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Ingenol Disoxate: A Novel 4-Isoxazolecarboxylate Ester of Ingenol with Improved Properties for Treatment of Actinic Keratosis and Other Non-Melanoma Skin Cancers

View Article: PubMed Central - PubMed

ABSTRACT

Introduction: Ingenol mebutate gel (Picato®, LEO Pharma A/S) is approved for the field treatment of actinic keratosis and is characterized by high sustained clearance of actinic lesions. The inherent propensity of ingenol mebutate towards chemical rearrangement necessitates refrigeration of the final product. We sought to identify novel ingenol derivatives with enhanced chemical stability and similar or improved in vitro potency and in vivo efficacy.

Methods: A number of ingenol esters were synthesized with full regiocontrol from ingenol. Chemical stability was determined in aqueous buffer at physiological pH and hydroalcoholic gel at lower pH. Acute cytotoxicity was determined in HeLa or HSC-5 cells. Keratinocyte proliferation, viability and caspase 3/7 activation was measured in primary epidermal keratinocytes. Relative gene expression levels were determined by real-time quantitative PCR. Evaluation of in vivo tumor ablating potential was performed in the murine B16 melanoma mouse model and in the UV-induced skin carcinogenesis model in hairless SKH-1 mice following topical treatment for two consecutive days with test compounds formulated at 0.1% in a hydroalcoholic gel.

Results: This work resulted in the identification of ingenol disoxate (LEO 43204) displaying increased stability in a clinically relevant formulation and in aqueous buffer with minimal pH-dependent acyl migration degradation. Ingenol disoxate exhibited a significantly higher cytotoxic potency relative to ingenol mebutate. Likewise, cell growth arrest in normal human keratinocyte was more potently induced by ingenol disoxate, which was accompanied by protein kinase C dependent transcription of markers of keratinocyte differentiation. Most notably, ingenol disoxate possessed a superior antitumor effect in a B16 mouse melanoma model and significantly increased median survival time relative to ingenol mebutate. A significant effect on tumor ablation was also observed in a murine model of ultraviolet irradiation-induced skin carcinogenesis.

Conclusion: These data illustrate that the favorable in vitro and in vivo pharmacological properties driving ingenol mebutate efficacy are either preserved or improved in ingenol disoxate. In combination with improved chemical stability to potentially facilitate storage of the final product at ambient temperatures, these features support further development of ingenol disoxate as a convenient and efficacious treatment modality of non-melanoma skin cancers.

Funding: LEO Pharma A/S.

Electronic supplementary material: The online version of this article (doi:10.1007/s13555-016-0137-2) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus

Comparison of antitumor effects in a UV-induced skin cancer model. Hairless SKH1 mice were irradiated 3× weekly for 10 weeks with a minimally erythemal dose of UV, leading to appearance of skin tumors starting at week 11. Skin tumors were treated topically for two consecutive days with ingenol disoxate or ingenol mebutate in hydroalcoholic gel formulations. Treatment of tumors was done individually starting 1–2 weeks after their appearance. Kaplan–Meyer survival curves are shown with ‘survival’ time defined as the time until a visible tumor appears within the tattooed area. Due to the skin reaction induced by the compounds it was not possible to assess the tumors until 2 weeks after treatment
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Fig11: Comparison of antitumor effects in a UV-induced skin cancer model. Hairless SKH1 mice were irradiated 3× weekly for 10 weeks with a minimally erythemal dose of UV, leading to appearance of skin tumors starting at week 11. Skin tumors were treated topically for two consecutive days with ingenol disoxate or ingenol mebutate in hydroalcoholic gel formulations. Treatment of tumors was done individually starting 1–2 weeks after their appearance. Kaplan–Meyer survival curves are shown with ‘survival’ time defined as the time until a visible tumor appears within the tattooed area. Due to the skin reaction induced by the compounds it was not possible to assess the tumors until 2 weeks after treatment

Mentions: A widely used model for skin carcinogenesis is UV-induced skin cancer in hairless mice, where repeated UV irradiations over several months lead to the development of skin tumors, primarily papillomas that finally may progress into SCC. Previous work has shown that field treatment with ingenol mebutate gel after completion of UV irradiation but before development of skin lesions leads to a reduction in tumor incidence [31]. We have used a slightly adapted model to compare the ability of ingenol mebutate and ingenol disoxate to treat UV-induced skin tumors. In our model, we treated individual tumors after they appeared with ingenol mebutate or ingenol disoxate in hydroalcoholic gel for two consecutive days. Ingenol mebutate 0.1% and ingenol disoxate 0.1% showed comparable effect, whereas ingenol mebutate 0.05% displayed a slightly weaker effect (Fig. 11). None of the tumors in untreated mice regressed spontaneously.Fig. 11


Ingenol Disoxate: A Novel 4-Isoxazolecarboxylate Ester of Ingenol with Improved Properties for Treatment of Actinic Keratosis and Other Non-Melanoma Skin Cancers
Comparison of antitumor effects in a UV-induced skin cancer model. Hairless SKH1 mice were irradiated 3× weekly for 10 weeks with a minimally erythemal dose of UV, leading to appearance of skin tumors starting at week 11. Skin tumors were treated topically for two consecutive days with ingenol disoxate or ingenol mebutate in hydroalcoholic gel formulations. Treatment of tumors was done individually starting 1–2 weeks after their appearance. Kaplan–Meyer survival curves are shown with ‘survival’ time defined as the time until a visible tumor appears within the tattooed area. Due to the skin reaction induced by the compounds it was not possible to assess the tumors until 2 weeks after treatment
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC5120626&req=5

Fig11: Comparison of antitumor effects in a UV-induced skin cancer model. Hairless SKH1 mice were irradiated 3× weekly for 10 weeks with a minimally erythemal dose of UV, leading to appearance of skin tumors starting at week 11. Skin tumors were treated topically for two consecutive days with ingenol disoxate or ingenol mebutate in hydroalcoholic gel formulations. Treatment of tumors was done individually starting 1–2 weeks after their appearance. Kaplan–Meyer survival curves are shown with ‘survival’ time defined as the time until a visible tumor appears within the tattooed area. Due to the skin reaction induced by the compounds it was not possible to assess the tumors until 2 weeks after treatment
Mentions: A widely used model for skin carcinogenesis is UV-induced skin cancer in hairless mice, where repeated UV irradiations over several months lead to the development of skin tumors, primarily papillomas that finally may progress into SCC. Previous work has shown that field treatment with ingenol mebutate gel after completion of UV irradiation but before development of skin lesions leads to a reduction in tumor incidence [31]. We have used a slightly adapted model to compare the ability of ingenol mebutate and ingenol disoxate to treat UV-induced skin tumors. In our model, we treated individual tumors after they appeared with ingenol mebutate or ingenol disoxate in hydroalcoholic gel for two consecutive days. Ingenol mebutate 0.1% and ingenol disoxate 0.1% showed comparable effect, whereas ingenol mebutate 0.05% displayed a slightly weaker effect (Fig. 11). None of the tumors in untreated mice regressed spontaneously.Fig. 11

View Article: PubMed Central - PubMed

ABSTRACT

Introduction: Ingenol mebutate gel (Picato®, LEO Pharma A/S) is approved for the field treatment of actinic keratosis and is characterized by high sustained clearance of actinic lesions. The inherent propensity of ingenol mebutate towards chemical rearrangement necessitates refrigeration of the final product. We sought to identify novel ingenol derivatives with enhanced chemical stability and similar or improved in vitro potency and in vivo efficacy.

Methods: A number of ingenol esters were synthesized with full regiocontrol from ingenol. Chemical stability was determined in aqueous buffer at physiological pH and hydroalcoholic gel at lower pH. Acute cytotoxicity was determined in HeLa or HSC-5 cells. Keratinocyte proliferation, viability and caspase 3/7 activation was measured in primary epidermal keratinocytes. Relative gene expression levels were determined by real-time quantitative PCR. Evaluation of in vivo tumor ablating potential was performed in the murine B16 melanoma mouse model and in the UV-induced skin carcinogenesis model in hairless SKH-1 mice following topical treatment for two consecutive days with test compounds formulated at 0.1% in a hydroalcoholic gel.

Results: This work resulted in the identification of ingenol disoxate (LEO 43204) displaying increased stability in a clinically relevant formulation and in aqueous buffer with minimal pH-dependent acyl migration degradation. Ingenol disoxate exhibited a significantly higher cytotoxic potency relative to ingenol mebutate. Likewise, cell growth arrest in normal human keratinocyte was more potently induced by ingenol disoxate, which was accompanied by protein kinase C dependent transcription of markers of keratinocyte differentiation. Most notably, ingenol disoxate possessed a superior antitumor effect in a B16 mouse melanoma model and significantly increased median survival time relative to ingenol mebutate. A significant effect on tumor ablation was also observed in a murine model of ultraviolet irradiation-induced skin carcinogenesis.

Conclusion: These data illustrate that the favorable in vitro and in vivo pharmacological properties driving ingenol mebutate efficacy are either preserved or improved in ingenol disoxate. In combination with improved chemical stability to potentially facilitate storage of the final product at ambient temperatures, these features support further development of ingenol disoxate as a convenient and efficacious treatment modality of non-melanoma skin cancers.

Funding: LEO Pharma A/S.

Electronic supplementary material: The online version of this article (doi:10.1007/s13555-016-0137-2) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus