Treatment of liver failure

Abstract

The present invention stems from the unexpected discovery that a combination of a stem cell mobilizer, such as G-CSF, and a TLR4 antagonist is beneficial for patients with liver failure, such as acute liver failure (ALF) or acute exacerbation of chronic liver failure (ACLF). The present invention utilizes these discoveries to provide a stem cell mobilizer, such as G-CSF, and a TLR4 antagonist that can be used in the treatment or prevention of liver failure, such as ALF or ACLF. The provision of stem cells and a TLR4 antagonist for use in the treatment or prevention of liver failure, such as ALF or ACLF, is also encompassed by the present invention.

Description

[Technical Field]

[0001] The present invention stems from the unexpected finding that stem cell mobilizers, such as G-CSF, have deleterious effects in liver failure, e.g., acute liver failure (ALF) or acute-on-chronic liver failure (ACLF), by exacerbating inflammation and tissue injury, but that their combination with an antagonist of TLR4 acts synergistically by preventing tissue injury-driven inflammation and promoting stem cell-associated pro-regenerative properties.

[0002] The present invention takes advantage of these findings to provide stem cell mobilizers, such as G-CSF, and antagonists of TLR4 that can be used in the treatment or prevention of liver failure, such as ALF or ACLF.

[0003] Also encompassed by the present invention is the provision of stem cells and TLR4 antagonists for use in the treatment or prevention of liver failure, eg, ALF or ACLF. [Background technology]

[0004] Acute decompensation events (AD), either alone or in combination with bacterial infection, massive ascites, GI bleeding, or hepatic encephalopathy, are the most common hospital-presenting manifestations of cirrhotic liver disease and can be successfully managed in most cases. However, 30% of patients will present with or develop rapidly progressive hepatic and / or extrahepatic organ failure, a condition called acute onset of chronic liver failure (ACLF). Approximately 40% of these patients will progress to multiple organ failure and death. There is currently no specific treatment for ACLF (1).

[0005] Acute-on-chronic liver failure (ACLF) affects approximately one in three patients hospitalized with complications of cirrhosis. ACLF is diagnosed by use of the Chronic Liver Failure (CLiF) Consortium criteria, which subdivide ACLF severity into grades 1, 2, and 3 depending on the number of organ failures. The CLiF Consortium Acute-on-chronic Liver Failure (CLiF-C ACLF) score is superior to traditional scores, such as the Child-Pugh score or the MELD score, in predicting mortality in this cohort (2).

[0006] The pathobiology of ACLF is characterized by immunopathological features involving two opposing mechanisms as the primary drivers of organ failure. The first is systemic and hepatic inflammation induced by pathogen-associated molecular patterns (PAMPs) and cell death molecules (damage-associated molecular patterns (DAMPs)) mediated through the TLR4 receptor pathway. PAMPs are derived from the portal vein-mediated translocation of gut-derived bacteria and bacterial products (e.g., lipopolysaccharide - LPS) to the liver, promoting proinflammatory responses via the Toll-like 4 (TLR4)-dependent canonical inflammasome in immune cells and the TLR4-independent non-canonical inflammasome in hepatocytes (3). TLR4 antagonists have been shown to be effective in treating conditions such as ACLF (WO2011GB01227) (4). The second is long-term organ injury, immune cell paralysis, and regenerative failure, which promote long-term organ dysfunction and secondary infections, ultimately resulting in a vicious cycle leading to multiple organ failure and death. Stem cells and other regulatory immune cells are known to modulate immune activation, thereby modulating inflammatory responses and supporting tissue repair (5). Granulocyte-colony-stimulating factor (G-CSF) is recognized to mobilize stem cells and immune cells and possess immunomodulatory and pro-regenerative properties. Data from a single-center study in India demonstrated the efficacy of G-CSF in patients with ACLF, improving 60-day survival from 30% to 70% (6). Similar data from other Asian centers confirmed the clinical benefit, but also included only small numbers of patients (7, 8). These results could not be confirmed in more rigorously conducted European clinical trials (9, 10). The discrepancy in results was attributed to unbalanced patient randomization, inadequate clinical classification included in the Asian trials, and possibly type I statistical error. This discrepancy set the stage for the GRAFT trial, a large multicenter study in Germany supported by the German Research Foundation (DFG). The GRAFT trial (NCT02669680) recruited 163 patients with ACLF according to the European CLIF criteria, and a planned interim analysis was performed.Data clearly showed that G-CSF had no effect on mortality in patients with ACLF and may be harmful in subgroups of patients, e.g., patients with ACLF according to APASL criteria and alcoholic hepatitis (11). Summary of the Invention

[0007] The present invention is based on the discovery that inhibiting TLR4 prevents G-CSF-mediated mortality and promotes G-CSF-dependent liver tissue repair. Thus, the present invention utilizes a combination of a stem cell mobilizer, such as G-CSF, and a Toll-like receptor 4 antagonist in the treatment and prevention of liver disease, such as ALF or ACLF, and symptoms and conditions associated with liver disease. The synergistic effect consists of enhanced hepatocyte proliferation and reduced liver injury.

[0008] Thus, the present invention provides: A stem cell mobilizer and a TLR4 antagonist for use in a method for treating or preventing liver failure in an individual in need thereof.

[0009] The present invention also provides: A method of treating or preventing liver failure in an individual in need thereof, comprising administering to said individual a stem cell mobilizer and an antagonist of TLR4; A stem cell mobilizer for use in a method for treating or preventing liver failure in an individual in need thereof, the method comprising the additional administration of an antagonist of TLR4 to the individual.

[0010] The present invention also provides: an antagonist of TLR4 for use in a method for treating or preventing liver failure in an individual in need thereof, said method also comprising administration of stem cells to the individual in need thereof; A method of treating or preventing liver failure in an individual in need thereof, comprising administering to said individual stem cells and an antagonist of TLR4; Stem cells for use in a method for treating or preventing liver failure in an individual in need thereof, said method comprising the additional administration of an antagonist of TLR4 to the individual.

[0011] The present invention provides a combination of a stem cell mobilizer and a TLR-4 antagonist for use in a method for treating or preventing liver failure, e.g., ALF or ACLF, or a method for treating an individual suffering from liver failure, e.g., ALF or ACLF. The present invention provides a composition comprising a stem cell mobilizer and a TLR-4 antagonist for use in a method for treating or preventing liver failure, e.g., ALF or ACLF, or a method for treating an individual suffering from liver failure, e.g., ALF or ACLF. The present invention provides a method for treating or preventing liver failure, e.g., ALF or ACLF, or a method for treating an individual suffering from liver failure, e.g., ALF or ACLF, comprising administration of a stem cell mobilizer and a TLR4 antagonist.

[0012] The present invention provides a stem cell mobilizer for use in a method for treating or preventing liver failure, e.g., ALF or ACLF, in an individual, or a method for treating an individual suffering from liver failure, e.g., ALF or ACLF, which method also includes administering to the individual an antagonist of TLR4.

[0013] The present invention provides antagonists of TLR4 for use in methods of treating or preventing liver failure, e.g., ALF or ACLF, in an individual, or methods for treating an individual suffering from liver failure, e.g., ALF or ACLF, which methods also include administering a stem cell mobilizer to the individual.

[0014] In a preferred embodiment, the stem cell mobilizer is G-CSF.

[0015] In a preferred embodiment, the liver failure is ACLF.

[0016] In particular, the present invention provides a G-CSF and a TLR-4 antagonist for use in a method of treating or preventing liver failure, e.g., ALF or ACLF, or treating an individual with liver failure, e.g., ALF or ACLF. The present invention provides a combination of a G-CSF and a TLR-4 antagonist for use in a method of treating or preventing liver failure, e.g., ALF or ACLF, or treating an individual with liver failure, e.g., ALF or ACLF.

[0017] Similarly, the invention provides the use of antagonists of G-CSF and TLR-4 in the manufacture of a medicament for use in the treatment or prevention of liver failure, e.g., ALF or ACLF, or in treating an individual suffering from liver failure, e.g., ALF or ACLF.

[0018] Similarly, the present invention provides a method of treating or preventing liver failure, such as ALF or ACLF, in an individual in need thereof, said method comprising administering to said individual G-CSF and an antagonist of TLR-4.

[0019] Similarly, the present invention provides a method of treating or preventing liver failure, e.g., ALF or ACLF, in an individual in need thereof, said method comprising administering to said individual an antagonist of TLR-4, wherein G-CSF is also administered to the patient.

[0020] The present inventors have shown that TLR4 antagonists inhibit inflammation and organ injury, reducing liver inflammation and thereby allowing hepatic stem cells to engraft. Accordingly, the present invention also contemplates a combination of stem cells and a TLR-4 antagonist for use in a method for treating or preventing liver failure, e.g., ALF or ACLF, or for treating an individual suffering from liver failure, e.g., ALF or ACLF. The present invention provides a composition comprising stem cells and a TLR-4 antagonist for use in a method for treating or preventing liver failure, e.g., ALF or ACLF, or for treating an individual suffering from liver failure, e.g., ALF or ACLF. The present invention provides a method for treating or preventing liver failure, e.g., ALF or ACLF, or for treating an individual suffering from liver failure, e.g., ALF or ACLF, comprising administering stem cells and a TLR4 antagonist.

[0021] The present invention provides stem cells for use in a method of treating or preventing liver failure, e.g., ALF or ACLF, in an individual or a method for treating an individual suffering from liver failure, e.g., ALF or ACLF, which method also includes administering to the individual an antagonist of TLR4.

[0022] The present invention provides antagonists of TLR4 for use in methods of treating or preventing liver failure, e.g., ALF or ACLF, in an individual, or methods for treating an individual suffering from liver failure, e.g., ALF or ACLF, which methods also include administering stem cells to the individual.

[0023] In a preferred embodiment, the stem cells are human stem cells.

[0024] In a preferred embodiment, the stem cells are hepatic stem cells.

[0025] In a preferred embodiment, the liver failure is ACLF.

[0026] The individual to be treated may suffer from liver cirrhosis, e.g., alcoholic cirrhosis. The individual to be treated may suffer from liver failure. The individual to be treated may suffer from paracetamol overdose. The individual may suffer from hepatorenal syndrome (HRS). The individual may suffer from or be at risk of one or more of the following, when compared to a subject without liver disease: renal dysfunction; renal failure; HRS; cerebral dysfunction and swelling; increased plasma creatinine; increased plasma ammonia; increased liver enzyme concentrations; increased inflammation, injury, or dysfunction in the liver and / or kidney and / or brain and / or blood circulation; liver tissue damage due to liver failure; acute liver failure, alcoholic hepatitis, and / or liver reperfusion injury. In a preferred embodiment, the individual suffers from ACLF. In a preferred embodiment, the individual suffers from ALF. In a preferred embodiment, the individual suffers from alcoholic hepatitis (AH). In a preferred embodiment, the individual has non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH). Individuals with AH, NAFLD, or NASH may also have ACLF.

[0027] A TLR4 antagonist for use according to the present invention may lead to (a) a decrease in the expression of TLR4 in the liver and / or kidneys and / or brain of an individual, and / or (b) a decrease in the level of TLR4 in the liver and / or kidneys and / or brain of an individual, and / or (c) a decrease in the activity of TLR4 in the liver and / or kidneys and / or brain of an individual, which would result in less inflammation and pro-inflammatory cytokine production and less dysfunction of organs, e.g., the liver and / or kidneys and / or brain, and / or (d) a decrease in the level of TLR4 in the urine of an individual. [Brief explanation of the drawings]

[0028] [Figure 1A-B]Figure 1 shows that G-CSF increases mortality in rodents with ACLF. A) C57B / 6 mice were gavaged with carbon tetrachloride (CCL4) for 6 weeks (n=6) to induce chronic liver injury. Subsequently, an injection of LPS (Klebsiella, 4 mg / kg, i.p.) served as a second hit to induce organ injury. Mice were treated with recombinant murine G-CSF (250 μg / kg, sc) for 5 consecutive days. B) A total of 50% of all G-CSF-treated animals died within 48 hours after LPS injection, whereas all other animals survived the entire treatment episode. [Figure 1C] C) Five days of G-CSF treatment in this ACLF mouse model had a profibrotic effect in the liver (Sirius Red). Similarly, G-CSF treatment maintained macrophage infiltration (F4 / 80+) after five days. CCL4 = carbon tetrachloride; G-CSF = granulocyte colony-stimulating factor. The data show that in the CCl4 + LPS model, G-CSF increased mortality, liver fibrosis, and inflammatory cell infiltrates. [Figure 2A-B]Figure 1 shows organ sensitization to endotoxin due to TLR4 upregulation in liver fibrosis. A) TLR4 expression in liver tissue (immunohistochemistry) from rats in the ALF and ACLF models (10x magnification (n = 4 per group)). Liver tissue from control animals showed low TLR4 expression (A1, A5). When chronic injury was induced by BDL, there was clear TLR4 upregulation in hepatocytes, whereas regenerating areas did not express TLR4 (A2). LPS injection did not significantly alter this pattern (A3). Pretreatment with recombinant alkaline phosphatase (recAP) (1000 U / kg, i.p.), a drug that dephosphorylates and inactivates LPS, for 4 days reduced TLR4 expression in hepatocytes (A4). In the ALF model, neither GalN (A6, A7) alone or in combination with LPS nor pretreatment with recAP altered TLR4 expression (A8). B) TUNEL staining (cell death) of liver tissue confirmed that chronic liver injury is associated with organ sensitization to endotoxin, likely through TLR4 upregulation, as LPS injection after BDL leads to an expansion of the area of ​​cell death (7), whereas the same LPS dose in animals with untreated livers showed only a low degree of cell death (3). The LPS effect could be reversed by recAP pretreatment (8). [Figure 2C-D] C) TLR4+ liver-infiltrating CD45+ cells were increased after G-CSF treatment. D) Using the TLR4 inhibitor (TAK-242) (10 mg / kg, i.p.), we were able to provide evidence that organ sensitization associated with chronic liver injury is mediated by TLR4. Injection of TAK-242 before or after LPS injection after BDL significantly reduced liver injury, as indicated by liver cell death (TUNEL) and ALT levels. [Figure 3A-B]Figure 1 shows that a TLR4 antagonist prevents G-CSF-associated mortality and ameliorates liver injury. A) C57B / 6 mice were gavaged with CCL4 for 6 weeks (n = 4-10) to induce chronic liver injury. Subsequently, an injection of LPS (Klebsiella, 4 mg / kg, i.p.) served as a second hit to induce organ injury. Mice were treated with recombinant murine G-CSF (250 μg / kg, sc) + / - TAK-242 (10 mg / kg, i.p.) twice for 24 hours. B) LPS injection + / - G-CSF in animals after CCL4 gavage led to a significant increase in ALT levels and cell death (TUNEL), which was prevented by the addition of TAK-242. [Figure 3C-D] C) Liver-infiltrating macrophages (F4 / 80+) and neutrophil granulocytes (Ly6G+) were significantly reduced by adding TAK-242 to G-CSF. D) Adding TAK-242 to G-CSF improved animal survival from 50% to 100% after 48 hours. [Figure 4-1] Figure 1 shows the synergistic regenerative effects of G-CSF and TAK-242 on ACLF. A) C57B / 6 mice were gavaged with CCL4 for 6 weeks (n = 10) to induce chronic liver injury. Subsequently, an injection of LPS (Klebsiella, 4 mg / kg, i.p.) served as a second hit to induce organ injury. Mice were treated with recombinant murine G-CSF (250 μg / kg, sc) + / - TAK-242 (10 mg / kg, i.p.) for either 24 hours or 5 days. [Figure 4-2]B) CDKN2A (p16), a key marker of cell cycle arrest (cellular senescence), was upregulated at the mRNA level after LPS injection but gradually decreased with G-CSF, TAK-242, and then combination therapy. As highlighted by the white-shaded area, LPS injection in CCL4 animals led to hepatocellular injury surrounded by Trp53 (p53)-positive (senescent) hepatocytes. G-CSF alone failed to reduce organ injury (white-shaded area) but numerically reduced the number of p53-positive hepatocytes. After 5 days of treatment with G-CSF + / - TAK-242, the number of senescent hepatocytes plummeted significantly. [Figure 4-3] C) Cyclin A is a marker of cell cycle progression (proliferation). It primarily regulates the transition from G2 to M phase. In control animals, the number of proliferating hepatocytes (white arrowheads) was low. After CCL4 gavage, and subsequently after LPS injection, there was no increase in cyclin A-positive hepatocytes, despite increasing amounts of proliferating immune cells. Treatment with G-CSF for 5 days, with or without TAK-242, significantly increased the number of proliferating hepatocytes, but with a single TAK-242 treatment, the cells were not proliferative. [Figure 4-4] D) Hepatic cytokine expression (mRNA) (IL6, TNFα) was increased in ACLF, but a single treatment with G-CSF could be reduced by TAK242. Thus, combinatorial therapy could significantly reduce liver inflammation. [Figure 4-5] E) BCL2 is an effector molecule of the STAT3 pathway that possesses anti-apoptotic and antibacterial properties. Combination therapy increased BCL2 expression in the liver (Western blot). F) Combination therapy with TAK-242 and G-CSF activated the STAT3 pathway and increased the expression of anti-apoptotic BCL2 (pooled liver lysates from all animals per group). Image quantification was performed using Image J, and group comparisons were performed by one-way ANOVA with post-hoc Tukey's multiple comparisons. mRNA data are expressed as ddCt values. Western blots were performed using pooled protein lysates from all animals per group. [Figure 4-6] G) Hepatic protein expression of markers of inflammation, cell death, and regeneration / senescence (CCl4-LPS 24-hour treatment model). Liver lysates from four randomly selected animals per group were pooled and applied to one profiler membrane. Densitometry was measured using Image J, and values ​​are expressed as mean pixel density. After sample pooling, statistical comparisons were not possible. [Figure 4-7] H) Liver aSMA expression (CCl4-LPS 24-hour treatment model). Liver sections were stained for aSMA as a marker of astrocyte activation (n = 4 per group). LPS, when combined with G-CSF, increased aSMA-positive area from 1.4% ± 0.6 to 8.6% ± 1.9 (p < 0.001) and 9% ± 4.1 (p < 0.001) in CCl4 animals. Addition of TAK-242 to G-CSF significantly reduced astrocyte activation (aSMA-positive area 3.3% ± 1 compared to CCl4 + LPS + G-CSF, p < 0.001). Image quantification was performed using Image J, and group comparisons were performed by one-way analysis of variance with post-hoc Tukey's multiple comparisons. [Figure 4-8]I) THP1 response to LPS after G-CSF stimulation. THP1 cells were PMA-activated to a macrophage-like phenotype and stimulated with LPS (10 ng / mL) ± G-CSF (100 ng / mL) (two batches in two replicates). Treatment was performed with 200 nM TAK-242 in two individuals per group. RNA extraction for measuring cytokine responses was performed 3 hours after LPS incubation. LPS induced a 60.2-fold upregulation of IL-6 mRNA expression in THP1 monocytes and a 6.2-fold upregulation in macrophages, which was enhanced by preincubation with G-CSF (71-fold in monocytes and 6.7-fold in macrophages). TAK-242 reduced IL-6 mRNA expression by 5.1-fold in monocytes and 2.8-fold in macrophages compared to unstimulated cells, a finding of similar biological significance. In monocytes, LPS incubation increased IL-1b expression by 1.8-fold and reduced expression by 0.8-fold in macrophages. G-CSF exaggerated IL-1b expression by 2.5-fold compared to unstimulated cells, but remained unchanged in macrophages (1.1-fold compared to unstimulated cells). TAK-242 reduced the IL-1b response in both cell lines (1.3-fold in monocytes and 0.7-fold in macrophages compared to unstimulated cells). [Figure 4-9]J) Proliferative effect of G-CSF after CCl4. To confirm that G-CSF exerts its proliferative effect in an environment without LPS-driven inflammation, G-CSF was used as a treatment after CCl4 administration. C57B / 6 mice were gavaged with 0.5 ml / ml of carbon tetrachloride (CCl4) for 6 weeks to induce chronic liver injury. G-CSF (250 μg / kg, sc) was injected once daily for 5 days without prior LPS administration. Results were compared with the LPS-treated group. G-CSF treatment enhanced cyclin A2-expressing hepatocytes from 1.4% ± 0.7 in CCl4 + LPS + G-CSF-treated animals to 2.1% ± 1.3 in CCl4 + G-CSF animals (p < 0.05). Ki67 expression, a marker of hepatocyte division, increased from 0.6% ± 0.2 after CCl4 + LPS + G-CSF to 4.1% ± 2.9 after CCl4 + G-CSF (p < 0.001). Image quantification was performed using Image J, and group comparisons were performed by one-way ANOVA with post-hoc Tukey's multiple comparisons. [Figure 5] Figure 1 shows the use of galactosamine (GalN) to induce a non-inflammatory second hit in a newly developed ACLF mouse model. Effect of G-CSF + TAK-242 on liver injury. A) To confirm that G-CSF exacerbates the inflammatory response in ACLF and induces a positive treatment effect in a non-inflammatory environment, we developed an alternative non-inflammatory ACLF model. C57B / 6 mice were gavaged with CCL4 for 6 weeks (n = 8) to induce chronic liver injury. Subsequently, galactosamine injection (GalN) (1000 mg / kg, i.p.) served as the primary non-inflammatory second hit to induce liver injury. Mice were treated with either recombinant mouse G-CSF (250 μg / kg, sc) + / - TAK-242 (10 mg / kg, i.p.) for 48 hours. B) TUNEL staining and ALT levels demonstrated that GalN injection was associated with significant liver injury. Combinatorial therapy consisting of G-CSF and TAK-242 was superior to treatment with either agent individually in inhibiting liver injury and reducing cell death (TUNEL). [Figure 6]Figure 1 shows necroptotic liver cell death in liver tissue. The CCl4-GalN model characterized necroptotic cell death. After GalN injection, cells expressed RIPK3, a mediator of necroptosis, while caspase 3 / 7 enzyme activity, a mediator of apoptotic cell death, remained unchanged. Treatment with G-CSF and TAK-242 significantly reduced hepatic RIPK3 expression. [Figure 7] Figure 1 shows BCL2 expression (STAT3 pathway) liver tissue. Combinatorial treatment of ACLF with TAK-242 and G-CSF increased hepatic expression of anti-apoptotic BCL2. [Figure 8]This figure shows the effect of TAK-242 / G-CSF in a non-inflammatory ACLF model. GalN induced a regenerative response not only in proliferating hepatocytes [Ki67: CCl4 0.1% ± 0.1 vs. CCl4 + GalN 2% ± 1.7, (p<0.01); cyclin A2: CCl4 0.1% ± 0.2 vs. CCl4 + GalN 1.9% ± 1.6, (p<0.05)] but also in cell cycle-arrested hepatocytes [p21: CCl4 0.5% ± 0.2 vs. CCl4 + GalN 6.4% ± 4.7, (p<0.01)]. G-CSF alone or in combination with TAK-242 reduced cell death and the subsequent regenerative response. TAK-242 alone maintained the abundance of proliferative (cyclin A2, Ki67) and senescent (p21) hepatocytes [(Ki67 CCl4+GalN+TAK-242 3% ± 2.1 vs. CCl4+GalN+TAK-242+G-CSF 1.2% ± 1.4 (p<0.05); cyclin A2 CCl4+GalN+TAK-242 1.9% ± 1.6 vs. CCl4+GalN+TAK-242+G-CSF 0.3% ± 0.2 (p<0.001); p21 CCl4+GalN+TAK-242 7.1% ± 4.5 vs. CCl4+GalN+TAK-242+G-CSF 1.7% ± 2.3 (p<0.001)]. Image quantification was performed using Image. Group comparisons were performed by one-way analysis of variance with post-hoc Tukey's multiple comparisons using J. mRNA data are expressed as ddCt values, and a greater than two-fold change in expression is considered biologically significant. Western blots were performed using pooled protein lysates from all animals per group. [Figure 9-1]Figure 1 shows that prevention of liver cell death by RIPA56 prevents the proliferative response after CCl4 + GalN. C57B / 6 mice were gavaged with 0.5 ml / ml carbon tetrachloride (CCl4) for 6 weeks to induce chronic liver injury (n = 8 per group). Galactosamine (GalN) (1000 mg / kg) was then administered to induce non-inflammatory liver injury, and treatment with the RIPK1 inhibitor, RIPA56, 3 mg / kg ip was initiated every 12 hours. RIPA56 treatment reduced GalN-induced cell death (TUNEL p < 0.001 (n = 4) and RIPK3 expression p < 0.001 (n = 3) compared to CCl4 + GalN). Compared to CCl4 + GalN, reduced liver injury was associated with decreased hepatocyte proliferation (Ki67 (n=3): 2% ± 1.7 vs. 0.2% ± 0.2, p<0.001; Cyclin A2 (n=3): 1.9% ± 1.6 vs. 0.03% ± 0.1, p<0.001). Image quantification was performed using Image J, and group comparisons were performed by one-way ANOVA with post-hoc Tukey's multiple comparisons. [Figure 9-2] Continued from Figure 9-1. [Figure 10]Figure 1 shows the ratio between regeneration and liver cell death. The ratio between hepatocyte proliferation markers (cyclin A2, Ki67) and liver cell death (TUNEL staining) was calculated. An increase in the ratio determines an enhanced regenerative response associated with the degree of liver injury, and vice versa. LPS injection with or without G-CSF led to a complete abrogation of hepatocyte proliferation despite the high degree of liver injury (cyclin A2 / TUNEL ratio: CCl4, 0.42 vs. CCl4 + LPS, 0.02 vs. CCl4 + LPS + G-CSF, 0.04). In contrast, hepatocyte proliferation was prevented after GalN injection (cyclin A2 / TUNEL ratio: CCl4 + GalN, 0.54). TAK-242 therapy enhanced hepatocyte regeneration in both short-term models (24-hour LPS treatment, 48-hour GalN treatment) (cyclin A2 / TUNEL ratio: CC14 + LPS + TAK-242, 1.4; CC14 + GalN + TAK-242, 0.9). G-CSF was able to demonstrate its pro-regenerative properties in the long-term LPS model after 5 days of treatment (cyclin A2 / TUNEL ratio: CC14 + LPS + TAK-242 + G-CSF, 1.7). DETAILED DESCRIPTION OF THE INVENTION

[0029] It should be understood that various applications of the disclosed methods can be tailored to the particular needs of the art. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only and is not intended to be limiting.

[0030] Furthermore, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the content clearly dictates otherwise. Thus, for example, reference to "an antagonist" includes "the antagonist," reference to "an antibody" includes two or more such antibodies, etc.

[0031] All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0032] The present inventors have unexpectedly found that ACLF can be treated by the use of a combination of a stem cell mobilizer, such as G-CSF, and an antagonist of TLR4.

[0033] Also encompassed by the present invention is the use of a combination of stem cells and a TLR4 antagonist to treat liver diseases, such as ALF and ACLF.

[0034] stem cells Stem cells have the ability to differentiate into various cell types in response to appropriate signals. These properties provide stem cells with the potential for tissue repair, replacement, and regeneration. Therefore, human stem cells, more specifically human embryonic stem cells (hESCs), are of particular interest in medical research. Embryonic stem cells have the potential to differentiate into more cell types than adult stem cells, and thus have great potential in therapy. Differentiation is triggered in vivo by various factors, some of which can be replicated in in vitro stem cell cultures. Induced pluripotent stem cells (iPSCs) are a form of stem cell that are often used in autologous procedures because they are generated from the tissue of the same patient receiving the transplant, thus avoiding immune rejection. iPSCs obtained in this manner do not have the ethical considerations of stem cells derived from embryos. In one embodiment of the present invention, the stem cells used are autologous and therefore derived from the individual being treated. In one embodiment of the present invention, the stem cells used are xenogeneic and therefore not derived from the individual being treated. In a preferred embodiment, the stem cells used are human stem cells. In a preferred embodiment, the stem cells used are hepatic stem cells. In a preferred embodiment, the stem cells used are human hepatic stem cells.

[0035] Stem Cell Mobilizer Stem cells and other regulatory immune cells are known to modulate immune activation, thereby modulating inflammatory responses and supporting tissue repair. (5) Stem cell mobilizers are compounds that stimulate stem cells to proliferate and migrate from the bone marrow into the circulation. Examples of such compounds include granulocyte colony-stimulating factor (G-CSF) and its analogs, such as filgrastim, lenograstim, and pegfilgrastim, GM-CSF, M-CSF, plerixafor (AMD3100), stem cell factor (SCF), vascular endothelial growth factor (VEGF), erythropoietin, and placental growth factor (PGF), CXCL12 / CXCR4 modulators, SIP agonists, VCAM / VLA-4 inhibitors, parathyroid hormone, proteasome inhibitors, and stabilizers of Groβ and HIF.

[0036] G-CSF Granulocyte colony-stimulating factor (G-CSF) is a glycoprotein recognized to mobilize stem cells and immune cells and possess immunomodulatory and pro-regenerative properties. It is also known as colony-stimulating factor 3 (CSF3). G-CSF is produced by a variety of different tissues and stimulates the bone marrow to produce stem cells and granulocytes, which are released into the bloodstream. G-CSF also acts to stimulate the survival, proliferation, differentiation, and function of neutrophil precursors and mature neutrophils. It is contemplated that recombinant analogs of G-CSF or biosimilars of G-CSF may be utilized in place of G-CSF in the present invention. The term "G-CSF" is intended to encompass G-CSF analogs or biosimilars of G-CSF.

[0037] TLR4 Toll-like receptor 4 (TLR4) is a key pattern recognition receptor for lipopolysaccharide (LPS) and other Gram-negative endotoxins and is expressed in many non-parenchymal and parenchymal cells, including hepatocytes and hepatic stellate cells. Its activation leads to NfKB-mediated inflammatory responses.

[0038] The present invention relates to the use of a combination of a stem cell mobilizer, such as G-CSF and an antagonist of TLR4, in the treatment, prevention and / or diagnosis of liver failure, such as acute onset of chronic liver failure (ACLF).

[0039] ALF and ACLF Acute liver failure (ALF) occurs in individuals with previously normal livers and results from overwhelming liver injury, usually caused by a hepatotoxic insult.

[0040] Acute-on-chronic liver failure (ACLF) is a distinct clinical entity encompassing acute deterioration of liver function in patients with cirrhosis, often decompensated cirrhosis, usually associated with an inciting event, resulting in failure of one or more organs and high short-term mortality. Uncontrolled inflammation is thought to be a major contributing factor. Distinctive features of ACLF are its rapid progression, the need for multiple organ support, and a high incidence of short- and mid-term mortality of 40-90%.

[0041] Thus, the present invention stems from the inventors' discovery of the synergistic effects of combining a TLR4 antagonist and the stem cell mobilizer G-CSF, and takes advantage of these effects by proposing a combination of a stem cell mobilizer, e.g., G-CSF, and a TLR4 antagonist as a therapeutic agent for use in the treatment or prevention of liver diseases, e.g., ALF or ACLF.

[0042] TLR4 antagonists The present invention relates to antagonism of Toll-like receptor 4 (TLR4). A TLR4 antagonist can be any compound or molecule that inhibits or reduces the activity, function, or amount of TLR4. Preferably, the antagonist functions in the liver and / or kidney and / or brain of a patient with liver failure. The antagonist may act preferentially in the liver and / or kidney, or may act in several locations, including the liver and / or kidney and / or brain. Preferably, the antagonist leads to a decrease in TLR4 activity, function, or amount in an organ of an individual to which the antagonist is administered, for example, in one or more of the individual's liver, kidney, brain, and heart. The antagonist may be targeted to the liver, kidney, or other organs, such as those listed above, upon administration as discussed further below.

[0043] Preferred antagonists are those that reduce the activity or amount of TLR4 by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to the activity or amount of TLR4 seen in the absence of the antagonist.

[0044] A TLR4 antagonist can reduce the activity or amount of TLR4 to an amount or activity that is the same as, similar to, or equivalent to that found in individuals without liver disease. For example, as exemplified herein, TLR4 expression has been found to be increased in association with models of liver cirrhosis. The use of a TLR4 antagonist according to the present invention can lead to a reduction in TLR4 expression in the liver and / or kidney and / or brain of an individual being treated relative to normal levels, e.g., levels found or predicted in individuals without chronic liver disease or cirrhosis.

[0045] The antagonist may specifically act to antagonize TLR4. That is, the effect of the antagonist on TLR4 may be greater than any other biological effect of the antagonist. Such an antagonist may be specific for inhibiting TLR4, i.e., reduce the activity of TLR4, but not other receptors, e.g., other Toll-like receptors. Such an antagonist may additionally or alternatively be specific for the expression of TLR4, i.e., reduce the expression of TLR4, but not other receptors, e.g., other Toll-like receptors. The antagonist for use in accordance with the present invention may be an antagonist of TLR4 as described herein that does not act as an antagonist of other Toll-like receptors. The antagonist for use in accordance with the present invention may act on TLR4 preferentially over other Toll-like receptors. For example, a TLR4 antagonist for use in accordance with the present invention may have one or more characteristics of a TLR4 antagonist as described herein, but may not have such characteristics in relation to other Toll-like receptors, or may have such characteristics to a lower degree in relation to other Toll-like receptors as compared to TLR4. For example, an antagonist that reduces the activity of TLR4 may also reduce the activity of other Toll-like receptors, or may reduce the activity of other Toll-like receptors to a lesser extent, e.g., a lower percentage reduction, than its effect on TLR4. An antagonist that reduces the expression or amount of TLR4 may not reduce the expression or amount of other Toll-like receptors, or may reduce the expression of other Toll-like receptors to a lesser extent, e.g., a lower percentage reduction, than its effect on TLR4. A TLR4 antagonist as described herein may have an effect on other Toll-like receptors, e.g., antagonism of the activity, signaling or expression of one or more other Toll-like receptors, that is less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, or less than 0.1% of the effect of the antagonist on the activity, signaling or expression of TLR4.

[0046] In this specification, "other Toll-like receptor" refers to any Toll-like receptor other than TLR4. At least 13 groups of Toll-like receptors have been identified in mammals. The other Toll-like receptor can be any such Toll-like receptor other than TLR4. The other Toll-like receptor can be one or more of these Toll-like receptors. The other Toll-like receptor can be any other Toll-like receptor other than TLR4.

[0047] The specificity of the TLR4 antagonist may be applied within the entire body of the individual being treated, i.e., the action of the TLR4 antagonist may be specific as discussed above throughout the individual's body. The specificity of the TLR4 antagonist may be applied within a particular tissue of the individual, such as the liver, kidney, and / or heart and / or brain. That is, in one embodiment, the TLR4 antagonist may act specifically to antagonize TLR4 as discussed above within the liver and / or kidney and / or other organs of the individual being treated.

[0048] Thus, the TLR4 antagonist may be a specific antagonist of TLR4, as described above, For example, the TLR4 antagonist may not be an antagonist of other Toll-like receptors or may have no significant effect on the activity or expression of other Toll-like receptors.

[0049] Any agent capable of inhibiting the activity or function of TLR4 may be suitable for use in the methods of the present invention. Antagonists for use according to the present invention may be direct or indirect antagonists of TLR4.

[0050] A direct antagonist is an agent whose activity is directly directed against TLR4. For example, a direct antagonist can be an agent that acts directly on the TLR4 receptor to reduce its activity. A direct antagonist can be an agent that disrupts TLR4 function or destabilizes the TLR4 receptor. A direct antagonist can reduce the amount of TLR4 in a patient by destroying or disrupting the TLR4 molecule. A direct antagonist can be an agent that acts on the TLR4 gene, promoter, or other gene regulatory region to reduce TLR4 expression. A direct antagonist can reduce TLR4 expression by preventing or reducing expression from the endogenous TLR4 gene.

[0051] A TLR4 antagonist can act to disrupt the activity of TLR4. For example, the antagonist can act by preventing the activation of TLR4 or by preventing the formation of a functional complex that includes TLR4.

[0052] Any agent or molecule having the above properties can be used as a TLR4 antagonist according to the present invention. Test agents can be or include, for example, peptides, polypeptides, proteins, antibodies, polynucleotides, small molecules, or other compounds that can be designed by rational drug design starting from known antagonists of TLR4.

[0053] Examples of TLR4 antagonists or inhibitors that can be used in accordance with the present invention include: peptide STM28 as described by Sugiyama et al. (European Journal of Pharmacology 594 (2008) 152-156); ethyl (6R)-6-[N-(2-chloro-4-fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate (TAK-242), which acts by blocking signaling mediated by the intracellular domain of TLR4, but not the extracellular domain; Tetrasodium [(2R,3R,4R,5S,6R)-4-decoxy-5-hydroxy-6-[[(2R,3R,4R,5S,6R)-4-[(3R)-3-methoxydecoxy]-6-(methoxymethyl)-3-[[(Z)-octadec-11-enoyl]amino]-5-phosphonatooxyoxan-2-yl]oxymethyl]-3-(3-oxotetradecanoylamino)oxan-2-yl]phosphate (eritoran), which may be provided as E5564. E5564 contains eritoran tetrasodium as the active ingredient. E5564 blocks receptor signaling and inhibits the release of inflammatory cytokines IL-1 and TNF.

[0054] NI-0101 is an anti-TLR4 monoclonal antibody that binds to an epitope on TLR4, interfering with its dimerization, which is required for intracellular signaling and the induction of pro-inflammatory pathways. NI-0101 is a product of NovImmuno SA.

[0055] OxPAPC (1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine), an oxidized phospholipid that has been shown to inhibit signaling induced by bacterial lipopeptides and lipopolysaccharide (LPS).

[0056] IAXO compounds, such as IAXO-101 (methyl 6-deoxy-6-N-dimethyl-N-cyclopentylammonium-2,3-di-O-tetradecyl-α-D-glucopyranoside iodide), IAXO-102 methyl 6-deoxy-6-amino-2,3-di-O-tetradecyl-α-D-glucopyranoside, or IAXO-103 (N-(3,4-bis-tetradecyloxy-benzyl)-N-cyclopentyl-N,N-dimethylammonium iodide).

[0057] Compounds that target TLRs, such as TLR4, are reviewed in Hennessy et al. (2010) Nature Reviews Drug Discovery 9:293-307.

[0058] Preferably, the TLR4 antagonist is not LPS.

[0059] A TLR4 antagonist can be a molecule that is capable of binding to TLR4 and preventing or disrupting the activity of TLR4.

[0060] Thus, one group of TLR4 antagonists for use in accordance with the present invention are anti-TLR4 antibodies. Such antibodies may be monoclonal or polyclonal, or may be antigen-binding fragments thereof. For example, antigen-binding fragments may be or include F(ab)2, Fab, or Fv fragments, i.e., fragments of the "variable" region of an antibody that contains the antigen-binding site. The antibody or fragment thereof may be a single-chain antibody, a chimeric antibody, a CDR-grafted antibody, or a humanized antibody.

[0061] The antibody can be directed against the TLR4 molecule, i.e., it can bind to an epitope present on TLR4 and thus selectively and / or specifically bind to TLR4. The antibody can be directed against another molecule involved in TLR4 expression and / or activity. For example, polyclonal antibodies can be generated that have broad activity against one or more epitopes on TLR4 and / or one or more other molecules involved in TLR4 expression and / or activity.

[0062] Antibodies can be generated by any suitable method. Means for preparing and characterizing antibodies are well known in the art; see, e.g., Harlow and Lane (1988) "Antibodies: A Laboratory Manual," Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. For example, antibodies can be generated by raising antibodies in a host animal against the entire polypeptide or a fragment thereof, e.g., an antigenic epitope thereof, hereinafter referred to as the "immunogen."

[0063] An antibody or other compound "specifically binds" to a molecule if it binds preferentially or with high affinity to the molecule for which it is specific, but does not bind substantially, or binds with low affinity, to other molecules. A variety of competitive binding or immunoradiometric protocols for determining the specific binding ability of an antibody are well known in the art (see, e.g., Maddox et al., J. Exp. Med. 158:1211-1226, 1993). Such immunoassays typically involve the formation of complexes between a specific protein and its antibody and the measurement of complex formation.

[0064] The TLR4 antagonist can be an antisense oligonucleotide, for example, an antisense oligonucleotide against a gene encoding a TLR4 protein. The term "antisense oligonucleotide" as used herein refers to a nucleotide sequence that is complementary to the mRNA of a desired gene. Such an antisense oligonucleotide can selectively hybridize with a desired gene. In the context of the present invention, the desired gene can be a gene encoding TLR4.

[0065] The TLR4 antagonist can modulate the expression of the TLR4 gene. For example, the TLR4 antagonist can be a short interfering nucleic acid (siRNA) molecule, a double-stranded RNA (dsRNA), a microRNA, a deoxyribose nucleic acid interference (DNAi) or a short hairpin RNA (shRNA) molecule.

[0066] The term "selectively hybridize" as used herein refers to the ability of a nucleic acid to detectably and specifically bind to a second nucleic acid. Oligonucleotides selectively hybridize to target nucleic acid strands under hybridization and wash conditions that minimize appreciable amounts of detectable binding to nonspecific nucleic acids. High stringency conditions can be used to achieve selective hybridization conditions as known in the art. Typically, hybridization and wash conditions are performed at high stringency according to conventional hybridization procedures. Washing conditions are typically 1-3x SSC, 0.1-1% SDS, 50-70°C, with the wash solution replaced after approximately 5-30 minutes.

[0067] The TLR4 antagonist may be a nucleic acid molecule, such as an antisense molecule or an aptamer. The nucleic acid molecule may bind to a specific target molecule.

[0068] Aptamers can be fully engineered in vitro, easily produced by chemical synthesis, have desirable storage properties, and induce little or no immunogenicity in therapeutic applications - characteristics that make them particularly useful in pharmaceutical and therapeutic applications.

[0069] The terms "nucleic acid molecule" and "polynucleotide" are used interchangeably herein and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Nucleic acids can contain conventional bases, sugar moieties, and internucleotide linkages, but can also contain modified bases, modified sugar moieties, or modified linkages. Nucleic acid molecules can be single-stranded or double-stranded.

[0070] Generally, aptamers can comprise oligonucleotides that are at least 5, at least 10, or at least 15 nucleotides in length. Aptamers can comprise sequences that are up to 40, up to 60, or up to 100 or more nucleotides in length. For example, aptamers can be 5-100 nucleotides, 10-40 nucleotides, or 15-40 nucleotides in length. Where possible, aptamers of shorter length are preferred, as they often lead to less interference with other molecules or materials.

[0071] Aptamers can be generated using conventional methods, such as the sequential evolution of nucleic acids (SELEX) procedure. SELEX is a method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules. For example, see US Pat. No. 5,654,151, US Pat. No. 5,503,978, US Pat. No. 5,567,588, and WO 96 / 38579. The SELEX method involves selecting nucleic acid aptamers, particularly single-stranded nucleic acids, capable of binding to a desired target from a collection of oligonucleotides. A collection of single-stranded nucleic acids (e.g., DNA, RNA, or variants thereof) is contacted with the target under conditions favorable for binding, nucleic acids bound to the target in the mixture are separated from those that are not bound, the nucleic acid-target complex is dissociated, and the target-bound nucleic acids are amplified to obtain a collection or library enriched for nucleic acids with the desired binding activity. This series of steps is then repeated as necessary to generate a library of nucleic acids (aptamers) with specific binding affinity to related targets.

[0072] Thus, any of the antagonists described herein can be used to antagonize TLR4, i.e., reduce the amount of TLR4 present and / or the activity or function of TLR4. Preferably, these antagonizing effects occur in the liver and / or kidney and / or brain.

[0073] A TLR4 antagonist can be an agent that reduces the production of endogenous TLR4. For example, the agent can act in a subject's cells to inhibit or prevent the expression of TLR4. Such an agent can be a transcription factor or enhancer that acts on the TLR4 gene to inhibit or prevent gene expression.

[0074] Preferably, the TLR4 antagonist is an agent capable of reducing injury and / or organ dysfunction caused by the administration of a hepatotoxin, such as acetaminophen. For example, this ability can be tested in a suitable animal model, such as a non-human animal (e.g., mouse or rat), treated with such a hepatotoxin. The effect of a potential TLR4 antagonist on such an animal can be evaluated. The TLR4 antagonist can be administered before, simultaneously with, or after the administration of the hepatotoxin to the animal. The effect of the hepatotoxin in the presence of the antagonist can be compared to the effect of the hepatotoxin in the absence of the TLR4 antagonist, for example, in vehicle-treated animals. A suitable TLR4 antagonist for use according to the present invention can reduce injury or organ dysfunction in an animal compared to that seen in the absence of the TLR4 antagonist. This reduced injury or dysfunction can be characterized using any of the criteria discussed further herein, such as a reduction in liver enzymes, a reduction in plasma creatinine and / or ammonia levels, a change in inflammatory modulator levels, such as NF6B or TNF-alpha levels, a reduction in interleukin-1a levels in the liver, a reduction in brain water, a reduction in tissue damage in an organ, or other characteristics of injury or organ dysfunction predicted to result from treatment with a hepatotoxin. The organ may be, for example, the liver, kidney, heart, and / or brain. A suitable TLR4 antagonist is predicted to have such an ameliorative effect compared to the effect seen with administration of a hepatotoxin in the absence of a TLR4 antagonist.

[0075] Pharmaceutical preparations Suitable TLR4 antagonists as described herein are usually formulated for administration with pharmaceutically acceptable carriers or diluents.Therefore, antagonists can be formulated as pharmaceuticals with standard pharmaceutically acceptable carrier(s) and / or excipient(s), as is conventional in the pharmaceutical field.The exact nature of the formulation will depend on several factors, including the desired route of administration.Usually, antagonists can be formulated for oral, intravenous, intragastric, intravascular or intraperitoneal administration.

[0076] When stem cells are to be administered, they are typically formulated for administration with a pharmaceutically acceptable carrier or diluent. Formulation of cells using standard pharmaceutically acceptable carriers and / or excipients can be carried out using conventional methods in the pharmaceutical field. The exact nature of the formulation will vary depending on several factors, including the cells to be administered and the desired route of administration. Suitable formulations are fully described in Remington's Pharmaceutical Sciences, 19th Edition, Mack Publishing Company, Eastern Pennsylvania, USA. The composition can be prepared with a physiologically acceptable carrier or diluent. Typically, such compositions are prepared as a liquid suspension of cells. The cells can be mixed with a pharmaceutically acceptable excipient that is compatible with the active ingredient. Suitable excipients include, for example, water, saline, dextrose, glycerol, etc., and combinations thereof.

[0077] In addition, if desired, the pharmaceutical compositions of the present invention may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and / or adjuvants which enhance the effectiveness of the compositions.

[0078] Stem cell mobilizers, such as G-CSF, are typically formulated for administration with a pharmaceutically acceptable carrier or diluent. Thus, stem cell mobilizers, such as G-CSF, can be formulated as pharmaceuticals with standard pharmaceutically acceptable carrier(s) and / or excipient(s), as is conventional in the pharmaceutical field. The exact nature of the formulation will depend on several factors, including the desired route of administration. Typically, stem cell mobilizers, such as G-CSF, can be formulated for oral, intravenous, intragastric, intravascular, or intraperitoneal administration.

[0079] The stem cell mobilizer, e.g., G-CSF, and the TLR4 antagonist can be present in a single formulation as described herein. The stem cell mobilizer, e.g., G-CSF, and the TLR4 antagonist can be present in a single combination, composition, or pharmaceutical composition.

[0080] The stem cell mobilizer, e.g., G-CSF, and the TLR4 antagonist may be formulated for simultaneous, subsequent, or sequential delivery. The TLR4 antagonist may be administered before, simultaneously with, or after the stem cell mobilizer, e.g., G-CSF. The stem cell mobilizer, e.g., G-CSF, may be administered before, simultaneously with, or after the TLR4 antagonist. The pharmaceutical composition of the present invention may contain G-CSF and a TLR4 antagonist.

[0081] The stem cell mobilizer, e.g., G-CSF, and the TLR4 antagonist can be present in a single formulation as described herein. The stem cell mobilizer, e.g., G-CSF, and the TLR4 antagonist can be present in a single combination, composition, or pharmaceutical composition.

[0082] The stem cells and the TLR4 antagonist may be formulated for simultaneous, subsequent, or sequential delivery. The TLR4 antagonist may be administered before, simultaneously with, or after the stem cells. The stem cells can be administered before, simultaneously with, or after the TLR4 antagonist. The pharmaceutical composition of the present invention may comprise stem cells and a TLR4 antagonist.

[0083] Pharmaceutical carriers or diluents can be, for example, isotonic solutions, such as physiological saline.Solid oral dosage forms can contain, together with the active compound, diluents such as lactose, dextrose, saccharose, cellulose, corn starch or potato starch; lubricants such as silica, talc, stearic acid, magnesium or calcium stearate and / or polyethylene glycol; binders such as starch, gum arabic, gelatin, methylcellulose, carboxymethylcellulose or polyvinylpyrrolidone; disaggregating agents such as starch, alginic acid, alginate or sodium starch glycolate; effervescent mixtures; dyes; sweeteners; wetting agents such as lecithin, polysorbate, lauryl sulfate; and non-toxic, pharmacologically inactive substances generally used in pharmaceutical preparations.Such pharmaceuticals can be produced by known methods, for example, by mixing, granulating, tableting, sugar-coating or film-coating processes.

[0084] Liquid dispersions for oral administration may be syrups, emulsions or suspensions. Syrups may contain carriers such as saccharose or saccharose with glycerine and / or mannitol and / or sorbitol.

[0085] Suspensions and emulsions may contain, as a carrier, for example, a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol. Suspensions or solutions for intramuscular injection may contain, together with ornithine and at least one of phenyl acetate and phenyl butyrate, a pharmaceutically acceptable carrier, for example, sterile water, olive oil, ethyl oleate, a glycol, for example, propylene glycol, and, if necessary, an appropriate amount of lidocaine hydrochloride.

[0086] When the administered TLR4 antagonist is a nucleic acid molecule, e.g., when the antagonist is in the form of an expression vector, certain facilitators of nucleic acid uptake and / or expression ("transfection-facilitating agents"), such as facilitators such as bupivacaine, cardiotoxins, and sucrose, as well as transfection-facilitating vehicles, e.g., liposomes or lipid preparations conventionally used to deliver nucleic acid molecules, may also be included in the composition.

[0087] Pharmaceutical formulations according to the present invention may further comprise one or more additional therapeutic agents. For example, the formulation may comprise one or more TLR4 antagonists and a stem cell mobilizer, such as G-CSF, as defined herein. The formulation may comprise two or more stem cell mobilizers. The formulation may comprise one or more TLR4 antagonists as described herein and also one or more additional therapeutic agents. Preferably, the additional therapeutic agent(s) are agents that assist in the treatment or prevention of the individual being treated. For example, one or more agents effective in treating liver disease may be administered as part of a formulation as described herein. One or more agents effective in treating an underlying liver condition or its symptoms in a patient may be administered as part of a formulation as described herein.

[0088] Furthermore, the formulation may comprise one or more TLR4 antagonists and stem cells, e.g., hepatic stem cells, as defined herein. The formulation may comprise stem cells and a stem cell mobilizer. The formulation may also comprise one or more TLR4 antagonists and one or more additional therapeutic agents as described herein. Preferably, the additional therapeutic agent(s) are agents that assist in the treatment or prevention of the individual being treated. For example, one or more agents effective in treating liver disease may be administered as part of a formulation as described herein. One or more agents effective in treating an underlying liver condition or its symptoms in a patient may be administered as part of a formulation as described herein.

[0089] treatment The present invention provides methods for the treatment of individuals with liver failure, eg, ACLF, and in particular for the treatment or prevention of symptoms and conditions associated with or resulting from liver failure, eg, ALF or ACLF.

[0090] Thus, the present invention provides a method of treating an individual with liver failure, e.g., ALF or ACLF, comprising administering to the subject a stem cell mobilizer, e.g., G-CSF, and a TLR4 antagonist. Similarly, a stem cell mobilizer, e.g., G-CSF, and a TLR4 antagonist can be provided for use in a method of treating an individual with liver failure, e.g., ALF or ACLF. Also provided is the use of a stem cell mobilizer, e.g., G-CSF, and a TLR4 antagonist, in the manufacture of a medicament for use in treating an individual with liver failure, e.g., ALF or ACLF.

[0091] Thus, the present invention provides a method of treating an individual with liver failure, e.g., ALF or ACLF, comprising administering to the subject a stem cell mobilizer, e.g., G-CSF, and a TLR4 antagonist. Similarly, a stem cell mobilizer, e.g., G-CSF, and a TLR4 antagonist for use in a method of treating an individual with liver failure, e.g., ALF or ACLF, can be provided. Also provided is the use of a stem cell mobilizer, e.g., G-CSF, and a TLR4 antagonist, in the manufacture of a medicament for use in treating an individual with liver failure, e.g., ALF or ACLF.

[0092] The present invention also provides a method of treating an individual with liver failure, e.g., ALF or ACLF, comprising administering to the subject stem cells, e.g., hepatic stem cells, and a TLR4 antagonist. Similarly, stem cells, e.g., hepatic stem cells, and a TLR4 antagonist for use in methods of treating an individual with liver failure, e.g., ALF or ACLF, can be provided. Also provided is the use of stem cells, e.g., hepatic stem cells, and a TLR4 antagonist in the manufacture of a medicament for use in treating an individual with liver failure, e.g., ALF or ACLF.

[0093] The G-CSF and TLR4 antagonists can be provided in a formulation as described herein. Thus, to treat or prevent liver failure, such as ALF or ACLF, or particularly symptoms or conditions associated with liver failure, such as ALF or ACLF, in a subject, the G-CSF and TLR4 antagonists as described herein can be administered to the subject. Thus, to improve the condition of a subject, for example, a subject suffering from liver failure, such as ALF or ACLF, the G-CSF and TLR4 antagonists as described herein can be administered. The G-CSF and TLR4 antagonists as described herein can be administered to alleviate the symptoms of a subject, for example, symptoms associated with liver failure, such as ALF or ACLF.

[0094] The G-CSF and TLR4 antagonists described herein can be administered to combat or delay the onset of liver failure, such as ALF or ACLF, or any symptoms associated therewith. Thus, the present invention can prevent the medical consequences of liver failure, such as ALF or ACLF. The use of the G-CSF and TLR4 antagonists described herein can extend the lifespan of patients with liver failure, such as ALF or ACLF.

[0095] Treatment of liver failure, for example, ALF or ACLF, refers to the treatment of individuals who have or are at risk of having liver failure, for example, ALF or ACLF. The individual may also suffer from chronic liver disease, for example, cirrhosis or alcoholic cirrhosis. The patient may also suffer from liver disease or cirrhosis associated with or caused by infection, for example, hepatitis virus infection, for example, hepatitis C virus infection. The patient may also suffer from liver disease or cirrhosis associated with or caused by treatment with hepatotoxins, for example, acetaminophen (paracetamol). The patient may also suffer from AH, NAFLD, or NASH. The methods described herein can be used in the treatment of any such disease.

[0096] An individual may suffer from one or more symptoms or conditions caused by or associated with liver failure, e.g., ALF or ACLF. Any one or more of these conditions or symptoms may be treated according to the present invention. For example, an individual may suffer from or be at risk of suffering from one or more of the following as a result of their liver failure, e.g., ACLF: renal dysfunction; renal failure; HRS; increased plasma creatinine; cerebral dysfunction and swelling; increased plasma ammonia; increased liver enzyme concentrations (e.g., increased ALT and / or AST concentrations in the liver); increased inflammation, injury, and / or dysfunction in the liver and / or kidneys and / or brain and / or blood circulation; or liver tissue damage due to liver failure, e.g., due to acetaminophen (APAP) toxicity. An individual may suffer from or be at risk of acute liver failure, alcoholic hepatitis, and / or liver reperfusion injury. These conditions may be caused by the individual's ALF and / or ACLF. The methods and uses described herein may be useful in the treatment or prevention of any one or more of these symptoms or conditions, particularly in individuals suffering from liver failure, eg, ACLF.

[0097] In particular, the methods described herein can be used in treating patients with liver failure, such as ALF or ACLF. For example, the patient may have or be at risk of renal failure. Liver failure, such as ALF or ACLF, can be caused by infection and / or inflammation. Liver failure, such as ACLF, can be caused by exposure to a hepatotoxin, such as acetaminophen (paracetamol), for example, exposure to high levels of a hepatotoxin, for example, an overdose of paracetamol. The methods described herein can be used to treat or prevent any of these conditions or symptoms.

[0098] As described herein, an antagonist of TLR4 can lead to decreased expression and / or decreased levels of TLR4 in the liver and / or kidney of a subject. For example, the antagonist can be an agent that inhibits transcription of TLR4 in cells of a subject.

[0099] As described herein, an antagonist of TLR4 can lead to a decrease in the activity of TLR4 in the liver and / or kidney of an individual.

[0100] The subject is treated with a stem cell mobilizer and a TLR4 antagonist as described herein. In a preferred embodiment, the subject is treated with a G-CSF and a TLR4 antagonist as described herein. As described above, the stem cell mobilizers, e.g., G-CSF and a TLR4 antagonist, can be administered alone, after each other, sequentially, and / or in the form of a pharmaceutical formulation. The formulation can include one or more additional therapeutic or prophylactic agents.

[0101] The subject is treated with stem cells and a TLR4 antagonist as described herein. In a preferred embodiment, the subject is treated with hepatic stem cells and a TLR4 antagonist as described herein. As described above, the stem cells, for example, hepatic stem cells, and the TLR4 antagonist can each be administered alone, after each other, sequentially, and / or in the form of a pharmaceutical preparation. The preparation can include one or more additional therapeutic or prophylactic agents.

[0102] In addition to the use of stem cells, two or more different TLR4 antagonists as described herein may be used in combination to treat a subject. The two or more antagonists may be administered together, in a single formulation, simultaneously, in two or more separate formulations, or separately or sequentially as part of a combined administration regimen.

[0103] The stem cells and TLR4 antagonist may be administered in combination with another agent known to be useful in the treatment or prevention of liver failure, such as ALF or ACLF. The stem cells and TLR4 antagonist and the other agent may be administered together in a single formulation, simultaneously, in two or more separate formulations, or separately or sequentially as part of a combined administration regimen.

[0104] In addition to the use of G-CSF, two or more different TLR4 antagonists as described herein can be used in combination to treat a subject. The two or more antagonists can be administered together, in a single formulation, simultaneously, in two or more separate formulations, or separately or sequentially as part of a combined administration regimen.

[0105] The G-CSF and TLR4 antagonist may be administered in combination with another agent known to be useful in the treatment or prevention of liver failure, e.g., ALF or ACLF. The G-CSF and TLR4 antagonist and the other agent may be administered together, in a single formulation, simultaneously, in two or more separate formulations, or separately or sequentially as part of a combined administration regimen.

[0106] The pharmaceutical composition or formulation of the present invention can be administered by any suitable route. Preferably, it is administered orally, intravenously, intragastrically, intraperitoneally, or intravascularly. The antagonist or formulation can be administered directly to the liver of a subject.

[0107] The pharmaceutical composition or formulation is administered in a therapeutically effective amount. The appropriate dose of the pharmaceutical composition or formulation of the present invention can be determined according to various parameters, such as the age, weight, and condition of the subject to be treated; the type and severity of liver disease; the route of administration; and the required regimen. The appropriate dose of each antagonist can be determined. For example, for some pharmaceutical compositions or formulations, the usual dose may be about 0.1 mg / kg / day to 30 g / kg / day. A physician will be able to determine the required dose of the antagonist for any particular subject.

[0108] The present invention is broadly applicable to methods of treatment and is relevant to the development of prophylactic and / or therapeutic treatments. It will be understood that all references herein to treatment include curative, symptomatic and prophylactic treatment.

[0109] Prevention or therapy includes, but is not limited to, inducing an effective reduction in the amount, function, or activity of TLR4 to cause a reduction in one or more symptoms or conditions associated with or resulting from ACLF. The symptoms or conditions may be, for example, any of those discussed above. For example, prevention or therapy may result in a reduction in symptoms of renal dysfunction, prevention or reduction of symptoms of liver failure, reduction in levels of plasma creatinine, plasma ammonia, liver enzyme concentrations (e.g., reduction in ALT and / or AST concentrations in the liver), reduction in inflammation in the liver and / or kidney and / or brain and / or blood circulation, and / or reduction in liver tissue damage resulting from liver failure, for example, due to acetaminophen (APAP) toxicity. Prophylaxis or therapy may result in maintenance of certain levels of renal dysfunction, renal failure, plasma creatinine, brain dysfunction and / or swelling, plasma ammonia, liver enzyme concentrations (e.g., ALT and / or AST concentrations in the liver), inflammation in the liver and / or kidneys and / or brain and / or circulation, and / or liver tissue damage due to liver failure, e.g., due to acetaminophen (APAP) toxicity, in patients who have or are predicted to have increased symptoms as a result of ACLF. Prophylaxis or therapy may result in such changes in symptoms or conditions in such individuals changing at a reduced rate compared to the changes that would have been seen or predicted in the absence of such treatment.

[0110] Prevention or therapy may have a similar effect with respect to any of the symptoms or consequences of liver failure, e.g., ALF or ACLF, described herein, i.e., treatment in accordance with the present invention may lead to a decrease in the severity of such symptoms or consequences, a maintenance of existing levels of such symptoms or consequences, or a slowing or reduction in the worsening of such symptoms or consequences.

[0111] Patients to be treated The present invention relates to the treatment or prevention of liver failure, such as ALF or ACLF, in an individual in need thereof. An individual treated according to the present invention may therefore have liver failure, such as ALF or ACLF, or may be at increased risk of liver failure, such as ALF or ACLF. For example, the subject may have liver failure. The subject may have immune dysfunction or insufficiency, systemic inflammation, renal failure, or brain dysfunction and / or brain swelling. The subject may have AH, NAFLD, or NASH.

[0112] Methods for diagnosing liver failure, immune dysfunction, kidney dysfunction, brain dysfunction, brain swelling or immune deficiency are well known in the art, especially to physicians and veterinarians in the art.For example, kidney dysfunction is characterized by a reduction or loss of kidney function, which can be evaluated by monitoring urine volume or sodium concentration and osmolality of urine.Hepatorenal syndrome is also associated with a reduction in renal blood flow.Preferably, the subject is diagnosed with liver failure by, for example, a medical or veterinary specialist.The subject may show one or more symptoms associated with liver failure, kidney dysfunction or kidney failure.

[0113] Methods for diagnosing liver failure, such as ACLF, are well known in the art, particularly to physicians and veterinarians in the art. ACLF is diagnosed using the Chronic Liver Failure (CLiF) Consortium criteria, the NACSELD criteria, or the APASL criteria. Pre-validated scores for assessing disease severity include the Child-Pugh (CP) classification, the Model for End-Stage Liver Disease (MELD), and the CLiF Consortium Acute Decompensation (CLIF-C AD) score.

[0114] The treated individual may have increased expression of TLR4 in the liver compared to a healthy individual, e.g., an individual without liver failure, e.g., ALF or ACLF. The treated individual may have increased serum or plasma TLR4 compared to a healthy individual, e.g., an individual without liver failure, e.g., ALF or ACLF.

[0115] The individual to be treated may have been diagnosed, for example, by any of these methods, with liver failure, e.g., ALF or ACLF, or one or more symptoms or conditions, such as those described herein, that may be associated with liver failure, e.g., ALF or ACLF. The individual to be treated may have been diagnosed as being at risk for liver failure, e.g., ALF or ACLF. For example, the individual may have been diagnosed with liver failure, cirrhosis, renal failure, and / or one or more symptoms associated with renal failure. For example, the individual to be treated may have cirrhosis, AH, NASH, idiopathic non-cirrhotic portal hypertension, congenital hepatic fibrosis, segmental nodular disease, Budd-Chiari syndrome, portal vein thrombosis, right heart failure, or schistosomiasis infection.

[0116] The subject to be treated can be any individual who is susceptible to liver failure, such as ALF or ACLF. The subject can be male or female. Females may be more susceptible to the harmful effects of alcohol than males. Females may develop chronic liver disease from smaller amounts of alcohol in a shorter time frame than males.

[0117] The subject to be treated may be a human. The subject to be treated may be a non-human animal. The subject to be treated may be a livestock animal, such as a cow or bull, sheep, pig, ox, goat, or horse, or may be a domestic animal, such as a dog or cat. The subject may or may not be an animal model for liver disease. The animal may be of any age, but is often a mature adult subject.

[0118] [Example] [Example 1] Stem cell mobilization has adverse consequences in acute onset of chronic liver failure (ACLF) Three small randomized trials in Asia showed that stem cell mobilization significantly increased survival, improved organ function, and reduced infection rates, thus suggesting stem cell mobilization as a novel treatment option in ACLF. However, we unexpectedly generated data showing that stem cell mobilization in the context of ACLF is harmful.

[0119] First, they conducted the first multicenter randomized controlled trial to test G-CSF in ACLF in Germany, funded by the German Research Foundation (DFG) (NCT02669680). They recruited 163 patients and randomized them into two groups: G-CSF treatment and standard care. G-CSF did not improve survival, and there was a trend toward increased mortality in the subgroup of patients with alcoholic hepatitis and ACLF according to the APASL criteria (11).

[0120] Second, due to conflicting data regarding the therapeutic benefits of G-CSF, we conducted preclinical studies in a well-established rodent model of ACLF to clarify the effects of stem cell mobilization. C57BL / 6 mice were gavaged with carbon tetrachloride (CCL4) for 6 weeks to induce liver fibrosis, and then injected with lipopolysaccharide (LPS) to induce tissue injury and organ failure. One hour after LPS injection, mice were treated with G-CSF subcutaneously at 250 μg / kg once daily for 5 consecutive days (Figure 1A). CCl4 induced significant bridging fibrosis and severe endotoxin-driven liver injury, with an expanded TUNEL-positive area indicating apoptotic / necroptotic parenchymal cells. All animals without treatment survived the 5-day follow-up after LPS injection. However, G-CSF therapy led to a 50% mortality rate and a higher degree of liver fibrosis (Sirius Red) after 48 hours (Figure 1B, C). This was accompanied by increasing liver fibrosis and macrophage infiltration of the liver (immunohistochemistry F4 / 80+) (Fig. 1C), which persisted 5 days after G-CSF treatment.

[0121] Further repetition by the scientists led to a 66% mortality rate in all ACLF animals treated with G-CSF within 48 hours after LPS injection, while all other animals survived the entire treatment period.

[0122] Treatment with G-CSF increased mortality, exacerbated hepatic macrophage infiltration and local inflammatory response (IL6) (FIG. 4D), and enhanced liver fibrosis.

[0123] Thus, G-CSF exacerbates endotoxin-driven ACLF and ACLF-associated mortality, which is associated with exacerbating hepatic macrophage infiltration.

[0124] [Example 2] Stem cell mobilization and toll-like receptor 4 inhibition act synergistically as treatments for acute exacerbation of chronic liver failure We investigated whether Toll-like receptor 4 (TLR4) inhibition prevents stem cell mobilization-induced harm in acute exacerbations of chronic liver failure and whether exacerbated inflammatory responses and organ dysfunction are associated with stem cell mobilization. All stem cell mobilization agents, such as G-CSF, GM-CSF, and AMD3100, are effective in mobilizing stem cells but also release pro-inflammatory cells, such as neutrophils and monocytes. We hypothesized that once released into the circulation during acute exacerbations of chronic liver failure, these cells immediately encounter circulating danger molecules, such as damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs), which activate immune cells via Toll-like receptor 4 (TLR4). The exaggerated inflammatory response then exacerbates tissue injury.

[0125] We hypothesized that increased G-CSF-associated mortality in ACLF may be due to a TLR4-dependent mechanism based on the following observations: Sensitization to endotoxin through upregulation of TLR4 in the liver is a key event in liver cirrhosis (Figure 2A, B). G-CSF increases TLR4 expression, as it has been shown that once administered to a mouse model of liver failure, G-CSF increased hepatic TLR4 expression and exacerbated liver injury in this model (12). We found that the number of TLR4-positive CD45+ immune cells increased in the liver (Figure 2C).

[0126] To investigate this hypothesis, we used the TLR4 antagonist, TAK-242, in a rodent ACLF model with or without G-CSF. TAK-242 was effective in reducing cell death in the liver (Figure 2D) and improved plasma ALT levels (Figure 2D).

[0127] We next tested whether TLR4 inhibition (e.g., TAK-242) rescued G-CSF-driven injury and inflammation in a preclinical model of ACLF. C57B / 6 mice were gavaged with CCL4 for 6 weeks, then injected with LPS and treated with either G-CSF or TAK-242, or their combination, for 24 hours and 5 days (Figures 3A and 4A). Results showed that in the LPS-driven inflammatory LPS model, animals were protected from G-CSF-associated death (Figure 3D), had reduced plasma ALT levels (Figure 3B), and rescued LPS-induced organ injury was evidenced by reduced liver cell death (TUNEL (Figure 3B), BCL2 (Figure 4E)) and inflammatory responses (macrophage infiltration, liver neutrophils (Figures 3C and 3D), TNF-alpha, and IL-6 (Figure 4D)).

[0128] The STAT3 pathway attenuates apoptosis by releasing BCL2, which antagonizes BAX. We also observed that the protective effect of TAK-242 / G-CSF was associated with activation of the STAT3 pathway (increased pSTAT3) (Figure 4F), possibly induced by IL-22 secretion (Figure 4G). This was accompanied by increased expression of anti-apoptotic BCL2 and a higher BCL2 / BAX ratio (Figure 4F). Similarly, as shown by protein profiling after pooling liver lysates, injury markers (e.g., lipocalin / NGAL and IL-13) were increased with G-CSF but reduced with the addition of TAK-242 (Figure 4G). LPS injection was also associated with astrocyte activation (aSMA expression), which could be significantly suppressed by TAK-242 (Figure 4H).

[0129] Monocytes and macrophages are major sources of cytokine release in liver disease. Therefore, we examined in vitro the extent to which LPS and G-CSF modulate cytokine responses in PMA-activated and naive THP1 cells. These experiments demonstrated that G-CSF incubation of PMA-activated THP1 macrophage-like cells and naive THP1 monocyte-like cells prior to LPS stimulation resulted in exacerbated cytokine responses, particularly upregulation of IL6 mRNA expression [THP1 macrophages + LPS 10 ng / mL: 6.2-fold upregulation vs. LPS 10 ng / mL + G-CSF 100 ng / mL: 6.7-fold upregulation; THP1 monocytes + LPS 10 ng / mL: 60.2-fold upregulation vs. LPS 10 ng / mL + G-CSF 100 ng / mL: 71-fold upregulation (Figure 4I)]. These in vitro findings are consistent with the 2.8-fold increased hepatic IL6 levels in G-CSF-treated animals (Figure 4D). These changes with LPS alone or in combination with G-CSF were completely prevented in the presence of TAK-242, clearly demonstrating that TAK-242 prevents the overwhelming inflammatory response driven by LPS and G-CSF. The data help explain the reduction in markers of organ injury in ACLF animals treated with G-CSF / TAK-242.

[0130] We observed that combinatorial therapy using TAK-242 and G-CSF interacted with each other on liver regeneration and injury. G-CSF combined with TAK-242 reduced hepatocyte senescence after 24 hours and significantly improved hepatocyte proliferation after 5 days of treatment, whereas individual treatment with TAK-242 did not enhance hepatocyte-driven tissue repair (Figure 4B, C, D). In CCL4 cirrhotic animals that had not received LPS, we observed a similar G-CSF effect on hepatocyte proliferation, thereby providing evidence that preventing LPS-TLR4 signaling is important for unmasking the proliferation-promoting potential of G-CSF.

[0131] As further evidence that G-CSF exerts a growth-promoting effect in a non-inflammatory environment, we tested 5 days of G-CSF therapy after CCl4 administration without LPS. In this setting, G-CSF injection significantly increased the number of proliferating hepatocytes; Ki67 staining was observed at 4.1% ± 2.9 (p < 0.001 vs. CCl4 + LPS + G-CSF 5d) and cyclin A2 staining was observed at 2.1% ± 1.3 (p < 0.05 vs. CCl4 + LPS + G-CSF 5d) (Figure 4J).

[0132] The protein expression of other regenerative markers was assessed in pooled liver lysates using a proteome profiler. TAK-242 alone administered for 24 hours in CCl4-LPS mice reduced both hepatic markers of vascular regeneration (e.g., angiopoietin 2, proliferin, and PDGF) and other markers involved in liver regeneration, such as IL-22, Flt3-ligand, and IGFBP-1 (Figure 4G, Revascularization and Regeneration). Addition of G-CSF to TAK-242 significantly increased hepatic protein expression of these pro-regenerative markers (Figure 4G, Revascularization and Regeneration).

[0133] These findings confirmed the hypothesis that G-CSF requires a non-inflammatory environment to exert its proliferative effects on hepatocytes and, therefore, that anti-inflammatory therapy with TAK-242 allows G-CSF to exert its pro-regenerative potential.

[0134] [Example 3] Mechanism of synergy between G-CSF and TAK-242 in a non-inflammatory ACLF model It is well known that G-CSF has growth-promoting properties, and liver cirrhosis is associated with cellular senescence and a lack of hepatocyte proliferation. We therefore evaluated whether the mechanism of the beneficial effects of TAK-242 and G-CSF as a combination therapy is through modulation of inflammation while increasing hepatocyte proliferation.

[0135] Several preclinical models of liver injury have been well documented in which stem cell recruitment and homing to the liver induces parenchymal regeneration. However, ACLF is a distinct disease entity, as it is primarily associated with sepsis, a systemic inflammatory response accompanied by tissue injury, in contrast to models that demonstrate hepatotoxic injury. Inflammation may mask regeneration induced by stem cells and regulatory immune cells. Therefore, we tested whether adding TAK-242 to G-CSF not only prevents the G-CSF-driven inflammatory response but also allows stem cell recruitment to demonstrate its tissue repair potential. Combination treatment of G-CSF and TAK-242 was tested in a separate model of ACLF consisting of gavage of CCL4 for 6 weeks followed by injection of galactosamine (a hepatotoxic, non-inflammatory hit) (Figure 5). In the GalN model, TAK-242 plus G-CSF was superior to the individual therapies in reducing tissue injury (TUNEL (Fig. 5B), BCL2 (Fig. 7), ALT levels (Fig. 5B), and RIPK3 (Fig. 6A, B)).

[0136] In the GalN-induced non-inflammatory model, the addition of TAK-242 to G-CSF was significantly superior to the individual therapies in reducing liver injury (TUNEL, RIPK3) through BCL2 upregulation (Figure 7).

[0137] In the GalN-induced non-inflammatory model, GalN injection induced significant liver injury, with elevated ALT levels [27.3 U / L (range 24.2-63.4) vs. 288 U / L (range 46-807) (p<0.001)], enlarged areas of cell death (TUNEL) [0.23% (range 0.17-0.29) vs. 2.6% (range 1.3-5.2)], and RIPK3 positivity [1% (range 0.75-1.1) vs. 3.2% (range 1.6-4.4), p<0.001)], a marker of necroptotic cell death. In contrast to the inflammatory LPS model, treatment with G-CSF alone reduced ALT levels [101 U / L (range 51-284)], liver cell death (TUNEL 1.2% (range 0.4-1.9), p<0.001), and RIPK3 expression [1.9% (range 0.9-2.5)]. TAK-242 alone tended to reduce ALT levels to 96.5 U / L (range 45-229), RIPK3 expression [1.7% (range 0.9-2.1)], and liver cell death [TUNEL 1.4% (range 0.4-3.3)], but did not reach statistical significance. The combination of G-CSF and TAK242 was superior to both single treatments, improving ALT levels (74.5 U / L, range 44-297), liver cell death (TUNEL 0.45% (0.11-0.95), p<0.001 compared with both individual therapies), and RIPK3 expression (1.23% (1-1.78), p<0.01 vs. CCl4+GalN). Furthermore, treatment with TAK242+G-CSF was also associated with activation of the STAT3 pathway and a trend toward increased anti-apoptotic BCL2 expression as assessed in pooled liver samples by Western blot.

[0138] We also evaluated whether hepatocyte proliferation was modulated by treatment with G-CSF ± TAK-242 in this model. CCl4-treated animals injected with GalN exhibited higher levels of proliferating hepatocytes [Ki67: CCl4 0.1% ± 0.1 vs. CCl4 + GalN 2% ± 1.7, (p < 0.01); cyclin A2: CCl4 0.1% ± 0.2 vs. CCl4 + GalN 1.9% ± 1.6, (p < 0.05)] and cell cycle-arrested hepatocytes (p21: CCl4 0.5% ± 0.2 vs. CCl4 + GalN 6.4% ± 4.7, (p < 0.01). Treatment with G-CSF with or without TAK-242 reduced the degree of proliferative and senescent hepatocytes, whereas TAK-242 alone did not alter the response to injury [(Ki67: CCl4 + GalN + TAK-242 - 3% ± 2.1 vs. CCl4 + GalN + TAK-242 + G-CSF: 1.2% ± 1.4 (p < 0.05); Cyclin A2: CCl4 + GalN + TAK-242 - 1.9% ± 1.6 vs. CCl4 + GalN + TAK-242 + G-CSF 0.3% ± 0.2 (p < 0.001); p21: CCl4 + GalN + TAK-242 7.1% ± 4.5 vs. CCl4 + GalN + TAK-242 + G-CSF 1.7% ± 2.3 (p < 0.001)] (Figure 8).

[0139] Taken together, these data suggest that in this non-inflammatory model, the combination of G-CSF and TAK-242 positively impacts liver injury and regeneration, and reduces markers of cellular senescence, confirming that the regenerative effects of G-CSF are exhibited in a non-inflammatory environment.

[0140] [Example 4] Relationship between cell death and liver regeneration and the effects of G-CSF and TAK-242 To investigate the role of cell death as a modulator of regeneration, we used RIPA56, a selective RIPK1 inhibitor, to prevent GalN-induced necroptotic cell death in various treatment groups. RIPA56 treatment effectively prevented GalN-driven cell death (TUNEL p<0.001 and RIPK3 p<0.001, both compared with CCl4 + GalN) and was associated with reduced hepatocyte proliferation compared with CCl4 + GalN (Ki67 2% ± 1.7 vs. 0.2% ± 0.2, p<0.001; Cyclin A2 1.9% ± 1.6 vs. 0.03% ± 0.1, p<0.001) (Figure 9), suggesting that in this non-inflammatory environment, the regenerative response directly correlates with the extent of injury.

[0141] We then calculated the ratio between the expression of cyclin A2, a marker of cell cycle progression, and the amount of liver cell death (TUNEL), which represents the regenerative potential of both drugs, in all models studied. Therefore, an increased ratio between cyclin A2 and TUNEL indicates enhanced regenerative activity and less cell death, and vice versa (Figure 10). In the 24-hour CCl4-LPS model, LPS injection, with or without G-CSF, completely inhibited the regenerative response (cyclin A2 / TUNEL ratio: CCl4 0.42 vs. CCl4 + LPS 0.02 vs. CCl4 + LPS + G-CSF 0.04). In contrast, in the CCl4-GalN model, the regenerative response was maintained after GalN injection (cyclin A2 / TUNEL ratio: CCl4 + GalN 0.54). In both "short-term" models, TAK-242 administration was associated with enhanced hepatocyte regenerative responses (cyclin A2 / TUNEL ratio: CCl4 + LPS + TAK-242: 1.4; CCl4 + GalN + TAK-242: 0.9), supporting the hypothesis that creating an anti-inflammatory environment is important for restoring regenerative potential. Furthermore, the anti-inflammatory environment (in the presence of TAK-242) enabled G-CSF to exert its positive effects on liver injury, particularly in the CCl4-LPS model, enhancing regeneration after 5 days of therapy (cyclin A2 / TUNEL ratio: CCl4 + LPS + TAK-242 + G-CSF: 1.7), thus supporting the regenerative potential of G-CSF, particularly after long-term treatment.

[0142] conclusion Data demonstrate synergy between G-CSF and TLR4 inhibition using TAK-242 by modulating the severity of inflammation while impacting liver proliferation, thereby improving survival in a model of ACLF. G-CSF is an approved treatment for post-chemotherapy neutropenia and for bone marrow stimulation in healthy stem cell donors. G-CSF has a long-standing and proven safety record and can be used for other purposes. It is available for clinical use and has a TRL of 9. TAK-242, a TLR4 antagonist, has been tested in clinical trials up to Phase 3 with a proven safety record. TAK-242 recently achieved TRL 8 status and is being tested in Phase 2a / 2b clinical trials.

[0143] Thus, the inventors have made the following unexpected findings: (i) In clinically relevant animal models, the stem cell mobilizer, G-CSF, is harmful because it induces systemic inflammation and increases mortality; (ii) TAK-242 inhibits inflammation and organ injury, (ii) reduces liver inflammation, thereby allowing hepatic stem cells to engraft; (iii) G-CSF mobilizes stem cells, but these cells fail to engraft in the liver due to liver inflammation, and G-CSF can lead to exacerbated inflammation and increased mortality in animal models. (iv) The combination of TAK242 with G-CSF, a stem cell mobilizer, acts synergistically to (i) reduce hepatic and systemic inflammation, (ii) mobilize stem cells without inducing inflammation, and (iii) these cells have improved ability to engraft in the liver. The present invention encompasses, for example, the following embodiments: [Embodiment 1] A stem cell mobilizer and a TLR4 antagonist for use in a method for treating or preventing liver failure in an individual in need thereof. [Embodiment 2] A TLR4 antagonist for use in a method for treating or preventing liver failure in an individual in need thereof, the method also comprising administering stem cells to the individual in need thereof. [Embodiment 3] The TLR4 antagonist for use according to embodiment 2, wherein the stem cells are hepatic stem cells. [Embodiment 4] A stem cell mobilizer and a TLR4 antagonist for use according to embodiment 1, or a TLR4 antagonist for use according to embodiment 2 or 3, wherein the liver failure is acute liver failure (ALF) or acute exacerbation of chronic liver failure (ACLF). [Embodiment 5] An individual has the following when compared to a subject not suffering from ACLF: (a) renal dysfunction and / or (b) renal failure and / or (c) brain dysfunction and / or (d) brain swelling and / or (e) inflammation, injury, or dysfunction in the kidney and / or brain; and / or (f) liver failure and / or (g) Immunodeficiency The stem cell mobilizer and TLR4 antagonist for use according to embodiment 4, or the TLR4 antagonist for use according to embodiment 4, in patients suffering from or at risk of having one or more of the following: [Embodiment 6] A stem cell mobilizer and TLR4 antagonist for use according to any of embodiments 1, 4, and 5, wherein the stem cell mobilizer and / or TLR4 antagonist is administered in combination with an additional agent useful in the treatment or prevention of liver failure, e.g., ALF or ACLF. [Embodiment 7] The TLR4 antagonist for use according to any of embodiments 2 to 5, wherein the stem cells and / or TLR4 antagonist are administered in combination with an additional agent useful in the treatment or prevention of liver failure, such as ALF or ACLF. [Embodiment 8] A stem cell mobilizer and TLR4 antagonist for use according to any one of embodiments 1 and 4 to 6, wherein the stem cell mobilizer is G-CSF. [Embodiment 9] A method for treating or preventing liver failure in an individual in need thereof, comprising administering to the individual a stem cell mobilizer and a TLR4 antagonist. [Embodiment 10] A method for treating or preventing liver failure in an individual in need thereof, comprising administering to the individual stem cells and a TLR4 antagonist. [Embodiment 11] The method of embodiment 9 or 10, wherein the liver failure is ALF or ACLF. [Embodiment 12] The method of embodiment 9 or 11, wherein the stem cell mobilizer is G-CSF. [Embodiment 13] A stem cell mobilizer for use in a method for treating or preventing liver failure in an individual in need thereof, the method comprising additionally administering to the individual a TLR4 antagonist. [Embodiment 14] Stem cells for use in a method for treating or preventing liver failure in an individual in need thereof, the method comprising additionally administering to the individual an antagonist of TLR4. [Embodiment 15] A stem cell mobilizer for use as described in embodiment 13 or stem cells for use as described in embodiment 14, wherein administration of a TLR4 antagonist precedes administration of the stem cell mobilizer or stem cells to the individual, is simultaneous with administration of the stem cell mobilizer or stem cells to the individual, is sequential to administration of the stem cell mobilizer or stem cells to the individual, or is subsequent to administration of the stem cell mobilizer or stem cells to the individual. [Embodiment 16] A stem cell mobilizer for use according to embodiment 13 or 15, wherein the stem cell mobilizer is G-CSF. [Embodiment 17] A stem cell mobilizer and TLR4 antagonist for use according to any one of embodiments 1, 4, 5, 6 and 8, wherein the TLR4 antagonist is TAK-242; a method according to any one of embodiments 9, 11 and 12, or a stem cell mobilizer for use according to any one of embodiments 13, 15 and 16. [Embodiment 18] A TLR4 antagonist for use according to any one of embodiments 2, 3, 4, 5 and 7, a method according to any one of embodiments 9, 10 and 11, or a stem cell for use according to any one of embodiments 14 or 15, wherein the TLR4 antagonist is TAK-242.

[0144] References TIFF0007798774000001.tif225148

Claims

1. A composition comprising a stem cell mobilizer and a TLR4 antagonist for use in a method for treating or preventing liver failure in an individual in need thereof, wherein the stem cell mobilizer is granulocyte colony-stimulating factor (G-CSF), filgrastim, lenograstim, or pegfilgrastim, the liver failure is acute liver failure (ALF) or acute-on-chronic liver failure (ACLF), and the TLR4 antagonist is TAK-242.

2. If an individual, when compared to a subject without ACLF, has: (a) renal dysfunction and / or (b) renal failure and / or (c) brain dysfunction and / or (d) brain swelling and / or (e) inflammation, injury, or dysfunction in the kidney and / or brain; and / or (f) liver failure and / or (g) Immunodeficiency The composition of claim 1, for use in a patient suffering from or at risk of one or more of the following:

3. 3. The composition of claim 1 or 2, wherein the stem cell mobilizer and / or TLR4 antagonist is administered in combination with an additional agent useful in the treatment or prevention of liver failure, e.g., ALF or ACLF.

4. The composition of any one of claims 1 to 3, wherein the stem cell mobilizer is G-CSF.

5. Use of a stem cell mobilizer and a TLR4 antagonist in the manufacture of a medicament for treating or preventing liver failure in an individual in need thereof, wherein the stem cell mobilizer is granulocyte colony-stimulating factor (G-CSF), filgrastim, lenograstim, or pegfilgrastim, the liver failure is acute liver failure (ALF) or acute-on-chronic liver failure (ACLF), and the TLR4 antagonist is TAK-242.

6. If an individual, when compared to a subject without ACLF, has: (a) renal dysfunction and / or (b) renal failure and / or (c) brain dysfunction and / or (d) brain swelling and / or (e) inflammation, injury, or dysfunction in the kidney and / or brain; and / or (f) liver failure and / or (g) Immunodeficiency 6. The use according to claim 5, wherein the patient is suffering from or at risk of having one or more of the following:

7. 7. The use of claim 5 or 6, wherein the stem cell mobilizer and / or TLR4 antagonist is administered in combination with a further agent useful in the treatment or prevention of liver failure, such as ALF or ACLF.

8. The use according to any one of claims 5 to 7, wherein the stem cell mobilizer is G-CSF.

9. A composition comprising a stem cell mobilizer for use in a method for treating or preventing liver failure in an individual in need thereof, the method comprising additionally administering to the individual a TLR4 antagonist, wherein the stem cell mobilizer is granulocyte colony-stimulating factor (G-CSF), filgrastim, lenograstim, or pegfilgrastim, the liver failure is acute liver failure (ALF) or acute-on-chronic liver failure (ACLF), and the TLR4 antagonist is TAK-242.

10. The composition of claim 9, wherein administration of the TLR4 antagonist precedes administration of the stem cell mobilizer to the individual, is simultaneous with administration of the stem cell mobilizer to the individual, is sequential to administration of the stem cell mobilizer to the individual, or is subsequent to administration of the stem cell mobilizer to the individual.

11. The composition of claim 9 or 10, wherein the stem cell mobilizer is G-CSF.

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