REvIEwS. Novel perspectives in the management of decompensated cirrhosis

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1 REvIEwS Novel perspectives in the management of decompensated cirrhosis Mauro Bernardi * and Paolo Caraceni Abstract The current approaches to the management of patients with decompensated cirrhosis are based on targeted strategies aimed at preventing or treating specific complications of the disease. The improved knowledge of the pathophysiological background of advanced cirrhosis, represented by a sustained systemic inflammation strictly linked to a circulatory dysfunction, provides a novel paradigm for the management of these patients, with the ambitious target of modifying the course of the disease by preventing the onset of complications and multiorgan failure; these interventions will eventually improve patients quality of life, prolong survival and reduce health- care costs. Besides aetiological treatments, these goals could be achieved by persistently antagonizing key pathophysiological events, such as portal hypertension, abnormal bacterial translocation from the gut, liver damage, systemic inflammation, circulatory dysfunction and altered immunological responses. Interestingly, in addition to strategies based on new therapeutic agents, these targets can be tackled by employing drugs that are already used in patients with cirrhosis for different indications or in other clinical settings, including non- absorbable oral antibiotics, non- selective β- blockers, human albumin and statins. The scope of the present Review includes reporting updated information on the treatments that promise to influence the course of advanced cirrhosis and thus act as disease-modifying agents. Department of Medical and Surgical Sciences, Alma Mater Studiorum University of Bologna, Bologna, Italy. *e- mail: mauro.bernardi@ unibo.it s Despite the underlying progression of portal hypertension and architectural derangements of liver structure, compensated cirrhosis generally remains asymptomatic, as in the preceding disease stages. Complications, such as ascites, bleeding from gastro- oesophageal varices, hepatic encephalopathy and severe jaundice, develop at a yearly rate of 5 7% and mark the transition to the decompensated stage 1, after which cirrhosis becomes a systemic disease. Indeed, the clinical picture of decompensated cirrhosis and its prognosis are influenced by not only the progressive decline of liver function but also, and sometimes pre- eminently, multiorgan or system dysfunction and ultimately failure 2. The course of decompensated cirrhosis is often abruptly accelerated by the development of acute- on-chronic liver failure (ACLF), a distinct clinical syndrome characterized by the acute deterioration of cirrhosis, the development of extrahepatic organ failure and high short- term mortality, despite its potential reversibility 3. A common precipitating event of ACLF is bacterial infection, to which patients with cirrhosis are highly susceptible owing to a complex dysfunction of both innate and acquired immunity 4. Generally speaking, decompensation is a prognostic breaking point: although the median survival of compensated cirrhosis exceeds 12 years, it decreases to ~2 years after decompensation 1. Treatments able to improve survival in patients with decompensated cirrhosis are still an important unmet need. The present Review discusses the paradigm shift occurring in the management of these patients from the sum of treatments specifically targeting each single complication to the search for disease- modifying agents able to slow down the progression of decompensated cirrhosis. Pathophysiological background Arterial vasodilation Since its publication in 1988, the peripheral vasodilation hypothesis has been considered the pathophysiological background of many, but not all, complications of cirrhosis 5. Arteriolar vasodilation, mainly arising in the splanchnic circulation, leads to a reduction of effective volaemia, which evokes the compensatory activation of vasoconstrictor systems and sodium- retaining and water-retaining systems, such as the renin angiotensin aldosterone axis, the sympathetic nervous system and arginine vasopressin release 5. The enhanced arterial inflow to the splanchnic circulation contributes to portal hypertension and increased intrahepatic vascular Nature Reviews Gastroenterology & Hepatology

2 Key points The management of decompensated cirrhosis currently addresses the prevention or treatment of specific complications; disease- modifying agents able to modify the course of decompensated cirrhosis still represent an unmet need. Removing aetiological factors can halt the progression of chronic liver disease, yet a substantial portion of patients with decompensated cirrhosis do not benefit from even successful aetiological treatments. Portal hypertension, abnormal translocation of bacterial products from the gut ( upstream events) and the consequent systemic inflammation and circulatory dysfunction ( downstream events) represent the main targets for mechanistic approaches. Transjugular portosystemic shunt and non- selective β- blockers can be seen as upstream treatments, as they lower portal hypertension and might prevent bacterial translocation. Bacterial translocation can be antagonized by antibiotic- based and non-antibioticbased interventions, but the former entail the risk of antibiotic resistance, whereas the efficacy of the latter still needs to be demonstrated. Human albumin and statins, which are able to simultaneously target several downstream pathophysiological mechanisms, represent promising disease- modifying agents, as they have proved their efficacy in prospective randomized trials. resistance 6. Moreover, the arterial vasodilation hypothesis can explain the development of cardinal features of decompensated cirrhosis, including renal retention of sodium and water, ascites formation, reduced renal perfusion and increased cardiac output. More recently, it became clear that, in the most advanced stages of cirrhosis, a relative decline of cardiac output favoured by cirrhotic cardiomyopathy aggravates effective hypovolaemia, ultimately leading to hyponatraemia and hepatorenal syndrome (HRS) 7,8. Systemic inflammation The molecular mechanism responsible for arterial vasodilation has been identified as abnormal endothelial synthesis of vasodilating substances, such as nitric oxide, carbon monoxide, prostacyclin and endocannabinoids 9. However, the causes of this derangement remained poorly defined until it became clear that it was attributable to the sustained inflammatory and pro- oxidant state that characterizes the interior milieu of patients with decompensated cirrhosis 10 (Fig. 1). A key event underlying this abnormality is abnormal bacterial translocation (BT) from the gut, which is defined as the migration of bacteria or bacterial products from the intestinal lumen to mesenteric lymph nodes or other extraintestinal organs and sites, a process that represents a disruption of the normal host flora equilibrium 11. By increasing intestinal permeability, portal hypertension is a major determinant of BT, to which quantitative and qualitative changes in the microbiome and impaired immune defence mechanisms in the intestinal wall and regional lymph nodes also contribute 11. The systemic spread of bacteria and bacterial products, known as pathogen- associated molecular patterns (PAMPs), activates immune cells when these PAMPs bind with innate recognition receptors, which leads to the release of pro- inflammatory cytokines and chemokines, such as TNF, IL-6, IL-8 or CX 3 C- chemokine ligand 1 (CX 3 CL1), as well as highly reactive oxygen and nitrogen species 12. Similar mechanisms are primed by the systemic spread of endogenous danger- associated molecular patterns (DAMPs) from the diseased liver, where inflammation and cell apoptosis and necrosis take place 13. Systemic inflammation can directly induce cell and tissue damage independent of cardiovascular abnormalities 14. Thus, both circulatory dysfunction and inflammation co- occur in the development of the multiorgan failure of decompensated cirrhosis 10. The exact contribution of each pathway and their chronological order are far from clear, but there is evidence that the deleterious effects of systemic inflammation have a pre- eminent role in the development of ACLF 15. Treatment of decompensated cirrhosis The current approaches to the management of patients with decompensated cirrhosis rely on targeted strategies aimed at preventing or treating specific complications (Box 1). In many cases, they are directed at attenuating or correcting effective hypovolaemia to prevent paracentesis- induced circulatory dysfunction and renal dysfunction induced by spontaneous bacterial peritonitis (SBP), and treating HRS 16. Established medical treatments, such as non- selective β- blockers (NSBBs) and terlipressin, designed to prevent or treat variceal bleeding are also directed against haemodynamic abnormalities, as they reduce portal hypertension by decreasing splanchnic arterial inflow 17. Moreover, the direct consequences of portal hypertension, such as bleeding from gastro- oesophageal varices or ascites formation, can be tackled by inserting a transjugular intrahepatic portosystemic shunt (TIPS) 18. Finally, attempts are being made to antagonize ammonia production to prevent or treat hepatic encephalopathy or intestinal dysbiosis and bacterial overgrowth in patients at high risk of bacterial infection 19,20. The improved knowledge of the pathophysiology of decompensated cirrhosis provided by the systemic inflammation hypothesis 10 offers a novel paradigm for the management of these patients, with the ambitious objective of modifying the course of the disease by preventing the onset of clinical complications and multiorgan failure. The achievement of this goal would improve patients quality of life, prolong survival and reduce health- care costs. Means to restore hepatic architecture by efficiently counteracting intrahepatic inflammation and fibrosis are not yet available 21. Thus, aetiological treatments, whenever possible, or mechanistic strategies that are able to interrupt pathophysiological circuits can be attempted (Box 1). Aetiological treatments Any treatment that is able to remove aetiological factor(s) can be expected to halt the progression of a chronic liver disease or even to lead to a regression of its pathological and functional abnormalities. Indeed, this is the case for compensated cirrhosis, irrespective of its causes, in which successful aetiological treatments prevent decompensation, reduce the incidence of hepatocellular carcinoma, markedly improve survival or even induce a regression of established cirrhosis

3 reduction of the functional liver mass and an increase in portal hypertension beyond critical levels. A baseline MELD score 18 in patients with HCV- related decompensated cirrhosis receiving DAAs independently predicted a worse survival 32. Similarly, 24% of patients waitlisted for liver transplantation that had achieved SVR after interferon- free treatment of HCV infection were delisted owing to improvement, but none of them had a baseline MELD score >20 (ref. 33 ). The 2-year survival of patients with HBV- related decompensated cirrhosis and sustained suppression of viral replication was 83%, but this fell to 64% in those belonging to Child Pugh class C 34. Finally, both abstinence and MELD score independently influenced survival in a large cohort of patients with decompensated alcoholic cirrhosis 35. Thus, aetiological treatments do not exhaust the topic of managing decompensated cirrhosis, and other strategies directed against the pathophysiological mechanisms are needed. Fig. 1 pathophysiological background of clinical manifestations in decompensated cirrhosis. The systemic spread of bacteria and bacterial products, owing to abnormal gut translocation, and danger- associated molecular patterns (DAMPs) from the diseased liver activate innate host immunity. The consequent release of pro- inflammatory cytokines and chemokines, and oxidative and nitrosative species, leads to circulatory dysfunction, to which splanchnic arterial vasodilation induced by portal hypertension also contributes. The direct effects of systemic inflammation and circulatory abnormalities ultimately lead to multiorgan dysfunction. BT, bacterial translocation; PAMPs, pathogen- associated molecular patterns; RNS, reactive nitrogen species; ROS, reactive oxygen species. Adapted with permission from Ref. 10, Elsevier. Aetiologic treatments can also be successful in decompensated cirrhosis, but their effect on the disease course is far more variable. Suppression of HBV replication can improve the Child Pugh score in 49 76% of patients, but a reduction in mortality was not achieved in all studies, and approximately one- third of patients died within a mean follow- up of ~2 years 26. The excellent efficacy and safety profile of direct- acting antiviral agents (DAAs) has made it possible to treat patients with HCV- related decompensated cirrhosis 27,28. Within a few months, Model for End- Stage Liver Disease (MELD) and Child Pugh scores markedly improve in 30 50% of patients, suggesting that sustained virologic response (SVR) can be followed by an improvement in survival in a proportion of patients. In the remainder, however, the severity of cirrhosis does not show any attenuation or even continues to progress up to death or liver transplantation Finally, although hard to attain, abstinence remains the cornerstone of management in patients with decompensated alcoholic cirrhosis, as its achievement slows disease progression and increases candidacy for effective treatments, including transplantation 31. However, like patients with viral cirrhosis, only ~60% of those who achieve abstinence improve their disease, as defined by a downward Child Pugh class migration 31. On the basis of these results, it would seem that even successful aetiological treatments do not arrest the progression of cirrhosis once the disease has reached a 'point of no return'. Even though there is currently no clear watershed, this threshold is probably linked to a Pathophysiological treatments The therapeutic approaches discussed in Fig. 2 should be directed at lowering portal pressure, reducing BT, modulating systemic inflammation, improving circulatory dysfunction, restoring the immunological response and neutralizing oxidative injury. Facing such a complex pathophysiological network with many interacting and often redundant pathways, successful interventions would require counteracting either the upstream initiating 'core' mechanisms or several downstream events simultaneously (Box 2). At present, candidate disease- modifying agents are interventions already used to prevent or treat specific complications of cirrhosis and drugs widely employed in other disease settings, while additional agents are still in development. Only a few interventions that are currently available, such as TIPS or non- absorbable antibiotics, actually address a single well- defined event, whereas others simultaneously hit different targets located either upstream or downstream in the pathophysiological cascade. In the following sections, these agents are classified according to their prevalent effect. Targeting upstream mechanisms In all likelihood, there is no single core factor whose antagonism can be expected to silence the whole pathophysiological cascade and its clinical manifestations. However, there is little doubt that portal hypertension and BT play central roles in triggering the mechanisms leading to systemic inflammation 10,11 (Box 2). Targeting portal hypertension Transjugular intrahepatic portosystemic shunts. The most effective treatment to decrease portal pressure is TIPS insertion, which decompresses the portal system by shunting an intrahepatic portal branch into a hepatic vein. Besides the haemodynamic consequences, increasing evidence indicates that the inflammatory response is decreased after TIPS insertion 36,37. Moreover, markers of systemic inflammation and BT, rather than portosystemic pressure gradients, are independent predictors of post- TIPS mortality 37,38. The major current clinical Nature Reviews Gastroenterology & Hepatology

4 indications for TIPS insertion are bleeding from gastro- oesophageal varices and recidivant (recurring on at least three occasions in the past 12 months) or refractory ascites, and there is no doubt that TIPS insertion succeeds in resolving or easing the management of these complications 18. However, in patients with variceal bleeding, an improvement in survival has been clearly demonstrated for early TIPS insertion only in high-risk patients, as identified by a Child Pugh score B with active bleeding at endoscopy or a Child Pugh score C <14 points within 72 h after bleeding or with hepatic venous pressure gradient (HVPG) >20 mmhg (refs 37,39 ). When TIPS insertion is used as a therapy for difficult- to-treat ascites, improved survival can be achieved in patients with recurrent ascites but not in those with refractory ascites Furthermore, the favourable effects of TIPS insertion in these patients have to be weighed against potential adverse effects of the procedure, the most frequent being the development of hepatic encephalopathy 39. Therefore, careful patient selection is crucial to maximize the beneficial effects of TIPS insertion, as detrimental consequences can occur in the most advanced stages of cirrhosis 18,40,41. This risk Box 1 strategies for treatment of patients with decompensated cirrhosis This figure depicts the natural history of an evolving chronic liver disease. The occurrence of clinical manifestations such as ascites, gastrointestinal (GI) bleeding, hepatic encephalopathy and severe jaundice mark decompensation, a watershed beyond which cirrhosis becomes a systemic disease. The progressive deterioration of these patients can be dramatically accelerated by acute- on-chronic liver failure (ACLF), a clinical syndrome characterized by hepatic and extrahepatic organ failure and often precipitated by bacterial infections, to which patients with cirrhosis are highly susceptible. The current strategies for prophylaxis and treatment of acute decompensation and organ failure rely on measures targeting each complication. However, besides improving already established treatments for specific complications of decompensated cirrhosis, we should consider more comprehensive management of the disease. Such an approach, if successful, could modify the natural history of the disease, thus preventing organ failure and ACLF, reducing hospitalizations and the burden on health care and improving patient survival and quality of life. An aetiological treatment, whenever possible, would represent the first choice. The advancements in the knowledge of the pathophysiological background of decompensated cirrhosis offer a network in which complementary or alternative strategies could find their targets through a mechanistic approach acting at the root of pathophysiological abnormalities. BT, bacterial translocation; CAID, cirrhosis- associated immune dysfunction; ETOH, ethanol; LT, liver transplantation; NASH, nonalcoholic steatohepatitis. prevents TIPS placement from being used to modify disease progression in a high number of patients with decompensated cirrhosis. Non- selective β- blockers. These drugs lower portal pressure and represent the mainstay for the primary and secondary prophylaxis of variceal bleeding 43. This effect is mainly achieved via blockade of β 1 -adrenergic receptors, which reduces splanchnic arterial inflow by lowering cardiac output. β 2 - adrenergic receptor blockade also contributes by leaving α- adrenergic tone unopposed, thereby promoting arterial vasoconstriction 44,45. In addition to their influence on haemodynamics, NSBBs have several other potentially important effects on all the pathophysiological mechanisms involved in BT, including increased intestinal permeability, slowed intestinal motility, bacterial overgrowth and dysbiosis and impaired immunological defences of the intestinal barrier 46,47. As shown in experimental models of cirrhosis, reducing portal pressure improves the congestion and effective blood flow of gut mucosa. Moreover, attenuation of sympathetic tone accelerates intestinal transit time and improves host defence against BT 48,49. These mechanisms are probably also displayed in patients with cirrhosis, in which propranolol administration restored intestinal permeability and reduced the circulating levels of lipopolysaccharide (LPS)-binding protein (LBP) and IL-6. Interestingly, these results were partially independent of haemodynamic effects 50. A meta- analysis showed that NSBB administration as primary or secondary prophylaxis of variceal bleeding is associated with reduced incidence of SBP, an effect that again was independent of the haemodynamic response 51. This study, however, evaluated retro spective analyses of patient cohorts. A prospective investigation enrolling 400 patients hospitalized with cirrhosis reported that the independent predictors of bacterial infections were advanced disease and chronic treatment with PPIs, whereas NSBB therapy was a protective factor. Moreover, infection- related morbidity and mortality were lower in patients taking NSBBs than in those not taking them 52. Although of great interest, these findings should be confirmed through adequately powered prospective studies with hard primary end points, such as survival, before the use of NSBBs can be extended from bleeding prophylaxis to the management of decompensated cirrhosis. In any case, their administration to patients with end- stage liver disease might be problematic owing to their potential adverse influence on survival in certain patient groups 53. Although the patient population(s) at risk of reduced survival have not yet been clearly identified, current indications suggest that patients who develop SBP or those with refractory ascites who develop systolic blood pressure <90 mmhg, hyponatraemia (<130 milliequivalents (meq)/l) or acute kidney injury should receive NSBBs with the greatest caution, if at all 43,54. Nonetheless, a prospective observational investigation in ACLF, including patients with ascites and SBP, showed that the ongoing administration of NSBBs was associated with a less severe syndrome and with improved 28-day and 3-month survival with respect to patients who did not receive or discontinued

5 β β β Fig. 2 potential pathophysiological treatments in decompensated cirrhosis. The main targets of and potential interventions for mechanistic pathophysiological treatments in decompensated cirrhosis are presented. Interventions are indicated in bold in relation to their main target. Of note, norfloxacin, rifaximin, simvastatin and human albumin (HA) are the interventions that have been evaluated in large multicentre, randomized clinical trials that used survival as the primary end point or as part of a composite primary end point. BT, bacterial translocation; FXR, farnesoid X- activated receptor ; TIPS, transjugular intrahepatic portosystemic shunt. NSBBs, a beneficial effect that vanished at 6 months and 12 months 55. It should also be noted that the patients who stopped NSBBs were more likely to have circulatory and lung failure and a higher Chronic Liver Failure Consortium (CLIF- C) ACLF score than those who did not stop NSBBs, indicating a more severe disease. However, when the probability of death was estimated by matching patients by CLIF- C ACLF score, mortality was lower in those receiving NSBBs. Interestingly, these patients had a lower white blood cell count than those not receiving NSBBs, suggesting that these drugs temper systemic inflammation. The mechanisms underlying this outcome need to be elucidated, but they could involve a reduction of BT, which is known to sensitize end organs to subsequent inflammatory injury 56. The effects of NSBBs in patients with ACLF have more recently been assessed by a randomized controlled trial published in abstract form in 2017, which only partially confirmed the benefits reported earlier 57. Patients with ACLF with no or small oesophageal varices were randomly assigned to carvedilol or placebo: carvedilol reduced the incidence of acute kidney injury and sepsis and improved survival at day 28 but not at day 90. Targeting bacterial translocation An obvious response to the need to counteract BT is the use of non- absorbable or poorly absorbable oral antibiotics (Box 2). Antibiotic- based interventions. Non- absorbable or poorly absorbable oral antibiotics induce quantitative and qualitative changes in the microbiota with no or minimal systemic effects. They have been used for decades as primary and secondary prophylaxis of bacterial infections in patients with decompensated cirrhosis. Norfloxacin is the most frequently used drug, being active against the Gram- negative bacteria that are responsible for most infections in advanced cirrhosis 16,58. Data from experimental models of cirrhosis in rats have convincingly shown that norfloxacin reduces the BT rate towards the regional lymph nodes 59,60. Norfloxacin also reduces BT in humans, as its administration to patients with cirrhosis and ascites led to a reduction of the circulating levels of LBP and soluble CD14, a component of the LPS receptor expressed on the membrane of myeloid lineage cells 61. This effect was associated with decreased levels of circulating pro- inflammatory cytokines, such as TNF, IL-12 and interferon-γ, and nitric oxide metabolites. Interestingly, the attenuation of systemic inflammation was linked to the improvement of circulatory dysfunction, as peripheral vascular resistance and arterial pressure rose 61. These changes occurred only in patients with increased serum LBP levels, suggesting that they were due to bowel decontamination rather than the direct immunomodulatory or vasoactive activities of the drug. Long- term selective intestinal decontamination with norfloxacin is currently used in specific settings such as secondary prophylaxis in patients recovering from SBP or primary prophylaxis in patients at high risk of developing SBP 16. Moreover, norfloxacin is administered to patients with gastrointestinal bleeding because of the high risk of these individuals developing bacterial infections 62. In all these contexts, the goal of reducing the incidence of bacterial infections has been achieved, yet a favourable effect on survival was only found from meta- analyses 63,64. However, a study devoted to the primary prophylaxis of SBP in patients with a low protein content in ascitic fluid (<1.5 g/dl) and severe cirrhosis, as defined by Child Pugh score 9 with serum bilirubin level 3 mg/dl or impaired renal function (serum creatinine level 1.2 mg/dl, blood urea nitrogen level 25 mg/dl or serum sodium level 130 meq/l) 65, demonstrated that norfloxacin increased 1-year survival compared with placebo (60% versus 48%) in addition to reducing 1-year risk of SBP (7% versus 61%). The first prospective, randomized, placebo-controlled trial designed to assess the effect of norfloxacin on the course of decompensated cirrhosis, with survival as the primary end point, was reported in abstract form in 2017 (ref. 66 ). The administration of norfloxacin (400 mg per day) in patients with Child Pugh C cirrhosis decreased 6-month mortality by 9.3% and reduced the risk of bacterial infections by 10.5%. Notably, this favourable effect vanished after stopping norfloxacin treatment. Unfortunately, long- term norfloxacin administration has led to selection of Gram- positive gut microorganisms in both experimental and clinical settings 59,67. Moreover, the emergence of quinolone- resistant strains carries the risk of infections associated with increased complication rates and mortality 68. In the light of these potential drawbacks, rifaximin, an antibiotic belonging to the ansamycins family, has been proposed as an interesting alternative. The drug is minimally absorbed, has a broad- spectrum activity against both Gram- negative and Gram- positive aerobic and anaerobic bacteria and has a low risk of inducing Nature Reviews Gastroenterology & Hepatology

6 Box 2 targets for disease-modifying agents in decompensated cirrhosis Because it occurs in many other advanced chronic diseases, the pathophysiology of decompensated cirrhosis results from a very complex network of many interacting, often redundant, molecular mechanisms. This state means that the development of an effective and safe disease- modifying agent could be extremely difficult. The first theoretical approach (part a of the figure) is based on the elimination of the aetiological cause(s) of cirrhosis. A second theoretical approach (part b of the figure) is to develop an intervention that is able to antagonize a single core event of the pathogenetic network that is localized upstream of the entire process. According to the systemic inflammation hypothesis, portal hypertension and abnormal bacterial translocation can be seen as core mechanisms. The third theoretical approach (part c of the figure) is based on interventions mainly acting on downstream pathophysiological events. Given the complexity of the pathophysiological network, these interventions could be successful only if they are able to hit several targets or if they combine agents acting at different levels. bacterial resistance 69,70. Moreover, rifaximin also exerts beneficial effects on the gut microbiota, as it downregulates the inflammatory response triggered by gut microorganisms, attenuates bacterial virulence by inhibiting adhesion, internalization and translocation and modifies bacterial metabolism Similar to norfloxacin, the administration of 1,200 mg per day of rifaximin for 4 weeks to 13 patients with alcoholic cirrhosis and ascites substantially reduced levels of circulating endotoxin and pro- inflammatory cytokines and improved systemic haemodynamics and glomerular filtration rate 75. The results of this pilot study were not confirmed by a placebo- controlled, prospective, randomized study in patients with cirrhosis and ascites (n = 54), which was published in The changes in systemic haemodynamics, glomerular filtration rate and circulating vasoactive hormones after rifaximin administration (1,100 mg per day for 4 weeks) were not statistically significantly different from those occurring in the placebo 76. Surprisingly, none of the 28 patients tested showed small intestine bacterial overgrowth at baseline, an abnormality reported in most patients with advanced cirrhosis 77. Conflicting results have also emerged in the clinical setting. Rifaximin is certainly effective in treating and preventing recurrence of hepatic encephalopathy and has proved to be safe after long- term administration up to 24 months 78. However, its effect on other complications and survival is far less clear. In general, retrospective and case control studies have shown that rifaximin reduces the incidence of hepatic encephalopathy, SBP, acute kidney injury, HRS and variceal bleeding and can even improve survival Conversely, prospective studies have yielded contrasting results. Systemic antibiotic prophylaxis, but not rifaximin, reduced the incidence of SBP in a cohort of patients with cirrhosis and ascites receiving antibiotic prophylaxis (either rifaximin or systemic antibiotic prophylaxis) 82. A large randomized open-label trial compared the effect of rifaximin combined with midodrine, an α 1 -adrenergic receptor agonist, and diuretics versus diuretics alone in patients with refractory ascites 83. Patients in the rifaximin midodrine arm had a reduced need for paracentesis and an improvement in mean arterial pressure, renal function and survival. However, weaknesses in patient selection and data interpretation prevent these results from being generalized. The findings from a large- scale trial (n = 518) assessing the effects of different formulations of soluble oral rifaximin dispersion versus placebo in patients with cirrhosis and ascites were published in abstract form in 2016 (ref. 84 ). Only patients receiving the lowest dosage of the immediate release formulation achieved the composite primary end point: a reduction in mortality risk or hospitalizations as a result of cirrhosis complications. Finally, a meta- analysis published in 2017 suggests that rifaximin in patients with cirrhosis has a protective effect against SBP and HRS, but the quality of the evidence was poor, and the effect of rifaximin on survival was not assessed 85. At present, whether norfloxacin or rifaximin is more effective in reducing BT in patients with cirrhosis remains unsettled. Experimental data published in 2018 suggest that BT is reduced more effectively by norfloxacin than rifaximin in rats with CCl 4 -induced cirrhosis, probably owing to a more efficient depletion of pathobionts from the gut microbiota 86. The observation that 4 weeks of rifaximin administration to patients with decompensated cirrhosis decreased faecal bacterial richness, but did not exert effects on particular species, seems to agree with these findings 87.

7 κ Non- antibiotic-based interventions. The risk of selecting antibiotic- resistant bacterial strains warrants the search for other strategies to suppress BT and its clinical consequences. This goal could be pursued with pharmacological and non- pharmacological agents, such as low- molecular-mass heparin, obeticholic acid and prebiotics and probiotics. Even though their clinical efficacy has not been definitively demonstrated, results emerging from experimental or clinical studies are promising. A randomized, placebo- controlled trial demonstrated the efficacy of low- molecular-mass heparins in preventing portal vein thrombosis in patients with cirrhosis in Child Pugh classes B and C (n = 70) 88. This study also showed that administration of 4,000 international units (IU) per day of enoxaparin for 48 weeks reduced the occurrence of decompensation, as defined by the development of ascites, portosystemic encephalopathy, SBP, portal hypertensive bleeding and bacterial infections. Lastly, increased survival was found in the active arm of the trial. The concomitant reductions of circulating levels of 16S ribosomal DNA, CD14 and IL-6 suggest that enoxaparin attenuates BT and the consequent systemic pro- inflammatory state. These beneficial effects might be related to the prevention of microthrombosis in the gut microcirculation, thus preserving blood flow and ultimately improving the effectiveness of the intestinal barrier. Indeed, serum levels of intestinal- type fatty acid- binding protein (I- FABP; also known as FABP2), a protein made exclusively by enterocytes and released into the circulation upon their necrosis, were reduced in patients receiving enoxaparin. However, variable results have emerged from experimental cirrhosis in rats. In one study, enoxaparin administration lowered portal pressure by improving the structural component of increased liver resistance. As fibrin deposition was reduced, Fig. 3 pleiotropic effects of statins. AP-1, activator protein 1; AT 1, angiotensin II type 1 receptor (also known as AGTR1); CCL2, CC- chemokine ligand 2; CRP, C- reactive protein; enos, endothelial NOS; NF- κb, nuclear factor- κb; NO, nitric oxide; RNS, reactive nitrogen species; ROS, reactive oxygen species; T H, helper T. an inhibition of intrahepatic microthrombosis can be hypothesized 89. Contrasting findings were reported in 2018 by a study in three rat models of cirrhosis, which showed that enoxaparin did not lower or even worsened portal pressure 90. As a result, no beneficial effects were seen on liver function, ascites development and infection, serum IL-6 levels or survival. The nuclear receptor farnesoid X- activated receptor (FXR; also known as NR1H4) acts as a transcription factor for activated bile acids and has an important role in regulating their metabolism together with that of lipids and carbohydrates 91. FXR also carries out important functions in the intestine, as it induces genes influencing gut permeability and modulating inflammation 92. The administration of obeticholic acid, a potent semisynthetic bile acid and FXR agonist, attenuates intestinal inflammation and BT to mesenteric lymph nodes in bile duct- ligated rats and in rats with CCl 4 -induced cirrhosis with ascites 93,94. Interestingly, the rs G > T polymorphism in the FXR gene (NR1H4), a mutation that leads to reduced translation of NR1H4 mrna and decreased FXR transcription factor activity, is associated with a fourfold increased risk of SBP in patients with cirrhosis 95. Finally, obeticholic acid reduced portal hypertension in two experimental rat models of cirrhosis, bile duct ligation and thioacetamide intoxication by increasing intrahepatic endothelial NOS (enos; also known as NOS3) activity through different pathways depending on the model employed 96. At present, obeticholic acid is approved to treat patients with primary biliary cholangitis 97. No studies addressing the effects of FXR agonists on clinical manifestations of decompensated cirrhosis are available, but its properties certainly warrant further investigation in this field. Intestinal bacterial overgrowth and dysbiosis are common in patients with cirrhosis. In general, the abundance of autochthonous bacteria like Bacteroidetes spp. is decreased, whereas numbers of potentially pathogenic bacteria like those in the phyla Proteobacteria and Fusobacteria are increased 77,98. However, substantial differences in microbiota composition emerge, depending on patient ethnicity or the aetiology of liver disease 99. Several mechanisms cooperate to induce such changes, including impaired gut motility, reduced bile flow, impaired gastric acid secretion and impaired intestinal mucosa immune defences 100. The possibility of modifying the taxonomy of intestinal microorganisms by administering prebiotics and probiotics is highly attractive. Several experimental and clinical studies have addressed this topic, but unfortunately, no clearcut results have been provided as yet. Although a favourable effect of probiotic administration on hepatic encephalopathy seems to emerge from systematic reviews and meta- analyses, data suggesting an effect on BT, systemic inflammation and, above all, clinically relevant events attributable to these abnormalities are scant and contradictory 99, Targeting downstream events Strategies influencing the pathophysiological network of decompensated cirrhosis might also target pathways downstream of BT, such as immune cell activation, pro- inflammatory cytokine release and oxidative injury. Nature Reviews Gastroenterology & Hepatology

8 Fig. 4 Functional properties of human albumin and potential target mechanisms in decompensated cirrhosis. Human albumin (Protein Data Bank ID: 1AO6) can exert a range of oncotic and non- oncotic effects that mediate its therapeutic efficacy. DAMPs, danger- associated molecular patterns; PAMPs, pathogen- associated molecular patterns; PGE 2, prostaglandin E 2. In this case, however, they should be able to simultaneously address several mechanisms owing to the extreme complexity of downstream event inter actions (Box 2). Statins and HA can be seen as prototypes of such agents. Statins. In addition to their effect on lipid metabolism, statins possess pleiotropic properties, as they exert antioxidant, antifibrotic and anti- inflammatory activities and improve endothelial dysfunction 104 (Fig. 3). Experimental and clinical studies have convincingly shown that statins reduce portal pressure by improving endothelium- dependent vasorelaxation of the intrahepatic circulation, an effect achieved by increasing the bioavailability of nitric oxide Despite these premises, a randomized controlled trial showed that the addition of simvastatin (20 mg/day for the first 15 days and 40 mg/day thereafter for 24 months) to standard therapy (NSBBs and band ligation) in patients with cirrho sis who had suffered from variceal haemorrhage did not reduce rebleeding 109. However, the study provided the important finding that patients who received simvastatin had a reduced mortality. Further analysis showed that the survival benefit was seen in the group including patients belonging to Child Pugh classes A and B but not in the group including those belonging to Child Pugh class C. Although the mechanisms responsible for these results remain to be ascertained, it is conceivable that the pleiotropic effects of the drug had a role. Several other studies based on the retrospective analysis of large- sized national registries have confirmed that statins favourably influence the course of chronic liver diseases of different aetiologies, as they reduce the risk of developing cirrhosis and hepatocellular carcinoma in patients with chronic hepatitis or subsequent decompensation in patients with cirrhosis The effects of statins seemed to be dose- dependent, and atorvastatin and fluvastatin were associated with the most favourable outcomes 112. As a whole, these results indicate that statins are promising agents for slowing down the progression of chronic liver disease. However, whether patients in the most advanced stages of cirrhosis benefit from their administration remains to be determined through specifically targeted randomized trials. Another matter that merits attention is the potentially increased risk of rhabdomyolysis, especially in patients with advanced cirrhosis, in whom statin metabolism might be severely impaired 116. Indeed, a greater than expected incidence of this complication has been reported in patients receiving 40 mg/day of simvastatin 109. These unfavourable effects might be overcome by a new class of drugs called nitrostatins, which combine a statin with a nitric oxide- donating moiety. Indeed, the hepatic and muscular toxicity induced by atorvastatin administration to rats with bile duct- ligation or CCl 4 - induced cirrhosis was attenuated by the use of equivalent doses of NCX 6560, a nitric oxide- releasing derivative of atorvastatin 117. Human albumin. HA is currently used in specific settings of decompensated cirrhosis, such as the prevention of paracentesis- induced circulatory dysfunction and SBP- induced renal dysfunction 16. In both contexts, a reduced rate of complications and a short- term survival benefit have been demonstrated 118,119. In addition, HA is an essential component, along with vasoconstrictors, in the treatment of HRS type 1 (refs 16,120 ). These favourable effects have traditionally been attributed to the capacity of HA to improve effective volaemia, as it is a potent plasma expander. However, HA also boasts properties unrelated to the regulation of fluid compartmentalization, some of which assume particular importance in relation to the chronic inflammatory state of decompensated cirrhosis. These non- oncotic properties include antioxidant and scavenging activities, as albumin is a major source of extracellular sulfhydryl groups; binding and transport of many endogenous and exogenous substances, including PAMPs, DAMPs and prostaglandin E 2 (PGE 2 ); regulation of endothelial function, thereby contributing to the maintenance of the normal capillary permeability; and modulation of inflammatory and immunological responses 121,122 (Fig. 4). In advanced cirrhosis, these functions are hampered not only by the reduction of serum HA concentrations but also, and possibly mainly, by post- transcriptional abnormalities of the molecule, such as cysteinylation or sulfinylation of the Cys34 residue, truncations at the amino terminus or carboxy terminus and glycosylation Evidence has emerged that the beneficial effects of HA administration in decompensated cirrhosis are also related to its non- oncotic properties. In rats with cirrhosis and ascites, HA, but not hydroxyethyl starch or saline, improved left ventricular contractility ex vivo, counteracting the negative effects of oxidative stress and TNF activation 127. In patients with cirrhosis and SBP, the amelioration of effective volaemia seen with HA, but not hydroxyethyl starch, was related to not only plasma expansion but also a striking rise in peripheral vascular resistances 128. This effect can be related only to the non-oncotic properties of HA. Plasma levels of coagulation factor VIII and von Willebrand factor-related antigen were reduced, and the increase in circulating nitric oxide metabolites seen with hydroxyethyl starch was prevented in the HA group, suggesting that HA was able

9 to attenuate endothelial activation. Finally, HA binds PGE 2, a property that is relevant in advanced cirrhosis, in which strikingly elevated circulating PGE 2 levels induce immune suppression by allowing for PGE 2 binding to macrophages. HA supplementation, by lowering free PGE 2, might contribute to restoring immune competence and reduce the risk of infections 129. The combined oncotic and non- oncotic properties would make HA an ideal multitarget agent able to exert beneficial effects at different steps of the vicious circle that links circulatory dysfunction, inflammatory response and oxidative stress in decompensated cirrhosis. However, a favourable effect on complications and survival could only derive from long- term HA administration, a matter on which little data were available until recently. In patients with cirrhosis and ascites, HA administration at a dose of 25 g per week for 1 year and 25 g every 2 weeks for the following 2 years reduced the probability of developing ascites and hospital readmissions but did not influence survival, which, instead, improved after a median follow- up of 84 months 130,131. However, the relatively small patient sample size (n = 126) precluded any definitive conclusion. The ANSWER study, a multicentre, randomized, open- label, controlled trial published in 2018, compared the effectiveness of HA (40 g twice a week for the first 2 weeks and 40 g once a week for 18 months) plus standard medical treatment (SMT) with SMT alone in 431 patients with cirrhosis and uncomplicated ascites 132. The cumulative incidence of paracentesis and the incidence of refractory ascites were approximately halved in patients enrolled in the HA with SMT arm. Furthermore, the incidence of complications such as SBP, non- SBP bacterial infections, hepatic encephalopathy grade III IV, renal impairment (as defined by serum creatinine >1.5 mg/dl), hyponatraemia (<130 mmol/l) and HRS type 1 were reduced. Finally, the most important finding was that the hazard ratio of overall 18-month mortality was reduced by 38%. However, conflicting results emerged from another multicentre, double- blind, randomized trial that enrolled 196 patients with cirrhosis awaiting liver transplantation, which was reported in abstract form in 2017 (ref. 133 ). Patients received either midodrine (15 30 mg per day) and HA (40 g per 15 days) or matching placebos for 1 year or until liver transplantation. Contrary to the ANSWER study, no beneficial effects of HA were seen on the incidence of complications or survival. Such divergent results might be explained by differences in the patient populations, as patients enrolled in the second trial had more advanced cirrhosis, as suggested by a higher MELD score. Moreover, the divergent dosages and administration schedules of HA could have influenced the serum albumin concentration to different extents. In the ANSWER study, in which roughly double the amount of HA was administered, serum albumin concentration steadily increased by ~0.8 g/dl throughout the entire follow- up period. Even though the optimal HA dosage and administration schedule and the identification of patient popu lations that would benefit more from long- term HA treatment need to be further investigated, the results of the ANSWER study strongly suggest that this treatment prevents the occurrence of complications and hence improves the survival of patients with decompensated cirrhosis. Targeting immunity and regeneration An innovative approach to the treatment of decompensated cirrhosis is bone marrow stem cell activation or bone marrow- derived mesenchymal stem cell (BM- MSC) transplantation, as these cells have potential immunomodulatory and/or liver- regenerative effects. A prospective study randomly assigned 55 patients with decompensated cirrhosis to receive either subcutaneous granulocyte colony- stimulating factor (G-CSF; 12 doses of 5 mg/kg per day for 5 days and then every third day) and darbopoietin alfa (40 μg per week for 4 weeks) or placebo 134. A greater improvement in liver severity scores (Child Turcotte Pugh: 48.6% versus 39.1%; MELD: 40.4% versus 33.0%) was seen in patients given the growth factor combination, and these patients also had a reduced incidence of sepsis and a better 12-month survival (68.6% versus 26.9%). More recently, another prospective trial, published in 2017, randomly assigned 65 patients with decompensated cirrhosis to either SMT plus G- CSF (5 μg/kg subcutaneously twice a day for 5 days then every 3 months for 3 days) and growth hormone (1U subcutaneously daily for 12 months), SMT plus G- CSF or SMT alone 135. Both arms receiving the additional treatments had a better 12-month transplant- free survival, in addition to improved nutrition, reduced liver stiffness, amelioration of quality of life scores and ascites control, and a reduced incidence of bacterial infections without any additional benefit from growth hormone administration. A different approach consisted of one- time or two- time hepatic arterial injections of BM- MSCs or SMT to 72 patients who were abstinent with Child Pugh class B or Child Pugh class C alcoholic cirrhosis. At 6 months, patients in the BM- MSC groups had reduced liver fibrosis, as assessed by histology, and an improvement in Child Pugh score irrespective of the number of injections received 136. Both these approaches seem promising, but the relatively small number of patients enrolled in these studies warrants further investigation before recommending these interventions to prevent complications and death in decompensated cirrhosis. Another trial published in 2018 that randomly assigned patients with predominantly compensated cirrhosis (MELD score ranging from 11 to 15) to either SMT, G- CSF (15 μg/kg daily for 5 consecutive days) or G- CSF plus stem cell infusion (CD133 + haemopoietic stem cells; cells per kg bodyweight on days 5, 30 and 60) failed to show an improvement in liver function or fibrosis 137. Moreover, an increased frequency of adverse events was seen in patients receiving G- CSF and stem cell infusion. Thus, the most effective doses, timing and schedule of administration of haematopoietic growth factor and/or stem cells are still far from established. Conclusions Experimental and clinical research in the past decade have improved our knowledge of the pathophysiological background underlying the clinical manifestations of decompensated cirrhosis, paving the way for novel Nature Reviews Gastroenterology & Hepatology