High Blood-brain Barrier Research Articles

Protein Toxin Chaperoned by LRP-1-Targeted Virus-Mimicking Vesicles Induces High-Efficiency Glioblastoma Therapy In Vivo.

Glioblastoma is a most intractable and high-mortality malignancy because of its extremely low drug accessibility resulting from the blood-brain barrier (BBB). Here, it is reported that angiopep-2-directed and redox-responsive virus-mimicking polymersomes (ANG-PS) (angiopep-2 is a peptide targeting to low-density lipoprotein receptor-related protein-1 (LRP-1)) can efficiently and selectively chaperone saporin (SAP), a highly potent natural protein toxin, to orthotopic human glioblastoma xenografts in nude mice. Unlike chemotherapeutics, free SAP has a low cytotoxicity. SAP-loaded ANG-PS displays, however, a striking antitumor activity (half-maximal inhibitory concentration, IC50 = 30.2 × 10-9 m) toward U-87 MG human glioblastoma cells in vitro as well as high BBB transcytosis and glioblastoma accumulation in vivo. The systemic administration of SAP-loaded ANG-PS to U-87 MG orthotopic human-glioblastoma-bearing mice brings about little side effects, effective tumor inhibition, and significantly improved survival rate. The protein toxins chaperoned by LRP-1-targeted virus-mimicking vesicles emerge as a novel and highly promising treatment modality for glioblastoma.

Targeted Covalent Inhibition of Prolyl Oligopeptidase (POP): Discovery of Sulfonylfluoride Peptidomimetics

Prolyl oligopeptidase (POP), a serine protease highly expressed in the brain, has recently emerged as an enticing therapeutic target for the treatment of cognitive and neurodegenerative disorders. However, most reported inhibitors suffer from short duration of action, poor protease selectivity, and low blood-brain barrier (BBB) permeability, which altogether limit their potential as drugs. Here, we describe the structure-based design of the first irreversible, selective, and brain-permeable POP inhibitors. At low-nanomolar concentrations, these covalent peptidomimetics produce a fast, specific, and sustained inactivation of POP, both invitro and in human cells. More importantly, they are >1,000-fold selective against two family-related proteases (DPPIV and FAP) and display high BBB permeability, as shown in both lipid membranes and MDCK cells.

Molecular modeling of inhibitors against fructose bisphosphate aldolase from Candida albicans.

Candida albicans is an opportunistic pathogen that causes from vulvovaginal and oropharyngeal candidiasis to systemic infections. The enzyme 1,6-fructose bisphosphate aldolase class II (FBA II), is a macromolecule existing only in lower organisms, being essential for the survival of the pathogen due to its function of maintaining the glycolysis process. The aim of this paper was to evaluate the inhibitors of FBA II regarding their physicochemical, pharmacokinetic and toxicological properties and apply concepts of rational drug development to propose new compounds for the treatment of fungal infections of C. albicans. Physicochemical (HyperChem software and the webserver cactus) and ADME/Tox (PreADMET webserver) properties were calculated to four inhibitors described in the literature and three analogues. None of the compounds presented in this study violated RO5, however all inhibitors demonstrated low or moderate human intestinal absorption (HIA), as well as low or moderate permeability in Caco-2 and MDCK, poor plasma proteins binding (PPB) and low permeability of the blood-brain barrier (BBB); however, Compound 4 is the exception for BBB permeability, being also the only non-mutagenic compound, and therefore, used as a lead compound. Analogues B and C presented high HIA, weak PPB and low BBB permeability, as well as a positive prediction for carcinogenicity in rats and mouse and non-mutagenicity in the Ames test. Through the evaluations carried out, it was concluded that the analogues B and C have proved to be promising candidates for oral administration drugs in the treatment of fungal infections of the genus Candida.

Pulsed Electromagnetic Field (PEMF) Mitigates High Intracranial Pressure (ICP) Induced Microvascular Shunting (MVS) in Rats.

High-frequency pulsed electromagnetic field (PEMF) stimulation is an emerging noninvasive therapy that we have shown increases cerebral blood flow (CBF) and tissue oxygenation in the healthy rat brain. In this work, we tested the effect of PEMF on the brain at high intracranial pressure (ICP). We previously showed that high ICP in rats caused a transition from capillary (CAP) to non-nutritive microvascular shunt (MVS) flow, tissue hypoxia and increased blood brain barrier (BBB) permeability. Using in vivo two-photon laser scanning microscopy (2PLSM) over the rat parietal cortex, and studied the effects of PEMF on microvascular blood flow velocity, tissue oxygenation (NADH autofluorescence), BBB permeability and neuronal necrosis during 4 h of elevated ICP to 30 mmHg. PEMF significantly dilated arterioles, increased capillary blood flow velocity and reduced MVS/capillary ratio compared to sham-treated animals. These effects led to a significant decrease in tissue hypoxia, BBB degradation and neuronal necrosis. PEMF attenuates high ICP-induced pathological microcirculatory changes, tissue hypoxia, BBB degradation and neuronal necrosis.

Rapid screening of brain-penetrable antioxidants from natural products by blood-brain barrier specific permeability assay combined with DPPH recognition

Screening brain-penetrable antioxidants from natural products is a promising way for neuroprotective drug discovery. However, there is no screening methodology enables simultaneous investigation of antioxidant activity and blood-brain barrier (BBB) permeability of compounds from complex samples. Here we propose a novel strategy by combining BBB specific parallel artificial membrane permeability assay with 1,1-diphenyl-2-picrylhydrazyl recognition (BBB-PAMPA–DPPH) to achieve rapid multicomponent screening. First, BBB specific artificial membrane was constructed to separate the compounds with high BBB permeability in herbal extracts. The antioxidant activity of the isolated compounds could be optically recognized through the bleaching of the purple-colored DPPH. By off-line combined HPLC–UV/Q-TOF-MS analysis, the exact BBB-penetrable compounds responsible for the antioxidant activity could be rapidly screened. With this approach, compound 2,6,4′-trihydroxy-4-methoxybenzophenon in Rhizoma Anemarrhena was found to be an antioxidant with very high BBB permeability, which could also be detected in rat plasma and brain tissue after oral administration. Our findings suggested the BBB-PAMPA–DPPH method could be a powerful tool for neuroprotective drug discovery from natural products.

A novel LRP1-binding peptide L57 that crosses the blood brain barrier

The blood-brain barrier (BBB) is a major obstacle to drug delivery into the central nervous system (CNS), in particular for macromolecules such as peptides and proteins. However, certain macromolecules can reach the CNS via a receptor-mediated transcytosis (RMT) pathway, and low-density lipoprotein receptor-related protein 1 (LRP1) is one of the promising receptors for RMT. An LRP1 ligand peptide, Angiopep-2, was reported to pass through the BBB and deliver covalently conjugated drugs into the CNS. While conjugation of LRP1 ligands with drugs would be an effective approach for drug delivery to the CNS, no other reliable LRP1 ligands have been reported to date. In this study, we aimed to identify novel LRP1 ligands to further investigate LRP1-mediated RMT. Using phage display technology, we obtained a novel peptide, L57 (TWPKHFDKHTFYSILKLGKH-OH), with an EC50 value of 45nM for binding to cluster 4 (Ser3332–Asp3779) of LRP1. L57 was stable in mouse plasma for up to 20min. In situ brain perfusion assay in mice revealed the significantly high BBB permeability of L57. In conclusion, we discovered L57, the first artificial LRP1-binding peptide with BBB permeability. Our findings will contribute to the development of RMT-based drugs for the treatment of CNS diseases.

Anticonvulsant activity of resveratrol-loaded liposomes in vivo

Resveratrol (3,5,4′-stilbenetriol), a natural polyphenol produced by various plants, has attracted attention over the past decade because of its multiple beneficial properties, including anti-inflammatory, anti-oxidant and chemopreventive, yet, there is limited information about its antiepileptic effects. Moreover, its poor solubility in water and low bioavailability are the challenging issues. In the present study, we aimed to investigate effects of free resveratrol and resveratrol delivered in amphipathic liposomal delivery system, which has a high blood–brain barrier crossing potential, on penicillin-induced epileptic seizure model. For this purpose, adult male Sprague–Dawley rats were divided into four groups as saline (Control), liposome (LIP), free resveratrol (RES) and resveratrol+liposome (RES+LIP). Penicillin-induced epileptic activity was recorded for 120 min by electrocorticography. Glutathione S-transferase (GST), Glutathione (GSH), Superoxide dismutase (SOD) and Malondialdehyde (MDA) assays were performed in brain tissues collected. Our results showed that RES+LIP was the most effective anticonvulsant treatment on penicillin-induced epileptic seizures when compared to control, as RES+LIP immediately decreased the number of spikes per minute. GST and SOD activity, as well as the GSH levels, were significantly increased in the RES+LIP group as compared with the control group. Also, the MDA levels were significantly higher in the RES+LIP compared to RES and control groups. In conclusion, RES+LIP treatment was more effective on the decrease in spike frequency and spike amplitudes than other treatments. Our results suggest that the RES+LIP is more effective than RES on penicillin-induced epileptiform activity.

Low-basicity 5-HT7 Receptor Agonists Synthesized Using the van Leusen Multicomponent Protocol

A series of 5-aryl-1-alkylimidazole derivatives was synthesized using the van Leusen multicomponent reaction. The chemotype is the first example of low-basicity scaffolds exhibiting high affinity for 5-HT7 receptor together with agonist function. The chosen lead compounds 3-(1-ethyl-1H-imidazol-5-yl)-5-iodo-1H-indole (AGH-107, 1o, Ki 5-HT7 = 6 nM, EC50 = 19 nM, 176-fold selectivity over 5-HT1AR) and 1e (5-methoxy analogue, Ki 5-HT7 = 30 nM, EC50 = 60 nM) exhibited high selectivity over related CNS targets, high metabolic stability and low toxicity in HEK-293 and HepG2 cell cultures. A rapid absorption to the blood, high blood-brain barrier permeation and a very high peak concentration in the brain (Cmax = 2723 ng/g) were found for 1o after i.p. (5 mg/kg) administration in mice. The compound was found active in novel object recognition test in mice, at 0.5, 1 and 5 mg/kg. Docking to 5-HT7R homology models indicated a plausible binding mode which explain the unusually high selectivity over the related CNS targets. Halogen bond formation between the most potent derivatives and the receptor is consistent with both the docking results and SAR. 5-Chlorine, bromine and iodine substitution resulted in a 13, 27 and 89-fold increase in binding affinities, respectively, and in enhanced 5-HT1AR selectivity.

Fast-onset antidepressant potentials of essential oil of herbs

The existing antidepressants demonstrated delayed onset of clinical effects, so fast-onset antidepressants are required. Essential oil of herbs showed potentials fast-onset antidepressant potential. First, its aromatic odor can directly activate olfactory nerves; its high lipophilicity causes a high blood-brain barrier penetration rate; and its high volatility is suitable for nasal-brain targeting and inhalation delivery. Therefore, essential oils can rapidly regulate brain functions by multiple ways, suggesting a fast-onset antidepressant potential. Second, the advance of studies on chemistry and pharmacology of antidepressant essential oils demonstrated chemical substances, antidepressant effects and possible action mechanisms of antidepressant essential oils. Third, the effect of essential oils' antidepressant components on fast-onset antidepressant targets was investigated. It was found that chemical constituents of essential oils antagonized NMDA receptor activities, suggesting that essential oils have fast-onset antidepressant effect. Finally, characteristics of essential oils, fast-onset antidepressant targets and drug delivery methods are integrated to give full play to essential oils' fast-onset antidepressant advantage and provide a new direction for new drug discovery.

Dynamic contrast-enhanced MRI in the evaluation of blood-brain barrier permeability following acute cerebral ischemia in rats

Objective To investigate the dynamic changes of blood-brain barrier(BBB) permeability after acute cerebral ischemia in rats with middle cerebral artery occlusion (MCAO) by dynamic contrast-enhanced(DCE)-MRI. Methods Sixty MCAO rat models were established by suture-occlusion method. All rats were divided randomly into twelve groups with different ischemia duration (3 hours, 6 hours, permanent) and reperfusion times (2, 6, 12 and 24 hours after reperfusion). Each group was examined by MRI at the time points. The BBB permeability parameters(Ktrans, Ve, Kep, rKtrans, rVe, rKep) were calculated by Siemens workstation and compared with Evans blue(EB) extravasation results. Multivariate analysis of variance (M-ANOVA), one-way analysis of variance (one-way ANOVA), Pearson analysis were respectively used to verify the influences of ischemia duration and reperfusion time on BBB permeability parameters, EB extravasation and relationships between parameters. Results In 3 hours and 6 hours ischemia duration groups, change of BBB permeability after reperfusion appeared biphasic. At 2 hours and 6 hours after reperfusion, BBB permeability increased, while rKtrans values and rVe values rose and rKep values dropped. BBB permeability decreased at 12 hours and increased again at 24 hours after reperfusion. The highest BBB permeability was observed at 6 hours after reperfusion. However, BBB permeability in permanent ischemia groups had uniphasic change, as its increase was rather mild as ischemia time went on. rKtrans values(1.99±0.79)were positively correlated with rVe values(2.88±1.78)(r=0.93, P<0.01) and negatively correlated with rKep values(0.66±0.21)(r=-0.84, P<0.01). The negative correlation between rVe values and rKep valueswas also significant(r=-0.80, P<0.01). EB extravasation results were consistent with MRI findings. Conclusions BBB permeability change was biphasic in reperfusion groups, while it was uniphasic in permanent ischemia groups. DCE-MRI may accurately reflect the changes of BBB permeability after acute cerebral ischemia. Both ischemic duration and reperfusion time had influences on BBB permeability. With prolongation of ischemic time, the duration of BBB permeability increase became shorter, BBB damage appeared earlier, with increased degree of ischemic damage. Key words: Blood brain barrier; Reperfusion injury; Infarction, middle cerebral artery; Magnetic resonance imging

Characterization of 11C-GSK1482160 for Targeting the P2X7 Receptor as a Biomarker for Neuroinflammation.

The purinergic receptor subtype 7 (P2X7R) represents a novel molecular target for imaging neuroinflammation via PET. GSK1482160, a potent P2X7R antagonist, has high receptor affinity, high blood-brain barrier penetration, and the ability to be radiolabeled with 11C. We report the initial physical and biologic characterization of this novel ligand. Methods:11C-GSK1482160 was synthesized according to published methods. Cell density studies were performed on human embryonic kidney cell lines expressing human P2X7R (HEK293-hP2X7R) and underwent Western blotting, an immunofluorescence assay, and radioimmunohistochemistry analysis using P2X7R polyclonal antibodies. Receptor density and binding potential were determined by saturation and association-disassociation kinetics, respectively. Peak immune response to lipopolysaccharide treatment in mice was determined in time course studies and analyzed via Iba1 and P2X7R Western blotting and Iba1 immunohistochemistry. Whole-animal biodistribution studies were performed on saline- or lipopolysaccharide-treated mice at 15, 30, and 60 min after radiotracer administration. Dynamic in vivo PET/CT was performed on the mice at 72 h after administration of saline, lipopolysaccharide, or lipopolysaccharide + blocking, and 2-compartment, 5-parameter tracer kinetic modeling of brain regions was performed. Results: P2X7R changed linearly with concentrations or cell numbers. For high-specific-activity 11C-GSK1482160, receptor density and Kd were 1.15 ± 0.12 nM and 3.03 ± 0.10 pmol/mg, respectively, in HEK293-hP2X7R membranes. Association constant kon, dissociation constant koff, and binding potential (kon/koff) in HEK293-hP2X7R cells were 0.2312 ± 0.01542 min-1⋅nM-1, 0.2547 ± 0.0155 min-1, and 1.0277 ± 0.207, respectively. Whole-brain Iba1 expression in lipopolysaccharide-treated mice peaked by 72 h on immunohistochemistry, and Western blot analysis of P2X7R for saline- and lipopolysaccharide-treated brain sections showed a respective 1.8- and 1.7-fold increase in signal enhancement at 72 h. Biodistribution of 11C-GSK1482160 in saline- and lipopolysaccharide-treated mice at 72 h was statistically significant across all tissues studied. In vivo dynamic 11C-GSK1482160 PET/CT of mice at 72 h after administration of saline, lipopolysaccharide, or lipopolysaccharide + blocking showed a 3.2-fold increase and 97% blocking by 30 min. The total distribution volumes for multiple cortical regions and the hippocampus showed statistically significant increases and were blocked by an excess of authentic standard GSK1482160. Conclusion: The current study provides compelling data that support the suitability of 11C-GSK1482160 as a radioligand targeting P2X7R, a biomarker of neuroinflammation.

Gold nanoparticle-capped mesoporous silica-based H2O2-responsive controlled release system for Alzheimer's disease treatment.

Due to the low ability to cross the blood-brain barrier (BBB) and non-specific interactions with metal ions necessary for normal cellular processes of metal chelator or Aβ inhibitors, we created a novel gold nanoparticle-capped mesoporous silica (MSN-AuNPs)-based H2O2-responsive controlled release system for targeted delivery of the metal chelator CQ and AuNPs (Aβ inhibitor). In this system, CQ and AuNPs are released only upon exposure to conditions in which H2O2 levels are high, such as those in Aβ plaques. The AuNPs on the surface of MSN also help in decrease the Aβ self-assembly, due to this, MSN-CQ-AuNPs were more efficient than MSN-CQ in inhibiting Cu2+-induced Aβ40 aggregation. Furthermore, MSN-CQ-AuNPs reduced the cell membrane disruption, microtubular defects and ROS-mediated apoptosis induced by Aβ40-Cu2+ complexes. Our data suggest that this controlled release system may have widespread application in the field of medicine for Alzheimer's disease.

Mucosal and blood-brain barrier transport kinetics of the plant N-alkylamide spilanthol using in vitro and in vivo models

BackgroundN-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.MethodsSpilanthes 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-MS2 method.ResultsSpilanthol 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).ConclusionsSpilanthol 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12906-016-1159-0) contains supplementary material, which is available to authorized users.

Role of hepcidin and its downstream proteins in early brain injury after experimental subarachnoid hemorrhage in rats.

Early brain injury (EBI) is a major cause of mortality from subarachnoid hemorrhage (SAH). We aimed to study the pathophysiology of EBI and explore the role of hepcidin, a protein involved in iron homeostatic regulation, and its downstream proteins. One hundred and thirty-two male Sprague-Dawley rats were assigned into groups (n=24/group): sham, SAH, SAH+hepcidin, SAH+hepcidin-targeting small interfering ribonucleic acid (siRNA), and SAH+scramble siRNA. Three hepcidin-targeting siRNAs and one scramble siRNA for hepcidin were injected 24h before hemorrhage induction, and hepcidin protein was injected 30min before induction. The rats were neurologically evaluated at 24h and euthanized at 72h. Hepcidin, ferroportin-1, and ceruloplasmin protein expression were measured by immunohistochemistry and Western blotting. Brain water content, blood-brain barrier (BBB) leakage, non-heme tissue iron and Garcia scale were evaluated. Hepcidin expression increased in the cerebral cortex and hippocampus after experimental SAH (P<0.05 compared to sham), while ferroportin-1 and ceruloplasmin decreased (P<0.05). Hepcidin injection lowered the expression of ferroportin-1 and ceruloplasmin further but siRNA reduced the levels of hepcidin (P<0.05 compared to SAH) resulting in recovery of ferroportin-1 and ceruloplasmin levels. Apoptosis was increased in SAH rats compared to sham (P<0.05) and increased slightly more by hepcidin, but decreased by siRNA (P<0.05 compared to SAH). SAH rats had lower neurological scores, high brain water content, BBB permeability, and non-heme tissue iron (P<0.05). In conclusion, downregulation of ferroportin-1 and ceruloplasmin caused by hepcidin enhanced iron-dependent oxidative damage and may be the potential mechanism of SAH.

Protective Effects of Edaravone in Adult Rats with Surgery and Lipopolysaccharide Administration-Induced Cognitive Function Impairment.

Postoperative cognitive dysfunction (POCD) is a clinical syndrome characterized by cognitive declines in patients after surgery. Previous studies have suggested that surgery contributed to such impairment. It has been proven that neuroinflammation may exacerbate surgery-induced cognitive impairment in aged rats. The free radical scavenger edaravone has high blood brain barrier permeability, and was demonstrated to effectively remove free radicals from the brain and alleviate the development of POCD in patients undergoing carotid endarterectomy, suggesting its potential role in preventing POCD. For this reason, this study was designed to determine whether edaravone is protective against POCD through its inhibitory effects on inflammatory cytokines and oxidative stress. First, Sprague Dawley adult male rats were administered 3 mg/kg edaravone intraperitoneally after undergoing a unilateral nephrectomy combined with lipopolysaccharide injection. Second, behavioral parameters related to cognitive function were recorded by fear conditioning and Morris Water Maze tests. Last, superoxide dismutase activities and malondialdehyde levels were measured in the hippocampi and prefrontal cortex on postoperative days 3 and 7, and microglial (Iba1) activation, p-Akt and p-mTOR protein expression, and synaptic function (synapsin 1) were also examined 3 and 7 days after surgery. Rats that underwent surgery plus lipopolysaccharide administration showed significant impairments in spatial and working memory, accompanied by significant reductions in hippocampal-dependent and independent fear responses. All impairments were attenuated by treatment with edaravone. Moreover, an abnormal decrease in superoxide dismutase activation, abnormal increase in malondialdehyde levels, significant increase in microglial reactivity, downregulation of p-Akt and p-mTOR protein expression, and a statistically significant decrease in synapsin-1 were observed in the hippocampi and prefrontal cortices of rats at different time points after surgery. All mentioned abnormal changes were totally or partially reversed by edaravone. To our knowledge, few reports have shown greater protective effects of edaravone on POCD induced by surgery plus lipopolysaccharide administration from its anti-oxidative stress and anti-inflammatory effects, as well as maintenance of Akt/mTOR signal pathway activation; these might be closely related to the therapeutic effects of edaravone. Our research demonstrates the potential use of edaravone in the treatment of POCD.

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

The 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.

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.

Exploring the physicochemical profile and the binding patterns of selected novel anticancer Himalayan plant derived active compounds with macromolecular targets

Abstract Plants are vital source of compounds offering plethora of therapeutic effects against various ailments without much side effects. Due to wide spread prevalence and drug resistance in cancer; there is an urgent need for discovery of new anti-cancer drugs. In the present study, selected novel anti-cancer plants derived compounds (cmpd1 to cmpd15) from Himalayan region were docked with defined molecular targets that regulate cell proliferation and apoptosis. The binding energies of best docked compounds ranged between −8.0 kcal/mol and −11.71 kcal/mol. Further analysis revealed critical hydrogen bonds and hydrophobic interactions between compounds and targets. The best docked compounds viz., cmpd15 against cyclin-dependent kinase-2 (CDK-2), cmpd8 against CDK-6 and cmpd9 against Topoisomerase I and II showed higher binding affinities than the native co-crystal ligands. The root mean square deviation (RMSD) and potential energy plot clearly indicates the stability of the complexes during 20 ns molecular dynamics (MD) simulation. The Molecular Mechanics/Poisson Boltzmann Surface Area (MM/PBSA) binding energy analysis revealed Van der Waals energy component which is the principal stabilizing energy for their interactions except CDK-2/cmpd15 complex. The polar solvation energy did not have favorable contribution to their stabilization. The binding energy decomposition analysis revealed per residue contribution for each docked complexes. Physicochemical profile studies showed that majority of the compounds conform to Lipinski's rule of five (ROF) having low to high blood brain barrier (BBB) penetration, human intestinal absorption, plasma binding protein inhibition and P glycoprotein inhibition.

Sialic acid (SA)-modified selenium nanoparticles coated with a high blood–brain barrier permeability peptide-B6 peptide for potential use in Alzheimer’s disease

The blood–brain barrier (BBB) is a formidable gatekeeper toward exogenous substances, playing an important role in brain homeostasis and maintaining a healthy microenvironment for complex neuronal activities. However, it also greatly hinders drug permeability into the brain and limits the management of brain diseases. The development of new drugs that show improved transport across the BBB represents a promising strategy for Alzheimer’s disease (AD) intervention. Whereas, previous study of receptor-mediated endogenous BBB transport systems has focused on a strategy of using transferrin to facilitate brain drug delivery system, a system that still suffers from limitations including synthesis procedure, stability and immunological response. In the present study, we synthetised sialic acid (SA)-modified selenium (Se) nanoparticles conjugated with an alternative peptide-B6 peptide (B6-SA-SeNPs, a synthetic selenoprotein analogue), which shows high permeability across the BBB and has the potential to serve as a novel nanomedicine for disease modification in AD. Laser-scanning confocal microscopy, flow cytometry analysis and inductively coupled plasma-atomic emission spectroscopy ICP-AES revealed high cellular uptake of B6-SA-SeNPs by cerebral endothelial cells (bEnd.3). The transport efficiency of B6-SA-SeNPs was evaluated in a Transwell experiment based on in vitro BBB model. It provided direct evidence for B6-SA-SeNPs crossing the BBB and being absorbed by PC12 cells. Moreover, inhibitory effects of B6-SA-SeNPs on amyloid-β peptide (Aβ) fibrillation could be demonstrated in PC12 cells and bEnd3 cells. B6-SA-SeNPs could not only effectively inhibit Aβ aggregation but could disaggregate preformed Aβ fibrils into non-toxic amorphous oligomers. These results suggested that B6-SA-SeNPs may provide a promising platform, particularly for the application of nanoparticles in the treatment of brain diseases. Statement of SignificanceAlzheimer’s disease (AD) is the world’s most common form of dementia characterized by intracellular neurofibrillary tangles in the brain. Over the past decades, the blood–brain barrier (BBB) limits access of therapeutic or diagnostic agents into the brain, which greatly hinders the development of new drugs for treating AD. In this work, we evaluated the efficiency of B6-SA-SeNPs across BBB and investigated the interactions between B6-SA-SeNPs and amyloid-β peptide (Aβ). We confirm that B6-SA-SeNPs could provide a promising platform because of its high brain delivery efficiency, anti-amyloid properties and anti-oxidant properties, which may serve as a novel nanomedicine for the application in the treatment of brain diseases.

Use of in-silico assays to characterize the ADMET profile and identify potential therapeutic targets of fusarochromanone, a novel anti-cancer agent.

PurposeFor 30 years nature has provided a plethora of natural products with potential meaningful anti-cancer activity. Fusarochromanone (FC101a) is a small molecule fungal metabolite exhibiting potent in-vitro growth inhibitory effects and is capable of inducing apoptosis, suppressing angiogenesis and tumorigenesis, and inhibiting endothelial cell growth in multiple cancer cell lines. Despite all we know regarding FC101a, the mechanism of action and molecular target(s) of this compound have remained an enigma. Furthermore, modest in-vivo activity has been documented and requires addressing.MethodEarly stage pharmacokinetics (PK) assessment is vital to successful drug development. Herein, we aimed to use in-silico assays to i) characterize an in-depth ADMET profile of FC101a and ii) to probe for possible therapeutic targets. Two-dimensional SDF files of FC101a and 13 analogs were introduced into ADMET Predictor Version 7.1 that parses the structures in order to calculate molecular descriptors, which are used to estimate ADMET properties. Calculated ADMET values were analyzed and subjected to multiple drug-like indices, delivering a PK profile of each analog. To probe for possible targets, a total of 49 proteins were introduced into SYBYL-X Version 2.0 platform and the deepest binding pocket of each protein was virtually docked with parent compound, FC101a; with the negative control, FC101b; and with the model compound, kynurenine.ResultsEach analog showed promising ADMET qualities, although FC101 Oxazole was identified as the most optimized analog. Despite FC101a having a desirable ADME and toxicity profile, areas of concern were identified and must be addressed in-vitro. These include potential mutagenic properties and estrogen receptor toxicity. We provide potential avenues medicinal chemists could use to achieve higher effective permeation, higher blood brain barrier (BBB) penetration, and higher aqueous solubility in FC101a. Molecular docking assays revealed procaspase-8 - cFLIP(L) complex as a potential biological target and led to proposed mechanisms of action by which FC101a facilitates procaspase-8 heterodimerization, thereby increasing proteolytic activity and up regulating extrinsic apoptosis.ConclusionOur data revealed both potential mechanisms of action and a promising ADMET profile of FC101a. These attributes render FC101a a promising lead candidate for development into a low toxic anti-cancer agent effective against a broad range of cancers.Electronic supplementary materialThe online version of this article (doi:10.1186/s40203-015-0010-5) contains supplementary material, which is available to authorized users.