U.S. patent application number 10/804223 was filed with the patent office on 2005-04-21 for use of lactobacillus salivarius.
This patent application is currently assigned to Enterprise Ireland (trading as BioResearc Ireland). Invention is credited to Collins, John Kevin, O'Mahony, Liam, O'Sullivan, Gerald Christopher, Shanahan, Fergus.
Application Number | 20050084482 10/804223 |
Document ID | / |
Family ID | 26320235 |
Filed Date | 2005-04-21 |
United States Patent
Application |
20050084482 |
Kind Code |
A1 |
Collins, John Kevin ; et
al. |
April 21, 2005 |
Use of lactobacillus salivarius
Abstract
Lactobacillus salivarius is useful in the prophylaxis or
treatment of undesirable inflammatory activity, especially
gastrointestinal inflammatory activity such as inflammatory bowel
disease or irritable bowel syndrome. The inflammatory activity may
also be due to cancer. The Lactobacillus salivarius is of human
origin isolated from resected and washed human gastrointestinal
tract. One such strain is UCC 118 described in WO-A-9835014.
Inventors: |
Collins, John Kevin;
(Doughcloyne, IE) ; O'Sullivan, Gerald Christopher;
(Cork, IE) ; O'Mahony, Liam; (Cork, IE) ;
Shanahan, Fergus; (Kinsale, IE) |
Correspondence
Address: |
JACOBSON HOLMAN PLLC
400 SEVENTH STREET N.W.
SUITE 600
WASHINGTON
DC
20004
US
|
Assignee: |
Enterprise Ireland (trading as
BioResearc Ireland)
National University of Ireland, Cork.
|
Family ID: |
26320235 |
Appl. No.: |
10/804223 |
Filed: |
March 19, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10804223 |
Mar 19, 2004 |
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10241797 |
Sep 12, 2002 |
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10241797 |
Sep 12, 2002 |
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09903590 |
Jul 13, 2001 |
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09903590 |
Jul 13, 2001 |
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PCT/IE00/00007 |
Jan 17, 2000 |
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Current U.S.
Class: |
424/93.45 |
Current CPC
Class: |
A61P 43/00 20180101;
C12N 1/205 20210501; Y10S 435/822 20130101; C12R 2001/01 20210501;
A61P 1/12 20180101; C12N 1/20 20130101; A61P 37/02 20180101; A61K
2039/52 20130101; A61K 2039/542 20130101; A61P 19/04 20180101; A61P
37/04 20180101; A23L 33/135 20160801; A61P 1/04 20180101; A61P 1/00
20180101; A61P 35/00 20180101; A61P 31/04 20180101; A61P 31/12
20180101; A23Y 2220/77 20130101; A61P 29/00 20180101; A61P 37/00
20180101; Y10S 435/853 20130101; A61P 19/02 20180101; A61P 31/14
20180101; A23C 9/1234 20130101; A61P 37/06 20180101; A61K 39/09
20130101; A23V 2002/00 20130101; A23V 2002/00 20130101; A23V
2200/3204 20130101; A23V 2200/324 20130101 |
Class at
Publication: |
424/093.45 |
International
Class: |
A61K 045/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 15, 1999 |
IE |
99 0033 |
Sep 20, 1999 |
IE |
99 0782 |
Claims
1-51. (canceled)
52. A method of treating or preventing inflammation or an
inflammatory disease in a subject which comprises administering to
the subject a preparation comprising a Lactobacillus salivarius
strain of Lactobacillus salivarius wherein the Lactobacillus
salivarius strain is isolated from resected and washed human
gastrointestinal tract.
53. The method of claim 52 wherein the inflammation is
gastrointestinal inflammation.
54. The method of claim 52, wherein the inflammatory disease is
selected from any one or more of inflammatory bowel disease,
Crohn's disease, ulcerative colitis, irritable bowel syndrome,
pouchitis, post infection colitis, diarrhoeal disease, and a
systemic inflammatory disease.
55. The method of claim 54, wherein the diarrhoeal disease is
associated with Clostridium difficile.
56. The method of claim 54, wherein the diarrhoeal disease is
associated with Rotovirus.
57. The method of claim 54, wherein the diarrhoeal disease is post
infective diarrhoeal disease.
58. The method of claim 53, wherein the inflammation is due to a
cancer or an autoimmune disorder.
59. The method of claim 52, wherein the systemic inflammatory
disease is rheumatoid arthritis.
60. The method of claim 58, wherein the cancer is gastrointestinal
cancer.
61. The method of claim 52, which further comprises administering
to the subject another probiotic material.
62. The method of claim 52, which further comprises administering
to the subject a prebiotic material.
63. The method of claim 52, wherein the preparation is a
formulation comprising an ingestible carrier.
64. The method of claim 63, wherein the ingestible carrier is a
pharmaceutically acceptable carrier.
65. The method of claim 64, wherein the formulation is a
pharmaceutical formulation in the form of a tablet, a capsule, or a
powder.
66. The method of claim 63, wherein the ingestible carrier
comprises a protein, a polypeptide, a peptide, or a combination
thereof.
67. The method of claim 66, wherein the protein, the polypeptide,
or the peptide is rich in glutamine, glutamate, or both.
68. The method of claim 63, wherein the ingestible carrier
comprises a lipid, a carbohydrate, a vitamin, a mineral, a trace
element, or a combination thereof.
69. The method of claim 63, wherein the ingestible carrier is a
food product.
70. The method of claim 69, wherein the food product is selected
from any one or more of acidified milk, yoghurt, a frozen yoghurt,
a milk powder, a milk concentrate, a cheese spread, a dressing, and
a beverage.
71. The method of claim 52, wherein the Lactobacillus salivarius
strain is present at more than 10.sup.6 cfu per gram of
preparation.
72. The method of claim 52, wherein the preparation comprises an
adjuvant, bacterial components, a drug, a biological compound, or a
combination thereof.
73. The method of claim 52, wherein the subject is a mammal.
74. The method of claim 73, wherein the mammal is human.
75. The method of claim 52, wherein the Lactobacillus salivarius
strain is in the form of viable cells.
76. The method of claim 52, wherein the Lactobacillus salivarius
strain is in the form of non-viable cells.
77. The method of claim 52, wherein the Lactobacillus salivarius
strain inhibits a broad range of Gram positive and Gram negative
microorganisms.
78. The method of claim 52, wherein the Lactobacillus salivarius
strain secretes a product having anti-microbial activity into a
cell-free supernatant, said activity being produced only by growing
cells and being destroyed by proteinase K, pronase E, or both.
79. The method of claim 52, wherein the Lactobacillus salivarius
strain is Lactobacillus salivarius subspecies salivarius.
80. The method of claim 52, wherein the Lactobacillus salivarius
strain is UCC118 [NCIMB 40829] or a mutant or variant thereof.
81. The method of claim 52, wherein the Lactobacillus salivarius
strain is a genetically modified mutant strain.
82. The method of claim 52, wherein the Lactobacillus salivarius
strain is a naturally occurring variant of Lactobacillus
salivarius.
83. A method of treating or preventing inflammation or an
inflammatory disease in a subject comprising administering to the
subject a preparation comprising a Lactobacillus salivarius strain
of Lactobacillus salivarius wherein the Lactobacillus salivarius is
characterised by changing an immunological marker when introduced
into a system comprising cells which interact with the immune
system and cells of the subject.
84. The method claim 83 wherein the strain of Lactobacillus
salivarius is isolated from resected and washed human
gastrointestinal tract.
85. The method of claim 83 wherein the cells which interact with
the immune system are epithelial cells.
86. The method of claim 83, wherein the immunological marker is a
cytokine.
87. The method of claim 86, wherein the cykotine is TNF.alpha..
88. The method of claim 83, wherein the cells which interact with
the immune system and the immune system cells are of matched
origin.
89. The method of claim 83, wherein the cells which interact with
the immune system are of gastrointestinal, respiratory, or
genitourinary origin.
90. The method of claim 83, wherein the cells of the immune system
are of gastrointestinal, respiratory, or genitourinary origin.
91. A method of treating or preventing inflammation or an
inflammatory disease in a subject which comprises administering to
the subject a preparation comprising a strain of Lactobacillus
salivarius UCC118 [NCIMB 40829] or a mutant or variant thereof.
92. The method of claim 91, wherein the Lactobacillus salivarius
strain is a genetically modified mutant strain.
93. The method of claim 91, wherein the Lactobacillus salivarius
strain is a naturally occurring variant of Lactobacillus
salivarius.
94. The method of claim 91, wherein the Lactobacillus salivarius
strain is in the form of viable cells.
95. The method of claim 91, wherein the Lactobacillus salivarius
strain is in the form of non-viable cells.
96. A method of treating or preventing cancer in a subject which
comprises administering to the subject a Lactobacillus salivarius
strain of Lactobacillus salivarius or a mutant or variant thereof.
Description
[0001] The invention relates to the use of strains of Lactobacillus
salivarius.
[0002] The defense mechanisms to protect the human gastrointestinal
tract from colonization by intestinal bacteria are highly complex
and involve both immunological and non-immunological aspects (V. J.
McCracken and H. R Gaskins, `Probiotics a critical review`, Horizon
Scientific Press, UK, 1999, p 278.). Innate deference mechanisms
include the low pH of the stomach, bile salts, peristalsis, much
layers and anti-microbial compounds such as lysozyme (D. C. Savage,
`Microbial Ecology of the Gut`, Academic Press, London, 1997,
p.278.). Immunological mechanisms include specialised lymphoid
aggregates, underlying M cells, called peyers patches which are
distributed throughout the small intestine and colon (M. F.
Kagnoff. Gastrocaterol. 1993, 105, 1275). Luminal antigens
presented at these sites result in stimulation of appropriate T and
B cell subsets with establishment of cytoline networks and
secretion of antibodies into the gastrointestinal tract (M. R.
Neutra and J- P Kraehenbuhl, `Essentials of mucosal immunology`,
Academic Press, San Diego, 1996, p.29., M. E. Lamm. Ann. Rev.
Mirobiol. 1997, 51, 311). In addition, antigen presentation may
occur via epithelial cells to intraepithelial lymphocytes and to
the underlying lamina propria immune cells (S. Raychaudhur et al.
Nat Biotecinol., 1998, 16, 1025). Therefore, the host invests
substantially in immunological defense of the gastrointestinal
tract. However, as the gastrointestinal raucous is the largest
surface at which the host interacts with the external environment,
specific control mechanisms must be in place to regulate immune
responsiveness to the 100 tons of food which is handled by the
gastrointestinal tract over an average lifetime (F. Shanahan,
`Physiology of the gastrointestinal tract`, Raven Press, 1994,
p.643.). Furthermore, the gut is colonised by over 500 species of
bacteria numbering 10.sup.11-10.sup.12 /g in the colon. Thus, these
control mechanisms must be capable of distinguishing non-pathogenic
adherent bacteria from invasive pathogens which would cause
significant damage to the host. In fact, the intestinal flora
contributes to defense of the host by competing with newly ingested
potentially pathogenic microorganisms.
[0003] Consumption of non-pathogenic, or probiotic, bacteria has
resulted in enhancement of immune parameters in healthy volunteers.
Examples of these immune modulatory effects are given in Table
1.
1TABLE 1 Immune Enhancing Effects Following Oral Consumption of
Probiotic Bacteria. Observed Effect Reference Increased Macrophage
Phagocytosis 10 Increased Natural Killer Cell Activity 11 Increased
IFN.gamma. serum levels 12 Increased B cell and NK cell numbers 12
Promotion of IgA responses 11, 13-15 Increased DTH responses 16
[0004] Bacteria present in the human gastrointestinal tract can
promote inflammation Aberrant immune responses to the indigenous
ricroflora have been implicated in certain disease states, such as
inflammatory bowel disease (Brandzeag P. et al. Springer Semin.
Immunopathol., 1997, 18, 555). Antigens associated with the normal
flora usually lead to immunological tolerance and failure to
achieve this tolerance is a major mechanism of mucosal inflammation
(Stallmach A. et al., Immunol. Today, 1998, 19, 438). Evidence for
this breakdown in tolerance includes an increase in antibody levels
directed against the gut flora in patients with IBD.
[0005] WO-A-98/35014 describes strains of Lactobacillus salivarius
isolated from resected and washed human gastrointestinal tract
which inhibits a broad range of Gram positive and Gram negative
microorganisms and which secretes a product having anti-microbial
activity into a cell-free supernatant.
STATEMENTS OF INVENTION
[0006] The immune system is designed to defend host tissue and
destroy invading pathogens. Upon recognition of the presence of a
bacterial cell, cells of the immune system become activated and
eliminate the bacterial threat. The production of inflammatory
mediators promote cellular activation and pathogen destruction.
[0007] Surprisingly, we have found that strains of Lactobacillus
salivarius elicit an anti-inflammatory effect in vitro and an vivo.
We have found that the immune perception of Lactobacillus
salivarius results in the suppression of inflammatory activity. The
deliberate consumption in large numbers of Lactobacillus salivarius
results in the suppression of inflammatory activity. The invention
is therefore of major potential therapeutic value in the
prophylaxis or treatment of undesirable inflammatory responses,
such as inflammatory bowel disease.
[0008] Lactobacillus salivarius is a commensal microorganism
originally isolated from the microbial flora within the human
gastrointestinal tract. The immune system within the
gastrointestinal tract cannot have a pronounced reaction to members
of this flora as the resulting inflammatory activity would also
destroy host cells and tissue function. Therefore, some
mechanism(s) exist whereby the immune system can recognise
commensal non-pathogenic members of the gastrointestinal flora as
being different to pathogenic organisms. This ensures that damage
to host tissues is restricted and a defensive barrier is still
maintained.
[0009] According to the invention there is provided use of a strain
of Lactobacillus salivarius in the prophylaxis and/or treatment of
undesirable inflammatory activity.
[0010] The invention the undesirable inflammatory activity may be
undesirable gastrointestinal inflammatory activity such as
inflammatory bowel disease, eg. Crohns disease, ulcerative colitis,
irritable bowel syndrome, pouchitis or post infection colitis.
[0011] The gastrointes inflammatory activity may also be diarrhoeal
disease. The diarrhoeal disease may be associated by Clostridium
difficile or be associated with Rotoviius. The diarrhoeal disease
may also be post infective diarrhoeal disease.
[0012] The inflammatory activity may be due to gastrointestinal
cancer or systemic inflammatory disease such as rheumatoid
arthritis.
[0013] In another instance the undesirable inflammatory activity
may be due to an autoimmune disorder.
[0014] In yet another instance the undesirable inflammatory
activity may be due to cancer.
[0015] In one embodiment the invention provides use of a strain of
Lactobacillus salivarius in the prophylaxis of cancer.
[0016] In another embodiment the invention provides use of a strain
of Lactobacillus salivarius wherein the Lactobacillus salivarius is
contained in a formulation.
[0017] Preferably the formulation includes another probiotic
material. Alternatively or additionally the formulation includes a
prebiotic material.
[0018] Ideally the formulation includes an ingestive carrier. The
ingestable carrier may be a pharmaceutically acceptable carrier
such as a tablet, capsule or powder.
[0019] Preferably the ingestable carrier is a protein and/or a
peptide, in particular proteins and/or peptides that are rich in
glutamine/glutamate; a lipid; a carbohydrate; a vitamin; mineral
and/or trace element.
[0020] Most preferably the ingestable carrier is a food product
such as acidified milk, yoghurt, frozen yoghurt, milk powder, milk
concentrate, cheese spreads, dressings or beverages.
[0021] In one embodiment the Lactobacillus salivarius is present in
the formulation at more than 10.sup.6 cfu per gram of delivery
system.
[0022] In another embodiment the formulation includes an adjuvant.
The formulation may include a bacterial component. The formulation
may alternatively or additionally include a drug entity. The
formulation may also include a biological compound.
[0023] In one embodiment the invention provides use of a strain of
Lactobacillus salivarius wherein the strain or formulation is for
administration to animals. Preferably the animal is a mammal, most
preferably a human.
[0024] In another embodiment the invention provides use of a strain
of Lactobacillus salivarius wherein the Lactobacillus salivarius
effects changes in an immunological marker when introduced into a
system comprising cells which interact with the immune system and
cells of the immune system.
[0025] Preferably the cells which interact with the immune system
are epithelial cells. Most preferably the immunological marker is a
cytokine especially TNF.alpha..
[0026] Preferably the cells which interact with the immune system
and the immune system cells are of matched origin.
[0027] In one embodiment the cells which interact with the immune
system are of gastrointestinal, respiratory or genitourinary
origin.
[0028] In another embodiment the cells of the immune system are of
gastrointestinal, respiratory or genitourinary origin.
[0029] In a further embodiment the invention provides use of a
strain of Lactobacillus salivarius wherein the Lactobacillus
salivarius strain is Lactobacallius salivarius subspecies
salivarius. Preferably the Lactobacallius salivarius is of human
origin, most preferably from resected and washed human
gastrointestinal tract.
[0030] Preferably the Lactobacillus salivarius inhibits a broad
range of Gram positive and Gram negative micro-organisms. Most
preferably it secretes a product having anti-microbial activity
into a cell-free supernatant, said activity being produced only by
growing cells and being destroyed by proteinase K and pronase
E.
[0031] An especially preferred strain of Lactobacillus salivarius
is Lactobacallius salivarius strain UCC188 or mutant or variant
thereof.
[0032] A deposit of Lactobacillus salivarius strain UCC118 was made
at the NCIB on Nov. 27, 1996 and accorded the accession number NCMB
40829. The strain of Lactobacillus salivarius is described in
WOA-98/35014.
[0033] The Lactobacillus salivarius may be a genetically modified
mutant or it may be a naturally occurring van ant of Lactobacillus
salivarius.
[0034] Preferably the Lactobacillus salivarius is in the form of
viable cells. Alternatively the Lactobacillus salivarius may be in
the form of non-viable cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] FIG. 1 is a graph of C. perfingens levels in the mice
consuming UCC118 compared to a placebo group (p<0.05). Results
are plotted as the mean log values.+-.standard error for each of
the groups
[0036] FIG. 2 is a bar chart of inflammatory scores for mice
consuming UCC118 in comparison to control mince. Results are shown
as the mean.+-.standard error for each of the groups.
[0037] FIG. 3 is a graph of TNF.alpha. levels over six weeks that
patients consume UCC118. Results are plotted as the mean pg/ml
TNF.alpha. level for each time point (n=22).
[0038] FIG. 4 is a graph of CDAI scores for patients consuming
UCC118 over the course of probiotic feeding. CDAI scores decreased
from an average of 180 to 160.
[0039] FIG. 5 is a graph of cytokine production in vivo following
exposure to UCC118, Results are expressed as pg/ml.
[0040] FIG. 6 is bar chart of extracellular TNF.alpha., IL-1 RA,
IL-6, sIL-6R, and IFN.alpha. levels following exposure to
Lactobacillus salivarius UCC118.
[0041] FIG. 7 is a gene array with specific gene sequences for 268
cytokines and related molecules to examine the immune response to
UCC118. The bottom panel illustrates the control culture while the
top panel illustrates cytokine gene expression by PBMCs following
exposure to UCC118; and
[0042] FIG. 8 is a bar chart of TNF.alpha. levels in the presence
of various bacterial strains.
DETAILED DESCRIPTION
[0043] We have developed criteria for in vitro selection of
probiotic bacteria that reflect certain in vivo effects on their
host, such as modulation of the GIT microflora and modulation of
the mucosal immune response resulting in the production of
secretory antibodies specific to the consumed strain. We have found
that Lactobacillus salivarius subsp. salivarius UCC118 not only
survives passage through the gastrointestinal tract and adheres to
human intestinal cell lines but also, surprisingly has
anti-inflammatory effects.
[0044] The general use of probiotic bacteria is in the form of
viable cells. However, it can also be extended to non-viable cells
such as killed cultures or -compositions containing beneficial
factors expressed by the probiotic bacteria. This could include
thermally killed micro-organisms or micro-organisms killed by
exposure to altered pH or subjection to pressure. With non-viable
cells product preparation is simpler, cells may be incorporated
easily into pharmaceuticals and storage requirements are much less
limited than viable cells. Lactobacillus casei YIT 9018 offers an
example of the effective use of heat killed cells as a method for
the treatment and/or prevention of tumour growth as described in US
Patent -No. U.S. Pat. No. 4,347,240.
[0045] It is unknown whether intact bacteria are required to exert
an anti-inflammatory effect or if individual active components of
the invention can be utllised alone. Proinflammatory components of
certain bacterial strains have been identified. The proinflammatory
effects of gram-negative bacteria are mediated by
lipopolysaccharide (LPS). LPS alone induces a proinflammatory
network, partially due to LPS binding to the CD14 receptor on
monocytes. It is assumed that components of probiocic bacteria
possess anti-inflammatory activity, due to the effects of the whole
cell. Upon isolation of these components, pharmaceutical grade
manipulation is anticipated.
[0046] The invention will be more clearly understood from the
following examples.
EXAMPLE 1
Detailed Description of the In Vivo Demonstration of the
Anti-Inflammatory Effects of Lactobacillus salivarius Especially
Subspecies salivarius UCC118.
[0047] Murine Model of Gastrointestinal Inflammation
[0048] Aberrant immune responses to the indigenous microflora have
been implicated in certain disease states, such as inflammatory
bowel disease (Brandzeag P., et al. Springer Semin. Immunopathol.,
1991, 18, 555). Antigens associated with the normal flora usually
lead to immunological tolerance and failure to achieve this
tolerance is a major mechanism of mucosal inflammation (Stallmach
A., et al. Immunol. Today, 1998, 19, 438). Evidence for this
breakdown in tolerance includes an increase in antibody levels
directed against the gut flora in patients with IBD. In addition,
certain mouse models predisposed to inflammatory lesions in the
gastrointestinal tract remain disease free when housed in germ free
conditions or when treated with antibiotics (Kuhn R., et al. Cell,
1993, 75, 263; Panwala C. M., et al. J. Immunol., 1998, 161,
5733).
[0049] C57BL/6 Interleukin-10 knockout mice are predisposed to
developing enterocolitis in the presence of an enteric bacterial
flora. When maintained in germ free conditions, IL-10 knock out
mice do not develop the disease (Kuhn R., et al. Cell, 1993, 75,
263). Since the pathogenesis of this disease has been linked with
the enteric flora, elimination of specific components of this flora
may have a beneficial effect on the severity of this disease.
[0050] Lactobacillus salivarius subsp. salivarius UCC118 is a
probiotic bacteria, which was isolated from a healthy human ileum.
It is suited to gastrointestinal colonization as it fulfills many
criteria set down for the selection of probiotic strains. These
include traits such as bile tolerance, acid resistance and in vitro
adherence to human colonic cell lines. Feeding trials in healthy
humans have been conducted and considerable modification of the
gastrointestinal flora was noted. In addition, UCC118 was perceived
by the mucosal immune system resulting in the production and
secretion of IgA specific to UCC118.
[0051] Thus, UCC118 survives passage through the gastrointestinal
tract, modulates the gut flora and is perceived by the mucosal
immune system. The influence of this probiotic bacteria in
modulating inflammatory responses within the gastrointestinal tract
was examined using a murine model of enterocolitis. In addition, we
examined the role of Lactobacillus salivarius subsp. salivarius
UCC118 in reducing the rate of neoplastic change within the
gastrointestinal tract.
[0052] Twenty IL-10 KO mice were studied (ten consumed probiotic
organisms in milk and 10 consumed unmodified milk) for 16 weeks.
Fecal microbial analysis was performed weekly to enumerate
excretion of lactobacilli, Clostridium perfringens, bacteroides,
coliforms, bifidobacteria and enterococci. At sacrifice, small and
large bowel were microbiologically and histologically assessed.
[0053] Fecal coliform and enterococci levels were significantly
reduced in test animals compared to the controls. At sacrifice, a
significant reduction in C. perfringens numbers was observed in the
test mice (FIG. 1). There were no fatalities in the test group
compared to two deaths from fulminant colitis in the control group.
Only one test mouse developed colonic adenocarcinoma compared to
five in the control group. Test animal mucosal inflammation
consistently scored lower than that of the control mice (FIG. 2).
The reduction in tumour incidence following consumption of UCC118
may be related to the reduced level of inflammation within the
gastrointestinal tract or may be due to elimination of
pro-carcinogenic members of the gastrointestinal flora (Rumney C.
J., et al. Carcinogenesis, 1993, 14, 79; Rowland I. R. (1995). In:
Gibson G. R. (ed). Human colonic bacteria: role in nutrition.
physiology and pathology, pp 155-174. Boca Raton CRC Press: Darveau
D. Nat. Biotech., 1999, 17, 19).
[0054] In conclusion, consumption of Lactobacillus salivarius
UCC118 results in a significant modulation of the gut flora and an
improvement in mortality rate, cancer incidence and disease
score.
EXAMPLE 2
Human Trial with UCC118 in Patients with Active Crohn's
Disease.
[0055] Inflammatory bowel disease (IBD) encompasses a number of
inflammatory disorders of the gastrointestinal tract, including
both Crohn's disease and ulcerative colitis.
[0056] Patients suffering from active Crohn's disease have been
treated with UCC118. Briefly, UCC118 was consumed in a fermented
milk product for 6 weeks by 22 patients. Microbiological and
immunological determinations were made at week 0, week 1, week 3
and week 6. This was not a placebo-controlled trial.
[0057] A number of systemic cytoline levels were measured over the
course of feeding. In particular, tumour necrosis factor .alpha.
(TNF.alpha.), a proinflammatory cytokine that has been implicated
in the pathogenesis of many inflammatory disease states, including
inflammatory bowel disease. Current therapies for inflammatory
bowel disease specifically aim to reduce TNF.alpha. levels
(Present. D. H., et al. New Eng. J. Med., 1999, 340, 1398). In this
trial, systemic TNF.alpha. levels were reduced following
consumption of UCC118 (FIG. 3).
[0058] In addition, patients were assessed regarding their Crohn's
Disease Activity Index (CDAI) over the six week trial period. This
index assesses the general health and well being of each patient
(FIG. 4). Overall, the disease activity index improved slightly for
the majority of individuals in the trial. These are patients with
moderately active disease and their CDAI scores would be expected
to increase. However, following treatment with UCC118, CDAI scores
did not increase and in fact they improved from a mean of 180 to
160.
EXAMPLE 3
Detailed Description of the In Vitro Demonstration of the
Mechanisms Underlying the Anti-Inflammatory Effects of
Lactobacillus salivarius Especially Subspecies salivarius
UCC118.
[0059] A number of methodologies have been utilised for these
studies including ELISAs (extracellular protein determination),
flow cytometry (intracellular protein determination) and cDNA
expression arrays (mRNA expression). In particular, examination of
the expression of tumour necrosis factor a has been targeted, due
to its clinical importance, and suppression of the production of
this cytoline, following exposure to UCC118, has been noted using
all three methodologies.
[0060] Using a transwell assay system, with epithelial cells and
peripheral blood mononuclear cells, extracellular cytoline levels
were measured by ELISAs. Following to-incubation with UCC118, the
amount of TNF.alpha. produced, was significantly reduced compared
to control cultures. Furthermore, IL-IRA and IFN.gamma. levels
dropped while IL6 and soluble IL-6 receptor levels increased (FIG.
5). Intracellular staining for TNF.alpha. confirmed the ELISA
result as TNF.alpha. levels were lowering the UCC118 stimulated
sample compared to controls.
[0061] FIG. 6 demonstrates the tricellular signalling that occurs.
To-incubation of PBMCs and Lactobacillus salivarius strain UCC118
results in the stimulation of TNF.alpha. production. However,
to-incubation of PBMCs, Lactobacillus salivarius strain UCC118 and
epithelial cells (CaCo-2 cells) results in a significant inhibition
of TNF.alpha. production. Thus, a significantly different pattern
of signalling is present in the tricellular model compared to
bacteria and PBMCs alone.
[0062] Gene arrays measure the quantity of mRNA in a population of
cells. We stimulated peripheral blood mononuclear ceUs with UCC118
for 24 hours and we examined the effect on cyeoline gene expression
(FIG. 7). Considerable modification of cytokine gene expression was
noted. For example, genes encoding the proinflammatory cytokines
IL-1 P and TNF.alpha. were turned off while genes encoding Th2 type
cytokines, such as IL-6, were enhanced.
[0063] In vitro models have demonstrated that UCC118 is capable of
inducing Th2 type cytoldnes (i.e. IL-6 and IL-6 soluble receptor)
while suppressing the production of inflammatory cytokines such as
TNF.alpha. and IL-1.beta.. Thus, these results suggest that
consumption of UCC118 would be of benefit to patients suffering
from inflammatory diseases, such as IBD.
EXAMPLE 4
Test for Anti-Inflammatory Bacterial Strains
[0064] A number of lactic acid bacteria, which have been isolated
from the human gastrointestinal tract, were examined in this novel
assay system for anti-inflammatory effect. All bacterial strains
were taken from -20.degree. C. glycerol stocks and incubated
anaerobically overnight in MRS broth and washed in antibiotic
containing medium. Epithelial cell monolayers were grown for 6
weeks prior to the addition of PBMCs and bacterial cells.
[0065] The results of these stimulations can be observed in FIG. 8.
Relative to control cultures, two bacterial strains suppressed
TNF.alpha. production. The two strains Lactobacillus salivarius
strain UCC118, which suppressed production of TNF.alpha., is the
subject of WO-A-9835014. The Bifdobacterium longum infantis strain
UCC 35624 is the subject of a PCT Application filed concurrently
with the present application.
[0066] Inflammation
[0067] Inflammation is the term used to describe the local
accumulation of fluid, plasma proteins and white blood cells at a
site that has sustained physical damage, infection or where there
is an ongoing immune response. Control of the inflammatory response
is exerted on a number of levels (for review see Henderson B., and
Wilson M. 1998. In "Bacteria-Cytokine interactions in health and
disease. Portland Press, 79-130). The controlling factors include
cytokines, hormones (e.g. hydrocortisone), prostaglandins, reactive
intermediates and leukotrienes. Cytokines are low molecular weight
biologically active proteins that are involved in the generation
and control of immunological and inflammatory responses, while also
regulating development, tissue repair and haematopoiesis. They
provide a means of communication between leukocytes themselves and
also with other cell types. Most cytolines are pleiotrophic and
express multiple biologically overlapping activities. Cytokine
cascades and networks control the inflammatory response rather than
the action of a particular cytokine on a particular cell type (Aral
K I, et al., Annu Rev Biochem 1990;59:783-836). Warning of the
inflammatory response results in lower concentrations of the
appropriate activating signals and other inflammatory mediators
leading to the cessation of the inflammatory response. TNF.alpha.
is a pivotal proinflammatory cytolone as it initiates a cascade of
cytokines and biological effects resulting in the inflammatory
state. Therefore, agents which inhibit TNF.alpha. are currently
being used for the treatment of inflammatory diseases, e.g.
inliximab.
[0068] Pro-inflammatory cytokines are thought to play a major role
in the pathogenesis of many inflammatory diseases, including
inflammatory bowel disease (IBD). Current therapies for treating
IBD are aimed at reducing the levels of these pro-inflammatory
cytokines, including IL-8 and TNF.alpha.. Such therapies may also
play a significant role in the treatmentc of systemic inflammatory
diseases such as rheumatoid arthritis.
[0069] In view of the anti-inflammatory properties of Lactobacillus
salivarius that we have discovered these strains may have potential
application in the treatment of a range of inflammatory diseases,
particularly if used in combination with other anti-inflammatory
therapies, such as non-steroid anti-inflammatory drugs (NSAIDs) or
Infliximab.
[0070] Diarrhoeal Disease.
[0071] The barrier function of the intestinal epithelium can be
diminished during nervous (acetylcholine) and immune (histamine)
mediated secretion. Certain bacterial toxins may also induce Ca2+
and PKC dependent secretion and thereby can disturb the epithelial
barrier (Ganguly N K and Kaur T. Indian J Med Res 1996;104:28-37,
Groot J A. Vet Q 1998;20(S3):45-9). Several studies have examined
the prevention and treatment of diarrhoeal using probiotic
bacteria. Prospective studies have demonstrated the efficacy of
lactic acid bacteria administration for both prophylactic and
therapeutic use against diarrhoea in pre-mature infants, new borns,
children (Isolauri E, et al., Dig Dis Sci 1994
December;39(12):2595-600) and in the treatment of antibiotic
related diarrhoea (Siitonen S, et al., Ann Med 1990
February;22(1):57-9) and traveller's diarrhoea (Oksanen P J, et
al., Ann Med 1990 February;22(1):53-6).
[0072] In view of the anti-inflammatory effect we have discovered
Lactobacillus salivarius may also produce an anti-diarrhoeal
effect, possibly medicated via cAMP modulation. Cyclic
AMP-dependent Cl-secretion is the major secretory pathway in the
human intestine (Brzuszczak I M, et al., J. Gastroenterol. Hepatol.
1996;11(9):804-10). The anti-diarrhoeal effect may not be
restricted just to diarrhoea resulting from gastrointestinal
inflammation, but can be applied to the general treatment of
diarrhoeal disease.
[0073] Autoimmune Disease
[0074] The immune system has a large repertoire of specificities
expressed by B and T cells. Some of these specificities will be
directed to self-components. Self-recognition is normally
controlled by clonal deletion and inactivation of self-reactive
lymphocytes. However, there is a constant background of
autoimmunity with antibodies to many proteins being found in serum.
A breakdown in the self-nonself recognition system results in
autoimmunity. When autoimmune disease does occur, the resulting
immune response damages the tissue bearing the offending antigen.
Immune complex deposition, type II hypersensitivity and
cell-mediated reactions are the most important mechanisms by which
immunopathological damage occurs. Examples of autoimmune diseases
include, but are not limited to, systemic lupus erythematosus,
rheumatoid arthritis, insulin dependent diabetes melitus,
myasthenia gravis and pernicious anaenua. We have found that
Lactobacillus salivarius is an immunomodulatory bacterium. Thus,
consumption either as a single component or in combination with
other bacteria by patients suffering from autoimmune disease may
restrict organ damage and help restore normal body homeostasis.
[0075] Inflammation and Cancer
[0076] The production of multifunctional cytolines across a wide
spectrum of tumour types suggests that significant inflammatory
responses are ongoing in patients with cancer. It is currently
unclear what protective effect this response has against the growth
and development of tumour cells in vivo. However, these
inflammatory responses could adversely affect the tumour bearing
host. Complex cytokine interactions are involved in the regulation
of cytokine production and cell proliferation within tumour and
normal tissues (McGee D W, et al., Immunology 1995
September;86(l):6-11, Wu S, et al.,. Gynecol Oncol 1994
April;53(.1):59-63). It has long been recognized that weight loss
(cachexia) is the single most common cause of death in patience
with cancer (Inagaki J. et al. Cancer 1974 February;33(2):568-73)
and initial malnutrition indicates a poor prognosis (Van Eys J.
Nutr Rev 1982 December;40(12):353-9). For a tumour to grow and
spread it must induce the formation of new blood vessels and
degrade the extracellular matrix. The inflammatory response may
have significant roles to play in the above mechanisms, thus
contributing to the decline of the host and progression of the
tumour. Due to the anti-inflammatory properties of Lactobacillus
salivarius these bacterial strains they may reduce the rate of
malignant cell transformation. Furthermore, intestinal bacteria can
produce, from dietary compounds, substances with genotoxic,
carcinogenic and tumour-promoting activity and gut bacteria can
activate pro-carcinogens to DNA reactive agents (Rowland I. R.
(1995). Toxicology of the colon: role of the intestinal microflora.
In: Gibson G. R. (ed). Human colonic bacteria: role in nutrition,
physiology and pathology, pp 155-174. Boca Raton CRC Press). In
general, species of Lactobacillus have low activities of xenobiotic
metabolising enzymes compared to other populations within the gut
such as bacteroides, eubacteria and clostridia (Saito Y., et al.,
Microb. Ecol. Health Dis., 1992;5, 105-110). Therefore, increasing
the number of Lactobacillus bacteria in the gut could beneficially
modify the levels of these enzymes.
[0077] Prebiotics
[0078] The introduction of probiotic organisms is accomplished by
the ingestion of the microorganism in a suitable carrier. It would
be advantageous to provide a medium that would promote the growth
of these probiotic strains in the large bowel. The addition of one
or more oligosaccharides, polysaccharides, or other prebiotics
enhances the growth of lactic acid bacteria in the gastrointestinal
tract (Gibson, G R. Br. J. Nutr. 1998;80 (4):S209-12). Prebiotics
refers to any non-viable food component that is specifically
fermented in the colon by indigenous bacteria thought to be of
positive value, e.g. bifidobacteria, lactobacilli. Types of
prebiotics may include those which contain fructose, xylose, soya,
galactose. glucose and mannose. The combined administration of a
probiotic strain with one or more prebiotic compounds, may enhance
the growth of the administered probiotic in vivo resulting in a
more pronounced health benefit, and is termed symbiotic.
[0079] Other Active Ingredients
[0080] It will be appreciated that the Lactobacillus salivarius may
be administered prophylactically or as a method of treatment either
on its own or with other probiotic and/or prebiotic materials as
described above. In addition, the bacteria may be used as part of a
prophylactic or treatment regime using other active materials such
as those used for treating inflammation or other disorders,
especially those of the gastrointestinal tract. Such combinations
may be administered in a single formulation or as separate
formulations administered at the same or different times and using
the same or different routes of administration.
[0081] The invention is not limited to the embodiments hereinbefore
described which may be varied in detail.
* * * * *