Judit Müller

Applicability of a blood-brain barrier specific artificial membrane permeability assay at the early stage of natural product-based CNS drug discovery

While numerous natural products (NPs) possess activity on central nervous system (CNS) targets, there has been no analytical approach to effectively identify compounds with high brain penetration potential in complex mixtures at the early stage of drug discovery. To overcome this issue, the performance of an in vitro parallel artificial membrane permeability assay for the blood-brain barrier (PAMPA-BBB) for natural products and for plant extracts has been validated and characterized. It was found that the PAMPA-BBB assay preserves its predictive power in the case of natural products and provides high phytochemical selectivity, which enables its use as a unique filtering tool in terms of selecting brain-penetrable compounds from plant extracts. Moreover, the present study has demonstrated that simple modifications in the assay design allow the direct use of PAMPA-BBB filtered samples in a dereplication process, as performed by NMR and LC-MS. The applicability of this procedure was demonstrated using extracts prepared from Tanacetum parthenium, Vinca major, Salvia officinalis, and Corydalis cava, representing different types of chemical diversity and complexity. Thus, the proposed protocol represents a potentially valuable strategy in the NP-based CNS drug discovery environment with a high-throughput screening platform.

In vitro dissolution–permeation evaluation of an electrospun cyclodextrin-based formulation of aripiprazole using μFlux™

Since it is a well-known fact that among the newly discovered active pharmaceutical ingredients the number of poorly water soluble candidates is continually increasing, dissolution enhancement of poorly water soluble drugs has become one of the central challenges of pharmaceutical studies. So far the preclinical studies have been mainly focused on formulation methods to enhance the dissolution of active compounds, in many cases disregarding the fact that the formulation matrix not only affects dissolution but also has an effect on the transport through biological membranes, changing permeation of the drug molecules. The aim of this study was to test an electrospun cyclodextrin-based formulation of aripiprazole with the novel μFlux apparatus, which monitors permeation together with dissolution, and by this means better in vitro-in vivo correlation is achieved. It was evinced that a cyclodextrin-based electrospun formulation of aripiprazole has the potential to ensure fast drug delivery through the oral mucosa owing to the ultrafast dissolution of the drug from the formulation and the enhanced flux across membranes as shown by the result of the novel in vitro dissolution and permeation test.

pH-gradient PAMPA-based in vitro model assay for drug-induced phospholipidosis in early stage of drug discovery

In the present study we validated a widely used, high-throughput in vitro permeability model (PAMPA) to be used at the early stage of drug discovery for the phospholipidosis (PLD) prediction of drug-like compounds. Regarding the mechanism of action of PLD, our pH-gradient PAMPA system is the first noncell based model to mimic one-way transport of cationic amphiphilic drugs (CADs) from cytosol to the lysosome. Moreover, due to the fact that PLD can mainly occur in lung, liver, brain, kidney and heart, we have used similar commercially available original tissue-derived lipid fractions (heart, liver, brain), and in the case mimicking membrane of kidney and lung tissue we prepared tissue-mimetic artificial lipid mixtures in house. Metabolism of a drug can change the degree of PLD depending on the physicochemical properties of metabolites and the rate of metabolism. Our data from 57 drugs and 4 metabolites of earlier and 2 metabolites of newly recognized outliers (phenacetin and bupropion) using our pH-gradient PAMPA system show a good correlation with in vivo PLD data. Moreover, predictive ability of our best system, the lung specific pH-gradient PAMPA model was significantly better than widely used in silico models and it was also slightly better than that of the known noncell based models on our selection of compounds. Our pH-gradient PAMPA systems therefore offer mechanistically alternative, accurate and cost-effective screening tools for the early prediction of PLD potential of drug-like compounds.

BBB penetration-targeting physicochemical lead selection: Ecdysteroids as chemo-sensitizers against CNS tumors.

&NA; The anticancer potential of ecdysteroids, especially their chemo‐sensitizing activity has recently gained a substantial scientific interest. A comprehensive physicochemical profiling was performed for a set of natural or semi‐synthetic ecdysteroids (N = 37) to identify a lead compound against central nervous system (CNS) tumors. Calculated properties, such as lipophilicity (clogP), topological polar surface area (TPSA), brain‐to‐plasma ratio (clogBB) along with the measured blood‐brain barrier specific in vitro permeability (logPe) were evaluated in parallel. Compounds with the highest CNS‐availability predicted (clogBB > 0.0 and logPe > −6.0) showed moderate to high lipophilicity (clogP = 3.89−5.25), relatively low TPSA (94.45 Å2), and shared a common apolar 2,3‐ and 20,22‐diacetonide motif (25, 30–33). These ecdysteroids were selected for testing their capacity to sensitize SH‐SY5Y neuroblastoma cells to vincristine. All of the five tested compounds exerted a remarkably strong, dose dependent chemo‐sensitizing activity: at 2.5–10.0 &mgr;M ecdysteroids increased the cytotoxic activity of vincristine one to three orders of magnitude in (e.g., from IC50 = 39.5 ± 2.9 nM to as low as 0.056 ± 0.03 nM). Moreover, analysis of the combination index (CI) revealed outstanding synergism between ecdysteroids and vincristine (CI50 = 0.072−0.444). Thus, based on drug‐likeness, physchem character and in vitro CNS activity, compound 25 was proposed as a lead for further in vivo studies. Graphical Abstract Figure. No caption available.

Palladium‐Catalyzed 2,2,2‐Trifluoroethoxylation of Aromatic and Heteroaromatic Chlorides Utilizing Borate Salt and the Synthesis of a Trifluoro Analogue of Sildenafil

A simple and convenient method was developed for the introduction of a 2,2,2-trifluoroethoxy group to various aromatic and heteroaromatic systems. The novel process utilizes aromatic chlorides as substrates, and tetrakis(2,2,2-trifluoroethoxy) borate salt as an inexpensive and readily available fluoroalkoxy source in a palladium-catalyzed cross-coupling reaction. The power of the developed methodology was demonstrated in the synthesis of a fluorous derivative of Sildenafil.

Blood-brain barrier specific permeability assay reveals N-methylated tyramine derivatives in standardised leaf extracts and herbal products of Ginkgo biloba

The linkage between the central nervous system availability and neuropharmacological activity of the constituents of Ginkgo biloba L. extracts (GBE) is still incomplete. In this study, the in vitro blood-brain barrier (BBB) permeability profile of the standardised GBE was investigated by the parallel artificial membrane permeability assay (PAMPA). Biomarkers, such as terpene trilactones, flavonoid aglycones and ginkgotoxin exerted moderate or good BBB-permeability potential (BBB+), while glycosides and biflavones were predicted as unable to pass the BBB. N-methyltyramine (NMT) and N,N-dimethyltyramine or hordenine (Hor) were identified among BBB+ compounds, while subsequent direct HRMS analysis revealed tyramine (Tyr) and N,N,N-trimethyltyramine or candicine (Can) in GBE as trace constituents. Distribution of Tyr, NMT, Hor and Can was determined by a validated ion-exchange mechanism-based liquid chromatography-electrospray ionisation-mass spectrometry (LC-ESI-MS) method in G. biloba samples, such as herbal drugs and dietary supplements. The total content of the four tyramine derivatives in various GBEs ranged from 7.3 up to 6357μg/g dry extract with NMT and Hor as most abundant ones. Considering the pharmacological activities and the revealed fluctuation in the concentration of the analysed adrenergic protoalkaloids, the presented rapid LC-ESI-MS method is proposed for monitoring of the levels of Tyr, NMT, Hor and Can in G. biloba products.

Biomimetic synthesis and HPLC-ECD analysis of the isomers of dracocephins A and B

Starting from racemic naringenin ((±)-1), a mixture of dracocephin A stereoisomers 6-(2”-pyrrolidinone-5”-yl)naringenin (±)-2a–d and its regioisomer, dracocephin B 8-(2”-pyrrolidinone-5”-yl)naringenin (±)-3a–d originally isolated from Dracocephalum rupestre, have been synthesized in a one-pot reaction. The separation of 2a–d and 3a–d was achieved by preparative HPLC. The four stereoisomers of each natural product were separated by analytical chiral HPLC and their absolute configuration was studied by the combination of HPLC–ECD measurements and TDDFT–ECD calculations. The synthesized flavonoid alkaloids were further characterized by physicochemical and in vitro pharmacological studies.

Three newly identified lipophilic flavonoids in Tanacetum parthenium supercritical fluid extract penetrating the Blood-Brain Barrier

HighlightsOptimized supercritical fluid extraction of lipohilic flavonoids and parthenolid from Tanacetum parthenium L.Three newly identified lipophilic flavonoids (aceronin, sudachitin, nevadensin) in Tanacetum parthenium L.PAMPA‐BBB penetration of flavonoids and sesquiterpene lactones from feverfew. ABSTRACT Feverfew (Tanacetum parthenium L.) as a perennial herb has been known for centuries due to its medicinal properties. The main sesquiterpene lactone, parthenolide is considered to be responsible for the migraine prophylactic effect, however the pharmacological benefits of the lipophilic flavonoid components can not be neglected. Supercritical fluid extraction (7% ethanol, 22 MPa, 64 °C) was carried out on the leaves of Tanacetum parthenium L. from which the presence of methylated flavonoids beside parthenolide and other sesquiterpene lactones were indicated by preliminary LC–MS analyses. Specific Parallel Artificial Membrane Permeability Assay (PAMPA) was applied to identify the components capable to cross the Blood‐Brain Barrier (BBB). Three lipophilic flavonoids were detected on the acceptor side, that were isolated (Prep‐HPLC) and identified as sudachitin, aceronin and nevadensin (LC–MS/MS, NMR). These flavonoids were also characterized individually by PAMPA‐BBB model. The presence of sudachitin and nevadensin was proven in the Asteraceae family, but neither of the three flavonoids were reported in Tanacetum parthenium L.