RESUMEN
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.
RESUMEN
Ingenol 3-benzoates were investigated with respect to chemical stability, pro-inflammatory effects, cell death induction and PKCδ activation. A correlation between structure, chemical stability and biological activity was found and compared to ingenol mebutate (ingenol 3-angelate) used for field treatment of actinic keratosis. We also provided further support for involvement of PKCδ for induction of oxidative burst and cytokine release. Molecular modeling and dynamics calculations corroborated the essential interactions between key compounds and C1 domain of PKCδ.
Asunto(s)
Antineoplásicos/farmacología , Benzoatos/farmacología , Diterpenos/farmacología , Queratosis Actínica/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Neoplasias Cutáneas/tratamiento farmacológico , Antineoplásicos/síntesis química , Antineoplásicos/química , Benzoatos/síntesis química , Benzoatos/química , Muerte Celular/efectos de los fármacos , Citocinas/metabolismo , Diterpenos/síntesis química , Diterpenos/química , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Queratosis Actínica/metabolismo , Queratosis Actínica/patología , Modelos Moleculares , Simulación del Acoplamiento Molecular , Estructura Molecular , Proteína Quinasa C-delta/antagonistas & inhibidores , Proteína Quinasa C-delta/metabolismo , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Relación Estructura-ActividadRESUMEN
Ingenol mebutate is the active ingredient in Picato® a new drug for the treatment of actinic keratosis. A number of derivatives related to ingenol mebutate were prepared by chemical synthesis from ingenol with the purpose of investigating the SAR and potency in assays relating to pro-inflammatory effects (induction of PMN oxidative burst and keratinocyte cytokine release), the potential of cell death induction, as well as the chemical stability. By modifications of the ingenol scaffold several prerequisites for activity were identified. The chemical stability of the compounds could be linked to an acyl migration mechanism. We were able to find analogues of ingenol mebutate with comparable in vitro properties. Some key features for potent and more stable ingenol derivatives have been identified.