film containing 57.7 mg S-ketamine hydrochloride (S-ketamine HCL). The Sketamine HCL is dispersed within a matrix to produce a film corresponding to 50 mg S-ketamine free base. The film(s) was/were placed either under the tongue or buccally on the mucosa. After placement of the films, the subject was not allowed to swallow for 10 min. The randomization sequence was determined by the randomization option in the Electronic Data Capture system CASTOR (www.castoredc.com). The oral thin films were provided by LTS Lohmann Therapie-Systeme AG (Andernach, Germany) and were dispensed by the hospital trial pharmacy on the morning of dosing. To calculate the bioavailability of the OTF, six hours after placement of the oral thin film, all subjects received an intravenous S-ketamine (Ketanest-S, Pfizer, the Netherland) infusion of 20 mg over 20 min. The intravenous dose of 20 mg given was based on a previous study on the pharmacokinetics of inhaled Sketamine in which a 20 mg intravenous dose was administered over 20 min. This dose was well accepted by the volunteers.8 We waited 6 h before giving the intravenous dose to ensure that most of the pharmacodynamic effects (i.e. the topic of our accompanying paper)4 had dissipated. Blood Sampling and S-Ketamine Measurement Blood samples were obtained at t = 0 (= oral thin film placement) 5, 10, 20, 40, 60, 90, 120, 180, 240, 300, 360 min, and at the following time periods following the start of the intravenous administration: 2, 4, 10, 15, 20, 30, 40, 60, 75, 90 and 120 min. 3-mL samples were obtained from a 22G arterial line placed in the radial artery of the non-dominant arm and collected in lithium heparin tubes. All heparin samples were centrifuged at 1,500 g for 10 min, within 15 min after withdrawal and plasma was separated and stored in two aliquots at -80 oCuntil analysis. For analysis the samples were thawed and 200 µL was transferred into glass tubes and 10 µL internal standard was added. After mixing, 250 µL buffer was added. After again mixing, 4 mL methyl-tertiair-butylether followed by 15 min and 15 min centrifugation. The upper organic layer was pipetted into another tube that contained 0.6 mL of 0.4 mol/L hydrochloric acid in methanol, and dried under a gentle stream of nitrogen at 35 oC. The residue was re-dissolved in 100 µL mobile phase (6.8 % methanol in water with 0.1 % formic acid) by vortexing and ultrasonication for 3 min and 5 µL sample was injected on the chromatographic system with a C18 column. All reference standards (ketamine and norketamine) and internal standards ketamine-D4 (K-D4), norketamine-D4 (NK-D4) were HCl salts and purchased from LGC Standards GmbH (Germany); cis-6-hydroxynorketamine (6-HNK) was purchased from Syncom BV (the Netherlands); and the internal standard hydroxy- norketamine-13C6 (HNK-13C6) was purchased from Alsachim SAS (France). 16
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