Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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MICROPARTICLES AND THEIR USE IN WOUND THERAPY
Field of the Invention
This invention relates to microparticles that may be
produced by spray-drying, and to their therapeutic use. In
particular, the invention relates to fibrin sealant.
Backcrround of the Invention
Fibrin sealant is a biological adhesive composed of
fibrinogen and thrombin which is used extensively to assist
wound healing and to provide sutureless closure of surgical
wounds. Fibrinogen is the main structural protein in
blood, responsible for forming clots.
For clot formation to occur, fibrinogen must be
proteolytically cleaved and converted into fibrin monomer
by thrombin, a serine protease that is converted to its
active form by Factor Xa. Fibrin monomers assemble into
fibrils and eventually form fibres in a three-dimensional
network. The formation of a clot also requires the
activity of Factor XIII. Factor XIII is a serine protease
which is converted to its active form by thrombin in the
presence of calcium. Activated Factor XIII (FXIIIa) then
converts the non-covalent bonds between the assembled
fibrin monomers into covalent bonds by transamination.
This renders the fibrin gel less susceptible to proteolytic
digestion by plasmin and also increases the overall
strength and stiffness of the gel. Fibrin gel is readily
resorbed by enzymatic and phagocytic pathways.
To reproduce this coagulation process in the form of
a biological adhesive, the fibrinogen component of fibrin
. sealant usually contains Factor XIII, and the thrombin
component is prepared in calcium chloride solution. The
two components are applied either sequentially or
simultaneously to the repair site, typically by syringe or
by spraying. Fibrin sealant readily adheres to wet
surfaces and, once polymerised, becomes a semi-rigid,
haemostatic, fluid-tight adhesive capable of holding
tissues or materials in the desired configuration.
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Currently, fibrin sealant products are applied to the
wound site as solutions, e.g. using a dual syringe device,
which mixes the fibrinogen and thrombin as they exit. The
main drawback of such delivery systems is clot formation
within the device, resulting in needle and tube blockages.
_.. The dual syringe systems are also awkward to fill and
manipulate. Further, if there is inadequate mixing of the
fibrinogen and thrombin solutions, only weak clots may
form.
Many wound sites ooze, and this can result in
significant accumulation of fluid at the site. When
solutions of the components of fibrin sealant are applied
to such sites, they are often flushed away.
US-A-4427651 discloses a sprayable preparation for
accelerated haemostasis and optimised biochemical control
of wound closure, containing a powdery mixture of 15-60% by
weight of thrombin, 5-80% by weight of a desiccating and
stabilising agent (albumin, globulin and/or fibrinogen),
and 1-10 % by weight of fibrinolysis inhibitor. The powdery
mixture is suspended in a low-boiling, anhydrous solvent
which is used as a propellant.
WO-A-9213495 discloses a lyophilised fibrinogen powder
prepared by precipitation, without control of particle
size. The powder is usually hydrated prior to use, with
the disadvantages described above. It is also proposed
that the powder can be used directly when the vessel or
wound to be closed is small, and the blood loss is not
rapid. In this case, the reaction is dependent on the
. presence of endogenous thrombin. In larger wounds, the
heavier blood flow will wash away the endogenous material,
and clotting will not take place.
Summary of the Invention
It has now been realised that spray-drying is useful
as a means to give novel, soluble microparticles (including
microcapsules) comprising fibrinogen or thrombin.
Respective fibrinogen-containing and thrombin-
containing soluble microparticles can be formulated
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together, in stable, dry form. This formulation can be
subsequently activated, as desired, to give a fibrin
sealant that is useful in wound therapy and surgical
repair. It can meet the primary objectives of achieving
good flow properties, enhanced, effective delivery to the
active site, and dissolution only at the site, not in the
delivery system.
Describtion of the Invention
Microparticles comprising fibrinogen or thrombin may
be prepared by the procedures described in WO-A-9218164,
WO-A-9609814 and WO-A-9618388. These spray-drying and
associated particle manipulation processes enable the
production of soluble protein microcapsules with defined
size distribution, e.g. of up to 50 ~m in diameter. For
example, as described in those documents, the
microparticles may be produced reproducibly, e.g. with 90%
or more (by mass) up to 20 ~,m, e.g. 1 to ZO ~,m, or up to 5
~,m, in size, or of submicron size if desired.
Microparticles of the invention may be prepared by
spray-drying a solution of the active component alone. An
alternative procedure comprises co-spray-drying, in which
fibrinogen or thrombin and another wall-forming material
are formulated and spray-dried, to give a microparticle in
which the active component is incorporated in the wall of
the particle.
The fibrinogen or thrombin may be full-length or any
active fragment thereof. Fragments are known; sse Coller
et a1, J. Clin. Invest. 89:546-555 (1992). Fibrinogen raw
material may be a frozen solution, although a lyophilised
powder which requires reconstitution prior to spray-drying
may be used.
The spray-drying of proteins in the presence of
excipients such as sugars (e.g. sucrose, lactose or
mannitol) or other proteins stabilises the protein of
interest and also ensures its effective dilution where low
doses are required. The sugar may have a beneficial
effect, in wound therapy.
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Suitable other proteins may be naturally-occurring or
recombinant. They may act as "wall-forming materials", as
described in WO-A-9218164, where various examples are
given. A preferred material is HSA (human serum albumin).
For example, fibrinogen is spray-dried alone or in the
presence of varying amounts of excipients such as HSA (e. g.
fibrinogen: HSA ratios of 1:1, 1:3, 3:1) and sugars (e. g.
mannitol).
The microparticles of this invention may have the
physical characteristics described in the three
publications identified above, e.g. being smooth and
spherical, although size is not so critical since
respirability is not a consideration. Known conditions can
be used to produce, for example, microparticles of, say, c.
1.5 ,um or c. 10 ~,m diameter, which can ensure optimum
recovery of either of the two proteins of interest.
It has been found that microparticles produced by
spray-drying fibrinogen are surprisingly active and
soluble. The use of spray-dried fibrinogen preparations
may therefore be particularly advantageous in the hospital
setting, where the solubilisation of freeze-dried
fibrinogen can take 25 minutes or more, and usually
requires heating. This is a rate-limiting step, and can
cause considerable delay in the administration of fibrin
sealant preparations to patients.
The concentration of the thrombin component in fibrin
sealant is relatively low (e.g. 150 ~g thrombin per 40 mg
fibrinogen). Preferably, therefore, thrombin is spray-
dried with excipients such as HSA, sucrose, lactose or
mannitol in varying proportions. This provides a
homogeneous formulation, as described in more detail in
Application No. PCT/GB97/00953.
Calcium ion, e.g. as calcium chloride, may be
incorporated in the thrombin feedstock. Alternatively,
calcium chloride may be added to the microcapsules after
processing.
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Microparticles of the invention may be sterilised, if
necessary or desired. Sterile processing, ~y-irradiation
and ethylene oxide are examples of suitable techniques.
Although the components of the microcapsules in a
5 fibrin sealant of the invention are preferably water
soluble, and the microparticles are preferably obtained by
spray-drying a suitable solution, the microparticles that
are obtainable may be free-flowing, discrete and
substantially anhydrous. This means that the components of
fibrin sealant in accordance with this invention are not
activated until they are wetted, e.g. by coming into
contact with liquid at a wound site. The active components
may therefore be delivered as a dry mixture, although
separate application of the different microparticles is
also envisaged.
A dry powder fibrin sealant product may be of
particular value where application to a large surface area
is required. This includes surgery and repair of traumatic
injuries to various organs such as the liver and spleen.
A further advantageous application is in skin grafting for
burns patients, and specifically where skin epidermal
sheets are cultured in vitro and then transferred to the
wound site. The use of fibrin sealant in the latter
indication has proved to be particularly effective in
patients with extensive burns, providing a biocompatible
anchorage for skin grafts. It may also be suitable in the
treatment of topical ulcers.
Products of the invention may be substantially dry.
This means that they can be formulated with absorbent
materials which, inter alia, can have the advantage over
liquid fibrin sealants of drying and concentrating the
product at the site of action, e.g. for haemostasis. A
suitable such material is carboxymethylcellulose.
A NO scavenger may also be included in the formulation
or, more generally, any material that promotes the
aggregation of clots, inhibits their breakdown, or inhibits
fibrin lysis. A material such as albumin has SH groups.
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These may remove NO from the site of aggregation, and thus
increase clot formation.
As described in more detail in WO-A-9609814, spray
dried microparticles may retain functional groups available
for the binding of therapeutic agents. In this invention,
a drug may be bound to the microparticles, if desired at
the site of application. Thus, for example, a cytotoxic
drug may be used where it is desired to treat skin cancer.
Other drugs that may be included in products of the
invention, e.g. those containing fibrinogen, are
coagulation factors such as Factors VII, VIII, IX, X and
XIII, and von Willebrand's factor. This may be
incorporated by co-spray-drying.
It has recently been observed that denatured albumin
microbubbles preferentially attach themselves to damaged
endothelium. This suggests that products of the invention
will accumulate at wound sites, not only because of the
activation of fibrinogen but also if there is an albumin
component of the microparticle.
The following Example illustrates the invention.
Example
A fibrin sealant was prepared. This comprised a dry
powder blend of microparticles respectively comprising
fibrinogen and thrombin.
Fibrinogen (SNBTS) was formulated with 600 mg sucrose.
Spray-drying was performed using a Mini Spray Dryer with a
collecting vessel. The conditions were as follows:
Inlet Temperature: 100°C
Outlet Temperature: 65°C
Atomisation Pressure: 1.0 bar
Atomisation Type: Schlick 970/0
Feed Rate: 1 g/min
A 20% final excipient loading was achieved. The
activity detected using a kinetic assay was 13.88 mg/100
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mg. The theoretical activity is 10 mg/100 mg. This
indicated full retention of the fibrinogen bioactivity.
Separately, 1 g D-mannitol (Roquette, ESEX4) was
dissolved in 10 ml of 40 mM CaCl2. The resultant solution
was used to reconstitute 1 vial of thrombin (SNBTS). The
spray-drying conditions used were as above, except that the
outlet temperature was c. 62°C, and the feed rate was
reduced to 0.75 g/min.
A thrombin clotting assay revealed a thrombin activity
l0 of 91.86 units/l00 mg. This compared favourably with the
theoretical activity, of 93 units/100 mg.
The respective microparticles containing fibrinogen
and thrombin were mixed to form a 50:50 blend, in a glass
vial. The vial was placed on a roller mixer for 20 min.
The blend was evaluated in a meat adhesion assay, in
various blend sizes. Each assay requires two sections of
liver (2.5 cm x 2.5 cm). One liver section is stapled to
a piece of cardboard. Both sections are wrapped in
aluminium foil and incubated at 37°C for 20 minutes.
The loose liver section is threaded with cotton. A
solution of 5o human serum albumin is applied to the
surface of both liver sections, followed by the dry powder
blend of fibrinogen and thrombin ( fibrin sealant) . The two
liver sections are placed together and incubated at 37°C
for 10 minutes.
The liver sections are then suspended from a clamp,
and a hook is attached to the cotton. Weights are placed
on the hook and the total weight suspended is used to
calculate the tensile strength of the dry powder fibrin
sealant blend in mg/mmZ. Results are given in the
following Table.
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Blend Size Fibrinogen Thrombin Tensile
(mg) (mg) (units) Streng~h
m mm
0 0 0 0
100 5 45 29.6
200 10 90 23.7
300 15 135 36.0
400 20 180 46.4
500 25 225 50.6
