Use of compounds for preparing drug for treating liver failure

Abstract

This invention relates to the treatment or prevention of liver failure.

Description

Technical Field

[0001] This invention relates to the treatment or prevention of liver failure. Prior Technology

[0002] Liver failure is a condition in which the liver is severely unable to perform its normal metabolic functions. Manifestations of liver failure include acute liver failure, cirrhosis, and acute exacerbations of chronic liver failure.

[0003] Acute liver failure (ALF)

[0004] The term ALF describes a condition characterized by acute loss of liver function in the absence of pre-existing chronic liver disease. ALF is also known as fulminant hepatic failure, acute liver necrosis, fulminant hepatic necrosis, and fulminant hepatitis.

[0005] ALF is a rare but severe outcome of sudden hepatocellular injury, which can progress to a fatal outcome over days or weeks. Various forms of hepatocellular damage result in a consistent pattern: rapidly occurring elevations in aminotransferases, altered mental status, and coagulation disorders. No existing liver disease distinguishes ALF from liver failure caused by end-stage chronic liver disease (decompensated cirrhosis and acute-on-chronic liver failure, ACLF). In ALF, the substances causing hepatocellular damage result in direct toxic necrosis or apoptosis and immune-mediated damage, a slower process. The time from symptom onset to the onset of hepatic encephalopathy distinguishes different forms of acute liver failure: direct, extremely rapid damage (within hours), termed hyperacute liver failure; and slower, immune-based damage (days to weeks), considered acute or subacute. As used herein, "hepatic encephalopathy" or HE refers to confusion, altered level of consciousness, and coma resulting from liver failure. In its later stages, it is called hepatic coma or hepatic coma.

[0006] In developed countries, the five most common causes of ALF are acetaminophen poisoning, local ischemia, drug-induced liver injury, hepatitis B, and autoimmune diseases, accounting for almost 80% of cases. In developing countries, hepatitis A, B, and E are the leading causes of ALF. Other causes of ALF account for less than 15% of all cases and include heatstroke, pregnancy-related injuries (such as acute fatty liver of pregnancy and HELLP syndrome [hemolysis, elevated liver enzymes, and low platelet count]), Budd-Chiari syndrome, non-hepatic viral infections such as herpes simplex, and diffuse invasive malignancies.

[0007] Without treatment, the prognosis is very poor; therefore, timely identification and management of patients with acute liver failure are crucial. Whenever possible, patients with acute liver failure should be managed in the intensive care unit of a liver transplant center.

[0008] Cirrhosis

[0009] As used in this article, "cirrhosis" refers to a condition characterized by the replacement of liver tissue by fibrosis and regenerative nodules, leading to loss of liver function and eventual decompensation. Ascites (fluid retention in the abdominal cavity) is the most common complication associated with decompensated cirrhosis. It is associated with poor quality of life, increased risk of infection, and poor long-term outcomes. Other potentially life-threatening complications include hepatic encephalopathy (confusion and coma) and bleeding from esophageal varices. Cirrhosis can present with many different signs and symptoms. These signs and symptoms may be a direct result of hepatocellular failure or secondary to the resulting portal hypertension. Due to the formation of collateral veins between the portal system and the periumbilical veins caused by portal hypertension, the effects of portal hypertension include splenomegaly, gastroesophageal varices, and portal collateral circulation.

[0010] Cirrhosis is clinically classified into two types: compensated cirrhosis and uncompensated cirrhosis.

[0011] As used in this article, "compensated cirrhosis" refers to severe scarring of the liver that still allows it to perform many vital bodily functions. Patients with compensated cirrhosis experience very few or no symptoms and may live without serious clinical complications. Early-stage compensated cirrhosis is characterized by low-grade portal hypertension and the absence of esophageal varices. Late-stage compensated cirrhosis is characterized by high-grade portal hypertension and the presence of esophageal varices, but without ascites or bleeding.

[0012] As used in this article, "uncompensated cirrhosis" refers to extensive scarring of the liver and its inability to function properly. Patients with uncompensated cirrhosis may experience a variety of symptoms, such as fatigue, loss of appetite, jaundice, weight loss, ascites and / or edema, hepatic encephalopathy, and / or hemorrhage. Early-stage uncompensated cirrhosis is characterized by the presence of ascites in patients who have never experienced bleeding, with or without esophageal varices. Late-stage uncompensated cirrhosis is characterized by more severe ascites, either alone or in combination with hemorrhage, bacterial infection, and / or hepatic encephalopathy. Complications associated with uncompensated cirrhosis may occur, such as ascites, edema, bleeding problems, bone and bone density loss, hepatomegaly, menstrual irregularities in women and gynecomastia in men, impaired mental status, pruritus, renal failure, and muscle atrophy.

[0013] Acute exacerbation of chronic liver failure (ACLF)

[0014] ACLF is also an important liver condition observed in patients with known chronic liver disease, in which liver function deteriorates acutely.

[0015] ACLF is a sudden and life-threatening deterioration of the clinical condition in patients with advanced cirrhosis or cirrhosis caused by chronic liver disease. This syndrome is characterized by three main features: it generally occurs in the context of severe systemic inflammation; it often occurs in close temporal relation to pro-inflammatory precipitating events (such as infection or alcoholic hepatitis); and it is associated with single or multiple organ failure affecting liver, kidney, brain, coagulation, and / or cardiovascular function. Organ failure is identified using the Modified Continuous Organ Failure Assessment Scale (EASL-CLIF Consortium Organ Failure Scoring System), which considers liver, kidney, and brain function, as well as coagulation, circulation, and respiration, allowing patients to be stratified into subgroups with different mortality risks. Based on the number of organ failures at diagnosis, patients are stratified into four prognostic levels (acute exacerbation of chronic liver failure without chronic liver failure, and acute exacerbations of grade 1, 2, and 3 chronic liver failure). Susceptibility to ACLF is related to the severity of the underlying chronic liver disease (i.e., progression of fibrosis to cirrhosis) and its etiology. Regardless of the underlying cause of chronic liver disease, compensated cirrhosis is the primary condition associated with the development of ACLF. Cholestatic liver disease, metabolic liver disease, chronic viral hepatitis, and non-alcoholic steatosis (NASH) are also classified as underlying chronic liver diseases. In Western countries, alcoholic cirrhosis accounts for 50%-70% of all underlying liver diseases associated with ACLF, while viral hepatitis-related cirrhosis accounts for approximately 10%-30% of all cases.

[0016] The severity of underlying diseases can be assessed using the Model for End-Stage Liver Disease (MELD) score.

[0017] ACLF requires a triggering event within a context of cirrhosis and / or chronic liver disease, and rapidly progresses to multiple organ failure with a high mortality rate. Triggering events can include reactivated hepatitis B or superimposed viral hepatitis, alcohol, drugs, ischemia, surgery, sepsis, or idiopathic conditions.

[0018] During disease flare-ups, bacterial translocation can play a crucial role in the progression of autocompensated cirrhosis to decompensated cirrhosis (marked by ascites, variceal hemorrhage, and the development of encephalopathy) via systemic inflammatory response syndrome. However, approximately 40% of patients with ACLF do not experience precipitating events.

[0019] The host response determines the severity of the damage. Inflammation and neutrophil dysfunction are crucial in the pathogenesis of ACLF, and a prominent pro-inflammatory cytokine profile contributes to the transition from stable cirrhosis to ACLF. In these patients, the inflammatory response may lead to immune dysregulation, which can increase susceptibility to infection, subsequently exacerbating the pro-inflammatory response and creating a vicious cycle. Several cytokines play important roles in ACLF. Elevated serum levels of several cytokines have been described in ACLF patients, including tumor necrosis factor (TNF)-α, sTNF-αR1, sTNF-αR2, interleukin (IL)-2, IL-2R, IL-4, IL-6, IL-8, IL-10, and interferon-α.

[0020] Hyperbilirubinemia is almost always present, and jaundice is considered a fundamental criterion for ACLF. Different authors have used different jaundice cutoff points, varying within the range of 6-20 mg / dL serum bilirubin. Besides jaundice, another hallmark of liver dysfunction is coagulopathy. Coagulation tests are often abnormal in patients with cirrhosis, due to impaired synthesis and increased consumption of clotting factors. Continued liver damage eventually leads to an unstoppable spiral of decline and death.

[0021] Besides the liver, the kidney is the most common organ to fail. Kidney failure can be classified into four types: hepatorenal syndrome, parenchymal disease, hypovolemia-induced kidney failure, and drug-induced kidney failure. Bacterial infection (such as spontaneous bacterial peritonitis) is the most common cause of kidney failure in cirrhosis, followed by hypovolemia (secondary to gastrointestinal bleeding or excessive diuretic treatment).

[0022] Hepatic encephalopathy (HE) is a common manifestation of ACLF. HE can be a contributing factor or consequence of ACLF. Ammonia is central to the pathogenesis of HE. In fact, numerous studies have emphasized that hyperammonemia plays a key role in the development of HE in patients with cirrhosis and other liver diseases. Due to liver failure, a large amount of serum ammonia escapes hepatic metabolism and can reach the brain, where high ammonia concentrations are closely associated with cerebral edema and the incidence of herniation.

[0023] In addition, brain swelling is an important characteristic of ACLF, which is similar to ALF.

[0024] One of the hallmarks of ACLF is cardiovascular collapse, which is similar to cardiovascular collapse seen in patients with ALF. This cardiovascular abnormality is associated with an increased risk of death, especially in patients who also present with renal dysfunction.

[0025] Respiratory complications of ACLF can be classified as acute respiratory failure (e.g., pneumonia) and respiratory complications due to cirrhosis (e.g., portal hypertension and hepatopulmonary syndrome). Patients with cirrhosis are at increased risk of pneumonia.

[0026] Patients with ACLF have a statistically higher mortality rate than non-ACLF patients at the same MELD score. Regardless of the precipitating event, the common final pathway leading to acute deterioration of liver function and multiple organ failure appears to be the expanded activation of systemic inflammation, followed by a period of immune system paralysis. The initial interstitial cytokine storm causes significant alterations in the macrocirculation and microcirculation, as well as the disruption of normal organ function, leading to multiple organ failure.

[0027] Early intervention to mitigate or correct injury is crucial. ACLF management is currently based on supportive care for organ failure, primarily in intensive care settings.

[0028] However, a significant proportion of patients with ACLF experience prior acute decompensation events (ascites, encephalopathy, gastrointestinal bleeding, bacterial infection). In fact, the development of liver failure in cirrhotic patients represents a decisive point in medical management, as this condition is typically associated with rapidly progressing multiple organ dysfunction. The lack of hepatic detoxification, metabolic and regulatory functions, as well as altered immune responses, can lead to life-threatening complications such as renal failure, increased susceptibility to infection, hepatic coma, and systemic hemodynamic dysfunction. Furthermore, only 20% of patients with advanced cirrhosis are eligible for liver transplantation.

[0029] Appropriate treatment or prevention is needed for liver failure, especially ACLF, ALF and cirrhosis, and even more so for decompensated cirrhosis.

[0030] Nitazoxanide (NTZ), first described in 1975, has been shown to be highly effective against anaerobic protozoa, worms, and a broad spectrum of microorganisms, including both anaerobic and aerobic bacteria. NTZ also exhibits antiviral activity and has been shown to possess broad anticancer properties by interfering with key metabolic and pro-death signaling pathways.

[0031] A pilot study of hepatic encephalopathy (HE) patients given NTZ and lactulose provided results regarding improvements in clinical presentation. Patients showed improved mental status and good tolerance to the medication (Elrakaybi et al., The clinical effects of nitazoxanide in hepatic encephalopathy patients: a pilot study, IJPSR, 2015; Vol. 6(11): 4657-4667). However, the authors of this study did not report any improvement in liver function or any other organ function. Furthermore, ammonia levels did not increase after NTZ administration.

[0032] This article unexpectedly demonstrates that NTZ can be used to treat individuals with or at risk of developing liver failure. Summary of the Invention

[0033] This invention is based on the following unexpected observations: NTZ protects hepatocytes from ammonia-induced toxicity and improves liver function. Furthermore, this paper unexpectedly demonstrates that NTZ prevents decompensation in an ACLF animal model.

[0034] Therefore, this invention relates to nitrozonide (NTZ), tizoxanide (TZ), tizoxanide glucuronic acid (TZG), or a pharmaceutically acceptable salt thereof, in a method for treating or preventing liver failure in an individual in need. In a particular embodiment, liver failure is ACLF, ALF, or cirrhosis, particularly compensated cirrhosis or uncompensated cirrhosis, and more particularly uncompensated cirrhosis. In a particular embodiment, liver failure is ACLF or ALF.

[0035] In one specific embodiment, the present invention relates to NTZ, TZ, TZG, or their pharmaceutically acceptable salts in a method for treating liver failure selected from ACLF and ALF. In another specific embodiment, the present invention relates to NTZ, TZ, TZG, or their pharmaceutically acceptable salts in a method for treating ACLF.

[0036] This invention further relates to NTZ, TZ, TZG, or their pharmaceutically acceptable salts in a method for treating or preventing liver failure caused by ACLF, ALF, cirrhosis, or decompensated cirrhosis in an individual in need. In a particular embodiment, NTZ, TZ, TZG, or their pharmaceutically acceptable salts are used in a method for treating or preventing liver failure caused by ACLF or ALF.

[0037] In one particular embodiment, an individual is at risk of ACLF, ALF, or cirrhosis, especially decompensated cirrhosis (also referred to herein as "individual at risk").

[0038] In one particular embodiment, the individual suffers from ACLF. Therefore, NTZ, TZ, TZG, or their medically acceptable salts are used in methods for treating ACLF.

[0039] In yet another embodiment, the individual suffers from ALF. Therefore, NTZ, TZ, TZG, or their medically acceptable salts are used in methods for treating ALF. In a particular embodiment, the method is used to treat drug-induced ALF, especially acetaminophen-induced ALF.

[0040] In yet another embodiment, the individual has cirrhosis. In another specific embodiment, the individual has compensated cirrhosis or uncompensated cirrhosis. In another embodiment, the individual has uncompensated cirrhosis. In another specific embodiment, the individual has compensated cirrhosis, and the method is used to prevent compensated cirrhosis from progressing to uncompensated cirrhosis.

[0041] In yet another embodiment, NTZ, TZ, TZG, or a pharmaceutically acceptable salt thereof are used in methods for preventing decompensated cirrhosis or reversing decompensated cirrhosis into compensated cirrhosis.

[0042] In another embodiment, the individual has ACLF and cirrhosis. In another specific embodiment, the individual has ACLF and compensated cirrhosis or uncompensated cirrhosis. In another embodiment, the individual has ACLF and uncompensated cirrhosis. In another specific embodiment, the individual has ACLF and compensated cirrhosis, and the method is used to prevent compensated cirrhosis from progressing to uncompensated cirrhosis.

[0043] In yet another embodiment, an individual at risk of ACLF has cirrhosis, particularly compensated or uncompensated cirrhosis, especially uncompensated cirrhosis.

[0044] In yet another specific embodiment, the individual has ACLF, which carries a higher risk of death within 28 days of hospitalization.

[0045] In one particular embodiment, the individual suffers from grade 1, 2 or 3 ACLF, especially grade 2 or 3 ACLF, according to the CANONIC study of the EASL-CLIF consortium.

[0046] In one specific embodiment, NTZ, TZ(G), or a pharmaceutically acceptable salt thereof are used in a method for preventing ACLF in an individual with cirrhosis. In another specific embodiment, the individual has alcoholic cirrhosis. In another specific embodiment, the individual has compensated alcoholic cirrhosis. In yet another specific embodiment, the individual has decompensated alcoholic cirrhosis.

[0047] In another embodiment, NTZ, TZ(G), or a pharmaceutically acceptable salt thereof are used in a method for preventing ACLF in an individual with chronic liver disease. In one particular embodiment, the individual has chronic liver disease with or without cirrhosis. In another particular embodiment, the individual has chronic liver disease with cirrhosis. In yet another particular embodiment, the cirrhosis is compensated cirrhosis or uncompensated cirrhosis, more particularly uncompensated cirrhosis.

[0048] In another specific embodiment, in a method for using NTZ, TZ(G), or a pharmaceutically acceptable salt thereof to improve liver function in an individual suffering from liver failure selected from the group consisting of ACLF, ALF, and cirrhosis, particularly compensated or uncompensated cirrhosis, and even more particularly uncompensated cirrhosis. In another specific embodiment, the method is used to improve liver function in an individual suffering from liver failure selected from the group consisting of ACLF and ALF.

[0049] In another specific embodiment, in a method for using NTZ, TZ(G), or a pharmaceutically acceptable salt thereof to improve the liver detoxification function of an individual suffering from liver failure selected from the group consisting of ACLF, ALF, and cirrhosis, particularly compensated cirrhosis or uncompensated cirrhosis, and even more particularly uncompensated cirrhosis. In one specific embodiment, the method is used to improve the liver detoxification function of an individual suffering from liver failure selected from the group consisting of ACLF and ALF.

[0050] In another specific embodiment, NTZ, TZ(G), or a medically acceptable salt thereof are used in a method for preventing liver decompensation in an individual with ACLF.

[0051] In one particular embodiment, an individual has ACLF and has at least one precipitating event, such as infection (e.g., viral, fungal, or bacterial infection) or alcoholic hepatitis, sepsis, poisoning, visceral bleeding, and drug-induced liver insufficiency (DILI).

[0052] In yet another specific embodiment, the individual suffers from ACLF caused by hepatic conditions (e.g., alcohol), extrahepatic conditions (e.g., viral, fungal or bacterial infection, sepsis, viral reactivation), or both.

[0053] In another specific embodiment, the individual is at risk of ACLF and has experienced a precipitating event. For example, the individual may have chronic liver disease and experience a precipitating event. In one specific embodiment, the individual has chronic liver disease and experiences a precipitating event, with or without cirrhosis. In one specific embodiment, the individual has chronic liver disease and experiences both a precipitating event and cirrhosis. In another specific embodiment, the cirrhosis is compensated cirrhosis or uncompensated cirrhosis, more particularly uncompensated cirrhosis.

[0054] In yet another embodiment, NTZ, TZ(G), or a medically acceptable salt thereof are used in a method for preventing extrahepatic organ failure in individuals with cirrhosis, particularly those with compensated or decompensated cirrhosis, especially those with decompensated cirrhosis. In one specific embodiment, the prevented extrahepatic organ failure is renal failure. In another specific embodiment, the prevented extrahepatic organ failure is brain failure. In another specific embodiment, the prevented extrahepatic organ failure is heart failure. In another specific embodiment, the prevented extrahepatic organ failure is lung failure.

[0055] In yet another embodiment, NTZ, TZ(G), or a medically acceptable salt thereof are used in a method for preventing extrahepatic organ failure in individuals with chronic liver disease and cirrhosis, particularly those with compensated or decompensated cirrhosis, especially those with decompensated cirrhosis. In one specific embodiment, the prevented extrahepatic organ failure is renal failure. In another specific embodiment, the prevented extrahepatic organ failure is brain failure. In another specific embodiment, the prevented extrahepatic organ failure is heart failure. In yet another specific embodiment, the prevented extrahepatic organ failure is lung failure.

[0056] In yet another embodiment, NTZ, TZ(G), or a medically acceptable salt thereof are used in a method of treating extrahepatic organ failure in an individual suffering from ACLF. In one specific embodiment, the extrahepatic organ failure being treated is renal failure. In another specific embodiment, the extrahepatic organ failure being treated is brain failure. In another specific embodiment, the extrahepatic organ failure being prevented is heart failure. In another specific embodiment, the extrahepatic organ failure being prevented is lung failure.

[0057] In yet another embodiment, NTZ, TZ(G), or a medically acceptable salt thereof are used in a method for preventing extrahepatic organ failure in an individual suffering from ACLF. In one specific embodiment, the prevented extrahepatic organ failure is kidney failure. In another specific embodiment, the prevented extrahepatic organ failure is brain failure. In another specific embodiment, the prevented extrahepatic organ failure is heart failure. In yet another specific embodiment, the prevented extrahepatic organ failure is lung failure.

[0058] In another specific embodiment, in a method of using NTZ, TZ(G) or a medically acceptable salt thereof to protect an individual from ammonia-induced damage, the individual has liver failure of the group selected from or is at risk of liver failure of the group selected from: ACLF, ALF and decompensated cirrhosis.

[0059] In another specific embodiment, NTZ, TZ(G), or a medically acceptable salt thereof are used in a method for treating or preventing HE. In one specific embodiment, the method is used to treat or prevent HE and further prevent cerebral edema. In one specific embodiment, in a method for treating or preventing HE in an individual suffering from liver failure such as selected from the group consisting of ACLF, ALF, and cirrhosis, especially compensated cirrhosis or uncompensated cirrhosis, and even more especially uncompensated cirrhosis. In yet another specific embodiment, in a method for treating or preventing HE in an individual at risk of liver failure such as selected from the group consisting of ACLF, ALF, and cirrhosis, especially compensated cirrhosis or uncompensated cirrhosis, and even more especially uncompensated cirrhosis. In yet another embodiment, in a method of using NTZ, TZ(G), or a medically acceptable salt thereof to prevent an individual with liver failure such as those selected from the group consisting of ACLF, ALF, and cirrhosis, especially compensated or uncompensated cirrhosis, and even more particularly uncompensated cirrhosis. In another specific embodiment, in a method of using NTZ, TZ(G), or a medically acceptable salt thereof to treat or prevent an individual with liver failure by protecting the brain from ammonia-induced damage, the individual with or at risk of liver failure selected from the group consisting of ACLF, ALF, and cirrhosis, especially compensated or uncompensated cirrhosis, and even more particularly uncompensated cirrhosis.

[0060] In another specific embodiment, NTZ, TZ(G), or a medically acceptable salt system thereof are used in a method for treating or preventing cerebral edema. In one specific embodiment, in a method for treating or preventing cerebral edema in an individual suffering from liver failure such as selected from the group consisting of ACLF, ALF, and cirrhosis, especially compensated cirrhosis or decompensated cirrhosis, and even more especially decompensated cirrhosis. In yet another specific embodiment, in a method for treating or preventing cerebral edema in an individual at risk of liver failure such as selected from the group consisting of ACLF, ALF, and cirrhosis, especially compensated cirrhosis or decompensated cirrhosis, and even more especially decompensated cirrhosis. In yet another embodiment, in a method of using NTZ, TZ(G), or a medically acceptable salt thereof to prevent cerebral edema in an individual suffering from liver failure such as those selected from the group consisting of ACLF, ALF, and cirrhosis, particularly compensated or uncompensated cirrhosis, and even more particularly uncompensated cirrhosis. In another specific embodiment, in a method of using NTZ, TZ(G), or a medically acceptable salt thereof to treat or prevent cerebral edema in an individual by protecting the brain from ammonia-induced damage, the individual suffering from or at risk of liver failure selected from the group consisting of ACLF, ALF, and cirrhosis, particularly compensated or uncompensated cirrhosis, and even more particularly uncompensated cirrhosis. Simple Explanation of the Diagram

[0061] Figure 1 shows the protease activity of dead cells after NTZ treatment and without NTZ treatment. The protective effect of NTZ against ammonium chloride (NH4Cl)-induced cell death in HepG2 cells was assessed. The dose-response of NTZ to low (60 mM) and high (120 mM) NH4Cl concentrations was evaluated. Cell death rate was assessed by protease activity assays. Bar graphs represent the mean and standard deviation. Compared with the NH4Cl-free control, using the non-parametric Mann-Whitney test, p < 0.05. Compared with the same dose of NH4Cl in the absence of NTZ (0 µM NTZ), using the non-parametric Kruskal-Wallis test, p < 0.05, p < 0.01, p < 0.001. Figure 2 shows representative images of liver sections from rats with TAA-induced cirrhosis. Liver sections stained with hematoxylin, eosin, and safranin were taken from rats 16 weeks after receiving TAA treatment for the last 4 weeks, with or without NTZ treatment. The fibrous diaphragm surrounding the liver nodules (gray) (black) is a marker of cirrhosis. The portal venous system and vascular system are shown in white. Figure 3 shows the plasma total bilirubin content in cirrhotic rats treated with and without NTZ. The bar chart represents the mean and standard deviation. Compared with the TAA control, the nonparametric Mann-Whitney test was used, §, §§, §§§: p < 0.05, p < 0.01, p < 0.001. Figure 4 shows the plasma albumin levels in cirrhotic rats treated with and without NTZ. The bar chart represents the mean and standard deviation. Compared with the TAA control, the Student t-test was used; *, **: p < 0.05, p < 0.01. Figure 5 is a schematic diagram of the ACLF induction and NTZ treatment regimen. Figure 6 shows the survival curves of cirrhotic rats treated with or without NTZ (vehicle) after LPS injection. The Mantel-Cox test was used to compare the survival curves; P = 0.058. Figure 7 shows the plasma total bile acids in cirrhotic rats after ACLF induction, treated with or without NTZ (mediator, veh). Stuttgart t-test was used; *p < 0.05. Figure 8 shows the effect of NTZ on reducing cerebral edema in rats with BDL+LPS-induced ACLF. Bar graphs represent the mean and standard deviation. Stuttgart t-test was used; *p < 0.05, **p < 0.01. Figure 9 shows the effect of NTZ on renal function in rats with BDL+LPS-induced ACLF. Bar graphs represent the mean and standard deviation. Mann-Whitney test was used, #p < 0.05, ##p < 0.01. Figure 10 shows the reduction of plasma AST by NTZ after acetaminophen poisoning. Bar charts represent the mean and standard deviation. ANOVA and Dunnett's multiple comparisons test were used. * p < 0.05, ** p < 0.01. Figure 11 shows the effect of NTZ treatment on the prevention of kidney injury. The bar graphs represent the mean and standard deviation of plasma creatinine, urea, and cystatin C concentrations. Stuttgart's T test and Welsh correction were used. * p < 0.05, ** p < 0.01. Figure 12 shows the significant reduction in ALT and AST levels in animals treated with CCl4 + LPS by NTZ. Results are presented as mean ± standard deviation and plotted as a bar chart. For all variables following a normal distribution, the Stuttgart T test was used to compare results between groups (#: p < 0.05; ##: p < 0.01; ###: p < 0.001). Where there were different variances between groups, the Wells test was used (¤: p < 0.05; ¤¤: p < 0.01; ¤¤¤: p < 0.001). Logarithmic transformation was applied to plasma ALT and AST in all samples to obtain normal data distributions. Figure 13 shows the significant reduction in GGT levels in animals treated with CCl4 + LPS by NTZ. Results are presented as mean ± standard deviation and plotted as a bar graph. The nonparametric Mann-Whitney test was applied ($:p < 0.05; $$:p < 0.01; $$$:p < 0.001). Figure 14 shows the effect of NTZ in preventing LPS-induced liver function changes, as indicated by its ability to reduce total bilirubin and restore albumin production. Experimental results are presented as mean ± standard deviation and plotted as a bar graph. In cases of varying variance among groups, the Wells test was used (¤: p < 0.05;¤¤: p < 0.01;¤¤¤: p < 0.001). The nonparametric Mann-Whitney test was used ($: p < 0.05; $$$: p < 0.01; $$$$: p < 0.001). Figure 15 shows the effect of NTZ on LPS-induced kidney injury as demonstrated by reducing plasma creatinine, urea, and cystatin C. Experimental results are presented as mean ± standard deviation and plotted as a bar graph. The nonparametric Mann-Whitney test was applied ($:p < 0.05; $$$:p < 0.01; $$$$:p < 0.001). Figure 16 illustrates the inflammatory effect of NTZ in the ACLF model, as demonstrated by its ability to reduce LPS-induced serum IFNγ levels. Experimental results are presented as mean ± standard deviation and plotted as a bar graph. For all variables following a normal distribution, the Stuttgart T-test was used to compare results between groups (#: p < 0.05; ##: p < 0.01; ###: p < 0.001). Implementation

[0062] Individuals requiring the treatment provided herein are patients with liver failure. In one particular embodiment, the individual is a patient with liver failure selected from the group consisting of: ACLF, ALF, and cirrhosis, such as compensated cirrhosis or uncompensated cirrhosis, and especially uncompensated cirrhosis.

[0063] Alternatively, individuals requiring the treatment described herein are patients at risk of liver failure selected from the following: ACLF, ALF, and cirrhosis, especially compensated or uncompensated cirrhosis, and even more so uncompensated cirrhosis. Specifically, individuals may be patients at risk of uncompensated cirrhosis or ACLF due to chronic liver disease.

[0064] As used in this article, the terms "individual" or "patient" refer to mammals, preferably humans.

[0065] ACLF is a multi-organ syndrome that typically occurs in individuals with cirrhosis and at least one organ failure with a high short-term mortality rate. ACLF in patients with chronic liver disease occurs in response to a combination of precipitating factors.

[0066] In one particular embodiment, the individual has chronic liver disease with cirrhosis and is at risk of developing ACLF.

[0067] The term "chronic liver disease" is used in this article to refer to liver disease associated with chronic liver injury, regardless of its underlying cause. Chronic liver disease can be caused by, for example, alcohol abuse (alcoholic hepatitis), viral infectious processes (e.g., hepatitis A, B, C, and E), or autoimmune processes (autoimmune hepatitis), nonalcoholic steatosis (NASH), cancer, or long-term exposure to mechanical or chemical damage to the liver. Chemical damage to the liver can be caused by a variety of substances such as toxins, alcohol, carbon tetrachloride, trichloroethylene, iron, or drugs.

[0068] In one particular embodiment, the individual has chronic liver disease with cirrhosis. In another particular embodiment, the individual has cirrhosis followed by: - Alcohol abuse, - Viral hepatitis (such as hepatitis caused by infection with hepatitis A, B, C, D, E, or G viruses), The use of drugs - Metabolic diseases, - Biliary tract diseases, - Primary biliary cholangitis, - Primary sclerosing cholangitis, or - NASH.

[0069] This invention is particularly suitable for preventing ACLF recurrence or managing ACLF.

[0070] In one particular embodiment, individuals with uncompensated cirrhosis or ACLF exhibit high MELD scores. As used herein, the terms "MELD score" or "end-stage liver disease model" refer to a scoring system used to assess the severity of liver dysfunction. MELD predicts survival using a patient's serum bilirubin, serum creatinine, and international proportion of prothrombin time (INR) values. It is calculated according to the following formula: MELD = 3.78 [Ln serum bilirubin (mg / dL)] + 11.2 [Ln INR] + 9.57 [Ln serum creatinine (mg / dL)] + 6.43, where Ln stands for Napierian logarithm.

[0071] Bilirubin is a yellow breakdown product of normal blood matrix metabolism. It is excreted in bile and urine. Most bilirubin (70%-90%) originates from heme degradation, with a smaller proportion from other heme proteins. In serum, bilirubin is typically measured as both direct bilirubin and total bilirubin. Direct bilirubin relates to conjugated bilirubin and includes both conjugated bilirubin and bilirubin covalently bound to albumin. Indirect bilirubin relates to unconjugated bilirubin. Serum bilirubin levels can be measured by any suitable method known in this art. Illustrative and non-limiting examples of methods for determining serum bilirubin include methods using diazo reagents, methods using DPD, methods using bilirubin oxidase, or methods using direct spectrophotometry of bilirubin. In short, the method for determining serum bilirubin using diazo reagents is based on the formation of azobilirubin by adding a mixture of p-aminobenzenesulfonic acid and sodium nitrite, with azobilirubin acting as an indicator. The method for determining serum bilirubin using DPD is based on the fact that bilirubin reacts with 0.1 mol / HCl containing 2,5-dichlorobenzidine (DPD) to form azobilirubin, which has maximum absorbance at 540-560 nm. The staining intensity is proportional to the bilirubin concentration. Unbound bilirubin reacting in the presence of a detergent (e.g., Triton TX-100) is used for total bilirubin determination, while only bound bilirubin reacts in the absence of a detergent. The method for determining serum bilirubin using bilirubin oxidase is based on the reaction catalyzed by bilirubin oxidase, an enzyme with maximum absorbance at 405-460 nm, which oxidizes bilirubin to biliverdin. Bilirubin concentration is proportional to the measured absorbance. Total bilirubin concentration is determined by adding sodium dodecyl sulfate (SDS) or sodium cholate, which causes unbound bilirubin to separate from albumin and precipitate. Serum bilirubin levels can also be determined by direct spectrophotometry at 454 nm and 540 nm. Measurements at these two wavelengths are used to reduce heme interference.

[0072] As used herein, the terms "International Prothrombin Time Ratio" or "INR" refer to a parameter used to measure the blood's clotting tendency. INR is the ratio of a patient's prothrombin time to that of a normal (control) sample, raised to the power of the ISI value used in the analytical system employed. Prothrombin time (PT) is measured by factors I (fibrinogen), II (prothrombin), V, VII, and X, and is used in conjunction with activated partial thromboplastin time. Prothrombin time is the time it takes for plasma to clot after the addition of tissue factor. This measures the extrinsic pathway of clotting. The result of INR-standardized prothrombin time is calculated using the following formula: INR = (PT test / PT normal) <isi>. The ISI value in the formula is the International Sensitivity Index for any tissue factor and indicates how well a particular batch of tissue factors can be compared to an international reference tissue factor. The ISI is typically between 1.0 and 2.0.

[0073] MELD scores are strongly correlated with short-term mortality; lower MELD scores are associated with lower mortality, while higher MELD scores are associated with higher mortality. Therefore, patients with low MELD scores, such as below 9, have a 3-month mortality rate of approximately 1.9%, while patients with high MELD scores, such as 40 or higher, have a 3-month mortality rate of approximately 71.3%.

[0074] As used herein, the term "high MELD score" refers to a patient with a MELD score greater than 9, for example, at least 10, at least 15, at least 19, at least 20, at least 25, at least 29, at least 30, at least 35, at least 39, at least 40, at least 45, or higher. In one particular embodiment, the invention is applied to individuals with a MELD score greater than 20.

[0075] In another specific embodiment, the patient to be treated exhibits impaired kidney function. As used herein, "impairment of kidney function," also referred to as "impairment of renal function," "kidney damage (condition)," "kidney failure," "kidney impairment," and "kidney failure" refer to a medical condition in which the kidneys are unable to adequately filter waste products from the blood. Kidney failure is primarily measured by the glomerular filtration rate, which is the reduced rate at which blood is filtered in the glomeruli of the kidneys. In kidney failure, there may be increased body fluids (leading to swelling), increased acid levels, elevated potassium levels, decreased calcium levels, increased phosphate levels, and, in later stages, anemia.

[0076] Individuals suffering from liver failure and associated HE who may benefit from this invention can be identified by the polymer vesicles and their method of use disclosed in WO2019053578.

[0077] As used herein, the term "treatment" refers to both therapeutic and preventative or therapeutic measures aimed at preventing or mitigating (alleviating) unwanted physiological changes or symptoms. Beneficial or desired clinical outcomes include, but are not limited to, symptom relief, stabilization of the pathological state (especially non-deterioration), slowing or halting disease progression, and improving or alleviating pathology. Specifically, for the purposes of this invention, treatment aims to slow the progression of liver damage and reduce the risk of additional complications. If no treatment is received, treatment may also involve extending survival compared to expected survival.

[0078] In the context of this invention, a therapeutically effective amount of NTZ, TZ(G), or a pharmaceutically acceptable salt thereof is administered to an individual. In one particular embodiment, NTZ or TZ or a pharmaceutically acceptable salt thereof is administered. In another embodiment, NTZ or a pharmaceutically acceptable salt thereof is administered to an individual, particularly NTZ.

[0079] "Therapeutic effective amount" refers to the amount of a drug that effectively achieves the desired therapeutic effect. Therapeutic effective amount can vary depending on factors such as an individual's disease state, age, sex, weight, and the drug's ability to elicit the desired response in the individual. Therapeutic effective amount is also the amount in which the beneficial therapeutic effect outweighs any toxic or harmful effects of the drug. The effective dosage and dosage regimen of the drug depend on the disease or condition to be treated and can be determined by someone skilled in this technique. Physicians generally skilled in this technique can easily determine and prescribe the effective amount of the pharmaceutical composition required. For example, a physician may start with a dosage of the drug used in the pharmaceutical composition below the level required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. Generally, the appropriate dosage of the composition of the present invention will be the amount of the compound that is the minimum dose required to effectively produce a therapeutic effect according to a specific dosing regimen. Such effective dosage will generally depend on the factors described above.

[0080] NTZ, TZ(G) or their pharmaceutically acceptable salts may be incorporated into a pharmaceutical composition which further comprises one or more pharmaceutically acceptable excipients or mediators (e.g., saline solution, physiological solution, isotonic solution, etc.) that are compatible with pharmaceutical use and are well known to those generally skilled in the art.

[0081] These compositions may further include one or more reagents or mediators selected from dispersants, solubilizers, stabilizers, preservatives, etc. Reagents or mediators suitable for these formulations (liquid and / or injectable and / or solid) are particularly methylcellulose, carboxymethylcellulose, polysorbate 80, mannitol, gelatin, lactose, vegetable oils, gum arabic, liposomes, etc.

[0082] These compositions can ultimately be formulated as injectable suspensions, syrups, gels, oils, ointments, pills, tablets, suppositories, powders, gel caps, capsules, aerosols, etc., using either herbal formulations or devices that ensure prolonged and / or slow release. For such formulations, reagents such as cellulose, carbonates, or starches can be advantageously used.

[0083] NTZ or TZ(G) may be in the form of pharmaceutically acceptable salts, particularly acid or base salts compatible with pharmaceutical applications. Salts of NTZ and TZ(G) include pharmaceutically acceptable acid addition salts, pharmaceutically acceptable base addition salts, pharmaceutically acceptable metal salts, ammonium salts, and alkylated ammonium salts. These salts may be obtained during the final purification step of the compound or by incorporating the salt into a previously purified compound.

[0084] NTZ, TZ(G), or their pharmaceutically acceptable salts can be administered via various routes and in various forms. For example, the compound can be administered systemically, orally, non-enterally, by inhalation, by nasal spray, by nasal drops, or by injection, such as intravenously, intramuscularly, subcutaneously, percutaneously, locally, or intra-arterially. Of course, the route of administration will be adapted to the drug form according to procedures familiar to those skilled in this art.

[0085] In one specific embodiment, the compound is formulated as a tablet. In another specific embodiment, the compound is administered orally.

[0086] The frequency and / or dosage of administration can be adapted by those skilled in the art to the patient, pathology, and form of administration. Typically, NTZ or TZ(G) can be administered at doses ranging from 0.01 mg / day to 4000 mg / day, such as 50 mg / day to 2000 mg / day, such as 100 mg / day to 2000 mg / day, and particularly between 100 mg / day and 1000 mg / day. In a particular embodiment, NTZ, TZ(G), or a pharmaceutically acceptable salt thereof is administered at a dose of about 1000 mg / day, particularly 1000 mg / day. In a particular embodiment, NTZ, TZ(G), or a pharmaceutically acceptable salt thereof is administered orally at a dose of about 1000 mg / day, particularly 1000 mg / day, particularly in tablet form. If necessary, administration can be performed daily or even several times daily. In one embodiment, the compound is administered at least once a day, such as once a day, twice a day, or three times a day. In one particular embodiment, the compound is administered once or twice daily. Specifically, oral administration can be performed once daily during meals, such as breakfast, lunch, or dinner, by taking a tablet containing the compound at a dose of about 1000 mg, particularly 1000 mg. In another embodiment, oral administration of tablets twice daily is performed, for example by taking a first tablet containing the compound at a dose of about 400 mg, about 500 mg, or about 600 mg, particularly 500 mg, during a meal, and a second tablet containing the compound at a dose of about 500 mg, particularly 500 mg, during another meal on the same day.

[0087] In the context of this invention, the term "about" applied to numerical values ​​means a value + / - 10%. For clarity, "about 100" refers to a value within the range of 90-110. Furthermore, in the context of this invention, the term "about X," where X is a numerical value, specifically reveals the value of X, and also reveals the lower and higher values ​​of the range thus defined, more specifically the value of X.

[0088] Suitablely, the treatment process using NTZ, TZ(G), or a medically acceptable salt thereof lasts for at least one week, particularly for at least two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, or twenty-four weeks or longer. In one particular embodiment, the treatment process lasts for at least one month, at least two months, or at least three months. In one particular embodiment, the treatment process lasts for at least one year or longer, depending on the individual's condition.

[0089] In one particular embodiment, NTZ, TZ(G), or a pharmaceutically acceptable salt thereof (the “medicine”) is used as the sole active ingredient for the treatment or prevention disclosed herein.

[0090] In yet another embodiment, the drug is used in combination therapy.

[0091] In one particular embodiment, the drug is used in combination with a therapy targeting the precipitating event.

[0092] In one particular embodiment, the drug is used in combination with a therapy targeting hepatic or extrahepatic conditions.

[0093] In one particular embodiment, the inducing event is a bacterial, fungal, or viral infection. Therefore, the drug may be combined with an antimicrobial or antiviral agent. As is well known in the art, the most suitable agent will be selected depending on the organism or virus causing the invention. In one particular embodiment, the inducing event is hepatitis B virus reactivation. In that case, the drug may be combined with a nucleoside or nucleoside analogue. Illustrative antiviral drugs include, but are not limited to, tenofovir, tenofovir alafenamide, and entecavir.

[0094] In another specific embodiment, the precipitating event is acute varicose bleeding. Therefore, the drug can be combined with vasoconstrictors such as terlipressin or somatostatin, or analogs such as octreotide or vapreotide, especially octreotide. This treatment can be accompanied by endoscopic therapy (preferably endoscopic varicose ligation performed during diagnostic endoscopy within 12 hours of admission). Short-term antibiotic prophylaxis, such as with ceftriaxone, can also be administered.

[0095] In another specific embodiment, the precipitating event is alcoholic hepatitis. Therefore, the drug can be combined with prednisolone, which is designated for use in patients with severe alcoholic hepatitis.

[0096] In another specific embodiment, the drug is used in combination with supportive therapy.

[0097] In one particular embodiment, the supportive therapy is cardiovascular support. For example, the drug may be combined with therapies for acute kidney injury, such as discontinuation diuretics or volume expansion (using intravenous albumin). The drug may also be combined with vasoconstrictors such as terlipressin or norepinephrine, especially in cases where there is no response to volume expansion.

[0098] In one specific embodiment, the supportive therapy is encephalopathy treatment. For example, the drug may be combined with lactulose. Depending on the situation, lactulose therapy may be further completed with the administration of an enema to cleanse the bowels. In cases of severe hepatic encephalopathy that is difficult to treat with lactulose, albumin dialysis may be used. In yet another specific embodiment, the drug may be combined with rifaximin. In yet another embodiment, the drug may be combined with lactitol.

[0099] In another specific embodiment, the drug is used in combination with a liposome-based toxin scavenger, such as an ammonia scavenger. Specifically, the liposome-based toxin scavenger may be a liposome-based intraperitoneal fluid, which can be particularly used to enhance the clearance of ammonia, especially ammonia accumulated during decompensated cirrhosis.

[0100] Intraperitoneal administration of vesicles (e.g., liposomes) with distal loading capacity (e.g., transmembrane pH-gradient liposomes) has been described as a method of concern for treating drug overdose and endogenous metabolite poisoning (e.g., hyperammonemia) (Forster et al., Sci Transl Med 2014; 6: 258ra141). As mentioned above, hyperammonemia is associated with hemocyaninemia (HE). Therefore, in the context of this invention, such intraperitoneal administration in combination with NTZ, TZ(G), or a pharmaceutically acceptable salt thereof, and a liposome-based toxin scavenger may be advantageous.

[0101] Specifically, WO 2014 / 023421 describes liposome vesicles for peritoneal dialysis in patients suffering from endogenous or exogenous poisoning, particularly hyperammonemia, wherein the pH within the liposomes differs from the pH within the peritoneal cavity, and wherein the pH within the liposomes produces charged toxins encapsulated by the liposomes. The liposome formulation may comprise vesicles of various properties, compositions, sizes, and characteristics coated with an aqueous medium having different compositions, pH, and osmotic strength. In a preferred embodiment, the liposome-like vesicles are made of polymers and do not contain lipids; for this reason, they are not considered liposomes in form but are referred to as polymeric vesicles. These polymeric vesicles allow for the delivery of liposomes or liposome-like vesicles, such as polymeric vesicles or nonionic niosomes, in any form or mode that makes them bioavailable in the peritoneal cavity. It is possible to add a step of extracting liposomes from the peritoneal cavity after and / or simultaneously with the delivery step. In one specific embodiment, the pH within the liposome vesicles is from 1 to 6.5, and charged toxins encapsulated in the liposomes are produced. The liposome bilayer comprises natural or synthetic phospholipids as the main component, and the diameter of the liposome vesicles is greater than 700 nm. In another specific embodiment, the natural or synthetic phospholipid is a long saturated phospholipid. In yet another specific embodiment, the natural or synthetic phospholipid is a long saturated phospholipid having an alkyl chain with more than 12 carbon atoms. In still another embodiment, the natural or synthetic phospholipid is a long saturated phospholipid having an alkyl chain with more than 14 carbon atoms.In another specific embodiment, the natural or synthetic phospholipid is 1,2-dilauryl-sn-glycerol-3-phosphate choline (DLPC); 1,2-dimyristyl-sn-glycerol-3-phosphate choline (DMPC); 1,2-dispalmityl-sn-glycerol-3-phosphate choline (DPPC); 1,2-distearatel-sn-glycerol-3-phosphate choline (DSPC); 1,2-dioleyl-sn-glycerol-3-phosphate choline (DOPC); 1,2-dimyristyl-sn-glycerol-3-phosphate ethanolamine (DMPE); 1,2-dispalmityl-sn-glycerol-3-phosphate ethanolamine (DPPE); 1,2 -Distearyl-sn-glycerol-3-phosphate ethanolamine (DSPE); 1,2-Diolyl-SA7-glycerol-3-phosphate ethanolamine (DOPE); 1-Myristyl-2-palmityl-sn-glycerol-3-phosphate choline (MPPC); 1-palmityl-2-myristyl-sn-glycerol-3-phosphate choline (PMPC); 1-Stearyl-2-palmityl-sn-glycerol-3-phosphate choline (SPPC); 1-palmityl-2-stearyl-sn-glycerol-3-phosphate choline (PSPC); 1,2-Dimyristyl-sn-glycerol-3-[dioxophosphoryl-racemic-(1-glycerol)] (DMPG); 1,2-Dipalmitoyl-sn-glycerol-3-[dioxophosphoryl-racemic-(1-glycerol)] (DPPG); 1,2-Distearatel-sn-glycerol-3-[dioxophosphoryl-racemic-(1-glycerol)] (DSPG); 1,2-Dioleoyl-sn-glycerol-3-[dioxophosphoryl-racemic-(1-glycerol)] (DOPG); 1,2-Dimyristyl-sn-glycerol-3-phosphate (DMPA); 1,2-Dipalmitoyl-sn-glycerol-3-phosphate (DPPA); 1,2-Dipalmitoyl-sn-glycerol-3-[dioxophosphoryl-L-serine] (DPPS); or La-phosphatidylcholine (EPC) from eggs or La-phosphatidylcholine (SPC) from soybeans. In yet another embodiment, the natural or synthetic phospholipid is 1,2-dispalmitoyl-sn-glycerol-3-phosphocholine (DPPC). In another embodiment, the liposome bilayer further comprises a steric hindrance stabilizer, wherein the concentration of the steric hindrance stabilizer, which may be a polyethylene glycol-modified lipid, in the liposome composition varies between 0 mol% and 30 mol%. In another embodiment, the liposome bilayer comprises a polyethylene glycol-modified lipid between 0.5 mol% and 20 mol%. In another embodiment, the polyethylene glycol-modified lipid is 1,2-distearyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG). In yet another embodiment, the diameter of the liposome vesicles is greater than 800 nm.In another embodiment, the liposome vesicles have a diameter of 700 nm to 10 μm. In another embodiment, the pH within the liposome vesicles is 1.5 to 5, more preferably 1.5 to 4.

[0102] These liposome vesicles can be produced according to the methods disclosed in WO 2014 / 023421 and WO 2016 / 177741.

[0103] WO 2018 / 033856 describes transmembrane pH-gradient polymeric vesicles and their use for scavenging ammonia and its methylated analogs (e.g., trimethylamine (TMA)). Methods for preparing such polymeric vesicles are also disclosed in WO 2018 / 033856. NTZ, TZ(G), or pharmaceutically acceptable salts thereof may be incorporated into such polymeric vesicles as liposome-based toxin scavengers. In one particular embodiment, the polymeric vesicle comprises: (a) a membrane comprising a block copolymer of poly(styrene) (PS) and poly(ethylene oxide) (PEO), wherein the PS / PEO molecular weight ratio is greater than 1.0 and less than 4.0; and (b) a core coated with an acid. In yet another embodiment, the block copolymer is a diblock copolymer. In another embodiment, when the polymeric vesicle is hydrated, the acid is present at a concentration between pH 1 and 6. In one embodiment, the acid is in an acidic aqueous solution. In yet another embodiment, the pH of the acidic aqueous solution is between 1 and 6, particularly between 2 and 5, or preferably between 2 and 4. In yet another embodiment, the acid is a hydroxy acid, preferably citric acid. In yet another embodiment, the polymer vesicles are prepared by a method comprising mixing an organic solvent containing the copolymer with an aqueous phase containing the acid. In yet another embodiment, the organic solvent is immiscible with water or partially miscible with water. In one embodiment, the core of the polymer vesicle is further coated with ammonia or a methylated analog thereof, preferably TMA. In yet another embodiment, the polymer vesicles are in a composition comprising at least one pharmaceutically acceptable excipient. In yet another embodiment, the composition is in liquid, semi-solid, or solid form.

[0104] In one particular embodiment, the supportive therapy is extracorporeal liver support. For example, an extracorporeal liver support device containing hepatocytes may be used. In another embodiment, in addition to administering the drugs provided herein, plasma exchange may also be performed. In yet another embodiment, extracorporeal liver support is albumin exchange or endotoxin removal.

[0105] The following examples are used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Example

[0106] Example 1: NTZ-induced expression of glutamic acid synthase involved in ammonia detoxification

[0107] Preclinical models

[0108] Liver injury was induced in 5-6 week old male C57Bl / 6 mice (Janvier Labs, France) by feeding them a cholesterol-supplemented (1%) choline-deficient L-amino acid-defined (CDAA / c) diet (Ssniff, Germany) for 12 weeks (n=12). Some animals received an NTZ-supplemented CDAA / c diet (Interchim, France) (100 mg / kg / day) from day 1 throughout the study (n=8). Mice receiving a choline-supplemented L-amino acid-defined (CSAA) diet served as additional controls (n=6).

[0109] All animal procedures were performed in accordance with standard protocols and standard recommendations for the proper care and use of laboratory animals.

[0110] Total transcript analysis

[0111] Total RNA was isolated from mouse livers (n=5) using the Nucleospin® 96 RNA kit (ref 740709.4, Macherey Nagel, France). RNA sample concentrations were measured using a Multiskan™ GO spectrophotometer (Thermo Scientific, France), and quality was assessed using an Agilent 2100 Bioanalyzer (USA). Libraries were prepared using the Illumina TruSeq LT mRNA kit (ref RS-122-2101, Illumina, USA), and mRNA was sequenced using a NextSeq 500 device (paired end sequences, 2 × 75 bp) in High Output flow cells (ref FC-404-2002 - NextSeq® 500 / 550 High Output kit version 2 (150 cycles), Illumina, USA).

[0112] Readings were cleared using Trimmomatic version 0.36 with the following parameters: SLIDINGWINDOW: 5:20 LEADING: 30 TRAILING: 30 MINLEN: 60 (Bolger et al., Bioinformatics, 2014). Subsequently, readings were compared with a reference genome (mice GRCm38.90-gene bank collection GCA_000001635.2) using an rnacocktail and hisat2 version 2.1.0 as the alignment tool, with preset parameters (Sahraeian et al., Nature Communications 2017).

[0113] Use featureCounts version 1.5.3 to generate a count table with preset parameters (Liao Y et al. Bioinformatics 2014).

[0114] To identify differentially expressed genes (DE genes), we used R (version 3.4.3) and the DESEq2 library (version 1.18.1). Gene annotations were retrieved using the AnnotationDbi library (version 1.40.0). In short, the count matrix generated by FeatureCounts was analyzed using the DESeqDataSetFromMatrix() function from the DESeq2 library, followed by the DEseq() function. For each condition (i.e., comparing NTZ+CDAA / c with CDAA / c), the fold change and p-value were retrieved using the results() function from DESeq2.

[0115] result

[0116] Analysis of genes showing differential expression in total liver transcripts of NTZ-treated mice compared to mice receiving only a CDAA / c diet revealed that Glul mRNA expression was significantly different (Table 1).

[0117] Table 1: Top 10 genes most significantly affected by NTZ exposure Sort symbol Average performance Change factor (log2) After adjusting the p-value Gene name 1 Gsta2 4272 1,652 7,42E-24 Glutathione S-transferase, α2 (Yc2) 2 Abcc4 1873 1,354 3,03E-17 ATP-binding cartridge, subfamily C (CFTR / MRP), member 4 3 Prodh 10649 0.756 1,46E-15 proline dehydrogenase 4 Slc1a2 18107 0,820 8,57E-15 Solute carrier family 1 (glial high-affinity glutamic acid transporters), member 2 5 Ldhd 14329 0,659 1,35E-14 Lactate dehydrogenase D 6 Ctsk 911 -0,930 8,09E-14 cathepsin K 7 Glul 105162 0.672 2,61E-13 Glutamate-aminoconjugation enzyme (glutamate synthase) 8 Abat 21598 0,792 1,84E-12 4-Aminobutyrate aminotransferase 9 Sugct 3484 0.664 3,14E-12 Succinyl-CoA glutarate-CoA transferase 10 Arhgef26 2664 1,108 4,46E-12 ρ-guanine nucleotide exchange factor (GEF) 26 Ranking: All differentially expressed genes are ranked according to their adjusted p-values. Change factor: log2 of NTZ+CDAA / c relative to CDAA / c. Adjusted p-value: The p-value representing the change factor adjusted for multiple tests.

[0118] Glul encodes a glutamic acid synthase involved in hepatic ammonia detoxification (Zhou et al., Neurochemistry International 2020), which is a known molecule that induces hepatocellular damage and liver fibrosis. In mouse livers, basal expression of Glul is extremely high (mean count 105,162, Table 1), and Glul expression is reduced by 40% in the CDAA / c diet compared to the CSAA control (adjusted p = 7 × 10⁻¹⁰). NTZ robustly restored basal Glul expression by inducing a 1.6-fold increase in basal expression in CDAA / c-fed mice (adjusted p = 2.6 × 10⁻¹³).

[0119] Therefore, this data demonstrates the important role of NTZ in preventing hepatocellular damage by stimulating ammonia detoxification through glutamic acid production.

[0120] Example 2: NTZ protects hepatocytes from ammonia-induced toxicity.

[0121] Patients with acute or chronic liver disease often present with hyperammonemia due to changes in ammonia metabolism and detoxification. This ammonia accumulation subsequently induces hepatocyte damage, scar tissue formation (fibrosis), and accelerates disease progression.

[0122] To evaluate the effect of NTZ on human hepatocytes subjected to ammonia-induced cellular stress, the human hepatoblastoma-derived HepG2 cell line (number 85011430, ECACC, UK) was cultured in a 5% CO2 incubator at 37°C in high glucose DMEM medium (Gibco, France) supplemented with 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, 1% sodium pyruvate, 1% L-glutamic acid, and 1% MEM non-essential amino acids.

[0123] To assess cell tolerance to ammonium chloride (NH₄Cl), 1 × 10⁵ cells were plated in 96-well plates, with NTZ doses ranging from 0 to 5 µM in the same cell culture medium. After cell adhesion (10 h), cells were washed with PBS and cultured for 14 h in DMEM without L-glutamic acid and FBS in the presence of 0, 60, or 120 mM NH₄Cl (Fluka, France).

[0124] Cytotoxicity was measured using the CytoTox-Glow™ assay (G9291, Promega, USA). In short, 100 µL of reagent was added to each well, and the cells were incubated in the dark at room temperature for 15 minutes. The CytoTox-Glow™ assay is a luminescent cytotoxicity assay that measures the relative number of dead cells in a cell population. Luminescence was measured using a Spark microquantitative disc reader (30086376, Tecan, USA). Luminescence intensity (RLU) was directly correlated with the percentage of cells experiencing cytotoxic stress.

[0125] The results are summarized in Figure 1.

[0126] In HepG2 cells, NH4Cl induced high mortality rates at both low and high concentrations (18-fold and 23-fold at 60 mM and 120 mM, respectively). Furthermore, NTZ reduced mortality in a dose-dependent manner at both NH4Cl concentrations (Figure 1). These results indicate that NTZ has a direct protective effect on hepatocytes.

[0127] Example 3: NTZ improves liver function in cirrhotic rats

[0128] Preclinical liver cirrhosis model

[0129] Male Sprague Dawley rats (250-275 g) (Janvier, France) were induced to have cirrhosis by intraperitoneal injection of thioacetamide (TAA) (ref 163678, Sigma) at a dose of 150 mg / kg during the first week and then twice weekly at a dose of 200 mg / kg during the 11th week (total induction period of 12 weeks). Rats were stratified into treatment groups based on mean plasma α-2 macroglobulin (a marker of fibrosis) and total plasma bilirubin (a marker of liver function) obtained from sublingual blood samples. Subsequently, rats received a standard diet of 30 mg / kg / day (n=12) or a diet supplemented with NTZ (n=10) for a 4-week intervention period. Rats that received intraperitoneal injection of NaCl during the 16th week served as additional healthy controls (n=10).

[0130] Animals were caged in pairs in a controlled environment with a 12:12-hour light-dark cycle, and the animals had free access to food and water. Their weight and food intake were monitored twice a week.

[0131] On the last day of treatment, plasma samples were obtained by sublingual blood collection, and rats were sacrificed after a 6-hour fasting period. The liver was rapidly removed for biochemical and histological analysis.

[0132] All animal procedures were performed in accordance with standard protocols and standard recommendations for the proper care and use of laboratory animals.

[0133] Tissue embedding and dissection

[0134] First, liver sections were fixed in 4% formalin solution (Merck Sigma, France) for 12–24 hours. Then, the liver sections were dehydrated in ethanol solutions (continuous baths of 70% and 100% ethanol). Next, the liver sections were incubated in isopropanol, followed by two baths in liquid paraffin (60°C). The liver sections were then placed in a rack (ref 11670990, Fisher Scientific, USA) and filled with Histowax® (ref F / 00403, Microm, France) to completely cover the tissue. The paraffin block containing the tissue sections was removed from the rack and stored at room temperature. The liver sections were then cut into 3 µm sections.

[0135] Sirius Red Dye

[0136] Liver sections were deparaffinized, rehydrated, and incubated for 15 minutes in 0.04% Fast Green solution (Sigma, catalog number F7258-25G). Subsequently, the liver sections were rinsed in 0.5% acetic acid solution and incubated for 30 minutes in a mixture of 0.1% Sirius Red (Direct Red, Alfa Aesar, Germany, catalog number B21693-25G; and picric acid solution, Sigma, catalog number P6744) and 0.04% Fast Green solution. The sections were dehydrated and mounted on CV Mount medium (Leica, US, catalog number 14046430011). Collagen proportionality area was assessed by morphological quantification of the Sirius positive area relative to the liver section area.

[0137] Histological examination

[0138] Virtual slides were generated using a Pannoracic 250 scanner from 3D Histech (Hungary). For each animal, fibrosis was assessed on Sirius Red and Fast Green stained sections based on the stage of fibrosis classification according to collagen reservoir location and hepatic structural pathological patterns, providing an indication of relative severity and disease progression. F0: No fibrosis; F1: Perisinusoidal or periportal fibrosis; F2: Perisinusoidal and portal / periportal fibrosis; F3: Bridging fibrosis; F4: Cirrhosis.

[0139] plasma analysis

[0140] Plasma concentrations of α-2 macroglobulin were determined by ELISA using the Abcam kit (catalog number ab157730, UK). The color intensity measured at 450 nm was proportional to the amount of a2M bound in the initial step. Sample values ​​were then derived from a standard curve. Results are expressed in ng / mL.

[0141] Total bilirubin concentration was measured using the Randox kit (Randox, catalog number BR 8377) for Daytona Enhanced Automation. In short, bilirubin is oxidized by vanadate at approximately pH 2.9 to produce biliverdin. In the presence of detergent and vanadate, both bound and unbound bilirubin are oxidized. This oxidation reaction causes a decrease in the optical density of the yellow color, specific to bilirubin. The decrease in optical density at 450 / 546 nm is proportional to the total bilirubin concentration in the sample.

[0142] Albumin concentration was measured using the Randox kit (Randox, catalog number AB 8301) for Daytona Enhanced Automation. In short, albumin measurement is based on its quantitative binding with the indicator 3,3',5,5'-tetrabromo-m-cresolphthalide (bromocresol green). The albumin-BCG complex exhibits maximum absorption at 578 nm.

[0143] result

[0144] TAA administration at 16 weeks induced cirrhosis in rats, as shown by histological scoring of F4 in all animals (Figure 2).

[0145] Bilirubin is a result of blood matrix metabolism (primarily derived from heme in red blood cells). The liver is responsible for clearing bilirubin from the blood. High plasma total bilirubin is a marker of liver dysfunction. As expected, plasma total bilirubin increased after 16 weeks of TAA administration. NTZ treatment significantly reduced plasma total bilirubin (Figure 3).

[0146] Albumin is synthesized in the liver. In any liver disease, a decrease in plasma albumin reflects impaired synthesis. As expected, plasma albumin decreased after 16 weeks of TAA administration, but was salvaged by NTZ treatment (Figure 4).

[0147] Of concern, these beneficial effects on liver function were independent of fibrosis, because the 4-week intervention with NTZ was too short to observe a significant effect on liver fibrosis (7.53% ± 0.64% collagen-proportional area in the NTZ case compared to 7.74% ± 0.38% in the TAA control group, p = 0.77). Multivariate analysis confirmed the independent effects of NTZ on both liver function (bilirubin or albumin) and fibrosis.

[0148] In summary, these results indicate that NTZ has a protective effect on liver detoxification and synthesis functions in animals with cirrhosis, independent of its antifibrotic effect.

[0149] Example 4: NTZ prevention of decompensation in an acute exacerbation model of chronic liver failure (ACLF)

[0150] Preclinical ACLF model

[0151] Male Sporgodory rats (175-200 g) (Janvier Labs, France) were induced to have cirrhosis by intraperitoneal injection of thioacetamide (TAA) at a dose of 150 mg / kg during the first week and then at a dose of 200 mg / kg three times a week for the next 14 weeks. Rats were stratified into treatment groups (n=5 / group) based on mean plasma levels of fibrosis markers (α-2 macroglobulin and TIMP1) obtained from sublingual blood samples and liver function tests (total bile acids and albumin).

[0152] Animals were caged in pairs in a controlled environment with a 12:12-hour light-dark cycle, and the animals had free access to food and water. Their weight and food intake were monitored twice a week.

[0153] Following the induction of cirrhosis, rats were given a single intraperitoneal injection of 0.05 mg / kg lipopolysaccharide (LPS from E. coli O111:B4, Sigma-Aldrich) to induce ACLF, which was discontinued one week after TAA administration. Rats were administered NTZ or a mediator at a dose of 50 mg / kg / day via oral gavage for 3 days, followed by a final dose 30 minutes before ACLF induction (Figure 5). Plasma was collected from sublingual blood samples shortly before sacrifice.

[0154] All animal procedures were performed in accordance with standard protocols and standard recommendations for the proper care and use of laboratory animals.

[0155] plasma analysis

[0156] As described in Example 3, plasma concentrations of α-2 macroglobulin were determined by ELISA using the Abcam kit (catalog number ab157730), and plasma concentrations of albumin were measured using the Randox kit (Randox, catalog number AB 8301) for Daytona Enhanced Automation.

[0157] The concentration of total bile acids was measured using a suitable Randox kit (Randox, catalog number BI 3863) for Daytona Enhanced Automation. In the presence of thioNAD, the enzyme 3-α-hydroxysteroid dehydrogenase (3-a HSD) converts bile acids to 3-ketosteroids and thioNADH. The reaction is reversible, and 3-a HSD converts 3-ketosteroids and thioNADH to bile acids and thioNAD. In the presence of excess NADH, the enzyme cycle occurs efficiently, and the rate of thioNADH formation was determined by measuring specific changes in absorbance at 405 nm.

[0158] Plasma TIMP-1 levels were measured using a quantitative sandwich ELISA assay from R&D Systems (catalog number RTM100). Sample values ​​were then calculated from the standard curve.

[0159] result

[0160] In cirrhotic rats, administration of LPS induced liver failure and death within 24 hours. In this experiment, NTZ treatment delayed death by 1 hour and allowed 1 in 5 rats to survive, while all animals in the mediator group died within 8 hours (Figure 6). Furthermore, NTZ reduced circulating bile acid levels, indicating improved liver function (Figure 7). Therefore, these results suggest that short-term NTZ treatment improves liver function and protects against decompensation in a liver failure model.

[0161] Example 5: NTZ improves cerebral edema in an ACLF rodent model induced by bile duct ligation and LPS injection.

[0162] Acute exacerbations of chronic liver failure are characterized by deterioration of organs other than acute hepatic decompensation. This study investigated the effect of NTZ on cerebral edema in a rat model of ACLF induced by bile duct ligation (BDL) – which induces liver injury and late fibrosis within 3 weeks – and by administration of lipopolysaccharide (LPS).

[0163] As described by Kountouras et al. (British journal of Experimental Pathology 1984), rats that underwent BDL (Brain Dysplasia) developed severe liver damage with late-stage fibrosis and bile duct hyperplasia 22 days after surgery.

[0164] Thirty-seven Sporgodory rats, weighing approximately 270 g, from the Charles River Laboratory (France) underwent bile duct surgery (BDL). After anesthesia with isoflurane (Vetflurane, Alcyon, France) and skin administration of 2% lidocaine (Alcyon), a medium-sized laparotomy was performed to expose the liver and duodenum. The main bile duct was identified and dissected. Subsequently, the bile duct was ligated in two sections: a first ligation in the middle of the bile duct and a second ligation above the entrance to the pancreatic duct. The bile duct was then dissected in the middle to prevent recanalization.

[0165] Animals were pretreated with 0.05 mg / kg butylpropionine (Buprecare, Alcyon) and 5 mg / kg carprofen (Carprofelican, Alcyon) before surgery. Animals received 0.05 mg / kg butylpropionine 4 hours after surgery and 5 mg / kg carprofen 24 and 48 hours after surgery.

[0166] Subsequently, after a minimum of 5 days of postoperative recovery, the animals were transferred to Genfit's animal facility (France). Fifteen days postoperatively, blood samples were collected via the sublingual vein under mild isoflurane (Isoflurin, Axience, France) anesthesia to measure markers of liver injury (serum aspartate aminotransferase (AST) and alkaline phosphatase (ALP)), thus stratifying the animals into treatment groups. Two BDL rats were excluded at this stage due to abnormal blood parameters, and four died or were euthanized for ethical reasons (a 10%-20% early mortality rate is expected with BDL surgery). Unmanipulated animals were included as healthy controls in the study.

[0167] Twenty-two days after BDL surgery, acute decompensation was induced by intraperitoneal administration of 1 µg / kg LPS (E. coli O111:B4, Sigma-Aldrich, Germany). Four BDL rats received phosphate-buffered saline (PBS, Fisher Scientific, USA) instead of LPS as a BDL control. Animals were continuously observed after LPS injection to assess the severity of inflammation and pain. In these rats, LPS injection induced a clearly visible hyperinflammatory response, including a reddening of the ears.

[0168] Treatment was administered via tube feeding with 100 mg / kg NTZ (Interchim, France) or a mordant (1% carboxymethyl cellulose (C4888, Sigma-Aldrich), 0.1% tween 80 (P8074, Sigma-Aldrich)) prior to LPS injection.

[0169] Three hours after LPS injection, blood samples were taken via the sublingual vein under light anesthesia, as previously described, and the animal was euthanized by cervical dislocation. Liver, spleen, kidney, and brain were collected.

[0170] Animals were handled carefully to minimize stress. All experiments were conducted in accordance with the French Ministry of Agriculture guidelines (Law 87-848) for laboratory animal experiments. The studies were conducted in accordance with the Animal Health Regulation (Directive No. 2010 / 63 / UE of the meeting of 22 September 2010 on animal protection and French Decree No. 2013-118 of 1 February 2013). [Group] [Compound] [animal / ] [Group] [Notes] healthy none 4 Untreated healthy control group BDL+PBS none 4 The liver fibrosis control group was discontinued 22 days after BDL. BDL+LPS+Carrier none 13 The ACLF control group was terminated 22 days after BDL and 3 hours after LPS. BDL+LPS+NTZ NTZ (100 mg / kg) 13 NTZ treatment was discontinued 22 days after BDL and 3 hours after LPS.

[0171] To assess cerebral edema, fresh brains were weighed, dehydrated by heating for 4 hours, and weighed again to calculate the water percentage.

[0172] result

[0173] Liver injury was significantly associated with increased cerebral edema, as assessed by the percentage of water in the brain (Figure 8). Administration of NTZ accompanied by LPS injection significantly reduced cerebral edema. Therefore, this experiment suggests that NTZ can be used to treat or prevent cerebral edema.

[0174] Example 6: NTZ improves renal function in an ACLF rodent model induced by bile duct ligation and LPS injection.

[0175] This study investigates the effects of NTZ on renal function markers in a rat model of ACLF induced by bile duct ligation (BDL) – which induces liver injury and advanced fibrosis within 3 weeks – and by administration of lipopolysaccharide (LPS).

[0176] Materials and Methods

[0177] As in previous examples, BDL surgery is typically performed on rats and in cases of acute decompensation. Group compound Animals / Groups Notes healthy none 4 Untreated healthy control group BDL+PBS none 4 The liver fibrosis control group was discontinued 22 days after BDL. BDL+LPS+Carrier none 13 The ACLF control group was terminated 22 days after BDL and 3 hours after LPS. BDL+LPS+NTZ NTZ (100 mg / kg) 13 NTZ treatment was discontinued 22 days after BDL and 3 hours after LPS.

[0178] Kidney damage was assessed by measuring serum cystatin C concentration using an ELISA (Quantikine® ELISA, MSCTC0, R&D Systems, mouse / rat).

[0179] result

[0180] LPS injection induced renal changes, as shown by an increase in serum cystatin C levels (Figure 9). NTZ treatment significantly reduced cystatin C, almost to the level of healthy controls. Taken together, these results confirm the efficacy of NTZ in improving peripheral organ (particularly brain and kidney) damage in ACLF.

[0181] Example 7: NTZ reduces liver damage in acetaminophen-induced acute liver failure.

[0182] Acute liver failure was induced in mice by administration of toxic doses of acetaminophen (APAP). After a 5-day acclimatization period, 8-week-old male C57BL / 6J mice (Janvier, France) were stratified into three groups of 10 mice each based on their body weight. All mice were fasted overnight before intraperitoneal injection of 300 mg / kg APAP. Thirty minutes prior to APAP poisoning, mice were administered via gavage with a mordant (1% carboxymethyl cellulose (C4888, Sigma-Aldrich), 0.1% Tween 80 (P8074, Sigma-Aldrich)), 50 mg / kg NTZ (Interchim, France), or 1200 mg / kg N-acetylcysteine ​​(NAC), serving as a reference treatment for APAP poisoning. Immediately after APAP injection, mice had free access to food and water.

[0183] Six hours after APAP administration, blood was collected from all mice via posterior orbital sinus puncture under anesthesia. Liver injury was assessed using a Horiba Pentra 400 instrument and related Pentra analysis kit (Horiba France SAS, France) by plasma aspartate aminotransferase (AST) measurement. Twenty-four hours after APAP administration, animals were euthanized by cervical dislocation, bled with saline, and liver samples were collected and weighed.

[0184] Animals were handled carefully to minimize stress. All experiments were conducted in accordance with the French Ministry of Agriculture guidelines (Law 87-848) for laboratory animal experiments. Research was conducted in accordance with animal health regulations (Directive No. 2010 / 63 / UE of the meeting of 22 September 2010 on animal protection and French Decree No. 2013-118 of 1 February 2013).

[0185] result

[0186] The results showed that APAP poisoning induced a significant increase in plasma AST (>6000 U / L in healthy mice, compared to <100 U / L). Unexpectedly, NTZ treatment drastically reduced AST to levels similar to those in the NAC (Non-Acute Acute) reference treatment for APAP poisoning (Figure 10). These results demonstrate the effectiveness of NTZ treatment in patients with drug-induced acute liver failure (ALF).

[0187] Example 8: NTZ improves renal function markers

[0188] Uncompensated cirrhosis is often associated with altered renal function. This study investigated the role of NTZ in preventing renal injury in an acute exacerbation of chronic liver failure (ACLF) model. In short, ACLF was induced in rats with advanced liver fibrosis / cirrhosis induced by repeated carbon tetrachloride (CCl4) administration for 15 weeks.

[0189] The study program consists of four phases: 1) Pre-sensitization period: Phenobarbital (P5178, Sigma Aldrich) was added to the drinking water (35 g / dl) of 32 four-week-old male Sporgodory rats (~200 g, Janvier Lab) for 2 weeks to activate cytochrome enzymes that metabolize CCl4. 2) Induction of liver fibrosis: Chronic liver damage was induced by repeated (twice a week) oral administration of CCl4 (ref: 289116, Sigma Aldrich; diluted in olive oil) (starting at 0.10 ml / kg, increasing the dose by 0.05-0.10 ml / kg at each administration, reaching 0.85 ml / kg at week 7 and thereafter) for up to 15 weeks. The final dosage is as follows: Week 1: 0.10 ml / kg 0.20 ml / Kg Week 2: 0.25 ml / kg 0.30 ml / Kg Week 3: 0.35 ml / kg 0.40 ml / Kg Week 4: 0.45 ml / kg 0.50 ml / Kg Week 5: 0.60 ml / kg 0.70 ml / Kg Week 6: 0.80 ml / kg 0.85 ml / Kg 0.85 ml / kg from week 7 onwards 3) Treatment period: After 15 weeks of CCl4 administration, discontinue CCl4 tube feeding one week before LPS administration. Administer mediator (1% carboxymethyl cellulose (C4888, Sigma-Aldrich), 0.1% Tween 80 (P8074, Sigma-Aldrich)) or NTZ (50 mg / kg BID) orally for 3 days prior to LPS challenge. Administer the final dose (50 mg / kg) of mediator or NTZ 30 minutes before the scheduled LPS challenge. 4) LPS challenge: Intraperitoneal injection of 0.03 mg / kg of LPS (L2630, Sigma Aldrich) from Escherichia coli O111:B4. Group compound Animals / Groups Notes Olive Oil W10 none 6 Non-pathological controls were terminated at week 10. CCl 4W10 none 12 The liver fibrosis control group was terminated at week 10. ACLF-mediator none 10 LPS attack after CCl 415 weeks ACLF-NTZ NTZ (50 mg / kg BID) 10 LPS attack after CCl 415 weeks

[0190] Animals were continuously observed for several hours after LPS administration to assess the severity of inflammation and pain. Animal appearance (fur, skin color changes), activity and alertness (response to stimuli, eyelid opening, movement, etc.), gait, and posture were monitored. Any animal showing clear signs of suffering and impending death was euthanized. In both the NTZ and carcass groups, 6 out of 10 animals survived the LPS injection. All surviving animals were euthanized 24 hours after LPS attack for plasma and tissue analysis. All animal procedures were performed according to standard protocols and in accordance with standard recommendations for the appropriate care and use of laboratory animals.

[0191] Renal injury was assessed using plasma creatinine, urea, and cystatin C concentrations. Plasma creatinine and urea concentrations were measured using the appropriate Randox kits (CR 8316 and UR 8334, respectively) for Daytona Enhanced Automation (Randox, catalog number BR 8377) according to the manufacturer's instructions. Plasma cystatin C concentrations were determined by ELISA (Quantikine® ELISA for mice / rat, MSCT0, R&D Systems).

[0192] The results showed that LPS injection 15 weeks after CCl4 administration impaired renal function, as evidenced by increases in plasma creatinine, urea, and cystatin C concentrations (Figure 11). NTZ treatment significantly prevented renal damage by restoring creatinine, urea, and cystatin C levels to those of the olive oil control.

[0193] Example 9: Evaluation of the efficacy of NTZ in rats against acute exacerbations of CCl4+ LPS-induced chronic liver failure.

[0194] The objective of this study was to evaluate the efficacy of NTZ in preventing LPS-induced liver and kidney damage in Sporgodory rats with CCl4-induced severe fibrosis.

[0195] This study comprises four phases: 1) Pre-sensitization period: Phenobarbital (35 g / dL) (ref P5178-25G, Sigma-Aldrich) was added to the drinking water of 4-week-old male Sporgodory rats (~200 g) for 2 weeks before CCl4 administration. 2) Induction period: Severe liver fibrosis was induced by repeated oral administration of tetrachloride (CCl 4) for 10 or 15 weeks using the same regimen as described in Example 8 (twice a week, increasing the dose from 0.10 ml / kg to 0.85 ml / kg). 3) Treatment period: After CCl4 administration for 15 weeks, discontinue tube feeding one week before LPS administration. Administer the carrier or NTZ orally (50 mg / kg twice daily (BID)) for 3 days prior to ACLF induction. Administer the final dose (50 mg / kg) 30 minutes before ACLF induction. 4) ACLF induction: administer 0.03 mg / kg LPS via intraperitoneal injection.

[0196] For oral administration, NTZ powder (ref RQ550, Interchim) was dissolved at a concentration of 10 mg / mL in a 1% CMC aqueous solution (sodium carboxymethyl cellulose ref C48 88-500G, Sigma Aldrich) and a 0.1% Tween 80 aqueous solution in an amber glass bottle. The solution was homogenized with a polytron and treated with a 10% power sonicator for 10 seconds. NTZ was kept under magnetic stirring until it was administered to rats at a dose of 10 mL / kg in the dark.

[0197] LPS solution (E. coli O111:B4, Sigma-Aldrich, Germany) was prepared under a microbiological safety cabinet and dissolved in phosphate-buffered saline (PBS) at 15 µg / mL. The solution was aliquoted and frozen until the day of the experiment. LPS was administered to rats at a dose of 2 mL / kg.

[0198] Thirty-eight male Sporgodory rats (Janvier Labs, France) have been allocated to this study.

[0199] Throughout the study, animals had free access to water and food (ref E15000-04, Ssniff, Germany). After 49 weeks of CCl4 administration, animals were randomly assigned to two groups based on plasma ammonia and albumin levels (to ensure similar procedures in both groups).

[0200] All surviving animals were euthanized 24 hours after LPS injection for plasma and tissue analysis (60% survived in both the causative agent and NTZ groups). At the end of the study, ACLF was assessed by biochemical analysis of liver and kidney injury or functional biomarkers.

[0201] All animal procedures were performed in accordance with standard protocols and standard recommendations for the proper care and use of laboratory animals in accordance with European Directive 2010 / 63UE.

[0202] Throughout the study, weight and food intake were monitored twice a week.

[0203] During the fibrosis induction period, animals were observed once daily to monitor clinical signs. Observations included changes in skin, fur, eyes, the incidence of secretions and excretions, and spontaneous activity (lacrimation, piloerection, abnormal breathing patterns). Changes in gait, posture, stereotypes (e.g., excessive grooming, repetitive circling), or bizarre behaviors (self-mutilation, walking backwards, etc.) were also monitored. Animals were euthanized if they experienced a weight loss of more than 25% for more than three consecutive days, lack of feeding, deterioration of general condition, or vocalization.

[0204] Following administration of LPS, animals were continuously observed for 7 hours to assess the severity of inflammation and pain. Assessment considered the animal's appearance (fur, skin color changes), activity and alertness (response to stimuli, eyelid opening, activity level, etc.), gait, and posture using a rating scale. Animals were euthanized if their general condition deteriorated (cold to the touch, inability to stand, vocalization, difficulty breathing).

[0205] At the end of the study, blood was collected via sublingual vein puncture under mild isoflurane anesthesia and transferred to EDTA, serum gel, and lithium heparin tubes. The EDTA and lithium heparin tubes containing blood were rapidly centrifuged, and the plasma fraction was collected. The lithium heparin plasma aliquot was flash-frozen in liquid nitrogen and stored at -80°C, the EDTA-plasma aliquot was stored at -20°C, and the serum-gel tubes were kept at room temperature for 30 minutes, centrifuged, and stored at -20°C.

[0206] The animals were subsequently euthanized via cervical dislocation, and their heads were removed for brain resection and weighing. The liver, spleen, and right kidney were collected and weighed. Liver sections were fixed and embedded in paraffin for histological analysis. The remaining liver was flash-frozen in liquid nitrogen and maintained at -80°C for biochemical analysis.

[0207] Histological sample processing and analysis:

[0208] First, liver sections were fixed in 4% formalin solution (Merck Sigma) for 10 or 24 hours (weekly protocol). Then, the liver sections were dehydrated in ethanol solutions (70% and 100% ethanol baths). The liver sections were then incubated (60°C) in two baths of isopropanol, followed by two baths of liquid paraffin. These steps were performed on a LOGOS One (Milestone, MicromMicrotech). The liver sections were then placed in a rack (Fisher Scientific, ref 11670990) and gently filled with Histowax® (Microm, ref F / 00403) to completely cover the tissue. The paraffin block containing the tissue sections was removed from the rack and stored at room temperature. The liver sections were then cut into 3 µm sections using an HM340E electronic rotary microtome (ThermoScientific, MicromMicrotech).

[0209] Sirius red and Fast Green staining was performed on an automated staining system ST5030 (Leica). Liver sections were deparaffinized, rehydrated, and incubated for 15 minutes in 0.04% Fast Green solution (Sigma, catalog number F7258-25G). Subsequently, liver sections were rinsed in 0.5% acetic acid solution and incubated for 30 minutes in a Sirius red 0.1% solution (Direct Red, Alfa Aesar, catalog number B21693-25G; and picric acid solution, Sigma, catalog number P6744)-Fast Green 0.04% solution. Sections were dehydrated and mounted on CV Mount medium (Leica, catalog number 14046430011) on an automated coverslip CV5030 (Leica). Histological examination and scoring were performed on the treatment group without differentiation. Images were acquired using a Pannoramic 250 Flash II digital slide scanner (3DHistech) and analyzed in QuantCenter software.

[0210] It is worth noting that only animals that survived 24 hours after LPS (60%) could be analyzed.

[0211] Experimental results are presented as mean ± standard deviation and plotted as bar charts. For all variables following a normal distribution, the Stulton T test was used to compare results between groups (#: p < 0.05; ##: p < 0.01; ###: p < 0.001). In cases of varying variance between groups, the Wells test was applied (¤: p < 0.05; ¤¤: p < 0.01; ¤¤¤: p < 0.001). Logarithmic transformation was applied to plasma ALT and AST in all samples to obtain a normal distribution. For other variables with non-normal distributions, the nonparametric Mann-Whitney test was applied ($: p < 0.05; $$$: p < 0.01; $$$$: p < 0.001). Unless otherwise specified, differences from the control group at week 10 (W10) were reported.

[0212] result

[0213] Repeated CCl4 administration induced severe liver fibrosis with an F3 score in most animals.

[0214] LPS-induced ACLF resulted in a significant 40% mortality rate within 24 hours in both the mediator-treated and NTZ-treated groups (4 / 10 rats in both groups). At 24 hours post-LPS, liver and kidney injury and functional markers were assessed in surviving animals. CCl4 significantly increased plasma ALT and AST levels, markers of hepatocellular injury, while NTZ treatment drastically reduced ALT by 80% (p=0.007) and AST by 87% (p=0.005) (Figure 14). CCl4 administration did not alter plasma GGT, another marker of liver injury (undetectable), while LPS injection increased plasma GGT levels to 11.2 U / L (Figure 15). NTZ treatment neutralized this LPS-induced GGT increase (< 6.15 U / L).

[0215] Plasma total bilirubin and albumin—liver function markers—were also altered in the presence of CCl4 and LPS (Figure 16). NTZ treatment prevented LPS-induced liver function changes, as shown by an 80% reduction in total bilirubin (p=0.02) and a 95% recovery in albumin production (p=0.04).

[0216] LPS injection also alters renal function, as evidenced by increases in plasma creatinine, urea, and cystatin C concentrations (Figure 17), confirming that organs outside the liver are also affected in this ACLF model. NTZ treatment also prevents LPS-induced renal damage by reducing plasma creatinine (-102%, p=0.009), urea (-92%, p=0.002), and cystatin C (-166%, p=0.02).

[0217] Serum IFNγ was measured in animals treated with CCl4 for 415 weeks and LPS, while other intercytokines (IL6, TNFα, IL1β) may not have been detected (not shown). NTZ treatment significantly reduced serum IFNγ by 56% (p=0.04), confirming the anti-inflammatory effect of NTZ treatment in the ACLF model (Figure 18).

[0218] in conclusion

[0219] In rats with severe fibrosis (F3 / F4), a 3-day pretreatment with NTZ at 100 mg / kg / day alleviated liver damage (ALT, AST, GGT) and prevented LPS-induced changes in liver (total bilirubin, albumin) and kidney (creatinine, urea, cystatin C) function.

[0220] none< / isi>

Claims

1. Use of a compound selected from nitazoxanide (NTZ), tizoxanide (TZ), tizoxanide glucuronic acid (TZG), and pharmaceutically acceptable salts thereof in the preparation of a medicament for the treatment or prevention of liver failure in individuals of need, selected from the group consisting of acute exacerbations of chronic liver failure (ACLF) and acute liver failure (ALF).

2. For the purposes described in claim 1, it is used for the treatment or prevention of ACLF.

3. For the purposes of request item 1 or 2, it is used to treat ACLF.

4. For the purposes of claim 1 or 2, it is used to prevent individuals with ACLF from progressing from compensated cirrhosis to decompensated cirrhosis.

5. For the purposes of claim 1, wherein the individual suffers from ACLF and compensated cirrhosis.

6. For the purposes of claim 1, wherein the individual suffers from ACLF and decompensated cirrhosis.

7. As requested in claim 1, it is used to reverse decompensated cirrhosis in an individual with ACLF to compensated cirrhosis.

8. For the purposes of request item 1 or 2, where the individual suffers from grade 2 or 3 ACLF.

9. As used in request item 1 or 2, wherein the individual has at least one ACLF-inducing event.

10. As claimed in claim 9, wherein the precipitating event is selected from alcoholic hepatitis; bacterial, fungal or viral infection; sepsis, poisoning; visceral bleeding and drug-induced liver failure.

11. For the purposes of request 1 or 2, wherein the individual suffers from ACLF caused by a hepatic or extrahepatic condition.

12. For the purposes of claim 1 or 2, it is used to prevent liver decompensation in individuals with ACLF.

13. For the purposes described in claim 1, it is used for the treatment or prevention of ALF.

14. For the purposes described in claim 13, it is used to treat or prevent drug-induced ALF.

15. For the purposes described in claim 14, it is used to treat or prevent acetaminophen-induced ALF.

16. Use of a compound selected from nitrozonidine (NTZ), TZ (G) and their pharmaceutically acceptable salts in the preparation of a medicament for the prevention of extrahepatic organ failure in individuals with cirrhosis.

17. For the purposes of claim 16, wherein the individual suffers from compensated cirrhosis or uncompensated cirrhosis.

18. For the purposes of claims 16 or 17, where the individual suffers from decompensated cirrhosis.

19. For the purposes of claims 16 or 17, where the individual suffers from alcoholic cirrhosis.

20. As requested in paragraphs 16 or 17, it is for the prevention of kidney failure.

21. As requested in item 16 or 17, where the individual has ACLF but does not have kidney failure.

22. For the purposes of request 16 or 17, wherein the individual suffers from ACLF and is accompanied by non-renal organ failure and renal dysfunction.

23. For the purposes of claim 1, it is used for further prevention of hepatic encephalopathy.

24. For the purposes described in claim 1, it is used for further treatment or prevention of cerebral edema.

25. For the purposes of claim 1, it is used for further prevention of cerebral edema.

26. As requested in claim 1, it is used in combination with a liposome-based toxin scavenger.

27. As claimed in claim 26, wherein the liposome-based toxin scavenger is an ammonia scavenger.

28. As claimed in claim 26 or 27, wherein the liposome-based toxin scavenger is intended for delivery to the peritoneum.

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