Hyperbilirubinemia is a well-known condition in the clinical setting; however, the causes of elevated serum bilirubin are diverse, as are the clinical ramifications of this condition. For example, diagnoses of individuals vary depending on whether they exhibit an unconjugated or conjugated hyperbilirubinemia. Diagnoses can include conditions of disordered bilirubin metabolism (Gilbert's, Crigler-Najjar, Rotor, or Dubin-Johnson syndromes) or an acquired disease, including alcoholic/non-alcoholic fatty liver disease, hepatotropic hepatitis, cirrhosis, or hepato-biliary malignancy. Assessment of bilirubin concentrations is typically conducted as part of routine liver function testing. Mildly elevated total bilirubin with normal serum activities of liver transaminases, biliary damage markers, and red blood cell counts, however, may indicate the presence of Gilbert's syndrome (GS), a benign condition that is present in $5-10% of the population. In this case, mildly elevated unconjugated bilirubin in GS is strongly associated with "reduced" prevalence of chronic diseases, particularly cardiovascular diseases (CVD) and type 2 diabetes mellitus (and associated risk factors), as well as CVD-related and all-cause mortality. These reports challenge the dogma that bilirubin is simply a potentially neurotoxic by-product of heme catabolism and emphasize the importance of understanding its potential beneficial physiologic and detrimental pathophysiologic effects, in order to appropriately consider bilirubin test results within the clinical laboratory setting. With this information, we hope to improve the understanding of disorders of bilirubin metabolism, emphasize the diagnostic importance of these conditions, and outline the potential impact GS may have on resistance to disease.

This clinical trial (ACTRN12619001296123) investigated the impact of silymarin (Legalon®) on circulating bilirubin concentration, lipid status, systemic inflammation, and antioxidant status. The study design was a randomized, placebo‐controlled, single‐blind crossover trial of healthy men (18‐65 years), conducted at Griffith University, Gold Coast, Australia. Participants were recruited from Griffith University and were randomized to silymarin (140 mg silymarin capsules thrice daily) or placebo (3 capsules containing mannitol taken daily) for 14 days followed by a ≥4‐week washout and crossover to the other arm. The main outcomes were whether silymarin treatment would increase serum bilirubin concentration by >0.29 mg/dL, change serum lipid status (cholesterol and triglycerides), inflammation (c‐reactive protein), and antioxidant capacity (ferric reducing ability of plasma) compared with baseline. Silymarin consumption (n = 17) did not affect serum concentrations of unconjugated bilirubin (0.73 versus 0.67 mg/dL, P = .79), cholesterol (185 versus 189 mg/dL, P = .19), triglycerides (94.2 versus 92.3 mg/dL, P = .79), c‐reactive protein (0.17 versus 0.09 mg/dL, P = .23), or antioxidant status (6.61 versus 6.67 mg Fe2+/dL, P = .40). These findings challenge previous reports and manufacturer claims of hyperbilirubinemia following silymarin treatment and are critical to guiding researchers toward an effective means to mildly elevate bilirubin, which evidence suggests could protect from cardiovascular disease.

Biliverdin (BV) possesses antioxidant and anti-inflammatory properties, with previous reports identifying protection against oxidant and inflammatory injury in animal models. Recent reports indicate that intra-duodenal administration of BV results in the formation of an uncharacterised metabolite, which is potently absorbed into the blood and excreted into the bile. This compound may be responsible for protection against inflammatory responses. This study aimed to identify novel, enterally-derived BV metabolites and determine the source of their metabolic transformation. Rat duodena and bacterial cultures of Citrobacter youngae were treated with BV and subsequently analysed via high performance liquid chromatography/high resolution tandem mass spectrometry to identify and characterise metabolites of BV. A highly abundant metabolite was detected in duodenal wash and bacterial culture supernatants with a 663.215 m/z (3 ppm mass accuracy) and a composition of C 33 N 4 O 9 H 36 S, which conformed to the predicted structure of bilirubin-10-sulfonate (BRS) and possessed a λ max of 440 nm. Bilirubin-10-sulfonate was then synthesized for comparative LCMS/MS analysis and matched with that of the biologically formed BV metabolite. This report confirms the formation of a previously undocumented metabolite of BV in mammals, indicating that a new metabolic pathway likely exists for BV metabolism requiring enteric bacteria, Citrobacter youngae . These data may have important implications with regard to understanding and harnessing the therapeutic efficacy of oral BV administration.

Background: Biliverdin, a by-product of haem catabolism, possesses potent endogenous antioxidant and anti-inflammatory properties. Bilirubin-C10-sulfonate (BRS), an active metabolite formed after enteral administration of BV in the rat, also possess antioxidant properties. Therefore, we investigated the anti-inflammatory and antioxidant activity of BV and BRS in an in vivo model of monosodium urate induced sterile inflammation.Methods: Subcutaneous air pouches were created on the dorsal flanks of Wistar rats (10-12 weeks of age). Prior to stimulation of the 6-day old pouch with monosodium urate (25 mg), groups were pre-treated with intraperitoneal BRS (27 mg/kg) and BV (27 mg/kg). Total and differential leukocyte counts were determined in pouch fluid aspirate at 1, 6, 12, 24 and 48 h after monosodium urate stimulation. Biliverdin (BV), BRS and unconjugated bilirubin (UCB) concentrations in the serum and pouch fluid were quantified using liquid chromatographymass spectrometry. Pouch fluid cytokine concentrations (IL-1β, IL-1⍺, TNF-⍺, IL-17A, IL-12, GM-CSF, IL-33, IFN-, IL-18, IL-10, MCP-1, CXCL-1 and IL-6) were assessed after 6 h. In addition, 24 h protein carbonyl and chloramine concentrations were assessed in pouch fluid using ELISA and spectrophotometry, respectively.Results: BRS and BV significantly (p < 0.05) inhibited leukocyte (total, neutrophil and macrophage) infiltration into the pouch fluid from 6 to 48 h. For example, after 6 h neutrophil counts decreased following BRS (0.32 ± 0.11 × 10 6 cells mL -1 ) and BV (0.17 ± 0.03 × 10 6 cells mL -1 ) compared to MSU only (3.51 ± 1.07 × 10 6 cells mL -1 ). Both BV and BRS significantly (p < 0.05) reduced pouch GM-CSF (

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Human papilloma virus (HPV) is the main culprit in cervical cancers. Although the HPV vaccine is now available, the slow and gradual process for HPV cancers to form means little will change, even for vaccinated individuals. This warrants the development of new therapeutic strategies in both the newly diagnosed and recurrent patients. We have previously shown that Alisertib (MLN8237), an Aurora A kinase inhibitor, potently and selectively kills HPV-positive cervical cancer cells. However, Alisertib is known for its unfavorable side effects when administered systemically. A targeted delivery approach is therefore warranted. The topical delivery of drugs to the cervix for the treatment of cervical cancer is an underexplored area of research that has the potential to significantly improve therapeutic outcome. Here, we design a novel topical drug delivery system for localized delivery in the vaginal tract using intravaginal silicone rings loaded with Alisertib. We assessed the suitability of the drug for the application and delivery method and develop a high-performance liquid chromatography method, then show that the vaginal rings were effective at releasing Alisertib over an extended period of time. Furthermore, we showed that Alisertib-loaded vaginal rings did not induce overt inflammation in the mouse vaginal tract. Our work has major translational implications for the future development of vaginal ring devices for the topical treatment of cervical cancer.

HPLC MethodBriefly, HPLC-PDA analysis was performed using a Shimadzu Prominence HPLC system consisting of an online degassing unit (DGU20A5R), solvent delivery unit (LC-20AT), autosampler (SIL-20 AC), column oven (CTO-20) and photo diode-array (SPD-M20A), connected in series. Separation was achieved via reverse phase C18 column (GraceSmart™ RP-C18, 150mm x 4.6mm, 3µm; Grace Davidson, Australia), which was preceded by a guard column (GraceSmart™ RP-C18, 3µm; Grace Davidson, Australia) and an UltraLine HPLC in-line filter (Restek, 0.5µm; Shimadzu, Sydney, Australia), respectively. The column oven and autosampler were set to 45˚C and 4˚C, respectively, with an initial flow rate of 1.6mL•min −1 . Starting mobile phase consisted of 20% organic B (100% HPLC grade MeOH) and 80% aqueous A (10mM NH4OAc in 25% HPLC grade MeOH and 75% Milli-Q purified H2O). A linear gradient was applied, reaching 90% B at 4.5 minutes, remaining until 7 minutes. From 7 to 11.5 minutes, the mobile phase was set at 20% B. To assist equilibration, flow rate remained at 1.6mL•min −1 until 6 minutes where it increased to 2.3mL•min −1 at 6.35 minutes, remaining until 8.5 minutes. From 8.5 to 9.5 minutes, the flow rate decreased to 1.6mL•min −1 and remained until 11.5 minutes. The total run time including adequate column re-equilibration was 11.5 minutes. For UCB and BRT extraction, 160μL of extractant (1:4 DMSO:MeOH) was added to 40μL of PPM sample, mixed via vortex for 10 seconds before centrifugation (21 500RCF; 10 minutes) to pellet protein. The supernatant was diluted (2:1) with Milli-Q H2O upon addition to a HPLC vial, prior to being placed in the autosampler where 40µL was injected for analysis. Commercially prepared BRT and UCB (Frontier Scientific Inc. Logan, UT, USA) served as external standards with retention times of 4.1 and 5.1 minutes respectively. Extraction efficiencies of these compounds from PPM was 95% and 91% for BRT and UCB, with standard curves indicating great linearity over concentration ranges of 1.5625µM -100µM (r 2 =0.9993) and 0.15625µM -10µM (r 2 =0.9995)

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