阅读说明：本技术 植物乳杆菌组合物及其用途 (Lactobacillus plantarum compositions and uses thereof ) 是由 B·耶普松 于 2019-06-13 设计创作，主要内容包括：本发明涉及一种用于治疗和/或预防人的年龄相关性全身性炎症的植物乳杆菌的至少一种益生菌菌株。(The present invention relates to at least one probiotic bacterial strain of lactobacillus plantarum for the treatment and/or prevention of age-related systemic inflammation in humans.)

1. A method for the treatment and/or prevention of age-related systemic inflammation in a human, the method comprising administering to a human in need thereof a therapeutically effective dose of at least one probiotic bacterial strain of Lactobacillus plantarum (Lactobacillus plantarum).

2. The method of claim 1, wherein the human is over the age of 60, 65, 70, 75, 80, 85, or 90 years.

3. The method of claim 1 or 2, wherein the human is a male.

4. The method of claim 1 or 2, wherein the human is a female.

5. The method of claim 4, wherein said female is a postmenopausal female.

6. The method according to any one of claims 1 to 5, wherein the effective dose of the at least one probiotic strain of Lactobacillus plantarum is administered at least once daily.

7. The method according to any one of claims 1 to 6, wherein the effective dose of the at least one probiotic bacterial strain of Lactobacillus plantarum is about 10 per dose⁶To about 10¹⁴Colony Forming Units (CFU), preferably about 10 per dose⁸To about 10¹²CFU or more preferably about 10 per dose⁹To about 10¹¹CFU。

8. The method according to any one of claims 1 to 7, wherein the effective dose or doses of the at least one probiotic strain of Lactobacillus plantarum is/are administered within one day, and wherein the daily dose of the at least one probiotic strain of Lactobacillus plantarum is about 10 per day⁶To about 10¹⁴CFU, preferably about 10 per day⁸To about 10¹²CFU or more preferably about 10 per day⁹To about 10¹¹CFU。

9. The method of any one of claims 1 to 8, wherein said treatment and/or prevention of age-related systemic inflammation involves reducing and/or preventing an increase in C-reactive protein (CRP) levels and/or reducing and/or preventing an increase in calprotectin levels.

10. The method according to any one of claims 1 to 9, wherein the at least one probiotic strain of lactobacillus plantarum is selected from the group consisting of lactobacillus plantarum 299(DSM 6595), lactobacillus plantarum 299v (DSM 9843), lactobacillus plantarum HEAL 9(DSM 15312), lactobacillus plantarum HEAL 19(DSM 15313), lactobacillus plantarum HEAL 99(DSM 15316) and lactobacillus plantarum GOS42(DSM 32131).

11. The method according to claim 10, wherein the at least one probiotic strain of lactobacillus plantarum is lactobacillus plantarum HEAL 9(DSM 15312).

12. The method according to any one of claims 1 to 11, wherein the at least one probiotic bacterial strain is administered in a composition comprising at least one carrier selected from the group consisting of a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, a diluent and a food product.

13. The method of claim 12, wherein the composition is provided in the form of a solution, suspension, emulsion, tablet, granule, powder, capsule, lozenge, chewing gum, or suppository.

14. The method of claim 12, wherein the food product is a cereal-based product, a dairy product, a fruit juice beverage, or a fermented food product.

15. At least one probiotic strain of lactobacillus plantarum for use in the treatment and/or prevention of age-related systemic inflammation in a human.

16. A pharmaceutical composition comprising at least one probiotic bacterial strain according to claim 15 and one or more pharmaceutically acceptable excipients, for use in the treatment and/or prevention of age-related systemic inflammation in a human.

17. Use of a composition comprising at least one probiotic bacterial strain according to claim 15 or use of a pharmaceutical composition according to claim 16 for the treatment and/or prevention of age-related systemic inflammation in a human.

18. The method or use according to any one of claims 1 to 17 for treating age-related systemic inflammation in a human, wherein the systemic inflammation in the human is indicated by serum CRP levels of 2-10mg/L or 3-10 m/L.

Technical Field

The present invention relates to at least one probiotic bacterial strain of Lactobacillus plantarum for the treatment and/or prevention of age-related systemic inflammation in humans. The invention also relates to a pharmaceutical composition thereof. Further, the present invention relates to methods and uses of at least one probiotic bacterial strain of lactobacillus plantarum and/or a pharmaceutical composition thereof.

Background

The aging process in humans involves almost all organs of the body with a gradual decline in function. Aging is associated with higher levels of low grade systemic inflammation that has no direct impact on the elderly's daily life, but may increase the risk of developing other diseases, such as cardiovascular disease, insulin resistance and diabetes, osteoporosis, cognitive decline and dementia, and various cancers, and may lead to increased mortality. Age-related low-grade systemic inflammation (also known as "inflammatory Aging" (France schi et al, 2000, Ann N Y Acad Sci 908:244- "254") in New York academy of sciences) is typically characterized by elevated levels of C-reactive protein (CRP) and proinflammatory cytokines such as interleukin 6(IL-6) and tumor necrosis factor alpha (TNF α) and reduced levels of anti-inflammatory cytokines such as interleukin-10 (IL-10) (Barettlt et al, 2012, aged Cell (Aging Cell) 11:912- "915).

The reasons for the above changes are unknown, nor is effective treatment or prevention of age-related systemic inflammation known in the art.

Surprisingly, the inventors have shown that administration of a specific species of probiotic, lactobacillus plantarum, has a significant effect on age-related systemic inflammation in otherwise healthy elderly individuals.

Disclosure of Invention

According to a first aspect, the present invention provides at least one probiotic strain of lactobacillus plantarum for use in the treatment and/or prevention of age-related systemic inflammation in a human.

Age-related systemic inflammation

"systemic inflammation" includes the meaning of systemic inflammation that is typically caused by the release of pro-inflammatory cytokines by immune-related cells and the activation of the innate immune system. Specifically included is the meaning of chronic systemic inflammation, which is generally a process when activation of the innate immune system persists beyond the initial acute phase and becomes "chronic".

"age-related systemic inflammation" includes the meaning that systemic inflammation is chronic and is associated with the aging process. Thus, age-related systemic inflammation does not include acute systemic inflammation caused by a single trauma (e.g., snake bite, burn, heart attack, or infection such as pneumonia). In general, age-related systemic inflammation may be present in otherwise healthy individuals, particularly the elderly.

Thus, systemic inflammation is indicated by markers in the blood and is distinct from local inflammation occurring in tissues/organs of the body.

It is believed that age-related systemic inflammation may contribute to the development or progression of other conditions and/or be a risk factor for other conditions, including cardiovascular disease, insulin resistance and diabetes, osteoporosis, cognitive decline and dementia, and various cancers. For example, there is now a scientific view that serum levels of CRP above 3mg/L are associated with an increased risk of cardiovascular disease.

In general, systemic inflammation can be classified as "low-grade systemic inflammation" when markers of inflammation (primarily CRP) cannot be attributed to viral or bacterial infection.

In the following examples, serum CRP levels of 2-10mg/L were used to define the low-grade systemic inflammatory group of patients to be treated according to the invention.

Treatment and prevention

"use in therapy and/or prophylaxis" comprises the meaning of the use in a subject to produce the following effects: preventing, delaying, protecting from, reducing the severity of, and/or eliminating one or more symptoms and/or other markers associated with a disease or condition.

"treating" or "treatment" includes indicating that the event or condition being treated is improved, reduced in severity, eliminated, prevented from further occurring, delayed and/or caused to cease. Such treatment is typically performed after an event (or the like) has occurred or the pathology has emerged. It is also to be understood that such terms may include the meaning that the event or condition is maintained in the current state without worsening or further progression.

"preventing (prevention, presence, or presence)" includes protecting the event or condition being prevented from occurring, delaying, reducing (e.g., reducing in severity), being prevented from occurring, or being caused to cease. Such prevention is typically performed before the occurrence of an event or manifestation of a condition, but it is understood that this may also mean preventing the further occurrence of the same kind of event. It is also to be understood that such terms may include the meaning that the event or condition is maintained in the current state without worsening or further progression.

For example, the symptoms of age-related systemic inflammation may be improved by at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% after administration of the at least one probiotic strain of lactobacillus plantarum according to the first aspect of the invention compared to the at least one probiotic strain of lactobacillus plantarum without administration.

For example, treating and/or preventing age-related systemic inflammation may involve reducing and/or preventing an increase in C-reactive protein (CRP) levels and/or may involve reducing and/or preventing an increase in calprotectin levels.

C-reactive protein and calprotectin

C-reactive protein (CRP) is an acute phase protein produced by the liver that increases after secretion of interleukin-6 by macrophages and T cells. Thus, CRP levels in plasma are elevated in response to the presence of inflammation in vivo. The physiological role of CRP is to bind to lysolecithin expressed on the surface of dead or dying cells (and certain types of bacteria) in order to activate the complement system via C1 q.

The level of CRP can be measured by any suitable method known in the art. CRP is typically measured by conventional blood tests to determine the concentration of CRP in plasma, for example, using antibodies specific for CRP. Examples of tests for measuring CRP include the tests described in domiinici et al (2004) journal of clinical laboratory analysis (J Clin Lab Anal) 18(5), 280-284, immunochromatographic assays and ELISA tests, such as the Eurolyser CRP assay using a photodynamic determination of the reaction between plasma CRP and an immobilized anti-CRP antibody. High sensitivity C-reactive protein (hs-CRP) assays may also be used (Pearson et al, 2003, Circulation 107(3):499-511), which are common in determining the risk of heart disease.

Serum CRP levels in healthy adults are usually as high as10 mg/L (Shine et al, 1981, Clin Chim Acta 117(1):13-23), and in one study, serum CRP levels in 90% of 468 healthy adult volunteers were less than 3mg/L (Shine et al, 1981, Proc. Clin Chim Acta 117(1): 13-23). CRP levels above 10mg/L (usually much higher) are often indicative of severe infection, trauma or chronic disease.

Serum CRP levels in individuals with age-related systemic inflammation are typically 2 to 10mg/L, e.g., 2 to 10mg/L, 3 to 10mg/L, 4 to 10mg/L, 5 to 10mg/L, 6 to 10mg/L, 7 to 10mg/L, 8 to 10mg/L, 9 to 10mg/L, 2 to 3mg/L, 2 to 4mg/L, 2 to 5mg/L, 2 to 6mg/L, 2 to 7mg/L, 2 to 8mg/L, or 2 to 9 mg/L. Individuals with serum CRP levels below 2mg/L may be considered without systemic inflammation.

Thus, it is to be understood that the level of CRP in the serum of a subject/patient may be improved by at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% after administration of at least one probiotic strain of lactobacillus plantarum according to the first aspect of the invention compared to the level of CRP in at least one probiotic strain of lactobacillus plantarum not administered.

Calprotectin is a protein which is released into the intestinal lumen by neutrophils in response to inflammation of the gastrointestinal tract. During intestinal inflammation, neutrophils migrate to the intestinal mucosa. The level of calprotectin in a fecal sample is elevated in response to the presence of inflammation in the gastrointestinal tract, including individuals with Inflammatory Bowel Disease (IBD) (e.g., ulcerative colitis or crohn's disease) or some bacterial infection of the gastrointestinal tract. In particular, calprotectin may be used to help distinguish inflammatory bowel conditions (e.g., IBD) from non-inflammatory bowel conditions (e.g., irritable bowel syndrome).

The level of calprotectin may be measured by any suitable method known in the art.Calprotectin is typically measured in a faecal sample, for example to determine the concentration of calprotectin using an antibody specific for calprotectin. Examples of tests for measuring calprotectin include the tests described in Acevedo et al (2018) J Clin Med Res. 10(5) 396-404, immunochromatographic assays and ELISA tests, e.g.ELISA(Bühlmann)、QuantumfCAL (Buhlmann) or(Eurospital)。

Faecal calprotectin levels up to 110 μ g/g faeces are generally considered normal. Fecal calprotectin levels between 110 and 1800 μ g/g feces are generally considered "elevated" and indicate inflammation. However, moderately elevated levels of fecal calprotectin in excess of 110 μ g/g of stool may still be normal.

Thus, it will be appreciated that the level of calprotectin may be improved by at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% after administration of at least one probiotic strain of lactobacillus plantarum according to the first aspect of the invention compared to the level of calprotectin of at least one probiotic strain of lactobacillus plantarum not administered.

Probiotic bacterial strain

Probiotics are defined as "live microorganisms that, when administered in sufficient amounts, confer a health benefit to the host" (Hill et al, Nature reviews: gastroenterology and hepatology (Nat Rev Gastroenterol Heatotel), 2014,11(8): 506-514). Bacteria of the genera Lactobacillus (Lactobacillus) and Bifidobacterium (Bifidobacterium) are the most commonly used bacteria in probiotic products. These bacteria are generally safe because probiotic products are based on these organisms. In order for bacteria to comply with the definition of probiotic, they must generally be able to survive and colonize the intestine, survive the production and storage processes, and have evidence to have a positive effect on consumer health.

The at least one probiotic strain of lactobacillus plantarum according to the first aspect of the invention may be any probiotic strain of lactobacillus plantarum.

Preferably, the at least one probiotic bacterial strain of lactobacillus plantarum according to the first aspect of the invention is selected from the group comprising lactobacillus plantarum 299(DSM 6595), lactobacillus plantarum 299v (DSM 9843), lactobacillus plantarum HEAL 9(DSM 15312), lactobacillus plantarum HEAL 19(DSM 15313), lactobacillus plantarum HEAL 99(DSM 15316) or lactobacillus plantarum GOS42(DSM 32131).

Most preferably, the at least one probiotic strain of lactobacillus plantarum according to the first aspect of the invention is lactobacillus plantarum HEAL 9(DSM 15312).

Lactobacillus plantarum 299(DSM 6595) was deposited in 1991 on 2.7.1 in the DSM-German Collection of microorganisms and cell cultures (DSM-DEUTSCHE SAMMLUNG VON MIKROORGANISMEN UND Zellkulturen GmbH) under the name Probi.

Lactobacillus plantarum 299v (DSM 9843) was deposited by Probi AB on 16.3.1995 in the DSM-German Collection of microorganisms and cell cultures, Mascheroder Weg 1b, D-38124, Braunschweig, Germany.

Lactobacillus plantarum HEAL 9(DSM 15312), Lactobacillus plantarum HEAL 19(DSM 15313) and Lactobacillus plantarum HEAL 99(DSM 15316) were deposited by Probi AB on DSMZ-German Collection of microorganisms and cell cultures (DSMZ-DEUTSCHE MLUNG VON MIKROORGANISMEN UND ZELLKULTUREN GmbH), Mascherder Weg 1b, D-38124, Broccoder, Blaker, Germany, at 11/27/2002.

Lactobacillus plantarum GOS42(DSM 32131) was deposited by Probi AB on day 9 and 2 of 2015 at the Debrelucelix research Institute DSMZ-Germany Collection of Microorganisms and Cell Cultures (Leibnizz Institute DSMZ-German Collection of Microorganisms and Cell Cultures), Inhoffentr.7B, D-38124.

The composition of the invention may comprise the specified probiotic strain or strains of lactobacillus plantarum, but preferably it consists of the specified probiotic strain or strains, without another effective amount of any other probiotic strain of lactobacillus and/or bifidobacterium or other microorganism.

The probiotic bacterial strain according to the first aspect of the present invention may be live, attenuated, inactivated or dead. Preferably, the probiotic bacterial strain is live. For example, preferably, the probiotic bacterial strain is freeze-dried.

Patient group

The at least one probiotic strain of lactobacillus plantarum according to the first aspect of the invention must be suitable for use in humans. For example, the age of the person may be over 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 years old.

Preferably, the at least one probiotic strain of lactobacillus plantarum is for use in elderly people, such as people over the age of 70, 75, 80, 85 or 90 years.

At least one probiotic strain of lactobacillus plantarum may be used for men.

The at least one probiotic strain of lactobacillus plantarum may be for use in women (including postmenopausal women). At least one probiotic strain of lactobacillus plantarum may be used in women just beginning to menopause. The at least one probiotic bacterial strain of lactobacillus plantarum may be for women up to 10 years after the onset of menopause, e.g. women up to 6, 7, 8, 9 or 10 years after the onset of menopause.

Menopause is the time during which the menstrual period is permanently stopped and women will no longer be fertile during most women's lifetime. Menopause usually occurs between the ages of 49 and 52. Menopause is generally defined by the health professional as the absence of vaginal bleeding that occurs in women for one year. Thus, the date of menopause itself is usually determined retrospectively once 12 months have elapsed after the last appearance of menstrual blood.

Composition comprising a metal oxide and a metal oxide

The at least one probiotic bacterial strain according to the first aspect of the present invention may be present in a composition comprising at least one suitable carrier. For example, the carrier may be a diluent or excipient. The composition may be a solid formulation or a liquid formulation and thus the at least one carrier may be solid or liquid or may comprise both at least one solid component and at least one liquid component.

Examples of suitable liquid carriers include water, milk, coconut water, fruit beverages and juices, milk substitutes (soy beverages, oat beverages, nut drinks and other plant based beverages), foaming beverages, glycerin, propylene glycol and other aqueous solvents.

Examples of suitable solid carriers or excipients include maltodextrin, inulin, cellulose (such as microcrystalline cellulose (MCC), Hydroxypropylmethylcellulose (HPMC) or Hydroxypropylcellulose (HPC)), sugar alcohols, high molecular weight polyethylene glycols, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants (such as starch (preferably corn starch, potato starch, tapioca starch or other plant starches)), sodium starch glycolate, croscarmellose sodium and certain complex silicates, and particulate binders (such as polyvinylpyrrolidone, sucrose, gelatin and acacia). Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

In embodiments according to the first aspect of the invention, the carrier may be selected from pharmaceutically acceptable carriers, pharmaceutically acceptable excipients, diluents and foodstuffs.

Examples of suitable pharmaceutically acceptable carriers, excipients and diluents include those well known to those skilled in the art, for example as described in remington: pharmaceutical sciences and practices (Remington: The Science and Practice of Pharmacy), 19 th edition, volumes 1 and 2 (Gennaro, eds., 1995, Mack Publishing Company).

"food" includes any consumable substance that provides a nutritional benefit or support to an organism. Examples of suitable food carriers include beverages (e.g., fruit juices), dairy products (e.g., yogurt, cheese, ice cream, infant formula, and spreads such as margarine), dairy substitute products (e.g., soy, nut or other plant-based beverages, yogurt, and spreads), cereal-based products (e.g., breads, biscuits, breakfast cereals, pasta, and dry food bars such as health bars), and infant food (e.g., fruit purees and/or vegetables).

The composition according to the first aspect of the invention may be a dried non-fermented composition, a fermented composition or a dried fermented composition. In this context, fermentation specifically comprises lactic acid fermentation by lactic acid bacteria under anaerobic conditions. In the case of a dry, non-fermented composition, substantially no fermentation occurs prior to ingestion by the subject, and thus fermentation occurs only in the gastrointestinal tract after ingestion of the composition by the subject.

Thus, in some embodiments according to the first aspect of the invention, the composition is in the form of a food product, wherein the food product is a cereal-based product, a dairy product, a fruit juice beverage or a fermented food product.

Examples of fermented food products include fermented dairy products (such as yoghurt, kefir or rassi), fermented milk-free milk substitutes (such as kefir from coconut milk), fermented cereal-based products (such as oats, oatmeal, corn, sorghum, wheat), fermented vegetables (such as sauerkraut (sauerkraut), kimchi (kimchi) or sauerkraut), fermented beans or soybeans (such as natto or fermented soybeans) and fermented teas (such as black tea fungus (kombucha)).

In some embodiments according to the first aspect of the present invention, the at least one probiotic bacterial strain is present in a non-naturally occurring composition, e.g. the composition comprises more than one or more probiotic bacterial strains and water.

In use, the at least one probiotic bacterial strain or the composition comprising said at least one probiotic bacterial strain according to the first aspect of the present invention may be mixed with a liquid carrier or a solid carrier prior to administration to a mammal. For example, the subject may mix the at least one probiotic bacterial strain or composition thereof with a carrier comprising one or more liquids selected from water, milk, coconut water, fruit and fruit juices, milk substitutes (soy, oat, nut and other plant based beverages), foaming beverages, or some other aqueous solvent or beverage prior to ingestion. Similarly, at least one probiotic bacterial strain or composition thereof may be mixed with a carrier consisting of one or more food products. Suitable food carriers include oatmeal carriers, barley carriers, fermented or non-fermented dairy products (e.g. yoghurt, ice cream, milkshakes), juices, beverages, soups, bread, biscuits, pasta, breakfast cereals, dried food bars including health bars, plant based foods (e.g. soy products), spreads, baby foods, infant nutrition, infant formula or breast milk substitute at birth.

Preferably, the formulation is a unit dose containing a daily dose or unit, daily sub-dose or an appropriate fraction thereof of the composition comprising the probiotic bacterial strain.

The composition according to the first aspect of the invention may be a dietary supplement. Dietary supplements encompass the meaning of an article of manufacture in e.g. pill, capsule, tablet or liquid form intended to supplement the diet when taken orally. Dietary supplements may contain substances that are vital to life and/or substances that have not been identified as vital to life but may have beneficial biological effects. When the composition according to the first aspect of the invention is in the form of a dietary supplement, the carrier or carriers to be added comprise those well known to the person skilled in the art, for example as described in remington: those given in pharmaceutical sciences and practices, 19 th edition, volumes 1 and 2 (Gennaro eds, 1995, Mike publishing Co.). Any other ingredients commonly used in dietary supplements are known to those skilled in the art and may also be added conventionally with at least one probiotic bacterial strain.

The composition according to the first aspect of the invention may be provided in the form of a solution, suspension, emulsion, tablet, granule, powder, capsule, lozenge, chewing gum or suppository.

In an embodiment according to the first aspect of the invention, the at least one probiotic bacterial strain is present at about 1 x 10⁶To about 1X 10¹⁴CFU/dose, preferably about 1X 10⁸To about 110¹²CFU/dose, more preferably about 1X 10⁹To about 1X 10¹¹CFU/dose, and most preferably about 1 × 10¹⁰CFU/dose amount is present (e.g., present in the composition). Such amount means the combined total CFU/dose of the probiotic strain, if at least one probiotic strain consists of more than one probiotic strain. For example, the at least one probiotic bacterial strain may be at least about 1 x 10⁶、1×10⁷、1×10⁸、1×10⁹、1×10¹⁰、1×10¹¹、1×10¹²Or about 1X 10¹³CFU/dose amount is present. The at least one probiotic bacterial strain may be present in an amount up to about 1 x 10¹⁴、1×10¹³、1×10¹²、1×10¹¹、1×10¹⁰、1×10⁹、1×10⁸Or about 1X 10⁷CFU/dose amount is present. The at least one probiotic bacterial strain according to the first aspect of the present invention may also be used alone in water or any other aqueous vehicle to which it is added or mixed prior to ingestion.

The composition according to the first aspect of the invention may be administered orally, buccally or sublingually in the form of tablets, capsules, powders, beads, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate, delayed or controlled release applications. The composition may be administered in the form of a powder composition, such as a fast-melting microbial composition, for example those described in WO 2017/060477 and british patent application 1708932.7, the entire contents of which are incorporated herein by reference.

The composition according to the first aspect of the invention may be formulated as a controlled release solid dosage form, for example any of those described in WO03/026687 and U.S. patent nos. 8,007,777 and 8,540,980, the entire contents of which are incorporated herein by reference. The compositions may be formulated in layered dosage forms, such as any of those described in WO 2016/003870, the entire contents of which are incorporated herein by reference.

Tablets may be prepared by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine at least one probiotic bacterial strain (e.g. freeze-dried) in free-flowing form (such as a powder or granules), optionally mixed with a binder (e.g. povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (e.g. sodium starch glycolate, crospovidone, croscarmellose sodium), surfactant or dispersing agent. Molded tablets may be prepared by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein, for example, using hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile.

Pharmaceutical composition

A second aspect of the invention provides a pharmaceutical composition comprising at least one probiotic bacterial strain according to the first aspect of the invention and one or more pharmaceutically acceptable excipients for use in the treatment and/or prevention of age-related systemic inflammation in a human.

The pharmaceutical composition according to the second aspect of the invention may be a composition as described above in relation to the first aspect of the invention. The term "pharmaceutically acceptable" encompasses that the excipient or excipients must not be deleterious to the recipient thereof and must be compatible with the at least one probiotic bacterial strain according to the first aspect of the invention. Examples of such pharmaceutically acceptable excipients are well known in the art and include those described above in relation to the first aspect of the invention, for example in ramington: those described in pharmaceutical sciences and practices, 19 th edition, volumes 1 and 2 (Gennaro eds, 1995, Micke publishing Co.).

For example, the pharmaceutical composition may be formulated as a controlled release solid dosage form, such as any of those described in WO03/026687 and U.S. patent nos. 8,007,777 and 8,540,980, or the pharmaceutical composition may be formulated as a layered dosage form, such as any of those described in WO 2016/003870.

The one or more pharmaceutically acceptable excipients may be sterile pyrogen-free water or saline.

Preferably, the pharmaceutical composition according to the second aspect of the invention may be administered by any conventional method, including oral and tube feeding. Administration may consist of a single dose or multiple doses over a period of time.

Methods of treatment

A third aspect of the invention provides a method for the treatment and/or prevention of age-related systemic inflammation in a human, the method comprising administering to a human in need thereof a therapeutically effective amount of at least one probiotic bacterial strain according to the first aspect of the invention or a pharmaceutical composition according to the second aspect of the invention.

In particular, the methods according to the third aspect of the invention comprise those wherein the prevention of age-related systemic inflammation in a human is indicated by reducing the serum level of one or more markers of age-related systemic inflammation compared to the absence of administration of said probiotic bacterial strain.

The method according to the third aspect of the invention may be performed by any person as defined above in relation to the first aspect of the invention. For example, the age of the person may be over 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 years old.

Preferably, the method according to the third aspect of the invention is performed on elderly people, for example people over the age of 70, 75, 80, 85 or 90 years.

The method according to the third aspect of the invention may be performed on a human.

The method according to the third aspect of the invention may be performed on a female comprising a postmenopausal woman. The method according to the third aspect of the invention may be performed on a female having just started menopause. The method according to the third aspect of the invention may be performed on women up to 10 years after the onset of menopause, for example women up to 6, 7, 8, 9 or 10 years after the onset of menopause.

Preferably, the serum level of CRP is reduced to less than 3mg/L, more preferably less than 2 mg/L.

Administration according to the method of the third aspect of the invention may comprise oral, buccal or sublingual administration as described above in relation to the first aspect of the invention.

The administration of the method according to the third aspect of the invention is preferably performed at least once daily.

Administration according to the method of the third aspect of the invention may comprise administration repeated for up to one, two, three, four or five weeks, up to one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve months, or more than one, two or three years or more. Preferably, administration is repeated for at least one week, two weeks, three weeks, more preferably at least four weeks, one month, two months or three months, and even more preferably at least six months, nine months or one year.

According to the first aspect of the invention, preferably, the unit dose administered according to the method of the third aspect of the invention is about 1 x 10⁶To about 1X 10¹⁴CFU/unit dose, preferably about 1X 10⁸To 1X 10¹²CFU per unit dose, and more preferably about 1X 10⁹To about 1X 10¹¹CFU/unit dose, and most preferably about 1X 10¹⁰CFU/unit dose. Application of the method according to the third aspect of the invention preferably results in about 1 x 10⁶To about 1X 10¹⁴CFU/unit dose, preferably about 1X 10⁸To about 1X 10¹²CFU/unit dose, more preferably about 1X 10⁹To about 1X 10¹¹CFU/unit dose, and most preferably about 1X 10¹⁰Effective dose of CFU per unit dose. Preferably, one unit dose is administered to each subject per day. Thus, application of the method according to the third aspect of the invention preferably results in about 1 × 10⁶To about 1X 10¹⁴CFU/day, preferably about 1X 10⁸To about 1X 10¹²CFU/day, more preferably about 1X 10⁹To about 1X 10¹¹CFU/day, and most preferably about 1 × 10¹⁰Daily dose of CFU/dayAmount of the compound (A).

It will be appreciated that a preferred daily dose may also be achieved by administering more than one sub-dose, for example by administering a unit dose comprising half of the preferred daily dose twice daily. Thus, the preferred range of effective dosages may also represent the preferred daily dosage to be achieved in any practical unit dose.

The subject may be instructed to consume a therapeutically effective amount of at least one probiotic bacterial strain according to the first aspect of the invention or a pharmaceutical composition according to the second aspect of the invention in combination with water, another aqueous solvent or a food product (e.g. yoghurt).

Therapeutic and/or prophylactic use

A fourth aspect of the invention provides the use of a composition comprising at least one probiotic bacterial strain according to the first aspect of the invention or a pharmaceutical composition according to the second aspect of the invention in the treatment and/or prevention of age-related systemic inflammation in a human.

The listing or discussion of a document in this specification to be expressly disclosed herein is not to be taken necessarily as an admission that the document is part of the state of the art or is common general knowledge.

The invention will now be described in more detail with reference to the following examples and the accompanying drawings.

Drawings

Figure 1 shows the mean change from baseline in the actual/absolute mg/L serum values of C-reactive protein (CRP) levels after four weeks of treatment for each of the three treatment groups.

Detailed Description

Exemplary dosage forms

In addition to the formulations referenced above (and incorporated herein by reference), the following examples illustrate pharmaceutical formulations according to the present invention.

Example a: tablet formulation

Tablets are prepared from the above ingredients by wet granulation followed by compression.

Example B: tablet formulation

The following formulations a and B were prepared by wet granulating the ingredients with a solution of povidone, then adding magnesium stearate and by compression.

Formulation A

Formulation B

Formulation C

The following formulations D and E were prepared by direct compression mixing of the ingredients. The lactose used in formulation E was of the directional compression type.

Formulation D

One or more probiotic strains 1 × 10⁹CFU

Pregelatinized starch NF 15150 mg

Formulation E

One or more probiotic strains 1 × 10⁹CFU

Lactose 150mg

100mg

Formulation F (controlled Release formulation)

The formulations were prepared by wet granulating the (below) ingredients with a solution of povidone, then adding magnesium stearate and compressing.

The release took a period of about 6-8 hours and was complete after 12 hours.

Example C: capsule formulation

Formulation A

A capsule formulation was prepared by mixing the ingredients of formulation D in example B above and filling into two hard gelatin capsules. Formulation B (below) was prepared in a similar manner.

Formulation B

Formulation C

(a) One or more probiotic strains 1 × 10⁹CFU

(b) Polyethylene glycol (Macrogol)4000 BP 350mg

The capsules were prepared by: melting polyethylene glycol 4000 BP; dispersing one or more probiotic bacterial strains in the melt; and the melt was filled into two hard gelatin capsules.

Formulation D (controlled Release Capsule)

The following controlled release capsule formulations were prepared by extruding ingredients a, b and c using an extruder, and then spheronizing and drying the extrudates. The dried pellets are then coated with a release controlling membrane (d) and filled into two-piece hard gelatin capsules.

Experimental example 1

Materials and methods

In a randomized, double-blind, placebo-controlled trial, 66 healthy participants with low systemic inflammation (defined by C-reactive protein, serum levels 2-10mg/L) aged >70 years were evaluated for possible anti-inflammatory activity of the probiotic product.

Exclusion criteria for this study were:

intake of antibiotic treatment within the last four weeks prior to inclusion in the study;

currently receiving corticosteroid therapy;

the presence of chronic inflammatory disease.

Subjects were randomly assigned to one of three groups:

1. lactobacillus plantarum Heal 9(Lp Heal 9)

2. Lactobacillus plantarum Heal 9+ berries (II)+Lp Heal 9)

3. Placebo

Each study product was formulated as a 10 g/dose powder and mixed with yogurt/yogurt and consumed once daily for a four week period. Test product A (group 2) was composed of 10 billion colony forming units (10) at a daily dose⁹CFU/dose) of freeze-dried lactobacillus plantarum HEAL9 probiotic, freeze-dried berries (blackberry and blackcurrant), and maltodextrin. Test product B (group 1) was composed of 10 billion colony forming units (10) at a daily dose⁹CFU/dose) of freeze-dried lactobacillus plantarum HEAL9 probiotic and maltodextrin treated to resemble test product a in appearance and taste. The placebo product is maltodextrinComposition, treated with coloring and flavoring/perfuming agents to resemble test product a in appearance and taste.

Participants were also asked to record study diaries throughout the study period to record their gut health status and as a means of checking compliance and to avoid taking other probiotic containing products.

Blood and fecal samples were collected at baseline and at the end of the study for analysis of the following parameters:

1. stool samples were analyzed for calprotectin (a marker of intestinal inflammation) and zonulin (a protein that modulates the permeability of intercellular tight junctions of the intestinal wall and serves as a marker of increased intestinal permeability);

2. blood samples were analyzed for the systemic inflammation markers CRP and fibrinogen.

Commercially available methods and equipment may be used (e.g., using Afinion from Alere/Abbott (see www.alere.com))^TMAlere Afinion by AS100 Analyzer^TMCRP assay) to determine CRP levels in blood, serum, and plasma.

This test is an in vitro method using a solid phase immunochemical assay based on a membrane coated with an anti-human CRP antibody that reacts with CRP in a sample. The analyzer measures the color intensity of the film, and this is proportional to the amount of CRP in the sample.

CRP levels in serum can be tested in a sensitive manner by a variety of methods (see Pearson TA et al (2003) Markers of inflammation and cardiovascular Disease: clinical and public health practice applications: Disease Control and Prevention center and statement by American Heart Association healthcare professionals (Markers of inflammation and cardiovascular Disease: application to clinical and public health practice: A state for health care subjects from the center for Disease Control and Prevention, circulation 511: 107; 499-

Results

Statistical analysis was performed in the study using Wilcoxon Rank sum Test (Wilcoxon Rank-sum Test).

No differences in the levels of zonulin and fibrinogen were detected between the probiotic group and placebo.

However, the level of the inflammatory marker CRP increased with time in the placebo group (group 3) and was receiving Lp HEAL9 and berry-Decrease in group of + Lp Heal9 (group 2) (fig. 1). The effect was even more pronounced in the group consuming probiotics alone without berry addition (group 1) (fig. 1).

The level of calprotectin, expressed as mean change over time, was not different between the probiotic group and the placebo. However, significantly fewer participants (p 0.028) were shown to increase calprotectin levels over time in lactobacillus plantarum (Lp HEAL9 only group) (group 1) compared to placebo (group 3) (table 1).

Table 1: analysis of the number of participants with stable or reduced calprotectin levels versus the number of participants with increased calprotectin levels

Conclusion

The results obtained with CRP and calprotectin show that lactobacillus plantarum, in particular lactobacillus plantarum HEAL9, has efficacy in the treatment and/or prevention of age-related systemic inflammation in otherwise healthy elderly.