Note: Descriptions are shown in the official language in which they were submitted.
WO 95/22316 ~ ~ ~ PCTIUS95/01792
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0
BIOLOGIC BIOADHESIVE COMPOSITIONS CONTAEVING FIBRIN GLUE
AND LIPOSOMES, METHODS OF PREPARATION AND USE
FIELD OF THE SON
' S The invention describes bioadhesive sealant compositions containing
fibrin glue comprising human-source components in combination or admixture
with
liposomes, and methods for making the compositions. Compositions and methods
of the invention are suitable for accelerating and ameliorating the healing
process
~r various types of surgical and nonsurgical procedures or wound healing in
mammals, including humans, and for maintaining hemostasis.
BACKGROUND OF THE INVENTION
Bioadhesive Fibrin Glue
Increased experience has been gained in the use of fibrin glue among
various surgical disciplines (I,erner, R. and Binur, N.S., 1990, J. Surg.
Res., 48:
165-181; Gibble, J.W. and Ness, P.M., 1990, Transfusion, 30:741-747; Sierra,
D.H., 1993, J. Biometer. Applic. 7, 309-352; Brennan, M., 1991, Blood Reviews,
5:240-244; Dresdale A., et al., 1985, Surg_,erv, 97:750-755; Sponitz W., et
al.,
1987, Amer. Sure., 59:460-462; Schlag G. and Redl H (Eds), 1986, Gynecology
and Obstetrics-Urology. Fibrin Sealant in Operative Medicine, Vol 3, Springer
Verlag (Berlin); Burnouf Radosevich, M. et al., 1990, Vox Sang, 58:77-84)'.
For
e~ple, surgeons, dentists and hematologists have reported that fibrin glue is
an
effective bioadhesive. Experience in animals and humans suggests that an
advantage of using fibrin glue rather than synthetic plastics (e.g.,
cyanoacrylate) or
sutures is that fibrin glue promotes local coagulation, thereby preventing
bleeding
even in hemophiliacs. Fibrin glue also appears to support regrowth of new
tissue
~d ~e extracellular matrix.
Fibrin glue is formed by mixing two components, human fibrinogen
(or a source of fibrinogen, such as a freeze-dried plasma protein concentrate
of
fibrinogenlfactor XIa//fibronectin) and an activating enzyme such as thrombin.
Prior to use, the plasma protein concentrates arse conventionally solubilized
in the
WO 95/22316 218 3 4 3 0 PCT/US95/01792
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presence of calcium chloride. Thrombin-induced activation of fibrinogen
results in
the formation of fibrin. Factor XIB and calcium participate in the cross-
linking
and stabilization of fibrin to become a tight mesh of polymeric fibrin glue.
Applied to tissue, the fibrin clot adheres to the site of application. The
rate of
coagulation and mechanical properties of the clot are dependent on the
concentration of fibrinogen as well as thrombin. Traditional fibrin glue
preparations are described in International Application No. W093/05067 to
Baxter
International, Inc.; W092/13495 to Fibratek, Inc.; and W091/09641 to Cryolife,
Inc.
Thrombin is a common physiological instigator of clotting.
Thrombin from a number of mammalian sources, most commonly bovine, is
routinely used in commercially-available fibrin glues. Human thrombin can be
employed in the formulation of the liposome-containing fibrin glue
bioadhesive, as
~ other appropriate catalyzing enzymes, such as reptilase or select venoms
(Fenton II, J.W. et al., 1977, J. Biol. Chem., 252:3587-3598; Gaffney P.J. et
al.,
1992, Thrombos. Haemostas. , 67:424-427; European Patent Application No. EP 0
439 156 Al , 1991; Stocker K. , et al. , 1982, Toxicon. , 20:265-273; Pickle
H. and
Stocker K., 1991, Thrombos. Haemostas., 65:444-450).
Fibrinogen may be in an intimate admixture with other proteins that
are typically found in anti-coagulated whole blood, in platelet-rich plasma,
in
plasma, in cryoprecipitate, or in precipitates of plasma obtained by a method
such
as Cohn precipitation of plasma. Such additional protein components may
include
fibronectin, immunoglobulin, particularly IgG, factor XIB, plasminogen, and
albumin.
The fibrinogen preparations used in the fibrin glue and liposome
compositions can be virally inactivated by one or more methods prior to their
employment in the invention (e.g. Examples 1-3).
Both fibrinogen and thrombin are derived from blood plasma by the
fractionation of plasma. Comprehensive reviews on the preparative techniques
of
each have been published and are the basis for most commercial plasma
fractionation procedures used by those skilled in the art and suitable for use
in the
WO 95/22316 2 ~ g 3 4 3 p PCT/US95/01792
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invention (For fibrinogen: Blomback, B. and Blomback, M. , 1956, Ark Kemi,
10:415-443; Stryker, M.H. & Waldman, A.A., 1978, Kirk-Othmer Encyclopedia
of Chemical TechnoloQV, Vol 4, 3rd ed. , pp 25-61, John Wiley; Lowe G. D. O.
et
al., 1987, Fibrinogen 2: Biochemistry, Physiology and Clinical Relevance.
Excerpts Medicus, Elsevier Science Publishers; For thrombin: Fenton II, J.W.
et
al., 1977, J. Biol. Chem., 252:3587-3598; Gaffney P.J. et al., 1992, Thrombos.
Haemostas., 67:424-427; Ward, G., 1991, European Patent Application No. EP 0
439 156 Al; and U.S. Patent No. 5,143,838 to Kraus et al.).
Alternative sources of human fibrinogen are also envisioned. For
e~ple, fibrinogen made by recombinant techniques could also be employed in
the fibrin glue and liposome composition. Molecular techniques available for
the
production of recombinant fibrinogen include the use of COS-1 or Hep G2 cells
transfected with DNA vectors containing isolated genes encoding normal or
mutant
human fibrinogen (Roy S.N. et al., 1991, J. Biol. Chem., 266:4758-4763; Roy
S.N. et al., 1994, J. Biol. Chem., 269:691-695). It is expected that future
developments will lead to the ability to produce usable amounts of fibrinogen
by
such techniques in other types of cells. Normal or mutant recombinant
fibrinogens
may be employed in fibrin glue compositions formulated with the types of
liposomes as described herein.
Despite the effectiveness and successful use of fibrin glue by medical
practitioners in Europe, neither fibrin glue nor its essential component
fibrinogen is
widely used in the United States at the present time because of the general
risks
~d p~blems of infection from pooled blood products contaminated with lipid-
enveloped viruses such as HIV, associated with AIDS, and the hepatitis-causing
viruses such as HBV and HCV (also known as non A-non B hepatitis virus), as
well as cytomegalovinls (CMV), Epstein-Ban virus, and the herpes simplex
viruses
in fibrinogen preparations. For similar reasons, human thrombin is not
currently
authorized for human use in the United States. Bovine thrombin, which is
licensed
for human use in the United States, is obtained from bovine sources which do
not
appear to carry significant risks for HIV and hepatitis, although other bovine
pa~ogens may be present.
WO 95/22316 21 ~ 3 4 3 0 pCT/US95/01792
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° Both human fibrinogen and human thrombin can be virally
inactivated against lipid coat viruses by treatment with organic solvent and
detergent (SD process) (U.S. Patent No. 4,540,573 to Neurath A.R. and Horowitz
B. , 1985; Horowitz, B, et al. , 1985, Transfusion, 25:516-522; Horowitz, B.
et al. ,
1992, Blond, 79:826-831;- Piet, M.P.J. et al., 1990, Transfusion, 30:591-598;
Burnouf Radosevich et al., 1990, Vow, Sg:77-84; Horowitz, B. et al., 1992,
Blood, 79:826-831). Other viral inactivation procedures for fibrinogen and
thrombin blood products include UV irradiation or heating (U.S. Patent No.
5,116,590 to Miyano, K. et al.).
I,;~omes
Liposomes are unilamellar or multilamellar lipid vesicles which
entrap a significant fraction of aqueous solution. The vesicular
microreservoirs of
liposomes can contain a variety of water-soluble materials, which are thus
suspended within the emulsion (reviewed in G. Gregorius (Ed.), 1991, Liposome
Tech, Vols. I, II, III, CRC Press, Boca Raton, Florida; M.J. Ostro (Ed.),
1983, Linosome Preoarations~ Methods & Mechanisms, Marcel Dekker Inc. New
York; Davis S.S. and Walker LM., 1987, Methods in Enz~molo$x, 149:51-64;
MaYhew E. et al., 1987, Methods in EnzvmoloQV, 149:64-77; Shafer-Korting M.
et al., 1989, J. Am. Acad. Dermatol , 21:1271-1275; Szoka F. and
Papahadjiopoulos D., 1980, Ann. Rev Biophvs Bioen in , 9:467-508; Harrigan
P.R. et al., 1990, Chem. & Ph s Lipids, 52:139-149; Patel H.M., 1985, Trans.
Biochem. Soc., 13:513-516; Ostro M.J., 1987 (Jan.), Sci. Am., 91). The
prep~hon of liposomes and the variety of uses of liposomes in biological
systems
have been disclosed in U. S . Patent No. 4, 708, 861 to M. C . Popescu et al.
, U. S .
Patent No. 4,224,179 to M. Schneider, U.S. Patent No. 4,235,871 to D.P.
Papahadjopoulos and F. C. Szoka, Jr. , P. R. Cullis et al. , 1987, In:
Liposomes as
Pharmaceuticals, M.J. Ostro, Ed., Marcel Dekker, New York, 39-72, and H.G.
Weder et al., 1986, In: Linosomes as drug carriers, K.H. Schmidt, Ed., Thieme,
Stuttgart, 26-39.
Liposomes are formed by mixing long chain carboxylic acids,
Mmes, and cholesterol, as well as phospholipids, in aqueous buffers. The
organic
WO 95/22316 218 3 4 3 0 pCT~S95/01792
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components spontaneously form multilamellar bilayer structures (i.e.
liposomes).
Depending on their composition and storage conditions, liposomes exhibit
varying
stabilities. Liposomes serve as models of cell membranes and also have been
used
as dnig delivery systems (M. Schafer-Korting et al., 1989, J. Am. Acad.
Dermatol., 21:1271-1275). Most attempts to use liposomes as drug delivery
vehicles have envisioned liposomes as entities which circulate in blood, to be
taken
up by certain cells or tissues in which their degradation would slowly release
their
internal aqueous drug-containing contents. In an effort to aid in their up-
take by a
given target tissue, some liposomes have been "tailored" by binding specific
~~~es or antigens to the outer surface. Liposomes have also been devised as
controlled release systems for delivery of their contents in vivo (H. M.
Patel, 1985,
Biochem. Soc. Transactions, 13:513-516). Compositions in which liposomes
containing biologically active agents are maintained and immobilized in
polymer
matrices, such as methycellulose, collagen, and agarose, for sustained release
of
the liposome contents, are described in U.S. Patent No. 4,708,861 to M.C.
Popescu et al.
fibrin Glue
Fibrin glue has the potential to be prepared in virally sterilized form
by treating the fibrinogen and thrombin by viral inactivation processes, such
as the
solvent-detergent or SD process. There is ample opportunity to improve fibrin
glue, fibrin glue compositions, and the utility of fibrin glue in wound
healing.
Various strategies have been used in attempts to improve the effectiveness of
e~sting and commercially available fibrin glues by adding water soluble
components to the fibrin glue preparation. Selective additives such as
antibiotics
(e.g. tobramycin or sisomicin), growth factors (e.g. EGF, TGF-B), peptides,
proteins, fatty acid derivatives, vitamins, hormones, steroids, and trace
elements
(e.g. calcium phosphate) have been included directly in the fibrin glue
formulation
in an effort to influence cell growth and wound healing, and to prevent
infection.
However, because the extraneous additives were supplied directly in the fibrin
glue
or in an adhesive, the half life of the additive may be affected. Direct
addition of
additives to fibrin glue may not be optimal for delayed, controlled, or long-
term
WO 95/22316 21 B 3 4 3 0 PCT/iJS95/01792
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release of an agent.
Other difficulties encountered by mixing extraneous additives
directly into fibrin glue components occur because the added materials may
affect
the rate of gelation or the mechanical properties of the fully-formed fibrin
glue.
Additives such as peptides may become cross-linked to the glue, and thus not
be
biologically available or effective in cross-linked form. Some additives might
alter
the enzymatic or biologic properties of thrombin. Alternatively, the additives
might increase the susceptibility of the fibrin glue matrix to plasmin-induced
degradation. Further, some additives might simply diffuse out of the fibrin
matrix
t~ repidly, thereby decreasing the "window" of their pharmacologic
effectiveness.
Such problems bespeak the need for different techniques for combining fibrin
glue
with exogenously-added substances.
The present invention affords a new generation of virally inactivated
bioadhesive sealant compositions comprising fibrin glue and liposomes whose
advantages and uses will become apparent from the following objectives of the
invention and disclosure. The present invention establishes a safe and unique
fibrin
glue and liposome formulation for widespread use and numerous surgical and
nonsurgical applications.
Further, topical therapies call for a need for drug delivery systems in
order to more effectively deliver active ingredients to the site of disease.
The
present invention provides a solution for several problems in the realm of
drug
delivery and improves upon extant methods. The present invention provides a
wholly biologically compatible bioadhesive system while concurrently providing
efficient and sustained delivery of therapeutic agents directly at and amund
the
required site of administration.
SUMMARY OF TREE INVENTION
It is an object of the present invention to provide a biological
composition in which fibrin glue and liposomes are combined together to form a
distinctive and novel bioadhesive for administration to animals, including
humans.
The final fibrin glue and liposome bioadhesive formulation may be achieved in
a
WO 95/22316 21 ~ 3 4 3 0 PCT/iJS95/01792
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number of different ways, such as by premixing at least one of the fibrin glue
components with liposomes prior to application or administration, and then
adding
the remaining components) to form in situ the final liposome-containing fibrin
glue.
It is another object of the invention to provide liposomes tailored for
use in conjunction with fibrin glue in various modes. In one mode, a
fibrinogen
solution can be mixed with liposomes and stored in the cold (i.e., at
4°C), or
frozen (i.e., at -30~C), or lyophilized. When desired, the fibrinogen/liposome
mixture is thawed or reconstituted with buffer, and then mixed with thrombin
at
~e site of a surgical or non-surgical opening or wound, thereby forming
liposome-
containing fibrin glue. Alternatively, liposomes can be pre-mixed with a
solution
of thrombin and stored cold, frozen, or lyophilized. When desired, thawed,
warmed, or reconstituted fibrinogen is mixed with a thrombin and liposome
mere at the site of administration, thereby forming liposome-containing fibrin
glue.
It is a further object of the invention to provide a stable fibrin glue
and liposome sealing matrix which is inexpensive and safe and can be easily
applied over a surgical or nonsurgical wound or opening, or injury site, or a
graft
site in a mammal, including humans, to promote, accelerate, and protect
sealing
and healing at and around the site. The fibrin glue and liposome formulation
remains at the site of application long enough to promote and protect the
healing
process. The fibrin glue is generally metabolized during wound healing and
does
not trigger an adverse reaction, toxicity, or an immune response in the
recipient
animal.
It is another object of the invention to provide a fibrin glue-
containing liposome composition in which medicaments or bioacdve additives are
encapsulated or entrapped in the liposomes. The liposomes serve as vehicles or
carriers of the medicaments and additives at an injury site, surgical or
nonsurgical
opening, or wound. The contents of the liposomes are released at the site
after
application, either in a spontaneous or controlled fashion.
bother object of the invention is to provide a novel method for
WO 95/22316 21 a 3 4 3 0
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° formulating fibrin glues to improve upon existing methods for better
protection and
treatment of surface wounds and surgical and nonsurgical openings.
Yet another object of the invention is to provide fibrin glue and
liposome bioadhesive compositions in which the liposomes add desirable
properties
to the glue components. The storage characteristics of both thrombin and
fibrinogen may be modulated and significantly improved by liposome components.
Biophysical properties of the fibrin glue composition, such as rate of
gelation,
viscoelasticity, and tensile strength, may be modulated and further improved
by the
liposome components of the composition.
A further object of the invention is to provide fibrin glue as the basis
for clot formation at the site of injury, surgery, or a wound while
simultaneously
providing the slow or rapid release of bioactive ingredients which are
contained in
the liposomes of the fibrin glue/liposome formulation. The formulation may
~mprise a number of different types of liposomes, each containing a different
bioacdve agent. Alternatively, the formulation may comprise a number of
liposomes of a particular type, each containing different additives.
Another object of the invention is to provide a biologically
compatible sealing agent comprising fibrin glue combined with liposomes to
favor
and maintain hemostasis following its use, even in heparinized individuals and
in
individuals suffering from coagulopathies. In addition, the fibrin glue and
liposome-containing bioadhesive system of the invention promises to reduce the
incidence of fistula formation and to decrease postoperative infections,
tissue
n~~sis, and toxicity.
Yet another object of the invention is to provide fibrin glue
compositions containing virally inactivated fibrinogen and thrombin
preparations
admixed with liposomes and other glue components from human and animal
sources to yield a virally inactivated fibrin glue and liposome preparation.
Such
virally inactivated fibrin glue and liposome compositions afford safer and
contaminant-free preparations which can be admixed and employed in accordance
with the invention.
CA 02183430 2005-12-O1
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BRIEF DESCRIPTION OF THE DRAWIrTGS
Figure 1. Size (i.e., diameter in microns) distribution of neutral liposomes
(Type
A), determined in a CoulterTM particle analyzer. The size distribution of the
prepared
liposomes was not significantly altered when the liposomes were formed in a
buffer
in the presence of 2 mM zinc chloride.
Figure 2. Schematic diagram for experimentally determining the breaking or
tensile strength ("BS" or "TS") of fibrin glue gel. See also Figure 10.
~~~ 3, Breaking strength (i.e. , tensile strength, "TS ") of fibrin glue (made
from cryoprecipitate ("Cryo") as the source of fibrinogen and thrombin with
and
without 5 °& (v/v) of neutral (Type A) liposomes ("Lipo A"), amine
(Type B)
liposomes ("Lipo B"), or carboxylic acid (Type C) liposomes ("Lipo C"). Fibrin
glue was formulated using 10 U/mL of thrombin and 10 mM Ca2+.
Figure 4. Breaking strength (i.e., tensile strength, "TS") of fibrin glue
formulated
with purified fibrinogen (i.e., Fraction I paste of Cohn preparation) and
thrombin
a'i~out ("Cohn 1 ") and with 5 ~ (vlv) of neutral (Type A) liposomes, "Lipo A"
;
amino (Type B) liposomes, "Lipo B"; or carboxylic acid (Type C) liposomes,
"Lipo C". Fibrin glue was formulated using 10 U/mL of thrombin and 10 mM
CaZ+,
~~ g, Viscoelasticity development (expressed as thromboelastograph ("TEG")
amplitude) of fibrin glue formulated with cryoprecipitate ("Cryo") and
thrombin
without (open circles) and with 5 ~ (v/v) of neutral (Type A) liposomes
("Cryo+Lipo A", filled circles), amino (Type B) liposomes ("Cryo+Lipo B",
filled triangles), and carboxylic acid (Type C) liposomes ("Cryo+Lipo C",
filled
squares).
Figure 6. Viscoelasticity development (expressed as thromboelastograph ("TEG")
~pli~de) of fibrin glue formulated with pure fibrinogen ("Fib", 3.6 mg/mL
final
WO 95/22316 218 3 4 3 ~ PCT/LTS95/01792
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° concentration) and thrombin without (open circles) and with 50 ~,L
(filled circles)
or 100 ~L (filled squares) of Type A liposomes ("Lipo A") added to the fibrin
glue
mixture (300 ~cL total).
figure 7. Mean wound breaking strength ("Wound BS") of mouse skin incision
closed with staples ("Control"), with fibrin glue formulated from
cryoprecipitate
("Cryo") as a fibrinogen source, and with fibrin glue formulated from
cryoprecipitate as a fibrinogen source in combination with Type A liposomes
("Cryo & Lipo A").
1 figure 8. Breaking strength (BS) of fibrin glue film without ("Neat") and
with
Type A, B, or C liposomes (8~b by volume), ("Lipo A, Lipo B, Lipo C",
respectively). Dimensions of fibrin glue film: 2 mm thick, 1 cm wide. Fibrin
glue formulation: 28 mg/mL or 45 mg/mL fibrinogen ("Fib"); 10 U/mL thrombin;
15 mM Ca(li).
figure 9. Percent elongation (" ~ Elong") prior to breaking of fibrin glue
film
without and with Type A, B, or C liposomes (8 ~ by volume), ("Lipo A, Lipo B,
Lipo C", respectively). Initial dimensions: 2 mm thick, 1 cm wide. Fibrin glue
formulation: 28 mg/mL or 45 mg/mL fibrinogen ("Fib"); 10 U/mL thrombin; 15
mM Ca(11).
~~~ 10. Photograph of fibrin glue (45 mg/mL fibrinogen) containing Type A
liposomes (8 ~ by volume) formulated into a film. The strip of fibrin glue and
liposome filin is being twisted 90°.
ire 11. Photograph of fibrin glue (45 mg/mL fibrinogen) containing Type A
liposomes (8 °b by volume) flexibly coating aluminum foil as a solid
substrate.
figure 12. Breaking strength (BS) of fibrin glue ("FG") mixed with bone
fm~ents ("Bone") without or with Type A liposomes, 8°b by volume,
("Lipo
WO 95/22316 21 a 3 4 3 0 pCT/L1S95/01792
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A"). Bone fragments not longer than 2 mM were mixed with fibrin glue without
or with liposomes and the matrix was allowed to set for 1 hour at 37~C in a
moist
environment. Fibrin glue components: fibrinogen (45 mg/mL), thrombin (2
U/mL), Ca(1'17 (15 mM).
DETAILED DESCRIPTION OF THE IhIVENTION
The fibrinogen for use in producing the fibrin glue-containing
liposome composition of the invention may be prepared by employing starting
materials of varying purities and by following a number of procedures known to
~o~ skilled in the art (Blomback, B. and M. Blomback, 1956, Ark. Kemi.,
10:415-443; Stryker, M.H. and Waldman, A.A., 1978, Kirk-Othmer En~clopedia
Qf Chemical Technolo~r, Vol. 4, 3rd ed. , John Wiley, pp 25-61; Lowe G. D. O.
et
al., 1987, Fibrino~een 2: Biochemistry, Physiology and Clinical Relevance,
~~rP~ M~icus, Elsevier Science Publishers; Dresdale A. , et al. , 1985,
Surgery,
97:750-755; Sponitz W., et al., 1987, Amer. SurQ., 59:460-462; and Burnouf
Radosevich, M. et al., 1990, Vox Sang 58:77-84). Fibrinogen may be purified
from human plasma as a by-product of anti-coagulated red blood cell
concentrates
from one individual or pooled from many individuals. Cryoprecipitate from
fresh
frozen plasma is frequently the source of concentrated fibrinogen (A. Dresdale
et
al., 1985, Surgery, 97:750-755) and is suitable for use in the present
invention.
Preferably, for the invention, fibrinogen is prepared from the fractionation
of
plasma by an adaptation of the Cohn technique (Blomback, B. and M. Blomback,
1956, Ark. Kemi., 10:415-443; Stryker, M.H. and Waldman, A.A., 1978, Kirk-
Othmer Encyclopedia of Chemical Technology, Vol. 4, 3rd ed., John Wiley, pp
25-61). The adaptation comprises viral inactivation using solvent-detergent or
other techniques, such as UV irradiation, lyophilization, or heating (U.S.
Patent
No. 5,116,950 to Miyano, K. et al.).
Despite careful blood donor selection and donor blood screening, a
primary concern in the preparation of fibrinogen from human sources is the
inactivation of infectious viruses. Lipid coat viruses which are insidiously
present
m human plasma protein samples are effectively inactivated by treating the
plasma
WO 95/22316 ~ PCT/US95/01792
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° or the blood component with organic solvent and detergent mixtures
(i.e., SD
mixtures), (U.S. Patent No. 4,540,573 to Neurath A.R. and Horowitz B., 1985;
Horowitz, B. et al., 1985, Transfusion, 25:516-522; Horowitz, B. et al., 1992,
Blood, 79:826-831; Piet, M.P.J. et al., 1990, Transfusion, 30:591-598; Burnouf
Radosevich et al., 1990, Vox Sane, 58:77-84; Horowitz, B. et al., 1992, Blood,
79:826-831). If desired, fibrinogen or thrombin sterilized by the SD process
may
be subjected to additional virucidal procedures and sterilization techniques,
such as
high temperature treatment (i.e dry heat at about 60°C to 68°C
for up to 96 hours)
or treatment with quenchers (e.g., 13-propriolactone or flavinoids such as
rutin) and
ultraviolet radiation. Further, it is envisioned that procedures which
effectively
eradicate or inactivate non-lipid coat viruses may also be used to treat the
fibrinogen, thrombin, and blood protein components. Although the SD process is
highly effective, it is cautioned that not all virvcidal treatments are
equally
e~~~ve; nonkilled or non-inactivated viruses are a threat to the safety of the
final
product. Care must also be taken to ensure that minimal protein degradation
occurs during viral inactivation, since the physical properties of the fibrin
glue
depend upon the absence of degradation or denaturation of the very protein
components responsible for its clotting activity (e.g. fibrinogen and
thrombin).
In accordance with the invention, human-derived virally inactivated
fibrin glue is prepared from several integral and interactive components,
which
include human fibrinogen and thrombin components that are virtually free from
active lipid coat viruses.
To formulate the fibrin glue for use in the invention, virally
inactivated, (VI) fibrinogen and thrombin from human sources are present in
the
fibrin glue composition in appropriate concentrations. For example, Table 1
describes the range of amounts, and the preferred and more preferred amounts
of
fibrinogen and thrombin (or other activating enzymes), liposomes, and calcium
used to formulate fibrin glue in which liposomes are embedded. The resulting
concentration of fibrinogen in the final fibrin glue composition is about 10-
90
mg/mL, preferably about 30-60 mg/mL, and more preferably about 40 mg/mL,
while the concentration of thrombin or other enzyme in the final glue
composition
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° is about 1-200 U/mL, preferably about 5-20 U/mL, and more preferably
about 10
U/mL.
Table 1
Calcium
fibrinogenThrombin Liposomes(Ca(In)
mg/mL U/mL ~cL/mL mM
Suitable Range 10-90 1-200 20-300 1-30
Preferred Range 30-60 5-100 50-100 10-15
Optimal Range 40 10 75 10
The fibrin glue and liposome bioadhesive composition of the
invention constitutes a new generation of biological and bioactive sealants,
adhesives, or material for fabricating films or coatings. As a result, there
is less
risk of contaminating agents (e.g. viruses and the like) found in the final
composition. In addition, the production of the present fibrin glue containing
liposomes is economical to make and use.
Liposomes for use in fibrin glues in general, and in the mixture with
fibrinogen in particular, may be formed by a variety of techniques. It is
important
that the liposomes, however they are produced, do not adversely modify the
physical properties of the fibrin glue once they are embedded in the glue. As
will
be clear from the present invention, the fibrin glue-containing liposome
composition is designed not to deter from the clot forming and bioadhesive
a~butes of the fibrin glue. In fact, the liposomes themselves may favorably
modulate the biophysical properties of the fibrin glue by increasing or
decreasing
the rate of clot formation and gelation, by strengthening or weakening the
clot, or
by improving the viscoelasdcity of the fibrin clot as needed or desired. The
techniques available for testing these biophysical properties of fibrin glue
have been
described (Mans G., 1988, Thrombos. ~Iaemostas., 59:500-503; Marx G. and
Blankenfeld, A., 1993, Blood Coag. and Fibrinolysis, 4:73-78). It is
envisioned
that the liposomes may be formulated to entrap a component or components which
could modulate the final fibrin glue characteristics. For example, some or all
of
the liposomes for use in the fibrin glue composition of the invention may be
WO 95/22316 218 3 4 3 0 pCT~S95/01792
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prepared to contain aprotinin or other anti-proteases, such as e-amino caproic
acid
(EACA) in their aqueous phase compartment, which, when released at the site of
fibrin glue-liposome composition during glue degradation, would slow the rate
of
protein degradation, thus prolonging the lifetime and viability of the fibrin
glue
sealant.
The fibrin glue formulated in the presence of liposomes maintains its
mechanical properties as determined by breaking strength or tensile strength
studies
and viscoelasdcity studies (e.g., Figs. 2-6). In the present invention the
quality of
the biophysical parameters is not significantly modified by the liposomes in
the
bioactive composition. Indeed, the liposomes are even suitable for improving
the
quality of the fibrin glue formulated to include such liposomes.
The basic constituents of liposomes are various saturated or
unsaturated lipids or phospholipids, with or without the addition of
cholesterol and
other constituents, such as aliphatic compounds having either amino or
carboxylic
acid groups. Nonlimiting examples of phospholipids that are suitable for use
in
formulating liposomes are phosphotidylcholine or lecithin, phosphatidyl
ethanolamine, phosphatidic acid, phosphatidylserine, phosphatidylinositol,
phosphatidylglycerol, sphingomyelin, cardiolipin, and hydrogenated products
thereof. Liposomes of several types suitable for use in the present invention
may
be prepared by a variety of techniques that may ultimately influence liposome
morphology, type, and size. Many techniques for preparing liposomes have been
described (Szoka Jr. and Papahaddjopoulos, D. (1980), Ann. Rev. Biophvs.
Bioengineering, 9:467-508; Gregorius G. (Fd). (1983) Liposome TechnoloQV,
Vols. I. II. I/1, CRC Press, Boca Raton, FL. (1991); Ostro (F.d)., Liposome
P-gr parations: Methods & Mechanisms, Marcel Dekker Inc. NY; Davis, 5.5. and
Walker, LM., (1987), Methods in EnzlrmOlog3i 149: 51-64). These methods are
applicable for producing liposomes for embedding into the fibrin glue to be
applied
to a specific tissue site. As but one example, a technique for producing
liposomes
for use in the invention is by ethanol injection. In this technique, equimolar
quantities of cholesterol and hydrogenated lecithin are mixed and warmed to
60~C
m ~°ol to form a solution, and the solution is injected in an aqueous
buffer at
WO 95/22316 ~ ~ PCT/LTS95/01792
-15-
60~C containing the material to be encapsulated. The emulsion is incubated
60~C
for one hour, centrifuged at 2000 x g for 5 minutes, and the supernatant is
removed. The liposomes are suspended in Tris-saline buffer and stored at 4~C.
Liposomes of several types are suitable for use in the present
invention. For example, neutral liposomes designated as Type A (described in
Example 5) are formulated with equimolar amounts of cholesterol and
hydrogenated lecithin and are referred to as neutral liposomes. As described
in
Example 6, Type B liposomes are formulated with 50 °b cholesterol,
40 h
hydrogenated lecithin, and 10 °6 stearyl amine on a molar basis. As a
consequence
of their composition, Type B liposomes have frex amine groups on their
surfaces;
such groups on Type B liposomes are potentially capable of affecting certain
biophysical parameters of the fibrin glue composition. Type C liposomes,
described in Example 7, are formulated with 50 ~ cholesterol, 40 ~
hydrogenated
l~ithin, and 10 ~ stearic acid on a molar basis. Type D liposomes are
formulated
with 50 °~ cholesterol, 40 °.& hydrogenated lecithin, and 10
°b diethylstearylamine on
a molar basis. Depending on pH, exemplary liposomes of the B and C types
contain electrically-charged chemical moieties on their surfaces.
Large liposomes (e.g. multilamellar vesicles having a diameter size
range of 0.1 to 5 to > 10 ~cm, and large unilamellar vesicles having a
diameter size
of 20.06 Vim) and small liposomes (e.g. small unilamellar vesicles having a
diameter size of about 0.02 to 0.05 ~,m) may be employed in the present
invention
and may be produced by conventional methods as previously indicated. Although
~o~ ~~ ~ the art will appreciate that virtually all types of liposomes are
suitable for use in the present invention, some liposome types may have
particular
pmperties which make them especially conducive to forming a stable and
effective
fibrin glue-liposome bioadhesive. For example, as described above, neutral
liposomes of 5 ~cm diameter with a good load capacity (e.g. at least about 10~
to
20 °.b aqueous phase) may be preferred so as to provide adequate
amounts of
entrapped materials at the site of action and so as not to interfere
substantially with
the crosslinking properties of the fibrin glue after application at the site.
~ a~°~~ with the invention, the contents of the liposomes are
WO 95/22316 2 ~ g 3 4 3 0 pCT~S95101792
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routinely entrapped in the aqueous phase, rather than within the membrane
bilayer
of the liposome. The amount of aqueous phase containing bioactive material
incorporated into the aqueous interior compartments of the liposomes for
effective
use in the invention was tested by forming Type A liposomes in Tris buffer
(Tris-
saline buffer, pH 7.4) containing 2 mM Znz+. The Zn2+ which remained after
washing the liposomes was measured by X-ray fluorescence (Gorodetsky, R. ,
Mou,
X. , Blankenfeld, A. , and Marx, G. , 1993, Amer. J. Hematol. , 42:278-283) .
From
these measurements, it was estimated that the liposomes contained, on a volume
per volume basis, about 10 °& to 20 ~ aqueous phase, which is
indicative of the
"load" of the liposomes. The loaded liposomes were stable at both 4~C and
22~C,
and they retained their aqueous phases quite well over a period of several
weeks.
Such Zn2+ containing liposomes were used in in vivo studies in accordance with
the
invention (Example 10).
Knowing the liposome load allows the calculation of the effective
concentration or fraction of reagents) entrapped (i.e., contained) within the
aqueous phase for effecting delivery and deposit of the load at the site of a
wound
or opening. A typical load is calculated on the basis of the orginal amount of
the
aqueous starting material used and the final amount of material that is
contained in
the liposome. As a specific but non-limiting example, the Zn2+ solution served
as
a quantifiable marker for the entrapped contents of the liposomes. Thus, the
amount of Zn2+ in the starting solution is determined (e.g., 130 ppm). The
liposomes are formulated to entrap a portion of the Zn2+ solution. The
fractional
liposome volume is determined from a particle counter. The liposomes so
formulated are washed in buffer. After washing, the aqueous phase is removed
and the amount of Znz+ that has been entrapped into the liposome is measured.
Using 300 ~,L of liposomes containing 2 mM Zn2+ solution in their
interiors would translate overall into 60 ~cM Zn2+ in 1 mL of fibrin glue at
and
around the site of the wound. The liposomes can fuse with cells at the site of
the
wound and thereby merge their aqueous contents with the contents of cells.
Thus,
the aqueous phase of the liposome is delivered to the wound site where it
subsequently diffuses into and around the site of application.
WO 95/22316 21 ~ 3 4 3 0 pCT~S95/01792
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° Another aspect of the invention involves the use of fibrin glue
containing light sensitive or photoactivatable liposomes (LSL), which may
provide
more controlled release of their contents into the environment over time. Such
liposomes can also be prepared to contain bioactive materials, additives, and
medicaments, and are kept in a light-protected (e.g. filtered) container until
use.
When used in fibrin glue, a light source (e.g., ultraviolet light or laser)
are
focussed on the LSL, thereby causing them to release their contents into the
surroundings at the site of application. Light sensitive and photoactivatable
liposomes are prepared essentially as described for non-light sensitive
liposomes;
fey ~ v~~y identical, but they provide for manipulated, light-controlled
release of their contents. Light sensitive liposomes may be prepared by
formulating liposomes to contain on their surfaces compounds having at least
one
light sensitive chemical double bond, which predisposes the compounds to
undergo
~~o~ational change when exposed to light, thereby resulting in
photoactivatible
liposomes. Such light-sensitive liposomes are prepared, for example, by using
lecithins of retinoic acid (such as 1,2-diretinoyl-sn-3-glycerophosphocholine
(DRPC), 2-retinoylysolectin (LRPC) or 1-palmitoyl-2-retinoyl-sn-3-
glycerophosphocholine (PRPC) in the lipid and cholesterol mixture, as
described
(Pidgeon, C. and Hunt, C.A., 1987, Methods in Enz,~. 149:99-111). Mixtures
of DRPC/LRPC in ratio ranges of about 70:30 to about 30:70 are used to
formulate light sensitive liposomes. Some formulations might include up to 40
~
added a-tocopherol (a-T) to help in forming the light-sensitive liposomes.
prep~~ons of LSL would necessarily be kept in the dark to prevent light
induced
degradation of the liposome structures and the release of their aqueous
compartments. The light-sensitive liposomes are mixed with fibrin glue
components (r. e. , fibrinogen or thrombin) of the fibrin glue and liposome
composition to allow their compartments to be released when desired by
exposure
to the chosen light source.
Alternatively, the glue component, thrombin, is incorporated into the
LSL. Such thrombin-containing liposomes are then mixed directly with
fibrinogen
m a light filtered container. The release of thrombin would be instigated by
WO 95/22316 218 3 4 3 0 pCT~S95/01792
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° exposing the mixture to the appropriate light source. Thus, a single-
compartment
delivery system could be devised for fibrin glue application, with clotting
only
initiated by exposure to the activating light source.
Bioactive agents contained in liposomes of the fibrin glue-containing liposome
composition
The lipvsomes of the invention are designed to contain, carry, and
release biologically active agents in accordance with the internal load or
capacity of
the liposomes. It is envisioned that liposomes containing biologically active
substances and medicaments and embedded in the fibrin glue will carry and
release
their contents at a wound site or surgical or nonsurgical opening in animals,
including humans, to aid in the healing and protection process following all
types
of surgical or wound healing procedures and applications. Examples of
applications for the "loaded" liposomes in fibrin glue include, but are not
limited
to, partial or complete replacement of sutures in skin grafts, burns or
ulcers, or
surgical and nonsurgical openings; nerve and vessel anastomoses; surgery of
soft
tissues, such as parenchyma) tissues of liver, lung, or spleen; microsurgeries
in all
areas of the body; orthopedic surgeries for tendon repair and bone or
cartilage
grafting, general surgeries, such as cuts and laceration repair;
cardiovascular
surgery for vascular grafts and anastomoses; thoracic surgery to seal duct
leaks and
esophageal anastomoses; otolaryngology-head and neck surgery; ophalmological
surgeries, general dental use and surgeries; general surgeries in various
anatomical
~y pas ~d sites.
A wide variety of biologically active agents as well as medicines and
pharmaceuticals may be contained within the liposomes of the fibrin glue-
liposome
formulation. Examples of various agents to be entrapped in the liposomes
include,
but are not limited to, drugs, neuroleptics, vitamins (e.g. Vitamin C, (i.e.
ascorbic
acid or ascorbate), Vitamin A, Vitamin E, Vitamin D, Vitamin B, or derivatives
thereof), growth factors (e.g. lymphokines, cytokines), hormones, steroids,
glucocorticosteroids, antibiotics (e.g. penicillin, gentimycin, erythromycin,
adriamycin, tobramycin), antibacterial compounds, including bacteriocidal and
2183430
WO 95/22316 PCT/LTS95101792
-19-
bacteriostatic compounds, antiviral compounds, antifungal compounds,
antiparasitic
compounds, tumoricidal compounds, tumoristatic compounds, toxins, enzymes,
enzyme inhibitors, proteins, peptides, minerals (such as zinc or copper),
neurotransmitters, lipoproteins, glycoproteins, immunomodulators,
immunoglobulins and fragments thereof, dyes, radiolabels, radiopaque
compounds,
fluorescent compounds, fatty acid derivatives, polysaccharides, cell receptor
binding molecules, anti-inflammatories, antiglaucomic compounds, mydriatic
compounds, anesthetics, nucleic acids (e.g. RNA and DNA fragments), and
polynucleotides. It is also envisioned that selected fragments, portions,
derivatives,
or analogues of some or all of the above may be used, when practical, as
additives
in the aqueous phase of the liposomes of the invention. In addition,
lipophilic
drugs or other compounds may be incorporated into the phospholipid membrane of
the liposomes.
The invention is suitable for multiple bioactive agents to be
contained in liposomes used in the fibrin glue bioadhesive composition. Should
such a utility be desired, two or more bioactive agents may be entrapped in
one
liposome type which forms an integral part of the fibrin glue, and becomes
subsequently embedded or deposited in the glue clot. Alternatively, two or
more
different types of liposomes or mixtures of liposome populations, each of
which
entraps the same or different bioactive agents, may be embedded in the fibrin
glue-
liposome composition. Different preparations of liposomes may comprise
monophasic lipid vesicles (i. e. those having unilamellar lipid bilayers) or
plurilamellar vesicles (i.e. those having multilamellar lipid bilayers), such
as have
been described previously (M. Schafer-Korting et al., 1989, J. Am. Acad.
Dermatol., 21:1271-1275; U.S. Patent No. 4,708,861 to M.C. Popsecu et al.). As
envisioned for use in the present fibrin glue and liposome composition, one
type of
lipsomes (e.g. neutral liposomes) is formulated to entrap a particular
bioactive
material and a second type of liposomes (either the same type as the first or
a
different type) is formulated to entrap another bioactive material. Both types
of
liposomes containing their respective bioactive contents are mixed with the
c°mp°nents comprising fibrin glue, and the resulting fibrin glue
and liposome
WO 95/22316 2 ~ g 3 4 3 p PCT/US95/01792
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° composition contains two types of liposomes capable of delivering
their respective
bioactive contents at the incision or wound or opening site. It is apparent
that
mixtures of different types of liposomes containing a variety of bioactive
materials
may be formulated and embedded in the composition.
F fibrin glue-containing Gposome formulations
As described hereinbelow, liposomes may be suspended in either a
fibrinogen or thrombin solution and stored at temperatures from about
4°C to about
37°C prior to use. Alternatively, individual mixtures of liposomes and
either
fibrinogen or thrombin preparations may be frozen at -70~C or lyophilized by
drying in vacuo at about -30~C. Prior to use, the mixtures are reconstituted
in
water or buffer such as Tris-saline. All of the methods of storage result in
viable,
long-lasting liposome glue compositions following reconstitution of the stored
materials.
In accordance with the invention, liposomes formulated with fibrin
glue in a variety of modes result in liposome-containing fibrin glue in which
liposomes are embedded and deposited in the clotted fibrin glue bioadhesive.
Because fibrin glue forms the environment for deposition of the liposomes, the
glue
localizes the liposomes at the site or sites of application.
An advantage of the fibrin glue of the invention is that it is
physiologically compatible with biological systems for in vivo use, such that
both it
and the liposomes contained therein, provide beneficial effects for the
recipient
~~ without being toxic. Similarly, another advantage of the fibrin glue-
liposome compositions is that the liposome-glue will remain in clotted form in
the
environment in which it is administered or applied due to the formulations of
present compositions of the invention, and will withstand physiological body
temperatures and the conditions of the host environment in vivo. Because they
comprise phospholipids and cholesterol, the liposomes of the composition will
also
be naturally metabolized over time by absorption by cells and tissue (reviewed
by
Schater-Korting M., Korting HC. and Braun-Falco_, O.J., 1989, Amer. Acad.
~a 1 21: 1271-1275). It is clear that the fibrin glue allows for controlled
WO 95/22316 218 3 4 3 0 PCT/US95/01792
-21 -
localization and the release of the contents of the embedded liposomes into
the
desired tissue site.
In one embodiment of the invention, fibrinogen, thrombin, and
liposomes are each stored separately, and then are mixed together when
desired,
for use to form the fibrin glue-liposome composition at the site of the
surgical or
nonsurgical wound or opening. By way of example, lyophilized fibrinogen (about
50-70 mg) and thrombin (about 20-40 il) were each reconstituted in an
appropriate
buffer (e.g., about 1 milliliter of 10 mM Tris-saline, pH 7.4) to form a
fibrinogen
solution and a thrombin solution. Thereafter, about 200 ~cL of liposomes were
added to the fibrinogen solution which was mixed gently to form a fibrinogen-
liposome suspension. The above solutions were formulated in syringes and the
steps to mix the component solutions were carried out in syringes. The
fibrinogen
and liposome suspension were applied simultaneously, along with the thrombin
solution, to the site of the wound. The fibrin glue that formed within minutes
contained about 10 °.b embedded liposomes and provided a liposome-
containing
bioadhesive at the wound site. On average, about 1 °6 to about 20 % (by
volume),
more preferably about 2 % to about 15 ~ (by volume), and most preferably,
about
5 °b to about 10 ~ (by volume) of liposomes were embedded in the fibrin
glue to
produce the fibrin glue and liposome bioadhesive of the invention. The amount
of
liposomes in the composition represent the volume per volume percentage of
liposomes in the final fibrin glue and liposome formulation.
In another embodiment, liposomes are pre-mixed with a solution of
fibrinogen, and stored at 4°C. The liposome and fibrinogen mixture can
be
lyophilized, if desired, prior to storage. Prior to or at the time of use, the
mixture
of liposomes and fibrinogen is warmed to 37~C and mixed with thrombin solution
at the site of injury or opening, thereby forming the fibrin glue composition
in
which the liposomes are entrapped.
In another embodiment, liposomes are pre-mixed with a solution of
thrombin, and stored at 4~C. Prior to or at the time of use, the mixture of
liposomes and thrombin is mixed with reconstituted fibrinogen solution at the
site
of the surgical or nonsurgical wound or opening, thereby forming liposome-
WO 95/22316 2 ~ g 3 4 3 0 PCT/IJS95/01792
-22-
° containing fibrin glue.
In accordance with the invention, the fibrinogen and thrombin
solutions are each stored in separate receptacles or containers (e.g.
syringes) prior
to use or mixing in the fibrin glue-liposome composition. Liposomes may be
suspended in a fibrinogen solution in a first syringe, and then mixed with the
thrombin solution from a second syringe on or around the site of the wound or
opening. Alternatively, liposomes may be suspended in a thrombin solution in a
first syringe, and then mixed with fibrinogen solution from a second syringe
at or
around the wound site or opening.
In vivo and other uses of fibrin glu~containing Gposome composition
The fibrin glue-liposome composition of the present invention may
be used for immediate or sustained release of a biologically active substance
or
moment both in vitro and in vivo. For in vivo use at a surgical or nonsurgical
site, the fibrin glue-liposome composition may be formulated in the number of
ways elucidated above. In brief, the fibrin glue components and liposomes,
however they are pre-mixed, may be added together at or over the wound site at
~e desired time of use. Consequently, the fibrin glue-liposome bioadhesive is
formed in situ following the admixture and administration of all of the
components
at the site. Administration is preferably topical and includes, but is not
limited to,
application on, at, around, or near areas such as eyes, skin, ears, or on
afflictions
such as wounds, burns, surgical and nonsurgical openings, fissures, ulcers,
blisters,
one breaks, and the like. The present invention is particularly useful for
such
treatments in which the release over time of antibiotics or healing,
prophylactic, or
therapeutic medicaments would assist in the healing and recovery process. In
addition, because the biochemical action of fibrin glue mimics a part of a
normal
biological process, the fibrin glue-liposome composition may be used to
promote
hemostasis by controlling hemorrhaging, to seal and bond tissue, and to
support
wound healing. Similarly, the fibrin glue-containing liposome composition may
be
topically administered at the site of burns in which the release of
antimicrobials,
~ll ~°~ factors, and/or medicaments is also of critical importance in
the
WO 95/22316 ~ ~ ~ PCT/US95/01792
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° promotion and speed of the healing process.
Fibrin glue containing liposomes can be also be used to bind bone
fragments. The bone-binding ability of the fibrin glue and liposome
composition is
very useful in bone reconstruction, as in plastic surgery or the repair of
major bone
breaks. For example, a bone fracture can be sealed with the fibrin glue and
liposome composition so that the glue both seals the break and entraps and
localizes the liposomes which are formulated to contain bone-specific growth
factors. Upon slow dissolution of the fibrin glue at the site of the bone
fracture,
the liposomes release their entrapped growth factors and thus improve the rate
and
qty of the healing bone.
For facial reconstruction, autologous bone from a patient can be
ground or made into powder or the like, added to fibrinogen mixed with
liposomes,
and mixed into a paste. Thrombin is then mixed with the fibrinogen and
liposome
paste in an amount sufficient (i. e. , 1 U/mL) to allow the paste to be
applied to the
desired locale, where the fibrin glue and liposome composition finally
congeals.
The amount of time for the congealing of the composition to occur can be
controlled by adjusting the level of thrombin used. The liposomes can be
formulated to contain bone growth factors (for example, as described in
Sampath
T.K. et al., 1992, J. Biol. Chem., 267:20352 and Wang E. et al., 1990, Proc.
Natl. Acad. Sci. USA, 87:220) or antibiotics in their aqueous phases. One
skilled
in the art will appreciate that the types of liposomes (e.g., neutral or
charged) and
the choice of aqueous phase components can be chosen as desired.
Fibrin glue containing liposomes can also be fabricated as a film or
membrane. Such films or membranes are advantageous to cover large surface
areas. In addition, the fibrin glue and liposome compositions can be employed
to
fabricate implantable devices which include not only films, but also foams or
chunks of the congealed fibrin glue and liposome composition. The films and
devices may be formed ex vivo by application as liquids or sprays for
subsequent
implantation or use in vivo after gelation. Such fibrin glue-containing
liposome
compositions may be also be used to coat devices, such as prosthethic devices,
~~e~~~ or valves, and the like, which would be temporarily inserted or
CA 02183430 2005-12-O1
-24-
permanently implanted into an animal or human patient.
For the purposes of the invention, the fibrin glue film (or membrane)
is defined as a thin layer whose thickness can range between 0.1 mm to 5 mm.
Such fibrin glue films exhibit a high degree of viscoelasticity and can be
reversibly
twisted and stretched up to 4 times their initial dimensions before breaking.
Fibrin
glue films containing Type A, B, or C liposomes which entrap biologically
active
compounds within their aqueous phases, are suitable for use in the invention.
For
example, fibrin glue film containing Type A, B, or C liposomes can be sprayed
onto a hydrophobic surface, such as ParafilmTM (American Can Co.) to form a
fibrin
glue and liposome film or membrane, which does not adhere permanently to the
surface. After setting or cross-linking for about 1 hour, the fibrin glue film
formulated with its entrapped liposomes is peeled away from the parafilm
surface
and exhibits physical characteristics virtually identical to fibrin glue film
formulated without liposomes. The combination of fibrin glue film and
liposomes
can impart beneficial biological effects to such films used as described above
for
formation in situ. The results of producing a film comprising the fibrin glue
and
liposome compositions is described in Example 12.
Fibrin glue film containing liposomes can also be used to coat or to
layer over a variety of materials used to make prosthetic devices for
implantation.
In an embodiment of this invention, fibrin glue containing liposomes can be
sprayed or applied as liquid onto a metal surface or other substrate onto
which the
composition adheres tightly. For example, a fibrin glue film containing Type A
liposomes sprayed onto aluminum foil bound very tightly. Alternately, the film
could be formed by layering the fibrin glue and liposome mixture onto the
surface
or substrate. When aluminum foil was used as the substrate, the film (about 1
mm
thick) could not be easily peeled or removed from the aluminum surface (Fig.
10),
while the same film deposited on the a hydrophobic surface was easily removed.
These examples serve to illustrate, but not to limit, the further embodiments
of the
invention in which Types A, B, or C liposomes are incorporated into fibrin
glue
film deposited onto a synthetic surface prior to use in animals or humans.
WO 95/22316 218 3 4 3 0 PCTIUS95101792
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EXAMPLES
The examples herein are meant to exemplify the various aspects of
carrying out the invention and are not intended to limit the invention in any
way.
EXAMPLE 1
Preparation of Fibrin~en from Cryoprecipitate
Although fibrinogen for use in the bioadhesive composition of the
invention may be prepared by several techniques as disclosed above, specific
and
non-limiting examples of fibrinogen preparation are provided. Using
cryoprecipitate as a source of fibrinogen is suitable for formulating the
fibrin glue
and liposome composition of the invention. Alternatively and oftentimes more
preferably, fibrinogen is desired in a more purified or concentrated form, and
thus
is prepared in accordance with the Cohn fractionation method described in
Example
2~
Cryoprecipitate was prepared according to the American Association
of Blood Bank (AABB) protocol as published in Walker, R.H. et al. , 1990,
Technical Manual American Association of Blood Banks, 10th Edition, Arlington,
VA.. Briefly, a unit of fresh-frozen human plasma was placed in a 0-4~C water
bath for 1 to 11h hours (or until thawed). The thawed plasma was centrifuged
at
5000 x g for 5 minutes 4°C. The supernatant was removed, leaving about
10 to
20 mL of plasma mixed with the cryoprecipitate, or concentrated fraction
containing a complex mixture of clotting proteins, including fibrinogen,
albumin,
haemophilic factors, and other proteins (see Table 2). 1fie cryoprecipitate
was
warmed to 37~C for 15 minutes prior to use in the fibrin glue and liposome
composition.
Table 2
Bi~emical Composition of Cryoprecipitate Prepared from Fresh-Frozen
Plasma ("FFP Cryo")
WO 95/22316 ~ PCT/US95/01792
-26-
0
Protein (mg/g) 60-80
Fibrinogen (mg/g) 9-30
1gG (mg/g) 10-14
Factor XIII (U/g) 4-9
Mean values n =1 a
One skilled in the art will appreciate that cryoprecipitate prepared
from fresh-frozen plasma is suitable for use in the bioadhesive composition
~n~g liPosomes, as is a more purified preparation of fibrinogen such as that
obtained from, but not limited to, the Fraction I paste of the Cohen
fractionation
method as described above and known to those in the art.
EXAMPLE 2
Preparation of Fibrinogen from Fraction I Paste of Cohen Fractionation
Fibrinogen was prepared from Fraction I paste cold ethanol plasma
fractionation (Blomback, B. and Blomback, M., 1956, Ark Kemi, 10:415-443;
Stryker, M.H. & Waldman, A.A., 1978, Kirk-Othmer Encyclopedia of Chemical
T~~ologv, Vol 4, 3rd ed., pp 25-61, John Wiley; Lowe G.D.O. et al., 1987,
Fibrinogen 2: Biochemistry, Physiology and Clinical Relevance. Excerpts
Medicus,
Elsevier Science Publishers). The Fraction I paste was slurried in cold (4~C)
Tris-
saline buffer containing 2 mM EDTA, pH 6.5, and the supernatant discarded. The
residual paste was dissolved in warm (37~C) Tris-saline buffer containing 2 mM
EDTA to form a solution, and the solution was either filtered or centrifuged
at
5000 x g for 15 minutes. The solution was cooled to 14~C. Cold 50~ ethanol
was then added slowly and the ethanol-containing mixture was cooled to 4~C
over
a 1 hour period. The precipitated fibrinogen was collected by centrifugation
at
3000 x g for 15 minutes, and was dissolved in Tris-saline buffer, pH 7.4.
Following this step, the purified fibrinogen was either stored at -30~C or was
lyophilized. Thawed fibrinogen or fibrinogen reconstituted by adding water or
buffer was mixed with liposomes of Types A, B, C, or D, as desired, without
p~ipitation or gelling of the resulting fibrinogen and liposome mixture.
CA 02183430 2005-12-O1
-27-
° EXAMPLE 3
P~,aration of Viiall~r-Inactivated ~VI), Fibrinogen and Thrombin
Viral inactivation of Lipid-coat viruses was achieved by employing
the solvent detergent (SD) method. To achieve SD viral inactivation, 1 ~ Tween
80TM (Triton X-I OOTM, sodium cholate, or other nonionic detergents may also
be used
and 0.3 °& tri(n-butyl)phosphate (TNBP) were added to the fibrinogen
preparation
and kept at 24-30 °C for 4 hours to result in SD fibriteogen. In the
Fraction I
paste method, viral inactivation was performed prior to precipitation with 7-
10 ~ ,
by volume, of cold ethanol. The TNBP solvent and Tween 80TM detergent reagents
were removed by repeated (2 times) precipitation of the fibrinogen with 7-10 ~
, by
volume, of cold ethanol (Burnouf Radosevich et al., 1990, Vox Sane, 58:77-84)
and resolubilized in a buffer of physiologic pIi and ionic strength. The
complete
SD procedure resulted in acceptable vital kill (on the order of greater than
lOs
logs) and Low amounts of residual viral inactivating reagents in the final SD
fibrinogen preparation.
Laboratory scale preparations of fibrinogen, including SD
fibrinogen, for use in the bioadhesive liposome and fibrin glue compositions
typically contained the following constituents as indicated. The fibrinogen
constituents are provided as a guide and are not meant to limit the invention
in any
way. For example: Fibrinogen (Fib): 20-80 mg/mL; Factor XIIi (FX~: 2-12
U/mL; Fibronectin (FIB: < 1 ~; Albumin (Alb): < 19to.
Bovine or human thrombin were employed for inducing gelation or
clotting of fibrin glue. Those skilled in the art will appreciate that, at the
present
time in the U.S., bovine thrombin is commercially available and is the type of
thrombin that is licensed for clinical use in the U.S. However, human thrombin
sources are also suitable for use in the invention provided that the human
thrombin
is appropriately purified and virally inactivated.
The human thrombin used for instigating cross-linking of the
components of the fibrin glue in the invention was obtained by activating
prothrombin from Cohn Fraction III paste by established techniques (Fenton II,
J.W. et al., 1977, J. Biol. Chem., 252:3587-98; Crowley, C., European Patent
CA 02183430 2005-12-O1
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° Application No. 0 439 156 Al; U.S. Patent No. 4,696,812 to Silbering
S.B. et al.
(1987); U.S. Patent No. 4,965,023 to Silbering S.B. et al., (1990)). Briefly,
prothrombin, prepared fmm Cohn fraction III paste, was activated by incubation
with 20-30 mM Ca(II) and a prothrombin activating amount of thromboplastin.
The resulting solution was filtered and passed over a DEAE-SepharoseTM column
to
remove contaminating proteins. The eluate was then passed over a CM-
SepharoseTM
column and the eluate was discarded. The bound thrombin was eluted with higher
ionic strength buffer (e.g. 0.5 N NaCl in PBS, pH 6.5 to 8.0). Other
variations of
this process have been described (see Crowley et al., European Patent
Application
No. 0 439 156 Al). Other methods of activating prothrombin may be employed.
The resulting purified thrombin can be virally inactivated by a variety of
methods
including SD, heat, or UV irradiation, with or without quenchers.
The purified thrombin maintained its enzymatic activity, even following viral
salivation treatment. In addition, mixing purified thrombin alone with
liposomes
of Types A-C as disclosed herein did not significantly alter its enzymatic or
clot-
inducing activity.
When mixed together, the virally inactivated fibrinogen and
thrombin components became coagulated and formed fibrin glue.
EXAMPLE 4
~aration of Li somes
To prepare liposomes containing a different lipids and cholesterol via
the ethanol injection technique, equimolar quantities of cholesterol (Chol)
and
hydrogenated lecithin (HI.) were dissolved in 100 ~,I. of absolute ethanol 100
~cL
chloroform and mixed at 60°C for 10 minutes and then injected into a 10-
fold
larger concentration of Tris-saline buffer, pH 7.4, containing the material to
be
entrapped in the aqueous phase of the liposome. Some liposomes were also made
with stearyl amine (B) or stearic acid (C) or diethylstearylamine added to the
alcohol phase in one-tenth molar quantifies relative to the amounts of Chol
and HL
used: Following the addition of all of the liposome reagents to the aqueous
phase,
~e mixture was incubated an additional 1 hour at 60°C, and then treated
in an
WO 95/22316 218 3 4 3 0 PCT/US95/01792
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° ultrasonic bath for 5 minutes. After ultrasonification, liposomes
were cooled to
22°C for 1 hour, centrifuged at 2000 x g for 10 minutes, washed in Tris-
saline
buffer, pH 7.4, and re-centrifuged two times more before storage at
4°C.
Evaluation of the prepared liposomes in a Coulter particle sizer (the Coulter
Company) showed unimodal distribution of particles with a diameter range of
from
about 0.9 to 10 microns, with a mean diameter of about 2.5 microns (Fig. 1)
EXAMPLE 5
Preparation of "Neutral" or "Tape A" Li spo omen
For the preparation of neutral or type A liposomes containing zinc,
160 milligrams (mg) of hydrogenated phosphatidylcholine (HPC) or L-a-lecithin,
I;Avanti Polar Lipids. Birmingham. AL) were mixed with 40 mg of cholesterol
(Chol), absolute ethanol (100 ~cL), and chloroform (100 ~,L) and incubated at
60°C
for 15 minutes. For preparation of exogenous materials to be entrapped in the
liposome, a solution of 5 mL of aqueous buffer (e.g. Tris-saline buffer: 20 mM
Tris, 0.15 N NaCI, pH 7.4) containing the material to be entrapped (e.g. 2 mM
zinc chloride) was warmed to 60°C. While this solution was stirred with
a
magnetic stir-bar, the HPC and Chol solution was added and vortexed for about
1
minute. The resulting mixture was incubated at 60°C for about 1 hour..
The
mixture was then cooled to 22°C and centrifuged at 2000 x g for 5
minutes to
settle the liposomes to the bottom of the tube. The supernatant solution was
removed after centrifugation, and the liposomes were washed in Tris-saline
buffer,
pH ~.4. After washing, the liposomes were again centrifuged and the wash
supernatant was removed. The prepared liposomes were analyzed in a cell
counter
or particle analyzer to determine that their size (i.e. in terms of liposome
volume)
was in the range of about 4 to about 12 fL, with an mean of about 7 fL. The
washed liposome zinc content was determined by x-ray fluorescence
spectrometry.
A liposome suspension, in which liposomes constituted 14 °6 of the
volume,
resulted in a zinc value of 20.5 ppm zinc compared with the wash buffer
control
supernatant which gave a value of around 3 ppm zinc. The prepared neutral
~°s°mes were stored at 4°C until use.
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° EXAMPLE 6
Preparation of "Amine" or "Type B" Liposomes
For the preparation of amine or type B liposomes, 160 milligrams
(mg) of hydrogenated phosphatidylcholine (HPC) or lecithin, (Avanti Polar
Lipids,
Birmingham, AL) were mixed with 40 mg of cholesterol (Chol), and 2.7 mg of
stearyl amine, absolute ethanol (100 ~cL), and chloroform (100 ~cL). The
resulting
mixture was incubated at 60°C for 15 minutes. For preparation of
exogenous
materials to be entrapped in the liposome, a solution of 5 mL of aqueous
buffer
(e.g. 20 mM Tris, 0.15 N NaCI, pH 7.4) containing the material to be entrapped
(e.g. zinc chloride) was warmed to 60°C. While this solution was
stirred with a
magnetic stir-bar, the HPC and Chol solution was added and vortexed for about
1
minute. The resulting mixture was incubated at 60°C for about 1 hour.
The
mixture was then cooled to 22°C and centrifuged at 2000 x g for 5
minutes to
settle the liposomes to the bottom of the tube. The supernatant solution was
removed after centrifugation and the liposomes were washed in Tris-saline
buffer,
pH 7.4. After washing, the liposomes were again centrifuged and the wash
supernatant was removed. The prepared liposomes were analyzed in a cell
counter
or particle analyzer to determine that their size (i.e. in terms of liposome
volume)
was in the range of about 4 to about 12 fL, with an mean of about 7 fL. The
prepared amine liposomes were stored at 4°C until use.
EXAMPLE 7
prep~tion of "Carbo_xylic acid" or "Tape C" Li spo omen
For the preparation of carboxylic acid or type C liposomes, 160
milligrams (mg) of hydrogenated phosphatidylcholine (IBC) or lecithin, Avanti
Polar Lipids. Birmingham. ALl were mixed with 40 mg of cholesterol (Chol), and
2.7 mg of stearic acid, absolute ethanol (100 ~,L), and chloroform (100 ~cL).
The
resulting mixture was incubated at 60°C for 15 minutes. For preparation
of
exogenous materials to be entrapped in the liposome, a solution of 5 mL of
aqueous buffer (e.g. Tris-saline buffer: 20 mM Tris, 0.15 N NaCI, pH 7.4)
contaitung the material to be entrapped (e.g. 2 mM zinc chloride) was warmed
to
WO 95/22316 ~ ~ PCT/LJS95/01792
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60°C. While this solution was stirred with a magnetic stir-bar, the HPC
and Chol
solution mixture was added and vortexed for about 1 minute. The final mixture
was incubated at 60°C for about 1 hour, cooled to 22°C, and
centrifuged at 2000 x
g for 5 minutes to settle the liposomes to the bottom of the tube. The
supernatant
solution was removed after centrifugation and the liposomes were washed in
Tris/saline pH 7.4 buffer. After washing, the liposomes were again centrifuged
and the wash supernatant was removed. The prepared liposomes were analyzed in
a cell counter or particle analyzer to determine that their size (i.e. in
terms of
liposome volume) was in the range of about 4 to about 12 fL, with an mean of
abut 7 fL. The prepared carboxylic acid liposomes were stored at 4°C
until use.
F.XA1VIPLE 8
Effects of Liposomes on the Breaking Strength. BS. (or Tensile Stren tg-hl of
Fibrin
Glue
The breaking strength of the fibrin glue in the present fibrin glue and
liposome composition was measured by mixing fibrin glue components in a
plastic
test tube and pipeting the still-liquid mixture into the interface of two
pieces of
coarse synthetic mesh (0.4 thick by 1 cm wide), (Fig. 2). The fibrin glue was
bowed to gel, such that the resulting glue as formulated was totally
interwoven
between the two pieces of coarse mesh (Marx, G. and Blankenfeld, A., 1993,
Blood Coag. Fibrin., 4:73-78). After 2 hours, the Factor ~a-induced cross-
linking reaction had occurred and the mesh-fibrin-mesh ensemble was pulled
apart.
The breaking strength was measured as grams per 0.4 cm2 cross-section. In such
a
test system, the breaking strength of fibrin glue that had been activated with
thrombin exhibited a linear correlation with the concentration of fibrinogen,
i. e. ,
[Fib], as described by the following equation:
BS = slope x [Fib]
The effects of ionic strength and pH on fibrin glue breaking strength were
examined. It was found that breaking strength plateaued above 0.1 N NaCI and
was maximum at pH 7.4. It was also determined that even using cryoprecipitate,
which is known to be less pure than purified fibrinogen, the breaking strength
was
y rested to the levels of fibrinogen.
WO 95/22316 2 ~ g 3 4 3 p PCT/US95101792
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This system of measuring and determining the breaking or tensile
strength of fibrin glue was useful for measuring the effects of the various
types of
liposomes on the mechanical properties of fibrin glue formed from
cryoprecipitate
or fibrin glue formed using pure fibrinogen (e.g., from Cohen Fraction I
paste),
(Figs. 3, and 4). The results showed that a particular type of liposome was
made
and added to the fibrin glue components to form the fibrin glue composition at
levels which did not significantly affect the mechanical properties and
integrity of
the fibrin glue formulation. Thus, liposomes can be added to the fibrin glue
to
produce the compositions of the invention in which the glue has a particular
m~hanical strength that is suitable for wound and incision closure and
healing.
EXAMPLE 9
Viscoelastic Effect of Liposomes in Fibrin Glue
After the onset of gelation, fibrin glue develops viscoelastic
properties which can be monitored in a thromboelastograph as TEG amplitude.
Tests with Type A, B and C liposomes, as described above, indicated that
depending on the composition and proportions of components in the final fibrin
glue composition, the liposomes may or may not significantly affect the
viscoelasticity of the fibrin glue.
For example, with low levels of thrombin (e.g. 0.5 U/mL final)
mixed with fibrinogen from cryoprecipitate, Types A, B and C liposomes did not
significantly increase the early phase of the development of viscoelasticity.
However, after 60 minutes, Type A and Type C liposomes increased the final
viscoelasticity of the fibrin glue, while Type B liposomes had no significant
effect
(Fig. 5). The results indicate that the amine gmups on the surfaces of Type B
liposomes may diminish the mechanical properties of the fibrin glue, probably
by
interfering with the cross-linking of fibrin instigated by factor XITIa. At
high
levels of thrombin (e.g. )20 U/mL final), coagulation was essentially
instantaneous,
measurable TEG amplitude was maximized within 2 minutes, and no significant
difference could be detected among any of the fibrin glue and liposome
formulations. These results indicate that liposomes with different surface
moieties,
WO 95/22316 ~ ~ ~ PCT/US95/01792
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such as the amine or carboxylic acid groups of the Type B and Type C
liposomes,
respectively, can be formulated, as desired, to affect the mechanical
properties of
fibrin glue as desired or needed in using the bioadhesive liposome-containing
fibrin
glue compositions.
In another series of experiments, the development of viscoelasticity
was measured for pure fibrinogen, i. e. Cohn Fraction I purified fibrinogen,
(final
concentration: 3.6 mg/mL; final volume: 300 uL) mixed with different volumes
(either 50 ~.L or 100 uL) of Type A liposome suspensions. Clotting (i.e.,
gelation)
was initiated by low levels of thrombin (e.g. 0.5 U/mL). Here, a small but
significant increase of the TEG amplitude was observed with added liposome
volume (Fig. 6). These results indicate that fibrin glue may be formulated by
altering the volume of liposomes added in a manner which does not
significantly
interfere with the viscoelastic properties of the final fibrin glue and
liposome
composition.
The ability of liposomes to modulate or not interfere with the
viscoelastic properties of fibrin glue can be advantageous, such as when the
fibrin
glue and liposome composition is used to prepare films or membranes that need
to
rem~ flexible during use, or when the composition is used to coat the surface
of a
prosthetic device which itself flexes or changes shape during its intended
use.
Often, it is desired that such devices, coatings, or membranes be resident in
vivo
for long periods of time. However, normal lytic processes could degrade the
fibrin
glue rather rapidly. For such uses, liposomes could be prepared with
proteolytic
~bitors encapsulated within their aqueous compartments. With the onset of
degradation of the glue, liposomes would be exposed and slowly release their
entrapped proteolytic inhibitors. This process would thereby decrease the rate
of
degradation of the fibrvl glue and liposome film or membrane. Thus, liposomes
would minimally affect and even augment the desired mechanical properties of
fibrin glue and would ultimately increase the effective lifetime of the fibrin
glue
membrane, coating, or film.
E'E 10
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° In Ytvo Animal Studies
Liposome and fibrin glue composition used in wound healing of skin incisions
To demonstrate the utility of liposomes entrapped in fibrin glue and
used in animals, in vivo experiments were performed. For surgical incisions, a
2
cm longitudinal, full skin thickness, paraspinal incision was made on the
dorsal
region of adult (e.g., 6-8 week old) Sprague-Dawley mice. Fascia was cut away
from the skin which was washed with physiological saline and dried with gauze
to
remove any blood from the field. The incision was either stapled or was sealed
with fibrin glue without or with added liposomes which had been prepared to
contain entrapped zinc as a type of bioadditive in accordance with the
invention.
Essentially, the Type A liposomes (i.e., neutral) were prepared by
injecting a warmed solution of cholesterol and lecithin into Tris-saline
buffer, pH
7.4, which contained 2 mM Zn(11) salt, and the resulting liposomes were
incubated
and washed as described. A Zn(11) solution entrapped in liposomes was used as
an
exemplary bioadditive in the aqueous compartment of the liposome. One skilled
in
the art will appreciate that other bioadditives, and solutions containing such
additives, are equally and particularly suitable for entrapment in the
liposomes of
the composition, as described in the Detailed Description of the Invention.
The
Zn(1T)-loaded liposomes were analyzed by X-ray fluorescence and found to have
encapsulated about 10-20 % aqueous phase of their total volume. The Zn(11)-
loaded
liposomes were added to fibrinogen in cryoprecipitate (10 % by volume) prior
to
mixing with thrombin at the site of incision, i.e., forming a fibrin glue and
Zn(ll)-
loaded liposome matrix at the wound site.
The animals were allowed to heal and were sacrificed after 14 days.
The skin incisions that had been sealed either with the fibrin glue and
liposome-
containing composition or with staples were excised and analyzed. The incision
area was analyzed for the presence of zinc in the scar area utilizing the x-
ray
fluorescence technique exactly as described in the reference by Gorodetsky R.
,
Sheskin J. , and Weinreb, 1986, Int. J. Determatol. 25:440-445. In contrast to
the
control stapled wound that contained 6 t 2 ppm zinc as a normal background
level, the wound tissue to which fibrin glue with zinc-entrapped liposomes had
WO 95/22316 ~ ~ PCTIiTS95101792
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been applied, contained double the zinc level (i. e. , 15 t 2 ppm) . The level
of
zinc found at the site of the stapled wound and at the site of application of
the
fibrin glue and zinc-containing liposome composition was compared with the
level
of zinc normally present in a non-cut region of the animal's skin or tissue.
The
results showed that a significantly increased level of zinc was released from
liposomes relative to that of the normal controls and of normal undamaged
skin.
This experiment demonstrated that the liposomes delivered zinc (or
other entrapped materials) to the tissue of an animal with a healing incision.
These
data showed that liposomes entrapped in fibrin glue delivered their
encapsulated
aqueous contents to a tissue site without interfering with the adhesion and
sealing
functions of the fibrin glue. Both the encapsulation of bioactive material in
the
liposomes and their fixation at a tissue site by fibrin glue were demonstrated
to
work in accordance with the invention as described.
EXAMPLE 11
Woundin~~ and Tensile Strength of Fibrin Glue at Wound Site
On the same tissue that was assayed for the presence of zinc, an
analysis of the breaking strength of the healed wound incision was performed
concomitantly. Mice (C3H strain) were shaved and a full depth incision
(approximately 2 cm in length) was made dorsally. The wound cavity was sealed
with fibrin glue: 1 mL fibrinogen (20 mg/ml) was placed in one syringe and 1
mL
thrombin 0.5 U/ml, 2 mM Ca(In and up to 200 tcL liposomes were placed in a
send syringe. The source of fibrinogen for preparation of the fibrin glue was
from cryoprecipitate. As a control, fibrin glue was also formulated in the
absence
of liposomes and used to seal the incision. The contents of the syringes were
released at the site of the incision to formulate the fibrin glue with or
without
added liposomes in situ. In other control animals, an incision was made as
above,
and the wound was closed with 4 surgical staples which were removed after 3
days. All of the mice were sacrificed after 3 days. A square of skin around
the
healed wound or control incision was excised immediately after sacrifice and
was
sliced perpendicular to the wound into 8 equivalent sections with a multiblade
razor
CA 02183430 2005-12-O1
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apparatus. The wound tensile strength (WTS) of the strips was measured in an
Accuforce M 100 Tensile Strength Apparatus (AmetekTM), with the values
expressed
in grams per 2 mm wide strip. Each point generally represented the mean of
about
28 to 42 measurements (e.g. 7 wound strips in 4 to 6 mice) with error bars
representing the standard error of the mean (SEM). The WTS results obtained
from control animals with stapled incisions, from contml animals with wounds
sealed with fibrin glue formulated without liposomes, and from experimental
animals with wounds sealed with fibrin glue formulated with liposomes were
compared (Fig. 'n. These findings indicate that liposomes can augument the
wound healing properties of fibrin glue.
EXAMPLE 12
Films or Membranes Produced by Fibrin Glue and Liposome Compositions
The addition of liposomes to fibrin glue can significantly and
advantageously modify the physical characteristics of the fiLn or membrane
formed
from the fibrin glue and liposome composition as described herein. For
example, a
2 mm thick by 1 cm wide fibrin glue film made from fibrin glue containing 28
mg/mL fibrinogen, 10 U/mL thrombin, 15 mM Ca(In solution, and liposomes
( 10 ~ by volume) exhibited a breaking strength of 11 grams and became
elongated
by more than 200 °& . For convenience, the components used to formulate
the fibrin
glue and liposome composition were placed in an appropriate r eceptacle or
container and sprayed onto the film or membrane; the spraying process mixed
the
~mponents prior to their application to the substrate. After gelling or
coagulation
of the fibrin glue and liposome composition, the film or membrane of fibrin
glue
and liposomes was cleanly peeled away from the substrate film or membrane
without the problems of sticking to the substrate or breakage during removal.
For
synthetic surfaces onto which the fibrin glue containing liposome film does
adhere,
the mechanism of adherence has not been completely elucidated, although ionic
interactions are presumably involved. For films formulated with 45 mg/mL
fibrinogen, 10 U/mL thrombin, and 15 mM Ca(~, an increase in film breaking
strength is noted (e.g., to 18 g) (Fig. 8). In addition, Type A liposomes
(i.e.,
CA 02183430 2005-12-O1
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° neutral) and Type C liposomes (i.e., carboxylic acid) formulated in
the fibrin glue
composition increased the relative breaking strength and percent or degree of
elongation, while Type B liposomes (i.e., amine) decreased the relative
breaking
strength and the percent of elongation before breaking (Figs. 8 and 9). This
example demonstrates that liposomes incorporated into fibrin glue films can
modulate its physical parameters in a controlled manner, and also indicates
that
films to be used as wound dressings or membrane devices can be fabricated from
fibrin glue films which contain liposomes.
E~LE 13
Fibrin Glue and Liposome Composition for Sealing Bone Breaks
To illustrate the technique of using the fibrin glue containing
liposome composition to seal and repair bone breaks, 50 mg of sheep femur bone
fm~ents (not longer than 2 mIvn were mixed with fibrin glue composed of 1 mL
of 50 mg/mL fibrinogen without or with 5 ~ (by volume) Type A liposomes, 300
uL of thrombin (10 U/mL), and 50 mM Ca(I>7. The fibrin glue, liposomes, and
bone matrix was allowed to set for 1 hour and the breaking strength (BS) was
measured using the techniques described above. The results indicated that Type
A
liposomes did not significantly decrease the mechanical properties of the
fibrin glue
and liposome composition which had been admixed with bone fragments (see Fig.
12).
Although the foregoing invention has been described in some detail
by way of illustration and example for purposes of clarity of understanding,
it will
be obvious to those skilled in the art that certain changes and modifications
may be
practiced without departing from the spirit and scope thereof, as described in
the
specification and as defined in the appended claims.
