WO2019039937A1

WO2019039937A1 - Therapeutic combination for cancer treatment

Info

Publication number: WO2019039937A1
Application number: PCT/NL2018/050547
Authority: WO
Inventors: Gerrit Jansen; Godefridus Johannes PETERS
Original assignee: Vrije Universiteit Medisch Centrum VUMC
Current assignee: Vrije Universiteit Medisch Centrum VUMC
Priority date: 2017-08-21
Filing date: 2018-08-20
Publication date: 2019-02-28
Anticipated expiration: 2020-02-21
Also published as: WO2019039937A8

Abstract

The invention relates to a composition comprising a statin and an aminopeptidase inhibitor. Said composition may be used as a medicament, including in a method of treating cancer, such as a hematological cancer. The invention further relates to methods of treating a cancer in an individual, and to a use of an aminopeptidase inhibitor in a method for the preparation of a medicament for the treatment of a cancer.

Description

THERAPEUTIC COMBINATION FOR CANCER TREATMENT

FIELD: The invention is in the field of treatment of cancer patients. More specifically, the invention provides a novel combination for improved treatment of cancer patients.

INTRODUCTION

Targeting of protein degradation pathways has provided new therapeutic opportunities for haematological malignancies. Proteasome inhibitors, with

Bortezomib (Velcade®) as a prototypical representative, have gained an established place in chemotherapeutic treatment regimens for multiple myeloma (Anderson,

2012. J Clin Oncol 30: 445-452; Niewerth et al., 2013. Expert Rev Anticancer Ther 13: 327-337). Aminopeptidases operating downstream of the proteasome were also identified as druggable targets with Bestatin as the founding representative displaying activity mainly against solid tumors (Hitzerd et al., 2014. Amino Acids 46: 793-808). Tosedostat represents a next generation aminopeptidase inhibitor that displays activity as monotherapy as well as in combination with various chemotherapeutic drugs, including cytarabine, daunorubicin and histone deacetylase (HDAC) inhibitors (Krige et al., 2008. Cancer Res 68: 6669-6679;

Cortes et al., 2013. Lancet Oncol 14: 354-362; Smith et al., 2015. Oncotarget 6: 17314-17327). Moreover, Tosedostat demonstrated promising clinical activity in phase I-III combination chemotherapy for acute myeloid leukemia (Cortes et al.,

2013. Lancet Oncol 14: 354-362; Jenkins et al., 2011. Leuk Res 35: 677-681;

Lowenberg et al., 2010. J Clin Oncol 28: 4333-4338; DiNardo and Cortes, 2014. Expert Opin Investig Drugs 23: 265-272; DiNardo and Cortes, 2015. Expert Opin Pharmacother 16: 95-106) and multiple myeloma (Moore et al., 2009. Mol Cancer Ther 8: 762-770), and has been evaluated for treatment of solid tumors (Reid et al., 2009. Clin Cancer Res 15: 4978-4985; van Herpen et al., 2010. Br J Cancer 103: 1362- 1368).

Tosedostat, and a close structural analogue CHR2863, are aminopeptidase inhibitor prodrugs with an esterase-sensitive motif (Krige et al., 2008. Cancer Res 68: 6669-6679). As hydrophobic drugs they can freely diffuse into cells where they are converted by intracellular esterases to their hydrophilic acid active metabolites that enhance their cellular retention and promote targeting of multiple

aminopeptidases. The inhibition of aminopeptidases provokes an amino acid deprivation response, inhibition of niTOR activity and blockade of protein synthesis (Krige et al., 2008. Cancer Res 68: 6669-6679), resulting in growth arrest and apoptosis.

Prolonged exposure to aminopeptidase inhibitors such as Tosedostat may provoke the onset of drug resistance. Recently, it was demonstrated (Verbrugge et al., 2016. Oncotarget 7: 5240-5257) that the cytotoxic activity of CHR2863 against U937 myeloid cells relies on carboxylesterase 1 (CES1) activity. Consistently, down-regulation of CES1 and abolishment of CHR2863 conversion to its

hydrophilic active metabolite constituted a dominant mechanism of acquired resistance to CHR2863 (Verbrugge et al., 2016. Oncotarget 7: 5240-5257).

There is thus a need for methods and means that may enhance the efficacy of aminopeptidase inhibitors such as Tosedostat and that may prevent or diminish resistance to prolonged exposure to aminopeptidase inhibitors such as Tosedostat.

SUMMARY OF THE INVENTION

The invention therefore provides a composition comprising a statin and an aminopeptidase inhibitor. Said composition can be used as a medicament, for example as a medicament for use in a method of treating cancer, including for example hematological cancers such as acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS).

As is shown in the examples, non-toxic concentrations of statins, which are inhibitors of the mevalonate-cholesterol pathway, markedly potentiate the growth inhibitory effects of either a prodrug (CHR2863) or a direct inhibitor (bestatin) of aminopeptidases. The potentiating effects of statins on aminopeptidase inhibitors were observed with various statins (simvastatin, fluvastatin, lovastatin and pravastatin) in a broad spectrum of human AML cell lines. The synergistic effects with statins were specific for aminopeptidase inhibitors (i.e. CHR2863 and

Bestatin) but not for other cytotoxic agents. Statin potentiation of CHR2863 activity was abrogated by co- administration of mevalonate or

farnesylpyrophosphate, and partly by geranylgeranylpyrophosphate, suggesting involvement of protein prenylation. Simvastatin impaired Rheb prenylation which is required for lysosomal membrane association and activation of mTOR. Hence, without being bound by theory, it is proposed that the dual inhibitory effect of impaired Rheb prenylation and CHR2863-induced mTOR inhibition may impose a potent synergistic inhibition on human cancer cells such as AML cells. These findings uncover a potent therapeutic combination of statins and aminopeptidase inhibitors for the treatment of cancers, including hematological cancers such as AML.

A composition according to the invention is either for simultaneous administration, for example in a tablet, or for separate administration of said statin and said aminopeptidase inhibitor, for example as two separate

pharmaceutical compositions.

A statin may be selected from atorvastatin, pravastatin, rosuvastatin, simvastatin, fluvastatin, lovastatin and pravastatin.

An aminopeptidase inhibitor may inhibit a human aminopeptidase of the Ml zinc-aminopeptidases subfamily, such asaminopeptidase N, leucine

aminopeptidase, and/or puromycin-sensitive aminopeptidase. In embodiments, the aminopeptidase inhibitor is a phenylethanoic acid cyclopentyl ester derivative, which in a particular embodiment is Tosedostat, or a structural analogue thereof.

A composition according to the invention may be a pharmaceutical

composition comprising a statin and an aminopeptidase inhibitor, and further comprising at least one pharmaceutically acceptable excipient.

The invention further provides a method of treating a cancer in an individual, said method comprising providing the individual with an effective dose of a composition comprising a statin and an aminopeptidase inhibitor. The cancer to be treated can be a hematological cancer such as AML and MDS The statin and the aminopeptidase inhibitor may be administered simultaneously or separately.

A statin and an aminopeptidase inhibitor may be administered in a tablet, optionally with differential release properties for the two compounds.

The invention further provides a use of an aminopeptidase inhibitor in a method for the preparation of a medicament for the treatment of a cancer in an individual, whereby said medicament further comprises a statin. Said medicament may be a tablet, optionally with differential release properties for the two compounds.

FIGURE LEGENDS

Figure 1. Growth inhibitory effects of CHR2863 for (A) U937/WT cells, (B)

U937/CHR2863(200) cells and (C) U937/CHR2863(5jiM) cells in the absence and presence of maximal non-toxic concentrations of simvastatin (2 μΜ, 2.5 μΜ and 2.5 μΜ, respectively). Cell growth inhibition was determined after 72 hours drug exposure. Results depicted are the mean ± SE of 6-10 separate experiments. (D) Combination index - fraction affected plot from (A-C) of the combination

simvastatin (fixed concentration) and CHR2863 for U937/WT, U937/CHR2863(200) and U937/CHR2863^M) cells.

Figure 2. Potentiating effect of maximal non- toxic concentrations of various statins on the CHR2863 activity in U937/WT, U937/CHR2863(200) and

υ937/("ΗΈ28β3(5μΜ) cells. Concentrations of simvastatin, lovastatin, pravastatin and fluvastatin were 2 μΜ, 2.5 μΜ, 100 μΜ and 0.5 μΜ, respectively, for U937/WT cells, and 2.5 μΜ, 5 μΜ, 200 μΜ and 1 μΜ, respectively, for U937/CHR2863(200) and ΙΙ937/ϋΗΉ2863(5μΜ) cells. Statin potentiating factor is defined as the ratio of IC50 (50% growth inhibition) of cell culture without statins vs IC50 of cell cultures in the presence of statins. Cell growth inhibition was determined after 72 hours drug exposure. Results depicted are the mean ± SD of 3-4 separate experiments.

Figure 3. Selectivity of simvastatin-potentiating effect for aminopeptidase inhibitors. Effect of non-toxic concentrations of simvastatin (2-2.5 μΜ) on the growth inhibitory activity of the aminopeptidase inhibitors CHR2863 and bestatin, HDAC inhibitor prodrug CHR2875, and daunorubicin in U937/WT,

U937/CHR2863(200) and ΙΤ937/ϋΒ¾2863(5μΜ) cells. Simvastatin potentiation factor is defined as the ratio of IC50 (50% growth inhibition) of cell culture without statins vs IC50 of cell cultures in the presence of statins. Cell growth inhibition was determined after 72 hours drug exposure. Results depicted are the mean of 2 separate experiments (for bestatin) and the mean ± SD of 3-4 separate experiments for CHR2863, CHR2875 and daunorubicin.

Figure 4. Simvastatin potentiation of CHR2863 activity in human AML cell lines versus human lymphoid and solid tumor cell lines. Cell growth inhibition was determined after 72 hours drug exposure in the absence or presence of maximal non-toxic concentrations of simvastatin, being (between brackets) for: U937 (2 μΜ), THP1 (2.5 μΜ), MV4- 11 (2.5 μΜ), KG1 (10 μΜ), CCRF-CEM (2.5 μΜ), CEM/Vbl (2.5 μΜ), SW1573 (0.2 μΜ), 2008 (0.75 μΜ), 2008/MRP1 (2.5 μΜ), MCF7 (1 μΜ), MCF7/MR (2.5 μΜ) and KB (1 μΜ). Simvastatin potentiation factor is defined as the ratio of IC50 (50% growth inhibition) of cell culture without statins vs IC50 of cell cultures in the presence of statins. IC50 values (between brackets) for

CHR2863 for the various cell lines (in the absence of simvastatin) were: U937 (61 ± 16 nM), THP1 (1172 ± 807 nM), MV4-11 (282 ± 51 nM), KG1 (394 ± 144 nM), CCRF-CEM (11, 170 ±5, 100 nM), CEM/Vbl (29, 100 ± 5,900 nM), SW1573 (6,625 ± 3,020 nM), 2008 (2,020 ± 1,080 nM), 2008/MRP1 (6,700 ± 2,560 nM), MCF7 (453 ± 400 nM), MCF7/MR (386 ± 64 nM), and KB (132 ± 50 nM). Results depicted are the mean ± SD of 3-5 separate experiments.

Figure 5. Effect of simvastatin and CHR2863 combinations on cell viability, apoptosis induction and cell cycle distribution in U937/WT, U937/CHR2863(200) and ΙΙ937/( :ΗΈ2863(5μΜ) cells. Cells (3 x 105/ml in 10 ml medium) were incubated for 48 hours with the indicated concentrations of simvastatin, CHR2863 and their combination and assessed for the impact on (A) cell viability, (B) apoptosis induction, (C) sub-Gl fraction and (D) cell cycle distribution. Cells incubated for 24 hours with Bortezomib or 48 hours with 6 μΜ CHR2863 served as control for cell growth inhibition and apoptosis induction. Percentages of apoptotic cells in control untreated U937/WT, U937/CHR2863(200) and υ937/ϋΒ¾2863(5μΜ) cells were 4.6 ± 1.9%, 5.2 ± 1.2% and 6.1 ± 0.9%, respectively. Sub-Gl fractions in control untreated U937/WT, U937/CHR2863(200) and U937/CHR2863^M) cells were 4.2 ± 2.0%, 9.1 ± 6.5% and 8.2 ± 2.2%, respectively. Results depicted are the mean ± SD of 4-5 separate experiments.

*: Statistically significant different compared to controls without drugs.

Figure 6. Effects of mevalonic acid, farnesylpyrophosphate and

geranylgeranylpyro-phosphate on simvastatin potentiation of CHR2863 activity. U937/WT, U937/CHR2863(200) and U937/CHR2863^M) cells were incubated for 72 hours with the indicated concentrations of CHR2863 and non-toxic

concentration of simvastatin in the presence of increasing concentrations of (A) mevalonic acid, (B) farnesylpyrophosphate (FPP) and (C) geranylgeranylpyrophosphate (GGPP). Results, depicted are presented as cell growth relative to control, are the mean ± SD of 4 separate experiments. The dashed line indicates the mean growth inhibition by CHR2863 alone.

Figure 7. Effect of simvastatin and CHR2863 combinations on Rheb prenylation. U937/WT, U937/CHR2863(200) and υ937/ΟΙ4Έ2863(5μΜ) cells were incubated for 48 hours with simvastatin, CHR2863, and their combination (as described in Figure 5), with or without additions of mevalonic acid (100 μΜ), FPP (2 μΜ), GGPP (1 μΜ) or FTI-277 (10 μΜ). The slower (upper) migrating band represents unprenylated Rheb, the faster (lower) migrating band represent prenylated Rheb.

Figure 8. Proposed model for synergistic action of statins and aminopeptidase inhibitor CHR2863. (A): Peptide breakdown by aminopeptidases generates amino acids for re-utilizing in protein synthesis. According to previously described models (Zoncu et al., 2011. Science 334: 678-683; Jewell et al., 2013. Nat Rev Mol Cell Biol 14: 133- 139; Dibble and Manning, 2013. Nat Cell Biol 15: 555-564; Duran and Hall, 2012. EMBO Rep 13: 121-128; Groenewoud and Zwartkruis, 2013. Biochem Soc Trans 41: 951-955), an increased intralysomal amino acid content triggers dissociation of V-ATPase and Ragulator-Rag-mTORCl complex. Binding of latter complex to (prenylated) Rheb (in the lysosomal membrane) will then induce mTOR activation and initiation of protein synthesis (B): Inhibition of aminopeptidases by CHR2863 (or bestatin) will reduce the intralysomal amino acid content and a dissociation of the Ragulator-Rag complex from mTORCl. By a different

mechanism, statins may block Rheb prenylation and hamper its lysosomal membrane localization. The combined effect of CHR2863 and statins may then synergize in impairing mTOR activation, protein synthesis and inhibiting cell growth.

Figure 9. Kaplan-Meier (KM)-curves of patients who received tosedosat and a statin versus those that received tosedosat without a statin. DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term "composition", as used herein, refers to a product that comprises a combination of a statin and an aminopeptidase inhibitor. The term "combination" is not intended to imply that said statin and said aminopeptidase inhibitor are formulated in one package for jointly administration, or that they must be administered at the same time, although these methods of administration are within the scope of this term. Said statin may be administered concurrently with, prior to, or after said aminopeptidase inhibitor. In general, said statin and aminopeptidase inhibitor may be administered at a dose and/or in a regimen that is determined as effective for that particular agent.

The term ^"statin^", as used herein, refers to an inhibitor of 3-hydroxy-3- methyl- glutaryl-CoA reductase (HMG-CoA reductase), and to salt or derivatives thereof. Suitable statins include, for example, atorvastatin, pravastatin, rosuvastatin, simvastatin, fluvastatin, lovastatin and pravastatin. All of these statins have well-defined chemical structures that are known to a person skilled in the art (e.g. Srinivasa et al., 2011. Int J Pharm Sciences Drug Res 3: 178-183).

As used herein, the term "aminopeptidase inhibitor^", refers to a compound which inhibits, reduces or represses the activity of one or more aminopeptidases of the class EC 3.4.11, and to salt or derivatives thereof. A preferred aminopeptidase inhibitor inhibits a human aminopeptidase of the Ml zinc- aminopeptidases subfamily. Said aminopeptidase inhibitor mayinhibit one or more of

aminopeptidase N (CD13; EC 3.4.11.2; UniProtKB P15144 (AMPN_HUMAN), leucine aminopeptidase 3 (LAP 3; EC 3.4.11.1; UniProtKB - P28838

(AMPL_HUMAN), aminopeptidase puromycm sensitive (NPEPPS; EC:3.4.11.14; UniProtKB - P55786 (PS A_HUMAN) , and leukotriene A4 hydrolase (LTA4H;

EC:3.3.2.6; UniProtKB - P09960 (LKH A4_HUMAN) . Compounds which inhibit reduce or repress the activity of an aminopeptidase, for example an

aminopeptidase set out above, may be identified using techniques which are well- known in the art. For example, methods to determine the ability of a compound to inhibit the cleavage of a peptide substrate by an aminopeptidase, such as the peptide Leu-Gly- Gly by LAPS, the fluorogenic peptide substrate Ala-AMC by NPEPPS, or the fluorogenic peptide substrate Arg-AMC by LTA4H, are known to a person skilled in the art, for example as described in Krige et al., 2008 (Krige et al., 2008. Cancer Res 68:6669-6679). Examples of suitable aminopeptidase inhibitors include bestatin (CAS 65391-42-6; N- (3R-amino-2S-hydroxy-oxo-4-phenylbutyl)-L- leucine); bestatin analogues such as sulfur-containing amino acid and peptide analogues (e.g. Ocain and Rich, 1988. J Med Chem 31: 2193-2199), lapstatin (3- amino-2-hydroxy-4-methylpentanoylvaline), and actinonin (CAS 13434- 13-4; 3-((l- ((2-[hydroxymethyl]- 1-pyrrolidinyl) carbonyl)-2-methylpropyl) carbamoyl) octanohydroxamic acid)) .

Further suitable aminopeptidase inhibitors are phenylethanoic acid cyclopentyl ester derivatives, for example as described in W09946241, which is incorporated herein by reference. A further suitable aminopeptidase inhibitor is CHR-2863 (cyclopentyl (2S)-2-[[(2R)-2-[(lS)-2-(hydroxyamino)-l-methoxy-2- oxoethyl] -4-methylpentanoyl] amino] -2-phenylacetate) .

An additional suitable aminopeptidase inhibitor is the aminopeptidase inhibitor CHR-2797 (Tosedostat; 2S-[2R-(S-Hydroxy-hydroxycarbamoyl-methyl)-4- methylpentanoylamino]-2-phenylethanoic acid cyclopentyl ester; CAS Registry Number 238750-77- 1). Tosedostat is described as a matrix metalloproteinase inhibitor or an aminopeptidase inhibitor. Tosedostat inhibits members of the Ml and M17 classes of aminopeptidases. Tosedostat is an orally bioavailable agent, which has been investigated in clinical trials for treatment of patients with leukemia (e.g., relapsed/refractory acute myeloid leukemia (AML)) and cancers that are solid tumors.

A further suitable aminopeptidase inhibitor is the aminopeptidase Bestatin, also known as ubenimex. Bestatin is a competitive, reversible protease inhibitor that is known to specifically inhibit cytosol aminopeptidase, aminopeptidase N, zinc aminopeptidase and amino peptidase B. It is derived from Streptomyc.es olivoreticuli. Ubenimex is an orally available agent that is being studied for use in the treatment of acute myelocytic leukemia and lymphedema,

As used herein, the term "treatment" or "treating" refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and may be performed during the course of clinical pathology. Desirable effects of the treatment include preventing occurrence or recurrence of the illness, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the illness, and/or amelioration or palliation of the state of the illness. In the present invention, "treatment" or "treating" is understood to mean amelioration or palliation of a human suffering from a cancer, such as a hematological cancer, by administering a pharmaceutical composition comprising a statin and an aminopeptidase inhibitor.

The term "therapeutically effective amount" refers to a quantity of a specified agent sufficient to achieve a desired effect in a subject being treated with that agent. Ideally, a therapeutically effective amount of an agent is an amount sufficient to inhibit or treat the disease or condition without causing a substantial cytotoxic effect in the subject. The therapeutically effective amount of an agent will be dependent on the subject being treated, the severity of the affliction, and the manner of administration of the therapeutic agent. The term in particular refers to (i) an amount sufficient to inhibit, block or counteract an aminopeptidase inhibitor, when combined with a statin, and/or (ii) an amount sufficient to treat a human suffering from a cancer, which may be a hematological cancer. It is within the knowledge and capabilities of the skilled practitioner to determine therapeutically effective dosage regimens.

The term "administering", as used herein, refers to the physical introduction of an agent or therapeutic compound or composition to a human suffering from a cancer such as a hematological cancer, using any of the various methods and delivery systems known to those skilled in the art. The agent or therapeutic compound or composition inhibits, blocks or counteracts an aminopeptidase, for example a human aminopeptidase of the Ml zinc-aminopeptidases subfamily,.

As used herein, the terms cancer or tumor are clinically descriptive terms that encompass diseases typically characterized by cells that exhibit abnormal cellular proliferation. The term cancer is generally used to describe a malignant tumor or the disease state arising from the tumor. Alternatively, an abnormal growth may be referred to in the art as a neoplasm. The term tumor, such as in reference to a tissue, generally refers to any abnormal tissue growth that is characterized, at least in part, by excessive and abnormal cellular proliferation. A tumor may be metastatic and capable of spreading beyond its anatomical site of origin and initial colonization to other areas throughout the body of the subject. A cancer may be characterized as a solid tumor or liquid tumor (e.g., a leukemia). Formulations and modes of administration

According to the invention, a composition comprising a statin and an aminopeptidase inhibitor may be administered to an individual in need thereof. A skilled person is aware of suitable methods for administration and dosage forms. Suitable dosage forms include, for example, sterile aqueous or non-aqueous solutions, suspensions, emulsions and various oral dosage forms such as tablets, capsules, caplets and the like. In general, the statin and aminopeptidase inhibitor are administered to a subject via a route and at a dose and frequency that are appropriate for the particular agent. Each of the statin and the aminopeptidase inhibitor may be administered by any one of several different routes that effectively delivers an effective amount of the compound. The routes may be the same or different. Such administrative routes include, for example, oral, intravenous, subcutaneous, enteral, rectal, intranasal, buccal, sublingual, intramuscular, topical intradermal, subdermal, and transdermal. An appropriate dose and a suitable duration and frequency of administration for the statin and the aminopeptidase inhibitor may be determined by such factors as the subject's condition, for example, stage of the cancer, severity of symptoms caused by the cancer, general health status, as well as age, gender, and weight, the particular form of the active antineoplastic agent, and the method of administration. Optimal doses of an agent may generally be determined using experimental models and/or clinical trials if not already established in the relevant art. The optimal dose may depend upon the body mass, weight, or blood volume of the subject. Administration of small molecules described herein can be performed by non-parenteral

administration such as by oral and enteral administration. Administering can be performed, for example, once, a plurality of times, and/or over one or more extended periods of time. A regimen may comprises daily administration, or twice daily administration, of said statin and aminopeptidase inhibitor, for a period of at least two months, such as 2-12 months.

A daily dosage of a statin may comprise between 0.5 mg and 100 mg of said statin. For example, routine daily dosages of atorvastatin and fluvastatin are between 10 mg and 80 mg, of lovastatin and pravastatin are between 10 mg and 80 mg, of pravastatin is between 1 mg and 4 mg, of rosuvastatin is between 5 mg and 40 mg, and of simvastatin is between 5 mg and 80 mg. A daily dosage of an aminopeptidase inhibitor may comprise between 0.5 mg and 500 mg of said inhibitor. For example, a routine daily dosage of bestatin is between 10 mg and 80 mg. A routine daily dosage of actinonin is between 1 mg and 200 mg. A routine daily dosage of Tosedostat is a between 100 mg and 500 mg, preferably between 120 mg and 240 mg.

Said daily dosage may be administered by a single tablet, or by a single procedure using other routes of administration such as, for example, injection. Alternatively, said daily dosage may be administered to the patient by two or more tablets, or two or more injections given simultaneously or sequentially to deliver the entire daily dosage to the patient. Alternatively, said daily dosage may be administered to the patient by a combination of routes to deliver the entire daily dosage to the individual.

The daily dosage may then be repeated at intervals of time such as daily, every other day, once a week, etc. Said daily dosage preferably is administered for a period of at least two months, such as 2-12 months.

A daily dosage of a statin and an aminopeptidase inhibitor can be

administered to individuals in such a way that the individual receives a loading dose followed by one or more maintenance doses. For example the loading dose may be a high dose in order to quickly reach a desired plasma concentration and then subsequent maintenance doses are at a lower dose, when compared to the loading dose, in order to maintain the required plasma concentration.

Said statin and an aminopeptidase inhibitor may beorally administered, for example as a tablet. The term "tablet" encompasses a "capsule" and a "caplet". A suitable size of a tablet ranges from a few millimeters to about one centimeter.

Said statin and an aminopeptidase inhibitor preferablyare administered as a pharmaceutical composition further comprising at least one pharmaceutically acceptable excipient. Such excipients are well known in the art and described, for example, in in Rowe et al., Handbook of Pharmaceutical Excipients: A

Comprehensive Guide to Uses, Properties, and Safety, 5^th Ed., 2006, and in

Remington: The Science and Practice of Pharmacy (Gennaro, 21^st Ed. Mack Pub. Co., Easton, PA (2005)). Suitable pharmaceutically acceptable excipients include, for example, preservatives, stabilizers, buffers, dyes, antioxidants, suspending agents, diluents, binders or granulating ingredients, a carbohydrate such as starch, a starch derivative such as starch acetate and/or maltodextrin, a polyol such as xylitol, sorbitol and/or mannitol, a lactose such as a-lactose monohydrate, anhydrous a-lactose, anhydrous β-lactose, spray-dried lactose, and/or agglomerated lactose, sugars such as dextrose, maltose, dextrate and/or inulin, glidants (flow aids) and lubricants to ensure efficient tabletting, sweeteners or flavours to enhance taste, and combinations thereof. In general, the type of excipient is selected based on the mode of administration, as well as the chemical composition of the active ingredient(s). Pharmaceutical compositions may be formulated for any appropriate manner of administration described herein and in the art.

Said tablet may release said statin and aminopeptidase inhibitor at different periods of time and/or in different compartments. Said statin and/or

aminopeptidase inhibitor may be quickly released in a first phase to provide maximum relief within a short time frame, which is followed by a sustained release phase to avoid a need for repeated, i.e. more than twice daily, administration.

Suitable devices for use as a biphasic release system are known in the art and encompass compressed double-layer tablets and "core-within-coating" systems, which involve the use of a sustained release tablet as a compressed core which is coated over the whole surface with a quickly disintegrating formulation. Both the core tablet and the outer coating contain said statin and/or aminopeptidase inhibitor.

Suitable biphasic release devices have been described in the art. For example, WO93/009771 describes a two pulse tablet of flutamide for the treatment of prostate cancer. The first pulse is obtained from an immediate release layer while the second pulse is obtained from a core which contains a solid dispersion of the flutamide in a carrier. The immediate release layer and the core are separated by a film layer of an enteric coating. WO94/12160 describes a capsule which contains a plurality of pellets with varying delay times for release of a drug. By mixing pellets of different delay times in one capsule, one can obtain pulsatile delivery of the drug.

Kits with unit doses of a statin and an aminopeptidase inhibitor are also contemplated by the present invention. Such kits may include a container containing the unit doses, an informational package insert describing the use and attendant benefits of the drugs in treating the cancer, and optionally an appliance or device for delivery of the composition. In certain embodiments, a kit is provided that comprises a pharmaceutical preparation wherein the pharmaceutical preparation comprises a pharmaceutical composition comprising a statin and an aminopeptidase inhibitor.

Treated individuals

The invention provides a composition comprising a statin and an

aminopeptidase inhibitor for use in a method of treating a cancer in an individual. Said cancer is a solid cancer or a liquid cancer.

A solid cancer is preferably selected from melanoma, prostate cancer, testicular cancer, breast cancer, brain cancer, pancreatic cancer, colon cancer, thyroid cancer, stomach cancer, lung cancer, ovarian cancer, Kaposi's sarcoma, skin cancer, squamous cell skin cancer, renal cancer, head cancer, neck cancer, throat cancer, squamous carcinoma that forms on moist mucosal linings, bladder cancer, osteosarcoma, cervical cancer, endometrial cancer, esophageal cancer, liver cancer, kidney cancer, an epithelial cell-derived cancer, and a mesenchymal cell- derived cancer. A preferred solid cancer is a lung cancer, more preferred a non- small cell lung cancer (NSCLC) such as a squamous cell lung cancer.

Said cancer preferably is a hematological cancer, also called blood cell tumor or liquid tumor. A hematological cancer in general occurs in blood, bone marrow, and/or lymph nodes and includes leukemias, such as myeloid leukemia and lymphocytic leukemia, lymphomas such as Hodgkin lymphoma and non-Hodgkin lymphoma, and myelomas e.g., multiple myeloma.

Leukemias include for example, acute leukemias such as acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML), and chronic leukemias such as chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), myelodysplastic syndrome (MDS), and hairy cell leukemia. In elderly patients, a myelodysplasia, which is a clonal disorder, could anticipate the development of AML of few months or years.

A cancer that may be treated with a composition comprising a statin and an aminopeptidase inhibitor according to the invention is AML. AML is a cancer of the myeloid lineage of blood cells, characterized by a rapid growth of myeloblasts, erythrocytes and/or platelets. Myeloblasts are unipotent stem cell that are able to differentiate into neutrophilic, basophilic or eosinophilic myelocytes. AML is the most common acute leukemia affecting adults, and its incidence increases with age. Although AML is a relatively rare disease, accounting for roughly 1.2% of cancer deaths in the United States, its incidence is expected to increase as the population ages.

The symptoms of AML are caused by replacement of normal bone marrow cells with leukemic cells. These symptoms include fatigue, shortness of breath, easy bruising and bleeding, and increased risk of infection. As an acute leukemia, AML progresses rapidly and is typically fatal within weeks or months if left untreated. AML presently is cured in 35-40% of people under 60 years old and 5— 15% over 60 years old. Older people who are not able to withstand intensive chemotherapy have an average survival of 5-10 months.

AML is generally treated initially with chemotherapy aimed at inducing remission. Subsequently, individuals with AML may go on to receive additional chemotherapy or to receive a hematopoietic stem cell transplant.

Inhibition of specific aminopeptidases in tumor cells such as AML may result in amino acid deprivation, inhibition of protein synthesis due to a decrease in the intracellular free amino acid pool, an increase in the level of the proapoptotic protein Noxa, a member of the BH3 (Bcl-2 homology 3)-only subgroup of the proapoptotic Bcl-2 (B-cell CLL/lymphoma 2) protein family, and ultimately cell death.

Aminopeptidase inhibitors such as Tosedostat and its close structural analogue CHR2863 are prodrugs that rely on esterase activities for their

conversion to active metabolites that can inhibit multiple aminopeptidases, thereby provoking amino acid depletion (Krige et al., 2008. Cancer Res 68: 6669-6679). Tosedostat is converted intracellularly into a poorly membrane-permeable active metabolite (CHR-79888) that inhibits the Ml family of aminopeptidases, particularly puromycin-sensitive aminopeptidase (PuSA), and leukotriene A4 (LTA4) hydrolase (see, e.g., Krige et al., 2008. Cancer Res 68: 6669-6679).

Tosedostat has also been reported to inhibit CD 13 (Aminopeptidase N), which is a Zn2+ dependent membrane-bound ectopeptidase that preferentially degrades proteins and peptides with a N-terminal neutral amino acid (see, e.g., Wickstrom et al., 2011. Cancer Sci 102: 501-508). Inhibition of these aminopeptidases in tumor cells such as AML may result in amino acid deprivation, resulting ultimately in death of said tumor cells.

It was recently demonstrated that CESl is the most likely candidate for bio- activation of the prodrugs Tosedostat and CHR2863, given the high CESl expression in myeloid cell lines and M4 and M5 FAB subtypes of AML clinical specimen (Verbrugge et al., 2016. Oncotarget 7: 5240-5257). The role of CESl in this enzymatic bio-activation was further implicated by the fact that acquired resistance to CHR2863 in AML cells was mediated by down-regulation of CESl expression.

CESl has an important physiological function in cholesterol metabolism via conversion of cholesteryl esters to cholesterol (Zhao et al., 2005. J Lipid Res 46: 2114-2121; Ghosh et al., 2010. Vascul Pharmacol 52: 1-10). Aberrant cholesterol metabolism is a characteristic feature of AML cells which has been exploited for therapeutic interventions with statins as inhibitors of HMG-CoA reductase, the key-enzyme in the MVA-cholesterol pathway (Wong et al., 2002. Leukemia 16: 508- 519; Berndt et al., 2011. Nat Rev Cancer 11: 775-791; Thurnher et al., 2012. Clin Cancer Res 18: 3524-35314). Both in vitro and in vivo studies have demonstrated that high concentrations of statins can induce apoptosis in AML cells through perturbations of prenylation and membrane anchoring of proteins involved in signal transduction pathways. In earlier studies, statins showed differential sensitization to AML cells (Xia et al, 2001. Leukemia 15: 1398-1407; van der Weide et al., 2012. Leukemia 26: 845-848; Sassano et al., 2007. Cancer Res 67: 4524-4532; Burke and Kukoly, 2008. Leuk Lymphoma 49: 322-330; de Jonge- Peeters et al., 2009. Ann Hematol 88: 573-580) but were also able to enhance the sensitivity of various anti-leukemic drugs, including cytarabine, daunorubicin and the cell cycle inhibitor UCN-01 (Kornblau et al., 2007. Blood 109: 2999-3006; Dai et al., 2007. Blood 109: 4415-4423).

As is shown in the examples, non-toxic concentrations of statins were able to markedly potentiate the growth inhibitory effects of either a prodrug (CHR2863) or a direct inhibitor (bestatin) of aminopeptidases in human AML cells. In addition, said non-toxic concentrations of statins were able to overcome acquired resistance to aminopeptidases inhibitors by sensitizing 14-fold resistant U937/CrIR2863(200) cells, hence restoring to WT sensitivity. Highly (270-fold) CHR2863 resistant υ937/ΟΗΙ 3863(5μΜ) cells could also be sensitized by co-administration of statins, albeit to a lower level (3-4 fold) even given the fact that active metabolite formation was almost 100-fold lower than in WT cells. Statin- dependent sensitization of CHR2863-resistant cells did not involve increased CES1 expression and/or enhanced active metabolite formation, suggesting that other mechanisms account for this potentiation effect. In drug combination experiments, simvastatin and CHR2863 achieved a significant enhancement of apoptosis induction as reflected in the accumulation of cells in sub-Gl fraction, whereas treatment with either drug alone had a minimal effect. Furthermore, following simvastatin- CHR2863 combinations, cell cycle analysis did not reveal any distinct cell cycle arrest in G1/G0-, S- or G2/M-phase, suggesting that the drug treatment did not interfere with specific cell cycle phases or check points. The potentiation and sensitization of an aminopeptidase inhibitor by statins may involve perturbations in Rheb prenylation as an essential complementary factor to mTOR inhibition by aminopeptidase inhibitors. These novel findings uncover a potent therapeutic combination of statins and aminopeptidase inhibitors for the treatment of AML.

It may be anticipated that the daily dosage of an aminopeptidase inhibitor may be reduced when it is combined with a statin, as said statin potentiates the anti-cancer efficacy of the aminopeptidase inhibitor. For example, said daily dosage of bestatin may be about 10 mg - 20 mg, when combined with a statin. Similarly, the daily dosage of Tosedostat may be about 50 mg - 100 mg, when combined with a statin. A person skilled in the art will understand that a reduction of the daily dosage may also be obtained by administration of the same dosage in a once every second day regimen.

It may be expected that a reduced daily dosage of an aminopeptidase inhibitor may help to reduce some adverse effects that may be observed after administration of an aminopeptidase inhibitor, especially to the elderly. A reduction of adverse effects may have a beneficial effect of treatment of these patients.

As used in the specification and claims, the singular forms "a", "an", "the" and

"said" include plural references unless the context clearly dictates otherwise. For example, the term a pharmaceutical excipient may refer to one or more pharmaceutical excipients for use in the presently disclosed formulations and methods.

For the purpose of clarity and a concise description, features are described herein as part of the same or separate embodiments, however, it will be appreciated that the scope of the invention may include embodiments having combinations of all or some of the features described.

EXAMPLES Example 1

Materials and methods

Chemicals

Simvastatin (430-104-M) was obtained from Alexis Biochemicals (San Diego, CA USA). Fluvastatin (10010337) and lovastatin (10010338) were purchased from Cayman Chemical Co (Ann Arbor, MI, USA). Pravastatin (P4498), R-mevalonic acid (50838), squalene (S3632), farnesylpyrophosphate (F6892),

geranylgeranylpyrophosphate (G6025), FTI-277 (F9803), bestatin (B8385) and daunorubicin (30450) were from Sigma Chemical Co (St Louis MO, USA).

Bortezomib was obtained from the VUmc Pharmacy department. CHR2863, CHR6768, and CHR2875 (Krige et al., 2008. Cancer Res 68: 6669-6679; Needham et al., 2011. J Pharmacol Exp 339: 132-142) were provided by Dr A Drummond (Chroma Pharmaceuticals Ltd, Abingdon, UK) and dissolved in dimethyl sulfoxide as a 10 niM stock solutions and stored at -20°C.

Antibodies

The following antibodies were used for Western blotting: CES1 monoclonal antibody (Lifespan Biosciences, Seattle, WA, USA, LS-C498701, 1: 1000 dilution). Rabbit polyclonal antibodies: total Akt (#9272), phospho-Akt (Ser308) (C31E5E) (#2965), phospho-Akt (Ser473) (#9271), total mTOR (7C10) (#2983), phospho- HITOR (Ser2448) (#2971), phospho-mTOR (Ser2481) (#2974), total S6K (#9202), phospho-S6Kp70 (Th389) (#9205) and Rheb (#4935), all from Cell Signaling- Technology, Danvers, MA, USA at 1: 1000 dilutions). β-Actin antibody was from Sigma- Aldrich, St. Louis, MO, USA (A2172, 1: 10,000 dilutions). Secondary antibodies included goat anti-mouse or goat anti-rabbit antibodies conjugated to IRDyeCR.SOOCW (1: 10.000, Odyssey; LI-COR, Biosciences, Nebraska, USA).

Cell culture

Human U937 myelomonocytic leukemia cells (ATCC, Manassas, VA, USA) and 2 sublines U937/CHR2863(200) and U937/CHR2863(5nM) with 14- and 270-fold acquired resistance to CHR2863, respectively, were isolated and cultured as described previously (Verbrugge et al., 2016. Oncotarget 7: 5240-5257). Other human myeloid leukemia cell lines (THP1, MV4- 11, and KG1), human

lymphoblastic cell lines (CEM and CEM Vbl), human ovarian carcinoma cell lines (2008 and 2008/MRPl), human breast carcinoma cell lines (MCF7/WT and

MCF7/MR), human lung cancer (SW1573) and human nasopharyngeal carcinoma cells (KB) were cultured as described previously (van Luijn et al., 2010.

Haematologica 95: 485-493; Hooijberg et al., 1999. Cancer Res 59: 2532-2535; Westerhof et al., 1995. Cancer Res 55: 3795-3802; Verbrugge et al, 2012. J

Pharmacol Exp Ther 341: 174- 182; Scheffer et al., 2000. Cancer Res 60: 5269-5277; Ifergan et al., 2005. Cancer Res 65: 10952-10958). In short, cells were grown in RPMI-1640 medium (Lonza, Verviers, Belgium) supplemented with 10% fetal calf serum (FCS, PAA Cell Culture Company, Pasching, Austria), 20 mM HEPES, 2 mM L-glutamine, and 100 U/ml penicillin/streptomycin (all from Lonza, Verviers, Belgium). The cell lines were cultured in 25 cm² culture flasks (Greiner Bio-One GmbH, Frickenhansen, Germany) in 10 ml medium at an initial density of 3 x 10⁵ cells/ml (or 1.25 x 104/cm² for adherent cells) and in a humidified atmosphere at 37°C and 5% C02. Cell cultures were refreshed every 3-4 days.

Western blotting

Western blot analysis was performed essentially as described by Verbrugge et al (Verbrugge et al., 2016. Oncotarget 7: 5240-5257). Briefly, cell lysates were prepared from 5 x 106 cells suspended in 150 μΐ ice-cold lysis buffer (Cell Signaling Technology, #9803) containing 4% Protease Inhibitor Cocktail and 1 mM NaV04. Supernatant fractions were collected by centrifugation (13,000 x g for 10 min, 4°C), and 30 pg protein aliquots were fractionated on a 4-20% TGX pre-cast SDS PAGE gels (Bio-Rad), followed by transfer onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA) suitable for chemoluminescent detection by the Odyssey Infrared Imaging System (PerkinElmer, Zaventem, Belgium). The membranes were pre-incubated for 1 hour in blocking buffer (Odyssey Blocking Buffer, LI-COR, Biosciences)) and incubated overnight at 4°C with specific primary antibodies and β-actin as a loading control. After 3 washing steps (phosphate- buffered saline (PBS)/0.05% TWEEN20®), the membranes were incubated for 1 hour with secondary antibodies. Detection of antibody binding was followed using the LI-COR Odyssey scanner (Biosciences) according to the manufacturers' instructions. Digital image acquisition and quantification was performed using the Odyssey infrared imaging system software (version 3.0.16, LI-COR Biosciences, Nebraska, USA).

Apoptosis assay

Cells were collected and washed three times with ice cold PBS. Early phase apoptosis was determined by the Annexin-V/7AAD Kit (PN IM3614, Beckman Coulter) using a FACSCalibur flow cytometer (Becton and Dickinson, San Jose, CA) using the manufacturers' protocol. In short, cells were washed and

resuspended in binding buffer. Annexin-V (1: 10) and 7-Amino-Actinomycin (7AAD, 1:20) were added and incubated for 15 min on ice in the dark. Binding buffer was added and analysis by flow cytometry followed within 1 hour. Flow cytometric analysis was performed using FACSCalibur flow cytometer (Becton and Dickinson, San Jose, CA) and Cell Quest software. Annexin-V-positive and 7AAD-positive cells were considered as apoptotic cells.

Cell cycle analysis

Cell cycle analysis was performed using a FACSCalibur flow cytometer (Becton and Dickinson, San Jose, CA) and propidium Iodide (PI) staining (Bijnsdorp et al., 2010. Cancer Sci 101: 440-447). Cells were washed three time with ice cold PBS and resuspended in medium. PI (5% Propidium Iodide dissolved in PBS with 1% trisodium citrate, 0.1% RNAse and 0.1% Triton X-100) was added, cells were vortexed and measured directly by flow cytometry. Fluorescence signal was detected through the FL2 channel. FACS analysis was performed using Cell Quest software.

Miscellaneous assays

Quantitative RT-PCR analysis to assess CES1 mRNA levels and LC-MS/MS analyses to determine the conversion of the prodrug CHR2863 to its metabolite CHR6768 were performed essentially as described before (Verbrugge et al

(Verbrugge et al, 2016. Oncotarget 7: 5240-5257).

Statistical analysis of synergism

Combination indexes (CI) for analysis of synergism between Simvastatin and CHR2863 were calculated by CalcuSyn software (Version 1.1.1, copyright Biosoft 1996) (Bijnsdorp et al., 2011. In "Methods and Protocols", Second edition, Methods in Molecular Biology (I A Cree (ed)) 731, 421-434) and the multiplicative model to predict effect of drug combinations (Valeriote and Lin, 1975. Cancer Chemother Rep 59: 895-900).

Statistics

For comparison between groups, a two-sided paired Student's t-test was used. Differences were considered to be significant at p < 0.05.

Results

Sim vastatin synergizes CH 2863 growth inhibition in paren tal and

CHR2863-resistant U937 cells

Growth inhibitory effects of CHR2863 were determined in human U937/WT cells and two recently established low (U937/CHR2863(200) and high

(U937/CHR2863^M)) CHR2863-resistant U937 cells (Verbrugge et al., 2016. Oncotarget 7: 5240-5257) in the absence or presence of a maximal non-toxic concentration of 2.0-2.5 μΜ Simvastatin (Figure 1). For U937/WT cells (Figure 1A), simvastatin potentiated the growth inhibitory effects of CHR2863 by 14-fold (from IC50: 60.9 ± 15.8 nM to 4.3 ± 1.3 nM). Similarly, simvastatin potentiated CHR2863 activity 18-fold in U937/CHR2863(200) cells (from IC50: 682 ± 182 nM to an IC50 of 37.8 ± 10.8 nM) which compares to CHR2863 sensitivity of U937/WT cells

(Figure IB). Lastly, simvastatin also potentiated the growth inhibitory effect of CHR2863 in U937/CHR2863^M) cells, albeit with a lower potentiation factor; 3.3-fold (from IC50: 12,900 ± 4,300 nM to 3,900 ± 2,200 nM) (Figure 1C). Analysis of dose response effect of drug interactions at a constant dose of simvastatin and fractional effect by CHR2863 revealed remarkable combination indices (CI) well below 1 for parental and CHR2863-resistant U937 cells, indicating a strong synergistic interactions (Figure ID), especially at the FA > 0.5, which is considered as relevant because growth is almost completely inhibited. Multiple statins synergize with CHR2863 in U937/WT and CHR2863- resistant U937 cells

It was next assessed whether or not statins other than simvastatin also had the ability to synergize with CHR2863 activity in U937AVT and CHR2863-resistant cells. Maximal non- toxic concentrations of the naturally- derived statins lovastatin (2.5-5 μΜ) and pravastatin (100-200 μΜ), as well as the synthetic statin fluvastatin (0.5- 1 μΜ) exhibited comparable capacities as simvastatin to potentiate CHR2863 activity as depicted by their potentiation factors (ratio IC50 CHR2863 with statin over IC50 CHR2863 without statin) (Figure 2).

Statin potentiation is selective for am inopeptidase inhibitors

To assess whether or not statin potentiation of the aminopeptidase inhibitor prodrug CHR2863 also extends to a direct aminopeptidase inhibitor, it was tested whether the growth inhibition of bestatin (Hitzerd et al., 2014. Amino Acids 46: 793-808; Ota and Uzuka, 1992. Biotherapy 4: 205-214) is potentiated by

simvastatin. Indeed, simvastatin potentiated both CHR2863 and bestatin activity with similar potentiation factors in U937AVT and CHR2863-resistant U937 cells (Figure 3). Moreover, statin potentiation appeared selective for aminopeptidase inhibitors as no potentiation was observed for two types of other drugs: CHR2875, which is activated similarly as CHR2863, but is HDAC inhibitor prodrug

(Needham et al., 2011. J Pharmacol Exp Ther 339: 132- 142, and daunorubicin used in combination chemotherapy with Tosedostat for AML (Figure 3).

Statin potentiation of CHR2863 activity is prim arily restricted to AML cells To determine whether statin potentiation of CHR2863 activity occurs in various human AML cells other than U937 cells, the potentiating effect of maximal nontoxic concentrations of simvastatin was examined in multiple AML cell lines, acute lymphocytic leukemia (ALL) CCRF-CEM cells as well as a panel of (multidrug resistance-related) solid tumor cell lines (Figure 4). As with U937 cells, CHR2863 growth inhibition was significantly potentiated by simvastatin in various AML cell lines including THP1, MV4- 11 and to a lower extent for KG1 cells. In contrast, simvastatin had no potentiating effect in CCRF-CEM cells and a P- glycoprotein/MDRl-overexpressing subline CEM VBL, although it should be noted that these cells had a low intrinsic sensitivity to CHR2863 (IC50 > 10 μΜ). The panel of solid tumor cell lines displayed variable sensitivity to CHR2863 (IC50: 0.13 - 6.7 μΜ), with the exception of MCF7 MR cells, however, none showed a potentiating effect by simvastatin. These results indicate that the simvastatin potentiating effect of CHR2863 is largely restricted to AML cells.

Sim vastatin - CHR286S combinations: impact on cell growth, apoptosis and cell cycle

An exposure of 48 h to maximal non-toxic concentrations of simvastatin and minimally cytotoxic (-IC10) concentrations of CHR2863 was tested for the impact on cell viability, apoptosis induction and sub-Gl fraction/cell cycle distribution of U937/WT, U937/CHR2863(200) and ΙΙ937/ΟΗΉ2863(5μΜ) cells (Figure 5).

Bortezomib (0.1 μΜ) and a high concentration of CHR2863 (6 μΜ) were included as a reference control. Single doses of CHR2863 and simvastatin had no effect on cell viability, whereas their combination significantly reduced cell viability in all three cell lines (Figure 5A) which was accompanied by a significantly increased apoptosis (Figure 5B) and an increase in the sub-Gl fraction (Figure 5C). No visible alterations in cell cycle distribution were noted at the tested concentrations of CHR2863, simvastatin or their combination (Figure 5D). The impact of

simvastatin and CHR2863 combinations on cell viability and apoptosis for the U937 cell lines were also found for 3 other AML cell lines including THP1 and MV4- 11 and to a lesser extent for KG1 cells (data not shown).

Reversal of simvastatin potentiation of CHR2863 activity by mevalonic acid, farnesylpyrophosphate and geranylgeranylpyrophosphate

To determine whether or not the statin-induced inhibition of HMG-CoA reductase is implicated in the simvastatin potentiation of CHR2863 cytotoxicity, it was assessed whether or not intermediates of the mevalonate pathway, i.e. mevalonic acid (MVA), farnesyl pyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP) were able to abrogate the potentiating effect of simvastatin. Increasing concentrations of MVA fully abrogated simvastatin potentiation of CHR2863 growth inhibition in U937/WT, U937/CHR2863(200) and U937/CHR2863(f^M) cells (Figure 6A). Likewise, increasing concentrations of FPP also abrogated the simvastatin potentiation effect of CHR2863 in U937/WT and U937/CHR2863(200) cells, albeit to a slightly lower extent than MVA (Figure 6B). Of note, FPP did not abrogate the simvastatin potentiation effect of CHR2863 in υ937/( ΗΈ2863(5μΜ) cells (Figure 6B). Lastly, GGPP abrogated the simvastatin potentiation effect of CHR2863 in U937/WT and U937/CHR2863(200) cells at an optimal concentration of 0.1 μΜ; above this concentration the abrogating effect was lost (Figure 6C). GGPP was unable to abrogate the potentiation effect of simvastatin in

υ937/ΟΗΈ2863(5μΜ) cells (Figure 6C).

Given the variable effects of FPP in abrogating the potentiating effect of simvastatin of CHR2863 in U937/WT and U937/CHR2863(200) cells vs.

υ937/ΟΗΈ2863(5μΜ) cells, we further examined whether a farnesyltransferase inhibitor (FTI-277) had similar effects on CHR2863 potentiation in these cells. Combinations of FTI-277 and CHR2863 were synergistic in U937/WT and additive in U937/CHR2863(200) cells, whereas U937/CHR2863^M) cells were resistant to FTI277 and no potentiation was found (data not shown).

Sim vastatin potentiation of CHR2863: mechanistic studies

To explore the mechanistic basis underlying the potentiation of CHR2863 growth inhibition by simvastatin, we first examined whether simvastatin upregulated the expression of carboxylesterase 1 (CES1), the enzyme involved in the conversion of CHR2863 to its active metabolite CHR6768 (Verbrugge et al., 2016. Oncotarget 7: 5240-5257). Western blot analysis revealed that expression of CES1 (as well as its other family members CES2 and CES3) in U937 WT, U937/CHR2863(200), and υ937/ΟΗΈ2863(5μΜ) cells was not altered with simvastatin and CHR2863 alone, in combination, and in combination with MVA (data not shown). Of note, υ937/ΟΚ¾2863(5μΜ) cells displayed markedly decreased CES1 expression levels as recently shown (Verbrugge et al., 2016. Oncotarget 7: 5240-5257). Consistent with unaltered CES1 expression levels in the presence of simvastatin, the ability of U937/WT and U937/CHR2863(200) cells to enzymatically convert CHR2863 to its active metabolite CHR6768 was unchanged, while U937/CHR2863(5jiM) cells had lower levels in line their lack of CES1 activity (data not shown).

Aminopeptidase inhibition can regulate mTOR activity (Krige ET AL., 2008.

Cancer Res 68: 6669-6679). Therefore we determined whether concentrations of simvastatin and CHR2863 which showed an enhanced growth inhibitory effect (after 48 hr drug incubation) were also associated with an altered cellular phosphorylation status of components of the ERK/Akt/mTOR pathway. Analysis of pERK(Thr202/Tyr204), pAkt(Ser473), pmTOR(Ser2448), pmTOR(Ser2481) and pS6Kp70(Th389) expression m U937/WT, U937/CHR2863(200), and υ937/ΟΗΙ 2863(5μΜ) cells showed no major differences upon exposure to

CHR2863, simvastatin, mevalonic acid, and their combinations (data not shown). This suggests that other mechanisms play a more prominent role in the synergy between statins and CHR2863.

Statins are known to impair the prenylation and membrane localization of various proteins (Berndt ET AL., 2011. Nat Rev Cancer 11: 775-791; Thurnher et al, 2013. Biochim Biophys Acta 1831: 1009- 1015; Greenwood et al., 2006. Nat Rev Immunol 6: 358-370). In the context of mTOR activation, it has been demonstrated that lysosomal membrane integration of Rheb protein is of relevance and is prenylation- dependent (Zoncu et al., 2011. Science 334: 678-683; Jewell et al, 2013. Nat Rev Mol Cell Biol 14: 133- 139; Dibble and Manning, 2013. Nat Cell Biol 15: 555-564). To this end, we examined whether under conditions that potentiated CHR2863 activity, simvastatin interfered with Rheb prenylation in U937/WT,

U937/CHR2863(200) and ΙΤ937/0,Ε¾2863(5μΜ) cells. Indeed, exposure to simvastatin resulted in a marked increase in unprenylated Rheb in all 3 tumor cell lines (Figure 7), as did the exposure to FTI-277. The level of unprenylated Rheb was retained in CHR2863-simvastatin combinations, whereas CHR2863 exposure alone had no effect on Rheb prenylation status. MVA and FPP, but not GGPP, abrogated the unprenylation impact of simvastatin alone and in combination with CHR2863. Hence, this profile of Rheb unprenylation parallels the statin and FTI- inhibitor induced potentiation of CHR2863 activity in U937/WT,

U937/CHR2863(200) and U937/CHR2863^M) cells.

A composite summary model which proposes a mechanistic basis for the synergistic action of aminopeptidase inhibitors and statins in AML cells is presented and discussed in Figure 8.

Example 2

Materials and methods

Clinical trial

Patients that had participated in a randomised phase 2 study (OPAL study;

ClinicalTrials.gov, number NCT00780598; Cortes et al., 2013. Lancet Oncol 14: 354-62), were analyzed for concomitant use of statins. Patients were aged 60 years or older with AML that had relapsed after a first complete remission lasting less than 12 months, or had achieved no previous complete remission. All 73 patients had received tosedosat, either as first salvage at 120 mg once daily for 6 months < at 240 mg once daily for 2 months followed by 120 mg for 4 months.

Results

The KM-curve shown in Figure 9 has the patients who were on a statin versus those that were not. Although the number of patients is rather small, the data seem to support a clinically useful interaction between tosedosat and statins.

Claims

1. A composition comprising a statin and an aminopeptidase inhibitor.

2. The composition according to claim 1, for use as a medicament.

3. The composition according to claim 1 or claim 2, for use in a method of treating cancerpreferably a hematological cancer..

4. The composition according to claim 3 wherein the hematological cancer is acute myeloid leukemia or myelodysplastic syndrome.

5. The composition according to any one of the previous claims, wherein said composition is for simultaneous or separate administration of said statin and said aminopeptidase inhibitor.

6. The composition according to any one of the previous claims, wherein said statin is selected from atorvastatin, pravastatin, rosuvastatin, simvastatin, fluvastatin, lovastatin and pravastatin.

7. The composition according to any one of the previous claims, wherein said aminopeptidase inhibitor specifically inhibits a human aminopeptidase of the Ml zinc-aminopeptidases subfamily, preferably aminopeptidase N, leucine aminopeptidase, and/or puromycin-sensitive aminopeptidase.

8. The composition according to any one of the previous claims, wherein said aminopeptidase inhibitor is a phenylethanoic acid cyclopentyl ester derivative.

9. The composition according to any one of the previous claims wherein the aminopeptidase inhibitor is tosedostat.

10. The composition according to any one of the previous claims, which is a pharmaceutical composition further comprising at least one pharmaceutically acceptable excipient.

11. The composition according to any one of the previous claims, which is a tablet such as a tablet with differential release properties for the statin and the aminopeptidase inhibitor.

12. A method of treating a cancer in an individual, said method comprising simultaneously or separately providing the individual with a therapeutically effective dose of a composition comprising a statin and an aminopeptidase inhibitor.

13. The method according to claim 12, wherein the statin and the aminopeptidase inhibitor are administered in a tablet, optionally with differential release properties for the two compounds.

14. The method according to claim 12 or claim 13, wherein the cancer is a hematological cancer such as acute myeloid leukemia or myelodysplastic syndrome.

15. A use of an aminopeptidase inhibitor in a method for the preparation of a medicament for the treatment of a cancer in an individual, whereby said medicament further comprises a statin.