Composition comprising alpha-lipoic acid and honokiol for treating neuropathies

Abstract

The present invention relates to a composition comprising alfa-lipoic acid, or a salt or complex thereof, and honokiol, wherein the weight quantity of said honokiol is between 1% and 30% with respect to the total weight of honokiol and alfa-lipoic acid.

Claims

1. A composition comprising alpha-lipoic acid, or a salt or complex thereof, and honokiol, in an amount sufficient to promote neurite extension and neuronal cell survival, wherein the weight amount of said honokiol is 1% to 30% with respect to the total weight of honokiol and alpha-lipoic acid in the composition.

2. The composition according to claim 1, wherein the weight amount of said honokiol is from 2% to 15% with respect to the total weight of honokiol and alpha-lipoic acid.

3. The composition according to claim 2, wherein the weight amount of said honokiol is from 3% to 10% with respect to the total weight of honokiol and alpha-lipoic acid.

4. The composition according to claim 1, further comprising gamma-linolenic acid or a salt or complex thereof.

5. The composition according to claim 4, wherein the amount of said gamma-linolenic acid is from 2% to 30% with respect to the total weight of honokiol and alpha-lipoic acid.

6. The composition according to claim 3, further comprising a physiologically acceptable source of selenium.

7. The composition according to claim 1, further comprising at least one component selected from the group consisting of vitamin C, vitamin E, and B vitamins.

8. A treatment of peripheral neuropathies, comprising administering an effective amount of a composition of claim 1 to a person in need thereof.

9. A dietary or pharmaceutical formulation for oral administration comprising a composition according to claim 1.

10. The formulation according to claim 9, in the form of tablets or capsules suitable for the daily administration of an amount from 200 mg to 1300 mg of alpha-lipoic acid and from 2.5 mg to 150 mg of honokiol.

11. The formulation according to claim 10 in the form of tablets or capsules suitable for the daily administration of an amount from 580 mg to 620 mg of alpha-lipoic acid and from 45 mg to 60 mg of honokiol, or suitable for the daily administration of an amount from 280 mg to 320 mg of alpha-lipoic acid and from 20 mg to 30 mg of honokiol.

12. The formulation according to claim 10, in the form of soft gelatin capsules suitable for the daily administration of an amount from 200 mg to 1300 mg of alpha-lipoic acid, from 2.5 mg to 150 mg of honokiol and from 10 mg to 300 mg of gamma-linolenic acid or a salt or complex thereof.

13. The formulation according to claim 11, in the form of soft gelatin capsules suitable for the daily administration of an amount from 580 mg to 620 mg of alpha-lipoic acid, of an amount from 45 mg to 60 mg of honokiol and from 125 mg to 140 mg of gamma-linolenic acid or suitable for the daily administration of an amount from 280 mg to 320 mg of alpha-lipoic acid, of an amount from 20 mg to 30 mg of honokiol and from 60 mg to 70 mg of gamma-linolenic acid.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The advantages of the compositions and formulations according to the present invention will become apparent to those skilled in the art from the following examples, with reference to the accompanying drawings in which:

(2) FIG. 1a shows a photograph of a primary cell culture of rat cortical neurons after 7 days in 0.5% EtOH;

(3) FIG. 1b shows a photograph of a primary cell culture of rat cortical neurons after 7 days in 0.5% EtOH additioned with 40 ng ml-1 bFGF;

(4) FIG. 1c shows a photograph of a primary cell culture of rat cortical neurons after 7 days in 0.5% EtOH additioned with a 10 M solution of 99.7% alpha-lipoic acid and 0.3% honokiol;

(5) FIG. 1d shows a photograph of a primary cell culture of rat cortical neurons after 7 days in 0.5% EtOH additioned with a 10 M solution of 95.5% alpha-lipoic acid and 4.5% honokiol;

(6) FIG. 1e shows a photograph of a primary cell culture of rat cortical neurons after 7 days in 0.5% EtOH additioned with a 10 M solution of 91% alpha-lipoic acid and 9% honokiol;

(7) FIG. 1f shows a photograph of a primary cell culture of rat cortical neurons after 7 days in 0.5% EtOH additioned with a 10 M solution of 46% alpha-lipoic acid and 54% honokiol;

(8) FIG. 1g shows a photograph of a primary cell culture of rat cortical neurons after 7 days of 0.5% EtOH additioned with a 10 M solution of alpha-lipoic acid alone;

(9) FIG. 1h shows a photograph of a primary cell culture of rat cortical neurons after 7 days of 0.5% EtOH additioned with a 10 M solution of honokiol alone;

(10) FIG. 2a shows a photograph of a primary cell culture of rat cortical neurons after 4 days in 0.5% EtOH;

(11) FIG. 2b shows a photograph of a primary cell culture of rat cortical neurons after 4 days in 0.5% EtOH additioned with a 10 M solution of 99.7% alpha-lipoic acid and 0.3% honokiol;

(12) FIG. 2c shows a photograph of a primary cell culture of rat cortical neurons after 4 days in 0.5% EtOH additioned with a 10 M solution of 91% alpha-lipoic acid and 9% honokiol;

(13) FIG. 2d shows a photograph of a primary cell culture of rat cortical neurons after 4 days in 0.5% EtOH additioned with a 10 M solution of 46% alpha-lipoic acid and 54% honokiol.

EXAMPLES

(14) Materials and Methods

(15) The purity of the used compounds was verified by high performance liquid chromatography (HPLC, single peak), by nuclear magnetic resonance (H.sup.1) and (C.sup.13) and by high resolution mass spectrometry.

(16) The culture medium was Dulbecco's modified culture medium (DMEM), fetal bovine serum (FBS) and B27 supplement were obtained from Gibco BRL (NY, USA). The reagents PD98059 [2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one], LY294002 [2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4one], and KN93 [N-[2-[N-(4-chlorocinnamyl)-N-methylaminomethyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide phosphate salt] were purchased from Sigma (MO, USA). Human recombinant fibroblast growth factors (bFGF) were supplied by Upstate Biotechnology Inc. (NY, USA). All other used reagents are reagents of highest purity available on the market.

Example 1

Morphological

(17) Eight primary cellular cultures were prepared as described in Abe K et al. Effects of recombinant human basic fibroblast growth factor and its modified protein CS23 on survival of primary cultured neurons from various regions of fetal rat brain., Jpn J Pharmacol 1990, 53 (2): 221-7. All operations were carried out under sterile conditions.

(18) Neuronal cells were separated from cerebral hemispheres of rat fetuses of 18 days (Japan SLC, Inc.) and suspended in 10% FBS/MEM, then seeded at 9000 cells cm.sup.2 in poly-L-lysine plates.

(19) After 24 hours, the culture medium was replaced with a serum-free medium, specific for the growth of neurons (Neurobasal Medium) supplemented with B27.

(20) Then, a first culture (A) was used as the control culture.

(21) A second culture (B) was additioned with 40 ng mL.sup.1 of bFGF.

(22) A third culture (C) was additioned with 99.7% by weight alpha-lipoic acid and 0.3% by weight honokiol to the overall concentration of 10 M.

(23) A fourth culture (D) was additioned with 95.5% by weight alpha-lipoic acid and 4.5% by weight honokiol to the overall concentration of 10 M.

(24) A fifth culture (E) was additioned with 91% by weight alpha-lipoic acid and 9% by weight honokiol to the overall concentration of 10 M.

(25) A sixth culture (F) was additioned with 46% by weight alpha-lipoic acid and 54% by weight honokiol to the overall concentration of 10 M.

(26) A seventh culture (G) was additioned with only alpha-lipoic acid to the overall concentration of 10 M.

(27) An eighth culture (H) was additioned with only honokiol to the overall concentration of 10 M.

(28) After 6 days incubation, the cells of the eight cultures were fixed with 4% formaldehyde.

(29) The eight cultures were then evaluated regarding the length of neurites at the microscopic level. The neurons were subjected to immunohistochemical staining for the microtubule-associated protein-2 (MAP2) according to the histochemical method for the length of neurites, as described in Fukuyama Y et al. Neurotrophic activity of honokiol on the cultures of fetal rat cortical neurons Bioorg. Med Chem. Lett 12, 1163-66 (2002). The length of the most extended neurite of the whole cellular body was measured and calculated by using software programs Lumina Vision and Mac-SCOPE, according to the above-mentioned method described in Y. Fukuyama et al. (2002). The morphometric analyses were performed by measuring the length of the longest neurite from each neuron using Lumina Vision and Mac-Scope software (Yoshiyasu Fukuyama et al. 2002).

(30) The results are shown in Table 1, in which p denotes statistical significance and ns means not significant.

(31) TABLE-US-00001 TABLE 1 Increase of Increase respect average neurite to control Culture ALA/honokiol ratio length (m) (culture A) A 280 1 B 295 (p = ns) 1.05 C ALA (99.7%) 450 (p = ns) 1.61 honokiol (0.3%) D ALA (95.5%) .sup.585.4 (p < 0.001) 2.09 honokiol (4.5%) E ALA (91%) .sup.630.7 (p < 0.001) 2.25 honokiol (9%) F ALA (46%) 289 (p = ns) 1.03 honokiol (54%) G ALA (100%) 285 (p = ns) 1.01 H honokiol (100%) 287 (p = ns) 1.02

(32) The length of neurites in cultures treated with the tested compositions according to the invention, with a concentration of honokiol between 4.5% and 9%, resulted significantly increased (p<0.001), while cultures treated with a composition containing alpha-lipoic acid 99.7% and 0.3% honokiol did not show a statistically significant increase in the length of neurites. The composition comprising 95.5% by weight alpha-lipoic acid and 4.5% by weight honokiol, and the one comprising 91% by weight alpha-lipoic acid and 9% by weight honokiol were found to be effective in promoting a significantly significant extension of neurites compared to control (culture A) and to the culture supplemented with growth factors (culture B), and especially also with respect to the culture additioned with a composition comprising 99.7% by weight alpha-lipoic acid and 0.3% by weight honokiol (culture C). It should be noted that the effect of the composition according to the invention, both in the proportions of 95.5% of alpha-lipoic acid and 4.5% honokiol, and of 91% alpha-lipoic acid and 9% honokiol, were found to be more significant than that obtained with the growth factor bFGF. The composition comprising 46% by weight alpha-lipoic acid and 54% by weight honokiol inhibited neurite outgrowth in culture F.

(33) The compositions comprising alpha-lipoic acid alone or honokiol alone stimulated neurite growth in a negligible way. The result is a value not significantly different from that of the control composition.

(34) FIG. 1 also shows that, after 7 days of cell culture, in cultures C, D and E (FIGS. 1c, 1d and 1e) neurons showed extended neurites that contained more prominent and dark neuronal somes compared to the control culture A (FIG. 1a), and were better, especially as regards the cultures D and E (FIGS. 1d and 1e), with respect to cultures B (FIG. 1b) respectively containing 40 ng ml.sup.1 of bFGF and cultures of individual alpha-lipoic acid (FIG. 1d) or honokiol (FIG. 1h).

(35) The test results show that the compositions according to the invention have an undoubted and unpredictable effect on the differentiation of cortical neurons.

Example 2

Assessment of Neuronal Survival

(36) Four primary cell cultures were prepared as described in Abe K et al. Effects of recombinant human basic fibroblast growth factor and its modified protein CS23 on survival of primary cultured neurons from various regions of fetal rat brain, Jpn J Pharmacol 1990, 53 (2): 221-7. All operations were carried out under sterile conditions. The neuronal cells were separated from cerebral hemispheres of rat fetuses of 18 days (Japan SLC, Inc.) and suspended in 10% FBS/MEM, then seeded at 20,000 cells cm.sup.2 in poly-L-lysine plates.

(37) After 24 hours, the culture medium was replaced with a serum-free medium, specific for the growth of neurons (Neurobasal Medium) supplemented with B27.

(38) The procedure of the cell cultures was essentially the same as implemented in the example with NBM/B27 except that, as a culture medium free from serum, DMEM supplemented with N2 was used (Cestelli A et al. Formulation of a novel synthetic medium for selectively culturing rat CNS neurons. Dev Brain Res 1985, 22.219); and also that plates had a cell density equal to 210.sup.5 cm.sup.2.

(39) Then, a culture (I) was used as the control culture.

(40) A culture (L) was additioned with 99.7% by weight alpha-lipoic acid and 0.3% by weight honokiol to the concentration of 10 M.

(41) A culture (M) was additioned with 91% by weight alpha-lipoic acid and 9% by weight honokiol to the concentration of 10 M.

(42) A culture (N) was additioned with 46% by weight alpha-lipoic acid and 54% by weight honokiol to the concentration of 10 M.

(43) After being incubated for 3 days, the cells of the four cultures were fixed with 4% formaldehyde.

(44) Neuronal survival was evaluated by the method of WST-8 H Tominaga et al. A water-soluble tetrazolium salt useful for colorimetric cell viability assay, Anal Commun 1999, 36.47. This method uses a salt of 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2.4-disolfofenil)-2H tetrazolium as a chromogen indicator to assess WST-8 cell viability, and produces results on cell viability that are in line with those obtained respectively with the MTT method using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide, and with the tritiated thymidine uptake method.

(45) The results are shown both in Table 2, in which the values are expressed as average, and in FIG. 2. In Table 2, p indicates statistical significance and ns means not significant.

(46) TABLE-US-00002 TABLE 2 % Absorbance at 450 nm with Culture respect to control (culture I) I 100.0 L 106.0 (p = ns).sup. M 143.1 (p < 0.001) N 29.7 (p < 0.001)

(47) Culture M has values that are significantly higher than the control I.

(48) Reference culture I, in the absence of active principles, determined the survival of a small number of neurons. On the contrary, in culture M the presence of the compositions according to the present invention at the concentration of 10 M, showed a high ability to grow and survive neurons.

Example 3

(49) Four primary cell cultures were prepared as described in Example 2 (like in Abe K et al. Effects of recombinant human basic fibroblast growth factor and its modified protein CS23 on survival of primary cultured neurons from various regions of fetal rat brain, Jpn. J. Pharmacol. 1990; 53 (2): 221-7).

(50) Then, a culture (0) was used as the control culture.

(51) A culture (P) was additioned with 91% by weight alpha-lipoic acid and 9% by weight honokiol to the overall concentration of 10 M.

(52) A culture (Q) was additioned with 91% by weight alpha-lipoic acid and 9% by weight honokiol to the concentration of 10 M and 0.003 M of Selenium.

(53) A culture (R) was additioned with 91% by weight alpha-lipoic acid and 9% by weight honokiol to the concentration of 10 M and 0.24 M of gamma linolenic acid.

(54) After being incubated for 3 days, the cells of the four cultures were fixed with 4% formaldehyde.

(55) The survival of neurons was assessed by WST-8 reduction test (Tominaga et al., 1999). The results are shown in Table 3 in which the values are expressed as averages. Furthermore, p denotes statistical significance.

(56) TABLE-US-00003 TABLE 3 % Absorbance at 450 nm with Culture respect to control (culture O) O 100.0 P 143.1 (p < 0.001) Q 178.9 (p < 0.001) R 176.5 (p < 0.001)

(57) The results reported in Table 3 show that, surprisingly, the compositions according to preferred embodiments of the invention, comprising selenium and/or gamma-linolenic acid are particularly effective in enhancing the survival of neuronal cultures. In particular, the compositions used in cultures Q and R are significantly more effective than those used for the cultivation of reference 0 and that used in culture P.