MSACL 2022 Abstract

Serum Bile Acid Profiling by UHPLC-MS/MS Predicts Cholesteric Pruritus Reduction in Maralixibat Treated Patients with Bile Salt Export Pump Deficiency

Xueheng Zhao (1), Pamela Vig (2), Cory Kostrub (2), Kenneth DR Setchell (1,3)
(1) Division of Pathology and Laboratory Medicine, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA; (2) Mirum Pharmaceuticals, Foster City, CA, USA; (3) College of Medicine, University of Cincinnati, Cincinnati, OH, USA

Kenneth Setchell, PhD (Presenter) Cincinnati Children’s Hospital Medical Center

Presenter Bio: Kenneth Setchell is Professor of Pediatrics and the Director of Clinical Mass Spectrometry in the Division of Pathology and Laboratory Medicine at the Cincinnati Children’s Hospital Medical Center and Department of Pediatrics of the University of Cincinnati College of Medicine. His expertise is in clinical and biomedical applications of mass spectrometry and allied techniques, primarily in small molecules and in the area of bile acids, steroids, sterols, lipids and small molecules with a focus on liver disease, gastroenterology and nutrition. Moving from the Medical Research Center’s Clinical Resarch Center in 1984 he established a mass spectrometry facility that provides clinical diagnostic testing, therapeutic drug monitoring and biomedical research support to investigators within CCHMC and the University of Cincinnati College of Medicine. This academic facility uniquely operates under GLP standards, is CAP/CLIA accredited, and consequently has provided analytical support for numerous Phase 1-3 clinical trials for the pharmaceutical industry, leading to 2 new drugs (Cholbam and Maralixibat) being FDA approved. He has published >430 peer-reviewed publications, is the inventor of 13 patents. He was awarded the 2004 Adolf Windaus Prize for research on bile acids following the discovery with mass spectrometry of 6 genetic defects in the cholesterol-bile acid synthetic pathway that cause liver disease in infants and children and is an internationally recognized center for the diagnosis of these defects. With the late James E. Heubi MD, they developed a successful therapy using oral cholic acid to reverse the disease in otherwise fatal forms of liver disease that was approved in 2015 by the FDA - the first drug to gain FDA approval in over 40 years for cholestatic liver disease. He is also recognized in the field of nutrition, specifically related to soy and isoflavones, having discovered the intestinally derived metabolite, S-(-)equol in human urine and making the association with soy food intake that has driven much of the interest in soy foods. He has received awards for his discoveries and in 2014 was listed by Thomson-Reuters as one of “The World’s Most Influential Scientific Minds” based on highly cited publications. He received the Distinguished Contributor Award 2016 at the Mass Spectrometry Applied to the Clinical Laboratory (MSACL) for lifetime contributions to the application of mass spectrometry to the clinical research. In 2023, named as one of the leading national and international medical researchers with 36,663 citations from 302 publications (from a total of >400 publications) with a 92 D-Index.

Relevant Financial Disclosures (within past 24 months, reported on Apr 05, 2025) Stock/Bonds Asklepion Pharmaceuticals; Aliveris srl Salary Mirum Pharmaceuticals; Travere Discussing Products? YES, presenter will present educational content related to products/services of one or more ineligible company(ies) listed above. Conflict mitigation IN PROCESS.

Background and Aims: Progressive familial intrahepatic cholestasis (PFIC) due to bile salt export pump (BSEP) deficiency is a distressing disease manifest by intractable pruritus, growth delay, and eventually culminating in liver failure. Bile acids have long been implicated in the pathogenesis of pruritus although the exact role they play is unclear. Maralixibat (MRX) is a nonabsorbable apical sodium-dependent bile acid (BA) transport inhibitor that interrupts the enterohepatic circulation of BAs. It was recently FDA approved for the treatment for pediatric cholestasis-associated pruritus because of is ability to reduce the accumulation of circulating and hepatic BAs. Not all patients respond to therapy for reasons that are unclear. We describe a targeted metabolomics approach to explore the association between the bile acid metabolome and cholestatic pruritus and for its potential to predict pruritus reduction in response to MRX treatment.

Methods: Total and individual serum BAs (sBA) including the major sub-species of TCA, TUDCA, TCDCA, TDCA, TLCA, GCA, GUDCA, GCDCA, GDCA, GLCA, CA, UDCA, CDCA, DCA, LCA and 7-hydroxy-4-cholesten-3-one (sterol-C4), a surrogate marker for BA synthesis, were measured using a validated ultraperformance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) assay. The internal standards, a cocktail of deuterium labeled standards were added to serum, calibrators and QC samples. sBA were extracted and separated on a Kinetex C18 (2.6 µm, 100 x 3.0 mm) column (Phenomenex, Torrance, CA) with gradient elution. Quantification of individual sBA was achieved by multiple reaction monitoring (MRM) of selected transitions. Total C24 bile acid concentrations were calculated from the sum of individual species. These assays were applied to the analysis of serum from 19 patients with genetically confirmed PFIC due to BSEP deficiency (open-label phase 2 INDIGO trial; NCT02057718) treated with MRX. Changes in the composition of sBA associated with pruritus improvement measured with the Itch Reported Outcome Observer (ItchRO[Obs]) score were monitored at intervals over 72 weeks, with pruritus responders, defined as ≥1 scale reduction, compared with nonresponders. Linear mixed model framework was also used to model longitudinal profiles of sBA and pruritus reduction over time.

Results: Of the 19 patients, 11 met the pruritus-response criteria, which correlated with a reduction from baseline in total sBA that was significantly greater than in nonresponders (p < 0.05) in the longitudinal data. Changes were also observed in the individual BA subspecies. Reductions in glycine and taurine-conjugated cholic acid (TCA) associated with pruritus reduction after MRX treatment and correlated with percentage ItchRO(Obs) score reduction (Pearson correlation coefficient: 0.58, 0.50, 0.53 for TCA, glycocholic acid, and total cholic acid [CA], respectively). A trend towards increased proportion of unconjugated BA in responders (9.84 ± 4.87%) was observed compared with nonresponders (0.61 ± 0.24%, p = 0.09). More importantly, longitudinal sBA profile changes, e.g. GUDCA, GCDCA, TCDCA significantly correlated with pruritus reduction (ꭕ2 =13.35, P<0.001; ꭕ2 =12.86, P<0.001; and ꭕ2 =19.50, P<0.001, respectively). Concomitant increases in serum sterol-C4 were observed in pruritus responders (ꭕ2 =4.70, P<0.05), consistent with the biological action of MRX in blocking the intestinal reabsorption of bile acids.

Conclusion: Targeted analysis of the bile acid metabolome by mass spectrometry revealed significant changes associated with pruritus response to MRX therapy in children with BSEP deficiency and measures of serum sterol-C4 further confirmed efficacy of the drug. These findings offer further interpretation on BA subspecies profile associated with reductions in cholestasis and pruritus, reinforcing data demonstrating that MRX is an effective pharmacological therapy for BSEP deficiency.