HK1072958B - New strain of lactic acid bacterium and edible compositions, drugs and veterinary products containing it - Google Patents

Description

Lactic acid bacterial strains and edible compositions, medicaments and veterinary products comprising such strains

The invention relates to a novel, biologically pure strain of Streptococcus thermophilus (Streptococcus thermophilus ssp. salivarius) (CD8) (deposited at the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany, accession number DSM 14667, 12.4.12.2001) in braun schviger, Germany, as well as progeny and mutants thereof.

The invention further relates to edible compositions, pharmaceutical and veterinary products containing said novel strain CD8 as active ingredient (or as one of the active ingredients) and to the use of the above-mentioned strain for the preparation of edible compositions, pharmaceutical and veterinary products for the prevention/treatment of various underlying pathological conditions or of the indicative and predictive signs of an evident pathology.

When the edible compositions and medicaments of the invention are used in humans, their preventive or rather therapeutic effect is mainly shown in: prevention of certain liver diseases, such as hepatic steatosis (fatty liver), particularly nonalcoholic liver steatosis, and hepatic encephalopathy; prevention of some endocrine and metabolic diseases such as hyperinsulinemia, insulin resistance and obesity; infant pathologies such as autism, Attention Deficit Disorder (ADD) and Attention Deficit Hyperactivity Disorder (ADHD) are also prevented (Attention deficiency/Hyperactive Disorder).

When used in animals, veterinary products are useful in the treatment of liver pathology and endocrine and metabolic diseases.

Briefly, in the remainder of this description, the prevention/treatment of hepatic steatosis (fatty liver) and non-alcoholic hepatic steatosis will be specifically described, as well as the specific prevention and treatment efficacy of the edible compositions and medicaments of the present invention against these pathologies.

Hepatic steatosis (or fatty liver) refers to the excessive accumulation of lipids in hepatocytes, by "excessive accumulation" is meant the accumulation of lipids that exceeds 5% of the normal liver weight. In macrovesicular (macrovesicular) hepatic steatosis, as occurs in adipocytes, large droplets of triglycerides swell the hepatocytes, translocating their nuclei to the cell borders. In vesicular (microviscular) hepatic steatosis, small droplets of triglycerides accumulate in hepatocytes, leaving the nucleus in the central position, and the hepatocytes appear foam-like.

Liver steatosis often results in a limited increase in serum transaminases (4-fold below the upper limit of normal) and can be reliably identified by imaging techniques such as ultrasonography, computerized tomography, and the like.

NonAlcoholic liver steatosis (often referred to as NonAlcoholic steatoHepatitis, NASH) is a clinical symptom of steatosis, with accompanying inflammation of the liver.

Non-alcoholic liver steatosis may be diagnosed by liver biopsy after confirmation of exclusion of other causes of liver disease (e.g. infection with hepatitis B virus and hepatitis C virus) and exclusion of abuse of alcohol (20 grams/day). Once the abuse factor is eliminated, the following possible etiological factors should be considered:

diet abnormality: obesity; total parenteral feeding; rapid weight loss

Medicine preparation: an estrogen; a corticosteroid; amiodarone

Metabolic diseases: atopy of beta-lipoproteinemia; hypolipoproteinemia beta; (ii) morbid Salson's disease;

wecker disease; lipodystrophy of limbs

Surgical modification of the gastrointestinal system: jejunal ileocecal bypass; large-area small intestine excision;

gastroplasty

The specific cause of nonalcoholic liver steatosis is not clear. In the past, the general patients suffering from this condition have been described as female, obese, hyperglycemic (possibly due to diabetes). In addition, patients may present with a blood triglyceride surplus and suffer from coronary disease, thyroid disorders or hypertension. It has recently been reported that patients with non-alcoholic liver steatosis do not always meet this description. One trial consisted of male and female subjects who were not overweight, who were not diabetic and who did not have an excess of blood triglycerides. Another group of patients diagnosed with non-alcoholic liver steatosis includes children aged 9 to 16 years. They are mostly overweight but only 2/30 has diabetes.

Without a blood test, this test would make possible a definitive diagnosis of steatosis and nonalcoholic liver steatosis.

An increase (and possibly no increase) in the transaminases aspartate transaminase AST and alanine transaminase ALT is the only biochemical marker, however they are common in both pathologies. Normal biochemical values have been found in pathologically obese individuals whose liver biopsies show progressive liver disease.

However AST/ALT ratios would be beneficial in distinguishing between alcoholic and non-alcoholic liver steatosis, where abuse of alcohol (ethanol) could lead to profound pathological anatomical changes. The AST/ALT ratio is generally higher than 2 in alcoholic steatosis, while ALT levels are higher than AST levels in non-alcoholic steatosis.

There is currently no specific method of treating nonalcoholic liver steatosis that is generally accepted. It is apparent that patients who are obese, have diabetes, and have elevated blood triglycerides are advised to lose weight and control their diabetes by taking a low-calorie, low-fat diet and taking insulin or hypoglycemic drugs. For a detailed review of the pathology, clinical aspects, diagnosis and treatment of NASH, see Sheth S G. et al, non-alcoholic steatohepatitis Ann. Intern. Med.1997, 127-.

The lack of effective and generally accepted treatment constitutes a risk factor because, although non-inflammatory steatosis is a benign condition, 10% to 50% of patients affected by non-alcoholic liver steatosis develop a progressive fibrosis that can degenerate into cirrhosis. Cirrhosis is the 11 th cause of death in western countries such as the united states.

It is therefore a need to have at our disposal a method for the effective prevention/treatment of liver steatosis, particularly nonalcoholic liver steatosis, and it is therefore an object of the present invention to make such a method available.

According to the invention, the method comprises a novel, biologically pure strain of Streptococcus thermophilus (CD8) (deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Brousswight, Germany on 4.12.2001, accession number DSM 14667) and progeny and mutants thereof.

The invention further relates to compositions containing the aforementioned strains of streptococcus as active ingredients, which compositions can be embodied in the form and activity of edible products or dietary supplements, or pharmaceuticals themselves, or veterinary products, for appropriate adjuvant or prophylactic or therapeutic effects, the particular function to be performed by the composition depending on the particular subject to which it is to be applied.

The invention also includes the use of the above-mentioned strains for the preparation of a composition suitable for the prevention/treatment of the liver, endocrine and metabolic disorders or autism and ADD or ADHD as previously listed, and a method of prevention/treatment based on the administration (preferably oral) of the above-mentioned composition

Although it is not necessary for the understanding and practical application of the present invention to explain the mechanism by which the neostreptococcal strain (CD8) exerts a preventive/curative effect, it can be hypothesized that the progression of benign and nonalcoholic liver steatosis (NASH) is inhibited by streptococcal-induced changes in the inflammatory cytokine signals, by an improvement in epithelial barrier function, and by preventing translocation of other intestinal bacteria. These mechanisms are not mutually exclusive and it is possible that new strains of streptococcus have multiple activities.

Lactic acid bacteria-based compositions that are protective against the intestinal tract are known, but compositions that alleviate or combat benign or non-alcoholic liver steatosis are not known.

The compositions of the invention comprising a prophylactically or therapeutically effective amount of streptococcus thermophilus (CD8) or progeny or mutants thereof, wherein the aforementioned streptococcus is the sole active ingredient or is admixed with one or more other active ingredients and/or one or more pharmacologically acceptable excipients, can be formulated as an edible composition or as a dietary supplement, as a pharmaceutical or veterinary product. The method of selecting the excipients and the most suitable preparation method according to the specific purpose of the composition belong to the category of those skilled in the food or pharmaceutical arts.

A particularly preferred composition comprises the aforementioned Streptococcus CD8 and Lactobacillus brevis CD 2. The latter is deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Bronsterger, Germany under the deposit number DSM 11988.

The aforementioned composition may further contain a strain selected from Bifidobacterium infantis (Bifidobacterium longum), Bifidobacterium breve (Bifidobacterium breve), Bifidobacterium longum (Bifidobacterium longum), Bifidobacterium bifidum (Bifidobacterium bifidum), Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus casei (Lactobacillus casei), Lactobacillus bulgaricus (Lactobacillus bulgaricus), Lactobacillus acidophilus (Lactobacillus acidophilus) and the like, or a mixture thereof.

The aforementioned composition may further comprise at least one of the following ingredients: vitamin E, choline, ursodeoxycholic acid, clofibrate, thioglitazone, gemfibrozil, betaine, N-acetylcysteine, rosiglitazone, a basic amino acid, L-carnitine or a lower alkyl L-carnitine or a pharmacologically acceptable salt thereof.

Preferred lower alkyl L-carnitines are acetyl, propionyl and isovaleryl L-carnitine. Pharmacologically acceptable salts of L-carnitine are the fumarate and tartrate salts, while pharmacologically acceptable alkyl L-carnitine salts are the halide, fumarate and galactarate salts.

Combinations suitable for oral administration may be presented in single dosage forms such as tablets, capsules and packets, or in powder and granule form, or in suspension or emulsion form.

Tablets and capsules may contain binders, lubricants, stabilizers, coloring substances, separating agents and the like. Tablets may be coated using well known techniques.

Liquid compositions may be presented as aqueous or oily suspensions, for example in edible oils, or may be prepared by dissolving or suspending the formulation in powder or granular form in water or other suitable solvent, as desired by the user.

These liquid compositions may contain suspending agents, emulsifiers, preservatives and the like.

The various dosage forms described above preferably contain an amount of Streptococcus thermophilus (CD8) sufficient to provide the user with 500 to 36000 million bacteria of said strain per day when the user follows an easy-to-use regimen.

We now describe a clinical study in which nonalcoholic liver steatosis patients were enrolled. Diagnosis of non-alcoholic liver steatosis must include exclusion of hepatitis C virus infection (i.e. hepatitis C virus antibodies) as well as hepatitis B virus infection (hepatitis B surface antigen). Ceruloplasmin and a-1-antitrypsin levels are generally normal in patients with nonalcoholic liver steatosis. Serological autoimmune parameters (anti-mitochondrial, anti-nuclear, anti-smooth muscle and anti-liver/kidney microsomal) were negative in patients affected by nonalcoholic liver steatosis, except for some patients with low positive titers of anti-nuclear antibodies (from 1: 40 to 1: 320).

Clinical study 1

24 patients diagnosed with nonalcoholic liver steatosis symptoms were recruited for clinical study, with the disorder determined by a compatible serum test showing elevated alanine aminotransferase. Every day, all patients ingested 1.8 trillion streptococcus thermophilus (CD8) lyophilized into particles.

Biochemical serum parameters were determined at the beginning of the study and 3 months after treatment, and body weight and lipid profiles were measured. 12 of 24 (50%) were obese (> 20% over ideal body weight). 11 patients were administered oral hypoglycemic agents and/or insulin or had fasting glucose values > 160 mg/dl.

Mean values of serum alkaline phosphatase (alk. phosph), alanine Aminotransferase (ALT) and gamma-glutamyl transpeptidase (GGT) after 3 weeks of treatment were significantly reduced from the allowable basal values (T < 0.01), as shown in the table below, where values are expressed as IU/l.

	Normal value	Before treatment	After treatment
				Alk.phosph.	98-275	322±83	214±44
ALT	0-42	96±21	38±12
				GGT	11-50	72±23	41±16

No significant changes in triglyceride, cholesterol and body weight were detected. The data presented in the table indicate that treatment of nonalcoholic liver steatosis with streptococcus thermophilus (CD8) results in significant improvement in levels of alkaline phosphatase, ALT and GGT, which are serum enzymes indicative of liver function such as cell lysis and cholestasis.

Clinical study 2

Five patients were recruited in the study: two men and three women with increased aspartate Aminotransferase (AST) and alanine Aminotransferase (ALT) levels, performed liver biopsies for them showed non-alcoholic liver steatosis developed during the six months prior to enrollment. These patients showed no signs of other chronic liver disease and were not taking triglyceride lowering drugs. After four months, each patient ingested 18000 million of the following species of bacteria per day:

streptococcus salivarius thermophilus (CD8)

Lactobacillus brevis (CD2)

Bifidobacterium infantis

Lactobacillus plantarum

Lactobacillus casei

Lactobacillus delbrueckii subspecies bulgaricus

Lactobacillus acidophilus.

Each gram of the composition comprises:

streptococcus salivarius thermophilus (CD8)1500 hundred million

Lactobacillus brevis (CD2)100 hundred million

Bifidobacterium infantis 1000 hundred million

Lactobacillus plantarum 100 hundred million

Lactobacillus casei 100 hundred million

300 hundred million of Lactobacillus delbrueckii subspecies bulgaricus

300 hundred million of lactobacillus acidophilus.

Serum levels of AST and ALT were determined at the beginning of the study and at 16 weeks.

The study was completed with five patients who did not show any negative response.

The results of the study are given in the following table, where the results are expressed as IU/l

	Normal value	Before treatment	After treatment
				AST	0-40	109±23	45±19
ALT	0-42	114±29	48±18

The present study did not reveal changes in the lipid profile (cholesterol, triglycerides), which confirmed the specific action of the aforementioned composition containing other lactic acid bacteria and streptococcus salivarius thermophilus (CD 8). The compositions are formulated so as to exhibit anti-inflammatory properties of Streptococcus thermophilus (CD8) and other lactic acid bacteria (e.g.Lactobacillus brevis CD2, see International patent application WO 99/42568).

International recognition of the deposit of microorganisms for the purposes of patent procedure under the Budapest treaty

Proof of deposit

INTERNATIONAL FORMVSL Pharma Ltd.1，Christchurch SquareDublin 8Ireland RECEIPT IN THE CASE OF AN ORIOINAL DEPOSIT

issued pursuant to Rule 7.1 by the

INTERNATIONAI，DEPOSITARY AUTHORITY

identified at the bottom of this page

¹ Where Rule 6.4(d)applia，such date is the date on whlch the status of intemational depositary authority was acauired.Form DSMA-BP/4(Sole page)0196

Claims (5)

1. A biologically pure Streptococcus thermophilus strain CD8, deposited at the Deutsche Sammlung, Germany, at 12.4.2001 with the deposit number DSM 14667, and its progeny.

2. A composition comprising a streptococcus thermophilus strain or progeny thereof, wherein said strain is deposited at the deutsche sammlung von mikroorganismen und zellkulturen gmbh of braunschweger, germany at 12.4.2001 with accession number DSM 14667.

3. The composition of claim 2, further comprising a Lactobacillus brevis strain CD2 deposited with the Deutsche Sammlung, Germany, under accession number DSM 11988, or its progeny.

4. The composition of claim 3, further comprising a strain selected from Bifidobacterium infantis, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium bifidum, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus delbrueckii subsp bulgaricus, Lactobacillus acidophilus, or a mixture thereof.

5. The composition of any one of claims 2-4, further comprising at least one of the following: vitamin E, choline, ursodeoxycholic acid, clofibrate, thioglitazone, gemfibrozil, betaine, N-acetylcysteine, rosiglitazone, a basic amino acid, L-carnitine or a lower alkyl L-carnitine or a pharmacologically acceptable salt thereof.