WO2022194926A1 - Nanoparticles comprising a core with a phenazine derivative and a shell with a nucleolipid and uses thereof - Google Patents
Nanoparticles comprising a core with a phenazine derivative and a shell with a nucleolipid and uses thereof Download PDFInfo
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- WO2022194926A1 WO2022194926A1 PCT/EP2022/056809 EP2022056809W WO2022194926A1 WO 2022194926 A1 WO2022194926 A1 WO 2022194926A1 EP 2022056809 W EP2022056809 W EP 2022056809W WO 2022194926 A1 WO2022194926 A1 WO 2022194926A1
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- group
- branched
- linear
- alkyl
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- ZSDSQXJSNMTJDA-UHFFFAOYSA-N trifluralin Chemical compound CCCN(CCC)C1=C([N+]([O-])=O)C=C(C(F)(F)F)C=C1[N+]([O-])=O ZSDSQXJSNMTJDA-UHFFFAOYSA-N 0.000 description 1
- 230000006663 ubiquitin-proteasome pathway Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 description 1
- 229940045145 uridine Drugs 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
- A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/498—Pyrazines or piperazines ortho- and peri-condensed with carbocyclic ring systems, e.g. quinoxaline, phenazine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Definitions
- Nanoparticles comprising a core with a phenazine derivative and a shell with a nucleolipid and uses thereof
- the present invention concerns nanoparticles with a core comprising a phenazine derivative and a shell comprising at least one layer of a nucleolipid, compositions comprising them and their pharmaceutical uses, especially in the prevention and/or treatment of diseases in which the interaction between the proteins Hsp27 and elF4E is involved.
- prostate cancer is one of the most frequent as well as the third cause of mortality due to cancer among men in France.
- the treatment involves surgery or radiotherapy but the androgenic removal, also called hormonotherapy, remains the only effective therapy of the locally advanced or metastatic prostate cancer.
- Flormonotherapy provides an objective answer for more than 80% of cases but in spite of its transitional effectiveness, it does not prevent the castration-resistant progression of the disease that usually arises within the 2 to 3 years after the beginning of the hormonal treatment.
- a strategy to improve the treatment of cancers consists in targeting the survival genes overexpressed in these diseases, in particular in the castration-resistant prostate cancer, so as to restore sensitivity to hormonotherapy and/or chemotherapy.
- the protein elF4E is part of a large multi-protein complex, elF4F, which plays a role in the translation initiation of eukaryotes and which is composed of: elF4E, that directly interacts with the cap structure of the mRNA, elF4A, a RNA helicase, and the scaffolding protein elF4G.
- Hsp27 is a chaperon protein belonging to the « Small Heat Shock Proteins » (Hsp) family. In the physiological conditions, the Hsp27 is expressed in a very low quantity in a ubiquitous form.
- Hsp27 During a cell stress, caused for instance by hormonotherapy or chemotherapy, Hsp27 accumulates in a large amounts in the cells, blocks the apoptosis, and thus permits cell survival.
- This deregulation of the proteic synthesis process is associated with the development and the progression of cancers. Flence, the protein translation becomes an oncogene process, which leads to cancers resistant to hormonotherapy and/or chemotherapy.
- the aim of the present invention is to provide an improved treatment of diseases wherein the interaction between Flsp27 and elF4E is involved, especially cancers.
- Another aim of the present invention is to provide an improved formulation allowing a satisfying solubilization and delivery of active ingredients targeting the interaction between Flsp27 and elF4E, in particular phenazine derivatives.
- One aim of the invention is also to provide a stable composition comprising a formulation of phenazine derivatives, in particular comprising nanoparticles containing phenazine derivatives.
- a particular aim of the present invention is to provide an improved formulation of phenazine derivatives, useful in the treatment of cancers.
- the invention thus relates to nanoparticles comprising:
- R I F3 ⁇ 4 2 and represent R 8 , -0R 8 or -OCOR 9 , with R 8 representing a linear or branched (Ci-C 2 o)alkyl group, and Rg representing a hydrogen atom or a linear or branched (C 2 -C 2 o)alkyl group;
- R 3 and R independently of each other, represent:
- halogen atom selected from the group consisting of: chlorine, fluorine, bromine, and iodine atom
- R 5 and R 6 representing independently of each other, a hydrogen atom, a linear or branched (Ci-C 2 o)alkyl group, a benzyl group or a -COR 10 group with Ri 0 representing a hydroxyl group or a linear or branched (Ci-C 2 o)alkoxy group;
- R 7 represents a linear or branched (Ci-C 2 o)alkyl group, or a aryl group;
- R 8 ' and Rg' representing a hydrogen atom or a linear or branched (Ci-C 2 o)alkyl group
- R 11 representing a hydrogen atom or a tert-butyloxycarbonyl group
- n and n' and m independently of each other, representing an integer with n and n', independently, ranging from 2 to 6 and m ranging from 1 to 2, and, when m is 2, each n value may be identical or different one from another, or one of its pharmaceutically acceptable salts; and a lipid shell comprising at least one layer of a nucleolipid having the following formula (A): wherein:
- ⁇ Re represents a purine or pyrimidine base such as uracil, adenine, guanine, cytosine, thymine, hypoxanthine, or their derivatives, or a non-natural mono- or bi- cyclic heterocyclic base each ring of which comprises 4 to 7 members, optionally substituted;
- Li and L 2 independently of each other, are selected from the group consisting of: hydrogen atom, -O-C(O)-, -0-C(S)-NH-, -0-C(0)-0-, -0-C(0)-NH-, an oxygen atom, a phosphate group, a phosphonate group or a heteroaryl comprising from 1 to 4 nitrogen atoms, said heteroaryl being unsubstituted or substituted by a linear or branched (C 2 - C 3 o)alkyl or a linear or branched (C 2 -C 3 o)alkenyl, or, Li and l_ 2 , together form a ketal group of formula: ; or, Li or l_ 2 is a hydrogen atom, and the other is a hydroxy group or a heteroaryl group comprising from 1 to 4 nitrogen atoms, said heteroaryl being unsubstituted or substituted by a linear or branched (C 2 -C 3 o)
- ⁇ Mi and M 2 independently of each other, represent:
- alkyl or alkenyl group being optionally completely or partially fluorinated, and/or substituted on the carbon at the end of the chain by a fluorine atom or by a benzyl or naphthyl ester or ether;
- each acyl chain is in C 2 -C 30 ;
- Li or l_ 2 represents hydrogen, and the other represents a hydroxy group or a heteroaryl group comprising 1 to 4 nitrogen atoms, then Mi and M 2 do not exist;
- ⁇ Ra is selected from the group consisting of:
- R b , R c and R d identical or different, represent a hydrogen atom, a linear or branched (Ci-Cio)alkyl or a linear or branched O- (Ci-Cio)alkyl;
- R a is bound by a covalent bond to another substituent R a , identical or different, of another compound of formula (A), identical or different, in order to form a compound of formula (A) in the form of a dimer.
- Phenazine derivatives are known as useful in the treatment of pancreatic and prostate cancers, as described in WO 2011/117830.
- the present inventors surprisingly discovered that the phenazine derivatives according to the general formula (I) as defined above are able to inhibit the specific Hsp27-elF4E interaction.
- the inventors surprisingly found a formulation allowing the administration of phenazine derivatives of general formula (I) to patients, which improves their efficacy against diseases in which the Hsp27-elF4E interaction is involved, in particular cancers.
- the nanoparticles according to the invention allow the administration of the phenazine derivatives of general formula (I) to mammal, especially humans.
- nanoparticles of the invention lead to a greater inhibition of the interaction between Hsp27 and elF4E in cells, compared to the phenazine derivatives of general formula (I) alone.
- the nanoparticles according to the invention lead to a decrease in cell viability and to an increase of apoptosis compared to phenazine derivatives of general formula (I) alone when administered to cancer cells, in particular in castration-resistant prostatic cancer cells.
- the nanoparticles according to the invention also reduce the tumor volume compared to phenazine derivatives of general formula (I) alone, in particular when administered in castration-resistant prostatic cancer.
- the nanoparticles according to the invention restore sensitivity to hormonotherapy and/or chemotherapy, such as in the castration-resistant prostate cancer.
- the inhibition of the Hsp27-elF4E interaction by the nanoparticles of the invention does not affect the other functions of the cells, whereas separate inhibition of Hsp27 and/or elF4E induces cytotoxicity, such as in non-cancer cells.
- the nanoparticles according to the invention also allow a homogenous cytoplasmic concentration of the phenazine derivatives in the cells, in particular when the nucleolipid (A) is DOUPEG-2000.
- alkyl means a saturated aliphatic hydrocarbon group which may be straight or branched. Preferred alkyl groups have 1 to 20 carbon atoms in the chain. Preferred alkyl groups are in particular methyl, ethyl, propyl or dodecyl groups. "Branched" means that one or lower alkyl groups such as methyl, ethyl or propyl are attached to a linear alkyl chain.
- alkoxy refers to a -O-alkyl group, with the alkyl group being as defined above. Among alkoxy, it may be cited, the methoxy, ethoxy, propoxy or isopropoxy groups
- alkylthio refers to a -S-alkyl group, with the alkyl group being as defined above.
- alkenyl refers to an alkyl group, comprising at least two carbon atoms and one double bond, preferably one or two double bonds. Alkenyl groups may have between 2 and 30 carbon atoms. Preferred alkenyl groups have between 10 and 20 carbon atoms in the chain, for example 17 carbon atoms.
- alkenylthio refers to a -S-alkenyl group, with the alkenyl group being as defined above.
- -(Ci-Cio)alkylene- means a saturated aliphatic hydrocarbon divalent radical which may be straight or branched having 1 to 10 carbon atoms in the chain.
- Preferred alkylene groups are methylene, ethylene or propylene groups.
- aryl refers to an aromatic monocyclic, bicyclic, or tricyclic hydrocarbon ring system comprising from 6 to 10 carbon atoms wherein any ring atom capable of substitution may be substituted by a substituent. In one embodiment, aryl groups are not substituted. Examples of aryl moieties include, but are not limited to phenyl or naphtyl.
- heteroaryl refers to an aromatic or partially unsaturated monocyclic or bicyclic hydrocarbon ring system comprising from 5 to 12 carbon atoms, wherein any ring atom capable of substitution may be substituted by a substituent and wherein one or more carbon atom(s) are replaced by one or more heteroatom(s) such as nitrogen atom(s), oxygen atom(s) and sulphide atom(s); for example 1 or 4 nitrogen atom(s), 1 or 2 oxygen atom(s), 1 or 2 sulphide atom(s) or a combination of different heteroatoms such as 1 nitrogen atom and 1 oxygen atom.
- Preferred heteroaryl groups comprise from 1 to 4 nitrogen atoms.
- heteroaryl examples include but are not limited to pyridine, pyrimidine, pyrazole, triazole, tetrazole or imidazole groups. In one embodiment, heteroaryl groups are not substituted.
- halogen refers to the atoms of the group 17 of the periodic table and includes in particular fluorine, chlorine, bromine, and iodine atoms, more preferably fluorine, chlorine and bromine atoms.
- purine or pyrimidine base is meant for example, a non-natural mono- or bi-cyclic heterocyclic base in which each cycle has 4 to 7 carbon atoms, preferably 5 or 6 carbon atoms, wherein any ring atom capable of substitution may be substituted by a substituent and wherein one or more carbon atom(s) are replaced by one or more heteroatom(s) such as nitrogen atom(s), oxygen atom(s) and sulphide atom(s); for example 1 , 2, 3 or 4 nitrogen atom(s), 1 or 2 oxygen atom(s), 1 or 2 sulphide atom(s) or a combination of different heteroatoms such as 1 nitrogen atom and 1 oxygen atom
- the purine or pyrimidine derivatives correspond respectively to substituted purine or pyrimidine base.
- the derivatives of the purine or pyrimidine base have an anti-tumoral activity and/or an anti-viral activity such as cytarabine (AraC), 5-fluoro-uracile (5-FU), iododeoxyuridine (IdU), 2’-deoxy-2’- methylidenecytidine (DMDC) or 5-chloro-6-azido-5,6-dihydro-2’-deoxyuridine.
- an anti-tumoral activity and/or an anti-viral activity such as cytarabine (AraC), 5-fluoro-uracile (5-FU), iododeoxyuridine (IdU), 2’-deoxy-2’- methylidenecytidine (DMDC) or 5-chloro-6-azido-5,6-dihydro-2’-deoxyuridine.
- non-natural mono- or bicyclic heterocyclic base is meant a base different from uracil, aden
- Protein interaction may be understood as a physical contact with molecular docking between proteins that occurs in a cell or in a living organism, preferably in particular biomolecular contexts.
- the interaction between Hsp27 and elF4E may thus be defined as a physical contact, preferably with molecular docking, between these two proteins.
- Hsp27 is phosphorylated when it interacts with elF4E.
- Tests for assessing the interaction between two proteins are well known.
- the interaction between two proteins may be assessed directly, for example by plasmon surface resonance, flow cytometry or beta-imager.
- the interaction between the two proteins Hsp27 and elF4E may be assessed by known methods such as co-immunoprecipitation or BRET assay (Bioluminescence Resonance Energy Transfer assay).
- the compounds of formula (I) and/or the compounds of formula (A) may comprise an unsaturation site and thus may be in their tautomeric forms and/or their Cis or Trans isomers.
- the compound of formula (I) is:
- R I R2 and represent R 8 , -OR 8 or -OCORg, with R 8 representing a linear or branched (Ci-C2o)alkyl group, and Rg representing a hydrogen atom or a linear or branched (Ci-C2o)alkyl group;
- ⁇ R3 and R4 independently of each other, represent:
- R 5 and R6 representing independently of each other: a hydrogen atom, a linear or branched (Ci-C2o)alkyl group, a benzyl group or a -COR10 group, R10 being a hydroxyl group or a linear or branched (Ci-C2o)alkoxy group ;
- each n value may be identical or different one from another, or one of its pharmaceutically acceptable salts.
- both Ri and R 2 are a linear or branched -0(Ci-C 2 o)alkyl group.
- R 3 and FU represent independently of each other: a hydrogen atom, -N(R 5 )(R 6 ) or -CO-[NH-(CH 2 ) n ] m -N(R 5 )(R 6 ), with n, m, R 5 and R 6 being as above.
- R 5 and R 6 are H or methyl, ethyl or propyl.
- n is 2 and/or m is 1.
- the compound of formula (I) has one of the following formulae:
- the compounds of general formula (I) herein described may have asymmetric centers.
- Compounds of the present invention containing an asymmetrically substituted atom may be isolated in optically active or racemic forms. It is well-known in the art how to prepare optically active forms, such as by resolution of racemic forms or by synthesis from optically active starting materials. All chiral, diastereomeric, racemic forms and all geometric isomeric forms of a compound are intended, unless the stereochemistry or the isomeric form is specifically.
- pharmaceutically acceptable salt refers to salts which retain the biological effectiveness and properties of the compounds of the invention and which are not biologically or otherwise undesirable.
- the compounds of general formula (I) may be provided in the form of a free base or in the form of addition salts with acids.
- Pharmaceutically acceptable acid addition salts may be prepared from inorganic and organic acids, while pharmaceutically acceptable base addition salts can be prepared from inorganic and organic bases.
- Pharmaceutically acceptable salts see Berge, et al. ((1977) J. Pharm. Sd, vol. 66, 1).
- the salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like, as well as salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, fumaric, methanesulfonic, and toluenesulfonic acid and the like.
- Li and l_ 2 independently of each other, are selected from the group consisting of: hydrogen atom, -O-C(O)-, -0-C(S)-NH-, -0-C(0)-0-, -0-C(0)-NH-, an oxygen atom, a phosphate group and a phosphonate group.
- R e represents a purine or pyrimidine base such as uracil, adenine, guanine, cytosine, thymine, or hypoxanthine, preferably uracil
- the nucleolipid of formula (A) has the following formula
- Mi and M 2 are independently of each other a linear or branched (C 2 -C3o)alkyl or a linear or branched (C 2 - C3o)alkenyl, preferably a (C 2 -C3o)alkenyl. More particularly in the nucleolipid of formula (A) or (A-l), Mi and M 2 are identical and preferably represent a linear (Cio-C 2 o)alkenyl, preferably comprising one double bond.
- R a is selected from the group consisting of:
- R b , R c and R d identical or different, represent a hydrogen atom or a linear or branched (Ci-Cio)alkyl group
- R a is a
- nucleolipid of formula (A) has one of the following formulae:
- the core comprises a compound of formula (I) having one of the following formulae:
- the lipid shell comprises at least one layer of a nucleolipid of formula (A) having one of the following formulae:
- r being an integer comprised between 10 and 20.
- the core of the nanoparticles comprising at least one compound of general formula (I) as defined above may be coated, preferably directly coated, by the lipid shell comprising at least one layer of a nucleolipid of general formula (A) as defined above.
- said lipid shell is coated by at least one other layer; for example by one or two layer(s) of another nucleolipid said layer(s) having an opposite charge compared to charge of the zeta potential of the core coated with the lipid shell as defined above.
- said another layer is a bilayer of the compound diC16dT, having the following formula:
- the core of the nanoparticles according to the invention comprises one compound of the general formula (I) as defined above.
- the core is hydrophobic.
- the core consists of an amorphous precipitate of at least one compound of general formula (I), preferably of one compound of general formula (I).
- the lipid shell consists of one layer of a nucleolipid of general formula (A) as defined above. In another embodiment, the lipid shell comprises two, three or more layers of nucleolipid(s) of general formula (A) as defined above, said nucleolipid(s) of general formula (A) being identical or different in each layer.
- the nanoparticles according to the invention are solid at room temperature, preferably between 15°C and 25°C.
- the nanoparticles of the invention are characterized by a mean size particle comprised between 50 nm and 150 nm, preferably between 60 nm and 130 nm.
- the nanoparticles of the invention are characterized by a polydispersity index inferior or equal to 0.6, preferably comprised between 0.1 and 0.5.
- the nanoparticles according to the invention have a low polydispersity index, preferably inferior to 0.5.
- the “polydispersity index” may refer to the measure of the width of the sizes of the nanoparticles.
- the polydispersity index may be measured by Dynamic light scattering.
- nanoparticles according to the invention thus have homogenous sizes, leading to stable compositions comprising them.
- the nanoparticles of the invention are characterized by a zeta potential comprised between 20 mV and 90 mV, preferably between 30 mV and 80 mV.
- the “zeta potential” may be defined as the potential difference between the dispersion medium and the stationary layer of the fluid attached to the dispersed particle and may be determined by Electrophoretic Light Scattering (ELS).
- ELS Electrophoretic Light Scattering
- the nanoparticles according to the invention advantageously lead to a stable colloidal dispersion, in an aqueous medium.
- the above mentioned nanoparticles are characterized by a mean size particle comprised between 60 nm and 80 nm and/or a polydispersity index comprised between 0.1 and 0.3 and/or a zeta potential comprised between 60 mV and 80 mV.
- the nanoparticles are characterized by a mean size particle comprised between 60 nm and 130 nm and/or a polydispersity index comprised between 0.1 and 0.5 and/or a zeta potential comprised between 30 mV and 50 mV.
- the mean size particle, the polydispersity index and the zeta potential as defined above may be determined thanks to the Zetasizer nanoserie (Malvern Instruments).
- the nanoparticles according to the invention may be prepared according to known techniques.
- the nanoparticles according to the invention may be prepared by nanoprecipitation or by formation of a lipid film.
- a process of preparation of the nanoparticles according to the invention comprises the following steps: i) solubilizing a nucleolipid of general formula (A) as defined above in an organic solvent, such as chloroform, thereby obtaining a mixture (N); ii) in another recipient solubilizing a compound of general formula (I) as defined above in an organic solvent, such as chloroform, thereby obtaining a mixture (P); iii) optionally stirring the mixture (N); iv) adding the mixture (N) in the mixture (P), thereby obtaining a mixture (M); v) removing the organic solvent from the mixture (M), thereby obtaining a nucleolipid film; vi) drying the nucleolipid film obtained at step v), preferably under vacuum; vii) rehydrating the dried nucleolipid
- step v) is performed by evaporating the organic solvent under reduced pressure or under a nitrogen flow.
- a process of preparation of the nanoparticles according to the invention comprises the following steps: i) solubilizing a nucleolipid of general formula (A) as defined above in an organic solvent, such as chloroform, thereby obtaining a mixture (N); ii) in another recipient solubilizing a compound of general formula (I) as defined above in an organic solvent, such as chloroform, thereby obtaining a mixture (P); iii) optionally stirring the mixture (N); iv) adding the mixture (N) in the mixture (P), thereby obtaining a mixture (M) by stirring; v) adding the mixture (M) dropwise to water; vi) concentrating the obtained mixture; vii) optionally removing the remaining organic solvent, preferably under reduced pressure; and vii) performing sonication.
- the invention also relates to the nanoparticles as defined above, for use as a medicament, more particularly for use in the prevention and/or treatment of diseases in which the Hsp27-elF4E interaction is involved.
- a disease wherein the Hsp27-elF4E interaction is involved it may be meant a disease wherein Flsp27 and/or elF4E are expressed, over-expressed and/or mutated, preferably overexpressed.
- the nanoparticles according to the invention are useful as inhibitors of the Flsp27-elF4E interaction.
- the invention also relates to a method of prevention and/or treatment of a disease in which the Flsp27-elF4E interaction is involved, comprising the administration to a mammal, preferably a human, in need thereof of a therapeutically effective amount of the nanoparticles according to the invention.
- the nanoparticles of the invention are intended for use in the prevention and/or treatment of cancers.
- Cancer means the uncontrolled, abnormal growth of cells and includes within its scope all the well-known diseases that are caused by the uncontrolled and abnormal growth of cells as well as metastasis.
- the cancer is chosen among the group consisting of lymphomas, angiosarcomas and the cancers of the lung, pancreas, breast, bladder, colon, skin, head and neck, ovarian, and prostate.
- the cancer is selected from the group consisting of: prostate cancer, breast cancer and pancreatic cancer, preferably prostate cancer such as resistant prostate cancer.
- the cancer is the hormono-resistant prostate cancer (also called castration resistant prostate cancer).
- resistant prostate cancer it may be meant a prostate cancer for which hormonotherapy and/or chemotherapy is(are) not sufficient to cure said prostate cancer.
- resistant prostate cancer is a prostate cancer for which hormonotherapy and/or chemotherapy do(es) not allow to:
- tumor stasis tumor stasis
- resistant prostate cancer may refer to:
- Hormonotherapy is also called androgen deprivation therapy (ADT) or androgen suppression therapy.
- ADT androgen deprivation therapy
- the goal of this treatment is to reduce levels of androgens (testosterone and dihydrotestosterone) in patients, or to stop them from affecting prostate cancer cells.
- LHRH agonists, LHRH antagonists, CYP17 inhibitors, anti-androgens, or estrogens may be used.
- Chemotherapy is a category of cancer treatment that uses chemical substances which are anti-cancer cells agents.
- chemical substances which are anti-cancer cells agents.
- docetaxel, cabazitaxel, mitoxantrone, or estramustine may be used in the treatment of prostaste cancer.
- 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 disorder or condition.
- the treatment of cancers may consist in destroying and/or depleting cancer cells and/or preventing resistance and/or restore sensitivity to hormonotherapy and/or chemotherapy of cancer cells.
- the term "patient” or “individual” to be treated is preferably intended for a human or non-human mammal (such as a rodent, for example a mouse or a rat, a feline, a canine, or a primate) affected or likely to be affected with a disease, in particular in which the interaction Hsp27-elF4E is involved.
- a human or non-human mammal such as a rodent, for example a mouse or a rat, a feline, a canine, or a primate
- the patient is a human.
- a “therapeutically effective amount” or “therapeutic dose” is an amount sufficient to obtain the desired clinical results (i.e., achieve therapeutic efficacy).
- a therapeutically effective dose can be administered in one or more administrations.
- a therapeutically effective dose is an amount that is sufficient to treat the disease as defined above, in particular cancers as defined above.
- the invention also relates to a pharmaceutical composition, preferably an aqueous pharmaceutical composition, comprising the nanoparticles as defined above and optionally one or more pharmaceutically acceptable excipient(s).
- the present invention also relates to a drug, comprising the nanoparticles as defined above. While it is possible for the nanoparticles of the invention to be administered alone, it is preferred to present them as pharmaceutical compositions.
- the pharmaceutical compositions, both for veterinary and for human use, useful according to the present invention comprise at least one nanoparticle as above defined, together with one or more pharmaceutically acceptable carriers and possibly other therapeutic ingredients.
- active ingredients necessary in combination therapy may be combined in a single pharmaceutical composition for simultaneous administration.
- compositions, carriers, diluents and reagents are used interchangeably and represent that the materials are capable of administration to or upon a mammal without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like.
- compositions that contains the nanoparticles as defined above dispersed therein are well understood in the art and need not be limited based on formulation.
- such compositions are prepared as injectables either as liquid solutions or suspensions; however, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared.
- the preparation can also be emulsified.
- the pharmaceutical compositions may be formulated in solid dosage form, for example capsules, tablets, pills, powders, dragees or granules.
- the choice of vehicle is generally determined in accordance with the physical and chemical properties of the nanoparticles, the particular mode of administration and the provisions to be observed in pharmaceutical practice.
- excipients such as lactose, sodium citrate, calcium carbonate, dicalcium phosphate and disintegrating agents such as starch, alginic acids and certain complex silicates combined with lubricants such as magnesium stearate, sodium lauryl sulphate and talc may be used for preparing tablets.
- lactose and high molecular weight polyethylene glycols When aqueous suspensions are used they can contain emulsifying agents or agents which facilitate suspension.
- Diluents such as sucrose, ethanol, polyethylene glycol, propylene glycol, glycerol and chloroform or mixtures thereof may also be used.
- compositions can be administered in a suitable formulation to humans and animals by topical or systemic administration, including oral, rectal, nasal, buccal, ocular, sublingual, transdermal, rectal, topical, vaginal, parenteral (including subcutaneous, intra-arterial, intramuscular, intravenous, intradermal, intrathecal and epidural), intracisternal and intraperitoneal.
- topical or systemic administration including oral, rectal, nasal, buccal, ocular, sublingual, transdermal, rectal, topical, vaginal, parenteral (including subcutaneous, intra-arterial, intramuscular, intravenous, intradermal, intrathecal and epidural), intracisternal and intraperitoneal.
- parenteral including subcutaneous, intra-arterial, intramuscular, intravenous, intradermal, intrathecal and epidural
- intracisternal including subcutaneous, intra-arterial, intramuscular, intravenous, intradermal, intrathecal and epidural
- the formulations can be prepared in unit dosage form by any of the methods well known in the art of pharmacy. In general, the formulations are prepared by uniformly and intimately bringing into association the nanoparticles with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product. Description of the figures
- Figures 1A and 1B Compound 14, 14+DOTAU (CAS Registry Number DOTAU: 868226- 06-6) and 14+DOUPEG-2000 (CAS Registry Number DOUPEG-2000: 1353570-75-8) at 100 mM inhibit the interaction of elF4E with Hsp27.
- the cells proteins PC-3 were extracted.
- FIG. 2 The treatment of PC-3 cells with 14+DOTAU and 14+DOUPEG-2000 inhibits the proliferation of PC-3 cells.
- the PC-3 cells had been treated at the doses of 25, 50 and 100 mM by the nanoparticles according to the invention. After 2 days of treatment, the cell viability was analyzed thanks to a test at MTT.
- Figure 2 shows the percentage of cell viability regarding the compounds and nanoparticles tested. The experiment was carried out in triplicate.
- Figure 4 14+DOTAU increases apoptosis of PC-3 cells in vitro.
- Figure 4 shows the percentage of cells undergoing the sub GO phase of the cellular cycle, which reflects cells undergoing apoptosis, when treated with control (PC-3), compound 14, DOTAU and 14+DOTAU.
- Figure 5 14+DOTAU significantly enhanced anticancer activity and reduced toxicity in tumor-xenograft mice.
- Figure 5 shows the tumor volume according to the duration of treatment.
- Figure 6. The treatment of PC-3 cells with 13+DOTAU and 13+DOU-PEG2000 inhibits the proliferation of PC-3 cells. The PC-3 cells had been treated at the doses of 25, 50 and 100 mM by the nanoparticles according to the invention. After 2 days of treatment, the cell viability was analyzed thanks to a test at MTT.
- Figure 6 shows the percentage of cell viability regarding the compounds and nanoparticles tested. The experiment was carried out in triplicate.
- NP D oTAu-com P ound 14 decreased tumor volume in vivo.
- Mice body weight was measured twice per week.
- FIG. 1 Distribution of tissue Ki-67 immunostaining intensity (measured as average optical density) according to the tumor treated with PBS, DOTAU, compound 14 and
- Example 1 Preparation of the nanoparticles according to the invention
- Compound 14 8,1 mg was kindly given by collaboration with Drs. M. Camplo and O. Siriteil Interdisciplinary Center for Nanosciences, CNRS UPR3118).
- DOTAU 8 mg was given kindly by Pr. P. Barthelemy. In an hemolyse tube, 8 mg of DOTAU (n) were solubilized in Chloroform (200 pL).
- DOTAU 8 mg
- DOTAU 8 mg
- Chloroform 200 mI_
- Compound 14 (n) was solubilized in Chloroform (200 mI_).
- the nucleolipid solution was added in the drug solution.
- the solvent was removed to yield a nucleolipid film.
- the nucleolipid film is thoroughly dried to remove residual organic solvent under vacuum 2 hours.
- the Nucleolipid layer is then rehydrated in 4 ml. of EuroBio Water (2mg/ml_).
- Example 2 Biological activity of the nanoparticles according to the invention
- the androgen-independent prostate cancer cell line PC-3 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in Dulbecco’s Modified Eagle’s Medium (Life Technology, Inc, Saint Aubin, France) supplemented with 10% fetal bovine serum (FBS). All cell lines were cultivated at 37°C in 5% C0 .
- the Phenazine Compounds The compound 14 as well as 14 encapsulated in the nucleolipids DOTAU and DOU-PEG 2000 were kindly given by collaboration with Drs. M. Camplo and O. Siri (Marseille Intended Center for Nanosciences, CNRS UPR3118). The compound 14 is dissolved in DMSO (dimethyl sulfoxide) at a stock concentration of 10 mM. The compound 14 was encapsulated in nanoparticles DOTAU and DOU-PEG2000 according to example 1 and had been dissolved in water. 3- Treatment of cells:
- PC-3 cells were seeding into 10cm dishes (1 250 OOOcells/well) or 12-well plates (50 000 to 100 OOOcells/well) according to the different experiments. The day after, the medium was changed to a new one containing DMSO (control) or derivative of phenazine 14 or phenazine 14 encapsulated in the nucleolipids DOTAU and DOU-PEG at a concentration of 100mM according to the different experiments. Effects of the treatment were analyzed 48 hours later. 4 Immunoprecipitation:
- PC-3 cells were seeded into a 12-well plate containing cover glasses covered by FBS, at a density of 100 000 cells/well. 24h later, cells were treated with compounds (DMSO, DOU-PEG2000, DOTAU, 14, 14+DOU-PEG2000 and 14+DOTAU at 100mM as indicated above. After 48h of treatment, cells were washed with PBS 1X and fixed with formaldehyde 4% (Thermo Fisher Scientific, lllkrich, France) during 15 min at RT. Several washes were done with PBS1X before mounting the cover glasses on glass slides by using Prolong Gold antifade reagent with DAPI (Life Technologies SAS, Villebon-Sur- Yvette, France).
- PC-3 cells were seeded into 6 cm dishes at a density of 250 000 cells/well. After 24h, cells were treated with compounds (DOU-PEG2000, DOTAU, 14, 14+DOU-PEG2000 and 14+DOTAU) at 100mM as indicated above. After 48h of treatment, dead and living cells were collected and washed with PBS1X, before being fixed in cold ethanol 70% (250mI/rqIIq ⁇ ). After 1 h incubation at 4°C, cells were washed with PBS1X before permeabilization using flow buffer (8 parts of citric acid 0.1 M, 192 parts of Na 2 HPC> 4 0.2M) (1 OOmI/pellet).
- flow buffer 8 parts of citric acid 0.1 M, 192 parts of Na 2 HPC> 4 0.2M
- PC3 cells were inoculated in the flank region of 2 weeks old male athymic (node scid). Tumors were measured weekly and their volume were calculated by the formula lengh * width * depth * 0.5236.
- mice were randomly selected for treatment with PBS (control) alone, compound 14 alone, DOTAU alone and 14+DOTAU.
- Each experimental group consisted of 6 mice of control and 8 mice for other groups.
- Compound 14 was tested at 1 mg/ml, which corresponds to its maximum of solubility or 14+DOTAU was tested at 2mg/ml. Injection lasted 8 weeks with two injections for weeks. Data points were expressed as average tumor volume levels ⁇ S.E.
- Figure 1A shows that the encapsulated compound 14 maintains its inhibiting capacity on the elF4E-Hsp27 interaction.
- the test at MTT shows that the treatment by the 14+DOTAU decreases cell viability of 70%. In fact, it goes from 100% for the untreated cells to 30% for the treated ones at a dose of 100 mM.
- the encapsulated compound 14 is more effective than the compound 14 alone whose effectiveness is only 50% for the same dose.
- phenazine derivatives have very low solubility, notably in aqueous medium and biological fluids. Therefore, the tested compound 14 alone cannot be administered as such to patients.
- the nanoparticles according to the invention maintain or improve the efficacy of the compounds of general formula (I), in particular decrease cell viability and can be administered to patients.
- C- The encapsulation of comoound#14 in nanooarticles improves the delivery of the compound for the cells:
- Compound#14 was shown to be internalized in cells and mainly localized in the cytoplasm with an heterogeneous distribution.
- the encapsulation of compound#14 by DOTAU was only slightly modified compared to compound #14, with a pedicaliar pattern of aggregates.
- Compound 14 DOU-PEG2000 staining pattern was much more intense and homogenous that the latter ones.
- the encapsulation of the compound#14 by par DOU-PEG2000 permits an homogenous and intense repartition of the compound#14, the intracellular localization of the compound remaining equally cytoplasmic.
- PC3 cells were inoculated in the flank region of 2 weeks old male athymic (Node Scids) (Figure 5). Tumors were measured weekly and their volume were calculated by the formula lengh * width * depth * 0.5236.
- mice were randomly selected for treatment with PBS (control) alone, the compound#14 alone, DOTAU alone and 14+DOTAU.
- Each experimental group consisted of 6 mice of control and 8 mice for other groups.
- the compound#14 was tested at 1 mg/ml, which corresponds to its maximum of solubility or 14+DOTAU was tested at 2mg/ml. Injection lasted 8 weeks with two injections for weeks. Data points were expressed as average tumor volume levels ⁇ S.E.
- the following BRET assay can be performed.
- Hsp27 or elF4E genes alone or with kozak sequence upstream were first inserted into a pCR8/GW/TOPO plasmid (Invitrogen). Kozak sequence was added each time the gene was found in N-ter in the final plasmid.
- the insertions into plasmids were realized by TOPO reaction according to manufacturer’s instructions (Invitrogen). Competent bacteria “MACH 1” (Invitrogen) were transformed using TOPO reaction products and the obtained clones were analysed by PCR with Go Taq enzyme (Promega). Positives clones were amplified by mini-culture and DNA was extracted with Wizard Plus SV Minipreps DNA Purification System kit (Promega), and sent for sequencing (M13 Forward primer) (GATC, Mulhouse, France).
- the second step was to transfer the genes from pCR8/GW/TOPO intermediate plasmids to destination plasmids containing luciferase and YFP by LR Gateway recombination reaction using LR clonase according to manufacturer’s instructions (Invitrogen).
- the destination plasmids pRLuc-C, pRLuc-N, pEYFP-N and pEYFP-C, were kindly given by Nelson Dusetti (UMR 1068, Centre mecanic Cancerologie Marseille, France). Competent bacteria “MACH 1” (Invitrogen) were transformed using LR reaction products.
- the clones obtained were amplified by mini-culture and DNA was extracted with the Wizard Plus SV Minipreps DNA Purification System kit (Promega). Enzymatic digestion by BsrGI (Promega) was performed in order to identify positives clones that were then amplified by maxi-culture and DNA was extracted using the PureYield Plasmid Midiprep System kit (Promega), and sent for sequencing (BRET primers) (GATC).
- HEK293T cells cultured in 12-well culture plates 400 000 cells/well were co-transfected with 0.2 pg of BRET donor plasmid N-ter luc/elF4E, and 0 to 1 pg of BRET acceptor plasmid Hsp27/YFP C-ter .
- the empty vector (pEYFP-C) was used to equalize DNA amounts to 1 .2 pg in each sample.
- the cell-permeable Rluc substrate coelenterazine-h (Interchim, Montlugon, France) diluted in phosphate buffered saline (PBS) 1X was added in culture medium to a final concentration of 5 pM, and cells were incubated 15 min at 37°C before reading. Readings were done using a LB 941 Tristar reader (Berthold France SA, Thoiry, France), with signal detection in the 470-490 nm (donor) and 520-540 nm (acceptor). BRET signal represents the BRET ratio of the Rluc and EYFP expression constructs compared to the BRET ratio for the Rluc expression construct alone.
- PBS phosphate buffered saline
- the BRET values were determined by using the following equation, expressed in milli-BRET unit (mBretll): 1000 J where E 0 corresponds to the ratio 530 nm acceptor signal/480 nm donor signal obtained with the Rluc construct alone in the same experiment.
- HEK293T cells were transfected separately with a BRET donor plasmid N-ter luc/elF4E or BRET acceptor plasmid Hsp27/YFP C-ter . Forty eight hours later, total cellular proteins were extracted following the manufacturer’s instructions of CelLytic NuCLEAR Extraction Kit (Sigma-Aldrich) and stored at -80°C.
- the present invention thus confirms that the nanoformulation did not impair the inhibition effect of compound 14 on Hsp27/elF4E interaction.
- NPDOTAU- compound 14 was the most effective compound.
- NP D oTAu- comPound 14 also demonstrated better pro-apoptotic activity compared to other agents.
- the in vivo experiments were also in favor of NPD OTA u- compound 14 and showed impressive anti-tumor effects on PC3 xenografts, which represents an aggressive AIPC model. Ki-67 was clearly decreased in N P DOT Au-compound 14 arm, explaining together with the increased pro-apopoptotic effect the therapeutic benefit.
- the positive effect is probably due to an increase of accumulation in the tumor area by passive targeting due to the EPR (enhanced permeability retention) effect. This behavior induces a local increase of drug concentration into the tumor resulting in a better antitumoral activity compared to free drug.
- the NPD OTA u- compound 14 diameter (75 nm) probably influences the tumor size growth inhibition since it avoids compound#! 4 renal clearance.
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Abstract
The present invention concerns nanoparticles with a core comprising a phenazine derivative and a shell comprising at least one layer of a nucleolipid, compositions comprising them and their pharmaceutical uses, especially in the prevention and/or treatment of diseases in which the interaction between the proteins Hsp27 and eIF4E is involved.
Description
Nanoparticles comprising a core with a phenazine derivative and a shell with a nucleolipid and uses thereof
The present invention concerns nanoparticles with a core comprising a phenazine derivative and a shell comprising at least one layer of a nucleolipid, compositions comprising them and their pharmaceutical uses, especially in the prevention and/or treatment of diseases in which the interaction between the proteins Hsp27 and elF4E is involved.
Numerous researches are dedicated to cancers and to their mechanisms of action Among cancers, prostate cancer is one of the most frequent as well as the third cause of mortality due to cancer among men in France. The treatment involves surgery or radiotherapy but the androgenic removal, also called hormonotherapy, remains the only effective therapy of the locally advanced or metastatic prostate cancer.
Flormonotherapy provides an objective answer for more than 80% of cases but in spite of its transitional effectiveness, it does not prevent the castration-resistant progression of the disease that usually arises within the 2 to 3 years after the beginning of the hormonal treatment.
A strategy to improve the treatment of cancers consists in targeting the survival genes overexpressed in these diseases, in particular in the castration-resistant prostate cancer, so as to restore sensitivity to hormonotherapy and/or chemotherapy.
Following this strategy, several studies have shown an increase of the expression of elF4E, a protein involved in the translation initiation, within numerous cancers making it a good therapeutic target (Jia, Y.,.Med Res Rev. 2012 Jul;32(4):786-814 and Rocchi et al. Advances in Protein Chemistry and Structural Biology, 2015, vol.101 ; Andrieu C., oncogene, 2010, Baylot, V., Cell death and disease, 2011).
The protein elF4E is part of a large multi-protein complex, elF4F, which plays a role in the translation initiation of eukaryotes and which is composed of: elF4E, that directly interacts with the cap structure of the mRNA, elF4A, a RNA helicase, and the scaffolding protein elF4G. Hsp27 is a chaperon protein belonging to the « Small Heat Shock Proteins » (Hsp) family. In the physiological conditions, the Hsp27 is expressed in a very low quantity in a ubiquitous form. During a cell stress, caused for instance by hormonotherapy or chemotherapy, Hsp27 accumulates in a large amounts in the cells, blocks the apoptosis, and thus permits cell survival.
The interaction between Hsp27 and elF4E protects elF4E of its own degradation through the ubiquitin-proteasome pathway and enables to maintain high levels of the protein synthesis and therefore cell survival (Andrieu C., Oncogene, 2010, Baylot, V., Cell death and disease, 2011). This deregulation of the proteic synthesis process is associated with the development and the progression of cancers. Flence, the protein translation becomes an oncogene process, which leads to cancers resistant to hormonotherapy and/or chemotherapy.
There is thus a need for an improved treatment of diseases wherein the interaction between Flsp27 and elF4E is involved, especially cancers. There is also a need for improved formulations of active ingredients targeting the interaction between Flsp27 and elF4E, more particularly in the treatment of cancers.
There is also a need for a treatment of cancers that restore sensitivity to hormonotherapy and/or chemotherapy. The aim of the present invention is to provide an improved treatment of diseases wherein the interaction between Flsp27 and elF4E is involved, especially cancers.
Another aim of the present invention is to provide an improved formulation allowing a satisfying solubilization and delivery of active ingredients targeting the interaction between Flsp27 and elF4E, in particular phenazine derivatives. One aim of the invention is also to provide a stable composition comprising a formulation of phenazine derivatives, in particular comprising nanoparticles containing phenazine derivatives.
A particular aim of the present invention is to provide an improved formulation of phenazine derivatives, useful in the treatment of cancers.
The invention thus relates to nanoparticles comprising:
- a core comprising at least one compound having the following general formula (I):
wherein:
• RI=F¾2 and represent R8, -0R8 or -OCOR9, with R8 representing a linear or branched (Ci-C2o)alkyl group, and Rg representing a hydrogen atom or a linear or branched (C2-C2o)alkyl group;
• R3 and R , independently of each other, represent:
- a hydrogen atom;
- a halogen atom selected from the group consisting of: chlorine, fluorine, bromine, and iodine atom;
- a cyano group;
- -NO2;
- -N(R5)(R6), with R5 and R6 representing independently of each other, a hydrogen atom, a linear or branched (Ci-C2o)alkyl group, a benzyl group or a -COR10 group with Ri0 representing a hydroxyl group or a linear or branched (Ci-C2o)alkoxy group;
- an amido group;
- -NHSC>2(R7), with R representing a linear or branched (Ci-C2o)alkyl group, or a aryl group;
- -ORs' or -OCORg', with R8' and Rg' representing a hydrogen atom or a linear or branched (Ci-C2o)alkyl group;
- a (Ci-C2o)alkyl, a (CrC2o)alkenyl, a (Ci-C2o)alkylthio or a (Ci-C2o)alkenylthio group, linear or branched;
- -COR10 with Ri0as defined above;
- -CO-[NH-(CH2)n]m-N(R5)(R6), -HN-CO-[(CH2)n-N(Rii)]p-(CH2)n'-N(Rii)(R6), or -O- CO-[(CH2)n-N(Rii)]p-(CH2)n’-N(Rii)(R6) with:
- R5 and R6 as defined above,
- R11 representing a hydrogen atom or a tert-butyloxycarbonyl group,
- p representing an integer ranging from 0 to 1 , and
- n, n' and m, independently of each other, representing an integer with n and n', independently, ranging from 2 to 6 and m ranging from 1 to 2, and, when m is 2, each n value may be identical or different one from another, or one of its pharmaceutically acceptable salts; and
a lipid shell comprising at least one layer of a nucleolipid having the following formula (A):
wherein:
■ Re represents a purine or pyrimidine base such as uracil, adenine, guanine, cytosine, thymine, hypoxanthine, or their derivatives, or a non-natural mono- or bi- cyclic heterocyclic base each ring of which comprises 4 to 7 members, optionally substituted;
■ Li and L2, independently of each other, are selected from the group consisting of: hydrogen atom, -O-C(O)-, -0-C(S)-NH-, -0-C(0)-0-, -0-C(0)-NH-, an oxygen atom, a phosphate group, a phosphonate group or a heteroaryl comprising from 1 to 4 nitrogen atoms, said heteroaryl being unsubstituted or substituted by a linear or branched (C2- C3o)alkyl or a linear or branched (C2-C3o)alkenyl,
or, Li and l_2, together form a ketal group of formula: ; or, Li or l_2 is a hydrogen atom, and the other is a hydroxy group or a heteroaryl group comprising from 1 to 4 nitrogen atoms, said heteroaryl being unsubstituted or substituted by a linear or branched (C2-C3o)alkyl;
■ Mi and M2, independently of each other, represent:
- a linear or branched (C2-C3o)alkyl;
- a linear or branched (C2-C3o)alkenyl; said alkyl or alkenyl group being optionally completely or partially fluorinated, and/or substituted on the carbon at the end of the chain by a fluorine atom or by a benzyl or naphthyl ester or ether;
- a diacyl chain in which each acyl chain is in C2-C30;
- a diacylglycerol;
- a sphingosine; and
- a ceramide group; when Li or l_2 represents hydrogen, and the other represents a hydroxy group or a heteroaryl group comprising 1 to 4 nitrogen atoms, then Mi and M2 do not exist;
■ Ra is selected from the group consisting of:
- hydroxy;
- amino;
- phosphate;
- phosphonate;
- phosphatidylcholine;
- phosphocholine;
- -0-(Ci-Cio)alkylene-phosphatidyicholine;
- thiophosphate;
- phosphonium;
- NH2-Rb;
- a -[NRbRcRd]+ group in which Rb, Rc and Rd, identical or different, represent a hydrogen atom, a linear or branched (Ci-Cio)alkyl or a linear or branched O- (Ci-Cio)alkyl;
- a linear or branched (C2-C3o)alkyl optionally substituted by a hydroxy group;
- a -0-C(0)-(Ci-Cio)aikylene-C(0)-0-[CH2-CH2-0]r-CH3 group or a -0-C(0)-(Ci- Cio)alkylene-S-S-[CH2-CH2-O]r-CH3, wherein r is an integrer comprised between 4 to 30;
- a cyclodextrin radical;
- a group of formula (B):
Rz
H-(-CH2-C-)w-H
CO-V- (B) in which V is -0-, -S-, or -NH- and Rz is H or CH3, and w=1 to 500;
- a -(CH2)W-V-RX group, in which Rx represents a (C2-C30) alkyl, and w=1 to 500;
- a heteroaryl group containing 1 to 4 nitrogen atoms, unsubstituted or substituted by a linear or branched (C2-C3o)alkyl, or by a (CH2)g-0-(CH2)h-Ri group in which g=1 to 6 and h=0 to 10 and R, represents a cyclic ketal group containing 5 to 7 carbon atoms, unsubstituted or substituted by at least one linear or branched (C2-C3o)alkyl or by a sterol radical, or
Ra is bound by a covalent bond to another substituent Ra, identical or different, of another compound of formula (A), identical or different, in order to form a compound of formula (A) in the form of a dimer.
Phenazine derivatives are known as useful in the treatment of pancreatic and prostate cancers, as described in WO 2011/117830.
The present inventors surprisingly discovered that the phenazine derivatives according to the general formula (I) as defined above are able to inhibit the specific Hsp27-elF4E interaction.
However, the greatest disadvantage of these phenazine derivatives remains their very low solubility, notably in aqueous medium and biological fluids. Due to this low solubility, the phenazine derivatives cannot be administered as such to patients.
The inventors surprisingly found a formulation allowing the administration of phenazine derivatives of general formula (I) to patients, which improves their efficacy against diseases in which the Hsp27-elF4E interaction is involved, in particular cancers.
Indeed, the nanoparticles according to the invention allow the administration of the phenazine derivatives of general formula (I) to mammal, especially humans. In particular, they protect the phenazine derivatives of general formula (I) from degradation, increase their solubility, and result in a sustained delivery.
Even more striking, the nanoparticles of the invention lead to a greater inhibition of the interaction between Hsp27 and elF4E in cells, compared to the phenazine derivatives of general formula (I) alone.
The nanoparticles according to the invention lead to a decrease in cell viability and to an increase of apoptosis compared to phenazine derivatives of general formula (I) alone when administered to cancer cells, in particular in castration-resistant prostatic cancer cells. The nanoparticles according to the invention also reduce the tumor volume compared to phenazine derivatives of general formula (I) alone, in particular when administered in castration-resistant prostatic cancer.
In a particular embodiment, the nanoparticles according to the invention restore sensitivity to hormonotherapy and/or chemotherapy, such as in the castration-resistant
prostate cancer. Advantageously, the inhibition of the Hsp27-elF4E interaction by the nanoparticles of the invention does not affect the other functions of the cells, whereas separate inhibition of Hsp27 and/or elF4E induces cytotoxicity, such as in non-cancer cells. In one embodiment, the nanoparticles according to the invention also allow a homogenous cytoplasmic concentration of the phenazine derivatives in the cells, in particular when the nucleolipid (A) is DOUPEG-2000.
Definitions
The term “alkyl” means a saturated aliphatic hydrocarbon group which may be straight or branched. Preferred alkyl groups have 1 to 20 carbon atoms in the chain. Preferred alkyl groups are in particular methyl, ethyl, propyl or dodecyl groups. "Branched" means that one or lower alkyl groups such as methyl, ethyl or propyl are attached to a linear alkyl chain.
The term “alkoxy” refers to a -O-alkyl group, with the alkyl group being as defined above. Among alkoxy, it may be cited, the methoxy, ethoxy, propoxy or isopropoxy groups The term “alkylthio” refers to a -S-alkyl group, with the alkyl group being as defined above.
The term “alkenyl” refers to an alkyl group, comprising at least two carbon atoms and one double bond, preferably one or two double bonds. Alkenyl groups may have between 2 and 30 carbon atoms. Preferred alkenyl groups have between 10 and 20 carbon atoms in the chain, for example 17 carbon atoms.
The term “alkenylthio” refers to a -S-alkenyl group, with the alkenyl group being as defined above.
The term “-(Ci-Cio)alkylene-” means a saturated aliphatic hydrocarbon divalent radical which may be straight or branched having 1 to 10 carbon atoms in the chain. Preferred alkylene groups are methylene, ethylene or propylene groups.
The term "aryl" refers to an aromatic monocyclic, bicyclic, or tricyclic hydrocarbon ring system comprising from 6 to 10 carbon atoms wherein any ring atom capable of substitution may be substituted by a substituent. In one embodiment, aryl groups are not substituted. Examples of aryl moieties include, but are not limited to phenyl or naphtyl. The term “heteroaryl” refers to an aromatic or partially unsaturated monocyclic or bicyclic hydrocarbon ring system comprising from 5 to 12 carbon atoms, wherein any ring atom capable of substitution may be substituted by a substituent and wherein one or more carbon atom(s) are replaced by one or more heteroatom(s) such as nitrogen atom(s), oxygen atom(s) and sulphide atom(s); for example 1 or 4 nitrogen atom(s), 1 or 2 oxygen atom(s), 1 or 2 sulphide atom(s) or a combination of different heteroatoms such as 1
nitrogen atom and 1 oxygen atom. Preferred heteroaryl groups comprise from 1 to 4 nitrogen atoms. Examples of heteroaryl include but are not limited to pyridine, pyrimidine, pyrazole, triazole, tetrazole or imidazole groups. In one embodiment, heteroaryl groups are not substituted. The term “halogen” refers to the atoms of the group 17 of the periodic table and includes in particular fluorine, chlorine, bromine, and iodine atoms, more preferably fluorine, chlorine and bromine atoms.
By “derivatives” of purine or pyrimidine base is meant for example, a non-natural mono- or bi-cyclic heterocyclic base in which each cycle has 4 to 7 carbon atoms, preferably 5 or 6 carbon atoms, wherein any ring atom capable of substitution may be substituted by a substituent and wherein one or more carbon atom(s) are replaced by one or more heteroatom(s) such as nitrogen atom(s), oxygen atom(s) and sulphide atom(s); for example 1 , 2, 3 or 4 nitrogen atom(s), 1 or 2 oxygen atom(s), 1 or 2 sulphide atom(s) or a combination of different heteroatoms such as 1 nitrogen atom and 1 oxygen atom In a particular embodiment, the purine or pyrimidine derivatives correspond respectively to substituted purine or pyrimidine base. For example, the derivatives of the purine or pyrimidine base have an anti-tumoral activity and/or an anti-viral activity such as cytarabine (AraC), 5-fluoro-uracile (5-FU), iododeoxyuridine (IdU), 2’-deoxy-2’- methylidenecytidine (DMDC) or 5-chloro-6-azido-5,6-dihydro-2’-deoxyuridine. By « non-natural mono- or bicyclic heterocyclic base » is meant a base different from uracil, adenine, guanine, cytosine, thymine or hypoxanthine.
“Protein interaction” may be understood as a physical contact with molecular docking between proteins that occurs in a cell or in a living organism, preferably in particular biomolecular contexts. The interaction between Hsp27 and elF4E may thus be defined as a physical contact, preferably with molecular docking, between these two proteins. In one embodiment, Hsp27 is phosphorylated when it interacts with elF4E.
Tests for assessing the interaction between two proteins, for example between the Hsp27 protein and the elF4E protein, are well known. The interaction between two proteins may be assessed directly, for example by plasmon surface resonance, flow cytometry or beta-imager. The interaction between the two proteins Hsp27 and elF4E may be assessed by known methods such as co-immunoprecipitation or BRET assay (Bioluminescence Resonance Energy Transfer assay).
The compounds of formula (I) and/or the compounds of formula (A) may comprise an unsaturation site and thus may be in their tautomeric forms and/or their Cis or Trans isomers.
Compounds of general formula (I) - phenazine derivatives
The process of preparation of the compounds of general formula (I) is described in WO 2011/117830, more specifically in page 13 and in pages 16 to 20 of WO 2011/117830.
In one embodiment, the compound of formula (I) is:
■ RI=R2 and represent R8, -OR8 or -OCORg, with R8 representing a linear or branched (Ci-C2o)alkyl group, and Rg representing a hydrogen atom or a linear or branched (Ci-C2o)alkyl group;
■ R3 and R4, independently of each other, represent:
- a hydrogen atom;
- a linear or branched (C1-C20) alkyl,
- a linear or branched (Ci-C2o)alkenyl,
- -N(R5)(R6), with R5 and R6 representing independently of each other: a hydrogen atom, a linear or branched (Ci-C2o)alkyl group, a benzyl group or a -COR10 group, R10 being a hydroxyl group or a linear or branched (Ci-C2o)alkoxy group ;
- -CO-[NH-(CH2)n]m-N(R5)(R6), -HN-CO-[(CH2)n-N(Rii)]p-(CH2)n-N(Rii)(R6), or -O- CO-[(CH2)n-N(Rii)]p-(CH2)n'N(Rii)(R6) with:
- R5 and R6 being as defined above,
- R11 representing a hydrogen atom or a tert-butyloxycarbonyl group,
- p representing an integer ranging from 0 to 1 , and
- n, n' and m, independently of each other, representing an integer with n and n', independently, ranging from 2 to 6 and m ranging from 1 to 2, and, when m is 2, each n value may be identical or different one from another, or one of its pharmaceutically acceptable salts.
In one embodiment, in the compound of the formula (I), both Ri and R2 are a linear or branched -0(Ci-C2o)alkyl group.
In another embodiment, in the compound of the formula (I), R3 and FU represent independently of each other: a hydrogen atom, -N(R5)(R6) or -CO-[NH-(CH2)n]m-N(R5)(R6), with n, m, R5 and R6 being as above. In a particular embodiment, R5 and R6 are H or methyl, ethyl or propyl. Preferably n is 2 and/or m is 1.
In one embodiment, the compound of formula (I) has one of the following formulae:
The compounds of general formula (I) herein described may have asymmetric centers. Compounds of the present invention containing an asymmetrically substituted atom may be isolated in optically active or racemic forms. It is well-known in the art how to prepare optically active forms, such as by resolution of racemic forms or by synthesis from optically active starting materials. All chiral, diastereomeric, racemic forms and all geometric isomeric forms of a compound are intended, unless the stereochemistry or the isomeric form is specifically. The term "pharmaceutically acceptable salt" refers to salts which retain the biological effectiveness and properties of the compounds of the invention and which are not biologically or otherwise undesirable.
The compounds of general formula (I) may be provided in the form of a free base or in the form of addition salts with acids. Pharmaceutically acceptable acid addition salts may be prepared from inorganic and organic acids, while pharmaceutically acceptable base addition salts can be prepared from inorganic and organic bases. For a review of pharmaceutically acceptable salts see Berge, et al. ((1977) J. Pharm. Sd, vol. 66, 1). For example, the salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like, as well as salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, fumaric, methanesulfonic, and toluenesulfonic acid and the like.
Nucleolipids of general formula (A)
The process of preparation of the nucleolipids of general formula (A) is described in WO 2005/116043 in pages 8 to 17 and in the examples. In one embodiment, Li and l_2, independently of each other, are selected from the group consisting of: hydrogen atom, -O-C(O)-, -0-C(S)-NH-, -0-C(0)-0-, -0-C(0)-NH-, an oxygen atom, a phosphate group and a phosphonate group.
In one embodiment, Re represents a purine or pyrimidine base such as uracil, adenine, guanine, cytosine, thymine, or hypoxanthine, preferably uracil In a particular embodiment, the nucleolipid of formula (A) has the following formula
(A-l):
wherein Ra, Mi and M2 are defined above.
In one embodiment, in the nucleolipid of formula (A) or (A-l), Mi and M2 are independently of each other a linear or branched (C2-C3o)alkyl or a linear or branched (C2- C3o)alkenyl, preferably a (C2-C3o)alkenyl. More particularly in the nucleolipid of formula (A) or (A-l), Mi and M2 are identical and preferably represent a linear (Cio-C2o)alkenyl, preferably comprising one double bond.
In another embodiment, in the nucleolipid of formula (A) or (A-l), Ra is selected from the group consisting of:
- a -[NRbRcRd]+ group in which Rb, Rc and Rd, identical or different, represent a hydrogen atom or a linear or branched (Ci-Cio)alkyl group, and
- a -0-C(0)-(Ci-Cio)alkylene-C(0)-0-[CH2-CH2-0]r-CH3 group, wherein r is an integer comprised between 4 and 30. In another embodiment, in the nucleolipid of formula (A) or (A-l), Ra is a
-[NRbRcRd]+ group in which Rb, Rc and Rd, identical or different, represent a hydrogen atom or a linear or branched (Ci-Cio)alkyl group, or a -0-C(0)-(C2-C6)alkylene-C(0)-0- [CH2-CH2-0]rCH3 group, wherein r is an integer comprised between 10 and 20.
In a particular embodiment, the nucleolipid of formula (A) has one of the following formulae:
In one embodiment, in the nanoparticles according to the invention: the core comprises a compound of formula (I) having one of the following formulae:
and
- the lipid shell comprises at least one layer of a nucleolipid of formula (A) having one of the following formulae:
with r being an integer comprised between 10 and 20.
Characterization of the nanoparticles according to the invention The core of the nanoparticles comprising at least one compound of general formula (I) as defined above may be coated, preferably directly coated, by the lipid shell comprising at least one layer of a nucleolipid of general formula (A) as defined above.
In one embodiment, said lipid shell is coated by at least one other layer; for example by one or two layer(s) of another nucleolipid said layer(s) having an opposite charge compared to charge of the zeta potential of the core coated with the lipid shell as defined above. In one embodiment, when the lipid shell consists of one layer of the nucleolipid DOTAU, said another layer is a bilayer of the compound diC16dT, having the following formula:
In one embodiment, the core of the nanoparticles according to the invention comprises one compound of the general formula (I) as defined above. In a particular embodiment, the core is hydrophobic. In another embodiment, the core consists of an
amorphous precipitate of at least one compound of general formula (I), preferably of one compound of general formula (I).
In one embodiment, the lipid shell consists of one layer of a nucleolipid of general formula (A) as defined above. In another embodiment, the lipid shell comprises two, three or more layers of nucleolipid(s) of general formula (A) as defined above, said nucleolipid(s) of general formula (A) being identical or different in each layer.
In a particular embodiment, the nanoparticles according to the invention are solid at room temperature, preferably between 15°C and 25°C.
In one embodiment, the nanoparticles of the invention are characterized by a mean size particle comprised between 50 nm and 150 nm, preferably between 60 nm and 130 nm.
By “mean size particle” it may be understood the mean of the diameter of the nanoparticles. The “mean size particles” may be measured by Dynamic light scattering (DLS). In one embodiment, the nanoparticles of the invention are characterized by a polydispersity index inferior or equal to 0.6, preferably comprised between 0.1 and 0.5. Advantageously, the nanoparticles according to the invention have a low polydispersity index, preferably inferior to 0.5.
The “polydispersity index” may refer to the measure of the width of the sizes of the nanoparticles. The polydispersity index may be measured by Dynamic light scattering.
The nanoparticles according to the invention thus have homogenous sizes, leading to stable compositions comprising them.
In another embodiment, the nanoparticles of the invention are characterized by a zeta potential comprised between 20 mV and 90 mV, preferably between 30 mV and 80 mV.
The “zeta potential” may be defined as the potential difference between the dispersion medium and the stationary layer of the fluid attached to the dispersed particle and may be determined by Electrophoretic Light Scattering (ELS). The nanoparticles according to the invention advantageously lead to a stable colloidal dispersion, in an aqueous medium.
In one embodiment, the above mentioned nanoparticles are characterized by a mean size particle comprised between 60 nm and 80 nm and/or a polydispersity index comprised between 0.1 and 0.3 and/or a zeta potential comprised between 60 mV and 80 mV. In another embodiment, the nanoparticles are characterized by a mean size particle
comprised between 60 nm and 130 nm and/or a polydispersity index comprised between 0.1 and 0.5 and/or a zeta potential comprised between 30 mV and 50 mV.
The mean size particle, the polydispersity index and the zeta potential as defined above may be determined thanks to the Zetasizer nanoserie (Malvern Instruments).
Method of preparation of the nanoparticles according to the invention
The nanoparticles according to the invention may be prepared according to known techniques. The nanoparticles according to the invention may be prepared by nanoprecipitation or by formation of a lipid film. In one embodiment, a process of preparation of the nanoparticles according to the invention comprises the following steps: i) solubilizing a nucleolipid of general formula (A) as defined above in an organic solvent, such as chloroform, thereby obtaining a mixture (N); ii) in another recipient solubilizing a compound of general formula (I) as defined above in an organic solvent, such as chloroform, thereby obtaining a mixture (P); iii) optionally stirring the mixture (N); iv) adding the mixture (N) in the mixture (P), thereby obtaining a mixture (M); v) removing the organic solvent from the mixture (M), thereby obtaining a nucleolipid film; vi) drying the nucleolipid film obtained at step v), preferably under vacuum; vii) rehydrating the dried nucleolipid film obtained in step vi) with water; and viii) performing sonication.
In one embodiment, step v) is performed by evaporating the organic solvent under reduced pressure or under a nitrogen flow. In one embodiment, a process of preparation of the nanoparticles according to the invention comprises the following steps: i) solubilizing a nucleolipid of general formula (A) as defined above in an organic solvent, such as chloroform, thereby obtaining a mixture (N); ii) in another recipient solubilizing a compound of general formula (I) as defined above in an organic solvent, such as chloroform, thereby obtaining a mixture (P); iii) optionally stirring the mixture (N); iv) adding the mixture (N) in the mixture (P), thereby obtaining a mixture (M) by stirring; v) adding the mixture (M) dropwise to water; vi) concentrating the obtained mixture;
vii) optionally removing the remaining organic solvent, preferably under reduced pressure; and vii) performing sonication. Uses of the nanoparticles according to the invention
The invention also relates to the nanoparticles as defined above, for use as a medicament, more particularly for use in the prevention and/or treatment of diseases in which the Hsp27-elF4E interaction is involved. By “a disease wherein the Hsp27-elF4E interaction is involved”, it may be meant a disease wherein Flsp27 and/or elF4E are expressed, over-expressed and/or mutated, preferably overexpressed. In one embodiment, the nanoparticles according to the invention are useful as inhibitors of the Flsp27-elF4E interaction.
The invention also relates to a method of prevention and/or treatment of a disease in which the Flsp27-elF4E interaction is involved, comprising the administration to a mammal, preferably a human, in need thereof of a therapeutically effective amount of the nanoparticles according to the invention.
In a particular embodiment, the nanoparticles of the invention are intended for use in the prevention and/or treatment of cancers.
“Cancer” means the uncontrolled, abnormal growth of cells and includes within its scope all the well-known diseases that are caused by the uncontrolled and abnormal growth of cells as well as metastasis.
In one embodiment, the cancer is chosen among the group consisting of lymphomas, angiosarcomas and the cancers of the lung, pancreas, breast, bladder, colon, skin, head and neck, ovarian, and prostate. In a particular embodiment, the cancer is selected from the group consisting of: prostate cancer, breast cancer and pancreatic cancer, preferably prostate cancer such as resistant prostate cancer. In a particular embodiment, the cancer is the hormono-resistant prostate cancer (also called castration resistant prostate cancer).
By “resistant prostate cancer” it may be meant a prostate cancer for which hormonotherapy and/or chemotherapy is(are) not sufficient to cure said prostate cancer. In particular, “resistant prostate cancer” is a prostate cancer for which hormonotherapy and/or chemotherapy do(es) not allow to:
- inhibit the tumor growth (tumor stasis); and/or
- decrease partially the tumor; and/or - suppress the tumor.
In one embodiment, “resistant prostate cancer” may refer to:
- metastatic prostate cancers
- prostate cancers which cannot be treated by surgery or radiation; and
- prostate cancers which remain or come back after treatment with surgery or radiation therapy.
Hormonotherapy is also called androgen deprivation therapy (ADT) or androgen suppression therapy. The goal of this treatment is to reduce levels of androgens (testosterone and dihydrotestosterone) in patients, or to stop them from affecting prostate cancer cells. In hormonotherapy, LHRH agonists, LHRH antagonists, CYP17 inhibitors, anti-androgens, or estrogens may be used.
Chemotherapy is a category of cancer treatment that uses chemical substances which are anti-cancer cells agents. In particular, docetaxel, cabazitaxel, mitoxantrone, or estramustine may be used in the treatment of prostaste cancer.
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 disorder or condition. In particular, the treatment of cancers may consist in destroying and/or depleting cancer cells and/or preventing resistance and/or restore sensitivity to hormonotherapy and/or chemotherapy of cancer cells.
According to the invention, the term "patient" or "individual" to be treated is preferably intended for a human or non-human mammal (such as a rodent, for example a mouse or a rat, a feline, a canine, or a primate) affected or likely to be affected with a disease, in particular in which the interaction Hsp27-elF4E is involved. Preferably, the patient is a human.
A "therapeutically effective amount" or "therapeutic dose" is an amount sufficient to obtain the desired clinical results (i.e., achieve therapeutic efficacy). A therapeutically effective dose can be administered in one or more administrations. In particular, a therapeutically effective dose is an amount that is sufficient to treat the disease as defined above, in particular cancers as defined above.
The invention also relates to a pharmaceutical composition, preferably an aqueous pharmaceutical composition, comprising the nanoparticles as defined above and optionally one or more pharmaceutically acceptable excipient(s).
The present invention also relates to a drug, comprising the nanoparticles as defined above.
While it is possible for the nanoparticles of the invention to be administered alone, it is preferred to present them as pharmaceutical compositions. The pharmaceutical compositions, both for veterinary and for human use, useful according to the present invention comprise at least one nanoparticle as above defined, together with one or more pharmaceutically acceptable carriers and possibly other therapeutic ingredients.
In certain embodiments, active ingredients necessary in combination therapy may be combined in a single pharmaceutical composition for simultaneous administration.
As used herein, the term "pharmaceutically acceptable" and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a mammal without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like.
The preparation of a pharmacological composition that contains the nanoparticles as defined above dispersed therein is well understood in the art and need not be limited based on formulation. Typically, such compositions are prepared as injectables either as liquid solutions or suspensions; however, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared. The preparation can also be emulsified. In particular, the pharmaceutical compositions may be formulated in solid dosage form, for example capsules, tablets, pills, powders, dragees or granules. The choice of vehicle is generally determined in accordance with the physical and chemical properties of the nanoparticles, the particular mode of administration and the provisions to be observed in pharmaceutical practice. For example, excipients such as lactose, sodium citrate, calcium carbonate, dicalcium phosphate and disintegrating agents such as starch, alginic acids and certain complex silicates combined with lubricants such as magnesium stearate, sodium lauryl sulphate and talc may be used for preparing tablets. To prepare a capsule, it is advantageous to use lactose and high molecular weight polyethylene glycols. When aqueous suspensions are used they can contain emulsifying agents or agents which facilitate suspension. Diluents such as sucrose, ethanol, polyethylene glycol, propylene glycol, glycerol and chloroform or mixtures thereof may also be used.
The pharmaceutical compositions can be administered in a suitable formulation to humans and animals by topical or systemic administration, including oral, rectal, nasal, buccal, ocular, sublingual, transdermal, rectal, topical, vaginal, parenteral (including subcutaneous, intra-arterial, intramuscular, intravenous, intradermal, intrathecal and
epidural), intracisternal and intraperitoneal. In a particular embodiment, the pharmaceutical composition as defined above is administered by intravenous injection.
It will be appreciated that the preferred route may vary with for example the condition of the recipient. The formulations can be prepared in unit dosage form by any of the methods well known in the art of pharmacy. In general, the formulations are prepared by uniformly and intimately bringing into association the nanoparticles with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product. Description of the figures
Figures 1A and 1B. Compound 14, 14+DOTAU (CAS Registry Number DOTAU: 868226- 06-6) and 14+DOUPEG-2000 (CAS Registry Number DOUPEG-2000: 1353570-75-8) at 100 mM inhibit the interaction of elF4E with Hsp27. The cells proteins PC-3 were extracted. (1A) The co-immunoprecipitation of elF4E had been carried out, and the rates of expression of elF4E and Hsp27 had been studied.
(1 B) The rates of elF4E, Hsp27 and vinculin had been analyzed by western blot.
Figure 2. The treatment of PC-3 cells with 14+DOTAU and 14+DOUPEG-2000 inhibits the proliferation of PC-3 cells. The PC-3 cells had been treated at the doses of 25, 50 and 100 mM by the nanoparticles according to the invention. After 2 days of treatment, the cell viability was analyzed thanks to a test at MTT. Figure 2 shows the percentage of cell viability regarding the compounds and nanoparticles tested. The experiment was carried out in triplicate.
Figure 3. Confocal Microscopic observation of the compound 14, 14+DOUPEG-2000 and 14+DOTAU in the PC-3 cells, where the nuclei had been marked at DAPI.
Figure 4. 14+DOTAU increases apoptosis of PC-3 cells in vitro. Figure 4 shows the percentage of cells undergoing the sub GO phase of the cellular cycle, which reflects cells undergoing apoptosis, when treated with control (PC-3), compound 14, DOTAU and 14+DOTAU.
Figure 5. 14+DOTAU significantly enhanced anticancer activity and reduced toxicity in tumor-xenograft mice. NOD SCID mice were treated with free compound 14, DOTAU and
14+DOTAU at doses of 1 and 2 mg/kg via i.v. administration (twice per week, n=8). PBS group was used as control (n=6). Figure 5 shows the tumor volume according to the duration of treatment. Figure 6. The treatment of PC-3 cells with 13+DOTAU and 13+DOU-PEG2000 inhibits the proliferation of PC-3 cells. The PC-3 cells had been treated at the doses of 25, 50 and 100 mM by the nanoparticles according to the invention. After 2 days of treatment, the cell viability was analyzed thanks to a test at MTT. Figure 6 shows the percentage of cell viability regarding the compounds and nanoparticles tested. The experiment was carried out in triplicate.
Figure 7. NPDoTAu-comPound 14 decreased tumor volume in vivo. NOD SCID mice were treated with free compound 14 (1 mg/kg), DOTAU and NPDoTAu-comPound 14 (2 mg/kg) via i.p. administration (twice per week, n = 8). PBS, group was used as control (n = 6). Mice body weight was measured twice per week.
Figure 8. Distribution of tissue Ki-67 immunostaining intensity (measured as average optical density) according to the tumor treated with PBS, DOTAU, compound 14 and
NPDOTAU-comPound 14-
EXAMPLES
Example 1 : Preparation of the nanoparticles according to the invention
Preparation A= 14+DOTAU (w:w, 2 ma/mU
Compound 14 = 8,1 mg was kindly given by collaboration with Drs. M. Camplo and O. Siri Marseille Interdisciplinary Center for Nanosciences, CNRS UPR3118).
DOTAU= 8 mg was given kindly by Pr. P. Barthelemy. In an hemolyse tube, 8 mg of DOTAU (n) were solubilized in Chloroform (200 pL).
In another hemolyse tube, 8 mg of Compound 14 (n) was solubilized in Chloroform (200 pL). After stirring, the nucleolipid solution was added in the drug solution (compound 14+ chloroform). After stirring, the mixed solution was then added dropwise in a large volume of water (15 mL of Eurobio Water). The resulted mixture was then concentrated and the remaining organic solvent was removed under reduced pressure. The final Volume was then fixed at 4 mL (2 mg/mL). After sonication: 4x15 min (37 kHz, 100%, 26°C), the particle size and zeta potential were determined by DLS (NanoZS, Malvern Zetasizer) (20 pL in 400 pL Eurobio Water) and are given in Table 1 below:
Preparation B=14 + DOU-PEG200Q (w:w, 2 mg/mL)
Compound 14=8 mg DOU-PEG2000=8 mg In a hemolyse tube, 8 mg of DOU-PEG2000 (n) were solubilized in Chloroform
(200 pL). In another hemolyse tube, 8 mg of Compound 14 (n) was solubilized in Chloroform (200 pL). After stirring, the nucleolipid solution was added in the drug solution. After stirring, the mixed solution was then added dropwise in a large volume of water (15 mL of Eurobio Water). The resulted mixture was then concentrated and the remaining organic solvent was removed under reduced pressure. The final Volume was then fixed at
4 ml. (2 mg/mL). After sonication: 4 x15 min (37 kHz, 100%, 26°C), the particle size and zeta potential were determined by DLS (NanoZS, Malvern Zetasizer) (20 pL in 400 pL Eurobio Water) and are given in Table 2 below.
Preparation C= 14 + DOTAU (w:w, 2 mg/mL)
Compound 14=8 mg DOTAU= 8 mg In a hemolyse tube, 8 mg of DOTAU (n) were solubilized in Chloroform (200 mI_). In another hemolyse tube, 8 mg of Compound 14 (n) was solubilized in Chloroform (200 mI_). After stirring, the nucleolipid solution was added in the drug solution. The solvent was removed to yield a nucleolipid film. The nucleolipid film is thoroughly dried to remove residual organic solvent under vacuum 2 hours. The Nucleolipid layer is then rehydrated in 4 ml. of EuroBio Water (2mg/ml_). After sonication: 4 x15 min (37 kHz, 100%, 26°C), the particle size and zeta potential were determined by DLS (NanoZS, Malvern Zetasizer) (20 mI_ in 400 mI_ Eurobio Water) and are given in Table 3 below.
Echantillon D= 14 + DOU-PEG200Q (w:w, 2 mg/mL)
Compound 14= 8 mg DOU-PEG2000= 8 mg
In a hemolyse tube, 8 mg of DOU-PEG2000 (n) were solubilized in Chloroform (200 pL). In another hemolyse tube, 8 mg of Compound 14 (n) was solubilized in Chloroform (200 pL). After stirring, the nucleolipid solution was added in the drug solution.
The solvent was removed to yield a nucleolipid film. The nucleolipid film is thoroughly dried to remove residual organic solvent under vacuum 2 hours. The Nucleolipid layer is then rehydrated in 4 ml. of EuroBio Water (2mg/ml_). After sonication: 5 x15 min (37 kHz, 100%, 26°C), the particle size and zeta potential were determined by DLS (NanoZS, Malvern Zetasizer) (20 pl_ in 400 mI_ Eurobio Water) and are given in Table 4 below.
The same preparations A, B, C and D were performed with compound 13, leading to 13+DOTAU and 13+DOUPEG2000.
Example 2: Biological activity of the nanoparticles according to the invention
I. Materials and Methods RT : Room temperature comprised between 15°C and 25°C.
1- Cell lines and cell culture:
The androgen-independent prostate cancer cell line PC-3 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in Dulbecco’s Modified Eagle’s Medium (Life Technology, Inc, Saint Aubin, France) supplemented with 10% fetal bovine serum (FBS). All cell lines were cultivated at 37°C in 5% C0 .
2- The Phenazine Compounds: The compound 14 as well as 14 encapsulated in the nucleolipids DOTAU and DOU-PEG 2000 were kindly given by collaboration with Drs. M. Camplo and O. Siri (Marseille Interdisciplinary Center for Nanosciences, CNRS UPR3118). The compound 14 is dissolved in DMSO (dimethyl sulfoxide) at a stock concentration of 10 mM. The compound 14 was encapsulated in nanoparticles DOTAU and DOU-PEG2000 according to example 1 and had been dissolved in water.
3- Treatment of cells:
PC-3 cells were seeding into 10cm dishes (1 250 OOOcells/well) or 12-well plates (50 000 to 100 OOOcells/well) according to the different experiments. The day after, the medium was changed to a new one containing DMSO (control) or derivative of phenazine 14 or phenazine 14 encapsulated in the nucleolipids DOTAU and DOU-PEG at a concentration of 100mM according to the different experiments. Effects of the treatment were analyzed 48 hours later. 4 Immunoprecipitation:
Cleared lysates with adjusted protein concentration (Pierce BCA Protein assay, Thermo Fisher scientific, lllkirch, France) were used for immunoprecipitation with 8 mI (1/50) of rabbit anti-elF4E antibody (Cell Signaling, Ozyme, Saint-Quentin-en-Yvelines, France) ON at 4°C. Lysis buffer was used to equalize amounts to 400mI in each sample. Immune complexes were precipitated after 1h incubation at 4°C with 30 mI of Trueblot anti-rabbit Ig IP beads (eBiosciences, Paris, France). After washing three times in cold lysis buffer, the complexes were re-suspended in 6mI of protein sample buffer (Bio-Rad, Marnes-la- Coquette, France) and boiled at 95°C for 5 min. 5- Western Blot:
Western blot was performed as described in Rocchi et al. (Cancer Res. 2004 Sep 15;64(18):6595-602) with 1/5000 rabbit Hsp27 antibody (Assay Designs, Villeurbanne, France), 1/1000 rabbit anti-elF4E antibody (Cell Signaling, Ozyme, Saint-Cyr-l’Ecole, France), 1/5000 anti-rabbit IgG HRP conjugate antibody (Santa Cruz Biotechnology, Heidelberg, Germany), 1/1000 anti-rabbit Trueblot IgG HRP conjugate antibody (eBiosciences). Loading levels were normalized using 1/2000 mouse anti-vinculin antibody (Sigma-Aldrich). Re-blot Plus Mild Solution (Millipore, Molsheim, France) was used for membrane stripping during 9 min at RT. 6- In vitro cell viability assay:
Cells were seeded in 12-well plates at the density of 50 000 to 100 000 cells / well. After 24h, cells were treated with:
- the compound 14,
- the compound 14 encapsulated in DOTAU (hereafter called 14+DOTAU),
- the compound 14 encapsulated in DOU-PEG2000 (hereafter called 14+DOU-PEG2000) in a total volume of 500mI / well. After 48h, cells were stained with 10OmI of MTT (3-(4,5- dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) for 2 hours at 37°C in an atmosphere of 5% CO2. After drying, the cells are resuspended in 500mI of DMSO for 30 min at room temperature. The optical density of each well is then read at 595 nm by spectrometry. Each assay was performed in triplicate.
The same test was made with compound#13, the compound#13 encapsulated in DOTAU (hereafter called 13+DOTAU), and the compounds 3 encapsulated in DOU- PEG2000 (hereafter called 13+DOU-PEG2000).
7 Statistical analysis for viability assays:
All viability assay results were expressed as mean +/- SEM. Statistical analysis was performed using one-way analysis of variance followed by Fisher’s protected least significant difference test (Statview 512, Brain Power Inc., Calabases, CA, USA). *P£0.05 was considered significant, with **P£0.01 and ***P£0.001
8- Confocal microscopy:
PC-3 cells were seeded into a 12-well plate containing cover glasses covered by FBS, at a density of 100 000 cells/well. 24h later, cells were treated with compounds (DMSO, DOU-PEG2000, DOTAU, 14, 14+DOU-PEG2000 and 14+DOTAU at 100mM as indicated above. After 48h of treatment, cells were washed with PBS 1X and fixed with formaldehyde 4% (Thermo Fisher Scientific, lllkrich, France) during 15 min at RT. Several washes were done with PBS1X before mounting the cover glasses on glass slides by using Prolong Gold antifade reagent with DAPI (Life Technologies SAS, Villebon-Sur- Yvette, France). Glass slides were kept to dry in the dark at RT during 24h and cover glasses were then immobilized with nail polish. Fluorescent images of compound#14 (absorption; 452, emission; 478) and DAPI (absorption; 350, emission; 450-490) were captured with a Zeiss 510 META fluorescence confocal microscope plan 40X/1.4 (Le Pecq, France).
9 Flow Cytometry:
PC-3 cells were seeded into 6 cm dishes at a density of 250 000 cells/well. After 24h, cells were treated with compounds (DOU-PEG2000, DOTAU, 14, 14+DOU-PEG2000 and 14+DOTAU) at 100mM as indicated above. After 48h of treatment, dead and living cells were collected and washed with PBS1X, before being fixed in cold ethanol 70%
(250mI/rqIIqΐ). After 1 h incubation at 4°C, cells were washed with PBS1X before permeabilization using flow buffer (8 parts of citric acid 0.1 M, 192 parts of Na2HPC>4 0.2M) (1 OOmI/pellet). After another washing step, RNA was degraded by incubation (30 min at 37°C) into PBS1X containing RNAase (Sigma-Aldrich, Saint-Quentin-Fallavier, France) at 0.5mg/ml (200pl/pellet). Supernatant was thrown and pellet was incubated (30 min, RT, dark) in PBS1X containing propidium iodide at 0.05mg/ml (1 ml/pellet) in order to stain DNA. Samples were passed into FACS tubes and DNA content was determined by flow cytometry using LSRII SORP (Becton Dickinson, Le Pont de Claix, France) machine. Rates of apoptosis were then measured using FlowJo software (Tree Star, Inc.).
10- In vivo tumor growth evaluation:
For in vivo study, 106 PC3 cells were inoculated in the flank region of 2 weeks old male athymic (node scid). Tumors were measured weekly and their volume were calculated by the formula lengh*width*depth*0.5236. When PC3 tumors reached 300-500 mm3’ mice were randomly selected for treatment with PBS (control) alone, compound 14 alone, DOTAU alone and 14+DOTAU. Each experimental group consisted of 6 mice of control and 8 mice for other groups. Compound 14 was tested at 1 mg/ml, which corresponds to its maximum of solubility or 14+DOTAU was tested at 2mg/ml. Injection lasted 8 weeks with two injections for weeks. Data points were expressed as average tumor volume levels ±S.E.
II. Results:
A- The encapsulation of the compound 14 bv the nucleolipids DOU-PEG200Q and DOTAU maintains its inhibiting capacity of the Hsp27-elF4E interaction: Some PC-3 cells were treated through the compound 14, DOTAU, DOU-
PEG2000, 14+DOTAU and 14+DOU PEG2000, at a dose of 100mM. The proteins of the cells have then been extracted and co-immunoprecipitated through the antibodies Hsp27 and elF4E. The immunoprecipitation has shown that the compound 14 kept its ability to inhibit the interaction between elF4E and Hsp27, even when encapsulated in nanoparticles according to the invention.
Figure 1A shows that the encapsulated compound 14 maintains its inhibiting capacity on the elF4E-Hsp27 interaction.
The Western Blot carried out in parallel (Figure 1 B) confirms that this inhibition of interaction is neither due to a degradation of proteins, nor to the effect of DOTAU or DOUPEG-2000 used as control.
The sum of these data shows that 14+DOTAU and 14+DOU-PEG2000 maintain their property of inhibition of the elF4E-Flsp27 interaction.
B- The compounds#13 and#14 encapsulated by DOTAU and DOUPEG-2000 inhibit the viability of PC-3 cells:
Cell viability assays were carried out in order to evaluate the effect of the inhibition of this interaction by compound 14 on CRPC (castration resistant prostate cancer) cell survival. PC-3 cells were treated with compound 14 during 48h at different concentrations (50, 100mM) and MTT assay was performed. The inhibition of Hsp27-elF4E interaction by compound 14 decreased the cells viability in a dose-dependent manner.
To evaluate the effect of the inhibition of the elF4E-Hsp27 interaction by the 14+DOTAU on the cellular proliferation of PC-3, a proliferation test at MTT (3-(4,5- dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium) was carried out. The cells were put into cultivation and treated at different doses 25, 50, and 100 mM by DOTAU, DOU-PEG2000, 14+ DOTAU and 14+DOU-PEG2000.
The results in Figure 2 show that the nanoparticles according to the invention decrease cell viability, compared to control.
The test at MTT shows that the treatment by the 14+DOTAU decreases cell viability of 70%. In fact, it goes from 100% for the untreated cells to 30% for the treated ones at a dose of 100 mM. The encapsulated compound 14 is more effective than the compound 14 alone whose effectiveness is only 50% for the same dose.
The same test was performed with compound 13, 13+DOTAU and 13+DOU- PEG2000 and the results are shown in Figure 6. The results in Figure 6 show that the nanoparticles according to the invention decrease cell viability, compared to control. The treatment with 13+DOUPEG-2000 leads to a cell viability of about 60% at 25 and 50 pM and of about 50% at 100 pM whereas compound 13 alone leads to a cell viability of about 80% at 25 and 50 pM and 50% at 100 pM.
It has to be noted that phenazine derivatives have very low solubility, notably in aqueous medium and biological fluids. Therefore, the tested compound 14 alone cannot be administered as such to patients.
Thus, the nanoparticles according to the invention maintain or improve the efficacy of the compounds of general formula (I), in particular decrease cell viability and can be administered to patients.
C- The encapsulation of comoound#14 in nanooarticles improves the delivery of the compound for the cells:
The effect of encapsulation by DOU-PEG 2000 or DOTAU on the intracellular tracing of the compound#14 has been studied with the auto-fluorescence of the compound#14 which absorbs to l = 452 nm, to emit a fluorescence to l = 478 nm.
Within the frame of this experiment of confocal microscopy (Figure 3), the cell nuclei are marked through the help of DAPI. The fluorescence visible in green on the images is just due to the chemical properties of the compound#14. We can equally observe that the nucleolipids alone: DOU-PEG2000 and DOTAU do not emit any fluorescence.
It can be seen that the compound 14 alone absorbed by the cells gets localized in the cytoplasm in a heterogeneous way.
Compound#14 was shown to be internalized in cells and mainly localized in the cytoplasm with an heterogeneous distribution. The encapsulation of compound#14 by DOTAU was only slightly modified compared to compound #14, with a pedicaliar pattern of aggregates. Compound 14 DOU-PEG2000 staining pattern was much more intense and homogenous that the latter ones.
The encapsulation of the compound#14 by par DOU-PEG2000 permits an homogenous and intense repartition of the compound#14, the intracellular localization of the compound remaining equally cytoplasmic.
D- The encapsulation increases the apoptosis:
The inhibition of Hsp27-elF4E interaction by compound#14, 14+DOTAU and 14+DOUPEG-2000 decreased the cells viability in a dose-dependent manner (Figure 4). To decipher the mechanism responsible for this viability inhibition, the cell cycle and more specifically the sub GO phase of the cellular cycle, which reflects cells undergoing apoptosis has been studied. The PC-3 cells were treated with compound#14, DOTAU, DOU-PEG 2000, 14+DOTAU and 14+DOUPEG-2000 at 100mM during 48h and labelled with propidium iodide to determine the different phases of cell cycle. Figure 4 shows that 14+DOTAU highly increases the number of cells undergoing apoptosis compared to compound 14 alone.
E- In vivo study.
For in vivo study, 106 PC3 cells were inoculated in the flank region of 2 weeks old male athymic (Node Scids) (Figure 5). Tumors were measured weekly and their volume were calculated by the formula lengh*width*depth*0.5236. When PC-3 tumors reached 300-500 mm3, mice were randomly selected for treatment with PBS (control) alone, the compound#14 alone, DOTAU alone and 14+DOTAU. Each experimental group consisted of 6 mice of control and 8 mice for other groups. The compound#14 was tested at 1 mg/ml, which corresponds to its maximum of solubility or 14+DOTAU was tested at 2mg/ml. Injection lasted 8 weeks with two injections for weeks. Data points were expressed as average tumor volume levels ±S.E.
The results show that 14+DOTAU significantly reduces the tumor volume in tumor grafted mice, compared to compounds 4 alone and to DOTAU alone.
The in vivo test was carried out on mice xenografted with androgen-independent PC-3 prostate cancer cells. DOTAU, compound#14 and NPDoTAu-comPound 14 were administrated by i.p. twice a week for 8 weeks. At the end of the experiment, after 8 weeks we observed that: 1- DOTAU cargo induced a 30% decrease of tumor growth, 2-compound 14 induced a 50% decrease of tumor growth and 3- the formulated NPDOTAU- compound 14 induced a 70% decrease of tumor growth. These results were consistent with in vitro experiments. The formulation of the NPDoTAu-comPound 14 significantly reduced androgen- independent PC-3 tumor growth (***P £ 0.01) (Figure 5), without any significant toxicity as shown mice weights monitoring along the experiment compared to control group (Figure 7). The assessment of Ki-67 immunoexpression and optical density in the harvested tumors was correlated with the inhibition of growth tumor (13,7 ± 0.18 for compound 14 vs 6.81 ± 0.14 for NPDoTAu-comPound i4, p < 0.0001) (Figure 8).
Statistical analysis. All the results were expressed as mean±S.E. Statistical analysis was performed by one-way ANOVA followed by Fisher’s protected least significant difference test. PrO.05 was considered significant (*); PrO.01 (**); PrO.001 (***).
Example 3: Bioluminescence Resonance Energy Transfer (BRET) assay
To determine the presence of the interaction between the Hsp27 and the elF4E proteins, the following BRET assay can be performed.
Construction of BRET plasmids
Hsp27 or elF4E genes alone or with kozak sequence upstream were first inserted into a pCR8/GW/TOPO plasmid (Invitrogen). Kozak sequence was added each time the gene was found in N-ter in the final plasmid. For the insertion into pCR8/GW/TOPO plasmid, genes were first amplified by PCR using Phusion enzyme (Finnzymes, Thermo Fisher Scientific, lllkirch, France) and the following BRET primers: Hsp27: Forward=5’-ATG ACC GAG CGC CGC GTC CC-3’ (SEQ ID NO: 1), Forwardk0zak= 5’-GCC ACC ATG ACC GAG CGC CGC GTC CC-3’ (SEQ ID NO: 2), Reverse=5’-CTT GGC GGC AGT CTC ATC GG- 3’ (SEQ ID NO: 3), elF4E: Forward=5’-ATG GCG ACT GTC GAA CCG GAA AC-3’ (SEQ
ID NO: 4), Forwardkozak=5’- GCC ACC ATG GCG ACT GTC GAA CCG GAA AC-3’ (SEQ ID NO: 5), Reverse=5’-AAC AAC AAA CCT ATT TTT AGT GGT GG-3’ (SEQ ID NO: 6). Then, dATP with Go Taq enzyme (Promega, Charbonnieres, France) were added at the ends of the gene (PCR product) to allow the matching between gene and plasmid. Genes were then purified from the PCR product with the Wizard SV Gel and PCR Clean-Up System kit (Promega).
The insertions into plasmids were realized by TOPO reaction according to manufacturer’s instructions (Invitrogen). Competent bacteria “MACH 1” (Invitrogen) were transformed using TOPO reaction products and the obtained clones were analysed by PCR with Go Taq enzyme (Promega). Positives clones were amplified by mini-culture and DNA was extracted with Wizard Plus SV Minipreps DNA Purification System kit (Promega), and sent for sequencing (M13 Forward primer) (GATC, Mulhouse, France). The second step was to transfer the genes from pCR8/GW/TOPO intermediate plasmids to destination plasmids containing luciferase and YFP by LR Gateway recombination reaction using LR clonase according to manufacturer’s instructions (Invitrogen). The destination plasmids pRLuc-C, pRLuc-N, pEYFP-N and pEYFP-C, were kindly given by Nelson Dusetti (UMR 1068, Centre Recherche Cancerologie Marseille, France). Competent bacteria “MACH 1” (Invitrogen) were transformed using LR reaction products. The clones obtained were amplified by mini-culture and DNA was extracted with the Wizard Plus SV Minipreps DNA Purification System kit (Promega). Enzymatic digestion by BsrGI (Promega) was
performed in order to identify positives clones that were then amplified by maxi-culture and DNA was extracted using the PureYield Plasmid Midiprep System kit (Promega), and sent for sequencing (BRET primers) (GATC).
BRET in living cells
HEK293T cells cultured in 12-well culture plates (400 000 cells/well) were co-transfected with 0.2 pg of BRET donor plasmid N-terluc/elF4E, and 0 to 1 pg of BRET acceptor plasmid Hsp27/YFPC-ter. The empty vector (pEYFP-C) was used to equalize DNA amounts to 1 .2 pg in each sample. The day after, cells were harvested and distributed in 96-well white microplates («40 000 cells/well). On the following day, the cell-permeable Rluc substrate coelenterazine-h (Interchim, Montlugon, France) diluted in phosphate buffered saline (PBS) 1X was added in culture medium to a final concentration of 5 pM, and cells were incubated 15 min at 37°C before reading. Readings were done using a LB 941 Tristar reader (Berthold France SA, Thoiry, France), with signal detection in the 470-490 nm (donor) and 520-540 nm (acceptor). BRET signal represents the BRET ratio of the Rluc and EYFP expression constructs compared to the BRET ratio for the Rluc expression construct alone. To assess signal variation, the BRET values were determined by using the following equation, expressed in milli-BRET unit (mBretll): 1000
J where E0 corresponds to the ratio 530 nm acceptor signal/480 nm donor signal obtained with the Rluc construct alone in the same experiment.
BRET in cell extracts
HEK293T cells were transfected separately with a BRET donor plasmid N-terluc/elF4E or BRET acceptor plasmid Hsp27/YFPC-ter. Forty eight hours later, total cellular proteins were extracted following the manufacturer’s instructions of CelLytic NuCLEAR Extraction Kit (Sigma-Aldrich) and stored at -80°C. 1 pg of lysate containing a BRET donor N- erluc/elF4E (or 4pg for CCND3/luc) was mixed with 0 to 30 pg of lysate containing a BRET acceptor Hsp27/YFPC-ter (or CDK6/YFP) and with lysis buffer in a 96-well white plate. After 30 min of incubation at RT, coelenterazine-h (Interchim) was added to a final concentration of 5 pM in lysis buffer, just before reading. Readings and analysis were made as above described.
In order to overcome the solubility issue, compound#14 was formulated with 2 cationic nucleolipids for encapsulation as nanoparticle formulation. DOTAU and DOU-PEG2000 are nucleoside-based cationic lipids derived from uridine.
The encapsulation of compound#14 with both NLs were obtained following film hydration method leading to obtain monodisperse NPs without observed any aggregation during the process. The tt-p stacking and hydrogen bonding interaction enhanced the stability of the formed NPs. In both cases is the second layer formed by the NL of the NP enhanced the water solubility either due to the positive charge of the DOTAU or the miscibility in water properties for the DOU-PEG2000. The analysis by dynamic light scattering show NPs size around 75-78nm and their positive zeta potential (+71 mV for NPDOTAU- compound 14 and + 42 mV for NPDou-PEG2ooo-comPound i4) indicate that the NL surround the NPs.
The present invention thus confirms that the nanoformulation did not impair the inhibition effect of compound 14 on Hsp27/elF4E interaction. On proliferation studies, NPDOTAU- compound 14 was the most effective compound. NPDoTAu-comPound 14 also demonstrated better pro-apoptotic activity compared to other agents. The in vivo experiments were also in favor of NPDOTAu-compound 14 and showed impressive anti-tumor effects on PC3 xenografts, which represents an aggressive AIPC model. Ki-67 was clearly decreased in N P DOT Au-compound 14 arm, explaining together with the increased pro-apopoptotic effect the therapeutic benefit. The positive effect is probably due to an increase of accumulation in the tumor area by passive targeting due to the EPR (enhanced permeability retention) effect. This behavior induces a local increase of drug concentration into the tumor resulting in a better antitumoral activity compared to free drug.
The NPDOTAu-compound 14 diameter (75 nm) probably influences the tumor size growth inhibition since it avoids compound#! 4 renal clearance.
Claims
1. Nanoparticles comprising:
- a core comprising at least one compound having the following general formula (I):
wherein:
• RI=F¾2 and represent R8, -OR8 or -OCOR9, with R8 representing a linear or branched (Ci-C2o)alkyl group, and Rg representing a hydrogen atom or a linear or branched (C2-C2o)alkyl group;
• R3 and R , independently of each other, represent:
- a hydrogen atom;
- a halogen atom selected from the group consisting of chlorine, fluorine, bromine and iodine atom;
- a cyano group;
- -NO2;
- -N(R5)(R6), with R5 and R6 representing independently of each other, a hydrogen atom, a linear or branched (Ci-C2o)alkyl group, a benzyl group or a -COR10 group with Ri0 representing a hydroxyl group or a linear or branched (Ci-C2o)alkoxy group;
- an amido group;
- -NHSC>2(R7), with R representing a linear or branched (Ci-C2o)alkyl group, or a aryl group;
- -ORs' or -OCORg', with R8' and Rg' representing a hydrogen atom or a linear or branched (Ci-C2o)alkyl group;
- a (Ci-C2o)alkyl, a (CrC2o)alkenyl, a (Ci-C2o)alkylthio or a (Ci-C2o)alkenylthio group, linear or branched;
- -COR10 with Ri0as defined above;
- -CO-[NH-(CH2)n]m-N(R5)(R6), -HN-CO-[(CH2)n-N(Rii)]p-(CH2)n'-N(Rii)(R6), or -O- CO-[(CH2)n-N(Rii)]p-(CH2)n’-N(Rii)(R6) with:
- R5 and R6 as defined above,
- Rii representing a hydrogen atom or a tert-butyloxycarbonyl group,
- p representing an integer ranging from 0 to 1 , and
- n, n' and m, independently of each other, representing an integer with n and n', independently, ranging from 2 to 6 and m ranging from 1 to 2, and, when m is 2, each n value may be identical or different one from another, or one of its pharmaceutically acceptable salts; and a lipid shell comprising at least one layer of a nucleolipid having the following formula (A):
wherein:
■ Re represents a purine or pyrimidine base such as uracil, adenine, guanine, cytosine, thymine, hypoxanthine, or their derivatives, or a non-natural mono- or bi- cyclic heterocyclic base each ring of which comprises 4 to 7 members, optionally substituted;
■ Li and L2, independently of each other, are selected from the group consisting of: hydrogen atom, -O-C(O)-, -0-C(S)-NH-, -0-C(0)-0-, -0-C(0)-NH-, an oxygen atom, a phosphate group, a phosphonate group or a heteroaryl comprising from 1 to 4 nitrogen atoms, said heteroaryl being unsubstituted or substituted by a linear or branched (C2- C3o)alkyl or a linear or branched (C2-C3o)alkenyl,
or, Li and l_2, together form a ketal group of formula: or, Li or l_2 is a hydrogen atom, and the other is a hydroxy group or a heteroaryl group comprising from 1 to 4 nitrogen atoms, said heteroaryl being unsubstituted or substituted by a linear or branched (C2-C3o)alkyl;
■ Mi and M , independently of each other, represent:
- a linear or branched (C2-C3o)alkyl;
- a linear or branched (C2-C3o)alkenyl; said alkyl or alkenyl group being optionally completely or partially fluorinated, and/or substituted on the carbon at the end of the chain by a fluorine atom or by a benzyl or naphthyl ester or ether;
- a diacyl chain in which each acyl chain is in C2-C30;
- a diacylglycerol;
- a sphingosine; and
- a ceramide group; when Li or l_2 represents hydrogen, and the other represents a hydroxy group or a heteroaryl group comprising 1 to 4 nitrogen atoms, then Mi and M2 do not exist;
■ Ra is selected from the group consisting of:
- hydroxy;
- amino;
- phosphate;
- phosphonate;
- phosphatidylcholine;
- phosphocholine;
- -0-(Ci-Cio)alkylene-phosphatidylcholine;
- thiophosphate;
- phosphonium;
- NH2-Rb;
- a -[NRbRcRd]+ group in which Rb, Rc and Rd, identical or different, represent a hydrogen atom, a linear or branched (Ci-Cio)alkyl or a linear or branched O- (Ci-Cio)alkyl;
- a linear or branched (C2-C3o)alkyl optionally substituted by a hydroxy group;
- a -0-C(0)-(Ci-Cio)alkylene-C(0)-0-[CH2-CH2-0]rCH3 group or a -0-C(0)-(Ci- Cio)alkylene-S-S-[CH2-CH2-0]rCH3, wherein r is an integrer comprised between 4 to 30;
- a cyclodextrin radical;
- a group of formula (B):
Rz
H-(-CH2-C-)w-H
CO-V- (B) in which V is -0-, -S-, or -NH- and Rz is H or CH3, and w=1 to 500;
- a -(CH2)W-V-RX group, in which Rx represents a (C2-C30) alkyl, and w=1 to 500; - a heteroaryl group containing 1 to 4 nitrogen atoms, unsubstituted or substituted by a linear or branched (C2-C3o)alkyl, or by a (CH2)g-0-(CH2)h-Ri group in which g=1 to 6 and h=0 to 10 and R, represents a cyclic ketal group containing 5 to 7 carbon atoms, unsubstituted or substituted by at least one linear or branched (C2-C3o)alkyl or by a sterol radical, or Ra is bound by a covalent bond to another substituent Ra, identical or different, of another compound of formula (A), identical or different, in order to form a compound of formula (A) in the form of a dimer.
2. The nanoparticles according to claim 1 , wherein in formula (I): ■ RI=R2 and represent Rs, -ORs or -OCOR9, with R8 representing a linear or branched (Ci-C2o)alkyl group, and Rg representing a hydrogen atom or a linear or branched (Ci-C2o)alkyl group;
■ R3 and R4, independently of each other, represent:
- a hydrogen atom; - a linear or branched (C1-C20) alkyl,
- a linear or branched (Ci-C2o)alkenyl,
- -N(R5)(R6), with R5 and R6 representing independently of each other: a hydrogen atom, a linear or branched (Ci-C2o)alkyl group, a benzyl group or a -COR10 group, R10 being a hydroxyl group or a linear or branched (Ci-C2o)alkoxy group; - -CO-[NH-(CH2)n]m-N(R5)(R6), -HN-CO-[(CH2)n-N(Rn)]p-(CH2)n'-N(Rii)(R6), or -O-
CO-[(CH2)n-N(Rii)]p-(CH2)n'N(Rii)(R6) with:
- R5 and R6 being as defined above,
- R11 representing a hydrogen atom or a tert-butyloxycarbonyl group,
- p representing an integer ranging from 0 to 1 , and
- n, n' and m, independently of each other, representing an integer with n and n', independently, ranging from 2 to 6 and m ranging from 1 to 2, and, when m is 2, each n value may be identical or different one from another, or one of its pharmaceutically acceptable salts.
3. The nanoparticles according to claim 1 or 2, wherein in the compound of the formula (I), both Ri and R2 are a linear or branched -0(Ci-C2o)alkyl group.
4. The nanoparticles according to anyone of claims 1 to 3, wherein in the compound of the formula (I), R3 and R4 represent independently of each other: a hydrogen atom, -N(R5)(R6) or -CO-[NH-(CH2)n]m-N(R5)(R6), with n, m, R5 and R6 being as defined in claim 1 .
5. The nanoparticles according to anyone of claims 1 to 4, wherein the compound of formula (I) has one of the following formulae:
6. The nanoparticles according to anyone of claims 1 to 5, wherein the nucleolipid of formula (A) has the following formula (A-l):
wherein Ra, Mi and M2 are defined in claim 1 .
7. The nanoparticles according to anyone of claims 1 to 6, wherein in the nucleolipid of formula (A) or (A-l), Mi and M2 are independently of each other a linear or branched (C2-C3o)alkyl or a linear or branched (C2-C3o)alkenyl, preferably a (C2- C3o)alkenyl.
8. The nanoparticles according to anyone of claims 1 to 7, wherein in the nucleolipid of formula (A) or (A-l), Ra is selected from the group consisting of:
- a -[NRbRcRd]+ group in which Rb, Rc and Rd, identical or different, represent a hydrogen atom or a linear or branched (Ci-Cio)alkyl group, and - a -0-C(0)-(Ci-Cio)alkylene-C(0)-0-[CH2-CH2-0]rCH3 group, wherein r is an integer comprised between 4 and 30.
9. The nanoparticles according to anyone of claims 1 to 8, wherein the nucleolipid of formula (A) has one of the following formulae:
10. The nanoparticles according to claim 1 , wherein: the core comprises a compound of formula (I) having one of the following formulae:
and the lipid shell comprises at least one layer of a nucleolipid of formula (A) having one of the following formulae:
with r being an integer comprised between 10 and 20.
11. The nanoparticles according to anyone of claims 1 to 10, characterized by a mean size particle comprised between 50 nm and 150 nm, preferably between 60 nm and 130 nm.
12. The nanoparticles according to anyone of claims 1 to 11 , for use as a medicament.
13. The nanoparticles according to anyone of claims 1 to 11 , for use in the prevention and/or the treatment of diseases wherein the Hsp27-elF4E interaction is involved, preferably cancers.
14. The nanoparticles for use according to claim 13, wherein the cancer is selected from the group consisting of: lymphomas, angiosarcomas and the cancers of the lung, pancreas, breast, bladder, colon, skin, head and neck, ovarian, and prostate.
15. A pharmaceutical composition, preferably an aqueous pharmaceutical composition, comprising the nanoparticles according to anyone of claims 1 to 14 and optionally one or more pharmaceutically acceptable excipient(s).
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