Pharmacodynamic properties for inhibition of cAMP- and cGMP elimination by pentoxifylline remain unaltered in vitro during hypothermia

Pentoxifylline (Sigma-Aldrich, St. Louis, MO, USA) was used in all experiments. Detailed methods for assessing intracellular access, enzyme inhibition and cellular efflux were first described in earlier publications [14, 15].

Temperatures

According to the European Resuscitation Council, hypothermia is divided into mild (35–32 °C), moderate (32–28 °C) and severe (below 28 °C) [19]. To get a broad view of the pharmacodynamics of pentoxifylline, we included several temperatures ranging from normothermia through all stages of hypothermia. The included temperatures were 37 °C, 34 °C, 32 °C, 28 °C, 24 °C and 20 °C.

Intracellular access

A review of the literature was performed to get an estimate of relevant therapeutic concentration of pentoxifylline. We searched PubMed with the following mesh terms: (Pentoxifylline) AND (intravenous) AND (plasma concentration) OR (serum concentration). The articles had to report adult human data and cardiovascular condition to be considered. Pentoxifylline was incubated at a final concentration of 100 µM as this corresponded to the highest concentrations found in the review [14, 15].

Blood was provided by Blodbanken UNN (Department of Immunohematology and Transfusion Medicine, University Hospital of North Norway) where all participants (n = 18) were pre-screened and only admitted as donors if they were healthy. Each parallel only included blood from one donor. Experiments were initiated by washing and centrifuging recently (< 24 h) drawn EDTA-blood 3 times with Krebs–Ringer-Phosphate-Buffer containing glucose (KRPB/G, pH ~ 7,4). Final Hct was of 0.40 in the incubate solution. Blood suspension was added to tubes containing 50 µL of either pentoxifylline or MQ-water (negative control). Each experiment contained triplicates of both drugs and control, and 3 experiments at each temperature were conducted – in total 9 parallels. The reactions were stopped after 30 min by putting the tubes on ice and adding 4 mL ice cold KRPB/G. The solutions were washed and centrifugated 3 times with ice cold KRPB/G. 50 µL of the remaining solution was then added to Eppendorf tubes along with 50 µL internal standard (IS), containing [13C,2H3]-Pentoxifylline, 250 nM (Alsachim, Illkirch Graffenstaden, France). 5 samples contained 50 µL known concentrations of pentoxifylline, and 50 µL IS, and served as controls for accurate analysis. All samples were added 200 mL 0.1 M ZnSO4, to lyse the erythrocytes, and then centrifugated. 30 µL was taken from Eppendorf tubes for measurements of protein concentration before adding 500 µL acetonitrile. 100 µL from each tube was collected for analysis using mass spectrometry (MS) [14, 15].

Enzyme inhibition

Assessment of pentoxifyllines ability to inhibit the phosphodiesterase enzymes were performed by incubating cAMP or cGMP with seven different concentrations of pentoxifylline. The concentrations were increasing by a factor of 10 ranging from 1.00 nM to 1.00 mM. For PDE3 assessment, the incubation solution included cAMP and for PDE5 assessment it included cGMP. Experiments were performed in triplicates each day at three separate days - in total 9 parallels. The reaction was started by adding either a solution containing 0.016 units/µg protein of PDE3 (Abcam, Cambridge UK), or 0.022 units/µg protein of PDE5 (Sigma-Aldrich, St. Louis, USA), to the Eppendorf tubes. Control samples were free of drug and was either with or without PDE3 or PDE5. This was done to assure that only the relevant PDE was responsible for breakdown of the cyclic nucleotide, as no other enzyme nor cellular material was added to the incubations. The incubation time was 30 min. Reaction was stopped by adding methanol to the tubes. Internal standard of cGMP/GMP or cAMP/AMP (Sigma-Aldrich, St. Louis, MO, USA, Germany and Toronto Research Chemicals Inc., Ontario, Canada) were added to each sample. 5 samples contained only known concentrations of cGMP/GMP or cAMP/AMP and served as calibrators. Samples were analyzed for cGMP/GMP and cAMP/AMP content, using MS [14, 15].

Cellular efflux inhibition

Cellular efflux was estimated with inside-out vesicles (IOV)s where erythrocytes from healthy, human donors were sampled. Donors were pre-screened and only admitted as donors by Blodbanken UNN (Department of Immunohematology and Transfusion Medicine, University Hospital of North Norway) if they were healthy. The erythrocytes were separated from plasma by centrifugation and washed. Inside-out vesicles were prepared according to Orvoll et al. [20] with minor modifications. Percentage IOV was verified using acetylcholinesterase accessibility test [21]. Batches of IOVs used in the parallels were made 8 times, including blood from a total of 35 healthy donors [14, 15].

IOVs were then incubated with or without 2 mM ATP and 7 different concentrations of pentoxifylline increasing by a factor of 10 ranging from 1.00 nM to 1.00 mM. The incubation solutions also included radioactive labeled [3H]-cGMP or [3H]-cAMP (Perkin Elmer, Boston, MA, USA), at a concentration of respectively 2 µM and 20 µM. [3H]-cAMP was used to assess ABCC4-inhibition and [3H]-cGMP for ABCC5-inhibition. The assays were performed in triplicates at 3 different days: In total 9 parallels were performed to calculate results for each concentration of pentoxifylline at all temperatures. Incubation time of 60 min was chosen to ensure sufficient quality of the samples for each parallel. The transport was stopped by adding ice cold buffer. The IOVs were then filtered through a nitrocellulose membrane (Bio-Rad Laboratories, Feldkirchen Germany), and the membrane was dried. The dried membranes were later added scintillation fluid and radioactivity was measured using a Packard TopCount NXT (Packard, Downers Grove, IL, USA) [14, 15].

Mass spectrometry (MS) analysis

Quantification of cAMP/AMP, cGMP/GMP and pentoxifylline in PDE- and intracellular access experiments were performed with liquid chromatography tandem mass spectrometry (LC–MS/MS). Preparation of samples for LC–MS/MS-analysis is described in paragraphs above. The method was found to be linear from 0.2 nM to at least 2000 nM (r2 > 0.998) for cAMP, cGMP and AMP. For GMP the method was linear from 2 nM to at least 2000 nM (r2 > 0.998), and 10 nM to at least 5000 nM for pentoxifylline (r2 > 0.998). Lower limit of quantification (LLOQ) was found to be 0.2 nM for cAMP, cGMP and AMP, 2 nM for GMP and 10 nM for pentoxifylline (2 µl injection volume) [14, 15].

Data analysis

Analysis and graph production were performed in SigmaPlot 14.0 (Systat Software, San Jose, CA, USA.). Ability to inhibit the different elimination pathways, enzymes and efflux pumps, were calculated as IC50-values, as according to Chou [22]. Ki-values were obtained by using the methods described by Cheng and Prustoff [23]. Measurement of intracellular concentrations of drugs were adjusted for protein concentrations in each sample. The incubation concentrations were also adjusted for protein concentration in each sample to evaluate the access in percentage. Analysis of variance (ANOVA) with Holm-Sidak multiple comparison post-hoc test was performed to evaluate changes in IC50-values compared to baseline (37 °C) for all elimination pathways. The same analysis was performed for intracellular concentrations of pentoxifylline during hypothermia compared to baseline (37 °C). When data was not normally distributed ANOVA on ranks and Dunn`s post hoc test was performed. Data are presented as mean ± standard error of the mean (SEM). p-values were considered significant when < 0.05.

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