WO2020021483A1

WO2020021483A1 - Composition for the treatment of oral cavity inflammations

Info

Publication number: WO2020021483A1
Application number: PCT/IB2019/056352
Authority: WO
Inventors: Grzegorz Kalbarczyk
Original assignee: Grzegorz Kalbarczyk Arkona Laboratorium Farmakologii
Priority date: 2018-07-27
Filing date: 2019-07-25
Publication date: 2020-01-30
Also published as: PL426472A1

Abstract

The object of the invention is a composition with domiphen bromide and pharmaceutically acceptable adjuncts, which is claimed to contain (per 100 ml of the solution): - domiphen bromide in an amount of 0.01-0.10%, preferably 0.01-0.03%; - purified water from 90 to 99.9%; - mineral salts (buffering, responsible for isotonicity), preferably NaCl, KCl, CaCl₂, MgCl₂, NaH₂PO₄,KH₂PO₄,Na₂HPO₄, K₂HPO₄, Na₂CO₃, NaHCO₃, in an amount of 0.5 to 5.0%, preferably 0.5 - 1.5%; - optionally aromas, preferably mint, apple, anise, lime, lemon, raspberry, cinnamon, clove in an amount of 0.1 to 0.9%.

Description

Composition for the treatment of oral cavity inflammations

The object of the invention is a composition, containing domiphen bromide, intended for the treatment of inflammatory conditions within the oral cavity.

Domiphen bromide belongs to the group of quaternary ammonium salts and has been known for over 50 years as a compound showing a marked antimicrobial activity against a wide range of bacteria and fungi. Domiphen bromide has bactericidal properties mainly against Gram (+) bacteria and, at higher concentrations, against certain Gram (-) bacteria. Domiphen bromide (like other cationic surfactants) is ineffective against bacterial spores, has variable antifungal activity and it is effective against certain viruses. The mechanism of action consists of joining with the bacterial cell membrane and creating channels in it through which cations migrate. Damage to the bacterial cell membrane is a bactericidal action. At present, domiphen bromide is approved by the European Union regulations as a component of preparations and cosmetics, mainly for tooth care.

Sore throat accompanying common colds is most often caused by viruses that invade the nasopharyngeal cavity (instead of the olfactory epithelium of the nose) or they affect the throat directly. In most cases, such infections are not serious and tend to self-limit. Patients, by choice, go for supporting medications (adjunctive therapy) that reduce infections and discomfort associated with them. Medical appointments often lead to antibiotic therapy, which is not only ineffective against viruses, but in addition it results in the growth of drug resistance of bacteria.

The second cause of infective sore throat may be bacterial infections [Eccles R. Understanding the symptoms of the common cold and influenza. Lancet Infect Dis. 2005 Nov; 5 (11): 718-25. Review] It is generally believed that about 70% of acute pharyngitis cases have a viral etiology, the remaining 30% are bacterial infections, mainly caused by b- hemolytic streptococcal group A - Streptococcus pyogenes (GAS). It is often accompanied by Staphylococcus aureus and Candida albicans fungus.

The first stage of infection is the colonisation of the mucous membranes of the upper respiratory tract. It is known, however, that the mucous membranes of the throat are protected from such colonisation due to their surface being constantly rinsed with saliva. Therefore, bacteria colonising mucous membranes must have adhesive properties (in order to ensure adhesion to mucosal cells). For many years it was supposed that the main adhesins of purulent streptococci are fimbriae that form M protein and teichoic acids. It turned out, however, that the major streptococcus adhesin is the surface F protein responsible for the adhesion of bacteria to the cells of the mucous membrane of the throat. F protein has the ability to bind epithelial cell fibronectin. The M protein is, most likely, responsible for the binding of purulent streptococcus to skin keratocytes. One of the late complications of streptococcal angina is rheumatoid arthritis. It has been shown that the strains that cause this complication have a thick coat of hyaluronic acid, which causes for the colonies of streptococcus on solid media to have a mucous appearance.

On the medical market, there can be found products dedicated for the treatment or supporting the treatment (adjunctive therapy) of upper respiratory tract infections (including pharyngitis and tonsillitis), but they are registered in various categories, i.e. as:

- foodstuffs - containing plant extracts or essential oils with a soothing or inhaling effect;

- medical devices, mainly class I - acting mechanically as a barrier preventing adhesion and penetration of bacteria in x the mucous membrane of the upper respiratory tract;

- medicinal products - containing active substances with antibacterial effect;

- medicinal products - containing substances with effect of local anaesthesia

It is worth noting that both foodstuffs and class I medical devices are products placed on the market without clinical trials proving their efficacy and safety of use; additionally they are usually of poorly documented quality.

At present, on the medical device market, there are only two products containing domiphen bromide (NUROFEN FORTE for children and MAALOX 200 mg for hyperacidity), in both cases bromide is used only as a preservative.

No medical device for rinsing gingival pockets containing any active substance used as an adjunct is available on the dental market. Also, on the throat spray market there is no product which contains domiphen bromide - an active substance - used as an adjunct or as a preservative.

In the state of prior art an invention US2017281490 is known focusing on compositions which, in addition to cationic surfactants (quaternary ammonium salts such as cetylpyridinium chloride, domiphen bromide, etc.) that can cause discoloration of teeth, contain other compounds that are to prevent these discolorations (the object of the invention).

There are also tablets /sublingual tablets known from the description of CN106822000 containing domiphen bromide in 1-200 parts by weight, for the treatment of thrush, pharyngitis, tonsillitis or ulcerative stomatitis.

There is also known from the prior art invention No. US6579513, concerning an orally administered oral care composition comprising a bactericide which reduces the amount of bacteria in the oral cavity and is effective against gingivitis and oral malodour. As a bactericide it contains cetylpyridinium chloride and domiphen bromide. The formulation may also contain one or more of the following: an alcohol component, a sweetening component and a flavour system.

Dodecyldiemethyl-2-phenoxyethylammonium bromide - chemical formula: C₂₂H₄₀BrNO Molar mass: 414.5 g / mol

CAS: 13900-14-6 (domiphen); 538-71-6 (domiphen bromide)

Pharmacopoeias: In Br. China includes the monohydrate.

ATC: A01AB06

Structural formula:

Domiphen bromide can be obtained for e.g. by boiling an aqueous solution of (2- phenoxyethyl) dimethylamine with dodecyl bromide:

The subject of the invention is a composition based on domiphen bromide for the treatment of oral inflammatory conditions, especially of the gingival pockets, that contains per 100 ml of solution (in wt%):

- domiphen bromide in an amount of 0.01-0.10%, preferably 0.01-0.03%;

- purified water in an amount of 90 to 99.9%;

mineral salts (buffering, responsible for isotonicity), preferably NaCl, KC1, CaCl₂, in an amount

of 0.5-5.0%;

and optionally aromas, preferably mint, apple, anise, lime, lemon, raspberry, cinnamon, clove in an amount of 0.1 to 0.9%.

The subject of the invention is also a variant of the composition based on domiphen bromide for the treatment of oral inflammatory conditions, particularly useful for the treatment of the throat that contains per 100 ml of solution (in wt%):

domiphen bromide in an amount of 0.01-0.10%, preferably 0.01-0.03%;

- purified water in an amount of 90 to 99%;

mineral salts (buffering, responsible for isotonicity), preferably NaCl, KC1, CaCl₂, MgCl₂, NaH₂P0_4, KH₂P0_4, Na₂HP0_{4, ,} K₂HP0₄ in an amount 0, 1 -0,5%

- aroma in an amount of 0.1 to 0.9%, preferably peppermint oil, clove oil, lemon, eucalyptus, anise;

moisturising substance, preferably glycerine 0,1 to 0,5%;

natural plant extract (of anti-inflammatory, protecting effect), preferably containing mucus, rich in mineral salts, preferably Cetraria islandica, i.e. Icelandic lichen, Chondurus crispus (curly filament), marshmallow ( Althaea officinalis L.), Black mallow flower ( Alcea rosea L. var. Nigra), mullein flower ( Verbascum L.), Coltsfoot leaf ( Tussilago farfara L.), Lime inflorescence (Tilia cordata Mill.);

- optionally a sweetener, preferably xylitol, sorbitol, maltitol, erythritol in an amount of 1 to 5%;

- optionally acacia or tragacanth gum in an amount of 0.1 % to 2.0 %.

Selection of domiphen bromide concentration

The study aimed to determine the optimal concentration of domiphen bromide, at which it shows observable antimicrobial activity with simultaneous lack of cytotoxicity (admissible in class III medical products cytotoxicity value is 2 on a scale of 0-4, where 0 means a non- cytotoxic product, and 4 - a highly cytotoxic product).

Eventually, concentration - 0.01% by weight was considered as minimal, which is effective in the treatment of oral cavity inflammations. Cytotoxicity studies of domiphen bromide preparations (0.01% by weight) carried out at the National Medicines Institute in Warsaw confirmed the mild cytotoxicity of the products (value 1 - a completely acceptable result).

Confirmation of the antimicrobial efficacy of the preparation for rinsing gingival pockets. Methods

In the study reference strains of the following microorganisms were used:

C. albicans ATCC 10231

Staphylococcus aureus ATCC 6538

Escherichia coli ATCC 8739

Pseudomonas aeruginosa ATCC 9027.

Microbiological media:

LT 100 Broth (proliferation medium)

- Tryptic Soy Agar (TSA)

The preparation effectiveness tests were conducted using two methods:

1) The contact plate method - on the medium inoculated with the above mentioned microorganisms using the surface method, 2 drops of the tested solution were applied and incubated at an appropriate temperature.

2) The well plate method - in the medium inoculated with the above mentioned microorganisms using the surface method 50 mL wells were excised and then a dose of 50 mL of the tested solution was transferred to each well and incubated at an appropriate temperature.

Study of the dynamics of antimicrobial activity were conducted using the microbroth dilution-based testing method:

The microbroth dilution-based testing method - the tested product was inoculated with the culture of a given microorganism in the ratio 9:1 (product: culture). Subsequently, the surface inoculation of the tested solution and its decimal dilutions were performed within a specified time from the beginning of the study. The inoculations were incubated at an appropriate temperature, the results were read and the logarithm of reduction was calculated.

Materials

The tested material was a formulation containing domiphen bromide at a concentration of

0.01%.

Conducting preparation efficacy tests

The first stage of the research was the multiplication of microorganisms in LT 100 Broth proliferation medium:

A single colony of a specified microorganism was transferred with a sterile inoculation loop onto LTB medium. The inoculated medium was incubated for 21 hours ± 3 hours at 32.5°C ± 2.5°C. After the incubation, depending on the chosen test method, the following activities were performed:

The contact plate method

Decimal dilution of LTB culture with microorganism in sterile physiological saline solution was performed.

TSA medium was inoculated with the obtained dilution in a volume of 0.1 ml with a sterile glass spreader using the surface method. The covered inoculated plate was left to stand for 15 minutes so that the surface of the plate could dry. After 15 minutes, 50 mL of the tested material was applied onto the surface of the plate. The plate was then left to stand until the material was completely absorbed. After drying, the plates were incubated at 32.5°C ± 2.5°C for 21 hours ± 3 hours.

The tests were carried out in parallel to the control, where sterile physiological saline was used instead of the tested material. After the incubation, the results were read. The grading system used:

- total growth inhibition,

- partial growth inhibition,

- no growth inhibition.

The well plate method

The obtained dilution was inoculated on TSA medium in a volume of 0.1 ml with a sterile glass spreader using the surface method.

50 mL wells were excised in the plate using a sterile punch. A dose of 50 mL of the tested material was transferred to each well. Such prepared medium was left so that the tested material was completely absorbed. After drying, the plate was incubated at 32.5°C ± 2.5°C for 2lh ± 3h. The tests were carried out in parallel to the control, where sterile physiological saline was used instead of the tested material. After incubation, the results were read by measuring the diameter of growth inhibition.

Study of the dynamics of the antimicrobial activity of the preparation

The first stage was the inoculation of LT 100 Broth medium with microorganism against which the dynamics of antimicrobial activity was examined. The inoculated liquid medium was incubated at 32.5°C ± 2.5°C for 21 h ± 3 h. After incubation, decimal dilution of the obtained culture was made in sterile saline. The dilution obtained was used in the studies. 9 ml of the tested material was transferred into a sterile tube and 1 ml of the decimated culture was added.

Immediately after inoculation of the tested material, a further decimal dilution (up to 10 inclusively) was made and surface inoculation of 0.1 ml of the inoculated tested material and its dilutions were performed in one repetition on TSA medium. The material was then incubated at 32.5°C ± 2.5°C.

In 0.5 an hour, 1.5 hours, 3 hours and 24 hours of the commencement of incubation, decimal dilutions (up to 10 ³) were made and TSA medium was inoculated with all solutions in one repetition using the surface method (0.1 ml). All solid media were incubated at 32.5°C ± 2.5°C for 21 hours ± 3 hours. The tests were carried out in parallel to the control, where sterile physiological saline was used instead of the tested material. After the incubation, the results were read by calculating the number of microorganisms in the dilutions within a given time of exposure of the tested material to those microorganisms. On the basis of the difference in the number of microorganisms - in the tested sample and the control - the logarithm of reduction (LogR) for a given microorganism at a given time was calculated using the formula.

Results

Table 1 - Study of the antimicrobial activity of the preparation using the contact plate method

Table 2 - Study of the antimicrobial activity of the preparation using the well plate method

Table 3 - Study of the dynamics of the antimicrobial activity of the preparation using the microbroth dialution-based method, described through the logarithm of reduction (Log R)

Summary and conclusions

The preparation containing domiphen bromide for rinsing gingival pockets shows very good antimicrobial properties towards C. albicans and S. aureus, which is visible in the contact plate method studies (complete inhibition of microbial growth in the place of contact of the tested material with microbiological medium) and the well plate method studies (inhibition zones 17.25 mm and 13.25 mm respectively). The preparation shows good antimicrobial activity towards E. coli and P. aeruginosa, where microbial growth in the contact plate method is partially inhibited.

Referring to the dynamics of the antimicrobial activity of the preparation, in the case of microorganisms: C. albicans, E. coli and S. aureus, immediate action to inhibit the growth of microorganisms of the tested material is evident. In the case of P. aeruginosa, a slower rate of inhibition of microbial growth is noticeable, however, the extend and intensity of its action is sufficient for the specificity of the product.

In conclusion, a medical device containing 0.01% by weight of domiphen bromide has good microbial growth inhibitory properties and the dynamics of this activity is satisfactory.

CYTOTOXICITY in vitro studies

According to the results of in vitro cytotoxicity tests using the agar diffusion method in accordance with PN-EN ISO 10993-5: 2009, the fluid containing 0.01% by weight domiphen bromide is non- cytotoxic (found cytotoxicity level - "1 ", interpretation - weak cytotoxicity). The studies were carried out on the NCTC mouse cell line fibroblasts clone 929, ATCC, on EMEM medium from Lonza, serial number 5MB018, FBS foetal bovine serum from Euroclone, serial number EUS00116, using the Lonza antibiotic solution serial number l6J2l5309.The study was carried out using negative control, positive control, culture and a carrier.

The below examples of the product composition, in accordance with the invention for gingival pockets don’t exclude examples with further variants under invention

Example 1

Purified water 99.25 g

Domiphen bromide 0.01 g

Sodium chloride 0.24 g

Potassium chloride 0.25 g

Calcium chloride 0.05 g

Mint aroma 0.20 g Example 2

Purified water 98.61 g

Domiphen bromide 0.03 g

Potassium chloride 0.86 g

Sodium dihydrogen phosphate 0.04 g

Potassium hydrogen phosphate 0.16 g

Lemon aroma 0.20 g

Mint aroma 0.10 g

Example 3

Purified water 97.48 g

Domiphen bromide 0.01 g

Sodium chloride 1.29 g

Magnesium chloride 0.48 g

Potassium dihydrogen phosphate 0.08 g

Sodium hydrogen phosphate 0.16 g

Flavor aroma 0.50 g

Example 4

Purified water 98.77 g

Domiphen bromide 0.01 g

Potassium chloride 0.58 g

Magnesium chloride 0.14 g

Apple aroma 0.25 g

Mint aroma 0.25 g

Study of the antimicrobial activity of the preparation for the throat

The study aimed at determining the degree of antimicrobial efficacy of a medical device containing domiphen bromide and used as an adjunctive therapy in throat inflammatory conditions.

Methods

The preparation effectiveness tests were carried out using two methods:

the diffusion-contact method - 2 drops of the tested preparation were applied onto the medium surface-inoculated with microbes, which was then incubated at an appropriate temperature;

the diffusion-well method - 50 mL wells were excised in the medium surface- inoculated with microbes; then a dose of 50 mL of the tested solution was transferred ¹ into each well and incubated at an appropriate temperature.

Materials

The tested material was a preparation containing domiphen bromide. The studies used reference strains of microorganisms:

- Candida albicans ATCC 10231 Staphylococcus aureus ATCC 6538

Escherichia coli ATCC 8739

Pseudomonas aeruginosa ATCC 9027

Microbiological media:

- LT 100 Broth - proliferation medium

- Tryptic Soy Agar (TSA) - growth medium

Methodology

The first stage of the study for both methods was the multiplication of microorganisms in LT 100 Broth medium:

A single colony of the given microorganism was transferred onto the sterile LT 100 medium using a sterile inoculation loop. Such inoculated medium was incubated for 21 h ± 3 h at 32.5°C ± 2.5°C. After incubation, depending on the chosen method, the following activities were performed:

In the diffusion-contact method:

A decimal dilution of the microbial culture grown in LT 100 Broth medium using a sterile physiological saline solution (0.1 ml LT 100 Broth: 0.9 ml 0.9% NaCl) was made. Surface inoculation was performed by applying 0.1 ml of the dilution obtained onto the TSA plate. The plate with inoculated medium was covered and left for 15 minutes to dry. After 15 minutes, 50 mL of the formulation was applied onto the surface of the medium and allowed to be completely absorbed. The plates were incubated for 21 h ± 3 h at 32.5°C ± 2.5°C. The study was repeated for each microorganism, in parallel to the control, where sterile physiological saline was used instead of the tested preparation.

After the incubation, the results were read.

In the diffusion-well method:

A decimal dilution of the microbial culture grown in LT 100 Broth medium using a sterile physiological saline solution (0.1 ml LT 100 Broth: 0.9 ml 0.9% NaCl) was made. Surface inoculation was performed by applying 0.1 ml of the dilution obtained to the plate with TSA medium. The plate with the inoculated medium was covered and left for 15 minutes to dry. After 15 minutes, 50 mL wells were excised in the medium using a sterile punch. A dose of 50 mL of the preparation was transferred to each well and allowed to be completely absorbed. The plates were incubated for 21 h ± 3 h at 32.5°C ± 2.5°C. The study was repeated for each microorganism, in parallel to the control, where sterile physiological saline was used instead of the tested preparation. After the incubation, the results were read.

RESULTS

Table 4 Study of the antimicrobial activity of the preparation for the throat using the diffusion-contact method

Table 5 Study of the antimicrobial activity of the preparation for the throat using the diffusion-well method

* the avarage size of the zone of inhibition; macroscopic data

Summary and conclusions

The preparation with domiphen bromide shows very good antimicrobial properties towards all tested microbial strains, which is particularly evident in the diffusion-contact method studies. Total inhibition of microbial growth was observed in the place of contact of the tested material with the microbiological medium and formation of inhibition zones around the contact zone, which may indicate a high surface activity of the preparation and translate into its effectiveness in contact with the nasal pharyngeal mucosa.

In the diffusion-well method, clear zones of growth inhibition against C albicans and S. aureus (16 mm and 25 mm, respectively) and no inhibition of E. coli growth were observed.

In conclusion, the formulation containing domiphen bromide shows good antimicrobial properties against the tested strains, visibly inhibiting their growth.

CYTOTOXICITY in vitro studies

The tests were carried out in accordance with PN-EN ISO 10993-5: 2009, on the NCTC mouse cell line fibroblasts clone 929, ATCC, on EMEM medium from Lonza, serial number 5MB018, FBS foetal bovine serum from Euroclone, serial number EUS00116, using the Lonza antibiotic solution, serial number 16J215309. The observed cytotoxicity level of the preparation was "1 " (weak cytotoxicity), which means that the product is safe.

It was found that medical devices containing domiphen bromide as the only active substance effectively inhibit microbial growth and the dynamics of this activity is satisfactory. As a single active substance, it triggers the expected efficacy in the treatment of oral inflammation at an optimal dose of 0.01 to 0.03% by weight.

The below examples of the composition in the form of a throat spray don’t exclude examples with further variants under invention.

Example 1

Purified water 88,96 g

Domiphen bromide 0.01 g

Cetraria islandica extract 2,50 g

Sodium chloride 0,12 g

Potassium chloride 0.08 g Calcium chloride 0.03 g

Glycerin 5,00 g

Xylitol 3,00 g

Mint aroma 0.30 g

Example 2

Purified water 90.48 g

Domiphen bromide 0.03 g

Tussilago farfara L. extract 1.50 g

Sodium chloride 0.22 g

Potassium dihydrogen phosphate 0.02 g

Sodium phosphorus 0.10 g

Glycerin 2.00 g

Sorbitol 5.00 g

Sour acidic oil 0.65 g

Example 3

Purified water 91.23 g

Domiphen bromide 0.01 g

Althaea officinalis L. extract 2.50 g

Sodium dihydrogen phosphate 0.08 g

Potassium hydrogen phosphate 0.08 g

Glycerin 2.00 g

Maltitol 4.00 g

Eucalyptus oil 0.10 g

Example 4

Purified water 90.20 g

Domiphen bromide 0.03 g

Alcea rosea L. var Nigra extract 1.00 g

Sodium chloride 0.12 g

Potassium chloride 0.08 g

Magnesium chloride 0.03 g

Potassium hydrogen phosphate 0.14 g

Glycerin 4.00 g

Sorbitol 4.00 g

Peppermint oil 0.40 g

As shown, domifen bromide, when used for the throat and gingival pockets as a single active substance and present in a composition in accordance with the invention, has antibacterial effect in the treatment of throat and gingival pocket inflammations. Domiphen bromide is used in medical devices as a preservative or, rarely, it is an active substance used as an adjunct in a system with other quaternary ammonium salts, such as, for example, cetylpyridinium chloride.

Domiphen bromide has not yet been used in medical devices intended for the oral cavity, as the only active substance, which would be - in addition - used at such low concentration.

Domiphen bromide in oral compositions in pressurised containers (i.e. in a form of a throat spray and liquid for rinsing gingival pockets) has been shown to have effective antibacterial properties as a single substance, despite its low concentration (0.01-0.03% by weight) necessary to ensure the safety of the invention.

Claims

Patent claims

1. A pharmaceutical composition with domiphen bromide and pharmaceutically ac- ceptable adjuncts, characterised in that it contains per 100 ml of solution (in wt%):

- domiphen bromide in an amount of 0.01-0.10%, preferably 0.01-0.03%;

- purified water from 90 to 99.9%;

- mineral salts (buffering, responsible for isotonicity), preferably NaCl, KCl, CaCI₂, MgCI₂, NaH₂PO₄, KH₂PO₄, Na₂HPO₄, K₂HPO₄, Na₂CO₃, NaHCO₃, in an amount of 0.5 to 5.0%, preferably 0.5-1.5%;

- and optionally flavours, preferably mint, apple, anise, lime, lemon, raspberry, cinnamon, clove in an amount of 0.1 to 0.9%.

2. A pharmaceutical composition with domiphen bromide and pharmaceutically ac ceptable adjuncts, characterised in that it contains per 100 ml of solution (in wt%):

- domiphen bromide in an amount of 0.01-0.10%, preferably 0.01-0,03%

- purified water from 90% to 99%;

- mineral salts (buffering, responsible for isotonicity), preferably NaCl, KCl, CaCb, MgCh, NaH₂PO₄, KH₂PO₄, Na₂HPO₄, , K2HPO4 in an amount of 0-1 to 5.0%, preferably 0.5-1.5%;

- moisturising substance preferably glycerine from 0,1 to 0,5%;

- aroma in an amount of 0.1 to 0.9%, preferably peppermint oil, clove oil, lemon oil, eucalyptus oil, anise oil;

- natural plant extract.

3. The composition according to claim 2, characterised in that it is in the form of a spray.

4. The composition according to claim 2, characterised in that it contains the following plant extracts: Cetraria islandica, i.e., Iceland moss), Chondunts crispus (Irish moss or carrageen moss), marshmallow (Althaea officinalis L), black mallow flower (Alcea rosea L. var. Nigra), mullein flower (Verbascum L.), coltsfoot leaf (Tussilago farfaxa L.), linden inflorescence (Tilia cordata Mill.).

5. The composition according to claim 2, characterised in that it contains a sweetener, preferably xylitol, sorbitol, maltitol, erythritol in an amount of 1 to 5%.

6. The composition according to claim 2, characterised in that it contain an acacia or tragacanth gum in an amount of 0.1 g to 2.0 g / 100 ml.