WO2004004711A1 - Use of at least two anticancer compounds, one of which at least modulates proteasome activity for making a medicine for cancer treatment - Google Patents

Abstract

The invention concerns the use of at least two anticancer compounds, one of which at least modulates proteasome activity for making a medicine for cancer treatment, in particular colon cancer. The invention also concerns a pharmaceutical composition comprising at least two anticancer compounds, one of which at least modulates proteasome activity in combination with at least one pharmaceutically acceptable carrier.

Description

The present invention relates to the use of at least two anti-cancer compounds, of which at least one is at least one which modulates the activity of the proteasome for the manufacture of a medicament intended to treat cancer. a module for the proteasome activity for obtaining a medicament intended for treating cancer, as well as pharmaceutical compositions containing the combination of the abovementioned compounds.

Cancer is one of the biggest causes of death in developed countries. One of the therapies used to fight cancer is the use of anticancer drugs (or chemotherapy), which interfere with metabolism and cell life, making it possible to inhibit tumor growth.

The anti-cancer compounds can in particular be anti-mitotic compounds, which make it possible to prevent the proliferation of cancer cells. Thus the periwinkle alkaloids (or vinca-alkaloids), and their derivatives obtained by chemical modification target the tubulin of the chromatic spindle of mitosis whose polymerization they inhibit (Review in Rahmani & Zhou, Cancer Surveys, vol 17, 1993 ). Among the vinca alkaloids currently used in chemotherapy, there may be mentioned vincristine, used in the treatment of cancers and leukemias since 1960. Like the other vinca alkaloids, vincristine binds in the cell on tubulin and prevents the formation of microtubules. It thus blocks the dynamics of the mitotic spindle along which the chromosomes migrate and stops the cells in metaphase. Mention may also be made of vindesine which is a semi-synthetic alkaloid derived from periwinkle, which also binds to tubulin and prevents the formation of microtubules, in particular the microtubular system of the mitotic spindle, which leads to the division of division. cellular. This compound is therefore active on cells already involved in the cell cycle. Vinorelbine is the latest drug in the vinca alkaloid family introduced in human therapy. Like the other periwinkle alkaloids, vinorelbine is a depolymerizing agent for tubulin, blocking cell division by altering the mitotic spindle and causing cell apoptosis. However, tubulin also constituting nerve fibers, vinca alkaloids have nerve toxicity, especially sensitive, and induce a depressive tendency. Thus, during administration of vincristine, the peripheral nervous system may be affected in the form of sensory disturbances of the extremities and a decrease in reflexes. When administering vindesine, if the neurological complications are less marked than those caused by vincristine, blood toxicity is the main limiting factor of this drug, inducing a temporary and generally moderate decrease in granulocytes. In the case of vinorelbine, the main toxicity of this drug is the decrease in white blood cells and granulocytes, and neurological toxicity is uncommon. The anticancer compounds can also be intercalants which are inserted between the nitrogenous bases of two DNA chains and thus block its normal activity ("Cancers from A to Z" Ed. Frison Roche). Thus, anthracyclines, anticancer drugs of natural origin, isolated like antibiotics from microorganisms, are inserted between the nitrogen base pairs of DNA and inhibit the activity of topoisomerase II, the enzyme that controls spatial structure of DNA. These compounds also form oxygenated free radicals responsible for their toxicity. Among the anthracyclines currently used in chemotherapy, mention may be made of doxorubicin. The formation of DNA-topo-isomerase II-doxorubicin complexes which fix DNA breaks, block its functions and lead to cell death. However, anthracyclines, and more particularly doxorubicin also promote the formation of free radicals which can cut DNA and damage membranes, inducing toxicity of these compounds on the heart.

More recently, it has been suggested that the proteasome may play a role in the treatment of cancers (reviewed in Pajonk & Me Bride, 2001, Radiation Research, 156: 447-459). The proteasome is a multicatalytic complex presenting in particular multiple peptidasic activities. In general, the pepteas activities of the proteasome allow the degradation of denatured, abnormal, renewal proteins or proteins regulating essential cellular functions. The proteasome could also play a role in the transport of certain molecules since it has been suggested that the transport of doxorubicin from the cytoplasm to the cell nucleus could be carried out by the proteasome (reviewed in Pajonk & Me Bride, 2001, Radiation Research, 156: 447-459). Thus, the proteasome could be a target in chemotherapy (Almond & Cohen, Leukemia; 16 (4): 433-433, 2002), suggesting that compounds modulating the activity of the proteasome could also be used to fight cancer.

Among the compounds capable of modulating the activity of the proteasome, there may be mentioned in particular certain inhibitors of the protease of the human immunodeficiency virus (HIV). Application FR 98/07373, filed by the “Plaintiffs” describes in particular that ritonavir or saquinavir, compounds usually intended to fight against AIDS, are capable of modulating the activity of the proteasome, by inhibiting the activity of chymotrypsin type and by increasing the trypsin-like activity of the proteasome, and could therefore be used to fight against other diseases such as hepatitis C or cancer.

If these anticancer drugs act on cancer cells, they all remain highly toxic on certain non-cancer cells, making it difficult to administer very effective doses without causing excessive toxicity. Currently, to fight cancer more effectively, pharmaceutical compositions are used which concentrate on the tumor the effectiveness of several anticancer drugs while dispersing their different toxicities on various organs. However, patients may exhibit over time a certain tolerance towards these pharmaceutical compositions, which reduces their effectiveness. Some studies have been carried out in HIV positive patients developing parallel cancer. In particular, a study carried out on HIV positive patients developing non-Hodkin lymphoma, subjected to two treatments (a first antiretroviral treatment intended to fight against the HIV virus and a second treatment intended to reduce the lymphoma) did not show any influence of the first treatment on the effectiveness of the second treatment (Vaccher et al, 2001, Cancer, vol 91 n ° l pl55- 163). Similarly, Nannan Panday et al (cancer chemoth pharmacol, 1999, vol 43 pages 516-519) have shown, in an HIV positive patient developing Kaposi syndrome subjected to a first antiretroviral treatment intended to fight against the HIV virus, and a second treatment (based on paclitaxel) intended to combat Kaposi syndrome, than the pharmacokinetics of paclitaxel observed in this patient was comparable to that of non-HIV positive control patients, again suggesting that the first antiretroviral treatment would have no effect on the second treatment. Thus, the problem that the invention wishes to solve is to obtain a new pharmaceutical composition making it possible to synergistically increase the effectiveness of known anti-cancer compounds.

Surprisingly, the inventors have discovered that by associating at least two anticancer compounds, at least one of which modulates the activity of the proteasome, a synergy of these compounds is obtained on the inhibition of the proliferation of growing cells. The invention thus relates to the use of at least two anti-cancer compounds, at least one of which modulates the activity of the proteasome, for the manufacture of a medicament intended for treating cancer. The invention also relates to a pharmaceutical composition comprising at least two anti-cancer compounds, at least one of which modulates the activity of the proteasome, in combination with at least one pharmaceutically acceptable excipient, for the manufacture of a medicament intended for treating cancer.

Within the meaning of the invention, the term “anticancer compound” means a compound which interferes in metabolism and cell life, making it possible to inhibit tumor growth. This anti-cancer compound can in particular be an antimitotic compound, which hinders or prohibits the reproduction of cells and their division, by acting in particular at the time of mitosis by altering the structure of the spindle, which prevents the migration of chromosomes to each end of the dividing cell, thereby blocking mitosis to result soon after cell death. Mention may in particular be made of vegetable alkaloids, extracts of periwinkle (vinca alkaloids) or of yew (paclitaxel), vinblastine, vincristine, vinorulbine. This anticancer compound can also be an intercalator which is inserted between two nitrogen base pairs of DNA. Mention may in particular be made of anthracyclines, such as in particular doxorubicin, daunomycin.

At least one of the compounds used in the invention is a compound which modulates the activity of the proteasome. The term “compound which modulates the activity of the proteasome” is intended to mean a compound which modifies one or more enzymatic activities of the proteasome. Unlike proteasome inhibition, modulation of the proteasome is compatible with cell life. These compounds can in particular be those described in particular in WO 94/14436 and EP 432 695 such as in particular ritonavir or saquinavir, or their pharmaceutically acceptable salts, in particular saquinavir mesylate. It has now been found that the combination of a compound modulating the activity of the proteasome with at least one other anti-cancer compound makes it possible to considerably improve the results obtained for combating cancer.

According to the invention, two anti-cancer compounds will preferably be used, one modulating the activity of the proteasome, the other fighting against cancer according to another mode of action, such as for example an anti-mitotic compound, preferably a vinca alkaloid, such as vincristine, or an intercalator preferably belonging to the family of anthracyclines, such as doxorubicin.

As a compound modulating the activity of the proteasome, an HIV protease inhibitor compound such as ritonavir or saquinavir or their pharmaceutically acceptable salts will advantageously be used.

The combination of vincristine with ritonavir as well as the combination of doxorubicin with ritonavir are particularly preferred.

The synergistic effect of such associations has been highlighted, in particular by demonstrating their effect on the proliferation of colon adenocarcinoma cell lines LoVo and HT-29 and on the mitochondrial membrane permeability of HT-29 cells.

The tests used are described in the following examples, given by way of illustration and which are in no way limiting. They will allow a better understanding of the invention with reference to the attached figures. Figure 1 shows the effects of ritonavir on cell proliferation

LoVo in culture, in the presence or absence of vincristine.

Figure 2A shows the effects of ritonavir on the proliferation of HT-29 cells in culture, in the presence or absence of vincristine.

Figure 2B shows the effects of ritonavir on the proliferation of HT-29 cells in culture, in the presence or absence of doxorubicin.

Figure 3 shows the effects of ritonavir on the mitochondrial membrane permeability of HT-29 cells, in the presence or absence of vincristine. Example 1 Effect of Ritonavir and Vincristine on Cell Proliferation

LoVo in culture (figure 1)

In order to study the effects of a proteasome modulator compound on the action of vincristine, a growth test MTT (3- [4,5-Dimethylthiazol-2-yl] -2,5-diphenyl-tetrazolium bromide) cell is carried out on LoVo cells in culture (cell line of colon adenocarcinoma; ATCC n ° CCL-229).

For this, 20,000 cells are seeded in microplate wells, in 100 μl of Ham'S F12 culture medium (BioWest Cat N val-LOl 35-500), 2mM L- Glutamine (Gibco Cat N 25030-024), supplemented by 0 , 01M HEPES (Gibco Cat N 15630-056), 25μg / ml of Gentamycin (Gibco Cat N 15750-045) and 10% Fetal Calf Serum, in the presence or absence of 10μg / ml of Ritonavir (Norvir® oral solution Abbot Laboratories Limited Queenborough Kent), and cultivated in an oven at 37 ° C and 5% CO ₂ . After 18h, a solution of Vincristine Sulfate (ICN Cat N. 190687; final concentration in the culture medium: 0.8ng / ml) is added to the culture medium. After 54 hours of incubation, 20 μl of 5 mg / ml MTT solution (Sigma M2128 5 mg / ml) are added. The medium is then aspirated 1.5 hours after the addition of MTT. The colored product is then dissolved in lOOμl of 40mM HC1 isopropanol. The optical density of this colored product, proportional to the number of living cells present in the medium, is measured at 590 irai. The values indicated in FIG. 1 are averages of optical density, obtained on 3 measurements. Thus, ritonavir associated with vincristine strongly reduces the proliferation of LoVo cells in culture compared to the action of each of these two compounds present in isolation in the culture medium. Indeed, the theoretical value of optical density expected during the presence of the two compounds in the culture medium, calculated as being the sum of the decreases observed on the OD when adding vincristine or ritonavir to the culture medium , should be around 0.16 while the measured value is around 0.04. These results show that vincristine acts in synergy with ritonavir to inhibit the proliferation of LoVo cells in culture. Example 2 Effect of Ritonavir, Doxorubicin and Vincristine on the Proliferation of HT-29 Cells in Culture (FIGS. 2A and 2B)

A protocol comparable to that developed in Example 1 is carried out in order to study the effects of a proteasome-modulating compound on the action of two anticancer agents on another cell line, on HT-29 cells in culture (cell line colon adenocarcinoma; ATCC # HTB-38). For this, 20,000 cells are seeded in microplate wells, in 100 μl of McCoy's 5A culture medium with Glutamax (Gibco Cat N 36600-021), supplemented by 0.01M HEPES (Gibco Cat N 15630-056), 25 μg / ml of Gentamycin (Gibco Cat N 15750-045) and 10% of Fetal Calf Serum, in the presence or absence of 10 μg / ml of Ritonavir (Norvir® oral solution Abbot Laboratories Limited Queenborough Kent) and cultured in an oven at 37 ° C and 5% CO2. After 6 p.m., a solution of Vincristine Sulfate (ICN Cat N. 190687; final concentration in the culture medium: 0.8ng / ml) or Doxorubicin Hydrochloride (ICN Cat N. 159101; final concentration in the culture medium: 0.125 μg / ml) is added to the culture medium.

After 54 hours of incubation, 20 μl of 5 mg / ml MTT solution (Sigma M2128 5 mg / ml) are added. The medium is then aspirated 1.5 hours after the addition of MTT. The colored product is then dissolved in lOOμl of 40mM HC1 isopropanol. The optical density of this colored product, proportional to the number of living cells, is measured at 590 nm. The values indicated in FIGS. 2A and 2B are averages obtained over 3 measurements.

As shown in FIG. 2A, vincristine, added in isolation to the culture medium, decreases the proliferation of HT29 cells in culture. Surprisingly, this effect is greatly increased when vincristine acts in the presence of ritonavir in the culture medium. Indeed, the theoretical value of optical density expected during the presence of the two compounds in the culture medium is of the order of 0.52 while the measured value is of the order of 0.32. As shown in FIG. 2B, ritonavir associated with doxorubicin greatly reduces the proliferation of HT-29 cells in culture compared to the action of each of these two compounds present in isolation in the culture medium. Indeed, the theoretical value of optical density expected during the presence of the two compounds in the culture medium is of the order of 0.57 while the measured value is of the order of 0.32.

These results show that doxorubicin and vincristine act in synergy with ritonavir to decrease the proliferation of HT-29 cells in culture.

Example 3 Effect of Ritonavir and Vincristine on the Mitochondrial Membrane Permeability of HT-29 Cells in Culture (FIG. 3)

In order to determine the mitochondrial membrane permeability, representative of cell apoptosis, 10 ⁵ of HT-29 cells (colon adenocarcinoma cell line; ATCC n ° HTB-38) are seeded in 1 ml of McCoy's 5A culture medium with Glutamax (Gibco Cat N 36600-021), supplemented by 0.01M HEPES (Gibco Cat N 15630-056), 25μg / ml of Gentamycin (Gibco Cat N 15750-045) and 10% of Fetal Calf Serum, in the presence or in the absence of 10 μg / ml of Ritonavir (Norvir® oral solution Abbot Laboratories Limited Queenborough Kent) After 6 pm, a solution of Vincristine Sulfate (ICN Cat N. 190687; final concentration in the culture medium: 1 ng / ml) or Doxorubicin Hydrochloride (ICN Cat N. 159101; final concentration in the culture medium: 0.125 μg / ml) is added to the culture medium. After 48 hours of incubation, the cells are then harvested by trypsination (GibcoBRL Cn 25090-028) and incubate for 15 minutes at 37 ° C with 40 nM of 3,3'-dihexyloxacarbocyanine iodide (Dioc6, Sigma, 31,842-6). At the end of the incubation, propidium iodide (PI, Sigma P4170, 1 μg / ml) is added to the culture medium, and the cells are immediately analyzed by flow cytometry. Dioc6 weakly marks cells with a drop in mitochondrial membrane potential, compared to intact cells. PI strongly marks dead cells compared to living cells. The combination of these two markers thus makes it possible to distinguish 4 populations of cells:

• Living cells when strong Dioc6 labeling and negative PI labeling are observed • Necrotic cells when strong Dioc6 labeling and positive PI labeling are observed • Cells in early apoptosis when weak Dioc6 labeling and negative PI labeling are observed

• Cells in late apoptosis when a weak Dioc6 labeling and a positive PI labeling are observed The results, expressed as a percentage of weak Dioc6 - positive PIs are presented in FIG. 3. When ritonavir and vincristine act simultaneously, the cells exhibit a very high percentage (21.78%), suggesting the presence of cells in late apoptosis, compared to control cells (3.54%), cells cultured in the presence of ritonavir alone (7.63%) or vincristine alone ( 7.36%).

These results show that vincristine acts in synergy with ritonavir to increase apoptosis of HT-29 cells in culture.

These examples thus demonstrate the synergistic effects of ritonavir, vincristine and / or doxorubicin, on the inhibition of cell proliferation and on the increase in apoptosis of cells in culture.

Thus, the combinations according to the invention are particularly advantageous for their anticancer activity and could be used for the manufacture of anticancer drugs intended more particularly for non-HIV positive patients. The present invention also relates to a method of potentiating an anti-cancer compound, by association with a compound modulating the activity of the proteasome. This potentiating method can use any of the above-mentioned associations or combinations of anticancer agents, at least one of which modulates the activity of the proteasome. The drugs obtained according to the invention are intended in particular to fight against cancer, that is to say against all diseases due to an abnormal multiplication of cells, in particular without limitation, myelomas, lymphomas, leukemias, carcinomas of the kidney, of the brain, colon, prostate, rectum, pancreas, ovaries, lung, liver, breast, skin cancers chosen from keratinomas and carcinomas, melanomas. It is understood that the manufacture of a medicament intended to treat the human immunodeficiency virus (HIV) is in no way intended by the present invention. The combinations according to the invention can be formulated in pharmaceutical compositions, in combination with at least one pharmaceutically acceptable excipient well known to those skilled in the art.

In the pharmaceutical compositions according to the invention, for oral, sublingual, subcutaneous, intramuscular, intravenous, topical, intratracheal, rectal, transdermal administration, the anticancer compounds can be administered in unit administration forms or in mixture with conventional pharmaceutical carriers, and intended for oral administration, for example in the form of a tablet, a capsule, an oral solution, etc., or rectally, in the form of a suppository, parenterally, in particular in the form of an injectable solution, in particular intravenously, intradermally, subcutaneously, etc., according to conventional protocols well known to those skilled in the art. For topical application, anti-cancer compounds can be used in creams, ointments, lotions, eye drops. When preparing a solid composition in the form of tablets, the anti-cancer compounds are mixed with a pharmaceutically acceptable excipient also known as a pharmaceutical vehicle, such as gelatin, starch, lactose, magnesium stearate, talc, gum Arabic or the like. The tablets can be coated with sucrose, a cellulose derivative, or other suitable materials. They can also be treated in such a way that they have a prolonged or delayed activity and that they continuously release a predetermined quantity of anti-cancer compounds. A preparation in capsules can also be obtained by mixing the anti-cancer compounds with a diluent and by pouring the mixture into soft or hard capsules. A preparation can also be obtained in the form of a syrup or for administration in the form of drops, in which the anticancer compounds are present together with a sweetener, an antiseptic, such as in particular methylparaben and propylparaben, as well as an agent giving taste or a suitable color. The water-dispersible powders or granules may contain the anti-cancer compounds in admixture with dispersing agents or wetting agents, or suspending agents, well known to those skilled in the art. For parenteral administration, aqueous suspensions, saline solutions are used isotonic or sterile and injectable solutions which contain dispersing agents, pharmacologically compatible wetting agents, such as in particular propylene glycol or butylene glycol.

The medicament or the pharmaceutical composition according to the invention may also comprise an activating agent which induces the effects of a medication or reinforces or supplements the effects of the main medication, in particular by increasing the bioavailability of the main medication.

The dosage depends on the severity of the condition. In the case of a pharmaceutical composition comprising an antimitotic such as in particular vincristine, and a compound modulating the activity of the proteasome such as in particular ritonavir, the administration can in particular be administered from 1 to 3 times per day so as to administer a daily dosage of 1 to 3 mg, preferably 1.5 to 2.5 mg of antimitotic and 100 to 1500 mg, preferably 600 to 1200 mg of compound modifying the activity of the proteasome, in combination with a pharmaceutically acceptable excipient. In the case of a pharmaceutical composition comprising an intercalating agent such as in particular doxorubicin, and a compound modulating the activity of the proteasome such as in particular ritonavir, the administration can in particular be carried out from 1 to 3 times per day so as to administer a daily dosage of 20 to 60 mg, preferably 30 to 50 mg of intercalator and 100 to 1500 mg, preferably 600 to 1200 mg of proteasome activity modulating compound, in combination with a pharmaceutical excipient acceptable.

Claims

I - Use of at least two anti-cancer compounds, at least one of which modulates the activity of the proteasome, for the manufacture of a medicament intended for treating cancer, in particular colon cancer. 2 - Use according to claim 1 characterized in that two anti-cancer agents are used, one of which modulates the activity of the proteasome. 3 - Use according to claim 1 or 2 characterized in that one of the anticancer compounds is an anti-mitotic compound. 4 - Use according to claim 3 characterized in that the anti-mitotic compound is a vinca-alkaloids, such as vincristine. 5 - Use according to claim 1 or 2, characterized in that one of the anticancer compounds is an intercalant. 6 - Use according to claim 5 characterized in that the intercalant belongs to the family of anthracyclines, such as in particular doxorubicin. 7 - Use according to any one of claims 1 to 6 characterized in that the compound modulating the activity of the proteasome is a compound inhibiting the protease of the human immunodeficiency virus (HIV). 8 - Use according to claim 7 characterized in that the compound is ritonavir or saquinavir, or their pharmaceutically acceptable salts. 9 - Use according to claim 4 or 8, characterized in that vincristine is used in combination with ritonavir or one of its acceptable pharmaceutical salts. 10 - Use according to claim 6 or 8, characterized in that doxorubicin is used in combination with ritonavir or one of its pharmaceutically acceptable salts. II - Pharmaceutical composition comprising at least two anti-cancer compounds, at least one of which modulates the activity of the proteasome in combination with at least one pharmaceutically acceptable excipient. 12 - Composition according to claim 11, characterized in that two anti-cancer agents are used, one of which modulates the activity of the proteasome. 13 - Composition according to claim 11 or 12, characterized in that one of the anticancer compounds is an anti-mitotic compound. 14 - Composition according to claim 13, characterized in that the anti-mitotic compound is a vinca-alkaloid, such as in particular vincristine. 15 - Composition according to claim 11 or 12 characterized in that one of the anticancer compounds is an intercalant. 16 - Composition according to claim 15 characterized in that the intercalant belongs to the family of anthracyclines, such as in particular doxorubicin. 17 - Composition according to any one of claims 11 to 16 characterized in that the compound modulating the activity of the proteasome is a compound inhibiting the protease of the human immunodeficiency virus (HIV). 18 - Composition according to claim 17 characterized in that the compound is ritonavir or saquinavir, or their pharmaceutically acceptable salts. 19 - Composition according to claim 14 or 18, characterized in that it combines vincristine and ritonavir or one of its pharmaceutically acceptable salts. 20 - Composition according to claim 16 or 18, characterized in that it combines doxorubicin and ritonavir or one of its pharmaceutically acceptable salts. 21 - Composition according to any one of claims 11 to 20 for the manufacture of medicament intended to treat cancer, in particular colon cancer.