Despite the constant development of new antiepileptic drugs (AEDs), more than 30% of patients develop refractory epilepsy (RE) characterized by a multidrug-resistant (MDR) phenotype. The "transporters hypothesis" indicates that the mechanism of this MDR phenotype is the overexpression of ABC transporters such as P-glycoprotein (P-gp) in the neurovascular unit cells, limiting access of the AEDs to the brain. Recent clinical trials and basic studies have shown encouraging results for the use of cannabinoids in RE, although its mechanisms of action are still not fully understood. Here, we have employed astrocytes and vascular endothelial cell cultures subjected to hypoxia, to test the effect of cannabidiol (CBD) on the P-gp-dependent Rhodamine-123 (Rho-123) efflux. Results show that during hypoxia, intracellular Rho-123 accumulation after CBD treatment is similar to that induced by the P-gp inhibitor Tariquidar (Tq). Noteworthy, this inhibition is like that registered in non-hypoxia conditions. Additionally, docking studies predicted that CBD could behave as a P-gp substrate by the interaction with several residues in the α-helix of the P-gp transmembrane domain. Overall, these findings suggest a direct effect of CBD on the Rho-123 P-gp-dependent efflux activity, which might explain why the CBD add-on treatment regimen in RE patients results in a significant reduction in seizure frequency.
Despite the constant development of new antiepileptic drugs (AEDs), more than 30% of patients develop refractory epilepsy (RE) characterized by a multidrug-resistant (MDR) phenotype. The -transporters hypothesis‖ indicates that the mechanism of this MDR phenotype is the overexpression of ABC transporters such as P-glycoprotein (P-gp) in the neurovascular unit cells, limiting access of the AEDs to the brain. Recent clinical trials and basic studies have shown encouraging results for the use of cannabinoids in RE, although its mechanisms of action are still not fully understood. Here, we have employed astrocytes and vascular endothelial cell cultures subjected to hypoxia, to test the effect of cannabidiol (CBD) on the P-gp-dependent Rhodamine-123 (Rho-123) efflux. Results show that during hypoxia, intracellular Rho-123 accumulation after CBD treatment is similar to that induced by the P-gp inhibitor Tariquidar (Tq). Noteworthy, this inhibition is like that registered in non-hypoxia conditions. Additionally, docking studies predicted that CBD could behave as a P-gp substrate by the interaction with several residues in the α-helix of the P-gp transmembrane domain. Overall, these findings suggest a direct effect of CBD on the Rho-123 P-gp-dependent efflux activity, which might explain why the CBD add-on treatment regimen in RE patients results in a significant reduction in seizure frequency.
Phoneutria nigriventer spider can cause severe envenomation in humans principally due to its venom toxin δ-ctenitoxin-Pn2a. Current low yielding antivenom production is extremely complicated and dangerous. Furthermore, δ-ctenitoxin-Pn2a cystine-knot motif provides exceptional stability hampering immune response activation. Here, epitopes from δ-ctenitoxin-Pn2a were identi ed, and antigenic peptides were designed for their potential use in antivenom production. The Immune Epitope Database Analysis Resource was used to identify the G 34 YFWIAWYKLANCKK 48 epitope and used to design antigenic peptides. The Cys was replaced by α-aminobutyric acid (Abu) to avoid disul de bonds formation. To increase their immunogenicity, branched and N-palmitoylated peptides were synthesized.Ac-GYFWIAWYKLAN-Abu-KKG-NH 2 (A), (Ac-GYFWIAWYKLAN-Abu-KK) 2 -KG-NH 2 (B), Palm-GYFWIAWYKLAN-Abu-KKG-NH 2 (C) and (Palm-GYFWIAWYKLAN-Abu-KK) 2 -KG-NH 2 (D) were synthesized using solid-phase peptide synthesis (SPPS) techniques and analyzed by ESI-MS demonstrating their identity. Also, they were evaluated by RP-HPLC, and all the chromatograms showed only one principal peak except that of the N-palmitoylated branched peptide which showed two principal peaks probably due to the presence of two conformations in slow interconversion. Cytotoxicity was evaluated on the murine macrophage cell line RAW264.7 by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay in the presence of increasing doses of each peptide (0.25-10.0 µM). Peptide A did not exhibit cytotoxicity between 0.25-10.0 µM, while B, C and D showed cytotoxicity over 10.0, 5.0 and 2.5 µM respectively. NF-κB cellular distribution was evaluated by immuno uorescence, after exposing macrophages to 0.5 µM of each peptide. An early activation was observed for all the assayed peptides demonstrating that they are promising candidates for their in vivo evaluation as immunogens in antivenom production.
Phoneutria nigriventer spider can cause severe envenomation in humans principally due to its venom toxin δ-ctenitoxin-Pn2a. Current low yielding antivenom production is extremely complicated and dangerous. Furthermore, δ-ctenitoxin-Pn2a cystine-knot motif provides exceptional stability hampering immune response activation. Here, epitopes from δ-ctenitoxin-Pn2a were identified, and antigenic peptides were designed for their potential use in antivenom production. The Immune Epitope Database Analysis Resource was used to identify the G34YFWIAWYKLANCKK48 epitope and used to design antigenic peptides. The Cys was replaced by α-aminobutyric acid (Abu) to avoid disulfide bonds formation. To increase their immunogenicity, branched and N-palmitoylated peptides were synthesized. Ac-GYFWIAWYKLAN-Abu-KKG-NH2 (A), (Ac-GYFWIAWYKLAN-Abu-KK)2-KG-NH2 (B), Palm-GYFWIAWYKLAN-Abu-KKG-NH2 (C) and (Palm-GYFWIAWYKLAN-Abu-KK)2-KG-NH2 (D) were synthesized using solid-phase peptide synthesis (SPPS) techniques and analyzed by ESI-MS demonstrating their identity. Also, they were evaluated by RP-HPLC, and all the chromatograms showed only one principal peak except that of the N-palmitoylated branched peptide which showed two principal peaks probably due to the presence of two conformations in slow interconversion. Cytotoxicity was evaluated on the murine macrophage cell line RAW264.7 by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay in the presence of increasing doses of each peptide (0.25-10.0 µM). Peptide A did not exhibit cytotoxicity between 0.25-10.0 µM, while B, C and D showed cytotoxicity over 10.0, 5.0 and 2.5 µM respectively. NF-κB cellular distribution was evaluated by immunofluorescence, after exposing macrophages to 0.5 µM of each peptide. An early activation was observed for all the assayed peptides demonstrating that they are promising candidates for their in vivo evaluation as immunogens in antivenom production.
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