Pharmacokinetics of sifuvirtide, a novel anti-HIV-1 peptide, in monkeys and its inhibitory concentration in vitro

Aim: To study the pharmacokinetics of sifuvirtide, a novel anti-human immunodeficiency virus (HIV) peptide, in monkeys and to compare the inhibitory concentrations of sifuvirtide and enfuvirtide on HIV-1-infected-cell fusion. Methods: Monkeys received 1.2 mg/kg iv or sc of sifuvirtide. An on-line solid-phase extraction procedure combined with liquid chromatography tandem mass spectrometry (SPELC/MS/MS) was established and applied to determine the concentration of sifuvirtide in monkey plasma. A four-I-127 iodinated peptide was used as an internal standard. Fifty percent inhibitory concentration (IC50) of sifuvirtide on cell fusion was determined by co-cultivation assay. Results: The assay was validated with good precision and accuracy. The calibration curve for sifuvirtide in plasma was linear over a range of 4.88-5000 mu g/L, with correlation coefficients above 0.9923. After iv or sc administration, the observed peak concentrations of sifuvirtide were 10626 +/- 2886 mu g/L and 528 +/- 191 mu g/L, and the terminal elimination half-lives (T,12) were 6.3 +/- 0.9 h and 5.5 +/- 1.0 h, respectively. After sc, T-max was 0.25-2 h, and the absolute bioavailability was 49% +/- 13%. Sifuvirtide inhibited the syncytium formation between HIV-1 chronically infected cells and uninfected cells with an IC50 of 0.33 mu g/L. Conclusion: An on-line SPE-LC/MS/MS approach was established for peptide pharmacokinetic studies. Sifuvirtide was rapidly absorbed subcutaneously into the blood circulation. The T-1/2 of sifuvirtide was remarkably longer than that of its analog, enfuvirtide, reported in healthy monkeys and it conferred a long-term plasma concentration level which was higher than its IC50 in vitro.

S-DABO 类衍生物及AZT-氟喹喏酮类偶联物体外抗HIV 活性及机制研究

本论文由2 个相对独立的部分组成: S-DABO 类衍生物体外抗HIV 活性及 其机制研究和AZT－氟喹喏酮类偶联物体外抗HIV-1 活性及其机制研究。 HIV 逆转录酶抑制剂一直是抗HIV 药物研发的热点。该类抑制剂靶定在病 毒复制周期早期，为HAART 疗法提供了很多新的药物组合。目前FDA 批准上 市的逆转录酶抑制剂虽然有很多，但由于较严重的毒副作用、HIV 病毒易变异、 耐药性的出现等问题还需要开发更多的新的逆转录酶抑制剂。本论文对23 个 S-DABO 类化合物和8 个AZT－氟喹喏酮类偶联物的体外抗HIV 活性进行检 测，并对其中活性较高的化合物进行靶点和机制研究。 23 个S-DABO 类化合物采用对C8166 细胞的毒性试验，对HIV-1ⅢB 诱导的 合胞体形成的抑制试验和对HIV-1ⅢB 急性感染的MT-4 细胞的保护试验进行抗 HIV-1 活性初步筛选。试验结果发现所有化合物均对多种HIV 宿主细胞毒性小， 其中22 个化合物具有抗HIV-1 活性，特别是化合物RZK-4 和RZK-5，其对 HIV-1ⅢB 诱导的合胞体形成的SI 值（Selective index）分别为>16666 和>38462； RZK-4 和RZK-5 对HIV-1ⅢB 急性感染的MT-4 细胞的保护的SI 值分别为2666.67 和2150.54，与相应的阳性对照药品NVP（Nevirapine）的SI 值相接近。以p24 抗原水平为指标，对其中20 个化合物的抗HIV-1 活性进行确证，发现这20 个 化合物均能抑制 HIV-1ⅢB p24 抗原的产生，其中RZK-4 和RZK-5 的EC50 值分 别为5.93 和5.74ng/ml，比相应的阳性对照药品NVP（Nevirapine）的EC50 值 要低（27.3ng/ml）。这些化合物对试验株HIV-1MN、临床分离株HIV-1KM018、非 核苷类抑制剂耐药株HIV-1ⅢB A17 也有较好的抑制效果。但这23 个S-DABO 类化合物对HIV-2 病毒株均无抑制作用。通过检测化合物对感染与未感染细胞的 融合的抑制、对HIV-1 逆转录酶和蛋白酶活性的抑制、对慢性感染H9 细胞 （H9/HIV-1ⅢB）中病毒复制的抑制等试验来探讨化合物的抗HIV-1 机制。结果 显示：有20 个化合物对HIV-1 蛋白酶（PR）有抑制作用，其中有17 个化合物 对HIV-1 逆转录酶（RT）有抑制作用；但所有化合物均不能抑制感染与未感染 细胞的融合，也不能抑制慢性感染H9 细胞中病毒的复制。试验结果表明，这 23 个S-DABO 类化合物主要通过抑制HIV-1 逆转录酶来发挥作用，它们是典型 的非核苷类RT 抑制剂。 8 个AZT－氟喹喏酮类偶联物采用对C8166 细胞的毒性试验，对HIV-1ⅢB 诱导的合胞体形成的抑制试验和对HIV-1ⅢB急性感染的MT-4 细胞的保护试验 进行抗HIV-1 活性初步筛选。试验结果发现其中2 个化合物SRLZ 和SROZ 有 较显著的抗HIV-1 活性，其对HIV-1ⅢB诱导的合胞体形成抑制的SI 值分别为 >41667 和>105263；对HIV-1ⅢB 急性感染的MT-4 细胞的保护的SI 值分别为 30162 和 6368，与AZT（Zidothymidine）的SI 值相近似。以p24 抗原水平为 指标，对其抗HIV-1活性进行确证，发现化合物SRLZ和SROZ均能抑制HIV-1ⅢB p24 抗原的产生，其EC50 值分别为 0.71 和2.1ng/ml,比相应的阳性对照药品AZT 的EC50 值要低（3.5ng/ml）。化合物SRLZ 和SROZ 对临床分离株HIV-1KM018 也有较好的抑制活性，其EC50 值分别为1.4 和22ng/ml。通过检测化合物对慢 性感染H9 细胞（H9/HIV-1ⅢB）中病毒复制的抑制试验来探讨化合物的抗HIV-1 机制，结果表明化合物SRLZ 和SROZ 均不能抑制慢性感染H9 细胞中病毒的 复制。通过检测化合物对金黄色葡萄球菌的抑制作用来检测其抗菌活性，化合 物SRLZ 和SROZ 对金黄色葡萄球菌有较好的抑制作用，其MIC（Minimum inhibitory concentration）值分别为14.65 和7.32μg/ml，与其相应的阳性对照药 物的MIC 值相类似。试验结果表明：药物—药物偶连这种化学修饰方法并没有 改变AZT－氟喹喏酮类偶联物的抗HIV 作用靶点，但也没有较大地影响到其体 外抗病毒活性和抗微生物活性。

In vitro anti-HIV and -HSV activity and safety of sodium rutin sulfate as a microbicide candidate

Sodium rutin sulfate (SRS) is a sulfated rutin modified from the natural flavonol glycoside rutin. Here, we investigated its in vitro anti-HIV and -HSV activities and its cytotoxic profile. Fifty percent inhibitory concentration (IC50) values of SRS against HIV-1 X4 virus IIIB, HIV-1 R5 isolates Ada-M and Ba-L were 2.3 +/- 0.2, 4.5 +/- 2.0 and 8.5 +/- 3.8 mu M with a selectivity index (SI) of 563, 575 and 329, respectively. Its IC50 against primary R5 HIV-1 isolate from Yunnan province in China was 13.1 +/- 5.5 mu M, with a Sl of 197. In contrast, unsulfated rutin had no activity against any of the HIV-1 isolates tested. Further study indicated that SRS blocked viral entry and virus-cell fusion likely through interacting with the HIV- I envelope glycoprotein. SRS also demonstrated some activity against human herpes simplex virus (HSV) with an IC50 of 88.3 +/- 0.1 mu M and a Sl of 30. The 50% cytotoxicity concentration (CC50) of SRS was >3.0 mM, as determined in human genital ME 180, HeLa and primary human foreskin fibroblast cells. Minimum inhibitory concentration of SRS for vaginal lactobacilli was >3.0 mM. These results collectively indicate that SRS represents a novel candidate for anti-HIV-1/HSV microbicide development. (C) 2007 Elsevier B.V. All rights reserved.

Novel cathelicidin-derived antimicrobial peptides from Equus asinus

In the present study, EA-CATH1 and EA-CATH2 were identified from a constructed lung cDNA library of donkey (Equus asinus) as members of cathelicidin-derived antimicrobial peptides, using a nested PCR-based cloning strategy. Composed of 25 and 26 residues, respectively, EA-CATH1 and EA-CATH2 are smaller than most other cathelicidins and have no sequence homology to other cathelicidins identified to date. Chemically synthesized EA-CATH1 exerted potent antimicrobial activity against most of the 32 strains of bacteria and fungi tested, especially the clinically isolated drug-resistant strains, and minimal inhibitory concentration values against Gram-positive bacteria were mostly in the range of 0.3-2.4 mu g center dot mL-1. EA-CATH1 showed an extraordinary serum stability and no haemolytic activity against human erythrocytes in a dose up to 20 mu g center dot mL-1. CD spectra showed that EA-CATH1 mainly adopts an alpha-helical conformation in a 50% trifluoroethanol/water solution, but a random coil in aqueous solution. Scanning electron microscope observations of Staphylococcus aureus (ATCC2592) treated with EA-CATH1 demonstrated that EA-CATH could cause rapid disruption of the bacterial membrane, and in turn lead to cell lysis. This might explain the much faster killing kinetics of EA-CATH1 than conventional antibiotics revealed by killing kinetics data. In the presence of CaCl2, EA-CATH1 exerted haemagglutination activity, which might potentiate an inhibition against the bacterial polyprotein interaction with the host erythrocyte surface, thereby possibly restricting bacterial colonization and spread.

Anti-HIV-1 activity of trichobitacin, a novel ribosome-inactivating protein

AIM: To determine whether trichobitacin, a novel ribosome-inactivating protein purified from the root tubers of Trichosanthes kirilowii, possesses the anti-HIV activity. METHODS: The inhibition of syncytial cell formation induced by human immunodeficiency virus type 1 (HIV-1),was determined under microscope, reduction of HIV-1 p24 antigen expression level was measured by ELISA, and decrease in numbers of HIV-1 antigen positive cells in acutely and-chronically infected cultures were detected by indirect immunofluorescence assay. RESULTS: Trichobitacin Was-found to greatly suppress syncytial cell formation induced by HIV-1 and to markedly reduce both expression of HIV-1 p24 antigen and the number of HIV antigen positive cells in acutely but not chronically HIV-1 infected culture. The median inhibitory concentration (IC50) in inhibition of syncytial cell formation and HIV antigen positive cells were 5 mu g.L-1 (95 % confidence limits: 1.3 - 20 mu g.L-1) and 0.09 mg.L-1 (95 % confidence limits: 0.011 - 0.755 mg.L-1), respectively. CONCLUSION: Trichobitacin is a novel ribosome-inactivating protein with anti-HIV-l activity.

In vitro activities of small peptides from snake venom against clinical isolates of drug-resistant Mycobacterium tuberculosis

The recent re-emergence of tuberculosis, especially the multidrug-resistant cases, has highlighted the importance of screening effective novel drugs against Mycobacterium tuberculosis. In this study, the in vitro activities of small peptides isolated from snake venom were investigated against multidrug-resistant M. tuberculosis. Minimum inhibitory concentrations (MICs) were determined by the Bactec TB-460 radiometric method. A small peptide with the amino acid sequence ECYRKSDIVTCEPWQKFCYREVTFFPNHPVYLSGCASECTETNSKWCCTTDKCNRARGG (designated as vgf-1) from Naja atra (isolated from Yunnan province of China) venom had in vitro activity against clinically isolated multidrug-resistant strains of M. tuberculosis. The MIC was 8.5 mg/l. The antimycobacterial domain of this 60aa peptide is under investigation. (C) 2003 Elsevier Science B.V. and the International Society of Chemotherapy. All rights reserved.

Microarray-based Raman spectroscopic assay for kinase inhibition by gold nanoparticle probes

In this paper, a microarray-based surface-enhanced Raman spectroscopic (SERS) assay for detection of kinase functionality and inhibition has been reported. Biotinylated anti-phosphoserinen antibodies mark the phosphorylation and inhibition events and gold nanoparticles are attached to the antibodies by standard avidin-biotin chemistry, followed by silver deposition for SERS signal enhancement. The avidin conjugated fluorescein is used as SERS probe. The alpha-catalytic subunit of cyclic adenosine 5'-monophosphate (cAMP) dependent protein kinase (PKA), its well known substrate, kemptide, and three inhibitors, H89, HA1077, and KN62 have been chosen here to establish the SERS assay. As expected, highly selective inhibition of PKA is demonstrated with the inhibitor H89 and the inhibition assay enable to detect kinase inhibition as well as derive IC50 (half maximal inhibitory concentration) plots.

Microarray-based kinase inhibition assay by gold nanoparticle probes

We report on the development of a new class of kinase microarray for the detection of kinase inhibition based on marking peptide phosphorylation/biotinylation events by attachment of gold nanoparticles followed by silver deposition for signal enhancement. The alpha-catalytic subunit of cyclic adenosine 5'-monophosphate-dependent protein kinase (PKA), and its well-known substrate, kemptide, were used for the purpose of monitoring phosphorylation and inhibition. As expected, highly selective inhibition of PKA is demonstrated with the four inhibitors: H89, HA1077, mallotoxin, and KN62. Furthermore, an inhibition assay demonstrates the ability to detect kinase inhibition as well as derive IC50 (half-maximal inhibitory concentration) plots.

Extraction kinetics of cerium(IV) from sulfuric acid medium by the primary amine N1923 using a constant interfacial area cell with laminar flow

BACKGROUND: Thermodynamic studies on Ce(IV) extraction with primary amine N1923 demonstrate that primary amine N1923 is an excellent extractant for separation of Ce(IV) from Re(III). In order to clarify the mechanism of extraction and to optimize the parameters in practical extraction systems used in the rare earth industry, the extraction kinetics was investigated using a constant interfacial area cell with laminar flow in the present work.RESULTS: The data indicate that the rate constant (k(ao).) becomes constant when stirring speed exceeds 250 rpm. The apparent forward extraction rate is calculated to be 10(-1.70). The activation energy (E.) was calculated to be 20.5 kJ/mol from the slope of log kao against 1000/T. The minimum bulk concentration of the extractant necessary to saturate the interface (C-min) is lower than 10(-5) mol L-1.CONCLUSION: Studies of interfacial tension and the effects of stirring rate and specific interfacial area on the extraction rate show that the extraction rate is kinetically controlled, and a mass transfer model has been proposed. The rate equation has been obtained as: -d[Ce(IV)]/dt = 10(-1.70)[Ce(IV)] [(RNH3)(2)SO4](1.376). The rate-controlling step has been evaluated from analysis of the experimental results.

Interfacial behavior of primary amine N1923 and the kinetics of thorium(IV) extraction in sulfate media

The influences of additive, diluents, temperature, acidity of the aqueous phase on the interfacial behavior of primary amine N1923 in sulfate media have been investigated using the Du Nouy ring method. In addition, the effect of concentration of thorium(IV) loaded in the organic phase on the interfacial tension has also been studied. The interfacial tension isotherms are processed by matching different adsorption equations such as the Gibbs and the Szyszkowski. The surface excess at the saturated interface (Gamma (max)) and the minimum bulk concentration of the extractant necessary to saturate the interface (C-min) under different conditions are calculated according to two adsorption equations to be presented in comprehensive tables and figures. It appears that primary amine N1923 has strong interfacial activity and behaves very differently in various diluents systems. The surface excess at saturated interface increase with the type of diluerits in the following order: chloroform < aromatic hydrocarbons < aliphatic hydrocarbons. The relationship between the interfacial activity and kinetics of thorium extraction by primary amine N1923 has been discussed by considering different factors. However, the interfacial activity of primary amine N1923 is only a qualitative parameter suggesting the interfacial mechanism for thorium extraction, it cannot give strong evidence quantitatively supporting this mechanism.

Interfacial behavior of DEHEHP and the kinetics of cerium(IV) extraction in nitrate media

The effects of diluents, temperature, acidity, and ionic strength of the aqueous phase on the interfacial properties of DEHEHP have been extensively investigated using the Du Nouy ring method. In addition, the effect of cerium(IV) concentration loaded in the organic phase on the interfacial tension has also been studied. With the increase of DEHEHP concentration, the value of interfacial tension (gamma) decreases in the studied system, which shows that DEHEHP has interfacial activity as a kind of surfactant. The surface excess at the saturated interface (Gamma(max)) and the minimum bulk concentration of the extractant necessary to saturate the interface (C-min) under the different conditions are calculated according to two adsorption equations such as the Gibbs and Szyszkowski functions to be presented in comprehensive tables and figures. The relationship between the interfacial activity of DEHEHP and cerium(IV) extraction kinetics by DEHEHP has been discussed by considering different factors such as the effects of diluents and temperature. However, the interfacial activity parameter of extractant only is a qualitative parameter, but cannot provide strong enough evidence to quantitatively explain the relationship between extraction kinetics and interfacial properties of an extractant.

Crystallographic and NMR studies on sterols from green alga Chaetomorpha basiretorsa Setchell

Chemical investigation of the ethanol extract of the marine green alga Chaetomorpha basiretorsa Setchell led to the isolation of a new sterol stigmast-4,28-dien-3 alpha 6 beta-diol 1 in addition to the five known sterols of beta-lawsaritol 2, saringosterol 3, 24-hydroperoxy-24-vinyl - cholesterol 4, beta-stigmasterol 5, 29-hydroxystigmasta-5, 24(28) -dien-3 beta-ol 6. Compounds were isolated by normal phase silica gel and Sephadex LH - 20 gel colum chromatography, reverse phase HPLC and recrystalization. Their structures were elucidated by spectroscopic methods including MS, IR 1D/2D NMR and X-ray analysis. Cytotoxicity of compounds was screened by using the standard WIT method. All these compounds were isolated from the green alga Chaetomorpha basiretorsa Setchell for the first time and they were inactive (50% inhibitory concentration was greater than 10 mu g /cm(3)) against KB, Bel -7402, PC - 3M, Ketr 3 and MCF - 7 cell lines.

Conduction band offset and electron effective mass in GaInNAs/GaAs quantum-well structures with low nitrogen concentration

We have investigated the optical transitions in Ga1-yInyNxAs1-x/GaAs single and multiple quantum wells using photovoltaic measurements at room temperature. From a theoretical fit to the experimental data, the conduction band offset Q(c), electron effective mass m(e)*, and band gap energy E-g were estimated. It was found that the Q(c) is dependent on the indium concentration, but independent on the nitrogen concentration over the range x=(0-1)%. The m(e)* of GaInNAs is much greater than that of InGaAs with the same concentration of indium, and increases as the nitrogen concentration increases up to 1%. Our experimental results for the m(e)* and E-g of GaInNAs are quantitatively explained by the two-band model based on the strong interaction of the conduction band minimum with the localized N states. (C) 2001 American Institute of Physics.

Inhibitory effects and mechanisms of high molecular-weight phlorotannins from Sargassum thunbergii on ADP-induced platelet aggregation

We evaluated the effects of high molecular-weight phlorotannins from Sargassum thunbergii (STP) on ADP-induced platelet aggregation and arachidonic acid (AA) metabolism in New Zealand white rabbits and Wistar rats. The inhibition of STP on platelet aggregation was investigated using a turbidimetric method, and the levels of the terminal products of AA metabolism were measured using the corresponding kits for maleic dialdehyde (MDA), thromboxane B-2 (TXB2) and 6-keto-prostaglandin F-1 alpha (6-keto-PGF(1 alpha)) by colorimetry and radioimmunoassay, as appropriate. We found that STP could inhibit ADP-induced platelet aggregation, and the inhibitory ratio was 91.50% at the STP concentration of 4.0 mg/mL. Furthermore, STP markedly affected AA metabolism by decreasing the synthesis of MDA (P < 0.01) and increasing the synthesis of 6-keto-PGF(1 alpha), thus changing the plasma TXB2/6-keto-PGF(1 alpha) balance when the platelets were activated (P < 0.01). Therefore, STP altered AA metabolism and these findings