WO1998042371A1

WO1998042371A1 - New application for antithrombin iii

Info

Publication number: WO1998042371A1
Application number: PCT/AT1998/000078
Authority: WO
Inventors: Hans-Peter Schwartz; Anton Philapitsch; Ludwig Pichler; Wilfried Auer; Yendra Linnau
Original assignee: Baxter Aktiengesellschaft
Priority date: 1997-03-25
Filing date: 1998-03-24
Publication date: 1998-10-01
Also published as: ATA51797A; AT405905B; EP0971732A1; AU6385298A; CA2284820A1

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

New use of antithrombin III

The invention relates to a new use of antithrombin III.

Antithrombin III (AT III) is a plasmatic protein that has an anticoagulant effect by inhibiting thrombin, the factors IXa, Xa, XIa, Xlla and plasmin.

AT III deficiency (or hereditary thrombophilia) is an autosomal dominant hereditary disease with a tendency to thrombosis or embolism due to reduced formation of AT III.

Acquired AT III deficiency can e.g. with consumption coagulopathy (DIC), sepsis, cirrhosis of the liver or with nephrotic syndrome.

Likewise, AT III deficiency symptoms can occur with heart valve prostheses, postoperative, thromboembolic complications, with estrogen therapy or with asparagine therapy.

All of these syndromes are currently treated with AT III administration, the application always being intravenous (see Kries et al., Eur. J. Pediatr. 144, 191-194 (1985)).

Heparin is a coagulation-inhibiting polymer made of D-glucuronic acid and D-glucosamine that occurs in the body (lungs, liver, thymus, spleen and basophilic mast cells) and contains several molecules of sulfuric acid per structural unit. Heparin inhibits the effect of thrombin on, for example, fibrinogen by binding to AT III, which is why heparin is also referred to as a cofactor for AT III. Furthermore, heparin also inhibits the effects of thrombokinase and thereby the conversion of prothrombin to thrombin, as well as the Agglomera ^¬ tion of the platelets and the clotting reaction.

Heparin AT III has an inhibitory effect on the arterial coagulation factors Va, IXa, Xa and Xlla and has an activating effect on lipoprotein lipase. However, heparin treatment can also cause serious side effects. Thus, the cause of a heparin-induced thrombocytopania and the associated risk of thrombosis are assumed to be immunological reactions that are not predictable. Due to the inhibition of platelet aggregation, bleeding often occurs as part of heparin therapy.

Clinically, heparins are routinely administered primarily for prophylaxis but also for the treatment of thrombosis and embolism. Hematomas ("bruises") often occur especially when heparins are administered subcutaneously, which patients often find very painful.

Methods for obtaining heparin have been described in numerous cases; an example of a heparin preparation suitable for subcutaneous use is disclosed in CA-PS 1 108 989.

The object of the present invention is therefore to provide an improved prophylaxis and therapy treatment of thromboses and embolisms, which can be used primarily for routine use in hospitals and no longer has the disadvantages of conventional heparin administration.

This object is achieved according to the invention by using AT III for the manufacture of a pharmaceutical preparation for preventing bleeding during anticoagulation therapy, in particular for the manufacture of a pharmaceutical preparation for prophylaxis or treatment of thrombosis.

Although the use of AT III is paradoxical in itself to prevent bleeding during therapy with heparin because of the known reinforcing anticoagulant effect of heparins on AT III (because an increased anticoagulant effect should actually be expected), the use according to the invention surprisingly shows extremely positive effects:

The formation of hematomas - compared to conventional borrowed heparin - were greatly reduced, the subcutaneous injection was also far less painful and, moreover, the treatment according to the invention also had an unexpected depot effect, as a result of which the functional half-life of AT III and heparin, particularly in the case of subcutaneous injection, could be greatly extended. This makes it possible for the first time not to perform adequate thrombosis prophylaxis not by the usual daily injections of heparin, but to carry out the treatments at longer intervals, for example at intervals of 2, 3, 4 days to one week, depending on the dose to be administered.

According to the invention, the inhibition of platelet aggregation can also be prevented or treated, and the bleeding time can be shortened.

The risk of thrombocytopenia and undesired bleeding as part of an anticoagulant therapy by the preparation or use according to the invention is considerably reduced. It could be demonstrated in vitro that the platelet aggregation is not influenced by AT III / heparin, while heparin shows a clearly inhibitory effect.

With the inventive indication for AT III that is, the use of AT is also provided only once ^¬ a pharmaceutical preparation with AT III as an effective component are available which may be provided for subcutaneous administration, III for the preparation of a subcutaneous medicament to be administered.

The present invention therefore also relates to a pharmaceutical preparation for subcutaneous injection, which preparation contains AT III as the or one of the essential active components. The subcutaneous preparation contains AT III in a relatively high concentration, corresponding to at least 2 times the concentration usual for in vitro products. A concentration in the range from 100 to 5000 ATH HI / ml is usually chosen, preferably between 500 and 3000 ATH HI / ml, depending on the purity of the protein used. In addition, AT III is also used as an IV or IM product for Intended use suggested.

The AT III used according to the invention can be contained in various types of preparations, for example in combination with high-affinity heparins or heparinoids with affinity for AT III, preferably as an AT III-heparin (or heparinoid) complex (heparinoids is a collective term for natural occurring, semi-synthetic and synthetic mucopolysaccharides with heparin-like effects).

A preferred production process for such a complex is described in AT-PS 379 310, wherein human plasma or AT III-containing plasma fractions are mixed with heparin or heparinoid and the heparin or heparinoid complex formed is purified by adsorption on an anion exchanger. Such a preparation is commercially available under the designation Atheplex ^® (Fa. Immuno, AT). Of course, this method can also be carried out with starting solutions which have been produced by the recombinant DNA technology.

The use of AT III according to the invention is preferably used in the context of an anticoagulation therapy which comprises a heparin treatment.

As mentioned, the pain associated with the heparin injection (as a side effect of the anticoagulation therapy) can be greatly reduced or even prevented with the combined administration of AT III and heparin or of AT III complexed with heparin. In addition, there is a significantly reduced hematoma formation at the injection site.

In particular, according to the invention, patients at risk of heparin-induced thrombocytopania can be treated.

The preparation for subcutaneous administration according to the invention can contain different types of heparin. For example, heparins in low molecular weight with a molecular weight of about 1500 to 10,000 or in high molecular weight with a Molecular weight of about 10,000 to 30,000 daltons may be included.

The preparation according to the invention has a surprisingly high functional half-life. Due to its novel absorption and pharmacokinetics, the preparation according to the invention is characterized by an exceptional bioavailability of AT III or heparin. The components of the preparation are preferably mixed and administered by the choice of the heparins or the heparinoids or their AT III affinity such that the heparins or heparinoids contained in vivo have a functional half-life of more than 10 hours, in particular of more than 20 hours, in certain cases even more than 30 hours.

The pharmaceutical preparation according to the invention is produced by completing a purified preparation containing AT III and optionally heparin, according to methods known per se, by combining it with suitable buffer, auxiliary, preservative and / or stabilizing substances or protease inhibitors and is filled in a form suitable for administration and is preferably stored in a stable manner, optionally in a lyophilized or frozen state.

The effective dosage of the preparation when used depends on the individual patient (height, body weight, condition, blood count, ...).

The AT III concentration in the preparation according to the invention or for the application according to the invention is preferably selected such that administration of a dose of 5 to 10,000 U AT HI / kg body weight in a volume of less than 20 ml, in particular less than 10 ml, preferably less than 5 ml, most preferably less than 2 ml.

The preparation according to the invention is preferably subjected to a method for inactivating infectious or pathogenic material, in particular viruses. These inactivation treatment is preferably ensured with a surfactant and / or heat treatment, for example by a heat treatment in the solid state, in particular a steam treatment according to EP-0 159 311, or EP-0 519 901 or EP-0 674 531.

Other treatments for inactivating viruses also include treatment with chemical or chemical / physical methods, e.g. with chaotropic substances according to W094 / 13329, DE 44 34 538 or EP-0 131 740 (solvent) or the photo inactivation.

Irradiation or nanofiltration is also a preferred physical method for depleting viruses in the context of the present invention.

Finally, the invention also relates to a kit for subcutaneous injection comprising a pharmaceutical preparation containing AT III, instructions for the use of the preparation for subcutaneous injection, and optionally an application device suitable for subcutaneous injection, preferably a syringe. This syringe has one in relation to i. m. -Spray very thin needle to facilitate penetration under the skin.

The pharmaceutical preparation in the kit is preferably provided in a container in lyophilized form, but it can also be provided in liquid form, in which case, however, it is then preferably stored in deep-frozen form.

The invention is explained in more detail with reference to the following examples and the drawing figures, to which, however, it should not be restricted.

Show it:

1A and 1B: the local tolerance of heparin and Atheplex ^® on the mouse; 2: the pain-inducing effect of heparin in the rat paw model;

3: the pain-triggering effect of Atheplex ^® in the rat model;

FIG. 4 shows the comparison of the ristocetin-dependent Plättenaggrega- tion of heparin and Atheplex ^®; and

Fig. 5: the determination of the half-life of heparin and Atheplex ^® .

Example:

Example 1: Haematoma formation in mice after subcutaneous injection of heparin or the preparation according to the invention (currently the best way of carrying out the invention in the opinion of the applicant)

In three runs, respectively 15 male NMRI mice are a total of g in weight of 25 to 30 having heparin (I MUNO AG, Wien) or preparation according to the invention (Atheplex ^®, obtainable from IMMUNO AG, Vienna.) Treated as follows:

1.5 IU heparin 1.5 heparin units Atheplex ^®

15 IU heparin 15 heparin units Atheplex ^®

Treatment (0.5 ml) was performed subcutaneously in the abdomen. The mice were examined daily for 4 days for discoloration of the subcutis. Changes were assessed as follows:

Score size not available 0 to 0.5 cm ² 1

0.6 - 4 cm ² 2

<4 cm ² 3 - o

Score discoloration none 0 low intensity 1 medium intensity 2 high intensity 3

Swelling none 0 slight 1 moderate 2 severe 3

The animals were sacrificed on day 4 and the subcutaneous changes were assessed pathologically and anatomically.

Average results are shown in Table 1 and Fig. 1.

TABLE 1A

Score

1.5 IU 1 2 3 4 day

Heparin 2.86 2.66 2.26 1.73

Atheplex® 1.20 0.40 0.20 0.20

TABLE 1B

Score

15 IU 1 2 3 ^' 4 Tacr

Heparin 3.66 4.13 3.33 2.33

Atheulex® 2, 86 1.80 1.40 1.33

As can be seen (Fig. 1A, B), there is a relatively significant difference between the two tested preparations in favor of Atheplex ^® at 1.5 and 15 units of heparin.

Control animals, which had received isotonic saline analogue, never showed hematomas. The section confirmed the visual assessment of the 4th day.

Example 2: Examination of the pain-triggering effect of a subcutaneous injection of heparin and the preparation according to the invention in the rat paw model

This effect was determined on the inflamed rat paw by determining the pain threshold according to Randall & Selitto (Arch. Int. Pharmacodyn. 111 (1957), 409-419).

For this purpose, a mercury monometer was connected to a 10 ml syringe, the piston of which was equipped with a short, bullet-shaped plug. This syringe applied increasing pressure to the rat paw (26.7 mbar / s). The pain threshold was given as the pressure in mbar that is necessary to trigger a defense reaction of the animal.

Female Sprague-Dawley rats weighing between 250 and 350 g were used for the study. The pain threshold was measured before the injection (previous value) and every hour up to 6 hours after the intraplantar injection of heparin (multiple doses) or Atheplex ^® (multiple doses) or isotonic saline (100 μl, negative control). The values after injection of test or control substances were expressed as a percentage of the previous value.

The results are shown in Figs. 2 (heparin) and Fig. 3 (Atheplex ^®) produced (x + SJJ-). On the ordinate, the percen ^¬ Tuelle pain threshold is applied: 100% = no pain; 0% = maximum pain. The abscissa represents the time axis (hours after intraplantar injection). The top curve (triangles) represents the control animals (isotonic saline, n = 15; no pain).

Heparin (Fig. 2) was tested in doses 1, 3, 10, 40 and 130 IU on 10 animals each. From 10 IU, progressive, severe pain occurs, with no further dose dependency being discernible. Atheplex ^® (Fig. 3) was tested in doses 10, 40, 130 and 200 units of heparin (n respectively 10). Here too there is initial pain, which, however, diminishes as the duration progresses. The timing and extent of this decrease are dose-dependent:

10 heparin units: reversal after the 2nd hour, complete freedom from pain after 6 hours.

200 heparin units: reversal after the 5th hour, residual pain at the 6th hour: 46.9 ± 3.0%.

These results show a significantly lower pain development due to the preparation according to the invention in contrast to conventional heparin therapy.

Example 3: In vitro investigation of the inhibition of platelet aggregation by heparin and by the preparation according to the invention

The influence of the substances on the platelet aggregation takes place by means of a turbidometric method using an aggregometer (from Chronolog). To 400 μl of a formaldehyde-fixed platelet preparation (250,000 cells per μl), 25 μl of an FVIII-vWF preparation and 25 μl sample or buffer to be tested are added as a control. After an incubation of 3 min at 37 ° C., the aggregation is started with 50 μl ristocetin sulfate (10 mg / ml). The aggregation is evaluated via the increase in the aggregation curve. The reference value is the aggregation with buffer addition.

In contrast to heparin, ristocetin sulfate-induced platelet aggregation was not inhibited by AT III-complexed heparin. The experiments were also repeated with a cleaned, recombined von Willebrand factor. The results with the vWF-dependent aggregation are identical to the FVIII-vWF-dependent reaction. Both can be reduced by adding heparin, but not with the addition of Atheplex ^® . factor evaluation units heparin in 500 ul test mixture Heparin Atheplex ^®

2.5 0, 47 1, 01

0.25 0, 84 0, 99

0.025 1, 09 1, 07 buffer control (without heparin) 1

The results show that heparin inhibits the aggregation in a dose-dependent manner, whereas the reaction is not or not significantly influenced by Atheplex ^® .

Example 4: Determination of the half-life of subcutaneously injected heparin and subcutaneously injected preparation according to the invention

6 awake rabbits (White New Zealanders, approx. 2.5 kg, of both sexes) are administered 6,000 units of unfractionated heparin per kg subcutaneously in the heparin group. In the comparison group, 6000 units of heparin with 1000 units of AT III as a complex (Atheplex ^® ) are injected subcutaneously. Citrate blood was drawn via the ear vein 6, 12, 18, 24, 30, 36, 42, 48, 54, 60 hours after the application. The heparin level in the plasma obtained is determined by means of an adapted thrombin inhibition determination using a chromogenic substrate (Coa-Chrom Diagnostika, Chromogenix). The calculations are based on biostatistical criteria. All tests of the heparin 6000 U / kg and Atheplex ^® application 1000 U / kg (= 6000 heparin units / kg) in the rabbit test were included in the calculation.

Heparin 6000 U / kg Atheplex ^® 1000 U / kg subcutaneously subcutaneously

Hep. Best. (Ila-Inh.) Hep.Best. (Ila-Inh.) AT III (Ag) lag time (h) 0.0006 6.02 10.7

Adsorption (l / h) 0.15 0.17 0.42

Elimination (l / h) 0.15 0.062 0.031 T / 2 adsorption (h) 4, 76 4.1 1, 66 from application 2.4 11, 5 17, 8

T / 2 elimination (h) 4.59 11.2 22.7 from application 16.9 31.7 42.4

AUC (E / ml.h) 1939 2850

Maximum (E / ml) 4.1 8.2 4.2 Maximum time (h) 12 18 18

The present data show the superiority of complexed heparin with AT III over uncomplexed heparin. The same superiority can also be seen with IV administration of Atheplex ^{® in} terms of half-life and recovery (recovery).

Claims

Patent claims:

1. Use of antithrombin III (AT III) for the manufacture of a pharmaceutical preparation for preventing bleeding during anticoagulant therapy.

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

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

4. Use according to one of claims 1 to 3, characterized in that AT III is used in combination with highly affine heparin.

5. Use according to one of claims 1 to 4, characterized in that an AT III-heparin complex is used.

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

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

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

9. Use according to one of claims 1 to 8, characterized labeled in ^¬ characterized that patients thrombocytopenia be treated with a risk of induced heparin.

10. Use according to one of claims 1 to 9, characterized labeled in ^¬ characterized in that AT III is administered subcutaneously. WO 98/42371_, _Λ PCT / AT98 / 00078

- 14 -

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

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

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

14. Preparation according to one of claims 11 to 13, characterized in that heparins with a molecular weight of 1500 to 10,000 are included.

15. Preparation according to one of claims 11 to 13, characterized in that heparins with a molecular weight of 10,000 to 30,000 are contained.

16. Preparation according to one of claims 11 to 15, characterized in that it contains heparins or heparinoids with a functional half-life of more than 10 hours, preferably more than 20 hours.

17. Preparation according to one of claims 11 to 16, characterized in that it contains an AT III concentration, which means the administration of a dose of 5 to 5000 U / kg in a volume of less than 20 ml, in particular less than 10 ml, preferably less than 5 ml allowed.

18. Kit for subcutaneous injection, characterized in that it comprises the preparation according to one of claims 11 to 17 and instructions for the use of the preparation for subcutaneous injection.

19. Kit for subcutaneous injection, characterized in that it comprises the preparation according to one of claims 11 to 18 and an application device suitable for subcutaneous injection, preferably a syringe.

20. Kit according to claim 18 or 19, characterized in that the preparation is in a container in lyophilized form.

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