f~ 0 2 ~ 9 8 7 2 ~
WO 96/06623 t~ ~! PCT/IB95/00405 A~llL~KOMBOTIC AND NON-HAEMORRHAGIC HEPARIN-BASED
COMPOSITIONS, PROCESS FOR THEIR PREPARATION AND
The present invention relates to heparin-based compositions as well as to a process for their prepara-tion and to their therapeutic applications.
The invention relates more particularly to heparin-based compositions neutralized with protamine, having antithrombotic activity but largely devoid of haemorrhagic and anticoagulant activities.
Heparins have been known and used for many decades for the preparation of medicaments with anti-thrombotic and/or anticoagulant activity intended in particular for the preventive and curative treatment of venous and arterial thromboses or alternatively for preventing the activation of coagulation in extracorporeal circulations.
It has for many years been known how to prepare low molecular weight heparins, which always have anti-thrombotic activity but whose anticoagulant activitiesare reduced.
Nevertheless, as regards either non-fractionated heparins or low molecular weight heparin, the risk of haemorrhage remains the main complication of heparin-based treatments. As a result, there is a considerablelimit on the use of heparins, which are contraindicated in particular in patients with a predisposition towards haemorrhaging, patients suffering from duodenal or gastric ulcers or alternatively patients who have recently undergone a surgical intervention, in whom antithrombotic treatment with heparin may lead to haemorrhaging.
Consequently, the advantageous properties of heparins, namely their antithrombotic or anticoagulant activity, cannot be correctly exploited on account of their considerable side effects associated with this REPLAC~M~NT SHEET (RULE 26) 02~ 98722 permanent risk of haemorrhaging.
When haemorrhages occur during treatments with heparin, the treatment makes use of protamine sulphate which bringæ about the in vivo neutralization of the heparin.
Although protamine has been used in this way for many years, the mechanism by which heparin is neutralized by protamine i8 not well known. A relatively recent study has shown simply that low molecular weight heparins were neutralized to a lesser degree than non-fractionated heparins ("In Vitro Protamine Neutralization Profiles of Heparine Differing in Source and Molecular Weight", ST~'MTN~ IN THROMBOSIS AND IN HEMOSTASIS, vol. 15 No. 4, 1989).
The problem which the present invention aims to solve is thus one of reducing the considerable risk of haemorrhaging outlined above, which limits the thera-peutic use of heparins.
The aim of the invention is, more precisely, to eliminate the risk of haemorrhaging asæociated with heparins as much as possible while at the same time ret~; n; ng their main properties, in particular the antithrombotic activity.
Thus, the aim of the present invention is to provide heparin compositions which have very advantageous pharmacological properties, in particular antithrombotic properties, which are essentially equivalent to those of the heparins used hitherto, without therewith exhibiting the major drawback which lies in the considerable risk of haemorrhaging.
Another aim of the present invention is also to provide a procesR for the preparation of such composi-tions which is simple to carry out and inexpensive, moreover allowing their therapeutic appIications to be developed.
The invention is also directed towards the therapeutic applications of these compositions.
These aims are achieved using heparin REPT.~ SHEET (RULE 26) . ~
compositions according to the invention which have antithrombotic activity and are substantially free of haemorrhagic activity. These compositions are charac-terized by the fact that they consist essentially of heparin fractions as obtained by the in vitro neutra-lization of heparin with protamine.
The expression heparin fraction neutralized with protamine is understood to refer to any fraction derived from a native or already fractionated heparin, or from a synthetic heparin, whose haemorrhagic power has been neutralized by the action of protamine or any analogue or equivalent thereof having a similar capacity to reduce the haemorrhagic power.
The compositions according to the invention advantageously consist of heparin fractions as obtained by the in vitro neutralization of a non-fractionated heparin or of a low molecular weight heparin, with protamine.
According to one embodiment of the invention, the composition consists of heparin fractions, 25% of which have a molecular mass of less than 2.5 kDa and 40% of which have a molecular mass of greater than 20 kDa.
According to another embodiment of the invention, the composition consists solely of heparin fractions having a molecular mass of less than 2.5 kDa.
In other embodiments the heparin fractions have a molecular mass spectrum which depends on the modes of neutralization with protamine that are used.
The compositions in accordance with the invention are substantially free of protamine.
The invention also provides a process for the preparation of the abovementioned compositions, charac-terized in that it comprises a step of in vitro neutra-lization of heparin with protamine.
The inventors have discovered, surprisingly, that the haemorrhagic activity of heparin can be neutralized in vitro, in particular using protamine, while at the same time retaining its antithrombotic properties.
REPLACEMENT SHEET (RULE 26) 0 2 7 9 8 7 2 ~
More precisely, the process according to the invention consists in reacting, in solution, a heparin with protamine, in particular in the form of a protamine salt, according to variable heparin/protamine ratios.
According to a preferred embodiment of the invention, a heparin solution is mixed with a solution of protamine salt, preferably at room temperature, the mixture obtained i8 centrifuged and the supernatant is collected.
According to the invention, the term heparin solution refers to a solution of native or already fractionated heparin, or of synthetic heparin.
The protamine salt advantageously consists of protamine sulphate.
According to the invention, any protamine analogue or equivalent which has a similar capacity to neutralize heparin and thus to reduce the haemorrhagic power can be used.
The supernatant may then be freeze-dried.
The heparin to be treated and the protamine may be used in different ratios which lead essentially to elimination of the risk of haemorrhaging which is associated with heparins.
The process comprises the step of neutralizing a heparin with protamine or an equivalent, preferably in heparin/protamine proportions of from 2/1 to 1/2.
According to one embodiment of this process, the heparin/protamine ratio is about 1/1. In this case, heparin compositions comprising fractions, at least 25%
of which have a molecular mass of less than 2.5 kDa and at least 40% of which have a molecular mass of greater than 20 kDa, are obtained.
According to another embodiment of the invention, the heparin/protamine ratio is about 1/2. In this case, heparin compositions essentially comprising fractions having a molecular mass of less than 2.5 kDa are obtained.
According to the process in accordance with the REPT~T~T SHEET (RULE 26) ~2~ ~72~
present invention, protamine-free heparin compositions are obtained.
Pharmacological study of the heparin compositions of the invention has made it possible to demonstrate, surprisingly, that they are substantially free of haemorrhagic activity and, in parallel, retain their antithrombotic property.
This pharmacological study also demonstrated, surprisingly, that the heparin fractions obtained by neutralization with protamine in accordance with the invention exert antithrombotic activity which increases as the doses administered increase, without increasing in parallel their haemorrhagic or anticoagulant activity.
Another experimental procedure made it possible to show that the compositions according to the invention are capable of inhibiting the hydrolytic activity of human leucocyte elastase more effectively than non-fractionated heparin. The suppression of the risk of haemorrhaging, in accordance with the invention, makes it possible to envisage administration via a parenteral route or via a broncho-pulmonary route as an aerosol, in the treatment of certain broncho-pulmonary complaints which may involve an excess of leucocyte elastase, such as acute respiratory distress syndromes, mucoviscidosis, and obstructive chronic bronchopneumopathies.
The heparin compositions according to the inven-tion, which are stable and non-toxic, may be employed for the preparation of medicaments which are useful in various therapeutic applications. These applications are those of heparin and of its standard derivatives, includ-ing cases where heparin is contraindicated on account of the risk of haemorrhaging which the patient presents.
They may serve in particular for the preparation of medicaments for the treatment and prevention of venous or arterial thromboses or alternatively for preventing the activation of coagulation in extracorporeal circulation.
The invention thus relates also to pharmaceutical compositions comprising a therapeutically effective REPLACEMENT SHEET (RULE 26) ~2~ ~87~
amount of a heparin composition according to the inven-tion as described above, in combination with a pharma-ceutically acceptable vehicle.
These may be, for example, antithrombotic pharma-ceutical compositions or alternatively compositions forinhibiting the hydrolytic activity of human leucocyte elastase.
The heparin fractions of these compositions may be placed in the form of a pharmaceutically acceptable salt according to stAn~Ard processes.
The pharmaceutical compositions according to the invention are advantageously injectable formulations intended in particular for parenteral administration.
For other applications, such as the inhibition of leucocyte elastase, formulations which are suitable for broncho-pulmonary administration are advantageously provided.
Other characteristics and advantages of the invention will become apparent on re~; ng the examples given below by way of non-limiting guide, with reference to the attached drawings, in which:
- Figure 1 is a comparative graph of the haemorrhagic activity of non-fractionated heparin, of low molecular weight and non-haemorrhagic heparin of the heparin compositions according to the invention (Sl, S2, S3);
- Figure 2 is a comparative graph of the anti-thrombotic activity of non-fractionated heparin, of low molecular weight heparin and of heparin compositions
0 according to the invention (Sl, S2, S3).
Products used: st~n~rd heparin (LEO), protamine sulphate (CHOAY) and low molecular weight heparin, "Enoxaparine", marketed under the name "Lovenox"
- EXAMPLE 1: Preparation of ~upernatant Sl 14.4 ml of a stAn~rd heparin solution having a titre of 72,000 IU (480 mg) and 48 ml of a protamine REPT~CFMF~T SHEET (RULE 26) 8 7 2 ~
_ sulphate solution having a titre of 48,000 HAU are prepared. These solutions are mixed together at room temperature. The hep~rin/protamine ratio is then 1:1, that is to say that 1 mg of heparin i8 neutralized with 1 mg of protamine sulphate.
The mixture thus obtained is centrifuged for 10 minutes and the supernatant is recovered and freeze-dried.
- EXAMPLE 2: Preparation of the supernatant S2 The process is carried out as described in Example 1, using 9 ml of a stAn~Ard heparin solution (i.e. 45,000 IU, 300 mg) and 60 ml of protamine sulphate (i.e. 60,000 HAU). The heparin/protamine ratio is then 1/2, that is to say that 1 mg of heparin is neutralized with 2 mg of protamine sulphate.
- EXAMPLE 3: Preparation of the supernatant S3 The process is performed as described in Example 1, using 4 ml of a solution of low molecular weight heparin, "Enoxaparine" (Lovenox), (i.e. 400 mg) and 40 ml of protamine sulphate (i.e. 40,000 HAU). The heparin/protamine ratio is then 1/1, that is to say that 1 mg of low molecular weight heparin is neutralized with 1 mg of protamine sulphate.
- Molecular mass distribution REPT~CT~NT SHEET (RULE 26) 1)2~ ~72~
Supernatant S1 obtained according to Example 1 - Molecular ma~ distribution expressed as a percentage 5Molecular mass W RI
~ 20 kDa 43.3 47.3 16-20 kDa 2.7 4.45 12-16 kDa 4.5 7.65 8-12 kDa 9.7 13.85 105-8 kDa 8.13 11.8 2.5-5 kDa 6.36 10.3 2.5 kDa 25.16 4.65 = 99.85 ~ = 100 TABLE II
Supernatant S2 obtained according to Example 2 - Molecular mass distribution expressed as a percentage Molecular mass W RI
~ 20 kDa 0 0 16-20 kDa 0 0 2012-16 kDa 0 0 8-12 kDa 0 0 5-8 kDa 0 0 2.5-5 kDa 0 0 2.5 kDa 100 100 ~= 100 ~- = 100 - Ultraviolet absorption spectrum for the supernatant S1 (Example 1) A solution diluted to 1/20 shows two absorption REPT.~FM~T SHEET (RULE 26) 8 7 7 ~
g peaks at the following wavelengths:
212 nm: OD = 3.47 and 271.5 nm: OD=2.22 - Titration of the 3upernatant S1 (Example 1) Before freeze-drying the supernatant S1 prepared according to Example 1, each flask contains 0.7 ml of supernatant solution. 12 identical assays were carried out in order to check the reproducibility: the results are given in Table III below:
FLASK No. Ma~o of heparin AZ~RE A A-Xa A-IIa per flask (IU/mg/ml) (I~/mg/ml) (IU/mg/ml) (mg/0.7ml) 1 23.43 84 61 32 2 23.43 83 54 29 3 22.41 84 62 32 4 22.S3 83 58 30 23.50 86 63 30 6 24.20 83 56 28 7 21.70 86 62 29 8 23.30 83 61 31 9 22.00 85 63 30 20 lo 20.70 83 58 32 11 21.30 85 57 28 12 20.80 80 54 30 M + DS 22 + 1.2 84 + 2.259 ~ 3.330 + 1.4 AZURE A: Method of Klein M.D. et al.
A-Xa: Chronometric assay of the heparin (Hépadot Laboratoire Stago) A-IIa: Aminolytic method - Protein assay The proteins in the supernatants S1 and S2, prepared according to Examples 1 and 2 respectively, are assayed according to the Pierce method (Pierce Laboratory reagent kit).
The results are given in Table IV below:
REPT.~CT~ T SHEET (RULE 26) - ~2~i ~872~
SOLUTIONS PROTEIN CON~NlKATIONS
(~g/mi) S1 (2 mg/ml) 3.2 Sl (1 mg/ml) ~ 1 5 S2 (2 mg/ml) 29.9 S2 (1 mg/ml) 11.2 LOVENOX (2 mg/ml) 8.6 LOVENOX (1 mg/ml) 6.7 - Electrolyte composition (mE~/l) The electrolyte composition of the supernatants S1 and S2 is gi~en in Table V below:
S1 24 0.55 S2 18 0.21 - Deter~; n~ tion of the pH
S1 5.57 S2 4.52 P~M~COLOGICAL STu~Y
A. Experimental studies in rats in a model of venous thrombosis induced by stasis and a model of induced haemorrhage:
Studies were carried out according to the method described by C. Doutremepuich et al., "Experimental venous thrombosis in rats treated with heparin and a low REPLACEMENT SHEET (RULE 26) 7 ~ ~
molecular weight heparin fraction~, Haemostasis, 13, 109-112 (1983).
a. Curative model (subcutaneous injections two hours after induction of the thrombosiæ).
Two studie6 were carried out according to the following procedure:
T0: ligation of the ~ena cava T0+2H: subcutaneous injection of the solutions T0+5H30: induction of the haemorrhage T0+6H : samples taken (blood and clot) The results obtained after the first study are collated in Tables VII and VIII below:
Weight of IHT (sec) CRT (sec) DTT (sec) clot (mg) Control 5.54_1.54 108+2019.6_1.319.4_0.5 Heparin 1.76_0.53 * 420+00 *180.0_0 * 180.0_0 *
(2 mg) S1 2.90_0.88 * 141_3623.2+1.8 20.5_1.5 (2 mg) S2 4.19_1.07 123_3221.4_2.119.6_1.4 (2 mg) Lovenox 3.03+0.72 * 153_4825.2_2.1 20.8_0.9 (2 mg) Heparin 4.18_1.06 144_6025.9_2.621.5_1.0 (1 mg) S1 4.68+0.91 122_2823.1_1.720.5_1.5 (1 mg) S2 4.55_1.48 123_3421.3_2.119.7_1.2 (1 mg) Lovenox 4.84_0.94 146_4021.6_2.020.0_1.5 (1 mg) IHT : Induced haemorrhage time CRT : Cephalin kaolin time DTT : Dilute thrombin time * = p ~ 0.05 (Mann Whitney test) REpT.~ L SHEET (RULE 26) q~ ~87 2~
- T~3LE VIII
Platelets White Red ~x 109/l) corpuscles corpuscles (x 109/l) (X lol2/1) Control 538+220 5.70+3.27 7.40+0.39 Heparin 684+241 4.25+1.80 7.71il.06 (2 mg) S1 589+222 4.07+2.06 7.61+0.71 (2 mg) S2 546+155 4.61+2.73 7.53+0.97 (2 mg) Lovenox 606+113 3.45~1.98 7.81+0.99 (2 mg) Heparin 692+263 5.02+3.25 7.71+1.05 (1 mg) S1 575+200 4.23+1.68 7.26+0.39 (1 mg) S2 600+242 4.21+2.70 7.46+1.15 (1 mg) Lovenox 621+188 5.47+2.62 7.88+0.81 (1 mg) This first study shows that heparin neutralized in vitro with protAm;ne in a heparin/protamine ratio of 1/1 leA~;ng to the supernatant S1 exerts, at a dose of 2 mg, quite considerable antithrombotic activity which is comparable to that of non-neutralized heparin and to that of Lovenox (low molecular weight heparin), whereas the anticoagulant activity and the haemorrhagic activity are only weakly increased.
The supernatant S1 has no effect on the blood cells.
Moreover, the supernatant S2, obtained by neutralization according to a heparin/protamine ratio of 1/2, exerts no haemorrhagic activity but possesses reduced antithrombotic activity.
The results of the second study are given in Table IX below:
REPLACEMENT SHEET (RULE 26) 8~ ~ 2 TABLE IX
GROUPS Clot wt. I~T (~) C~tT (8) DTT (~) (mq) PLACEBO 6.91 + 1.09 126 + 49 22 ~ 1.8 19.6 + 1.0 HEPARIN 3.41~1.08~ ~ 420 ~ ~ 180 ~ ~ 180 2 mg SNl 5.22+2.17 124~58 21.2~1.14 19.5~1.3 SN2 5.63~1.93 145+38 22.1~2.80 20.0+1.2 LOVENOX 4.33+1.06~ 182+56 ~ 24.9+0.70 21.3+1.3 HEPMIN 3.38~0.55 ~ 420 ~ ~ 180 ~ ~ 180 3 mg SNl 4.73+1.77 151~24 22.0~1.6 19.5+0.5 SN2 5.26~1.24 131+47 20.4~1.8 20.8+1.8 LOVENOX 3.62~0.90~ 140+38 ~ 29.0+1.6 22.0+1.7 ~D5PMIN 2.75~0.91 ~ 420 ~ ~ 180 * ~ 180 4 mg SNl 4.09~1.10 155~43 22.5+1.9 19.7+0.9 SN2 4.72~2.33 136+48 18.6+5.7 19.2+0.5 LOVENOX 3.33~0.98 198~76 ~ 50.9~3.9 ~ 39.4+1.9 ~EPMIN 2.15+0.83~ ~ 420 ~ ~ 180 ~ ~ 180 5 mg SNl 3.70~1.28~ 142~33 29.5+3.6 ~ 24.2+9.3 SN2 4.84~1.12 145+48 22.0~1.3 20.3+0.7 SN3 3.52~0.30~ 121+15 20.8+1.8 19.6~1.75 LOVENOX 2.85~1.14~ 389~86 ~ ~ 180 ~ ~ 180 8EPMIN 0.98~0.82~ ~ 420 ~ ~ 180 ~ ~ 180 mg SNl 3.15~1.21~ 171~64 ~ 30.2+2.5 ~ 32.6+6.9 SN2 4.09~1.16~ 136+74 23.8~1.7 20.0+0.7 SN3 2.29~0.40~ 157+21 27.0~2.0 23.0+1.0 LOVENOX 1.44+0.48~ ~ 420 ~ ~ 180 ~ ~ 180 Clot wt. : Weight of the experimental clot IHT : Induced haemorrhage time CRT : Cephalin-kaolin time DTT : Dilute thrombin time It i8 seen from the results obtained that the antithrombotic activity of fractions Sl, S2 and S3 according to the invention increases as the doses administered increase.
If we refer to the dose-effect curves, estab-lished for doses ranging from 2 mg/kg to 10 mg/kg, it is seen, in Figure 1, that irrespective of the type of heparin treated in accordance with the invention, the heparin fraction obtained has haemorrhagic activity similar to that of the control group, even at the highest doses. The non-fractionated heparin and the low molecular weight heparin (Lovenox), which is not neutralized with protamine, according to the invention, have considerable haemorrhagic activity when compared with the heparin fractions of the invention.
Figure 2 shows that the heparin fractions obtained according to the invention have advantageous REPT.~C~M~T SHEET (RULE 26) ~ ~ ~ 9 8 ~ ~ ~
antithrombotic activity. In the case of fraction S1 (heparin/protamine ratio of 1/1), this activity is comparabie to that of non-neutralized hepar,ns ir. accor-dance with the invention.
b. Preventive model (subcutaneous administration one hour before induction of the thrombosis).
The study was carried out with the supernatant S1 (Example 1) according to the following procedure:
T0 : subcutaneous injection of the solutions T0+1 hour : induction of the stasis T0+24 hours : samples taken (blood and clot) The results obtained are given in Table X below:
Weight of C~T (sec) DTT (sec) Ti (sec) clot (mg) Control 5.13+1.03 19.5+0.4 18.7+0.6 19.8_0.83 Heparin 3.40_0.70 * 20.7+0.4 19.3_0.8 19.8+1.30 (4 mg) S1 3.23_0.61 * 20.5+1.1 19.4+0.8 19.3+0.83 (4 mg) Lovenox 3.48+0.94 * 19.6+0.8 19.7+0.9 19.7_1.09 (4 mg) C~T : Cephalin kaolin time DTT : Dilute thrombin time Ti : Titrarin (Stago Laboratory) time * = p c 0.05 (Mann Whitney test) The results obtained show that, for preventive purposes, S1 exerts antithrombotic activity which is comparable to that of heparin and Lovenox, 24 hours after induction of the thrombosis.
B. Experimental study in rats in a model of thrombosis induced by generation of free radicals (reference: Doutremepuich - In press - Annales de Cardiologie et Angiologie) The study was carried out with S1 (Example 1) according to the following procedure:
REPT~CT~T~'~T SHEET (RULE 26) 7 ~ ~
(T0 : subcutaneous injection of the solutions) T0+25 min : injection of rose bengal at a dose of 5 mg/kg T0+30 min : induction of free radicals in the first arteriole by photochemical reaction T0:55 min : injection of rose bengal at the same dose T0+60 min : induction of free radicals in the second arteriole T0+85 min : injection of rose bengal at the same dose T0+90 min : induction of free radicals in a venule.
After the final thrombosis, a blood sample is taken intracardially.
The excitation time is set at 2 minutes and the observation time at 10 minutes.
The results given in Table XI below are obtained:
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Arteriole T0 + 30' CONTROL S1 (2 mg/kg) HEPARIN
(2 mg/kg) Duration of 4.50_0.82 9.68_0.44* 6.80_2.32 embolization (min) Number of12.00_2.454.00+3.56* 3.56_2.12*
emboli Arteriole T0 + 60' Duration of 3.49_0.36 9.81_0.25* 5.9_3.6 embolization (min) Number of7.33_0.47 5.00_2.45 4.56_3.6 emboli Venule T0 + 90' CONTROL S1 (2 mg/kg) HEPARIN
(2 mg/kg) Duration of 4.53_2.04 3.68+2.06 embolization (min) Number of7.00+4.32 4.00_1.41*
emboli Duration of embolization: time between the first embolus and the final embolus detaching from the clot.
Number of emboli: number of ~mholi detaching from the clot.
In this model of thrombosis induced by free radicals, the supernatant S1 (Example 1) exerts signifi-cant antithrombotic activity when compared with the placebo group, which persists after 90 minutes (TOt90 min). This acti~ity i~ higher than that of heparin injected at the same dose, after 30 and 60 minutes (T0+30 and T0+60 min).
C. Experimental study in rats in a model of thrombosis induced by endothelial lesion with a laser (Ref.: Vesvres, Haemostasis 1993, 23, 8-12) a. Study 1 REPT~CFM~T SHEET (RULE 26) 7 ~ 8 . .
The study was carried out according to the following procedure:
T0 : subcutaneous injection of the test sub-stance at a dose of 2 mg/kg T0+35 min: induction of the arterial thrombosis using a laser beam.
The observation time is set at 10 minutes.
The results obtained are given in Table XII
T0 + 35' (ARTERIAL THROM3OSIS) CONTROL S1 (2 mg/kg) HEPARIN
(2 mg/kg) Number of1.2+0.4 2.0_1.4 2.5+3.3 laser strikes Number of10.2_2.7 1.5+0.7 * 3.3+2.4 *
emboli Duration of 6.3+1.8 1.0+0.0 * 2.1_1.8 *
~holization (min) S1 exerts antithrombotic activity comparable to that of non-neutralized heparin injected at the same dose and reduces the number of ~holi as well as the duration of embolization in a statistically significant manner.
b. Study 2 The study was carried out according to the following procedure:
T0 : ~ubcutaneous injection of the test sub-stances at a dose of 2 mg/kg.
T0+lh : induction of the first arterial thrombosis T0+3h : induction of the second arterial thrombosis T0+6h : induction of the third arterial thrombosis.
The observation time is set at 10 minutes.
The results obtained are given in Table XIII
REPr~C~M~T SHEET (RULE 26) 7 ~ ~
TAB~ Xlll TO + 1 h TO + 3 h TO + 6 h S1 Hep S1 Hep S1 Hep Number of 1.6+0.5 1.6+0.5 2.0+0.0 1.6+0.5 1.6~0.5 1.0~0.0 laser strikes Number of 3.0~1.0 5.Oil.7 5.3+3.5 6.7~1.2 8.3~3.0 7.5+2.1 emboli Duration 1.3+0.5 2.6il.2 2.3~1.5 3.Oil.O 4.3~2.4 3.0+1.4 o~
emboli-zation Sl exerts antithrombotic activity comparable to that of heparin which has not been neutralized with protamine.
In conclusion, the studies described above show that antithrombotic activity is observed in the three models of experimental thrombosis, namely the venous model induced by stasis, the model of arterial thrombosis induced by free radicals and the model of arterial thrombosis induced by endothelial lesion with a laser.
According to the invention, the heparin fraction obt~ine~ from a low molecular weight heparin, "Enoxaparine" (Lovenox), has higher antithrombotic activity than that of the same low molecular weight heparin not treated in vitro with protamine and also higher than that of fractions obtained from non-fractionated heparins not treated in vitro with protamine, while at the same time no longer presenting any risk of haemorrhaging.
The process according to the invention makes it possible, in a simple and inexpensive manner, to substantially eliminate the haemorrhagic activity of heparins while at the same time ret~i~ing their antithrombotic activity.
REPLACEMENT SHEET (RULE 26)