Skin, mucosal and blood-brain barrier kinetics of model cyclic depsipeptides : the mycotoxins beauvericin and enniatins

This research takes off by giving an introduction to the wonderful world of the chemically very diverse cyclic depsipeptides (CDPs). This family of compounds is biosynthetically produced by an extreme variety of natural organisms, such as bacteria and fungi, which can be found all over the world. CDPs exhibit multiple biological activities, serving as interesting drug leads for the development of medicines. For the first time a classification system for CDPs was introduced. Some CDPs synthesised by fungi, however, can also be toxic and are potentially harmful. In scientific literature, there is a lack of a clear definition for these so-called mycotoxins, although very important for risk analyses. Therefore, we presented a new, quantitative expressed ‘mycotoxin’ definition, by means of the philosophical concept of explication. Recently, beauvericin and enniatins are recognised as ‘emerging’ mycotoxins. Exposure to these CDPs should not be considered as trivial, given their potential biological effects and presence as food and feed contaminants. Subsequent to the successful development of a bioanalytical method for the determination of beauvericin and enniatins in diverse matrices, their kinetic transport across the skin, oral mucosal and blood-brain barrier was quantitatively evaluated. Finally, a pharmaceutical preparation, containing enniatins as active ingredient and used in the treatment of upper respiratory tract infections, was investigated. Because of the negative risk-benefit ratio, we questioned its use in these innocent respiratory infections. For the same reason, the European Medicines Agency (EMA) has very recently endorsed the recall of these medicines from the European market

Adsorption of cyclic depsipeptide mycotoxins to glass

During analytical processes, adsorption of peptides, which is believed to mostly be due to non-covalent interactions and depending upon the experimental conditions, cannot only lead to significant loss of the analyte, but also to increased analytical variability. This undesirable aspect, however, has been given scant attention [1-2]. Some cyclic depsipeptides are considered as mycotoxins, i.e. beauvericin (BEA) and enniatins (ENNs). To quantitatively evaluate this transdermal behaviour, ex-vivo in-vitro Franz Diffusion Cell (FDC) experiments are performed. The adsorption of the cyclic depsipeptide analytes to the FDC glass wall, of which the quality differs from analytical volumetric glassware, was not yet investigated. Therefore, BEA and ENNs were solubilised in six different solvent mixtures (i.e. different concentrations of ACN and EtOH in H2O) and exposed to FDC glass (in duplicate). After 24h equilibration at 25°C, an aliquot of each solution was assayed with a newly developed and validated UHPLC-MS/MS method in multiple reaction monitoring (MRM) mode. Responses were analysed using loglinear models to obtain recoveries. These were fitted using generalised estimating equations with unstructured covariance to account for correlation within the duplicates [3]. QQ-plots confirmed the normality of the raw residuals in these models. Bonferroni corrected p-values and 95% confidence intervals were determined to account for multiplicity in the analysis of each compound separately. Our results showed that at an EtOH or ACN concentration of ≥ 50% there is no statistical or biomedical significant adsorption effect. At lower levels of organic solvents, however, a significant adsorption effect cannot be excluded, i.e. up to approximately 50% adsorption at 10% ACN. References: [1] Herath, H.M.D.R.; Kim, R.-R.; Cabot, P.J.; Shaw, P.N.; Hewavitharana, A.K. Inaccuracies in the quantification of peptides – A case study using β-endorphin assay. LCGC North America, 2013, 31(1): 58-61. [2] Pezeshki, A.; Vergote, V.; Van Dorpe, S.; Baert, B.; Burvenich, C.; Popkov, A.; De Spiegeleer, B. Adsorption of peptides at the sample drying step: influence of solvent evaporation technique, vial material and solution additive. Journal of Pharmaceutical and Biomedical Analysis, 2009, 49: 607-612. [3] Liang, K-Y.; Zeger, S.L. Longitudinal data analysis using generalized linear models. Biometrics, 1986, 78(1): 13-22

Transdermal penetration enhancing effect of the N-alkylamide spilanthol

The dermal penetration of compounds may be influenced by other compounds when mixtures are presented to the skin. Plant extracts, often used in cosmeceuticals, are complex mixtures of wanted bio-actives as well as undesirable impurities like nanoparticles and mycotoxines. A major question is if plant bio-actives (like spilanthol) can significantly alter the dermal penetration of other compounds which can be actives (like testosterone) or impurities (like mycotoxins). If so, the qualification assessment of the product quality needs to include this influence within the Quality-by-Design strategy. Therefore, the concentration-dependent penetration promoting effect of spilanthol was investigated on the three CART transdermal model compounds i.e. caffeine, ibuprofen and testosterone [1]. It was shown that spilanthol has a compound and concentration dependent penetration enhancing effect. No significant penetration enhancing effect for ibuprofen has been observed. However, with increasing spilanthol concentration (from 0 up to 1%, m/V), the permeability of caffeine increases, resulting in an enhancing ratio (ER) of 4.60 ± 0.49 (mean ± SEM, n=4). For testosterone, a maximal penetration enhancing concentration of 0.5% spilanthol was found (ER = 4.13 ± 0.44 (mean ± SEM, n=3)). Our findings with these model compounds are also confirmed with mycotoxins [2]. In conclusion, the existence of a significant mutual influence of compounds towards skin penetration should always be considered as part of the functional quality evaluation or in topical product development. References [1] B. Baert, E. Deconinck, M. Van Gele, M. Slodicka, P. Stoppie, S. Bode, G. Slegers, Y. Vander Heyden, J. Lambert, J. Beetens, B. De Spiegeleer. Transdermal penetration behaviour of drugs: CART-clustering, QSPR and selection of model compounds, 2007, Bioorganic & medicinal chemistry, 15(22): 6943-6955. [2] B. De Spiegeleer, J. Boonen, L. Veryser, L. Taevernier, S.V. Malysheva, J. Diana Di Mavungu, S. De Saeger, N. Roche, P. Blondeel. Skin penetration enhancing properties of the plant N-alkylamide spilanthol, 2012, manuscript in preparation

Human skin kinetics of cyclic depsipeptide mycotoxins

Cyclic depsipeptides (CDPs) are an emerging group of naturally occurring bioactive peptides, some of which are already developed as pharmaceutical drugs, e.g. valinomycin. They are produced by bacteria, marine organisms and fungi [1]. Some CDPs are secondary fungal metabolites, which can be very toxic to humans and animals, and are therefore called mycotoxins. Currently, dermal exposure data of CDP mycotoxins is scarce and fragmentary with a lack of understanding about the local skin and systemic kinetics and effects, despite their widespread skin contact and intrinsic hazard. Moreover, mechanistic pathways and models are completely absent. Therefore our general goal was to provide a quantitative characterisation of their dermal kinetics. The emerging cyclic depsipeptide mycotoxins enniatins (ENNs) and beauvericin (BEA) were used as model compounds and their transdermal kinetics were quantitatively evaluated using human skin in an in vitro Franz diffusion cell set-up and UPLC-MS analytics [2]. BEA and ENNs are non-ionised cyclic hexadepsipeptides, with ionophoric and lipophilic properties, causing serious health problems [3-6]. Overall, immunosuppressive effects are thought to play a role in their toxicity. This knowledge is needed not only for the risk assessment due to skin-contact of CDP-contaminated food, feed, indoor surfaces and airborne particles, but also for the development of topically applied drugs with CDP-like structure, treating dermatological diseases or having systemic functions after transdermal penetration. References: [1] R. Lemmens-Gruber, M.R. Kamyar, R. Dornetshuber, Current Medicinal Chemistry, 16, 1122 (2009). [2] L. Taevernier, L. Veryser, N. Roche, S. Vansteelandt, B. De Spiegeleer, manuscript in preparation. [3] M. Jestoi, Critical Reviews in Food Science and Nutrition, 48, 21 (2008). [4] M. Celik, H. Aksoy, S.Yilmaz, Ecotoxicology and Environmental Safety, 73, 1553 (2010). [5] R. Dornetshuber, P. Heffeter, M.R. Kamyar, Chemical Research in Toxicology, 20, 465 (2007). [6] A. Tonshin, V. Teplova, M. Andersson, Toxicology, 276, 49 (2010)

N-alkylamides : from plant to brain

Background: Plant N-alkylamides (NAAs) are bio-active compounds with a broad functional spectrum. In order to reach their pharmacodynamic targets, they have to overcome several barriers of the body in the absorption phase. The permeability kinetics of spilanthol (a diene NAA) and pellitorine (a triene NAA) across these barriers (i.e. skin, oral/gut mucosa, blood-brain barrier) were investigated. Methods: The skin and oral mucosa permeability were investigated using human skin and pig mucosa in an ex vivo in vitro Franz diffusion cell set-up. The gut absorption characteristics were examined using the in vitro Caco-2 cell monolayer test system. The initial blood-brain barrier transport kinetics were investigated in an in vivo mice model using multiple time regression and efflux experiments. Quantification of both NAAs was conducted using HPLC-UV and bio-analytical UPLC-MS methods. Results: We demonstrated that spilanthol and pellitorine are able to penetrate the skin after topical administration. It is likely that spilanthol and pellitorine can pass the endothelial gut as they easily pass the Caco-2 cells in the monolayer model. It has been shown that spilanthol also crosses the oral mucosa as well as the blood-brain barrier. Conclusion: It was demonstrated that NAAs pass various physiological barriers i.e. the skin, oral and gut mucosa, and after having reached the systemic circulation, also the blood-brain barrier. As such, NAAs are cosmenutriceuticals which can be active in the brain

Quantitative in vitro and in vivo evaluation of intestinal and blood-brain barrier transport kinetics of the plant N-alkylamide pellitorine

Objective. To evaluate the gut mucosa and blood-brain barrier (BBB) pharmacokinetic permeability properties of the plant N-alkylamide pellitorine. Methods. Pure pellitorine and an Anacyclus pyrethrum extract were used to investigate the permeation of pellitorine through (1) a Caco-2 cell monolayer, (2) the rat gut after oral administration, and (3) the BBB in mice after intravenous and intracerebroventricular administration. A validated bioanalytical UPLC-MS2 method was used to quantify pellitorine. Results. Pellitorine was able to cross the Caco-2 cell monolayer from the apical-to-basolateral and from the basolateral-to-apical side with apparent permeability coefficients between 0.6 center dot 10(-5) and 4.8 center dot 10(-5) cm/h and between 0.3 center dot 10(-5) and 5.8 center dot 10(-5) cm/h, respectively. In rats, a serum elimination rate constant of 0.3 h(-1) was obtained. Intravenous injection of pellitorine in mice resulted in a rapid and high permeation of pellitorine through the BBB with a unidirectional influx rate constant of 153 mu L/(g center dot min). In particular, 97% of pellitorine reached the brain tissue, while only 3% remained in the brain capillaries. An efflux transfer constant of 0.05 min(-1) was obtained. Conclusion. Pellitorine shows a good gut permeation and rapidly permeates the BBB once in the blood, indicating a possible role in the treatment of central nervous system diseases

Mucosal and blood-brain barrier transport kinetics of the plant N-alkylamide spilanthol using in vitro and in vivo models

Background: N-alkylamides (NAAs) are a large group of secondary metabolites occurring in more than 25 plant families which are often used in traditional medicine. A prominent active NAA is spilanthol. The general goal was to quantitatively investigate the gut mucosa and blood-brain barrier (BBB) permeability pharmacokinetic properties of spilanthol. Methods: Spilanthes acmella (L.) L. extracts, as well as purified spilanthol were used to investigate (1) the permeation of spilanthol through a Caco-2 cell monolayer in vitro, (2) the absorption from the intestinal lumen after oral administration to rats, and (3) the permeation through the BBB in mice after intravenous injection. Quantification of spilanthol was performed using a validated bio-analytical UPLC-MS2 method. Results: Spilanthol was able to cross the Caco-2 cell monolayer in vitro from the apical-to-basolateral side and from the basolateral-to-apical side with apparent permeability coefficients P-app between 5.2.10(-5) and 10.2.10(-5) cm/h. This in vitro permeability was confirmed by the in vivo intestinal absorption in rats after oral administration, where an elimination rate constant k(e) of 0.6 h(-1) was obtained. Furthermore, once present in the systemic circulation, spilanthol rapidly penetrated the blood-brain barrier: a highly significant influx of spilanthol into the brains was observed with a unidirectional influx rate constant K-1 of 796 mu l/(g.min). Conclusions: Spilanthol shows a high intestinal absorption from the gut into the systemic circulation, as well as a high BBB permeation rate from the blood into the brain