CA2263800C - A thrombin blood fraction for use in a medical procedure - Google Patents
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Abstract
The subject invention relates to the use of thrombin in a medical procedure in an animal.
More specifically, the subject invention relates to such use of thrombin wherein the thrombin is a thrombin blood fraction, as defined hereinbelow.
More specifically, the subject invention relates to such use of thrombin wherein the thrombin is a thrombin blood fraction, as defined hereinbelow.
Description
wo 94/00s66 PCI/GB93/Ot323 A THROMBIN BLOOD FRACTION FOR USE IN A MEDICAL PROCEDURE.
1. FIELD OF TH~ INvENTIoN
The ~ubject invention relates to the use cf thrombin in a medical procedure in an anlmal. More s eciflcally, the subject invention relates to such u~ o~ thrombin ~herein the thrombin is a thro~bin blood fraction, as defined hereinbelow.
1. FIELD OF TH~ INvENTIoN
The ~ubject invention relates to the use cf thrombin in a medical procedure in an anlmal. More s eciflcally, the subject invention relates to such u~ o~ thrombin ~herein the thrombin is a thro~bin blood fraction, as defined hereinbelow.
2. ~CK~,~OUND OF TH~ INVENTION
One ~rh~r im for hemo~tasis, i.e., prevention o~ blood ios~, of an anlmal is the formation of a blood clot. Clot ~ormation, i.e., blood coagulation, occur~ by means of a complex cascade of reactions with the final steps being the conversion of fibrino~en ~y thrombin, calcium ions and activated factor XIII to form the fibrin clot. For a review of the mechanisms o~ blood coagulation and the ~tructure of fi~rinogen, see C.~. Jackson, Ann. Rev.
~iochem., 49:765-811 (1980) and B. Furie and B.C.
Furie, Cell, 53:505-518 (1988).
Thrombin, which is a proteolytic enzyme, is derived from prothro~3bin. Prochrombin i5 converted to t~rombin by calclu~ and prothrombinase.
Prothrombinase is ~ormed th~ough a c~scade of reactions that begins with the proteins factor XI and factor XII.
A fibrin sea~ant is a biolo~ical adhesive whose effect imitate~ the final stages of coagulation, thereby resulting in a fibrin clot. Conventional fibrin sealants generally consist of concentrated human fibrlnogen, bovine aprotinin and factor XIII, as the first component and bo~ine thrombin and ca}clum chloride as t~e fiecond component. Application i~
generally carried out with a double-barrelled syringe, wh~ch permits simultaneous applicatlon of both components to the site where one wants to form the fibrin clot. Aprotinin is a fibrinolytic lnhibitor added to promote stability of fibrin sealants.
S The ~ibrinogen component of the f}brin sealant i~ prepared from pooled human plasma. The ~ibrinogen can be concentrated from the human plasma by cryoprecipitation and precipitation using various reagents, e.g., polyethylene glycol, ether, ethanol, ammonium sulfate or glycine. For an ~xcellent review of f i~rin sea~ant~, $ee M. Brennan, Bl~od Reviews, 5:240-244 t1991); J.W. Gibble and P.N. Ness, Tran~fusion, 30:741-747 (1990); ~. Matras, J. Oral Maxillofac surg., 43:605-611 (1985) and R. Lerner and lS N. Binur, J. o~ Surgical Research, 48:165-181 ~1990).
Recently, there has also been an interest in the preparation of fibrin sealants that utilize autologous fibrin. An autologous fibrin sealant is a fibrin sealant whe~ein the fibrinogen component o~ the fibrin sealant is extracted from the patients own bl~od. The use of an autologous ribrin seAlant is preferred because it eliminates the risk of transmisslon of blood-transmitted infections, e.g., hepati~is B, non ~, non B hepatitis and acquired 2S immune deficiency ~yndrome (AIDS), that could otherwise be present in the fibrinogen component extracted from pooled human plasma. See L.E.
Silberstein et al., Transfusion, 28:319-321 ~1988); K.
Laita~ari a~d J. Luo~onen, Laryngoscope, 99:974-976 (1989) and A. Dresdale et al., The Annals of Thoraclc Surgery, 40:385-387 (1985).
An infection can be transmitted by a fibrin sealant not only by means of the fibrincgen but also by means of the bovine aprotinin and the bovine ~s thrombin component. Bovine thrombin has been known to carry the in~ectious agent bovine spongiform encephalitis (~S~) and o~her viruses pathogenic to m~m~l S .
Furthermore, bo~ine thro~bin i~ a potential antigen, which can cause immllnological reactions in hl-m~n5 Thus, the use o~ bo~ine thrombin could re~ult in the recipient of the bo~ine thro~bin being ad~ersely a~fected. See D M. Taylor, J. of Hospital ln~ection, 18(Supplement A):141-146 ll991), S.B. Prusiner et al., Cornell Vet, 81 No. 2: 85-96 (1991) and D. Matthews, ~ Roy. Soc. Health,CI~
One ~rh~r im for hemo~tasis, i.e., prevention o~ blood ios~, of an anlmal is the formation of a blood clot. Clot ~ormation, i.e., blood coagulation, occur~ by means of a complex cascade of reactions with the final steps being the conversion of fibrino~en ~y thrombin, calcium ions and activated factor XIII to form the fibrin clot. For a review of the mechanisms o~ blood coagulation and the ~tructure of fi~rinogen, see C.~. Jackson, Ann. Rev.
~iochem., 49:765-811 (1980) and B. Furie and B.C.
Furie, Cell, 53:505-518 (1988).
Thrombin, which is a proteolytic enzyme, is derived from prothro~3bin. Prochrombin i5 converted to t~rombin by calclu~ and prothrombinase.
Prothrombinase is ~ormed th~ough a c~scade of reactions that begins with the proteins factor XI and factor XII.
A fibrin sea~ant is a biolo~ical adhesive whose effect imitate~ the final stages of coagulation, thereby resulting in a fibrin clot. Conventional fibrin sealants generally consist of concentrated human fibrlnogen, bovine aprotinin and factor XIII, as the first component and bo~ine thrombin and ca}clum chloride as t~e fiecond component. Application i~
generally carried out with a double-barrelled syringe, wh~ch permits simultaneous applicatlon of both components to the site where one wants to form the fibrin clot. Aprotinin is a fibrinolytic lnhibitor added to promote stability of fibrin sealants.
S The ~ibrinogen component of the f}brin sealant i~ prepared from pooled human plasma. The ~ibrinogen can be concentrated from the human plasma by cryoprecipitation and precipitation using various reagents, e.g., polyethylene glycol, ether, ethanol, ammonium sulfate or glycine. For an ~xcellent review of f i~rin sea~ant~, $ee M. Brennan, Bl~od Reviews, 5:240-244 t1991); J.W. Gibble and P.N. Ness, Tran~fusion, 30:741-747 (1990); ~. Matras, J. Oral Maxillofac surg., 43:605-611 (1985) and R. Lerner and lS N. Binur, J. o~ Surgical Research, 48:165-181 ~1990).
Recently, there has also been an interest in the preparation of fibrin sealants that utilize autologous fibrin. An autologous fibrin sealant is a fibrin sealant whe~ein the fibrinogen component o~ the fibrin sealant is extracted from the patients own bl~od. The use of an autologous ribrin seAlant is preferred because it eliminates the risk of transmisslon of blood-transmitted infections, e.g., hepati~is B, non ~, non B hepatitis and acquired 2S immune deficiency ~yndrome (AIDS), that could otherwise be present in the fibrinogen component extracted from pooled human plasma. See L.E.
Silberstein et al., Transfusion, 28:319-321 ~1988); K.
Laita~ari a~d J. Luo~onen, Laryngoscope, 99:974-976 (1989) and A. Dresdale et al., The Annals of Thoraclc Surgery, 40:385-387 (1985).
An infection can be transmitted by a fibrin sealant not only by means of the fibrincgen but also by means of the bovine aprotinin and the bovine ~s thrombin component. Bovine thrombin has been known to carry the in~ectious agent bovine spongiform encephalitis (~S~) and o~her viruses pathogenic to m~m~l S .
Furthermore, bo~ine thro~bin i~ a potential antigen, which can cause immllnological reactions in hl-m~n5 Thus, the use o~ bo~ine thrombin could re~ult in the recipient of the bo~ine thro~bin being ad~ersely a~fected. See D M. Taylor, J. of Hospital ln~ection, 18(Supplement A):141-146 ll991), S.B. Prusiner et al., Cornell Vet, 81 No. 2: 85-96 (1991) and D. Matthews, ~ Roy. Soc. Health,CI~
3-5 (~ebruary 1991).
Accordingly, there is the need for a sealant that utilizes throm~in that can be delivered to a patient without the risk o~ ~iral contamination or oeher ad~erse a~ects 3. SUMMARY OF TXB INvENTIO~
The subject invention relates to a composition ~or use in a medical procedure in an ~n i ma 1, which composition is a thrombin cont~; ni n~ blood fraction which 20 comprises:
(a) a thro~bin co~centration of from about 1 NIH unit/~l to about 2,000 ~I~ unit~/ml, and (b) a specific acti~ity o~ thrombin o~ from ~ 25 about 1 NI~ unit/mg o~ blood protein tO
about 200 NIH units/mg of blood protein, wherein said thrombin blood fraction ls substantially free o~ active antithrombin III.
The subject invention also relates to a method for preparing a prothrombin blood ~ractlon ~rom whole blood comprising:
.
W094/00566 PCT/GB93~01323 ~a) diluting whole blood to an ionic 5trength of less than about 100 millimolar;
(b) separating plasma from said whole blood;
(c) lowering the pH of said pla~ma to precipitate a prothrombin blood fraction; and (d) separating ~aid pro~hrombin blood ~raction from said plasma.
The prothrombin blood fraction is then redissolved and converted to thrombin, i.e., the thrombin blood fracti~n i~ formed, which can then be utilized i~ a medical procedure in an animal, e.g., as 15 a component of a fibrin sealant .
4. DE'rAILED DESt:~RIPTION OF ~HE INVENTION
The ~ubject invention relates to the use of a thrombin blood fraction, as deflned hereinbelow, in 2 a a medical procedure, e.g., as a co~ponent of a flbrin sealan~, in an ~ni~al, preferably a mammal. Suitable mammals include a human, a cow, a pig, a dog and a rabbit, or other ~a~mals that have an adequate blood volume to prepare the thrombin blood fraction. The thrombin blood fraction can be prepared ~rom whole blood and is impure ~n that it contains blood proteins other than thrombin. However, the thrombin blood fraction can be prepared very simply and rapidly, e. g ., in less than about one or two hours, and is believed to be as efficacious as a highly pure thrombin preparation.
The thro~bin blood Yraction can be prepared from whols blood. It is Preferred that the whole blood be obtalned from a single individual animal.
~5 Also, it i~ preferred that the ~hrombin blood rraction W094/00s66 PCT/GB93/01323 be administered to the 6ame ind~ ~idual animal Yrom which the whole blood was taksn. Thus, one aspect of the ~nven~ion is the use of an autologous thrombin blood fraction in a medical procedure. ~n this S embodiment, there l~ also no ri~k o~ transmlssion of blocd-transmitted lnfections becaUse the throm~in blood ~raction i~ to be admini5tered to the same individual animal that donated the whole blood. Al~o, for the ~ame rea60n, the blood proteins other t~an thrombin ~hat are present in the thrombin blood fraction would not be antigenic.
Furthermore, since the thrombin blood ~raction is pre~erred to be prepared from a ~ingle individual animal and used in the same, general~y small volume~ of whole blood are required, especially since it is also pre~erred to prepare a thrombin blood fraction ~or a single use. Also, it is prefe~red to prepare the ~raction within ~everal hours of the time of use. Tt i~ pre~erred that from about lO ml to about 50 ml, more pre~erably from about lO ml to about 30 ml and most preferably from about l~ ml to about 20 ml of whole blood be utilized to prepare the thrombin blood fraction of the subject invention.
The throm~in blood fraction of the subject invent~on ha~ a thrombin concentration of from about l NIH unit to about 2,000 NIH units, preferably from about lO0 NI~ units to about 800 NIH units and most preferably from about lO0 NIH un~ts to about 500 NIH
units per ml of the thrombin blood fraction.
It is believed that at such concentrations the thrombin blood fraction possesses a ~ufficient thrombin concentration ~or the desired medlcal u~e.
of course, the preferred thrombin concentration depends on the medical use o~ the thrombin blood ~5 frac~ion.
~ .
W094/00566 PCT~GB93/Ot323 The thrombin concentration oY the thrombin blood fraction can be determined by measuring the coagu~ation time of a 8tandard fibrlnoqen solut1on after addition o~ the thrombin blood fraction in a suitable diluted form. As a reference, st~ d thrombln ~olution6, containing from 2 to 15 NIH
unit~/ml, can be utilized.
The throm~in blood fraction of the subject invention has a speci~ic activity of thrombin o~ from about 1 NIH unit to about 200 NIH units, preferably from about 5 NIH units ~o about 100 NIH units and most prefera~y from ab~ut 5 NIH units to about 50 NIH
units per mg. of total blood protein. Such lower specific activities o~ the thrombin blood fraction are believed to be as effective in a medical procedure as the more purs thrombin blood fractions. ~ow~ver, such lower specific activity of the thrombin blood fractions can be prepared more readlly.
The specific activity of the thrombin blo~d fraction is, in essence, a ~e2surement of the amount o~ thrombin per amount of ~lood protein in the thrombin blood fraction. Thus, the specific activity of the throm~in blood fraction o~ the subject -invention is quite low relative to thrombin preparations that have been prepared heretofore. F~r example, United States Patent No. 5,143,838 discloses a throm~in preparation with a specific activity of at least 800 NIH units per mg. of total protein. Also, United States Patent No. 5,151,355 discloses a thrombin preparation with a ~peclfic activity of greater than 1,000 NIH units/mg. of total protein.
Although the speclflc activity of the thrombin blood fractlon of the sU~iect is low, never the less it is believed that the blood fracti~n is e~icacious for use in medical procedures.
_, Thc ~pecific activity of the thrombin blood fractlon o~ the sub~ect invention can be calculatad by mea~uring t~e thrombin concentration ~H un~ts/ml) and by dividing that number by the protein s concentration (mg/ml) mea~ured by a~y ~tandard proteln a~say, e.g., W abcor~ance.
The thrombin blood fraction i5 also ~ub~tantially ~ree of active antithrombin I~I. For the purp~se of the subject invention, "substantially free of active antithrombin III" means that the thrombin blood fraction per unit volume contains an amount of active antithrombin III that is le~ than about 50~ by activi~y ~f antithrombin I~I in ~ormal pla~ma per unit volum~. It is preferred that ~uch percentage be les~ than about 30%, more preferably less than about lO~ and mo~t preferably less than about 5~. It is essential that the antithrombin III
elther be removed from or in-_~ivated in the thrombin blood fraction. Otherwi~e, antithrombin I~I will prevent the conversion of the p~othrombin in the blood fraction to thrombin and/or inacti~ate the thrombin that is formed.
It is preferred that the thro~bin blood fraction also be ~ubstantially free of fibrinogen and fi~rin. For the purpose o~ the sub~ect invention, substantially free of fibrinogen and fibrin means that the thrombin blood fraction per u~it volume contains an amount of fibrinogen plus fibrin that is less than about 1% by weight of fibrinogen in normal plasma per unit volume. It is preferred that fibrinogen not be present in the thrombin blood fraction because the thrombln will convert --he fibrinogen to fibrin, which will polymerize to fo; a clot, thereby rendering the thrombin blood fraction lmpractlcable. The f ibrinogen ~s itsel~ ~an be removed from the thrombin blood fractlon W094/00566 PCT~GB93/01323 or the ~ibrinogen can be removed a5 the fibrin clot, there~y, o~ course, also removing the fibrin.
However, the fibrinogen need not be removed if, for example, the thrombin is inactivated as described in PCT publication No. W09l/09641.
The throm~in blood ~raction can be ut~llzed Lmmediately after it ls p~epared. I~ the fraction is not utllized i~ediate~y after it5 preparation, the fraction c~n be stored. Storage of the fraction requires that the fraction be preser~Pd ~y, ~or example, freezing or lyophilizin~ the fraction or holdin~ the composition at 4~C. The fraction in frozen or ~yophilized form wil} ~e stable for a period o~ months. When the fraction is held at ~~C, it is stable for at lea~t a period of days.
I~ the fraction is frozen, the fraction must be thawed at the time o~ use. I~ the ~raction is lyoph~lized, at time of use, it is pre~erred that the ~raction be reconstituted by addirion of ~istilled water.
The thrombin ~lood fraction can be in vlrtually any form, for example, ~ solution, suspension, emulsion or solid, with a solution being pre~erred. Thus, ~or example, such fraction can ke a liquid, gel, paste or salve. Also, of course, the ~raction can be in the form o~ a ~ranule.
If the thrombin blood Çractlon is in a solid for~, ~hen the concentration of the thrombir. blood fraction can be deternined ~y dissolving i~ in a solution and then measuring the thrombln concent-ation. If the resulting t~r~.mDi.
concentration is from about l NI~ ~nit~ o abou~
2,000 NIH units/ml, then the thrombin blood fractlon is wlthln the scope of the subjec~ invention.
W094~00s66PCT/GB93/01323 _ 9 _ 4.1. MET~OD FOR PREPA~ATIO~ OF THE
THROMRIN ~QOO FRACTION
The thrombin blood frac~ion of the ~ubject inYention can be prepared by any method known or to be deYeloped. Alco, the throm~in ~lood ~raction can be prepa~ed in a device as descr$bed ln the PCT publication No. WO91/17778.
4.1.1. MET~OD FO~ PREPARATION OF TXE THROMBI~
B~OOD FRACTION BY FORM~TION OF T~E
EuGLo8uLIN FRACTION FROM PLASMA
Whole blood can be withdrawn from an indiYidual ani~al, e.g., a human, and preferably in the prese~ce of an anticoagulant. Any anticoagulant ~5 can b~ utilized so long as it does not ac~ by dlrec~ly inactivating thro~tn~ Su~tabla anticoagulants are hepari~, EDTA, citrate or any other agent that can, direc~y or indirectly, prevent the fornation of thrombin, with citrate being preferred.
The plasma, wh~ch contains the prothro~hin, is then separated ~rom the whole blood. Any separation technique can be utilized, ~or example, ~ediment~tion, cen~ uga~ion or i ltration.
Centri~ugation can be car~ied out at about 1,500 to 25 about 3,0~0 g. ror a~out 10 minutes. T~e supernatant, which contains the plasma, can be re~oved by standard ~echniques. If it is desired to obtain a thrombin blood fraction that contains srowt~ ~actors, then such centrirugation should ~e at about ~25 g. for about 20 30 mlnutes or 1,000 g. for abou~ 2 to 3 ~i~utes. The thrombin blood fracLion cf t~e subjec~ ~nvention will then contain gro~h ~actors, which are released from the platelets during the converslon o. p-othrombin to thrombln.
W094/00s66 PCT/GB93/01323 The plasma in then treated, for examp~e, by dilution with distil~ed water, followed by the addition of acid, e.g., citric acid, to lower the ionic strength to les~ than about 100 milli~olar, preferably less than about 50 millimolar and mo~t preferably ~rom about 20 to about 40 millimolar and lower the pH to about 4.5 to a~out 6 and preferably to from abo~t 5 to a~out 5.5. Lactic acid or acetic acid are also 6uitable acids. Generally, a weight ratio of the plasma: distilled water or acid of from about 1:5 to about 1:50, with 1:10 being preferred.
This treatment, i.e., the lowering o~ the ionic ctrength and pH o~ the plasma, results in t~e for~ation of a precipitate that i~ generally referred SS to as the "euglobulin fraction." The euglobulin fraction contains the prothrom~in, fibrinogen and many other blood proteins, but is substantially f~ee of an~ithrombin II~.
Rather than diluting the plasma to lower the ionic strength, and before acidifying ths plasma, the ionic strength can be lowered by dialyzing the pla~ma by placing the plasma in a dialysi~ bag, which is then placed in distilled water. The dialysis permits the ions to di~fuse out o~ the plasma, thereby lowerin~
the ionic strength. A ~;uitable dialy~is bag i6 composed of cellulo6e nitrate. Also, the ionic strength can be lower~d by dia~iltration or occlusion chromatography.
The euglobulin fraction can be prepared as described in A. Quick, Production o~ ~hrombin From Precipitate Obtained by Acidification of ~iluted Plasma, Am. J. Physiol., 1~3:114-118 (1955) and R.
Biggs and R.G. Macfarlane, ~luman Blood Coagu~ation, pages 3î5-376, Blackwell6 Scientific Publica~ions, W094t00566 PCT/GB93tOt32 Oxford, 3rd Edition (1962).
T~e AYrecc fluid can then be ~eparated ~rom the euglobulin fraction ~y, for example, centrifugation, ~iltration or sedim~ntat~ on.
Centrifugatlon can be carried at about 1,500 g. for about 2 to abou~ 5 mi~ute~.
The pro~hrom~in of the euglobulin f raction ia ~hen redissolvsd and conver~ed to thro~bin, thereby 1~ forming a throm~in blood fraction o~ the subject in~entlon. This c~n be carried out by solubi~izing the euglobulin fraction in a physiologically accepta~le solutio~, e.g., saline, in an amount equal to or preferably less t~an (about 10~ the original amount o~ plasma. An alkaline hu~er can ~e added in an amount to raise the pH of the 601ution to a~out 6 ~o about 8 and preferably to about 6.5 to about 7.5.
N~nlimi~ing examples o~ suita~le alkaline ~uf~ers include sodium hydroxide, potas~ium hydroxide, calcium hydroxide, bicarbonate buff ers such as sodium bicar~onate and potassium bicar~onate, salts o~ acetic acid a~d salts of sulfuric acid. Preferred alkaline buffers include: Sodium carbonate/bicarbona~e p~ 7.o, Sodium bicarbonate~Na~H pH 7.~, 1.5M Glycine/NaOH pH
6.5-7.5, Bi6 hydroxyet~ylaminoethane ~ulphonic acid (BES) pH 7.5, ~.ydroxyethylpiperazine propane sulphonic acid tEPPS) pH 7.5, Tricine pH 7.5, Morpholino propane sulphonic acid (MOPS) pH 7.0, Trishydroxymethyl aminoethane sulphonic acid (TES) p~ 7.0 and ~o Cyclohexylaminoethane sulphonic acid (CHES) pH 7.0;
with Sodium carbonate/bicar~onate pH 6.5 - 7.5 Bis hydroxethylaminoethane sulphonic acid (BE5) pH 7.5, ~ydroxyet~ylpiperazine propane sulphonic acid (EPPS) pH 7.5 and Trishydroxy~eth~l aminoet~ane sulphonic ~5 dcid (TES) pH 7.5 being most preferred.
W094/00566 PCTtGB93~01323 Calcium i~ added to the neutral solution in order to convert the prothrombin to thrombin. ~f course, ~he calcium can be part of the alkaline buffer. Calcium can be added in the form of, for S example, calcium chloride. Th2 amount of calcium added should be sufficient to convert an amount of prothror~in to thrombin that i6 6ufficlent for the intended medical use. Furthérmore, the reaction should be permitted to occur ~or a period of ti~e ~0 suf~ici~nt to convert enough of the prothrombin to thrombin that is suf~icicnt for the inte~ded medical use. Generally, from about 5 millimolar to about 50 millimolar calcium chloride i~ suf~icient.
~lso, rather than adding a soUrce of calcium ~5 ions, prothrombin activating enzymes from snake venoms can be utilized. For example, snake venom fro~ Eccis carinatu6 or the Australian Tiger snake can be utilized.
As the thrombin forms, i~ convert~ the fibrinogen to fi~rin, which forms a fibrin clot. It is preferred to remove the fibrin clot, which can be carried out by, for example, wrapplng the fibrin clot around a ~tirring rod or collecting the fibrin onto glass beads.
4.1.2. METHOD FOR PREPARATION OF THROMBIN BLOOD
FR~CTIoN BY DILUTING WHOLE BLOOD
In a preferred embodiment, the thrombin b}ood fraction can ~e prepared by initially preparing a prothrombin blood fraction from whole blood comprising:
(a) dilu~ing whole blood to an ionic ~trenqth of less than about 100 millimolar;
(b) separating plasma fro~ said whole blood;
(c) lowering the pH o~ ~aid plasma to precipitate a prot~rombin blood fraction; and (d) separating said prothrombin blood 5 fraction from said pla~ma.
The prothro~bin blood fraction is then redissolved and converted to thrombln, i.e., the thrombin blood fraction of the subjec~ invention is formed, which can then be utilized in a medical proce~re in an animal, e.g., as a component of a ribrin ~ealant. This method provide8 a thrombin blood fraction of the subject invention, which can be prepared in only about 45 minutes!
Specifically, who~e ~lood is drawn from an individual animal. The whole blood is then i e~iately (within about five minutes) ~iluted in order to lower the ionic strength to less than about lO0 millimolar, preferably less than about 50 millimslar and preferably to from about 20 to about 40 millimolar. It should be noted that since the whole blood i~ diluted immediately, there i~ no need ~or the u~e of an anticoagulant. Any physiologically acceptable solution at physiological osmotic préssure can be utilized to lower the ionic strength, e.g., a glucose aqueous ~olutlon such as a 5.5% isotonic a~ueous glucose ~olutio~.
The plasma is then separated from the whole blood. Any separation technique can be utilized, for example, ~edtmentation~ centrifugation or filtratlon.
Centrlfugation can be carried out at about 1,500 to about 3,000 g. ~or about 5 to about lO minutes. The ~upernatant, which contains the plasma, can be removed by standard technlques.
The plasma fractlon is ~hen acidified, thereby resulting in the formation of a prothrombin blood fraction, which is generally referred to a~ the euglobulin fracSion, which is a precipitate. The plasma fraction can be acidi~ied with, for exa~ple, citric acid, lactic acid or acetic acid. The pH
S should be lowered to from about 4.5 to about 6 and pref erably to ~rom about 5 to about 5.5.
The excess ~luid can then be separated from the prothrom~in blood ~raction by, for example, centrifugatlon, filtration or se~i~entation.
Centrifugation can be carried out at about ~,SOO g for about 2 to about 5 minutes. This prothrombin blood ~raction contains prothrombin, fibrinogen and many other blood proteins, but does not contain antithrom~in III. The prothrombin blood fraction is then redis~olved and the prothrombin is converted ~o thrombin. Any physiologically acceptable solution, e.~ aline, can be utilized to redissolve the proth~ombin fraction. Furthermore, only a cmall ~olume of solution is required to redissolve the prothrombin fraction. It is believed that only from about O.4 ml to abo~1t 1 ~1 o~ solution i8 required i~
about 1~ ml of whole blood was initially drawn. An alkaline buffer can be added in an amount to raise the pH of the ~olution to about 6 to a~out 8 an~
preferably to about 6.5 to abo~t ~.5. Nonlimiting examples of suitable alkaline buffers include sodium hydroxide, potassium hydroxide, calcium hydroxide, bicarbonate buffer~ such as sodium b~carbonate and potassium bicar~onate, tri-metal salts of cltric acid, salts Or acetic acid and salt~ o~ sul~uric acid.
Preferred alkaline buffers include: Sodium carbonate/bicar~onate pH 6-~, Sodium bicarbonate/NaOH
pH 6-8, Glycine/NaO~ pH 6-8, Bis hydroxyethylaminaethane sulphonic acid (BES) pH 6-8, Hydroxyethylpiperazine propane sulphonic acid (EPPS) _, W094/00~66 PCT/GB93/01323 pH 6-8, Tricine pH 6-8, Morpholino propane sulphonic acid (~O~S) pH 6-8, Trishydroxymethyl amlnoethane ~ulphonic acid (TES) pH 6-8 and Cyclohexylaminoethane ~ulphonic acid (CH~S) pH 6-8; with Sodium S carbonate/bicarbonate pH 6-8 Bis hydroxethylaminoethane sUlphonic acid (~ES) pH 6-8, Hy~roxyethylpiperaZine propane sulphonic acid (EPPS) pH 6-8 and TrishydroXymet~yl aminoethane ~ulphonic acid (TES) pH 6-8 being most pre~erred.
Calcium ls added to the neutral solutlon in order to convert the prothrombin to thrombin. of course the calcium can be part of the alkaline buffer.
Calcium can be added in the form of form of, for example, calcium chloride. The amount of calclum added should be sufficient to convert an amount o~
prothrombin to thrombin that i~ sufficient for the intended medical use. Furthermore, the reaction should be permitted to occur for a period of time sufficient to convert enough of the proth~ombin to thrombin that is sufficient for the intended medical u~e. Generally, from about 5 millimolar to about 50 millimolar calcium chloride is sufficient. It has been observed that at a~out 25 minutes of reactlon sufficient amounts of thrombin are formed ~or most 2s medical uses of the thrombin blood fraction of the subject in~entio~.
Also, rather tha~ adding a source of calcium ions, prothrombin activatin~ enzymes from snaXe venoms can be utilized. For example, snake venom from Eccis carinatus or the Australian Tiger sna~e can be uti}ized.
A~; the thrombin forms, it converts the ~ibrinogen to fibrin, which forms a fibrin clot. ~t is preferred to remove the ~ibrin clot, which can be ~s carried out by, for example, wrapping the fibrin clot W094/00566 PCT~GB93/01323 around a stirring rod or coll~ction of the fibrin onto glasa beads.
4.l.3. PREPARATION OF THROMBIN BLOOD
F~ACTIO~ ~Y REMOVING OR INACTIVATING
ANTIT~RoMBIN ~I~ FROM PLAS~
In an alternative method, the thrombin ~lood fraction of the ~ubject invention can be prepared by withdrawing whole blood ~rom an individual ~nimal, e.g., a human, and preferably in the presence o~ an l anticoagulant. Any anticoagulant can be utilized.
Suitable anticoagulants are heparin, EDTA, citrate or any other agent that can, directly or indirectly, preve~t the for~ation of thrombin, with citrate ~eing preferred.
'rhe plasma, which contains the prothrom~in, is then separated from the whole blood. Any separation technique can be utilized, for example, sedimentation, centrifuqation or filtration.
Centrifugation can be carried out at a~out 3,000 g.
2 for about lO mlnutes. The supernatant, which contains the pla5ma, can be removed by st~ rd techniques.
Antithrombin III is then removed from the plasma or is inactivated. For example, the pH of the plasma can be lowered to at least to about 5. Such lowerinq of pH inactivates antithrombin III; otherwise antithro~bin III would prevent the conversion of prothrombin to thrombin. Antithrombin III can be inactivated by any technique. For example, by the addition of 0.05 ml o~ 5 mol./liter ~Cl per l.o ml plasma. After about l0 to about 20 minutes of incubation, the plasma can be neutralized with 0.05 ml of 5 mol.Jliter ~aOH per ml oY plasma.
Rather than inactivating the antithrombin II', the an~lthrombin III can be removed from the plasma by pas~ing the pla~ma throu~h a column that CA 02263800 l999-03-l7 binds antithrombin III, e.g., a heparin column or a column with anti~o~le~ to antithrombin III.
The plasma fraction, which contains prothrombin, is then treated to convert prothrom~in to thrombin. This can be carried out by, for example, the addition of a source o~ calcium ions, as described above, or by the addition o~ O.l ml CaCl, of 0.36 mol.~liter per ml of plasma. Also, rather than adding a source of calcium lons, prothro~bin acti~ating enzymes from snake venoms can be utilized. For example, snake venom ~rom Eccis carinatus or the Australian Tiger snake.
As the throm~in ~orms, it converts the fibrinogen to fibrin, whlch forms a fibrin clot. It i5 preferred to remove the fibrin clot, wh~ch can be carried out ~y, ~or example, wrapping the fibrin clot around a stirring rod or collection o~ the fibrin onto glass heads.
The resulting plasma is a thrombin blood fraction of the subject invention.
4.l.4. METHOD FOR THE PREPARATION OF A
THRO~BIN BLOOD FRACTION BY DILU~ING
WIT~ AN ACID sorl~T~o~ ~RECTLY
By the known process ~or preparing thrombin 2 from plasma, plasma i5 diluted in the ratio l:lO with water, wherea~ter a pH-red~cing acid is added, such as - acetic acid, with the result that the pH-value is about 5.0 to 5.3. The mixture is then centri~uged ~or 20 ~in. at 2,0~ ~. The resulting precipitate contains d~fferent coaqulation ractors, such as inter alia prothrombin and fibrinogen. When the excess fluid has been removed, the precipitate i5 dissolved in a p~ysiological solutlon, pre~erably a 0.9~ sodium chloride solution, wherearter a p~-value-increasing a~ent, such as sodium carbonate is adc~ed unti~ the W094/00~66 PCT/GB93/01323 pH-value i5 about 7.9. When the precipitate hac been ~issolved, calcium chloride is added and cause~ the conversion of the prothrombin into throm~in by a conventional, so-called coagulation cascade. The resulting thrombin causes a converslon o~ rincgen into fibrin, wherea~ter the thrombin is separated by centrifuging and then su~jected to a s~ccee~tng purirication ~column purification).
Another a~pect o~ the subject invention ts that the diluting step is performed directly with a diluted acid solution, e.g., 0.4~ acetic acid, ~y the centrifuging step being performed in a container with a relatively large flat precipitation surface, by the physiological solution, with. the agent for increasing pH, and the calcium chloride being added at the came time to the precipitate as a mixture, whereby ~aid precipitate is dis~olved and then coagulates while f~rming ~ibrin, and by the throm~in then being removed .
As a result, it is now possible to prepare thrombin ~rom plasma of autologous blood in a relatively quick way, which is in particular due to the fact that the precipitation is per~ormed in a container with a relatively large flat precipitation 2s surface. A quick and complete dissolution is thereby ensured of the precipitate containing prothrombin and ~i~rinogen. The complete dissolution i~ important before the presence of calcium chloride causes the conversion of prothrombin to thrombin. A too early ~o thrombin formation leads to fibrin formation and the fibrin will ~ind the added fluid, whereby the dissolution of the precipitate ~tops and cannot continue untll ~aid fluid has again been passed out of the ~i~rin. The relatively small precipitate with a ~5 relatively large surface area relative to the amount causes a complete dissolving of ~aid precip~tate by addition of the combined uixture of physiological solution, Wi~h the agent for increasing pH, and calcium chlorids before the thrombin has been formed S through ~aid coagulation cascade and starts the forma~ion of fibrin.
Moreo~er ac~ording to the invention, the mixture may be admixed with a plasminogen cataly~t, ~uch as streptokinase, before being added to the precipitate with the effect that the ~ollowing separation of thrombin from fibr~n is promoted.
According to the invention, the ~ixture added to the precipitate can advantageously be set to increase the pH-value to 6.5, whereby the resulting active thrombin is found to obtain the best keeping qualities as the thrombin has a tendency to become inactive on s~anding, which is usually the case in connection with enzymes. In addition, the dissolved precipitate can be transferred to a f~exible material before the coagulation starts, said f~exi~le material presenting a relatively large surface upon which the fibrin resulting ~rom the coagulation can be deposited, whereafter the thrombin may be pressed OUt of the fibrin by the flexible material being subjected to a compaction. Exactly the obtained complete dissolving o~ the precipitate prior to formation of fibrin turned out to allow a quick transfer of the solution to a flexible material prior to said formation of ~ibrin. The flexible material ensures that the fibrln is deposited across a particularly large surface, the compaction of which facilitates the separation of thrombin from fibrin.
It is particularly prererred when the flexible material i~ a sponge with open pores .
WO94fOo566 PCT/GB93/01323 -- Zo --The ~lexible material can be placed in a syringe, into which the dis301ved precipitate can be absorbed and from which the throm~ln can be squeezed out by an acti~ation of the piston of the syrlnge. In this manner, a particularly easy separation of the thrombin from the flexible material is en~ured, Moreover, the centri~uging may according to the invention suitably be per~ormed at about l,Soo g for about 5 min, which algo accelerates the preparing 1~ of thrombin.
Thus, the sub~ect invention co~prises a process for preparing throm~in f~o~ human blood plasma, whereby the blood plas~a is diluted to about 10 to 17% with water or another ion-intensity-reducing ~5 fluid and an acid to reduce pH to a~out 5 . O to 5 . 3, whereafter the mixture is centri~uged, and ~hereby the precipitate resulting from the centrifuging is admixed a physiological solution and calcium chloride, ~aid physiological solution increasing pH to about 6 to 8, characterized by the diluting step being performed directly with a diluted acid solution, by the centrifuging step being performed in a contalner with a relatively large plane precipitation sur~ace, by the physiological solution, with the agen~ for increasing pH, and t~e calcium chloride being added at the same time to the precipitate as a mixture, whereby said ~ precipitate is dissolved and then coagulates while formin~ fibrin, and by the thrombin then being removed from the fibrin.
3~
W094/00566 PCT/GB93tOl323 4.2. ACCELERATIO~ OF TXROMBIN FORMATIO~
BY CONTACTING BLOOD OR ~LASMA WITH
A SU~FACE THAT ACTIVATES aLOOD
COAGUT~TIO~ F~CTORS XI .~ND XII
Another aspect of the sub~ect in~entlon is the preparation of a thrombin blood fractlon wherein whole blood or pla~ma is contacted with a surface that activates blood coagulation factors XI and XII. It is believed that a negatively charged surface such as glass or kaolin, with glass being preferred, ca~
~0 provide such activation. Nonlimiting examples of glass sur~aces are ~la~s beads, glass wool, glass filters and glass capillary tubes.
The whole blood or plasma should contact the surface for a period of time sufficient to activate such blood coagulation ~actors, e.g., for a~out 5 to a~out 10 minutes. If the whole blood or pla~ma has not been treated with an a..;.coagulant, then the whole blood or plasma should be in contact with such a surface for not ~ore than about 60 seconds, preferably less than about 30 seconds and more prefera~ly about 15 seconds. Without the anticoagulant, and i~ the contact of the whole blood or ~lasma wit~ the surface is too long, then fibrin clots will form prematurely.
It is preferred that p~as3a be exposed to a z5 surface of about 4 cm2 to a~out 60 c~l, pre~erably ~rom about lO cm' to about 30 cm~ and most preferably about - 20 cm2 of such surface for each millili~er of plasma.
Also, it is preferred that whole ~lood be exposed to a surface of from about 2 cm- to about 30 cm-, preferably fro~ about 5 cm2 to about 15 cm2 and most pre~erably about lO cm- of such surface for each ~illiliter of whole blood.
It is believed that the con~acting o~ t~e plasma or whole blood with a ~ur~ace that can activate fac~ors XI and XII accelerates the conversion time of prothrombin to throm~in. Thus, a thrombin blood fraction can be prepared in an extremely chort period o~ time.
This activation of factors XI and XII can be S utilized to accelerate the preparation of any thrombin blood fraction, regardleSs of how the ~raction is ma~e and regardless of its purity and specif~c activity.
~lowever, it i5 essent~al that the thrombln blood fraction be substantially free of active antithro~bin III~ It is eqsential that the antithrombin III either be removed from or inactivated in the thrombin blood ~raction prior to converting the prothrombin to thrombin. Otherwise, antithrombin III will prevent the conversion of the pr~thrombin to thrombln and/or ~S inactivate the thrombin. It is also preferred that the thrombin blood fraction be ~ub~tantially free of ~ibrinogen and fibrin. For example, with respect to the activation when the thrombin blood fraction is prepared by mean~ o~ the euglobulin fraction as described in Section 4.1.1., the contacting of the plasma shou~d be carried out immediately prior to the dilution of the plasma. When a thrombin blood fraction is prepared by diluting whole blood, as described in Section 4.1.2., the contacting o~ the 2s whole blood should be carried out immediately prior to the dil~tion of the whole blood. For example, this contacting step can be carried out by withdraw~ng who~e blood into a syringe that contains, ~or example, glass beads. After not more than about 60 seconds, the whoie b~ood is discharged from the syringe and the thrombin blood ~raction o~ the subject invention i~
then prepared, as descrlbed above.
wos4/00s66 PCT/G~93/01323 4.3. THE USES OF T~E 'rXROM~N BLOOD
FRAC'rION OF T~E SUBJECT INVE~TIO~
~ he thrombin blood fracti~n o~ the su~ject invention can be utilized in any medical procedure, known or to be developed, in an an t mal, t ncluding vetinary procedures. Any species of animal is suitable, but, o~ course, humans are preferred.
The thrombin blood fraction can be employed as a component of a ~ibrin sealant or can be employed alone just as conventlonal th~ombin preparations have been employed. The thrombin blood fraction is utilized by contacting the des t red site of the animal with the thrombin blood fraction. For the purpose of the subject tnvention, the "desired site" is that location in or on an animal where one desires to for~
a ~i~rin clot. What or where the desired site is depends on the use of the thrombin blood ~raction of the subject invention.
The us~ of the thrombin ~lood fract~on as a component of a fibrin sealant, can be utilized for connecting tissues or organs, stopping bleeding, healing wounds, sealinq a surgical wound, use in vascular surgery lnclude providin~ hemostasis for stitch hole bleeding of distal coronary artery anastomoses; le~t ventricular suture lines; aortotomy and cannulation sites; diffuse epimyocardial bleeding seen in reoperatlons; and oozing from venous bleeding sites, e.g. at atrial, caval, or riqh~ ventricular levels. The subject invention is also useful for sealing of dacron artery grafts prior to grafting, sealing tissues ou~side the body, producing fibrin rafts for cell growth, stopping ble~ding ~rom damaqed spleens (there~y saving the organ), livers, and o~her parenchymatous orqans; sealing tracheal and bronchial 3g anastomoses and air leaks or lacera~.ions or the lung, sealing bronchial stumps, bronchial fistula~3 and WO 91/00566 PCTJGBg3/01323 ~orh~eal f~stula~; for suturele~s ~eamless healln~
(~Zipper" technique), and embolizatlon 1~ v~sc~ r radio~ogy of intracerebral AV~'s, llver AV~'s, ang$0dy6plasia of colon, esophagsal varices, "pumping"
GI bleeders secondary to peptic ulcers, etc. The su~ect inve~tion is further u~eful for prcvid~ng hemostasis ~n corneal transplants, no~ebleeds, post ton~llectomie~, teeth extractions and other applications. See G.~, Gestrin~ and R. Lermer, V~-~r~ r Surgery, 294-304, Sept./Oct. 1983, The thrombin blood fraction of the subject invention can be emp~oyed alone to staunch oozinq hemorrhages or hemorrhages Ln hollow or~ans. The t~rom~in blood fraction can also be uti~ized in the ~5 treatment or dama~ed li~e ani~al tissue by utilizinq the fraction to actiYate the release of the materials, i.e., plated-derived factors, ~ro~ platelets, wherein such materiAls can be utilized to heal damaged tlssue.
See United States Patent No. 5,165,938. The thrombin ~lood fraction can also ~e utll~zed to assist in the cell culture srowt~ of keratinocytes and to assist in t~e auto~ogous transplantation o~ keratinocytPs, or any other skin-derived cells, e.q., fi~roblasts. See V. Ron~ard e~ al., Burns 17:~81-18~ (1991), H. 9ro~y, 2s Canadian Patent ~o. 2,018,020 and ~. Hu~yadi et al., J. Der~atol. Sur~. oncol. 1:~5-78.(1988).
Also, the thrombin blood Frac~-n can ~e placed on a solid support, e.g., bandage, su~ure, prosthesis, or dressing, that ~ill be in contact ~~ith 3~ the deslred site. Such support is then place~ ir.
con~act ~lth the desired site until, ~or example, -he ~ibrin clot ~orms.
The dosage of the thrombi~ blood fraction depends on its particular use, but the dosage should be an e~ective amount ~or the composition to perform its intended u~e. Generally, it is believed that from S a~out 0.5 ml to a~out 5 ml of' the thrombin blood fraction is sU~icient. However, depending on the use, the dosage can range fro~ about 0.05 ml to a~out 40 ml.
If the thro~in ~lood fraction is utilized as a component o~ a fibrin sealant, then the fibrin soalant can be applied to the des~red slte with, for example, a double-barrelled syringe. The double-barrelled ~yringe can be Y-shaped, thereby permitting the mixing of fibrinogen and the thrombin lS blood fraction immediately prior to the contacting step. Also, rather than a Y-shaped double-barrelled syringe a dou~le-barrelled syringe with two openings can be utilized. This permits the simultaneous contacting of the desired site. Also, the compositions of the double-barrelled syringe can be sprayed onto the de~ired site. See H.B. Kram et ~1., The Amcrican Surgeon, 57;3~1 (1591). Also, if the blood fraction is emp}oyed as a component of a fibrin sealant, then autologous fibrinogen ca~ be utilized, thereby rendering the entire fibrin sealant autologous. Also, if the thrombin blood fraction is employed alone, then the fraction can be applied to the desired site with a single-barrelled syringe.
It should also be noted that the ~hrombin blood frac~ion of the subject invention can further comprise a source of calcium ions, e.g., calcium chloride. The source o~ calcium ions assists in the conversion o~ fibrinogen to the ~ibrin clot. The amount of calcium ions should be the same a~ that utilized in conventional ~ibrin sealants. However, W094/00~66 PCT/GB93/01323 since the throm~$n blood fraction may contain a source o~ calcium ions already due to the conver~ion of prothrom~in to thrombin, an additional sour~e of calciu~ ions may not be required. But, if more calcium iB needed to form the fibrin clot than to form thrombin, then, as an option, PYc~c~ calcium from what is required to forrD thrombin can be utilized 80 that no addi~ion~l calcium need be added when the thrombin blood ~raction is utilized in the medical procedure, e.g., as a component of a fibrin sealant.
5. ~MP~
EXaMPLE I
Preparatlon of a Composition Containing a Throm~in ~lood Fraction Obtained From 17 ml of Fresh Blood From a Human Adult Donor A puncture of the vein of a human was performed by a needle and 17 ml o~ ~lood was drawn into an e~pty 30 ml syri~ge. Tmmediately after drawing the blood, ~t was transferred to a 50 ml test tube cont~in;~ 3~ ml of a solution containing s.5 glucose. The 50 ml test tube was placed in a centrifuge and centrlfuged for 5 minutes at },500 x g at room temperatur~. After centrifugation, 40 ml of 2 the supernatant plasma/glucose solution was removed by a syringe, and tran~ferred to a new SO ml test tube.
By means of l.O7 ml of a 2.8% citric acid solution, the p~ in the plasma~glucose 501ution was lowered to 5.2 and after a period of lO minutes at room temperature, the solution was centrifuged at 18~C
at 1,500 x g ~or 5 minutes. After centrifugation, the supernatant was drained off and the precipitate was dissolved in 0.424 ml o~ a solution containing 14 mmole/L of ~aHCO3 and 8 gram~L of NaCl. This precipitate contains f ibrino~en, prothrombin and oth~r blood proteins, but is ~ubstantially rree of antlthrombi~ III. The pH of this di~solved prothrombin containing euglo~ulin solution was 7.~5.
Activation of the prothrombin was perfor~ed S by the addition o~ ~.027 ml of a solUtion cont~ining CaCl2, 0.5 mole~. From 12 to 17 minute~ after the addition of the CaCl2, the fibrinogen in t~e ~olution started to coagulate, and the fibrin thus for.~ed was r~moved by means of a polystyrene ~patula. Small lC samples were removed at dirferent intervals and the thrombin c~ncentration was measured. The re~ult~ from the example are shown i~ Table I.
Table I
15 time after lO 20 30 60 120 27 Ca-addition min min min min min hour NIH uJml 0 180 368 540 600 7~7 EXAMPLE II
Preparation of a Composit~on Containing a Thrombin Blood Fract~on Obtalned From 17 ml of Fresh Glass Activated Blood From a Human Adult Donor In this experi~ent, the donor and the day ~or the performance was the same as used i~ Example I.
A puncture o~ the vein of a human was performed by a needle and 20 ml o~ blood was drawn into a 30 ml syringe containing 20 grams of glass beads with a diameter of approximately 2 mm. The total surface area of the beads was approximately 2~0 cm~ and, therefore, the ~urrace area was about ll.5 cm2 per ml o~ whole blood.
Immediately a~ter drawinq the blood, the syringe was turned gently for lO to 15 seconds before 17 ml of the blood was transferred to a 50 ml test CA 02263800 l999-03-l7 W094/00s66 PCT/GB93/01323 tu~e containing 34 ml o~ a solution contAlni~g 5.
glucose. The 50 ml test tube was placed in a centri~uge and centrifuged ror 5 minutes at 1,500 x g at room temperature. ~fter centrifugation, 40 ml of the supernatant pla~ma/qlucose ~olut~on was removed by a syringe, and trans~erred to another 50 ml te8t tube.
By means o~ 1.07 ml o~ a 2.8% citric acid solution, the pH was lowered to 5.2 and after a period of 10 minutes at room temperature, the solution was lo centri~uged at 18~C at 1,500 x g for 5 minute6.
After centrifugation the supernatant was drained o~ and the precipitate was dis~olved in 0.424 ml o~ a solution containing 14 mmole/L of NaHCO3 and 8 gramJL of NaC1. This precipitate contains prothrombin, ~ibrinogen and other blood proteins, but is substantially free of antithrombin III. The pH of this dissolved prothrom~in containing euglobulin solution was 7.35.
Acti~ation of the prothrombin was performed by the ~ddition o~ a 0.027 ml of a solutlon containing CaCl2, 0.5 mole/L. From 4 to 9 minutes a~ter the addition of the CaCl2, the fibrinogen in the solution started to coagulate, and the fibrin thus for3ed was removed by ~eans of a polystyrene spatul~. Small 2s samples were removed at dif~erent intervals and the thrombin concentration was ~easured. The results are shown in Table II.
Table II
~C
time after 10 Z0 30 60 120 27 Ca-addition min min min min min h~ur NIH u/~l 130 444 560 720 695 8ao Thus, from Table II it i5 readily apparent that thq glass activation accelerates the time required for the conversion of the prothrombln to thrombin. For example, at only l0 minutes after the addition of a source of calcium ions, 130 NIH units/ml of thrombin activity was measured. In con~rast, without qlass activation, as in Example I, at l0 minutes after the addition Of a source o~ calcium ions, there was no detectable throm~ln activlty.
EXAMPLE III
Preparation of a Composition Containing a Thrombin Fraction Obtained From 17 ml of Fresh Blo~d From ~uman Adult Donors, Characterized by Low Specific Thrombin A~iVlty ~5 In four experiments, perfor~ed as described in Example I, the thrombin concentration and the specific activity of thrombin were measured. The results are given in Ta~les III and IV where the pH
value is the pH in the dissolved euglobulin fractions.
Table III
A thrombin measured from 15 min to 2 hours a~ter dissolution of the euglobulin fraction. NIH u/ml donor pH E-280 15 min 30 min 60 min 2 hr JH 6.96 25.8 142 483 661 779 ~JS 6.35 26.7 0 56 942 1,238 LK 6.98 l9.5 30 249 430 s62 K~ 6.21 31.5 0 142 616 725 (E-280 is a measurement of ~he total prote~n concentra~ion in mg/ml).
W094/00~66 PCT/GB93/01323 Table IV
B SpeciSic activity of thrombin measured from 15 min to 2 hours after dissolution of the euglo~ulin fraction. NIH u~mq protein donor 15 ~iA 30 min 60 ~in 2 hr JH 5.5 lg 26 30 HJS 0 2.1 35 46 LR 1.5 13 22 29 ~N O 4.5 20 23 T~us, the preparation of a thrombin blood rraction as descri~ed in Example I results 1~ a thrombin blood fraction o~ a concentration and spec~fic actiY1ty of t~e subject inventlon.
~XAMPLE ~V
Preparation of Throm~ln Fro~ Whole Blood Usin~ the Device of WO91/17778 The device described in WO91/17778 was used ~or the preparation of throm~in. ~efore the ~lood was in~roduced into the device, 40 m1 o~ a _.5 glucose solution was in~roduced i~to ~he first chambe (}4) through the filter (43) mounted on the tubins (39).
Blood, lO ml, was c~llected from human donors into a syringe and immedia~el~ ~hereafter transferre~ through the tubing (39) in~o the glucose solution. The device was placed in a centri~uge 2nd ~0 centrifuged for 5 mln at 1,500 x g. ~fter centrifuga~on, the p1asma/g1uoose solution was transferred i~to the second chamber (30) with the red cells re~aining in the chamber (1~). Through the sterile filter ln tu~e (60) 0.6 ml o~ 2.8% c~trlc ac~~
solution was introduced into the plasr.a/~lucose -~ . .
W O 94/00~66 PC~rtG B93/01323 - 3~ -solution. After 5 t~ lO min, th~ device was placed in a centrifuge and centri~uged ror 5 min at 1,500 x y.
A~ter cen~r~fugatlon the supernatant was transferred to the first chamber (14) and the precipitate, the s euglobu~in fraction, rP~e~ in ~he s~nA r~mh~r (30). Through the sterile filter in tube ~60) 0.85 ml of a colution containing 7.5 mM ~laHco3~ 52 ~ NaCl and 30 mM CaCl2 was introduced into the 6econd rh~mh~r ~30). The euglobulin precipitate wag dissolved within ~0 1-2 ~inutes, and transferred to the 6yringe (5-) connected to the ~econd rhArher ~30). The syrinye contained a polyurethane sponge ~acilitating the removal o~ the forned _ibrin. Thro~n concentrations were measured after 30 min to 22 hours. The results lS are recited ~n Table v.
Ta~le V
thron~in concentration NIH u/ml ~onor pla-cma 30 min 1 hour 2 hour 22 hour dilution RH-A 11.9% 186 Z40 231 2?9 RH-~ 12.3% 5~ l30 130 132 RH-C 11.7% l7a 186 180 19~
2s RH-D 12.5~ 104 12~ 92 123 EXAMPLE V
Preparat~on of ~h-o~in and Fibrinoqen rrc,.
Whole Blood Using 2 Device System ~'ade Fro-Two Inter-Connected Devices of 30 W091/17778, and the Use of Thrombin and Fi~rinoaen ! n a ri ~ri~ Glue The devlce syster. consists of two devices as desc-i~ed in W091/17778. The -wo tubings (39) from tne two devices ~ere connec~ed to the same .
W094/00566 PCT/GB93/Ot323 can~ula (4~) by means of a three-way connector. In the device used for the thrombin precipitation 5 glass beads, 3 mm in diameter, were placed in the second chamber (30). The syringe in the thrombi~ device was S changed from being a 3 ml syringe in the f ibrino~en device to a 1 ml syringe.
Before the ~lood was collected from the donor, cltrate and glucose solutions were ~illed into the two device~. citra~e, 5 ml of a 3.8% solution, was introduced into the first chAmhPr (14) o~ the fibrinogen de~ice (herea~ter named DeYice-F) through the filter 143) ~oun~ed on the tubing (39). Glucose, 34 ml of a 5.5~ solution was i~troduced into the firs~
chamber (14) of the thrombin device (hereafter named lS Device-T) through the filter t43) mounted on the tubing t39).
~ lood, 45 ml was collected through the cannula lnto the first chamber (14) in DeYlce-F
containing the citrate solution, and 17 ml was collected into the ~irst chamber (14) in the Device-T
containing the glucose solution. After collection of the blood, the separator-system was disconnected ~rom the donor, and the tubing (39) was sealed close to inlet ~38). Both devices were placed in a centr~fuge and centrifuged for lO min at 1,500 x g.
The separated plasma in Device-F was transferred to the second chamber ~30) and 2.5 ml of a 96% ethanol solution was introduced into the ~econd chamber (30) through the sterile filter in tube (60).
The dev~ce was now placed into a ice-water bath for 20 minutes to reduce the temperature in the plasmaJethanol solution to approximately O to 4~C. At this temperature, 85% of the fibrino~en in plasma was precipltated. The device was now placed in a centrifuge and centrifuged for 5 min at 1,500 x g.
W094/00~66 PCT/GB93/01323 The supernatant serum was transferred to the fir~t chamber ~14), and the ~olid fibrlnogen wa~ dissolved by incu~ation for 5 minutes at 37~C. The dissolved ~olution was transferred to the 5terile syrlnge (51).
The concentration of fibrino~en was measured to be 31 mg/ml.
The separated p~asma/gluco6e in Device-T was transferred into the second ch~mhPr (30) with the red cells r~in~n~ in the ~irst chamber (14). Through the sterile filter in tube t60) 1.2 ml of a 2.8 citric acid solution was introduced into the plasma/gluco~e solution. After 5 to 10 min the device was placed in a centrifuge and centri~uged for 5 min at 1,500 x g. ~fter centrifugation, the supernatant lS was transferred to the ~irst rhAmh~r (14) and the precipitate, the euglo~ulin ~raction, remained in the second chamber (30]. Through the sterile fllter in the tube (60), 0.85 ml of a solution containing 7.5 mM
Na~CO3, 52 mM ~aC1 and 30 mM CaCl. was introduced into the second chamber (30). The euglobulin precipitate was dissolved within ~-2 minutes, and the fi~rin ~ormed durin~ the activation of prothro~in to thrombin was collected onto S glass beads placed ln the second cha~ber (30). After 15 minutes the thrombin solution was transferred to the syringe (51) connected to the second chamber (30). Thro~bin concentration was measured to ~e 248-340-372 NIH u/ml a~ter 15-30-60 minutes, respectively.
The two syrin~es containing the fibrinogen and the thrombln were used as a double barrelled syringe. The two solutions were expelled from the syringes and formed immediately a ~irm ~i~rin clot.
., ~XAMPLE VI
54 ml of 0.04~ HAc were added to 6 ml of pla~ma. This mixture was placed in a flat-botto~ed container of the type known from the above S PCT/DK91/00131. The contalner is of a circular cross section with an inner diameter of 4.S cm. The pH-value was 5.3. The pH-value and the relatively low concentration of ions in the provided mixture ensure the ~ollowing precipitati~n of the coagul~tion lo factors, inter alia prothrombin, as a precipitate by a centrifu~in~. The centri~uging was performed at ~,500 g for 5 min. Thus the centrifuging was relatively ~uickly terminated, which is due to the relatively short falling height and large precipitation surface.
~5 After removal of excess ~luid, an 0.75 ml a~ueous solutlon o~ 0.9% NaC1, 0.03% Na~CO3 and 25 mM CaCl2 was added t~ the precipitate. After dissolving o~ the precipitate, the ~olution was ~ucked into a 2.5 ml syringe conta~nin~ a polyurethane spon~e. The ~ormation of fibrin did not start u~til about 1 to 2 min. afte~ the precipitate had hec~e completely dtssolved, and accord~ngly more than enough time for the suc~ing procedure. After termi~ation of the formation o~ fibrin in the syringe, the thro~bin 2s solution could be expelled by squeezin~ the sponge by means of the piston of the ~yringe, the fibrln remaining depositing on the large surface of the sponge.
The dissolved precipitate had a pH-value of 6.5. other amounts of Na2C03 or another b~se or a buffer system can be used provided the pH-value is between 6.0 and 7.5, but an optimum balance between the keeping ~alities of the thrombin and the capacity of the t~rombin to accelerate the coagulation process is found at 6.5.
..
Wog~tO0566 PCTtCB93/01323 T~e thrombin was expelled ~rom the ~yringe aftPr 30 min, and a concantratlon Or 256 NIH units per ml was obtained. The thrombin concentration lncreaged by t~me, but after 30 to 60 min a suf~iciant amount of s thro~in was obtained ~or a conYentlonal use in a ~ibrin sealant.
T~e preparation of thrombin was in the present Example produced ~rom 12 ml Or autolo~ous blood and was terminated over a per$od or 45 to 60 min, i.e., almost simultaneously with the termination Or the preparation Or ~ibrinogen. ~he pro~uced amount o~ thrombin was ~ufficient for being used in combination with fibrinogen produced from 45 ml of autologous blood in the manner described in lS WO9l/17778.
The fibrin formed during the thrombin preparation has always a tendency to bind the thrombin. The release o~ this thro~bin can, h2wever, be promoted by the addition o~ a plasminogen catalyst, 5UC~l as streptokinase, urokinase or t-PA ~tissue plasminogen catalyst) optionally ad~ixed with a physiological solution.
Accordingly, there is the need for a sealant that utilizes throm~in that can be delivered to a patient without the risk o~ ~iral contamination or oeher ad~erse a~ects 3. SUMMARY OF TXB INvENTIO~
The subject invention relates to a composition ~or use in a medical procedure in an ~n i ma 1, which composition is a thrombin cont~; ni n~ blood fraction which 20 comprises:
(a) a thro~bin co~centration of from about 1 NIH unit/~l to about 2,000 ~I~ unit~/ml, and (b) a specific acti~ity o~ thrombin o~ from ~ 25 about 1 NI~ unit/mg o~ blood protein tO
about 200 NIH units/mg of blood protein, wherein said thrombin blood fraction ls substantially free o~ active antithrombin III.
The subject invention also relates to a method for preparing a prothrombin blood ~ractlon ~rom whole blood comprising:
.
W094/00566 PCT/GB93~01323 ~a) diluting whole blood to an ionic 5trength of less than about 100 millimolar;
(b) separating plasma from said whole blood;
(c) lowering the pH of said pla~ma to precipitate a prothrombin blood fraction; and (d) separating ~aid pro~hrombin blood ~raction from said plasma.
The prothrombin blood fraction is then redissolved and converted to thrombin, i.e., the thrombin blood fracti~n i~ formed, which can then be utilized i~ a medical procedure in an animal, e.g., as 15 a component of a fibrin sealant .
4. DE'rAILED DESt:~RIPTION OF ~HE INVENTION
The ~ubject invention relates to the use of a thrombin blood fraction, as deflned hereinbelow, in 2 a a medical procedure, e.g., as a co~ponent of a flbrin sealan~, in an ~ni~al, preferably a mammal. Suitable mammals include a human, a cow, a pig, a dog and a rabbit, or other ~a~mals that have an adequate blood volume to prepare the thrombin blood fraction. The thrombin blood fraction can be prepared ~rom whole blood and is impure ~n that it contains blood proteins other than thrombin. However, the thrombin blood fraction can be prepared very simply and rapidly, e. g ., in less than about one or two hours, and is believed to be as efficacious as a highly pure thrombin preparation.
The thro~bin blood Yraction can be prepared from whols blood. It is Preferred that the whole blood be obtalned from a single individual animal.
~5 Also, it i~ preferred that the ~hrombin blood rraction W094/00s66 PCT/GB93/01323 be administered to the 6ame ind~ ~idual animal Yrom which the whole blood was taksn. Thus, one aspect of the ~nven~ion is the use of an autologous thrombin blood fraction in a medical procedure. ~n this S embodiment, there l~ also no ri~k o~ transmlssion of blocd-transmitted lnfections becaUse the throm~in blood ~raction i~ to be admini5tered to the same individual animal that donated the whole blood. Al~o, for the ~ame rea60n, the blood proteins other t~an thrombin ~hat are present in the thrombin blood fraction would not be antigenic.
Furthermore, since the thrombin blood ~raction is pre~erred to be prepared from a ~ingle individual animal and used in the same, general~y small volume~ of whole blood are required, especially since it is also pre~erred to prepare a thrombin blood fraction ~or a single use. Also, it is prefe~red to prepare the ~raction within ~everal hours of the time of use. Tt i~ pre~erred that from about lO ml to about 50 ml, more pre~erably from about lO ml to about 30 ml and most preferably from about l~ ml to about 20 ml of whole blood be utilized to prepare the thrombin blood fraction of the subject invention.
The throm~in blood fraction of the subject invent~on ha~ a thrombin concentration of from about l NIH unit to about 2,000 NIH units, preferably from about lO0 NI~ units to about 800 NIH units and most preferably from about lO0 NIH un~ts to about 500 NIH
units per ml of the thrombin blood fraction.
It is believed that at such concentrations the thrombin blood fraction possesses a ~ufficient thrombin concentration ~or the desired medlcal u~e.
of course, the preferred thrombin concentration depends on the medical use o~ the thrombin blood ~5 frac~ion.
~ .
W094/00566 PCT~GB93/Ot323 The thrombin concentration oY the thrombin blood fraction can be determined by measuring the coagu~ation time of a 8tandard fibrlnoqen solut1on after addition o~ the thrombin blood fraction in a suitable diluted form. As a reference, st~ d thrombln ~olution6, containing from 2 to 15 NIH
unit~/ml, can be utilized.
The throm~in blood fraction of the subject invention has a speci~ic activity of thrombin o~ from about 1 NIH unit to about 200 NIH units, preferably from about 5 NIH units ~o about 100 NIH units and most prefera~y from ab~ut 5 NIH units to about 50 NIH
units per mg. of total blood protein. Such lower specific activities o~ the thrombin blood fraction are believed to be as effective in a medical procedure as the more purs thrombin blood fractions. ~ow~ver, such lower specific activity of the thrombin blood fractions can be prepared more readlly.
The specific activity of the thrombin blo~d fraction is, in essence, a ~e2surement of the amount o~ thrombin per amount of ~lood protein in the thrombin blood fraction. Thus, the specific activity of the throm~in blood fraction o~ the subject -invention is quite low relative to thrombin preparations that have been prepared heretofore. F~r example, United States Patent No. 5,143,838 discloses a throm~in preparation with a specific activity of at least 800 NIH units per mg. of total protein. Also, United States Patent No. 5,151,355 discloses a thrombin preparation with a ~peclfic activity of greater than 1,000 NIH units/mg. of total protein.
Although the speclflc activity of the thrombin blood fractlon of the sU~iect is low, never the less it is believed that the blood fracti~n is e~icacious for use in medical procedures.
_, Thc ~pecific activity of the thrombin blood fractlon o~ the sub~ect invention can be calculatad by mea~uring t~e thrombin concentration ~H un~ts/ml) and by dividing that number by the protein s concentration (mg/ml) mea~ured by a~y ~tandard proteln a~say, e.g., W abcor~ance.
The thrombin blood fraction i5 also ~ub~tantially ~ree of active antithrombin I~I. For the purp~se of the subject invention, "substantially free of active antithrombin III" means that the thrombin blood fraction per unit volume contains an amount of active antithrombin III that is le~ than about 50~ by activi~y ~f antithrombin I~I in ~ormal pla~ma per unit volum~. It is preferred that ~uch percentage be les~ than about 30%, more preferably less than about lO~ and mo~t preferably less than about 5~. It is essential that the antithrombin III
elther be removed from or in-_~ivated in the thrombin blood fraction. Otherwi~e, antithrombin I~I will prevent the conversion of the p~othrombin in the blood fraction to thrombin and/or inacti~ate the thrombin that is formed.
It is preferred that the thro~bin blood fraction also be ~ubstantially free of fibrinogen and fi~rin. For the purpose o~ the sub~ect invention, substantially free of fibrinogen and fibrin means that the thrombin blood fraction per u~it volume contains an amount of fibrinogen plus fibrin that is less than about 1% by weight of fibrinogen in normal plasma per unit volume. It is preferred that fibrinogen not be present in the thrombin blood fraction because the thrombln will convert --he fibrinogen to fibrin, which will polymerize to fo; a clot, thereby rendering the thrombin blood fraction lmpractlcable. The f ibrinogen ~s itsel~ ~an be removed from the thrombin blood fractlon W094/00566 PCT~GB93/01323 or the ~ibrinogen can be removed a5 the fibrin clot, there~y, o~ course, also removing the fibrin.
However, the fibrinogen need not be removed if, for example, the thrombin is inactivated as described in PCT publication No. W09l/09641.
The throm~in blood ~raction can be ut~llzed Lmmediately after it ls p~epared. I~ the fraction is not utllized i~ediate~y after it5 preparation, the fraction c~n be stored. Storage of the fraction requires that the fraction be preser~Pd ~y, ~or example, freezing or lyophilizin~ the fraction or holdin~ the composition at 4~C. The fraction in frozen or ~yophilized form wil} ~e stable for a period o~ months. When the fraction is held at ~~C, it is stable for at lea~t a period of days.
I~ the fraction is frozen, the fraction must be thawed at the time o~ use. I~ the ~raction is lyoph~lized, at time of use, it is pre~erred that the ~raction be reconstituted by addirion of ~istilled water.
The thrombin ~lood fraction can be in vlrtually any form, for example, ~ solution, suspension, emulsion or solid, with a solution being pre~erred. Thus, ~or example, such fraction can ke a liquid, gel, paste or salve. Also, of course, the ~raction can be in the form o~ a ~ranule.
If the thrombin blood Çractlon is in a solid for~, ~hen the concentration of the thrombir. blood fraction can be deternined ~y dissolving i~ in a solution and then measuring the thrombln concent-ation. If the resulting t~r~.mDi.
concentration is from about l NI~ ~nit~ o abou~
2,000 NIH units/ml, then the thrombin blood fractlon is wlthln the scope of the subjec~ invention.
W094~00s66PCT/GB93/01323 _ 9 _ 4.1. MET~OD FOR PREPA~ATIO~ OF THE
THROMRIN ~QOO FRACTION
The thrombin blood frac~ion of the ~ubject inYention can be prepared by any method known or to be deYeloped. Alco, the throm~in ~lood ~raction can be prepa~ed in a device as descr$bed ln the PCT publication No. WO91/17778.
4.1.1. MET~OD FO~ PREPARATION OF TXE THROMBI~
B~OOD FRACTION BY FORM~TION OF T~E
EuGLo8uLIN FRACTION FROM PLASMA
Whole blood can be withdrawn from an indiYidual ani~al, e.g., a human, and preferably in the prese~ce of an anticoagulant. Any anticoagulant ~5 can b~ utilized so long as it does not ac~ by dlrec~ly inactivating thro~tn~ Su~tabla anticoagulants are hepari~, EDTA, citrate or any other agent that can, direc~y or indirectly, prevent the fornation of thrombin, with citrate being preferred.
The plasma, wh~ch contains the prothro~hin, is then separated ~rom the whole blood. Any separation technique can be utilized, ~or example, ~ediment~tion, cen~ uga~ion or i ltration.
Centri~ugation can be car~ied out at about 1,500 to 25 about 3,0~0 g. ror a~out 10 minutes. T~e supernatant, which contains the plasma, can be re~oved by standard ~echniques. If it is desired to obtain a thrombin blood fraction that contains srowt~ ~actors, then such centrirugation should ~e at about ~25 g. for about 20 30 mlnutes or 1,000 g. for abou~ 2 to 3 ~i~utes. The thrombin blood fracLion cf t~e subjec~ ~nvention will then contain gro~h ~actors, which are released from the platelets during the converslon o. p-othrombin to thrombln.
W094/00s66 PCT/GB93/01323 The plasma in then treated, for examp~e, by dilution with distil~ed water, followed by the addition of acid, e.g., citric acid, to lower the ionic strength to les~ than about 100 milli~olar, preferably less than about 50 millimolar and mo~t preferably ~rom about 20 to about 40 millimolar and lower the pH to about 4.5 to a~out 6 and preferably to from abo~t 5 to a~out 5.5. Lactic acid or acetic acid are also 6uitable acids. Generally, a weight ratio of the plasma: distilled water or acid of from about 1:5 to about 1:50, with 1:10 being preferred.
This treatment, i.e., the lowering o~ the ionic ctrength and pH o~ the plasma, results in t~e for~ation of a precipitate that i~ generally referred SS to as the "euglobulin fraction." The euglobulin fraction contains the prothrom~in, fibrinogen and many other blood proteins, but is substantially f~ee of an~ithrombin II~.
Rather than diluting the plasma to lower the ionic strength, and before acidifying ths plasma, the ionic strength can be lowered by dialyzing the pla~ma by placing the plasma in a dialysi~ bag, which is then placed in distilled water. The dialysis permits the ions to di~fuse out o~ the plasma, thereby lowerin~
the ionic strength. A ~;uitable dialy~is bag i6 composed of cellulo6e nitrate. Also, the ionic strength can be lower~d by dia~iltration or occlusion chromatography.
The euglobulin fraction can be prepared as described in A. Quick, Production o~ ~hrombin From Precipitate Obtained by Acidification of ~iluted Plasma, Am. J. Physiol., 1~3:114-118 (1955) and R.
Biggs and R.G. Macfarlane, ~luman Blood Coagu~ation, pages 3î5-376, Blackwell6 Scientific Publica~ions, W094t00566 PCT/GB93tOt32 Oxford, 3rd Edition (1962).
T~e AYrecc fluid can then be ~eparated ~rom the euglobulin fraction ~y, for example, centrifugation, ~iltration or sedim~ntat~ on.
Centrifugatlon can be carried at about 1,500 g. for about 2 to abou~ 5 mi~ute~.
The pro~hrom~in of the euglobulin f raction ia ~hen redissolvsd and conver~ed to thro~bin, thereby 1~ forming a throm~in blood fraction o~ the subject in~entlon. This c~n be carried out by solubi~izing the euglobulin fraction in a physiologically accepta~le solutio~, e.g., saline, in an amount equal to or preferably less t~an (about 10~ the original amount o~ plasma. An alkaline hu~er can ~e added in an amount to raise the pH of the 601ution to a~out 6 ~o about 8 and preferably to about 6.5 to about 7.5.
N~nlimi~ing examples o~ suita~le alkaline ~uf~ers include sodium hydroxide, potas~ium hydroxide, calcium hydroxide, bicarbonate buff ers such as sodium bicar~onate and potassium bicar~onate, salts o~ acetic acid a~d salts of sulfuric acid. Preferred alkaline buffers include: Sodium carbonate/bicarbona~e p~ 7.o, Sodium bicarbonate~Na~H pH 7.~, 1.5M Glycine/NaOH pH
6.5-7.5, Bi6 hydroxyet~ylaminoethane ~ulphonic acid (BES) pH 7.5, ~.ydroxyethylpiperazine propane sulphonic acid tEPPS) pH 7.5, Tricine pH 7.5, Morpholino propane sulphonic acid (MOPS) pH 7.0, Trishydroxymethyl aminoethane sulphonic acid (TES) p~ 7.0 and ~o Cyclohexylaminoethane sulphonic acid (CHES) pH 7.0;
with Sodium carbonate/bicar~onate pH 6.5 - 7.5 Bis hydroxethylaminoethane sulphonic acid (BE5) pH 7.5, ~ydroxyet~ylpiperazine propane sulphonic acid (EPPS) pH 7.5 and Trishydroxy~eth~l aminoet~ane sulphonic ~5 dcid (TES) pH 7.5 being most preferred.
W094/00566 PCTtGB93~01323 Calcium i~ added to the neutral solution in order to convert the prothrombin to thrombin. ~f course, ~he calcium can be part of the alkaline buffer. Calcium can be added in the form of, for S example, calcium chloride. Th2 amount of calcium added should be sufficient to convert an amount of prothror~in to thrombin that i6 6ufficlent for the intended medical use. Furthérmore, the reaction should be permitted to occur ~or a period of ti~e ~0 suf~ici~nt to convert enough of the prothrombin to thrombin that is suf~icicnt for the inte~ded medical use. Generally, from about 5 millimolar to about 50 millimolar calcium chloride i~ suf~icient.
~lso, rather than adding a soUrce of calcium ~5 ions, prothrombin activating enzymes from snake venoms can be utilized. For example, snake venom fro~ Eccis carinatu6 or the Australian Tiger snake can be utilized.
As the thrombin forms, i~ convert~ the fibrinogen to fi~rin, which forms a fibrin clot. It is preferred to remove the fibrin clot, which can be carried out by, for example, wrapplng the fibrin clot around a ~tirring rod or collecting the fibrin onto glass beads.
4.1.2. METHOD FOR PREPARATION OF THROMBIN BLOOD
FR~CTIoN BY DILUTING WHOLE BLOOD
In a preferred embodiment, the thrombin b}ood fraction can ~e prepared by initially preparing a prothrombin blood fraction from whole blood comprising:
(a) dilu~ing whole blood to an ionic ~trenqth of less than about 100 millimolar;
(b) separating plasma fro~ said whole blood;
(c) lowering the pH o~ ~aid plasma to precipitate a prot~rombin blood fraction; and (d) separating said prothrombin blood 5 fraction from said pla~ma.
The prothro~bin blood fraction is then redissolved and converted to thrombln, i.e., the thrombin blood fraction of the subjec~ invention is formed, which can then be utilized in a medical proce~re in an animal, e.g., as a component of a ribrin ~ealant. This method provide8 a thrombin blood fraction of the subject invention, which can be prepared in only about 45 minutes!
Specifically, who~e ~lood is drawn from an individual animal. The whole blood is then i e~iately (within about five minutes) ~iluted in order to lower the ionic strength to less than about lO0 millimolar, preferably less than about 50 millimslar and preferably to from about 20 to about 40 millimolar. It should be noted that since the whole blood i~ diluted immediately, there i~ no need ~or the u~e of an anticoagulant. Any physiologically acceptable solution at physiological osmotic préssure can be utilized to lower the ionic strength, e.g., a glucose aqueous ~olutlon such as a 5.5% isotonic a~ueous glucose ~olutio~.
The plasma is then separated from the whole blood. Any separation technique can be utilized, for example, ~edtmentation~ centrifugation or filtratlon.
Centrlfugation can be carried out at about 1,500 to about 3,000 g. ~or about 5 to about lO minutes. The ~upernatant, which contains the plasma, can be removed by standard technlques.
The plasma fractlon is ~hen acidified, thereby resulting in the formation of a prothrombin blood fraction, which is generally referred to a~ the euglobulin fracSion, which is a precipitate. The plasma fraction can be acidi~ied with, for exa~ple, citric acid, lactic acid or acetic acid. The pH
S should be lowered to from about 4.5 to about 6 and pref erably to ~rom about 5 to about 5.5.
The excess ~luid can then be separated from the prothrom~in blood ~raction by, for example, centrifugatlon, filtration or se~i~entation.
Centrifugation can be carried out at about ~,SOO g for about 2 to about 5 minutes. This prothrombin blood ~raction contains prothrombin, fibrinogen and many other blood proteins, but does not contain antithrom~in III. The prothrombin blood fraction is then redis~olved and the prothrombin is converted ~o thrombin. Any physiologically acceptable solution, e.~ aline, can be utilized to redissolve the proth~ombin fraction. Furthermore, only a cmall ~olume of solution is required to redissolve the prothrombin fraction. It is believed that only from about O.4 ml to abo~1t 1 ~1 o~ solution i8 required i~
about 1~ ml of whole blood was initially drawn. An alkaline buffer can be added in an amount to raise the pH of the ~olution to about 6 to a~out 8 an~
preferably to about 6.5 to abo~t ~.5. Nonlimiting examples of suitable alkaline buffers include sodium hydroxide, potassium hydroxide, calcium hydroxide, bicarbonate buffer~ such as sodium b~carbonate and potassium bicar~onate, tri-metal salts of cltric acid, salts Or acetic acid and salt~ o~ sul~uric acid.
Preferred alkaline buffers include: Sodium carbonate/bicar~onate pH 6-~, Sodium bicarbonate/NaOH
pH 6-8, Glycine/NaO~ pH 6-8, Bis hydroxyethylaminaethane sulphonic acid (BES) pH 6-8, Hydroxyethylpiperazine propane sulphonic acid (EPPS) _, W094/00~66 PCT/GB93/01323 pH 6-8, Tricine pH 6-8, Morpholino propane sulphonic acid (~O~S) pH 6-8, Trishydroxymethyl amlnoethane ~ulphonic acid (TES) pH 6-8 and Cyclohexylaminoethane ~ulphonic acid (CH~S) pH 6-8; with Sodium S carbonate/bicarbonate pH 6-8 Bis hydroxethylaminoethane sUlphonic acid (~ES) pH 6-8, Hy~roxyethylpiperaZine propane sulphonic acid (EPPS) pH 6-8 and TrishydroXymet~yl aminoethane ~ulphonic acid (TES) pH 6-8 being most pre~erred.
Calcium ls added to the neutral solutlon in order to convert the prothrombin to thrombin. of course the calcium can be part of the alkaline buffer.
Calcium can be added in the form of form of, for example, calcium chloride. The amount of calclum added should be sufficient to convert an amount o~
prothrombin to thrombin that i~ sufficient for the intended medical use. Furthermore, the reaction should be permitted to occur for a period of time sufficient to convert enough of the proth~ombin to thrombin that is sufficient for the intended medical u~e. Generally, from about 5 millimolar to about 50 millimolar calcium chloride is sufficient. It has been observed that at a~out 25 minutes of reactlon sufficient amounts of thrombin are formed ~or most 2s medical uses of the thrombin blood fraction of the subject in~entio~.
Also, rather tha~ adding a source of calcium ions, prothrombin activatin~ enzymes from snaXe venoms can be utilized. For example, snake venom from Eccis carinatus or the Australian Tiger sna~e can be uti}ized.
A~; the thrombin forms, it converts the ~ibrinogen to fibrin, which forms a fibrin clot. ~t is preferred to remove the ~ibrin clot, which can be ~s carried out by, for example, wrapping the fibrin clot W094/00566 PCT~GB93/01323 around a stirring rod or coll~ction of the fibrin onto glasa beads.
4.l.3. PREPARATION OF THROMBIN BLOOD
F~ACTIO~ ~Y REMOVING OR INACTIVATING
ANTIT~RoMBIN ~I~ FROM PLAS~
In an alternative method, the thrombin ~lood fraction of the ~ubject invention can be prepared by withdrawing whole blood ~rom an individual ~nimal, e.g., a human, and preferably in the presence o~ an l anticoagulant. Any anticoagulant can be utilized.
Suitable anticoagulants are heparin, EDTA, citrate or any other agent that can, directly or indirectly, preve~t the for~ation of thrombin, with citrate ~eing preferred.
'rhe plasma, which contains the prothrom~in, is then separated from the whole blood. Any separation technique can be utilized, for example, sedimentation, centrifuqation or filtration.
Centrifugation can be carried out at a~out 3,000 g.
2 for about lO mlnutes. The supernatant, which contains the pla5ma, can be removed by st~ rd techniques.
Antithrombin III is then removed from the plasma or is inactivated. For example, the pH of the plasma can be lowered to at least to about 5. Such lowerinq of pH inactivates antithrombin III; otherwise antithro~bin III would prevent the conversion of prothrombin to thrombin. Antithrombin III can be inactivated by any technique. For example, by the addition of 0.05 ml o~ 5 mol./liter ~Cl per l.o ml plasma. After about l0 to about 20 minutes of incubation, the plasma can be neutralized with 0.05 ml of 5 mol.Jliter ~aOH per ml oY plasma.
Rather than inactivating the antithrombin II', the an~lthrombin III can be removed from the plasma by pas~ing the pla~ma throu~h a column that CA 02263800 l999-03-l7 binds antithrombin III, e.g., a heparin column or a column with anti~o~le~ to antithrombin III.
The plasma fraction, which contains prothrombin, is then treated to convert prothrom~in to thrombin. This can be carried out by, for example, the addition of a source o~ calcium ions, as described above, or by the addition o~ O.l ml CaCl, of 0.36 mol.~liter per ml of plasma. Also, rather than adding a source of calcium lons, prothro~bin acti~ating enzymes from snake venoms can be utilized. For example, snake venom ~rom Eccis carinatus or the Australian Tiger snake.
As the throm~in ~orms, it converts the fibrinogen to fibrin, whlch forms a fibrin clot. It i5 preferred to remove the fibrin clot, wh~ch can be carried out ~y, ~or example, wrapping the fibrin clot around a stirring rod or collection o~ the fibrin onto glass heads.
The resulting plasma is a thrombin blood fraction of the subject invention.
4.l.4. METHOD FOR THE PREPARATION OF A
THRO~BIN BLOOD FRACTION BY DILU~ING
WIT~ AN ACID sorl~T~o~ ~RECTLY
By the known process ~or preparing thrombin 2 from plasma, plasma i5 diluted in the ratio l:lO with water, wherea~ter a pH-red~cing acid is added, such as - acetic acid, with the result that the pH-value is about 5.0 to 5.3. The mixture is then centri~uged ~or 20 ~in. at 2,0~ ~. The resulting precipitate contains d~fferent coaqulation ractors, such as inter alia prothrombin and fibrinogen. When the excess fluid has been removed, the precipitate i5 dissolved in a p~ysiological solutlon, pre~erably a 0.9~ sodium chloride solution, wherearter a p~-value-increasing a~ent, such as sodium carbonate is adc~ed unti~ the W094/00~66 PCT/GB93/01323 pH-value i5 about 7.9. When the precipitate hac been ~issolved, calcium chloride is added and cause~ the conversion of the prothrombin into throm~in by a conventional, so-called coagulation cascade. The resulting thrombin causes a converslon o~ rincgen into fibrin, wherea~ter the thrombin is separated by centrifuging and then su~jected to a s~ccee~tng purirication ~column purification).
Another a~pect o~ the subject invention ts that the diluting step is performed directly with a diluted acid solution, e.g., 0.4~ acetic acid, ~y the centrifuging step being performed in a container with a relatively large flat precipitation surface, by the physiological solution, with. the agent for increasing pH, and the calcium chloride being added at the came time to the precipitate as a mixture, whereby ~aid precipitate is dis~olved and then coagulates while f~rming ~ibrin, and by the throm~in then being removed .
As a result, it is now possible to prepare thrombin ~rom plasma of autologous blood in a relatively quick way, which is in particular due to the fact that the precipitation is per~ormed in a container with a relatively large flat precipitation 2s surface. A quick and complete dissolution is thereby ensured of the precipitate containing prothrombin and ~i~rinogen. The complete dissolution i~ important before the presence of calcium chloride causes the conversion of prothrombin to thrombin. A too early ~o thrombin formation leads to fibrin formation and the fibrin will ~ind the added fluid, whereby the dissolution of the precipitate ~tops and cannot continue untll ~aid fluid has again been passed out of the ~i~rin. The relatively small precipitate with a ~5 relatively large surface area relative to the amount causes a complete dissolving of ~aid precip~tate by addition of the combined uixture of physiological solution, Wi~h the agent for increasing pH, and calcium chlorids before the thrombin has been formed S through ~aid coagulation cascade and starts the forma~ion of fibrin.
Moreo~er ac~ording to the invention, the mixture may be admixed with a plasminogen cataly~t, ~uch as streptokinase, before being added to the precipitate with the effect that the ~ollowing separation of thrombin from fibr~n is promoted.
According to the invention, the ~ixture added to the precipitate can advantageously be set to increase the pH-value to 6.5, whereby the resulting active thrombin is found to obtain the best keeping qualities as the thrombin has a tendency to become inactive on s~anding, which is usually the case in connection with enzymes. In addition, the dissolved precipitate can be transferred to a f~exible material before the coagulation starts, said f~exi~le material presenting a relatively large surface upon which the fibrin resulting ~rom the coagulation can be deposited, whereafter the thrombin may be pressed OUt of the fibrin by the flexible material being subjected to a compaction. Exactly the obtained complete dissolving o~ the precipitate prior to formation of fibrin turned out to allow a quick transfer of the solution to a flexible material prior to said formation of ~ibrin. The flexible material ensures that the fibrln is deposited across a particularly large surface, the compaction of which facilitates the separation of thrombin from fibrin.
It is particularly prererred when the flexible material i~ a sponge with open pores .
WO94fOo566 PCT/GB93/01323 -- Zo --The ~lexible material can be placed in a syringe, into which the dis301ved precipitate can be absorbed and from which the throm~ln can be squeezed out by an acti~ation of the piston of the syrlnge. In this manner, a particularly easy separation of the thrombin from the flexible material is en~ured, Moreover, the centri~uging may according to the invention suitably be per~ormed at about l,Soo g for about 5 min, which algo accelerates the preparing 1~ of thrombin.
Thus, the sub~ect invention co~prises a process for preparing throm~in f~o~ human blood plasma, whereby the blood plas~a is diluted to about 10 to 17% with water or another ion-intensity-reducing ~5 fluid and an acid to reduce pH to a~out 5 . O to 5 . 3, whereafter the mixture is centri~uged, and ~hereby the precipitate resulting from the centrifuging is admixed a physiological solution and calcium chloride, ~aid physiological solution increasing pH to about 6 to 8, characterized by the diluting step being performed directly with a diluted acid solution, by the centrifuging step being performed in a contalner with a relatively large plane precipitation sur~ace, by the physiological solution, with the agen~ for increasing pH, and t~e calcium chloride being added at the same time to the precipitate as a mixture, whereby said ~ precipitate is dissolved and then coagulates while formin~ fibrin, and by the thrombin then being removed from the fibrin.
3~
W094/00566 PCT/GB93tOl323 4.2. ACCELERATIO~ OF TXROMBIN FORMATIO~
BY CONTACTING BLOOD OR ~LASMA WITH
A SU~FACE THAT ACTIVATES aLOOD
COAGUT~TIO~ F~CTORS XI .~ND XII
Another aspect of the sub~ect in~entlon is the preparation of a thrombin blood fractlon wherein whole blood or pla~ma is contacted with a surface that activates blood coagulation factors XI and XII. It is believed that a negatively charged surface such as glass or kaolin, with glass being preferred, ca~
~0 provide such activation. Nonlimiting examples of glass sur~aces are ~la~s beads, glass wool, glass filters and glass capillary tubes.
The whole blood or plasma should contact the surface for a period of time sufficient to activate such blood coagulation ~actors, e.g., for a~out 5 to a~out 10 minutes. If the whole blood or pla~ma has not been treated with an a..;.coagulant, then the whole blood or plasma should be in contact with such a surface for not ~ore than about 60 seconds, preferably less than about 30 seconds and more prefera~ly about 15 seconds. Without the anticoagulant, and i~ the contact of the whole blood or ~lasma wit~ the surface is too long, then fibrin clots will form prematurely.
It is preferred that p~as3a be exposed to a z5 surface of about 4 cm2 to a~out 60 c~l, pre~erably ~rom about lO cm' to about 30 cm~ and most preferably about - 20 cm2 of such surface for each millili~er of plasma.
Also, it is preferred that whole ~lood be exposed to a surface of from about 2 cm- to about 30 cm-, preferably fro~ about 5 cm2 to about 15 cm2 and most pre~erably about lO cm- of such surface for each ~illiliter of whole blood.
It is believed that the con~acting o~ t~e plasma or whole blood with a ~ur~ace that can activate fac~ors XI and XII accelerates the conversion time of prothrombin to throm~in. Thus, a thrombin blood fraction can be prepared in an extremely chort period o~ time.
This activation of factors XI and XII can be S utilized to accelerate the preparation of any thrombin blood fraction, regardleSs of how the ~raction is ma~e and regardless of its purity and specif~c activity.
~lowever, it i5 essent~al that the thrombln blood fraction be substantially free of active antithro~bin III~ It is eqsential that the antithrombin III either be removed from or inactivated in the thrombin blood ~raction prior to converting the prothrombin to thrombin. Otherwise, antithrombin III will prevent the conversion of the pr~thrombin to thrombln and/or ~S inactivate the thrombin. It is also preferred that the thrombin blood fraction be ~ub~tantially free of ~ibrinogen and fibrin. For example, with respect to the activation when the thrombin blood fraction is prepared by mean~ o~ the euglobulin fraction as described in Section 4.1.1., the contacting of the plasma shou~d be carried out immediately prior to the dilution of the plasma. When a thrombin blood fraction is prepared by diluting whole blood, as described in Section 4.1.2., the contacting o~ the 2s whole blood should be carried out immediately prior to the dil~tion of the whole blood. For example, this contacting step can be carried out by withdraw~ng who~e blood into a syringe that contains, ~or example, glass beads. After not more than about 60 seconds, the whoie b~ood is discharged from the syringe and the thrombin blood ~raction o~ the subject invention i~
then prepared, as descrlbed above.
wos4/00s66 PCT/G~93/01323 4.3. THE USES OF T~E 'rXROM~N BLOOD
FRAC'rION OF T~E SUBJECT INVE~TIO~
~ he thrombin blood fracti~n o~ the su~ject invention can be utilized in any medical procedure, known or to be developed, in an an t mal, t ncluding vetinary procedures. Any species of animal is suitable, but, o~ course, humans are preferred.
The thrombin blood fraction can be employed as a component of a ~ibrin sealant or can be employed alone just as conventlonal th~ombin preparations have been employed. The thrombin blood fraction is utilized by contacting the des t red site of the animal with the thrombin blood fraction. For the purpose of the subject tnvention, the "desired site" is that location in or on an animal where one desires to for~
a ~i~rin clot. What or where the desired site is depends on the use of the thrombin blood ~raction of the subject invention.
The us~ of the thrombin ~lood fract~on as a component of a fibrin sealant, can be utilized for connecting tissues or organs, stopping bleeding, healing wounds, sealinq a surgical wound, use in vascular surgery lnclude providin~ hemostasis for stitch hole bleeding of distal coronary artery anastomoses; le~t ventricular suture lines; aortotomy and cannulation sites; diffuse epimyocardial bleeding seen in reoperatlons; and oozing from venous bleeding sites, e.g. at atrial, caval, or riqh~ ventricular levels. The subject invention is also useful for sealing of dacron artery grafts prior to grafting, sealing tissues ou~side the body, producing fibrin rafts for cell growth, stopping ble~ding ~rom damaqed spleens (there~y saving the organ), livers, and o~her parenchymatous orqans; sealing tracheal and bronchial 3g anastomoses and air leaks or lacera~.ions or the lung, sealing bronchial stumps, bronchial fistula~3 and WO 91/00566 PCTJGBg3/01323 ~orh~eal f~stula~; for suturele~s ~eamless healln~
(~Zipper" technique), and embolizatlon 1~ v~sc~ r radio~ogy of intracerebral AV~'s, llver AV~'s, ang$0dy6plasia of colon, esophagsal varices, "pumping"
GI bleeders secondary to peptic ulcers, etc. The su~ect inve~tion is further u~eful for prcvid~ng hemostasis ~n corneal transplants, no~ebleeds, post ton~llectomie~, teeth extractions and other applications. See G.~, Gestrin~ and R. Lermer, V~-~r~ r Surgery, 294-304, Sept./Oct. 1983, The thrombin blood fraction of the subject invention can be emp~oyed alone to staunch oozinq hemorrhages or hemorrhages Ln hollow or~ans. The t~rom~in blood fraction can also be uti~ized in the ~5 treatment or dama~ed li~e ani~al tissue by utilizinq the fraction to actiYate the release of the materials, i.e., plated-derived factors, ~ro~ platelets, wherein such materiAls can be utilized to heal damaged tlssue.
See United States Patent No. 5,165,938. The thrombin ~lood fraction can also ~e utll~zed to assist in the cell culture srowt~ of keratinocytes and to assist in t~e auto~ogous transplantation o~ keratinocytPs, or any other skin-derived cells, e.q., fi~roblasts. See V. Ron~ard e~ al., Burns 17:~81-18~ (1991), H. 9ro~y, 2s Canadian Patent ~o. 2,018,020 and ~. Hu~yadi et al., J. Der~atol. Sur~. oncol. 1:~5-78.(1988).
Also, the thrombin blood Frac~-n can ~e placed on a solid support, e.g., bandage, su~ure, prosthesis, or dressing, that ~ill be in contact ~~ith 3~ the deslred site. Such support is then place~ ir.
con~act ~lth the desired site until, ~or example, -he ~ibrin clot ~orms.
The dosage of the thrombi~ blood fraction depends on its particular use, but the dosage should be an e~ective amount ~or the composition to perform its intended u~e. Generally, it is believed that from S a~out 0.5 ml to a~out 5 ml of' the thrombin blood fraction is sU~icient. However, depending on the use, the dosage can range fro~ about 0.05 ml to a~out 40 ml.
If the thro~in ~lood fraction is utilized as a component o~ a fibrin sealant, then the fibrin soalant can be applied to the des~red slte with, for example, a double-barrelled syringe. The double-barrelled ~yringe can be Y-shaped, thereby permitting the mixing of fibrinogen and the thrombin lS blood fraction immediately prior to the contacting step. Also, rather than a Y-shaped double-barrelled syringe a dou~le-barrelled syringe with two openings can be utilized. This permits the simultaneous contacting of the desired site. Also, the compositions of the double-barrelled syringe can be sprayed onto the de~ired site. See H.B. Kram et ~1., The Amcrican Surgeon, 57;3~1 (1591). Also, if the blood fraction is emp}oyed as a component of a fibrin sealant, then autologous fibrinogen ca~ be utilized, thereby rendering the entire fibrin sealant autologous. Also, if the thrombin blood fraction is employed alone, then the fraction can be applied to the desired site with a single-barrelled syringe.
It should also be noted that the ~hrombin blood frac~ion of the subject invention can further comprise a source of calcium ions, e.g., calcium chloride. The source o~ calcium ions assists in the conversion o~ fibrinogen to the ~ibrin clot. The amount of calcium ions should be the same a~ that utilized in conventional ~ibrin sealants. However, W094/00~66 PCT/GB93/01323 since the throm~$n blood fraction may contain a source o~ calcium ions already due to the conver~ion of prothrom~in to thrombin, an additional sour~e of calciu~ ions may not be required. But, if more calcium iB needed to form the fibrin clot than to form thrombin, then, as an option, PYc~c~ calcium from what is required to forrD thrombin can be utilized 80 that no addi~ion~l calcium need be added when the thrombin blood ~raction is utilized in the medical procedure, e.g., as a component of a fibrin sealant.
5. ~MP~
EXaMPLE I
Preparatlon of a Composition Containing a Throm~in ~lood Fraction Obtained From 17 ml of Fresh Blood From a Human Adult Donor A puncture of the vein of a human was performed by a needle and 17 ml o~ ~lood was drawn into an e~pty 30 ml syri~ge. Tmmediately after drawing the blood, ~t was transferred to a 50 ml test tube cont~in;~ 3~ ml of a solution containing s.5 glucose. The 50 ml test tube was placed in a centrifuge and centrlfuged for 5 minutes at },500 x g at room temperatur~. After centrifugation, 40 ml of 2 the supernatant plasma/glucose solution was removed by a syringe, and tran~ferred to a new SO ml test tube.
By means of l.O7 ml of a 2.8% citric acid solution, the p~ in the plasma~glucose 501ution was lowered to 5.2 and after a period of lO minutes at room temperature, the solution was centrifuged at 18~C
at 1,500 x g ~or 5 minutes. After centrifugation, the supernatant was drained off and the precipitate was dissolved in 0.424 ml o~ a solution containing 14 mmole/L of ~aHCO3 and 8 gram~L of NaCl. This precipitate contains f ibrino~en, prothrombin and oth~r blood proteins, but is ~ubstantially rree of antlthrombi~ III. The pH of this di~solved prothrombin containing euglo~ulin solution was 7.~5.
Activation of the prothrombin was perfor~ed S by the addition o~ ~.027 ml of a solUtion cont~ining CaCl2, 0.5 mole~. From 12 to 17 minute~ after the addition of the CaCl2, the fibrinogen in t~e ~olution started to coagulate, and the fibrin thus for.~ed was r~moved by means of a polystyrene ~patula. Small lC samples were removed at dirferent intervals and the thrombin c~ncentration was measured. The re~ult~ from the example are shown i~ Table I.
Table I
15 time after lO 20 30 60 120 27 Ca-addition min min min min min hour NIH uJml 0 180 368 540 600 7~7 EXAMPLE II
Preparation of a Composit~on Containing a Thrombin Blood Fract~on Obtalned From 17 ml of Fresh Glass Activated Blood From a Human Adult Donor In this experi~ent, the donor and the day ~or the performance was the same as used i~ Example I.
A puncture o~ the vein of a human was performed by a needle and 20 ml o~ blood was drawn into a 30 ml syringe containing 20 grams of glass beads with a diameter of approximately 2 mm. The total surface area of the beads was approximately 2~0 cm~ and, therefore, the ~urrace area was about ll.5 cm2 per ml o~ whole blood.
Immediately a~ter drawinq the blood, the syringe was turned gently for lO to 15 seconds before 17 ml of the blood was transferred to a 50 ml test CA 02263800 l999-03-l7 W094/00s66 PCT/GB93/01323 tu~e containing 34 ml o~ a solution contAlni~g 5.
glucose. The 50 ml test tube was placed in a centri~uge and centrifuged ror 5 minutes at 1,500 x g at room temperature. ~fter centrifugation, 40 ml of the supernatant pla~ma/qlucose ~olut~on was removed by a syringe, and trans~erred to another 50 ml te8t tube.
By means o~ 1.07 ml o~ a 2.8% citric acid solution, the pH was lowered to 5.2 and after a period of 10 minutes at room temperature, the solution was lo centri~uged at 18~C at 1,500 x g for 5 minute6.
After centrifugation the supernatant was drained o~ and the precipitate was dis~olved in 0.424 ml o~ a solution containing 14 mmole/L of NaHCO3 and 8 gramJL of NaC1. This precipitate contains prothrombin, ~ibrinogen and other blood proteins, but is substantially free of antithrombin III. The pH of this dissolved prothrom~in containing euglobulin solution was 7.35.
Acti~ation of the prothrombin was performed by the ~ddition o~ a 0.027 ml of a solutlon containing CaCl2, 0.5 mole/L. From 4 to 9 minutes a~ter the addition of the CaCl2, the fibrinogen in the solution started to coagulate, and the fibrin thus for3ed was removed by ~eans of a polystyrene spatul~. Small 2s samples were removed at dif~erent intervals and the thrombin concentration was ~easured. The results are shown in Table II.
Table II
~C
time after 10 Z0 30 60 120 27 Ca-addition min min min min min h~ur NIH u/~l 130 444 560 720 695 8ao Thus, from Table II it i5 readily apparent that thq glass activation accelerates the time required for the conversion of the prothrombln to thrombin. For example, at only l0 minutes after the addition of a source of calcium ions, 130 NIH units/ml of thrombin activity was measured. In con~rast, without qlass activation, as in Example I, at l0 minutes after the addition Of a source o~ calcium ions, there was no detectable throm~ln activlty.
EXAMPLE III
Preparation of a Composition Containing a Thrombin Fraction Obtained From 17 ml of Fresh Blo~d From ~uman Adult Donors, Characterized by Low Specific Thrombin A~iVlty ~5 In four experiments, perfor~ed as described in Example I, the thrombin concentration and the specific activity of thrombin were measured. The results are given in Ta~les III and IV where the pH
value is the pH in the dissolved euglobulin fractions.
Table III
A thrombin measured from 15 min to 2 hours a~ter dissolution of the euglobulin fraction. NIH u/ml donor pH E-280 15 min 30 min 60 min 2 hr JH 6.96 25.8 142 483 661 779 ~JS 6.35 26.7 0 56 942 1,238 LK 6.98 l9.5 30 249 430 s62 K~ 6.21 31.5 0 142 616 725 (E-280 is a measurement of ~he total prote~n concentra~ion in mg/ml).
W094/00~66 PCT/GB93/01323 Table IV
B SpeciSic activity of thrombin measured from 15 min to 2 hours after dissolution of the euglo~ulin fraction. NIH u~mq protein donor 15 ~iA 30 min 60 ~in 2 hr JH 5.5 lg 26 30 HJS 0 2.1 35 46 LR 1.5 13 22 29 ~N O 4.5 20 23 T~us, the preparation of a thrombin blood rraction as descri~ed in Example I results 1~ a thrombin blood fraction o~ a concentration and spec~fic actiY1ty of t~e subject inventlon.
~XAMPLE ~V
Preparation of Throm~ln Fro~ Whole Blood Usin~ the Device of WO91/17778 The device described in WO91/17778 was used ~or the preparation of throm~in. ~efore the ~lood was in~roduced into the device, 40 m1 o~ a _.5 glucose solution was in~roduced i~to ~he first chambe (}4) through the filter (43) mounted on the tubins (39).
Blood, lO ml, was c~llected from human donors into a syringe and immedia~el~ ~hereafter transferre~ through the tubing (39) in~o the glucose solution. The device was placed in a centri~uge 2nd ~0 centrifuged for 5 mln at 1,500 x g. ~fter centrifuga~on, the p1asma/g1uoose solution was transferred i~to the second chamber (30) with the red cells re~aining in the chamber (1~). Through the sterile filter ln tu~e (60) 0.6 ml o~ 2.8% c~trlc ac~~
solution was introduced into the plasr.a/~lucose -~ . .
W O 94/00~66 PC~rtG B93/01323 - 3~ -solution. After 5 t~ lO min, th~ device was placed in a centrifuge and centri~uged ror 5 min at 1,500 x y.
A~ter cen~r~fugatlon the supernatant was transferred to the first chamber (14) and the precipitate, the s euglobu~in fraction, rP~e~ in ~he s~nA r~mh~r (30). Through the sterile filter in tube ~60) 0.85 ml of a colution containing 7.5 mM ~laHco3~ 52 ~ NaCl and 30 mM CaCl2 was introduced into the 6econd rh~mh~r ~30). The euglobulin precipitate wag dissolved within ~0 1-2 ~inutes, and transferred to the 6yringe (5-) connected to the ~econd rhArher ~30). The syrinye contained a polyurethane sponge ~acilitating the removal o~ the forned _ibrin. Thro~n concentrations were measured after 30 min to 22 hours. The results lS are recited ~n Table v.
Ta~le V
thron~in concentration NIH u/ml ~onor pla-cma 30 min 1 hour 2 hour 22 hour dilution RH-A 11.9% 186 Z40 231 2?9 RH-~ 12.3% 5~ l30 130 132 RH-C 11.7% l7a 186 180 19~
2s RH-D 12.5~ 104 12~ 92 123 EXAMPLE V
Preparat~on of ~h-o~in and Fibrinoqen rrc,.
Whole Blood Using 2 Device System ~'ade Fro-Two Inter-Connected Devices of 30 W091/17778, and the Use of Thrombin and Fi~rinoaen ! n a ri ~ri~ Glue The devlce syster. consists of two devices as desc-i~ed in W091/17778. The -wo tubings (39) from tne two devices ~ere connec~ed to the same .
W094/00566 PCT/GB93/Ot323 can~ula (4~) by means of a three-way connector. In the device used for the thrombin precipitation 5 glass beads, 3 mm in diameter, were placed in the second chamber (30). The syringe in the thrombi~ device was S changed from being a 3 ml syringe in the f ibrino~en device to a 1 ml syringe.
Before the ~lood was collected from the donor, cltrate and glucose solutions were ~illed into the two device~. citra~e, 5 ml of a 3.8% solution, was introduced into the first chAmhPr (14) o~ the fibrinogen de~ice (herea~ter named DeYice-F) through the filter 143) ~oun~ed on the tubing (39). Glucose, 34 ml of a 5.5~ solution was i~troduced into the firs~
chamber (14) of the thrombin device (hereafter named lS Device-T) through the filter t43) mounted on the tubing t39).
~ lood, 45 ml was collected through the cannula lnto the first chamber (14) in DeYlce-F
containing the citrate solution, and 17 ml was collected into the ~irst chamber (14) in the Device-T
containing the glucose solution. After collection of the blood, the separator-system was disconnected ~rom the donor, and the tubing (39) was sealed close to inlet ~38). Both devices were placed in a centr~fuge and centrifuged for lO min at 1,500 x g.
The separated plasma in Device-F was transferred to the second chamber ~30) and 2.5 ml of a 96% ethanol solution was introduced into the ~econd chamber (30) through the sterile filter in tube (60).
The dev~ce was now placed into a ice-water bath for 20 minutes to reduce the temperature in the plasmaJethanol solution to approximately O to 4~C. At this temperature, 85% of the fibrino~en in plasma was precipltated. The device was now placed in a centrifuge and centrifuged for 5 min at 1,500 x g.
W094/00~66 PCT/GB93/01323 The supernatant serum was transferred to the fir~t chamber ~14), and the ~olid fibrlnogen wa~ dissolved by incu~ation for 5 minutes at 37~C. The dissolved ~olution was transferred to the 5terile syrlnge (51).
The concentration of fibrino~en was measured to be 31 mg/ml.
The separated p~asma/gluco6e in Device-T was transferred into the second ch~mhPr (30) with the red cells r~in~n~ in the ~irst chamber (14). Through the sterile filter in tube t60) 1.2 ml of a 2.8 citric acid solution was introduced into the plasma/gluco~e solution. After 5 to 10 min the device was placed in a centrifuge and centri~uged for 5 min at 1,500 x g. ~fter centrifugation, the supernatant lS was transferred to the ~irst rhAmh~r (14) and the precipitate, the euglo~ulin ~raction, remained in the second chamber (30]. Through the sterile fllter in the tube (60), 0.85 ml of a solution containing 7.5 mM
Na~CO3, 52 mM ~aC1 and 30 mM CaCl. was introduced into the second chamber (30). The euglobulin precipitate was dissolved within ~-2 minutes, and the fi~rin ~ormed durin~ the activation of prothro~in to thrombin was collected onto S glass beads placed ln the second cha~ber (30). After 15 minutes the thrombin solution was transferred to the syringe (51) connected to the second chamber (30). Thro~bin concentration was measured to ~e 248-340-372 NIH u/ml a~ter 15-30-60 minutes, respectively.
The two syrin~es containing the fibrinogen and the thrombln were used as a double barrelled syringe. The two solutions were expelled from the syringes and formed immediately a ~irm ~i~rin clot.
., ~XAMPLE VI
54 ml of 0.04~ HAc were added to 6 ml of pla~ma. This mixture was placed in a flat-botto~ed container of the type known from the above S PCT/DK91/00131. The contalner is of a circular cross section with an inner diameter of 4.S cm. The pH-value was 5.3. The pH-value and the relatively low concentration of ions in the provided mixture ensure the ~ollowing precipitati~n of the coagul~tion lo factors, inter alia prothrombin, as a precipitate by a centrifu~in~. The centri~uging was performed at ~,500 g for 5 min. Thus the centrifuging was relatively ~uickly terminated, which is due to the relatively short falling height and large precipitation surface.
~5 After removal of excess ~luid, an 0.75 ml a~ueous solutlon o~ 0.9% NaC1, 0.03% Na~CO3 and 25 mM CaCl2 was added t~ the precipitate. After dissolving o~ the precipitate, the ~olution was ~ucked into a 2.5 ml syringe conta~nin~ a polyurethane spon~e. The ~ormation of fibrin did not start u~til about 1 to 2 min. afte~ the precipitate had hec~e completely dtssolved, and accord~ngly more than enough time for the suc~ing procedure. After termi~ation of the formation o~ fibrin in the syringe, the thro~bin 2s solution could be expelled by squeezin~ the sponge by means of the piston of the ~yringe, the fibrln remaining depositing on the large surface of the sponge.
The dissolved precipitate had a pH-value of 6.5. other amounts of Na2C03 or another b~se or a buffer system can be used provided the pH-value is between 6.0 and 7.5, but an optimum balance between the keeping ~alities of the thrombin and the capacity of the t~rombin to accelerate the coagulation process is found at 6.5.
..
Wog~tO0566 PCTtCB93/01323 T~e thrombin was expelled ~rom the ~yringe aftPr 30 min, and a concantratlon Or 256 NIH units per ml was obtained. The thrombin concentration lncreaged by t~me, but after 30 to 60 min a suf~iciant amount of s thro~in was obtained ~or a conYentlonal use in a ~ibrin sealant.
T~e preparation of thrombin was in the present Example produced ~rom 12 ml Or autolo~ous blood and was terminated over a per$od or 45 to 60 min, i.e., almost simultaneously with the termination Or the preparation Or ~ibrinogen. ~he pro~uced amount o~ thrombin was ~ufficient for being used in combination with fibrinogen produced from 45 ml of autologous blood in the manner described in lS WO9l/17778.
The fibrin formed during the thrombin preparation has always a tendency to bind the thrombin. The release o~ this thro~bin can, h2wever, be promoted by the addition o~ a plasminogen catalyst, 5UC~l as streptokinase, urokinase or t-PA ~tissue plasminogen catalyst) optionally ad~ixed with a physiological solution.
Claims (7)
1. A method for preparing a prothrombin blood fraction from whole blood comprising:
(a) diluting whole blood to an ionic strength of less than about 100 millimolar;
(b) separating plasma from said whole blood;
(c) lowering the pH of said plasma to precipitate a prothrombin blood fraction; and (d) separating said prothrombin blood fraction from said plasma.
(a) diluting whole blood to an ionic strength of less than about 100 millimolar;
(b) separating plasma from said whole blood;
(c) lowering the pH of said plasma to precipitate a prothrombin blood fraction; and (d) separating said prothrombin blood fraction from said plasma.
2. The method of claim 1 wherein an anticoagulant is not added to said whole blood.
3. The method of claim 2 wherein prior to said diluting step, said whole blood is contacted with a surface for not more than about 60 seconds, wherein said surface activates blood coagulation factors XI and XII.
4. The method of claim 3 wherein said surface is a negatively charged surface.
5. The method of claim 4 wherein said negatively charged surface is glass.
6. The method of claim 1 which further comprises forming a thrombin blood fraction by converting the prothrombin of said prothrombin blood fraction to thrombin.
7. The method of claim 6 which further comprises removing any fibrin clot from said thrombin blood fraction.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DK92830A DK83092D0 (en) | 1992-06-24 | 1992-06-24 | PROCEDURE FOR THE EXTRACTION OF THROMBIN |
| DK0830/92 | 1992-06-24 | ||
| CA002131316A CA2131316C (en) | 1992-06-24 | 1993-06-24 | A thrombin blood fraction for use in a medical procedure |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002131316A Division CA2131316C (en) | 1992-06-24 | 1993-06-24 | A thrombin blood fraction for use in a medical procedure |
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| Publication Number | Publication Date |
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| CA2263800A1 CA2263800A1 (en) | 1994-01-06 |
| CA2263800C true CA2263800C (en) | 2002-04-09 |
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| CA 2367024 Expired - Fee Related CA2367024C (en) | 1992-06-24 | 1993-06-24 | A thrombin blood fraction for use in a medical procedure |
| CA 2263800 Expired - Fee Related CA2263800C (en) | 1992-06-24 | 1993-06-24 | A thrombin blood fraction for use in a medical procedure |
| CA 2367124 Expired - Fee Related CA2367124C (en) | 1992-06-24 | 1993-06-24 | A thrombin blood fraction for use in a medical procedure |
| CA 2367105 Expired - Fee Related CA2367105C (en) | 1992-06-24 | 1993-06-24 | A thrombin blood fraction for use in a medical procedure |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2367024 Expired - Fee Related CA2367024C (en) | 1992-06-24 | 1993-06-24 | A thrombin blood fraction for use in a medical procedure |
Family Applications After (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2367124 Expired - Fee Related CA2367124C (en) | 1992-06-24 | 1993-06-24 | A thrombin blood fraction for use in a medical procedure |
| CA 2367105 Expired - Fee Related CA2367105C (en) | 1992-06-24 | 1993-06-24 | A thrombin blood fraction for use in a medical procedure |
Country Status (1)
| Country | Link |
|---|---|
| CA (4) | CA2367024C (en) |
-
1993
- 1993-06-24 CA CA 2367024 patent/CA2367024C/en not_active Expired - Fee Related
- 1993-06-24 CA CA 2263800 patent/CA2263800C/en not_active Expired - Fee Related
- 1993-06-24 CA CA 2367124 patent/CA2367124C/en not_active Expired - Fee Related
- 1993-06-24 CA CA 2367105 patent/CA2367105C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CA2367024C (en) | 2006-06-06 |
| CA2367105C (en) | 2006-06-06 |
| CA2367024A1 (en) | 1994-01-06 |
| CA2367105A1 (en) | 1994-01-06 |
| CA2263800A1 (en) | 1994-01-06 |
| CA2367124C (en) | 2006-11-14 |
| CA2367124A1 (en) | 1994-01-06 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| MKLA | Lapsed | ||
| MKLA | Lapsed |
Effective date: 20090625 |