TREATMENT FOR NON-ALCOHOLIC FATTY LIVER DISEASES

Abstract

The present invention relates to the use of mixtures of vitamin E and polyunsaturated fatty acids (PUFAs) as agents for the prevention, control and/or treatment of conditions associated with excessive fat accumulation in the liver which is not caused by alcohol abuse. This includes prevention, control and/or treatment of non-alcoholic steatosis in the liver—known as non-alcoholic fatty liver disease (NAFLD)—and/or non-alcoholic steatohepatitis (NASH) in a subject in need thereof. In particular, the present invention relates to the use of such compounds comprising vitamin E and PUFAs as active ingredients in the manufacture of medicaments for the prevention, control and/or treatment of conditions related to NAFLD.

Claims

1. Combination of vitamin E and polyunsaturated fatty acid (PUFA) in the use of prevention, control or treatment of non-alcoholic fat accumulation in the liver in a subject in need thereof, preferably for prevention, control or treatment of non-alcoholic fatty liver disease or non-alcoholic steatohepatitis, wherein the ratio of vitamin E and polyunsaturated fatty acids is administered in the range of about 1:1 to about 1:5, or in the range of about 4:1 to about 20:1, wherein the vitamin E is calculated as α-tocopherol and the ratio is calculated as weight ratio.

2. Combination according to claim 1, wherein the vitamin E is selected from the group consisting of α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol and mixtures thereof, preferably α-tocopherol and/or γ-tocopherol.

3. Combination according to claim 1, wherein the polyunsaturated fatty acid is selected from the group consisting of ω-3, ω-6, ω-9 polyunsaturated fatty acids and oxylipins derived therefrom, preferably ω-6 or ω-3 PUFAs, more preferably selected from ω-3 α-linolenic acid, ω-3 eicosapentaenoic acid, ω-3 docosapentaenoic acid, ω-3 docosahexaenoic, ω-3 docosatetraenoic acid, ω-6 γ linolenic acid, ω-6 linoleic acid, ω-6 conjugated linoleic acid, ω-6 arachidonic acid, ω-6 docosapentaenoic acid, and combinations thereof.

4. Combination according to claim 1, wherein the ratio of vitamin E and polyunsaturated fatty acids is administered in the range of about 1:1 to about 1:5, or in the range of about 4:1 to about 20:1, wherein the vitamin E is calculated as α-tocopherol and the ratio is calculated as weight ratio.

5. Combination according to claim 1, wherein the amount of vitamin E is from about 5 to about 2000 mg per day and the amount of PUFA is from about 10 mg to about 4000 mg per day.

6. A composition substantially consisting of vitamin E and PUFA for prevention, control or treatment of non-alcoholic fatty liver disease or non-alcoholic steatohepatitis, wherein a combination according to claim 1 is used.

7. A composition with hepatoprotective effect in the form of a tablet, soft drink or gelatin capsule comprising a combination according to claim 1.

8. A process for the preparation of a tablet, soft drink or gelatin capsule having hepatoprotective effect, said process comprising the step of mixing a combination according to claim 1 with one or more of the following ingredients selected from water, vegetable oils, gelatin, lubricants, and/or cellulose.

9. A method for the treatment or prevention of diseases associated with non-alcoholic fat accumulation in the liver, which comprises administering to the subject in need thereof an effective amount of PUFA and vitamin E according to claim 1.

Description

EXAMPLES

Example 1: Synergistic Effect of Vitamin E and PUFA in Leucocytes

Experimental Procedures:

[0039] Leukocytes are obtained from healthy donors. Mononuclear cells (MNC) are purified by Ficoll-Isopaque gradient centrifugation. Cells (at 3-8×106 cells/mL) are cultured in phenol-red free RPMI 1640, supplemented with 0.25% FBS, 0.1 mM NEAA, 50 U/mL penicillin, 50 μg/mL streptomycin and 5×10-5 M 2-mercaptoethanol. Cells are stimulated with LPS (100 ng/mL) and IFN-γ (20 U/mL) for 2-24 h. Multiparametric kits for determination of cytokines and chemokines are purchased from BIO-RAD Laboratories (Hercules, Calif.) and used in the LiquiChip Workstation IS 200 (Qiagen, Hilden, Germany). The data are evaluated with the LiquiChip Analyser software (Qiagen).

[0040] The algorithm developed by Chou and Talalay is used to calculate synergistic effects (Chou, T-C. & Talalay, P. A simple Biol.Chem. 252, 6438-6442, 1977; Chou, T-C.& Talalay, P. Analysis of combined drug effects—A new look at a very old problem. Trends in Biological Sciences. November 1983, p 450-454, 1983). Interactions are quantified by the Combination Index (CI). Briefly, the % of inhibition of the concentration of each single substance alone or the mixture of both are determined. The affected fraction (fa) (values between 0 and 1) and unaffected fraction (fu) (1−fa), respectively, is calculated. For median-effect plots, log (fa/fu) is plotted against log (D), where D represents the concentration of each single compound alone or the mixture of both. Using CalcuSyn software (Biosoft, Ferguson, Mo.), which is based upon the method by Chou & Talalay, a CI is computed for every fraction affected: a CI<1 reflects synergistic inhibition of the respective inflammatory parameter; if CI=1 the substances have additive interactions; when CI>1 the interaction of substances reflects antagonism. It has also been observed that substances can have synergistic or antagonistic interactions at given concentrations or ratios, respectively (see e.g. Pappa et al. Quantitative combination effects between sulforaphane and 3,3′-generalized equation for the analysis of multiple inhibitions in Michaelis-Menten kinetic systems. J. diindolylmethane on proliferation of human colon cancer cells in vitro. Carcinogenesis 28, 1471-77, 2007).

Results:

[0041] Cells are stimulated with LPS, an pathogen-derived component that induces inflammatory response, which is reflected in the expression of inflammatory genes and the secretion of cytokines, interleukins and chemokines (Table 1). LPS induces a massive increase of the secretion of inflammatory mediators such as TNF-alpha. Concomitantly, inflammatory mediators are secreted. These parameters are modulated by pre-incubating cells with α-tocopherol or ω-3 PUFA prior to the stimulation with LPS. For instance, the pro-inflammatory cytokine TNF-γ is reduced by 29% and 64%, respectively (Table 1a). IL-1beta and CCL4/MIP-1b are also significantly reduced by the two substances (Tables 1b, 1c). Thus, the substances reduces the extent of the inflammatory stress in cells that respond to inflammatory stimuli.

[0042] When α-tocopherol and ω-3 PUFA s are combined at different concentrations and ratios, significant synergistic effects are observed in the inhibition of the production of the pro-inflammatory cytokine TNF-alpha, but also of the pro-inflammatory interleukin IL-1beta, as well as the chemokine CCL4 (see Tables 1a-c, right column). The synergistic effects are most prominent for TNF-alpha.

TABLE-US-00001 TABLE 1 Synergistic effects of α-tocopherol and ω-3 PUFA in the inflammatory response of leukocytes. 1a: synergism between AT and ω-3 PUFA in inhibiting TNF-alpha % p Com- pg/mL ± SD inhibition (vs bined Treatment TNF-alpha (vs LPS) LPS) index LPS 5290 ± 240 — — — AT 10 μM + LPS 5505 ± 177 −4 0.415 AT 50 μM + LPS 3755 ± 21  29 0.012 AT 200 μM + LPS − α 3870 ± 14  27 0.014 — DHA 10 μM + LPS 3190 ± 14  60 0.007 — DHA 20 μM + LPS 3400 ± 523 64 0.043 — DHA 50 μM + LPS 3105 ± 163 59 0.009 — (AT 10 + DHA 1810 ± 269 34 0.005 0.04 10) + LPS (AT 10 + DHA 2845 ± 516 54 0.026 0.06 20) + LPS (AT 10 + DHA 3025 ± 417 57 0.022 0.16 50) + LPS (AT 50 + DHA 1800 ± 14  34 0.002 0.18 10) + LPS (AT 200 + DHA 1705 ± 431 32 0.009 0.70 10) + LPS 1b: synergism between AT and ω-3 PUFA in inhibiting CCL4/MIP-1beta % p Com- pg/mL ± SD inhibition (vs bined Treatment CCL4 (vs LPS) LPS) index LPS 99000 ± 11341 — — — AT 10 μM + LPS 91400 ± 5657  7 0.485 AT 50 μM + LPS 83900 ± 3111  15 0.210 AT 200 μM + LPS − α 87950 ± 3165  11 0.315 — DHA 10 μM + LPS 79995 ± 3465  32 0.151 — DHA 20 μM + LPS 67000 ± 7523  62 0.079 — DHA 50 μM + LPS 37600 ± 778  62 0.024 — (AT 10 + DHA 61700 ± 6930  38 0.058 0.44 10) + LPS (AT 50 + DHA 67800 ± 283  32 0.062 0.56 10) + LPS (AT 200 + DHA 75359 ± 495  24 0.098 0.77 10) + LPS 1c: synergism between AT and ω-3 PUFA in inhibiting IL-1beta % p Com- pg/mL ± SD inhibition (vs bined Treatment IL-1beta (vs LPS) LPS) index LPS 6310 ± 170 — — — AT 10 μM + LPS 6770 ± 438 −7 0.301 AT 50 μM + LPS 5735 ± 134 9 0.064 AT 200 μM + LPS − α 5430 ± 113 14 0.026 — DHA 10 μM + LPS 5405 ± 742 14 0.235 — DHA 20 μM + LPS  4865 ± 1633 23 0.339 — DHA 50 μM + LPS 1440 ± 71  77 0.001 — (AT 10 + DHA 10) + LPS 3520 ± 71  44 0.002 0.41 (AT 10 + DHA 20) + LPS 3645 ± 884 42 0.053 0.83 (AT 10 + DHA 50) + LPS 796 ± 41 87 0.001 0.63 (AT 50 + DHA 10) + LPS 3570 ± 113 43 0.003 0.54 (AT 50 + DHA 50) + LPS 1013 ± 95  83 0.001 0.77 (AT 200 + DHA 10) + LPS 3455 ± 106 45 0.002 0.95 (AT 200 + DHA 50) + LPS 1080 ± 28  83 0.001 0.95 AT = α-tocopherol; for more details see text.

Example 2: Soft Gelatin Capsule

[0043] Soft gelatin capsules are prepared by conventional procedures providing a dose of vitamin E of 5 to 1000 mg (e.g. α-tocopherol), such as e.g. 50 mg, and at least one compound selected from the group of PUFAs as defined above of 10 to 1000 mg (e.g. DHA), such as e.g. 200 mg, wherein the amounts are the recommended daily doses. Other ingredients to be added: glycerol. Water, gelatine, vegetable oil.

Example 3: Hard Gelatin Capsule

[0044] Hard gelatin capsules are prepared by conventional procedures providing a dose of vitamin E of 5 to 1000 mg (e.g. α-tocopherol) and at least one compound selected from the group of PUFAs as defined above of 10 to 1000 mg (e.g. DHA), wherein the amounts are the recommended daily doses. Other ingredients to be added: fillers, such as, e.g., lactose or cellulose or cellulose derivatives q.s.; lubricant, such as, e.g., magnesium stearate if necessary (0.5%).

Example 4: Tablet

[0045] Tablets are prepared by conventional procedures providing as active ingredient 5 to 1000 mg of vitamin E (e.g. α-tocopherol), such as e.g. 20 mg, per tablet and at least one compound selected from the group of PUFAs as defined above of 10 to 1000 mg (e.g. DHA), and as excipients microcrystalline cellulose, silicone dioxide (SiO2), magnesium stearate, crospovidone NF (which is a disintegratent agent) ad 500 mg.

Example 5: Soft Drink

[0046] An orange juice drink colored with beta-Carotene 10% CWS and with vitamin E (e.g. α-tocopherol) and at least one compound selected from the group of PUFAs as defined above (e.g. DHA) may be prepared according to Table 2 and 3.

TABLE-US-00002 TABLE 2 Soft drink ingredients Sugar syrup 64° Brix 156.2 g  Sodium benzoate  0.2 g Ascorbic acid, fine powder  0.2 g Citric acid 50% w/w  5.0 g Pectin solution 2% w/w 10.0 g Vitamin E 2-500 mg  PUFA 5-1000 mg Juice compound (see Table 3) 30.0 g Water to 250.0 g 

[0047] First, sodium benzoate is dissolved in water whilst stirring. Stirring is continued and sugar syrup, ascorbic acid, citric acid, pectin solution and juice compound are added one after the other. Do not use a high speed mixer. The bottling syrup is diluted with (carbonated) water to one liter of beverage.

TABLE-US-00003 TABLE 3 Ingredients Juice compound Orange juice concentrate 65° Brix 483.3 g Lemon juice concentrate 45° Brix 173.3 g Oily orange flavor  5.0 g Beta-carotene 10% CWS as 10% stock solution  10.0 g Deionized water 328.4 g

[0048] For preparation of the juice compound, deionized water is first added to the juice concentrates with gently stirring to allow the juice concentrates to hydrate. Then, the oily flavor and beta-carotene 10% CWS stock solution are added and pre-emulsified in a rotor-stator-homogenizer. Homogenization is performed in a high-pressure homogenizer at 200 bar.

[0049] Typical serving of a soft drink can be 240 ml, with an amount in Vitamin E (e.g. α-tocopherol) which is about 5-100 mg/serving and an amount in PUFA (e.g. DHA) which is about 5-500 mg/serving

Example 6: Synergistic Effect of Vitamin E and PUFA in Kupffer Cells

[0050] The present study is extended to macrophages isolated from murine or human liver: Adherent cells (i.e. Kupffer cells) are obtained from liver tissue. Isolation and cultivation is done according to the method described by Li et al., Immonological Letters 158, 52-56, 2014 or according to any method known in the art. These cells are treated with compounds as described above (see Example 1). These cells will be activated with an inflammatory stimulus and the effect of substances and combination thereof on the reduction of the inflammatory response is determined. With this set-up, identical effects as those described for blood cell derived macrophages (see Ex. 1) are obtained.