AU616555B2

AU616555B2 - The use of plasminogen activator inhibitor (pai-2) for immunosuppression

Info

Publication number: AU616555B2
Application number: AU40905/89A
Authority: AU
Inventors: Norbert Heimburger; Hans Ulrich Schorlemmer; Thomas Stief
Original assignee: Behringwerke AG
Current assignee: Siemens Healthcare Diagnostics GmbH Germany
Priority date: 1988-08-31
Filing date: 1989-08-30
Publication date: 1991-10-31
Anticipated expiration: 2009-08-30
Also published as: PT91584B; DK428089D0; EP0356945B1; KR900002791A; DE3829523A1; ATE97576T1; ES2061842T3; EP0356945A2; DE58906241D1; PT91584A; JPH02108633A; AU4090589A; DK428089A; EP0356945A3

Abstract

The possibility of using plasminogen activator inhibitor (PAI-2) for producing a medicament for immunosuppression is described. Also described is a medicament containing PAI-2 and an ornithine decarboxylase inhibitor.

Description

F" I I, COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952-69 616 55 COMPLETE

SPECIFICATION

(ORIGINAL)

Class Application Number: Lodged: Int. Class Complete Specification Lodged: 0 Accepted: 0 Published: *Priority: 0 0 00 Oelated Art a 9044 MKame of Applicant: Aqdress of Applicant BEHRINGWERKE AKTIENGESEL-iLSCHAFT D-3550 Marburg, Federal Republic of Germany Actual Inventor: THOMAS STIEF, NORBERT 1-EIMBURGER and HANS ULRICH SCHORLEMMER XXA0WES3Maemr Patent Trademark Attorneys 50 QUEEN STREET, MELBOURNE, AUSTRALIA, 3000.

4 4 tAddress for Service Complete Specification for the invention entitled: THE USE OF PLASMINOGEN ACTIVATOR INHIBITOR (PAI-2) FOR IMMUNOSUPPRE SSION The following statement is a full description of this invention, including the best method of performing it known to u 7 4 BEHRINGWERKE AKTIENGESELLSCHAFT 88/B 025 Ma 708 Dr. Ha/Bi The use of plasminogen activator inhibitor (PAI-2) for immunosuppression The invention relates to the use of plasminogen activator inhibitor (PAI-2) as an immunosuppressant.

Macrophage and polymorphonuclear neutrophilic leukocytes (PMN) are essentially involved in the immune response of the body. They have an attack system which comprises proteases on the one hand and potent oxidizing agents (Nt chloroamines and oxygen radicals) on the other. They Ott influence and enhance the immune response via these pro- °0 teases and oxidizing agents.

0 9 By immune response is meant the reaction of immunocomoo: petent cells such as the monocyte/macrophage system to stimulators of inflammation, infection, tissue transformation and tissue repair. The monocyte/macrophage system performs a wide variety of tasks throughout the body ,20 because monocytes migrate from the blood into a wide variety of tissues where they undertake, not least, specific immunological tasks. Thus, the monocytes differentiate into alveolar macrophages in the lung, into Kupffer's cells in the liver, into osteoclasts in bone, into microglial cells in the central nervous system S(CNS), into mesangial macrophages in the kidney, into surface cells in the synovial membrane, and into pleural 4 or peritoneal macrophages in the body cavities.

Inhibitors of these macrophages and PMN are of great clinical interest because a large number of human diseases is associated with increased macrophage activity.

There may be circumstances in which suppression of the function of one or more compartments of the immune system .0 I- 2 is desirable. Possible circumstances of this nature are a reaction of the immune system which goes beyond the normal extent (hyperergic state) or a reaction of the immune system against the body's own tissue.

Substances which are able to inhibit the biological action of macrophages can act as immunosuppressants.

There is a great need for immunosuppressants which a) have few side effects and b) are highly specific. The corticoid derivatives which are widely used clinically comply with neither a) nor b).

It has been found, surprisingly, that plasminogen activator inhibitor (PAI-2) displays an action inhibiting V, C the secretion of oxygen radicals in in vitro test systems with macrophages and the secretion products thereof.

a According to Thrombosis and Hemostasis 56, 415-416 g*od (1986), the properties of PAI-2 include the following: molecular weight 47,000 (unglycosylated), 60,000 (glycosylated); isoelectric point 5 inhibition constant 0 for urokinase: 9 x 10M-s s 1 0 6 to a It is surprising that PAI-2 is able to inhibit urokinase in an oxidative environment, i.e. PAI-2 is an oxidation- Sresistant PAI in contrast to the PAI of the endothelial type (PAI-1).

l It has been found, surprisingly, that PAI-2 in physiolo- I 25 gical concentrations has a potent inhibitory action on r the chemiluminescence reaction of macrophages which derives from the release of oxygen radicals. Associated with this it has been found that PAI-2 has an immunosuppressant action in vivo in transplantation models.

The invention therefore relates to the use of placental plasminogen activator inhibitor (PAI-2) in a process for the preparation of an agent for immunosuppression.

r" I 3 Examples of diseases in which an agent of this type can be used are autoimmune diseases such as psoriasis, glomerulonephritis, preferably of the mesangial fields, multiple sclerosis, Guillain-Barr6 syndrome, collagenoses, allergic disorders, Sch6nlein-Henoch syndrome, degenerative disorders such as Parkinson's disease, arteriosclerosis, disturbances of wound and bone healing and transformation, or shock syndrome such as ARDS (adult respiratory distress syndrome) post-burn disorder and consumption coagulopathy. An agent of this type might also be of importance for infectious diseases such as

AIDS.

C

It f t I 151 44.e 4 V 6 4 4 The transplantation of exogenous tissue is also followed by an immunological reaction against the transplanted organ which is recognized as foreign or against the recipient organism in the nature of a graft-versus-host reaction, which leads to rejection. Rejection crises of this nature may also arise with placental tissue which in some cases is also recognized by the mother's body as foreign and may lead to placenta/trophoblast disturbances and premature detachment of the placenta. It is therefore necessary to suppress the immune system of the organ recipient or of the transplant in order to ensure the survival of the transplant or of the recipient.

Complications (after-bleeding) after subarachnoid hemorrhage has taken place are likewise partly caused by increased activity of white blood cells.

In contrast to the superoxide dismutase which is employed clinically for disorders such as Crohn's disease, ulcerative colitis, collagenoses or multiple sclerosis, and which is able to neutralize oxygen radicals which have been produced, PAI-2 prevents the production of oxygen radicals. PAI-2 also displays an action against allergic encephalomyelitis AE is regarded as a model of the human disorder multiple sclerosis.

4 -4- Reactions of the immune system going beyond the normal, and pathologically elevated release of oxygen radicals associated therewith, also occur in reperfusion states, i.e. a sudden flow of blood through a previously hypoperfused tissue, when breathing a high percentage of oxygen, on clinical use or poisoning with, for example, paraquat, use of cisplatin, adriamycin, nitrofurantoin, bleomycin, streptozotocin and other diabetogenic substances, irradiation therapy and tissue damage associated therewith.

Measures known to be suitable for suppressing the immune system include treatment with antibodies against lymphatic tissue, with ionizing radiation and with chemical substances. An aggressive treatment of this nature is associated with pronounced side effects: organotoxicity, 15 sterility with cytostatics and ionizing radiation, 7e" cerebral edema with tranexamic acid, anti-antibody formation with the risk of a serum disease on treatment with antibodies.

By contrast, use of human physiological PAI has none of the side effects and acts considerably more effectively S. even at low doses. High concentrations of PAI-2 have been described even in the plasma of, for example, healthy pregnant women. It emerges from this that the therapeutic index of this protein is high, with at least up to urokinase-inhibiting units/ml of plasma (200 300 89'o ng/ml).

t6 a 4 09 An effective amount of PAI-2 for immunosuppression and inhibition of the release of oxygen radicals, for example to inhibit the damage to tissue at risk during reperfusion, subarachnoid hemorrhages, autoimmune diseases, multiple sclerosis, collagenoses, allergic disorders, Sch6nlein-Henoch syndrome, degenerative disorders, arteriosclerosis, disturbances of wound and bone healing and transformation, post-burn disorders, transplant incompatibility, premature detachment of the placenta and pre-eclampsia, shock syndrome, depends on a number of

I

5 factors, for example the age and the weight of the patient and the clinical condition.

An effective dose is, in general, in the range 0.7 30,000 and preferably 7 3000 urokinase-inhibiting units/kg/24 h, i.e. 525 225,000 units, which can be administered i.v. or, solubilized, i.m. for a patient weighing 75 kg. PAI-2 can be stabilized by adding stabilizers such as albumin, polygeline or glycine. f Aose .tL\e r-c o* 5'2l to a% a5 rr 'Ikl'on ?A\\tS o 1 Al- a cQ" c>\So \ce AseA PAI-2 can also be administered topically for local therapy and prophylaxis of disturbances of wound healing, burns, transplant rejections, allergic respiratory S"0 disorders and other disorders associated with elevated macrophage activity.

o a S° There are medical indications for combinations of PAI-2 a.5 and a fibrinolytic such as tissue plasminogen activator (tPA), urokinase, streptokinase, or plasminogen streptokinase activator complex for lysis therapy preferably of arterial occlusions such as in myocardial infarkt or stroke. Combined use of PAI-2 and fibrinolytic makes it 20 possible immediately to inhibit tissue damage such as, o*o for example, necroses during and after acute reperfusion ao je., \e o c.es.

owing to hyperactive white blood cells\ If one of the natural plasminogen activators tPA or urokinase is used in combination with PAI-2 as fibrinolytic it is preferably the single-chain forms of these substances which are I used, because they, in contrast to the two-chain forms, form no or virtually no inactive complexes with PAI-2.

The effective dose of fibrinolytic in combination with PAI-2 is 1 200 mg of tPA or urokinase or 100,000 3 million units of streptokinase or activator complex for systemic administration in a patient weighing 75 kg.

Further medical indications are for combinations of PAI- 2 and an ornithine decarboxylate inhibitor with a low molecular weight, such as a derivative of a polyamine, or 6 Swith a high molecular weight, such as a transglutaminase.

Sft cose. o 5Da to '59.5) i O on C o Aec-to I'\_oci r use-cA Particularly preferred for use as an immunosuppressant is the combination of PAI-2 with a transglutaminase such as F XIII derived from the placenta or plasma or by genetic manipulation. If F XIII is used in combination with PAI- 2, the effective dose of F XIII is in general 0.7 300 U/kg/24 h for systemic administration to a patient weighing 75 kg.

The effect of the substances according to the invention on the immune response of mice and of humans is illustrated by way of example hereinafter in selected standard in vivo and in vitro test methods which are known to be used for assessing immunosuppressors.

Example 1 Effect of PAI-2 (urokinase inhibitor) on the stimulation of human phagocytes in vitro Polymorphonuclear granulocytes and mononuclear phagocytes were obtained from the peripheral blood of healthy donors and tested for various functions after administration of 0 20 the product. The parameters of phagocyte function inves- Stigated were the chemiluminescence reaction (formation of oxygen radicals) and the secretion of lysosomal enzymes.

The effect of PAI-2 both on non-activated phagocytes and on phagocytes activated by immune complexes was measured. PAI-2 was obtained as described in J.

Biol. Chem. 262, 3646-3653 (1987).

Compared with phagocytes from the corresponding control groups (untreated cells) the chemiluminescence reaction (generation of 02 radicals) was significantly and dosedependently reduced with both human granulocytes and monocytes (phagocytes) (Table 1).

u p .13 7 Example 2 Effect of PAI-2 on the activity of mouse peritoneal macrophages in vivo Female NMRI mice (18 20 g) received intraperitoneal administration of urokinase inhibitor (PAI-2) in various concentrations from 5 to 100 U/animal. The controls received the same volumes (0.5 ml) of the solvent (physiologically buffered saline solution, pH Two hours and 24 hours later the mice were sacrificed, the macrophages were removed from their abdominal cavities, and the determination of the activity was carried out by means of chemiluminescence as described in Example 1. As S' is evident from Table 2, PAI-2 reduces the activity of *macrophages taken from mice previously treated with the product. The suppression was observed in the chemio luminescence both 2 hours and 24 hours after administration of the substance with and without addition of immune complexes in vitro.

Example 3 Effect of chloramine T oxidation on the activity of plasminogen activator inhibitor (PAI) in plasma a) Various chloramine T concentrations 50 pl of PAl-deficient plasma (Biopool, Umea, Sweden), PAI-1-rich plasma (15 urokinase-inhibiting units (U)/ml) and PAI-deficient plasma supplemented with 15 U of PAI-

S

4 2/ml were incubated with 50 pl of 100 m iol/l tris, 100 4 mmol/l NaC1, 1 RHaemaccel, 0.1 RTriton X 100 buffer S(TSHT), pH 8.5, and 50 pl of chloramine T (CT) of various concentrations in distilled water at room temperature for 10 min. Then 200 pl of TSHT buffer with 5 IU of urokinase (uPA) and for zero balancing with 0 IU of uPA/ml were added, and incubation at room temperature was continued for 10 min in order to generate uPA/PAI complexes. The remaining uPA activity was subsequently determined with the method described by Stief et al. (Thromb. Res. 559-573, 1988) by adding 200 pl of 10 mmol/l chloramine 8 T, 3 mmol/l tranexamic acid in distilled water, incubating at room temperature for 15 min and starting the detection reaction by adding 500 pl of 0.6 mmol/1 chromogenic plasmin substrate HD-Nv&-CHA-Lys-pNA (pNA paranitroanilide) in 480 mmol/l NaCI.

After incubation at room temperature for 10 minutes, the substrate reaction was stopped by addition of 100 pl of 8.4 mol/l acetic acid, and the extinction at 405 nm was determined. See Table 3 for the result.

Losses of activity of PAI-1 occurred at and above 0.1 pmol of CT, whereas PAI-2 is about 5 times as resis- S. tant to CT oxidation.

As little as 0.5 pmol of CT in 50 pl of plasma results in 0 more than 50 loss of active PAI-1, whereas the same dose of CT still leaves 77 of the PAI-2 activity *ao intact.

b) Effect of various PAI-2, RFibrogammin and ascorbic acid concentrations on loss of control, caused by oxidation, of plasma via plasminogen activator activity.

op o 100 pl of standard human plasma anticoagulated with citrate were incubated with 50 pl of a) PAI-2, b) RFibrogammin, c) ascorbic acid solution of various concentrations in distilled water with 250 l of TSHT buffer with 1.25 IU of uPA, 100 pl of 10 mmol/l CT and 100 pl of 25 3 mmol/l HD-Nva-CHA-Lys-pNA at 37 0 C for 60 min. The sub- S* strate conversion was then stopped by addition of 500 pl of 3.4 mol/l acetic acid, and the resulting extinction at 405 nm was determined. See Table 4 for the result.

It is seen that PAI-2 (10 U) and RFibrogammin (0.5 Fibrogammin units) and vitamin C (20 pmol) lead to a restoration o2 the loss of control via the uPA/plasmin system.

The initially elevated extinctions decrease as far as 0 on supplementation of the plasma with the substances.

-L1 U Tr 9 It is possible to replace vitamin C by N-acetylcysteine or N-acetylmethionine.

Example 4 Effect of various CT concentrations on the uPA/plasminogen/plasmin system, neutralization of the effect by

R

PAI-2 and/or Antagosan in combination with vitamin C (ascorbic acid) 100 pl of standard human plasma anticoagulated with EDTA are added to 250 pl of TSHT buffer containing 1.25 IU of uPA, and 100 pl of distilled water containing a) 10 mmol/l ascorbic acid, b) 2.5 U of PAI-2, c) 10 mmol/l ascorbic acid, 2.5 U of PAI-2, d) 2.5 KIU of RAntagosan, e) 2.5 KIU of "Antagosan, 2.5 U of PAI-2, o CO f) 2.5 KIU of RAntagosan, 10 mmol/1 ascorbic acid, 2.5 U of PAI-2, h) (control). Then 100 pl of CT in various concentrations in distilled water were added, and the 00 44** mixture was incubated at 37°C for 45 min. Addition of 0000 500 pl of 0.6 mmol/l HD-Nva-CHA-Lys-pNA in 480 mmol/l NaC1 started the substrate reaction. The substrate conversion was stopped after 6 min (37 0 C) by addition of 500 pl of 20 acetic acid. See Table 5 for the result.

So o a 0 It is seen that increasing amounts of CT result in proportional increases in the extinction measured at 405 nm. The physiological control in the plasma via the serine protease activity of plasmin and urokinase has been lost. The control can be restored by supplementation of human plasma with Antagosan and/or PAI-2, and con- SA siderably less plasmin activity can be detected. The use 1 of vitamin C enhances the effect of PAI-2/Antagosan in a synergistic manner.

Example Effect of PAI-2 on the survival time of transplanted skin from the rat tail In this design of experiment, pieces, about 0.5 x 1.0 cm i 10 in size, of tail skin from Lewis rats were transplanted onto the tail of Fischer rats. The transplanted pieces of skin are recognized as foreign by the immune system of the recipient animals and are rejected. As is evident from Table 6, the survival time of transplants in the Lewis/Fischer rat model was between 16 and 18 days in the control groups which were treated only with the solvent.

PAI-2 (50 U/animal per injection) was administered intraperitoneally on 7 consecutive days starting either on day 1 or on day 10 after transplantation. It emerged from this, surprisingly, that PAI-2 in the concentration used increased the survival times of the transplants from 17.0 1.4 to 26.0 2.0 and 23.0 2.4.

t I I I I 15

I

44 I Table 1 Effect of PAI-2 on oxidative metabolism (chemiluminescence) with and without stimulation by immune complexes (50 pg/ml) in vitro Chemiluminescence Integrated RLU/15 min (x10 3 Cell type PAI-2 (U/ml) .4 20

Q

4 a 444 4 -IC +IC Human 0 2478 354 18516 1571 monocytes 0.07 1321 92 9347 709 (phagocytes) 0.15 1061 35 4825 403 1x10 6 cells 0.7 811 68 2640 235 633 49 1580 221 6.0 452 31 1040 163 25.0 247 75 695 125 Human 0 3110 127 48547 2372 granulocytes 0.05 1656 252 28633 4394 2x10 5 cells 0.1 1172 131 15567 1762 1018 94 12267 709 422 48 6587 604 135 22 4550 665 r T Table 2 Effect of PAI-2 (urokinase inhibitor) on macrophage activity (1 x 106 cells) in vivo.

P AI1-2 U/animal 1 x i. p.

Chemiluminescence Integrated RLU/15 min. (X 103) Ic Ic 24 h 2 h 24 h 2 h a a.

a aoa a a 0 a a a. a a 0000 0 5 50 100 2775± 1655± 1145± 6 00± 46 6± 375 106 70 118 75 4480 ±453 344 0 ±142 1467 ±85 915 ±19 488 ±166 51800 ±1286 2 7175 ±1859 19267 ±1550 13833 ±2346 9 783 2 2188 72500 1556 23950 1625 17200 ±1407 8953 ±287 4960 ±392 a. a.

o a a 00 0 o 0, o a a o o* o a, a a a a o.a a a a a., *a aa a a a o Table 3 12 CT dose 4l) Extinction PAI-def. PAI-1

(A

405 X 1000) Inhibition of uPA PAI-2 PAI-i PAI-2 M% M% PAI-1 PAI-2 0 0.01 0.05 0.1 0.175 0.25 0.375 0 0.5 0.7 of 5 1.0 1.5 0 2.0 1011 1016 1033 1050 1063 1099 1060 1049 1001 982 921 834 350 377 351 459 541 596 668 735 777 485 498 503 509 528 549 593 626 696 100 97 101 86 75 66 57 46 34 27 21 17 100 98 98 100 96 77 52 42 38 4 00 000 04 044 049 0 09 000 00 0*00 807 716 792 718 738 667 Extinction for PAl plasmaa (1 x 100 Extinction for PAl-deficient plasma 6 4..

of 0 Amol of CT distilled water control 4 *4 0 0 O 00 ii 4404 41 04 0 0 4 0 13 Table 4 Addition of uPA inhib.U.

PAl -2

A

405 units (x 1000)

A

405 rimol/1 (x 1000) Vitamin C

A

405 nm (x 1000) 0 1 579 467 343 144 61 0.0 0.0 0.0 0 0.1 0.25 0.5 1 2 4 8 579 325 168 63 20 25 9 0.0 579 363 219 112 62 36 o2O 44 a 0 0 00 0 O 4 044 4 4 Table Addition of Control Plasmin activity generated CT (nmol/l) (A 40 5 .X 1000) of control) h) a) b) c) d) e) f) g) 0 0.0 5 32 56 53 65 40 36 37 22 10 121 30 48 60 26 22 26 9 456 33 50 64 16 21 32 11 30 870 54 43 62 10 35 29 40 837 100 48 60 47 62 27 29 14 Table 6 Effect of PAI-2 on skin transplantation in Fisciier rats Substance Days of transplant rejection Mean (days) (x s) 17.0 1.4 Control PAl -2 U/anim.al 7 x i. p.

day 1-7 PAl -2 U/animal 7 xi.p.

day 10-17 19, 18, 16, 16, 16 29, 27, 24, 26, 24 22, 27, 24, 22, 21 26 .0 2. 0 23.2 2.4 4 *4 0 4 4, 0 40 S S 9, 0 00* 1* 00

A

Claims

1. Method of inducing immunosuppression in a patient comprising administering an effective amount of plasminogen activator inhibitor (PAI-2) to a patient requiring immunosuppression.

2. Methods of therapy or prophylaxis of transplant- and placenta-rejection crises or graft versus host reactions in a patient comprising administering an effective amount of PAl- 2.

3. Method of treatment of autoimmune disease in a patient comprising administering an effective amount of PAI-2.

4. Method of therapy and prophylaxis of disturbances in wound and bone healing in a patient comprising administering an effective amount of PAI-2. Method of treatment of pathological processes associated with increased formation of S oxygen radicals in a patient comprising administering an effective amount of PAI-2.

6. Method of therapy or prophylaxis of tissue damage during and after acute reperfusion in a patient comprising administering an effective amount of PAI-2.

7. Method of therapy or prophylaxis of after-bleeding complications of subarachnoid j haemorrhage in a patient comprising administering an effective amount of PAI-2.

8. Method of therapy or prophylaxis of disorders associated with increased leucocyte activity in a patient comprising administering an effective amount of PAI-2.

9. Method of treatment of burns in a patient comprising administering an effective amount of PAI-2 in the form of a spray said spray optionally containing fibrin adhesive. 16 Method of promoting wound healing in a patient comprising administering an effective amount of PAI-2 in the form of a spray said spray optionally containing a fibrin adhesive.

11. Method of treatment of asthma in a patient comprising administering an effective amount of PAI-2 in the form of a spray.

12. Method of any one of the preceding claims wherein 52 to 2.25 million urokinase- inhibiting units per dose (75 kg body weight) of PAI-2 is administered.

13. Method of claim 12 wherein a stabiliser, preferably albumin or degraded cross- linked collagen is administered in adjunct with PAI-2.

14. Method of any of the preceding claims wherein, an ornithine decarboxylate inhibitor is also administered.

15. Method of claim 14 wherein the ornithine decarboxylate inhibitor is factor XIII. S: 16. Method of claim 14 or claim 15 wherein 52 to 225,000 units per dose (75 kg body o weight) of ornithine decarboxylate inhibitor and 52 to 225,000 units of PAI-2 is administered. DATED this 10th day of July 1991. BEHRINGWERKE AKTIENGESELLSCHAFT WATERMARK PATENT TRADEMARK ATTORNEYS S THE ATRIUM 290 BURWOOD ROAD HAWTHORN VICTORIA 3122 AUSTRALIA DBM/JMW (CH)