Analytical, pharmacokinetic and regulatory characterisation of selected plant N-alkylamides

N-alkylamides (NAAs) are secondary metabolites occurring in many plants. These bioactive compounds, with many structural variations, are known to exert various pharmacological properties. This group of bioactive plant compounds was investigated in this doctoral research. Achillea millefolium and Achillea ptarmica are plants used in indigenous cultures to treat various stomach disorders. It was already demonstrated that some NAAs are responsible for the pharmacological properties. Therefore, the NAAs were analytically characterised in both plant extracts using two different analytical techniques (HPLC-ESI-MS and GC-EI-MS). Furthermore, the transdermal and intestinal properties of the plant NAAs pellitorine and spilanthol were evaluated. In this research, these NAAs were topically applied on human skin and orally given to rats to investigate if these NAAs were able to reach the blood circulation. Once in the blood circulation, the blood-brain barrier permeability properties were quantitatively evaluated. The use of a single quadrupole MS detector was shown in high-throughput transdermal research. The advantages of this type of detector were demonstrated, compared to an ultraviolet detector, e.g. a gain in selectivity and lower limit of detections were obtained. Lastly, the regulatory status of N-alkylamide containing health products was discussed. Products containing NAAs are widely available on the market as e.g. food supplements, medicinal products, cosmetics and medical devices. Currently, no unambiguous legal product classification for these products exist. The classification is dependent on the dose of the bioactive NAAs, their (claimed) use and their biological functionalities

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)

The blood-brain barrier (BBB) permeability properties of plant N-alkylamides

Background: N-alkylamides (NAAs), a large group of secondary metabolites occurring in more than 25 plant families, are potential drug candidates as several biological and medical functions are known such as central nervous activities [1]. Objective: Our general goal was to investigate if NAAs with a different structure, e.g. spilanthol and pellitorine, are able to pass the blood-brain barrier (BBB) and if so, to what extent. Methods: A blood to brain (multiple time regression (MTR)), as well as a brain to blood (efflux) transport experiment were conducted in an in vivo mice model to investigate the initial BBB rate kinetics. The mice were anesthetized, after which the NAA dose solution was injected. Blood was obtained at regular time points after injection, and thereafter, the mice were decapitated and the brains collected. Quantification of the NAAs was done using a bio-analytical LC-MS method. Results: The MTR experiment indicated that spilanthol was able to cross the BBB in mice. A rapid but highly significant influx of spilanthol into the brains was observed with an unidirectional influx rate of 217.0 µl/(g×min). The curve reached a plateau-phase after about 10 minutes exposure time and can be explained by efflux of spilanthol out of the brain or distribution or elimination of spilanthol. The efflux transfer constant calculated for spilanthol was 0.1 min-1. This equals a brain half-time disappearance of 6.4 min. The comparative results of on-going BBB studies, including the permeability properties of pellitorine, will be reported as well. Seen the different possible pharmacological targets of these plant NAAs and their pharmaco-ethnological use, our BBB results may trigger further exploration into the medicinal use of these important plant metabolites. References [1] Boonen J, Bronselaer A, Nielandt J, Veryser L, De Tré G, De Spiegeleer B. Alkamid database: Chemistry, occurance and functionality of plant N-alkylamides. J Ethnopharmacol 2012;142(3):563-59

Penetration enhancing effect of phytoceramides

Ceramides are essential components in the stratum corneum barrier function. Different classes of ceramides are present in human skin, differing in the nature of sphingosine and acyl moieties with respect to chain length, degree of saturation and the presence of an OH group [1]. Ceramides with a saturated sphingosine base containing a hydroxyl function at C4 are known as phytoceramides. A few studies demonstrated the penetration enhancing properties of ceramides [2-5], however, systematic studies using phytoceramides are lacking. This led us to assess the penetration enhancing effect of phytosphingosine and a series of nine phytoceramides via transdermal experiments using in vitro Franz diffusion cells. As transdermal model compounds, testosterone, caffeine and ibuprofen were tested in a 50:50 (V/V) EtOH:H2O dose formulation [6]. Results showed that the penetration enhancing effect of the phytoceramides depends on the used model compound. Selected phytoceramides exhibited a penetration enhancing ratio of more than two. References [1] Janůšová, B.; Zbytovská, J.; Lorenc, P.; Vavrysová, H.; Palát K.; Hrabálek, A.; Vávrová, K. (2011). Effect of ceramide acyl chain length on skin permeability and thermotropic phase behavior of model stratum corneum lipid membranes. Biochimica et Biophysica, 1811, 129–137. [2] Vávrová, K.; Hrabálek, A.; Dolezal, P.; Holas, T.; Zbytovská, J. (2003). L-serine and glycine based ceramide analogues as transdermal permeation enhancers: polar head size and hydrogen bonding. Bioorganic & medicinal chemistry letters, 13, 2351-2353. [3] Vávrová, K.; Zbytovská, J.; Hrabálek, A. (2005). Amphiphilic transdermal permeation enhancers: structure-activity relationships. Current Medicinal Chemistry, 12, 2273-2291. [4] Novotý, J.; Janůšová, B.; Novotý, M.; Hrabálek, A.; Vávrová, K. (2009). Short-chain ceramides decrease skin barrier properties. Skin pharmacology and Physiology, 22, 22-30. [5] Sinko, B.; Kökösi, J.; Avdeef, A.; Takács-Novák, K. (2009). A PAMPA study of the permeability-enhancing effect of new ceramide analogues. Chemistry & Biodiversity, 6, 1867-1874. [6] Baert, B.; Deconinck, E.; Van Gele, M.; Slodicka, M.; Stoppie, P.; Bode, S.; Slegers, G.; Vander Heyden, Y.; Lambert, J.; Beetens, J.; De Spiegeleer, B. (2007). Transdermal penetration behaviour of drugs: CART-clustering, QSPR and selection of model compounds. Bioorganic & medicinal chemistry, 15(22), 6943-6955

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

The use of UHPLC coupled to single quad MS detection in Franz diffusion cell methodologies

C

Transdermal evaluation of caffeine in different formulations and excipients

Background: The stratum corneum(SC) forms adifficultphysical barrier fordrugs to pass through the skin. Several strategieswere developed to overcome this barrier.Optimization of topical drug formulations by selected excipients may facilitate the penetration of drugs through the SC into the viable skin cells and ultimately into the systemic circulation. Methods: Here, both the influence of two formulations (a classic carbomer-based gel and a novel Pluronic® lecithin organo gel (PLO gel)) and selected excipients (ethanol, propylene glycol, diethylene glycol monoethyl ether, isopropyl myristate (IPM), and water) with or without the penetration enhancer miconazole nitrate on the transdermal penetration characteristics of caffeine were determined using an in vitro Franz diffusion cell setup. Results: Higher fluxes were observed for the carbomer-based gel compared to the PLO gel. Among the commonly used excipients, IPM showed the best penetration enhancing properties, while the presence of the penetration enhancer miconazole nitrate did not significantly alter the apparent skin permeation characteristics for caffeine. Conclusion: The high ethanol percentage in the carbomer-based gel could explain the results as supported by our excipient data.Moreover, IPMcould play a beneficial role in topical formulations as this excipient was responsible for a significant increase in the amount of caffeine penetrated through the skin. No overall statistical significant effect of the presence of miconazole nitrate as a penetration enhancer was observed