Study on Pharmacokinetics of Metabolites of Adefovir in Rats after Intragastric Administration of Adefovir Derivatives / 中国药房

OBJECTIVE： To establish a method for the determination of adefovir （PMEA） and study the pharmacokinetics of metabolites PMEA in rats after intragastric administration of PMEA derivatives [PMEA prodrug， adefovir-ursodeoxycholic acid-3-propyl ester， L-leucine-3-propyl ester （PMEA-1c）] in rats. METHODS： HPLC-MS/MS method was adopted. The determination was performed on BEH C18 column with mobile phase consisted of 0.1％ formic acid acetonitrile-0.1％ formic acid water （gradient elution） at the flow rate of 0.25 mL/min. The column temperature was 30 ℃， and sample size was 1 μL. The quantitative ions were PMEA m/z 274.1→162.1， puerarin （internal standard） m/z 417.1→267.1. 12 rats were randomly divided into adefovir dipivoxil （ADV） group （positive control， 90 mg/kg） and PMEA-1c group （160 mg/kg）， with 6 rats in each group. They were given relevant medicine once intragstrically， and the blood samples were collected from tail vein 0.083， 0.25， 0.5， 0.75， 1， 2， 4， 6， 8， 10， 12， 24 h after administration to determine the plasma concentration of PMEA. Relevant pharmacokinetic parameters were calculated by using DAS 2.0 software. RESULTS： The linear range of PMEA was 6.1-440.0 ng/mL （r＝0.998 5）. RSDs of intra and inter day of precision and stability tests were all less than 10％ （n＝3， 5， 6）， and the accuracy was 82.16％-97.33％ （RSD≤6.4％， n＝5）. Matrix effects ranged from 95.96％-106.35％ （RSD≤4.9％， n＝5）. The pharmacokinetic parameters of PMEA in ADV group and PMEA-1c group were as follows as t1/2 were （1.762±0.117） and （2.548±0.174） h； AUC0-24 h were （2 170.059±146.091） and （4 704.257±176.792） μg·h/L； cmax were （613.092±9.504） and （697.295±15.275） μg/L， respectively. Compared with ADV group， t1/2， AUC0-24 h， AUC0-∞ and cmax of rats in PMEA-1c group were increased significantly （P＜0.01 or P＜0.001）. CONCLUSIONS： Established method is accurate and reliable. The trial indicates that PMEA-1c metabolism is single compartment model， show that can be used as a potential prodrug for adefovir， which lay a foundation for the further study of adefovir prodrug.

Synthesis and bioactivity of mixed phosphonate derivatives of adefovir with hepatic targeting property / 中国药科大学学报

In order to search for new adefovir analogues as anti-HBV agents with enhanced antiviral activity and hepatotrophic property,adefovir bis L-amino acid ester was used as lead compound to produce ten adefovir mono L-(thio)amino acid ester, mono bile acid ester derivatives(6a-6j). The design based on bile acid prodrug strategy,which can improve drug oral bioavaliability and liver-targeted enrichment by using enterohepatic circula-tion of bile acid.Sub-structure combination method was adopted to introduce L-(thio)amino acid ester and bile acid ester fragments on the phosphonate functionality of adefovir. The structures of target compounds were confirmed by 1H NMR, 13C NMR,ESI-MS and ESI-HRMS.HepG 2.2.15 cell were used for in vitro anti-HBV activity assessment.Compound 6c with high antiviral activity(EC500.92μmol/L,SI 512.63)was further investi-gated for its tissue distribution in mice.The results showed that content of compound 6c in liver was higher than that of adefovir dipivoxil,and in contrast its content in kidney was lower than that in positive control at all time points(0.25-12 h).Compound 6c exhibits higher antiviral activity,selective index and higher liver distribution,making it a potential anti HBV agent for further investigation.

In vitro metabolism and stability of adefovir mixed phosphonate derivative Q3-I2 / 中国药科大学学报

@#A, UPLC-QTOF-MS/MS and UPLC-MS/MS were used to investigate the derivative of adefovir mixed phosphonate Q3-I2. Stability and in vitro metabolites of Q3-I2, and the control drug adefovir dipivoxil were co-inculbated with artificial gastric juice, artificial intestinal juice, rat blank plasma and rat liver microsomes, using UPLC-MS/MS and UPLC-QTOF-MS/MS measure the residual concentration of the compounds in each incubation system and the metabolites in the liver microsomal system, respectively, and calculate the half-life and clearance rate by the substrate elimination method. The compounds designed and synthesized in this experiment are stable in the gastrointestinal tract, prolonging t1/2 of plasma and liver microsomes and rapidly degrading the active meta-bolites. In the liver microsomal system, a total of 8 metabolites were detected by positive and negative ion mode, including hydrolysis, oxidation, acetylation, and glucuronidation.

Comparison of hPepT1 transfected MDCK cells to hPepT1 transfected HeLa cells / 中国药理学通报

Aim To screen a more suitable transfection recep-tor,and improve the efficiency of constructing cell lines highly expressing human peptide transporters 1 (hPepT1 ).Methods The recombinant plasmid pcDNA3.1 (+)-hPepT1 was transfect-ed into MDCK cells and HeLa cells by LipofectamineTM 2000 transfection reagent,respectively.The monoclonal cells were se-lected and cultured.Expression of hPepT1 mRNA and protein were determined by qRT-PCR and Western blot,respectively. The uptake capacity of Glysar in transfected cells was examined. Results Compared with wild type cells,the expression of hPepT1 and the uptake of Glysar in transfected MDCK cells and HeLa cells significantly increased (P <0.05).Although the up-take of Glysar in HeLa cells was higher than that of MDCK cells,on the contrary,the expression of hPepT1 and the uptake of Glysar in MDCK-hPepT1 cells was higher than that of HeLa-hPepT1 cells.Conclusion MDCK cells may serve as a more suitable transfected receptor for the construction of a cellular model with high expression of hPepT1 ,which would make the construction of a cell model highly expressing hPepT1 more effi-cient.

Establishment and identification of HEK293 cell lines with stable and high expression of NTCP / 中国药理学通报

Abstrate:Aim To construct HEK293 cell line with stable and high expression of sodium taurocholate cotransporting polypeptide (NTCP ) efficiently and rapidly.Method Vector expressing EGFP-NTCP fusion protein was constructed and verified by DNA sequencing.The pEGFP-NTCP expression vector was transfected into HEK293 cells by FuGENE 6 transfection reagent. The transfected cells with high expression of green fluorescent protein were selected using fluorescence microscope for screening of G418 for 14 days to obtain cell lines stably and highly expressing NTCP.NTCP expression was detected by RT-PCR,qRT-PCR, Western-blot and the uptake experiment of taurocholic acid.Re-sult RT-PCR,qRT-PCR,Western-blot and the uptake experi-ment revealed that compared to the control cells,the expression of NTCP was significantly positive (P<0.01)in stable trans-fected cells showing green fluorescence (P<0.05 ).Conclusion The HEK293 cell line with stable and high expression of NTCP has been established efficiently and rapidly,which provides a cellular model for the study of the mechanism of the uptake of bile acid derivatives.

Establishment of an in vitro blood-brain barrier model by co-culturing rat brain microvascular endothelial cells,pericytes and astrocytes / 中国药理学通报

Aim To establish in vitro blood-brain barrier (BBB) model with characteristics of simulation of in vivo BBB by primi-tive co-culture of brain-microvessel endothelial cells (BMECs) with brain-microvessel pericytes (BMPC)and astrocytes (AS). Methods BMECs,BMPC and AS from SD rats were primitively isolated,purified and cultured,and then primitive culture cells were identified by cellular morphological and immunocytochemi-cal staining methods.Five types of in vitro BBB models were es-tablished by using Millicell culture insert (pore diameter 0.4μm)and their barrier functions were evaluated by detection of transendothelial electrical resistance (TEER),permeability of sodium fluorescent (Na-FLU ),expression of alkaline phospha-tase (AKP)and γ-glutamyl transpeptidase (γ-GT1 ),and simi-larity of permeation amount for positive drugs in vitro and in vivo BBB conditions.Results Primitive culture of BMECs presented typical pebbles-like structure,BMPC presented larger soma with branching property,AS presented slender synapse and shallower cytoplasm.Moreover,immunocytochemical staining results iden-tified primitive cells were targeted cells.TEER value for co-cul-ture of BMECs,BMPC and AS reached (478 ±25 )Ω· cm2 , permeability coefficients (Papp )value of Na-FLU was [(8.23 ± 0.78) ×10 -6 ]cm·s-1 ,expression of AKP and γ-GT1 were (6.90 ±0.27 )King unit · g-1 Pro and (4.39 ±0.32 )μg · g-1 Pro respectively.Moreover,good correlation could be found in Papp for positive controls in vitro and in vivo BBB models (R2=0.92).Conclusion The established in vitro BBB model by using primitive co-culture of BMECs with BMPC and AS posses-ses in vivo BBB properties in cell morphology,structures and barrier functions,and can be used as a powerful tool for studying physiology,pathology of BBB and screening candidate com-pounds.

A highly purified and active extract and primitive culture method for rat microvascular endothelial cells (BMECs) / 中国药理学通报

Aim To establish a highly purified,active and prac-tical extract and primitive culture method for rat brain microvas-cular endothelial cells ( BMECs) for providing materials for con-struction in vitro blood-brain barrier ( BBB) model. Methods Cerebral cortex of 1-2 week SD rats was collected,and successive digestion with typeⅡcollagenase and collagenase/dispase, sieve filtration and then twice gradient centrifugation in 20% BSA and 44% Percoll condition were used to obtain brain microvascular section. After that brain microvascular section was seeded in cul-ture bottle and then primarily cultured. Inverted microscope and factor-VIII relative antigen immunostaining methods were used for cellular morphological observation and identification. Results BMECs climbed out the vessel segment and proliferated with ad-herence after 2 h in vitro culture,and they became typical peb-bles structure after further 3-4 d culture. Morever, factor-VIII relative antigen immunostaining identified that expression for the endothelial cells was positive,cytoplasm was brown and positive cells account for more than 99%. Conclusion Rat BMECs with high purity could be extracted and cultured by using the above methods,and it has great potential for construction in vitro BBB model and in depth studies on biological characteristics and func-tions of BMECs.

Design, synthesis and anti-oxidative evaluation of L-amino acid prodrugs of scutellarein / 药学学报

To design and synthesize a series of novel scutellarein 4'-L-amino acid prodrugs with more potent anti-oxidative activity and improved physicochemical properties. Scutellarein was used as lead compound, according to successful experience of improving bioavailability of oral administration drugs by active transport mechanism, principle of hybridization was used to introducing L-amino acid structural fragments at 4'-position of scutellarein to design and synthesize target scutellarein 4'-L-amino acid prodrugs. The synthetic compounds were tested on their physicochemical properties and in vitro anti-oxidative activity against H202 induced oxidative damage in PC12 cells. Five compounds were found to have more potent anti-oxidative activity than positive control VE. Moreover the physicochemical properties of synthesized compounds were evaluated, and the results revealed that L-amino acid ether derivatives are more stable (t1/2 9-92 h) than their corresponding ester derivatives (t1/2 0.5 h). Water solubility of scutellarein 4'-L-amino acid ester and ether derivatives were 1 796-4 100 microg.mL-1 and 27.7-81.1 microg.mL-1 respectively, in comparison with scutellarin, the solubility of compounds 18, 19 and 22, 24-27 increased about 120-280 fold and 2-6 fold respectively. All these results suggested that L-amino acid prodrug strategy has significant potential in scutellarein prodrug design.

Design, synthesis and anti-hBV evaluation of adefovir mono-L-amino acid ester, mono non-steroidal anti-inflammatory drugs carboxylic ester prodrugs / 药学学报

A series of adefovir mono-L-amino acid esters, mono non-steroidal anti-inflammatory drugs carboxylic ester prodrugs with more potent anti-HBV activity and lower nephrotoxicity were designed and synthesized. Adefovir bis (L-amino acid) ester was used as lead compound, according to pathological and pharmacological findings that non-steroidal anti-inflammatory drugs can effectively inhibit the organic anion transporter 1 (hOAT1)-mediated adefovir phosphonic acid pairs of anion transport across tubular basement membrane thereby reducing the nephrotoxicity of adefovir. Flatten design principle was used to introducing non-steroidal anti-inflammatory drugs structural fragments to design and synthesize target adefovir mixture ester prodrugs. HepG2 2.2.15 cell line was used as in vitro anti-HBV activity evaluation model. Five compounds exhibited antiviral activity, and compound 18 showed the most potent anti-HBV activity and relatively high selective index (EC50 3.92 micromol L(-1), SI 9.97). HK-2 cell line was used as in vitro model to evaluate nephrotoxicity. Results suggested the target compounds have lower cytotoxicity than the positive control. Moreover, by analyzing the primary structure and activity relationship of these compounds, it could suggest that mono-L-amino acid ester, mono non-steroidal anti-inflammatory drugs carboxylic ester prodrugs strategy has significant potential in the acyclic nucleoside phosphonates prodrug design.

Simultaneous determination of eight components in Polygonum orientale by ultra performance liquid chromatography (UPLC) method / 中国中药杂志

In this study, a UPLC-PDA method for simultaneous determination of catechin(1), isoorientin(2), orientin(3), quercetin-3-O-(2"-O-alpha-rhamnopyranosyl)-beta-glucarono-pyranoside(4), taxifoliol(5), luteolin(6), quercitrin(7) and kaempferol-3-O-beta-D-glucoside(8) in Polygonum orientale in Guizhou province was developed. Analysis was performed at 45 degrees C, on ACQUITY UPLC BEH C18 column (2.1 mm x 150 mm, 1.7 microm), eluted with a gradient program of acetonitrile-0.1% H3PO4 aqueous solution as the mobile phases. The flow rate was of 0.30 mL x min(-1), and the detection wavelength was 260 nm. The method validation proved that the linearity, instrument precision, repeatability, intermediate precision and accuracy accorded with the requirement for anassay. The established method had been used for determination above eight components in twenty lots of P. orientale in different places and harvest time in Guizhou province, and the results showed that the chemical composition was basically the same in herbs, but there were some difference in content of the tested components. the contents of the tested ingredients were relatively high and stable in the sample collected in August.