Regulatory status of N-alkylamide containing health products.

N-alkylamides (NAAs) are secondary metabolites occurring in more than 25 plant families. Plants containing NAAs are traditionally used in food for flavouring, tingling, pungent and saliva-enhancing properties but also to treat various diseases. NAA containing products are abundantly available on the market as food, cosmetics, medical devices and medicinal products. However, no unambiguous legal product classification is applied for these products. In this study, the different health product classes from a European viewpoint are discussed in relation to the pharmacokinetic and pharmacodynamic properties of the NAAs, their applied dosage and claimed usage.

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.

Fish Hydrolysates: A Regulatory Perspective of Bioactive Peptides.

For the first time introduced on the Japanese market, bioactive fish hydrolysates are now available all over the world as food supplements, functional food ingredients or nutricosmeceuticals. They are generally produced from low value fish waste, an almost inexhaustible source of raw material, and are sold as high value products, making them economically interesting from a manufacturer's view point. Most of these products have health or structure/function claims on their packages with different actions like antihypertensive, blood-glucose lowering, anxiolytic, and skin anti-aging activities. Although the different regional legislations all aim to assure consumer safety and prevent misleading of the consumer, the number of legally approved fish hydrolysate containing products drastically differs among different regions. This is because products that have been positively evaluated based on safety and efficacy in one region were found to have not enough evidence for efficacy in another region. These findings call for further international harmonization of the regulation and classification of these products. Moreover, interaction studies of these bioactive products with the normal diet or medicines are generally not performed, keeping the consumer uninformed of the possible risks of combining these products with medicinal products or other food ingredients.

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.

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.

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.

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.

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.

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.

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.

Gas chromatographic method for the determination of lumefantrine in antimalarial finished pharmaceutical products.

A simple method has been developed and validated for quantitative determination of lumefantrine in antimalarial finished pharmaceutical products using gas chromatography coupled to flame ionization detector. Lumefantrine was silylated with N,O-bis(trimethyl-silyl)trifluoro-acetamide at 70°C for 30 minutes, and chromatographic separation was conducted on a fused silica capillary (HP-5, 30 m length × 0.32 mm i.d., 0.25 µm film thickness) column. Evaluation of the method within analytical quality-by-design principles, including a central composite face-centered design for the sample derivatization process and Plackett-Burman robustness verification of the chromatographic conditions, indicated that the method has acceptable specificity toward excipients and degradants, accuracy [mean recovery = 99.5%, relative standard deviation (RSD) = 1.0%], linearity (=0.9986), precision (intraday = 96.1% of the label claim, RSD = 0.9%; interday = 96.3% label claim, RSD = 0.9%), and high sensitivity with detection limits of 0.01 µg/mL. The developed method was successfully applied to analyze the lumefantrine content of marketed fixed-dose combination antimalarial finished pharmaceutical products.

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.

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.

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%.

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.

Risk evaluation of impurities in topical excipients:The acetol case / 药物分析学报

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 mg/(day?person), the toxicity-qualified specification limits of acetol in topical excipients were calculated to be 90 mg/mL and 180 mg/mL for propylene glycol and glycerol, respectively.

Skin penetration enhancing properties of the plant N-alkylamide spilanthol.

ETHNOPHARMACOLOGICAL RELEVANCE: Plants are often used for skin diseases in different ethnopharmacological systems. Local and systemic effects of topically applied compounds can be significantly increased by plant constituents having skin penetration enhancers. MATERIALS AND METHODS: In this study, we examined the proposed penetration enhancing properties of spilanthol, an N-alkylamide abundantly present in several Asteraceae plants like Spilanthes acmella L., on three model drugs (caffeine, testosterone and ibuprofen). Moreover, as plants are frequently contaminated with toxic environmental substances, the mutual influence on the transdermal behavior between spilanthol and six model mycotoxins (aflatoxin B1, ochratoxin A, fumonisin B1, citrinin, zearalenone, T-2 toxin) was investigated. RESULTS: Spilanthol exhibits component and concentration dependent penetration enhancing effects. No significant penetration enhancing effect for ibuprofen has been observed, but with increasing spilanthol concentration (from 0 up to 1w/V%), the permeability of caffeine increased, resulting in an enhancing ratio (ER) of 4.60. For testosterone, a maximal penetration enhancing concentration of 0.5% spilanthol was found (ER=4.13). Next to its beneficial applicability to increase local as well as systemic pharmacological effects of dermally co-administrated drug, this N-alkylamide negatively influences human health risk if spilanthol containing formulations are polluted with mycotoxins: the presence of spilanthol (0.3w/V%) induced a significant increase of permeability coefficient Kp of five investigated mycotoxins, with ER values ranging between 1.57 and 6.37. On the other hand, mycotoxins themselves do not significantly influence the transdermal behavior of spilanthol. CONCLUSIONS: The existence of a significant mutual influence of compounds towards skin penetration should always be considered during the development or as part of the functional quality evaluation of topical products.

A critical quality parameter in quantitative fused-core chromatography: The injection volume.

As part of the method development, the injection volume as a critical quality attribute in fast fused-core chromatography was evaluated. Spilanthol, a pharmaceutically interesting N-alkylamide currently under investigation in our laboratory, was chosen as the model compound. Spilanthol was dissolved in both PBS and MeOH/H2O (70/30, v/v) and subsequently analyzed using a fused-core system hereby selecting five chromatographic characteristics (retention time, area, height, theoretical plates and symmetry factor) as responses. We demonstrated that the injection volume significantly influenced both the qualitative and quantitative performance of fused-core chromatography, a phenomenon which is confounded with the nature of the used sample solvent. From 2 µL up to 100 µL injection volume with PBS as solvent, the symmetry factor decreased favorably by 20%. Moreover, the theoretical plates and the quantitative parameters (area and height) increased up to 30%. On the contrary, in this injection volume range, the theoretical plates for the methanol-based samples decreased by more than 60%, while the symmetry factor increased and the height decreased, both by 30%. The injection volume is thus a critical and often overlooked parameter in fused-core method description and validation.

A critical quality parameter in quantitative fused-core chromatography：The injection volume / 药物分析学报

As part of the method development, the injection volume as a critical quality attribute in fast fused-core chromatography was evaluated. Spilanthol, a pharmaceutically interesting N-alkylamide currently under investigation in our laboratory, was chosen as the model compound. Spilanthol was dissolved in both PBS and MeOH/H2O (70/30, v/v) and subsequently analyzed using a fused-core system hereby selecting five chromatographic characteristics (retention time, area, height, theoretical plates and symmetry factor) as responses. We demonstrated that the injection volume significantly influenced both the qualitative and quantitative performance of fused-core chromatography, a phenomenon which is confounded with the nature of the used sample solvent. From 2 mL up to 100 mL injection volume with PBS as solvent, the symmetry factor decreased favorably by 20%. Moreover, the theoretical plates and the quantitative parameters (area and height) increased up to 30%. On the contrary, in this injection volume range, the theoretical plates for the methanol-based samples decreased by more than 60%, while the symmetry factor increased and the height decreased, both by 30%. The injection volume is thus a critical and often overlooked parameter in fused-core method description and validation.

Alkamid database: Chemistry, occurrence and functionality of plant N-alkylamides.

ETHNOPHARMACOLOGICAL RELEVANCE: N-Alkylamides (NAAs) are a promising group of bioactive compounds, which are anticipated to act as important lead compounds for plant protection and biocidal products, functional food, cosmeceuticals and drugs in the next decennia. These molecules, currently found in more than 25 plant families and with a wide structural diversity, exert a variety of biological-pharmacological effects and are of high ethnopharmacological importance. However, information is scattered in literature, with different, often unstandardized, pharmacological methodologies being used. Therefore, a comprehensive NAA database (acronym: Alkamid) was constructed to collect the available structural and functional NAA data, linked to their occurrence in plants (family, tribe, species, genus). MATERIALS AND METHODS: For loading information in the database, literature data was gathered over the period 1950-2010, by using several search engines. In order to represent the collected information about NAAs, the plants in which they occur and the functionalities for which they have been examined, a relational database is constructed and implemented on a MySQL back-end. RESULTS: The database is supported by describing the NAA plant-, functional- and chemical-space. The chemical space includes a NAA classification, according to their fatty acid and amine structures. CONCLUSIONS: The Alkamid database (publicly available on the website http://alkamid.ugent.be/) is not only a central information point, but can also function as a useful tool to prioritize the NAA choice in the evaluation of their functionality, to perform data mining leading to quantitative structure-property relationships (QSPRs), functionality comparisons, clustering, plant biochemistry and taxonomic evaluations.