U.S. patent application number 12/210559 was filed with the patent office on 2009-01-08 for chemotherapeutic compositions.
Invention is credited to Graham John Hamilton MELROSE.
Application Number | 20090011023 12/210559 |
Document ID | / |
Family ID | 3787618 |
Filed Date | 2009-01-08 |
United States Patent
Application |
20090011023 |
Kind Code |
A1 |
MELROSE; Graham John
Hamilton |
January 8, 2009 |
CHEMOTHERAPEUTIC COMPOSITIONS
Abstract
The present invention relates to the treatment of
gastrointestinal and/or cancer, and a method of weight gain, via
the ingestion of polymeric compositions in humans, animals or birds
in need of said treatment. The invention provides methods for the
treatment of cancer, the treatment and/or prevention of
gastrointestinal disease and/or infection and/or diarrhea, and a
method for increasing weight gain in humans, animals or birds
comprising administering to said humans, animals or birds an
effective amount of a pharmaceutical or veterinary composition, or
feed additive, comprising an effective amount of a polymer and/or
copolymer, having the repeating polymeric unit (I), wherein R is H
or alkyl, usually C.sub.1 to C.sub.4, or this unit in hydrated,
hemiacetal or acetal form, together with a pharmaceutically or
veterinarally acceptable carrier, diluent, adjuvant, excipient
and/or controlled release system. ##STR00001##
Inventors: |
MELROSE; Graham John Hamilton;
(Nedlands, AU) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Family ID: |
3787618 |
Appl. No.: |
12/210559 |
Filed: |
September 15, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10792867 |
Mar 5, 2004 |
7438899 |
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12210559 |
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08952648 |
Nov 24, 1997 |
6723336 |
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PCT/AU96/00328 |
May 30, 1996 |
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10792867 |
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Current U.S.
Class: |
424/484 ;
424/78.31 |
Current CPC
Class: |
A61P 31/00 20180101;
C08L 2205/02 20130101; C08L 31/04 20130101; C08L 33/00 20130101;
A61P 1/00 20180101; A61P 35/00 20180101; C08L 29/12 20130101; C08L
29/04 20130101; Y02A 50/473 20180101; C08L 29/14 20130101; Y02A
50/30 20180101; A61K 31/765 20130101; C08L 29/12 20130101; C08L
2666/04 20130101; C08L 29/14 20130101; C08L 2666/04 20130101 |
Class at
Publication: |
424/484 ;
424/78.31 |
International
Class: |
A61K 9/10 20060101
A61K009/10; A61K 31/74 20060101 A61K031/74; A61P 31/00 20060101
A61P031/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 30, 1995 |
AU |
PN3276 |
Claims
1. A composition for treatment of gastrointestinal disease wherein
said composition comprises: (a) a chemotherapeutic active compound
comprising a polymer and/or copolymer of acrolein, having a
structure according to Formula I: ##STR00006## as the repeating
polymeric unit, wherein R is H or alkyl, preferably C.sub.1 to
C.sub.4 or this unit in hydrated, hemiacetal or acetal form; and
(b) a pharmaceutically or veterinarally acceptable carrier.
2. The composition according to claim 1 in the form of pellets
comprising a controlled release system of a crosslinked water
absorbent polymer matrix adapted to form a gel when wet.
3. The composition according to claim 1 wherein the carrier is
selected from the group consisting of polymers and copolymers of
one or more of acrylic acid, methacrylic acid, esters of acrylic
acid, esters of methacrylic acid, amides of acrylic acid and amides
of methacrylic acid, polyvinyl alcohol and polyvinyl acetate.
4. The composition according to claim 1 wherein the carrier is
selected from the group consisting of polymers and copolymers of
one or more of acrylic acid, methacrylic acid, esters of acrylic
acid, esters of methacrylic acid, amides of acrylic acid and amides
of methacrylic acid, polyvinyl alcohol and polyvinyl acetate.
5. The composition according to claim 1 wherein the carrier
comprises a polymer of acrylic acid and/or a copolymer of acrylic
acid and acrylamide.
6. The composition according to claim 1 wherein the active agent is
present in an amount of from 5 to 50% by weight of the
composition.
7. The composition according to claim 1 comprising a liquid carrier
selected from the group consisting of water, olive oil, peanut oil,
sesame oil, sunflower oil, arachis oil, coconut oil, liquid
paraffin, ethylene glycol, propylene glycol, polyethylene glycol,
polypropylene glycol, ethanol, propanol, isopropanol, glycerol,
fatty alcohols, triglycerides and mixtures thereof.
8. The composition according to claim 1 wherein the liquid carrier
is selected from the group consisting of ethylene glycol, propylene
glycol, polyethylene glycol, polypropylene glycol, ethanol
propanols, isopropanols, glycerol, fatty alcohols, triglycerides
and mixtures thereof.
9. The composition according to claim 1 wherein the carrier
comprises polyethylene glycol and water.
10. The composition according to claim 1 wherein the carrier
comprises controlled release pellets wherein said pellets are added
to animal feed to provide a feed containing from 1% to 20% by
weight of said polymer or copolymer of acrolein.
11. The composition according to claim 1 wherein said polymer or
copolymer of acrolein has a molecular weight greater than 1000
Daltons.
12. The composition according claim 1 wherein the polymer or
copolymer of acrolein comprises in the range from 0.1 to 5 moles of
carboxyl groups per kilogram of said polymer or copolymer of
acrolein.
13. The composition according to claim 1 wherein the repeating
polymeric unit is in the hydrated diol form, the hemiacetal or
acetal form, as formed from the condensation of diol form with the
aldehyde or diol form, the tetrahydropyran or fused tetrahydropyran
formed from condensation of the diol form, the aldol-Michael
self-condensation form or mixtures thereof illustrated,
non-comprehensively by the following formulae: ##STR00007## wherein
n is one or more.
14. A chemotherapeutic composition in the form of pellets
comprising: (a) an active agent comprising polymers and/or
copolymers of acrolein having the repeating polymeric unit:
##STR00008## wherein R is H or alkyl, preferably C.sub.1 to
C.sub.4, or this unit is hydrated, hemiacetal or acetal form,
together with a pharmaceutically or veterinarally acceptable
carrier, and (b) a polymeric matrix within which the active agent
is contained; and wherein the pellets are formed by a process
comprising the step of adding a polymeric matrix to a solution
and/or suspension and/or emulsion of said polymers and/or
copolymers.
15. The chemotherapeutic composition according to claim 14, wherein
adding said polymeric matrix to a solution and/or suspension and/or
emulsion of the said polymers and/or copolymers, comprises the
steps of: (i) dissolving said polymers or copolymers in an aqueous
alkaline or basic solution; (ii) neutralising said solution with
acid; (iii) adding to said neutralised solution insoluble,
crosslinked, absorbent polymers of acrylic acid and/or copolymers
of acrylamide and acrylic acid to form wet swollen pellets.
16. The chemotherapeutic composition according to claim 14 wherein
the wet, swollen pellets are at least partly dried.
17. A method of treatment or prevention of a gastrointestinal
dysfunction in a human, animal or bird comprising administering to
said human, animal or bird the composition according to claim
1.
18. The method according to claim 17 wherein the polymer or
copolymer comprises carboxyl groups at a concentration of from 0.1
to 5 moles of carboxyl groups per kilogram of polymer.
19. The method according to claim 17 wherein the gastrointestinal
dysfunction is a gastrointestinal disease, gastrointestinal
infection or diarrhea resulting from an infection of the
gastrointestinal tract.
20. The method according to claim 17 wherein the gastrointestinal
dysfunction is a bacterial disease or infection or cancer.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a Divisional of U.S. application
Ser. No. 10/792,867 filed on Mar. 5, 2004, which is a divisional of
U.S. application Ser. No. 08/952,648, for which priority is claimed
under 35 U.S.C. .sctn. 120, which is the national phase of PCT
International Application No. PCT/AU96/00328 filed May 30, 1996,
which claims priority on Australian Application No. PN 3276 filed
May 30, 1995 for which priority is claimed under 35 U.S.C. .sctn.
119, the entire contents of all of which are hereby incorporated by
reference.
TECHNICAL FIELD
[0002] The present invention relates to the treatment of
gastrointestinal disease, and/or cancer, and a method of weight
gain, via the ingestion of polymeric compositions in humans,
animals or birds in need of said treatment.
BACKGROUND ART
[0003] Under the conditions of intensive pig-rearing, post weaning
colibacillosis is ubiquitous. For example, intensive pig-rearing is
used throughout Europe, the United States and Australia. For
example, in the United States, post-weaning colibacillosis is the
most common cause of mortality in the weaner pig (Leman et al.,
1986). The disease is associated with the proliferation of
Escherichia coli bacteria in the anterior small intestine after
weaning, and gives rise to either death, or the young weaner
failing to make normal weight-gains. Other microbiological diseases
are common and often accompany colibacillosis in pigs as well as in
other animals in intensive rearing conditions, especially
poultry.
[0004] In the past, attempts have been made to control disease by
the ingestion of antimicrobial compositions or by vaccination,
neither of which has proved entirely effective.
[0005] The rationale behind the lack of success surrounding
ingestion of antimicrobial compositions has been the fact that
proteinaceous feedstuffs and the contents of the gastrointestinal
tract present a reactive and hazardous environment to any
chemotherapeutic agent and hence, in vitro active antimicrobial
agent will often not be effective when used in vivo within the
gastrointestinal tract.
[0006] Further, to be antimicrobially effective in a practical way,
there is often the restrictive demand that the gastrointestinal
disease controlling agent should reasonably maintain the very low
microbiological content in the duodenum, yet the very high content
in the lower parts of the intestine.
[0007] As well, it does not follow that even in vivo effective
antimicrobials will give weight-gains, since it is common that even
a cocktail of several such commercially used antimicrobials only
gives rise to variable, partial or even negative relative
weight-gains over control animals.
[0008] Accordingly, there is a need for improvements in the
prevention and/or treatment of disease via ingestion of
antimicrobial compositions in humans and/or animals and/or birds in
need of said treatment. Furthermore, there is the need for the
attainment of weight gains of said humans and/or animals and/or
birds undergoing said treatment.
[0009] The present invention provides polymers and/or copolymers
preferably derived from acrolein and having the polymeric repeating
unit:
##STR00002##
wherein R is H or alkyl, usually C.sub.1 to C.sub.4 or this unit in
hydrated, hemiacetal or acetal form and illustrated
non-comprehensively of all possible structures, by the following
formulae:
##STR00003##
wherein n is one or more and R is as defined above; hereinafter
referred to as the "Subject Polymers", as broad spectrum in vivo
antimicrobials and/or anti-cancer agents suitable for treatment by
the oral route.
[0010] It is known that the rate of passage of molecules across
membranes has an inverse relationship to their molecular weights.
Thus, it is widely and generally accepted in the art that molecules
taken by the oral route and having molecular weight <1000, will
have their passage across the gut so restricted, and consequently
any potential toxicity will be effectively minimized.
[0011] The Subject Polymers, being aldehydic, are especially
reactive with protein. (In fact, in microbiological assays, the
routine and facile method of quenching/destroying the activity of
the Subject Polymers is to add protein.) Hence, the Subject
Polymers would not be expected to exhibit significant
microbiological activity in the intestine, especially in the
presence of proteinaceous feed.
[0012] Therefore, the present invention provides a method of
prevention and/or treatment of gastrointestinal disease in humans,
animals or birds resulting from the microbial infection of the
gastrointestinal tract, and a method for increasing weight gain in
humans, animals or birds having gastrointestinal disease and/or
infection, comprising administering an effective amount to said
humans, animals or birds of a pharmaceutical composition or feed
additive, comprising an effective amount of the Subject Polymers
together with a pharmaceutically or veterinarally acceptable
carrier, diluent, adjuvant, excipient or controlled release
system.
OBJECT OF THE INVENTION
[0013] It is an object of the present invention to provide methods
for the prevention and treatment of dysfunctional/pathological
states in a human, animal or bird, especially, those states within
the gastrointestinal tract associated with colibacillosis,
diarrhea, mortality and/or reduced weight gains.
DISCLOSURE OF THE INVENTION
[0014] The present invention is based upon a series of discoveries
herein:
[0015] First, it has now been discovered that the Subject Polymers,
being of high molecular weight are inhibited in passage through
biological membranes, and have facilitated applications in the
gastrointestinal tract, since passage through the gut and into the
bloodstream to potentially cause toxicity is minimized. In
particular, it has been discovered that the Subject Polymers have
molecular weights >1000, and generally have molecular weights
>2000, which effectively minimize their passage across the
intestinal membranes and as a result, it has been found herein that
whilst the Subject Polymers exhibit toxicity following intra-venous
injection, they exhibit no toxicity when administered orally. Hence
it has been discovered that the Subject Polymers have minimized
toxicities and that they have associated advantages when used for
chemotherapeutic purposes, in any species having gut membranes, for
example, humans, animals or birds.
[0016] Generally, the Subject Polymers formed by ionic
initiation/catalysis are more hydrophilic than those formed by free
radical initiation/catalysis, and hydrophilicity of the Subject
Polymers may generally be increased by inclusion within them of
hydrophilic groups, especially carboxyl groups, or of hydrophilic
monomers, especially acrylic acid. Particularly, carefully heating
the Subject Polymers formed by ionic initiation/catalysis with
ample air from room temperature to up to 100.degree. C., and
preferably up to between 80-85.degree. C., produces the Subject
Polymers having 0.1-5 moles of carboxyl groups/kg, aqueous soluble
at the pH of the duodenum especially, and preferred for the
applications in gastrointestinal tracts described and envisaged
herein.
[0017] The Subject Polymers have the following properties:
[0018] The Subject Polymers have an unusually broad in vivo
antimicrobial profile, providing a method of treatment of
gastrointestinal disease in humans, animal or birds.
[0019] It has been shown that the Subject Polymers provide
increased weight gains in humans, animals or birds.
[0020] It has also been shown that the Subject Polymers are in vivo
anti-cancer agents.
[0021] According to a first embodiment of the invention there is
provided a method of in vivo cancer treatment in humans, animals or
birds comprising administering to said humans, animals or birds an
effective amount of a pharmaceutical or veterinary composition or
feed additive, comprising an effective amount of polymers and/or
copolymers having the repeating polymeric unit:
##STR00004##
wherein R is H or alkyl, usually C.sub.1 to C.sub.4, or this unit
in hydrated, hemiacetal or acetal form and illustrated
non-comprehensively of all possible structures, by the following
formulae:
##STR00005##
wherein n is one or more and R is as defined above; together with a
pharmaceutically or veterinarally acceptable carrier, diluent,
adjuvant, excipient and/or controlled release system.
[0022] According to a second embodiment of the invention there is
provided a method for the treatment and/or prevention of
gastrointestinal disease and/or infection and/or diarrhea in
humans, animals or birds resulting from a microbial infection of
the gastrointestinal tract in said humans, animals or birds,
comprising administering to said humans, animals or birds an
effective amount of a pharmaceutical or veterinary composition or
feed additive, comprising an effective amount of a polymer and/or
copolymer as defined in the first embodiment of the invention,
together with a pharmaceutically or veterinarally acceptable
carrier, diluent, adjuvant, excipient and/or controlled release
system.
[0023] According to a third embodiment of the invention there is
provided a method of increasing weight gain in humans, animals or
birds having cancer and/or gastrointestinal microbial disease
and/or infection, in said humans, animals or birds, comprising
administering to said humans, animals or birds an effective amount
of a pharmaceutical or veterinary composition or feed additive,
comprising an effective amount of a polymer and/or copolymer as
defined in the first embodiment of the invention, together with a
pharmaceutically or veterinarally acceptable carrier, diluent,
adjuvant, excipient and/or controlled release system.
[0024] In terms of the Subject Polymers being a prevention and/or
treatment for gastrointestinal disease, it has now been shown that
farm-piglets given the Subject Polymers ad libitum in their
drinking water, have reduced numbers of diarrhea days and lower
counts of E. coli in their excreta. Moreover, the effect as
proposed, is markedly greater than the partial effect exhibited by
the administration of commercial antimicrobial agents together with
vaccination.
[0025] In addition, it has now been discovered that the Subject
Polymers in a polymeric controlled-release system as well as
leading to fewer diarrhea days in the animal, also gave rise to
extraordinarily high weight-gains. As exemplified in Example 17,
piglets displayed lower mortality, and weight gains of some 46%
above control piglets.
[0026] Therefore, it is proposed that the Subject Polymers should
be provided as an additive to the animal's solid feed, in the form
of a polymeric controlled-release system, since then, the Subject
Polymers would be released slowly and mainly in the duodenum.
Further, it is now proposed that having the Subject Polymers in a
controlled-release system will reduce any destructive chemical
reaction between the Subject Polymers and either constituents of
the gastrointestinal tract or the proteinaceous feed, especially
during the essential pre-heat sterilization of the feed.
Furthermore, any rejection by the animal due to taste, will be
reduced.
[0027] When using the Subject Polymers for the treatment of disease
and/or infection in humans, animals or birds in need of treatment,
for example in piglets having diarrhea associated with
colibacillosis, it is preferred to begin dosing immediately after
weaning and to always continue for the next 5 days, sometimes for
the next 30 days, but preferably at least for the next 15 days
after weaning; at any time that such symptoms of diarrhea are
observed. Treatment in a controlled-release form is preferred and
containing theoretically 5%-50% w/w Subject Polymers but
preferably, 20%-30% w/w. Preferably the controlled-release
form/pellet is added to the feed, either during production of the
feed or during feeding such that the feed contains 1%-20% w/w and
preferably 2%-8% w/w of pellet. If for example a piglet of 5 kg
daily eats 500 g of feed with 4% w/w controlled-release pellet
containing 25% w/w Subject Polymers--this calculates to a dose rate
of Subject Polymers of 1000 mg/kg(liveweight)/day; in a
controlled-release form a dose rate of 50-5000 mg/kg/day is
practical but 500-2500 mg/kg/day is preferred. Smaller dose rates
of 25-500 mg/kg/day should be used when the Subject Polymers are
given in a non-controlled-release form. Dose protocols and rates
are similar for other microbiological infections and for
cancer.
[0028] Both the in vivo antimicrobial activity in the presence of
relatively huge quantities of reactive proteins in the
gastro-intestinal tract, and the range of chemotherapeutic
activities of the Subject Polymers, revealed herein, are
surprising. Also, in unpublished work, the Subject Polymers have
been found to exhibit significant anti-coagulant activities in
human or a range of various animal bloods. This particular range of
surprising in vivo activities suggests that unexpectedly, the
Subject Polymers have such activities related to
lectin-selectin-integrin type adsorptive inter-actions.
[0029] Compositions for administration in the method of the
invention may be prepared by means known in the art for the
preparation of compositions (such as in the art of veterinary and
pharmaceutical compositions) including blending, grinding,
homogenizing, suspending, dissolving, emulsifying, dispersing and
where appropriate, mixing of the Subject Polymers together with
selected excipients, diluents, carriers and adjuvants.
[0030] For oral administration, the pharmaceutical or veterinary
composition may be in the form of tablets, lozenges, pills,
troches, capsules, elixirs, powders, including lyophilized powders,
solutions, granules, suspensions, emulsions, syrups and tinctures.
Slow-release, or delayed-release, forms may also be prepared, for
example in the form of coated particles, multi-layer tablets or
microgranules.
[0031] It is preferred that the controlled release system comprises
a pH-sensitive, crosslinked, water-absorbent pellet, which when wet
is a gel.
[0032] Solid forms for oral administration may contain
pharmaceutically or veterinarally acceptable binders, sweeteners,
disintegrating agents, diluents, flavorings, coating agents,
preservatives, lubricants and/or time delay agents. Suitable
binders include gum acacia, gelatin, corn starch, gum tragacanth,
sodium alginate, carboxymethylcellulose or polyethylene glycol.
Suitable sweeteners include sucrose, lactose, glucose, aspartame or
saccharine. Suitable disintegrating agents include corn starch,
methylcellulose, polyvinylpyrrolidone, xanthan gum, bentonite,
alginic acid or agar. Suitable diluents include lactose, sorbitol,
mannitol, dextrose, kaolin, cellulose, calcium carbonate, calcium
silicate or dicalcium phosphate. Suitable flavoring agents include
peppermint oil, oil of wintergreen, cherry, orange or raspberry
flavoring. Suitable coating agents include polymers or copolymers
of acrylic acid and/or methacrylic acid and/or their esters, and/or
their amides, waxes, fatty alcohols, zein, shellac or gluten.
Suitable preservatives include sodium benzoate, vitamin E,
alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben or
sodium bisulphite. Suitable lubricants include magnesium stearate,
stearic acid, sodium oleate, sodium chloride or talc. Suitable time
delay agents include glyceryl monostearate or glyceryl
distearate.
[0033] Liquid forms for oral administration may contain, in
addition to the above agents, a liquid carrier. Suitable liquid
carriers include water, oils such as olive oil, peanut oil, sesame
oil, sunflower oil, safflower oil, arachis oil, coconut oil, liquid
paraffin, ethylene glycol, propylene glycol, polyethylene glycol,
ethanol, propanol, isopropanol, glycerol, fatty alcohols,
triglycerides or mixtures thereof.
[0034] Suspensions for oral administration may further comprise
dispersing agents and/or suspending agents. Suitable suspending
agents include sodium carboxymethylcellulose, methylcellulose,
hydroxypropylmethyl-cellulose, polyvinyl-pyrrolidone, sodium
alginate or cetyl alcohol. Suitable dispersing agents include
lecithin, polyoxyethylene esters of fatty acids such as stearic
acid, polyoxyethylene sorbitol mono- or di-oleate, -stearate or
-laurate, polyoxyethylene sorbitan mono- or di-oleate, -stearate or
-laurate and the like.
[0035] The emulsions for oral administration may further comprise
one or more emulsifying agents. Suitable emulsifying agents include
dispersing agents as exemplified above or natural gums such as gum
acacia or gum tragacanth.
[0036] According to a fourth embodiment of the invention there is
provided a method of producing pellets, the pellets comprising
polymers and/or copolymers as defined in the first embodiment of
the invention, together with a pharmaceutically or veterinarally
acceptable carrier, diluent, adjuvant, excipient and/or controlled
release system, mainly within a polymeric matrix. The method
comprising adding a polymeric matrix to a solution, suspension or
emulsion of the polymers and/or copolymers as defined in the first
embodiment of the invention.
[0037] According to a fifth embodiment of the invention there is
provided a method of producing pellets or like solid composition,
the pellets comprising polymers and/or copolymers as defined in the
first embodiment of the invention, mainly within a polymeric
matrix, said method as defined in the fourth embodiment of the
invention and comprising the steps of:
[0038] (i) dissolving said polymers and/or copolymers in an aqueous
alkaline or basic solution;
[0039] (ii) neutralizing said solution with acid;
[0040] (iii) adding to said neutralized solution insoluble,
cross-linked, absorbent polymers of acrylic acid and/or copolymers
of acrylamide and acrylic acid, to form wet, swollen pellets;
and
[0041] (iv) optionally, wholly or partially drying said wet swollen
pellets.
[0042] The so-formed wet, swollen pellets may be used either wet,
partially dried or wholly dried, as an additive to, for example,
animal feed. This system is further designed so that the
carboxyl-containing groups of the outer polymeric matrix cause the
Subject Polymers to remain essentially contained within the matrix
when in the acidic environment of the stomach. However, in the
alkaline environment of the duodenum, the carboxyl groups of the
matrix become ionized and mutually-repelling, and the pellet
rapidly swells to allow the Subject Polymers, aided by repulsion
among their own ionic groups, to be excluded by a diffusion
process, approximately matching the speed of passage of feed
through the duodenum.
[0043] In this invention, the term, "controlled release system" is
used in the same context as that in, and includes the same range of
examples as quoted in "Controlled Drug Delivery" (Robinson &
Lee, 1987). Many other pH-sensitive controlled-release systems
which are known in the art (Robinson and Lee, 1987) may be
substituted for the polymer of acrylic acid or copolymer of
acrylamide and acrylic acid. For example, soluble and anionic or
insoluble, cross-linked and anionic, cellulosic systems or soluble
and anionic, or insoluble, cross-linked and anionic polymers
derived from any generic acrylic acid and/or its derivatives. Such
cross-linked and insoluble polymers are preferred since they swell
and also, are less likely to be metabolized.
[0044] In summary, the compositions described herein may be used to
control cancer and/or microbiological disease and especially result
in greater weights of humans, animals or birds having
gastrointestinal infections. The invention is applicable in humans,
animals or birds having microbiological diseases of the
gastrointestinal tract for example, Escherichia coli. A preferred
use is to bring about greater weights in newly-weaned piglets
having diarrhea and associated with the proliferation of
Escherichia coli bacteria in the anterior small intestine. The
invention may be applicable in humans having gastrointestinal
disease, for example, from Staphylococcus aureus and/or
Helicobactor pylori bacteria
[0045] The invention will now be described with reference to
specific Examples, which should not be construed as limiting on the
scope thereof.
EXAMPLES 1 TO 13
Preparations and Structures of Subject Polymers
Example 1
[0046] (a) Using a free radical initiator/free radical catalyst:
9.64 g distilled acrolein and 25 g methanol were placed in a 100 ml
round bottom flask and purged with nitrogen. 0.525 g benzoyl
peroxide was added and the solution stirred under nitrogen at
60.degree. C. The reaction was allowed to continue for a total of
ca. 88 hours. After this time the reaction solution had become
strongly yellow in color and had a solids content of 30.5%.
.sup.13C-NMR (300 MHZ) .delta. (relative to d.sub.4-methanol at
49.00): 33.27 (CH); 33.53 (CH); 33.79 (CH); 33.87 (CH.sub.2); 37.03
(CH); 37.29 (CH); 37.54 (CH); 37.64 (CH.sub.2); 97.15 (CH); 103.37
(CH); 104.34 (CH); 139.36 (CH); 139.78 (CH.sub.2); 196.72 (CH). The
.sup.13C-NMR spectrum shows some residual acrolein with the
aldehyde carbon at 6196.72 and the vinylic CH.sub.2 and CH at
6139.78 and 6139.36, respectively; apart from the 6196.72 (CH)
resonance absorption, there was no other attributed to --CHO. The
spectrum is consistent with polymeric acrolein consisting of fused
tetrahydropyran rings and some free dihydroxy methyl groups. The
rings exist in either the boat or chair conformations giving rise
to more chemical shifts than may be expected.
[0047] Typically, as an indication of molecular weight, the
polyacrolein was found to have a retention time which was shorter
than that of polyethyleneglycol 2,000 on a Porasil GPC 60 Angstrom
column using a Waters Associated Model ALC/GPC 204 liquid
chromatograph fitted with a differential refractometer R401.
[0048] (b) Using an ionic initiator/catalyst: 1.6 g distilled
acrolein was made up to 20 ml with demonized water in a 200 ml
beaker and then, ca. 0.5 ml of 0.2M sodium hydroxide added with
stirring to pH ca. 10-11. The solution became cloudy and a white
precipitate began to form. The contents were stirred for a further
2 hours and then filtered. The precipitate was washed thoroughly
with demonized water until the filtrate was neutral. The product
may be carefully heat-dried in contact with ample air, initially at
ambient temperatures and then at temperatures up to about
100.degree. C. Alternatively, as in this case, the product may be
dried under vacuum to a white-pale yellow, fine powder; dissolved
in methanol, and it may be evaporated down to dryness and then
again, dissolved in methanol or other solvents. Often, the Subject
Polymers have 0.1-5 moles of carboxyl groups/kg and were found to
have GPC retention times which were mainly shorter than that of
polyethylene glycol 2,000. .sup.13C-NMR (300 MHz) .delta. (relative
to d.sub.4-methanol at 49.00): 19-31 (CH.sub.2); 35.95 (CH.sub.2);
37-42 (CH); 62-73 (CH.sub.2); 73-81 (CH); 92-95 (CH); 96-103 (CH);
114-120 (CH.sub.2); 134-141 (CH); 196.0 (CH).
[0049] In experiments using in each case, 2 groups of 10 Swiss
white mice (treated and control, respectively) it was found that
solutions of the Subject Polymers in aqueous triethanolamine
adjusted to pH 8, had an acute intra-venous toxicity of 320 mg/kg,
and acute oral toxicity of >5000 mg/kg (no deaths nor abnormal
signs were apparent over 14 days).
Example 2
[0050] 6.489 g distilled acrylic acid, 0.56 g distilled acrolein
and 15 g methanol were placed in a 50 ml round bottom flask and
purged with nitrogen. 0.33 g benzoyl peroxide was added and the
solution stirred under nitrogen at 60-65.degree. C. The reaction
was continued for ca. 66 hours. After this time the contents of the
flask had become very viscous, having a solids content of 57.7%
(indicating 100% conversion).
[0051] A sample of the viscous material was placed on a petri dish
and dried on a hot plate to remove solvent. Drying was completed in
an oven at 80.degree. C. and a transparent, slightly yellow colored
polymer was obtained. The copolymer is completely soluble in warm
water (ca. 50.degree. C.) and once dissolved remains so, even on
cooling the solution.
[0052] In order to ensure that the solids obtained were polymeric,
a simple dialysis experiment was performed: 10 g of an aqueous
solution containing 0.65% solids was placed in a dialysis tube.
This was irrigated with water continuously for ca. 66 hours. The
solution in the dialysis tube was then recovered and the solids
content determined at 0.68%. Since the solids were completely
retained and the lower limit for solids penetration through the
dialysis tube is 2000 mwt, it is concluded that the solids are
polymeric.
Example 3
[0053] 2.8 g of acrolein diethyl acetal was placed in a 100 ml
round bottom flask and the contents purged with nitrogen. A
solution of 0.216 g potassium persulphate in 7.5 g water was added
with stirring, under nitrogen. The flask was placed in an oil bath
at 60-70.degree. C. and stirred for ca. 20 hours. A yellow solid
was recovered and dried at 50.degree. C.; weight 0.915 g.
Example 4
[0054] 4 g distilled acrylic acid, 4.81 g acrolein diethyl acetal
and 15 g methanol were placed in a 50 ml round bottom flask and
purged with nitrogen. Then 0.3 g benzoyl peroxide was added and
stirring continued under nitrogen at 60-65.degree. C. for 70 hours
(solids determination indicated a 50% conversion). .sup.13C-NMR
(300 MHz) .delta. (relative to d.sub.4-ethanol at 49.00): 15.58
(CH.sub.3); 18.31 (CH.sub.3); 35.52 (CH.sub.2); 36.24 (CH.sub.2);
37.07 (CH.sub.2); 42.36 (CH); 42.85 (CH); 58.32 (CH.sub.2); 130.00
(CH); 131.57 (CH.sub.2); 178.51 (CH).
Example 5
[0055] 3.8 g of acrolein diethyl acetal, 3.3 g vinyl pyrrolidone
and 10 g methanol were placed in 50 ml round bottom flask and
thoroughly purged with nitrogen. 0.71 g azobisisobutyronitrile was
added and the flask heated in an oil bath at 60-65.degree. C., with
stirring under nitrogen for 72 hours when the conversion was 44%.
The copolymer was found to be soluble in methanol.
Example 6
[0056] In a similar technique to the above, 3.9 g acrolein diethyl
acetal, 1.16 g acrylic acid, 7.5 ml water and 0.216 g potassium
persulphate were heated under nitrogen, with stirring in an oil
bath at 60-70.degree. C. for ca. 24 hours when a white waxy
material was recovered; it was insoluble in water, but swelled in
methanol, acetone, tetra-hydrofuran or methyl ethyl ketone.
Example 7
[0057] A similar result was achieved through heating and stirring
in the usual way to the above: 14.5 g methanol, 3.62 g distilled
acrolein, 1.21 g distilled acrylic acid and 0.265 g benzoyl
peroxide. After 40 hours the conversion was 40%.
Examples 8-11
[0058] A 50:50 mixture of monomers was treated as follows: 2.35 g
distilled acrolein, 2.88 g distilled acrylic acid and 14.5 g
methanol were placed in a 50 ml round bottom flask and flushed with
nitrogen. 0.2625 g benzoyl peroxide was added and after heating at
60-70.degree. C. for 48 hours the conversion was 70%. The polymer
swelled in methanol but was insoluble in water.
[0059] Similar preparations were achieved with different ratios of
the monomers acrolein: acrylic acid namely, 30:70 (Example 9),
10:90 (Example 10), 2.5:97.5 (Example 11). The products from
Examples 10 and 11 were soluble in water and retained by dialysis
tubing of exclusion 2,000 mwt.
Example 12
[0060] In a similar preparation to the above, 42% conversion was
achieved of a polymer which swelled in methanol or water, from 1.8
g acrolein, 3.3 g vinyl pyrrolidone and 0.071 g
azobisisobutyronitrile.
Example 13
[0061] 30 mg benzoyl peroxide was added to a solution of 1.02 g
polyethyleneglycol acrylate and 0.5 ml acrolein in 5 ml methanol.
The mixture was stirred and heated to reflux for 48 hours and gave
90% conversion; the residual oil (1.2 g) was chromatographed on
Sephadex LH-20 (18 g) in methanol. The structure of the resulting
polymer was confirmed by NMR analysis.
Example 14
[0062] Young adult female mice were inoculated intra-peritoneally
with 2.3.times.10.sup.6 Ehrlich ascites tumour cells and one week
later, those mice with swollen abdomens were selected for further
experimentation: 15 of such mice were treated twice a day for three
days and 15 once a day for the next three days, with
intra-peritoneal injections of the Subject Polymers in 0.5% aqueous
sodium carbonate at a dose rate of 150 mg/kg mouse; a control group
of 15 mice having Ehrlich ascites tumour cells was treated with the
sodium carbonate solution, only. All mice in both groups were then
sacrificed, the peritoneal fluids were collected and the number of
tumour ceils counted:
TABLE-US-00001 Control Group Mouse Treated Group Number Peritoneal
cells .times. 10.sup.9 Mouse Number Peritoneal cells .times.
10.sup.9 1 2.30 16 0.97 2 3.55 17 0.54 3 2.13 18 0.14 4 0.05 19
0.06 5 2.55 20 0.11 6 2.27 21 0.07 7 2.93 22 0.27 8 1.93 23 0.93 9
2.34 24 0.36 10 5.58 25 1.04 11 2.23 26 0.12 12 1.65 27 0.64 13
4.76 28 0.16 14 2.01 29 0.67 15 3.36 30 0.54 x = 2.64 x = 0.44 s =
1.31 s = 0.35
[0063] A t-test showed the control and treated groups to be
different at greater than 99% confidence level.
Example 15
[0064] The Subject Polymers were suspended/dissolved in the
drinking water of piglets, at 0.1% w/v for the first 2-3 days and
then at 0.05% w/v for the next 7 days; drinking was ad libitum;
consumption of Subject Polymers was approximately 200 mg/kg of
piglet/day. Three groups of 28-day, newly-weaned piglets (mean
weight 5.6 kg) were studied:
[0065] 1. Treated with Subject Polymer, only;
[0066] 2. Treated with prior vaccination protocol, only;
[0067] 3. No treatment at all (control).
[0068] During the 14-day trial, in keeping with usual practice at
the farm, those piglets which were observed during the daily
inspection to have diarrhea, were given an injection of
antibiotic.
TABLE-US-00002 Treatment: Subject Treatment: No Treatment: Polymers
Vaccination Only Control No. 28-day piglets in 15 19 10 group No.
diarrhea days 0.47 1.11 1.8 (mean/piglet) No. injections of 0.6
2.53 4.6 antibiotic (mean/piglet) No. piglets with heavy 0.53 0.79
0.9 E. coli in faeces, day 7 (mean/piglet) Weight-gain, 14 days 33
32 37 mean %/piglet)
Example 16
[0069] In a typical small-scale experiment, Subject Polymers (35 g)
were dissolved with stirring in water (638 g) containing sodium
carbonate (19 g), and then the solution was immediately neutralized
to pH 7.2 with 10% hydrochloric acid; Sucrose (32 g; sweetening
agent) which may be discretionally included, was added with
continued stirring and then either an absorbent polymer of acrylic
acid (64 g; CARBOPOL 934, B.F. Goodrich, USA) or an absorbent
copolymer of acrylamide-acrytic acid (64 g; ALCOSORB AB 3S, Allied
Colloids, England) was added. The resulting swollen beads took-up
the typically pale-yellow color of the Subject Polymers in
solution. The beads/pellets may be used wet, but were typically,
either partially or wholly dried at temperatures up to 45.degree.
C., before use. However, higher temperatures, up to about
90-100.degree. C. may be used. On the basis of solid substances
added, theoretically, the pellets contain approximately 25% w/w of
Subject Polymers.
[0070] Two experiments were undertaken to assess the rate of
release of the Subject Polymers at simulated pHs of the stomach and
the duodenum, respectively. First, the above fully-dried beads (200
mg) were gently stirred in 0.1M hydrochloric acid (100 mL). An
aliquot (10 mL) was periodically removed and diluted in excess 0.2M
aqueous sodium bicarbonate; measurement of a peak near 265 nm
indicated a maximum release, in 0.1M hydrochloric acid, of
approximately 35% after about forty five minutes. Second, by
contrast, a comparable experiment in 0.1M aqueous sodium
bicarbonate solution revealed 35% release in about twenty minutes,
and 90%-100% after three to five hours. These properties of the
beads were unaffected by heating conditions which may be used
commercially, to reduce microorganisms in feed (90.degree. C./30
minutes).
Example 17
[0071] In one experiment, two groups, each of ten newly-weaned
21-day-old pigs from a commercial piggery were given an oral dose
of E. coli (50 mL; 10.sup.9 CFU/mL) of haemolytic E. coli, cultured
from a field case of post-weaning colibacillosis. On the same day,
one group of the pigs was given ad libitum feeding of 19% crude
protein feed containing the wholly-dried beads/pellets sprinkled in
the feed at the rate of 3.75% w/w of feed. The pellets contained
theoretically, approximately 25% w/w of the Subject Polymers.
Furthermore, a "No Treatment" group was given feed, only. Each pig
consumed about 500 g of feed, daily. Swabs were taken of rectal
faeces initially and then again, 48 hours after dosage with the E.
coli. Three days after this regime (during which time two pigs of
the untreated group died with acute secretory diarrhea), all
remaining pigs were sacrificed and the E. coli estimated
semi-quantitatively in sections of the duodenum (after swabbing the
mucosa and then using a scale of 4 highest) to 0 (lowest) within
the usual microbiologists' dilution-strike technique on culture
plates). Post-mortem examination did not reveal any gastric
inflammation in any pig.
[0072] Scores for haemolytic E. coli in the rectal faeces of pigs
in the two groups on the day of inoculation, and 48-hours later,
and are illustrated in the following table:
TABLE-US-00003 Initial 48 hours later No Treatment Treatment No
Treatment Treatment 1 2 1 0 0 0 3 1 1 1 4 2 1 0 4 0 0 1 3 1 0 2 2 1
0 1 1 1 1 1 4 0 0 0 4 2 0 0 2 2 Total 4 8 28 10
[0073] After 48 hours the number of haemolytic E. coli in the
faeces of the pigs receiving the Subject Polymers was significantly
less than that in the untreated pigs (.chi..sup.2=16.24;
p=<0.001).
[0074] Furthermore, the distribution of haemolytic E. coli in the
upper (25%) and mid (50%) jejunum of the treated and untreated
pigs, after sacrifice, is recorded below:
TABLE-US-00004 25% 50% No Treatment Treatment No Treatment
Treatment 1 1 2 1 0 0 1 0 1 1 4 4 3* 0 4* 0 2 0 3 1 1 1 2 1 1 3 2 3
4 0 4 0 3* 20 4* 0 1 0 2 0 Total 17 6 28 9 *Dead pigs
[0075] The treated pigs had significantly fewer haemolytic E coli
than the untreated pigs, at both the 25% site (.chi.*=7.38;
p=<0.01) and the 50% site (.chi..sup.2=16.24; p=<0.001).
[0076] Another experiment was executed identically, except for the
fact that 15 newly-weaned pigs were in each group. The initial
inoculation of E. coli in the experimental group was reduced to
one-third of previously described, and the controlled-release
pellets theoretically contained 20% w/w of the Subject Polymers.
Observations of diarrhea days and weight gain were made of the pigs
over 21 days. The results showed the treated group had
statistically significant fewer diarrhea days and statistically
significant (46%) greater weight-gain over the control group.
TABLE-US-00005 Live Weight Gains and Days of Diarrhea Group Control
Treated Number 15 15 Mean Initial weight (kg), day 1 4.77 4.43
Standard Error 0.22 0.19 Relative (%) 100.00 92.00 Mean Final
weight (kg), day 21 6.63 7.31 Standard Error 0.35 0.36 Relative (%)
100.00 110.30 Mean Daily gain (g), 0-21 days 94.18 137.33 Standard
Error 10.27 13.16 Relative (%)* 100.00 146.00 Days of diarrhea** 32
13 *t = 2.47; p = 0.02 (two tail) **No diarrhea in any pig, after
day 10.
REFERENCES
[0077] Leman, A. D., Straw, B., Glock, R. D., Mengeline, W. L.,
Penny, R. H. C. and Scholl, E. (1986). "Diseases of Swine", 6th
Edition, Iowa State University Press. [0078] Robinson, J. R. and
Lee, V. H. L. (1987). "Controlled Drug Delivery". Marcell
Dekker.
* * * * *