Note: Descriptions are shown in the official language in which they were submitted.
wo s3/06sss 2 1 2 10 2 8 Pcr/DKs2/002s6
HEMt:)STATIC COMPOSlTiON FOR LOCAL HEMOSTASIS
FIELD OF INVENTION
The present invention relates to a method for arresting local bleedings by topical use
of FVlla and a hemostatic composition containing FVlla.
5 B~CKGROUND OF THE INVENTION
When blood vessels are injured by physical traumas including surgical interventions
bleeding will occur. If bleedings are left alone they will ev~ntually be arrested by a
normally occurring physiolo~ical process characterized by a chain of events
involving the combined activity of vascular, platelet, and plasma factors, leading to
10 the formation of a blood clot. This process is referred to as physiological
hemostasis (blood coagulation3, which is describ~d in details below. In the case of
a minor superficial bleeding this physiological hemostasis is adequate for the arrest.
There are two separate systsms which can promote blood coagulation. These
systems are referred to as the intrinsic and the extrinsic coagulation pathways.
15 In the intrinsic pathway, only blood clotting factors present in piasma are u~ilized.
An intermediate event in the intrinsic pathway is the activation of Factor IX to Factor
IXa, a reaction catalyzed by Fa~tor Xla and calcium ions. Factor IXa then
participates in the activation of Factor X to Factor Xa in the presence of Factor Vllla,
phospholipid and calcium ions~
20 The extrinsic pathway involves plasma factors as well as components present in
tissue extracts. Factor Vll, a proenzyme present in plasma, participates also in the
WO 93/06855 PCl /DK92/00296
212~ 02Ç~ 2
extrinsic pathway of blood coagulation by converting (upon its activation to Vlla)
Factor X to Xa in the presence of tissue factor and calcium ions.
Factor Xa in turn then converts prothrombin to thrombin in ths pres~nce of Factor
Va, calcium ions and phospholipid. Finally, thrombin converts the plasma fibrinogen
into fibrin, which in the presence of Factor Xllla and calcium ions is cross-linked and
thus forming the blood clot.
Blood factors such as Factor Vlll:C (see US Patents 4,831,119; 4,868,112; 4,886,876;
4,657l894; Re. 32,011 and 4,649,132) and Factor Vlla (see US Patents 4,784,950;
4,382,083; 4,479,938 and 4,357,321) purified from natural sources or made via
10 recombinant techniques have been used for treating patients, such as hemophiliacs,
having blood-clotting deficiencies or inhibi`tors to blood-clotting factors. These
blood-clotting factors have been delivered to the patient needing treatme~t as an
aqueous solution by infusion or bolus injection depending on the blood factor to be
dslivered and the condition of the patient. Cessation of the bleeding is expected to
15 occur typically between 15 minutes to 3 hours or more after the delivery of the
blood-clotting factor.
However, faster arresting of the bleeding is necessary in th~ case of severe
bleedings emerging from more extensive injuries involving larger arteries or when
seeping bleedings occur from larger mucosal surfaces or on cavities without
20 drainage. If the bleeding continues in even a shorter period it may result inextensive losses of blood which may have an adverse effect on the normal function
of the body. Also, in the case of bleeding occurring in osseous non-expandable
cavities, the accumulation of extravasated blood may cause local damages of softtissues due to increased pressure. The usual treatment of such conditions involve
25 the adaption of surgical and/or medical hemostatic measures.
Surgical arrest of bleeding comprises ligation or suture of disrupted blood vessels,
plugging by using tampons in cavities, coagulating tissue surfaces inclu~ing their
wo s3/068ss 2 1 2 1 0 2 8 Pcr/DKg2/002g6
exposed disrupted blood vessels by heated instruments or by the application of
cauterizing agents or heated air.
Surgical hemostasis may also be aided by the appliGation at the injured site of
appropriately sized blocks, plates, or films of biologically absorbable hemostatic
5 sponges. ~:
Pharmaceutical preparations containing bovine thrombin or other blood clotting
factors such as Factor Vlll, Factor Xlll or calcium i~ns are currently used in some
places as hemostatic adjuncts in surgery, said adjuncts being administered e.g. by
spraying a suitable solution thereof onto the site of bleeding such as in US
10 4,298,598. Also textile materials such as gauze or cotton wool fabrics or biologically
absorbable sponges, which prior to the application have been soaked in a solution
of one or more of said hemostatic compounds, are used such as in US 4,363,319.
US patent 2,558,395 discloses a ready-to-use undenatured gelatine hemostatic
sponge containing thrombin. US 4,265,233 discloses wound healing material
15 comprising a structure made from compounds such as gelatine, collagen,
polyglycolic and polylactic acid to which FXIII has been fixed by covalent bir~ding.
EP 277096 A discloses a wound dressing comprising a stable thrombin composition
and a substrate such as hemostaticl porous sponges of collagen or denatured
gelatine and WO 90/13320 discloses a porous sponge containing a hemostatically
20 effective amount of thrombin, and hemostatically effective amounts of one or more
blood coagulation factors other than thrombin. US patent 4,563,387 and US patent4,642,111 relate to, respectively, a methvd and device for tr~ating cancer and which
disclose an anti-cancer drug and a blood coagulation factor being fixed to a
structure, such as a polymer, capable of being delivered by injection to the site of
25 bleeding directly causecl by the cancer treatment.
Japanese published patent application No. 59-116213 discloses an aerosol
containing FXIII and thrombin and Japanese published patent application No.
WO 93/06855 PCr/DK92/00296
212~02~
02-167234 discloses adhesive for living tissues containing fibrinogen, prothrombin,
FVII, FIX, FX, FXIII, antithrombin, protease inhibitor and calcium ions.
In the recent years increasing concern has however arisen regarding the safe useof bovine derived products e.g. thrombin or prothrombin in pharrnaceutical products
5 for human use. Several reports describe th~ possible risk of transmitting an
infectious agent causing Bovine Spongiform Encephalopathy (BSE) in cattle into
humans, where the virus-like agent may be the reason for one or more well known
diseases characterized by degenerative encephalopathy e.g. Creutzfeldt-Jacob
disease and Kuru. Furthermore, clinical investigators have obs~lved thc~ the topical
10 IJse of bovine thrombin in humans may cause the development of an~ibodies
cross-reacting to human thrombin and causing bleeding problems.
It is, therefore) an object of the present invention to provide a safe and,effective
means to topically arrest bleedings at the site of an injury.
SUMMARY OF THE INVENTION
15 The present invention is based on the surprising recognition that FVlla is capable of
momentarily arrest of bleedings when appiied topicaily to the site of injury without
the presence of thrombin or other coaguiation factors and when FVlla is in
association, together with or incorporated into a biologically compatible carrier
(which, as used herein, is intended to include a composition or material) capable of
20 preventing FVlla from being washed away from the site of injury.
According to the present invention, FVlla is incorporated into a biologically
compatible carrier which does not contain thrombin and is unaccornpanied or
uncontaminated by any other blood clotting factors.
wo 93/068~5 2 1 2 1 0 ~ ~ PCI'/DK92/00296
The present invention is thus related to a hemostatic composition comprising a
hemostatically effective amount of FVlla incorporated into biologically compatible
carrier said composition containing no thrombin.
More specifically, this invention provides a hemostatic composition for inducing5 hemostasis at a bleeding wound comprising a hemostatically effective amount ofFVlla which is uncontaminated or unaccompanied by other blood cl~tting factors
and which has sufficient activity alone to produce a hemostatic effect, together with
a biologically compatible carrier which permits said factor Vlla to remain affixed to,
in association with or contacting said wound site.
10 DEFINITIONS
Prior to setting forth the invention, it may be helpful to an understanding thereof to
set forth definition of certain terms to be used hereinafter.
Hemostat or Hemostatic Agent: An agent that arr~sts hemorrhage.
Hemos~atic Composition: A composition that contains a Hemostat or Hemostatic
15 Agent.
Blood clot: The final uutcome of the blood coagulation cascade, formed by
conversion of soluble plasma fibrinogen into insoluble fibrin, which physically stops
the bleeding. The blood clot covers the surface, keeps the wound edges together
and forms the matrix for the following cell proliferation and wound healing
20 Blood clotting factors: Plasma proteins which participate in the blood coagulation
cascade.
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212102~ 6
Activated blood ctotting factors: Blood clotting factors converted to active enzymes
by the action of an activator, often itseif being an activated blood clotting factor.
They are generally designated by the addition of a lower case postscript "a" (e.g.
Factor Vlla).
5 Proenzymes: An enzyme precursor that in general has reduced or no activity as
compared to the mature enzyme.
Biologically absorbable: Material which can be degraded in the body to smaller
molecules having a size which allows transport into the blood stream and gradualremoval from the site of application.
10 S~onge: A porous structure being reticulate and having an inner surface
considerably larger than the outer surface. The porous structure will contain hollow
spaces within the reticulate structure and can absorb many times its own weight in
liquids. -
Covalent binding: A bond between two atoms in which both of the atoms concerned
1~ contribute the elec~ron or electrons.
Dressing: Material apptied to a wound and fastened in place to provide protection
and to promote healing.
Topical: Local.
Biologically Compatible: The ability to be accepted in the body and remain functional
20 for a period without rejection.
Gel: A colloidal system comprising a solid and a liquid phase which exists as a solid
or semisolid mass.
wo 93~068~ 2 1 2 ~ 0 2 ~ pcr/DK92/oo296
Paste: An ointment-like preparation of one or more substances in a hydrogel or fatty
base. It is less greasy and better absorbed than an aintment.
Granule: A minute particle or mass.
Film: Any thin coYering, coating, or layer.
5 Plaster: A substance intended for external application, made of such material and
of such consistency as to adhere to the skin.
Bandage: A strip of gauze, muslin, flannel, or other material used to hold dr~ssings
in place, or to check hemorrhage.
DETAILED DESCRIPTION
10 FVlla is to be used in a hemostatically effective amount. By hemostatically sffective
arnount is mean~ an amount which will preferably cause arrest of the bleeding if kept
in association with or contacting the site of the injury for a sufficient amount of ~ime,
preferably from about 60 seconds in patients not having an impaired hemostatic
mechanism to less than about 10 minutes in patients having an irnpaired hemostatic
15 mechanism. FVlla should be used in an amount ranging from about û.2 to about
2.0 mg, preferably from about 0.5 to about 1.5 mg and more preferably from about0~9 to about 1.1 mg per application.
FVlla may be derived from plasrna as described in EP 00821 82B or by recombinantDNA-technology as described in EP 0200421A. Human purified factor Vlla is
20 preferably made by the methods described by Broze and Majerus, J. Bio. Chem.
255, 4: 1242-1247, 1980, and Hedner and Kisiel, J. Clin. Invest. 71: 1836-1841, 1983.
These methods yield factor Vll without detectable amounts of other blood
coagulation factors.
wo 93/06855 pcr/DKs2/oois6
212~02'~ 8
An even further purified factor Vll preparation may be obtained by including an
additional gel filtration as the final purification step. Factor Vll is then converted into
activated factor Vlla by known means, e.g. by several different plasma proteins, such
as factor X~IA, IXA OR XA. Alternatively, as described by Bjoern et al., ~"Activation
S of Coagulation Factor Vll to Vlla", Research Disclosure 269:564-565, 1986) factor Vll
may be activated by passing it through an ion-exchange chromatography column,
such as MonoQ (Pharmacia Fine Chemicals, Uppsala, Sweden) or the like.
It will be appreciated by those skilled in the art that a suitable factor Vlla for use in
the present invention may also be produced by recombinant DNA technology, e.g.,
10 by insertion of the cDNA or gene encoding factor Vll (Hagen et al., Proc. Natl. Acad.
Sci. USA 83: 2412-2416, 1986) in a suitable vector, transformir~g of suitable cell lines
with the vector and growing the transformed cells in an appropriate medium
whereupon the expressed product is isolated and activated into factor Vlla.
Production of FVII by recombinant DNA technology is also described in US Patent
15 4,784,950 which is incorporated herein by reference in its entirety. Factor Vlla
produced by recombinant DNA technology may be authentic factor Vlla or a more
or less modified factor Vlla, provided that such mod-~ied factor Vlla has substantially
the same biological activity for blood coagulation as authentic factor Vlla. Such
modified factor Vlla may be prepared by modifying the DNA sequence encoding
20 factor Vil sither by altering the amino acid codons or by removal of some of the
amino acid codons in the natural gene by known means, e.g., by site-specific
mutagenesis.
It is evident that the practice of the methods described herein is independent of how
the factor Vlla is derived and, therefore, the present invention is contemplated to
25 cover the use of any factor Vlla preparation suitable for use herein.
The carrier material may be a gel, a paste, a solid or other suitable biologically
compatible/acceptable carrier for topical application of pharmaceuticals or other
biologically active compositions.
W093/0685~ 21 21 0 2 8 PCI'/DK92/00296
The viscosity of the gel or paste will preferably be from about 200 cps to about30,000 cps.
The biologically compatible carrier will typically be made of natural macromolecules
such as gelatine, collagen, alginic acid, cellulose, chitin, fibrinogen, fibrin, fibrin split
5 products, fibronectin, fibronectin fragments, globulin, myoglobulin, casein, keratin,
albumin, polysaccharides e.g. dextrans, glycosaminglycans, agar, pectin, starch or
from chemical modified natural molecules such as denatured gelatine,
alginicacid-alginates e.g. calcium alginate, oxidized cellulose, substituted cellulose
ethers e.g. glycol cellulose, methyl cellulose, ethyl cellulose, hydroxymethyl cellulose,
10 hydroxyethyl cellulose, hydroxypropylmethyl cellulose, substituted cellulose esters
e.g. acetylated cellulose, substituted cellulose ether-esters e.g. acetylated ethyl
cellulose, chit~osan or from synthetic polymers such as vinyl polymers, e.g.
polyacrylic acid, polymethacrylic acid, polyvinyl pyrrolidone and polyviny~alcohol,
polyglycolic acid, polylactic acid, polydextroses or copolymers such as
15 polyo~yethylene-polyoxypropylene copolymers orfrom naturalfibers, syntheticfibers
or mixtures of any of the above materials/compounds.
A solid biologically compatible carrier will preferably be a granule, powder, spo~ge,
tilm~ plaster, surgical dressing or a bandage.
Solid biologically compatible carriers will typically be selected frorn those-already
20 used as hemostats such as modified cellulose, collagen, gelatine, alginate orsynthetic polymers.
The biologically compatible carrier may furthermore contain a fibrinolysis inhibitor,
such as aprotinin, epsilon-aminocaproic acid or tranexamic acid. It may also contain
a stabi!izer, such as naturally occurring amino acids, mono- or disaccharides,
2~ polyglycols, glycerol, proteins or a metal salt, such as calcium salts, and mixtures
thereof. Also buffering salts may be added, such as alkaline metal acetates, alkaiine
metal carbonates or hydrogen carbonates, alkaline metal citrates, alkaline metal
WO 93/06855 PCr/DK92/00296
2~2~0?.8
phosphates or hydrogen phosphates, alkaline metal succinates, imidazole, TRIS, and
zwitteranionic buffering systems, and mixtures thereof. Furthermore, antimicrobial
or bacteriostatic agents, such as antibiotics, sulphonamides, antimycotic agents,
antiviral compounds, and preservatives may be added.
5 The present method and hemostatic composition will be useful for enhancing thearrest of bleedings in several instances of surgical interventions or other injuries such
as in the accidental injury of the skin and/or adjacent tissues or of larger abdominal
organs (liver, spleen, or intestines); in lung surgery; in neurosurgery to prevent
pressure damages of the cerebral or nerve tissues; in orthopedic surgcry during
10 which extensive hemorrhages frequently occur which are difficult to arrest by other
means; in vascular surgery to arrest seepage of blood from the sites of suturing; in
oral or dental surgery such as extraction of teeth; and in nose-bleeding (epistaxis).
- In a ready-to-use product incorporation of FVlla into the carrier material may be
done by various known methods, such as co-precipitation, swelling, dispersion,
15 mixing, soaking, spraying, embedding, injection or a combination thereof.
If the carrier is a gel or a paste, FVlla is preferably incorporated into the c~rrier
material under aseptical conditions. This may be carried out by adding a suitable
solution of FVlla to the carrier material whiCh is then stirre~ gently by suitable means
to obtain a uniform distribution of FVlla within the gei or paste. The FVlla Ioaded
20 carrier material is then transferred to a suitable package form e.g. a tube, a plastic
container or a syringe. Terminal sterilization may be carried out by means of, for
instance, heat or ionizing irradiation.
If the carrier is solid it may be loaded with FVlla by placing the material in a suitable
solution of FVlla for a period sufficient to ensure that the carrier material is25 adequately soaked with the FVlla solution. FVlla may also be incorporated into the
solid carrier by means of spraying, embedding or multiple injections. After vacuum
drying or freeze drying to evaporate excess of water the FVlla impregnated carrier
W093J06855 212 1 0 2 8 PCI`/DK92/00296
is transferred to a suitable package, such as paper bags or a blister paGkage and
terminally sterilized by means of, for instance, heat, ethyleneoxide or ionizingirradiation.
FVlla may be fixed to the carrier by electrostatic interaction between FVlla and the
5 carrier material.
FVlla may also be covalently bound to the carrier by means of chemical crosslinking
reagents, such as bifunctional N-hydroxy succinimide esters or other bifunctional
chemical crosslinking reagents.
Finally, FVlla may be fixed to the carrier by physical means such as absorption,10 dispersion or adsorption.
FVlla may also be added to the carrier just before use, e.g. by spraying a suitable
solution of FVlla onto the carrier material or by embedding the carrier into a FVlla
solution. Alternatively, ths FVlla solution may be injected into the carrier.
A preferred carrier is a biodegradable sponge material known in the prior art ~s15 hemostatic sponges.
Materials for the preparation of hemostatic sponges are conventionally selected from
biodegradable or biologicaJly absorbable compounds such as collagenl gelatine,
chitin, cellulose, polyglycolic acid and polyacetic acid. Such absorbable hemostats
can be left at the site of bleeding even after suturing of internal injuries and will exert
~0 their effect over a period of time, dependent on their water solubility, degradability,
and size.
The characteristics of the above materials may be conditioned by various chemical
or physical treatments resulting in e.g. a preferred improved mechanical strength of
WO 93/0685~ PCr/DK92/00296
21210~.8 12
the structure or in rendering the material less water soluble thereby retarding the rate
of absorption which may extend the period of hemostatic activity.
As an example, gelatine may be denatured by treatment at temperatures in the
range of 100 - 160C for several hours. After such treatment the originally water
5 soluble gelatine will become substantially water insoluble but can still be degraded
to absorbable molecules by proteolytic enzymes present in the body.
In contrast, hemostatic sponges prepared from undenatured gelatine will dissolverather rapidly and turn into a soft gel when brought into contact with aqueous
solutions or bleeding wounds.
10 The FVlla containing dry hemostatic sponge may be prepared either by forming a
foam of undenatured gelatine and FVlla which is thereafter freeze-dried or by
saturating a preformed dried sponge with a solution of FVlla, the wet sponge
thereafter being freeze-dried.
The latter technique implies the possibility to apply water insoluble sponge material
15 which may be advantageQus because Such sponges ret~in their physical.structure
- after application to the site of bleeding for considerably longer tirne than
undenatured sponges.
In a preferred embodiment of the present invention the carrier is a ready-to-usehemostatic sponge to which FVlla has been added prior to packaging and terminal
20 sterilization.
WO 93/06$~iS 2 1 2 1 0 2 8 PCI/DK92/00296
13
EXAMPLES
Example 1:
Four 5 mrn cores of a gelatine sponge (Spongostan commercially available from
Novo Nordisk A/S~ were cut using a punch. Two of these were soaked in sterile
5 water and the other two were soaked in two ml of sterile water in which was
dissolved 1.13 mg of Factor Vlla. The soaking time was approxim~tely five minutes
before application to bleeding sites which were made as described below.
A 450 gram Spraque-Dawley rat was anesthetized with halothane, followed by 0.2
ml/kg of a stock anesthetic solution, which was given intraperitoneally.
10 The rat was placed on a warrning pad and the abdomen was opened udth a long,
mid-line incision to expose the liver. Gut contents were packed with warm salineswabs.
A piece of steel was placed behind the liver to provide a firm bed. Four 5 mm
biopsies were cored through the full thickness of the liver and removed and the four
15 prepared pieces of gelatine spon~ were placed into the holes.
These four sites were observed for 20 minutes and at the end of the time the iiver
was excised and an attempt was made to remove the gelatine plugs by grasping
with fine toothed forceps and pulling gently.
The two sites which were plugged with geiatine sponge plugs impregnated with
20 Factor Vlla stopped bleeding, while the two other sites continued to ooze~ It was not
difficult to remove any of the four plugs from the liver biopsy sites, but it appeared
more difficult to remove those soaked in Factor Vlla.
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2121 028 14
Example 2:
Prior to the surgical intervention, a small piece (45 x 20 x 10 mm) was cut out of a
dry, gelatine sponge (Spongostan Standard~. The size stated was chosen to ensurethat the sponge would exactly cover an incision 25 mm in length with an overlap of
5 10mm.
Also an aqueous solution (1.0 mg/ml) of freeze dried Factor Vlla, containing calcium
ions (concentration of 10.0 mMol~, was made and kept at room temperature prior
to the operation.
In an anaesthetized pig, laparotomy was performed through a midline incision and10 the spleen was delivered into the wound. Incisions were made 3.0 mm deep and 25
mm in length ~sing a special device made from a scalpel mounted with a stop block
and a pattern with a linear groove. The first incision was a control incision left for
free bieeding for 12 minutes to ensure that coagulation did not occur spontaneously.
Another incision was then made 30 mm apart from the first incision and allowed ~o
15 bleed freely ~or 60 seconds. A pieee of gelatin~ sponge was then carefully piace
upon ~he incision and 1.0 ml of Fa~:tor Vlla solution was dropped onto and gently
massaged into the sponge under light finger pressure for 30 seconds. Complete
hemostasis was obtained momentarily.
The test series did also include four different, commercially available hemostatic
20 sponges moistened with an isotonic Sodium chloride solution. The individual time
for hemostasis ranged from 1.8 minutes to 7.5 minutes.
Using the same test procedure bovine thrombin, applied in a watery solution (50 NIH
Units/ml), also provoked momentary hemostasis.
wo 93/06ss~ 2 :1 2 1 0 2 8 pcr/DKs2~oo296
Example 3:
Without being incorporated into a matrix, an aqueous solution of Factor Vlla wasapplied topically as a spray to control venous bleeding from the gallbladder bed and
from abdominal surgical incisions. The investigation was divided into two parts
5 involYing a total of 8 patients. The study was designed as a doubls-blind
randomized placebo controlled study.
Vials containing 562.5 ,I~g of Iyophilized Factor Vlla or placebo preparations
resembling Factor Vlla were reconstituted with 3.7 ml of sterile water immediately
before use and transferred into syringes with sprinkler needles. All 3.7 ml were10 syringed at each administration.
Four patients undergoing cholecystectomy were investigatecl, two receiving Factor
Vlla and two matchlng placebo. After removal of the gallbladder, Fac~or Vlla or
placebo was syringed on to the ~allbladder bed. Efficacy was assessed 2 minutes
later. In each case the efficacy of the pr~paration was rated comparing ooze before
15 and after application.
Four other patients undergoing general elective abdominal surgery were
investigated. Each incision was extended down to but not through the peritoneum
with arterial "spurters" being controlled using the surgeon~s usual technique.
Immediately after the surgical incision the middle of the wound was cover~d with a
20 thick swab and Factor Vlla was syringed on to tne one end of the wound and
matching placebo to the other. Efficacy was assessed 3 minutes after the wound
was syringed. The surgeon judged blindly which half of the wound was bleeding
less.
In these studies, Factor Vlla had no effect in the control of venous bleeding. The
25 likely reason was considered to be that Factor Vlla was washed away from the
wound when applied only in an aqueous solution and not incorporated into a rnatrix
WO 93/06855 PCI /DK92/00296
2 l 21028 16
or a biologically compatible carrier which would have allowed factor Vlla to remain
in contact with the bleeding wound.
The present invention is not to be limited in scope by the above examples since they
are intended as single illustrations of the invention. Indeed, various modifications of
5 the invention in addition to those shown and described herein will become apparent
to those skilted in the art from the foregoing description. Such modifications are
intended to fall within the scope of the appended claims.
