Botanical oleander extract and oleandrin have superior effects on innate immune functions pertaining to dermal allergic reactions in canine cells when compared to oclacitinib

Theresa W. Fossum Phoenix Animal Wellness, San Antonio, TX

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Gitte S. Jensen NIS Labs, Klamath Falls, OR

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Robert A. Newman MD Anderson Cancer Center, University of Texas, Houston, TX

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Jose R. Matos Innovar LLC, Plano, TX

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Abstract

OBJECTIVE

To perform testing for cytokines involved in dermal inflammatory reactions and to document and compare the effects of an oleander extract (OE), oleandrin, and oclacitinib on biomarkers relevant to allergic reactions. The effects of these compounds under inflamed culture conditions are of direct importance to the treatment of canine atopic dermatitis.

METHODS

Testing involved primary canine dermal fibroblasts and the canine DH82 macrophage cell line; both cell types are important for initiating, regulating, and resolving dermal allergic reactions via cytokine communication.

RESULTS

Under inflamed conditions, OE and oleandrin downregulated key cytokines secreted by canine dermal fibroblasts and the DH82 macrophage cell line; all of which are treatment targets in dermatitis. In the DH82 macrophage cultures, the most noteworthy reductions involved IL-6, IL-12/IL-23p40, interferon-γ, tumor necrosis factor-α, VEGF, and nerve growth factor-β. Oclacitinib triggered reductions of some cytokines involved in allergic reactions, including TGF-β1, IL-12/IL-23p40, and tumor necrosis factor-α; however, these reductions were less robust than the reductions triggered by OE and oleandrin and accompanied by increases in other cytokines involved in dermal inflammation, including IL-6, interferon-γ, and nerve growth factor-β. In cultures of primary dermal fibroblasts, OE and oleandrin reduced the levels of IL-8 and monocyte chemoattractant protein-1, whereas oclacitinib had little or no effect.

CONCLUSIONS

Oleander extract and oleandrin directly modulate immune responses under inflamed conditions. Moreover, OE and oleandrin appear to provide a more beneficial overall cytokine regulation than oclacitinib under inflamed culture conditions.

CLINICAL RELEVANCE

These results suggest that OE and oleandrin are efficacious agents to treat canine atopic dermatitis. Future studies should evaluate the efficacy of these compounds in dogs affected by atopic dermatitis.

Abstract

OBJECTIVE

To perform testing for cytokines involved in dermal inflammatory reactions and to document and compare the effects of an oleander extract (OE), oleandrin, and oclacitinib on biomarkers relevant to allergic reactions. The effects of these compounds under inflamed culture conditions are of direct importance to the treatment of canine atopic dermatitis.

METHODS

Testing involved primary canine dermal fibroblasts and the canine DH82 macrophage cell line; both cell types are important for initiating, regulating, and resolving dermal allergic reactions via cytokine communication.

RESULTS

Under inflamed conditions, OE and oleandrin downregulated key cytokines secreted by canine dermal fibroblasts and the DH82 macrophage cell line; all of which are treatment targets in dermatitis. In the DH82 macrophage cultures, the most noteworthy reductions involved IL-6, IL-12/IL-23p40, interferon-γ, tumor necrosis factor-α, VEGF, and nerve growth factor-β. Oclacitinib triggered reductions of some cytokines involved in allergic reactions, including TGF-β1, IL-12/IL-23p40, and tumor necrosis factor-α; however, these reductions were less robust than the reductions triggered by OE and oleandrin and accompanied by increases in other cytokines involved in dermal inflammation, including IL-6, interferon-γ, and nerve growth factor-β. In cultures of primary dermal fibroblasts, OE and oleandrin reduced the levels of IL-8 and monocyte chemoattractant protein-1, whereas oclacitinib had little or no effect.

CONCLUSIONS

Oleander extract and oleandrin directly modulate immune responses under inflamed conditions. Moreover, OE and oleandrin appear to provide a more beneficial overall cytokine regulation than oclacitinib under inflamed culture conditions.

CLINICAL RELEVANCE

These results suggest that OE and oleandrin are efficacious agents to treat canine atopic dermatitis. Future studies should evaluate the efficacy of these compounds in dogs affected by atopic dermatitis.

Canine atopic dermatitis is a common allergic skin disease in dogs that is typically caused by a hypersensitivity reaction to environmental allergens. Dogs with atopic dermatitis frequently display symptoms such as itching, redness, and skin lesions. This condition can significantly impact a dog's quality of life and may require ongoing (life-long) management to control symptoms and improve comfort.

A spectrum of pharmacological agents are commonly used to address the multifaceted manifestations of this allergic skin disorder including corticosteroids (prednisone or prednisolone); antihistamines; immunosuppressants (ie, cyclosporine); Apoquel (oclacitinib; works through inhibition of the Janus kinase [JAK]-signal transducer and activator of transcription [STAT] pathway)13; Cytopoint (lokivetmab; monoclonal antibody against IL-31); essential fatty acid supplements; and topical treatments including sprays, medicated shampoos, and ointments. While these treatments may mitigate inflammation, alleviate pruritus, and combat secondary skin infections in dogs afflicted with atopic dermatitis, long-term administration of these agents is often problematic for pet owners due to lack of efficacy, high cost, and/or undesirable side effects. Thus, research leading to the development of new, safer, and less expensive alternatives to treat dogs with atopic dermatitis is warranted.

Janus kinase inhibitors are a class of medications that work by targeting the JAK enzymes (JAK1, JAK2, JAK3, and TYK2).3 These enzymes are involved in transmitting signals from various cytokines and growth factors, which play crucial roles in immune responses and inflammation. Specifically, JAK inhibitors interfere with the activation of STAT proteins downstream of cytokine receptors. In atopic dermatitis, cytokines such as IL-4, IL-13, and IL-31 play key roles in promoting inflammation and pruritus of the skin. These cytokines signal through JAK-STAT pathways, particularly involving JAK1 and JAK3. By inhibiting JAK enzymes, JAK inhibitors disrupt the transmission of signals from these cytokines, thereby reducing the inflammatory response and alleviating symptoms associated with canine atopic dermatitis. This leads to a decrease in skin inflammation, pruritis, and overall improvement in coat condition. Angiogenesis may be involved in the pathogenesis of atopic dermatitis, where resident inflammatory mast cells and macrophages are major sources of VEGF production. Expression of VEGF in inflammatory skin lesions indicates that inhibition of angiogenesis is a useful strategy for treatment of chronic, inflammatory skin disorders.4

Oleandrin is a highly lipid-soluble cardiac glycoside isolated from the plant Nerium oleander (Apocynaceae) that has been used as a traditional herbal medicine. At high concentrations, it is known to be cardiotoxic; however, its safe use in dogs when administered orally has recently been reported.5 Recent studies6 have documented that oleandrin rapidly affects human immune cell function, both directly and in the context of immune challenges with known bacterial and viral mimetics. The aforementioned study used an in vitro model of human peripheral blood mononuclear cells to document effects under 3 different culture conditions: normal, challenged with the viral mimetic polyinosinic:polycytidylic acid poly I:C, and inflamed by lipopolysaccharide (LPS). Cells were evaluated for immune activation markers CD69, CD25, and CD107a, and culture supernatants were tested for cytokines.6 The study6 demonstrated that oleander extract (OE) and oleandrin directly activate human innate immune cells enhance antiviral immune responses through natural killer cell activation and interferon-γ (IFN-γ) levels but in contrast modulate immune responses under inflamed conditions.

Other natural glycosides have shown immune-modulating effects,7 and an aqueous extract of Nerium oleander has also shown immune-related effects in vitro.8 The objective of this study was to determine whether oleandrin and an OE may interfere with production of canine proinflammatory cytokines that are involved in allergic reactions and to compare those results with that of a known JAK inhibitor, oclacitinib, currently marketed for treatment of atopic dermatitis in dogs.

Methods

Oleandrin (Sigma-Aldrich), oclacitinib (Sigma-Aldrich), and an OE (PBI 05204; Phoenix Biotech­nology Inc) were prepared in DMSO and then diluted in PBS such that the highest concentration in the cell-based assays was 500 ng/mL. PBI-05204 is an extract of the Nerium oleander plant obtained using supercritical (meaning above 31 °C and 71 bar [100 kPa]) CO2 extraction technology and was provided by Phoenix Biotechnology Inc. Characterization of PBI-05204 was carried out using an AccuTOF-DART mass spectrometer (Jeol).6 Specific content of the extract was previously reported.6 The specific batch of OE tested had an oleandrin content of approximately 3%. Because prior dose-normalized in vitro and in vivo assays across a wide range of therapeutic areas have demonstrated that oleandrin is the key active component of PBI-05204, the concentration of OE in the cell cultures was normalized according to its oleandrin content, spanning a dose range of 0.03 to 0.5 µg/mL oleandrin. The concentration in cell cultures was determined based on previously published data on human immune cells6 and initial viability testing on the 2 cell types used here with the Invitrogen CyQUANT MTT Cell Viability Assay. This testing for cellular relative metabolic activity was conducted before cytokine testing to ensure that the cells were viable and active when treated with test products.

We tested 3 different concentrations of oclacitinib: 10 nM, 500 nM, and 10 µM, to cover the dose range in published literature. DMSO was used as a control.

Canine cell cultures

Two types of cells were used (1) primary canine dermal fibroblasts (Cell Biologics), and (2) the canine DH82 macrophage cell line (Distributor [Millipore Sigma] and Origin [Culture Collections UK Health Security Agency]). The DH82 cell line has been shown to be a useful tool in evaluating inflammatory reactivity in canine macrophages, with similarities in phenotype and function to primary canine monocyte-derived macrophages.9 DH82 cells have been used to study macrophage polarization toward M1 versus M2 subtypes relevant for allergic reactivity, as the M2a subtype of macrophages plays an important role in immunoglobulin E–mediated allergies and other T-helper 2 (Th2)–type immune reactions.10

The test products OE, oleandrin, and oclacitinib were compared in canine cell cultures under unstressed and inflamed culture conditions to expand knowledge of their anti-inflammatory activities as they pertain to allergies.  Both dermal fibroblast and macrophage cell cultures were tested under normal or inflamed conditions, where the inflammatory conditions were induced by LPS (Sigma-Aldrich). After 24 hours, cells were pelleted by centrifugation, and the culture supernatants were collected and used for testing cytokine levels using Bio-Plex protein arrays (Bio-Rad Laboratories Inc) and utilizing xMAP technology (Luminex). The following cytokine panels were used: (1) human 15-plex for Th17 cytokines: IL-1β, IL-4, lL-6, IL-10, IL-17A, IL-17F, IL-21, IL-22, IL-23, IL-25, IL-31, IL-33, IFN-γ, sCD40L, and tumor necrosis factor-α (TNF-α); and 2) canine 11-plex: IL-2, IL-6, IL-8, TGF-β1, IL-12/IL-23p40, nerve growth factor-β, IFN-γ, TNF-α, monocyte chemoattractant protein-1 (MCP-1; CCL2), VEGF-A, and stem cell factor (SCF). The use of a human cytokine multiplex panel of antibodies in addition to a canine cytokine multiplex panel was necessary to try to determine the broadest array of cytokine response data relevant to atopic dermatitis benefiting from the antibody cross-reactivity between human and canine cytokines.

Data analysis

Average and SD for each data set were calculated using Microsoft Excel, version 2405. The analysis involved calculation of statistical significance when comparing the data from cell cultures treated with test products to DMSO-treated control cultures, matching the dose of DMSO control to the dose of DMSO in each dose of test product, using the 2-tailed unpaired t test. Statistical significance was indicated if P < .05, and a high level of significance was indicated if P < .01.

Results

Effects on canine dermal fibroblast cytokine secretion

Culture supernatants from the canine dermal fibroblasts were tested for cytokine levels using a canine 11-plex detection kit. Under the culture conditions used, the following cytokines were detectable in supernatants from dermal fibroblasts: IL-6, IL-8, MCP-1 (CCL2), VEGF-A, and SCF. As shown (Figure 1), IL-8, MCP-1, and SCF levels were reduced by all 3 test products. For IL-8, a similar reduction was seen for each of the test products, while for MCP-1, OE triggered the most robust reduction in secretion of this cytokine by the dermal fibroblasts. For SCF, oleandrin triggered the greatest reduction in this cytokine expression, and in contrast, OE showed the mildest reduction.

Figure 1
Figure 1

Cytokine secretion by canine dermal fibroblasts: heat map showing the levels of significance for changes to cytokine levels in cell cultures treated with oleandrin extract (OE), oleandrin, and oclacitinib. A—Direct effects of the test products under normal culture conditions. B—Effects of the test products under inflamed culture conditions, where inflammation was induced by lipopolysaccharide. The numbers in the heat maps indicate the percent change from untreated cultures (A) and from lipopolysaccharide-treated control cultures (B) and show the largest change across the dose range of 0.03 to 0.5 µg/mL oleandrin and 0.01 to 10 µM oclacitinib. The dose range of the botanical OE was matched to oleandrin for oleandrin content. The color coding displays the level of significance of a change. IFN-γ = Interferon-γ. MCP-1 = Monocyte chemoattractant protein-1. NGF-β = Nerve growth factor-β. SCF = Stem cell factor. TNF-α = Tumor necrosis factor-α.

Citation: American Journal of Veterinary Research 2024; 10.2460/ajvr.24.05.0153

Under LPS-mediated inflamed conditions, the secretion of IL-8 was reduced by all 3 test products, where robust reductions were mediated by OE and oleandrin, in contrast to only a mild reduction produced by oclacitinib (Figure 1). VEGF-A levels were reduced by both OE and oleandrin, reaching statistical significance for oleandrin for both cytokines and for OE for MCP-1. While MCP-1 levels were not affected by treatment of the dermal fibroblasts with oclacitinib, OE and oleandrin triggered statistically significant reductions in the secretion of this cytokine (Figure 2). The anti-inflammatory effects of OE and oleandrin, specifically for IL-8 and MCP-1, were seen at the low dose of 0.03 μg/mL cell culture, whereas the low dose of 0.01 μM oclacitinib was not efficacious in reducing the secretion of these 2 cytokines from dermal fibroblasts.

Figure 2
Figure 2

Cytokine secretion by canine dermal fibroblasts: changes to cytokine production by canine DH82 macrophage cells under inflamed culture conditions. Detection antibodies (IL-8 [A] and MCP-1 [B]) toward canine cytokines are shown. Data for the middle dose of each test product is shown (oleandrin, 0.125 µg/mL; oclacitinib, 0.5 µM). For both cytokines, OE and oleandrin triggered robust reductions in the cytokine levels, where IL-8 secretion was significantly reduced by oleandrin and highly significantly by OE and MCP-1 secretion was significantly reduced by oleandrin and the reduction triggered by OE was near (P < .01) but did not reach statistical significance. In contrast, oclacitinib reduced IL-8 by only a few percent and had no effect on MCP-1 secretion. When comparing the test products to controls, statistical significance is indicated with (*)P < .1; *P < .05; **P < .01.

Citation: American Journal of Veterinary Research 2024; 10.2460/ajvr.24.05.0153

Effects on canine DH82 macrophage cytokine secretion using canine detection kit

The culture supernatants from the canine DH82 macrophage cell line were tested for cytokine levels using a canine 11-plex detection kit, both under normal and inflamed culture conditions. As shown (Figure 3), data produced under normal culture conditions to document the direct immune-modulating effects of the test products were as follows. Levels of IL-6 were increased by OE and oleandrin but not oclacitinib. The levels of IL-8 and MCP-1 were reduced by oclacitinib but not affected by either OE or oleandrin. Oclacitinib triggered increased levels of SCF but was not statistically significant (P < .1), in contrast to no significant effects by either OE or oleandrin. The production of IFN-γ, TGF-β, and IL-12/IL-23 by the DH82 macrophages was not significantly changed by the test products. Finally, IL-2 was not detectable in supernatant solutions from the DH82 macrophages.

Figure 3
Figure 3

Cytokine secretion by the canine DH82 macrophage cell line: heat map showing the levels of significance for changes to cytokine levels in cell cultures treated with OE, oleandrin, and oclacitinib. A—Direct effects of the test products under normal culture conditions. B—Effects of the test products under inflamed culture conditions, where inflammation was induced by lipopolysaccharide. The numbers in the heat maps indicate the percent change from untreated cultures (A) and from lipopolysaccharide-treated control cultures (B) and show the largest change across the dose range of 0.03 to 0.5 µg/mL oleandrin and 0.01 to 10 µM oclacitinib. The dose range of the botanical OE was matched to oleandrin for oleandrin content. The color coding displays the level of significance of a change.

Citation: American Journal of Veterinary Research 2024; 10.2460/ajvr.24.05.0153

Under inflamed conditions (Figure 4), IL-6 levels were robustly reduced by the lower concentrations of OE and moderately reduced by oleandrin, which was in contrast to a mild increase triggered by oclacitinib. Oclacitinib induced increased levels of IFN-γ and SCF, in contrast to reduced levels by OE and oleandrin. In addition, OE and oleandrin reduced levels of NGF-β and VEGF, but these were not affected by oclacitinib. The levels of TGF-β, IL-12/IL-23, and TNF-α were reduced by all 3 test products with oleandrin triggering the most robust reduction in TGF-β levels.

Figure 4
Figure 4

Cytokine secretion by the canine DH82 macrophage cell line: changes to cytokine production by canine DH82 macrophage cells under inflamed culture conditions, using detection antibodies toward canine cytokines. Data for the lowest doses of the test products are shown. For all 6 cytokines (A–F), OE and oleandrin triggered robust reductions in the cytokine levels, reaching a high level of statistical significance for IL-6 and significance for IL-12/IL-23p40 and TNF-α. In contrast, only the 2 cytokines IL-12/IL-23p40 and TNF-α were reduced by oclacitinib and not to the same level as for OE and oleandrin. Oclacitinib triggered increased levels of IL-6 and IFN-γ. Statistical significance is indicated if P < .05, and a high level of significance is indicated if P < .01. When comparing the test products to controls, statistical significance is indicated with (*)P < .1; *P < .05; **P < .01

Citation: American Journal of Veterinary Research 2024; 10.2460/ajvr.24.05.0153

Effects on canine DH82 macrophage cytokine secretion using human Th17 cytokine detection kit

Due to the limitations on cytokine detection antibodies toward canine cytokines, human detection antibodies were used to test for secretion of cytokines that were not available in canine detection kits. Culture supernatants from canine DH82 macrophage cells were tested for 5 additional cytokines of relevance to atopic dermatitis using the human detection kits. Five cytokines were detectable, and the results are presented for IL-1β, IL-10, IL-22, IL-23, and sCD40L (Figure 5).

Figure 5
Figure 5

Cytokine secretion by the canine DH82 macrophage cell line: a human cytokine detection kit was used to capture data on cytokines where such kits are not available for canine research: heat map showing the levels of significance for changes to cytokine levels in cell cultures treated with OE, oleandrin, and oclacitinib. A—Direct effects of the test products under normal culture conditions. B—Effects of the test products under inflamed culture conditions, where inflammation was induced by lipopolysaccharide. The numbers in the heat maps indicate the percent change from untreated cultures (A) and from lipopolysaccharide-treated control cultures (B) and show the largest change across the dose range of 0.03 to 0.5 µg/mL oleandrin and 0.01 to 10 µM oclacitinib. The dose range of the botanical OE was matched to oleandrin for oleandrin content. The color coding displays the level of significance of a change.

Citation: American Journal of Veterinary Research 2024; 10.2460/ajvr.24.05.0153

Under normal unstressed culture conditions, oclacitinib triggered increases for all 5 of these cytokines with the increase in IL-22 being significant (44% increase; Figure 5). The increased levels of TNF-α, IL-1β, and IL-23 at low concentrations of oclacitinib were not significant. In contrast, under normal unstressed conditions, neither OE nor oleandrin triggered significant changes for any of the 5 proinflammatory cytokines.

Under inflamed culture conditions, oclacitinib triggered reduced IL-1b secretion (not significant) and only minor changes to the other cytokines examined (Figure 6). For IL-10, the low doses of all 3 test products triggered decreased levels; this was most robust for OE and oleandrin. For IL-1β, the lowest concentrations of all 3 test products resulted in decreased levels.

Figure 6
Figure 6

Changes to cytokine production by canine dermal fibroblasts under inflamed culture conditions: using detection antibodies toward human cytokines, changes to cytokine production by canine dermal fibroblasts under inflamed culture conditions using data for the lowest dose of each test product are shown (A–D). While a similar magnitude of IL-1β reduction was seen for all 3 test products (A). Oleandrin and OE showed more effect on IL-10 (B) secretion.

Citation: American Journal of Veterinary Research 2024; 10.2460/ajvr.24.05.0153

Discussion

The goal of this work was to examine whether an OE and oleandrin triggered downregulation of cellular communication compounds (cytokines) involved in allergic reactions and to compare these results with that of a known medication for atopic dermatitis in dogs, oclacitinib. The testing involved dermal fibroblasts and immune cells, the latter representing immune cells that are present in the microcirculation of the skin, as well as immune cells residing in the skin tissue. Canine dermal fibroblasts were tested in parallel to the canine macrophage cell line DH82.

The cellular production of cytokines was tested under normal versus inflamed culture conditions, where the inflamed conditions are of interest for the chronic inflammatory conditions in atopic dermatitis. The results showed a reduction of proinflammatory cytokines of relevance to atopic dermatitis in cultures treated with OE and oleandrin. Microscopic observations did not show reduced cell viability, and the data were interpreted as the cells being highly activated.

Using human detection kits, under normal unstressed culture conditions, neither OE nor oleandrin triggered detectable changes to any of the 6 cytokines. In contrast, oclacitinib triggered increased levels of IL-1β, IL-10, IL-22, IL-23, sCD40L, and TNF-α, the first 3 reaching statistical significance. Elevation of the cytokines may result in an undesirable inflammatory response in vivo. Indeed, it has been reported that Apoquel is more efficacious when it is initially given with corticosteroids for 4 days.11 The data reported here suggest that anti-inflammatory adjunct therapy will not be necessary when treating canine atopic dermatitis with OE or oleandrin. Under inflamed conditions, OE and oleandrin triggered reduced levels of TNF-α (human kit) at the lowest concentration of both products. Only for the highest concentration of oclacitinib was a reduction seen for this cytokine; this concentration may not be clinically relevant. The proinflammatory cytokine IL-1β was reduced by all 3 test products. None of the 3 test products triggered changes to the levels of IL-22, IL-23, or sCD40L. While a similar magnitude of IL-1β reduction was seen for all 3 test products, only OE and oleandrin reduced TNF-α levels.

While the direct effects of OE and oleandrin are of interest in understanding normal healthy skin functions, the effects of OE and oleandrin using canine detection kits under inflamed culture conditions are of direct importance to treatment of canine atopic dermatitis. Under inflamed conditions, both OE and oleandrin downregulated key cytokines secreted by both canine dermal fibroblasts and DH82 macrophages. The overall pattern of reduced cytokine production under inflamed conditions was more favorable for OE and oleandrin than for the leading pharmaceutical drug for canine dermatitis, oclacitinib. Oclacitinib triggered reductions of some cytokines, including TGF-β, IL-12/IL-23p40, and TNF-α; however, these reductions were less robust than the reductions triggered by OE and oleandrin. Furthermore, oclacitinib also triggered increases in other cytokines involved in dermal inflammation, including IL-6, IFN-γ, and SCF. For all 6 cytokines, OE and oleandrin triggered robust reductions in the cytokine levels. In contrast, only the 2 cytokines IL-12/IL-23p40 and TNF-α were reduced by oclacitinib and not to the same level as for OE or oleandrin. Oclacitinib triggered increased levels of IL-6 and IFN-γ. These data suggest that OE or oleandrin may provide a therapeutic advantage over oclacitinib. Moreover, in cultures of canine dermal fibroblast, under inflamed conditions, both OE and oleandrin triggered reduced levels of IL-8 and MCP-1. This was in contrast to oclacitinib, which did not trigger a change in MCP-1 and only triggered a very minor reduction in IL-8 levels that may not be clinically relevant.

The robust and significant reduction in IL-12/IL-23p40 by both OE and oleandrin is important in the context of dermatitis. The cytokines IL-12 and IL-23 share a common IL-12/IL-23p40 subunit in structure and play a central role in T-cell–mediated responses in inflammation. Overactivated IL-12 and IL-23 signaling drives aberrant Th1 and Th17 immune responses and contributes to immune-mediated diseases.12 The reduction in NGF-β by OE and oleandrin is also noteworthy. In a human clinical trial of patients with atopic dermatitis, the level of NGF-β was significantly higher in affected patients than in healthy controls and correlated with the severity of itch, erythema, scale/xerosis, and eosinophil count.13 The growth factor VEGF and the inflammatory cytokines TNF-α and IL-8 were reduced by OE and oleandrin under inflamed conditions. These cytokines are known to be upregulated in chronic skin disorders and induce angiogenesis.4 In addition, SCF influences mast cell activation and the release of inflammatory mediators and is elevated in tissues undergoing allergic inflammation.14

In conclusion, the overall pattern of reduced cytokine production under inflamed conditions was favorable for OE and oleandrin. We have previously shown that both OE and oleandrin directly activate natural killer cells.6 For some of the cytokines, all 3 compositions exhibited dose-dependent proinflammatory versus anti-inflammatory effects. Oclacitinib, under both unstressed and inflamed culture conditions, induced cytokines that are thought to be undesirable in dermal allergies. Because of the broader and generally more favorable cytokine response seen with OE and oleandrin as compared to oclacitinib, it may be interesting to evaluate combination therapy with products such as oclacitinib; however, these data suggest that sole therapy with OE or oleandrin may reduce both the pruritis and inflammation noted in affected dogs. Further evaluation of OE and oleandrin in the treatment of canine atopic dermatitis appears to be warranted. A limitation of the study was that canine IL-31 was not commercially available and there was limited cross-reactivity between human IL-31 and canine cells. As this is a very important cytokine relative to the expression and severity of canine atopic dermatitis, the future testing of the ability of products to reduce expression levels of IL-31 will be important.

Acknowledgments

None reported.

Disclosures

Dr. Fossum is the CEO of Phoenix Animal Wellness and Dr. Fossum's Pet Care. The former is a subsidiary of Phoenix Biotechnology Inc, which owns the patents for the oleander extracts used in these studies. Drs. Newman and Matos are members of the Board of Directors for Phoenix Animal Wellness.

No AI-assisted technologies were used in the generation of this manuscript.

Funding

This research was supported in part by funds from Phoenix Biotechnology Inc.

References

  • 1.

    Fukuyama T, Ehling S, Cook E, Bäumer W. Topically administered janus-kinase inhibitors tofacitinib and oclacitinib display impressive antipruritic and anti-inflammatory responses in a model of allergic dermatitis. J Pharmacol Exp Ther. 2015;354(3):394405. doi:10.1124/jpet.115.223784

    • Search Google Scholar
    • Export Citation
  • 2.

    Banovic F, Tarigo J, Gordon H, Barber JP, Gogal RM Jr. Immunomodulatory in vitro effects of oclacitinib on canine T-cell proliferation and cytokine production. Vet Dermatol. 2019;30(1):17-e6. doi:10.1111/vde.13037

    • Search Google Scholar
    • Export Citation
  • 3.

    Gonzales AJ, Bowman JW, Fici GJ, Zhang M, Mann DW, Mitton-Fry M. Oclacitinib (APOQUEL(®)) is a novel Janus kinase inhibitor with activity against cytokines involved in allergy. J Vet Pharmacol Ther. 2014;37(4):317324. doi:10.1111/jvp.12101

    • Search Google Scholar
    • Export Citation
  • 4.

    Lee HJ, Hong YJ, Kim M. Angiogenesis in chronic inflammatory skin disorders. Int J Mol Sci. 2021;22(21):12035. doi: 10.3390/ijms222112035

    • Search Google Scholar
    • Export Citation
  • 5.

    Fossum T, Newman R, Matos J. Preliminary investigation of the safe dose of oleandrin when administered orally to Beagles. J Am Vet Med Assoc. 2024;262(7):966972. doi:10.2460/javma.23.11.0651

    • Search Google Scholar
    • Export Citation
  • 6.

    Jensen G, Yu L, Iloba I, Cruickshank D, Matos J, Newman R. Differential activities of the botanical extract PBI-05204 and oleandrin on innate immune functions under viral challenge versus inflammatory culture conditions. Molecules. 2023;16;28(12):4799. doi:10.3390/molecules28124799

    • Search Google Scholar
    • Export Citation
  • 7.

    Xie S, Spelmink L, Codemo M, et al. Cinobufagin modulates human innate immune responses and triggers antibacterial activity. PLoS One. 2016;11(8):e0160734. doi:10.1371/journal.pone.0160734

    • Search Google Scholar
    • Export Citation
  • 8.

    Benson K, Newman R, Jensen G. Antioxidant, anti-inflammatory, anti-apoptotic, and skin regenerative properties of an Aloe vera-based extract of Nerium oleander leaves (NAE-8®). Clin Cosmet Investig Dermatol. 2015;8:239248 doi:10.2147/CCID.S79871

    • Search Google Scholar
    • Export Citation
  • 9.

    Barnes A, Bee A, Bell S, et al. Immunological and inflammatory characterization of three canine cell lines: K1, K6 and DH82. Vet Immunol Immunopathol. 2000;75(1-2):925. doi:org/10.1016/S0165-2427(00)00184-7

    • Search Google Scholar
    • Export Citation
  • 10.

    Herrmann I, Gotovina J, Fazekas-Singer J, et al. Canine macrophages can like human macrophages be in vitro activated toward the M2a subtype relevant in allergy. Dev Comp Immunol. 2018;82:118127. doi:10.1016/j.dci.2018.01.005

    • Search Google Scholar
    • Export Citation
  • 11.

    Olivry T, Lokianskiene V, Blanco A, Del Mestre P, Bergvall K, Beco L. A randomised controlled trial testing the rebound-preventing benefit of four days of prednisolone during the induction of oclacitinib therapy in dogs with atopic dermatitis. Vet Dermatol. 2023;34(2):99106. doi:10.1111/vde.13134

    • Search Google Scholar
    • Export Citation
  • 12.

    Chyuan IT, Lai JH. New insights into the IL-12 and IL-23: from a molecular basis to clinical application in immune-mediated inflammation and cancers. Biochem Pharmacol. 2020;175:113928. doi:10.1016/j.dci.2018.01.005

    • Search Google Scholar
    • Export Citation
  • 13.

    Yamaguchi J, Aihara M, Kobayashi Y, Kambara T, Ikezawa Z. Quantitative analysis of nerve growth factor (NGF) in the atopic dermatitis and psoriasis horny layer and effect of treatment on NGF in atopic dermatitis. J Dermatol Sci. 2009;53(1):4854. doi:10.1016/j.jdermsci.2008.08.011

    • Search Google Scholar
    • Export Citation
  • 14.

    Hammerberg B, Olivry T, Orton SM. Skin mast cell histamine release following stem cell factor and high-affinity immunoglobulin E receptor cross-linking in dogs with atopic dermatitis. Vet Dermatol. 2001;12(6):339346. doi:10.1046/j.0959-4493

    • Search Google Scholar
    • Export Citation
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