FR2867189A1 - New compound comprising protein kinase regulator and photoactivatable molecule, useful for treating protein kinase-related diseases, e.g. tumors and inflammation - Google Patents

Abstract

Compound (A) comprises one or molecules (II) that inhibit, regulate and/or modulate activity of a protein kinase (PK), where (the new feature) (II) is/are associated with at least one photoactivatable molecule (I) is new. ACTIVITY : Cytostatic; Antiarteriosclerotic; Ophthalmological; Antidiabetic; Antiinflammatory. A conjugate (GP) of the known kinase inhibitor STI571 with a carboxylated porphyrin was tested on K562 cells (chronic myeloid leukemia, carrying the Philadelphia translocation). A concentration of 10 mu g/ml GP gave 50% kill, 48 hours after a 2 hour exposure, but this was increased to 65% kill when the cells were also irradiated for 14 seconds with 10 J/cm2> of light energy (at 514 nm). MECHANISM OF ACTION : Protein kinase inhibitor.

Description

s New drug compounds for the treatment of pathologies

  dependent on protein kinase activity

  The present invention relates to novel compounds having an action of inhibiting, regulating and / or modulating protein kinase activity as well as drugs and pharmaceutical compositions for the treatment of pathologies dependent on protein kinase activity.

  Protein kinases (PK) play a central role in the transmission of intracellular signals such as: metabolism, differentiation, cell and tissue proliferation, and apoptosis. It is estimated that there are approximately 2000 distinct protein kinases in humans. They produce, thanks to the specific phosphorylation of proteins, a stable modification of the biological activity which will be canceled only under the action of protein phosphatases. The balance between phosphorylation by protein kinases and dephosphorylation by protein phosphatases regulates many important cellular processes. A large number of proto-oncongene products are part of protein kinases.

  The large family of protein kinases can be subdivided according to the type of activation and the type of substrate. Protein kinases A are activated by cAMP, protein kinases G by cGMP, protein kinases C by diacylglycerol (DAG), and Cal + dependent Cal + / calmodulin kinases. The classification is based on the nature of the phosphorylated amino acid residue. There are tyrosine kinases and serine / threonine kinases. Kinases can have a broad substrate specificity or be specialized for some proteins.

  The development of selective PK inhibitors that can block or modulate pathologies in which this signaling pathway is defective is a promising approach for the development of new anti-cancer drugs.

  However, since the activity of these inhibitors is enzymatic, this imposes a constant blocking of the site and therefore constant drug intake. The object of the present invention is to propose associating a protein kinase inhibitor with a photoactivatable compound for the purpose of using the inhibitor for the recognition of the site to be treated, and the photoactivatable compound for the destruction of the receptor site by a photochemical action lo (photochemotherapy) for a more durable and / or more effective blocking of the targeted kinase activity.

  Photochemotherapy is based on activation using a light radiation of wavelength adapted to the absorption spectrum of a photoactivatable compound. The light radiation absorbed by the photoactivatable compound causes reactions of type 1 (production of hydroxyl radicals) or type 2 (production of singlet oxygen) by an intersystem conversion mechanism. These radical species lead to oxidation and peroxidation reactions leading to the destruction or inactivation of the treated site.

  For this purpose, the compound according to the present invention associates one or more photoactivatable molecules (I) and one or more molecules (II) having an action of inhibition, regulation and / or modulation of the activity of a protein kinase. .

  We thus discovered that these compounds containing at least one photosensitive molecule could be given the possibility of specifically recognizing protein kinase receptor sites to inhibit them by means of a photochemical reaction. This inhibition proved to be more durable than that obtained with the protein kinase inhibitors, alone, that is to say, not associated with photosensitive molecules.

  Advantageously, the photoactivatable molecule (I), that is to say having a photochemical activity under light irradiation, is a photosensitizing agent chosen from: porphyrins or their derivatives, such as hematoporphyrin, dihematoporphyrin ethers, hematoporphyrin dimers, hematoporphyrin trimers, benzoporphyrin, green porphyrins, merocyanines, porphycenes, sodium porphimers, verteporfin, chlorins, preferably chosen from families of diphenyl chlorine molecules (SIM), tetrahydroporphyrins, such as m-THPC, bacterioclorins, zinc phthalocyanine, purpurins and their derivatives, such as ethyl etopurpurin tin, pheophorbides or a combination of several of these molecules.

  The photoactivatable molecule (I) may also be a dye preferably chosen from: acridine orange, bengal rose, methylene blue, indocyanine green, fluorescein and rubrene, or one of their polymers reacting to light, or chlorophyll a, riboflavin, phenaleone or a combination of several of these molecules.

  Similarly, advantageously, the molecule (II) of the compound according to the present invention is chosen from the following inhibitors of the following target kinases: STI 571 (Bcr-Abl inhibitors) - ZD-1839, OSI-774 , PKI 166, EKB-569, GW 57 2016 (EGF-R inhibitors) 25 - CEP 2563 (PKC / Trk inhibitor) - UCN-01, GCP 41251 (STI 412), safingol, perifosine (PKC inhibitors SU 5416, CGP 79787, CP-564959, ZD 6474, ZD 2171, SU-11248 (VEGF-R inhibitors) - flavopiridol, CI-202 (CDK inhibitors).

  These examples of inhibitors are ATP-competitive low molecular weight protein kinases.

  The association between the molecules (I) and the molecules (II) of the compound of the present invention can be carried out: either by a covalent bond according to the formula (I) n (II) n and m being understood between 1 and 5, or by means of a connecting element L according to formula (1), L- (11) n and ms being between 1 and 5, and L being chosen from: an alkyl group or substituted or unsubstituted acyl; a polymer, for example poly-1-glutamic acid, poly-d-glutamic acid, poly-dl-glutamic acid, poly-1-aspartic acid, poly-d-aspartic acid , poly-dl-aspartic acid, polylysine, polysaccharides, polyhydroxypropylmethacrylamide, dextran, poly (hydropropylglutamine), poly (hydroethylglutamine), hyaluronic acid, carboxymethyl dextran, polyacrylic acid chitosan, polyglycine, polytyrosine, polyphenylalanine, polypropylene oxide (PPO), polyethylene glycol (PEG), copolymers of PEG with PPO, polyglycolic acid, polyvinylpyrrolidone, polylactic acid, polyvinyl alcohol; an antibody or a fragment thereof, a peptide or a protein.

  The linkage between molecules (I) and (II) through L can be cleavable by an enzyme, sensitive to pH, destroyed by light or any other physical or chemical process.

  The compound according to the invention finds an interesting application in photochemotherapy.

  In this regard, the present invention also relates to a medicament containing, as active ingredient, a compound as described above. Such a medicament is in particular intended for the treatment of pathologies dependent on the protein kinase activity, preferably intended for the treatment of dysregulation of angiogenesis, tumor growth, cancers, atherosclerosis, macular degeneration linked to the age, diabetic retinopathy or inflammatory diseases.

  The present invention also relates to a pharmaceutical composition, characterized in that it contains a compound of the aforementioned type associated with a pharmaceutically acceptable excipient.

  The compounds of the invention may be presented in any form of composition administrable to the human or animal body and suitable for enteral, parenteral or transdermal administration, such as tablet, dragee, dermal cream, capsule, capsule, suspension or oral solution or injectable, such as syrup or ampoule, transdermal patch, liposomal formulation (nanoparticles) etc. associated with suitable excipients and dosed to allow one or more courses comprising one or more daily administrations for a day or several days at a dose for example between 0.1 and 20 mg / kg of weight.

  The excipients may be aqueous or lipid-containing solutions optionally containing agents for suspending, stabilizing, preserving and / or dispersing. The compounds may also be in a lyophilized form such as a powder which is added before use with a sterile liquid, such as sterile water for injection.

  Preferred administrable forms are injectable forms, particularly in the form of intramuscular or intravenous injection or in the form of transcutaneous application.

  The compounds of the invention may also be used for the ex-vivo treatment of cells, for example in the context of bone marrow purges in the treatment of leukemias and more generally for the implementation of the methods included under the term cell therapy. .

  The invention will be better understood on reading illustrative embodiments.

Example I

  The following compound and the effects it induces with and without light illustrate, by way of example, the preparation of a compound according to the invention consisting of a fraction of the STI571 tyrosine kinase inhibitor (Gleevec, Imatinib ) and a porphyrin and its effects.

  The synthesis of this compound, corresponding to the following general formula: CH 3 CH 3 (GP) and which will be called thereafter to simplify GP, comprises the following steps. io

  Step 1: cleavage of the amide bond of ST1571 Hydrolysis

NH NH2

  Step 2: A carboxylated porphyrin is then used to reform the amide bond thereby regenerating part of the STI571 structure.

HO CH3 CH3 N H2 CH3 CH3

  The tests described below relate to the activity of the GP molecule compared to the activity of STI571 alone. The activity of this new GP molecule has been tested in vitro with toxicity and phototoxicity tests.

  GP shows, in an absolute alcohol solution, 5 main absorption peaks respectively at 415, 513, 548, 590 and 646 nm. The solution was obtained as follows: One milligram of GP was dissolved in 1 mL of absolute alcohol.

  The ST1571 molecule does not show any absorption in the visible.

  To study the toxicity and phototoxicity of the GP molecule, it was proceeded as follows: K562 cells (chronic myeloid leukemia, CML) were used, as they express the Philadelphia chromosome. In fact, the Philadelphia chromosome found in the leukemic cells of patients with CML is produced by translocation between chromosome 9 and chromosome 22, resulting in the expression of a Bcr chimeric protein and Abl kinase in the body. cytoplasm, which makes this cell type susceptible to treatment with antityrosine kinase such as STI 571.

  The cells were cultured in 25 cm 2 Falcon flasks (Polylabo, Strasbourg, France) in RPMI medium (Roswell Park Memorial Institute medium) supplemented with 10% (VN) fetal calf serum (FCS), 100 penicillin 100 mg streptomycin and 2 mM glutamine.

  Cells were subcultured in culture at a 1: 3 dilution, which maintained these cells in the exponential growth phase. The absence of mycoplasma contamination has been regularly monitored.

  The cells thus cultured were subjected to a photodynamic treatment.

  STI571 was dissolved in phosphate buffer. For the GP molecule, a stock solution was made at 1 mg / ml in a buffer (50% water, 30% PEG, 20% ethanol), the successive dilutions being carried out in phosphate buffer. Aliquots (11 μl) of the photosensitizing solution GP or STI571 were added to K562 cells in 96 well plate wells. The cell concentration was 5 × 10 4 cells / ml (100 μl per well). The final concentration of GP or STI571 solutions was in the range of 0.5 to 20 μg / ml. Immediately after addition of GP or STI571 photosensitizer, the cell plaques were kept in complete darkness until PDT (Photodynamic therapy or photochemotherapy) treatment. Fresh media containing FCS but free of GP or STI571 were prepared and added after centrifugation at 1800 rpm for 5 minutes, before laser irradiation. An argon laser emitting at 514 nm coupled to an optical fiber was used to irradiate the cells. The light emission power at the end of the fiber was set using a power meter (Coherent, France) at 200 mW. The light was transmitted to the target by an optical fiber at a distance of 20 mm to irradiate cells in a 6 mm diameter well within a single field providing illumination of the entire area. . The exposure time was 14 seconds per well at 514 nm providing an energy density of 10 J / cm 2.

  The cells were incubated with GP or STI571 for 2 hours. For the phototoxicity assays, the cells were washed and the medium was replaced immediately before laser irradiation.

  The cell counts were performed 24, 48 and 72 hours after the end of the experiments. At the time of counting, 15 μL of PBS-MTT solution (5 mg / mL) was added to the wells. After 4 hours, 150 μl of 0.04 N isopropanol HCI was added according to the method described by Mosmann (T. Mosmann.

  Rapid colorimetric assay for cell growth and survival: application to proliferation and cytotoxicity assay. J. Immunol. Methods, 65, 55-63, 1985).

  The optical density of each well of the microplates was then read at 570 nm using an Opsys MR apparatus (Dynex Technologies, Chantilly, USA).

  One well containing RPMI without phenol red and 15 μl MU solution (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide) was used to determine the blanks for the measurements. absorbance.

  The results are shown in FIGS. 1 to 3: FIG. 1 represents the toxicity of STI571 measured at 24, 48 and 72 hours on K562 cells after incubation for 2 hours; Figure 2 shows the toxicity of GP measured at 24, 48 and 72 hours on K562 cells after incubation for 2 hours; Figure 3 shows the toxicity and phototoxicity (irradiation at 514nm and 10 J / cm2) of GP measured at 48 hours on K562 cells after incubation for 2 hours; Through these figures, the STI571 molecule alone shows concentration-dependent growth inhibition on K562 cells. The GP molecule used in the absence of light causes comparable growth inhibition, although it is slightly lower than ST1571 alone. This suggests that the STI571 moiety coupled to porphyrin is indeed the inhibitory fraction of anti-tyrosine kinase activity, and that the presence of porphyrin does not prevent site recognition.

  The K562 cells incubated with GP and subjected to light irradiation have a statistically stronger inhibition of growth than when they are incubated with GP in the absence of light.

  No significant difference was observed between irradiated and non-irradiated cells with STI571 alone, not associated with porphyrin (results not shown here).

  In order to confirm the presence of the GP molecule at the level of the K562 cells, the in vitro location of the GP molecule was visualized by fluorescence imaging in order to confirm that the abovementioned compounds were able to penetrate the cells. . Thus, K562 cells were seeded at 105 cells / ml on circular glass slides. After 2 hours, cells were incubated with GP (20 μg / mL) at 37 ° C for 4 hours and then washed in PBS (Phosphate Buffer Saline Solution) (pH 7.2). The analysis of the GP fluorescence (emitting at 630 nm) was carried out after excitation between 450 nm and 480 nm produced by a 150 W Xenon lamp using a highly sensitive black-and-white photon detection video camera (Kappa CF 8 Fischer Scientific SA, France) connected to an optical microscope (Olympus BX 40, France) equipped with a 100-fold magnifying oil immersion objective.

  The strongest fluorescence intensity (Figure 4) was localized mainly in the cytoplasm of K562 cells. The images produced immediately after incubation for 4 hours with GP clearly demonstrate that the compounds are able to enter the cells. No residual fluorescence was detected in K562 cells alone or incubated with STI571 (results not shown here).

Example II

  The following compound consists of a STI571 tyrosine kinase inhibitor (Gleevec, Imatinib) fraction and a SIM01 chlorin (described in WO 0210173) 2,3 dihydro 5,15-bis (3,5-dihydroxy) 1-phenyl) porphyrin.

  The synthesis method is identical to that of Example I. The resulting compound has the following general formula (GC): and will be called later to simplify GC.

  The tests concerning the toxicity and phototoxicity of the GC molecule are identical to those carried out in Example I. The GC molecule shows results comparable to those described in Example I, that is to say for the GC molecule used. in the absence of light results in a similar growth inhibition of K562 cells although a little lower than STI571 alone.

  The K562 cells incubated with GC and subjected to light irradiation have a strong statistically greater inhibition of growth than when they are incubated with GC in the absence of light, more important than GP in the presence of equivalent dose light. In fact, SIM is more powerful than porphyrin because of a greater singlet oxygen quantum yield, thus producing a more intense photochemical reaction.

Example III

  The following compound consists of a fraction of the inhibitor ZD1839 (Iressa) and a porphyrin.

  ZD1839 is an inhibitor of intracellular phosphorylation of many cell surface transmembrane receptor tyrosine kinases, including epidermal growth factor receptor tyrosine kinases (EGFR-TK). EGFR is expressed on the cell surface of many normal and cancerous cells. ZD1839 works by blocking the transduction signal involved in cancer cell proliferation, survival and host-dependent processes promoting tumor growth.

  The compound prepared corresponds to the following general formula: ## STR2 ## and will be called later to simplify IP.

  The synthesis of this compound comprises the following steps.

  Step 1: cleavage of the amide bond of ZD1839 H3C.

HN

  Hydrolysis O / NH2 H3C Step 2: A carboxylated porphyrin is then used to reform the amide bond thus regenerating part of the structure of ZD1839. CH3

HO CH3 CH3O NH2 H3C ,,

HN CH3 CH3

  The tests concerning the toxicity and phototoxicity of the IP molecule are identical to those carried out in Example I. The IP molecule shows results comparable to those described in Example 1, ie the IP molecule used. in the absence of light results in a similar growth inhibition of K562 cells although somewhat less than ZD1839 alone.

  The K562 cells incubated with IP and subjected to a light irradiation exhibit a strong inhibition of statistically greater growth than when they are incubated with IP in the absence of light.

Claims (10)

  A compound comprising one or more molecules (II) having an action of inhibiting, regulating and / or modulating the activity of a protein kinase, characterized in that said molecule (s) (II) is (are) associated with one or more photoactivatable molecules (I).   2. Compound according to claim 1, characterized in that the photoactivatable molecule (I) is a photosensitizing agent chosen from: porphyrins or their derivatives, such as hematoporphyrin, dihematoporphyrin ethers, hematoporphyrin dimers, hematoporphyrin trimers, benzoporphyrin, green porphyrins, merocyanines, porphycenes, sodium porphimers, verteporfin, chlorins, preferably chosen from families of chlorinated biphenyl molecules (SIM), tetrahydroporphyrins, such as mTHPC , bacterioclorins, zinc phthalocyanine, purpurins and their derivatives and pheophorbides.   3. Compound according to claim 1, characterized in that the photoactivatable molecule (I) is a dye, preferably chosen from: acridine orange, bengal rose, methylene blue, indocyanine green, fluorescein and rubrene, or a light-reactive polymer thereof, or chlorophyll a, riboflavin, phenaleone, or a combination of several of these molecules.   4. Compound according to any one of the preceding claims, characterized in that the molecule (II) is chosen from the following inhibitors of the following target kinases: STI 571 (Bcr-Abl inhibitors) ZD-1839, OSI -774, PKI 166, EKB-569, GW 57 2016 (EGF-R inhibitors) CEP 2563 (PKCITrk inhibitor) - UCN-01, GCP 41251 (STI 412), safingol, perifosine (PKC inhibitors) - SU 5416, CGP 79787, CP-564959, ZD 6474, ZD 2171, SU-11248 (VEGF-R inhibitors) - flavopiridol, CI-202 (CDK inhibitors).   5. Compound according to one of the preceding claims, characterized in that the association between the molecules (I) and (II) is carried out by a covalent bond according to formula (1) n (II) m, n and m being between 1 and 5.   6. Compound according to any one of claims 1 to 4, characterized in that the association between the molecules (I) and (II) is carried out by means of a connecting element L according to formula (1) n L411 m, n and m being between 1 and 5, and L being chosen from: a substituted or unsubstituted alkyl or acyl group; a polymer, for example poly-1-glutamic acid, poly-d-glutamic acid, poly-dl-glutamic acid, poly-1-aspartic acid, poly-d-aspartic acid poly-dl-aspartic acid, polylysine, polysaccharides, polyhydroxypropylmethacrylamide, dextran, poly (hydropropylglutamine), poly (hydroethylglutamine), hyaluronic acid, carboxymethyl dextran, polyacrylic acid, chitosan, polyglycine, polytyrosine, polyphenylalanine, polypropylene oxide (PPO), polyethylene glycol (PEG), copolymers of PEG with PPO, polyglycolic acid, polyvinyl pyrolidone, polylactic acid, polyvinyl alcohol; an antibody or a fragment thereof, a peptide or a protein.   10. Compound according to claim 6, characterized in that the bond between (I) and (II) through L can be cleavable by an enzyme, sensitive to pH, destroyed by light or any other physical or chemical process .   11. Medicament, characterized in that it contains, as active ingredient, a compound according to one of claims 1 to 8.   9. Drug according to claim 8, s characterized in that it is intended for the treatment of pathologies dependent on the protein kinase activity, in preference to the treatment of dysregulation of angiogenesis, tumor growth, cancers, cancer. atherosclerosis, age-related macular degeneration, diabetic retinopathy or inflammatory diseases. i0   10. Pharmaceutical composition, characterized in that it contains a compound according to one of claims 1 to 7 combined with a pharmaceutically acceptable excipient.