In vivo efficacy study of the anti-inflammatory properties of Surolan

Abstract

This study evaluated the anti-inflammatory properties of Surolan with the use of a pinna model of inflammation in 80 mice and a randomized complete block design. Within each of 8 blocks, 10 treatments, consisting of different combinations of the constituents of Surolan with and without the prednisolone acetate component, were randomly assigned to the 9-wk-old CD-1 mice. The treatments were administered as a single dose 30 min after pinna inflammation was induced with tetradecanoylphorbol acetate. Ear-skin edema and erythema were assessed and measured 4 and 6 h thereafter. Treatment effects were evaluated with repeated-measures analysis of variance. Inclusion of prednisolone acetate with the Surolan vehicle, either alone or in combination with the nonsteroidal constituents of the suspension, resulted in a significant reduction in mean ear-skin thickness and erythema. The inflammation-reducing properties of prednisolone were not significantly affected by the other components of Surolan.

Otitis externa in dogs, characterized by acute or chronic inflammation of the epithelium of the external auditory canal, is a common cause of veterinary visits in North America (1,2). Dogs with otitis externa often present with swelling and erythema of the epithelial tissue of the ear canal, increased discharge from the ceruminous glands in the ear, and behavior suggesting otic pain and pruritus (1,3).

Primary causes of otitis externa include parasite infections, foreign bodies, neoplasia, hypersensitivity diseases, disorders of keratinization, glandular diseases, and autoimmune diseases (1,2,4,5). Some dogs may be predisposed to otitis externa if they have abnormally small or restrictive ear canals or excessive moisture in the ear, or if they suffer trauma to the ear (4,6). Infection with gram-positive or gram-negative organisms or yeast can aggravate and perpetuate otitis externa. Organisms frequently isolated from ears with otitis externa include Malassezia pachydermatis, Candida spp., Staphylococcus intermedius, S. aureus, Proteus spp., Pseudomonas spp., coli, and Streptococcus spp. (2,6–9).

Treatment of otitis externa depends on the primary cause. Once the primary cause has been identified and dealt with, other factors that either predispose the animal to ear problems or perpetuate the disease should be identified and managed. If microbial agents are isolated from affected ears, appropriate antimycotic or antibacterial medications (4,10) should be used. Topical medications containing steroidal anti-inflammatory agents such as hydrocortisone, prednisolone, dexamethasone, and betamethasone are indicated in the management of a number of causes of otitis externa. The addition of a topical anti-inflammatory agent reduces glandular secretions and inflammation in the ear, reduces pruritus and pain, and can lessen recovery time.

Surolan (Janssen Pharmaceutica, Beerse, Belgium) is a topical agent indicated for the treatment of otitis externa in dogs. The product contains, per milliliter of suspension, 23 mg of the antimycotic agent miconazole nitrate, 5500 IU of the antibiotic polymyxin B sulfate, and 5 mg of the corticosteroid prednisolone acetate. Field trials have demonstrated that this product is efficacious at reducing inflammation and treating infections caused by many types of pathogens (7,8,11,12). Treatment regimens in these trials varied from 9 d to 1 mo in duration and from applying the suspension once a day to applying it twice a day. All studies demonstrated that Surolan treatment was successful, with a minimal rate of recurrence of otitis externa in the study populations.

Although Surolan appeared to reduce inflammation, it was unknown whether this was due to the steroid component or was simply a result of eliminating the infectious agents that were causing tissue damage. A concern was raised that there might be interaction between the nonsteroidal components and the prednisolone that would render the latter ineffective. The objectives of this study were to determine the anti-inflammatory efficacy of Surolan in a mouse inflammation model and to determine whether the product’s nonsteroidal components interfered with the anti-inflammatory effects of prednisolone in the suspension.

We assigned 80 female CD-1 mice, each 9 wk old and weighing between 17 and 26 g, to 8 blocks in a randomized complete block design. A block consisted of 10 mice, housed in adjacent individual cages (25 × 25 × 30 cm). Each block was handled as a unit every time the animals were manipulated, and animals within each block were treated as similarly as possible throughout the study. The mice were acclimated to the laboratory facility for 7 d, during which time they were monitored daily. Throughout the study, the mice received a commercial pelleted mouse diet and water ad libitum.

Ten treatments were randomly assigned within each block (Table I): Surolan suspension, Surolan vehicle alone or with 1 or 2 of the suspension components, acetone (control), and no treatment (control). Owing to the large number of animals, those in half of the blocks were treated on day 0 and the remainder the next day.

Table I.

Mean ear-skin thickness and erythema scores in mice at 0, 4, and 6 h after induction of ear-skin inflammation with tetradecanoylphorbol acetate (TPA) dissolved in acetone and topical treatment 30 min later with Surolan or its components

	Mean ear-skin score; assessment time (h)
	Thickness (mm)			Erythemab
Treatment groupa	0	4	6	0	4	6
Acetone control	0.24	0.30	0.38	0.00	0.50	0.63
TPA control	0.25	0.57	0.60	0.00	1.50	1.88
Vehicle	0.25	0.47	0.51	0.00	1.50	1.63
Vehicle + miconazole	0.25	0.46	0.51	0.00	1.75	2.00
Vehicle + polymyxin	0.26	0.50	0.54	0.00	1.25	1.75
Vehicle + miconazole + polymyxin	0.25	0.44	0.48	0.00	2.00	2.13
Vehicle + prednisolone	0.24	0.34	0.41	0.00	1.75	1.75
Vehicle + prednisolone + miconazole	0.24	0.34	0.40	0.00	1.25	1.63
Vehicle + prednisolone + polymyxin	0.24	0.34	0.40	0.00	1.50	1.13
Surolan	0.25	0.36	0.38	0.00	1.75	1.38

^aAcetone alone was applied to the ears of the 1st control group and TPA in acetone to the ears of the 2nd control group; neither received the treatment combinations subsequently

^b0 — normal ear color; 1 — mild redness; 2 — moderate redness; 3 — severe redness

To induce inflammation of the ear skin, all mice except those in the acetone-control group received 4 μL of tetradecanoylphorbol acetate (TPA) dissolved in 20 μL of acetone on the pinna of the right ear at time 0 (13). The dose was equally divided between the lateral and medial surfaces of the pinna. The acetone group received 20 μL of acetone applied to the ear skin.

The ears were assessed for abnormalities, erythema, and edema before the trial began. Treatments were administered to the applicable groups 30 min after inflammation induction as a single 0.02-mL dose applied to the inner and outer surfaces (0.01 mL per side) of the right ear by means of an automatic microliter pipette.

Ear edema and erythema were evaluated 4 and 6 h after the application of TPA. Edema was measured with an electronic digital micrometer; measurements were taken just inside the outer edge of the pinna. Erythema was assessed by inspection of the earflap and scored as follows: 0 — normal ear color; 1 — mild redness; 2 — moderate redness; and 3 — severe redness. All personnel involved in measuring and scoring the study variables were blinded to treatment assignment.

The mean ear-skin thickness and erythema scores for each treatment group were determined at 0, 4, and 6 h. Repeated-measures analysis of variance was used to study the difference in means between the groups treated with or without prednisolone, excluding the control groups. To evaluate the effect of miconazole nitrate and polymyxin B sulfate on the anti-inflammatory effects of prednisolone, pairwise comparisons with the use of Turkey’s W procedure were undertaken between the means for a) Surolan vehicle and prednisolone, b) the vehicle, prednisolone, and miconazole, c) the vehicle, prednisolone, and polymyxin B, and d) Surolan suspension. Frequency distributions of the residuals from each model, as well as plots of the residuals by predicted value and treatment, were examined for normality and homogeneity of variance. All analyses were conducted at the individual animal level with SAS PROC MIXED (SAS Institute, Cary, North Carolina, USA) (14). The error value for determining statistical significance was P ≤ 0.05. The study was conducted according to US Good Laboratory Practice regulations (15) and scientific principles and followed the guidelines of the Canadian Council on Animal Care (16).

The ears of all the mice appeared to be normal before the application of TPA. The mean ear-skin thickness and erythema scores at time 0 were similar in all the treatment groups. The irritant TPA induced a clear and consistent inflammation, with skin redness and swelling. Four hours after its application, the skin of the ears of the TPA control group was erythematous and swollen to more than twice its initial thickness. The edema and erythema persisted through the 6-h assessments. In comparison, the ears of the acetone control group showed minimal swelling and minor erythema during the same period.

The ears of the mice in the TPA control group had the most pronounced swelling and had erythema scores between 1 (mild) and 2 (moderate), with the highest at the 6-h assessment (Table I). The ears exposed only to acetone had a mean erythema score of less than 1. Among the remaining groups, those not treated with prednisolone generally had higher edema and erythema scores than those treated with prednisolone. The overall mean erythema score for ears treated with prednisolone (1.52) was significantly lower (P ≤ 0.0382) than the mean for ears not treated with prednisolone (1.75) when the values for the 2 control groups were excluded. Similarly, the overall mean edema score (ear-skin thickness) for ears treated with prednisolone (0.37) was significantly lower (P ≤ 0.0001) than the overall mean for ears not treated with prednisolone (0.48) when the values for the 2 control groups were excluded.

Pairwise comparisons of the mean edema and erythema scores demonstrated no significant differences between the groups treated with vehicle + prednisolone and those treated with vehicle + prednisolone + miconazole, vehicle + prednisolone + polymyxin, or Surolan.

This study demonstrated that the steroid component of Surolan was associated with attenuation of inflammation in this murine model. Furthermore, it was evident that the other 2 active ingredients in the product, miconazole nitrate and polymyxin B sulfate, did not interfere with the anti-inflammatory effect of the prednisolone.

In the presence of the vehicle and the other individual and combined nonsteroidal constituents of Surolan, prednisolone significantly reduced edema and erythema in this model. This finding is consistent with that of a study done in dogs with clinical otitis externa by Cieslicki (8) in which the effects of Surolan with its steroidal component were compared with the effects of the product without the prednisolone component. The results suggested that prednisolone increased the rate of healing.

In this study, applying TPA to the ears of mice produced 2 components of the inflammatory process: edema and erythema. Adding prednisolone to the various nonsteroidal components of Surolan and applying the suspensions topically to the ears resulted in a reduction in edema and erythema. When the values for all the groups treated with prednisolone were compared with the values for those without prednisolone (excluding the control groups), there was a significant difference (P ≤ 0.05) in the means for both edema and erythema. Furthermore, pairwise comparisons demonstrated that the inflammation-reducing effect of prednisolone was not significantly affected by the other components of Surolan, added singly or in combination. This observation, combined with the fact that field studies have found Surolan to be effective against many causes of otitis externa (11,12), suggests that prednisolone contributes to the anti-inflammatory properties of Surolan and is an important component of the product.

In many cases of canine otitis externa there are multiple infectious agents complicating a primary cause, and the condition is often painful and pruritic. The results of this study suggest that including a steroidal anti-inflammatory agent in the treatment of otitis externa likely hastens the healing process and helps to alleviate discomfort.