U.S. patent application number 17/840923 was filed with the patent office on 2022-09-29 for method for selection of agents influencing intestinal motility disorders and pain.
The applicant listed for this patent is BioGaia AB. Invention is credited to John Bienenstock, Eamonn Connolly, Wolfgang Kunze.
Application Number | 20220306984 17/840923 |
Document ID | / |
Family ID | 1000006402605 |
Filed Date | 2022-09-29 |
United States Patent
Application |
20220306984 |
Kind Code |
A1 |
Connolly; Eamonn ; et
al. |
September 29, 2022 |
METHOD FOR SELECTION OF AGENTS INFLUENCING INTESTINAL MOTILITY
DISORDERS AND PAIN
Abstract
A method is provided for evaluating agents for the treatment of
different intestinal motility disorders, using distinct
methodological parts related to musculature and nerves of the GI
tract which communicate with the brain. In particular, the present
invention provides a method for the selection of an agent effective
for the treatment of an intestinal motility disorder, wherein said
method comprises: a) a step of spatiotemporal (ST) mapping carried
out on a gastrointestinal segment to analyse the effect of said
agent on gastrointestinal motility; and b) a step of ex vivo nerve
bundle recording carried out on a gastrointestinal segment to
analyse the effect of said agent on mesenteric afferent nerve
firing. Bacterial strains selected by the methods of the invention
and the use of said bacterial strains in the treatment of
intestinal motility disorders are also provided.
Inventors: |
Connolly; Eamonn; (Lidingo,
SE) ; Kunze; Wolfgang; (Hamilton, CA) ;
Bienenstock; John; (Toronto, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BioGaia AB |
Stockholm |
|
SE |
|
|
Family ID: |
1000006402605 |
Appl. No.: |
17/840923 |
Filed: |
June 15, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16217507 |
Dec 12, 2018 |
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17840923 |
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15705922 |
Sep 15, 2017 |
10174388 |
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16217507 |
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15389577 |
Dec 23, 2016 |
9816150 |
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15705922 |
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14425470 |
Mar 3, 2015 |
9555065 |
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PCT/EP2013/068202 |
Sep 3, 2013 |
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15389577 |
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61696277 |
Sep 3, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G06T 2207/30092
20130101; G06T 7/13 20170101; G06T 7/0012 20130101; A61K 35/74
20130101; A61K 49/0004 20130101; A61K 35/747 20130101; A61K 2035/11
20130101; C12R 2001/225 20210501; C12N 1/20 20130101; A23V 2002/00
20130101; A23L 33/135 20160801; C12N 1/205 20210501 |
International
Class: |
C12N 1/20 20060101
C12N001/20; A61K 49/00 20060101 A61K049/00; G06T 7/13 20060101
G06T007/13; A61K 35/74 20060101 A61K035/74; G06T 7/00 20060101
G06T007/00; A23L 33/135 20060101 A23L033/135; A61K 35/747 20060101
A61K035/747 |
Claims
1. A method of treating poor nutritional uptake without increasing
the risk for constipation in a subject having poor nutritional
uptake, the method comprising administering to the subject a
Lactobacillus reuteri bacterial strain that has differential
effects on transit time in the jejunum and in the colon, wherein
the L. reuteri bacterial strain decreases MMC frequency and MMC
velocity in the jejunum, increases MMC frequency and MMC velocity
in the colon, thereby increasing transit time of material through
the jejunum, resulting in increased nutritional uptake in the
jejunum and decreasing the transit time of material through the
colon, resulting in reduced risk for constipation, thereby treating
poor nutritional uptake without increasing the risk for
constipation.
2. The method of claim 1, wherein the L. reuteri bacterial strain
decreases mesenteric afferent nerve firing in both jejunum and
colon, thereby decreasing pain signalling in the jejunum and
colon.
3. The method of claim 1, wherein decreasing MMC frequency and MMC
velocity in the jejunum of the subject reduces jejunum motility and
increasing MMC frequency and MMC velocity in the colon of the
subject increases colon motility.
4. The method according to claim 1, wherein said L. reuteri
bacterial strain is L. reuteri DSM 17938, having been deposited
under DSMZ Accession No. DSM 17938.
5. The method of claim 1, wherein the subject having or at risk of
having poor nutritional uptake is a newborn, an infant, a toddler
or a child.
6. The method of claim 1, wherein the subject having or at risk of
having poor nutritional uptake is an elderly subject.
7. A method of decreasing migrating motor complex (MMC) frequency
and MMC velocity in the jejunum and increasing MMC frequency and
MMC velocity in the colon of a subject diagnosed with poor
nutritional uptake, the method comprising administering to said
subject diagnosed with poor nutritional uptake a Lactobacillus
reuteri bacterial strain that has differential effects on MMC
frequency and velocity in the jejunum and colon, wherein the L.
reuteri bacterial strain decreases MMC frequency and MMC velocity
in the jejunum of the subject and increases MMC frequency and MMC
velocity in the colon of the subject, thereby increasing transit
time of material through the jejunum and decreasing transit time of
material through the colon.
8. The method of claim 7, wherein decreasing MMC frequency and MMC
velocity in the jejunum of the subject reduces jejunum motility and
increasing MMC frequency and MMC velocity in the colon of the
subject increases colon motility.
9. The method of claim 7, wherein increasing transit time results
in increased nutritional uptake in the jejunum and decreasing
transit time of material through the colon reduces the risk for
constipation.
10. The method of claim 7, wherein the L. reuteri bacterial strain
decreases mesenteric afferent nerve firing in both jejunum and
colon, thereby decreasing pain signalling in the jejunum and
colon.
11. The method according to claim 7, wherein said L. reuteri
bacterial strain is L. reuteri DSM 17938, having been deposited
under DSMZ Accession No. DSM 17938.
12. The method of claim 7, wherein the subject diagnosed with poor
nutritional uptake is a newborn, an infant, a toddler or a
child.
13. The method of claim 7, wherein the subject diagnosed with poor
nutritional uptake is an elderly subject.
Description
STATEMENT OF PRIORITY
[0001] This application is a continuation application of U.S.
application Ser. No. 16/217,507, filed Dec. 12, 2018, which is a
continuation application of U.S. application Ser. No. 15/705,922,
filed Sep. 15, 2017, allowed, which is a continuation application
of U.S. application Ser. No. 15/389,577, filed Dec. 23, 2016, now
U.S. Pat. No. 9,816,150, which is a divisional application of U.S.
application Ser. No. 14/425,470, filed Mar. 3, 2015, now U.S. Pat.
No. 9,555,065, which is a 35 U.S.C. .sctn. 371 national phase
application of International Application Serial No.
PCT/EP2013/068202, filed Sep. 3, 2013, which claims the benefit of
U.S. Provisional Patent Application No. 61/696,277; filed Sep. 3,
2012, the entire contents of each of which are incorporated by
reference herein.
BACKGROUND OF THE INVENTION
Field of the Invention
[0002] The term intestinal motility disorders applies to abnormal
intestinal contractions often associated with pain, constipation or
diarrhea. This phrase is used to describe a variety of disorders in
which the gut has not developed properly or lost its ability to
coordinate muscular activity due to various causes. Such disorders
may manifest in a variety of ways, and includes but are not limited
to the following: [0003] Abdominal distention [0004] Recurrent
obstruction [0005] Abdominal colicky pain [0006] Constipation
[0007] Gastroesophageal reflux disease [0008] Intractable,
recurrent vomiting [0009] Diarrhea [0010] Irritable bowel syndrome
(IBS) [0011] Inflammatory bowel disease [0012] Fecal incontinence
[0013] Infantile colic [0014] Frequent recurrent abdominal pain
(FRAP) [0015] Regurgitation [0016] Food intolerance
[0017] In a broad sense, any significant alteration in the transit
of foods and secretions into the digestive tract may be considered
an intestinal motility disorder.
[0018] Proper coordinated movements of the stomach and intestines
are required to digest and propel intestinal contents along the
digestive tract. The patterns of contraction and relaxation
necessary for proper motility of the gastrointestinal (GI) tract
are complex and uses the nerves and muscles within the GI walls.
Every day, at any time, many factors can influence GI motility
(e.g., physical exercise, emotional distress). Newborn infants have
to develop the complex system of motility in the GI tract. The
pathogenesis of intestinal motility disorders is in most cases
multifactorial.
[0019] Although the overall structural organization of nerves and
musculature is similar throughout the small and large intestine,
each part has distinct motor activities. Stationary or short
propagated contractions with occasional longer peristaltic and
antiperistaltic complexes occur during and after feeding and this
allows the food bolus to be broken up and mixed with digestive
juices for proper absorption. After digestion the motor program
changes to one with longer propagated contractions that move
digested products in the anal direction. Other motor activity
occurs, which is more regular, making the peristaltic waves (i.e.,
contractions of the circular musculature of the small intestine)
that allow progression of undigested food through the
intestines.
[0020] Aging, dementia, stroke, Parkinson disease, spinal cord
injuries, rectal tears during birthing, diabetes, surgical
complications, and neuromuscular disorders (e.g., myasthenia
gravis) may cause motility disorders.
[0021] IBS, a commonly diagnosed disorder of intestinal motility,
has been considered a disease of the colon for decades, but
research on GI motility has demonstrated that underlying motility
disturbances can occur also in the small bowel. IBS may be
accompanied by abdominal pain.
[0022] The causes of irritable bowel syndrome (IBS) remain unknown.
According to some reports, the small intestine and colon of
patients with IBS are more sensitive and reactive to mild stimuli
than usual.
[0023] Occasionally, fecal incontinence may occur after ingestion
of certain foods. Sugars, insoluble fibers, and starches (except
rice) are normally broken down in the intestines, forming a
variable amount of gas that must be expelled. Most people who have
lactase deficiency cannot digest lactose, a sugar common in several
foods (e.g., milk, cakes). People who have lactose deficiency may
experience uncontrolled liquid diarrhea after lactose
ingestion.
[0024] Constipation is the most common digestive complaint in the
United States but despite its frequency, often remains unrecognized
until the patient develops secondary disorders, such as anorectal
disorders or diverticular disease.
[0025] There is no widely accepted clinically useful definition of
constipation. Health care providers usually use the frequency of
bowel movements (i.e., less than 3 bowel movements per week) to
define constipation. However, the Rome criteria, initially
introduced in 1988 and subsequently modified twice to yield the
Rome III criteria, have become the research-standard definition of
constipation.
[0026] According to the Rome III criteria for constipation, a
patient must have experienced at least 2 of the following symptoms
over the preceding 3 months: [0027] Fewer than 3 bowel movements
per week [0028] Straining [0029] Lumpy or hard stools [0030]
Sensation of anorectal obstruction [0031] Sensation of incomplete
defecation [0032] Manual maneuvering required to defecate
[0033] Constipation is frequently chronic, can significantly affect
an individual's quality of life, and may be associated with
significant health care costs. It is considered chronic if it
occurred for at least 12 weeks (in total, not necessarily
consecutively) during the previous year.
[0034] Constipation commonly has several causes, either primary or
secondary. The most frequent of these are the following: [0035]
Diet that is very poor in fiber [0036] Pregnancy [0037]
Psychological constipation and clinical depression related to
lifestyle changes (e.g., travel, a new job, or divorce), in which
the patient ignores the urge to defecate [0038] Hypothyroidism
[0039] Electrolyte imbalance, especially if it involves Ca.sup.++
or K.sup.+ [0040] Tumors producing mechanical compression on an
intestinal tract, either internally or externally [0041] Nervous
system injuries [0042] Aging [0043] Parkinson's disease [0044]
Intoxication from lead, mercury, phosphorus, or arsenic
[0045] Constipation is quite common during pregnancy. The muscle
contractions that normally move food through the intestines slow
down because of higher levels of the hormone progesterone and
possibly extra iron taken as prenatal vitamin. This is sometimes
also accompanied by lower abdominal pain.
[0046] Constipation is also associated with increased age and the
so called "the aging gut" commonly found especially in people over
70 and in chronic care institutions. This condition is accompanied
by: [0047] Reduced number of neurons in the myenteric plexus
impaired response to direct stimulation leading to myenteric
dysfunction [0048] Increased collagen deposition of the left colon,
leading to abnormalities in colonic and rectal compliance and
dysmotility [0049] Reduction in the amplitude of inhibitory nerve
input to the circular muscle layer of the colon, resulting in lack
of segmental motor coordination [0050] Increased binding of plasma
endorphins to intestinal receptors in persons 60 and older
[0051] At the other end of the aging spectrum intestinal motility
disorders, persistent or excessive crying from infant colic is one
of the most distressing problems of infancy. It is distressing for
the infant, the parents, and the involved healthcare professionals.
The parents of the irritable infant may view the crying as an
indictment of their caregiving ability or as evidence of illness in
their child. Infantile colic is a condition that resolves with
time.
[0052] The most widely accepted definition for colic is the Wessel
criteria or the "rule of three": crying that lasts for more than
three hours per day, occurs on more than three days per week, and
persists for more than three weeks. This definition also requires
that the infant is "otherwise healthy and well fed".
[0053] Intestinal hypermotility secondary to a presumed autonomic
imbalance also has been proposed as one etiology for colic. Many of
the mechanisms that regulate motor activity are immature in
infants. The immaturity of these mechanisms may result in increased
vulnerability to feeding intolerance. Thus, colic may be a common
clinical manifestation in the subpopulation of infants who have
maturational dysfunction in one or more of the aspects of motility
regulation.
[0054] According to some epidemiologic reports, as many as 30
million Americans have intestinal motility disorders. Available
data from the medical literature indicate that worldwide, 30-45% of
all GI conditions are referable to intestinal motility disorders.
[0055] Present treatments and recommendations vary depending on the
type of [0056] motility disorders, for constipation some examples
are: [0057] Fiber or bulking agents (not useful for slow transit
constipation) [0058] Stool softeners [0059] Stimulant laxatives
[0060] Osmotic laxatives (for example polyethylene glycol) [0061]
Chloride-channel activator (for example Lubiproston) [0062]
Serotonergic agents (5-HT4 receptor agonists)
[0063] Further the use of Lactic Acid Bacteria (LAB) has been
studied in a randomized clinical trial: Efficacy of Lactobacillus
paracasei-enriched Artichokes in the Treatment of Patients With
Functional Constipation (Riezzo et al; Aliment Pharmacol Ther. 2012
February; 35(4):441-50)
[0064] Also, 90 breastfed colicky infants were randomly assigned to
treatment with L. reuteri or simethicone in a trial. At baseline,
median daily crying time was 197 minutes in both groups. Among the
83 infants who completed the trial, median daily crying time was
lower in the L. reuteri than in the simethicone group on day 7 (159
versus 177 minutes) and day 28 (51 versus 145 minutes). (Savino F
et al, Pediatrics. 2007; 119(1):e124).
[0065] Intestinal motility disorders applies to abnormal intestinal
contractions often associated with pain, there are many different
kinds of treatments and recommendations for the different
disorders, some which work better than many others.
[0066] So there is an overall need and specific problems to solve
for various motility disorders namely; How to best select agents to
normalize or treat intestinal motility disorders and gut/intestinal
pain?
SUMMARY OF THE INVENTION
[0067] The inventors of the invention herein have developed a new
method for evaluating agents for the treatment of different
intestinal motility disorders, using distinct methodological parts
related to musculature and nerves of the GI tract and their
communication with the brain.
[0068] Thus, the present invention relates to a method for the
selection of an agent effective for the treatment of an intestinal
motility disorder, wherein said method comprises:
[0069] a) a step of spatiotemporal (ST) mapping carried out on a
gastrointestinal segment to analyse the effect of an agent on
gastrointestinal motility; and
[0070] b) a step of ex vivo nerve bundle recording carried out on a
gastrointestinal segment to analyse the effect of an agent on
mesenteric afferent nerve firing.
[0071] In preferred embodiments the ST mapping in step a) is
carried out by video imaging or recording. In the methods of the
invention, step a) preferably comprises using the ST map generated
in step a) to measure migrating motor complex (MMC) frequency
and/or migrating motor complex (MMC) velocity in said
gastrointestinal segment.
[0072] In the methods of the invention, step a) preferably further
comprises the measurement of intraluminal pressure, for example
intraluminal peak pressure (PPr).
[0073] In the methods of the invention, preferably step b)
comprises measuring the spontaneous firing frequencies of
mesenteric afferent nerve bundles.
[0074] In preferred aspects, the present invention discloses a
two-step method for selection of agents effective in motility
disorders. In the first step the inventors combined intraluminal
pressure recordings with spatiotemporal maps to analyze effects of
different agents on motility. In this step intraluminal peak
pressure (PPr) is preferably measured and video recordings made of
mouse ex vivo jejunum and colon segments before and after
intraluminal applications of agents. Migrating motor complex
frequency and velocity are also calculated in the first step of the
method. In the second step intestinal nerve signaling is analyzed
and the spontaneous firing frequencies of mesenteric afferent nerve
bundles is measured. Thus, the present invention discloses a method
for selection of agents effective in motility disorders comprising
a first step using simultaneous recording of intraluminal pressure
changes in addition to video recording and spatiotemporal maps and
a second step which is an analysis of nerve signaling, for example
by taking mesenteric, e.g. extracellular mesenteric, nerve
recordings.
[0075] Thus, in a yet further embodiment, the present invention
provides a method for the selection of an agent effective for the
treatment of an intestinal motility disorder, comprising the
steps:
[0076] (a) recording intraluminal pressure, creating a
spatiotemporal (ST) map to enable an analysis of the effects of
different agents on gastrointestinal motility wherein said
spatiotemporal map is used to measure migrating motor complex
frequency and velocity, in an intestinal sample in the presence and
absence of a test agent; and
[0077] (b) analysing intestinal nerve signalling by measuring the
spontaneous firing frequencies of mesenteric afferent nerve bundles
in an intestinal sample in the presence and absence of a test
agent.
[0078] In preferred embodiments of this method in step (a)
intraluminal peak pressure (PPr) is measured.
[0079] The present invention relates to the described two-step
method and also to new bacterial strains.
DESCRIPTION OF FIGURES
[0080] FIG. 1 Organ motility recordings
[0081] FIG. 2 Picture and data output from motility recordings
[0082] FIG. 3 Picture and data output from motility recordings
[0083] FIG. 4 Mesenteric nerve recording; innervation of the
intestine
[0084] FIG. 5 Extracellular recordings
[0085] FIG. 6 Details of the StMAP plug-in software
[0086] FIGS. 7A-7G Graphs showing the results of step a). Agents
tested are (A) L. gasseri 345A (DSM 27123), (B) L. gasseri 621A
(DSM 27126), (C) L. gasseri T1, (D) L. gasseri T2, (E) L. reuteri
ATCC PTA 6475, (F) L. reuteri DSM 17938 and (G) L. rhamnosus
(JB-1).
[0087] FIG. 8 Graphs showing the results of step b), agents tested
are L. rhamnosus (JB-1), L. reuteri DSM 17938, L. reuteri ATCC PTA
6475, L. gasseri 345A (DSM 27123), L. gasseri 621A (DSM 27126), L.
gasseri T1 and L. gasseri T2.
DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS
THEREOF
[0088] To facilitate understanding of the invention, a number of
terms are defined below.
[0089] As used herein the term "intraluminal peak pressure" (PPr)
is based on intraluminal pressure recordings, where intraluminal
pressure changes are measured at the midpoint of the longitudinal
axis of the gut segment. The pressure signal is analyzed and
intraluminal peak pressure (PPr) is identified and measured.
[0090] As used herein the term "migrating motor complex frequency"
(MMC frequency) is calculated by counting the number of dark MC
bands in the spatiotemporal maps.
[0091] As used herein the term "migrating motor complex velocity"
(MMC velocity) is measured from the slope(s) of each band in the
spatiotemporal map generated by migrating motor complexes.
[0092] As used herein the term "firing frequency" is used to
measure the sensory spike trains to the brain.
[0093] As used herein the term "agent" is used to mean any
substance or material including whole cells, microorganisms,
proteins, peptides, enzymes, molecules or other biological or
chemical material that can be used to influence motility and or
pain in the gastro intestinal system of a mammal. Preferred agents
are bacterial strains, e.g. probiotic bacterial strains.
[0094] The method of the invention herein is a two step method,
based on a spatio-temporal (ST) mapping method (step 1) combined
with an improved technology for nerve bundle recording ex vivo
(step 2) in which a gastro intestinal segment is excised with or
without the mesenteric arcade containing the nerve bundle supplying
the segment made up of both spinal and vagal fibers.
[0095] In the first step of the presented preferred method
intraluminal pressure recordings are combined with spatiotemporal
(ST) maps to analyze the effects of different agents on
motility.
[0096] ST maps provide a tool for visualizing a temporally evolving
and spatially varying field, which can be used in the analysis of
gastrointestinal motility including behaviors in health and
motility disorders. Using spatiotemporal video imaging the
inventors herein have developed a part of the new method using
computer analysis of spatiotemporal maps of changes in gut
diameter. This reveals motor patterns that are very difficult to
detect with any other technique. The parameters of these patterns
directly correlate results obtained with mouse ex vivo intestinal
segments with human gut pharmacology. The peristaltic propagated
motor complexes (MCs) or mixing movements (stationary MCs) are
digitally recorded in real-time and the images stored. The analysis
software, as described further down, detects the edges of the
intestine and is able to automatically calculate the diameter along
the length of the segment over time. This data is used by the
program to construct spatiotemporal maps that are able to extract
highly specific information about motor patterns; for example,
migrating motor complex (MMC) frequency (neurally dependent MMC
frequency), MMC velocity and myogenic (slow wave related)
contraction rates of contraction.
[0097] Further, it is beginning to be accepted that intestinal
microorganisms signal to the brain as part of the so called
microbiome-gut-brain axis. However, very little is known about the
role of the gut microbiome in the development or function of the
nervous system. Nothing is presently known about the quantitative
nature of the nervous signal relayed from gut to the central
nervous system.
[0098] Single sensory neurons, including those among the vagus
fibers, represent continuous physical stimuli as patterned spike
trains that encode the nature and intensity of the stimulus. In
addition to this, stimuli may be represented in a population code
determined by the number of active fibres in the bundle. All the
information reaching the brain via primary afferents has to be
encoded in the language of neuronal spike trains. Therefore,
knowing how the sensory spike trains are affected by commensals,
probiotic strains and different substances enable us to identify
new beneficial gut microorganisms and their active molecules by
their effects on primary afferent firing as well as new drugs and
other compounds that in various fashion can intervene in this
signaling system.
[0099] The new method of the invention herein for evaluating agents
for the treatment of different intestinal motility disorders allows
differentiation of specific effects of the agents, for example a
probiotic bacterial strain or another biological or chemical agent,
on both muscle and on nerves. Experimental data confirms that it is
not necessarily the same agent that is able to modulate for example
mixing pattern or propulsion pattern in a certain part of the GI
tract that also is the most effective in modulating pain associated
with enteric and/or the central nervous system.
[0100] Furthermore, to investigate the effects of certain molecules
in different diseases, our method allows different studies of
luminal perfusion and diffusion to and across the epithelial
barrier.
Two fundamental patterns of motility are conducted by the digestive
tube:
[0101] Propulsion: foods must be propelled along the full length of
the digestive tube in order to be subjected to the sequential
series of processing involved in disassembly and absorption. The
principal type of propulsive motility, seen particularly in the
esophagus and small intestine, is peristalsis--a ring of muscle
contraction moves from the oral side of a bolus of ingesta and
toward the anus, propelling the contents of the lumen in that
direction; as the ring moves, the muscle on the other side of the
distended area relaxes, facilitating smooth passage of the
bolus.
[0102] Mixing: If ingested materials were simply propelled through
the digestive tube, you would expect very poor digestion and
absorption, because the digestive enzymes would not be adequately
mixed with the ingesta and the bulk of the ingesta would not come
in contact with the epithelial cells that absorb nutrients,
including water. Segmentation contractions are a common type of
mixing motility seen especially in the small intestine--segmental
rings of contraction chop and mix the ingesta. Alternating
contraction and relaxation of the longitudinal muscle in the wall
of the gut also provides effective mixing of its contents.
[0103] Using the new method herein, changed gut motility, seen as
either the mixing pattern (post feeding) or the propulsive pattern,
seen clearly using the ST-map method, in combination with changes
in pain caused by different motility disorders, and manifested
centrally or peripherally, can be detected and recorded.
[0104] One kind of pain is visceral pain that results from the
activation of nociceptors of the abdominal viscera (organs).
Visceral structures are highly sensitive to distension (stretch),
ischemia and inflammation, but relatively insensitive to other
stimuli that normally evoke pain. Visceral pain is diffuse,
difficult to localize and often referred to a distant, usually
superficial, structure. It may be accompanied by symptoms such as
nausea, vomiting, changes in vital signs as well as emotional
manifestations. The pain may be described as sickening, deep,
squeezing, and dull. Distinct structural lesions or biochemical
abnormalities explain this type of pain in only a proportion of
patients. These diseases are sometimes grouped under
gastrointestinal neuromuscular diseases (GINMD). Persons can also
experience visceral pains, often very intense in nature, without
any evidence of structural, biochemical or histolopathologic reason
for such symptoms.
[0105] A nociceptor is a sensory receptor that responds to
potentially damaging stimuli by sending action potentials to
specific nociceptive neurons (A.delta. or C) which transmit to the
anterolateral tracts of the spinal cord (plus a minor vagal
projection) and then to the thalamus, and prosencephalon including
the insular and cingulate cortices. Critical for pain perception
originating in the gut pathology is the activation of pain messages
from the gut to the central nervous system via extrinsic primary
afferent fibres that travel in mesenteric afferent nerve
bundles.
[0106] In the system of the invention herein, changes in motility
and pain, are read outs as alterations in motility patterns (for
example PPr, MMC velocity and/or MMC frequency) or contraction
amplitudes or increased afferent mesenteric nerve spinal traffic
for specific and selected parts of the gut. It has been surprising
to find that different agents can promote mixing in one part of the
GI tract and propulsion in another part, or have no effect at all,
and influence pain signaling systems in one part, but not in
another part of the GI-tract, and via different nerve pathways such
as vagal or for visceral pain through the dorsal root ganglion.
Such precise regional specificity highlights the power and
specificity of our approach in assessing the effects of test
molecules.
[0107] It is therefore an object of the present invention to find
agents suitable for treatment, prevention or modulating specific
motility disorders, by using the model herein.
[0108] The two steps in the method presented will provide data on
gastrointestinal motility and mesenteric afferent nerve firing.
Analyzing these parameters will result in a method of selecting for
agents effective in the treatment of an intestinal motility
disorder. Thus, the analysis of the effect of an agent in step a)
and step b) of the provided methods is used to determine whether
the agent is effective for the treatment of an intestinal motility
disorder.
[0109] Preferred parameters to be measured in step a) are MMC
frequency and/or MMC velocity and this is conveniently done by way
of an STmap which is preferably generated by video imaging, e.g.
using a video recording, and preferably comprises a pattern of
alternating bands of light and dark hues that contains 3 sets of
information: position along the gut (ordinate), time (abscissa) and
gut diameter (applicate or z-axis). Using these variables, the
spatiotemporal map can become a motility "fingerprint" whose
sensitivity is important in defining the detailed and nuanced
effects that specific probiotic strains and other agents have on
motility and the ability to distinguish between them.
[0110] Other preferred parameters to be measured in step a) are
intraluminal pressure, in particular intraluminal peak pressure
(PPr). Other parameters, for example as described in the Examples,
can optionally be measured from the ST map and in step a) of the
method.
[0111] A preferred parameter to be measured in step b) is the
spontaneous firing frequencies of mesenteric afferent nerve
bundles. This technique can be used to determine changes in the
excitability of the mesenteric nerve fibres induced by different
treatments.
[0112] Analyzing one or more of these parameters will result in a
method of selecting agents effective in motility disorders.
Appropriate methods and apparatus for generating the ST map and
measuring these parameters are described in the experimental
Examples and Figures.
[0113] Thus, in preferred methods, the two steps in the method
presented will give data on intraluminal peak pressure (PPr), MMC
frequency, MMC velocity and spontaneous firing frequencies of
mesenteric afferent nerve bundles. Analyzing all of these
parameters will result in a preferred method of selecting agents
effective in motility disorders.
[0114] The methods of the invention may be used to find suitable
agents for different motility disorders. Agents are chosen to
affect the MMC velocity and/or MMC frequency and/or PPr and nerve
firing frequency of the patient in a beneficial way in order to
influence, prevent or treat the motility disorder.
[0115] Step a) of the method uses ST mapping (ST analysis) of a
gastrointestinal segment to analyse the effect of an agent on
gastrointestinal motility. To do this preferably the MMC frequency
and/or MMC velocity are measured using the ST map. Conveniently the
ST map shows images of MC bands from which the frequency and
velocity can be measured, for example as described in the
experimental Examples. In addition, preferably the intraluminal
pressure and, more preferably, the intraluminal peak pressure (PPr)
is measured within the gastrointestinal segment.
[0116] The gastrointestinal segment for use in the methods of the
present invention can be from any appropriate part of the
gastrointestinal tract, for example can be a segment from the small
intestine (e.g. jejunum) or from the large intestine (e.g. colon).
Appropriate segments for step b) of the method will require the
presence of an appropriate nerve bundle to enable the measurement
of mesenteric afferent nerve firing. This can conveniently be
provided by having a gastrointestinal segment with attached
mesenteric tissue, for example as described in the experimental
Examples. Thus, the methods of the invention are conveniently
carried out on ex vivo segments from an appropriate experimental
animal, for example on mouse gastrointestinal segments (e.g. mouse
colon or jejunum segments). Surprisingly, the methods of the
present invention can detect differences in the action of the same
agent on both types of intestinal section. The ability to carry out
a comparison of the effect of an agent on the small versus the
large intestine could be advantageous, particularly as, depending
on the intestinal motility disorder to be treated and the clinical
stage and symptoms thereof, a treatment which is region-specific,
e.g. specific for either the small or large intestine might be
beneficial.
[0117] Changes in gastrointestinal motility induced by an agent can
be detected as a read out in step a) of the method, for example as
an alteration in motility pattern or contraction amplitudes. Some
agents will have no effect at all. An agent which can increase
gastrointestinal motility, for example by increasing the MMC
frequency and/or MMC velocity and/or intraluminal pressure such as
PPr will likely be useful to treat disorders in which it would be
advantageous to increase the propulsive motility along the
digestive tube such as constipation and colic. If an agent is shown
to reduce or not significantly change gastrointestinal motility,
for example by reducing or not significantly changing the MMC
frequency and/or MMC velocity and/or intraluminal pressure such as
PPr, then such an agent would be unlikely to be useful to treat
such disorders and would not be selected, particularly if there is
a reduction or no significant change in all the parameters
measured. However, an agent which can result in a reduction in
gastrointestinal motility, for example by reducing one or more of
MMC frequency and/or MMC velocity and/or intraluminal pressure such
as PPr will likely be useful to treat disorders in which it would
be advantageous to decrease the propulsive motility along the
digestive tube, such as IBS or diarrhea.
[0118] An increase in gastrointestinal motility, for example an
increase in the MMC frequency and/or MMC velocity and/or
intraluminal pressure such as PPr in a segment of large intestine,
by an agent in the methods of the invention is particularly
preferred to select an agent for use to treat constipation or
colic.
[0119] Step b) of the methods of the invention analyses the effect
of an agent on mesenteric afferent nerve firing (pain signaling)
and can thus be used as a read out for pain, i.e. whether or not an
agent is likely to have an effect on pain, e.g. visceral pain. An
increase or no significant effect on afferent nerve firing is
indicative of an agent which is likely to result in an increase in
pain, or no significant effect on pain, respectively, whereas a
decrease in afferent nerve firing is indicative of an agent which
will reduce pain. Preferred agents are thus those that result in a
decrease in afferent nerve firing, e.g. a decrease in the
spontaneous firing frequency of afferent nerve bundles.
Alternatively, an agent which results in no significant change in
nerve firing might be selected if the strain shows advantageous
properties as measured by step a) of the method.
[0120] The agent to be tested is added to the chosen
gastrointestinal segment in any appropriate manner. Conveniently,
the agents are added to the intraluminal space of the segment, i.e.
are applied intraluminally. In order to analyse the effect of the
agent on motility (in step a) or pain signalling (in step b), the
steps are conveniently carried out in the presence and the absence
of the agent. For example, steps a) and b) are carried out before
and after the agent is applied. Thus, in such methods the effect of
the agent is compared to an appropriate control, for example the
results in the presence of the test agent are compared with the
results in the absence of a test agent, e.g. results with buffer
alone as opposed to buffer plus agent (e.g. buffer plus a bacterial
strain).
[0121] Thus, a yet further aspect of the invention provides an
agent selected by the methods of the invention. A preferred agent
is a microorganism, more preferably a bacterial strain, preferably
a probiotic bacterial strain. The agents selected by the methods of
the invention may take the form of a pharmaceutical compound or
composition or a nutritional compound or composition.
[0122] As described above, depending on the motility disorder which
it is desired to be treated, the selection criteria will vary. For
example, if the intestinal motility disorder is one in which it is
desired to reduce the transit time of material through the
intestine (or to increase the propulsive motility along the
digestive tube), e.g. constipation, regurgitation or colic, then in
step a) of the method, an agent of interest will act to increase
gastrointestinal motility, for example by increasing MMC frequency
or MMC velocity or intraluminal pressure (e.g. PPr). Preferred
agents will increase two or more of these parameters, for example
will increase MMC frequency and MMC velocity or will increase MMC
frequency and intraluminal pressure (e.g. PPr), or will increase
MMC velocity and intraluminal pressure (e.g. PPr). Most preferred
agents will increase all of these parameters, for example will
increase MMC frequency, MMC velocity and intraluminal pressure
(e.g. PPr). Step a) of the method can be assessed on an appropriate
gastrointestinal segment from the small or large intestine, for
example a jejunal segment for the small intestine or a colon
segment for the large intestine. In some embodiments, the use of
large intestine, e.g. colon segments, is preferred.
[0123] For the treatment of such disorders, an agent will be
selected which does not effect or does not increase pain
signalling, or preferably acts to decrease pain signalling. In step
b) of the method such agents will have no effect or will preferably
act to reduce or decrease mesenteric afferent nerve firing.
Preferably said agent will have no effect on or will preferably act
to reduce the spontaneous firing frequencies of mesenteric afferent
nerve bundles. Step b) of the method can be assessed on any
appropriate gastrointestinal segment from the small or large
intestine, for example a jejunum segment for the small intestine or
a colon segment for the large intestine. In some embodiments, the
use of small intestine, e.g. jejunum segments is preferred.
[0124] Using the method of the present invention, the inventors
have selected a new bacterial strain, Lactobacillus gasseri 345A
(LG345A), which has been shown to increase gastrointestinal
motility by way of being shown to increase MMC frequency, MMC
velocity and intraluminal peak pressure. This strain has also been
shown to decrease pain signalling by decreasing mesenteric afferent
nerve firing (by decreasing the frequency of firing of mesenteric
afferent nerve bundles). This isolated bacterial strain (and other
strains, e.g. L. gasseri strains, with the characteristics, e.g.
the ability to induce the above mentioned effects on
gastrointestinal motility and pain signalling, of this deposited
strain) forms a preferred aspect of the invention and can be used
to treat intestinal motility disorders, particularly those in which
it is desired to reduce the transit time of material through the
intestine. Thus, preferred conditions to be treated with this
strain are constipation or colic. Strains, e.g. L. gasseri strains,
which can increase gastrointestinal motility by having the ability
to increase each of MMC frequency, MMC velocity and intraluminal
peak pressure, and which can decrease pain signalling, e.g. by
having an ability to decrease mesenteric afferent nerve firing
(e.g. by decreasing the frequency of firing of mesenteric afferent
nerve bundles) form preferred aspects of the present invention.
[0125] Alternatively, if for example, the intestinal motility
disorder for treatment is one in which it is desired to increase
the transit time of material through the intestine (e.g. disorders
involving rapid passage transit), e.g. IBS or diarrhea, then in
step a) of the method, an agent of interest will act to decrease
gastrointestinal motility, for example by decreasing MMC frequency
or MMC velocity or intraluminal pressure, e.g. PPr. Preferred
agents will decrease at least MMC velocity. Preferred agents will
decrease two or more of these parameters, for example will decrease
MMC velocity and MMC frequency or will decrease MMC frequency and
intraluminal pressure (e.g. PPr) or will decrease MMC velocity and
intraluminal pressure (e.g. PPr). Most preferred agents will
decrease all of these parameters, for example will decrease MMC
frequency, MMC velocity and intraluminal pressure (e.g. PPr). Step
a) of the method can be assessed on an appropriate gastrointestinal
segment from the small or large intestine, for example a jejunal
segment for the small intestine or a colon segment for the large
intestine. In some embodiments, the use of large intestine, e.g.
colon, segments is preferred.
[0126] For the treatment of such disorders, an agent will be
selected which does not effect or does not increase pain
signalling, or preferably acts to decrease pain signalling. In step
b) of the method such agents will have no effect or will preferably
act to reduce or decrease mesenteric afferent nerve firing.
Preferably said agent will have no effect on or will preferably act
to reduce the spontaneous firing frequencies of mesenteric afferent
nerve bundles. Step b) of the method can be assessed on an
appropriate gastrointestinal segment from the small or large
intestine, for example a jejunal segment for the small intestine or
a colon segment for the large intestine. In some embodiments, the
use of small intestine, e.g. jejunum, segments is preferred.
[0127] Using the method of the present invention, the inventors
have selected a new strain, Lactobacillus gasseri 621A (LG621A),
which has been shown to decrease gastrointestinal motility by way
of being shown to decrease MMC velocity and have no effect on MMC
frequency or intraluminal peak pressure. This strain has also been
shown to decrease pain signalling by decreasing mesenteric afferent
nerve firing (by decreasing the frequency of firing of mesenteric
afferent nerve bundles). This isolated bacterial strain (and other
strains, e.g. L. gasseri strains, with the characteristics, e.g.
the ability to induce the above mentioned effects on
gastrointestinal motility and pain signalling, of this deposited
strain) forms a preferred aspect of the invention and can be used
to treat intestinal motility disorders, particularly those in which
it is desired to increase the transit time of material through the
intestine (e.g. disorders involving rapid passage transit). Thus,
preferred conditions to be treated with this strain are IBS or
diarrhea. Strains, e.g. L. gasseri strains, which can decrease
gastrointestinal motility, e.g. by having the ability to decrease
one or more of MMC frequency, MMC velocity and intraluminal peak
pressure (preferably MMC velocity), and which can decrease pain
signalling, e.g. by having an ability to decrease mesenteric
afferent nerve firing (e.g. by decreasing the frequency of firing
of mesenteric afferent nerve bundles) form preferred aspects of the
present invention.
[0128] It is clear from the above that the methods of the invention
can also be used to select or identify agents which are not
appropriate for the treatment of intestinal motility disorders, for
example agents which do not show any effect on one or more, or two
or more, or all three of MMC velocity or MMC frequency or
intraluminal pressure may not be suitable for use in the treatment
of intestinal motility disorders, especially if such agents do not
have a beneficial effect on reducing pain signalling. In particular
those agents which show no effect on any of these parameters are
unlikely to be suitable for the treatment of an intestinal motility
disorder. In addition, those agents which have an effect of
increasing pain signalling as measured by an increase in mesenteric
afferent nerve firing in step b) of the method are unlikely to be
suitable for the treatment of an intestinal motility disorder.
[0129] Thus, a yet further preferred aspect of the invention
provides an agent, preferably a bacterial strain, selected by the
method of the invention for use in therapy, more particularly for
use in the treatment of an intestinal motility disorder such as
those described herein. Appropriate intestinal motility disorders
to be treated are described elsewhere herein. Preferred intestinal
motility disorders to be treated by the agents or bacterial strains
of the invention are those in which it is desired to reduce the
transit time of material through the intestine, for example colic,
regurgitation or constipation. Other preferred intestinal motility
disorders are those in which it is desired to increase the transit
time of material through the intestine, for example IBS or
diarrhea.
[0130] Methods of treatment of a subject with an intestinal
motility disorder are also provided by the present invention, said
methods comprising the administration of an agent, preferably a
bacterial strain, selected by the method of the invention, to said
subject in an amount effective to treat said intestinal motility
disorder.
[0131] Methods of treatment of a subject with an intestinal
motility disorder are also provided by the present invention, said
methods comprising the administration of a bacterial strain of the
invention to said subject in an amount effective to treat said
intestinal motility disorder. Preferred strains are LG345A or
LG621A for the treatment of intestinal motility disorders as
described elsewhere herein.
[0132] Preferred intestinal motility disorders and other preferred
features are as described elsewhere herein for other aspects of the
invention.
[0133] Also provided by the present invention is the use of an
agent, preferably a bacterial strain, selected by the method of the
invention, in the manufacture of a composition or medicament for
use in the treatment of an intestinal motility disorder.
[0134] Also provided by the present invention is the use of a
bacterial strain of the invention, in the manufacture of a
composition or medicament for use in the treatment of an intestinal
motility disorder. Preferred strains are LG345A or LG621A for the
treatment of intestinal motility disorders as described elsewhere
herein.
[0135] Preferred intestinal motility disorders and other preferred
features are as described elsewhere herein for other aspects of the
invention.
[0136] In the methods and uses of the present invention described
herein, the terms "increase", "decrease", "reduce", etc., refer to
a measurable change in levels, preferably a significant change in
levels, more preferably a statistically significant change,
preferably with a probability value of .ltoreq.0.05.
[0137] Preferred subjects for treatment using the methods of the
invention are mammals, more preferably humans. Where the intestinal
motility disorder to be treated is constipation then preferred
subjects are elderly patients or pregnant women. An elderly patient
will generally be understood to be a patient aged 70 or over. Where
the intestinal motility disorder to be treated is colic, preferably
this is infantile colic.
[0138] A yet further aspect of the invention provides a product
comprising the agents or strains of the invention (e.g. agents or
strains selected by the methods of the invention) for the
therapeutic uses as defined elsewhere herein, wherein said use
further comprises the administration of at least one further
therapeutic or nutritional agent. In such embodiments, the further
therapeutic agent can be any further agent which is useful in the
treatment of the intestinal motility disorder in question. The
further nutritional agent can be any appropriate nutritional
component, e.g. a foodstuff or a food supplement.
[0139] Said further agents can be administered together with the
agent or strain of the invention (e.g. as a composition) or can be
administered separately. In addition, said further agents can be
administered at the same time as the agent or strain of the
invention or at different time points. Suitable administration
regimes and timings can readily be determined by the skilled person
depending on the further agent in question.
[0140] The present invention also provides a composition
comprising:
(i) an agent, preferably a bacterial strain, obtainable by the
selection method of the invention, or a bacterial strain of the
invention as otherwise defined herein; and (ii) at least one
additional component selected from the group consisting of a
pharmaceutically acceptable carrier, diluent or excipient, a
foodstuff or food supplement, or a further therapeutic or
nutritional agent. Thus, said compositions can be formulated as
pharmaceutical compositions or as nutritional compositions, e.g. as
a food product.
[0141] The therapeutic uses of the agents, strains and compositions
of the invention as defined herein include the reduction,
prevention or alleviation of the relevant disorder or symptoms of
disorder (e.g. can result in the modulation of disease symptoms).
Such reduction, prevention or alleviation of a disorder or symptoms
thereof can be measured by any appropriate assay. Preferably the
reduction or alleviation of a disorder or symptoms is statistically
significant, preferably with a probability value of <0.05. Such
reduction or alleviation of a disorder or symptoms are generally
determined compared to an appropriate control individual or
population, for example a healthy subject or an untreated or
placebo treated subject.
[0142] An appropriate mode of administration and formulation of the
agents, strains, compositions, etc., is chosen depending on the
site of disease. A preferred mode of administration is oral,
however, equally for some treatments intravenous or intramuscular
injection will be appropriate.
[0143] Appropriate doses of the agents, strains and compositions of
the invention as defined herein can readily be chosen or determined
by a skilled person depending on the disorder to be treated, the
mode of administration and the formulation concerned.
[0144] As described above, using the invention herein the inventors
have obtained two new strains, Lactobacillus gasseri 345A and
Lactobacillus gasseri 621A. These strains have been deposited at
DSMZ (Leibniz Institute DSMZ--German Collection of Microorganisms
and Cell Cultures, Inhoffenstr. 7B, D-38124 Braunschweig, Germany)
on 18 Apr. 2013 and have been designated the numbers DSM 27123 and
DSM 27126, respectively.
[0145] The discovery by the inventors herein that effects of an
agent influencing motility disorders are in most cases multi
factorial and are different in various part of the GI-tract and
consequently have to be studied in that way to find the best agent
for the specific disorder, led to the method of the invention.
[0146] It is therefore an object of the present invention to find
agents suitable for treatment, prevention or modulating specific
motility disorders, by using the model herein.
[0147] In one embodiment of the invention the object is to select a
compound or other agent, with potential uses as either a
nutritional compound or a pharmaceutical, that is effective in
preventing or to treat constipation in humans, especially
elderly.
[0148] In one embodiment of the invention the object is to select a
probiotic bacterial strain that can be effective in preventing or
treating constipation in humans, especially elderly subjects or
pregnant women.
[0149] In another embodiment, the object of the invention is to
select an agent, for example a probiotic strain, that can be
effective in preventing or treating infantile colic.
[0150] In another embodiment, the object of the invention is to
select an agent, for example a probiotic strain, that can be
effective in preventing or treating diarrhea or other motility
disorders wherein it is beneficial to decrease velocity and thereby
increase transit time.
[0151] In one embodiment of the invention the object is to select
an agent, for example a probiotic strain, that can be effective in
treating or modulating symptoms of Irritable bowel syndrome (IBS).
Such an agent is selected to increase transit time, preferably
without causing any pain and preferably to reduce pain.
[0152] In yet another embodiment, the object of the invention is to
select an agent, for example a probiotic strain, that can be
effective in preventing fecal incontinence.
[0153] In yet another embodiment, the object of the invention is to
select an agent, for example a probiotic strain, that can be
effective to increase mixing in the small and large intestine
without causing any pain and preferably reducing pain, to
facilitate or result in better nutritional uptake in a subject,
especially in elderly.
[0154] The following are some examples of the invention, which are
not meant to be limiting of the use of the invention herein but to
show practical examples in detail how the invention may be used.
Example 1 relates to the description of the spatiotemporal mapping
(StMap) software plugin and Example 2 relates to the steps of the
selection of effective agents and Example 3 to a selection of
agents for a specific motility disorder.
Example 1
Description of the StMap Plug-In for the NIH ImageJ Software;
[0155] ImageJ is a public domain Java image processing program
inspired by NIH Image for the Macintosh. It runs, either as an
online applet or as a downloadable application, on any computer
with a Java 1.4 or later virtual machine. Downloadable
distributions are available for Windows, Mac OS, Mac OS X and
Linux.
[0156] ImageJ was designed with an open architecture that provides
extensibility via Java plugins. Custom acquisition, analysis and
processing plugins can be developed using ImageJ's built in editor
and Java compiler. User-written plugins make it possible to solve
almost any image processing or analysis problem.
[0157] We developed the plug-in following the description of the
software available from the National Institutes of Health (NIH),
9000 Rockville Pike, Bethesda, Md., USA) according to the details
in FIG. 6.
[0158] ROI stands for "Region of Interest"
Example 2
Selection of Agents Effective in Motility Disorders.
[0159] This selection comprises a two-step method. The first step
using simultaneous recording of intraluminal pressure changes in
addition to video recording and spatiotemporal maps. The second
step is an analysis of nerve signaling.
[0160] All agents are compared to a control and the agents are not
compared to each other, this will result in one graph per agent
tested.
First Step, Spatiotemporal Analysis
[0161] We used adult male Swiss Webster mice (20-30 g) bought from
Charles River Laboratories (Wilmington, Mass., USA). The mice were
killed by cervical dislocation, in line with McMaster guidelines
for the use and care of animals. All ensuing procedures were ex
vivo.
Organ Bath Motility Recordings
[0162] 4-cm-long jejunal or distal colon segments were excised and
the contents allowed to empty by flushing the segment with Krebs
saline under 2 hPa gravity pressure head. Each segment was mounted
in a 20-ml organ bath chamber and submerged in oxygenated Krebs
(FIG. 1A). Oral and anal ends were cannulated, and the lumen was
gravity perfused with (95% 02 and 5% CO.sub.2)--gassed Krebs using
several Mariotte bottles. The intraluminal compartment was perfused
at 0.5 ml/min with room temperature buffer (19 to 22.degree. C.).
The organ chamber (serosal compartment) was perfused with
pre-warmed (34.degree. C.), carbogen-gassed, Krebs solution at a
rate of 5 ml/min). Oxygenated Krebs buffer was of the following
composition (mM): 118 NaCl, 4.8 KCl, 25 NaHCO.sub.3, 1.0
NaH.sub.2PO.sub.4, 1.2 MgSO.sub.4, 11.1 glucose, and 2.5
CaCl.sub.2) bubbled with carbogen gas (95% 02 and 5% CO.sub.2). At
the beginning of the experiment, intraluminal pressure was adjusted
to 3 hPa and the recordings were made at this filling pressure.
Bacteria were applied by switching the oral luminal inflow from
Marriotte bottles containing Krebs to ones containing Krebs plus
bacteria by closing and opening the appropriate stopcocks, as
illustrated in FIG. 1A. Intraluminal pressure changes were measured
at the midpoint of the longitudinal axis of the gut segment. The
pressure signal was amplified, digitized, stored on a PC computer
and analyzed off-line using PClamp 9 software (Molecular Devices,
Sunny Vale, Calif., USA).
[0163] Images were recorded using a video camcorder (JVC Everio
Hard Disk Camcorder Model GZ-MG155U) which was placed 10 cm above a
gut segment (FIG. 1A). Recording was started in synchrony with the
pressure recording using an 8 to 12 cm field of view for the
duration of the experiment. The camera output was in raw video
format (MOD) at 30 frames per second (fps). 10-min long video clips
were excised from the MOD file using video editing software
(Avidemux version 2.5.0, Open source software available from
Avidemux). The clips were then converted into the MOV format using
a video converter (Zune converter version 1.1, Open source software
available at the FFmpeg website). The final video clips were
resampled to a resolution of 384.times.256 pixels and 25 fps.
[0164] Video recordings were analyzed using in-house image
processing software (StMap, see example 1) developed as a plug-in
for NIH ImageJ (version 1.43c, Open source software available from
the NIH). The software converts the image (1B) in each frame of the
video into a black-and-white silhouette (1C) and generates a
spatiotemporal map using an edge detection routine. The routine
first measures the diameter at each position along the gut and it
then represents the physical diameter at each position as a hue
value (ranges from 0-255, black to white). As gut diameter
decreases during contractions, the hue value is reduced towards 0
and will be shown as toward darker values. As the software reads
through each 10 minute clip, it generates a spatiotemporal map--a
pattern of alternating bands of light and dark hues that contains 3
sets of information: position along the gut (ordinate), time
(abscissa) and gut diameter (applicate or z-axis) (FIG. 1D). Using
these variables, the spatiotemporal map becomes a motility
"fingerprint" whose sensitivity is critically important in defining
the detailed and nuanced effects that specific probiotic strains
and other agents have on motility and the ability to distinguish
between them.
[0165] Since the StMap measures the diameter changes at each
position, StMap can be interpreted as a stacking of numerous 2D
diameters versus time graphs. In fact, for map, if the location of
the pressure transducer were identified (dotted line in FIG. 2A)
and were shown as a grey scale versus time graph (FIG. 2B), this
graph would be in register with the simultaneously recorded
pressure vs. time recording at that locus (FIG. 2C).
[0166] Motility parameters were measured directly from the
spatiotemporal map using the StMap plugin. In this system we are
able to test (using tetrodotoxin neuron silencing) whether
contractions depend on the enteric nervous system or are myogenic
(driven by rhythmic muscle contractions but neuron independent)
Neurally generated, anally propagating, migrating motor complexes
MCs generated thick dark bands that slant diagonally from left to
right; the propagation velocity (mm s.sup.-1) is measured from the
slope(s) of each band (FIG. 3). For each treatment, slopes of 3-5
successive MCs were averaged to calculate the propagation velocity.
MC frequency (mHz) was gauged by counting the number of dark MC
bands during a 10 min segment. MC amplitude was measured directly
from the baseline diameter (FIG. 2B) or pressure (FIG. 2C) to the
next peak with 3 values being averaged for each treatment. The rate
of diameter change (cm/s) was calculated from the time taken for
the diameter to constrict maximally from baseline (downstroke of
MCs in (FIG. 2B).
Agents to be Tested
[0167] Lactobacillus rhamnosus (JB-1), Lactobacillus reuteri (DSM
17938), Lactobacillus reuteri ATCC PTA 6475, Lactobacillus gasseri
345A (DSM 27123), Lactobacilllus gasseri 621A (DSM 27126),
Lactobacillus gasseri T1 and Lactobacillus gasseri T2 were all
tested in the first step of the methods of the invention.
[0168] Cell numbers were determined optically and viability was
always checked by ability to grow after plating on growth medium
agar plates. Cells from frozen stocks were thawed and centrifuged
at 2000 rpm for 15 min, and the pellet was suspended in equal
volume of Krebs buffer. Then the suspension was again centrifuged,
and the cells were removed and resuspended in Krebs at the original
concentration. Just prior to use, bacteria were diluted to working
concentrations with fresh Krebs buffer. Krebs containing bacteria
were fed to the intraluminal compartment while ion channel
modulating drugs added to the Krebs buffer perfusing the serosal
compartment. The time required for the drug solution to flow from
the tap to the recording chamber was 30 s. The IK.sub.Ca ion
channel blocker 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole
(TRAM-34) (Tocris Bioscience, Ellisville, Mo., USA) was dissolved
with pure dimethyl sulfoxide (DMSO) to make 10-mM stock solutions,
and these were diluted in oxygenated Krebs to make working
concentrations 30 min before use. The IK.sub.ca channel opener,
5,6-dichloro-1-ethyl-2-benzimidazolinone (DCEBIO) (Tocris) was
dissolved in DMSO to make a 10-mM stock solution; the final working
concentration in Krebs buffer was 0.1 or 1 .mu.M.
[0169] The agents are analyzed according to the presented step 1
above and the results are shown in FIG. 7
Statistics
[0170] Statistics were calculated using GraphPad Prism 5.0
(GraphPad Software, San Diego, Calif., USA). Descriptive statistics
are given as means.+-.SD, but in concentration-response plots,
sampling errors are displayed using SEM; the sample size is denoted
by n. The statistically discernible difference for tests of
significance was set at P=0.05; all tests were 2-tailed.
Second Step, the Analysis of Nerve Signaling
[0171] All procedures adhered to Canadian Council on Animal Care
guidelines and were approved by the Animal Research Ethics Board of
McMaster University (Permit #08-08-35).
Extracellular Recordings
[0172] Adult male Swiss Webster mice (20-30 g) were procured from
Charles River Laboratories (Wilmington, Mass., USA). The mice were
sacrificed by cervical dislocation. All ensuing procedures were ex
vivo. Segments of excised distal jejunum (.about.2.5 cm) with
attached mesenteric tissue were removed from euthanized animals
(FIG. 4) and immediately placed in a Sylgard-coated recording Petri
dish filled with Krebs buffer of the following composition (mM):
118 NaCl, 4.8 KCl, 25 NaHCO.sub.3, 1.0 NaH2PO4, 1.2 MgSO4, 11.1
glucose and 2.5 CaCl2 bubbled with carbogen (95% O2/5% CO2). The
oral and anal ends of the gut segment were cannulated with plastic
tubing and gently emptied using an attached syringe filled with
oxygenated Krebs buffer; then, the segment and mesenteric tissue
were pinned out and the mesenteric nerve bundle isolated by careful
dissection under a stereo microscope (FIG. 5) as described by
Barbara et al (Gastroenterology, Volume 132, Issue 1, Pages 26-37,
January 2007). The preparation was transferred to an inverted
microscope and the lumen gravity perfused at 0.5-1 ml/min with room
temp (-22.degree. C.) oxygenated Krebs and/or additives using
several Mariotte bottles. The serosal compartment was separately
perfused with prewarmed (34.degree. C.) Krebs solution at 3-5
ml/min. The nerve bundle was gently drawn by suction into a glass
pipette attached to a patch clamp electrode holder (CV-7B;
Molecular Devices, Sunnyvale, Calif., USA) and extracellular nerve
recordings were made using a Multi-Clamp 700B amplifier and
Digidata 1440A signal converter (Molecular Devices). Electrical
signals were bandpass-filtered at 0.1 to 2 kHz, sampled at 20 kHz
and stored on a personal computer running pClamp 10 software
(Molecular Devices) for post-hoc analysis.
Vagotomy
[0173] For some experiments, a subdiaphragmatic vagotomy was
carried out as previously described (van der Kleij H., Am J Physiol
Regul Integr Comp Physiol 295:1131-1137, 2008)). Animals were
allowed to recover for 10-14 days before harvesting the jejunum and
mesenteric tissue for electrophysiological experiments. Sham
vagotomy was performed in 3 animals. Post-operatively, the body
weight and general health of the mice were measured daily. We found
no evidence of significant differences in weight gain 1 week
post-surgery in either vagotomized or sham-treated animals. All
vagotomized mice were tested for completeness of the procedure by
recording after each experiment the responses to the serosal
application of cholecystokinin (CCK).
[0174] Vagotomy was deemed to have been effective when we found
that CCK did not increase mesenteric nerve firing rate.
Integrity of Jejunal Segments
[0175] To test the integrity of the jejunal segments with respect
to translocation of bacteria across the epithelium during an
experiment, we labeled JB-1 with 5-(6)-carboxyfluorescein
succinimidyl ester (CFSE), placed these at a concentration of
10.sup.9/ml Krebs into the lumen and after incubation for 75 min,
fixed the tissue in paraformaldehyde, and examined sections for
their presence below the epithelium in confocal microscopy
(dual-laser microscopy, LSM 510, Carl Zeiss, Germany). JB-1 were
washed twice in PBS and suspended in a final concentration of CFSE
of 5 .mu.M in PBS supplemented with 5% fetal calf serum and
incubated for 25 min at 37.degree. C. The tissues (n=3) were fixed
in 4% paraformaldehyde at 4.degree. C. overnight, then washed 3
times in PBS for 10 min each, sectioned at 10 and 30 .mu.m and
transverse sections transferred to microscope slides and mounted.
These were then reviewed in optical slices by Z-stacking
methodology.
Agents to be Tested
[0176] Lactobacillus rhamnosus (JB-1), Lactobacillus reuteri (DSM
17938), Lactobacillus reuteri ATCC PTA 6475, Lactobacillus gasseri
345A (DSM 27123), Lactobacilllus gasseri 621A (DSM 27126),
Lactobacillus gasseri T1 and Lactobacillus gasseri T2 were all
tested in the second step of the methods of the present
invention.
[0177] Cell numbers were determined optically, and viability
checked by the ability to grow after plating on growth medium agar
plates. Live L. rhamnosus were grown from frozen (-80.degree. C.) 1
ml aliquots, which consisted of 5.times.10.sup.9 cells in
Man-Rogosa Sharpe broth (Difco Laboratories, Sparks, Md., USA).
Cells from frozen stocks were thawed and centrifuged at 2000 rpm
for 15 min, and the pellet was suspended in equal volume of Krebs
buffer. The suspension was again centrifuged, and the cells removed
and resuspended in Krebs at the original concentration. Just prior
to use, the cells were diluted to working concentrations with Krebs
buffer. For some experiments the bacteria were diluted directly to
working concentrations after thawing (in broth); the bacteria were
always applied into the lumen of the jejunal segment.
Cholecystokinin (25-33) sulphated (CCK; AnaSpec, Fremont, Calif.)
was dissolved in DMSO to make a 1 mM stock solution. Aliquots were
diluted on the day of the experiment to working concentration in
Krebs buffer, with a final DMSO concentrations .ltoreq.0.0001%.
Off-Line Data Analysis
[0178] Multi- and single-unit spontaneous firing frequencies of
mesenteric afferent nerve bundles were measured using Clampfit 10.2
(Molecular Devices). Both methods (multi-unit spike discharge and
waveform analysis) of measurement are routinely used to determine
changes in the excitability of the mesenteric nerve fibers induced
by different treatments. The timing of multi-unit spikes was
determined using the peak detection module of Clampfit, and average
frequency calculated from spike intervals. Single-unit activity was
isolated from the multi-unit activity using the spike shape
automatic template detection tool of Clampfit (computerized
waveform analysis). After template detection, discrimination was
always checked by visual inspection and visually non-spike shapes
events were discarded (<0.2%). The results are presented in FIG.
8.
Statistics
[0179] Data are expressed as means.+-.SEM with n referring to the
total number of the jejunal segments recorded; the maximum number
of segments recorded from the same animal was two. The Wilcoxon
test was used for paired data comparisons and Friedman test with
Dunn's post hoc test for repeated measures analysis of variance
were performed using Prism software 5.0 (GraphPad Software, Inc.,
San Diego, Calif.). Because large variations in spontaneous
activity may occur between one preparation and another in
multi-unit neural activity, all comparisons were paired with before
and after treatment recordings made where each nerve bundle served
as its own control. Differences were considered significant if
P.ltoreq.0.05.
Example 3
[0180] A. Selection of Optimal Probiotic Bacterial Agents to Help
Treat Motility Disorders (e.g. Constipation in the Elderly) Using
the Defined Steps of the Present Invention;
[0181] L. rhamnosus (JB-1), Lactobacillus reuteri (DSM 17938),
Lactobacillus reuteri ATCC PTA 6475, Lactobacillus gasseri 345A
(DSM 27123), Lactobacilllus gasseri 621A (DSM 27126), Lactobacillus
gasseri T1 and Lactobacillus gasseri T2 is analyzed according to
example 2. The results are summarized in table 1.
TABLE-US-00001 Motility Pain Agent Jejunum Colon signaling JB-1 MMC
frequency MMC velocity PPr DSM 17938 MMC frequency MMC velocity PPr
.smallcircle. .smallcircle. ATCC PTA MMC frequency -- .smallcircle.
.smallcircle. 6475 MMC velocity -- .smallcircle. PPr --
.smallcircle. LG345A MMC frequency -- MMC velocity -- PPr -- LG621A
MMC frequency -- .smallcircle. MMC velocity -- PPr -- .smallcircle.
T1 MMC frequency -- .smallcircle. .smallcircle. MMC velocity --
.smallcircle. PPr -- T2 MMC frequency -- .smallcircle. MMC velocity
-- .smallcircle. PPr -- .smallcircle.
Table 1
[0182] L. reuteri DSM 17938 and L. gasseri 345A increase the MMC
velocity in the colon and also increases at least one of the
parameters PPr or MMC frequency in the colon (L. reuteri DSM 17938
increases the MMC frequency and L. gasseri 345A increases the PPr).
Both these strains decrease the frequency of spontaneous firing of
mesenteric afferent nerve bundles. Therefore these strains are
selected for treatment of colic, e.g. infantile colic, since these
strains increase the speed of MC propagation flow and at the same
time decrease the frequency of firing of mesenteric afferent nerve
bundles.
[0183] The increase in MMC velocity and the decrease of the
frequency of spontaneous firing of mesenteric afferent nerve
bundles caused by L. retueri DSM 17938 and L. gasseri 345A makes
them suitable for treatment of constipation, especially in pregnant
women where a reduction is wanted, or in the elderly.
[0184] L. rhamnosus JB-1 and L. gasseri 621A decrease the MMC
velocity. L. gasseri 621A also decreases the frequency of the
firing of mesenteric afferent nerve bundles and can therefore be
used for treating disorders involving rapid passage transit, for
example IBS. L. rhamnosus JB-1 has no effect on the frequency of
firing of mesenteric afferent nerve bundles and is therefore not
applicable for treatment of motility disorders.
[0185] L. reuteri ATCC PTA 6475, L. gasseri T1 and L. gasseri T2
does not show any effect on neither MMC velocity nor the
spontaneous frequency of firing of mesenteric afferent nerve
bundles and is therefore not recommended for use in motility
disorders.
DEPOSIT INFORMATION
[0186] A deposit of the proprietary bacterial strains Lactobacillus
gasseri 345A and Lactobacillus gasseri 621A has been made with the
Leibniz Institute DSMZ (DSMZ)--German Collection of Microorganisms
and Cell Cultures, Inhoffenstr. 7B, D-38124 Braunschweig, Germany.
The date of deposit for Lactobacillus gasseri 345A and
Lactobacillus gasseri 621A was Apr. 18, 2013. Upon issuance of a
patent, all restrictions upon the deposits will be removed, and the
deposits are intended to meet all of the requirements of 37 C.F.R.
.sctn. 1.801-1.809. The DSMZ has issued accession number DSM 27123
for Lactobacillus gasseri 345A and accession number DSM 27126 for
Lactobacillus gasseri 621A. These deposits will be maintained in
the depository for a period of 30 years, or 5 years after the last
request, or for the effective life of the patent, whichever is
longer, and will be replaced as necessary during that period.
Applicants do not waive any infringement of their rights granted
under this patent or under the Plant Variety Protection Act (7
U.S.C. 2321 et seq.).
* * * * *