U.S. patent application number 15/265706 was filed with the patent office on 2017-01-19 for effervescent composition in solid form for use in vaginal applications for the treatment of vaginal infections.
The applicant listed for this patent is PROBIOTICAL S.P.A.. Invention is credited to Giovanni MOGNA, Luca MOGNA, Gian Paolo STROZZI.
Application Number | 20170014335 15/265706 |
Document ID | / |
Family ID | 43975441 |
Filed Date | 2017-01-19 |
United States Patent
Application |
20170014335 |
Kind Code |
A1 |
MOGNA; Giovanni ; et
al. |
January 19, 2017 |
EFFERVESCENT COMPOSITION IN SOLID FORM FOR USE IN VAGINAL
APPLICATIONS FOR THE TREATMENT OF VAGINAL INFECTIONS
Abstract
The present invention relates to an effervescent composition in
solid form for use in vaginal applications for the treatment of
vaginal infections.
Inventors: |
MOGNA; Giovanni; (NOVARA
(NO), IT) ; STROZZI; Gian Paolo; (NOVARA (NO),
IT) ; MOGNA; Luca; (NOVARA (NO), IT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PROBIOTICAL S.P.A. |
Novara (NO) |
|
IT |
|
|
Family ID: |
43975441 |
Appl. No.: |
15/265706 |
Filed: |
September 14, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13982255 |
Nov 12, 2013 |
9492377 |
|
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PCT/IB2012/000095 |
Jan 24, 2012 |
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15265706 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/0034 20130101;
A61K 47/02 20130101; A61K 9/2054 20130101; A61P 33/02 20180101;
Y02A 50/473 20180101; A61K 47/12 20130101; A61P 15/02 20180101;
A61K 35/742 20130101; A61P 31/10 20180101; A61K 9/205 20130101;
A61K 9/0007 20130101; A61P 31/04 20180101; A61P 31/22 20180101;
A61K 35/747 20130101; Y02A 50/30 20180101 |
International
Class: |
A61K 9/46 20060101
A61K009/46; A61K 9/00 20060101 A61K009/00; A61K 35/747 20060101
A61K035/747; A61K 47/12 20060101 A61K047/12; A61K 47/02 20060101
A61K047/02 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 28, 2011 |
IT |
MI2011A000107 |
Mar 1, 2011 |
IT |
MI2011A000316 |
Claims
1-14. (canceled)
15. A medicament comprising an effervescent composition in solid
form comprising: an acid-base system comprising an organic acid and
a salt of the carbonate and/or bicarbonate anion; said salt being
present in an amount comprised from 1 to 15% by weight, relative to
the total weight of the composition, a mixture comprising
microcrystalline cellulose and arabinogalactan, at least one
probiotic bacterial strain having the ability to reduce and/or
eliminate 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 Haemophilus ducreyi, the
effervescent composition being formulated for treatment of a
vaginal infection.
16. The medicament in accordance with claim 15, wherein said
composition is in the form of a tablet, ovule, lozenge or
granules.
17. The medicament in accordance with claim 15, wherein the salt of
carbonate and/or bicarbonate anion is present in an amount
comprised from 3 to 13% by weight, relative to the total weight of
the composition.
18. The medicament in accordance with claim 15, wherein the
acid-base system consists of sodium bicarbonate and citric acid,
and wherein the sodium bicarbonate is present in an amount
comprised from 3 to 13% by weight, relative to the total weight of
the composition.
19. The medicament in accordance with claim 15, wherein said at
least one probiotic bacterial strain belongs to at least one
species selected from the group consisting of: Lactobacillus
plantarum, Lactobacillus pentosus, Lactobacillus casei,
Lactobacillus casei ssp. paracasei, Lactobacillus rhamnosus,
Lactobacillus acidophilus, Lactobacillus delbrueckii, Lactobacillus
delbrueckii ssp. bulgaricus, Lactobacillus delbrueckii ssp.
delbrueckii, Lactobacillus fermentum, Lactobacillus gasseri,
Lactobacillus reuteri, Bifidobacterium longum, Bifidobacterium
bifidum, Bifidobacterium breve, Bifidobacterium animalis ssp.
lactis, Bifidobacterium adolescentis, Bifidobacterium
pseudocatenulatum, Bifidobacterium catenulatum or Bifidobacterium
infantis; said at least one bacterial strain is preferably selected
from among: Lactobacillus salivarius CRL 1328, Lactobacillus
paracasei CRL 1289, Lactobacillus gasseri CRL 1259, Lactobacillus
crispatus CRL 1251, Lactobacillus crispatus CRL 1266, Lactobacillus
acidophilus CRL 1294, Lactobacillus paracasei LPC 00, Lactobacillus
plantarum LP 02 and Lactobacillus fermentum LF 10.
20. A medicament comprising an effervescent tablet in solid form
comprising: an acid-base system comprising sodium bicarbonate and
citric acid; said bicarbonate being present in an amount comprised
from 1 to 15% by weight, relative to the total weight of the
composition, a mixture comprising microcrystalline cellulose and
arabinogalactan in a ratio by weight comprised from 1:1 to 3:1, at
least one probiotic bacterial strain having the ability to reduce
and/or eliminate 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 Haemophilus ducreyi, the
effervescent composition being formulated for treatment of a
vaginal infection.
Description
[0001] The present invention relates to an effervescent composition
in solid form for use in vaginal applications for the treatment of
vaginal infections.
[0002] It is well known that the composition of intestinal and
urogenital microflora represents a critical point for an
individual's health and wellbeing. The vaginal ecosystem consists
of epithelial cells that line the vagina and uterus, glandular
cells that secrete into the lumen of the organ and complex
bacterial flora represented by different species of
microorganisms.
[0003] These microorganisms have the ability to ferment glycogen
originating from the decomposition of parabasal cells of the
eutrophic vaginal mucosa, with a consequent production of lactic
acid, whose final effect is the establishment and maintenance of an
acidic vaginal environment (with pH values of 4-4.5 under
physiological conditions).
[0004] The H.sup.+ deriving from lactic acid contribute to the
formation of hydrogen peroxide. This molecule is toxic for a large
number of bacterial species which lack the enzyme catalase. At the
level of vaginal secretions, concentrations of 0.75-5 .mu.g/ml are
easily reached and are abundantly sufficient in order for the toxic
effect co be expressed.
[0005] The combined action of hydrogen peroxide, uterine peroxidase
(produced by the cervix and endometrium) and Cl.sup.+ and I.sup.+
ions also limits bacterial growth by directly activating
polymorphonucleates, which exert a bactericidal action in
epithelial intercellular spaces. In women, due to a variety of
exogenous and endogenous factors, such as the intake of
antibiotics, states of stress, hormonal modulations associated with
pregnancy and the menstrual cycle or the intake of high
concentrations of estrogen, an imbalance frequently occurs in the
vaginal ecosystem. The alteration in the balance of the vaginal
ecosystem leads to a prevalence of so-called "opportunistic"
microorganisms (e.g. Candida albicans and glabrata) and/or
pathogenic microorganisms (e.g. Neisseria gonorrheae and
Trichomonas vaginalis) which can lead to candidiasis, vaginitis or
forms of vaginosis.
[0006] Epidemiological data show that vaginal infections today
affect over a billion women worldwide every year, with serious
repercussions from a socioeconomic standpoint. The therapy
generally adopted is an antibiotic and/or fungicidal one, which
usually gives good results in the shore term, but shows to be
incapable of preventing recurrent infections due to the ever
increasing resistance of pathogens. Moreover, not all subjects who
need to be treated are able to undertake and tolerate an antibiotic
or fungicidal therapy.
[0007] The use of microorganisms capable of restoring the correct
composition of vaginal microflora is known. However, the mode of
delivery of the microorganisms into the vaginal environment
represents a very critical factor. It is known, for example, that
soft gelatin capsules, usually known as soft gels, entail
suspending the probiotic component in an oily matrix which, after
the product's application, tends to constitute a physical barrier
capable of slowing down or almost totally inhibiting the capacity
of said probiotic component to colonize the vaginal mucosa.
Furthermore, the oily matrix could exert a toxic effect on the
microorganisms, to such a degree as to considerably reduce the
number and viability thereof.
[0008] For example, the capsules may take a relatively long time to
dissolve and in any case they do not assure an adequate dispersion
of the active ingredient throughout the vaginal environment.
[0009] For example, hydrophilic suspensions, or hydrogels, entail a
mode of application that is neither easy nor very comfortable. In
fact, the subjects must remain lying down for at least 20-30
minutes after applying them so as to prevent the product from
leaking out.
[0010] Finally, traditional vaginal tablets pose a problem of high
mortality of the microorganisms during their manufacture and
moreover they are not able to ensure an adequate distribution of
microorganisms in the vaginal environment.
[0011] Thus, there remains a need to have a composition capable of
delivering probiotic bacteria into the vaginal environment and
ensuring a complete dispersion/distribution thereof.
[0012] Moreover, there remains a need to have a pharmaceutical form
that can be easily administered and is practical to use.
[0013] In particular, there remains a need to have a composition
which represents a valid alternative to antibiotic and/or
fungicidal therapy and at the same represents an improvement over
the known forms of administration.
[0014] The Applicant has surprisingly found that a probiotic
formulation in the form of a suitably prepared solid composition is
capable of solving the problems persisting in the prior art.
[0015] The subject matter of the present invention is an
effervescent composition in solid form, as set forth in the
appended independent claim.
[0016] Other preferred embodiments of the present invention are set
forth in the appended dependent claims.
[0017] Table 1 shows the bacterial strains tested by the Applicant
and which form the subject matter of the present invention.
[0018] Table 2 shows an example of a composition according to the
invention.
[0019] Table 3 shows the mortality data for the probiotic bacteria
according to the different pressures exerted and stability of the
tablets (expressed as the half-life of the bacterial load) after 2
years of storage thereof at 25.degree. C.
[0020] Table 4 shows a composition that is not in accordance with
the present invention.
[0021] Table 5 shows a composition in tablet form that is not in
accordance with the present invention.
[0022] The Applicant conducted lengthy, intense experimental
research activity with the aim of identifying the optimal
qualitative and quantitative composition for preparing the
composition of the present invention, as well as the operating
conditions for the preparation thereof.
[0023] The composition of the present invention is an effervescent
composition. The effervescence is due to the formation of carbon
dioxide which occurs when an acid-base system comprising an organic
acid and a salt of the carbonate and/or bicarbonate anion comes
into contact with the water/moisture present in the vaginal cavity.
The acid-base system is capable of maintaining the intravaginal pH
stable within an interval comprised from 3 to 5.5, preferably from
4 to 5. Advantageously, the intravaginal pH is comprised from 4.2
to 4-5.
[0024] The composition of the present invention is an effervescent
composition in solid form. In a preferred embodiment, the organic
acid is in solid form, preferably a powder or granules, and the
salt of the carbonate and/or bicarbonate anion is in solid form,
preferably a powder or granules.
[0025] In a preferred embodiment, the effervescent composition in
solid form is in the form of a tablet, ovule, lozenge or
granules.
[0026] The organic acid is selected from the group consisting or,
or alternatively comprising, citric acid, malic acid, tartaric
acid, fumaric acid, lactic acid and mixtures thereof. In a
preferred embodiment, the organic acid is citric acid.
[0027] The Applicant has found that the use of adipic and/or
ascorbic acid as the organic acid present in the aforesaid
acid-base system provokes a toxic effect on the microorganisms,
resulting in the mortality thereof already at the time of
preparation of the composition in solid form, for example as a
powder or granules. In the case of the preparation of a tablet,
said mortality occurs after the components are mixed to give a
composition in solid form, for example as a powder or granules, but
prior to compression. Therefore, the composition of the present
invention does not contain adipic acid and/or ascorbic acid.
[0028] The salt of the carbonate and/or bicarbonate anion is
selected from the group consisting of or, alternatively,
comprising: sodium carbonate, potassium carbonate, calcium
carbonate, magnesium carbonate, sodium glycine carbonate, sodium
bicarbonate, potassium bicarbonate, calcium bicarbonate, magnesium
bicarbonate, sodium lactate, potassium lactate, carbonate lactate
and mixtures thereof. Preferably, said salt is selected from the
group consisting of or, alternatively, comprising a salt of the
bicarbonate anion. In a preferred embodiment, the salt is sodium
bicarbonate.
[0029] In a preferred embodiment, the amount of the salt of the
carbonate or bicarbonate anion is comprised from 1 to 15% by
weight, relative to the total weight of the composition.
Preferably, it is comprised from 3 to 13% by weight, relative to
the total weight of the composition; even more preferably, it is
comprised from 4 to 12% by weight, relative to the total weight of
the composition. Advantageously, it is comprised from 5 to 10% by
weight, relative to the total weight of the composition.
[0030] In a preferred embodiment, the amount of sodium bicarbonate
is comprised from 1 to 15% by weight, relative to the total weight
of the composition. Preferably, it is comprised from 3 to 13% by
weight, relative to the total weight of the composition; even more
preferably it is comprised from 5 to 10% by weight, relative to the
total weight of the composition. Advantageously, it is comprised
from 5 to 10% by weight, relative to the total weight of the
composition.
[0031] In a preferred embodiment, the acid-base system consists of
citric acid and sodium bicarbonate in an amount by weight as
described above.
[0032] The Applicant has found that when the salt of the carbonate
or bicarbonate anion, e.g. sodium bicarbonate, is more than 15% by
weight, relative to the total weight of the composition, a toxic
effect is manifested against the microorganisms due to an osmotic
effect.
[0033] The amount of organic acid to be used in the acid-base
system is calculated once the amount of the salt of the carbonate
or bicarbonate anion used in accordance with the above-described
concentrations has been determined. The amount of organic acid used
is stoichiometric and is a function of the salt used and solely
serves the purpose of forming carbon dioxide. In the presence of
said salt, the organic acid is capable of developing effervescence,
due to the formation of carbon dioxide, after hydration inside the
vaginal cavity.
[0034] The effervescent tablet, suitably formulated, is capable of
delivering a large population of lactic bacteria for rapid
colonization of the entire epithelium and vaginal mucosa. Thanks to
the pH-controlled and stabilized effervescent system, the lactic
bacteria are delivered into the vaginal cavity in a good
physiological condition which enables them to grow and
multiply.
[0035] Advantageously, the composition of the present invention, in
the form of an effervescent tablet, is capable of ensuring an
optimal distribution of the bacteria over the vaginal mucosa.
Moreover, said composition is capable of ensuring and maintaining
an anaerobic or microaerophilic environment inside the vaginal
cavity thanks to the formation of carbon dioxide.
[0036] The Applicant has found that a mixture comprising or,
alternatively, consisting of [microcrystalline
cellulose:arabinogalactan], preferably in a ratio by weight of 1:1
to 3:1, is capable of preserving the number and viability of the
cells of the microorganisms when it is desired to obtain a tablet
from the composition in powder form.
[0037] In a preferred embodiment, the composition of the present
invention comprises a mixture containing [microcrystalline
cellulose:arabinogalactan] in a ratio by weight of 1:1 to 3:1.
[0038] Preferably, arabinogalactan derived from plants is used.
Advantageously, arabinogalactan derived from larch (e.g.
FiberAid.RTM., from the Larex.RTM. range of products distributed by
Lonza Inc. USA) is used.
[0039] The Applicant has likewise found that using corn starch as
the aggregating substance in the composition of the present
invention provides unsatisfactory results when the composition in
powder form is used to prepare tablets, since the tablets obtained
following compression of the powder are capped.
[0040] A mixture comprising or, alternatively, consisting of
[microcrystalline cellulose:arabinogalactan] in a ratio by weight
of 1:1 Co 1:3, on the contrary, allows tablets to be obtained with
no occurrence of capping. Therefore, the tablets obtained from the
composition of the present invention do not contain corn starch. In
place of corn starch, a mixture containing [microcrystalline
cellulose:arabinogalactan] is used in a ratio by weight preferably
comprised from 2:1 to 3:2. The microcrystalline cellulose can be
either partly or completely replaced by cellulose in powder form.
Both microcrystalline cellulose and cellulose in powder form are
present in the list of food additives as E460.
[0041] Arabinogalactan is a biopolymer consisting of arabinose and
galactose monomers. There are two classes of arabinogalactans:
those derived from plants and chose of microbial origin.
[0042] The microcrystalline cellulose (or cellulose in powder form)
is present in amount comprised from 2 to 45% by weight, relative to
the total weight of the composition, preferably from 5 to 25%.
[0043] The arabinogalactan is present in amount comprised from 5 to
30% by weight, relative to the total weight of the composition,
preferably from 10 to 20%.
[0044] Preferably, the composition of the present invention can
further comprise at least one additional component selected from
the group comprising: sodium carboxymethyl cellulose, anhydrous
calcium hydrogen phosphate and hydroxypropyl methylcellulose.
[0045] Said at least one additional component is present in the
composition of the present invention in a total amount that ranges
from 3 co 70% w/w, more preferably from 6 to 40% w/w, and even more
preferably from 10 to 25% w/w, relative to the total weight of the
composition.
[0046] According to a preferred embodiment of the invention, the
composition of the present invention further comprises magnesium
stearate in an amount of 0.5% to 7% w/w, preferably 1% to 3.5% w/w,
relative to the total weight of the composition.
[0047] According to a preferred embodiment of the invention, the
composition of the present invention can further comprise silicon
dioxide in an amount of 0.5% to 4% w/w, preferably 1% to 2% w/w,
relative to the total weight of the composition.
[0048] According to a preferred embodiment of the invention, the
composition of the present invention can further comprise sucrose
palmitate in an amount of 0.5% to 7% w/w, preferably 1% to 3.5%
w/w, relative to the total weight of the composition.
[0049] According to a preferred embodiment of the invention, the
composition of the present invention can further comprise a mixture
of glycerides (saponifiable fats) in an amount of 0.5% to 10% w/w,
preferably 2% to 7% w/w, relative to the total weight of the
composition.
[0050] According to a preferred embodiment of the invention, the
composition can further comprise from 0.5% to 30% w/w, preferably
from 1% to 5% w/w, of sucrose ester, the percentages being
expressed relative to the total weight of the composition.
[0051] The composition of the invention is for vaginal applications
for the treatment of vaginal infections such as vaginitis,
vaginosis, candidiasis, gonorrhoea, herpes and venereal ulcer.
[0052] In a preferred embodiment, the composition is in the form of
a tablet. Alternatively, the tablet can be coated with one or more
polymer materials known in the art.
[0053] With regard to the microbial population of the formulations
according to the present invention, it is advantageous to use
probiotic microorganisms, used individually or in a mixture, even
one consisting of different genera or species.
[0054] Probiotic microorganisms are by definition microorganisms,
prevalently bacteria of human origin, which are capable of inducing
beneficial effects if taken regularly in a sufficient quantity and
for an adequate amount of time.
[0055] The composition of the present invention comprises at least
one probiotic bacterial strain having the ability to reduce and/or
eliminate 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, Herpex simplex and Haemophilus ducreyi.
[0056] Preferably, said bacterial strain belongs to at least one
species selected from the group consisting of: Lactobacillus
plantarum, Lactobacillus pentosus, Lactobacillus easel,
Lactobacillus casei ssp. paracasei, Lactobacillus rhamnosus,
Lactobacillus acidophilus, Lactobacillus delbrueckii, Lactobacillus
delbrueckii ssp. bulgaricus, Lactobacillus delbrueckii ssp.
delbrueckii, Lactobacillus fermentum, Lactobacillus gasseri,
Lactobacillus reuteri, Bifidobacterium longum, Bifidobacterium
bifidum, Bifidobacterium breve, Bifidobacterium animalis asp.
lactis, Bifidobacterium adolescentis, Bifidobacterium
pseudocatenulatum, Bifidobacterium catenulatum or Bifidobacterium
infantis.
[0057] Among the above-mentioned bacterial species, the bacterial
strains listed in Table 1 have demonstrated to be particularly
preferred.
[0058] In a preferred embodiment, the bacterial strains are
selected from among: Lactobacillus salivarius CRL 1328,
Lactobacillus paracasei CRL 1289, Lactobacillus gasseri CRL 1259,
Lactobacillus crispatus CRL 1251, Lactobacillus crispatus CRL 1266,
Lactobacillus acidphilus CRL 1294, Lactobacillus paracasei LPC 00,
Lactobacillus plantarum LP 02 and Lactobacillus fermentum LF
10.
[0059] In a preferred embodiment, the composition of the present
invention, preferably in the form of a tablet, comprises at least
two strains selected from the above-mentioned group.
[0060] In a preferred embodiment, the composition of the present
invention, preferably in the form of a tablet, comprises at least
three strains selected from the above-mentioned group.
[0061] In a preferred embodiment, the composition of the present
invention, preferably in the form of a tablet, comprises at least
four strains selected from the above-mentioned group.
[0062] The strains selected from among: Lactobacillus salivarius
CRL 1328, Lactobacillus paracasei CRL 1289, Lactobacillus gasseri
CRL 1259, Lactobacillus crispatus CRL 1266 and Lactobacillus
fermentum LP 10 have demonstrated to foe advantageous.
[0063] In the tablets according to the invention, the
microorganisms, preferably used in the form of a lyophilized
culture having a viable count generally comprised from 10 to 200
billion colony forming units (CFU)/gram, are preferably present in
an amount of 0.5 to 20% w/w, preferably 1 to 15% w/w, even more
preferably 3 to 10% w/w, relative to the total weight of the
tablet.
[0064] In one of the preferred embodiments, in order to enhance the
probiotic effectiveness of the formulations according to the
present invention, specific prebiotic components are introduced
into the powder mixture, so that a symbiotic composition is
obtained. The prebiotic component is generally a non-digestible
mixture of a saccharidic nature, at least partially soluble in
water or in an aqueous solution, which stimulates the growth and/or
activity of one or more probiotic bacterial strains as described
above. Among these prebiotic agents, dietary fibres are
preferred.
[0065] Preferably, said prebiotic fibre is selected from the group
comprising: fructo-oligosaccharides (POS), galacto-oligosaccharides
(GOS), trans-galactooligosaccharides (TOS), xylo-oligosaccharides
(XOS), chitosan oligosaccharides (COS), .alpha.-galactosides (such
as raffinose and stachyose), pectins, gums, partially hydrolyzed
gums, inulin, psyllium, acacia, carob, oat or bamboo fibre, citrus
fibres and, in general, fibres containing a soluble and an
insoluble portion, in a variable ratio to each other.
[0066] Advantageously, said prebiotic fibre is selected from among
fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS) and
xylo-oligosaccharides (XOS). These fibres are not used by yeasts of
the Candida genera, which thus lends a competitive advantage to the
bacterial strains present in the composition of the present
invention.
[0067] Preferably, the prebiotic component is present in the
composition in an amount of 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 total weight of the composition.
[0068] In a preferred embodiment, the composition according to the
invention can contain additional active components, e.g. vitamins,
minerals, vegetable extracts or other compounds with an effect that
is synergistic with or complementary to that of the population of
microorganisms present in the formulations according to the
invention.
[0069] Preferably, said additional active components are present in
the composition in an amount of 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 total weight of the composition.
[0070] The combination of technological excipients of compression
and effervescence according to the present invention assures an
adequate cohesiveness of the powders of the tablet and brings about
the desired kinetics of release of the active ingredients after the
intake thereof.
[0071] The mixing of the various components, generally in powder
form, can be done by adding the components in any order whatsoever,
taking care to add the culture of microorganisms, preferably in the
form of a lyophilizate, as the last ingredient, to prevent the
possibility that an excessive mixing time may induce a mechanical
shock in the wall of the bacterial cells, with consequent suffering
and reduced stability of the final product.
[0072] Formulations produced in accordance with the method of the
present invention are characterized by excellent flowability, a
parameter that favours uniform loading of the dies (i.e. the space
dedicated to accommodating the specific amount of powder to be
compressed) in the compression machines. Said compression machines
typically comprise a device for loading the dies (generally
represented by a hopper for delivering the powder mixture) and a
metal disk of suitable radius, in whose thickness the dies
themselves are obtained; these are present in variable number
depending on the machine, and the step of compressing the powder
takes place therein, thanks to the simultaneous, synergistic
movement of two moving parts of the machine, defined as
punches.
[0073] During the compression step, the lower punch defines, with
its position, the volume of the die (and thus the amount of powder
loaded each time), whereas the upper punch, positioned outside the
die the during loading thereof with the powder mixture, is able to
be lowered during the compression step and enter into contact with
the powder present in the die, thus imposing, thanks to the force
exerted, a more or less marked mechanical deformation on the
particles making up the powder. At the end of the compression step,
the upper punch rises, thus moving away from the die, and
subsequently the lower punch also rises, expelling the newly formed
tablet from the die.
[0074] Said compression machines further comprise a number of
devices for controlling the main operating parameters (position of
the lower punch at the start of the compression cycle, lowest
position reached by the upper punch during the compression cycle,
rotation speed of the disk in which the dies are present and so
forth). There exist alternative and rotary compression machines,
according to the machine's overall operating modes and,
consequently, the number of tablets it is able to produce in a
given unit of time. Tablets can thus be produced using compression
machines known in the art and using punches of varying shape,
preferably double-radius round convex and oval punches.
[0075] The compression force applied during manufacture is
evaluated by quantifying, with a hardness tester, the force
required to break a tablet. Said force is measured in Kp
(Kiloponds) (1 Kp=9.807 Newtons (N)). The negative effect of
compression on the population of microorganisms is manifested both
when the applied forces are high, for example in tablets with a
hardness of 10-12 Kp, and when they are low, such as, for example,
in tablets with a hardness of 5-6 Kp. The disadvantage of using
high forces is a larger reduction in the number of microorganisms,
whereas with low forces a reduced cohesion of the tablet is
generally obtained. Advantageously, the tablets of the present
invention show a compression force comprised from 5 to 12 Kp,
depending on the type of tablet it is desired to obtain.
[0076] In manufacturing tests on tablets containing L. paracasei
LPC 00, L. acidphilus LA 02 and L. salivarius CRL 1328 (table 2),
the Applicant obtained very positive results both in terms of
survival and in terms of subsequent stability.
[0077] In particular, a mortality ranging from 4 to 15% was
observed when the compression force applied was such as to obtain
tablets with a hardness comprised from 5 to 6 Kp and a mortality
ranging from 10 to 25% when the compression force applied was such
as to obtain tablets with a hardness comprised from 10 to 12 Kp.
Another advantage of the method according to the invention is that
this enables the production of sufficiently cohesive tablets even
at low compression forces, for example tablets with a hardness of
5-6 Kp.
[0078] The tablets prepared in accordance with the present
invention were tested in order to evaluate the disintegration time
according to the European Pharmacopoeia, Ed. X, ref. 2.9.1. The
friability of uncoated tablets was tested in accordance with ref.
2.9-7 and the resistance to crushing according to ref. 2.9.8.
[0079] Table 2 shows an example of a composition in accordance with
the invention.
[0080] Table 3 shows the mortality data for the probiotic bacteria
(composition shown in table 2) according to the different pressures
exerted and the stability of the tablets (expressed as the
half-life of the bacterial load) after 2 years of storage thereof
at 25.degree. C. The tablets obtained with the formulations
according to the invention had a thickness of about 7.7 mm in the
case of tablets with a high degree of hardness and about 8.4 mm in
the case of tablets with a low degree of hardness.
[0081] In table 3, stability is expressed as the half-life of the
probiotic component of the formulation, i.e. the time that elapses
before the initial viable cell count is halved.
[0082] The following were also measured:
(1) Number of viable cells 1.times.10.sup.9 CFU/g of powder
expected at time zero (2) Number of viable cells 1.times.10.sup.9
CFU/g of powder found at time zero (3) % mortality due to
compression (4) Number of viable cells 1.times.10.sup.9 CPU/tablet
after 2 years at 25.degree. C. (5) half-life in days
[0083] The above data were obtained by evaluating the number of
probiotic bacteria present in the powder composition and in the
composition after compression using the viable plate count method,
in accordance with methods known to persons skilled in the art.
[0084] In the case of the powder composition, an amount comprised
from 1 to 4 grams of sample was resuspended in a suitable volume of
a sterile liquid medium, generally a 0.85% sodium chloride saline
solution, to which bacteriological peptone is added in a proportion
of 1 g/litre.
[0085] After dissolution of the powder and subsequent
homogenization with a suitable piston-driven instrument, the number
of cells/ml is reduced by means of subsequent base 10 serial
dilutions.
[0086] In practice, 1 ml of the most concentrated dilution is
transferred each time, using a sterile pipette, into 9 ml of
diluent; this operation is carried out a number of times that is
sufficient to bring the quantity of microorganisms present per ml
of diluent to a number comprised from 10 to 300, so that following
their transfer into a Petri dish and the addition of a suitable
agarized culture medium they form separate, and thus countable,
colonies.
[0087] In the case of the tablets, a protocol was observed which
provides for prior crushing of three to five tablets and
continuance of the analysis as described above for the powder
composition.
[0088] The experimental data demonstrate a low mortality both in
tablets with a high degree of hardness (approximately 16.4% of the
initial population) and in those with a lower degree of hardness
(approximately 8.2% of the initial population). A further
advantageous aspect of the composition of the present invention is
improved stability, over time, of the probiotic microorganisms
(probiotic component) present therein compared to the typical
stability of a prior art formulation. This advantage is due to the
use of a mixture containing [microcrystalline
cellulose:arabinogalactan] in a ratio by weight preferably
comprised from 1:1 to 3:1. This mixture is capable of greatly
limiting the mechanical damage caused to the probiotic bacterial
cells by the compression step and allows tablets to be obtained
with no occurrence of capping.
[0089] The Applicant has found that the half-lives of the probiotic
component in the powder composition and in the tablets of varying
hardness manufactured with a formulation according to the invention
are very similar, demonstrating the fact that the technological
components used are able to minimize the mechanical damage caused
by the compression step.
[0090] Advantageously, the tablets according to the invention
display a high rate of survival of the probiotic microorganisms
even following the compression step.
[0091] A further advantageous aspect is the ability to release
carbon dioxide as the tablet disintegrates, resulting in the
creation of an anaerobic or microaerophilic environment
particularly favourable to vaginal colonization by the probiotics
used.
[0092] The effervescence is further capable of assuring an adequate
and homogeneous distribution of the probiotic active ingredient in
the vaginal environment by increasing the effectiveness of
colonization.
[0093] The compositions of the present invention make it possible
to obtain tablets containing probiotic microorganisms which are
effective and stable over time, even at non-refrigerated
temperatures, because the components forming the composition have
been chosen and selected with the aim of reducing mortality due to
mixing and mortality due to compression without impairing the
cohesion of the powder. For this reason, the composition of the
present invention, preferably in the form of a tablet, contains
neither adipic acid nor ascorbic acid, the amount of the salt of
the carbonate or bicarbonate anion present is lower than 15% by
weight, relative to the total weight of the composition, and a
mixture of microcrystalline cellulose and arabinogalactan is used
instead of corn starch.
[0094] The Applicant arrived at these results starting off from a
composition that demonstrated to be inadequate (table 4).
[0095] The composition of table 4 shows: 80.8% mortality due to
mixing, 40.5% mortality due to compression and an overall mortality
(mixing+compression) of 88.6%. The composition of table 4 shows
marked mortality already at the powder level, prior to compression.
The pH of dissolution (in saline in a 1:10 ratio) is not a problem,
as it is equal to 5.22 for the first formulation and 5.53 for the
second one.
[0096] Adipic acid was found to be toxic. But this toxicity
accounts for only a certain portion of the mortality observed
(about 60% of the total mortality). Ascorbic acid was found to be
toxic. But this toxicity accounts for a further portion of
mortality. Removing ascorbic acid from the composition of table 4
reduces mortality from 73.8 to 26.7%. Adipic acid on its own is in
any case toxic (45.8% at pH 4.24); its toxicity seems to increase
in the presence of ascorbic acid. Ascorbic acid thus seems to show
a toxicity synergistic with that of adipic acid.
[0097] However, it is important to note that the sum of the
toxicity due to adipic acid and ascorbic acid does not account for
the overall mortality observed.
[0098] For this reason, the Applicant investigated the presence of
other components capable of having a toxic effect with the aim of
justifying the total toxicity observed in the composition of table
4.
[0099] Therefore, some other component (apart from pH, which was
already considered) must justify the remaining toxicity in the same
manner as adipic and ascorbic acid.
[0100] A degree of mortality was ascertained to be due to osmotic
pressure, given by the bicarbonate present in an excessive amount.
This mortality revealed to be equal to about 20%. Adipic acid,
ascorbic acid and the osmotic effects mediated by bicarbonate are
responsible for the toxicity observed. Bicarbonate in itself is not
toxic: it becomes so only because of the osmotic effect.
[0101] Therefore, a composition (table 5) was prepared without
adipic acid and ascorbic acid and with an amount of bicarbonate
anion (e.g. sodium bicarbonate) less than 15% by weight, relative
to the total weight of the composition.
[0102] For example, 84 mg of sodium bicarbonate constitutes 1
millimole and the CO.sub.2 that is released occupies a total volume
of 25.4 ml at 37.degree. C. and under atmospheric pressure. The
quantity of citric acid was chosen in such a manner as to ensure
complete release of the CO.sub.2 from the sodium bicarbonate at the
pH values observed after the tablet's dissolution.
[0103] The composition of table 5 showed problems when subjected to
a compression step in order to prepare the tablets.
[0104] The powder (table 5) showed poor cohesive properties due to
the use of corn starch in a formulation with a low moisture
content. All of the tablets output by the compression machine were
capped even before being passed through the deduster.
[0105] Following this compression test the Applicant proceeded to
replace corn starch with a mixture containing [microcrystalline
cellulose:arabinogalactan] in a ratio by weight preferably
comprised from 1:1 to 3:1.
TABLE-US-00001 TABLE 1 Deposit Deposit Selected strain number date
Depositor Lactobacillus salivarius DSM 24441 04.Jan.2011
Probiotical SpA CRL 1328 (under license from Cerela) Lactobacillus
paracasei DSM 24440 04.Jan.2011 Probiotical SpA CRL 1289 (under
license from Cerela) Lactobacillus gasseri DSM 24512 25.Jan.2011
Probiotical SpA CRL 1259 (under license from Cerela) Lactobacillus
crispatus DSM 24438 04.Jan.2011 Probiotical SpA CRL 1251 (under
license from Cerela) Lactobacillus crispatus DSM 24439 04.Jan.2011
Probiotical SpA CRL 1266 (under license from Cerela) Lactobacillus
acidophilus DSM 24513 25.Jan.2011 Probiotical SpA CRL 1294 (under
license from Cerela) Lactobacillus fermentum CNCM 21.Jul.1988
Probiotical LF 5 I-789 SpA Lactobacillus fermentum DSM 19187
20.Mar.2007 Probiotical LF 10 SpA Lactobacillus fermentum DSM 18298
24.May.2006 Probiotical LF 09 SpA Lactobacillus fermentum DSM 19188
20.Mar.2007 Probiotical LF 11 SpA Lactobacillus paracasei LMG
31.Jan.2002 Probiotical LPC 00 P-21380 SpA Lactobacillus plantarum
LMG 16.Oct.2001 Laboratorio LP 01 P-21021 Microbiologico Grana
Provolone SRL Lactobacillus plantarum LMG 16.Oct.2001 Laboratorio
LP 02 P-21020 Microbiologico Grana Provolone SRL Lactobacillus
pentosus DSM 21980 14.Nov.2008 Probiotical LPS 01 SpA Lactobacillus
acidophilus DSM 21717 06.Aug.2008 Probiotical LA 02 SpA
Lactobacillus rhamnosus DSM 16605 20.Jul.2004 Probiotical LR 04 SpA
Lactobacillus rhamnosus DSM 19739 27.Sep.2007 Probiotical LR 05 SpA
Lactobacillus rhamnosus DSM 21981 14.Nov.2008 Probiotical LR 06 SpA
Lactobacillus paracasei DSM 21717 06.Aug.2008 Probiotical LPC 08
SpA Lactobacillus delbrueckii DSM 22106 10.Dec.2008 Probiotical LDD
01 (MB 386) SpA (Steve Jones srl) Lactobacillus reuteri DSM 17938
BioGaia Lactobacillus reuteri DSM 23877 05.Aug.2010 Probiotical LRE
01 SpA Lactobacillus reuteri DSM 23878 05.Aug.2010 Probiotical LRE
02 SpA Lactobacillus reuteri DSM 23879 05.Aug.2010 Probiotical LRE
03 SpA Lactobacillus reuteri DSM 23880 05.Aug.2010 Probiotical LRE
04 SpA Lactobacillus salivarius DSM 22776 23.Jul.2009 Probiotical
LS03 SpA Lactobacillus plantarum LMG 16.Oct.2001 Laboratorio PR ci
P-21022 Microbiologico Grana Provolone SRL Lactobacillus plantarum
LMG 16.Oct.2001 Laboratorio 776/2 hi P-21023 Microbiologico Grana
Provolone SRL Lactobacillus pentosus LMG 16.Oct.2001 Laboratorio
9/1 ei P-21019 Microbiologico Grana Provolone SRL
TABLE-US-00002 TABLE 2 Composition mg/tablet Bacterial strains: L.
paracasei LPC 00, 58 L. acidphilus LA 02 and L. salivarius CRL 1328
with a concentration comprised from 20 to 100 .times. 10.sup.9
CFU/g) Fructo-oligosaccharides (FOS) 327 Arabinogalactan 250
Microcrystalline cellulose 270 Anhydrous calcium hydrogen
phosphaste 95 Mixture of glycerides 65 Citric acid 64 Sodium
bicarbonate 56 Insoluble dietary fibre 43 Sucrose palmitate 26
Silicon dioxide 26 Magnesium stearate 20 TOTAL (1 tablet) 1,300
TABLE-US-00003 TABLE 3 (1) (2) (3) (4) (5) Formulation powder 5.9
6.1 / 1.3 327 Table 2 tablets w/ / 5.6 8.2 1.2 328 low grade of
hardness (5-6 kp) tablets w/ / 5.1 16.4 1 292 high degree of
hardness (10-12 kp)
TABLE-US-00004 TABLE 4 inadequate composition Composition mg/tablet
Bacterial strains: (L. paracasei LPC 00, 291 L. acidophilus LA 02,
L. paracasei CRL 1289 with a concentration comprised from 20 to 100
.times. 10.sup.9/gram) Corn startch 335 Adipic acid 268 Sodium
bicarbonate 210 Ascorbic acid 100 Magnesium stearate 42 Anhydrous
lactose 34 Stearic acid 12 Silicon dioxide 8 TOTAL: (1 tablet )
1,300
TABLE-US-00005 TABLE 5 1,300 mg composition Components mg/tablet
Bacterial strains: (L. paracasei LPC 00, 199 L. acidophilus LA 02,
L. paracasei CRL 1328 with a concentration comprised from 20 to
10.sup.9/gram) Arabinogalactan 300 Galacto-oligosaccharides (GOS)
315 Corn startch 315 Insoluble dietary fibre 43 Citric acid 38
Magnesium stearate 34 Sodium bicarbonate 28 Silicon dioxide 18
Stearic acid 10 TOTAL (1 tablet) 1,300
* * * * *