ATP disodium salt was purchased from Pharma Waldhof GmbH, Düsseldorf, Germany. Adenosine 5′-diphosphate (ADP) disodium salt, adenosine 5′-monophosphate (AMP) sodium salt, adenine, inosine, hypoxanthine, uric acid and nitric acid were purchased from Sigma Chemical Co., St. Louis, USA. Adenosine and lithium carbonate (Li2CO3) were obtained from Fagron BV., Uitgeest, The Netherlands. Perchloric acid (PCA) 70% solution in water was purchased from Sigma-Aldrich, Steinheim, Germany. KOH, KH2PO4, K2CO3, K2HPO3*3H2O and NaOH were obtained from Merck, Darmstadt, Germany and 0.9% saline from Braun, Melsungen, Germany. Bengmark-type naso-duodenal tubes were from Flocare, Zoetermeer, The Netherlands. NH4NO3 was obtained from Fluka, Steinheim, Germany. Trichloroacetic acid (TCA) 20% solution in water was from Serva, Heidelberg, Germany. Citric acid suitable for human consumption was obtained from the pharmacy of Maastricht University Medical Centre.
Production of pellets
ATP pellets were produced at Ghent University, Faculty of Pharmaceutical Science, Belgium as described by Huyghebaert et al. , with minor modifications to obtain an ATP concentration of >40% (wt:wt) after coating. Placebo pellets were produced in the same manner, but without ATP. To verify the timing of intestinal release, Li2CO3 (60 mg per administration) was added to the pellets. The proximal-release pellets were coated with 30% Eudragit® L30D-55 (ATP or placebo pellets), and the distal-release pellets (ATP only) were coated with 15% Eudragit® FS 30 D (Röhm Pharma, Darmstadt, Germany), mixed with anionic copolymers of methacrylic acid and ethylacrylate (1:1). After coating, the pellets were cured overnight at room temperature at 60% (proximal-release pellets) or 20% (distal-release pellets) humidity, packed in aluminum foil sachets (VaporFlex®, LPS, NJ, USA), sealed at their respective humidity and stored at room temperature. Pellets were used within 3 months after production.
To test whether the coating of the pellets was adequate, a dissolution test (n = 3 for each type of coating) was performed using the reciprocating cylinder method (USP apparatus 3 from Bio-Dis, VanKel, NJ, USA) at a dip rate of 21 dips per minute using 3 g pellets per vessel (250 mL) with two consecutive media: 0.1 N HCl (37°C), and a 0.2 M KH2PO4 buffer (37°C) with a pH that was adjusted to 6.5 for the proximal-release pellets, and pH 7.4 for the distal-release pellets. Samples were collected after 2 h in HCl and after 2, 5, 10, 20, 30 and 60 min in buffer as described in Huyghebaert et al. . ATP and metabolite concentrations were measured by HPLC separation and UV-analysis as previously described .
Sample collection during the intervention
Venous blood was collected from the antecubital vein by a 20 gauge intravenous catheter (Terumo-Europe NV, Leuven, Belgium), connected to a three-way stopcock (Discofix®, Braun Melsungen AG, Melsungen, Germany). Blood was collected into 4 mL EDTA tubes (Venosafe, Terumo-Europe NV) by inserting a 21 gauge multisample needle (Venoject Quick Fit, Terumo-Europe NV) into the membrane of a closing cone (IN-Stopper, Braun Melsungen AG) that was attached directly to the stopcock. The anticoagulant EDTA inhibits the extracellular hydrolysis of ATP by Ca2+- and Mg2+-activated enzymes such as plasma membrane-bound CD39 . To avoid clotting after each blood collection, approximately 1.5 mL of heparinized (50 I.E./mL) 0.9% saline was used to rinse the blood collection set-up. It was removed before the next blood collection.
Three baseline blood samples were collected at 30, 20 and 10 min before administration. Starting 30 min after pellet administration or 15 min after naso-duodenal administration, blood samples were collected every 15 min. Between 210 and 420 min (pellets) or 270 min (naso-duodenal tube) after administration, samples were collected every 30 min. Total volume collected per day was 92 mL.
After blood collection, the tubes were inverted three times and put on ice. Five hundred μL of blood was added to 500 μL ice-cold PCA (8% wt:v), vortex-mixed and frozen in liquid nitrogen. Untreated plasma samples (centrifugation at 3000 rpm, 10 min, 4°C) were collected for assessment of lithium release from the pellets. All samples were stored at -80°C awaiting analysis.
ATP measurement in whole blood by HPLC
Equipment, sample preparation and measurement conditions have been previously described and validated . Briefly, after thawing, the protein fraction was precipitated (12,000 g, 10 min, 4°C) and 40 μL 2 M K2CO3 in 6 M KOH was added to 650 μL supernatant to neutralize the pH. The resulting insoluble perchlorate was removed by centrifugation (12,000 g, 10 min, 4°C), and 40 μL supernatant was mixed with 160 μL 0.05 M phosphate buffer pH 6.0 in HPLC vials.
Lithium measurement in plasma
To investigate the timing of pellet disintegration, plasma concentrations of the lithium marker were measured using a modified Trapp protocol . Following thawing on ice, 50 μL plasma was vortex-mixed with 10 μL trichloroacetic acid (20% v:v) and centrifuged (14,000 rpm, 10 min) to precipitate the proteins. The supernatant was diluted 20 times in 0.1 M nitric acid, which also served as the blank. Two replicate measurements per sample were performed on a SpectrAA 400 graphite tube atomic absorption spectrophotometer (AAS) (Varian, Palo Alto, CA, USA) with a lithium hollow-cathode lamp, operated at 5 mA and a 1.0 nm slit. Peak height measurements at 670.8 nm wavelength were compared with values for standards of known concentrations (ranging from 2 to 10 ng/mL). Initially, 20 μL sample and 5 μL modifier solution (1.2 M NH4NO3) were injected into the top hole of the graphite tube. Then, fluids were evaporated at 95°C for 40 s and at 120°C for 10 s. The ash time was 15 s at 700°C, followed by atomization at 2300°C with a 3 s read time. If the obtained signal exceeded the standard concentration range (0–10 ng/mL), samples were diluted with blank and measured again.
The area under the concentration vs. time curve (AUC) was calculated using the linear trapezoidal rule from time zero until the last time point of sampling t (AUC0-t). C
were defined as the minimum and maximum observed concentrations, respectively. t
was the time at which C
was reached. AUC of the five conditions were compared and analyzed by paired-samples t-tests. A P-value < 0.05 was considered statistically significant. Analyses were performed with the SPSS software package version 16.0 for Windows.