WO2015007869A1 - Local anesthetics and use thereof in cancer - Google Patents

Abstract

The present invention relates to a compound which is an inhibitor of cellular energy transduction processes, preferably a local anesthetic, for use in the treatment of cancer or for use in prevention of cancer recurrence. Optionally, the local anesthetic is used in combination with an inhibitor of autophagy.

Description

LOCAL ANESTHETICS AND USE THEREOF IN CANCER

FIELD OF THE INVENTION:

The present invention relates to a compound which is an inhibitor of cellular energy transduction processes for use in the treatment of cancer or for use in prevention of cancer recurrence.

BACKGROUND OF THE INVENTION:

Cancer recurrence is a major health problem and depends on cancer type and other parameters such as tumor size, stage and grade, hypoxia, intrinsic radioresistance or chemoresistance of the tumor cells, the vasculature, vasculogenesis and other host factors such as tumor infiltration of inflammatory cells, the presence of circulating tumor cells or that of dormant cancer cells. Local anesthetics, widely used during cancer surgery for intraoperative anesthesia or postoperative analgesia, could interact with cancer cell metabolism, potentially leading to cancer cell proliferation arrest by different pathways.

A series of articles show a toxic effect of local anesthetics on cancer cells viability in vitro (see for example Jose C et al, 2012 or Karniel M et al 2000). However no study compared the specificity of this effect on cancer cells versus non cancer cells originating from the same tissue. In addition, it is not clear whether this effect is cytotoxic or cytostatic and how it relates to cell cycle progression, energy states and redox signalling.

The inventors reasoned that cancer cells present with peculiar modalities of energy transduction, so that the specificity of action of levobupivacaine, a local anesthetic with previously demonstrated interactions with the bioenergetic systems, namely glycolysis and oxidative phosphorylation, might be related to the deviant bioenergetic profile of cancer cells and their high energy demand. In tumors, the intense anabolic needs of cancer cells as well as the activation of different oncogenes and the inhibition of tumor suppressors typically destabilize the balance between glycolysis and oxidative phosphorylation. Local anesthetics- induced destabilization of this optimum bioenergetic equilibrium could impact cancer cells physiology and reduce their growth. SUMMARY OF THE INVENTION:

The inventors found a potent and specific antiproliferative effect of levobupivacaine on human prostate cancer cells as compared to corresponding non cancer cells. The mode of action included a two-site inhibition of cellular energy transduction processes, also referred to as "bioenergetics". Levobupivacaine anti-Cancer specificity was explained by a higher energy demand of the cancer cells, as revealed by the elevated ADP/ATP ratio at steady-state as compared to the corresponding non-cancer cells. Levobupivacaine inhibits both the glycolysis and oxidative phosphorylation process (at the level of respiratory chain complex I ) to a specific extent, respectively, which triggers the anti-proliferative effect. This was observed by an alteration of cell cycle progression in cells treated with levobupivacaine (arrest at Gl phase before the replicative S-Phase). Moreover, such bioenergetic impairment caused by levobupivacaine activated compensatory autophagy in the prostate cancer cells. Thus, the inventors used levobupivacaine in combination with a blocker of autophagy and killed the prostate cancer cells with little impact on the non-cancer prostate epithelial cells.

Thus, the invention relates to a compound which is an inhibitor of cellular energy transduction processes for use in the treatment of cancer or for use in prevention of cancer recurrence.

DETAILED DESCRIPTION OF THE INVENTION:

Definitions:

Throughout the specification, several terms are employed and are defined in the following paragraphs.

Local anesthetic and uses thereof

A first object of the invention relates to a compound which is an inhibitor of cellular energy transduction processes for use in the treatment of cancer or for use in prevention of cancer recurrence. In another embodiment, the invention relates to a compound which is a dual moderate inhibitor of glycolysis and oxidative phosphorylation for use in the treatment of cancer or for use in prevention of cancer recurrence.

In another embodiment, the invention relates to an inhibitor which impacts the ADP/ATP ratio for use in the treatment of cancer or for use in prevention of cancer recurrence.

In another embodiment, the invention relates to an inhibitor which impacts both glycolysis and oxidative phosphorylation, leading to an increase in the ADP/ATP ratio and a decrease of cell cycle progression for use in the treatment of cancer or for use in prevention of cancer recurrence.

As used herein, the term "cellular energy transduction processes" denotes the systems of energy transduction which ultimately generate ATP in cells (bioenergetic system). These processes include, but are not limited to mitochondrial oxidative phosphorylation and glycolysis. Thus, "a dual-site bioenergetic inhibitor" denotes an inhibitor of the production of ATP by both mitochondria (mitochondrial oxidative phosphorylation) and glycolysis.

Thus, in a particular embodiment, the invention relates to a multi-site bioenergetic inhibitor of ATP production by mitochondria and glycolysis for use in the treatment of cancer or for use in prevention of cancer recurrence.

In one embodiment, the inhibitor of the cellular energy transduction processes may be a local anesthetic.

Thus, in a particular embodiment, the invention relates to a local anesthetic for use in the treatment of cancer or for use in prevention of cancer recurrence.

As used herein, the term "local anesthetic (LA)" denotes a drug that causes reversible local anesthesia, generally for the aim of having a local analgesic effect that is, inducing absence of pain sensation. Clinical local anesthetics belong to one of two classes: aminoamide and aminoester anesthetics. Aminoamides may be Articaine, Bupivacaine, Cinchocaine, Dibucaine, Etidocaine, Levobupivacaine, Lidocaine, Lignocaine, Mepivacaine, Prilocaine, Ropivacaine or Trimecaine. Aminoester may be Benzocaine, Chloroprocaine, Cocaine, Cyclomethycaine, Dimethocaine, Larocaine, Piperocaine, Propoxycaine, Procaine, Novocaine, Proparacaine, Tetracaine or Amethocaine.

In one embodiment, the local anesthesic is an aminoamide anesthesia. In a particular embodiment the aminoamide anesthesic is the Levobupivacaine.

According to the invention the cancer may be selected from the group consisting of adrenal cortical cancer, anal cancer, bile duct cancer (e.g. periphilar cancer, distal bile duct cancer, intrahepatic bile duct cancer), bladder cancer, bone cancer (e.g. osteoblastoma, osteochrondroma, hemangioma, chondromyxoid fibroma, osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of the bone, chordoma), brain and central nervous system cancer (e.g. meningioma, astocytoma, oligodendrogliomas, ependymoma, gliomas, medulloblastoma, ganglioglioma, Schwannoma, germinoma, craniopharyngioma), breast cancer (e.g. ductal carcinoma in situ, infiltrating ductal carcinoma, infiltrating lobular carcinoma, lobular carcinoma in situ, gynecomastia), Castleman disease (e.g. giant lymph node hyperplasia, angio follicular lymph node hyperplasia), cervical cancer, colorectal cancer, endometrial cancer (e.g. endometrial adenocarcinoma, adenocanthoma, papillary serous adnocarcinoma, clear cell), esophagus cancer, gallbladder cancer (mucinous adenocarcinoma, small cell carcinoma), gastrointestinal carcinoid tumors (e.g. choriocarcinoma, chorioadenoma destruens), Hodgkin's disease, Kaposi's sarcoma, kidney cancer (e.g. renal cell cancer), laryngeal and hypopharyngeal cancer, liver cancer (e.g. hemangioma, hepatic adenoma, focal nodular hyperplasia, hepatocellular carcinoma), lung cancer (e.g. small cell lung cancer, non-small cell lung cancer), mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer (e.g. esthesioneuroblastoma, midline granuloma), nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma (e.g. embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, pleomorphic rhabdomyosarcoma), salivary gland cancer, skin cancer (e.g. melanoma, nonmelanoma skin cancer), stomach cancer, testicular cancer (e.g. seminoma, nonseminoma germ cell cancer), thymus cancer, thyroid cancer (e.g. follicular carcinoma, anaplastic carcinoma, poorly differentiated carcinoma, medullary thyroid carcinoma,), vaginal cancer, vulvar cancer, and uterine cancer (e.g. uterine leiomyosarcoma) .

In one embodiment, the cancer is a prostate cancer or a skin cancer.

In a particular embodiment, the prostate cancer is a prostate adenocarcinoma.

In another particular embodiment, the skin cancer is basal cell carcinoma, squamous cell carcinoma or a melanoma. According to the invention, the local anesthesic may be formulated of a topical, oral, intranasal, intravenous, in the vicinity of the nerve, intramuscular or subcutaneous administration as well as wound infiltration and the like.

In one embodiment, the local anesthesic according to the invention is administrated locally.

In a particular embodiment, the local anesthesic according to the invention is delivered continuously with a pump. The pump may be useful to deliver the local anesthesic with a controlled concentration and flux. Thus method may be useful to have a better effect of the therapeutic compound. The pump or any means which permit a controlled delivery of the local anesthesic may be used to administrate the local anesthesic during a period of 24-72 hours to 1 week. The pump used according to the invention may have a flow of 1 ml/hour to 15 ml/hour for a human. In one embodiment, the flow can be 2 ml/hour, 3 ml/hour, 4 ml/hour, 5 ml/hour, 6 ml/hour, 7 ml/hour, 8 ml/hour, 9 ml/hour, 10 ml/hour, 11 ml/hour, 12 ml/hour, 13 ml/hour or 14 ml/hour.

In a particular embodiment and for a mouse model, the flow of the pump is Ιμΐ/hour of levobupivacaine 5mg/ml which corresponds to a levobupivacaine dose of 0.16mg/kg/h.

Thus, in a human, the dose may be lOml/hour of levobupivacaine at l,25mg/ml.

In one embodiment, the local anesthetic according to the invention is used after chirurgical excision of the primary tumor to prevent cancer recurrence.

Typically said local anesthetic may be used in combination with radiotherapy and hormone therapy as well as autophagy blockers.

Inhibitor of the bioenergetics and inhibitor of autophagy and uses thereof

A second object of the invention relates to a combination containing an inhibitor of the cellular energy transduction processes and an inhibitor of autophagy for use in the treatment of cancer or for use in prevention of cancer recurrence.

In a particular embodiment, the inhibitor of cellular energy transduction processes is a local anesthetic according to the invention. Thus, in another embodiment, the invention relates to a combination containing a local anesthetic and an inhibitor of autophagy for use in the treatment of cancer or for use in prevention of cancer recurrence. As used herein, an "autophagy" denotes a catabolic mechanism that involves cell degradation of unnecessary or dysfunctional cellular components through the lysosomal machinery. This mechanism involves the PI3K/AKT/mTOR pathway.

Thus, according to the invention, an inhibitor of the autophagy may be an inhibitor of the PBK/AKT/mTOR pathway.

As used herein, the term "PI3K" used for "Phosphatidylinositide 3-kinases" denotes a family of enzymes involved in cellular functions such as cell growth, proliferation, differentiation, motility, survival and intracellular trafficking, which in turn are involved in cancer. PI3Ks are a family of related intracellular signal transducer enzymes capable of phosphorylating the 3 position hydroxyl group of the inositol ring of phosphatidylinositol (Ptdlns).

As used herein, the term "AKT" for "Protein Kinase B" denotes a serine/threonine- specific protein kinase that plays a key role in multiple cellular processes such as glucose metabolism, apoptosis, cell proliferation, transcription and cell migration.

As used herein, the term "mTOR" for "mammalian Target Of Rapamycin" denotes a serine/threonine protein kinase that regulates cell growth, cell proliferation, cell motility, cell survival, protein synthesis, and transcription.

In one embodiment, the inhibitor of the autophagy may be an inhibitor of PI3K.

In a particular embodiment, the PI3K inhibitor may be but is not limited to Wortmannin, demethoxyviridin, LY29400 or BKM120 [see for example http://en.wikipedia.org/wiki/PI3K___inhibitor].

In another embodiment, the inhibitor of the autophagy may be an inhibitor of AKT. In a particular embodiment, the PI3K inhibitor may be but is not limited to Perifosine or Miltefosine [see for example hu ://c n . w i k i cd i a . o rg/w i k i A T i n h i b i to # A T i n h i b i to rs] . In another embodiment, the inhibitor of the autophagy may be an inhibitor of mTOR. In a particular embodiment, the mTOR inhibitor may be but is not limited to Everolimus, Rapamycin, Temsirolimus or Ridaforolimus [see for example http://en.wikipedia.Org/wiki/MTOR_inhibitor#mTOR_inhibitors_as

In another embodiment, the inhibitor of the autophagy may be the Chloroquine [see for exemple http ://www.clinicaltrials. gov/ ct2/ show/NCTQQ969306?term=chloroquine&rank= 1 ] .

In another embodiment, the invention relates to i) an inhibitor of bio energetics and ii) an inhibitor of autophagy according to the invention as a combined preparation for simultaneous, separate or sequential use in the treatment of cancer or in the prevention of cancer recurrence in a patient.

In a particular embodiment, the inhibitor of the bioenergetics is a local anesthetic.

Thus, in another embodiment, the invention relates to i) a local anesthetic and ii) an inhibitor of autophagy according to the invention as a combined preparation for simultaneous, separate or sequential use in the treatment of cancer or in the prevention of cancer recurrence in a patient.

Therapeutic methods

A third object of the invention relates to a method of treating cancer or of preventing the recurrence of cancer comprising administering to a subject in need thereof a therapeutically effective amount of an inhibitor of cellular energy transduction processes or an inhibitor of cellular energy transduction processes and an inhibitor of autophagy according to the invention.

In a particular embodiment, the inhibitor of cellular energy transduction processes is a local anesthetic according to the invention.

Thus, in another embodiment, the invention relates to a method of treating cancer or of preventing the recurrence of cancer comprising administering to a subject in need thereof a therapeutically effective amount of a local anesthetic or a local anesthetic and an inhibitor of autophagy according to the invention. In the context of the invention, the term "treating" or "treatment", as used herein, means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such a disorder or condition.

As used herein, the term "subject" denotes a mammal, such as a rodent, a feline, a canine, and a primate. Preferably a subject according to the invention is a human.

By a "therapeutically effective amount" of the local anesthetic or the local anesthetic and the inhibitor of autophagy according to the invention is meant a sufficient amount of the ligand to treat said cancer, at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the small molecule, the polypeptide or the nucleic acid 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 polypeptide or the nucleic acid employed; the specific composition employed, the age, body weight, general health, sex and diet of the patient, the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed, and like factors well known in the medical arts. For example, it is well known 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.

The local anesthetic or the local anesthetic and the inhibitor of autophagy according to the invention may be used in combination with any other therapeutic strategy for treating the disorders or conditions as above described (e.g. external radiotherapy, chemotherapy or cytokine therapy).

In another embodiment, the local anesthetic according to the invention and the inhibitor of the autophagy according to the invention may be administrated simultaneously, separately or sequentially to the subject.

Pharmaceutical composition A fourth object of the invention relates to a pharmaceutical composition comprising an effective amount of an inhibitor of cellular energy transduction processes or an inhibitor of cellular energy transduction processes and an inhibitor of autophagy for use in the treatment of cancer or for use in the prevention of cancer recurrence and pharmaceutically acceptable excipients or carriers.

In a particular embodiment, the inhibitor of the bioenergetics is a local anesthetic according to the invention.

Thus, in another embodiment, the invention relates to a pharmaceutical composition comprising an effective amount of a local anesthetic or a local anesthetic and an inhibitor of autophagy for use in the treatment of cancer or for use in the prevention of cancer recurrence and pharmaceutically acceptable excipients or carriers.

Any therapeutic agent of the invention as above described may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.

"Pharmaceutically" or "pharmaceutically acceptable" refers 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.

The form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc.

The pharmaceutical compositions of the invention can be formulated for a topical, oral, intranasal, intraocular, intramuscular or subcutaneous administration and the like.

Preferably, 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 doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment. To prepare pharmaceutical compositions, an effective amount of a local anesthetic or of an inhibitor of autophagy according to the invention may be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

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.

Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The local anesthetic or the inhibitor of autophagy according to the invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various 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.

The preparation of more, or highly concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small tumor area.

Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.

For parenteral administration in an aqueous solution, for example, the solution may be suitably buffered and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, perinervous, wound infiltration, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

In addition to the compounds formulated for parenteral administration, such as intramuscular injection, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; time release capsules; and any other form currently used.

In a particular embodiment, a composition according to the invention may be formulated as a cream, microencapsulated local anesthetic, microsphere, nanosphere, sustained-release local anesthetics particles, gel, nanocapsule, which can be used locally. In a particular embodiment, the cream may be used after chirurgical excision of the primary tumor to prevent cancer recurrence. In another particular embodiment, the cream may be useful in the case of skin cancer or prostate cancer.

Compositions of the present invention may comprise a further therapeutic active agent.

The present invention also relates to a kit comprising a local anesthetic and/or an inhibitor of autophagy according to the invention and a further therapeutic active agent.

In one embodiment said therapeutic active agent is an anticancer agent. For example, said anticancer agents include but are not limited to fludarabine, gemcitabine, capecitabine, methotrexate, taxol, taxotere, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, platinum complexes such as cisplatin, carboplatin and oxaliplatin, mitomycin, dacarbazine, procarbazine, etoposide, teniposide, campathecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, L-asparaginase, doxorubicin, epimbicm, 5-fluorouracil, taxanes such as docetaxel and paclitaxel, leucovorin, levamisole, irinotecan, estramustine, etoposide, nitrogen mustards, BCNU, nitrosoureas such as carmustme and lomustine, vinca alkaloids such as vinblastine, vincristine and vinorelbine, imatimb mesylate, hexamethyhnelamine, topotecan, kinase inhibitors, phosphatase inhibitors, ATPase inhibitors, tyrphostins, protease inhibitors, inhibitors herbimycm A, genistein, erbstatin, and lavendustin A. In one embodiment, additional anticancer agents may be selected from, but are not limited to, one or a combination of the following class of agents: alkylating agents, plant alkaloids, DNA topoisomerase inhibitors, anti-folates, pyrimidine analogs, purine analogs, DNA antimetabolites, taxanes, podophyllotoxin, hormonal therapies, retinoids, photosensitizers or photodynamic therapies, angiogenesis inhibitors, antimitotic agents, isoprenylation inhibitors, cell cycle inhibitors, actinomycins, bleomycins, anthracyclines, MDR inhibitors and Ca²⁺ ATPase inhibitors.

Additional anticancer agents may be selected from, but are not limited to, cytokines, chemokines, growth factors, growth inhibitory factors, hormones, soluble receptors, decoy receptors, monoclonal or polyclonal antibodies, mono-specific, bi-specific or multi-specific antibodies, monobodies, polybodies.

Additional anticancer agent may be selected from, but are not limited to, growth or hematopoietic factors such as erythropoietin and thrombopoietin, and growth factor mimetics thereof. In the present methods for treating cancer the further therapeutic active agent can be an antiemetic agent. Suitable antiemetic agents include, but are not limited to, metoclopromide, domperidone, prochlorperazine, promethazine, chlorpromazine, trimethobenzamide, ondansetron, granisetron, hydroxyzine, acethylleucine monoemanolamine, alizapride, azasetron, benzquinamide, bietanautine, bromopride, buclizine, clebopride, cyclizine, dunenhydrinate, diphenidol, dolasetron, meclizme, methallatal, metopimazine, nabilone, oxypemdyl, pipamazine, scopolamine, sulpiride, tetrahydrocannabinols, thiefhylperazine, thioproperazine and tropisetron. In a preferred embodiment, the antiemetic agent is granisetron or ondansetron.

In another embodiment, the further therapeutic active agent can be an hematopoietic colony stimulating factor. Suitable hematopoietic colony stimulating factors include, but are not limited to, filgrastim, sargramostim, molgramostim and epoietin alpha.

In still another embodiment, the other therapeutic active agent can be an opioid or non-opioid analgesic agent. Suitable opioid analgesic agents include, but are not limited to, morphine, heroin, hydromorphone, hydrocodone, oxymorphone, oxycodone, metopon, apomorphine, nomioiphine, etoipbine, buprenorphine, mepeddine, lopermide, anileddine, ethoheptazine, piminidine, betaprodine, diphenoxylate, fentanil, sufentanil, alfentanil, remifentanil, levorphanol, dextromethorphan, phenazodne, pemazocine, cyclazocine, methadone, isomethadone and propoxyphene. Suitable non-opioid analgesic agents include, but are not limited to, aspirin, celecoxib, rofecoxib, diclofinac, diflusinal, etodolac, fenoprofen, flurbiprofen, ibuprofen, ketoprofen, indomethacin, ketorolac, meclofenamate, mefanamic acid, nabumetone, naproxen, piroxicam and sulindac.

In yet another embodiment, the further therapeutic active agent can be an anxiolytic agent. Suitable anxiolytic agents include, but are not limited to, buspirone, and benzodiazepines such as diazepam, lorazepam, oxazapam, chlorazepate, clonazepam, chlordiazepoxide and alprazolam.

In another embodiment, the invention is a product of a) at least one local anesthetic according to the invention and b) at least one inhibitor of the autophagy according to the invention, as a combined preparation for simultaneous, separate or sequential use in the treatment of cancer or in the prevention of cancer recurrence. 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. Impact of levobupivacame and etoposide on proliferation. Cell number of BHP and DU145 after 24 h treatments with 1 mM of levobupivacame or 50 μΜ etoposide. Results are expressed as the percentage of the control. All the data shown correspond to the mean value ± SD of N > 3 different experiments. Significantly different from the untreated cells at: ** P < 0.01, *** P < 0.001.

Figure 2. Impact of levobupivacame 1 mM on mitochondria. (A): Complex I activity on DU145 after 24h of treatment with 1 mM levobupivacame before and after the adjunction of 100 nM rotenone. (B): Fluorescence of CM-H2DCFDA on untreated and treated cells with 1 mM levobupivacame for 24 hours, with or without co-treatment with resveratrol (rsv) and/or H202. (C): Proliferation after 24 hours of 1 mM levobupivacame with or without antioxidants (NAC, Resveratrol and a-tocopherol). H202 level in DU145 cells after 24 h treatment with 1 mM levobupivacame (positive and negative controls are shown). All the data shown correspond to the mean value ± SD of N > 3 different experiments. Significantly different from the untreated cells at: * P < 0.05, ** P < 0.01.

Figure 3. Impact of levobupivacame on the bioenegetic state of BHP and DU145 cells. Total ATP steady-state of untreated and treated (A): BHP and (B): DU145 cells with 1 mM levobupivacame. Contribution of mitochondria and glycolysis in ATP content was assessed with specific inhibitors (oligomycin and iodoacetate, respectively) for 10 min. (C): Energetic status of BHP and DU145 cell lines was determined with the ratio ATP/ADP at steady state without treatment. (D): Proliferation of cells with rotenone + iodoacetate and levobupivacame 1 mM after 24 hours of treatment. All the data shown correspond to the mean value ± SD of N > 3 different experiments. Significantly different from the untreated cells at: * P < 0.05.

Figure 4. Impact of levobupivacame of autophagy induction. (A): Proliferation of BHP and DU145 cell lines after 24h of treatment with 1 mM levobupivacame co-treated or not with 100 nM Wortmannin. Results are expressed as percentage of control. All the data shown correspond to the mean value ± SD of N > 3 different experiments. Significantly different from the untreated cells at: * P < 0.05, ** P < 0.01, *** P < 0.001. (B): Impact of levobupivacaine of autophagy induction: Proliferation of PC3 cell line after 24h of treatment with 1 mM levobupivacaine co-treated or not with 100 nM Wortmannin. Results are expressed as percentage of control. All the data shown correspond to the mean value ± SD of N > 3 different experiments. Significantly different from the untreated cells at: *** P < 0.001.

Figure 5. Effect of levobupivacaine on apoptose, Akt and cell cycle. Different effect of 24 hours of treatment with 1 mM levobupivacaine on DU145 and BHP cells viability and proliferation. Cell death was assessed with (A): the expression level of PARP and (B): Caspase activity on untreated (Untr) and treated (Levo) cells. The effect of 50 μΜ etoposide on Caspase activity has also been assessed. Cell survival pathway was assessed with (C): the expression level of P-AKT. Cell cycle arrest in DU145 was determined with (D): flux cytometry analysis of BrDU and 7-AAD staining. (E): Medium term effect of levobupivacaine on proliferation rate was determined over 48 h of growth after 24 h of treatment and subsequent wash-off. All the data shown correspond to the mean value ± SD of N > 3 different experiments. Significantly different from the untreated cells at: * P < 0.05, ** P < 0.01, *** P < 0.001.

Figure 6. In- vivo results. The size of the tumors was measured at different days (0, 15, 21 and 28) after treatment of the mice with saline (PHY, negative control) or levobupivacaine (AL; local anesthetics = levobupivacaine)). The points at day 0 are circles, triangles at day 15, squares at day 21 and diamonds at day 28. At each day, the data set of treated mice "AL" was compared to the corresponding dataset "PHY" and the p value of the test is given on the top of the points. For instance, D15 AL must be compared with D15 PHY. At day 21 the reduction in tumor size is statistically significant with p=0.009, and also at J28 (p=0.023).

EXAMPLES:

Example 1: in-vitro example

Material & Methods Chemicals

Levobupivacaine hydrochloride 0.5% was purchased from ABBOTT (Rungis, France). All other reagents were purchased from Sigma, at the exception of the ATP monitoring kit (ATP Bio luminescence Assay Kit HS II from Roche Diagnostics GmbH, Mannheim, Germany), the ATP/ADP ratio kit (Abeam, Paris, France), the Caspase-Glo® 3/7 Assay (Promega, Madison, WI, USA) and the primary antibodies (Complex I NDUFB8 sub- unit antibody from MitoSciences, Eugene, OR, USA; LC3B and AKT-P from Abeam, Paris, France; and PARP from Santa Cruz Biotechnology, Santa Cruz, NM, USA).

Cell line and cell culture conditions

Human prostate cancer cells (DU145 - carcinoma from metastatic site, and PC3 - adenocarcinoma Grade IV) were obtained from ATCC (Bethesda, MD, USA). BHP primary epithelial prostate cells were obtained from P. Pourquier. The cancer cells were grown in Dulbecco's Modified Eagle Media (DMEM) containing 25mM glucose and supplemented with 10% fetal bovine serum, 100 U/ml penicillin and lOOU/ml of streptomycin. BHP cells were grown in optimal PrEGM Bullet Kit medium containing 25mM Glucose and 2mM glutamine provided by Lonza, Walkersville, MD, USA. All of the cells were grown in 5%> C02 at 37°C. For all experiments, the cells were harvested during the exponential phase of growth at 70%> confluency.

Cell viability and enumeration

Cell proliferation rate was evaluated by counting the cells during the exponential phase of growth in the growing medium using a Malassez hemocytometer. The cells were seeded in 6-well plates (100,000 cells per well), treated or not with 1 mM levobupivacaine, 50μΜ etoposide, rotenone (0.5, 5, 50 or 500 nM) or 2.5μΜ iodoacetate as precised in the text. Cells were counted manually on a Mallassez counting chamber (n > 3 for each condition). To investigate the role of reactive oxygen species in the cytotoxic effect of levobupivacaine 1 mM, we repeated the enumeration procedure in the presence of the following antioxidants: 20 μΜ resveratrol, ImM N-acetyl-L-cysteine or 0.4mM a-tocopherol (n > 3 for each condition). We verified that in these concentrations the different antioxidants had no impact on cell viability (data not shown). To determine the role of autophagy on levobupivacaine induced- cytotoxicity, we repeated the enumeration procedure in the presence of ΙΟΟηΜ wortmannin. After 6, 48 or 72 hours of local anesthetic exposure, the DU145 cells were washed with PBS and trypsinized, and 25 μΐ sample of this cell suspension was used for staining with Trypan Blue (1 : 1). A live/dead cell count was performed. Caspase activity was assessed to evaluate cell death induction with the Caspase-Glo® 3/7 Assay (Promega, Madison, WI, USA) according to the manufacturer recommandation after 24h of treatment with or without ImM levobupivacaine or 50μΜ etoposide. DU145 cell line proliferation was also evaluated after 24h of treatment with 1 mM levobupivacaine and subsequent wash-out of the cells with DMEM medium for 24 to 48h.

Cell respiration and adenosine triphosphate measurements

Endogenous respiratory rate was assayed in intact cells using high-resolution respirometry (Oroboros Instruments, Innsbruck, Austria). Respiration was measured at 37 °C with 2 x 106 cells/mL in DMEM.15 Levobupivacaine (0.1 μΜ to 3mM) or rotenone (0.5nM to 3.5nM) was titrated at steady state, and the respiratory rates were expressed as ng atom O/min/1 x 106 cells. The intracellular ATP content was measured using the bio luminescent ATP kit HS II (Roche Applied Science). 50uL of a cell suspension of 2 x 106 cells/mL were plated in a white 96-well plate. When attached to the plate, they were treated or not with ImM levobupivacaine for 2 hours. When precised in the text, cells were pre-adapted for 2h in galactose medium before levobupivacaine treatment. For each condition, four wells were used to measure the total ATP content, and four wells were treated with 2 μg/mL oligomycin A for 10 min to block mitochondrial ATP synthesis and allow ATP turnover via ATP consuming processes. In a third subset of four wells, glycolysis was blocked with 2.5μΜ iodoacetate over a 10-min period to evaluate the participation of glycolysis to cellular ATP synthesis. Then, cells were immediately lysed to release the intracellular ATP using the lysis buffer provided with the kit (volume ratio 1 : 1) for 5 min at room temperature. ATP concentration is determined with the light-emitting, luciferase-catalyzed oxidation of luciferin with ATP. A total of ΙΟΟμί of luciferase reagent provided by the kit was injected into the wells and bio luminescence was measured (10 s integration time) on a lumino meter (Luminoskan, Labsystems, Finland). Standardization was performed with known quantities of standard ATP provided with the kit measured under the same conditions. To evaluate the ATP/ADP ratio, we used the Abeam ATP/ADP Ratio Assay Kit (Bio luminescent) and followed the protocol of the manufacturer. The ratio of ATP to ADP in the cell is a measure of the available metabolic energy.

Western blotting Total cell lysis was performed by sonication (total time of 5 min with a cycle of 30 s sonication, 30 s rest and 45°C amplitude at 4°C) on a Epishear multi-sample sonicator (Active Motif, La Hulpe, Belgium). Samples were diluted in an SDS-PAGE tricine sample buffer (Bio-Rad Laboratories) containing 2% β-mercaptoethanol by incubation for 30 min at 37°C. The samples were then separated on a 4-20 % SDS polyacrylamide gradient mini-gel (Bio- Rad Laboratories) at 150 V. Proteins (30 μg of proteins per well) were transferred electrophoretically to 0.45 μιη polyvinylidine difluoride membranes for 2 hours at 100mA in CAPS buffer (3.3 g CAPS, 1.5 L 10 % methanol, pH 11) on ice. The membranes were blocked overnight in 5% milk-PBS + 0.02 % azide and incubated for 4 hours with the primary antibodies. After six washes with PBS with 0.05% Tween 20, the membranes were incubated for 1 hour with horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse (Bio- Rad Laboratories) diluted in PBS with 5% milk. This secondary antibody was detected in a Chemidoc (Bio-Rad Laboratories) using the chemiluminescent ECL PlusTM reagent (Amersham Biosciences, GE Healthcare, Uppsala, Sweden). The signal was quantified by densitometric analysis using Image J (National Institutes of Health, Bethesda, MD, USA) software.

Flow cytometric cell cycle assay

The cells were treated with 1 mM levobupivacaine and allowed to incorporate BrdU for 3 hours at 37°C before the end of the 24 hour treatment. The cells were trypsinized and counted, and 2 x 106 cells/mL were fixed with the solution provided in the manufacturer's kit (BrdU Flow Kit, BD Biosciences, France) for 25 min at 0°C. Then, the cells were incubated with DNase 30 % overnight at 0°C and stained with 7-AAD. Subsequent detection of BrdU was accomplished using antibodies for BrdU (1 : 100) according to the manufacturer's instructions and visualized by flow cytometry (CANTO).

Fluorometric measurement of cellular ROS production

The cells were trypsinized, counted, and incubated in presence of 1 mM levobupivacaine for 24 hours in DMEM with or without the presence of 20μΜ resveratrol. Changes in cytosolic H202 levels were monitored using the CM-H2DCFDA probe. The probe was added to the cell suspension in the presence of levobupivacaine and incubated for 30 min at 37°C, according to the manufacturer's protocol. The cells were then washed twice in PBS, and fluorescence was measured in a quartz cuvette on a Xenius spectrofluorometer (SAFAS, Monaco, France). A second reading was performed with the addition of ΙΟΟμΜ H202 in the cuvette to verify the response and absence of saturation of the CM-H2DCFDA probe. The signal increased immediately after the addition of H202 in a dose-dependent manner (data not shown). Mitochondrial complex I and catalase activity enzylmatic assays

The enzymatic activity of complex I was assessed by monitoring the oxidation of nicotinamide-adenine-dinucleotide disodium-salt (NADH).16 Briefly, the oxidation of NADH by complex I was recorded using the ubiquinone analogue decylubiquinone (Sigma D-7911) as electron acceptor. Enzyme activity was measured by starting the reaction with 80 μg of total cell lysate. The decrease in absorbance due to NADH oxidation was measured at 340 nm. The extinction coefficient used for NADH was 6.22 Mm/cm. The enzymatic activity of catalase was monitored as described previously.17 The decrease in NADH absorbance due to H202 deoxydation was measured at 340 nm. The extinction coefficient used was 40 mM/cm. Statistical analysis

All of the data presented in this study correspond to the mean value of n experiments ± SD, with a minimum of n > 3. Comparison of the data sets (control versus treated cells) was performed with Student's t-test using SigmaStat 3.1 (Systat Software Inc., San Jose, CA, USA). Two sets of data were considered statistically different when P < 0.05.

Results

Levobupivacaine inhibits prostate cancer cell proliferation

We performed cell enumeration analyses on BHP and on DU145, in the presence of either 50μΜ etoposide, a validated chemotherapeutic agent, or ImM of the local anesthetic levobupivacaine for 24 hours (Figure 1). Etoposide, widely used in prostate cancer treatment, induces apoptosis in cancer 18 and normal cells, albeit to a minor extent in the latter.19 In our study, both drugs delivered during 24 hours induced a significant (P<0.005) reduction in cell number in the cancer cell line (Figure 1) and levobupivacaine even showed a higher cancer- specificity. Similar data were obtained with another prostate cancer cell line (PC3 adenocarcinoma) (data not shown). To determine whether the reduction in cancer cell growth mediated by 1 mM levobupivacaine was caused by cell death or by cell growth arrest, we evaluated the former by measuring the cellular content of active (ADP)-Ribose Polymerase (PARP) by western blot (normalized to tubulin) (data not shown). No significant increase in PARP was observed in DU145 cells treated with 1 mM levobupivacaine over the 24-h period compared to the untreated control (P=0.1975). Likewise, in treated DU145 cells, no significant increase of the caspase 3/7 activity was observed while etoposide induced apoptosis (data not shown). When the incubation period with 1 mM levobupivacaine was raised to 72 hours, no further loss of cell viability was measured with the trypan blue exclusion method (4.95% ± 2.4 of dead cells compared to 0.01% ± 0.002 in the untreated control (P=0.2137), suggesting that ImM levobupivacaine decreased the rate of cancer cell proliferation rather than induced cell death. In contrast, we observed a significant (32.7 ± 3.9 %) increase in active PARP content in the primary cell line BHP treated with ImM levobupivacaine during 24 hours (data not shown) in good correspondance with the 31.3 ± 3.4 % higher caspase 3/7 activity in these cells (data not shown). Such increase in apoptosis markers observed in BHP cells was also in good correspondence with the decrease in cell number (-28.7 ± 5.3 %) induced by 1 mM levobupivacaine (Figure 1). Previous work reported that levobupivacaine was able to reduce Akt activity and subsequentely to activate apoptosis in C2C12 non-cancer cells. Accordingly, we found a significant reduction of the active form of Akt kinase in primary BHP cells for which apoptosis was observed (data not shown). This effect was not observed in DU145 cancer cells for which levobupivacaine failed to activate apoptosis. Levobupivacaine arrests cancer cell cycle progression prior to the S-phase

The impact of ImM levobupivacaine on DU145 cell cycle progression was investigated by flow cytometry and western blot. First, we analyzed the proportion of cancer cells entering the different stages of the cell cycle, using BrdU and 7-AAD as fluorescent indicators of DNA active synthesis and DNA content, respectively. We found a significant difference in the distribution of the cell population between the cell cycle phases (P=0.0441 for the G0-G1 phase and P=0.0054 for the S phase) (data not shown). Indeed, ImM levobupivacaine led to the accumulation of the prostate cancer cells in the non-proliferating G0/G1 phase indicative of a potential failure to enter the high energy demanding S phase. This observation illustrates the anti-pro liferative potency of ImM levobupivacaine on DU145 prostate cancer cells. This effect occurred after removal of the drug as DU145 cells required a longer time to perform cell doubling over 48 hours (data not shown).

Levobupivacaine alters complex I activity and generates ROS We assayed the direct effect of levobupivacaine on mitochondrial function by performing a titration of cellular oxygen consumption with levobupivacaine concentrations ranging from ΙΟμΜ to 3mM on DU145 intact cells (data not shown). Levobupivacaine inhibited cell respiration in a dose-dependent manner with an apparent Ki of 600μΜ while considering the complex I-dependent respiration. Titration of cell respiration with rotenone revealed a significant part of oxygen consumption independent of complex I (potentially supported by complex II). The impact of levobupivacaine on cell respiration was explained by the direct inhibition of respiratory chain complex I (P=0.0068) as shown by the measurement of complex I specific activity by spectrophotometry (Figure 2A). In addition to this direct effect of levobupivacaine on complex I enzyme activity, we analyzed the impact of this drug on mitochondrial biogenesis, based on previous analyses which revealed an additional reduction of mitochondrial content in the muscle of rats exposed to bupivacaine. Accordingly, the western blot analysis of DU145 cells treated with ImM levobupivacaine showed a significant decrease in complex I NDUFB8 subunit content normalized to tubulin (P<0.05) (data not shown). Several studies have indicated that the inhibition of CI enzyme activity can trigger the excessive production of ROS. Using the intracellular fluorescent indicator CM- H2DCFDA, we estimated the potential increase in cellular H202 concentration induced by exposure of the DU145 cells to ImM levobupivacaine over a 24-hour period (Figure 2B). We observed a significant increase of approximately 22 % of CM-H2DCFDA fluorescence in the treated cells as compared the untreated control (P=0.0285). To identify the eventual activation of the antioxidant systems in cells treated with 1 mM levobupivacaine, we measured catalase activity following 24 hours of treatment (data not shown). No difference was observed in this enzyme activity suggesting that oxidative stress caused by levobupivacaine was not sufficient to activate the antioxydant defense program. To further evaluate the possible impact of the ROS increase on cancer cell proliferation, we performed a co-treatment of DU145 cells with 1 mM levobupivacaine and the antioxidant resveratrol used at 20μΜ. As expected, this co- treatment prevented the increase in ROS production (Figure 2B) but no diminution of the antiproliferative effect of levobupivacaine was noticed. Similar results were obtained with the antioxydants a-tocopherol and N-Acetyl-L-cystein (NAC) (Figure 2C). These findings suggest that levobupivacaine-mediated ROS overproduction does not contribute to its antiproliferative property.

The anti-proliferative potency of levobupivacaine on DU145 cancer cells is mediated by multi-site inhibition of energy production To decipher the mechanisms by which levobupivacaine altered the growth of DU145 cancer cells we measured the impact of this drug on overall energy metabolism. First, we assessed the changes in cellular ATP content in DU145 and BHP cells exposed for 2 hours to ImM levobupivacaine (Figure 3 A and B, respectively). The total ATP content was reduced by 20% in DU145 cells and 57% in BHP cell line (P=0.0264 and P = 0.0059, respectively). In DU145 cancer cells (Figure 3A), this reduction of ATP content had two components: mitochondrial ATP production was diminished by approximately 50% and glycolytic ATP production by 17%. In BHP cells mitochondrial ATP production was diminished by 60% and glycolytic ATP production by 55% (Figure 3B). To verify whether the observed reduction of intracellular total ATP content induced by levobupivacaine was the primary cause of cell cycle arrest, we mimicked the dual inhibition of the two energy production systems, namely glycolysis and OXPHOS, by using specific inhibitors of those two pathways. Rotenone was used as a specific inhibitor of respiratory chain complex I, and iodoacetate as an inhibitor of glycolysis. In order to accurately reproduce the bioenergetic impact of levobupivacaine we had first to determine the doses of rotenone and of iodoacetate, which could reduce mitochondrial and glycolytic ATP production to the same levels as levobupivacaine did. We chose to use rotenone at ΙΟΟηΜ as this dose mimicked the inhibitory effect of levobupivacaine on complex I-dependant respiration. Iodoacetate decreased the total ATP levels in a dose-dependent manner in DU145 cells (data not shown) and the combination of 100 nM rotenone with 2.5μΜ iodoacetate mimicked the inhibition of 20% of the total ATP (as obtained with levobupivacaine in DU145 cells) (data not shown). This combination of inhibitors triggered a strong decrease in DU145 cell growth after 24 hours of treatment, as also obtained with levobupivacaine (Figure 3D). To understand the basis of the cancer specific effect of levobupivacaine we considered that the repercussion of a given reduction of ATP production could have different consequences on cell proliferation, in accordance with the metabolic state of the cells and the metabolic control analysis. To evaluate the energy state of DU145 and BHP cells we measured the ATP/ADP ratio at steady-state which gives a measure of the balance between ATP synthesis and ATP consumption. We found a twice lower ATP/ADP ratio in DU145 cells as compared to BHP cells indicative of a higher rate of ATP consumption in the cancer cells (Figure 3C) which also showed a higher rate of proliferation (doubling time of 10.4 h as compared to BHP (19.8 h)). Although those two cell types showed similar steady-state ATP content values (Figure 3A and B) the higher ATP demand of DU145 cancer cells was indicated by the twice higher steady- state ADP content. Influence of tumor-like microenvironmental conditions on levobupivacaine antiproliferative effect

To investigate the impact of levobupivacaine on energy production in challenging conditions as occur in tumors we placed the DU145 cells in situations of reduced glucose concentration and oxygen tension. Cell culture is typically performed under 21% oxygen while in vivo oxygen tension varies between 1 to 10%> according to the tumor type. So, we placed DU145 cells under 1% 02 and treated with levobupivacaine for 24 h. We found no difference in the proliferation of cells grown in 25mM glucose in normoxia or hypoxia (data not shown). As most cancer cells are typically grown in high glucose media (25mM glucose) as performed above, we also analyzed the effect of levobupivacaine in DU145 cells grown in a 5mM glucose medium (data not shown). The results indicate a higher anti-proliferative effect of levobupivacaine in those conditions more representative of physiology (P=0.0303). Conversely, at low oxygen (1%) this effect was no longer observed (data not show), indicating the influence of the microenvironment on cancer cell sensitivity to levobupivacaine .

Levobupivacaine anti-cancer cytotoxic effect is potentialized by blockade of autophagy with Wortmannin

Autophagy is a survival pathway activated in response to nutrient deprivation, metabolic stress and exposure to anticancer drugs. The reduction in respiratory chain protein content in DU145 cells exposed to levobupivacaine led us to investigate the possible activation of autophagy by this drug. Levobupivacaine strongly induced the splicing- activation of LC3B, a marker of autophagy in the DU145 cancer cells while no significant change was detected in the primary prostate cell line BHP (data not shown). To further evaluate whether such autophagy was potentially used as a death or a survival pathway in these cells, we blocked autophagy with lOOnM Wortmannin and reassessed the impact of lmM levobupivacaine on cell enumeration after 24h. Wortmannin synergized the cytotoxic effect of levobupivacaine on the prostate cancer cell lines while it had no significant effect on BHP cells (Figure 4A). Similar findings were obtained with the PC3 cell line (Figure 4 B)

Example 2: in- vivo example

Material & Methods 26 mice SKH-1 immunocompetent were exposed (submitted) to UVB (3 irradiations per weeks of 150mJ / cm-2 UVB during 6 months) until obtaining at least one cutaneous tumor > 2mm of diameter. At this stage, mice were randomized in two groups: L group and SSI Group. In every group, mice benefited: i) from a counting and from a measure of the total tumoral volume of the cutaneous tumors, ii) of a surgical excision of tumors, iii) of a local infiltration at the level of the wound of the surgical sections, iv) of the implementation of a multi holed catheter linked with an osmotic pump (Alzet) allowing a wound continuous infiltration (1 micro litre / hour of for 7 days).

In the group L, the wound infiltration was performed with levobupivacaine (3mg/kg levobupivacaine 0.25mg/ml, i.e 300-400 microl/mouse); The osmotic pump contained 5mg/ml levobupivacaine. In SSI GROUP, saline serum was in the osmotic pump and wound infiltration was perfomed with saline serum. Both pump and catheter were removed during general anesthesia with isoflurane, 7 days after. Mice were evaluated once a week, during 28day period. Recurrence was defined by a new skin tumor >2 mm. The number of tumor >2mm was noted, and volume (VTT) was calculated at Day7, Dayl4, Day21 and Day28. The results were given as median [25%-75%] and groups were compared using Mann Withney Tes, P<0.05 was considered as significant

Results

26 mice have been included: 14 in L Group, 12 SSI group, 4 mice have been excluded: 3 for error in measures and one was dead. At day 0, no significant diffrence was noted between groups on the number and the volume (VTT). At day 28, the volume (VTT) was significantly decrease in L group [39.300 [13.100-53.100] mm3) in comparison with SSI group (80.600 [42.000-190.700] mm3) ((p=0.023). At Day 21, the volume (VTT) was significantly decrease in L group (9.2 [0-24] mm3) in comparison with in SSI group (35.60[ 19.000-63.800] mm3) (p=0.009). No significant difference was observed between groups if number of tumor was compared at Day 28 as well as Day 21. Example 3 : additional in-vitro example

A key result of the in vitro study is the deciphering of the mode of action of levobupivacaine on the cell cycle of prostate cancer cells. For instance, in figure 5 we analyzed the proportion of cancer cells entering the different stages of the cell cycle, using BrdU and 7-AAD as fluorescent indicators of DNA active synthesis and DNA content, respectively. We found a significant difference in the distribution of the cell population between the cell cycle phases (P=0.0441 for the G0-G1 phase and P=0.0054 for the S phase) (Figure 5D). Indeed, ImM levobupivacame led to the accumulation of the prostate cancer cells in the non-proliferating GO/Gl phase indicative of a potential failure to enter the high energy demanding S phase. This observation illustrates the anti-pro liferative potency of ImM levobupivacame on DU145 prostate cancer cells. This effect occurred after removal of the drug as DU145 cells required a longer time to perform cell doubling over 48 hours.

Example 4: additional in-vivo example

Figure 6 shows the levobupivacame antitumor effect in vivo, without precedent. The size of the tumor is reduced by 40% on a standardized model of skin tumor induced by UVs. This is full novelty and the first in vivo demonstration of an anti-cancer effect of this drug.

Conclusions:

The inventors present in vivo data on a standardized mice model (levobupivacame treated versus saline) where levobupivacame reduces skin cancer (SCC) tumor size. Such data can't be found in the state of the art.

The inventors present data in support of the specificity of levobupivacame anti-cancer effect by comparing, for example, epithelial prostate cancer cells (DU145) with corresponding epithelial non-cancer cells (BHP). The results show a specificity of the anti-cancer effect which makes the distinction with a non-specific cellular toxic effect. Such data (cancer versus tissue-paired non cancer cells) can't be found in the state of the art.

Moreover, the inventors show that levobupivacame induces its anti-cancer effect by triggering the combined dual inhibition of glycolysis (by 17%) and oxidative phosphorylation (by 50%), each to a moderate extent. This is the first time that "a dual moderate bioenergetic inhibition strategy" is proposed.

At last, the inventors demonstrate for the first time that levobupivacame alters cell cycle progression and maintain the cancer cells in the Gl phase. 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.

Jose C, Bellance N, Chatelain EH, Benard G, Nouette-Gaulain K, Rossignol R. Antiproliferative activity of levobupivacaine and amino imidazole carboxamide ribonucleotide on human cancer cells of variable bioenergetic profile. Mitochondrion. 2012 Jan;12(l): 100-9. doi: 10.1016/j.mito.2011.03.010.

Karniel M, Beitner R. Local anesthetics induce a decrease in the levels of glucose 1 , 6- bisphosphate, fructose 1,6-bisphosphate, and ATP, and in the viability of melanoma cells. Mol Genet Metab. 2000 Jan;69(l):40-5.

Claims

CLAIMS:

1. A compound which is an inhibitor of cellular energy transduction processes for use in the treatment of cancer or for use in prevention of cancer recurrence.

2. A compound for use according to the claim 1 wherein the inhibitor is a local anesthetic.

3. The local anesthetic for use according to claim 2 wherein the cancer is a prostate cancer or a skin cancer.

4. The local anesthetic for use according to claim 3 wherein the prostate cancer is a prostate adenocarcinoma.

5. The local anesthetic for use according to claim 3 wherein the skin cancer is basal cell carcinoma, squamous cell carcinoma or a melanoma.

6. The local anesthesic for use according to claims 2 to 5 wherein the local anesthesic is administrated locally.

7. The local anesthesic according to claims 6 wherein the local anesthesic is administrated thank to a pump.

8. The local anesthesic according to claims 2 to 7 wherein the local anesthesic is an aminoamide anesthesic.

9. The local anesthesic according to claims 2 to 7 wherein the aminoamide anesthesic is the Levobupivacaine.

10. The local anesthetic for use in the prevention of cancer recurrence according to claim 2 wherein the local anesthetic is used after chirurgical excision of the primary tumor.

11. The local anesthetic for use according to claims 2 to 10 wherein the local anesthetic is used in combination with an inhibitor of autophagy.

12. The local anesthetic for use according to claim 11 wherein the inhibitor of autophagy is the Wortmannin.

13. A i) local anesthetic according to claim 2 to 10 and ii) an inhibitor of autophagy according to claims 11 to 12 as a combined preparation for simultaneous, separate or sequential use in the treatment of cancer or in the prevention of cancer recurrence in a patient.

14. A pharmaceutical composition comprising an effective amount of an inhibitor of cellular energy transduction processes or inhibitor of cellular energy transduction processes and an inhibitor of autophagy for use in the treatment of cancer or for use in the prevention of cancer recurrence and pharmaceutically acceptable excipients or carriers.