一种新型外用二酯糖皮质激素喷雾对特应性皮炎马尔济斯-比格犬的 ...

王帆

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

一种新型外用二酯糖皮质激素喷雾对特应性皮炎马尔济斯-比格犬的速发和迟发皮肤过敏反应的影响:一项安慰剂对照研究

 

作者：Petra Bizikova, Keith E. Linder, Judy Paps and Thierry Olivry

 

Abstract

摘要

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 immediate and late-phase IDT reactions, and prolonged application caused skin atrophy at treated sites. A 2-week withdrawal period prior to IDT is proposed.

研究了0.0584%氢化可的松醋丙酯喷雾剂对10只特应性皮炎马尔济斯-比格犬速发、迟发皮肤过敏反应的抑制作用及停药后的抑制持续时间。所有受试者在腋窝和腹股沟区域以及随机选择的一侧胸腔喷药，每天1次，连续14天(第1期)或7天(第2期)。治疗前、治疗后7天和治疗后14天分别在双侧胸腔皮内注射(IDT)组胺和抗犬IgE抗血清。在第二阶段，IDT每周进行一次，持续5周。每个IDT由一名对积极治疗部位不知情的研究者进行评估。治疗前和治疗后14天分别采集双胸侧抗IgE相关迟发反应24小时皮肤活检，测定炎症细胞数量和真皮厚度。第1阶段:治疗部位组胺和抗IgE诱导的整体评分在7天和14天后显著降低，90%以上的犬出现阴性反应。与基线相比，双方的迟发反应也显著减少，这与炎症细胞内流减少有关。此外，14天后，治疗侧皮肤厚度显著减少。第2阶段:治疗2周后，所有犬的组胺反应在未治疗侧变为阳性。综上所述，0.0584%氢化可的松醋丙酯喷雾剂可显著降低速发和迟发IDT反应，且长期使用可导致治疗部位皮肤萎缩。建议在IDT之前有2周的戒断期。

 

Introduction

介绍

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.

特应性皮炎(AD)是一种慢性瘙痒性皮肤疾病，需要长期治疗以减轻和预防临床症状的复发。出于这个原因，兽医经常采取过敏原特异性免疫治疗(ASIT)的做法，同时或不同时使用抗炎药物。

 

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.Although the knowledge about the effect of anti-inflammatory drugs on allergen-specific IgE serology results is incomplete in veterinary medicine, there is evidence suggesting the suppressive effect of systemic antihistamines on IDT immediate reactions . Moreover, previous studies have suggested a variable effect of topical and systemic glucocorticoid (GC) on such a reaction. For this reason, before performing IDT, dogs need to be taken off these anti-allergic medications for drug-specific durations. 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), bacterial urinary tract infections and ⁄ or iatrogenic Cushing’s syndrome.

选择纳入ASIT的过敏原，依赖于通过皮内试验(IDT)或过敏原特异性IgE血清学证明IgE介导的超敏反应。虽然兽医学中关于抗炎药对过敏原特异性IgE血清学结果的影响的知识尚不完整，但有证据表明全身抗组胺药对IDT速发反应有抑制作用。此外，先前的研究表明，外用和全身糖皮质激素(GC)对这种反应的影响是可变的。因此，在进行IDT之前，犬需要在特定的药物持续时间内停用这些抗过敏药物。不幸的是，许多犬都患有严重的AD，因此不可能在不显著影响生活质量的情况下停用GC。此外，GC的使用通常伴随着不良反应，如皮肤萎缩(特别是外用GC)，细菌性尿路感染和医源性库欣综合征。

 

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.

因此，兽医皮肤病学的一个重要进展是实施一种干预措施，提供有效的皮肤抗炎作用，而不会对IDT的结果产生任何相关干扰，而且副作用的风险很低。

 

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 effect at skin sites requiring treatment and, an IDT could be performed at an untreated area without the need for discontinuing therapy.

虽然理想的GC尚未合成，但含有0.0584%氢化可的松醋丙酯(皮乐美)以其高效、渗透性好、在真皮中快速失活和最小的全身效应而闻名，可能是解决上述一些临床难题的独特药物。事实上，这种喷雾剂的特性可以在需要治疗的皮肤部位提供足够的抗炎效果，并且可以在未经治疗的区域进行IDT而无需停止治疗。

 

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.

为了验证这一假设概念，我们决定评估0.0584%氢化可的松醋丙酯喷雾剂对治疗和未治疗皮肤区域皮下注射组胺和抗犬- IgE抗血清后的速发和迟发反应(LPRs)的影响。此外，如果要观察到临床相关的IDT反应抑制，则需要在第二个实验中评估这种抑制的持续时间。

 

Materials and methods

材料与方法

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

实验设计的所有阶段都事先得到了北卡罗莱纳州立大学机构动物护理和使用委员会(IACUC)的批准。

 

Study subjects

研究对象

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:

本研究选择了10只特应性皮炎马尔济斯-比格杂交犬(MBA)。这个数字被确定为足以提供95%的功率来检测两组值之间50%的差异(30%的标准差)，P = 0.05 (InStat;GraphPad, San Diego, CA, USA)。有4只未去势雄性和4只未绝育雌性和2只已绝育雌性。这些动物的平均年龄为9.4岁(8-10岁)，体重在4.4 - 6.8公斤之间。实验的两个阶段如下:

 

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;

第一阶段:评价0.0584%氢化可的松醋丙酯喷雾剂对治疗和未治疗皮肤部位皮内注射组胺和抗犬IgE抗体的速发和迟发皮肤反应的影响;

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.

第二阶段:评估0.0584%氢化可的松醋丙酯喷雾在直接治疗和未治疗的皮肤部位皮下注射组胺对速发皮肤反应的抑制时间。

 

Phase 1

第一阶段

Experimental design

实验设计

The study design was a randomized, blinded and placebo (vehicle)- controlled experiment.

研究设计为随机、盲法、安慰剂(载体)对照实验。

 

Application of GC spray

GC喷雾的应用

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.

两阶段使用的活性药物均为市售的0.0584%氢化可的松醋丙酯喷雾(皮乐美)。不含氢化可的松醋丙酯的相同喷雾剂被用作安慰剂。

 

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).

如图1(第一阶段)所示，在10只MBA犬的腋窝和腹股沟区域每天喷洒一次，持续14天，这两个区域是AD犬皮肤病变常患病的区域。根据制造商的说明，使用的剂量是两泵(260IL)覆盖10*10厘米的皮肤正方形表面，或者换句话说，每个选定部位两泵(所有四个部位总共八泵)。

 

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.

通过随机方式，每只犬随机接受两次主动喷雾，喷洒在左胸或右胸10*10平方厘米的剃光区域，而另一侧则使用对照喷雾(每10*10平方厘米剃光区域两次)。一名研究人员(JP)确定每只犬的随机化序列，对其他研究人员保密直到研究结束，并独立于其他研究人员使用GC和对照喷雾剂。

 

Intradermal challenges

皮内注射的激发

On days -5, 7 and 14, one of the investigators (PB) performed IDT with 0.05 mL of 0.001% histamine phosphate, 0.05 mL of anticanine-IgE polyclonal antibodies (0.08 mg ⁄ mL) 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.

在第5、7和14天，1名研究人员(PB)在胸两侧分别用0.05 mL 0.001%磷酸组胺、 0.05 mL抗犬免疫球蛋白IgE多克隆抗体(0.08 mg / mL)和0.05 mL磷酸盐缓冲盐水进行IDT。测试区域的毛发在前一天被剪掉。

 

Clinical evaluation

临床评价

Twenty minutes after each injection, the same investigator (PB) assessed the extent and severity of the wheals as previously described. For extent determination, the average diameter (D) inorthogonal 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.

每次注射20分钟后，同一位研究者(PB)评估如前所述的风疹的严重程度。为确定程度，以毫米为单位测量正交方向的平均直径(D)。对于严重程度，红斑(E)和紧致度(F)以3分制主观评估如下:1 =无红斑，无到小而柔软的红斑，2 =弱红斑，紧致性红斑或3 =强红斑，紧致性红斑。最后，计算全局每轮评分(GWS): 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.

此外，同一研究人员使用传统的(0到4+)评分系统主观地将皮肤反应分为阳性或阴性。如果得分为2+或更高，则认为反应是阳性，得分为1+或0则认为反应是阴性。

 

Six hours following IDT with anticanine-IgE antiserum, the macroscopic characteristics of the LPRs were evaluated as described previously. 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.

用抗犬- IgE抗血清IDT后6小时，按前面描述的方法评估LPR的宏观特征。红斑程度(E)分为1 ~ 3级，1 =无红斑，2 =轻度红斑，3 =强烈红斑。同样，皮肤变硬程度(I)主观评分为1 ~ 3分:1 =无硬结，2 =轻度硬结，3 =硬结或强硬结。总体LPR评分(GLS)计算公式为:GLS = E·i。记录实验过程中观察到的所有不良反应。

 

 

 

Collection of skin biopsy specimens

采集皮肤活检样本

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.

每次IDT刺激后24小时，所有犬都用美托咪定，剂量为750 lg / m2静脉注射利多卡因2%在每个部位皮下注射抗犬- IgE抗血清。用载体和活性药物治疗的每个部位进行一次8毫米打孔活检。活检部位用手术订书钉缝合。2%红霉素凝胶在采样后使用，以防止活检部位的继发性感染。随后肌内注射阿替美唑，与美托咪定相同的体积。

 

Histopathology and immunohistochemistry

组织病理学和免疫组织化学

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).

采集所有活检样本均固定在中性缓冲福尔马林中，等分并包埋于石蜡中。2个5-lm切片切片，用血红素和伊红(H&E)染色，改良Luna技术检查嗜酸性粒细胞，低pH甲苯胺蓝染色计数真皮肥大细胞，免疫组织化学方法与抗cd3兔抗血清。

 

Enumeration of the cell infiltrate and dermal thickness measurement

细胞浸润计数及真皮厚度测定

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.

所有组织学切片随机、编码并以盲法检查。在所有微观评估结束时，代码被打破了。采用H&E染色、Luna染色、甲苯胺蓝染色、CD3染色分别计数真皮内炎性细胞、嗜酸性细胞、肥大细胞和T淋巴细胞的总数。

 

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 .

所有计数均在Nikon Eclipse E200显微镜上进行，放大倍数为×400。这些数字是通过对20个连续的0.25 mm × 0.1 mm的真皮浅层(表皮和毛囊峡部之间的区域)进行计数获得的，不包括附件结构、内皮细胞、血管腔内的炎症细胞和成纤维细胞。每张载玻片的总表面计数为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.

若真皮浅层总表面积小于0.5 mm2，则计数整个真皮浅层细胞面积，归一化为0.5 mm2。计数加倍以获得真皮每平方毫米的计数。皮肤厚度测量是在福尔马林固定，HE染色的组织学切片上进行的，使用带校准眼千分尺的明场显微镜。测量收集在两个组织学切片上，在每个切片的三个位置，每个样本总共测量六次。测量垂直于皮肤表面在毛囊间区域的毛囊之间的中点，测量范围从表皮基底膜区延伸到真皮胶原束和真皮底部的脂膜之间的界面。计算得到的6个部位的皮肤厚度值取平均值进行统计分析。

 

Phase 2

第二阶段

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).

只有第7天和第14天的皮肤反应临床评价与治疗前相比在5%水平上有显著差异(P = 0.05)，才进行第2期。

 

Experimental design

实验设计

Phase 2 was designed as randomized, blinded and placebo (vehicle)- controlled experiment initiated 3 months after phase 1 was finished.

第二阶段设计为随机、盲法、安慰剂(载体)对照实验，在第一阶段结束后3个月开始。

 

Application of GC spray

GC喷雾的应用

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.

GC喷雾的使用与第一阶段相同，只是使用时间缩短至总共7天(图1;值得注意的是，这个较短的持续时间对应于该制剂的“每个标签”批准建议。

 

Intradermal challenges

皮内注射的激发

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.

在第1、7、14、21、28和35天，一名研究人员(PB)在胸两侧用0.05 mL 0.001%磷酸组胺和0.05 mL磷酸盐缓冲盐水行IDT。测试区域的毛发在前一天被剪掉。

 

Clinical evaluation

临床评价

The clinical evaluation of the immediate skin reactions was performed as described for phase 1.

速发皮肤反应的临床评估按照第一阶段的描述进行。

 

Statistical analysis

统计分析

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.

将临床评估结果(GWS和GLS)以及每侧胸腔和每个时间点的总真皮细胞、嗜酸性粒细胞、肥大细胞和T淋巴细胞计数与活性(GC)和载体产品(安慰剂)治疗前的值进行比较。统计学比较采用非参数重复测量单因素方差分析(Friedman检验)，采用计算机软件。Dunn’s post-test用于确定两组值之间的差异。

 

Results

结果

Phase 1

第一阶段

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.

所有的犬都能很好地耐受喷雾剂的使用。不良事件没有记录，除了临床可见的轻度皮肤萎缩，10只犬中的8只在腹股沟和腋窝区域在喷雾应用的第二周结束。

 

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).

与干预前相比，治疗侧组胺和抗犬- IgE抗血清GWS在7天和14天后显著降低(图2a,b;表1)。与基线相比，未治疗侧的GWS仅在14天后显著降低(图2a,b;表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).

使用主观评分系统对皮肤反应进行临床评估，10只犬治疗前对组胺和抗犬- IgE抗血清IDT的皮肤反应均为阳性。每天使用GC喷雾7天后，治疗胸侧组胺和抗犬- IgE抗血清的阳性反应分别为0和1(图2c);而在未经治疗的胸侧，组胺和抗犬- IgE抗血清分别有5 / 10和6 / 10的犬呈阳性反应(图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 GLS were significantly lower at both treated and untreated sides after 7 and 14 days of treatment compared with that at baseline (Table 1).

进一步喷施7天，进一步增加了胸腔两侧和两种注射类型阴性结果的数量(图2c,d)。治疗7天和14天后，治疗组和未治疗组的GLS均显著低于基线(表1)。

 

The histological assessment confirmed the existence of superficial perivascular pattern of dermal inflammation at the sites of anticanine-IgE antiserum injection; it was dominated by eosinophils with fewer lymphocytes. Histiocytes ⁄ dendritic cells were less commonly seen, whereas neutrophils and plasma cells were rare. This inflammatory reaction was visibly diminished after 14 days of application of 0.0584% hydrocortisone aceponate compared with the pretreatment samples (Figure 3). Enumeration of cells revealed a statistically significant reduction in the total leukocyte, eosinophil and CD3+ lymphocyte counts at treated sides compared with pretreatment values. A difference in dermal mast cell numbers was not identified. A statistically significant decrease in dermal thickness was observed at treated thoracic sides after 14 days compared with the pretreatment values. Moreover, a statistically non-significant reduction in the total leukocyte counts, eosinophil counts and dermal thickness values was recorded at untreated sides (Figure 4; Table 2). The reduction in dermal thickness was associated with atrophy of dermal collagen and partial atrophy of adnexa. Treated sides had decreased numbers of primary and secondary hair follicles in the anagen phase of the hair cycle. Reduction in dermal thickness was not attributable to decrease in oedema, as oedema was minimal in pretreatment and post-treatment biopsy samples obtained 24 h after anticanine-IgE antiserum injection. Inflammation was more pronounced in pretreatment biopsies compared with that in controls, but it contributed only partially to differences in dermal thickness values.

组织学检查证实抗犬- IgE抗血清注射部位存在浅表血管周围型皮肤炎症;以嗜酸性粒细胞为主，淋巴细胞较少。组织细胞/树突状细胞较少见，而中性粒细胞和浆细胞罕见。与预处理样品相比，应用0.0584%氢化可的松醋丙酯14天后，这种炎症反应明显减轻(图3)。细胞计数显示，与预处理值相比，处理侧的白细胞总数、嗜酸性粒细胞和CD3+淋巴细胞计数有统计学意义的减少。未发现真皮肥大细胞数量的差异。与预处理值相比，治疗后14天胸侧皮肤厚度有统计学意义的减少。此外，在未处理的一侧，记录了白细胞总数、嗜酸性粒细胞计数和真皮厚度值的统计学上无显著减少(图4;表2)真皮厚度的减少与真皮胶原的萎缩和附件的部分萎缩有关。在毛发周期的生长期，治疗侧的原发性和继发性毛囊数量减少。皮肤厚度的减少并不是由于水肿的减少，因为在注射抗犬- IgE抗血清24小时后获得的预处理和治疗后活检样本中，水肿最小。与对照组相比，炎症在预处理活检中更为明显，但它仅部分地导致了真皮厚度值的差异。

 

Phase 2

第二阶段

As seen in Figure 5a (left), histamine-induced GWS at treated sides were significantly lower on day 7 (the end of the daily application of 0.0584% hydrocortisone aceponate spray) and 7 days after the treatment was discontinued (day 14). Although the histamine-induced GWS at untreated sides were also reduced, this reduction was not significantly different to the pretreatment values (Figure 5a, right). The clinical assessment of skin reactions using the standard 0 to 4+ scoring system yielded positive skin reactions to IDT with histamine at sides treated with the active ingredient in one of ten and zero of ten dogs on the last day of spray application (day 7) and 1 week later (day 14 in Figure 5b) respectively (Figure 5b, left). Weekly repeated IDT with histamine demonstrated the return of the histamine immediate reactivity in more than 50% of dogs after 21 days of withdrawal period (day 28 in Figure 5b). The clinical assessment of histamine-induced skin reactions at untreated sides showed positive responses in eight of ten dogs on the last day of application and 1 week later (days 7 and 14; Figure 5b, right). Positive skin reactions to histamine at distant untreated sides were seen in all dogs, 14 days after ending the application of the spray (day 21 in Figure 5b).

如图5a(左)所示，组胺诱导的治疗侧GWS在第7天(每天使用0.0584%氢化可的松醋丙酯喷雾剂结束)和停止治疗后第7天(第14天)显著降低。虽然未处理侧组胺诱导的GWS也有所减少，但这种减少与预处理值没有显著差异(图5a，右)。使用标准的0到4+评分系统对皮肤反应的临床评估显示，在喷雾剂使用的最后一天(第7天)和1周后(图5b中的第14天)，在使用活性成分处理的一侧，有十分之一的犬和十分之一的犬对组胺IDT皮肤反应阳性(图5b，左)。每周用组胺重复IDT，在停药21天后，超过50%的犬恢复了组胺的速发反应性(图5b中的第28天)。组胺诱导皮肤反应的临床评估显示，10只犬中有8只在应用的最后一天和1周后(第7天和第14天;图5b，右)。在停止使用喷雾剂14天后(图5b中的第21天)，所有犬的远端未处理侧皮肤对组胺的阳性反应均可见。

 

 

 

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 (<2+) skin reaction to histamine and anticanine-IgE antiserum injection at treated and untreated sides of the thorax.

图2。复合图片，一期-在治疗组和未治疗组胸侧每日应用含0.0584%氢化可的松醋丙酯喷雾剂(皮乐美) 7和14天前后皮内注射组胺和抗犬- IgE抗血清后速发皮肤反应的GWS (a、b)和临床评估(c、d)。(a,b)数据代表了整体风疹评分的中位数和四分位数范围(IQR)。** p < 0.01;* p < 0.05。(c,d)数据表示在治疗侧和未治疗侧胸腔注射组胺和抗犬- IgE抗血清后皮肤反应呈阳性(2+及以上)和阴性(<2+)的犬的数量。

 

 

 

Discussion

讨论

In this study, the daily application of the novel diester GC spray containing 0.0584% hydrocortisone aceponate (Cortavance) significantly reduced immediate- and latephase histamine and anticanine-IgE-associated skin reactions at directly treated skin sites after 7 and 14 days. Moreover, an additional evaluation of immediate skin reactions by the conventional – subjective – 0 to 4+ scoring system revealed negative responses in most dogs during this period.

在这项研究中，每天使用含有0.0584%氢化可的松醋丙酯(皮乐美）的新型二酯GC喷雾，在7天和14天后，在直接治疗的皮肤部位显著减少了速发和迟发组胺和抗犬Ige相关的皮肤反应。此外，通过传统的-主观- 0到4+评分系统对速发皮肤反应进行的额外评估显示，在此期间，大多数犬的反应为阴性。

 

Reviewing the veterinary literature, we found only three studies that tested the effect of topical GC-containing formulations on IDT reactions. Two of them evaluated the effect of the low-potency GC 1% hydrocortisone leave-on conditioner ‘Resicort’ (Virbac, St. Louis, MO, USA). The first study used this 1% hydrocortisone-containing formulation twice daily for 6 weeks without showing a significant reduction in histamine-induced wheals. In the second placebo-controlled blinded study, the daily application of the same product for 3 days resulted in a significant reduction in immediate skin reactions following intradermal injection of anticanine-IgE antibodies, but the median decrease in diameter (1 mm; 11%) was deemed not to be of any clinical relevance. The clinical assessment of the immediate reactions performed in our study, however, revealed a lack of histamine and anticanine-IgE skin reactivity in the majority of dogs at sites treated with the active ingredient. This variation of results is probably due to the longer duration of treatment and the higher potency of the hydrocortisone aceponate used in our study.

回顾兽医文献，我们发现只有三个研究测试了外用含GC制剂对IDT反应的影响。其中两项研究评估了低效GC 1%氢化可的松免洗护发素Resicort (Virbac)的效果。第一项研究使用含有1%氢化可的松的配方，每天两次，持续6周，没有显示组胺诱导的风疹明显减少。在第二项安慰剂对照盲法研究中，连续3天每天使用相同的产品，在皮内注射抗犬- Ige抗体后，速发皮肤反应显著减少，但直径中位数减少(1 mm;11%)被认为没有任何临床相关性。然而，在我们的研究中进行的速发反应的临床评估显示，在使用活性成分治疗的部位，大多数犬缺乏组胺和抗犬- IgE皮肤反应性。这种结果的差异可能是由于在我们的研究中使用的氢化可的松醋丙酯的治疗持续时间较长和效力较高。

 

DeBoer and Cooley, using a moderately potent 0.015% triamcinolone solution-containing spray applied twice daily for 8 days (Genesis, Virbac USA), con-firmed a significant GC-induced reduction in reaction sizes after intradermal injections of mast cell degranulating substances (compound 48 ⁄ 80, anti-IgE monoclonal antibodies and substance P) compared with vehicle-treated sites. Interestingly, such reduction was not observed for histamine-induced immediate reactions. These findings were similar to observations reported in the human medical literature and supported the theory that topical GC are capable of reducing the number – and⁄ or activation – of dermal mast cells as well as the tissue content of histamine. Although mast cell numbers were not measured in any of the previous veterinary studies evaluating topical GC, DeBoer and Cooley were able to demonstrate a concurrent reduction in LPR-associated dermal cellular infiltrates after triamcinolone compared with vehicle applications. Similarly, in our study, we detected a significant reduction in LPR-associated dermal cellular infiltrate scores after a 14-day application of 0.0584% hydrocortisone aceponate at directly treated skin sites compared with baseline values, and this reduction correlated with the significant inhibition of macroscopic LPR. Despite the significant inhibition of LPR and previous data from human studies, the number of dermal mast cells in our study did not differ between baseline values and those obtained after 14 days of daily treatment on both treated and untreated skin sites. The absence of mast cell inhibition could have been due to our inability to identify degranulated mast cells in pretreatment samples resulting in the underestimation of the total number of these cells. Indeed, Pucheu-Haston et al.8 reported a clear decrease in the number of intact mast cells and an increase in the number of degranulated mast cells in tissues from LPR 24 h after injections of anticanine-IgE antiserum. Moreover, the same investigator identified a lack of appreciable difference in either the number or appearance of mast cells between pretreatment samples and samples after a 3-day administration of oral prednisolone.

DeBoer和Cooley使用含有0.015%曲安奈德溶液的中等效度喷雾，每天两次，持续8天(Genesis, Virbac USA)，证实皮内注射肥大细胞脱粒物质(化合物48⁄80，抗IgE单克隆抗体和P物质)与载体处理部位相比，GC诱导的反应大小显著减少。有趣的是，在组胺诱导的速发反应中没有观察到这种减少。这些发现与人类医学文献中报道的观察结果相似，并支持了外用GC能够减少真皮肥大细胞的数量和/或激活以及组织中组胺含量的理论。尽管肥大细胞数量在之前评估外用GC的任何兽医研究中都没有测量，但DeBoer和Cooley能够证明，与载体应用相比，曲安奈德应用后，LPR相关的真皮细胞浸润同时减少。同样，在我们的研究中，我们检测到与基线值相比，在直接治疗的皮肤部位，应用0.0584%氢化可的松醋丙酯14天后，LPR相关的真皮细胞浸润评分显著降低，并且这种降低与宏观的LPR显著抑制有关。尽管LPR有明显的抑制作用，而且之前的人类研究数据也显示，在我们的研究中，皮肤肥大细胞的数量在基线值和在治疗和未治疗的皮肤部位每天治疗14天后没有差异。肥大细胞抑制的缺失可能是由于我们无法在预处理样品中识别脱颗粒肥大细胞，导致低估了这些细胞的总数。事实上，Pucheu-Haston等人8报道，注射抗犬IgE抗血清24小时后，LPR组织中完整肥大细胞数量明显减少，脱颗粒肥大细胞数量增加。此外，同一研究人员还发现，在口服泼尼松龙3天后，预处理样本和口服泼尼松龙3天后的样本在肥大细胞的数量或外观上没有明显的差异。

 

 

 

Figure 3. Reduction in dermal inflammation: the pictures on the left represent the anticanine-IgE antiserum-induced late-phase reactions (LPRs) in two of the ten dogs before the application of glucocorticoid spray. In both dogs, there is moderate-to-severe, superficial perivascular dermatitis dominated by eosinophils and lymphocytes. Mild oedema is present in dog 1. The pictures on the right represent the anticanine-IgE antiseruminduced LPRs at the treated sides in the same two dogs after 14 days of treatment with the 0.0584% hydrocortisone aceponate spray. A marked reduction to an absence of perivascular inflammation is observed in both samples. H&E stain, ×200 magnification, bar = 100 um.

图3。皮肤炎症减轻:左图为10只犬中2只在使用糖皮质激素喷雾剂前的抗犬- IgE抗血清诱导的迟发反应(LPRs)。在这两只犬中，都有中度至重度的浅表血管周围皮炎，主要是嗜酸性粒细胞和淋巴细胞。1号犬出现轻度水肿。右图为0.0584%氢化可的松醋丙酯喷雾剂治疗14天后，同一两只犬治疗侧抗犬IgE抗血清诱导的LPR。在两个样本中观察到血管周围炎症的明显减少。H&E染色，×200放大，标尺 = 100 um。

 

 

 

Figure 4. Composite picture – histological evaluation of total dermal cellular infiltrate of 24-h late-phase reaction and dermal thickness before and after 14 days of daily application of 0.0584% hydrocortisone aceponate containing spray (CortavanceTM) at treated and untreated thoracic sides. Data represent the median and interquartile range (IQR) of the total dermal cell infiltrate per mm2 and skin thickness in um. ***P < 0.001; **P < 0.01; *P < 0.05.

图4。复合图片-每日应用0.0584%含氢化可的松醋丙酯喷剂(皮乐美)前后14天对治疗组和未治疗组胸侧24小时反应后期真皮细胞浸润总量和真皮厚度的组织学评价。数据代表每平方毫米真皮细胞浸润总量和皮肤厚度的中位数和四分位数范围(IQR)。*** p < 0.001;** p < 0.01;* p < 0.05。

 

 

Table 2. Dermal cellular infiltrate of 24-h late-phase reaction and dermal thickness prior to the 0.0584% hydrocortisone aceponate application and after 14 days of daily application at directly treated and untreated thoracic sides

表2。应用0.0584%氢化可的松醋丙酯前和每日应用14天后，直接治疗组和未治疗组胸侧24小时反应后期真皮细胞浸润和真皮厚度

 

 

 

Figure 5. Composite picture, phase 2 – duration of inhibition of immediate skin reactions by a 0.0584% hydrocortisone aceponate spray at directly treated and untreated skin sites. (a) Global wheal scores (GWS) after intradermal injection of histamine before (day )1), after 7 days of daily application of 0.0584% hydrocortisone aceponate spray (Cortavance) and 7, 14, 21, 28 days after discontinuation of the treatment (days 14, 21, 28, 35). Data represent the median and interquartile range (IQR) of the GWSs. ***P < 0.001; **P < 0.01; *P < 0.05. (b) Data represent number of dogs with positive (2+ and more) and negative (<2+) skin reaction to histamine injection at treated and untreated sides of the thorax.

图5。复合图，第二阶段- 0.0584%氢化可的松醋丙酯喷雾对直接治疗和未治疗皮肤部位的速发皮肤反应的抑制持续时间。(a)在(第1天)、每天使用0.0584%氢化可的松喷雾剂(皮乐美) 7天后，以及停药后7、14、21、28天(第14、21、28、35天)皮内注射组胺后的总体风疹评分(GWS)。数据表示GWS的中位数和四分位数范围。*** p < 0.001;** p < 0.01;* p < 0.05。(b)数据表示治疗侧和未治疗侧胸腔组胺注射皮肤反应阳性(2+及以上)和阴性(<2+)的犬的数量。

 

Altogether, the above-mentioned observations from our study confirmed that the 0.0584% hydrocortisone aceponate spray represents a potent topical GC potentially suitable for treatment of skin areas affected by AD. Indeed, hydrocortisone aceponate (0.1% w ⁄ w) is categorized as a moderately potent GC in the medical literature, as it exhibits an anti-inflammatory effect comparable with that of betamethasone-17-valerate, albeit with fewer side effects that allow its usage for larger body areas in children.

总之，我们研究的上述观察结果证实，0.0584%氢化可的松醋丙酯喷雾是一种有效的外用GC，可能适用于治疗AD患病的皮肤区域。事实上，氢化可的松醋丙酯(0.1% w / w)在医学文献中被归类为中等效力的GC，因为它显示出与倍他米松-17-戊酸相当的抗炎作用，但副作用更少，允许其用于儿童较大的身体区域。

 

The unique properties of this novel diester GC brought up the question of whether this medication could be used to alleviate signs of AD in canine patients awaiting the performance of IDT at an untreated skin site. Indeed, in a randomized placebo-controlled study, human patients with hay fever hypersensitive to pollen allergens were treated on one arm with 0.05% clobetasol 17-propionate – a very potent topical GC – twice daily for 1 week, while the other arm received an identical placebo. A significant reduction in prick test immediate reaction and LPR to pollen allergens was found on GC-treated arm compared with baseline and control-treated sites. Interestingly, the wheal diameter of histamine reactions was not reduced with clobetasol compared with control.

这种新型二酯 GC的独特性质提出了一个问题，即这种药物是否可以用于缓解患犬在未经治疗的皮肤部位等待IDT的症状。事实上，在一项随机安慰剂对照研究中，对花粉过敏原过敏的人类花粉热患者在一只手臂上接受0.05%氯倍他索17-丙酸治疗，每天两次，持续一周，这是一种非常有效的外用GC，而另一只手臂则接受相同的安慰剂。与基线和对照处理部位相比，GC处理组对花粉过敏原的点刺试验速发反应和LPR显著降低。有趣的是，与对照组相比，氯倍他索组胺反应的风疹直径没有减少。

 

In our study, dogs sprayed daily with the 0.0584% hydrocortisone aceponate spray in the axillary and inguinal regions and on one side of the thorax exhibited reduced histamine and anticanine-IgE antiserum associated IDT reactions after 7 days of application at distant untreated sites. This reduction, however, was not statistically significant. Clinical assessment using the subjective 0 to 4+ scoring system showed that an average of 50% and 40% of dogs had positive (‡2+) reactions to histamine and anticanine-IgE injections respectively. The prolonged application of the spray 1 week beyond the recommended time led to a further statistically significant decrease in GWS after both types of injection. At that time, only 30% and 20% of dogs had positive immediate skin reactions to intradermal injections of histamine and anticanine-IgE antiserum administered at untreated thoracic sides respectively. Additionally, for both time points, the LPRs (GLS) were significantly reduced. This inhibition of LPR scores was accompanied by a reduction, although not statistically significant, in total cell, and eosinophil counts in samples from untreated sites after intradermal injection of anticanine-IgE antibody.

在我们的研究中，每天在犬的腋窝和腹股沟区域以及胸部一侧喷洒0.0584%氢化可的松醋丙酯喷雾剂，在远处未治疗部位应用7天后，组胺和抗犬- IgE抗血清相关IDT反应降低。然而，这种减少在统计学上并不显著。采用主观0 ~ 4+评分系统的临床评估显示，平均50%和40%的犬分别对组胺和抗犬Ige注射有阳性反应(‡2+)。在超过推荐时间1周后延长喷雾剂的使用，两种类型的注射后GWS的进一步统计学显著降低。当时，只有30%和20%的犬在未经治疗的胸侧分别皮内注射组胺和抗犬- IgE抗血清后立即出现阳性皮肤反应。此外，在两个时间点，LPRs (GLS)均显著降低。这种LPR评分的抑制伴随着皮内注射抗犬IgE抗体后未经处理部位样品中总细胞计数和嗜酸性粒细胞计数的减少，但没有统计学意义。

 

One can only hypothesize the mechanism for the antiinflammatory effect seen at the distant untreated thoracic sides in our dogs. A simple explanation would be that the hydrocortisone aceponate was systemically absorbed without sufficient degradation and subsequently generated a generalized anti-inflammatory effect at distant untreated areas. This theory, however, cannot be proved by this study due to the lack of laboratory data (pharmacology, biochemistry, haematology and ACTH stimulation tests) in tested dogs. However, it is known that the hydrocortisone aceponate, transformed into potent 17-mono-ester (hydrocortisone 17-propionate) in the epidermis, is heavily metabolized to the less active 21- mono-ester (hydrocortisone 21-propionate) as soon as the active molecule reaches the dermis. The weaker metabolites are then progressively released into the blood where they undergo further degradation and inactivation resulting in minimal systemic side effects. This lack of systemic effect is further supported by an unpublished experimental study in which the once daily application of the same 0.0584% hydrocortisone aceponate spray on six healthy beagles for 14 days did not cause any clinically or statistically significant changes in haematological and biochemical values and the ACTH stimulation test results were comparable between placebo and actively treated groups. A similar observation was reported in human medicine, where hypothalamic-pituitary-adrenal suppression appeared to be a rare complication in young people treated with mild to moderately potent topical diester GC for short as well as long durations.

我们只能假设在我们的犬的远端未治疗的胸侧看到的抗炎作用的机制。一个简单的解释是氢化可的松醋丙酯在没有充分降解的情况下被全身吸收，随后在远处未治疗的区域产生了广泛的抗炎作用。然而，由于缺乏试验犬的实验室数据(药理学、生物化学、血液学和ACTH刺激试验)，本研究无法证明这一理论。然而，众所周知，氢化可的松醋丙酯在表皮中转化为强效的17-单酯(氢化可的松17-丙酸酯)，一旦活性分子到达真皮，就会被大量代谢为活性较低的21-单酯(氢化可的松21-丙酸酯)。然后，较弱的代谢物逐渐释放到血液中，在那里它们进一步降解和失活，导致最小的全身副作用。一项未发表的实验研究进一步支持了这种缺乏系统性作用的说法，该研究表明，在6只健康的比格犬身上每天使用一次相同的0.0584%氢化可的松醋丙酯喷雾剂，持续14天，没有引起血液学和生化值的任何临床或统计学上的显著变化，ACTH刺激试验结果在安慰剂组和积极治疗组之间是相当的。在人类医学中也报道了类似的观察结果，下丘脑-垂体-肾上腺抑制似乎是一种罕见的并发症，在使用轻度至中度有效的外用二酯GC治疗的年轻人中，无论是短期还是长期。

 

Other possible explanations for the observed distant anti-inflammatory effect at untreated sites could be the cutaneous diffusion of the active drug from treated to untreated areas or the accidental exposure of untreated skin to the active medication due to dog–dog contact in the runs. The latter hypothesis, however, appears to be less likely because of the rapid absorption of the spray after application with only minimal residual effect at the skin surface.  Finally, we cannot exclude that the dogs may have licked some of the active product on their groin, thereby producing a systemic rather than localized cutaneous anti-inflammatory effect.

对于在未治疗部位观察到的远距离抗炎作用，其他可能的解释可能是活性药物从治疗部位扩散到未治疗部位，或者由于犬在跑步中接触而使未治疗的皮肤意外暴露于活性药物。然而，后一种假设似乎不太可能，因为喷雾在应用后迅速吸收，在皮肤表面只有最小的残留效应。最后，我们不能排除犬可能舔了一些腹股沟上的活性产品，从而产生了全身而不是局部的皮肤抗炎作用。

 

An unexpected finding in our study was the visible skin atrophy in the axillary and inguinal regions of most dogs during phase 1, which was associated with a significantly reduced dermal thickness measured histologically at treated thoracic sides. This observation was surprising in the light of the novel diester GC considered to have minimal atrophogenic potential in human medicine due to the limited toxicity to epidermal cells and effect on collagen synthesis. Because of this low capacity to induce skin atrophy, these novel topical diester GC are used to treat sensitive areas including facial or scrotal skin or large body areas in children. Interestingly, a tolerance study completed prior to the registration of this 0.0584% hydrocortisone aceponate spray in Europe revealed the lack of significant difference in skin thickness between treated and untreated beagles after 14 days of application of active and placebo sprays respectively.31 Similarly, a recently finished study from the same group yielded an absence of skin atrophy at directly treated areas after several weeks of application of the same product.

在我们的研究中，一个意想不到的发现是，在第一阶段，大多数犬的腋窝和腹股沟区域可见皮肤萎缩，这与经治疗的胸侧皮肤厚度的组织学测量显着减少有关。由于对表皮细胞的毒性有限和对胶原合成的影响，新型二酯GC被认为在人类医学中具有最小的萎缩潜力，因此这一观察结果令人惊讶。由于这种诱导皮肤萎缩的能力较低，这些新型的外用二酯 GC用于治疗敏感区域，包括面部或阴囊皮肤或儿童的大片身体区域。有趣的是，在欧洲注册0.0584%氢化可体酸钠喷雾剂之前完成的一项耐受性研究显示，在分别使用活性喷雾剂和安慰剂喷雾剂14天后，接受治疗和未接受治疗的比格犬之间的皮肤厚度没有显著差异同样，最近完成的一项来自同一组的研究表明，在使用相同的产品几周后，直接治疗的区域没有皮肤萎缩。

 

The reason for the significant reduction in the dermal thickness observed in our dogs is unknown. Altered dermal metabolism of the hydrocortisone aceponate and increased absorption of the active drug due to the small size of our dogs resulting in a greater ratio of surface area to body weight could have played a role in the observed changes. Indeed, the higher surface area-to-body volume ratio in infants and young children is considered to be responsible for increased risk of systemic side effects in this population.  Additionally, we used a line of Maltese–beagle atopic dogs, and not normal laboratory beagles, and it is possible that our atopic dogs may have a defective epidermal barrier that could lead to increased absorption of GC in the dermis. Indeed, a pilot study showed that dogs with spontaneous AD may have a defective lipid barrier.34 At this time, however, we do not know whether or not our Maltese–beagle atopic dogs exhibit abnormal skin barrier. It is worth noting that this study design did not permit to determine whether the degree of dermal atrophy recorded in our dogs would be present after a 7-day application, the per label recommended duration of treatment.

在我们的犬身上观察到的皮肤厚度显著减少的原因尚不清楚。由于我们的犬体型小，皮肤对氢化可的松醋丙酯的代谢发生了改变，活性药物的吸收增加，导致表面积与体重的比例更大，这可能是观察到的变化的原因。事实上，婴幼儿较高的体表面积与身体体积比被认为是导致这一人群系统性副作用风险增加的原因。此外，我们使用了一系列马尔济斯-比格犬，而不是正常的实验室比格犬，我们的特应性皮炎犬可能具有缺陷的表皮屏障，这可能导致真皮中GC的吸收增加。事实上，一项初步研究表明，患有自发性AD的犬可能有缺陷的脂质屏障然而，在这个时候，我们不知道我们的马尔济斯-比格犬特应性皮炎犬是否表现出异常的皮肤屏障。值得注意的是，这项研究设计不允许确定我们的犬在使用7天后是否会出现皮肤萎缩的程度，这是每个标签推荐的治疗时间。

 

Importantly, a 7-day application of the spray once daily in the axillary and inguinal region and on one side of the thorax caused non-significant reductions in immediate skin reactions at untreated areas, with a clinically negative skin test (<2+) in one-third of our dogs. This information has practical relevance for dermatologists wishing to perform IDT in dogs treated with this novel diester GC-containing spray. In case of negative or unacceptably weak reaction to IDT with positive controls, the application of the spray should be discontinued and the patient should be tested at least 2 weeks later.

重要的是，在腋窝和腹股沟区域以及胸腔一侧每天使用一次喷雾剂，持续7天，未治疗区域的速发皮肤反应没有显著减少，三分之一的犬的临床皮肤试验呈阴性(<2+)。这一信息具有实际意义的皮肤科医生希望进行IDT治疗的犬与这种新型的含二酯GC喷雾。如果IDT阳性对照阴性或反应弱到不可接受的程度，则应停止喷雾剂的应用，并至少在2周后对患犬进行检测。

 

However, clinicians should bear in mind that the prolonged application of the spray beyond the time recommended by the manufacturer further decreased histamine reactions in our dogs, suggesting that prolonged treatment should lead to longer pre-IDT withdrawal times. In conclusion, the 0.0584% hydrocortisone aceponate spray was shown to be a potent GC that exerts some influence on IDT reactions performed at distant untreated skin areas even after short (7 days) treatment duration. While clinicians may still be able to perform IDT in most dogs when they are still being treated, a withdrawal time of 2 weeks is recommended to assure lack of effect on IDT reactions. Furthermore, if this spray were to be used for longer than 7 days, clinicians should be aware that IDT reactions are likely to be affected, even at untreated areas, and that withdrawal times are likely to be longer than 2 weeks.

然而，临床医生应该记住，长时间使用喷雾剂超过制造商推荐的时间会进一步降低我们犬的组胺反应，这表明延长治疗应该导致更长的IDT前停药时间。综上所述，0.0584%氢化可的松醋丙酯喷雾剂被证明是一种有效的GC，即使在短时间(7天)治疗后，也能对远距离未治疗皮肤区域发生的IDT反应产生一定影响。虽然临床医生可能仍然能够对大多数仍在接受治疗的犬进行IDT，但建议停药时间为2周，以确保对IDT反应没有影响。此外，如果这种喷雾剂的使用时间超过7天，临床医生应该意识到IDT反应很可能受到影响，即使在未经治疗的地区，并且停药时间可能超过2周。

 

杨先生