Note: Descriptions are shown in the official language in which they were submitted.
CA 02234936 1998-04-17
COMPOSITION FOR AND TREATMENT OF
INFLAMMATORY BOWEL DISEASE BY COLON
AD~STRATION OF N-ACETYLGLUCOSANIINE
FIELD OF THE INVENTION
This invention relates to novel compositions and novel
methods of treating inflammatory bowel disease (IBD). More particular-
1y, this invention pertains to novel compositions containing N-acetyl-
glucosamine (NAG) as an active IBD treating agent and a pharmacologi-
cally and colonically suitable carriers for the NAG, and a method ~ of
administering the compositions to the colon to treat IBD in a person
afflicted with IBD.
BACKGROUND OF THE INVENTION
In general terms the "bowel" extends from the stomach to the
anus and comprises the small intestine and the large intestine. The small
intestine comprises three main sections, the duodenum (which is adjacent
to the stomach), the jejunum (which is intermediate) and the ileum (which
is distant to the stomach). The large intestine (which is termed the colon)
is joined to the remote end of the ileum of the small intestine by the
ileocecal valve. The large intestine (colon) comprises two main sections,
the caecum (which is connected to the ileum of the small intestine), and
the rectum, which is the remote part of the large intestine (colon). The
remote end of the rectum is connected to the anus.
The term "inflammatory bowel disease" (IBD) is a generic
expression which encompasses a number of specific bowel diseases
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including ulcerative colitis, chronic proctitis and Crohn's disease. These
diseases may or may not overlap with one another to a certain extent.
When they do, they blur the boundaries between the various types of IBD
afflictions. Ulcerative colitis is a chronic, non-specific inflammatory
bowel disease which involves ulcerative lesions of the colon. Chronic
proctitis comprises inflammation of the colon and often the rectum.
Crohn's disease appears as several types of intestinal inflammation but
most often the term refers to inflammation of the terminal portion of the
ileum. Synonyms of Crohn's disease are regional enteritis (the intestine)
or regional iletis, although the latter is not always apt since Crohn's
disease is not limited to the ileum. Crohn's disease often involves
diseases, fistulas (deep sinnous passages or tracts in the colon), perianal
ulcerations and narrowing of the intestinal lumen (strictures). Surgical
removal of the diseased portion of the intestine (termed a colectomy, or
ileectomy) is reserved for those cases which are most resistant to
treatment, since about half of those treated by surgery experience a
recurrence of the disease in another segment of the intestine. The most
common clinical symptoms of Crohn's disease include abdominal pain,
fever, anorexia, weight loss and a right lower quadrant "fullness" .
Inflammatory bowel disease (IBD) can involve inflammatory
disruption of vascular and matrix glycosaminoglycans (GAGS), specifi-
cally the loss of GAG's from the intestinal wall which results in fibrosis
of the bowel and a loss of function (see S.H. Murch, et al., Reference 3
in Bibliography). Organ culture shows this disruption is due to the
presence of matrix degrading enzymes (see S.L. Pender, et al., Reference
6 in Bibliography).
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Statistics indicate that approximately 1 percent of the North
American population suffer from inflammatory bowel disease (IBD) of
one form or another. The annual incidence of ulcerative colitis (UC) is
estimated at between six to eight cases per 100,000 population per year,
with the onset of the disease occurring most frequently between the ages
of 30 and 50. Since its recognition several decades ago, Crohn's Disease
has increased in incidence and it is estimated to affect between twenty
and seventy persons per year per 100,000 population, with 15,000 to
30,000 new cases occurring each year in the United States. By age 85,
it is estimated that nearly two-thirds of the population have developed
some diverticula in their colon.
A recent survey in France found an incidence of 4.9 per
100,000 for Crohn's Disease and 3.2 for ulcerative colitis. The highest
age specific incidence for Crohn's Disease was 20 to 29 years, whereas
for ulcerative colitis, it was between 20 and 39 years. The incidence of
Crohn's Disease in France was similar to northern European studies, but
the incidence of ulcerative colitis was lower.
Historically, inflammatory bowel disease (IBD) has
conventionally been treated by the use of steroidal anti-inflammatory
drugs such as cortisone or sulphasalazine, or antispasmodic agents such
as propantheline and dicyclomine, supplemented with laxative or anti-
diarrheal drugs, as well as drugs which reduce intestinal motility,
depending on symptoms. More recently, 5-aminosalicylic acid (5-ASA)
and related drugs, which are considered the active moiety of
sulphasalazine, have been found to be effective in the treatment of
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inflammatory bowel disease. Their action may be related to an ability to
remove irritating superoxide radicals formed in inflamed tissues. In
severe, refractory disease the use of immunosuppressant drugs, such as
azathioprine, may be used. In many cases, surgery to remove severely
damaged intestinal tissue is required.
Canadian Patent No. Issue Date Inventor
1, 318, 592 June 1993 Burton
U.S. Patent No. Issue Date Inventor
5,229,374 January 1992 Burton and Freeman
5,192,750 March 1993 Burton and Gislason
5,217,962 June 1993 Burton and McLean
SUMMARY OF INVENTION
The invention is directed to a composition for treating
inflammatory bowel disease in a patient suffering from inflammatory
bowel disease comprising: (a) a therapeutic amount of N-
acetylglucosamine; and (b) a pharmacologically and bowel acceptable
carrier, adapted for delivery of the N-acetylglucosamine to the bowel of
said patient.
The N-acetylglucosamine can be present in the composition
in an amount ranging from 0. 5 gram to 5 grams . The carrier can be any
medium which delivers the N-acetylglucosamine to the diseased area of
the bowel. The carrier can be distilled or purified water, a foam suitable
for rectal administration, a suppository base which can surround the N-
acetylglucosamine, or an orally ingestible time-release substance which
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can withstand degradation by the gastric acids of the stomach and can
release the N-acetylglucosamine in the bowel or colon. The water can
be present in sufficient quantity to comprise an analeptic enema.
The foam can comprise N-acetylglucosamine, a surfactant,
an adjuvant and a blowing agent. The carrier can comprise one or more
substances selected from the group consisting of propylene glycol,
emulsifying wax, polyoxyethylene-10-stearyl ether, ethoxylated cetyl and
stearyl alcohols, stearath-10, cetyl alcohol, methyl paraben, propyl
paraben, trolamine, purified water, cetyl alcohol, ethoxylated stearyl
alcohol, polyoxyethylene-10-stearyl ether, methyl and propyl parabens,
propylene glycol, dry ethanol amine, de-ionized water and suitable
propellents .
The suppository base can be selected from the group
consisting of theobroma oil, glycerinated gelatin, hydrogenated vegetable
oil, polyalkyl glycol, fattyacid ester of polyalkylene glycol, coconut oil
base, hydrogenated fatty acid, hydrogenated vegetable oil,
monoglyceride, cocoa butter, petroleum oil, beeswax, glycerine,
polyethylene glycol 600 dilaurate, hydrogenated cocoa glyceride and
polyethylene glycol.
The time release substance can be selected from the group
consisting of an acrylic-based resin coating, a methacrylic acid
copolymer, an acrylic-based resin mixed with a suitable non-medicinal
carrier such as lactose, magnesium stearate, polyethylene glycol,
polyvinyl pyrolidone, or sodium starch glycolate, cellulose or ethyl
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cellulose, a matrix composition comprised of a hydrophilic polymer and
an enteric polymer, a cellulose derivative, polyvinyl acetate phthalate, or
polyvinyl acetate phthalate mixed with a plasticizer, a polysaccharide
which is decomposable in the bowel, a locust bean gum or a guar gum,
a film-forming polymer having hydrophilic groups, a film-forming acrylic
polymer in admixture with a polysaccharide comprising from 30 to 100
by weight of at least one monomer selected from the group consisting of
lower alkyl esters of acrylic acid and lower alkyl esters of methacrylic
acid, a hydrocolloid gum obtained from a higher plant, and an anionic
carboxylic polymer which does not dissolve at a pH below about 4 but
is soluble at a pH ranging from about 4 to about 7.5.
The foam can comprise 0.5 to 5 grams of N-acetyl-
glucosamine as the active ingredient and 20 grams of a foam containing
propylene glycol, emulsifying wax, polyoxyethylene-10-stearyl ether,
cetyl alcohol, methylparaben and propylparaben, trolamine, purified
water and inert propellents, dichlorodifluoromethane or
dichlorotetrafluoroethane.
The composition can comprises between about 0.1 % by
weight to about 90 % by weight of N-acetylglucosamine, coated with
about 5 % by weight to about 29 % by weight of a hydrophilic polymer,
and from about 0.5 % by weight to about 25 % by weight of an acrylic
polymer which dissolves at a pH in the range of about 5.0 to about 7.5.
The invention is also directed to a method of treating a
patient suffering from inflammatory bowel disease comprising administer-
CA 02234936 1998-04-17
ing to the bowel of said patient a composition comprising a therapeutic
amount of N-acetylglucosamine and a bowel compatible and pharma-
cologically acceptable carrier. The N-acetylglucosamine can be present
in an amount between about 0.5 gram and 5 grams per enema.
The pharmacologically acceptable carrier can be distilled or
purified water, a bowel compatible foam which can coat the colon, a
suppository which can be solid at room temperature but melt at body
temperature, or an orally ingestible time-release substance which can
withstand degradation by the gastric acids of the stomach and release the
N-acetylglucosamine in the bowel.
The enema can be administered to said patient by an
enemator.
DRAWINGS
In drawings which illustrate specific embodiments of the
invention, but which should not be construed as restricting the spirit or
scope of the invention in any way:
Figure 1 depicts a histological section of rectal tissue prior
to treatment with N-acetylglucosamine.
Figure 2 depicts a histological section of rectal tissue after
treatment with N-acetylglucosamine.
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_ g _
Figure 3 depicts a higher magnification of the histological
section depicted in Figure 1.
Figure 4 depicts a higher magnification of the histological
section depicted in Figure 2.
DESCRIPTION
We have discovered that certain forms of inflammatory
bowel disease (IBD) can be treated with good success by the administra-
tion to the afflicted area of the upper bowel or the colon of a composition
containing N-acetylglucosamine (NAG) in an effective amount, and a
bowel and pharmacological acceptable carrier, which can be distilled or
purified water, a foam enema, a suppository, or a time-release carrier
which can be ingested orally and passes largely unscathed through the
stomach and releases the N-acetylglucosamine in the upper bowel or
colon. The time release coating protecting the N-acetylglucosamine from
attack by the gastric stomach acids can be an acrylic-based resin coating
which delays release of the N-acetylglucosamine until the acrylic-based
resin coated N-acetylglucosamine reaches the upper intestine or lower
intestine, depending on the nature of the carrier. A suitable acrylic acid
can be Eudragit STM, which is a methacrylic acid copolymer. The N-
acetylglucosamine which is coated by the acrylic-based resin can be
mixed with a suitable non-medicinal carrier such as lactose, magnesium
stearate, polyethylene glycol, polyvinyl pyrolidone, or sodium starch
glycolate. Further suitable coatings which withstand attack by stomach
gastric acids can be cellulose or ethyl cellulose. The gastric acid resistant
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coating can also be a matrix composition which is comprised of a
hydrophilic polymer and an enteric polymer. The enteric polymer is
impermeable to gastric fluids of the stomach and aids in retarding release
of the N-acetylglucosamine in regions of low pH, thereby allowing lower
levels of N-acetylglucosamine to be employed. At the higher pH range
of intestinal fluids, the hydrophilic polymer and enteric polymer dissolve
and thereby release the N-acetylglucosamine in the bowel.
A specific formulation can comprise between about 0.1 % by
weight to about 90 % by weight of the N-acetylglucosamine, coated with
about 5 % by weight to about 29 % by weight of a hydrophilic polymer,
and from about 0.5 % by weight to about 25 % by weight of an acrylic
polymer which dissolves at a pH in the range of about 5.0 to about 7.5.
The acrylic resin should be formulated so that it withstands
dissolution and attack at a pH of less than 5, but decomposes at a pH
ranging from about 5 to about 8. A further suitable coating can comprise
a cellulose derivative such as polyvinyl acetate phthalate, or polyvinyl
acetate phthalate, mixed with a plasticizer. The latter is normally
decomposable at a pH in the range pH of about 3 to about 6.
A further suitable coating can be a polysaccharide, which is
decomposable in the colon, such as a locust bean gum or a guar gum,
and a film-forming polymer preferably having hydrophilic groups. Such
coatings are decomposed under the influence of glycosidic enzymes in the
colon. The polysaccharide, which is decomposable in the colon, should
contain galactose and mannose units. The film-forming acrylic polymer
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in admixture with the polysaccharide should comprise from about 30 to
100 % by weight of at least one monomer selected from the group
consisting of lower alkyl esters of acrylic acid and lower alkyl esters of
methacrylic acid.
A further suitable coating used in the form of a membrane
surrounding the core of the N-acetylglucosamine, and suitable non-
medicinal carriers, can be a pharmaceutically acceptable, film-forming
anionic carboxylic polymer which is difficult to dissolve at a low pH of
less than 4, but is soluble at a higher pH ranging from about 4 to about
7.5. A further suitable gastric acid resistant coating can be a
hydrocolloid gum obtained from higher plants, admixed with a suitable
pharmaceutically acceptable binder.
It will be understood that in addition to selecting one or more
appropriate gastric acid resistant coating(s), it is important to select an
appropriate thickness of the water-insoluble coating. As will be readily
understood, a thin coating will be more readily penetrated by the gastric
acids than a thick coating.
The suppository can be a small solid body shaped for ready
introduction into the rectum, made of the N-acetylglucosamine enclosed
in a carrier substance which is hydrophilic and solid at ordinary ambient
temperatures but melts at body temperature. The bases for the supposi-
tory can be theobroma oil, glycerinated gelatin, hydrogenated vegetable
oil, a mixture of polyethylene glycols of various molecular weights,
and/or fatty acid esters of polyethylene glycol.
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The body temperature melting coatings which envelope the
N-acetylglucosamine can be selected from a wide group of materials and
substances such as coconut oil base, hydrogenated fatty acids, or
hydrogenated vegetable oils, formulated to be solid at ambient room
temperature, but which dissolve at body temperature. Other suitable
coatings can be monoglycerides, cocoa butter, vegetable and petroleum
oils, beeswax, glycerine, polyethylene glycol 600 dilaurate, hydrogenated
cocoa glycerides, polyethylene glycol. The polymeric material should be
dissolvable in body fluids, and preferably be capable of slowly releasing
the N-acetylglucosamine into the rectal area while the polymeric material
is dissolving in the body fluids. The film can comprise a water-soluble
polymer which is bio-erodable and contains the active N-acetyl-
glucosamine.
The rectal foam can typically be 0.5 to 5 grams of N-
acetylglucosamine as the active ingredient in 20 g of a foam containing
propylene glycol, emulsifying wax, polyoxyethylene-10-stearyl ether,
cetyl alcohol, methylparaben and propylparaben, trolamine, purified
water and inert propellants, dichlorodifluoromethane and dichlorotetra-
fluoroethane.
The foam should be hydrophilic and can be made up of
propylene glycol, ethoxylated cetyl and stearyl alcohols, stearath-10, cetyl
alcohol, methyl paraben, propyl paraben, trolamine and purified water,
with dichlorodifluoromethane and dichlorotetrafluoroethane as propellents
or blowing agents. Other foam causing carriers for the N-acetylglucosa-
mine comprising a water miscible mucoadhesive foam base can be
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formulated with cetyl alcohol, ethoxylated stearyl alcohol, polyoxyethyl-
ene-10-stearyl ether, methyl and propyl parabens, propylene glycol, dry
ethanol amine, de-ionized water and suitable propellents . In general, the
liquid vehicle which can create the foam should include at least one
surfactant, a foaming propellent such as an environmentally benign
halocarbon, and an adjuvant for the active ingredients, namely, N-
acetylglucosamine, which is capable of suspending or solubilizing the N-
acetylglucosamine in the foam.
N-acetylglucosamine, and its precursor, glucosamine, are
classified biochemically as amino sugars. Amino sugars are essential
biochemical compounds formed from blood glucose by most cells of the
body through a series of biochemical reactions, which were elucidated
many years ago. Other amino sugars include galactosamine and
mannosamine and their N-acetyl-derivatives. N-acetylglucosamine is the
precursor of N-acetylgalactosamine and N-acetylmannosamine and thus,
metabolically, N-acetylglucosamine plays an important role in the
availability of all amino sugars which are incorporated into functional
complex carbohydrates, such as glycosaminoglycans (GAG's). However,
unlike galactosamine and mannosamine, N-acetylglucosamine is available
in commercial quantities at reasonable cost.
N-acetylglucosamine and related amino sugars are compo-
nents of a large number of high molecular weight complex polysacchar-
ides, including the functionally important glycosaminoglycans and
proteoglycans, formerly referred to as mucupolysaccharides.
Glycosaminoglycans are composed of repeating disaccharide units of N-
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acetylglucosamine or related amino sugars, and glucuronic or iduronic
acid. Examples of glycosaminoglycans include hyaluronic acid (which
is made up of repeating units of N-acetylglucosamine and glucuronic
acid), chondroitin sulphate, dermatan sulphate, keratan sulphate and
heparin, all of which contain either N-acetylglucosamine or the amino
sugar N-acetylgalactosamine, which is synthesized from N-acetyl-
glucosamine. Glycosaminoglycans are also present in proteoglycans,
which are structures containing a number of glycosaminoglycans chains
linked to a polypeptide or protein core.
N-acetylglucosamine plays an important role in the normal
maintenance of cell function, including the permeability of cell mem-
branes, the structural integrity of connective tissues such as skin and
cartilage, and joint lubrication. In the intestines, glycosaminoglycans are
essential to the formation of the glycocalyx of gastrointestinal cells and
the mucous secreted by the intestinal goblet cells. As well, they are
essential components of the extracellular fluid or "glue" which holds cells
together, immunoglobulins, blood group antigens and a wide diversity of
other biochemically and immunologically important substances.
The extracellular matrix, composed largely of proteoglycans
containing N-acetylglucosamine, is present in the basement membrane,
as well as the lamina propia, and the submucosa of the gastrointestinal
tract. Proteoglycans containing N-acetylglucosamine play an important
function in anchoring cells to the mucopolysaccharide layer on the
epithelial cells lining the gastrointestinal tract, as well as cell repair and
intestinal permeability.
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Although N-acetylglucosamine is found in all cells and
tissues of the body as a component of glycosaminoglycans and proteo-
glycans, it appears that, unlike glucose, significant concentrations of free
N-acetylglucosamine or its precursor, glucosamine, are not available in
the blood or intracellular fluids. Under normal conditions, the body
utilizes all the available N-acetylglucosamine it produces. In disease
states, such as those involving inflammatory reactions of the
gastrointestinal tract (IBD), the body reserves of N-acetylglucosamine
may be inadequate to support cellular repair and function at an optimal
level. It has been demonstrated (see Burton et al., Bibliography
Reference 1) that intestinal tissue from patients with inflammatory bowel
disease preferentially incorporate N-acetylglucosamine into the bowel
wall rather than glucosamine, which lends support to the need for
additional N-acetylglucosamine in inflammatory bowel disease (IBD).
When N-acetylglucosamine is given to animals or humans,
either intravenously or by mouth, it is quickly distributed throughout the
body and incorporated within minutes into glycosaminoglycans and
proteoglycans in most tissues and organs, including the intestinal tract
and articular cartilage of joints. Free N-acetylglucosamine has a half life
in blood of less than ten minutes, but when incorporated into
glycosaminoglycans, the N-acetylglucosamine contained in these complex
molecules turns over very slowly, with a half life measured in hours, and
even days.
N-acetylglucosamine is a natural amino sugar belonging to
a class of compounds that are usually very well tolerated. Mono-
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saccharides, and their simple derivatives, are widely used in medicine
even at high doses. Even when given by intravenous administration, N-
acetylglucosamine is considered to be very safe. Specifically, no
mutagenicity, carcinogenicity or teratogenicity are to be expected.
Single intravenous or oral doses of N-acetylglucosamine of
over 25 grams/kg body weight administered to mice or rats have resulted
in no observed toxic effects. Intravenous dosing of 20 grams of N-
acetylglucosamine to humans had no adverse effects, did not affect blood
glucose or insulin levels, and is therefore considered safe for use by
diabetics .
Studies in animals and humans have demonstrated that N-
acetylglucosamine is non-toxic at doses substantially greater than those
used for most medicinal purposes. Oral administration at doses of up to
3 to 6 grams/kg body weight per day for 30 days had no toxic effects,
and resulted in no gross or microscopic changes in various tissues and
organs. N-acetylglucosamine has been used orally, as a human nutri-
tional supplement, at doses of up to 3 grams per day without adverse
effects. For the past several months, we have administered up to 4.5
grams per day orally, or 3 grams daily rectally, to children with no
adverse consequences.
In inflamed bowel tissue, it has been demonstrated by Burton
et al. (see Reference 1 in Bibliography) that there is a preferential
incorporation of N-acetylglucosamine over glucosamine. This may be the
result of a biochemical deficiency in the process by which glucosamine
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is converted to N-acetylglucosamine in the intestinal cells. The
acetylation step in the conversion of glucosamine to N-acetylglucosamine
may be deficient. This would result in a reduction of the protective
glycocalyx or proteoglycans covering the intestinal cells, as well as a
decrease in mucus production and the slowing of diseased tissue repair.
As a result of the loss of these protective components, the intestinal wall
would be more susceptible to damage, inflammation and ulceration.
While we do not wish to be bound adversely by any theories, it appears
that N-acetylglucosamine is more readily absorbed into cells as a result
of its passive diffusion across cell membranes, as compared to the
apparent active transport mechanism (a complex, energy requiring
system) required for the intracellular distribution of glucosamine. We
have observed that in inflammatory bowel disease, there is a loss of N-
acetylglucosamine-containing glycosaminoglycans from the intestinal wall
which results in the bowel wall becoming abnormally thick, or fibrotic.
When this occurs, the permeability and function of the gut is impaired.
Although symptoms of ulcerative colitis and Crohn's Disease
are similar and are often hard to differentiate, a gross pathological
difference is that Crohn's Disease is an inflammatory condition in which
inflammation and ulcerations may be distributed along any part of the
entire gut. In Crohn's Disease, fibrosis of the gut may be extensive
causing luminal narrowing, resulting in bowel obstruction. Both
ulcerative colitis and Crohn's Disease' can become serious and may
require intensive drug therapy or surgery. Patients with inflammatory
bowel disease exhibit high cellular turnover in the gut wall, often leading
to ulceration or fistulas. The chronic nature of these inflammatory
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conditions, as well as the tendency to affect younger people with the peak
incidence of ulcerative colitis occurring between 15 and 30 years of age,
makes inflammatory bowel disease a serious medical problem. Onset of
the disease most often occurs during adolescence or early adulthood.
Diverticulitis is a more common, but less severe
gastrointestinal condition that is often categorized with inflammatory
bowel disease. This disorder is an inflammation of one or more
diverticular, which are sac-like outpouchings of the colon wall that occur
at weak points. In diverticulitis, the cell wall of the colon is primarily
affected and the symptoms range from relatively minor discomfort to
painful perforation of the intestinal lining. Diet has been implicated as
' a causative or contributing factor to the development of diverticulitis,
which is also associated with increasing age, being uncommon in
individuals under the age of 35. The disorder is progressive, developing
from mild symptoms to painful inflammation. Early stages of
diverticulitis may respond to a modified diet, but later stages require
medication or surgery.
In inflammatory bowel disease, including Crohn's Disease,
there is a significant loss of glycosaminoglycans (GAG's) from the
intestinal wall, providing further evidence of the involvement of
glycosaminoglycans in the pathology of inflammatory bowel disease. We
do not wish to be bound by any incorrect theories. However, the
following is offered as a possible explanation of the foundation for the
invention. The finding of loss of GAG's from the bowel well supports
the rationale for the beneficial effects of N-acetylglucosamine in the
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treatment of inflammatory bowel disease, since N-acetylglucosamine
would provide the necessary substrate or amino sugar components needed
to replace, or prevent the loss of glycosaminoglycans. The normal loss
and turnover of intestinal cells is about half a million cells per minute.
This increases by as much as three to five times in inflammatory bowel
disease. This may explain the need for additional amino sugars in order
to support cellular regeneration since the systemic pool of free amino
sugars is very limited. If the loss of epithelial cells in the gut exceeds the
rate of production, it follows that the integrity of the mucosa would be
compromised. To further support the concept of N-acetylglucosamine
preventing or replacing the bowel wall glycosaminoglycans in inflamma
tory bowel disease, we have observed that N-acetylglucosamine has a
significant effect in stimulating the synthesis of hyaluronic acid when
applied to the intestinal epithelium or to human mesothelial cells isolated
from the intestinal peritoneum.
Example 1
Preliminary research conducted at The University of British
Columbia has . demonstrated that N-acetylglucosamine is a preferential
precursor in the synthesis of the constituent glycosaminoglycans and
proteoglycans in the inflamed and damaged tissues occurring in inflam-
matory bowel disease. This research suggests that the oral administration
of N-acetylglucosamine supplements the body's supply of the essential
amino sugars when under the stress of excessive tissue breakdown and
loss.
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Example 2
The effect of N-acetylglucosamine on digestive tissue has
also been demonstrated in a confidential animal model of inflammatory
bowel disease carried out at the Department of Biology at Queen's
University, Kingston, Ontario. Trinitrobenzenesulfonic acid and alcohol
instilled intrarectally in rats caused a chronic inflammatory bowel
reaction with ulcers, bowel wall fibrosis or thickening, and pathological
and histological changes similar to those seen in ulcerative colitis. It was
found that a single intrarectal instillation of N-acetylglucosamine (NAG),
prior to challenge with trinitrobenzenesulfonic acid (TNB), had a
significant, dose-related effect in reducing the number and severity of the
ulcerative lesions, as well as reducing bowel wall thickening (see Table
1 below).
Table 1: Bowel Wall Fibrosis in
Trinitrobenzenesulfonic Acid (TNB)-Ethanol Treated Rats
Colon Fibrosis (weigh~,g_m, of 8 cm, of colon)
Intrarectal Administration Mean + sum
Control (20 mg TNB* in 0.25 mL ethanol) 2.301 f 0.222
mg NAG/kg Bwt 1 hr. before TNB/EtHO 1.669 ~ 0.142
50 mg NAG/kg Bwt 1 hr. before TNB/EtHO 1.339 f 0.155*
100 mg NAG/kg Bwt 1 hr. before TNB/EtHO 1.150 ~ 0.068*
*Statistically significant different from control group
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Example 3
Additional evidence that N-acetylglucosamine stimulates
glycosaminoglycans synthesis was observed in an ex vivo rat gastric
model, in which the blood supply to the stomach is maintained but the
stomach is opened and placed in a plastic chamber, allowing drugs to be
placed on the inside, mucosal surface. When N-acetylglucosamine was
placed on the mucosal surface of the stomach, copious quantities of
mucus were observed being released from the goblet cells of the stomach.
On the basis of this observation, additional experiments were carried out
in which it was found that in the presence of ethanol (which is a gastric
irritant), N-acetylglucosamine, but not glucosamine, caused a significant
reduction in the potential difference between the mucosal and serosal
surfaces. This phenomenon may be a result of N-acetylglucosamine
stimulating the synthesis of sulphated glycosaminoglycans. The effect of
N-acetylglucosamine in stimulating mucus release by the stomach may
explain the anecdotal clinical claims that N-acetylglucosamine is effective
in the treatment of non-erosive gastritis.
In inflammatory bowel disease, there is also a disruption of
vascular and matrix glycosaminoglycans. Organ culture shows this to be
due to matrix degrading enzymes. N-acetylglucosamine is a substrate for
glycosaminoglycans production, but in addition to its role as a fuel in
fibroblast matrix synthesis, there is evidence that N-acetylglucosamine
can act intracellularly as an antagonist of O-phosphorylation and may
thereby regulate many inflammatory pathways.
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Examples 4 to 23
Clinical Evidence of the Effect of
N-acetyl~lucosamine in Inflammatory Bowel Disease
Research has been conducted at the Royal Free Hospital in
London, U.K., and it was noted that there is a loss of
glycosaminoglycans from the intestinal wall in patients with inflammatory
bowel disease, which is associated with bowel wall thickening or fibrosis.
Ethics approval was obtained to treat children with serious, intractable
upper or lower inflammatory bowel disease with oral N-acetyl-
glucosamine. To date, over twenty children have been treated with N-
acetylglucosamine at doses of up to 12 grams a day. The children were
generally resistant to other therapy, including such toxic drugs as
azathioprine and intravenous steroids, and therefore there were minimal
expectations that N-acetylglucosamine would provide a significant clinical
benefit. Of the initial six patients treated, three with upper inflammatory
bowel disease have responded well, with one child doing better than on
any previous therapy. Three children with lower inflammatory bowel
disease did not demonstrate significant therapeutic response with oral N-
acetylglucosamine. This is not unexpected since only a small fraction of
each oral dose would be expected to reach the diseased tissue in the
lower bowel.
In order to expose the inflamed colon to a higher concentra-
tion of N-acetylglucosamine, patients with lower inflammatory bowel
disease have been administered N-acetylglucosamine by rectal enema at
CA 02234936 1998-04-17
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a dose of 1 to 2 grams three times a day. The initial three patients had
symptomatic clinical improvement within forty-eight hours of initiating
therapy. Pre- and post-biopsies of the colon of several children indicated
that, after six weeks of rectal N-acetylglucosamine therapy, there was a
significant improvement in the histopathology of the bowel wall.
A representative example of this effect is shown in Figures
1 and 2. Figure 1 is a section from the pre-N-acetylglucosamine
treatment biopsy, and clearly demonstrates that prior to N-acetyl-
glucosamine therapy, an atrophic epithelium with heavy inflammatory
infiltrate and fibrosis was present. Pre-treatment there was an almost
complete void of villi and the epithelial membrane was virtually absent.
The post-N-acetylglucosamine treatment biopsy (Figure 2) indicates the
presence of significantly less inflammatory infiltrate, as well as an
increase in the number of villi and an almost normal epithelial membrane.
Figures 3 and 4 are pictures of the same biopsies depicted in Figures 1
and 2, but , at a higher magnification. An important finding is the
significant reduction in the fibrosis of the bowel wall.
Rectal administration of NAG ( 1.5-2 g bd), as a single
therapy, to nine children with therapy-resistant proctitis of distal colitis
induced clinical remission in four of those children, improvement in three
of the children, and no effect in two of the children. Biopsies before and
after treatment have shown histological improvement in four of five
children, with a striking increase in glycosaminoglycan (GAG) density in
the bowels of such children (which has not seen with other treatments)
and intraepithelial wheat germ agglutin (WGA) staining. Epithelial
CA 02234936 1998-04-17
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morphology was particularly improved. Oral NAG (1-1.5 g bds) was
added to the treatment of eleven children with severe small intestinal and
colonic disease, including seven with critical strictures. Eight of the
eleven children showed improvement, while three did not and required
surgical resection. Six of the seven children with treatment-resistant
strictures showed marked resolution of symptoms. Endoscopic or
radiological assessment confirmed substantial improvement in four of the
six children. GAG and WGA staining was again denser in the NAG
treated children than in children treated with steroids or enteral nutrition.
This first uncontrolled trial suggests that NAG is of
therapeutic efficacy in resistant IBD, with a mode of action distinct from
conventional therapies. Its lack of known adverse effects makes it
particularly suitable for therapeutic use.
Commencing on or about April, 1996, an extensive study of
the use of prescribed dosages of N-acetylglucosamine (NAG) on
paediatric patients of the Royal Free Hospital suffering from inflamma-
tort' bowel disease has been conducted. Since commencing the tests
with NAG on or about April, 1996, extensive case histories on those
children tested with NAG have been compiled. Details of biopsies and
other relevant information taken from such case histories are reproduced
in summary form as follows. The patient in each case is identified by his
or her initials.
CA 02234936 1998-04-17
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Rectal Administration in Ulcerative Colitis (UC) and
Chronic Proctitis Following Colectomy (Removal of Part
of Colon Leaving Rectal Stump) and Ileostomy (Artificial
Opening, in Ileum to Evacuate Feces through Abdomen)
UC with previous subtotal colectomy, ileostomy and mucous
fistula, now with chronic proctitis (presumed due to defunction and
underlying UC).
I0 1. ZY. Intractable proctitis within rectal stump, failed
previously on metronidazole and corticosteroids: Commenced NAG
December 1996, dose 2 grams once daily p.r. Clinical response within
2 days, with cessation of blood loss. Post-treatment biopsy at 2 months
showed significant improvement, particularly in epithelial morphology
and integrity; with 30-50 % reduction in inflammatory infiltrate. Has
been treated with NAG continually since, with only minimal symptoms.
2. SG. Clinical problem, inflammation of ileo-anal pouch,
with additional problems due to failure of pouch growth with age (thus
symptoms only partly due to inflammation). Had failed to respond
previously to cyclosporine, interferon-«, corticosteroids, long-chain fatty
acid enemas, metronidazole, codeine phosphate. Commenced NAG
March 1997, dose 2 grams once daily pr (unhappy with twice daily
enema regime). Modest clinical response, but histology after 1 month
showed definite improvement compared to pre-treatment biopsy, with
overall reduction in histological Pouchitis Index from 9/ 12 to 6/ 12.
Continues with rectal NAG in view of histological response. Has
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subsequently achieved complete clinical remission with NAG in
combination with antibiotics (metron, diazole and ciprofloxacin).
3. SB. Severe long-term inflammation of rectal stump,
unresponsive to corticosteroid enemas, metronidazole and ciprofloxacin.
Commenced NAG enemas April, 1997, dose 1 gram twice daily rectally.
Only minor clinical improvement reported at 2 months, but significant
histological response compared to pre-treatment biopsy. In this case, the
inflammatory infiltrate was significantly reduced, while epithelial changes
. were less marked.
4. MS. Intractable inflammation of rectal stump,
unresponsive to cyclosporine, corticosteroids and antidiarrhoeals.
Commenced NAG enemas (1 gram three times daily) initially in October,
1996, with substantial improvement. Biopsies were not performed at that
time. The enemas were stopped because of good clinical progress in
December, 1996, and his symptoms gradually returned. He was
recommenced on NAG enemas in June, 1997 (1 gram twice daily) after
pre-treatment biopsy.
5. LE. (possible diagnosis Crohn's, but with similar post-
colectomy rectal stump inflammation, unresponsive to corticosteroids and
cyclosporine). Commenced NAG enemas (1.5 grams twice daily) June,
1997, following pre-treatment biopsy. Follow-up clinical assessment and
biopsies in August, 1997 have shown significant improvement.
CA 02234936 1998-04-17
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6. SE-K. Severe persistent inflammation of rectal stump,
resistant to topical and systemic steroids, metronidazole, ciprofloxacin.
Commenced on rectal NAG (1.5 grams twice daily) in June, 1997. Pre-
treatment biopsy showed anal mucosa thus post-treatment biopsy will be
difficult to interpret. However the clinical response has been prompt and
marked, and she has shown complete resolution of bleeding and
discharge.
7. RM. Persistent inflammation of rectal stump, with
severe perianal inflammation. Treatment (1 gram twice daily) discon-
tinued at child's request after brief period because of pain on inserting
enema catheter.
Rectal Administration in Distal Intestinal Disease
(Without Surg_ery Involving Removal of
Part of Colon Leaving Rectal Stump (Colectomy))
8. MI. Chronic distal proctitis, of indeterminate
histology, resistant to corticosteroids and ciprofloxacin. The proctitis
was limited to the first 5 cm of the colon only, and did not involve the
rectum. NAG enemas (1.5 grams bd) commenced February, 1997,
discontinued after 2 months without effect.
9. SE. Severe ulcerative peri colitis, almost requiring
emergency surgery in July, 1996. During this episode, she had received
intravenous azathioprine, intravenous heparin, intravenous corticosteroids
and was additionally commenced on NAG during the convalescent phase.
CA 02234936 1998-04-17
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This was at a dose of 1g three times daily orally and 1g twice daily
rectally. She has subsequently been maintained on azathioprine, variable
corticosteroid doses and aminosalicylates. She has shown an extremely
good clinical response, in the circumstances of her severe disease, which
we would nbrmally have treated surgically, but in this case it was
avoided. Although it is impossible to determine the individual contribu-
tion of NAG, it is very noticeable that her rectal biopsies showed signs
of relatively improved histology, which were not detectable before the
NAG enemas were commenced. She is currently well and continues oral
NAG, azathioprine and low dose heparin.
Crohn's Disease (With or Without Surgery
10. AT. This boy has Crohn's disease and a tight anal
stricture, which due to the boy's growth has required regular surgical
dilatation for the last three years. He is maintained on a low dose oral
prednisolone and aminosalicylates for his disease, and commenced NAG
enemas (1g twice daily) in December, 1996. Since commencement of
NAG he has shown marked clinical improvement, and subsequent
examination under anaesthesia in March, 1997 showed clear improvement
in his anal stricture, with substantially less dilatation needed. Mucosal
inflammation also was noted to have decreased. He subsequently
remained entirely asymptomatic.
11. HS. A girl with long-standing Crohn's disease,
previous hemicolectomy and surgery for multiple strictures. Previously
treated with enteral nutrition, corticosteroids, azathioprine; cyclosporin
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and aminosalicylates. Previous barium examinations had shown extensive
stricturing, and she had become grossly cushingoid because of steroid
treatment. NAG was commenced in July, 1996 ( 1 g three times daily by
mouth), and this was associated with clear improvement in stricture
related symptoms and reduction in inflammatory markers. She has had
one minor relapse in January, 1997, but has subsequently been well.
Follow up barium examination in April, 1997 was normal.
12. GC. A boy with severe left-sided Crohn's colitis,
(which is clinically notorious as a variant of the disease virtually resistant
to treatment) resistant to enteral nutrition, azathioprine and
corticosteroids. In addition, he had an associated inflammatory anal
stricture . He was commenced on NAG ( 1 g three times daily by mouth
and SOOmg twice daily rectally) in April, 1996, in addition to his
intensive therapeutic regime. Although subsequent endoscopy did show
evidence of rectal sparing, that had not been evident previously, his
symptoms remained intractable and he underwent subtotal colectomy in
February, 1997.
13. GN. A boy with a similar history to GC (above) of
intractable left sided Crohn's colitis. He had failed to respond to
corticosteroids, aminosalicylates, enteral nutrition and azathioprine. He
was corrimenced on NAG ( 1 g three times daily by mouth) in April 1996.
He had minimal response to this, and underwent subtotal colectomy in
March, 1997.
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14. LC. The third patient with severe left sided Crohn's
colitis. She has been managed on enteral nutrition, aminosalicylates,
corticosteroids and azathioprine. Four months after commencement on
azathioprine, she remained severely unwell with persistent rectal
bleeding, severe anaemia and marked increase in inflammatory markers.
She was commenced on NAG in February, 1997 ( 1 g three times daily by
mouth) in addition to azathioprine and steroids. She showed marked
clinical improvement within two weeks of commencing NAG, and has
subsequently gone into complete remission. The only complicating factor
in interpreting this case is that azathioprine may show a delayed onset of
efficacy, but the temporal association of improvement with NAG was
very striking, and she has avoided the colectomy that we had thought
previously would .be inevitable. There was recently a NAG supply
problem and her symptoms have recurred.
15. DB. This boy has relentlessly severe Crohn's disease
affecting the terminal ileum, with stricture. He had failed on treatment
with corticosteroids, enteral nutrition, azathioprine and aminosalicylates.
He was commenced on NAG (1g twice daily by mouth) in April, 1996.
He showed limited clinical response, and underwent hemicolectomy in
June, 1996. The severity of his disease was shown by a rapid early
relapse and currently he pursues a relentless course of the disease.
16. RP. This girl has a stricturing form of Crohn's
disease, and had undergone previous removal of two small intestinal
strictures, when the surgeons noted multiple other strictures throughout
the small intestine (bowel) (January, 1995). She had been managed with
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enteral nutrition and aminosalicylates, plus intermittent corticosteroids.
In view of severe stricture-related symptoms, confirmed by barium follow
through in March, 1996, when she had multiple small intestinal stric-
tures, she was commenced on NAG in March, 1996, (initial dose 1g
three times daily, increased to 2g three times daily in August, 1996).
Barium assessment showed reduction in stricturing after one month of
NAG treatment, associated with very marked clinical improvement.
Subsequent barium examinations in September and December, 1996
showed no evidence of stricture. Thus we have radiological and clinical
evidence of NAG improvement in stricturing small intestinal Crohn's
disease.
17. EH. This girl has a long stricture of her terminal
ileum, associated with long-standing Crohn's disease. This was
confirmed by barium follow through in December, 1996 when there was
marked pre-synoptic dilatation and she was complaining of obstructive
pain after taking solids. She had been managed with enteral nutrition,
aminosalicylates and steroids. She was commenced on NAG (1g three
times daily by mouth) in November, 1996 in addition to her current
treatment. Within three to four weeks she became asymptomatic and was
able to eat an unrestricted diet. She remains asymptomatic and has
weaned off steroids. Follow up barium examination in June, 1997
showed decrease in the stricturing, although she still has radiologically
a significant stricture: Undoubtedly, without the NAG treatment she
would have required hemicolectomy and removal of her stricture as this
was at a critical level in November, 1996. At this time, we are uncertain
CA 02234936 1998-04-17
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whether she will entirely avoid surgery, but she certainly will remain on
NAG in the long term because of its obvious benefits to her.
18. ML. This boy has stricturing Crohn's disease, with
history of a previous hemicolectomy, resection of strictures, and then the
development of gastric outlet obstruction due to stricturing duodenal
disease. He underwent endoscopic dilatation in May, 1996 and was
commenced on NAG at that time (1g twice daily). His additional
treatment was based on enteral nutrition. Salazopyrine and azathioprine
in addition to Omeprazole (a steroid). He underwent re-dilatation in
July, 1996, when the stricture had improved, and this was confirmed by
barium swallow in August, 1996. Repeat endoscopy in March, 1997
showed significant improvement, with almost no evidence of residual
stricture. However he was readmitted with recurrence of his stricture,
and has now undergone surgery for this. He has continued with the
NAG treatment in the meantime, in addition to his other treatment. Thus
the early apparent improvement in stricturing was not maintained but
there is a benefit because he has been taken off Omeprazole in the
meantime.
19. IJ. This boy also has multiple strictures of his small
intestine, with very aggressive Crohn's disease. He has previously failed
on enteral nutrition, azathioprine and high dose corticosteroids and has
also previously undergone hemicolectomy. He was demonstrated to have
multiple critical strictures throughout the small bowel (small intestine),
which represents a major therapeutic problem. He was commenced on
NAG (1.5g three times daily by mouth) in February, 1997, in addition
CA 02234936 1998-04-17
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to continuing his previous treatment with azathioprine and enteral
nutrition. He has shown gradual improvement, both in terms of his
stricture related symptoms and his inflammatory indices. An early
barium examination in March, 1997 showed no significant improvement
in the strictures after one month on NAG. A follow up barium study will
probably be performed in the autumn.
20. NBL. This girl has stricturing small bowel Crohn's
disease, with a long terminal deal stricture with pre-synoptic dilatation.
She has been managed with enteral nutrition and aminosalicylates
previously. She was commenced on NAG (1.5g three times daily) in
April, 1997, in association with enteral nutrition. She is significantly
improved clinically, with no current stricture associated symptoms when
reviewed in June, 1997. We plan to perform a follow up barium study.
Overall Assessment
The foregoing evidence confirms that NAG has definite
therapeutic effect in the treatment of inflammatory bowel disease. There
are certain clear areas where NAG appears to out-perform conventional
treatments. NAG clearly shows efficacy by rectal administration in the
defunctioning colitis following subtotal colectomy. Several of the NAG
treated children reviewed have shown very prompt clinical improvement.
Follow up histological examination in three NAG treated children shows
definite improvement. In addition, the children who had more extensive
colitis developed evidence of rectal sparing which had not been there
previously .
CA 02234936 1998-04-17
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NAG also appears to be of benefit in the stricturing variant
of inflammatory bowel disease (IBD), particularly in upper small
intestinal strictures. Only a single long term treatment failure occurred
in the last month because of a temporary NAG supply problem, and all
other NAG treated children suffering from small intestinal strictures have
shown benefit. Certainly NAG has been instrumental in avoiding early
stricture surgery in two children, who had critical strictures before
commencing NAG treatment and they are currently asymptomatic.
The cases where NAG appears to be less successful have
been in the treatment of terminal ileal and upper colonic disease, when
the NAG was given by mouth. In contrast, rectal treatment by NAG has
shown marked improvement. The latter success is probably due to the
fact that rectally administered NAG does not have to pass through the
digestive process of the gut. It follows that administering NAG with a
suitable carrier that can deliver the NAG to the IBD area will be
successful in treating the IBD. Suitable carriers for rectal administration
can be distilled or purified water, a foam suitable for rectal administra-
tion, or a suppository. A suitable carrier for oral administration is a
coating, or a double coating, in which the NAG is protected, the coating
or double coating withstanding acidic and enzymatic degradation by the
stomach and small intestine, until the afflicted IBD area is reached. Then
the coating is digested and the NAG is delivered unscathed to the
afflicted area.
As will be apparent to those skilled in the art in the light of
the foregoing disclosure, many alterations and modifications are possible
CA 02234936 1998-04-17
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in the practice of this invention without departing from the spirit or scope
thereof. Accordingly, the scope of the invention is to be construed in
accordance with the substance defined by the following claims.
CA 02234936 1998-04-17
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BIBLIOGRAPHY
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Glucosamine Relative to 3H-N-Acetyl Glucosamine in the Intestinal
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Chem. 78: 19, 1983.
2. Vantrappen, G. and Geboes, K. Glycosaminoglycans and the gut.
Lancet 341: 730, 1993.
3. Murch, S.H. Disruption of sulphated glycosaminoglycans in
intestinal inflammation. Lancet 341: 711, 1993.
4. Murch, S.H., et al. Congenital enterocyte heparan sulphate
deficiency with massive albumin loss, secretory diarrhoea, and
malnutrition. Lancet 347: 1299, 1996.
5. Murch, S.H. Sulphation of proteoglycans and intestinal function.
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