CA2284820A1 - A new use of antithrombin iii - Google Patents

Abstract

This invention describes the application of AT III in producing a pharmaceutical preparation for preventing bleeding during anticoagulant therapy and a pharmaceutical preparation for subcutaneous injection which contains AT III as an active ingredient.

Description

SEP-21-99 04:23 +43 1 512 98 05 P.02 R-233 Job-951 21/fl9 ' 99 10 : 20 FAX +45 1 512 98 05 PAT. ATT. VIENNA f~ 002 F I L E, PAN-tN~T H I S A M~#B~EB
#f~ TRANSLATION
A New Use of Antit.hrombin zzI
The invention relates to a new use of antithrombin ZIZ.
Ant~.thrombin III (AT III) is a plasma protein which acts to inhibit coagulation by inhibiting thrombin, factors IXa, Xa, XTs, XIIa and plasmin_ AT III deficiency (or hereditary thromboph:ilia) is an autosomal-dominant hereditary disease with a thx-ombosis and embolism tendency because of a reduced formation of AT III.
An acquired AT III deficier~.cy may, a . g.., occur in disseminated intravasal cvagulopathies (DIC), sepsis, cirrhosis of the liver or with nephx~otic syndrome_ Likewise, AT III deficiency symptoms may occur in case of cardiovalvular prostheses, postoperative, thromboembolic complications, in. estrogen therapy or in asparaginase therapy.
1~t present, all these syndrome are treated i.a. by administering AT rII, application always having been effected intravenously (cf. von Kries et al., Eur. J.
Pediatr. 144, 191-194 (19BS)).
Heparin is a coagulation-inhibiting polymer occurring in the body (lungs, liver, thymus, spleen and basophilic mast cells) and obtained from p-glucuronic acid and D-glucoaamine, and it comprises several molecules of su7.furic acid per structural un~.t_ Heparin SEP-21-99 04:23 +43 1 512 98 05 P.03 R-233 Job-951 21/09 '99! 10:20 FAZ +4a 1 512 98 05 PAT.ATT.VIENNA ~ 005 inhibits the activity of thrombin vn e.g. fibrinogen by binding to AT III, and therefore heparin is also called .
a ao-factor for AT III. Furthermore, heparin also inhibits the activity of thrambolcinase and, thus, the conversion of prothrombin to thrombin as well ass the agglomeration. of thrombocytes and the blood coagulation reaction.
Heparin/AT IIr has an inhibiting effect on the arterial blood coagulation factors Va, IXa, Xa az~.d xlla and has an activating effect on lipoprotein lipase_ Heparin treatment may, however, also have serious side effects. It is assumed to be the cause of heparin-induced thrombocytopenia and a consequent risk of thrombosis of immunologieal reactions which cannot be foreseen. Due to an a.mhibition of platelet aggregation, hemorrhages also occur Frequently in the course of a heparin therapy:
Clinically, heparines are routinely administered primarily fox the prevention but also for the therapy of thromboses and embolisms. Primarily ~.f heparins are administered subcutaneously, which is often experienced by patients to be very painful, frequently hematomas ( "bruises ~~ ) occur .
rlethods of recovering heparin hare been described in large numbers; one example for a heparin preparation suitable for subcutaneous application is disclosed in CA patent 1,108,989.

SEP-21-99 04:23 +43 1 512 98 05 P.04 R-233 Job-951 21/09 '99, 10:21 FAX +4~ 1 512 98 05 PAT.ATT.VIENNA ~J004 rt is the object. of the present invention to provide an improved prevention and therapeutical treatment of thromboses anal embolisms which above all can be used routinely in hospitals az~.d which no longer has the. disadvantages of conventional heparizz administration.
According to the invention, this object is achieved by using AT III fox producing a pharmaceutical preparation for preventing hemorrhages during an anticoagulation therapy, in particular for producing a pharmaceutical preparation for thrombosis prevention ox treatment.
Although the use of AT TII particularly for the prevention of hemorrhages during the therapy with lr~eparin as such ie a paradox because of the known intensifying anticoagulative effect of heparins on AT
zzr (because as such an intensified coagulation-inhibiting action had to be expected), the use according to the invention surprisingly has yielded extremely positive effects:
Thus, the formation of hematomas - as compared tv conventional heparin admi.nistratzon - could be greatly reduced, subcutanous injection was also much less painful arid therebeyond, the treatment according to the invention also exhibited a depot effect wh~.ch was not to be expected, whereby it was possib7.e to highly increase the functional half life of AT III and SEP-21-99 04:23 +43 1 512 98 05 P.05 R-233 Job-951 21/09 '99! 10:21 FAZ +45 1 512 98 05 PAT.ATT.VIENNA ~ 005 heparin, primarily in case of subcutaneous izxjection.
Thus it has become possible for the first time to carry out a sufficient thrombosis prevention not by means of the common daily injection of hepdril~., hut to carry out the treatments at longer time intervals, e.g. at intervals of 2, 3, 4 days up to one week, depending on the dose to be admi.nistared.
According to the invention, furthermore, lIl this manner the inhibition of platelet aggregation can be prevented or treated, respectively, and also the bleeding time caxl be shortened.
The rielk of a thrombocytopenia a~ well as of undesired hemorrhages in the course of an anticoagulant therapy with the preparation of the invention or the use thereof, respectively, is markedly reduced. It could be demonstrated in vitro that thrombocyte aggregation is not affected by AT III/heparin, whereas heparin does clearly show an inhibitory effect.
with the indication for AT III according to the ixwentio~n, for the first time a pharmaceutical preparation comprising AT III as an active component has been provided which can be provided for subcutaneous admini.sl.ration, i . a . the use of A'T zII for produc~.ng a medicament which is to be administered ~subcutaneously.
Thus, the present ~.nvention also relates tv a pharmaceutical preparation for subcutaneous injection, SEP-21-99 04:23 +43 1 512 9B 05 P.06 R-233 Job-951 21/n9 '99 10:21 FAZ +43 1 512 98 05 PAT.ATT.VIENNA X1008 which prepa..ration comprises AT IzI as the or rune of the essential active component(s). The subcutaneous preparation comprises AT zzs at a relatively high concentration, corresponding to at :Least a two-fvJ.d concentration of that common for in vitro-products.
Usually, a concentration ranging from 100 to 5,000 U of AT III/ml will be chosen, preferably between 500 and 3,000 U of AT III/ml, depending on the purity of the protein used. Be9ide9, AT III is also suggested as an i.v. or i.m. product to be used according to the invention.
AT III used according to the invention may be contained in various preparations, e.g. in combination with heparins or heparinoids having a high affinity or having an affinity for AT III, respectirrely, preferably as an AT III-heparin (or heparinoid)-complex (heparinoids being a collective term for naturally occurring, semisynthelric and synthetic mucopoly-saccharides of heparin-like action).
A preferred production method for such a complex has been described in AT patent 379 310, wherein human plasma or AT m-containing plasma fractions are admixed with heparin or heparinvi.d, respectively, and the heparin- or heparinoid-complex, respectively, formed is purified by adsorption on an anion exchanger.
Such a prepartion is commercially available under the name Atheplexa (from Baxter, AT). This method may, of - s -SEP-21-99 04:23 +43 1 512 98 05 P.OZ R-233 Job-A51 21/0,9 ' 99, 10: 22 FAZ +43 1 512 98 05 PAT. ATT. VIENNA ~J 007 course, alBO be carried out with starting solutions prepared by means of recombinant D1~TA technology.
preferably, the use of A'f III acaoxding to the invention takes place within the frame of an anticoagulation therapy comprising a heparin treatment.
As it has been mentioned, the pain associated with the heparin injection (as a side effect of the anticoagulation therapy) can be greatly reduced or even prevented by the combined administration of AT ITI and heparin or of AT III camplexed on heparin. seside9, hematoma fvarmation at the site of injection is clearly reduced.
In particular, patients having a risk of heparin-induced thrombocytopenia can be treated according to the invention.
The preparation according to the invention. for the subcutaneous administration may comprise different types of heparin_ For instance, hepaxins in low-molecular form having a molecular weight of approximately 7.,5po to 10,000 Daltons or in, high-molecular ~vrm having a molecular weight of approximate~.y 10,000 try 30,000 Daltons may be GQntain~:d .
The preparation according to tre invention has a surprisingly high functional half-life_ Due to its novel resorption and pharmaco>cinetics, the preparation according to the invention is characterized by an SEP-21-99 04:23 +43 1 512 98 05 P.OB R-233 Job-951 21/09 '99. 10:22 FAZ +49 1 512 98 05 PAT.ATT.VIENNA X1008 extraordinary bivavailability of AT III and heparin, respectively. By selecting the heparine or the heparinoids and their AT III affinzty. respectively, the components of the preparation preferably are mixed and adminisCered such that the heparins or hepa..r.i.noids, reepectively, contained have a functional half-life in Wvo of more than 10 hours, in particular more than 20 hours, in certain cases also more than 30 hours_ The pharmaceutical preparation according to the invention is produced by finishing a puri.f.ied AT IIZ
and, optionally, heparin-containing preparation according to methods known per ee, by optionally combining it with suitable buffer, auxiliary, preservative and/or stabilizing substances and/or protease inhibitors, respectively, and filling it into containers in a form suitable for administration and preferably packing 1t in storage-stable, optionally lyophilized or frozen state.
When being used, the effective dosage of the preparation will depend quite individually on the respective patient tsiae, body weight, condition, blood COUnt,...).
Preferably, the AT IIT concentration in the preparation of the invention or for the use acooxding to the invention will be chosen such that an administration of a dose of from 5 to 10,000 U of AT 2II/><g body weight in a volume of less than 20 ml, -SEP-21-99 04:23 +43 1 512 98 05 P.09 R-233 Job-B51 21/09 '99 10:22 FAX +45 1 512 98 05 PAT.ATT.VIENNA ~J009 in particular less than 10 ml, preferably less than ml, most preferred less than 2 ml, will be enabled.
Preferably, the preparation accord~.zlg to the invention is subjected to a method for irxactivating infectious or pathogenic material, in particular viruses. This inactivation treatment is preferably ensured by a tenside and/or heat treatment, e.g. by a heat treatment in the solid grate, in particular a vapor treatment according to EP-o 159 311, or EP-0 ~i9 901 ar EP-0 674 531.
Further treatments for inactivating viruses also include the treatment with chemical or chemical/physical methods, e.g. with chaotropic substances according to W094/13329, DE 44 34 538 car Fp-0 131 740 (solvents) or photoinactivation.
Irradiation oz~ nanofiltration also is a preferred phys~.cal method for depleting viruses withixi the scope of the present invention.
Finally, the inventiaxi relates to a kit for subcutaneous injection comprising an AT III-containing pharmaceutical preparation, instructions for using the preparation for subcutaneous injection, as well ae optionally an application device suitable fOr subcutaneous injection, preferably a syringe. Compared to i.m. syringes, this syringe has a very thin needle so as to facilitate penetration below the skin.
Preferably, the pharmaceutical preparation in the _ g SEP-21-99 04:23 +43 1 512 98 05 P.10 8-233 Job-951 21/0.9 '99. 10:22 FAg +43 1 512 98 05 PAT.ATT.VIENNA ~JO10 kit is provided in a container in lyophilized foam, it may, however, also be provided in liquid form. in which case, however, it is preferably stored in deep-frozen form.
The invention will now be explained in more detail by way of the following Examples and the drawing figures to wh~.ch, however; it shall not be restricted.
Figs. 2A and 1$ show the local tolerance of heparin and Atheplexa in mouse;
Fig_ 2 shows the pain-triggering et:~ect of heparin in the rat paw model;
Fig. 3 shows the pain-tr~.ggering effet of Atheplex~' in the rat model;
Fig_ 4 shows the comparison of the Rlstoce~-irl-dependent platelet aggregation of heparin and Atheplex~; and Fig. 5 shows the determination of the half-life of heparin and Atheplex°.
E x a m p 1 a s .
g x a m p 1 a 1 . $ematoma for~$tiosx on mice after subcut~eous injeati,oa of heparia aad of the preparation acco:cding to the inveatioa. respectsvely (at present cQneidercd by Applicant to be the beet mode of carrysug out the inverWion) In three runs, each using a total of z5 male NMRI
mice havi,mg a weight of fxwm 25 to 3o g are treated with heparin (IMMT1N0 AG, "Vienna) or with the SEP-21-99 04:23 +43 1 512 98 05 P.11 R-233 Job-951 21/'09 ' 99. 10 : 23 FAa +45 1 512 98 05 PAT. ATT. VIENNA ~J 011 preparation according to the invention, respec.~ively (Atheplexm, obtainable from IMMUNO AG,,Vienna), as follows 1.5 IU of heparin 1.5 heparin units of Atheplexe 15 IU of heparin 25 heparin units of Atheplex~
Treatment (0.5 ml) was effected subcutaneously in the xegion of the abdomen. The mice were examined daily for 4 days for a discoloration of the subcutis. changes were e~raluated as follows:
SGOre Syze none presexxt 0 up to 0.5 cm' 0.6 - 4 cm' 2 4 cm~ 3 L~iscolorativn none slight intensity 1 middle intensity high intensity Swelling none o low degree middle degree 2 high degree SEP-21-99 04:23 +43 1 512 98 05 P.12 R-233 Job-951 21/09 '99 10:23 FAZ +45 1 512 98 05 PAT.ATT.VIENNA X1012 On the 4th day, the animals were sacri.Liced, and the subcutaneous changes were pathologically-anatomically evaluated.
mean results are apparent from Table 1 and 1~ig. 1.
TABLE lA
Score 1 5 IU i 2 3 4 Da.v Heparin 2.86 2.66 2.26 1.73 At ex~ 1.20 0.40 0.20 0.0 .SC:OrPr.
i5 IU 1 2 3 4 Heparin 3_66 4.13 3.33 2.33 Athet~lex~ 2 86 1 80 1.40 1.33 As apparent (Figs. 3.A, B) . at 7..5 and ~.5 heparin units there i.s a relati~rely clear difference between the two tested preparations in tavor of Atheplex~_ control animals which had received analogously isotonic saline did not show hematomas at any point of time.
pi.saection confirmed the respective visual evaluation of the 4th day.
E x a m p 1 a 2 . Testing of the pain-triggering effect of a subcutaneous injection of hepari~a and of SEP-21-99 04:23 +43 1 512 98 05 P.13 R-233 Job-951 21/09 '99 10:25 FA7C +45 1 512 98 05 PAT.ATT.VIENNA ~J015 the preparation according to the invention in the. rat paw model this effect was determined on the izzflamed rat paw by determining the pain threshold according to Randall & Selitto (Arch. Int. Pharmacodyn. 111 (1957), 409-419 ) .
Fvr this purpose, a mercury manometer was connected with a l0 ml syringe whose piston was equipped with a short, projectile-type peg. with this syringe, an increasing pressure was exerted on the rat paw (26.7 mbar/s). The pain threshold was defined ag that pressure in mbar which is necessary to trigger a defense reaction of the animal.
For the tests, female Sprague-Dawley rats having weight between 250 and 35o g wexe used. The pain threshold was measured each prior to injection (initial value) and hourly up to 6 hours after intraplantar injection of heparin (several doses) or Ath.eplexe (several doses), respectively, or isotonic saline (100 ~tl, negative control) . The values after injection of test or control substances were c-expressed in peroent of the initial value.
The results are apparent from Fig. 2 (heparin) and Fig. 3 (Atheplex~) (x ~ sg). On the oxdinate, the pain threshold expressed in percent. is entered: 100 % = no .
pain; (7 % = maximum pain. The abscissa represents the time axis (hours after intraplantar injection). The - 1~ -SEP-21-99 04:23 +43 1 512 98 05 P.14 R-233 Job-A51 21/'09 '99 10:24 FA7C +4~ 1 512 98 05 PAT.ATT.VIENNA f~1014 uppermost Curve (triangles) in each case z~epresmic_s the control animals (isotonic saline, n= 25; no pain)_ Heparin (Fig. 2) was tested on 10 animals each, at.
the doses 1, 3, 10, 40 and 130 IU. Starting from ZO IU, a progressive, strong pain occurs, no further dose dependence being recognizable.
AtYieplex~ (Fig. 3) was tested at the dosis 10, 40, 130 and 200 a 'n units (n = 10 in each case). Here, too, pain occurs initially which, however, decreases again in the course of time. The point pf time and the extent of such decrease are dose-dependent:
- 7.0 heparin units: reversion after the 2nd hour, complete freedom from pain after 6 hours.
- 200 heparin units: reversion after the 5th hour, residual pain at the 6th hour: 46.9 ~ 3.0 °s.
These results show a markedly lower pain development by the preparation according to the invention in contrast to the conventional heparin therapy.
S x a m p 1 a 3 . Z'n virs~o teetiug' of the inhibition of platelet aggregation by Heparin and by the preparation aacorda.ng to the in~rention The influence of the substances on platelet aggregation is caused by means of a turbidometric method by using an aggregometer (from Chronology. To 400 ~1 of a formaldehyde-fixed thrombocyte preparation (250, 000 cells per ~C1) , 25 ~.l of a FVIII-vWF-SEP-21-99 04:23 +43 1 512 98 05 P.15 R-233 Job-951 21Y09 '98 10:24 FAZ +45 1 512 98 05 PAT.ATT.VIENNA ~J015 preparation and 25 ~.1 of the sample to be tested, or of buffer as control, respectively, are admixed. After an incubation of 3 min at 37°C, aggregation is sr~trted with SO ul of Ristocetin sulfate (10 mg/ml). Evaluation of the aggregation ie effected via the rise of the aggregation curve. The reference value is the aggregation with buffer addition.
In contrast to heparin, the RisGocetin sulfate-induced platelet aggregation was not inhibited by AT
III-complexed heparin. The tests were also repeated with a purified, recombinant van Willebrand Factor. The results with the vGlF-dependent aggregation are identical with the Fvzzr-vWF-dependent reaction. Both can be reduced by the adda.tion of heparin, yEt not by the addition of .Atheplex~.
Factorial Evaluation Uriits of heparin in 5d0 1 test mixture Heparin Atheplexe

2.5 0.47 1.01 0.25 0.84 0.99 0.025 1_09 1_07 Buffer corxtrvl (without hepar7_n) 1 The results show that heparin inhibts the aggregation in dote-dependence, urhereas the reaction is not yr not substantially influenced by Atheplexe.
8 x a m p 1 a g . Determ~.natioYl raf the half-life pf gy~~~staneoualy ix~jeated heparin axed subcutan~ously SEP-21-99 04:23 +43 1 512 9A 05 P.16 R-233 Job-951 21/09 '9» 10:24 FA% +4a 1 512 98 05 PAT.ATT.VIENNA X1016 inj Bated preparation aacord~.ng to tk~e inventir~n To & awake rabbits each (White New Zealasld, ca. 2_5 kg, o~ Dither sex) in the heparin group, E~,000 units r~~
unfractionated heparin per kg are administered subcutaneouely. In. ChB comparison group, 6,000 units of_ heparin are subcutaneously injected with 1,000 units of AT III, as a complex (Atheplex°). rltrated blood was drawn from the ear ve~.zl 6, 22, 18, 24, 3Q, 36, 42, 48, 54, 60 hours after application. mhe heparin level is determined a.n the recovered plasma via an adapted thrombin inhibition determination by means of chromogenic substrate (from Coa-Chrom Diagnoetika, Chromogenix}. The calculations are carried out according to bioStatiG Criteria. All the tests of the heparin-6000 L1'/kg and Atheplex'~-applicatiora 1000 u/I~g (~ 60a0 heparin units/kg) in the rabbit test were included in the calculation.

SEP-21-99 04:23 +43 1 512 98 05 P.1T R-233 Job-951 21109 '99 10:24 FAg +45 1 512 98 05 PAT.ATT.VIENNA ~J017 Heparin 6000 U/kg AthepJ.er. iono IT/kg subcutaneous Bubautaneous Hep.det.(zza-Inh) Hep.det.

(IIa-Inh.) AT III(Aa).

Lag' time (h) O.OOOS 6.02 10.7 Adsorption (1/h) 0.15 0.27 0.42 Sl~.mination (1/h) 0.15 O.OS2 0.031 T/2 adscarption (h) 4.76 4.1 1.66 as of application 2.4 11.5 17.8 t/2 elimination (h) 4.59 11_2 22.7 as of application 16.9 31.7 42.4 AUC (U/ml.h) 1939 2850 Maximum (U/ml) 4.1 8.2 4.2 Time maximum (h) 12 1$ Z8 The present data demonstrate the superiority of complexed heparin with AT III over the non~comp7.exed heparin. An equal superiority is also shown. with an i.v. administration of AthepJ.ex° as regards halt-life arid recovez~y.

Claims (21)

Claims:

1. The use of antithrombin III (AT III) for producing a pharmaceutical preparation for the prevention of hemorrhages during an anticoagulant therapy.

2. The use according to claim 1, characterized in that the inhibition of platelet aggregation is prevented or treated, respectively.

3. The use according to claim 1 or 2, characterized in that the bleeding time is shortened.

4. The use according to any one of claims 1 to 3, characterized in that AT III is employed in combination, with high affinity heparin.

5. The use according to any one of claims 1 to 4, characterized in that an AT III-Heparin-complex is employed.

6. The use according to any one of claims 1 to 5, characterized in that the anticoagulant therapy comprises the treatment with heparins.

7. The use according to any one of claims 1 to 6, characterized in that pain as a side effect of an anticoagulant therapy is prevented.

8. The use according to any one of claims 1 to 7, characterized in that hematoma formation at the site of injection is reduced.

9. The use according to any one of claims 1 to 8, characterized in that patients are treated who run a risk of heparin-induced thrombocytopenia.

20. The use according to any one of claims 1 to 9, characterized in that AT III is administered subcutaneously.

11. A pharmaceutical preparation for subcutaneous injection, characterized in that it comprises AT III.

12. A preparation according to claim 11, characterized in that the AT III is contained in a complex with heparins or heparinoids.

13. A preparation according to claim 11 or 12, characterized in that it contains heparins or heparinoids with a high affinity to AT III.

14. A preparation according to any one of claims 11 to 13, characterized in that heparins having a molecular weight of from 1,500 to 10,000 are contained therein.

15. A preparation according to any one of claims 11 to 13, characterized in that heparins having a molecular weight of from 10,000 to 30,000 are contained therein.

16. A preparation according to any one of claims 11 to 15, characterized in that it comprises heparins or hgparinoids, respectively, having a functional half-life of more than 10 hours, preferably more than 20 hours.

17. A preparation according to any one of claims 11 to 16, characterized in that it comprises an AT III
concentration which enables the administration of a dose of from 5 to 5,000 U/kg in a volume of less than 20 ml, in particular less than 10 ml, preferably less than 5 ml.

18. A kit for subcutaneous injection, characterized in that it comprises the preparation according to any one of claims 11 to 17 as well ae instructions for using the preparation for subcutaneous injection.

19. A kit for subcutaneous injection, characterized in that it comprises the preparation according to any one of claims 11 to 18 as well as an application device suitable for subcutaneous injection, preferably a syringe.

20. A kit according to claim 18 or 19, characterized in that the preparation is present in lyophilized farm in a container.

21. A kit according to claim 18 or 19, characterized in that the preparation is present in liquid form and, optionally, deep-frozen in a container.