Title: Treatment of C. difficile toxin B associated
United States Patent: 6,465,435
Issued: October 15, 2002
Inventors: Heerze; Louis D. (Edmonton, CA); Armstrong; Glen
D. (Edmonton, CA)
Assignee: SYNSORB Biotech, Inc. (Calgary, CA)
Appl. No.: 593040
Filed: June 13, 2000
This invention relates to prevention and/or treatment of antibiotic
associated diarrhea, including Clostridium difficile associated diarrhea (CDAD),
pseudomembranous colitis (PMC) and other conditions associated with C.
difficile infection, using oligosaccharide compositions which bind C.
difficile toxin B. More specifically, the invention concerns neutralization
of C. difficile toxin B associated with such conditions.
DETAILED DESCRIPTION OF THE INVENTION
As used herein the following terms have the following meanings:
The term "antibiotic-associated bacterial diarrhea" refers to the condition
wherein antibiotic therapy disturbs the balance of the microbial flora of
the gut, allowing pathogenic organisms such as Clostridium difficile to
flourish. These organisms cause diarrhea. Antibiotic-associated bacterial
diarrhea includes such conditions as Clostridium difficile associated
diarrhea (CDAD) and pseudomembranous colitis (PMC).
The term "biocompatible"refers to chemical inertness with respect to human
tissues or body fluids.
The terms "compatible linker arm" or "linker arm" refer to a moiety which
serves to space the oligosaccharide structure from the biocompatible support
and which is bifunctional wherein one functional group is capable of binding
to a reciprocal functional group of the support and the other functional
group is capable of binding to a reciprocal functional group of the
oligosaccharide structure. Compatible linker arms preferred in the present
invention are non-peptidyl spacer arms. The oligosaccharide may be linked
via an 8-methoxycarbonyloctyl linker or via another appropriate non-peptidyl
linker, such as a urea-like linker arm of the formula --NH--(CH2)m
--NHC(O)NH--, where m is an integer of from about 2 to about 10.
The term "oligosaccharide" means saccharides comprising 1 to about 20
saccharide moieties. Saccharide derivatives may also be used as saccharide
moieties included in the term oligosaccharide [67-69].
The term "pseudomembranous colitis" (PMC), also known as pseudomembranous
enterocolitis or enteritis, refers to the inflammation of the mucous
membrane of both small and large intestine with the formation and passage of
pseudomembranous material (composed of fibrin, mucous, necrotic epithelial
cells and leukocytes) in the stools.
The term "support" refers to an inert material to which the oligosaccharide
sequences may be bound or immobilized via a compatible linker arm. Where use
is in vivo, the support will be biocompatible.
The term "SYNSORB" refers to 8-methoxycarbonyloctyl oligosaccharide
structures covalently coupled to Chromosorb P.TM. (Manville Corp., Denver,
Colo.) , a derivatized silica particle material. Where indicated, the
SYNSORB may use a urea-like linker arm rather than the
The term "toxin A" refers to an enterotoxin of Clostridium difficile which
initiates CDAD and related conditions. This toxin has a lectin-like
The term "toxin B" refers to a cytotoxin of Clostridium difficile which
causes destruction of intestinal cells and induces the release of
For purpose of this application, all sugars are referenced using
conventional three letter nomenclature. All sugars are assumed to be in the
D-form unless otherwise noted, except for fucose, which is in the L-form.
Further all sugars are in the pyranose form.
B. Pharmacology Amino acid sequences in C. difficile toxin A and B that are
similar to sequences responsible for oligosaccharide binding in
Streptococcal glucan binding proteins have been reported [49-51]. Although,
as noted above, the receptor for toxin B is not known, the oligosaccharide
binding specificity for these glucan binding proteins is for repeated
glucose units linked together as shown below :
.alpha.Glc(1.fwdarw.6).alpha.Glc(1.fwdarw.6).alpha.Glc . . .
The oligosaccharide isomaltotriose (.alpha.Glc(1-6).alpha.Glc(1-6)Glc) was
immobilized by attachment onto Chromosorb P using a linker arm, and tested
in toxin B neutralization experiments. The results from these experiments
are presented graphically in FIGS. 1A and 1B, where concentration and time
dependent neutralization of C. difficile toxin B cytotoxic activity using
immobilized isomaltotriose SYNSORB (n=3) is shown. Concentration
neutralization experiments were performed by incubating immobilized
isomaltotriose (10, 20 or 40 mg) with 1 mL of toxin B for 2 hours at room
temperature. The amount of toxin activity in each sample was measured using
Chinese hamster ovary (CHO) cells.
The results are presented as the percent activity remaining relative to
control toxin solutions that had not been incubated with SYNSORB. Time
dependent neutralization experiments were performed by incubating toxin B
with 20 mg samples of immobilized isomaltotriose SYNSORB for 1, 2 and 4 h at
room temperature. A control incubation (4 h) of toxin B with Chromosorb P
was included to determine the extent of background binding to the support.
The results are presented as the percent activity remaining relative to
control toxin solutions that had not been incubated with SYNSORB and
indicate that toxin B bound to isomaltotriose SYNSORB in a concentration and
time dependent manner. The results also indicated that toxin B binds to the
support slowly, requiring up to 4 hours to achieve significant toxin B
binding under these conditions. Further, these data show that
oligosaccharides which contain .alpha.(1-6)-linked repeating units of
glucose are effective at binding toxin B and can serve as a therapeutic for
C. difficile-mediated diarrhea.
SYNSORBs which incorporate oligosaccharides terminating in glucose or N-acetylglucosamine
were also examined for toxin B binding by measuring the cytotoxic activity
of toxin B with or without SYNSORB in CHO cells. Results of these studies
are shown in Table 1, where * indicates SYNSORBs using the urea-like linker
Toxin B Neutralization Studies
SYNSORB Percent Presence of
Number Common Name Oligosaccharide Structure Neutralization
23 -- .beta.Glc 0 0
38 -- .alpha.Glc(1-2).beta.Gal 78 + 16 80
-74* maltose .alpha.Glc(1-4).beta.Glc 96 + 0 80
3-76* cellobiose .beta.Glc(1-4).beta.Glc 93 + 5 80
5-128* isomaltotriose .alpha.Glc(1-6).alpha.Glc(1-6).beta.Glc 96
+ 0 80
179A* isomaltose .alpha.Glc(1-6).beta.Glc 96 + 9 N.D.
78 chitobiose .beta.GlcNAc(1-4).beta.GlcNAc 93 + 5
All SYNSORBS tested except SYNSORB 23 effectively neutralized toxin B
cytotoxicity. By comparison, toxin A did not bind to the SYNSORBs 5-128 (isomaltotriose)
and 179A (isomaltose). The other SYNSORBs in Table 1 were not tested against
toxin A. This observation confirms that there are differences in the binding
specificity of toxin A and toxin B, even though there is some amino acid
homology (60% amino acid homology) between the two toxins. Oligosaccharides
which bind toxin A have been identified [65-66].
We also utilized a SYNSORB derivative that incorporates two different
oligosaccharide ligands. The ligands selected for the dual labelling of
Chromosorb P.TM. were based on previous results which revealed differential
oligosaccharde binding specificities for toxins A and B. Since the
oligosaccharide .alpha.Gal(1-3).beta.Gal(1-4).beta.Glc (Cd) binds toxin A
but not toxin B, it was selected for use as the toxin A neutralizing
component, and was immobilized onto amino derivatized Chromosorb P using an
8-methoxycarbonyl octyl linker arm. Toxin B but not toxin A binds to
isomaltose (.alpha.Glc(1-6)Glc). Utilizing the amino derivatized Chromosorb
that already incorporated the Cd oligosaccharide, isomaltose was immobilized
onto the support using the recently developed "Instasorb" linker arm
technology as disclosed in PCT/CA97/00851 . The resulting SYNSORB (SYNSORB
5174, which has both oligosaccharides covalently bound by their respective
linkers) was then tested for toxin A and B binding. SYNSORB Cd and
isomaltose SYNSORB (SYNSORB 179A) were included as controls. The results,
presented in FIG. 3, show that SYNSORB 5174 neutralized both toxin A and B
activity. The results also indicate the toxin neutralizing capacity of
SYNSORB 5174 was comparable to SYNSORB Cd and SYNSORB 179A. Thus, a support
comprising more than one oligosaccharide ligand can be used to bind both
toxin A and toxin B.
Chemical methods for the synthesis of oligosaccharide structures can be
accomplished by methods known in the art. These materials are generally
assembled using suitably protected individual monosaccharides.
The specific methods employed are generally adapted and optimized for each
individual structure to be synthesized. In general, the chemical synthesis
of all or part of the oligosaccharide glycosides first involves formation of
a glycosidic linkage on the anomeric carbon atom of the reducing sugar or
monosaccharide. Specifically, an appropriately protected form of a naturally
occurring or of a chemically modified saccharide structure (the glycosyl
donor) is selectively modified at the anomeric center of the reducing unit
so as to introduce a leaving group comprising halides, trichloroacetimidate,
acetyl, thioglycoside, etc. The donor is then reacted under catalytic
conditions well known in the art with an aglycon or an appropriate form of a
carbohydrate acceptor which possesses one free hydroxyl group at the
position where the glycosidic linkage is to be established.
A large variety of aglycon moieties are known in the art and can be attached
with the proper configuration to the anomeric center of the reducing unit.
Appropriate use of compatible blocking groups, well known in the art of
carbohydrate synthesis, will allow selective modification of the synthesized
structures or the further attachment of additional sugar units or sugar
blocks to the acceptor structures.
After formation of the glycosidic linkage, the saccharide glycoside can be
used to effect coupling of additional saccharide unit(s) or chemically
modified at selected positions or, after conventional deprotection, used in
an enzymatic synthesis. In general, chemical coupling of a naturally
occurring or chemically modified saccharide unit to the saccharide glycoside
is accomplished by employing established chemistry well documented in the
The supports to which the oligosaccharide structures of the present
invention are bound or immobilized include a wide variety of biocompatible
materials known in the art. Water soluble biocompatible polymers such as
hydrogels, carboxymethyl celluloses, synthetic polymers, and the like are
particularly preferred. In particular, these supports are useful for
delivery to the gut, especially prolonged delivery. Useful supports are
non-absorbable, that is to say that they may be soluble or insoluble, so
long as they are not absorbed by the body.
Solid supports are particularly useful for certain applications. Such solid
supports to which the oligosaccharide structures of the present invention
are bound may be in the form of sheets or particles. A large variety of
biocompatible solid support materials are known in the art. Examples thereof
are silica, synthetic silicates such as porous glass, biogenic silicates
such as diatomaceous earth, silicate-containing minerals such as kaolinite,
and synthetic polymers such as polystyrene, polypropylene, and
polysaccharides. Preferably the solid supports have a particle size of from
about 10 to 500 microns for in vivo use. In particular, particle sizes of
100 to 200 microns are preferred.
The oligosaccharide structure(s) is covalently bound or noncovalently
(passively) adsorbed onto the support so as to be immobilized. The covalent
bonding may be via reaction between functional groups on the support and the
compatible linker arm of the oligosaccharide structure. It has unexpectedly
been found that attachment of the oligosaccharide structure to the
biocompatible support through a compatible linking arm provides a product
which, notwithstanding the support, effectively removes toxin. Linking
moieties that are used in indirect bonding are preferably organic
bifunctional molecules of appropriate length (at least one carbon atom)
which serve simply to distance the oligosaccharide structure from the
surface of the support.
The compositions of this invention are preferably represented by the
where OLIGOSACCHARIDE represents an oligosaccharide group of at least 1
sugar unit which group binds to toxin B or toxins A and B, Y is oxygen,
sulfur or nitrogen, R is an aglycon linking arm of at least 1 carbon atom,
SUPPORT is as defined above, and n is an integer greater than or equal to 1.
Oligosaccharide sequences containing about 2 to 10 saccharide units may be
used. Sequences with about 2 to 4 saccharide units are preferred. In some
instances, more than one oligosaccharide group may be linked to the support,
e.g., one oligosaccharide group which binds toxin B and another which binds
toxin A, to provide a composition which binds to more than one toxin moiety.
Numerous aglycon linking arms are known in the art. For example, a linking
arm comprising a para-nitrophenyl group (i.e., --OC6 H4 pNO2)
has been disclosed . At the appropriate time during synthesis, the nitro
group is reduced to an amino group which can be protected as N-trifluoroacetamido.
Prior to coupling to a support, the trifluoroacetamido group is removed
thereby unmasking the amino group.
A linking arm containing sulfur has been disclosed . Specifically, the
linking arm is derived from a 2-bromoethyl group which, in a substitution
reaction with thionucleophiles, has been shown to lead to linking arms
possessing a variety of terminal functional groups such as, --OCH2
CH2 SCH2 CO2 CH3 and --OCH2 CH2 SC6
H4 -pNH2. These terminal functional groups permit reaction to
complementary functional groups on the support, thereby forming a covalent
linkage to the support. Such reactions are well known in the art.
A 6-trifluoroacetamido-hexyl linking arm, (--O--(CH2)6
--NHCOCF3) has been disclosed in which the trifluoroacetamido
protecting group can be removed, unmasking the primary amino group used for
Other exemplifications of known linking arms include the
7-methoxycarbonyl-3,6,dioxaheptyl linking arm  (--OCH2
--CH2)2 OCH2 CO2 CH3); the
2-(4methoxycarbonylbutan-carboxamido) ethyl  (--OCH2 CH2 NHC(O)(CH2)4
CO2 CH3); the allyl linking arm  (--OCH2 CH.dbd.CH2)
which, by radical co-polymerization with an appropriate monomer, leads to
copolymers; other allyl linking arms  are known (--O(CH2 CH2
O)2 CH2 CH.dbd.CH2). Additionally, allyl linking arms can be
derivatized in the presence of 2-aminoethanethiol  to provide for a
linking arm --OCH2 CH2 CH2 SCH2 CH2 NH2. Other
suitable linking arms have also been disclosed [21-23, 25, 26]. The
particular linking employed to covalently attach the oligosaccharide group
to the support is not critical.
We have found that synthetic oligosaccharide sequences covalently attached
to a biocompatible support, e. g., Chromosorb P.TM. (SYNSORB) may be used to
bind toxin B. These compositions are useful to treat or prevent CDAD, PMC
and other conditions associated with C. difficile infection. When a solid
support is to be used, SYNSORB is particularly preferred for these
compositions because it is non-toxic and resistant to mechanical and
In studies using rats (a widely accepted model for preclinical studies,
since they are predictive of human response), SYNSORBs have been found to
pass unaffected through the rat gastrointestinal tract. They were found to
be eliminated completely and rapidly (99%.eliminated in 72 hours) following
oral administration. Additionally, the high density of oligosaccharide
moieties on SYNSORBs is particularly useful for binding toxins which have
carbohydrate binding affinity. For example, toxin A is thought to possess
multiple oligosaccharide binding sites .
Non-peptidyl linking arms are preferred for use as the compatible linking
arms of the present invention. The use of glycopeptides is not desirable
because glycopeptides contain several, often different, oligosaccharides
linked to the same protein. Glycopeptides are also difficult to obtain in
large amounts and require expensive and tedious purification. Likewise, the
use of BSA or HSA conjugates is not desirable due to questionable stability
in the gastrointestinal tract when given orally.
Covalent attachment of an oligosaccharide group containing a toxin B binding
unit through a non-peptidyl spacer arm to an inert support permits efficient
binding and removal of toxin B or toxins A and B from a sample to be
analyzed for the presence of toxin B (or toxins A and B) or from the
intestine of a patient suffering from or susceptible to CDAD, PMC or another
condition associated with C. difficile infection. When the oligosaccharide
is synthesized with this compatible linker arm attached (in non-derivatized
form), highly pure compositions may be achieved which can be coupled to
D. Pharmaceutical Compositions
The methods of this invention are achieved by using pharmaceutical
compositions comprising one or more oligosaccharide structures which bind
toxin B attached to a support.
When used for oral administration, which is preferred, these compositions
may be formulated in a variety of ways. It will preferably be in liquid or
semisolid form. Compositions including a liquid pharmaceutically inert
carrier such as water may be considered for oral administration. Other
pharmaceutically compatible liquids or semisolids, may also be used. The use
of such liquids and semisolids is well known to those of skill in the art.
(See, e.g., Remington's Pharmaceutical Sciences, 18th edition, 1990.)
Compositions which may be mixed with liquid or semisolid foods such as
enteral nutritional formulas, applesauce, ice cream or pudding may also be
preferred. Formulations, such as SYNSORBs, which do not have a disagreeable
taste or aftertaste are preferred. A nasogastric tube may also be used to
deliver the compositions directly into the stomach.
Solid compositions may also be used, and may optionally and conveniently be
used in formulations containing a pharmaceutically inert carrier, including
conventional solid carriers such as lactose, starch, dextrin or magnesium
stearate, which are conveniently presented in tablet or capsule form. The
(OLIGOSACCHARIDE-Y-R)n -SUPPORT composition itself may also be used
without the addition of inert pharmaceutical carriers, particularly for use
in capsule form.
Doses are selected to provide neutralization and elimination of toxin B
found in the gut of effected or at risk subjects. Useful doses are from
about 0.25 to 1.25 micromoles of oligosaccharide/kg body weight/day,
preferably about 0.5 to 1.0 micromoles of oligosaccharide/kg body
weight/day. Using SYNSORB compositions, this means about 0.5 to 1.0 gram
SYNSORB/kg body weight/day, which gives a concentration of SYNSORB in the
gut of about 20 mg/ml. For subjects with clinical symptoms, administration
is expected to be 3 or 4 times daily, for a period of one week or until
clinical symptoms are resolved. For at risk subjects, prolonged prophylactic
administration, e.g., in enteral nutritional formulas, is indicated. The
dose level and schedule of administration may vary depending on the
particular oligosaccharide structure used and such factors as the age and
condition of the subject.
As discussed previously, oral administration is preferred, but formulations
may also be considered for other means of administration such as per rectum.
The usefulness of these formulations may depend on the particular
composition used and the particular subject receiving the treatment. These
formulations may contain a liquid carrier that may be oily, aqueous,
emulsified or contain certain solvents suitable to the mode of
Compositions may be formulated in unit dose form, or in multiple or subunit
doses. For the expected doses set forth previously, orally administered
liquid compositions should preferably contain about 1 micromole
We have found that C. difficile toxin B may be neutralized by certain
oligosaccharide sequences which bind the toxin. In particular, synthetic
oligosaccharides covalently attached to supports via non-peptidyl compatible
linker arms have been found to neutralize toxin B or toxins A and B
effectively. Examples of such compositions are certain SYNSORBs, which
neutralize the activity of toxin B or toxins A and B.
We have tested the ability of several oligosaccharide sequences attached to
Chromosorb P via 8-methoxylcarbonyloctyl (MCO) or urea-like spacer arms to
neutralize toxin B or toxins A and B. The oligosaccharde sequences attached
to supports useful in the present invention are those which bind toxin B
and, in some cases, both toxins A and B.
The binding affinity of an oligosaccharide to toxin B is readily detectable
by a simple in vitro test, as for example, set forth in Example 3 below. For
the purposes of this invention, oligosaccharide sequences attached to
supports which bind toxin B means those compositions which reduce
cytotoxicity in CHO cell assays by at least 50%.
Several different oligosaccharide sequences attached to supports via
compatible linker arms have been found to have the ability to neutralize
toxin B (and in some cases, toxin A and B) activity. These sequences, and
others that also bind toxin B, may be used to treat or prevent CDAD, PMC and
other conditions associated with C. difficile infection. The optimal time
for complete removal of toxin B activity was found to be about 4 hours at
37oC., using a concentration of SYNSORB of 20 mg in 1 ml sample.
Since each gram of SYNSORB contains approximately 0.25 to 1.0 micromoles
oligosaccharide, the total amount of oligosaccharide to be given in a daily
dose would range from 7.5 to 30 micromoles, using a gut volume of four
Treatment or prevention of CDAD, PMC or other conditions associated with C.
difficile infection may be accomplished by oral administration of
compositions containing oligosaccharide sequences covalently bound to a
support via a compatible linker arm (e.g., SYNSORBs). For example, SYNSORBs
have been found to pass through the stomach of rats intact. This means that
they are intact when they contact toxin B in the intestinal tract.
Subsequent elimination of intact SYNSORB with toxin B bound to it results in
elimination of toxin B from the patient.
Oligosaccharide sequences covalently attached via compatible linker arms to
a support, e.g., SYNSORBs, are useful to treat individuals who suffer from
multiple episodes of diarrhea. Upon initial reoccurrence of diarrhea,
patients would be treated with SYNSORB to remove toxin B or both toxin A and
toxin B from the intestine. The removal of toxin A prevents the initial
tissue damage to the intestinal lining, which leads to prevention or
reduction of diarrhea. Removal of toxin B prevents the cytotoxicity of this
toxin to the intestinal and colonic cells, which also leads to prevention or
reduction of diarrhea. No further treatment with antibiotics need be given,
allowing the re-establishment of the normal intestinal microflora within the
gut. The advantage of such treatment is that it does not affect the
recolonization of the intestinal tract by normal microflora. Treatment until
discontinuance of diarrhea would allow complete recovery.
In addition to its usefulness in patients suffering from recurring diarrhea,
treatment with oligosaccharide sequences covalently attached via compatible
linker arms to supports, e.g., SYNSORBs, may be used to treat all
individuals who suffer from or are prone to develop CDAD, PMC or other
conditions associated with C. difficile infection. The use of the
oligosaccharide-support compositions of the present invention in combination
with antibiotic therapy will be able to reduce the diarrhea more
effectively, leading to more rapid recovery.
Toxin B and/or toxin A may be measured directly on the surface of the
oligosaccharide-containing support using any suitable detection system. For
example, radioactive, biotinylated or fluorescently labelled monoclonal or
polyclonal antibodies specific for the toxin may be used to determine the
amount of toxin bound to the support. A wide variety of protocols for
detection of formation of specific binding complexes analogous to standard
immunoassay techniques is well known in the art.
Claim 1 of 12 Claims
What is claimed is:
1. A pharmaceutical composition useful in treating or preventing Clostridium
difficile associated diarrhea (CDAD) and related conditions initiated by C.
difficile toxin B, which composition comprises:
a) at least one oligosaccharide, sequence covalently attached to a
pharmaceutically acceptable inert support through a non-peptidyl compatible
linker arm, wherein said oligosaccharide sequence binds C. difficile toxin
b) a pharmaceutically acceptable carrier, wherein said composition is
capable of being eliminated from the gastrointestinal tract.
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