Eye health composition

Abstract

Ophthalmic nutraceutical composition comprising: vitamins; trace elements; carotenoids; omega-3 fatty acids; and resveratrol.

Claims

1. A method of treating an eye pathology caused by an overexpression of a vascular endothelial growth factor (VEGF) pathway in a subject in need thereof, the method comprising: administering to the subject a daily dose composition consisting of: 120 mg to 240 mg of vitamin C; 10 mg to 500 mg of vitamin E; 5 mg to 100 mg of zinc; 0.2 mg to 10 mg of copper; 2 mg to 50 mg of the lutein; 0.5 mg to 10 mg of zeaxanthin; fish oil comprising: 172 mg to 380 mg of EPA; and 190 to 366 mg of DHA; and 30 to 200 mg of resveratrol, wherein said eye pathology is selected from the group consisting of exudative age-related macular degeneration (AMD), retinal venous occlusion, macular edema, diabetic macular edema, myopia, diabetic retinopathy, neovascular glaucoma, and corneal neovascularization.

2. The method according to claim 1, wherein the composition consists of: 120 mg to 240 mg of vitamin C; 30 mg of vitamin E; 12.5 mg of zinc; 1 mg of copper; 10 mg of the lutein; 2 mg of zeaxanthin; fish oil comprising: 172 mg to 380 mg of EPA; and 190 to 366 mg of DHA; and 30 mg of resveratrol.

3. The method according to claim 1, wherein said subject is suffering from symptoms of said pathology only in one eye.

4. The method according to claim 1, wherein the composition is administered orally.

5. The method according to claim 1, wherein the composition is formulated as a capsule, a tablet, or a pill.

Description

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

(1) FIGS. 1A and 1B compare, in undifferentiated (pathological) retinal cells incubated (A) in the presence of a composition according to the invention (formula A) or (B), and in the presence of a composition similar to the one of the invention but without resveratrol (formula B), the expression of VEGFR receptors (VEGFR1 and VEGFR2) and their phosphorylation state (p-VEGF2 Y1054 and Y951), as well as VEGF, by western blot (image) and normalization by -actin (diagram).

(2) FIGS. 2A and 2B compare, in differentiated (healthy) retinal cells incubated (A) in the presence of a composition according to the invention (formula A) or (B) and in the presence of a composition similar to the one of the invention but without resveratrol (formula B), the expression of VEGFR receptors (VEGFR1 and VEGFR2) and their phosphorylation state (p-VEGF2 Y1054 and Y951), as well as VEGF, by western blot (image) and normalization by -actin (diagram).

(3) FIGS. 3A and 3B compare by western blot (A) and in diagram form after normalization by -actin (B) the expression of VEGFR receptors (VEGFR1 and VEGFR2) and their state of phosphorylation (p-VEFG2 Y951) in the mouse eye having or not having (Co) received an injection of LPS (LPS), when they are treated with the composition according to the invention (A), with resveratrol alone (R) or with a composition without resveratrol (B).

(4) FIGS. 4A and 4B compare by western blot (A) and in diagram form after normalization by -actin (B) the expression of VEGFR receptors (VEGFR1 and VEGFR2) and their state of phosphorylation (p-VEFG2 Y951) in the eye of the Control mouse (Co) or having received an injection of LPS (LPS), when they are treated with the composition according to the invention (A), with resveratrol alone (R) or with a composition without resveratrol (B).

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

I/COMPOSITION ACCORDING TO THE INVENTION

I-1/Formula of the Composition (A)

(5) TABLE-US-00003 TABLE 1 formula of a composition according to the invention (A) Ingredient Quantity (mg)/day Quantity (mg)/day Vitamin C 240 Vitamin E 30 Zinc 12.5 Copper 1 Lutein 10 Zeaxanthin 2 Fish oil* 950 DHA content 190 EPA content 380 Resveratrol. 30 *Fish oil comprising 70% by weight omega-3 fatty acid including EPA and DHA.

I-2/Producing the Composition in the Form of Capsules

(6) The quantities shown in table 1 correspond to a daily dose. In practice, the composition according to the invention is produced in the form of 2 capsules.

(7) For each capsule, the following are added to the master batch: glycerol monostearate; beeswax; soy lecithin.

(8) The composition of the capsule jacket is as follows: bovine gelatin red iron oxide; black iron oxide.

(9) The capsules are then covered with a PVC/PVDC shell and an aluminum film. Alternatively, they can be packaged in a pill organizer.

(10) It is recommended that these two capsules be taken with a little water before or during the main meal.

II/IN VITRO STUDY OF THE COMPOSITION ACCORDING TO THE INVENTION (FORMULA A)

II-1/Preparation of Solutions in Different Concentrations of Resveratrol

(11) The stock solution is prepared in DMSO (Dimethyl Sulfoxide), in the amount of 0.115 g of formula A in 1 mL of DMSO, which corresponds to 10 mM of resveratrol.

(12) Solutions of 1, 5, 10 and 20 M resveratrol equivalent, denoted A1, A5, A10 and A20 respectively, are prepared from this stock solution. The dilutions are performed in a culture medium composed of DMEM/F12+1% FBS (fetal bovine serum without phenol red).

II-2/Effect of the Composition According to the Invention on Pathological Cells

(13) A/Protocol:

(14) Cells studied: culture of undifferentiated ARPE-19 (ATCC CRL-2302) (human retinal pigment epithelium) cells.

(15) These cells can be equated with damaged or pathological retinal cells.

(16) Experimental Protocol:

(17) a) Cell Culture:

(18) The cells were maintained at 37 C., and in the presence of 5% CO2 in the medium DMEM/F12 (GIBCO; 31331), completed with 10% FBS (Lonza) and 15 mM HEPES (pH 7.4; GIBCO).

(19) b) Incubation Between the Compositions (Formula A or B) and the Cells: 1, 5, 10 or 20 M. The 10 M of Resveratrol Corresponding to 0.115 mg/mL of Formula A in DMEM/F12+1% FBS w/o Phenol Red.

(20) The 10 M equivalent from formula B also corresponding to 0.115 mg/mL of formula B in DMEM/F12+1% FBS w/o phenol red.

(21) The day prior to treatment, the medium was eliminated and replaced by DMEM/F12 without phenol red (GIBCO; 21041), completed with 1% FBS (Lonza) and 15 mM HEPES (pH 7.4; GIBCO). After 24 hours, the cells were processed for 24 h with the different formulas (Formula A, Formula B and resveratrol) in the medium DMEM/F12 without phenol red (GIBCO; 21041), completed with 1% FBS (Lonza) and 15 mM HEPES (pH 7.4; GIBCO).

(22) c) Western Blot:

(23) After 24 h of stimulation, the media were eliminated, the cells were washed with PBS 1 then scrubbed in PBS 1. The cells were centrifuged and the remainder was resuspended in the RIPA buffer along with a cocktail of protease inhibitors (Roche) and phosphatase inhibitors. The cells were lysed in ice for 30 minutes, then centrifuged. The protein concentration was determined by means of a protein quantification kit (Lowry kit, biorad). 45 g of proteins were mixed with an equal volume of concentrated Laemmli 5, denatured then loaded onto an acrylamide gel. After electrophoresis, the proteins were transferred to a nitrocellulose membrane (Amersham). The blots were then blocked with milk at 5%+ PBS-T 0.1% for 1 hour at ambient temperature. After blocking, the membranes were incubated with cell-signaling primary antibodies (VEGFR-1, VEGFR-2 (55B11) or Phospho-VEGF Receptor 2 (Tyr951) (15D2)) or Abcam (Anti-VEGF antibody [VG-1]). The membranes were washed 3 times with 0.1% PBS-T then incubated with corresponding HRP conjugated antibodies for 1 hour at ambient temperature. The membranes were again washed 3 times in 0.1% PBS-T and the signal was detected by ECL (Tebu bio). The images were recorded with the ChemiDoc imaging system and analyzed with Quantity One Software (Biorad) software.

(24) d) Quantification of the Level of Expression and Activity of the Receptors:

(25) The -actin was also quantified in order to relativize the levels of expression.

(26) e) Controls:

(27) Co=control sample corresponding to DMSO (used for solubilization of the formulae) in the culture medium. R20=Resveratrol at 20 M alone diluted in the culture medium (=DMEM/F12+1% FBS (fetal bovine serum without phenol red).
B/Results:
1) Evidence of the Effect of the Resveratrol

(28) To provide evidence of the effect of resveratrol on the expression of the VEGFR receptors, the composition according to the invention (formula A) is compared to a composition characterized by the absence of resveratrol, formula B given in table 2 below:

(29) TABLE-US-00004 TABLE 2 composition of formula B without resveratrol Ingredient Quantity (mg) Vitamin B1 1.4 Vitamin B2 1.6 Vitamin B3 18 Vitamin B6 2 Vitamin B9 0.2 Vitamin B12 0.001 Vitamin C 60 Vitamin E 10 Zinc 7.5 Manganese 1 Selenium 0.025 Copper 1 Lutein 10 Zeaxanthin 2 Fish oil 400 EPA content 160 Glutathion 1

(30) Dilutions of this composition, denoted B1, B5, B10 and B20, were produced under conditions similar to those described in point II-1: 0.115 grams of formula B in 1 mL of DMSO, then equivalent dilutions in the culture medium.

(31) FIG. 1 illustrates the results obtained on the pathological cells, treated with the composition according to the invention (A) or the equivalent thereof without resveratrol (B).

(32) It can be seen from this figure that on this type of cell, the presence of resveratrol causes a decrease in the expression of receptors (VEGFR1 and VEGFR2), as well as their phosphorylation: the composition according to the invention (A) makes it possible to suppress the expression of the VEGF and of the VEGF receptors, and in a way that is dose-dependent; the phosphorylation of the receptors present also decreases, and in a way that is dose-dependent, which indicates a reduction of activation of these receptors.

(33) The composition according to the invention thus causes a quantitative reduction, but also qualitative reduction of the activation of the VEGF receptors in pathological cells, and consequently of neovascularization.

(34) Insofar as the composition without resveratrol (formula B) does not enable a reduction in the expression of VEGF receptors, nor their phosphorylation (FIG. 1B), this effect would therefore be related to the presence of resveratrol.

(35) 2) Evidence of Synergy in the Composition According to the Invention

(36) The dose-dependent effect of the composition according to the invention on pathological cells was compared to that of the R20 solution, containing only 20 M resveratrol.

(37) FIG. 1A shows that resveratrol alone makes it possible to reduce the expression of VEGF receptors. However, this effect is amplified in the presence of other ingredients of the composition according to the invention (compare A20 and R20).

II-3/Effect of the Composition According to the Invention on Healthy Cells

(38) A/Protocol:

(39) The protocol used is the same as the one described in the example II-2, except for the cells studied:

(40) Cells studied: culture of differentiated ARPE-19 (human retinal pigment epithelium) cells.

(41) Protocol for Obtaining Differentiated Cells:

(42) The undifferentiated cells are seeded in T75 flasks. The cells are maintained at 37 C., and in the presence of 5% CO2 in the medium DMEM/F12 (GIBCO; 31331), completed with 10% FBS (Lonza) and 15 mM HEPES (pH 7.4; GIBCO). When the cells reach confluence, the medium is replaced by DMEM/F12 (GIBCO; 31331), completed with 1% FBS (Lonza) and HEPES (15 mM pH 7.4; GIBCO). The media were changed 2 times per week for 8 weeks.

(43) Said cells can be equated with undamaged or healthy retinal cells.

(44) B/Results:

(45) 1) Evidence of the Activity of the Resveratrol

(46) FIG. 2 illustrates the effect of the composition according to the invention (A), or the equivalent thereof without resveratrol (B), on healthy cells.

(47) Contrary to what was observed in pathological cells, the composition according to the invention (A) increases the expression of the VEGF receptors in a way that is dose-dependent. Moreover, the phosphorylation of the receptors also shows an increase. There is therefore a quantitative as well as qualitative increase of the activation, and consequently of the neovascularization of healthy cells.

(48) However, no effect was observed with the formulation without resveratrol (see FIG. 2: compare FIG. 2A and FIG. 2B).

(49) It can be seen from FIG. 2 that this increase is dose-dependent and significant for the highest concentrations of resveratrol.

(50) 2) Evidence of Synergy in the Composition According to the Invention

(51) The dose-dependent effect of the composition according to the invention on healthy cells was compared to that of a solution containing only resveratrol in the amount of 20 M (FIG. 2, R20): this reveals that resveratrol alone at the concentration tested has no effect on healthy cells, just like the formula without resveratrol (FIG. 2B).

III/EFFECT OF THE COMPOSITION ACCORDING TO THE INVENTION IN VIVO IN THE MOUSE

(52) In order to validate the preceding results obtained in vitro on a cellular model, experiments were performed in the animal, using C57BL/6 mice as model. In order to reproduce the symptoms of AMD, the mice (apart from the control group of mice) received an injection of 0.5 g lipopolysaccharide (LPS) in one of the two eyes (right eye).

(53) AMD is characterized by chronic inflammation. Lipopolysaccharides are well known for inducing local inflammation, resulting in stimulating the expression of cytokines in the damaged tissues. Since the administration of LPS is by injection and is confined to the eye, it is expected that the LPS acts only locally in the injected (right) eye, and is not diffused to other tissues, particularly into the left eye.

(54) Prior to the injection of LPS and to test the efficacy of the different compositions, the mice received orally, once per day for 15 days, either resveratrol alone (R), or the composition A according to the invention described in table 1 (A), or the comparative composition B without resveratrol as described in table 2 (B).

(55) Upon completion of the treatments, the retinas of the eyes of the mice are removed and the levels of expression of the VEGF, VEGF-R1, and VEGF-R2, and of the activated form (phosphorylated on tyrosine 951) of the VEGF-R2 were analyzed.

(56) A/Protocol:

(57) C57BL/6 mice were separated into 5 groups: 1 control group (Co) received only the carrier; 1 group (LPS) received a retro-orbital injection of 0.5 g LPS (E. Coli) in the right eye on the 12.sup.th day; 1 group (R) received, by oral administration, resveratrol alone in a dose of 35 mg/kg/day for 15 days, and 0.5 g LPS was administered in the right eye by retro-orbital injection on the 12.sup.th day, or 72 h before the end of the experiment; 1 group (A) received, by oral administration, the composition A from table 1 in a dose of 314.5 mg/kg/day for 15 days, and 0.5 g LPS was administered in the right eye by retro-orbital injection on the 12.sup.th day, or 72 h before the end of the experiment; 1 group (B) received, by oral administration, the composition B from table 2 in a dose of 314.5 mg/kg/day for 15 days, and 0.5 g LPS was administered in the right eye by retro-orbital injection on the 12.sup.th day, or 72 h before the end of the experiment.

(58) At the end of the experiment, the mice are anesthetized and euthanized in order to remove the retinas from the right eye (having received the LPS injection and imitating the diseased eye) and of the left eye (imitating the healthy eye). The retinas are then lysed in order to study by Western-blot the expression of the proteins of the VEGF pathway.

(59) In parallel, different organs were removed to verify the non-toxicity of the tested compositions.

(60) B/Results:

(61) The results obtained for the retinas of the right eye (imitating the damaged eye) are presented in FIG. 3. The results obtained for the retinas of the left eye (imitating the healthy eye) are presented in FIG. 4.

(62) 1/Eye Treated by LPS Injection (Right):

(63) In the right eye, it is expected that the LPS injection causes an inflammatory response, particularly the overexpression of cytokines and therefore activation of the VEGF pathway, as described by Nagineni et al. (Aging and Disease, 2014; 5(2): 88-100). It can be seen from FIG. 3 that the levels of expression of VEGF-R1, VEGF-R2, the presence of VEGF-R2 in phosphorylated form, and even the level of VEGF are very low in the mice labeled LPS, while a higher expression of these proteins (in comparison with the untreated control Co) was expected due to the induced inflammation. These results tend to show that treatment by 0.5 g LPS (LPS specimen of FIG. 3) is very toxic and very poorly supported by the retinas of the mice. The small quantity of proteins is therefore explained by the toxicity of the LPS which causes cellular death and consequently the cells cannot express the VEGF receptors or secrete VEGF.

(64) In comparison, the retinas of the right eye of the mice having received an injection of LPS but having consumed either resveratrol (R), the composition A or the composition B, show very high levels of proteins, which seems to indicate that the consumption of these products has made it possible, at least to a certain degree, to protect the cells of the retina from the toxic effect of the LPS.

(65) Moreover, it will be noted that taking the composition A daily for 15 days causes a significant decrease in the expression of VEGF receptors such as VEGF-R1, VEGF-R2 and activated phospho-VEGF-R2 Y951 form thereof compared to the control mice, and this is more significant than what is observed with the composition B without resveratrol or with resveratrol alone (R). These results, therefore, are coherent with what was observed in vitro in FIG. 1, showing the value of the composition according to the invention for inhibiting more effectively the activation pathway of the VEGF receptors in a pathological, inflammatory context.

(66) 2/Contralateral Eye not Treated by Injection of LPS (Left):

(67) The results obtained with the retinas of the left eye, not having had contact with the LPS and imitating a healthy eye, are shown in FIG. 4.

(68) The data obtained show that the effect of resveratrol alone (R), as expected, is to decrease the expression of VEGF-R1 and VEGF-R2. Significantly, the composition A according to the invention makes it possible to maintain a level of VEGF-R1, VEGF-R2 and of the activated phospho-VEGF-R2 Y951 form, comparable to the control mice (Co). There is a lesser effect for the composition B without resveratrol. In other words, and as observed in vitro (FIG. 2), the combination of resveratrol and vitamins, trace elements, carotenoids and omega-3 fatty acids of the composition according to the invention has a synergistic effect in comparison with resveratrol alone and the composition B.

(69) It can be seen from the results above that the composition A is capable of decreasing the VEGF-R activation pathway in diseased retinal cells but is also capable of maintaining a normal level of functioning of said pathway in normal retinas, which is essential for maintaining sufficient blood flow to the retina and avoiding the degeneration processes.

(70) Thus, and contrary to resveratrol alone, the composition A can advantageously be administered to subjects in whom only one of the two eyes is damaged, with benefit to the damaged eye without affecting the healthy eye.

CONCLUSIONS

(71) Surprisingly and for the first time in the field of ophthalmology, in relation to retinal cells, it has been shown that a composition according to the invention containing resveratrol has a differential action at VEGFR receptors necessary for neovascularization, depending on the type of cells: repression in pathological cells and no repression, and even a reverse effect, in healthy cells.

(72) Thus, the resveratrol in the context of a composition according to the invention would make it possible to reestablish a balance within the deficient cells but would not modify in any way, and would even keep fit the healthy cells by means of the supplemental contribution of the essential elements provided by blood circulation.

(73) In the same way, in patients at risk, the administration of the composition A would make it possible to prevent the appearance of the symptoms, allowing the first anomalies to be treated by preserving the health potential while waiting for the appearance of anomalies. Indeed, the retinal cells will still receive the supply of necessary oxygen, nutrients and vitamins via physiological blood flow supported by a physiological level of VEGF, which will not decrease as could be the case with resveratrol alone.