Determining therapeutic trough ranges for linezolid

Linezolid (LZD) is an oxazolidinone approved for the treatment of gram-positive infections. Therapeutic drug monitoring is increasingly used to optimize LZD dosing. The therapeutic target for LZD is to achieve an area under the concentration-time curve over 24 h divided by the MIC (AUC/MIC) > 100. In this study, we determined the trough ranges associated with this therapeutic AUC. Concentration-time profiles for 999 virtual patients were simulated using a previously published pharmacokinetic model for LZD. AUC was estimated for each virtual patient using the trapezoidal method. We determined the trough ranges that achieve the therapeutic target of AUC/MIC > 100 at different MIC values of 1, 2 and 4 μg/mL. Trough samples correlated well with LZD AUC (R² = 0.87). For trough concentration of 2–5 μg/mL, 99% had an AUC_0–24 > 100 µg⋅h⋅ml⁻¹, 23% had an AUC_0–24 > 200 µg⋅h⋅ml⁻¹ and none had an AUC_0–24 > 400 µg⋅h⋅ml⁻¹. For trough concentrations of 5–8 µg/ml, 87% of the patients had an AUC_0–24 > 200 µg⋅h⋅ml⁻¹ and none had an AUC_0–24 > 400 µg⋅h⋅ml⁻¹ To achieve the therapeutic target of an AUC/MIC > 100, it is suggested that trough ranges be set at 2–5 µg/ml if the MIC < 2 and 5–8 µg/ml if the MIC = 2; however, at an MIC of 4 µg/ml, it is difficult to achieve an AUC/MIC > 100 without increasing the risk of LZD toxicity.     

A validated HPLC method for the simultaneous determination of naftidrofuryl oxalate and its degradation product (metabolite), naftidrofuryl acid: applications to pharmaceutical tablets and biological samples

A simple, sensitive, and selective reverse phase‐high performance liquid chromatography (RP‐HPLC) method was developed and validated for the simultaneous determination of naftidrofuryl oxalate (NF) and its hydrolytic degradation product (metabolite), naftidrofuryl acid (NFA). Chromatographic separation was achieved on Spheri‐5 RP‐C8 (5 µm) (220 × 4.6 mm i.d.) column using a mobile phase composed of acetonitrile, 0.05 M sodium acetate and triethylamine (40 : 60 : 0.1, by volume) adjusted to pH 5.5 using glacial acetic acid. The mobile phase was pumped at flow rate 1.5 ml/min. The UV detector was set at 225 nm and quantification of the analytes was based on measuring the peak areas. The method was proved to be accurate and precise with linearity ranges of 0.1–25 and 0.2–25 µg ml^‐1 for NF and NFA, respectively. The limits of detection were 0.03 and 0.04 µg ml^‐1 for NF and NFA, respectively. The method was applied to serve three goals: (1) stability‐indicating assay of the parent drug NF in its pharmaceutical formulation, (2) determination of the degradation product NFA down to a level of 0.005% in the presence of large excess of the parent drug, and (3) drug monitoring of naftidrofuryl and its metabolite, naftidrofuryl acid, in human plasma/urine samples taken from a healthy volunteer treated with 200 mg oral dose of naftidrofuryl oxalate. The proposed method proved to be accurate, precise, and reliable in all these application fields. Copyright © 2012 John Wiley & Sons, Ltd.     

A sensitive spectrophotometric method for the determination of propranolol HCl based on oxidation bromination reactions

Three new, simple, sensitive, rapid and economical spectrophotometric methods (A, B and C) have been developed for the determination of propranolol hydrochloride (PRO) in bulk drug and dosage forms. These methods are based on oxidation‐bromination reaction of PRO by bromine, generated in situ by the action of acid on a bromate‐bromide mixture, followed by determination of unreacted bromine by three different reaction schemes. In method A, the determination of the residual bromine is based on its ability to bleach the indigo carmine dye and by measuring the absorbance at 610 nm. The residual bromine (in method B), is treated with excess of iron(II) and the resulting iron(III) is complexed with thiocyanate and the absorbance is measured at 480 nm. Method C involves treating the unreacted bromine with a measured excess of iron(II) and the remaining iron(II) is complexed with 1,10‐phenanthroline and the increase in absorbance is measured at 510 nm. In all three methods, the amount of bromine reacted corresponds to the drug content. The different experimental parameters affecting the development and stability of the colour are carefully studied and optimized. Beer's Law is valid within a concentration range of 1–13, 4–12 and 2–9 µg ml⁻¹ for methods A, B, and C, respectively. The molar absorptivity, Sandell's sensitivity, detection and quantification limits are calculated. Common excipients used as additives in pharmaceutical preparations do not interfere in the proposed methods. The proposed methods have been successfully applied to the determination of PRO in pharmaceutical preparations and the results were statistically compared with those of the official method by applying the Student's t‐test and F‐test. Copyright © 2010 John Wiley & Sons, Ltd.     

Interaction between silver nanoparticles of 20 nm (AgNP20) and human neutrophils: induction of apoptosis and inhibition of de novo protein synthesis by AgNP20 aggregates

Cytotoxic and proinflammatory properties of silver nanoparticles (AgNPs) have been reported in few studies but the direct interaction between AgNPs and neutrophils, which play a key role in inflammation, has never been documented. Here, we examined the role of AgNPs with a starting size of 20 nm (AgNP₂₀) in human neutrophils. Using dynamic light scattering for the characterization of NPs suspended under identical conditions to those used for in vitro experiments, we found that, at 10 µg ml^–1, 92% of AgNP₂₀ possess a diameter of 17.1 nm but, at 100 µg ml^–1, a tri‐modal size distribution with large aggregates was observed (> 500 nm). Neutrophil cell size increased when treated with AgNP₂₀ and transmission electronic microscopy experiments revealed that AgNP₂₀ can rapidly interact with the cell membrane, penetrate neutrophils, localize in vacuole‐like structures, and be randomly distributed in the cytosol after 24 h. Treatment with 100 µg ml^–1 AgNP₂₀ for 24 h (but not 10 µg ml^–1) increased the neutrophil apoptotic rate and inhibited de novo protein synthesis. We conclude that AgNP₂₀ induced apoptosis and can act as potent inhibitors of de novo protein synthesis at 100, but not 10 µg ml^–1 in human neutrophils. Copyright © 2013 John Wiley & Sons, Ltd.     

Investigation on cobalt‐oxide nanoparticles cyto‐genotoxicity and inflammatory response in two types of respiratory cells

The increasing use of cobalt oxide (Co₃O₄) nanoparticles (NPs) in several applications and the suggested genotoxic potential of Co‐oxide highlight the importance of evaluating Co₃O₄ NPs toxicity. Cyto‐genotoxic and inflammatory effects induced by Co₃O₄ NPs were investigated in human alveolar (A549), and bronchial (BEAS‐2B) cells exposed to 1–40 µg ml^–1. The physicochemical properties of tested NPs were analysed by transmission electron microscopy (TEM) and dynamic light scattering (DLS). Cytotoxicity was studied to analyze cell viability (WST1 test) and membrane damage (LDH assay), direct/oxidative DNA damage was assessed by the Formamido‐pyrimidine glycosylase (Fpg)‐modified comet assay and inflammation by interleukin (IL)‐6, IL‐8 and tumor necrosis factor‐alpha (TNF‐α) release (ELISA). In A549 cells, no cytotoxicity was found, whereas BEAS‐2B cells showed a viability reduction at 40 µg ml^–1 and early membrane damage at 1, 5 and 40 µg ml–1. In A549 cells, direct and oxidative DNA damage at 20 and 40 µg ml^–1 were detected without any effects on cytokine release. In BEAS‐2B cells, significant direct DNA damage at 40 µg ml^–1 and significant oxidative DNA damage with a peak at 5 µg ml^–1, that was associated with increased TNF‐α release at 1 µg ml^–1 after 2 h and increased IL‐8 release at 20 µg ml^–1 after 24 h, were detected. The findings show in the transformed alveolar cells no cytotoxicity and genotoxic/oxidative effects at 20 and 40 µg ml^–1. In normal bronchial cells, moderate cytotoxicity, direct DNA damage only at the highest concentration and significant oxidative‐inflammatory effects at lower concentrations were detected. The findings confirm the genotoxic‐oxidative potential of Co₃O₄ NPs and show greater sensitivity of BEAS‐2B cells to cytotoxic and oxidative‐inflammatory effects suggesting the use of different cell lines and multiple end‐points to elucidate Co₃O₄ NPs toxicity. Copyright © 2015 John Wiley & Sons, Ltd.     

Indirect fibre‐optic colorimetric determination of ascorbic acid using 2‐(5‐bromo‐2‐pyridylazo)‐5‐diethylaminophenol and cloud point extraction

A new method has been developed for the indirect determination of ascorbic acid (AA) in commercial syrup preparations based on cloud point extraction (CPE) separation and preconcentration, and determination by molecular absorption spectrometry. The colorimetric method was based on the reduction of Fe(III) to Fe(II) and complexation of Fe(II) with 2‐(5‐bromo‐2‐pyridylazo)‐5‐diethylaminophenol (Br‐PADAP), followed by its extraction into Triton X‐114. Selectivity of the method was increased with the use of EDTA as a masking agent. The absorbance was measured at 742 nm. Various influencing factors on the separation and preconcentration of AA have been investigated systematically, and the optimized operation conditions were established. The proposed method allows the determination of AA in the range 5–200 µg L^?1 with a relative standard deviation of 3.0%. The detection limit was found to be 0.9 µg L^?1 for AA. This method has been applied to the determination of ascorbic acid in commercial pharmaceutical preparations. Copyright © 2011 John Wiley & Sons, Ltd.     

PVC membrane sensor for potentiometric determination of iron (II) in some pharmaceutical formulations based on a new neutral ionophore

A novel poly (vinyl chloride) PVC membrane sensor for Fe²⁺ ions is described. The sensor is based on the use of newly synthesized chiral 2,6‐bis‐(carboxamide methyl ester)pyridine derivative as neutral ionophore in plasticized PVC membrane. The sensor display a fast, stable and near‐Nernstian response over a relative wide ferrous concentration range (1 × 10^?3 to 6 × 10^?6 M), with cationic slope of 31.5 ± 0.5, mV per concentration decade over a pH range of 5.0–9.0. The direct determination of 0.25–56.0 µg/ml of ferrous in aqueous solution shows an average recovery of 98.5% and a mean relative standard deviation of 1.5% at 20.0 µg/ml. The sensor displays long life‐span, long‐term stability, high reproducibility, and short response time. Selectivity coefficients for Fe(II) relative to a number of interfering substances were investigated. The sensor shows high significantly for Fe²⁺ over Fe,³⁺ Cu,²⁺ Zn,²⁺ Cd,²⁺ Hg,²⁺ Pb,²⁺ Ni,²⁺ Co,²⁺ Mn,²⁺ Al,³⁺ alkaline earth and alkali metal ions. The sensor is successfully applied for measurement of ferrous in drug formulations. The results obtained for the determination of ferrous using the proposed sensor are comparable favourably with those obtained using the spectrophotometric method. Copyright © 2010 John Wiley & Sons, Ltd.     

Electrochemical study of spiramycin and its determination in pharmaceutical preparation

Spiramycin (SPY) is a medium‐spectrum antibiotic with high effectiveness against Gram‐positive bacteria. The voltammetric behaviour of spiramycin was studied using differential pulse polarography (DPP) and square wave polarography (SWP). The drug in Britton‐Robinson buffer (pH 11.5) is reduced at ? 1.45 V, giving rise to a well‐defined cathodic peak using hanging mercury drop electrode (HMDE) versus Ag/AgCl electrode. This peak is attributed to the reduction of the aldehyde group. The results proved that the reduction of SPY is an irreversible diffusion‐controlled process. The diffusion current‐concentration relationship was shown to be rectilinear over the range of 20–80 and 0.8–80 µg ml^?1 using DPP and SWP modes, respectively, with detection limit of 8.5 µg ml^?1 (1.01 × 10^?5 M) and 0.46 µg ml^?1 (5.46 × 10^?7 M) for DPP and SWP modes, respectively. A mechanism is postulated for the reduction of SPY. The proposed techniques were successfully applied to the determination of the studied compound either in pure form or in its formulation. Copyright © 2010 John Wiley & Sons, Ltd.     

Lipid peroxidation in the kidney of rats treated with V and/or Mg in drinking water

Spontaneous and stimulated lipid peroxidation (LPO) after vanadate and magnesium treatment was studied in kidney supernatants obtained from outbred 5‐month‐old, albino male Wistar rats. The 2‐month‐old animals daily received: group I (control), deionized water to drink; group II, water solution of sodium metavanadate, NaVO₃ (SMV, 0.125 mg V ml^?1); group III, water solution of magnesium sulfate, MgSO₄ (MS, 0.06 mg Mg ml^?1); and group IV, water solution of SMV‐MS at the same concentrations as in groups II and III for V and Mg, respectively, over a 12‐week period. FeSO₄, NaVO₃ and MgSO₄ were selected as agents that may modify LPO process in in vitro conditions. Spontaneous malondialdehyde (MDA) levels in kidney supernatants increased significantly in the rats in groups II and IV, compared with groups I and III; and they were also significantly higher in all the groups of rats compared with the liver supernatants. The total antioxidant status (TAS) in groups II and IV tended to be higher too. Vanadium concentration in the kidney of the rats in groups II and IV increased, whereas the kidney Mg content in groups II, III and IV decreased, compared with levels in the liver. As the two‐way ANOVA indicated, the changes in the basal MDA level, TAS and Mg concentration in the liver of rats at combined V and Mg application only resulted from independent action of V. As far as the in vitro results are concerned, in the supernatants obtained from the rats in groups II and IV, a significant increase in MDA level was demonstrated in the presence of 30 µm of exogenous FeSO₄ as well as 30, 100, 200 and 400 µm NaVO₃ and 100, 200, 400, 600, 800 and 1000 µm MgSO₄, compared with groups I and III. The 600, 800 and 1000 µm of exogenous MgSO₄ also significantly elevated MDA production in the supernatants obtained from the rats in group III, compared with spontaneously formed MDA in the same supernatants. The three‐way ANOVA showed that the changes in LPO induced by in vitro treatment of kidney supernatants with exogenous Fe or V or Mg (600, 800 and 1000 µm ) were a consequence of independent action of those metals and they also resulted from the interactions between exogenous Fe (Fe_exog) and endogenous V (V_end) and between V_end and exogenous V (V_exog). In conclusion, V (as NaVO₃) consumed by the rats with drinking water at a dose of 12.9 mg V kg^?1 b.w. per 24 h for 12 weeks increased the basal LPO and markedly enhanced TAS in the renal tissue. Its pro‐oxidant potential was also found in in vitro conditions. The Mg dose (6 mg Mg kg^?1 b.w. per 24 h) ingested by the rats together with V (12.7 mg V kg^?1 b.w. per 24 h) neither reduced nor intensified the spontaneous LPO, compared with V‐only intoxicated animals; however, the stimulating effect of Mg on LPO was revealed in in vitro conditions.     

In vitro evaluation of the effects of perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) on IL‐2 production in human T‐cells

Perfluorinated compounds, such as perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), have been shown to alter various immune functions suggesting they are immunotoxic. This study assessed the effects of PFOS and PFOA on interleukin (IL)‐2 production in the human Jurkat T‐cell line and PFOS in healthy human primary T cells. Jurkat cells were stimulated with phytohemagglutinin (PHA)/phorbol myristate acetate (PMA), anti CD‐3/anti CD‐28, or anti CD‐3, and dosed with 0, 0.05, 0.1, 0.5, 1, 5, 10, 50, 75, or 100 µg ml^?1 PFOS or 0, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, or 10 µg ml^?1 PFOA. Jurkat cells stimulated with PHA/PMA or anti CD‐3 exhibited decreased IL‐2 production beginning at 50 µg PFOS ml^?1 and 5 µg PFOS ml^?1 respectively, but stimulation with anti‐CD3/anti‐CD28 resulted in no changes compared with the control. Addition of the PPAR‐alpha antagonist GW6471 to PFOS‐dosed cells stimulated with PHA/PMA resulted in decreases in IL‐2 production starting at 50 µg PFOS ml^?1, which suggests PFOS affected T‐cell IL‐2 production via PPAR‐alpha‐independent mechanisms. Exposure to PFOA, PFOA + GW6471, or PFOS + PFOA in Jurkat cells resulted in no significant differences in IL‐2 production. In vitro dosing studies using healthy primary human CD4+ T cells were consistent with the Jurkat results. These data demonstrated that PFOA did not impact IL‐2 production, but PFOS suppressed IL‐2 production in both a human cell line and human primary cells at dose levels within the high end of the human exposure range. A decrease in IL‐2 production is characteristic of autoimmune diseases such as systemic lupus erythematosus and should be further investigated. Copyright © 2014 John Wiley & Sons, Ltd.     

Chromatographic determination of drugs of abuse in vitreous humor using solid‐phase extraction

A simple method is presented for the simultaneous determination of morphine, 6‐acetylmorphine, codeine, cocaine, benzoylecgonine, cocaethylene, methadone and 2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine (EDDP) in vitreous humor by high‐performance liquid chromatography with photodiode array detector after solid‐phase extraction with Oasis® HLB cartridges and dichloromethane as eluent. The chromatographic process was carried out using an XTerra® RP8 column (250 × 4.6 mm i.d., 5 µm particle size) and a mobile phase composed of acetonitrile and pH 6.5 phosphate buffer in gradient mode. A linear response from the detector was obtained within the concentration range of 0.1–4 µg ml^?1, with correlation coefficients higher than 0.99. The limits of detection were lower than 30 ng ml^?1 for all the drugs studied, the coefficients of variation fluctuated between 0.1 and 12.4%, and the average recoveries were higher than 78% for all the drugs except for EDDP, with a value of 66.4%. Finally, the proposed method was applied to 15 vitreous humor samples coming from individuals who had died from opiate and/or cocaine overdose, showing consumption of cocaine in 14 cases, methadone in five cases and heroin in three cases. Average concentrations of 0.30 µg ml^?1 for morphine, 0.24 µg ml^?1 for 6‐acetylmorphine, 0.10 µg ml^?1 for codeine, 0.81 µg ml^?1 for cocaine, 1.26 µg ml^?1 for benzoylecgonine, 0.15 µg ml^?1 for cocaethylene, 0.11 µg ml^?1 for methadone and 0.68 µg ml^?1 for EDDP were obtained. Copyright © 2012 John Wiley & Sons, Ltd.     

Combined antitumor effects of bee venom and cisplatin on human cervical and laryngeal carcinoma cells and their drug resistant sublines

In the present study, we investigated the possible combined anticancer ability of bee venom (BV) and cisplatin towards two pairs of tumour cell lines: parental cervical carcinoma HeLa cells and their cisplatin‐resistant HeLa CK subline, as well as laryngeal carcinoma HEp‐2 cells and their cisplatin‐resistant CK2 subline. Additionally, we identified several peptides of BV in the BV sample used in the course of the study and determined the exact concentration of MEL. BV applied alone in concentrations of 30 to 60 µg ml^–1 displayed dose‐dependent cytotoxicity against all cell lines tested. Cisplatin‐resistant cervical carcinoma cells were more sensitive to BV than their parental cell lines (IC₅₀ values were 52.50 µg ml^–1 for HeLa vs. 47.64 µg ml^–1 for HeLa CK cells), whereas opposite results were obtained for cisplatin‐resistant laryngeal carcinoma cells (IC₅₀ values were 51.98 µg ml^–1 for HEp‐2 vs. > 60.00 µg ml^–1 for CK2 cells). Treatment with BV alone induced a necrotic type of cell death, as shown by characteristic morphological features, fast staining with ethidium‐bromide and a lack of cleavage of apoptotic marker poly (ADP‐ribose) polymerase (PARP) on Western blot. Combined treatment of BV and cisplatin induced an additive and/or weak synergistic effect towards tested cell lines, suggesting that BV could enhance the killing effect of selected cells when combined with cisplatin. Therefore, a greater anticancer effect could be triggered if BV was used in the course of chemotherapy. Our results suggest that combined treatment with BV could be useful from the point of minimizing the cisplatin concentration during chemotherapy, consequently reducing and/or postponing the development of cisplatin resistance. Copyright © 2013 John Wiley & Sons, Ltd.     

In vitro exposure to cigarette smoke induces oxidative stress in follicular cells of F1 hybrid mice

This study assessed the influence of cigarette smoke condensate (CSC) and benzo(a)pyrene [B(a)P] on the levels of two oxidative stress biomarkers [8‐isoprostane (8‐IsoP) and 8‐hydroxy‐2‐deoxy Guanosine (8‐OH‐dG)], in in‐vitro spent media of follicle cells. Follicles (100–130 µm) isolated from ovaries of F₁ hybrid (C57Bl/6j × CBA/Ca) mice were cultured for 13 days in media exposed to B(a)P [0 ng ml^–1 (control) to 45 ng ml^–1] or CSC [0 µg ml^–1 (control) to 130 µg ml^–1]. The concentrations of oxidative stress biomarkers in spent media were quantified by enzyme‐linked immune sorbent assays (ELISA). CSC and B(a)P treatment induced a significant, dose‐dependent increase in the concentrations of 8‐IsoP and 8‐OH‐dG in the spent media. We conclude that CSC and B(a)P exposure can induce oxidative stress in ovarian follicles, an effect that may contribute to the previously documented decline in follicle development and premature ovarian failure in women who smoke. © Her Majesty the Queen in Right of Canada 2013.     

Lipid peroxidation in the liver of rats treated with V and/or Mg in drinking water

The effect of V⁵⁺ and Mg treatment on spontaneous and stimulated lipid peroxidation (LPO) was studied in liver supernatants obtained from outbred 5‐month‐old, albino male Wistar rats. The 2‐month‐old animals daily received deionized water to drink (control, group I); group II — water solution of NaVO₃ (SMV) at a concentration of 0.125 mg V ml^?1; group III — water solution of MgSO₄ (MS) at a concentration of 0.06 mg Mg ml^?1, group IV — water solution of SMV‐MS at the same concentrations as in groups II and III for V and Mg, respectively, over a 12‐week period. Three metal salts were selected as agents that may modify the LPO process (FeSO₄, NaVO₃ and MgSO₄). V‐intoxicated rats and those treated with V and Mg in combination had higher liver spontaneous malondialdehyde (MDA) formation, compared with the control and Mg‐supplemented animals. In the same groups of animals the total antioxidant status (TAS) was also significantly lowered, in comparison with the control. In the supernatants obtained from the above‐mentioned groups of rats a significant increase in MDA concentration was found in the presence of exogenous 30 µm FeSO₄ as well as 30, 100, 200 and 400 µm NaVO₃, compared with groups I and III. Significantly elevated MDA production was also observed in the supernatants obtained from the rats exposed to V and Mg in combination in the presence of exogenous 100 and 200 µm MgSO₄ in comparison with the control and group III as well as in the presence of exogenous 400 and 600 µm MgSO₄ compared only with group III. In vitro treatment with 1000 µm MgSO₄ of control liver supernatants and those obtained from group III significantly enhanced MDA level, compared with spontaneous MDA formation. The two‐way ANOVA indicated that the changes in the basal MDA level and in TAS in the rats at combined V and Mg application, were not due to V–Mg interaction, but resulted from independent action of V. In addition, the three‐way ANOVA revealed that the changes in LPO induced by in vitro treatment of liver supernatants with exogenous Fe or V or Mg (600, 800 and 1000 µm ) were a consequence of independent action of those metals and they also resulted from the interactions between Fe_exog and V_end and between V_end and V_exog. In conclusion, V consumed by the rats with drinking water at a dose of 12 mg V kg^?1 body weight per 24 h for 12 weeks decreased TAS and enhanced spontaneous LPO in the hepatic tissue, which confirms its pro‐oxidant potential, was also found in in vitro conditions with regard to LPO. Mg administered to rats in combination with V, at the concentration used, neither reduced nor intensified the basal LPO, compared with V‐only treated animals; however, its stimulating effect on LPO was revealed in in vitro conditions, which requires further study. Copyright © 2009 John Wiley & Sons, Ltd.     

Error structure for the HPLC analysis for atenolol,metoprolol and propranolol: a useful weighting method in parameter estimation

Three reversed-phase high performance liquid chromatography (HPLC) methods with UV detection were developed and fully validated for the quantification of three β-blockers: atenolol, metoprolol and propranolol. After validation, error structures for the HPLC analysis were established using a convenient and practical procedure. The mean percentage of relative standard deviation (RSD) of the experimental concentrations (C), were less than 4.29% for proportionality and less than 3.68% for precision for any of the drugs, which allowed the quantitation of β-blockers assayed at concentrations in the range 25–0.78 μg·ml⁻¹. The error structures for the HPLC analysis were: SD (μg·ml⁻¹)=5.02×10⁻²+0.65×10⁻² C for atenolol, SD (μg·ml⁻¹)=4.55×10⁻²+0.63×10⁻² C−7.58×10⁻⁶ C³ for metoprolol and SD (μg·ml⁻¹)=2.73×10⁻²+1.46×10⁻² C−3.49×10⁻⁴ C² for propranolol. The reciprocal of the square of the SD of the drug concentrations measured within the calibration curve could be used as weighting methods in parameter estimation by non-linear regression.     

Spectrophotometric study of diclofenac-Fe(III) complex

A multifactor optimisation technique is successfully applied to develop a new spectrophotometric method in which diclofenac sodium is analysed and determined as it's Fe(III) complex. The effect of simultaneously varying the pH, ionic strength and concentration of colour reagents in the reaction mixture were studied. A four-variable two-level factorial design was used to investigate the significance of each variable and interactions between them. A response surface design was used to optimise complex formation and extraction. It was established that diclofenac reacts with Fe(III) chloride, in the presence of ammonium thiocyanate, in the pH range 4.2–6.5, forming a red chloroform extractable (2:1) complex with maximum absorbance at 481 nm. By applying the methods of Sommer and Job involving non-equimolar solutions the conditional stability constant of the complex, at the optimum pH of 6.0 and an ionic strength μ = 0.19M, was found to be 10^6.4. Good agreement with Beer's law was found for diclofenac concentrations up to mmol l⁻¹. The nominal percent recovery of diclofenac was 98.8% (n = 10). The lower limit of sensitivity of the method was found to be 14.7 μg ml⁻¹.     

PXR–ABC drug transporters/CYP-mediated ursolic acid transport and metabolism in vitro and vivo

The transporting kinetics and metabolic kinetics of ursolic acid were studied in transgenic cell models. Then, the pharmacokinetics features of ursolic acid and the expression of ATP-binding cassette transporters (ABC transporter) and cytochrome P450 (CYP) enzymes in tissues after pregnane X receptor (PXR) activation by 5-pregnen-3β-ol-20-one-16α-carbonitrile (PCN) were investigated in rats. After silencing of PXR in Caco2–siRNA–PXR cells, there was a decrease in the protein abundance of P-glycoprotein, breast cancer-resistant protein, multidrug resistance-associated protein 2 (MRP2), and CYP2C9. The apparent permeability (PDR) values of 10, 20, and 50 µM ursolic acid in Caco2 cells were 2.19 ± 0.44, 1.40 ± 0.17, and 1.25 ± 0.07, respectively, whereas in Caco2–siRNA–PXR cells, they were 1.85 ± 0.36, 1.24 ± 0.11, and 1.19 ± 0.04, respectively. PXR–RXRα would significantly activate ABC transporter expression in Caco2 cells. Compared with Caco2 cells, when the concentrations of ursolic acid were 10, 20, and 50 µM, the PDR values increased in Caco2–PXR–RXRα cells after PXR activation: 1.60 ± 0.31 versus 1.97 ± 0.21, 1.46 ± 0.08 versus 2.01 ± 0.19, and 1.32 ± 0.26 versus 2.09 ± 0.22, respectively. Simultaneously, PXR–RXRα would activate the expression of CYP2C9; metabolic kinetics of ursolic acid in CYP metabolizing enzyme lysate of Caco2 cells and Caco2–PXR–RXR cells was studied and it was found that the K_m values were 81.99 ± 44.32 and 60.05 ± 29.62 µg/ml, and V_max values were 3.77 ± 0.86 and 3.41 ± 0.96 µg · ml⁻¹ · min⁻¹, respectively. However, in human CYP metabolizing recombinase, we found that both CYP2C9 and CYP34A were involved in the metabolism of ursolic acid. V_m and K_m values for CYP3A4 and CYP2C9 were 3.57 ± 1.12 µg · ml⁻¹ · min⁻¹ and 81.71 ± 18.38 µg/ml, 3.85 ± 1.46 µg · ml⁻¹ · min⁻¹ and 62.18 ± 14.56 µg/ml, respectively. As a strong agonist for mouse pxr, PCN could significantly affect pharmacokinetics of ursolic acid in rats, and it showed discrepant effects on messenger RNA expression of cyp and transporters in tissues.     

In vitro evaluation of the cytotoxic and genotoxic effects of artemether,an antimalarial drug,in a gastric cancer cell line (PG100)

Artemisinin is a sesquiterpene lactone endoperoxide, obtained from Artemisia annua, and extensively used as an antimalarial drug. Many studies have reported the genotoxic and cytotoxic effects of artemisinins; however, there are no studies that compare such effects between cancer cell lines and normal human cells after treatment with artemether, an artemisinin derivative. Gastric cancer is the fourth most frequent type of cancer and the second highest cause of cancer mortality worldwide. Thus, the aim of this study was to evaluate the in vitro genotoxic and cytotoxic effects induced by artemether in gastric cancer cell line (PG100) and compare them with the results obtained in human lymphocytes exposed to the same conditions. We used MTT (3‐(4,5‐methylthiazol‐2‐yl)‐2, 5‐diphenyl‐tetrazolium bromide) assay, comet assay and ethidium bromide/acridine orange viability staining to evaluate the cytotoxic and genotoxic effects of artemether in PG100. MTT assay showed a decrease in the survival percentages for both cell types treated with different concentrations of artemether (P < 0.05). PG100 also showed a significant dose‐dependent increase in DNA damage index at concentrations of 119.4 and 238.8 µg ml^?1 (P < 0.05). Our results showed that artemether induced necrosis in PG100 at concentrations of 238.8 and 477.6 µg ml^?1, for all the tested harvest times (P < 0.05). In lymphocytes, artemether induced both apoptosis and necrosis at concentrations of 238.8 and 477.6 µg ml^?1, for all the tested harvest times (P < 0.05). In conclusion, human lymphocytes were more sensitive to the cytotoxic effects of the antimalarial drug than the gastric cancer cell line PG100. Copyright © 2011 John Wiley & Sons, Ltd.     

High-performance liquid chromatographic determination of cis-dichlorodiammineplatinum(II) in plasma ultrafiltrate

A reproducible, simple and sensitive high-performance liquid chromatographic method was described for the quantitative analysis of cis-diamminedichloroplatinum(II) (CDDP) in ultrafiltrate plasma in the presence of nickel chloride as internal standard. CDDP and the internal standard were chelated by exchange with diethyldithiocarbamate. After derivatization, the mixture was directly injected into the column. Chromatography was performed on an Ultrasphere column and the eluent measured spectrophotometrically at 260 nm for CDDP and at 250 nm for the internal standard. The peak area ratio of CDDP to the internal standard varied linearly with concentration over the range 0.05–10 μg ml⁻¹. Precision and reproducibility were both excellent and the limit of quantification was 0.03 μg ml⁻¹ using only 0.5 ml of ultrafiltrate. The present method, without extraction, should be entirely automated. This assay may be suitable for therapeutic drug monitoring in patients receiving CDDP.     

Scintillation proximity radioimmunoassay for the measurement of acyclovir

A homogeneous, single-tube scintillation proximity radioimmunoassay (SPRIA) to quantitate acyclovir (Zovirax^®), ACV, (9-[(2[hydroxyethoxy)]methylguanine)} in human plasma is described. The reagents for the SPRIA are an anti-ACV monoclonal antibody (WACO4 MAb), tritiated ACV, and scintillation proximity reagent (goat anti-mouse immunoglobulin G (IgG) coupled to fluoromicrospheres). The ACV standard curve range in the SPRIA is from 0.7 ng ml⁻¹ (3.0 nmol l⁻¹) to 90.0 ng ml⁻¹ (0.4 μmol l⁻¹) with a 50% inhibitory concentration of 5.0 ng ml⁻¹ (22.2 nmol l⁻¹). However, the lower limit of quantification is 7 ng ml⁻¹ at 1:10 dilution of plasma. Analytical recovery of ACV in spiked human plasma controls ranges between 90–110%. Intra- and inter-assay relative standard deviations were < 8%. This high throughput homogeneous assay is a rapid, convenient and simple alternative to the current radioimmunoassay that uses ammonium sulfate precipitation as the separation method. This technique is particularly attractive because it requires neither separation of bound from free drug nor use of scintillation fluid. The procedure was applied to quantitate ACV in samples from pre-clinical and clinical studies after the administration of valaciclovir, a prodrug of ACV (256U87, Valtrex^®, l-valyl ester of ACV). Automation of this assay will further improve efficiency in processing a larger number of samples.