The mycotoxin definition reconsidered towards fungal cyclic depsipeptides.

Currently, next to the major classes, cyclic depsipeptides beauvericin and enniatins are also positioned as mycotoxins. However, as there are hundreds more fungal cyclic depsipeptides already identified, should these not be considered as mycotoxins as well? The current status of the mycotoxin definition revealed a lack of consistency, leading to confusion about what compounds should be called mycotoxins. Because this is of pivotal importance in risk assessment prioritization, a clear and quantitatively expressed mycotoxin definition is proposed, based on data of widely accepted mycotoxins. Finally, this definition is applied to a set of fungal cyclic depsipeptides, revealing that some of these should indeed be considered as mycotoxins.

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-MS(2) 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 Papp 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 ke 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 K1 of 796 µl/(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.

The influence of the acyl chain on the transdermal penetration-enhancing effect of synthetic phytoceramides.

BACKGROUND/AIMS: The skin has become very attractive as a route for drug administration. Optimization of topical drug formulations by the addition of penetration enhancers may facilitate the passage of drugs through the stratum corneum. METHODS: In this paper, the skin penetration effect of phytosphingosine and 9 derived phytoceramides (PCERs) on 3 transdermal model drugs (i.e. caffeine, testosterone, ibuprofen) was investigated via Franz diffusion cell experiments using split-thickness human skin. Azone was included as a positive control. RESULTS: The main finding in our study was that the PCERs exerted a compound-dependent penetration-enhancing effect. Some of the investigated PCERs exhibited a penetration-enhancing ratio of more than 2 (mean ± SE): for caffeine PCER1 (2.48 ± 0.44), PCER2 (2.75 ± 0.74), PCER3 (2.62 ± 0.93) and PCER6 (2.70 ± 0.45) and for testosterone PCER1 (2.08 ± 0.56), PCER2 (2.56 ± 0.13), PCER3 (3.48), PCER4 (2.53), PCER5 (2.04 ± 0.14), PCER6 (2.05 ± 0.48) and PCER10 (4.84 ± 0.79), but none of them had an influence on ibuprofen. CONCLUSION: The investigated PCERs exhibited a penetration-enhancing effect on caffeine and testosterone but not on ibuprofen.

Chemical Classification of Cyclic Depsipeptides.

Cyclic depsipeptides (CDPs) are a family of cyclic peptide-related compounds, of which the ring is mainly composed of amino- and hydroxy acid residues joined by amide and ester bonds (at least one), leading to a wide diversity of fascinating chemical structures. They differ in both their ring structure and their side chains, especially by the nature of the unusual and non-amino acid building blocks. To date, however, there is no overall uniform chemical classification system available for CDPs and naming of the diverse family members is done rather arbitrarily. Therefore, a broad evaluation of different CDP structures is done, i.e., 1348 naturally occurring CDPs were included, and a straightforward chemical classification system using apparent chemical characteristics is proposed in order to organize the currently scattered CDP data. The overall validity of the classification approach is verified and the compounds categorized in the same groups are considered to be structurally related. This evaluation also revealed that traditionally formed CDP subfamilies, like the dolastatins, might be misleading from a chemical point of view given the structural differences in this subfamily. This up-to-date CDP overview enables peptide and natural product scientists to study the wide diversity in CDP structures, their chemical interrelationships and identification of existing and newly found CDPs. Together with the available information on the species producing these CDPs and their reported biological activities, this paper provides a useful tool to gain new insights into this diverse group of peptides.

N-alkylamide profiling of Achillea ptarmica and Achillea millefolium extracts by liquid and gas chromatography-mass spectrometry.

Achillea millefolium and Achillea ptarmica are both plants belonging to the Asteracea family and are traditionally used for their medicinal properties. It has already been shown that some N-alkylamides (NAAs) are responsible for these pharmacological actions. Therefore, in the present study, the NAA content of the two plants was analytically characterised. Different extracts were prepared from the roots, the leaves, the stems and the flowers. The structures of NAAs have been assigned in ethanolic extracts of Achillea millefolium and Achillea ptarmica using high performance liquid chromatography - electrospray ionisation - mass spectrometry (HPLC-ESI-MS) and gas chromatography - electron impact - mass spectrometry (GC-EI-MS). Using both analytical techniques, the structures of 14 and 15 NAAs have been assigned in Achillea ptarmica and Achillea millefolium, respectively. Structures of two new NAAs, previously never observed in Achillea ptarmica, were assigned: deca-2E,6Z,8E-trienoic acid 2-methylbutylamide (homospilanthol) or a related isomeric compound and deca-2E,4E-dienoic acid N-methyl isobutylamide. The structure of homospilanthol or a related isomeric compound was also assigned in Achillea millefolium for the first time.

Exploration of the Medicinal Peptide Space.

The chemical properties of peptide medicines, known as the 'medicinal peptide space' is considered a multi-dimensional subset of the global peptide space, where each dimension represents a chemical descriptor. These descriptors can be linked to biofunctional, medicinal properties to varying degrees. Knowledge of this space can increase the efficiency of the peptide-drug discovery and development process, as well as advance our understanding and classification of peptide medicines. For 245 peptide drugs, already available on the market or in clinical development, multivariate dataexploration was performed using peptide relevant physicochemical descriptors, their specific peptidedrug target and their clinical use. Our retrospective analysis indicates that clusters in the medicinal peptide space are located in a relatively narrow range of the physicochemical space: dense and empty regions were found, which can be explored for the discovery of novel peptide drugs.

Regulatory development of geriatric medicines: To GIP or not to GIP?

Geriatric patients represent the main users of medicines, but are historically often minimally included in clinical trials, resulting in a gap in the knowledge of the benefit/risk balance of medicines in this heterogeneous population. As the worldwide population is aging, the need for safe and effective medicines for older patients is proportionally increasing. The aim of this review is to provide an overview of the current regulatory status of the development of geriatric medicines, the encountered challenges and the view of the involved stakeholders, coming to the conclusion whether it is necessary or not to implement a Geriatric Investigation Plan (GIP), by analogy with pediatrics.

Blood-brain barrier transport kinetics of the cyclic depsipeptide mycotoxins beauvericin and enniatins.

The cyclic depsipeptide mycotoxins beauvericin and enniatins are capable of reaching the systemic circulation through various routes of exposure and are hence capable of exerting central nervous system (CNS) effects, if they are able to pass the blood-brain barrier (BBB), which was the main objective of this study. Quantification of the mycotoxins was performed using an in-house developed and validated bio-analytical UHPLC-MS/MS method. Prior to the BBB experiments, the metabolic stability of the mycotoxins was evaluated in vitro in mouse serum and brain homogenate. The BBB permeation kinetics of beauvericin and enniatins were studied using an in vivo mice model, applying multiple time regression for studying the blood-to-brain influx. Additionally, capillary depletion was applied to obtain the fraction of the peptides really entering the brain parenchyma and the fraction loosely adhered to the brain capillary wall. Finally, also the brain-to-blood efflux transport kinetics was studied. Metabolic stability data indicated that the investigated mycotoxins were stable during the duration of the in vivo study. The brain influx study showed that beauvericin and enniatins are able to cross the blood-brain barrier in mice: using the Gjedde-Patlak biphasic model, it was shown that all investigated mycotoxins exert a high initial influx rate into the brain (K1 ranging from 11 to 53µL/(g×min)), rapidly reaching a plateau. After penetration, the mycotoxins reached the brain parenchyma (95%) with only a limited amount residing in the capillaries (5%). Negligible efflux (<0.005min(-1)) from the brain was observed in the 15min post-intracerebroventricular injection.

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-MS(2) 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 · 10(-5) and 4.8 · 10(-5) cm/h and between 0.3 · 10(-5) and 5.8 · 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 µL/(g·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.

Human skin permeation of emerging mycotoxins (beauvericin and enniatins).

Currently, dermal exposure data of cyclic depsipeptide mycotoxins are completely absent. There is a lack of understanding about the local skin and systemic kinetics and effects, despite their widespread skin contact and intrinsic hazard. Therefore, we provide a quantitative characterisation of their dermal kinetics. The emerging mycotoxins enniatins (ENNs) and beauvericin (BEA) were used as model compounds and their transdermal kinetics were quantitatively evaluated, using intact and damaged human skin in an in vitro Franz diffusion cell set-up and ultra high-performance liquid chromatography (UHPLC)-MS analytics. We demonstrated that all investigated mycotoxins are able to penetrate through the skin. ENN B showed the highest permeation (kp,v=9.44 × 10(-6) cm/h), whereas BEA showed the lowest (kp,v=2.35 × 10(-6) cm/h) and the other ENNs ranging in between. Combining these values with experimentally determined solubility data, Jmax values ranging from 0.02 to 0.35 µg/(cm(2) h) for intact skin and from 0.07 to 1.11 µg/(cm(2) h) for damaged skin were obtained. These were used to determine the daily dermal exposure (DDE) in a worst-case scenario. On the other hand, DDE's for a typical occupational scenario were calculated based on real-life mycotoxin concentrations for the industrial exposure of food-related workers. In the latter case, for contact with intact human skin, DDE's up to 0.0870 ng/(kg BW × day) for ENN A were calculated, whereas for impaired skin barrier this can even rise up to 0.3209 ng/(kg BW × day) for ENN B1. This knowledge is needed for the risk assessment after skin exposure of contaminated food, feed, indoor surfaces and airborne particles with mycotoxins.

Characterization data on the topical carrier DDC642.

This article contains original data, figures and methods used in the characterization of the liposomal carrier 'DDC642' for topical applications, described in "An elastic liposomal formulation for RNAi-based topical treatment of skin disorders: proof-of-concept in the treatment of psoriasis" (Desmet et al., 2016) [1]. Several elastic liposomal formulations have been evaluated for their ability to encapsulate and deliver RNA interference (RNAi) molecules to cultured primary skin cells. The efficiency and effectiveness of these liposomes were compared to that of our previously characterized liposomes, the 'SECosomes' (SEC) (Geusens et al., 2010) [2]. After selection of a potential superior carrier, based on encapsulation and transfection efficiency data (Desmet et al., 2016) [1], the selected DDC642 liposomes were characterized more in-depth. Herein, a detailed characterization of the DDC642 liposome and RNAi-loaded lipoplexes is given, including the matching protocols.

An elastic liposomal formulation for RNAi-based topical treatment of skin disorders: Proof-of-concept in the treatment of psoriasis.

RNA interference (RNAi) is a rapidly emerging approach for targeted gene silencing to alleviate disease pathology. However, lack of efficient carriers for targeted delivery delays the clinical translation of RNAi. An interesting target for local RNAi therapeutics is the skin as it allows direct access to target cells. Still, applications are limited due to the effective skin barrier which hinders penetration. Herein, a description is given of a liposomal carrier, called 'DDC642', capable of delivering RNAi molecules to the epidermis of impaired and intact human skin, without targeting the dermis or circulatory system. In a psoriasis tissue model, down-regulation of the psoriasis marker human beta-defensin 2 by DDC642-delivered siRNA was confirmed, providing proof-of-concept. These liposomes thus hold great potential as topical delivery system for RNAi therapeutics in the treatment of numerous skin diseases.

Enniatin-containing solutions for oromucosal use: Quality-by-design ex-vivo transmucosal risk assessment of composition variability.

Fusafungine, a mixture of the cyclic hexadepsipeptides enniatins, is currently on the market for the treatment of upper respiratory tract diseases because of its bacteriostatic and anti-inflammatory effects. In this study, a quality-by-design risk assessment was performed with two objectives: (i) investigate whether enniatins are able to permeate the mucosa and reach blood circulation, as the summary of product characteristics indicates this is not the case, and if so, to quantify their transmucosal kinetics and (ii) study the influence of excipient concentration variability on mucosal permeation. First, the concentration of the two main excipients isopropyl myristate and ethanol, known penetration enhancers, in several marketed samples was determined using GC-FID. Then, the transmucosal kinetics of the enniatins were quantitatively evaluated for different dose solutions, using porcine buccal mucosa in an ex-vivo in-vitro Franz diffusion cell set-up, with UHPLC-MS/MS bioanalytics. This study demonstrated that enniatins are capable of permeating the mucosa. However, no risk of a significant different transmucosal permeability with varying excipient concentrations was detected.

Implementation of a single quad MS detector in high-throughput transdermal research of plant extracts.

In this study, a new type of single quadrupole mass spectrometric detector was implemented in transdermal research. The local skin pharmacokinetic properties of the plant N-alkylamides (NAAs) pellitorine and anacycline, present in an Anacyclus pyrethrum extract, and spilanthol, present in a Spilanthes acmella extract were investigated. This single quad MS detection method showed great advantages compared to the traditional UV detector. The NAAs could be identified and quantified in the samples with an ultra performance liquid chromatography (UPLC)-single quad MS detection system, even if they were not separated, which is a requirement when using an UV-detector. Another advantage of the UPLC-MS system is that lower limit of detection values could be obtained allowing a more accurate and precise determination of the experimental lag time in the in vitro skin permeation experiments. To conclude, this single quad MS detector coupled to UPLC is a useful analytical tool with improved performance compared to high performance liquid chromatography (HPLC)-UV for biomedical-pharmaceutical purposes in transdermal research.

Analytical quality-by-design approach for sample treatment of BSA-containing solutions.

The sample preparation of samples containing bovine serum albumin (BSA), e.g., as used in transdermal Franz diffusion cell (FDC) solutions, was evaluated using an analytical quality-by-design (QbD) approach. Traditional precipitation of BSA by adding an equal volume of organic solvent, often successfully used with conventional HPLC-PDA, was found insufficiently robust when novel fused-core HPLC and/or UPLC-MS methods were used. In this study, three factors (acetonitrile (%), formic acid (%) and boiling time (min)) were included in the experimental design to determine an optimal and more suitable sample treatment of BSA-containing FDC solutions. Using a QbD and Derringer desirability (D) approach, combining BSA loss, dilution factor and variability, we constructed an optimal working space with the edge of failure defined as D<0.9. The design space is modelled and is confirmed to have an ACN range of 83±3% and FA content of 1±0.25%.

Quantitative transdermal behavior of pellitorine from Anacyclus pyrethrum extract.

The plant Anacyclus pyrethrum (AP) consists of several N-alkylamides with pellitorine as main constituent. AP extracts are known to be biologically active and some products for topical administration containing AP plant extracts are already commercially available with functional cosmeceutical claims. However, no transdermal data for pellitorine are currently available. Therefore, our general goal was to investigate the local skin pharmacokinetics of the plant N-alkylamide pellitorine using a Franz diffusion cell set-up. Two different forms were applied on human skin: purified pellitorine and the AP extract. Our study demonstrated that pellitorine is able to cross the stratum corneum and the subsequent skin layers. A significantly higher permeability coefficient was observed when the AP extract (Kp=2.3 × 10(-4)cm/h) was administered, compared to purified pellitorine (Kp=1.1 × 10(-4)cm/h). With the obtained pellitorine concentrations in the skin layers and the receptor fluid, it is concluded that local and systemic effects can be expected after topical application. Due to these findings and as a regulatory consequence, products containing reasonable concentrations of pellitorine are recommended to be classified as a medicinal product.

Risk evaluation of impurities in topical excipients: The acetol case.

Pharmaceutical excipients for topical use may contain impurities, which are often neglected from a toxicity qualification viewpoint. The possible impurities in the most frequently used topical excipients were evaluated in-silico for their toxicity hazard. Acetol, an impurity likely present in different topical pharmaceutical excipients such as propylene glycol and glycerol, was withheld for the evaluation of its health risk after dermal exposure. An ex-vivo in-vitro permeation study using human skin in a Franz Diffusion Cell set-up and GC as quantification methodology showed a significant skin penetration with an overall Kp value of 1.82×10-3 cm/h. Using these data, limit specifications after application of a dermal pharmaceutical product were estimated. Based on the TTC approach of Cramer class I substances, i.e. 1800 µg/(day∙person), the toxicity-qualified specification limits of acetol in topical excipients were calculated to be 90 µg/mL and 180 µg/mL for propylene glycol and glycerol, respectively. It is concluded that setting specification limits for impurities within a quality-by-design approach requires a case-by-case evaluation as demonstrated here with acetol.

Related impurities in peptide medicines.

Peptides are an increasingly important group of pharmaceuticals, positioned between classic small organic molecules and larger bio-molecules such as proteins. Currently, the peptide drug market is growing twice as fast as other drug markets, illustrating the increasing clinical as well as economical impact of this medicine group. Most peptides today are manufactured by solid-phase peptide synthesis (SPPS). This review will provide a structured overview of the most commonly observed peptide-related impurities in peptide medicines, encompassing the active pharmaceutical ingredients (API or drug substance) as well as the finished drug products. Not only is control of these peptide-related impurities and degradants critical for the already approved and clinically used peptide-drugs, these impurities also possess the capability of greatly influencing initial functionality studies during early drug discovery phases, possibly resulting in erroneous conclusions. The first group of peptide-related impurities is SPPS-related: deletion and insertion of amino acids are related to inefficient Fmoc-deprotection and excess use of amino acid reagents, respectively. Fmoc-deprotection can cause racemization of amino acid residues and thus diastereomeric impurities. Inefficient deprotection of amino acid side chains results into peptide-protection adducts. Furthermore, unprotected side chains can react with a variety of reagents used in the synthesis. Oxidation of amino acid side chains and dimeric-to-oligomeric impurities were also observed. Unwanted peptide counter ions such as trifluoroacetate, originating from the SPPS itself or from additional purification treatments, may also be present in the final peptide product. Contamination of the desired peptide product by other unrelated peptides was also seen, pointing out the lack of appropriate GMP. The second impurity group results from typical peptide degradation mechanisms such as ß-elimination, diketopiperazine, pyroglutamate and succinimide formation. These SPPS- and degradation-related impurity types can also found in the finished peptide drug products, which can additionally contain a third group of related impurities, i.e. the API-excipient degradation products.

Dry heat forced degradation of buserelin peptide: kinetics and degradant profiling.

Buserelin is a GnRH agonist peptide drug, comprising a nine amino acid sequence (pGlu-His-Trp-Ser-Tyr-D-Ser(tBu)-Leu-Arg-Pro-NH-Et) and most commonly known for its application in hormone dependent cancer therapy, e.g. prostate cancer. In order to evaluate its hot-melt extrusion (HME) capabilities, buserelin powder in its solid state was exposed to elevated temperatures for prolonged time periods. A stability indicating UPLC-PDA method was used for quantification of buserelin and the formed degradants. Different solid state kinetic models were statistically evaluated of which the Ginstling-Brounshtein model fitted the data best. Extrapolation to and experimental verification of typical HME-related conditions, i.e. 5 min at 100°C and 125°C, showed no significant degradation, thus demonstrating the HME capabilities of buserelin. Mass spectrometric identification of the buserelin-related degradants formed under solid state heat stress was performed. Based upon the identity of these degradants, different degradation hypotheses were raised. First, direct ß-elimination of the hydroxyl moiety at the serine residue, followed by fragmentation into an amide (pGlu-His-Trp-NH2) and pyruvoyl (pyruvoyl-Tyr-D-Ser(tBu)-Leu-Arg-Pro-NH-Et) peptide fragments, was postulated. Alternatively, internal esterification due to nucleophilic attack of the unprotected serine residue, followed by ß-elimination or hydrolysis would yield pGlu-His-Trp, pGlu-His-Trp-NH2 and the pyruvoyl peptide fragment. Degradant pGlu-His-Trp-Ser-Tyr-NH2 is believed to be formed in a similar way. Secondly, direct backbone hydrolysis would yield pGlu-His-Trp and Tyr-D-Ser(tBu)-Leu-Arg-Pro-NH-Et peptide fragments. Moreover, the presence of Ala-Tyr-D-Ser(tBu)-Leu-Arg-Pro-NH-Et can be explained by hydrolysis of the Trp-Ser peptide bond and conversion of the serine moiety to an alanine moiety. Third and finally, isomerisation of aforementioned peptide fragments and buserelin itself was also observed.

UHPLC-MS/MS method for the determination of the cyclic depsipeptide mycotoxins beauvericin and enniatins in in vitro transdermal experiments.

Currently, dermal exposure data of cyclic depsipeptide mycotoxins beauvericin and enniatins are completely absent with a lack of local skin and systemic kinetics, despite their widespread skin contact and intrinsic hazard. Therefore a sensitive and specific bioanalytical high-throughput UHPLC-MS/MS method was developed for the quantitative and simultaneous determination of cyclic depsipeptide mycotoxins beauvericin and enniatins (A, A1, B, B1, D, E, C/F) in human skin Franz diffusion cell samples. The limits of detection ranged between 10 and 17pg/ml, while the total run time was only 4.5min. There was no significant effect of endogenous skin compounds on the mycotoxin MS signal observed, and the accuracy (0.68-24.68% bias) and precision (0.57-10.70% RSD) were considered acceptable for our purposes. Moreover, it was demonstrated that these cyclic depsipeptides are stable for at least 7 days when formulated in different organic or aqueous mixtures. Finally, adsorption to glass did occur: at least 50% ethanol or acetonitrile is required to prevent significant adsorption effects, which could be as high as 45%.