WO2014076453A1 - Adrenergic agonists for use in treating liver damage - Google Patents

Abstract

The invention relates to liver damage, and to pharmaceutical compositions for use in treating, preventing or ameliorating liver damage or disease, especially acute liver damage. The invention is particularly, although not exclusively, concerned with treating or preventing liver damage caused by paracetamol poisoning. The invention also extends to methods of treating such conditions.

Description

ADRENERGIC AGONISTS FOR USE IN TREATING LIVER DAMAGE

The invention relates to liver damage, and to pharmaceutical compositions for use in treating, preventing or ameliorating liver damage or disease, especially acute liver damage. The invention is particularly, although not exclusively, concerned with treating or preventing liver damage caused by paracetamol (also known as acetaminophen) poisoning. The invention also extends to methods of treating such conditions.

Paracetamol (Acetaminophen, APAP) overdose, either deliberate, through suicide attempts, or unintentionally, because of consumption of multiple-drug preparations containing APAP, is a major public health problem worldwide because it causes much morbidity which frequently progresses to fulminant liver failure (FLF). This is despite the presence of N-Acetyl Cysteine (NAC) as an antidote, and Governmental attempts to reduce the non-prescription availability of APAP. FLF may result in death if a suitable liver for transplantation cannot be found, with about 200 such deaths per year, in England and Wales alone. Besides these deaths, there is the fiscal cost of liver transplantation and subsequent maintenance of these transplanted patients, with total such costs having being estimated worldwide at billions of dollars per year. A shortage of donor livers for transplantation as a treatment for liver disease, including FLF, drives the search to understand the factors that regulate liver regeneration.

There is therefore a need to provide an improved means of treating liver disease or damage. The inventors have surprisingly demonstrated that the activation of oc- adrenergic receptors and/or β-adrenergic receptors, which are present on hepatic progenitor cells (stem cells, HPC), promotes the expansion of these stem cells and can therefore be used to treat liver damage.

Thus, in a first aspect of the invention, there is provided an adrenergic receptor agonist, for use in treating, preventing or ameliorating liver damage. In a second aspect, there is provided a method of treating, ameliorating or preventing liver damage in a subject, the method comprising administering, to a subject in need of such treatment, a therapeutically effective amount of an adrenergic receptor agonist.

Hepatic progenitor cells (HPC) are bi-potential liver resident stem cells that can differentiate into hepatocytes or bile duct cells. They are activated to promote hepatic regeneration and replace lost liver tissue after acute massive hepatocyte loss or when mature hepatocyte replication is impaired, as in chronic liver inflammatory conditions, such as non-alcoholic steatohepatitis. Emerging evidence suggests that the sympathetic nervous system (SNS) maybe involved in liver repair, either directly or through effects on liver cells, such as myofibroblastic hepatic stellate cells (HSC), which are regulated positively by the SNS. Also, it has previously been shown the ai-adrenoceptor antagonist, prazosin (PRZ), expanded liver progenitors and reduced injury in a chronic model of liver disease.

Therefore, in a further series of experiments with the β-adrenergic receptor antagonist propranolol (PRL), the inventor's starting hypothesis was that the homeostatic effect of SNS signalling on HPC expansion is inhibitory and that PRL would, as with PRZ, expand HPC numbers and reduce liver injury. Initial results, under in vivo conditions simulating non-alcoholic steatohepatitis (NASH), showed emphatically that PRL, like PRZ, expanded the HPC population. However, PRL unlike PRZ, significantly increased biochemical and histological markers of liver injury and cell death. Mechanistic studies showed that PRL induced hepatocyte death, as evidenced by increased release of ALT, LDH, TNF-a and FAS ligand, through both the extrinsic and intrinsic apoptotic pathways as judged by upregulation of FAS receptor, caspase-8 proteins, and cytochrome C. These PRL results caused the inventors to modify their working hypothesis and, as a result, they postulated that surprisingly, the basal action of SNS agonist signalling in liver injury maybe to promote HPC expansion.

This modified hypothesis was supported by the finding that infusion of the SNS agonists Norepinephrine (NE) and Isoprenaline (ISO) into spontaneously

steatohepatitic ob/ob mice induced increases in HPC number, and a parallel reduction in liver injury. Furthermore, in the complete absence of the SNS in mice lacking

Dopamine β-hydroxylase (Dbh ) and which therefore cannot synthesize SNS neurotransmitters, a diet inducing NASH led to a loss of the hepatomegaly and expansion of HPC normally associated with this diet and observed in the controls. This reduction was reversed by infusion of ISO. Moreover, although HPC are acknowledged to play only a minor role in liver regeneration after a partial hepatectomy, in the absence of agents that inhibit replication of mature hepatocytes, the inventors surprisingly also observed a clear reduction in HPC numbers in the Dbh ¹ mice post hepatectomy. These surprising results suggested unequivocally and for the first time, that direct SNS agonist signalling is required to expand the HPC compartment after acute and chronic liver injury.

In support of the above there is evidence showing that the SNS regulates stem cell physiology in other organs, such that pharmacological manipulation of the SNS has been shown to modulate haematopoietic stem cell proliferation and egress. Moreover, adrenergic agents have been shown to induce proliferation of neuronal stem cells and embryonic stem cells have also been shown to respond to adrenergic stimulation. Given the clinical importance of APAP poisoning and evidence suggesting that the SNS regulates HPC and reduces liver injury, the inventors hypothesized that SNS stimulation by ISO would expand the HPC population and reduce acute liver injury induced by APAP. They also sought to investigate the mechanisms through which ISO affected HPC. The results comprehensively show that HPC are markedly expanded by the SNS β-adrenoceptor agonist ISO through the β-catenin-Wnt pathway and that ISO drastically reduces APAP induced injury. Since there is a possibility that ISO may cause abnormal cardiac rhythms in patients with acute APAP poisoning, the inventors then sought to determine if the ai-adrenoceptor agonist phenylephrine, which may induce less abnormal rhythms, also caused an expansion of HPC with reduced liver injury. Accordingly, the adrenergic receptor agonist may be used for treating, preventing or ameliorating any kind of liver damage or failure. For example, the agonist may be used to treat fulminant liver failure (FLF). The liver damage which is treated maybe acute liver damage. For example, the liver damage may have been caused by administration or consumption of a poison, for example paracetamol (i.e. APAP) or alcohol. The liver damage may have been caused by ingestion of Khat plant, which like APAP, may also cause acute liver failure (ALF).

Adrenergic receptors are metabotopic G-protein coupled receptors (GPCRs) that are activated by catecholamines, especially noradrenaline and adrenaline. These receptors are generally classified as either alpha(oc)-adrenoceptors or beta( )-adrenoceptors. Accordingly, in one embodiment, the adrenergic receptor agonist may be an oc- adrenergic receptor agonist. In another embodiment, the adrenergic receptor agonist may be a β-adrenergic receptor agonist. The term "agonist" can mean a molecule that selectively binds to either the a- or the β- adrenergic receptor to initiate the signal transduction reaction. Preferably, the agonist is operable, in use, to selectively activate the desired adrenergic receptor, i.e. the agonist activates the target adrenoceptor to a greater extent, or at lower doses, than other types of adrenergic receptors.

Alpha-adrenergic receptors may further be characterized as either oci-adrenoceptors or ofc-adrenoceptors. Therefore, the adrenergic receptor agonist may be either an or an ^-adrenergic receptor agonist. Activation of alphai-adreonceptors promotes the activation of the G protein, G_q, which, in turn leads to the activation of the

phoshpolipase C signaling pathway, whereas activation of oc₂ adrenoceptors promotes the activation of the G protein, Gi, which in turn leads to the activation of the adenylate cyclase signaling pathway. Hence, a suitable oci-adrenergic receptor agonist maybe selected from a group consisting of: Noradrenaline, Xylometazoline, Phenylephrine, and Methoxamine.

A preferred oci-adrenergic receptor agonist is Phenylephrine, as described in Example 7. A suitable oc₂-adrenergic receptor agonist may be selected from a group consisting of: Clonidine, Dexmedetomidine, Medetomidine, and Romifidine.

The skilled person will appreciate that oci-adrenoceptors may be further subcategorized as ocia-, O - or ocid-adrenoceptors. oc₂-adrenoceptors may be further subcategorized as oc or oc_2c-adrenoceptors. Beta-adrenergic receptors may be further characterized, as betai-adrenoceptors, beta₂- adrenoceptors or beta₃-adrenoceptors. Therefore, the adrenergic receptor agonist may be a βι-, a β₂- or a ₃-adrenergic receptor agonist. However, in some embodiments, the agonist may not be a ₃-adrenergic receptor agonist. Stimulation of either of the three β-adrenergic receptors promotes the activation of the G protein, G_s, which in turn leads to the activation of the adenylate cyclase signaling pathway. A suitable βι-adrenergic receptor agonist may be selected from a group consisting of: Dobutamine, Isoprenaline, and Noradrenaline. A preferred βι-adrenergic receptor agonist is Isoprenaline, as described in the Examples. A suitable

receptor agonist may be selected from a group consisting of: Isoprenaline and Salbutamol. As described in the Examples, these agonists will be useful in the treatment of acute liver disease/damage.

In some embodiments of the invention, it may be desirable to administer an oc- adrenoceptor agonist and a β-adrenoceptor agonist simultaneously. For example, an - adrenergic receptor agonist such as Noradrenaline, Xylometazoline, Phenylephrine, or Methoxamine may be administered together with a βι-adrenergic receptor agonist such as Dobutamine, Isoprenaline, and Noradrenaline. Preferably, Phenylephrine is administered with Isoprenaline.

Classification of oc-adrenoceptors and β-adrenoceptors, and their subtypes, may be achieved by comparing the potency of the catecholamines, isoprenaline, adrenaline and noradrenaline at each of these receptors, and possibly also by determining the type of intracellular signaling pathway which is activated by the action of an agonist at the receptor.

Adrenergic receptor agonists used according to the invention may achieve their functional effect through promoting the expansion of hepatic progenitor cells (HPC's). Although not wishing to be bound by any hypothesis, the inventors believe that adrenoceptor agonists promote expansion/proliferation of hepatic progenitor cells through activation of the HPC Wnt pathway, which leads to the expression of various Wnts. Wnts are a family of signaling proteins which pass signals from receptors found on the surface of cells to their nuclei to regulate gene expression. Accordingly, the agonist may be operable in use to enhance HPC expansion, preferably by activating the Wnt pathway.

Therefore, in a third aspect, there is provided an adrenergic receptor agonist, for use in inducing the expression of Wnt by hepatic progenitor cells.

Preferably, expression of Wnt l, 3a, 6 or 10a maybe induced by the agonist compared to the level of expression in the absence of the agonist. The term "expression" can relate to the detection of a Wnt protein in any compartment of the cell (e.g. in the nucleus, cytosol, the Endoplasmic Reticulum or the Golgi apparatus); or detection of the mRNA encoding a Wnt. It will be appreciated that adrenoceptor agonists according to the invention may be used in a medicament, which may be used in a monotherapy, i.e. use of only an adrenoceptor agonist (e.g. an antibody or a catecholamine) for treating, ameliorating, or preventing acute liver damage/disease. Alternatively, adrenoceptor agonists according to the invention maybe used as an adjunct to, or in combination with, known therapies for treating, ameliorating, or preventing acute liver damage/disease. For example, adrenoceptor agonists of the invention may be used in combination with known agents for treating acute liver damage/disease, such N-Acetyl Cysteine etc.

The adrenoceptor agonists according to the invention maybe combined in

compositions having a number of different forms depending, in particular, on the manner in which the composition is to be used. Thus, for example, the composition may be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micellar solution, transdermal patch, liposome suspension, or any other suitable form that may be administered to a person or animal in need of treatment. It will be appreciated that the vehicle of medicaments according to the invention should be one which is well-tolerated by the subject to whom it is given.

The composition may comprise liver-targeting means, arranged, in use, to target the adrenoceptor agonist at least adjacent the liver. For example, the adrenoceptor agonist may be formulated within a liposome or liposome suspension, which liposome comprises a ligand which targets the liver. Advantageously, such liver targeting significantly improves delivery of the active agent to the treatment site increasing efficacy. Medicaments comprising adrenoceptor agonists according to the invention may be used in a number of ways. For instance, oral administration may be required, in which case the adrenoceptor agonists may be contained within a composition that may, for example, be ingested orally in the form of a tablet, capsule or liquid. Compositions comprising adrenoceptor agonists of the invention may be administered by inhalation (e.g. intranasally). Compositions may also be formulated for topical use. For instance, creams or ointments maybe applied to the skin, for example, adjacent the treatment site, e.g. the liver.

Adrenoceptor agonists according to the invention may also be incorporated within a slow- or delayed-release device. Such devices may, for example, be inserted on or under the skin, and the medicament may be released over weeks or even months. The device maybe located at least adjacent the treatment site. Such devices may be particularly advantageous when long-term treatment with adrenoceptor agonists used according to the invention is required and which would normally require frequent administration (e.g. at least daily injection).

In a preferred embodiment, adrenoceptor agonists and compositions according to the invention may be administered to a subject by injection into the blood stream or directly into a site requiring treatment. Injections maybe intravenous (bolus or infusion) or subcutaneous (bolus or infusion), or intradermal (bolus or infusion).

It will be appreciated that the amount of the adrenoceptor agonist that is required is determined by its biological activity and bioavailability, which in turn depends on the mode of administration, the physiochemical properties of the adrenoceptor agonist and whether it is being used as a monotherapy or in a combined therapy. The frequency of administration will also be influenced by the half-life of the adrenoceptor agonist within the subject being treated. Optimal dosages to be administered maybe determined by those skilled in the art, and will vary with the particular adrenoceptor agonist in use, the strength of the pharmaceutical composition, the mode of

administration, and the advancement of the disease being treated. Additional factors depending on the particular subject being treated will result in a need to adjust dosages, including subject age, weight, gender, diet, and time of administration.

Generally, a daily dose of between o.o^g/kg of body weight and o.5g/kg of body weight of the adrenoceptor agonist according to the invention may be used for treating, ameliorating, or preventing liver damage/disease, depending upon which adrenoceptor agonist is used, e.g. catecholamine or antibody. More preferably, the daily dose of the adrenoceptor agonist is between o.oimg/kg of body weight and 500mg/kg of body weight, more preferably between o.img/kg and 200mg/kg body weight, and most preferably between approximately lmg/kg and loomg/kg body weight. As discussed in the examples, particularly Examples 6 and 7, the adrenoceptor agonist may be administered before, during or after onset of acute liver disease/damage. For example, the agonist may be administered immediately after a subject has ingested a toxic amount of paracetamol. Daily doses may be given as a single administration (e.g. a single daily injection). Alternatively, the adrenoceptor agonist may require administration twice or more times during a day. As an example, adrenoceptor agonists may be administered as two (or more depending upon the severity of the disease being treated) daily doses of between 25mg and 7000 mg (i.e. assuming a body weight of 70 kg). A patient receiving treatment may take a first dose upon waking and then a second dose in the evening (if on a two dose regime) or at 3- or 4-hourly intervals thereafter. Alternatively, a slow release device may be used to provide optimal doses of

adrenoceptor agonist according to the invention to a patient without the need to administer repeated doses. In another embodiment, the adrenoceptor agonist may be administered before the onset of liver damage. For example, in cases where a subject is undergoing clinical trials or being treated with a drug which is known to, or likely to, cause acute liver damage (for example an anticancer drug), then it may be advantageous to protect the liver by per-administering the adrenoceptor agonist of the invention.

Known procedures, such as those conventionally employed by the pharmaceutical industry (e.g. in vivo experimentation, clinical trials, etc.), may be used to form specific formulations comprising the adrenoceptor agonist according to the invention and precise therapeutic regimes (such as daily doses of the adrenoceptor agonist and the frequency of administration). The inventors believe that they are the first to describe a pharmaceutical composition for treating acute liver disease/damage, based on the use of the agonist of the invention.

Hence, in a fourth aspect of the invention, there is provided a liver damage treatment composition, comprising an adrenergic receptor agonist and a pharmaceutically acceptable vehicle.

Liver damage or disease which may be treated with the composition may be acute. In addition, the liver disease may be caused by a variety of factors, which can include paracetamol or Acetaminophen (APAP) overdose, alcoholism, or other diseases, such as Malaria. The agonist may comprise an a- or a β-adrenergic receptor agonist. In one embodiment, the agonist may be either an % or an oc₂-adrenergic receptor agonist. A suitable oci-adrenergic receptor agonist may be selected from a group consisting of: Noradrenaline, Xylometazoline, Phenylephrine, and Methoxamine. Preferably, the agonist is Phenylephrine. A suitable oc₂-adrenergic receptor agonist may be selected from a group consisting of: Clonidine, Dexmedetomidine, Medetomidine, and

Romifidine. In another embodiment, the agonist may be a βι-, a β₂- or a p₃-adrenergic receptor agonist. A suitable βι-adrenergic receptor agonist may be selected from a group consisting of: Dobutamine, Isoprenaline, and Noradrenaline. Preferably, the agonist is Isoprenaline. A suitable βι-adrenergic receptor agonist may be selected from a group consisting of: Isoprenaline and Salbutamol.

The invention also provides in a fifth aspect, a process for making the composition according to the fourth aspect, the process comprising contacting a therapeutically effective amount of an adrenergic receptor agonist and a pharmaceutically acceptable vehicle.

A "subject" maybe a vertebrate, mammal, or domestic animal. Hence, compositions and medicaments according to the invention may be used to treat any mammal, for example livestock (e.g. a horse), pets, or maybe used in other veterinary applications. Most preferably, however, the subject is a human being.

A "therapeutically effective amount" of the adrenoceptor agonist is any amount which, when administered to a subject, is the amount of medicament or drug that is needed to treat liver disease/damage or produce the desired effect.

For example, the therapeutically effective amount of adrenergic receptor agonist used maybe from about o.oi mg to about 8oo mg, and preferably from about o.oi mg to about 500 mg. It is preferred that the amount of adrenoceptor agonist is an amount from about o.i mg to about 250 mg, and most preferably from about 0.1 mg to about 20 mg.

A "pharmaceutically acceptable vehicle" as referred to herein, is any known compound or combination of known compounds that are known to those skilled in the art to be useful in formulating pharmaceutical compositions. In one embodiment, the pharmaceutically acceptable vehicle maybe a solid, and the composition may be in the form of a powder or tablet. A solid pharmaceutically acceptable vehicle may include one or more substances which may also act as flavouring agents, lubricants, solubilisers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coatings, or tablet- disintegrating agents. The vehicle may also be an encapsulating material. In powders, the vehicle is a finely divided solid that is in admixture with the finely divided active agents according to the invention. In tablets, the active agent (e.g. the adrenoceptor agonist) may be mixed with a vehicle having the necessary compression properties in suitable proportions and compacted in the shape and size desired. The powders and tablets preferably contain up to 99% of the active agents. Suitable solid vehicles include, for example calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins. In another embodiment, the pharmaceutical vehicle maybe a gel and the composition may be in the form of a cream or the like.

However, the pharmaceutical vehicle may be a liquid, and the pharmaceutical composition is in the form of a solution. Liquid vehicles are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The

adrenoceptor agonist according to the invention may be dissolved or suspended in a pharmaceutically acceptable liquid vehicle such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats. The liquid vehicle can contain other suitable pharmaceutical additives such as solubilisers, emulsifiers, buffers,

preservatives, sweeteners, flavouring agents, suspending agents, thickening agents, colours, viscosity regulators, stabilizers or osmo-regulators. Suitable examples of liquid vehicles for oral and parenteral administration include water (partially containing additives as above, e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g.

glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil). For parenteral administration, the vehicle can also be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid vehicles are useful in sterile liquid form compositions for parenteral administration. The liquid vehicle for pressurized compositions can be a halogenated hydrocarbon or other pharmaceutically acceptable propellant. Liquid pharmaceutical compositions, which are sterile solutions or suspensions, can be utilized by, for example, intramuscular, intrathecal, epidural, intraperitoneal, intravenous and particularly subcutaneous injection. The adrenoceptor agonist maybe prepared as a sterile solid composition that maybe dissolved or suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium.

The adrenoceptor agonist and pharmaceutical compositions of the invention may be administered orally in the form of a sterile solution or suspension containing other solutes or suspending agents (for example, enough saline or glucose to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monoleate, polysorbate 8o (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and the like. The adrenoceptor agonists according to the invention can also be administered orally either in liquid or solid composition form. Compositions suitable for oral

administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixirs, and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.

All of the features described herein (including any accompanying claims, abstract and drawings), and/ or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and/ or steps are mutually exclusive.

For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying Figures, in which: -

Figure lA shows the mean number of CK19 positive HPCs in the liver of mice control

Dbh⁺ , Dbh , and Dbh mice infused with Isoprenaline (Dbh +ISO) at 20 mg/kg/day to induce activation of the SNS. Data are mean ± s.e.m, n= 5 mice per group. *p < 0.05 in Dbh mice compared to control mice and #p < 0.05 in Dbh + ISO compared to Dbh (one-way AN OVA with Tukey's post hoc test);

Figure lB shows the results of a duplex PCR performed on isolated EpCAM+ cells (EpCAM+ cells) and EpCAM depleted non-parenchyma cells (EpCAM-cells) from normal mouse liver. Total liver extract served as control;

Figure lC are representative flow cytometry plots of side population (SP) cells in total NPC isolated from normal mice liver. The same samples treated with verapamil which inhibit the function of the ABC transporter lost the SP population. EpCAM positive cells were highly enriched in the SP cells. Inset number indicates percentage of positive cells in total NPC;

Figure lD shows adrenoceptor mRNA expression of isolated EpCAM+ cells and the liver progenitor cell line (603B cells) using RT-PCR;

Figure lE (Left panel) are the results of a cell proliferation assay which show the fold- increase in the number of 603B cells at different doses of isoprenaline (ιοορΜ - ιομΜ). Results are expressed as fold change± s.e.m from 6 biological replicates relative to control (basal medium). **p<o.ooi compared to basal medium control (one-way ANOVA with Tukey's post hoc test);

Figure lE (Right panel) are the results of a cell proliferation assay which show the fold- increase in the number of 603B cells treated with basal medium (basal) as control, ιθμΜ of isoprenaline, and ιθμΜ of isoprenaline (ISO) after pre-treatment with ιομΜ of Propranolol (ISO+PRL). Results are expressed as fold change± s.em, relative to basal from 3 biological replicates; *p<0.05 compared to basal; #p<0.05 compared to ISO; Figure lF shows the percentage of EpCAM+ cells (determined using flow cytometry), the number of CK19 cells (determined using Immunohistochemistry) and the number of EpCAM cells in (the livers of mice) mice which received either a control treatment (Con) or isoprenaline (Iso) at a dose of 2.5mg/kg. Control mice received PBS vehicle (Con). Data are representative from 2 independent experiments. Data are mean ± s.e.m. (n=4 per group);

Figure lG are representative images of HPCs detected in mice livers using

immunohisotchemistry. Mice received either a control treatment (Con) or isoprenaline (ISO);

Figure 2A is a representative western blot and densitometric analyses showing indicated protein expression in 603B cells treated with either ιομΜ isoprenaline (ISO) or basal media (Con). An antibody to β-actin was used as a loading control. Data are mean ± s.e.m, n= 3;

Figure 2B are immunoflurorescent immunocytochemistry images of 603B cells treated with isoprenaline showing cell membrane localisation of β-catenin (left) and nuclear localization of β-catenin (right). Nuclei were stained with dapi (blue);

Figure 2C is a proliferation assay showing the fold-increase in the number of 603B cells stimulated with ιομΜ isoprenaline (ISO) or ιομΜ ISO in the presence (or absence) of ΐμΜ of the specific Wnt/β catenin inhibitors XAV939 (XAV) and PNU-74654 (PNU). Data are mean ± s.e.m. (n= 3); **p<o.ooi compared to basal medium; #p<o.05 compared to ISO; Figure 2D shows the level of Wnt ligand (Wnt l, 3a & 6) mRNA expression, assessed by real- time PCR, in 603B cells treated with ιομΜ of isoprenaline or control (Con). Data are mean ± s.e.m. (n= 4), *p<0.05;

Figure 2E are the results of a CCK8 proliferation assay which show the effect of the Wnt antagonist (recombinant Dkk 1, 0.1 μg/ml) on isoprenaline (lC^M)-stimulated 603B cells. Data are mean ± s.e.m (n= 3);

Figure 2F are immunohistochemical stains for active β-catenin in a mouse liver 24 hours after administration of vehicle (Con) or Isoprenaline (ISO). Left panel = lower magnification; Right panel = higher magnification;

Figure 3A is a graph showing that ALT (top ) and % necrosis (bottom ) are cumulative for all animals either injected with vehicle, isoprenaline (2.5mg/kg), APAP (375mg/kg) or APAP with subsequent administration of isoprenaline (A+I) at 24h after first administration; n=n to 22 per group;

Figure 3B is a representative histological image of mice liver 24 hours after injection with either vehicle (control), isoprenaline (2.5mg/kg), APAP (375mg/kg) or APAP with subsequent administration of isoprenaline (A+I); left hand panel in each figure is the lower magnification and the right hand panel the higher magnification;

Figure 3C shows the ALT 3h of mice after APAP administration. Data are mean ± s.e.m, n=4/group; *p=o.05, by 2- tailed unpaired t- test;

Figure 3D is the flow cytometric analysis of CD45-/EpCAM+ve cells in non-arenchymal cell fraction. Data are mean ± s.e.m, n=4/group; *p<0.05, **p<o.ooi;

Figure 3E is the immunohisotchemical analyses of progenitor cells using EpCAM (left) and CK19 (right). Data are mean numbers of EpCAM and CK19 +ve cells per portal tract ± s.e.m (n= 5/group);

Figure 3F shows CK19 positive cell density confined to small portal tracts; *p<0.05,

**p<o.ooi;

Figure 3G shows liver injury judged by ALT elevation (left) and HPC number determined by CK19 positive cells density (right) in mice treated with APAP alone or APAP with subsequent administration of PRL (lomg/kg). Data are mean ± s.e.m, n=4 each, *p<o.05, ns = not significant;

In Figures 3A-3G, Con = vehicle treated, APAP = 375mg/kg of APAP and subsequent vehicle treatment, A+ISO = 375mg/kg of APAP and subsequent 2.5mg/kg of ISO treatment;

Figure 4A is a representative western blot (upper) and densitometric (lower) analyses of β-catenin in the livers of mice treated with APAP or APAP + ISO. ISO was administered l hour after APAP and the livers were harvested 24.I1 after APAP initial administration; n=4 per group. *p<0.05;

Figure 4B are representative micrographs of immunohistochemiacal staining of active β-catenin in the livers of mice treated with APAP or APAP/ISO (A+I) 24h after initial administration as in (a) above. Upper panel = lower magnification, Lower panel= higher magnification;

Figure 4C shows wnt ligand expression in total liver 24h after initial administration were analyzed in vehicle injected (Con), APAP injected (APAP), and APAP with subsequent ISO injected (A+I) mice. Data are mean ± s.e.m, n=4 each. *p<o.05, **p<o.ooi;

Figure 4D shows the level of wnt ligand expression in EpCAM+ve cells, the EpCAM depleted non-parenchymal fraction, and hepatocytes isolated from mice livers treated with APAP and ISO 24h after initial APAP administration;

Figure 4E are the results of a LDH cell cytotoxicity assay. Isolated hepatocytes from normal mice liver were treated with lomM APAP or control medium in the presence or absence of ISO (ιθθρΜ - ιομΜ), 20mM of N-acethylsysteine (NAC), loong/ml recombinant mouse Wnt3a, Wnt3a with recombinant mouse Dkki (0.1 μg/ml), 603B conditioned medium from cells stimulated with ISO (CM) and CM with Dkki. Each bar represents replicates from 6 wells of the same treatment. Results are expressed as fold change± s.e.m. relative to triton X treated hepatocyte as controls. *p<0.05 compared to APAP alone;

Figure 5A shows the affect of recombinant TWEAK, 0.04 μg/g on liver injury, assessed using ALT;

Figure 5B shows the affect of recombinant TWEAK, 0.04 μg/g on CKi9+ve HPC cell numbers;

Figure 5C are representative images of immunohistochemical staining with CK19 (DAB chromogen, brown), upper panels; and CK19 with K167 (AEC chromogen, red), double staining (middle panels) and NFKB p65 immunostaining (lower panels). Insert = higher magnification, arrow head indicates positive staining of M67 in CK 19 +ve HPCs. White arrow head indicates nuclear localization of NFKb p65 in periportal ductular cells; Figure 5D shows the experimental design of the TWEAK study;

Figure 5E shows the % necrosis (left) and serum ALT (right) from mice treated with APAP and TWEAK pre-treated mice with subsequent APAP administration. Data are mean ± s.e.m, n=4 each. *p<0.05, 24h after initial APAP administration;

Figure 5F are representative histological images of APAP and TWEAK/APAP mice livers; Figure 5G shows the experimental design of EpCAM positive cell administration. Mice were administered APAP. One and half hours later they given EpCAM +ve cells, or EpCAM +ve cells with/without DKKi, EpCAM depleted NPC or vehicle. EpCAM +ve cells were isolated from APAP + ISO treated mice;

Figure 5H shows the production of the liver injury marker, ALT, 24.I1 after APAP administration. Serum ALT (left) and % necrosis (right) were analyzed in APAP + vehicle (APAP), APAP and EpCAM depleted non-parenchymal cells (A+NPC), APAP + EpCAM +ve (A+Epc), and APAP + EpCAM +ve (A+Epc+DKKi). Data are mean ± s.e.m, n=4/group. *p<0.05;

Figure 6A shows the experimental design of the study used to obtain the results of Figure 6B;

Figure 6B shows the ALT of mice which received APAP and 1 or 3hrs later were received NAC. An alternative batch of mice was treated with APAP followed by ISO 3hrs after initial APAP;

Figure 6C shows the immunohistochemical staining of mice which were used in Figure 6B;

Figure 7a shows the ALT of mice treated with APAP followed 1 hr later by

phenylephrine (PE, 3mg/kg or lomg/kg) and sacrifice 24hrs after APAP

administration; and

Figure 7b shows the affect of PE (ιορΜ to ιθμΜ) on the proliferation of 603B cells.

Data are mean ± s.e.m. (n= 3). **p<o.oi compared to control (Con).

Figure 8 shows that ISO induces β catenin activation on HPCs in vivo.

lomg/kg of ISO treated liver were subjected to analysis of active β-catenin (red) expression in pan-cytokeratin positive HPCs (green). Positive β-catenin nuclear staining is as shown on HPCs (yellow).

Materials & Methods

Animals

Male C57BL/6j mice with a mean weight 25 to 30g were from our Biological Services colony. Male dopamine β-hydroxylase deficient (Dbh^~/-) and Dbh⁺/^~ mice (30-40 weeks) were also from our colony as previously described (Oben, J.A., et ah, 2004). All animals were housed in an environmentally controlled room with 12-h light/dark cycle and allowed free access to food and water. All animals were treated in accordance with The Animals (Scientific Procedures) Act, UK, 1986 guidelines. Materials

Culture medium was obtained from Invitrogen. All other chemicals were from Sigma unless otherwise stated. Cell line

Immature murine cholangiocyte cell line (603B cells) were a kind gift from Professor Diehl.

Animal experiments

All mice are fasted overnight before APAP administration. APAP was dissolved in warm phosphate buffered saline (PBS) and administered intra-peritoneally (IP) with APAP at a dose of 375mg/kg, 500mg/kg or PBS as control. One hour after APAP injection, either Propranolol in water (4mg/kg), Isoproterenol (ISO) in water (2.5mg/kg) or water were administered IP. 24 hours after APAP treatment, mice were sacrificed with carbon dioxide. Dbhrl^~ mice were administered ISO as previously described (Mackintosh, C.A., et al. 2000). Mouse recombinant TWEAK (R&D systems) was administered IP at 0.04 μg/g body weight.

Cell isolation

Hepatocytes were isolated as previously reported (Schwabe, R.F., et al, 2001). Hepatic stellate cells, Kupffer cell and hepatic sinusoidal lining cell were extracted by optiprep gradient and subsequent selective adherence method as previously reported (Oben, J.A., et al, 2004; Li, Z., et al. 2002; and Williams, J.M., et al. 2010). Purity of HSC, KC, SEC was assessed by immunocytochemistry using GFAP, aSMA, F4/80 and vWF antibody and revealed 98%, 92%, 87% purity respectively. EpCAM+ cell were isolated by BD Magnet according to the manufacturer's instructions.

Assessment of liver injury

The degree of liver injury was assessed by histology and serum ALT. All liver sections were stained with haematoxylin and eosin (H&E) and scanned by NanoZoomer

(Hamamatsu, Japan). Necrotic area was measured and expressed as a percentage of necrotic tissue in whole area of liver section using NDP.view (Hamamatsu, Japan).

Cell culture experiments

603B cell were cultured as previously described (Omenetti, A., et al 2009). For proliferation assay, FBS was reduced to 1% and used as a basal state. LSEC were cultured on collagen coated plate and other liver cell fractions were cultured on normal dish using RPMI-1640 containing io%FBS. Primary hepatocytes were cultured on either collagen coated 96 well plate or 60mm dish using Williams E medium

supplemented with 10% FBS, insulin-trasferrin- selenium G cocktail and ιοοηΜ dexamethasone. After 4 hours plating, cells were washed and replaced with basic media containing the reagents and incubated for a further 2 hours. After 2 hour of reagents treatment, APAP containing media adjusted to lomM of final concentration was added. Concentration of the drugs we used in this experiment was decided on the basis of preliminary experiments (data not shown).

Proliferation assay

CCk-8 assay

Cell proliferation assay was performed using the Cell counting Kit-8 (CCK-8) according to the manufacturer's protocol.

Direct cell counts

To further confirm the CCK-8 assay we also directly counted the cell numbers in some experiments. Adherent cells were treated with 0.25% trypsin solution containing 0.02% EGTA in Ca²⁺ and Mg²⁺ free phosphate-buffered saline at 37 °C for 5 min, and the viable cell number and dead cell number was determined using Nucleocounter

(Chemometec).

Cytotoxic assay

LDH released from cells were assessed by LDH assay kit (Cayman) with 0.1% Triton X treated cells as positive controls.

Immunohistochemistry (IHC)

Formalin-fixed paraffin-embedded tissue were cut at 4μπι onto glass slides coated with poly-1 -lysine. For chromogenic IHC, antibody binding was visualized using the

ImmPRESS Peroxidase Polymer Detection Reagents (Vector lab, UK). For double chromogenic IHC, microwave heat treatment in citric based solution (Vector lab, UK) were applied after the first color development. For immunofluorescence IHC or immunocytochemistry, Alexa Fluor 555 and Alexa Fluor 488 conjugated secondary antibody were used. Nuclei were stained with DAPI (Vector). All images were captured using a Nikon Eclipse e6oo microscope and camera (DXM1200F) and acquired with NIS-Elements Advance software (Nikon). HPC numbers were counted by an expert liver pathologist unaware of the identity of the groups as previously described (Oben, J.A., et al. 2003).

PCR and semi-quantitative real time PCR

RNA was isolated using TRIzol (Invitrogen), according to the manufacturer's instructions. cDNA was synthesized with the Qiagen QuantiTect Reverse Transcription kit (Qiagen).

Duplicate PCR reactions were performed with multiplex PCR kit (Qiagen) using mixed primer (GAPDH and target primer). Semi-quantitative real time PCR was done with Rotor-Gene 3000 (Corbett Robotics) and QuantiFast SYBR Green PCR kit (Qiagen). All real-time PCR reactions were performed in triplicate with GAPDH as an internal control. Target gene levels in treated samples are presented as a ratio to levels detected in corresponding control samples, according to the ΔΔΟ: method.

Western blotting

Western blotting was performed as described (Soeda, J et al., 2012). Western blots shown are representative of 2 or 3 independent repeats. Semi-quantitative analysis of western blots by densitometry was carried out using Lab Works 4.6 software (UVP, USA).

Flow cytometry

Total NPC were extracted and analysed as previously described (Okabe, M et al. 2009;

Yovchev, M.I., et al. 2008; and Lin, K.K. and M.A. Goodell, 2011). Hoechst.3332 staining was performed as described (Lin, K.K. and M.A. Goodell, 2011; and Goodell,

M.A., et al., 1996) with minor modifications. Briefly, total NPC were adjusted to 10⁶ cells/ml in pre-warmed RPMI complete media (io%FBS, P/S, galutamate), incubated for 90 minutes at 37 degree with 5ug/ml of Hoechst with verapamil as (50UM) control.

Samples were then washed with ice-cold PBS and incubated with Fc blocker (Cdi6/32 mouse monoclonal antibody: BD) and stained with PE conjugated EpCAM (Biolgend) and Alexa -fluor 700 conjugated CD45. Data were analyzed by FlowJo software

(version and company).

Statistical analyses

All data were expressed as mean ± s.e.m. and means were compared by the Student's t- test or AN OVA as appropriate. Sample size per group, n = />3 per group. Example 1 - The SNS Regulates Hepatic Progenitor Cell (HPC) Expansion

To determine if the SNS regulates HPC expansion, it was first confirmed whether Dbh ^/ (Dopamine β-hydroxylase) mice, which are genetically deficient in the SNS

neurotransmitters norepinephrine (NE) and epinephrine, have a significantly attenuated HPC population compared to their heterozygote controls. HPC populations were enumerated by the immunohistochemical presence of CK-19. As shown in Figure la, treatment with isoprenaline (ISO), a non-specific β-adrenoceptor agonist, significantly recovered HPC numbers in Dbh ^/ mice.

To confirm the expression of adrenoceptors on HPCs, EpCAM+ve cells were isolated from the livers of control C57BL mice. Expression of EpCAM (epithelial cell adhesion molecule) has been shown to be a reliable marker of HPCs in mice (Schmelzer, E et al 2007; Tanaka, M et al. 2009; Okabe, M et al. 2009; Yovchev, M.I., et al 2007). These EpCAM+ve cells expressed other known HPC markers, for example CK19, Sox9,

TROP2, and Oct4, as shown in Figure lb. The EpCAM+ve cells also showed Hoechst 33342 extruding properties, i.e. they were side population (SP) cells, as shown in Figure lc. Moreover, these HPCs, expressed cab-, aic-, 02a-, 02b-, 02c-, plus βι- and β2- adrenergic receptor subtypes at the mRNA level, as show in Figure id.

The above results were corroborated by double immunofluorescent staining with pan- cytokeratin (another accepted HPC marker (Yin, L., et al 2002)) and βι and β2 adrenoceptor. This confirmed that βι and β2 adrenoceptors are expressed on HPCs at the protein level. Therefore, there is an association between the expansion of HPC populations and the SNS, mediated via adrenoceptor.

To further delineate the role of adrenergic stimulation in HPC proliferation, the 603B cell line was used. 603B cells, like HPCs, are derived from the terminal branches of the biliary tree (Ueno, Y et al 2003; Omenetti, A., et al 2007). As shown in Figure id, 603B cells possess the same adrenoceptor profile as isolated EpCAM+ve cells. This finding validated their further use in this study. Treatment with ISO induced 603B cell proliferation and their pre-treatment with the β-adrenoceptor antagonist, propranolol (PRL), inhibited ISO induced proliferation, as shown in Figure le. Surprisingly, ISO also increased the number of the HPC cells in normal C57BL/6J mice, as determined by expression of EpCAM (flow cytometry) and CK19 (immunohistochemistry), as shown in Figure if and Figure lg. Therefore, these findings suggested direct expansion of HPCs by ISO in murine liver.

Example 2 - ^-adrenoceptor stimulation activates the canonical Wnt pathway on HPCs

To elucidate the molecular pathway through which stimulation of β-adrenoceptors induces proliferation of HPCs, the canonical Wnt pathway was investigated. As shown in Figure 2a, total β-catenin expression was significantly increased in ISO treated 603B cells. Expression of dephophorelated β-catenin (activated β-catenin) and cyclin Di which is a known β-catenin target gene were also significantly upregulated in ISO treated 603B cells. Furthermore, immunofluorescence cytochemistry showed accumulation of β-catenin in the nuclei of ISO treated 603B cells, as shown in Figure 2b. This data suggests that ISO treatment activated the canonical Wnt pathway in 603B cells.

To further elucidate the molecular pathway through which stimulation of β- adrenoceptors induces proliferation of HPCs, the effect of β-catenin specific inhibitors were used in proliferation assays with 603B cells. As shown in Figure 2c, ISO-induced proliferation was partially but significantly inhibited by β-catenin specific inhibitors. This indicates that the effect of ISO on 603B proliferation is partly mediated by β- catenin. ISO treatment also significantly increased Wnti, 3a, 6 and 10a mRNA expression in 603B cells, as shown in Figure 2d. These Wnt ligands are known to activate the canonical Wnt pathway (Koch, S., et al 2011) and thus suggest that ISO- induced 603B proliferation is partly autocrine in nature.

To confirm the findings of Example 6 (above), the Wnt antagonist DKKi (Koch, S., et al 2011) was used in the presence of ISO. Figure 2e shows that there was a trend towards statistical difference between the proliferation of ISO-treated and ISO plus DKKi- treated 603B cells.

The effect of β-adrenoceptor stimulation on the canonical Wnt pathway was studied further in vivo. As shown in Figure 2f, mice treated with ISO showed upregulation of Wnt6 mRNA in total liver at 24.I1 after injection and strong β-catenin immunoreactivity on periportal ductular cell detected by immunohistochemistry. Double

immunoflurorescence confirmed these cells were HPCs (see Figure 8). These results indicate that ISO treatment also activates the canonical Wnt pathway on HPC in vivo. Example z - ISO protects against APAP-induced liver injury and enhances HPCs expansion

To determine whether above-mentioned findings have any relevance with respect to liver disease, an APAP induced liver injury model which results in massive hepatic necrosis and progenitor cell proliferation was used (Williams, CD. et al 2011; Kofman, A.V et al 2005).

Mice were initially administered APAP at 500mg/kg intraperitoneally. This resulted in a significant number of deaths, and was reduced by ISO treatment. Therefore, the dose of APAP was reduced to 375mg/kg. lh after administration with APAP, mice were treated with either ISO or PBS vehicle. As shown in Figures 3a and 3b, APAP treatment induced massive hepatic necrosis as judged by histology and ALT 24.I1 after APAP treatment. ISO treatment significantly reduce the ALT (3332±402.9 vs. 674.1±173 IU/L, p<o.oooi) and hepatic necrosis (35.25i3.745 vs. i8.48±i.935%, p<o.oooi). Figure 3C shows that a significant elevation in ALT was detected as early as 3h after APAP treatment, and this elevation in ALT was significantly attenuated by treatment with ISO. The number of HPCs in the livers of the various treatment groups was analyzed using flow cytometry and immunohistochemistry. As shown in Figure 3d and 3e, ISO treatment significantly increased the number of HPCs even though injury, as shown in Figure 3c, was far less compared to the APAP alone group. The density of HPC in the smallest portal tract, was also analyzed by CK19 positivity, as it is reported that in APAP induced liver injury models the density of CK19 positive cells in the smallest portal tract is a more precise quantification compared to the absolute number. Figure 3f shows that the HPC density was significantly increased in the APAP+ISO group compared to the APAP alone group. To clarify the significance of β-adrenoceptor signalling in this model, the β-adrenoceptor antagonist PRL was used. Figure 3g shows that PRL treatment markedly increased injury and resulted in reduced numbers of HPCs.

Example 4 - Expanding Hepatic progenitor cells are the main source of Wnt

The inventors then decided to determine how ISO treatment protects the liver from APAP induced injury. In order to do this, the canonical Wnt pathway was investigated. Canonical Wnt signalling is reported to be hepatoprotective against APAP induced liver injury in addition to its role in HPC proliferation. Mice were initially administered APAP at 500mg/kg intraperitoneally. This resulted in a significant number of deaths, and was reduced by ISO treatment. Therefore, the dose of APAP was reduced to 375mg/kg. lh after administration with APAP, mice were treated with either ISO or PBS vehicle. As shown in Figure 4a, Western blotting showed that β-catenin expression was significantly increased in the livers of ISO treated mice compared to those treated with APAP alone and controls at 24h after injection.

Immunohistochemistry using activated β-catenin antibody also showed strong β- catenin staining in the livers of APAP+ISO treated groups. Analysis of 16 known various Wnt ligands in the APAP and APAP+ISO treated livers showed that Wnt6, Wntioa, Wntu and Wnti6 were upregulated in the APAP alone and APAP+ISO groups, with Wnt6 showing significantly higher expression in the APAP+ISO group compared to APAP only group, see Figure 4c. At this time, significant HPCs expansion was also detected in the livers of APAP+ISO mice. Importantly, Wnt 6, 10a, 11 and 16 were significantly upregulated in APAP and APAP+ISO group at 2h after APAP

administration. Among these Wnt ligands, Wnt 10a showed significantly higher expression in the APAP+ISO group. Moreover, significant β-catenin activation in the APAP+ISO group was also detected at 3h after APAP administration. These data suggested that ISO treatment enhanced the canonical Wnt pathway.

To further define the cell types in the liver responsible for these ligand upregulation, hepatocytes, EpCAM positive cells, and EpCAM depleted non-parenchymal cells were isolated from the livers of mice treated with APAP+ISO. Wnt ligand expression in these fractions was then anlayzed. As shown in Figure 4d, among the Wnt ligands which was upregulated in vivo, the EpCAM positive cell fraction showed significant higher Wnt6, 10a, and 16 expression compared to the other fractions. In addition, the EpCAM positive fraction showed the highest expression of Wnti and Wnt3a. Among these upregulated Wnt ligands, Wnti, 3a, 6 and 10a are known to induce the canonical Wnt pathway.

To evaluate the possible influence of ISO induced Wnt upregulation on liver cells, Wnt expression in the various liver cell types in the presence and absence of ISO was anlayzed ex vivo. Wnt 6 expression was detected in isolated hepatic stellate cells (HSC) and Kupffer cells (KC). Culture activation significantly upregulated Wnt 6 expression in HSC compared to freshly isolated HSC. However, ISO did not induce upregulation of Wnt 6 in HSC or KC. These data suggest that the major source of Wnt is HPCs. The inventors also investigated several cytokines which can induce HPC proliferation but they could not detect any ISO specific significant elevation in these cytokines. These results suggested that ISO treatment increases HPC number as well as their expression of Wnt to subsequently activate the canonical Wnt pathway in hepatocytes and protect from APAP toxicity.

To further support this postulate, primary hepatocytes were extracted from mice livers and treated with APAP. This treatment significantly induced their death as judged by release of LDH. ISO pre-treatment did not protect the hepatocytes from APAP induced death at any dose investigated. However, recombinant Wnt3a pre-treatment significantly protected the hepatocytes against APAP. As shown in Figure 4e, the conditioned media from 603B stimulated with ISO significantly protected the hepatocytes, an effect reversed by recombinant DKKi. Western blotting showed that ISO did not increase β-catenin expression on hepatocytes but rWnt3a and ISO stimulated conditioned media induced increased β-catenin expression (data not shown). These results strongly suggested that ISO protects hepatocytes from APAP induced cell death not directly but through paracrine activation of the canonical Wnt pathway. Example - Hepatic progenitor cell expansion is hepatoprotective

The above Examples indicate that expanding HPCs are the source of Wnt and that these HPCs have a protective role in APAP induced liver injury. To test this hypothesis, Tumour associated weak inducer of apoptosis (TWEAK) was used together with direct HPC administration. TWEAK has been reported to specifically promote progenitor cell expansion in the liver with no effect on hepatocytes (Jakubowski, A., et al 2005). To take advantage of this property, recombinant TWEAK was administered before APAP treatment and expansion of the endogenous HPCs.

As shown in Figure 5a and 5b, TWEAK administration induced HPC proliferation without any evidence of hepatocyte cell death, as judged by ALT and active caspase-3 immunostaining. The effect of TWEAK is mediated by NF-kB signaling in HPCs (Tirnitz-Parker, J.E et al 2010) and NF-kB p65 immunostaining has revealed a strong cytoplasmic and nuclear expression of the protein, especially in periportal ductular cells, see Figure 5c. Furthermore, expansion of HPC by TWEAK administration protected from APAP induced liver injury, as shown in Figures 5d, 5e and 5f. As described in Figure 5g, pooled EpCAM positive cells were from the livers of mice treated with APAP+ISO 2h after APAP administration were then administered to mice which has been treated with APAP. EpCAM positive cells injection significantly ameliorated liver injury compared to vehicle and EpCAM depleted non-parenchymal cells. DKKi treatment reversed the effect of EpCAM positive cell administration, as shown in Figure 5h.

Example 6 - Delayed administration of isoprenaline (ISO

The effects of ISO in combination with the current gold-standard treatment, NAC, were also investigated. Figure 6A shows the experimental design of the study. As shown in Figure 6B, administration of I50mg/kg of NAC markedly reduces hepatocyte injury when administered l hour following overdose. However, NAC did not have a protective role if it was administered 3hrs post APAP. Conversely, ISO markedly reduced APAP induced-liver injury even at 3hrs post APAP.

Example 7 - Effect of the ai-adrenoceptor Phenylephrine agonist on APAP induced liver injury

To determine if the ^-adrenoceptor agonist, phenylephrine, induces effects similar to isoprenaline, the protocol of Example 16 (above) was repeated with ISO.

APAP was administered at 375mg/kg and either phenylepherine (PE) or PBS vehicle were given lh after APAP. As shown in Figure 7a, APAP alone induced substantial liver injury reflected by increased ALT (3500±750). This effect was moderately reduced by PE 3mg (2000±i20o) and significantly reduced by PE lomg (450±200, p<0.005).

To investigate the pathways through which PE protects from APAP induced liver injury, 603B cells were cultured in the presence and absence of PE with ISO. As shown in Figure 7B, it was found that PE, at ιορΜ to ιθμΜ, induced moderate but significant proliferation of 603B cells.

References

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Claims

Claims

l. An adrenergic receptor agonist, for use in treating, preventing or ameliorating liver damage.

2. An agonist according to claim l, wherein the liver damage which is treated is acute liver damage.

3. An agonist according to either claim 1 or claim 2, wherein the liver damage is caused by administration or consumption of a poison, for example paracetamol, alcohol, or Khat plant.

4. An agonist according to any preceding claim, wherein the agonist is an oc- adrenergic receptor agonist.

5. An agonist according to any preceding claim, wherein the agonist is either an or an ofc-adrenergic receptor agonist.

6. An agonist according to claim 5, wherein the oci-adrenergic receptor agonist is selected from a group consisting of: Noradrenaline, Xylometazoline, Phenylephrine, and Methoxamine.

7. An agonist according to either claim 5 or claim 6, wherein the oci-adrenergic receptor agonist is Phenylephrine.

8. An agonist according to claim 5, wherein the ^-adrenergic receptor agonist is selected from a group consisting of: Clonidine, Dexmedetomidine, Medetomidine, and Romifidine.

9. An agonist according to claim 1, wherein the agonist is a β-adrenergic receptor agonist.

10. An agonist according to claim 9, wherein the adrenergic receptor agonist is a βι-, a β₂- or a ₃-adrenergic receptor agonist.

11. An agonist according to claim 10, wherein the βι-adrenergic receptor agonist is selected from a group consisting of: Dobutamine, Isoprenaline, and Noradrenaline.

12. An adrenergic receptor agonist according to claim n, wherein the βι-adrenergic receptor agonist is Isoprenaline.

13. An agonist according to claim 10, wherein the ₂-adrenergic receptor agonist is selected from a group consisting of: Isoprenaline and Salbutamol.

14. An agonist according to any preceding claim, wherein the agonist is operable, in use, to enhance HPC expansion, preferably by activating the Wnt pathway.

15. An adrenergic receptor agonist, for use in inducing the expression of Wnt by hepatic progenitor cells.

16. An agonist according to claim 15, wherein expression of Wnt 1, 3a, 6 or 10a is induced by the agonist compared to the level of expression in the absence of the agonist.

17. A liver damage treatment composition, comprising an adrenergic receptor agonist and a pharmaceutically acceptable vehicle.

18. A composition according to claim 17, wherein the agonist is as defined in any one of claims 1-16.

19. A composition according to either claim 17 or claim 18, wherein the composition comprises liver-targeting means, arranged, in use, to target the adrenoceptor agonist at least adjacent the liver.

20. A process for making the composition according to any one of claims 17-19, the process comprising contacting a therapeutically effective amount of an adrenergic receptor agonist and a pharmaceutically acceptable vehicle.