In vitro screening of clinical drugs identifies sensitizers of oncolytic viral therapy in glioblastoma stem-like cells.

Oncolytic viruses (OV) have broad potential as an adjuvant for the treatment of solid tumors. The present study addresses the feasibility of clinically applicable drugs to enhance the oncolytic potential of the OV Delta24-RGD in glioblastoma. In total, 446 drugs were screened for their viral sensitizing properties in glioblastoma stem-like cells (GSCs) in vitro. Validation was done for 10 drugs to determine synergy based on the Chou Talalay assay. Mechanistic studies were undertaken to assess viability, replication efficacy, viral infection enhancement and cell death pathway induction in a selected panel of drugs. Four viral sensitizers (fluphenazine, indirubin, lofepramine and ranolazine) were demonstrated to reproducibly synergize with Delta24-RGD in multiple assays. After validation, we underscored general applicability by testing candidate drugs in a broader context of a panel of different GSCs, various solid tumor models and multiple OVs. Overall, this study identified four viral sensitizers, which synergize with Delta24-RGD and two other strains of OVs. The viral sensitizers interact with infection, replication and cell death pathways to enhance efficacy of the OV.

Synthesis and radiopharmaceutical preparation of (ethylenediamine) (1-carbon-11-malonate) platinum(II) for PET studies.

Interest in the distribution, biotransformation, and mechanism of action of anticancer platinum complexes has led to the microscale, semi-automated and remote-controlled synthesis of (ethylenediamine) (1-[11C]malonate) platinum(II) [( 11C]Ptenmal, EDMAL, JM40) from cyclotron-produced [11C]cyanide. Carbon-11 cyanoacetate is produced by reacting [11C]cyanide with bromoacetate. After hydrolysis, the resulting [11C]malonic acid is purified and complexed to (diaquo) (ethylenediamine) platinum(II). Each step of the synthesis was optimized by studying the influence of different variables like reaction time and temperature, pH, necessary purification of intermediates, concentration and ratios of the reactants. Purification of the endproduct is achieved using preparative high performance liquid chromatography. The total incorporation of the [11C]cyanide in the final product was 17-40%. After approximately 1 hr, approximately 40 mCi of [11C]Ptenmal are produced in 10 ml sterile and isotonic dextrose solution ready for i.v. injection. The specific activity is approximately 200 mCi/mumol at EOB.

Microscale synthesis of nitrogen-13-labeled cisplatin.

A microscale synthesis of [13N]cisplatin (cis-dichlorodiammineplatinum(II), cis-DDP) from cyclotron-produced [13N]ammonia is presented. Temperature, reaction time, ratios, and concentration of reactants have been optimized for each step of the synthesis. Purification is performed by ion exchange chromatography. Radiochemical purity and optimization processes are controlled by high performance liquid chromatography and high performance thin layer chromatography--22 mCi [13N]cisplatin in 10 ml of solution is produced. The entire procedure takes approximately 15 min and the specific activity is approximately 300 mCi/mumole at EOB.

Optimization of capillary electrophoretic separation of several inhibitors of the angiotensin-converting enzyme.

Capillary electrophoretic separation of eight inhibitors of the angiotensin-converting enzyme, viz., enalapril, lisinopril, quinapril, fosinopril, perindopril, ramipril, benazepril and cilazapril, was investigated with respect to the following parameters: pH of the running buffer, organic modifiers and surfactants. The most critical parameter is the pH of the running buffer. The addition of sodium dodecyl sulfate had a negative influence on the peak symmetry, and selectivity was not improved. The separation of the eight compounds can be performed by means of two phosphate buffers (each 100 mM) at pH 7.0 and pH 6.25, respectively. This combination is necessary for the selective identification of structurally related substances because of their similar pKa values.

Optimization and validation of a capillary zone electrophoretic method for the analysis of several angiotensin-II-receptor antagonists.

We optimized a capillary zone electrophoretic method for separation of six angiotensin-II-receptor antagonists (ARA-IIs): candesartan, eprosartan, irbesartan, losartan potassium, telmisartan, and valsartan. A three-level, full-factorial design was applied to study the effect of the pH and molarity of the running buffer on separation. Combination of the studied parameters permitted the separation of the six ARA-IIs, which was best carried out using 60 mM sodium phosphate buffer (pH 2.5). The same system can also be applied for the quantitative determination of these compounds, but only for the more soluble ones. Some parameters (linearity, precision and accuracy) were validated.

Optimization and validation of a capillary zone electrophoretic method for the simultaneous analysis of four atypical antipsychotics.

A capillary zone electrophoretic method has been developed and optimized for separation of four atypical antipsychotics (AAPs): clothiapine (cT), clozapine (cZ), olanzapine (O), and quetiapine (Q). A three-level full-factorial design was applied to study the effect of the pH and molarity of the running buffer on separation. Combination of the studied parameters permitted the separation of the four AAPs, which was best carried out using 80 mM sodium phosphate buffer (pH 3.5). The same system can also be applied for the quantitative determination of these compounds. The method was then validated regarding linearity, precision, and accuracy. Especially, the possibility of simultaneous quantification and identification of the active ingredient in the finished product is very attractive.

Simultaneous determination of hydrochlorothiazide and several inhibitors of angiotensin-converting enzyme by capillary electrophoresis.

A capillary electrophoresis method was developed for the simultaneous determination of hydrochlorothiazide and several angiotensin-converting enzyme (ACE) inhibitors: enalapril, lisinopril, quinapril, fosinopril, ramipril, and cilazapril. The most critical parameter is the pH of the running buffer. Separation was performed on a fused-silica capillary (52 cm total length x 75 microm I.D.) using a sodium phosphate buffer (pH 7.25; 100 mM). The method was successfully applied to the quantitative determination of these compounds in their corresponding pharmaceutical formulation. The method was validated in terms of linearity of response, reproducibility and accuracy.

Optimisation by experimental design of a capillary electrophoretic method for the separation of several inhibitors of angiotensin-converting enzyme using alkylsulphonates.

A statistical experimental design was used to optimise a capillary electrophoretic separation method for eight inhibitors of the angiotensin-converting enzyme: enalapril, lisinopril, quinapril, fosinopril, perindopril, ramipril, benazepril, and cilazapril. Because a free solution capillary electrophoresis system did not achieve a complete separation of these eight compounds in one run, the usefulness of alkylsulphonates as ion-pairing agents was investigated. After preliminary investigations to determine the experimental domain and the most important factors, a three-level full-factorial design was applied to study the impact of the pH and the molarity of the ion-pairing agent on the separation. Improved separations were obtained suggesting a favourable effect of ion-pairing interactions between analytes and the additive; however, it remained impossible to separate them all in one run. A combination of two systems was still necessary for the selective identification of these structurally-related substances.

Optimization and validation of a micellar electrokinetic chromatographic method for the analysis of several angiotensin-II-receptor antagonists.

We have optimized a micellar electrokinetic capillary chromatographic method for the separation of six angiotensin-II-receptor antagonists (ARA-IIs): candesartan, eprosartan mesylate, irbesartan, losartan potassium, telmisartan, and valsartan. A face-centred central composite design was applied to study the effect of the pH, the molarity of the running buffer, and the concentration of the micelle-forming agent on the separation properties. A combination of the studied parameters permitted the separation of the six ARA-IIs, which was best carried out using a 55-mM sodium phosphate buffer solution (pH 6.5) containing 15 mM of sodium dodecyl sulfate. The same system can also be applied for the quantitative determination of these compounds, but only for the more stable ARA-IIs (candesartan, eprosartan mesylate, losartan potassium, and valsartan). Some system parameters (linearity, precision, and accuracy) were validated.

Optimization and validation of a micellar electrokinetic chromatographic method for the analysis of florfenicol.

We have optimized a micellar electrokinetic capillary chromatographic method for the separation of florfenicol and florfenicol amine, its degradation product. The separation was carried out using a 50mM sodium borate buffer (pH 9.0) containing 25mM of sodium dodecyl sulphate. The method selectivity was proven by the simultaneous separation of florfenicol and two structural antibiotics, chloramphenicol and thiamphenicol. The same system can also be applied for the quantitative determination of these antibiotics. The method was then validated regarding linearity, precision and accuracy.

Simultaneous determination of hydrochlorothiazide and several angiotensin-II-receptor antagonists by capillary electrophoresis.

We have investigated the capability of the capillary zone electrophoretic (CZE) and micellar electrokinetic capillary chromatographic (MEKC) methods to simultaneously separate hydrochlorothiazide and six angiotensin-II-receptor antagonists (ARA-IIs): candesartan, eprosartan mesylate, irbesartan, losartan potassium, telmisartan, and valsartan. The CZE and MEKC methods are suitable for the qualitative and quantitative determination of combined HCT/ARA-IIs in pharmaceutical formulations. Depending on the ARA-II, at least one of the two methods can be used for each combination. The two methods have been validated in terms of their linearity of response, reproducibility, and accuracy.

Determination of captopril and its degradation products by capillary electrophoresis.

Captopril, an antihypertensive agent, and its degradation products have been quantified in pharmaceutical formulations by capillary zone electrophoresis (CZE). A method using cetyltrimethylammonium bromide (CTAB) added to a sodium phosphate buffer (pH 5.5; 100 mM) as running buffer and using N-acetyl-L-tyrosine as an internal standard has been developed and validated for the quantitative determination of captopril in tablets. The method is an indicator of compound stability and can also be applied to the purity control of the raw material and for the determination of the degradation products. For this purpose, salicylic acid is used as an internal standard.

The quantitative determination of several inhibitors of the angiotensin-converting enzyme by CE.

Capillary electrophoresis (CE) was applied to the study of several inhibitors of the angiotensin-converting enzyme. Separation of the compounds was performed by means of two phosphate buffers (each 100 mM) at pH 7.0 and 6.25, respectively [S. Hillaert, W. Van den Bossche, J. Chromatogr. A, 895 (2000) 33-42.]. Due to the highest selectivity of the first mentioned running buffer, the same system has been applied for the quantification of enalapril, lisinopril, quinapril, fosinopril, perindopril and benazepril in their corresponding pharmaceutical formulation. Especially, the possibility of simultaneous identification and quantification of the active ingredient in the finished product is very attractive. Excipients do not adversely affect the results. This paper deals with the validation of some parameters of the quantitative analysis: linearity, precision, accuracy and robustness.

The qualitative and quantitative determination of quinolones of first and second generation by capillary electrophoresis.

Capillary electrophoresis (CE) was applied to the study of 10 quinolones of first and second generation--nalidixic acid, oxolinic acid, pipemidic acid, cinoxacin, norfloxacin, ciprofloxacin, ofloxacin, pefloxacin, fleroxacin, and flumequine. Separation was performed on a fused silica capillary (75 microm-60 cm) using a phosphate buffer (pH 7.0, 125 mM). Detection was at 214 nm. Only norfloxacin and ciprofloxacin cannot be separated in this way. Because of the specificity of the method, the identification of the individual quinolones by their migration time was possible. The same system has been applied for the quantitative determination of quinolones in tablets and capsules. Excipients do not adversely affect the results. Some parameters (linearity, precision, accuracy) were validated. Especially the possibility of simultaneous quantification and identification of the active ingredient in the finished product is very attractive.

Separation of 2-arylpropionic acids on a cellulose based chiral stationary phase by RP-HPLC.

The enantiomers of eight 2-arylpropionic acids, a group of chiral non steroidal antiinflammatory drugs, were resolved as their benzylamide derivatives on a high-performance liquid chromatographic chiral stationary phase consisting of a covalently bound tris (4-methylbenzoate) cellulose layer on silica gel. The column was used under reversed-phase conditions using methanol as the main mobile phase component, with a perchlorate buffer pH 2.0. A compromise for derivatization with a water soluble carbodiimide and 1-hydroxybenzotriazole of a group of eight analytes was obtained. The derivatives were identified by IR- and MS-spectroscopy.

Enantiomeric separation of amide derivatives of some 2-arylpropionic acids by HPLC on a cellulose-based chiral stationary phase.

A reversed phase high-performance liquid chromatographic method has been developed for the determination of the R- and S-enantiomers of ibuprofen, flurbiprofen, ketoprofen and tiaprofenic acid. Separation has been achieved using a tris(4-methylbenzoate)cellulose phase after derivatization into their amides. Flurbiprofen could also be partially resolved into its enantiomers without prior derivatization.

Simultaneous effects of amines and alkylsulphonates in reversed-phase ion-pair liquid chromatography--application to the separation of 2-imidazoline drugs.

The reversed-phase liquid chromatographic separation of several pharmaceutically important 2-imidazoline derivatives using eluents containing both amines and alkylsulphonates, is described. The addition of N, N-dimethyloctylamine and sodium octanesulphonate to an acidic aqueous methanolic eluent, resulted in an eluent with much higher separation power than mobile phases containing only one modifier. The combined effect of the amine and of the alkylsulphonate enabled baseline (or nearbaseline) separations of all the 2-imidazolines studied. The use of such eluents is described in terms of efficiency, selectivity, peak symmetry and separation time and the mechanism of retention is discussed. Some separation examples are given to demonstrate the applicability of the developed technique in the quality control of these drugs in pharmaceuticals.

The determination of non-steroidal antiinflammatory drugs in pharmaceuticals by capillary zone electrophoresis and micellar electrokinetic capillary chromatography.

Ibuprofen, indomethacin, ketoprofen, piroxicam and diclofenac have been quantified in dragees, suspension, suppositories, capsules, injection solutions and tablets by capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MEKC). The experiments were performed without specific sample pretreatment. The reproducibility of the method was investigated. Good quantitation was obtained in short analysis times. CE and MEKC are found to offer a good alternative to conventional HPLC methods.

Capillary zone electrophoresis and micellar electrokinetic capillary chromatography of some non-steroidal antiinflammatory drugs (NSAIDs).

The possibilities of capillary electrophoresis (CE) and micellar electrokinetic capillary chromatography (MEKC) were investigated for the qualitative analysis of some non-steroidal antiinflammatory drugs. In CE the influence of the pH of the buffer and its ionic strength were investigated for a test mixture of six compounds. Also the influence of organic modifiers was studied. The best conditions were applied to the separation of 15 drugs. In MEKC the influence of the concentration of SDS in buffers with pH ranges of 8.0-9.0 was investigated. The influence of an organic modifier, namely acetonitrile was discussed, whereby an interesting phenomenon of change in retention behaviour was noted. A combination of CE and MEKC allows the separation of the 15 above-mentioned compounds and forms an interesting alternative to HPLC.

Determination of captopril on a coated capillary by capillary electrophoresis.

Capillary electrophoresis with a coated capillary was applied to the determination of captopril, an inhibitor of the angiotensin-converting enzyme. The proposed method using a 100 mM sodium phosphate buffer pH 5.5 and salicylic acid as an internal standard was developed for the quantitative determination of captopril in commercially available pharmaceuticals.