Dissolvable microneedles for transdermal drug delivery showing skin pentation and modified drug release.

Topical therapies for chronic skin diseases suffer from a low patient compliance due to the inconvenient treatment regimens of available products. Dissolvable microneedles (MN) with modified release offer an interesting possibility to increase the compliance by acting as a depot in the skin and thereby decreasing the dosing frequency. Furthermore, the bioavailability can be increased significantly by bypassing the barrier of the skin by the direct penetration of the MN into the skin. In this study the depot effect and skin penetration of an innovative dissolvable MN patch was assessed by insertion in ex vivo human skin and in vivo using minipigs. The MN patches are based on biodegradable polymers and the active pharmaceutical ingredients calcipotriol (Calci) and betamethasone-17-21-dipropionate (BDP) used to treat psoriasis. Using computed tomography (CT) and Coherent anti-Stokes Raman scattering (CARS) microscopy it was possible to visualize the skin penetration and follow the morphology of the MN as function of time in the skin. The depot effect was assessed by studying the modified in vitro release in an aqueous buffer and by comparing the drug release of a single application of a patch both ex vivo and in vivo to daily application of a marketed oleogel containing the same active pharmaceutical ingredients. The CT and CARS images showed efficient penetration of the MN patches into the upper dermis and a slow swelling process of the drug containing tip over a period of 8 days. Furthermore, CARS demonstrated that it can be used as a noninvasive technique with potential applicability in clinical settings. The in vitro release studies show a release of 54% over a time period of 30 days. The pharmacological relevance of MNs was confirmed in human skin explants and in vivo after single application and showed a similar response on calcipotriol and BDP mediated signaling events compared to daily application of the active oleogel. Altogether it was demonstrated that the MN can penetrate the skin and have the potential to provide a depot effect.

Evaluating Dermal Pharmacokinetics and Pharmacodymanic Effect of Soft Topical PDE4 Inhibitors: Open Flow Microperfusion and Skin Biopsies.

PURPOSE: To investigate the difference in clinical efficacy in AD patients between two topical PDE4 inhibitors using dermal open flow microperfusion and cAMP as a pharmacodynamic read-out in fresh human skin explants. METHODS: Clinical formulations were applied to intact or barrier disrupted human skin explants and both skin biopsy samples and dermal interstitial fluid was sampled for measuring drug concentration. Furthermore, cAMP levels were determined in the skin biopsies as a measure of target engagement. RESULTS: Elevated cAMP levels were observed with LEO 29102 while no evidence of target engagement was obtained with LEO 39652. In barrier impaired skin the dISF concentration of LEO 29102 was 2100 nM while only 33 nM for LEO 39652. For both compounds the concentrations measured in skin punch biopsies were 7-33-fold higher than the dISF concentrations. CONCLUSIONS: Low unbound drug concentration in dISF in combination with minimal target engagement of LEO 39652 in barrier impaired human skin explants supports that lack of clinical efficacy of LEO 39652 in AD patients is likely due to insufficient drug availability at the target. We conclude that dOFM together with a pharmacodynamic target engagement biomarker are strong techniques for establishing skin PK/PD relations and that skin biopsies should be used with caution.

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

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.

Synthesis, biological evaluation and SAR of 3-benzoates of ingenol for treatment of actinic keratosis and non-melanoma skin cancer.

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.

Syntheses, biological evaluation and SAR of ingenol mebutate analogues for treatment of actinic keratosis and non-melanoma skin cancer.

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.

Ingenol mebutate: induced cell death patterns in normal and cancer epithelial cells.

We investigated the proposed necrotic mechanism of ingenol mebutate, a natural compound with anti-cancer properties in human keratinocytes, the human squamous cell carcinoma cell line HSC-5, and HeLa cervix carcinoma cells. Topical application of a clinical dose of ingenol mebutate 0.05% (1.15 mM) gel to human reconstituted full-thickness skin equivalents strongly reduced epidermal, but not dermal viability. Ingenol mebutate showed cytotoxic potency between 200-300 M on normal and cancer cells. When keratinocytes were induced to differentiate, they became significantly less sensitive to ingenol mebutate and half-maximal induction of cell death required more than 300 M ingenol mebutate. Cytotoxic concentrations of ingenol mebutate caused rupture of the mitochondrial network within minutes paralleled by cytosolic calcium release in all cells. Subsequently, plasma membrane integrity was lost as seen by propidium uptake into the cells. This was in sharp contrast to lysis of cells with low concentrations of the detergent Triton X-100 that permeabilized the plasma membrane within minutes without affecting organelle morphology. Buffering of intracellular calcium and inhibition of the mitochondrial permeability transition pore reduced the cytotoxic effect of ingenol mebutate in cancer cells, but not in normal keratinocytes. However, these inhibitors could not prevent cell death subsequent to prolonged incubation. Our findings reveal that ingenol mebutate does not mediate cytotoxicity by a simple lytic, necrotic mechanism, but activates distinct processes involving multiple cell organelles in a cell-type and differentiation-dependent manner. These data improve our understanding of ingenol mebutate-target cell interactions and offer new insights relevant to the removal of aberrant cells in human skin.

Differential effects of levetiracetam, carbamazepine, and lamotrigine on reproductive endocrine function in adults.

Animal studies have shown endocrine changes after levetiracetam treatment. The present study investigated reproductive and sexual function in patients with epilepsy (aged 18-45) treated with levetiracetam (LEV: 30 men/26 women), carbamazepine (CBZ: 63 men/30 women), or lamotrigine (LTG: 37 men/40 women) monotherapy and in healthy controls (36 men/44 women). In women, no endocrine changes were observed during LEV treatment, whereas steroid hormone-binding globulin levels were greater and progesterone levels lower in women using CBZ. Dehydroepiandrosterone sulfate levels were higher and androstenedione levels lower in LTG-treated women. Arizona Sexual Experience Scale scores, which were significantly lower in females using LTG or LEV, suggesting they have better sexual function than CBZ users and controls. In men, no drug-specific hormonal pattern was observed after LEV treatment. Male patients in all treatment groups had lower androstenedione and free testosterone. Those using CBZ had lower free androgen indices and dehydroepiandrosterone sulfate levels, and higher steroid hormone-binding globulin, follicle-stimulating hormone, and luteinizing hormone levels. Arizona Sexual Experience Scale scores for men were similar in all groups. In conclusion, LEV treatment apparently has no drug-specific sexual or endocrine side effects in men or women in this age group.

TAB1 modulates IL-1alpha mediated cytokine secretion but is dispensable for TAK1 activation.

Biochemical evidence indicates that TGF-beta-activated kinase 1 (TAK1), a key modulator of the inflammatory response, exists in a complex with various adaptor proteins including the TAK1 binding protein 1 (TAB1). However, the physiological importance of TAB1 in TAK1 activation, and in the subsequent induction of proinflammatory mediators, remains unclear. In this study, a critical role for TAK1 in IL-1alpha or TNFalpha stimulated MAPK and NFkappaB activation was confirmed by inhibition of the nuclear accumulation of NFkappaB p65 and phosphorylated forms of c-Jun and p38 following siRNA mediated TAK1 silencing. These effects were associated with significant reductions in IL-1alpha stimulated levels of secreted IL-6, IL-8, MCP-1 and GM-CSF. In contrast, IL-1alpha or TNFalpha dependent cellular redistribution of NFkappaB p65 and phosphorylated c-Jun and p38 was not affected by 80% siRNA mediated knockdown of TAB1 protein levels. Interestingly, IL-6, IL-8 and GM-CSF release from TAB1 siRNA transfected cells was significantly reduced following IL-1alpha treatment, but was unchanged after TNFalpha stimulation, suggesting differential roles for TAB1 in IL-1alpha and TNFalpha signalling pathways. These findings may imply an as yet unidentified role for TAB1 in the inflammatory response, which is independent of the activation of classical TAK1 associated signalling cascades.

Multiplex analysis of inflammatory signaling pathways using a high-content imaging system.

This chapter describes a robust high-content cellular screening assay to simultaneously analyze the spatiotemporal activation of three different kinase-associated signaling pathways involving NF-kappaB, JNK, and p38, all of which are closely implicated in proliferative and proinflammatory responses. Signal transduction is dependent on the translocation of NF-kappaB p65 and phosphorylated c-Jun and p38 from the cytosol to the nucleus, and fluorescent immunolabeling was used to monitor changes in their cellular distribution. Cellular screening, data acquisition, and data interpretation were conducted on the ArrayScan HCS Reader (Cellomics Inc., Pittsburgh, PA). Assay adaptation to various cellular systems is feasible when sufficient separation of the nuclear and cytosolic compartment can be achieved and if cell adhesion properties permit proper attachment to the culture plates. Substitution of NF-kappaB p65 and phosphorylated forms of c-Jun and p38 as targets to analyze other translocating components is possible and is limited primarily by antibody specificity and the risk of fluorescent bleed-through between emission channels. Because assay validity is particularly confounded by inadequate spectral separation of the detection dyes in multicolor labeling assays, means of eliminating or counterbalancing staining artifacts are illustrated. Also, protocol parameter settings important for imaging and image processing are described, including object identification, image exposure settings, separation of cytosolic and nuclear regions, number of cells sufficient for analysis, and the use of gating thresholds critical for cell sorting and subpopulation analysis. This assay is a useful tool to investigate the interplay between signaling pathways and the mode of action, potency, and selectivity of compound inhibition of specific target molecules in a cellular context.

Inflammatory pathway analysis using a high content screening platform.

High content cellular screening assays are useful tools to investigate the interplay between signaling pathways and offer valuable platforms to determine the mode of action, potency, and selectivity of potential drug candidates in a biological setting. We describe a cell-based multiplex fluorescent imaging assay that permits concurrent detection and quantification of the distribution of nuclear factor kappaB (NFkappaB) p65/RelA and phosphorylated forms of p38 and c-Jun between the cytosol and nucleus. Cellular screening, data acquisition, and data interpretation were conducted on the ArrayScan HCS Reader (Cellomics Inc., Pittsburgh, PA). A significant window between untreated and interleukin-1alpha (IL-1alpha) stimulated HeLa cells for all three targets was achieved with low variability. Staining specificity was confirmed with blocking peptides and pharmacological inhibition of p38, c-Jun-N-terminal kinase (JNK), and inhibitory kappaB kinase 2, and channel bleed-through was eliminated or counterbalanced by the use of fixed exposure times together with careful reporter channel selection. The JNK inhibitor SP600125 was used as a demonstration compound because in addition to inhibiting nuclear accumulation of phosphorylated c-Jun it reduced nuclear translocation of phosphorylated p38 and NFkappaB p65/RelA in a dose-dependent manner, indicating a lack of SP600125 selectivity. This was supported by RNA interference where co-transfection of small interfering RNA targeting both JNK1 and JNK2, to limit signaling redundancy, significantly inhibited IL-1alpha-stimulated translocation of phosphorylated c-Jun without altering phosphorylated p38 and NFkappaB p65/RelA redistribution. This image analysis application is a valuable and information-rich screening tool to investigate compound selectivity and/or cross-talk between key signaling pathways involved in the inflammatory response.