Comparative study of four immortalised human brain capillary endothelial cell lines, hCMEC/D3, hBMED, TY10, and BB19, and optimization of culture conditions, for an in vitro blood-brain barrier model for drug permeability studies

BACKGROUND: Reliable human in vitro blood–brain barrier (BBB) models suitable for high-throughput screening are urgently needed in early drug discovery and development for assessing the ability of promising bioactive compounds to overcome the BBB. To establish an improved human in vitro BBB model, we compared four currently available and well characterized immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, with respect to barrier tightness and paracellular permeability. Co-culture systems using immortalized human astrocytes (SVG-A cell line) and immortalized human pericytes (HBPCT cell line) were designed with the aim of positively influencing barrier tightness. METHODS: Tight junction (TJ) formation was assessed by transendothelial electrical resistance (TEER) measurements using a conventional epithelial voltohmmeter (EVOM) and an automated CellZscope system which records TEER and cell layer capacitance (C(CL)) in real-time. Paracellular permeability was assessed using two fluorescent marker compounds with low BBB penetration (sodium fluorescein (Na-F) and lucifer yellow (LY)). Conditions were optimized for each endothelial cell line by screening a series of 24-well tissue culture inserts from different providers. For hBMEC cells, further optimization was carried out by varying coating material, coating procedure, cell seeding density, and growth media composition. Biochemical characterization of cell type-specific transmembrane adherens junction protein VE-cadherin and of TJ proteins ZO-1 and claudin-5 were carried out for each endothelial cell line. In addition, immunostaining for ZO-1 in hBMEC cell line was performed. RESULTS: The four cell lines all expressed the endothelial cell type-specific adherens junction protein VE-cadherin. The TJ protein ZO-1 was expressed in hCMEC/D3 and in hBMEC cells. ZO-1 expression could be confirmed in hBMEC cells by immunocytochemical staining. Claudin-5 expression was detected in hCMEC/D3, TY10, and at a very low level in hBMEC cells. Highest TEER values and lowest paracellular permeability for Na-F and LY were obtained with mono-cultures of hBMEC cell line when cultivated on 24-well tissue culture inserts from Greiner Bio-one® (transparent PET membrane, 3.0 μm pore size). In co-culture models with SVG-A and HBPCT cells, no increase of TEER could be observed, suggesting that none of the investigated endothelial cell lines responded positively to stimuli from immortalized astrocytic or pericytic cells. CONCLUSIONS: Under the conditions examined in our experiments, hBMEC proved to be the most suitable human cell line for an in vitro BBB model concerning barrier tightness in a 24-well mono-culture system intended for higher throughput. This BBB model is being validated with several compounds (known to cross or not to cross the BBB), and will potentially be selected for the assessment of BBB permeation of bioactive natural products

Use and caregiver-reported efficacy of medical cannabis in children and adolescents in Switzerland.

Evidence on the use and efficacy of medical cannabis for children is limited. We examined clinical and epidemiological characteristics of medical cannabis treatment and caregiver-reported effects in children and adolescents in Switzerland. We collected clinical data from children and adolescents (< 18 years) who received Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), or a combination of the two between 2008 and 2019 in Switzerland. Out of 205 contacted families, 90 agreed to participate. The median age at the first prescription was 11.5 years (interquartile range (IQR) 6-16), and 32 patients were female (36%). Fifty-one (57%) patients received CBD only and 39 (43%) THC. Patients were more likely to receive THC therapy if one of the following symptoms or signs were present: spasticity, pain, lack of weight gain, vomiting, or nausea, whereas seizures were the dominant indication for CBD therapy. Improvements were reported in 59 (66%) study participants. The largest treatment effects were reported for pain, spasticity, and frequency of seizures in participants treated with THC, and for those treated with pure CBD, the frequency of seizures. However, 43% of caregivers reported treatment interruptions, mainly because of lack of improvement (56%), side effects (46%), the need for a gastric tube (44%), and cost considerations (23%).Conclusions: The effects of medical cannabis in children and adolescents with chronic conditions are unknown except for rare seizure disorders, but the caregiver-reported data analysed here may justify trials of medical cannabis with standardized concentrations of THC or CBD to assess its efficacy in the young. What is Known: • The use of medical cannabis (THC and CBD) to treat a variety of diseases among children and adolescents is increasing. • In contrast to adults, there is no evidence to support the use of medical cannabis to treat chronic pain and spasticity in children, but substantial evidence to support the use of CBD in children with rare seizure disorders. What is New: • This study provides important insights into prescription practices, dosages, and treatment outcomes in children and adolescents using medical cannabis data from a real-life setting. • The effects of medical cannabis in children and adolescents with chronic conditions shown in our study support trials of medical cannabis for chronic conditions

Establishment and validation of an immortalized in vitro human blood-brain barrier (BBB) model for drug permeability studies, and application to natural product derived leads

In the human brain, the endothelial cells lining the cerebral microvessels form a uniquely tight cellular layer separating the brain tissue from the bloodstream. This cellular barrier, designated as “blood-brain barrier” (BBB), prevents the entry of xenobiotics and neurotoxic metabolites into the central nervous system (CNS) and thus protects the nerve tissue from chemical damage. More than 98% of small molecule drugs have been estimated to not cross the BBB. For drugs targeting the CNS, however, low BBB permeability may lead to limited brain penetration, culminating in insufficient drug concentrations at the target sites and thus therapeutic failure. On the other hand, low BBB permeation is desirable for non-CNS drugs, as this reduces the risk of CNS-related side effects. Regardless of the therapeutic area, drug lead candidates should therefore be screened for their ability to permeate the BBB already at an early stage of the drug development process, in order to reduce their attrition rate at a later stage. In the past years, a broad spectrum of cell-based in vitro BBB models has been developed and implemented in academia and industry to bring forward molecules with high potential for CNS exposure. Despite considerable efforts, there is still an urgent need for reliably predictive BBB models, in particular human ones. Primary human cells are difficult to obtain on ethical grounds, are laborious to cultivate, suffer from batch-to-batch variation, and are thus suitable only for low throughput screenings. To overcome these limitations, immortalized human brain microvascular endothelial cell lines have been generated by transfection with tumor genes. Unlike primary cultures, immortalized cells are easy to cultivate, proliferate indefinitely, and maintain their differentiating properties even after repeated passaging. These properties render them highly suitable for standardized screenings amenable to higher throughput. Regrettably, currently available immortalized human brain capillary endothelial cell lines often show deficiencies such as low barrier tightness, relatively high leakage of barrier integrity markers, and insufficient expression of key transporter systems. Consequently, careful optimization and validation of human cell line-based in vitro BBB models have to be carried out prior to their application to permeability screening of drug candidates. The aim of this thesis was to establish a human in vitro BBB model based on an immortalized human brain capillary endothelial cell line, to validate it with a representative series of drug substances known to cross the BBB to a varying extent, and to apply it to BBB permeability studies of promising lead compounds of natural origin. To establish an improved in vitro human BBB model, we evaluated in a first step four currently available immortalized human brain capillary endothelial cell lines (hCMEC/D3, hBEMC, TY10, and BB19) regarding their ability to produce endothelial cell monolayers with sufficient barrier tightness in a 24-well Transwell system. Transendothelial electrical resistance (TEER) values were recorded in real-time using an automated CellZscope system to obtain highly standardized data. Culture conditions (growth medium composition, tissue culture insert material, coating material and procedure, cell seeding density) were systematically optimized, the impact of co-cultured immortalized human astrocytes (SVG-A cell line) and pericytes (HBPCT cell line) on barrier integrity of endothelial cell monolayers was investigated, and biochemical and immunocytochemical characterization of cell-type specific cellular junction proteins was performed. Under the conditions examined in our experiments, mono-cultures of hBMEC cell line exhibited highest TEER values (around 40 Ωcm2) and lowest leakage of two fluorescent barrier integrity markers (sodium fluorescein, Na-F; and lucifer yellow, LY) (apparent permeability coefficients (Papp) in the range of 3–5 x 10-6 cm/s). Furthermore, hBMEC cells were shown to express the tight junction proteins ZO-1 and claudin-5, and the endothelial marker protein VE-cadherin, confirming their endothelial lineage. Thus, we concluded that hBMEC cell line was the most suitable cell line in terms of barrier tightness for the establishment of an immortalized in vitro human BBB model. The hBMEC cell line-based in vitro human BBB model was validated in a next step with a representative series of structurally diverse compounds known to cross the BBB to a different extent. Antipyrine, caffeine, diazepam, and propranolol were selected as positive controls, while atenolol, cimetidine, quinidine, and vinblastine served as negative controls. For each compound, a quantitative ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) assay in Ringer HEPES buffer (RHB) was developed and validated in terms of selectivity, precision, and reliability according to current international guidelines. During method validation, numerous biological and analytical challenges were encountered, demonstrating that major precautions have to be taken prior to quantification, and underlining the importance of careful method development. All compounds were screened in the in vitro human BBB model with the barrier integrity marker Na-F in parallel, and endothelial permeability coefficients (Pe) across hBMEC monolayers were determined by means of the validated UHPLC-MS/MS methods. The in vitro human BBB model correctly predicted BBB permeability of the selected compounds, with the exception of one negative control (quinidine, a small basic lipophilic P-glycoprotein (P-gp) inhibitor and substrate). A limitation of the model may thus be the lack of discrimination between passively diffusing compounds and substrates of active efflux. Complementary assays to determine efflux pump interaction are therefore recommended. Nevertheless, we conclude that our model represents a promising tool for early BBB permeability assessment of lead candidates in drug discovery, as it is of human origin (thus reducing the risk for data confounded by species differences), easy and fast to set up, and thus amenable to moderate to higher throughput screening. After validation, we screened the alkaloid piperine from black pepper (Piper nigrum L.) and five selected piperine analogs with positive allosteric γ-aminobutyric acid type A (GABAA) receptor modulatory activity for their ability to permeate the BBB in the immortalized in vitro human BBB model. Since GABAA receptors are expressed in the CNS, lead compounds modulating this target need to cross the BBB to reach their sites of action. For comparative purposes, the compounds were screened in parallel in a human stem cell-derived and in a well-established primary animal (bovine endothelial/rat astrocytes co-culture) in vitro BBB model. For each compound, a quantitative UHPLC-MS/MS assay in the corresponding matrix was developed, and permeability coefficients in each model were determined. In vitro predictions from both human models were in good agreement, while permeability data from the animal model differed to some extent. In all three BBB models, piperine and the semisynthetic analog SCT-64 displayed highest BBB permeability, which was corroborated by in silico prediction data. For the other piperine analogs, BBB permeability was low to moderate in the two human models, and moderate to high in the animal model. Efflux ratios (ER) calculated from bidirectional permeability experiments indicated that the compounds were not substrates of active efflux transporters. In addition to GABAA receptor modulating compounds, the indolinone derivative (E,Z)-3-(4-hydroxy-3,5-dimethoxybenzylidene)indolin-2-one (indolinone) from woad (Isatis tinctoria L.) was screened in our immortalized in vitro human BBB model. The compound had previously been shown to possess potent histamine release inhibitory and anti-inflammatory properties, and thus represents a promising lead candidate for the development of new anti-allergic drugs. In vitro data from the immortalized in vitro BBB model indicated a high BBB permeation potential for indolinone, which was corroborated by in vitro permeability data obtained from two well-established primary animal models, and by in silico prediction data. Furthermore, P-gp interaction of the compound was assessed with the aid of two specific efflux pump interaction assays. Both assays suggested that no active mediated transport mechanism was involved for the compound. In conclusion, we have successfully established and validated an easy and fast to set up human in vitro BBB model, and applied it to in-house BBB drug permeability assays of promising lead candidates of natural origin

Validation of an immortalized human (hBMEC) in vitro blood-brain barrier model

We recently established and optimized an immortalized human in vitro blood-brain barrier (BBB) model based on the hBMEC cell line. In the present work, we validated this mono-culture 24-well model with a representative series of drug substances which are known to cross or not to cross the BBB. For each individual compound, a quantitative UHPLC-MS/MS method in Ringer HEPES buffer was developed and validated according to current regulatory guidelines, with respect to selectivity, precision, and reliability. Various biological and analytical challenges were met during method validation, highlighting the importance of careful method development. The positive controls antipyrine, caffeine, diazepam, and propranolol showed mean endothelial permeability coefficients (P e) in the range of 17-70 × 10(-6) cm/s, indicating moderate to high BBB permeability when compared to the barrier integrity marker sodium fluorescein (mean P e 3-5 × 10(-6) cm/s). The negative controls atenolol, cimetidine, and vinblastine showed mean P e values < 10 × 10(-6) cm/s, suggesting low permeability. In silico calculations were in agreement with in vitro data. With the exception of quinidine (P-glycoprotein inhibitor and substrate), BBB permeability of all control compounds was correctly predicted by this new, easy, and fast to set up human in vitro BBB model. Addition of retinoic acid and puromycin did not increase transendothelial electrical resistance (TEER) values of the BBB model

Development and validation of a LC–MS/MS method for assessment of an anti-inflammatory indolinone derivative by in vitro blood–brain barrier models

International audienceThe compound (E,Z)-3-(4-hydroxy-3,5-dimethoxybenzylidene)indolin-2-one (indolinone) was identified from lipophilic woad extracts (Isatis tinctoria L., Brassicaceae) as a compound possessing potent histamine release inhibitory and anti-inflammatory properties [1]. To further evaluate the potential of indolinone in terms of crossing the blood-brain barrier (BBB), we screened the compound in several in vitro cell-based human and animal BBB models. Therefore, we developed a quantitative LC-MS/MS method for the compound in modified Ringer HEPES buffer (RHB) and validated it according to FDA and EMA guidelines [2,3]. The calibration curve of indolinone in the range between 30.0 and 3000ng/ml was quadratic, and the limit of quantification was 30.0ng/ml. Dilution of samples up to 100-fold did not affect precision and accuracy. The carry-over was within acceptance criteria. Indolinone proved to be stable in RHB for 3h at room temperature (RT), and for three successive freeze/thaw cycles. The processed samples could be stored in the autosampler at 10°C for at least 28h. Moreover, indolinone was stable for at least 16 days in RHB when stored below -65°C. This validation study demonstrates that our method is specific, selective, precise, accurate, and capable to produce reliable results. In the immortalized human BBB mono-culture model, the apparent permeability coefficient from apical to basolateral (PappA→B), and the Papp from basolateral to apical (PappB→A) were 19.2±0.485×10(-6)cm/s and 21.7±0.326×10(-6)cm/s, respectively. For the primary rat/bovine BBB co-culture model a PappA→B of 27.1±1.67×10(-6)cm/s was determined. In the primary rat BBB triple co-culture model, the PappA→B and the PappB→A were 56.2±3.63×10(-6)cm/s and 34.6±1.41×10(-6)cm/s, respectively. The data obtained with the different models showed good correlation and were indicative of a high BBB permeation potential of indolinone confirmed by in silico prediction calculations. P-glycoprotein (P-gp) interaction for indolinone was studied with the aid of a calcein-AM uptake assay, and by calculation of the efflux ratio (ER) from the bidirectional permeability assays. For both bidirectional BBB models an ER below 2 was calculated, indicating that no active mediated transport mechanism is involved for indolinone. In porcine brain capillary endothelial cells (PBCECs), the calcein-AM uptake assay demonstrated that indolinone is neither a P-gp substrate nor a P-gp inhibitor and is accumulated into cells at high extent

Pharmacokinetics and In Vitro Blood-Brain Barrier Screening of the Plant-Derived Alkaloid Tryptanthrin

International audienceThe indolo[2,1-b]quinazoline alkaloid tryptanthrin was previously identified as a potent anti-inflammatory compound with a unique pharmacological profile. It is a potent inhibitor of cyclooxygenase-2, 5-lipooxygenase-catalyzed leukotriene synthesis, and nitric oxide production catalyzed by the inducible nitric oxide synthase. To characterize the pharmacokinetic properties of tryptanthrin, we performed a pilot in vivo study in male Sprague-Dawley rats (2 mg/kg bw i. v.). Moreover, the ability of tryptanthrin to cross the blood-brain barrier was evaluated in three in vitro human and animal blood-brain barrier models. Bioanalytical UPLC-MS/MS methods used were validated according to current international guidelines. A half-life of 40.63 ± 6.66 min and a clearance of 1.00 ± 0.36 L/h/kg were found in the in vivo pharmacokinetic study. In vitro data obtained with the two primary animal blood-brain barrier models showed a good correlation with an immortalized human monoculture blood-brain barrier model (hBMEC cell line), and were indicative of a high blood-brain barrier permeation potential of tryptanthrin. These findings were corroborated by the in silico prediction of blood-brain barrier penetration. P-glycoprotein interaction of tryptanthrin was assessed by calculation of the efflux ratio in bidirectional permeability assays. An efflux ratio below 2 indicated that tryptanthrin is not subjected to active efflux

Andrographolide-loaded nanoparticles for brain delivery: formulation, charcterization and in vitro permeability using hCMEC/D3 cell line

Andrographolide (AG) is a major diterpenoid of the Asian medicinal plant Andrographis paniculata which has shown exciting pharmacological potential for the treatment of inflammation-related pathologies including neurodegenerative disorders. Conversely, the low bioavailability of AG still represents a limiting factor for its use. To overcome these limitations, AG was loaded into human serum albumin based nanoparticles (HSA NPs) and poly ethylcyanoacrylate nanoparticles (PECA NPs). HSA NPs were prepared by thermal (HSAT AG NPs) and chemical cross-linking (HSAC AG NPs), while PECA AG NPs were produced by emulsion-polymerization. NPs were characterized in terms of size, zeta (ζ)-potential, polydispersity, and release studies of AG. In addition, the ability of free AG and AG-loaded in PECA and HSAT NPs to cross the blood-brain barrier (BBB) was assessed using an in vitro BBB model based on human cerebral microvascular endothelial cell line (hCMEC/D3). For BBB drug permeability assays, a quantitative UPLC-MS/MS method for AG in Ringer HEPES buffer was developed and validated according to international regulatory guidelines for industry. Free AG did not permeate the BBB model, as also predicted by in silico studies. HSAT NPs improved by two-fold the permeation of AG while maintaining the integrity of the cell layer, while PECA NPs temporarily disrupted BBB integrity

In vitro blood–brain barrier permeability predictions for GABAA receptor modulating piperine analogs

International audienceThe alkaloid piperine from black pepper (Piper nigrum L.) and several synthetic piperine analogs were recently identified as positive allosteric modulators of γ-aminobutyric acid type A (GABAA) receptors. In order to reach their target sites of action, these compounds need to enter the brain by crossing the blood-brain barrier (BBB). We here evaluated piperine and five selected analogs (SCT-66, SCT-64, SCT-29, LAU397, and LAU399) regarding their BBB permeability. Data were obtained in three in vitro BBB models, namely a recently established human model with immortalized hBMEC cells, a human brain-like endothelial cells (BLEC) model, and a primary animal (bovine endothelial/rat astrocytes co-culture) model. For each compound, quantitative UHPLC-MS/MS methods in the range of 5.00-500ng/mL in the corresponding matrix were developed, and permeability coefficients in the three BBB models were determined. In vitro predictions from the two human BBB models were in good agreement, while permeability data from the animal model differed to some extent, possibly due to protein binding of the screened compounds. In all three BBB models, piperine and SCT-64 displayed the highest BBB permeation potential. This was corroborated by data from in silico prediction. For the other piperine analogs (SCT-66, SCT-29, LAU397, and LAU399), BBB permeability was low to moderate in the two human BBB models, and moderate to high in the animal BBB model. Efflux ratios (ER) calculated from bidirectional permeability experiments indicated that the compounds were likely not substrates of active efflux transporters