Patent Application
20230381218
The present disclosure relates to a prebiotic formula, a dietary fiber, a food and a pharmaceutical composition including the same, and an application thereof.
Prebiotics, as a nutrient source for probiotics, have an influence on the composition of the probiotics in the intestine and confer health benefits. Among them, water-soluble polysaccharides are appropriate raw materials due to their various sources and well-established manufacturing processes. Their physicochemical properties vary by size, chemical structure, solubility, viscosity, and fermentability, each of which influences microbial competence.
The water-soluble polysaccharides that is resistant to hydrolysis by digestive enzymes is commonly selected as prebiotic formula, such as inulin, fructo-oligosaccharides, glucan, gluco-oligosaccharides, galacto-oligosaccharides, and lacto-oligosaccharides.
However, oligosaccharides are easily consumed by facultative anaerobic Lactobacilli or oxygen-tolerant anaerobic Bifidobacteria while passing through the small intestine, resulting in small intestine bacterial overgrowth and leading to digestive abnormalities. Currently, product is still few on the market in design for metabolized by obligate anaerobes in the colon.
In addition, patients often discontinue immune checkpoint blockade (ICB) therapy due to limited response to the treatment or adverse events. Fortunately, recent findings suggest that modifying the intestinal microbiota can substantially improve efficacy of ICB therapy. Moreover, intestinal toxicity, causing adverse events such as diarrhea or colitis, is necessary to find a way to overcome. Therefore, the prebiotic formula is a suitable choice.
Accordingly, the present disclosure provides a solution to the above questions. In addition, the disclosure also aims to provide a dietary fiber, a food and a pharmaceutical composition made from the prebiotic formula, and the applications.
According to one or more embodiment of the present disclosure, a prebiotic formula comprises glucomannan polysaccharides with a main distribution range of molecular weight at 0.6-107 kDa, said low molecular weight glucomannan polysaccharides, having β-1,4-D-glucopyranose units and β-1,4-D-mannopyranose units in backbone, and a portion of mannosyl units acetylated.
Moreover, the present disclosure provides a dietary fiber, a food and a pharmaceutical composition including said prebiotic formula.
Furthermore, the prebiotic formula of the present disclosure mediates the functions of intestinal mucosa protection and intestinal immunity modulation. Said prebiotic formula is fermented by inducible microbiota in the intestine after ingestion. The type of said inducible microbiota includes the clostridial IV or XIV clusters or the genera of Clostridium, Bacteroides, Alistipes, Bifidobacterium, Lactobacillus, Enterococcus or Akkermansia.
Said intestinal mucosa protection includes, by said inducible microbiota, maintaining cell density of the intestinal epithelium, reducing the aberrancy of the intestinal mucosa, maintaining or improving integrity of the intestinal mucosal barrier, or increasing the immune tolerance of the intestinal mucosa.
Said intestinal immunity modulation includes, by said inducible microbiota, stimulating the activation of innate immune cells, facilitating the development and maturation of isolated lymphoid follicles (ILFs), or elevating the number of ILFs in the intestinal mucosa.
Additionally, the disclosed prebiotic formula mediates the functions of assisting ICB therapy in improving efficacy of ICB therapy, or reducing or preventing intestinal toxicity in the therapy. Said prebiotic formula is fermented by the inducible microbiota in the intestine after ingestion so as to elevate the number and facilitate the development and maturation of ILFs to provide mature adaptive CD4+ T, CD8+ T cells for the purpose of therapy.
In view of the above description, the disclosed prebiotic formula can protect intestinal mucosa, modulate intestinal immunity, or assist ICB therapy in improving efficacy of ICB therapy, or reducing or preventing the intestinal toxicity in the therapy.
The present disclosure will become better understood from the detailed description given hereinbelow and the accompanying drawings which are given by way of illustration only and thus are not intended to limit the present disclosure and wherein:
In the following detailed description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the disclosed embodiments. It will be apparent, however, that one or more embodiments may be practiced without these specific details.
Moreover, the verification of the experimental results in the embodiments of the present disclosure involves sacrifice of the experimental animals. In order to conform to the international “3Rs” initiative of animal welfare, the numbers of the experimental animals used in the present disclosure is limited, but it is sufficient to scientifically demonstrate the experimental results. Therefore, it should be understood that these embodiments mentioned below are not Intended to restrict the extent of the present disclosure.
The disclosed prebiotic formula comprises glucomannan polysaccharides with a main distribution range of molecular weight at 0.6-107 kDa, said low molecular weight glucomannan polysaccharides, having β-1,4-D-glucopyranose units and β-1,4-D-mannopyranose units in backbone, and a portion of mannosyl units acetylated.
Natural glucomannan polysaccharides are not readily fermented in the intestine owing to high molecular weight, and artificial hydrolysis is required.
Artificial hydrolysis consisting of acidic, enzymatic, ultrasonic or microwave hydrolysis is well established. The hydrolyzed are classified as oligosaccharides (<4 kDa) having 3 to 20 sugar units, low molecular weight polysaccharides (4-200 kDa) having 20 to 1,000 sugar units, less-high molecular weight polysaccharides (<1,000 kDa) having less than sugar units, and high molecular weight polysaccharides having more than 5,000 sugar units, depending on demand.
The disclosed prebiotic formula eschews being consumed by the facultative anaerobic Lactobacilli or the oxygen-tolerant anaerobic Bifidobacterium while passing through the small intestine, which enables them to reach the colon and be metabolized by obligate anaerobes.
In addition, since linear structure in aqueous solution, the disclosed prebiotic formula is reacted with a plurality of endo-1,4-β-mannosidase simultaneously, which is advantageously fermented by the intestinal bacteria. If the polysaccharide becomes curved, folded, or tangled due to elongation, the resulting steric hindrance may hinder the reaction.
Furthermore, a portion of mannosyl units is acetylated, and thus it facilitates hydration.
The disclosed prebiotic formula may be incorporated into a dietary fiber. In the subject, it may include other ingredients or food additives, which conform to the regulations and do not have negative influence on the effect of the prebiotic formula.
The disclosed prebiotic formula may be incorporated into a nutrient or supplement composition as food. In the subject, it may include other ingredients or food additives, which conform to the regulations and do not have negative influence on the effect of the prebiotic formula.
The disclosed prebiotic formula may also be incorporated into a pharmaceutical composition. In the subject, it may include other additives or active ingredients, which conform to the regulations and do not have negative influence on the effect of the prebiotic formula.
The disclosed prebiotic formula may be fermented by the inducible microbiota in the intestine after ingestion, which mediate the mechanisms of initiating intestinal mucosa protection and intestinal immunity modulation. The applications will be described in detail below.
The disclosed prebiotic formula is designed on the basis of the enzymatic characteristics of the metabolisms amongst the known intestinal bacteria. The composition of the inducible microbiota has the clostridial IV or XIV clusters, or the genera of Clostridium, Bacteroides, Alistipes, Bifidobacterium, Lactobacillus, Enterococcus or Akkermansia.
For example, the disclosed prebiotic formula in the intestine may be directly detected by glycan sensor/transcriptional regulator on the surface of Bacteroides ovatus (B. ovatus) or Bacteroides thetaiotaomicron (B. thetaiotaomicron) etc., inducing specific endo-1,4-β-mannosidase on the membrane leading to the primary hydrolysis. Meanwhile, acetyl-mannan esterase and β-glucosidase are also encoded to hydrolyze the acetyl groups and the glucosyl units, respectively. Moreover, Faecalibacterium prausnitzii (F. prausnitzii) or Roseburia intestinalis (R. intestinalis) may perceive the primary hydrolysate of free sugar with 5 to 7 units, expressing exo-1,4-β-mannosidase in the cytoplasm for the secondary hydrolysis. The residual hydrolysate is mainly non-reducing-end mannotriose or mannobiose, which may be further hydrolyzed by β-mannanase and metabolized to butyric acid by Clostridium butyricum (C. butyricum) or Bifidobacterium adolescentis (B. adolescentis) and so on. The symbiotic community is dominated rapidly depending on a cascade expanding.
Moreover, the primary hydrolysate may be fermented by the resident bacteria in mucus, such as Akkermansia muciniphila (A. muciniphila) or Bifidobacterium pseudolongum (B. pseudolongum), resulting in their population growth and inosine production. Mannotriose of the secondary hydrolysate may be fermented by the genera of Alistipes. The free disaccharides or monosaccharides may be fermented by the genera of Bifidobacterium or Lactobacillus for acetic acid or lactic acid production and their population growth. They may also be fermented by the genera of Enterococcus for lactic acid production to compete the population growth against the phylum of Proteobacteria.
The disclosed prebiotic formula may be fermented by the inducible microbiota in the intestine to modulate the innate immunity to protect the intestinal mucosa, thereby strengthening the intestinal mucosal barrier.
The term “intestinal mucosal barrier” herein refers to the barrier formed by the collective of epithelial cells and goblet cells together with the mucus secreted from them, and
Paneth cells together with the antimicrobial peptides produced by them, and it separates the intestinal microbiota from the immune cells in the lamina propria.
Although the detailed mechanism by which the inducible microbiota impacts the intestinal mucosa remains unclear, promoting the renewal of the epithelial cells is an effective way to maintain the intestinal mucosal barrier function. Autophagy of the epithelial cells is a self-restoring process, allowing amino acids or fatty acids to undergo oxidative phosphorylation in the mitochondria, which leads to the rapid consumption of oxygen and nutrients in the cell. Moreover, under the circumstance of hypoxia in the intestinal mucosa, the inflammatory factors expressed by immune cells may be reduced, alleviating the mucosal inflammation and further triggering more autophagy of the epithelial cells.
As an example of promoting autophagy, Enterococci may avoid the attacks from the host macrophages so that they may opportunistically infect the host. As a response to Enterococci infection, if the epithelial cells are infected with Enterococci, the epithelial cells will be stimulated to activate the autophagy-related gene, inducing autophagy in the epithelial cells so as to clear the bacterial infections within the cells.
Furthermore, autophagy may also help to recycle the cellular nutrients, thereby preventing the nutrients from leaking into the mucosa and causing E. coli infection.
The communication between the inducible microbiota and the microfold (M) cells in the intestinal mucosa may induce the type 3 innate lymphoid cells (ILC3) to regulate the differentiation of naive CD4+ T cells into either pro-inflammatory helper T cells (Th17) or anti-inflammatory regulatory T cells (Tregs). By repetitive stimulation of the balance between pro- and anti-inflammatory responses, the immune tolerance may be enhanced, and the integrity of the intestinal mucosal barrier may be strengthened, thereby defending against bacterial invasion in the intestines.
Furthermore, some of the lymphocytes in the lamina propria are attracted by the inducible microbiota to migrate to the epithelium and become the intraepithelial lymphocytes (IELs), thereby involved in the renewal of the epithelial cells and the surveillance of change in the mucosal bacteria. The IELs release cytokine IL-6 to stimulate the proliferation and differentiation of the crypt stem cells in the intestinal epithelium, leading to a tight alignment of the neighboring epithelial cells and an increase in the secretion of mucus by goblet cells so as to protect the intestinal mucosa from various bacteria, food antigens, or chemical substances. As such, this protects the intestinal mucosa against acute or chronic intestinal mucosal inflammation, polyps, or tumorigenesis.
Butyrate is a metabolite from butyric acid producing bacteria such as Clostridium butyricum (C. butyricum) or Faecalibacterium prausnitzii (F. prausnitzii) among the inducible microbiota. Since there lacks glucose and oxygen in the colon, the aerobic glycolysis of the epithelial cells is restricted, thereby relying on butyrate as their primary energy source.
The epithelial cells absorb and transport butyrate to the mitochondria, implementing β-oxidation and oxidative phosphorylation to generate energy. This process consumes the last oxygen under hypoxia, creating a predominantly anaerobic environment in the intestinal mucosa. Therefore, none of oxygen can effectively diffuse into the intestinal lumen, thereby maintaining the oxygen-exhausted condition so as to promote the growth and metabolism of the obligate anaerobes.
Under the anaerobic environment, hypoxia-inducible factor, HIF-1α, of ILC3 may be stabilized, facilitating the synthesis of the transcription factor, HIF-1, to promote the proliferation of ILC3 and express the retinoic-acid-related-orphan-receptor-gamma-t (RORγt), thereby becoming RORγt+ ILC3 cells.
In addition, with the communication by microfold cells, the resident mononuclear phagocytes within the lamina propria secrete IL-1β to stimulate the expression of Csf-2 (also known as GM-CSF) by RORγt+ ILC3 cells, promoting the proliferation per se and then differentiation into dendritic cells or macrophages. Among them, the dendritic cells secrete IL-10 to suppress inflammation and generate retinoic acid to activate RORγt+ ILC3 cells, thereby facilitating development of the cryptopatches and then maturation into the ILFs.
Since cancer cells inhibit immune checkpoints carried by cytotoxic T lymphocytes (CTLs), they cannot be recognized by CTLs generally, leading CTLs unable to trigger cytotoxic effects. Under ICB therapy, artificial monoclonal antibodies are applied to blockade the immune checkpoints, significantly increasing the sensitivity of the immune cells such as CTLs to recognize cancer cells and exert cytotoxic effects. Therefore, the quantity of the adaptive CD8+ T cells in the patient during ICB therapy is a factor influencing the efficacy.
The retinoic-acid-activated RORγt+ ILC3 cells can express the major histocompatibility complex II, and present non-specific peptides to naive CD8+ T cells, thereby aiding the development of the adaptive CD8+ T. Therefore, the ILFs may provide corresponding cells for the need in ICB therapy.
Furthermore, inosine, the metabolite produced by the inducible microbiota, may serve as nutrition for lymphocytes survived in the ILFs under glucose deprivation. This improves the metabolic adaptability of T cells so as to improve ICB therapy.
In summary, the disclosed prebiotic formula may increase the number and facilitate the development and maturation of ILFs in the intestinal mucosa to help the differentiation, development, and maturation of the adaptive CD4+ T, CD8+ T cells. As a result, improved efficacy may be obtained during ICB therapy.
The ILFs which become mature after stimulated by the inducible microbiota may provide an environment for the differentiation, development and maturation of the adaptive CD4+ T, CD8+ T cells. In the germinal center of the mature ILFs, the number of B cells is far more than that of the resident innate T lymphocytes. B cells primarily carry out the tasks regarding memory and antigen presentation to assist the innate T cells in modulating the development of the adaptive CD4+ T, CD8+ T cells.
The resident and mature adaptive CD8+ T cells within the ILFs avoid triggering toxicity, and they are called memory effector T lymphocytes meanwhile. After they enter peripheral circulation and infiltrate into lymphoid tissues of tumor, they can implement cytotoxic effects after induced by ICB. As a result, intestinal toxicity may be reduced or prevented in ICB therapy.
Moreover, to assess whether an individual's response to ICB therapy is favorable or not, the type and relative abundance of the patient's fecal microbiota may be analyzed and compared to the inducible microbiota.
The term “relative molecular weight” herein means the molecular weight calculated on the basis of the standards by gel permeation chromatography.
Table 1 shows the molecular weight of the standards S1-S6.
The mice are sacrificed on day 28 and subjected to routine pathological evaluations to assess the condition of the epithelial cells and the immune cells in the colonic mucosa.
The “aberrancy score” (AS) herein refers to the evaluation of tissue abnormalities or inflammation in the intestinal mucosa. The higher the score means the more aberrancy. The aberrancy score is calculated by the following equation, and the feature and grade for evaluation are shown in Table 2.
Aberrancy score=Inflammatory severity×Involvement+Inflammatory extent×Involvement+Epithelium regeneration×Involvement+Crypt damage×Involvement (30 sessions for each group are evaluated in blinded fashion by clinical pathology consultants.)
Table 2 shows histological scoring scheme for colitis.
Collectively, it is evident that the Formula/DSS group has an intact intestinal mucosal barrier function compared to the DSS group, thereby avoiding damage to the intestinal epithelium and inflammation caused by DSS.
In addition, in the Control and Formula groups, the number of resident CD8+ T cells within the ILFs is far less than that of CD4+ T cells. Compared to the DSS group, the Formula/DSS group has significantly more CD8+ T cells in the ILFs, suggesting that the adaptive CD8+ T cells will accelerate proliferation and differentiation when the mucosa is damaged by DSS.
Moreover, compared to the Control group, the Formula group has a greater number and larger size of the ILFs in the intestinal mucosa, suggesting that the inducible microbiota stimulates the activation of the innate T cells and the aggregation of B cells.
Furthermore, since the Formula group has a greater number of more mature ILFs compared to the Control group, suggesting that the ILFs have better efficient of providing CD4+ T or CD8+ T during ICB therapy.
Prior to ICB therapy, continuous consumption of the disclosed prebiotic formula may facilitate the inducible microbiota to stimulate the ILFs mature, facilitating the differentiation, development and maturation of the adaptive CD4+ T, CD8+ T cells, which may assist ICB therapy. Moreover, the assessment of the type and relative abundance of the fecal microbiota of a patient ingesting the prebiotic formula of the present disclosure may serve as an indicator of an individual's response to ICB therapy.
This non-provisional application claims priority under 35 U.S.C. § 119(a) on Patent Application No. 63/345,095 filed in U.S. on May 24, 2022, the entire contents of which are hereby incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| 63345095 | May 2022 | US |
