Maralixibat in progressive familial intrahepatic cholestasis (MARCH-PFIC): a multicentre, randomised, double-blind, placebo-controlled, phase 3 trial.

BACKGROUND: Progressive familial intrahepatic cholestasis (PFIC) is a group of autosomal recessive disorders, the most prevalent being BSEP deficiency, resulting in disrupted bile formation, cholestasis, and pruritus. Building on a previous phase 2 study, we aimed to evaluate the efficacy and safety of maralixibat-an ileal bile acid transporter inhibitor-in participants with all types of PFIC. METHODS: MARCH-PFIC was a multicentre, randomised, double-blind, placebo-controlled, phase 3 study conducted in 29 community and hospital centres across 16 countries in Europe, the Americas, and Asia. We recruited participants aged 1-17 years with PFIC with persistent pruritus (>6 months; average of ≥1·5 on morning Itch-Reported Outcome [Observer; ItchRO(Obs)] during the last 4 weeks of screening) and biochemical abnormalities or pathological evidence of progressive liver disease, or both. We defined three analysis cohorts. The BSEP (or primary) cohort included only those with biallelic, non-truncated BSEP deficiency without low or fluctuating serum bile acids or previous biliary surgery. The all-PFIC cohort combined the BSEP cohort with participants with biallelic FIC1, MDR3, TJP2, or MYO5B deficiencies without previous surgery but regardless of bile acids. The full cohort had no exclusions. Participants were randomly assigned (1:1) to receive oral maralixibat (starting dose 142·5 µg/kg, then escalated to 570 µg/kg) or placebo twice daily for 26 weeks. The primary endpoint was the mean change in average morning ItchRO(Obs) severity score between baseline and weeks 15-26 in the BSEP cohort. The key secondary efficacy endpoint was the mean change in total serum bile acids between baseline and the average of weeks 18, 22, and 26 in the BSEP cohort. Efficacy analyses were done in the intention-to-treat population (all those randomly assigned) and safety analyses were done in all participants who received at least one dose of study drug. This completed trial is registered with ClinicalTrials.gov, NCT03905330, and EudraCT, 2019-001211-22. FINDINGS: Between July 9, 2019, and March 4, 2022, 125 patients were screened, of whom 93 were randomly assigned to maralixibat (n=47; 14 in the BSEP cohort and 33 in the all-PFIC cohort) or placebo (n=46; 17 in the BSEP cohort and 31 in the all-PFIC cohort), received at least one dose of study drug, and were included in the intention-to-treat and safety populations. The median age was 3·0 years (IQR 2·0-7·0) and 51 (55%) of 93 participants were female and 42 (45%) were male. In the BSEP cohort, least-squares mean change from baseline in morning ItchRO(Obs) was -1·7 (95% CI -2·3 to -1·2) with maralixibat versus -0·6 (-1·1 to -0·1) with placebo, with a significant between-group difference of -1·1 (95% CI -1·8 to -0·3; p=0·0063). Least-squares mean change from baseline in total serum bile acids was -176 µmol/L (95% CI -257 to -94) for maralixibat versus 11 µmol/L (-58 to 80) for placebo, also representing a significant difference of -187 µmol/L (95% CI -293 to -80; p=0·0013). The most common adverse event was diarrhoea (27 [57%] of 47 patients on maralixibat vs nine [20%] of 46 patients on placebo; all mild or moderate and mostly transient). There were five (11%) participants with serious treatment-emergent adverse events in the maralixibat group versus three (7%) in the placebo group. No treatment-related deaths occurred. INTERPRETATION: Maralixibat improved pruritus and predictors of native liver survival in PFIC (eg, serum bile acids). Maralixibat represents a non-surgical, pharmacological option to interrupt the enterohepatic circulation and improve the standard of care in patients with PFIC. FUNDING: Mirum Pharmaceuticals.

Predictors of 6-year event-free survival in Alagille syndrome patients treated with maralixibat, an ileal bile acid transporter inhibitor.

BACKGROUND AND AIMS: Refractory pruritus and other complications of cholestasis are indications for liver transplantation (LT) in patients with Alagille syndrome (ALGS). We evaluated predictors of event-free survival and transplant-free survival in patients with ALGS treated with maralixibat (MRX), an ileal bile acid transporter inhibitor. APPROACH AND RESULTS: We assessed patients with ALGS from 3 clinical trials of MRX with up to 6 years of follow-up. Event-free survival was defined as the absence of LT, surgical biliary diversion, hepatic decompensation, or death; transplant-free survival was the absence of LT or death. Forty-three potential predictors were evaluated, including age, pruritus (ItchRO[Obs] 0-4 scale), biochemistries, platelets, and serum bile acids. Harrell's concordance statistic assessed goodness-of-fit, and then, Cox proportional hazard models confirmed the statistical significance of the predictors identified. A further analysis was performed to identify cutoffs using a grid search. Seventy-six individuals met the criteria of receiving MRX for ≥48 weeks with laboratory values available at week 48 (W48). The median duration of MRX was 4.7 years (IQR: 1.6-5.8); 16 had events (10 LT, 3 decompensation, 2 death, and 1 surgical biliary diversion). The 6-year event-free survival improved with a clinically meaningful >1-point ItchRO(Obs) reduction from baseline to W48 (88% vs. 57%; p = 0.005), W48 bilirubin < 6.5 mg/dL (90% vs. 43%; p < 0.0001), and W48 serum bile acid < 200 µmol/L (85% vs. 49%; p = 0.001). These parameters were also predictive of 6-year transplant-free survival. CONCLUSIONS: Improvement in pruritus by 48 weeks, and lower W48 bilirubin and serum bile acid levels were associated with fewer events. These data may help identify potential markers of disease progression for ALGS patients treated with MRX.

Maralixibat Treatment Response in Alagille Syndrome is Associated with Improved Health-Related Quality of Life.

OBJECTIVE: The objective of this study was to assess the impact of treatment response to the ileal bile acid transporter inhibitor maralixibat on health-related quality of life (HRQoL) in children with Alagille syndrome. STUDY DESIGN: This analysis used data from the ICONIC trial, a phase 2 study with a 4-week double-blind, placebo-controlled, randomized drug withdrawal period in children with Alagille syndrome with moderate-to-severe pruritus. Clinically meaningful treatment response to maralixibat was defined a priori as a ≥1-point reduction in the Itch-Reported Outcome (Observer) score, from baseline to week 48. HRQoL was assessed using the Pediatric Quality of Life Inventory Generic Core, Family Impact, and Multidimensional Fatigue scale scores, which were collected via the caregiver. The minimal clinically important difference for HRQoL ranged from 4 to 5 points, depending on the scale. RESULTS: Twenty of the 27 patients (74%) included in this analysis achieved an Itch-Reported Outcome (Observer) treatment response at week 48. The mean (SD) change in Multidimensional Fatigue score was +25.8 (23.0) for responders vs -3.1 (19.8) for nonresponders (P = .03). Smaller and non-statistically significant mean changes were observed for the Pediatric Quality of Life Inventory Generic Core and Family Impact scores. Controlling for baseline Family Impact score, responders' Family Impact scores increased an average of 16.9 points over 48 weeks compared with non-responders (P = .05). Smaller and non-statistically significant point estimates were observed for the Pediatric Quality of Life Inventory Generic Core and Multidimensional Fatigue scores. CONCLUSION: The significant improvements in pruritus seen with maralixibat at week 48 of the ICONIC study are clinically meaningful and are associated with improved HRQoL. TRIAL REGISTRATION: ClinicalTrials.gov: NCT02160782.

Efficacy and safety of maralixibat treatment in patients with Alagille syndrome and cholestatic pruritus (ICONIC): a randomised phase 2 study.

BACKGROUND: Alagille syndrome is a rare genetic disease that often presents with severe cholestasis and pruritus. There are no approved drugs for management. Maralixibat, an apical, sodium-dependent, bile acid transport inhibitor, prevents enterohepatic bile acid recirculation. We evaluated the safety and efficacy of maralixibat for children with cholestasis in Alagille syndrome. METHODS: ICONIC was a placebo-controlled, randomised withdrawal period (RWD), phase 2b study with open-label extension in children (aged 1-18 years) with Alagille syndrome (NCT02160782). Eligible participants had more than three times the normal serum bile acid (sBA) levels and intractable pruritus. After 18 weeks of maralixibat 380 µg/kg once per day, participants were randomly assigned (1:1) to continue maralixibat or receive placebo for 4 weeks. Subsequently, all participants received open-label maralixibat until week 48. During the long-term extension (204 weeks reported), doses were increased up to 380 µg/kg twice per day. The primary endpoint was the mean sBA change during the RWD in participants with at least 50% sBA reduction by week 18. Cholestastic pruritus was assessed using observer-rated, patient-rated, and clinician-rated 0-4 scales. The safety population was defined as all participants who had received at least one dose of maralixibat. This trial was registered with ClinicalTrials.gov, NCT02160782, and is closed to recruitment. FINDINGS: Between Oct 28, 2014, and Aug 14, 2015, 31 participants (mean age 5·4 years [SD 4·25]) were enrolled and 28 analysed at week 48. Of the 29 participants who entered the randomised drug withdrawal period, ten (34%) were female and 19 (66%) were male. In the RWD, participants switched to placebo had significant increases in sBA (94 µmol/L, 95% CI 23 to 164) and pruritus (1·7 points, 95% CI 1·2 to 2·2), whereas participants who continued maralixibat maintained treatment effect. This study met the primary endpoint (least square mean difference -117 µmol/L, 95% CI -232 to -2). From baseline to week 48, sBA (-96 µmol/L, -162 to -31) and pruritus (-1·6 pts, -2·1 to -1·1) improved. In participants who continued to week 204 (n=15) all improvements were maintained. Maralixibat was generally safe and well tolerated throughout. The most frequent adverse events were gastrointestinal related. Most adverse events were self-limiting in nature and mild-to-moderate in severity. INTERPRETATION: In children with Alagille syndrome, maralixibat is, to our knowledge, the first agent to show durable and clinically meaningful improvements in cholestasis. Maralixibat might represent a new treatment paradigm for chronic cholestasis in Alagille syndrome. FUNDING: Mirum Pharmaceuticals.

Comparison of ADAPT, FIB-4 and APRI as non-invasive predictors of liver fibrosis and NASH within the CENTAUR screening population.

BACKGROUND & AIMS: The development of accurate non-invasive tests to detect and measure the extent of fibrosis and disease activity in patients with non-alcoholic steatohepatitis (NASH) - the progressive phenotype of non-alcoholic fatty liver disease (NAFLD) - is of great clinical importance. Herein, we aimed to validate the performance of PRO-C3 and ADAPT for the detection of moderate/severe fibrosis within the CENTAUR screening population. METHODS: PRO-C3 was assessed in plasma from the screening population of the phase IIb CENTAUR study (NCT02217475) in adults with NASH and liver fibrosis. The relation between PRO-C3 and histologic features of NASH was evaluated, as well as the demographics of patients with high and low levels of PRO-C3. The diagnostic ability of PRO-C3, as a standalone marker or incorporated into ADAPT, to identify patients with F≥2 and NASH was estimated using receiver-operating characteristic analysis and logistic regression models. RESULTS: A total of 517 individuals with matched biopsy and PRO-C3 measurements were included. Patients with PRO-C3 levels ≥20.2 ng/ml showed increased levels of insulin, HOMA-IR, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase, and platelet count compared to patients with low PRO-C3 (p <0.05). PRO-C3 increased stepwise with increasing liver fibrosis, lobular inflammation, hepatocyte ballooning, steatosis, and NAFLD activity score (p <0.05), and could distinguish between NAFL and NASH (p <0.0001). PRO-C3 was independently associated with fibrosis and NASH when adjusted for clinical confounders. ADAPT outperformed Fibrosis-4, AST-to-platelet ratio index, and AST/ALT ratio as a predictor of advanced fibrosis and NASH (p <0.001). CONCLUSION: PRO-C3 was associated with NAFLD activity score and fibrosis. ADAPT outperformed other non-invasive scores for detecting NASH. These data support the use of PRO-C3 and ADAPT as diagnostic tools to identify patients with NASH eligible for inclusion in clinical trials. CLINICAL TRIAL NUMBER: NCT02217475 LAY SUMMARY: PRO-C3 is a serological biomarker associated with liver disease activity and fibrosis. Its performance for the detection of disease activity and fibrosis is improved when it is incorporated into the ADAPT score. Herein, we showed that ADAPT was better at selecting patients with non-alcoholic steatohepatitis for inclusion in clinical trials than other non-invasive scores.

Pharmacological Inhibition of CCR2/5 Signaling Prevents and Reverses Alcohol-Induced Liver Damage, Steatosis, and Inflammation in Mice.

Kupffer cell and macrophage (MØ) activation contributes to steatosis, inflammation, and fibrosis in alcoholic liver disease (ALD). We found increased frequency of MØ, T cells, and expression of C-C chemokine receptor type 2 (Ccr2) and C-C chemokine receptor type 5 (Ccr5) in the livers of patients with ALD, and increased circulating chemokines, C-C chemokine ligand types 2 (CCL2), and C-C chemokine ligand types 5 (CCL5) in patients with alcoholic hepatitis. We hypothesized that inhibition of CCL2 signaling with the dual CCR2/5 inhibitor, cenicriviroc (CVC), would attenuate ALD. In a mouse model of ALD, liver injury (alanine aminotransferase [ALT]) and steatosis were prevented by CVC whether administered as "prevention" throughout the alcohol feeding or as "treatment" started after the development of ALD. Alcohol-induced increases in early liver fibrosis markers (sirius red, hydroxyproline, and collagen-1) were normalized by both modes of CVC administration. We found that prevention and treatment with CVC reversed alcohol-related increases in liver mRNA and protein expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, and CCL2. CVC administration regimens prevented the increase in infiltrating MØ (F4/80lo CD11bhi ) and reduced proinflammatory Ly6Chi MØ in livers of alcohol-fed mice. CVC increased liver T-cell numbers and attenuated Il-2 expression without an effect on CD69+ or CD25+ T-cell expression. In vitro, CVC inhibited CCL2-induced increases in hepatocyte fatty acid synthase (Fasn) and adipose differentiation-related protein (Adrp), whereas it augmented acyl-coenzyme A oxidase 1 (Acox-1), proliferator-activated receptor gamma co-activator alpha (Pgc1α) and uncoupling protein 2 expression, suggesting mechanisms for attenuated hepatocyte steatosis. We found that CCL2 and CCL5 sensitized hepatocytes to lipopolysaccharide-induced liver injury (TNF-α, ALT, and lactate dehydrogenase release). Alcohol feeding induced apoptosis (poly ADP-ribose polymerase [PARP] and caspase-3 [CASP-3] cleavage) and pyroptosis (gasdermin D [GSDMD] cleavage) in livers, and CVC prevented both of these forms of cell death. Conclusion: Together, our data demonstrate preclinical evidence for CCR2/CCR5 inhibition with CVC as a potent intervention to ameliorate alcohol-induced steatohepatitis and liver damage.

The Critical Role of Chemokine (C-C Motif) Receptor 2-Positive Monocytes in Autoimmune Cholangitis.

The therapy of primary biliary cholangitis (PBC) has lagged behind other autoimmune diseases despite significant improvements in our understanding of both immunological and molecular events that lead to loss of tolerance to the E2 component of pyruvate dehydrogenase, the immunodominant autoepitope of PBC. It is well known that Ly6Chi monocytes are innate immune cells infiltrating inflammatory sites that are dependent on the expression of C-C motif chemokine receptor 2 (CCR2) for emigration from bone marrow. Importantly, humans with PBC have a circulating monocyte pro-inflammatory phenotype with macrophage accumulation in portal tracts. We have taken advantage of an inducible chemical xenobiotic model of PBC and recapitulated the massive infiltration of monocytes to portal areas. To determine the clinical significance, we immunized both CCR2-deficient mice and controls with 2OA-BSA and noted that CCR2 deficiency is protective for the development of autoimmune cholangitis. Importantly, because of the therapeutic potential, we focused on inhibiting monocyte infiltration through the use of cenicriviroc (CVC), a dual chemokine receptor CCR2/CCR5 antagonist shown to be safe in human trials. Importantly, treatment with CVC resulted in amelioration of all aspects of disease severity including serum total bile acids, histological severity score, and fibrosis stage. In conclusion, our results indicate a major role for Ly6Chi monocytes and for CCR2 in PBC pathogenesis and suggest that inhibition of this axis by CVC should be explored in humans through the use of clinical trials.

Macrophages contribute to the pathogenesis of sclerosing cholangitis in mice.

BACKGROUND & AIMS: Macrophages contribute to liver disease, but their role in cholestatic liver injury, including primary sclerosing cholangitis (PSC), is unclear. We tested the hypothesis that macrophages contribute to the pathogenesis of, and are therapeutic targets for, PSC. METHODS: Immune cell profile, hepatic macrophage number, localization and polarization, fibrosis, and serum markers of liver injury and cholestasis were measured in an acute (intrabiliary injection of the inhibitor of apoptosis antagonist BV6) and chronic (Mdr2-/- mice) mouse model of sclerosing cholangitis (SC). Selected observations were confirmed in liver specimens from patients with PSC. Because of the known role of the CCR2/CCL2 axis in monocyte/macrophage chemotaxis, therapeutic effects of the CCR2/5 antagonist cenicriviroc (CVC), or genetic deletion of CCR2 (Ccr2-/- mice) were determined in BV6-injected mice. RESULTS: We found increased peribiliary pro-inflammatory (M1-like) and alternatively-activated (M2-like) monocyte-derived macrophages in PSC compared to normal livers. In both SC models, genetic profiling of liver immune cells identified a predominance of monocytes/macrophages; immunohistochemistry confirmed peribiliary monocyte-derived macrophage recruitment (M1>M2-polarized), which paralleled injury onset and was reversed upon resolution in acute SC mice. PSC, senescent and BV6-treated human cholangiocytes released monocyte chemoattractants (CCL2, IL-8) and macrophage-activating factors in vitro. Pharmacological inhibition of monocyte recruitment by CVC treatment or CCR2 genetic deletion attenuated macrophage accumulation, liver injury and fibrosis in acute SC. CONCLUSIONS: Peribiliary recruited macrophages are a feature of both PSC and acute and chronic murine SC models. Pharmacologic and genetic inhibition of peribiliary macrophage recruitment decreases liver injury and fibrosis in mouse SC. These observations suggest monocyte-derived macrophages contribute to the development of SC in mice and in PSC pathogenesis, and support their potential as a therapeutic target. LAY SUMMARY: Primary sclerosing cholangitis (PSC) is an inflammatory liver disease which often progresses to liver failure. The cause of the disease is unclear and therapeutic options are limited. Therefore, we explored the role of white blood cells termed macrophages in PSC given their frequent contribution to other human inflammatory diseases. Our results implicate macrophages in PSC and PSC-like diseases in mice. More importantly, we found that pharmacologic inhibition of macrophage recruitment to the liver reduces PSC-like liver injury in the mouse. These exciting observations highlight potential new strategies to treat PSC.

The sources of parenchymal regeneration after chronic hepatocellular liver injury in mice.

After liver injury, parenchymal regeneration occurs through hepatocyte replication. However, during regenerative stress, oval cells (OCs) and small hepatocyte like progenitor cells (SHPCs) contribute to the process. We systematically studied the intra-hepatic and extra-hepatic sources of liver cell replacement in the hepatitis B surface antigen (HBsAg-tg) mouse model of chronic liver injury. Female HBsAg-tg mice received a bone marrow (BM) transplant from male HBsAg-negative mice, and half of these animals received retrorsine to block indigenous hepatocyte proliferation. Livers were examined 3 and 6 months post-BM transplantation for evidence of BM-derived hepatocytes, OCs, and SHPCs. In animals that did not receive retrorsine, parenchymal regeneration occurred through hepatocyte replication, and the BM very rarely contributed to hepatocyte regeneration. In mice receiving retrorsine, 4.8% of hepatocytes were Y chromosome positive at 3 months, but this was frequently attributable to cell fusion between indigenous hepatocytes and donor BM, and their frequency decreased to 1.6% by 6 months, as florid OC reactions and nodules of SHPCs developed. By analyzing serial sections and reconstructing a 3-dimensional map, continuous streams of OCs could be seen that surrounded and entered deep into the nodules of SHPCs, connecting directly with SHPCs, suggesting a conversion of OCs into SHPCs. In conclusion, during regenerative stress, the contribution to parenchymal regeneration from the BM is minor and frequently attributable to cell fusion. OCs and SHPCs are of intrinsic hepatic origin, and OCs can form SHPC nodules.

Plastic adult stem cells: will they graduate from the school of hard knocks?

Notwithstanding the fact that adult bone marrow cell engraftment to epithelial organs seems a somewhat uncommon event, there is no doubt it does occur, and under appropriate conditions of a strong and positive selection pressure these cells will expand clonally and make a significant contribution to tissue replacement. Likewise, bone-marrow-derived cells can be amplified in vitro and differentiated into a multitude of tissues. These in essence are the goals of regenerative medicine using any source of stem cells, be it embryonic or adult. Despite such irrefutable evidence of what is possible, a veritable chorus of detractors of adult stem cell plasticity has emerged, some doubting its very existence, motivated perhaps by more than a little self-interest. The issues that have led to this state of affairs have included the inability to reproduce certain widely quoted data, one case where the apparent transdifferentiation was due to contamination of the donor tissue with haematopoietic cells and, most notoriously, extrapolating from the behaviour of embryonic stem cells to suggest that adult bone marrow cells simply fuse with other cells and adopt their phenotype. While these issues need resolving, slamming this whole new field because not everything is crystal clear is not good science. The fact that a phenomenon is quite rare in no way mitigates against its very existence: asteroid collisions with the Earth are rare, but try telling the dinosaurs they do not occur! When such events do occur (transdifferentiation or collision), they certainly can make an impact.

Adult stem cell plasticity: new pathways of tissue regeneration become visible.

There has recently been a significant change in the way we think about organ regeneration. In the adult, organ formation and regeneration was thought to occur through the action of organ-or tissue-restricted stem cells (i.e. haematopoietic stem cells making blood; gut stem cells making gut, etc.). However, there is a large body of recent work that has extended this model. Thanks to lineage tracking techniques, we now believe that stem cells from one organ system, for example the haematopoietic compartment, can develop into the differentiated cells within another organ system, such as liver, brain or kidney. This cellular plasticity not only occurs under experimental conditions, but has also been shown to take place in humans following bone marrow and organ transplants. This trafficking is potentially bi-directional, and even differentiated cells from different organ systems can interchange, with pancreatic cells able to form hepatocytes, for example. In this review we will detail some of these findings and attempt to explain their biological significance.

Hepatic stem cells.

The liver in an adult healthy body maintains a balance between cell gain and cell loss. Though normally proliferatively quiescent, hepatocyte loss such as that caused by partial hepatectomy, uncomplicated by virus infection or inflammation, invokes a rapid regenerative response to restore liver mass. This restoration of moderate cell loss and 'wear and tear' renewal is largely achieved by hepatocyte self-replication. Furthermore, hepatocyte transplants in animals have shown that a certain proportion of hepatocytes can undergo significant clonal expansion, suggesting that hepatocytes themselves are the functional stem cells of the liver. More severe liver injury can activate a potential stem cell compartment located within the intrahepatic biliary tree, giving rise to cords of bipotential so-called oval cells within the lobules that can differentiate into hepatocytes and biliary epithelial cells. A third population of stem cells with hepatic potential resides in the bone marrow; these haematopoietic stem cells can contribute to the albeit low renewal rate of hepatocytes, make a more significant contribution to regeneration, and even completely restore normal function in a murine model of hereditary tyrosinaemia. How these three stem cell populations integrate together to achieve a homeostatic balance is not known. This review focuses on two major aspects of liver stem cell biology: firstly, the identity of the liver stem cells, and secondly, their potential value in the treatment of major liver disease.