Effect of 1022C>T and 1222A>G genetic polymorphisms of SLC22A1 gene on substrates binding to human organic cation transporter 1

Human organic cation transporter 1 (hOCT1) is a transmembrane influx transporter protein encoded by the SLC22A1gene. hOCT1 plays a pivotal role in the hepatocellular and renal uptake of several xenobiotics and endogenoussubstrates. The human SLC22A1 gene is highly polymorphic. Non-synonymous single nucleotide polymorphisms(SNPs) of the human SLC22A1 gene tend to impair the transmembrane conductance of substrates by hOCT1. Herein,we describe the effect of 1022C>T and 1222A>G variations in the human SLC22A1 gene on hOCT1 structure andsubstrate binding. The three-dimensional (3D) structures of hOCT1 variants were ab initio models using the iTASSERserver, and drug-binding residues of the transmembrane domain were predicted using the Prankweb server. Substratebinding was analyzed by molecular docking using AutoDock 4.2.6. Amino acid residues, crucial for substrate bindingand transport, were altered in Met408Val and Pro341Leu variants and were suggestive of conformational changeinduced by 1022C>T and 1222A>G SNPs. Moreover, a statistically significant difference was observed betweenthe binding affinities of substrates to wild and mutant variants. Therefore, it is evident that 1022C>T and 1222A>Gnon-synonymous SNPs impair the drug uptake process of hOCT1, and hence patients with the former variants need tobe closely monitored for idiosyncratic adverse drug reactions or sub-therapeutic responses while being initiated intotherapy with hOCT1 substrates.

Influences of molecular weight and particle size to intranasal drug delivery based on cell model of "nose-brain" / 中草药

Objective: To explore the factors affecting the nasal entry of the pharmaceutical preparations into the brain based on the established model of the "nose-brain" pathway in vitro. Methods: Calu-3 cells and OECs cells were co-cultured to construct a "nasal-brain" pathway cell model group. Taking fluorescein isothiocyanate dextran (FD) and fluorescent silver nanoparticles (AgNPs) as model drugs, the effects of drug molecular weight (Mw) factors and preparation particle size factors on the drug transnasal transport into the brain were explored. Results: The apparent permeability coefficient (Papp) of transcellular monolayer transport of FD decreased with the increase of molecular weight. The uptake of fluorescein isothiocyanate dextran with different molecular weights by OECs tended to be saturated after 90 min. As the molecular weight of FD increased, the uptake of OECs decreased significantly during the same uptake time. The apparent permeability coefficient of fluorescent AgNPs with different particle sizes in the "nose-brain" multi-channel cell model group of calu-3 monolayer decreased with the increase of the particle size of the nanoparticles. When the particle size was less than 40 nm, its transport characteristics in Calu-3 were shown as medium absorption (1 × 10-6 < Papp < 10 × 10-6), and when the particle size of nanoparticles was more than 60 nm, its transport characteristics were shown as difficult to absorb (Papp < 1 × 10-6). The uptake of OECs of fluorescent AgNPs with different particle sizes tended to be saturated at 60 min, and with the increase of the particle size of fluorescent AgNPs, the uptake of OECs at the same uptake time showed a significant decline. Conclusions The Mw of the drug and the particle size of the nano-formulation have an important influence on the nasal transport of the drug into the brain. Drugs with a molecular weight of < 4 000 and nano particles with a particle size of less than 40 nm have better transport and uptake characteristics.

Insulin-like growth factor 1 modulates the phosphorylation, expression, and activity of organic anion transporter 3 through protein kinase A signaling pathway

Organic anion transporter 3 (OAT3) plays a vital role in removing a broad variety of anionic drugs from kidney, thus avoiding their possible toxicity in the body. In the current study, we investigated the role of insulin-like growth factor 1 (IGF-1) in the regulation of OAT3. We showed that IGF-1 induced a dose- and time-dependent increase in OAT3 transport activity, which correlated well with an increase in OAT3 expression. The IGF-1-induced increase in OAT3 expression was blocked by protein kinase A (PKA) inhibitor H89. Moreover, IGF-1 induced an increase in OAT3 phosphorylation, which was also blocked by H89. These data suggest that the IGF-1 modulation of OAT3 occurred through PKA signaling pathway. To further confirm the involvement of PKA, we treated OAT3-expressing cells with PKA activator Bt2-cAMP, followed by examining OAT activity and phosphorylation. We showed that OAT3 activity and phosphorylation were much enhanced in Bt2-cAMP-treated cells as compared to that in control cells. Finally, linsitinib, an anticancer drug that blocks the IGF-1 receptor, abrogated IGF-1-stimulated OAT3 transport activity. In conclusion, our study demonstrated that IGF-1 regulates OAT3 expression and transport activity through PKA signaling pathway, possibly by phosphorylating the transporter.

Development of Human in vitro Brain-blood Barrier Model from Induced Pluripotent Stem Cell-derived Endothelial Cells to Predict the in vivo Permeability of Drugs / 神经科学通报·英文版

An in vitro blood-brain barrier (BBB) model is critical for enabling rapid screening of the BBB permeability of the drugs targeting on the central nervous system. Though many models have been developed, their reproducibility and renewability remain a challenge. Furthermore, drug transport data from many of the models do not correlate well with the data for in vivo BBB drug transport. Induced-pluripotent stem cell (iPSC) technology provides reproducible cell resources for in vitro BBB modeling. Here, we generated a human in vitro BBB model by differentiating the human iPSC (hiPSC) line GM25256 into brain endothelial-type cells. The model displayed BBB characteristics including tight junction proteins (ZO-1, claudin-5, and occludin) and endothelial markers (von Willebrand factor and Ulex), as well as high trans-endothelial electrical resistance (TEER) (1560 Ω.cm ± 230 Ω.cm) and γ-GTPase activity. Co-culture with primary rat astrocytes significantly increased the TEER of the model (2970 Ω.cm to 4185 Ω.cm). RNAseq analysis confirmed the expression of key BBB-related genes in the hiPSC-derived endothelial cells in comparison with primary human brain microvascular endothelial cells, including P-glycoprotein (Pgp) and breast cancer resistant protein (BCRP). Drug transport assays for nine CNS compounds showed that the permeability of non-Pgp/BCRP and Pgp/BCRP substrates across the model was strongly correlated with rodent in situ brain perfusion data for these compounds (R = 0.982 and R = 0.9973, respectively), demonstrating the functionality of the drug transporters in the model. Thus, this model may be used to rapidly screen CNS compounds, to predict the in vivo BBB permeability of these compounds and to study the biology of the BBB.

Evaluation of antimicrobial activity and cell viability of Aloe vera sponges

Background: Aloe vera L., member of the Liliaceae family, has been shown to stimulate cell proliferation and contribute to healing and angiogenesis, has anti-bacterial, anti-fungal and anti-inflammatory activity. In addition, Aloe vera can be used as a support for drug transport. Our objective is to evaluate antimicrobial activity and cytotoxicity of sponges of Aloe vera L. for use as a carrying support of drugs. Results: In this work, sponge of free Aloe vera (AV) loaded with amoxicillin (AMX) or nystatin (NYS) at 1% w/w, were prepared and physico-chemically characterized via X-ray diffraction, Fourier Transform Infrared Spectroscopy and thermal analysis. Antimicrobial potency of AV sponge alone, loaded with AMX or NYS, against strains of Streptococcus mutans, Staphylococcus aureus, Aggregatibacter actinomycetemcomitans, Enterococcus faecalis and Candida albicans was determined. Osteoblasts and human gingival fibroblasts were cultivated on AV, Aloe vera loaded with amoxicillin (AV/AMX) and Aloe vera loaded with nystatin (AV/NYS) and cellular viability was assessed. The physico-chemical characterization performed suggested that the loaded drugs were dispersed in the sponge and those interactions between the AV sponge and the loaded drugs were weak. Furthermore, AV loaded with AMX or NYS demonstrated antimicrobial potency and osteoblasts and fibroblasts were viable after 24 hrs on free AV, and AV loaded with AMX or NYS. Conclusions: Our results indicate that sponges of free AV, loaded with AMX or NYS, are biocompatible and exhibit antimicrobial activity.

Pulmonary Peptidomimetic Drug and Peptide Transportand Peptide Transporters / 昆明医科大学学报

Recent studies show that peptide transporters expressed in respiratory tract play a major role in pulmonary peptidomimetic drug and peptide transport.In this article we reviewed the structure,localization,function and the transport mechanism of peptide transporters.