title: Ingenol Disoxate: A Novel 4-Isoxazolecarboxylate Ester of Ingenol with Improved Properties for Treatment of Actinic Keratosis and Other Non-Melanoma Skin Cancers.
abstract: BACKGROUND
  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.
  
  
  CONCLUSIONS
  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.
  
  
  BACKGROUND
  LEO Pharma A/S.
authors: Skin Research, LEO Pharma A/S, Ballerup, Denmark. MVBDK@leo-pharma.com.
authors: Malene
authors: http://orcid.org/0000-0002-3331-866X
authors: M
authors: Bertelsen
authors: Skin Research, LEO Pharma A/S, Ballerup, Denmark.
authors: Martin
authors: M
authors: Stahlhut
authors: Drug Design, LEO Pharma A/S, Ballerup, Denmark.
authors: Gunnar
authors: G
authors: Grue-Sørensen
authors: Drug Design, LEO Pharma A/S, Ballerup, Denmark.
authors: Xifu
authors: X
authors: Liang
authors: Skin Research, LEO Pharma A/S, Ballerup, Denmark.
authors: Gitte Bach
authors: GB
authors: Christensen
authors: Skin Research, LEO Pharma A/S, Ballerup, Denmark.
authors: Kresten
authors: K
authors: Skak
authors: Pharmaceutical Design and Development, LEO Pharma A/S, Ballerup, Denmark.
authors: Karen Margrethe
authors: KM
authors: Engell
authors: Drug Design, LEO Pharma A/S, Ballerup, Denmark. THGDK@leo-pharma.com.
authors: Thomas
authors: T
authors: Högberg
keywords: Animal tumor models; Antiproliferative; Chemical stability; Cytotoxicity; Ingenol derivatives; Pro-inflammatory; Regiocontrolled syntheses; Skin cancer