OF NASOPHARYNGEAL CARCINOMA

FIELD OF THE INVENTION:

The present invention relates to methods and pharmaceutical compositions for the treatment of nasopharyngeal carcinoma.

BACKGROUND OF THE INVENTION:

Nasopharyngeal carcinoma (NPC), also known as cavum cancer, develops in the upper part of the pharynx. Described for the first time in 1921 it is a heterogeneous tumor composed of large epithelial cells and of lymphocytes, reason why this tumor has first been presented as a lymphoepithelioma. In the 1970's, graft experiments in nude mice have revealed that the epithelial cells were the only malignant part of these lymphoepitheliomas. In 1973, the presence of Epstein-Barr virus (EBV) genomes in the tumor cells was reported and it is now clear that the association between this tumor and EBV is very tight, as all tumor cells of nearly 100% of the cases contain the viral genome. Moreover, the structure of the terminal repeat sequences of the virus indicates that in each tumor, the virus is clonal, and was thus present in the initial cell of which the transformation gave rise to the tumor. This observation suggests that the virus could play a causal role. However, despite huge efforts for more than 40 years, the role of the virus is still elusive. Moreover, it is likely that environmental and genetic factors are also involved. Surprisingly, despite the fact that EBV is an ubiquitous virus, infecting in a latent manner more than 95% of the adult human world population, NPC presents a remarkable geographic distribution with three endemic regions: South China and South Eastern Asia (30-80 cases/100000 inhabitants/year), North Africa, Maghreb and Mashreq (8-10 cases/100000 inhabitants/year) and Inuit populations in the Great North (3-5 cases/ 100000 inhabitants/year). In the rest of the world, this pathology is rare and 2 to 3 orders of magnitude less frequent than in the endemic zones.

From a clinical point of view, the tumor localization precludes surgery and for a long time, radiotherapy has been the treatment of choice. However, even if this treatment is efficient, it does not protect against metastases nor relapses in a great proportion of cases, most often resulting in a fatal issue. Indeed about 50% of patients develop recurrence or metastasis after radiotherapy and at this stage there is still no effective treatment. Metastases are often cervical lymph node metastasis, but there are also lung metastases, bone, liver or distal lymph nodes. At present, different protocols of chemotherapy, combined or not with radiotherapy, are under evaluation and interferon and cisplatin seem promising. However, the numerous therapeutic failures and the scarcity of available efficient treatments largely justify the search for new therapeutic molecules against this very frequent (in some regions of the world) although orphan tumor.

The inventors have previously searched for molecules with differential toxicity on normal cells and metastatic melanoma cells and in particular for molecules that could be able to reduce the development of metastases. The inventors found that macrocyclic lactones kill melanoma cells in vitro and decrease lung metastatic implantation of murine and human melanoma cells in immunodepressed mice. Ivermectin, 22, 23-dihydroavermectin Bla, is a macrocyclic lactone, widely used as anthelmintic and insecticidal agent that has also shown protective effects against cancers. Furthermore we have also demonstrated that PAKl is the macrocyclic lactones target involved in their cytotoxicity against melanoma. P21 -activated Kinase 1 (PAK-1), (also known as Serine/threonine-protein kinase PAK 1 or P21 protein (Cdc42/Rac)-activated kinase 1) contributes to MAPK pathway activation. PAKl phosphorylates CRAF at Ser 338 and MEKl at Ser 298. PAKl is a critical effector that link the Rho GTPases to cytoskeleton reorganization and nuclear signaling and has been implicated in a wide range of biological activities, including roles in cell transformation, cell motility and morphology, tumor growth and tumorigenesis.

SUMMARY OF THE INVENTION:

The present invention relates to methods and pharmaceutical compositions for the treatment of nasopharyngeal carcinoma. In particular, the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

Here the inventors show that macrocyclic lactones and PAKl inhibitors are cytotoxic in vitro and in vivo against nasopharyngeal carcinoma (NPC) tumor cells. Given the need for new pharmacological drugs against NPC tumors the findings have important implications for the development of new strategies for the treatment of this orphan disease.

Accordingly a first object of the present invention relates to a method of treating nasopharyngeal carcinoma in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a PAK-1 inhibitor.

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

As used herein, the term "PAK-1" has its general meaning in the art and refers to P21- Activated Kinase 1, also known as Serine/threonine-protein kinase PAK 1, or P21 protein (Cdc42/Rac)-activated kinase 1. PAK-1 is a member of p21 -activated kinases family (PAKs) involved in the ERK activation, MAPK pathway activation and that are critical effectors that link the Rho GTPases to cytoskeleton reorganization and nuclear signalling and have been implicated in a wide range of biological activities.

As used herein, the term "PAK-1" inhibitor refers to any compound that is able to inhibit the activity or expression of PAK-1. In particular the PAK-1 inhibitor blocks PAK-1 interaction with proteins involved in ERK pathway and MAPK pathway such as RAF-1 (CRAF), inhibits its phosphorylation, or blocks MAPK cascade. The term "PAK-1 antagonist" refers to a compound that selectively blocks or inactivates PAK-1. As used herein, the term "selectively blocks or inactivates" refers to a compound that preferentially binds to and blocks or inactivates PAK-1 with a greater affinity and potency, respectively, than its interaction with the other sub-types or isoforms of the PAKs family.

Example of PAK-1 inhibitors include the compounds described in WO2004007504, WO2006072831, WO2007023382, WO2007072153, WO2009086204, WO2010071846, WO2011044264, WO2011044535, WO2011156640, WO2011156646, WO2011156775, WO2011156780, WO2011156786, and WO 2013026914.

Additional examples of PAK-1 inhibitors include, but are not limited to, staurosporine, 3-hydroxystaurosporine, K252a, CEP-1347, OSU-03012, DW12, FL172 (disclosed in Yi et al., Biochemical Pharmacology, 2010, 80:683-689, the disclosure of which with respect to Pakl inhibitor compounds is hereby incorporated herein by reference), IP A3 (commercially available from Tocris), PF-3758309, PAK10 (available from Calbiochem), EKB569, TKI258, FRAX-597 and SU-14813.

In some embodiments, the PAK-1 inhibitor is a macrocyclic lactone. As used herein, the term "macrocyclic lactones" has its general meaning in the art and refers to macrocyclic lactones and macrocyclic lactones derivatives described in Lespine A. Lipid-like properties and pharmacology of the anthelmintic macrocyclic lactones. Expert Opin Drug Metab Toxicol. 2013 Dec; 9(12): 1581-95. Macrocyclic lactones, like ivermectin, are capable of inhibiting PAK-1 activity (e.g. HASMIMOTO ET AL: "Ivermectin inactivates the kinase PAK1 and blocks the PAK1 dependent growth of human ovarian cancer and NF2 tumor cell lines", DRUG DISCOV. THERAPEUTICS, vol. 3, no. 6, 2009, - 2009, pages 243-246). Examples of macrocyclic lactones include those described in WO 2012078605, WO 2012150543, WO2011075592, W0199316189, and WO2012028556. In some embodiments, examples of macrocyclic lactones include but are not limited to Ivermectin (Stromectol), Doramectin, Selamectin, Moxidectin, Milbemycin, Abamectin, Nemadectin and Eprinomectin.

In some embodiments, the PAK-1 inhibitor is an inhibitor of PAK-1 expression. An "inhibitor of expression" refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene. In a preferred embodiment of the invention, said inhibitor of gene expression is a siRNA, an antisense oligonucleotide or a ribozyme. For example, anti- sense oligonucleotides, including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of PAK-1 mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of PAK-1, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding PAK-1 can be synthesized, e.g., by conventional phosphodiester techniques. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566, 135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732). Small inhibitory RNAs (siR As) can also function as inhibitors of expression for use in the present invention. PAK-1 gene expression can be reduced by contacting a subject or cell with a small double stranded R A (dsPvNA), or a vector or construct causing the production of a small double stranded R A, such that PAK-1 gene expression is specifically inhibited (i.e. RNA interference or RNAi). Antisense oligonucleotides, siRNAs, shRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid to the cells and typically cells expressing PAK-1. Typically, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art. In some embodiments, the inhibitor consists in a vector that comprises the CRISPR/cas 9 protein and the appropriate RNA guide for disrupting the expression level of the gene encoding for PAK- 1.

By a "therapeutically effective amount" of the PAK-1 inhibitor as above described is meant a sufficient amount of the compound. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidential with the specific inhibitor employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

Typically, the PAK-1 inhibitor of the present invention is combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Typically, the pharmaceutical compositions contain vehicles, which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Sterile injectable solutions are prepared by incorporating the PAK-1 inhibitor at the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

In some embodiments, the P AK- 1 inhibitor of the present invention is administered to the subject in combination with at least one chemotherapeutic agent. The term "chemotherapeutic agent" refers to chemical compounds that are effective in inhibiting tumor growth. Examples of chemotherapeutic agents include alkylating agents such as Dacarbazine, thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaorarnide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a carnptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estrarnustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimus tine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as the enediyne antibiotics (e.g. calicheamicin, especially calicheamicin (11 and calicheamicin 21 1 , see, e.g., Agnew Chem Intl. Ed. Engl. 33:183-186 (1994); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, canninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idanrbicin, marcellomycin, mitomycins, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomgrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophospharnide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pento statin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; sizofiran; spirogennanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylarnine; trichothecenes (especially T-2 toxin, verracurin A, roridinA and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobromtol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.].) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-1 1 ; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are antihormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and phannaceutically acceptable salts, acids or derivatives of any of the above.

In some embodiments, the PAK-1 inhibitor is administered to the subject in combination with radiotherapy. In some embodiments, the radiation is one of x-ray and gamma ray. For example, but not by way of limitation, x-ray radiation can be administered; in particular, high-energy megavoltage (radiation of greater that 1 MeV energy) can be used for deep tumors, and electron beam and orthovoltage x-ray radiation can be used for the treatment of NPC. Gamma ray emitting radioisotopes, such as radioactive isotopes of radium, cobalt and other elements may also be administered to expose tissues to radiation. However, any radiation therapy protocol can be used. Radiation therapy as used herein includes both ionizing and non-ionizing radiation. Non-ionizing radiation may be used, for example, in connection with photodynamic therapy ("PDT") and PDT-photosensitizing agents.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: Cytotoxicity of macrocyclic lactones and PAK1 inhibitor on NPC cells.

(A, B and C) Three human NPC cell lines (HONE-1, CNE-1 and C666-1) were cultivated 72h with increasing concentrations of four macrocyclic lactones: Ivermectin, Selamectin, Moxidectin and Abamectin, with the PAK1 inhibitor FRAX-597 or with vehicle alone (RPMI/DMSO 0,25%). Surviving cells were counted using a BD Accuri C6 FACS analyzer. Macrocyclic lactones and PA 1 inhibitor show similar cytotoxicity on EBV positive (C666-1) and EBV negative (HONE-1 and CNE-1) NPC cells.

Figure 2: Macrocyclic lactones cytotoxicity on NPC cells

HONE-1 , C666-1 and CNE-1 cells were seeded at 5xl0 ³ /well and treated for 72h with increasing concentration of Ivermectin, Selamectin, Moxidectin, or Abamectin. Surviving cells were quantified using a Cellomics Arrayscan microscope. Results show that Ivermectin and the three other macrocyclic lactones are effective to kill EBV negative and EBV positive NPC cells.

Figure 3: Pictures illustrating Ivermectin cytotoxicity on NPC cells.

HONE-1 and CNE-1 cells were treated with increasing concentrations of Ivermectin (0,6; 1,25; 2,5, 5 and 10 μΜ) for 72h. After treatment pictures were taken using a Cellomics Arrayscan microscope (10X objective). These pictures illustrate Ivermectin cytotoxicity on HONE-1 and CNE-1 cells.

Figure 4: Macrocyclic lactones and PAK1 inhibitor are not cytotoxic on normal thymocytes

Freshly extracted mouse thymocytes were cultivated 72h with increasing concentrations of four macrocyclic lactones: Ivermectin, Selamectin, Moxidectin and Abamectin, with the PAK1 inhibitor FRAX-597 or with vehicle alone (RPMI/DMSO 0,25%). Surviving cells were counted using a BD Accuri C6 FACS analyzer. Results show that thymocytes are resistant to macrocyclic lactones and PAKl inhibitor at least up to a dose of 10 mM.

Figure 5: Intra-peritoneal Ivermectin injections reduce local HONE-1 tumors growth in immunodepressed mice.

(A) HONE-1 cells were injected s.c. (Ixl0 ⁶ /mouse) in 10 NMRI nude mice and left untreated for 48h. Then mice were separated in two groups of 5 mice and daily IP injected for 18 days with either 0,1 mL of vehicle alone (PBS/DMSO 0,25%) or with 3.25mg/kg of Ivermectin. Tumors surfaces were measured using digital caliper every 2-3 days and mice were sacrificed at day 21. (B) HONE-1 cells were injected s.c. (2xl0 ⁶ /mouse) in 8 NMRI nu/nu mice and left untreated for 48h. Then mice were separated in two groups, one of 2 and one of 6 mice. These mice were daily IP injected for 12 days with either 0,1 mL of vehicle alone (PBS/DMSO 0,25%) for the group of 2 mice or with 3.25mg/kg of Ivermectin for the group of 6 mice. Tumors growth, measured every 2-3 days using digital caliper, is shown in comparison with the tumors surfaces at day 5. Mice were sacrificed at day 15.

Figure 6: Intra-peritoneal Ivermectin injections reduce local CNE-1 tumors growth in immunodepressed mice.

CNE-1 cells were injected s.c. (2xl0 ⁶ /mouse) in 11 NMRI nude mice and left untreated for 48h. Then mice were separated in two groups of 4 and 7 mice and daily IP injected for 15 days. The group of 4 mice was injected with 0,1 mL of vehicle alone (PBS/DMSO 0,25%)) and the group of 7 mice with 3.25mg/kg of Ivermectin. Tumors surfaces were measured every 2-4 days using digital caliper. Tumors growth is illustrated in comparison with the tumors surfaces at day 5. Mice were sacrificed at Day 18.

Figure 7: Intra-peritoneal Ivermectin injections are not toxic for immunodepressed mice.

Ivermectin at 3,25 mg/kg or vehicle (PBS/DMSO 0,25%) were daily administered for

20 days by intra-peritoneal (IP) injections in 13 NMRI nude mice. Mice were weighed before and after injections showing that Ivermectin injections do not induce any significant weight loss.

Figure 8: In NPC cells Ivermectin treatment reduces MAPK pathway activation and RAF1 phosphorylation.

(A) Quantifications of phosphorylated RAFl and ERK1/2 protein expression. CNE-1 cells were treated for 72 h with Ivermectin at 0,3 and 3 μΜ or with vehicle alone and then lysed for analysis of p-ERKl/2, ERK1/2 and p-RAFl expression by Western Blot. (B) Quantifications of phosphorylated ERK1/2, RAFl and MEK proteins expression as compared to expression of corresponding total proteins expression. HONE-1 cells were treated for 72 h with Ivermectin at 0,3 and 3 μΜ or with vehicle alone, then lysed for analysis of p-ERKl/2, ER 1/2, p-RAFl, RAF1, p-MEK and MEK expression by Western Blot. Actin was used as a loading control.

EXAMPLE:

Material & Methods

Mice

Six- to eight-wk-old female NMRI nu/nu mice (JANVIER Labs) were used for in vivo experiments. These immunodepressed mice were used to limit the in vivo immune response against NPC cells. All the experiments involving mice were done using appropriate conditions of husbandry experimentation and care, under the control of the Regional Comity of Midi-Pyrenees (France).

Cell lines

Three human NPC cell lines were provided by Dr. P. Busson (UMR 8126 - Gustave Roussy 94805 Villejuif - France): HONE-1 (EBV negative), CNE-1 (EBV negative) and C666-1 (EBV positive). These cell lines were maintained in culture by serial passages in culture medium composed of RPMI 1640 medium supplemented with 10% fetal bovine serum (FCS), ImM glutamine and 1% penicillin-streptomycin-amphotericin B. They were monthly tested to be mycoplasm-free.

Thymuses were harvested from four-wk-old female C57BL/6 mice, minced and single-cell suspensions of thymocytes were prepared and cultivated at 5xl0 ⁶ cells/well in 48 wells culture plates.

Evaluation of macrocyclic lactones and anti-PAKl inhibitor toxicity on human NPC cells and on normal murine thymocytes

HONE-1, CNE-1 and C666-1 NPC cells were treated in vitro for 72h with four macrocyclic lactones: Ivermectin, Selamectin, Moxidectin or Abamectin (purchased from Sigma-Aldrich) and one PAK1 inhibitor: FRAX-597 (purchased from CliniScience) at different doses from 1 to 10 μΜ. Normal thymocytes were similarly treated with these macrocyclic lactones and PA 1 inhibitor. The cells were also cultivated 72h with vehicle alone (RPMI/DMSO 0,25%). Surviving cells were counted after 72h of culture with a BD Accuri C6 FACS analyzer or with a Cellomics Arrayscan microscope.

In vivo NPC subcutaneous tumor growth

To evaluate the impact of repeated intra-peritoneal (IP) Ivermectin injections on local subcutaneous NPC tumor growth, NMRI-nu/nu mice were injected subcutaneously (s.c.) into the flank with lxl 0 ⁶ or 2xl0 ⁶ HONE-1 or CNE-1 tumor cells. Two days later and every following day, mice were IP injected with 0,1 mL of vehicle (PBS/DMSO 0,25%) or 0,1 mL of vehicle containing 3,25 mg/kg of Ivermectin. This dose was chosen because it corresponded approximately to the dose given to humans as anthelmintic agent. Mice were monitored for tumor growth every 2-4 days by palpation and tumor surfaces were measured using digital caliper. Tumor-bearing mice were sacrificed at day 15, 18 or 21 after tumor injection. At this time all mice were alive, behaved normally and did not loose weight, while the tumors did not display ulcerations. Two groups of mice, IP injected with vehicle or Ivermectin, were compared. After s.c. injection of lxlO ⁶ HONE-1 cells, palpable tumors appeared around day 6 and they grew progressively. On the other hand after s.c. injection of 2xl0 ⁶ HONE-1 or CNE-1 cells, substantial tumors were already present at day 5 and their subsequent growth was reduced.

Western blot analyses

HONE-1 and CNE-1 cells were lysed in lysis buffer (20 mM Tris pH 7.6, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, Triton, 1% proteases, and phosphatase inhibitor cocktail), and protein extracts were prepared by the standard procedure and then separated on sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel electrophoresis (30 μg protein/lane). Proteins were blotted onto polyvinylidendifluoride membranes. The filters were incubated at 4°C overnight with the different primary antibodies against: p-ERK (Cell Signaling, rabbit monoclonal antibody diluted at 1/3000); ERK1/2 (Santa Cruz Biotechnology, rabbit monoclonal antibody, diluted at 1/1000), p-RAFl (Merck Millipore, mouse monoclonal antibody, diluted at 1/1000), RAF1(R&D Systems, mouse monoclonal antibody, diluted at 1/1000), MEK 1/2 (rabbit monoclonal antibody from Cell Signaling, diluted at 1/1000) and p- ME (MEK 1/2 S217/221, rabbit monoclonal antibody from Cell Signaling, diluted at 1/1000). Actin was used as a loading control (Chemicon, Merck Millipore). The Hybond-p membranes (GE Healthcare) were then incubated with HRP-labeled secondary antibodies (R&D System and Cell Signaling) for lh at room temperature and then detected with a chemiluminescence detection ECL kit (Thermo Scientific Pierce). Band intensities were quantified using ImageLab software, Bio-Rad.

Results:

Macrocyclic lactones and PAK1 inhibitor are cytotoxic for EBV positive and EBV negative NPC cells

The capacity of four macrocyclic lactones: Ivermectin, Selamectin, Moxidectin and Abamectin and the PAK1 inhibitor FRAX-597 to kill NPC cells was tested using three human NPC cell lines cell lines, HONE-1, CNE-1 and C666-1. The cells were cultivated for 72h with increasing concentrations of macrocyclic lactones, PA 1 inhibitor or with vehicle alone (RPMI/DMSO 0,25%). Surviving cells were counted using a BD Accuri C6 FACS analyzer (Figure 1) or a Cellomics Arrayscan microscope (Figure 2). Death of CNE-1 and HONE-1 cells, induced in vitro by Ivermectin, is illustrated by pictures taken on the microscope (Figure 3). Among the NPC cells tested, only C666-1 is EBV positive, but the three cell lines are similarly sensitive to the macrocyclic lactones and the PAK1 inhibitor (Figure 1, 2). The data allowed us to evaluate between 2 and 7 μΜ the IC50 of all compounds, for the three NPC cell lines. All these data show that macrocyclic lactones and PAK1 inhibitor are cytotoxic in vitro to NPC cells independently of EBV presence.

Macrocyclic lactones and PAK1 inhibitor are not cytotoxic in vitro on normal thymocytes

Freshly extracted mouse thymocytes were used as normal control cells. They were used as control because T lymphocytes are very sensitive to anticancer therapy and white blood cells often collapse in patients receiving chemotherapy. As for NPC cells, thymocytes were cultivated for 72h with increasing concentrations of macrocyclic lactones, PAK1 inhibitor or with vehicle alone (RPMI/DMSO 0,25%). Surviving thymocytes were counted using a BD Accuri C6 FACS analyzer (Figure 4). The data show that these control cells are resistant to macrocyclic lactones and PA 1 inhibitor up to the dose of 10 μΜ.

Ivermectin intra-peritoneal injections decrease NPC subcutaneous tumor growth in nude mice

In order to test the efficacy of Ivermectin against NPC development in vivo, the two EBV negative NPC cell lines were used. C666-1 cell line has not been used to avoid the risk of introducing the EBV virus in an unadapted animal facility. We initially used HONE-1 cells that were injected subcutaneously (s.c.) in the flank of immunodepressed NMRI nude mice at a dose of lxlO ⁶ cells/mouse to produce local subcutaneous tumors. After cells injection, mice were left untreated for 48 h, in order to allow HONE-1 cells to implant. Then, 5 mice were injected intra-peritoneally (IP) lx/day for 18 days with 0,1 mL of vehicle and 5 mice with 3.25mg/kg of Ivermectin. Tumors growth was measured with a digital caliper, showing that all mice injected with vehicle alone developed growing tumors whereas only 3 on 5 mice injected with Ivermectin have developed tumors, and two mice subsequently rejected their tumors (Figure 5A).

Then two groups of nude mice were injected s.c. with 2xl0 ⁶ HONE-1 cells in order to avoid the initial rejection of tumors in the Ivermectin-treated group. Unexpectedly in these conditions substantial tumors were already present in all mice at day 5 and the tumor growth was stopped on day 8. Tumor growth was observed until day 8 in the 2 mice injected with vehicle alone whereas among the 6 Ivermectin-treated mice, 4 mice reduced their tumor and one of them completely rejected its tumor (Figure 5B).

To confirm that IP injections of Ivermectin could lead to a reduced NPC tumor development in immunodepressed nude mice, CNE-1 cells were s.c. injected in 11 nude mice at a dose of 2xl0 ⁶ cells/mouse to produce local subcutaneous tumors. As in previous experiments, mice were left untreated for 48 h, in order to allow CNE-1 cells to implant. Then mice were divided in two groups: 4 mice were subsequently daily injected with vehicle and 7 mice with Ivermectin at 3.25mg/kg. In the group of mice injected with vehicle the tumors initially grew and then decreased slowly whereas in the group of mice injected with Ivermectin, 6 on 7 mice reduced their tumor. In both groups one mouse rejected its tumor (Figure 6).

Altogether these results show that IP Ivermectin injections decrease local development of NPC tumors.

Ivermectin intra-peritoneal injections are not toxic in vivo in nude mice

To determine whether Ivermectin could be used in preclinical phases for the treatment of NPC cells, we investigated the toxicity of this molecule in mice. The molecule was daily administered by intra-peritoneal (IP) injections in NMRI-nu/nu mice (Nude).

Thirteen NMRI-nu/nu mice were weighed before and after the repeated daily IP injections for 18 days with vehicle (PBS/DMSO 0,25%) or with Ivermectin at 3,25 mg/kg. Before injections the average weight of mice was 25,8 g. After vehicle injections the average weight was 27,7 g and after Ivermectin injections the average weight was 26,7 g. These results show that weight loss associated with Ivermectin injections compared to vehicle injections is not significant (less than 4%) (Figure 7).

Furthermore, mice behavior was not modified by these IP injections, and the quantification of white blood cells in peripheral blood of mice after these injections showed no decrease in circulating white blood cells (data not shown). Altogether these results confirm that Ivermectin, an FDA approved molecule, is not toxic in vivo at the concentration used in our experiments.

The kinase PAK1 as a target of Ivermectin involved in NPC cells killing

It was previously shown that Ivermectin inactivates the kinase PAK1 in tumor cells, leading to reduced tumor growth of ovarian and NF2 human cancer cells. PAK1 phosphorylates Rafl on Ser338 and MEKl on Ser 298, which contributes to MAPK pathway activation and tumor growth. To test whether these molecules are involved in the Ivermectin- induced NPC cells death, we have tested by western-blot analyses the effect of Ivermectin on MEKl/2, ERKl/2 and RAFl phosphorylation. HONE-1 and CNE-1 cells treated for 72h with Ivermectin at 0,3 and 3 μΜ were lysed and their extracts analyzed by western-blot experiments. Inhibitions of MAPK pathway and RAFl activation were detected. These inhibitions are very limited at 0,3 μΜ of Ivermectin but drastic at 3 μΜ (Figure 8A and 8B). These results strongly suggest the involvement, in NPC cells death, of Ivermectin-induced inactivation of PAK1, resulting in p-RAFl and p-MEK expression decrease after Ivermectin treatment. Moreover the capacity of another PAK1 inhibitor, FRAX-597, to similarly kill these NPC cells (Figure 1) confirmed this involvement of PAK1 inhibition in Ivermectin- induced NPC cells death.

Conclusion:

Ivermectin is a well tolerated and clinically approved compound (EMEA- and FDA- approved) used to treat parasite infections. Ivermectin and other macrocyclic lactones actions against parasites have been ascribed to their inhibition of parasite-specific Glutamate-gated chloride channels. Here, we show, both in vitro and in vivo, that Ivermectin and other macrocyclic lactones, used at very low doses (3,25 mg/kg) corresponding to the doses used in human to treat helminthic infections, are efficient to kill NPC cells. This cytotoxicity involves inactivation of PAK1 and is effective in vivo with no discernable side effects. Moreover Ivermectin and other macrocyclic lactones are cytotoxic to NPC cells regardless of their EBV expression. Our results open the field for new therapies of NPC cancer and are particularly interesting because of the lack of available efficient treatments against this orphan tumor.

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.