WO2022175670A1 - Lovastatin for use in the treatment of neuroblastoma - Google Patents

Abstract

The present invention relates to compositions comprising lovastatin, or a pharmaceutically acceptable salt thereof, for use in the treatment of neuroblastoma.

Description

TREATMENT Field of the invention This invention relates to new uses of lovastatin. Background of the invention Neuroblastoma is a childhood cancer originating from the neuroblasts of the sympathetic nervous system. It is the most common solid tumour in children after brain tumours. The incidence of neuroblastoma is about 10.54 cases per 1 million per year in children younger than 15 years old.90% of neuroblastoma cases are diagnosed by age 5. The genetic causation of neuroblastoma, like most cancers, is complex and heterogeneous. A common event in patients with high risk neuroblastoma and associated with poor prognosis is the amplification of the MYCN gene. MYCN is a transcription factor expressed during development that plays an important role in brain and neural development. Other genetic aberrations associated with neuroblastoma include: ALK activation, TERT activation, deletion at 1p36 or 11q14-23, 17q gain and changes in expression or mutations of KIF1B, PHOX2B, LIN28B, TRKA, TRKB and RAS in relapsed tumours. The most common primary tumour site is adrenal (47%), followed by the paraspinal sympathetic ganglia in the abdomen/retroperitoneum (24%), thorax (15%), pelvis (3%) and neck (3%). Neuroblastoma usually occurs sporadically, but familial cases with a prevalence of about 1-2% are also reported. Based on a number of factors including age at diagnosis, histopathological classification and the genetic profile of the tumour, patients can be classified into four groups: extremely low risk, low risk, moderate risk and high risk. Patients that are in the low or moderate risk groups can achieve an overall survival rate >95% with the current treatment paradigm. However, patients in the high risk group have a survival rate of around 40-50%. At present there is no effective therapy to treat neuroblastoma patients who are in the high risk category. The current treatment path for patients that have high risk tumours is intensive and multimodal; they receive high-dose chemotherapy, radiotherapy, surgery, myeloablative therapy and stem cell transplant, isotretinoin differentiation therapy and immunotherapy (anti-GD2, IL-1/GM-CSF). Despite multiple therapeutic options for these patients the survival rate is still 40-50%, meaning there is a high unmet need for new therapeutics for patients in this risk category, that can prolong life with reduced toxicity. Lovastatin is a member of the statin class of drugs, it is a fermentation product of Aspergillus terreus. Lovastatin is a lipid lowering drug that acts via inhibiting HMG- CoA reductase, an early and rate limiting step of cholesterol biosynthesis. It is approved for treatment of hyperlipidemias, atherosclerosis, coronary disease and hypercholesterolemia. Lovastatin is formulated as a prodrug in the form of an inactive but cell permeant beta lactone. In vivo and in cells the beta lactone is converted to the active beta hydroxy acid form which is less able to pass through the cell membrane. Lovastatin has the following name: (2S)- (1S,3R,7S,8S,8aR)-1,2,3,7,8,8a-hexahydro- 3,7-dimethyl-8-[2-[(2R,4R)-tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl]ethyl]-1- naphthalenyl ester, 2-methylbutanoic acid. Summary of the invention The present invention provides a new therapy for neuroblastoma. As will be evident from the in vitro data presented below, lovastatin is effective in treating neuroblastoma. Accordingly, a first aspect of the invention is a composition comprising lovastatin, or a pharmaceutically acceptable salt thereof, for use in the treatment of neuroblastoma. A second aspect of the invention is a kit comprising (i) at least one dose of lovastatin, or a pharmaceutically acceptable salt thereof; and optionally (ii) at least one dose of trifluoperazine or prochlorperazine, or pharmaceutically acceptable salts thereof, for simultaneous, separate or sequential use in the treatment of neuroblastoma. A third aspect of the invention is a use of a composition comprising lovastatin, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for use in the treatment of neuroblastoma. A fourth aspect of the invention provides a method of treating neuroblastoma comprising administering to a patient a composition comprising lovastatin, or a pharmaceutically acceptable salt thereof. Description of the figures Figures 1A-F show the results from the lovastatin monotherapy in in vitro testing. Figures 2A-F show the results from the trifluoperazine monotherapy in in vitro testing. Figures 3A-F show the results from the prochlorperazine monotherapy in in vitro testing. Figures 4A-C show the cell death and viability results from lovastatin and trifluoperazine in in vitro combination testing. Figures 5A-C show the cell death and viability results from lovastatin and prochlorperazine in vitro combination testing. Detailed description Xenografts based on conventional cancer cell lines have been used for decades and while this model system can provide valuable data, cultured cell lines that have adapted to the in vitro microenvironment often differ from the original tumour found in patients. Patient-derived xenografts (PDXs) are generated from the subcutaneous or orthotopic implantation of intact patient tumor fragments directly into immunodeficient mice or rats, thereby avoiding in vitro adaptation. Importantly, PDXs have been found to recapitulate the histopathological hallmarks, genetic pathways and mutational patterns of the corresponding patient tumors. The in vitro data herein has been generated using the PDX1, PDX2, and PDX3 cell models, as described in Braekeveldt et al, Int. J. Cancer, 2015, E252-E261. In the present invention, and as demonstrated by the in vitro data herein, lovastatin inhibits cell viability and increases cell death in neuroblastoma patient- derived xenograft tumours, and is therefore effective at treating and neuroblastoma. By the term “treatment” or “treating” as used herein, we refer to therapeutic (curative) treatment, which includes reducing the size of a neuroblastoma tumour or stopping a neuroblastoma tumour increasing in size. “Patient” and “subject” are used interchangeably and refer to the subject that is to be administered the composition comprising lovastatin. Preferably the subject is a human. In one embodiment the patient is a paediatric patient, preferably a paediatric patient 15 years of age and younger, more preferably 10 years of age or younger, most preferably 5 years of age and younger. The methods of classification of neuroblastoma tumours are complex and are based on a combination of different factors. Described below is a method of classifying neuroblastoma tumours according to the INRGSS system. The characteristics described below for each stage are exemplary only. There are other established systems for classifying the tumours, such as the INSS system, among others. The medical practitioner is skilled in diagnosing and would be able to determine the stage of the tumour.According to the INRGSS system, the tumours of neuroblastoma may be described as follows: Stage L1: The tumour is located only in the area where it started; no image defined risk factors (IDRFs) are found on imaging scans, such as CT or MRI. Stage L2: The tumour has spread to the tissue nearby the area it started; IDRFs are found on imaging scans, such as CT or MRI. Stage M: The tumour has spread to other parts of the body (except stage MS, see below). Stage MS: The tumour has spread to only the skin, liver, and/or bone marrow (less than 10% bone marrow involvement) in patients younger than 18 months. Suitably the compounds and compositions of the invention can be used to treat any of the tumours described above. Suitably the subject may be classified as very low, low, moderate or high risk. Preferably, the subject is classified as moderate or high risk, more preferably high risk. Very low-risk neuroblastoma refers to Stage L1/L2 with ganglioneuroma maturing or ganglioneuroblastoma intermixed histology, Stage L1 with non-amplified MYCN, or Stage MS in children younger than 18 months of age with no chromosome 11q aberration. Low-risk neuroblastoma refers to Stage L2 in children younger than 18 months of age with no 11q aberration, Stage L2 in children older than 18 months of age with ganglioneuroblastoma nodular or neuroblastoma with differentiating histology and no 11q aberration, Stage M in children younger than 18 months without MYCN amplification and hyperdiploidy. Moderate-risk neuroblastoma refers to Stage L2 in children younger than 18 months without MYCN amplification with 11q aberration, Stage L2 in children older than 18 months with ganglioneuroblastoma nodular or neuroblastoma with differentiating histology with 11q aberration, Stage L2 in children older than 18 months with ganglioneuroblastoma nodular or neuroblastoma with poorly differentiated or undifferentiated histology, Stage M in children younger than 12 months with diploidy, or Stage M in children 12 months to 18 months with diploidy. High-risk neuroblastoma refers to Stage L1 with MYCN amplification, Stage L2 with MYCN amplification, Stage M in children younger than 18 months of age with MYCN amplification, Stage M in children with older than 18 months, Stage MS in children younger than 18 months with 11q aberration, or Stage MS in children younger than 18 months of age with MYCN amplification. In one embodiment, a composition comprising lovastatin is used for the treatment of neuroblastoma, wherein the patient has had or is going to have surgery to remove some or all of the neuroblastoma tumour. This may be particularly advantageous if the neuroblastoma is large and/or expands across tissue boundaries, so it is difficult to remove it all by surgery and/or a quick removal of at least some of it is desired/beneficial. The term “surgery” has its normal meaning in the art. Surgery is an invasive technique with the fundamental principle of physical intervention on organs/organ systems/tissues for diagnostic or therapeutic reasons. As used herein, a pharmaceutically acceptable salt is a salt with a pharmaceutically acceptable acid or base. Pharmaceutically acceptable acids include both inorganic acids such as hydrochloric, sulphuric, phosphoric, diphosphoric, hydrobromic or nitric acid and organic acids such as fendizoic, citric, fumaric, maleic, malic, ascorbic, succinic, tartaric, benzoic, acetic, methanesulfonic, ethanesulfonic, ethanedisulfonic, salicylic, stearic, benzenesulfonic or p-toluenesulfonic acid. Pharmaceutically acceptable bases include alkali metal (e.g. sodium or potassium) and alkali earth metal (e.g. calcium or magnesium) hydroxides and organic bases such as alkyl amines, aryl amines or heterocyclic amines. In an alternative embodiment, the present invention is directed to a composition comprising lovastatin, or a pharmaceutically acceptable salt thereof, for use in the treatment of neuroblastoma, wherein lovastatin is the only active agent in the composition. By only active agent it is meant that the composition does not contain other components which may be used in the treatment of neuroblastoma. In an alternative embodiment, the present invention is directed to a composition comprising lovastatin, or a pharmaceutically acceptable salt thereof, and trifluoperazine, or a pharmaceutically acceptable salt thereof for use in the treatment of neuroblastoma. As shown by the below in vitro data, trifluoperazine has been identified as showing synergistic effects in the treatment of neuroblastoma, when used in combination with lovastatin. In an alternative embodiment, the present invention is directed to a composition comprising lovastatin, or a pharmaceutically acceptable salt thereof, and prochlorperazine, or a pharmaceutically acceptable salt thereof, for use in the treatment or of neuroblastoma. As shown by the below in vitro data, prochlorperazine has been identified as showing synergistic effects in the treatment of neuroblastoma, when used in combination with lovastatin. In an alternative embodiment, the present invention is directed to a composition comprising lovastatin, or a pharmaceutically acceptable salt thereof, for use in combination with a second composition comprising trifluoperazine, or a pharmaceutically acceptable salt thereof, or prochlorperazine, or a pharmaceutically acceptable salt thereof, wherein the two compositions are administered to the subject simultaneously, separately or sequentially. In a preferred embodiment the pharmaceutically acceptable salt of trifluoperazine is hydrochloride. In a preferred embodiment the pharmaceutically acceptable salt of prochlorperazine is maleate. In an alternative preferred embodiment, the pharmaceutically acceptable salt of prochlorperazine is ethanedisulfonate (edisylate). In a further preferred embodiment, the pharmaceutically acceptable salt of prochlorperazine is methanesulfonate (mesylate). Trifluoperazine is a typical antipsychotic of the phenothiazine class. It blocks the dopaminergic D1 and D2 receptors in the brain, depresses the release of hypothalamic and hypophyseal hormones and is believed to depress the reticular activating system thus affecting basal metabolism, body temperature, wakefulness, vasomotor tone, and emesis. It is approved for the treatment of schizophrenia and anxiety disorders. Trifluoperazine has the systematic name 10-[3-(4-methylpiperazin- 1-yl)propyl]-2-(trifluoromethyl)-10H-phenothiazine. Prochlorperazine is a piperazine phenothiazine that blocks the dopaminergic D2 receptors in the brain. It is approved for the treatment of severe nausea and vomiting as well as short term management of anxiety and schizophrenia. Prochlorperazine has the systematic name 2-chloro-10-[3-(4-methyl-1-piperazinyl)propyl]- 10H- phenothiazine. The compositions of the invention may contain a pharmaceutically acceptable carrier. By “pharmaceutically acceptable carrier” is meant any diluent or excipient, such as fillers or binders, that is compatible with the other ingredients of the compositions, and which is not deleterious to the recipient. The pharmaceutically acceptable carrier can be selected on the basis of the desired route of administration, in accordance with standard pharmaceutical practices. In the present invention, the compositions may be administered in a variety of dosage forms. In one embodiment, the compositions may be formulated in a format suitable for oral, rectal, parenteral, intranasal or transdermal administration or administration by inhalation or by suppository. The compositions may be administered orally, for example as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules. Preferably, the compositions are formulated such that it is suitable for oral administration, for example tablets and capsules. Tablets and capsules may be prepared with binding agents, for example, syrup, acacia, gelatin, sorbitol, tragacanth, celluloses or polyvinylpyrrolidone; fillers, such as lactose, sucrose, corn starch, calcium phosphate, sorbitol, or glycine; lubricants, such as magnesium stearate, talc, polyethylene glycol, or silica; and surfactants, such as sodium lauryl sulfate. Liquid compositions may contain conventional additives such as suspending agents, for example sorbitol syrup, methyl cellulose, sugar syrup, gelatin, carboxymethyl-cellulose, or edible fats; emulsifying agents and surfactants such as lecithin, or acacia; vegetable oils such as almond oil, coconut oil, cod liver oil, or peanut oil; preservatives such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT). Liquid compositions may be encapsulated in, for example, gelatin to provide a unit dosage form. The compositions may also be administered parenterally, whether subcutaneously, intravenously, intramuscularly, intrasternally, transdermally or by infusion techniques. The compositions may also be administered by inhalation. An advantage of inhaled medications is their direct delivery to the area of rich blood supply in comparison to many medications taken by oral route. Thus, the absorption is very rapid as the alveoli have an enormous surface area and rich blood supply and first pass metabolism is bypassed. The present invention also provides an inhalation device containing the compositions of the present invention. Typically said device is a metered dose inhaler (MDI), which contains a pharmaceutically acceptable chemical propellant to push the medication out of the inhaler. The compositions may also be administered by intranasal administration. The nasal cavity’s highly permeable tissue is very receptive to medication and absorbs it quickly and efficiently. Nasal drug delivery is less painful and invasive than injections, generating less anxiety among patients. By this method absorption is very rapid and first pass metabolism is usually bypassed, thus reducing inter-patient variability. Further, the present invention also provides an intranasal device containing the composition according to the present invention. The compositions may also be administered by transdermal administration. For topical delivery, transdermal and transmucosal patches, creams, ointments, jellies, solutions or suspensions may be employed. The present invention therefore also provides a transdermal patch containing the compositions. The compositions may also be administered by sublingual administration. The present invention therefore also provides a sub-lingual tablet comprising the compositions. The compositions may also be formulated with an agent which reduces degradation of the substance by processes other than the normal metabolism of the patient, such as anti-bacterial agents, or inhibitors of protease enzymes which might be the present in the patient or in commensural or parasite organisms living on or within the patient, and which are capable of degrading the compound. Liquid dispersions for oral administration may be syrups, emulsions and suspensions. Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol. The suspension or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride. Solutions for injection or infusion may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions. In an embodiment of the invention, the compositions are administered in an effective amount to treat neuroblastoma. An effective dose will be apparent to one skilled in the art, and is dependent on a number of factors including age, sex, weigh, which the medical practitioner will be capable of determining. The compositions and kits of the present invention provide for the administration of more than one drug, and they can be administered simultaneously, sequentially or separately. It is not necessary that they are packed together (but this is one embodiment of the invention). It is also not necessary that they are administered at the same time. As used herein, “separate” administration means that the drugs are administered as part of the same overall dosage regimen (which could comprise a number of days), but preferably on the same day. As used herein “simultaneously” means that the drugs are to be taken together or formulated as a single composition. As used herein, “sequentially” means that the drugs are administered at about the same time, and preferably within about 1 hour of each other. Preferably, the drugs are administered simultaneously i.e. taken together or formulated as a single composition. Most preferably, they are formulated as a single composition. In a preferred embodiment, the composition comprises 1 mg to 100 mg, preferably 5 mg to 90 mg, more preferably 10 mg to 80 mg, yet more preferably 20 mg to 60 mg lovastatin. The composition may be administered once a day, twice a day, three times a day or four times a day. In an embodiment of the invention, the composition is administered at least once a day. Preferably it is administered as a single daily dose. Preferably the single daily dose is 1 mg to 100 mg, preferably 5 mg to 90 mg, more preferably 10 mg to 80 mg, yet more preferably 20 to 60 mg of lovastatin. Exemplary doses are 10, 20, 40, 60 or 80 mg of lovastatin. In another embodiment, the composition further comprises trifluoperazine or a pharmaceutically acceptable salt thereof, wherein the composition comprises 2 mg to 40 mg, preferably 4 mg to 20 mg, more preferably 15 to 20 mg of trifluoperazine. Alternatively the trifluoperazine or a pharmaceutically acceptable salt thereof is administered in a second composition, wherein the second composition comprises 2 mg to 40 mg, preferably 4 mg to 20 mg, more preferably 15 to 20 mg of trifluoperazine. In another embodiment, the composition further comprises prochlorperazine, or a pharmaceutically acceptable salt thereof, wherein the composition comprises 1 mg to 40 mg, preferably 5 to 35 mg, more preferably 10 mg to 30 mg of prochlorperazine. Alternatively the prochlorperazine or a pharmaceutically acceptable salt thereof is administered in a second composition, wherein the second composition comprises 1 mg to 40 mg, preferably 5 to 35 mg, more preferably 10 mg to 30 mg of prochlorperazine. Suitably the composition comprising lovastatin and the second composition are a single daily dose. Suitably the two compositions are administered simultaneously i.e. lovastatin and trifluoperazine or prochlorperazine are taken together. The compositions may also be administered sequentially i.e. at about the same time, and preferably within about 1 hour of each other. In an embodiment of the invention, the composition is administered twice daily. Preferably each dose is 0.5 mg to 50 mg, more preferably 2.5 mg to 45 mg, more preferably 5 mg to 40 mg, yet more preferably 10 to 30 mg lovastatin. Exemplary doses are 5, 10, 20, 40, or 50 mg of lovastatin. When the composition further comprises trifluoperazine or prochlorperazine, or pharmaceutically acceptable salts thereof, as described above, each dose comprises 1 mg to 20 mg, more preferably 2 mg to 10 mg, yet more preferably 7.5 mg to 10 mg of trifluoperazine or 0.5 mg to 20 mg, more preferably 2.5 mg to 17.5 mg, yet more preferably 5 mg to 15 mg of prochlorperazine. Alternatively, when the composition is administered with the second composition comprising trifluoperazine or prochlorperazine, or pharmaceutically acceptable salts thereof, as described above, each dose comprises 1 mg to 20 mg, more preferably 2 mg to 10 mg, yet more preferably 7.5 mg to 10 mg of trifluoperazine or 0.5 mg to 20 mg, more preferably 2.5 mg to 17.5 mg, yet more preferably 5 mg to 15 mg of prochlorperazine. Each dose or composition may be administered separately or sequentially i.e. at about the same time, and preferably within about 1 hour of each other. In an embodiment of the invention, the composition is administered three times daily. Preferably each dose is 0.3 mg to 33 mg, more preferably 1.5 mg to 30 mg, more preferably 3 mg to 30 mg, yet more preferably 6 to 20 mg lovastatin. Exemplary doses are 5, 10, 20, or 30 mg of lovastatin. When the composition further comprises trifluoperazine or prochlorperazine, or pharmaceutically acceptable salts thereof, as described above, each dose comprises 0.6 mg to 15 mg, more preferably 1.3 mg to 6.5 mg, yet more preferably 5 mg to 6.5 mg of trifluoperazine or 0.3 mg to 13 mg, more preferably 1.7 mg to 12 mg, more preferably 3 mg to 10 mg of prochlorperazine. Alternatively, when the composition is administered with the second composition comprising trifluoperazine or prochlorperazine, or pharmaceutically acceptable salts thereof, as described above, each dose comprises 0.6 mg to 15 mg, more preferably 1.3 mg to 6.5 mg, yet more preferably 5 mg to 6.5 mg of trifluoperazine or 0.3 mg to 13 mg, more preferably 1.7 mg to 12 mg, more preferably 3 mg to 10 mg of prochlorperazine. Each dose or composition may be administered separately or sequentially i.e. at about the same time, and preferably within about 1 hour of each other. Preferably, the dosage regime is such that the total daily dosage of lovastatin does not exceed 100 mg. Suitably the effective dose of lovastatin results in a concentration of 1 to 10 μM, preferably 1 to 5 μM, more preferably 1 to 3 μM in cells. In order to treat neuroblastoma, the composition comprising lovastatin is used in a chronic dosage regime i.e. chronic, long-term treatment. Suitably the regime lasts for at least one month, suitably at least two months, such as at least three months. The present invention also relates to a kit comprising: (i) at least one dose of lovastatin, or a pharmaceutically acceptable salt thereof; and optionally (ii) at least one dose of trifluoperazine, or a pharmaceutically acceptable salt thereof, or prochlorperazine, or a pharmaceutically acceptable salt thereof, for simultaneous, separate or sequential use in the treatment of neuroblastoma. The present invention also relates to the use of a composition comprising lovastatin, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for use in the treatment of neuroblastoma. This embodiment of the invention may have any of the preferred features described above. The present invention also relates to a method of treating neuroblastoma comprising administering to a patient a composition comprising lovastatin or a pharmaceutically acceptable salt thereof. This embodiment of the invention may have any of the preferred features described above. The method of administration may be according to any of the routes described above. The present invention further relates to a method of treating neuroblastoma comprising administering to the patient a composition comprising lovastatin or a pharmaceutically acceptable salt thereof, and a second composition comprising trifluoperazine, or a pharmaceutically acceptable salt thereof, or prochlorperazine, or a pharmaceutically acceptable salt thereof, wherein the two compositions are administered to the patient simultaneously, separately or sequentially. This embodiment of the invention may have any of the preferred features described above. The method of administration may be according to any of the routes described above. For the avoidance of doubt, the present invention also embraces prodrugs which react in vivo to give a compound of the present invention. Experimental Section Example 1: Monotherapy in vitro drug testing of lovastatin, trifluoperazine and prochlorperazine The individual efficacies of lovastatin, trifluoperazine and prochlorperazine were investigated and their ability to inhibit cell growth utilizing neuroblastoma patient- derived xenograft tumours grown in vitro (PDX1, PDX2 and PDX3). Methods All in vitro preclinical models were derived from patient derived xenograft tumours and cultured ex vivo. The establishment of PDX 1, 2 & 3 was described in Braekeveldt et al, Int. J. Cancer, 2015, E252-E261. All models had MYCN amplification and are therefore representative of high-risk patients. PDX 1, 2 & 3 tumours were grown in male or female NSG mice, mice were housed under pathogen free conditions and given access to food and water ab libitum. Animals were sacrificed, tumour tissues excised and digested for 45 mins at 37^oC with Liberase (0.15mg/ml). Dissociated tissue was passed through a 70Njm cell strainer and cultured in non-adherent plates as tumour spheroids at 37^oC in a humidified atmosphere with 5% CO2 in the following culture medium: Dulbecco's Modified Eagle's medium DMEM/F12 medium with glutamine, 100 IU/ml penicillin and 100 Njg/ml of streptomycin, 2% B27 supplement, 40 ng/ml of basic fibroblast growth factor (FGF) 2 and 20 ng/ml of epidermal growth factor (EGF). Directly upon seeding cells were treated with the compounds solubilised in DMSO, then cultured for 3 or 7 days. Cell viability was quantified using the CellTiter-Glo^® luminescence Cell Viability Assay. Using this assay the number of metabolically active viable cells present in a culture can be determined based on the concentration of ATP. The luminescent signal produced is proportional to the amount of ATP present. Lovastatin, trifluoperazine hydrochloride and prochlorperazine maleate compounds had a purity of >95%. Drugs were tested in triplicate, at a minimum of 6 concentrations, top concentration of 10NjM (free-base equivalent). Cell viability readouts were normalized to DMSO treated controls, concentration-response curves were plotted and IC50 and R2 values were calculated using GraphPad Prism 8.4.3. Results Reduction in cell viability by lovastatin Results for lovastatin treatment in neuroblastoma cell lines can be seen in Figures 1A-F. Lovastatin inhibited the growth of PDX 1, 2 & 3 after 3 days in culture. After 7 days of culture lovastatin inhibited the viability of all three cell lines at an IC50 of 0.84NjM (PDX 1), 2.67NjM (PDX 2) and 0.15NjM (PDX3). Reduction in cell viability by trifluoperazine Results for trifluoperazine treatment in neuroblastoma cell lines can be seen in Figures 2A-F. Trifluoperazine inhibited the growth of PDX 1, 2 and 3 after 3 and 7 days in culture. Trifluoperazine inhibited cell viability by 50% in all cell models at both timepoints at concentrations of 4NjM or less. Reduction in cell viability by prochlorperazine Results for prochlorperazine treatment in neuroblastoma cell lines can be seen in Figures 3A-F. Prochlorperazine inhibited the growth of PDX 1, 2 and 3 after 3 and 7 days in culture. Prochlorperazine inhibited cell viability by 50% in all cell models at both timepoints at concentrations of 3.9NjM or less. Example 2 - Fixed Concentration Combination Study of lovastatin and trifluoperazine or prochlorperazine Materials and Methods Cell culture conditions Fixed concentration combination studies were carried out using PDX 1 & 2 cell models. Cells were cultured using the same protocol as described for the monotherapy studies. Cells were treated for 7 days with lovastatin and trifluoperazine or prochlorperazine at a fixed concentration indicated in Table 1. The fixed concentrations were equivalent to the IC50 values of each agent at 7 days as monotherapies in the two models. Table 1 – Concentrations of lovastatin, trifluoperazine, and prochlorperazine used in the combination study Concentrations (μM)

Trypan blue staining assay Viable cells in the culture were quantified using the trypan blue staining assay.

an blue is an azo dye that is cell membrane impermeable that can only enter dead cells that have a permeable membrane. The dye binds to intracellular proteins and therefore dead cells are stained blue. The number of live (unstained) and dead (stained) cells were then quantified, and the percentage of viable cells calculated. Flow cytometry Cell death was quantified by Annexin V/Propidium iodide (PI) and caspase 3 staining using flow cytometry. For Annexin V/PI staining cells were incubated with fluorescent annexin V and PI, a human vascular anti-coagulant that binds to phospholipid phosphatidylserine (PS). PS is normally found on the inner leaflets of the plasma membrane, during apoptosis these move to the outer leaflet of the plasma membrane. Therefore, annexin V only binds to apoptotic cells. PI is a red fluorescent stain that binds to DNA and is impermeable to live cells, therefore only binds to dead cells. Annexin V/PI assay was carried out according to manufacturer’s instructions. Caspase 3 an apoptosis marker was quantified using the NucView® Caspase-3 assay kit in live cells according to manufacturer’s instructions. Cells were counted on a FACSverse flow cytometer and populations gated and quantified. Data Analysis Graphs of live and dead cell values were plotted using GraphPad Prism 8.4.2. Results Lovastatin and trifluoperazine The results can be seen in Figures 4A-C. A reduction in cell viability and increase in cell death (annexin V/PI) was observed in both PDX 1 and PDX 2 cells treated with lovastatin and trifluoperazine as single agents. When the agents were combined there was a further reduction in cell viability with no viable cells being present in PDX 2 cell cultures. In line with this reduction in cell viability, there was a large increase in cell death in the combination treated cell cultures quantified by annexin V/PI staining. The cell death induced by the combination was via the apoptotic pathway, as indicated by an increase in caspase 3 positive cells. The results for the combination of lovastatin and trifluoperazine were greater than an expected additive effect based on the single agent results, demonstrating a synergistic benefit to their combination. Lovastatin and prochlorperazine The results can be seen in Figures 5A-C. A reduction in cell viability and increase in cell death (annexin V/PI) was observed in both PDX 1 and PDX 2 cells treated with lovastatin and prochlorperazine as single agents. When the agents were combined there was a further reduction in cell viability in both PDX 1 and PDX 2. In line with this reduction in cell viability, there was a large increase in cell death via the apoptotic pathway in the combination treated cell cultures quantified as determined by annexin V/PI and caspase 3 staining. The results for the combination of lovastatin and prochlorperazine were greater than an expected additive effect based on the single agent results, demonstrating a synergistic benefit to their combination. Example 3 - Concentration Response Combination Studies of lovastatin and trifluoperazine or prochlorperazine Materials and Methods Cell culture conditions Concentration response combination studies were carried out in the PDX 1, 2 and 3 cell models. Cells were cultured using the same protocol as described for the monotherapy studies. At seeding, cells were treated with lovastatin combined with either trifluoperazine or prochlorperazine in a 6x6 concentration matrix of increasing concentrations of both drugs. Cells were cultured for 3 or 7 days; cell viability was quantified using the CellTiter-Glo® luminescence Cell Viability Assay. Drugs Lovastatin, trifluoperazine hydrochloride and prochlorperazine maleate all had a purity of >95%. Drugs were tested in triplicate, the top concentration of Lovastatin was 20NjM, top concentration of trifluoperazine and prochlorperazine was 5NjM (free- base equivalent). Data analysis Cell viability readouts were normalized to DMSO treated controls and a viability matrix was produced. ZIP combination synergy scores were calculated from this matrix using ZIP Synergy Finder ver 2.0 https://synergyfinder.fimm.fi/ (Ianevski et al, Nucleic Acids Research, 2020, Pages W488–W493). Scores of -10 to 10 indicate an additive combination effect, scores of >10 indicate a synergistic combination effect. Results The results of the combined treatment of lovastatin and trifluoperazine can be seen in Table 2. There was a synergistic reduction in cell viability of both PDX 1 and PDX 2 when lovastatin was combined with trifluoperazine indicated by ZIP scores of >10. The results of the combined treatment of lovastatin and prochlorperazine can be seen in Table 2. There was an additive reduction in cell viability when lovastatin was combined with prochlorperazine in PDX 1 and PDX 2 after 3 days of treatment, indicated by ZIP scores between -10 and 10. There was a synergistic reduction in cell viability of both PDX 1 and PDX 2 when lovastatin was combined with prochlorperazine indicated by ZIP scores of >10 after 7 days of treatment. Table 2 – ZIP Synergy scores for combinations of lovastatin and trifluoperazine or prochlorperazine (Scores of -10 to 10 indicate an additive combination effect, scores of >10 indicate a synergistic combination effect) ZIP Synergy Scores

Conclusions Lovastatin inhibits the cell viability and increases cell death in neuroblastoma cell lines, and is therefore effective at treating neuroblastoma. The reduction in cell viability can be further enhanced when a combination of lovastatin and either trifluoperazine or prochlorperazine is used. The combination treatment leads to synergistic increases (i.e. greater than the expected additive values) in cell death and reduction in cell viability, and this synergy is quantified by the ZIP synergy scores.

Claims

Claims 1. A composition comprising lovastatin or a pharmaceutically acceptable salt thereof, for use in the treatment of neuroblastoma.

2. The composition for use according to any preceding claim wherein the subject of the treatment is a moderate or high risk subject, preferably high risk.

3. The composition for use according to any preceding claim, wherein the subject of the treatment is a human.

4. The composition for use according to any preceding claim, wherein the subject of the treatment is a paediatric patient.

5. The composition for use according to any preceding claim, wherein the composition comprises 1 mg to 100 mg, preferably 5 mg to 90 mg, more preferably 10 mg to 80 mg, yet more preferably 20 mg to 60 mg of lovastatin.

6. The composition for use according to any preceding claim, wherein administration is by a dose two times per day.

7. The composition for use according to claim 6, wherein the dose comprises 0.5 mg to 50 mg, preferably 2.5 mg to 45 mg, more preferably 5 mg to 40 mg, yet more preferably 10 mg to 30 mg of lovastatin.

8. The composition for use according to any of claims 1 to 4, where administration is by a dose three times per day.

9. The composition for use according to claim 8, wherein the dose comprises 0.3 mg to 33 mg, preferably 1.5 mg to 30 mg, more preferably 3 mg to 30 mg, yet more preferably 6 mg to 20 mg of lovastatin.

10. The composition for use according to any preceding claim, to be administered orally or intravenously.

11. The composition for use according to any of claims 1 to 9, to be administered by parenteral, transdermal, sublingual, rectal or inhaled administration.

12. The composition for use according to any preceding claim, wherein lovastatin is the only active agent in the composition.

13. The composition for use according to any of claims 1 to 11 wherein the composition further comprises trifluoperazine, or a pharmaceutically acceptable salt thereof, or prochlorperazine, or a pharmaceutically acceptable salt thereof.

14. The composition for use according to any of claims 1 to 11 in combination with a second composition comprising trifluoperazine, or a pharmaceutically acceptable salt thereof, or prochlorperazine, or a pharmaceutically acceptable salt thereof, wherein the two compositions are to be administered to a subject simultaneously, separately or sequentially.

15. The composition for use according to claims 13 or 14, wherein the amount of trifluoperazine is 2 to 40 mg, or the amount of prochlorperazine is 1 to 40 mg.

16. The composition for use according to claim 13 or 14 wherein the pharmaceutically acceptable salt of trifluoperazine is hydrochloride.

17. The composition for use according to claim 13 or 14 wherein the pharmaceutically acceptable salt of prochlorperazine is maleate.

18. A kit comprising: (i) at least one dose of lovastatin, or a pharmaceutically acceptable salt thereof; and (ii) at least one dose of trifluoperazine, or a pharmaceutically acceptable salt thereof, or prochlorperazine, or a pharmaceutically acceptable salt thereof, for simultaneous, separate or sequential use in the treatment of neuroblastoma.

19. A kit according to claim 18, having any of the additional features of claims 2 to 17.

20. Use of lovastatin, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for use in the treatment of neuroblastoma.

21. Use according to claim 20, having any of the additional features of claims 2 to 17.

22. A method of treating neuroblastoma comprising administering to a patient a composition comprising lovastatin or a pharmaceutically acceptable salt thereof.

23. The method according to claim 22, having any of the additional features of claims 2 to 13, 16 and 17.

24. The method according to claims 22 or 23 wherein the method further comprises administering to the patient a second composition comprising trifluoperazine, or a pharmaceutically acceptable salt thereof, or prochlorperazine, or a pharmaceutically acceptable salt thereof, wherein the two compositions are administered simultaneously, separately or sequentially.