5 and 1.0 h post-spike. The aliquots were immediately treated with 200 µL cold acetonitrile containing 0.4% citric acid and stored at −80 °C until analysed as described in Section 2.7. An emulsion for intravenous administration containing each of the three compounds (i.e., aripiprazole, N-hydroxymethyl-aripiprazole or aripiprazole lauroxil) in equimolar SCH 900776 purchase concentrations equivalent to 1 mg aripiprazole was produced. The emulsions

consisted of compound, 20% w/w fractionated coconut oil, 1.2% w/w lecithin, 2% w/w glycerol and q.s. water. The amount of each compound added was 1 mg aripiprazole/mL, 1.2 mg N-hydroxymethyl-aripiprazole/mL or 1.87 mg aripiprazole lauroxil/mL, i.e., equimolar. Each of the three compounds was dissolved in the oil together with lecithin and gently heated to 50 °C with continuous stirring. Glycerol was added to the aqueous phase as an isotonic agent and the aqueous phase was heated to 50 °C. The two phases were mixed and homogenised to a pre-emulsion by rapid stirring for 1 min. The pre-emulsion was placed on ice and the droplet size was further reduced by means of a homogeniser equipped with a standard microtip at a power output of 5

(Sonifier Cell Disruptor, Model B15, Branson, Pusan, Korea) for 10 min. The formulation was then filtered through a 0.45 µm sterile filter into a sterilised glass bottle with a rubber membrane and a crimped lid. The protocol used for the in vivo study in rats was approved by the institutional animal ethics committee in accordance with Danish law regulating experiments on animals MEK inhibitor and in compliance with EC directive 2010/63/EU, and the NIH guidelines on animal welfare. Female Sprague Dawley rats, weighing 248–276 g on the day of administration, obtained from Charles Amino acid River (Sulzfeld, Germany) were used for the pharmacokinetic studies (n = 6 per group). The animals were acclimatised for a minimum of 5 days in groups of 2 on wooden

bedding (Tapvei, Kortteinen, Finland) in plastic cages, 595 × 380 × 200 mm3, with a stainless steel lid (Scanbur, Sollentuna, Sweden) in humidity- and temperature-controlled ventilation cupboards (Scantainers, Scanbur Technology, Karlslunde, Denmark), relative humidity 40–60%, temperature 20 ± 1 °C, light from 6:00–18:00 h. The animals had free access to a standard rodent diet (Altromin 1325, Brogaarden, Denmark) and water ad libitum during the study. The animals were randomly assigned to three groups (n = 6 per group) receiving either aripiprazole, N-hydroxymethyl-aripiprazole or aripiprazole lauroxil molar equivalent to 5 mg aripiprazole/kg. The animals were dosed by injection into the tail vein with a submicron emulsion containing a molar concentration equivalent to 1 mg aripiprazole/mL. Blood samples of 100 µL were obtained from the lateral tail vein by individual vein puncture and collected into potassium–EDTA tubes (Microvette 500 K3E, Saarstedt, Nümbrecht, Germany).