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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

PKC-dependent induction of keratinocyte differentiation markers. Primary human epidermal keratinocytes were incubated with 100 nM ingenol disoxate or 100 nM ingenol mebutate in low calcium medium (0.06 mM CaCl2) or exposed to high calcium medium (1.8 mM CaCl2) for 72 h. When indicated cells were pre-incubated with 500 nM bisindolylmaleimide I (BIS) for 30 min prior to exposure to 100 nM ingenol derivatives. Markers of keratinocyte differentiation were analyzed by qPCR. Bars depict relative expression compared to vehicle-treated keratinocytes from one representative experiment (mean ± standard error of mean). The experiment was performed in two donors with similar results
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Fig6: PKC-dependent induction of keratinocyte differentiation markers. Primary human epidermal keratinocytes were incubated with 100 nM ingenol disoxate or 100 nM ingenol mebutate in low calcium medium (0.06 mM CaCl2) or exposed to high calcium medium (1.8 mM CaCl2) for 72 h. When indicated cells were pre-incubated with 500 nM bisindolylmaleimide I (BIS) for 30 min prior to exposure to 100 nM ingenol derivatives. Markers of keratinocyte differentiation were analyzed by qPCR. Bars depict relative expression compared to vehicle-treated keratinocytes from one representative experiment (mean ± standard error of mean). The experiment was performed in two donors with similar results

Mentions: To further explore the mechanism underlying the antiproliferative effect we investigated if treatment could impact keratinocyte differentiation. In the epidermis of healthy skin, keratinocytes proliferate in the basal layer but commit to terminal differentiation in response to a vertical calcium gradient as they move outwards through the supra-basal layers before forming the stratum corneum barrier. This strictly regulated balance between proliferation and differentiation is profoundly disturbed in non-melanoma skin cancer. At a concentration (100 nM) inducing complete growth arrest both ingenol disoxate and ingenol mebutate stimulated transcription of intermediate (INVL) and late (TGM1, FLG, LOR) keratinocyte differentiation markers (Fig. 6).Fig. 6


Ingenol Disoxate: A Novel 4-Isoxazolecarboxylate Ester of Ingenol with Improved Properties for Treatment of Actinic Keratosis and Other Non-Melanoma Skin Cancers
PKC-dependent induction of keratinocyte differentiation markers. Primary human epidermal keratinocytes were incubated with 100 nM ingenol disoxate or 100 nM ingenol mebutate in low calcium medium (0.06 mM CaCl2) or exposed to high calcium medium (1.8 mM CaCl2) for 72 h. When indicated cells were pre-incubated with 500 nM bisindolylmaleimide I (BIS) for 30 min prior to exposure to 100 nM ingenol derivatives. Markers of keratinocyte differentiation were analyzed by qPCR. Bars depict relative expression compared to vehicle-treated keratinocytes from one representative experiment (mean ± standard error of mean). The experiment was performed in two donors with similar results
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5120626&req=5

Fig6: PKC-dependent induction of keratinocyte differentiation markers. Primary human epidermal keratinocytes were incubated with 100 nM ingenol disoxate or 100 nM ingenol mebutate in low calcium medium (0.06 mM CaCl2) or exposed to high calcium medium (1.8 mM CaCl2) for 72 h. When indicated cells were pre-incubated with 500 nM bisindolylmaleimide I (BIS) for 30 min prior to exposure to 100 nM ingenol derivatives. Markers of keratinocyte differentiation were analyzed by qPCR. Bars depict relative expression compared to vehicle-treated keratinocytes from one representative experiment (mean ± standard error of mean). The experiment was performed in two donors with similar results
Mentions: To further explore the mechanism underlying the antiproliferative effect we investigated if treatment could impact keratinocyte differentiation. In the epidermis of healthy skin, keratinocytes proliferate in the basal layer but commit to terminal differentiation in response to a vertical calcium gradient as they move outwards through the supra-basal layers before forming the stratum corneum barrier. This strictly regulated balance between proliferation and differentiation is profoundly disturbed in non-melanoma skin cancer. At a concentration (100 nM) inducing complete growth arrest both ingenol disoxate and ingenol mebutate stimulated transcription of intermediate (INVL) and late (TGM1, FLG, LOR) keratinocyte differentiation markers (Fig. 6).Fig. 6

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