Reverse micelle system comprising metal ions and use thereof

Abstract

The present invention relates to reverse micelle system based on sterols, acylglycerols, phospholipids or sphingolipids and metal ions. Reverse micelle system of the invention is able to cross mucosa and cellular membranes. It thus allows vectorization of metal ions to target sites. It is advantageously useful in the pharmaceutical and dietetic fields.

Claims

1. A reverse micelle system comprising 150-1500 g/ml of at least one metal ion, a sterol, an acylglycerol, lecithin, an alcohol and water, wherein the weight ratio of lecithin to acylglycerol is from 0.05 to 0.40.

2. The reverse micelle system according to claim 1, wherein the micelles have aqueous cores of: a) 4 nm; b) from 3 to 5 nm; c) from 3.5 to 5 nm; or d) from 3.7 to 4.5 nm.

3. The reverse micelle system according to claim 1, obtainable by the following method: (a) contacting (i) a sterol, (ii) an acylglycerol, (iii) lecithin, (iv) an alcohol, (v) water, and (vi) at least one metal ion, and (b) stirring the mixture obtained in (a) at 40 C. or less for a time sufficient to obtain formation of reverse micelles.

4. The reverse micelle system according to claim 1, wherein the weight ratio of sterol/acylglycerol ranges from 0.015 to 0.05 or from 0.03 to 0.04.

5. The reverse micelle system according to claim 3, wherein the stirring of step (b) is carried out at a temperature ranging from 15 C. to 40 C., from 25 C. to 40 C., or from 30 C. to 37 C.

6. The reverse micelle system according to claim 1, wherein the acylglycerol is selected from the group consisting of 1,2-diolein and 1-oleoyl-2-acetyl glycerol.

7. The reverse micelle system according to claim 1, wherein the sterol is sitosterol or cholesterol.

8. The reverse micelle system according to claim 1, wherein the metal ion is selected from the group consisting of lithium, zinc, niobium, vanadium, selenium, molybdenum, chromium, antimony, tin, gold, ruthenium, palladium, platinum, strontium, arsenic, manganese and mixtures thereof.

9. A pharmaceutical composition comprising a reverse micelle system according to claim 1 and at least a pharmaceutically acceptable carrier, excipient or support.

10. The pharmaceutical composition according to claim 9, wherein the metal ion is lithium.

11. The pharmaceutical composition according to claim 9, wherein the metal ion is vanadium.

12. The pharmaceutical composition according to claim 9, wherein the metal ion is strontium.

13. The pharmaceutical composition according to claim 9, wherein the metal ion is manganese.

14. The pharmaceutical composition according to claim 9, wherein the metal ions in the reverse micelle system cross the blood brain barrier.

15. A method for the delivery of at least one metal ion to a mammal, wherein said method comprises mucosal administration to said mammal of the pharmaceutical composition according to claim 9.

16. A method for the treatment or improvement of the symptoms of a disease or disorder selected from pathologies of the central nervous system (CNS), neurodegenerative diseases, autoimmune diseases, type 2 diabetes, insulin resistance, metabolic syndrome, cancers, respiratory tumors, acute promyelocytic leukemia, acquired immunodeficiency syndromes, osteoporosis, diseases linked to oxidative stress, bipolar disorders, depression, and inflammatory disorders, wherein said method comprises administration to a mammal in need of such treatment and/or improvement of the pharmaceutical composition according to claim 9.

17. The method according to claim 16, wherein the disease or disorder is selected from pathologies of the CNS and neurodegenerative diseases.

18. The method according to claim 16, wherein the disease or disorder is selected from type 2 diabetes, insulin resistance and metabolic syndrome.

19. The method according to claim 16, wherein the disease or disorder is osteoporosis.

20. The method according to claim 16, wherein the disease or disorder is selected from disorders linked to oxidative stress.

21. The method according to claim 16, wherein the disease or disorder is selected from genetic, tumoral, viral and degenerative diseases in the central nervous system.

22. The reverse micelle system according to claim 8, wherein the metal ion is arsenic.

23. The reverse micelle system according to claim 1, said reverse micelle system consisting of 150-1500 g/ml of at least one metal ion, a sterol, an acylglycerol, lecithin, an alcohol and water, wherein the weight ratio of lecithin to acylglycerol is from 0.05 to 0.40.

24. The reverse micelle system according to claim 1, wherein the water is present in an amount, by weight, ranging from 5 g to 15 g per 100 ml total volume of said system.

25. The reverse micelle system according to claim 1, wherein the alcohol is present in an amount, by weight, ranging from 5 g to 12 g per 100 ml total volume of said system.

26. The reverse micelle system according to claim 1, wherein the water is present in an amount, by weight, ranging from 5 g to 15 g per 100 ml total volume of said system and the alcohol is present in an amount, by weight, ranging from 5 g to 12 g per 100 ml total volume of said system.

27. The reverse micelle system of claim 1, wherein the system comprises 180 g/ml of at least one metal ion.

28. The reverse micelle system of claim 1, wherein the system comprises 300 g/ml of at least one metal ion.

29. The reverse micelle system of claim 1, wherein the system comprises 600 g/ml of at least one metal ion.

30. The pharmaceutical composition according to claim 10, wherein the composition comprises 150-1200 g/ml of lithium.

31. The pharmaceutical composition according to claim 30, wherein the composition comprises 600 g/ml of lithium.

32. The reverse micelle system of claim 1, wherein the acylglycerol has the following formula (I): ##STR00002## in which: R.sub.1 is an acyl residue of a linear or branched, saturated or unsaturated fatty acid having between 14 and 24 carbon atoms, a hydrogen atom, or a mono-, di- or tri-galactose or glucose; R.sub.2 is an acyl residue of a linear or branched, saturated or unsaturated fatty acid having between 2 and 18 carbon atoms; and R.sub.3 is an acyl residue of a linear or branched, saturated or unsaturated fatty acid having between 14 and 24 carbon atoms, or a hydrogen atom.

33. The reverse micelle system of claim 1, wherein the acylglycerol has the following formula (I): ##STR00003## in which: R.sub.1 is an acyl residue of a linear or branched, saturated or unsaturated fatty acid having between 14 and 24 carbon atoms or a hydrogen atom; R.sub.2 is an acyl residue of a linear or branched, saturated or unsaturated fatty acid having between 2 and 18 carbon atoms; and R.sub.3 is a hydrogen atom.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1: Evaluation of the impact of the rate of incorporated water in absence of lecithin on diffraction curves (FIG. 1a) and size (FIG. 1b) of reverse micelles.

(2) FIG. 2: Evaluation of the impact of the rate of incorporated water in presence of lecithin on diffraction curves (FIG. 2a) and size (FIG. 2b) of reverse micelles.

(3) FIG. 3: Evaluation of the impact of the sterol origin on diffraction curves of reverse micelles.

(4) FIG. 4: In vivo evaluation of lithium distribution in blood components after administration of lithium in reverse micelles formulated with or without lecithin.

(5) FIG. 5: In vivo evaluation of efficacy of lithium in reverse micelles on EAE clinical scores.

(6) FIG. 6: In vivo evaluation of efficacy of lithium in reverse micelles on EAE axonal loss.

(7) FIG. 7: In vivo evaluation of efficacy of lithium in reverse micelles on R6/2 motor coordination (rotarod test).

(8) FIG. 8: In vivo evaluation of efficacy of lithium in reverse micelles on R6/2 motor performances (swimming tank test).

(9) FIG. 9: In vivo evaluation of efficacy of lithium in reverse micelles on R6/2 cognitive performances (swimming tank test).

(10) FIG. 10: In vivo evaluation of efficacy of lithium in reverse micelles on survival of mice inoculated with prion.

(11) FIG. 11: In vivo evaluation of efficacy of lithium in reverse micelles on mice behavioural model of depression (tail suspension test).

EXAMPLES

Example 1

Evaluation of Water Incorporation Impact on Formation and Size of Reverse Micelles in Absence of Lecithin

(12) The aim of this study was to evaluate by X ray diffraction method and visual determination the impact of water content on the formation of thermodynamically stable microemulsions and the size of reverse micelles dispersed therein.

(13) 10 formulations of reverse micelles with different percentages of water were prepared according to the procedure below.

(14) 0.7 g of phytosterol were dissolved in 1.4 g of absolute ethanol by magnetic stirring at 300 r/min for 15 minutes at 37 C. Glycerol monooleate was added thereto and magnetic stirring was carried out at 500 r/min for 45 minutes at 37 C. Purified water was added to this oil mixture and stirred between at 700 rpm for 30 minutes at 37 C. to form empty reverse micelles.

(15) The different formulations are summarized in the table below.

(16) TABLE-US-00001 Sample Water content (%) Glycerol monooleate Water 1 1 25.8 g 0.3 g 2 2 25.5 g 0.6 g 3 3 25.2 g 0.9 g 4 4 24.9 g 1.2 g 5 5 24.6 g 1.5 g 6 6 24.3 g 1.8 g 7 7 24.0 g 2.1 g 8 8 23.7 g 2.4 g 9 9 23.4 g 2.7 g 10 10 23.1 g 3.0 g

(17) Empty reverse micelles were prepared by increasing quantity of water from 1% to 10% with increment of 1% (the percentage of water is expressed by weight of water/total volume of the composition, density of 0.94). The percentage of absolute ethanol (5%) and phytosterol (2.5%) (weight/total weight of the composition) were unchanged for all these products.

(18) The formation of thermodynamically stable microemulsions was evaluated by the visual determination of their limpidity.

(19) Lattice parameters are obtained by X-ray diffraction and they are assumed to correspond to the size of reverse micelles of the invention. Samples were introduced in 1.5 mm diameter glass capillaries and a transmission configuration was used. A cupper rotating anode X-Ray source (functioning at 4 kW) with a multilayer focusing Osmic monochromator giving high flux (10.sup.8 photons/sec) and punctual collimation were employed. An Image plate 2D detector was used. Diffraction curves were obtained giving diffracted intensity as a function of the wave vector q. Diffracted intensity was corrected by exposition time, transmission and intensity background coming from diffusion by an empty capillary. Reverse micelle sizes were calculated with the formula: d=2 /qmax (q max is the wave vector corresponding to the maximal diffracted intensity).

(20) Diffraction curve of 10 samples prepared according to above procedure are shown in FIG. 1a, which clearly demonstrates that between 1% and 6% of incorporated water, the qmax value decreases when the percentage of water increases. FIG. 1b shows that between 1% and 6% of incorporated water, the size of reverse micelles increases from 3.1 to 3.7 nm when the percentage of water increases. In contrast, from 7% of incorporated water, the size of reverse micelles stops increasing.

(21) Furthermore, the visual analysis shows that from 1 to 5% of incorporated water, the products are limpid. From 6% of water, the products become more and more turbid.

(22) These results clearly show that formulations formed in absence of lecithin are unstable over a certain amount of water (6%). They additionally show that the micelles formulated without lecithin cannot exceed a given size even when increasing the amount of water in the formulation.

Example 2

Evaluation of Water Incorporation Impact on Formation and Size of Reverse Micelles in Presence of Lecithin

(23) The aim of this study was to evaluate by X ray diffraction method and visual determination the impact of water content on the formation of thermodynamically stable microemulsions and the size of reverse micelles dispersed therein in presence of increasing rate of lecithin.

(24) 3 formulations of reverse micelles with different percentages of water and lecithin were prepared according to the procedure below.

(25) Commercially available lecithin was dissolved in 8.5 g absolute ethanol by magnetic stirring at 300 r/min for 10 minutes at room temperature. 2.3 g of phytosterol were added to the mixture and stirred in the same conditions. Glycerol monooleate was added thereto and magnetic stirring was carried out at 500 r/min for 45 minutes at 37 C. to form an oil mixture. Purified water was added to the oil mixture and stirred at room temperature by magnetic stirring at 700 r/min for 30 minutes to form empty reverse micelles.

(26) The different formulations are summarized in the table below.

(27) TABLE-US-00002 Glycerol Sample Lecithin monooleate Water Oil mixture 11 0 g (0%) 79.3 g 20.4 mg (4%) 453.9 mg 12 9.4 g (10%) 64.8 g 45.0 mg (9%) 433.8 mg 13 14.1 g (15%) 57.0 g 60.0 mg (12%) 423.3 mg

(28) Empty reverse micelles were prepared by varying quantity of water from 4% (sample 11) to 12% (sample 13) and lecithin from 0% (sample 11) to 15% (sample 13). Lecithin content is calculated from weight of lecithin/total weight of the composition and water content from weight of water/total volume of the composition (density of 0.94). The percentage of phytosterol was 2.5% (weight of phytosterol/total weight of the composition) and of absolute ethanol was 9% (weight of absolute ethanol/total weight of the composition) for all these samples.

(29) The formation of thermodynamically stable microemulsions was evaluated by the visual determination of their limpidity.

(30) The size of reverse micelles of these formulations was evaluated by X-ray diffraction experiments as described in example 1.

(31) Diffraction curves of samples 11, 12, and 13 are shown in FIG. 2a which clearly demonstrates that the diffracted intensity increases and the qmax value decreases when the percentage of lecithin increases from 0 to 15%. FIG. 2b demonstrates that the size of reverse micelles increases from 3.1 to 4.5 nm when the percentage of lecithin increases from 0 to 15%. The visual analysis shows that these formulations are limpid.

(32) Consequently, these experiments show that the addition of 15% of lecithin allows the formation of thermodynamically stable microemulsions with reverse micelle size of 4.5 nm and high percentages of water (12%). Addition of lecithin thus solves the drawbacks of reverse micelles formulated in absence of lecithin described in example 1.

Example 3

Reverse Micelles According to the Invention

(33) The aim of this study was to evaluate by X ray diffraction method and visual determination the stability of a microemulsion according to the invention and the size of reverse micelles dispersed therein.

(34) Sample A: 5.7 g of commercially available lecithin were dissolved in 8.5 g of absolute ethanol by magnetic stirring at 300 r/min for 10 minutes at room temperature. 2.4 g of phytosterol were added to the mixture and stirred in the same conditions. 68.6 g of glycerol monooleate were added thereto and magnetic stirring was carried out at 500 r/min for 1 hour at 37 C. 9.0 g of purified water containing 148.7 mg of vanadium sulfate (30.0 mg of metal vanadium) were added to this oil mixture and stirred at room temperature at 700 r/min for 15 minutes to form reverse micelles containing 300 g metal vanadium/ml (density of 0.94). The formulation contains 6% of lecithin.

(35) Lecithin content is calculated from weight of lecithin/total weight of the composition. The percentage of water was 9% (weight of water/total volume of the composition), of phytosterol was 2.5% (weight of phytosterol/total weight of the composition) and of absolute ethanol was 9% (weight of absolute ethanol/total weight of the composition).

(36) Visual analysis showed that the microemulsion formulated with 6% of lecithin was limpid. The size of reverse micelles of this formulation was determined by X-ray diffraction experiments as described in example 1 to be 4 nm.

Example 4

Evaluation of the Impact of Sterol Origin on Formation and Size of Reverse Micelles

(37) The aim of this study was to evaluate by X ray diffraction method and visual determination the impact of the origin of the sterol (cholesterol or phytosterol) incorporated in the formulation on the formation of thermodynamically stable microemulsions and the size of reverse micelles dispersed therein.

(38) 4 formulations of reverse micelles containing vanadium or empty with phytosterol or cholesterol were prepared according to the procedures below:

(39) Sample B: 84.5 g of lecithin were dissolved in 141.0 g of absolute ethanol by magnetic stirring at 150 r/min for 15 minutes at room temperature. 35.2 g of phytosterol were added to the mixture and stirred in the same conditions. 1074.3 g of glycerol monooleate were added thereto and magnetic stirring was carried out at 200 r/min for 1 h and 20 minutes at 37 C. 75.0 g of purified water containing 1.3 g of vanadium sulfate (0.27 g of metal vanadium) were added to this oil mixture and stirred at room temperature at 240 r/min for 15 minutes to form reverse micelles containing 180 g metal vanadium/ml (density of 0.94).

(40) Sample 14: 75.0 g of purified water were added to of the oil mixture prepared according to sample B and stirred at room temperature at 240 r/min for 15 minutes to form empty reverse micelles.

(41) Sample C: 1.7 g of lecithin were dissolved in 2.8 g of absolute ethanol by magnetic stirring at 300 r/min for 10 minutes at room temperature. 0.7 g of cholesterol were added to the mixture and stirred in the same conditions. 21.5 g of glycerol monooleate were added thereto and magnetic stirring was carried out at 500 r/min for 45 minutes at 37 C. 0.5 g of purified water containing 8.5 mg of vanadium sulphate (1.8 mg of vanadium metal) were added to 8.9 g of this oil mixture and stirred at room temperature at 700 r/min for 15 minutes to form reverse micelles containing 180 g metal vanadium/ml (density of 0.94).

(42) Sample 15: 0.5 g of purified water were added to 8.9 g of the oil mixture prepared according to sample C and stirred at room temperature at 700 r/min for 15 minutes to form empty reverse micelles.

(43) Reverse micelles containing 180 g metal vanadium/ml (samples B and C) or without metal (samples 14 and 15) were formulated with phytosterol or cholesterol according to the above procedures. The percentage of water was 5% (weight of water/total volume of the composition), that of sterol was 2.5% (weight of sterol/total weight of the composition), that of lecithin was 6% (weight of lecithin/total weight of the composition) and that of absolute ethanol was 10% (weight of absolute ethanol/total weight of the composition) for all these samples.

(44) The formation of thermodynamically stable microemulsions was evaluated by the visual determination of their limpidity.

(45) The size of reverse micelles of these formulations was evaluated by X-ray diffraction experiments as described in example 1.

(46) Diffraction curve of samples B, C, 14 and 15 are shown in FIG. 3 which demonstrates that the qmax values are the same for all the formulations. Then, the reverse micelle size is calculated at 3.3 nm. Furthermore, the visual observation of these products shows that all samples are limpid.

(47) These experiments show that neither the sterol origin (phytosterol or cholesterol) nor the addition of vanadium at 180 g/ml has an impact on the formation of thermodynamically stable microemulsions, nor on the size of reverse micelles dispersed therein.

Example 5

Evaluation of In Vivo Blood Repartition of Lithium Formulated in Reverse Micelles in Presence and Absence of Lecithin

(48) The aim of this study was to evaluate the repartition in blood components (plasma versus blood cells) of lithium formulated in 2 different reverse micelles formulations prepared according to procedures below (samples D and E) when delivered by rectal route.

(49) Sample D: 1.4 g of phytosterol were dissolved in 4.0 g of absolute ethanol by magnetic stirring at 300 r/min for 10 minutes at room temperature. 48.9 g of glycerol monooleate were added thereto and magnetic stirring was carried out at 500 r/min for 45 minutes at 37 C. 2.4 g of purified water containing 25.7 mg of lithium carbonate (4.8 mg of metal lithium) were added to this oil mixture and stirred at room temperature at 700 r/min for 15 minutes to form reverse micelles containing 80 g metal lithium/ml (density of 0.94).

(50) Sample E: 3.4 g of lecithin were dissolved in 5.1 g of absolute ethanol by magnetic stirring at 300 r/min for 10 minutes at room temperature. 1.5 g of phytosterol were added to the mixture and stirred in the same conditions. 41.1 g of glycerol monooleate were added thereto and magnetic stirring was carried out at 500 r/min for 45 minutes at 37 C. 5.4 g of purified water containing 65.6 mg of lithium citrate (4.8 mg of metal lithium) were added to this oil mixture and stirred at room temperature at 700 r/min for 15 minutes to form reverse micelles containing 80 g metal lithium/ml (density of 0.94).

(51) Administered Products: Sample D: reverse micelles prepared according to above procedure at 80 g metal lithium/ml, delivered at 1 ml/kg by rectal route Sample E: reverse micelles prepared according to above procedure at 80 g metal lithium/ml, delivered at 1 ml/kg by rectal route

(52) Spragues Dawley rats, 5 rats per group, were administered with 160 g metal lithium/kg (80 g/kg twice, 15 minutes interval) by rectal route with 2 formulations of lithium in reverse micelles (samples D and E). These products were administered slowly with a pipette into the lower rectum, immediately after the anal sphincter.

(53) Animals were sacrificed 2 hours after the last treatment and blood samples were collected by intra-cardiac puncture. Centrifugation was performed at 4500 r/m for 5 minutes to separate plasma from blood cells.

(54) Lithium concentration in plasma and blood cells was determined by Inductively Coupled Plasma Mass Spectrometry (ICPMS).

(55) Results are shown in FIG. 4 which demonstrates that the addition of lecithin in reverse micelles of sample E did not decrease the delivery of lithium and did not change the repartition of lithium between plasma and blood cells. Reverse micelles according to the invention are thus as efficient as those not containing lecithin for delivery of low quantities of metal ions. They additionally allow delivery of greater amounts of metal ions than reverse micelles without lecithin.

Example 6

Evaluation of In Vivo Efficacy of Reverse Micelles Formulated with Lithium in EAE Mice Model of Multiple Sclerosis

(56) Multiple Sclerosis is the most common autoimmune inflammatory disease in the CNS. It is characterized by immune mediated demyelinisation and neurodegeneration of the CNS.

(57) Experimental autoimmune encephalomyelitis (EAE) is a standard widely used experimental model of the clinical, immunological and neuropathological features of Multiple Sclerosis.

(58) The aim of this study was to evaluate the efficacy of a pre-treatment with reverse micelles formulated with lithium according to procedures below (samples F and 16) in a chronic EAE model induced in mice.

(59) Sample F: 3.4 g of lecithin were dissolved in 5.0 g absolute ethanol by magnetic stirring at 300 r/min for 10 minutes at room temperature. 1.4 g of phytosterol were added to the mixture and stirred in the same conditions. 41.2 g of glycerol monooleate were added thereto and magnetic stirring was carried out at 500 r/min for 45 minutes at 37 C. 5.4 g of water containing 32.7 mg of lithium citrate (2.4 mg of metal lithium) were added to this oil mixture and stirred at room temperature at 700 r/min for 15 minutes to form reverse micelles containing at 40 g metal lithium/ml (density of 0.94).

(60) Sample 16: 1.7 g of lecithin were dissolved in 2.5 g absolute ethanol by magnetic stirring at 300 r/min for 10 minutes at room temperature. 0.7 g of phytosterol were added to the mixture and stirred in the same conditions. 20.6 g of glycerol monooleate were added thereto and magnetic stirring was carried out at 500 r/min for 45 minutes at 37 C. 2.7 g of water were added to this oil mixture and stirred at room temperature at 700 r/min for 15 minutes to form empty reverse micelles.

(61) Administered Products: Sample F: reverse micelles prepared according to above procedure at 40 g metal lithium/ml, delivered at 1 ml/kg by rectal route from day 0 to day 50 Sample 16: empty reverse micelles prepared according to above procedure, delivered at 1 ml/kg by rectal route from day 0 to day 50

(62) To induce EAE, C57b1/6 were injected subcutaneously on days 0 and 7 with 150 g of MOG peptide emulsified in complete Freund's adjuvant (Difco laboratories) supplemented with Mycobacterium tuberculosis H37 RA (Difco laboratories) at a final concentration of 5 mg/ml. In addition, on days 0 and 2 post-immunization (p.i.), mice were given 500 ng pertussis toxin (Sigma Aldrich Corporation) intraperitonally.

(63) These mice (4 mice per group) were treated 5 days a week by rectal route with lithium formulated in reverse micelles (sample F) from day 0 to day 50 post immunization. Sample F at 40 g metal lithium/kg was delivered slowly with a pipette into the lower rectum, immediately after the anal sphincter. Mice treated with empty reverse micelles (sample 16) in the same conditions (4 mice per group) were used as control.

(64) Animals were weighed and scored for clinical signs of disease from day 10 post-immunization until day 50 (week-ends excluded). Clinical assessment of EAE was performed daily according to the following criteria: 0=no disease signs, 1=tail weakness, 2=tail paralysis, 3=incomplete paralysis of one or two hind legs, 4=complete hind limb paralysis, 5=moribund, 6=death.

(65) Animals were sacrificed on day 50 and spinal cords were harvested and frozen.

(66) Quantification of axonal loss was performed on frozen spinal cord after neurofilament immunostaining. Cryostated sections of 14 m were incubated firstly with an antibody directed against the 200 kDa neutrofilament protein (Serotec). They were incubated after with a secondary antibody labelled with biotin (Vector laboratories) and after with streptavidin-alexafluor (Molecular Probes). The axonal density was determined by visualisation of the neurofilament immunostaining using a fluorescent microscope (Zeiss Axioplan II).

(67) Results of clinical scores are shown in FIG. 5 which demonstrates that lithium in reverse micelles formulation containing lecithin (sample F) is effective at preventing the apparition of clinical signs during the acute phase of the disease and facilitates partial recovery during the chronic phase of the disease. In contrast empty reverse micelles are ineffective at decreasing clinical score (sample 16).

(68) These results are confirmed by immunostaining assays presented in FIG. 6 which demonstrates that only the treatment with lithium in reverse micelles (sample F) prevents the axonal loss in spinal cord of EAE induced mice.

Example 7

Evaluation of In Vivo Efficacy of Reverse Micelles Formulated with Lithium in the R6/2 Mice Model of Huntington Disease

(69) Huntington disease (HD) is an inherited fatal neurologic disorder caused by an expansion of a CAG repeat in exon 1 of the huntingtin gene. The selective loss of a subset of brain cells (neurons) involves psychiatric, motor and cognitive disturbances.

(70) R6/2 mice (Jackson Laboratory) are a transgenic model widely used as Huntington's disease model. They express human HD gene carrying approximately 120+/5 (CAG) repeat expansions. Transgenic mice exhibit a progressive neurological phenotype that mimics many of the features of HD, including deficits of motor coordination, altered locomotor activity, impaired cognitive performance and seizures.

(71) The aim of this study was to evaluate by a standardized battery of analysis the neuroprotective effect of lithium formulated in reverse micelles according to procedures below (samples G and H) in R6/2 mice compared to unformulated lithium administered by oral route. Motor coordination and balance were evaluated by rotarod test and swimming tank test. Cognitive performances were evaluated by swimming tank test.

(72) Sample G: 1.0 g of phytosterol were dissolved in 3.0 g of absolute ethanol by magnetic stirring at 300 r/min for 10 minutes at room temperature. 36.5 g of glycerol monooleate were added thereto and magnetic stirring was carried out at 500 r/min for 45 minutes at 37 C. 1.8 g of purified water containing 9.7 mg of lithium carbonate (1.8 mg of metal lithium) were added to this oil mixture and stirred at room temperature at 700 r/min for 15 minutes to form reverse micelles containing 40 g metal lithium/ml (density of 0.94).

(73) Sample H: 9.4 g of lecithin were dissolved in 8.5 g of absolute ethanol by magnetic stirring at 300 r/min for 10 minutes at room temperature. 2.3 g of phytosterol were added to the mixture and stirred in the same conditions. 64.8 g of glycerol monooleate were added thereto and magnetic stirring was carried out at 500 r/min for 45 minutes at 37 C. 4.0 g of purified water containing 24.3 mg of lithium citrate (1.8 mg of metal lithium) were added to 38.2 g of this oil mixture and stirred at room temperature at 700 r/min for 15 minutes to form reverse micelles containing 40 g metal lithium/ml (density of 0.94).

(74) Sample 17: 4.0 g of purified water were added to 38.2 g of the oil mixture prepared according to sample H and stirred at room temperature at 700 r/min for 15 minutes to form empty reverse micelles.

(75) Administered Products: Sample G: reverse micelles prepared according to procedure above at 40 g metal lithium/ml, delivered at 1 ml/kg/d by rectal route for at least 8 weeks Sample H: reverse micelles prepared according to procedure above at 40 g metal lithium/ml, delivered at 1 ml/kg/d by rectal route for at least 8 weeks Sample 17: empty reverse micelles prepared according to procedure above, delivered at 1 ml/kg by rectal route for at least 8 weeks Unformulated lithium: lithium in solution at 3.2 mg metal lithium/ml, delivered at 5 ml/kg/d by oral route (gavage) for at least 8 weeks

(76) As described below, R6/2 at 8-9 weeks old (apparition of the first clinical signs) were treated 5 days a week for at least 8 weeks with lithium in solution (group 4, 10 mice per group respectively) or formulated in reverse micelles (samples G and H). Lithium in solution was delivered by gavage at 16 mg metal lithium/kg using a plastic syringe fitted with a metal curved gavage tube. Samples G and H at 40 g metal lithium/kg were delivered slowly with a pipette into the lower rectum, immediately after the anal sphincter (groups 1 and 2, 10 and 9 mice per group). R6/2 mice and wild type mice treated in the same conditions with empty reverse micelles (sample 17) (group 3 and 5, 8 and 6 mice per group) were used as control.

(77) Rotarod tests were performed before and during the 4.sup.th and 6.sup.th week of treatment using a Rota Rod apparatus (UGO Basile 47600, rotating rod diameter 3 cm). After an acclimation test of 15 minutes, mice performed 2 trials at 5 rpm. The latency at which each mouse falls of the rod was recorded.

(78) Swimming tests were performed using a tank filled to a depth of 20 cm with water and a visible escape platform located at the end of the tank side. During the 4.sup.th week of treatment, the latency to reach the platform and the swimming speed were recorded 5 times per day during 5 consecutive days.

(79) Results of rotarod test are shown in FIG. 7 which clearly demonstrates that both formulations of metal lithium in reverse micelles (samples G and H) maintain motor coordination of R6/2 mice. Furthermore, the effect of sample H formulated with lecithin administered at a dosage of 40 g/kg was greater than those obtained with lithium by oral gavage at 16 mg/kg. In contrast, the treatment with reverse micelles containing no metal (sample 17) was ineffective at maintaining motor coordination of R6/2 mice.

(80) Results of swimming speed shown in FIG. 8 confirm that the R6/2 mice treated with lithium in both formulations of reverse micelles (samples G and H) have better motor performances than R6/2 mice treated with lithium by oral gavage at 16 mg/kg or with reverse micelles containing no metal (sample 17).

(81) Results of cognitive performance are shown in FIG. 9 which clearly demonstrate that both formulations of metal lithium in reverse micelles (samples G and H) maintain the capacity of R6/2 to learn a simple task: choose the good direction to reach the platform. In contrast, reverse micelles containing no metal (sample 17) are ineffective at maintaining cognitive performances.

(82) Reverse micelles according to the invention are thus as efficient as those not containing lecithin for delivery of low quantities of metal ions. They additionally allow delivery of greater amounts of metal ions than reverse micelles without lecithin.

(83) TABLE-US-00003 Delivered Delivered Mouse Animal dose volume strain Groups Number Treatment (g/kg/d) (ml/kg/d) R6/2 1 10 Sample G 40 1 2 9 Sample H 40 1 3 8 Sample 17 0 1 4 10 Lithium solution 16000 5 Wild 5 6 Sample 17 0 1 type

Example 8

Formulation of Reverse Micelles with High Concentration of Lithium

(84) The aim of this study was to formulate lithium in reverse micelles at 600 g/ml according to the invention.

(85) Sample I: 6.6 g of lecithin were dissolved in 5.9 g of absolute ethanol by magnetic stirring at 300 r/min for 10 minutes at room temperature. 1.6 g of phytosterol were added to the mixture and stirred in the same conditions. 45.3 g of glycerol monooleate were added thereto and magnetic stirring was carried out at 500 r/min for 45 minutes at 37 C. 6.3 g of purified water containing 567.7 mg of lithium citrate (41.9 mg of metal lithium) were added to this oil mixture and stirred at room temperature at 700 r/min for 15 minutes to form reverse micelles containing 600 g metal lithium/ml (density of 0.94).

(86) The formation of thermodynamically stable microemulsion according to the procedure above was evaluated by the visual determination of its limpidity after 3 months at 40 C.

(87) This experiment shows that the addition of lecithin at 10% allows the formation of a limpid thermodynamically stable microemulsion with 600 g/ml of metal lithium.

Example 9

Evaluation of In Vivo Efficacy of Reverse Micelles Formulated with Lithium in a Mice Model of Prion Disease

(88) Prion diseases or transmissible spongiform encephalopathies (TSEs) are a family of fatal and rare progressive neurodegenerative disorders caused by an infectious agent (Prion) composed of protein of misfolded form. This agent is able to induce abnormal folding of normal cellular prion proteins in the brain, leading to brain damage (neuronal loss, . . . ) and associated symptoms (dementia, . . . ).

(89) C57b1/6J mice intracerebrally inoculated with brain homogenates infected with ME7 prion typically die between 165 and 175 days after inoculation. The disease is characterised by vacuolisation and neuronal loss, typically with a bilateral, symmetrical distribution as well as asctrocyte proliferation and prion protein accumulation.

(90) The aim of this study was to evaluate the efficacy of treatment with reverse micelles formulated with lithium according to procedure below (sample J) on survival of mice infected with prion compared to unformulated lithium administered by oral route at higher dose.

(91) Sample J: 23.5 g of lecithin were dissolved in 21.2 g of absolute ethanol by magnetic stirring at 300 r/min for 15 minutes at room temperature. 5.9 g of phytosterol were added to the mixture and stirred in the same conditions. 162.0 g of glycerol monooleate were added thereto and magnetic stirring was carried out at 500 r/min for 45 minutes at 37 C. 10.8 g of purified water containing 259.0 mg of lithium citrate (19.2 mg of metal lithium) were added to 102.0 g of this oil mixture and stirred at room temperature at 700 r/min for 30 minutes to form reverse micelles containing 160 g metal lithium/ml (density of 0.94).

(92) Sample 18: 10.8 g of purified water were added to 102.0 g of the oil mixture prepared according to sample J and stirred at room temperature at 700 r/min for 30 minutes to form empty reverse micelles.

(93) Administered Products: Sample J: reverse micelles prepared according to procedure above at 160 g metal lithium/ml, delivered at 1 ml/kg/d by rectal route from 90 days after inoculation until death Sample 18: empty reverse micelles prepared according to procedure above, delivered at 1 ml/kg by rectal route from 90 days after inoculation until death Unformulated lithium: lithium in solution at 3.2 mg metal lithium/ml, delivered at 5 ml/kg/d by oral route (gavage) from 90 days after inoculation until death

(94) 20 l of brain homogenates with 1% of ME7 prion were inoculated under general anaesthesia in brain of 5 weeks old C57b1/6 mice to induce disease. These mice were treated 5 days a week (week-ends excluded) with lithium in solution (14 mice per group) or formulated in reverse micelles (sample J) (14 mice per group) from 90 days after inoculation (at the post symptomatic phase) until death. Lithium in solution was delivered by gavage at 16 mg metal lithium/kg using a plastic syringe fitted with a metal curved gavage tube. Sample J at 160 g metal lithium/kg was delivered slowly with a pipette into the lower rectum, immediately after the anal sphincter. Prion inoculated mice treated in the same conditions with empty reverse micelles (sample 18) (10 mice per group) were used as control.

(95) Results of survival are shown in FIG. 10 which clearly demonstrates that treatment with lithium in solution at 16 mg/kg or formulated in reverse micelles at 160 g/kg (sample J) increase survival of prion infected mice compared to the treatment with reverse micelles containing no metal (sample 18).

(96) This experiment shows that, with an equivalent efficacy, the formulation in reverse micelles according to the invention affords a lithium dose decrease by 100 times compared to the dose of lithium in solution.

Example 10

Evaluation of In Vivo Efficacy of Reverse Micelles Formulated with Lithium in a Behavioral Mice Model of Depression

(97) Bipolar disorder is a psychiatric disease describe as a mood disorder. It is defined by the alternation of mania and depression episodes. The treatment to stabilize mood is based on administration of high doses of lithium which has acute antimanic and antidepressant effects and long term prophylactic effects.

(98) The aim of this study was to evaluate the efficacy of a pre-treatment with reverse micelles formulated with lithium according to procedure below (sample K) on a standard widely used behavioural mice model of depression (tail suspension test) compared to unformulated lithium administered by oral route at higher dose.

(99) Sample K: 28.2 g of lecithin were dissolved in 24.5 g of absolute ethanol by magnetic stirring at 300 r/min for 10 minutes at room temperature. 7.0 g of phytosterol were added to the mixture and stirred in the same conditions. 194.4 g of glycerol monooleate were added thereto and magnetic stirring was carried out at 500 r/min for 45 minutes at 37 C. 7.2 g of purified water containing 161.6 mg of lithium citrate (12.0 mg of metal lithium) were added to 68.0 g of this oil mixture and stirred at room temperature at 700 r/min for 15 minutes to form reverse micelles containing 150 g metal lithium/ml (density of 0.94).

(100) Sample 19: 7.2 g of purified water were added to 68.0 g of the oil mixture prepared according to sample K and stirred at room temperature at 700 r/min for 15 minutes to form empty reverse micelles.

(101) Administered Products: Sample K: reverse micelles prepared according to procedure above at 150 g metal lithium/ml, delivered at 1 ml/kg/d by rectal route during 4 weeks before the test Sample 19: empty reverse micelles prepared according to procedure above, delivered at 1 ml/kg by rectal route during 4 weeks before the test Unformulated lithium: lithium in solution at 3.2 mg metal lithium/ml, delivered at 5 ml/kg/d by oral route (gavage) during 4 weeks before the test

(102) C57b1/6 mice were treated 5 days a week (week-ends excluded) during 4 weeks with lithium in solution (12 mice per group) or formulated in reverse micelles (sample K) (12 mice per group). Lithium in solution was delivered by gavage at 16 mg metal lithium/kg using a plastic syringe fitted with a metal curved gavage tube. Sample K at 150 g metal lithium/kg was delivered slowly with a pipette into the lower rectum, immediately after the anal sphincter. Mice treated in the same conditions with empty reverse micelles (sample 19) (12 mice per group) were used as control.

(103) After a 4-week treatment, mice were tails suspended during 6 min and depression-like behaviour was characterized by time spent to be immobile. The total time of immobility was recorded.

(104) Results of tail suspension test are shown in FIG. 11 which clearly demonstrates that times of immobility of mice treated with lithium in solution at 16 mg/kg or formulated in reverse micelles at 150 g/kg (sample K) are significantly lower than the time of immobility of mice treated with reverse micelles containing no metal (sample 19).

(105) This experiment shows that, the depression-like behaviour induced by tail suspension test is alleviated by both lithium treatment. Furthermore, the formulation of lithium in reverse micelles according to the invention affords a dose decrease by 100 times compared to the dose of lithium in solution.

Example 11

Formulation of Reverse Micelles with High Concentration of Lithium

(106) The aim of this study was to formulate lithium in reverse micelles at 1200 g/ml according to the invention.

(107) Sample L: 569.9 g of lecithin were dissolved in 342.1 g of absolute ethanol by magnetic stirring at 100 r/min for 15 minutes at room temperature. 95.2 g of phytosterol were added to the mixture and stirred in the same conditions. 2313.4 g of glycerol monooleate were added thereto and magnetic stirring was carried out at 200 r/min for 120 minutes at 37 C. 480.0 g of purified water containing 6478.1 mg of lithium citrate (480.0 mg of metal lithium) were added to this oil mixture and stirred at room temperature at 200 r/min for 15 minutes to form reverse micelles containing 1200 g metal lithium/ml (density of 0.95).

(108) The formation of thermodynamically stable microemulsion according to the procedure above was evaluated by the visual determination of its limpidity.

(109) This experiment shows that the addition of lecithin at 15% allows the formation of a limpid thermodynamically stable microemulsion with 1200 g/ml of metal lithium.