Title: Implantable particles
for the treatment of gastroesophageal reflux disease
United States Patent: 7,060,298
Issued: June 13, 2006
Inventors: Vogel; Jean
Marie (Boxborough, MA); Thomas; Richard (Elmont, MA); Boschetti; Egisto (Croissy
sur Seine, FR)
Medical, Inc. (Rockland, MA)
Appl. No.: 029294
Filed: December 28, 2001
Executive MBA in Pharmaceutical Management, U. Colorado
The invention encompasses the treatment
of urinary incontinence, gastroesophageal reflux disease and the
amelioration of skin wrinkles using biocompatible hydrophilic cationic
microparticles and a cell adhesion promoter.
OF THE INVENTION
The present invention uses microparticles,
particularly microbeads, having a positive charge on its surface and a
cell adhesion promoter and optionally, a cell growth promoting agent, to
treat GERD, urinary incontinence, and skin wrinkles. The microparticles of
the invention are preferably used with autologous cells. In other words,
the microparticles of the invention are colonized with the appropriate
cells prior to implantation. This pre-implantation step has been shown to
reduce or eliminate immunological responses and implantation rejection
reactions. Further, the use of non-biodegradable biologically compatible
microbeads with positive charges and autologous cells, whether
tissue-specific or not, improves tissue acceptance and overall treatment.
According to the methods of the present invention, treatment of GERD,
urinary incontinence, and skin wrinkles is achievable while avoiding or
substantially reducing adverse tissue reactions, including implantation
rejection, degradation of particles, resorption, migration and other
adverse events. The methods of the invention also involve increased
connective tissue response.
Microbeads or microparticles for use in the present invention are based on
a biocompatible non-toxic polymer coated with agents which promote cell
adhesion. Living cells attach to the microparticles forming layered cells
therein which link with surrounding tissues to enhance long term stability
of the beads.
Microparticles intended to be implanted in various locations of the body
according to the present invention are composed of a non-resorbable
hydrophilic polymer containing the appropriate material for cell adhesion,
and may additionally contain radiopaque molecules or other marking agents,
to facilitate localization by radiology prior to or during intervention.
Hydrophilic copolymers usable for this application are those of the
acrylic family such as polyacrylamides and their derivatives,
polyacrylates and their derivatives as well as polyallyl and polyvinyl
compounds. All of these polymers are crosslinked so as to be stable and
non-resorbable, and can contain within their structure other chemicals
displaying particular properties, such as chemotactic effects, promotion
of cell adhesion to cells or tissues, such as cells of the esophagus wall
or the urethra wall, or skin cells, and/or marking agents.
The microparticles for use in the present invention are non-toxic to
tissues and cells, biocompatible, and adhesive to various cells and
tissues at the site of implantation by means of the cell growth they
promote. In addition, these microparticles are non-resorbable and
non-biodegradable, and thus are stable, durable, and will maintain their
general shape and position once implanted at a desired site.
In general, microparticles for use in the present invention may have any
shape, with microparticles which are spherical in shape being preferred.
Microparticles for use in the present invention may have diameters ranging
between about 10 .mu.m to about 1000 .mu.m. Preferably, microparticles for
use in the present invention which have cells adhered to the surface
thereof will have diameters ranging between 50 .mu.m and 1000 .mu.m.
Possible variations of the present invention include replacing the
microparticles with any biocompatible, non-toxic non-resorbable polymeric
particles, membrane, fibers or other solid substrates treated with an
agent promoting cell adhesion. The invention also includes linear soluble
polymers which, after injection, crosslink in situ to constitute a solid,
cell adhesion promoting filling agent. Preparation and/or injection of
empty microparticles (microbubbles) that are prepared in advance or are
generated in place via the use of appropriate catheters, are also
contemplated in this invention.
The microparticles, or other solid substrates, for use in the present
invention are flexible, such that they can easily pass into and through
injection devices and small catheters without being permanently altered,
but the microparticles are also resistant to the muscle contraction stress
generated during and after the implantation process. They are also
thermally stable which allows for easy, convenient sterilization, and
The microparticles, or other solid substrates, for use in the present
invention are also stable in suspension which allows the microparticles or
other solid substrates to be formulated and stored in suspension and
injected with different liquids. More specifically, the hydrophilic nature
of the microparticles permits placing them in suspension, and in
particular, in the form of sterile and pyrogenic (pyrogen-free) injectable
solutions, while avoiding the formation of aggregates or adhesion to the
walls of storage containers and implantation devices, such as catheters,
syringes, needles, and the like. Preferably, these injectable solutions
contain microparticles or other solid substrates distributed approximately
in caliber segments ranging between about 10 .mu.m and about 2000 .mu.m.
The microparticles of the present invention are both hydrophilic and
cationic. The microparticles preferably comprise a copolymer of a neutral
hydrophilic monomer, a difunctional monomer, one or more monomers having a
cationic charge, and optionally, a functionalized monomer capable of
rendering the microparticle detectable. The microparticles may also
comprise one or more cell adhesion promoters and a marking agent.
The copolymer is preferably a hydrophilic acrylic copolymer which
comprises in copolymerized form about 25 to about 98% neutral hydrophilic
acrylic monomer by weight, about 2 to about 50% difunctional monomer by
weight and about 0 to about 50% by weight of one or more monomers having a
By way of example, the copolymers described in French Patent 2,378,808,
which is incorporated herein by reference, can be used in accordance with
this invention to prepare the base microparticle copolymer.
As hydrophilic acrylic monomer, acrylamide and its derivatives,
methacrylamide and its derivatives or hydroxymethylmethacrylate can be
Examples of difunctional monomer, include but are not limited to the
N,N'-methylene-bis-acrylamide, N',N'-diallyltartiamide or
Further, the monomer having a cationic charge, includes but is not limited
to those carrying a tertiary or quaternary amine function, preferably
diethylaminoethyl acrylamide, methacrylamidopropyl trimethylammonium or
In a particularly preferred embodiment, a copolymer comprising about 25 to
about 98% methacrylamide by weight, about 2 to about 50%
N,N-methylene-bis-acrylamide by weight is used.
In one particularly advantageous embodiment of the invention, it is
possible to increase the stability of the microspheres by reticulating the
adhesion agent. By way of example, in the case of gelatin, the
reticulating agent can be chosen among the difunctional chemical agents
reacting on the gelatin amines (e.g., glutaraldehyde, formaldehyde,
glyoxal, and the like).
The functionalized monomer is generally obtained by chemical coupling of
the monomer with a marker, which can be: a chemical dye, such as Cibacron
Blue or Procion Red HE-3B, making possible a direct visualization of the
microspheres (Bosahetti, J. Biochem-Biophys. Meth., 19:21 36 (1989)).
Examples of functionalized monomer usable for this type of marking N-acryloyl
hexamethylene Cibacrone Blue or N-acryloyl hexamethylene Procion Red
HE-3B; a magnetic resonance imaging agent (erbium, gadolinium or
magnetite); a contrasting agent, such as barium or iodine salts,
(including for example acylamino-e-propion-amido)-3-triiodo-2,4,6-benzoic
acid, which can be prepared under the conditions described by Boschetti et
al. (Bull. Soc. Chim., No. 4 France, (1986)). In the case of barium or
magnetite salts, they can be directly introduced in powered form in the
initial monomer solution.
As indicated above it is also possible to mark the microspheres after
their synthesis. This can be done, for example, by grafting of fluorescent
markers derivatives (including for example fluorescein isothiocyanate (FITC),
rhodamine isothiocyanate (RITC) and the like).
Various types of cell adhesion promoters well known in the art may be used
in the present invention. In particular, cell adhesion promoters can be
selected from collagen, gelatin, glucosaminoglycans, fibronectins, lectins,
polycations (such polylysine, chitosan and the like), or any other natural
or synthetic biological cell adhesion agent.
Preferably, the cell adhesion promoter is present in the microparticle, or
other solid substrate, in an amount between about 0.1 to 1 g per ml of
Microparticles are prepared by suspension polymerization, drop-by-drop
polymerization or any other method known to the skilled artisan. The mode
of microparticle preparation selected will usually depend upon the desired
characteristics, such as microparticle diameter and chemical composition,
for the resulting microparticles. The microparticles of the present
invention can be made by standard methods of polymerization described in
the art (see, e.g., E. Boschetti, Microspheres for Biochromatography and
Biomedical Applications. Part I, Preparation of Microbands In:
Microspheres, Microencapsulation and Liposomes, John Wiley & Sons, Arshady
R., Ed., 1998 (in press) which is incorporated herein by reference).
Microspheres are prepared starting from an aqueous solution of monomers
containing adhesion agents such as collagen (gelatin is a denatured
collagen). The solution is then mixed with a non-aqueous-compatible
solvent to create a suspension of droplets, which are then turned into
solid gel by polymerization of monomers by means of appropriate catalysts.
Microspheres are then collected by filtration or centrifugation and
Cell adhesion promoters or marking agents are introduced on microbeads by
chemical coupling procedures well known in affinity chromatography,
referred to by the term "ligand immobilization". Another method of
introduction is by diffusion within the gel network that constitutes the
bead and then trapping the diffused molecules in place by precipitation or
chemical cross-linking. Therapeutic agents, drugs or any other active
molecules that are suitable for transportation by the beads can also be
introduced into the microbeads prior to bead implantation according to
this last method.
The microspheres of the invention can also be obtained by standard methods
of polymerization described in the art such as French Patent 2,378,808 and
U.S. Pat. No. 5,648,100, each of which is incorporated herein by
reference. In general, the polymerization of monomers in solution is
carried out at a temperature ranging between about 0.degree. C. and about
100.degree. C. and between about 40.degree. C. and about 60.degree. C., in
the presence of a polymerization reaction initiator.
The polymerization initiator is advantageously chosen among the redox
systems. Notably, it is possible to use combinations of an alkali metal
persulfate with N,N,N',N'-tetramethylethylenediamine or with
dimethylaminopropionitrile, organic peroxides such as benzoyl peroxides or
The quantity of initiator used is adapted by one skilled in the art to the
quantity of monomers and the rate of polymerization sought.
Polymerization can be carried out in mass or in emulsion.
In the case of a mass polymerization, the aqueous solution containing the
different dissolved constituents and the initiator undergoes
polymerization in an homogeneous medium. This makes it possible to access
a lump of aqueous gel which can then be separated into microspheres, by
passing, for example, through the mesh of a screen.
Emulsion or suspension polymerization is the preferred method of
preparation, since it makes it possible to access directly microspheres of
a desired size. It can be conducted as follows: The aqueous solution
containing the different dissolved constituents (e.g., different monomers,
cell adhesion agent), is mixed by stirring, with a liquid organic phase
which is not miscible in water, and optionally in the presence of an
emulsifier. The rate of stirring is adjusted so as to obtain an aqueous
phase emulsion in the organic phase forming drops of desired diameter.
Polymerization is then started off by addition of the initiator. It is
accompanied by an exothermic reaction and its development can then be
followed by measuring the temperature of the reaction medium.
It is possible to use as organic phase vegetable or mineral oils, certain
petroleum distillation products, chlorinated hydrocarbons or a mixture of
these different solutions. Furthermore, when the polymerization initiator
includes several components (redox system), it is possible to add one of
them in the aqueous phase before emulsification.
The microspheres thus obtained can then be recovered by cooling, decanting
and filtration. They are then separated by size category and washed to
eliminate any trace of secondary product.
The polymerization stage can be followed by a stage of reticulation of the
cell adhesion agent and possibly by a marking agent stage in the case of
microspheres rendered identifiable by grafting after synthesis.
Microparticles of the present invention which have the specific properties
of cell adhesion and growth promotion can be used directly for tissue
bulking. Moreover, the microparticles of the present invention can have
specific autologous cells grown on their surface in vitro, thereby making
the microparticles particularly useful for tissue bulking.
Prior to the present invention, the injection of implantable substances
suspended in a physiological solution into a tissue resulted in the
formation of discrete aggregates inside the muscle mass. These discrete
aggregates can constitute various amounts of the implanted substance which
stays together, however, the substance does not become attached to or a
part of the tissue itself. This detachment allows the implanted substance
to move from the original implantation site.
According to the present invention, in order to avoid this problem, the
microparticles may be injected individually and separately, or more
preferably, the surface of the microparticles may be colonized by a layer
of cells for better integration and long term stability of the implant.
Microparticles of the present invention demonstrate superior ability to
grow cells on their surfaces. For example, primary muscle cells have been
successfully adhered to the surface of the microparticles of the present
invention thereby allowing for a better integration within a muscle
tissue. In addition, since the ultimate goal of tissue bulking is to
artificially increase tissue mass, preadipocytes have also been used to
colonize the surface of the microparticles prior injection. In this case,
the preadipocytes have a volume similar to any other regular cell, but
after implantation when the preadipocytes are subject to in vivo
physiological conditions, they accumulate go droplets of fats thereby
increasing the mass of the implant by more than 10% in volume.
According to the present invention, one means of performing tissue bulking
in a patient can be described as follows: a) Primary cells are extracted
from the patient by a simple biopsy and isolated; b) These cells are grown
on the surface of the microparticles under growth promoting conditions
(e.g., possibly using a nutrient media which contains autologous serum
(drawn from the patient), until confluence); c) the microparticles having
the patient's cells grown on the top are injected into the patient's
target tissue to be bulked.
For the treatment of GERD, the microparticles, or other solid substrates,
are introduced via the esophagus, either by endoscopic delivery or by
laparoscopic technique, and are injected into the walls of the sphincter
where the esophagus meets the stomach, i.e., the lower esophageal
sphincter. This decreases the internal lumen of the sphincter muscle thus
permitting easier contraction of the muscle with reduced regurgitation of
the gastric fluids into the esophagus. In addition, this treatment reduces
the inflammation of the lower esophagus. The microparticles, or other
solid substrates, may also be loaded with X-ray opaque dye or other
imaging agents for subsequent X-ray visualization.
In another embodiment, microparticles injected into the sphincter at the
junction of the esophagus and stomach in order to treat GERD may also
include an amount of a drug used to treat GERD, such as H.sub.2 histamine
antagonists including cimetidine, ranitidine, famotidine and nizatidine;
inhibitors of H.sup.+,K.sup.+-ATPase including omeprazole and lansoprazole;
antacids including e.g., Al(OH).sub.3, Mg(OH).sub.2, and CaCO.sub.3. As
with the treatment of urinary incontinence and skin wrinkles, the
microspheres may also be used with anti-inflammatory agents, angiogenesis
inhibitors, radioactive elements, and antimitotic agents.
Other therapeutic agents to be used in combination with the microspheres
or microparticles of the present invention include those for the treatment
of skin disorders, GERD and urinary incontinence as reported in Goodman &
Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., McGraw-Hill
(1996) and The Physicians's Desk Reference.RTM. 1997.
The primary advantages of the method of treating GERD according to the
present invention over the prior art methods are:
a) Less invasive effects on the patient compared to surgery;
b) More permanent effects over antacids or other drugs;
c) Good biocompatibility with chemotactic effects; and
d) Ability to use X-ray visualization or MRI to assist in follow-up
evaluation of the patient.
For the treatment of urinary incontinence, the microparticles, or other
solid substrates, are introduced via the urethra and injected into the
walls of the bladder sphincter, decreasing the internal lumen of the
sphincter muscle thus permitting easier contraction of the muscle with
reduced likelihood of incontinence. The microparticles, or other solid
substrate, may also be loaded with X-ray opaque dye, or other imaging
agents for subsequent X-ray visualization.
In another embodiment, microparticles injected into the bladder sphincter
in order to treat urinary incontinence may also include an amount of a
drug used to treat urinary incontinence, such as antidiuretics,
anticholinergics, oxybutynin and vasopressins.
Injected microparticles can generate some transient adverse reactions such
as local inflammation, therefore the microparticles can contain or be
injected with anti-inflammatory drugs, such as salicylic acid derivatives
including aspirin; para-aminophenol derivatives including acetaminophen;
non-steroidal anti-inflammatory agents including indomethacin, sulindac,
etodolac, tolmetin, diclodfenac, ketorolac, ibuprofen, naproxen,
flurbiprofen, ketoprofen, fenoprofen, oxaprozin; anthranilic acids
including mefenamic acid, meclofenamic acid; enolic acids such as
piroxicam, tenoxicam, phenylbutazone, oxyphenthatrarone; and nabumetone.
These anti-inflammatories are preferably adsorbed on the microparticle's
network and released slowly over a short period of time (a few days).
The microparticles may also be used to release other specific drugs which
can be incorporated within the microparticle network before injection into
the patient. The drug would be released locally at the site of
implantation over a short period of time to improve the overall treatment.
Incorporation of active molecules, such as drugs, into the microparticles
of the present invention can be accomplished by mixing dry microparticles
with solutions of said active molecules or drugs in an aqueous or
hydro-organic solution. The microparticles swell by adsorbing the
solutions and incorporate the active molecule of interest into the
microparticle network. The active molecules will remain inside the
microparticle due to an active mechanism of adsorption essentially based
on ion exchange effect. The microparticles by their nature carry cationic
groups and have the ability to adsorb anionic molecules, such as well
known anti-inflammatory drugs, and these anionic molecules are then
released slowly upon injection into the patient due to the action of
physiological salt and pH. The ability of various types of microparticles
to adsorb drug molecules may be readily determined by the skilled artisan,
and is dependent on the amount of cationic monomers present in the initial
solution from which the microparticles are prepared.
Some of the primary advantages of treating urinary incontinence according
to the present invention over prior art methods are:
a) More permanent effect than the use of regular viscous solutions of
b) Good biocompatibility with chemotactic effect;
c) Visualization under X-ray or MRI to assist in follow-up evaluation; and
d) Preventing repeated treatments with resorbable naturally occurring
substances like collagen.
The primary advantages of the method of treating skin wrinkles according
to the present invention are:
(a) less invasive effects on the patient compared to surgery;
(b) more permanent effects than the use of collagen injections; and
(c) good biocompatibility with chemotactic effects.
For treatment of skin wrinkles, the microparticles may be introduced via
injection. The microparticles may also include one or more
Claim 1 of 17 Claims
1. A method for treating
gastroesophageal reflux disease, which comprises implanting into the lower
esophageal sphincter or the diaphragm of a mammal in need of such treatment
a therapeutically effective tissue-bulking amount of microparticles, wherein
the microparticles comprise a biocompatible, non-toxic hydrophilic
copolymer, which comprises in copolymerized form about 25% to about 99% by
weight of neutral hydrophilic acrylic monomer, about 2% to about 30% by
weight of one or more monomers having a cationic charge, and about 1% to
about 30% by weight of a functionalized monomer.
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