Composition comprising vitamin A and non digestible oligosaccharides

Abstract

The present invention relates to a combination of Vitamin A and non-digestible oligosaccharides for use in treating and/or preventing allergy.

Claims

1.-15. (canceled)

16. A method for: a. treating allergy, b. preventing allergy, c. reducing the risk for allergy, and/or d. inducing and/or enhancing oral immune tolerance to an allergen, in an infant, the method comprising administering to the infant a combination of (i) preformed Vitamin A selected from the group consisting of retinol, retinal, retinoic acid and retinyl esters, and (ii) at least one non-digestible oligosaccharide selected from the group consisting of galacto-oligosaccharide and fructo-oligosaccharides.

17. The method according to claim 16, wherein the combination is in the form of a nutritional composition comprising 5 to 10 μg Retinol Equivalent (RE) of said preformed Vitamin A per gram dry weight and/or 100 to 200 μg RE of said preformed Vitamin A per 100 kcal.

18. The method according to claim 16, wherein the at least one non-digestible oligosaccharide is a mixture of galacto-oligosaccharides and fructo-oligosaccharides.

19. The method according to claim 16, wherein said preformed Vitamin A comprises or are retinyl esters.

20. The method according to claim 16, wherein the subject is at risk of or suffering from a food allergy, in particular a cow's milk protein allergy.

21. The method according to claim 17, wherein the composition comprises 6 to 9 μg RE preformed Vitamin A per g dry weight and/or 120 to 180 μg per 100 kcal.

22. The method according to claim 17, wherein the composition comprises 15 to 250 mg non-digestible oligosaccharides per g dry weight.

23. The method according to claim 17, wherein the composition comprises a source of protein, the protein source being selected from hydrolysed protein or free amino acids.

24. The method according to claim 17, wherein the composition is an infant formula, a follow on formula or a young child formula.

25. The method according to claim 17, wherein said composition comprises two or more non-digestible oligosaccharides selected from the group consisting of galacto-oligosaccharides, fructo-oligosaccharides and uronic acid oligosaccharides.

26. Infant formula, follow on formula or young child formula, comprising: i. 6 to 9 μg RE preformed Vitamin A per g dry weight and/or 120 to 180 μg RE preformed Vitamin A per 100 kcal, wherein the preformed Vitamin A is selected from the group consisting of retinol, retinal, retinoic acid and retinyl ester, ii. 15 to 250 mg of at least one non-digestible oligosaccharide selected from the group consisting of galacto-oligosaccharides and fructo-oligosaccharides, per gram dry weight of the formula, and iii. hydrolysed protein and/or free amino acids.

27. Infant formula, follow on formula or young child formula according to claim 26, wherein the at least one non-digestible oligosaccharide is a mixture of galacto-oligosaccharides and fructo-oligosaccharides.

28. Infant formula, follow on formula or young child formula according to claim 26, comprising two or more non-digestible oligosaccharides selected from the group consisting of galacto-oligosaccharides, fructo-oligosaccharides and uronic acid oligosaccharides.

29. Infant formula, follow on formula or young child formula according to claim 26, wherein the preformed vitamin A is retinol palmitate, and the formula (iii) comprises hydrolysed protein.

30. A method for a. treating allergy, b. preventing allergy, c. reducing the risk of allergy, and/or d. inducing/enhancing oral immune tolerance to an allergen, in a subject, wherein the subject is an infant or young child that is at risk of or suffering from a food allergy, wherein the subject is administered with an infant formula, follow on formula or young child formula comprising a combination of (i) and (ii), wherein the formula comprises, i. 6 to 9 μg RE preformed Vitamin A per g dry weight and/or 120 to 280 μg RE preformed Vitamin A per 100 kcal, wherein the preformed Vitamin A is selected from the group consisting of retinol, retinal, retinoic acid and retinyl ester, ii. 15 to 250 mg of at least one non-digestible oligosaccharide selected from the group consisting of galacto-oligosaccharides and fructo-oligosaccharides, per gram dry weight of the formula, and iii. hydrolysed protein and/or free amino acids.

31. The method according to claim 30, wherein the allergy is a cow's milk protein allergy.

32. The method according to claim 30, wherein the preformed vitamin A is retinol palmitate, and the formula (iii) comprises hydrolysed protein.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0200] FIG. 1. Clinical symptoms in control and whey sensitized mice fed Vitamin A and a mixture of non-digestible oligosaccharides GFA. The acute allergic skin response measured one hour after intra-dermal injection. Values are means±SEM, n=8-10, a one-way ANOVA and Bonferroni post hoc was used. Δ μm Indicates ear thickness before i.d. injection deducted from thickness after i.d. injection.

[0201] FIG. 2: The anaphylactic symptom scores measured at thirty minutes after challenge. Values are means±SEM, n=8-10. Data is calculated with the Kruskal-Wallis and Dunn's post hoc test.

[0202] FIG. 3: The body temperature was measured 30 minutes after i.d. injection via transponder read out. Values are means±SEM, n=8-10, a one-way ANOVA and Bonferroni post hoc was used. Asterix indicates a significant difference compared to sham, **** p<0.0001, *** p<0.001, * p<0.05. The ‘&’ sign indicates a significant difference compared to the VitA.sup.+ group. GFA, short-chain Galacto-oligosaccharides/long-chain Fructo-oligosaccharides/pectin derived Acidic-oligosaccharides; s, standard 4000 ILl/kg vitamin A; +, enriched vitamin A 8000 ILl/kg.

EXAMPLES

Example 1: A Diet with Non-Digestible Oligosaccharides and an Increased Level of Vitamin a Results in a Reduced Allergic Reaction in Mice Sensitized to Whey Protein

[0203] Material and methods: The semi-synthetic cow's milk free AlN93G chow (Research Diet Services, Wijk bij Duurstede, the Netherlands) was used as control diet. In all diets casein and whey were replaced by soy protein. The diets were composed, with ‘standard’ 4000 IU Vitamin A/kg or enriched with 8000 IU Vitamin A/kg (VitA.sup.+). For the latter supplementation, twice the standard amount of Vitamin A is chosen, for which it is known that no adverse effects occur for this dose and time frame. Before start of dietary intervention, mice were born and weened by their mothers which received standard chow (without whey or casein) containing the standard Vitamin A dose of 4000 IU/kg. Beyond Vitamin A, diets were either supplemented or not supplemented with a mixture of non-digestible oligosaccharides. One percent iso-caloric supplementation of scGOS (Vivinal GOS, Friesland Campina Domo, Zwolle, The Netherlands), IcFOS (Raftiline HP, Orafti, Wijchen, The Netherlands), and pAOS (Südzucher, Mannheim, Germany) in a 9:1:2 wt ratio was added at the expense of cellulose and lactose.

[0204] Four weeks old and body weight >11 gram, specific pathogen-free female C3H/HeOuJ mice (Charles River, Sulzfeld, Germany) were housed under conventional housing at the animal facility (Intravacc, Bilthoven, the Netherlands). Mice were held in a light/dark cycle of 12 h/12 h, controlled 65-70% relative humidity and 22±2° C. temperature with ad libitum access to tap water and pelleted food. Upon arrival, mice were fed the specific diets which was continued throughout the study: control diet (s), enriched vitamin A (VitA+) diet, diet supplemented with GFA (GFA) and diet supplemented with GFA and enriched Vitamin A (VitA.sup.+/GFA). Mice (n=10-15) were housed in groups and kept in two cohorts (5/cage) in Makrolon III-H cages. For cage enrichment mice were provided with two red-transparent polycarbonate igloo's on 9kGy irradiated sawdust bedding (Lignocel 9s, J. Rettenmaier & Söhne GmbH, Germany).

[0205] Following 13 days of pre-treatment with the diets (day −14 to 0), mice were sensitized weekly for five times (day 0, 7, 14, 21, 28) by means of oral gavage, with 10 μg Cholera Toxin (CT, List Biological Laboratories, USA) or 10 μg CT/20 mg whey protein concentrate (sweet whey protein concentrate 60 (sWPC60), Milei, Germany) per 500 μl Dulbecco's phosphate-buffered saline (DPBS) (Life Technologies, Inc., Invitrogen, USA) per oral gavage using a blunt needle (stainless steel) (Kent Scientific Corporation, USA). Temperature transponders (IPTT-300, BMDS, USA) were injected subcutaneously under isoflurane gas anesthesia, between the 3.sup.rd and 4.sup.th sensitization.

[0206] To measure the acute allergic skin response whey protein was injected intradermally (i.d.) (10 μg whey /20 μl PBS) in the ear pinnae at day 35. The acute allergic skin response was determined as delta (A) ear swelling (expressed as Δ μm) by subtraction the ear thickness measured just before i.d. injection from the ear thickness measured one hour after i.d. challenge using a digital micrometer (Mitutoyo, Veenendaal, the Netherlands). The body temperature and the anaphylactic shock symptoms were scored according to the method previously described by van Esch et al Toxicology Letters 220 (2013) 95-102.

[0207] Eighteen hours before the end of the study, mice were challenged intragastrically (i.g). with 50 mg whey/500 μl DPBS to activate the mucosal gastrointestinal immune response. At day 37 under terminal isoflurane gas anesthesia, blood and MLN were isolated and stored for further analysis.

[0208] One centimetre of the intestinal mid small intestine was collected after sacrifice and stored in RNAlater™ (Qiagen GmbH, Hilden, Germany) at 4° C. until further processing, described previously by Kerperien et al. (Pediatr Allergy Immunol. 2014; 25(8):747-54). mRNA levels were calculated with CFX Manager software (version 1.6) and corrected for the expression of Ribosomal protein S13(Rps13) with 100×2.sup.{circumflex over ( )}(Rps13-gene of interest) as described previously (Garcia-Vallejo J J et al. Analytical biochemistry. 2004; 329(2):293-932). Validated primers for Il10, Ifnγ, Foxp3 were purchased from SAbioscience (Qiagen, German Town, Md., USA).

[0209] The splenocyte suspension was lysed to remove red blood cells as described previously (Kostadinova et al, Front Immunol. 2016; 7:673). MLN and spleen cell suspensions were made with a 70 μm cell strainers (Thermo Fisher Scientific, Amsterdam, the Netherlands), resuspended in RPMI 1640, 10% fetal bovine serum and penicillin (100 U/mL)/streptomycin (100 μg/mL) and quantified using a Coulter Z1 particle counter (Beckmann Coulter, Brea, USA). One million cells per well were added to polypropylene V-bottom 96 well plates (BD Biosciences, Germany) and blocked with rat anti-mouse CD16/32 (BD Biosciences, Germany) for 20 minutes at 4° C. to block non-specific binding sites. For surface staining the cells were incubated with the different antibodies of the staining panels for 30 minutes at 4° C. After the antibody incubation, the cells were fixed using BD cell fix (Becton Dickinson, Belgium). Directly after the staining procedure for extracellular markers, these cells were permeabilized using a fixation/permeabilization buffer (eBioscience, San Diego, USA) overnight. The next day, the cells were blocked again and incubated with the intracellular marker antibodies for 30 minutes at 4° C. Then the cells were fixed with the BD cell fix. Fluorescence Minus Ones (FMOs) and unstained cells were used as controls. In addition, compensation staining with UltraComp beads (eBioscience, San Diego, USA) was performed to correct for the spectral overlap, which occurs in multi-color staining. Cell surface staining antibodies used were: CD8a-APC-Cy7, CD11c-PerCP-Cy5.5 and CD25-Pe-Cy7 from BD biosciences (San Jose, Calif., USA) and CD4-PerCP-Cy5.5, CD69-Pe-Cy7, CD11b-PE, CD103-APC, CX3CR1-FITC, B220-APC from Ebiosciences (San Diego Calif., USA). Cytokines and intracellular staining for IFNγ-APC, came from Ebiosciences (San Diego Calif., USA). The analysis of the stained cells was performed using FACS Canto II cytometer (BD Biosciences, USA) and FACS Diva software (BD Biosciences, Germany).

[0210] To determine the sample size historical data with sWPC60 as whey source and ear swelling of allergic mice (123±20 μm) as primary outcome parameter were used. The estimated effect size was set at 20% with 80% power and an a of 5%. Statistical analysis was performed using GraphPad Prism 7 (GraphPad Software, San Diego, Calif., USA). Not normal distributed numerical data was LOG transformed. Results are shown in FIG. 1. The data are represented as the mean±SEM plots. One-way ANOVA and post hoc Bonferroni multiple comparisons test with selected groups were used to analyze the data. Selected groups are sham vs. allergic mice (as model control), allergic mice fed a diet with standard level of Vitamin A vs. allergic mice fed a diet enriched in Vitamin A (VitA.sup.+,) allergic mice fed a diet with standard level of Vitamin A vs. allergic mice fed a diet with a standard level of Vitamin A and a mixture of non-digestible oligosaccharides (GFA), allergic mice fed VitA.sup.+ vs. allergic mice fed VitA.sup.+/GFA and allergic mice fed a diet with standard level of Vitamin A and GFA vs. allergic mice fed VitA.sup.+/GFA. Kruskal-Wallis was used to analyze the anaphylactic shock score (categorial data), followed by Dunn's multiple comparison post-test. P-value <0.05 was considered significant.

[0211] Results

[0212] The allergic symptoms measured were the acute allergic skin response, anaphylaxis and body temperature. Table 1 shows that the % anaphylactic shock was lowest in the group consuming the diet with the non-digestible oligosaccharides and enriched in Vitamin A (VitA+/GFA).

[0213] FIG. 1 shows the acute allergic skin response measured one hour after intra-dermal injection. Values are means±SEM, n=8-10, a one-way ANOVA and Bonferroni post hoc was used. Δ μm Indicates ear thickness before i.d. injection deducted from thickness after i.d. injection. FIG. 2 shows the anaphylactic symptom scores measured at thirty minutes after challenge. Values are means±SEM, n=8-10. Data is calculated with the Kruskal-Wallis and Dunn's post hoc test. FIG. 3: The body temperature was measured 30 minutes after i.d. injection via transponder read out. Values are means±SEM, n=8-10, a one-way ANOVA and Bonferroni post hoc was used. Asterix indicates a significant difference compared to sham, **** p<0.0001, *** p<0.001, * p<0.05. The ‘&’ sign indicates a significant difference compared to the VitA.sup.+ group. GFA, short-chain Galacto-oligosaccharides/long-chain Fructo-oligosaccharides/pectin derived Acidic-oligosaccharides; s, standard 4000 IU/kg vitamin A; +, enriched vitamin A 8000 IU/kg.

[0214] The acute allergic skin response was significantly different between the sham sensitized mice fed control diet (sham) and whey sensitized mice fed control diet (allergic mice) (p<0.0001) (See FIG. 1).

[0215] In addition, all other treatment groups had a significantly higher acute allergic skin response than the sham (p<0.0001) (See FIG. 1). Surprisingly, only whey sensitized mice supplemented with a diet comprising a combination of non-digestible oligosaccharides GFA and enriched levels of Vitamin A (allergic mice fed VitA.sup.+/GFA) had a significant lower acute allergic skin response compared to the whey sensitized mice supplemented with elevated levels of Vitamin A without GFA (allergic mice fed VitA.sup.+) (p<0.05). The allergic skin response in the VitA+/GFA group was also lower when compared to the group receiving a diet with GFA and a standard level of vitamin A or the group receiving a diet with standard level of vitamin A and without non-digestible oligosaccharides.

[0216] Measured at thirty minutes, the anaphylactic symptom score was significantly higher in the allergic mice, allergic mice fed elevated levels of Vitamin A (VitA.sup.+) and allergic mice fed non-digestible oligosaccharides GFA and standard level of Vitamin A compared to the sham (p<0.001), while surprisingly this score was not significant higher for the allergic mice fed elevated levels of Vitamin A and non-digestible oligosaccharides GFA (See FIG. 2). Additionally, there was a trend of a lower body temperature (which is indicative for allergic reaction) in the groups with standard levels of vitamin A (with or without the non-digestible oligosaccharides GFA), but this lowering of the body temperature was not observed in the group that had consumed a diet with GFA and enriched levels of Vitamin A (See FIG. 3).

TABLE-US-00001 TABLE 1 Anaphylactic shock symptoms at one hour after the intradermal ear challenge of all mice. Data are represented in percentages per treated group. % humane % anaphylactic endpoint Amount in chow; shock (mice (mice/mice Vitamin A intake GFA total) with shock) Normal 4000 IU/kg ≈20 − 90 (9/10) 0 (0/9) IU/day + 80 (8/10) 0 (0/8) Elevated 8000 IU/kg; ≈40 − 80 (8/10) 0 (0/8) IU/day + 40 (4/10) 0 (0/4)

[0217] To measure markers of local T helper cell responses, mRNA levels for Ifnγ (Th1), Il10 and Foxp3 (Treg) were measured in the mid small intestine. For Ifnγ and Foxp3 mRNA the highest levels were observed in the VitA+/GFA group and the increase was statistically significant compared to allergic mice fed VitA+(p<0.05). For Il10 mRNA the same trends were shown. Ifnγ and Foxp3 mRNA expression in allergic mice fed VitA.sup.+/GFA was also increased compared to sham (p<0.05), and a similar trend was observed for IL-10 mRNA. Foxp3 mRNA was decreased in allergic mice fed an elevated level of Vitamin A compared to allergic mice (p<0.05).

[0218] Intracellular cytokine expression of IFNγ was measured in CD11c.sup.+/CD4.sup.− phagocytes of the spleen. Compared to sham, the allergic mice fed VitA.sup.+/GFA showed an increase in CD11c.sup.+CD4.sup.− IFNγ.sup.+ phagocytes (p=0.064). In the other groups the level was lower. IFNγ could play a role as an autocrine regulator for DC function. Exogenous IL12 or CD40 ligation combined with high levels of IL18 can induce IFNγ production in immature BALBc derived DC in vitro. One study shows that human monocytes derived from buffy coats pre-treated with IFNγ become regulatory DC and subsequently these IFNγ′ DC produced more IL10 upon stimulation, and expressed more inhibitory molecules and downregulated T cell migration in vitro (Svajger et al. J Leukoc Biol. 2014; 95(1):33-46.7). This suggests that the IFNγ′ phagocytes observed in the spleen could be a more regulatory DC phenotype and the frequency of these cells increases in the mice fed VitA.sup.+/GFA. This is indicative for induction of immune tolerance.

[0219] The results in this example are indicative for increased levels of dietary Vitamin A supporting the non-digestible oligosaccharides to reduce the allergic symptom development in a mouse model for allergic sensitization, and thus of an effect of the combination of dietary vitamin A and non-digestible oligosaccharides on treating and/or preventing allergy, reducing the risk for allergy and inducing and/or enhancing oral immune tolerance to an allergen, in a subject.

Example 2

[0220] Packed powdered Infant Formula, intended for infants suffering from cow's milk allergy, with instructions to reconstitute with water.

[0221] After reconstitution the formula, by adding 13.66 g powder to an end volume of 100 ml, the formula comprises 66 kcal per 100 ml. Per 100 ml there is: [0222] 3.4 g fat (vegetable fat, fish oil, microbial oil), [0223] 7.1 g digestible carbohydrates (mainly lactose and starch), [0224] 670 mg non-digestible oligosaccharides (galacto-oligosaccharides and fructo-oligosaccharides in a wt/wt ratio 9/1. Source of galacto-oligosaccharides is VivinalGOS, source of fructo-oligosaccharides is Raftiline HP), [0225] 1.6 g protein (extensively hydrolysed whey protein) [0226] 100 μg Retinol Equivalent Vitamin A in the form of retinyl palmitate [0227] Minerals, trace elements, other vitamins and micro-ingredients as known in the art and according to directives for infant formula.

Example 3

[0228] Packed powdered Follow On Formula, i) intended for infants that are more prone to developing an allergy or ii) that reduces the risk of developing milk-allergy for infants with a family history of atopy, with instructions to reconstitute with water.

[0229] After reconstitution (14.7 g powder to an end volume of 100 ml) the formula comprises 68 kcal per 100 ml. Per 100 ml there is [0230] 3.2 g fat (vegetable fat), [0231] 8.2 g digestible carbohydrates (mainly lactose and starch), [0232] 800 mg non-digestible oligosaccharides (galacto-oligosaccharides and fructo-oligosaccharides in a wt/wt ratio 9/1. Source of galacto-oligosaccharides is VivinalGOS, source of fructo-oligosaccharides is Raftiline HP), [0233] 1.6 g protein (partially hydrolysed whey protein) [0234] 75 μg Retinol Equivalent Vitamin A in the form of retinyl palmitate [0235] Minerals, trace elements, other vitamins and micro-ingredients as known in the art and according to directives for infant formula.

Example 4

[0236] Packed powdered Infant Formula, for the dietary management of cow's milk allergy, multiple food protein allergies and other where an amino acid based diet is recommended.

[0237] The pack contains instructions to reconstitute with water.

[0238] After reconstitution, 14.7 g powder to an end volume of 100 ml, the formula comprises 68.2 kcal per 100 ml. Per 100 ml there is [0239] 3.4 g fat (mainly vegetable fat), [0240] 8.2 g digestible carbohydrates (mainly from glycose syrup), [0241] 1.6 g protein equivalent (based on free amino acids) [0242] 110 μg RE vitamin A in the form of retinyl acetate, [0243] 800 mg non-digestible oligosaccharides (short chain fructo-oligosaccharides and long chain fructo-oligosaccharides in a wt/wt ratio 9/1. Source of short chain fructo-oligosaccharides is Raftilose P95, source of long chain fructo-oligosaccharides in Raftiline HP), [0244] Minerals, trace elements, other vitamins and micro-ingredients as known in the art and according to directives for infant formula