Biophysical investigation of liposome systems decorated with bioconjugated copolymers in the presence of amantadine.

Liposome-based technologies derived from lipids and polymers (e.g., PEGylated liposomes) have been recognized because of their applications in nanomedicine. However, since such systems represent myriad challenges and may promote immune responses, investigation of new biomaterials is mandatory. Here, we report on a biophysical investigation of liposomes decorated with bioconjugated copolymers in the presence (or absence) of amantadine (an antiviral medication). First, copolymers of poly(N,N-dimethylacrylamide-co-fluoresceinacrylate-co-acrylic acid-N-succinimide ester)-block-poly(N-isopropylacrylamide) (PDMA-b-PNIPAM) containing a fluorescence label were biofunctionalized with short peptides that resemble the sequence of the loops 220 and 130 of the binding receptor of the hemagglutinin (HA) protein of the influenza A virus. Then, the bioconjugated copolymers were self-assembled along with liposomes composed of 1,2 dimyristoyl-sn-glycero-3-phosphocholine, sphingomyelin, and cholesterol (MSC). These biohybrid systems, with and without amantadine, were systematically characterized using differential scanning calorimetry (DSC), dynamic light scattering (DLS), and cryogenic transmission electron microscopy (cryoTEM). Finally, the systems were tested in an in vitro study to evaluate cytotoxicity and direct immunofluorescence in Madin Darbin Canine Kidney (MDCK) cells. The biohybrid systems displayed long-term stability, thermo-responsiveness, hydrophilic-hydrophobic features, and fluorescence properties and were presumable endowed with cell targeting properties intrinsically integrated into the amino acid sequences of the utilized peptides, which indeed turn them into promising nanodevices for biomedical applications.

Amantadine interactions with phase separated lipid membranes.

Amantadine, a small amphilphic organic compound that consists of an adamantane backbone and an amino group, was first recognized as an antiviral in 1963 and received approval for prophylaxis against the type A influenza virus in 1976. Since then, it has also been used to treat Parkinson's disease-related dyskinesia and is being considered as a treatment for corona viruses. Since amantadine usually targets membrane-bound proteins, its interactions with the membrane are also thought to be important. Biological membranes are now widely understood to be laterally heterogeneous and certain proteins are known to preferentially co-localize within specific lipid domains. Does amantadine, therefore, preferentially localize in certain lipid composition domains? To address this question, we studied amantadine's interactions with phase separating membranes composed of cholesterol, DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine), and DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), as well as single-phase DPhPC (1,2-diphytanoyl-sn-glycero-3-phos-phocholine) membranes. From Langmuir trough and differential scanning calorimetry (DSC) measurements, we determined, respectively, that amantadine preferentially binds to disordered lipids, such as POPC, and lowers the phase transition temperature of POPC/DSPC/cholesterol mixtures, implying that amantadine increases membrane disorder. Further, using droplet interface bilayers (DIBs), we observed that amantadine disrupts DPhPC membranes, consistent with its disordering properties. Finally, we carried out molecular dynamics (MD) simulations on POPC/DSPC/cholesterol membranes with varying amounts of amantadine. Consistent with experiment, MD simulations showed that amantadine prefers to associate with disordered POPC-rich domains, domain boundaries, and lipid glycerol backbones. Since different proteins co-localize with different lipid domains, our results have possible implications as to which classes of proteins may be better targets for amantadine.

Determination of Acetylamantadine by γ-Cyclodextrin-Assisted α-HL Nanopore for Potential Cancer Prediagnosis.

The high expression of Spermidine/spermine N1-acetyltransferase (SSAT-1) is an important indicator in early cancer diagnosis. Here, we developed a nanopore-based methodology with γ-cyclodextrin as an adaptor to detect and quantify acetylamantadine, the specific SSAT-1-catalyzed product from amantadine, to accordingly reflect the activity of SSAT-1. We employ γ-cyclodextrin and report that amantadine cannot cause any secondary signals in γ-cyclodextrin-assisted α-HL nanopore, while its acetylation product, acetylamantadine, does. This allows γ-cyclodextrin to practically detect acetylamantadine in the interference of excessive amantadine, superior to the previously reported ß-cyclodextrin. The quantification of acetylamantadine was not interfered with even a 50-fold amantadine and displayed no interference in artificial urine sample analysis, which indicates the good feasibility of this nanopore-based methodology in painless cancer prediagnosis. In addition, the discrimination mechanism is also explored by 2-D nuclear magnetic resonance (NMR) and nanopore experiments with a series of adamantane derivatives with different hydrophilic and hydrophobic groups. We found that both the hydrophobic region matching effect and hydrophilic interactions play a synergistic effect in forming a host-guest complex to further generate the characteristic signals, which may provide insights for the subsequent design and study of drug-cyclodextrin complexes.

Ultrasensitive SERS aptasensor using Au@Ag bimetallic nanorod SERS tags for the selective detection of amantadine in foods.

Herein, a novel surface enhanced Raman spectroscopy (SERS) aptasensor was developed for amantadine (AMD) detection, based on magnetite nanoparticles coated with polyethylenimine, silver nanoclusters and aptamers (Fe3O4@PEI@AgNC-apt) as the capture probe and complementary DNA-modified gold nanorods (AuNRs@4-MPBA@Ag-c-DNA containing 4-mercaptophenylboric acid molecules) as the reporter probe. In the presence of AMD, the AMD and the reporter probe competed for the aptamer on the surface of the capture probe, resulting in the reporter probe detaching from the capture probe leading to a decrease in intensity of the SERS signal at 1067 cm-1 for 4-MPBA. Under optimal conditions, a good linear relationship was established between the SERS intensity at 1067 cm-1 and the logarithm of the AMD concentration over the range 10-6-102 mg L-1, with a LOD of 0.50 × 10-6 mg L-1. The AMD levels in spiked samples were evaluated using the SERS aptasensor, with good recoveries ranging from 90.57% to 113.49% being obtained.

Exploring Amantadine Derivatives as Urease Inhibitors: Molecular Docking and Structure-Activity Relationship (SAR) Studies.

This article describes the design and synthesis of a series of novel amantadine-thiourea conjugates (3a-j) as Jack bean urease inhibitors. The synthesized hybrids were assayed for their in vitro urease inhibition. Accordingly, N-(adamantan-1-ylcarbamothioyl)octanamide (3j) possessing a 7-carbon alkyl chain showed excellent activity with IC50 value 0.0085 ± 0.0011 µM indicating that the long alkyl chain plays a vital role in enzyme inhibition. Whilst N-(adamantan-1-ylcarbamothioyl)-2-chlorobenzamide (3g) possessing a 2-chlorophenyl substitution was the next most efficient compound belonging to the aryl series with IC50 value of 0.0087 ± 0.001 µM. The kinetic mechanism analyzed by Lineweaver-Burk plots revealed the non-competitive mode of inhibition for compound 3j. Moreover, in silico molecular docking against target protein (PDBID 4H9M) indicated that most of the synthesized compounds exhibit good binding affinity with protein. The compound 3j forms two hydrogen bonds with amino acid residue VAL391 having a binding distance of 1.858 Å and 2.240 Å. The interaction of 3j with amino acid residue located outside the catalytic site showed its non-competitive mode of inhibition. Based upon these results, it is anticipated that compound 3j may serve as a lead structure for the design of more potent urease inhibitors.

Conjugation of Aminoadamantane and γ-Carboline Pharmacophores Gives Rise to Unexpected Properties of Multifunctional Ligands.

A new series of conjugates of aminoadamantane and γ-carboline, which are basic scaffolds of the known neuroactive agents, memantine and dimebon (Latrepirdine) was synthesized and characterized. Conjugates act simultaneously on several biological structures and processes involved in the pathogenesis of Alzheimer's disease and some other neurodegenerative disorders. In particular, these compounds inhibit enzymes of the cholinesterase family, exhibiting higher inhibitory activity against butyrylcholinesterase (BChE), but having almost no effect on the activity of carboxylesterase (anti-target). The compounds serve as NMDA-subtype glutamate receptor ligands, show mitoprotective properties by preventing opening of the mitochondrial permeability transition (MPT) pore, and act as microtubule stabilizers, stimulating the polymerization of tubulin and microtubule-associated proteins. Structure-activity relationships were studied, with particular attention to the effect of the spacer on biological activity. The synthesized conjugates showed new properties compared to their prototypes (memantine and dimebon), including the ability to bind to the ifenprodil-binding site of the NMDA receptor and to occupy the peripheral anionic site of acetylcholinesterase (AChE), which indicates that these compounds can act as blockers of AChE-induced ß-amyloid aggregation. These new attributes of the conjugates represent improvements to the pharmacological profiles of the separate components by conferring the potential to act as neuroprotectants and cognition enhancers with a multifunctional mode of action.

In Silico Analysis Revealed a Unique Binding but Ineffective Mode of Amantadine to Influenza Virus B M2 Channel.

The M2 proton channel of influenza A (AM2) and B (BM2) have a highly conserved function motif, considered as the effective target. As yet, there is no effective drug against BM2. Research showed that AM2 channel blocker, amantadine (AMT), was able to bind to BM2 channel, but AMT lacked inhibition against BM2. Nevertheless, the study of the binding but ineffective mode of AMT to BM2 is challenging. To resolve the challenge and obtain more information for drug design of inhibitors targeting BM2, multiple molecular dynamics simulations were performed. We discovered AMT mainly adopted up binding mode in BM2, involved in a transition flipping from down mode to up mode. Furthermore, we discovered a new key factor to explain ineffective inhibition of AMT to BM2 because of the unmatched spatial geometry between AMT and BM2. Our work could enrich structural feature information on BM2 and provide a new perspective for rational drug design of anti-influenza B.

Microhydration of substituted diamondoid radical cations of biological relevance: infrared spectra of amantadine+-(H2O)n = 1-3 clusters.

Hydration of biomolecules and pharmaceutical compounds has a strong impact on their structure, reactivity, and function. Herein, we explore the microhydration structure around the radical cation of the widespread pharmaceutical drug amantadine (C16H15NH2, Ama) by infrared photodissociation (IRPD) spectroscopy of mass-selected Ama+Wn = 1-3 clusters (W = H2O) recorded in the NH, CH, and OH stretch range of the cation ground electronic state. Analysis of the size-dependent frequency shifts by dispersion-corrected density functional theory calculations (B3LYP-D3/cc-pVTZ) provides detailed information about the acidity of the protons of the NH2 group of Ama+ and the structure and strength of the NHO and OHO hydrogen bonds (H-bonds) of the hydration network. The preferred sequential cluster growth begins with hydration of the two acidic NH protons of the NH2 group (n = 1-2) and continues with an extension of the H-bonded hydration network by forming an OHO H-bond of the third W to one ligand in the first hydration subshell (n = 3), like in the W2 dimer. For n = 2, a minor population corresponds to Ama+W2 structures with a W2 unit attached to Ama+via a NHW2 H-bond. Although the N-H proton donor bonds are progressively destabilized by gradual microhydration, no proton transfer to the Wn solvent cluster is observed in the investigated size range (n ≤ 3). Besides the microhydration structure, we also obtain a first impression of the structure and IR spectrum of bare Ama+, as well as the effects of both ionization and hydration on the structure of the adamantyl cage. Comparison of Ama+ with aliphatic and aromatic primary amine radical cations reveals differences in the acidity of the NH2 group and the resulting interaction with W caused by substitution of the cycloalkyl cage.

Structure and drug binding of the SARS-CoV-2 envelope protein transmembrane domain in lipid bilayers.

An essential protein of the SARS-CoV-2 virus, the envelope protein E, forms a homopentameric cation channel that is important for virus pathogenicity. Here we report a 2.1-Å structure and the drug-binding site of E's transmembrane domain (ETM), determined using solid-state NMR spectroscopy. In lipid bilayers that mimic the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) membrane, ETM forms a five-helix bundle surrounding a narrow pore. The protein deviates from the ideal α-helical geometry due to three phenylalanine residues, which stack within each helix and between helices. Together with valine and leucine interdigitation, these cause a dehydrated pore compared with the viroporins of influenza viruses and HIV. Hexamethylene amiloride binds the polar amino-terminal lumen, whereas acidic pH affects the carboxy-terminal conformation. Thus, the N- and C-terminal halves of this bipartite channel may interact with other viral and host proteins semi-independently. The structure sets the stage for designing E inhibitors as antiviral drugs.

Photoregulation of Gene Expression with Amantadine-Modified Caged siRNAs through Host-Guest Interactions.

RNA interference is an essential and powerful tool for targeting and verifying specific gene functions. Conditional control of small interfering RNA (siRNA) activity, especially using light activation, is a potential method for regulating target gene expression and functions. In this study, a series of photolabile siRNAs with amantadine modification have been rationally designed and developed through host-guest interactions between amantadine and ß-cyclodextrin derivatives to enhance the blocking effect of siRNA binding and/or RNA-induced silencing complex processing. These caged siRNAs with amantadine modification at the 5' end of antisense-strand RNA were efficiently inactivated through the host-guest interactions between amantadine and ß-cyclodextrin. Photomodulation of the gene silencing activity of these amantadine-modified caged siRNAs targeting both exogenous and endogenous genes was successfully achieved, which indicates that host-guest interactions could be a new strategy for developing new caged siRNAs for gene photoregulation with low leaking activity.

Virtual Screening Identifies Chebulagic Acid as an Inhibitor of the M2(S31N) Viral Ion Channel and Influenza A Virus.

The increasing prevalence of drug-resistant influenza viruses emphasizes the need for new antiviral countermeasures. The M2 protein of influenza A is a proton-gated, proton-selective ion channel, which is essential for influenza replication and an established antiviral target. However, all currently circulating influenza A virus strains are now resistant to licensed M2-targeting adamantane drugs, primarily due to the widespread prevalence of an M2 variant encoding a serine to asparagine 31 mutation (S31N). To identify new chemical leads that may target M2(S31N), we performed a virtual screen of molecules from two natural product libraries and identified chebulagic acid as a candidate M2(S31N) inhibitor and influenza antiviral. Chebulagic acid selectively restores growth of M2(S31N)-expressing yeast. Molecular modeling also suggests that chebulagic acid hydrolysis fragments preferentially interact with the highly-conserved histidine residue within the pore of M2(S31N) but not adamantane-sensitive M2(S31). In contrast, chebulagic acid inhibits in vitro influenza A replication regardless of M2 sequence, suggesting that it also acts on other influenza targets. Taken together, results implicate chebulagic acid and/or its hydrolysis fragments as new chemical leads for M2(S31N) and influenza-directed antiviral development.

Host-Guest Protein Assembly for Affinity Purification of Methyllysine Proteomes.

Protein-protein interactions drive self-assembly of biomacromolecules and thus enable important physiological functions at a cellular level. Supramolecular chemists have developed artificial host-guest interactions that are similar with, yet distinct from and orthogonal to, the natural protein-protein interactions. For instance, cucurbit[n]urils are synthetic receptors that can specifically recognize proteins with N-terminal aromatic residues with high affinities, yet this interaction can be reversed by the competition of small molecules such as amantadine. Herein, we develop a site-specific, oriented protein-display method by combining the host-guest interaction based on cucurbit[7]uril and a covalent protein-peptide reaction. A methyllysine-binding protein HP1ß chromodomain (CD) is immobilized via host-guest interactions and used as the "bait" to capture methyllysine proteomes from cancer cells. The captured "fish"-methyllysine-containing proteins-can be released via competitive displacement by amantadine in a nondenaturing and traceless manner. This affinity purification method found 73 novel methyllysine sites from 101 identified sites among 66 methylated proteins from 255 HP1ß CD-binding proteins in cancer cells via subsequent mass spectrometric analysis. This work thereby presents a new strategy of artificial host-guest protein assembly in affinity purification of methyllysine proteins in coupling to mass spectrometry.

Amantadine hydrochloride monitoring by dried plasma spot technique: High-performance liquid chromatography-tandem mass spectrometry based clinical assay.

Amantadine plasma concentrations correlate well with desired therapeutic effects and adverse outcomes; information on amantadine exposure could be useful when multiple amantadine clearance pathways are impaired or non-compliance is suspected. Micro-sampling strategies, like dried plasma spot, would be particularly useful because ambulatory patients that do not attend a clinic can easily sample a few drops of blood by themselves at the required time of the dosing interval. We developed and validated a dried-plasma-spot-based high performance liquid chromatography-tandem mass spectrometry assay to quantify amantadine. This assay met relevant validation requirements within a hematocrit range of 20-50% and was linear from 100 to 2000 ng/mL. Amantadine was stable in dried plasma spots for up to 21 days at room temperature, regardless of whether the dried plasma spot was protected from light or not. The correlation between paired dried and wet plasma concentrations was assessed in 52 patients. Deming regression coefficients between wet plasma and simultaneously pipetted dried plasma spots were used to predict plasma concentrations. Bland-Altman plots revealed a strong agreement between dried and wet plasma concentrations, supporting the clinical usefulness of dried plasma spots for amantadine monitoring with a self-sampling strategy at a convenient time and place for the patient.

Site-directed mutations of anti-amantadine scFv antibody by molecular dynamics simulation: prediction and validation.

A recombinant single-chain variable fragment (scFv) antibody was produced from a hybridoma cell strain secreting the monoclonal antibody for amantadine (AMD), and then its recognition mechanisms for AMD were studied using the molecular docking and molecular dynamics. Complex dockings revealed that three regions are involved in antibody recognition; framework 2 of the VL chain (LFR2) GLU40 and TYR42, complementarity-determining region of the VL chain (LCDR3) TYR116, and framework 2 of the VH chain (HFR2) HIS40 and TRP52 were the key amino acid residues. The results of molecular dynamics show that the most important amino acid residues in the interaction between AMD and scFv are HIS40 and TYR116. On the basis of the results of virtual mutation, the scFv antibody was evolved by directional mutagenesis of amino acid residue GLY107 to PHE. Indirect competitive ELISA (icELISA) results indicated that the scFv mutant had highly increased affinity for AMD with up to 3.9-fold improved sensitivity. Thus, the scFv antibody can be applied for mechanistic studies of intermolecular interactions, and our work offered affinity maturated antibodies by site mutations, which were beneficial for valuable anti-AMD antibody design and preparation in future.

Supra-blot: an accurate and reliable assay for detecting target proteins with a synthetic host molecule-enzyme hybrid.

In accordance with the rapid increase in demand for selective and spatial chemical tagging, and accurate detection of proteins of interest, we develop a sensitive protein detection method, termed "Supra-blot" capitalizing on high-affinity host-guest interaction between cucurbit[7]uril (CB[7]) and adamantylammonium (AdA). The method can directly detect chemically tagged proteins without false-positive signals caused by endogenous biomolecules. Not only a single specific protein, but also spatially localized proteins in cells were labeled with AdA, and selectively detected by a host molecule-enzyme hybrid, CB[7]-conjugated horseradish peroxidase (CB[7]-HRP) generating amplified chemiluminescence signals. This study shows the great potential of Supra-blot for accurate and reliable detection of proteins of interest in cells.

The boundary lipid around DMPC-spanning influenza A M2 transmembrane domain channels: Its structure and potential for drug accommodation.

We have investigated the perturbation of influenza A M2TM in DMPC bilayers. We have shown that (a) DSC and SAXS detect changes in membrane organization caused by small changes (micromolar) in M2TM or aminoadamantane concentration and aminoadamantane structure, by comparison of amantadine and spiro[pyrrolidine-2,2'-adamantane] (AK13), (b) that WAXS and MD can suggest details of ligand topology. DSC and SAXS show that at a low M2TM micromolar concentration in DPMC bilayers, two lipid domains are observed, which likely correspond to M2TM boundary lipids and bulk-like lipids. At higher M2TM concentrations, one domain only is identified, which constitutes essentially all of the lipid molecules behaving as boundary lipids. According to SAXS, WAXS, and DSC in the absence of M2TM, both aminoadamantane drugs exert a similar perturbing effect on the bilayer at low concentrations. At the same concentrations of the drug when M2TM is present, amantadine and, to a lesser extent, AK13 cause, according to WAXS, a significant disordering of chain-stacking, which also leads to the formation of two lipid domains. This effect is likely due, according to MD simulations, to the preference of the more lipophilic AK13 to locate closer to the lateral surfaces of M2TM when compared to amantadine, which forms stronger ionic interactions with phosphate groups. The preference of AK13 to concentrate inside the lipid bilayer close to the exterior of the hydrophobic M2TM helices may contribute to its higher binding affinity compared to amantadine.

Development and validation of an LC-MS/MS method for the quantitative analysis of the anti-influenza agent camphecene in rat plasma and its application to study the blood-to-plasma distribution of the agent.

A method of quantitative determination of camphecene, a new anti-influenza agent, in rat blood plasma based on LC-MS/MS was developed, validated and used to study the distribution of the agent between blood cells and blood plasma. The method was validated according to FDA and EMA recommendations in terms of selectivity, linearity, accuracy, precision, recovery, stability and carry-over. Plasma samples were precipitated with methanol followed by the addition of a methanolic solution of 2-adamantylamine hydrochloride (internal standard). HPLC analysis was performed on a reversed-phase column; the total time of analysis was 11 min, including column equilibration. MS/MS detection was performed on a 3200 QTRAP triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode. Transitions 196.4 → 122.2/153.3 and 152.2 → 93.1/107.2 were monitored for camphecene and the internal standard, respectively. The calibration curve was built in the concentration range of 10-5000 ng/ml. The intra-day and inter-day accuracy and precision, carry-over and recovery were within the acceptable limits. It was found that, after spiking blood with camphecene and separating plasma, the concentration of the substance in the latter was close to its initial concentration in the blood. This property of the substance may be useful for clinical trials of the agent. It has also been established that the process of camphecene distribution (adsorption) between blood cells and blood plasma is reversible, and the amount of adsorbed substance is linearly dependent on its initial concentration in the blood for a wide range of concentrations, temperatures and hematocrit values.

Supramolecular Surface Charge Regulation in Ionic Covalent Organic Nanosheets: Reversible Exfoliation and Controlled Bacterial Growth.

Poor control on the exfoliation of covalent organic frameworks (COFs) remains a disadvantage for their application as two-dimensional nanosheets. An equally important problem is the reversible control at the available surface charges on COFs. Herein, a strategy for the reversible exfoliation, re-stacking, and surface-charge control of a propidium iodide based ionic covalent organic framework, PI-TFP, using cucurbit[7]uril (CB[7]) induced molecular recognition, is reported. The surface charge on PI-TFP facilitates its initial self-exfoliation. However, complexation with CB[7] resulted in re-stacking with concomitant decrease in zeta potential from +28±3.0 to +0.004±0.003 mV. Addition of 1-adamantylamine hydrochloride (AD) facilitates decomplexation of PI-TFP from CB[7], resulting in exfoliation and an increase in zeta potential to +24±3.0 mV. Such control on the exfoliation, re-stacking, and the associated regulation of the surface charge in PI-TFP was exploited for controlling bacterial growth. Thus, the activity of E. coli and S. aureus bacteria obtained with the self-exfoliated PI-TFP could be reversibly controlled by the CB[7]/AD pair.

De novo design of a homo-trimeric amantadine-binding protein.

The computational design of a symmetric protein homo-oligomer that binds a symmetry-matched small molecule larger than a metal ion has not yet been achieved. We used de novo protein design to create a homo-trimeric protein that binds the C3 symmetric small molecule drug amantadine with each protein monomer making identical interactions with each face of the small molecule. Solution NMR data show that the protein has regular three-fold symmetry and undergoes localized structural changes upon ligand binding. A high-resolution X-ray structure reveals a close overall match to the design model with the exception of water molecules in the amantadine binding site not included in the Rosetta design calculations, and a neutron structure provides experimental validation of the computationally designed hydrogen-bond networks. Exploration of approaches to generate a small molecule inducible homo-trimerization system based on the design highlight challenges that must be overcome to computationally design such systems.

Automated Stopped-Flow Fluorimetric Sensor for Biologically Active Adamantane Derivatives Based on Zone Fluidics.

A zone-fluidics (ZF) based automated fluorimetric sensor for the determination of pharmaceutically active adamantine derivatives, i.e., amantadine (AMA), memantine (MEM) and rimantadine (RIM) is reported. Discrete zones of the analytes and reagents (o-phthalaldehyde and N-acetylcysteine) mix and react under stopped-flow conditions to yield fluorescent iso-indole derivatives (λex/ λem = 340/455 nm). The proposed ZF sensor was developed and validated to prove suitable for quality control tests (assay and content uniformity) of commercially available formulations purchased from the Greek market (EU licensed) and from non-EU web-pharmacies at a sampling rate of 16 h-1. Interestingly, a formulation obtained through the internet and produced in a third-non-EU-country (AMA capsules, 100 mg per cap), was found to be out of specifications (mean assay of 85.3%); a validated HPLC method was also applied for confirmatory purposes.