U.S. patent application number 16/526715 was filed with the patent office on 2019-11-21 for therapy for use in the treatment of tumors, acquired immunodeficiency syndrome and leukemias by dual immune biostimulation.
The applicant listed for this patent is Chiara BENASSAI, Elena MOGNA, Vera MOGNA. Invention is credited to Giovanni MOGNA.
Application Number | 20190350989 16/526715 |
Document ID | / |
Family ID | 51136602 |
Filed Date | 2019-11-21 |
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United States Patent
Application |
20190350989 |
Kind Code |
A1 |
MOGNA; Giovanni |
November 21, 2019 |
THERAPY FOR USE IN THE TREATMENT OF TUMORS, ACQUIRED
IMMUNODEFICIENCY SYNDROME AND LEUKEMIAS BY DUAL IMMUNE
BIOSTIMULATION
Abstract
A composition for human and animal use as a therapy for the
treatment of tumors, acquired immunodeficiency syndrome and
leukemias is described. The composition, for human and animal use
as an antitumor agent, has a strain of bacteria Lactobacillus
reuteri LRE 03 DSM 23879 which is able to strongly stimulate the
production of pro-inflammatory cytokines (Th1) interferon
INF-gamma, the cytokines exhibiting a marked antitumor activity,
and/or a strain of bacteria Lactobacillus salivarius LS06 DSM 26037
which is able to strongly stimulate the production of dendritic
cells, the dendritic cells also exhibiting a marked antitumor
activity.
Inventors: |
MOGNA; Giovanni; (NOVARA,
IT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BENASSAI; Chiara
MOGNA; Elena
MOGNA; Vera |
Novara
Novara
Novara |
|
IT
IT
IT |
|
|
Family ID: |
51136602 |
Appl. No.: |
16/526715 |
Filed: |
July 30, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15307756 |
Oct 28, 2016 |
10413576 |
|
|
PCT/IB2015/000602 |
Apr 30, 2015 |
|
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16526715 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 31/18 20180101;
A61P 35/00 20180101; A61K 35/74 20130101; A61P 35/02 20180101; A61K
35/745 20130101; A61K 2035/115 20130101; A61K 35/747 20130101; A61K
36/886 20130101 |
International
Class: |
A61K 35/747 20060101
A61K035/747; A61K 36/886 20060101 A61K036/886; A61K 35/74 20060101
A61K035/74; A61K 35/745 20060101 A61K035/745 |
Foreign Application Data
Date |
Code |
Application Number |
May 5, 2014 |
IT |
MI2014A000816 |
Claims
1. A composition comprising a mixture consisting of: a strain of
bacteria belonging to the species Lactobacillus reuteri identified
as Lactobacillus reuteri LRE03 with deposit number DSM 23879,
deposited on 5 Aug. 2010 by Probiotical SpA at DSMZ--Deutsche
Sammlung von Mikroorganismen und Zellkulturen GmbH, and/or a strain
of bacteria belonging to the species Lactobacillus salivarius
identified as Lactobacillus salivarius LS06 with deposit number DSM
26037, deposited on 6 Jun. 2012 by Probiotical SpA at
DSMZ--Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH,
in an amount effective for antitumor chemotherapeutic treatment,
acquired immunodeficiency syndrome treatment and leukemia
treatment.
2. The composition according to claim 1, wherein said mixture has a
bacterial concentration comprised from 1.times.10.sup.8 UFC/g of
mixture to 1.times.10.sup.12 UFC/g of mixture.
3. The composition according to claim 1, wherein said mixture
comprises: a strain of bacteria Lactobacillus reuteri LRE03 DSM
23879 and a strain of bacteria Lactobacillus salivarius LS06 DSM
26037, in a weight ratio comprised from 1:5 to 5:1.
4. The composition according to claim 1, wherein said composition
further comprises an Aloe gel or a derivative thereof.
5. The composition according to claim 1, wherein said composition
further comprises a freeze-dried Aloe arborescens in an amount
comprised from 1 to 25% by weight, relative to the weight of the
composition.
6. The composition according to claim 1, wherein said composition
further comprises a tyndallized bacterial product of
Bifidobacterium lactis Bb 1 with deposit number DSM 17850,
deposited at DSMZ on 23 Dec. 2005, by BioMan S.r.l. Company.
7. The composition according to claim 6, wherein said tyndallized
bacterial product of the strain Bifidobacterium animalis ssp.
lactis Bb1 DSM 17850 is in an amount comprised from 10 to 50 mg/g
of the composition.
8. The composition according to claim 1, wherein said composition
further comprises one or more food grade excipients or pharma grade
excipients, and/or additives acceptable by the body, and/or
co-formulants acceptable by the body.
9. The composition according to claim 1, wherein said strain of
bacteria belonging to the species Lactobacillus reuteri and strain
of bacteria belonging to the species Lactobacillus salivarius are
in an amount effective to treat tumors in human and animal.
10. The composition according to claim 1, wherein said composition
has a bacterial concentration from 1.times.10.sup.9 UFC/g to
1.times.10.sup.11 UFC/g.
11. The composition according to claim 1, wherein said composition
comprises a strain of bacteria Lactobacillus reuteri LRE03 DSM
23879 and a strain of bacteria Lactobacillus salivarius LS06 DSM
26037, in a weight ratio from 1:3 to 3:1.
12. The composition according to claim 1, wherein said composition
comprises a strain of bacteria Lactobacillus reuteri LRE03 DSM
23879 and a strain of bacteria Lactobacillus salivarius LS06 DSM
26037, in a weight ratio from 1:2 to 2:1
13. The composition according to claim 1, wherein said composition
comprises a strain of bacteria Lactobacillus reuteri LRE03 DSM
23879 and a strain of bacteria Lactobacillus salivarius LS06 DSM
26037, in a weight ratio of 1:1.
14. The composition according to claim 1, wherein said composition
further comprises an Aloe arborescens gel.
15. The composition according to claim 14, wherein the Aloe
arborescens gel is freeze-dried.
16. The composition according to claim 1, wherein said composition
further comprises a freeze-dried Aloe arborescens in an amount from
5 to 15% by weight, relative to the weight of the composition.
17. The composition according to claim 6, wherein said tyndallized
bacterial product of the strain Bifidobacterium animalis ssp.
lactis Bb1 DSM 17850 is in an amount of 20 mg/g of composition.
18. The composition according to claim 1, wherein said composition
further comprises fructooligosaccharides (FOS), green tea,
sucralose, and/or maltodextrins.
19. The composition according to claim 1, wherein the composition
further comprises highly bioavailable zinc.
20. The composition according to claim 19, wherein the highly
bioavailable zinc is internalized inside a tyndallized bacterial
cell of the strain Bifidobacterium animalis ssp. lactis Bb1 DSM
17850.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is the U.S. National Stage of
International Patent Application No. PCT/IB2015/000602 filed on
Apr. 30, 2015, the corrected version of which was published on Nov.
12, 2015, which, in turn, claims priority to Italian Application
No. MI2014A000816 filed on May 5, 2014.
[0002] The present invention relates to a composition for human and
animal use as a therapy for the treatment of tumors, acquired
immunodeficiency syndrome and leukemias. The composition of the
present invention, for human and animal use as antitumor agent,
comprises a mixture comprising or, preferably, consisting of a
strain of bacteria Lactobacillus reuteri LRE 03 DSM 23879 which is
able to strongly stimulate the production of pro-inflammatory
cytokines (Th1) interferon INF-gamma, said cytokines exhibit a
marked antitumor activity, and/or a strain of bacteria
Lactobacillus salivarius LS06 DSM 26037 which is able to strongly
stimulate the production of dendritic cells, said dendritic cells
also exhibit a marked antitumor activity.
[0003] As regards oncology, the current medical therapy is known to
comprise chemotherapy, endocrine therapy, the treatment with immune
response modifiers and the treatment with molecular-targeted drugs.
The main purpose of antitumor chemotherapy is to kill, at any cell
cycle phases, neoplastic cells and thus, reduce both primary tumor
and metastasis masses.
[0004] It is known that antitumor chemotherapeutic treatments
decrease the immune system activity and that a compromised immune
system is unable to protect organisms against viral and bacterial
infections.
[0005] In addition, it is known that chemotherapy (chemo) primarily
affects the tumor but, unfortunately, it also causes side-effects
on healthy tissues, specifically those with a fast proliferation
and turnover, such as esophageal, gastric and intestinal mucosae,
resulting in mucositis, nausea, vomiting, diarrhea, nutrient
malabsorption and, thus, malnutrition.
[0006] Therefore, the common denominator of all chemotherapeutics
is: bone marrow toxicity, which, in turn, leads to immunodepression
and consequent infections, mainly caused by Gram-negative bacteria
and fungi such as Candida, gastrointestinal epithelium toxicity and
intestinal microflora toxicity (chemotherapeutic antibiotics).
[0007] Accordingly, it would be desirable to have a natural,
effective and well-tolerated composition so that to lessen the
adverse effects, typical of a chemotherapeutic treatment.
[0008] Thus, there is still a strong need for having an adjunctive
therapy in order to prevent and/or reduce both symptoms and side
effects of chemotherapy being used for the treatment of tumors.
[0009] Furthermore, there is still a need for having an adjunctive
therapy to chemotherapy, being able to act on immune system by
stimulating it (immunostimulation), in order to restore its
efficacy, since chemotherapy is well known to entail a reduction of
the immune system efficacy.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 shows a plot reporting the cell proliferation index
(P.I.) of PHA and LRE 03 in comparison to the baseline. The PBMC
proliferative response under all the stimulation conditions
resulted significantly higher than in absence of stimulation
(baseline).
[0011] FIG. 2 shows a plot reporting the percentage of total
dendritic cell in PHA and LRE 03 relative to the baseline.
[0012] FIG. 3 shows a plot reporting the percentage of helper
T-lymphocytes in PHA and LRE 03 relative to the baseline.
[0013] FIG. 4 shows three plot reporting the amount of cytokine
IL-12p70, IFN-.gamma. and IL-4 being released in PHA, LPS and LRE
03 relative to the baseline.
[0014] FIG. 5 shows a plot reporting the increase relative to the
baseline by LRE 03 strain. The bacterial strain Lactobacillus
reuteri LRE 03 DSM 23879 increased by 6-fold and 47-fold the
secretion of cytokine IL-12p70 and cytokine IFN-.gamma.,
respectively, relative to non-stimulation conditions.
[0015] FIG. 6 shows two plots reporting in panel A the increase of
total dendritic cell (Lineage-/HLA-DR+) percentage induced by the
strain LS 06 compared to the baseline and LPS and in panel B the
increase relative to the baseline by LS 06. In particular, the
bacterial strain L. salivarius LS 06 (DSM 26037) increased by
7-fold the percentage of total dendritic cells, relative to
non-stimulation conditions (baseline) (panel B).
[0016] After a long and intensive research and development activity
on a wide group of bacterial strains belonging to different
species, the Applicant identified and selected specific bacterial
strains, which suitably meet the above-cited needs.
[0017] It is an object of the present invention: [0018] a strain of
bacteria belonging to the species Lactobacillus reuteri identified
as Lactobacillus reuteri LRE03 with deposit number DSM 23879,
deposited on 5 Aug. 2010 by Probiotical SpA at DSMZ--Deutsche
Sammlung von Mikroorganismen und Zellkulturen GmbH, under the
Budapest Treaty, and/or [0019] a strain of bacteria belonging to
the species Lactobacillus salivarius identified as Lactobacillus
salivarius LS06 with deposit number DSM 26037, deposited on 6 Jun.
2012 by Probiotical SpA at DSMZ--Deutsche Sammlung von
Mikroorganismen und Zellkulturen GmbH, under the Budapest
Treaty.
[0020] The Applicant found that the strain of bacteria
Lactobacillus reuteri LRE03 DSM 23879 shows a proven and surprising
ability (see experimental part) to stimulate the production of
pro-inflammatory cytokines (Th1) interferon INF-gamma. The strain
of bacteria Lactobacillus reuteri LRE03 DSM 23879 shows a
surprising immunostimulatory activity towards the endogenous
production of interferon gamma IFN-gamma. The strain of bacteria
Lactobacillus reuteri LRE03 DSM 23879, selected by the Applicant,
exhibits a surprising immunomodulatory activity, since it activates
the immune system through the stimulation of pro-inflammatory
cytokines (Th1) interferon INF-gamma. The endogenous cytokine
stimulation/production does not cause toxicity, as opposed to the
infusion administration of said cytokines, as in the case of
exogenous cytokines.
[0021] By virtue of the strong stimulation of the production of
pro-inflammatory cytokines (Th1) interferon INF-gamma, the strain
of bacteria Lactobacillus reuteri LRE03 DSM 23879 is able to exert
an effective antitumor action, by counteracting and reducing tumor
cell proliferation.
[0022] The Applicant also found that the strain of bacteria
Lactobacillus salivarius LS06 DSM 26037 exhibits a proven and
surprising ability (see experimental part) to stimulate the
dendritic cell production. Dendritic cells assist the immune system
in protecting organisms from outside attacks of dangerous
microorganisms, such as viruses and bacteria.
[0023] Because of its strong stimulation of dendritic cell
production, the strain of bacteria Lactobacillus salivarius LS06
DSM 26037 is able to exert an effective antitumor action, by
counteracting and reducing tumor cell proliferation.
[0024] It is an object of the present invention a (i) mixture of
bacteria comprising or, alternatively, consisting of: a strain of
bacteria Lactobacillus reuteri LRE03 DSM 23879, and/or a strain of
bacteria Lactobacillus salivarius LS06 DSM 26037, for human or
animal use as antitumor agent.
[0025] In an embodiment, said mixture (i) comprises or,
alternatively, consists of: [0026] a strain of bacteria
Lactobacillus reuteri LRE03 DSM 23879, and/or [0027] a strain of
bacteria Lactobacillus salivarius LS06 DSM 26037, for human or
animal use in tumor treatment for counteracting and/or reducing
tumor cell proliferation, in acquired immunodeficiency syndrome
treatment and leukemia treatment.
[0028] In another embodiment, the (i) mixture of bacteria comprises
or, alternatively, consists of: [0029] a strain of bacteria
Lactobacillus reuteri LRE03 DSM 23879 and a strain of bacteria
Lactobacillus salivarius LS06 DSM 26037, in a weight ratio
comprised from 1:5 to 5:1, preferably from 1:3 to 3:1, even more
preferably from 1:2 to 2:1 or 1:1.
[0030] The (i) mixture of bacteria has a bacterial cell
concentration comprised from 1.times.10.sup.8 UFC/g of mixture to
1.times.10.sup.12 UFC/g of mixture, preferably from
1.times.10.sup.9 UFC/g of mixture to 1.times.10.sup.11 UFC/g of
mixture. Within the context of the present invention, all the
above-cited mixtures are referred to, for the sake of brevity, as
"the mixture of bacteria or the mixtures of bacteria of the present
invention".
[0031] It is another object of the present invention a
pharmaceutical composition or a medical device composition, which
is meant as a substance in compliance with the directive 93/42/EEC
definition, hereinafter referred to, for the sake of brevity, as
"the composition or compositions of the present invention", said
composition comprises or, alternatively, consists of:
(i) a mixture of bacteria of the present invention, as described
above, and/or (ii) a mixture comprising or, alternatively,
consisting of a gum, preferably an alginate or a derivative
thereof, and/or a gel, preferably an Aloe gel or a derivative
thereof, and/or (iii) a source of highly assimilable zinc, and/or
(iv) one or more food grade or pharma grade excipients and/or
additives and/or co-formulants, acceptable by the body, such as
preferably fructooligosaccharides (FOS), green tea, sucralose
and/or maltodextrins.
[0032] In an embodiment, which is an object of the present
invention, the composition of the present invention comprises or,
alternatively, consists of: (i) a mixture of bacteria of the
present invention and (iv) one or more food grade or pharma grade
excipients and/or additives and/or co-formulants, acceptable by the
body, said composition being for human and animal use in antitumor
chemotherapeutic treatments, acquired immunodeficiency syndrome
treatments and leukemia treatments.
[0033] In another embodiment, which is an object of the present
invention, the composition of the present invention comprises or,
alternatively, consists of: (i) a mixture of bacteria of the
present invention, (ii) a mixture comprising or, alternatively,
consisting of a gel, preferably an Aloe gel or a derivative thereof
and (iv) one or more food grade or pharma grade excipients and/or
additives and/or co-formulants, acceptable by the body, said
composition being for human and animal use in antitumor
chemotherapeutic treatments, acquired immunodeficiency syndrome
treatments and leukemia treatments. Said (ii) mixture comprises or,
alternatively, consists of a gel, preferably an Aloe gel or a
derivative thereof. The Aloe product, or a derivative thereof, is
preferably Aloe arborescens; preferably in freeze-dried form. Aloe
arborescens is preferably in freeze-dried form and exerts an
anti-inflammatory action.
[0034] Furthermore, the Applicant found that the immune system (IS)
activation by the composition of the present invention takes place
through a "dual biostimulation". The "dual biostimulation" consists
of a first and a second biostimulations. The first biostimulation
is obtained due to the presence, in the composition of the present
invention, of very highly bioavailable zinc. This biologically
available form of zinc stimulates the thymus to producing a greater
amount (number) of lymphocytes. Said T-lymphocytes "overproduced"
by the thymus produce non-toxic endogenous cytokines, such as
interferon-gamma and dendritic cells.
[0035] The second biostimulation, combined with the first one, is
promoted by the strain of bacteria Lactobacillus reuteri LRE03 DSM
23879 and/or the strain of bacteria Lactobacillus salivarius LS06
DSM 26037 which, in turn, stimulate lymphocytes, now in a greater
amount due to the previous thymus stimulation by the zinc, to
producing more cytokines (specifically, INF-gamma and dendritic
cells). In addition to the above, there is also a basal
anti-inflammatory effect ensured by the presence of Aloe or a
derivative thereof, preferably Aloe arborescens; preferably in
freeze-dried form.
[0036] The very high bioavailability of zinc derives from the fact
that it is in the form of zinc internalized inside a tyndallized
bacterial cell of a bacterial strain belonging to the species
Bifidobacterium lactis, preferably the strain selected by the
Applicant is the strain of bacteria Bifidobacterium lactis Bb 1 DSM
17850 deposited at DSMZ on 23 Dec. 2005, being the object of the
European Patent Application No. 08789404, herein incorporated by
reference.
[0037] Basically, the Applicant found that the highly bioavailable
zinc internalized into a tyndallized cell (inactivated cell) is
able to activate the immune system (IS), specifically the thymus
responsible for the production of T-lymphocytes, which produce
non-toxic endogenous cytokines, such as interferon-gamma and
dendritic cells.
[0038] In another embodiment, which is an object of the present
invention, the composition of the present invention comprises or,
alternatively, consists of: (i) a mixture of the present invention,
(ii) a mixture comprising or, alternatively, consisting of a gel,
preferably an Aloe gel or a derivative thereof, (iii) a source of
highly assimilable and bioavailable zinc in the form of zinc
internalized inside a tyndallized bacterial cell of a strain of
bacteria belonging to the species Bifidobacterium lactis,
preferably said strain is the strain of bacteria Bifidobacterium
lactis Bb 1 DSM 17850 and (iv) one or more food grade or pharma
grade additives and/or co-formulants and/or formulation
technological ingredients, acceptable by the body, said composition
being for human and animal use in antitumor chemotherapeutic
treatments, acquired immunodeficiency syndrome treatments and
leukemia treatments. Said (iv) source of highly assimilable and
bioavailable zinc is present as organic zinc in the form of
bacterial tyndallized product of the strain Bifidobacterium lactis
Bb1 DSM 17850 (ProbioZinc.RTM., deposited on 23 Dec. 2005 at
DSMZ--Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH,
by BioMan S.r.l. Company (Italy). The tyndallized bacterial product
of the strain Bifidobacterium animalis ssp. lactis Bb1 DSM 17850 is
in an amount comprised from 10 to 50 mg/g of composition,
preferably 20 mg/g of composition.
[0039] All the above-cited compositions of the present invention
are effectively applicable for use as adjunctive therapy to
antitumor chemotherapeutic treatments, acquired immunodeficiency
syndrome treatments and leukemia treatments.
[0040] Finally, the composition of the present invention comprises
food grade or pharma grade excipients and/or additives and/or
co-formulants, which allow the manufacture of pharmaceutical forms
as powders, granules, tablets or capsules. It can also contain, for
example, fructooligosaccharides FOS and/or green tea and/or
sucralose and/or maltodextrins.
[0041] The composition of the present invention comprises from
1.times.10.sup.8 to 1.times.10.sup.12 of viable bacterial cells
UFC/g of composition, preferably from 1.times.10.sup.9 to
1.times.10.sup.11 of viable bacterial cells UFC/g of composition.
The composition of the present invention is preferably recommended
to be administered 1-2 times daily for 4-12 weeks.
[0042] The composition of the present invention comprises said
Aloe, advantageously freeze-dried Aloe arborescens in an amount
comprised from 1 to 25% by weight, relative to the weight of the
composition; preferably from 5 to 15% by weight, relative to the
weight of the composition. The composition of the present invention
may contain freeze-dried Aloe arborescens (Alagel.TM.) for example
1.5 gram/dose.
[0043] In light of its overall mechanism of action, the composition
of the present invention is able to make the side effects of a
chemotherapeutic treatment more tolerable in individuals with a
tumor disease.
[0044] In an embodiment, the composition of the present invention
comprises the (i) mixture of the present invention (having a
bacterial cell concentration comprised from 1.times.10.sup.8 UFC/g
of mixture to 1.times.10.sup.12 UFC/g of mixture, preferably from
1.times.10.sup.9 UFC/g of mixture to 1.times.10.sup.11 UFC/g of
mixture) comprising or, alternatively, consisting of: [0045] a
strain of bacteria Lactobacillus reuteri LRE03 DSM 23879, or [0046]
a strain of bacteria Lactobacillus salivarius LS06 DSM 26037, or
[0047] a strain of bacteria Lactobacillus reuteri LRE03 DSM 23879
and a strain of bacteria Lactobacillus salivarius LS06 DSM 26037,
in a weight ratio comprised from 1:5 to 5:1, preferably from 1:3 to
3:1, even more preferably from 1:2 to 2:1 or 1:1, and/or
fructooligosaccharides FOS and/or green tea and/or sucralose and/or
maltodextrins.
[0048] Indeed, the strain L. reuteri LRE03 DSM 23879 is able to
significantly stimulate the endogenous production of
interferon-gamma (IFN-.gamma.). The ability of the strain L.
reuteri LRE03 DSM 23879 to induce the release of cytokines, in
particular interferon-gamma INF-gamma, by the primary cells of
immune system was quantified by co-incubation thereof with PBMCs
(Peripheral Blood Mononuclear Cells) isolated from peripheral blood
of healthy adult individuals. The results showed a stimulation of
IFN-gamma secretion with a concentration of 480 pg/ml, namely
47-fold greater than the control. The IFN-gamma production was
assessed in the culture supernatant after 5 days of stimulation
relative to non-stimulation conditions (baseline). Interferon-gamma
(IFN-.gamma.) has hindrance properties against viral and bacterial
infections, similarly to the other interferons, and
non-physiological cell proliferation, which is mediated by changes
of cytoskeleton and cell membrane, modulations of oncogene product
expression and regulation of the cell differentiation process
(protraction of almost all the phases of mitosis both in normal and
tumor cells). IFN-.gamma. also shows a pivotal and characteristic
immunomodulatory effect, by stimulating the activity of both cells
specialized in the body's immune response such as macrophages,
monocytes, neutrophils and unspecialized cells such as platelets,
endothelial and epithelial cells, fibroblasts and parenchymal
cells.
[0049] The composition of the present invention ensures a suitable
amount (2 mg/dose) of highly assimilable zinc internalized by the
microorganism Bifidobacterium lactis Bb1. The highly bioavailable
(internalized) zinc is in the form of a tyndallized (inactivated)
cell. This form of zinc is very bioavailable and, thus, more easily
assimilable by the organism. The zinc ion, being bioavailable and
readily assimilable by the organism, plays a pivotal role and a
direct action towards the thymus, which is responsible for the
stimulation/production of lymphocytes, which produce more
cytokines.
[0050] The strain of bacteria Bifidobacterium lactis Bb 1 DSM 17850
was deposited at DSMZ on 23 Dec. 2005, by BioMan S.r.l. Company
(Italy). Indeed, the strain of bacteria Bifidobacterium lactis Bb 1
DSM 17850 is able to accumulate zinc inside the cell during its
growth in a liquid culture medium. The dietary zinc accumulated
inside the cell (ProbioZinc.RTM.) has an assimilability 17-fold
greater than zinc gluconate and 31.5-fold greater than zinc
sulfate, as shown in an in vitro study carried out on Caco-2 cells
in a Transwell system. The high assimilability of the trace element
zinc allows to effectively counterbalancing deficiency conditions
even at very low dosages. Furthermore, zinc is known for playing an
important role on the immune system, specially the thymus, the
organ where the production of T-lymphocytes takes place which, when
differentiate to CD4+ T-lymphocytes (helper T-lymphocytes), secrete
a series of cytokines such as IL-12 and IFN-.gamma.. Said zinc
mechanism of action is synergistic with that of the strain L.
reuteri LRE03 DSM 23879.
[0051] In light of its overall mechanism of action, the composition
being object of the present invention is effectively applied for
use as adjunctive therapy in individuals with tumor diseases and
undergoing chemotherapy, as well as in antiretroviral treatments in
individuals with Acquired Immunodeficiency Syndrome (AIDS) and
leukemia treatments.
Experimental Part
[0052] The Applicant tested the immunomodulatory properties of the
strain of bacteria Lactobacillus reuteri LRE 03 (ID1777) DSM 23879,
as described below.
[0053] Specifically, the investigation was conducted after
different times of stimulation, so that to analyze both cytokines
involved in innate immunity and those responsible for acquired
immunity.
a) Bacterial Cultures and Growth Conditions
[0054] Firstly, bacterial cultures of the strain of bacteria
Lactobacillus reuteri LRE 03 DSM 23879 were prepared under specific
growth conditions. The strain was cultured in Man Rogosa Sharpe
(MRS) medium, in a thermostatic bath at 37.degree. C. As regards
the immunomodulatory experiments, after a growth period of
approximately 16 hours, bacteria were subcultured for 6 hours,
under the above-cited conditions, in order to reach the exponential
growth phase. Then, they were washed twice with sterile
phosphate-buffered saline (PBS, pH 7.2); the physiological status
and the number of cells were determined with a cytofluorimetric
technique by using the commercial kit "Cell Viability Kit with
liquid beads", marketed by Becton Dickinson Company, following the
manufacturer's instructions. The cells were thus brought to the
optimal concentration established in preliminary experiments and
used in subsequent tests.
b) Peripheral Blood Mononuclear Cell Separation
[0055] Next, peripheral blood mononuclear cells were separated. The
peripheral blood mononuclear cells (PBMC) were separated by density
gradient centrifugation. For this aim, 20 ml of "buffy coat" of
healthy donors from the Immuno-transfusion Service of Ospedale di
Borgomanero (Italy) were used for each experiment, thus obtaining
an average yield of 200.times.10.sup.6 PBMC/buffy. The amount of
separated cells was determined by cell count in Burker's chambers,
using Turk's dye, which allows to discriminating between
mononuclear and polymorphonuclear cells. Cells were brought to a
concentration of 2.times.10.sup.6 cells/ml in RPMI-1640 growth
medium (Invitrogen), supplemented with 10% heat inactivated Bovine
fetal serum (FCS, Gibco), 1% glutamine and 25 mM Hepes.
c) PBMC Stimulation
[0056] Next, the peripheral blood mononuclear cells (PBMC) were
stimulated with the bacterial strain. After separation, PBMCs were
stimulated with the bacterial strain for 1 and 5 days. The internal
controls for each experiment were as follows:
Negative control: PBMCs alone 1 day control: PBMCs stimulated with
1 .mu.g/ml Lipopolysaccharide (LPS; Escherichia coli, serotype
055:B5, Sigma Chemicals Co., St. Louis, Mo.). 5 days control: PBMCs
stimulated with 1 .mu.g/ml Phytohaemagglutinin (PHA-P; Sigma
Chemicals Co., St. Louis, Mo.).
[0057] At the different times of analysis, cultures were
centrifuged at 1500 rpm for 10 minutes. Supernatants were taken and
stored at -20.degree. C. until analysis. The cells were used for
subsequent tests.
d) Cell Proliferation Analysis
[0058] Then, the cell proliferation analysis was performed. Cell
proliferation was assessed with a cytofluorimetric technique by
using the bromodeoxyuridine (BrdU) nuclear labeling protocol. This
method was developed as alternative to the more traditional
radiolabeling system with tritiated thymidine. Particularly, cell
cultures were added with a mixture of 5-bromo-2'-deoxyuridine
(BrdU) and 2'-deoxycytidine (dC), either at 20 .mu.M final
concentration. Following to 16-hour incubation at 37.degree. C.
under humidified atmosphere, 5% CO.sub.2, the cell proliferation
was analyzed by a cytofluorimetric technique. The culture
supernatants were harvested and stored at -20.degree. C. until
analysis. Following to fixation and cell wall permeabilization, the
cellular DNA was labeled with anti-BrdU FITC-conjugated monoclonal
antibody (mAb) (Becton Dickinson). The cells were analyzed within
24 hours from their preparation by using a cytofluorimeter
FACScalibur from Becton Dikinson Company and the analysis program
CellQuest.
[0059] Results were expressed as cell proliferation index (P.I.),
being calculated as ratio of the percentage of proliferating cells
in the presence of stimulus and the percentage of the same in the
absence of stimulation. A P.I. value greater than 2 was considered
acceptable (cut-off value). In all the experiments, stimulation
with the mitogen (PHA) as control always resulted greater than the
cut-off value, confirming that PBMCs were viable and with
proliferative capability.
e) Analysis of Molecules Characterizing Individual Cell
Subpopulations
[0060] Next, the analyses of molecules characterizing the
individual cell subpopulations were performed. As regards the
immunophenotypic characterization, the cells were incubated for 30
minutes in the dark, with different combinations of the following
monoclonal antibodies (mAb) conjugated to fluorescein
isothiocyanate (FITC), phycoerythrin (PE) or peridinin chlorophyll
protein (PerCP): CD3, CD4, CD8, CD14, CD16, CD19, CD20, CD56,
HLA-DR. After incubation, the samples were washed, fixed with a
solution containing 1% paraformaldehyde and stored at 4.degree. C.
Within 24 hours from preparation, the samples were analyzed by a
cytofluorimeter FACScalibur, the cells being selected so that to
exclude contaminant cellular debris from analysis.
f) Cytokine Dosage
[0061] Next, the cytokine dosage was performed. Cytokine
concentration in the culture supernatants was determined by
E.L.I.S.A. assay (Enzyme-Linked Immunoabsorbent Assay).
Specifically, for cytokine (IL-4, IL-10, IFN-.gamma. and IL-12p70)
dosage, the kits "Human ELISA Ready-SET-Go" from eBioscence
Company, San Diego Calif. were used.
g) Statistical Analysis
[0062] A statistical analysis by using the paired Student's t test
was performed. A p<0.05 value was considered significant.
Results
[0063] i) The proliferative response induced by the strain of
bacteria Lactobacillus reuteri LRE 03 DSM 23879 was determined. In
vitro analysis of cell proliferation is a very useful biological
parameter for investigating the immune system functioning. In order
to analyze whether the tested bacterial strain could affect the
induction of lymphocyte proliferation, peripheral blood mononuclear
cells (PBMC) were stimulated with the bacterial strain
Lactobacillus reuteri LRE 03 DSM 23879. Phytohaemagglutinin (PHA),
which is a mitogenic stimulus able to induce T-lymphocyte
polyclonal proliferation, was used as positive control. PBMCs were
separated from peripheral venous blood samples of 4 healthy male
donors, average age of 40 years (range 21-52 years), from the
Transfusion Service of Ospedale S.S. Trinita, Borgomanero
(Novara).
[0064] As shown in FIG. 1, where the cell proliferation index
(P.I., see the above-described methods) is reported, the PBMC
proliferative response under all the stimulation conditions
resulted significantly higher than in absence of stimulation
(baseline).
[0065] In FIG. 1, the Mean.+-.standard error of the mean (S.E.M.)
of 4 independent experiments is shown. The statistical significance
was calculated by using the Student's t test. p<0.05 values have
to be considered statistically significant, as compared to the
baseline (non-stimulated PBMCs).
[0066] ii) The effects of the bacterial strain Lactobacillus
reuteri LRE 03 DSM 23879 on the different cell subpopulations were
assessed. In order to detect which cell subpopulations were induced
to proliferate following to stimulation with the tested probiotic
strain, a multiparametric flow cytometry analysis was performed.
The subsequent figures (FIG. 2 and FIG. 3) show the percentage of
the main cell subpopulations involved both in natural and acquired
immune response.
[0067] iia) Natural Immunity. After one day, the stimulation with
the bacterial strain Lactobacillus reuteri LRE 03 (DSM 23879)
caused a change in total dendritic cell (Lineage-/HLA-DR+)
percentage.
[0068] In FIG. 2, the proliferative response Mean.+-.S.E.M. of 4
independent experiments is shown. The statistical significance was
calculated by using the Student's t test. p<0.05 values have to
be considered statistically significant, as compared to the
baseline (non-stimulated PBMCs).
[0069] iib) Acquired Immunity. After five days, the stimulation
with the bacterial strain Lactobacillus reuteri LRE 03 DSM 23879
caused a significant increase in helper T-lymphocytes (CD3+/CD4)
percentage.
[0070] In FIG. 3, the Mean.+-.S.E.M. of 12 independent experiments
is shown. The statistical significance was calculated by using the
Student's t test. p<0.05 values have to be considered
statistically significant, as compared to the basal condition
(non-stimulated PBMCs).
[0071] iii) Cytokine secretion. The different spectrum of cytokines
secreted by cell subpopulations involved in immune responses plays
a pivotal role in selecting the effector system to be used in
response to a specific antigenic stimulus. T-lymphocytes represent
the main effector and regulatory cells of cell-mediate immunity. In
response to an antigen or pathogenic agent, T-cells synthetize and
secrete a variety of cytokines required for growth and
differentiation and as activating factors of other immunocompetent
cells. In order to investigate whether the tested bacterial strain
would induce a different cytokine secretion by PBMCs, said cells
were activated for 1 and 5 days. The amount of cytokines (IL-12p70,
IFN-.gamma. and IL-4) being released in the culture supernatants
was measured by E.L.I.S.A. assay.
[0072] iv) Cytokines with predominantly pro-inflammatory action.
The induction of cytokines IL-12p70 and IFN-.gamma., as main
representatives of cytokines with predominantly pro-inflammatory
action was assessed. As shown in FIG. 4, the bacterial strain
Lactobacillus reuteri LRE 03 (DSM 23879) is able to induce a
significant increase of both the tested cytokines, relative to
basal conditions.
[0073] v) Cytokines with predominantly immunoregulatory action. The
induction of cytokines IL-4, as main representatives of cytokines
with predominantly immunoregulatory action was assessed. As shown
in FIG. 4, the tested bacterial strain Lactobacillus reuteri LRE 03
(DSM 23879) shown to be able to induce a reduction of cytokine IL-4
secretion, relative to basal conditions.
[0074] In FIG. 4 the Mean.+-.S.E.M. of 4 independent experiments is
shown. The statistical significance was calculated by using the
Student's t test. p<0.05 values have to be considered
statistically significant, as compared to the baseline
(non-stimulated PBMCs). The production of cytokines IL-12p70 was
assessed in culture supernatants after 1 day of stimulation.
IFN-.gamma. and IL-4 production was assessed in culture
supernatants after 5 days of stimulation.
[0075] Data relative to the dosage of cytokines secreted by PBMCs
following to stimulation with the bacterial strain Lactobacillus
reuteri LRE 03 DSM 23879 underlined the capability of the strain
itself to significantly increase pro-inflammatory cytokines.
Specifically, the bacterial strain Lactobacillus reuteri LRE 03 DSM
23879 increased by 6-fold and 47-fold the secretion of cytokine
IL-12p70 and cytokine IFN-.gamma., respectively, relative to
non-stimulation conditions (baseline, FIG. 5).
[0076] Considering the marked ability of the bacterial strain
Lactobacillus reuteri LRE 03 DSM 23879 to stimulate the cytokine
IFN-.gamma. secretion, the results of the present study were
compared to those obtained from experiments with other bacterial
strains, all belonging to Probiotical S.p.A. collection.
Specifically, the increase relative to the baseline was compared,
namely the fold change of the IFN-.gamma. amount, relative to
non-stimulation conditions (baseline).
[0077] As shown in table 1, the bacterial strain Lactobacillus
reuteri LRE 03 (DSM 23879) resulted the best IFN-.gamma. inducing
agent relative to both strains of the same species and strains of
different species, all belonging to the genus Lactobacillus.
[0078] In Table 1, the increase of cytokine IFN-.gamma. induced by
stimulation with different lactobacilli and bifidobacteria,
compared to the baseline, is shown.
Experimental Part
[0079] The Applicant tested the immunomodulatory properties of the
bacterial strain Lactobacillus salivarius LS 06 DSM 26037, as
described below.
[0080] Specifically, the immunomodulatory properties towards total
circulating dendritic cells of the probiotic strain Lactobacillus
salivarius LS 06 DSM 26037, previously characterized from both the
microbiological and molecular point of views, were assessed. In
particular, after 24 hours of stimulation, a multiparametric flow
cytometry analysis, by selecting the DCs in peripheral blood
mononuclear cells from healthy adults donors, was carried out.
a) Bacterial Cultures and Growth Conditions
[0081] The strain was cultured in Man Rogosa Sharpe (MRS) medium,
in a thermostatic bath at 37.degree. C. As regards the
immunomodulatory experiments, following to a growth period of
approximately 16 hours, the bacteria were subcultured for 6 hours,
under the above-cited conditions, in order to reach the exponential
growth phase. Thus, they were washed twice with sterile
phosphate-buffered saline (PBS, pH 7.2); the physiological status
and the number of cells were determined with a cytofluorimetric
technique by using the commercial kit "Cell Viability Kit with
liquid beads", marketed by Becton Dickinson Company, following the
manufacturer's instructions. The cells were then brought to the
optimal concentration established in preliminary experiments and
used in subsequent tests.
b) Peripheral Blood Mononuclear Cell Separation
[0082] Peripheral blood mononuclear cells (PBMC) were separated by
density gradient centrifugation. For this aim, 20 ml of "buffy
coat" of healthy donors from the Immuno-transfusion Service of
Ospedale di Borgomanero were used for each experiment, thereby
obtaining an average yield of 200.times.10.sup.6 PBMC/buffy. The
amount of separated cells was determined by cell count in Burker's
chambers, by using Turk's dye, which allows to discriminating
between mononuclear cells and polymorphonuclear cells. The cells
were brought to a concentration of 2.times.10.sup.6 cells/ml in
RPMI-1640 growth medium (Invitrogen), supplemented with 10% heat
inactivated Bovine fetal serum (FCS, Gibco), 1% glutamine and 25 mM
Hepes.
c) PBMC Stimulation
[0083] After separation, PBMCs were stimulated for 24 hours with
the bacterial strain. The internal controls for each experiment
were as follows: Negative control: PBMCs alone; 1 day control:
PBMCs stimulated with 1 .mu.g/ml Lipopolysaccharide (LPS;
Escherichia coli, serotype 055:B5, Sigma Chemicals Co., St. Louis,
Mo.).
[0084] After stimulation, the cultures were centrifuged at 1500 rpm
for 10 minutes. Then the supernatant was discharged and the cells
used for subsequent tests.
d) Total Dendritic Cells Analysis
[0085] As regards the immunophenotypic characterization, cells were
incubated for 30 minutes in the dark with different combinations of
the following monoclonal antibodies (mAb) conjugated to fluorescein
isothiocyanate (FITC) or peridinin chlorophyll protein (PerCP):
CD3, CD14, CD16, CD19, CD20, CD56 and HLA-DR. After incubation, the
samples were washed, fixed with a solution containing 1%
paraformaldehyde and stored at 4.degree. C. Within 24 hours from
preparation, the samples were analyzed by a cytofluorimeter
FACScalibur, the cells being selected so that to exclude
contaminant cellular debris from analysis.
e) Statistical Analysis
[0086] A statistical analysis by using the paired Student's t test
was performed. A p<0.05 value was considered significant.
Results: Bacterial Strain Effect on Dendritic Cells
[0087] In order to determine the effect of the tested probiotic
strain to the dendritic cell modulation, a multiparametric flow
cytometric analysis was carried out.
[0088] As shown in FIG. 6, panel A, after 24 hours, the stimulation
with the strain LS 06 induced a significant increase of total
dendritic cell (Lineage-/HLA-DR+) percentage.
[0089] Specifically, the bacterial strain L. salivarius LS 06 (DSM
26037) increased by 7-fold the percentage of total dendritic cells,
relative to non-stimulation conditions (baseline, FIG. 6, panel
B).
[0090] In FIG. 6, panel B, the Mean.+-.S.E.M. of 12 independent
experiments is shown. The statistical significance was calculated
by using the Student's t test. p<0.05 values have to be
considered statistically significant, as compared to the baseline
(non-stimulated PBMCs).
[0091] In Table 2, the increase of dendritic cells induced by
stimulation with different lactobacilli and bifidobacteria,
relative to the baseline, is shown.
CONCLUSIONS
[0092] Data demonstrated that the bacterial strain L. salivarius LS
06 induced a significant increase of total DC percentage, relative
to the standard basal conditions. In particular, the bacterium LS
06 increased by 7-fold the total DC percentage.
[0093] The intestinal colonization by bacteria capable to
modulating the dendritic cells, such as the strain L. salivarius LS
06 being characterized in the present study, is a very important
factor in diseases characterized by an immunological imbalance.
TABLE-US-00001 TABLE 1 No. of Single strains Abbreviation ID
Deposit No. individuals IFN-g L. casei subsp. LPC 08 1696 DSM 21718
8 7.83 .+-. 0.56 paracasei L. fermentum LF 11 1639 DSM 19188 8 8.67
.+-. 1.05 L. paracasei LPC 00 1076 LMG P-21380 8 7.83 .+-. 0.56 L.
paracasei LPC 00 1076 LMG P-21380 4 8.06 .+-. 0.95 L. plantarum LP
09 1837 DSM 25710 4 22.29 .+-. 4.09 L. pentosus LPS 01 1702 DSM
21980 8 19.54 .+-. 1.68 L. reuteri LRE 01 1775 DSM 23877 4 2.79
.+-. 0.61 L. reuteri LRE 03 1777 DSM 23879 4 47.02 .+-. 4.38 L.
rhamnosus LR 05 1602 DSM 19739 10 4.16 .+-. 1.06 L. salivarius LS
06 1727 DSM 26037 4 4.95 .+-. 0.92 (L166) L. salivarius DL V8 1813
DSM 25545 4 2.74 .+-. 0.57 B. animalis BS 01 1195 LMG P-21384 10
6.84 .+-. 0.81 subsp lactis Bifidobacterium DL BL07 1820 DSM 25669
4 15.25 .+-. 4.01 longum Bifidobacterium DL BL08 1823 DSM 25670 4
8.95 .+-. 1.77 longum Bifidobacterium DL BL09 1821 DSM 25671 4
12.01 .+-. 2.75 longum Bifidobacterium DL BL10 1824 DSM 25672 4
11.35 .+-. 2.09 longum Bifidobacterium DL BL11 1825 DSM 25673 4
11.85 .+-. 3.78 longum Bifidobacterium BL01 1239 None 4 21.9 .+-.
4.67 longum Bifidobacterium BL02 1295 None 4 20.84 .+-. 0.89 longum
Bifidobacterium BL03 1152 DSM 16603 4 24.44 .+-. 5.45 longum
Bifidobacterium BL04 1740 DSM 23233 4 19.11 .+-. 5.38 longum
Bifidobacterium W11 1114 None 4 26.01 .+-. 7.40 longum
Bifidobacterium W11 wt 1161 None 4 27.42 .+-. 6.78 longum
Bifidobacterium PCB133 1687 DSM 24691 4 29.09 .+-. 8.25 longum
Bifidobacterium BL05 1352 DSM 23234 4 14.94 .+-. 2.28 longum
Bifidobacterium BL06 no ID DSM 24689 4 31.90 .+-. 3.96 longum L.
acidophilus LA02 1688 DSM 21717 8 4.91 .+-. 0.70 L. deldrueckii
LDD01 1391 DSM 22106 8 6.46 .+-. 0.92 subsp. delbrueckii L.
fermentum LF09 1462 DSM 18298 8 0.80 .+-. 0.15 L. fermentum LF10
1637 DSM 19187 8 4.25 .+-. 0.4 L. plantarum LP01 1171 LMG P-21021 8
1.77 .+-. 0.42 L. plantarum LP02 91 LMG P-21020 8 4.59 .+-. 0.59 L.
reuteri LRE02 1774 DSM 23878 4 1.19 .+-. 0.12 L. reuteri LRE04 1779
DSM 23880 4 1.72 .+-. 0.39 L. reuteri DLLRE07 -- DSM 25683 4 1.00
.+-. 0.07 L. reuteri DLLRE08 1841 DSM 25684 4 0.93 .+-. 0.06 L.
reuteri DLLRE09 1842 DSM 25685 4 1.22 .+-. 0.29 L. rhamnosus LR06
1697 DSM 21981 10 2.64 .+-. 0.83 L. salivarius LS01 1797 DSM 22775
10 1.44 .+-. 0.13 L. salivarius LS04 -- DSM 24618 L. salivarius
LS03 1382 DSM 22776 10 0.72 .+-. 0.26 L. salivarius DLV1 1806 DSM
25138 8 1.40 .+-. 0.13 L. salivarius LS05 1719 DSM 26036 4 1.80
.+-. 0.09 (L66) L. salivarius LS02 1468 DSM 20555 8 1.32 .+-. 0.67
B. lactis BA05 1518 DSM 18352 8 1.24 .+-. 0.08 B. breve BR03 1270
DSM 16604 10 2.20 .+-. 0.20 B. breve BR03 1270 DSM 16604 8 6.92
.+-. 1.02 B. pseudolongum BPS01 1812 None 8 0.64 .+-. 0.33 subsp.
globosum B. longum B1975 1742 DSM 24709 4 2.99 .+-. 0.71
TABLE-US-00002 TABLE 2 No. of Dendritic Single strains Abbreviation
ID Deposit No. individuals cells L. fermentum LF 11 1639 DSM 19188
8 0.98 .+-. 0.12 L. paracasei LPC 00 1076 LMG P-21380 8 1.47 .+-.
0.24 L. pentosus LPS 01 1702 DSM 21980 8 1.46 .+-. 0.28 L. reuteri
LRE 03 1777 DSM 23879 4 2.11 .+-. 0.59 L. rhamnosus LR 05 1602 DSM
19739 10 1.84 .+-. 0.29 L. salivarius LS06 1727 DSM 26037 4 7.35
.+-. 3.52 (L166) B. animalis BS 01 1195 LMG P-21384 10 1.32 .+-.
0.12 subsp. lactis L. acidophilus LA02 1688 DSM 21717 8 0.76 .+-.
0.16 L. deldrueckii LDD01 1391 DSM 22106 8 1.14 .+-. 0.19 subsp.
Delbrueckii L. fermentum LF09 1462 DSM 18298 8 1.23 .+-. 0.12 L.
fermentum LF10 1637 DSM 19187 8 1.03 .+-. 0.19 L. plantarum LP01
1171 LMG P-21021 8 0.86 .+-. 0.18 L. plantarum LP02 91 LMG P-21020
8 1.27 .+-. 0.18 L. salivarius LS01 1797 DSM 22775 10 3.93 .+-.
2.01 L. salivarius LS04 -- DSM 24618 L. salivarius LS03 1382 DSM
22776 10 5.96 .+-. 3.53 L. salivarius LS02 1468 DSM 20555 8 3.97
.+-. 1.80 B. lactis BA05 1518 DSM 18352 8 1.34 .+-. 0.47 B. breve
BR03 1270 DSM 16604 10 1.83 .+-. 0.29
* * * * *