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Title:
LACTIC ACID BACTERIAL COMPOSITION FOR THE SIMULTANEOUS TREATMENT OF VAGINAL INFECTIONS OF FUNGAL AND BACTERIAL ORIGIN
Document Type and Number:
WIPO Patent Application WO/2017/163217
Kind Code:
A1
Abstract:
The present invention relates to a pharmaceutical composition or a composition for a medical device or a composition for a food supplement (briefly, the composition (s) of the present invention) based on lactic acid bacteria belonging to the species Lactobacillus fermentum for vaginal and oral use for the simultaneous treatment of vaginal infections, disorders or diseases of fungal and bacterial origin. Specifically, the present invention relates to a said composition comprising pharmaceutical- and/or food- grade excipients and a mixture, which comprises or, alternatively, consists of at least a strain of bacteria belonging to the species Lactobacillus fermentum for vaginal and oral use for the simultaneous treatment of vaginal infections, disorders or diseases selected from candidiasis, vaginitis, vulvovaginitis or bacterial vaginosis.

Inventors:
MOGNA GIOVANNI (IT)
Application Number:
IB2017/051711
Publication Date:
September 28, 2017
Filing Date:
March 24, 2017
Export Citation:
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Assignee:
PROBIOTICAL SPA (IT)
International Classes:
A61K35/747; A61P15/02
Domestic Patent References:
WO2012101500A12012-08-02
WO2010133761A12010-11-25
WO2014184643A12014-11-20
WO2013093941A22013-06-27
Foreign References:
EP2158916A12010-03-03
EP2000530A12008-12-10
Other References:
FRANCESCA DEIDDA ET AL: "In Vitro Activity of Lactobacillus fermentum LF5 Against Different Candida Species and Gardnerella vaginalis", JOURNAL OF CLINICAL GASTROENTEROLOGY., vol. 50, 1 November 2016 (2016-11-01), US, pages S168 - S170, XP055324073, ISSN: 0192-0790, DOI: 10.1097/MCG.0000000000000692
FRANCO VICARIOTTO ET AL: "Effectiveness of the Two Microorganisms Lactobacillus fermentum LF15 and Lactobacillus plantarum LP01, Formulated in Slow-release Vaginal Tablets, in Women Affected by Bacterial Vaginosis", JOURNAL OF CLINICAL GASTROENTEROLOGY., vol. 48, 1 January 2014 (2014-01-01), US, pages S106 - S112, XP055324076, ISSN: 0192-0790, DOI: 10.1097/MCG.0000000000000226
FILIPPO MURINA ET AL: "Can Lactobacillus fermentum LF10 and Lactobacillus acidophilus LA02 in a Slow-release Vaginal Product be Useful for Prevention of Recurrent Vulvovaginal Candidiasis?", JOURNAL OF CLINICAL GASTROENTEROLOGY., vol. 48, 1 January 2014 (2014-01-01), US, pages S102 - S105, XP055324098, ISSN: 0192-0790, DOI: 10.1097/MCG.0000000000000225
FRANCO VICARIOTTO ET AL: "Effectiveness of the Association of 2 Probiotic Strains Formulated in a Slow Release Vaginal Product, in Women Affected by Vulvovaginal Candidiasis: A Pilot Study", JOURNAL OF CLINICAL GASTROENTEROLOGY, 1 October 2012 (2012-10-01), United States, pages S73 - S80, XP055145436, Retrieved from the Internet [retrieved on 20141009], DOI: 10.1097/MCG.0b013e3182684d71
STRUS M ET AL: "Studies on the effects of probiotic Lactobacillus mixture given orally on vaginal and rectal colonization and on parameters of vaginal health in women with intermediate vaginal flora", EUROPEAN JOURNAL OF OBSTETRICS & GYNECOLOGY AND REPRODUCTIVE BIOLOGY, ELSEVIER IRELAND LTD, IE, vol. 163, no. 2, 1 August 2012 (2012-08-01), pages 210 - 215, XP002712127, ISSN: 0301-2115, [retrieved on 20120619], DOI: 10.1016/J.EJOGRB.2012.05.001
HOESL C E ET AL: "The Probiotic Approach: An Alternative Treatment Option in Urology", EUROPEAN UROLOGY, ELSEVIER BV, NL, vol. 47, no. 3, 1 March 2005 (2005-03-01), pages 288 - 296, XP027606331, ISSN: 0302-2838, [retrieved on 20050301]
PROBIOTICAL: "OUR PROBIOTIC STRAINS", 1 January 2016 (2016-01-01), XP055382106, Retrieved from the Internet [retrieved on 20170616]
Attorney, Agent or Firm:
HOFFMANN EITLE S.R.L. et al. (Piazza Sigmund Freud 1, Torre 2 Piano 22, Milano, 20154, IT)
Download PDF:
Claims:
CLAIMS

1. Pharmaceutical composition or composition for a medical device or composition for a food supplement or composition for a food product comprising pharmaceutical- and/or food-grade excipients and a mixture comprising or, alternatively, consisting of at least a strain of bacteria belonging to the species Lactobacillus fermentum; said composition being for vaginal or oral use for the treatment of at least a vaginal infection, wherein said at least a vaginal infection is selected from candidiasis, vaginitis, vulvovaginitis or bacterial vaginosis, caused by at least a pathogen selected from the group comprising: Candida glabrata, Candida parapsilosis, Candida krusei, Candida tropicalis, Gardnerella vaginalis, Trichomonas vaginalis, Neisseria gonorrhoeae, Escherichia coli, Herpes simplex and Hemophilus Ducreyie and wherein the bacteria belong to the strain Lactobacillus fermentum (LF05) with deposit number DSM 32277 or CNCM I-789 (depositor Probiotical SpA).

2. The composition for use according to claim 1 , wherein said mixture further comprises or, alternatively, consists of at least a strain of bacteria belonging to the species Lactobacillus fermentum selected from the group comprising or, alternatively, consisting of the strains of bacteria from 3 to 14 below:

L. fermentum

No. 12 LF16 1853 DSM 26956 Probiotical SpA

L. fermentum

No. 13 LF18 1897 DSM 29197 Probiotical SpA

L. fermentum

No.14 LF 25 1956 DSM 32275 Probiotical SpA

L. fermentum

3. The composition for use according to claim 2, wherein said mixture comprising or, alternatively, consisting of at least a strain of bacteria belonging to the species Lactobacillus fermentum selected from the group comprising or, alternatively, consisting of the strains of bacteria No. 7, 8, 11 , 12, 13 and 14.

4. The composition for use according to claim 3, wherein said mixture comprising or, alternatively, consisting of at least a strain of bacteria belonging to the species Lactobacillus fermentum selected from the group comprising or, alternatively, consisting of the strains of bacteria No. 8, 11 , 13 and 14.

5. The composition for use according to at least any one of the preceding claims, wherein said vaginal infection is selected from candidiasis, vaginitis, vulvovaginitis or bacterial vaginosis due to at least a pathogen selected from the group comprising: Candida glabrata, Candida parapsilosis, Candida krusei, Candida tropicalis and Gardnerella vaginalis.

6. The composition for use according to any one of the preceding claims, wherein said composition is for the treatment of vaginal infections, disorders or diseases due to the pathogens Candida albicans and Gardnerella vaginalis.

7. The composition for use according to any one of the preceding claims, wherein said composition is in a solid form or liquid form, preferably in the form of powder, granules, tablet, lozenge, solution, vaginal douche, dispersion or gel.

8. The composition for use according to any one of the preceding claims, wherein said mixture contains said strains of bacteria at a concentration comprised from 1x108 to 1x1012 CFU/g, preferably from 1x109 to 1x1011 CFU/g.

9. The composition for use according to any one of the preceding claims, wherein said composition contains said strains of bacteria at a concentration comprised from 1x10s to 1x1010 CFU/g, preferably from 1x107 to 1x10s CFU/g.

10. The composition for use according to any one of the preceding claims, wherein said mixture of bacteria is present in said composition in a weight ratio comprised from 1 :2 to 1 : 10, preferably in a weight ratio comprised from 1 :3 to 1 :5.

Description:
DESCRIPTION of the invention entitled: Lactic acid bacterial composition for the simultaneous treatment of vaginal infections of fungal and bacterial origin.

The present invention relates to a pharmaceutical composition or a composition for a medical device or a composition for a food supplement (briefly, the composition (s) of the present invention) based on lactic acid bacteria belonging to the species Lactobacillus fermentum for vaginal and oral use for the simultaneous treatment of vaginal infections, disorders or diseases of fungal and bacterial origin. Specifically, the present invention relates to a said composition comprising pharmaceutical- and/or food- grade excipients and a mixture, which comprises or, alternatively, consists of at least a strain of bacteria belonging to the species Lactobacillus fermentum for vaginal and oral use for the simultaneous treatment of vaginal infections, disorders or diseases selected from candidiasis, vaginitis, vulvovaginitis or bacterial vaginosis.

The composition of the intestinal and urogenital microflora is known to represent a critical point for women's health and wellbeing. The vaginal ecosystem consists of epithelial cells lining the vagina and uterus, gland cells secreting in the lumen of the organ and a complex bacterial flora represented by different species of microorganisms.

Such microorganisms are able to fermenting the glycogen deriving from the decomposition of parabasal cells of the eutrophic vaginal mucosa, resulting in the production of lactic acid, the final effect of which is the establishment and maintenance of an acidic vaginal environment (with pH values of approximately 4- 4.5 under physiological conditions).

Hydrogen ions (H + ) deriving from lactic acid contribute to the formation of hydrogen peroxide. Such a peroxide molecule is toxic for a large number of bacterial species lacking the enzyme catalase. In vaginal secretions, concentrations of about 0.75-5 g/ml are readily reached, which are more than sufficient for the toxic effect to be exerted.

The combined action of hydrogen peroxide, uterine peroxidase (produced by the cervix and endometrium) and chloride (CI ) and iodide (I ) ions limits the bacterial growth even by directly activating polymorphonuclear cells, which exert a bactericidal action in the epithelial intercellular spaces.

Due to various exogenous and endogenous factors, such as the use of antibiotics, stress conditions, hormonal modulations related to pregnancy, menstrual cycle and/or taking high estrogen concentrations, an unbalance of the vaginal ecosystem often occurs in women. An alteration in the balance of the vaginal ecosystem leads to a prevalence of microorganisms such as Candida albicans and glabrata and/or

Gardnerella vaginalis, which cause candidiasis, vaginitis, vulvovaginitis and bacterial vaginosis.

It is known that approximately 75% of women suffered or will suffer, at some point in their life, from at least an episode of vulvovaginal mycosis, due to a fungus (yeast) called Candida albicans (Candida vaginitis or vulvovaginitis) in 90% of cases. Moreover, it has to be noted that a relapse, namely a further infectious event, occurs in about 40-50% of women.

Furthermore, another important aspect of vaginal infections concerns bacterial vaginosis. A bacterial vaginosis is known to be a vaginal inflammation of bacterial origin and is the most common cause of vaginal disorders in childbearing and menopausal women. Bacterial vaginosis is caused by an alteration in the balance of the vaginal microflora, with abnormal development of commensal germs, which, following to an alteration of the vaginal ecosystem, turn into pathogens due to many causes. The main agent responsible for most of the episodes of bacterial vaginosis, almost 80% of cases, is Gardnerella vaginalis. Bacterial vaginosis can be treated with antibiotics such as for example metronidazole or clindamycin. However the bacterial, as well as the fungal vaginosis, can also relapse even after therapy. Therefore, the antibiotic treatment does not represent in any way a definitive treatment, is ineffective in avoiding one or more relapses, nor is a treatment suitable for all the categories of patients.

At present, epidemiological data show that vaginal infections affect more than one billion of women per year worldwide, with serious socio-economic consequences. Furthermore, vaginosis has been related to preterm delivery and a high incidence of necrotizing enterocolitis (NEC). A link between autism and perinatal inflammation has been suggested.

The use of antibiotics during pregnancy has been associated with the onset of asthma.

Vulvovaginal candidiasis (WC) is usually caused by C. albicans but it can also be due to other species of

C. albicans or yeasts. Typical WC symptoms comprise itching, vaginal irritation, dyspareunia, external dysuria and abnormal vaginal secretions. None of these symptoms is WC-specific.

C. glabrata is a pathogenic yeast with various and unique biological characteristics. It could be isolated from the skin and urine, and represents one of the most common yeasts found on mucous membranes. It is considered as an opportunistic pathogen causing surface and systemic infections, in particular in immunocompromised patients. In Europe, the percentage of overall resistance to fluconazole of C. glabrata strains is 16.5%.

C. parapsilosis is an exogenous pathogen. It can lead to sepsis, onychomycosis and dermatomycosis, is found also in mucosae, and has biochemical characteristics different from the other species.

C. tropicalis is the most common pathogen of the genus Candida. It is found in mixed cultures along with

C. albicans and is very resistant to antifungal therapies. This pathogen can proliferate on mucous membranes and can be observed even in the absence of specific signs of a disease.

C. krusei is a fungus naturally resistant to fluconazole, and can be isolated from beer, milk and derivatives, and skin, saliva and feces from animals. It is related to some forms of infantile diarrhea and occasionally to systemic diseases. Such a fungus is able to colonizing the intestine, the respiratory and urinary tracts of patients with granulocytopenia. Bacterial vaginosis (BV) is a clinical polymicrobial syndrome due to the replacement of the normal lactobacilli capable to produce hydrogen peroxide by high concentrations of anaerobic bacteria (e.g. the species Prevotella and Mobiluncucus), ureaplasma, mycoplasma and several and annoying kinds of anaerobic organisms. Among women in need of treatment, BV is the main cause of vaginal secretions and odor. However, in a study conducted in USA, most of women with BV were found to be asymptomatic. The percentage of women, which experienced at least an episode of BV ranges between 5 and 70%. BV is characterized by the Gardnerella vaginalis infection in 80% of reported cases.

After a gynecological diagnosis subsequent to a laboratory fungal analysis, an antibiotic and/or fungicidal therapy is generally chosen, which usually leads to good results in the short term, but is unable to prevent recurrent infections, due to the increasing resistance of pathogens. Furthermore, not all the subjects in need of treatment are able to take and tolerate an antibiotic or fungicidal therapy.

Miconazole and fluconazole represent the most used active ingredients for treating candidiasis: miconazole is mainly used topically for infections due to C. albicans and species other than C. albicans. Among triazoles, fluconazole is the molecule of choice for the treatment of candidiasis, but its efficacy is variable: in fact from 29% to 43% of patients with systemic infections obtain no satisfactory results with these molecules. In addition, these molecules have a highly variable effectiveness against different species of yeasts.

It is also known the use of microorganisms, such as for example lactic acid bacteria, able to restoring a proper composition of the vaginal microflora. Lactic acid bacteria can be used as active ingredient in a composition for vaginal use, which can be for example in a solid form as an oral tablet, lozenge, granules or powder or solid form as a vaginal tablet or in a liquid form as a vaginal douche, solution or gel.

However, thus far, there is still a need for having a fast and effective treatment against vaginal infections, disorders or diseases independent from a gynecologic diagnosis and microbiological analyses, in order to achieve rapid and beneficial results from the treatment of a bacterial vaginosis and/or Candida vaginitis. Basically, it would be very useful to provide a treatment, which, for its application, does not need a diagnosis, which would imply for the patient to be subjected to medical examinations and sample withdrawals, is devoid of side effects typical of the commonly used antibiotics/antifungal drugs, avoids the development of cross-resistance to antibiotics and is highly tolerable and effective.

Specifically, there is still a need for having a composition for vaginal and oral use for the simultaneous treatment of vaginal infections, disorders or diseases of fungal and bacterial origin which is (i) effective and/or (ii) easy to prepare and/or (iii) able to definitively act on the balance of the vaginal ecosystem and/or (iv) effective in avoiding one or more relapses and suitable for all the categories of patients, said composition having a broad-spectrum activity against pathogenic microorganisms among which, but not limited to, yeasts of the genus Candida, responsible for fungal candidiasis, vaginitis and vulvovaginitis, and the bacterium Gardnerella vaginalis, which accounts for 80% of cases of bacterial vaginosis. Furthermore, there is still a need for having a composition for vaginal and oral use for the simultaneous treatment of vaginal infections, disorders or diseases of fungal (namely, due to fungi and/or yeasts) and bacterial origin, which represents a valid alternative to an antibiotic and/or fungicidal therapy and, at the same time, represents an effective treatment relative to the known administration forms against both Candida and Gardnerella vaginalis infections.

The Applicant, after an extended and demanding research and development activity, developed a composition for vaginal or oral use based on lactic acid bacteria being able to effectively solve the common problems of the known technique.

It is an object of the present invention a composition for vaginal or oral use, as set forth in the appended independent claim.

Other preferred embodiments of the present invention are set forth in the appended dependent claims. Graph 1 relates to an illustrative histogram of the effect of the co-growth of lactobacilli and C. albicans in MRS.

Graph 2 relates to histograms representing the mortality of different species of Candida co-grown with LF5.

Graph 3 relates to histograms representing the mortality of different species of Candida co-grown with LF5.

II Graph 4 relates to histograms representing the mortality of different species of Candida co-grown with LF5.

Figure 1 relates to the action exerted by LF5 on C. albicans ATCC 90028 during the co-growth of the two microorganisms.

Figure 2 relates to the inhibition halos of C. albicans ATCC 90028 on agar medium induced by a broth culture of the strain LF5: disc technique.

Figure 3 relates to the inhibition halos of the growth of C. albicans ATCC 90028 on agar medium induced by the freeze-dried concentrate of the supernatant from the disc and spot techniques.

Figures 4, 5 and 6 relate to the mechanism of action of the strain LF5 against C. albicans.

Graph 5 relates to the inhibition of C. albicans ATCC 90028: Graphs of overall data, at experiment 1 and experiment 2.

Table 11 relates to experiments on strains of bacteria L. Fermentum showing the MICs at 24 and 48 hours.

Figure 7 shows the results of the clinical study for the efficacy and tolerability of LF5 compared with placebo in patients with Candida albicans.

Figure 8a shows the trend of the overall average number of symptoms and signs (+ESM) related to the fungal vaginal colonization, during and after the treatment with LF5 or miconazole. Figure 8b shows the trend of the total average intensity of symptoms and signs (+ESM) related to the fungal vaginal colonization, during and after the treatment with LF5 or miconazole.

Figure 9 shows the activity of different strains of Lactobacillus fermentum against several species of

Candida.

Figure 10 shows the activity of Lactobacillus fermentum LF5 against several species of Candida.

The Applicant carried out a long and intense experimental research activity, during which tested a first group of bacterial strains belonging to the species below:

- L plantarum, such as for example the strain of bacteria L. plantarum (LP 01) LMG P-21021 ;

- L pentosus, such as for example the strain of bacteria L. pentosus (LPS 01) DSM 21980;

- L delbrueckii ssp. delbrueckii, such as for example the strain of bacteria L. delbrueckii ssp. delbrueckii (DSMZ 20074 LDD01) DSM 22106;

- L rhamnosus, such as for example the strain of bacteria L rhamnosus (LR 06) DSM 21981 ;

- B. breve, such as for example the strain B. breve (BR 03) DSM 16604.

Said bacterial strains were tested in order to assess the inhibition data on Gardnerella vaginalis (responsible for 80% of cases of bacterial vaginosis in women) as well as the activity and efficacy thereof against Candida albicans. The results of said inhibition tests were negative for the pathogen Gardnerella vaginalis. The tests came back negative, since none of the tested strains was able to inhibit the pathogen Gardnerella vaginalis.

The strain of the pathogen Gardnerella vaginalis being used in the tests of the present invention is the type Gardnerella vaginalis ATCC 14018, also referred to as Haemophilus vaginalis, as reported below: Gardnerella vaginalis ATCC ® Number: 14018™, Preceptrol® Culture. Organism: Gardnerella vaginalis (Gardner and Dukes) Greenwood and Pickett deposited as Haemophilus vaginalis Gardner and Dukes. ATCC Medium: 814 GC Medium.

The Applicant then tested a second group of vaginal bacterial strains belonging to the species below:

(i) L crispatus, such as for example the strain of bacteria L. crispatus CRL 1266 ID1626 DSM 24439;

(ii) L. crispatus, such as for example the strain of bacteria L crispatus CRL 1251 ID1606 DSM 24438;

(iii) L paracasei, such as for example the strain of bacteria L paracasei LPC 08 ID1696 DSM 21718;

(iv) L paracasei, such as for example the strain of bacteria L paracasei CRL 1289 ID1608 DSM 24440;

(v) L. fermentum, such as for example the strain of bacteria L. fermentum LF 11 ID1639 DSM 19188.

Table 1

I Amount of fresh MRS I 0 0.5 1 2 4

G. vaginalis 0.497 1.251 1.534 1.811 1.807

Table 1 reports the results of the inhibition test (Analytical method infra) of G. vaginalis by the strains of vaginal bacteria mentioned above from (i) to (v).

Results in Table 1 refer to the optical density, which was detected at the wavelength OD600 after 24 hours of microaerophilic growth. The test was performed with a starting inoculum at 2% from fresh broth culture in TH Broth. It was also observed that adding the MRS medium increased the growth ability of the pathogen.

Then, the Applicant tested a third group of strains of bacteria belonging to the species Lactobacillus fermentum, in order to assess the inhibition data on Gardnerella vaginalis (responsible for 80% of cases of bacterial vaginosis in women) as well as the activity and efficacy thereof against Candida albicans and other yeasts of the genus Candida. The Applicant only selected the strains of bacteria belonging to the species L. fermentum, which shown to be active and effective against both the pathogens Candida albicans and Gardnerella vaginalis.

The results of absorbance OD600, at 24 hours and 48 hours, are reported in Table 2 and Table 3, respectively.

Table 2: inhibition test (Analytical method infra)

1637

No. 8 0.683 0.725 0.688

L Fermentum LF 11 ID

1639

No. 9 0.783 0.735 0.669

L Fermentum DPPMA

114 ID 1757

No. 10 0.827 0.884 0.761

L. Fermentum Lb2 ID

1753

No. 11 0.765 0.628 0.723

L Fermentum LF 15 ID

1852

No. 12 0.716 0.580 0.652

L. Fermentum LF 16 ID

1853

No. 13 0.673 0.715 0.668

L. Fermentum LF 18 ID

1897

No. 14 0.775 0.648 0.733

L. Fermentum LF 25 ID

1956

Table 3: inhibition test (Analytical method infra)

No. 2 0.574 0.337 0.233

L. Fermentum

LF 5 ID 686

CNCM 1-789

No. 3 0.617 0.463 0.435

L. Fermentum

LF 06 ID 1456

No. 4 0.489 0.474 0.379

L. Fermentum

LF 07 ID 1459

No. 5 0.510 0.532 0.372

L. Fermentum

LF 08 ID 1460

No. 6 0.797 0.759 0.374

L Fermentum LF 09 ID

1462

No. 7 0.618 0.696 0.447

L Fermentum LF 10 ID

1637

No. 8 0.466 0.488 0.357

L. Fermentum LF 11 ID

1639

No. 9 0.499 0.588 0.473

L. Fermentum DPPMA

114 ID 1757

No. 10 0.532 0.601 0.583

L. Fermentum Lb2 ID

1753

No. 11 0.594 0.367 0.263

L. Fermentum LF 15 ID

1852

No. 12 0.588 0.676 0.427

L Fermentum LF 16 ID

1853 No. 13 0.496 0.498 0.377

L. Fermentum LF 18 ID

1897

No. 14 0.601 0.377 0.273

L. Fermentum LF 25 ID

1956

Therefore, it is an object of the present invention a pharmaceutical composition or a composition for a medical device or a composition for a food supplement or a composition for a food product (briefly, the composition(s) of the present invention) comprising pharmaceutical- and/or food-grade excipients and a mixture consisting of or, alternatively, comprising at least a strain of bacteria belonging to the species Lactobacillus fermentum; said composition being for vaginal or oral use for the treatment of vaginal infections, disorders or diseases selected from candidiasis, vaginitis, vulvovaginitis and/or bacterial vaginosis.

An embodiment of the present invention relates to a pharmaceutical composition or a composition for a medical device or a composition for a food supplement or a composition for a food product (briefly, the composition(s) of the present invention) comprising pharmaceutical- and/or food-grade excipients and a mixture comprising or, alternatively, consisting of at least a strain of bacteria belonging to the species Lactobacillus fermentum selected from the group comprising or, alternatively, consisting of the bacterial strains from 1 to 14 (listed in Table 4); said composition being for vaginal or oral use for the treatment of vaginal infections, disorders or diseases selected from candidiasis, vaginitis, vulvovaginitis and/or bacterial vaginosis.

Table 4

No. 6 LF 09 1462 DSM 18298 Anidral Sri

L. fermentum

No. 7 LF 10 1637 DSM 19187 Anidral Sri

L. fermentum

No. 8 LF 11 1639 DSM 19188 Anidral Sri

L. fermentum

No. 9 DPPMA 114 1757 DSMZ 23757 Probiotical SpA

L. fermentum

No. 10 Lb2 1753 DSM 16143 Probiotical SpA

L. fermentum

No. 11 LF 15 1852 DSM 26955 Probiotical SpA

L. fermentum

No. 12 LF16 1853 DSM 26956 Probiotical SpA

L. fermentum

No. 13 LF18 1897 DSM 29197 Probiotical SpA

L. fermentum

No. 14 LF 25 1956 DSM 32275 Probiotical SpA

L. fermentum

An embodiment of the present invention relates to a pharmaceutical composition or a composition for a medical device or a composition for a food supplement or a composition for a food product (briefly, the composition(s) of the present invention) comprising pharmaceutical- and/or food-grade excipients and a mixture comprising or, alternatively, consisting of at least a strain of bacteria belonging to the species Lactobacillus fermentum; said composition being for vaginal or oral use for the treatment of a vaginal infection, disorder or disease selected from candidiasis, vaginitis, vulvovaginitis or bacterial vaginosis caused by at least a pathogen selected from the group comprising: Candida glabrata, Candida parapsilosis, Candida krusei, Candida tropicalis, Gardnerella vaginalis, Trichomonas vaginalis, Neisseria gonorrhoeae, Escherichia coli, Herpes simplex and Hemophilus Ducreyie, wherein the bacteria belong to the strain Lactobacillus fermentum (LF05) with deposit number DSM 32277 or CNCM I-789 (depositor Probiotical SpA).

A preferred embodiment of the present invention relates to the pharmaceutical composition or composition for a medical device or composition for a food supplement or composition for a food product (briefly, the composition(s) of the present invention), as defined above, comprising pharmaceutical- and/or food-grade excipients and a mixture which, in addition to the bacteria belonging to the strain Lactobacillus fermentum (LF05) with deposit number DSM 32277 or CNCM 1-789 (depositor Probiotical SpA), comprises or, alternatively, consists of at least a strain of bacteria belonging to the species Lactobacillus fermentum selected from the group comprising or, alternatively, consisting of the strains of bacteria No. 7, 8, 11 , 12,

13 and 14 (listed in Table 4); said composition being for vaginal or oral use for the treatment of vaginal infections, disorders or diseases selected from candidiasis, vaginitis, vulvovaginitis or vaginosis. Advantageously, said at least a strain of bacteria belonging to the species Lactobacillus fermentum is selected from the group comprising or, alternatively, consisting of the strains of bacteria No. 8, 11 , 13 and

14 (listed in Table 4).

Preferably, in the composition for use as described above, said vaginal infection is selected from candidiasis, vaginitis, vulvovaginitis or bacterial vaginosis due to at least a pathogen selected from the group comprising: Candida glabrata, Candida parapsilosis, Candida krusei, Candida tropicalis and Gardnerella vaginalis.

Advantageously, the inventors found that the bacteria of the strain Lactobacillus fermentum (LF05) with deposit number DSM 32277 or CNCM I-789 (depositor Probiotical SpA) act against different species of yeasts of the genus Candida, as well as against bacteria, such as Gardnerella vaginalis, which are the main causes of bacterial vaginitis. In this respect, the bacteria of the strain Lactobacillus fermentum (LF05) with deposit number DSM 32277 or CNCM I-789 can be used for the treatment of disorders due to the concurrent presence of yeasts of the genus Candida, among which, inter alia, Candida albicans, and bacteria, among which, but not limited to, Gardnerella vaginalis.

Advantageously the composition of the present invention is for vaginal or oral use for the treatment of vaginal infections, disorders or diseases due to the association of the pathogens Candida albicans and Gardnerella vaginalis, in that Lactobacillus fermentum (LF05) was found to be active against yeasts, such as Candida albicans and, surprisingly, also against Candida species other than albicans, and the bacterium Gardnerella vaginalis.

Advantageously, the composition of the present invention is for vaginal or oral use for the treatment of vaginal infections, disorders or diseases selected from candidiasis, among which fungal candidiasis, vaginitis and bacterial vaginosis.

An embodiment of the present invention relates to a pharmaceutical composition or a composition for a medical device or a composition for a food supplement or a composition for a food product comprising pharmaceutical- and/or food-grade excipients and a mixture comprising or, alternatively, consisting of at least a strain of bacteria belonging to the species Lactobacillus fermentum; said composition being for vaginal or oral use for the treatment of at least a vaginal infection, wherein said at least a vaginal infection comprises or consists of bacterial vaginosis, caused by the pathogenic bacterium Gardnerella vaginalis and wherein the bacteria belong to the strain Lactobacillus fermentum (LF05) with deposit number DSM 32277 or CNCM I-789 (depositor Probiotical SpA). Preferably, in the composition of the invention, said mixture further comprises or, alternatively, consists of at least a strain of bacteria belonging to the species Lactobacillus fermentum selected from the group comprising or, alternatively, consisting of the strains of bacteria from 3 to 14 below:

In a preferred embodiment, said mixture further comprises or, alternatively, consists of at least a strain of bacteria belonging to the species Lactobacillus fermentum selected from the group comprising or, alternatively, consisting of the strains of bacteria No. 7, 8, 11, 12, 13 and 14. In a preferred embodiment, said mixture further comprises or, alternatively, consists of at least a strain of bacteria belonging to the species Lactobacillus fermentum selected from the group comprising or, alternatively, consisting of the strains of bacteria No. 8, 11 , 13 and 14.

In a preferred embodiment, the composition according to the present invention is for use in the treatment of at least a vaginal infection comprising, in addition to a bacterial infection caused by the pathogenic bacterium Gardnerella vaginalis, a vaginal infection, vaginitis, vulvovaginitis or bacterial vaginosis due to at least a pathogen selected from the group comprising: Candida albicans, Candida glabrata, Candida parapsilosis, Candida krusei, Candida tropicalis, Trichomonas vaginalis, Neisseria gonorrhoeae, Escherichia coli, Herpes simplex and Hemophilus Ducreyi.

In a preferred embodiment, the composition according to the present invention is for use in the treatment of at least a vaginal infection comprising, in addition to a bacterial infection caused by the pathogenic bacterium Gardnerella vaginalis, a vaginal infection due to the pathogen Candida albicans.

The composition of the present invention can be in a solid form or in a liquid form. As a solid form it can be in the form of powder, granules, tablet or lozenge, whereas as a liquid form it can be in the form of solution, vaginal douche, dispersion or gel.

Preferably, said mixture contains said strains of bacteria at a concentration comprised from 1x10 8 to 1x10 12 CFU/g, preferably from 1x10 9 to 1x10 11 CFU/g.

Preferably, said composition contains said strains of bacteria at a concentration comprised from 1x10 s to 1x10 10 CFU/g, preferably from 1x10 7 to 1x10 9 CFU/g.

Preferably, said mixture of bacteria is in said composition in a weight ratio comprised from 1 :2 to 1 :10, preferably in a weight ratio comprised from 1 :3 to 1 :5.

The composition of the present invention is for vaginal use and oral use and is effectively applied for the treatments of vaginal infections, disorders or diseases, in that it has a wide-spectrum activity against pathogenic microorganisms such as the microorganism Candida albicans, a yeast responsible for candidiasis, vaginitis, vulvovaginitis and the bacterium Gardnerella vaginalis, which causes bacterial vaginosis. Said composition can also be useful for vaginal applications for the treatment of vaginal infections, disorders or diseases such as gonorrhea, herpes and chancroid.

The strains of bacteria listed in Table 4 can be in said bacterial mixture either as live or dead bacteria, or in the form of sonicated, tyndallized or freeze-dried bacteria.

In a preferred embodiment, the strains of bacteria belonging to the species L. fermentum being present in said bacterial mixture contained in said composition of the present invention are in a coated form with one (single coating) or more (for example double coating) plant materials for lipid coating having a melting point comprised from 30°C to 80°C, preferably from 40°C to 60°C.

The composition of the present invention comprises at least a strain of probiotic bacteria belonging to the species L fermentum able to reducing and/or eliminating the presence of pathogenic agents selected from the group comprising: Candida albicans, Candida glabrata, Candida parapsilosis, Candida krusei, Candida tropicalis, Gardnerella vaginalis, Trichomonas vaginalis, Neisseria gonorrhoeae, Escherichia coli, Herpes simplex and Hemophilus Ducreyi.

As an example, in the tablets according to the invention, the strains of bacteria, preferably used in the form of freeze-dried culture having a viable count usually comprised from 10 to 200 billions of colony-forming units (CFU)/gram, are preferably in an amount from 0.5 to 20% w/w, preferably from 1 to 15% w/w, even more preferably from 3 to 10% w/w, relative to the overall weight of the tablet.

In one of the preferred embodiments, in order to enhance the efficacy of the formulations according to the present invention, specific prebiotic components are added to the powdery mixture thus obtaining a symbiotic composition. The prebiotic component is usually a not digestible saccharide material, at least partially soluble in water or aqueous solution, which stimulates the growth and/or activity of one or more strains of probiotic bacteria as described above. Among these prebiotic agents, food fibers are preferred. Preferably, said prebiotic fiber is selected from the group comprising: fructooligosaccharides (FOS), galactooligosaccharides (GOS), trans-galactooligosaccharides (TOS), xylooligosaccharides (XOS), chitosan oligosaccharides (COS), a-galactosides (such as raffinose and stachyose), pectins, gums, partially hydrolyzed gums, inulin, psyllium, acacia, carob, oat, bamboo fiber, citrus fibers and, in general, fibers containing a soluble and an insoluble portions, with variable ratios each other.

Advantageously, said prebiotic fiber is selected from fructooligosaccharides (FOS), galactooligosaccharides (GOS) and xylooligosaccharides (XOS). These fibers are not exploited by yeasts of the genus Candida, thus providing a competitive advantage for the bacterial strains of the composition of the present invention.

Preferably, the prebiotic component is present in the composition in an amount up to 70% w/w, preferably comprised from 5 to 50% w/w, even more preferably from 10 to 30% w/w, relative to the overall weight of the composition.

In a preferred embodiment, the composition according to the invention can contain additional active components, for example vitamins, mineral salts, plant extracts or other compounds with synergistic or complementary effect to that of the microorganism population of the formulations according to the invention.

Preferably, said additional active components are in an amount up to 70 w/w, preferably comprised from 0.5 to 40% w/w, even more preferably from 1 to 20% w/w, relative to the overall weight of the composition. In an embodiment (E1) the present invention relates to a pharmaceutical composition or a composition for a medical device or a composition for a food supplement or a composition for a food product comprising pharmaceutical- and/or food-grade excipients and a mixture comprising or, alternatively, consisting of at least a strain of bacteria belonging to the species Lactobacillus fermentum; said composition being for vaginal or oral use for the treatment of vaginal infections. In a preferred embodiment (E2), the present invention relates to the composition for use according to embodiment E1 , wherein said vaginal infections are selected from candidiasis, vaginitis, vulvovaginitis or bacterial vaginosis.

In a preferred embodiment (E3), the present invention relates to the composition for use according to embodiment E1 or E2, wherein said mixture comprising or, alternatively, consisting of at least a strain of bacteria belonging to the species Lactobacillus fermentum selected from the group comprising or, alternatively, consisting of the strains of bacteria from 1 to 14 below:

L. fermentum

No. 14 LF 25 1956 DSM 32275 Probiotical SpA

L. fermentum

In a preferred embodiment (E4), the present invention relates to the composition for use according to embodiment E3, wherein said mixture comprising or, alternatively, consisting of at least a strain of bacteria belonging to the species Lactobacillus fermentum selected from the group comprising or, alternatively, consisting of the strains of bacteria No. 1 , 2, 7, 8, 11 , 12, 13 and 14.

In a preferred embodiment (E5), the present invention relates to the composition for use according to embodiment E4, wherein said mixture comprising or, alternatively, consisting of at least a strain of bacteria belonging to the species Lactobacillus fermentum selected from the group comprising or, alternatively, consisting of the strains of bacteria No. 1 , 2, 8, 11 , 13 and 14.

In a preferred embodiment (E6), the present invention relates to the composition for use according to any one of embodiments E1-E5, wherein said composition is for the treatment of vaginal infections, disorders or diseases due to the pathogens Candida albicans and Gardnerella vaginalis.

In a preferred embodiment (E7), the present invention relates to the composition for use according to any one of embodiments E1-E6, wherein said composition is in a solid form or liquid form, preferably in the form of powder, granules, tablet, lozenge, solution, vaginal douche, dispersion or gel.

In a preferred embodiment (E8), the present invention relates to the composition for use according to any one of embodiments E1-E7, wherein said mixture contains said strains of bacteria at a concentration comprised from 1x10 8 to 1x10 12 CFU/g, preferably from 1x10 9 to 1x10 11 CFU/g.

In a preferred embodiment (E9), the present invention relates to the composition for use according to any one of embodiments E1-E8, wherein said composition contains said strains of bacteria at a concentration comprised from 1x10 6 to 1x10 10 CFU/g, preferably from 1x10 7 to 1x10 9 CFU/g.

In a preferred embodiment (E10), the present invention relates to the composition for use according to any one of embodiments E1-E9, wherein said mixture of bacteria is present in said composition in a weight ratio comprised from 1 :2 to 1 :10, preferably in a weight ratio comprised from 1 :3 to 1 :5.

[Experimental part

Biological anti-Cand/ /a spp. activity specific of the strain L fermentum LF5 - "in vitro" study.

1. Selection of the optimal liquid and agar substrate

for the growth of microorganisms: Candida albicans and L. fermentum.

Growth tests for C. albicans ATCC 90028 and the strains L. fermentum LF5 (Table 5) were conducted on several liquid and agar substrates, under different conditions and for more or less long periods of incubation. The growth was assessed based on the turbidity of the culture medium by reading the absorbance at 560 nm of the cultures themselves, see table 5.

Table 5 Growth of C. albicans ATCC 90028 and L. fermentum LF5 on several substrates under various incubation conditions.

Therefore, the following conditions, as the perfect compromise for co-growing the strains L. fermentum and C. albicans ATCC 90028, were detected:

a) liquid or agar MRS as the growth substrate;

b) 37°C as the incubation temperature (preferential for lactobacillus);

c) under aerobiosis (a condition observed for a good growth of the yeast). 2. Assessment of the biological activity of the strain L fermentum LF5 co-grown with C. albicans.

2.1 Assessment of the biological activity against Candida albicans

Growth tests in which Candida albicans ATCC 90028 was inoculated (10 4 cell/ml) in liquid medium inoculated with the strain L. fermentum (10 3 cell/ml) were established. The inhibition of the yeast induced by the bacterium was assessed by directly counting it in agar medium after 24 and 48 hours of co-growth, (a) In view of the average of the values from the counts, the strain LF5 (Table 4) can be affirmed to determine an inhibition of C. albicans ATCC 90028 equal to 99.9% after 24 hours and 99.999% after 48 hours. Therefore, when the number ratio between inoculated cells of C. albicans and L. fermentum is 10 to 1 respectively, a MCB99.9 after 24 hours of incubation and a MCB99.999 after 48 hours of contact are obtained.

b) The specific inhibition, induced by L. fermentum LF5 against C. albicans, is not equally found in the co- growth of the yeast with other strains of lactobacillus. The number of Candida colonies being counted in these samples is in any case lesser than that obtained when growing the yeast alone in MRS. This phenomenon can be ascribed to the spontaneous acidification of the medium occurring during the growth of lactobacilli, as well as definitely to the competition for nutrients and the presence of various catabolic products from lactic acid bacteria. In the substantial inhibition observed for C. albicans by L fermentum LF5 and overall quantified above, a specific activity of the strain LF5 takes place, which distinguishes it from other acidifying lactobacilli (Graph 1).

c) It is further demonstrated that L fermentum LF5 maintains the capability of inhibiting the growth of C. albicans in a liquid medium even after several passages in a culture thereof starting from MCB (Master Cell Bank), up to even the eighth sub-culture, it was therefore possible to start the production of the strain LF5 in a pilot plant and then realize the scaling-up at an industrial level. It was then proven the maintenance of the specific activity against the yeast even in the final freeze-dried product.

2.2 Assessment of the biological activity against other strains belonging to the genus Candida spp.

According to the same operating modes as above, co-growth tests for the strain L. fermentum LF5 along with other strains of Candida: Candida glabrata ATCC 90030, Candida Krusei ATCC 6258, Candida parapsilosis ATCC 22019 were performed.

The strain L fermentum LF5 is able to inducing a substantial degree of mortality (Graphs 2, 3, 4), slightly varying according to the species, as well as the conditions under which it is assessed, of the above different strains of Candida. On average, an inhibition of about 99.9% during the first 24 hours of incubation is observed, with a mortality reaching 99.99 - 100% during the 48 hours depending on the given strain. In view of the fact that the species Candida parapsilosis and glabrata are opportunistic pathogens, which can lead to both infections at the skin surface level, and systemic disorders it can be supposed an application of the strain LF5 as active ingredient for future pharmaceutical formulations, not for systemic use only with urogenital and intestinal efficacy, but also for topical use at the skin surface level.

2.3 Hypothesis for the possible mechanism of action of L fermentum LF5 against C. albicans ATCC 90028

By microscope observation of the co-growth in LF5 and C. albicans liquid medium and digital image capture, it was possible to evaluate the action of the lactobacillus, which seems to perform a kind of yeast "opsonization", likely followed by lysis of the membrane through a specific, presumably protein factor bound to the membrane and possibly secreted (Figure 1).

3. Assessment of the biological activity of the supernatant of fermentation

Growth tests for C. albicans ATCC 90028, inoculated into the fermented broth from LF5 (supernatant) were performed. The supernatant represents the culture medium in which L fermentum LF5 (10 6 cell/ml), under optimum conditions for the lactobacillus, was developed and then cell deprived through filtration (Table 6).

Table 6 C. albicans ATCC 90028 population after 24- and 48-hour growth in LF5 supernatant, with a positive control represented by the count of Candida inoculated into 10 ml MRS after 24 h (5.7x10 6 cell/ml). a) The "activity" leading to the inhibition of Candida, being observed in the co-growth experiments (item 2.1) is maintained in the supernatant, presumably through a specific "factor" released from LF5 even in the culture broth.

b) Such an inhibition activity against the yeast is directly proportional to the concentration of supernatant into which it is inoculated and thus presumably to the concentration of the "factor" released by the lactobacillus in the culture broth (i.e. only supernatant and diluted 1 :2 Table 6).

c) This concentration increases during the 48 hours of L fermentum growth (i.e. No. of yeast colonies counted after 24 hours from inoculation, in the 24- and 48-hour supernatant).

d) Given the same concentration, the inhibition activity increases as contacting time increases (i.e. No. of colonies counted in the 24-hour supernatant after 24 and 48 hours of incubation).

e) Specifically, the inhibition induced by the broth fermented by LF5, after 24 hours of incubation and cell deprived through filtration, against C. albicans ATCC 90028 is 99.99% at 24 hours and 99.999% at 48 hours. Such an inhibition is due to the specific activity of LF5 as well as definitely to the acidity of the growth substrate into which the yeast (supernatant) is inoculated, and to some nutrient deficiency. In any case, additional inhibition tests for Candida, inoculated into the LF5 supernatant, by adjusting the pH and adding glucose and peptone in advance, shown the presence therein of a specific activity, which determines by itself a yeast inhibition of about 96% and 99% at 24 and 48 hours, respectively, f) The inhibition efficiency of the LF5 supernatant increases when it is sub-coltured in MRS at temperatures greater than 37°C; in particular 42°C results to be the optimal condition for producing a higher concentration of the specific an\i-Candida "factor". Conversely, the growth of LF5 at controlled pH of 5 or 5.5 seems to not affect such a concentration/productivity.

The growth of C. albicans ATCC 90028 in agar medium, seeded by spread plating (10 4 cells), contacted with the broth culture and LF5 supernatant, deposited on agar by the spot or impregnated disc technique was assessed.

a) In the plates with the disc (Figure 2) in correspondence of that impregnated with broth culture of LF5 TQ, an inhibition of the Candida growth around the disc occurs, with the appearance of a well-detectable halo of about 5 mm from the disc edge.

b) The technique of the impregnated disc, put on agar medium previously spread plated with the yeast, generally leads to a more naked-eye detectable inhibition halo and/or lysis by L. fermentum, relative to the spot technique: in the plates with spots, however, within the drop of LF5 TQ, single cells of yeast with not well-defined profiles can be microscopically observed, as if cell debris is present and the area among the cells is dark and inhomogeneous. The condition outside the drop is different, where the yeast cells have well-defined profiles and the agar bottom is clearer and almost homogeneous. What is detectable inside the drop can be ascribed to an inhibition of the yeast growth, with the presence of possible debris due to the lysis, along with lactobacillus cells which grown anyway.

c) The agar tests carried out with discs and/or spots of the supernatant, harvested from a LF5 culture broth, determined no formation of detectable halos probably because of the difficulty for the "factor" with specific activity to spreading into the agar. The freeze-dried and reconstituted supernatant, in order to concentrate, even up to 20 times, such a factor, leads to the occurrence of clear inhibition halos (Figure 3) with both the disc and spot techniques as well as by directly using the not reconstituted powder (Figure 4- 5-6).

4. Clinical study for the efficacy and tolerability of LF5 vaginal capsules compared with placebo in patients with Candida albicans

100 patients with vaginitis or vulvovaginitis due to Candida albicans, average age of 32.7 years (range 18- 64 years) randomly assigned to two groups, which resulted homogeneous, each of 50 patients, treated with placebo o LF5 dosed at≤10 9 in a vaginal capsule formulation at a dosage of 1 vaginal capsule at night for three consecutive days, according to the experimental double-blind design were treated.

No discontinuations during the treatment nor during the two weeks of post-treatment observation occurred, thus two groups of 50 observations for efficacy and tolerability are provided.

The treatment with LF5 produced microbiological eradication at the end of the three-day treatment in a proportion of patients significantly greater than placebo, along with a very low risk of relapses during the next two weeks. Furthermore, even the symptom remission was significantly greater with LF5 compared with placebo.

The efficacy data relative to the comparison LF5 vs placebo are reported below in Table 7a and figure 7. Table 7a Results of the study for LF5 efficacy compared with placebo

**P<0.01 ; ***0.001

1Fisher

2 Mann-Whitney

Table 7b: Patient's opinion about efficacy

***0.001

1 Chi-square approximation

4.1 Tolerability study The clinical tolerability was good with both the preparations, with 3 cases of poor local reactions with LF5

(6%) and 1 with placebo (2%), with a not statistically relevant difference between the two groups. No clinically relevant changes for peripheral hemodynamics or routine hematology were observed.

The tolerability data relative to the comparison LF5 vs placebo are reported below in Table 7b and figure

7.

Table 7c Results of the study for LF5 tolerability compared with placebo

1 Chi-square approximation

2 Fisher's exact test

The strain LF5 has an efficacy and tolerability profile in the treatment of vaginal colonization by Candida albicans which is definitely favorable and, in any case, significantly more favorable relative to placebo, because of a statistically higher microbiological and clinical efficacy, with the same risk of poor, not clinically relevant local reactions. LF5 in vaginal capsules is thus suggested as a valid alternative to synthetic antifungal drugs for the treatment of vaginal candidiasis.

Examples of tested compositions

Composition of LF5 proprietary medicinal products

Each vaginal capsule contains:

Active ingredient:

Freeze-dried Lactobacillus Fermentum No. 1 and 2 (Table 4) NLT 10 9 CFU

Excipients:

Medium-chain triglycerides 1.964 mg Silica (Aerosil 300) 36 mg

Shell composition: Gelatin F.U. 400 mg

Glycerol F.U. 200 mg

Dimethylpolysiloxane 1000 90 mg

Titanium dioxide E 171 9 mg

Each capsule of dermatological oily suspension contains: Active ingredient:

Freeze-dried Lactobacillus Fermentum No. 1 and 2 (Table 4) NLT 10 9 CFU Excipients:

Medium-chain triglycerides 1.964 mg Silica (Aerosil 300) 36 mg Shell composition:

Gelatin F. U. 400 mg

Glycerol F.U. 200 mg

Titanium dioxide E 171 9 mg

Dimethylpolysiloxane 1000 90 mg

5 Clinical study for the efficacy and tolerability of LF5 vaginal capsules compared with miconazole in patients with Candida albicans

100 patients with vaginal candidiasis, average age of 38.0 years (range 19-61 years) randomly assigned to two groups, which resulted homogeneous, each of 50 patients treated with miconazole or LF5 dosed at ≤ 10 9 in vaginal capsule formulation at a dosage of 1 vaginal capsule at night for three consecutive days, according to the single-blind experimental design were treated.

No discontinuations during the treatment nor during the two weeks of post-treatment observation occurred; thus two groups of 50 observations for efficacy and tolerability are provided.

Both the treatments produced, at the end of the three-day treatment, microbiological eradication of Candida in almost all the patients (96% vs. 94%). The risk of relapse during the two weeks following to the treatment also resulted very low with both the treatments. However, the relapse risk with miconazole (8/47 patients; 17%) was appreciably higher relative to LF5 (5/48 cases; 10%). The symptom remission was very favorable with both the treatments as well.

The clinical tolerability was good with both the preparations. The frequency of local adverse events, however, was three-fold greater with miconazole (6 cases; 12%) relative to LF5 (2 cases; 4%). No clinically relevant changes for peripheral hemodynamics or routine hematology were observed.

The efficacy data relative to the comparison LF5 vs miconazole are reported below in Table 8a and figure 8a and 8b. Table 8a Results of the study for the LF5 efficacy compared with placebo

***0.001

1Fisher

2 Mann-Whitney

Table 8b: Patient's opinion about efficacy

***0.001

1 Chi-square approximation

The tolerability data relative to the comparison LF5 vs miconazole are reported below in Table 8c. Table 8c Results of the study for LF5 tolerability compared with miconazole

1 Chi-square approximation 2 Fisher's exact test

In conclusion, LF5 presents an efficacy and tolerability profile in the treatment of vaginal candidiasis, which is definitely favorable and substantially equal, as regards the profile of microbiological and clinical efficacy, to a well-established drug such as miconazole. However, the LF5 tolerability, even without reaching a statistical significance, was highly greater relative to that of the reference drug. Accordingly, LF5 vaginal capsules result a valid alternative to synthetic antifungal drugs for the treatment of vaginal candidiasis.

6 LF5: study for local tolerability and activity in patients with Candida albicans

100 patients with vaginitis, vulvovaginitis o more extensive Candida albicans colonization, average age of 34.0 years (range 17-65 years) with LAB dosed at≤10 9 in vaginal capsule formulation at a dosage of 1 vaginal capsule at night for 14 consecutive days, according to the open experimental design were treated. No treatment discontinuations occurred, therefore 100 observations for efficacy and tolerability are provided.

The treatment with LF5 produced a microbiological eradication at the end of the first three days of treatment in 92% of patients, and in an additional 5% during the following period of treatment. Overall, only 3% of patients failed the therapy.

During the same observation period, a remarkable remission of the symptoms, in particular during the first three days of treatment was detected as well as, being statistically significant, during the remaining period of observation.

The clinical tolerability was good, with 5 total reports of poor local reactions. No clinically relevant changes in routine hematology were observed.

In conclusion, LF5 has an efficacy and tolerability profile in the treatment of vaginal colonization by Candida albicans which is definitely favorable, in that it already has a statistically significant microbiological and clinical efficacy during the first three days of application, with a further improvement when the therapy is continued, without relevant risks of local, always slight, clinically irrelevant and spontaneously reversible adverse reactions, nor detectable risks of alterations of the lab parameters. Therefore, LF5 vaginal capsules are suggested as a valid alternative to synthetic antifungal drugs for the treatment of vaginal candidiasis, even when it spreads to the proximal structures and in patients with particular conditions such as pregnancy.

Methods The aim of the method is to verify if a given bacterium is able to inhibit the broth growing of Gardnerella vaginalis by the production of substances with bacteriostatic/bactericidal action.

Materials and reagents:

- MRS Broth (Difco)

- Cysteine chlorohydrate sol. 5% aqueous solution, in sterile pure water. Sterilize through syringe filters 0.22 μιη of diameter

- THB (Todd Hewitt Broth Sigma - Aldrich)

- Peptone saline solution (METJNT 119-version in force)

- NaOH 30%

- Gardnerella vaginalis ATCC 14018 (Biogenetics) Sampling and preparation of the sample: Preparation of the inhibitory strain

Two sequential inoculations of the probiotic bacterium into 15 ml MRS broth (5.1) 0.1% and incubated at 37±1 °C overnight are performed. When the bacterium belongs to the genus Bifidobacterium the culture broth is supplemented with 1 % cysteine HCI (5.2).

A third inoculation with the same modes, except for a broth amount of 100 ml is carried out.

The incubation is continued up to 30±3 hours.

The broth culture is thus centrifuged and the supernatant separated from cells is brought to pH 6.6+0.2 by adding NaOH cone.

The supernatant is then filtered through 0.2 μιη filters and stored at a temperature of 4-5°C. Preparation of G. vaginalis

A pellet of G. vaginalis ATCC 14018 is inoculated into 10 ml THB medium supplemented with 1 % Cysteine

HCI (5.2) and incubated at 37±1 °C overnight.

A sequential inoculation at 1% 37±1 °C overnight is performed.

The fresh broth culture thus prepared is used for the inhibition experiment

Procedure:

The sterile tubes being prepared contain:

10ml 9.5ml 9ml 8ml

0ml 0.5ml 1ml 2ml Bacterial supernatant Oml 0.5ml 1ml 2ml 4ml

(7.5)

All the tubes thus prepared are added with 1% Cysteine HCI (5.2).

They are then inoculated at 1 % with the fresh broth culture of G. vaginalis and incubated at 37±1 °C for 24 + 24 hours.

At 24 and 48 hours spectrophotometric readings (wavelength A600) by using for each set a blank with the same percentage composition as the two culture broths are conducted.

7. Comparative study for the efficacy relative to azole therapies

In view of the above, a combination of Lactobacillus fermentum strains was selected in vitro based on the ability thereof to fight different species of Candida and then compared with miconazole and fluconazole as regards the inhibition efficacy. Specifically, the possible use of Lactobacillus strains as microorganisms for the prophylaxis and/or adjuvant therapy of acute vulvovaginal candidiasis (WC) and other vaginal infections due to Candida yeasts was studied.

Materials and methods

Strains and conditions of bacterial growth.

The strains of Candida being used in the present study were purchased from the American Type Culture Collection (ATCC). Specifically, the following biotypes were used: C. albicans ATCC 10231 , C. parapsilosis ATCC 22019, C. krusei ATCC 6258, C. glabrata ATCC 2001 and C. tropicalis ATCC 750 (14). The strains of lactobacilli of this study, all isolated from vaginal swabs of healthy female subjects or directly brushing the intestinal mucosa of healthy humans, were classified based on their phenotypic and genotypic characteristics: L. fermentum LF5 (DSM 32277), L. fermentum LF09 (DSM 18298), L. fermentum LF10 (DSM 19187) and L. fermentum LF11 (DSM 19188).

The probiotic strains were cultured overnight in De Man, Rogosa and Sharpe (MRS) medium (Difco, BD, Maryland). In all the experiments, fresh culture media for all the strains of Candida were used. Before the experiment, the single strains of Candida were cultured in Sabouraud Dextrose Broth (Difco, BD, Maryland), an optimal medium for yeasts, for 48 hours under aerobic conditions.

Every probiotic was then co-cultured in the same broth (MRS) with any one of said Candida microorganisms. The ratio between the inoculum of probiotic and yeast was 1 :100 in favor of the latter. Incubation was carried out under aerobiosis at 37°C for 24 hours. Each culture (1 ml_) was sampled after 24 hours of incubation for the selective enumeration of the yeasts, conducted on yeast extract glucose chloramphenicol (YGC) agar (Sigma-Aldrich, Milan, Italy). The YGC agar plates were incubated under aerobic conditions at 37°C for 4 days in order to allow the growth of any present yeast. The colonies were then counted and the results expressed as the number of colony forming units (CFU) / ml_.

Evaluation of the fungicidal activity. The minimum inhibitory concentration (MIC) for fluconazole was determined by E-test (bioMerieux Italy, Florence) with MHE agar. The plates were incubated under aerobic conditions at 30°C and read after 24 hours. Furthermore, since there is no specific E-test for miconazole, the MIC was determined by using the broth macrodilution method. We assessed the activity of serially diluted fluconazole and miconazole (Sigma-Aldrich, Missouri, USA) (from 1 mg / ml to 1 mg / ml) against the Candida strains. Serial dilutions of azoles were prepared in Sabouraud Dextrose Broth in the presence of strains of Candida and broths were incubated under aerobic conditions at 30°C and then read at an optical density (DO) of 560 nm. The MIC was defined as the lowest concentration of antibiotic, which completely inhibited the visible growth.

Results

Inhibitory activity of azoles against Candida strains. As it can be inferred from tables 8-9, a marked resistance for all the species of Candida was detected, except for C. parapsilosis ATCC 22019. As regards this biotype, a MIC of 1 mg/ml was obtained, thus confirming the slight sensitivity to fluconazole and miconazole.

Table 9 Assessment of the inhibitory activity of miconazole by using a macrodilution test of the culture medium (optical density, OD)

Table 10

MIC (minimum inhibitory concentration) of fluconazole from EJest (range 0.016-256 pg/ml). *p≤0.05

Candida species MIC (Mg/ml) Candida parapsilosis ATCC 22019 1*

Candida albicans ATCC 10231 >256

Candida tropicalis ATCC 750 >256

Candida krusei ATCC 6258 >256

Candida glabrata ATCC 2001 >256

Antagonistic effects of L. fermentum against Candida.

Once the resistance of most of the Candida species to the two reference drugs is detected, the ability of Lactobacillus fermentum to inhibit the yeast growth was assessed.

As shown in Fig. 9, all the tested strains of Lactobacillus, namely LF5, LF09, LF10 and LF11 , shown the capability to significantly inhibit the growth of the five species of Candida by at least four logarithms. Furthermore, the best result obtained with miconazole against C. parapsilosis is even two logarithms lower. The results for LF5 are reported in Figure 10.

Interestingly, the dose of antibiotic in humans generally provides the management of a maximum of 400 mg/die: such a per os concentration peaks an average plasma concentration of 30-40 pg / ml, a value which is much lower than the efficacy cut-off in the case of resistant strains.

Discussion

The possible role of L fermentum compared with miconazole and fluconazole against different species of Candida was tested in vitro.

Specifically, the growth capability of Candida species in the presence of increasing concentrations of miconazole and fluconazole by two distinct tests, the broth macrodilution test for miconazole and E-test for fluconazole, was quantified.

The present results are highly surprising and represent the first proof for an in vitro relatively poor efficacy of azoles, which are widely used in counteracting the growth of Candida species and for a high effectiveness of lactic acid bacteria belonging to the species Lactobacillus fermentum. The only inhibition test was detected with C. parapsilosis ATCC 22019, even though the overall results are little relevant. The poor efficacy of azoles could account for the frequent relapses of Candida infections.

Actually, a complete eradication of the yeast from the vaginal environment could be difficult to obtain, thus maintaining the conditions for a subsequent new development and clinical manifestation of unfavorable symptoms.

On the other side, the selected strains of L fermentum according to the invention confirmed the possibility to not only effectively and directly counteract the growth, but also the viability of several strains of Candida. One of these strains of L fermentum, namely the strain LF5 (identified with deposit number CNCM 1-789) was used in women with acute WC, but without any correlation with a specific species of Candida and not in the simultaneous presence of bacterial infection due to Gardnerella vaginalis, thus confirming the capability thereof in resolving the symptoms and rapidly eradicating the infection (Vicariotto F. et al. "Effectiveness of the association of 2 probiotic strains formulated in a slow release vaginal product, in women affected by vulvovaginal candidiasis: a pilot stud . J Clin Gastroenterol. 2012;46 Suppl:S73-80).

8. Inhibition of Gardnerella vaginalis by L. fermentum LF5

This study was conducted in order to assess the ability of L. fermentum LF5 to directly inhibit Gardnerella vaginalis in vitro, so that to evaluate the potential efficacy thereof even in the event of mixed vaginal infections or BV.

Materials and methods

Bacterial strains and growth conditions.

The strain Gardnerella vaginalis being used was purchased from the American Type Culture Collection (ATCC). Before the experiment, G. vaginalis ATCC 14018 was cultured in a heart-brain infusion (BHI) broth (Oxoid, Milan, Italy) consisting of 2% gelatin (weight / weight), 0.5% yeast extract, 0.1 % starch, and 0.1% glucose (7).

The strain Lactobacillus fermentum LF5 of this study was isolated from the vaginal swab of a healthy female subject and classified according to the phenotypic and genotypic characteristics thereof.

It was cultured overnight in De Man, Rogosa and Sharpe (MRS) broth (Difco, BD, Maryland).

Assessment of the antagonistic activity of LF5 against G. vaginalis.

LF5 supernatants were prepared as follows: an overnight culture in MRS broth was centrifuged at 5,000 x g. The resulting supernatant was neutralized at pH 6.5 with NaOH 1 N, sterilized by filtration through syringe filters (Ministart pore size 0.22 mm), and analyzed for the presence of any inhibitory molecule in the broth. Neutralized LF5 supernatants were then added with different percentages of fresh BHI broth, prepared as described above, inoculated with G. vaginalis. The growth of Gardnerella alone (positive control) and in the presence of different concentrations of neutralized supernatants ranging from 5% to 20%, after 24 and 48 hours of incubation at 37°C under microaerophilic conditions, was quantified by optical density at 600 nm (OD600). The growth of the positive control (Gardnerella alone) was assessed in BHI broth added to an amount of fresh MRS broth ranging from 5% to 20%. Thus, the positive controls were as similar as possible to the tubes inoculated with LF5. This test was repeated three times in order to ensure reliability and reproducibility. Results

The in vitro inhibition results of LF5 against G. vaginalis ATCC 10231 are reported in Table 11. L. fermentum LF5 shown a significant activity after both 24 and 48 hours (46% and 82% with 20% neutralized supernatant, respectively). A significant inhibition of the dose-dependent growth was detected with the neutralized supernatants in particular after 48 hours of incubation, up to a growth inhibition of G. vaginalis of even 80% (Tab. 9).

Table 11

L. fermentum LF5 - biological activity against G. vaginalis.

LF5 supernatant (0.5, 1 and 2 mL) is able to inhibit the growth of G. vaginalis even after 24 hours, but the best inhibition is after 48 hours. Positive control = G. vaginalis - growth in an optimal medium plus an equal volume of MRS (5%, 10% and 20%) (n = 3).

The study shows that L. fermentum LF5 is able to exert a fundamental inhibitory activity against G. vaginalis, thus revealing a multipurpose application. The above-cited data show that the strain LF5 is also useful for the treatment of vulvovaginal candidiasis (WC), due to different strains, among which, inter alia, Candida albicans; accordingly it can be used even in the clinical management of mixed vaginitis.