EP1276489A1

EP1276489A1 - Novel medicines based on sesquiterpene mixtures

Info

Publication number: EP1276489A1
Application number: EP01929759A
Authority: EP
Inventors: Odile Rougereau-Person; André Rougereau
Original assignee: Individual
Current assignee: Individual
Priority date: 2000-04-27
Filing date: 2001-04-27
Publication date: 2003-01-22
Also published as: AU2001256445A1; CA2407442A1; US20040072915A1; WO2001080868A1; FR2808196A1

Abstract

The invention concerns pharmaceutical compositions characterised in that they contain, in association with a pharmaceutically inert carrier, a therapeutically efficient amount of a mixture of farnesol α-bisabolol and/or geraniol and/or derivatives thereof, in particular functional derivatives such as their esters, for example methohexate, their ethers, in particular with hydroxylated compounds such as adriamycin, C1-C4 alkyl ethers, or glycosylated derivatives. The invention is useful in particular for preventing and treating cancers.

Description

NEW DRUGS BASED ON MIXTURES OF SESQUITERPENES

The invention relates to new drugs based on mixtures of sesquiterpenes and / or sesquiterpene derivatives.

Sesquiterpenes are formed from repetitions of C5 isoprenoid units and for many of them are active ingredients of natural essences.

Previous work by the inventors had shown the existence of derivatives of such compounds, more particularly geranyl-farnesyl, in the extraction products obtained from tumor masses (tumor source itself or metastatic source).

These results were closer to experimental data showing the value of isoprenoids C ₁ C 5 and n ₂ in the formation of protein derivatives linked to the tumor cell, such as ras protein 21. Thus, blocking the farnesylation of proteins could make it possible to transform the oncogenic activity of protein 21 ras and consequently to destroy the tumor cell by apoptosis.

The inventors have now found a synergistic effect of certain mixtures of sesquiterpenes of great interest in oncology, or derivatives of sesquiterpenes, for specific therapeutic indications. These therapeutic indications are linked to the specific accumulation of these sesquiterpenes in rapidly renewing tissue masses, of physiological or pathological type, such as tumor masses.

The invention therefore aims to provide new drugs to specific anti-tumor effect based on mixtures of sesquiterpenes and / or sesquiterpene derivatives. The term sesquiterpene, as used in the description and the claims, will denote either sesquiterpenes alone or their derivatives.

According to another aspect, it also relates to the use of these compositions for the preparation of medicaments for the prevention and / or the treatment of epithelial tissues or of connective tissues.

The pharmaceutical compositions according to the invention are characterized in that they contain, where appropriate in combination with a pharmaceutically inert vehicle, a therapeutically effective amount of a mixture of α-bisabolol and farnesol and / or geraniol and / or their derivatives, in particular functional derivatives such as their esters, for example with methohexate, their ethers, in particular with hydroxylated compounds such as adriamycin, alkyl ethers of 1 to 4 carbon atoms, or glycosyl derivatives. It will be noted with interest that the sesquiterpene derivatives with antimitotics potentiate the effects of the latter. Such sesquiterpene derivatives defined above with antimitotic agents are therefore targeted by the invention.

Cx-bisabolol is a cyclic sesquiterpene of formula:

Farnesol is a linear sesquiterpene corresponding to the formula: (CH j) - C - CH - (CH _C) "- C (CH ₃₎ = CH - _(CH:) ₂ - C (CH ₃₎ = CH -

CH OH.

Geraniol is also a linear sesquiterpene and corresponds to the formula:

(CH,) - C ≈ CH - (CH ~ (CH ₃ ) = CH - OH

Advantageous pharmaceutical compositions according to the invention contain, in their active principle, from 99 to 1% of α-bisabolol and, respectively, from 1 to 99% of farnesol and / or geraniol, and in particular from 90 to 10% of 'α-bisabolol and, respectively, from 10 to 90' .. of farnesol and / or geraniol, where appropriate in the form of their derivatives, as defined above.

In the mixtures formed from α-bisabolol and farnesol and / or geraniol, the α-bisabolol is advantageously present in proportions at least equal to those of farnesol, or higher.

The invention thus relates in particular to pharmaceutical compositions containing in their active principle 99 to 50% of α-bisabolol and, respectively 1 to 5 _. 0% farnesol, and in particular 90 to 50% α-bisabolol and, respectively, 10 to 50% farnesol, as with geraniol.

In the compositions containing, in admixture with the α-bisabolol, farnesol and geraniol, the geraniol is generally in lower proportions, or at most equal to those of farnesol. These sesquiterpenes are thus for example in particular in ratios of 35.35.30% to 40.20.20% or 20.40.40% respectively in farnesol, geraniol and α-bisabolol. The pharmaceutical compositions defined above can also contain one or more other linear or cyclic sesquiterpenes.

These are, for example, sesquiterpenes with a bisabolan nucleus such as the lanceol, or else with a guaiane nucleus, with the group of guaïanolides and derivatives with a patchoulane nucleus.

These compounds can represent from 1 to 5% in the compositions defined above, it being understood that their proportion is always lower than that of farnesol, geraniol and α-bisabolol or their derivatives.

The enantiomeric or diastereoisomeric forms of sesquiterpenes used in the mixtures of the invention also form part of the embodiments of the latter.

The levorotatory forms are particularly preferred for therapeutic applications, the other forms being more especially used as laboratory reagents.

It is known that the sesquiterpenes of the mixtures according to the invention are widely used in perfumery and are therefore readily available as commercial products. As mentioned above, they can also be obtained by synthesis or by extraction from plants.

By chemical synthesis, one or more of their -OH groups can be advantageously blocked by a protective group by operating according to conventional techniques. As a variant, or if appropriate in combination with the preceding arrangement, one or more of these -OH groups can be functionalized to confer particular properties on the compounds, and in particular to allow their use as prodrugs. Examples that may be mentioned are esters, in particular acetates, or esters with methohexate and, as indicated above, ethers, in particular with hydroxylated compounds such as adriamycin, alkyl ethers of 1 to 4 carbon atoms. , or glycosyl derivatives.

The toxicological studies of these compounds have made it possible to demonstrate their great safety. It should be noted in this regard that α-bisabolol responds to the GRAS system in the United States. The acute toxicity measurements in male and female mice, as well as in male and female rats (administered doses of 600, 800 and 1,200 g of sesquiterpene mixture per kg bodyweight) show that the LD ₅₀ is significantly higher than 5g / kg.

By oral route, the LD ₅₀ is thus 7.5 g / kg in the male mouse, greater than 9.75 g / kg in the male rat or the female rat, and 12 g / kg in the female mouse.

By ip route, the LD ₅ o measured is greater than lg / kg (1.25 g / kg in the male mouse, 1.3 g / kg in the female mouse and 1.80 g / kg in the female rat, 1 , 90 g / kg in male rats).

At the autopsy of the animals, no organ damage was observed.

Long-term nutritional toxicity studies have also been performed in rats, with monitoring of food intake, and evaluation of animal weight.

These are rats which received a normal diet over a period of 3, 12 and 24 months with the incorporation of 0.400 g of sesquiterpene-based composition as defined above per 100 g of food. The results of anatomical studies pathological did not identify any physiological or pathological abnormalities. In particular, no spontaneous tumor development was observed, making it possible to conclude that there is no long-term nutritional toxicity.

These advantageous properties make it possible to take full advantage of the synergistic effects, demonstrated in the examples, of the anti-tumor activities of the sesquiterpene mixtures defined above, in specific anti-tumor applications in humans and animals.

In these applications, the pharmaceutical compositions contain, where appropriate, active principles from other drugs. Mention will be made in particular of their association with the water-soluble and liposoluble vitamins, amino acids such as D-alanine and with anti-tumor agents. Examples of antimitotic agents which can be used are alkylating agents, nitrosoureas, organoplastins, antifolics, antipurics, antipyriids, topoisomerase inhibitors, spindle agents, and hormonal and non-hormonal cytostatic agents. These combinations may also include antibiotics such as penicillins, cephalosporins, beta-lactams, aminoglycosides, lincosamides, polymyxins, quinolones, nitroso-5-amidazoles, sulfonamides, teicoplanins, vincomycin (or any other antitumor agent active).

The pharmaceutical compositions of the invention will also be used with advantage in combination with compounds facilitating their assimilation, such as sugars such as glucose, proteins for example hydrolysates of animal proteins, in particular fish, or lipids, such as oils. food, such as rapeseed, sunflower or olive. It is also advantageous to combine, depending on the location of the tumor, a surgical treatment and / or a chemotherapy or radiotherapy therapy with the treatment with administration of compositions according to the invention.

The use of the various mixtures has the consequence of reducing the active doses in chemotherapy or in radiotherapy, and consequently of decreasing the more or less toxic iatrogenic side effects.

The pharmaceutical compositions of the invention can be administered in different forms. They can thus be administered orally, dermally, or injectable, subcutaneously, venously, or intramuscularly, or even rectally.

For oral administration, use is in particular of ampoules, tablets, pills, tablets, capsules, drops, syrup. These compositions advantageously contain from 7 to 25 g of active principle per intake unit, preferably from 5 to 10 g (corresponding to intakes ranging from 0.1 to 0.7 to 0.8 g / kg of body weight)

For administration by the cutaneous route, in the form of ointments, cream, gel or any other suitable topical dosage form, the doses are between 2 and 15 g in one or more applications.

Other forms of administration include solutions for injection by intravenous, subcutaneous or intramuscular route, prepared from solutions, sterile or sterilizable, and distributed in different packages, for example 10, 50, 100, 500 and 1000 ml. It can also be suspensions or emulsions.

These injectable forms contain, per dosage unit, from 10 to 50 g of active principle, preferably from 10 to 25 g.

For rectal administration, suppositories are used as dosage forms.

The study of the effects of the compositions according to the invention in normal rats has shown that the mixtures of sesquiterpenes of the active principles are fixed and accumulate in tissues with rapid cell renewal.

The compositions of the invention are therefore especially suitable for the treatment of any cancer: digestive, uterine, vaginal cancers, tumors of the pharynx and larynx, melanomas of the skin, pancreatic cancers, lymphoma, lungs, prostate, bladder, bone , etc.

The application of the pharmaceutical compositions of the invention to prevent any potential tumor development in the cancers listed above also falls within the scope of the invention.

As an indication, the dosage usable in humans, for the treatment of mammary or pulmonary tumors, corresponds to the following doses: 7 to 25 g / day (0.1 to 0.7 g / kg) of active ingredient at base of mixtures of sesquiterpenes, in one or more doses.

According to another aspect, the invention relates to the use of mixtures of sesquiterpenes defined above for the development of anti-tumor drugs. Eue also targets their use as laboratory reagents serving as a reference in studies of anti-tumor activities of products to be tested.

Other characteristics and advantages of the invention will appear in the examples which follow, with reference to FIGS. 1 to 6, which represent the effect, on cells in culture, respectively:

- Figures 1 to 3, of a mixture of farnesol and α-bisabolol on the one hand, of its constituents on the other hand, at 0.5mM (Figure 1) and 0.1mM (Figure 2, scale of 0 to 14000 and figure 3, scale from 0 to 400),

FIG. 4, of a mixture of farnesol and α-bisabolol at different concentrations, compared to farnesol alone,

FIG. 5, of a mixture of geraniol and α-bisabolol at different concentrations, compared to geraniol alone, and

- Figure 6, a farnesol / geraniol / α-bisabolol mixture at different concentrations.

Example 1 Comparative study of the activity on the MCF7 line of farnesol (F) on the one hand and of a mixture according to the invention of farnesol and α-bisabolol (F + B) on the other hand

The cells of the MCF7 line are rapidly growing breast adenocarcinoma cells.

The doses tested during these tests are 0.1.10 ^ό and 0, 5.10 ^" 'M.

1/100 dilutions are made extemporaneously in the RPMI alone and stirred so as to obtain a homogeneous emulsion at the time of use.

The average number of cells seeded is approximately 10 million for each of the trials.

First test: search for an effect on MCF7 cells at doses of 0.5 mM.

During dull ^®1 " ⁰ subculturing of the cells, were seeded:

- a box of MCF7 cells for culture,

- a box of control MCF7 cells,

- a dish with the same number of cells + (F), 0.5mM, - a dish with the same number of cells + (F + B), 0.5mM.

- a box with the same number of cells + B, 0.5 mM.

The boxes were placed in the oven, the caps unscrewed, the media changed every two days. On the eighth day after washing with 5 ml of PBS, the cells are detached with

PBS-EDTA, recovered using 10 ml of RPMI 10% FCS, then counted using a Malassez cell.

Second test: search for an effect on MCF7 cells at a dose of 0.1 lMM.

The procedure is as above, but by seeding with the _ _2 ^th transplanting of the cells and using doses of 0.1 mM

(F), (F + B) and (B).

Results

First test: Test at a dose of 0.5 mM (the results are given in FIG. 1) H

the cells of cells in culture have a homogeneous adherent bed of cells with a few dead cells, the medium is orange in color.

- the box containing F 0.5 mM shows few adherent cells and a large number of floating cells (dead), the medium is of purple hue.

- the box containing a 0.5 mM F + B mixture shows agglomerated cells, floating (dead) in large numbers, the medium is of purple hue. There are only rare adherent cells.

- the box containing B 0.5 mM shows few adherent cells and a large number of floating cells (dead), the medium is of purple hue. This aspect is comparable to that of culture with F alone.

After counting, the following results are obtained:

- subculture indicator: 12570.10 ³

- test indicator: 13500.10 ^J

- F only 0.5 mM: 370.10 ³

- mixture F + B 0.5 mM (50/50): 70.10 ³

- B 0.5 mM: 250.10 ³

These results are shown in Figure 1.

Second test: Test at a dose of 0.1 mM

- the dish of cells in culture (control) has a homogeneous adherent bed of cells with a few dead cells, the medium is of orange tint.

- the boxes containing B have a homogeneous adherent bed of cells with a few dead cells, the medium is orange in color. This aspect is comparable to that of the control box.

- the box containing only 0.1 mM F shows few adherent cells and a large number of floating cells (dead), the medium is of purple hue.

- the box containing a 0.1 mM F + B mixture shows few adherent cells and a large number of floating cells (dead), the medium is of purple hue.

After counting, the following results are obtained:

- test indicator: 12780.10 ³ - F only 0.1 mM: 385.10 ³

- mixture F + B 0.1 mM (50/50): 190.10 ³

- B 0.5 mM: 8930.10 ³

The results obtained demonstrate an alteration in the adhesion of MCF7 cells in culture by F alone, the mixture F + B, and B alone, at a dose of 0.5 mM. This alteration is found during the test carried out at a dose of 0.1 mM for F alone and the mixture. Concerning B, the alteration is very weak. The count shows a number of cells lower than controls, but the macroscopic appearance of the ^'culture dish was comparable to the control box.

During the two tests, the mixture F + B shows a more significant alteration than that obtained with F alone whatever the dose observed.

During these tests, the activity of the F + B mixture with respect to MCF7 cells of mammary adenocarcinoma in culture appears to be greater than that of F alone or of B alone at the same dose,

These results are shown in the graphs of Figures 2 and 3 with scales, respectively, from 0 to 14,000 and from 0 to 400.

Example 2 Comparative study of the activity on 5mF RIN cells of farnesol (F) or geraniol (G) on the one hand and of mixtures according to the invention, respectively, of farnesol and of α-bisabolol (F + B) or geraniol and α-bisabolol (G + B)

The operation is carried out under the conditions reported in Example 1. The results obtained are as follows:

a - Effect of F and F + B: product (0.1 mM) number of cells (xlO- *)

- F 0, ImM 70. 10 ³

- F + B (90/10) 62. 10 ³

- F + B (50/50) 60. 10 ³ - witness 12000. 10 ³

It will be noted on examining these results, also illustrated in FIG. 4, that the mixture according to the invention, at different doses, makes it possible to reduce the number of cells by at least 10% under the conditions of the test by compared to farnesol alone.

b - Effect of G and G + B produced (0.1 mM) number of cells (x10 ³ )

- G 6900. 10 ³ - G + B (90/10) 1240. 10 ³

- G + B (50/50) 245. 10 ³

- witness 11600. 10 ³

Here again, the advantageous effect of the mixtures according to the invention in particular in 50/50 proportions (see also Figure 5).

It will be noted with interest that during studies in cell culture, where pancreatic tumor cells have been associated with non-tumor pancreatic cells, the various compositions of farnesol, geraniol and α-bisabolol, have selectively destroyed only the pancreatic cells tumor.

c - Effect of F + G + B produced (0, lmM) number of cells (xlO ³ )

- F + G + B (35/35/30) 80. 10 ³

- F + G + B (40/20/40) 30. 10 ³ - F + G + B (20/40/40) 2. 10 ³

- witness 10850. 10 ³

These results demonstrate the antimitotic effect of the mixtures of the invention, which are particularly advantageous with ternary compositions rich in α-bisabolol.

Example 3 Application of the mixtures according to the invention to the treatment of patients suffering from cancers

Chop Treatments. : (over 1 week)

The patient is administered the following products:

- Oncovin ^R : 0.6 to 1.4 mg / m ²

(S)

- Adriamycin: 50 to 300 mg / day

- Endoxan ^® : 100 to 500 mg / m ² - Prednisone ^® : usual treatment

Coupled treatment: (over 1 week)

- Deticene ^® : 50 to 250 g / m ² - Doxorubicin ^® : 10 to 75 mg / nr

Short cycle: (3 to 4 days)

Zophrène ^® : 1 or tablet of 4 to 8 mg / d combined with: - Holoxan: 0.5 and 3 g / irr

- Cis-platinum: 10 to 120 mg / itr

- Pionalide ^® : 100 and 400mg / d

The beneficial effects of the mixtures according to the invention have been observed in the context of clinical trials carried out on cancer patients with their informed consent. These tests confirmed the non-toxicity of the mixtures over 6 months of treatment and showed the disappearance of bone metastases in 4 to 6 months (MRI analysis and CT scan).

We will for example report cases of recovery in 6 months, in six patients suffering, respectively, from generalized cancer and subjected to chemotherapy for 1 year, from cancer of the vocal cords, from breast cancer with bone metastases (at the end of the treatment, the analyzes carried out showed the absence of nodules and the disappearance of the metastases), a ly phoma, and a tumor of the bladder with metastases to the sacrum.

Claims

1. Pharmaceutical compositions, characterized in that they contain, where appropriate in association with a pharmaceutically inert vehicle, a therapeutically effective amount of a mixture of α-bisabolol and farnesol and / or geraniol and / or their derivatives, in particular of functional derivatives such as their esters, for example with methohexate, their ethers, in particular with hydroxylated compounds such as adriamycin, alkyl ethers of 1 to 4 carbon atoms, or glycosyl derivatives.

2. Pharmaceutical compositions according to claim 1, characterized in that they contain, in their active principle, from 99 to 1% of α-bisabolol and, respectively, from 1 to 99% of farnesol and / or geraniol, and in particular from 90 to 10% of α-bisabolol and, respectively, from 10 to 90% of farnesol and / or geraniol.

3. Pharmaceutical compositions according to claim 2, characterized in that they contain in their active principle a mixture of α-bisabolol and farnesol, _ the α-bisabolol being present in proportion at least equal to that of farnesol or higher .

4. Pharmaceutical compositions according to claim 3, characterized in that they contain in their active principle

99 to 50% of α-bisabolol and, respectively, 1 to 50% of farnesol, and in particular 90 to 50% of α-bisabolol and, respectively, 10 to 50% of farnesol and or of geraniol.

5. Pharmaceutical compositions according to claim 2, characterized in that the proportion of geraniol is lower, or at most equal to that of farnesol in the compositions containing these two sesquiterpenes.

6. Pharmaceutical compositions according to claim 5, characterized in that they contain farnesol, geraniol, and 1 'α-bisabolol in ratios of 35/35/30, or 40/20/40, or 20/40 / 40.

7. Pharmaceutical compositions according to any one of claims -1 to 6, characterized in that they also contain one or more linear or cyclic sesquiterpenes.

8. Pharmaceutical compositions according to any one of claims 1 to 7, in which said sesquiterpenes are in the form of derivatives with antimitotic agents.

9. Pharmaceutical compositions according to any one of claims 1 to 8, characterized in that the sesquiterpenes are in their enantiomeric and / or diastereoisomeric forms, and in particular in levorotatory form.

10. Pharmaceutical compositions according to any one of claims 1 to 9, characterized in that they can be administered by oral, cutaneous, injectable or, rectal route.

11. Pharmaceutical compositions according to claim 10, characterized in that they are intended for oral administration and are in the form of tablets, tablets, capsules, pills, drops, syrup and that they contain from 7 to 25 g of active ingredient per unit of intake, preferably from 5 to 10g.

12. Pharmaceutical compositions according to claim 10, characterized in that they are intended for administration by the cutaneous route, and are in the form of ointments, cream or gel, the doses being between 2 and 15 g, in one or more applications.

13. Pharmaceutical compositions according to claim 10, characterized in that they are injectable solutions, intravenously, subcutaneously or intramuscularly, these solutions advantageously containing per unit of intake from 7 to 50 of active principle, preferably from 10 to 25 g.

14. Pharmaceutical compositions according to claim 10, characterized in that they are intended for administration by the rectal route and are in the form of suppositories.

15. Pharmaceutical compositions according to any one of claims 1 to 14, used as anti-tumor drugs to treat any type of epithelial and connective tissue cancers.

16. Use of mixtures of sesquiterpenes defined in any one of claims 1 to 9, for the preparation of medicaments for treating any type of epithelial and connective tissue cancers.