Nutritional composition for improving intestinal barrier integrity, preparation of the composition and method of treatment

Abstract

The invention concerns composition comprising 2′-fucosyllactose for use in the treatment of virus induced diarrhea.

Claims

1. A method for the treatment of rotavirus induced intestinal barrier disruption in an infant or toddler by administering a nutritional composition that comprises 2′-fucosyllactose, wherein the nutritional composition is selected from an infant formula and a follow-on and which is not human milk.

2. The method according to claim 1, wherein the virus induced intestinal barrier disruption is virus induced disruption of intestinal epithelial cells.

3. The method according to claim 1, wherein the nutritional composition further comprises at least one non-digestible oligosaccharides with a DP in the range of 2 to 250, wherein the non-digestible oligosaccharides are selected from the group consisting of fructo-oligosaccharides, galacto-oligosaccharides, xylo-oligosaccharides, arabino-oligosaccharides, arabinogalacto-oligosaccharides, gluco-oligosaccharides, chito-oligosaccharides, glucomanno-oligosaccharides, galactomanno-oligosaccharides and mannan-oligosaccharides.

4. The method according to claim 3, wherein the non-digestible oligosaccharides are selected from the group consisting of fructo-oligosaccharides and galacto-oligosaccharides.

5. The method according to claim 1, wherein the nutritional composition further comprises at least one long chain polyunsaturated fatty acids (LC-PUFA) selected from the group consisting of eicosapentaenoic acid (EPA, 20:5 n3), docosahexaenoic acid (DHA, 22:6 n3), arachidonic acid (ARA, 20:4 n6) and docosapentaenoic acid (DPA, 22:5 n3).

6. The method according to claim 1, wherein the human subject is an infant.

7. The method according to claim 1, wherein the nutritional composition is an infant formula.

8. The method according to claim 1, wherein the nutritional composition further comprises at least two non-digestible oligosaccharides with a DP in the range of 2 to 250, wherein the non-digestible oligosaccharides are selected from the group consisting of fructo-oligosaccharides, galacto-oligosaccharides, xylo-oligosaccharides, arabino-oligosaccharides, arabinogalacto-oligosaccharides, gluco-oligosaccharides, chito-oligosaccharides, glucomanno-oligosaccharides, galactomanno-oligosaccharides and mannan-oligosaccharides.

9. The method according to claim 8, wherein the nutritional composition further comprises fructo-oligosaccharides and galacto-oligosaccharides.

10. The method according to claim 1, wherein the nutritional composition further comprises at least one long chain polyunsaturated fatty acids (LC-PUFA) selected from the group consisting of docosahexaenoic acid (DHA, 22:6 n3), eicosapentaenoic acid (EPA, 20:5 n3) and arachidonic acid (ARA, 20:4 n6).

11. A method for improving intestinal barrier integrity in an infant or toddler by administering a nutritional composition selected from an infant formula or a follow-up formula and which is not human milk, that comprises a 2′-fucosyllactose.

12. The method according to claim 11, wherein the improvement of intestinal barrier integrity is a reduction in the permeability of the intestinal barrier.

13. The method according to claim 11, wherein the nutritional composition further comprises at least one non-digestible oligosaccharides with a DP in the range of 2 to 250, wherein the non-digestible oligosaccharides are selected from the group consisting of fructo-oligosaccharides, galacto-oligosaccharides, xylo-oligosaccharides, arabino-oligosaccharides, arabinogalacto-oligosaccharides, gluco-oligosaccharides, chito-oligosaccharides, glucomanno-oligosaccharides, galactomanno-oligosaccharides and mannan-oligosaccharides.

14. The method according to claim 13, wherein the non-digestible oligosaccharides are selected from the group consisting of fructo-oligosaccharides and galacto-oligosaccharides.

15. The method according to claim 11, wherein the nutritional composition further comprises at least one long chain polyunsaturated fatty acids (LC-PUFA) selected from the group consisting of eicosapentaenoic acid (EPA, 20:5 n3), docosahexaenoic acid (DHA, 22:6 n3), arachidonic acid (ARA, 20:4 n6) and docosapentaenoic acid (DPA, 22:5 n3).

16. The method according to claim 11, wherein the human subject is an infant.

17. The method according to claim 11, wherein the nutritional composition is an infant formula.

Description

DESCRIPTION OF THE FIGURE

(1) The FIGURE shows the alpha-1 antitrypsin (A1AT) concentration in the gut wash was analyzed by ELISA as a measure of the intestinal barrier disruption; results are expressed as mean±S.E.M. (n=4-8); * p<0.05 compared to REF group; #p<0.05 compared to RV group (by MWU test).

EXAMPLES

Example 1

(2) Newborn rats were distributed into five groups of 24 animals each: the reference (REF) group, rotavirus-infected (RV) group, and 3 rotavirus-infected groups supplemented with: a) a mixture of scGOS and lcFOS (RV+GOS/FOS group); b) 2′-FL (RV+2′-FL group); and c) both scGOS/lcFOS and 2′-FL (RV+GOS/FOS+2′-FL group).

(3) Suckling rats were orally administered once daily with the same normalized volume/body weight of all products (4.5 μL/g/day), from the second to the sixteenth day of life, corresponding to the strict lactation period. The RV+GOS/FOS group was supplemented with 0.8 g of scGOS/lcFOS/100 g of body weight. The RV+2′-FL group was supplemented with 0.2 g of 2′-FL/100 g of body weight. The RV+GOS/FOS+2′-FL group received both products at the same doses as when given separately and maintaining the volume of administration (4.5 μL/g/day). The REF and RV groups were administered with a matched volume of water.

(4) The RV (simian SA-11) was obtained as previously described (Perez-Cano et al. Pediatr Res 2007; 62:658-63), and inoculated at day 5 of life (4×10.sup.8 Tissue Culture Infectious Dose 50 [TCID50]/rat) in all the experimental groups with the exception of the REF group, which received the same volume of phosphate-buffered solution (PBS) under the same conditions.

(5) Body weight was recorded daily throughout the study to assess weight gain. Half (n=12) of each group of animals were sacrificed at day 8, to analyze variables associated with the peak of diarrhea, and the other half (n=12 per group) at day 16, to analyze the effects of the supplementations once the diarrhea was resolved. Moreover, the naso-anal and tail lengths were measured to determine the body/tail ratio, the body mass index (BMI), calculated as body weight/length.sup.2 (g/cm.sup.2) and the Lee Index, calculated as (weight.sup.0.33/length)×1000 (g.sup.0.33/cm).

(6) Sample Collection and Processing

(7) At days 8 and 16, the half of each litter were intramuscularly anesthetized with ketamine (90 mg/kg) (Merial Laboratories S.A., Barcelona, Spain) and xylazine (10 mg/kg) (Bayer A.G., Leverkusen, Germany), exsanguinated and their intestines were obtained. The intestines were opened lengthwise, cut into 5 mm pieces and incubated with 2 mL of PBS in a shaker (10 min, 37° C.) to obtain the gut wash (GW). After centrifugation, supernatants were stored at −20° C. and −80° C. until alpha-1 antitrypsin (A1AT) analysis.

(8) The quantification of A1AT in the gut wash, as a marker of intestinal permeability, was performed with the rat SERPINA1/Alpha 1 Antitrypsin ELISA kit (LifeSpan Biosciences Inc., Seattle, Wash., USA) following the manufacturer's instructions. The standard concentrations ranged from 100 to 1.563 ng/mL. Assay sensitivity was 1.56 ng/mL.

(9) Statistical Analysis

(10) The Statistical Package for the Social Sciences (SPSS v22.0) (IBM, Chicago, Ill., USA) was used for statistical analysis. Data was tested for homogeneity of variance and normality distribution by the Levene's and Shapiro-Wilk tests, respectively. When data was homogeneous and had a normal behavior, conventional one-way ANOVA test followed by the post hoc Bonferroni was performed. Otherwise, the nonparametric Kruskal-Wallis test followed by the post hoc Mann-Whitney U (MWU) test were performed. Finally, the chi-square test was used to compare frequencies of diarrhea incidence. Significant differences were established when p<0.05.

(11) Results

(12) Growth

(13) The RV infection did not produce any significant change in growth either at the peak of diarrhea (day 8) or at the end of the study (day 16), as shown by the results in body weight, body/tail ratio, BMI and Lee Index (Table 1). The group supplemented with scGOS/lcFOS had a slightly higher body weight at the end of the study (day 16, p<0.05), and although none of these growth changes modified the BMI, some differences were seen in the body/tail length ratio and the Lee Index. All supplementations increased the body/tail length ratio compared to REF or RV at some time point. Moreover, the Lee Index was decreased exclusively at the peak of diarrhea compared to REF.

(14) TABLE-US-00001 TABLE 1 Growth-associated variables REF RV RV + GOS/FOS RV + 2′-FL RV + GOS/FOS + 2′-FL Body weight (g) day 8   13.52 ± 0.25  13.60 ± 0.19 14.08 ± 0.17 13.51 ± 0.19  13.67 ± 0.19  day 16  31.40 ± 0.89  31.91 ± 0.65   34.16 ± 0.55*.sup.# 32.79 ± 0.50  33.53 ± 0.40  Body/tail length ratio day 8    2.14 ± 0.07   2.15 ± 0.03    2.26 ± 0.03*.sup.#   2.32 ± 0.03*.sup.# 2.22 ± 0.03 day 16   1.77 ± 0.02   1.77 ± 0.02    1.92 ± 0.04*.sup.# 1.86 ± 0.05  1.84 ± 0.03* BMI (g/cm.sup.2) day 8    0.30 ± 0.01   0.29 ± 0.00  0.29 ± 0.00 0.28 ± 0.01 0.28 ± 0.00 day 16   0.35 ± 0.01   0.36 ± 0.00  0.37 ± 0.01 0.37 ± 0.01 0.36 ± 0.00 Lee Index day 8  354.76 ± 3.42 348.80 ± 1.73 345.21 ± 1.34* 343.49 ± 2.46*.sup.# 344.52 ± 1.43*  day 16 334.44 ± 2.51 335.88 ± 2.41 337.12 ± 3.36  337.93 ± 3.48   332.44 ± 1.26   Results are expressed as mean ± S.E.M. (n = 12); *p < 0.05 compared to REF (by MWU test); .sup.#p < 0.05 compared to RV (by MWU test).
Intestinal Barrier Function

(15) Alpha-1 antitrypsin (A1AT) belongs to the serpin superfamily and is known as a protease inhibitor. It is resistant to degradation by digestive enzymes and is an endogenous marker for the presence of blood proteins in the intestinal tract.

(16) The A1AT levels in the gut wash (see FIGURE) were determined by ELISA to assess the changes in the gut permeability. The serum level of A1AT is significantly elevated during the inflammatory response and can be transported across the intestinal epithelial layer into the intestinal lumen due to the increased permeability of the intestinal epithelial barrier (Yang et al. Sci Rep 2015; 5: 15004). The RV group displayed higher levels of A1AT in the gut wash, but they did not differ statistically from the REF group. However, the supplementation with 2′-FL and GOS/FOS+2′-FL reduced the concentration of A1AT in the gut wash compared to both the RV and REF (p<0.05), indicating that not only were they able to treat the gut permeability disruption induced by RV, but they also promoted an enhanced gut barrier function evidenced by the reduced extravasation of A1AT protein and its trafficking towards the gut lumen. The treatment of intestinal barrier disruption was studied on the 3.sup.rd day of the diarrheal period which corresponds to the observed peak in the severity of diarrhea in the studied animals.

Example 2: Infant Formula for the Treatment of Virus Induced Intestinal Barrier Disruption

(17) An infant formula according to the invention comprising per 100 ml (13.9 dry weight):

(18) 1.4 g protein (whey and casein)

(19) 7.3 g digestible carbohydrates (including lactose)

(20) 3.6 g fat (vegetable fat, fish oil)

(21) 0.6 g non-digestible oligosaccharides of which 60 mg 2′-fucosyllactose and 480 mg beta-galacto-oligosaccharides, and 60 mg fructo-oligosaccharides

(22) Further are included: choline, myo-inositol, taurine, minerals, trace elements, and vitamins in accordance with guidelines.