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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.


Stimulation of IL-8 secretion from keratinocytes. a Secreted IL-8 levels were quantified in the supernatant from primary human epidermal keratinocytes incubated with increasing concentrations of ingenol disoxate (closed circles) or ingenol mebutate (open circles) for 6 h. Plots are composites of four individual experiments (mean ± standard error of the mean). b Half-maximal stimulatory concentrations [EC50 with 95% confidence interval (CI 95%)] of IL-8 release for ingenol disoxate, ingenol mebutate and the 5- and 20-isomers of ingenol disoxate. For N < 3 individual EC50 estimates are given
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Fig7: Stimulation of IL-8 secretion from keratinocytes. a Secreted IL-8 levels were quantified in the supernatant from primary human epidermal keratinocytes incubated with increasing concentrations of ingenol disoxate (closed circles) or ingenol mebutate (open circles) for 6 h. Plots are composites of four individual experiments (mean ± standard error of the mean). b Half-maximal stimulatory concentrations [EC50 with 95% confidence interval (CI 95%)] of IL-8 release for ingenol disoxate, ingenol mebutate and the 5- and 20-isomers of ingenol disoxate. For N < 3 individual EC50 estimates are given

Mentions: The recruitment of neutrophil granulocytes into ingenol mebutate treatment fields has been shown to be critical for the eradication of engrafted tumors in a mouse tumor model [30]. Ingenol mebutate is believed to facilitate neutrophil granulocyte tissue infiltration via PKC-dependent mechanisms including upregulation of adhesion molecules on endothelial cells as well as cytokine release, in particular IL-8, from keratinocytes and other cells residing in the skin [14, 30]. We wanted to test if ingenol disoxate was associated with a similar pro-inflammatory response at nontoxic concentrations. Therefore, we investigated the capacity to stimulate IL-8 release from keratinocytes and found that ingenol disoxate stimulated release of this cytokine with a potency (EC50 = 3.3 nM) and maximum efficacy comparable to ingenol mebutate (Fig. 7).Fig. 7


Ingenol Disoxate: A Novel 4-Isoxazolecarboxylate Ester of Ingenol with Improved Properties for Treatment of Actinic Keratosis and Other Non-Melanoma Skin Cancers
Stimulation of IL-8 secretion from keratinocytes. a Secreted IL-8 levels were quantified in the supernatant from primary human epidermal keratinocytes incubated with increasing concentrations of ingenol disoxate (closed circles) or ingenol mebutate (open circles) for 6 h. Plots are composites of four individual experiments (mean ± standard error of the mean). b Half-maximal stimulatory concentrations [EC50 with 95% confidence interval (CI 95%)] of IL-8 release for ingenol disoxate, ingenol mebutate and the 5- and 20-isomers of ingenol disoxate. For N < 3 individual EC50 estimates are given
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5120626&req=5

Fig7: Stimulation of IL-8 secretion from keratinocytes. a Secreted IL-8 levels were quantified in the supernatant from primary human epidermal keratinocytes incubated with increasing concentrations of ingenol disoxate (closed circles) or ingenol mebutate (open circles) for 6 h. Plots are composites of four individual experiments (mean ± standard error of the mean). b Half-maximal stimulatory concentrations [EC50 with 95% confidence interval (CI 95%)] of IL-8 release for ingenol disoxate, ingenol mebutate and the 5- and 20-isomers of ingenol disoxate. For N < 3 individual EC50 estimates are given
Mentions: The recruitment of neutrophil granulocytes into ingenol mebutate treatment fields has been shown to be critical for the eradication of engrafted tumors in a mouse tumor model [30]. Ingenol mebutate is believed to facilitate neutrophil granulocyte tissue infiltration via PKC-dependent mechanisms including upregulation of adhesion molecules on endothelial cells as well as cytokine release, in particular IL-8, from keratinocytes and other cells residing in the skin [14, 30]. We wanted to test if ingenol disoxate was associated with a similar pro-inflammatory response at nontoxic concentrations. Therefore, we investigated the capacity to stimulate IL-8 release from keratinocytes and found that ingenol disoxate stimulated release of this cytokine with a potency (EC50 = 3.3 nM) and maximum efficacy comparable to ingenol mebutate (Fig. 7).Fig. 7

View Article: PubMed Central - PubMed

ABSTRACT

Introduction: Ingenol mebutate gel (Picato&reg;, 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.