FR2877224A1 - Use of composition containing eucalyptol, linalool, itaconic or methylsuccinic anhydride, terpinen-4-ol, alpha-terpineol, camphor, bornyl acetate, geranyl formate, geraniol and coumarin to treat cancer - Google Patents

Abstract

Composition comprising ethanol, ethyl acetate, eucalyptol, dipropylene glycol, linalool, linalool acetate, itaconic or methylsuccinic anhydride, terpinen-4-ol, alpha -terpineol, camphor, bornyl acetate, geranyl formate, geraniol or geranyl acetate, coumarin and diethyl phthalate is used to produce a pharmaceutical composition for treating cancer. ACTIVITY : Cytostatic. In a test, a 30% solution of a composition comprising ethanol (20%), ethyl acetate (1%), eucalyptol (1%), dipropylene glycol (20%), linalool (13%), linalool acetate (2%), itaconic or methylsuccinic anhydride (1%), terpinen-4-ol (5%), alpha -terpineol (5%), camphor (1%), bornyl acetate (4%), geranyl formate (2%), geraniol or geranyl acetate (5%), coumarin (1%) and diethyl phthalate (20%) exhibited an IC50 of 0.74% against proliferation of KB cells. MECHANISM OF ACTION : None given.

Description

The subject of the present invention is the use of a composition for the

  manufacture of a medicament for the treatment of cancers and more particularly for inhibiting tumor growth or the proliferation of metastases.

  The cells are constantly renewed, some of them die and are replaced by new ones.

  Cancer is characterized by uncontrolled growth of abnormal cells. They proliferate anarchically to form a tumor that invades and destroys the surrounding tissues. The tumor may be benign (ie, safe) or malignant (ie, dangerous, causing metastasis).

  The metastatic process involves a series of steps: after invasion of the surrounding tissue, cancer cells can enter the blood or lymphatic vessels and move with the circulation. They can stop in a new vascular bed, migrate into the environment of the new organ and then proliferate to form a micro metastasis. The latter will develop a new vascular network to grow.

  The causes of cancer can be of viral origin, genetic (dysfunction of certain genes), immune or hormonal. Other factors such as smoking or exposure to certain types of radiation also favor their appearance.

  The standard treatments for cancer are: surgery, radiotherapy, chemotherapy and / or hormone therapy. Other less demanding treatments for the patient may be considered to inhibit tumor growth or proliferation of metastases.

  Angiogenesis is a biological process of neovascularization that develops around the tumor, allowing the tumor to stock up and grow. More and more drugs include angiogenesis inhibitors forming a new type of treatment for cancer that specifically interrupts the formation and growth of new blood vessels and thereby cuts off the blood supply to asphyxiate the tumor.

  Gene therapy is also used to replace a defective gene, responsible for the formation of cancer cells, with an intact gene.

  Another therapy is to develop the immune system of the body to defend against the intrusion of cancer cells.

  The present invention therefore aims at providing another means for inhibiting tumor growth or the proliferation of metastases and in particular a composition containing only natural compounds or very low cost.

  The applicant has demonstrated that a composition comprising ethanol, ethyl acetate, eucalyptol, dipropylene glycol, linalool, linalool acetate, itaconic anhydride or methyl succinic acid, terpinene-4-ol, α-terpineol, camphor, bornyl acetate, geranyl formate, geraniol or geranyl acetate, coumarin, diethyl phthalate, surprising antiproliferative activity of cancer cells.

  The toxicological profile of these different compounds is not likely to induce a risk to human health and the formulation does not present an innovation likely to cause adverse reactions to humans. Moreover, it is not known to date of harmful interaction between these compounds.

  According to the invention, this composition is used for the manufacture of a pharmaceutical composition intended for the treatment of cancers and more particularly for inhibiting tumor growth or the proliferation of metastases in cancer patients.

  This composition is particularly suitable for application to a human patient, but it can also be used for other mammals.

  Preferably, the compounds are assayed in proportions close to the following values: ethanol 20%; ethyl acetate 1% eucalyptol 1%; dipropylene glycol 20%; linalool 13%; linalool acetate 2%; itaconic anhydride or methyl succinic 1%; terpinene-4-ol 5% a-terpineol 5%; camphor 1%; bornyl acetate 4%; Geranyl formate 2%; geraniol or geranyl acetate 5%; coumarin 1% diethyl phthalate 20%.

  The usable quantity depends on the desired effect and the composition can be used pure or diluted with a pharmaceutically acceptable vehicle, advantageously between 0.01 and 0.3%, preferably at 0.7%.

  Most of the compounds at the base of the composition can be distilled off from natural products, for example: ethanol (fermented grapes), eucalyptol (eucalyptus radiata), / ina / ol (occimum basilicum), linalool acetate (citrus aurantium) , terpinene-4-ol (malaleuca alternifolia), a-terpineol (citrus aurantifolia), camphor (rosmarinus officinalis), bornyl acetate (cistus ladaniferus), geranyl formate (pelargonium graveolens), geraniol (citrus lemonum), geranyl acetate (cymbopogon nardus), coumarin (Tonka bean). The other compounds can be obtained by simple and inexpensive manufacturing processes, for example, ethyl acetate (WO 01/46117A1), dipropylene glycol (US 3574772), itaconic anhydride (GB 854999), methyl succinic anhydride (FR 2735775). ), diethyl phthalate (US 2618651).

  According to one characteristic of the invention, these compounds are advantageously extracted by distillation from a mixture of iodized salt, lemon juice, fresh onion, citric acid and alcohol, assayed in the proportions of: % iodized salt, 15% lemon juice, 50% fresh onion, 5% citric acid, 20% alcohol.

  These different characteristics make it possible to obtain a composition containing only natural compounds of very low cost and easy to prepare.

  The routes of administration of the composition according to the present invention, in the form of an oral solution or in an appropriate medicinal form, will be established according to the pathology to be treated, for example by the oral route, locally by topical application to the skin. and mucous membranes or by intravenous, intramuscular, intraperitoneal or subcutaneous injection. These compositions may be in any of the pharmaceutical forms usually used in human medicine: tablets, capsules, drops, granules, injectables, ointments, creams or gels. Preferably, the composition is in the form of a solution for injection parenterally.

  The compounds of the invention may be conventionally associated with vehicles, carriers or excipients conventionally used in the medical and pharmaceutically acceptable medium: preservatives, antioxidants, solvents, stabilizers, etc.

  In addition, the composition may be used in combination with other anti-cancer agents such as antimicrotubule agents (vinblastine, vincristine, vindesine, etc.), alkylating agents (ifosfamide, lomustine, cyclophosphamide, chlorambucil, busulfan, .. .), anthracyclines (doxorubicin, epirubicin, losoxantrone, ...), antibiotic agents (bleomycin, mitomycin, etc.), platinum derivatives (cisplatin or carboplatin, etc.) or antiangiogenic agents ( platelet factor 4 + heparin, medroxyprogesterone, SCM-chitin, ...).

  As regards the administration concentrations, it will be necessary to adapt the concentration to the targeted pathology and possibly to the patient. Optimum concentrations are between 0.01 and 0.3% and will preferably be around 0.7%.

  The dosage will be adapted according to the concentration of the basic mixture, the pathology to be treated and the profile of the patient. Preferably, the solution is prepared in such a way as to inject from 0.01 to 1000 ml, said solution containing 0.7% of the composition according to the invention.

  Other characteristics and advantages of the present invention will emerge more clearly on reading the following examples, given by way of non-limiting indication and with reference to the appended drawings, in which: FIG. 1 shows the effects of a treatment using increasing concentrations of the applicant's composition (0.01 to 5% final) on the survival of the KB cell line. The effects are determined after 96h of treatment and revealed by an MTT test. The results are expressed as percentage of survival. Each value is the average of four determinations. (Predetermination experience).

  FIG. 2 shows the effects of treatment with increasing concentrations of the applicant's composition (0.01 to 5% final) on the survival of the KB cell line. The effects are determined after 96h of treatment and revealed by a MU test. The results are expressed as percentage of survival. Each value is the average of four determinations. (Determination experiments 1 to 3). FIG. 3 summarizes the effects of treatment with increasing concentrations of the applicant's composition (0.01 to 5% final) on the survival of the KB cell line. The effects are determined after 96h of treatment and revealed by a MU test. Three independent experiments were conducted.

  EXAMPLE 1 Determination of the concentrations of the composition according to the invention making it possible to obtain 50% inhibition of the proliferation (IC50) of the human cell line KB.

1. Materials and methods.

  The composition of the Applicant was provided as a solution (30% v / v in water).

  the compounds of the base composition are dosed in proportions close to the following values: ethanol 20%; ethyl acetate 1%; eucalyptol 1%; dipropylene glycol 20%; linalool 13%; linalool acetate 2%; itaconic anhydride or methyl succinic 1%; terpinene-4-ol 5%; a-terpineol 5%; camphor 1%; bornyl acetate 4%; Geranyl formate 2%; geraniol or geranyl acetate 5%; coumarin 1%; diethyl phthalate 20%.

  The culture medium was used as a vehicle.

  The KB cell line as well as the appropriate culture medium were provided by Oncodesign. The cells were cultured in monolayer in an oven at 37 C and in a humid atmosphere (5% CO2, 95% air). The culture medium was RPMI 1640 (Ref BE12-702F, Cambrex) containing 2 mM L-glutamine and supplemented with 10% fetal calf serum (Ref DE14-801E, Cambrex). For the experiments, the cells were detached from the support by a 10-minute incubation with a solution of trypsineversene (Ref 02-007E, Cambrex), diluted 10-fold in Hank's buffer containing neither calcium nor magnesium (Ref BE10-543F, Cambrex). The cells were enumerated using a haematimeter and their viability was assessed by a 0.25% trypan blue exclusion test.

  Detection of mycoplasmas was performed by the Mycotect test (Ref 15672-017, Invitrogen). Detection of mycoplasmas was performed in duplicate from culture supernatants and compared to positive and negative controls. No contamination by mycoplasmas has been demonstrated in line KB.

  2. Experimental design: study of cytotoxicity in vitro.

  KB cells (10,000 cells per well) were dispensed 24h in 96-well flat-bottomed microplates (Ref 055260, Nunc). The cells were incubated 24 h before treatment at 37 ° C. in 100 μl of culture medium containing no substance to be tested.

  at. STEP 1: Predetermination of ICso The KB cells were incubated for 96 hours with 5 different concentrations of the applicant's composition (between 0.01% and 5% in final concentration). The cells (100 μl) were incubated in a final volume of 200 μl of culture medium containing the applicant's composition. The experiment was carried out only once, each concentration being tested in quadruplicate, the control cells being incubated with culture medium without the composition of the Applicant. At the end of the incubation, the cytotoxic activity was evaluated by a MU test.

  b. STEP 2: Determination of ICso The KB cells were incubated for 96 hours with 10 different concentrations of the applicant's composition (between 0.01% and 5% in final concentration). The cells (100 μl) were incubated in a final volume of 200 μl of culture medium containing the composition of the applicant. The experiment was carried out three times, each concentration being tested in quadruplicate, the control cells being incubated with culture medium without the composition of the Applicant. At the end of the incubation, the cytotoxic activity is evaluated by a MU test.

  vs. MU Assay The in vitro cytotoxic activity of the applicant's composition was revealed by the MTT assay (CARMICHAEL et al., Cancer Res., 1987, 47: 936-942). Assuming that ELISA tests were to be performed, 50 μl of culture supernatants were taken from each well before revelation by the MU test and stored at -80 ° C. At the end of the incubation, 20 μl of a solution of tetrazolium salts at 5 mg / ml (Ref M2003, Sigma) diluted in phosphate buffer (Ref BE17-517Q, Cambrex) and filtered at 0.22 μm were added to each well. The microplates were incubated for 4 h at 37 ° C. The supernatants were then removed and the formazan crystals were solubilized with 200 μl of DMSO (Sigma). The absorbance (OD) at 570 nm was measured using a Multiskan spectrophotometer (Labsystem). The results were recorded using the Genesis software (Labsystem). The results are expressed as the concentration of the Applicant's composition required to inhibit 50% of cell growth (IC50).

3. Results.

  at. Determination of IC50 The dose-response curve of inhibition of proliferation (IC) is expressed as follows: OD wells exposed to the drug IC = X 100 OD wells without drug IC50: concentration of the composition of the applicant inhibiting 50 % of cell proliferation.

  The dose-response curve was calculated using the XLFit 3 (IDBS) software. The IC50 values corresponding to the pre-determination and determination steps were calculated using XLFit 3 software from semi-log curves.

  b. In vitro cytotoxicity study The result of the predetermination study is presented in figure 1, the results of the determination study are presented in figure 2. The table summarizing the IC50s determined during the determination study is presented in figure 3.

  The KB cell line is sensitive in vitro to the composition of the Applicant. The IC50 of the Applicant's composition on this line is 0.74 0.14%. This value is not significantly changed if the solution is filtered.

4. Conclusion.

  An antiproliferative activity of the Applicant's composition was observed on the KB cell line.

  A use for the manufacture of a pharmaceutical composition for inhibiting tumor growth or the proliferation of metastases in cancer patients can therefore be envisaged.

  EXAMPLE 2 Evaluation of the Tolerance of the Applicant's Composition in the Nude Mouse

1. Protocol.

  Three healthy Nude mice were injected with 100 μl of the applicant's composition at 5% for five consecutive days. The survival and weight of the animals were recorded twice a week. The animals were sacrificed 14 days after the last treatment and autopsies.

2. Results.

  No change in animal weight was observed following the subcutaneous injection of the Applicant's composition at 5% for five consecutive days. Nothing special was observed at the injection site.

  The animals were sacrificed 14 days after the last treatment. Nothing remarkable was observed during the autopsy of the animals.

3. Conclusion.

  The composition of the applicant is perfectly tolerated in the Nude mouse.

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Claims (9)

  1. Use of a composition comprising ethanol, ethyl acetate, eucalyptol, dipropylene glycol, linalool, linalool acetate, itaconic anhydride or methyl succinic anhydride, terpinene-4-ol, α-terpineol, camphor, bornyl acetate, geranyl formate, geraniol or geranyl acetate, coumarin, diethyl phthalate, for the manufacture of a pharmaceutical composition for the treatment of cancers.   2. Use according to claim 1 for the manufacture of a pharmaceutical composition for inhibiting tumor growth in cancer patients.   3. Use according to claim 1 for the manufacture of a pharmaceutical composition for inhibiting the proliferation of metastases in patients with cancer.   4. Use according to one of the preceding claims, characterized in that the compounds are assayed in the proportions of: 20% ethanol; ethyl acetate 1%; eucalyptol 1%; dipropylene glycol 20%; linalool 13%; linalool acetate 2%; itaconic anhydride or methyl succinic 1%; terpinene-4-ol 5%; a-terpineol 5%; camphor 1%; bornyl acetate 4%; Geranyl formate 2%; geraniol or geranyl acetate 5%; coumarin 1%; diethyl phthalate 20%.   5. Use according to one of the preceding claims, characterized in that the composition is diluted with a pharmaceutically acceptable vehicle between 0.01 and 0.3%.   - 11 - 6. Use according to claim 5, characterized in that the composition is diluted to about 0.7%.   7. Use according to one of the preceding claims, characterized in that the composition is used in combination with other anti-cancer agents such as antimicrotubules, alkylating agents, anthracyclines, antibiotic agents, platinum derivatives , anti-angiogenic agents.   8. Use according to one of the preceding claims, characterized in that the various components are extracted from a composition comprising iodized salt, lemon juice, fresh onion, citric acid and water. alcohol.   9. Use according to claim 7, characterized in that the components are dosed in the proportions of: 10% iodized salt, 15% lemon juice, 50% fresh onion, 5% citric acid, 20% alcohol.