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

Abstract

IntroductionIngenol 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.MethodsA 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.ResultsThis 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.ConclusionThese 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.FundingLEO Pharma A/S.Electronic supplementary materialThe online version of this article (doi:10.1007/s13555-016-0137-2) contains supplementary material, which is available to authorized users.

Chemical Proteomics Identifies SLC25A20 as a Functional Target of the Ingenol Class of Actinic Keratosis Drugs

“…Most of these proteins did not interact with ingenol (Figure C), which instead competitively blocked Ing-DAyne labeling of a mostly distinct subset of proteins in human cells (Figure S1D). We also performed an additional competition study in HSC-5 cells with a biologically active analogue of IngMebingenol disoxate ( 10 , IngDsx, Figure S2A)that shows enhanced chemical stability and is currently in phase 3 clinical development . We observed that the majority of high-occupancy protein targets for IngMeb were also targets of IngDsx (Figure S2B, Table S1), which tended to show higher competition ratios for these shared targets.…”

“…Previous SAR studies demonstrated that ingenol itself is pharmacologically inactive 42 , 43 and the methylation of C5- or C20-hydroxyl groups of IngMeb also eliminates biological activity. 44 In addition, simultaneous removal of the C18/C19 methyl groups and dimethylcyclopropane moiety resulted in substantial loss in activity.…”

“…Previous SAR studies demonstrated that ingenol itself is pharmacologically inactive , and the methylation of C5- or C20-hydroxyl groups of IngMeb also eliminates biological activity . In addition, simultaneous removal of the C18/C19 methyl groups and dimethylcyclopropane moiety resulted in substantial loss in activity. , Recent studies have further revealed that IngMeb activity requires hydroxyl groups at the C4 and C5 positions and is maintained with various esters at the C3 position. , The 4-, 5-, and 20-hydroxyl groups, however, also form a framework for acyl migration of the ester, leading to subsequent loss in IngMeb potency .…”

“…With azido-ingenol ( 6 ) in hand, we generated an alkynylated dialkyl diazirine fully functionalized probeIng-DAyne, 9 via CuAAC chemistry with the corresponding diazirine diyne S4 (Figures C and D). We next examined the activity of the Ing-DAyne probe in previously described assays that include in vitro activation of recombinant PKCδ, stimulation of IL-8 release in human primary keratinocytes, and induction of oxidative burst in human polymorphonuclear leukocytes (PMNs, i.e., neutrophils) . The Ing-DAyne probe showed ∼10–100-fold reductions in biological activity compared to IngMb, but still maintained submicromolar potency in both in vitro and cell-based assays (Figure D), prompting us to move forward with the Ing-DAyne probe to identify cellular targets of IngMeb.…”

Total Synthesis as a Vehicle for Collaboration

“… (+)-Ingenol ( 11 ) was first isolated from Euphorbia ingens in 1968, and its derivatives have since been employed as effective treatments for the precancerous skin condition actinic keratosis. The potent analog, ingenol mebutate ( 46 ), marketed as Picato by LEO Pharma, is an FDA-approved topical treatment for the disease, whereas isoxazole derivative ingenol disoxate ( 47 ) is currently undergoing clinical trials for the same ailment . The preparation of these therapeutic agents presented a major obstacle for LEO Pharma, as only 275 mg of ingenol ( 11 ) can be isolated per kilogram of plant material .…”