EP3265086A1

EP3265086A1 - A lysosomal inhibitor and a multikinase inhibitor in the treatment of liver cancer

Info

Publication number: EP3265086A1
Application number: EP16707535.7A
Authority: EP
Inventors: Rajiv Jalan; Fausto ANDREOLA; Francesco DI CHIARA
Original assignee: UCL Business Ltd
Current assignee: UCL Business Ltd
Priority date: 2015-03-04
Filing date: 2016-02-25
Publication date: 2018-01-10
Also published as: GB201503668D0; WO2016139455A1

Abstract

The invention relates to the treatment of liver cancer, and provides novel pharmaceutical formulations and novel treatment regimes for this condition.

Description

A LYSOSOMAL INHIBITOR AND A MULTIKINASE INHIBITOR IN THE TREATMENT OF LIVER CANCER

The present invention relates to the treatment of liver cancer, and provides novel pharmaceutical formulations and novel treatment regimes for this condition. Hepatocellular carcinoma (HCC) is one of the most common solid malignancies with over a million new cases diagnosed annually worldwide and it accounts for 90% of all primary cancers that arise in the liver. In the absence of treatment, HCC is associated with a poor prognosis with less than 10% 5-year overall survival rate. HCC occurs in an advanced stage and most patients are not suitable to potentially curative treatments (e.g. hepatic resection or liver transplantation).

Sorafenib, the only approved palliative drug for HCC, is expensive, not well-tolerated, and has limited efficacy. Its use is limited to patients with well-compensated cirrhosis and at the doses administered for clinical effect, its toxicity prevents use in more advanced patients. In the doses used, it is cytostatic in vitro and in vivo. Therefore, if the drug dosage can be reduced, it can be administered to a larger group of patients. In view of the above, there is clearly a need to provide improved compositions and treatment regimes for the treatment of liver cancer. The inventors focused their attention on the multikinase inhibitor, Sorafenib, and have demonstrated a surprisingly synergistic effect with Chloroquine, a 4-aminoquinolin lysosomal inhibitor drug that is normally used in the treatment or prevention of malaria. Chloroquine, when used alone, almost completely prevents the development of HCC in vivo causing cellular ultrastructural damage. The inventor's in vitro studies show that if HCC cells, once rendered oncostatic by Chloroquine-sensitisation, are exposed even to sub-pharmacological doses of Sorafenib, they become oncolytic and are unable to use the key molecular pathways that are necessary to repair the cell damage and recover. This is clear evidence of synergy between Chloroquine and Sorafenib, and the inventors have shown that this synergistic effect can be harnessed as a novel approach for the treatment of liver cancer, in particular hepatocellular carcinoma (HCC).

Hence, in a first aspect of the invention, there is provided a lysosomal inhibitor and a multikinase inhibitor, for use in the treatment, prevention or amelioration of liver cancer in a subject, wherein the lysosomal inhibitor is administered to the subject for a first time interval sufficient to achieve sensitisation thereto, and wherein, following the first interval, the multikinase inhibitor thereof is administered to the subject for a second time interval.

In a second aspect, there is provided a method of treating, preventing or ameliorating liver cancer in a subject, the method comprising administering, to a subject in need of such treatment, a therapeutically effective amount of a lysosomal inhibitor for a first time interval sufficient to achieve sensitisation thereto, and then, following the first interval, administering a therapeutically effective amount of a multikinase inhibitor for a second time interval.

As described in the Examples and as shown in Figures 8-10, the inventor's have demonstrated that administration of the lysosomal inhibitor, Chloroquine, during the first time interval surprisingly sensitises the subject to the Chloroquine such that the subject's tumour cells become oncostatic. Then, during the second time interval, administration to the subject of sub-pharmacological doses of the multikinase inhibitor, Sorafenib, renders the subject's tumour cells oncolytic, because they cannot use the key molecular pathways required to repair the cell damage caused by

Chloroquine-sensitisation. The synergistic effect between the lysosomal inhibitor, Chloroquine, and the multikinase inhibitor, Sorafenib, could not have been predicted, and there is nothing in the prior art to suggest that pre-treatment with Chloroquine would have been beneficial.

Advantageously, as Chloroquine and Sorafenib are already in clinical practice, the barrier to performing a clinical trial is minimal. The invention allows the treatment of patients with advanced liver disease by using sub-therapeutic doses of Sorafenib for which there is no known treatment at present. It results in the significant reduction of the cost of Sorafenib to ι/ιο^Λ the price by reducing the dose required. As a result, the dosage of the multikinase inhibitor can be significantly reduced, and so it can be administered to a larger group of patients.

The invention may be used for treatment of any liver or hepatic cancer, and preferably advanced liver cancer. Preferably, the invention is used for the treatment of hepatocellular carcinoma (HCC). Advantageously, and preferably, the invention described herein maybe used as adjuvant therapy in patients undergoing locoregional therapies, such as transarterial embolisation. Furthermore, preferably the invention described herein maybe used to treat patients with HCC who are not suitable for resection or transplantation with a Pugh score >7 who are not suitable for hepatic resection.

The term "sensitisation" in pharmacological terms refers to a situation when exposure to one drug can render an individual hypersensitive to another drug. In the present case, therefore, exposure to the lysosomal inhibitor renders the subject hypersensitive to the multikinase inhibitor. This can also be referred to as "cross-sensitisation". As the rational to use Chloroquine as a 'priming' or sensitizing drug is to cause an impairment of the endo/lysosmal compartment leading to a transient cytostatic effect on cancer cells, the inventors believe that other lysosomal inhibitors may also be used to achieve similar effects.

Preferably, the lysosomal inhibitor is selected from a group consisting of: Leupeptin (a nontoxic thiol protease inhibitor, proposed to have therapeutic use in hereditary muscular dystrophies); Bafilomycin A (an inhibitor of lysosomal v-ATPase); Rapamycin (a lipophilic macrolide antibiotic inhibitor of mTOR); and Chloroquine.

The preferred lysosomal inhibitor is Chloroquine. The structure of Chloroquine is shown in Figure 12.

Preferably, the lysosomal inhibitor is administered to the subject during the first time interval in the absence of the multikinase inhibitor.

Preferably, the first time interval is at least 12 hours, more preferably at least 24 hours, even more preferably at least 36 hours, and most preferably at least 48 hours.

Preferably, the first time interval is at least one week, more preferably at least two weeks, even more preferably at least three weeks, and most preferably at least four weeks. Preferably, the first time interval is less than ten weeks, more preferably less than eight weeks, and most preferably less than six weeks. Accordingly, it is preferred that the first time interval is between 12 hours and ten weeks, more preferably between one week and eight weeks, and most preferably between four weeks and six weeks. It will be appreciated that any of the timings described herein can be combined in any combination. The dosage of the lysosomal inhibitor administered during the first time interval may be between 50 and 500mg lysosomal inhibitor once per day, preferably between 75 and 300mg lysosomal inhibitor once per day, more preferably between 100 and 200mg lysosomal inhibitor once per day, and most preferably between 125 and 175 mg lysosomal inhibitor once per day. Most preferably, the first time interval is a period of four to six weeks. Most preferably, the lysosomal inhibitor is Chloroquine.

Multikinase inhibitors work by inhibiting multiple intracellular and cell surface kinases, some of which are implicated in tumour growth and metastatic progression of cancer, thereby decreasing tumour growth and replication. Examples of suitable multikinase inhibitors include Foretinib (an oral multikinase inhibitor targeting MET, RON, AXL, TIE-2, and VEGFR) and Sorafenib.

Preferably, the multikinase inhibitor is Sorafenib, the structure of which is shown in Figure 13. In one preferred embodiment, the multikinase inhibitor is administered to the subject during the second time interval in the absence of the lysosomal inhibitor.

In another preferred embodiment, the multikinase inhibitor is administered to the subject during the second time interval in the presence of the lysosomal inhibitor. Advantageously, by continuing to administer the lysosomal inhibitor during the second time interval, the tumour cells continue to remain sensitised thereto.

Preferably, the second time interval is at least 12 hours, more preferably at least 24 hours, even more preferably at least 36 hours, and most preferably at least 48 hours. Preferably, the second time interval is at least one week, more preferably at least two weeks, even more preferably at least three weeks, and most preferably at least four weeks. It is preferred however that the multikinase inhibitor (either alone or in combination with the lysosomal inhibitor) is administered to the subject in the second time interval for as long as is necessary in order to kill the tumour, at which point administration of the multikinase inhibitor can stop. In the event that the tumour reestablishes itself, the treatment regime described maybe repeated, in which the lysosomal inhibitor maybe again administered to the subject for a first time interval sufficient to achieve sensitisation thereto, and then, following the first interval, the multikinase inhibitor may be again administered to the subject for a second time interval. This process may be repeated in a pulsed therapy technique until the tumour is completed removed. It may be preferred to subject the subject to a recovery interval period in between each repeating cycle of treatment with the lysosomal inhibitor followed by the multikinase inhibitor.

The dosage of multikinase inhibitor administered during the second time interval may be between 5 and 400 mg multikinase inhibitor once or twice daily, preferably between 10 and 300mg lysosomal inhibitor once or twice per day, more preferably between 20 and 200mg lysosomal inhibitor once or twice per day, and most preferably between 30 and 100 mg lysosomal inhibitor once or twice per day. Most preferably, the above doses of multikinase inhibitor are administered twice daily. Most preferably, about 4omg twice daily is administered. Preferably, the multikinase inhibitor is Sorafenib.

In an embodiment in which the lysosomal inhibitor continues to be administered during the second time interval, the dosage may be about 100 to 200mg daily. The inventors have found that the synergistic effect of the combination of a lysosomal inhibitor (preferably, Chloroquine) and a multikinase inhibitor (preferably, Sorafenib) means that much lower dosages of the multikinase inhibitor are needed to treat the subject. As such, they have produced a novel composition comprising Chloroquine and Sorafenib which can formulated into a pharmaceutical composition.

Thus, in a third aspect of the invention, there is provided a pharmaceutical composition comprising Chloroquine and Sorafenib, and a pharmaceutically acceptable vehicle.

Preferably, the Chloroquine is present as Chloroquine base. Preferably, the composition comprises more than lomg Chloroquine base, more preferably more than 50 mg

Chloroquine base, even more preferably more than 100 mg Chloroquine base, and still more preferably more than i2omg Chloroquine base.

Preferably, the composition comprises less than 450mg Chloroquine base, more preferably less than 400 mg Chloroquine base, even more preferably less than 300 mg Chloroquine base, and still more preferably less than 200 mg Chloroquine base.

Preferably, the composition comprises between 10 and 450 mg Chloroquine base, more preferably between soand 400 mg Chloroquine base, even more preferably between 100 and 300 mg Chloroquine base, and still more preferably between 120 and 200 mg Chloroquine base. Most preferably, the composition comprises about 150 mg

Chloroquine base.

Preferably, the composition comprises more than i.6mg Sorafenib, more preferably more than 8mg Sorafenib, even more preferably more than i6mg Sorafenib, and still more preferably more than 24mg Sorafenib, and most preferably more than 32mg Sorafenib.

Preferably, the composition comprises less than i8omg Sorafenib, more preferably less than i4omg Sorafenib, even more preferably less than loomg Sorafenib, and still more preferably less than 8omg Sorafenib.

Preferably, the composition comprises between 1.6 and i8omg Sorafenib, more preferably between 16 and i4omg Sorafenib, even more preferably between 24 and 96mg Sorafenib, and still more preferably between 32 and loomg Sorafenib. Most preferably, the composition comprises about 4omg Sorafenib.

In a fourth aspect, there is provided the pharmaceutical composition according to the third aspect, for use as a medicament.

In a fifth aspect, there is provided the pharmaceutical composition according to the third aspect, for use in the treatment, prevention of amelioration of liver cancer.

In a sixth aspect, there is provided a method of treating, preventing or ameliorating liver cancer in a subject, the method comprising administering, to a subject in need of such treatment, a therapeutically effective amount of the pharmaceutical composition according to the third aspect.

It will be appreciated that the lysosomal inhibitor and multikinase inhibitor (i.e. both of which are referred to hereinafter as "agents") may be used in a medicament which may be used in a monotherapy, or as an adjunct to, or in combination with, known therapies for treating, ameliorating, or preventing liver cancer. The agents according to the invention may be 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 etc. 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.

Medicaments comprising the agents according to the invention (i.e. the complex or aggregate) may be used in a number of ways. For instance, oral administration may be required, in which case the agents may be contained within a composition that may, for example, be ingested orally in the form of a tablet, capsule or liquid. Compositions comprising agents of the invention may be administered by inhalation (e.g. intranasally). Compositions may also be formulated for topical use. For instance, creams or ointments may be applied to the skin.

Agents 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 may be located at least adjacent the treatment site. Such devices may be

particularly advantageous when long-term treatment with agents used according to the invention is required and which would normally require frequent

administration (e.g. at least daily injection). In a preferred embodiment, agents 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 agent 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 agent, 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 agent within the subject being treated. Optimal dosages to be administered may be determined by those skilled in the art, and will vary with the particular agent in use, the strength of the pharmaceutical composition, the mode of administration, and the advancement of the disease. 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 500mg/kg of body weight of the agent according to the invention may be used. More preferably, the daily dose is between o.oimg/kg of body weight and 400mg/kg of body weight, and more preferably between o.img/kg and 200mg/kg body weight.

As discussed in the Examples, the composition maybe administered before, during the or after the onset of liver disease. For example, the lysosomal inhibitor and multikinase inhibitor may be administered immediately after a subject has developed a disease. Daily doses maybe given systemically as a single administration (e.g. a single daily injection). Alternatively, the agents may require administration twice or more times during a day. As an example, the agents 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 agents acccording to the invention to a patient without the need to administer repeated doses.

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 Chloroquine and Sorafenib according to the invention and precise therapeutic regimes.

The invention also provides, in a seventh aspect, a process for making the

pharmaceutical composition according to the third aspect, the process comprising contacting a therapeutically effective amount of Chloroquine and Sorafenib, and a pharmaceutically acceptable vehicle.

A "subject" maybe a vertebrate, mammal, or domestic animal. Hence, agents, 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 agent is any amount which, when

administered to a subject, is the amount of drug that is needed to treat the target disease, or produce the desired effect, e.g. result in liver tumor killing. For example, the therapeutically effective amount of agent used may be from about o.oi mg to about 8oo mg, and preferably from about o.oi mg to about 500 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 may be 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 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 may be 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 agents 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 agents may be prepared as a sterile solid composition that may be dissolved or suspended at the time of

administration using sterile water, saline, or other appropriate sterile injectable medium.

The agents 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 agents 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 is a graph showing the time (24-96 hours)- and dose (0.1, 1, 15, 30, 60, 120 and 24θμΜ, respectively)-dependent response of HCC cell lines (Huh7 and HepG2) to Chloroquine in order to determine the IC50. Figure lB shows the effect of single or multiple doses (ΐ5μΜ) of Chloroquine on HCC cell lines for up to 96 hours;

Figure 2A are electron microscopy images of HCC cells (Huh7) treated with

Chloroquine for 24hrs (2Ab) and following 96h of recovery (2Ac). Figure 2B shows uptake of neutral red and staining of endo/lysosomal compartment in HCC cells treated with Chloroquine for 24hrs (2Bb) and following 96h of recovery (2Bc).Figure 2C shows graphs showing the speed of cell recovery following exposure to Chloroquine for Huh7 cells (left) and HepG2 (right);

Figure 3A are electron microscopy images of HCC cells (Huh7) treated with

Chloroquine for 48hrs (3Ab) and following 96h of recovery (3Ac). Figure 2B shows uptake of neutral red and staining of endo/lysosomal compartment in HCC cells treated with Chloroquine for 48hrs (3Bb) and following 96h of recovery (3Bc). Figure 3C shows graphs showing the speed of cell recovery following exposure to Chloroquine for Huh7 cells (left) and HepG2 (right);

Figure 4A are electron microscopy images of HCC cells (Huh7) treated with

Chloroquine for 72hrs (4Ab) and following 96h of recovery (4Ac). Figure 2B shows uptake of neutral red and staining of endo/lysosomal compartment in HCC cells treated with Chloroquine for 72hrs (4Bb) and following 96h of recovery (4Bc). Figure 4C shows graphs showing the speed of cell recovery following exposure to Chloroquine for Huh7 cells (left) and HepG2 (right); Figures 5A and 5B show when time-dependent CQ-damage occurred during the cell cycle;

Figure 6A is a graph showing time (24-96 hours)- and dose (0.1, 1, 2.5, 5, 10, 20, 40 and 5θμΜ, respectively)-dependent responses of HCC cell lines (Huh7 and HepG2) to Sorafenib in order to determine the IC50. Figure 6B shows the effect of single or multiple doses (3μΜ) of Sorafenib on HCC cell lines for up to 96 hours;

Figure 7A shows the effect of Sorafenib, when used alone at 3μΜ, causing a transient cytostatic effect on HCC cells (Huh7, HepG2) following 24 hours treatment. Figure 7B shows the effect of Sorafenib causing a transient cytostatic effect on HCC cells (Huh7, HepG2) following 48 hours treatment. Figure 7C shows the effect of Sorafenib causing a transient cytostatic effect on HCC cells (Huh7, HepG2) following 72 hours treatment. Figure 7D are electron microscope images of HepG2 cells following 72 hours treatment with Sorafenib, and following g6 of recovery; Figure 8A shows the effect of Sorafenib for 24.I1 on HCC cells (Huh7, HepG2) that have been made cytostatic by 72 h chloroquine-sensitisation, and also following 96 hours of recovery time. Figure 8B shows corresponding electron microscope images of the treated HepG2 cells; Figure 9A shows the effect of Sorafenib for 48h on HCC cells (Huh7, HepG2) that have been made cytostatic by 72 h chloroquine-sensitisation, and also following 96 hours of recovery time. Figure 9B shows corresponding electron microscope images of the treated HepG2 cells; Figure 10A shows the effect of Sorafenib for 72h on HCC cells (Huh7, HepG2) that have been made cytostatic by 72 h chloroquine-sensitisation, and also following 96 hours of recovery time. Figure 10B shows corresponding electron microscope images of the treated HepG2 cells; Figure 11 shows a time-dependent increase in cytoplasmic release of low-molecular weight nucleosomal DNA, as a marker of apoptosis (peaking to 20-fold increase in cells exposed to the proposed treatment regimen);

Figure 12 shows the structure of Chloroquine; and

Figure 13 shows the structure of Sorafenib. Materials and Methods Cell culture

HCC lines (HuH7 and HepG2) were maintained in MEM medium supplemented with 10% foetal bovine serum, lX L-glutamine, lX Non-Essential Amino acids, 100 U/ml penicillin, and 100 μg/ml Streptomycin in a 5% C0₂ incubator at 37°C. Chloroquine and Sorafenib treatments, cell viability assay and determination of ICy> For dose- and time-dependent cell response to Chloroquine (Invivogen) and Sorafenib (Novaxar™), HCC cell lines were seeded (2xio⁴cells/cm²) on 96-well plates and allowed to grow overnight prior to incubation for o to 96 hours in the presence of θ.ι-24θμΜ Chloroquine, or ο.ι-5θμΜ Sorafenib, respectively. Cell viability was measured using the CellTiter 96Aqueous™ Non-Radioactive Cell Proliferation Assay (Promega, UK) according to the manufacturer's protocol. IC₅₀ values were calculated using GraphPad Prism® software.

In combination treatment experiments (Chloroquine + Sorafenib), cells were treated with ΐ5μΜ Chloroquine for the indicated times. After CQ treatment, media was replaced daily with fresh media containing 2.5μΜ Sorafenib and cells returned to the incubator for the indicated times. In the recovery experiments, after treatments, media was replaced with fresh media and cells allowed to growth for up to further 96 hours.

Transmission electron microscopy

After treatment, cells were fixed in 1.5% glutaraldehyde, 2% paraformaldehyde in 0.1M Cacodylate buffer (PH7.4), post-fixed in 1% osmium tetroxide for 1 hour, embedded in Spurr's resin, sectioned, double stained with uranyl acetate and lead citrate, and analysed by using a JEM 1010 - transmission electron microscope (Jeol -UK). Neutral Red Uptake Assay

Treated HCC cells were assayed for neutral red uptake (a supravital dye incorporated and bound in the acid compartment/lysosomes of viable cells) according to Repetto et al (Nature Protocols, 2008; 3(7):ii25-3i). Briefly, after treatment, cells were incubated for 2 h with a medium containing Neutral Red (Sigma), washed in iX PBS and differences in Neutral Red staining/ distribution were examined under a phase-contrast inverted microscope.

Cell Cycle Analysis

To prepare cells for cell cycle analysis, cells were fixed in 70% ethanol for 30 min on ice and washed twice with lXPBS. DNA was stained with propidium iodide

(Molecular Probes) containing RNaseA for 30 min at room temperature. Flow cytometry was performed on a LSR Fortessa B cytometer (BD Biosciences) using FACSDiva software (Becton Dickinson). Cell cycle analysis was performed using ModFit LT™ software. Detection of apoptosis

Cell Death Detection ELISA^PLUS (Roche) was used to assess apoptotic cell death according to manufacturer's protocol. Examples

Example ι - Response of HCC cell lines to Chloroquine fCO) as single treatment:

Determination of IC o and modality of treatment

Time (24-96 hours)- and dose (0.1, 1, 15, 30, 60, 120 and 24θμΜ, respectively)- dependent response of HCC cell lines (Huh7 and HepG2) to Chloroquine (structure shown in Figure 12) was performed to determine the IC50. As shown in Figure lA, the IC50 at 96 hours was ΐ5μΜ.

To determine the best modality of treatment, cells were treated for up to 96h with CQ (ΐ5μΜ) either as single dose or repeated dose (daily replenishment). Figure lB shows that CQ has a cytostatic effect on HCC cells irrespective of the modality of treatment.

Example 2 - Cytostatic response of HCC cell lines to Chloroquine (CQ) is associated with a reversible and time-dependent disorganization of the intracellular compartment resulting in cell cycle arrest (G2/M phase)

As chloroquine prevents the acidification and maturation of endosomes, the inventors reasoned that the observed cytostatic effect of CQ treatment (15 μΜ) could be due a progressive impairment of cytoplasmic compartmentalization. Ultrastructural analyses of CQ-treated cells showed indeed a time-dependent cytoplasmic disorganization mostly due to damage to the endo-lysomal compartment (see Figures 2Ab, 3Ab, 4Ab, electron microscopy data of HCC cells treated with CQ for 24, 48 and 72h, respectively). Neutral red uptake experiments (a dye retained by the lysosomes) confirmed CQ effect on deregulation of the lysosomal compartment and its restoration after recovery (see Figures 2B, 3B, 4B).

As both mitochondria and nuclei preserved their structure and no sign of apoptosis was observed even after 72 hours of CQ-treatment, the inventors reasoned that the cells could overcome the CQ-induced damage. Recovery experiments showed that when CQ treatment was interrupted, HCC cells regained the ability to grow (Figures 2C, 3C, 4C) by fully overcoming the cellular ultrastructural damage (Figures 2Ac, 3AC, 4Ac) associated with normalization of the endo-lysomal compartment (Figures 2Bc, 3Bc, 4BC, neutral red uptake after g6h recovery).

The time-dependent CQ-damage was also associated with a progressive arrest of HCC cells in G2/M phase (see Figure 5A). The cell cycle was restored to control levels after 96h from withdrawal of CQ-treatment (see Figure 5B).

Example 3 - Response of HCC cell lines to Sorafenib (Sb) as single treatment:

Determination of IC50 and modality of treatment

Time (24-96 hours)- and dose (0.1, 1, 2.5, 5, 10, 20, 40 and 5θμΜ, respectively)- dependent response of HCC cell lines (Huh7 and HepG2) to Sorafenib (structure shown in Figure 13) was performed to determine the IC50. As shown in Figure 6A, the IC50 at 96 hours was 3μΜ, which represents the minimal concentration to achieve a cytostatic effect (comparable to a sub-pharmacological concentration, with pharmacological in vitro use ranging 20-ιθθμΜ).

To determine the best modality of treatment, cells were treated for up to 96h with Sb (3μΜ) either as single dose or repeated dose (daily replenishment). As expected for multikinase inhibitors, Sb has a cytostatic effect on HCC cells but required, to sustain its effect, a daily replenishment (see Figure 6B).

Example 4 - Removal of Sorafenib after short-term treatment with minimal dose results in ability of HCC cells to restore growth

Sorafenib, when used alone at 3μΜ for up to 72 hours, caused a transient cytostatic effect on HCC cells (Huh7, HepG2) (Figure 7A, B, C; 24, 48, 72 hours treatment, respectively); when Sorafenib was removed, only short-term treated cells were able to overcome the cytostatic effect over a period of 96h. No ultrastructural changes were observed (see Figure 7D). Example 5 - HCC cells, made cytostatic by CO-sensitisation. and exposed to minimal dose of Sorafenib become cytophatic

HCC cells, once made cytostatic by 72 h CQ-sensitisation, were then exposed to sub- pharmacological daily doses of Sorafenib for up to 72h. When using this treatment schedule, the cells became progressively cytophatic and unable to recover growth (Figure 8A, 9A, 10A) and ultrastructural cell damage (Figure 8B, 9B, 10B). Importantly, cells exposed to longer sorafenib-treatment showed irreversible cellular damage with presence of autophagic bodies (see Figure 9B) and apoptosis (see Figure 10B), respectively. This was further confirmed by using a cell death detection ELISA; Figure 11 shows a time-dependent increase in cytoplasmic release of low- molecular weight nucleosomal DNA, as a marker of apoptosis (peaking to 20-fold increase in cells exposed to the proposed treatment regimen).

Summary

The inventors have demonstrated that if HCC cells, once rendered cytostatic by chloroquine-sensitisation (e.g. ΐ5μΜ for 24-72 hours), are exposed to sub- pharmacological doses of Sorafenib (e.g. 3μΜ for 24-72 hours), they become cytophatic, and are unable to use the key molecular pathways that are necessary to repair the cell damage and recover. This is clear evidence of synergy between these two compounds, and the inventors have shown that this synergistic effect between chloroquine and sorafenib can be harnessed as a novel approach for the treatment of hepatocellular carcinoma (HCC).

Claims

1. A lysosomal inhibitor and a multikinase inhibitor, for use in the treatment, prevention or amelioration of liver cancer in a subject, wherein the lysosomal inhibitor is administered to the subject for a first time interval sufficient to achieve sensitisation thereto, and wherein, following the first interval, the multikinase inhibitor thereof is administered to the subject for a second time interval.

2. A lysosomal inhibitor and a multikinase inhibitor, for use according to claim l, wherein advanced liver cancer is treated.

3. A lysosomal inhibitor and a multikinase inhibitor, for use according to either claim 1 or claim 2, wherein hepatocellular carcinoma (HCC) is treated.

4. A lysosomal inhibitor and a multikinase inhibitor, for use according to any preceding claim, wherein the lysosomal inhibitor is selected from a group consisting of: Leupeptin; Bafilomycin A; Rapamycin; and Chloroquine.

5. A lysosomal inhibitor and a multikinase inhibitor, for use according to any preceding claim, wherein the lysosomal inhibitor is Chloroquine.

6. A lysosomal inhibitor and a multikinase inhibitor, for use according to any preceding claim, wherein the lysosomal inhibitor is administered to the subject during the first time interval in the absence of the multikinase inhibitor.

7. A lysosomal inhibitor and a multikinase inhibitor, for use according to any preceding claim, wherein the first time interval is at least 12 hours, or at least 24 hours, or at least 36 hours, or at least 48 hours.

8. A lysosomal inhibitor and a multikinase inhibitor, for use according to any preceding claim, wherein the first time interval is at least one week, or at least two weeks, or at least three weeks, or at least four weeks.

9. A lysosomal inhibitor and a multikinase inhibitor, for use according to any preceding claim, wherein the first time interval is between 12 hours and ten weeks, or between one week and eight weeks, or between four weeks and six weeks.

10. A lysosomal inhibitor and a multikinase inhibitor, for use according to any preceding claim, wherein the dosage of the lysosomal inhibitor administered during the first time interval is between 50 and 500mg lysosomal inhibitor once per day, or between 75 and 300mg lysosomal inhibitor once per day, or between 100 and 200mg lysosomal inhibitor once per day, or between 125 and 175 mg lysosomal inhibitor once per day.

11. A lysosomal inhibitor and a multikinase inhibitor, for use according to any preceding claim, wherein the multikinase inhibitor is Foretinib or Sorafenib.

12. A lysosomal inhibitor and a multikinase inhibitor, for use according to any preceding claim, wherein the multikinase inhibitor is Sorafenib.

13. A lysosomal inhibitor and a multikinase inhibitor, for use according to any preceding claim, wherein the multikinase inhibitor is administered to the subject during the second time interval in the absence of the lysosomal inhibitor.

14. A lysosomal inhibitor and a multikinase inhibitor, for use according to any one of claims 1-12, wherein the multikinase inhibitor is administered to the subject during the second time interval in the presence of the lysosomal inhibitor.

15. A lysosomal inhibitor and a multikinase inhibitor, for use according to any preceding claim, wherein the second time interval is at least 12 hours, or at least 24 hours, or at least 36 hours, or at least 48 hours.

16. A lysosomal inhibitor and a multikinase inhibitor, for use according to any preceding claim, wherein the second time interval is at least one week, or at least two weeks, or at least three weeks, or at least four weeks.

17. A lysosomal inhibitor and a multikinase inhibitor, for use according to any preceding claim, wherein the dosage of multikinase inhibitor administered during the second time interval is between 5 and 400 mg multikinase inhibitor once or twice daily, or between 10 and 300mg lysosomal inhibitor once or twice per day, or between 20 and 200mg lysosomal inhibitor once or twice per day, or between 30 and 100 mg lysosomal inhibitor once or twice per day.

18. A lysosomal inhibitor and a multikinase inhibitor, for use according to any preceding claim, wherein the multikinase inhibitor is administered twice daily.

19. A lysosomal inhibitor and a multikinase inhibitor, for use according to any preceding claim, wherein when the lysosomal inhibitor continues to be administered during the second time interval, the dosage is about 100 to 200mg daily.

20. A pharmaceutical composition comprising Chloroquine and Sorafenib, and a pharmaceutically acceptable vehicle.

21. A pharmaceutical composition according to claim 20, wherein the Chloroquine is present as Chloroquine base.

22. A pharmaceutical composition according to either claim 20 or claim 21, wherein the composition comprises between 10 and 450 mg Chloroquine base, or between 50 and 400 mg Chloroquine base, or between 100 and 300 mg Chloroquine base, or between 120 and 200 mg Chloroquine base.

23. A pharmaceutical composition according to any one of claims 20-22, wherein the composition comprises between 1.6 and i8omg Sorafenib, or between 16 and i4omg Sorafenib, or between 24 and 96mg Sorafenib, or between 32 and loomg Sorafenib.

24. The pharmaceutical composition according to any one of claims 20-23, for use as a medicament.

25. The pharmaceutical composition according to any one of claims 20-23, for use in the treatment, prevention of amelioration of liver cancer.

26. A process for making the pharmaceutical composition according to any one of claims 20-23, the process comprising contacting a therapeutically effective amount of Chloroquine and Sorafenib, and a pharmaceutically acceptable vehicle.