WO2009154463A2

WO2009154463A2 - Butyrate as a medicament to improve visceral perception in humans

Info

Publication number: WO2009154463A2
Application number: PCT/NL2009/050365
Authority: WO
Inventors: Steven Alfons Lieven William Vanhoutvin; Frederik Jan Troost; Robert Jan Maria Brummer; Konraad Venema; Henrike Maria Hamer; Dorothea Maria Agnes Elisa Jonkers
Original assignee: Stichting Top Institute Food And Nutrition
Priority date: 2008-06-20
Filing date: 2009-06-19
Publication date: 2009-12-23
Also published as: WO2009154463A3

Abstract

The invention relates to a new use of a butyrate source in human for improving visceral perception.

Description

BUTYRATE AS A MEDICAMENT TO IMPROVE VISCERAL PERCEPTION

IN HUMANS

Field of the invention The invention relates to a new use of butyrate in human to improve visceral perception.

Background of the invention

The human large intestine harbours a complex diversity of micro-organisms, which exert both positive and negative effects on gut physiology. The gut microbiota affects, the local and systemic immune system, has a role in the intestinal defense against pathogens and is important in the metabolism of nutrients and toxic compounds [I]. The two main types of bacterial metabolism that occur in the gut are saccharolytic and proteolytic fermentation. Saccharolytic fermentation is favorable for the host because it produces short chain fatty acids (SCFAs), such as butyrate, acetate and propionate. A variety of health promoting properties have been attributed to SCFAs. End products of proteolytic fermentation are less favorable, including nitrogenous metabolites, some of which are carcinogenic. The proximal colon is predominantly a site of saccharolytic fermentation, whereas in the distal colon, where carbohydrates are present only in minute amounts, mainly proteolytic fermentation takes place. This may partly explain why many gastrointestinal disorders may occur mainly in the distal colon.

Dietary ingredients which can increase the amount of saccharolytic fermentation throughout the gut and reduce proteolysis as treatment for intestinal disorders are currently under investigation. There is growing interest in functional foods which affect the composition and activity of the gut microbiota. One approach is the ingestion of live microorganisms (probiotics) that enter the gut and persist long enough to have beneficial effects on the host. Another one is to use non-digestible carbohydrates (prebiotics) that are selectively fermented by indigenous beneficial bacteria [2]. The health-promoting effects that have been attributed to probiotics and prebiotics are modulation of the immune system, improved digestion and absorption, vitamin synthesis, protection against the development of colon cancer, cholesterol reduction and inhibition of the growth and colonization of potential pathogens [3]. Short chain fatty acids produced during bacterial fermentation of non-digestible carbohydrates were reported to have several beneficial effects [4] . Increased colonic butyrate formation has often been proposed as one of the protective mechanisms of high fiber diets [5-7]. Butyrate is the major energy source for colonocytes [2], and it may act as a signal metabolite affecting epithelial cell proliferation and differentiation [8]. Besides, there is some evidence that butyrate beneficially affects several inflammatory parameters including nuclear factor kappa B (NF-kB), secretory IgA (SIgA), cytokines and myeloperoxidase (MPO) activity [9]. Furthermore, butyrate stimulates intestinal mucus production supporting the mucosal barrier function [10, 11], possesses anti- oxidative capacity [12, 13], increases mucosal blood flow [14], and may decrease colonic epithelial permeability [15, 16]. Paradoxically, a rat study suggested that butyrate causes an increase in colonic sensitivity[17].

Evidence for the beneficial effects of butyrate from human clinical studies is limited. Hallert et al. [18] instructed 22 patients with quiescent ulcerative colitis to add 60 g oat bran to this daily diet. During this 4-weeks intervention, the fecal butyrate concentration increased significantly by 36%; abdominal symptoms improved significantly at 12 weeks and returned to baseline 3 months later. No patient showed signs of colitis relapse in this short period of observation. Several other clinical studies have been published using rectal enemas containing butyrate or a mixture of SCFAs (acetate, propionate and butyrate) in patients with diversion colitis [19, 20], distal ulcerative colitis [21-29], radiation colitis [30]. In 1989, the first study that showed a positive effect of SCFA enemas in patients with diversion colitis was published by Harig et al. [19]. In this cross-over study, SCFA enemas (acetate 6OmM, propionate 3OmM, butyrate 4OmM) were administered twice daily to 5 patients for 2-6 weeks. Endoscopic appearance of the mucosa improved after 2 weeks of SCFA treatment and even more after 4-6 weeks. Histological findings paralleled endoscopic observations. With saline placebo, no consistent change in inflammation was observed. In another study, 10 patients with distal ulcerative colitis were treated for 2 weeks with sodium butyrate enemas (10OmM) and 2 weeks with placebo in random order (single blind cross over trial) and found a regression of clinical symptoms in the butyrate group but not in the placebo group [24]. In a follow-up study [25], 47 patients with distal ulcerative colitis received either a SCFA mixture enema (acetate 6OmM, propionate 3OmM, butyrate 4OmM), butyrate enema (10OmM) or placebo twice daily for 8 weeks. This study failed to show a significant advantage of SCFA or butyrate over placebo with regard to the disease activity index. In conclusion, two studies [21, 28] could not demonstrate beneficial effects of SCFA enemas on disease activity, others did find clinical, endoscopical and/or histological improvement using enemas with butyrate or a combination of SCFA in colitis patients [19, 23-25, 31].

Until present, the potentially beneficial effects of butyrate in humans have been studied in patients with distal ulcerative colitis. Although most studies in animals and human showed no adverse effects, two studies in rats showed that butyrate instillation in the colon increases visceral sensitivity [17, 32]. It was not known whether a comparable effect of butyrate enemas can be observed in human subjects. We recently carried out a double-blind randomized cross-over study in healthy subjects, and surprisingly showed a concentration dependent decrease of visceral sensitivity, i.e. an improvement of visceral perception, due to butyrate treatment in healthy volunteers. Effects of different dosages of butyrate were shown to decrease subjective scores of pain, urge and discomfort in all subjects in a dose-dependant way. Therefore, the present invention demonstrates for the first time that butyrate can be used as a new treatment in human for improving visceral perception and/or decreasing visceral hypersensitivity. According to the concept of the gut-brain axis, explaining the physiology of visceral perception, the effect of butyrate on visceral perception can be evoked anywhere along this axis ranging from the intestinal microbiota to the brain cortex.

Description of the invention

A butyrate source

In a first aspect of the invention, there is provided a butyrate source for use as a medicament for improving visceral perception and/or inducing a detectable decrease of visceral sensitivity in a human subject.

As defined herein, a "butyrate source" means any agent, chemical agent or living agent leading to the production of butyrate. Alternatively or in combination with previous definition, a "butyrate source" may also refer to a given concentration of butyrate directly administered to a human subject as later defined herein.

Bacterium/ non-digestible carbohydrate/digestible carbohydrate In a preferred embodiment, butyrate is produced by the action of a living agent such as a bacterium (i.e. via bacterial digestion or fermentation) which is administered to a human subject and/or which is already present in the intestine of a human subject. In a second preferred embodiment, butyrate is produced from a chemical agent preferably a carbohydrate. A carbohydrate may be a non-digestible, non-digested or a digestible carbohydrate. A carbohydrate is preferably an oligosaccharide or a polysaccharide. An oligosaccharide is defined herein as a saccharide polymer with a small number (usually 3 to 10) of components sugars, also known as simple sugars. A polysaccharide is a relatively more complex carbohydrate than an oligosaccharide. Polysaccharides are polymers made up of many monosaccharides joined together by glycosidic bonds. More preferably, a carbohydrate is a non-digestible carbohydrate, even more preferably a non-digestible oligosaccharide (NDO) or a non-digestible polysaccharide which is administered to a human subject. In a third preferred embodiment, butyrate is produced by the combined action of a bacterium and a carbohydrate, wherein both are preferably administered to a human being or wherein only a carbohydrate is administered to a human being. Within the context of the invention, "non-digestible" carbohydrate preferably means that the human being does not have the intestinal enzymes to digest this carbohydrate. However, this carbohydrate is expected to be digested or fermented by (endogenous) intestinal bacteria. This digestion or fermentation mainly occurs in the colon. Preferred NDO include fructo-oligosaccharide (FOS), galacto-oligosaccharide (GOS). Inuline is a preferred non-digestible polysaccharide. Resistant starch and lactulose are other preferred non-digestible carbohydrates. Therefore in a preferred embodiment, a source of butyrate comprises a non-digestible carbohydrate, more preferably comprises a NDO, which will be converted at least partly into butyrate in the colon of a human being by the endogenous intestinal bacteria. In another preferred embodiment, a non-digested or a digestible carbohydrate is used. When one refers herein to a digestible carbohydrate, it may be that under specific circumstances, this digestible carbohydrate is a non-digested carbohydrate. A non- digested carbohydrate is a carbohydrate which is potentially a digestible carbohydrate as defined below, but which is not digested due to certain circumstances such as transit through the upper intestines, inaccessibiltiy by digestive enzymes (e.g. due to enclosure within a plant cell), or other reasons known to the skilled person in the art. To optimize the production of butyrate, a non-digested or a digestible carbohydrate is preferably formulated so that it will be as less as possible digested or degraded by the enzymes of the human being and will be as much as possible digested or fermented by intestinal bacteria. Such formulations are known to the skilled person as enteric formulations, encapsulations (coatings), and/or slow-release formulations are more extensively described later herein. Such types of formulation may also be used for any other butyrate source as defined herein: for example bacteria and/or non-digestible carbohydrate. Examples of digestible carbohydrate are lactose, starch and oligosaccharides derived from starch. In this preferred embodiment, a given amount of a digestible carbohydrate, a non-digested carbohydrate, a non-digestible carbohydrate and/or a given amount of a bacterium is administered to a human being. This amount of digestible carbohydrate, non-digested carbohydrate, non-digestible carbohydrate and/or of bacterium is preferably such that an increase of the internal concentration of butyrate in the lumen is inducible and/or detectable (detectable increase compared to the physiological concentration of butyrate) as defined herein. The concentration of butyrate in the lumen is preferably assessed using GC-MS (Gas Chromatograph- Mass Spectrometry) as described in Venema K et al (Venema K, et al (2005), Microbial

Ecology in Health and Disease, 17:47-57). More preferably, the increase is detectable when it is of at least 1%, 2%, 5%, 7%, 10%, 15%, 20%, 25%, or more. Usually, the physiological concentration of butyrate in the lumen of a human being is ranged between 11 and 25 mM (40). Therefore the administration of a butyral source may lead to a concentration of butyrate in the lumen which is ranged between 1.5 and 20 mM, or of at least 2OmM, or of at least 25mM, or of at least 3OmM, or of at least 35mM, or of at least 4OmM, or of at least 45mM, or of at least 5OmM, or of at least 55mM, or of at least 6OmM, or of at least 65mM, or of at least 7OmM, or of at least 75mM, or of at least 8OmM, or of at least 85mM, or of at least 9OmM, or of at least 95mM, or of at least 10OmM.

Preferred bacteria include one or more of the following: a food-grade bacterium, a commensal bacterium or a (attenuated) pathogenic bacterium. In one embodiment, a bacterium is a food-grade bacterium, particularly a gram-positive bacterium, and more preferably a lactic acid bacterium. A bacterium may also be a probiotic bacterium, which in itself has a beneficial effect when ingested by a subject. A preferred bacterium belongs to a genus selected from the group consisting of Lactobacillus, Lactococcus, Leuconostoc, Carnobacterium, Streptococcus, Bifidobacterium, Bacteroides, Eubacterium, Clostridium, Fusobacterium, Propionibacterium, Enterococcus, Staphylococcus, Peptostreptococcus, and Escherichia. A further preferred bacterium is a Lactobacillus or Bifidobacterium species selected from the group consisting of L. reuteri, L. fermentum, L. acidophilus, L. crispatus, L. gasseri, L. johnsonii, L. plantarum, L. paracasei, L. murinus, L. jensenii, L. salivarius, L. minutis, L. brevis, L. gallinarum, L. amylovorus, B. bifidum, B. longum, B. infantis, B. breve, B. adolescente, B. animalis, B. gallinarum, B. magnum, and B. thermophilum. It is to be noted that each of these bacteria is probably not able to directly produce butyrate. However, they indirectly induce butyrate production in the colon by changing the colon environment, so that other bacteria would be able to produce butyrate. It is for example known that lactic acid bacteria produce lactate, which will assist endogenous bacteria in a cross-feeding mechanism to product butyrate.

An amount of a bacterium ranged between 10⁷ and 10¹³ may be administered. Preferably, between 10⁹ and 10¹¹. As to the total quantity administered of non-digestible, non-digested and digestible carbohydrate, for example, quantities of 5 to 30 g of may be orally administered on a daily basis. Preferably, 5 to 15 g a day taken 1-3 times a day. In a preferred embodiment, at least 5 g of carbohydrate is administered per day to induce a production of at least 2OmM of butyrate in the lumen. In a more preferred embodiment, at least 10 to 20 g of carbohydrate is administered per day to induce a production of at least 50 to 100 mM of butyrate in the lumen. An amount of a bacterium ranged between approximately 10⁷ and approximately 10¹³ may be administered. Preferably, between approximately 10⁹ and approximately 10¹¹. As to the total quantity administered of non-digestible, non-digested and digestible carbohydrate, for example, quantities of approximately 5 to approximately 30 g of may be orally administered on a daily basis. Preferably, approximately 5 to approximately 15 g a day taken 1-3 times a day. In a preferred embodiment, at least approximately 5 g of carbohydrate is administered per day to induce a production of approximately at least 2OmM of butyrate in the lumen. In a more preferred embodiment, approximately at least 10 to 20 g of carbohydrate is administered per day to induce a production of at least approximately 50 to 100 mM of butyrate in the lumen. A preferred carbohydrate in this context is a non-digestible carbohydrate, more preferably an oligosaccharide, even more preferably a FOS or a GOS.

Depending on the identity of the digestible carbohydrate, non-digested carbohydrate, non-digestible carbohydrate and/or bacterium used, the skilled person may have to adapt the quantity used to reach the desired effect (i.e. increased quantity of butyrate produced in the lumen).

Preferably, a bacterium and/or a digestible carbohydrate and/or a non-digested carbohydrate and/or a non-digestible carbohydrate are suitable for convenient (oral) administration in one or more doses per day or per week.

Butyrate

Alternatively or in combination with earlier preferred embodiments, in another embodiment, a given quantity of butyrate is directly introduced or administered into a human being. Butyrate is a SCFA with a carbon chain length of 4. The term butyrate may encompass the linear unbranched butyrate (n-butyrate) or a branched chain or substituted butyrate. Depending on the desired effect, a butyrate source may comprise an amount of butyrate of at least 1 mmol per day administered to a human being or at least 2 mmol/day, or at least 3 mmol/day, or at least 4 mmol/day, or at least 5 mmol/day, or at least 6 mmol/day, or at least 7 mmol/day, or more. Depending on the desired effect, a butyrate source may comprise an amount of butyrate of at least approximately 1 mmol per day administered to a human being or at least approximately 2 mmol/day, or at least approximately 3 mmol/day, or at least approximately 4 mmol/day, or at least approximately 5 mmol/day, or at least approximately 6 mmol/day, or at least approximately 7 mmol/day, or more.

In a preferred embodiment, a butyrate source is a butyrate composition and is preferably for rectal administration, more preferably by rectal enema. An enema is a small bottle (typically 60ml as used in the example) with a long tip, used to infuse a solution (butyrate). Usually, a solution of a butyrate source is infused into the rectum using a rectal enema. Rectal enema has been extensively defined in the example. Rectal administration of a butyrate source may also be achieved using suppositories. Other modes of administrations of a butyrate source are not excluded: oral administration using a capsule or a tablet. Such tablet or capsule is preferably an enteric and/or a slow release formulation as later defined herein. Alternatively a butyrate source may be locally administered via a catheter or a pump.

As already mentioned herein, any butyrate source may be used to induce a detectable increase of the internal concentration of butyrate in the lumen as defined herein. An agent that may be used to reach such increase is by accelerating intestinal transit (35).

Within the context of the invention, "visceral sensitivity" is defined as being a subjective sensation thought to originate from the gut and felt by a human being, said sensation being associated with pain such as pain, urge or discomfort. The fact that a subjective sensation associated with pain (i.e. pain, urge or discomfort) is thought to originate from the gut is named "Visceral perception". Visceral sensitivity or visceral perception is preferably assessed by quantifying at least one of urge pain and discomfort as described in the example by a standardised barostat protocol. Alternatively or in combination with previous preferred embodiment, visceral sensitivity or visceral perception is preferably assessed by measuring the compliance as extensively described in the experimental part. Compliance is preferably assessed using a staircase distension protocol with pressure steps of about 3 mmHg, preferably 3 mmHg with a duration of 30 seconds each, preferably 30 seconds each and a range of 0-33 mmHg. Compliance is a parameter which represents the elasticity of the intestinal wall. It is measured as the volume increase as a result of an increase of lmmHg in intraluminal pressure (expressed as ml/mmHg). The slope of the pressure-volume curve is calculated at the steepest part of the curve. This slope is considered as the dynamic compliance, which may be affected by muscle tone and relaxation due to adaptation. Compliance may also be assessed by using the slope in the highest pressure range of the curve. This slope represents the static compliance, which may be less depending on muscle tone but more on elasticity of connective tissue. In the context of the invention, compliance is synonymous of dynamic compliance, which is inversely related to abdominal complaints.

As defined herein, visceral perception is preferably not linked with a specific disease or condition. Visceral perception may be altered as a result of e.g. an alteration in the composition of the intestinal intra-luminal content, changes in serotonergic activity of either the intestine and/or the brain, physiological and/or psychological stress and any alteration of intestinal barrier function. Within the context of the invention, a use of a butyrate source as defined herein may lead to an improvement of visceral perception or a detectable decrease of visceral sensitivity in a treated individual. It preferably means that at least one of urge, pain and discomfort is statistically significantly decreased and/or compliance is statistically significantly increased in said individual as assessed in the standardised barostat protocol as defined in the example. Preferably, a subject wherein a detectable decrease of visceral sensitivity has been reached was a healthy subject at the onset of the treatment.

Preferably, a pain score higher or equal to 10% at a pressure of 23mmHg or lower indicates that a subject suffers from altered visceral perception. The decrease is preferably measured by comparison with the value of the corresponding parameter in a healthy or control subject. In a preferred embodiment, a subject has an altered visceral perception and as a result of a use of a butyrate as defined in the invention, said subject has an improved visceral perception.

In a preferred embodiment, improving visceral perception is distinct from treating visceral hypersensitivity. Visceral hypersensitivity may be caused by any disease or condition originating from anywhere along the brain-gut axis and its associated systems such as intermediary metabolism and the immune system. Visceral hypersensitivity may be caused by a disease or condition of the gut. Visceral hypersensitivity may also be caused by an alteration in brain function. The use of a butyrate source as defined herein, preferably an amount of butyrate of at least 1 or at least 2 mmol/day or approximately at least 1 or at least 2 mmol/day and/or of a butyrate source which is able to induce a detectable increase of the amount of butyrate in the lumen is said to be functional (i.e. to be used for improving visceral perception ) when such butyrate source induces a statistically significant decrease of at least one of urge, pain and discomfort and/or a statistically significant increase of compliance as assessed in the standardised barostat protocol as defined in the example.

Alternatively or in combination with previous embodiment, a butyrate source is said to be functional in the lumen when such butyrate source induces a detectable decrease of visceral sensitivity or an improvement of visceral perception, which is synonymous of inducing a statistically significant decrease of at least one of urge, pain and discomfort and/or a statistically significant increase of compliance as assessed in the standardised barostat protocol as defined in the example. The decrease of at least one of pain urge and discomfort is preferably assessed by comparison with the initial value of a parameter in the subject before treatment. In a preferred embodiment, a decrease means that at least one of these parameters will approximately reach a physiological level of a corresponding control healthy subject not suffering from altered visceral perception. Approximately in this context may mean at least 1% difference with the corresponding value of a healthy subject or at least 5%, 10% difference with the corresponding value of a corresponding healthy subject not suffering from altered visceral perception..

The increase of compliance is preferably assessed by comparison with the initial value of said parameter in a subject before treatment. In a preferred embodiment, an increase means that said parameter will approximately reach a physiological level of a corresponding control healthy subject not suffering from altered visceral perception.. Approximately in this context may mean at least 1% difference with the corresponding value of a healthy subject or at least 5%, 10% difference with the corresponding value of a corresponding healthy subject not suffering from altered visceral perception..

A statistically significant increase or decrease as used herein preferably means that using the standardised barostat protocol as defined in the example with 11 subjects, based on the linear part of the curve of figures 3, 4 ,5, and 6 we obtain:

- a change of pain of at least 16% on the VAS score (figure 3), - a change of discomfort of at least 15,4% (figure 5) and/or

- a change of a minimum of one category on the 6 point scale for urge (figure 4)

- a change of compliance of -at least 0.49 ml/mmHg— (figure 6). Preferably, pain is used as parameter. The skilled person will understand that if one uses more than 11 subjects in said test, one would be able to detect smallest statistically significant increase or decrease . The use of a test with 11 subjects is thought to be representative enough to be able to assess the functionality of a butyrate source of the invention.

Alternatively, a subject does not suffer from an altered visceral perception and the use of a source of butyrate as defined herein will induce a decrease of the perception of the visceral sensitivity of said subject (decrease of at least one of urge, pain and discomfort and/or increase of compliance) or improvement of visceral perception. The term "decrease" has already be defined herein.

In the context of the invention, an organism or an individual or a subject is an human being. Preferably, an organism treated is suspected to have a high risk of developing an alteration of visceral perception due for example to potential genetic predisposition, and/or to the age of the subject and/or to the lifestyle of a subject (for example nutritional habit and/or to the absence of physical activity). Alternatively, a subject to be treated may be healthy.

The term "prevention" shall be understood to include complete prevention, prophylaxis, as well as statistically significantly decreasing the individual's risk of getting an altered visceral perception, preferably statistically significantly decreasing at least one of urge, pain and discomfort. The term shall also be understood to include alleviation of any of the symptoms associated with altered visceral perception (i.e. urge, pain and/or discomfort and/or compliance) already developed.

The term "treatment" or "treating" is understood the management and care of a patient for the purpose of combating the altered visceral perception. The term "treating" is also herein synonymous of "delaying" the development of any of the symptoms associated with altered visceral perception (i.e. urge, pain and/or discomfort and/or compliance), preferably associated with increased visceral sensitivity. Usually, one may expect that the delay in the development of a symptom is of approximately at least one day, one week or more or of at least one day, one week or more. In a preferred embodiment, a source of butyrate comprises at least one of: at least 5 g of a carbohydrate as defined herein, an amount of butyrate of approximately at least 1 or at least 2 mmol and - between approximately 10⁷ and approximately 10¹³ bacteria.

In a preferred embodiment, a source of butyrate comprises at least one of: at least 5 g of a carbohydrate as defined herein, an amount of butyrate of at least 1 or at least 2 mmol and - between 10⁷ and 10¹³ bacteria.

Said source of butyrate is preferably designed to be administered on a daily basis. Each of these components has been extensively defined earlier on.

Composition In a second aspect, there is provided a composition, preferably a food composition or a pharmaceutical composition comprising a butyrate source as defined herein. More preferably, a pharmaceutical composition comprises a butyrate source and at least one inert excipient.

Optionally, an additional active ingredient may be present in a food or pharmaceutical composition.

As additional active ingredient in a food or pharmaceutical composition, one may cite: an additional SCFA selected from the group comprising of acetate, propionate, valerate and caproate.

Other additional active ingredients in a pharmaceutical composition may be an ingredient which is normally classically used for treating an altered visceral perception such as a laxating agent, a spasmolithicum, a painkiller such as paracetamol and/or an anti-inflammatory agent.

Compositions for enteral or oral administration may be either food compositions or pharmaceutical compositions whereas compositions for rectal administration will usually be pharmaceutical compositions. Pharmaceutical compositions will usually comprise a pharmaceutical carrier in addition to a source of butyrate. The formulation of a composition depends on the intended mode of administration and (therapeutic) application and on the source of butyrate chosen. A pharmaceutical carrier can be any compatible, non toxic substance suitable to deliver a butyrate source to the GI-tract of a subject. E.g. sterile water, or inert solids or excipient may be used as the carrier usually complemented with pharmaceutically acceptable adjuvants, buffering agents, dispersing agents, and the like. Compositions will either be in liquid, e.g. a stabilized suspension of a butyrate source, or in solid and/or dry forms, e.g. a powder of lyophilized bacteria. E.g. for oral and rectal administration, the bacteria can be administered in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions. Rectal administration may be via a rectal enema as earlier defined herein. In this case, a butyrate source or composition is liquid or semi liquid. Alternatively, rectal administration may be using suppositories. In this case, a butyrate source or composition is solid or semi solid. In each type of rectal formulation of the composition, bacteria may be present in a solid dosage form. The bacteria as used in the invention may be dried and later on mixed with inactive ingredients and/or excipients and optionally encapsulated with for example gelatin to form gelatine capsules. Alternatively, the bacteria may be tabletted. Inactive ingredients or inert excipients are such as flavoring agents, stabilizers, sugars or other energy sources, buffering agents, thickeners, diluents, dispersing aids, emulsifiers, and/or binders. Examples of inactive agents are: glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate and the like. The bacteria may also be first added in a liquid form, after which the combination is dried. The use of a carbohydrate enriched media such as a dairy product, preferably milk may be used to this end. A preferred composition according to the invention is suitable for consumption by a subject, preferably a human. Such compositions may be in the form of a food supplement or a food or food composition, which besides a butyrate source also contains a suitable food base. A food or food composition is herein understood to include liquids for human consumption, i.e. a drink or beverage. The food or food composition may be a solid, semi-solid and/or liquid food or food composition, and in particular may be a dairy product, such as a fermented dairy product, including but not limited to a yoghurt, a yoghurt-based drink or buttermilk. Such foods or food compositions may be prepared in a manner known per se, e.g. by adding a butyrate source to a suitable food or food base, in a suitable amount. In such butyrate source or composition intended for oral administration, at least part or all of a butyrate source or composition may be formulated in a capsule, an enteric formulation and/or a slow- release formulation. Such capsule and/or formulation are well known to the skilled person (36, 37, 38, 39). This is a preferred embodiment, since it will protect at least part or all of a butyrate source or composition from degradation in the intestinal tract before reaching the intestine wherein butyrate will exert its action. If a digestible carbohydrate is present in a butyrate source or composition, it is preferred that it is formulated in such a capsule, enteric formulation and/or slow release formulation. In a further preferred embodiment, the bacteria are micro-organisms that are used in or for the preparation of a food or food composition, e.g. by fermentation. Examples of such bacteria include lactic acid bacteria, such as probiotic lactic acid strains as earlier exemplified herein. In doing so, the bacteria as used in the invention may be used in a manner known per se for the preparation of such fermented foods or food compositions, e.g. in a manner known per se for the preparation of fermented dairy products using lactic acid bacteria. In such methods, the bacteria as used in the invention may be used in addition to the micro-organism usually used, and/or may replace one or more or part of the micro-organism usually used. For example, in the preparation of fermented dairy products such as yoghurt or yoghurt-based drinks, a food grade lactic acid bacterium as used in the invention may be added to or used as part of a starter culture or may be suitably added during such a fermentation or may be added at the end of a fermentation. Preferably, a lactic acid bacterium is added at the end of the fermentation.

Use

In a further aspect, there is provided the use of a butyrate source or a composition comprising a butyrate source for the manufacture of a medicament for improving visceral perception in a human subject. All features of this use concerning the identity of the butyrate source, the types of composition used, the definition of visceral perception have already be defined herein.

Method

Accordingly, in a further aspect, there is provided a method for improving visceral perception using a butyrate source.

All features of this method concerning the identity of butyrate source, the types of composition used, the definition of visceral perception have already be defined herein.

In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition the verb "to consist" may be replaced by "to consist essentially of meaning that butyrate or a composition as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one". The word " approximately" or " about" when used in combination with a numerical value preferably means that said value +/- 1% of said value is encompassed by the present invention. The word " approximately" or " about" when used in combination with an integer (as example: approximately 5) preferably means that the invention also encompasses said integer minus 1 or said integer plus 1 (as example: 4 or 5 or 6 are encompassed by the present invention). Each embodiment as described herein may be combined with other embodiment(s) as described herein unless otherwise indicated.

All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety. The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.

Description of the figures

Figure 1. Design of barostat study consisting of 3 randomised cross-over test periods of 7 days, interspaced with 14 days wash-out period.

Figure 2. Catheter used. A commercially available catheter dedicated for rectal barostat measurements, (non-compliant poly-ethylene balloon with a volume of ca 600ml, connected to a double lumen catheter enabling intra-balloon pressure measurements)

Figure 3. The effect of 60 ml of a placebo, 5OmM or 10OmM butyrate (0, 3, 6 mmol of butyrate) intervention on pain scores measured on 100mm visual analogue scales.

Figure 4. The effect of 60 ml of a placebo, 5OmM or 10OmM butyrate (0, 3, 6 mmol of butyrate) intervention on urge scores measured on a six-point scale ranging from no urge (0) to maximal urge/ emergency stop (5)

Figure 5. The effect of 60 ml of a placebo, 5OmM or 10OmM butyrate (0, 3, 6 mmol of butyrate) intervention on discomfort scores measured on 100mm visual analogue scales.

Figure 6. Pressure- Volume relationship (compliance) for each dose of butyrate. No difference was found between 0 and 5OmM. Dynamic compliance was calculated at the inflection point (steepest point)

Examples Materials and methods

Subjects:

12 healthy volunteers (3 male and 8 female), participated in this study. None of the subjects had a history of gastrointestinal disease or previous abdominal surgery. This study was approved by the Ethics Committee of University Hospital Maastricht and all volunteers gave their written informed consent.

Study design: The study design consisted of three periods of one week, with a wash-out period of 2 weeks in between two test weeks, in which the volunteers self-administered rectal enemas containing a 60 ml solution of either, butyrate (10OmM), butyrate (5OmM) or placebo (saline) daily. The enemas were administered just prior to sleeping. The first 20 minutes after administration of the enema, the volunteers were instructed to stay on their left side. The enemas were made isotonic with sodium chloride at a pH of 7. Barostat measurements were performed at the start and the end of each test week (Figure 1). Prior to the study onset, each volunteer performed a "screening" barostat measurement to exclude possible learning effects that could bias the study measurements.

Test protocol:

Visceral sensitivity was measured using a highly standardised barostat protocol composed of several components. After arrival, the volunteer self-administered a rectal enema containing 60ml of saline. Five minutes after administration of the enema, subjects were instructed to void rectal contents in order to clean the rectum.

The volunteers were placed on a bed in a left lateral position and remained in this position during the entire test procedure. A commercially available barostat balloon (Mui Scientific, part: C7-2CB-R, see figure 2) was lubricated with KY gel (Johnsson & Johnsson) and inserted rectally 3 cm proximal to the anal sphincter. After a 5 minutes habituation period, the balloon was attached to the barostat equipment (Distender II, G&J electronics, Ontario, Canada) and the barostat procedure was started. The controlled distensions of the balloon were programmed using the standard software package of the barostat equipment (Protocol Plus Deluxe, version 6_7 ). All compositions and specifications of the catheter and the specific protocols used have already been extensively described in Whitehead et al 1997 (Whitehead WE et al, Dig. Dis. Sci., (1997), 42:223-241). The barostat protocol consisted of four parts, each designed for the measurement of specific parameters of interest: Balloon unfolding:

This very short part of the protocol consists of 1 single distension of 2020 mmHg during 1 minute and is needed to ensure the balloon is placed correctly without folds and wrinkles that may impair the airflow.

Minimal Distension Pressure (MDP) (The minimal balloon pressure needed to overcome the intra abdominal pressure) The next part of the protocol consisted of a stepwise distension protocol with pressure steps of 1 mmHg with a duration of 30 seconds and a range from 0-20 mmHg. The MDP was defined as the first pressure at which respiratory curves were present in the volume recording of the balloon. The entire protocol was followed, independent from the height of the measured MDP and therefore directly served as a sensitisation step prior to the compliance and perception measurements. The obtained MDP value was set as a reference point (set to zero).

Compliance (elasticity of the rectum in ml/mmHg)

Directly after finishing the MDP measurements the next step of the protocol was initiated. This part of the protocol, designed for compliance measurement, which represents the elasticity of the rectum in ml/mmHg, consisted of a staircase distension protocol with pressure steps of 3 mmHg with a duration of 30 seconds each and a range of 0-33 mmHg

Visceral perception

After the compliance measurement, the protocol for the visceral perception measurements was initiated. The distension protocol consisted of semi-random distensions (4,13,10,19,16,25,22,31,28,37,34,43,40,49,46,55,52,61,58,67,64,71 mmHg), with a duration of 1 minute, interspaced with 30 second intervals at MDP. Thirty seconds after the start of each distension, volunteers scored the sensation of pain and discomfort on a 10 cm Visual Analogue Scale (VAS) and urge on a 6-point scale (0= no feeling, 1= just sensible, 2= cleary sensible/ light urge, 3= normal urge, 4= strong urge/ have to run to toilet, 5= maximum/stop) represented by 6 buttons on an electronic control panel (disternder II perception panel) directly linked to the barostat equipment. The program was followed until the maximal score for pain, urge or discomfort was reached.

Data handling:

The pain and discomfort data were analyzed using a Gaussian non-linear regression. On the other hand, the urge data is a six level ordinal variable of interest. It therefore was analyze using a mixture of a logistic distribution (parameterized as a proportional-odds) and a gamma distribution (to introduce a frailty dependence) (33). The mean regression was imposed through the time variable to follow a logistic ('S-shape') curve. The model included pressure, 'mdp', first sensation, procedure (treated or not), and carry over effect as explanatory variables. The inference criterion used for comparing the models is their ability to predict the observed data, i.e. models are compared directly through their minimized minus log-likelihood. When the numbers of parameters in models differed, they were penalized by adding the number of estimated parameters, a form of the Akaike information criterion (AIC)(34). For each variable of interest, the dose as a continuous variable was then added to the model. The quality of life variable under consideration was found to be affected by the drug if the AIC decreased compared to the model not containing the treatment.

Results

Butyrate significantly affected/decreased/improved visceral perception compared to placebo in a dose-dependent way. For all three parameters (pain, urge, or discomfort) butyrate treatment resulted in a significant reduction of scores throughout the entire pressure range of the protocol (see figures 3, 4 and 5). The effect of butyrate on painscores is a factor 1.8 larger in the 100 mM concentration compared to the 50 mM concentration. For discomfort scores this factor is pressure dependent and varies from 1.5 at a pressure of 4 mmHg to 2.7 at a pressure of 70 mmHg. Only the highest dosage of butyrate increased the dynamic compliance, when measured at a pressure of 12mmHg, which was in the linear part of the graph (Fig 6). At a pressure of 12mmHg, the volume increased from 200ml (CI 197 -203ml) after placebo and 5OmM butyrate to 215ml (CI 212-218ml) after 10OmM. The dynamic compliance for 0, 50 and 10OmM of butyrate, measured at the inflection point was 14.31 ml/mmHg in all three treatments. Butyrate however lowered the pressure at which the inflection point occurred. For 0 and 5OmM of butyrate, the inflection point was 9.7 - 9.8 mmHg whereas a dose of 10OmM of butyrate significantly decreased the inflection point to 8.6 - 8. Compliance of the rectal wall significantly increased after one week 10OmM butyrate treatment, which suggests that butyrate beneficially affects the ability of the colon to adapt to intraluminal volume changes.

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Claims

1. A butyrate source for use as a medicament for improving visceral perception in a human subject.

2. A butyrate source according to claim 1, wherein the butyrate source induces a statistically significant decrease of at least one of urge, pain and discomfort and/or a statistically significant increase of compliance as assessed in the standardised barostat protocol as defined in the example.

3. A butyrate source according to claim 1 or 2, wherein the butyrate source comprises at least 1 or at least 2 mmol butyrate per day and/or is able to induce a detectable increase of the amount of butyrate in the lumen of the treated human subject.

4. A butyrate source according to any one of claim 1 to 3, wherein the butyrate source is a butyrate source for administration by rectal enema.

5. A butyrate source preferably according to any one of claim 1 to 4, wherein the butyrate source is present in a composition for oral consumption, said composition comprising a non-digestible carbohydrate and/or a digestible carbohydrate and/or a non-digested carbohydrate and/or a bacterium and/or butyrate.

6. A butyrate source according to any one of claims 1 to 5 comprising at least one of: at least 5 g of a carbohydrate as defined herein, an amount of butyrate of at least 1 or at least 2 mmol and between 10⁷ and 10¹³ bacteria.

7. A composition comprising a butyrate source as defined in any one of claim 1 to 6, said composition being a pharmaceutical composition or said composition being a food composition.

8. A food composition according to claim 7, comprising a butyrate source preferably according to claim 1 and 2, wherein the butyrate source is present in a composition for oral consumption, said composition comprising a non-digestible carbohydrate and/or a non-digested carbohydrate and/or a digestible carbohydrate and/or a bacterium and/or butyrate.

9. A pharmaceutical composition according to claim 7, wherein the composition comprises at least one inert excipient.

10. A pharmaceutical composition according to claim 7 or 9, wherein the composition comprises an additional active ingredient.

11. Use of a butyrate source or a composition comprising a butyrate source for the manufacture of a medicament for improving visceral perception in a human subject.

12. Use of a butyrate source according to claim 11, wherein the butyrate source comprises at least 1 or at least 2 mmol butyrate per day and/or is able to induce a detectable increase of the amount of butyrate in the lumen of the treated human subject.

13. Use of a butyrate source according to claim 11 or 12, wherein the butyrate source is a butyrate composition for administration by rectal enema.

14. Use of a butyrate source preferably according to any one of claims 11 to 13. wherein the butyrate source is present in a composition for oral consumption, said composition comprising a non-digestible carbohydrate and/or a non-digested carbohydrate and/or a digestible carbohydrate and/or a bacterium and/or butyrate.

15. Method for improving visceral perception using a butyrate source.