A randomised, double-blinded, controlled trial to determine the efficacy of combined therapy of oclacitinib and marine oil extract PCSO-524 in dogs with atopic dermatitis
Authors:
INTRODUCTION
The pathogenesis of canine atopic dermatitis (cAD) is complex and multifactorial, and usually requires life-long medical care in order to maintain an acceptable quality of life.1 During the past decade, a number of proinflammatory and pruritogenic cytokines have been identified as targets for therapy of cAD. However, even with the advent of new-generation medications, such as oclacitinib and lokivetmab, which have good efficacy and a relatively low risk of adverse reactions, multimodal therapeutic strategies are recommended for management of this disease as reviewed in published treatment guidelines.2, 3
Polyunsaturated fatty acids (PUFAs) appear to have a synergistic effect when used in combination with antihistamines, corticosteroids or ciclosporin, allowing for improved control of clinical signs or dose reduction of medication.4 PCSO-524 (Antinol; V and P Co., Ltd) is a patented marine oil extract from the New Zealand green-lipped mussel. The mixture is composed of several types of lipids, including sterol esters, triglycerides, free fatty acids, free sterols and polar lipids. It contains up to 91 fatty acids, with DHA and EPA being the most abundant types of omega-3 polyunsaturated fatty acids.5 In addition, furan fatty acids (containing a furan ring) in PCSO-524 are considered to possess potent anti-inflammatory properties via the inhibition of both the cyclooxygenase-2 and 5-lipooxygenase pathways, which metabolise arachidonic acid into pro-inflammatory prostanoids and leukotrienes.6, 7 Anecdotal feedbacks from pet owners suggested that PCSO-524 might alleviate the clinical signs of cAD, although the product was developed as an anti-inflammatory for canine osteoarthritis.8 To the best of the authors’ knowledge, there are no published data to evaluate the clinical efficacy of PCSO-524 in dogs with cAD. We hypothesised that combining PCSO-524 with oclacitinib would provide a synergistic effect, particularly when reducing the dosage of oclacitinib in dogs with cAD.
MATERIALS AND METHODS
Animals and diagnostic procedures
The study was conducted in compliance with the Procedures of Good Clinical Practice guidelines issued by the Japanese Ministry of Health, Labour and Welfare. Owners’ written consent for their dogs to participate was obtained before the study.
Client-owned dogs with chronic pruritus were recruited in two veterinary facilities between September 2019 and January 2020. The diagnosis of cAD was fulfilled with the clinical criteria proposed by Favrot et al. and followed by compatible history, and other pruritic dermatoses were ruled out based on the standard guidelines.9, 10 Screening examinations consisted of Wood’s lamp examination, multiple trichograms and skin scrapings to rule out ectoparasites and cytological evaluation to screen for microbial infections; these were identified by intracellular bacteria based on multiple cytological examinations. Ruling out Malassezia overgrwoth was done in the same manner. Exclusion criteria were systemic and dermatological diseases other than AD. Routine isoxazoline ectoparasiticide prophylaxis was administered during the study. An elimination diet trial was performed on all dogs enrolled in the study and patients that responded either partially or completely after eight weeks of the trial were excluded. Before inclusion, anti-inflammatory and antipruritic medications, such as glucocorticoids (oral or topical), ciclosporin and antihistamines were discontinued for at least two weeks. Dogs treated with long-acting glucocorticoids were excluded. In addition, all topical medications were withdrawn for the same period of time and also prohibited during the whole study. The dogs were continued on their regular diet throughout the study.
Treatment allocation and study protocol
Each dog was randomly assigned to one of two groups using a computer-generated randomisation table. Dogs either received oclacitinib combined with PCSO-524 or were administered oclacitinib combined with the control, sunflower oil capsules. Each PCSO-524 capsule contained a proprietary mix of PCSO-524 (50 mg), olive oil (100 mg) and vitamin E (D-alpha-tocepherol; 0.225 mg). The control capsules contained sunflower oil (139.5 mg/capsule), water and glycerin. The capsules in the PCSO-524 and control groups both contained granules of the same colour and size, so that the owners and investigators were unable to identity the product. Dogs were assessed on the day of enrolment, which was classified as Day (D)0, and then on D14, D28 and D42 by the same veterinary surgeon. All patients received oclacitinib at an initial dose of 0.4–0.6 mg/kg twice daily for 14 days, followed by once daily until D42. Concurrently, dogs in both the PCSO-524 and sunflower oil groups were given capsules twice a day for the first two weeks and once a day thereafter until D42, with the dosage determined based on their body weight (one capsule per 10 kg). Clinical evaluation and pruritus were determined by Canine Atopic Dermatitis Extent and Severity Index, 4th iteration4 (CADESI-04) and a validated owner-assessed pVAS at each visit.4 Moreover, trans epidermal water loss (TEWL) was measured by a closed chamber device (VAPOSCAN; Asahi-Techno Co. Ltd) at D0 and each time point. Dogs were allowed to acclimatise to the examining area for 30 min before the TEWL measurement. Measurements were made on the inguinal region in a controlled environment (temperature range 20–25°C and humidity range 50%–70%) and repeated three times at each site. The mean of six measurements was used as a representative value. However, if the variation between three measurements exceeded 15%, the results were deemed unreliable and the measurement had to be redone. Adverse events were reported by the owner and if any additional medical treatment was needed, the dog would be withdrawn from the study.
Statistical analysis
All statistical analyses were calculated based on STATVIEW software v5.0 (HULINKS, Inc.). For all statistical analysis, the significant level was set at 5% (p ≤ 0.05) for the adjusted p-value. The pVAS, CADESI-04 and TEWL of two groups before and after treatment were analysed by a paired t-test, and intergroup differences were analysed with Students t-test.
RESULTS
A total of 17 atopic dogs were recruited in the present study. Group assignment, breed, sex, age, weight and assessment of clinical changes are summarised in Table 1. All dogs completed the study and no adverse events over the course of the study were reported by the owners. Nine dogs were in the PCSO-524 group and eight dogs were in the control group.
| Group | Age (month) | Body weight (kg) | Breed | Sex | Clinical score | Pruritus score | Trans epidermal water loss (g/m2/h) | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| D0 | D14 | D28 | D42 | D0 | D14 | D28 | D42 | D0 | D14 | D28 | D42 | |||||
| PCSO-524 | 177 | 3.8 | Papillon | FS | 43 | 10 | 10 | 8 | 4 | 0 | 0 | 0 | 8.2 | 5.4 | 4.2 | 4 |
| PCSO-524 | 106 | 6.2 | Miniature Dachshund | MC | 49 | 46 | 36 | 31 | 6 | 3 | 1 | 2 | 16.4 | 6.7 | 6 | 6 |
| PCSO-524 | 76 | 8.3 | French bulldog | FS | 65 | 14 | 12 | 12 | 7 | 1 | 1 | 1 | 10.6 | 4.1 | 2.3 | 2.1 |
| PCSO-524 | 124 | 4.2 | Toy poodle | FS | 44 | 12 | 8 | 8 | 6 | 1 | 1 | 2 | 8.5 | 3.1 | 2.5 | 2.6 |
| PCSO-524 | 53 | 5.4 | Shiba inu | MC | 38 | 16 | 10 | 8 | 6 | 1 | 0 | 0 | 12.1 | 4.2 | 2.3 | 1.8 |
| PCSO-524 | 104 | 2.5 | Chihuahua | FS | 40 | 18 | 6 | 2 | 7 | 1 | 0 | 0 | 18.2 | 10.2 | 4.8 | 3.6 |
| PCSO-524 | 76 | 7.5 | French bulldog | FS | 41 | 13 | 10 | 8 | 5 | 2 | 1 | 1 | 11.1 | 4.2 | 3.7 | 2.4 |
| PCSO-524 | 28 | 12.4 | Miniature bulldog | FS | 34 | 13 | 13 | 9 | 7 | 1 | 0 | 0 | 17.5 | 7.8 | 9.5 | 5.4 |
| PCSO-524 | 161 | 7.8 | Shiba inu | FS | 91 | 78 | 37 | 37 | 4 | 1 | 1 | 1 | 21 | 8.6 | 6.6 | 5.7 |
| Placebo | 67 | 3.5 | Toy poodle | FS | 37 | 18 | 22 | 18 | 5 | 1 | 2 | 3 | 10.4 | 8.7 | 6.2 | 6.9 |
| Placebo | 96 | 5.1 | Bichon frise | MC | 42 | 24 | 38 | 40 | 5 | 1 | 3 | 4 | 7.2 | 5.6 | 8.4 | 8.8 |
| Placebo | 96 | 7.6 | Shiba inu | FS | 86 | 59 | 43 | 27 | 7 | 4 | 4 | 4 | 13.1 | 5.8 | 6 | 6.4 |
| Placebo | 185 | 3.9 | Toy poodle | FS | 41 | 33 | 27 | 26 | 5 | 1 | 0 | 0 | 9.1 | 9.3 | 9.4 | 7.4 |
| Placebo | 24 | 3 | Chihuahua | MC | 14 | 10 | 16 | 10 | 6 | 1 | 7 | 7 | 7.9 | 8 | 8.7 | 8.4 |
| Placebo | 174 | 6.8 | Miniature Dachshund | MC | 26 | 16 | 20 | 22 | 5 | 2 | 3 | 3 | 8.6 | 7.8 | 8.2 | 9.4 |
| Placebo | 164 | 4.4 | Shiba inu | MC | 18 | 16 | 22 | 28 | 4 | 4 | 4 | 5 | 9.6 | 6.2 | 8.8 | 9.2 |
| Placebo | 76 | 8.5 | French bulldog | FS | 52 | 26 | 38 | 46 | 6 | 3 | 5 | 6 | 12.4 | 10.8 | 11.4 | 13.6 |
- Abbreviations: D, Day; FS, female spayed; MC, male castrated.
CADESI-04
Means ± standard deviation (SD) of the CADESI-04 for both groups across all time points are presented in Figure 1. At D14, both groups showed a significant decrease compared to the baseline (both p < 0.001). The mean CADESI-04 reduced from 49.4 ± 17.9 to 24.4 ± 22.8 in the PCSO-524 group, and from 39.5 ± 21.2 to 14 ± 3 in the control group. No significant differences were found between the PCSO-524 and control groups at D0 and D14 (p = 0.33 and p = 0.93). However, there was a significant improvement in the PCSO-524 group at D28 and D42 (15.7 ± 11.9 and 13.7 ± 11.9, respectively; p = 0.004 and p = 0.03) compared to the control group (28.2 ± 9.3 and 27.1 ± 10.7). Additionally, the PCSO-524 group exhibited a significant decrease over time when compared to D0 (p = 0.003, p < 0.001 and p < 0.001 for D14, D28 and D42, respectively).
FIGURE 1
pVAS
The mean pVAS and SD for two groups is illustrated in Figure 2. Both groups showed a significantly decline in pVAS score at D14 compared to the baseline while on twice-daily oclacitinib (p < 0.001 and p < 0.001). In the control group, mean pVAS reduced from 5.4 ± 0.8 at D0 to 3.5 ± 1.9 at D28 and 4.0 ± 2.0 at D42, compared to the baseline (p = 0.04 and p = 0.12). In the PCSO-524 group, the mean pVAS was 5.8 ± 1.2 at D0 and reduced to 0.5 ± 0.5 at D28 and 0.7 ± 0.7 at D42, both of which were statistically significant compared to D0 (p < 0.001 and p < 0.001). There was a significant different in pVAS between the two groups at D28 and D42 (p < 0.001 and p < 0.001) (Figure 2).
FIGURE 2
TEWL
Changes over time in TEWL with mean and SD are shown in Figure 3. There was a significant difference in the TEWL between the PCSO-524 group and the control at D0 (p = 0.04). In the control group, there was no statistical significance in mean values when D0 was compared with D14, D28 and D42 (p = 0.06, p = 0.17 and p = 0.31). However, a consistent reduction in TEWL was observed in the PCSO-524 group, reducing from 13.7 ± 4.7 at D0 to 6.0 ± 2.4 at D14, 4.6 ± 2.4 at D28 and 3.7 ± 1.6 at D42 as compared to D0 (all p < 0.001).
FIGURE 3
DISCUSSION
This randomised, double-blinded, controlled clinical trial demonstrates a significant improvement in pruritus, clinical signs (as assessed by CADESI-04) and TEWL in dogs treated with oclacitinib and PCSO-524 concurrently when compared with the control group of oclacitinib and sunflower oil. Furthermore, the outcome suggests that PCSO-524 combined with oclacitinib may compensate for a potential rebound in pruritus when oclacitinib was reduced to once daily dosing after D14.
Oclacitinib is an immunomodulatory drug that blocks the pro-inflammatory and pruritogenic cytokines through the Janus kinase (JAK) signalling pathway. The ‘rebound phenomenon’ of pruritus was reported in clinical cases when the dose was tapered from twice to once daily.11 Recently, a clinical study of client owned dogs with cAD demonstrated that 33 of 53 (73%) were unable to taper the dose of oclacitinib from twice daily to once daily without the dogs experiencing an increase in pruritus.12 To manage the pruritus effectively, prolonged twice-daily therapy was recommended, which is associated with an extra burden of routine blood tests and an increased risk of adverse effects. In our study, we also observed a rebound in pruritus at D14 after reducing to once daily oclacitinib in the control group; however, the dogs in the PCSO-524 group remained well controlled after 14 days. In the current study, the CADESI-04 scores of the PCSO-524 group at D28 and D42 did not show a significant difference (p = 0.28 and p = 0.18) compared to D14, yet they were significantly different when compared to the control group at D28 and D42 (p = 0.04 and p = 0.03). In addition, only one dog achieved pVAS <2 (a normal score) in the control group at D42, while all dogs showed pVAS <2 in the PCSO-524 group at D28 and D42. Despite being a relatively short-term study compared to other clinical studies on fatty acids, the current study aimed to observe the efficacy of synergistic therapy of PCSO-524 and oclacitinib. A review paper has reviewed 25 studies that evaluated the efficacy of various commercial fatty acid supplements containing omega-3 and/or omega-6 FA for treating cAD.13 The studies were categorised based on the duration of treatment, with short-term studies lasting 2–8 weeks and long-term studies lasting 8–16 weeks. During short-term observation, the focus is primarily on changes in pruritus and clinical improvement. By contrast, long-term observation focuses primarily on the medical-sparing effect or the use of fatty acid supplements as a sole therapy. Under these conditions, a 42 day trial period would be sufficient for observing the effect of combined therapy with PCSO-524 and oclacitinib. In 2019, a blinded, placebo-controlled study recruited a group of dogs with spontaneous cAD and fed a diet containing polyphenols, antioxidants and omega-3 fatty acid alongside with oclacitinib (0.4–0.6 mg/kg twice daily for 14 days then tapered to once a day) for a 60 day trial.14 The study found that the combination of fatty acids and oclacitinib led to a significant improvement in clinical scores, specifically the CADESI-04 and pruritus scores, compared to the placebo group at both D30 and D60.14 These findings were similar to ours and suggest that fatty acid combined with oclacitinib can enhance the anti-inflammatory effect and benefit clinical scores after reducing the dosage. The anti-inflammatory efficacy of PCSO-524 can be attributed to the supplement’s functional ingredient furan fatty acids.6 Furan fatty acids have been examined in various assay systems for anti-inflammatory activity, including in vitro and in vivo studies, modifying the leukotriene and prostaglandin production in the lipoxygenase and cyclooxygenase pathways.8
TEWL is one physiological parameter to evaluate skin barrier function in dogs, yet its usefulness has remained controversial as a result of the lack of a consistent methodology.15, 16 Several studies have examined TEWL as a means of evaluating treatment efficacy in lokivetmab, ciclosporin and hydrocortisone aceponate (HCA) spray,17–19 yet comparing the results across different studies can be difficult. In the study by Marsella et al., eight dogs were randomly assigned to receive either an oclacitinib or control for four weeks and followed by a wash-out period of four weeks, and then crossed-over to the other group. The study recruited a colony of atopic beagle dogs that were exposed to allergens in a controlled manner, in an attempt to replicate the natural occurring AD in dogs. Interestingly, the use of oclacitinib led to a significant increase in TEWL in the inguinal area, which is different from our findings as we did not observe significant changes in control group.16 It could be challenging to interpret the differences as both studies have a limited sample size, various level of inflammation and individual physiological changes. Despite the limitations in clinical studies, an animal model combining in vivo and in vitro methods could offer more comprehensive insights into this phenomenon by biomolecular evidence. A microarray analysis using a mouse/human skin model revealed that the inflammatory cytokines interleukin (IL)-4/IL-13 may impair skin barrier function by downregulating epidermal differentiation via the JAK-signal transducers and activators of transcription (STAT) signalling pathway.20 This suggests that oclacitinib, as a JAK inhibitor, may have the potential to improve abnormal keratinocyte differentiation by reversing the gene expression alteration observed in AD lesions. Despite the proposed hypothesis, it is important to remember that TEWL is merely one physiological parameter and should not be overemphasised in its role in skin barrier function.
Sunflower oil is not considered a significant source of omega-3 fatty acid, yet it is a good source of omega-6 fatty acid, particularly linoleic acid which is an important component of the stratum corneum in dogs. In a previous study involving client-owned dogs, it was demonstrated that sunflower oil had no impact on TEWL and the activity of various chemokines and cytokines during a four week wash-in period.21 As a result, we employed sunflower oil as the control in our study. Based on current evidence, the inclusion of vitamine E in the capsule has shown no significant impact on skin barrier function. Vitamine E is not suggested as a treatment option for cAD. Therefore, we do not regard it as an actvie ingreident in this product.
The limitations of this study are the small number of dogs, the lack of dietary fatty acid standardisation and its relatively short duration. In addition, the TEWL did not shown a normal distribution at the inclusion at D0. Despite the improvement seen in the PCSO-524 group, a 12 week study period with controlled diet might provide a more precise conclusion.
In conclusion, based on the results from this study on the 17 atopic dogs examined, we postulate that the anti-inflammatory effects of PCSO-524 compensated for the rebound effect which is often seen when oclacitinib is reduced to once daily dosing after D14. PCSO-524 supplementation also reduced the TEWL and this finding warrants further investigation.
AUTHOR CONTRIBUTIONS
Takeo Nishiyama: Writing—original draft; project administration; software; resources; investigation; data curation; formal analysis. Masashi Kusakabe: Project administration. Ichiro Imanishi: Resources. Tadashi Hisano: Project administration. Teruyasu Fukamachi: Project administration. Norihito Taguchi: Project administration. Keita Iyori: Supervision; funding acquisition; methodology. Yun–Hsia Hsiao: Writing—review & editing; writing—original draft; validation.
ACKNOWLEDGEMENTS
The authors thank the owners and their canine companions that participated in the clinical trial.
FUNDING INFORMATION
This study was partly financially supported by V and P Co., (https://vetzpetz.jp/pages/company) which also supplied the capsules used in the trial. The funders had no role in study design, data collection and analysis, in writing the manuscript or decision to publish.
CONFLICT OF INTEREST STATEMENT
The authors declare no conflicts of interest.