Note: Descriptions are shown in the official language in which they were submitted.
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DESCRIPTION of the invention entitled: Lactic acid bacterial composition for
the treatment of
bacterial vaginal infections by Gardnerella vaginalis and, if present, of
concurrent fungal
infections.
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 ug/m1 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 (Cl-) 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 alb/cans and
glabrata and/or
Gardnerella vagina/is, 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
alb/cans (Candida vaginitis or
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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 vagina/is.
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 (\NC) is usually caused by C. albicans but it can
also be due to other species of
C. albicans or yeasts. Typical \NC 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.
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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 vagina/is 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 vagina/is, which accounts for 80% of cases of
bacterial vaginosis.
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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 vagina/is 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.
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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 vagina/is
(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
vagina/is. The tests came back negative, since none of the tested strains was
able to inhibit the pathogen
Gardnerella vagina/is.
The strain of the pathogen Gardnerella vagina/is being used in the tests of
the present invention is the
type Gardnerella vagina/is ATCC 14018, also referred to as Haemophilus
vagina/is, as reported below:
Gardnerella vagina/is ATCC Number: 14018TM, Preceptrol Culture. Organism:
Gardnerella vagina/is
(Gardner and Dukes) Greenwood and Pickett deposited as Haemophilus vagina/is
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
Amount of fresh MRS
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0 0.5 1 2 4
G. vagina/is 0.497 1.251 1.534 1.811 1.807
Amount of supernatant
Neutralized supernatant
0.5 1 2 4
of the strain:
L. crispatus CRL 1266
ID 1626 0.505 0.511 0.673 1.035
DSM 24439
L. crispatus CRL 1251
ID 1605 0.531 0.444 0.422 0.491
DSM 24438
L. paracasei LPC08
ID 1696 0.653 1.130 1.538 1.724
DSM 21718
L. paracasei CRL 1289
ID 1608 / / 1.476 1.774
DSM 24440
L. fermentum
LF 11
/ 0.339 0.302 /
ID 1639
DSM 19188
Ecocillin INOCULUM 0.794 1.074 1.391 1.690
Table 1 reports the results of the inhibition test (Analytical method infra)
of G. vagina/is 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 0D600 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 vagina/is
(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.
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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 vagina/is.
The results of absorbance 0D600, at 24 hours and 48 hours, are reported in
Table 2 and Table 3,
respectively.
Table 2: inhibition test (Analytical method infra)
Amount of fresh MRS
24 hours 0.5 1 2
Gardnerella vagina/is 1.379 1.542 1.567
Amount of supernatant
Strain
0.5 1 2
No. 1 0.745 0.608 0.703
L Fermentum LF 5 ID
686
DSM 32277
No. 2 0.745 0.608 0.703
L Fermentum
LF 51D 686
CNCM 1-789
No. 3 0.727 0.541 0.591
L Fermentum
LF 06 ID 1456
No. 4 0.677 0.530 0.589
L Fermentum
LF 07 ID 1459
No. 5 0.681 0.583 0.717
L Fermentum
LF 08 ID 1460
No. 6 0.715 0.551 0.615
L Fermentum LF 09 ID
1462
No. 7 0.736 0.610 0.682
L Fermentum LF 10 ID
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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)
Amount of fresh MRS
48 hours 0.5 1 2
Gardnerella vagina/is 1.384 1,554 1,562
Amount of supernatant
Strain
0.5 1 2
No. 1 0.574 0,337 0,233
L Fermentum LF 5 ID
686
DSM 32277
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No. 2 0.574 0.337 0.233
L Fermentum
LF 5ID 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
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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
List of tested strains Abbreviation ID Deposit
Depositor
No. 1 LF 05 686 DSM 32277 Probiotical SpA
L fermentum
No. 2 LF 05 686 CNCM 1-789 Probiotical SpA
L fermentum
No. 3 LF 06 1456 DSM 18295 Anidral Srl
fermentum
No. 4 LF 07 1459 DSM 18296 Anidral Srl
L fermentum
No. 5 LF 08 1460 DSM 18297 Anidral Srl
L fermentum
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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 vagina/is,
Trichomonas vagina/is, 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 1-789 (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
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(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 vagina/is.
Advantageously, the inventors found that the bacteria of the strain
Lactobacillus fermentum (LF05) with
deposit number DSM 32277 or CNCM 1-789 (depositor Probiotical SpA) act against
different species of
yeasts of the genus Candida, as well as against bacteria, such as Gardnerella
vagina/is, 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 1-789 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 vagina/is.
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 vagina/is, 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 vagina/is.
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 vagina/is
and wherein the bacteria belong to the strain Lactobacillus fermentum (LF05)
with deposit number DSM
32277 or CNCM 1-789 (depositor Probiotical SpA).
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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:
List of tested strains Abbreviation ID Deposit
Depositor
No. 3 LF 06 1456 DSM 18295 Anidral Srl
L fermentum
No. 4 LF 07 1459 DSM 18296 Anidral Srl
L fermentum
No. 5 LF 08 1460 DSM 18297 Anidral Srl
L fermentum
No. 6 LF 09 1462 DSM 18298 Anidral Srl
L fermentum
No. 7 LF 10 1637 DSM 19187 Anidral Srl
L fermentum
No. 8 LF 11 1639 DSM 19188 Anidral Srl
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
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.
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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 vagina/is, 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 vagina/is,
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 vagina/is, 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 1x108 to
1x1012 CFU/g, preferably from 1x109 to 1x1011 CFU/g.
Preferably, said composition contains said strains of bacteria at a
concentration comprised from 1x108 to
1x101 CFU/g, preferably from 1x107 to 1x109 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
vagina/is, 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
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the group comprising: Candida albicans, Candida glabrata, Candida
parapsilosis, Candida krusei, Candida
tropicalis, Gardnerella vagina/is, Trichomonas vagina/is, 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 (El) 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.
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In a preferred embodiment (E2), the present invention relates to the
composition for use according to
embodiment El, 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 El 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:
List of tested strains Abbreviation ID Deposit
Depositor
No. 1 LF 05 686 DSM 32277 Probiotical SpA
L fermentum
No. 2 LF 05 686 CNCM 1-789 Probiotical SpA
L fermentum
No. 3 LF 06 1456 DSM 18295 Anidral Srl
L fermentum
No. 4 LF 07 1459 DSM 18296 Anidral Srl
L fermentum
No. 5 LF 08 1460 DSM 18297 Anidral Srl
L fermentum
No. 6 LF 09 1462 DSM 18298 Anidral Srl
L fermentum
No. 7 LF 10 1637 DSM 19187 Anidral Srl
L fermentum
No. 8 LF 11 1639 DSM 19188 Anidral Srl
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
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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 El-ES, wherein said composition is for the treatment of
vaginal infections, disorders
or diseases due to the pathogens Candida alb/cans and Gardnerella vagina/is.
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 1x108 to 1x1012 CFU/g, preferably from 1x108 to 1x1011 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 1x108 to 1x101 CFU/g, preferably from 1x107 to 1x108 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-Candida 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 alb/cans and L. fermentum.
Growth tests for C. alb/cans 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
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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.
Candida albicans L. fermentum LF5
ATCC 90028
MRS liquid medium 37 C
aerobiosis pH 6 +++ +++
pH 5.5 ++(+) +++
pH 5 ++(+) n.p.
pH 4.5 ++ n.p.
pH 4 + n.p.
Sabouraud liquid medium 37 C
aerobiosis pH 5.5 +++ ++(+)
pH 5 +++ ++
pH 4.5 +++ n.p.
pH 4 ++(+) n.p.
Sabouraud agar medium 30 C
aerobiosis (spread plating) -I--H- -
aerobiosis (inclusion) -I--H- -
anaerobiosis (inclusion) n.p. -
MRS agar medium 30 C
aerobiosis (spread plating) -I--H- ++ (48h iti 1mm)
anaerobiosis (spread plating) +/- `- (24h)/+++ (48h 0 2mm)
MRS agar medium 37 C
aerobiosis (spread plating) -I--H- -H-F (24h 0 1mm)
aerobiosis (spread plating) - -H-F (24h 0 2mm)
Table 5 Growth of C. alb/cans 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. alb/cans 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).
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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 alb/cans
Growth tests in which Candida alb/cans ATCC 90028 was inoculated (104 cell/m1)
in liquid medium
inoculated with the strain L fermentum (103 cell/m1) 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. alb/cans 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.
alb/cans and L fermentum is 10 to
1 respectively, a MCB999 after 24 hours of incubation and a MCB99999 after 48
hours of contact are
obtained.
b) The specific inhibition, induced by L fermentum LF5 against C. alb/cans, 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. alb/cans 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.
alb/cans in a liquid medium even after several passages in a culture thereof
starting from MOB (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 Kruse/
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
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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. alb/cans
ATCC 90028
By microscope observation of the co-growth in LF5 and C. alb/cans 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. alb/cans ATCC 90028, inoculated into the fermented broth
from LF5 (supernatant)
were performed. The supernatant represents the culture medium in which L.
fermentum LF5 (106 cell/ml),
under optimum conditions for the lactobacillus, was developed and then cell
deprived through filtration
(Table 6).
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!'''''''''''''''' 244-svperrit:sti a at ha
irvotilig awl Bitra i iitm: 1 .: i
1 i.NOCULUM 10000 calfirrti of C a/bit-aro
--......,... ___________________________ --------',------:------- --....i.
,..i=-, io r0:1 __________ ao pern want ..':ri 5 ro; supernatant + 5 nil
TAR$
tosiitArjr* 24 '.F of goz.v4 1 0 P-i: of gri,,t,44 i ;e:4 h af 96,40 1
s.S.# fl of grok40
-4 ..............................................
T.n I
= ....1 ...
i 5
......................... [
4/lit-sopirilateint harvesting and filtration
t c-c Lulu hg: -i oot.) c&i,'s-t; 0 r: albi.pori ''
_______________________________________________________________ I
mi supernatant 1::in 5 mi supernatant + 5 tTi1 keli.i$.
..=:DilLit.1,0:-.G 24: ifµT Of WOVV.rti .4. h sv gsKswth ====4
h csiglawi:f
.................................................... ¨I-
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 (5.7x106
cell/m1).
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. alb/cans 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
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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 anti-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. alb/cans ATCC 90028 in agar medium, seeded by spread plating
(104 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 alb/cans, average
age of 32.7 years (range 18-
64 years) randomly assigned to two groups, which resulted homogeneous, each of
50 patients, treated
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with placebo o LF5 dosed at 109 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
Parameter Placebo LAB (LF5) Statistics
Positive/negative Baseline 50/0 50/0 -
3 Days 14/36 2/48 0.002**1
Final 40/10 5/45 <0.001'1
Chi square 164.967 122.750***
Eradication Failure 14 2 44.673'2
Relapse 26 3
Success 10 45
**P<0.01; '0.001
1Fisher
2Mann-Whitney
Table 7b: Patient's opinion about efficacy
Opinion Placebo LAB (LF5) Mann-Whitney1
Excellent 4 42 53.322***
Good 6 3
Acceptable 26 3
None 14 2
'0.001
1Chi-square approximation
4.1 Tolerability study
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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
Opinion Placebo LAB (LF5) Mann-Whitney1
Tolerability -
Excellent 49 47 0.002**1
Good 3 1 1.031
Acceptable 0 0
None 0 0
Adverse effects 0.6172
Present 1 3
Adverse 49 47
1Chi-square approximation
2 Fisher's exact test
The strain LF5 has an efficacy and tolerability profile in the treatment of
vaginal colonization by Candida
alb/cans 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 109CFU
Excipients:
Medium-chain triglycerides 1.964 mg
Silica (Aerosil 300) 36 mg
Shell composition:
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Gelatin F. U. 400 mg
Glycerol F.U. 200 mg
Di methyl polysiloxane 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 109 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
Di methyl polysiloxane 1000 90 mg
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
109 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.
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Table 8a Results of the study for the LF5 efficacy compared with placebo
Parameter LAB (LF5) Miconazole Statistics
Positive/negative Baseline 50/0 50/0 -
3 days 2/48 3/47 1.0001
Final 5/45 8/42 0.5541
Chi square 122.750 110.462***
Eradication Failure 2 3 0.774'2
Relapse 3 5
Success 45 42
'0.001
1Fisher
2Mann-Whitney
Table 8b: Patient's opinion about efficacy
Opinion LAB (LF5) Miconazole Mann-Whitney1
Excellent 40 38 53.322***
Good 5 4
Acceptable 3 5
None 1 3
'0.001
1Chi-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
Opinion LAB (LF5) Miconazole Mann-Whitney1
Tolerability -
Excellent 48 44 2.148
Good 1 3
Acceptable 1 3
None 0 0
Adverse events
Present 1 3 0.2692
Adverse 49 47
1Chi-square approximation
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2Fisher'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 alb/cans
colonization, average age of
34.0 years (range 17-65 years) with LAB dosed at 109 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 alb/cans 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
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The aim of the method is to verify if a given bacterium is able to inhibit the
broth growing of Gardnerella
vagina/is 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 pm of diameter
- THB (Todd Hewitt Broth Sigma ¨ Aldrich)
- Peptone saline solution (MET_INT 119-version in force)
- NaOH 30%
- Gardnerella vagina/is 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 conc.
The supernatant is then filtered through 0.2 pm filters and stored at a
temperature of 4-5 C.
Preparation of G. vagina/is
A pellet of G. vagina/is 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:
THB 10m1 9.5m1 9m1 8m1 6m1
MRS Oml 0.5m1 1m1 2m1 4m1
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Bacterial supernatant Oml 0.5m1 1m1 2m1 4m1
(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. vagina/is
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
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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)
Miconazole C. parapsilosis C. albicans C. tropicalis C. krusei
C. glabrata
(pg/ml) ATCC 22019 ATCC 10231 ATCC 750 ATCC 6258 ATCC 2001
0 1.610 1.912 1.972 1.987 2.246
1 1.515 1.906 1.950 1.990 2.260
2.50 1.115 1.910 1.927 1.900 2.260
5.00 0.975 1.901 1.908 1.893 2.246
50 0.894 1.905 1.846 1.994 2.239
250 0.701 1.916 1.942 2.004 2.212
300 0.415 1.889 1.937 1.999 2.215
500 0.314 1.863 1.903 1.988 2.213
1.000 0.295 1.464 1.931 1.972 2.198
Table 10
MIC (minimum inhibitory concentration) of fluconazole from E_test (range 0.016-
256 pg/ml). *Ip0.05
Candida species MIC (pg/ml)
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Candida parapsilosis ATCC 22019 1*
Candida alb/cans 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
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CNCM 1-789) was used in women with acute \NC, but without any correlation with
a specific species of
Candida and not in the simultaneous presence of bacterial infection due to
Gardnerella vagina/is, 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 study. J Clin
Gastroenterol. 2012;46
Suppl:S73-80).
8. Inhibition of Gardnerella vagina/is by L. fermentum LF5
This study was conducted in order to assess the ability of L fermentum LF5 to
directly inhibit Gardnerella
vagina/is in vitro, so that to evaluate the potential efficacy thereof even in
the event of mixed vaginal
infections or By.
Materials and methods
Bacterial strains and growth conditions.
The strain Gardnerella vagina/is being used was purchased from the American
Type Culture Collection
(ATCC). Before the experiment, G. vagina/is 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. vagina/is.
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. vagina/is. 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 (0D600). 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.
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Results
The in vitro inhibition results of LF5 against G. vagina/is 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.
vagina/is of even 80% (Tab. 9).
Table 11
L fermentum LF5 - biological activity against G. vagina/is.
LF5 supernatant (0.5, 1 and 2 mL) is able to inhibit the growth of G.
vagina/is even after 24 hours, but the
best inhibition is after 48 hours. Positive control = G. vagina/is - growth in
an optimal medium plus an
equal volume of MRS (5%, 10% and 20%) (n = 3).
24h incubation 48h incubation
Sample OD = 600 nm
+5% +10% +20% +5% +10% +20%
G. vaginalis 1.379 1.542 1.557 1.384 1.554 1.562
+ MRS (%) 0.01 0.02 0.015 0.014 0.01 0.016
G. vaginalis 0.764 0.645 0.843 0.562 0.336
0.275
+ LF5 (%) 0.008 0.006 0.007 0.006 0.004 0.003
The study shows that L. fermentum LF5 is able to exert a fundamental
inhibitory activity against G.
vagina/is, thus revealing a multipurpose application. The above-cited data
show that the strain LF5 is also
useful for the treatment of vulvovaginal candidiasis (\NC), due to different
strains, among which, inter alia,
Candida albicans; accordingly it can be used even in the clinical management
of mixed vaginitis.
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