Prospective Evaluation of the Use of Lokivetmab in Dogs With Atopic Dermatitis in Brazil

Background: Lokivetmab (Cytopoint®, Zoetis) is a caninized anti-IL-31 monoclonal antibody licensed for treating clinical manifestations of canine atopic dermatitis in varying degrees of severity. The objective of this 30-day prospective study was to evaluate the level of improvement in cases of atopic dermatitis after the application of lokivetmab, using the Canine Atopic Dermatitis Extent and Severity Index (CADESI-4) variables, Visual Analogue Scale (VAS) of pruritus, owner assessment of the condition, and incidence of adverse events. A total of 110 animals recruited from two private clinics in the State of São Paulo each received 2 mg of lokivetmab/kg of body weight by subcutaneous injection on day 0. Dogs were observed by owners who provided reports of their observations by telephone or e-mail on days 2 and 14 and were observed by clinicians on day 30. Results: Baseline mean VAS of pruritus was 7.68 (median = 7), decreasing to 3.82 (median = 2) by day 2 (P<0.001). CADESI-4 variables decreased from 36.5 at baseline to 16.3 on day 30 (P<0.001). Reports of treatment responses submitted by owners and evaluation of CADESI-4 variables after treatment were signicantly similar (P<0.001). Owners rated the response to treatment as good to excellent for 75% of the dogs. Adverse events, including vomiting, diarrhea, and/or lethargy, were observed in 18 dogs (16.4%). All events were transient, and none required treatment. Conclusions: Based on ndings in this study, lokivetmab was determined to provide a rapid and effective response for control of canine atopic dermatitis. Lokivetmab was well tolerated with only mild and transient adverse events observed.


Background
Atopic dermatitis (AD) is a chronic allergic and in ammatory disease, associated with cytokines. The age of onset is generally around 6 months to 3 years of age, and multiple breeds have a genetic predisposition to AD, with the incidence within those breeds variable according to the genetic pool and the region [1]. Pruritus is the primary characteristic of the condition, with secondary lesions occurring around the face, ears, ventral abdomen, exor aspects of elbow, carpal, and tarsal joints, interdigital skin, and perineal area [2][3][4][5]. It is caused by different allergens called atopens and can be controlled but cannot be cured [6].
Dogs react to the condition in several ways, including scratching, biting, licking, or rubbing. If intense, the itching often leads to episodes of self-trauma [3,7]. The Visual Analogue Scale (VAS) of pruritus created by Rybnicek et al [8] is a vertical line with descriptions of the levels of symptom severity ranging from 0 to 10. The scale is applied based on the dog owner's assessment of the level of pruritus.
Interleuken-31 (IL-31) is responsible for inducing pruritus and plays an important role in the pathogenesis of canine AD [4,9]. In one evaluation of several antipruritic therapeutics in an IL-31-induced model, it was possible to determine relative speed of onset of reduced IL-31-induced pruritis as well as duration of action [10]. Neutralization of IL-31 with a monoclonal antibody (mAb) has been found to provide relief from itching related to AD and in ammation, with a greater margin of safety than for less targeted therapies such as corticosteroids or cyclosporine [10][11][12].
Lokivetmab is a caninized mAb that binds selectively and neutralizes IL-31 [4,10,13]. In one study, a signi cant reduction in pruritus was observed for at least 1 month following administration of a single subcutaneous (SC) injection of lokivetmab at 0.5 mg/kg or 2 mg/kg compared to placebo (P<0.05), and the level of response duration increased with additional doses [4]. In a clinical trial in client-owned dogs with AD, monthly administration of lokivetmab at 2 mg/kg SC (actual doses ranged from 1.0-3.3 mg/kg) was noninferior to daily administration with oral cyclosporine at 5 mg/kg for pruritus reduction [13].
Mean Canine Atopic Dermatitis Extent and Severity Index (CADESI-03) scores were not signi cantly different between the treatments at any time points [13]. The bene ts of this mAb therapy include its rapid onset of activity, infrequent dosing, lack of age restriction for its use, safety, and compatibility with other drugs [11,14]. Lokivetmab is approved in several countries for treatment of canine AD at a minimum dose of 1 or 2 mg/kg SC, and with expected effectiveness for at least 1 month [11,15,16].
The objective of the present study was to evaluate the e cacy of lokivetmab (2 mg/kg) in the control of canine AD through the clinical evolution of lesions using CADESI-4 and the reduction of pruritus measured by VAS. The safety assessment included recording of all adverse events. Dog owners also provided their perception of the treatment response.
The majority of dogs (96.4%) had previously been treated with other antipruritic medications, without satisfactory success. Treatments given for control of itching and in ammation as reported by the owners are shown in Fig. 2. The medications reported with the highest frequency were de azacort, oclacitinib, prednisolone, and cyclosporine. Some dogs had received 2 or more medications.
A total of 4 dogs were withdrawn during the study. Reasons for withdrawal included diagnosis with giardiasis, emergency orthopedic surgery, onset of an adverse event, and worsening of pruritus (at the tutor's request).
VAS data are shown in Table 1. The baseline mean VAS score was 7.68 and decreased by approximately 50% to 3.82 at day 2 (P<0.001). Scores on days 14 and 30 remained signi cantly lower than those on day 0 (P<0.001). Median scores followed a similar pattern, ranging from 7 on day 0 to 3 on days 14 and 30. The mean CADESI-4 score on day 30 (16.3) was less than half of the baseline mean score (36.5), a statistically signi cant reduction (P <0.001) ( Table 2). There was also a similar reduction in the median score from 25.5 on day 0 to 10.5 on day 30. There was a positive and signi cant correlation (P<0.001) for the change from baseline for VAS and CADESI-4 scores (Spearman's r = 0.593) (Fig. 3). Overall, 75% of owners rated the e cacy of treatment good to excellent (Fig. 4).
Bacterial foliculitis with Staphylococcus spp were diagnosed in 38 dogs (34.5%) at baseline (Table 3), and 21 of the total population (19.8%) were positive for Staphylococcus spp on day 30 (P = 0.024 compared with day 0), which represents 55.3% of the 38 dogs positive for Staphylococcus spp on day 0. A total of 7 dogs (6.7%) negative for any bacterial or fungal skin infection on day 0 developed infection with Malassezia spp during the study. Also, 2 additional dogs (1.9%) that were negative for any skin infection on day 0 were positive for mixed infection with Staphylococcus spp and Malassezia spp on day 30. treatment with lokivetmab at 2 mg/kg SC, with 8 dogs (7.4%) experiencing an event on the day of treatment, 2 (1.9%) reported the next day, and 8 (7.4%) reported on day 2 ( Table 4). The events were transient and considered to be mild, without the need for additional support treatment.

Discussion
Of the 110 dogs diagnosed with atopic dermatitis in the study, the majority (84.5%) were of a de ned breed. This nding is consistent with previously published data [7,17], showing that dogs of many de ned breeds are genetically predisposed to developing atopic dermatitis, likely due to a combination of genetic characteristics as well as certain environmental factors [3]. The most frequent breed represented in the present study was shih tzu, which is the most popular dog breed in Brazil [18]. Body weight of the animals ranged from 2.5 to 39.8 kg (mean 13.8 kg), with more than half of the dogs each weighing between 2.5 and 10 kg, demonstrating a current predominance of miniature and small client-owned dogs in Brazil [18]. Approximately 60% of the dogs in this study were females. Earlier studies do not predict a predisposition for the disease according to sex [5,13]. Although there were more female dogs than male dogs presented with atopic dermatitis in the present study, no conclusions were drawn regarding this observation.
A limitation of this study is the absence of a control group. Initially, the study was to be carried out as a randomized clinical trial with a test group (lokivetmab) and a negative (placebo) or positive (corticosteroids) control group. However, with regard to a negative control group, we faced an ethical dilemma when we determined there was a severe degree of pruritus and lesions from itching and rubbing. Thus, it was decided that it would be very di cult to convince the owners that their dog might only receive a placebo for its severe infection. The other option of using a corticosteroids as a positive control treatment could be problematic for dogs with a long history of using prednisolone or other steroid treatments associated with adverse reactions.
Based on American laboratory dose titration and clinical dose determination studies with different dosages [4,12], the minimum effective dosage was determined to be 2 mg/kg. Pharmacokinetic studies also predicted the 2 mg/kg dose would have mAb concentrations above EC 50 for 28 days [12]. In the present study, enrollment of animals with more severe AD (VAS ≥7 and CADESI-4 ≥20) provided an opportunity to evaluate the level of e cacy over the 30-day period for these cases, noting that many other therapeutic options do not provide prolonged e cacy, as demonstrated by the daily administration of cyclosporine over 28 days of evaluation, compared with a single SC administration of lokivetmab on day 0 [13]. The 30-day e cacy period also provides a sensible, convenient period for dog owners to increase treatment adherence and compliance.
The time it takes for treatment to demonstrate its effect is an extremely important factor. In the present study, the mean VAS on day 0 was 7.68, which demonstrated a signi cant reduction within 2 days after treatment, remaining stable at the improved level throughout the study. Similarly, there was a satisfactory reduction in severity of lesion scores. CADESI-4 scores at baseline were reduced by 55% on day 30, in agreement with the study by Michels et al [4] where CADESI-4 scores were improved as early as 7 days after treatment.
There was relatively high variability among the animals in the severity of lesions at baseline. Despite the heterogeneity of lesion severity, the enrollment criteria required each dog to have a CADESI-4 score ≥20; therefore, none of the dogs had a skin condition on day 0 that would be considered mild. When evaluating Spearman's correlation between VAS and CADESI-4, there was a positive and signi cant correlation between these 2 variables, which indicates that as the pruritus improved, so did the skin lesions. This phenomenon probably occurs due to an indirect anti-in ammatory effect of lokivetmab which, despite speci cally blocking IL-31 (pruritic) rather than targeting other agents of the in ammatory cascade, its role in reducing self-trauma from scratching is reduction of in ammation. This therapeutic aspect of lokivetmab was also highlighted by Moyaert et al. [13].
Associated with clinical improvements, there was also a reduction in Staphylococcus spp infections present on day 30. However, 7 dogs developed Malassezia spp infections despite the use of the antiseptic shampoos, and 2 animals developed a mixed infection of Staphyloccocus spp and Malassezia spp). Lokivetmab treatment appeared to facilitate a reduction of the bacterial infection, but not for the fungal infection. The reduction in bacterial infections was likely due to the reduction in the act of scratching, but no a plausible explanation for the appearance of Malassezia infections. Future studies will include evaluations to attempt to determine the role of AD and treatment, if any, in the development of these fungal infections.
The adverse events observed following administration of lokivetmab were mild and required no additional treatment or drug intervention. Of the 4 patients that were withdrawn from the present study by the owner, only 1 was due to diarrhea and severe vomiting on day 0, following administration of lokivetmab. The appearance and incidence of vomiting, diarrhea, or lethargy reported in the present study are similar to those reported previously in randomized clinical trials of e cacy and safety of lokivetmab in client-owned dogs, including one large safety study in dogs with reported comorbidities and only minimal restrictions for concurrent medications during the study [4,13,14]. As stated in the conclusions of the researchers in those earlier controlled studies, we found that lokivetmab demonstrated a good safety pro le when administered by SC injection to dogs with atopic dermatitis.
OGATE is a relevant, but subjective, tool in assessing adherence and success of the therapeutic regimen [19]. In our sample, 75% of the dog owners rated the treatment as good to excellent, indicating a high level of satisfaction. Despite the majority of positive responses and the signi cant clinical improvement of pruritus and lesion scores achieved in this study, ares related to exposure to allergens are common and may cause some owners to feel that treatment did not provide the expected response [17]. In their development of a core outcome set for evaluating and comparing outcomes of treatments administered to dogs with atopic dermatitis, Olivry and others acknowledge the subjective nature of OGATE, but advise that it is a useful criterion when assessed in concert with results of clinical assessments [19].
It may be possible to provide acceptable e cacy, perhaps with even fewer adverse reactions, with lower dosages of lokivetmab. Results of one clinical study indicated that lokivetmab at 0.5 mg/kg provided signi cant reductions in lesion scores, although the treatment effect was observed later, and the duration of e cacy was shorter than for the dogs treated with lokivetmab at 2 mg/kg [4]. Future studies may be undertaken to determine whether lower doses will provide adequate e cacy against the clinical effects of AD for dogs in Brazil.

Conclusion
Lokivetmab administered SC at 2 mg/kg to dogs with AD was effective in controlling signs and symptoms of canine AD, providing signi cant improvement of pruritus and clinical lesions and high level of owner satisfaction, with a low incidence of adverse events.

Overview
This study protocol was approved and followed the ethical principles of CEUA -UNISA and Plataforma Brasil. The owners or handlers responsible for care of the animals gave written informed consent for each dog to participate in the study.
This was a prospective study conducted from May 2019 to July 2020, with dogs followed for 30 days after treatment. The dogs enrolled were patients from 2 veterinary clinics specializing in veterinary dermatology: Cãopanheiro (Sorocaba-SP) and Dermatoclínica (São Paulo-SP). History was documented for each dog, and each dog was given a complete physical examination. Dermatological examinations were performed when appropriate, including parasitological examination of skin scrapings, cytology for bacteria and/or yeast, microbiological cultures, or skin biopsies.

Inclusion criteria
Dogs of any breed or sex, weighing at least 2 kg, and diagnosed with AD according to meeting at least 6 of the 8 Favrot's 2010 criteria [5] and having a minimum CADESI-4 of 20 and a minimum VAS score of 7 were eligible to be enrolled in the study. Dogs diagnosed with cutaneous adverse food reactions remained on the same diet throughout the study. All dogs were free of ectoparasites on day 0 and prevention of new infestations was maintained throughout the study.
Dogs currently receiving other treatments for AD must have discontinued those treatments as follows before receiving treatment in the study: oclacitinib: 3 days; oral corticosteroids or cyclosporine: 4 weeks;antimicrobial therapy;. Dogs with super cial pyoderma were able to use topical antiseptic formulations for 15 days before and into the study period as needed. to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Ethics declarations
This study protocol was approved and followed the ethical principles of CEUA -UNISA, Plataforma Brasil and is reported in accordance with ARRIVE guidelines 2.0. The owners or handlers responsible for care of the animals gave written informed consent for each dog to participate in the study. Treatments were administered by veterinarians specializing in treatment of dermatological conditions in dogs and other animal species.

Availability of Date and Materials
The datasets analysed during the current study are not publicly available due to the presence of additional data that were not used in the present study. Probably it will be useful for future publications, but are available from the corresponding author on reasonable request.  Distribution of responses provided by owners regarding their perception of treatment e cacyn in dogs with AD treated with lokivetmab day 0(N = 106).