Atenolol

A total of 180 male Swiss mice (Janvier Labs, France, 25–37 g, 5–7 weeks old) were used in this study. A single intraperitoneal (i.p.) injection of LPS (22 mg/kg, from Escherichia coli O55:B5, L2880, Merck, Darmstadt, Germany) was used to create our preclinical endotoxemic shock model. Mice were divided into different groups: the LPS group (n = 49); the LPS + atenolol group: a selective β1-adrenergic blocker (atenolol, 0.1 mg/kg i.p, AstraZeneca, Courbevoie, France) was injected 6 hours post LPS injection (n = 33); and the LPS + sivelestat group: a selective ET inhibitor (sivelestat, ab14618, Abcam, Cambridge, UK) was injected 4 hours (10 mg/kg, i.p.), 8 hours (20 mg/kg, i.p.), and 12 hours (20 mg/kg, i.p.) post LPS injection (n = 16). Control groups were also used for each condition: the saline group (NaCl 0.9%, i.p.; n = 56), the atenolol group (atenolol, 0,1 mg/kg i.p., 6 hours post saline injection; n = 14), and the sivelestat group (NaCl 0.9%, i.p. + 3 sivelestat injections at 4, 8, and 12 hours post saline injection; n = 12) (Supplemental Figure 1).
Twenty-two hours post LPS injection, the mice were used for gastrocnemius MEP recordings or harvested for tissue analysis. This delay is sufficient to induce a severe inflammatory response but does not cause the death of the animals which occurs from the twenty-fourth hour.

Atenolol (purity ≥98%; CAS 29122-68-7), metoprolol succinate (purity ≥98%; CAS 98418-47-4) and propranolol hydrochloride (purity 99%; CAS 318-98-9) were obtained from AstraZeneca (Alderley Park, UK). Diclofenac sodium salt (purity ≥98%; CAS 15307-79-6), phenylbutazone (purity ≥98%; CAS 50-33-9), carbamazepine (purity ≥98%, CAS 298-46-4) and diazepam (purity ≥98%; CAS 439-14-5) were purchased from Sigma-Aldrich (Poole, UK). All chemicals and reagents for tissue culture procedures were obtained from Life Technologies Ltd (Paisley, UK). Pharmaceuticals were prepared fresh on the day of exposure in solvent (DMSO) and diluted in Leibovitz’s L-15 medium (no serum or antibiotic addition) to a concentration of 200 μg L^-1 (0.2% DMSO). A final well concentration of 100 μg L^-1 (0.1% DMSO) for each pharmaceutical was used. Pharmaceuticals that carried a salt weight were accounted for when calculating exposure concentrations i.e. final well concentrations refer to parent chemical minus the counter ion.

Four model compounds were selected: Atenolol, Enalaprilat, ketoprofen, and metoprolol. Biopharmaceutical classification (BCS) and some physicochemical properties for the four drugs are summarized in Table 1. Four PEs with different mechanisms of action were selected: SDS (anionic surfactant), sodium caprate (fatty acid), chitosan (polysaccharide), and EDTA (chelating agent). Atenolol and metoprolol tartrate were provided by AstraZeneca AB (Mölndal, Sweden). Enalaprilat, ketoprofen, sodium caprate, SDS, bovine albumin (A2153), EDTA, and inactin (thiobutabarbital) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium phosphate dibasic dihydrate (Na₂HPO₄·2H₂O), potassium dihydrogen phosphate (KH₂PO₄), sodium hydroxide (NaOH), and sodium chloride (NaCl) were purchased from Merck KGaA (Darmstadt, Germany). ⁵¹Cr-EDTA was purchased from PerkinElmer Life Sciences (Boston, MA, USA). Chitosan hydrochloride (molecular mass 40-300 kDa, degree of acetylation 8.8%) was purchased from Kraeber and Co GmbH (Ellerbek, Germany). Parecoxib (dynastat) was obtained from Apoteket AB, Uppsala, Sweden.