U.S. patent application number 14/025531 was filed with the patent office on 2014-07-24 for methods of diagnosing and treating small intestinal bacterial overgrowth (sibo) and sibo-related conditions.
This patent application is currently assigned to CEDARS-SINAI MEDICAL CENTER. The applicant listed for this patent is Cedars-Sinai Medical Center. Invention is credited to Henry C. Lin, Mark Pimentel.
Application Number | 20140206636 14/025531 |
Document ID | / |
Family ID | 25275454 |
Filed Date | 2014-07-24 |
United States Patent
Application |
20140206636 |
Kind Code |
A1 |
Lin; Henry C. ; et
al. |
July 24, 2014 |
METHODS OF DIAGNOSING AND TREATING SMALL INTESTINAL BACTERIAL
OVERGROWTH (SIBO) AND SIBO-RELATED CONDITIONS
Abstract
Disclosed is a method of treating small intestinal bacterial
overgrowth (SIBO) or a SIBO-caused condition in a human subject.
SIBO-caused conditions include irritable bowel syndrome,
fibromyalgia, chronic pelvic pain syndrome, chronic fatigue
syndrome, depression, impaired mentation, impaired memory,
halitosis, tinnitus, sugar craving, autism, attention
deficit/hyperactivity disorder, drug sensitivity, an autoimmune
disease, and Crohn's disease. Also disclosed are a method of
screening for the abnormally likely presence of SIBO in a human
subject and a method of detecting SIBO in a human subject. A method
of determining the relative severity of SIBO or a SIBO-caused
condition in a human subject, in whom small intestinal bacterial
overgrowth (SIBO) has been detected, is also disclosed.
Inventors: |
Lin; Henry C.; (Albuquerque,
NM) ; Pimentel; Mark; (Los Angeles, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Cedars-Sinai Medical Center |
Los Angeles |
CA |
US |
|
|
Assignee: |
CEDARS-SINAI MEDICAL CENTER
Los Angeles
CA
|
Family ID: |
25275454 |
Appl. No.: |
14/025531 |
Filed: |
September 12, 2013 |
Related U.S. Patent Documents
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Application
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13782504 |
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8562952 |
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14025531 |
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13315671 |
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13315671 |
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11348995 |
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7048906 |
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11348995 |
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09374142 |
Aug 11, 1999 |
6861053 |
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09837797 |
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09546119 |
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Current U.S.
Class: |
514/39 ; 514/152;
514/195; 514/398 |
Current CPC
Class: |
A23V 2002/00 20130101;
A61K 38/2271 20130101; A61K 31/353 20130101; A61K 31/4164 20130101;
A61P 3/02 20180101; G01N 33/497 20130101; A61K 9/0087 20130101;
A61K 38/54 20130101; Y10T 436/145555 20150115; Y10T 436/214
20150115; A61K 9/0053 20130101; A61K 51/1206 20130101; A61K 38/225
20130101; A61K 31/4184 20130101; Y10T 436/22 20150115; A61K 31/138
20130101; A61B 5/418 20130101; A61K 36/534 20130101; A61K 49/0004
20130101; A61K 31/7036 20130101; A61K 31/43 20130101; G01N 33/6893
20130101; A61K 31/7048 20130101; A61K 2300/00 20130101; A61K
2300/00 20130101; A61K 38/1796 20130101; Y10T 436/18 20150115; Y10T
436/147777 20150115; A61B 5/412 20130101; A61K 38/22 20130101; A61K
31/198 20130101; G01N 2033/4977 20130101; A61K 31/4045 20130101;
Y10S 426/80 20130101; A61B 5/0836 20130101; A61K 31/201 20130101;
A61K 31/424 20130101; A61B 5/415 20130101; A61K 31/65 20130101;
A61K 38/22 20130101; A61K 31/135 20130101; G01N 2333/70571
20130101; A61K 38/225 20130101; G01N 2800/06 20130101; Y10S 426/801
20130101; A61K 45/06 20130101; A23L 33/40 20160801; A61K 31/496
20130101 |
Class at
Publication: |
514/39 ; 514/398;
514/195; 514/152 |
International
Class: |
A61K 31/7036 20060101
A61K031/7036; G01N 33/497 20060101 G01N033/497; A61K 31/65 20060101
A61K031/65; A61K 31/4164 20060101 A61K031/4164; A61K 31/43 20060101
A61K031/43 |
Goverment Interests
[0002] The U.S. Government has a paid-up license in this invention
and the right in limited circumstances to require the patent owner
to license others on reasonable terms as provided for by the terms
of Grant NIH DK 46459.
Claims
1. A method of treating small intestinal bacterial overgrowth
(SIBO) or a SIBO-caused condition in a human subject, said method
comprising: detecting in the subject by suitable detection means,
the presence of SIBO, wherein a population of proliferating
bacteria is present in the small intestine of the subject, or
detecting with said means the absence of SIBO; and, if the presence
of SIBO is detected in the subject, depriving the bacterial
population or nutrient(s) sufficiently to inhibit the growth of
said bacteria in the small intestine, and thereby at least
partially eradicating SIBO in the human subject.
2-45. (canceled)
Description
[0001] This application claims the benefit of priority under 35
U.S.C. .sctn.120 as a continuation of U.S. application Ser. No.
13/782,504, filed on Mar. 1, 2013 and issued as 8,562,952 on Oct.
22, 2013, which is a continuation of U.S. patent application Ser.
No. 13/315,671, filed on Dec. 9, 2011 and issued as U.S. Pat. No.
8,388,935 on Mar. 5, 2013, which is a continuation of U.S. patent
application Ser. No. 12/768,531, filed on Apr. 27, 2010 and issued
as 8,110,177 on Feb. 7, 2012, which is a continuation of U.S.
patent application Ser. No. 11/348,995, filed on Feb. 7, 2006 and
issued as 7,736,622 on Jun. 15, 2010, which is a divisional of U.S.
patent application Ser. No. 09/837,797, filed on Apr. 17, 2001 and
issued as U.S. Pat. No. 7,048,906 on May 23, 2006, which is a
continuation-in-part of U.S. patent application Ser. No.
09/374,142, filed on Aug. 11, 1999 and issued as U.S. Pat. No.
6,861,053 on Mar. 1, 2005, a continuation-in-part of U.S. patent
application Ser. No. 09/374,143, filed on Aug. 11, 1999 and issued
as U.S. Pat. No. 6,562,629 on May 13, 2003, and a
continuation-in-part of U.S. patent application Ser. No.
09/546,119, filed on Apr. 10, 2000 and issued as U.S. Pat. No.
6,558,708 on May 6, 2003.
BACKGROUND OF THE INVENTION
[0003] Throughout this application various publications are
referenced within parentheses. The disclosures of these
publications in their entireties are hereby incorporated by
reference in this application in order to more fully describe the
state of the art to which this invention pertains.
[0004] 1. The Field of the Invention
[0005] This invention relates to the medical arts. It relates to a
method of diagnosing and treating small intestinal bacterial
overgrowth (SIBO), and conditions caused by SIBO.
[0006] 2. Discussion of the Related Art
[0007] Small intestinal bacterial overgrowth (SIBO), also known as
small bowel bacterial overgrowth (SBBO), is an abnormal condition
in which aerobic and anaerobic enteric bacteria from the colon
proliferate in the small intestine, which is normally relatively
free of bacterial contamination. SIBO is defined as greater than
10.sup.6 CFU/mL small intestinal effluent (R. M. Donaldson, Jr.,
Normal bacterial populations of the intestine and their relation to
intestinal function, N. Engl. J. Med. 270:938-45 [1964]).
Typically, the symptoms include abdominal pain, bloating, gas and
alteration in bowel habits, such as constipation and diarrhea.
[0008] Irritable bowel syndrome, Crohn's disease, chronic fatigue
syndrome, chronic pelvic pain syndrome, fibromyalgia, depression,
attention deficit/hyperactivity disorder, autism, and autoimmune
diseases, e.g., multiple sclerosis and systemic lupus
erythematosus, are all clinical conditions of unclear etiology. No
association has been made heretofore between any of the afore-going
diagnostic categories and SIBO.
[0009] Irritable bowel syndrome (IBS) is the most common of all
gastrointestinal disorders, affecting 11-14% of adults and
accounting for more than 50% of all patients with digestive
complaints. (G. Triadafilopoulos et al., Bowel dysfunction in
fibromyalgia, Digestive Dis. Sci. 36(1):59-64 [1991]; W. G.
Thompson, Irritable Bowel syndrome: pathogenesis and management,
Lancet 341:1569-72 [1993]). It is thought that only a minority of
people with IBS actually seek medical treatment. Patients with IBS
present with disparate symptoms, for example, abdominal pain
predominantly related to defecation, alternating diarrhea and
constipation, abdominal distention, gas, and excessive mucus in the
stool.
[0010] A number of possible causes for IBS have been proposed, but
none has been fully accepted. (W. G. Thompson [1993]). These
hypotheses included a fiber-poor Western diet, intestinal motility
malfunction, abnormal pain perception, abnormal psychology or
behavior, or psychophysiological response to stress.
[0011] A high fiber diet increases stool bulk and shortens gut
transit time. However the presence of IBS in non-Western countries,
such as China and India, and the failure of dietary fiber
supplements to treat IBS in double-blind clinical trials are
inconsistent with the Afiber hypothesis.apprxeq.for the causation
of IBS. (W. Bi-zhen and P. Qi-Ying, Functional bowel disorders in
apparently healthy Chinese people, Chin. J. Epidemiol. 9:345-49
[1988]; K. W. Heaton, Role of dietary fibre in irritable bowel
syndrome. In: R. W. Read [ed.], Irritable bowel syndrome, Grune and
Stratton, London, pp. 203-22 [1985]; W. G. Thompson et al.,
Functional bowel disorders and functional abdominal pain,
Gastroenterol. Int. 5:75-92 [1992]).
[0012] Those experiencing chronic IBS pain are often depressed and
anxious. Treatment with tricyclic antidepressants has been used to
raise the pain threshold of some IBS patients. (W. G. Thompson
[1993]). Abreu et al. and Rabinovich et al. taught the use of
corticotropin-releasing factor antagonists to relieve
stress-related symptoms, including depression and anxiety, in IBS,
anorexia nervosa, and other disorders. (M. E. Abreu,
Corticotropin-releasing factor antagonism compounds, U.S. Pat. No.
5,063,245; A. K. Rabinovich et al., Benzoperimidine-carboxylic
acids and derivatives thereof, U.S. Pat. No. 5,861,398). Becker et
al. taught the use of serotonin antagonists to treat depression and
anxiety associated with IBS and other conditions. (D. P Becker et
al., Meso-azacyclic aromatic acid amides and esters as serotonergic
agents, U.S. Pat. No. 5,612,366).
[0013] Those with IBS symptoms have not been shown to have a
different psychological or psychosocial make-up from the normal
population. (W. E. Whitehead et al., Symptoms of psychologic
distress associated with irritable bowel syndrome: comparison of
community and medical clinic samples, Gastroenterol. 95:709-14
[1988]). But many IBS patients appear to perceive normal intestinal
activity as painful. For example, IBS patients experience pain at
lower volumes of rectal distention than normal or have a lower than
normal threshold for perceiving migrating motor complex phase III
activity. (W. E. Whitehead et al., Tolerance for rectosigmoid
distension in irritable bowel syndrome, Gastroenterol. 98:1187-92
[1990]; J. E. Kellow et al., Enhanced perception of physiological
intestinal motility in the irritable bowel syndrome, Gastroenterol.
101(6): 1621-27 [1991]).
[0014] Bowel motility in IBS patients differs from normal controls
in response to various stimuli such as drugs, hormones, food, and
emotional stress. (D. G. Wangel and D. J. Deller, Intestinal
motility in man, III: mechanisms of constipation and diarrhea with
particular reference to the irritable bowel, Gastroenterol.
48:69-84 [1965]; R. F. Harvey and A. E. Read, Effect of
cholecystokinin on colon motility on and symptoms in patients with
irritable bowel syndrome, Lancet i:1-3 [1973]; R. M. Valori et al.,
Effects ofdifferent types of stress and "prokinetic drugs" on the
control of the fasting motor complex in humans, Gastroenterol.
90:1890-900 [1986]).
[0015] Evans et al. and Gorard and Farthing recognized that
irritable bowel syndrome is frequently associated with disordered
gastro-intestinal motility. (P. R. Evans et al., Gastroparesis and
small bowel dysmotility in irritable bowel syndrome, Dig. Dis. Sci.
42(10):2087-93 [1997]; DA. Gorard and M. J. Farthing, Intestinal
motor function in irritable bowel syndrome, Dig. Dis. 12(2):72-84
[1994]). Treatment directed to bowel dysmotility in IBS includes
the use of serotonin antagonists (D. P Becker et al.,
Meso-azacyclic aromatic acid amides and esters as serotonergic
agents, U.S. Pat. No. 5,612,366; M. Ohta et al., Method of
treatment of intestinal diseases, U.S. Pat. No. 5,547,961) and
cholecystokinin antagonists (Y. Sato et al., Benzodiazepine
derivatives, U.S. Pat. No. 4,970,207; H. Kitajima et al.,
Thienylazole compound and thienotriazolodiazepine compound, U.S.
Pat. No. 5,760,032). But colonic motility index, altered
myoelectrical activity in the colon, and small intestinal
dysmotility have not proven to be reliable diagnotic tools, because
they are not IBS-specific. (W. G. Thompson [1993]).
[0016] Because there has been no known underlying cause for IBS,
treatment of IBS has been primarily directed to symptoms of pain,
constipation or diarrhea symptoms.
[0017] For example, administration of the polypeptide hormone
relaxin, used to relax the involuntary muscles of the intestines,
is a treatment taught to relieve the pain associated with IBS. (S.
K. Yue, Method of treating myofascial pain syndrome with relaxin,
U.S. Pat. No. 5,863,552).
[0018] Borody et al. taught the use of a picosulfate-containing
laxative preparation to treat constipation in IBS, small intestinal
bacterial overgrowth, and acute or chronic bacterial bowel
infections. (T. J. Borody et al., Picosulfate-containing
preparation for colonic evacuation, U.S. Pat. No. 5,858,403).
Barody also taught the use of an anti-inflammatory agent to treat
IBS. (T. J. Barody, Treatment of non-inflammatory and
non-infectious bowel disorders, U.S. Pat. No. 5,519,014). In
addition, constipation in IBS has been treated with amidinourea
compounds. (J. Yelnosky et al., Amidinoureas for treating irritable
bowel syndrome, U.S. Pat. Nos. 4,701,457 and 4,611,011).
[0019] Kuhla et al. taught the use of triazinone compounds to
relieve IBS symptoms of constipation, diarrhea, and abdominal pain.
(D. E. Kuhla et al., Triazinones for treating irritable bowel
syndrome, U.S. Pat. No. 4,562,188). And Kitazawa et al. taught the
use of napthy- and phenyl-sulfonylalkanoic acid compounds to treat
IBS symptoms. (M. Kitazawa et al., Naphthysulfonylalkanoic acid
compounds and pharmaceutical compositions thereof, U.S. Pat. No.
5,177,069; M. Kitazawa et al., Phenylsulfonylalkanoic acid
compounds and pharmaceutical compositions thereof, U.S. Pat. No.
5,145,869). Day taught an IBS treatment involving the
administration of an anion-binding polymer and a hydrophilic
polymer. (C. E. Day, Method for treatment of irritable bowel
syndrome, U.S. Pat. No. 5,380,522). And Borody et al. taught the
use of salicylic acid derivatives to treat IBS. (T. J. Borody et
al., Treatment of non-inflammatory and non-infectious bowel
disorders, U.S. Pat. No. 5,519,014).
[0020] A probiotic approach to the treatment of IBS has also been
tried. For example, Allen et al. described the use of a strain of
Enterococcus faecium to alleviate symptoms. (W. D. Allen et al.,
Probiotic containing Enterococcus faecium strain NCIMB 40371, U.S.
Pat. No. 5,728,380 and Probiotic, U.S. Pat. No. 5,589,168). Borody
taught a method of treating irritable bowel syndrome by at least
partial removal of the existing intestinal microflora by lavage and
replacement with a new bacterial community introduced by fecal
inoculum from a disease-screened human donor or by a composition
comprising Bacteroides and Escherichia coli species. (T. J. Borody,
Treatment of gastro-intestinal disorders with a fecal composition
or a composition of bacteroides and E. coli, U.S. Pat. No.
5,443,826).
[0021] Fibromyalgia (FM) is a syndrome of intense generalized pain
and widespread local tenderness, usually associated with morning
stiffness, fatigue, and sleep disturbances. (F. Wolfe,
Fibromyalgia: the clinical syndrome, Rheum. Dis. Clin. N. Amer.
15(1):1-17 [1989]). Fibromyalgia is often associated with IBS
(34-50% of FM cases) or other gastrointestinal symptoms, Raynaud's
phenomenon, headache, subjective swelling, paresthesias,
psychological abnormality or functional disability, sometimes with
overlapping symptoms of coexisting arthritis, lower back and
cervical disorders, and tendonitis. Fibromyalgia affects 1-5% of
the population and is more prevalent among women than men. (G.
Triadafilopoulos et al. [1991]).
[0022] As in IBS, a diagnosis of FM correlates with a decreased
pain threshold among FM patients compared to non-patients. (F.
Wolfe et al., Aspects of Fibromyalgia in the General Population:
Sex, Pain Threshold, and Fibromyalgia Symptoms, J. Rheumatol.
22:151-56 [1995]). But other conventional laboratory evaluations of
FM patients are uniformly normal. (G. Triadafilopoulos et al.
[1991]). The symptoms of FM patients are typically treated with
anti-inflammatory agents and low dose tricyclic antidepressants.
Administration of relaxin for involuntary muscle dysfunction is
also a treatment taught to relieve the pain associated with
fibromyalgia. (S. K. Yue, Method of treating myofascial pain
syndrome with relaxin, U.S. Pat. No. 5,863,552). However, there has
been no known cause of FM to which diagnosis and/or treatment could
be directed.
[0023] Chronic fatigue syndrome (CFS) affects more than a half
million Americans. (P. H. Levine, What we know about chronic
fatigue syndrome and its relevance to the practicing physician, Am.
J. Med. 105(3A):100S-03S [1998]). Chronic fatigue syndrome is
characterized by a sudden onset of persistent, debilitating fatigue
and energy loss that lasts at least six months and cannot be
attributed to other medical or psychiatric conditions; symptoms
include headache, cognitive and behavioral impairment, sore throat,
pain in lymph nodes and joints, and low grade fever. (M. Terman et
al., Chronic Fatigue Syndrome and Seasonal; Affective Disorder:
Comorbidity, Diagnostic Overlap, and Implications for Treatment,
Am. J. Med. 105(3A):1155-24S [1998]). Depression and related
symptoms are also common, including sleep disorders, anxiety, and
worsening of premenstrual symptoms or other gynecological
complications. (A. L. Komaroff and D. Buchwald, Symptoms and signs
of chronic fatigue syndrome, Rev. Infect. Dis. 13:S8-S11 [1991]; B.
L. Harlow et al., Reproductive correlates of chronic fatigue
syndrome, Am. J. Med. 105(3A):94S-99S [1998]). Other physiologic
abnormalities are also associated with CFS in many patients,
including neurally-mediated hypotension, hypocortisolism, and
immunologic dysregulation. (P. H. Levine [1998]). A subgroup of CFS
patients complain of exacerbated mood state, diminished ability to
work and difficulty awakening during winter months, reminiscent of
seasonal affective disorder. (M. Terman et al. [1998]).
[0024] The etiology of CFS has been unknown, and the heterogeneity
of CFS symptoms has precluded the use of any particular diagnostic
laboratory test. (P. H. Levine [1998]). Symptomatic parallels have
been suggested between CFS and a number of other disease
conditions, resulting from viral infection, toxic exposure,
orthostatic hypotension, and stress, but none of these has been
shown to have a causal role in CFS. (E.g., I. R. Bell et al.,
Illness from low levels of environmental chemicals: relevance to
chronic fatigue syndrome and fibromyalgia, Am. J. Med.
105(3A):74S-82S [1998]; R. L. Bruno et al., Parallels between
post-polio fatigue and chronic fatigue syndrome: a common
pathophysiology?, Am. J. Med. 105(3A):66S-73S [1998]; R. Glaser and
J. K. Kiecolt-Glaser, Stress-associated immune modulation:
relevance to viral infections and chronic fatigue syndrome, Am. J.
Med. 105(3A):35S-42S [1998]; P. C. Rowe and H. Calkins, Neurally
mediated hypotension and chronic fatigue syndrome, Am. J. Med.
105(3A):15S-21S [1998]; L. A. Jason et al., Estimating the
prevalence of chronic fatigue syndrome among nurses, Am. J. Med.
105(3A):91S-93S [1998]). One study reported that there was no
support for an etiological role in CFS of Yersinia enterocolitica
infection. (C. M. Swanink et al., Yersinia entercolitica and the
chronic fatigue syndrome, J. Infect. 36(3):269-72 [1998]).
Accordingly, there has been no known cause to which diagnosis
and/or treatment of CSF could be directed.
[0025] Consequently, the diagnosis and treatment of CFS have
continued to be directed to symptoms, rather than to an underlying
treatable cause. For example, the use of relaxin has been described
for relaxing the involuntary muscles and thus relieve pain
associated with CFS. (S. K. Yue, Method of treating myofascial pain
syndrome with relaxin, U.S. Pat. No. 5,863,552).
[0026] Attention deficit/hyperactivity disorder (ADHD) is a
heterogeneous behaviorial disorder of unknown etiology that always
appears first in childhood, affecting 3-20% of elementary
school-age children, and continues to affect up to 3% of adults.
(Reviewed in L. L. Greenhill, Diagnosing attention
deficit/hyperactivity disorder in children, J. Clin. Psychiatry 59
Suppl 7:31-41 [1998]). Those affected with ADHD symptoms typically
exhibit inattentiveness and distractability (AD type), hyperactive
and impulsive behavior (HI type), or a combination of these, to a
degree that impairs normal functioning and is often socially
disruptive. (M. L. Wolraich et al., Examination of DSM-IV criteria
for attention deficit/hyperactivity disorder in a county-wide
sample, J. Dev. Behay. Pediatr. 19(3):162-68 [1998]; J. J. Hudziak
et al., Latent class and factor analysis of DSM-IV ADHD: a twin
study of female adolescents, J. Am. Acad. Child Adolesc. Psychiatry
37(8):848-57 [1998]). Often prescribed are central nervous system
stimulants, tricyclic antidepressants, antihypertensives,
analgesics, or antimanic drugs, but there has been no known cause
of ADHD to which diagnosis and/or treatment could be directed. (S.
C. Schneider and G. Tan, Attention deficit/hyperactivity disorder.
In pursuit of diagnostic accuracy, Postgrad. Med. 101(4):231-2,
235-40 [1997]; W. J. Barbaresi, Primary-care approach to the
diagnosis and management of attention deficit/hyperactivity
disorder, Mayo Clin. Proc. 71(5):463-71 [1996]).
[0027] There has also been no known cause for autoimmune diseases,
including multiple sclerosis and systemic lupus erythematosus.
Multiple sclerosis (MS) is a neurologic disease that primarily
strikes teens and young adults under 35 years. Affecting 350,000
Americans, MS is the most frequent cause of neurologic disability
except for traumatic injuries; MS affects twice as many females
compared to males. (S. L. Hauser, Multiple Sclerosis and other
demyelinating diseases In: Harrison's Principles of Internal
Medicine, 13th ed., K. J. Isselbacher et al. (eds.), McGraw-Hill,
pp. 2287-95 [1994]). The disease is characterized by chronic
inflammation, scarring, and selective destruction of the myelin
sheath around neural axons of the central nervous system, and is
thought to be caused by autoimmune responses. A treatment for MS
taught by Weiner et al. is related to oral administration of
autoantigens to the patient to suppress the autoimmune response by
eliciting suppressor T-cells specific for myelin basic protein
(MBP). There are no specific diagnostic tests for MS; diagnosis is
based on clinical recognition of destructive patterns of central
nervous system injury that are produced by the disease. (S. L.
Hauser [1994]) Nerve damage may be mediated by cytokines,
especially TNF-.alpha., which has been found to be selectively
toxic to myelin and to oligodendrocytes in vitro. Elevated levels
of TNF-.alpha. and IL-2 were measured in MS patients. (J. L.
Trotter et al., Serum cytokine levels in chronic progressive
multiple sclerosis: interleukin-2 levels parallel tumor necrosis
factor-alpha levels, J. Neuroimmunol. 33(1):29-36 [1991]; H. L.
Weiner et al., Treatment of multiple sclerosis by oral
administration of autoantigens, U.S. Pat. No. 5,869,054). Another
treatment for MS involves the administration of a vitamin D
compound. (H. F. DeLuca et al., Multiple sclerosis treatment, U.S.
Pat. No. 5,716,946). However, there has been no known cause of MS
to which diagnosis and/or treatment could be directed.
[0028] Systemic lupus erythematosus (SLE) is an autoimmune
rheumatic disease characterized by deposition in tissues of
autoantibodies and immune complexes leading to tissue injury (B. L.
Kotzin, Systemic lupus erythematosus, Cell 85:303-06 [1996]). In
contrast to autoimmune diseases such as MS and type 1 diabetes
mellitus, SLE potentially involves multiple organ systems directly,
and its clinical manifestations are diverse and variable. (Reviewed
by B. L. Kotzin and J. R. O'Dell, Systemic lupus erythematosus, In:
Samler's Immunologic Diseases, 5th ed., M. M. Frank et al., eds.,
Little Brown & Co., Boston, pp. 667-97 [1995]). For example,
some patients may demonstrate primarily skin rash and joint pain,
show spontaneous remissions, and require little medication. At the
other end of the spectrum are patients who demonstrate severe and
progressive kidney involvement that requires therapy with high
doses of steroids and cytotoxic drugs such as cyclophosphamide. (B.
L. Kotzin [1996]).
[0029] The serological hallmark of SLE, and the primary diagnostic
test available, is elevated serum levels of IgG antibodies to
constituents of the cell nucleus, such as double-stranded DNA
(dsDNA), single-stranded DNA (ss-DNA), and chromatin. Among these
autoantibodies, IgG anti-dsDNA antibodies play a major role in the
development of lupus glomerulonephritis (GN). (B. H. Hahn and B.
Tsao, Antibodies to DNA, In: Dubois=Lupus Erythematosus, 4th ed.,
D. J. Wallace and B. Hahn, eds., Lea and Febiger, Philadelphia, pp.
195-201 [1993]; Ohnishi et al., Comparison of pathogenic and
nonpathogenic murine antibodies to DNA: Antigen binding and
structural characteristics, Int. Immunol. 6:817-30 [1994]).
Glomerulonephritis is a serious condition in which the capillary
walls of the kidney's blood purifying glomeruli become thickened by
accretions on the epithelial side of glomerular basement membranes.
The disease is often chronic and progressive and may lead to
eventual renal failure.
[0030] The mechanisms by which autoantibodies are induced in these
autoimmune diseases remains unclear. As there has been no known
cause of SLE, to which diagnosis and/or treatment could be
directed, treatment has been directed to suppressing immune
responses, for example with macrolide antibiotics, rather than to
an underlying cause. (E.g., Hitoshi et al., Immunosuppressive
agent, U.S. Pat. No. 4,843,092).
[0031] Another disorder for which immunosuppression has been tried
is Crohn's disease. Crohn's disease symptoms include intestinal
inflammation and the development of intestinal stenosis and
fistulas; neuropathy often accompanies these symptoms.
Anti-inflammatory drugs, such as 5-aminosalicylates (e.g.,
mesalamine) or corticosteroids, are typically prescribed, but are
not always effective. (Reviewed in V. A. Botoman et al., Management
of Inflammatory Bowel Disease, Am. Fam. Physician 57(1):57-68
[1998]). Immunosuppression with cyclosporine is sometimes
beneficial for patients resistant to or intolerant of
corticosteroids. (J. Brynskov et al., A placebo-controlled,
double-blind, randomized trial of cyclosprorine therapy in active
chronic Crohn's disease, N. Engl. J. Med. 321(13):845-50
[1989]).
[0032] Nevertheless, surgical correction is eventually required in
90% of patients; 50% undergo colonic resection. (K. Leiper et al.,
Adjuvant post-operative therapy, Baillieres Clin. Gastroenterol.
12(1):179-99 [1998]; F. Makowiec et al., Long-term follow-up after
resectional surgery in patients with Crohn's disease involving the
colon, Z. Gastroenterol. 36(8):619-24 [1998]). The recurrence rate
after surgery is high, with 50% requiring further surgery within 5
years. (K. Leiper et al. [1998]; M. Besnard et al., Postoperative
outcome of Crohn's disease in 30 children, Gut 43(5):634-38
[1998]).
[0033] One hypothesis for the etiology of Crohn's disease is that a
failure of the intestinal mucosal barrier, possibly resulting from
genetic susceptibilities and environmental factors (e.g., smoking),
exposes the immune system to antigens from the intestinal lumen
including bacterial and food antigens (e.g., Soderholm et al.,
Epithelial permeability to proteins in the non-inflamed ileum of
Crohn's disease?, Gastroenterol. 117:65-72 [1999]; D. Hollander et
al., Increased intestinal permeability in patients with Crohn's
disease and their relatives. A possible etiologic factor, Ann.
Intern. Med. 105:883-85 [1986]; D. Hollander, The intestinal
permeability barrier. A hypothesis to its involvement in Crohn's
disease, Scand. J. Gastroenterol. 27:721-26 [1992]). Another
hypothesis is that persistent intestinal infection by pathogens
such as Mycobacterium paratuberculosis, Listeria monocytogenes,
abnormal Escherichia coli, or paramyxovirus, stimulates the immune
response; or alternatively, symptoms result from a dysregulated
immune response to ubiquitous antigens, such as normal intestinal
microflora and the metabolites and toxins they produce. (R. B.
Sartor, Pathogenesis and Immune Mechanisms of Chronic Inflammatory
Bowel Diseases, Am. J. Gastroenterol. 92(12):5S-11S [1997]). The
presence of IgA and IgG anti-Sacccharomyces cerevisiae antibodies
(ASCA) in the serum was found to be highly diagnostic of pediatric
Crohn's disease. (F. M. Ruemmele et al., Diagnostic accuracy of
serological assays in pediatric inflammatory bowel disease,
Gastroenterol. 115(4):822-29 [1998]; E. J. Hoffenberg et al.,
Serologic testing for inflammatory bowel disease, J. Pediatr.
134(4):447-52 [1999]).
[0034] In Crohn=s disease, a dysregulated immune response is skewed
toward cell-mediated immunopathology. (S. I. Murch, Local and
systemic effects of macrophage cytokines in intestinal
inflammation, Nutrition 14:780-83 [1998]). But immunosuppressive
drugs, such as cyclosporine, tacrolimus, and mesalamine have been
used to treat corticosteroid-resistant cases of Crohn=s disease
with mixed success. (J. Brynskov et al. [1989]; K. Fellerman et
al., Steroid-unresponsive acute attacks of inflammatory bowel
disease: immunomodulation by tacrolimus [FK506], Am. J.
Gastroenterol. 93(10):1860-66 [1998]). An abnormal increase in
colonic permeability is also seen in patients with Crohn's disease.
(Vermeire S. et al., Anti-Saccharomyces cerevisiae antibodies
(ASCA), phenotypes of IBD, and intestinal permeability: a study in
IBD families, Inflamm Bowel Dis. 7(1):8-15 [2001]).
[0035] Recent efforts to develop diagnostic and treatment tools
against Crohn=s disease have focused on the central role of
cytokines (S. Schreiber, Experimental immunomodulatory therapy of
inflammatory bowel disease, Neth. J. Med. 53(6):S24-31 [1998]; R.
A. van Hogezand and H. W. Verspaget, The future role of anti-tumour
necrosis factor-alpha products in the treatment of Crohn's disease,
Drugs 56(3):299-305 [1998]). Cytokines are small secreted proteins
or factors (5 to 20 kD) that have specific effects on cell-to-cell
interactions, intercellular communication, or the behavior of other
cells. Cytokines are produced by lymphocytes, especially T.sub.H1
and T.sub.H2 lymphocytes, monocytes, intestinal macrophages,
granulocytes, epithelial cells, and fibroblasts. (Reviewed in G.
Rogler and T. Andus, Cytokines in inflammatory bowel disease, World
J. Surg. 22(4):382-89 [1998]; H. F. Galley and N. R. Webster, The
immuno-inflammatory cascade, Br. J. Anaesth. 77:11-16 [1996]). Some
cytokines are pro-inflammatory (e.g., tumor necrosis factor
[TNF]-.alpha., interleukin [IL]-1(.alpha. and .beta.), IL-6, IL-8,
IL-12, or leukemia inhibitory factor [LIF]); others are
anti-inflammatory (e.g., IL-1 receptor antagonist [IL-1ra], IL-4,
IL-10, IL-11, and transforming growth factor [TGF]-.beta.).
However, there may be overlap and functional redundancy in their
effects under certain inflammatory conditions.
[0036] In active cases of Crohn=s disease, elevated concentrations
of TNF-.alpha. and IL-6 are secreted into the blood circulation,
and TNF-.alpha., IL-1, IL-6, and IL-8 are produced in excess
locally by mucosal cells. (Id.; K. Funakoshi et al., Spectrum of
cytokine gene expression in intestinal mucosal lesions of Crohn=s
disease and ulcerative colitis, Digestion 59(1):73-78 [1998]).
These cytokines can have far-ranging effects on physiological
systems including bone development, hematopoiesis, and liver,
thyroid, and neuropsychiatric function. Also, an imbalance of the
IL-1.beta./IL-1ra ratio, in favor of pro-inflammatory IL-1.beta.,
has been observed in patients with Crohn=s disease. (G. Rogler and
T. Andus [1998]; T. Saiki et al., Detection of pro-and
anti-inflammatory cytokines in stools of patients with inflammatory
bowel disease, Scand. J. Gastroenterol. 33(6):616-22 [1998]; S.
Dionne et al., Colonic explant production of IL-1 and its receptor
antagonist is imbalanced in inflammatory bowel disease (IBD), Clin.
Exp. Imunol. 112(3):435-42 [1998]; But see S. Kuboyama, Increased
circulating levels of interleukin-1 receptor antagonist in patients
with inflammatory bowel disease, Kurume Med. J. 45(1):33-37
[1998]). One study suggested that cytokine profiles in stool
samples could be a useful diagnostic tool for Crohn=s disease. (T.
Saiki et al. [1998]).
[0037] Treatments that have been proposed for Crohn=s disease
include the use of various cytokine antagonists (e.g., IL-1ra),
inhibitors (e.g., of IL-1.beta. converting enzyme and antioxidants)
and anti-cytokine antibodies. (G. Rogler and T. Andus [1998]; R. A.
van Hogezand and H. W. Verspaget [1998]; J. M. Reimund et al.,
Antioxidants inhibit the in vitro production of inflammatory
cytokines in Crohn=s disease and ulcerative colitis, Eur. J. Clin.
Invest. 28(2):145-50 [1998]; N. Lugering et al., Current concept of
the role of monocytes/macrophages in inflammatory bowel
disease-balance of pro-inflammatory and immunosuppressive
mediators, Ital. J. Gastroenterol. Hepatol. 30(3):338-44 [1998]; M.
E. McAlindon et al., Expression of interleukin 1 beta and
interleukin 1 beta converting enzyme by intestinal macrophages in
health and inflammatory bowel disease, Gut 42(2):214-19 [1998]). In
particular, monoclonal antibodies against TNF-.alpha. have been
tried with some success in the treatment of Crohn=s disease. (S. R.
Targan et al., A short-term study of chimeric monoclonal antibody
cA2 to tumor necrosis factor alpha for Crohn=s disease. Crohn=s
Disease cA2 Study Group, N. Engl. J. Med. 337(15):1029-35 [1997];
W. A. Stack et al., Randomised controlled trial of CDP571 antibody
to tumour necrosis factor-alpha in Crohn disease, Lancet
349(9051):521-24 [1997]; H. M. van Dullemen et al., Treatment of
Crohn=s disease with anti-tumor necrosis factor chimeric monoclonal
antibody (cA2), Gastroenterol. 109(1):129-35 [1995]).
[0038] Another approach to the treatment of Crohn=s disease has
focused on at least partially eradicating the bacterial community
that may be triggering the inflammatory response and replacing it
with a non-pathogenic community. For example, McCann et al. (McCann
et al., Method for treatment of idiopathic inflammatory bowel
disease, U.S. Pat. No. 5,599,795) disclosed a method for the
prevention and treatment of Crohn=s disease in human patients.
Their method was directed to sterilizing the intestinal tract with
at least one antibiotic and at least one anti-fungal agent to kill
off the existing flora and replacing them with different, select,
well-characterized bacteria taken from normal humans. Borody taught
a method of treating Crohn=s disease by at least partial removal of
the existing intestinal microflora by lavage and replacement with a
new bacterial community introduced by fecal inoculum from a
disease-screened human donor or by a composition comprising
Bacteroides and Escherichia coli species. (T. J. Barody, Treatment
of gastro-intestinal disorders with a fecal composition or a
composition of bacteroides and E. coli, U.S. Pat. No. 5,443,826).
However, there has been no known cause of Crohn=s disease to which
diagnosis and/or treatment could be directed.
[0039] Pain is a common symptom associated with irritable bowel
syndrome, fibromyalgia, chronic fatigue syndrome, chronic pelvic
pain syndrome, depression, ADHD, autoimmune diseases, and Crohn=s
disease. While the experience of pain is intertwined with a
person's emotions, memory, culture, and psychosocial situation (D.
A. Drossman and W. G. Thompson, Irritable bowel syndrome: a
graduated, multicomponent treatment approach, Ann. Intern. Med.
116:1009-16 [1992]), evidence shows that certain cytokine
mediated-immune responses can influence the perception of pain.
Cytokines can be released in response to a variety of irritants and
can modulate the perception of pain. For example, exposure of human
bronchial epithelial cells to irritants, including acidic pH,
results in a receptor-mediated release of inflammatory cytokines
IL-6, IL-8, and TNF-.alpha.. (B. Veronesi et al., Particulate
Matter initiates inflammatory cytokine release by activation of
capsaicin and acid receptors in a human bronchial epithelial cell
line, Toxicol. Appl. Pharmacol. 154:106-15 [1999]). Irritant
receptors on cell surfaces, e.g., receptors sensitive to noxious
stimuli, such as capsaicin and pH, mediate the release of cytokines
and also mediate the release of neuropeptides from sensory nerve
fibers, which is known to result in a neurogenic inflammatory
processes and hyperalgesia (excessive sensitivity to pain). (Id.;
R.O.P. de Campos et al., Systemic treatment with Mycobacterium
bovis bacillus calmett-guerin (BCG) potentiates kinin B.sub.1
receptor agonist-induced nociception and oedema formation in the
formalin test in mice, Neuropeptides 32(5):393-403 [1998]).
[0040] The perception of pain, is also influenced by the mediation
of kinin B.sub.1 and B.sub.2 receptors, which bind peptides called
kinins, e.g., the nonapeptide bradykinin or the decapeptide
kallidin (lysyl bradykinin) While the precise mechanism of action
is unknown, kinins cause the release of other pro-inflammatory and
hyperalgesic mediators such as neuropeptides. Cytokines
IL-1(.alpha. and .beta.), IL-2, IL-6, and TNF-.alpha. are thought
to activate kinin B.sub.1 receptor, and thus can contribute to
enhanced perception ofpain. (R.O.P. de Campos et al. [1998]. The
endotoxin of Escherichia coli significantly activated kinin B.sub.1
receptor-mediated neurogenic and inflammatory pain responses in
animals. (M. M. Campos et al., Expression of B.sub.1 kinin
receptors mediating paw oedema formalin-induced nociception.
Modulation by glucocorticoids, Can. J. Physiol. Pharmacol.
73:812-19 [1995]).
[0041] It has also been shown that IL-1.beta., IL-6, and
TNF-.alpha., administered to the mammalian brain, can modulate pain
perception via prostaglandin-dependent processes. (T. Hori et al.,
Pain modulatory actions of cytokines and prostaglandin E.sub.2 in
the Brain, Ann. N.Y. Acad. Sci. 840:269-81 [1998]). Granulocytes,
which accumulate in nearly all forms of inflammation, are
non-specific amplifiers and effectors of specific immune responses,
and they can also modulate the perception of pain. Neutrophils, a
type of granulocyte cell, are known to accumulate in response to
IL-1.beta., and neutrophil accumulation plays a crucial positive
role in the development of nerve growth factor (NGF)-induced
hyperalgesia. (G. Bennett et al., Nerve growth factor induced
hyperalgesia in the rat hind paw is dependent on circulating
neutrophils, Pain 77(3):315-22 [1998]; see also E. Feher et al.,
Direct morphological evidence of neuroimmunomodulation in colonic
mucosa of patients with Crohn=s disease, Neuroimmunomodulation
4(5-6): 250-57 [1997]).
[0042] Visceral hyperalgesia, or pain hypersensitivity, is a common
clinical observation in small intestinal bacterial overgrowth
(SIBO), Crohn=s disease, chronic pelvic pain syndrome, and
irritable bowel syndrome (IBS). As many as 60% of subjects with IBS
have reduced sensory thresholds for rectal distension compared to
normal subjects. (H. Mertz et al., Altered rectal perception is a
biological marker of patients with the irritable bowel syndrome,
Gastroenterol. 109:40-52 [1995]). While the experience of pain is
intertwined with a person's emotions, memory, culture, and
psychosocial situation (D. A. Drossman and W. G. Thompson,
Irritable bowel syndrome: a graduated, multicomponent treatment
approach, Ann. Intern. Med. 116:1009-16 [1992]) and the etiology
for this hyperalgesia has remained elusive, evidence shows that
certain cytokine mediated-immune responses can influence the
perception of pain. Cytokines, including IL-1(.alpha. and .beta.),
IL-2, IL-6, and TNF-.alpha., can be released in response to a
variety of irritants and can modulate the perception of pain,
possibly through the mediation of kinin B.sub.1 and/or B.sub.2
receptors (see, M. M. Campos et al., Expression of B.sub.1 kinin
receptors mediating paw oedema formalin-induced nociception.
Modulation by glucocorticoids, Can. J. Physiol. Pharmacol.
73:812-19 [1995]; R.O.P. de Campos et al., Systemic treatment with
Mycobacterium bovis bacillus calmett-guerin (BCG) potentiates kinin
B.sub.1 receptor agonist-induced nociception and oedema formation
in the formalin test in mice, Neuropeptides 32(5):393-403 [1998]).
Cytokine and neuropeptide levels are altered in IBS. An increase in
substance P (neuropeptide)-sensitive nerve endings has been
observed in subjects with IBS. (X. Pang et al., Mast cell substance
P-positive nerve involvement in a patient with both irritable bowel
syndrome and interstitial cystitis, Urology 47:436-38 [1996]). It
has also been hypothesized that there is a sensitization of
afferent pathways in IBS. (E. A. Mayer et al., Basic and clinical
aspects of visceral hyperalgesia, Gastroenterol 1994; 107:271-93
[1994]; L. Bueno et al., Mediators and pharmacology of visceral
sensitivity: from basic to clinical investigations, Gastroenterol.
112:1714-43 [1997]).
[0043] Fibromyalgia, typically involving global musculoskeletal
and/or cutaneous pain, is, by definition; a hyperalgesic state
since the American College of Rheumatology defines fibromyalgia as
a history of global pain in the setting of 11 out of 18 predefined
tender points. (F. Wolfe et al., The American College of
Rheumatology 1990 criteria for the classification of fibromyalgia,
Arthritis Rheum. 33:160-72 [1990]). Evidence implies that the
hyperalgesia of fibromyalgia is not simply trigger point-related
but rather a global hyperalgesia. (L. Vecchiet et al., Comparative
sensory evaluation of parietal tissues in painful and nonpainful
areas in fibromyalgia and myofascial pain syndrome, In: Gebhart G
F, Hammond D L, Jensen T S, editors, Progress in Pain Research and
Management, Vol. 2, Seattle: IASP Press, pp. 177-85 [1994]; J.
Sorensen et al., Hyperexcitability in fibromyalgia, J. Rheumatol.
25:152-55 [1998]).
[0044] Cytokine and neuropeptide levels are altered in IBS,
fibromyalgia, and Crohn=s disease. It has been shown that levels of
substance P, a neuropeptide associated with nociception, are
elevated in the cerebrospinal fluid of subjects with fibromyalgia.
(H. Vaeroy et al., Elevated CSF levels of substance P and high
incidence of Raynaud's phenomenon in patients with fibromyalgia:
new features for diagnosis, Pain 32:21-26 [1988]; I. J. Russell et
al., Elevated cerebrospinal fluid levels of substance P in patients
with the fibromyalgia syndrome, Arthritis Rheum. 37:1593-1601
[1994]). And an increase in substance P-sensitive nerve endings has
been observed in subjects with IBS and Crohn's disease. (X. Pang et
al., Mast cell substance P-positive nerve involvement in a patient
with both irritable bowel syndrome and interstitial cystitis,
Urology 47:436-38 [1996]; (C. R. Mantyh et al., Receptor binding
sites for substance P, but not substance K or neuromedin K, are
expressed in high concentrations by arterioles, venules, and lymph
nodules in surgical specimens obtained from patients with
ulcerative colitis and Crohn's disease, Proc. Natl. Acad. Sci.
85:3235-39 [1988]; S. Mazumdar and K. M. Das, Immunocytochemical
localization of vasoactive intestinal peptide and substance P in
the colon from normal subjects and patients with inflammatory bowel
disease, Am. J. Gastrol. 87:176-81 [1992]; C. R. Mantyh et al.,
Differential expression of substance P receptors in patients with
Crohn's disease and ulcerative colitis, Gastroenterol. 1995;
109:850-60 [1995]).
[0045] Patients with chronic pelvic pain are usually evaluated and
treated by gynecologists, gastroenterologists, urologists, and
internists, but in many patients with chronic pelvic pain the
examination and work-up remain unrevealing, and no specific cause
of the pain, such as endometriosis, can be identified. In these
cases the patient is commonly said to be suffering from a "chronic
pelvic pain syndrome." Once the diagnosis of chronic pelvic pain is
made, treatment is typically directed to symptomatic pain
management, rather than to an underlying cause. (Wesselmann U,
Czakanski P P, Pelvic pain: a chronic visceral pain syndrome, Curr.
Pain Headache Rep. 5(1):13-9 [2001]).
[0046] Mental functioning and feelings of fatigue or depression can
also be influenced by immune responses. Peripherally released
pro-inflammatory cytokines, such as IL-1, IL-6 and TNF-.alpha., act
on brain cellular targets and have been shown to depress
spontaneous and learned behavior in animals; the vagus nerve has
been shown to mediate the transmissions of the immune message to
the brain, resulting in production of pro-inflammatory cytokines
centrally in the brain. (R. Dantzer et al., Cytokines and sickness
behavior, Ann. N.Y. Acad. Sci. 840:586-90 [1998]). In addition,
there is bidirectional interplay between neurotransmitters and the
immune system; lymphocytes and macrophages bear surface receptors
for the stress hormone corticotrophin releasing hormone (CRH), and
they respond to CRH by enhanced lymphocyte proliferation and
feedback upregulation of hypothalamic CRH production. (S. H. Murch
[1998]).
[0047] Pituitary production of proopiomelanocortins, such as
endorphins and enkephalins, is upregulated by IL-1 and IL-2,
possibly mediated by CRH, and lymphocytes and macrophages recognize
these endogenous opiates via surface receptors. (S. H. Murch
[1998]). Lymphocytes (T.sub.H2) and macrophages also produce and
process enkephalin to an active form. Macrophage-derived cytokines,
such as TNF-.alpha., IL-1, and IL-6, are known to modulate
neurotransmitter release and to affect overall neural activity;
cytokines can induce classic illness behavior such as somnolence,
apathy, depression, irritability, confusion, poor memory, impaired
mental concentration, fever and anorexia.
[0048] While immunological responses of various severities can lead
to symptoms characteristic of irritable bowel syndrome,
fibromyalgia, chronic pevic pain syndrome, chronic fatigue
syndrome, impaired mentation and/or memory, depression, autism,
ADHD, autoimmune diseases, and Crohn=s disease, there has been a
definite need to determine a causal factor, for each of these
diagnostic categories, to which diagnostic testing and treatment
can be directed effectively.
[0049] SIBO has, until recently, mostly been suspected in subjects
with significant malabsorptive sequelae. Most of the described
cases of SIBO involve anatomic alterations such as physical
obstruction (E. A. Deitch et al., Obstructed intestine as a
reservoir for systemic infection, Am. J. Surg. 159:394 [1990]),
surgical changes (e.g., L. K. Enander et al., The aerobic and
anaerobic microflora of the gastric remnant more than 15 years
after Billroth II resection, Scand. J. Gastroenterol. 17:715-20
[1982]), direct communication of the small intestine with colonic
contents such as fistulae (O. Bergesen et al., Is vitamin B12
malabsorption in bile fistula rats due to bacterial overgrowth? A
study of bacterial metabolic activity in the small bowel, Scand. J.
Gastroenterol. 23:471-6 [1988]) and ileocecal valve dysfunction
(surgical or otherwise) (W. O. Griffin, Jr, et al., Prevention of
small bowel contamination by ileocecal valve, S. Med. J. 64: 1056-8
[1971]; P. Rutgeerts et al., Ileal dysfunction and bacterial
overgrowth in patients with Crohn's disease, Eur. J. Clin. Invest.
11:199-206 [1981]). Less commonly, SIBO has been associated with
chronic pancreatitis (E. Trespi and A. Ferrieri, Intestinal
bacterial overgrowth during chronic pancreatitis, Curr. Med. Res.
Opin. 15:47-52 [1999]), hypochlorhydria (e.g., S. P. Pereira et
al., Drug-induced hypochlorhydria causes high duodenal bacterial
counts in the elderly, Aliment. Pharmacol. Ther. 12:99-104 [1998]),
and immunodeficiency (C. Pignata et al., Jejunal bacterial
overgrowth and intestinal permeability in children with
immunodeficiency syndromes, Gut 31:879-82 [1990]; G. M. Smith et
al., Small intestinal bacterial overgrowth in patients with chronic
lymphocytic leukemia, J. Clin. Pathol. 43:57-9 [1990]).
[0050] SIBO has been associated with infections of the abdominal
cavity in cases of alcoholic cirrhosis. (F. Casafont Morencos et
al., Small bowel bacterial overgrowth in patients with alcoholic
cirrhosis, Dig. Dis. Sci. 40(6):1252-1256 [1995]; J. Chesta et al.,
Abnormalities in proximal small bowel motility in patients with
cirrhosis, Hepatology 17(5):828-32 [1993]; C. S. Chang et al.,
Small intestine dysmotility and bacterial overgrowth in cirrhotic
patients with spontaneous bacterial peritonitis, Hepatology
28(5):1187-90 [1998]). SIBO has also been associated with symptoms
of chronic diarrhea, anorexia or nausea in elderly patients, and
the prevalence of overgrowth in subjects over 75 years old is
reported to be as high as 79% even in the absence of clinically
evident clues of overgrowth or achlorhydria. (S. M. Riordan et al.,
Small intestinal bacterial overgrowth in the symptomatic elderly,
Am. J. Gastroenterol. 92(1):47-51 [1997]). SIBO is also associated
with chronic digestive symptoms in children, especially infants
under two years of age (D. De Boissieu et al., Small-bowel
bacterial overgrowth in children with chronic digestive diarrhea,
abdominal pain, or both, J. Pediatr. 128(2):203-07 [1996]), and
with chronic diarrhea after liver transplantation in children. (D.
R. Mack et al., Small bowel bacterial overgrowth as a cause of
chronic diarrhea after liver transplantation in children, Liver
Transpl. Surg. 4(2):166-69 [1998]).
[0051] Although diabetic enteropathy (F. Goldstein et al., Diabetic
diarrhea and steatorrhea. Microbiologic and clinical observations,
Ann. Intern. Med. 1970; 72:215-8 [1970]), idiopathic intestinal
pseudo-obstruction (A. J. Pearson et al., Intestinal
pseudo-obstruction with bacterial overgrowth in the small
intestine, Am. J. Dig. Dis. 14:200-05 [1969]) and scleroderma (I.
J. Kahn et al., Malabsorption in intestinal scleroderma: Correction
with antibiotics, N. Engl. J. Med. 274: 1339-44 [1966]) are all
known to produce motility disturbances leading to SIBO. Two
previous reports have examined small bowel motility among
anatomically and medically naive SIBO subjects. (G. Vantrappen et
al., The interdigestive motor complex of normal subjects and
patients with bacterial overgrowth of the small intestine, J. Clin.
Invest. 59: 1158-66 [1977]; P. O. Stotzer et al., Interdigestive
and postprandial motility in small-intestinal bacterial overgrowth,
Scand. J. Gastroenterol. 31:875-80 [1996]). These authors suggest
that the majority of subjects with SIBO in the absence of other
predisposing conditions, lack the phase III of interdigestive
motility during short term recordings.
[0052] Phase III of interdigestive motility is a period of phasic
contractions propagating through the length of the small intestine,
approximately once every 87.2.+-.5.4 minutes in the fasting state.
(E. E. Soffer et al., Prolonged ambulatory duodeno-jejunal
manometry in humans: Normal values and gender effect, Am. J.
Gastrol. 93:1318-23 [1998]). This fasting event is responsible for
sweeping residue including small bowel contaminants, such as
accumulated bacteria, into the colon in preparation for the next
meal. (V. B. Nieuwenhujuijs et al., The role of interdigestive
small bowel motility in the regulation of gut microflora, bacterial
overgrowth, and bacterial translocation in rats, Ann. Surg. 228:
188-93 [1998]; E. Husebye, Gastrointestinal motility disorders and
bacterial overgrowth, J. Intem. Med. 237:419-27 [1995]). The
endogenous peptide, motilin, is involved in the mediation of this
event. (G. Vantrappen et al., Motilin and the interdigestive
migrating motor complex in man, Dig. Dis. Sci. 24:497-500 [1979]).
Other prokinetic agents, such as erythromycin, are believed to act
on the motilin receptor and have been shown to rapidly induce an
interdigestive motility event in dogs and humans. (M. F. Otterson
and S. K. Sarna, Gastrointestinal motor effect of erythromycin, Am.
J. Physiol. 259:G355-63; T. Tomomasa et al., Erythromycin induces
migrating motor complex in human gastrointestinal tract, Dig. Dis.
Sci. 31:157-61 [1986]).
[0053] In general, the speed of transit through the small intestine
is normally regulated by inhibitory mechanisms located in the
proximal and distal small intestine known as the jejunal brake and
the ileal brake. Inhibitory feedback is activated to slow transit
when end products of digestion make contact with nutrient sensors
of the small intestine. (E.g., Lin, H. C., U.S. Pat. No. 5,977,175;
Dobson, C. L. et al., The effect of oleic acid on the human ileal
brake and its implications for small intestinal transit of tablet
formulations, Pharm. Res. 16(1):92-96 [1999]; Lin, H. C. et al.,
Intestinal transit is more potently inhibited by fat in the distal
(Ileal brake) than in the proximal (jejunal brake) gut, Dig. Dis.
Sci. 42(1):19-25 [1997]; Lin, H. C. et al., Jejunal brake:
inhibition of intestinal transit by fat in the proximal small
intestine, Dig. Dis. Sci., 41(2):326-29 [1996a]).
[0054] Specifically, jejunal and ileal brakes slow transit by the
release of gut peptides such as peptide YY and by the activation of
neural pathways such as those involving endogenous opioids. (Lin,
H. C. et al., Fat-induced ileal brake in the dog depends on peptide
YY, Gastroenterol. 110(5):1491-95 [1996b]). Transit is then slowed
by the stimulation of nonpropagative intestinal contractions which
inhibit movement of the lumenal content. The removal or impairment
of these inhibitory mechanisms can lead to abnormally rapid
transit. For example, in patients with a history of resection of
the terminal ileum, intestinal transit can become uncontrolled and
abnormally accelerated when the ileal brake is no longer intact.
Time for processing of food can then be so reduced that few end
products of digestion are available to trigger the jejunal brake as
the remaining inhibitory mechanism.
[0055] Peptide YY and its analogs or agonists have been used to
manipulate endocrine regulation of cell proliferation, nutrient
transport, and intestinal water and electrolyte secretion. (E.g.,
Balasubramaniam, Analogs of peptide yy and uses thereof, U.S. Pat.
No. 5,604,203; W09820885A1; EP692971A1; Croom et al., Method of
enhancing nutrient uptake, U.S. Pat. No. 5,912,227; Litvak, D. A.
et al., Characterization of two novel proabsorptive peptide YY
analogs, BIM-43073D and BIM-43004C, Dig. Dis. Sci. 44(3):643-48
[1999]). A role for peptide YY in the regulation of intestinal
motility, secretion, and blood flow has also been suggested, as
well as its use in a treatment of malabsorptive disorders (Liu, C.
D. et al., Peptide YY: a potential proabsorbtive hormone for the
treatment of malabsorptive disorders, Am. Surg. 62(3):232-36
[1996]; Liu, C. D. et al., Intralumenal peptide YY induces colonic
absorption in vivo, Dis. Colon Rectum 40(4):478-82 [1997]; Bilchik,
A. J. et al., Peptide YY augments postprandial small intestinal
absorption in the conscious dog, Am. J. Surg. 167(6):570-74
[1994]).
[0056] Lin et al. immuno-neutralized peptide YY in vivo to block
the ileal brake response and, thus, showed that it is mediated by
peptide YY. (Lin, H. C. et al., Fat-induced ileal brake in the dog
depends on peptide YY, Gastroenterology, 110(5):1491-95 [1996b]).
Serum levels of peptide YY increase during the ileal brake response
to nutrient infusion into the distal ileum. (Spiller, R. C. et al.,
Further characterisation of the `ileal brake` reflex in man--effect
of ileal infusion of partial digests of fat, protein, and starch on
jejunal motility and release of neurotensin, enteroglucagon, and
peptide YY, Gut, 29(8):1042-51 [1988]; Pironi, L. et al.,
Fat-induced ileal brake in humans: a dose-dependent phenomenon
correlated to the plasma levels of peptide YY, Gastroenterology,
105(3):733-9 [1993]; Dreznik, Z. et al., Effect of ileal oleate on
interdigestive intestinal motility of the dog, Dig. Dis. Sci.,
39(7):1511-8 [1994]; Lin, C. D. et al., Intralumenal peptide YY
induces colonic absorption in vivo, Dis. Colon Rectum, 40(4):478-82
[April 1997]). In contrast, in vitro studies have shown peptide YY
infused into isolated canine ileum dose-dependently increased
phasic circular muscle activity. (Fox-Threlkeld, J. A. et al.,
Peptide YY stimulates circular muscle contractions of the isolated
perfused canine ileum by inhibiting nitric oxide release and
enchancing acetylcholine release, Peptides, 14(6):1171-78
[1993]).
[0057] Kreutter et al. taught the use of .beta..sub.3-adrenoceptor
agonists and antagonists for the treatment of intestinal motility
disorders, as well as depression, prostate disease and dyslipidemia
(U.S. Pat. No. 5,627,200).
[0058] Bagnol et al. reported the comparative immunovisualization
of mu and kappa opioid receptors in the various cell layers of the
rat gastrointestinal tract, including a comparatively large number
of kappa opioid receptors in the myenteric plexus. (Bagnol, D. et
al., Cellular localization and distribution of the cloned mu and
kappa opioid receptors in rat gastrointestinal tract, Neuroscience,
81(2):579-91[1997]). They suggested that opioid receptors can
directly influence neuronal activity in the gastrointestinal
tract.
[0059] Kreek et al. taught the use of opioid receptor antagonists,
such as naloxone, naltrexone, and nalmefene, for the relief of
gastrointestinal dysmotility. (Kreek et al., Method for controlling
gastrointestinal dysmotility, U.S. Pat. No. 4,987,136). Riviere et
al. taught the use of the opioid receptor antagonist fedotozine in
the treatment of intestinal obstructions (Riviere, P. J. M. et al.,
U.S. Pat. No. 5,362,756). Opioid-related constipation, the most
common chronic adverse effect of opioid pain medications in
patients who require long-term opioid administration, such as
patients with advanced cancer or participants in methadone
maintenance, has been treated with orally administered
methylnaltrexone and naloxone. (Yuan, C. S. et al.,
Methylnaltrexone for reversal of constipation due to chronic
methadone use: a randomized controlled trial, JAMA 283(3):367-72
[2000]; Meissner, W. et al., Oral naloxone reverses
opioid-associated constipation, Pain 84(1):105-9 [2000];
Culpepper-Morgan, J. A., et al., Treatment of opioid-induced
constipation with oral naloxone: a pilot study, Clin. Pharmacol.
Ther. 52(1):90-95 [1992]; Yuan, C. S. et al., The safety and
efficacy of oral methylnaltrexone in preventing morphine-induced
delay in oral-cecal transit time, Clin. Pharmacol. Ther.
61(4):467-75 [1997]; Santos, F. A. et al., Quinine-induced
inhibition of gastrointestinal transit in mice: possible
involvement of endogenous opioids, Eur. J. Pharmacol.,
364(2-3):193-97 [1999]. Naloxone was also reported to abolish the
ileal brake in rats (Brown, N. J. et al., The effect of an opiate
receptor antagonist on the ileal brake mechanism in the rat,
Pharmacology, 47(4):230-36 [1993]).
[0060] Receptors for 5-hydroxytryptamine (5-HT) have been localized
on various cells of the gastrointestinal tract. (Gershon, M. D.,
Review article: roles played by 5-hydroxytryptamine in the
physiology of the bowel, Aliment. Pharmacol. Ther., 13 Suppl
2:15-30 [1999]; Kirchgessner, A. L. et al., Identification of cells
that express 5-hydroxytryptaminel A receptors in the nervous
systems of the bowel and pancreas, J. Comp. Neurol.,
15:364(3):439-455 [1996]). Brown et al. reported that subcutaneous
administration of 5-HT3 receptor antagonists, granisetron and
ondansetron, in rats delayed intestinal transit of a baked bean
meal but abolished the ileal brake induced by ileal infusion of
lipid. They postulated the presence of 5-HT3 receptors on afferent
nerves that initiate reflexes that both accelerate and delay
intestinal transit. (Brown, N.J. et al., Granisetron and
ondansetron: effects on the ileal brake mechanism in the rat, J.
Pharm. Pharmacol. 45(6):521-24 [1993]). Kuemmerle et al. reported
neuro-endocrine 5-HT-mediation of motilin-induced accelerated
gastrointestinal motility. (Kuemmerle, J. F. et al., Serotonin
neural receptors mediate motilin-induced motility in isolated,
vascularly perfused canine jejunum, J. Surg. Res., 45(4):357-62
[1988]).
[0061] Ninety-five percent of the human body's stores of
5-hydroxyltryptamine (5-HT), also known as serotonin, are found in
the gastrointestinal tract. (Gershon, M. D., The Second Brain, New
York: Harper Collins [1998]). In the intestines, the vast majority
of 5-HT is located in the enterochromaffin (EC) cells of the mucosa
(Gershon [1998]). 5-HT is also released by myenteric 5-HT neurons
in the myenteric plexus. (Gershon, M. D., The enteric nervous
system, Annu Rev Neurosci 4: 227-272 [1981]; Gershon, M. D. et al.,
Serotonin: synthesis and release from the myenteric plexus of the
mouse intestine, Science 149: 197-199 [1965]; Holzer, P., and G.
Skofitsch, Release of endogenous 5-hydroxytryptamine from the
myenteric plexus of the guinea-pig isolated small intestine, Br J
Pharmacol 81: 381-386 [1984]; Penttila, A., Histochemical reactions
of the enterochromaffin cells and the 5-hydroxytryptamine content
of the mammalian duodenum, Acta Physiol Scand Suppl 281: 1-77
[1966]). These intrinsic 5-HT neurons receive input from
parasympathetic and sympathetic fibers (Gershon, M. D., and D. L.
Sherman, Noradrenergic innervation of serotoninergic neurons in the
myenteric plexus, J Comp Neurol 259: 193-210 [1987]) and provide
input to the motor neurons in their vicinity to suggest that they
are interneurons. 5-HT3 receptors are widely expressed by these
myenteric 5-HT neurons as well as their neighboring neurons
(Galligan, J. J., Electrophysiological studies of
5-hydroxytryptamine receptors on enteric neurons, Behav Brain Res
73: 199-201 [1996]; Zhou, X., and J. J. Galligan, Synaptic
activation and properties of 5-hydroxytryptamine(3) receptors in
myenteric neurons of guinea pig intestine, J Pharmacol Exp Ther
290: 803-10 [1999]). However, the physiologic function of these
myenteric 5-HT neurons is not known. (E. G., Gershon, M. D. Review
article: roles played by 5-hydroxytryptamine in the physiology of
the bowel, Aliment Pharmacol Ther 13 Suppl 2: 15-30, 1999]; Grider,
J. R. et al., 5-HT released by mucosal stimuli initiates
peristalsis by activating 5-HT4/5-HT1p receptors on sensory CGRP
neurons, Am J Physiol 270: G778-G782 [1996]).
[0062] Regardless of the source of 5-HT (mucosal vs. neuronal or
both), the signaling role of this molecule is facilitated by the
availability of a 5-HT reuptake transporter called SERT that
terminates the signal with its removal. (Wade, P. R. et al.,
Localization and function of a 5-HT transporter in crypt epithelia
of the gastrointestinal tract, J Neurosci 16: 2352-64 [1996]).
Since SERT is a part of the plasma membrane of serotonergic neurons
(Blakely, R. D. et al., Cloning and expression of a functional
serotonin transporter from rat brain, Nature 354: 66-70 [1991]),
these transporters are ideally positioned to remove neuronal 5-HT
after signaling is completed. Serotonergic nerves are, however,
absent from the intestinal mucosa. (Furness, J. B., and M. Costas,
The enteric nervous system, New York: Churchill Livingston [1987]).
Instead, mucosal 5-HT from EC cells is removed by SERT expressed by
neighboring epithelial cells. (Chen, J. X. et al., Guinea pig 5-HT
transporter: cloning, expression, distribution, and function in
intestinal sensory reception, Am J Physiol 275: G433-G448
[1998]).
[0063] The action of SERT is blocked by drugs that inhibit the
reuptake transporter. These serotonin-selective reuptake inhibitors
(S SRI) are widely used as antidepressants. The most commonly
prescribed example is fluoxetine (Prozac). These agents
significantly alter the peristaltic response. Wade et al. reported
that fluoxetine initially acclerated the passage of a pellet
through an isolated segment of guinea pig colon to suggest
potentiation of the peristaltic effect of 5-HT when the removal of
this molecule was inhibited (Wade et al. [1996]). However, as the
dose of the SSRI was increased, the transit of the pellet became
slower and slower. This observation with fluoxetine suggested to
Gershon that 5-HT receptors became desensitized when an excess of
5-HT stayed around for a longer period of time and traversed
further away from its mucosal source (Gershon [1998]). These are
then the current concepts to explain the common gastrointestinal
side effects of SSRIs including nausea (excess 5-HT acting on
extrinsic sensory nerves) and diarrhea (excess 5-HT acting on
intrinsic primary afferent neurons to initiate peristalsis; Gershon
[1998]).
[0064] The current scientific foundation for understanding the role
of serotonin in normal and abnormal motility of the small intestine
has been based on the role of mucosal serotonin in two enteric
functions. The first is as the neurotransmitter, via the activation
of intrinsic primary afferent neurons (IPAN), for the peristaltic
reflex, which mediates colonic evacuation, and for the mucosal
secretory reflex. (E.g., Grider, J. R. et al., 5-Hydroxytryptamine4
receptor agonists initiate the peristaltic reflex in human, rat,
and guinea pig intestine, Gastroenterology, 115(2):370-80 [1998];
Jin, J. G. et al., Propulsion in guinea pig colon induced by
5-hydroxytryptamine (HT) via 5-HT4 and 5-HT3 receptors, J.
Pharmacol. Exp. Ther., 288(1):93-97 [1999]; Foxx-Orenstein, A. E.
et al., 5-HT4 receptor agonists and delta-opioid receptor
antagonists act synergistically to stimulate colonic propulsion, Am
J. Physiol., 275(5 Pt. 1):G979-83 [1998]; Foxx-Orenstein, A. E.,
Distinct 5-HT receptors mediate the peristaltic reflex induced by
mucosal stimuli in human and guinea pig intestine, Gastroenterology
111(5):1281-90 [1996]; Wade, P. R. et al., Localization and
function of a 5-HT transporter in crypt epithelia of the
gastrointestinal tract, J. Neurosci., 16(7):2352-64 [1996];
Grinder, J., Gastrin-releasing peptide (GRP) neuron are excitatory
neurons in the descending phase of the peristaltic reflex,
Gastronenterology 116: A1000 [1999]; Cooke, H., M. Sidhu, and Y.
Wang, 5-HT activates neural reflexes regulating secretion in the
guinea pig colon, Neurogastroenterol Motil 9: 181-6 [1997]; Cooke,
H. J., and H. V. Carey, Pharmacological analysis of
5-hydroxytryptamine actions on guinea-pig ileal mucosa, Eur J
Pharmacol 111: 329-37, [1985]; Frieling, T., J. Wood, and H. Cooke,
Submucosal reflexes: distension-evoked ion transport in the guinea
pig distal colon, Am J Physiol 263: G91-96 [1992]; Hardcastle, J.,
and P. Hardcastle, Comparison of the intestinal secretory responses
to 5-hydroxytryptamine in the rat jejunum and ileum in-vitro, J
Pharm Pharmcacol 49: 1126-31 [1997]; Kinsman, R. I., and N. W.
Read, Effect of naloxone on feedback regulation of small bowel
transit by fat, Gastroenterology 87: 335-337 [1984]).
[0065] The second enteric role for 5-HT is as the signal to the
brain about lumenal conditions, linking mucosal stimuli with the
brain via extrinsic primary sensory neurons. (Blackshaw, L. A., and
D. Grundy, Effects of 5-hydroxytryptamine on discharge of vagal
mucosal afferent fibres from the upper gastrointestinal tract of
the ferret, J Auton Nery Syst 45: 41-50 [1993]). On the basis of
this understanding, concepts have evolved to explain the irritable
bowel syndrome as a condition of serotonin excess (leading to
diarrhea from excessive peristalsis) (Gershon [1998]), even as the
constipation typical of this syndrome remains puzzling. Similar
explanations have also been used to explain the diarrhea reported
by patients taking SSRI (e.g. Prozac).
[0066] The intestinal response to 5-HT has previously been
described in terms of the peristaltic reflex in in vitro models.
Bulbring and Crema first showed that lumenal 5-HT resulted in
peristalsis. (Bulbring et al., J. Physiol. 140:381-407 [1959];
Bulbring et al., Brit. J. Pharm. 13:444-457 [1958]). Since the
stimulation of peristalsis by 5-HT was unaffected by extrinsic
denervation (Bulbring et al., QJ Exp. Physiol. 43:26-37 [1958]),
the peristaltic reflex was considered to be intrinsic to the
enteric nervous system. Using a modified Trendelenburg model that
compartmentalized the peristaltic reflex into the sensory limb, the
ascending contraction limb (orad to stimulus) and the descending
relaxation limb (aborad to stimulus), Grider, et al. reported that
(1) mucosal stimulation but not muscle stretch released 5-HT to
activate a primary sensory neuron to release calcitonin
gene-related peptide (CGRP)(Grider et al., Am. J. Physiol.
270:G778-G782 [1996]) via 5-HT4 receptors in humans and rats (also
5-HT1p in rats) and 5-HT3 receptors in guinea pigs; (2) cholinergic
interneurons are then stimulated by CGRP to initiate both ascending
contraction via an excitatory motor neuron that depends on
substances P and K and acetylcholine (Grider et al., Am. J.
Physiol. 257:G709BG714 [1989]) and descending relaxation (Grider,
Am. J. Physiol. 266:G1139-G1145 [1994]; Grider et al. [1996], Jin
et al., J. Pharmacol. Exp. Ther. 288:93-97 [1999]) via an
inhibitory motor neuron that depends on pituitary adenylate
cyclase-activating peptide (PACAP), nitric oxide and vasoactive
inhibitory peptide (VIP)(Grider et al., Neuroscience 54:521-526
[1993]; Grider et al., J. Auton. Nerv. Syst. 50:151-159 [1994]);
and (3) peristalsis is controlled by [a] an opioid pathway that
inhibits descending relaxation by suppressing the release of VIP;
[b] a somatostatin pathway that inhibits this opioid pathway
(Grider, Am. J. Physiol. 275:G973-G978 [1998]); and [c] a GABA
(Grider, Am. J. Physiol. 267:G696-G701 [1994]) and a gastrin
releasing peptide (GRP) (Grider, Gastroenterol. 116:A1000 [1999])
pathway that stimulate VIP release. An opioid pathway that inhibits
the excitatory motor neurons responsible for ascending contraction
has also been described (Gintzler et al., Br. J. Pharmacol.
75:199-205 [1982]; Yau et al., Am. J. Physiol. 250:G60-G63 [1986]).
These observations are consistent with neuroanatomic and
electrophysiological observations.
[0067] In addition, mucosal stroking has been found to induce 5-HT
release by intestinal mucosal cells, which in turn activates a
5-HT4 receptor on enteric sensory neurons, evoking a neuronal
reflex that stimulates chloride secretion (Kellum, J. M. et al.,
Stroking human jejunal mucosa induces 5-HT release and Cl.sup.-
secretion via afferent neurons and 5-HT4 receptors, Am. J. Physiol.
277(3 Pt 1):G515-20 [1999]).
[0068] Agonists of 5-HT4/5, 5-HT3 receptors, as well as opioid
.DELTA. receptor antagonists, were reported to facilitate
peristaltic propulsive activity in the colon in response to
mechanical stroking, which causes the endogenous release of 5-HT
and calcitonin gene-related protein (CGRP) in the stroked mucosal
area. (Steadman, C. J. et al., Selective 5-hydroxytrypamine type 3
receptor antagonism with ondansetron as treatment for
diarrhea-predominant irritable bowel syndrome: a pilot study, Mayo
Clin. Proc. 67(8):732-38 [1992]). Colonic distension also results
in CGRP secretion, which is associated with triggering the
peristaltic reflex. 5-HT3 receptor antagonists have been used for
the treatment of autism. (E.g., Oakley et al., 5-HT3 receptor
antagonists for the treatment of autism, U.S. Pat. No.
5,225,407).
[0069] Improved methods of detecting or diagnosing SIBO and
SIBO-caused conditions are also a desideratum. Typically, detection
of SIBO is done by detecting hydrogen and/or methane exhaled in the
the breath. (E.g., P. Kerlin and L. Wong, Breath hydrogen testing
in bacterial overgrowth of the small intestine, Gastroenterol.
95(4):982-88 [1988]; A. Strocchi et al., Detection of malabsorption
of low doses of carbohydrate: accuracy of various breath H.sub.2
criteria, Gastroenterol. 105(5):1404-1410 [1993]; D. de Boissieu et
al., [1996]; P. J. Lewindon et al., Bowel dysfunction in cystic
fibrosis: importance of breath testing, J. Paedatr. Child Health
34(1):79-82 [1998]). Hydrogen is a metabolic product of the
fermentation of carbohydrates and amino acids by bacteria normally
found in the colon. While the hydrogen that is produced in the
colonic lumen may be excreted via the lungs (exhaled breath) and
the anus (flatus), these routes of excretion are responsible for
the elimination of only a fraction of the total amount of hydrogen
(10%) that is produced in the gut (Levitt, M. D. et al., Hydrogen
(H.sub.2) catabolism in the colon of the rat, J Lab din Med
84:163-167 [1974]).
[0070] The major mechanism for the removal of hydrogen produced by
bacterial fermentation is the utilization of this gas by colonic
bacteria that competes to use hydrogen via one of three hydrogen
disposal pathways that are mutually exclusive. These pathways
depend on the metabolism of methanogenic bacteria (Levitt, M. D. et
al., H.sub.2 excretion after ingestion of complex carbohydrates,
Gastroenterology 92:383-389 [1987]), acetogenic bacteria (Lajoie,
R. et al., Acetate production from hydrogen and [c13] carbon
dioxide by the microflora of human feces, Appl Environ Microbiol
54:2723-2727 [1988]) and sufate-reducing bacteria (Gibson, G. R. et
al., Occurrence of sulphate-reducing bacteria in human faeces and
the relationship of dissimilatory sulphate reduction to
methanogenesis in the large gut, J Appl Bactereriol 65:103-111
[1988]). Methanogenic bacteria are more efficient than the other
colonic bacteria in the elimination of lumenal hydrogen. (Strocchi,
A. et al., Methanogens outcompete sulphate reducing bacteria for
H.sub.2 in the human colon, Gut 35:1098-1101 [1994]). Acetogenic
bacteria are uncommon, being found in the intestinal populations of
<5% of humans.
[0071] In the colon, sulfate-reducing bacteria reduces sulfate to
hydrogen sulfide. (MacFarlane, G. T. et al., Comparison of
fermentation reactions in different regions of the human colon, J
Appl Bacteriol 72:57-64 [1992]). Hydrogen sulfide is more damaging
to tissues than anionic sulfide or sulfhydryl compounds. Intestinal
bicarbonate facilitates the conversion of hydrogen sulfide produced
by sulfate-reducing bacteria in the gut to anionic sulfide.
(Hamilton W A: Biocorrosion: The action of sulphate-reducing
bacteria, in Biochemistry of Microbial Degradation, C. Ratlidge
(ed.) Dordrecht, Kluwer Academic Publishers, pages 555-570 [1994]).
Since sulfate-reducing bacteria are more common in patients with
the diagnosis of ulcerative colitis (Pitcher, M. C. L. et al.,
Incidence and activities of sulphate-reducing bacteria in gut
contents of healthy subjects and patients with ulcerative colitis,
FEMS Microbiol Ecol 86:103-112 [1991]), sulfate-reducing bacteria
have been considered for a possible role in the pathogenesis of
ulcerative colitis. (Florin, R. H. J. et al., A role for sulfate
reducing bacteria in ulcerative colitis?, Gastroenterology 98:A170
[1990]). This link has been postulated to be related to the
injurious effect of hydrogen sulfide in impairing the use of short
chain fatty acids as fuel by colonic epithelial cells. (Roediger,
W. E. W. et al., Sulphide impairment of substrate oxidation in rat
colonocytes: a biochemical basis for ulcerative colitis?, Clin Sci
85:623-627 [1993]; Roediger, W. E. et al., Reducing sulfur
compounds of the colon impair colonocyte nutrition: implication of
ulcerative colitis, Gastroenterology 1993; 104:802-809).
[0072] Currently, clinical detection of sulfur-containing gases is
limited to the detection of halitosis or bad breath. (Rosenberg, M.
et al., Reproducibility and sensitivity of oral malodor
measurements with a protable sulphide monitor, J Dent Res. 1991
November; 70(11):1436-40). After garlic ingestion, the presence of
allyl methyl sulfide differentiates the intestine rather than the
mouth as the source of the sulfur-containing volatile gas (Suarez,
F. et al., Differentiation of mouth versus gut as site of origin of
odoriferous breath gases after garlic ingestion, Am J Physiol 276(2
pt 1):G425-30 [1999]).
[0073] The role of sulfate-reducing bacteria in small intestinal
bacterial overgrowth has not been studied, and the presence of
sulfate-reducing bacteria are not detected using the standard
breath testing method which typically detects only the presence of
hydrogen, methane and carbon dioxide.
[0074] There remains a need for an underlying causal factor, to
which diagnostic testing and treatment can be directed, for SIBO
and SIBO-caused conditions, such as irritable bowel syndrome;
fibromyalgia; chronic pelvic pain syndrome; chronic fatigue
syndrome; autism; depression; impaired mentation and/or memory;
sugar craving; ADHD; MS, SLE and other autoimmune diseases; and
Crohn=s disease. This and other benefits of the present invention
are described herein.
SUMMARY OF THE INVENTION
[0075] The present invention relates to the diagnosis or treatment
of small intestinal bacterial overgrowth (SIBO) and SIBO-caused
conditions. SIBO-caused conditions, as described herein, include
irritable bowel syndrome (IBS), Crohn's disease (CD), fibromyalgia
(FM), chronic pelvic pain syndrome (CPPS), chronic fatigue syndrome
(CFS), depression, impaired mentation, impaired memory, halitosis,
tinnitus, sugar craving, autism, attention deficit/hyperactivity
disorder (ADHD), drug sensitivity, and autoimmune diseases, for
example, multiple sclerosis (MS), systemic lupus erythematosus
(SLE).
[0076] In particular, the present invention relates to a method of
treating small intestinal bacterial overgrowth (SIBO) or a
SIBO-caused condition in a human subject. The method involves
detecting in the subject by any suitable detection means, the
presence or absence of SIBO in the subject. If SIBO is detected in
the subject, the method further involves depriving the bacterial
population, which constitutes the overgrowth in the small
intestine, of nutrient(s), sufficiently to inhibit the further
growth of the bacteria in the small intestine. With the growth of
the bacteria constituting the SIBO condition thus inhibited, SIBO
is at least partially eradicated, as the subject's phase III
interdigestive motility is better able to clear the small intestine
of the overgrowth and sweep the bacteria into the colon for
eventual elimination from the body. In addition, the at least
partial eradication of the SIBO condition also decreases the
occurrence or magnitude of bacteria-related toxicity, sepsis (in
more severe or advanced SIBO), and/or the subject's own immune
responses, which are continually triggered by the presence of SIBO
in non-immunocompromised subjects. The clinical symptoms of the
subject associated with SIBO or the SIBO-caused condition are,
consequently, ameliorated by the at least partial eradication of
SIBO.
[0077] In an alternative aspect of the present invention, the
method involves inhibiting the growth of the bacteria in the
subject's small intestine, which bacteria constitute a SIBO
condition that has been detected, by introducing into the lumen of
the small intestine, a pharmaceutically acceptable disinfectant or
antibiotic composition in an amount sufficient to inhibit the
growth of the bacteria, thereby at least partially eradicating SIBO
in the human subject.
[0078] In still another alternative aspect of the present
invention, the method of treating small intestinal bacterial
overgrowth (SIBO), or a SIBO-caused condition, in a human subject
involves administering to the subject a pharmaceutically acceptable
composition comprising a stabilizer of mast cell membranes in the
lumenal wall, in an amount sufficient to inhibit a mast
cell-mediated immune response to SIBO in the human subject.
[0079] The present invention also relates to a method of screening
for the abnormally likely presence of SIBO in a human subject. The
method involves obtaining a serum sample from the subject, and then
quantitatively determining a concentration in the serum sample of
serotonin, one or more unconjugated bile acid(s), and/or folate. An
abnormally elevated serum concentration of one or more of these
substances is indicative of a higher than normal probability that
SIBO is present in the subject. Thus, if the method of screening
for the presence of SIBO is employed as part of a blood work-up,
either as part of a routine physical or by way of investigating a
particular clinical complaint of the subject's, the practitioner
can be made aware that SIBO is more than normally likely to be
present. The practitioner can then elect to pursue a less
convenient, but more diagnostically powerful, detection means for
SIBO.
[0080] The present invention also relates to such a diagnostically
powerful SIBO detection means. In particular, this inventive method
of detecting small intestinal bacterial overgrowth in a human
subject involves detecting the relative amounts of methane,
hydrogen, and at least one sulfur-containing gas in a gas mixture
exhaled by the human subject, after the subject has ingested a
controlled quantity of a substrate. The exhaled gas mixture is at
least partially produced by the metabolic activity of the
intestinal microflora of the subject.
[0081] The present invention is also directed to a method of
determining the relative severity of SIBO or a SIBO-caused
condition in a human subject in whom SIBO has been detected. The
method involves detecting in the subject by suitable detection
means, the presence or absence of SIBO, and, if the presence of
SIBO is detected in the subject, the method further involves
detecting in the subject by suitable detection means a relative
level of intestinal permeability, abnormally high intestinal
permeability indicating a relatively severe SIBO or SIBO-caused
condition in the subject.
[0082] The present invention also relates to a kit for the
diagnosis of SIBO or a SIBO-caused condition, comprising: at least
one breath sampling container, a pre-measured amount of a
substrate, and instructions for a user in detecting the presence or
absence of SIBO by determining the relative amounts of methane,
hydrogen, and at least one sulfur-containing gas in a gas mixture
exhaled by the subject, after ingestion of a controlled quantity of
the substrate. Thus, the kit is particularly useful in practicing
the inventive method of detecting small intestinal bacterial
overgrowth in a human subject.
[0083] These and other advantages and features of the present
invention will be described more fully in a detailed description of
the preferred embodiments which follows. The present invention is
further described by the disclosures of related applications U.S.
patent application Ser. No. 09/374,142, filed on Aug. 11, 1999;
U.S. patent application Ser. No. 09/546,119, filed on Apr. 10,
2000; U.S. patent application Ser. No. 09/420,046, filed Oct. 18,
1999; U.S. patent application Ser. No. 09/359,583, filed on Jul.
22, 1999; U.S. patent application Ser. No. 08/832,307, filed on
Apr. 3, 1997 and issued as U.S. Pat. No. 5,977,175 on Nov. 2, 1999;
and U.S. patent application Ser. No. 08/442,843, filed on May 17,
1995, which are all incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0084] FIG. 1 shows visual analog scores reported by subjects with
IBS and SIBO before and after antibiotic treatment.
[0085] FIG. 2 shows visual analog scores from subjects with IBS and
SIBO in a pilot study, before and after antibiotic treatment.
[0086] FIG. 3 shows visual analog scores reported by subjects with
fibromyalgia and SIBO before and after antibiotic treatment.
[0087] FIG. 4 shows the correlation between the degree of
improvement in symptoms and residual breath hydrogen production
after antibiotic treatment in subjects with fibromyalgia and
SIBO.
[0088] FIG. 5 shows visual analog scores reported by subjects with
Crohn=s disease and SIBO before and after antibiotic treatment.
[0089] FIG. 6 shows the correlation between degree of improvement
in symptoms and residual breath hydrogen production after
antibiotic treatment in subjects with Crohn=s disease.
[0090] FIG. 7 shows that the severity of diarrheal symptoms is
comparatively less in SIBO patients who excrete methane.
[0091] FIGS. 8A and 8B show a typical effect of total enteral
nutrition (TEN) regimen in the eradication of SIBO as detected by
LBHT. In FIG. 8A (pre-treatment), SIBO was initially detected.
After 14 days of the TEN regimen, follow-up LBHT shows that SIBO
had been at least partially eradicated (FIG. 8B).
[0092] FIG. 9 demonstrates that slowing of the rate of intestinal
transit by fat depends on peptide YY (PYY), which is a
physiological fat signal molecule.
[0093] FIG. 10 demonstrates that demonstrates that slowing of the
rate of intestinal transit by fat depends on a serotonergic
pathway.
[0094] FIG. 11 illustrates that the fat induced ileal brake depends
on an ondansetron-sensitive, efferent serotonergic 5-HT3-mediated
pathway.
[0095] FIG. 12 shows that ondansetron abolishes the fat-induced
ileal brake in a dose-dependent fashion.
[0096] FIG. 13 shows that ondansetron abolishes the fat-induced
ileal brake when administered luminally but not intravenously.
[0097] FIG. 14 illustrates that the slowing of intestinal transit
by distal gut 5-HT depends on an ondansetron-sensitive
5-HT-mediated pathway in the proximal (efferent) and distal
(afferent) gut.
[0098] FIG. 15 shows that lumenal 5-HT, delivered to the proximal
gut, slows intestinal transit in a dose-dependent fashion.
[0099] FIG. 16 illustrates that lumenal 5-HT slows intestinal
transit via activation of an intestino-intestinal reflex.
[0100] FIG. 17 illustrates that slowing of intestinal transit by
distal gut fat depends on an extrinsic adrenergic neural
pathway.
[0101] FIG. 18 illustrates that slowing of intestinal transit by
PYY depends on an extrinsic adrenergic neural pathway.
[0102] FIG. 19 illustrates that slowing of intestinal transit by
5-HT in the distal gut depends on a propranolol-sensitive extrinsic
adrenergic neural pathway.
[0103] FIG. 20 illustrates that intestinal transit is slowed by
norepinephrine (NE) in a 5-HT-mediated neural pathway.
[0104] FIG. 21 illustrates that the fat-induced jejunal brake
depends on the slowing effect of a naloxone-sensitive, opioid
neural pathway.
[0105] FIG. 22 illustrates that the fat-induced ileal brake depends
on the slowing effect of an efferent, naloxone-sensitive, opioid
neural pathway.
[0106] FIG. 23 shows that slowing of intestinal transit by distal
gut 5-HT depends on a naloxone-sensitive, opioid neural
pathway.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0107] The present invention is directed to a method of treating
small intestinal bacterial overgrowth (SIBO) or a SIBO-caused
condition in a human subject, including a juvenile or adult, of any
age or sex.
[0108] The upper gastrointestinal tract of a human subject includes
the entire alimentary canal, except the cecum, colon, rectum, and
anus. While some digestive processes, such as starch hydrolysis,
begin in the mouth and esophagus, of particular importance as sites
of digestion are the stomach and small intestine (or "small
bowel"). The small intestine includes the duodenum, jejunum, and
the ileum. As the term is commonly used in the art, the proximal
segment of the small bowel, or proximal gut, comprises
approximately the first half of the small intestine from the
pylorus to the mid-gut. The distal segment, or distal gut includes
approximately the second half, from the mid-gut to the ileal-cecal
valve.
[0109] As used herein, "digestion" encompasses the process of
breaking down large biological molecules into their smaller
component molecules, for example, proteins into amino acids.
"Predigested" means that the process of digestion has already begun
or has occurred prior to arrival in the upper gastrointestinal
tract.
[0110] As used herein, "absorption" encompasses the transport of a
substance from the intestinal lumen through the barrier of the
mucosal epithelial cells into the blood and/or lymphatic
systems.
[0111] Small intestinal bacterial overgrowth (SIBO), is an abnormal
condition in which aerobic and anaerobic enteric bacteria from the
colon proliferate in the small intestine, which is normally
relatively free of bacterial contamination. SIBO is defined as
greater than 10.sup.6 CFU/mL small intestinal effluent. (R. M.
Donaldson, Jr., Normal bacterial populations of the intestine and
their relation to intestinal function, N. Engl. J. Med. 270:938-45
[1964]). Typically, the symptoms of SIBO include abdominal pain,
bloating, gas and alteration in bowel habits, such as constipation
and diarrhea. SIBO-caused conditions is used herein interchangeably
with the term "SIBO-related conditions," and regardless of ultimate
causation, is a condition associated with the presence of SIBO in
the subject. SIBO-caused conditions include other common symptoms,
such as halitosis ("bad breath"), tinnitus (the experience of noise
in the ears, such as ringing, buzzing, roaring, or clicking, which
may not be associated with externally produced sounds), sugar
craving, i.e., an intense desire for sweet foods or flavors, which
can result in abnormally large consumption of sweet foods and
beverages and frequently leads to health-threatening obesity. Drug
sensitivity is another common SIBO-caused condition, in which the
subject is hypersensitive to medications, such as non-steroidal
anti-inflammatory medications, anti-insomniacs, antibiotics, or
analgesics, and can suffer an unpredictable allergic-type reaction
to medications at doses that normally do not adversely affect the
vast majority of patients. It is a benefit provided by the present
invention that it provides a useful solution in the present tense,
for many patients, to the problem of drug sensitivity, without
requiring complex pharmacogenetic research and customized drug
development.
[0112] Other SIBO-caused conditions, as described herein, can
include those falling in the diagnostic categories of irritable
bowel syndrome, Crohn's disease, fibromyalgia, chronic pelvic pain
syndrome, chronic fatigue syndrome, depression, impaired mentation
(including impairment of the ability to concentrate, calculate,
compose, reason, and/or use foresight or deliberate judgment),
impaired memory, autism, attention deficit/hyperactivity disorder,
and/or autoimmune diseases, such as systemic lupus erythematosus
(SLE) or multiple sclerosis (MS).
[0113] In accordance with the invention, the SIBO-caused condition
can be, but need not be, previously diagnosed or suspected. The
skilled medical practitioner is aware of suitable up-to-date
diagnostic criteria by which a suspected diagnosis is reached.
These diagnostic criteria are based on a presentation of symptom(s)
by a human subject. For example, these criteria include, but are
not limited to, the Rome criteria for IBS (W. G. Thompson,
Irritable bowel syndrome: pathogenesis and management, Lancet
341:1569-72 [1993]) and the criteria for CFS established by the
Centers for Disease Control and Prevention (CDC). (K. Fukuda et
al., The chronic fatigue syndrome: a comprehensive approach to its
definition and study, Ann. Intern. Med. 121:953-59 [1994]). The
diagnostic criteria for fibromyalgia of the American College of
Rheumatology will also be familiar (F. Wolfe et al., The American
College of Rheumatology 1990 Criteria for the Classification of
Fibromyalgia: Report of the Multicenter Criteria Committee,
Arthritis Rheum. 33:160-72 [1990]), as will be the criteria for
depression or ADHD provided for example, by the Diagnostic and
Statistical Manual (DSM)-IV or its current version. (E.g., G. Tripp
et al., DSM-IV and ICD-10: a comparison of the correlates of ADHD
and hyperkinetic disorder, J. Am. Acad. Child Adolesc. Psychiatry
38(2):156-64 [1999]). Symptoms of systemic lupus erythematosus
include the 11 revised criteria of the American College of
Rheumatology, such as a typical malar or discoid rash,
photosensitivity, oral ulcers, arthritis, serositis, or disorders
of blood, kidney or nervous system. (E. M Tan et al., The 1982
revised criteria for the classification of systemic lupus
erythematosus [SLE], Arthritis Rheum. 25:1271-77 [1982]).
Appropriate diagnostic criteria for multiple sclerosis are also
familiar (e.g., L. A. Rolak, The diagnosis of multiple sclerosis,
Neuronal Clin. 14(1):27-43 [1996]), as are symptoms of Crohn=s
disease useful in reaching a suspected diagnosis. (e.g., J. M.
Bozdech and R. G. Farmer, Diagnosis of Crohn=s disease,
Hepatogastroenterol. 37(1):8-17 [1990]; M. Tanaka and R. H.
Riddell, The pathological diagnosis and differential diagnosis of
Crohn=s disease, Hepatogastroenterol. 37(1):18-31 [1990]; A. B.
Price and B. C. Morson, Inflammatory bowel disease: the surgical
pathology of Crohn's disease and ulcerative colitis, Hum. Pathol.
6(1):7-29 [1975]). The practitioner is, of course not limited to
these illustrative examples for diagnostic criteria, but should use
criteria that are current in the art.
[0114] Detection of the presence of SIBO in the human subject also
corroborates the suspected diagnosis of the SIBO-caused condition,
held by a qualified medical practitioner who, prior to the
detection of SIBO in the human subject, suspects from more limited
clinical evidence that the human subject has, for example,
irritable bowel syndrome, fibromyalgia, chronic fatigue syndrome,
chronic pelvic pain syndrome, depression, autism, ADHD, an
autoimmune disease, or Crohn=s disease. By applying the inventive
diagnostic method the suspected diagnosis is corroborated, i.e.,
confirmed, sustained, substantiated, supported, evidenced,
strengthened, affirmed or made more firm.
[0115] The inventive method of treating SIBO, or a SIBO-caused
condition, involves first detecting the presence or absence of SIBO
in the subject by suitable detection means. Detecting the presence
or absence of SIBO is accomplished by any suitable means or method
known in the art. For example, one preferred method of detecting
SIBO is breath hydrogen testing. (E.g., P. Kerlin and L. Wong,
Breath hydrogen testing in bacterial overgrowth of the small
intestine, Gastroenterol. 95(4): 982-88 [1988]; A. Strocchi et al.,
Detection of malabsorption of low doses of carbohydrate: accuracy
of various breath H.sub.2 criteria, Gastroenterol. 105(5):1404-1410
[1993]; D. de Boissieu et al., [1996]; P. J. Lewindon et al., Bowel
dysfunction in cystic fibrosis: importance of breath testing, J.
Paedatr. Child Health 34(1):79-82 [1998]). Breath hydrogen or
breath methane tests are based on the fact that many obligately or
facultatively fermentative bacteria found in the gastrointestinal
tract produce detectable quantities of hydrogen or methane gas as
fermentation products from a substrate consumed by the host, under
certain circumstances. Substrates include sugars such as lactulose,
xylose, lactose, sucrose, or glucose. The hydrogen or methane
produced in the small intestine then enters the blood stream of the
host and are gradually exhaled.
[0116] Typically, after an overnight fast, the patient swallows a
controlled quantity of a sugar, such as lactulose, xylose, lactose,
or glucose, and breath samples are taken at frequent time
intervals, typically every 10 to 15 minutes for a two- to four-hour
period. Samples are analyzed by gas chromatography or by other
suitable techniques, singly or in combination. Plots of breath
hydrogen in patients with SIBO typically show a double peak, i.e.,
a smaller early hydrogen peak followed by a larger hydrogen peak,
but a single hydrogen peak is also a useful indicator of SIBO, if
peak breath hydrogen exceeds the normal range of hydrogen for a
particular testing protocol. (See, G. Mastropaolo and W. D. Rees,
Evaluation of the hydrogen breath test in man: definition and
elimination of the early hydrogen peak, Gut 28(6):721-25
[1987]).
[0117] A variable fraction of the population fails to exhale
appreciable hydrogen gas during intestinal fermentation of
lactulose; the intestinal microflora of these individuals instead
produce more methane. (G. Corazza et al., Prevalence and
consistency of low breath H.sub.2 excretion following lactulose
ingestion. Possible implications for the clinical use of the
H.sub.2 breath test, Dig. Dis. Sci. 38(11):2010-16 [1993]; S. M.
Riordan et al., The lactulose breath hydrogen test and small
intestinal bacterial overgrowth, Am. J. Gastroentrol. 91(9);
1795-1803 [1996]). Consequently, in the event of an initial
negative result for breath hydrogen, or as a precaution, methane
and/or carbon dioxide contents in each breath sample are optionally
measured, as well as hydrogen, or a substrate other than lactulose
is optionally used. Also, acting as a check, the presence of SIBO
is demonstrated by a relative decrease in peak hydrogen exhalation
values for an individual subject after antimicrobial treatment, in
accordance with the present invention, compared to pretreatment
values.
[0118] Another preferred method of detecting bacterial overgrowth
is by gas chromatography with mass spectrometry and/or radiation
detection to measure breath emissions of isotope-labeled carbon
dioxide, methane, or hydrogen, after administering an
isotope-labeled substrate that is metabolizable by gastrointestinal
bacteria but poorly digestible by the human host, such as
lactulose, xylose, mannitol, or urea. (E.g., G. R. Swart and J. W.
van den Berg, .sup.13C breath test in gastrointestinal practice,
Scand. J. Gastroenterol. [Suppl.] 225:13-18 [1998]; S. F. Dellert
et al., The 13C-xylose breath test for the diagnosis of small bowel
bacterial overgrowth in children, J. Pediatr. Gastroenterol. Nutr.
25(2):153-58 [1997]; C. E. King and P. P. Toskes, Breath tests in
the diagnosis of small intestinal bacterial overgrowth, Crit. Rev.
Lab. Sci. 21(3):269-81 [1984]). A poorly digestible substrate is
one for which there is a relative or absolute lack of capacity in a
human for absorption thereof or for enzymatic degradation or
catabolism thereof.
[0119] Suitable isotopic labels include .sup.13C or .sup.14C. For
measuring methane or carbon dioxide, suitable isotopic labels can
also include .sup.2H and .sup.3H or .sup.17O and .sup.18O, as long
as the substrate is synthesized with the isotopic label placed in a
metabolically suitable location in the structure of the substrate,
i.e., a location where enzymatic biodegradation by intestinal
microflora results in the isotopic label being sequestered in the
gaseous product. If the isotopic label selected is a radioisotope,
such as .sup.14C, .sup.3H, or .sup.15O, breath samples can be
analyzed by gas chromatography with suitable radiation detection
means. (E.g., C. S. Chang et al., Increased accuracy of the
carbon-14 D-xylose breath test in detecting small-intestinal
bacterial overgrowth by correction with the gastric emptying rate,
Eur. J. Nucl. Med. 22(10):1118-22 [1995]; C. E. King and P. P.
Toskes, Comparison of the 1-gram [.sup.14C]xylose, 10-gram
lactulose-H.sub.2, and 80-gram glucose-H.sub.2 breath tests in
patients with small intestine bacterial overgrowth, Gastroenterol.
91(6):1447-51 [1986]; A. Schneider et al., Value of the
.sup.14C-D-xylose breath test in patients with intestinal bacterial
overgrowth, Digestion 32(2):86-91 [1985]).
[0120] Another preferred method of detecting small intestinal
bacterial overgrowth is direct intestinal sampling from the human
subject. Direct sampling is done by intubation followed by scrape,
biopsy, or aspiration of the contents of the intestinal lumen,
including the lumen of the duodenum, jejunum, or ileum. The
sampling is of any of the contents of the intestinal lumen
including material of a cellular, fluid, fecal, or gaseous nature,
or sampling is of the lumenal wall itself. Analysis of the sample
to detect bacterial overgrowth is by conventional microbiological
techniques including microscopy, culturing, and/or cell numeration
techniques.
[0121] Another preferred method of detecting small intestinal
bacterial overgrowth is by endoscopic visual inspection of the wall
of the duodenum, jejunum, and/or ileum.
[0122] The preceding are merely illustrative and non-exhaustive
examples of methods for detecting small intestinal bacterial
overgrowth.
[0123] Another suitable, and most preferred, means for detecting
the presence or absence of SIBO is the present inventive method of
detecting small intestinal bacterial overgrowth in a human subject,
which involves detecting the relative amounts of methane, hydrogen,
and at least one sulfur-containing gas in a gas mixture exhaled by
said human subject, after the subject has ingested a controlled
quantity of a substrate. The inventive method of detecting small
intestinal bacterial overgrowth is more likely than conventional
breath tests described above to detect the presence of SIBO,
because in some subjects a pattern exists that is termed
"non-hydrogen, non-methane excretion" (see, e.g., Example 9c
hereinbelow). This pattern is the result of the subject having a
bacterial population constituting the SIBO condition, in which a
sulfate-reducing metabolic pathway predominates as the primary
means for the disposition of dihydrogen. In that condition, the
removal of the hydrogen can be so complete that there is little
residual hydrogen or methane gas to be detected in the exhaled
breath, compared to the amount of sulfur-containing gas, such as
hydrogen sulfide or a volatile sulfhydryl compound detectable by
the inventive method of detecting small intestinal bacterial
overgrowth.
[0124] In accordance with the inventive method of detecting small
intestinal bacterial overgrowth, the substrate is preferably a
sugar, as described hereinabove, and more preferably a poorly
digestible sugar or an isotope-labeled sugar. The at least one
sulfur-containing gas is methanethiol, dimethylsulfide, dimethyl
disulfide, an allyl methyl sulfide, an allyl methyl sulfide, an
allyl methyl disulfide, an allyl disulfide, an allyl mercaptan, or
a methylmercaptan. Most preferably, the sulfur-containing gas is
hydrogen sulfide or a sulfhydryl compound.
[0125] The detection or determination of the relative amounts of
methane, hydrogen, and at least one sulfur-containing gas in the
exhaled gas mixture is accomplished by means or systems known in
the art, preferably by means of gas chromatography (e.g., Brunette,
D. M. et al., The effects of dentrifrice systems on oral malodor, J
Clin Dent. 9:76-82 [1998]; Tangerman, A. et al., A new sensitive
assay for measuring volatile sulphur compounds in human breath by
Tenax trapping and gas chromatography and its application in liver
cirrhosis, Clin Chim Acta 1983; May 9; 130(1):103-110 [1983])
and/or a radiation detection system, if appropriate. Most
preferably, mass spectrometry is employed to detect the relative
amounts of methane, hydrogen, and at least one sulfur-containing
gas in the exhaled gas mixture. (E.g., Spanel P, Smith D.,
Quantification of hydrogen sulphide in humid air by selected ion
flow tube mass spectrometry, Rapid Commun Mass Spectrom
14(13):1136-1140 [2000]). Combined gas chromatography and mass
spectrometry (GC/MS) is also useful. (E.g., Chinivasagam, H. N. et
al., Volatile components associated with bacterial spoilage of
tropical prawns, Int J Food Microbiol 1998 Jun. 30; 42(1-2):45-55).
Most preferably, but not necessarily, the detection system employed
requires only a single sample of exhaled gas mixture for the
detection of methane, hydrogen, and at least one sulfur-containing
gas. Detection methods that separately detect methane, hydrogen,
and/or at least one sulfur containing gas are also useful.
[0126] Thus, thin-layer chromatography or high pressure liquid
chromatography can be useful for detection of volatile
sulfur-containing compounds. (E.g., Tsiagbe, V. K. et al.,
Identification of volatile sulfur derivatives released from
feathers of chicks fed diets with various levels of
sulfur-containing amino acids, J Nutr 1987 117(11): 18859-65
[1987]).
[0127] Direct-reading monitors for sulfides based on the use of an
electrochemical voltametric sensor or polarographic cell can also
be employed. Typically, gas is drawn into a sensor equipped with an
electrocatalytic sensing electrode. An electrical current is
generated by an electrochemical reaction proportional to the
concentration of the gas. The quantity of the gas is typically
determined by comparing to a known standard.
[0128] In some embodiments of the inventive method of detecting
SIBO in a human subject, before detection, volatile
sulfur-containing gases are trapped in Tenax absorbent (e.g.,
Tangerman, A. et al., Clin Chim Acta May 9; 130(1):103-110 [1983];
Heida, H. et al., Occupational exposure and indoor air quality
monitoring in a composting facility, Am Ind Hyg Assoc J
56(1):39-43[1995]) or other solvent/absorbent system such as
dinitrophenyl thioethers (Tsiagbe, V. K. et al. [1987]).
[0129] It generally takes about 2 to 3 hours of the subjects's time
and a pre-test fast to accomplish breath testing for SIBO; thus, a
quicker and more convenient screening method to determine those
subjects most likely to have SIBO is desirable. Such a screening
test allows the clinician to make a more informed decision as to
which patients would likely benefit from more definitive SIBO
testing, as described above. This pre-screening reduces unnecessary
inconvenience and expense for subjects who are unlikely to have
SIBO.
[0130] Hence, the present invention provides a method of screening
for the abnormally likely presence of SIBO in a human subject. By
abnormally likely is meant a likelihood of SIBO greater than
expected in the general population. The inventive screening method
involves obtaining a serum sample from the subject, which
conventionally involves a blood draw, followed by separation of the
serum from the whole blood. Conventional immunochemical techniques,
such as ELISA, employing commercially available reagents, are used
to quantitatively determine a concentration in the serum sample of
serotonin (5-HT), one or more unconjugated bile acids (e.g., total
bile acids or individual bile acids, e.g., deoxycholic acid),
and/or folate, an abnormally elevated serum concentration of one or
more of these being indicative of a higher than normal probability
that SIBO is present in the subject. Such quantitative
immunochemical determinations of serum values are also made
commercially (e.g., Quest Diagnostics-Nichols Institute, 33608
Ortega Highway, San Juan Capistrano, Calif. 92690).
[0131] For example, a normal range for serum 5-HT is up to about
0.5 nanograms per milliliter. The normal range for total bile acids
in serum is about 4.0 to about 19.0 micromole per liter, and for
deoxycholic acid the normal range is about 0.7 to about 7.7
micromoles per liter. Normal ranges for other unconjugated bile
acids are also known. The normal range for serum folate is about
2.6 to about 20.0 nanograms per milliliter. In accordance with the
inventive method of screening, subjects with at least one serum
value beyond the normal range are thus more than normally likely to
have SIBO present and are candidates for further diagnostic SIBO
detection procedures.
[0132] The present invention also relates to a method of
determining the relative severity of SIBO or a SIBO-caused
condition in a human subject in whom SIBO has been detected by a
suitable detection means, as described herein above. If the
presence of SIBO is detected in the subject, then suitable
detection means are employed to detect in the subject a relative
level of intestinal permeability, compared to normal. Abnormally
high intestinal permeability indicates a relatively severe SIBO or
SIBO-caused condition in the subject, which alerts the clinician
that a more aggressive SIBO treatment regimen is desirable.
[0133] Techniques for detecting intestinal permeability and normal
intestinal permeability ranges are known. (E.g., Haase, A. M. et
al., Dual sugar permeability testing in diarrheal disase, J.
Pediatr. 136(2):232-37 [2000]; Spiller, R. C. et al., Increased
rectal mucosal endocrine cells, T lymphocytes, and increased gut
permeability following acute Campylobacter enteritis and in post
dysenteric irritable bowel syndrome, Gut 47(6):804-11 [2000];
Smecuol, E. et al., Sugar tests detect celiac disease among
first-degree relatives, Am. J. Gastroenterol. 94(12):3547-52
[1999]; Cox, M. A. et al., Measurement of small intestinal
permeability markers, lactulose and mannitol in serum: results in
celiac disease, Dig. Dis. Sci. 44(2):402-06 [1999]; Cox, M. A. et
al., Analytical method for the quantitation of mannitol and
disaccharides in serum: a potentially useful technique in measuring
small intestinal permeability in vivo, Clin. Chim. Acta
263(2):197-205 [1997]; Fleming, S. C. et al., Measurement of sugar
probes in serum: an alternative to urine measurement in intestinal
permeability testing, Clin. Chem. 42(3):445-48 [1996]).
[0134] Briefly, intestinal permeability is typically accomplished
by measuring the relative serum or urine levels of two sugars,
after ingestion of controlled amounts by the subject. One of the
sugars, for example mannitol, is chosen because it is more
typically more easily absorbed through the intestinal mucosa than
the other sugar, for example, lactulose. Then about two hours after
ingestion, a serum or urine sample is taken, and the ratio of the
two sugars is determined. The closer the ratio of the two sugars in
the sample approaches the ratio originally ingested, the more
permeable is the subject's intestine.
[0135] After the presence of SIBO has been detected in the subject,
in accordance with the inventive method of treating small
intestinal bacterial overgrowth (SIBO) or a SIBO-caused condition
in a human subject, the proliferating bacterial population
constituting the SIBO is deprived of nutrient(s) sufficiently to
inhibit the growth of the bacteria in the small intestine, which
results in at least partially eradicating SIBO in the human
subject.
[0136] Depriving the bacterial population of nutrient(s) is
accomplished by any of a number of means.
[0137] For example, in some embodiments of the method of treating
SIBO or a SIBO-caused condition, the subject consumes for a
sustained period, a diet consisting essentially of nutrients that
upon arrival in the upper gastrointestinal tract of the subject,
are at least partially predigested. The sustained period being
sufficient to at least partially eradicate SIBO in the human
subject is at least about three days, preferably about 7 to about
18 days, and more preferably about 10 to about 14 days.
[0138] In some embodiments of the method, the at least partially
predigested nutrient(s) are contained in a commestible total
enteral nutrition (TEN) formulation, which is also called an
"elemental diet." Such formulations are commercially available, for
example, Vivonex.RTM. T.E.N. (Sandoz Nutrition, Minneapolis, Minn.)
and its variants, or the like. (See, e.g., Example 11 hereinbelow).
A useful total enteral nutrition formulation satisfies all the
subject's nutritional requirements, containing free amino acids,
carbohydrates, lipids, and all essential vitamins and minerals, but
in a form that is readily absorbable in the upper gastrointestinal
tract, thus depriving or "starving" the bacterial population
constituting the SIBO of nutrients of at least some of the nutients
they previously used for proliferating. Thus, bacterial growth in
the small intestine is inhibited.
[0139] In another embodiment of the inventive method, a pancreatic
enzyme supplement is administered to the subject before or
substantially simultaneously with a meal, such that nutrients
contained in the meal are at least partially predigested upon
arrival in the upper gastrointestinal tract of the subject by the
activity of the pancreatic enzyme supplement. Useful pancreatic
enzyme supplements are commercially available, commonly called
"Pancreatin"; such supplements contain amylase, lipase, and/or
protease. Representative methods of administering the pancreatic
enzyme supplement include giving, providing, feeding or
force-feeding, dispensing, inserting, injecting, infusing,
prescribing, furnishing, treating with, taking, swallowing,
ingesting, eating or applying.
[0140] In a preferred embodiment, depending on the formulation, the
pancreatic enzyme supplement is administered up to a period of 24
hours prior to ingestion of the food or nutrient comprising the
meal, but most preferably between about 60 to 0 minutes before
ingestion, which is substantially simultaneosly with the meal. The
period of time prior to ingestion is determined on the precise
formulation of the composition. For example, a controlled release
formulation can be administered longer before the meal. Other quick
release formulations can be taken substantially simultaneously with
the meal.
[0141] In other embodiments of the method of treating small
intestinal bacterial overgrowth or a SIBO-caused condition,
depriving the bacterial population of nutrient(s) involves
enhancing the digestion and/or absorption of the nutrient(s) in the
upper gastrointestinal tract of the human subject by slowing
transit of the nutrient(s) across the upper gastrointestinal tract
of the human subject, thereby at least partially depriving the
bacterial population of the nutrient(s). These embodiments of the
inventive take advantage of a novel understanding of the peripheral
neural connections that exist between the enteric nervous system of
the upper gastrointestinal tract, including an intrinsic
serotonergic neural pathway, and the vertebral ganglia, and thence
to the central nervous system. The present invention provides a
means to enhance region-to region (e.g., intestino-intestinal
reflex) communications by way of replicating 5-HT as a signal (or
releasing 5-HT at a distance as a surrogate signal). Thus, the
present invention provides a way to increase 5-HT in locations in
the central nervous by transmitting a neural signal from the gut,
or to transmit a 5-HT-mediated neural signal originating in one
location in the gut via an intrinsic cholinergic afferent neural
pathway to a second distant location in the gut where a
serotonergic signal of the same or greater intensity is
replicated.
[0142] The present technology, therefore, allows neurally mediated
modulation of the rate of upper gastrointestinal transit in the
human subject. The present invention allows the artificially
directed transmission and/or amplification of nervous signals from
one location in the enteric nervous system to another via a
prevertebral ganglion, bypassing the central nervous system. The
invention takes advantage of an intrinsic serotonergic neural
pathway involving an intrinsic cholinergic afferent neural pathway
that projects from peptide YY-sensitive primary sensory neurons in
the intestinal wall to the prevertebral celiac ganglion. The
prevertebral celiac ganglion is in turn linked by multiple
prevertebral ganglionic pathways to the central nervous system, to
the superior mesenteric ganglion, to the inferior mesenteric
ganglion, and also back to the enteric nervous system via an
adrenergic efferent neural pathway that projects from the
prevertebral celiac ganglion to one or more enterochromaffincells
in the intestinal mucosa and to serotonergic interneurons that are,
in turn, linked in the myenteric plexus or submucous plexus to
opioid interneurons. The opioid interneurons are in turn linked to
excitatory and inhibitory motoneurons. The opioid interneurons are
also linked by an intestino-fugal opioid pathway that projects to
the prevertebral celiac ganglion, with one or more neural
connections therefrom to the central nervous system, including the
spinal cord, brain, hypothalamus, and pituitary, and projecting
back from the central nervous system to the enteric nervous
system.
[0143] In particular, the present invention employs a method of
manipulating the rate of upper gastrointestinal transit of food or
nutrinet substance(s). The method involves administering by an oral
or enteral delivery route a pharmaceutically acceptable composition
comprising an active agent to the upper gastrointestinal tract. To
slow the rate of upper gastrointestinal transit, the active agent
is an active lipid; a serotonin, serotonin agonist, or serotonin
re-uptake inhibitor; peptide YY or a peptide YY functional analog;
calcitonin gene-related peptide (CGRP) or a CGRP functional analog;
an adrenergic agonist; an opioid agonist; or a combination of any
of any of these, which is delivered in an amount and under
conditions such that the cholinergic intestino-fugal pathway, at
least one prevertebral ganglionic pathway, the adrenergic efferent
neural pathway, the serotonergic interneuron and/or the opioid
interneuron are activated thereby. This results in the rate of
upper gastrointestinal transit in the subject being slowed, which
is the basis for prolonging the residence time of orally or
enterally administered food or nutrient substances, thus promoting
or enhancing their dissolution and/or absorption in the upper
gastrointestinal tract.
[0144] The inventive pharmaceutically acceptable compositions limit
the presentation of a food or nutrient substance to the proximal
region of the small intestine for absorption.
[0145] Depending on the desired results, useful active agents
include, active lipids; serotonin, serotonin agonists, or serotonin
re-uptake inhibitors; peptide YY or peptide YY functional analogs;
CGRP or CGRP functional analogs; adrenergic agonists; opioid
agonists; or a combination of any of any of these; antagonists of
serotonin receptors, peptide YY receptors, adrenoceptors, opioid
receptors, CGRP receptors, or a combination of any of these. Also
useful are antagonists of serotonin receptors, peptide YY
receptors, CGRP receptors; adrenoceptors and/or opioid
receptors.
[0146] Serotonin, or 5-hydroxytryptamine (5-HT) is preferably used
at a dose of about 0.03 to about 0.1 mg/kg of body mass. 5-HT3 and
5-HT4 serotonin receptor agonists are known and include HTF-919 and
R-093877 (Foxx-Orenstein, A. E. et al., Am. J. Physiol. 275(5 Pt
1):G979-83 [1998]); prucalopride;
2-[1-(4-Piperonyl)piperazinyl]benzothiazole;
1-(4-Amino-5-chloro-2-methoxyphenyl)-3-[1-butyl-4-piperidinyl]-1-propanon-
e; and
1-(4-Amino-5-chloro-2-methoxyphenyl)-3-[1-2-methylsulphonylamino)et-
hyl-4-piperidinyl]-1-propanone. Serotonin re-uptake inhibitors
include Prozac or Zoloft.
[0147] Useful serotonin receptor antagonists include known
antagonists of 5-HT3, 5-HT1P, 5-HT1A, 5-HT2, and/or 5-HT4
receptors. Examples include ondansetron or granisetron, 5HT3
receptor antagonists (preferred dose range of about 0.04 to 5
mg/kg), deramciclane (Varga, G. et al., Effect of deramciclane, a
new 5-HT receptor antagonist, on cholecystokinin-induced changes in
rat gastrointestinal function, Eur. J. Pharmacol. 367(2-3):315-23
[1999]), or alosetron. 5-HT4 receptor antagonists are preferably
used at a dose of about 0.05 to 500 picomoles/kg. 5-HT4 receptor
antagonists include 1-Piperidinylethyl 1H-indole-3-carboxylate
(SB203186);
1-[4-Amino-5-chloro-2-(3,5-dimethoxyphenyl)methyloxy]-3-[1-[2methylsulpho-
nylamino]ethyl]piperidin-4-yl]propan-1-one (RS 39604);
3-(Piperidin-1-yl)propyl 4-amino-5-chloro-2-methoxybenzoate.
[0148] Peptide YY (PYY) and its functional analogs are preferably
delivered at a dose of about 0.5 to about 500 picomoles/kg. PYY
functional analogs include PYY (22-36), BIM-43004 (Liu, C D. et
al., J. Surg. Res. 59(1):80-84 [1995]), BIM-43073D, BIM-43004C
(Litvak, D. A. et al., Dig. Dig. Sci. 44(3):643-48 [1999]). Other
examples are also known in the art (e.g., Balasubramaniam, U.S.
Pat. No. 5,604,203).
[0149] PYY receptor antagonists preferably include antagonists of
Y4/PP 1, Y5 or Y5/PP2/Y2, and most preferably Y1 or Y2. (E.g.,
Croom et al., U.S. Pat. No. 5,912,227) Other examples include
BIBP3226, CGP71683A (King, P. J. et al., J. Neurochem. 73(2):641-46
[1999]).
[0150] CGRP receptor antagonists include human CGRP(8-37) (e.g.,
Foxx-Orenstein et al., Gastroenterol. 111(5):1281-90 [1996]).
[0151] Useful adrenergic agonists include norepinephrine.
[0152] Adrenergic or adrenoceptor antagonists include
.beta.-adrenoceptor antagonists, including propranolol and
atenolol. They are preferably used at a dose of 0.05-2 mg/kg.
[0153] Opioid agonists include delta-acting opioid agonists
(preferred dose range is 0.05-50 mg/kg, most preferred is 0.05-25
mg/kg); kappa-acting opioid agonists (preferred dose range is
0.005-100 microgram/kg); mu-acting opioid agonists (preferred dose
range is 0.05-25 microgram/kg); and episilon-acting agonists.
Examples of useful opioid agonists include deltorphins (e.g.,
deltorphin II and analogues), enkephalins (e.g., [d-Ala(2),
Gly-ol(5)]-enkephalin [DAMGO]; [D-Pen(2,5)]-enkephalin [DPDPE]),
dinorphins,
trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl-]benzeneaceta-
mide methane sulfonate (U-50, 488H), morphine, codeine, endorphin,
or .beta.-endorphin.
[0154] Opioid receptor antagonists include mu-acting opioid
antagonists (preferably used at a dose range of 0.05-5
microgram/kg); kappa opioid receptor antagonists (preferably used
at a dose of 0.05-30 mg/kg); delta opioid receptor antagonists
(preferably used at a dose of 0.05-200 microgram/kg); and epsilon
opioid receptor antagonists. Examples of useful opioid receptor
antagonists include naloxone, naltrexone, methylnaltrexone,
nalmefene, H2186, H3116, or fedotozine, i.e., (+)-1-1
[3,4,5-trimethoxy)benzyloxymethyl]-1-phenyl-N,N-dimethylpropylamine.
Other useful opioid receptor antagonists are known (e.g., Kreek et
al., U.S. Pat. No. 4,987,136).
[0155] The active agents listed above are not exhaustive but rather
illustrative examples, and one skilled in the art is aware of other
useful examples.
[0156] As used herein, "active lipid" encompasses a digested or
substantially digested molecule having a structure and function
substantially similar to a hydrolyzed end-product of fat digestion.
Examples of hydrolyzed end products are molecules such as
diglyceride, monoglyceride, glycerol, and most preferably free
fatty acids or salts thereof.
[0157] In a preferred embodiment, the active agent is an active
lipid comprising a saturated or unsaturated fatty acid. Fatty acids
contemplated by the invention include fatty acids having between 4
and 24 carbon atoms (C4-C24).
[0158] Examples of fatty acids contemplated for use in the practice
of the present invention include caprolic acid, caprulic acid,
capric acid, lauric acid, myristic acid, oleic acid, palmitic acid,
stearic acid, palmitoleic acid, linoleic acid, linolenic acid,
trans-hexadecanoic acid, elaidic acid, columbinic acid, arachidic
acid, behenic acid eicosenoic acid, erucic acid, bressidic acid,
cetoleic acid, nervonic acid, Mead acid, arachidonic acid,
timnodonic acid, clupanodonic acid, docosahexaenoic acid, and the
like. In a preferred embodiment, the active lipid comprises oleic
acid.
[0159] Also preferred are active lipids in the form of
pharmaceutically acceptable salts of hydrolyzed fats, including
salts of fatty acids. Sodium or potassium salts are preferred, but
salts formed with other pharmaceutically acceptable cations are
also useful. Useful examples include sodium- or potassium salts of
caprolate, caprulate, caprate, laurate, myristate, oleate,
palmitate, stearate, palmitolate, linolate, linolenate,
trans-hexadecanoate, elaidate, columbinate, arachidate, behenate,
eicosenoate, erucate, bressidate, cetoleate, nervonate,
arachidonate, timnodonate, clupanodonate, docosahexaenoate, and the
like. In a preferred embodiment, the active lipid comprises an
oleate salt.
[0160] The active agents suitable for use with this invention are
employed in well dispersed form in a pharmaceutically acceptable
carrier. As used herein, "pharmaceutically acceptable carrier"
encompasses any of the standard pharmaceutical carriers known to
those of skill in the art. For example, one useful carrier is a
commercially available emulsion, Ensure.sup.7, but active lipids,
such as oleate or oleic acid are also dispersible in gravies,
dressings, sauces or other comestible carriers. Dispersion can be
accomplished in various ways. The first is that of a solution.
[0161] Lipids can be held in solution if the solution has the
properties of bile (i.e., solution of mixed micelles with bile salt
added), or the solution has the properties of a detergent (e.g., pH
9.6 carbonate buffer) or a solvent (e.g., solution of Tween). The
second is an emulsion which is a 2-phase system in which one liquid
is dispersed in the form of small globules throughout another
liquid that is immiscible with the first liquid (Swinyard and
Lowenthal, "Pharmaceutical Necessities" REMINGTON'S PHARMACEUTICAL
SCIENCES, 17th ed., AR Gennaro (Ed), Philadelphia College of
Pharmacy and Science, 1985 p. 1296). The third is a suspension with
dispersed solids (e.g., microcrystalline suspension). Additionally,
any emulsifying and suspending agent that is acceptable for human
consumption can be used as a vehicle for dispersion of the
composition. For example, gum acacia, agar, sodium alginate,
bentonite, carbomer, carboxymethylcellulose, carrageenan, powdered
cellulose, cholesterol, gelatin, hydroxyethyl cellulose,
hydroxypropyl cellulose, hydroxypropyl methylcellulose,
methylcellulose, octoxynol 9, oleyl alcohol, polyvinyl alcohol,
povidone, propylene glycol monostearate, sodium lauryl sulfate,
sorbitan esters, stearyl alcohol, tragacanth, xantham gum,
chondrus, glycerin, trolamine, coconut oil, propylene glycol, thyl
alcohol malt, and malt extract.
[0162] Any of these formulations, whether it is a solution,
emulsion or suspension containing the active agent, can be
incorporated into capsules, or a microsphere or particle (coated or
not) contained in a capsule.
[0163] The pharmaceutically acceptable compositions containing the
active agent, in accordance with the invention, is in a form
suitable for oral or enteral use, for example, as tablets, troches,
lozenges, aqueous or oily suspensions, dispersible powders or
granules, emulsions, hard or soft capsules, syrups, elixirs or
enteral formulas. Compositions intended for oral use are prepared
according to any method known to the art for the manufacture of
pharmaceutical compositions. Compositions can also be coated by the
techniques described in the U.S. Pat. Nos. 4,256,108; 4,160,452;
and 4,265,874, to form osmotic therapeutic tablets for controlled
release. Other techniques for controlled release compositions, such
as those described in the U.S. Pat. Nos. 4,193,985; and 4,690,822;
4,572,833 can be used in the formulation of the inventive
pharmaceutically acceptable compositions.
[0164] An effective amount of active lipid is any amount that is
effective to slow gastrointestinal transit and control presentation
of a food or nutrient substance to a desired region of the small
intestine. For example, an effective amount of active lipid, as
contemplated by the instant invention, is any amount of active
lipid that can trigger any or all of the following reflexes:
intestino-lower esophageal sphincter (relaxation of LES);
intestino-gastric feedback (inhibition of gastric emptying);
intestino-intestinal feedback (ileo jejunal feedback/ileal brake, j
ejuno-jejunal feedback/jejunal brake, intestino-CNS feedback (for
example, intensifying intestinal signalling of satiety');
intestino-pancreatic feedback (control of exocrine enzyme output);
intestino-biliary feedback (control of bile flow);
intestino-mesenteric blood flow feedback (for the control of
mucosal hyperemia); intestino-colonic feedback (so called
gastro-colonic reflex whereby the colon contracts in response to
nutrients in the proximal small intestine).
[0165] Methods of administering are well known to those of skill in
the art and include most preferably oral administration and/or
enteral administration. Representative methods of administering
include giving, providing, feeding or force-feeding, dispensing,
inserting, injecting, infusing, perfusing, prescribing, furnishing,
treating with, taking, swallowing, eating or applying. Preferably
the pharmaceutically acceptable composition comprising the active
agent is administered in the setting of a meal, i.e., along with or
substantially simultaneously with the meal, most preferably an hour
or less before the meal. It is also useful to administer the active
agent in the fasted state, particularly if the pharmaceutical
composition containing the active agent is formulated for long
acting or extended release. In some embodiments, such as the
inventive method for manipulating post-prandial blood flow, the
pharmaceutical composition is also usefully administered up to an
hour after a meal, and most preferably within one hour before or
after the meal.
[0166] In order to stretch biologic activity so that one has a
convenient, daily dosage regimen, the present invention
contemplates that the inventive compositions can be administered
prior to ingestion of the food, nutrient and/or drug.
[0167] In a preferred embodiment, the inventive compositions
(depending on the formulation) are administered up to a period of
24 hours prior to ingestion of the food, nutrient and/or drug, but
most preferably between about 60 to 5 minutes before ingestion. The
period of time prior to ingestion is determined on the precise
formulation of the composition. For example, if the formulation
incorporates a controlled release system, the duration of release
and activation of the active lipid will determine the time for
administration of the composition. Sustained release formulation of
the composition is useful to ensure that the feedback effect is
sustained.
[0168] In a preferred embodiment, the pharmaceutically acceptable
composition of the invention contains an active lipid and is
administered in a load-dependent manner which ensures that the
dispersion of active lipid is presented to the entire length of the
small intestine. Administration is in one or more doses such that
the desired effect is produced. In some preferred embodiments, the
load of active lipid per dose is from about 0.5 grams to about 2.0
grams, but can range up to about 25 grams per dose as needed.
Generally, patients respond well to the most preferred amount of
active lipid, which is in the range of about 1.6 to 3.2 grams. For
patients who fail to respond to this dose range, a dose between 6
and 8 grams is typically effective.
[0169] Sequential dosing is especially useful for patients with
short bowel syndrome or others with abnormally rapid intestinal
transit times. In these patients, the first preprandial
administration of the active lipid occurs in a condition of
uncontrolled intestinal transit that can fail to permit optimal
effectiveness of the active lipid. A second (or more) preprandial
administration(s) timed about fifteen minutes after the first or
previous administration and about fifteen minutes before the meal
enhances the patient=s control of intestinal lumenal contents and
the effectiveness of the active lipid in accordance with the
inventive methods. Normalization of nutrient absorption and bowel
control throughout the day, including during the patient's extended
sleeping hours, is best achieved by a dietary regimen of three
major meals with about five snacks interspersed between them,
including importantly, a pre-bedtime snack; administration of a
dose of the inventive composition should occur before each meal or
snack as described above.
[0170] Treatment with the inventive compositions in accordance with
the inventive methods can be of singular occurrence or can be
continued indefinitely as needed. For example, patients deprived of
food for an extended period (e.g., due to a surgical intervention
or prolonged starvation), upon the reintroduction of ingestible
food, can benefit from administration of the inventive compositions
before meals on a temporary basis to facilitate a nutrient adaptive
response to normal feeding. On the other hand some patients, for
example those with surgically altered intestinal tracts (e.g.,
ileal resection), can benefit from continued pre-prandial treatment
in accordance with the inventive methods for an indefinite period.
However, clinical experience with such patients for over six years
has demonstrated that after prolonged treatment there is at least a
potential for an adaptive sensory feedback response that can allow
them to discontinue treatment for a number of days without a
recurrence of postprandial diarrhea or intestinal dumping.
[0171] The use of pharmaceutically acceptable compositions of the
present invention in enteral feeding contemplates adding the
composition directly to the feeding formula. The composition can
either be compounded as needed into the enteral formula when the
rate of formula delivery is known (i.e., add just enough
composition to deliver the load of active lipids). Alternatively,
the composition of the invention can be compounded at the factory
so that the enteral formulas are produced having different
concentrations of the composition and can be used according to the
rate of formula delivery (i.e., higher concentration of composition
for lower rate of delivery).
[0172] If the inventive composition were to be added to an enteral
formula and the formula is continuously delivered into the small
intestine, the composition that is initially presented with the
nutrient formula allows slowing the transit of nutrients that are
delivered later. Except for the start of feeding when transit can
be too rapid because the inhibitory feedback from the composition
has yet to be fully activated, once equilibrium is established, it
is no longer logistically an issue of delivering the composition as
a premeal although the physiologic principle is still the same.
[0173] Before dietary fats can be absorbed, the motor activities of
the small intestine in the postprandial period must first move the
output from the stomach to the appropriate absorptive sites of the
small intestine. To achieve the goal of optimizing the movement of
a substance through the small intestine, the temporal and spatial
patterns of intestinal motility are specifically controlled by the
nutrients of the lumenal content.
[0174] Without wishing to be bound by any theory, it is presently
believed that early in gastric emptying, before inhibitory feedback
is activated, the load of fat entering the small intestine can be
variable and dependent on the load of fat in the meal. Thus, while
exposure to fat can be limited to the proximal small bowel after a
small load, a larger load, by overwhelming more proximal absorptive
sites, can spill further along the small bowel to expose the distal
small bowel to fat. Thus, the response of the duodenum to fat
limits the spread of fat so that more absorption can be completed
in the proximal small intestine and less in the distal small
intestine. Furthermore, since the speed of movement of lumenal fat
must decrease when more fat enters the duodenum, in order to avoid
steatorrhea, intestinal transit is inhibited in a load-dependent
fashion by fat. This precise regulation of intestinal transit
occurs whether the region of exposure to fat is confined to the
proximal gut or extended to the distal gut.
[0175] In accordance with the present invention it has been
observed that inhibition of intestinal transit by fat depends on
the load of fat entering the small intestine. More specifically,
that intestinal transit is inhibited by fat in a load-dependent
fashion whether the nutrient is confined to the proximal segment of
the small bowel or allowed access to the whole gut.
[0176] As described above, the inventive technology can also
operate by transmitting to and replicating at a second location in
the upper gastrointestinal tract a serotonergic neural signal
originating at a first location in the proximal or distal gut of a
mammal. For example, the first location can be in the proximal gut
and the second location can be elsewhere in the proximal gut or in
the distal gut. Or conversely, the first location can be in the
distal gut and the second location can be elsewhere in the distal
gut or in the proximal gut.
[0177] Employing this inventive technology to slow the rate of
upper gastrointestinal transit, during and after a meal, nutrient
absorption in the upper gastrointestinal tract is enhanced,
depriving bacterial populations in the lower small intestine of
nutrients. In response to luminal fat in the proximal or distal
gut, a serotonin (5-HT)-mediated anti-peristaltic slowing response
is normally present. Therefore, some embodiments of the method
involve increasing 5-HT in the gut wall by administering to the
mammal and delivering to the proximal and/or distal gut, an active
lipid, or serotonin, a serotonin agonist, or a serotonin re-uptake
inhibitor.
[0178] Alternatively, the active agent is PYY, or a PYY functional
analog. PYY or the PYY analog activates the PYY-sensitive primary
sensory neurons in response to fat or 5-HT. Since the predominant
neurotransmitter of the PYY-sensitive primary sensory neurons is
calcitonin gene-related peptide (CGRP), in another embodiment, CGRP
or a CGRP functional analog is the active agent.
[0179] In other embodiments the point of action is an adrenergic
efferent neural pathway, which conducts neural signals from one or
more of the celiac, superior mesenteric, and inferior mesenteric
prevertebral ganglia, back to the enteric nervous system. The
active agent is an adrenergic receptor (i.e., adrenoceptor) agonist
to activate neural signal transmission to the efferent limb of the
anti-peristaltic reflex response to luminal fat.
[0180] Since adrenergic efferent neural pathway(s) from the
prevertebral ganglia to the enteric nervous system stimulate
serotonergic interneurons, which in turn stimulate enteric opioid
interneurons, in other embodiments of the method, the active agent
is 5-HT, 5-HT receptor agonist, or a 5-HT re-uptake inhibitor to
activate or enhance neural signal transmission at the level of the
serotoneregic interneurons.
[0181] Alternatively, the active agent is an opioid receptor
agonist to activate or enhance neural signal transmission via the
opioid interneurons.
[0182] In accordance with the invention, pharmaceutically
acceptable compositions containing the active agent can be in a
form suitable for oral use, for example, as tablets, troches,
lozenges, aqueous or oily suspensions, dispersible powders or
granules, emulsions, hard or soft capsules, syrups, elixirs or
enteral formulas. Compositions intended for oral use can be
prepared according to any method known to the art for the
manufacture of pharmaceutical compositions and such compositions
can contain one or more other agents selected from the group
consisting of a sweetening agent such as sucrose, lactose, or
saccharin, flavoring agents such as peppermint, oil of wintergreen
or cherry, coloring agents and preserving agents in order to
provide pharmaceutically elegant and palatable preparations.
Tablets containing the active ingredient in admixture with
non-toxic pharmaceutically acceptable excipients can also be
manufactured by known methods. The excipients used can be, for
example, (1) inert diluents such as calcium carbonate, lactose,
calcium phosphate or sodium phosphate; (2) granulating and
disintegrating agents such as corn starch, potato starch or alginic
acid; (3) binding agents such as gum tragacanth, corn starch,
gelatin or acacia, and (4) lubricating agents such as magnesium
stearate, stearic acid or talc. The tablets can be uncoated or they
can be coated by known techniques to delay disintegration and
absorption in the gastrointestinal tract and thereby provide a
sustained action over a longer period. For example, a time delay
material such as glyceryl monostearate or glyceryl distearate can
be employed. They can also be coated by the techniques described in
the U.S. Pat. Nos. 4,256,108; 4,160,452; and 4,265,874, to form
osmotic therapeutic tablets for controlled release. Other
techniques for controlled release compositions, such as those
described in the U.S. Pat. Nos. 4,193,985; and 4,690,822; 4,572,833
can be used in the formulation of the inventive pharmaceutically
acceptable compositions.
[0183] In some cases, formulations for oral use can be in the form
of hard gelatin capsules wherein the active ingredient is mixed
with an inert solid diluent, for example, calcium carbonate,
calcium phosphate or kaolin. They can also be in the form of soft
gelatin capsules wherein the active ingredient is mixed with water
or an oil medium, for example, peanut oil, liquid paraffin, or
olive oil.
[0184] In one embodiment of the present invention, the
pharmaceutically acceptable composition is an enterically coated or
a sustained release form that permits intestinal transit to be
slowed for a prolonged period of time.
[0185] In an alternative aspect of the method of treating small
intestinal bacterial overgrowth (SIBO) or a SIBO-caused condition
in a human subject, after the presence of SIBO is detected in the
human subject by suitable detection means, as described above, a
pharmaceutically acceptable disinfectant composition is introduced
into the lumen of the small intestine so as to conatct the bacteria
constituting the SIBO condition. The disinfectant composition is
introduced in an amount sufficient to inhibit the growth of the
bacteria in the small intestine, thereby at least partially
eradicating SIBO in the human subject.
[0186] Preferably, the pharmaceutically acceptable disinfectant
composition consists essentially of hydrogen peroxide; a
bismuth-containing compound or salt; or an iodine-containing
compound or salt. The pharmaceutically acceptable disinfectant
(i.e., bacteriocidal) composition can also contain other
non-bacteriocidal ingredients, such as any suitable
pharmaceutically acceptable carrier, excipient, emulsant, solvent,
colorant, flavorant, and/or buffer, as described hereinabove.
Formulations for oral or enteral delivery are useful, as described
hereinabove with respect to known delivery modalities for active
agents, e.g., tablets, troches, lozenges, aqueous or oily
suspensions, dispersible powders or granules, emulsions, hard or
soft capsules, syrups, elixirs or enteral formulas.
[0187] Embodiments of disinfectant or bacteriocidal compositions
containing hydrogen peroxide are known for internal use in
vertebrates (e.g., Ultradyne, Ultra Bio-Logics Inc., Montreal,
Canada). Preferably, an aquesous solution of about 1% to about 3%
(v/v) hydrogen peroxide is introduced orally or otherwise enterally
to the lumen, most conveniently by ingestion.
[0188] Embodiments of disinfectant or bacteriocidal compositions
containing bismuth compounds or salts are also known, for example,
bismuth-2-3-dimercaptopropanol (BisBAL), bismuth thiols (e.g.,
bismuth-ethanedithiol), or bismuth-3,4-dimercaptotoluene (BisTOL),
and in over the counter preparations, such as PeptoBizmol. (See,
e.g., Domenico, P. et al., Activity of Bismuth Thiols against
Staphylococci and Staphylococcal biofilms, Antimicrob. Agents
Chemother. 45(5):1417-21 [2001]).
[0189] Embodiments of disinfectant or bacteriocidal compositions
containing iodine compounds or salts are also known, for example,
povidone-iodine solutions.
[0190] In still another alternative aspect of the method of
treating small intestinal bacterial overgrowth (SIBO) or a
SIBO-caused condition in a human subject, after the presence of
SIBO is detected in the human subject by suitable detection means,
as described above, a pharmaceutically acceptable composition is
administered to the subject. The pharmaceutically acceptable
composition contains a stabilizer of mast cell membranes in the
lumenal wall of the small intestine, in an amount sufficient to
inhibit a mast cell-mediated immune response in the human subject.
This embodiment is a relatively aggressive treatment and is most
useful in more severe or advanced SIBO, for example, as confirmed
by high intestinal permeability in the subject (see hereinabove).
Suitable mast cell stabilizers include oxatamide or chromoglycate
(potassium or sodium salts preferred). (e.g., Pacor, M. L. et al.,
Controlled study of oxatomide vs disodium chromoglycate for
treating adverse reactions to food, Drugs Exp Clin Res 18(3):119-23
[1992]; Stefanini, G. F. et al., Oral cromolyn sodium in comparison
with elimination diet in the irritable bowel syndrome, diarrheic
type, Multicenter Study of 428 patients, Scand. J. Gastroenterol.
30(6):535-41 [1995]; Andre, F. et al., Digestive permeability to
different-sized molecules and to sodium cromoglycate in food
allergy, Allergy Proc. 12(5):293-98 [1991]; Lunardi, C. et al.,
Double-blind cross-over trial of oral sodium cromoglycate in
patients with irritable bowel syndrome due to food intolerance,
Clin Exp Allergy 21(5):569-72 [1991]; Burks, A. W. et al.,
Double-blind placebo-controlled trial of oral cromolyn in children
with atopic dermatitis and documented food hypersensitivity, J.
Allergy Clin. Immunol. 81(2):417-23 [1988]).
[0191] After the SIBO condition is at least partially eradicated,
typically within a couple of weeks, there is an improvement in the
symptom(s) of irritable bowel syndrome, fibromyalgia, chronic
fatigue syndrome, chronic pelvic pain syndrome, autism, impaired
mentation, impaired memory, depression, ADHD, an autoimmune
disease, or Crohn=s disease. It is a benefit of the inventive
treatment method that after treatment, subjects routinely report
feeling better than they have felt in years.
[0192] The inventive method of treating small intestinal bacterial
overgrowth (SIBO) or a SIBO-caused condition in a human subject, as
decribed above, can be optionally combined, simultaneously or in
sequence, with other suitable methods of at least partially
eradicating small intestinal bacterial overgrowth, such as the
following.
[0193] For example, at least partially eradicating the bacterial
overgrowth is accomplished by administering an antimicrobial agent,
including but not limited to a natural, synthetic, or
semi-synthetic antibiotic agent. For example, a course of
antibiotics such as, but not limited to, neomycin, metronidazole,
teicoplanin, doxycycline, tetracycline, ciprofloxacin, augmentin,
cephalexin (e.g., Keflex), penicillin, ampicillin, kanamycin,
rifamycin, rifaximin, or vancomycin, which may be administered
orally, intravenously, or rectally. (R. K. Cleary [1998]; C. P.
Kelly and J. T. LaMont, Clostridium difficile infection, Annu. Rev.
Med. 49:375-90 [1998]; C. M. Reinke and C. R. Messick, Update on
Clostridium difficile-induced colitis, Part 2, Am. J. Hosp. Pharm.
51(15):1892-1901 [1994]).
[0194] Alternatively, an antimicrobial chemotherapeutic agent, such
as a 4- or 5-aminosalicylate compound is used to at least partially
eradicate the SIBO condition. These can be formulated for
ingestive, colonic, or topical non-systemic delivery systems or for
any systemic delivery systems. Commercially available preparations
include 4-(p)-aminosalicylic acid (i.e., 4-ASA or
para-aminosalicylic acid) or 4-(p)-aminosalicylate sodium salt
(e.g., Nemasol-Sodium.RTM. or Tubasal.RTM.). 5-Aminosalicylates
have antimicrobial, as well as anti-inflammatory properties (H. Lin
and M. Pimentel, Abstract G3452 at Digestive Disease Week,
100.sup.th Annual Meeting of the AGA, Orlando, Fla. [1999]), in
useful preparations including 5-aminosalicylic acid (i.e., 5-ASA,
mesalamine, or mesalazine) and conjugated derivatives thereof,
available in various pharmaceutical preparations such as
Asacol.RTM., Rowasa.RTM., Claversal.RTM., Pentasa.RTM.,
Salofalk.RTM., Dipentum.RTM. (olsalazine), Azulfidine.RTM. (SAZ;
sulphasalazine), ipsalazine, salicylazobenzoic acid, balsalazide,
or conjugated bile acids, such as ursodeoxycholic
acid-5-aminosalicylic acid, and others.
[0195] Another preferred method of at least partially eradicating
small intestinal bacterial overgrowth, particularly useful when a
subject does not respond well to oral or intravenous antibiotics or
other antimicrobial agents alone, is administering an intestinal
lavage or enema, for example, small bowel irrigation with a
balanced hypertonic electrolyte solution, such as Go-lytely or
fleet phosphosoda preparations. The lavage or enema solution is
optionally combined with one or more antibiotic(s) or other
antimicrobial agent(s). (E.g., J. A. Vanderhoof et al., Treatment
strategies for small bowel bacterial overgrowth in short bowel
syndrome, J. Pediatr. Gastroenterol. Nutr. 27(2):155-60 [1998])
[0196] Another preferred method of at least partially eradicating
small intestinal bacterial overgrowth employs a probiotic agent,
for example, an inoculum of a lactic acid bacterium or
bifidobacterium. (A. S. Naidu et al., Probiotic spectra of lactic
acid bacteria, Crit. Rev. Food Sci. Nutr. 39(1):13-126 [1999]; J.
A. Vanderhoof et al. [1998]; G. W. Tannock, Probiotic propertyies
of lactic acid bacteria: plenty of scope for R & D, Trends
Biotechnol. 15(7):270-74 [1997]; S. Salminen et al., Clinical uses
of probiotics for stabilizing the gut mucosal barrier: successful
strains and future challenges, Antonie Van Leeuwenhoek
70(2-4):347-58 [1997]). The inoculum is delivered in a
pharmaceutically acceptable ingestible formulation, such as in a
capsule, or for some subjects, consuming a food supplemented with
the inoculum is effective, for example a milk, yoghurt, cheese,
meat or other fermentable food preparation. Useful probiotic agents
include Bifidobacterium sp. or Lactobacillus species or strains,
e.g., L. acidophilus, L. rhamnosus, L. plantarum, L. reuteri, L.
paracasei subsp. paracasei, or L. casei Shirota, (P. Kontula et
al., The effect of lactose derivatives on intestinal lactic acid
bacteria, J. Dairy Sci. 82(2):249-56 [1999]; M. Alander et al., The
effect of probiotic strains on the microbiota of the Simulator of
the Human Intestinal Microbial Ecosystem (SHIME), Int. J. Food
Microbiol. 46(1):71-79 [1999]; S. Spanhaak et al., The effect of
consumption of milk fermented by Lactobacillus casei strain Shirota
on the intestinal microflora and immune parameters in humans, Eur.
J. Clin. Nutr. 52(12):899-907 [1998]; W. P. Charteris et al.,
Antibiotic susceptibility of potentially probiotic Lactobacillus
species, J. Food Prot. 61(12):1636-43 [1998]; B. W. Wolf et al.,
Safety and tolerance of Lactobacillus reuteri supplementation to a
population infected with the human immunodeficiency virus, Food
Chem. Toxicol. 36(12):1085-94 [1998]; G. Gardiner et al.,
Development of a probiotic cheddar cheese containing human-derived
Lactobacillus paracasei strains, Appl. Environ. Microbiol.
64(6):2192-99 [1998]; T. Sameshima et al., Effect of intestinal
Lactobacillus starter cultures on the behaviour of Staphylococcus
aureus in fermented sausage, Int. J. Food Microbiol. 41(1):1-7
[1998]).
[0197] Optionally, after at least partial eradication of small
intestinal bacterial overgrowth, use of antimicrobial agents or
probiotic agents can be continued to prevent further development or
relapse of SIBO.
[0198] Another preferred method of at least partially eradicating
small intestinal bacterial overgrowth is by normalizing or
increasing phase III interdigestive intestinal motility between
meals with any of several modalities to at least partially
eradicate the bacterial overgrowth, for example, by suitably
modifying the subject's diet to increase small intestinal motility
to a normal level (e.g., by increasing dietary fiber), or by
administration of a chemical prokinetic agent to the subject,
including bile acid replacement therapy when this is indicated by
low or otherwise deficient bile acid production in the subject.
[0199] For purposes of the present invention, a prokinetic agent is
any chemical that causes an increase in phase III interdigestive
motility of a human subject's intestinal tract. Increasing
intestinal motility, for example, by administration of a chemical
prokinetic agent, prevents relapse of the SIBO condition, which
otherwise typically recurs within about two months, due to
continuing intestinal dysmotility. The prokinetic agent causes an
in increase in phase III interdigestive motility of the human
subject's intestinal tract, thus preventing a recurrence of the
bacterial overgrowth. Continued administration of a prokinetic
agent to enhance a subject=s phase III interdigestive motility can
extend for an indefinite period as needed to prevent relapse of the
SIBO condition.
[0200] Preferably, the prokinetic agent is a known prokinetic
peptide, such as motilin, or functional analog thereof, such as a
macrolide compound, for example, erythromycin (50 mg/day to 2000
mg/day in divided doses orally or I.V. in divided doses), or
azithromycin (250-1000 mg/day orally).
[0201] However, a bile acid, or a bile salt derived therefrom, is
another preferred prokinetic agent for inducing or increasing phase
III interdigestive motility. (E. P. DiMagno, Regulation of
interdigestive gastrointestinal motility and secretion, Digestion
58 Suppl. 1:53-55 [1997]; V. B. Nieuwenhuijs et al., Disrupted bile
flow affects interdigestive small bowel motility in rats, Surgery
122(3):600-08 [1997]; P. M. Hellstrom et al., Role of bile in
regulation of gut motility, J. Intern. Med. 237(4):395-402 [1995];
V. Plourde et al., Interdigestive intestinal motility in dogs with
chronic exclusion of bile from the digestive tract, Can. J.
Physiol. Pharmacol. 65(12):2493-96 [1987]). Useful bile acids
include ursodeoxycholic acid and chenodeoxycholic acid; useful bile
salts include sodium or potassium salts of ursodeoxycholate or
chenodeoxycholate, or derivatives thereof.
[0202] A compound with cholinergic activity, such as cisapride
(i.e., Propulsid.RTM.; 1 to 20 mg, one to four times per day orally
or I.V.), is also preferred as a prokinetic agent for inducing or
increasing phase III interdigestive motility. Cisapride is
particularly effective in alleviating or improving hyperalgesia
related to SIBO or associated with disorders caused by SIBO, such
as IBS, fibromyalgia, or Crohn=s disease.
[0203] A dopamine antagonist, such as metoclopramide (1-10 mg four
to six times per day orally or I.V.), domperidone (10 mg, one to
four times per day orally), or bethanechol (5 mg/day to 50 mg every
3-4 hours orally; 5-10 mg four times daily subcutaneously), is
another preferred prokinetic agent for inducing or increasing phase
III interdigestive motility. Dopamine antagonists, such as
domperidone, are particularly effective in alleviating or improving
hyperalgesia related to SIBO or associated with disorders caused by
SIBO, such as IBS, fibromyalgia, or Crohn=s disease.
[0204] Also preferred is a nitric oxide altering agent, such as
nitroglycerin, nomega-nitro-L-arginine methylester (L-NAME),
N-monomethyl-L-arginine (L-NMMA), or a 5-hydroxytryptamine (HT or
serotonin) receptor antagonist, such as ondansetron (2-4 mg up to
every 4-8 hours I.V.; pediatric 0.1 mg/kg/day) or alosetron. The
5-HT receptor antagonists, such as ondansetron and alosetron, are
particularly effective in improving hyperalgesia related to SIBO,
or associated with disorders caused by SIBO, such as IBS,
fibromyalgia, or Crohn=s disease.
[0205] An antihistamine, such as promethazine (oral or I.V. 12.5
mg/day to 25 mg every four hours orally or I.V.), meclizine (oral
50 mg/day to 100 mg four times per day), or other antihistamines,
except ranitidine (Zantac), famotidine, and nizatidine, are also
preferred as prokinetic agents for inducing or increasing phase III
interdigestive motility.
[0206] Also preferred are neuroleptic agents, including
prochlorperazine (2.5 mg/day to 10 mg every three hours orally; 25
mg twice daily rectally; 5 mg/day to 10 mg every three hours, not
to exceed 240 mg/day intramuscularly; 2.5 mg/day to 10 mg every
four hours I.V.), chlorpromazine (0.25 mg/lb. up to every four
hours [5-400 mg/day] orally; 0.5 mg/lb. up to every 6 hours
rectally; intramuscular 0.25/lb. every six hours, not to exceed
75/mg/day), or haloperidol (oral 5-10 mg/day orally; 0.5-10 mg/day
I.V.). Also useful as a prokinetic agent, for purposes of the
present invention, is a kappa agonist, such as fedotozine (1-30
mg/day), but not excluding other opiate agonists. The opiate
(opioid) agonists, such as fedotozine, are particularly effective
in alleviating or improving hyperalgesia related to SIBO or
associated with disorders caused by SIBO, such as IBS,
fibromyalgia, or Crohn=s disease.
[0207] The preceding are merely illustrative of the suitable means
by which small intestinal bacterial overgrowth is at least
partially eradicated by treatment in accordance or in combination
with the inventive methods. These means can be used separately, or
in combination, by the practitioner as suits the needs of an
individual human subject.
[0208] Optionally, treating further includes administering to the
human subject an anti-inflammatory cytokine or an agonist thereof,
substantially simultaneously with or after at least partially
eradicating the bacterial overgrowth of the small intestine, to
accelerate or further improve the symptom(s) of irritable bowel
syndrome, fibromyalgia, chronic fatigue syndrome, depression, ADHD,
or an autoimmune disease, or Crohn=s disease. Useful
anti-inflammatory cytokines include human IL-4, IL-10, IL-11, or
TGF-.beta., derived from a human source or a transgenic non-human
source expressing a human gene. The anti-inflammatory cytokine is
preferably injected or infused intravenously or subcutaneously.
[0209] Optionally, when the suspected diagnosis is irritable bowel
syndrome, fibromyalgia, chronic fatigue syndrome, depression, ADHD,
or an autoimmune disease, such as multiple sclerosis or systemic
lupus erythematosus, symptoms are improved by administering an
antagonist of a pro-inflammatory cytokine or an antibody that
specifically binds a pro-inflammatory cytokine The antagonist or
antibody is administered to the human subject substantially
simultaneously with or after treatment to at least partially
eradicate the bacterial overgrowth. The antagonist or antibody is
one that binds to a pro-inflammatory cytokine or antogonizes the
activity or receptor binding of a pro-inflammatory cytokine.
Pro-inflammatory cytokines include TNF-.alpha., IL-1.alpha.,
IL-1.beta., IL-6, IL-8, IL-12, or LIF. The cytokine antagonist or
antibody is preferably derived from a human source or is a chimeric
protein having a human protein constituent. The cytokine antagonist
or antibody is preferably delivered to the human subject by
intravenous infusion.
[0210] Optionally, the method of treating irritable bowel syndrome,
fibromyalgia, chronic fatigue syndrome, depression, attention
deficit/hyperactivity disorder, an autoimmune disease, or Crohn=s
disease, further comprises administering an agent that modifies
afferent neural feedback or sensory perception. This is
particularly useful when, after at least partial eradication of
SIBO, the subject experiences residual symptoms of hyperalgesia
related to SIBO or associated with a disorder caused by SIBO, such
as IBS, fibromyalgia, or Crohn=s disease. Agents that modify
afferent neural feedback or sensory perception include 5-HT
receptor antagonists, such as ondansetron and alosetron; opiate
agonists, such as fedotozine; peppermint oil; cisapride; a dopamine
antagonist, such as domperidone; an antidepressant agent; an
anxiolytic agent; or a combination of any of these. Useful
antidepressant agents include tricyclic antidepressants, such as
amitriptyline (Elavil); tetracyclic antidepressants, such as
maprotiline; serotonin re-uptake inhibitors, such as fluoxetine
(Prozac) or sertraline (Zoloft); monoamine oxidase inhibitors, such
as phenelzine; and miscellaneous antidepressants, such as
trazodone, venlafaxine, mirtazapine, nefazodone, or bupropion
(Wellbutrin). Typically, useful antidepressant agents are available
in hydrochloride, sulfated, or other conjugated forms, and all of
these conjugated forms are included among the useful antidepressant
agents. Useful anxiolytic (anti-anxiety) agents include
benzodiazepine compounds, such as Librium, Atavin, Xanax, Valium,
Tranxene, and Serax, or other anxiolytic agents such as Paxil.
[0211] Eradication of the bacterial overgrowth is determined by
detection methods described above, particularly in comparison with
recorded results from pre-treatment detection. After at least
partially eradicating the bacterial overgrowth, in accordance with
the present method, the symptom(s) of irritable bowel syndrome,
fibromyalgia, chronic fatigue syndrome, depression, ADHD, an
autoimmune disease, or Crohn=s disease are improved. Improvement in
a symptom(s) is typically determined by self-reporting by the human
subject, for example by VAS scoring or other questionnaire.
Improvement in academic, professional, or social functioning, e.g.,
in cases of ADHD or depression can also be reported by others or
can be observed by the clinician. Improvement (increase) in pain
threshold, e.g., in subjects diagnosed with fibromyalgia, can be
measured digitally, for example, by tender point count, or
mechanically, for example, by dolorimetry. (F. Wolfe et al.,
Aspects of Fibromyalgia in the General Population: Sex, Pain
Threshold, and Fibromyalgia Symptoms, J. Rheumatol. 22:151-56
[1995]). Improvement in visceral hypersensitivity or hyperalgesia
can be measured by balloon distension of the gut, for example, by
using an electronic barostat. (B.D. Nabiloff et al., Evidence for
two distinct perceptual alterations in irritable bowel syndrome,
Gut 41:505-12 {1997]). Some improvement(s) in symptoms, for example
systemic lupus erythematosus symptoms, such as rashes,
photosensitivity, oral ulcers, arthritis, serositis, or
improvements in the condition of blood, kidney or nervous system,
can be determined by clinical observation and measurement.
[0212] The present invention also relates to a kit for the
diagnosis of SIBO or a SIBO-caused condition. The kit comprises at
least one breath sampling container, a pre-measured amount of a
substrate, and instructions for a user in detecting the presence or
absence of SIBO by determining the relative amounts of methane,
hydrogen, and at least one sulfur-containing gas in a gas mixture
exhaled by the human subject, after the human subject has ingested
a controlled quantity of the substrate. The present kit is useful
for practicing the inventive method of detecting SIBO in a human
subject, as described hereinabove.
[0213] The kit is a ready assemblage of materials or components for
facilitating the detection of small intestinal bacterial
overgrowth, in accordance with the present invention. The kit
includes suitable storage means for containing the other components
of the kit. The kit includes at least one, and most preferably
multiple, air-tight breath sampling container(s), such as a bag,
cylinder, or bottle, and at least one pre-measured amount of a
substrate, which is preferably an isotope-labeled substrate or
substrate that is poorly digestible by a human. Preferably the
substrate is a sugar, such as lactulose (e.g., 10-20 g units) or
xylose, or a sugar, such as glucose (e.g., 75-80 g units), lactose,
or sucrose, for measuring breath hydrogen, methane, and at least
one sulfur-containing gas, such as hydrogen sulfide, a sulfhydryl
compound, methanethiol, dimethylsulfide, dimethyl disulfide, an
allyl methyl sulfide, an allyl methyl sulfide, an allyl methyl
disulfide, an allyl disulfide, an allyl mercaptan, or a
methylmercaptan.
[0214] The present kit also contains instructions for a user in how
to use the kit to detect small intestinal bacterial overgrowth
(SIBO) or to corroborate a suspected diagnosis of irritable bowel
syndrome, fibromyalgia, chronic fatigue syndrome, chronic pelvic
pain syndrome, autism, impaired mentation, impaired memory,
depression, ADHD, an autoimmune disease, or Crohn=s disease, in
accordance with the present methods.
[0215] Optionally, the kit also contains compositions useful for at
least partially eradicating SIBO, as described above.
[0216] The components assembled in the kits of the present
invention are provided to the practitioner stored in any convenient
and suitable way that preserves their operability and utility.
[0217] For example the components can be in dissolved, dehydrated,
or lyophilized form; they can be provided at room, refrigerated or
frozen temperatures.
[0218] The foregoing descriptions for the methods and kits of the
present invention are illustrative and by no means exhaustive. The
invention will now be described in greater detail by reference to
the following non-limiting examples.
EXAMPLES
Example 1
Composition of the Database
[0219] Data were assembled from 202 human subjects from the
Cedars-Sinai Medical Center GI Motility Program who completed an
extensive questionnaire of health history. These patients were all
referred for lactulose breath hydrogen testing (LBHT) by more than
30 private gastroenterologists. These patients were selected by
their gastroenterologists to undergo breath testing, because they
had symptoms compatible with SIBO. However, the questionnaire
focused on general risk factors, associated conditions, and
symptoms found in these patients and not specifically the incidence
of SIBO. After antibiotic therapy, 59 subjects actually returned
for a follow up LBHT and a follow-up questionnaire. This likely
resulted in an underestimate of responsiveness to treatment, since
only those who failed to respond adequately were likely to return
to assess eradication of SIBO.
Example 2
Breath Hydrogen Testing
[0220] Subjects were tested after an overnight fast. At time zero,
each subject swallowed 15 ml of Chronulac formula, delivering 10 g
of lactulose; every 5-20 min thereafter, for 2-4 hours, a 50
cm.sup.3 end-expiratory breath sample was taken with an airtight
sampling bag. Each breath sample was then analyzed for hydrogen
content with a gas chromatograph (Quintron Model DP, Quintron
Instrument Co., Division of E. F. Brewer Co, Menomonee Falls, Wis.
53051), standardized using a QuinGas standard as instructed by the
manufacturer. Hydrogen peaks were plotted before and after an
antimicrobial treatment regimen for comparison. The normal range
for the second hydrogen peak was 0 to 20 ppm.
Example 3
Diagnosis and Antibiotic Treatment of Irritable Bowel Syndrome
[0221] The two hundred-two (202) human subjects were assessed for
SIBO with LBHT. Of the 202 subjects in the database, 95 claimed to
have been given a diagnosis of IBS. In addition, a symptom
questionnaire was used to determine whether these subjects
fulfilled Rome criteria for IBS, and four of the subjects failed to
meet the Rome criteria. Crohn=s disease was present in 14 of the
subjects and four had a history of ulcerative colitis. After these
22 subjects were excluded, 73 subjects remained.
[0222] Among the 107 subjects who stated that they had not
previously been given a diagnosis of IBS, 78 met Rome criteria.
After the 21 who had Crohn=s disease, five who had ulcerative
colitis and one with short bowel transit were excluded, 51 subjects
remained. Data gathered from these subjects were pooled with data
from the previous 73 subjects with suspected IBS, yielding a total
of 124 of the original 202 (61%) subjects with a suspected
diagnosis of IBS.
[0223] Of the 124, 92 (74%) were positive for SIBO. However, of the
32 subjects meeting the Rome criteria, who were negative for SIBO,
14 had been treated with antibiotics within 3 months prior to LBHT.
Therefore, the incidence of SIBO among the 110 untreated subjects
was 92 (84%), showing a strong association between a suspected
diagnosis of IBS and the presence of SIBO. After neomycin treatment
(500 mg twice daily for ten days), 23 of these 92 returned for
follow-up testing. On a visual analog scores (VAS), subjects were
asked to rate their degree of post-treatment improvement. These 23
subjects reported a 60.+-.31% improvement, although 17 had only
partial eradication of SIBO, based on their LBHT results. (FIG.
1).
[0224] There was a likely selection bias in the database due to the
fact that subjects were referred for LBHT, because their physicians
suspected they had SIBO. To correct for this bias, a pilot study
was also conducted looking at the incidence of bacterial overgrowth
in patients with IBS. All patients between the ages of 18 and 65
referred to the Cedars-Sinai GI Motility Program who met Rome
criteria for IBS, and who had had a previous upper GI (small bowel)
with follow-through (i.e., barium or Gastrograffin imaging
analysis) ruling out Crohn=s disease and ulcerative colitis, were
asked to present to the GI motility laboratory for LBHT. Eight
human subjects with a suspected diagnosis of IBS, based on the Rome
criteria, were tested for SIBO, using LBHT as described in Example
2. Seven of these patients (87.5%) were found to have SIBO based on
hydrogen peaks in a range of 80-250 ppm of hydrogen. Six of the 7
subjects testing positive for SIBO returned approximately 10 days
after completion of a 10 day course of neomycin as described above.
Neomycin treatment completely eradicated the SIBO in each of the
six subjects, based on post-treatment breath hydrogen peaks in the
normal range of 0-20 ppm. The six subjects reported an average
improvement in their IBS symptoms of 65.+-.28% (Range: 20-100%) on
VAS scoring. FIG. 2 shows VAS for the six subjects, based on a
scale of 0-5, with 0 implying no pain and 5 the most pain of
life-time. It is clear from these results that at least partial
eradication of bacterial overgrowth results in an improvement in
gastrointestinal symptoms including bloating, gas, diarrhea,
abdominal pain, sensation of incomplete evacuation and even
constipation, associated with IBS. Additionally, significant
extraintestinal symptoms of IBS, such as joint pain and fatigue,
were also substantially improved, and the degree of improvement was
greater in subjects who had complete eradication of SIBO.
Comparison of Efficacies of Various Antibiotic Regimes for Treating
SIBO.
[0225] Subjects referred to the Cedars-Sinai GI Motility Program
for a lactulose breath hydrogen test (LBHT) to assess SIBO were
entered into a database. Those that tested positive for SIBO were
given antibiotic treatment by their referring physician and in some
cases, returned for a follow-up LBHT to assess eradication of SIBO.
During the follow-up LBHT, subjects were asked which antibiotic
they were given to treat their SIBO. The eradication rate of each
antibiotic was evaluated.
[0226] Of the 771 subjects in the database, 561 (73%) tested
positive for SIBO. Of the 170 subjects who returned for a follow-up
LBHT, 65 subjects were excluded because they did not specify or
could not remember which antibiotic they took. Based on the
remaining 105 subjects, neomycin, augmentin, and ciprofloxacin were
the most commonly prescribed, with neomycin being most successful.
(See Table 1 below). Flagyl was a relatively poor choice by itself.
None of the commonly used antibiotics was universally successful in
eradicating overgrowth. Thus, Table 1 shows that, while a number of
antibiotics are able to eradicate SIBO, neomycin was most
effective.
TABLE-US-00001 TABLE 1 Comparison of efficacies of various
antibiotic regimes for treating SIBO Number % Patients of Patients
Total with SIBO SIBO Eradicated Number Eradicated Neomycin 42 76 55
Flagyl 2 8 25 Ciprofloxacin 3 6 50 Augmentin 2 4 50 Flagyl +
Ciprofloxacin 1 2 --* Tetracycline 2 2 --* Doxycycline 1 1 --*
Trovan 0 1 --* Neomycin/Biaxin + Amoxicillin 1 1 --* Neomycin +
Ciprofloxacin 1 1 --* Tetracycline + Flagyl 1 1 --* Neomycin +
Flagyl 0 1 --* Biaxin 0 1 --* *Too few subjects to determine
percent success.
Prevalence of SIBO in Normal Controls.
[0227] The prevalence of SIBO in IBS compared to normal controls
was determined as defined by the lactulose hydrogen breath test.
Fifty-seven IBS subjects enrolled in a double blind placebo
controlled trial and 9 normal controls underwent a lactulose breath
hydrogen test (LBHT) to diagnose SIBO. IBS subjects had to meet
Rome I criteria. Control subjects had to have none of the Rome I
criteria, based on telephone or in-person interviews. SIBO was
defined as a greater than 20 ppm rise in H.sub.2 concentration
during the first 90 minutes of lactulose breath hydrogen testing.
The prevalence of SIBO in IBS subjects and controls was compared
using Chi-square.
[0228] Of the 57 IBS subjects, 41 (72%) had SIBO. Of the 9 normal
controls, only 1 subject (11%) had SIBO (.chi..sup.2=9.9, OR=20.5,
CI:2.2-481.8, p<0.01). These results confirm the association
between IBS and SIBO as there is a much higher prevalence of SIBO
in IBS compared to normal controls.
Example 4
Diagnosis and Treatment of Fibromyalgia and Chronic Fatigue
Syndrome
Fibromyalgia:
[0229] Of the 202 patients in the database, 37 (18%) had a
suspected diagnosis of fibromyalgia. Of these 37, 28 tested
positive for SIBO. However, of the nine who tested negative for
SIBO, six had taken antibiotics within the preceding 3 months, and
were excluded. Therefore, 28 out of 30 (93%) of subjects with
suspected fibromyalgia had SIBO, demonstrating a strong association
between a suspected diagnosis of fibromyalgia and the presence of
SIBO.
[0230] After neomycin treatment (500 mg twice daily, 10-day
course), ten of these 28 subjects returned, and post-treatment LBHT
confirmed that SIBO had been at least partially eradicated. These
ten subjects reported a 63.+-.19% overall improvement in their
symptoms by VAS scoring. FIG. 3 compares the VAS scores for various
symptoms reported by the subjects with a suspected diagnosis of
fibromyalgia before and after neomycin treatment. Symptoms included
bloating, gas, diarrhea, joint pain and fatigue to treatment.
Subjects were asked to identify the symptom most improved. Five
subjects reported that pain was the most improved; three subjects
reported that the level of fatigue was most improved, and two
others reported that their abdominal complaints improved the most.
There was a negative correlation between the degree of improvement
in the VAS scoring and the amount of residual hydrogen peak seen in
LBHT. (Pearson=-0.689, p=0.02; FIG. 4).
[0231] Subsequently, forty-six human subjects with FM (ACR
criteria) entered a double blind randomized placebo controlled
trial. Each subject underwent LBHT, a tender point examination and
completed a questionnaire at the initial (baseline) and at every
subsequent visit. Subjects were randomized to receive neomycin (500
mg twice daily in liquid form) or a matched placebo, for 10 days.
After completion of this treatment, subjects with persistent SIBO
received antibiotics (open label) until at least partially
eradication was confirmed by LBHT. T-test was used to compare the
symptom scores of patients whose SIBO condition was at least
partially eradicated with those whose SIBO was not at least
partially eradicated. Forty-two of the 46 FM patients (91.3%) were
found to have SIBO. Six out of 20 patients (30%) in the neomycin
group achieved complete at least partially eradication in the
blinded arm. Only 6 subjects showed no difference in the symptom
score before and after the 10 d treatment. Twenty-eight subjects
went on to open label treatment with 17 (60.7%) achieving complete
at least partially eradication of SIBO. When symptom scores after
at least partially eradication of SIBO on double blind or open
treatment were compared to baseline, there was significant
improvement in Tender Points, Tender Point Score, Hamilton
Depression Scale, Fibromyalgia Impact Questionnaire (FIQ), Beck
Depression Scale, Health Assessment Questionnaire (HAQ), VAS-Pain,
VAS-Memory/Concentration and IBS-Quality of Life (QOL). (Initial
data in Table 1a). These results confirm that SIBO is associated
with fibromyalgia, and that at least partially eradication of SIBO
improves symptoms in fibromyalgia.
TABLE-US-00002 TABLE 1a Selected Symptom Scores Double Blind
Randomized Placebo Controlled Trial with Subjects Diagnosed with
Fibromyalgia. SIBO SIBO not eradicated eradicated eradicated vs.
not (n = 25) (P = 15) eradicated Observation Baseline eradicated
P-value Baseline eradicated P-value P-value Tender Points 13.3 .+-.
2.9 10.3 .+-. 4.2 0.01 13.6 .+-. 2.0 12.1 .+-. 4.1 NS NS (TP) TP
Score 20.3 .+-. 7.0 15.0 .+-. 9.1 0.01 23.7 .+-. 8.0 19.9 .+-. 9.7
NS NS FIQ 66.8 .+-. 18.2 49.5 .+-. 17.7 0.0001 72.7 .+-. 19.9 64.1
.+-. 20.9 0.04 0.02 VAS-pain(mm) 80.7 .+-. 22.7 52.4 .+-. 28.5
0.00005 87.5 .+-. 19.6 76.2 .+-. 25.2 NS 0.01 HAQ 42.4 .+-. 10.5
37.7 .+-. 10.1 0.005 45.1 .+-. 11.2 43.9 .+-. 12.1 NS NS
Chronic Fatigue Syndrome:
[0232] Thirty of 202 subjects in the database (15.9%) had received
a diagnosis of chronic fatigue syndrome. Of these 30 subjects, 21
(70%) had SIBO as indicated by LBHT, but four out of the nine
without SIBO had recently taken antibiotics. Therefore, the
prevalence of SIBO was 21 out of 26 (81%) subjects with a diagnosis
of CFS. After treatment with neomycin (500 mg twice daily, 10-day
course), nine of the 21 subjects diagnosed with CFS, returned for
follow-up LBHT and questionnaire. LBHT showed that all nine
subjects experienced at least partially eradication of SIBO, and
important symptoms of CFS were substantially improved after
treatment. (Table 2).
TABLE-US-00003 TABLE 2 VAS scores by CFS patients reporting before
and after anti-biotic treatment. Symptom Before Antibiotic After
Antibiotic P-value Bloating 4.3 .+-. 1.0 2.3 .+-. 1.7 0.002 Fatigue
4.6 .+-. 1.0 3.5 .+-. 1.4 0.02
Example 5
Autoimmune Diseases, Depression, ADHD, Autism, Mentation and
Memory
SLE.
[0233] Fifteen of the 202 (7.4%) subjects in the database had been
diagnosed with SLE. Of these 15 subjects, 13 (87%) had bacterial
overgrowth, as indicated by LBHT. Four of the 15 subjects with SLE
returned for follow-up LBHT and questionnaire after treatment with
neomycin (500 mg twice daily for 10 days). LBHT results for these
four were negative for SIBO, and other significant symptoms were
significantly improved after treatment. (Table 3).
TABLE-US-00004 TABLE 3 VAS scores by SLE patients reporting before
and after anti-biotic treatment. Symptom Before Antibiotic After
Antibiotic P-value Bloating 3.0 .+-. 2.0 1.3 .+-. 1.3 0.1 Joint
Pains 2.5 .+-. 1.5 0.5 .+-. 0.6 0.04 Gas 3.3 .+-. 1.7 1.9 .+-. 1.7
0.3 Fatigue 4.6 .+-. 1.0 3.5 .+-. 1.4 0.3
Multiple Sclerosis:
[0234] A 22-year-old female who presented with a history of
multiple sclerosis symptoms and with plaques demonstrated on MRI
imaging. A suspected diagnosis of multiple sclerosis had been made
by a neurologist was based on various neuropathies of the
peripheral nervous system, including numbness, tingling, and
weakness in the lower extremities, but this subject also had
associated bloating, gas, distension and alteration in bowel
habits. The subject also complained of a significant fatigue and
nausea. The subject underwent LBHT, which detected SIBO. She was
subsequently treated with neomycin (500 mg twice daily for 10
days), which at least partially eradicated the bacterial
overgrowth. This was followed by complete resolution of her nausea,
fatigue, bloating, gas distension and alteration in bowel habits.
In addition, the subject showed dramatic improvement and resolution
of her neuropathies. She no longer had numbness or tingling in the
hands or feet and was functioning quite well. Approximately 6-8
weeks after this initial response, the patient had a relapse of her
symptoms, including bloating, gas, distension and neuropathy. She
had a repeat LBHT that confirmed a recurrence of SIBO. Upon
re-treatment with neomycin (500 mg twice daily for 10 days), she
once again experienced complete resolution of her symptoms.
Depression:
[0235] A 73-year-old female presented with bloating, gas, abdominal
distention, and cramping for a period of 3 years prior to LBHT.
Symptoms of depression first appeared concurrently with the first
appearance of bowel symptoms, and were serious enough that
psychiatric hospitalization had been considered by her attending
psychiatrist. The subject reported feeling very depressed and was
convinced that life was not worth living. The subject=s LBHT
indicated the presence of a SIBO condition. After treatment with
neomycin (500 mg twice daily for 10 days), the subject stated that
she felt A100% better. .apprxeq.She reported that her depression
was completely resolved and that her energy was back to normal. In
addition, her bowel symptoms were also completely improved. The
subject had been prescribed eight different anti-depressant
medications, all of which were discontinued as a result of her
improvement.
ADHD:
[0236] A 13 year-old female was brought in by her mother with a
suspected diagnosis of attention deficit/hyperactivity disorder (AD
type), made by a pediatrician. Concurrently, she also had
significant bloating, gas and some alteration in bowel habits. She
had initially been referred for diagnosis by her teachers and
school counselors, because she had been having difficulty
performing in school for the previous two to three years, after
having previously been a very good student. Prior to the detection
of SIBO, the subject had been treated with multiple pharmacologic
agents for depression, including amitryptiline, with no noticeable
improvement in her symptoms.
[0237] The subject underwent LBHT that demonstrated the presence of
SIBO. The subject was treated with neomycin (500 mg twice daily for
10 days) and after complete at least partially eradication of the
bacterial overgrowth, she had resolution of her bowel symptoms.
Additionally, she started to get AA.apprxeq.averages in school
again after being in the AC.apprxeq.range. She was able to
concentrate better, and her teachers noticed a difference in her
focus and attitude. Approximately two months later the subject had
a relapse in her attention problem which was concurrent with a
recurrence of the bacterial overgrowth, as detected by LBHT. After
repeat treatment with neomycin (500 mg twice daily for 10 days),
the subject again responded with improved concentration and
resolution of bowel symptoms.
Autism:
[0238] The patient was a 6-year-old female with a history of autism
after having failed development after the age of one year. Before
treatment, the patient was categorized as having a developmental
age of 15 months. She also complained of abdominal distension, gas,
bloating and altered bowel habits. The patient was treated with
Augmentin (500 mg twice a day for ten days), which resulted in a
substantial improvement in bowel habits altogether. The bloating,
gas, distension and diarrhea resolved. In addition, there were some
positive concentration and behavioral changes. The patient was more
responsive and cognitively appreciative of her parents' wishes, and
there was some advancement in intellectual behavior. For example,
after treatment she was able to tolerate clothing and had improved
concentration.
Memory/Mentation/Concentration:
[0239] The patient was a 72-year-old female with a history of
chronic intestinal complaints over several years. She experienced
altered bowel habits with alternating diarrhea and constipation
with bloating, gas, distension and abdominal pain. Also, she had
been diagnosed by several psychiatrists as having psychiatric
problems due to decreased mentation from mild senility, and she
contemplated psychiatric hospitalization.
[0240] SIBO was detected in this patient by LBHT. A subsequent
course of antibiotics completely eradicated the SIBO condition, and
she returned to report joyfully that she no longer needed the
psychotropic medications that she had been prescribed, because she
feels completely normal, including her bowels. She is now able to
drive a car again, which was previously prevented from doing due to
her impaired memory and difficulty in concentrating on the road.
Treatment of her SIBO condition (neomycin, 500 mg twice a day for
ten days) has produced a dramatic improvement in her quality of
life.
Example 6
Diagnosis and Treatment of Crohn=s Disease
[0241] Of the 202 subjects in the database, 39 (19%) had a
suspected diagnosis of Crohn=s disease. Of these 39, eight
demonstrated short bowel transit and one subject produced neither
hydrogen nor methane in LBHT; these nine were excluded. Of the 30
remaining subjects, 22 had SIBO. However, of the eight subjects who
had a negative LBHT result, five had been treated with antibiotics
within the preceding 3 months. If these subjects are excluded, 22
out of 25 (88%) subjects with a suspected diagnosis of Crohn=s
disease had SIBO, which shows a strong association between a
suspected diagnosis of Crohn=s disease and the presence of
SIBO.
[0242] Of the 22 patients testing positive for the presence of
SIBO, nine returned after neomycin treatment (10-day course of 500
mg twice/daily) for LBHT, which showed at least partially
eradication of SIBO. These nine patients reported a 57.+-.32% (n=8
because one patient failed to report percent improvement) overall
improvement in their symptoms by VAS. If these subjects remained
positive after antibiotic treatment with neomycin, metronidazole
(Flagyl.sup.7), or ciprofloxacin, their improvement was only
20.+-.0% as opposed to 69.+-.27% if the breath test was negative
(p<0.05). FIG. 5 shows a dramatic improvement in the patients
symptoms after treatment. There was an especially notable reduction
in bloody stools, diarrhea and fatigue.
[0243] As with the subjects with fibromyalgia, there was a negative
correlation between the degree of improvement in the VAS scoring
and the amount of residual hydrogen production (Pearson=-0.787,
p=0.02; FIG. 6).
[0244] To correct for selection bias, a pilot study was conducted
to determine the incidence of SIBO in subjects who had received a
suspected diagnosis of Crohn=s disease at Cedars-Sinai Medical
Center's IBD Center within the preceding three months. Six of these
subjects underwent LBHT, of whom five (83%) were positive for
SIBO.
[0245] Two of the six subjects returned for follow-up after
antibiotic therapy (10-day course of neomycin). Post-treatment
LBHTs showed that SIBO had been completely at least partially
eradicated in both subjects. They reported, respectively, a 60% and
80% overall improvement in their symptoms. This improvement was
stated to include substantial reduction in diarrhea, gas and
bloating.
Example 7
Response Stratification
[0246] There is a stratification in the degree of overgrowth and
production of hydrogen among the various diagnostic categories. For
example, during the double blind study in the treatment of SIBO in
fibromyalgia (Example 4), it was noted that the level of hydrogen
production during the LBHT was much higher in this group of
subjects as compared to those in subjects in the IBS incidence
study described in Example 3. Given that the bacterial load is
related to the level of hydrogen production, this implies that the
degree of overgrowth is higher in patients with fibromyalgia
compared to subjects with IBS.
[0247] The stratification of breath hydrogen levels with respect to
diagnostic categories is as follows: IBS/Crohn=s Disease (40-70 ppm
of hydrogen); CFS (50-100 ppm of hydrogen); and FM (100-250 ppm of
hydrogen).
Example 8
Intestinal Dysmotility Associated with IBS and FM
[0248] Clinical experience showed that SIBO tends to recur after
anti-biotic treatment within about 2 months. To demonstrate that a
lack of phase III interdigestive motility is responsible for SIBO
in subjects with IBS or fibromyalgia, antreduodenal manometry was
conducted in human subjects diagnosed with IBS or FM.
Antreduodenal Manometry.
[0249] PhaseIII interdigestive (fasting) motility was assessed in
15 human subjects. An antreduodenal manometry was performed by
placing an 8-channel small bowel manometry catheter (each channel
spaced 5 cm apart) into the small bowel using fluoroscopic
guidance. After placement of the catheter, manometric recordings
were made with an Arndorffer perfusion system with signals
collected using Medtronics/Synectics Polygraf and associated
Polygram software. Data were assessed for the characteristics of
interdigestive motility.
IBS.
[0250] Phase III interdigestive motility was assessed for a
six-hour period in 15 human subjects having a suspected diagnosis
of IBS, as defined by Rome Criteria, corroborated by concomitant
SIBO. Of these 15 subjects, 13 (86%) had no detectable phase III
interdigestive motility during the period of study. One subject
(7%) had phase III interdigestive motility of short duration (<3
minutes), and one subject (7%) had normal phase III interdigestive
motility.
Fibromyalgia.
[0251] Phase III interdigestive motility was assessed in seven
human subjects having a suspected diagnosis of fibromyalgia
corroborated by the presence of SIBO. Of these seven subjects, six
(86%) lacked detectable phase III interdigestive motility, and one
subject (14%) had motility of less than normal peristaltic
amplitude. The duration of study in the patients with fibromyalgia
averaged 216.+-.45 minutes in the fasting state.
Example 9a
Treatment of SIBO-Related IBS with a Prokinetic Agent
[0252] Erythromycin, as a motilin agonist, can induce phase III of
interdigestive motility. (E.g., M. J. Clark et al., Erythromycin
derivatives ABT229 and GM 611 act on motilin receptors in the
rabbit duodenum, Clin. Exp. Pharmacol. Physiol. 26(3):242-45
[1999]). Therefore, two subjects with recurrent IBS symptoms
received prokinetic treatment with erythromycin.
[0253] The two subjects were a 55-year-old female and a 43-year-old
female, both diagnosed with IBS. SIBO was detected in these
subjects by LBHT. Antibiotic treatment of the SIBO resulted in
greater than 90% improvement in symptoms. However, IBS symptoms
recurred three to four weeks later, concurrent with a return of the
SIBO condition. Subsequent courses of antibiotic treatment resulted
in a similar pattern of improvement followed by a rapid recurrence
of IBS symptoms in both subjects. Antreduodenal manometry was
performed, demonstrating a lack of phase III of interdigestive
motility, and erythromycin (50 mg daily) was prescribed to the
subjects. The two subjects subsequently remained free of IBS
symptoms and SIBO for at least 18 months and six months,
respectively.
[0254] These results demonstrate the effectiveness of prokinetic
treatment with erythromycin in preventing the recurrence of SIBO
and IBS symptoms in subjects diagnosed with IBS.
Example 9b
Treatment of SIBO-Related IBS with a Supplemental Pancreatic
Enzyme
[0255] Supplementing food with pancreatic enzymes facilitates more
efficient absorption and digestion of food nutrients, thus allowing
ingested food nutrients to be absorbed higher up in the small
intestine than otherwise. This leads to a relative deprivation of
nutrients to the bacteria involved in the SIBO condition. An
example of this treatment modality occurred in the case of a
19-year-old male who had longstanding history of altered bowel
habits, bloating, gas, distension and significant urge to evacuate.
All of these symptoms were consistent with irritable bowel syndrome
(IBS). The patient was diagnosed as having SIBO based on the
results of LBHT. Subsequent to treatment with antibiotics, the
patient had significant improvement in his symptoms. However, his
SIBO condition became difficult to manage due to antibiotic
resistance. An alternative treatment regimen was prescribed, which
involved the addition of a pancreatic enzyme to the patient's food
(10,000 Units human pancrease in capsules ingested immediately
before each meal). With this therapy, the patient reported that his
gastrointestinal complaints have improved by approximately 30-40%,
corresponding to partial eradication of his SIBO condition.
Treatment was continued for at least eight months with a
continuation of the improvement in symptoms during that period.
Example 9c
Excessive Methane Production in Subjects with Small Intestinal
Bacterial Overgrowth is Associated with Less Diarrhea
[0256] Bacterial metabolism is the major mechanism for the removal
of hydrogen that is produced during fermentation reactions of
intestinal bacteria. Specifically, hydrogen is consumed in the
production of methane and in the reduction of sulfates to sulfides,
with the 2 pathways being mutually exclusive. Since intestinal
sulfides are known to be damaging to intestinal epithelium, it was
hypothesized that diarrhea may be a less prevalent symptom among
patients with small intestinal bacterial overgrowth (SIBO) who test
positive for methane (no damaging sulfides produced).
[0257] Subjects referred to the Cedars-Sinai GI Motility Program
for LBHT were entered into a database. Subjects were asked to rate
symptoms of bloating, diarrhea, constipation, abdominal pain,
mucous in stool, incomplete evacuation, straining and urgency, on
visual analogue scales (0-5, with 0 representing no symptoms). An
ANOVA was used to compare symptom scores between subjects producing
no measured gases (only sulfide producing bacteria), H.sub.2 only,
H.sub.2 and CH.sub.4, and CH.sub.4 only, on the LBHT.
[0258] Of the 771 subjects in the database, 48 were excluded
because they demonstrated rapid transit on the LBHT. Of the 723
subjects remaining, 514 were positive for SIBO and 43 were
considered non-methane, non-hydrogen producers. Among the 514 who
had SIBO, 435 (85%) produced H.sub.2 only, 68 (13%) produced both
H.sub.2 and CH.sub.4, and 11 (2%) produced CH.sub.4 only. The
severity of diarrhea was highest in the non-H.sub.2, non CH.sub.4
and H.sub.2 only group with less in the H.sub.2 and CH.sub.4 group,
and CH.sub.4 only group. There was a significant difference between
the three groups for diarrhea (p<0.00001 after Boneferroni
correction). Urgency demonstrated the same trend, but was not
significantly different. All other symptoms were no different. The
severity of diarrheal symptoms is less in SIBO patients who excrete
methane (FIG. 7). In the non-methane-producers, greater severity of
diarrheal symptoms likely reflects the reduction of sulfates to
sulfides as the alternate pathway for the removal of hydrogen.
Example 10
Treatment of SIBO-Related Hyperalgesia
[0259] An adult male subject with a suspected diagnosis of IBS was
found to have SIBO, as detected by LBHT. Anorectal manometry
revealed rectal hypersensitivity in this subject. After eradication
of his SIBO condition with antibiotic treatment, a repeat anorectal
manometry showed that his rectal hyperalgesia had resolved.
[0260] Two adult female subjects with IBS required additional
pharmacologic manipulations to treat their SIBO-related
hyperalgesia. In the first case, SIBO was eradicated by antibiotic
treatment. However, the subject complained of persistent feelings
of rectal distension, consistent with residual hyperalgesia related
to SIBO. The subjected was then administered Colpermin (peppermint
oil) capsules and Elavil (5 mg taken at night) that alleviated her
SIBO-related hyperalgesic symptoms, presumably by reducing
intestinal wall tension and decreasing mechanoreceptor
activation.
[0261] The second female subject with a diagnosis of IBS was also
found to have SIBO, as detected by LBHT. Her SIBO was eradicated by
a combined treatment with antibiotic, intestinal lavage with
Go-Lytely, and cisapride (10 mg tid) to increase her abnormally low
phase III interdigestive motility. After eradication of SIBO, this
subject similarly complained of persistent SIBO-related
hyperalgesic symptoms of the bowel. Administration of Colpermin
(peppermint oil) then successfully alleviated the hyperalgesia,
presumably by reducing the mechanoreceptor feedback for rectal
distension.
Example 11
Treatment of SIBO Using Predigested Nutritional Formula
[0262] Based on the hypothesis that SIBO is promoted by nutritional
components in food arriving at the distal gut, where they are used
for carbon and energy by bacterial populations responsible for the
SIBO condition, ten patients (8 female; 2 male; age range 17-64
years old; none having had a bowel resection) each diagnosed with
IBS in accordance with with the Rome Criteria, and each having SIBO
as determined by LBHT, were treated with a total enteral nutrition
(TEN) formula, which is absorbed in the proximal gut (Vivonex.RTM.
T.E.N.; Sandoz Nutrition, Minneapolis, Minn.). Vivonex is a
glutamine-enriched total enteral nutrition product, containing
protein as free amino acids in a 56:44 essential to nonessential
amino acid ratio, and inter alia, carbohydrate as maltodextrin and
modified starch, safflower oil, and all essential vitamins and
minerals. Vivonex is available as a powder for aqueous
reconstitution (2.84 oz. packet;. 1 packet mixed with 250 mL
H.sub.2O delivers 300 mL of formula). Each patient was administered
an amount of reconstituted Vivonex to meet daily caloric needs
according to the manufacturer's instructions, based on the each
patient's weight, height and other relevant factors. The patient's
were allowed no other nutritional intake, but water was allowed
freely. After 14 days of the TEN regimen, each patient resumed his
or her normal diet.
[0263] FIG. 8 shows a representative result. In FIG. 8A
(pre-treatment), SIBO was initially detected by LBHT. After 14 days
of the TEN regimen, follow-up LBHT shows that SIBO had been at
least partially eradicated (FIG. 8B). Eradication was complete in
eight of the patients with a greater than 80% improvement in IBS
symptoms. Two of the patients had only partial eradication of SIBO
with <20% improvement in IBS symptoms. The eradication of SIBO
was maintained for up to two months after the TEN regimen was
discontinued and normal nutrition had been resumed.
Example 12
Use of Active Lipids to Treat SIBO-Related Conditions
[0264] Oleate and Oleic Acid Slow Upper Gut Transit and Reduce
Diarrhea in Patients with Rapid Upper Gut Transit and Diarrhea
[0265] Rapid transit through the upper gut can result in diarrhea,
maldigestion and absorption, and weight loss; and pharmacologic
treatment with opiates or anticholinergics often is required. It
was tested whether fatty acids could be used to slow upper gut
transit and reduce diarrhea in patients with rapid transit and
diarrhea.
[0266] In a preliminary study, five patients with persistent
diarrhea for 3 to 22 months, (one each due to vagal denervation,
ileal resection for Crohn's disease, and vagotomy and antrectomy,
and two due to idiopathic causes) were studied. Each patient
demonstrated rapid upper gut transit on routine lactulose breath
hydrogen testing (or variations thereof measuring labelled carbon
dioxide)(Cammack et al. Gut 23:957-961 [1982]). This test relies on
the metabolism of certain carbohydrate materials (e.g. lactulose)
by the microbial flora within the caecum. By generating gas which
can be detected in the expired air, it is possible to make some
estimation about the initial arrival of the administered material
within the colon.
[0267] Each patient received orally in random order, 0, 1.6 or 3.2
g of sodium oleate in 25 mL Ensure (Ross), followed by 100 mL
water. Thirty minutes after each dose of oleate, patients received
10 g lactulose orally, followed by 25 mL water. Breath samples were
collected in commercially available breath testing bags (Quintron,
Menomonee Falls, Wis.) every 10-15 minutes, and the hydrogen
content of the samples was measured using a breath analyzer
(Microlyzer Model 12, Quintron Instruments, Menomonee Falls, Wis.),
calibrated against gas samples of known hydrogen concentration.
With a syringe, a 40-mL sample of the expired breath was withdrawn
from the collection bag and analyzed immediately for hydrogen
concentration (ppm). The hydrogen concentration value from each
sample was plotted against time. Upper gut transit time was defined
as the time in minutes from ingestion of lactulose (t.sub.0) until
a rise of H.sub.2 of >10 ppm. Data were further analyzed using
1-way repeated measures analysis of variance (ANOVA)(See Table
4).
TABLE-US-00005 TABLE 4 Effect of oleate on upper gut transit time
(mean .+-. SE). Oleate (g) 0 1.6 3.2 Transit time (min) 46 .+-. 8.6
116 .+-. 11.1 140 .+-. 11.5
[0268] Upper gut transit was significantly prolonged by oleate in a
dose-dependent fashion (p<0.005, significant trend). During
prolonged ingestion of oleate 15-30 minutes prior to meals, all
patients reported reduced diarrhea. The patient with Crohn's
disease reported complete resolution of chronic abdominal pain as
well as post prandial bloating and nausea, and gained 22 lbs. In
addition, the patient with vagotomy and antrectomy reported
resolution of postprandial dumping syndrome (flushing, nausea,
light-headedness).
[0269] The effect of an active lipid on transit time was determined
in 8 normal human subjects (1 male and 7 females with a mean age of
35.+-.2.6 years [SE]) and 45 patients (20 males and 25 females with
a mean age of 49.1.+-.2.5 [SE], age range from 18 to 90 years) with
chronic diarrhea (i.e., continuous diarrhea for more than two
months) associated with a wide variety of diagnoses and conditions
(e.g., Crohn=s disease; irritable bowel syndrome; short bowel
syndrome; Indiana pouch; AIDS; ulcerative colitis; vagotomy;
antrectomy; ileostomy; partial and complete colectomy; colon
cancer; diabetes mellitus type 1; pancreatic insufficiency;
radiation enteropathy; esophagectomy/gastric pull-up; total and
subtotal gastrectomy; gastrojejunostomy), made by referring
gastroenterologists. The method was the same as described above,
except oleic acid (Penta Manufacturing, Livingston, N.J.) replaced
sodium oleate in 50 mL of Ensure emulsion. All subjects refrained
from taking antibiotics for at least two weeks before each testing
date and during stool measurement periods. Patients were also
instructed to refrain from anti-diarrheal drugs, laxatives,
somatostatin analogues or anticholinergics for at least 48 hours
before each test. In both the normal and patient groups, there was
a significant slowing of upper gut transit time in response to
oleic acid, as summarized in Table 5 below (p<0.001).
TABLE-US-00006 TABLE 5 Effect of Oleic Acid on upper gut transit
time. Transit time (min) (mean .+-. SE) Oleic Acid (g) 0 1.6 3.2
Normal 105.2 .+-. 12.1 116 .+-. 11.1 140 .+-. 11.5 Patients 29.3
.+-. 2.8 57.2 .+-. 4.5 83.3 .+-. 5.2
[0270] Continuing oleic acid treatment at home was offered to
Aresponders.apprxeq. (i.e., patients who experienced a greater than
100% increase in baseline transit time with 3.2 g oleic acid). Of
the 36 responders out of the original 45 patients, 18 provided
records of stool volume and frequency on- and off-treatment for
comparison. The inconvenient and unappealing nature of stool
collection and measurement were the primary reasons reported by
responders who chose not to participate in stool collection. After
completing a set of three preliminary breath hydrogen tests, each
participating responder was asked to refrain from taking oleic acid
for two days in order to measure off-treatment stool output for a
24-hour period. Patients were issued a stool pattern record form
and a stool collection container with graduated volume markings to
record the frequency and volume of bowel movements. After two days
without oleic acid, each patient took 3.2 g of oleic acid mixed
with 25 mL of Ensure emulsion three times a day, 30 minutes before
breakfast, lunch and dinner. After taking oleic acid for two days,
patients recorded stool output for another 24-hour period. With
this oleic acid emulsion treatment, stool frequency decreased from
6.9.+-.0.8 to 5.4.+-.0.9 bowel movements per 24-hour period
(p<0.05), and stool volume decreased from 1829.0.+-.368.6 to
1322.5.+-.256.9 per 24-hour period (p<0.05). A slight and
transient burning sensation in the mouth or throat was the only
adverse effect reported by any patient taking the oleic acid
treatment.
[0271] These experiments demonstrate that active lipids, such as
oleate and oleic acid, are effective in slowing upper gut transit
in a dose-dependent manner, thus enabling longer residence time for
food in the upper gut and a concomitant greater nutrient absorption
there.
Fat in Distal Gut Inhibits Intestinal Transit More Potently than
Fat in Proximal Gut.
[0272] In 4 dogs equipped with duodenal (10 cm from pylorus) and
mid-gut (160 cm from pylorus) fistulas, as described hereinbelow
(Example 14), intestinal transit was compared across an isolated
150 cm test segment (between fistulas) while 0, 15, 30 or 60 mM
oleate was delivered into either the proximal or distal segment of
the gut as a solution of mixed micelles in pH 7.0 phosphate buffer
at 2 mL/min for 90 minutes. The segment of gut not receiving oleate
was perfused with phosphate buffer, pH 7.0, at 2 mL/min. 60 minutes
after the start of the perfusion, -20 .mu.Ci of .sup.99mTc-DTPA
(diethylenetriaminepentaacetic acid) was delivered as a bolus into
the test segment. Intestinal transit was then measured by counting
the radioactivity of 1 ml samples collected every 5 minutes from
the diverted output of the mid-gut fistula.
[0273] Intestinal transit was calculated by determining the area
under the curve (AUC) of the cumulative percent recovery of the
radioactive marker. The square root values of the AUC (Sqrt AUC),
where 0=no recovery by 30 minutes and 47.4=theoretical,
instantaneous complete recovery by time 0, were compared across
region of fat exposure and oleate dose using 2-way repeated
measures ANOVA (see Table 6 below).
TABLE-US-00007 TABLE 6 Effect of Oleate and oleic acid on
intestinal transit. Region of Oleate dose (mM) (mean .+-. SE) fat
exposure 15 30 60 Proximal 1/2 of gut 41.6 .+-. 1.4 40.6 .+-. 10.2
34.4 .+-. 3.0 Distal 1/2 of gut 25.6 .+-. 1.4 18.9 .+-. 1.5 7.0
.+-. 3.8 Control: buffer into both proximal and distal 1/2 of gut =
41.4 .+-. 4.6.
[0274] These experiments demonstrate that intestinal transit is
slower when fat is exposed in the distal 1/2 of gut (region effect
p<0.01). These experiments also demonstrate that oleate is
effective to inhibit intestinal transit in a dose-dependent fashion
(dose effect, p<0.05); and that dose dependent inhibition of
intestinal transit by oleate depends on the region of exposure
(interaction between region and dose, p<0.01).
Case Study Showing Successful Treatment of Diarrhea-Predominant
Irritable Bowel Syndrome with Oleic Acid.
[0275] The patient was a 39-year old male with a history of
adolescent-onset, persistent diarrhea. After a routine
gastrointestinal work-up failed to provide an explanation for his
symptoms, he was given the diagnosis of diarrhea-predominant
irritable bowel syndrome. He presented with complaints of excessive
gas, postprandial bloating, diarrhea and urgency, and 3 to 7 liquid
bowel movements per day. His upper gut transit times were (min) 30
(0 g oleic acid), 117 (1.6 g oleic acid) and 101 (3.2 g oleic
acid). With continuing oleic acid treatment as described above, he
reported his bowel frequency reduced to a single, solid bowel
movement per day. He also reported complete relief from the
symptoms of gaseousness, bloating and rectal urgency.
Relatively Rapid Basal Upper Gut Transit in Patients with
Inflammatory Bowel Disease (IBD).
[0276] The mean upper gut transit time for IBD patients (n=18) at 0
grams of oleic acid was 79.1.+-.11.0 min., compared to 118.7.+-.9.8
min for normal subjects (n=5)(p=0.04, t-test).
Active Lipid Increases Upper Gut Transit Time.
[0277] The mean transit time for normal subjects (n=5) at 0 grams
of oleic acid was 118.7.+-.9.8 min, at 4 grams of Oleic acid was
136.0.+-.15.4 min. (P<0.05, t-test). The mean AUC for normal
subjects at 0 grams of oleic acid was 1438.9.+-.208.5; at 4 grams
of oleic acid it was 1873.3.+-.330.5 (p<0.05, t-test). The mean
transit time for IBD patients (n=18) at 0 grams of oleic acid was
79.1.+-.11.0 min; at 4 grams of oleic acid it was 114.6.+-.16.0
min. (p<0.05, t-test). The mean AUC for IBD patients at 0 grams
of oleic acid was 687.3.+-.98.2; at 4 grams of oleic acid it was
1244.9.+-.250.4. (p<0.05, t-test).
[0278] These data show that oleic acid slowed gut transit time and
thus substantially increased the opportunity for absorption of food
nutrients in the upper gut region in both normal and IBD groups.
Thus, the in individuals having SIBO a condition, treatnment in
accordance with the method of deprives the bacteria of much of the
nutrient supply required for growth.
Example 13
Eradication of SIBO in Subjects with Irritable Bowel Syndrome
Lowers their Serum Levels of 5-HT
[0279] Previous studies have shown that patients with irritable
bowel syndrome (IBS) have elevated plasma 5-hydroxytryptamine
(5-HT) levels. Since it was shown hereinabove that IBS is
associated with small intestinal bacterial overgrowth (SIBO) and
symptoms of IBS are reduced by antibiotic eradication of SIBO, the
hypothesis was tested that eradication of SIBO will reduce plasma
5-HT levels in IBS patients to provide further evidence of the
relationship between IBS and SIBO.
[0280] The plasma 5-HT levels of 7 human subjects diagnosed with
IBS were compared before and after successful eradication of SIBO,
as part of a double blind placebo controlled trial. A lactulose
breath hydrogen test (LBHT) was performed to diagnose SIBO at
baseline and when eradication was achieved. Fasting blood samples
were taken at baseline and on the day that eradication of SIBO was
confirmed. The plasma 5-HT level (ng/mL) was determined in each
sample by ELISA (Kit-Research Diagnostics Inc., Flanders, N.J.). A
paired t-test was performed to compare 5HT levels (mean.+-.SE)
before and after eradication of SIBO.
[0281] The results indicated that the amount of plasma 5-HT was
reduced from 0.7.+-.0.4 ng/mL before eradication to 0.5.+-.0.5
ng/mL after eradication of SIBO in the subjects (p<0.05). Thus,
eradication of SIBO in IBS subjects decreases fasting plasma 5-HT
levels, which provides further evidence for the relationship
between IBS and SIBO.
Example 14
Neural Regulation of the Rate of Upper Gastrointestinal Transit
[0282] The experiments described below are based on a previously
described chronic multi-fistulated dog model, employing surgically
fistulated male or female mongrel dogs weighing about 25 kg each.
(Lin, H. C. et al., Inhibition of gastric emptying by glucose
depends on length of intestine exposed to nutrient, Am. J. Physiol.
256:G404-G411 [1989]). The small intestines of the dogs were each
about 300 cm long from the pylorus to the ileal-cecal valve. The
duodenal fistula was situated 15 cm from the pylorus; the mid-gut
fistula was situated 160 cm from the pylorus. Occluding Foley
catheters (balloon catheters that are inflated to produce a
water-tight seal with the lumenal surface) were placed into the
distal limb of a duodenal fistula and a mid-gut fistula, fat or
other test agents were administered lumenally to the thus
compartmentalized Aproximal.apprxeq.section of the gut, i.e.,
between the fistulas, or to the compartmentalized
Adistal.apprxeq.section of the gut, i.e., beyond the mid-gut
fistula. Perfusate was pumped into a test section through the
catheter at a rate of 2 mL/minute. Test agents were administered
along with buffer perfusate, but some test agents were administered
intravenously, where specifically noted.
[0283] Intestinal transit measurements were made by tracking the
movement of a liquid marker across the approximately 150 cm
intestinal test segment by delivering about 20 .mu.Ci .sup.99mTc
chelated to diethyltriamine pentaacetic acid (DTPA)(Cunningham, K.
M. et al., Use of technicium-99m (V)thiocyanate to measure gastric
emptying of fat, J. Nucl. Med. 32:878-881 [1991]) as a bolus into
the test segment after 60 minutes of a 90-minute perfusion. The
output from the mid-gut fistula was collected every 5 min
thereafter for 30 minutes, which period is illustrated in FIGS.
9-23. Using a matched dose of .sup.99mTc to represent the original
radioactivity (Johansson, C., Studies of gastrointestinal
interactions, Scand. J. Gastroenterol. 9(Suppl 28):1-60 [1974];
Zierler, K., A simplified explanation of the theory of indicator
dilution for measurement of fluid flow and volume and other
distributive phenomena, Bull. John Hopkins 103:199-217 [1958]), the
radioactivity delivered into the animal as well as the
radioactivity of the recovered fistula output were all measured
using a gamma well counter. After correcting all counts to time
zero, intestinal transit was calculated as the cumulative percent
recovery of the delivered .sup.99mTc-DTPA. This method has been
well validated over the years and appreciated for its advantage of
minimal inadvertent marker loss. To demonstrate this point, we
perfused phosphate buffer, pH 7.0, through the proximal gut and
followed the cumulative recovery of this marker (% recovery) over
time (n=1). There was a very high level of marker recovery, with
90% of the marker recovered by 30 minutes and 98% of the marker
recovered by 45 minutes.
(1) Slowing of Intestinal Transit by PYY Depends on
Ondansetron-Sensitive 5-HT-Mediated Pathway.
[0284] Peptide YY (PYY) slows transit and is a signal for lumenal
fat (Lin, H. C. et al., Fat-induced ileal brake in the dog depends
on peptide YY, Gastroenterol. 110(5):1491-95 [1996b]; Lin, H. C. et
al., Slowing of intestinal transit by fat in proximal gut depends
on peptide YY, Neurogastroenterol. Motility 10:82 [1998]). Since
serotonin (5-HT) can also be a signal for fat (Brown, N. J. et al.,
The effect of a 5HT3 antagonist on the ileal brake mechanism in the
rat, J. Pharmacol. 43:517-19 [1991]; Brown, N. J. et al. [1993]),
the hypothesis was tested that the slowing of transit by PYY can
depend on a 5-HT-mediated pathway by comparing the rate of marker
transit during the administration of PYY in the presence or absence
of ondansetron (Ond; a 5-HT receptor antagonist) in the proximal
versus distal gut (n=2 for each treatment).
[0285] Normal saline (0.15 M NaCl) or PYY (0.8 .mu.g/kg/h) was
administered intravenously over a 90 minute period, while phosphate
buffer, pH 7.0, was perfused into the lumen of the proximal gut
through the duodenal fistula at a rate of 2 mL/min for the 90
minutes and was recovered from the output of the mid-gut fistula.
The results are summarized in FIG. 9. Transit was slowed by
intravenous PYY, with recovery of the marker decreased from
75.1.+-.3.6% (control: IV normal saline [NS]+lumenal normal saline,
i.e., NS-NS in FIG. 9) to 17.1.+-.11.0% (IV PYY+lumenal normal
saline, i.e., PYY-NS in FIG. 9). This effect was abolished by
adding the specific 5-HT3 receptor antagonist ondansetron (0.7
mg/kg/h) to the buffer introduced into the proximal gut so that
recovery increased to 78.3.+-.4.8% (IV PYY+lumenal Ond proximal,
i.e., PYY-Ond in prox in FIG. 9) but not by ondansetron in the
distal gut, which decreased recovery to 12.9.+-.12.9% (IV PYY+Ond
in Distal, i.e., PYY-Ond in Dist). These results imply that slowing
of transit by PYY depended on a 5-HT-mediated pathway located in
the segment of the small intestine where transit was measured.
(2) the Fat Induced Jejunal Brake Depends on an
Ondansetron-Sensitive Serotonin (5-HT)-Mediated Pathway.
[0286] The hypothesis was tested that slowing of transit by fat
depends on a serotonergic pathway by comparing intestinal transit
during perfusion with buffer or oleate in the presence or absence
of ondansetron, a 5-HT3 receptor antagonist, in the proximal gut
(n=3 each treatment). Buffer or 60 mM oleate was perfused through
the duodenal fistula into the lumen of the proximal gut for a
90-minute period, in the manner described in Example 14(1), along
with a bolus of normal saline.+-.ondansetron (0.7 mg/kg) at the
start of transit measurement. The rate of intestinal transit was
slowed by the presence of oleate (p<0.05) in an
ondansetron-sensitive manner. (p<0.05). The results are
summarized in FIG. 10.
[0287] Specifically, ondansetron increased recovery of marker in
the perfusate from 41.6.+-.4.6% (mean.+-.SE) (lumenal
oleate+lumenal normal saline, i.e., Oleate-NS in FIG. 10) to
73.7.+-.10.6% (lumenal oleate+lumenal ondansetron, i.e., Oleate-Ond
in FIG. 10) during oleate perfusion but decreased recovery from
96.0.+-.4.0% (lumenal phosphate buffer+lumenal normal saline, i.e.,
Buffer-NS in FIG. 10) to 57.9.+-.15.9% (lumenal buffer+lumenal
ondansetron, i.e., Buffer-Ond in FIG. 10) during buffer perfusion.
These results imply that slowing of intestinal transit by the
fat-induced jejunal brake and the acceleration of intestinal
transit by buffer distension both depended on an
ondansetron-sensitve 5-HT3-mediated pathway.
(3) the Fat-Induced Ileal Brake Depends on an
Ondansetron-Sensitive, Efferent Serotonin (5-HT)-Mediated
Pathway.
[0288] The fistulated dog model allows for the ileal brake (oleate
in distal gut, buffer in proximal gut) to be separated into the
afferent (distal) vs. efferent (proximal) limb of the response.
Since 5-HT3 receptors are found on extrinsic primary sensory
neurons (afferent limb) and on intrinsic 5-HT neurons of the
myenteric plexus (5-HT interneuron)(efferent limb), the
identification of the location of the 5-HT3 pathway (afferent vs.
efferent limb) can localize the serotonergic pathway responsible
for the slowing of transit by fat in the distal gut (ileal brake).
Using occluding Foley catheters, the small intestine was
compartmentalized into the proximal gut and the distal gut as
described hereinabove. Intestinal transit was measured across the
proximal gut (between fistulas) as described hereinabove. By
perfusing buffer through the proximal gut while fat was perfused
through the distal gut to trigger the fat-induced ileal brake, the
distal gut represented the afferent limb of the response and the
proximal gut represented the efferent limb of the response. To test
for the location of the serotonergic pathway, 5-HT3 receptor
antagonist ondansetron was then mixed with the appropriate
perfusate and adminstered into either the proximal or distal gut.
Control=buffer in proximal and distal gut. Four dogs were
tested.
[0289] Delivering ondansetron lumenally into either the proximal or
distal gut, intestinal transit was slowed by the ileal brake
(76.3.+-.3.1% [Control in FIG. 11] vs. 22.9.+-.3.8% [Ileal Brake in
FIG. 11]; p<0.005). But the ileal brake was abolished by
ondansetron delivered to the proximal gut (68.5.+-.2.7%; Ond in
Prox in FIG. 11; n=4) but not distal gut (22.8.+-.2.6%; Ond in Dist
in FIG. 11; n=4).
[0290] Since ondansetron delivered with the fat in the distal gut
had no effect, but ondansetron delivered with the buffer in the
proximal gut abolished the ileal brake, the slowing of intestinal
transit by fat in the distal gut depended on an
ondansetron-sensitive, serotonergic pathway located on the efferent
rather than afferent limb of the response. And since ondansetron
abolished the jejunal brake in Example 14(2) when delivered with
fat and abolished the ileal brake in Example 14(3) when delivered
with buffer, this region-specific result cannot be explained by
inactivation of drug by fat, differences in permeability or
absorption.
(4) Ondansetron Abolishes the Fat-Induced Ileal Brake in a
Dose-Dependent Manner.
[0291] The fat-induced ileal brake was abolished by the 5-HT
receptor antagonist ondansetron in a dose-dependent manner.
Perfusion of buffer was through both the duodenal and mid-gut
fistulas (2 mL/min over 90 minutes); the buffer administered to the
mid-gut fistula contained buffered normal saline (pH=7.0; Buffer
Control in FIG. 12) or 60 mM oleate to induce the ileal brake
response (Ileal Brake in FIG. 12). During the ileal brake response,
ondansetron was added at t.sub.o as a single bolus in the following
doses (mg): 6.25; 12.5; and 25. Results are shown in FIG. 12.
[0292] Oleate induced the ileal brake (24. 1% marker recovery
[Ileal brake in FIG. 12] vs. 81.2% marker recovery for the Buffer
Control). The ileal brake was abolished by ondansetron delivered
into the proximal gut in a dose-dependent manner (35.4% marker
recovery at 6.25 mg ondansetron, 55.8% marker recovery at 12.5 mg
ondansetron, and 77.6% marker recovery at 25 mg ondansetron).
(5) Fat in the Distal Gut Causes the Release of 5-HT from the
Proximal Gut.
[0293] To test the hypothesis that fat in the distal gut causes the
release of 5-HT in the proximal gut, the amount of 5-HT collected
from the output of the mid-gut fistula (proximal gut 5-HT) over a
90-minute period of buffer perfusion through both the duodenal and
mid-gut fistulas (2 mL/min); buffer (control) or oleate (60 mM) was
administered to the distal gut (n=1). The amount of 5-HT was
determined using an ELISA kit specific for 5-HT (Sigma;
Graham-Smith, D. G., The carcinoid syndrome, In: Topics in
Gastroenterology, Truclove, S. C. and Lee, E. (eds.), Blackwells,
London, p. 275 [1977]; Singh, S. M. et al., Concentrations of
serotonin in plasma--a test for appendicitis?, Clin. Chem.
34:2572-2574 [1988]). The amount of 5-HT released by the proximal
gut increased in response to fat in the distal gut from 100 .mu.g
in the control (buffer minus oleate) to 338 .mu.g (buffer plus
oleate to distal gut), showing that 5-HT is released in the
proximal gut in response to fat in the distal gut. Thus, the
release of 5-HT by the proximal gut can serve as a relayed signal
for fat in the distal gut. The relayed release of 5-HT in the
proximal gut in response to fat in the distal gut is consistent
with Example 14(2), showing that slowing of intestinal transit by
fat depends on an efferent 5-HT-mediated pathway to the proximal
gut.
(6) Ondansetron Abolishes the Fat-Induced Ileal Brake when
Administered Lumenally but not Intravenously.
[0294] To confirm that the reversal of the slowing of transit by
ondansetron was peripheral, i.e., enteric, rather than systemic,
the effect of ondansetron was compared when delivered luminally
(through the duodenal fistula into the proximal gut) versus
intravenously. Ondansetron was either delivered lumenally into the
proximal gut (0.7 mg/kg/h; Ond in prox in FIG. 13) or administered
intravenously (0.15 mg/kg/1.5h; iv Ond in FIG. 13) during
fat-induced ileal brake (60 mM oleate input through the mid-gut
fistula into the distal gut as described above). Two dogs were
tested (n=2).
[0295] Results are shown in FIG. 13. Compared to the ileal brake
(20.+-.1.8% marker recovery), the marker recovery increased to
78.+-.2.4% with lumenal ondansetron (p<0.005). Intravenous
ondansetron had no substantial effect on the ileal brake
(13.+-.2.0% marker recovery). These results imply that the 5-HT3
receptor antagonist worked enterically rather than
systemically.
(7) the Slowing of Intestinal Transit by Distal Gut 5-HT Depends on
an Ondansetron-Sensitive 5-HT3-Mediated Pathway in the Proximal Gut
(Efferent) and Distal Gut (Afferent).
[0296] To test the hypothesis that lumenal 5-HT may slow intestinal
transit via 5-HT3 receptors similar to fat, 0.7 mg/kg ondansetron,
a 5-HT3 receptor antagonist or buffered saline (pH 7.0) was
delivered into either the proximal or distal gut as a bolus at the
start of the transit measurement. Four dogs were tested.
[0297] Results are shown in FIG. 14. The slowing of intestinal
transit by 5-HT (0.1 mg/kg/h) administered to the distal gut
(35.2.+-.2.2% marker recovery) (vs. 76.1.+-.4.7% marker recovery
for buffer control) was abolished by ondansetron added to the
proximal or distal gut as shown by % marker recovery of
73.8.+-.9.5% (Ond-Prox in FIG. 14) vs. 79.5.+-.2.4% (Ond-Dist in
FIG. 14), respectively (p<0.001).
[0298] This shows that in the conscious whole animal, the slowing
of intestinal transit by luminal 5-HT depended on an
ondansetron-sensitive serotonergic pathway located on both the
afferent and efferent limb of the intestino-intestinal reflex. (See
also, Brown, N. J. et al., Granisetron and ondansetron: effects on
the ileal brake mechanism in the rat, J. Pharm. Pharmacol.
45(6):521-24 [1993]). In contrast, the slowing of intestinal
transit by distal gut fat (Example 14[3]) depended on a 5-HT3
pathway localized specifically on the efferent limb to suggest that
5-HT is not the stimulus for the afferent limb of the fat-induced
ileal brake, but rather involves a signal other than 5-HT, such as
PYY. However, 5-HT is the stimulus for the afferent limb of the
slowing of intestinal transit by 5-HT in the distal gut.
(8a) 5-HT in the Distal Gut Slows Intestinal Transit in a
Dose-Dependent Manner.
[0299] In a preliminary experiment, intestinal transit during
buffer perfusion of both the proximal and distal guts (81.2%
recovery) was slowed by 5-HT in distal gut so that marker recovery
decreased to 73.8% at 2 mg 5-HT (0.033 mg 5-HT/kg/h), 53.1% at 3 mg
(0.05 mg 5-HT/kg/h) and 11.6% at 4 mg (0.066 mg 5-HT/kg/h) dose
over a 90 minute period (n=1).
[0300] The dose-dependent effect of 5-HT in slowing intestinal
transit was confirmed in an additional experiment. The cumulative %
recovery of the radioactive marker was reduced in a dose-dependent
fashion as the 5-HT perfusion increased from 0 to 0.1 mg/kg/h to
suggest that intestinal transit is slowed by lumenal 5-HT. However,
the speed of transit was markedly accelerated when the 5-HT dose
was increased to 0.3 mg/kg/h. (Table 7).
TABLE-US-00008 TABLE 7 Effect of 5-HT delivered to distal gut on
intestinal transit time (min) in multi-fistulated dogs (n = 2
dogs). 5-HT dose (mg/kg/h .times. 90 min) 0 0.033 0.05 0.066 0.1
0.3 68.5 .+-. 1.0 69.6 .+-. 4.2 33.5 .+-. 1.5 15.2 .+-. 0.5 16.1
.+-. 4.9 73.8 .+-. 0.6
(8b) Lumenal 5-HT, Delivered to the Proximal Gut, Slows Intestinal
Transit in a Dose-Dependent Fashion in the Conscious Whole Animal
Model.
[0301] In in-vitro models, lumenal 5-HT applied to an isolated
bowel loop accelerated transit by triggering the peristaltic
reflex. In contrast, in the conscious whole animal model applied
herein (with extrinsic nerves intact), 5-HT applied lumenally
slowed intestinal transit (Example 14[8a] above). In further
experiments, 5-HT was delivered at a rate of 0, 0.033, 0.066, 0.05
and 0.1 mg/kg/h into the proximal gut. Four dogs were tested.
[0302] Results are shown in FIG. 15. Intestinal transit was
significantly slowed by 5-HT in the proximal gut in a
dose-dependent fashion (p<0.00001). Marker recovery during
buffer perfusion was 75.0.+-.4.4% while at the dose of 0.066
mg/kg/h marker recovery was reduced to 16.9.+-.+3.7%, and was not
significantly different from the dose of 0.1 mg/kg/h. At the
intermediate dose of 0.05 mg/kg/h, marker recovery was
33.2.+-.14.0%; buffer vs 0.05 mg/kg/h; p<0.005) and at the
lowest dose of 0.033 mg/kg/h, marker recovery was not significantly
different from the buffer control.
(8c) Slowing of Intestinal Transit by 5-HT is not Dependent on
Volume of the Output of the Midgut Fistula.
[0303] 5-HT stimulates small bowel and colonic secretion. We have
observed a slowing effect of 5-HT on intestinal transit (Example
14[8a-b]). As a control, to determine whether intestinal transit
correlates with volume of the output of the midgut fistula. Varying
doses of 5-HT (0, 0.033, 0.1, 0.3 mg/kg/h) were perfused into the
proximal gut, .sup.99mTc was delivered into the test segment as a
bolus for transit measurement. The volume of the output of the
midgut fistula was collected during the last 30 minutes of the 90
min perfusion experiment (n=21). Transit was plotted against output
volume. There was no correlation between transit during 5-HT
treatment and the volume of the output of the midgut fistula (data
not shown).
[0304] Therefore, the observed transit effect of 5-HT cannot be
explained solely on the basis of volume effect related to 5-HT
induced intestinal secretion. The observed transit effect of 5-HT
must depend on transit-specific regulation.
[0305] Together, the results in Example 14(8) and show that,
contrary to the effect of 5-HT in an in-vitro model, lumenally
administered 5-HT slows intestinal transit in a dose-dependent
fashion in the conscious whole animal model, which implies that the
slowing of intestinal transit depends on extrinsic nerves.
(9a) 5-HT in the Distal Gut Causes Release of 5-HT in the Proximal
Gut.
[0306] To test the hypothesis that 5-HT in the distal gut causes
the release of 5-HT in the proximal gut, the amount of 5-HT
collected from the output at the mid-gut fistula (Proximal gut
5-HT) over a 90-minute period of buffer perfusion through both the
duodenal and mid-gut fistulas (2 mL/min each) was compared in the
presence or absence of 5-HT (0.05 mg/kg/h) administered to the
distal gut (n=1). 5-HT concentration was determined using an ELISA
kit specific for 5-HT (Sigma). The amount of 5-HT released by the
proximal gut increased from 156 .mu.g in the control (minus distal
5-HT) to 450 .mu.g (plus 5-HT to distal gut), implying that 5-HT is
released by the proximal gut in response to 5-HT in the distal gut.
Thus, the release of 5-HT by the proximal gut can serve as a
relayed signal for distal gut 5-HT. This relayed release of 5-HT in
the proximal gut explains the results of Example 14(6) showing that
the slowing of intestinal transit by distal gut 5-HT was abolished
by ondansetron in the proximal gut (efferent limb of response) as
well as in the distal gut (afferent limb of response).
(9b) Fat in Distal Gut Releases 5-HT from Proximal Gut.
[0307] To test the hypothesis that the proximal gut releases 5-HT
in response to lipid in the distal gut, we compared the amount of
5-HT in the output of the midgut fistula (i.e., proximal gut 5-HT)
with buffered saline (control) or oleate in the distal gut. The
amount of 5-HT collected over 90 min was measured using a
5-HT-specific ELISA test kit, as described herein above. Four dogs
were tested.
[0308] The amount of proximal gut 5-HT increased from 82.7.+-.20.53
ng to 211.75.+-.35.44 ng (p<0.005) when the distal gut perfusate
was switched from buffer to oleate, implying that 5-HT is released
from the proximal gut in response to fat in the distal gut, as a
relayed signal for fat.
[0309] Fat is also a chemical trigger for the release of 5-HT, thus
these results are consistent with the release of 5-HT via a long
distance, intestino-intestinal communications, or reflex.
(9c) Luminal 5-HT Slows Intestinal Transit Via Activation of the
Intestino-Intestinal Reflex.
[0310] To confirm that 5-HT, delivered lumenally, slowed intestinal
transit via the activation of an intestino-intestinal reflex, we
compared intestinal transit across the proximal one-half of gut
while 0 (pH 7.0 buffered saline control) or 0.1 mg/kg/h of 5-HT was
delivered into either the proximal or distal one-half of the gut.
Four dogs were tested.
[0311] Results are shown in FIG. 16. Intestinal transit across the
proximal gut was slowed by 5-HT in either the proximal or distal
gut, demonstrated by the marker recovery decreasing from
85.0.+-.7.3% (Saline-Prox in FIG. 16)(p<0.005) to 20.1.+-.4.5%
for proximal gut 5-HT (5-HT-Prox in FIG. 16) and 76.1.+-.1.3%
(Saline-Dist in FIG. 16) to 35.2.+-.2.3% (5-HT-Dist in FIG. 16)
(p<0.005) for distal gut 5-HT.
[0312] These results imply that the slowing of intestinal transit
by 5-HT depends on a long-distance, region-to-region reflex, since
5-HT administered into the distal gut slowed intestinal transit
through the physically separate proximal gut.
(10) Intravenous PYY Causes Release of 5-HT in the Proximal
Gut.
[0313] The amount of 5-HT released from the proximal gut in
response to intravenous PYY or buffered saline (Control) during
buffer perfusion (2 mL/min over 90 minutes) through both the
duodenal and mid-gut fistulas was measured to test the hypothesis
that intravenous PYY (0.8 mg/kg/h) causes the release of 5-HT in
the proximal gut. 5-HT was measured as in Example 14(9) above. The
amount of 5-HT released by the proximal gut increased from 140.1
.mu.g (Control) to 463.1 .mu.g in response to intravenous PYY.
[0314] This result was comparable with the response when 60 mM
oleate was administered to the distal gut (buffer only to the
proximal gut) during the perfusion without intravenous PYY (509.8
.mu.g of 5-HT; n=1), which implies that the release of 5-HT in the
proximal gut stimulated by fat in the distal gut can be mediated by
PYY.
(11) Slowing of Intestinal Transit by Fat in the Distal Gut Depends
on an Extrinsic Adrenergic Neural Pathway.
[0315] A distension-induced intestino-intestinal inhibitory neural
reflex projects through the celiac prevertebral celiac ganglion via
a cholinergic afferent and an adrenergic efferent (Szurszewski, J.
H. and King, B. H., Physiology of prevertebral ganglia in mammals
with special reference to interior mesenteric ganglion, In:
Handbook of Physiology: The Gastrointestinal System, Schultz, S. G.
et al. (eds.), American Physiological Society, distributed by
Oxford University Press, pp. 519-592 [1989]). Intestinal transit
was measured during fat perfusion of the distal small intestine in
the presence or absence of intravenous propranolol (50 .mu.g/kg/h;
n=2 dogs), a .beta.-adrenoceptor antagonist, to test the hypothesis
that the slowing of intestinal transit by fat in the distal gut
also depends on an adrenergic pathway. Perfusion of buffer was
through both the duodenal and mid-gut fistulas (2 mL/min over 90
minutes); the buffer administered to the mid-gut fistula contained
60 mM oleate. The results are illustrated in FIG. 17.
[0316] Intestinal transit was slowed by distal gut fat
(79.7.+-.5.8% marker recovery [Buffer Control in FIG. 17] compared
to 25.8.+-.5.2% recovery with fat perfusion into the distal gut
[Oleate-NS in FIG. 17]). Intravenous propranolol abolished this
jejunal brake effect so that recovery increased to 72.1.+-.4.7%
(oleate+propanolol, i.e., Oleate-Prop in FIG. 17), implying that
the slowing of transit by fat in the distal gut depends on a
propranolol-sensitive, adrenergic pathway. This result supports the
hypothesis that the response to fat involves an adrenergic
efferent, such as the extrinsic nerves projecting through the
prevertebral ganglia.
(12) Slowing of Intestinal Transit by PYY Depends on an Extrinsic
Adrenergic Neural Pathway.
[0317] Intestinal transit during buffer perfusion of the proximal
and distal small intestine in the presence or absence of
intravenous propranolol (50 .mu.g/kg/h; n=2) was measured, to test
the hypothesis that the slowing of intestinal transit by PYY (a fat
signal) also depends on an adrenergic pathway. Perfusion was
through both fistulas as described in Example 14(11) except that
oleate was not administered to the distal gut, and, instead, 30
.mu.g PYY (0.8 mg/kg/h) was administered intravenously during the
90 minute perfusion period. The results are summarized in FIG.
18.
[0318] Slowing of intestinal transit by PYY (78.1.+-.2.2% marker
recovery minus PYY [Buffer Control in FIG. 18] vs. 11.8.+-.5.4%
recovery with intravenous PYY [PYY-NS in FIG. 18]) was abolished by
intravenous propranolol. In the presence of propanolol, marker
recovery increased to 66.3.+-.3.1% (PYY-Prop in FIG. 18). This
result, consistent with the results of Example 14(11), implies that
the slowing of transit by PYY depends on a propranolol-sensitive,
adrenergic pathway, which supports the hypothesis that the response
to PYY involves an adrenergic efferent such as the extrinsic nerves
projecting through the prevertebral ganglia.
(13) Slowing of Intestinal Transit by 5-HT in the Distal Gut
Depends on a Propranolol-Sensitive Extrinsic Adrenergic Neural
Pathway.
[0319] Intestinal transit during buffer perfusion of the proximal
and distal small intestine in the presence or absence of
intravenous propranolol (50 .mu.g/kg/h; n=2) was measured, to test
the hypothesis that the slowing of intestinal transit by 5-HT in
the distal gut also depends on an adrenergic pathway. Buffer
perfusion was through both fistulas as described in Example 14(12)
except that 5-HT (0.05 mg/kg/h) was administered to the distal gut
during the 90 minute perfusion period. The results are summarized
in FIG. 19.
[0320] Slowing of intestinal transit by 5-HT (83.3.+-.3.3% marker
recovery minus 5-HT [Buffer Control in FIG. 19] vs. 36.1.+-.2.3%
recovery with administration of 5-HT to the distal gut [5-HT-NS in
FIG. 19]) was abolished by intravenous propranolol. In the presence
of propanolol, marker recovery increased to 77.7.+-.7.6% (5-HT-Prop
in FIG. 19). This result implies that the slowing of transit by
5-HT depends on a propranolol-sensitive, extrinsic adrenergic
pathway, perhaps similar to that responsible for the response to
distal gut fat.
[0321] Enterochromaffin cells of the intestinal mucosa and
myenteric 5-HT neurons are innervated by adrenergic nerves.
(Gershon M D, Sherman D L., Noradrenergic innervation of
serotoninergic neurons in the myenteric plexus, J Comp Neurol. 1987
May 8; 259(2): 193-210 [1987]). To test the hypothesis that the
slowing of intestinal transit by distal gut fat (ileal brake) and
5-HT depended on an adrenergic pathway, five dogs were equipped
with duodenal (10 cm from the pylorus) and midgut (160 cm from the
pylorus) fistulas as described above. Using occluding Foley
catheters, the small intestine was compartmentalized into the
proximal (between fistulas) and distal (beyond midgut fistula)
one-half of gut. Buffer (pH 7.0) was perfused into the proximal gut
while 60 mM oleate was perfused into the distal gut at 2 ml/min for
90 min. Intestinal transit across the proximal gut was compared
during intravenous administration of 50 .mu.g/kg/h propranolol or
saline. In addition, the effect was also determined of 5-HT
administered at 0.1 mg/kg/h on intestinal transit with and without
i.v. propranolol. Intestinal transit (mean.+-.SE) was measured by
.sup.99mTc-DTPA marker recovery in the output of the midgut fistula
during the last 30 min of the 90 min experiment. The cumulative %
marker recovered was compared using ANOVA and additional analyses
by paired t-test.
[0322] Results are shown in Table 8 below. Oleate (p<0.002) and
5-HT (p<0.005) perfused into the distal gut slowed transit
through the proximal gut as compared to buffer control. The slowing
of intestinal transit by distal gut fat or 5-HT was both abolished
by iv propranolol (p<0.01). These results provide further
evidence that the slowing of intestinal transit by distal gut fat
or 5-HT depends on an adrenergic efferent nerve.
TABLE-US-00009 TABLE 8 Effect of 5-HT and propranolol on proximal
intestinal transit. i.v. Agent Perfusate Saline (i.v.) Propranolol
(i.v.) Buffer Control 70.11 .+-. 6.51 -- Oleate (Ileal brake) 26.62
.+-. 5.36 66.42 .+-. 8.26 5-HT distal gut 28.27 .+-. 5.03 63.85
.+-. 8.76
(14) Intestinal Transit is Slowed by Norepinephrine in a
5-HT-Mediated Neural Pathway.
[0323] Intestinal transit during buffer perfusion of the proximal
and distal small intestine with intravenous norepinephrine (NE;
adrenergic agent) in the presence or absence of the 5-HT receptor
antagonist ondansetron was measured, to test the hypothesis that
the slowing of intestinal transit also depends on an adrenergic
efferent pathway. Perfusion of buffer was through both the duodenal
and mid-gut fistulas (2 mL/min over 90 minutes); norepinephrine
(0.12 .mu.g/kg/h) was administered intravenously during the 90
minute perfusion period; and normal saline with or without
ondansetron (0.7 mg/kg/h; n=2) was administered in the perfusate to
the proximal gut. The results are summarized in FIG. 20.
[0324] Intestinal transit was slowed by NE so that marker recovery
was reduced from 76.9% (Buffer Control in FIG. 20) to 13.3% (NE-NS
in FIG. 20). Ondansetron abolished this slowing effect with marker
recovery increased to 63.4% (NE-Ond in FIG. 20), to implies that NE
(adrenergic efferent) slows transit via a 5-HT-mediated pathway.
This result confirms that slowing of intestinal transit is mediated
by an adrenergic efferent projecting from the prevertebral ganglion
to the gut action on a 5-HT-mediated pathway.
[0325] To test the hypothesis that norepinephrine slows intestinal
transit via 5-HT3 receptors, buffer transit across the proximal gut
was compared during intravenous administration of norepinephrine
with and without lumenally-perfused ondansetron. Five dogs were
equipped with duodenal (10 cm from the pylorus) and midgut (160 cm
from the pylorus) fistulas as described above. Using occluding
Foley catheters, the small intestine was compartmentalized into the
proximal (between fistulas) and distal (beyond midgut fistula)
one-half of gut. Buffer (pH 7.0) was perfused into the proximal gut
at 2 ml/min for 90 min. Intestinal transit of buffer across the
proximal gut was compared during intravenous administration of 50
mg norepinephrine/30 ml/1.5 h with and without ondansetron perfused
lumenally (0.7 mg/kg/h). Intestinal transit (mean.+-.SE) was
measured by .sup.99mTc-DTPA marker recovery in the output of the
midgut fistula during the last 30 min of the 90 min experiment. The
cumulative % marker recovered was compared using ANOVA and
additional analyses by paired t-test.
[0326] Results are shown in Table 9 below. These results show that
both an adrenergic and serotonergic pathways are involved in the
slowing of intestinal transit.
TABLE-US-00010 TABLE 9 Effects of norepinephrine (NE) and
ondansetron (Ond) on proximal intestinal transit. Transit Across
Proximal Gut (Cumulative % Marker Recovered) Buffer Control 68.5
.+-. 5.0.sup.a Buffer + NE 16.3 .+-. 3.4.sup.ab Buffer + NE + Ond
63.0 .+-. 4.4.sup.b .sup.ap < 0.003 .sup.bP < 0.0009
(15) the Fat-Induced Jejunal Brake Depends on the Slowing Effect of
a Naloxone-Sensitive, Opioid Neural Pathway.
[0327] To test the hypothesis that the slowing of intestinal
transit depended on an opioid pathway, the proximal gut was
perfused (2 mL/minute for 90 minutes) with buffer containing 60 mM
oleate and 0 (normal saline), 3, 6, or 12 mg of naloxone mixed
therein, an opioid receptor antagonist. As shown in FIG. 21, the
fat-induced jejunal brake response depended on the dose of naloxone
mixed with the oleate (p<0.05, 1-way ANOVA)(n=7). Specifically,
marker recovery was 30.0.+-.3.6% with 0 mg naloxone, 41.0.+-.5.2%
with 3 mg naloxone, 62.8.+-.8.2% with 6 mg naloxone and
60.6.+-.6.1% with 12 mg naloxone. This result demostrates that
proximal gut fat slows intestinal transit via opioid pathway.
(16) the Effect of Naloxone was Specific for Fat-Triggered
Feedback.
[0328] Intestinal transit was compared during perfusion of the
proximal gut with buffer containing 0 (normal saline) or 6 mg
naloxone (n=3). The rate of intestinal transit was not
significantly affected by the opioid receptor antagonist naloxone
when fat was not present in the proximal gut. Marker recovery was
88.0.+-.1.3% with naloxone and 81.3.+-.6.1% without naloxone. This
implies that the accelerating effect of naloxone was specific for
reversing the jejunal brake effect of fat.
(17) the Fat-Induced Ileal Brake Depends on the Slowing Effect of
an Efferent, Naloxone-Sensitive, Opioid Neural Pathway.
[0329] The fistulated dog model allowed for the
compartmentalization of the afferent limb (distal gut) from
efferent limb (proximal gut) of the fat-induced ileal brake. To
test for the location of the opioid pathway involved in the slowing
of transit by fat, perfusion of buffer was through both the
duodenal and mid-gut fistulas (2 mL/min over 90 minutes); the
buffer administered through the mid-gut fistula to the distal gut
contained 60 mM oleate to induce the ileal brake; 6 mg naloxone was
delivered into either the proximal or distal gut (n=11). The
results are summarized in FIG. 22.
[0330] Naloxone delivered to the proximal gut increased marker
recovery from 34.6.+-.4.8% to 76.2.+-.5.2% (Naloxone in Prox in
FIG. 21), but naloxone delivered to the distal gut had no effect on
the ileal brake (marker recovery of 29.4.+-.5.4% [Naloxone in Dist
in FIG. 21]). This result implies that the fat-induced ileal brake
depends on an efferent, naloxone-sensitive opioid pathway, because
an identical amount of naloxone was delivered into either of the
two compartments, but the accelerating effect only occurred when
naloxone was delivered into the efferent compartment. Therefore, an
opioid pathway is involved that is located peripherally, rather
than systemically. The accelerating effect in response to the
opioid receptor antagonist is a result of the efferent location of
the opioid pathway. It cannot be explained on the basis of chemical
interaction with the perfusate, since the acceleration of transit
was seen when naloxone was mixed with oleate in Example 14(15), as
well as with buffer in this experiment.
(18) Mu and Kappa Opioid Antagonists Abolish Fat-Induced Ileal
Brake.
[0331] The fat-induced ileal brake (marker recovery 33. 1%) was
abolished by a mu antagonist (H2186, Sigma) delivered into the
proximal gut so that marker recovery increased to 43.8% at 0.037 mg
H2186, 88.2% at 0.05 mg H2186 and 66.8% at 0.1 mg H2186 over 90
minutes. A similar effect was seen when a kappa antagonist (H3116,
Sigma) was used (marker recovery increased to 73.2%% at 0.075 mg
H3116, 90.9% at 0.1 mg H3116, and 61.8% at 0.125 mg H3116 over 90
minutes; n=1).
(19) Slowing of Intestinal Transit by Distal Gut 5-HT Depends on a
Naloxone-Sensitive, Opioid Neural Pathway.
[0332] In Example 14(5), 5-HT in the distal gut slowed intestinal
transit, similar to the effect of fat in the distal gut. Since the
ileal brake induced by fat in the distal gut was shown to depend on
an efferent, naloxone-sensitive opioid pathway (Example 14(17), it
was tested whether the slowing of intestinal transit in response to
5-HT in the distal gut also depends on an efferent, opioid pathway.
Buffer was perfused into both the proximal and distal guts at 2
mL/minute for 90 minutes. Either normal saline (Buffer Control in
FIG. 23) or 5-HT (0.05 mg/kg/h; 5-HT in Dist in FIG. 23) was
administered to the distal gut over the 90 minute perfusion. When
the perfusate to the distal gut contained 5-HT (i.e., 5-HT in
Dist), naloxone (6 mg) was simultaneuosly delivered through the
duodenal fistula to the proximal gut over the 90 minutes (Naloxone
in Prox in FIG. 23). Results are summarized in FIG. 23.
[0333] First, intestinal transit was slowed by 5HT in the distal
gut. Marker recovery was reduced from 79.4.+-.4.1% (Buffer Control)
to 37.0.+-.1.8% (5-HT in Dist). Second, naloxone in proximal gut
abolished this slowing effect with marker recovery increased to
90.1.+-.4.6% (Naloxone in Prox). These results imply that slowed
intestinal transit in response to 5-HT in the distal gut, depends
on an efferent opioid pathway.
[0334] The foregoing examples being illustrative but not an
exhaustive description of the embodiments of the present invention,
the following claims are presented.
* * * * *