Nucleotide supplementation as a novel adjunctive therapy for canine atopic dermatitis

Nucleotide supplementation as a novel adjunctive therapy for canine atopic dermatitis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Nucleotide supplementation as a novel adjunctive therapy for canine atopic dermatitis Carolina D. N. Barroso, Vitória L. Cavalheiro, Louise M. Imamura, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3225374/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Atopic dermatitis (AD) is an important allergic condition in dogs, and treatment is lifelong. Alternatives for therapy are important, as dogs do not respond equally to commonly used therapies. Novel therapies on the market are also costly, which limits their adoption in low and middle-income countries. Nucleotides have been shown to improve innate barrier function and were therefore tested for their efficacy in canine atopic dermatitis. Two independent mouse trials were performed. The first trial (pilot) aimed to standardize the atopic dermatitis challenge model and to perform a proof-of-concept for the use of nucleotides for the treatment of AD. The second trial expanded on the results of the first by assessing the oral use of the experimental compound. Lastly, a clinical trial was run on 27 dogs with naturally occurring AD, for 8 weeks. The mouse trials revealed that use of nucleotides diminished the dermal inflammation that is common in AD. On histopathological analyses, scores of inflammatory cells, and fibrosis in the dermis of were lower in treated mice, resulting in diminished dermal thickness. Both oral and injectable administration routes proved effective. Differently from what occurred in the dermis, epidermal thickening was not reversed by the test compound. In dogs, oral treatment reduced owner-assessed pruritus and in vet-assessed erythema. These data indicate a possible use for oral nucleotide supplementation against canine AD. Atopic dermatitis canine allergy pruritus nucleotides mouse model Figures Figure 1 Figure 2 1. Introduction Canine atopic dermatitis (CAD) is one of the most prevalent dermatological affections in dogs. It is an allergic disease with a genetic predisposition for excessive IgE responses to innocuous stimuli (Halliwell, 2006 ; Lian & Halliwell, 1998 ). CAD occurs with overt inflammation and pruritus that occur due to exacerbated immune responses, mainly derived from CD4 + T helper 2 (T H 2) lymphocytes. Activated T CD4 + cells releasing T H 2 cytokines, such IL-4, IL-5, and IL-13, contribute to inflammation and development of injuries which commonly are located on the rostrum, axillary and inguinal regions, abdomen, and extremities of dogs (Brandt, 2011 ; Hensel et al., 2015 ; Jeong et al., 2003 ; T. J. Nuttall et al., 2019 ). No curative treatment is available for CAD, but some medications effectively reduce pruritus and severity of skin lesions. Glucocorticoids, cyclosporin, antihistamines, lokivetmab, oclacitinib and less often, recombinant interferon have been used. However, treatments show variable efficacy and important adverse effects. Treatment side effects may include vomiting, diarrhoea, alterations in bodyweight and immunosuppression (Elkholly et al., 2020 ). Pricing of treatment is also a problem, especially in low and middle-income countries, affecting the accessibility to some of the therapies (personal experience). Given the wide prevalence of the disease, its clinical importance, and chronicity, alternatives are needed to alleviate clinical signs. Nucleotides have widely been used in livestock with the goal of improving intestinal health and of modulating immunity by providing such nutrients for the rapid proliferation of immune cells (Jung & Batal, 2012 ; Peng et al., 2013 ; Superchi et al., 2012 ). Therefore, the goal of this study was to test oral supplementation of nucleotides as possible adjunctive in the treatment of CAD. 2. Material and Methods 2.1 Animal experimentation ethics All animal work was evaluated and approved by the Ethics Committee for Animal Research of Federal University of Paraná (protocol number 068/2017) and of Imunova Análises Biológicas LTDA (002.2018). Mice were maintained in line with good husbandry practices (Foster et al., 2014) . Dogs were clinically assessed by a veterinarian and were maintained with their owners [guardians] throughout the trial. Written informed consent was obtained from the dog guardians, who were instructed on the intervention, on their own role in decision-making, on the alternative treatments and on the possible risks of the trial. 2.2 Mouse trials Two independent mouse trials were performed. The first trial (pilot) aimed to standardize the atopic dermatitis challenge model and to perform a proof-of-concept for the use of nucleotides for the treatment of CAD. The second trial expanded on the results of the first by assessing the best route to deliver the experimental compound. To induce atopic dermatitis in mice, 1% of 2-chloro-1,3,5-trinitrobenzene (TCNB) was used diluted in acetone, according to the literature (Giménez-Rivera V.A., Metz M., 2014; Kitagaki et al., 1995) . TCNB is a hapten molecule commonly used in animal contact hypersensitivity model since it is a potentially allergenic substance that binds to proteins, eliciting an immune response. Thus, the animals received 20 µL of TCNB both on the internal and external faces of the right ear pinna. In the first week, it was administered on D1 and D7, and later every other day for 35 days. Four experimental groups of mice were used for each trial. In both, the negative control only received acetone and the positive control developed the induced AD but was not treated. In the first trial, one group was treated with betamethasone, an anti-inflammatory, as a treatment control. Dosage of nucleotides was as previously described for mice (Novak et al., 1994). The second trial evaluated nucleotide oral and injectable delivery along with an empirical dose reduction and the addition of the carrier aluminium hydroxide (AlOH 3 ) to one of the formulations as it has been shown to protect and slowly release nucleotides, improving its absorption (Nakayama et al., 2010a). The experimental design and further information for each trial are represented in Table 1. At the end of the study, mice were euthanized. Ears were sampled for histology after 50 days (first trial) and 40 days (second trial) of the first application of TCNB. Histological sections were blindly evaluated by an independent pathologist for histopathologic features (ear swelling, leucocyte infiltration and degenerative patterns). 2.3 Dog clinical trial From October to December of 2020, dogs were selected for the trial based on dermatological evaluation and presumptive AD diagnosis as described previously (Linek & Favrot, 2010) . Other pruritic diseases were ruled out clinically based on consistent history and physical examination alone. All dogs had also been diagnosed with AD by previous attending veterinarians. These animals had previously failed to respond to the common therapies or that were withdrawn from them due to adverse effects. A withdrawal period of 30 days for glucocorticoids, antibiotics, cyclosporin and other AD treatments was required before the trial. The test compound was an aqueous formulation of nucleotides (proprietary composition of Imunova Análises Biológicas LTDA.). The product can be provided by the authors for replication or further studies upon reasonable request. Two hundred microliters (200 µg) of nucleotides were used for each dose. The compound was administered orally with a 1 mL syringe by a veterinarian at the day of enrolment and then again 14 days later. Dogs were assessed by owners with an analogue pruritus scale (Hill et al., 2007). The veterinarian also assessed the dogs in the clinic using the CADESI-03 scale (Olivry et al., 2007). Assessments were performed 14 days after each dose. The trial was ended following the clinical assessment of the second dose. 2.4 Statistics Data distribution was assessed by the D’Agostino-Pearson omnibus normality test. Mouse ear pinnae were compared by unpaired t test with Welch´s correction (dermis) or one-way ANOVA (epidermis). Lesion scores of dogs were compared with Kruskal–Wallis test. P was attributed at 0.10 (Amrhein et al., 2017; Dahiru, 2008). GraphPad Prism 8 (GraphPad, Inc.) was used for statistics and for graphs. 3. Results 3.1 Mouse trials The repeated topical applications of TCNB induced a clinical condition on mouse pinnae that was similar to canine atopic dermatitis in both trials, as had been previously described (Kitagaki et al., 1995) . In the pilot study, as expected, challenged ears became visibly sensitive and with evident erythema and thickening (Figures 1A and 1B). The experimental challenge induced dermal thickening due to fibrosis, congestion, diffuse dermatitis with abundant neutrophils and fibroblasts, angiogenesis, and dermal edema, as assessed by a trained veterinary pathologist (descriptive data). The test product reverted the effect of dermatitis. The effect of the product was visually comparable to those seen with betamethasone, reducing dermis width in relation to the challenged control, as illustrated in Figure 1B. No statistical analyses were possible in the pilot trial due to the low number of animals. The second mouse trial assessing the use of the nucleotides via the oral route showed that dermis width was reduced with oral treatment in the presence of aluminium hydroxide. However, the lower dose of nucleotides (without AlOH 3 , T1) used in the main moue trial was not sufficient for controlling dermal oedema (Figure 1C). Epidermal thickening could not be reverted by any of the mouse treatments. 3.2 Dog clinical trial – description of enrolled dogs Out of 350 dogs enrolled, 27 fulfilled the inclusion criteria. 14/27 were female. Mongrel dogs were the most common (n = 8/27), followed by Lhasa Apso (n = 6/27) and Shitzu (n = 4/27). Mean age and weight were 6 ± 3 years and 8 ± 11.6 kg, respectively. 20/27 dogs had previously received corticosteroids and/or cyclosporin for the treatment of AD, and 17/20 were responsive the these. 3.3 Dog clinical trial – treatment results Pruritus was the main clinical complaint at study enrolment for all dogs. Excoriations on the skin were commonly seen on the inguinal region, abdomen, limb extremities and ear pinnae. All dogs had detectable erythema (Figure 2A). 37% of animals had chronic otitis. Veterinarian-assessed erythema and owner-assessed pruritus were reduced by the experimental treatment after 2 and 1 doses respectively (Figure 2A, B). Alopecia and excoriation were not significantly improved by the treatment (Figure 2A). Only 3 animals showed possible side effects of the experimental treatment. Fever (n = 1 animal), reduced activity (n = 1) and intestinal constipation (n = 1) were seen throughout the experiment, although their association to the treatment was not further investigated, since these clinical signs were transitory. 4. Discussion Canine AD is a disease manifested as chronic, pruritic, and inflammatory dermatitis, which requires long-term treatment. Immunologically, it is characterized by excessive Th2 lymphocyte responses, which coordinates IL-4, IL-5, and IL-13 release, leading to IgE production (Gedon & Mueller, 2018 ; T. Nuttall et al., 2013 ; Prelaud & Laprais, 2020 ). Cytokine release, neuronal itch stimuli and the pruritic behaviours of the animal establish a vicious cycle of itch that perpetuates and potentially exacerbates the skin lesions and defects in the skin barrier function (Marsella et al., 2012 ; Nagafuchi et al., 1997 ). The manifestations of AD described here for TNCB-sensitized mice were caused by recurrent exposure to the chemical, which is a potent hapten allergen, resulting in chronic inflammation of the ear skin. Therefore, this is a suitable model to study atopic dermatitis (Man et al., 2008 ). Our proposed nucleotide treatment reduced dermal thickness and markedly suppressed dermal inflammation and fibrosis in mice, possibly due to its capacity of interfering with inflammation via nucleotide receptors on immune cells (Chen et al., 2014 ; Thompson et al., 2021 ). Following a hapten challenge, T cells infiltrate the application site and produce cytokines that mediate the characteristic tissue oedema (Kish et al., 2011 ). This perturbation also develops barrier and epidermal abnormalities, consequently generating the typical histopathological profile (Man et al., 2008 ). The epidermis, however, was not reduced in the same manner after treatment. Blood vessels terminate in the dermis, while the epidermis is nourished via diffusion from there (Cevc & Vierl, 2007 ). This may account for the faster responses seen in the dermis. Furthermore, the mice in our study showed a reduction in pruritus intensity regardless of the route of administration, showing that the nucleotides – or intermediary effector molecules induced by nucleotide administration – reached the skin either way. It is possible that the parenteral route is more systemically effective than the oral route, which may especially target intestinal immune responses (Iijima et al., 1996 ; Usami et al., 1996 ). Nevertheless, mice that were challenged directly on the skin with TNCB had benefits from the oral administration of the test compound. This indicated that dogs with AD could also benefit from oral treatment, which is easier for owners to administer than frequent injections (Nakayama et al., 2010b ). In our preliminary clinical trial, dogs also benefited from the use of oral nucleotides. There were improvements in pruritus and erythema, but AD alopecia and excoriation were not reverted by the treatment. This may be due to the short treatment course and study follow-up, since alopecia and excoriation are usually inflicted by the dog. A behavioural change, which can take longer, may be needed for the reduction of these lesions (Harvey et al., 2019 ). The reduction in pruritus can improve quality of life for the affected dogs and their guardians (Linek & Favrot, 2010 ). This study indicates that oral nucleotide administration may bear such benefits. Nevertheless, more studies are necessary to better understand the efficacy, dosage variations, mechanisms, and long-terms effects from this product. Declarations Funding This project was funded by Imunova Análises Biológicas LTDA. No changes were made to the report due to commercial considerations. Conflict of Interest Statement All authors were associated to Imunova Análises Biológicas LTDA, which owns the nucleotide technology. Author Contribution Carolina D. N. Barroso: writing, investigation; Vitória L. Cavalheiro: writing, investigation; Louise Imamura: investigation; Caroline Cordeiro: investigation; Luiz Felipe Caron: conceptualization, writing - review & editing; Breno C. B. Beirão: writing – review & editing (lead), supervision, conceptualization. Data availability Raw data can be made available to interested parties under reasonable request. Acknowledgments The authors thank Carolina Trentin for assistance with veterinary consultations. References Amrhein V, Korner-Nievergelt F, Roth T (2017) The earth is flat (p > 0.05): significance thresholds and the crisis of unreplicable research. PeerJ 5(7):3544. https://doi.org/10.7717/PEERJ.3544 Brandt EB (2011) Th2 Cytokines and Atopic Dermatitis. 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N, number of animals in each experimental group. † In the pilot mouse trial, the treatment was conducted weekly for 3 weeks following the induction of atopic dermatitis (on D35). ‡ In the second mouse trial, treatment was conducted on D18 and on D25 (before the end of the protocol for induction of atopic dermatitis, on D35). Additional Declarations Competing interest reported. All authors were associated to Imunova Análises Biológicas LTDA, which owns the nucleotide technology. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3225374","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":223926981,"identity":"f51e1e50-24a3-4e89-a13c-4874a8ab87be","order_by":0,"name":"Carolina D. N. Barroso","email":"","orcid":"","institution":"Imunova Análises Biológicas LTDA. Curitiba","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Carolina","middleName":"D. 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Beirão","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIiWNgGAWjYBACAwh1gIENRH1gkAOSPCRoYZzBYEyCFhBg5iFGi7n04WMPPjDckeOTPvzwsW2bQZ55A++xD/i0WPalpRvOYHhmzMaXZmyc22ZQLHOAL3kGXoed4TGT5mE4nNjGw2Amndv2J3EGA48xfr+c4f8G0lLfxsP+TdqyzYAYLTxsIC0JbDxA6xiJ0WLZw2YmOcPgmWEbD0+xYc85g2IJZr5kvFrMeZifSXyouCMv38O+8cGPMoM8Cfbew3i1QJ2HYCYwMBOhAQUkkKphFIyCUTAKhj8AAGOMO77MR8GVAAAAAElFTkSuQmCC","orcid":"","institution":"Universidade Federal do Paraná","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Breno","middleName":"C. B.","lastName":"Beirão","suffix":""}],"badges":[],"createdAt":"2023-08-01 17:29:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3225374/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3225374/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":41314303,"identity":"e4123c34-e1f5-4742-b491-2d7397642e4e","added_by":"auto","created_at":"2023-08-09 15:15:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":14356030,"visible":true,"origin":"","legend":"\u003cp\u003eInduced atopic dermatitis (AD) and effect of nucleotides in mice. A) Experimental induction of AD lead to clinical signs that were compatible with the naturally occurring canine disease. The right ear showed reddening and sensitivity to the touch. The left ear was left unchallenged for comparison. B) Microscopic features of the induced AD. There was visible change in dermal and epidermal thickness. Representative results from the pilot and main mouse trials. The arrows indicate how these parameters were measured (10 measurements per ear section). C) Dermis and epidermis thickness from the main mouse trial. Graphs represent the median ± 95% CI from 10 individual measurements of 3 ear sections from each mouse. Statistical analysis by unpaired t with Welch´s correction (dermis) or one-way ANOVA (epidermis). Statistical comparison was not possible in the pilot test due to low N. 10× magnification, HE stain.\u003c/p\u003e","description":"","filename":"Figure1v02.png","url":"https://assets-eu.researchsquare.com/files/rs-3225374/v1/458bd953dd17f18c55bf60dc.png"},{"id":41314302,"identity":"cdbb68a1-7a72-4952-9287-9d93b7a91d96","added_by":"auto","created_at":"2023-08-09 15:15:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":14302306,"visible":true,"origin":"","legend":"\u003cp\u003eClinical trial of nucleotides in dogs. A) Scoring of lesions (veterinarian assessment) or of pruritus (owner [guardian] assessment). Bars show the median and dots show individual dog scoring pre- or post-treatment (evaluations performed 1 week after each treatment). Statistical analysis by Kruskal-Wallis comparing all groups simultaneously. B) Representative clinical result of a dog treated with the test compound. Arrows on the left image show areas of intense erythema pre-treatment. The image to the right shows the same region by the end of the trial.\u003c/p\u003e","description":"","filename":"Figure2v02.png","url":"https://assets-eu.researchsquare.com/files/rs-3225374/v1/a42b1386481522812a1a5e7b.png"},{"id":51447915,"identity":"62055d7b-788c-4327-b6ce-34595cf745cd","added_by":"auto","created_at":"2024-02-21 18:38:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1361438,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3225374/v1/c46cad2a-d490-4c04-b659-6f4ffb01b061.pdf"}],"financialInterests":"Competing interest reported. All authors were associated to Imunova Análises Biológicas LTDA, which owns the nucleotide technology.","formattedTitle":"Nucleotide supplementation as a novel adjunctive therapy for canine atopic dermatitis","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCanine atopic dermatitis (CAD) is one of the most prevalent dermatological affections in dogs. It is an allergic disease with a genetic predisposition for excessive IgE responses to innocuous stimuli (Halliwell, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Lian \u0026amp; Halliwell, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). CAD occurs with overt inflammation and pruritus that occur due to exacerbated immune responses, mainly derived from CD4\u003csup\u003e+\u003c/sup\u003e T helper 2 (T\u003csub\u003eH\u003c/sub\u003e2) lymphocytes. Activated T CD4\u003csup\u003e+\u003c/sup\u003e cells releasing T\u003csub\u003eH\u003c/sub\u003e2 cytokines, such IL-4, IL-5, and IL-13, contribute to inflammation and development of injuries which commonly are located on the rostrum, axillary and inguinal regions, abdomen, and extremities of dogs (Brandt, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Hensel et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Jeong et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; T. J. Nuttall et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNo curative treatment is available for CAD, but some medications effectively reduce pruritus and severity of skin lesions. Glucocorticoids, cyclosporin, antihistamines, lokivetmab, oclacitinib and less often, recombinant interferon have been used. However, treatments show variable efficacy and important adverse effects. Treatment side effects may include vomiting, diarrhoea, alterations in bodyweight and immunosuppression (Elkholly et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Pricing of treatment is also a problem, especially in low and middle-income countries, affecting the accessibility to some of the therapies (personal experience). Given the wide prevalence of the disease, its clinical importance, and chronicity, alternatives are needed to alleviate clinical signs.\u003c/p\u003e \u003cp\u003eNucleotides have widely been used in livestock with the goal of improving intestinal health and of modulating immunity by providing such nutrients for the rapid proliferation of immune cells (Jung \u0026amp; Batal, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Peng et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Superchi et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Therefore, the goal of this study was to test oral supplementation of nucleotides as possible adjunctive in the treatment of CAD.\u003c/p\u003e"},{"header":"2. Material and Methods","content":"\u003cp\u003e2.1 Animal experimentation ethics\u003c/p\u003e\n\u003cp\u003eAll animal work was evaluated and approved by the Ethics Committee for Animal Research of Federal University of Paran\u0026aacute; (protocol number 068/2017) and of Imunova An\u0026aacute;lises Biol\u0026oacute;gicas LTDA (002.2018). Mice were maintained in line with good husbandry practices \u003cspan lang=\"EN-US\"\u003e(Foster et al., 2014)\u003c/span\u003e. Dogs were clinically assessed by a veterinarian and were maintained with their owners [guardians] throughout the trial. Written informed consent was obtained from the dog guardians, who were instructed on the intervention, on their own role in decision-making, on the alternative treatments and on the possible risks of the trial.\u003c/p\u003e\n\u003cp\u003e2.2 Mouse trials\u003c/p\u003e\n\u003cp\u003eTwo independent mouse trials were performed. The first trial (pilot) aimed to standardize the atopic dermatitis challenge model and to perform a proof-of-concept for the use of nucleotides for the treatment of CAD. The second trial expanded on the results of the first by assessing the best route to deliver the experimental compound. To induce atopic dermatitis in mice, 1% of 2-chloro-1,3,5-trinitrobenzene (TCNB) was used diluted in acetone, according to the literature \u003cspan lang=\"EN-US\"\u003e(Gim\u0026eacute;nez-Rivera V.A., Metz M., 2014; Kitagaki et al., 1995)\u003c/span\u003e. TCNB is a hapten molecule commonly used in animal contact hypersensitivity model since it is a potentially allergenic substance that binds to proteins, eliciting an immune response. Thus, the animals received 20 \u0026micro;L of TCNB both on the internal and external faces of the right ear pinna. In the first week, it was administered on D1 and D7, and later every other day for 35 days.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFour experimental groups of mice were used for each trial. In both, the negative control only received acetone and the positive control developed the induced AD but was not treated. In the first trial, one group was treated with betamethasone, an anti-inflammatory, as a treatment control. Dosage of nucleotides was as previously described for mice (Novak et al., 1994). The second trial evaluated nucleotide oral and injectable delivery along with an empirical dose reduction and the addition of the carrier aluminium hydroxide (AlOH\u003csub\u003e3\u003c/sub\u003e) to one of the formulations as it has been shown to protect and slowly release nucleotides, improving its absorption (Nakayama et al., 2010a). The experimental design and further information for each trial are represented in Table 1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAt the end of the study, mice were euthanized. Ears were sampled for histology after 50 days (first trial) and 40 days (second trial) of the first application of TCNB. Histological sections were blindly evaluated by an independent pathologist for histopathologic features (ear swelling, leucocyte infiltration and degenerative patterns).\u003c/p\u003e\n\u003cp\u003e2.3 Dog clinical trial\u003c/p\u003e\n\u003cp\u003eFrom October to December of 2020, dogs were selected for the trial based on dermatological evaluation and presumptive AD diagnosis as described previously \u003csup\u003e(Linek \u0026amp; Favrot, 2010)\u003c/sup\u003e. Other pruritic diseases were ruled out clinically based on consistent history and physical examination alone. All dogs had also been diagnosed with AD by previous attending veterinarians. These animals had previously failed to respond to the common therapies or that were withdrawn from them due to adverse effects. A withdrawal period of 30 days for glucocorticoids, antibiotics, cyclosporin and other AD treatments was required before the trial.\u003c/p\u003e\n\u003cp\u003eThe test compound was an aqueous formulation of nucleotides (proprietary composition of Imunova An\u0026aacute;lises Biol\u0026oacute;gicas LTDA.). The product can be provided by the authors for replication or further studies upon reasonable request. Two hundred microliters (200 \u0026micro;g) of nucleotides were used for each dose. The compound was administered orally with a 1 mL syringe by a veterinarian at the day of enrolment and then again 14 days later. Dogs were assessed by owners with an analogue pruritus scale (Hill et al., 2007). The veterinarian also assessed the dogs in the clinic using the CADESI-03 scale (Olivry et al., 2007). Assessments were performed 14 days after each dose. The trial was ended following the clinical assessment of the second dose.\u003c/p\u003e\n\u003cp\u003e2.4 Statistics\u003c/p\u003e\n\u003cp\u003eData distribution was assessed by the D\u0026rsquo;Agostino-Pearson omnibus normality test. Mouse ear pinnae were compared by unpaired \u003cem\u003et\u003c/em\u003e test with Welch\u0026acute;s correction (dermis) or one-way ANOVA (epidermis). Lesion scores of dogs were compared with Kruskal\u0026ndash;Wallis test. \u003cem\u003eP\u0026nbsp;\u003c/em\u003ewas attributed at 0.10 (Amrhein et al., 2017; Dahiru, 2008). GraphPad Prism 8 (GraphPad, Inc.) was used for statistics and for graphs.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e3.1 Mouse trials\u003c/p\u003e\n\u003cp\u003eThe repeated topical applications of TCNB induced a clinical condition on mouse pinnae that was similar to canine atopic dermatitis in both trials, as had been previously described \u003cspan lang=\"EN-US\"\u003e(Kitagaki et al., 1995)\u003c/span\u003e. In the pilot study, as expected, challenged ears became visibly sensitive and with evident erythema and thickening (Figures 1A and 1B).\u0026nbsp;The experimental challenge\u0026nbsp;induced dermal thickening due to fibrosis, congestion, diffuse dermatitis with abundant neutrophils and fibroblasts, angiogenesis, and dermal edema, as assessed by a trained veterinary pathologist (descriptive data).\u0026nbsp;The test product reverted the effect of dermatitis. The effect of the product was visually comparable to those seen with betamethasone, reducing dermis width in relation to the challenged control, as illustrated in Figure 1B. No statistical analyses were possible in the pilot trial due to the low number of animals.\u003c/p\u003e\n\u003cp\u003eThe second mouse trial assessing the use of the nucleotides via the oral route showed that dermis width was reduced with oral treatment in the presence of aluminium hydroxide. However, the lower dose of nucleotides (without AlOH\u003csub\u003e3\u003c/sub\u003e, T1) used in the main moue trial was not sufficient for controlling dermal oedema (Figure 1C). Epidermal thickening could not be reverted by any of the mouse treatments.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e3.2 Dog clinical trial \u0026ndash; description of enrolled dogs\u003c/p\u003e\n\u003cp\u003eOut of 350 dogs enrolled, 27 fulfilled the inclusion criteria. 14/27 were female. Mongrel dogs were the most common (n = 8/27), followed by Lhasa Apso (n = 6/27) and Shitzu (n = 4/27). Mean age and weight were 6 \u0026plusmn; 3 years and 8 \u0026plusmn; 11.6 kg, respectively. 20/27 dogs had previously received corticosteroids and/or cyclosporin for the treatment of AD, and 17/20 were responsive the these.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e3.3 Dog clinical trial \u0026ndash; treatment results\u003c/p\u003e\n\u003cp\u003ePruritus was the main clinical complaint at study enrolment for all dogs. Excoriations on the skin were commonly seen on the inguinal region, abdomen, limb extremities and ear pinnae. All dogs had detectable erythema (Figure 2A). 37% of animals had chronic otitis.\u003c/p\u003e\n\u003cp\u003eVeterinarian-assessed erythema and owner-assessed pruritus were reduced by the experimental treatment after 2 and 1 doses respectively (Figure 2A, B). Alopecia and excoriation were not significantly improved by the treatment (Figure 2A). Only 3 animals showed possible side effects of the experimental treatment. Fever (n = 1 animal), reduced activity (n = 1) and intestinal constipation (n = 1) were seen throughout the experiment, although their association to the treatment was not further investigated, since these clinical signs were transitory.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eCanine AD is a disease manifested as chronic, pruritic, and inflammatory dermatitis, which requires long-term treatment. Immunologically, it is characterized by excessive Th2 lymphocyte responses, which coordinates IL-4, IL-5, and IL-13 release, leading to IgE production (Gedon \u0026amp; Mueller, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; T. Nuttall et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Prelaud \u0026amp; Laprais, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Cytokine release, neuronal itch stimuli and the pruritic behaviours of the animal establish a vicious cycle of itch that perpetuates and potentially exacerbates the skin lesions and defects in the skin barrier function (Marsella et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Nagafuchi et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1997\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe manifestations of AD described here for TNCB-sensitized mice were caused by recurrent exposure to the chemical, which is a potent hapten allergen, resulting in chronic inflammation of the ear skin. Therefore, this is a suitable model to study atopic dermatitis (Man et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Our proposed nucleotide treatment reduced dermal thickness and markedly suppressed dermal inflammation and fibrosis in mice, possibly due to its capacity of interfering with inflammation via nucleotide receptors on immune cells (Chen et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Thompson et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Following a hapten challenge, T cells infiltrate the application site and produce cytokines that mediate the characteristic tissue oedema (Kish et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). This perturbation also develops barrier and epidermal abnormalities, consequently generating the typical histopathological profile (Man et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The epidermis, however, was not reduced in the same manner after treatment. Blood vessels terminate in the dermis, while the epidermis is nourished via diffusion from there (Cevc \u0026amp; Vierl, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). This may account for the faster responses seen in the dermis.\u003c/p\u003e \u003cp\u003eFurthermore, the mice in our study showed a reduction in pruritus intensity regardless of the route of administration, showing that the nucleotides \u0026ndash; or intermediary effector molecules induced by nucleotide administration \u0026ndash; reached the skin either way. It is possible that the parenteral route is more systemically effective than the oral route, which may especially target intestinal immune responses (Iijima et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Usami et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). Nevertheless, mice that were challenged directly on the skin with TNCB had benefits from the oral administration of the test compound. This indicated that dogs with AD could also benefit from oral treatment, which is easier for owners to administer than frequent injections (Nakayama et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2010b\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn our preliminary clinical trial, dogs also benefited from the use of oral nucleotides. There were improvements in pruritus and erythema, but AD alopecia and excoriation were not reverted by the treatment. This may be due to the short treatment course and study follow-up, since alopecia and excoriation are usually inflicted by the dog. A behavioural change, which can take longer, may be needed for the reduction of these lesions (Harvey et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The reduction in pruritus can improve quality of life for the affected dogs and their guardians (Linek \u0026amp; Favrot, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). This study indicates that oral nucleotide administration may bear such benefits. Nevertheless, more studies are necessary to better understand the efficacy, dosage variations, mechanisms, and long-terms effects from this product.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project was funded by Imunova An\u0026aacute;lises Biol\u0026oacute;gicas LTDA. No changes were made to the report due to commercial considerations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors were associated to Imunova An\u0026aacute;lises Biol\u0026oacute;gicas LTDA, which owns the nucleotide technology.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCarolina D. N. Barroso: writing, investigation; Vit\u0026oacute;ria L. Cavalheiro: writing, investigation; Louise Imamura: investigation; Caroline Cordeiro: investigation; Luiz Felipe Caron: conceptualization, writing - review \u0026amp; editing; Breno C. B. Beir\u0026atilde;o: writing \u0026ndash; review \u0026amp; editing (lead), supervision, conceptualization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRaw data can be made available to interested parties under reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Carolina Trentin for assistance with veterinary consultations.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAmrhein V, Korner-Nievergelt F, Roth T (2017) The earth is flat (p \u0026gt; 0.05): significance thresholds and the crisis of unreplicable research. 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N, number of animals in each experimental group.\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e\u0026dagger;\u003c/sup\u003eIn the pilot mouse trial, the treatment was conducted weekly for 3 weeks following the induction of atopic dermatitis (on D35).\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e\u0026Dagger;\u003c/sup\u003eIn the second mouse trial, treatment was conducted on D18 and on D25 (before the end of the protocol for induction of atopic dermatitis, on D35).\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Atopic dermatitis, canine, allergy, pruritus, nucleotides, mouse model","lastPublishedDoi":"10.21203/rs.3.rs-3225374/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3225374/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAtopic dermatitis (AD) is an important allergic condition in dogs, and treatment is lifelong. Alternatives for therapy are important, as dogs do not respond equally to commonly used therapies. Novel therapies on the market are also costly, which limits their adoption in low and middle-income countries. Nucleotides have been shown to improve innate barrier function and were therefore tested for their efficacy in canine atopic dermatitis. Two independent mouse trials were performed. The first trial (pilot) aimed to standardize the atopic dermatitis challenge model and to perform a proof-of-concept for the use of nucleotides for the treatment of AD. The second trial expanded on the results of the first by assessing the oral use of the experimental compound. Lastly, a clinical trial was run on 27 dogs with naturally occurring AD, for 8 weeks. The mouse trials revealed that use of nucleotides diminished the dermal inflammation that is common in AD. On histopathological analyses, scores of inflammatory cells, and fibrosis in the dermis of were lower in treated mice, resulting in diminished dermal thickness. Both oral and injectable administration routes proved effective. Differently from what occurred in the dermis, epidermal thickening was not reversed by the test compound. In dogs, oral treatment reduced owner-assessed pruritus and in vet-assessed erythema. These data indicate a possible use for oral nucleotide supplementation against canine AD.\u003c/p\u003e","manuscriptTitle":"Nucleotide supplementation as a novel adjunctive therapy for canine atopic dermatitis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-08-09 15:14:58","doi":"10.21203/rs.3.rs-3225374/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5ae33cce-01eb-449c-b777-f1b62fc76bdf","owner":[],"postedDate":"August 9th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-02-21T18:30:13+00:00","versionOfRecord":[],"versionCreatedAt":"2023-08-09 15:14:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3225374","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3225374","identity":"rs-3225374","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}