The Pharmacokinetics and Anti-inflammatory Properties of Bile Pigments and their Derivatives in the Rat

Abstract

Background: Bile pigments (BPs) are endogenously produced molecules including biliverdin (BV), unconjugated bilirubin (UCB) and bilirubin glucuronides, and have historically been referred to as waste products of haem catabolism. New metabolic pathways of BP metabolism, including the formation of bilirubin-C10-sulfonate (BRS), have been discovered, which suggest a physiologically important role for BPs in mammals. For example, BPs possess potent, antioxidant and anti-inflammatory properties which may protect against inflammatory diseases such as cardiovascular disease, diabetes, ischemia/reperfusion injury, rheumatoid arthritis and ulcerative colitis. To understand the therapeutic utility of BPs however, the pharmacokinetics of these compounds must be investigated, followed by efficacy testing in models of inflammatory pathology. This thesis contains two experimental chapters, which aim to i) explore and compare the pharmacokinetics of BRS and BV in the rat and ii) to test the efficacy of intraperitoneal administration of BRS and BV in a rat pouch model of monosodium urate (MSU) crystal-induced sterile inflammation.

Methods: In the first experimental chapter, 10-12-week male Wistar rats were anaesthetised and BRS (n=5) and BV (n=5) were administered via intravenous (2.7 mg/kg), intraperitoneal (27 mg/kg) and intraduodenal (27 mg/kg) routes. Periodic blood samples were collected from the right jugular vein over 180 min. The concentration of BRS, BV and UCB were quantified in the serum using ultra-high-performance liquid chromatography coupled to mass spectrometry (UHPLC-MS) and pharmacokinetic parameters were calculated using a noncompartmental model to compare the pharmacokinetics of administered BRS and BV. The second experimental chapter involved inflating a subcutaneous air pouch (20 ml) on the dorsal flank of 10-12-week male Wistar rats to generate a pseudo-synovial capsule. On day 7, a sterile suspension of MSU crystals (25 mg in 5 mL saline) was introduced into the pouch to stimulate an acute inflammatory response, similar to that observed in gout. Groups were pre-treated (27 mg/kg, i.p.) with BRS (n=11) and BV (n=11) 30 min prior to MSU crystal infusion into the pouch. For comparison, an untreated group (MSU only, n=8) received an equivalent volume of i.p. PBS. Exudate was aspirated from the air pouch at 1, 6, 12, 24 and 48 hours. Total and differential leukocyte counts in the pouch fluid were determined using a haemocytometer and an exudate smear/rapid differential staining method, respectively. The concentration of inflammatory cytokines: IL-1⍺, IL-1β, TNF-⍺, IL-6, IL-10, IL-12, IL-17A IL-18, GM-CSF, CCL2, CXCL1 and IFN-γ were investigated in the 6-hour pouch fluid using a multiplexed bead-based flow cytometric immunoassay.

Results: After i.v. delivery, both pigments had similar tissue distribution (volume of distribution, Vd; BRS:0.0463±0.00820, BV:0.0560±0.00750 L/kg, P>0.05). However, BRS was less readily eliminated from the systemic circulation as indicated by its elimination half-life (BRS:190±52.1, BV:58.7±11.7 min, P<0.01) and area under the curve over 180 min (AUC180;BRS:7480±2059, BV:873 ±143 µMmin, P<0.0001), which were 3- and 8-fold greater than BV, respectively. Despite this, i.v. BV administration resulted in more pronounced increases in serum UCB (UCB AUC180; BRS:101±25.6, BV:246±30.1 µMmin, P<0.05). After i.p. delivery, the AUC180 (BRS:23300±2220, BV:2670±436 µMmin, P<0.0001) and peak concentrations (Cpeak; BRS:179±11.5, BV:22.1±2.94 µM) for BRS were considerably greater than those for BV. Moreover, the increase in serum UCB over 180 min was similar in both conditions (AUC180; BV:606±110, BRS:532±41.0 µMmin, P>0.05). After enteral administration, BRS had poor bioavailability (BA: 1.25 %, Cpeak 1.12 ±0.220 µM ) while BV was not bioavailable, and both treatments failed to increase serum UCB over 180 min.

Stimulation of the subcutaneous air pouch with MSU crystals resulted in a marked acute inflammatory response characterised by infiltration of leukocytes consisting of neutrophils, monocytes and lymphocytes. Both i.p. BRS and BV reduced (P<0.05) leukocyte infiltration across 48 hours. At 6 hours, GM-CSF concentrations in the pouch exudate were lower after BRS and BV treatment (MSU only:13.0±1.97, BRS:6.96±1.54, BV:5.83±1.21 pg/mL, both P<0.05 vs MSU only). Biliverdin additionally attenuated CCL2 (MSU only:17290±4500, BV:4270±2480 pg/mL, P<0.05) and IL-18 (MSU only:81.6±20.0, BV:25.7±8.15pg/mL, P<0.05) concentrations. Investigation of other cytokines after BRS and BV treatment showed no significant (P>0.05) differences compared to the MSU only group.

Conclusion: This study was the first to investigate the pharmacokinetics of BRS and anti-inflammatory properties of BRS and BV against MSU crystal-induced inflammation in the rat. BRS was retained within the circulation for longer after i.v. and i.p. administration, and therefore induced higher circulating concentrations, suggesting a potential advantage of BRS compared to BV, which was eliminated more rapidly. Against MSU crystal-induced inflammation, i.p pre-treatment with both pigments similarly inhibited leukocyte infiltration, however, BV exhibited a broader spectrum of inhibition of pro-inflammatory cytokines when compared to BRS. Despite this, exogenous delivery of BRS may induce elevations in BP concentrations over longer durations and therefore provide longer lasting antioxidant and anti-inflammatory properties which may prove clinically valuable for treatment of acute and/or chronic inflammatory diseases.