Note: Descriptions are shown in the official language in which they were submitted.
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METHOD AND COMPOSITIONS FOR
INHIBITING ADHESION FORMATION
Statement of Government Rights
The work described herein was supported in part by NIH grant
HD 34824. The Government of the United States of America may have certain
rights in the invention.
Field of the Invention
This invention relates to the field of medicine and inhibition of
post-operative/post-wounding adhesion formation.
Background of the invention
Surgical intervention involves wounding the patient in order to
effect a cure. One unwanted result from surgery is post-operative adhesion
formation. The term "adhesion" used in its medical sense refers to
conglutination, the process of adhering or uniting of two surfaces or parts.
For
example, the union of the opposing surfaces of a wound, or opposing surfaces
of peritoneum. Also, adhesions, in the plural, can refer to inflammatory bands
that connect opposing serous surfaces. The term adhesion, as used herein, also
includes fibrinous adhesions, which are adhesions that consist of fine threads
of
fibrin resulting from an exudate of plasma or lymph, or an extravasion of
blood.
Keloid, a smooth overgrowth of fibroblastic tissue that arises in an area of
injury or, occasionally, spontaneously is also a form of adhesion.
It has been reported that adhesion development is a major source
of postoperative morbidity and mortality. The most frequent surgical
procedures implicated in significant adhesion formation are gynecologic,
cardiovascular, and general abdominal surgery. This is true for traditional
surgery, as well as laparoscopic surgery.
Complications of intraperitoneal adhesions include intestinal
obstruction, chronic or recurrent pelvic pain, infertility in females, and
prolonged surgical time and postoperative complications (when additional
surgical procedures are needed). In extreme cases, debilitating adhesions can
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be treated by adhesiotomy, surgical section or lysis of the adhesion
(adhesiolysis). Thus, methods and compositions for inhibiting adhesion
formation in patients would be most useful.
Animal surgery has become more common as the value, both
sentimental and economic, of animals increases along with the skill and
practice
of veterinary surgeons. Animals are subject to the same problems with post-
surgical adhesion formation and the debilitating effects therefrom. Adequate
and humane care for animals subject to surgical procedures has prompted the
adoption of many technologies and procedures from human diagnosis and
surgery for use on animals. Thus, it would be useful to have methods and
compositions for inhibiting adhesion formation in animals as well.
Physical barriers have been tested for preventing adhesion
formation by limiting tissue apposition during peritoneal healing, when most
adhesions begin to form by the development of a fibrinous matrix between the
tissue surfaces. Generally, physical barriers, either membranes or gels, have
been used to decrease the apposition of injured peritoneum until
reperitonealization occurs and, thus, inhibit or prevent adhesion formation.
In
rat model systems, reperitonealization takes approximately seven (7) days. It
has been found, that in a rat model system, barrier placement for at least 36
hours is needed to prevent adhesion formation (Harris et al., 1995, Surgery,
117:663-9).
While many adjuvants have been tested in animal models for
preventing adhesions, currently only three barriers have been approved for use
in humans for reducing postoperative adhesion formation. InterceedTM,
(Johnson & Johnson Medical) is an oxidized regenerated cellulose; SeprafilmTM
(Genzyme Corp.), is a modified hyaluronic acid complexed with modified
carboxymethylcellulose and PrecludeTM, (W.L. Gore) is an expanded
polytetraflurorethylene. InterceedTM is properly used in the absence of blood.
PrecludeTM has been approved as a pericardial substitute, but is not
bioresorbable. SeprafilmTM has been approved for use in abdominal wall and
uterine incisions.
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Potential antiadhesion agents tested in a rat system include
Ringer's lactate, 32% dextran 70 solution, and 1 % and 2% modified
carboxymethylcellulose solution. One half of the solutions were applied to the
defect, and the excess allowed to pool within the peritoneal cavity of the
abdomen. Ringer's lactate was not effective since it was rapidly absorbed in
the
peritoneal cavity, and was, thus, not present during the full 36 hour critical
period. The other, slightly viscous solutions were more effective.
Hyaluronic acid (HA) has been used as a barrier in abdomino-
pelvic and orthopedic surgery. Modified HA as a resorbable gel and, a
hyaluronan-based gel of auto-crosslinked polysaccharides (ACP) have also been
tested. The ACP, hyaluronic acid derivatives, have been demonstrated to
prevent laparoscopic adhesion formation in the rabbit model system, and has
been reported by DeIaco et al., 1998, Fertility and Surgery, 69(2): 318-323.
A chemically modified hyaluronate and carboxymethylcellulose
(HA/CMC) gel formulation was applied in a rabbit models system by Leach et
al., 1998, Fertility and SurgerX, 69(3): 415-418.
The evaluation of a polyethylene glycol/polylactic acid (EG/LA)
film to prevent adhesion formation in the rabbit model system was conducted by
Rodgers et al. , 1998, Fertility and SurgerX, 69(3):403-408.
Thus, in the medical arts there continues to exist a need for
materials and methods useful for inhibiting or preventing adhesion formation
in
animals as well as in humans.
Summary of the Invention
The invention provides needed compositions and methods useful
for inhibiting or ameliorating adhesion formation in mammals, including
humans.
Adhesions resulting from wounding or surgical procedures can be
inhibited or at least ameliorated by treating a wound or surgical site with an
antagonist molecule that interacts with alpha V beta 3 (av~i3) integrin, or
with
the integrin binding site of an extracellular matrix protein to block an av~33
integrin from binding to an extracellular matrix protein in a mammalian body.
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Suitable antagonist molecules for this purpose are proteins, peptides (linear
as
well as cyclic) and peptidomimetics that mimic the av~33 integrin binding site
on
an extracellular matrix protein, e.g., fibronectin, or that bind to the av~33
integrin itself so as to interfere with its binding to an extracellular matrix
protein. Illustrative of such av~i3 antagonist molecules are monoclonal
antibodies and bioactive portions or fragments thereof that include, mimic, or
block the av~i3 binding site on an extracellular matrix protein, or that
specifically bind to an antigenic epitope of the av~33 molecule so that
inhibition
of av~33 integrin binding to an extracellular matrix protein is achieved. One
such monoclonal antibody is LM609 in its entirety, as well as its bioactive
antigen binding portions or fragments such as Fab, Fab2, Fv, and mixtures
thereof. Both the murine as well as the humanized versions of monoclonal
antibody LM609 are suitable for the present purposes.
In addition, fibronectin fragments that include amino acid residue
sequences corresponding to the av~33 integrin binding site on fibronectin, and
which may for example include the amino acid residue sequence Arg-Gly-Asp-
Ser (RGDS) or bioequivalents thereof, can be utilized as av~33 antagonist
molecules for the purposes of the present invention to interfere with an av~33
integrin binding to fibronectin or any other extracellular matrix protein that
has
a binding site defined by the amino acid residue sequence RGDS or
bioequivalents thereof.
Also suitable are the so-called peptidomimetics, i.e., non-peptidic
organic compounds that mimic the aforementioned peptides vis-a-vis interaction
with av~33 integrins so as to interfere with the av~33 integrin binding to an
extracellular matrix protein, e.g., fibronectin, and the like.
Adhesion formation is inhibited, or at least minimized, by
applying to the surgical site an adhesion-inhibiting amount of at least one of
the
aforementioned av~i3 antagonist molecules, directly or in a physiologically
compatible carrier vehicle. Application of these antagonists is readily
accomplished via intraperitoneal (i.p.) administration, subcutaneous (s.c.)
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injection, intravenous (i.v.) administration, or other suitable route of
administration.
Suitable carrier vehicles can be liquids as well as physiologically
acceptable absorbable pastes and solids. The antagonist molecule can be
applied
as an antagonist composition with the antagonist molecule dissolved in an
aqueous vehicle or present as a suspension therein, usually at a concentration
of
at least about 50 micrograms per milliliter (~,g/ml). Likewise, the antagonist
composition can be an absorbable paste or a solid constituted by the inhibitor
molecule in solution or suspension, and an absorbable gelatin sponge or
powder, or as an absorbable dried hydrogel, absorbable hyaluronic acid
derivatives, and the like, as the carrier vehicle. The av~33 antagonist
composition can be packaged in appropriately sized dosage forms provided with
a label which indicates that the inhibitor composition contained within the
package can be used to inhibit adhesion formation.
The av~i3 antagonists contemplated by the present invention can
either bind to the extracellular matrix protein, or to the av(33 integrin
molecule;
however, to be effective such binding must interfere with the normal
interaction
between av~i3 integrin and its binding site on an extracellular matrix
protein.
Illustrative of such extracellular matrix proteins are fibronectin,
fibrinogen,
vitronectin, von Willebrand Factor, laminin, collagen, tenascin, osteopontin,
thrombospondin, and the like.
A preferred method for inhibiting post-operative adhesion
formation comprises administering to a surgical patient (human as well as
veterinary) an adhesion inhibiting amount of an av~i3 integrin inhibitor
molecule. Such administration encompasses application of an aliquot of the
inhibitor composition to a tissue surface to be protected from adhesion
formation either directly, or as an aerosol spray, or via a pad, gel,
solution,
suspension, or the like, as a suitable carrier vehicle.
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Detailed Description of Preferred Embodiments
Adhesion formation occurs as an aberration of the wound healing
cascade involving cell adhesion and migration of fibroblasts. Adhesions often
occur as a result of trauma or bleeding at the time of surgery when the
interruption of a peritoneal surface results in the exposure of the underlying
stromal layers. Subsequently, this exposure leads to the release of kinins and
histamine which, in turn, increase capillary permeability and allow
serosanguinous exudate containing inflammatory cells to be released. This
exudate, rich in fibrin, leads to clotting on the injured surface. Without
dissolution of the clot, inflammatory cells and fibroblasts infiltrate the
fibrin
rich extracellular matrix which results in the formation of adhesions.
The extracellular matrix is composed of an interactive network of
proteins which forms the meshwork upon which cells adhere to organize tissues.
The macromolecules that constitute the extracellular matrix are mainly
secreted
locally by cells in the matrix. In most connective tissues these
macromolecules
are secreted largely by fibroblasts or cells from the fibroblast family, such
as
chondroblasts or osteoblasts. The two main classes of extracellular
macromolecules that make up the matrix are polysaccharide glycosaminoglycans
(GAGs), which are usually found covalently linked to protein in the form of
proteoglycans, and fibrous proteins. The fibrous proteins are usually
considered as one of two functional types: mainly structural (for example
collagen and elastin) or mainly adhesive (for example fibronectin and
laminin).
see for example The Molecular Biology of the Cell, 2nd ed. (Alberts et al. ,
editors, Garland Publishing, Inc., New York, 1989) 802-824.
Cell adhesion regulates cell migration, growth and differentiation
in embryonic tissues, and in the extracellular matrix, as well as contributes
to
the formation of malignancies, inflammation, immune regulation and
hemostasis. Mediators of cell adhesion, transmembrane cellular receptors,
include integrins, immunoglobulin supergene family, cadherins, selectins, CD-
44 related molecules, and transmembrane proteoglycans. (See Chothia &
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Jones, 1997, Ann. Rev. Biochem. , 66:823-62). Integrins are an important class
of binding protein which interact with many ligands. The av~33 integrin is a
membrane-bound glycoprotein identified as important for cell-cell adhesion and
migration. Integrins bind to diverse ligands including components of the
extracellular matrix, cell surface immunoglobulin (Ig) superfamily receptors,
surface components of microorganisms, and certain plasma proteins. For
review see Loftus, J.C. et al., 1994, J. Biol. Chem., 269(41):25235-25238.
Murine monoclonal (mAb) IgG antibody LM609, produced by the
hybridoma cell line LM 609, is specific for integrin av(33 (Cheresh et al.,
1987,
J. Biol. Chem., 262: 17703-17711). Murine hybridoma LM609 has been
deposited with the American Type Culture Collection (ATCC, Rockville, MD,
USA) as the International Depository Authority under the Budapest Treaty, and
assigned the ATCC Designation HB 9537, on September 15, 1987.
LM609 cDNA has been cloned, and soluble Fab portions thereof
have been made from transformed host cells. LM609 antibody also has been
humanized to reduce its immunogenicity, (WO 99/29888).
Proteins and peptides suitable for use as av~33 antagonist
molecules are those that include the av~i3 complementary binding site on
fibronectin, the amino acid residue sequence RGDS, or bioequivalents thereof.
The peptides can be linear or cyclic.
Also suitable are non-toxic, non-peptide organic compounds that
define a region that is substantially complementary to the av~33 integrin
binding site or to the binding site on the extracellular matrix protein for
the
av~i3 integrin.
In use, to inhibit adhesion formation, the av(33 integrin
antagonist molecule can be applied to a surgical site directly, as an aerosol
powder, or in a physiologically compatible carrier vehicle which can be a
liquid, such as water, alone or together with an absorbable powder in the form
of a paste. Illustrative of such absorbable powders is sterile gelatin powder
commercially available under the designation GELFOAM~ (Upjohn Co.).
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Alternatively, the av~i3 integrin antagonist molecule can be delivered to the
surgical site on a sterile gelatin foam or sponge, on a dried, absorbable
hydrogel of the type described in U.S. Patent No. 5,409,703 to McAualley et
al., or on a hyaluronic acid derivative. For intraperitoneal (i.p.),
subcutaneous
(s.c.) or intravenous (i.v.) administration the preferred carrier vehicle is
an
aqueous vehicle such as water, or an aqueous saline solution.
A rabbit sidewall model of adhesion formation has been
previously described (Rogers et al., 1996, J. Invest. Sure., 9:388-91; Rodgers
et al., 1998, supra). Rabbits were anesthetized with a mixture of 55 mg
ketamine hydrochloride per kg rabbit body weight, and 5 mg xylazine per kg
intramuscularly. Following preparation for sterile surgery, a midline
laparotomy was performed. The cecum and bowel were exteriorized, and
digital pressure was exerted to create subserosal hemorrhages over all
surfaces
that could be in contact with the area of sidewall injury. The damaged
intestine was then lightly abraded with 4" 4x4-ply sterile gauze until
punctate
bleeding was observed. The cecum and bowel were then returned to their
normal anatomical position.
This rabbit model is very similar to a standardized rat model for
adhesion formation described by Harris et al. (1995, supra). In this rat
model,
an abdominal wall defect and cecal abrasion were created, air dried for 10
minutes, and the two injured surfaces placed into contact before closure. The
rats were anesthetized with intraperitoneal sodium pentobarbital (43 mg/kg).
The ventral abdomen was prepared and given an iodophor scrub, and rinsed
with 70 % alcohol. A 6 cm midline skin incision was made, and the skin
retracted. A 4 cm midline abdominal wall incision was made, and the right
abdominal wall was rejected. A 1 x 2 cm segment of parietal peritoneum was
sharply excised from the wall including a superficial layer of underlying
muscle, 1 cm lateral to the midline incision. The cecum was then elevated and
positioned so that at closure the cecum would contact the abdominal wall
defect. Thereafter, the cecum was abraded in a standard manner by scraping
with a scalpel blade so that a homogeneous surface of petechial hemorrhages
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was created over a 1 x 2 cm area. The abdominal wall defect was also abraded.
Both the abdominal wall and cecal defects were exposed to air for 10 minutes.
The defects were then placed in contact, the midline incision was closed with
a
running 4-0 polypropylene suture and the skin closed with 4-0 silk (Harris et
al., 1995, supra).
Another rabbit system for studying reproductive organ adhesions
is the rabbit uterine horn model. In this model, a midline incision is made
and
the uterine horns are brought through the incision. An approximately 5 cm long
areas around the entire circumference of the uterine horns are abraded using
surgical gauze, and by scraping 12 times with a scalpel blade. This injury
results in generalized erythema without areas of active bleeding. The horns
are
then replaced in the abdominal cavity and the wound closed.
With the advent of laparoscopic surgical instruments and
techniques, such minimally invasive surgery has become more common. Yet,
adhesion formation is still a possible complication from such laparoscopic
surgery. A rabbit animal model has been used to examine laparoscopic
adhesion prevention (DeIaco, et al., 1998, supra). Briefly, a Verres needle
was
inserted into the abdominal cavity entering the abdominal wall at the midline,
for carbon dioxide gas insufflation using an automatic laparoinsufflator. A
trocar was then inserted, entering the abdominal wall at the same position. An
arthroscope was then inserted into the cavity through the trocar with the aid
of a
Xenon 300 W light source. All surgical procedures are performed by means of
an endoscopic microcamera. After inspection of the abdomen, laparoscopic
scissors and atraumatic forceps were inserted, without trocar, through two
lateral incisions.
A standardized injury to the peritoneum or internal surfaces can
be induced by denuding a 2 x 2 cm area of the right uterine horn for 30
seconds
with forceps, making a 1-cm incision in the distal right uterine horn,
denuding a
5 x 5 cm area of the peritoneum of the abdominal wall in front of the previous
lesions.
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Administration of whole murine LM 609 mAb to the surgical site
of a patient is possible, but may be precluded for long-term, or multiple,
uses
lest a host immune reaction is triggered against the murine LM 609 protein
(i.e., Human anti-mouse antibody, HAMA in human patients; Cat or Dog anti-
s mouse antibody and other reactions in other treated animals). This can be
partially minimized by using LM 609 that has been truncated, either into Fab,
Fab2, or Fv constructs or mixtures thereof. The generation of antibody
fragments such as Fab, Fab2 or Fv is known in the art and is taught for
example
by French (1998, Methods in Molecular Biolo~y, Immunochemical Protocols,
2nd Ed., 80:121-134). Also known is the use of recombinant DNA methods to
prepare these antigen binding proteins and artificial constructs (i.e. single
chain
Fv, scFv, scAb; Molecular Recognition Units, MRUs). see for example
Verhoeyen et al. "Advances in antibody engineering" in Molecular
Immunolo~y, (IRL Press at Oxford University Press, Oxford, 1996) chapter 7.
For use in treating humans, the murine LM 609 antibody, or
portions thereof, can be humanized by the judicious substitution of amino acid
residues in the protein structure to alter the immunogenic epitopes of the
antibody surface to appear as human protein to the treated host, wherein the
antigen specificity of the binding active site, including complementarity
determining region (CDR) amino acid residues, are conserved to maintain
antigen epitope binding specificity. Humanized monoclonal antibodies are
known, and have been previously described in the art. For example,
Waldmann et al. (U.S. Patent 5,502,167) describe a humanized antibody in
which the amino acid sequence of the CDRs is derived from the sequence of
CDRs of a monoclonal antibody having the specificity of binding to resting
and activated T-cells. Hoogenboom et al. (U.S. Patent 5,565,332) describe
methods for producing antibodies with increased human characteristics
involving selective mutation of either heavy or light chains, recombination of
the mutated chains, and antigen selective screening for binding activity.
Adair
et al., (U.S. Patent 5,859,205) describe specific methods for grafting the CDR
of antibody heavy and light chains to acceptor framework regions. Thus, it is
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possible to graft the amino acid residues of the LM 609 antibody encoding for
the active site forming residues to a human antibody framework, thereby
creating a chimeric antibody with the binding specificity of the LM 609
antibody but having the antigenic appearance of a human antibody. A unique
method for optimizing CDR grafting further involving chain reassortment and
antigen selection, has been described in W099/29888 (Barbas et al.).
Any resultant reduction in affinity that may occur by this process,
and, thus, reduction in binding efficiency, can be minimized by screening with
antigen to select better binding constructs, and by generating mufti-valent
binding constructs. For example, it may also be advantageous to cross-link two
or more LM 609 antigen binding active sites, or humanized active sites, using
chemical linkers as known in the art, so as to form bifunctional or multi-
functional groups of active sites that could act more efficiently as a
blocking
agent. Similarly, it is also possible to attach multiple active sites to solid
supports such as micro-particles and larger latex polymer or colloidal beads
or
the like. The attachment of whole antibody, or active site containing
fragments
or constructs thereof, to solid support systems is known in the art. For
example, Wang (1998, Methods in Molecular BioloQV, Immunochemical
Protocols, 2nd Ed., 80:365-376) describes the use of immunomagnetic beads
for cell sorting, where the beads have been coated with bioreactive molecules
such as antibodies, streptavidin, lectins, and peptides.
It is also possible to practice the methods of the invention or to
prepare equivalent compositions of the invention by generating antibodies that
interfere with, or otherwise block, the binding of av~33 integrin to
fibronectin
or other extracellular matrix protein, thus interfering with the formation of
the contacts needed for the development of adhesions. To that end, suitable
animals can be immunized to stimulate the generation of antibodies specific
for
the av~33 integrin or to the av~33 binding site of fibronectin or other
extracellular matrix protein. Suitable such antibodies can be selected by
antigen screening using routine methods known in the art. The generation of
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monoclonal antibodies from suitably selected clones is also well known and can
be utilized for the production of suitable av~33 inhibitor molecules.
Substitution of any of these extant, or subsequently generated
antibodies in the compositions or methods of the invention is within the
purview of this invention and is equivalent in result as well as means.
Antagonist molecules suitable for inhibition of av~33 binding in
addition to those described herein above can also be easily selected by
routine
screening. To that end, an in vitro screening assay can be constructed
utilizing cell adhesion to a prepared extracellular matrix comprising
fibronectin
or other suitable matrix component. In initial screening systems, candidate
molecules can be administered, and the subsequent behavior of the cells
scored. Reduction in binding to the substrate, or inhibition of proliferation
of
matrix components mirroring adhesion formation, indicates a promising
candidate for further study. The initial screening can be conducted with
mixtures of compounds so as to reduce screening time and effort by
performing batch screens. Specific binding inhibition of specific compound
mixtures can be individually assayed once any initial results indicate
success.
Such screening can be fully automated, if desired. Regardless of how such
inhibiting molecules are found, it is envisioned that they will be suitably
formulated into a composition of the invention for use in the method of the
invention. Ideally the optimal concentration for use of any such compound is
at least comparable to that for LM 609 antibody, and minimizes any potential
adverse or toxic effects.
Antagonist molecules that specifically bind to, or interfere with,
the av~i3 integrin binding site of an extracellular matrix protein molecule
are
also suitable for practicing the invention.
The invention and many of its embodiments are illustrated by the
following example.
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Example
Initial tests with solutions containing whole murine mAb LM 609
at a concentration from 0.1 to 10 mM were conducted using the Rabbit Uterine
Horn model. The advantage of using solutions for these tests is that, unlike
physical barriers, there is no need for precise placement of a physical
barrier,
or (in some cases) for the subsequent removal of the barrier. A solution can
be
placed within the peritoneal cavity, thus infusing the entire cavity, with no
restriction or localization. Thus, administration of a therapeutic composition
containing mAb LM 609 can be more easily accomplished than by the
placement of known physical barriers, and the effectiveness less dependent
upon physical factors.
Laparotomies were performed on twenty-one (21) anesthetized
New Zealand white rabbits, that were randomly assigned to have either an
"open and close" sham procedure (sham, n=3) or a procedure in which pelvic
abrasions were intentionally created in a standardized fashion (experimental,
n=18). A midline laparotomy was performed on the experimental animals,
and the sidewalk, bladder, uterus and fallopian tubes were abraded with 200
grit sandpaper until punctate bleeding occurred. After injury, the rabbits in
the
experimental groups were randomized to receive intraperitoneally 2 ml of
saline (saline control; group iii, n=6), 2 ml of anti-beta 1 antibody (anti-
beta 1;
1 mg/ml, group ii, n=6), or 2 ml of LM 609 (anti-av~33; 1 mg/ml; group i,
n=6). The abdominal wall was closed in two layers using 3.0 polyglactin 910
(Vicryl) for the muscle and 4.0 polyglactin 910 to close the skin in a
subcuticular fashion.
The animals were housed with a 12 hour light/dark cycle and fed
150g of food per day, water ad libitum. The rabbits were numbered with a 5
digit tag, and solutions were labeled A, B, and C, respectively. The animals
were sacrificed by three weeks post-operatively using 50 mg intravenous
pentobarbital, and the intraperitoneal adhesions were scored by a single
observer who was unaware of the treatment identity, using scoring system
modified from Blauer et al. (1988, Fertility & Sterility, 49:144-49).
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Table I. Adhesion Scoring System
Score Description
0 No adhesion
1 Single filmy adhesion
2 More than one filmy adhesion
3 Single dense adhesion
4 More than one dense adhesion
A filmy adhesion was defined as one that could easily be
disrupted. A dense adhesion was one that was not easily separated.
Statistical analysis was performed using STATA. The
differences of adhesion scores between the groups was determined using a
Kruskall-Wallis test for equality of populations with an a=0.05 (p=0.02).
Each group was then analyzed with respect to the other groups using a two
sample Wilcoxon rank sum analysis with an a=0.05.
The resulting scores in the LM609 treatment group were similar
to that seen in the sham operated group (p=0.11) demonstrating that the
treatment was effective in reducing adhesion formation. The results show that
the control treatments were not as effective when compared with the sham
operated group, saline (p=0.05) or anti-beta 1 integrin (p=0.03).
Table II. Adhesion Scores
Treatment Scores
Sham 0 0 1
Saline 444034
anti-b 1 4 3 2 3 4 2
LM609 1 2 2 0 1 3
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The invention, having been fully described in many of its aspects
and claimed herein can be made and executed without undue experimentation
according to the teaching herein. While the compositions and methods of this
invention have been described by way of example above, it will be apparent
that
S many variations and modifications may be applied to the compositions and
methods described herein without departing from the concept, spirit and scope
of the invention.
