparable study, ambient humidity ranged from 19% were obtained when compared to other study treatments to 44%, which may explain the higher sensitivity during the same time point, which may represent a values, as the same study found that a lower humidity

257 http://www.openveterinaryjournal.com R. Cantarella et al. Open Veterinary Journal, (2017), Vol. 7(3): 254-260 ______results in a higher corneal sensitivity measurement Seitz et al., 1996; Chen et al., 1997; Aragona et al., (increased filament length) (Dorbandt et al., 2017). 2000; Yamada et al., 2002; Acosta et al., 2005, 2007). With these results of humidity kept in mind, it can A limitation of the current study is that the low number explain why corneal sensitivity values (filament length) of individuals evaluated may have a profound effect on obtained in the present study are lower than those significance (Type II statistical error), and it is possible obtained in previous studies (Barrett et al., 1991; Good that a significant increase would have been achieved et al., 2003; Dorbandt et al., 2017). with higher study numbers. Topical administration of Although some previous studies in humans and dogs all eye drops tested (considered together) show a demonstrated the analgesic action of flurbiprofen and significant increase in corneal sensitivity values within ketorolac (Sigle and Nasisse, 2006; Maca et al., 2010), the initial 15-30 minutes. However, when evaluated the present study suggests that the ophthalmic individually only flurbiprofen (at 30 minutes) and application of these two drugs do not promote analgesic benzalkonium chloride (at 15 minutes) demonstrated an activity on corneal nociceptor activity in healthy canine increase in corneal sensitivity. For diclofenac, although eyes. Another recent study (Dorbandt et al., 2017) also not statistically significant, a trend exists towards an demonstrated a lack of analgesic activity after topical increase in corneal sensitivity values 30 minutes after application of flurbiprofen to canine eyes. topical application. Another limitation is that the only Nevertheless, Acosta et al. (2007) found that topical preservative evaluated was benzalkonium chloride. ketorolac 0.4% resulted in analgesic efficacy in healthy Given the result of benzalkonium chloride application feline eyes and that this drug was capable of decreasing alone, the initial increase in corneal sensitivity noted peripheral nociceptor activity. Although the study by between 15 and 30 minutes may suggest an irritant Acosta et al. (2007) also uses the Cochet-Bonnet action caused by different preservative agents present esthesiometer as mechanical stimuli, data collection in each of these ophthalmic solutions: thimerosal was performed using the evaluation of the evoked 0.005% for flurbiprofen; boric acid for diclofenac; potential of neuronal fibers. This methodology, benzalkonium chloride 0.01% for ketorolac. For previously developed by Chen et al. (1997), comprises ketorolac, the trend towards an increase in sensitivity a more quantifiable parameter for the evaluation of after the administration was an expected result given corneal nociceptors. In this way, the single use of a the significant increase in corneal sensitivity after blinking reflex in the current investigation as an benzalkonium chloride alone was observed. The fact indirect observational parameter of corneal sensitivity that benzalkonium chloride did not show a significant promoted by Cochet-Bonnet esthesiometry may have difference when used in combination with ketorolac resulted in insufficient data due to inherent variability may be due to the slightly higher variation of the in the blink response. corneal sensitivity means observed at baseline. This Studies in humans have found that topical application overall effect also might be responsible by the burning of diclofenac demonstrates comparatively higher sensation reported by several human patients that use corneal analgesic potential (Szerenyi et al., 1994; Seitz these drugs topically (Chen et al., 1997; Aragona et al., et al., 1996) which corroborates the results from 2000; Acosta et al., 2005). However, new Aragona et al. (2000). Studies have also demonstrated investigations are necessary to prove the mechanism of evidence for the interaction of diclofenac with β- action for these agents on the corneal surface. Although endorphin (endogenous inhibitory neurotransmitter) the subjectivity of the blink reflex by observational and substance P (nociceptors endogenous excitatory evaluation may have interfered directly in obtaining neurotransmitter). In these studies, there was an esthesiometry results, both baseline and experimental observed increase in plasma β-endorphins (Martini et values were always evaluated by the same observer, al., 1984) and a decrease in levels of substance P in thus minimizing the possibility of subjectivity on tears (Yamada et al., 2002), which may contribute to variable measurement. the potential analgesic action of this drug. Thus, the The results of the current study demonstrate that authors believe that topical diclofenac effectively corneal sensitivity of normal, nonbrachycephalic dogs caused inhibition of the eicosanoid production in the is decreased by topical diclofenac sodium 0.1% at 75 metabolic pathway in the canine and 90 minutes after application. However, the topical corneas. This mechanism was then responsible for administration of flurbiprofen sodium 0.03% and decreasing corneal nociceptor activity of polymodal benzalkonium chloride 0.01% result in an immediate nociceptors, which are present in the cornea and are increase in corneal sensitivity between 15 and 30 responsive to prostaglandins and bradykinin (Belmonte minutes after application while ketorolac tromethamine et al., 2004). This decrease in corneal nociception 0.5% has no effect. The results of topical diclofenac generated a clinically-detectable degree of topical demonstrated in the current study mimics those found analgesia (Bloom, 1996). This assumption corroborates in humans in that diclofenac was the only topical the findings from other authors (Szerenyi et al., 1994; NSAID observed to cause an immediate, but transient,

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