Concentration profiles and safety of topically applied betulinic acid and NVX-207 in eight healthy horses-A randomized, blinded, placebo-controlled, crossover pilot study.

The naturally occurring betulinic acid (BA) and its derivative NVX-207 show anticancer effects against equine malignant melanoma (EMM) cells and a potent permeation in isolated equine skin in vitro. The aim of the study was to determine the in vivo concentration profiles of BA and NVX-207 in equine skin and assess the compounds' local and systemic tolerability with the intent of developing a topical therapy against EMM. Eight horses were treated percutaneously in a crossover design with 1% BA, 1% NVX-207 or a placebo in a respective vehicle twice a day for seven consecutive days with a seven-day washout period between each formulation. Horses were treated at the neck and underneath the tail. Concentration profiles of the compounds were assessed by high-performance liquid chromatography in the cervical skin. Clinical and histopathological examinations and blood analyses were performed. Higher concentrations of NVX-207 were found in the skin compared to BA. Good systemic tolerability and only mild local adverse effects were observed in all three groups. This study substantiates the topical application of BA and NVX-207 in further clinical trials with horses suffering from EMM; however, penetration and permeation of the compounds may be altered in skin affected by tumors.

Investigation of ex vivo Skin Penetration of Coenzyme Q10 from Microemulsions and Hydrophilic Cream.

INTRODUCTION: Coenzyme Q10 (CoQ10) has been widely used in topical and cosmeceutical products due to its cutaneous antioxidant and energizer effects. CoQ10 is found in a higher concentration in the epidermis compared to dermis. The epidermal level of CoQ10 can be reduced due to several factors such as skin UV irradiation and photoaging. Various dermal nano-formulations have been investigated to overcome the skin barrier and enhance the poor penetration of CoQ10. The nanocarriers are designed to target and concentrate the CoQ10 in the viable epidermis. Most of these studies, however, failed to show the depth and extent of penetration of CoQ10 from the various carrier systems. OBJECTIVE: The distribution of CoQ10 across the various skin layers has to be shown using skin slices representing the different skin layers. METHODS: To realize this objective, a sensitive and selective HPLC method was developed and validated for the quantification of CoQ10 in the different skin slices. The method applicability to skin penetration (using excised human skin) as well as stability studies was investigated using CoQ10-loaded lecithin-based microemulsion (ME) and hydrophilic cream formulations. RESULTS: It could be shown that the highest concentration of CoQ10 in the viable epidermis, the target skin layer for CoQ10, was observed after application of the CoQ10 in the hydrophilic cream. This cream contains 10% of 2-ethylhexyl laurate which works obviously as a penetration enhancer for CoQ10. In contrast, the penetration of CoQ10 was lower from the ME. Just in the deeper dermis, a certain amount of CoQ10 could be detected. CONCLUSIONS: The HPLC method quantified the trace quantities of the CoQ10 distributed across the various skin layers and, hence, can be used to investigate the skin penetration of CoQ10 from various dermal standard and nano-formulations.

Mass spectrometry-based secretome analysis of non-small cell lung cancer cell lines.

Tyrosine kinase inhibitors, such as erlotinib, display reliable responses and survival benefits for the treatment of human non-small cell lung cancer (NSCLC) patients. However, primary or acquired resistance limits their therapeutic success. In this study, we conducted in-depth mass spectrometric analyses of NSCLC cell secretomes. To identify secreted proteins that are differentially regulated in erlotinib-sensitive (PC-9) and -resistant (PC-9ER) NSCLC cell lines, SILAC experiments were performed. On average, 900 proteins were identified in each sample with low variations in the numbers of identified proteins. Fourteen proteins were found to be differently regulated among erlotinib-sensitive and -resistant NSCLC cell lines, with five proteins (tissue-type plasminogen activator, epidermal growth factor receptor, urokinase-type plasminogen activator, platelet-derived growth factor D, and myeloid-derived growth factor) showing the most prominent regulation. Tissue-type plasminogen activator (t-PA) was up to 10-times upregulated in erlotinib-resistant NSCLC cells compared with erlotinib-sensitive cells. T-PA is an established tumor marker for various cancer types and seems to be a promising prognostic marker to differentiate erlotinib-sensitive from erlotinib-resistant NSCLC cells. To gain further insights into t-PA-regulated pathways, a t-PA variant was expressed in E. coli cells and its interactions with proteins secreted from erlotinib-sensitive and -resistant NCSLC cells were studied by a combined affinity enrichment chemical cross-linking/mass spectrometry (MS) approach. Fourteen proteins were identified as potential t-PA interaction partners, deserving a closer inspection to unravel the mechanisms underlying erlotinib resistance in NSCLC cells.

StavroX--a software for analyzing crosslinked products in protein interaction studies.

Chemical crosslinking in combination with mass spectrometry has matured into an alternative approach to derive low-resolution structural information of proteins and protein complexes. Yet, one of the major drawbacks of this strategy remains the lack of software that is able to handle the large MS datasets that are created after chemical crosslinking and enzymatic digestion of the crosslinking reaction mixtures. Here, we describe a software, termed StavroX, which has been specifically designed for analyzing highly complex crosslinking datasets. The StavroX software was evaluated for three diverse biological systems: (1) the complex between calmodulin and a peptide derived from Munc13, (2) an N-terminal ß-laminin fragment, and (3) the complex between guanylyl cyclase activating protein-2 and a peptide derived from retinal guanylyl cyclase. We show that the StavroX software is advantageous for analyzing crosslinked products due to its easy-to-use graphical user interface and the highly automated analysis of mass spectrometry (MS) and tandem mass spectrometry (MS/MS) data resulting in short times for analysis. StavroX is expected to give a further push to the chemical crosslinking approach as a routine technique for protein interaction studies.