Dataset Information

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

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.

SUBMITTER: Bertelsen M

PROVIDER: S-EPMC5120626 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Actinic keratosis is a common skin disease that may progress to invasive squamous cell carcinoma if left untreated. Ingenol mebutate has demonstrated efficacy in field treatment of actinic keratosis. However, molecular mechanisms on ingenol mebutate response are not yet fully understood. In this study, we evaluated the gene expression profiles of actinic keratosis lesions before and after treatment with ingenol mebutate using microarray technology. Actinic keratoses on face/scalp of 15 immunocompetent patients were identified and evaluated after treatment with topical ingenol mebutate gel 0.015%, applied once daily for 3 consecutive days. Diagnostic and clearance of lesions was determined by clinical, dermoscopic, and reflectance confocal microscopy criteria. Lesional and non-lesional skin biopsies were subjected to gene expression analysis profiled by Affymetrix microarray. Differentially expressed genes were identified, and enrichment analyses were performed using STRING database. At 8 weeks post-treatment, 60% of patients responded to ingenol mebutate therapy, achieving complete clearance in 40% of cases. A total of 128 differentially expressed genes were identified following treatment, and downregulated genes (114 of 128) revealed changes in pathways important to epidermal development, keratinocyte differentiation and cornification. In responder patients, 388 downregulated genes (of 450 differentially expressed genes) were also involved in development/differentiation of the epidermis, and immune system-related pathways, such as cytokine and interleukin signaling. Cluster analysis revealed two relevant clusters showing upregulated profile patterns in pre-treatment actinic keratoses of responders, as compared to non-responders. Again, differentially expressed genes were mainly associated with cornification, keratinization and keratinocyte differentiation. Overall, the present study provides insight into the gene expression profile of actinic keratoses after treatment with ingenol mebutate, as well as identification of genetic signatures that could predict treatment response.

Project description:OBJECTIVE: The purpose of this study was to evaluate the safety and efficacy of ingenol disoxate gel using a once-daily, three-day field treatment regimen in patients with actinic keratosis. DESIGN: This was a Phase II, multicenter, open-label trial (clinicaltrials.gov: NCT02305888). SETTING: The study was conducted in 20 trial sites in the United States. PARTICIPANTS: Participants included patients with 5 to 20 clinically typical actinic keratosis lesions on the full face/chest (250cm2), scalp (25-250cm2), or the trunk/extremities (250cm2). MEASUREMENTS: We measured incidence of dose-limiting events based on local skin responses. Percentage reduction in actinic keratosis lesion count from baseline, complete clearance, and partial clearance (?75%) of actinic keratosis lesions were assessed at Week 8. RESULTS: Nine of 63 (14.3%) patients in the face/chest group reported dose-limiting events; zero of 63 patients in the scalp group reported dose-limiting events; and 11 of 62 (17.7%) patients in the trunk/extremities group reported dose-limiting events. Mean composite local skin response scores peaked at Day 4, then rapidly declined, reaching or approaching baseline levels by Week 4. Less than five percent of patients reported severe adverse events; the most common treatment-related adverse events were application site pain and pruritus. The reduction in actinic keratosis lesion count was 78.9, 76.3, and 69.1 percent for the face/chest, scalp, and trunk/extremities groups, respectively. Complete clearance was achieved in 36.5, 39.7, and 22.6 percent of patients in the face/chest, scalp, and trunk/extremities groups, respectively. Partial clearance was achieved in 71.4, 65.1, and 50.0 percent of patients in the face/chest, scalp, and trunk/extremities groups, respectively. CONCLUSION: Ingenol disoxate demonstrated adverse events and local skin reaction profiles similar to results seen in trials evaluating shorter two-day regimens and was effective in patients with actinic keratosis. These data support the use of ingenol disoxate gel for actinic keratosis field treatment.