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Patent 2963582 Summary

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(12) Patent: (11) CA 2963582
(54) English Title: ORAL PREPARATION COMPRISING MUCOADHESIVE, BIOCOMPATIBLE HYDROPHILIC POLYMER FOR PREVENTING AND/OR TREATING INFLAMMATORY CONDITIONS OF THE GASTROINTESTINAL TRACT
(54) French Title: PREPARATION ORALE RENFERMANT UN POLYMERE MUCOADHESIF, HYDROPHILE, BIOCOMPATIBLE DESTINEE A LA PREVENTION ET AU TRAITEMENT D'ETATS INFLAMMATOIRES DU TRACTUS GASTRO-INTESTINAL
Bibliographic Data
(51) International Patent Classification (IPC):
  • A61K 31/785 (2006.01)
  • A61K 9/00 (2006.01)
  • A61K 31/74 (2006.01)
  • A61P 1/00 (2006.01)
  • A61P 1/04 (2006.01)
  • A61P 29/00 (2006.01)
(72) Inventors :
  • SEDLAKOVA, ZDENKA (Czechia)
  • POLAKOVA, LENKA (Czechia)
(73) Owners :
  • USTAV MAKROMOLEKULARNI CHEMIE AV CR, V.V.I. (Czechia)
(71) Applicants :
  • USTAV MAKROMOLEKULARNI CHEMIE AV CR, V.V.I. (Czechia)
(74) Agent: ROBIC
(74) Associate agent:
(45) Issued: 2017-12-05
(86) PCT Filing Date: 2015-11-20
(87) Open to Public Inspection: 2016-06-02
Examination requested: 2017-04-04
Availability of licence: N/A
(25) Language of filing: English

Patent Cooperation Treaty (PCT): Yes
(86) PCT Filing Number: PCT/CZ2015/050011
(87) International Publication Number: WO2016/082808
(85) National Entry: 2017-04-04

(30) Application Priority Data:
Application No. Country/Territory Date
PV2014-825 Czechia 2014-11-28

Abstracts

English Abstract

The present invention relates to a preparation for oral use destined for prevention and treatment of inflammatory affections of gastrointestinal tract, said preparation containing a mucoadhesive, biocompatible hydrophilic polymer containing: - at least one type of structural units A selected from a group comprising units derived from acrylic and methacrylic acids and their salts, hydroxyalkyl acrylates and methacrylates, hydroxyalkyl acrylamides and methacrylamides, N,N-dialkylaminoalkyl acrylates and methacrylates, N,N-dialkylaminoalkyl acrylamides and methacrylamides, N- vinyl-2-pyrrolidone and mixtures thereof; - at least one type of structural units B selected from a group comprising units derived from hydroxy terminated (polyoxyethylene) acrylates and methacrylates, alkyloxy terminated (polyoxyethylene) acrylates and methacrylates, hydroxy terminated (polyoxyethylene) acrylamides and methacrylamides, alkyloxy terminated (polyoxyethylene) acrylamides and methacrylamides and mixtures thereof; and - at least one type of structural units C derived from sterically hindered amines.


French Abstract

La présente invention concerne une préparation pour une utilisation par voie orale pour prévenir et traiter des affections inflammatoires du tractus gastro-intestinal, ladite préparation contenant un polymère hydrophile, biocompatible et muco-adhérent contenant : - au moins un type d'unités structurelles A choisies dans un groupe comportant des unités dérivées d'acides acrylique et méthacrylique et leurs sels, de méthacrylates et d'acrylates d'hydroxyalkyle, de méthacrylamides et d'acrylamides d'hydroxyalkyle, de méthacrylates et d'acrylates de N,N-dialkylaminoalkyle, de méthacrylamides et d'acrylamides de N,N-dialkylaminoalkyle, de N-vinyle-2-pyrrolidone et des mélanges de ceux-ci ; - au moins un type d'unités structurelles B choisies dans un groupe comprenant des unités dérivées de méthacrylates et d'acrylates (polyoxyéthylène) à terminaison hydroxy, de méthacrylates et d'acrylates (polyoxyéthylène) à terminaison alkyloxy, de méthacrylamides et d'acrylamides (polyoxyéthylène) à terminaison hydroxy, de méthacrylamides et d'acrylamides (polyoxyéthylène) à terminaison alkyloxy et des mélanges de ceux-ci ; - au moins un type d'unités structurelles C dérivées d'amines à encombrement stérique.

Claims

Note: Claims are shown in the official language in which they were submitted.


17
CLAIMS
1. A preparation for oral administration for prevention and/or treatment of
inflammatory conditions
of gastrointestinal tract, characterized in that it contains a mucoadhesive,
biocompatible
hydrophilic polymer containing:
- at least one type of structural units A selected from the group consisting
of units derived from
acrylic and methacrylic acids and their salts, hydroxyalkyl acrylates and
methacrylates,
hydroxyalkyl acrylamides and methacrylamides, N,N-dialkylaminoalkyl acrylates
and
methacrylates, N,N-dialkylaminoalkyl acrylamides and methacrylamides, N-vinyl-
2-pyrrolidone
and mixtures thereof;
- at least one type of structural units B selected from the group consisting
of units derived from
hydroxy terminated (polyoxyethylene) acrylates and methacrylates, alkyloxy
terminated
(polyoxyethylene) acrylates and methacrylates, hydroxy terminated
(polyoxyethylene)
acrylamides and methacrylamides, alkyloxy terminated (polyoxyethylene)
acrylamides and
methacrylamides and mixtures thereof; and
- at least one type of structural units C derived from monomers of general
formula:
Image
wherein R1 is ¨H or ¨OH or oxygen radical, R2 to R5 is (C1 - C4) alkyl, X is
¨CH(Y)¨ or ¨
CH(Y)CH2¨ and Y is a chemical group capable of undergoing radical
polymerization:
Image
wherein R is ¨H or ¨CH3 and Z is ¨O¨ or ¨NH¨.

18
2. The preparation according to claim 1, characterized in that the polymer
contains up to 98 mol.
% of units A, from 1 to 99 mol. % of units B, and at least 1 mol. % of units
C.
3. The preparation according to claim 1 or 2, characterized in that structural
units C are derived
from N-(2,2,6,6-tetramethylazinan-4-yl) methacrylamide or N-(2,2,6,6-
tetramethylazinan-4-yl)
methacrylate.
4. The preparation according to any one of claims 1 to 3, characterized in
that the polymer is in the
form of a linear polymer, a branched polymer or a covalently crosslinked
polymer.
5. The preparation according to any one of claims 1 to 4, characterized in
that it further contains
pharmaceutically acceptable auxiliary substances selected from the group
consisting of excipients
for easier tablet making, effervescent acid and base components,
disintegration agents, sweeteners
and aromatic compounds.
6. The preparation according to any one of claims 1 to 5, characterized in
that it is in the form of a
tablet, a capsule, syrup, solution, emulsion or suspension.
7. Use of the preparation as defined in any one of claims 1 to 6, for the
manufacture of a medicament
useful for the prevention and/or treatment of inflammatory conditions of the
gastrointestinal tract.

Description

Note: Descriptions are shown in the official language in which they were submitted.


1
ORAL PREPARATION COMPRISING MUCOADHESIVE, BIOCOMPATIBLE HYDROPHILIC POLYMER
FOR
PREVENTING AND/OR TREATING INFLAMMATORY CONDITIONS OF THE GASTROINTESTINAL
TRACT
Field of Art
The present invention relates to a polymeric preparation, which adjusts the
internal environment of
gastrointestinal tract (GIT) by removal of harmful substances, introduced into
the GIT together
with food or originated from processes taking place in GIT, mainly from
inflammatory processes.
The preparation has a beneficial influence on functioning of the mucose of the
gastrointestinal tract
and/or on inflammatory disease cure.
Background Art
Inner surfaces of GIT mucose are exposed to a very aggresive environment of
gastrointestinal
juices, characterized by extremely low pH values, enzymatic activity of
gastrointestinal juices and
actions of substances introduced into the GIT together with food. The inner
surface of stomach
wall and of other parts of GIT are covered by a layer of mucus, which has,
among others, a
protective function for the mucous membrane. When such layer is disrupted from
any reason, the
mucous membrane of the organ wall is irritated by contact with
gastrointestinal juices, which can
lead to inflammation. Nowadays, stomach ulcers, Crohn disease and ulcerative
colitis belong
among the most frequent inflammatory diseases of GIT.
No reliable prophylactic or therapeutic method exists at present to prevent
GIT mucous membrane
from inflammatory changes. The most frequently used therapeutic methods use
antibiotics and
steroid based anti-inflammatory drugs. However, their excessive use might
cause very harmful side
effects, e.g. malaise, hypertension, diabetes, osteoporosis, glaucoma.
Patients, who cannot use
antibiotics or steroid based anti-inflammatory drugs, are treated by
immunosuppressants which
weaken their immune system and therefore reduce the symptoms of the disease.
The effect of
immunosuppressive treatment of Crohn disease and ulcerative colitis, based on
weakening of the
autoimmunity reaction, is disadvantageous in long-term usage because it makes
the patient more
predisposed towards other diseases.
Excessive production of reactive oxygen free radicals (reactive oxygene
species, ROS) is a very
important factor during tissue damage in the first (inflammatory) phase,
caused by
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activities of anti-inflammatory mediators. Oxygen free radicals are for
example hydroxyl,
peroxyl and superoxide anionradicals. If the ROS concentration exceeds
antioxidation
capacity of the surrounding cells, the so-called oxidative stress occurs. The
oxidative stress
caused by ROS might cause further damage of the tissue, therefore worsen the
overall
progression of the inflammation, and importantly prolongate the course of
treatment.
Use of hindered amine stabilizers (HAS) and their oxidated derivatives such as
nitroxides
and hydroxylamines in inflammatory treatment is mentioned in patent documents.
CZ
293419 B6 relates to hydrophilic polymers in the form of a gel or a cover
foil, which
enhances the treatment process upon its application on a skin inflammation
wound. The
starting polymers contain a covalently bound sterically hindered amine or its
oxidated
derivative (nitroxide or hydroxylamine), which serve as very efficient free
radical
scavengers. Materials and preparations according to the above mentioned patent
can be
used on external injuries, wherein the localised effect is reached by applying
the
preparation directly on the injury. However, such preparations are not
suitable for the
complex and diverse GIT environment, where the effect of the preparation needs
to be
assured in a relatively large and changing volume of gastrointestinal juices,
and changing
properties and composition of the environment, eg. different pH values in
different parts of
the GIT. For oral use, the accessibility of functional groups to the noxas
present,
localization of the preparation effect on the GIT mucous membrane and minimal
systemic
effect on the organism, are most important. Such properties might be achieved
by a
combination of an antioxidant with a polymer.
JP 2012-111700 A relates to polymeric antioxidants for use in GIT. According
to this
document, the polymeric antioxidant is in the form of particles or micelles
and contains
derivatives of cyclic nitroxides in its structure. The described block
copolymers contain
chemically bound blocks of polyethylene glycol and polystyrene, whereas the
polystyrene
blocks contain styrene units functionalized in position 4 by functional groups
capable of
undergoing chemical modifications enabling introduction of cyclic nitroxides.
A
disadvantage of such solution is the binding of functional nitroxides on the
polystyrene
block of the copolymer, which forms the hydrophobic core of a micelle or a
particle.
respectively, therefore it is separated from its surroundings by a layer of
polyethylene
glycol which forms the hydrophilic coating of the micelle or the particle,
respectively. It
results in a limited accessibility of hydrophilic ROS to the antioxidant
placed in the core of
the micelle.

3
Another factor influencing the progression of an inflammatory disease in GIT
is the extremely low
pH value, e.g. in stomach juices. Compounds chemically binding hydrochloric
acid, contained in
stomach juices, can serve as a supporting agent to prevent the inflammation of
the stomach mucous
membrane and as a protection of the mucous membrane during the inflammatory
stage (gastritis,
irritated stomach, heartburn, reflux, gastroesophageal reflux disease, stomach
ulcer and duodenum
ulcer). It particularly concerns inorganic compounds based on bicarbonates,
hydroxides, metal
oxides (allurninium, magnesium), and optionally their combinations with
omeprazole, which acts
as a proton pump inhibitor. Low molecular weight compounds are usually
dispersed in a polymeric
carrier which swells in stomach juices and therefore ensures their controlled
release into the
surrounding medium. However, the presence of low molecular weight compounds
can cause a
whole range of side effects to sensitive patients, due to the absorption of
the compounds into the
bloodstream. Another disadvantage of the use of such metal containing
preparations is their
interaction with commonly used medicaments, especially with antibiotics -
their absorption is
significantly inhibited by those metal cations.
The above mentioned disadvantages of the present state of the art can be
eliminated by using the
oral preparation according to the present invention.
Disclosure of the Invention
Object of the invention is a preparation for oral administration destined for
prevention and
treatment of inflammatory affections of the gastrointestinal tract. The
preparation contains a
biocompatible, hydrophilic polymer with affinity towards the glycoproteins
present on the GIT
mucous membrane surface, said polymer containing functional groups in its
structure, which are
able to bind harmful substances from the GIT juice. Individual functional
components are
covalently bound within polymeric chains, therefore they are effectively
prevented from being
released and absorbed into the bloodstream, which would lead to adverse
systemic effects.
CA 2963582 2017-08-16

3a
According to a particular aspect, there is provided a preparation for oral
administration for
prevention and/or treatment of inflammatory conditions of gastrointestinal
tract, characterized in
that it contains a mucoadhesive, biocompatible hydrophilic polymer containing:
- at least one type of structural units A selected from the group consisting
of units derived from
acrylic and methacrylic acids and their salts, hydroxyalkyl acrylates and
methacrylates,
hydroxyalkyl acrylamides and methacrylamides, N,N-dialkylaminoalkyl acrylates
and
methacrylates, N,N-dialkylaminoalkyl acrylamides and methacrylamides, N-vinyl-
2-pyrrolidone
and mixtures thereof;
- at least one type of structural units B selected from the group consisting
of units derived from
hydroxy terminated (polyoxyethylene) acrylates and methacrylates, alkyloxy
terminated
(polyoxyethylene) acrylates and methacrylates, hydroxy terminated
(polyoxyethylene)
acrylamides and methacrylamides, alkyloxy terminated (polyoxyethylene)
acrylamides and
methacrylamides and mixtures thereof; and
- at least one type of structural units C derived from monomers of general
formula:
X
R3 ________________________________________
R4
R2 R5
I 1
wherein RI is ¨H or ¨OH or oxygen radical, R2 to R5 is (CI - C4) alkyl, X is
¨CH(Y)¨ or ¨
CH(Y)CH2¨ and Y is a chemical group capable of undergoing radical
polymerization:
H2C ______________________________________
_______________________________________________ 0
wherein R is ¨H or ¨CH3 and Z is ¨0¨ or ¨NH¨.
According to another particular aspect, there is provided the use of the
preparation as defined
herein, for the manufacture of a medicament useful for the prevention and/or
treatment of
inflammatory conditions of the gastrointestinal tract.
Said polymer contains three types of structural units which have different
functions in the
polymeric structure and in the functioning of the preparation. Those
structural units are:
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4
Structural units A, characterized by an ability to change their hydrophilic
properties
depending on the surrounding pH. These structural units ensure good solubility
of the
linear polymer, and, eventually, good swellability of branched and cross-
linked polymeric
structures in gastrointestinal tract juices in a wide range of pH (from 1.0 to
7.0). They are
also able to adjust the pH of gastric fluid. These units are selected from
acrylic and
methacrylic acids and their salts, hydroxyalkyl acrylates and methacrylates, N-
viny1-2-
pyrrolidone, N,N-dialkylaminoalkyl acrylates and methacrylates, N,N-
dialkylaminoalkyl
acrylamides and methacrylamides.
Structural units B are selected from a group of compounds characterized by
their affinity
towards glycoproteins present on the gastrointestinal tract mucous membrane
surface. The
presence of such units in the polymeric structure enhances the adhesion of the
polymer on
the mucous membrane surface, and therefore keeps the active substance,
incorporated in
the polymeric chain, in contact with the afflicted site of the mucous
membrane. Monomers
suitable for providing affinity towards gastrointestinal tract mucous membrane
surface are
selected from hydroxy terminated (polyoxyethylene) acrylates and
methacrylates, alkyloxy
terminated (polyoxyethylene) acrylates and methacrylates, hydroxy terminated
(polyoxyethylene) acrylamides and methacrylamides, alkyloxy terminated
(polyoxyethylene) acrylamides and methacrylamides, preferably of an average
molecular
mass /1411 in the range of 200 to 1000.
Preferably, the term õalkyl" means C1-C6 alkyls, if it is not stated
otherwise.
Salts include, in particular, salts of alkali metals and ammonium.
Structural units C contain chemical groups capable of binding or inactivating
substances
which are toxic to the gastrointestinal tract or which negatively influence
inflammatory
processes. Suitable hydrophilic monomers capable of binding and inactivating
free radicals
are selected from a group of sterically hindered amines and their oxidated
derivatives of
general formula:

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X
R3 __________________________________
R4
R2/N
N R5
I
wherein Rl is ¨H or ¨OH or oxygen radical, R2 to R5 is (C1 - C4) alkyl, X is
¨CH(Y)¨ or ¨
CH(Y)CH2¨ and Y is a chemical group capable of undergoing radical
polymerization:
5 H2C
0
wherein R is ¨H or ¨CH3 and Z is ¨0¨ or ¨NH¨, whereas the content of
structural units C
in the polymer is at least 1 %, preferably from 3 to 30 mol.%.
Structural units C are preferably derived from N-(2,2,6,6-tetramethylazinan-4-
y1)
methacrylamide or N-(2.2,6,6-tetramethylazinan-4-y1) methacrylate.
Preferably, the polymer contains up to 98 % of units A, from 1 to 99 % of
units B and at
least 1 % of units C (in molar %).
Hydrophilic polymer according to the invention is prepared by radical
polymerization of a
mixture of precursors of structural units A, B and C.
Initiators are used to initiate the radical copolymerization. Suitable
initiators of the radical
polymerization, as well as suitable polymerization procedures and conditions,
are generally
known to a skilled person. For example, radical thermic initiators such as
azoinitiators,
diacylperoxides and other peroxocompounds, UV initiators generating free
radicals by UV
irradiation or redox initiators, generating free radicals by oxidation-
reduction reaction, can
be suitable. Initiation does not have to be limited by the types of initiators
mentioned
above.
Polymers can be branched or crosslinked. Crosslinking reagents for the
preparation of
branched or crosslinked polymers can be, for example,
ethylenedi(meth)acrylate, diglycol

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6
and oligoglycol acrylates and methacrylates, ethylendiamindi(meth)acrylate,
1,1'-divinyl-
3 ,3 '-(ethan-1,1'-diy1)bis(pyrrolidin-2-one), 2,3-dihydroxybutan-1,4-diy1
diacrylate or
dimethacrylate, N,N'-methylenebisacrylamide or other crosslinkers commonly
used in the
field.
The hydrophilic polymer is, therefore, usually in the form of a high-molecular
weight
linear polymer, branched polymer or in the form of particles composed of
covalently
crosslinked gel. It is not absorbed by the gastrointestinal tract mucous
membrane, neither
does it enter the bloodstream, nor is it decomposed into low-molecular weight
compounds
which could be absorbed by the gastrointestinal tract. Non-absorbance of the
polymer
eliminates systemic contraindication. Moreover, the polymer is chemically
neutral and
does not undergo chain degradation in GIT juices.
The hydrophilic polymer is soluble or it has a high swelling ability in GIT
juices. The
polymer, upon its contact with juices of gastrointestinal tract, gradually
swells, which
makes the active structural units C, incorporated in polymeric chains,
accessible. Upon its
gradual swelling, the polymer at the same time adheres to the gastrointestinal
tract mucose
because of the presence of structural units B, showing affinity towards mucose
proteins.
The active substance gets therefore in contact with the afflicted site of the
mucous
membrane; therewithal, the time for which the polymer (together with its
active substance)
rests in the particular part of the gastrointestinal tract is prolongated due
to the adhesion of
the polymer to the mucose.
The preparation according to the present invention can preferably be in a form
of a tablet
(simple or multilayered, coated and non-coated, effervescent), capsule
suitable for oral use.
syrup, solution, emulsion or suspension.
Besides the polymer according to the present invention, the preparation can
further contain
other pharmaceutically acceptable auxiliary substances, for example excipients
for easier
tablet making, commonly used in the field; acid and base components ensuring
the
effervescent way of administration; disintegration agents, e.g.
microcrystalline cellulose.
starches (corn, potato and modified starches). The content of disintegration
agents in the
preparation can reach up to 20 wt. %, preferably from 2 to 10 wt. %.

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Sweeteners can be used as auxiliary substances in order to mask the bitter
taste of active
compounds or to enhance the sweet taste of the dosage form. Sweeteners, which
can be
used in the present invention, are selected from saccharides, such as
monosaccharides or
disaccharides (D-glucose, D-fructose, D-xylose, maltose, sucrose or sorbitol),
polyols
(glycerol, dulcitol, mannitol, sorbitol or xylitol), artificial sweeteners
(saccharin and its
sodium, potassium or calcium salts, cyclamate and its sodium or calcium salt,
aspartame,
acesulfame or their potassium salts). The content of sweeteners in the
preparation can
range from 2 to 60 wt. %, preferably from 5 to 10 wt. % (relative to the
weight of the
dosage form).
The auxiliary substances can include also aromatic compounds, which can be
used in the
present invention and which are not limited to the following examples: e.g.,
orange, lemon
or grape flavour, peppermint flavour etc. The aromatic compounds can be used
in the range
of 0.01 to 5 wt. % (relative to the weight of the dosage form).
The polymer structure is designed to enable preferential concentration of the
preparation
close to the gastrointestinal tract mucose, e.g. utilising the adhesion of the
preparation
towards the mucose surface. The protective effect of the preparation towards
the
gastrointestinal tract mucose is enhanced by its adhesion to the mucose, the
adjustment of
the local pH in the mucose proximity or by protection of the mucose cells
against harmful
substances. The preparation includes chemical structures which specifically
bind harmful
substances of gastrointestinal tract juices, e.g. structures which bind and
inactivate free
radicals, structures adjusting pH of the gastric fluid by binding hydrochloric
acid etc.
Functional structures specifically binding harmful substances are covalently
incorporated
into the material of the preparation. They are therefore prevented from
releasing into the
bloodstream, leading to potential adverse systemic effects. Preferably, such
specific
binding is via covalent bonding.
In comparison with JP 2012-111700 A, the present invention is a polymeric
preparation
composed of copolymers, in which different types of hydrophilic units (A, B,
C) are
statistically incorporated into the whole length of the polymeric chain. It
ensures an even
swelling of the polymer, which highly enhances the accessibility of the bound
ROS

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8
antioxidant and enables molecules of the antioxidant to act in a close
proximity of the
mucose surface.
Another advantage of the present invention is the method of incorporating the
molecules of
antioxidants into the polymer by radical copolymerization, in which the
antioxidant acts as
comonomer. It simplifies the production of the preparation on industrial
scale, which
increases the industrial applicability of the invention.
Brief Description of Drawings
Fig. 1: Time dependence of relative fluorescence intensity of the reaction
mixture
containing peroxyl free radicals and the polymer according to Example 4. The
curve a
corresponds to the reference sample without polymer, curves b, c, d, e
correspond to
samples containing 5 mg, 10 mg, 20 mg and 40 mg of the polymer.
Fig. 2: EPR spectrum of the polymer solution according to Example 5, after the
reaction
with in situ generated peroxyl free radicals.
Fig. 3: In vivo imaging of three mice 2 h, 5 h and 12 h after p.o. application
of polymer
solution labeled according to Example 8 (ca 50 MBq/mouse).
Fig. 4 Ex vivo biodistribution of the polymer according to Example 8 in DBA/2
mice; 2 h,
6 h and 24 h after the p.o. application, expressed as a percentage of the
total dosage (%
ID).
Fig. 5 A part of a large intestine of a BALB/c mouse before (left) and after
the exposure to
the ABC polymer suspension (right) under UV light.
Fig. 6 Time dependence of the polymer swelling according to Example 14 in
simulated
GIT juices (stomach juice ¨ pH=1.6, small intestine juice ¨ pH=6.5, large
intestine juice ¨
pH=7.0).
Fig. 7 Time dependence of the relative fluorescence intensity of the reaction
mixture
containing hydroxyl free radicals and the polymeric network according to
Example 14. The
continuous curve corresponds to a blank sample, other dependences belong to
samples
with polymer concentrations of 0.9 mg/mL (o). 4.5 mg/mL (A) and 9.0 mg/mL (0).
Fig. 8 Time dependence of the polymer swelling according to Example 17 in
simulated
GIT juices (stomach juice ¨ pH=1.6, small intestine juice ¨ pH=6.5, large
intestine juice ¨
pH=7.0).

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9
Fig. 9 Development of average weight of mice tested in individual groups in
the course of
the treatment described in Example 20.
Examples
Example 1
Mixture of 0.2 g of N-(2,2,6,6-tetramethylazinan-4-y1) methacrylate and 6 g of
N-viny1-2-
pyrrolidone, 0.1 g of 1.1--diviny1-3,3--(ethan-1.1--diy1)bis(pyrrolidin-2-one)
and 1.68 g of
poly(ethyleneglycol) methyl ether methacrylate (average M. = 500) was
dissolved in 120
ml of methanol. After an addition of 12 mg of 2,2'-azobis(2-
methylpropionitrile), the
mixture was heated under inert atmosphere to 55 C for 24 h. The resulting
copolymer was
extracted by ethanol and dried under vacuum. The powdered polymer has good
swelling
properties in water.
Example 2
Mixture of 0.35 g of N,N-(dimethylaminoethyl) methacrylate, 4.68 g of
poly(ethyleneglycol) methyl ether methacrylate (average M. = 300). 1.0 g of N-
(2,2,6,6-
tetramethylazinan-4-y1) methacrylamide and 35 mg of 2.2 r-azobis(2-
methylpropionitrile)
was dissolved in 20 mL of tetrahydrofurane (THF). The solution was heated at
60 C for
24 h. After the polymerization was finished, the viscose solution was
precipitated with
diethylether and repeatedly precipitated with diethylether from a THF
solution. After
drying, the resulting polymer has a good solubility in water.
Example 3
Mixture of 0.72 g of poly(ethyleneglycol) methyl ether methacrylate (average
M. = 300).
1.8 g of N,N-(diethylaminopropyl) methacrylamide, 0.4 g of N-(2,2,6,6-
tetramethylazinan-
4-y1) methacrylamide and 100 mg of 2,2'-azobis(2-methylpropionitrile) was
dissolved in
27 mL of toluene and heated at 70 C for 24 h. The resulting solution was
partially
evaporated, the polymer was precipitated with heptane and repeatedly
precipitated with

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heptane from a THF solution. After drying, the resulting polymer was obtained
in a powder
form, having good solubility in water.
Example 4
5
Mixture of 2.00 g of 2-hydroxyethyl methacrylate, 1.84 g of
poly(ethyleneglycol) methyl
ether methacrylate (average Mn = 300), 2.1 g of N-(2,2,6,6-tetramethylazinan-4-
y1)
methacrylamide and 0.25 g of 2.2'-azobis(2-methylpropionitrile) was dissolved
in 118 mL
of toluene and heated at 70 C for 24 h. The resulting polymer was filtered
off and re-
10 precipitated with ether from methanol solution. The resulting polymer
had a powder
consistence and a good water solubility.
Example 5
Antioxidation ability of a solution of the linear polymer obtained in Example
4 was
determined against in situ generated peroxyl radicals. The experiment was as
follows:
Stock buffered solutions of the polymer at concentrations 8 mg/mL, 16 mg/mL,
32 mg/mL
and 64 mg/mL were prepared. In each vial, 650 tL of the polymer stock solution
was
placed together with 1.3 mL of fluorescein solution (2.4 x 10-5 M) and 1.95 mL
of AAPH
solution (0,16 M). Directly after addition of all components, a continuous
decrease of
fluorescence intensity was measured during 60 minutes at 37 C. The
measurement was
repeated twice for each polymer concentration. Fig. 1 shows that the polymer
at the above
mentioned conditions acts as a very efficient peroxyl radical scavenger.
Example 6
EPR spectrum of the polymer solution from Example 5 was measured after a
reaction with
peroxyl radicals (Fig. 2). EPR spectrum confirmed the presence of the oxidated
form of a
sterically hindered amine.
Example 7

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11
0.17 g of N-(2,2,6,6-tetramethylazinan-4-y1) methacrylamide, 1.48 g of 2-
hydroxyethyl
methacrylate, 1.95 g of poly(ethyleneglycol) methyl ether methacrylate
(average 1110 =
300), 36 mg of N-methacryloyl tyrosinamide (a precursor for radioisotope
labeling) and
153 mg of 2,2'-azobis(2-methylpropionitrile) was dissolved in 20 mL of
dioxane. After an
addition of 70 mL of toluene the solution was heated at 70 C for 24 h. After
a partial
evaporation of solvents, the polymer was precipitated with diethylether. The
precipitation
was repeated several times from methanol solution to diethylether. The
resulting polymer
was dried under vacuum, and had a powder consistence and a good water
solubility. The
sample was purified using a desalting column.
Example 8
Polymer sample with incorporated tyrosinamide according to Example 7 was
labeled with
radioisotope 1311 according to the following procedure:
6 mg of the polymer was dissolved in 100 pL of phosphate buffer (PBS),
followed by an
addition of 50 pL, of Chloramine-T solution (20 mg chloramine / 1 mL PBS).
After 20
minutes of incubation, 260 pL of Na131I solution (corresponding to an activity
of 420
MBq) and another 3 [t1_, of Chloramine-T solution were added. After 90 minutes
of
incubation, the reaction was ended by adding 20 pL of ascorbic acid solution
(50 mg / 1
mL PBS), 50 pL of 2-amino-2-hydroxymethyl-propan-1,3-diole solution (100 mg /
1 mL
PBS) and 501..LL of freshly prepared NaBH4 solution (20 mg of hydride / 1 mL
PBS). High-
molecular fractions expressing overall ca 89 % of the total radioactivity were
lyophilised.
Example 9
5 mg of the lyophilised product from Example 8, having specific activity of 20
MBq/mg,
was dissolved in 2.5 mL of saline. In order to image in vivo biodistribution,
the solution of
the labeled polymer was administered per orally (p.o.) to three Balb/c mice in
a dose of
cca 50 MBq/mouse. The imaging was performed on In vivo MS FX PRO for small
animals. The experimental animals were under anesthesia during the whole time
of the
procedure. Static scanning was performed in time intervals of 2 h, 5 h and 12
h after
polymer administration. Already 12 hours after polymer solution
administration, almost all

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12
activity was eliminated (Fig. 3). The experiment proved that the polymer is
neither uptaken
by a bloodstream, nor specifically absorbed and cumulated in a particular
organ.
Example 10
5 mg of the lyophilised product from Example 8, having specific activity of 20
MBq/mg,
was dissolved in 2.5 mL of saline. Ex vivo biodistribution of the polymer
solution was
monitored in three time intervals: 2 h, 6 h and 24 h after p.o. application.
The ex vivo
biodistribution study of the radiolabeled polymer solution (dose ca 1
MBq/mouse) was
performed p.o. to nine DBA/2 mice. The experimental animals were sacrified
within the
predetermined time limits (three animals per each time limit). The following
organs were
then taken away: blood, spleen, pancreas, stomach, intestines, kidneys, liver,
heart, lungs,
muscle, bone and thyroid. The radioactivity was measured in the organs using
an automatic
gamma counter. The results were expressed as percentages of the originally
applied dose
(% ID). The ex vivo biodistribution study confirmed a complete elimination of
the polymer
via the gastrointestinal tract (Fig. 4).
Example 11
Mixture of 3.0 g of N-(2,2,6,6-tetramethylazinan-4-y1) methacrylamide. 2.91 g
of 2-
hydroxyethylmethacrylate, 2.67 g of poly(ethyleneglycol) methyl ether
methacrylate
(average /14-11 = 300). 45 mg of 5-1[3-(methacryloylamino)propyl]thioureidyl }
fluoresceine,
18 mg of N,N--methylenebisacrylamide and 366 mg of 2.2'-azobis(2-
methylpropionitrile)
was dissolved in 30 mL of toluene and 3.5 mL of methanol. The polymerization
was
carried out for 24 h at the temperature of 70 C. The precipitated polymer
(ABC) was
filtered off, extracted by dioxane and dried into a constant weight.
Analogical procedure
was used to prepare a polymeric sample (AC), which does not contain the
structural unit
based on poly(ethyleneglycol) methyl ether methacrylate.
Isolated parts of a large intestine of a male mouse BALB/c were exposed to
suspensions of
polymers ABC and AC (20 mg / 1 mL PBS; pH = 7.4) for 5 min. A strong
fluorescence of
the ABC polymer is observable under a UV lamp on the inner mucose of the
exposed
intestine. The fluorescence persists even after repeated washing with a buffer
(Fig. 5). The

CA 02963582 2017-04-04
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13
experiment proved markedly stronger mucoadhesive properties of the ABC polymer
in
comparison with the AC polymer, which does not contain the structural units B.
Example 12
Mixture of 2.6 g of poly(ethyleneglycol) methyl ether methacrylate (average Mn
= 950).
0.8 g of 2-hydroxypropyl methacrylamide, 0.8 g of N-(2,2,6,6-tetramethylazinan-
4-y1)
methacrylate and 32 mg of 2,2'-azobis(2-methylpropionitrile) was dissolved in
28 mL of
acetone and heated at 50 C for 24 h. The resulting polymer is water soluble.
Example 13
Mixture of 2.91 g of 2-hydroxyethyl methacrylate, 2.67 g of
poly(ethyleneglycol)
methacrylate (average Mt, = 300), 3.0 g of N-(2,2,6,6-tetramethylazinan-4-y1)
methacrylamide, 18 mg of N,N--methylenebisacrylamide and 366 mg of 2,2'-
azobis(2-
methylpropionitrile) was dissolved in 90 mL toluene and heated at 70 C for 24
h. The
polymer in a form of a powder precipitates during the reaction and has good
swelling
properties in water.
Example 14
Mixture of 0.78 g of N-(2,2,6,6-tetramethylazinan-4-ye methacrylate, 0.8 g of
2-
hydroxypropyl methacrylamide, 0.2 g of poly(ethyleneglycol) methacrylate
(average M11 =
360) and 0.15 g of 2,2'-azobis(2-methylpropionitrile) was dissolved in 12 mL
of toluene
and heated at 70 'V for 24 h. The resulting polymer was precipitated from
methanol
solution to diethylether. Dry polymer was in the form of a powder, soluble in
water.
Example 15
2.54 g of N,N-(dimethylaminopropyl) methacrylamide, 2.0 g of N-(2,2,6,6-
tetramethylazinan-4-ye methacrylamide, 1.78 g poly(ethyleneglycol) methyl
ether
methacrylate (average Mn = 300). 92 mg of N,N--methylenebisacrylamide and 0.24
g of
2,2'-azobis(2-methylpropionitrile) were heated in a mixture of 20 mL of
dioxane and 40

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14
mL of heptane at 70 C for 24 h. The resulting polymer network was filtered
off, several
times extracted with heptane and dried under vacuum.
Example 16
Swelling properties of the polymer network prepared in Example 15 were
determined at
the temperature of 37 C in a solution simulating gastrointestinal juices. 0.2
g of the
polymer was placed into a centrifuge tube together with the above mentioned
juice, giving
the overall mass of 10 g (including the tube). The tube was placed into a bath
temperated to
37 'V for the time listed in Fig. 6. The contents of the tube were
centrifuged, the upper
layer was taken off and the tube was weighted. The degree of swelling was
calculated
using the following formula:
swelling (%) = 100 x (mt ¨ 'no)/'no
wherein mt is the mass of the swollen polymer in time t, and mo is the mass of
the dry
polymer before the experiment. The polymer network shows from 800 to 1300% of
swelling in a wide range of pH of the simulated GIT juices (pH = 1.6 to 7.0;
Fig. 6).
Example 17
Antioxidation capacity against in situ generated hydroxyl free radicals in
solution of the
polymer network prepared in Example 15 was determined, according to the
following
procedure. Eight Eppendorf tubes containing appropriate amounts of the polymer
were
placed into a thermoblock and tempered to 37 'C. Then 900 [IL of a buffer, 200
litL of
Na2W04.2 FI70 solution in a buffer (7.3 mM), 10 0_, of fluorescein solution
(2.4 x 10 M)
were placed into each tube and the polymer was kept to swell in this mixture
for 5 minutes.
Hydroxyl radical generation was started by an addition of 10 L of hydrogen
peroxide
solution. The tubes were shaken and tempered to 37 C during the whole time of
the
reaction. Then Eppendorf tubes were opened in predetermined time intervals.
Their content
was filtered using a microne filter, and the fluorescence values of the clear
solutions were
determined. Each sample was measured twice. Fig. 7 shows that even very low

CA 02963582 2017-04-04
WO 2016/082808 PCT/CZ2015/050011
concentrations of the polymer network effectively scavenge the in situ
generated hydroxyl
radicals.
Example 18
5
4.0 g of N,N-(dipropylaminoethyl) methacrylate, 1.76 g of N-(2,2,6.6-
tetramethylazinan-4-
yl) methacrylate, 0.3 g of 2-hydroxyethyl acrylate, 0.28 g of
poly(ethyleneglycol) methyl
ether methacrylate (average Mll = 300), 15 mg of ethylenedimethacrylate and
0.22 g of
2,2'-azobis(2-methylpropionitrile) were heated in a mixture of dioxane and
heptane at 70
10 'V for 24 h. The resulting polymer network was filtered off, several
times extracted with
heptane and dried under vacuum.
Example 19
15 Swelling properties of the polymer network prepared in Example 18 were
determined
using the same procedure as in Example 16. The results of this experiment are
shown in
Fig. 8. The polymer network swells in the range of 1000 % to 1800 % in
simulated GIT
juices (pH = 1.6 to 7.0).
Example 20
The therapeutic effect of the preparation prepared in Example 15 in the GIT
was performed
with the Balb/C induced chronic colitis experimental model.
20 male Balb/C mice were divided into three groups:
K ¨ control group of healthy mice without colitis (n=4)
N ¨ non-treated group with induced colitis (n=8)
L ¨ treated group with induced colitis (n=8).
Testing was started by chronic colitis induction in the N and L groups:
experimental
animals drank 3.5% sodium dextran sulfate (DSS) solution in the following
scheme: 7 days
DSS solution, 7 days water, 7 days DSS solution, 7 days water, 7 days DSS
solution; then
treatment was started in the L group. The tested preparation was administered
to the
animals of the L group twice per day by stomach probe (2x250 jiL 2% solution)
for 7 days.

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16
The mice were regularly weighted during the experiment (weight loss is a
disease
indicator, weight gain is an indicator of therapeutic effect). The day after
the last
therapeutic dose, dissection was performed, colons were taken away and their
length was
measured (shortening of the colon is an indicator of disease, its lengthening
is an indicator
of therapeutic effect).
Fig. 9 shows the comparison of average weight of mice in individual groups
during the
course of the treatment. There is a clearly visible difference between the N
group and the L
group which shows slowing down (or even reversing) of the pathological process
in the L
group.
The results based on the colon length are in good agreement with the weight-
based results,
which demonstrate slowing down or even reversing of the pathological process
in the large
intestine. Healthy animals showed an average colon length of almost 10 cm, the
non-
treated (N) group animals showed an average colon length of about half of this
value. The
treated (L) group showed more than 20% extension of the colon length in
comparison to
the non-treated (N) group.

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Title Date
Forecasted Issue Date 2017-12-05
(86) PCT Filing Date 2015-11-20
(87) PCT Publication Date 2016-06-02
(85) National Entry 2017-04-04
Examination Requested 2017-04-04
(45) Issued 2017-12-05

Abandonment History

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Maintenance Fee

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Payment History

Fee Type Anniversary Year Due Date Amount Paid Paid Date
Request for Examination $800.00 2017-04-04
Application Fee $400.00 2017-04-04
Registration of a document - section 124 $100.00 2017-04-20
Maintenance Fee - Application - New Act 2 2017-11-20 $100.00 2017-08-28
Final Fee $300.00 2017-10-19
Maintenance Fee - Patent - New Act 3 2018-11-20 $100.00 2018-08-09
Maintenance Fee - Patent - New Act 4 2019-11-20 $100.00 2019-08-02
Maintenance Fee - Patent - New Act 5 2020-11-20 $200.00 2020-09-14
Maintenance Fee - Patent - New Act 6 2021-11-22 $204.00 2021-09-15
Owners on Record

Note: Records showing the ownership history in alphabetical order.

Current Owners on Record
USTAV MAKROMOLEKULARNI CHEMIE AV CR, V.V.I.
Past Owners on Record
None
Past Owners that do not appear in the "Owners on Record" listing will appear in other documentation within the application.
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