Liquid chromatography in combination with mass spectrometry (LC/MS) is a superior analytical technique for metabolite profiling and identification studies performed in drug discovery and development laboratories. In the early phase of drug discovery the analytical approach should be both time- and cost-effective, thus providing as much data as possible with only one visit to the laboratory, without the need for further experiments. Recent developments in mass spectrometers have created a situation where many different mass spectrometers are available for the task, each with their specific strengths and drawbacks. We compared the metabolite screening properties of four main types of mass spectrometers used in analytical laboratories, considering both the ability to detect the metabolites and provide structural information, as well as the issues related to time consumption in laboratory and thereafter in data processing. Human liver microsomal incubations with amitriptyline and verapamil were used as test samples, and early-phase 'one lab visit only' approaches were used with all instruments. In total, 28 amitriptyline and 69 verapamil metabolites were found and tentatively identified. Time-of-flight mass spectrometry (TOFMS) was the only approach detecting all of them, shown to be the most suitable instrument for elucidating as comprehensive metabolite profile as possible leading also to lowest overall time consumption together with the LTQ-Orbitrap approach. The latter however suffered from lower detection sensitivity and false negatives, and due to slow data acquisition rate required slower chromatography. Approaches with triple quadrupole mass spectrometry (QqQ) and hybrid linear ion trap triple quadrupole mass spectrometry (Q-Trap) provided the highest amount of fragment ion data for structural elucidation, but, in addition to being unable to produce very high-important accurate mass data, they suffered from many false negatives, and especially with the QqQ, from very high overall time consumption.
We describe the purification and characterization of two novel cysteine proteinase inhibitors found in Atlantic salmon skin. One of these, salmon kininogen, has a molecular mass of 52 kDa as determined by matrix-assisted laser desorption/ionization time-of-flight MS, is multiply charged with pI values of 4.0, 4.2 and 4.6 and shows homology to kininogens including the bradykinin motif. The other, salarin, has a molecular weight of 43 kDa, a pI of 5.1 and shows weak homology to cysteine proteinases. Both proteins are N-and O-glycosylated and inhibit papain and ficin but not trypsin.Keywords: Atlantic salmon; cysteine proteinase inhibitor; kininogen; salarin. [18,19] and their expression is changed in malignant processes [20]. At present two cystatin-related hereditary human diseases are known [21,22].In 1992 Rinne found that salmon skin extract contains cysteine proteinase inhibitors with considerably larger molecular weights than those described from other fish species, i.e. carp [23,24], chum salmon [25], and recently rainbow trout [26]. The larger molecular masses of salmon skin inhibitors suggested that they could be unique in structure and function. Thus we started in 1996 to purify and characterize these inhibitors further. During this work, some preliminary results were reported but now known to be partly erroneous [27]. In the present report we describe our results on purification and characterization of these inhibitors from the Atlantic salmon (Salmo salar L.) skin. The amino acid sequence results revealed that one of the inhibitors is homologous to kininogens whereas the other showed slight similarity to cysteine proteinases. M A T E R I A L S A N D M E T H O D S MaterialsThe starting material for the purification was the skin of Atlantic salmon (S. salar L.) weighing 2±3 kg. The skin (1 kg) was homogenized in 1 L of 10 mm Tris/HCl pH 7.4, 10 mm EDTA, 0.25 m sucrose with an inhibitor mixture for final concentration of 0.1 mm phenylmethanesulphonyl fluoride, 5 mm benzamidine and 15 mm sodium azide. The homogenate was centrifuged at 6000 g for 30 min at 14 8C, and the supernatant was collected. To clarify the extract further it was ultracentrifuged at 100 000 g for 2 h at 14 8C, the floating fat was removed and the clear supernatant collected. Chromatographic purification stepsPurification of the inhibitors from skin extract was carried out in four chromatographic steps: papain-affinity chromatography, gel filtration, anion-exchange chromatography and reversedphase chromatography. After each step the inhibitory activity towards papain was assayed as described in the sectioǹ Inhibition assay'. The clarified skin extract was subjected to affinity chromatography on a papain±Sepharose column [5]. The carboxymethyl-papain±Sepharose 4B Fast Flow (Pharmacia Biotech) was prepared as described earlier [28]. The affinity chromatography column (1.6 Â 8 cm) was equilibrated with 20 mm sodium phosphate, pH 7.5 and the skin extract applied to the column with a flow rate of 0.6 mL´min 21. The column was then washed wit...
(11)C-ORM-13070 was rapidly metabolized in human subjects after intravenous injection. The effective radiation dose of (11)C-ORM-13070 was in the same range as that of other (11)C-labelled brain receptor tracers. An injection of 500 MBq of (11)C-ORM-13070 would expose a subject to 2.0 mSv of radiation. This supports the use of (11)C-ORM-13070 in repeated PET scans, for example, in receptor occupancy trials with novel drug candidates.
Aims The aim of this study was to compare lung deposition of budesonide administered from two dry powder inhalers, Giona® Easyhaler® 200 µg/dose and Pulmicort® Turbuhaler® 200 µg/dose by utilizing a pharmacokinetic method. Methods This was an open, randomized, crossover study in 33 healthy subjects. The study consisted of four treatment periods separated by at least 4 wash‐out days. Equivalence in lung deposition was assessed after a single inhaled 1000 µg (5 × 200 µg) dose of budesonide from Giona® Easyhaler® and from Pulmicort® Turbuhaler®. Concomitant oral charcoal administration (40 g) was used to prevent gastrointestinal (GI) absorption of budesonide. The efficacy of the charcoal was studied after oral administration of a budesonide 2 mg capsule. The subjects were trained to inhale the study drugs with controlled flow rates, which resulted in an equal pressure drop (4 kPa) across both inhalers. Venous blood samples for the determination of budesonide concentrations in plasma were drawn before and at predetermined time points up to 8 h after drug administration. Budesonide concentrations in plasma were determined using liquid chromatography‐tandem mass spectrometry. Several pharmacokinetic parameters were estimated, the area under the budesonide concentration in plasma vs time curve from dosing to infinity (AUC(0, ∞)) being the primary response variable. Equivalence in lung deposition was concluded if the 90% confidence interval (CI) for the Easyhaler® : Turbuhaler® ratio of AUC(0, ∞) fell within the limits of 0.8–1.25. Results The mean AUC(0,∞) value after Easyhaler® treatment was 3.48 (standard deviation (SD) 0.93) ng ml−1 h and after Turbuhaler® treatment 3.46 (1.13) ng ml−1 h. The Easyhaler® : Turbuhaler® AUC(0, ∞) ratio was 1.02 and the 90% CI was from 0.96 to 1.09. The mean Cmax values (SD) for budesonide in plasma after Easyhaler® and Turbuhaler® treatments were 1.22 (0.41) ng ml−1 and 1.29 (0.44) ng ml−1, respectively. There was no statistically significant difference (P = 0.39) between the median tmax for Easyhaler® (30 min) and Turbuhaler® treatment (23 min). Charcoal impaired the GI absorption of budesonide by 96%. The occurrence of adverse events was similar during both treatments. Conclusions The results show that the lung deposition of budesonide from Giona® Easyhaler® 200 µg/dose and Pulmicort® Turbuhaler® 200 µg/dose dry powder inhalers is equivalent. The charcoal block used to prevent GI absorption of swallowed budesonide was found to be effective.
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