U.S. patent application number 10/404512 was filed with the patent office on 2004-10-07 for methods of determining efficacy of treatments of inflammatory diseases of the bowel.
This patent application is currently assigned to The Procter & Gamble Company and Alimentary Health, Ltd., The Procter & Gamble Company and Alimentary Health, Ltd.. Invention is credited to Chen, Ker-Sang, Luo, Fangyi, O'Mahony, Liam Diarmuid.
Application Number | 20040197304 10/404512 |
Document ID | / |
Family ID | 33096943 |
Filed Date | 2004-10-07 |
United States Patent
Application |
20040197304 |
Kind Code |
A1 |
Chen, Ker-Sang ; et
al. |
October 7, 2004 |
Methods of determining efficacy of treatments of inflammatory
diseases of the bowel
Abstract
Novel methods of determining efficacy of a treatment of
inflammatory diseases of the bowel in mammals are provided. The
methods are of use in screening and determining the efficacy of
treatments of inflammatory diseases of the bowel, and for
determining the efficacy response of individual sufferers of
inflammatory diseases of the bowel to a given regime. Kits for
carrying out the method are also provided.
Inventors: |
Chen, Ker-Sang; (West
Chester, OH) ; Luo, Fangyi; (Mason, OH) ;
O'Mahony, Liam Diarmuid; (Cork, IE) |
Correspondence
Address: |
THE PROCTER & GAMBLE COMPANY
INTELLECTUAL PROPERTY DIVISION
WINTON HILL TECHNICAL CENTER - BOX 161
6110 CENTER HILL AVENUE
CINCINNATI
OH
45224
US
|
Assignee: |
The Procter & Gamble Company
and Alimentary Health, Ltd.
Attention: Chief Patent Counsel 6090 Center Hill Road
Cincinnati
OH
45224
|
Family ID: |
33096943 |
Appl. No.: |
10/404512 |
Filed: |
April 1, 2003 |
Current U.S.
Class: |
424/85.1 ;
424/85.2 |
Current CPC
Class: |
G01N 33/5023 20130101;
G01N 33/5091 20130101; G01N 2800/52 20130101; G01N 33/6866
20130101; G01N 33/6869 20130101; G01N 33/6863 20130101; G01N
33/5044 20130101; G01N 2333/52 20130101; G01N 33/5008 20130101;
G01N 2800/065 20130101 |
Class at
Publication: |
424/085.1 ;
424/085.2 |
International
Class: |
A61K 038/19; A61K
038/20 |
Claims
What is claimed is:
1. A method of determining the efficacy of a treatment of
inflammatory diseases of the bowel in mammals comprising the steps
of: a) measuring the level of at least one anti-inflammatory
cytokine and at least one pro-inflammatory cytokine in a biological
sample from a mammalian subject; b) determining the ratio of the at
least one anti-inflammatory cytokine to the at least one
pro-inflammatory cytokine; c) administering said treatment; d)
measuring the level of the at least one anti-inflammatory cytokine
and the at least one pro-inflammatory cytokine in a biological
sample from said mammalian subject at a time following
administration of said treatment; e) determining the ratio of the
at least one anti-inflammatory cytokine to the at least one,
pro-inflammatory cytokine following administration of said
treatment; wherein an increase in the ratio of anti-inflammatory
cytokine to pro-inflammatory cytokine following the administration
of said treatment is indicative of an inhibitor of inflammatory
diseases of the bowel, and no change or a decrease in the ratio of
anti-inflammatory cytokine to pro-inflammatory cytokine following
the administration of said treatment is indicative said treatment
not being an inhibitor of inflammatory diseases of the bowel.
2. The method according to claim 1 wherein the anti-inflammatory
cytokine is selected from the group comprising interleukin-10,
transforming growth factor-.beta., interleukin-4, interleukin-5,
interleukin-13, and mixtures thereof.
3. The method according to claim 2 wherein the anti-inflammatory
cytokine is selected from the group comprising interleukin-10,
transforming growth factors, and mixtures thereof.
4. The method according to claim 1 wherein the pro-inflammatory
cytokine comprises interleukin-12, tumour necrosis factor-.alpha.,
interferon-.gamma., interleukin-2, and mixtures thereof.
5. The method according to claim 4 wherein the pro-inflammatory
cytokine comprises interleukin-12, tumour necrosis factor-.alpha.,
interferon-.gamma., and mixtures thereof.
6. The method according to claim 1, wherein said ratio of
anti-inflammatory cytokine to pro-inflammatory cytokine is the
ratio interleukin-10/interleukin-12.
7. The method according to claim 1, wherein said ratio of
anti-inflammatory cytokine to pro-inflammatory cytokine is the
ratio transforming growth factor-.beta./interleukin-12.
8. The method according to claim 1, wherein said ratio of
anti-inflammatory cytokine to pro-inflammatory cytokine is the
ratio interleukin-10/interferon-.gamma..
9. The method according to claim 1 wherein said biological sample
comprises urine, plasma, serum, saliva, tissue biopsies,
cerebrospinal fluid, peripheral blood mononuclear cells with in
vitro stimulation, peripheral blood mononuclear cells without in
vitro stimulation, gut lymphoid tissues with in vitro stimulation,
gut lymphoid tissues without in vitro stimulation, gut lavage
fluids, and mixtures thereof.
10. The method according to claim 9 wherein said biological sample
comprises serum, peripheral blood mononuclear cells with in vitro
stimulation, peripheral blood mononuclear cells without in vitro
stimulation, and mixtures thereof.
11. The method according to claim 10 wherein said biological sample
comprises peripheral blood mononuclear cells with in vitro
stimulation, peripheral blood mononuclear cells without in vitro
stimulation, and mixtures thereof.
12. The method according to claim 9 wherein said in vitro
stimulation comprises a mitogen, probiotic, anti-CD3 molecule, and
mixtures thereof.
13. The method according to claim 10, wherein said in vitro
stimulation comprises a mitogen.
14. The method according to claim 13 wherein said mitogen comprises
a lipopolysaccharide, lectin, superantigen, and mixture
thereof.
15. The method according to claim 14, wherein said lectin comprises
concanavalin A, phytohemagglutinin, pokeweed mitogen, and mixtures
thereof.
16. The method according to claim 1 wherein the means for measuring
the levels of said at least one anti-inflammatory cytokine in said
biological sample comprises ELISAs, radioimmunoassays, multiplexed
ELISAs on microarray platforms, multiplexed ELISAs using coded
microspheres coupled with a flow cytometer detection systems,
bioassays, Western blots, chromatograph-based separation systems,
RT-PCR, competitive reverse transcription PCR, Northern blots, gene
arrays, direct measurement of m-RNA, and mixtures thereof.
17. The method according to claim 16 wherein the means for
measuring the levels of anti-inflammatory cytokines in said
biological sample comprises ELISAs, RIAs, multiplexed ELISAs using
coded microspheres coupled with a flow cytometer detection systems,
and mixtures thereof.
18. The method according to claim 17 wherein the means for
measuring the levels of said at least one anti-inflammatory
cytokine in said biological sample comprises multiplexed ELISAs
using coded microspheres coupled with a flow cytometer detection
systems.
19. The method according to claim 1 wherein the means for measuring
the levels of said at least one pro-inflammatory cytokine in said
biological sample comprises ELISAs, radioimmunoassays, multiplexed
ELISAs on microarray platforms, multiplexed ELISAs using coded
microspheres coupled with a flow cytometer detection systems,
bioassays, Western blots, chromatograph-based separation systems,
RT-PCR, competitive reverse transcription PCR, Northern blots, gene
arrays, direct measurement of m-RNA, and mixtures thereof.
20. The method according to claim 19 wherein the means for
measuring the levels of said at least one pro-inflammatory cytokine
in said biological sample comprises ELISAs, RIAs, multiplexed
ELISAs using coded microspheres coupled with a flow cytometer
detection systems, and mixtures thereof.
21. The method according to claim 20, wherein the means for
measuring the levels of said at least one pro-inflammatory cytokine
in said biological sample comprises multiplexed ELISAs using coded
microspheres coupled with a flow cytometer detection systems.
22. A kit comprising a first measuring element or system for
measuring at least one anti-inflammatory cytokine in a biological
sample from a mammalian subject before treatment and at at least
one time point after or during treatment, a second measuring
element or system for measuring at least one pro-inflammatory
cytokine in a biological sample from said mammalian subject before
treatment, and at at least one time point after or during
treatment, wherein the change in ratio of anti-inflammatory to
pro-inflammatory cytokine after administration of the treatment can
be determined.
23. The kit according to claim 22, wherein said kit further
comprises language indicating that an increase in said ratio of
anti-inflammatory cytokine to said pro-inflammatory cytokine is
indicative of an inhibitor of inflammatory diseases of the bowel.
Description
TECHNICAL FIELD
[0001] The present invention relates to the field of inflammatory
diseases of the bowel, particularly to methods for determining
efficacy of treatments of these diseases. Furthermore, kits to
carry out the method of the present invention are also
provided.
BACKGROUND OF THE INVENTION
[0002] Inflammatory diseases of the bowel is the general term for
diseases that cause inflammation of the intestines such as
irritable bowel syndrome (IBS), and the inflammatory bowel diseases
(IBD) such as ulcerative colitis and Crohn's disease that are
chronic inflammatory disorders of the gastrointestinal (GI) tract.
For example, ulcerative colitis is an IBD that causes inflammation
of the mucosa lining of the large intestine, usually occurring in
the rectum and lower part of the colon, but it may affect the
entire colon. Crohn's disease may affect any section of the GI
tract (i.e. mouth, oesophagus, stomach, small intestine, large
intestine, rectum and anus), and may involve all layers of the
intestinal wall. The cause of many of these diseases is
unknown.
[0003] IBS is a functional gastrointestinal disorder in which
abdominal discomfort or pain is associated with defecation or
change in bowel habit, and with features of disordered defecation.
Theses symptoms represent a condition in which disturbances in
motor and/or sensory function of the gut may be associated with
psychosocial disorders, and the interaction leads to symptoms at
several levels of the gastrointestinal tract.
[0004] IBS is now considered to be the most common gastrointestinal
disorder. Prevalence in western world is estimated to be 15-20% of
the adolescent and adult population and the disorder accounts for
20-50% of the referrals to gastroenterology clinics.
[0005] Current approaches to management of IBS consist of
identification of symptoms consistent with the syndrome and the
exclusion of organic disease with similar presentation, followed by
non-pharmacological and pharmacological therapies, where
appropriate. Current pharmacological therapeutic options are
limited and the effectiveness of many is poorly documented. The
current pharmacological therapies aim at treating symptoms with the
rationale being either to modulate intestinal motility, decrease
visceral sensitivity or treat associated disorders, particularly
anxiety or depression.
[0006] The most common symptoms of IBD include abdominal pain,
tenesmus, fecal urgency and bloody diarrhoea. Sufferers may also
experience fatigue, weight loss, loss of appetite, rectal bleeding
and loss of body fluids and electrolytes. The symptoms of the
disease are usually progressive, and sufferers typically experience
periods of remission followed by severe flare-ups.
[0007] Despite the prevalence of IBD (it affects an estimated 2
million people in the United States alone), there is no cure and
the exact causes of the disease are not yet understood.
Conventional treatments for IBD have involved anti-inflammatory
drugs, immunosuppressive drugs and surgery. However, many of the
drugs used for treating the disease have negative side effects such
as nausea, dizziness, anaemia, leukopaenia, skin rashes and drug
dependence, and the surgical procedures are often radical
procedures, such as intestinal resectomy and colectomy.
[0008] This has led to several investigators to attempt to identify
new and novel drugs for treatment of the inflammatory diseases of
the bowel. Unfortunately, the very nature of the disease means that
measuring the efficacy of potential treatments in human subjects is
very difficult. Often, the results of human trials depend upon
subjective testimony from the trial candidates themselves, with
little or no biochemical or physiological data to substantiate
claims. Animal models may be used to allow tissue sections from
affected organs to be taken, but drugs effective in animal models
do not always have the same efficacy in humans.
[0009] The control of inflammatory diseases is exerted at a number
of levels. The controlling factors include hormones,
prostaglandins, reactive oxygen and nitrogen intermediates,
leukotrienes and cytokines. Cytokines are low molecular weight
biologically active proteins that are involved in the generation
and control of immunological and inflammatory responses. A number
of cell types produce these cytokines, with neutrophils, monocytes
and lymphocytes being the major sources during inflammatory
reactions due to their large numbers at the injured site.
[0010] Multiple mechanisms exist by which cytokines generated at
inflammatory sites influence the inflammatory response. Chemotaxis
stimulates homing of inflammatory cells to the injured site, whilst
certain cytokines promote infiltration of cells into tissue.
Cytokines released within the injured tissue result in activation
of the inflammatory infiltrate. Most cytokines are pleiotropic 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.
As uncontrolled inflammatory responses can result in diseases such
as inflammatory diseases of the bowel, it is reasonable to expect
that cytokine production has gone astray in individuals affected
with these diseases. However, as many cytokines may have both pro-
and anti-inflammatory activities, it is very difficult to attribute
disease symptoms, or recovery there from, with a particular
individual cytokine.
[0011] Based on the forgoing, it is desirable to provide methods
for measuring the efficacy of potential treatments for inflammatory
diseases of the bowel in humans or other mammals that generate
biochemical or physiological data. This data could be used to
evaluate the efficacy of the treatment. It is further desirable to
provide methods for measuring changes in the levels of specific
cytokines potentially involved in the pathogenesis of inflammatory
diseases of the bowel such that the prognosis and disease
progression of a subject with inflammatory diseases of the bowel
can be monitored.
SUMMARY OF THE INVENTION
[0012] The present invention provides novel methods for determining
the efficacy of a treatment of inflammatory diseases of the bowel
in mammals comprising the steps of:
[0013] a) measuring the level of at least one anti-inflammatory
cytokine and at least one pro-inflammatory cytokine in a biological
sample from a mammalian subject;
[0014] b) determining the ratio of the at least one
anti-inflammatory cytokine to the at least one pro-inflammatory
cytokine;
[0015] c) administering said treatment;
[0016] d) measuring the level of the at least one anti-inflammatory
cytokine and the at least one pro-inflammatory cytokine in a
biological sample from said mammalian subject at a time following
administration of said treatment;
[0017] e) determining the ratio of the at least one
anti-inflammatory cytokine to the at least one pro-inflammatory
cytokine following administration of said treatment;
[0018] wherein, an increase in the ratio of anti-inflammatory
cytokine to pro-inflammatory cytokine following the administration
of said treatment is indicative of an inhibitor of inflammatory
diseases of the bowel, and no change or a decrease in the ratio of
anti-inflammatory cytokine to pro-inflammatory cytokine following
the administration of said treatment is indicative of said
treatment not being an inhibitor of inflammatory diseases of the
bowel.
[0019] The present invention is also directed towards providing
further uses of the methods herein, and kits for carrying out the
method herein.
BRIEF DESCRIPTION OF THE FIGURES
[0020] FIG. 1 is a bar graph demonstrating the mean ratio of IL-10
to IL-12 generated from PBMCs from IBS patients with in vitro
stimulation both pre- and post-feeding with Bifidobacteria
infantis.
[0021] FIG. 2 is a bar graph demonstrating the mean ratio of IL-10
to TNF-.alpha. generated from PBMCs from IBS patients with in vitro
stimulation both pre- and post-feeding with Bifidobacteria
infantis.
[0022] FIG. 3 is a bar graph demonstrating the mean ratio of IL-10
to IFN-.gamma. generated from PBMCs from IBS patients with in vitro
stimulation both pre- and post-feeding with Bifidobacteria
infantis.
[0023] Table 1 demonstrates the Pearson's correlation coefficients
and p-values for testing the statistical significance of the
negative association between the change in IL-10 to IL-12 ratio and
the change in abdominal pain/discomfort score in IBS patients fed
with Bifidobacterium infantis. Note that the mean IL-10 to IL-12
ratio increased and the mean abdominal pain/discomfort score
decreased from pre- to post-feeding with Bifidobacterium
infantis.
DETAILED DESCRIPTION OF THE INVENTION
[0024] All weights, measurements and concentrations herein are
measured at 25.degree. C. on the composition in its entirety,
unless otherwise specified.
[0025] Unless otherwise indicated, all percentages of compositions
referred to herein are weight percentages and all ratios are weight
ratios.
[0026] Unless otherwise indicated, all molecular weights are weight
average molecular weights.
[0027] Unless otherwise indicated, the content of all literature
sources referred to within this text are incorporated herein in
full by reference.
[0028] Except where specific examples of actual measured values are
presented, numerical values referred to herein should be considered
to be qualified by the word "about".
[0029] As used herein, "inflammatory diseases of the bowel" include
"irritable bowel syndrome--IBS" and "inflammatory bowel
disease--IBD".
[0030] As used herein, "inflammatory bowel disease" or "IBD"
includes diseases that cause inflammation of the intestines such as
ulcerative colitis and Crohn's disease.
[0031] Methods and Use
[0032] The present invention is directed towards providing methods
of determining efficacy of a treatment of inflammatory diseases of
the bowel in mammals comprising the steps of:
[0033] a) measuring the level of at least one anti-inflammatory
cytokine and at least one pro-inflammatory cytokine in a biological
sample from a mammalian subject;
[0034] b) determining the ratio of the at least one
anti-inflammatory cytokine to the at least one pro-inflammatory
cytokine;
[0035] c) administering said treatment;
[0036] d) measuring the level of the at least one anti-inflammatory
cytokine and the at least one pro-inflammatory cytokine in a
biological sample from said mammalian subject at a time following
administration of said treatment;
[0037] e) determining the ratio of the at least one
anti-inflammatory cytokine to the at least one pro-inflammatory
cytokine following administration of said treatment;
[0038] wherein, an increase in the ratio of anti-inflammatory
cytokine to pro-inflammatory cytokine following the administration
of said treatment is indicative of an inhibitor of inflammatory
diseases of the bowel, and no change or a decrease in the ratio of
anti-inflammatory cytokine to pro-inflammatory cytokine following
the administration of said treatment is indicative said treatment
not being an inhibitor of inflammatory diseases of the bowel. The
method of the present invention may further comprise repeating
steps (d) and (e) at least once at similar time points following
the administration of said treatment. Furthermore, the method of
the present invention may comprise repeating steps (d) and (e) at
least once at similar time points whilst said mammalian subject is
still being administered said treatment. The method of the present
invention may be utilised to both screen and clinically evaluate
unknown or new treatments or compositions for efficacy in the
treatment of inflammatory diseases of the bowel. Furthermore, the
method of the present invention may also be used to monitor the
efficacy of a known treatment in an individual patient with
inflammatory disease of the bowel. Further still, the method herein
may be used to provide a predictive biomarker for inflammatory
diseases of the bowel helpful in diagnosis of the disorder.
[0039] The method herein is suitable for use in screening and
determining clinical efficacy of treatments and compositions for
the treatment of inflammatory diseases of the bowel. These diseases
include inflammatory gastro-intestinal disorders, some non-limiting
examples of which include irritable bowel syndrome (IBS), and IBDs
such as ulcerative colitis and Crohn's disease. Preferably, the
method herein is used to determine the efficacy of treatments for
irritable bowel syndrome (IBS).
[0040] The treatments herein include any treatment and/or
composition for use in the treatment of inflammatory diseases of
the bowel. The compositions may comprise one or more ingredients
that are to have their potential efficacy in the treatment of
inflammatory diseases of the bowel, preferably IBS, determined.
Non-limiting examples of such compositions include
anti-inflammatory drugs, probiotic compositions, new compositions
and compounds not known to have efficacy in the treatment of
inflammatory diseases of the bowel, compositions and compounds
known to alleviate the symptoms of inflammatory diseases of the
bowel including new delivery forms of known drugs useful in the
treatment of inflammatory diseases of the bowel and mixtures
thereof.
[0041] The method of the present invention is also of use in
determining the response of an individual sufferer of inflammatory
diseases of the bowel to a composition useful in the treatment of
inflammatory disease of the bowel. This allows the determination of
the efficacy of a given treatment in an individual sufferer of
inflammatory disease of the bowel, and enables physicians to
monitor the progress of patients, and to determine whether to
change drug type or delivery form in order to optimise the
treatment of the patient. This would result in the patient
receiving better treatment for their disease, and a decrease in the
amount of drugs and money wasted on treatments that are ineffective
on a population of sufferers of inflammatory diseases of the
bowel.
[0042] Biological Sample
[0043] The method of the present invention comprises measuring
cytokine levels in a biological sample obtained from a mammalian
subject both before and during or after administration of said
treatment. Non-limiting examples of mammalian subjects suitable for
use herein include human, simian, canine, feline, bovine, ovine,
porcine, rodent subjects including murine and rat species, rabbit
or equine subjects, preferably a human subject. Biological samples
useful herein will be well-known to one skilled in the art. As used
herein, "biological sample" includes urine, plasma, serum, saliva,
tissue biopsies, cerebrospinal fluid, peripheral blood mononuclear
cells with in vitro stimulation, peripheral blood mononuclear cells
without in vitro stimulation, gut lymphoid tissues with in vitro
stimulation, gut lymphoid tissues without in vitro stimulation, gut
lavage fluids, and mixtures thereof. Preferably, the biological
sample used in the method of the present invention comprises serum,
tissue biopsies, peripheral blood mononuclear cells with in vitro
stimulation, peripheral blood mononuclear cells without in vitro
stimulation, and mixtures thereof, more preferably peripheral blood
mononuclear cells with in vitro stimulation, peripheral blood
mononuclear cells without in vitro stimulation, and mixtures
thereof. Peripheral blood mononuclear cells (PBMC) may be harvested
from EDTA-treated, non-coagulated venous blood using methods known
to those skilled in the art, such as Ficoll-Hypaque density
centrifugation. More preferably still, the method of the present
invention utilizes a biological sample comprising peripheral blood
mononuclear cells with in vitro stimulation. As used herein,
"peripheral blood mononuclear cells" with or without in vitro
stimulation includes freshly harvested PBMC, whole cell homogenates
of freshly harvested PBMC, extracted protein fractions of freshly
harvested PBMC, mRNA transcripts from freshly harvested PBMC,
tissue culture medium supernatants of freshly harvested PBMC,
frozen PBMC, whole cell homogenates of frozen PBMC, extracted
protein fractions of frozen PBMC, mRNA transcripts from frozen
PBMC, tissue culture medium supernatants of frozen PBMC,, in vitro
cultures of harvested PBMC, whole cell homogenates of in vitro
cultures of harvested PBMC, extracted protein fractions of in vitro
cultures of harvested PBMC, mRNA transcripts from in vitro cultures
of harvested PBMC, tissue culture medium supernatants of in vitro
cultures of harvested PBMC, and mixtures thereof.
[0044] As used herein "in vitro stimulation" includes the
stimulation of biological samples outside of the donor's body,
typically in a laboratory tissue culture setting. Preferably, the
stimulus comprises a mitogen, probiotic, anti-CD3 molecules known
to those skilled in the art, and mixtures thereof. More preferably,
the stimulus comprises a mitogen, probiotic, and mixtures
thereof.
[0045] As used herein, "mitogen" includes materials that are
capable of inducing cell division in a high percentage of T or B
cells. Suitable of examples of mitogens useful herein include
lectins, bacterial lipopolysaccharides, super-antigens and mixtures
thereof. As used herein, "super-antigen" includes materials that
can bind to residues in the V (variable) domain of the T-cell
receptor and to residues in class II MHC molecules outside of the
antigen-binding cleft, even when the T-cell receptor does not
recognise the antigenic peptide bound to the class II MHC molecule.
Suitable examples of super-antigens useful herein include
staphylococcal enterotoxins, toxic shock syndrome toxin 1, and
mixtures thereof. Preferably, the mitogen comprises lectins,
bacterial lipopolysaccharides, and mixtures thereof. Suitable
examples of lectins useful herein include concanavalin A (isolated
from Jack beans), phytohemagglutinin (isolated from kidney beans),
pokeweed mitogen (isolated from pokeweed) and mixtures thereof,
preferably phytohemagglutinin (PHA). Suitable examples of bacterial
lipopolysaccharides useful herein include Escherichia coli (E.
coli) 0111:B4, E. coli 055:B5, E. coli K-235 (all available from
Sigma (St Louis, Mo.)), Salmonella Minnesota, Salmonella
typhimurium, Shigella flexneri, Klebisella pneumonia, Pseudomonas
aeruginosa, and mixtures thereof.
[0046] Probiotics are micro-organisms, or processed compositions of
micro-organisms which beneficially affect a host. How probiotics
beneficially affect the host is unknown. For the purpose of the
present invention, "probiotics" is further intended to include the
metabolites generated by the micro-organism during a fermentation
process, if they are not separately indicated. These metabolites
may be released to the medium of fermentation, or they may be
stored within the micro-organism. As used herein "probiotic" also
includes bacteria, bacterial homogenates, bacterial proteins,
bacterial extracts, bacterial supernatants, and mixtures thereof,
that perform beneficial functions for the host when given at a
therapeutic dose. Therefore, yeasts, moulds and bacteria may be
included. EP 0862863 lists some examples of probiotics presently
known. Suitable examples of probiotics useful herein comprise
strains of Bifidiobacterium longum infantis (NCIMB 35624) (See
also, WO 00/42168, Collins et al., published Jul. 20, 2000),
Lactobacillus johnsonii (CNCM 1-1225), Bifidobacterium lactis
(DSM20215), Lactobacillus paracasei (CNCM 1-2216), and mixtures
thereof. Further non-limiting examples of probiotics useful herein
are described in WO 03/010297 A1, WO 03/010298 A1, WO 03/010299 A1
(all published Feb. 6, 2003).
[0047] Cytokines
[0048] The method of the present invention comprises measuring at
least one anti-inflammatory and at least one pro-inflammatory
cytokine levels in a biological sample obtained from a mammalian
subject, both before and after treatment with the composition of
interest. It is known to those skilled in the art that cytokines
are pleiotropic, and express multiple biologically overlapping
activities. Accordingly, it should be understood that although the
cytokines useful herein are categorized by their inflammatory
action in the present system, some such ingredients can in some
instances provide more than one immune response action. Therefore,
the classifications herein are made for the sake of
convenience.
[0049] Anti-inflammatory cytokines useful in the present invention
comprise those known in the art. Non-limiting examples of
anti-inflammatory cytokines useful herein include interleukin-4,
interleukin-5, interleukin-13 interleukin-10, transforming growth
factor-, and mixtures thereof. Preferred anti-inflammatory
cytokines for use in the present invention include interleukin-10
(IL-10: accession number: CAA55201; GI accession ID: 14625940),
transforming growth factor-.beta. isoforms 1, 2, 3 and 4, and
mixtures thereof. Pro-inflammatory cytokines useful in the present
invention comprise those known in the art. Non-limiting examples of
pro-inflammatory cytokines useful herein include interleukin-2,
heterodimeric interleukin-12, tumour necrosis factor-.alpha.,
tumour necrosis factor-.beta., interferon-.gamma., and mixtures
thereof. Preferred pro-inflammatory cytokines useful herein include
heterodimeric interleukin-12 (IL-12: accession number: chain A;
1F45A, chain B; 1F45B; GI accession ID: chain A; 1479640; chain B;
1479641), tumour necrosis factor-.alpha. (TNF: accession number:
AAC03542; GI accession ID: 2905634), interferon-.gamma. (INF:
accession number: NP.sub.--000610; GI accession ID: 10835171), and
mixtures thereof.
[0050] Means for Measuring Levels
[0051] According to the method of the present invention, the levels
of at least one anti-inflammatory cytokine and at least one
pro-inflammatory cytokine in the biological sample are measured.
Means for measuring the levels of anti-inflammatory or
pro-inflammatory cytokines, or both, comprise methods known to
those skilled in the art. The levels of said anti-inflammatory and
said pro-inflammatory cytokines may be measured by measuring mRNA
expression or protein expression, as is known to one skilled in the
art. Non-limiting examples of such methods include immunosorbent
assays, enzyme-linked immunosorbent assays (ELISAs),
radioimmunoassays (RIAs), multiplexed ELISAs on microarray
platforms, multiplexed ELISAs using coded microspheres coupled with
a flow cytometer detection systems, bioassays, Western blots,
chromatograph-based separation systems, RT-PCR, competitive reverse
transcription PCR, Northern blots, gene arrays, direct measurement
of m-RNA, and mixtures thereof, preferably ELISAs, RIAs,
multiplexed ELISAs using coded microspheres coupled with a flow
cytometer detection systems, chromatograph-based separation
systems, western blots, and mixtures thereof. More preferably the
means comprises multiplexed ELISAs using coded microspheres coupled
with a flow cytometer detection systems. Suitable ELISAs for use in
the method of the present invention comprise those known to those
skilled in the art, non-limiting examples of which include direct
ELISAs, indirect ELISAs, direct sandwich ELISAs, indirect sandwich
ELISAs, and mixtures thereof. A non-limiting example of
commercially available multiplexed ELISAs using coded microspheres
coupled with a flow cytometer detection systems suitable for use
herein is the LINCOplex kit assay available from Linco Research
Inc., Missouri, USA coupled with a BIOPLEX BEAD FLOW CYTOMETER.TM.
from Bio Rad GmbH.
[0052] Ratio
[0053] The method of the present invention comprises the steps of
determining the ratio of at least one anti-inflammatory cytokine to
at least one pro-inflammatory cytokine before administration of the
treatment, and determining the same ratio either during the
treatment or following completion of the treatment. As used herein,
the ratio of anti-inflammatory cytokine to pro-inflammatory
cytokine means the level of anti-inflammatory cytokine divided by
the level of pro-inflammatory cytokine. Such ratio can be described
by the formula: 1 Ratio = Level of Anti - Inflammatory Cytokine
Level of Pro - Inflammatory Cytokine
[0054] Without wishing to be bound by theory, it is believed that
according to the present invention, if the ratio as determined
herein increases after or during treatment, when compared with the
ratio determined prior to the commencement of treatment, then the
treatment is considered to be an inhibitor of inflammatory diseases
of the bowel. The ratio herein can be determined using any
anti-inflammatory cytokine, and any pro-inflammatory cytokine
herein. Preferably, the ratios useful herein include the ratio
IL-10/IL-12, the ratio IL-10/IFN-.gamma., the ratio of
IL-10/TNF-.alpha., and mixtures thereof. Without wishing to be
bound by theory, it is believed that the specific ratios described
herein are pivotal to the progression or remission of inflammatory
diseases of the bowel, and therefore alterations in the ratios
herein are indicative of the inhibition or promotion of disease
effects by treatments being investigated. It appears that
individuals with inflammatory diseases of the bowel have a skewed
cytokine profile that is indicative of an inflammatory condition.
Similarly, the cytokines used herein that demonstrate the greatest
change following treatment with inhibitors of inflammatory diseases
of the bowel indicate that sufferers of these diseases have PBMC
that are biased towards greater Th-1 activity, altering the normal
Th-1/Th-2 cytokine balance. It has surprisingly been found that by
increasing the ratios described herein, the symptoms of
inflammatory diseases of the bowel can be alleviated. Without
wishing to be bound by theory, it is believed that this is due to
the fact that by increasing these ratios in sufferers of
inflammatory diseases of the bowel, the treatment brings the ratios
back up towards, or close to, those levels found in healthy
subjects.
[0055] Kits
[0056] According to the present invention, kits are provided for
carrying out the method of the present invention. Preferably the
kits comprise a first measuring element or system for measuring at
least one anti-inflammatory cytokine in a biological sample from a
mammalian subject before treatment and at at least one time point
after or during treatment, a second measuring element or system for
measuring at least one pro-inflammatory cytokine in a biological
sample from said mammalian subject before treatment, and at at
least one time point after or during treatment, wherein the change
in ratio of anti-inflammatory to pro-inflammatory cytokine after
administration of the treatment can be determined. Such measuring
elements or systems may include those known to one skilled in art,
non-limiting examples of which include immunosorbent assays,
enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays
(RIAs), multiplexed ELISAs on microarray platforms, multiplexed
ELISAs using coded microspheres coupled with a flow cytometer
detection systems, bioassays, Western blots, chromatograph-based
separation systems, RT-PCR, competitive reverse transcription PCR,
Northern blots, gene arrays, direct measurement of m-RNA, and
mixtures thereof, preferably ELISAs, RIAs, multiplexed ELISAs using
coded microspheres coupled with a flow cytometer detection systems,
chromatograph-based separation systems, western blots, and mixtures
thereof, more preferably multiplexed ELISAs using coded
microspheres coupled with a flow cytometer detection systems. The
kits of the present invention may further comprise means for
obtaining the biological sample from the subject. Such means may
comprise venous blood collection tubes and devices for collecting
said blood, sample tubes for urine or saliva, means for separating
PBMCs from venous blood, and mixtures thereof.
[0057] Furthermore, the kits of the present invention may comprise
instructions for use. The instructions for use may comprise
language indicating how to determine the ratio according to the
present invention, and/or language indicating what the results of
the performed method mean. A non-limiting example of such
instructions includes language indicating that an increase in the
ratio of anti-inflammatory cytokine to pro-inflammatory cytokine is
indicative of an inhibitor of inflammatory diseases of the
bowel.
EXAMPLES
[0058] Ten healthy adults and 13 self-reported irritable bowel
syndrome (IBS) patients were fed daily with probiotic preparation
of 100 ml milk containing 10.sup.8 colony forming units (CFUs)/ml
of Bifidobacterium infantis 35624 (a potential treatment of IBS)
for 3 weeks. Venous blood was drawn from each subject before and
after oral feeding with the probiotic preparation. Venous blood was
collected into blood collection tubes (CPT.TM. VACUTAINER cell
separation tubes containing sodium heparin, Becton Dickinson,
Franklin Lakes, N.J.). PBMC were isolated from the blood using by
centrifugation at 400.times.g, and subsequently cultured in vitro
in appropriate cell culture conditions as known to one skilled in
the art. Dulbecco's Minimum Essential Media (DMEM) supplemented
with 100 U/ml penicillin G/100 .mu.g/ml streptomycin (Gibco), 2.5
.mu.g/ml fungizone (Gibco), 292 .mu.g/ml L-glutamine (Gibco) and
10% foetal calf serum (#1103155, Gibco) was used herein for all
cell cultures except for the measurement of TGF-.beta., wherein the
foetal calf serum was left out. The isolated PBMC suspension was
adjusted to a viable cell count of 1.3.times.10.sup.6/ml and were
cultured either alone in medium (no stimulation), or with 0.1
.mu.g/ml E. coli 0111B4 lipopolysaccharide (Sigma, St Louis, Mo.),
1 .mu.g/ml phytohemagglutinin (PHA--Sigma, St Louis, Mo.) or
10.sup.7 CFU/ml whole Bifidobacterium infantis 35624 for 3 days.
Subsequently, cell suspensions were collected into microfuge tubes
and centrifuged in an MRX-152 microfuge (Tomy Tech, Palo Alto,
Calif.) at 10 000 rpm at 4.degree. C. for 3 minutes and
supernatants collected for analysis.
[0059] The quantity of cytokines in supernatants were analysed by
using the commercially available LINCOplex kit assay (from Linco
Research Inc., Missouri, US) in a BIOPLEX BEAD FLOW CYTOMETER.TM.
(Bio Rad, Hercules, Calif.). Twenty-five .mu.l of culture
supernatant were incubated with a panel of microsphere beads coated
with antibodies specific to the cytokines interleukin-10 (IL-10),
interleukin-12 (IL-12), tumour necrosis factor-.alpha. (TNF),
interferon-.gamma. (INF), and transforming growth factor-.beta.
(TGF) (LINCOplex kit assay from Linco Research Inc., Missouri, US).
Bead mixtures were washed and further incubated with
streptavidin-phycoerythrin at room temperature for 30 minutes.
Concurrently, controls and standards, prepared in serial dilutions
were also incubated with the aforementioned procedures. Bead
suspensions were washed and resuspended in buffer for reading by
the BIOPLEX system. The cytokine levels were quantitated in units
of .mu.g/ml. The cytokine levels in both populations and the
feeding effects were analysed statistically with Student's paired
t-test.
[0060] Daily oral feeding with probiotic Bifidobacterium infantis
35624 for 3 weeks increased the ratio IL-10/IL-12 from 11.2.+-.3.9
(mean.+-.standard error) to 409.5.+-.95.2 in IBS patients' PBMC
with in vitro stimulation by Bifidobacterium infantis 35624.
Similarly, when PBMC from the IBS population were stimulated in
vitro with other probiotics including Lactobacillus 299V and
Lactobacillus GG, similar increases in the IL-10/IL-12 ratio were
observed (see FIG. 1).
[0061] Additionally, probiotic stimulation (Bifidobaterium) of PBMC
from IBS patients after feeding demonstrated an increase from
0.1.+-.0.0 to 2.0.+-.0.5 in the ratio IL-10/TNF-.alpha.; similar
results were also seen following stimulation with other probiotics
including Lactobacillus 4331 and Lactobacillus 299V (see FIG. 2).
Also, stimulation with probiotic Bifidobacterium infantis 35624
resulted in an increase in the IL-10/IFN-.gamma. ratio from
0.7.+-.0.1 to 14.1.+-.2.3 following treatment (see FIG. 3).
[0062] Furthermore, it was observed that the mean abdominal
pain/discomfort decreased and the mean IL-10 to IL-12 ratio
increased in those IBS patients treated with Bifidobacterium
infantis. The negative correlation between the change in abdominal
pain/discomfort and the change in IL-10 to IL-12 ratio
indicatedthat the increase in IL-10 to IL-12 ratio was associated
with the relief from IBS symptom of abdominal pain/discomfort (see
Table 1).
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