Agents for use in a method of treating an acute liver disease subject

Abstract

The present invention relates to a senotherapeutic agent, population of isolated cells and / or anti- inflammatory agent for use in a method of treating an acute liver disease subject, wherein the acute liver disease subject has a p21 hepatocyte expression level obtained from a sample from the subject greater than a reference p21 hepatocyte expression level. Also provided are kits, method for selecting subjects for participation in an acute liver disease treatment clinical trial, and methods for selecting an acute liver disease subject for liver transplant.

Description

[0001] AGENTS FOR USE IN A METHOD OF TREATING AN ACUTE LIVER DISEASE SUBJECT

[0002] Field of the Invention

[0003] The present invention relates to a senotherapeutic agent, population of isolated cells, cell regenerative agent and / or anti-inflammatory agent for use in a method of treating an acute liver disease subject, wherein the acute liver disease subject has a p21 hepatocyte expression level obtained from a sample from the subject greater than a reference p21 hepatocyte expression level. Also provided are kits, methods for selecting subjects for participation in an acute liver disease treatment clinical trial, and methods for selecting an acute liver disease subject for liver transplant.

[0004] Background

[0005] Liver disease encompasses a broad spectrum of liver injury ranging from simple steatosis through alcohol- and non-alcohol- related hepatitis, to cirrhosis and hepatocellular carcinoma (HCC). The latter, liver cancer, is the third most common cause of cancer-related death worldwide and will develop in 1 in 3 subjects with cirrhosis. Despite some improvements in outcomes for those subjects in whom the disease is detected early, there remains a limited range of only minimally effective treatment options for the overwhelming majority of subjects who have their disease detected at a later stage. Precision medicine offers the potential to target more effective therapies to individuals with different forms of this disease, across this highly heterogeneous cancer.

[0006] The incidence of liver disease has increased by 400% since the 1970s, and is statistically the biggest cause of death in people aged between 35-49. Although liver disease is largely preventable, 75% of those diagnosed present with late-stage disease, at which point it is too late for lifestyle changes or intervention. Liver injury varies largely depending on the condition, which can be broken down into chronic disease including non-alcoholic fatty liver disease (NAFLD), alcohol-related liver disease (ALD), hepatocellular carcinoma (HCC), viral hepatitis (Hep A), and acute liver injury, for example poisoning.

[0007] The management of these different types of chronic liver injury also largely varies. ALD is the most prevalent of liver disorders, accounting for 60% of all liver disease, and -7,700 deaths per year in the UK. The first stage of ALD is a build-up of fat in the liver as a result of the by-products of alcohol being broken down by the liver, which then progresses to alcohol-related hepatitis and the formation of scar tissue (fibrosis), and finally cirrhosis, which is usually irreversible and can result in fatal liver failure. ALD is often diagnosed during blood tests for other health problems, or upon going to an accident and emergency hospital department with symptoms of serious liver disease or liver failure, at which point it is unlikely the damage can be reversed. The treatment for ALD is first and foremost to stop alcohol consumption, whilst also treating any complications of the disease. NAFLD is only usually diagnosed once it has become serious, and is discovered by testing the blood for alanine aminotransferase (ALT) and aspartate aminotransferase (AST). The management of NAFLD is primarily lifestyle changes such as eating a balanced diet and remaining physically active. However, NAFLD can progress to non-alcohol related steatohepatitis (NASH), which 5% of UK adults develop, and is much more serious and can lead to liver failure, and in some cases liver cancer. Around 6,000 cases of primary liver cancer are diagnosed in the UK every year, with the majority of these subjects having underlying advanced liver disease. HCC is the most common form of liver cancer, which has seen a 63% increase in incidence and 55% increase in mortality over the past decade, the highest of which is in Scotland. Diagnosis for HCC is usually by ultrasound, CT or MRI scans, or tissue biopsies. Management for HCC includes surgery to remove the tumour, and chemotherapy and I or radiotherapy.

[0008] Acute liver disease or liver injury is defined as the loss of liver function that occurs quickly (in days or weeks) in a person who has no pre-existing liver disease. Acute liver disease is less common than chronic disease, and is most commonly caused by a hepatitis virus or drug poisoning, for example with paracetamol (acetaminophen). Paracetamol overdose is the most common cause of acute liver failure, and can result from one very large dose, or an accumulation of higher than the recommended dose over a number of days. Acute liver failure can have serious complications including cerebral oedema, bleeding disorders, infections and kidney failure which is more common following a paracetamol overdose. In the UK, paracetamol is the most common cause of overdose accounting for 48% of all poisoning admissions, with -100,000 people presenting with paracetamol overdose each year, with 227 deaths as a result of paracetamol poisoning recorded last year. Currently, when a subject comes into hospital with acute liver disease, they require a biopsy whilst serological tests are being completed. Treatment options largely depend upon the source of the liver injury, but commonly involve treatment with corticosteroids in the short term which are not always effective and require the subject to be monitored, and decisions regarding liver transplant as a long-term solution.

[0009] Liver transplant is considered when a subject’s life becomes threatened by end-stage liver disease, and is usually only offered when other treatment options have been exhausted. To be offered a liver transplant, certain criteria must be met, such as completely stopping alcohol and drug use, and being well enough to recover from the surgery itself. Criteria for liver transplant selection among adults are discussed in Millson C, et a / . Frontline Gastroenterology 2020;0:1-10. doi:10.1136 / flgastro-2019-101215. In particular, Table 1 of Millson et al. outlines clinical features in paracetamol acute liver failure and non-paracetamol induced acute liver failure on which a decision to refer the subject to a liver transplant unit (LTU) are made. Currently scoring systems used clinically to define subject outcome and the need for liver transplantation employ markers during progressive disease in its later stage and incorporate multisystem dysfunction (including encephalopathy, respiratory and renal failure).

[0010] In general, for a transplant decision to be made, certain criteria need to be met by which point the subject is significantly unwell and will either receive a liver transplant or pass away from the disease. There remains a significant unmet need for a test to predict the severity of acute liver disease in a subject, and thus which treatment, if any, for acute liver disease would be most appropriate. In particular, there remains a significant unmet need for a test to predict if an acute liver disease subject is likely to require treatment or instead likely to be able to recover without therapeutic intervention. There also remains a need for a test to predict an acute liver disease subject’s requirement for potentially lifesaving liver transplant.

[0011] The present invention has been devised in light of the above considerations and aims to address this unmet need as well as providing certain related advantages.

[0012] Summary of the Invention

[0013] The present inventors studied a cohort of subjects with acute liver specific disease (acute indeterminant hepatitis) undergoing diagnostic biopsy in the early stages of acute severe liver disease. While routine biochemistry and clinical parameters at the time of biopsy did not define either outcome or subsequent multiorgan dysfunction (including both renal and cerebral failure - hepatic encephalopathy) in this cohort, the inventors surprisingly found that hepatocellular p21 expression on the baseline diagnostic biopsy predicts both survival and the future development of renal and cerebral dysfunction. The development of subsequent renal dysfunction was a predictor of survival. Hepatocellular p21 expression, therefore, provides a prognostic biomarker in acute liver disease that may be used for the rapid, early and informative stratification of subjects into treatment subgroups, and in particular for the selection of subjects requiring liver transplant.

[0014] According to a first aspect, the present invention provides a senotherapeutic agent for use in a method of treating an acute liver disease subject, wherein the acute liver disease subject has a p21 hepatocyte expression level obtained from a sample from the subject greater than a reference p21 hepatocyte expression level.

[0015] As noted above, the present inventors have found that hepatocellular p21 expression in an acute liver specific disease subject surprisingly correlates with the severity of the acute liver disease and the likely survival outcome of the patient without treatment. Based on this, the present inventors have identified an acute liver disease subject sub-group, for which rapid treatment is especially advantageous.

[0016] In some embodiments, the senotherapeutic agent comprises a senolytic agent or a senomorphic agent.

[0017] The term “senotherapeutic agent”, as used herein, will be understood to refer to a therapeutic agent which specifically targets cellular senescence. Targeting cellular senescence may comprise the inhibition or prevention of cellular senescence.

[0018] The term “senolytic agent” is a known term of the art which is understood to refer to an agent which is capable of selectively clearing senescent cells. In particular, the term “senolytic agent” will be understood to refer to a pharmacological agent which is capable of specifically targeting, typically downregulating, pro-survival and anti-apopotic cellular pathways. Thus, the administration of a senolytic agent typically leads to increased apoptosis / decreased survival of senescent cells, acting to clear the senescent cells. This may otherwise be referred to as senolysis. Various senolytic agents are known to those skilled in the art. Further information on senolytic agents can be found, for example, in Kirkland JL, Tchkonia T. Senolytic drugs: from discovery to translation. J Intern Med. 2020 Nov;288(5):518-536, which is herein incorporated by reference in its entirety.

[0019] In some embodiments, the senolytic agent comprises one or more of UBX1235, ABT-737, ABT-199 (Venetoclax), ABT-263 (Navitoclax), UBX0101 , Dasatinib, Quercitin, Fisetin, ALK4 / 5 / 7, AZD1260111 , and a cardiac glycoside.

[0020] By “senomorphic agent”, as used herein, this will be understood to refer to a pharmacological agent which is capable of specifically targeting and downregulating the SASP (senescence-associated secretory phenotype) pathway. Various senomorphic agents will be known to those skilled in the art. In some embodiments, the senomorphic agent comprises one or more of an insulin-like growth factor (IGF), rapamycin, everolimus, temsirolimus, and deforolimus. In some embodiments, the senomorphic agent comprises one or more of rapamycin, everolimus, temsirolimus, and deforolimus. In some embodiments, the senomorphic agent comprises an insulin-like growth factor (IGF). In some embodiments the IGF comprises IGF-1 or IGF-2. Preferably, the IGF comprises IGF-1. More preferably, the IGF comprises recombinant human IGF-1 (rhlGF-1). rhlGF-1 is available from various commercial providers under the generic name Mecasermin, and so the skilled person would readily be able to access this. One such example is Increlex, which is available from Ipsen Ltd. Further information about mecasermin is available from Drug Bank (https: / / go.drugbank.com / drugs / DB01277) which is incorporated herein by reference in its entirety.

[0021] According to a further aspect, the present invention provides a population of isolated cells for use in a method of treating an acute liver disease subject, wherein the acute liver disease subject has a p21 hepatocyte expression level obtained from a sample from the subject greater than a reference p21 hepatocyte expression level; and optionally wherein the population of cells comprise stem cells or hepatocytes.

[0022] Also provided by the present invention is an anti-inflammatory agent for use in a method of treating an acute liver disease subject, wherein the acute liver disease subject has a p21 hepatocyte expression level obtained from a sample from the subject greater than a reference p21 hepatocyte expression level; and optionally wherein the anti-inflammatory agent comprises a TGF-0 antagonist, a TLR4 antagonist or a CXCR4 antagonist.

[0023] As used herein, the term “anti-inflammatory agent” will be understood to refer to a pharmacological agent which is capable of inhibiting, decreasing or preventing inflammatory activity.

[0024] In some embodiments the method comprises administering the anti-inflammatory agent and a cell- regenerative agent to the subject. The cell-regenerative agent may comprise G-CSF or GM-CSF. In some embodiments, the anti-inflammatory agent comprises a TGF-0 antagonist, optionally wherein the TGF-0 antagonist comprises an anti- TGF-0 antibody.

[0025] The present invention also provides a method for selecting subjects for participation in an acute liver disease treatment clinical trial, the method comprising: a) measuring a p21 hepatocyte expression level in a sample obtained from the subject; b) comparing the measured p21 hepatocyte expression level to a reference p21 hepatocyte expression level; and c) selecting the subject for participation in the clinical trial when said measured p21 hepatocyte expression level is greater than said reference p21 hepatocyte expression level.

[0026] As the skilled person will appreciate, it can be difficult to accurately and rapidly diagnose a subject with acute liver disease. Given such difficulties, it can be challenging to recruit enough suitable subjects for participation in an acute liver disease treatment clinical trial. Advantageously, therefore, the present inventors have found that the measured p21 hepatocyte expression level is an accurate marker for stratifying acute liver disease subjects and thus to enable selection of suitable subjects for clinical trial participation.

[0027] According to a further aspect, the present invention provides a method for selecting an acute liver disease subject for liver transplant, the method comprising: a) measuring a p21 hepatocyte expression level in a sample obtained from the subject; b) comparing the measured p21 hepatocyte expression level to a reference p21 hepatocyte expression level; and c) making a prediction of whether the subject will require a liver transplant based on the comparison in step b), wherein said measured p21 hepatocyte expression level being greater than said reference p21 hepatocyte expression level indicates that the subject will require a liver transplant, and wherein said measured p21 hepatocyte expression level being equal to or lower than said reference p21 hepatocyte expression level indicates that the subject will not require a liver transplant.

[0028] In some embodiments, the sample comprises liver tissue obtained from the subject and the p21 hepatocyte expression level is measured as the number of p21 positive hepatocytes in a given area of the sample divided by the total number of hepatocytes in that area, optionally expressed as a percentage or fraction.

[0029] In some embodiments, the p21 hepatocyte expression level is measured by immunohistochemistry (IHC) or multiplex immunofluorescence.

[0030] In some embodiments, the sample has been formalin-fixed and paraffin-embedded prior to said measuring of the p21 hepatocyte expression level.

[0031] The reference p21 hepatocyte expression level may be about 10%, about 11 %, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19% about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39% or about 40% of the hepatocytes being p21 positive. The reference p21 hepatocyte expression level may be about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21 %, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39% or about 40% of the hepatocytes being p21 positive.

[0032] In some embodiments, the reference p21 hepatocyte expression level is of from about 10% to about 16% of the hepatocytes being p21 positive.

[0033] In some embodiments, the subject is a human having or suspected of having acute liver disease without any significant signs of chronic liver disease. The acute liver disease may comprise acute indeterminate hepatitis, acute autoimmune hepatitis, acute viral hepatitis, or drug-induced liver injury.

[0034] In some embodiments, yH2AX hepatocyte expression level in a sample obtained from the subject is also measured.

[0035] According to a further aspect, the present invention provides use of a kit comprising an anti-p21 antibody for: selecting subjects for participation in an acute liver disease treatment clinical trial or predicting whether an acute liver disease subject will require a liver transplant. In some embodiments, the kit further comprises one or more hepatocyte-specific reagents, optionally an anti-HNF4a antibody.

[0036] By “p21 hepatocyte expression level”, this will be understood to refer to the expression level of p21 in hepatocytes. Expression of p21 may comprise p21 mRNA expression and / or p21 protein expression.

[0037] As used herein, the term “acute liver disease” will be understood to refer to a sudden-onset reduction or loss in liver function. The term “acute liver disease” may be used interchangeably herein with the term “acute liver failure”. The sudden-onset reduction or loss in liver function in acute liver disease typically occurs within days or weeks. The subject may have no pre-existing liver disease. In particular, the subject may have had no recorded significant liver disease in the preceding six months. The acute liver disease subject may have symptoms of encephalopathy, jaundice and / or coagulopathy, but did not have these symptoms in the preceding six months. In the context of the present invention, the preceding six months will typically be understood to be calculated from the date at which the subject presented to a clinician with at least one symptom of acute liver disease. The date at which the subject presented to a clinician with at least one symptom of acute liver disease may therefore be termed “day 0”, and the preceding six months calculated as the six months preceding day 0.

[0038] The liver function of the subject may be determined using liver blood test results obtained from a blood sample from the subject. A normal function of the liver is to clear bilirubin, a blood waste product, from the blood. Therefore, liver blood tests may comprise testing the concentration of bilirubin in the blood. In some embodiments, liver blood tests comprise testing the concentration of one or more of alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), albumin and total protein, bilirubin, gamma-glutamyl transferase (GGT) and L-lactate dehydrogenase (LD) in the blood. Liver blood tests may further comprise a test for and prothrombin time (PT) of the blood. The skilled person will be aware of a normal range of liver blood tests, indicating that the liver function of a subject is normal, and an abnormal range of liver blood tests, indicating that the liver function of the subject is reduced. A reduction or loss in liver function, as used herein, may comprise a decrease in liver function of at least two fold, at least three fold, at least four fold, at least five fold, at least six fold, at least seven fold, at least eight fold, at least nine fold, or at least ten fold.

[0039] In some embodiments, the acute liver disease subject has a bilirubin expression level obtained from a sample from the subject greater than a reference bilirubin expression level. The sample may comprise a blood sample. In some embodiments, the acute liver disease subject has a creatinine expression level obtained from a sample from the subject greater than a reference creatinine expression level. Reference bilirubin and creatinine expression levels are disclosed, for example, in Millson C, et al. Frontline Gastroenterology 2020;0:1-10. doi:10.1136 / flgastro-2019-101215. In particular, Table 1 of Millson et al. outlines clinical features in paracetamol acute liver failure and non-paracetamol induced acute liver failure on which a decision to refer the subject to a liver transplant unit (LTU) are made. This reference is incorporated herein by reference in its entirety. Advantageously, the inventors have found that the combination of the p21 hepatocyte expression level and the bilirubin and / or creatinine expression level of the subject can provide even greater sensitivity in stratifying subjects.

[0040] As the skilled person will appreciate, coagulopathy will be understood to refer to the subject having an International Normalised Ratio (INR) of at least 1 .5, and preferably greater than 1 .5. The term “encephalopathy”, as used herein, will be understood to refer to any brain disease which alters the brain function or structure. Generally, encephalopathy in the context of the present invention refers to hepatic encephalopathy. Jaundice is a medical condition wherein the skin and / or the whites of the eyes turn yellow.

[0041] It will be appreciated that acute liver disease is not chronic liver disease. The skilled person will be aware that acute liver disease is distinct from chronic liver disease. Typically, a subject with chronic liver disease has at least one symptom of liver disease in the preceding six months. More typically, a subject with chronic liver disease has recorded significant signs of liver disease in the preceding six months. This contrasts with a subject with acute liver disease, wherein typically the subject has no liver disease or no signs of significant liver disease in at least the preceding six months, and optionally the subject has no pre-existing liver disease. Chronic liver disease may comprise cirrhosis (scarring of the liver). In contrast, acute liver disease typically does not comprise cirrhosis.

[0042] In the context of the present invention, “acute liver disease” may comprise acute liver failure and acute liver injury. Acute liver injury (ALI) may be defined as an acute derangement in liver function tests associated with liver-related coagulopathy in the absence of underlying chronic liver disease (CLD). Thus, ALI may be defined as the subject having an INR of at least 1 .5, wherein the subject does not have cirrhosis and wherein the subject has no liver disease or no significant signs of liver disease in at least the preceding six months, optionally wherein the subject has no pre-existing liver disease. In some embodiments, ALI does not comprise encephalopathy. Acute liver failure may be defined as the subject having an INR of at least 1 .5, wherein the subject does not have cirrhosis, wherein the subject has no liver disease in at least the preceding six months and wherein the subject optionally further has encephalopathy or signs of encephalopathy. Thus, in some embodiments, the subject has an INR of at least 1 .5. The inventors have advantageously found that stratifying subjects based on their p21 hepatocyte expression level and INR value can give increased sensitivity in prognosing outcome and determining treatment strategy.

[0043] As used herein, “p21” refers to Cyclin-dependent kinase inhibitor 1 (also known as CDK-interacting protein 1 or Melanoma differentiation-associated protein 6 (MDA-6)), which in humans is encoded by the gene CDKN1 A (also known as CAP20, CDKN1 , CIP1 , MDA6, PIC1 , SDI1 or WAF1). The amino acid sequence of human p21 is disclosed at UniProt Entry P38936 (last updated 2007-01-23 v3; Checksum: 98D1 E7C519ADFCA9), which is incorporated herein by reference in its entirety.

[0044] As the skilled person will appreciate, a hepatocyte is a primary parenchymal cell of the liver. The hepatocyte can be identified by those skilled in the art by morphological features identifiable from microscopy. For example, a hepatocyte may have a polygonal shape. The hepatocyte may comprise at least one substantially round nucleus. Hepatocytes typically comprise a plurality of Golgi membranes. Various markers for hepatocytes are also known to those skilled in the art. For example, markers including, but not limited to, cytokeratin 18, cytokeratin 19, HNF4a, ILK, albumin, transferrin, transthyretin, and a-1 -antitrypsin are known to be expressed at detectable levels in differentiated hepatocytes.

[0045] According to a further aspect, the present invention provides an insulin-like growth factor (IGF) for use in a method of treating liver disease in a subject. The IGF may comprise IGF-1 or IGF-2, preferably IGF-1. Optionally, the IGF comprises recombinant human IGF, preferably recombinant human IGF-1. In some embodiments, the subject is a human subject. The liver disease may comprise acute liver disease, as defined herein. Alternatively, the liver disease may comprise chronic liver disease. In some embodiments, the chronic liver disease comprises acute-on-chronic liver disease. By “acute-on-chronic liver disease”, this will be understood to refer to pre-existing chronic liver disease with a sudden further reduction or loss in liver function. The sudden-onset further reduction or loss in liver function in acute-on-chronic liver disease typically occurs within days or weeks. In some embodiments, the liver disease comprises acute liver disease or acute-on-chronic liver disease. In some embodiments, the subject has a p21 hepatocyte expression level obtained from a sample from the subject greater than a reference p21 hepatocyte expression level. The reference p21 hepatocyte expression level may be as defined herein. For example, the p21 hepatocyte expression level may be of from about 10% to about 16% of the hepatocytes being p21 positive. Also provided is a method of treating liver disease in a subject, the method comprising administering a therapeutically effective amount of an insulin-like growth factor (IGF) to the subject. It will be appreciated the embodiments of the above medical use aspect equally apply to the method of treatment aspect. The method may comprise subcutaneous administration. The invention also provides an insulin-like growth factor (IGF) for the manufacture of a medicament for the treatment of liver disease in a subject. The IGF may be as defined herein.

[0046] The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided. In particular, the first, second, and / or third aspects may be combined. For example, it will be appreciated that the present invention also provides a senotherapeutic agent, population of isolated cells and / or anti-inflammatory agent for use in a method of treating an acute liver disease subject, wherein the acute liver disease subject has a p21 hepatocyte expression level obtained from a sample from the subject greater than a reference p21 hepatocyte expression level. In embodiments comprising a combination of therapies (for example a senotherapeutic agent and an anti-inflammatory agent, or a senotherapeutic agent and an isolated population of cells), each therapy may be administered simultaneously, sequentially or separately. It will also be appreciated that any of the embodiments of the first, second and / or third aspects may be combined with any of the aspects concerning an insulin-like growth factor (IGF) for use in a method of treating liver disease in a subject, especially concerning p21 expression levels and acute liver disease. It will be appreciated that each embodiment disclosed herein can relate to any of the aspects disclosed herein. In particular, it will be appreciated that embodiments described in relation to the methods of treatment and medical uses disclosed herein may apply to any other method disclosed herein.

[0047] Summary of the Figures

[0048] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

[0049] Figure 1 : Loss of parenchymal cells and increased senescence in non-survivor liver biopsies Representative multiplex immunofluorescence (IF) images showing (A) Survivors (B) Non-survivor, showing quantification of liver (C&D) hepatocytes (HNF4a), (E&F) P21 expression, (G&H) yH2AX expression. Data are expressed as mean ±SD , n=17 per group. *P<0.05; **P<0.005; ***P<0.0005, (Mann-Whitney L) test).

[0050] Figure 2: Increased percentage of p21+ and YH2AX+ hepatocytes in liver samples from nonsurviving subjects

[0051] Representative graphs show (A) the percentage of p21+hepatocytes in area fields of images taken of liver biopsy samples and (B) the percentage of YH2AX+hepatocytes in area fields of images taken of liver biopsy samples. For both graphs, the area field was the field observed using a 10X magnification using a widefield microscope, and specifically the absolute number of HNF4a-positive cells in a particular field area.

[0052] Figure 3: Hepatic parenchymal senescence correlates with renal dysfunction

[0053] Representative plots show (A) baseline creatinine (B) creatinine-percent change from baseline to 28 days (C) baseline prothrombin time (D) prothrombin time-percent change from baseline to 28 days (E) correlation plots of DeltaCR (day 28- dayO) and %P21+vehepatocytes (F) Quantification of deltaCR (day 28- dayO) with recategorized hepatocytes based on P21 expression (G) Correlation plots of deltaCR (last day-0) and %P21+vehepatocytes. (H) Quantification of deltaCR delta CR (last -dayO) with recategorized hepatocytes based on P21 expression; data are expressed as mean±SD, n=17 per group. *P < 0.05; **P < 0.005; ***P < 0.0005, (Mann-Whitney U test ). Figure 4: Hepatic parenchymal senescence correlates with brain function

[0054] Representative plots show (A) HE cumulative % of subjects developing HE from baseline to 28-day follow-up (B) Percentage of p21 -positive senescent hepatocytes with and without HE (C) categorized hepatocytes based on p21 expression per field area in subjects with and without HE; data are expressed as mean±SD, n=17 per group. *P < 0.05; **P < 0.005; ***P < 0.0005, (Mann-Whitney U test ).

[0055] Figure 5: Bilirubin levels correlate with p21 hepatocyte expression levels

[0056] Representative plots show (A) bilirubin levels over time in survivors versus non-survivors (B) bilirubin levels at the time of liver biopsy in survivors versus non survivors and (C) bilirubin levels at the time of liver biopsy in subjects with a p21 hepatocyte expression level (at the time of liver biopsy) less than 10.71% or greater than or equal to 10.71 %.

[0057] Figure 6: Bilirubin levels correlate with p21 hepatocyte expression levels

[0058] Representative plots show (A) bilirubin levels over time in subjects with a p21 hepatocyte expression level (at the time of liver biopsy) less than 15.77% or greater than or equal to 15.77% (B) bilirubin levels at the time of liver biopsy in subjects with a p21 hepatocyte expression level (at the time of liver biopsy) less than 15.77% or greater than or equal to 15.77%.

[0059] Figure 7: INR and p21 expression

[0060] Representative plots show (A) INR values over time in survivors versus non-survivors (B) INR values at the time of liver biopsy in survivors versus non survivors (C) INR values over time in subjects with a p21 hepatocyte expression level (at the time of liver biopsy) less than 10.71% or greater than or equal to 10.71% and (D) INR values at the time of liver biopsy in subjects with a p21 hepatocyte expression level (at the time of liver biopsy) less than 10.71% or greater than or equal to 10.71%.

[0061] Figure 8: INR and p21 expression

[0062] Representative plots show (A) INR values over time in subjects with a p21 hepatocyte expression level (at the time of liver biopsy) less than 15.77% or greater than or equal to 15.77% (B) INR values at the time of liver biopsy in subjects with a p21 hepatocyte expression level (at the time of liver biopsy) less than 15.77% or greater than or equal to 15.77%.

[0063] Figure 9: Renal function and p21 expression

[0064] Representative plots show (A) Creatinine levels over time in survivors versus non-survivors (B) Creatinine levels at the time of liver biopsy in survivors versus non survivors (C) Creatinine levels over time in subjects with a p21 hepatocyte expression level (at the time of liver biopsy) less than 10.71% or greater than or equal to 10.71% and (D) Creatinine levels at the time of liver biopsy in subjects with a p21 hepatocyte expression level (at the time of liver biopsy) less than 10.71% or greater than or equal to 10.71 %. Figure 10: Renal function and p21 expression

[0065] Representative plots show (A) Creatinine levels over time in subjects with a p21 hepatocyte expression level (at the time of liver biopsy) less than 15.77% or greater than or equal to 15.77% (B) Creatinine levels at the time of liver biopsy in subjects with a p21 hepatocyte expression level (at the time of liver biopsy) less than 15.77% or greater than or equal to 15.77%.

[0066] Figure 11 : p21 hepatocyte expression in relation to hepatic encephalopathy

[0067] Representative plot to show p21 hepatocyte expression level (by percentage) in subjects with or without HE.

[0068] Figure 12: p21 hepatocyte expression in relation to acute kidney injury (AKI)

[0069] Representative plot to show p21 hepatocyte expression level (by percentage) in subjects with or without AKI.

[0070] Figure 13: p21 hepatocyte expression in relation to extra-hepatic organ dysfunction

[0071] Representative plot to show p21 hepatocyte expression level (by percentage) in subjects with HE and / or AKI or without HE and AKI.

[0072] Figure 14: Transplant-free survival in relation to p21 hepatocyte expression level

[0073] Representative plot to show p21 hepatocyte expression level (by percentage) in subjects who did or did not survive.

[0074] Figure 15: reference p21 hepatocyte expression level ROC curve

[0075] Representative ROC curve plotting sensitivity versus specificity for p21 hepatocyte expression level cutoff values

[0076] Figure 16: Cumulative survival of subjects with a p21 hepatocyte expression level at the time of liver biopsy of less than 10.7 or greater than or equal to 10.7

[0077] Representative Kaplan-Meier curve to show cumulative survival over time in days.

[0078] Figure 17: Schematics of animal models used

[0079] (A) is a schematic of the rat BDL model used to represent acute on chronic liver failure and treatment thereof with rhlGF-1 and (B) is a schematic of the mouse CCL4-LPS model used in Figure 18.

[0080] Figure 18: In vivo hepatic mRNA expression of the IGF system after intervention by TAK-242 (TLR4 antagonist), Nec-1 (necroptosis inhibitor) or G-TAK

[0081] Representative graphs show mRNA expression (A-D) in a BDL model with therapies of TAK-242 or Nec-1 and (E-H) in a CCL4 model with a therapy of G-Tak. (A) and (E) show IGF-1 mRNA expression, (B) and (F) show IGF-1 R mRNA expression, (C) and (G) show IGFBP1 mRNA expression and (D) and (H) show IGFBP3 mRNA expression. Figure 19: Liver function in a rat BDL model with rhlGF-1 therapy

[0082] Representative graphs show (A) ALT levels (B) AST levels (C) bilirubin levels and (D) ammonia levels in rats of BDL model with or without LPS and rhlGF-1 therapy.

[0083] Figure 20: Kidney function in a rat BDL model with rhlGF-1 therapy

[0084] Representative graphs show (A) glucose levels (B) creatinine levels and (C) urea levels in rats of BDL model with or without LPS and rhlGF-1 therapy.

[0085] Figure 21 : Mortality in a rat BDL model with rhlGF-1 therapy

[0086] Representative graph shows percentage mortality in rats of BDL model with or without LPS and rhlGF-1 therapy.

[0087] Figure 22: mRNA expression of genes in the p53 / pRb signalling, cell cycle and p53 effector pathways in a rat BDL model with rhlGF-1 therapy

[0088] Representative graphs show relative mRNA expression of (A) Cdkn2a, (B) Cited2, (C) IGF-1 R, (D) Myc, (E) SerpinEI and (F) SerpinE2.

[0089] Figure 23: mRNA expression of genes in the Interferon signalling pathway in a rat BDL model with rhlGF-1 therapy

[0090] Representative graphs show relative mRNA expression of (A) EGR1 , (B) IFNy, (C) NFKB1 and (D) PRKCD.

[0091] Figure 24: mRNA expression of genes involved in cell adhesion and cytoskeleton in a rat BDL model with rhlGF-1 therapy

[0092] Representative graphs show relative mRNA expression of (A)CD44 and (B) TSP-1 .

[0093] Figure 25: mRNA expression of a further gene in the senescence pathway, Twist 1 in a rat BDL model with rhlGF-1 therapy

[0094] Detailed Description of the Invention

[0095] The present invention provides a method of treating acute liver disease in a subject, the method comprising administering to the subject one or more of a senotherapeutic agent, population of isolated cells, anti-inflammatory agent and cell-regenerative agent, wherein the anti-inflammatory agent optionally comprises a TGF-0 antagonist, a TLR4 antagonist or a CXCR4 antagonist, wherein the population of isolated cells optionally comprise stem cells or hepatocytes and wherein the subject is known to have a p21 hepatocyte expression level greater than a reference p21 hepatocyte expression level.

[0096] In some embodiments, the method comprises administering to the subject a senotherapeutic agent. In some embodiments the method comprises administering to the subject a population of isolated cells. In some embodiments, the method comprises administering to the subject an anti-inflammatory agent. In some embodiments, the method comprises administering to the subject a cell-regenerative agent. In some embodiments, the method comprises administering to the subject a senotherapeutic agent and an anti-inflammatory agent. In some embodiments, the method comprises administering to the subject a senotherapeutic agent and a cell-regenerative agent. In some embodiments, the method comprises administering to the subject an anti-inflammatory agent and a cell-regenerative agent. In some embodiments, the method comprises administering to the subject the population of isolated cells and one or more of a senotherapeutic agent, cell-regenerative agent and anti-inflammatory agent. Any combination of the therapeutic agents / cells disclosed herein can be envisaged by the present invention.

[0097] As used herein, the term “cell-regenerative agent” will be understood to a pharmacological agent which is capable of inducing the production of cells in a subject, for example, the production of cells such as haematopoietic stem cells from the bone marrow. Exemplary cell-regenerative agents include, but are not necessarily limited to G-CSF and GM-CSF.

[0098] The present invention also provides any of the above agents, cells or combinations thereof for use in a method of treating acute liver disease in a subject, wherein the acute liver disease subject is known to have a p21 hepatocyte expression level greater than a reference p21 hepatocyte expression level. Also provided is any of the above agents, cells or combinations thereof for use in a method of treating an acute liver disease subject; wherein a p21 hepatocyte expression level has been measured in a sample obtained from the subject and wherein said p21 hepatocyte expression level is greater than a reference p21 hepatocyte expression level. Further provided by the present invention is any of the above agents, cells or combinations thereof for use in a method of treating acute liver disease in a subject, wherein the acute liver disease subject has a p21 hepatocyte expression level obtained from a sample from the subject greater than a reference p21 hepatocyte expression level.

[0099] Also provided by the present invention is a method of treating acute liver disease in a subject, the method comprising: a) measuring a p21 hepatocyte expression level in a sample obtained from the subject; b) comparing the measured p21 hepatocyte expression level to a reference p21 hepatocyte expression level; and c) administering to the subject one or more of a senotherapeutic agent, population of isolated cells, anti-inflammatory agent, and cell-regenerative agent, when the subject’s measured p21 hepatocyte expression level is greater than the reference p21 hepatocyte expression level, wherein the anti-inflammatory agent optionally comprises a TGF-0 antagonist, a TLR4 antagonist or a CXCR4 antagonist and wherein the population of isolated cells optionally comprises stem cells or hepatocytes.

[0100] The sample may comprise liver tissue obtained from the subject. In some embodiments, the p21 hepatocyte expression level is measured as the number of p21 positive hepatocytes in a given area of the sample divided by the total number of hepatocytes in that area, optionally expressed as a percentage or fraction. In some embodiments, the p21 hepatocyte expression level is measured as an average p21 intensity in a given area of the sample, optionally an average p21 fluorescence intensity in a given area of the sample. The average p21 fluorescence intensity may be determined by calculating the mean or median p21 fluorescence intensity of the hepatocytes in a given area of the sample.

[0101] A given area of the sample may be identified using microscopy. For example, a given area of the sample may comprise a microscopic field of view of the sample. The given area of the sample may comprise a selected area of a microscopic field of view of the sample. In some embodiments, a given area of the sample comprises a microscopic immunofluorescence field of view of the sample. In some embodiments, a given area of the sample comprises a widefield microscopic field of view of the sample, Preferably, the given area of the sample comprises a widefield microscopic immunofluorescence field of view of the sample. As the skilled person will appreciate, widefield microscopy is a form of microscopy wherein the entire specimen or sample is exposed to the microscope light source. In other embodiments, a given area of the sample comprises a confocal microscopic field of view of the sample. Preferably, the given area of the sample comprises a confocal microscopic immunofluorescence field of view of the sample. Confocal microscopy will be understood to refer to microscopy wherein the microscope light source is focused to a specific location in the sample, said specific location being the only location imaged by the microscope during a confocal scan.

[0102] The microscopic field of view may be at a magnification of at least 10X, at least 20X, at least 30X, at least 40X, at least 50X or at least 100X. In some embodiments, the microscopic field of view may be at a magnification of at least 10X. In some embodiments, the microscopic field of view may be at a magnification of 10X.

[0103] In other embodiments, a given area of the sample may be analysed using flow cytometry. For example, a selected area of the sample, which may otherwise be referred to as a given area of the sample, may be processed for subsequent analysis by flow cytometry. Processing may comprise centrifugation and then optional resuspension of the cells in the selected area. Processing may comprise digestion of the selected area of the sample in order to isolate cells within the selected area such as hepatocytes.

[0104] In some embodiments, the p21 hepatocyte expression level may be measured from the entire sample. For example, the p21 hepatocyte expression level may be measured as a cumulative or mean p21 hepatocyte expression level for the entire sample, preferably when the sample comprises liver tissue obtained from the subject. In some embodiments, the p21 hepatocyte expression level may be measured as the number of p21 positive hepatocytes in the entire sample divided by the total number of hepatocytes in the entire sample, optionally expressed as a percentage or fraction.

[0105] In some embodiments, the sample comprises liver tissue obtained from the subject and the p21 hepatocyte expression level is measured as the number of p21 positive hepatocytes in a given area of the sample divided by the total number of hepatocytes in that area, optionally expressed as a percentage or fraction. The p21 expression level may be measured by flow cytometry, immunohistochemistry (IHC) or multiplex immunofluorescence. In some embodiments, the p21 expression level is measured by immunohistochemistry (IHC) or multiplex immunofluorescence. In some embodiments, an anti-p21 antibody is used to measure the p21 expression level, preferably to measure the p21 protein expression level. By “antibody”, this will be understood to include a fragment or derivative thereof, or a synthetic antibody or synthetic antibody fragment.

[0106] In view of today's techniques in relation to monoclonal antibody technology, antibodies can be prepared to most antigens. The antigen-binding portion may be a part of an antibody (for example a Fab fragment) or a synthetic antibody fragment (for example a single chain Fv fragment [ScFv]). Suitable monoclonal antibodies to p21 may be prepared by known techniques, for example those disclosed in "Monoclonal Antibodies: A manual of techniques ", H Zola (CRC Press, 1988) and in "Monoclonal Hybridoma Antibodies: Techniques and Applications ", J G R Hurrell (CRC Press, 1982). Chimeric antibodies are discussed by Neuberger et a / . (1988, 8th International Biotechnology Symposium Part 2, 792-799). Monoclonal antibodies (mAbs) are useful in the methods and kits of the invention and are a homogenous population of antibodies specifically targeting a single epitope on an antigen. Thus, in some embodiments the anti-p21 antibody is monoclonal.

[0107] Polyclonal antibodies are useful in the methods and kits of the invention. Thus, in some embodiments the anti-p21 antibody is polyclonal. Monospecific polyclonal antibodies are preferred. Suitable polyclonal antibodies can be prepared using methods well known in the art.

[0108] Fragments of antibodies, such as Fab and Fab2 fragments may also be used / provided as can genetically engineered antibodies and antibody fragments. The variable heavy (VH) and variable light (VL) domains of the antibody are involved in antigen recognition, a fact first recognised by early protease digestion experiments. Further confirmation was found by "humanisation" of rodent antibodies. Variable domains of rodent origin may be fused to constant domains of human origin such that the resultant antibody retains the antigenic specificity of the rodent parented antibody (Morrison et al (1984) Proc. Natl. Acad. Sd. USA 81 , 6851-6855).

[0109] That antigenic specificity is conferred by variable domains and is independent of the constant domains is known from experiments involving the bacterial expression of antibody fragments, all containing one or more variable domains. These molecules include Fab-like molecules (Better et al. (1988) Science 240, 1041); Fv molecules (Skerra et al (1988) Science 240, 1038); single-chain Fv (ScFv) molecules where the VH and VL partner domains are linked via a flexible oligopeptide (Bird et al. (1988) Science 242, 423; Huston et al (1988) Proc. Natl. Acad. Sd. USA 85, 5879) and single domain antibodies (dAbs) comprising isolated V domains (Ward et al. (1989) Nature 341 , 544). A general review of the techniques involved in the synthesis of antibody fragments which retain their specific binding sites is to be found in Winter & Milstein (1991) Nature 349, 293- 299.

[0110] By "ScFv molecules", this will be understood to refer to molecules wherein the VH and VL partner domains are covalently linked, e.g. by a flexible oligopeptide.

[0111] Fab, Fv, ScFv and dAb antibody fragments can all be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of the said fragments. Whole antibodies, and F(ab')2 fragments are "bivalent". By "bivalent", this will be understood to mean that the said antibodies and F(ab')2 fragments have two antigen combining sites. In contrast, Fab, Fv, ScFv and dAb fragments are monovalent, having only one antigen combining site. Synthetic antibodies which bind to P21 may also be made using phage display technology as is well known in the art.

[0112] Various antibodies which are capable of specifically binding to p21 are already commercially available and known to those skilled in the art. Exemplary anti-p21 antibodies include, but are not necessarily limited to 2H2L13, HJ21 , WA-1 , DCS-60, R.229.6, EA10, GT1032 and GT8611 , amongst others. Such antibodies are readily available from suppliers including, but not necessarily limited to Invitrogen, Santa Cruz Biotechnology and Abeam. In some embodiments the anti-p21 antibody is selected from 2H2L13, HJ21 , WA-1 , DCS-60, R.229.6, EA10, GT1032 and GT8611 .

[0113] In some embodiments the antibody is detectably labelled or, at least, capable of detection. For example, the antibody may be labelled with a radioactive atom or a coloured molecule or a fluorescent molecule or a molecule which can be readily detected in any other way. Suitable detectable molecules include fluorescent proteins, luciferase, enzyme substrates, and radiolabels. The antibody may be directly labelled with a detectable label or it may be indirectly labelled. For example, the antibody may be unlabelled and can be detected by another antibody which is itself labelled. Alternatively, the second antibody may have bound to it biotin and binding of labelled streptavidin to the biotin is used to indirectly label the first antibody.

[0114] In embodiments wherein the sample comprises liver tissue, the liver tissue sample may have been formalin-fixed and paraffin-embedded prior to said measuring of the p21 hepatocyte expression level.

[0115] In embodiments where the sample comprises liver tissue obtained from the subject, the liver tissue may otherwise be referred to herein as a liver biopsy. The liver tissue may have been obtained from the subject at day 0 (i.e. the same day that the subject presented to a clinician with at least one symptom of acute liver disease). In embodiments where the liver tissue was obtained from the subject at day 0, the liver biopsy may otherwise be referred to herein as a baseline biopsy. In some embodiments, the liver tissue was obtained from the subject no more than about 96 hours after the subject presented to the clinician with at least one symptom of acute liver disease. In some embodiments, the liver tissue was obtained from the subject no more than about 72 hours, optionally no more than about 48 hours and further optionally no more than about 24 hours after the subject presented to a clinician with at least one symptom of acute liver disease. In some embodiments, the liver tissue was obtained from the subject no more than about 12 hours after the subject presented to a clinician with at least one symptom of acute liver disease. Advantageously, measurement of a p21 hepatocyte expression level in a sample obtained from the subject at an early time point (i.e. close or at substantially the same time to which the subject initially presented to a clinician with at least one symptom of acute liver disease), ensures that the subject can be stratified into a subgroup for treatment rapidly, thereby increasing the chance of the subject receiving the appropriate treatment at an early enough stage to improve their chance of survival.

[0116] The reference p21 hepatocyte expression level may comprise a p21 hepatocyte expression level from a subject who does not have liver disease. A subject who does not have liver disease typically has normal liver blood test results obtained from a blood sample from the subject. The blood sample may have been obtained at substantially the same time as a sample obtained from the subject from which the p21 hepatocyte expression level was determined. Alternatively, the blood sample may have been obtained within at least about 24 hours, at least 48 hours or at least about 72 hours of the obtaining of the sample from which the p21 hepatocyte expression level was determined. The skilled person will be aware of liver blood test result values which would be considered normal and liver blood test result values which would be considered abnormal. A subject who does not have liver disease may otherwise be referred to as a healthy control subject. In some embodiments the reference p21 hepatocyte expression level may comprise an average p21 hepatocyte expression level calculated from the p21 hepatocyte expression level from a plurality of healthy control subjects.

[0117] In some embodiments, the reference p21 hepatocyte expression level is at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19% or at least about 20% of the hepatocytes being p21 positive.

[0118] In some embodiments, the reference p21 hepatocyte expression level is at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19% or at least about 20% of the hepatocytes being p21 positive.

[0119] In some embodiments, the reference p21 hepatocyte expression level is at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19% or at least about 20% of the hepatocytes being p21 positive. In some embodiments, the reference p21 hepatocyte expression level is no more than about 40%, no more than about 39%, no more than about 38%, no more than about 37%, no more than about 36%, no more than about 35%, no more than about 34%, no more than about 33%, no more than about 32%, no more than about 31%, no more than about 30%, no more than about 29%, no more than about 28%, no more than about 27%, no more than about 26% or no more than about 25% of the hepatocytes being p21 positive.

[0120] In some embodiments, the reference p21 hepatocyte expression level is of from about 15% to about 40% of the hepatocytes being p21 positive. In some embodiments, the reference p21 hepatocyte expression level is of from about 15% to about 30% of the hepatocytes being p21 positive. In some embodiments, the reference p21 hepatocyte expression level is of from about 20% to about 30% of the hepatocytes being p21 positive.

[0121] In some embodiments, the reference p21 hepatocyte expression level is of from about 7% to about 40% of the hepatocytes being p21 positive. In some embodiments, the reference p21 hepatocyte expression level is of from about 7% to about 30% of the hepatocytes being p21 positive.

[0122] In some embodiments, the reference p21 hepatocyte expression level is of from about 10% to about 40% of the hepatocytes being p21 positive. In some embodiments, the reference p21 hepatocyte expression level is of from about 10% to about 30% of the hepatocytes being p21 positive. In some embodiments, the reference p21 hepatocyte expression level is of from about 10% to about 21 % of the hepatocytes being p21 positive. In some embodiments, the reference p21 hepatocyte expression level is of from about 10% to about 20% of the hepatocytes being p21 positive. In some embodiments, the reference p21 hepatocyte expression level is of from about 10% to about 16% of the hepatocytes being p21 positive. In some embodiments, the reference p21 hepatocyte expression level is of from about 10% to about 15% of the hepatocytes being p21 positive.

[0123] The reference p21 hepatocyte expression level may be about 10%, about 11 %, about 12%, about 13% or about 14% of the hepatocytes being p21 positive.

[0124] The reference p21 hepatocyte expression level may be about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21 %, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31 %, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39% or about 40% of the hepatocytes being p21 positive.

[0125] The reference p21 hepatocyte expression level may be about 20%, about 21 %, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31 %, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39% or about 40% of the hepatocytes being p21 positive.

[0126] The reference p21 hepatocyte expression level may be about 20%, about 21 %, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29% or about 30% of the hepatocytes being p21 positive.

[0127] In some embodiments, the reference p21 hepatocyte expression level is about 10% of the hepatocytes being p21 positive. In some embodiments, the reference p21 hepatocyte expression level is about 11 % of the hepatocytes being p21 positive. In some embodiments, the reference p21 hepatocyte expression level is about 15% of the hepatocytes being p21 positive. In some embodiments, the reference p21 hepatocyte expression level is about 16% of the hepatocytes being p21 positive.

[0128] In some embodiments, the reference p21 hepatocyte expression level may be about 20% of the hepatocytes being p21 positive. In some embodiments, the reference p21 expression level may be about 25% of the hepatocytes being p21 positive. In some embodiments, the reference p21 expression level may be about 30% of the hepatocytes being p21 positive. The present inventors have surprisingly found that a reference p21 hepatocyte expression level at the percentages described herein is an effective value at which to stratify subjects into those likely to survive and those not likely to survive (and hence urgently requiring a liver transplant). This represents a simple and effective method which can readily be utilised by clinicians and will advantageously enable the stratification of subjects into the appropriate treatment group at an earlier stage, thereby improving the chance of survival for subjects.

[0129] By “p21 positive”, this will be understood to mean that a hepatocyte comprises a detectable level of p21 mRNA and / or p21 protein. Preferably, p21 positive means that the hepatocyte comprises a detectable level of p21 protein. In some embodiments, the sample comprises a biological liquid sample comprising liver-derived protein and / or liver-derived nucleic acid and wherein measuring the p21 hepatocyte expression level comprises inferring or deriving the p21 hepatocyte expression level from a measurement of p21 protein and / or p21- encoding DNA or p21 -encoding RNA that is present in the biological liquid sample.

[0130] The biological liquid sample may comprise whole blood, serum, plasma, lymphatic fluid, synovial fluid, ascites fluid, interstitial or extracellular fluid, cerebrospinal fluid, saliva, mucus, semen, sweat, urine or any other bodily fluids. In some embodiments, the biological liquid sample comprises whole blood, serum or plasma. In some embodiments, the biological liquid sample comprises whole blood.

[0131] Administration of the agent / cells to the subject may be on substantially the same day as step b) and or step a) of the method. For example, administration of the agent / cells to the subject may be at day 0. Alternatively, administration of the agent / cells to the subject may be about 24 hours, about 48 hours or about 72 hours after the p21 hepatocyte expression level of the sample is measured or obtained.

[0132] Step a) of the method may comprise obtaining a sample from the subject and measuring a p21 hepatocyte expression level in the sample. The sample may be as defined herein, for example, liver tissue, such as a liver biopsy sample. Measuring of the p21 hepatocyte expression level may be on the same day as obtaining the sample from the subject. In some embodiments, measuring of the p21 hepatocyte expression level may occur no more than about 24 hours after the sample has been obtained from the subject.

[0133] The present inventors have also surprisingly found that other senescence markers can advantageously be used as additional prognosis markers for the subject. Exemplary senescence markers may include, but not necessarily be limited to yH2AX, P16, P53, beta galactosidase (Bgal), Ki67 / BrdU, LaminBI , Interleukin (IL)-1 , Interleukin (IL)-6, p53-binding protein 1 (53BP1), Aurora kinase A (Aurka), Aurora kinase B (Aurkb), Anillin (Anin), ArhgapHa, Assembly Factor For Spindle Microtubules (Aspm), Baculoviral IAP repeat containing 5 (Birc5), Cdc20, Cyclin-dependent kinase 2 (Cdk2),Cyclin-dependent kinase inhibitor 1a (Cdknla), Cyclin-dependent kinase inhibitor 3 (Cdkn3), Centromere protein F (Cenpf), Centromere protein M (Cenpm), Disks large-associated protein 5 (Dlgap5), Growth-Arrest Specific 2 Like 3 (Gas2l3), Hyaluronan Mediated Motility Receptor (Hmmr), Kinesin Family Member 11 ( Kif 11 ), CCL2, TGFB1 , TGFB2 and TGFB3.

[0134] An increased or detectable level of P16, P53, Bgal, LaminBI , IL-1 , IL-6, Arhgapl 1a, Birc5, Cyclin- dependent kinase inhibitor 1a (Cdknla), Cyclin-dependent kinase inhibitor 3 (Cdkn3), CCL2, TGFB1 , TGFB2 or TGFB3 may be understood to be a marker of senescence, and thus an additional prognosis marker.

[0135] As the skilled person will appreciate, Ki67 / BrdU is a nuclear antigen which is a marker of active cell proliferation. In the context of the present invention, an absence of Ki67 / BrdU (for example, an undetectable level of Ki67 / BrdU), may be understood to be a marker for senescence, and thus an additional prognosis marker. Anillin (Anin) is an actin-binding protein that, as the skilled person will appreciate, plays a role in cell growth, migration, and cytokinesis. In the context of the present invention, an absence of Anin (for example, an undetectable level of Anin), may be understood to be a marker for senescence, and thus an additional prognosis marker.

[0136] Assembly Factor For Spindle Microtubules (Aspm), as the skilled person will appreciate, is involved with mitotic spindle function in cells. In the context of the present invention, an absence of Aspm (for example, an undetectable level of Aspm), may be understood to be a marker for senescence, and thus an additional prognosis marker.

[0137] Another exemplary senescence marker is Cyclin A2 (CCNA2). CCNA2 is a member of the cyclin family that functions as a regulator of cyclin-dependent kinases (CDKs). In the context of the present invention, a reduction in the level of or an absence of CCNA2 (for example, an undetectable level of CCNA2), may be understood to be a marker for senescence and thus an additional prognosis marker.

[0138] In the context of the present invention, an absence of Cdc20, CdK2, Cenpf, Cenpm, Dlgap5, Gas2l3 or KIF11 may be understood to be a marker for senescence and thus an additional prognosis marker. For example, an undetectable level of Cdc20, CdK2, Cenpf, Cenpm, Dlgap5, Gas2l3 or KIF11 may be understood to be a marker for senescence and thus an additional prognosis marker.

[0139] In some embodiments the additional senescence marker is selected from CCNA2, yH2AX, P16, P53, beta galactosidase (Bgal), Ki67 / BrdU, LaminBI , Interleukin (IL)-1 , Interleukin (IL)-6, p53-binding protein 1 (53BP1), Chemokine ligand 2 (CCL2), TGFB1 , TGFB2 and TGFB3.

[0140] In some embodiments the additional senescence marker is selected from yH2AX, P16, P53, beta galactosidase (Bgal), Ki67 / BrdU, LaminBI , Interleukin (IL)-1 , Interleukin (IL)-6, p53-binding protein 1 (53BP1), Chemokine ligand 2 (CCL2), TGFB1 , TGFB2 and TGFB3. In some embodiments the additional senescence marker is selected from CCNA2, yH2AX, CCL2, TGFB1 , TGFB2 and TGFB3. In some embodiments the additional senescence marker is selected from CCNA2, CCL2, TGFB1 , TGFB2 and TGFB3.ln some embodiments the additional senescence marker is selected from yH2AX, CCL2, TGFB1 , TGFB2 and TGFB3. In some embodiments the additional senescence marker is selected from CCL2, TGFB1 , TGFB2 and TGFB3.

[0141] An exemplary additional senescence marker may comprise yH2AX. The present inventors have found that the senescence marker yH2AX can be used as a further prognosis marker. Thus, in some embodiments, measuring a p21 hepatocyte expression level in the sample may further comprise measuring a yH2AX hepatocyte expression level in a sample obtained from the subject. Optionally, the sample comprises liver tissue. Further optionally, the sample may be the same sample from which the p21 hepatocyte expression level is measured.

[0142] In some embodiments, the method comprises: a) measuring a p21 hepatocyte expression level and a yH2AX hepatocyte expression level in a sample obtained from the subject; b) comparing the measured p21 hepatocyte expression level to a reference p21 hepatocyte expression level and comparing the measured yH2AX hepatocyte expression level to a reference yH2AX hepatocyte expression level; and c) administering to the subject one or more of a senotherapeutic agent, population of isolated cells, anti-inflammatory agent and cell-regenerative agent when the subject’s measured p21 hepatocyte expression level is greater than the reference p21 hepatocyte expression level and the subject’s measured yH2AX hepatocyte expression level is greater than said reference yH2AX hepatocyte expression level, wherein the anti-inflammatory agent optionally comprises a TGF-0 antagonist, a TLR4 antagonist or a CXCR4 antagonist and wherein the population of isolated cells optionally comprises stem cells or hepatocytes.

[0143] The yH2AX hepatocyte expression level may be measured in the same or different way to that of p21 . For example, the yH2AX hepatocyte expression level may be measured as the number of yH2AX positive hepatocytes in a given area of the sample divided by the total number of hepatocytes in that area, optionally expressed as a percentage or fraction.

[0144] In some embodiments, the reference yH2AX hepatocyte expression level may comprise an undetectable yH2AX hepatocyte expression level. In such embodiments, any measured yH2AX hepatocyte expression level which is detectable will be understood to be greater than this reference yH2AX hepatocyte expression level.

[0145] In some embodiments, the reference yH2AX hepatocyte expression level is at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39% or at least about 40% of the hepatocytes being yH2AX positive. In some embodiments, the reference yH2AX hepatocyte expression level is no more than about 50%, no more than about 49%, no more than about 48%, no more than about 47%, no more than about 46%, no more than about 45%, no more than about 44%, no more than about 43%, no more than about 42% or no more than about 41% of the hepatocytes being yH2AX positive. In some embodiments, the reference yH2AX hepatocyte expression level is of from about 20% to about 50% of the hepatocytes being yH2AX positive. In some embodiments, the reference yH2AX hepatocyte expression level is of from about 30% to about 50% of the hepatocytes being yH2AX positive. In some embodiments, the reference yH2AX hepatocyte expression level is of from about 30% to about 40% of the hepatocytes being yH2AX positive. In some embodiments, the reference yH2AX hepatocyte expression level is about 30% of the hepatocytes being yH2AX positive. In other embodiments, the reference yH2AX hepatocyte expression level is about 35% of the hepatocytes being yH2AX positive.

[0146] In some embodiments, the method further comprises measuring at least one hepatocyte marker. In some embodiments, the hepatocyte marker comprises HNF4a. In such embodiments, the total number of hepatocytes may be determined to be the total number of HNF4a positive cells. In some embodiments, an anti-HNF4a antibody is used to measure the HNF4a. In some embodiments, the anti-HNF4a antibody is monoclonal. In other embodiments, the anti-HNF4a antibody is polyclonal. Various suitable anti-HNF4a antibody are commercially available and known to those skilled in the art.

[0147] In some embodiments, measuring a p21 hepatocyte expression level in the sample further comprises measuring an expression level of an inflammatory marker in a sample obtained from the subject. Optionally, the sample comprises liver tissue. Further optionally, the sample may be the same sample from which the p21 hepatocyte expression level is measured. Suitable inflammatory markers may include, but not necessarily be limited to, markers for immune cells such as, for example, macrophages and / or T- cells. In some embodiments the inflammatory marker comprises IBA1 , which is a macrophage-specific marker. In some embodiments the inflammatory marker comprises CD8, which is a CD8-T-cell-specific marker. Other inflammatory markers may include, for example. IFNy, NFKB1 , PRKCD or EGR1. The measured inflammatory marker expression level may be compared to a reference inflammatory marker expression level. The reference inflammatory marker expression level may be obtained from a subject who does not have acute liver disease.

[0148] One or more of a senotherapeutic agent, population of isolated cells, anti-inflammatory agent and cell- regenerative agent may be administered to the subject when the subject’s measured p21 hepatocyte expression level is greater than the reference p21 hepatocyte expression level and the subject’s measured inflammatory marker expression level is greater than said reference inflammatory marker expression level, wherein the anti-inflammatory agent optionally comprises a TGF-0 antagonist, a TLR4 antagonist or a CXCR4 antagonist and wherein the population of isolated cells optionally comprises stem cells or hepatocytes.

[0149] Alternatively, one or more of a senotherapeutic agent, population of isolated cells, anti-inflammatory agent and cell-regenerative agent may be administered to the subject when the subject’s measured p21 hepatocyte expression level is greater than the reference p21 hepatocyte expression level and the subject’s measured inflammatory marker expression level is lower than said reference inflammatory marker expression level, wherein the anti-inflammatory agent optionally comprises a TGF-0 antagonist, a TLR4 antagonist or a CXCR4 antagonist and wherein the population of isolated cells optionally comprises stem cells or hepatocytes.

[0150] The inflammatory marker expression level may be measured in the same or different way to that of p21 . For example, the inflammatory marker expression level may be measured as the number of inflammatory marker positive cells in a given area of the sample. Another example may be that the inflammatory marker expression may be measured as the percentage positive area of the whole slide. In this example, no cell count is required. Alternatively, the inflammatory marker expression level may be measured as mRNA in a sample, for example a liquid sample such as a blood sample, obtained from the subject.

[0151] The method typically comprises administering a therapeutically effective amount of the agent and / or population of isolated cells of the invention. A therapeutically effective amount is an amount which ameliorates one or more symptoms, such as all the symptoms, of the disease and / or abolishes one or more symptoms, such as all the symptoms, of the disease. The therapeutically effective amount preferably cures the acute liver disease.

[0152] The administration of the agent and / or population of isolated cells to the subject may increase liver function by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or even about 100%, when compared to the liver function of the subject prior to administration. The administration of the anti-inflammatory agent to the subject may restore liver function of the subject to normal levels. As the skilled person will appreciate, a “normal” level of liver function will be understood to mean that the subject’s liver blood test results, following treatment, are in what is considered a clinically normal range.

[0153] Any suitable form of administration may be used for the agent and / or population of isolated cells of the invention. For example, the agent and / or population of isolated cells may be administered subcutaneously, intranasally, orally, topically, intraperitoneally or intravenously. In some embodiments, the agent and / or population of isolated cells is orally or intravenously administered.

[0154] In some embodiments, the senotherapeutic agent comprises a senolytic agent or a senomorphic agent.

[0155] In some embodiments, the senolytic agent comprises one or more of UBX1235, ABT-737, ABT-199 (Venetoclax), ABT-263 (Navitoclax), UBX0101 , Dasatinib, Quercitin, Fisetin, ALK4 / 5 / 7, AZD1260111 , and a cardiac glycoside. Exemplary cardiac glycosides include, but are not necessarily limited to ouabain, ouabagenin and digoxin.

[0156] In some embodiments, the senomorphic agent comprises an agent which targets the mTOR pathway. For example, the senomorphic agent may comprise rapamycin or a rapalog. Exemplary rapalogs include, but are not necessarily limited to everolimus, temsirolimus, and deforolimus. In some embodiments, the senomorphic agent comprises one or more of rapamycin, everolimus, temsirolimus, and deforolimus.

[0157] In some embodiments, the anti-inflammatory agent comprises a TGF-0 antagonist, optionally wherein the TGF-P antagonist comprises an anti- TGF-0 antibody. Various suitable anti- TGF-0 antibodies are known to the skilled person and commercially available.

[0158] In some embodiments, the anti-inflammatory agent comprises a TLR4 antagonist. The TLR4 antagonist may comprise TAK-242.

[0159] In some embodiments the method comprises administering the anti-inflammatory agent and a cell- regenerative agent to the subject. The cell-regenerative agent may comprise G-CSF or GM-CSF.

[0160] In some embodiments, the method comprises administering an anti-inflammatory agent comprising a TLR4 antagonist and a cell-regenerative agent comprising G-CSF to the subject.

[0161] In some embodiments, the method comprises administering an anti-inflammatory agent comprising TAK- 242 and a cell-regenerative agent comprising G-CSF to the subject. This combination of agents may otherwise be referred to as “G-TAK”.

[0162] The population of isolated cells may comprise a population of stem cells or a population of hepatocytes.

[0163] In some embodiments, the population of isolated cells comprises a population of stem cells. In some embodiments, the stem cells comprise hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs) or induced pluripotent stem cells (iPSCs). In some embodiments, the stems cells comprise hematopoietic stem cells (HSCs).

[0164] In the context of the present invention, “population of isolated cells” will be understood to refer to a plurality of isolated cells. The isolated cells may comprise a substantially identical morphology and / or phenotype. Typically, a plurality of isolated cells may be understood to comprise at least about 1 x 105cells, optionally at least about 1 x 106cells, further optionally at least 1 x 107cells.

[0165] The population of isolated cells may comprise a population of primary isolated cells. By “primary cell” this will be understood to refer to a cell that has been obtained from a subject. Primary cells are not immortalised cells from a cell line.

[0166] The primary cell may be autologous. Alternatively, the primary cell may be allogeneic. In embodiments comprising a population of primary isolated cells, the population may comprise a mixture of autologous and allogenic cells.

[0167] As the skilled person will appreciate, autologous cells are cells from the same subject, i.e. cells which have been obtained from a subject which will be administered back to the same subject. Allogeneic cells are cells obtained from a different subject to the subject to which the cells will be administered. The different subjects are typically from the same species. Allogenic cells are thus genetically different to the subject to which they are administered.

[0168] The method may comprise administering of a pharmaceutical composition comprising any of the agents or populations of isolated cells as defined above and a pharmaceutically or physiologically acceptable diluent and / or carrier.

[0169] The carrier and / or diluent is generally selected to be suitable for the intended mode of administration and can include agents for modifying, maintaining, or preserving, for example, the pH, osmolarity, viscosity, clarity, colour, isotonicity, odour, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition. Typically, these carriers and / or diluents include aqueous or alcoholic / aqueous solutions, emulsions, or suspensions, including saline and / or buffered media.

[0170] Suitable further agents for inclusion in the pharmaceutical compositions include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antimicrobials, antioxidants (such as ascorbic acid, sodium sulphite, or sodium hydrogen-sulphite), buffers (such as borate, bicarbonate, Tris-HCI, citrates, phosphates, or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediamine tetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrins), proteins (such as free serum albumin, gelatin, or immunoglobulins), colouring, flavouring and diluting agents, emulsifying agents, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (such as sodium), preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (such as glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as pluronics; PEG; sorbitan esters; polysorbates such as Polysorbate 20 or Polysorbate 80; Triton; tromethamine; lecithin; cholesterol or tyloxapal), stability enhancing agents (such as sucrose or sorbitol), tonicity enhancing agents (such as alkali metal halides, such as sodium or potassium chloride, or mannitol sorbitol), delivery vehicles, excipients and / or pharmaceutical adjuvants.

[0171] The carrier and / or diluent may be a parenteral, optionally intravenous vehicle. Suitable parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride and lactated Ringer's. Suitable physiologically-acceptable thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates may be included. Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as those based on Ringer's dextrose. In some cases, one might include agents to adjust tonicity of the composition, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in a pharmaceutical composition. For example, in many cases it is desirable that the composition is substantially isotonic. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents, and inert gases, may also be present. The precise formulation will depend on the route of administration. Additional relevant principle, methods and components for pharmaceutical formulations are well known (see, e.g., Allen, Loyd V. Ed, (2012) Remington's Pharmaceutical Sciences, 22nd Edition).

[0172] The subject may be a mammal. For example, the subject may be a dog, cat, horse, human, goat, sheep, cow, rat or mouse. In some embodiments, the subject is a dog, cat, horse or human. In some embodiments, the subject is a human. In some embodiments, the subject is a human having or suspected of having acute liver disease without any significant signs of chronic liver disease. In some embodiments, the subject is male. In some embodiments, the subject is female.

[0173] The subject may have one or more of the following symptoms of acute liver disease: jaundice, encephalopathy, abdominal pain, nausea, vomiting, disorientation or confusion, drowsiness and tremors. In some embodiments, the subject does not have encephalopathy. In some embodiments, the subject has jaundice. In some embodiments, the subject doesn’t have encephalopathy and does have jaundice. In other embodiments, the subject has encephalopathy and jaundice.

[0174] In some embodiments, the acute liver disease comprises acute indeterminate hepatitis, acute autoimmune hepatitis, acute viral hepatitis, or drug-induced liver injury. Drug-induced liver injury may comprise acetaminophen-induced liver injury, NSAID (non-steroidal anti-inflammatory drugs)-induced liver injury, amiodarone-induced liver injury, anabolic steroid-induced liver injury, contraceptive pill- induced liver injury, chlorpromazine-induced liver injury, erythromycin-induced liver injury, halothane- induced liver injury, methyldopa-induced liver injury, isoniazid-induced liver injury, methotrexate-induced liver injury, statin-induced liver injury, sulfa drug-induced liver injury, tetracycline-induced liver injury, amoxicillin-clavulanate-induced liver injury, anti-seizure drug-induced liver injury, disulfiram-induced liver injury, niacin-induced liver injury, azathoaprine-induced liver injury or ketoconazole-induced liver injury. In some embodiments, the drug-induced liver injury comprises acetaminophen-induced liver injury. In some embodiments, the acute liver disease comprises acute indeterminate hepatitis or drug-induced liver injury. For example, the acute liver disease may comprise acute indeterminate hepatitis or acetaminophen-induced liver injury.

[0175] The present invention also provides a method for selecting subjects for participation in an acute liver disease treatment clinical trial, the method comprising: a) measuring a p21 hepatocyte expression level in a sample obtained from the subject; b) comparing the measured p21 hepatocyte expression level to a reference p21 hepatocyte expression level; and c) selecting the subject for participation in the clinical trial when said measured p21 hepatocyte expression level is greater than said reference p21 hepatocyte expression level.

[0176] Any of the embodiments above may apply to this aspect. In particular, the p21 hepatocyte expression level, reference p21 hepatocyte expression level, measurement step (for example, the measurement of further markers, such as, for example, yH2AX), subject and sample may be as defined above.

[0177] According to a further aspect, the present invention provides a method for selecting an acute liver disease subject for liver transplant. The method comprises: a) measuring a p21 hepatocyte expression level in a sample obtained from the subject; b) comparing the measured p21 hepatocyte expression level to a reference p21 hepatocyte expression level; and c) making a prediction of whether the subject will require a liver transplant based on the comparison in step b), wherein said measured p21 hepatocyte expression level being greater than said reference p21 hepatocyte expression level indicates that the subject will require a liver transplant, and wherein said measured p21 hepatocyte expression level being equal to or lower than said reference p21 hepatocyte expression level indicates that the subject will not require a liver transplant.

[0178] Any of the embodiments described for the above aspects may also apply to the method for selecting an acute liver disease subject for liver transplant. In particular, the method may further comprise measurement of a further marker, such as, for example, yH2AX.

[0179] The liver transplant may comprise a liver obtained from an allogeneic individual, a bio-printed liver, or an auxiliary liver transplant. In some embodiments the liver comprises a liver obtained from an allogeneic individual. The allogeneic individual may be human. In some embodiments, the allogeneic individual is related to the subject, for example a parent or sibling.

[0180] In some embodiments, the subject is predicted not to require a liver transplant. In such embodiments, the subject may be selected for corticosteroid treatment. Various suitable corticosteroids are known and suitable for this use. For example, the corticosteroid may be selected from prednisone, prednisolone, methylprednisolone, triamcinolone, cortisone, hydrocortisone, betamethasone and dexamethasone. In some embodiments, the corticosteroid comprises prednisone, prednisolone, methylprednisolone or triamcinolone.

[0181] According to a further aspect, the present invention provides use of a kit comprising an anti-p21 antibody for: a) selecting subjects for participation in an acute liver disease treatment clinical trial; or b) predicting whether an acute liver disease subject will require a liver transplant.

[0182] The anti-p21 antibody may be any anti-p21 antibody described above in relation to the above aspects. In some embodiments, the anti-p21 antibody is provided in an aqueous solution, optionally a buffered solution.

[0183] In some embodiments, the kit further comprises one or more hepatocyte-specific reagents, optionally an anti-HNF4a antibody. In some embodiments, the one or more hepatocyte-specific reagents are provided in an aqueous solution, optionally a buffered solution.

[0184] In some embodiments, the kit further comprises an anti-yH2AX antibody. In some embodiments, the anti- yH2AX antibody is provided in an aqueous solution, optionally a buffered solution.

[0185] The kit may further comprise instructions for use.

[0186] Aspects and embodiments of the present invention will now be discussed with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

[0187] Examples

[0188] Material and methods

[0189] Ethics

[0190] The study was approved by the London-Hampstead Research Ethics Committee (07 / Q0501 / 50) and was in accordance with the declaration of Helsinki as reported before1. This study reported multilobular necrosis as a predominant histopathological feature being significantly more frequent in non-survivors1.

[0191] Subject selection and Data collection

[0192] The study included 34 consecutive subjects with acute indeterminate hepatitis who were admitted to a single hospital and underwent transjugular liver biopsy or liver transplantation. Of the 34 subjects, 14 underwent liver transplantation and three died within three months. They were defined as non-survivors. Seventeen subjects who recovered spontaneously were defined as survivors. All 34 liver tissue biopsies analysed in this study were obtained at the time of enrolment (baseline biopsies). The following clinical data were collected from the subjects: Sex, age, date of histopathological examination, history of chronic disease, results of biochemical tests at baseline, and at follow-up up to 28 days or the last value before death. These include serum levels of alanine aminotransferase (ALT), aspartate transaminase (AST), bilirubin, alkaline phosphatase (ALP), international normalized ratio (INR), creatinine, prothrombin time, albumin, and hepatic encephalopathy.

[0193] Multiplex immunofluorescence

[0194] Multiplex immunofluorescence staining was performed as previously described1 3. Formalin-fixed, paraffin-embedded (FFPE) liver samples were deparaffinized and rehydrated in xylene (Roth, Germany) and ethanol (Roth, Germany). Antigen retrieval was performed with Tris-EDTA buffer (pH 9) or universal antigen retrieval (Abeam, Germany) in a water bath at 98 °C for 30 minutes, followed by a cooling period of 20-30 minutes. Tissues were blocked with 2% normal goat serum (Thermo Fisher Scientific, USA) to prevent non-specific antibody binding. Slides were incubated overnight at 4 °C with primary antibodies diluted in antibody dilution solution (Life Technology, USA) and stained for 30 minutes with fluorescently labelled secondary antibodies (Table 3) together with DAPI nuclear counterstain (Sigma Aldrich (USA). After scanning the entire slide with a Zeiss Axio Observer?, images were merged and background was subtracted. After each run, antibodies were stripped by using the 2-mercaptoethanol / SDS (2 ME I SDS) method2, and staining was repeated in multiple cycles over an 3-day period. Subsequently, all scans were aligned, hyperstacked and concatenated using the plugin FIJI HyperStackReg V5.6. For binary images, cell segmentation was performed using llastik software (v 1 .3.3). Cell identification and counting as well as fluorescence intensity measurement were performed using CellProfiler v3.1 .9 and plugin FIJI.

[0195] Statistical analysis

[0196] SPSS software (version 24.0, SPSS Inc, Chicago, USA) or Graphpad Prism software with Student t test (for normally distributed variables) or Mann-Whitney U test (for non-normally distributed variables) was used to manage and analyse the data in this study. Correlation analysis was performed using the Excel toolkit. A P value of <.05 was considered statistically significant.

[0197] EXAMPLE 1 : Non-survivor SIAH subjects showed loss of parenchymal cells and increased senescence

[0198] We have previously reported multilobular necrosis with increased serum parameters and reduced biosynthetic functions in subjects with acute liver disease (severe acute indeterminate hepatitis (siAH))1. In the current study, we investigated whether characteristics of hepatocyte senescence were associated with outcome and development of extrahepatic organ injury in these subjects.

[0199] We examined liver biopsies from subjects who had recovered and were discharged (hereafter designated as survivors) compared with subjects who had not survived or were transplanted (hereafter designated as non-survivors). Each liver biopsy was obtained at the time of enrolment and so may otherwise be referred to as baseline biopsies. Consistent with previous reports <1. 4, 5) routine biochemistry and clinical parameters typically used to determine if a subject requires a liver transplant did not define if the subject was not likely to survive or, conversely, likely to survive. In addition, the routine biochemistry and clinical parameters did not predict subsequent multiorgan dysfunction (including both renal and cerebral failure - hepatic encephalopathy) in this cohort. The routine biochemistry and clinical parameters comprised the measurement of bilirubin, arterial lactate (ALT), the age of the subject, creatinine levels, albumin levels and prothrombin time.

[0200] We then examined the presence of hepatocytes in the liver biopsies from the survivors and non-survivors using the HNF4a antibody. We found that the number of HNF4a-positive hepatocyte regions (hepatocyte islands surrounded by necrotic areas) was significantly lower in the non-survivors compared with the survivors (Figure 1-A&B). Therefore, we quantified HNF4a-positive hepatocytes per field area and found a significant reduction in the density and number of HNF4a positive cells per field area in non-survivors compared with survivors. (Figure1-C&D).

[0201] We further investigated the expression of two senescence markers (p21 and yH2AX) in areas of preserved hepatocyte to understand if the loss of hepatocytes and severity of necrosis correlated with the amount of senescent hepatocytes. We observed a significant increase in the percentage of p21+vesenescent hepatocytes (p21+vehepatocytes / total hepatocytes or p21+vehepatocytes / area field ) in non- survivors compared with survivors (Figure 1-E&F and Figure 2A). These results were also confirmed by another senescent marker, yH2AX, in which we consistently observed significantly increased expression of yH2AX+vesenescent hepatocytes (yH2AX+vehepatocytes / total hepatocytes or yH2AX+vehepatocytes / area field ) in non-survivors (Figure 1-G&H and Figure 2B). These results suggest that in severe acute indeterminate hepatitis, cellular senescence may be associated with hepatocyte loss, leading to poor prognosis in non-survivors.

[0202] EXAMPLE 2: Hepatic parenchymal senescence correlates with extrahepatic organ dysfunction

[0203] We then investigated the effects of hepatic senescence on extrahepatic organs. The development of extrahepatic organ failure such as the kidney was determined by calculating the differences in blood markers between baseline and follow-up, using for creatinine and prothrombin time the last available value before death (hereafter referred as last), if it occurred first. Both markers were not different at baseline but increased significantly during follow up in non-survivors (Figure 3-A-D).

[0204] We then correlated serum renal creatinine and prothrombin time with hepatic parenchymal senescence markers p21 to understand whether p21 expression is associated with development of renal injury and coagulation failure. The percent change in creatinine level (DeltaCR, day 28-day 0) correlated positively with the percentage of p21+ve senescent hepatocytes (survival encircled in green and non-survival in red) (Figure 3-E). We further categorized the relative number of p21 expressing hepatocytes and plotted them against percent change in creatinine level (DeltaCR, day 28-day 0) and found that subjects with a high percentage of p21+ve hepatocytes had significantly higher values for percent change in creatinine level than subjects with a low percentage of p21+ve hepatocytes (Figure 3-F). Similar results were observed for the percent change in creatinine deltaCR (last- day 0) compared with recategorized hepatocytes based on p21 expression (Figure 3-G&H). We then analysed whether the p21 expression in hepatocytes is also associated with the development of hepatic encephalopathy (HE), which is another type of extrahepatic organ failure. For this purpose, we analysed the HE grade from baseline to 28-day follow-up in both survivors and non-survivors. HE was observed in only one subject from the survivor cohort whereas in non-survivors, HE was observed in a greater number of subjects at 28 days (Figure 4-A). We calculated the percentage of senescent hepatocytes in subjects with and without HE and found that subjects with HE had increased expression of P21 -positive senescent hepatocytes compared with subjects without HE (Figure 4-B). Similar results were observed after categorization of hepatocytes based on p21 expression per field area in subjects with and without HE (Figure 4-C).

[0205] EXAMPLE 3: Stratification of subjects using a reference p21 hepatocyte expression level

[0206] Material and methods

[0207] Ethics and subject selection

[0208] The data for this example is from human subjects admitted to Royal Free Hospital, London, UK with severe acute hepatitis (sAH) between 2010 and 2023. The parameters used to define sAH are Jaundice + INR >1 .2. Acute hepatitis (AH) was defined as per EASL guidelines6.

[0209] Ethical approval for the study was gained through HRA Hampshire (IRAS 333200). An existing ethical approval (IRAS 254793) was in place for the first part of this study. The results presented here are from 27 human subjects with 28 liver biopsy or explant samples. Further subject details are presented in Table 1 below.

[0210]

[0211] Table 1 : Subject details

[0212] Immunohistochemistry

[0213] 4-micrometer-thick paraffin sections were stained for HNF4a (Abeam ab201460), p21 (Abeam ab109520), and IBA1 (Abeam 178846). All antibodies were first optimised in human colon and liver tissues. The primary and secondary conditions can be seen in Tables 2 and 3 below. Detection was performed with 3,3'-diaminobenzidine (DAB) (Abeam ab64238) followed by counterstaining with hematoxylin.

[0214] Images were obtained using Zeiss Axioskop microscope and Zeiss Axiocam ICc5 camera. 5-10 random consecutive non-overlapping images were taken per slide at x40 magnification. Cell counts were performed automatically for HNF4a using Imaged Fiji Package7and manually on blinded slides for p21 .The p21 slides were the sections which were cut immediately before and after the HNF4a section, putting a distance of 4pm between these to allow accurate and reproducible hepatocyte-specific p21 counts.

[0215] Table 2: Primary conditions for immunohistochemistry

[0216] Table 3: Secondary conditions for immunohistochemistry

[0217] Statistical analysis SPSS software (version 24.0, SPSS Inc, Chicago, USA) or Graphpad Prism software with Student t test (for normally distributed variables) or Mann-Whitney U test (for non-normally distributed variables) was used to manage and analyse the data in this study. A P value of <.05 was considered statistically significant.

[0218] Comparisons for normally distributed continuous variables were done by t-tests and by Chi-Square for categorical variables. Pearson correlation was used to assess the relationship between two normally distributed continuous variables. The statistical significance of event-free survival data was tested using log-rank test (Kaplan-Meier curves) and Cox-regression models. Receiver operating characteristic (ROC) curve analysis was used to assess the performance of p21+ hepatocyte percentage to predict survival. The cut-off was identified by Youden index. This was further adjusted to improve specificity. The percentage for p21 positive hepatocytes were calculated by adding all the individual readings for each image per slide. This was then divided by the sum of all the HNF4a reads from the same areas of the slides to obtain the antibody positive per total hepatocyte ratio.

[0219] Results

[0220] As detailed in Example 1 , routine biochemistry and clinical parameters typically used to determine if a subject requires a liver transplant do not determine if a subject was not likely to survive or, conversely, likely to survive.

[0221] We decided to further investigate if hepatocyte p21 expression, specifically a reference p21 hepatocyte expression level, could be used to more effectively stratify subjects. In particular, it was decided to analyse if stratification of p21 hepatocyte expression could provide increased sensitivity to the current routine biochemistry and clinical parameters typically used to determine if a subject is or is not likely to survive and hence their treatment regime.

[0222] We examined liver biopsies from survivor subjects compared to non-survivor subjects. Each liver biopsy was obtained at the time of enrolment and so may otherwise be referred to as baseline biopsies. In the context of these examples and as shown in Table 1 , the mean time to biopsy was 3.89 days, with a standard deviation of 6.61 days. For each biopsy, the p21 hepatocyte expression level was obtained as described above. The p21 hepatocyte expression level was therefore indicative of a single timepoint, this being the time at which the liver biopsy was taken.

[0223] The routine biochemistry and clinical parameters, which comprised the measurement of bilirubin, INR, and creatinine levels, were assessed at the time of admission of the subject to hospital (day 0), and then subsequently over time, for example, at days 3, 7, 14, and if possible, day 28. The presence or absence of hepatic encephalopathy and / or acute kidney injury was also determined. These parameters were assessed relative to the p21 hepatocyte expression level obtained from the biopsy.

[0224] We found that bilirubin levels on its own did not distinguish between survivors and non-survivors, both at the time of biopsy and when considered over time (Figure 5-A&B). However, we observed that subjects with higher p21 hepatocyte expression levels had a significantly higher bilirubin level (246.9 pmol vs 365.3 pmol, p = 0.0163) at the time of sample collection (Figure 5-C). For this analysis, we set the cut-off p21 hepatocyte expression value (which may otherwise be referred to as the reference p21 hepatocyte expression level) as 10.71%. p21 hepatocyte expression levels below this value were considered “low” and levels above or equal to this “high”. We found that a reference p21 hepatocyte expression level of 15.77% was also able to distinguish between bilirubin level - with a “high” p21 hepatocyte expression level associated with higher bilirubin levels both at the time of biopsy and over time (Figure 6-A&B). These results were not statistically significant.

[0225] In contrast, we observed that the INR value at the time of biopsy was significantly higher in subjects that did not survive compared to survivor subjects (Figure 7-B). This difference remained pronounced over time (Figure 7-A). We found that stratification of the subjects’ p21 hepatocyte expression level above / equal to or below a reference p21 hepatocyte expression level of 10.71% also correlated with INR levels - a high p21 hepatocyte expression level (above or equal to 10.71%) was associated with high INR levels, and a low p21 hepatocyte expression level (less than 10.71%) was associated with lower INR levels over time (Figure 7-C), although there was little difference at the time of biopsy (Figure 7-D). This stratification was also apparent but was less pronounced when using a higher reference hepatocyte expression level value of 15.77% (Figure 8-A&B).

[0226] We then considered creatinine levels, as a read-out of kidney function. Figures 9-A&B show that creatinine levels at the time of biopsy collection, and when analysed thereafter, were not significantly different between survivors and non-survivors. However, we observed a trend towards significance in increased creatinine levels of subjects with high p21 hepatocyte expression levels (defined as greater than or equal to 10.71%) compared to subjects with low p21 hepatocyte expression levels (less than 10.71%). This was observed both over time and at the time of biopsy collection (60.10 pmol vs 86.60 pmol, p = 0.0681) (Figures 9-C&D). Increasing the reference p21 hepatocyte expression level to 15.77% demonstrated a similar trend over time, although this was not significant (Figures 10-A&B).

[0227] Similarly to Example 1 , we also found that p21 hepatocyte expression levels were significantly higher in subjects who developed hepatic encephalopathy (HE) compared to subjects who did not develop HE (12.83% vs 27.41%, p = 0.0153, Figure 11). Cox-regression analysis confirmed this statistical significance, with a p value of 0.032 (Table 4 below).

[0228] Table 4: Cox-regression analysis of p21 hepatocyte expression levels and hepatic encephalopathy

[0229] Likewise, we found that p21 hepatocyte expression levels were higher in subjects who developed acute kidney injury (AKI) (Figure 12), although this was not statistically significant (14.88% vs 18.47%, p = 0.544). When these parameters were combined (i.e. subjects who developed acute kidney injury and / or HE), we observed a trend for higher p21 hepatocyte expression levels in subjects who had developed HE and / or AKI, although this was not statistically significant (Figure 13).

[0230] The clear association between p21 hepatocyte expression levels and clinical outcome was further demonstrated by analysis of the p21 hepatocyte expression levels in survivors who had not had a liver transplant (referred to as “transplant-free survivors”) versus non-survivors (who were defined as needing a transplant or died within 90 days of admission). Figure 14 shows that p21 hepatocyte expression levels were significantly higher in the non-survivors (11 .91 % vs 24.48%, p = 0.0176). This is further evidenced by the values shown in Table 5 for survivors versus non-survivors. In particular, we found that the mean p21 hepatocyte expression level for survivors was 11 .91%. In contrast, the mean p21 hepatocyte expression level for non-survivors was significantly higher - at 24.48%. The difference in value of all other parameters tested were found to be individually not significant.

[0231] Table 5: Table of characteristics for survivors and non-survivors

[0232] Further statistical analysis, using cox-regression for transplant-free survival (univariable) was carried out. Results are shown in Table 6. Briefly, we found that INR, creatinine levels and p21 hepatocyte expression levels were predictive of transplant free survival in the univariable cox regression analysis.

[0233] Table 6: Cox-regression analysis for transplant-free survival

[0234] EXAMPLE 4: Validation of the reference p21 hepatocyte expression level

[0235] Having considered the stratification ability of reference p21 hepatocyte expression levels of 10.71 and 15.77%, we sought to further explore potential reference p21 hepatocyte expression levels.

[0236] Table 7, below, shows AUROC (area under the receiver operating characteristics curve), sensitivity and specificity values for the reference p21 hepatocyte expression levels 10.707%, 15.769% and 20.967% in the prediction of transplant-free survival. The associated ROC curve is shown in Figure 15. It will be appreciated that since the AUROC relates to p21 hepatocyte expression, the AUROC value remains the same for each reference p21 hepatocyte expression level (AUROC 0.737).

[0237] Table 7: AUROC analysis of various cut-off values As Table 7 shows, a cut-off (which may otherwise be referred to as reference p21 hepatocyte expression level) value of 10.701 gave the greatest sensitivity of 0.889 (i.e. the greatest proportion of true positive results for subjects likely to have transplant free survival). A cut-off value of 20.967 gave the lowest sensitivity (0.333) but greatest specificity of (0.789 - i.e. the greatest proportion of true negatives, these being subjects not likely to have transplant free survival). The optimal reference p21 hepatocyte expression level was determined to be the value closest on the ROC curve in Figure 15 to where sensitivity is 1 and 0 is specificity. Having tested various cut-off values as shown in Table 8, we determined that the optimal cut-off value was likely to be between 10.707 and 15.77%, as validated in Example s.

[0238] Table 8: Full list of cut-off values tested, together with their sensitivity and specificities

[0239] The ability of these reference p21 hepatocyte expression levels to effectively stratify subjects was further explored using Kaplan-Meier cumulative survival curves. Figure 16 shows a strong trend towards survival being significantly lower in subjects with a p21 hepatocyte expression level of greater than or equal to 10.71%. The time shown represents days. These curves also demonstrated that non-survival typically occurred at an early stage for subjects having a p21 hepatocyte expression level of greater than or equal to 10.71%; the majority of deaths in this category occurred less than 20 days after admission. This highlights the need for urgent stratification and treatment in this subject group.

[0240] These examples demonstrate how we have identified that the p21 hepatocyte expression level is surprisingly able to stratify subjects into those who do not require treatment and those who do require treatment and / or a liver transplant. For this subgroup of subjects, failure to administer treatment is likely to be fatal. The present invention therefore lies in the previously unrecognised finding that the p21 hepatocyte expression level can effectively stratify subjects.

[0241] EXAMPLE 5: IGF system expression and therapeutic use thereof in a rodent model of acute liver disease Materials and Methods

[0242] Animal model

[0243] The rat model used to mimic acute on chronic liver failure (ACLF) in this study was published and described previously9and is shown in Figure 17A. Briefly, male Sprague-Dawley rats (weights 260 + / -20 g, age 8-10 weeks) were studied 4 weeks after either a sham-operation or bile duct ligation surgery (BDL). rhlGF-1 (INCRELEX® (mecasermin), Ipsen Biopharmaceuticals, Cambridge, MA, US) was administered i.p twice daily at a dose of 0.5mg / Kg bw from week 2 to 4 post surgery. LPS derived from Klebsiella pneumonia (0.025 mg / kg) (Sigma, UK) was injected intraperitoneally (i.p.) as a second hit to induce acute injury on day 28. Animals were culled 4 hours post LPS injection.

[0244] For the CCL4-LPS model, male C57BL / 6 mice (body weight: 30 g±4 g; age:8-10 weeks) were gavaged with carbon tetrachloride (CCI4 0.5 mg / ml dissolved in olive oil - dose 0.5 ml / kg bw) twice weekly for a total of 6 weeks to induce liver fibrosis, as described previously10. To induce an acute injury, LPS (Klebsiella pneumonia, Sigma, UK), dissolved in saline to a final concentration of 6.25 pg / pl was injected i.p to a final dose of 4 mg / kg. Therapeutic interventions with G-CSF (250 pg / kg, s.c.) and / or TAK-242 (10 mg / kg, i.p.) were started 1 hour after LPS injection and repeated 22 hours after LPS injection. Animals were sacrificed 24 hours after LPS injections and 2 hours after the last therapeutic intervention. A schematic of this model is shown in Figure 17B. Sampling and storage

[0245] Blood samples were taken from the abdominal aorta or right heart as appropriate. EDTA and / or lithium heparin plasma was centrifuged 2,500 rpm for 10 min and stored at -80°C for later analysis. All tissues (liver, brain, kidneys) were snap frozen in liguid nitrogen and stored at -80°C for further analysis. In addition, organs were harvested in formalin(48 h) for histological assessment.

[0246] Biochemistry

[0247] Plasma samples were analysed with Cobas Integra 400 multianalyser (Roche; UK) for alanine aminotransferase (ALT), Aspartate transaminase (AST), glucose, albumin, bilirubin, creatinine and urea levels. mRNA expression of Senescence-associated genes

[0248] Total RNA was extracted from rat liver tissues and then cleaned up by using QIAzol Lysis method. RNA samples were guantified and assessed using NanoDrop1000 System (Thermo Scientific, USA), followed by gel electrophoresis for evaluation of purity and integrity.

[0249] Expression status of the senescence pathways was assessed using the RT2Profiler™ PCR Array Rat Cellular Senescence panel (Cat No PARN-050Z, Qiagen, UK); list of genes included in this panel is shown in Table 9.

[0250] Data were analysed using the pathway signal flow (PSF) method. 1 pg of total RNA template was used for cDNA synthesis using RT2 first strand kit (Qiagen, UK), followed by gPCR reactions RT2 SYBR Green gPCR mastermix as per manufacturer’s protocol (Qiagen, UK). Real-time PCR reactions were performed using 7500 Fast Real-Time PCR System (Thermo Scientific, UK). mRNA expression of IGF system in the rodent animal models

[0251] Total RNA was extracted from rodent (mouse and rat) liver tissues and then cleaned up by using QIAzol reagent (Qiagen). RNA samples were guantified and assessed using NanoDrop1000 System (Thermo Scientific, USA), followed by gel electrophoresis for evaluation of purity and integrity. Gene expression was assessed by gPCR in individual samples. To this end, 0.2 pg of total RNA were retro-transcribed using the QuantiTect Reverse Transcription Kit (Qiagen, CA, USA) and 1 pl of the cDNA sample was then used to set up real-time PCR reactions using ABI 7500 Fast Real-Time PCR System (ThermoFisher, UK), as per manufacturer’s protocol; the individual gPCR assays used are outlined below (Integrated DNA Technology, Belgium). HPRT1 was used as housekeeping gene. Each sample was tested in duplicates. Target genes were normalized for housekeeping gene and their relative guantification was carried out with 2-AACt method (where Ct represents the threshold cycle) using the samples from the sham animals / control group as calibrator. gPCR probes details:

[0252] Igf1 , Mus_musculus: Assay no Mm. PT.58.5811533

[0253] Igfl r, Mus_musculus: Assay no Mm.PT.58.11619137 Igfbpl , Mus_musculus: Assay no Mm. PT.58.45852897

[0254] Igfbp3, Mus_musculus: Assay no Mm. PT.58.6744601

[0255] Hprtl , Mus_musculus: Assay no Mm. PT.39a.22214828

[0256] Igf1 , Rattus_norvegicus: Assay no Rn. PT.58.37138478

[0257] Igfl r, Rattus_norvegicus: Assay no Rn. PT.58.38098384

[0258] Igfbpl , Rattus_norvegicus: Assay no Rn. PT.58.6479202

[0259] Igfbp3, Rattus_norvegicus: Assay no Rn. PT.58.18302903

[0260] Hprtl , Rattus_norvegicus: Assay no Rn. PT.39a.22214832

[0261] Results

[0262] We next sought to identify further possible treatments of liver disease. Current treatment options for liver disease are limited. For many subjects, a liver transplant may be the only treatment option, but it can take weeks to decide upon this option and then even longer for a transplant to become available, by which stage the subject may be too unwell for the transplant, or has already passed away. We therefore sought to identify further possible treatments which can be administered and avoid the need for a liver transplant. In particular, we sought to investigate treatments which could be administered to subjects having a p21 hepatocyte expression level higher than a reference p21 hepatocyte expression level.

[0263] It was decided to investigate the Insulin-like growth factor (IGF) system, which includes IGF-1 , IGF-1 R and IGFBP1-7. The role of the IGF system in liver disease is not clear-cut, with previous studies giving mixed results. Indeed, the expression of IGF-1 R in hepatocytes remains unclear8. Therefore, we decided to assess if IGF system up- or downregulation was observed in a rat model of liver failure (BDL) and liver failure with acute injury (BDL + LPS).

[0264] Analysis of in vivo hepatic mRNA expression of the IGF system in a BDL rat model of liver failure indicated that IGF-1 R and IGFBP1 expression was elevated in liver failure (Figure 18-B&C, see “BDL” and “BDL+ LPS i.p.” columns). In contrast, IGF-1 expression was decreased compared to sham-treated mice (Figure 18-A). In other words, we observed deregulation of the IGF system in liver failure. Treatment with the TLR4 antagonist TAK-242 or the necroptosis inhibitor Nec-1 reduced IGF-1 R and IGFBP1 expression, indicating therapeutic potential of these agents in the treatment of liver failure.

[0265] We next sought to evaluate whether the observed deregulation of the IGF system plays a role in the inhibition of hepatic regeneration and / or promotion of hepatic senescence in our rat models of liver failure (BDL) and liver failure with acute injury (BDL+LPS). In particular, we wanted to determine whether rhlGF- 1 therapy can alleviate hepatic inflammation, reduce hepatic senescence, and promote liver regeneration in this rat model of liver failure.

[0266] Liver function was assessed using ALT, AST, bilirubin and ammonia levels (Figure 19). Reduced bilirubin and ammonia levels upon rhlGF-1 therapy (Figure 19-C&D, see “BDL_IGF-1_LPS”) indicated improved liver function. Kidney function was assessed by measuring glucose, creatinine and urea levels (Figure 20). No significant difference in kidney function was observed upon treatment.

[0267] Mortality was also assessed. The administration of rhlGF-1 was found to considerably reduce mortality in this acute liver injury model (BDL+ LPS, Figure 21).

[0268] We next sought to investigate changes in the expression of senescence-associated genes in the above liver failure rodent models, following administration of rhlGF-1. Expression of the senescence-associated genes Cdkn2a, Cited2, IGF-1 R, Myc, SerpinEI and SerpinE2 generally increased in the liver failure models (BDL and BDL_LPS, Figure 22). This expression was reduced with administration of rhlGF-1 (BDL_IGF-1_LPS, Figure 22). We also analysed expression of genes in the interferon signalling pathway (Figure 23), genes involved in cell adhesion and cytoskeleton regulation (Figure 24), as well as the senescence-associated gene Twistl (Figure 25). Expression of Twistl followed a similar trend to the other senescence-associated genes shown in Figure 22.

[0269] DISCUSSION

[0270] Current clinical scoring systems used to define subject outcome and the need for liver transplantation typically employ markers which are associated with progressive disease in its later stage and incorporate multisystem dysfunction (including encephalopathy, respiratory and renal failure). It can therefore be challenging to determine if a subject with acute liver disease is likely to require a liver transplant.

[0271] For acute liver disease, a decision concerning a liver transplant is typically based on routine biochemical and clinical parameters such as any history of paracetamol ingestion, pregnancy, other drugs, laboratory results and general comorbidity, amongst others. Laboratory results can include features such as PT, pH, arterial lactate, glucose, viral screen, autoantibodies and immunoglobulins and bilirubin concentration. Other features can include the presentation of symptoms such as an altered level of consciousness, hypoglycaemia and / or encephalopathy. However, as detailed in the Examples above, these routine biochemical and clinical parameters are often unable to predict if the subject is likely or unlikely to survive and hence require a liver transplant. Once the subject is presenting other significant symptoms such as encephalopathy and renal failure, they are typically significantly unwell and it can be too late for a liver transplant to occur, such that they may not survive.

[0272] As demonstrated in the Examples above, we have surprisingly found that hepatocellular p21 expression can be used as a baseline diagnostic to predict both survival and the future development of renal and cerebral dysfunction, thereby providing a prognostic biomarker in acute liver disease for the rapid, early and informative selection of subjects for a particular treatment, such as, for, example, a liver transplant.

[0273] In addition to our finding that hepatocellular p21 expression is an effective stand-alone diagnostic for predicting survival and future development and renal and cerebral dysfunction, we have found that the addition of hepatocellular p21 expression to other parameters already commonly used to stratify subjects (such as bilirubin, creatinine and INR), can be used to generate a more sensitive diagnostic for predicting survival. We have also demonstrated that rhlGF-1 therapy in liver failure can reduce hepatic senescence, improve liver function and reduce mortality.

[0274] References

[0275] A number of publications are cited above in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Full citations for these references are provided below. The entirety of each of these references is incorporated herein.

[0276] 1 Lin, S. et al. Prognostic Role of Liver Biopsy in Patients With Severe Indeterminate Acute Hepatitis. Clin Gastroenterol Hepatol 20, 1130-1141 e1137, doi:10.1016 / j.cgh.2O21 .08.008 (2022).

[0277] 2 Guillot, A., Kohlhepp, M. S., Bruneau, A., Heymann, F. & Tacke, F. Deciphering the Immune Microenvironment on A Single Archival Formalin-Fixed Paraffin-Embedded Tissue Section by An Immediately Implementable Multiplex Fluorescence Immunostaining Protocol. Cancers (Basel) 12, doi:10.3390 / cancers12092449 (2020).

[0278] 3 Engelmann, C. et al. Combination of G-CSF and a TLR4 inhibitor reduce inflammation and promote regeneration in a mouse model of ACLF. J Hepatol 77, 1325-1338, doi : 10.1016 / j.jhep.2022.07.006 (2022).

[0279] 4 De Martin et al. Early liver transplantation for corticosteroid non-responders with acute severe autoimmune hepatitis: The SURFASA score. J Hepatol. 2021 Jun;74(6):1325-1334. doi: 10.1016 / j.jhep.2020.12.033.

[0280] 5 Stravitz et al. Future Directions in acute liver failure. Hepatology. 2023 May 16. doi: 10.1097 / HEP.0000000000000458.

[0281] 6. Wendon, J., et al., EASL Clinical Practical Guidelines on the management of acute (fulminant) liver failure. J Hepatol, 2017. 66(5): p. 1047-1081

[0282] 7. Schindelin, J., Arganda-Carreras, I., Frise, E. et al. Fiji: an open-source platform for biological- image analysis. Nat Methods 9, 676-682 (2012). https: / / doi.org / 10.1038 / nmeth.2019

[0283] 8. Adamek and Kasprzak. Insulin-Like Growth Factor (IGF) System in Liver Diseases. Int. J. Mol. Sci.2018, 19, 1308 https: / / doi.org / 10.3390 / ijms19051308

[0284] 9. Harry, D. et al. Increased sensitivity to endotoxemia in the bile duct-ligated cirrhotic Rat

[0285] Hepatology, 30 (1999), pp. 1198-1205

[0286] 10. D.E. Carl, S.S. Ghosh, T.W. Gehr, A. Abbate, S. Toldo, A.J. Sanyal; A model of acute kidney injury in mice with cirrhosis and infection Liver Int, 36 (2016), 865-873 For standard molecular biology techniques, see Sambrook, J., Russel, D.W. Molecular Cloning, A Laboratory Manual. 3 ed. 2001 , Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press

[0287]

Claims

Claims:1 . A senotherapeutic agent for use in a method of treating an acute liver disease subject; wherein the acute liver disease subject has a p21 hepatocyte expression level obtained from a sample from the subject greater than a reference p21 hepatocyte expression level.

2. The senotherapeutic agent for use of claim 1 , wherein the senotherapeutic agent comprises a senolytic agent or a senomorphic agent.

3. The senotherapeutic agent for use of claim 2, wherein the senolytic agent comprises one or more of UBX1235, ABT-737, ABT-199 (Venetoclax), ABT-263 (Navitoclax), UBX0101 , Dasatinib, Quercitin, Fisetin, ALK4 / 5 / 7, AZD1260111 , and a cardiac glycoside.

4. The senotherapeutic agent for use of claim 2 or claim 3, wherein the senomorphic agent comprises one or more of an insulin-like growth factor (IGF), rapamycin, everolimus, temsirolimus and deforolimus,5. The senotherapeutic agent for use of claim 2 or claim 3, wherein the senomorphic agent comprises one or more of rapamycin, everolimus, temsirolimus, and deforolimus.

6. A population of isolated cells for use in a method of treating an acute liver disease subject; wherein the acute liver disease subject has a p21 hepatocyte expression level obtained from a sample from the subject greater than a reference p21 hepatocyte expression level; and optionally wherein the population of cells comprise stem cells or hepatocytes.

7. An anti-inflammatory agent for use in a method of treating an acute liver disease subject; wherein the acute liver disease subject has a p21 hepatocyte expression level obtained from a sample from the subject greater than a reference p21 hepatocyte expression level; and optionally wherein the anti-inflammatory agent comprises a TGF-0 antagonist, a TLR4 antagonist or a CXCR4 antagonist.

8. The anti-inflammatory agent for use of claim 7, wherein the method comprises administering the anti-inflammatory agent and a cell-regenerative agent to the subject.

9. The anti-inflammatory agent for use of claim 8, wherein the cell-regenerative agent comprises G- CSF or GM-CSF.

10. The anti-inflammatory agent for use of any one of claims 7 to 9, wherein the anti-inflammatory agent comprises a TGF-0 antagonist, optionally wherein the TGF-0 antagonist comprises an anti- TGF-P antibody.11 . A method for selecting subjects for participation in an acute liver disease treatment clinical trial, the method comprising: a) measuring a p21 hepatocyte expression level in a sample obtained from the subject;b) comparing the measured p21 hepatocyte expression level to a reference p21 hepatocyte expression level; and c) selecting the subject for participation in the clinical trial when said measured p21 hepatocyte expression level is greater than said reference p21 hepatocyte expression level.

12. A method for selecting an acute liver disease subject for liver transplant, the method comprising: a) measuring a p21 hepatocyte expression level in a sample obtained from the subject; b) comparing the measured p21 hepatocyte expression level to a reference p21 hepatocyte expression level; and c) making a prediction of whether the subject will require a liver transplant based on the comparison in step b), wherein said measured p21 hepatocyte expression level being greater than said reference p21 hepatocyte expression level indicates that the subject will require a liver transplant, and wherein said measured p21 hepatocyte expression level being equal to or lower than said reference p21 hepatocyte expression level indicates that the subject will not require a liver transplant.

13. The senotherapeutic agent, population of isolated cells or anti-inflammatory agent for use of any one of claims 1 to 10, the method for selecting subjects for participation of claim 11 or the method for selecting an acute liver disease subject for liver transplant of claim 12, wherein the sample comprises liver tissue obtained from the subject and wherein the p21 hepatocyte expression level is measured as the number of p21 positive hepatocytes in a given area of the sample divided by the total number of hepatocytes in that area, optionally expressed as a percentage or fraction.

14. The senotherapeutic agent for use, population of isolated cells for use, anti-inflammatory agent for use, the method for selecting subjects for participation or the method for selecting an acute liver disease subject for liver transplant of any one of claims 1 to 13, wherein the p21 hepatocyte expression level is measured by immunohistochemistry (IHC) or multiplex immunofluorescence.

15. The senotherapeutic agent for use, population of isolated cells for use, anti-inflammatory agent for use, the method for selecting subjects for participation or the method for selecting an acute liver disease subject for liver transplant of any one of claims 1 to 14, wherein the sample has been formalin-fixed and paraffin-embedded prior to said measuring of the p21 hepatocyte expression level.

16. The senotherapeutic agent for use, population of isolated cells for use, anti-inflammatory agent for use, the method for selecting subjects for participation or the method for selecting an acute liver disease subject for liver transplant of any one of claims 1 to 15, wherein the reference p21 hepatocyte expression level is about 10%, about 11 %, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19% about 20%, about 21 %, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39% or about 40% of the hepatocytes being p21 positive.

17. The senotherapeutic agent for use, population of isolated cells for use, anti-inflammatory agent for use, the method for selecting subjects for participation or the method for selecting an acute liver disease subject for liver transplant of any one of claims 1 to 16, wherein the reference p21 hepatocyte expression level is about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39% or about 40% of the hepatocytes being p21 positive.

18. The senotherapeutic agent for use, population of isolated cells for use, anti-inflammatory agent for use, the method for selecting subjects for participation or the method for selecting an acute liver disease subject for liver transplant of any one of claims 1 to 15, wherein the reference p21 hepatocyte expression level is of from about 10% to about 16% of the hepatocytes being p21 positive19. The senotherapeutic agent for use, population of isolated cells for use, anti-inflammatory agent for use, the method for selecting subjects for participation or the method for selecting an acute liver disease subject for liver transplant of any one of claims 1 to 18, wherein the subject is a human having or suspected of having acute liver disease without any significant signs of chronic liver disease.

20. The senotherapeutic agent for use, population of isolated cells for use, anti-inflammatory agent for use, the method for selecting subjects for participation or the method for selecting an acute liver disease subject for liver transplant of any one of claims 1 to 19, wherein the acute liver disease comprises acute indeterminate hepatitis, acute autoimmune hepatitis, acute viral hepatitis, or drug-induced liver injury.21 . The senotherapeutic agent for use, population of isolated cells for use, anti-inflammatory agent for use, the method for selecting subjects for participation or the method for selecting an acute liver disease subject for liver transplant of any one of claims 1 to 20, wherein yH2AX hepatocyte expression level in a sample obtained from the subject is also measured.

22. Use of a kit comprising an anti-p21 antibody for: a) Selecting subjects for participation in an acute liver disease treatment clinical trial; or b) Predicting whether an acute liver disease subject will require a liver transplant.

23. The use of claim 22, wherein the kit further comprises one or more hepatocyte-specific reagents, optionally an anti-HNF4a antibody.

24. An insulin-like growth factor (IGF) for use in a method of treating liver disease in a subject.

25. The IGF for use of claim 24, wherein the IGF comprises IGF-1 .

26. The IGF for use of claim 24 or claim 25, wherein the subject is a human subject.

27. The IGF for use of any one of claims 24 to 26, wherein the liver disease comprises acute liver disease.

28. The IGF for use of any one of claims 24 to 26, wherein the liver disease comprises chronic liver disease.

29. The IGF for use of claim 28, wherein the chronic liver disease comprises acute-on-chronic liver disease.

30. The IGF for use of any one of claims 24 to 29, wherein the subject has a p21 hepatocyte expression level obtained from a sample from the subject greater than a reference p21 hepatocyte expression level.

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