Preparation and evaluation of liposomes encapsulating synthetic MMP inhibitor (Ro 28-2653)—cyclodextrin complexes

In this study, preparation and evaluation of liposomes, intended for intravenous administration, encapsulating synthetic MMP inhibitor (Ro 28-2653) – cyclodextrin complexes were realized. An increase in Ro solubility, via formation of binary (Ro/HPβCD) or ternary (Ro/HPβCD/L-lysine) complexes, permitted a similar increase in encapsulation efficiency of liposomes (Table 1). Moreover, Ro release kinetics depend on the encapsulation efficiency.     

Determination of the N-methyl- -aspartate receptor NR2B subunit blocker Ro 63-1908 in rat, cynomolgus monkey and human plasma by high-performance liquid chromatography with column switching and fluorescence detection

A HPLC method with automated column switching was developed and validated for the determination of Ro 63-1908 in rat and cynomolgus monkey plasma. Human plasma was used for calibration and was also included in the validation process. Ro 63-1908 belongs to a class of neuroprotective N-methyl- -aspartate (NMDA) receptor blockers which were in development for the treatment of stroke and traumatic brain injury. The method involves deproteinisation of plasma samples with ethanol and direct injection of the supernatant (1.4 ml) into the HPLC column-switching system. To prevent a breakthrough of the analyte and the internal standard on the precolumn (Purospher RP-18, 75×4 mm) due to the high ethanol content, the injection solution was diluted, on-line, using an additional pump and a T-piece. 1% ammonium acetate–ethanol (100:2, v/v) was used as mobile phase for injection, as well as for on-line dilution, resulting in pre-concentration of the analyte and the internal standard on the precolumn. As Purospher RP-18 is a non-endcapped stationary phase with a special selectivity for amines, the analyte and the internal standard could then be selectively eluted with 30% acetonitrile (without any buffer in the mobile phase) and transferred to the analytical column [consisting of two coupled columns (125+250×4 mm) packed with Superspher 60 RP-select B], where they were separated by gradient elution and detected by fluorescence detection. Compared to the use of a 125 mm long precolumn and dilution of the supernatant with ammonium acetate prior to injection, the 75 mm precolumn and the on-line dilution procedure allowed about one third shorter run times (21 min) and, therefore, a higher sample throughput. The limit of quantification was 1 ng/ml using 0.4 ml plasma. The method was applied to more than 670 plasma samples from pharmacokinetic and toxicokinetic studies and is also suitable for other matrices and NMDA receptor blockers.     

Determination of Arotinoid Ro 15–0778 and its Metabolite in Plasma by HPLC

Abstract

A method is described for the quantitative analysis in plasma of Ro 15–0778, an arotinoid, (E)-1, 2, 3, 4-tetrahydro-1, 1, 4, 4-tetramethyl-6-(1-methyl-2-phenylethenyl) naphthalene and its metabolite Ro 14–6113, (E)-4- 2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-2 methylethenyl) phenol. Following a simple extraction, the compounds are determined by reversed-phase high-performance liquid chromatography (HPLC) and detection at 300 nm. The experimental error is below 12% in the concentration range 6–4350 ng/ml. The detection limit is about 2 ng/ml. This method was applied to plasma specimens collected from patients receiving single or multiple dose administration of this compound.     

19-失碳-1α, 25-二羟基维生素D~3A环合成子的合成

报道以价廉、易得的D-(-)-奎尼酸为手性源,经9步反应,有效地合成光学活性的19-失碳-1α,25-二羟基维生素D~3A环合成子4的合成方法。     

Contribution of cyclodextrins in the development of different pharmaceutical formulations of a new matrix metalloproteinase inhibitor

Ro 28-2653 is a new synthetic inhibitor of matrix metalloproteinases. The ability of these enzymes to degrade various components of the extracellular matrix seems to play a major role in tumors progression and is potentially effective against bronchial remodeling in asthma and BPCO. Ro 28-2653 is very poorly soluble in water. This low solubility estimated at about 0.56 μg/ml in water at 25 °C gives rise to difficulties in pharmaceutical formulation of oral, injectable or nebulizable solutions. The purpose of our study is to prepare and to characterize inclusion complexes between Ro 28-2653 and cyclodextrins and to investigate the biopharmaceutical repercussion of the inclusion of the active substance. The complex formation was investigated by phase solubility studies. ¹H-NMR spectroscopy and molecular modeling studies were carried out to elucidate the structure of the inclusion complex between Ro 28-2653 and cyclodextrin. Oral, intravenous and nebulizable solutions of Ro 28-2653 were developed with cyclodextrin. The in vivo studies were performed on healthy sheep for the pharmacokinetic evaluation of the oral and intravenous formulations while the nebulization of the complex solution was studied by using an asthma model in mouse.     

云南青刺果仁、叶微量元素成分分析

青刺果在云南地方民间具有悠久的药用历史。本文运用电感耦合氩等离子体发射光谱仪（ICP－AES），对云南青刺果仁、叶中常量及微量元素进行了分析。结果表明青刺果仁、叶中含有较为丰富的常量及微量元素，叶中的含量比仁中的含量要高。本文结果为进一步开放利用云南青刺果提供了依据。     

Determination of the cephalosporin Ro 13-99041 in plasma,urine, and bile by means of ion-pair reversed phase chromatography

An HPLC method has been developed for the determination of the cephalosporin antibiotic Ro 13-9904 in plasma, urine, and bile of dogs and of human volunteers using the technique of ion-pair chromatography with a LiChrosorb RP-18 column. The three mobile phases employed contained tetrapentyl-, tetraoctyl- and hexadecyltrimethyl-ammonium bromide, respectively, as lipophilic counterions. The chromatographic conditions chosen allowed simple and rapid sample preparation. Plasma was deproteinized with ethanol and the supernatant was directly injected onto the column; urine and bile were diluted with mobile phase and injected without any purification. The detection limit for the cephalosporin was about 0.5 μg/ml for plasma samples and approximately 5 μg/ml for bile and urine.     

Determination of 7-amino-flunitrazepam (Ro 20-1815) and 7-amino-desmethylflunitrazepam (Ro 5-4650) in plasma by high-performance liquid chromatography and fluorescence detection

A high-performance liquid chromatographic method for the simultaneous determination of 7-amino-flunitrazepam (Ro 20-1815) and 7-amino-desmethylflunitrazepam (Ro 5-4650) in plasma is described. After extraction with an organic solvent, the compounds and their internal standard (7-amino-methylclonazepam or Ro 5-3384) are derivatized with fluorescamine and chromatographed on a reversed-phase muBondapak C18 column using pH 8 buffer solution-acetonitrile (3:1) as mobile phase. The detection is performed by a fluorometer at excitation and emission wavelengths of 390 and 470 nm, respectively. The sensitivity limit is about 1 ng/ml of plasma for both 7-amino-flunitrazepam and 7-amino-desmethylflunitrazepam. The method has been applied to the determination of plasma levels of these substances during pharmacokinetic studies of flunitrazepam, desmethylflunitrazepam and 7-amino-flunitrazepam.     

酶化学合成一对对映体Ro 25-8210和Ro 25-6630

利用白地霉菌株和啤酒酵母催化还原α－氯代酮　４分别制得光学活性的（Ｒ）和（Ｓ）－α－氯代酶 ８，并以它们作为关键步骤合成了 Ｒｏ２５－８２１０（１）和 Ｒｏ２５－６６３０（２）．     

Crystal structure of a squalene cyclase in complex with the potential anticholesteremic drug Ro48-8071

Squalene-hopene cyclase (SHC) catalyzes the conversion of squalene into pentacyclic compounds. It is the prokaryotic counterpart of the eukaryotic oxidosqualene cyclase (OSC) that catalyzes the steroid scaffold formation. Because of clear sequence homology, SHC can serve as a model for OSC, which is an attractive target for anticholesteremic drugs. We have established the crystal structure of SHC complexed with Ro48-8071, a potent inhibitor of OSC and therefore of cholesterol biosynthesis. Ro48-8071 is bound in the active-center cavity of SHC and extends into the channel that connects the cavity with the membrane. The binding site of Ro48-8071 is largely identical with the expected site of squalene; it differs from a previous model based on photoaffinity labeling. The knowledge of the inhibitor binding mode in SHC is likely to help develop more potent inhibitors for OSC.     

A validated high-performance liquid chromatographic method for the determination of moclobemide and its two metabolites in human plasma and application to pharmacokinetic studies

A rapid and sensitive reversed-phase high-performance liquid chromatographic method (RP-HPLC) with ultraviolet detection has been developed for the determination of moclobemide and its metabolites, p-chloro-N-(-2-morpholinoethyl)benzamide N'-oxide (Ro 12-5637) and p-chloro-N-[2-(3-oxomorpholino)ethyl]-benzamide (Ro 12-8095), in human plasma. The assay was performed after single liquid-liquid extraction with dichloromethane at alkaline pH using phenacetin as the internal standard. Chromatographic separation was performed on a C(18) column using a mixture of acetonitrile and water (25:75, v/v), adjusted to pH 2.7 with ortho-phosphoric acid, as mobile phase. Spectrophotometric detection was performed at 239 nm. The method has been validated for accuracy, precision, selectivity, linearity, recovery and stability. The quantification limit for moclobemide and Ro 12-8095 was 10 ng/mL, and for Ro 12-5637 was 30 ng/mL. Linearity of the method was confirmed for the range 20-2500 ng/mL for moclobemide (r = 0.9998), 20-1750 ng/mL for Ro 12-8095 (r = 0.9996) and 30-350 ng/mL for Ro 12-5637 (r = 0.9991). Moreover, within-day and between-day precisions and accuracies of the method were established. The described method was successfully applied in pharmacokinetic studies of parent drug and its two metabolites after a single oral administration of 150 mg of moclobemide to 20 healthy volunteers.     

符合ISO/IEC指南25-1990的实验室质量体系向ISO/IEC 17025-1999标准过渡的文件修改

按照ISO／IEC指南25－1990建立起较完善的质量体系的实验室,可通过对质量体系文件进行适当增补或少量删减,以实现向ISO／IEC 17025－1999标准的过渡。对质量手册,按照ISO／IEC指南25－1990的13个要素逐个讨论了具体的增删内容;对程序文件,则按ISO／IEC 17025－1999要求逐个列出了应有的程序。     

Determination of the imidazo quinazoline derivative Ro 13-6438 in biological fluids by high-performance liquid chromatography

A high-performance liquid chromatographic assay has been developed for the imidazo quinazoline derivative Ro 13-6438 [D-(-)-6-chloro-1,5-dihydro-3-methylimidazo(2,1-b)-quinazolin++ +-2(3H)-one], which is under clinical investigation as a cardioactive drug. The drug is extracted from biological fluids into 1-chlorobutane--1-hexanol (90:10) and back-extracted into perchloric acid. This extract is chromatographed directly, using a reversed-phase high-performance liquid chromatographic system with ultraviolet detection at 254 nm. The detection limit in plasma is about 1 ng ml-1, using a 1-ml sample. The assay is rapid, accurate and sufficiently sensitive for the study of the single-dose kinetics of Ro 13-6438 in man following a 7.5-mg intravenous dose. No instability of the unchanged substance was observed in plasma during storage for one day at room temperature and for five months at --20 degrees C.     

The Structure of the Polycyclic Nonadecapeptide Ro 09-0198

The conventional determination of the amino-acid sequence of the peptide antibiotic Ro 09-0198 was prevented by four cyclizations via side chains. The joint application of NMR spectroscopy at highest field and automated Edman degradation yielded a complete determination of the connectivity pattern. The three-dimensional structure derived from NOE data exhibits an interesting separation of amino acids with hydrophilic and hydrophobic side chains.     

Rapid determination of the benzodiazepine antagonist flumazenil, Ro 15-1788, by high performance liquid chromatography

A sensitive (2 ng/mL) and specific method for the determination of the benzodiazepine antagonist Ro 15-1788 or flumazenil is described. Following a simple extraction, the compound is analyzed by reversed phase high performance liquid chromatography (HPLC) and detection at 245 nm. The method was applied to plasma specimens collected from patients receiving a single dose of this drug.     

液相色谱-串联质谱法检测干血点样本中25-羟基维生素D2和25-羟基维生素D3

建立了一种高通量液相色谱-串联质谱技术检测干血点(DBS)样本中25-羟基维生素D2[25(OH)D2]和25-羟基维生素D3[25(OH)D3]的方法.以DBS为样本,以4-苯基-1,2,4-三唑啉-3,5-二酮(PTAD)为试剂进行分析物衍生化,所需样本量仅约相当于6μL全血当量的DBS样本;使用甲醇直接超声提取分析物,避开了通常情况下DBS样本前处理中的全血复溶和蛋白质沉淀等繁琐步骤;整个前处理过程使用自动化液体处理平台实现自动化操作和高检测通量;以25(OH)D2-D6和25(OH)D3-D3为同位素内标,消除基质效应的影响.前处理后的样本进行LC-MS/MS分析,使用C18柱进行分离,流动相为甲醇(含5 mmol/L甲酸铵)-水(含5 mmol/L甲酸铵)(75:25,V/V),洗脱时间为4 min,使用多反应监测模式(MRM)定量.结果表明:25(OH)D2和25(OH)D3的检出限为0.12 ng/mL(S/N=3),定量限为0.94 ng/mL(S/N=10).25(OH)D2和25(OH)D3在0.94~120.00 ng/mL范围内线性关系良好,日内相对标准偏差(RSD)分别为1.4%~8.6%和3.7%~15.5%,日间RSD分别为4.0%~5.3%和3.8%~14.9%,平均回收率分别为91.7%±7.9%~108.5%±6.5%和94.8%±6.8%~101.3%±2.9%.DBS样本在不同温度(-20℃,22℃,37℃)下储存不同时间(0,1,2,3,5,7,14天)后的稳定性实验显示样本总体RSD°15%.以25(OH)D参考物质NIST SRM 972a中的Level 3制备标准DBS样本,25(OH)D2和25(OH)D3的回收率分别为110.3%和103.0%.     

Lanthiopeptin,a new peptide effective against herpes simplex virus: Structural determination and comparison with Ro 09-0198, an immunopotentiating peptide

The structure of lanthiopeptin, a new lanthionine peptide effective against Herpes virus, was newly determined. It has a unique tetracyclic sequence which is very similar to but different from the proposed structure for Ro 09-0198. However, Ro 09-0198 was found to be quite the same compound as lanthiopeptin in all respects.     

Determination of Ro 14-1761, a new third-generation cephalosporin, in the plasma and milk of cattle by column switching high-performance liquid chromatography

A sensitive and specific high-performance liquid chromatographic procedure was developed for the determination of the third-generation cephalosporin Ro 14-1761 in cow plasma and milk. The molecular structure of the new antimicrobial was very close to that of ceftriaxone, but the high-performance liquid chromatographic methods available for the latter could not be used as Ro 14-1761 adsorbed and/or degraded during the chromatographic process. Furthermore, the high-performance liquid chromatographic technique derived for ceftriaxone was not sensitive enough for our purposes. In the new assay, the plasma (milk) protein was precipitated with acetonitrile after dilution of the sample with water. For low concentrations (less than or equal to 10 micrograms/ml), the supernatant obtained after centrifugation was concentrated by extracting acetonitrile with methylene chloride. Quantification was performed by column switching high-performance liquid chromatography with UV detection (274 nm) using ion-pair reversed-phase chromatography. Ethylenediaminotetraacetic sodium salt had to be added to the mobile phase (1.2 mM) to prevent adsorption and/or degradation of the cephalosporin on the analytical column. The selectivity of the chromatographic separation was enhanced by heating the column to ca. 50 degrees C. The drug recovery was better than 85%. The limit for quantitative determination in both milk and plasma was 0.1 microgram of Ro 14-1761 per millilitre with an accuracy of 1% (coefficient of variation 10%). The overall accuracy and precision were 1-10% in the 0.1-100 micrograms/ml concentration range.     

Determination of antimicrobial drug Ro 12-6995 (a 2,4-diamino-5-benzylpyrimidine) in blood plasma by high-performance liquid chromatography

Summary A HPLC procedure for the determination of Ro 12-6995, a new antimicrobial drug, in blood plasma is described. Adsorption liquid chromatography with LiChrosorb Si 60 as stationary phase with fluorescence detection was used to quantify the drug, which is extracted from plasma with n-butyl acetate. Due to the high fluorescence of Ro 12-6995, the plasma extracts can be injected directly onto the column and no evaporation step in the sample preparation is necessary. The assay is rapid, accurate and sensitive, and easily applicable to the determination of plasma series.     

Chemical extraction and direct adsorptive purification of recombinant human antigen Ro52

The antigenic protein Ro52 was expressed in the E. coli system harboring a 6 x His tag in the form of insoluble inclusion bodies. Direct chemical extraction of the product using 6-8 M urea proved to be effective. Furthermore, the tagged protein was recovered by direct adsorption on Ni2+-loaded commercial adsorbents derivatized with iminodiacetic acid. Screening experiments in small packed columns revealed that selective binding and elution were possible using a denaturing buffer at pH 4.5. The hydrodynamic evaluation of scaled-up fluidized systems showed values for the phi (dynamic zone) parameter in the range 0.95-1.00 for fluidization in buffer and in the range 0.70-0.85 for the biomass-containing feedstock. Removal of macromolecular DNA released by the disrupted biomass was mandatory. Under optimized process conditions good recovery (60-70%) was achieved and a highly purified (95%) product obtained. The purified Ro52 retained its immunoreactivity against sera of patients with systemic lupus erythematosus (SLE) and Sjogren's syndrome-related disorders. The production and application of new recombinant antigens may contribute to increasing the sensitivity and specificity of the detection of anti-Ro antibodies in these autoimmune diseases.