ORAL DOSE COMPOSITIONS OF TRIBUTYRIN FOR THE GENERATION OF BUTYRATE IN THE GUT

Abstract

The present disclosure relates generally to compositions for a dietary supplement, a food supplement, and/or a nutritional supplement, useful in the generation of butyrate in the gastrointestinal track or gut of a mammal for effecting and improving health and wellness. The present disclosure also relates to the use of a dietary supplement provided in a therapeutically effective amount as a replacement for probiotics and prebiotic usage. Additionally, the present disclosure relates to a dietary supplement composition including a low therapeutic dose of tributyrin either alone, or in combination with prebiotics and/or probiotics to enhance health and wellness. Furthermore, the present disclosure also relates to a low dose dietary supplement composition which generates therapeutic amounts of butyrate in the gut of a mammal independent of the state of the microbiome to achieve desired health benefits of butyrate.

Claims

1. A dietary supplement comprising a prebiotic component provided in a dosage amount of between 10 and 100 mg per day, wherein the supplement generates a therapeutic amount of butyrate in a gastrointestinal tract of an adult human.

2. The dietary supplement of claim 1, wherein the prebiotic component is tributyrin.

3. The dietary supplement of claim 2, wherein the tributyrin is provided in a therapeutic adult human dosage amount up to 100 mg per day.

4. The dietary supplement of claim 1, wherein the tributyrin generates the therapeutic dose amount of butyrate independent of a composition of a microbiome in the gastrointestinal tract of the adult human.

5. The dietary supplement of claim 1, wherein the therapeutic amount of butyrate provides a plurality of health benefits to the adult human.

6. A dietary supplement composition for increasing a level of butyrate in the gut of a mammal; the composition comprising: a prebiotic component; a probiotic component; wherein the combination of the prebiotic and the probiotic provides a synergistic effective to increase the production of butyrate in the gut of the mammal for improved health benefits.

7. The dietary supplement composition of claim 6, wherein the prebiotic is tributyrin.

8. The dietary supplement composition of claim 7, wherein tributyrin is provided in a dosage amount of between 10 and 100 mg per day for an adult human.

9. The dietary supplement composition of claim 8, wherein the dosage amount of tributyrin provides a therapeutically effective increase in the production of butyrate in the gut of the mammal independent of a composition of a microbiome in the gut.

10. The dietary supplement composition of claim 6, wherein the probiotic is selected from the group consisting of Lacticaseibacillus rhamnosus GG and Limosilactobacillus reuteri.

11. A dietary supplement comprising tributyrin administered in a dosage amount of between 10 and 100 mg per day, wherein the tributyrin generates a therapeutic dose amount of butyrate in a gut of an adult human.

12. The dietary supplement of claim 11, wherein the tributyrin is provided as a replacement for a probiotic or a prebiotic.

13. The dietary supplement of claim 11, wherein the tributyrin generates the therapeutic dose amount of butyrate without addition of a probiotic or a prebiotic.

14. The dietary supplement of claim 11, wherein the adult dose of between 10 and 100 mg/day is capable of being administered to a human child or other mammal as modified based on a given weight of the child or mammal and other factors to determine the child or mammal dose is less than 100 mg/day.

15. A dietary supplement composition for increasing production of butyrate in a gut of a mammal, the composition comprising: between about 30 and 70% tributyrin; between about 5 and 15% partially hydrolyzed guar gum fiber; and between about 15-65% inert ingredients, wherein the composition generates a therapeutically effective amount of butyrate in the gut of the mammal for improving health in the mammal.

16. The dietary supplement composition of claim 15, wherein the composition has a water activity of less than 0.2.

17. The dietary supplement composition of claim 15, wherein dietary composition is provided as a powder for oral consumption.

18. The dietary supplement composition of claim 17, wherein the powder composition is combined with a probiotic or a prebiotic.

19. The dietary supplement composition of claim 15, wherein the therapeutically effective increase in the production of butyrate in the gut of the mammal is independent of a composition of a microbiome in the gut.

Description

DETAILED DESCRIPTION

[0033] The present disclosure relates generally to dietary supplements and compositions containing tributyrin administered in therapeutically effective amounts for effecting health in a mammal. Disclosed are dietary supplements and compositions comprising therapeutically effective dosage amounts of tributyrin which are lower than previously known, for use in effecting health as related to the production of butyrate in the body at therapeutic levels, and other health benefits resulting from the generation of butyrate. The present disclosure also relates to tributyrin as a replacement for probiotics and/or prebiotics for enhanced health benefits as related to the production of butyrate in the body. The present disclosure further relates to dietary compositions of tributyrin in combination with either probiotics and/or prebiotics for enhanced synergistic health benefits as related to the production of butyrate in therapeutic amounts in the body and resulting health benefits. Finally, the administration of tributyrin as a dietary supplement and/or composition and the generation of butyrate and resulting benefits, is not dependent on the state or condition of the microbiome of the subject receiving the supplement.

Methodology

[0034] In order to assess the action of tributyrin with and without prebiotics and probiotics, the present disclosure includes the use of two different model systems, which are described below. Currently, it is not possible to conveniently assess the production of butyrate in-situ, in the human body, or even in live animals. Therefore, the standard method for assessing both SCFA production and assessing the state and composition of the microbiome, is to measure and observe changes in the feces. Changes in the composition of feces provides an indicator of changes to the microbiome and gut.

[0035] Simply increasing bacteria in the microflora which are known to generate butyrate, in of itself, does not demonstrate a therapeutic benefit. An important aspect of the present disclosure is that the direct generation of butyrate provides a “therapeutic benefit” to the overall health of the subject mammal, including humans. In order to demonstrate the proposed “therapeutic benefit” of the generation of butyrate the present model systems compare butyrate generation in the gut to the generation of butyrate by known therapeutic prebiotics, such as inulin, provided at known therapeutic dosages. In this manner, the present disclosure demonstrates for the first time that tributyrin generates butyrate in amounts having a therapeutic benefit.

[0036] In the examples discussed below, three different prebiotics are used: (a) inulin, a well-known prebiotic which has been used therapeutically for gut health at a 4 g/day dose; XOS (xylooligosaccharide) a prebiotic derived from milk, with a demonstrated therapeutic benefit for gut health at 1 gram/day (a low dose prebiotic); and partially hydrolyzed guar gum (PHGG), with a therapeutic dose of 5-10 g/day. All three of these prebiotics are butyrate generators and all have demonstrated therapeutic benefits at these identified dose levels. In different examples, these known therapeutic prebiotics are used to compare against tributyrin butyrate generation in the present disclosure, and thus demonstrate a therapeutic benefit of the administration of tributyrin and the resulting generation of therapeutic amounts of butyrate.

[0037] In the first type of experiment, an ex-vivo model system is used. In this model system, an ex vivo simulation of the fermentation that occurs in the colon was used. Human feces from 6 volunteers were used as a gut microbiota starter cultures. The test materials were added to this system and butyrate generation was determined after 48 hours of fermentation, compared to controls for each set. Butyrate generation was measured using chromatography.

[0038] In this series of experiments the human equivalent dose (HED) was determined to be 1 gram/day of ingested prebiotic in-vivo corresponds to approximately 1 g/L in-vitro dose. Therefore a 10 mg/ml in-vitro dose is equivalent to 10 mg/day HED.

[0039] In the second type of experiment, an animal model system was used to study changes in the gut microbiome behavior. Caenorhabditis elegans (C. elegans), a nematode commonly used as an organism model, was used in the present study. Reduction of oxidative stress was determined in the C. elegans model. Butyrate has previously been shown in rodent and porcine studies to ameliorate oxidative stress in the intestine generated via hydrogen peroxide or Diquat dibromide exposure. Thus, an oxidative stress assay is a rapid indirect method of determining whether the tributyrin doses proposed in this study can produce beneficial butyrate in vivo.

[0040] Oxidative stress was induced in C. elegans by administering a lethal dose of 250 mM of the herbicide, Paraquat. Previous studies have shown that this concentration of Paraquat will kill adult C. elegans in liquid culture within 24 hours of exposure via the generation of superoxide anions that lead to oxidative damage of vital cellular components. The standard methods of growing the animals, treating with Paraquat, washing out the Paraquat, and observing survival at 12 hours post Paraquat treatment have been previously described. One group received only Paraquat as a positive control, while a second group received nothing as a negative control, and all others received the therapeutic treatments. At least 60 animals per group were used in the studies.

[0041] In the C. elegans series of studies, the human equivalent dose (HED) of tributyrin was determined in the following manner. A typical human stomach size of 250 ml was used as a baseline. 100 mg per serving per day of tributyrin for a 60 Kg adult is equivalent to 400 μg/ml (˜1.32 mM). Tributyrin is soluble in DMSO up to a maximum concentration of 100 mg/ml, which is equivalent to 330.73 mM. C. elegans can be exposed to a 1% solution of DMSO in aqueous standard nematode growth media without compromising its viability. Doses between 10 mg HED [40 μg/ml (˜0.13 mM)], and 100 mg HED [400 μg/ml (˜1.32 mM)] were used.

EXAMPLES

[0042] In addition to providing a method for determining a preferred therapeutic dose of tributyrin to effect health, the present disclosure also claims a novel therapeutic composition for tributyrin to effect health based on reduction to practice (Examples 1 and 2). In these examples, preferred human equivalent dosages of 10 to 100 mg per day were found to be y effective for generating therapeutic levels of butyrate and for enhancing health. An oxidative stress assay is a indirect method of determining whether the doses proposed in this study can produce beneficial butyrate in vivo (Example 3). Further, the present disclosure provides an enhanced or synergistic effect when tributyrin is combined with probiotics or prebiotics for the generation of butyrate (Example 4). The enhanced butyrate generation therapeutic effect demonstrated in these Examples is unique and not previously known.

Example 1

[0043] In this study using the fecal sample model system, low doses of tributyrin were used demonstrating (a) butyrate generation at HED between 50 and 100 mg/day oil, (b) equivalence of butyrate generation for tributyrin to a HED of 4 g/day of a prebiotic, i.e., inulin (common therapeutic dose). Since the therapeutic dose of inulin is known to be about 4 grams per day this experiment shows that tributyrin at HED of 50 mg and 100 mg/day are therapeutically effective butyrate generation dosages. This level of therapeutic dose for butyrate generation has not been known or demonstrated previously.

TABLE-US-00001 Human Butyrate Test Equivalent Generation Standard Test Dose Dose (per day) (mM) Deviation Blank 2.86 0.87 Inulin  4 g/L  4 g 3.62 1.52 Tributyrin  50 mg/L  50 mg 3.22 0.86 Tributyrin 100 mg/L 100 mg 3.73 0.88

[0044] The results from Example 1 show that low dose levels of tributyrin, between HED 50 and 100 mg/day generate about the same amount of butyrate as a therapeutic dose of inulin at an HED of 4 g/day. The differences in butyrate generation are not statistically different, but in all cases are statistically different from the blank (control).

Example 2

[0045] In this example, the same fecal sample method was used to study butyrate generation compared with a prebiotic XOS at a HED of 1 g/day (lower end therapeutic usage level). XOS (xylooligosaccharide) is a prebiotic derived from milk and is one of the lower therapeutic dose prebiotics available on the market. Therapeutic doses of XOS have been described as high as 2.8 g/day so the choice of 1 g/day is at the extreme lower end of a suitable prebiotic dose.

[0046] In this experiment, the results show equivalent butyrate generation between 100 mg/day HED tributyrin and 1 g/day HED XOS, indicating a therapeutic generation level with 100 mg/day tributyrin. The results for tributyrin and XOS are statistically significantly different from the control (blank), again demonstrating the therapeutic generation of butyrate by tributyrin at low doses.

TABLE-US-00002 Human Butyrate Test Equivalent Generation Standard Test Dose Dose (per day) (mM) Deviation Blank 2.78 0.67 XOS  1 g/L  1 g/L 3.30 0.64 Tributyrin 100 mg/L 100 mg 3.51 0.65

Example 3

[0047] In this example, the C. elegans model as described above was used to measure oxidative stress as a health condition, and specifically as an indirect measurement of butyrate generation in the nematodes C. elegans. An oxidative stress assay is a rapid indirect method of determining whether the doses proposed in this study can produce beneficial butyrate in vivo. The results illustrated below show significant reduction in oxidative stress with tributyrin HED of 10 and 100 mg/day. PHGG was used to demonstrate the comparative therapeutic benefit. In this series of experiments, butyrate was not measured directly in the organisms, but PHGG is a known butyrate generator which generates therapeutic levels of butyrate at a PHGG dose of 5-10 g/day. Oxidative stress reduction was used as a measure of butyrate generation and compared tributyrin at different dosages to PHGG. The results show that those organisms provided with tributyrin at a HED of 10 and 100 mg/day, survived as well as those organisms provided with PHGG, thus demonstrating a correlation between the benefits of butyrate generation from the tributyrin compared to a known probiotic.

TABLE-US-00003 Human % Survival Dose of Worms Equivalent at 12 Standard Test Test Dose (per day) hours Deviation Negative Control 98.69 1.33 Positive Control (Paraquat) 250 nM 67.63 9.99 PHGG  20 mg/mL  5 g 96.24 2.56 PHGG + Paraquat  20 mg/mL + 250 nM  5 g 49.39 3.98 PHGG  40 mg/mL  10 g 96.27 0.82 PHGG + Paraquat  40 mg/mL + 250 nM  10 g 62.99 3.98 Tributyrin  40 ug/mL  10 mg 97.24 2.85 Tributyrin + Paraquat  40 ug/mL + 250 nM  10 mg 68.86 4.07 Tributyrin 400 ug/mL 100 mg 99.23 1.25 Tributyrin + Paraquat 400 ug/mL + 250 nM 100 mg 73.63 2.90

Example 4

[0048] In this example, tributyrin is combined with two different probiotics in separate experiments specifically, Lacticaseibacillus rhamnosus GG (LGG) and Limosilactobacillus reuteri (REU). LGG is a gram-positive probiotic used to treat female urogenital tract infections and for the prevention of rotavirus diarrhea in children LGG is a known butyrate generator. REU is a lactic acid bacteria found in the GI tract and is used as a probiotic for the treatment of pediatric diarrheal disease. Although REU does not produce butyrate directly, it is capable of activating butyrate.

[0049] As in the other examples, the action of tributyrin (and in this case combined with probiotics), was compared with a prebiotic (XOS) with demonstrated therapeutic benefit. The results unexpectedly show a marked increase in butyrate generation versus tributyrin alone or the two probiotics—LGG and REU alone. The synergistic combination of tributyrin and each of the probiotics demonstrates an enhanced butyrate generation effect not previously observed. Coupled with the therapeutic dose of XOS, this example supports the assertion that tributyrin at a low dose, combined with a probiotic, produces enhanced butyrate generation with a therapeutic effect.

TABLE-US-00004 Human Butyrate Equivalent Generation Standard Test Test Dose Dose (mM) Deviation Blank 2.78 0.67 XOS  1 g/L  1 g 3.30 0.64 Tributyrin Oil 100 mg/L 100 mg 3.51 0.65 LGG 5 * 10{circumflex over ( )}7 CFU/mL 10{circumflex over ( )}7 CFU 3.15 0.67 Tributyrin Oil + LGG 100 mg/L + 5*10{circumflex over ( )}7 100 mg + 10{circumflex over ( )}7 9.03 0.90 REU 5 * 10{circumflex over ( )}7 CFU/mL 10{circumflex over ( )}7 CFU 2.72 0.66 Tributyrin Oil + REU 100 mg/L + 5*10{circumflex over ( )}7 100 mg + 10{circumflex over ( )}7 9.63 0.76

[0050] Powder Composition

[0051] Tributyrin is an oil. An oil form is suitable for applications such as with softgels and liquid capsules. However, the present disclosure also provides for a novel powder form of the present composition including tributyrin. This powder form contains partially hydrolyzed guar gum and other inert ingredients. Partially hydrolyzed guar gum is a prebiotic itself but has also been shown to improve absorption of active ingredients.

[0052] Unexpectedly, the addition of 10% partially hydrolyzed guar gum significantly lowered the water activity of the powder. The use of partially hydrolyzed guar gum for this purpose has not been previously known or described. The powder composition also contains acacia gum which has film forming properties, which is important for spray drying of powder. The powder composition contains between 30 and 70% tributyrin, so the quantity of powder used to meet the dose range for tributyrin is between 1.43×(70%) and 3.33×(30%) the amount of oil required. The powder form is suitable for other consumer formats such as capsules and tablets. Both the oil and powder forms are suitable for the therapeutic purpose provided in the present disclosure.

[0053] Two different tributyrin powder compositions were created as follows:

Composition A contains 50% tributyrin oil, 27% acacia gum, 10% partially hydrolyzed guar gum, 2% rosemary extract, 1% microcrystalline cellulose. Composition B contains 50% tributyrin oil, 37% acacia gum, 2% rosemary extract, 1% microcrystalline cellulose. The water activity for Composition A was measured to be 0.12. The water activity for composition B was measured to be 0.30. A water activity of less than 0.2 is necessary and desirable for combination with probiotics and/or other moisture sensitive dietary ingredients to create the powder composition.

[0054] The previous discussions focus generally on compositions of tributyrin for adult human oral consumption. However, with suitable dose modifications. these compositions are also suitable for animal (pet, livestock, etc.) oral consumption and human child oral consumption for the enhanced generation of butyrate to improve health, using known conversions.

[0055] The above disclosure relates primarily to the use of tributyrin for use in adult humans. However, tributyrin compositions between 10 and 100 mg per day human dose, based on a human weight of 70 kg, can be converted into animal doses, for pet or livestock applications, using known or suitable conversion factors. In dogs, for example, an oral equivalent dose range is calculated at approximately 0.257 to 2.57 mg/kg dog weight. Tributyrin compositions between 10 and 100 mg per day human dose, based on a human weight of 70 kg, can be converted into children's equivalent doses of approximately 0.143 to 1.43 mg/kg child weight. A slightly modified version of Clark's rule was used for this calculation—Clark's rule is based on a 68 kg adult; the present disclosure uses a 70 kg adult for the calculations. Other potential factors, including age, health conditions and/or disease/illness, other medications being administered, may also play a role to determine the dosage amount of tributyrin

[0056] It should be noted that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the spirit and scope of the present invention and without diminishing its attendant advantages. Further, references throughout the specification to “the invention” are nonlimiting, and it should be noted that claim limitations presented herein are not meant to describe the invention as a whole. Moreover, the invention illustratively disclosed herein suitably may be practiced in the absence of any element which is not specifically disclosed herein.

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