U.S. patent application number 10/229326 was filed with the patent office on 2003-08-07 for tissue treatment compositions.
This patent application is currently assigned to Biovitrum AB, a Swedish corporation. Invention is credited to Wadstrom, Jonas.
Application Number | 20030147878 10/229326 |
Document ID | / |
Family ID | 26661103 |
Filed Date | 2003-08-07 |
United States Patent
Application |
20030147878 |
Kind Code |
A1 |
Wadstrom, Jonas |
August 7, 2003 |
Tissue treatment compositions
Abstract
A tissue treatment composition, especially an adhesive
composition comprises (i) fibrin or fibrinogen and (ii) a
biodegradable and biocompatible polymer capable of forming a
viscous aqueous solution. In addition to glueing, the tissue
adhesive composition may be used for slow-release of a drug
incorporated into it or for anti-adherence purposes, for wound
healing, etc.
Inventors: |
Wadstrom, Jonas; (Uppsala,
SE) |
Correspondence
Address: |
FISH & RICHARDSON PC
225 FRANKLIN ST
BOSTON
MA
02110
US
|
Assignee: |
Biovitrum AB, a Swedish
corporation
|
Family ID: |
26661103 |
Appl. No.: |
10/229326 |
Filed: |
August 26, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10229326 |
Aug 26, 2002 |
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08999137 |
Dec 29, 1997 |
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6440427 |
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08999137 |
Dec 29, 1997 |
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08780442 |
Jan 8, 1997 |
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08780442 |
Jan 8, 1997 |
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08162078 |
Feb 7, 1994 |
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5631011 |
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08162078 |
Feb 7, 1994 |
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PCT/SE92/00441 |
Jun 17, 1992 |
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Current U.S.
Class: |
424/94.64 ;
514/54 |
Current CPC
Class: |
A61F 2/30767 20130101;
A61L 24/043 20130101; A61L 27/34 20130101; A61F 2002/30677
20130101; A61K 38/363 20130101; A61F 2210/0004 20130101; C08L 89/00
20130101; A61F 2/0077 20130101; C08L 89/00 20130101; A61F 2/28
20130101; A61F 2002/30062 20130101; A61L 26/0042 20130101; A61L
27/34 20130101; A61L 24/043 20130101; A61F 2/30756 20130101; A61F
2250/0067 20130101 |
Class at
Publication: |
424/94.64 ;
514/54 |
International
Class: |
A61K 038/48; A61K
031/715 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 17, 1991 |
SE |
9101853-1 |
Claims
1. A tissue treatment composition comprising (i) fibrin or
fibrinogen and (ii) a biodegradable and biocompatible polymer
capable of forming a viscous aqueous solution.
2. A tissue treatment composition according to claim 1,
characterized in that constituent (i) comprises fibrin or
fibrinogen and Factor XIII.
3. A tissue treatment composition according to claim 1 or 2,
characterized in that it is a two-component composition to be mixed
at the time of application thereof, one component comprising
fibrinogen and the other thrombin, said viscous polymer being
contained in one or both of said two components.
4. A tissue treatment composition according to claim 1, 2 or 3,
characterized in that said fibrinogen comprising component
additionally contains at least one additional member selected from
Factor XIII, fibronectin, a plasmin inhibitor, a plasminogen
activator inhibitor, and plasminogen.
5. A tissue treatment composition according to claim 3 or 4,
characterized in that said thrombin comprising component
additionally contains bivalent calcium.
6. A tissue treatment composition according to any one of claims 3
to 5, characterized in that said viscous polymer is contained in
the fibrin or fibrinogen comprising component.
7. A tissue treatment composition according to any one of claims 1
to 6, characterized in that said viscous polymer is a high
molecular polysaccharide or proteoglycan.
8. A tissue treatment composition according to claim 7,
characterized in that said high molecular polysaccharide is
selected from the group consisting of xanthan, dextran, cellulose
and glycosaminoglycans, and salts and derivatives thereof.
9. A tissue treatment composition according to claim 8,
characterized in that said high molecular glycosaminoglycan is
hyaluronic acid or a salt or derivative thereof.
10. A tissue treatment composition according to any one of claims 1
to 9, characterized in that the viscosity of the mixed composition
when applied is in the range of about 500 to about 1,000,000 cP,
preferably about 1,000 to about 500,000 cP.
11. A tissue treatment composition according to any one of claims 3
to 10, characterized in that one of said components is supported on
a solid matrix or carrier material.
12. A tissue treatment composition according to any one of claims 9
to 11 for the administration of hyaluronic acid or a salt or
derivative thereof.
13. A tissue treatment composition according to any one of claims 1
to 12, characterized in that it is in deep-frozen or lyophilized
form or in the form of a film or sheet which may be pre-formed.
14. A tissue treatment composition according to any one of the
preceeding claims, characterized in that it is a tissue adhesive
composition.
15. An anti-adherence composition, characterized in that it
comprises a tissue treatment composition according to any one of
claims 1 to 14.
16. A slow-release drug formulation, characterized in that it
comprises a tissue treatment composition according to any one of
claims 1 to 14.
17. A slow-release drug formulation according to claim 16,
characterized in that the therapeutical substance is said viscous
polymer itself or a species coupled thereto.
18. A composition for promoting wound healing, characterized in
that it comprises a tissue treatment composition according to any
one of claims 1 to 14.
19. A composition for the prevention of adhesion and scar
formation, characterized in that it comprises a composition
according to any one of claims 1 to 14.
20. A composition for coating prosthetic materials or as carrier of
cellular transplants, characterized in that it comprises a
composition according to any one of claims 1 to 14.
21. A composition according to any one of claims 17 to 20,
characterized in that it additionally comprises at least one wound
healing promoting protein.
22. A composition according to claim 21, wherein said at least one
wound healing promoting protein is selected from the group
consisting of serum proteins, tissue growth factors, hormones,
chemotactic proteins, and nutritional proteins.
23. Use of a composition according to any one of claims 1 to 14 as
an anti-adhesive composition.
24. Use of a composition according to any one of claims 1 to 14 as
s slow-release drug formulation.
25. Use of a composition according to any one of claims 1 to 14 as
a wound healing composition.
26. Use of a composition according to any one of claims 1 to 14 for
coating prosthetic materials.
27. Use of a composition according to any one of claims 1 to 14 as
a carrier of cell transplants.
Description
[0001] The present invention relates to a tissue treatment
composition, especially a tissue adhesive having improved
properties and to the use of such compositions as anti-adherence or
would healing-compositions, as slow-release drug formulations, for
coating tissues or prosthetic materials, and as carriers for cell
transplants.
[0002] The use of blood coagulating substances for stopping
bleedings and for sealing wounds has been known for a long time.
Thus, the hemostatic effect of fibrin powder was reported about 80
years ago, and attempts were made to employ fibrin or fibrin
patches to stop bleeding in brain and general surgery.
[0003] Today such use of fibrin as a biologic adhesive has been
widely accepted and found application in many fields of surgery.
Generally fibrin sealants are based upon the two components
fibrinogen and thrombin. As these components mix a fibrin coagulum
is formed in that the fibrinogen molecule is cleaved through the
action of thrombin to form fibrin monomers which spontaneously will
polymerize to form a three-dimensional network of fibrin, largely
kept together by hydrogen bonding. This corresponds to the last
phase of the natural blood clotting cascade, the coagulation rate
being dependent on the concentration of thrombin used.
[0004] In order to improve the tensile strength, covalent
crosslinking between the fibrin chains is provided for by including
Factor XIII in the sealant composition. The strength of the fibrin
clot is further improved by the addition of fibronectin to the
composition, the fibronectin being crosslinked and bound to the
fibrin network formed.
[0005] To prevent a too early degradation of the fibrin clot by
fibrinolys, the fibrin sealant composition may comprise a
plasminogen activator inhibitor or a plasmin inhibitor, such as
aprotinin. Such an inhibitor will also reduce the fibrinolytic
activity resulting from any residual plasminogen in the fibrinogen
composition.
[0006] Similarly, compositions according to the invention which
include hyaluronic acid (or other polysaccharides), may also
comprise a hyaluronidase inhibitor such as one or more flavonoids
(or corresponding inhibitors for other polysaccharides) in order to
prevent premature degradation (i.e. to prolong the duration) of the
hyaluronic acid component by hyaluronidase which is always present
in the surrounding tissues. The hyaluronic acid may, as mentioned
above, be crosslinked, a commercially available example being
Hylan.RTM. (trademark, available from Biomatrix, Ritchfield, N.Y.,
USA). The hyaluronic acid compositions may e.g. have the form of
gels, solutions, etc.
[0007] The results obtainable by fibrin sealants are basically:
[0008] (i) Hemostasis. The fibrin clot acts as a hemostatic barrier
and reduces the risk of serum, lymph and liquor leakage. The
hemostatic effect may be enhanced if the fibrin sealant is combined
with a biocompatible solid flat material such as collagen.
[0009] (ii) Glueing. Due to its adhesive properties the fibrin
sealant atraumatically connects tissues by forming a strong joint
between them and adapts uneven wound surfaces. This glueing effect
is increased by fibronectin being bound to exposed collagen.
[0010] (iii) Wound healing. The fibrin sealant promotes the
ingrowth of fibroblasts which in combination with efficient
hemostasis and adhesion between the wound surfaces provides for an
improved healing process. Wound healing promoted by fibrin sealants
results in strong scar formation and does not prevent the formation
of adhesions. The use of the compositions according to the
invention as an anti-adherence/wound healing composition does,
however, result in a normal (regenerative) tissue rather than scar
tissue, i.e. optimal wound heaing. Furthermore, such compositions
also reduce the inflammatory response as appears from the test
results reported in Table 4 below.
[0011] Fields of application include among others: ear, nose and
throat surgery, general surgery, dentistry, neurosurgery, plastic
surgery, thorax and vascular surgery, abdominal surgery,
orthopaedics, accident surgery, gynaecology, urology, and
opthalmology. Fibrin sealants have also been used for local
application of drugs, such as antibiotics, growth factors and
cytostatics.
[0012] Commerical fibrin glues (prepaed from human plasma) are
available under the trade names Tissucol, Tisseel and Fibrin-Kleber
Humano Immuno (Immuno AG, Vienna, Austria) as well as Beriplast
(Behringwerke AG, Marburg, Germany) (these trade names being
registered trademarks in several countries). Tisseel.TM. is a
two-component kit containing a fluid thrombin component including
calcium chloride and a somewhat more viscous fibrinogen component
including factor XIII, fibronectin, aprotinin and plasminogen. The
two components are delivered deep frozen in two separate syringes,
or as two lyphilized powders with corresponding aprotinin and
calcium solutions as solvents. As explained above the fibrin
sealant consolidates when the two components are combined due to
fibrin monomer aggregation. The setting rate is dependent on the
thrombin concentration and varies from a few seconds (high thrombin
concentration) to a couple of minutes (low thrombin
concentration).
[0013] However, an important and well known disadvantage of the
known preparations resides in the water-like fluidity of the
components when applied, which leads to considerable handling
difficulties of the glue. Efforts have been made to overcome this
problem and facilitate the mixing of the components by the
development of particular application modes such as a
double-syringe applicator (e.g. that supplied under the trade name
Duploject.RTM., Immuno AG, Vienna, Austria, and which is disclosed
in e.g. U.S. Pat. No. 4,359,049, or a special spray system as
disclosed in e.g. EP-A-156 098). The basic problem with a low
viscosity glue still remains, however. Firstly, a non-viscous or
low viscosity glue is unsuitable for use on non-horizontal surfaces
since it will run off before setting. Secondly, there is a definite
risk of a non-viscous or low vicosity glue running off to sites
where it is unwanted and where it might cause complications. This
is particularly the case in vascular surgery since the fluid glue
may reach inside the vessels before it sets and thereby cause
thromboembolic complications. An instantantaneously setting fibrin
glue (containing a high concentration of thrombin), on the other
hand, cannot be used where the parts to be sealed require
subsequent adaptation.
[0014] A different approach has been disclosed by i.a. Bass et al
in J. Vasc. Surg. May 11, 1990, (5):718-25, which is incorporated
herein by reference. This paper discloses a technique called laser
tissue soldering (or welding), wherein a laser energy absorbing dye
(chromophore) and fibrinogen are soldered by means of a laser to
produce a strong anastomosis which is said to be i.a. faster
healing than a conventional sutured anastomosis. Similar
coagulation and/or bonding effects can be achieved with other
proteins and energy sources.
[0015] It is an object of the present invention to provide an
improved fibrin glue which is devoid of the above low viscosity
problem, and which promotes wound healing without scar formation or
development of adhesions. This object is achieved by including in a
fibrin glue composition of the above mentioned type a viscosity
increasing amount of a biodegradable and biocompatible polymer
capable of forming a viscsous aqueous solution. In accordance with
the present invention it has thus been found that by the addition
of such a viscosity enhancing polymer, the glue composition will
obtain a viscosity adequat to facilitate and improve the handling
and application thereof, while not negatively affecting the
favourable properties of the fibrin glue. For wound healing and
anti-adherence purposes the adhesive properties may, however, be
less pronounced, or even missing.
[0016] Accordingly, the present invention relates to a tissue
treatment composition comprising (i) fibrin or fibrinogen and (ii)
a biodegradable and biocompatible polymer capable of forming a
viscous aqueous solution, optionally also other proteins.
[0017] One use form of the present tissue adhesive composition is
thus an improved fibrin sealant or glue which upon use exhibits
viscosity characteristics permitting easy and safe application
thereof at a desired location or site.
[0018] In another use form the present tissue treatment composition
comprises a therapeutical substance and constitutes a
pharmaceutical composition for local administration of the
therapeutical substance. In a particular embodiment the
therapeutical substance is the viscous polymer itself or a species
coupled thereto as will be described in more detail below.
[0019] Still another use form of the present tissue treatment
composition is a wound healing and an anti-adherence composition,
the high molecular composition conferring such adherence-preventing
properties to the composition that it may be used for preventing
the adherence of adjacent tissues in surgical procedures. Related
to such anti-adherence use is the use of the present tissue
treatment composition for wound healing. By, for example, glueing
wound edges with the tissue treatment, neat scars will be obtained.
Further, cellular transplants, in particular dermal transplants,
will heal faster. This would, of course, be of particular interest
in plastic surgery.
[0020] The above mentioned biodegradable and biocompatible polymer
capable of forming an aqueous solution may be selected from a wide
variety of substances (including substances which will be available
in the future) and the selection thereof can readily be made by the
person skilled in the art.
[0021] A preferred group of said biodegradable and biocompatible
polymers, hereinafter frequently referred to as viscosity enhancing
polymers, consists of high molecular polyglycans or
polysaccharides. Exemplary of such polysaccharides for the purposes
of the invention are xanthan, dextran, cellulose and proteoglycans,
especially hyaluronic acid, and salts and derivatives thereof. As
examples of cellulose derivatives may be mentioned methyl
cellulose, carboxymethyl cellulose (CMC) and hydroxy-propylmethyl
cellulose (HPMC), just to mention a few thereof. Examples of
viscosity enhancing polymers other than high molecular
polysaccharides are gelatin and polyvinylpyrrolidone.
[0022] A preferred polysaccharide/polyglycan is hyaluronic acid and
salts and derivatives thereof. Sodium hyaluronate is a high
molecular weight linear polysaccharide built up of repeating
disaccharide units. It exists in the extracellular space of all
tissues and has the same simple chemical structure in all species.
Hence, the application of a purified preparation of hyaluronate
results in but a temporary increase of the local concentration of
endogenous material and its utilization in the composition will
therefore not have any detrimental physiological effects. In
solution the hyaluronate adopts a conformation of very extended
random coils, that already at low concentrations entangle into a
flexible molecular network that gives hualuronate solutions
interesting Theological properties that are useful for the present
purposes.
[0023] The visco-elastic properties of sodium hyaluronate has lead
to its clinical use as spacer and to facilitate operative
procedures in the field of eye surgery. It has also been
demonstrated to be biologically active in enhancing epithelial
regeneration of the ear tympanic membrane and to inhibit the
ingrowth of vascular endothelial cells. Further, it plays a role in
wound healing, influencing the migration of granulation tissue
cells and reduces the amount of adhesions formed after surgery. The
bioavailability of sodium hyaluronate per se is, however, limited
due to its rapid turnover and short half-life.
[0024] When the tissue treatment composition is used as an improved
tissue adhesive, the proportion of the viscosity enhancing polymer
in the fluid fibrin glue as applied should be selected to provide
an appropriate viscosity for the intended application while not
adversely interfering with the fibrin clotting, and will depend on
the polymer and the particular tissue adhesive composition to be
produced. Suitable initial viscosities of the final solution
mixture of the total composition for each particular application
may readily be established by the skilled person, but will
generally be in the range of about 500 to about 1,000,000
centipoises (cP), preferably about 1,000 to about 500,000
centipoises. The term "final solution mixture" as used herein does
not necessary mean a homogeneous state. On the contrary, depending
on the mixing procedure, the mixture will in many cases not reach a
homogeneous or uniform state before clotting. As is well known to
the person skilled in the art, the viscosity is correlated to
concentration and limiting viscosity number, .eta..sub.0=(Conc. x
[.eta.]).sup.3,6/10. Modified after Morris et al, Carbohydrate
polymers, Vol. 1, 1981, p. 5-21. From [.eta.] we get the molecular
weight using Cleland's formula for [.eta.]=k x average molecular
weight.sup.k1, Cleland et al, Biopolymers, Vol. 9, 1970, p.
799-80.
[0025] Like the prior art fibrin sealants the tissue adhesive
composition of the present invention may comprise additional
constituents. Thus, in addition to sealer protein and viscosity
enhancing polymer, such as e.g. high molecular polysaccharide, the
composition will preferably comprise Factor XIII and/or fibronectin
and/or plasminogen. Advantageously, the composition will also
include clotting enzyme, i.e. thrombin, especially in combination
with bivalent calcium, such as calcium chloride. The concentration
of calcium chloride will then vary, e.g. between 40 mM to 0.2 M
depending on the specific purpose of the tissue adhesive
composition, high concentrations of calcium chloride inhibiting
fibroblast growth and therefore being preferred for anti-adherence
applications (along with absence of fibronectin which stimulates
the ingrowth of fibroblasts). It may further be valuable to include
a fibrinolysis inhibitor, such as a plasmin inhibitor, e.g.
aprotinin, aprilotinin, alpha-2-antiplasmin, alpha-2-macroglobulin,
alpha-1-antitrypsin, epsilon-aminocaproic acid or tranexamic acid,
or a plasmin activator inhibitor, e.g. PAI-1 or PAI-2.
[0026] While the proportions of the previously known ingredients in
the tissue adhesive compositions of the invention may be selected
with guidance of prior art compositions, the necessary amount of
the viscosity enhancing polymer can readily be determined by a
person skilled in the art depending on the particular polymer and
the intended use form. Thus, if the concentration and/or molecular
weight of the viscosity enhancing polymer is too low, the viscosity
increase will be insufficient, and a too high concentration and/or
molecular weight will inhibit the fibrin polymerization and the
adhesion to the tissue.
[0027] By increasing the thrombin concentration, the polymerization
of fibrinogen may be speeded up with a consequential influence on
the time until the glue sets.
[0028] At low thrombin concentrations the fibrin glue composition
will remain more or less fluid for several minutes after
application. A further beneficial effect of increasing the
viscosity with a viscosity enhancing polymer in accordance with the
invention is therefore the possibility to use lower concentrations
of thrombin, which is required in situations where the parts to be
sealed require subsequent adaptation even on non-horizontal
surfaces.
[0029] The tissue treatment composition of the present invention
may be presented in the same type of preparations as the prior art
fibrin sealants. In an advantageous embodiment the tissue adhesive
is therefore a two-component preparation, one component comprising
the blood clot protein(s) and the other comprising thrombin and
bivalent calcium as well as possible additives including
fibrinolysis inhibitors. The viscosity enhancing polymer may be
contained in one or both of the two components depending on the
intended use of the tissue adhesive. While in the case of a fibrin
glue the viscosity enhancing polymer may be contained in either or
both of the two components, it is for other applications preferably
associated with the fibrin or fibrinogen component. It is, of
course, at least theoretically, also possible to provide the
viscosity enhancing polymer as a separate component. The components
may be provided in deep frozen solution form or as lyophilized
powders, to be diluted prior to use with appropriate aqueous
solutions, e.g. containing aprotinin and calcium ions,
respectively.
[0030] The tissue treatment composition of the invention may also
be used in various combinations as is per se known in the art. For
example, with reference to the above mentioned two-component
embodiment, one component may be provided in a biocompatible solid
matrix material as a prefabricated unit and the other (activating)
component may be added at the time of use. The viscosity enhancing
polymer may then be provided together with any one or both of said
components.
[0031] In such an embodiment the tissue adhesive of the invention
may include a tissue-compatible flat matrix material, such as a
non-woven fabric, into which the blood coagulation substance, the
viscosity enhancing polymer, e.g. high molecular polysaccharide,
and optional additional constituents are impregnated. In a
variation the viscosity enhancing polymer is added together with
the thrombin. In another variation the matrix material is
impregnated with the thrombin, and the blood coagulation substance
is added together with the viscosity enhancing polymer at the time
of use. Such a non-woven fabric may, for example, be a
glycoprotein, such as collagen (preferably porous), globulin,
myoglobulin, casein or albumin; gelatin; silk fibroin or a
polysaccharide, such as cellulose; or mixtures thereof. Such an
embodiment will, for instance, be particularly useful for stopping
bleedings and covering wounds. It is to be noted, however, that, as
will be readily understood, for anti-adherence purposes a material
like collagen having adhesion enhancing properties would not be
appropriate; cellulose e.g. being a more suitable material in this
respect. Such an impregnated flat material is advantageously
provided in lyophilized form.
[0032] In another embodiment the tissue treatment composition is
provided as a film or sheet for surgical use comprising a
non-crosslinked combination of fibrin and viscosity enhancing
polymer.
[0033] The tissue treatment composition of the present invention
may, of course, be used in all other preparations in which the
prior art fibrin glues have been presented, e.g. as an implantation
material for joint cartilage and bone defect repair material in
combination with embryonic chondrocytes or mesenchymal cells, such
as described for a conventional fibrin glue in e.g. U.S. Pat. No.
4,642,120.
[0034] As already mentioned above the present tissue treatment
composition, e.g. in any one of the above described embodiments,
may be used for the application of a pharmaceutically active
substance. By incorporating a drug, such as an antibiotic, a growth
factor, etc. into the tissue adhesive it will be enclosed in the
fibrin network formed upon application of the tissue adhesive. It
will thereby be ensured that the drug is kept at the site of
application while being controllably released from the composition,
e.g. when used as ocular drops, a wound healing preparation,
etc.
[0035] As also mentioned above the pharmaceutically active
substance to be released from the present tissue adhesive
composition may be the viscosity enhancing polymer in itself or a
substance coupled thereto.
[0036] A specific example of such a viscosity enhancing polymer
fulfilling the viscosity enhancing requirement as well as having
therapeutical and pharmaceutical utility, and for which it may be
desired to sustain the bioavailability, is hyaluronic acid and
salts and derivatives thereof which are easily soluble in water
and, as mentioned previously, have an extremely short biological
half-life. The tissue treatment composition of this invention thus
constitutes an advantageous slow-release preparation for
proteoglycans such as hyaluronic acid and its salts and
derivatives, and considerably increases the bioavailability
thereof.
[0037] The tissue treatment composition of the present invention
may, for example, be prepared and provided in administration forms
in analogous manner as the prior art tissue adhesives.
[0038] It should be emphasized that the compositions are not
restricted to the adhesive properties, but non-adhesive
compositions are also included, especially when the compositions
primarily are intended for wound healing. The latter compositions
may in particular include non-adhesive proteins such as albumin
and/or growth factors. Substantially non-adhesive compositions may
also be obtained when the polymer part of the composition inhibits
the adhesive properties of the protein part. It should in this
context be emphasized that the invention comprises both adhesive
and substantially non-adhesive compositions, although it has for
simplicity reasons often has been referred to as an "adhesive" in
this speicification, including the Examples.
[0039] In the following the invention will be described in more
detail by way of non-limiting examples. In one example the gluing
properties of an embodiment of the tissue adhesive composition are
tested in animal experiments, reference being made to the
accompanying drawing in which the only figure is a schematic side
view of a clamped vessel with three sutures. A second example
describes the preparation of another embodiment of tissue adhesive
composition. A third example illustrates the use of the tissue
treatment composition as a controlled release preparation, and a
fourth example shows the properties of the tissue treatment
composition as an anti-adhesion and wound healing promoting
agent.
EXAMPLE 1
[0040] Animals
[0041] Twentyone male Sprague-Dawley rats with a body weight of
230-345 g were used. They were housed under standardized
environmental conditions, with free access to water and food, for
one week prior to the experiments.
[0042] Operative Procedure
[0043] The animals were anaesthetized with fluanisone; 0.75 mg/100
g b.w. and fenatyl; 0.024 mg/100 g b.w., (Hypnorm.RTM., Janssen,
Belgium), in combination with midazolan; 0.38 mg/100 g b.w.,
(Dormicum.RTM., Roche AG, Switzerland) given subcutaneously. An
additional dose was given after 30-45 minutes.
[0044] The animals were placed on a warming water blanket
(Aquamatic K-20-D, Hamilton, Cincinatti, USA) which was set at
37.degree. C.
[0045] The femoral vessels were exposed through an L-shaped groin
incision and the artery was mobilized from the inquinal ligament to
the epigastric vessels. The profunda artery was cauterized with
bipolar diathermia. A few .mu.l of papaverine, 40 mg/ml, (ACO),
Sweden), were administered topically to resolve vasospasm induced
by the operative trauma. Two to three minutes later two separate
microvascular clamps were applied and the artery divided with a
pair of scissors. The cut ends of the vessel were gently dilated
with a microdilator and the lumina washed out with a few
millilitres of saline. The artery was then sutured with three 10.0
nylon sutures (STT) placed at 120.degree. from each other. The
tension of the vessel caused a gap of 0.2-0.4 mm between each
suture as is schematically illustrated in FIG. 1, wherein reference
numeral 1 designates the vessel, 2 represents clamps, 3 designates
sutures, and 4 indicates said gaps.
[0046] Instead of completing the anastomosis, which generally
requires a total of 8-12 sutures, the gap between the stitches were
sealed with 0.2 ml of fibrin glue applied with a Duploject.RTM.
double-syringe system (Immuno AG, Austria). The glueing procedure
of the anastomosis was randomized to one of the three following
glue preparations:
[0047] I. Original Tisseel.RTM. (Immuno AG, Austria) in which one
syringe ("Fibrinogen component") contained 75-115 mg/ml of
fibrinogen, 2-9 mg/ml of plasma fibronectin, 10-50 U of Factor
XIII, 40-120 .mu.l of plasminogen and 3000 KIU/ml of aprotinin, and
had a viscosity of about 100 cP. The other syringe ("Thrombin
component") contained 500 IU/ml of thrombin and 40 mM CaCl.sub.2
and had a viscosity of 1.2 cP. The initial viscosity of the mixed
contents (1+1) of the two syringes was well below 100 cP.
[0048] II. The Tisseel.RTM. Fibrinogen component was mixed 1+1 with
sodium hyaluronate solution, 10 mg/ml, with an average molecular
weight of 4,000,000, zero shear viscosity 300,000 cP, dissolved in
0.002 M Na-phosphate, 0.145 M NaCl, pH 7.3 (Healon.RTM., Kabi
Pharmacia AB, Sweden); the addition of hyaluronate thus reducing
the final concentrations of aprotinin and fibrinogen by one half
and providing a viscosity of about 24,000 cP. The initial viscosity
of the solution obtained upon mixture (1+1) of the hyaluronate-
supplemented Fibrinogen component with the Thrombin component was
about 2,000 cP.
[0049] III. The Fibrinogen component was diluted 1+1 with 0.145 M
NaCl, leading to the same final concentrations of aprotinin and
fibrinogen as in preparation II.
[0050] Five minutes after glue application the vascular clamps were
removed, beginning with the distal one. Occasional bleeding was
controlled by gentle manual compression.
[0051] Test Procedure and Results
[0052] The patency was tested 20 minutes after completion of the
anastomosis with Aucland's patent test (Aucland R. D., Microsurgery
Practice Manual, 2nd Ed. 1980, Mosby, St. Louis). The skin was then
closed with interrupted sutures and the animals were allowed to
awaken from the anaesthesia.
[0053] The animals were re-evaluated 24 hours later in a blind
fashion. The animals were anaestetized and the patency of the
anastomosis was tested as described above. The rats were then
killed with an overdose of barbiturate and the patent arteries were
excised. Excessive fibrin glue was removed, and the vessel and the
anastomosis were then incised longitudinally and inspected from
inside. Intravascular thrombus material was gently removed by
flushing with saline and remaining wall adherent fibrin glue
deposits were semi-quantitatively assessed in percent of the
internal vessel diameter.
[0054] The effects of the three types of glue preparations on the
patency of the anastomosis and fibrin glue deposits were evaluated
by comparing groups I, II and III with the use of a one-way ANOVA
with multiple range testing according to the method of least
significant differences, 20 minutes and 24 hours, respectively,
after completion of the anastomosis. All data were given as
mean+SEM. A difference at the 5% level was regarded as signficant.
The results are summarized in Table 1 below.
1 TABLE 1 Experimental groups I II III Original Diluted with
Diluted with Tisseel.sup.R hyaluronate saline Patency rates (%)
35.7 .+-. 13.3 85.7 .+-. 9.7 35.7 .+-. 13.3 20 min postop. Fibrin
mass inside 37.5 .+-. 11.9 8.0 .+-. 4.2 47.9 .+-. 11.1 the patent
vessels in % of vessel diameter 24 h postop.
[0055] As appears form the table the patency 20 minutes after
completion of the anastomosis was significantly higher (p<0.01)
in group II than in grops I and III. The semi-quantitative
determination of the amount of fibrin mass which reached inside the
patent vessels was significantly lower in sodium hyaluronate
treated fibrin preparations than in groups I and III.
[0056] The patency rates achieved in the present study are
comparable to those previously reported on microvascular
anastomosis performed with a minimal number of sutures combined
with additional fibrin glue (50-75%), (see e.g. Aucland R. D.,
supra).
[0057] The patency rate 20 minutes after completion of the
anastomosis was thus significantly higher in the group where
Tisseel.RTM. was combined with sodium hyaluronate. This improvement
is due to an increased viscosity of the preparation and not a
dilution of fibrin, which is demonstrated by the fact that dilution
with saline did not influence the patency rates. This mechanism is
further demonstrated by the fact that less fibrin reached inside
the vessel in the sodium hyaluronate treated group.
EXAMPLE 2
[0058] A fibrin glue composition corresponding to composition II in
Example 1, but wherein xanthan was substituted for hyaluronic acid,
was prepared by mixing 1 part (volume) of the Fibrinogen component
of original Tisseel.RTM. (Immuno AG, Austria), which had the same
composition as given in Example 1, with 1 part (volume) of a 10
mg/ml aqueous xanthan solution having a zero shear viscosity of
1,000,000 cP. The resulting solution containing Tisseel.RTM. and
xanthan, and having a viscosity of 80,000 cP, was then mixed (1+1)
with the Tisseel.RTM. Thrombin component (500 IU/ml; viscosity 1.2
cP). The subsequent clotting of fibrin in the resulting mixture,
which had an initial viscosity of about 6,400 cP, was followed with
a rheometer at 37.degree. C. After 30 minutes the phase angle was
about 4.degree. C. and the elastic (storage) modulus was 1,300
Pa.
[0059] For comparison, 1 part (volume) of the Fibrinogen component
of original Tisseel.RTM. was mixed with 1 part (volume) of saline
(0.15 M NaCl). The resulting fibrinogen containing solution was
mixed with the Thrombin component (500 IU-/ml) and the fibrin
clotting was followed as above at 37.degree. C. After 30 minutes
the phase angle was about 20 and the elastic (storage) modulus was
1,800 Pa.
[0060] Thus, in both cases very elastic gels (phase angle close to
0.degree.) of approximately the same stiffness (elastic modulus)
were formed at 37.degree. C.
EXAMPLE 3
[0061] Test Procedure
[0062] 1% (10 mg/ml) sodium hyaluronate (NaHA) (Healon.RTM., Kabi
Pharmacia, Uppsala, Sweden) was mixed 1+1 with the aprotinin and
fibrinogen containing syringe of Tisseel.RTM. fibrin glue (Immuno
AG, Vienna, Austria) as described for glue preparation II in
Example 1. The second, thrombin containing component of the fibrin
glue was added with the Duploject applicator (Immuno AG, Vienna,
Austria) leading to a final concentraiton of 0.25% NaHA. 100 mg of
the final composition were placed at the bottom of a petri dish.
After a setting time of one minute (group A) and 10 minutes (group
B) later, 10 ml of 0.145 M NaCl were added. The petri disches were
either put in a shaking bath with a frequency of 1 Hz at 37.degree.
C., or at rest in a humidified tissue culture chamber set at
37.degree. C. (groups C and D, respectively).
[0063] The corresponding controls were 1% NaHA (Healon.RTM.)
diluted 1+3 with 0.145 M NaCl, leading to the same final
concentrations of NaHA as in the experimental groups (0.25%). The
controls were treated in the same manner as the experimental groups
with shaking (groups AC and BC) and without shaking (groups CC and
DC). Samples of the added NaCl solution were taken after 30 min, 60
min, 4 h, 8 h and 24 h for analysis of NaHA. The NaHA
concentrations of the NaCl solution were determined with the HA
Test 50 (Kabi Pharmacia AB, Uppsala, Sweden).
[0064] Results
[0065] In the control groups the NaCl solutions were almost
immediately saturated and there was practically no further increase
in NaHA concentration up to 24 h, irrespectively of whether the
samples were put at rest or shaken. In the experimental groups, on
the other hand, the concentration of NaHA in the NaCl solution
increased steadily. The dissolution rates were depending on whether
the fibrin clots were allowed to set for 10 min before adding the
NaCl solution, and whether they were put at rest or shaken. The
results are summarized in the following Table 2.
2TABLE 2 NaHA conc. in added NaCl-solution (.mu.g/ml) Experimental
Control groups groups Shake Rest Shake Rest Time A B C D AC BC CC
DC 30 min 2.4 0.7 1.3 0.5 23 27 26 27 24 h 21 25 23 15 33 29 28
36
[0066] To express how fast NaHA was dissolved, the time until 30%
of the final amount of A was found in the NaCl solution was
calculated. The results are summarized in Table 3 below.
3TABLE 3 Dissolution time until 30% of NaHA in solution (min.)
Experimental Control groups groups Shake Rest Shake Rest A B C D AC
BC CC DC 15 36 336 462 3 3 3 3
[0067] The above results clearly show that sodium hyaluronate
dissolves up to 150 times slower in a saline solution when it is
incorporated in a fibrin clot. The dissolving rate is very much
dependent upon how long the fibrin glue is allowed to set before
the solvent is added. It can be expected that the dissolution rate
will decrease further if the clot is allowed to set for still
longer periods of time since it will take several hours to complete
the crosslinkage of the fibrin bundles. It is further demonstrated
that the mixture of NaHA and fibrin should not be under motion if a
prolongation of the dissolution rate is desired.
EXAMPLE 4
[0068] Animals and Test Procedure
[0069] Ten male Sprague Dawley rats with a body weight of 440-480 g
were used. The environmental conditioning and the anaesthesia were
the same described in Example 1 above. A laparotomy was performed
through a midline incision. A titanium disc having a 11 mm diameter
circular hole in the middle was placed on the surface of the right
anterior liver lobe. The serosal surface of the liver exposed in
the hole of the disc was gently brushed with a nylon interdental
tooth brush until petechial bleeding was achieved. A somewhat
larger area of the parietal peritoneum was traumatized in the same
manner. The location of the traumatized parietal surface was chosen
so that the two brusched surfaces should be in direct contact with
each other. Previous studies have shown that solid adhesions
develop only at the location where both serosal surfaces are in
direct contact. Five animals were each allocated to either no
treatment or to treatment with a mixture of hyaluronate and fibrin
glue as described for glue preparation II in Example 1. In the
second case the circumscribed lesion on the liver surface was
covered with 0.4 ml of the hyaluronate and fibrin glue mixture.
Hyaluronate was added and mixed 1+1 with the contents of the
fibrinogen containing syringe of Tisseel.RTM. (glue preparation II
in Example 1 above).
[0070] The rats were kept on their backs for 20 minutes after
application of the mixture to assure that parietal and visceral
surfaces were not in contact during the setting of the glue. The
abdomen and skin were then closed in layers with a running 4.0
Dexon suture. The animals were sacrificed after 48 days. The
abdomen was re-exposed through a midline incision and the adhesions
were evaluated. The occurrence and the area of adhesions developed
were expressed in percent of the initial serosal damage.
[0071] The effect of the hyaluronate-fibrin glue on the development
of adhesions was evaluated by means of an unpaired tow-tailed t
test. Data are given as mean +SEM. A difference at the 5% level was
regarded as significant.
[0072] Results
[0073] All adhesions found were located between the liver and the
inner surface of the abdominal wall, localized to the area where
the liver surface had been traumatized with the brush. All but one
of the animals in the control group developed adhesions whereas
none of the animals of the treatment group developed any adhesions.
The results are presented in Table 4 below.
4 TABLE 4 Control group Treatment group Occurrence of adhesions 80
.+-. 20* 0 .+-. 0 (number of animals, %) Inflammatory reactions 1.2
.+-. 0.4 0.8 .+-. 0.2 (0-3) Adherence area (%) 65 .+-. 23.8* 0 .+-.
0 *significant at 5% level
[0074] The combined treatment with hyaluronate and fibrin glue thus
completely abolished the development of adhesions, something that
has so far not been achieved with either treatment alone
(Lindenberg S., et al., Acta Chir, Scand. 151:525-527, 1985; and
Amiel D., et al., J. Hand. Surg. (Am) 14:837-843, 1989). The
mechanisms behind this finding are unclear. The fact that
hyaluronate is kept at the location of trauma for a longer period
of time as well as the changed composition of the fibrin clot seems
to optimize the conditions for wound healing and prevent the
formation of excessive scar tissue.
[0075] Furthermore, the composition also markedly reduced the
inflammatory reaction, which indicates that the wound healing is
induced by regeneration rather than formation of scar tissue and
shrinkage.
[0076] It is to be understood that when reference is made herein to
e.g. "polymers", "proteins", "polysaccharides", "polyglucans", and
the like, then the invention also includes salts, derivatives and
other modifications thereof which are functionally equivalent as
regards the herein disclosed properties.
* * * * *