WO2018122762A1

WO2018122762A1 - Oral preparation comprising a probiotic preparation of lactobacilluls helveticus

Info

Publication number: WO2018122762A1
Application number: PCT/IB2017/058460
Authority: WO
Inventors: Cristiano Umberto RUMIO; Francesco Palladini; Davide NENZIONI; Domenico MANISCALCO
Original assignee: Metis Healthcare Srl
Current assignee: Metis Healthcare Srl
Priority date: 2016-12-28
Filing date: 2017-12-28
Publication date: 2018-07-05
Anticipated expiration: 2019-06-28
Also published as: IT201600131819A1

Abstract

The present invention relates to a pharmaceutical preparation comprising probiotics for treating oral cavity diseases.

Description

ORAL PREPARATION COMPRISING A PROBIOTIC PREPARATION OF LACTOBACI LLULS HELVETICUS

Description

Technical field of the invention

The present invention find application in the medical field, in particular, in the treatment of oral cavity diseases.

Background art

There are known the properties of probiotic micro^¬ organisms in counteracting the development of pathogens due to their antagonistic activity and to the capacity of modulating the immune system.

While the application to the intestines has long been known, the same approach has been followed only very recently for treating the oral cavity and pharynx.

Such a part of the body is certainly easier to be reached as compared to the intestines and offers the possibility of a direct therapeutic treatment.

Furthermore, since the passage in the gastrointestinal system is avoided, the drug is not subjected to changes which could alter and diminish its effectiveness .

The oral cavity and the pharynx are characterized by the absence of an abundant mucosal layer and by a lower microbial competition than the intestines, which factors partially prevent the activity of the probiotics at the l intestinal level.

Furthermore, the presence of several lymphatic structures, such as the tonsils of the Waldeyer' s lymphatic ring, allow a very intimate contact of the drug, thus favoring its activity.

The focus on the oral cavity and pharynx is justified by the rate of acute infections of the upper respiratory tract, for which treatment traditional anti^¬ inflammatory drugs and optionally antibiotics are normally taken.

Especially in the pediatric field, it should be noted that acute pharyngites caused by Streptococcus pyogenes are one of the main causes of illness in scholastic age.

Following the therapeutic approach which involves the use of probiotics, two micro-organisms have been selected, which are characterized by the capacity of antagonizing S. pyogenes and by the modulating properties on the immune responses both in the epithelial cells and in the cells of the lymphatic system; they are in particular Streptococcus salivarius and Lactobacillus helveticus .

Studies have further demonstrated that its S-layer surface protein (SlpA) may be involved in the mechanisms which mediate the immune activity of the bacterium in the host, probably by activating immunostimulating factors .

However, in order to be usefully used, the micro^¬ organisms described above must be formulated in a suitable pharmaceutical preparation.

Summary of the invention

The inventors of the present patent application have developed pharmaceutical formulations, whose components are capable of exerting a particularly favorable synergistic action for treating oral cavity diseases.

Object of the invention

The present invention relates to pharmaceutical preparations comprising probiotic micro-organisms.

According to another aspect, the invention describes the use of such pharmaceutical preparations for preventing and/or treating diseases of the oral cavity, nasal cavities, pharyngeal and laryngeal tracts, and middle ear.

According to another object, the present invention describes a method of preventing and/or treating diseases of the oral cavity, nasal cavities, pharyngeal and laryngeal tracts, and middle ear, comprising the administration of the described pharmaceutical preparations .

Detailed description of the invention The present invention relates to a pharmaceutical preparation comprising a preparation of the probiotic micro-organism Lactobacillus helveticus .

In particular, Lactobacillus helveticus MIMLh5 was isolated from a Grana Padano cheese whey-graft.

In a preferred aspect of the invention, Lactobacillus helveticus is Lactobacillus helveticus MIMLh5.

In an even more preferred aspect of the invention, Lactobacillus helveticus is the micro-organism deposited with the Leibniz-Lnstitut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH ( InhoffenstraBe 7B, 38124 Braunschweig - GERMANY) on February 7, 2017 with number DSM 32422.

Its conditions for culturing, long-term storage and for testing its viability are reported in the following table :

Transfer interval: weekly

CONDITIONS FOR LONG -TERM STORAGE

-80°C in the presence of 20% (v/v) glycerol

CONDITIONS FOR TESTING THE VIABILITY THeREOF

Growth in MRS medium at 37°C for 24-48 hours , anaerobically if cultured on M17 agar plates

In particular, Streptococcus salivarius was isolated from the pharyngeal mucosal of a healthy subject.

In a preferred aspect, it is Streptococcus salivarius ST3.

In an even more preferred aspect of the invention, Streptococcus salivarius is the micro-organism deposited with the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH ( InhoffenstraBe 7B, 38124 Braunschweig - GERMANY) on February 7, 2017 with number DSM 32423.

Its conditions for culturing, long-term storage and for testing the viability thereof are reported in the following table:

Incubation time: 24-48 hours

Short-term storage: 4°C

Transfer interval: weekly

CONDITIONS FOR LONG -TERM STORAGE

-80°C in the presence of 20% (v/v) glycerol

CONDITIONS FOR TESTING THE VIABILITY THEREOF

Growth in M17 1% (w/v) glucose medium at 37°C for 24-48 hours, anaerobically if cultured on M17 agar plates

In an aspect of the invention, the described preparation comprises Lactobacillus helveticus in combination with Streptococcus salivarius .

For the objects of the present invention, each probiotic preparation of the micro-organism, that is of Lactobacillus helveticus and/or Streptococcus salivarius, may be in active or inactive form.

In particular, this means that the micro-organism is active, i.e. live, or was inactivated by suitable methods, such as tyndallization, for example.

Therefore, in a preferred aspect, the probiotic micro-organism preparation of the invention is a tyndallized preparation.

The probiotic component is for example present in the preparation of the invention in an amount of about 0.6-1% (weight/total weight of the pharmaceutical preparation) .

In a preferred aspect, such a component represents about 0.8-0.9% (weight/weight) of the pharmaceutical preparation .

The preparation of the invention may further comprise hyaluronic acid.

As an alternative, a pharmaceutically acceptable salt thereof may be used, such as the sodium salt or potassium salt thereof, or a mixture of the two salts.

For the purposes of the present invention, the hyaluronic acid is hyaluronic acid of low molecular weight .

Preferably, the molecular weight of the hyaluronic acid used is of about 0.2-1 x 10⁶ Da and even more preferably of about 0.4-0.8 x 10⁶ Da.

The hyaluronic acid component is present in the preparation of the invention in an amount of about 0.01- 20% (weight/weight) , according to the type of pharmaceutical formulation.

For the preparation of a liquid pharmaceutical form, represented by an oral spray, a mouthwash or an oral wash or a syrup, for example, the hyaluronic acid may be preferably present in an amount of about 0.01-1% (weight/weight), preferably of about 0.1-0.4% (weight/weight), and more preferably of about 0.2% (weight/weight) .

The preparations of the invention comprise water, preferably demineralized water. In an aspect of the invention, the described preparation comprises further pharmacologically active components capable of increasing or modulating the activity of the preparation itself.

Such components may comprise for example one or more polysaccharides having inhibitory activity towards the adhesion of S. pyogenes to the oropharyngeal mucosa.

In a particular aspect of the invention, the described preparation may further comprise a preparation of the S-layer surface protein (SlpA) of Lactobacillus helveticus .

Furthermore, one or more medicinal plant extracts may be included, selected from the group comprising: an extract of Echinacea (for example, Echinace Angustifolia) , Eucalyptus globulus Labill, Prunus armeniaca L. Garcinia kola Heckel, Garcinia kola Heckel, Sisymbrium officinale Scop. (Syn. Erysimum officinale L. ) .

In a preferred aspect of the invention, the described formulations comprise an extract of: Echinace Angustifolia and/or Syn. Erysimum officinale L.

In an even more preferred aspect, each of such extracts is included, independently of one another, in an amount of about 0.01-5% (weight/weight) and preferably of about 1% (weight/weight) in the described preparations .

In particular, an apparent synergistic effect has been observed, given by the association with one or both of the above extracts.

Such a synergistic effect occurs with the extract of Echinace Angustifolia and Syn. Erysimum officinale L, even when both are used.

According to a preferred aspect of the invention, the pharmaceutical preparation is an oral spray.

Therefore, the preparation may include one or more components adapted to obtain an oral spray formulation.

For example, the preparation may comprise one or more of the components selected from: preservatives, antioxidants, chelating agents, buffers (for example sodium bicarbonate) , viscosity regulators, stabilizers, dispersers, sweeteners, flavorings, film-forming agents, etc. adapted to obtain an oral spray preparation.

Among the preservatives, the following are contemplated: ethyl alcohol, glycerin, propylenglycol , sorbic acid and sorbates, sodium benzoate, etc.

In a preferred aspect of the invention, the preparation comprises a combination of preservatives comprising: sodium benzoate and potassium sorbate.

In particular, the concentration of preservatives falls within the limits provided by the regulations. For example, each of the sodium benzoate and potassium sorbate may be present in amounts of about 0.05%-0.2% (weight/weight) and preferably in amounts of about 0.1% (weight/weight) .

In an aspect of the invention, the described preparation comprises Epsileen® (Siveele, Netherlands), an ε-polylysine homopolypeptide produced via bacterial fermentation and having antimicrobial and antibacterial activity against molds, yeasts, Gram-positive and Gram- negative bacteria.

Epsileen® may be included in an amount of about 0.05% (weight/weight) .

A further antimicrobial (preservative) which may be used is Biosecur®.

Such a preservative may be present in an amount of about 0.3% (weight/weight) .

Among the antioxidants, the following are contemplated: ascorbic acid and esters thereof, hydrosulfites and bisulfites, butylated hydroxyanisole (BHA) , tocopherols, lecithin, etc.

Among the chelating agents, the following are contemplated: ethylenediaminetetraacetic acid (EDTA) and salts thereof, citric acid and tartaric acid, etc.

Among the sweeteners, the following are contemplated: glucose, fructose, xylitol, sorbitol, sucrose, sucralose or other artificial sweeteners (saccharin, acesulfame K, etc. ) ,

4, 1' , 6' ,trichlorogalactosucrose.

In particular, the sweeteners may be present in an amount of about 0.04-0.1% (weight/weight ) and preferably of about 0.06-0.08% (weight/weight) .

In a particular aspect of the invention, the pharmaceutical preparation preferably does not comprise one or more of the following as an ingredient: glucose, fructose, sucrose, fructooligosaccharides (FOS) .

The flavorings comprise one or more of the flavoring compounds allowed for pharmaceutical preparations, in particular for oral use.

For this purpose, the following may be used: flavoring of mint, orange, strawberry and other generally appreciated flavorings.

In a preferred aspect of the invention, the described pharmaceutical spray preparation is a preparation for children.

Other liquid preparations for oral use comprise solutions, such as mouthwashes or oral washes, syrups or suspensions, in suitable aqueous or hydroalcoholic solutions .

To this end, the use of suitable excipients may be provided, such as for example: suspending agents, such as sorbitol syrup, cellulose derivatives or hydrogenated fats; emulsifiers, such as for example: lecithin or acacia, other non-aqueous carriers, such as for example: almond oil, ester oils, ethyl alcohol or fractioned vegetable oils; preservatives, such as for example: methyl- or propyl-p-hydroxybenzoate or sorbic acid; antibacterial agents.

A liquid preparation, for example for an oral spray, according to the present invention, is a preparation having the following composition:

or the same preparation was obtained by adding an extract of Echinace Angustifolia (extract) and/or Syn. Erysimum officinale L in an amount of about 1% (w/w) each .

In a preferred aspect, the preservatives may comprise potassium sorbate and sodium benzoate, each in an amount of about 0.1% (w/w) .

The present invention further describes solid pharmaceutical preparations for oral use, represented for example by lozenges, tablets, (hard or soft) capsules, granules or dragees, prepared according to methods known in the field and by using suitable and pharmaceutically acceptable excipients.

For example, binding agents may be included, such as for example: pregelatinized cornstarch, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cellulose, additives, such as for example: lactose, sorbitol, microcrystalline cellulose, calcium hydrogen phosphate; lubricants, such as for example: magnesium stearate, talc, silica; disintegrants , such as for example: potato starch or sodium starch glycolate; preservative agents, such as for example: sodium lauryl sulfate; stabilizers, such as for example: hydrophilic fumed silica; pH modifying agents, such as for example sodium bicarbonate.

Other components, such as sweeteners and flavors, for example, may be included as described above for liquid preparations.

For the purposes of the present invention, a solid pharmaceutical preparation, for example represented by a tablet, comprises hyaluronic acid or a salt thereof. Such a component may be preferably included in an amount of about 8-12% (weight/weight) and more preferably of about 10% (weight/weight) .

A solid preparation, for example a tablet, according to the present invention, is a preparation having the following composition:

or the same preparation was obtained with the addition of Echinace Angustifolia Extract and/or Syn. Erysimum officinale L in an amount of about 1% (w/w) each .

The preservatives may comprise potassium sorbate and sodium benzoate, each in an amount of about 0.1% (w/w) . A further pharmaceutical form which may be prepared according to the present invention is represented by toothpastes .

For this purpose, suitable excipients will be selected from: additives; binders, such as for example: carrageenan or derivatives thereof, xanthan gum, carboxymethylcellulose, carbopol; additives, such as for example: bentonite, magnesium aluminum silicate, microcrystalline cellulose, silica, sodium alginate; abrasive agents; humectants, such as for example: sorbitol, glycerol; surfactants; antibacterial agents; binding agents, such as for example: carboxymethyl cellulose alkali salts, hydroxymethyl cellulose, hydroxyethyl carboxymethyl cellulose, synthetic and natural gums, polyvinylpyrrolidone, starch, and others; preservatives, such as for example: sodium benzoate; antibacterial agents, such as for example: zinc citrate dihydrate, antibacterial agents, such as for example: triclosan; desensitizing agents; anti-tartar agents; sodium bicarbonate; antibacterial activity enhancers, such as for example: enzymes; whitening agents, such as for example: peroxides; plant extracts; water; lastly, a fluorine source may be included, represented for example by: sodium monofluorophosphate, stannous fluoride, sodium fluoride. Furthermore, flavorings, colorants and sweeteners known in the field and adapted to the particular pharmaceutical preparation may be included.

Those skilled in the art may refer to the handbook "Remington's Pharmaceutical Sciences Handbook", Mack Pub. Co., N.Y., USA, 17th edition, 1985.

According to another aspect, the invention describes the use of the pharmaceutical preparation for preventing and/or treating diseases of the oral cavity, nasal cavities, pharyngeal and laryngeal tracts, and middle ear .

In particular, diseases refers to inflammatory and infectious pathologies.

More in particular, such pathologies are bacterial by nature .

In particular, they are streptococci, group A beta- haemolytic streptococci, in more detail Streptococcus pyogenes, and group C_r G and F Streptococci .

In a particularly preferred aspect of the invention, the described preparation is used in treating oropharyngeal cavity infections in children, and especially in children aged 5-12 years.

According to another object, the present invention describes a method for preventing and/or treating diseases of the oral and pharyngeal cavity comprising the administration of an effective amount of one of the described pharmaceutical preparations.

Such a method comprises in particular the administration of the preparation of the invention several times a day.

For example, in the case of the oral spray mentioned above, this may be applied 2-3 times a day.

In particular, the amount of the administered probiotic preparation preferably comprises about 10¹⁰ micro-organisms (for each of the administered strain) .

In a preferred aspect, the described method is applied for preventing and/or treating oropharyngeal cavity infections in children, and especially in children aged 5-12 years.

EXAMPLE 1

Formulation

In the following example, an oral spray preparation according to the present invention is described.

Flavoring 0.1

Demineralized water q. s . to 100

*for example, Epsileen®

EXAMPLE 2

Formulation

In the following example, another oral spray preparation according to the present invention is described .

*for example, Biosecur®

The formulation indicated above may be modified adding the extract of:

Component Amount %

(weight/weight)

Echinace Angustifolia (extract) 1

and/or

Syn. Erysimum officinale L 1 thus proportionally reducing the amount of water.

EXAMPLE 3

Formulation

In the following example, a preparation for oral tablets according to the present invention is described.

The formulation indicated above may be modified by adding the extract

Component Amount %

(weight/weight)

Echinace Angustifolia (extract) 1

and/or

Syn. Erysimum officinale L 1 thus proportionally reducing the amount of water.

From the description above, the several advantages provided by the present invention will become apparent to those skilled in the art.

First, the preparation provides a very valid alternative to the use of drugs traditionally used for treating oropharyngeal cavity diseases, among which in primis non-steroidal anti-inflammatory drugs.

The possibility to use inactive micro-organisms further increases the safety of the pharmaceutical product, especially if it should be administered to patients of pediatric age.

The presence of inactive micro-organisms reduces or even eliminates stability problems of the formulation, with important technological advantages; moreover, the shelf-life of the product is usefully prolonged.

Among the most surprising aspects, the preventive and/or therapeutic effect was noted, produced by the combination of the probiotic preparation with hyaluronic acid, which effect goes well beyond the properties of the single components.

In particular, the association with one or both extracts of Echinace Angustifolia and Syn. Erysimum officinale L showed a further apparent synergistic effect . Moreover, the presence of sodium benzoate and potassium sorbate in the pharmaceutical preparations of the invention proved to be able to form a buffer solution with the hyaluronic acid capable of regulating the pH.

It was noted how such an effect further enhances the action of the probiotic component.

Claims

1. An oral pharmaceutical preparation comprising a probiotic preparation of Lactobacillus helveticus .

2. A pharmaceutical preparation according to claim 1, further comprising a probiotic preparation of Streptococcus salivarius .

3 . A pharmaceutical preparation according to claim 1 or 2, wherein such a probiotic preparation of Lactobacillus helveticus, and/or that of Streptococcus salivarius, is in active or inactive form.

. A pharmaceutical preparation according to the preceding claim, wherein such a probiotic preparation is a preparation of Lactobacillus helveticus MIMLh5 (DSM 32422) .

5. A pharmaceutical preparation according to the preceding claim, wherein said probiotic preparation is a preparation of Streptococcus salivarius ST3 (DSM 32423) .

6. A pharmaceutical preparation according to any one of the preceding claims, wherein each of such probiotic preparations is present, independently of one another, in an amount of about 0.6-1% (weight/total weight of the pharmaceutical preparation), preferably of about 0.8% (weight/total weight of the pharmaceutical preparation) .

7. A pharmaceutical preparation according to any one of the preceding claims, further comprising hyaluronic acid or a pharmaceutically acceptable salt thereof.

8. A pharmaceutical preparation according to claim 7, wherein the hyaluronic acid has a molecular weight of about 0.2-lxlO⁶ Da and preferably of about 0.4-0.8xl0⁶ Da.

9 . A pharmaceutical preparation according to claim 7 or 8, wherein the hyaluronic acid is present in an amount of about 0.01-20% (weight/weight) .

10. A pharmaceutical preparation according to any one of the preceding claims from 7 to 9, wherein said preparation is an oral spray and the hyaluronic acid is present in an amount of about 0.01-1% (weight/weight), preferably of about 0.1-0.4% (weight/weight), and more preferably of about 0.2% (weight/weight) .

11. A pharmaceutical preparation according to any one of the preceding claims from 7 to 9, wherein said preparation is a solid preparation and comprises hyaluronic acid in an amount of about 8-12%

(weight/weight) , and preferably of about 10%

(weight/weight) .

12. A pharmaceutical preparation according to any one of the preceding claims, further comprising a preparation of S-layer surface protein (SlpA) .

13. A pharmaceutical preparation according to any one of the preceding claims, further comprising one or more medicinal plant extracts selected from the group comprising: an extract of Echinacea, Eucalyptus globulus Labill, Prunus armeniaca L. Garcinia kola Heckel, Garcinia kola Heckel, Sisymbrium officinale Scop. (Syn. Erysimum officinale L.) .

14. A pharmaceutical preparation according to any one of the preceding claims, comprising an extract of: Echinace Angustifolia and/or an extract of Syn. Erysimum officinale L.

15. A pharmaceutical preparation according to claim 13 or 14, wherein each of said extracts is included in an amount of about 0.01%-5% (weight/weight ) and preferably of about 1% (weight/weight) .

16. A pharmaceutical preparation according to any one of the preceding claims, further comprising a combination of preservatives comprising: sodium benzoate and potassium sorbate.

17. A pharmaceutical preparation according to the preceding claim, wherein each of the sodium benzoate and potassium sorbate may be present in amounts of about 0.05%-0.2% (weight/weight), and preferably in amounts of about 0.1% (weight/weight) .

18. A pharmaceutical preparation for oral use according to any one of the preceding claims in liquid form, selected from the group comprising: sprays, mouthwashes or oral washes, syrups or suspensions; or in solid form, selected from the group comprising: lozenges, tablets, hard capsules or soft capsules, granules, dragees; or in toothpaste form.

19. A pharmaceutical preparation according to any one of the preceding claims represented by an oral spray having the following formulation:

20. A pharmaceutical preparation according to any one of the preceding claims represented by an oral spray having the following formulation:

Flavoring 0.1

Demineralized water q. s . to 100

21. A pharmaceutical preparation according to any one of the preceding claims, represented by an oral tablet having the following formulation:

22. A pharmaceutical preparation according to any one of the preceding claims, represented by an oral tablet having the following formulation:

Sorbitol (D-Glucitol) 80.42

Sodium benzoate 0.1

Potassium sorbate 0.1

Hyaluronic acid 10

Sweeteners 0.08

Sodium bicarbonate q.s. to pH

6/7

Hydroxypropyl cellulose 5

Magnesium stearate FU - Ph @@@EUR 2

Hydrophilic fumed silica 1

Flavoring 0.4

Demineralized water q.s. to 100

23. A pharmaceutical preparation according to any one of the preceding claims from 19 to 22, further comprising an extract of: Echinace Angustifolia and/or an extract of Syn. Erysimum officinale L.

24. A pharmaceutical preparation according to claim 23, wherein each of said extracts is included in an amount of about 0.01%-5% (weight/weight), and preferably of about 1% (weight/weight) , thus proportionally reducing the amount of water.

25. A pharmaceutical preparation according to any one of the preceding claims for use in preventing and/or treating diseases, in particular inflammatory and infectious diseases, of the oral cavity, nasal cavities, pharyngeal and laryngeal tracts, and middle ear.

26. A pharmaceutical preparation according to the preceding claim for use in the prevention and/or treatment of children.

27. A pharmaceutical preparation according to claim 25 or 26, for use in preventing and/or treating inflammatory and infectious diseases supported by streptococci, preferably beta-haemolytic streptococci.

28. A method of preventing and/or treating diseases, in particular inflammatory and infectious diseases, of the oral cavity, nasal cavities, pharyngeal and laryngeal tracts, and middle ear in a patient, comprising the step of administering an effective amount of a preparation according to any one of the claims from 1 to 24 to said patient .

29. A method according to the preceding claim, wherein said infections are supported by streptococci, preferably beta-haemolytic streptococci.

30. A method according to the preceding claim 28 or 29, wherein said patient is aged 5-12 years.