Note: Descriptions are shown in the official language in which they were submitted.
2 1 ~2~
Wo 9a/23605 PCTIUS95/02388
Descri~tion
~qethod For Treating And Inhibiting
Gastric And Duodenal Ulcers
This application claims the benef it of priority under
5 35 U.S.C. 120 to co-pending U.S. Application Serial No.
08/104,483 filed on September 28, 1993, which is a
Continuation-in-Part Application of U. S . Serial No.
07/922,519 file on July 31, 1992, now ;lhAnt~ n~d~
Technical Field:
The present invention relates a method for treating
and inhibiting gastric and ~ dF~n~ 1 ulcers in a patient .
Ba~:kuluu.ld Art:
Infection by the gram-negative, spiral,
microaerophilic bacterium E~elicobacter pylori (~. pylori),
formerly known as Campylobacter pylori (C. pylori), is a
primary cause of non-autoimmune gastritis, is a factor in
peptic ulcer disease and is more common in patients with
gastric carcinoma. First isolated by Warren (Lancet (1983)
1:1273) and Marshall (Lancet (1983~ 1:l273-5), H. pylori
has been isolated in gastric tissue biopsies in patients
throughout the world. While the precise r-^hiln; ::m of
inflammation is not well understood, ~. pylori iS found in
association with the apical surf aces of gastric mucous-
secreting cells.
Due to the site specificity of att~ ~, it has been
suggested that there are specific attachment sites for ~.
pylori which exist on gastric and ~ nAl mucous-secreting
cells. Nl .,us studies have been undertaken to attempt to
identify the specif ic binding site of ~. pylori .
Evans et al (Infection and Immunitv (1988) 56:2896-
2906) reported that ~. pylori binding to an erythrocyte
receptor, as measured by hemagglutination inhibition, is
preferentially inhibited by N-acetylneuraminyl-~(2 ~3)-Gal
WO 95/2360s 2 ~ 8 3 3 2 9 1 ~".J~ ~
--2--
,B1--4 Glc (hereill after NeuAc(2--3)-lactose) as compared with
N-acetylneuraminyl-~ (2--6) -Gal ~1--4 Glc (herein after
NeuAc (2--6) -lac-tose) . Sialoproteins which contain the
NeuAc(2--3)Gal isomer of NeuAc-lactose, i.e., human
5 erythrocyte glycophorin A, fetuin, and human ~2-
macroglobulin, al50 inhibited ~. pylori binding, but at
higher concentrations (mg/ml ) than that observed f or
NeuAc(2--3)-lactose, while no inhibition was observed for
the ,ULL~ 1;ng asialoglycoproteins.
Evans et al ibid, measured the hemagglutination
inhibiting ability (HIA) of several "- containing a
NeuAc-lactose structure. Based on the hemagglutination
inhibition activity, the re6earches de~rm; nerl that in
order to produce 100% IIAI, 1. 000 mg/ml Of ~2-Macroglobulin
was needed, 0.500 mg/ml o~ fetuin Was needed, 0.250 mg/ml
of Glycophorin A was needed and 0 . 078 mg/ml of bovine
NeuAc-lactose was needed. Based on their hemagglutination
inhibition st~dies the researches show fetuin to be about 2
times as effective as ~2-Macroglobulin but only 0.156 times
20 as effective as bovine NeuAc-lactose which comprises about
80% of NeuAc(2 ~3)-lactose and 20% of NeuAc(2--6)-lactose.
Evans et al (Infection and ImmunitY (1989) 57:2272-
2278) have also observed that H. pylori binds to monolayers
of Y-1 mouse adrenal cells. But, this adherence can be
25 prevented by pretreating the Y-1 cells with neur~mini~ e
and is blocked by fetuin. ~iowever, it should be noted that
there is no relat;nn~h;~ between Y-1 mouse adrenal cells
and gastric tissue.
Linawood et al ~Lancet (1989) 2:238-241) have reported
3 0 the isolation of a gastric glycerolipid material which they
observed to behave as a receptor f or ~ . pylori . The
material was isolated from red blood cells, and mucosal
scrapings of pig stomach and human stomach. ~he
investigators postulated that the material was a sulphated
35 alkylacylglycero-lipid, but the actual ~LLuuLuLc~ of this
W0 95/2360~ 2 7 8 ~ 3 ~ oo
--3--
material was not been reported. Subsequent investigations
(Linqwood et al., Infection and ImmunitY (1992) 60:2470-
2474 ) showed that this receptor i6
phosphatidylethanolamine .
Linqwood et al., Infection and Immunity (1992) 61:
2472-2478 report that Helico~acter pylori specifically
recognizes phosphatidylethanolamine,
gangliotriaosylceramide and gangliotetraosylceramide and
the isolation of an S-adhesin which is believed to be
responsible for the lipid-binding specificity of this
organism. However, none of the ~ c which are
reported as specifically recognized by H. pylori, are
sialylated oligo~c~ h~rides .
Tzovelekis çt al (Infection and ImmunitY (1991)
59:4252-4253) reported binding inhibition of H. pylori to
HEp-2 cells by gastric mucin. The investigators observed
that purif ied mucin showed the greatest inhibition of H .
pylori binding while asialomucin exhibits somewhat
tlimin;~hecl inhibition and periodate-n~ d mucin
20 exhibited the lowest level of binding. On these
observations, the researchers concluded that sialic acids
are at least partially responsible for the binding
interaction between H. pylori and human gastric mucin.
However, it should be noted that mucin contains a variety
25 of different saccharide groups and linkages.
Boren et al (Science (1993) 262:1892-1895) have
reported that Lewisb blood group and H type I antigens
mediate H. pylori att~ L to human gastric mucosa.
Fauchere et al Microbial Pathogenesis, 1990 9 427-439
30 report that H. pylori adherence can be assessed by
microtiter assays and involves a bacterial surface material
which co-purifies with urease and is different from the N-
acetyl-neuraminyl-lactose binding hemagglutinin.
Rob;ncnn et al report in J. Med. Microbiol. (1990) 33
35 277-284 that pre-treatment of human erythrocytes with
~ ~ 8332~
WO 95/23605 .
--4--
neurAm;niflAr-~ from Arthrobacter ureafaciens and Clostridium
perfringens abolished hemagglutination by the soluble, but
not the cell-asGociated hemagglutinin, which suggests that
sialic acid is not involved in binding inhibition of ~.
5 pylori.
Dunn et al Reviews of Infectious Diseases
1991;13 (Suppl 8): (S~57-64) report binding inhibition
studies by Nean Fluorescence Intensity by LLeai L of
materials with a neur~miniflAr~e. The researchers report a
10 16.8% decrease in MFI upon neurAm;n;flAre treatment of N-
acetylneuraminyllactose of 16.8%, a 29.8% reduction with
fetuin and an 8 . 6% reduction of asialofetuin. However, the
researchers report a 3 0% increase upon treatment of KAT0
cells with neur~m;n;flAge. Such results call into ~auestion
15 the role of sialylation in the site specif ic binding of H.
pylori .
Saitoh et al report a sulfate-containing glycerolipid
as a ligand which is specifically r~co~n; ~fl by ~. pylori.
While there have been ~/U6 studies into
20 with El. pylori binding inhibition, it clear that the
literature is replete with conflicting evidence.
Moreover, there is even a lack of a crln~pnqllc as to
the signif icance of the methods of testing f or ~ . pylori
binding inhibition. Hemagglutination assays have been used
25 by many different researchers (see for example Evans et a
(Infection Anfl ImmunitY (1988) 56:2896-2906), however
Fiaueroa et Al report in Journal of Infection (1992) 24
263-267, an adh~rence r- ' An;r-, which is nQ~ fl~-p~nfl;nq on
the expression of specific hemagglutinin antigen. This
30 report openly questions the relatinnr~h;~ between
hemagglutination inhibition and Er. pylori binding
inhibition. Furth ~, many of the cell surface adhesion
systems, used to test for }~. pylori binding inhibition,
have no relat;~nch;p to gastric tissue at all.
W095r23605 2 ~ 2 9 PCTrUSgSro2388
--5--
In addition to the numerous binding inhibition
studies, methods have been pursued to treat gastric and
duodenal ulcer patients.
Colloidal bismuth subcitrate (CBS~ has been used
successfully in treating both gastric and ~ n~ l ulcer
PA~ (for a review, see Lambert in Reviews of
Infectious ~iS~oc (1991) 13 (Suppl. 8) :S691-5. CBS has
proven effective as a histamine H~ antagonist and has been
associated with lower relapse rates after cessation of
therapy attributed to CBS's ability to eradicate ~. pylori.
Bismuth subsalicylate t8SS) has also been observed to
inhibit ~. pylori.
Coleman et al (U.S. Patent No. 4,935,406) reported a
method for relieving gastrointestinal disorder, resulting
from ~. pylori population, through the administration of
bismuth (phosph/sulf ) ated saccharide compositions. The
saccharide compositions according to this method are simple
phosphates and sulf ates of aldose and ketose
-- o ~ crh ~ rides .
~l; n1 r~1 trials have been reported (Evans et al, Ann.
Internal Med. (1991) August 15, 115(4) :266-9) in treating
Er. pylori using ranitidine in conjunction with a "triple
therapy" of r;ll;n or tetracycline, metronidazole (an
antiprotozoal), and BSS. The clinical studies suggested
that ulcer healing was more rapid in patients receiving
ranitidine plus the "triple therapy" than in patients
receiving ranitidine alone.
The strong role that ~. pylori plays in peptic ulcers
has led to an anno--n~ L in February 1994 by an
;n1~p~n~1~nt advisory panel of experts convened by the
National Institutes of Health, to advise that patients
nl~s~rl with peptic ulcers and ~. pylori be treated for
two weeks with a combination of antibiotics. A copy of the
Consensus Development Conf erence statement ~elicobacter
pylori ln Pe~tic l)lcer Disease is available from the
W0 95/23605 ~ 1 8 3 3 2 9 PcT/US95/02388
--6--
National Institutes of Health. There was no re~ '~tion
f or any other t~pe of therapy .
However, long-term eradication of this organism has
been difficult with these therapies. The antibiotic
5 approach runs the risk of the dev~ of new antibiotic
resistant strains. In addition, there are side affects
associated from long term antibiotic therapy, which are
unpleasant and make compliance with such a ~ a; L regime
more difficult. Thus, a method of treating H. pylori with
10 good long-term eradication has not yet been developed.
As evidenced by the prior art identified above, there
are a variety of structurally diverse ~ _ '¢ identified
as candidates for being r~¢p~n¢;hle for site specific
at~;~ L of Er. pylori. The state of the art is further
15 complicated by the variety of different in vitro assays
used for predicting Er~ pylori binding inhibition, for which
there is no identified correlation with effective El. pylori
binding inhibition in mammals (Fiqueroa et al Journal of
Infection (1992) 24 263-267). Even though 3' sialyl
20 lactose has previously been identified as having
hemagglutination inllibiting activity, and therefor
speculatively identif ied as being a gastric colonization
factor rEvans et al (Infection and Immunitv (1988) 56:2896-
2906~ ) it was o]lly one u~-d of many identified as
25 possible candidates. The same publication, also reports
the same activity, albeit only 0.156 times as great, for
the compound fetuin. Accordingly, the state of the art,
would not allow one to have selected 3 ' sialyl lactose from
the many other and structurally diverse compounds, as a
30 particularly effective means for inhibiting Er~ pylori
binding inhibition in mammals.
Based on the inventors ' studies, it has now been
discovered that 3 ' sialyl lactose is a surprisingly
effective inhibitor of Er~ pylori binding inhibition in
WO95/2360~ 2 ~ 9 I~"~, -"`7'~0Q
--7--
mammals. And this finding has been validated by the
inventors through in vivo l; i~n test data.
In addition, contrary to earlier reports, the
inventors o~ the present invention have disuuveLed that
5 fetuin has minimal activity in inhibiting binding of N.
pylori cells, in v~tro. The inventors have discovered that
the binding inhibition activity associated with fetuin,
appears to be attributable to a high molecular weight
impurity which is a contaminate of commercially available
10 fetuin. Based on their assay, the inventors of the present
invention have disc:uvere~ that 3 ' sialyl lactose has an
ability to inhibit binding of N. pylori to a degree far in
excess of what would have been expected in light of that
previously reported for fetuin. From previous reports, one
would expect that 0 .156 times as much 3 ' sialyl lactose
would be needed to achieve the same effectiveness, as
achieved with fetuin. But since the inventors of the
present invention have discovered that fetuin has minimal
effectiveness in binding inhibition of N. pylori cells,
20 their discovery that 3' sialyl lactose surprisingly
strongly inhibits N. pylori, provides that 3 ' sialyl
lactose can be used in an amount far below that which would
have been predicted from the prior art. It is on the basis
of this discovery that the present inventors have realized
25 that 3 ' sialyl lactose is unexpectedly superior in
inhibiting N. pylori in mammals.
Disclosure of the Invention
Accordingly, one object of the present invention is to
a method for treating and/or preventing gastric and/or
30 ~ o~f~n~l ulcers.
Another object of the present invention is to provide
a method for inhibiting Nelicobacter-pylori infection
and/or reinfection to r-r~ n tissue, including
WO s5l23605 2 i 8 3 3 ~ 9 P~
--8--
eliminating ~elir~hAnt~r pylori from the stomach and/or
rdr~nllm of a patient in need thereof.
Another obj ect of the present invention is to provide
a pharmaceutical composition f or inhibiting ~elicobacter
pylori infection or reinfection of r ~ n tissue,
lnr~ ;ng eliminating }~r~7;crhArter pylori from the stomach
and/or ~lnr-l~mlm of a patient in need thereof and for
treating and/or preventing gastric and/or ~ n~ 1 ulcers .
All of the above obj ects of the present invention and
other objects which are apparent from the description of
the invention given herein below have been discov~r ad by
the inventors to be satisf ied by administering a
composition comprising an oligosacrh~ride of Formula I
(NeuAc--~ (2--3 )--pGal--~ ( 1)--(--X--) m~ ( ~Y--) n~) p~Z
wherein
X = a rhr~m;ri~l bond or a group capable of lin7.~ing the
p galactose to either the lin7sing group Y or the
multivalent support Z;
wherein the C1 glycosidic oxygen of galactose may be
replaced by N, S or C;
Y = a linking group;
Z = a multivalent support;
m = 0 or 1;
n = 0 or 1; and
p = an integer of 2-1,000.
The present invention is also provided for by an
olig~sarrh~ride composition of Formula II
NeuAc-~ ( 2 - 3 ) -pGa 1- ~ ( 1 ) -A
wherein
A = a group capable of bonding to the p galactose;
wherein the Cl glycosidic oxygen of galactose may be
replaced by N, S or C.
Contrary to previous reports ( Evans et al ( Inf ertion
and ImmunitY _(1988) 56:2896-2906) ), the inventors of the
present invention have discovered that an oligns~rh~ride
21 83329
WO 95/23605 r~"~ uu
_9_
of Formula I or Formula II, specifically NeuAc ~2 ~3)Gal
,B1-4 Glc (herein after also as 3 ' sialyl lactose) is
dramatically more effective (more than 6. 41 times more
effective) at inhibiting ~elico~acter pylori than is
5 fetuin, when treating mammals. Specifically, Applicants
have discovered that 3 ' sialyl lactose has unexpectedly
uv~d activity in a method for treating ~. pylori
infections in mammals.
In addition, the inventors of the present invention
10 have discovered that a multivalent presentation of an
oligosaccharide (i.e. the oligoc~crh~ride of Formula I) is
unexpectedly superior, on a molar basis based on the
oligosaccharide groups, than the monovalent presentation of
the same oligosaccharide.
In addition, a method in which a pharmaceutical
composition comprising the oligos~- hAride of Formula I
and/or Formula II alone, or in combination with an ~I2
blocker, an antibiotic, oligosacrhAride ~ _ - and/or an
antiulcerative ~ __ is administered to a mammal, has
been found by the inventors to be effective at inhibiting
the binding of ~ellco~acter pylori to the gastric and
;-l mucosa and relieving the effects of gastric and
~llnd~nAl ulcers.
Best Mode for ~'Arryinq Out the Invention
The following abbreviations are used throughout the
text: "Gal" for galactose; "Glc" for glucose; "NeuAc" for
N-Acetylneuraminic acid.
The olign~acrhAride compound of Formula I
(NeuAc--~(2--3)--pGal--,B(l)--(--X--)m--(--Y--)n--)p--Z
3 0 wherein
X = a rhf~m; cAl bond or a group capable of linking the
p galactose to either the linking group Y or the
multivalent support Z;
Wo gsl2360S 3 3 2 9
--10--
wherein th~ C1 glycosidic oxygen of galactose may be
replaced by N, S or C;
Y = a linki.ng group;
Z G a multivalent support;
m = 0 or 1;
n = 0 or 1; and
p = an integer of 2-1, 000 is admini6tered according
to the present method.
For example X can be a substituted C1 20 alkyl group, a
substituted C1 20 alkyl carboxylic ester group, a substituted
C1 ,0 alkyl carbo~y amide group, a hydroxy terminated
polyether, an amine terminated polyether, inositol, an
oligos:~rrh~ridet a f~ rh~ride or a r ns~r~ rh:~lride with
the tl~rm;npll reducing end of the olign~rrh~ride,
fl; ~:Arrh~ride or ~ rh:~ride in the pyranose or open
chain form, an azaolignc Icrh~ride, an azadisaccharide or an
;:~7: ns;~ccharide wit_ the tPrm;nAl reducing end of the
azaoligns~-ch~ride, ~7~;s~crh~ride or ~ rh;~ride in
the pyranose or open chain form, wherein said substitution
is capable of reacting with the linking group of the
multivalent support, such as a hydroxyl group or an amine
group .
Preferably the group X is a monosaccharide hexose
group such as glucose, N-acetylglll~nr~m;n~, galactose, N-
acetylqalactn~m;n~, mannose, fucose, allose, altrose,
gulose, idose, talose and rh~mnnse In addition, a
suitable group X is a reduced form of the above-identif ied
hexose groups, such as glucitol.
when the group X is capable of bonding directly to the
multivalent support, then n is o.
When the Cl glycosidic oxygen of galactose is capable
of bonding directly to the multivalent support, then both m
and n are 0.
A suitable linker group has one tt~rm;n~l portion of
the Y group capable of bonding with the group X, while the
W0 95l23605 ~ 1 ~ 3 :~ 2 ~
other tPrmin~ end is capable of bonding with the
multivalent support.
The chemistry n~c~s!;~ry to link the group X and
linking group Y and to link linking group Y to the
5 multivalent support is well known in the f ield of linking
chemistry. For example when X is a saccharide such as an
olig~sacrh~ride, a q;c~rrh~ride or a - Ir~crh~ride, a bond
between X and Y can be formed by reacting an aldehyde or
carboxylic acid at Cl of the X group or any aldehyde or
10 carboxylic acid group introduced onto the X group by
oxidation, with the Y group, to form a suitable bond such
as -NH-, -N(R) - where R is Cl 2~ alkyl, a l-ydL~,~ycllkylamine,
a amide, an ester, a thioester, a th i o;`m; cl~ .
When X is a saccharide such as an olig~c~crh~ride, a
15 ~ crh~ride or a -~ crh~ride, a bond between X and Y
can be formed by reacting the Cl hydroxyl group, in the
pyranose form with an acylating agent and a molecular
halide, followed by reaction with a nllrleoph;lP to form a
suitable bond such as -NH-, -N(R) - where R is Cl 2~ alkyl, -
20 S- and -0-. This type of linking chemistry is described by
Stowell et al Advances in CaLbohydL~Ite Chemistry and
Biochemistry, 37 (1980) p 225+.
A suitable multivalent support is a cu~ .ul.d with
multiple binding sites to a t~rm;n:ll end of the linking
25 group, which is not bound to the group X of the linking
group, with multiple binding sites to the group X, or with
multiple binding sites to the Cl glycosidic oxygen of
galactose. Examples include but are not limited to a
polyol, a polysaccharide, polylysine, avidin, a
30 polyacrylamide, dextran, lipids, lipid emulsions,
1 ;roSI -~l a dendritomer, human serum albumin, bovine serum
albumin or a cyclodextrin.
The oligf~rrh~ride is provided as a multivalent
molecule according to Formula I. In this ~mho~ the
35 oligr~arrh~ride portion is bound to a multivalent support
WO 9~23605 2 ~ 8 3 3 2 9
using known technique6 60 as to produce a conjugate in
which more than one individual molecule of the
oligos~rrhAride is covalently attached through a linker to
the multivalent support. The multivalent support is
5 sufficiently long to provide a multivalent molecule leaving
from between 2-1,000 (i.e. p = an integer of 2-1,oOO),
preferably 2-100, more preferably 2-30 molecules of the
olig~sArrh Iride portion bound to the multivalent support.
The oligosaccharide portion can be bound to the
10 multivalent support via the free anomeric carbon of the
group X. Alternatively, the oligocArrhAride portion can be
bound via a phenethylamine-isothiocyanate derivative as
described by Smith et al. Complex Ca, ~ol~d~ates part C,
Methods in Enzymology, volume L, Ed by V. Ginsburg (1978),
p 169-171. It is preferable that the oligrcArrhAride of
Formula I remains soluble in water, however it is also
possible to administer the olig~s~A~crhAride of Formula I in
the form of polymer particle6.
For example, the oligr,~ArrhAride portion of Formula I
2 0 may be bound to a support to f orm a bead wherein the
surf ace of t_e bead is bound with the oligosaccharide
portion of Formula I.
The oligosacrhAride composition of Formula II
NeuAc-~ (2-3 ) -pGal-,~ ( 1) -A
wherein
A = a group capable of bonding to the p galactose;
wherein the Cl glycosidic oxygen of galactose may be
replaced by N, S or C;
i5 administered according to the present method.
For example A can be a Cl 20 alkyl group, a Cl 20 alkyl
carboxylic ester group, a Cl 20 alkyl carboxy amide group, a
polyether, inositol, an oli~nsArrhAride,- a ~l; cArrhAride or
a -~ -rh~ride with the t~rm;nAl reducing end of the
olignca~rhAride, disaccharide or monosaccharide in the
pyranose or open chain form, an azaoligosArrhAride, an
2 ~ 2 9
WO 9S/23605 P~1/l~ ~JI
--13--
A~At1icArrhAride or an A r,sAcrhAride with the terminal
reducing end of the azaoligncAcrhAride~ :~7A~l; cacrhAride or
A ~nosAcrhAride in the pyranose or open chain form,
Preferably the group A is a - - cr~hAride hexose
5 group such as glucose, N-acetylgl1lcosAm;n~, galactose, N-
acetylgalactosAmin~, mannose, fucose, allose, altrose,
gulose, idose, talose and rhamnose. In addition, a
suitable group A is a reduced form of the above-identified
hexose groups, such as glucitol.
The correspnn~l; ng N and S glycosides of galactose can
be prepared by conventional methods known to those of
ordinary skill in the art from galactose followed by
attachment of a sialyl acid group at the 3 position by
conventional methods . The corrc~cpnnr9; n~ C glycoside of
15 galactose can be made by conventional synthetic organic
techniques, followed by at~ L of a sialyl acid group
at the 3 position by conventional methods.
Any known suitable rhArr~ceutically acceptable cations
may be used with the oligoc~c rhArides of Formula I and
20 Formula II, to form a salt of the carboxylic acid group.
Suitable cations, include conventional non-toxic salts
including a metal salt such as an alkali metal salt (e.g.
sodium salt, potassium salt, etc. ) or an ~ilkAl ;n-~ earth
metal salt (e.g. calcium salt, magnesium salt, etc.), an
25 ammonium salt, an organic base salt (e.g. trimethylamine
salt, triethylamine salt, pyridine salt, picoline salt,
dicyclohexylamine salt, N, N'-dibenzylethyl-~n~;Am;ne salt,
etc. ), an organic acid salt (e.g. formate, acetate,
trifluoroacetate, maleate, tartrate, meth~n-~clllfonate,
30 benzenesulfonate, tol~l~n~clllfonatel etc.), an inorganic
acid salt (e.g. hydrochloride, 11YdL~)~L~ / sulfate,
phosphate, etc. ), a salt with an amino acid (e.g. arginine
salt, aspartic acid salt, glutamic acid salt, etc. ), and
the like.
Wo ss/7360s 2 T 8 3 ~ 2 9 r~
The oli~c~ ~hArides of the present invention may be
obtained using any known method, ;n~ n~ (1)
enzymatically, using one of the inventor's method described
in published international application W0 91/16449, (2)
5 synthetically, using rl~csif ~l organic chemistry, (3) by
degradation of a natural occurring oligoc~-rh~ride,
glycolipid, or glycopeptide or (4) isolation from natural
source such as bovine colostrum. The isolation of 3 '
sialyl lactose from bovine colostrum i6 described in Veh et
al, Journal of Chromatography, 212, (1981) 313-322.
The oligosaccharides of Formula I and Formula II may
be administered in Cull; u~ Lion with a known proton pump
inhibitor or a known H2 receptor antagonist. A
~:yLes~ Lative proton pump inhibitor is omeprazole, and
15 representative H2 antagonists include cimetidine,
ranitidine, nizatidine and famotidine. The amount of
proton pump inhibitor and H2 antagonist administered in
conjunction with the present oligocac~h~ride is about the
same amount administered f or their known therapy .
20 Accordingly, effective dosages of the proton pump inhibitor
and H, can be ~f~t~rm; n~ by routine experimentation.
Alternatively a known antiulcerative c~ l u~ may be
used in conjunction with or as a replacement for the H2
receptor antagonist. Suitable antiulceratives include
25 aceglutamide aluminum complex, ~-acetamidocaproic acid zinc
salt, acet n~ 1nn~, aL~I~Lu~il, benexate hydrochloride,
bismuth subcitrate 801, bismuth subsalicylate,
carbenoxolone, cetraxate, cimetidine, enprostil,
esaprazole, famotidine, ft~ ;d~, gefarnate, guaiazulene,
30 irsogladine, misoprostol, nazatidine, ornoprostil, y-
oryzanol, pifarnine, pirenzepine, plaunotol, ranitidine,
rioprostil, rosaprostol, luL~ e, roxatidine acetate,
sofalcone, spizofurone, sucralfate, teprenone,
tr;~ o~il, trithiozine, troxipide, and zol ;m;~;nc~. The
35 amount of antiulcerative administered in conjunction with
WO95/23605 2~83~ p~,"o,.,~ 7~
--15--
the present olig~c~c~h~ride is about the same amount
administered for its known therapy. Accordingly, effective
dosage of the antiulcerative can be det~rm;n~d by routine
experimentation .
Alternatively, the oligo~acrh~rides of Formula I and
Formula II may be administered in conjunction with an
antibiotic with activity against ~. pylorl. Suitable
antibiotics include metronidazole, tetracycline, bismuth,
erythromycin, a macrolide, a quinolone, a cephalosporin and
amoxicillin. The amount of antibiotic administered in
conjunction with the present oligosaccharide is about the
same amount administered f or its known therapy .
Accordingly, effective dosage of the antibiotic can be
det~rm;nF-~l by routine experimentation.
Alternatively, the oligosaccharides of Formula I and
Formula II may be administered in conjunction with a H-type
1 or Lewisb blood group antigen or an oligoc~ h~ride such
as NeuAc-rv(2 ~6)-Gal ,l~l ~4 Glc. Suitable H-type 1 and Lewisb
blood group antigens are reported in Boren et al (Science
(1993) 262:1892-1895).
The anti-Er. pylori compositions of the present
invention contains the olig--s~c~h~rides of Formula I and
Formula II in association with any suitable liquid or
solid, pharmaceutically acceptable carrier or excipient,
preferable in a form suitable for oral or enteral
administration. In addition, the pharmaceutical
compositions of the present invention are pref erably
pyrogen free.
The pharmaceutical compositions are usually
administered as a mixture with a carrier suitably selected
;ng upon the route for administration using standard
formulations. For example, the . _u.ld of the present
invention may be administered in the form of tablets which
may be prepared using known t~f-hn;ql~-oC by adding to a
powder of the active ingredient of the present invention an
wo gsl23605 2 l 8 3 ~ 2 ~ ~
--16--
excipient such as s~arch, lactose, sucrose, glucose,
crystalline cellulose, calcium carbonate or kaolin, a
ellulosel a glucose solution, a sucrose
solution, water or ethanol, a disintegrator such as starch,
5 agar, gelatin powder, caLl,u,~y t_hylc~ lose calcium (CMC-
Ca), callo,~y L1lylcQllulose sodium (CMC-Na), crystalline
cellulose, calcium carbonate or sodium llydL.,4~llcarbonate,
or a lubricant such as r~7nF~cjllm stearate, calcium
stearate, talc, macrogoal 4,000, macrogoal 6,000 or stearic
10 acid.
The mixtur,e is then subjected to compression molding
by a conventional tableting method, and if nec~cc~ryl
applying a sugar coating by means of a concentrated sugar
solution containing e.g. gum arabic, talc,
15 polyvinylpyrrolidone, polyethyleneglycol and/or titanium
oxide, applying a film coating by means of a film-forming
agent ~efl of e.g. polyvinyl acetal
diethyl~m;nnacetate, ilydLu~y~Lu~ylmethylcellulose,
1lydL~y~lu~ylcelllllnc~, ethylcellulose or
2 0 polyvinylpyrrolidone or applying an enteric coating by
means of a film-forming agent ~ Fe~ of e.g.
ethyl c~ l oc~ phthalate, cellulose acetate phthalate or
lly ~L u~sy pL upy lmethy 1 cel lulose phtha 1 ate .
These ph2rmaceutical compositions may be in the form
25 of granules or fine granules which may be prepared by
adding to the active ingredient of the present invention a
binder such as starch, gelatin, gum arabic,
methylc~ llnce, sodium caL~u,.y thylc~lllllnse, heavy
5ilicic anhydride or light silicic anhydride, followed by
30 kne~9;n~ and granulation by usual methods; or as a powder
of the active ingredient of the present invQntion by
itself; or as capsules which may be ~L~yaL~d by adding to
the active ingredient of the present invention an excipient
such as lactose, starch or crystalline cellulose and/or a
WO 95/23605 2 ~ g 3 ~ 2 q r~
--17--
lubricant such as magnesium stearate, calcium stearate or
talc, and filling the mixture into capsules.
A solution or sUcp~n~inn may be ~ ~dl~d by adding any
diluent customarily, used in the art. For example,
5 suitable diluents include water, ethyl alcohol, propylene
glycol, polyoxyethylene sorbitol, and sorbitan esters.
Sodium chloride, glucose or glycerol may be incorporated
into such a liquid preparation in an amount sufficient to
prepare an isotonic solution. The therapeutic composition
10 may also further contain ordinary dissolving aids, buffers,
pain-alleviating agents, art preservativeS, and optionally
coloring agents, fragrances, f lavors, sweeteners and other
pharmacologically active agents such are well known in the
art .
Suitable compositions may take the form of a solution,
6uspension, tablet, coated tabolet or any rh~rr--e~ltically
acceptable form suitable for delivery to the stomach or
duodenum .
According to a preferred '~ t of the present
invention, the oligosacoh~ride or rh:~r~ eutical
compo6itions are administered orally or enterally to a
patient in need thereof to inhibit ~. pylori binding or
eliminate E~. pylo~i colonies from the patient's stomach
and/or duodenum.
Typically, suitable patients are humans. However the
present method is also applicable to treatment of animals,
;nC~ ;n~ but not limited to mammals such as pigs, cows,
horses, sheep, goats, dogs, cats, rodents and non-human
primates .
~he method of the present invention is suitable for
preventing and treating patients with dllodl~n~ 1 ulcers,
gastric ulcers and the prevention of gastric cancers in
patients .
Suitable amounts of the pharmaceutical composition
containing the oligoc~rrh~rides of Formula I and/or Formula
~ ~ 8332~
Wo 95/2360~ r~
-18-
II to be administered include those which produce an
effective stomach ~;u~ lLL~tion of oligos~c~-h~ride of from
1 llg to 10,000 mg/ml per dose, preferably lO ~Lg to 1,000
mg/ml, more preferably 0. 5mg to 50 mg/ml, most preferably 1
5 to 10 mg/ml. For example, based on an average human
stomach volume of 500 ml, a dose of 3 gm would produce an
effective stomach ~u~ LLc-tion of about 6 mg/ml.
Administration of the pharmaceutical composition
comprising the oligo~Ar-h~ride o~ Formula II is performed
10 preferably to achieve a continuous effective stomach
concentration of from 1 ,~lg to 10,000 mg/ml per dose,
preferably 10 ~g to 1,000 mg/ml, more preferably 0.5mg to
50 mg/ml, most preferably 1 to 10 mg/ml. This can be
achieved by administration, at least daily, preferably
twice daily, more preferably three times a day and most
preferably four times a day.
When administered as a multivalent molecule a
pharmaceutical composition comprising the oligosaccharide
of Formula I is administered so as to achieve a continuous
effective stomach c ullcellLLrlLion of from 1 ~g to 1,000 mg/ml
per dose, preferably 10 ,~ to 100 mg/ml, more preferably 50
,ug to 5 mg/ml, most preferably 10 ,ug to 2 mg/ml. ~his can
be achieved by administration, at least daily, preferably
twice daily, more preferably three times a day and most
preferably four times a day.
When a proton pump inhibitor, H2 antagonist, or
antiulcerative is c~ tlm; n; qtered, the composition is
formulated to provide between 10-500 mg, preferably 100-300
mg of the proton pump inhibitor, H2 antagonist, or
antiulcerative daily. For example suitable therapies
include administration of tetracycline (500 mg four times
daily), bismuth subsalicylate (two tablets four times
daily, with meals and at bedtime), and metronidazole (250
mg three times daily, with meals) each taken for a 14 day
WO 9S/2360S ~ ~ 8 3 3 2 9 PCrlUS9/ilO2388
--19--
period . Dosage f orms include such unit dosage f orms such as
tablets, c~rs~llP~l solutions or suspensions.
After eradication of the El. pylori infection or
LL-~a~ L of the ulcer, maintenance dosages of are
5 administered so as to achieve a continuous effective
stomach u.lc~ LLcltion of from 1 ,~g to 1,000 mg/ml per dose,
preferably 10 ~g to 100 mg/ml, more preferably 50 ~g to 5
mg/ml, most preferably 10 ,ug to 2 mg/ml. This can be
achieved by administration, at least daily, preferably
10 twice daily, more preferably three times a day and most
preferably four times a day.
* * *
other features of the invention will become apparent
in the course of the following descriptions of exemplary
15 ~mhoA;r-nts which are given for illustration of the
invention and are not intended to be limiting thereof.
* * *
Examl~le 1.
Cell cultures, to test for the effectiveness of Er.
20 pylori binding inhibition were prepared from human
carcinomas stomach cancer epithelial cells ~uTu-80 obtained
from the American Type Culture Collection Rockville, MD,
according to a modif ied procedure from that reported in
Fauchere et al Microbial Pathogenesis 1990;9 427-439. The
25 cultures were maintained in Basal medium Eagle containing
1096 fetal calf serum in T-75 flasks, at 37C and a 5% CO2
ai ~ ^re. Cells were harvested by trypsin/EDTA release
and plated on 96-well flat bottom microtiter plates. The
microtiter plates were incubated for 2-3 days until the
30 monolayers grew to confluence. Prior to binding inhibition
tests, the monolayer was washed with Hanks Rz~ nrefl Salt
solution (HBSS) containing Ca~ and Mgl2, 0.1%BSA, 50mM
HEPES, O . 01 phenol red or HBHPR.
W09~2360~ 2 1 83~29 ~ 3~. ~
--20--
H. pylort bacteria isolates were obtained from B.
Marshall (from 1:he University of Virginia) and grown on
sheep blood agar, collected at 48 h, waghed and 5llcp~n~e~a~
in a binding buffer of HBSS + 0.1% bovine serum albumin +
5 50mM HEPES buffer + 0. 01% phenol red or HBHPR.
In order to test for ~. pylori binding inhibition, the
~:u~ lL- ~tion of ~. pylorl which bound to the monolayer was
~ccign~d an int~ te ODsgs (optical density at 595 nm)
(about 0 . 4 OD umits) . The same concentration of bacteria
10 and test ~ _ ' were combined for 10 minutes, then
transferred onto the monolayer. Binding was allowed to
occur for 20 min at room temperature under mild agitation.
The unbound bacteria was washed away with 1 wash of HBHPR,
then 2 washes of the same buf f er without HEPES buf f er
15 (HBPR).
The amount of bacterial adhesion to the monolayer was
measured by incubating with 50 ~l urea-phenol red (UPR)
solution (0.2% urea, 0.03% phenol red in 0.85% NaCl). The
presence of bound bacteria is indicated by the presence of
20 bacterial urease which generates NH3, which raises the pH
and changes the color to purple, near at ODsgs.
ICso in mg/ml was det~rm;n-~ for each _ a tested.
The test data is reported below in Table 1:
Table 1
Molar ICso mmol/ml
activity2
25 3 ' sialyl lactose 1 6 x 10-3
6 ' sialyl lactose -- > 1 x lo-
3 ' sialyl lactose-HSAl 3 . 45 x 10-3 2 x 10-5
lactose -- > 1 x 10-2
HSA ---- > 1 x 10-4
WO95/2360S ?3~3~ p~
--21--
1 3 ~ sialyl lactose-HSA i5 a complex of 3 ' sialyl lactose with
HSA, with about 20 molecules of 3'sialyl lactose bound to
the HSA.
' relative to 3 ' sialyl lactose
The data reveals that 3'sialyl lactose, when tested in
a multivalent form was 290 times more effective on a molar
basis than 3 ' sialyl lactose.
ExamPle 2:
The binding inhibiting activity of fetuin was
d~t~rm;n~d as follows:
C ~;ially available fetuin from Sigma Oh~mic~l was
purified on a SEPHACRYL S-100 column (from ph~ ) in
aqueous 0.15M NaCl plus 0.05M Tris-HCl, pH 7.0 plus 0.02%
NaN3 and the ICso det~rmin~d for each of the peaks isolated.
ICsos were determined using the HuTu-80 cell line
monolayers. The results are shown below in Table 2, where
fraction # 3 Cur~ dS with pure fetuin and fractions # 1
and # 2 correspond with unidentif ied high molecular weight
impurities .
Table 2
Fetuin ICso (mg/ml)
fraction
Expt A Expt B Expt C
# 1 0.5 0.5 0.3
# 2 0.6 0.5 0.4
25 # 3 * * 1.3
crude f etuin 1. 3 3 1. 4 1. 5
no means of irhibition observ d even at the highest
concentration tested of 2 mg/ml.
~ ~ 8~29
W095/2360s r~"~
--22--
In vivo An;r-l test:
Gnotobiotic derived piglets (delivered by cesarean
section and housed in a germ-f ree environment) were orally
treated with 100 mg of 3 ' sialyl lactose in 5 . 0 ml of water .
5 Ex~eriment A: _
Six day old gnotobiotic piglets were orally treated
with seven doses of loO mg each of 3 ' sialyl lactose, at
about 8 hour intervals. As a control, the piglets were
administered water. The third administration of 3 ' sialyl
10 lactose and control was ~c -n; Qll with 2 x 109 live ~.
pylorl. Two piglets were administered 3'sialyl lactose and
2 piglets were administered the control. The results are
shown below in Table 3.
Ex~eriment B:
Twenty one day old gnotobiotic piglets were orally
treated with seven doses of 100 mg each of 3 ' sialyl
lactose, at about 8 hour intervals. As a control, the
piglets were administered water. The third administration
of 3' sialyl lactose and control was ac -n~ed with 4 x
20 109 live ~. pylori. Four piglets were administered 3'sialyl
lactose and 2 piglets were administered the control. The
results are shown below in Table 3.
The piglets were evaluated by detQ~n;n;n~ bacterial
colonies in blood-agar as colony forming units/gram of
25 gastric epithelium (CFU/g). Gastric epithelium homogenates
were plated on agar in serial 1:10 dilutions and bacterial
colonies were counted on the plates, with 20-200
colonies/plate after 5 days.
Table 3
Experiment A Experiment B mean + SD
30 3'sialyl lactose 5.44, 0 4.46, 25.5, 6.9 i: 5.9
3.71, 2.48
WO 9Sl23605 ~ ~ 8 3 3 2 9 r~"~
--23--
¦¦ control ¦ 23.1, 28.8 ¦ 24.1, 6.5 ¦ 20.6 + 8.3 ¦¦
Example 3.
An anti~ c~hcl~ter composition is ~Lt:~ared by
sllcpF~n~l;n~ 1 g of the 3'sialyl lactose in a mixture of
water and propylene glycol.
Exam~le 4.
An anti-~elicobacter composition is prepared by mixing
1 g of 3 ' sialyl lactose with 250 mg of the H2 receptor
antagonist ranitidine. The mixture is then Cllcp~n~l~d in a
mixture of water and propylene glycol.
Exam~le 5.
An anti-Helicobacter composition is prepared by mixing
1 g of 3 ' sialyl lactose with 250 mg of the proton pump
inhibitor omeprazole. The mixture is then sllqp~n~lecl in a
mixture of water and propylene glycol.
ExamPle 6 .
An anti-~elicobacter composition is ~L_~ar~d by mixing
1 g of 3 ' sialyl lactose with 500 mg of a tetracycline.
The mixture is then uqr~n~l~d in a mixture of water and
2 0 propylene glycol .
Exam~le 7
As a therapeutic treatment, a patient infected with ~.
pylt~ri is treated with the composition of Example 3. The
patient is treated orally four times daily with each dosage
providing an effective stomach ~ullcel~LLc.tion of 2 mg/ml.
Therapy is continued for two weeks, after which examination
showed eradication of the ~. pylorl bacteria. After
eradication, maintenance therapy with the composition of
the present invention is continued to prevent recurrence.
* * * *
Obviously, ~ us modif ications and variations of
the present invention are possible in light of the above
t~rhin~s. It is therefore to be understood that within
~ ~ ~332~
Wo ss/2360s ~
--24--
the scope of the appended claims, the invention may be
practiced otherwise than as specif ically described herein.
