FIELD OF THE INVENTION

This invention pertains to a method for treatment or prevention of pulmonary hypertension in a patient. The invention further relates to supplements especially beneficial in stopping the progression of or reversing the symptoms of pulmonary hypertension. In particular the present invention provides a novel use for probiotic bacteria, either live or dead or non-viable, as well as food supplements, nutritive compositions and/or pharmaceutical compositions comprising these, for the treatment or prophylaxis of pulmonary lung diseases in a subject.

BACKGROUND OF THE INVENTION

Pulmonary hypertension (PH) is a condition that is characterized by thickened arterial walls of the vessels in the lung. The condition may be congenital PH, primary PH where the initial lesion is in the lungs or acquired PH from failure of other members of the cardiac-pulmonary axis. Congenital PH is generally due to a left-right shunt associated with ventricular or atrial septal defects or patent ductus arteriosus. Primary PH may be caused by emphysema, chronic asthma, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, and pulmonary embolism with parenchymal infarct, reactive pulmonary blood vessels or tissues. Some of the triggers for acquired PH are exposures to toxins, infections, smoking, and residence at high altitudes. Whatever the nature, congenital, primary or acquired, PH eventually leads to right ventricular hypertrophy with or without heart failure. Patients are able to compensate with conventional medical therapies and can stabilize for an indefinite time. Other patients afflicted with this condition die due to progressive heart decompensation or may require a heart/lung transplant or long term oxygen therapy.

Cor pulmonale is defined as right ventricular hypertrophy and dilatation or both, secondary to PH caused by respiratory disorders

While the underlying etiology of acquired PH may not yet be known, one common factor leading to the condition may be that the pulmonary blood vessels and lung tissue are very sensitive to certain factors that trigger the development of the disease. Among these suspected factors are a preexisting condition of Raynaud's syndrome, appetite suppressants, cocaine, and congestive heart failure. Certain disease states such as emphysema, reactive airway disease, chronic obstructive pulmonary disease, H IV infection, scleroderma and systemic lupus erythematosus are often accompanied by PH.

Treatment commonly is merely symptomatic. Methods of lowering of the vasculature's blood pressure in those patients with hypertension may give some relief.

US 6,218,366 discloses a therapeutic method comprising administering ribose to a mammal in an effective amount to increase the tolerance of the mammal to hypoxia, wherein the hypoxia is due to cardiovascular disease, myocardial stunning, anaesthesia, surgical procedures, peripheral vascular disease, intermittent claudication, pulmonary dysfunction, physical exertion, pharmaceutical treatment, tachycardia or poisoning.

US 6,420,342 discloses a nutritional composition, comprising ribose and folate in amounts which are effective to support total nucleotide metabolism, and 0.1-8 g orotate per 5 g of ribose.

WO 2006/050585 Discloses a food product comprising at least one betaine and L-arginine in a combined amount effective to induce and/or to sustain a physiological increase in nitric oxide production in a mammal after ingesting said food product. This is claimed to be beneficial for the treatment of pulmonary hypertension by lowering the blood pressure.

WO 2005/058335 discloses the use of LMG P-22110 (L rhamnosus NRCC L572) for the treatmen or prophylaxis of COPD.

SUMMARY OF THE INVENTION

The present inventors surprisingly found that probiotic bacteria can prevent heart disease caused by pulmonary dysfunction. In particular probiotic bacteria are lactic acid producing bacteria, preferably selected from the group consisting of Lactobacillus and Bifidobacterium. Strains of probiotic bacteria of these species are highly effective in the prevention of heart disease caused by pulmonary dysfunction. In a preferred embodiment the composition comprising probiotica bacteria is used for the treatment and prevention of pulmonary hypertension. In particular it was found that when measuring the effect on right ventricular hypertrophy (RVH) in hearts of LPS sensitised mice using several strains of lactic acid bacteria these strains of probiotic bacteria had significant beneficial effect on RVH. This was especially surprising as the probiotic bacteria were administered orally. RVH is the earliest sign of right ventricular pressure overload (hypertension) in patients with COPD. This structural adaptation of the heart does not alter right ventricular and left ventricular systolic function, see Vonk-Noordegraaf et al., Chest 2005; 127:1898-1903.

DETAILED DESCRIPTION OF THE INVENTION

The present invention thus concerns a method for the treatment or prevention of pulmonary heart disease, in particular pulmonary hypertension, said method comprising administering an effective amount of probiotc bacteria to a subject.

In other words the present invention concerns the use of probiotic bacteria for the preparation of a composition for the treatment and/or prevention of pulmonary heart disease, in particular pulmonary hypertension. In other words the invention concerns a composition comprising probiotic bacteria for use in the treatment and/or prevention of pulmonary heart disease, in particular pulmonary hypertension.

Herein "probiotic bacteria", "probiotics" or "probiotic strain(s)" refers to strains of live micro-organisms, preferably bacteria, which have a beneficial effect on the host when ingested (e.g. enterally or by inhalation) by a subject.

Normal pulmonary arterial pressure ( at sea level) has a mean value of 12-16 mm Hg. In the context of the present invention pulmonary hypertension (PH) is defined as a mean pulmonary artery pressure of >20 mm Hg and is placed in the heterogeneous group of PH associated with disorders of the respiratory system and/or hypoxaemia. The term "out-of-proportion" pulmonary hypertension, in particular in patients with COPD, is defined by a mean pulmonary artery pressure >35-40 mm Hg and a relatively preserved lung function (with low to normal arterial carbon dioxide tension) that cannot explain prominent dyspnoea and fatigue.

Probiotics

The probiotic bacteria used are preferably lactic acid producing bacteria, preferably of the genus

Lactobacillus or Bifidobacterium. The bacteria preferably are food-grade, i.e. they are preferably considered as not harmful, when ingested by a human or animal subject. It is understood that non-food grade bacteria, for example pathogenic bacteria, which have been modified so that they are no longer harmful when ingested by a subject, are included within the scope of the invention. The Lactobacillus strains may be of the following species: L. rhamnosus, L. casei, L. paracasei, L. helveticus, L. delbrueckii, L. reuteri, L. brevis, L. crispatus, L. sakei, L. jensenii, L. sanfransiscensis, L. fructivorans, L. kefiri, L. curvatus, L. paraplantarum, L. kefirgranum, L. parakefir, L. fermentum, L. plantarum, L. acidophilus, L. johnsonii, L gasseri, L xylosus, L salivarius etc. Preferred species are L rhamnosus, L casei, L paracasei, L. reuteri, L. crispatus, .L fermentum L. plantarum L. acidophilus, L. johnsonii L. gasseri L salivarius, more preferred are L. plantarum, L. casei or L. rhamnosus. Most preferred is to use Lactobacillus strains belonging to the species L. casei.

The Bifidobacterium strains may be of the following species: B. longum, B. breve, B. animalis, B. infantis, B. bifidum, B. adolescentis, B. pseudolongum, B. catenulatum, B. pseudocatenulatum, B. angulatum etc. Preferred species are B. breve and/or B. animalis (especially B. animalis subspecies lactis).

The species identity of micro-organisms can be determined biochemically or by sequencing (e.g. conserved regions) or by known methods such as pulse field gel electrophoresis. In general, strains of bacteria belong to the same species if they show at least 97 % nucleic acid sequence identity in the 16 S rRNA region (e.g. when optimally aligned by for example the programs GAP or BESTFIT using default parameters).

In one embodiment the probiotic bacteria used acoording to thepresent invention are selected from the group consisting of B. breve M-16V, L rhamnosus NRCC L572, DN 114 001 and DN 173 010.

L rhamnosus NRCC L572 has previously been deposited in accordance with the Budapest Treaty at the Belgian Co-ordination Collections of Microorganisms, BCCM™, Gent, Belgium, under Accession No. LMG P-22110.

L casei DN-114 001 has previously been deposited in accordance with the Budapest Treaty at the Collection Nationale de Cultures de Microorganisms (CNCM, lnstitut Pasteur, Paris, France) under the number 1-1518. Bifidobacterium animalis. DN- 173 010 also previously has been deposited in accordance with the Budapest Treaty at the Collection Nationale de Cultures de Microorganisms (CNCM, lnstitut Pasteur, Paris, France) and is registered under the number CNCM 1-2494. B. breve M-16V is from Morinaga Milk Industry, Kanagawa, Japan.

In one embodiment the present composition comprises 10 ² to 10 ¹³ colony forming units (cfu) of probiotic bacteria per gram (g) dry weight of the present composition, or 10 ² to 10 ¹² cfu, or 10 ³ to 10 ¹⁰ cfu, or from 10 ⁴ to 5xlO ⁹ cfu. In one embodiment the present composition comprises 10 ² to 10 ¹³ colony forming units (cfu) per gram (g) dry weight of the present composition, or 10 ² to 10 ¹² cfu, or 10 ³ to 10 ¹⁰ cfu, or from 10 ⁴ to 5xlO ⁹ cfu of one selected from the group consisting of B. breve M-16V, L rhamnosus NRCC L572, DN 114 001 and DN 173 010.

In a further embodiment dead or non-viable bacterial cells of the probitic bacteria are used in the above compositions, instead of or in addition to live (or viable) bacteria, as for example described in WO 01/95741. The amount of dead or non-viable cells used may, for example, be equivalent to that used for live bacteria. Suitable amounts can be easily determined by a skilled person. In such compositions, the amounts of cells are counted (e.g. using a flowcytometer) or measured in a different way as known to a skilled person, as measurement as 'colony forming units' is not feasible.

It is understood that when referring to compositions comprising living cells, this encompasses cells which are viable, such as for example lyophilised cells, which become active again after administration or reconstitution with liquid.

Dietary fibres

In one embodiment the present composition further comprises dietary fibres (prebiotics). Dietary fibres as used in the present invention are typically resistant to digestion and absorption in the human small intestine with preferably a complete or partial fermentation in the large intestine. Preferably the present composition comprises at least one dietary fibre selected from the group consisting of galactooligosaccharides, trans galactooligosaccharides, inulin, fructooligosaccharides (FOS) including long chain FOS (IcFOS) and short chain FOS (scFOS) and mixtures thereof, xylooligosaccharides, palatinoseoligosaccharide, soybean oligosaccharide, gentiooligosaccharide, pectin, pectin hydrolysate, pectate, alginate, chondroitine, hyaluronic acids, heparine, heparane, sialoglycans, fucoidan, fucooligosaccharides, carrageenan, xanthan gum, cellulose, polydextrose (PDX, a non-digestible carbohydrate that has been synthesized from randomly cross-linked glucose and sorbitol), guar gum, arabinoxylan preferably MGN-3 Rice Bran Arabinoxylan Compound according to US Patent: 5,560,914, xyloglycan, callose, lignin and/or degradation products thereof. All of these have beneficial prebiotic effects in the intestinal system. It is unknown how the immunological effects are induced by these dietary fibres. It is likely that a combination of induced changes in the flora in addition to potential direct effects on the immune system is responsible for the observed effects. Without being bound by theory, the inventors hypothesize that the prebiotic effect will enhance the probiotic effect of the composition since the prebiotics stimulate the growth of probiotic bacteria in the gut.

In one embodiment the dietary fibres are selected from galactooligosaccharides (GOS), trans- galactooligosaccharides (TOS), fructooligosaccharides (FOS) and pectin hydrolysate.

Composition

The composition made using one or more strain(s) according to the invention may be any type of composition, which is suitable for a human subject suffering from pulmonary heart diseases, especially pulmonary hypertension. The composition may be a food, a food supplement composition, nutritive (food) composition or pharmaceutical composition. Depending on the type of composition and its preferred administration method, the components and texture of the composition may vary. A food or food/nutritive composition comprises besides the bacterial strain(s) of the invention also a suitable food base. A food or food composition is herein understood to include solids (for example powders), semisolids and/or liquids (e.g. a drink or beverage) for human or animal consumption. A food or food/nutritive composition may be a dairy product, such as a fermented dairy product, including but not limited to yoghurt, a yoghurt-based drink or buttermilk. Such foods or food compositions may be prepared in a manner known per se, e.g. by adding the strain(s) of the invention to a suitable food or food base, in a suitable amount (see e.g. WO 01/82711). In a further embodiment, the strain(s) are used in or for the preparation of a food or food/nutrient composition, e.g. by fermentation. Examples of such strains include probiotic lactic acid producing bacteria of the invention. In doing so, the strain(s) of the invention may be used in a manner known per se for the preparation of such fermented foods or food/nutrition compositions, e.g. in a manner known per se for the preparation of fermented dairy products using lactic acid producing bacteria. In such methods, the strain(s) of the invention may be used in addition to the micro-organism usually used, and/or may replace one or more or part of the micro-organism usually used. For example, in the preparation of fermented dairy products such as yoghurt or yoghurt-based drinks, a live food grade lactic acid producing bacterium of the invention may be added to or used as part of a starter culture or may be suitably added during such a fermentation. Food supplement compositions

Apart from an effective amount of probiotic bacteria, a food supplement may comprise one or more carriers, stabilizers, prebiotics and the like. Preferably, the composition is in powder form, for enteral (preferably oral) administration, although nasal administration or inhalation may also be suitable. When using live cells of the strain(s), the cells may be present in an encapsulated form in order to be protected against the stomach. For example the composition may be in the form of a powder packed in a sachet which can be dissolved in water, fruit juice, milk or another beverage.

Food/nutrition composition

Apart from one or more probiotic bacteria in a suitable dosage, a nutrition composition preferably comprises carbohydrates and/or proteins and/or lipids suitable for human and/or animal consumption. The compositions may or may not contain other bioactive ingredients, such as other (probiotic) strains, and prebiotics (dietary fibers), which support the probiotic strains. When using living cells of the probiotic bacteria, the cells may be present in an encapsulated form in order to be protected against the stomach. The nutrition is preferably in liquid or powder form. The nutrition is preferably administered enterally, such as orally or by tube feeding.

The present composition in one embodiment provides nutrition and comprises a lipid component, a protein component and a carbohydrate component. The lipid component preferably provides 5 to 50% of the total calories, the protein component preferably provides 5 to 50% of the total calories, and the carbohydrate component preferably provides 15 to 90% of the total calories. For calculation of the % of total calories for the protein component, the total of energy provided by the proteins, peptides and amino acids needs to be taken and the energy provided by digestible carbohydrates.

Pharmaceutical composition

One or more probiotic bacteria in a suitable dosage may also be used to make a pharmaceutical composition for treatment or prevention pulmonary hea rt disease, in particu lar pu lmonary hypertension. Pharmaceutical compositions wi ll usually be used for entera l, prefera bly ora l, nasal/inhalation, vaginal or rectal administration. Pharmaceutical compositions will usually comprise a pharmaceutical carrier in addition to the probiotic bacteria. The preferred form depends on the intended mode of administration and (therapeutic) application. The pharmaceutical carrier can be any compatible, nontoxic substance suitable to deliver the strains(s) to the desired body cavity, e.g. the intestine of a subject. E.g. sterile water, or inert solids may be used as the carrier usually complemented with pharmaceutically acceptable adjuvants, buffering agents, dispersing agents, and the like. Pharmaceutical compositions may further comprise additional biologically or pharmaceutically active ingredients.

The compositions comprising one or more probiotic bacteria according to the invention are suitable to either treat patients already suffering from pulmonary heart disease, in particular pulmonary hypertension or may be administered prophylactically to subjects which are at risk of developing pulmonary heart disease, in particular pulmonary hypertension.

EXAMPLES

Example 1

In the present examples pulmonary hypertension was induced in mice by inducing lung emphysema by inhalation of lypopolysaccharide (LPS).

Animals:

Specific pathogen free male BALB/c byJlco mice were obtained from Charles River (Maastricht, the Netherlands). Food and water were provided ad libitum and the mice were used when 7-8 weeks of age. All experiments were approved by the animal ethics committee of the University of Utrecht, The Netherlands. LPS from E. coli, serotype O55:B5 was obtained from Sigma.

Sensitisation, treatment and challenge:

Lung emphysema was induced by intranasal administration of LPS (5 μg in 50 μl phosphate buffered saline (PBS)) or, as a control, PBS (50 μl) twice a week for four weeks (day 0, 3, 7, 10, 10, 14, 17, 21, and 24) followed by two weeks of rest. Mice were treated daily with 0.2 ml PBS containing 10 ⁹ (CFU) per strain lactic acid bacteria orally via gavage. Two different diet protocols were used in three separate experiments (table 1). In treatment protocol I, mice received a probiotic diet from day 14 untill the end of the experiment. In preventive protocol II, mice received a probiotic diet starting 14 days before the first LPS (or PBS) treatment untill the end of the experiment. In protocol III mice were treated with either the preventive protocol or the treatment protocol. Table 1 Diet strategies

NA = Not Applicable

Right Ventricular Hypertrophy

Hypertrophy of the right ventricular is an indication for pulmonary hypertension. The whole heart (of 4 out of 10 animals) was isolated and the right ventricular free wall (RV) was completely separated and removed under a dissecting microscope at day 42. The left ventricle and septa (LV+S) and RV were weighed separately after blotting dry. The ratio of RV weight to LV+S weight was used as an index of right ventricular hypertrophy. In case of experiment p281 the ratio of heart weight to body weight was used as an index for heart hypertrophy.

Results

Intranasal LPS administration as described in methods led to heart right ventricle hypertrophy as measured in experiments p235 and p240, in experiment p280 however LPS administration led to an increase in total heart size therefore data is represented as the ratio of heart weight to total body weight. Data from the experiments are depicted in table 2.

Table 2 Heart hypertrophy in Balb/c mice 2 weeks after the last PBS- or LPS-treatment

Results are expressed as arithmetic average ± SEM . p<0.05, p<0.01 and p<0.001 compared with PBS treated mice. * p<0.05, ** p<0.01 and *** p<0.001 compared with LPS-treated mice using Student's t-test.

These results are indicative for the beneficial effect of these probiotic bacteria on the treatment as well as the prevention of pulmonary heart disease, in particular pulmonary hypertension.