Effect of a novel topical diester glucocorticoid ... - Wiley Online Library

DOI: 10.1111/j.1365-3164.2009.00782.x

Effect of a novel topical diester glucocorticoid spray on immediate- and late-phase cutaneous allergic reactions in Maltese–beagle atopic dogs: a placebo-controlled study Petra Bizikova*, Keith E. Linder†‡, Judy Paps* and Thierry Olivry*† *Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, 4700 Hillsborough Street, Raleigh, NC, USA † Department of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA ‡ Center for Comparative Medicine and Translational Research, North Carolina State University, Raleigh, NC 27606, USA Correspondence: Thierry Olivry, Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, 4700 Hillsborough Street, Raleigh, NC 27606, USA. E-mail: thierry_olivry@ ncsu.edu Sources of Funding This study was funded by Virbac SA, Carros, France. Conflict of Interest The authors do not report any conflict of interest relevant to this study.

Abstract The inhibitory effect of 0.0584% hydrocortisone aceponate spray on immediate- and late-phase skin reactions and the duration of inhibition after medication withdrawal were studied in 10 Maltese-beagle atopic dogs. All subjects were sprayed on axillary and inguinal regions and on one randomly chosen side of the thorax once daily for 14 (phase 1) or 7 days (phase 2). Intradermal injections (IDT) of histamine and anticanine IgE antiserum were performed bilaterally on the thorax before, 7 and 14 days after treatment. During phase 2, IDT was performed once weekly for 5 weeks. Each IDT was evaluated by an investigator blinded to the site of active treatment. Skin biopsies of 24-h anti-IgE-associated late-phase reactions were collected from both thoracic sides before and 14 days after treatment to determine the number of inflammatory cells and dermal thickness. Phase 1: Histamine and anti-IgE-induced global wheal scores at treated sites were significantly lower after 7 and 14 days with negative reactions present in >90% of dogs. Late-phase reactions at both sides were also significantly decreased compared with that at baseline, and this was associated with reduced inflammatory cell influx. Moreover, a significant decrease in dermal thickness was recorded at treated sides after 14 days. Phase 2: Histamine reactions became positive at untreated sides in all dogs 2 weeks after treatment. In conclusion, the 0.0584% hydrocortisone

aceponate spray significantly decreased immediateand late-phase IDT reactions, and prolonged application caused skin atrophy at treated sites. A 2-week withdrawal period prior to IDT is proposed. Accepted 29 April 2009

Introduction Atopic dermatitis (AD), a chronic pruritic skin condition, requires long-term management to alleviate and prevent the recurrence of clinical signs. For this reason, veterinarians have often resorted to the practice of allergen-specific immunotherapy (ASIT) with or without concurrent usage of anti-inflammatory drugs.1 The selection of allergens to be included in ASIT relies on the demonstration of IgE-mediated hypersensitivity by either intradermal testing (IDT) or allergen-specific IgE serology.2 Although the knowledge about the effect of anti-inflammatory drugs on allergen-specific IgE serology results is incomplete in veterinary medicine,3 there is evidence suggesting the suppressive effect of systemic antihistamines on IDT immediate reactions.4,5 Moreover, previous studies have suggested a variable effect of topical and systemic glucocorticoid (GC) on such a reaction.6–9 For this reason, before performing IDT, dogs need to be taken off these anti-allergic medications for drug-specific durations.6 Unfortunately, many dogs suffer from AD of such severity that withdrawal of GC is not possible without a marked impact on quality of life. Furthermore, the administration of GC is often accompanied by undesirable adverse effects such as skin atrophy (especially with topical GC),10 bacterial urinary tract infections11 and ⁄ or iatrogenic Cushing’s syndrome.12 As a result, an important advance in veterinary dermatology would be to implement an intervention offering potent cutaneous anti-inflammatory effect without any relevant interference on results of IDT and with a low risk of side effects. Although ideal GC have not been synthesized yet, the novel topical diester GC containing 0.0584% hydrocortisone aceponate (Cortavance; Virbac SA, Carros, France), known for its high potency, good penetration, rapid inactivation in the dermis and minimal systemic effect, could represent a unique drug to resolve some of the abovementioned clinical dilemmas. Indeed, the properties of this spray could provide sufficient anti-inflammatory

ª 2010 The Authors. Journal compilation ª 2010 ESVD and ACVD, Veterinary Dermatology, 21, 71–80.

71

Bizikova et al.

effect at skin sites requiring treatment and, an IDT could be performed at an untreated area without the need for discontinuing therapy. To test this hypothetical concept, we decided to evaluate the effect of 0.0584% hydrocortisone aceponate spray on immediate- and late-phase reactions (LPRs) after intradermal injections of histamine and anticanine-IgE antiserum performed at treated and untreated skin areas. Moreover, if clinically relevant inhibition of IDT reactions were to be observed, the duration of this inhibition would be assessed in a second experiment.

Materials and methods All phases of the experimental design of the study were approved beforehand by North Carolina State University Institutional Animal Care and Use Committee (IACUC).

Study subjects Ten atopic Maltese–beagle crossbred dogs (MBA) were selected for this study. This number was determined to be sufficient to provide a 95% power to detect a 50% difference between two groups of values (standard deviation of 30%) at P = 0.05 (InStat; GraphPad, San Diego, CA, USA). There were four intact males and four intact and two spayed females. The mean age of the animals was 9.4 years (range 8–10 years), and they weighed between 4.4 and 6.8 kg. The two phases of the experiment are as follows: Phase 1: Evaluation of the effect of a 0.0584% hydrocortisone aceponate spray on immediate- and late-phase skin reactions to intradermal injection of histamine and anticanine-IgE antibodies at treated and untreated skin sites; Phase 2: Evaluation of the duration of inhibition of immediate skin reactions to intradermal injections of histamine by a 0.0584% hydrocortisone aceponate spray at directly treated and untreated skin sites.

Phase 1 Experimental design The study design was a randomized, blinded and placebo (vehicle)controlled experiment. Application of GC spray The active medication used for both phases was the commercially available 0.0584% hydrocortisone aceponate spray (Cortavance). The same spray without the hydrocortisone aceponate was used as a placebo. As indicated in Figure 1 (phase 1), the ten MBA dogs were sprayed once daily for 14 days in both axillary and inguinal regions, which are areas commonly affected by skin lesions in dogs with AD. In accordance with the manufacturer’s instruction, the dose used was two pumps (260 lL) to cover a square surface of 10 · 10 cm of skin or, in other words, two pumps per selected site (a total of eight pumps for all four sites). Using a coin toss, each dog was randomized to also receive two pumps of the active spray on a 10 · 10 cm2 shaved area on either the left or right thorax, while the other side was sprayed with the control spray (two pumps per 10 · 10 cm2 shaved area). One of the investigators (JP) determined the randomization sequence for each dog, kept the code blinded from the other investigators until the end of the study and applied the GC and control sprays independently from the other investigators. Intradermal challenges On days -5, 7 and 14, one of the investigators (PB) performed IDT with 0.05 mL of 0.001% histamine phosphate,6 0.05 mL of anticanine-IgE polyclonal antibodies (0.08 mg ⁄ mL)8,13 and 0.05 mL of phosphate-buffered saline on both sides of the thorax. The hair on the tested areas was clipped the day before. Clinical evaluation Twenty minutes after each injection, the same investigator (PB) assessed the extent and severity of the wheals as previously described.5 For extent determination, the average diameter (D) in

Figure 1. Study design.

72


Effect of a steroid spray on intradermal testing

orthogonal directions was measured in millimetres. For severity, erythema (E) and firmness (F) were assessed subjectively on a threepoint scale as follows: 1 = no erythema, none to small, softer wheal, 2 = weak erythema, firm wheal or 3 = strong erythema, firm wheal. Finally, a global wheal score (GWS) was calculated as follows: GWS = D · E · F. Additionally, the same investigator subjectively graded the skin reactions as positive or negative using the conventional (0 to 4+) scoring system. The reactions were considered positive if they were graded 2+ and higher, and negative with a score of 1+ or 0.6 Six hours following IDT with anticanine-IgE antiserum, the macroscopic characteristics of the LPRs were evaluated as described previously.5 The degree of erythema (E) was graded from 1 to 3 as follows: 1 = no erythema, 2 = mild slight erythema or 3 = strong erythema. Similarly, the degree of skin induration (I) was subjectively scored from 1 to 3 as follows: 1 = no induration, 2 = mild induration or 3 = firm or strong induration. The global LPR score (GLS) was determined as follows: GLS = E · I. All adverse effects observed during the experiment were recorded. Collection of skin biopsy specimens Twenty-four hours after each IDT challenge, all dogs were sedated with medetomidine (Domitor; Pfizer, Exton, PA, USA) at a dosage of 750 lg ⁄ m2 intravenously.14 Lidocaine 2% (Lidocaine 2%; Phoenix Pharmaceuticals, St. Joseph, MO, USA) was administered subcutaneously on each site previously injected with the anticanine-IgE antiserum. One 8-mm punch biopsy was obtained from each site treated with vehicle and active medication. The biopsy site was closed with surgical staples. A 2% erythromycin gel (Erythromycin topical gel 2% USP; Stiefel Laboratories, Coral Gables, FL, USA) was applied following sample collection to prevent secondary infection of the biopsy sites. The sedation was then reversed with intramuscular injection of atipamazole (Antisedan; Pfizer) at the same volume used for medetomidine. Histopathology and immunohistochemistry All biopsy specimens collected were fixed in neutral buffered formalin, bisected and embedded in paraffin. Two 5-lm sections were cut and stained using haematoxylin and eosin (H&E), modified Luna’s technique for examination of eosinophils,15 a low-pH toluidine blue stain to enumerate dermal mast cells16 and an immunohistochemical method with anti-CD3 rabbit antiserum (Dako, Carpentia, CA, USA).17 Enumeration of the cell infiltrate and dermal thickness measurement All histological slides were randomized, coded and examined in a blinded fashion. The code was broken at the end of all microscopic assessments. The total number of inflammatory cells, eosinophils, mast cells and T lymphocytes were counted in the dermis using H&E, Luna, toluidine blue and CD3 stains respectively. All counts were made on a Nikon Eclipse E200 microscope (Nikon Instruments Inc., Melville, NY, USA) at ·400 magnification. The numbers were obtained by counting 20 consecutive 0.25 mm · 0.1 mm fields of superficial dermis (the area between the epidermis and the beginning of a hair follicle isthmus), excluding adnexal structures, endothelial cells, inflammatory cells inside of the vessels lumen and fibroblasts. The total surface counted per slide was 0.5 mm2. If the total surface of the superficial dermis was less than 0.5 mm2, then cells were counted in the entire superficial dermal area and normalized to 0.5 mm2. Numbers were doubled to obtain the counts per mm2 of dermis. Dermal thickness measurements were performed on formalin-fixed, H&E-stained histological sections using bright field microscopy with a calibrated ocular micrometer. Measurements were collected over two histological sections at three positions per section for a total of six measurements per sample. Measurements were taken perpendicular to the skin surface at the mid-point between follicles in interfollicular areas, and they extended from the epidermal basement membrane zone to the interface between dermal collagen bundles and pannicular fat at the base of the dermis. Calculated dermal thickness values from the six sites were averaged for statistical analysis.

Phase 2 Phase 2 was to proceed only if the clinical evaluation of the skin reactions on days 7 and 14 was shown to be significantly different compared with the values before the treatment at the level of 5% (P = 0.05). Experimental design Phase 2 was designed as randomized, blinded and placebo (vehicle)controlled experiment initiated 3 months after phase 1 was finished. Application of GC spray The application of the GC spray was identical to the application described in phase 1, except for the shortened duration of the application to a total of 7 days (Figure 1; phase 2). Of note is that this shorter duration corresponds to the ‘per label’ approved recommendation for this formulation. Intradermal challenges On days 1, 7, 14, 21, 28 and 35, one of the investigators (PB) performed IDT with 0.05 mL of 0.001% histamine phosphate and 0.05 mL of phosphate-buffered saline on both sides of the thorax. The hair on the tested areas was clipped the day before. Clinical evaluation The clinical evaluation of the immediate skin reactions was performed as described for phase 1.

Statistical analysis Results of the clinical evaluation (GWS and GLS), as well as total dermal cell, eosinophil, mast cell and T lymphocyte counts, at each side of the thorax and for each time point, were compared with values obtained before treatment with active (GC) and vehicle products (placebo). Statistical comparison was performed by using a nonparametric repeated measures one-way ANOVA (Friedman test) utilizing a computer software (Prism 4.0; GraphPad Softwares, La Jolla, CA, USA). Dunn’s post-tests were used to determine differences between two groups of values.

Results Phase 1 All dogs tolerated the application of the spray well. Adverse events were not recorded except for mild skin atrophy clinically visible in eight of ten dogs in the inguinal and axillary regions at the end of the second week of spray application. Histamine and anticanine-IgE antiserum GWS at treated sides were significantly lower after 7 and 14 days compared with pre-intervention values (Figure 2a,b; Table 1). At untreated sides, the GWS were significantly lower only after 14 days compared with that at baseline (Figure 2a,b; Table 1). The clinical assessment of skin reactions using the subjective 0 to 4+ scoring system yielded positive skin reactions to IDT with histamine and anticanine-IgE antiserum in all ten dogs before treatment. After 7 days of daily application of the GC spray, zero of ten and one of ten dogs had positive responses to histamine and anticanine-IgE antiserum at the treated thoracic sides respectively (Figure 2c); while five of ten and six of ten dogs had positive responses to histamine and anticanine-IgE antiserum at untreated thoracic sides respectively (Figure 2d). The additional application of the spray for 7days further increased the number of negative results at both sides of the thorax and for both injection types (Figure 2c,d). The


73

Bizikova et al.

Figure 2. Composite picture, phase 1 – evaluation of the GWS (a,b) and clinical assessment (c,d) of the immediate skin reaction after intradermal injection of histamine and anticanine-IgE antiserum before and after 7 and 14 days of daily application of 0.0584% hydrocortisone aceponate containing spray (CortavanceTM) at treated and untreated thorax sides. (a,b) Data represent the median and interquartile range (IQR) of the global wheal scores. **P < 0.01; *P < 0.05. (c,d) Data represent number of dogs with positive (2+ and greater) and negative (