WO2017042337A1 - Short-chain fatty acids for use in the treatment of cardiovascular disease - Google Patents

Short-chain fatty acids for use in the treatment of cardiovascular disease Download PDF

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Publication number
WO2017042337A1
WO2017042337A1 PCT/EP2016/071317 EP2016071317W WO2017042337A1 WO 2017042337 A1 WO2017042337 A1 WO 2017042337A1 EP 2016071317 W EP2016071317 W EP 2016071317W WO 2017042337 A1 WO2017042337 A1 WO 2017042337A1
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Prior art keywords
scfa
chain fatty
short chain
fatty acid
treatment
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PCT/EP2016/071317
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French (fr)
Inventor
Dominik N. MÜLLER
Nicola WILCK
Andras Balogh
Hendrik BARTOLOMAEUS
Ralf Dechend
Lajos MARKO
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Max-Delbrück-Centrum Für Molekulare Medizin In Der Helmhotz-Gemeinschaft
Charité - Universitätsmedizin Berlin
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Publication of WO2017042337A1 publication Critical patent/WO2017042337A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

  • the invention relates to Short Chain Fatty Acids (SCFA), SCFA-derivatives and SCFA pro-drugs, for use in the treatment of cardiovascular disease.
  • SCFA Short Chain Fatty Acids
  • the invention further relates to the use of dietary fiber and/or isolated populations of intestinal SCFA-producing bacteria preferably in combination with SCFA for the prevention and treatment of cardiovascular disease, in particular to the treatment and/or prevention of cardiac arrhythmia and/or hypertensive heart disease.
  • Cardiovascular disease is a significant health problem in many countries and in 201 1 there were more than 150,000 deaths as a result of CVD in the United Kingdom. Cardiovascular diseases cause more than 800,000 deaths in the US each year. High blood pressure was listed as a primary or contributing cause of death for 326,000 Americans in 2006 and in 2010 it was estimated to cost the United States $76.6 billion in health care services, medications and missed days of work (Centers for Disease Control and Prevention, Lloyd-Jones et al, Circulation 2010; 121 :e1-e170). There is a clear need for new treatment strategies.
  • the gut microbiome is increasingly recognized as an important factor in human health. Lifestyle, diet and antibiotics are potent modulators of the microbiome. Gut bacteria release metabolites into the circulation of the host, where they influence important processes such as metabolism and inflammation. Short-chain fatty acids (SCFA), which are produced by certain bacteria in the colon, are known to induce regulatory T cells (Treg). Adoptive-transfer studies with Tregs demonstrated beneficial actions on cardiac structural and electrical remodeling, atherosclerosis and metabolic syndrome. To the knowledge of the inventors, the therapeutic potential of SCFA in treating cardiovascular or hypertensive heart disease has not been explored.
  • SCFA Short-chain fatty acids
  • the technical problem underlying the present invention is to provide novel or alternative means for treating and/or preventing cardiovascular disease.
  • the invention therefore relates to Short Chain Fatty Acid (SCFA) for use in the treatment of cardiovascular disease.
  • SCFA Short Chain Fatty Acid
  • the SCFA is selected from saturated fatty acids comprising six or less carbon atoms, or 5 or less carbon atoms, preferably 4 or less or 3 or less carbon atoms.
  • the Short Chain Fatty Acid (SCFA) for use according to the present invention is characterised in that the SCFA is propionic acid or propionate.
  • the Short Chain Fatty Acid (SCFA) for use according to the present invention is characterised in that the SCFA is butyric acid or butyrate.
  • the Short Chain Fatty Acid (SCFA) for use according to the present invention is characterised in that the SCFA is selected from the group consisting of formic acid, acetic acid, isobutyric acid, valeric acid, isovaleric acid or caproic acid, or formate, acetate, isobutyrate, valerate, isovalerate and caproate.
  • the Short Chain Fatty Acid (SCFA) for use according to the present invention is characterised in that the SCFA is administered as a salt, preferably a sodium, calcium or potassium salt.
  • the Short Chain Fatty Acid (SCFA) for use according to the present invention is characterised in that the SCFA is propionate and is administered as a sodium propionate, calcium propionate or potassium propionate.
  • the invention is based on the surprising finding that SCFA provided to a subject can have a therapeutic effect on various heart conditions. SCFA have been previously linked to immune modulation but a therapeutic effect on cardiovascular disease could not have been derived from the prior art.
  • the invention relates to Short Chain Fatty Acid (SCFA) for use as a medicament and/or food additive as described herein, wherein the treatment is a prophylactic and/or therapeutic treatment.
  • SCFA Short Chain Fatty Acid
  • the invention relates to Short Chain Fatty Acid (SCFA) for use as a medicament and/or food additive as described herein, wherein the prophylactic treatment relates to primary prophylaxis or secondary prophylaxis.
  • Prophylactic treatments for cardiovascular disease typically relate to following no-smoking regimes combined with healthy diets. Only limited possibilities exist for avoiding cardiovascular complications in patients at risk thereof.
  • the present invention provides a surprisingly effective means of reducing risk of cardiovascular disease through SCFA provision, either in the form of direct administration as a medication or by modulating intestinal bacteria to produce elevated levels of SCFA.
  • prophylaxis There are two main types of prophylaxis: primary and secondary. Any measure taken to prevent an illness before it occurs is referred to as primary prophylaxis. Secondary prophylaxis relates to procedures that help prevent illness recurrence after experiencing a disease. The present invention proves effective against the CVD described herein, and particularly effective against cardiac arrhythmia, which typically occurs after myocardial infarction. The treatment of a population of patients having suffered a heart attack, preventing future cardiac arrhythmia, is a preferred embodiment of the invention. The present invention therefore further relates to the use of Short Chain Fatty Acid (SCFA) as a medicament as described herein for the secondary prophylaxis of cardiac arrhythmia in subjects having previously experienced myocardial infarction.
  • SCFA Short Chain Fatty Acid
  • the invention relates to Short Chain Fatty Acid (SCFA) for use as a medicament as described herein, wherein the cardiovascular disease is characterised by an inflammation of cardiac tissue.
  • SCFA Short Chain Fatty Acid
  • the cardiovascular disease to be treated relates to hypertensive heart disease or hypertensive cardiac damage.
  • the cardiovascular disease to be treated relates to diastolic heart failure (heart failure with preserved ejection fraction; HFpEF).
  • the cardiovascular disease to be treated relates to systolic heart failure (heart failure with reduced ejection fraction (HFrEF).
  • Hypertensive heart disease includes a number of complications of high blood pressure that affect the heart. The symptoms and signs of hypertensive heart disease will depend on whether or not it is accompanied by heart failure. Patients can present acutely with heart failure and pulmonary edema due to sudden failure of pump function of the heart. Acute heart failure can be precipitated by a variety of causes including myocardial ischemia, marked increases in blood pressure, or cardiac arrhythmias, especially atrial fibrillation.
  • the present invention appears particularly effective in the treatment of hypertensive heart disease.
  • the invention relates to Short Chain Fatty Acid (SCFA) for use as a medicament as described herein, wherein the cardiovascular disease is a cardiac arrhythmia.
  • SCFA Short Chain Fatty Acid
  • the cardiac arrhythmia relates to sinus tachycardia, atrial tachycardia, atrial fibrillation, atrial flutter, ventricular fibrillation, ventricular tachycardia, non- sustained ventricular tachycardia and/or ventricular ectopic beats.
  • Cardiac arrhythmia also known as cardiac dysrhythmia or irregular heartbeat
  • cardiac dysrhythmia or irregular heartbeat is a group of conditions in which the heartbeat is irregular, too fast, or too slow.
  • Symptoms can include lightheadedness, passing out, shortness of breath, chest pain, and may predispose a person to complications such as stroke or heart failure, or even cardiac arrest.
  • Medical procedures for treating arrhythmia typically relate to the use of a pacemaker, and surgery. Novel means are required for arrhythmia treatment.
  • the present invention presents a surprising development in the field of arrhythmia treatment, no indication has been provided previously that arrhythmia could be treated effectively using the compounds or approaches described herein.
  • the invention relates to Short Chain Fatty Acid (SCFA) for use as a medicament as described herein, wherein the subject of treatment (patient) exhibits one or more of the following attributes:
  • a) shows symptoms of being at risk of developing a cardiovascular disease and/or heart failure; b) has elevated levels of risk markers in ex vivo tests;
  • d) exhibits a predisposition of developing cardiovascular disease, for example a genetic predisposition of developing cardiovascular disease
  • e exhibits borderline or established thickening of the left ventricular wall (hypertrophy);
  • f shows an abnormal electrocradiogram/Holter monitor or echocardiogram.
  • the subject to be treated is at risk of developing atrial and/or ventricular cardiac arrhythmias.
  • An intended subject of the present invention is, in particular, a subject who is at risk of developing heart failure with preserved ejection fraction (HFpEF).
  • HFpEF patients typically have an increased risk of developing various kinds of cardiac arrhythmias and typically exhibit cardiac hypertrophy.
  • the symptoms of being at risk of developing a cardiovascular disease are considered, without limitation, as the presence of arterial hypertension, metabolic syndrome, high LDL serum levels, low HDL serum levels, diabetes mellitus and/or insulin resistance.
  • the presence of left ventricular myocardial hypertrophy represents a symptom of being at risk of developing a cardiovascular disease.
  • the markers indicating risk of cardiovascular disease relate to high-sensitivity C-reactive protein, and/or atrial natriuretic peptides/peptide precursors (e.g. BNP and NTpro-BNP) as measured in serum and/or plasma.
  • high-sensitivity C-reactive protein and/or atrial natriuretic peptides/peptide precursors (e.g. BNP and NTpro-BNP) as measured in serum and/or plasma.
  • SCFA Short Chain Fatty Acid
  • cardiomyopathy myocarditis, valvular heart disease, congenital heart disease, coarctation, atrial and/or ventricular septal defects, cerebrovascular insult, pericardial disease and/or preferably combinations of two and more thereof.
  • the subject of treatment exhibits (i) relatively low levels of bacteria capable of SCFA-production in their intestine and/or (ii) relatively low levels of SCFA in their intestine, compared to a suitable control, for example a healthy subject and/or a population average.
  • the present invention is characterised by the relationship between SCFA and heart conditions, and is not limited to the administration of SCFA alone as a medication.
  • the production of SCFA by intestinal bacteria is a known concept; but the modulation of these bacteria in order to treat heart disease, in particular the various specific embodiments of cardiovascular disease disclosed herein, has not previously been proposed.
  • the present invention therefore provides a number of aspects linked by a unifying concept, these aspects relate to the use of SCFA as a medicament, the use of intestinal bacterial populations as a medicament, optionally in combination with dietary fiber, to treat or prevent cardiovascular illness.
  • the modulation of SCFA production in the intestine of subjects also is encompassed by the present invention.
  • the SCFA are orally administered to a subject.
  • Oral administration enables direct and effective delivery of SCFA to the intestine, where the SCFA are absorbed and distributed to the rest of the body.
  • the invention further relates to Short Chain Fatty Acid (SCFA) for use as a medicament as described herein, wherein SCFA are orally administered in nutritional (food and/or beverage) products.
  • SCFA Short Chain Fatty Acid
  • the invention relates to Short Chain Fatty Acid (SCFA) for use as a medicament or food additive as described herein, wherein the subject of treatment is coadministered with an isolated population of intestinal bacteria, preferably sources of bacterial SCFA production in the human gut, and/or dietary fiber. Suitable bacteria are disclosed herein.
  • SCFA Short Chain Fatty Acid
  • the invention relates to Short Chain Fatty Acid (SCFA) for use as a medicament or food additive as described herein, wherein the SCFA are administered in the form of dietary fiber as a substrate for SCFA production via bacteria present in the intestine of a subject.
  • SCFA Short Chain Fatty Acid
  • a further aspect of the invention therefore relates to dietary fiber for use as a substrate in SCFA production by intestinal bacteria in the treatment of cardiovascular disease. Suitable dietary fibers are disclosed herein.
  • a further aspect of the invention relates to an isolated population of intestinal bacteria for the treatment of cardiovascular disease, wherein said bacterial population produces elevated levels of SCFA compared to a suitable control.
  • a suitable control may relate to subject not having been treated with SCFA producing bacteria, or a population average.
  • the embodiments described above are bound by a common concept of SCFA production being linked to the treatment or prevention of cardiovascular disease.
  • the gut microbiota is widely termed as the bacterial community that lives within an individual's gastrointestinal tract or elsewhere on or in the body.
  • the gut alsowise referred to as the intestine
  • the collection of genes present in a gut microbiota, the microbiome exceeds the number of host genes by far.
  • Known physiologic functions of the gut microbiome include digestion and nutrient uptake, protection from pathogen invasion, promotion of tissue differentiation, and stimulation of the immune system.
  • microbiome for the development of diseases has been recently recognized.
  • the microbiome is sensitive to changes in lifestyle and diet.
  • widespread use of antibiotics substantially impact microbiome composition, with consequences for the metabolism of the host.
  • metabolic diseases e.g. rheumatoid arthritis, colitis
  • differences between the microbiomes from diseased and non- diseased individuals have been demonstrated.
  • metabolites produced by a given microbiome further adds information on the functional properties of a community.
  • Products of microbial metabolism act as signaling molecules and influence the host organism. Besides influencing intestinal function, microbial metabolites are resorbed and affect the liver, the brain, the lungs and even the heart.
  • Bacterial metabolites known to influence metabolism, immune cells and vascular function include secondary bile acids, trimethylamine and SCFA, respectively.
  • the enhancement of a function of the gut microbiome is therefore one aspect of the present invention, whereby the SCFA production by the microbiome can be enhanced and supplemented by administration of SCFA substrate, such as dietary fiber, thereby leading to enhanced production of SCFA in the gut of the subject, or by administering the SCFA directly to a patient in need thereof.
  • SCFA substrate such as dietary fiber
  • Short-chain fatty acids
  • SCFA carboxylic acids of bacterial origin that are produced by the gut microbiota through fermentation of partially and nondigestable polysaccharides (dietary fiber).
  • SCFA are a subset of saturated fatty acids, preferably comprising six or less carbon atoms that include acetate, propionate, butyrate, pentanoic and hexanoid acid. They are highly abundant in the colon but can also be detected in the blood. Despite being an energy source for certain bacteria and intestinal epithelial cells, their immunomodulatory effects have been highlighted in a number of studies.
  • SCFAs elicit their anti-inflammatory effects through binding to endogenous receptors such as the G-protein-coupled receptors (GPCRs) GPR41 , GPR43, GPR109 or so far unidentified unknown GPCRs and eventually through their capacity to inhibit histone deacetylase (HDAC) activity.
  • GPCRs G-protein-coupled receptors
  • HDAC histone deacetylase
  • SCFA alter gene expression in a variety of cells through inhibiting the removal of acetyl groups from histones by HDACs.
  • HDAC inhibition increases the expression of the transcription factor forkhead box P3 (Foxp3) in T cells, which subsequently increases proliferation and function of Tregs. Compared to butyrate and propionate, acetate seems to lack the inhibitory effect on HDACs.
  • the SCFA is selected from saturated fatty acids comprising six or less carbon atoms, or 5 or less carbon atoms.
  • the SCFA is formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid or caproic acid, or formate, acetate, propionate, butyrate, isobutyrate, valerate, isovalerate, caproate, preferably propionic acid or propionate.
  • the present invention relates to SCFAs in various forms, including SCFA-derivatives or SCFA- pro-drugs.
  • the preferred SCFA of the present invention including acetate, propionate, butyrate, pentanoic and hexanoid acid, may be derivatized to enable modified behavior of the SCFA compound with respect to in vivo half-life, packaging efficiency, production, modified taste or smell, for example by producing derivatives of SCFA as described below.
  • esterified compounds disclosed herein relate potentially to pro-drugs, enabling SCFA release post-ad m i n istration .
  • SCFA compounds are disclosed in WO 2012/131069, which is herein incorporated in its entirety, that may be administered according to the present invention.
  • SCFAs according to the present invention relate preferably to SCFA compounds of the following formula: wherein
  • X represents -0-, -S-. or -NH-, preferably -0-;
  • R represents hydrogen, alkyi, aryl, arylalkyl, polyalkylene glycol
  • R1 represents hydrogen, alkyi, hydroxyalkyl, arylalkylcarboxylic acid,
  • R2 represents hydrogen, alkyi, -0-R3;
  • R3 represents hydrogen, aryl, arylalkyl, hydroxyalkyl-carboxyl
  • cardiovascular disease or pharmaceutically acceptable salts thereof, for use in the treatment, prevention or attenuation of cardiovascular disease, or any particular cardiovascular disease as described herein.
  • the compound of formula (I) is a compound, wherein
  • X represents -0-, -S-, or -NH-, preferably -0-;
  • R represents hydrogen, alkyi, aryl, arylalkyl, polyalkylene glycol
  • R1 represents hydrogen, alkyi, hydroxyalkylcarboxylic acid
  • R2 represents hydrogen, alkyi, -0-R3;
  • R3 represents hydrogen, aryl, arylalkyl, hydroxyalkyl-carboxyl
  • the compound of formula (I) is a compound according to the invention and as described herein in the various embodiments, wherein
  • X represents -0-, -S-, or -NH-, preferably -0-;
  • R represents hydrogen, C1 -C6 alkyi, unsubstituted or substituted phenyl with one or more, same or different, substituents selected from the group consisting of nitro, halogen, amino, hydroxyl, cyano, C1 -C4 alkyloxy or trifluoro;
  • R1 represents hydrogen, carboxylic acid, C1-C6 alkyi, hydroxy-C1 -C6 alkyi wherein the alkyi group may be unsubstituted or substituted with one or more, same or different, substituents selected from the group consisting of hydroxyl, amino, carboxylic acid, halogen, cyano, or nitro;
  • R2 represents hydrogen, C1-C6 alkyi, -0-R3; and R3 represents hydrogen, unsubstituted or substituted phenyl with one or more, same or different, substituents selected from the group consisting of nitro, halogen, amino, hydroxyl, cyano, C1-C4 alkyloxy or trifluoro, phenyl-C1-C6 alkyl wherein the phenyl group may be unsubstituted or substituted with one or more, same or different, substituents selected from the group consisting of nitro, halogen, amino, hydroxyl, cyano, C1 -C4 alkyloxy or trifluoro, hydroxy-C1-C6 alkyl-carboxyl;
  • the compound of formula (I) is a compound according to the invention and as described herein in the various embodiments, wherein
  • X is -0-
  • R is hydrogen
  • R1 represents hydrogen, carboxylic acid, C1 -C4 alkyl. hydroxy-C1 -C4 alkyl wherein the alkyl group may be unsubstituted or substituted with one or more, same or different, substituents selected from the group consisting of hydroxyl, amino, or carboxylic acid, preferably hydroxyl and/or carboxylic acid; and
  • R2 is hydrogen or C1-C4 alkyl; or pharmaceutically acceptable salts thereof.
  • the compound of formula (I) is a compound according to the invention and as described herein in the various embodiments, wherein
  • X is -0-
  • R is hydrogen
  • R1 represents hydrogen, carboxylic acid, C1-C4 alkyl, hydroxy-C1 -C4 alkyl wherein the alkyl group may be unsubstituted or substituted with one or more, same or different, substituents selected from the group consisting of hydroxyl, amino or carboxylic acid, preferably hydroxyl and/or carboxylic acid; and
  • R2 is -OR3
  • R3 represents hydrogen, unsubstituted or substituted phenyl with one or more, same or different, substituents selected from the group consisting of nitro, halogen, amino, hydroxyl, cyano or methoxy, phenyl-C1 -C4 alkyl wherein the phenyl group may be unsubstituted or substituted with one or more, same or different substituents selected from the group consisting of nitro, halogen, amino, hydroxyl, cyano or methoxy, hydroxy-C1-C3 alkyl-carboxyl; or pharmaceutically acceptable salts thereof.
  • the compound of formula (I) is a compound according to the invention and as described herein in the various embodiments, wherein
  • X is -0-
  • R is hydrogen;
  • R1 represents hydrogen, carboxylic acid, C1-C3 alkyl, hydroxy-C1 -C3 alkyl wherein the alkyl group may be unsubstituted or substituted with one or more, same or different, substituents selected from the group consisting of hydroxyl and/or carboxylic acid; and
  • R2 is hydrogen or C1-C4 alkyl; or pharmaceutically acceptable salts thereof.
  • the compound of formula (I) is a compound according to the invention and as described herein in the various embodiments, wherein
  • X is -0-
  • R is hydrogen
  • R1 represents hydrogen, carboxylic acid, C1-C3 alkyl, hydroxy-C1 -C3 alkyl wherein the alkyl group may be unsubstituted or substituted with one or more, same or different, substituents selected from the group consisting of hydroxyl or carboxylic acid;
  • R2 is -OR3
  • R3 represents hydrogen, unsubstituted or substituted phenyl with one or more, same or different, substituents selected from the group consisting of nitro, halogen, amino, hydroxyl, cyano or methoxy, phenyl-C1 -C4 alkyl wherein the phenyl group may be unsubstituted or substituted with one or more, same or different, substituents selected from the group consisting of nitro, halogen, amino, hydroxyl, cyano, or methoxy, hydroxy-C1-C3 alkyl- carboxyl; or pharmaceutically acceptable salts thereof.
  • the compound of formula (I) is a compound according to the invention and as described herein in the various embodiments, wherein
  • X is -0-
  • R is hydrogen
  • R1 is selected from the group consisting of hydrogen, methyl, ethyl, n-propyl, isopropyl, hydroxy methyl, dihydroxymethyl, hydroxyethyldicarboxylic acid, carboxylic acid
  • R2 is selected from the group consisting of hydrogen, hydroxyl or methyl; or pharmaceutically acceptable salts thereof, or wherein
  • X is -0-
  • R is hydrogen
  • R1 is selected from the group consisting of hydrogen, methyl, ethyl, n-propyl, isopropyl;
  • R2 is -OR3;
  • R3 is selected from the group consisting of 1 -hydroxyethylcarbonyl, benzyl, nitrophenyl; or pharmaceutically acceptable salts thereof.
  • the compound of formula (I) is a compound according to the invention and as described herein in the various embodiments, wherein
  • X is -0-
  • R represents CrC4 alkyl, unsubstituted or substituted phenyl with one or more, same or different, substituents selected from the group consisting of nitro, halogen, amino, hydroxyl, cyano or methoxy, phenyl-C C4 alkyl wherein the phenyl group may be unsubstituted or substituted with one or more, same or different, substituents selected from the group consisting of halogen, nitro, amino, hydroxyl, cyano or methoxy, polyalkylene glycol;
  • R1 is carboxylic acid, C1 -C4 alkyl or hydroxy-C1 -C4 alkyl, wherein the alkyl group may be unsubstituted or substituted with one or more, same or different, substituents selected from the group consisting of hydroxyl, amino or carboxylic acid; and
  • R2 is hydrogen; or pharmaceutically acceptable salts thereof.
  • the compound of formula (I) is a compound according to the invention and as described herein in the various embodiments, wherein
  • X is -0-
  • R represents C1-C4 alkyl, unsubstituted or substituted phenyl with one or more, same or different, substituents selected from the group consisting of nitro, halogen, amino, hydroxyl, cyano or methoxy, phenyl-C1 -C4 alkyl wherein the phenyl group may be unsubstituted or substituted with one or more, same or different, substituents selected from the group consisting of halogen, nitro, amino, hydroxy], cyano or methoxy, polyalkylene glycol;
  • R1 is carboxylic acid, C1 -C3 alkyl or hydroxy-C1 -C3 alkyl wherein the alkyl group may be unsubstituted or substituted with one or more, same or different, substituents selected from the group consisting of hydroxy! and/or carboxylic acid; and
  • R2 is hydrogen; or pharmaceutically acceptable salts thereof.
  • the compound of formula (I) is a compound according to the invention and as described herein in the various embodiments, wherein
  • X is -0-
  • R is selected from the group consisting of methyl, ethyl, propyl, benzyl, nitrobenzyl, polyethylene glycol;
  • R1 is selected from the group consisting of ethyl, hydroxyethyl, methyl, hydroxymethyl;
  • R2 is hydrogen; or pharmaceutically acceptable salts thereof.
  • the compounds of this invention may contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and
  • the compounds of this invention may also contain linkages (e. g., carbon- carbon bonds) wherein bond rotation is restricted about that particular linkage, e. g. restriction resulting from the presence of a ring or double bond. Accordingly, all cis-trans and E/Z isomers are expressly included in the present invention.
  • the compounds of this invention may also be represented in multiple tautomeric forms, in such instances, the invention expressly includes all tautomeric forms of the compounds described herein, even though only a single tautomeric form may be represented (e. g.
  • the compounds of formula (I) of the invention as described herein in the various embodiments, or a pharmaceutically acceptable salt thereof, or a composition comprising the SCFA compound of formula (I) according to the invention and as described herein, or a pharmaceutically acceptable salt thereof, particularly in a therapeutically effective amount, optionally, together with a pharmaceutically acceptable carrier, is used in the treatment, prevention or attenuation of one or more of the cardiovascular diseases, as described herein.
  • Alkyl as such means a straight-chained or branched saturated aliphatic hydrocarbon having from 1 to 10 carbon atoms, wherein the alkyl group may be unsubstituted or substituted with one or more, same or different, substituents selected from the group consisting of hydroxyl, amino, carboxylic acid, halogen, cyano, or nitro.
  • C1 -C4 alkyl such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl.
  • “Hydroxyalkyl” stands for one of the above-defined alkyl groups wherein at least one hydrogen atom is replaced by a hydroxyl group and wherein the hydroxyalkyl group may be unsubstituted or substituted with one or more, same or different substituents selected from the group consisting of hydroxyl, amino, carboxylic acid, halogen, cyano, or nitro.
  • Typical representatives are -CH20H, -CH2CH20H, -CH(OH)-CH3, -CH(OH)CH2CH3, CH2CH(CH2CH20H)CH2CH3I etc..
  • Aryl means a monovalent, monocyclic, bicyclic or tricyclic, aromatic carbocyclic hydrocarbon radical, preferably a 6-14 member aromatic ring system.
  • Preferred aryl groups include, but are not limited to phenyl, naphthyl, phenanthrenyl, and anthracenyl, wherein the aryl group may be unsubstituted or substituted with one or more, same or different substituents selected from the group consisting of halogen; alkyl; alkyloxy, cyano, trifluoro, nitro, amino, hydroxyl.
  • Alkoxy means -O-alkyl, wherein alkyl has the meaning given above.
  • Halogen means fluorine, chlorine, bromine, or iodine, preferably fluorine, chlorine or iodine.
  • Polyalkylene glycol means a moiety that comprises at least two aklylene glycol units such as - O-alkyl-O-alkyl-O-moiety wherein alkyl have the meaning given above.
  • the polyalkylene glycol moiety may be solely comprised of polyalkylene glycol, or may be part of a larger structure, such as polyoxyalkylated glycerol and other polyoxyalkylated polyols such as polyoxyethylated sorbitol or polyoxyethylated glucose.
  • the number of alkylene units may vary and is greater than 1 .
  • polyalkylene glycol are polyethylene glycol (PEG) or polypropylene glycol (PPG). Most preferred polyalkylene glycol are PEG wherein the number of ethylene units may vary from 8 to 150.000 or more, particularly from 10 to 80.000, more particularly from 20 to 10.000.
  • propionate refers preferably to the pharmaceutically acceptable salt of propionic acid such as, for example, the sodium salt of propionic acid.
  • salts include salts of acidic or basic groups present in compounds of the invention.
  • Pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate.
  • Suitable base salts include, but are not limited to, aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, and
  • composition refers, for the purposes of the present invention, to a therapeutically effective amount of the active ingredient, i.e. the SCFA compound of formula (I) or a pharmaceutically acceptable salt thereof, optionally, together with a pharmaceutically acceptable carrier or diluent. It embraces compositions that are suitable for the curative treatment, the control, the amelioration, an improvement of the condition or the prevention of a disease or disorder in a human being or a non-human animal. Thus, it embraces pharmaceutical compositions for the use in the area of human or veterinary medicine.
  • the present invention can be administered intravenously, intradermally, intraarterially, intraperitoneal ⁇ , intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, topically, locally, inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990,
  • the compounds of the present invention and the pharmaceutical compositions containing said compounds may be administered preferably orally, and thus be formulated in a form suitable for oral administration, i.e. as a solid or a liquid preparation.
  • Suitable solid oral formulations include tablets, capsules, pills, granules, pellets and the like.
  • Suitable liquid oral formulations include solutions, suspensions, dispersions, emulsions, oils and the like. If formulated in form of a capsule, the compositions of the present invention comprise, in addition to the active compound and the inert carrier or diluent, a hard gelating capsule.
  • the present invention also provides a nutritional (food or beverage) product, such as an oral nutritional composition, for oral consumption and optionally for enteral adsorption, wherein the nutritional composition includes the compounds of the present invention.
  • a nutritional (food or beverage) product such as an oral nutritional composition, for oral consumption and optionally for enteral adsorption
  • the nutritional composition includes the compounds of the present invention.
  • the nutritional compositions may be a liquid oral nutritional supplement or a complete feeding.
  • the nutritional compositions may be administered in any known form including, for example, tablets, capsules, liquids, chewables, soft gels, sachets, powders, syrups, liquid suspensions, emulsions and solutions in convenient dosage forms.
  • a nutritional composition may be a food product intended for human consumption, for example, a beverage, a drink, a bar, a snack, an ice cream, a dairy product, for example a chilled or a shelf-stable dairy product, a fermented dairy product, a drink, for example a milk-based drink, an infant formula, a growing-up milk, a confectionery product, a chocolate, a cereal product such as a breakfast cereal, a sauce, a soup, an instant drink, a frozen product intended for consumption after heating in a microwave or an oven, a ready-to-eat product, a fast food or a nutritional formula.
  • Dietary fiber as referred to herein is the edible parts of plants or analogous carbohydrates that are resistant to digestion and absorption in the human small intestine with complete or partial fermentation in the large intestine.
  • Dietary fiber includes polysaccharides, oligosaccharides, lignin, and associated plant substances. Dietary fibers promote beneficial physiological effects including laxation, and/or blood cholesterol attenuation, and/or blood glucose attenuation.
  • the present invention therefore relates to the administration of dietary fiber as a medicament in the treatment of cardiovascular disease, as outlined in more detail herein. Dietary fiber may be administered either with or without SCFA and/or intestinal bacteria in combination, in order to provide a cardioprotective effect.
  • Dietary fibers are well known for their ability to alter the intestinal milieu, thereby modulating physiological processes along the entire length of the intestine, with effects differing in the small and large intestine.
  • the primary function of fiber in the small intestine is to enhance viscosity, while in the large intestine, it is to serve as a substrate for short-chain fatty acid (SCFA) production.
  • SCFA short-chain fatty acid
  • Fibers are carbohydrates and lignin that cannot be hydrolyzed by human digestive enzymes but are fermented by intestinal bacteria to produce hydrogen, methane, carbon dioxide, water, and SCFA. Fibers are typically classified based on their fermentability, solubility, and viscosity. Most fermentable fibers are soluble and viscous, and most insoluble fibers are non-viscous and not completely fermentable. Currently, nutrition labeling focuses on solubility to classify the types of dietary fiber. Insoluble fibers, including cellulose and lignin, are completely insoluble in water and are minimally fermented in the colon. They serve primarily as bulking agents, through their capacity to hold water. Insoluble fibers increase stool mass and promote the normal progression of contents through the intestine, attenuating constipation when liquid intake is adequate.
  • SCFA beneficial effects of fiber are associated with their fermentability and production of SCFA.
  • Acetate, propionate, and butyrate comprise 83% of the SCFA produced in the colon.
  • SCFA are readily absorbed by intestinal epithelial cells, providing energy and stimulating sodium transport, water absorption, and intestinal growth.
  • SCFA are known to promote a healthy gut environment by stimulating the growth of beneficial bacteria, such as bifidobacteria and lactobacilli, and inhibiting the growth of harmful bacterial strains.
  • beneficial bacteria promote intestinal health by stimulating a positive immune response and out-competing the growth of harmful bacteria. It is for these reasons that SCFA are so important following radiation therapy, the use of antibiotics, extreme changes in diet, and other events known to upset microbiota subpopulations in the intestine.
  • soluble fibers In association with cultivating a positive microbiota, soluble fibers have a mild laxative effect that can help prevent or alleviate constipation.
  • the present invention proposes for the first time that SCFA may
  • the human intestinal tract can be thought of as an ecosystem where bacterial subpopulations from about 800 species of bacteria interact with one another, host intestinal epithelial cells, and components of the host immune system. Specific bacterial subpopulations flourish when the intestinal environment is suitable. Nutrient availability, pH, physiological processes, and the absence of competing bacteria all affect the mix of bacteria that colonize the gut at a given time.
  • Beneficial bacteria generally referred to as probiotics
  • probiotics have been used for years to increase the proportion of beneficial bacteria in the intestine and to prevent or treat medical conditions.
  • probiotics include lesser frequency and shorter duration of diarrhea associated with antibiotics and chemotherapy, stimulation of positive immune response, and reduction of cancer-promoting enzymes in the colon.
  • a probiotic For a probiotic to promote the growth of beneficial bacteria in the intestine, it must survive passage through the stomach and retain its ability to colonize in the distal intestine and colon.
  • Commonly used probiotics include strains of lactobacilli and bifidobacteria.
  • Common food sources of probiotics are yogurt, buttermilk, kimchi, sauerkraut, and other cultivated and fermented foods.
  • the gut microbiome and cardiovascular disease The gut microbiome and cardiovascular disease:
  • the present invention is directed to multiple related aspects of modulating or enhancing SCFA provision in the body of a patient, for example by providing SCFA via direct administration and/or by modulating the gut microbiome to produce SCFA, as a means for the treatment and prevention of cardiovascular heart disease, in particular hypertensive heart disease.
  • Sources of bacterial SCFA production in the human gut/intestine relate to Actinobacteria, Bacteroidetes, Firmicutes-Lachnospiraceae, Firmicutes-Ruminococcaceae, Firmicutes-Negativicutes, Firmicutes-Peptostreptococcaceae, Firmicutes-Clostridiaceae or Verrucomicrobia.
  • the present invention therefore encompasses the administration of an isolated population of bacteria encompassing one or more bacteria of said families for the treatment of cardiovascular disease.
  • SCFA cardiovascular disease
  • Tregs regulatory T cells
  • RAAS renin- angiotensin-aldosterone system
  • Angll leads to vasoconstriction, endothelial dysfunction and increased vascular permeability.
  • Angll-induced pressure overload leads the myocardial hypertrophy and fibrosis.
  • Enhanced Angll expression precedes pathologic cardiac remodeling and ultimately hypertensive heart disease.
  • Plasma levels of Angll are significantly elevated in hypertensive and heart failure patients.
  • ACE inhibitors and Angll receptor blockers interfere with Angll production and Angll receptor binding, respectively, their efficacy in hypertension and heart failure treatment has been shown repeatedly.
  • Angll is also a potent activator of the immune system, a fact which is not sufficiently addressed by current treatments.
  • NF- ⁇ pro-inflammatory nuclear factor 'kappa-light-chain- enhancer' of activated B-cells
  • ROS reactive oxygen species
  • Angll activates immune cells.
  • the importance of inflammation in hypertension and target-organ damage is illustrated by previous animal experiments, in which immunosuppressants such as
  • mice which lack B and T cells develop only blunted hypertension during Angll infusion. Adoptive transfer of T, but not B cells, restores blood pressure increase. Similarly, mice with a severe combined immunodeficiency show an attenuated Angll response, less cardiac hypertrophy and fibrosis.
  • Tregs represent a T cell subset, characterized by the expression of CD4, CD25 and the transcription factor FoxP3. Tregs suppress effector T cells such as Th1 and Th17 cells, but also macrophages and dendritic cells and thereby contribute to self-tolerance and immune
  • Treg release inhibitory cytokines such as TGF- ⁇ and IL- 10. Enhancement of Treg function is a promising treatment option to limit Angll-elicited tissue inflammation.
  • This state-of-affairs was demonstrated in a study investigating the effect of an adoptive Treg transfer, where intravenous transfer of Tregs from donor mice did not alter Angll- induced hypertension, but ameliorated myocardial hypertrophy and immune cell infiltration.
  • the functional relevance of this treatment was illustrated by a reduced vulnerability to cardiac ventricular arrhythmias under programmed electrophysiological stimulation.
  • Treg-based immunosuppressive strategies has been recognized, safety issues and adverse effects of available treatment regimens prevented their use in clinical practice. Therefore, new Treg enhancing treatment options are needed.
  • the present invention relates to the provision of such means, shown by the experimental outlines provided herein.
  • treating a subject afflicted with a disorder shall mean slowing, stopping or reversing the disorder's progression.
  • treating a subject afflicted with a disorder means reversing the disorder's progression, ideally to the point of eliminating the disorder itself.
  • ameliorating a disorder and treating a disorder are equivalent.
  • the term “attenuation” as used herein refers to reduction of a disease in a subject or in a tissue of a subject. The particular degree or level of the reduction or clearance is in some embodiments at least 15%, 25%, 35%, 50%, 65%, 75%, 80%, 85%, 90%, 95%, 98% or more.
  • the treatment of the present invention may also, or alternatively, relate to a prophylactic administration of therapeutic agent.
  • a prophylactic administration may relate to the prevention of a medical disorder, or the prevention of development of said disorder, whereby prevention or prophylaxis is not to be construed narrowly under all conditions as absolute prevention.
  • Prevention or prophylaxis may also relate to a reduction of the risk of a subject developing any given medical condition, preferably in a subject at risk of said condition.
  • a "patient” or “subject” for the purposes of the present invention is used interchangeably and meant to include both humans and other animals, particularly mammals, and other organisms. Thus, the methods are applicable to both human therapy and veterinary applications.
  • the patient or subject is a mammal, and in the most preferred embodiment the patient or subject is a human.
  • Combined administration encompasses simultaneous treatment, co-treatment or joint treatment, and includes the administration of separate formulations of SCFA with intestinal bacteria and/or dietary fiber, whereby treatment may occur within minutes of each other, in the same hour, on the same day, in the same week or in the same month as one another. Sequential administration of any given combination of combined agents is also encompassed by the term "combined administration".
  • a combination medicament, comprising one or more of said SCFAs with another therapeutic agent, such as intestinal bacteria and/or dietary fiber, may also be used in order to co-administer the various components in a single administration or dosage.
  • a combined therapy with intestinal bacteria and/or dietary fiber may precede or follow treatment with SCFA by intervals ranging from minutes to weeks.
  • the intestinal bacteria and/or dietary fiber and SCFA are administered separately, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the intestinal bacteria and/or dietary fiber would be able to exert an advantageously combined effect.
  • Fig. 1 The gut microbiome influences Angll-induced cardiac damage.
  • a healthy microbiome is characterized by sufficient SCFA production.
  • Treg induction by SCFA potentially ameliorates cardiac remodeling.
  • HDAC inhibition and vasoactive properties potentially contribute to the beneficial action of SCFA.
  • Fig. 2 Experimental outline of experiments employing SCFA treatment (sodium propionate, C3) in Angll-induced cardiac damage in mice.
  • Fig. 3 Oral administration of sodium propionate (C3) significantly increases serum levels of propionate, as measured by gas chromatography mass spectrometry.
  • Fig. 4 Oral treatment with propionate (C3) reduces mortality in mice infused with Angll to induce cardiac damage.
  • Fig. 5 Oral treatment with propionate (C3) ameliorates Angll-induced cardiac hypertrophy, as measured by (C) heart weight /tibia length ratio and confirmed by (A) echocardiographic measurement of the left ventricular mass and (B) interventricular septal width and left ventricular posterior wall width in diastole.
  • D Effect of propionate (C3) on Ang ll-induced hypertension. Data are systolic blood pressure as measured by radiotelemetry.
  • E Effect of propionate (C3) on Ang ll-induced endothelial dysfunction. Endothelium-dependent vasorelaxation to acetylcholine of mesenteric arteries isolated from C3-treated mice and respective controls are shown.
  • Fig. 6 Propionate (C3) reduces expression of biomarkers for cardiac hypertrophy and cardiac fibrosis, as measured for mRNA expression of (A) BNP, (B) Beta-MHC, (C) NGAL and (D) CTGF.
  • Fig. 7 Propionate (C3) reduces vulnerability to harmful ventricular arrhythmias, as measured by in vivo cardiac electrophysiology. Representative surface and intracardiac recordings are shown (upper panel). Quantification presented (lower panel) in % of stimulation protocols applied leading to induction of ventricular arrhythmias.
  • Fig. 8 Propionate (C3) reduces interstitial and perivascular cardiac fibrosis, as measured by fibronectin and collagen I immunofluorescence of heart cryosections. Representative images are shown (upper panel), lower panel shows quantification.
  • Fig. 9 Propionate (C3) reduces effector memory T cells in experimental hypertension, indicative of an anti-inflammatory effect on Angll-induced inflammation. Flow cytometric analysis of splenic effector memory T cells and naive T cells are shown (upper panel: representative flow cytometry plots, lower panel: quantification).
  • Fig. 10 Propionate (C3) prevents splenic Th17 expansion, but not Th1 , in experimental hypertension as measured by flow cytometry.
  • Fig. 11 Propionate (C3) prevents cardiac immune cell infiltration in experimental hypertension, as measured using flow cytometry. The abundance T helper cells, the cytotoxic T cells and the macrophages per heart is shown.
  • Fig. 12 Propionate (C3) reduces cardiac Th17 cells, but not Th1 cells in experimental hypertension as measured by flow cytometry. ROR-gamma t+ and TBet+ cells in CD4+ lymphocytes are shown.
  • Fig. 13 Experimental outline of experiments employing anti-CD25 Antibody (PC61 ) for regulatory T cell (Treg) depletion.
  • Fig. 14 Degree of Treg depletion on day 14 of Angll infusion, induced by the injection of anti- CD25 Antibody (PC61 ), as measured by flow cytometry of splenocytes.
  • Fig. 15 Treg depletion reduces cardiac anti-hypertrophic effects of propionate, as measured by (A) echocardiographic measurement of the left ventricular wall and confirmed by (B)
  • Fig. 16 Treg depletion blocks anti-fibrotic effects of propionate, as measured by fibronectin and collagen I immunofluorescence in heart cryosections. Upper panel shows representative images, lower panel shows quantifications.
  • Fig. 17 Treg depletion prevents the propionate-induced reduction of splenic Th17 cells. Th1 cells remain unaffected. Flow cytometric measurements are shown.
  • Fig. 18 Treg depletion prevents the anti-inflammatory propionate effect in the spleen.
  • Fig. 19 Treg depletion prevents the anti-inflammatory propionate effect in the heart. Numbers of CD4+ and CD8+ cells were determined by immunofluorescence and analyzed per heart cryosection.
  • Fig. 20 Butyrate & Propionate ameliorate Angll-induced renal damage. Urine was collected and analyzed for albuminuria.
  • Fig. 21 Butyrate & Propionate ameliorate Angll-induced interstitial cardiac fibrosis, as analyzed by Fibronectin immunofluorescence of heart cryosections.
  • Fig. 22 Butyrate & Propionate ameliorate cardiac T cell infiltration. Immunofluorescence of CD4+ lymphocytes per heart section is shown. EXAMPLES
  • Microbiome-host communication occurs via gut bacterial metabolites which are resorbed by the host and target various organs.
  • Short-chain fatty acids (SCFA) are produced from bacterial fermentation, are highly abundant in the gut but can also be detected in the blood.
  • mice Male NMRI mice were subcutaneously infused with Angll (1.44 mg/kg/d) for two weeks and received either propionate (Angll+C3) or sodium-matched drinking water (Angll). To deplete endogenous SCFA production mice were fed a purified low- fiber diet. Body weight was similar among all groups. Propionate treatment significantly reduced albuminuria (Angll 1 143 ⁇ 193; Angll+C3 302 ⁇ 69 ⁇ g/d). Propionate significantly reduced cardiac hypertrophy as measured by heart-to-tibia ratio (Angll 10.1 ⁇ 0.4; Angll+C3 8.9 ⁇ 0.4 mg/mm) and was confirmed by echocardiography.
  • the present invention is based in part on demonstration of the influence of the microbiome on Angll-induced cardiac damage in mice, providing support that the gut microbiome, and in particular SCFA as microbial metabolites, influence Angll-induced cardiac damage (Fig. 1 ).
  • Short-chain fatty acids are produced from bacterial fermentation, are highly abundant in the gut but can also be detected in the blood.
  • the SCFA propionate can regulate T cell differentiation into effector and regulatory T cells in peripheral tissues. Activation of the immune system and particularly T cells may substantially contribute to hypertensive target organ damage, whereby anti-inflammatory strategies have been shown to be beneficial in animal models.
  • the inventors therefore assessed whether treatment with propionate is beneficial in angiotensin (Angll)-induced target organ damage.
  • Angll angiotensin
  • mice received a 14-day infusion of Angll or solvent (Sham) via osmotic minipumps.
  • Angll-infused mice received concomitant treatment with sodium-propionat (C3) vis drinking water or sodium- matched control drinking water.
  • Sodium propionate and control treatment started 14 days prior to Angll infusion.
  • the experimental outline is provided in Fig. 2.
  • BNP brain natriuretic peptide
  • Fig. 6A brain natriuretic peptide
  • Beta-MHC ⁇ -myosin heavy chain
  • NGAL Neutrophil gelatinase-associated lipocalin
  • NGAL Neutrophil gelatinase-associated lipocalin
  • CGF connective tissue growth factor
  • Ventricular arrhythmias Susceptibility to ventricular arrhythmias was significantly reduced by propionate treatment, as measured in % of positive stimulation protocols during in vivo cardiac electrophysiology examinations (Fig. 7).
  • Ventricular arrhythmias are typically considered as potentially harmful abnormal rapid heart rhythms that originate in the ventricles. Ventricular arrhythmias may represent life threatening conditions as such, or act as indicators for other heart disease, and are commonly associated with heart attacks.
  • the reduction in susceptibility to ventricular arrhythmias represents a promising medical effect induced by propionate (C3).
  • Myocardial fibrosis was significantly reduced by propionate (C3) treatment as measured by fibronectin and collagen I immunofluorescence stainings (Fig. 8). It is known that Ang l l-induced cardiac damage leads to increased matrix deposition like interstitial fibronectin deposition as well as increased perivascular collagen type I deposition, which are both typical markers for cardiac fibrosis. Propionate (C3) treatment leads to a significant reduction in fibronectin and collagen I deposition indicating a potentially therapeutic effect against cardiac fibrosis.
  • Flow cytometry analyses demonstrated that propionate (C3) also prevents local cardiac immune cell infiltration in experimental hypertension and by examining the number of T helper cells per heart, the cytotoxic T cells per heart and the macrophages per heart (Fig. 1 1 ).
  • Th17 cells are considered a pro-inflammatory T helper subtype, such that the propionate-induced reduction in Th17 cells indicates an anti-inflammatory treatment effect.
  • mice received Angll infusions for 14 days via osmotic minipumps to induce cardiac damage ( as described in experimental part I). Concomitantly, mice were administered sodium propionate (C3) via drinking water. In order to reduce Tregs in vivo, mice were treated with the anti-CD25 antibody (clone: PC61 ) by repeated subcutaneous injection (days -1 , day 1 and day 5 of Angll infusion). Similar subcutaneous injections of the respective lgG1 isotype control served as control condition without Treg depletion.
  • the experimental outline is provided in Fig. 13.
  • Treg depletion induced by the anti-CD25 antibody moderately reduces the anti-hypertrophic effects of propionate (C3) compared to the IgG control as shown by the hypertrophy indices(Fig. 15).
  • Treg depletion blocks the anti- fibrotic effects of propionate (C3) compared to the IgG control condition as shown by fibronectin and collagen I immunofluorescence (Fig. 16).
  • Treg depletion prevents the propionate (C3) effect on splenic Th17 cells as shown by flow cytometry, indicative of a loss of the antiinflammatory effect of propionate (Fig. 17).
  • the loss of the anti-inflammatory propionate (C3) effect becomes evident in the spleen (Fig. 18), where effector memory T cells increase upon Treg depletion despite proprionate (C3) treatment and compared to the IgG control (flow cytometry).
  • cardiac infiltration with CD4+ and CD8+ lymphocytes is enhanced in the Treg depleted condition compared to IgG control as shown by immunofluorescence using specific antibodies, confirming the loss of the anti-inflammatory propionate (3) effect on the heart (Fig. 19).
  • mice were infused with Angll or solvent (Sham) and received either sodium propionate (P), sodium butyrate (B) or sodium-matched drinking water (NaCI).
  • Angll-infusion lead to a profound end-organ damage in kidneys and hearts (Figs. 20-22).
  • Angll-induced renal damage was determined by analysis of albuminuria, which is a surrogate marker for hypertensive organ damage. Propionate and butyrate reduced Angll-induced albuminuria to a similar extent (Fig. 20).
  • cardiac Angll-induced cardiac fibrosis was analyzed by fibronectin
  • the data provided herein indicates that propionate and butyrate attenuate Angll-induced cardiac remodeling, cardiac hypertrophy, fibrosis and susceptibility to arrhythmias.
  • the Angl l-elicited inflammatory response in spleen and cardiac tissue is attenuated in propionate-treated animals.
  • the mode of action of propionate appears to be Treg dependent.
  • 0-1 aims to characterize Angl l/hypertensive cardiac target-organ damage in mice with different intestinal microbiomes.
  • the inventors submit that antibiotic depletion of intestinal SCFA producers will aggravate Angll-induced cardiac end-organ damage.
  • mice Male NMRI mice (12 weeks) are infused with Angll (1 .44 mg/kg/d for 14 days) via subcutaneous osmotic minipumps (Alzet), which causes hypertension and cardiac remodeling.
  • Alzet subcutaneous osmotic minipumps
  • mice are treated with different antibiotics (abx) before and during Angll infusion.
  • the selected antibiotics either deplete Gram-positive (vancomycin) or Gram- negative (polymyxin B) bacteria.
  • Another antibiotic cocktail produces an abiotic environment in the intestine.
  • vancomycin has been shown to efficiently deplete SCFA producers in the intestine and SCFA levels in feces.
  • antibiotics are administered orally ad libitum.
  • Oral antibiotics without systemic resorption are selected to avoid effects on host cells and to avoid potential systemic side-effects.
  • Control group normal drinking water w/o antibiotics + Angll.
  • Vancomycin group (dominant Gram-negative flora): to deplete Gram-positive bacteria and SCFA producers, vancomycin (500 mg/L) is administered in the drinking water + Angll infusion.
  • Polymyxin-group (dominant Gram-positive flora): to deplete the intestinal Gram- negative flora, polymxin B (100 mg/L) is administered in the drinking water + Angll infusion.
  • mice without detectable intestinal colonization an oral cocktail of 4 antibiotics (ampicillin 1 g/L, neomycin 1g/L, metronidazole 1 g/L and 500 mg/L vancomycin) is administered + Angll infusion.
  • antibiotics ampicillin 1 g/L, neomycin 1g/L, metronidazole 1 g/L and 500 mg/L vancomycin
  • Protocols for assessing these factors are disclosed in the context of Experimental parts II and III and in the methods below. Furthermore, a skilled person is aware of the experiments required to assess these factors.
  • Immune cells are isolated from:
  • Cells are incubated with antibodies and measured by flow cytometry (BD FACSCanto II).
  • Labeling protocols focus on regulatory, Th1 and Th17 cells.
  • Expression of marker cytokines is measured after PMA/ionomycin re-stimulation.
  • Surface markers may include CD3, CD4, CD8, ⁇ T cells, CD45R, CD45RB, CD1 1 b, CD1 1 c, CD69, CD25, CD44, CD62L, Intracellular markers (Helios, FOXP3), Cytokines (IFN- ⁇ , IL-17, IL-10)
  • SCFA concentrations acetate, propionate, butyrate
  • 0-2 aims to identify potent bacterial SCFA producers as potential candidates for probiotic treatment regimens. Therefore, different bacterial strains are tested in vitro and in vivo for their capacity to produce SCFA.
  • germ-free mice (bred in sterile isolators, devoid of any bacteria) are mono-colonized with the respective candidates. Respective bacteria are administered to germ-free mice by oral gavage of live bacterial cultures. Subsequently, mice are further maintained in a sterile environment to prevent exogenous contamination with other environmental bacteria. Fecal samples and serum samples collected from monocolonized mice are then analyzed for SCFA content and compared to results from the in vitro culture experiments.
  • Protocols for assessing these factors are disclosed in the context of Experimental parts II and III and in the methods below. Furthermore, a skilled person is aware of the experiments required to assess these factors.
  • mice Twelve-week old male NMRI mice (Janvier Labs) were used throughout the study. We followed the American Physiological Society guidelines for animal care, and the local Animal Review Board (Landes Eck, Germany) approved all protocols. To induce hypertension and cardiac end-organ damage, mice received a continuous subcutaneous Angiotensin (Ang) II (Merck, Millipore, Darmstadt, Germany) infusion (1.44 mg/kg/d) for 14 days via osmotic minipumps (Alzet Osmotic Pumps, Cupertino, CA, USA).
  • Ang Angiotensin II
  • osmotic minipumps Alzet Osmotic Pumps, Cupertino, CA, USA.
  • mice Three weeks prior to the implantation of the pump mice were fed a purified diet low in fibre (Ssniff, Soest, Germany) in order to reduce endogenous intestinal SCFA production.
  • SCFA propionate mice were either administered 200 mM sodium propionate (Sigma Aldrich) in drinking water ad libitum, control animals received sodium-matched drinking water.
  • On day 14 of Angll infusion mice were sacrificed in anesthesia, blood and organs were collected. In a subgroup of mice blood pressure was measured using implantable radiotelemetry devices (DSI, St.
  • mice received an anti-CD25 antibody or the respective isotype control by intraperitoneal injection. 250 ⁇ g of anti-CD-25 (clone: PC61 , kindly provided by T. Hunig, University of Wijrzburg, Germany) or rat lgG1 isotype control (Bio X Cell) was injected on day -1 , day 1 and day 5 of Angll infusion.
  • the programmed ventricular stimulation protocols included trains of 10 basal stimuli (S1 , cycle length 80/90/100 ms) followed by up to 3 extra-stimuli (S2-S4), delivered with a coupling interval decreasing in steps of 5 ms until ventricular refractoriness was reached. Additional short episodes of burst pacing with cycle lengths down to 60 ms were applied. Occurrence and duration of ventricular arrhythmias were documented. Only stimulation protocols with reproducible ventricular arrhythmias longer than five consecutive beats were considered positive. The percentage of positive protocols applied is presented as 'arrhythmia inducibility'.
  • Specimens were analysed using a Zeiss Axioplan-2 imaging microscope and AxioVision 4.8 software (Carl Zeiss Microscopy GmbH, Jena, Germany). The investigator had no knowledge of the treatment group assignment. Interstitial fibrosis was analyzed by fibronectin immunofluorescence. Cy-3 positive area was measured in high-power fields of crosscut cardiomyocytes. Quantification was performed using ImageJ software with a mean threshold for the Cy3-positve area. Perivascular fibrosis was quantified in collagen I stained heart sections. Cy3-positve perivascular width was measured using AxioVision software (Carl Zeiss GmbH, Germany) and normalized to the size of the vessel as measured by the media width.
  • Infiltrating cells lymphocytes were stained in heart sections after staining with CD4 and CD8 antibodies and counted under a microscope. CD4+ and CD8+ cells are presented as numbers per heart section. Cells positive for fibroblast-specific protein-1 (FSP-1 ) were assessed per high-power field.
  • FSP-1 fibroblast-specific protein-1
  • RNA analysis Total RNA was isolated from snap-frozen left ventricular heart tissue using the RNeasy Mini Kit (QIAGEN) following the manufacturer's protocol. RNA concentration and quality was assed using the NanoDrop-1000 Spectrophotometer (Thermo Fisher). 2 ⁇ g RNA was transcribed to cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biotech).
  • Target gene mRNA expression was quantified using real-time polymerase chain reaction (PCR). TaqMan and SYBR green analysis was conducted using an Applied Biosystems 7500 Sequence Detector (Applied Biosystems). Expression of target mRNA was normalized to the 18S gene as housekeeping gene. All primers and probes were synthetized by Biotez, Berlin, Germany.
  • mice were sacrificed in anesthesia, hearts were perfused with cold saline to remove residual blood and dissected from the thorax. The atria were removed and the remaining hearts were enzymatically digested and dissociated following Miltenyi's 'Preparation of single-cell suspension from mouse heart' protocol (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany). Harvested spleens were immediately put on ice in PBS/EDTA, gently smashed through a 70 ⁇ strainer, followed by erythrocyte lysis and subsequent filtering through a 40 ⁇ mesh to obtain single-cell solutions.
  • Isolated immune cells were either directly stained for surface markers using the respective fluorochrome-conjugated antibodies (incubation 30 mins on ice in PBS/EDTA/BSA), or restimulated with 50 ng/mL phorbol 12- myristate 13-acetate (PMA, Sigma-Aldrich) and 750 ng/mL lonomycin (Sigma Aldrich) for 4 hours at 37°C and 5% C02 in RPMI 1640 medium (Thermo Fisher) with 10% FBS, 1 %

Abstract

The invention relates to Short Chain Fatty Acids (SCFA), SCFA-derivatives and SCFA pro-drugs, for use in the treatment of cardiovascular disease. The invention further relates to the use of dietary fiber and/or isolated populations of intestinal bacteria preferably in combination with SCFA for the treatment of cardiovascular disease. The present invention relates further to the prophylaxis of cardiovascular disease, in particular to the treatment and/or prevention of cardiac arrhythmia and/or hypertensive heart disease.

Description

SHORT-CHAIN FATTY ACIDS FOR USE IN THE TREATMENT OF
CARDIOVASCULAR DISEASE
DESCRIPTION
The invention relates to Short Chain Fatty Acids (SCFA), SCFA-derivatives and SCFA pro-drugs, for use in the treatment of cardiovascular disease. The invention further relates to the use of dietary fiber and/or isolated populations of intestinal SCFA-producing bacteria preferably in combination with SCFA for the prevention and treatment of cardiovascular disease, in particular to the treatment and/or prevention of cardiac arrhythmia and/or hypertensive heart disease.
BACKGROUND OF THE INVENTION
Cardiovascular disease (CVD) is a significant health problem in many nations and in 201 1 there were more than 150,000 deaths as a result of CVD in the United Kingdom. Cardiovascular diseases cause more than 800,000 deaths in the US each year. High blood pressure was listed as a primary or contributing cause of death for 326,000 Americans in 2006 and in 2010 it was estimated to cost the United States $76.6 billion in health care services, medications and missed days of work (Centers for Disease Control and Prevention, Lloyd-Jones et al, Circulation 2010; 121 :e1-e170). There is a clear need for new treatment strategies.
Prevention and management of arterial hypertension represents a major challenge for healthcare systems worldwide. Arterial hypertension is regarded a 'silent killer' as it rarely causes initial symptoms. However, persistent hypertension damages organs such as the heart and leads to hypertensive heart disease. Hypertensive heart disease includes left ventricular hypertrophy, electrical remodeling and increased occurrence of cardiac arrhythmias, diastolic and systolic dysfunction and ultimately symptomatic heart failure. Despite a number of treatment options, the burden of hypertension and its sequelae on health care providers is substantial.
The gut microbiome is increasingly recognized as an important factor in human health. Lifestyle, diet and antibiotics are potent modulators of the microbiome. Gut bacteria release metabolites into the circulation of the host, where they influence important processes such as metabolism and inflammation. Short-chain fatty acids (SCFA), which are produced by certain bacteria in the colon, are known to induce regulatory T cells (Treg). Adoptive-transfer studies with Tregs demonstrated beneficial actions on cardiac structural and electrical remodeling, atherosclerosis and metabolic syndrome. To the knowledge of the inventors, the therapeutic potential of SCFA in treating cardiovascular or hypertensive heart disease has not been explored.
SUMMARY OF THE INVENTION
In light of the prior art the technical problem underlying the present invention is to provide novel or alternative means for treating and/or preventing cardiovascular disease.
This problem is solved by the features of the independent claims. Preferred embodiments of the present invention are provided by the dependent claims. The invention therefore relates to Short Chain Fatty Acid (SCFA) for use in the treatment of cardiovascular disease.
In a preferred embodiment the SCFA is selected from saturated fatty acids comprising six or less carbon atoms, or 5 or less carbon atoms, preferably 4 or less or 3 or less carbon atoms.
In one embodiment the Short Chain Fatty Acid (SCFA) for use according to the present invention is characterised in that the SCFA is propionic acid or propionate.
In one embodiment the Short Chain Fatty Acid (SCFA) for use according to the present invention is characterised in that the SCFA is butyric acid or butyrate.
In one embodiment the Short Chain Fatty Acid (SCFA) for use according to the present invention is characterised in that the SCFA is selected from the group consisting of formic acid, acetic acid, isobutyric acid, valeric acid, isovaleric acid or caproic acid, or formate, acetate, isobutyrate, valerate, isovalerate and caproate.
In one embodiment the Short Chain Fatty Acid (SCFA) for use according to the present invention is characterised in that the SCFA is administered as a salt, preferably a sodium, calcium or potassium salt.
In one embodiment the Short Chain Fatty Acid (SCFA) for use according to the present invention is characterised in that the SCFA is propionate and is administered as a sodium propionate, calcium propionate or potassium propionate.
The invention is based on the surprising finding that SCFA provided to a subject can have a therapeutic effect on various heart conditions. SCFA have been previously linked to immune modulation but a therapeutic effect on cardiovascular disease could not have been derived from the prior art.
In a preferred embodiment the invention relates to Short Chain Fatty Acid (SCFA) for use as a medicament and/or food additive as described herein, wherein the treatment is a prophylactic and/or therapeutic treatment. In one embodiment the invention relates to Short Chain Fatty Acid (SCFA) for use as a medicament and/or food additive as described herein, wherein the prophylactic treatment relates to primary prophylaxis or secondary prophylaxis.
Prophylactic treatments for cardiovascular disease typically relate to following no-smoking regimes combined with healthy diets. Only limited possibilities exist for avoiding cardiovascular complications in patients at risk thereof. The present invention provides a surprisingly effective means of reducing risk of cardiovascular disease through SCFA provision, either in the form of direct administration as a medication or by modulating intestinal bacteria to produce elevated levels of SCFA.
There are two main types of prophylaxis: primary and secondary. Any measure taken to prevent an illness before it occurs is referred to as primary prophylaxis. Secondary prophylaxis relates to procedures that help prevent illness recurrence after experiencing a disease. The present invention proves effective against the CVD described herein, and particularly effective against cardiac arrhythmia, which typically occurs after myocardial infarction. The treatment of a population of patients having suffered a heart attack, preventing future cardiac arrhythmia, is a preferred embodiment of the invention. The present invention therefore further relates to the use of Short Chain Fatty Acid (SCFA) as a medicament as described herein for the secondary prophylaxis of cardiac arrhythmia in subjects having previously experienced myocardial infarction.
In one embodiment the invention relates to Short Chain Fatty Acid (SCFA) for use as a medicament as described herein, wherein the cardiovascular disease is characterised by an inflammation of cardiac tissue.
In a preferred embodiment of the invention the cardiovascular disease to be treated relates to hypertensive heart disease or hypertensive cardiac damage.
In a preferred embodiment of the invention the cardiovascular disease to be treated relates to diastolic heart failure (heart failure with preserved ejection fraction; HFpEF).
In a preferred embodiment of the invention the cardiovascular disease to be treated relates to systolic heart failure (heart failure with reduced ejection fraction (HFrEF).
Hypertensive heart disease includes a number of complications of high blood pressure that affect the heart. The symptoms and signs of hypertensive heart disease will depend on whether or not it is accompanied by heart failure. Patients can present acutely with heart failure and pulmonary edema due to sudden failure of pump function of the heart. Acute heart failure can be precipitated by a variety of causes including myocardial ischemia, marked increases in blood pressure, or cardiac arrhythmias, especially atrial fibrillation.
In light of the experimental data provided herein the present invention appears particularly effective in the treatment of hypertensive heart disease.
In a preferred embodiment the invention relates to Short Chain Fatty Acid (SCFA) for use as a medicament as described herein, wherein the cardiovascular disease is a cardiac arrhythmia.
In a preferred embodiment of the invention the cardiac arrhythmia relates to sinus tachycardia, atrial tachycardia, atrial fibrillation, atrial flutter, ventricular fibrillation, ventricular tachycardia, non- sustained ventricular tachycardia and/or ventricular ectopic beats.
Cardiac arrhythmia, also known as cardiac dysrhythmia or irregular heartbeat, is a group of conditions in which the heartbeat is irregular, too fast, or too slow. Symptoms can include lightheadedness, passing out, shortness of breath, chest pain, and may predispose a person to complications such as stroke or heart failure, or even cardiac arrest. Medical procedures for treating arrhythmia typically relate to the use of a pacemaker, and surgery. Novel means are required for arrhythmia treatment. The present invention presents a surprising development in the field of arrhythmia treatment, no indication has been provided previously that arrhythmia could be treated effectively using the compounds or approaches described herein.
In further embodiments the invention relates to Short Chain Fatty Acid (SCFA) for use as a medicament as described herein, wherein the subject of treatment (patient) exhibits one or more of the following attributes:
a) shows symptoms of being at risk of developing a cardiovascular disease and/or heart failure; b) has elevated levels of risk markers in ex vivo tests;
c) has previously experienced cerebral or myocardial ischemia;
d) exhibits a predisposition of developing cardiovascular disease, for example a genetic predisposition of developing cardiovascular disease;
e) exhibits borderline or established thickening of the left ventricular wall (hypertrophy); and/or
f) shows an abnormal electrocradiogram/Holter monitor or echocardiogram.
In further embodiments of the invention the subject to be treated is at risk of developing atrial and/or ventricular cardiac arrhythmias. An intended subject of the present invention is, in particular, a subject who is at risk of developing heart failure with preserved ejection fraction (HFpEF). HFpEF patients typically have an increased risk of developing various kinds of cardiac arrhythmias and typically exhibit cardiac hypertrophy.
In a preferred embodiment of the invention the symptoms of being at risk of developing a cardiovascular disease are considered, without limitation, as the presence of arterial hypertension, metabolic syndrome, high LDL serum levels, low HDL serum levels, diabetes mellitus and/or insulin resistance. In particular, the presence of left ventricular myocardial hypertrophy represents a symptom of being at risk of developing a cardiovascular disease.
In a preferred embodiment of the invention the markers indicating risk of cardiovascular disease relate to high-sensitivity C-reactive protein, and/or atrial natriuretic peptides/peptide precursors (e.g. BNP and NTpro-BNP) as measured in serum and/or plasma.
A further preferred embodiment of the invention relates to Short Chain Fatty Acid (SCFA) for use as a medicament as described herein, wherein the cardiovascular disease to be treated, for example prophylactically, and/or the symptoms of being at risk of further cardiovascular disease, is selected from the group consisting of heart failure with reduced ejection fraction (HFrEF = systolic heart failure), heart failure with preserved ejection fraction (HFpEF = diastolic heart failure), hypertensive heart disease, coronary artery disease, myocardial infarction,
cardiomyopathy, myocarditis, valvular heart disease, congenital heart disease, coarctation, atrial and/or ventricular septal defects, cerebrovascular insult, pericardial disease and/or preferably combinations of two and more thereof.
In a preferred embodiment of the invention the subject of treatment, as described herein, exhibits (i) relatively low levels of bacteria capable of SCFA-production in their intestine and/or (ii) relatively low levels of SCFA in their intestine, compared to a suitable control, for example a healthy subject and/or a population average.
The present invention is characterised by the relationship between SCFA and heart conditions, and is not limited to the administration of SCFA alone as a medication. The production of SCFA by intestinal bacteria is a known concept; but the modulation of these bacteria in order to treat heart disease, in particular the various specific embodiments of cardiovascular disease disclosed herein, has not previously been proposed. The present invention therefore provides a number of aspects linked by a unifying concept, these aspects relate to the use of SCFA as a medicament, the use of intestinal bacterial populations as a medicament, optionally in combination with dietary fiber, to treat or prevent cardiovascular illness. The modulation of SCFA production in the intestine of subjects also is encompassed by the present invention.
In a preferred embodiment of the invention the SCFA are orally administered to a subject. Oral administration enables direct and effective delivery of SCFA to the intestine, where the SCFA are absorbed and distributed to the rest of the body.
The invention further relates to Short Chain Fatty Acid (SCFA) for use as a medicament as described herein, wherein SCFA are orally administered in nutritional (food and/or beverage) products. This embodiment enables effective prevention of heart conditions via a simple and straightforward administration regime.
In one embodiment the invention relates to Short Chain Fatty Acid (SCFA) for use as a medicament or food additive as described herein, wherein the subject of treatment is coadministered with an isolated population of intestinal bacteria, preferably sources of bacterial SCFA production in the human gut, and/or dietary fiber. Suitable bacteria are disclosed herein.
In one embodiment the invention relates to Short Chain Fatty Acid (SCFA) for use as a medicament or food additive as described herein, wherein the SCFA are administered in the form of dietary fiber as a substrate for SCFA production via bacteria present in the intestine of a subject. A further aspect of the invention therefore relates to dietary fiber for use as a substrate in SCFA production by intestinal bacteria in the treatment of cardiovascular disease. Suitable dietary fibers are disclosed herein.
A further aspect of the invention relates to an isolated population of intestinal bacteria for the treatment of cardiovascular disease, wherein said bacterial population produces elevated levels of SCFA compared to a suitable control.
A suitable control may relate to subject not having been treated with SCFA producing bacteria, or a population average. The embodiments described above are bound by a common concept of SCFA production being linked to the treatment or prevention of cardiovascular disease.
DETAILED DESCRIPTION OF THE INVENTION
The gut microbiota is widely termed as the bacterial community that lives within an individual's gastrointestinal tract or elsewhere on or in the body. However, the gut (otherwise referred to as the intestine) has received primary attention. The collection of genes present in a gut microbiota, the microbiome, exceeds the number of host genes by far. Known physiologic functions of the gut microbiome include digestion and nutrient uptake, protection from pathogen invasion, promotion of tissue differentiation, and stimulation of the immune system.
The relevance of the microbiome for the development of diseases has been recently recognized. The microbiome is sensitive to changes in lifestyle and diet. Moreover, widespread use of antibiotics substantially impact microbiome composition, with consequences for the metabolism of the host. For several inflammatory conditions (e.g. rheumatoid arthritis, colitis) and metabolic diseases (obesity, diabetes), differences between the microbiomes from diseased and non- diseased individuals have been demonstrated. Although the description of definite pathophysiologic mechanisms in this mutual host-microbiome relationship remains a challenge, the relevance of the microbiome is now widely recognized.
Community-wide gut microbiome analysis can be facilitated by culture-independent next- generation sequencing methods. Sequencing of DNA isolated from fecal material is widely used, preferably by metagenomic shotgun sequencing or 16S amplicon sequencing. Therefore, DNA is extracted from feces, the bacterial 16S rRNA marker gene is primer amplified and then sequenced. The resulting 16S sequences and the number of times each sequence is detected reliably describe the taxonomic composition of the bacterial community.
Analysis of metabolites produced by a given microbiome further adds information on the functional properties of a community. Products of microbial metabolism act as signaling molecules and influence the host organism. Besides influencing intestinal function, microbial metabolites are resorbed and affect the liver, the brain, the lungs and even the heart. Bacterial metabolites known to influence metabolism, immune cells and vascular function include secondary bile acids, trimethylamine and SCFA, respectively.
The enhancement of a function of the gut microbiome is therefore one aspect of the present invention, whereby the SCFA production by the microbiome can be enhanced and supplemented by administration of SCFA substrate, such as dietary fiber, thereby leading to enhanced production of SCFA in the gut of the subject, or by administering the SCFA directly to a patient in need thereof. Through these embodiments, potentially combined, a subject can receive the beneficial effect of the gut microbiome to an improved degree as would be usually possible under standard conditions, for example on an unspecialized diet.
Short-chain fatty acids:
Food components are metabolized by the gut microbiota to bioactive compounds, which are resorbed by the host and reach different organs via the blood. SCFA are carboxylic acids of bacterial origin that are produced by the gut microbiota through fermentation of partially and nondigestable polysaccharides (dietary fiber).
SCFA are a subset of saturated fatty acids, preferably comprising six or less carbon atoms that include acetate, propionate, butyrate, pentanoic and hexanoid acid. They are highly abundant in the colon but can also be detected in the blood. Despite being an energy source for certain bacteria and intestinal epithelial cells, their immunomodulatory effects have been highlighted in a number of studies.
SCFAs elicit their anti-inflammatory effects through binding to endogenous receptors such as the G-protein-coupled receptors (GPCRs) GPR41 , GPR43, GPR109 or so far unidentified unknown GPCRs and eventually through their capacity to inhibit histone deacetylase (HDAC) activity. SCFA alter gene expression in a variety of cells through inhibiting the removal of acetyl groups from histones by HDACs. Of note, HDAC inhibition increases the expression of the transcription factor forkhead box P3 (Foxp3) in T cells, which subsequently increases proliferation and function of Tregs. Compared to butyrate and propionate, acetate seems to lack the inhibitory effect on HDACs. Whether HDAC inhibition by SCFA occurs directly or indirectly through GPCRs remains to be elucidated. Taken together, SCFA (especially butyrate and propionate) are important microbial metabolites with immunomodulatory properties. To the knowledge of the inventors their role in treatment of hypertensive cardiac damage has not been previously disclosed.
In a preferred embodiment the SCFA is selected from saturated fatty acids comprising six or less carbon atoms, or 5 or less carbon atoms. In a further preferred embodiment the SCFA is formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid or caproic acid, or formate, acetate, propionate, butyrate, isobutyrate, valerate, isovalerate, caproate, preferably propionic acid or propionate.
Additional preferred SCFA relate to those provided in the table below:
Figure imgf000008_0001
The present invention relates to SCFAs in various forms, including SCFA-derivatives or SCFA- pro-drugs. The preferred SCFA of the present invention, including acetate, propionate, butyrate, pentanoic and hexanoid acid, may be derivatized to enable modified behavior of the SCFA compound with respect to in vivo half-life, packaging efficiency, production, modified taste or smell, for example by producing derivatives of SCFA as described below.
The esterified compounds disclosed herein relate potentially to pro-drugs, enabling SCFA release post-ad m i n istration .
For example, a number of SCFA compounds are disclosed in WO 2012/131069, which is herein incorporated in its entirety, that may be administered according to the present invention.
For example, SCFAs according to the present invention relate preferably to SCFA compounds of the following formula:
Figure imgf000009_0001
wherein
X represents -0-, -S-. or -NH-, preferably -0-;
R represents hydrogen, alkyi, aryl, arylalkyl, polyalkylene glycol;
R1 represents hydrogen, alkyi, hydroxyalkyl, arylalkylcarboxylic acid,
R2 represents hydrogen, alkyi, -0-R3; and
R3 represents hydrogen, aryl, arylalkyl, hydroxyalkyl-carboxyl;
or pharmaceutically acceptable salts thereof, for use in the treatment, prevention or attenuation of cardiovascular disease, or any particular cardiovascular disease as described herein.
In one embodiment, the compound of formula (I) is a compound, wherein
X represents -0-, -S-, or -NH-, preferably -0-;
R represents hydrogen, alkyi, aryl, arylalkyl, polyalkylene glycol;
R1 represents hydrogen, alkyi, hydroxyalkylcarboxylic acid;
R2 represents hydrogen, alkyi, -0-R3; and
R3 represents hydrogen, aryl, arylalkyl, hydroxyalkyl-carboxyl;
or pharmaceutically acceptable salts thereof.
In one embodiment the compound of formula (I) is a compound according to the invention and as described herein in the various embodiments, wherein
X represents -0-, -S-, or -NH-, preferably -0-;
R represents hydrogen, C1 -C6 alkyi, unsubstituted or substituted phenyl with one or more, same or different, substituents selected from the group consisting of nitro, halogen, amino, hydroxyl, cyano, C1 -C4 alkyloxy or trifluoro;
R1 represents hydrogen, carboxylic acid, C1-C6 alkyi, hydroxy-C1 -C6 alkyi wherein the alkyi group may be unsubstituted or substituted with one or more, same or different, substituents selected from the group consisting of hydroxyl, amino, carboxylic acid, halogen, cyano, or nitro;
R2 represents hydrogen, C1-C6 alkyi, -0-R3; and R3 represents hydrogen, unsubstituted or substituted phenyl with one or more, same or different, substituents selected from the group consisting of nitro, halogen, amino, hydroxyl, cyano, C1-C4 alkyloxy or trifluoro, phenyl-C1-C6 alkyl wherein the phenyl group may be unsubstituted or substituted with one or more, same or different, substituents selected from the group consisting of nitro, halogen, amino, hydroxyl, cyano, C1 -C4 alkyloxy or trifluoro, hydroxy-C1-C6 alkyl-carboxyl;
or pharmaceutically acceptable salts thereof.
In another specific embodiment, the compound of formula (I) is a compound according to the invention and as described herein in the various embodiments, wherein
X is -0-,
R is hydrogen;
R1 , represents hydrogen, carboxylic acid, C1 -C4 alkyl. hydroxy-C1 -C4 alkyl wherein the alkyl group may be unsubstituted or substituted with one or more, same or different, substituents selected from the group consisting of hydroxyl, amino, or carboxylic acid, preferably hydroxyl and/or carboxylic acid; and
R2 is hydrogen or C1-C4 alkyl; or pharmaceutically acceptable salts thereof.
In another specific embodiment, the compound of formula (I) is a compound according to the invention and as described herein in the various embodiments, wherein
X is -0-,
R is hydrogen;
R1 represents hydrogen, carboxylic acid, C1-C4 alkyl, hydroxy-C1 -C4 alkyl wherein the alkyl group may be unsubstituted or substituted with one or more, same or different, substituents selected from the group consisting of hydroxyl, amino or carboxylic acid, preferably hydroxyl and/or carboxylic acid; and
R2 is -OR3; and
R3 represents hydrogen, unsubstituted or substituted phenyl with one or more, same or different, substituents selected from the group consisting of nitro, halogen, amino, hydroxyl, cyano or methoxy, phenyl-C1 -C4 alkyl wherein the phenyl group may be unsubstituted or substituted with one or more, same or different substituents selected from the group consisting of nitro, halogen, amino, hydroxyl, cyano or methoxy, hydroxy-C1-C3 alkyl-carboxyl; or pharmaceutically acceptable salts thereof.
In another embodiment, the compound of formula (I) is a compound according to the invention and as described herein in the various embodiments, wherein
X is -0-,
R is hydrogen; R1 represents hydrogen, carboxylic acid, C1-C3 alkyl, hydroxy-C1 -C3 alkyl wherein the alkyl group may be unsubstituted or substituted with one or more, same or different, substituents selected from the group consisting of hydroxyl and/or carboxylic acid; and R2 is hydrogen or C1-C4 alkyl; or pharmaceutically acceptable salts thereof.
In another embodiment, the compound of formula (I) is a compound according to the invention and as described herein in the various embodiments, wherein
X is -0-,
R is hydrogen:
R1 represents hydrogen, carboxylic acid, C1-C3 alkyl, hydroxy-C1 -C3 alkyl wherein the alkyl group may be unsubstituted or substituted with one or more, same or different, substituents selected from the group consisting of hydroxyl or carboxylic acid;
R2 is -OR3; and
R3 represents hydrogen, unsubstituted or substituted phenyl with one or more, same or different, substituents selected from the group consisting of nitro, halogen, amino, hydroxyl, cyano or methoxy, phenyl-C1 -C4 alkyl wherein the phenyl group may be unsubstituted or substituted with one or more, same or different, substituents selected from the group consisting of nitro, halogen, amino, hydroxyl, cyano, or methoxy, hydroxy-C1-C3 alkyl- carboxyl; or pharmaceutically acceptable salts thereof.
In one embodiment the compound of formula (I) is a compound according to the invention and as described herein in the various embodiments, wherein
X is -0-,
R is hydrogen;
R1 is selected from the group consisting of hydrogen, methyl, ethyl, n-propyl, isopropyl, hydroxy methyl, dihydroxymethyl, hydroxyethyldicarboxylic acid, carboxylic acid
methylcarboxylic acid, hydroxymethylcarboxylic, ethylcarboxylic acid; and
R2 is selected from the group consisting of hydrogen, hydroxyl or methyl; or pharmaceutically acceptable salts thereof, or wherein
X is -0-,
R is hydrogen;
R1 is selected from the group consisting of hydrogen, methyl, ethyl, n-propyl, isopropyl; R2 is -OR3; and
R3 is selected from the group consisting of 1 -hydroxyethylcarbonyl, benzyl, nitrophenyl; or pharmaceutically acceptable salts thereof. In one embodiment, the compound of formula (I) is a compound according to the invention and as described herein in the various embodiments, wherein
X is -0-,
R represents CrC4 alkyl, unsubstituted or substituted phenyl with one or more, same or different, substituents selected from the group consisting of nitro, halogen, amino, hydroxyl, cyano or methoxy, phenyl-C C4 alkyl wherein the phenyl group may be unsubstituted or substituted with one or more, same or different, substituents selected from the group consisting of halogen, nitro, amino, hydroxyl, cyano or methoxy, polyalkylene glycol;
R1 is carboxylic acid, C1 -C4 alkyl or hydroxy-C1 -C4 alkyl, wherein the alkyl group may be unsubstituted or substituted with one or more, same or different, substituents selected from the group consisting of hydroxyl, amino or carboxylic acid; and
R2 is hydrogen; or pharmaceutically acceptable salts thereof.
In one embodiment, the compound of formula (I) is a compound according to the invention and as described herein in the various embodiments, wherein
X is -0-,
R represents C1-C4 alkyl, unsubstituted or substituted phenyl with one or more, same or different, substituents selected from the group consisting of nitro, halogen, amino, hydroxyl, cyano or methoxy, phenyl-C1 -C4 alkyl wherein the phenyl group may be unsubstituted or substituted with one or more, same or different, substituents selected from the group consisting of halogen, nitro, amino, hydroxy], cyano or methoxy, polyalkylene glycol;
R1 is carboxylic acid, C1 -C3 alkyl or hydroxy-C1 -C3 alkyl wherein the alkyl group may be unsubstituted or substituted with one or more, same or different, substituents selected from the group consisting of hydroxy! and/or carboxylic acid; and
R2 is hydrogen; or pharmaceutically acceptable salts thereof.
In one embodiment the compound of formula (I) is a compound according to the invention and as described herein in the various embodiments, wherein
X is -0-,
R is selected from the group consisting of methyl, ethyl, propyl, benzyl, nitrobenzyl, polyethylene glycol;
R1 is selected from the group consisting of ethyl, hydroxyethyl, methyl, hydroxymethyl; and
R2 is hydrogen; or pharmaceutically acceptable salts thereof.
The compounds of this invention may contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and
diastereomeric mixtures. All such isomeric forms of these compounds are expressly included in the present invention. The compounds of this invention may also contain linkages (e. g., carbon- carbon bonds) wherein bond rotation is restricted about that particular linkage, e. g. restriction resulting from the presence of a ring or double bond. Accordingly, all cis-trans and E/Z isomers are expressly included in the present invention. The compounds of this invention may also be represented in multiple tautomeric forms, in such instances, the invention expressly includes all tautomeric forms of the compounds described herein, even though only a single tautomeric form may be represented (e. g. alkylation of a ring system may result in alkylation at multiple sites, the invention expressly includes all such reaction products). All such isomeric forms of such compounds are expressly included in the present invention. All crystal forms of the compounds described herein are expressly included in the present invention.
The compounds of formula (I) of the invention as described herein in the various embodiments, or a pharmaceutically acceptable salt thereof, or a composition comprising the SCFA compound of formula (I) according to the invention and as described herein, or a pharmaceutically acceptable salt thereof, particularly in a therapeutically effective amount, optionally, together with a pharmaceutically acceptable carrier, is used in the treatment, prevention or attenuation of one or more of the cardiovascular diseases, as described herein.
"Alkyl" as such means a straight-chained or branched saturated aliphatic hydrocarbon having from 1 to 10 carbon atoms, wherein the alkyl group may be unsubstituted or substituted with one or more, same or different, substituents selected from the group consisting of hydroxyl, amino, carboxylic acid, halogen, cyano, or nitro. Preferred are C1 -C6 alkyl, such as methyl, ethyl, n- propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl (amyl), 2-pentyl (sec-pentyl), 3-pentyl, 2- methylbutyl, 3-methylbutyl (= iso-pentyl or iso-amyl), 3-methylbut-2-yl, 2-methylbut-2-yl, 2,2- dimethylpropyl (= neopentyl), n-hexyl, iso-hexyl, sec-hexyl, tert-hexyl and the like. Most preferred are C1 -C4 alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl.
"Hydroxyalkyl" stands for one of the above-defined alkyl groups wherein at least one hydrogen atom is replaced by a hydroxyl group and wherein the hydroxyalkyl group may be unsubstituted or substituted with one or more, same or different substituents selected from the group consisting of hydroxyl, amino, carboxylic acid, halogen, cyano, or nitro. Typical representatives are -CH20H, -CH2CH20H, -CH(OH)-CH3, -CH(OH)CH2CH3, CH2CH(CH2CH20H)CH2CH3I etc..
"Aryl" means a monovalent, monocyclic, bicyclic or tricyclic, aromatic carbocyclic hydrocarbon radical, preferably a 6-14 member aromatic ring system. Preferred aryl groups include, but are not limited to phenyl, naphthyl, phenanthrenyl, and anthracenyl, wherein the aryl group may be unsubstituted or substituted with one or more, same or different substituents selected from the group consisting of halogen; alkyl; alkyloxy, cyano, trifluoro, nitro, amino, hydroxyl.
"Alkoxy" means -O-alkyl, wherein alkyl has the meaning given above.
"Halogen" means fluorine, chlorine, bromine, or iodine, preferably fluorine, chlorine or iodine.
"Polyalkylene glycol" means a moiety that comprises at least two aklylene glycol units such as - O-alkyl-O-alkyl-O-moiety wherein alkyl have the meaning given above. The polyalkylene glycol moiety may be solely comprised of polyalkylene glycol, or may be part of a larger structure, such as polyoxyalkylated glycerol and other polyoxyalkylated polyols such as polyoxyethylated sorbitol or polyoxyethylated glucose. The number of alkylene units may vary and is greater than 1 .
Preferred, polyalkylene glycol are polyethylene glycol (PEG) or polypropylene glycol (PPG). Most preferred polyalkylene glycol are PEG wherein the number of ethylene units may vary from 8 to 150.000 or more, particularly from 10 to 80.000, more particularly from 20 to 10.000.
The term "propionate" refers preferably to the pharmaceutically acceptable salt of propionic acid such as, for example, the sodium salt of propionic acid.
The term "pharmaceutically acceptable salts" include salts of acidic or basic groups present in compounds of the invention. Pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate.
formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzensulfonate, p- to!uenesulfonate and pamoate salts. Certain compounds of the invention can form
pharmaceutically acceptable salts with various amino acids. Suitable base salts include, but are not limited to, aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, and
diethanolamine salts.
The expression "pharmaceutical composition" is used herein in the widest sense. It refers, for the purposes of the present invention, to a therapeutically effective amount of the active ingredient, i.e. the SCFA compound of formula (I) or a pharmaceutically acceptable salt thereof, optionally, together with a pharmaceutically acceptable carrier or diluent. It embraces compositions that are suitable for the curative treatment, the control, the amelioration, an improvement of the condition or the prevention of a disease or disorder in a human being or a non-human animal. Thus, it embraces pharmaceutical compositions for the use in the area of human or veterinary medicine.
The present invention can be administered intravenously, intradermally, intraarterially, intraperitoneal^, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, topically, locally, inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference).
The compounds of the present invention and the pharmaceutical compositions containing said compounds, may be administered preferably orally, and thus be formulated in a form suitable for oral administration, i.e. as a solid or a liquid preparation. Suitable solid oral formulations include tablets, capsules, pills, granules, pellets and the like. Suitable liquid oral formulations include solutions, suspensions, dispersions, emulsions, oils and the like. If formulated in form of a capsule, the compositions of the present invention comprise, in addition to the active compound and the inert carrier or diluent, a hard gelating capsule. Broadly, the present invention also provides a nutritional (food or beverage) product, such as an oral nutritional composition, for oral consumption and optionally for enteral adsorption, wherein the nutritional composition includes the compounds of the present invention. If the nutritional compositions are formulated to be administered orally, the compositions may be a liquid oral nutritional supplement or a complete feeding. In this manner, the nutritional compositions may be administered in any known form including, for example, tablets, capsules, liquids, chewables, soft gels, sachets, powders, syrups, liquid suspensions, emulsions and solutions in convenient dosage forms.
"A nutritional composition" may be a food product intended for human consumption, for example, a beverage, a drink, a bar, a snack, an ice cream, a dairy product, for example a chilled or a shelf-stable dairy product, a fermented dairy product, a drink, for example a milk-based drink, an infant formula, a growing-up milk, a confectionery product, a chocolate, a cereal product such as a breakfast cereal, a sauce, a soup, an instant drink, a frozen product intended for consumption after heating in a microwave or an oven, a ready-to-eat product, a fast food or a nutritional formula.
Dietary Fiber:
"Dietary fiber" as referred to herein is the edible parts of plants or analogous carbohydrates that are resistant to digestion and absorption in the human small intestine with complete or partial fermentation in the large intestine. Dietary fiber includes polysaccharides, oligosaccharides, lignin, and associated plant substances. Dietary fibers promote beneficial physiological effects including laxation, and/or blood cholesterol attenuation, and/or blood glucose attenuation. The present invention therefore relates to the administration of dietary fiber as a medicament in the treatment of cardiovascular disease, as outlined in more detail herein. Dietary fiber may be administered either with or without SCFA and/or intestinal bacteria in combination, in order to provide a cardioprotective effect.
Dietary fiber formulations are known in the art and have been described for example in
US8530447, which is disclosed herein in its entirety, that may be administered according to the present invention.
Dietary fibers are well known for their ability to alter the intestinal milieu, thereby modulating physiological processes along the entire length of the intestine, with effects differing in the small and large intestine. The primary function of fiber in the small intestine is to enhance viscosity, while in the large intestine, it is to serve as a substrate for short-chain fatty acid (SCFA) production. The different physical properties of fibers and their corresponding effects collectively promote normal intestinal function.
Fibers are carbohydrates and lignin that cannot be hydrolyzed by human digestive enzymes but are fermented by intestinal bacteria to produce hydrogen, methane, carbon dioxide, water, and SCFA. Fibers are typically classified based on their fermentability, solubility, and viscosity. Most fermentable fibers are soluble and viscous, and most insoluble fibers are non-viscous and not completely fermentable. Currently, nutrition labeling focuses on solubility to classify the types of dietary fiber. Insoluble fibers, including cellulose and lignin, are completely insoluble in water and are minimally fermented in the colon. They serve primarily as bulking agents, through their capacity to hold water. Insoluble fibers increase stool mass and promote the normal progression of contents through the intestine, attenuating constipation when liquid intake is adequate.
Additional beneficial effects of fiber are associated with their fermentability and production of SCFA. Acetate, propionate, and butyrate comprise 83% of the SCFA produced in the colon. SCFA are readily absorbed by intestinal epithelial cells, providing energy and stimulating sodium transport, water absorption, and intestinal growth. SCFA are known to promote a healthy gut environment by stimulating the growth of beneficial bacteria, such as bifidobacteria and lactobacilli, and inhibiting the growth of harmful bacterial strains. Beneficial bacteria promote intestinal health by stimulating a positive immune response and out-competing the growth of harmful bacteria. It is for these reasons that SCFA are so important following radiation therapy, the use of antibiotics, extreme changes in diet, and other events known to upset microbiota subpopulations in the intestine. In association with cultivating a positive microbiota, soluble fibers have a mild laxative effect that can help prevent or alleviate constipation. However, the present invention proposes for the first time that SCFA may be directly related in treating cardiovascular disease.
The human intestinal tract can be thought of as an ecosystem where bacterial subpopulations from about 800 species of bacteria interact with one another, host intestinal epithelial cells, and components of the host immune system. Specific bacterial subpopulations flourish when the intestinal environment is suitable. Nutrient availability, pH, physiological processes, and the absence of competing bacteria all affect the mix of bacteria that colonize the gut at a given time.
Beneficial bacteria, generally referred to as probiotics, have been used for years to increase the proportion of beneficial bacteria in the intestine and to prevent or treat medical conditions.
Beneficial effects attributed to probiotics include lesser frequency and shorter duration of diarrhea associated with antibiotics and chemotherapy, stimulation of positive immune response, and reduction of cancer-promoting enzymes in the colon. For a probiotic to promote the growth of beneficial bacteria in the intestine, it must survive passage through the stomach and retain its ability to colonize in the distal intestine and colon. Commonly used probiotics include strains of lactobacilli and bifidobacteria. Common food sources of probiotics are yogurt, buttermilk, kimchi, sauerkraut, and other cultivated and fermented foods.
The gut microbiome and cardiovascular disease:
Recent evidence suggests that the gut microbiome also plays a role in cardiovascular diseases. Although the importance of an intact intestinal barrier in heart failure patients has been recognized, we lack a conclusive understanding of how gut bacteria influence cardiovascular function. Modulation of the gut microbiome represents a promising therapeutic option in hypertension and cardiac end-organ damage.
In light of this, the present invention is directed to multiple related aspects of modulating or enhancing SCFA provision in the body of a patient, for example by providing SCFA via direct administration and/or by modulating the gut microbiome to produce SCFA, as a means for the treatment and prevention of cardiovascular heart disease, in particular hypertensive heart disease. Sources of bacterial SCFA production in the human gut/intestine (phyla/families) relate to Actinobacteria, Bacteroidetes, Firmicutes-Lachnospiraceae, Firmicutes-Ruminococcaceae, Firmicutes-Negativicutes, Firmicutes-Peptostreptococcaceae, Firmicutes-Clostridiaceae or Verrucomicrobia. The present invention therefore encompasses the administration of an isolated population of bacteria encompassing one or more bacteria of said families for the treatment of cardiovascular disease.
Inflammatory mechanisms in Angiotensin ll-induced (Angll-induced) hypertensive cardiac damage:
The administration, or modulation of the production, of SCFA presents an effective means for treating cardiovascular disease for a number of reasons, for example due to its potential effect on regulatory T cells (Tregs). As background information, long-term activation of the renin- angiotensin-aldosterone system (RAAS) in hypertension leads to enhanced systemic and local production of Angll and aldosterone, which induces a plethora of pathologic processes in different organs, such as the vasculature, the kidneys and the heart. In the vasculature, Angll leads to vasoconstriction, endothelial dysfunction and increased vascular permeability. In the heart, Angll- induced pressure overload leads the myocardial hypertrophy and fibrosis. Enhanced Angll expression precedes pathologic cardiac remodeling and ultimately hypertensive heart disease. Plasma levels of Angll are significantly elevated in hypertensive and heart failure patients. ACE inhibitors and Angll receptor blockers interfere with Angll production and Angll receptor binding, respectively, their efficacy in hypertension and heart failure treatment has been shown repeatedly.
Angll is also a potent activator of the immune system, a fact which is not sufficiently addressed by current treatments. Besides activation of pro-inflammatory nuclear factor 'kappa-light-chain- enhancer' of activated B-cells (NF-κΒ), promotion of reactive oxygen species (ROS)-production, expression of adhesion molecules, cytokines and chemokines, Angll activates immune cells. This includes the innate immune system (e.g. macrophages) as well as the adaptive immune system (e.g. T cells). The importance of inflammation in hypertension and target-organ damage is illustrated by previous animal experiments, in which immunosuppressants such as
dexamethasone, TNF-a antagonism or mycophenolate mofetilsignificantly ameliorate
hypertension and target-organ damage. Unfortunately the potential severe side effects of nonspecific immunosuppressive therapies prohibit their use in hypertension. Further research is warranted to provide targeted anti-inflammatory therapies in hypertension in the future.
Along this line, the role of T cells (as part of the adaptive immune system) in Angll hypertension has been addressed in previous animal studies. RAG-1 "'" mice, which lack B and T cells develop only blunted hypertension during Angll infusion. Adoptive transfer of T, but not B cells, restores blood pressure increase. Similarly, mice with a severe combined immunodeficiency show an attenuated Angll response, less cardiac hypertrophy and fibrosis.
Tregs represent a T cell subset, characterized by the expression of CD4, CD25 and the transcription factor FoxP3. Tregs suppress effector T cells such as Th1 and Th17 cells, but also macrophages and dendritic cells and thereby contribute to self-tolerance and immune
homeostasis. Among other mechanisms, Treg release inhibitory cytokines such as TGF-β and IL- 10. Enhancement of Treg function is a promising treatment option to limit Angll-elicited tissue inflammation. This state-of-affairs was demonstrated in a study investigating the effect of an adoptive Treg transfer, where intravenous transfer of Tregs from donor mice did not alter Angll- induced hypertension, but ameliorated myocardial hypertrophy and immune cell infiltration. The functional relevance of this treatment was illustrated by a reduced vulnerability to cardiac ventricular arrhythmias under programmed electrophysiological stimulation. Although the potential of Treg-based immunosuppressive strategies has been recognized, safety issues and adverse effects of available treatment regimens prevented their use in clinical practice. Therefore, new Treg enhancing treatment options are needed. The present invention relates to the provision of such means, shown by the experimental outlines provided herein.
As used herein, "treating" a subject afflicted with a disorder shall mean slowing, stopping or reversing the disorder's progression. In the preferred embodiment, treating a subject afflicted with a disorder means reversing the disorder's progression, ideally to the point of eliminating the disorder itself. As used herein, ameliorating a disorder and treating a disorder are equivalent. The term "attenuation" as used herein refers to reduction of a disease in a subject or in a tissue of a subject. The particular degree or level of the reduction or clearance is in some embodiments at least 15%, 25%, 35%, 50%, 65%, 75%, 80%, 85%, 90%, 95%, 98% or more.
The treatment of the present invention may also, or alternatively, relate to a prophylactic administration of therapeutic agent. Such a prophylactic administration may relate to the prevention of a medical disorder, or the prevention of development of said disorder, whereby prevention or prophylaxis is not to be construed narrowly under all conditions as absolute prevention. Prevention or prophylaxis may also relate to a reduction of the risk of a subject developing any given medical condition, preferably in a subject at risk of said condition.
A "patient" or "subject" for the purposes of the present invention is used interchangeably and meant to include both humans and other animals, particularly mammals, and other organisms. Thus, the methods are applicable to both human therapy and veterinary applications. In the preferred embodiment the patient or subject is a mammal, and in the most preferred embodiment the patient or subject is a human.
Combined administration encompasses simultaneous treatment, co-treatment or joint treatment, and includes the administration of separate formulations of SCFA with intestinal bacteria and/or dietary fiber, whereby treatment may occur within minutes of each other, in the same hour, on the same day, in the same week or in the same month as one another. Sequential administration of any given combination of combined agents is also encompassed by the term "combined administration". A combination medicament, comprising one or more of said SCFAs with another therapeutic agent, such as intestinal bacteria and/or dietary fiber, may also be used in order to co-administer the various components in a single administration or dosage.
A combined therapy with intestinal bacteria and/or dietary fiber may precede or follow treatment with SCFA by intervals ranging from minutes to weeks. In embodiments where the intestinal bacteria and/or dietary fiber and SCFA are administered separately, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the intestinal bacteria and/or dietary fiber would be able to exert an advantageously combined effect. In such instances, it is contemplated that one would contact the subject with multiple treatment agents within about 12-24 h of each other, or I onther embodiments within 48 hours, within 1 week, or within 1 month of each other. In some situations, it may be desirable to extend the time period for treatment significantly, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1 , 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.
FIGURES
The following figures are presented in order to describe particular embodiments of the invention, by demonstrating a practical implementation of the invention, without being limiting to the scope of the invention or the concepts described herein.
Short description of the figures:
Fig. 1 : The gut microbiome influences Angll-induced cardiac damage. A healthy microbiome is characterized by sufficient SCFA production. Treg induction by SCFA potentially ameliorates cardiac remodeling. HDAC inhibition and vasoactive properties potentially contribute to the beneficial action of SCFA.
Fig. 2: Experimental outline of experiments employing SCFA treatment (sodium propionate, C3) in Angll-induced cardiac damage in mice.
Fig. 3: Oral administration of sodium propionate (C3) significantly increases serum levels of propionate, as measured by gas chromatography mass spectrometry.
Fig. 4: Oral treatment with propionate (C3) reduces mortality in mice infused with Angll to induce cardiac damage.
Fig. 5: Oral treatment with propionate (C3) ameliorates Angll-induced cardiac hypertrophy, as measured by (C) heart weight /tibia length ratio and confirmed by (A) echocardiographic measurement of the left ventricular mass and (B) interventricular septal width and left ventricular posterior wall width in diastole. (D) Effect of propionate (C3) on Ang ll-induced hypertension. Data are systolic blood pressure as measured by radiotelemetry. (E) Effect of propionate (C3) on Ang ll-induced endothelial dysfunction. Endothelium-dependent vasorelaxation to acetylcholine of mesenteric arteries isolated from C3-treated mice and respective controls are shown.
Fig. 6: Propionate (C3) reduces expression of biomarkers for cardiac hypertrophy and cardiac fibrosis, as measured for mRNA expression of (A) BNP, (B) Beta-MHC, (C) NGAL and (D) CTGF. Fig. 7: Propionate (C3) reduces vulnerability to harmful ventricular arrhythmias, as measured by in vivo cardiac electrophysiology. Representative surface and intracardiac recordings are shown (upper panel). Quantification presented (lower panel) in % of stimulation protocols applied leading to induction of ventricular arrhythmias.
Fig. 8: Propionate (C3) reduces interstitial and perivascular cardiac fibrosis, as measured by fibronectin and collagen I immunofluorescence of heart cryosections. Representative images are shown (upper panel), lower panel shows quantification. Fig. 9: Propionate (C3) reduces effector memory T cells in experimental hypertension, indicative of an anti-inflammatory effect on Angll-induced inflammation. Flow cytometric analysis of splenic effector memory T cells and naive T cells are shown (upper panel: representative flow cytometry plots, lower panel: quantification).
Fig. 10: Propionate (C3) prevents splenic Th17 expansion, but not Th1 , in experimental hypertension as measured by flow cytometry.
Fig. 11 : Propionate (C3) prevents cardiac immune cell infiltration in experimental hypertension, as measured using flow cytometry. The abundance T helper cells, the cytotoxic T cells and the macrophages per heart is shown.
Fig. 12: Propionate (C3) reduces cardiac Th17 cells, but not Th1 cells in experimental hypertension as measured by flow cytometry. ROR-gamma t+ and TBet+ cells in CD4+ lymphocytes are shown.
Fig. 13: Experimental outline of experiments employing anti-CD25 Antibody (PC61 ) for regulatory T cell (Treg) depletion.
Fig. 14: Degree of Treg depletion on day 14 of Angll infusion, induced by the injection of anti- CD25 Antibody (PC61 ), as measured by flow cytometry of splenocytes.
Fig. 15: Treg depletion reduces cardiac anti-hypertrophic effects of propionate, as measured by (A) echocardiographic measurement of the left ventricular wall and confirmed by (B)
interventricular septal width and left ventricular posterior wall width in diastole and (C) heart weight /tibia length ratio.
Fig. 16: Treg depletion blocks anti-fibrotic effects of propionate, as measured by fibronectin and collagen I immunofluorescence in heart cryosections. Upper panel shows representative images, lower panel shows quantifications.
Fig. 17: Treg depletion prevents the propionate-induced reduction of splenic Th17 cells. Th1 cells remain unaffected. Flow cytometric measurements are shown.
Fig. 18: Treg depletion prevents the anti-inflammatory propionate effect in the spleen.
Frequencies of splenic effector memory T cells and naive T cells are shown.
Fig. 19: Treg depletion prevents the anti-inflammatory propionate effect in the heart. Numbers of CD4+ and CD8+ cells were determined by immunofluorescence and analyzed per heart cryosection.
Fig. 20: Butyrate & Propionate ameliorate Angll-induced renal damage. Urine was collected and analyzed for albuminuria.
Fig. 21 : Butyrate & Propionate ameliorate Angll-induced interstitial cardiac fibrosis, as analyzed by Fibronectin immunofluorescence of heart cryosections.
Fig. 22: Butyrate & Propionate ameliorate cardiac T cell infiltration. Immunofluorescence of CD4+ lymphocytes per heart section is shown. EXAMPLES
The following examples are presented in order to describe practical and in some cases preferred embodiments of the invention, without being limiting to the scope of the invention or the concepts described herein.
Background: Increasing evidence suggests that the gut microbiota critically influence host health and immune homeostasis. Microbiome-host communication occurs via gut bacterial metabolites which are resorbed by the host and target various organs. Short-chain fatty acids (SCFA) are produced from bacterial fermentation, are highly abundant in the gut but can also be detected in the blood.
Summary of Methods and Results: Male NMRI mice were subcutaneously infused with Angll (1.44 mg/kg/d) for two weeks and received either propionate (Angll+C3) or sodium-matched drinking water (Angll). To deplete endogenous SCFA production mice were fed a purified low- fiber diet. Body weight was similar among all groups. Propionate treatment significantly reduced albuminuria (Angll 1 143 ± 193; Angll+C3 302 ± 69 μg/d). Propionate significantly reduced cardiac hypertrophy as measured by heart-to-tibia ratio (Angll 10.1 ± 0.4; Angll+C3 8.9 ± 0.4 mg/mm) and was confirmed by echocardiography. Propionate treatment significantly reduced interstitial (Angll 16.5 ± 0.8; Angll+C3 6.6 ± 0.2%) and perivascular cardiac fibrosis (Angll 1.5 ± 0.06; Angll+C3 1 .1 ± 0.03 μιη/μιτι) as measured by fibronectin and collagen I immunofluorescence, respectively. In vivo cardiac electrophysiology studies showed a significantly reduced susceptibility to ventricular arrhythmias in propionate-treated mice (Angll 71 ± 14; Angll+C3 24 ± 16 %), indicating the functional relevance of the improved cardiac morphology. Flow cytometry of cardiac tissue and cardiac immunohistochemistry revealed a significant reduction of Angll- induced inflammatory cell infiltration. Furthermore, propionate significantly reduced the expression of splenic CD4+ effector memory T cells (Angll 43.5 ± 4.9; Angll+C3 28.9 ± 2.8 %) and lnterleukin-17 in CD4+ T cells (Angll 0.7 ± 0.12; Angll+C3 0.3 ± 0.06 %) as measured by flow cytometry.
Conclusion: The data indicate that propionate significantly attenuates Angll-induced cardiac remodeling and inflammation, and reduces susceptibility to arrhythmias. The gut microbiome is a promising target for treatment of hypertensive heart disease.
Role of the gut microbiome and SCFA in Angll-induced cardiac damage:
The present invention is based in part on demonstration of the influence of the microbiome on Angll-induced cardiac damage in mice, providing support that the gut microbiome, and in particular SCFA as microbial metabolites, influence Angll-induced cardiac damage (Fig. 1 ).
Increasing evidence suggests that the gut microbiota critically influence host health and immune homeostasis. Microbiome-host communication occurs via gut bacterial metabolites which are resorbed by the host and target various organs.
Short-chain fatty acids (SCFA) are produced from bacterial fermentation, are highly abundant in the gut but can also be detected in the blood. The SCFA propionate can regulate T cell differentiation into effector and regulatory T cells in peripheral tissues. Activation of the immune system and particularly T cells may substantially contribute to hypertensive target organ damage, whereby anti-inflammatory strategies have been shown to be beneficial in animal models. The inventors therefore assessed whether treatment with propionate is beneficial in angiotensin (Angll)-induced target organ damage.
Experimental Part I:
Mice received a 14-day infusion of Angll or solvent (Sham) via osmotic minipumps. Angll-infused mice received concomitant treatment with sodium-propionat (C3) vis drinking water or sodium- matched control drinking water. Sodium propionate and control treatment started 14 days prior to Angll infusion. The experimental outline is provided in Fig. 2.
Treatment with propionate (C3) elevated propionate serum levels (Fig. 3) and reduced mortality in Angll-infused mice (Fig. 4). Cardiac hypertrophy was significantly reduced by propionate treatment as measured by heart weight /tibia length ratio (Fig. 5C) and confirmed by
echocardiographic measurement of the left ventricular wall (Fig. 5A) and interventricular septal width and left ventricular posterior wall width in diastole (Fig. 5B). Furthermore, the effect of propionate (C3) on Ang ll-induced systolic blood pressure, as measured by radiotelemetry, was assessed, indicating a reduction in systolic blood pressure induced by propionate (C3) treatment (Fig. 5D). This was confirmed by an improved endothelium-dependent vasorelaxation of mesenteric arteries isolated from C3-treated mice compared to controls (Fig. 5E, right). The reduction of cardiac hypertrophy - a cardinal sign of hypertensive heart disease - by oral propionate (C3) treatment indicates a promising medical effect.
Cardiac mRNA expression of brain natriuretic peptide (BNP), a widely accepted biomarker for heart failure (Fig. 6A), Beta-MHC (β-myosin heavy chain), an indicator of pathological cardiac hypertrophy (Fig. 6B), Neutrophil gelatinase-associated lipocalin (NGAL), a biomarker of cardiovascular disease (Fig. 6C) and connective tissue growth factor (CTGF), a biomarker for cardiac fibrosis (Fig. 6D),were significantly reduced by propionate treatment. A molecular basis for a therapeutic effect induced by propionate (C3) is therefore also demonstrated, providing a sound case for an efficacious treatment.
Susceptibility to ventricular arrhythmias was significantly reduced by propionate treatment, as measured in % of positive stimulation protocols during in vivo cardiac electrophysiology examinations (Fig. 7). Ventricular arrhythmias are typically considered as potentially harmful abnormal rapid heart rhythms that originate in the ventricles. Ventricular arrhythmias may represent life threatening conditions as such, or act as indicators for other heart disease, and are commonly associated with heart attacks. The reduction in susceptibility to ventricular arrhythmias represents a promising medical effect induced by propionate (C3).
Myocardial fibrosis was significantly reduced by propionate (C3) treatment as measured by fibronectin and collagen I immunofluorescence stainings (Fig. 8). It is known that Ang l l-induced cardiac damage leads to increased matrix deposition like interstitial fibronectin deposition as well as increased perivascular collagen type I deposition, which are both typical markers for cardiac fibrosis. Propionate (C3) treatment leads to a significant reduction in fibronectin and collagen I deposition indicating a potentially therapeutic effect against cardiac fibrosis. Flow cytometry analyses demonstrated that propionate (C3) significantly reduced the abundance of effector memory T cells in the spleen as well as increased number of naive Th cells indicating a shift from pro-inflammation towards an anti-inflammatory status (Fig. 9). Propionate (C3) reduced the expression of IL-17 in CD4+ T cells in spleen and lymph nodes, as measured by flow cytometry (Fig. 10). Flow cytometry analyses demonstrated that propionate (C3) also prevents local cardiac immune cell infiltration in experimental hypertension and by examining the number of T helper cells per heart, the cytotoxic T cells per heart and the macrophages per heart (Fig. 1 1 ). Flow cytometry analyses of CD4+ T cells demonstrated that propionate (C3) reduces the numbers of cardiac RORgt-positive Th17 cells, but not TBet-positive Th1 cells in experimental hypertension (Fig. 12). Th 17 cells are considered a pro-inflammatory T helper subtype, such that the propionate-induced reduction in Th17 cells indicates an anti-inflammatory treatment effect.
Experimental Part II:
In order to more closely investigate the role of Treg in mediating the beneficial effects of propionate (C3) treatment, Treg depletion experiments in mice were performed. Therefore, mice received Angll infusions for 14 days via osmotic minipumps to induce cardiac damage ( as described in experimental part I). Concomitantly, mice were administered sodium propionate (C3) via drinking water. In order to reduce Tregs in vivo, mice were treated with the anti-CD25 antibody (clone: PC61 ) by repeated subcutaneous injection (days -1 , day 1 and day 5 of Angll infusion). Similar subcutaneous injections of the respective lgG1 isotype control served as control condition without Treg depletion. The experimental outline is provided in Fig. 13. Confirmation of persistent reduction in Treg frequencies by PC61 treatment at day 14 of Angll infusion is provided in Fig. 14. From this we conclude that Treg depletion by PC61 treatment is sufficient to draw conclusions on the role of Tregs in mediating the therapeutic effect of propionate (C3). To analyze the degree of cardiac end-organ damage with (PC61 ) or without (IgG) Treg depletion, analogous experiments were conducted to those described above for the initial Angll-induced cardiac damage model.
As is described in Figures 15 to 19, several beneficial aspects of propionate (C3) treatment on the heart are prevented when Tregs are depleted. First, Treg depletion induced by the anti-CD25 antibody moderately reduces the anti-hypertrophic effects of propionate (C3) compared to the IgG control as shown by the hypertrophy indices(Fig. 15). Importantly, Treg depletion blocks the anti- fibrotic effects of propionate (C3) compared to the IgG control condition as shown by fibronectin and collagen I immunofluorescence (Fig. 16). Moreover, Treg depletion prevents the propionate (C3) effect on splenic Th17 cells as shown by flow cytometry, indicative of a loss of the antiinflammatory effect of propionate (Fig. 17). Similarly, the loss of the anti-inflammatory propionate (C3) effect becomes evident in the spleen (Fig. 18), where effector memory T cells increase upon Treg depletion despite proprionate (C3) treatment and compared to the IgG control (flow cytometry). Finally, cardiac infiltration with CD4+ and CD8+ lymphocytes is enhanced in the Treg depleted condition compared to IgG control as shown by immunofluorescence using specific antibodies, confirming the loss of the anti-inflammatory propionate (3) effect on the heart (Fig. 19). Taken together, Treg depletion experiments in vivo suggest that propionate (C3)
substantially acts via Tregs to dampen systemic Angll-induced inflammation, as well as cardiac remodeling and inflammation under Angll infusion. Experimental Part III:
Additional experimentation was conducted using both propionate and butyrate in order to assess the efficacy of alternative SFCA in treatment/prevention of hypertensive end-organ damage.
Therefore, mice were infused with Angll or solvent (Sham) and received either sodium propionate (P), sodium butyrate (B) or sodium-matched drinking water (NaCI). Angll-infusion lead to a profound end-organ damage in kidneys and hearts (Figs. 20-22). First, Angll-induced renal damage was determined by analysis of albuminuria, which is a surrogate marker for hypertensive organ damage. Propionate and butyrate reduced Angll-induced albuminuria to a similar extent (Fig. 20). Second, cardiac Angll-induced cardiac fibrosis was analyzed by fibronectin
immunofluorescence. Propionate and butyrate reduced Angll-induced cardiac interstitial fibrosis to a similar extent (Fig. 21 ). Third, Angll-induced cardiac infiltration with CD4+ lymphocytes was analyzed by immunofluorescence of cryosections. Both, propionate and butyrate reduced CD4+ infiltration. Importantly, propionate reduced immune cell infiltration to a greater extent compared to butyrate.
Taken together, the examples described herein regarding albuminuria, interstitial cardiac fibrosis and cardiac T cell infiltration were carried out additionally for butyrate and surprisingly a beneficial therapeutic effect could be discerned for butyrate, thereby suggesting that alternative SCFAs also show similar effects as propionate.
The data provided herein indicates that propionate and butyrate attenuate Angll-induced cardiac remodeling, cardiac hypertrophy, fibrosis and susceptibility to arrhythmias. The Angl l-elicited inflammatory response in spleen and cardiac tissue is attenuated in propionate-treated animals. The mode of action of propionate appears to be Treg dependent.
Experimental Part IV:
Although the significance of the intestinal microbiome for cardiovascular health and disease of the host is becoming more recognized, to date nothing is known about the influence of the gut microbiome on hypertensive cardiac damage. The experiments described below provide support for the invention to the extent that gut microbiome composition and SCFA treatment influence Angll-induced cardiac end-organ damage. This inter-organ crosstalk of intestinal bacteria with the cardiovascular system occurs via bacterial metabolites, which are produced in the intestine and are resorbed by the host. SCFA (e.g. propionate, butyrate) are known microbial metabolites with immunoregulatory function and vasoactive properties. Therefore, the invention is based on SCFA and their effect on Angll-induced cardiac remodeling.
Outline 0-1 :
0-1 aims to characterize Angl l/hypertensive cardiac target-organ damage in mice with different intestinal microbiomes. The inventors submit that antibiotic depletion of intestinal SCFA producers will aggravate Angll-induced cardiac end-organ damage.
The experiments are based on the Angll-infusion mouse model. Male NMRI mice (12 weeks) are infused with Angll (1 .44 mg/kg/d for 14 days) via subcutaneous osmotic minipumps (Alzet), which causes hypertension and cardiac remodeling. In order to systematically investigate the influence of different microbiome compositions on hypertensive cardiac damage, mice are treated with different antibiotics (abx) before and during Angll infusion. The selected antibiotics either deplete Gram-positive (vancomycin) or Gram- negative (polymyxin B) bacteria. Another antibiotic cocktail (see below) produces an abiotic environment in the intestine. It is important to note that vancomycin has been shown to efficiently deplete SCFA producers in the intestine and SCFA levels in feces. To selectively target the intestine, antibiotics are administered orally ad libitum. Oral antibiotics without systemic resorption (vancomycin, polymyxin B) are selected to avoid effects on host cells and to avoid potential systemic side-effects.
Four defined microbiome conditions are characterized:
0-1 Control group: normal drinking water w/o antibiotics + Angll.
0-1 Vancomycin group (dominant Gram-negative flora): to deplete Gram-positive bacteria and SCFA producers, vancomycin (500 mg/L) is administered in the drinking water + Angll infusion.
- 0-1 Polymyxin-group (dominant Gram-positive flora): to deplete the intestinal Gram- negative flora, polymxin B (100 mg/L) is administered in the drinking water + Angll infusion.
0-1 Abiotic group: To generate mice without detectable intestinal colonization an oral cocktail of 4 antibiotics (ampicillin 1 g/L, neomycin 1g/L, metronidazole 1 g/L and 500 mg/L vancomycin) is administered + Angll infusion.
All groups are analyzed with focus on:
blood pressure and cardiac hypertrophy/fibrosis (incl. hypertrophy-relevant pathways) in vivo cardiac electrophysiology
- T helper cells (Treg, Th1 , Th17)
- SCFA levels and cardiac HDAC activity.
Protocols for assessing these factors are disclosed in the context of Experimental parts II and III and in the methods below. Furthermore, a skilled person is aware of the experiments required to assess these factors.
Immune cells are isolated from:
small and large intestine (Lamina propria)
mesenteric lymph nodes
- spleen
peripheral blood.
Cells are incubated with antibodies and measured by flow cytometry (BD FACSCanto II).
Labeling protocols focus on regulatory, Th1 and Th17 cells. Expression of marker cytokines is measured after PMA/ionomycin re-stimulation. Surface markers may include CD3, CD4, CD8, γδ T cells, CD45R, CD45RB, CD1 1 b, CD1 1 c, CD69, CD25, CD44, CD62L, Intracellular markers (Helios, FOXP3), Cytokines (IFN-γ, IL-17, IL-10) Bacteria-derived SCFA are produced in the colon and have been shown to have Treg-promoting and vasoactive properties. Therefore, measurement of SCFA is of interest. SCFA concentrations (acetate, propionate, butyrate) in feces and serum are measured using gas
chromatography/mass spectrometry.
Expected Outcome:
1 . Modulation of intestinal bacteria with antibiotics influences Angll-induced cardiac end- organ damage.
2. Depletion of SCFA producers with vancomycin aggravates Angll-induced cardiac
remodeling along with reduced frequencies of Tregs.
Outline 0-2:
0-2 aims to identify potent bacterial SCFA producers as potential candidates for probiotic treatment regimens. Therefore, different bacterial strains are tested in vitro and in vivo for their capacity to produce SCFA.
First, representative non-pathogenic isolates of the murine and human gastrointestinal tract are cultured under appropriate conditions using appropriate culture media. Culture supernatants are collected under standardized growth conditions and analyzed for SCFA content using gas chromatography mass spectrometry. Potent candidates are then used for further in vivo experiments.
Second, these candidate bacteria are tested for their in vivo capacity to produce SCFA.
Therefore, germ-free mice (bred in sterile isolators, devoid of any bacteria) are mono-colonized with the respective candidates. Respective bacteria are administered to germ-free mice by oral gavage of live bacterial cultures. Subsequently, mice are further maintained in a sterile environment to prevent exogenous contamination with other environmental bacteria. Fecal samples and serum samples collected from monocolonized mice are then analyzed for SCFA content and compared to results from the in vitro culture experiments.
Third, the ability of selected SCFA producers to rescue Angll-mediated cardiac damage is investigated. Therefore, the above mentioned Angll mouse model is used. Briefly, conventionally colonized NMRI mice receive Angll infusions via subcutaneous osmotic minipumps for 14 days with concomitant daily gavage of live bacteria of interest or control solution. After 14 days, cardiac end-organ damage is assessed using echocardiography, immunohistochemistry and flow cytometry. In addition, blood pressure is monitored by radiotelemetry. Susceptibility to cardiac arrhythmias is investigated by in vivo cardiac electrophysiology. Fecal samples and serum samples are analyzed for SCFA by gas chromatography mass spectrometry. DNA is extracted from fecal samples for 16S amplicon sequencing to determine the bacterial composition.
Protocols for assessing these factors are disclosed in the context of Experimental parts II and III and in the methods below. Furthermore, a skilled person is aware of the experiments required to assess these factors.
MATERIALS/METHODS Animal protocols. Twelve-week old male NMRI mice (Janvier Labs) were used throughout the study. We followed the American Physiological Society guidelines for animal care, and the local Animal Review Board (Landesgesundheitsamt Berlin, Germany) approved all protocols. To induce hypertension and cardiac end-organ damage, mice received a continuous subcutaneous Angiotensin (Ang) II (Merck, Millipore, Darmstadt, Germany) infusion (1.44 mg/kg/d) for 14 days via osmotic minipumps (Alzet Osmotic Pumps, Cupertino, CA, USA). Three weeks prior to the implantation of the pump mice were fed a purified diet low in fibre (Ssniff, Soest, Germany) in order to reduce endogenous intestinal SCFA production. To study the effects of the SCFA propionate (C3) mice were either administered 200 mM sodium propionate (Sigma Aldrich) in drinking water ad libitum, control animals received sodium-matched drinking water. On day 12 of Angll infusion fluid intake was measured, urine and stool were collected over 24 hours in metabolic cages. On day 13 of Angll infusion echocardiography in anesthesia was performed. On day 14 of Angll infusion mice were sacrificed in anesthesia, blood and organs were collected. In a subgroup of mice blood pressure was measured using implantable radiotelemetry devices (DSI, St. Paul, MN, USA), which were implanted subcutaneously prior to Angll infusion. For regulatory T cell (Treg) depletion experiments, mice received an anti-CD25 antibody or the respective isotype control by intraperitoneal injection. 250 μg of anti-CD-25 (clone: PC61 , kindly provided by T. Hunig, University of Wijrzburg, Germany) or rat lgG1 isotype control (Bio X Cell) was injected on day -1 , day 1 and day 5 of Angll infusion.
In vivo cardiac electrophysiology. Programmed electrical stimulation in mice was performed in the right ventricle under isoflurane anesthesia. In brief, body temperature of anesthesized mice was kept constant at 37°C. Surface ECG was recorded using standard limb leads. Via the right jugular vein, an octapolar 2-French electrode catheter (CIBer mouse cath, NuMed Inc.) was placed into the right ventricle, guided by the morphology of intracardiac electrical signals. Signals were recorded and programmed electrical stimulation was performed using a portable EP tracer system (CardioTek, Netherlands). Impulses were delivered at twice diastolic threshold with a pulse duration of 1.5 ms. To test for inducibility of arrhythmias, the programmed ventricular stimulation protocols included trains of 10 basal stimuli (S1 , cycle length 80/90/100 ms) followed by up to 3 extra-stimuli (S2-S4), delivered with a coupling interval decreasing in steps of 5 ms until ventricular refractoriness was reached. Additional short episodes of burst pacing with cycle lengths down to 60 ms were applied. Occurrence and duration of ventricular arrhythmias were documented. Only stimulation protocols with reproducible ventricular arrhythmias longer than five consecutive beats were considered positive. The percentage of positive protocols applied is presented as 'arrhythmia inducibility'.
Immunofluorescence of cardiac cryosections. Staining was performed on acetone-fixed 5μιη cryosections. Unspecific binding was blocked with 10% normal donkey serum (Jackson
ImmunoResearch, West Grove, USA) for 2h. Sections were incubated with the respective primary antibodies overnight in a humid chamber, followed by 2h staining with a Cy3-conjugated secondary antibody (Jackson ImmunoResearch).
Specimens were analysed using a Zeiss Axioplan-2 imaging microscope and AxioVision 4.8 software (Carl Zeiss Microscopy GmbH, Jena, Germany). The investigator had no knowledge of the treatment group assignment. Interstitial fibrosis was analyzed by fibronectin immunofluorescence. Cy-3 positive area was measured in high-power fields of crosscut cardiomyocytes. Quantification was performed using ImageJ software with a mean threshold for the Cy3-positve area. Perivascular fibrosis was quantified in collagen I stained heart sections. Cy3-positve perivascular width was measured using AxioVision software (Carl Zeiss GmbH, Germany) and normalized to the size of the vessel as measured by the media width. Infiltrating cells lymphocytes were stained in heart sections after staining with CD4 and CD8 antibodies and counted under a microscope. CD4+ and CD8+ cells are presented as numbers per heart section. Cells positive for fibroblast-specific protein-1 (FSP-1 ) were assessed per high-power field.
mRNA analysis. Total RNA was isolated from snap-frozen left ventricular heart tissue using the RNeasy Mini Kit (QIAGEN) following the manufacturer's protocol. RNA concentration and quality was assed using the NanoDrop-1000 Spectrophotometer (Thermo Fisher). 2μg RNA was transcribed to cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied
Biosystems). Target gene mRNA expression was quantified using real-time polymerase chain reaction (PCR). TaqMan and SYBR green analysis was conducted using an Applied Biosystems 7500 Sequence Detector (Applied Biosystems). Expression of target mRNA was normalized to the 18S gene as housekeeping gene. All primers and probes were synthetized by Biotez, Berlin, Germany.
Flow cytometry. To analyze immune cells infiltrating into the heart, mice were sacrificed in anesthesia, hearts were perfused with cold saline to remove residual blood and dissected from the thorax. The atria were removed and the remaining hearts were enzymatically digested and dissociated following Miltenyi's 'Preparation of single-cell suspension from mouse heart' protocol (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany). Harvested spleens were immediately put on ice in PBS/EDTA, gently smashed through a 70μιη strainer, followed by erythrocyte lysis and subsequent filtering through a 40μιη mesh to obtain single-cell solutions. Cells were counted by trypan blue exclusion and stained for flow cytometric analysis. Isolated immune cells were either directly stained for surface markers using the respective fluorochrome-conjugated antibodies (incubation 30 mins on ice in PBS/EDTA/BSA), or restimulated with 50 ng/mL phorbol 12- myristate 13-acetate (PMA, Sigma-Aldrich) and 750 ng/mL lonomycin (Sigma Aldrich) for 4 hours at 37°C and 5% C02 in RPMI 1640 medium (Thermo Fisher) with 10% FBS, 1 %
Penicillin/Streptomycin, followed by an additional hour with 0.75μΙ_/ιτιΙ_ BD GolgiStop™ (BD
Bioscience). For all measurements, dead cell exclusion was performed using the fixable viability dye for 405nm excitation (Thermo Fisher). For staining of intracellular antigens, cells were permeabilized and fixed using the FoxP3 Staining Buffer Kit (eBioscience) and then stained with the respective antibodies. Cells were analyzed on a BD FACSCanto II flow cytometer using BD FACSDiva software (BD Bioscience). Data analysis was performed with FlowJo (TreeStar).
Statistics. Outliers identified by Grubbs' test were excluded and normality was assessed by Kolgomorow-Smirnow test. To compare two groups we used t-test or Mann-Whitney U-test, as appropriate. To compare more than two groups we used one-way ANOVA followed by Tukey's test post-hoc or Kruskal-Wallis test with Dunn's test post-hoc, as appropriate. For comparison of blood pressure time courses from telemetry measurements we used two-way ANOVA. All statistical analysis was performed using GraphPad Prism 6.

Claims

Short Chain Fatty Acid (SCFA) for use in the treatment of cardiovascular disease.
Short Chain Fatty Acid (SCFA) for use according to the preceding claim, wherein the SCFA is propionic acid or propionate.
Short Chain Fatty Acid (SCFA) for use according to claim 1 , wherein the SCFA is butyric acid or butyrate.
Short Chain Fatty Acid (SCFA) for use according to claim 1 , wherein the SCFA is selected from the group consisting of formic acid, acetic acid, isobutyric acid, valeric acid, isovaleric acid or caproic acid, or formate, acetate, isobutyrate, valerate, isovalerate and caproate.
Short Chain Fatty Acid (SCFA) for use according to claim 2, wherein the SCFA is propionate and is administered as a sodium propionate, calcium propionate or potassium propionate.
Short Chain Fatty Acid (SCFA) for use according to any one of the preceding claims, wherein the treatment is a prophylactic treatment.
Short Chain Fatty Acid (SCFA) for use according to any one of the preceding claims, wherein the cardiovascular disease is characterised by an inflammation of cardiac tissue.
Short Chain Fatty Acid (SCFA) for use according to any one of the preceding claims, wherein the cardiovascular disease is hypertensive heart disease.
Short Chain Fatty Acid (SCFA) for use according to any one of the preceding claims, wherein the cardiovascular disease is diastolic heart failure (heart failure with preserved ejection fraction; HFpEF).
Short Chain Fatty Acid (SCFA) for use according to any one of the preceding claims, wherein the cardiovascular disease is systolic heart failure (heart failure with reduced ejection fraction (HFrEF).
Short Chain Fatty Acid (SCFA) for use according to any one of the preceding claims, wherein the cardiovascular disease is a cardiac arrhythmia.
Short Chain Fatty Acid (SCFA) for use according to any one of the preceding claims for the secondary prophylaxis of cardiac arrhythmia in subjects having previously
experienced myocardial infarction.
Short Chain Fatty Acid (SCFA) for use according to any one of the preceding claims, wherein the subject of treatment exhibits one or more of the following attributes:
a) shows symptoms of being at risk of developing a cardiovascular disease and/or heart failure;
b) has elevated levels of risk markers in ex vivo tests;
c) has previously experienced cerebral or myocardial ischemia; and/or
d) exhibits a predisposition of developing cardiovascular disease, for example a genetic predisposition of developing cardiovascular disease,
e) exhibits borderline or established thickening of the left ventricular wall
(hypertrophy), and/or f) shows an abnormal electrocradiogram/Holter monitor or echocardiogram.
14. Short Chain Fatty Acid (SCFA) for use according to any one of the preceding claims, wherein the subject of treatment exhibits (i) relatively low levels of bacteria capable of SCFA-production in their intestine and/or (ii) relatively low levels of SCFA in their intestine, compared to a suitable control, for example a healthy subject and/or a population average.
15. Short Chain Fatty Acid (SCFA) for use according to any one of the preceding claims, wherein SCFA are orally administered to a subject.
16. Short Chain Fatty Acid (SCFA) for use according to the preceding claim, wherein SCFA are orally administered to a subject as and/or in a nutritional product.
17. Short Chain Fatty Acid (SCFA) for use according to any one of the preceding claims, wherein the subject of SCFA treatment is co-administered with an isolated population of intestinal bacteria.
18. Short Chain Fatty Acid (SCFA) for use according to any one of the preceding claims, wherein the subject of SCFA treatment is co-administered with dietary fiber.
19. Short Chain Fatty Acid (SCFA) for use according to any one of the preceding claims, wherein the SCFA are administered in the form of dietary fiber as a substrate for SCFA production via bacteria present in the intestine of a subject.
20. Isolated population of intestinal bacteria for the treatment of cardiovascular disease, wherein said bacterial population produces elevated levels of SCFA compared to a suitable control.
21 Dietary fiber for the treatment of cardiovascular disease, wherein said dietary fiber produces elevated levels of SCFA compared to a suitable control.
PCT/EP2016/071317 2015-09-09 2016-09-09 Short-chain fatty acids for use in the treatment of cardiovascular disease WO2017042337A1 (en)

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US11065217B2 (en) 2017-01-27 2021-07-20 Temple University—Of the Commonwealth System of Higher Education Use of short chain fatty acids for the treatment and prevention of diseases and disorders
US11759442B2 (en) 2017-01-27 2023-09-19 Temple University-Of The Commonwealth System Of Higher Education Use of short chain fatty acids for the treatment and prevention of diseases and disorders
US10414755B2 (en) 2017-08-23 2019-09-17 Novartis Ag 3-(1-oxoisoindolin-2-yl)piperidine-2,6-dione derivatives and uses thereof
US10640489B2 (en) 2017-08-23 2020-05-05 Novartis Ag 3-(1-oxoisoindolin-2-yl)piperidine-2,6-dione derivatives and uses thereof
US10647701B2 (en) 2017-08-23 2020-05-12 Novartis Ag 3-(1-oxoisoindolin-2-yl)piperidine-2,6-dione derivatives and uses thereof
US11053218B2 (en) 2017-08-23 2021-07-06 Novartis Ag 3-(1-oxoisoindolin-2-yl)piperidine-2,6-dione derivatives and uses thereof
US11185537B2 (en) 2018-07-10 2021-11-30 Novartis Ag 3-(5-amino-1-oxoisoindolin-2-yl)piperidine-2,6-dione derivatives and uses thereof
US11192877B2 (en) 2018-07-10 2021-12-07 Novartis Ag 3-(5-hydroxy-1-oxoisoindolin-2-yl)piperidine-2,6-dione derivatives and uses thereof
US11833142B2 (en) 2018-07-10 2023-12-05 Novartis Ag 3-(5-amino-1-oxoisoindolin-2-yl)piperidine-2,6-dione derivatives and uses thereof
CN114177165A (en) * 2021-10-28 2022-03-15 华中科技大学同济医学院附属协和医院 Use of short chain fatty acids in vascular related disorders
CN115068458A (en) * 2022-07-21 2022-09-20 哈尔滨医科大学 Application of valeric acid in preparation of medicine for preventing and treating diabetes
CN115068458B (en) * 2022-07-21 2023-12-12 哈尔滨医科大学 Application of valeric acid in preparation of medicine for preventing and treating diabetes

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