WO2010136449A1

WO2010136449A1 - Akg composition and use thereof in the production of a drug

Info

Publication number: WO2010136449A1
Application number: PCT/EP2010/057150
Authority: WO
Inventors: Gildas Breton; Stéphane Lozachmeur; François Le Goffic; Alain Legrand; Anne Laure Schmid-Deniau
Original assignee: Polaris; Universite De Rennes 1
Priority date: 2009-05-26
Filing date: 2010-05-25
Publication date: 2010-12-02
Also published as: FR2945951A1; FR2945951B1

Abstract

The present invention relates to a composition of 1-O-alkylglycerol (AKG), characterised in that said composition comprises (in wt %): - at least 85% of a combination of AKG in C18:1 n-9 and C16:1; - less than 0.5% of AKG in C18:0; - less than 15% of AKG in C12:0 and C14:0. The invention also relates to the use of such a composition for the production of a drug for treating cancer.

Description

AKG composition and its use for the manufacture of a medicament

The present invention relates to a novel composition of 1-O-alkylglycerols. It also relates to a pharmaceutical preparation comprising such a composition, as well as the use of this composition for the manufacture of a medicament for the treatment of cancer. Shark liver oil is used ancestral medicine popular in the Scandinavian countries and Japan, for its fortifying properties, or healing. Its use has since been expanded and is now of growing interest. The chemical studies carried out on the shark liver oil made it possible to characterize there a certain type of ether lipids present in abundance, 1-0-alkylglycerols (AKG) in diacylated form: alkyldiacylglycerols (AKDAG).

In mammals and humans, AKDAGs are present in the bone marrow, spleen and liver. They are also present in smaller amounts in breast milk, and lymph nodes. But the most abundant and most used source of AKD AG is the liver oil of some sharks.

AKGs are composed of an aliphatic chain linked to a glycerol by an ether bond in position sn-1; this is called 1-O-alkylglycerol. The ether bond gives AKG high stability and makes them resistant to hydrolysis.

Below is the semi-developed formula of selachylic alcohol or AKG 18: 1 (S).

OH

Throughout the present application, AKG x: y is understood to be a 1-O-alkylglycerol having x carbon atoms in the O-alkyl chain and unsaturated (s). Thus, for the selachylic alcohol, eighteen carbon atoms and unsaturation are present. When this designation is followed by the designation "n-9", it means that the unsaturation is located on the ninth atom of carbon from the last carbon of the chain (on the seventh if one uses the designation "n-7", etc.).

The most common natural AKGs are as follows: AKG 12: 0 = 1-O-Dodecyl-sn-glycerol; AKG 14: 0 = 1-O-Tetradecyl-sn-glycerol;

AKG 16: 0 = 1-O-Hexadecyl-sn-glycerol = chemoalcohol; AKG 18: 0 = 1-O-octadecyl-sn-glycerol = batyl alcohol; AKG 16: 1 = 1-O- (Z) -9'-Hexadecenyl-sn-glycerol;

AKG 18: 1 n-9 = 1-O- (Z) -9'-octadecenyl-sn-glycerol = selachylic alcohol. Alkyldiacylglycerols are present in shark liver oil

(20% in an oil without squalene), as well as in chimera liver oil which contains 50%. Shark liver oil and chimera is already marketed as a dietary supplement.

The anti-tumor effect of alkylglycerols in general and of shark liver oil in particular is known.

The state of the art in the field can be illustrated by the documents:

- WO-2008/037538;

- WO-98/32447; - Pedrono F and garlic. (2004): "Natural alkylglycerols restricted growth and metastasis of grafted tumors in mice" and

- Brohult A (1986): "Reduced mortality in cancer patients after administration of alkoxyglycerols".

Reference may also be made to documents FR 3 809M, US 6177476, WO 98/32447 and to the article "KROTKIEWSKY M AND AL: Cytostatic and cytotoxic effects of alkylglycerols (Ecorner) MEDICAL SCIENCE MONITOR,

MEDISCIENCE PUBLIKACJE NAUKOWE, WARZAW, PL, vol. 9, no. 11, November 1, 2003 ".

The average composition of the purified fraction of natural AKG extracted from shark liver oil is as follows (percentages by weight):

- AKG 14: 0: 2 at 3%;

- AKG 16: 0: 11 at 16%;

AKG 16: 1: about 11%;

- AKG 18: 0: 3 at 6%; AKG C18: 1 n-9: about 60%;

AKG C18: 1 n-7: 7 to 10%. The present applicant has found that a particular composition of these basic AKGs, whether of natural or synthetic origin, has an amplified action for the treatment of cancer and that it acts more precisely, depending on the type of cells and the type of cancer, on: - the decrease in the number of metastases; the decrease of tumor growth; decrease in metastatic spread; the variation (decrease) in the weight of the spleen and the number of splenocytes; - and that it had an anti-angiogenesis effect.

Thus, the present invention relates in the first place to a composition of 1-O-alkylglycerol (AKG), characterized in that it consists of (percentages by weight):

at least 85% of a combination of AKG in Cl 8: 1 n-9 and C16: 1;

less than 0.5% of AKG in C 18: 0;

- less than 15% of AKG in C12: 0 and C14: 0.

The composition according to the invention is particularly active because it consists essentially of monounsaturated AKG, the other compounds being either inactive, or slightly active, or provided with a deleterious effect.

According to other advantageous and non-limiting characteristics of this composition:

it contains 90% AKG in C 18: 1 n-9 and C 16: 1;

the ratio C18: l / C16: 1 is between 1: 10 and 10: 1; said ratio is between 8: 1 and 9: 1.

said AKGs are of natural origin;

said AKGs come from shark liver oil;

said AKGs are synthetic AKGs, in their active form of the (S) enantiomer. The invention also relates to a pharmaceutical preparation comprising a composition as described above, the use of such a composition for the preparation of a medicament for the treatment of cancer, as well as its use as a dietary supplement or as a dietary food.

It also relates to a method of manufacturing a composition according to one of the preceding characteristics, from shark liver oil which consists in implementing the following steps: - desalinization of the oil;

transesterification of the oily residue and recovery of the fraction of interest;

- selective extraction of the fraction of interest; elimination of the esters of linear or branched C ₁ to C ₁₀ aliphatic alcohols by saponification;

separation of alkyl glycerols by urea

Other features and advantages of the present invention will appear on reading the following detailed description of some embodiments.

1. In vivo tests and results

Model: C57B1 / 6 female syngeneic mice, 6 to 8 weeks old, are grafted with LLC1 cancer cells (Lewis Lung

Carcinoma), at 1/10 ⁶ cells / 100 μl intramuscularly. They are tested in pet shops, with a normal cycle of light, at 20 ⁰ C, and a free access food consisting of a mixture of pellets + AKG + oils and vitamins (2g / d).

A control group receives olive oil. Groups consisting of 12 mice / group are formed. Nutritional treatment starts from the first day of the transplant and ends after 20 days.

The AKGs distributed are those constituting the purified fraction of natural AKG extracted from shark liver oil.

Analysis of the results After euthanasia of the mice, the lungs are removed and the metastases are counted in double blind under a binocular loupe.

The results are statistically treated with GraphPad software with the non-parametric Mann and Whitney t test because of the non-Gaussian distribution of the samples. The difference is considered significant when p <0.05.

^1st general study

The table below shows the effect of the purified fraction of AKG extracted from shark liver oil (dosed at 25 mg / day / mouse) on the number of lung metastases, determined at 21 days post transplant. Group treated with AKG vs. control group (olive oil).

Moreover, the results show that the AKGs administered at lower concentrations, ie between 12.5 and 18.75 mg / day / mouse, do not induce an effect either on tumor regression or on the reduction of pulmonary metastases.

It therefore appears that the dose of 25 mg natural AKG / mouse can not be reduced.

2nd study with a reconstituted AKG mixture The nutritional treatment based on synthetic AKG shows a more marked effect compared to the mixture with a significant reduction in the number of metastases, respectively 60% and 43% compared to the control group (olive ). These values can not be compared with the values obtained in the first general study because the protocol lasted 10 more days. But these results show that some components of the natural mixture have no effect or have adverse effects.

The tests described above are now renewed. However, this time, each AKG is tested individually (use of synthetic AKG).

Study of anticancer effects of synthetic AKG with saturated chain.

C 12: 0 18.7% decrease in tumor volume compared with the olive control group, no significant effect on the number of metastases.

C14: 0 no significant effect on tumor growth, number of metastases and metastatic spread.

C16: 0 significant decrease of 34.4% in the number of metastases compared to the control group olive, Cl 8: 0 no significant effect, tendency to increase the tumor volume and the number of metastases. Study of anticancer effects of unsaturated synthetic chain AKG.

These tests show the very clear efficiency of AKG C16: 1 and C18: 1 n-

9 compared to the olive control group, 25 mg / day / mouse.

Dose effect on pure AKGs:

Cl 8: 1 n-9 visible effect from 18.75 mg / d / mouse and plateau effect beyond 25 mg / d / mouse.

2. Effects of AKG on organ weights

The liver and spleen are organs whose weight can be modified in the presence of inflammation and / or treatment. Indeed, hepatosplenomegaly is one of the inflammatory symptoms due to the concomitant increase in the volume of the liver and spleen. The effects of AKG on these organs have therefore been observed.

Model: C57B1 / 6 syngeneic female mice aged 6 to 8 weeks are grafted with LLC1 (Lewis Lung Carcinoma) cancer cells at the rate of 1/10 ⁶ cells / 100 μl intramuscularly. They are raised in pet shops, with a normal cycle of light, at 20 ⁰ C, and a free access food consisting of a mixture of granules + AKG + oils and vitamins (2g / d).

Results analysis

After euthanasia of the mice, the rats and livers are removed and weighed. The results are statistically treated with GraphPad software with the non-parametric Mann and Whitney t test because of the non-Gaussian distribution of the samples. The difference is considered significant when p <0.05.

Study of the weight of the spleen

In the olive control group, the weight of the female rats tripled in grafted mice compared to healthy mice. For groups fed C16: 0 and Cl 8: 0, the weight of the female rats is almost identical to the control group. It is significantly greater in the Cl 8: 0 group.

On the other hand, we see a downward trend for the group fed with the purified AKG fraction extracted from shark liver oil. The effects are especially very significant for the groups fed on C16: 1 and C18: 1 n- 9 where the rats maintain a weight virtually identical to that of healthy mice. These results suggest that AKG C16: 1 and C18: 1 n-9 would have a significant effect in reducing spleen inflammation in cancer mice.

Study of the weight of the liver Concerning the study of the livers, we observe similar weights between the olive control group and the healthy mice. However, there was a significant decrease for groups fed C16: 1 and Cl 8: 1 n-9.

3. Results of the in vitro study

Tumor neoangiogenesis is a key step that allows the growth and development of the primary tumor (nutrient supply, development of a network that allows colonization of other tissues).

Model: HUVEC cells are isolated from human umbilical cords. The cells are cultured in endothelial cell growth medium supplemented with endothelial cell growth medium. They are cultured at 37 ° C in a humid atmosphere enriched with CO2.

The proliferation of HUVEC cells is studied in the presence or absence of AKG tested at different concentrations. The cells are labeled by the incorporation of tritiated thymidine, which makes it possible to measure the synthesis of DNA and therefore neoangiogenesis. AKG C16: 1 and C18: 1 very significantly inhibit the incorporation of [3H] -thymidine into HUVEC cells: respectively 2272 DPM against 8431 for the control, and 1647 DPM against 10279 for the control. In contrast, C12: 0 and C14: 0 have no significant effect at the same dose. AKG Cl 8: 0 tends to increase the incorporation of tritiated thymidine at the same dose.

4. Process for obtaining the specific AKG composition

Step 1: oil quenching

Desqualinization of the shark liver oil. This operation is carried out by short-path molecular distillation. The distillate obtained contains squalene (45% to 65%) having a purity greater than 99% while the residue (55% to 35%) is composed of triglycerides (75% to 80%), diacylated glycerol ethers ( 18% to 25%) as well as 5 to 10% of steroids, fatty acids and compounds of unknown structures. .

This desalination can also be achieved by other means such as supercritical CO2 extraction (Carlos F. Torres, Luis Vasquez, Francisco J. Senoras, Guillermo Reglero, J. Amer.Chem.Soc (2007) 84: 443-448). ).

Step 2: transesterification of the oily residue and recovery of the fraction of interest

Transesterification of the oily residue into butyl esters of fatty acids and glycerol ethers with n-butanol and potassium or sodium n-butoxide. After the usual treatments, namely acidification to pH 7, washing with pure water and evaporation of n-butanol at 50 ^° C. under 20mbars, the F1 fraction composed of: butyl esters of fatty acids (75 to 80%); alkyl glycerols (18 to 25%); sterols and minor derivatives (5 to 10%).

Step 3: Selective extraction of the fraction of interest F1 is selectively extracted with the mixture of heptane, methanol and water. Then the lower phase (methanol) is harvested. The upper phase is again extracted twice in succession with methanol and water. The methanolic phases are combined and washed with the same volume of water. The upper phase thus obtained is subjected to evaporation under vacuum (20mbars) under heat to remove methanol and water. The fraction F2 is then obtained composed of: butyric esters of fatty acids (11%); alkyl glycerols (78%); sterols and minority derivatives (11%).

Step 4: Elimination of the remaining butyl esters by saponification

In order to remove the remaining butyl esters, a saponification of F2 is carried out using potassium hydroxide. The medium is then extracted by a mixture

50/50 of ethyl ether and water. The upper phase is harvested and dried over sodium sulfate. The ethyl ether is evaporated. The F3 fraction composed of: butyl fatty acid esters (0%) is then obtained; alkyl glycerols (89%); cholesterol and minority derivatives (11%).

Step 5: Separation of alkylglvercols by urea

F3 is mixed with ethanol, methanol and urea. The mixture is heated to 70 ° C. and then the solution is allowed to return to room temperature for 4 h and then left at 4 ° C. for 18 h.

The solution is then filtered to collect: a chlatrate;

- the alcoholic phase; This is evaporated under vacuum.

The residue is taken up in 1N hydrochloric acid and ethyl acetate. After washing with 100 ml of water, the ethyl acetate is evaporated.

An oil is obtained whose alkyl glycerol composition is as follows (analysis by gas chromatography):

This process therefore makes it possible to obtain a particularly advantageous composition because it contains the two most active alkylglycerols (C 18: 1 n-9 and C16: 1) and contains the harmful alkylglycerol (C 18: 0) only in the form of trace. In step 2 described above for transesterification, n-butanol and n-butylate were used.

However, any other aliphatic alcohol having one to ten carbon atoms, linear or branched, and the corresponding ester could be used. Preferably, the C4 to C8 alcohols will be used. As the carbon chain of the alcohol elongates, the saturated AKGs tend to remain in the non-methanol phase, resulting in the formation of a final composition rich in unsaturated AKG.

It is of course possible to obtain a composition in accordance with the invention by synthesizing each of the AKGs mentioned above. Asymmetric synthesis:

Thus, to synthesize AKG Cl 8: 1 n-9 (selachyl alcohol) in its active form of the (S) enantiomer, the following steps can be carried out:

1) Oleic alcohol - oleyl paratoluene sulfonate also called: - (Z) -9octadecenyl p-toluene sulfonate; 2) oleyl sulfonate paratoluene + isopropylidene glycerol- (R) ->

(R) 1- [N - ((Z (-9-octadecenyl) -2,3-di-O-isopropylidene-sn-glycerol;

3) R) 1-O - ((Z (-9-octadecenyl) -2,3-di-O-isopropylidene glycerol + HCl - ^ selachylic alcohol (D)

Reference: Synthesis von Glycerylatherphosphatiden Georges Hirt, Herve Saroka, Wilhem Bannwarthund Richard Barner Helvetica Chimica Acta - Vol66.fasc.4 (1983) -Nrl20. Finally, it should be noted that the composition according to the invention can be used as a dietary supplement or dietary food for specialized medical purposes.

Claims

1. Composition of 1-O-alkylglycerol (AKG), characterized in that it consists of (percentages by weight): at least 85% of a combination of AKG C 18: 1 n-9 and d AKG C16: 1; less than 0.5% of AKG in C 18: 0;

- less than 15% of AKG in C12: 0 and C14: 0.

2. Composition according to claim 1, characterized in that it contains at least 90% AKG C 18: 1 n-9 and C16: 1.

3. Composition according to one of claims 1 and 2, characterized in that the ratio C18: 1 / C16: 1 is between 1: 10 and 10: 1.

4. Composition according to claim 3, characterized in that said ratio is between 8: 1 to 9: 1.

5. Composition according to any one of the preceding claims, characterized in that said AKG are of natural origin.

6. Composition according to claim 5, characterized in that said AKG come from shark liver oil.

7. Composition according to one of claims 1 to 4, characterized in that said AKG are synthetic AKG in their active form of the enantiomer (S).

8. Pharmaceutical preparation comprising a composition according to one of the preceding claims.

9. Use of a composition according to one of claims 1 to 7 for the manufacture of a medicament for the treatment of cancer, in particular to reduce the number of metastases, decrease tumor growth, reduce metastatic spread, reduce weight spleen, or anti-angiogenesis effect.

10. Use of a composition according to one of claims 1 to 7 as a dietary supplement or dietary food.

11. A method of manufacturing an AKG composition, from shark liver oil, characterized in that it consists in implementing the following steps:

- desalinization of the oil;

transesterification of the oily residue and recovery of the fraction of interest; - selective extraction of the fraction of interest;

elimination of the esters of linear or branched C ₁ to C ₁₀ aliphatic alcohols by saponification;

separation of alkyl glycerols by urea; until a composition consisting of (percentages by weight) is obtained: at least 85% of a combination of AKG in Cl 8: 1 n-9 and AKG C16: 1;

less than 0.5% of AKG in C18: 0;

- less than 15% of AKG in C12: 0 and C14: 0.