WO1988006037A1

WO1988006037A1 - Application of dextran sulphate in the production of a drug for the treatment and prophylaxis of vasoconstriction and vascular occlusion

Info

Publication number: WO1988006037A1
Application number: PCT/EP1988/000082
Authority: WO
Inventors: Elke Seewaldt
Original assignee: Dr. Karl Thomae Gmbh
Priority date: 1987-02-21
Filing date: 1988-02-04
Publication date: 1988-08-25
Also published as: DE3705669A1

Abstract

New application of dextran sulphate or its salts with a physiologically compatible base in the treatment and prophylaxis of vasoconstriction and vascular occlusion, which are caused by hyperplasia of the intima.

Description

Use of dextran sulfate for the manufacture of a medicament for the treatment and prophylaxis of vasoconstrictions and vascular occlusions

To prevent a re-occlusion after vascular surgery, high-molecular dextran or heparin and various combinations such as acetylsalicylic acid / dipyridamole, dextran / warfarin, etc. are administered today. - Here, when applying high-molecular dextran, e.g. Dextran

40,000 (dextran 40,000 is also referred to as low molecular weight in the literature, in contrast to high-molecular dextran 60,000), the viscosity-reducing property and the anti-aggregating effect on erythrocytes and platelets in the foreground.

A vascular occlusion after vascular surgery is a multifactorial process which, according to current knowledge, focuses on the formation of a neointimal hyperplasia in addition to thrombus formation (see, for example, Drug Development Research _6, 167-176 (1985), Progress in

Cardiovascular Diseases Vol. XXVI, 355-372 (1984) and Acta Chir. Scand. 149, 721-727 (1983)). However, it is still largely unknown which factors are decisive in the development of hyperplasia and how it can be influenced.

In the case of aorto-coronary artery bypass, the pathological changes in the vessel wall are described relatively well (for example LOWRIE, GM et al. In Am. J. Cardiol., 1976, 38: 865-872). After that, the "early occlusion" in the first three months is mostly caused by thrombus formation. In contrast, the "late occlusion" is the narrowing of the vascular lumen caused by hyperplasia of the intima. This intimal hyperplasia consists of smooth muscle cells (AJ MURDAY et al. In Cardiovasc. Res., 1983, 17: 446-451). It is assumed that the hyperplasia is caused by damage to the endothelium (stretching), the subsequent adherence of platelets to the subendothelial matrix, the release of a growth factor for smooth muscle cells from the platelets and the proliferation of the smooth muscle cells caused thereby.

A substance that inhibits the growth of smooth muscle cells would have to inhibit both the formation of atherosclerotic plaques and the narrowing of the vascular lumen by intimal thickening after vascular surgery.

Surprisingly, it has now been found that non-toxic dextran sulfate, in particular its salt with a physiologically compatible base such as its sodium salt, largely inhibits hyperplasia.

The term dextran sulfate here means dextrans in which the 3 free hydroxy groups per glucose unit are partially or completely replaced by hydroxysulfonyloxy radicals. For example, with a degree of substitution of 1 the sulfur content is 13.2%, with a degree of substitution of 2 with 19.9% and with a degree of substitution of 3 with 23.9% each based on the free sulfonic acid.

However, preferred dextran sulfates are their physiologically tolerable salts, in particular their alkali salts such as the sodium salt. For example, a degree of substitution of 1 has a sulfur content of. approx.12.1%, - 3 -

with a degree of substitution of 2 one of approximately 17.5% and with a degree of substitution of 3 one of approximately 20.5%. Preferred sodium salts of the dextran sulfates are those which have at least a sulfur content of 10%, but those sodium salts which have a sulfur content of 11.9% to 20%, preferably 12 to 19.5%, are particularly preferred. , exhibit.

For example, the following sodium salts of dextran sulfates are mentioned here:

M.G. of the dextran sulfate sulfur substitution

(Sodium salt) grad

3,400 (see AU-A-22856/83) approx. 12.1% 1 5,000 (Sig a, 32-F-0667) not determined

5,000 (Sigma, 65-F-0216) approx.16.1% ^X 5,000 (see CA.103, approx.17.0% 2.3 153669v (1985))

6,000 (M. Sangyo, N-2645) approx. 17.2% ^x

7,000 (see Bri .J.Pharma 1,3 col.8_, 476 (1953)) 7,300 (see Merck's Index approx. 17.0% No. 2907, 1976) 8,000 (Sigma, 74-F-0630) approx.13.0% ^x 11,000 (see CA.5_3_, approx.12.2%

1448a (1959)) 15,000 (see CA.103, approx.17.0% 2.3

153669v (1985))

^x = The sulfur content was determined by titration as sulfate after Schöninger combustion. Furthermore, those dextran sulfates with a molecular weight of up to 15,000 are preferred according to the invention, in particular those with a molecular weight between 1,000 and 10,000, but those with a molecular weight between 2,500 and 5,500 are particularly preferred.

The dextran sulfates used according to the invention are known from the literature or can be obtained by processes known from the literature by chlorosulfonation of dextrans (see US Pat. No. 2,715,091) or by cleavage and simultaneous sulfonation of cellulose (see AU-A-83.22856).

For example, the growth inhibitory effect of

Dextran sulfate 32-F-0667 (M.G.: 5,000) from Sigma,

Dextran sulfate 65-F-0216 (M.G.: 5,000) from Sigma,

Dextran sulfate N-2645 (M.G.: 6,000) from M.Sangyo and

Dextran sulfate 74-F-0630 (M.G.: 8,000) from Sigma

compared to that of dextran and heparin tested as follows:

1. Proliferation test

Smooth muscle cells (SMC) and endothelial cells (EC) were isolated from the porcine aorta and cultured under defined conditions in medium 199 with 10% FCS (fetal calf serum) at 37 ° C. and 5% CO ₂ .

Test procedure:

A

The cells are sown in a specific density (approx. 3x10 "2 cells / 25 cm) onto Roux 25 plates coated with gelatin. After the cells have adhered (after 4 1/2 hours) the medium is changed and the substance is added. The determination of the cell count at this point in time represents the initial value for the assessment of the substance effect. On the 4th day, the medium is changed again (with substance addition) and on the 6th day (EC may not be confluent, Since cell contact inhibits growth), the cells are detached enzymatically and the cell number is determined again with the cell counter. The mean value is determined from 3 culture dishes (4 counts per dish).

It is important that the two cell types undergo as many cell duplications as possible during the time of exposure to the substance. Under the test conditions mentioned, the SMC and EC double about 5 times.

To check the cell counter measurement, microscopic counts are carried out regularly. The morphology of the cells is continuously checked and documented.

Substance dose cell count + SE (% of control)

Dextran sulfate 5 45.5 + 0.8 ((MMGG 55000000)) 1 100 2 244 ,, 00 ++ 00 ,, 55 31.1 + 1.1

20 16.3 + 1.1 24, 5 + 1.0

SIGMA 40 16, 3 + 4, 7 20, 1 + 0, 4

32-F-0667 80 9.6 + 0.4

160 7.2 + 0.4 - 6 -

Substance dose cell count + SE (% of control)

Cμg / ml] SMC EC

Dextran sulfate 5 46.7 + 0.7 84.8 + 0.6

(MG 8000) 10 38.7 + 1.4 67.4 + 1.0

SIGMA 20 12.8 + 1.7 46.0 + 1.0

74-F-0630 40 6.3 + 0.2 36.6 + 1.6

Dextran 5 91, 4 + 4, 5

(MG 5000) 10 83, 5 + 1.4

20 97.2 + 0.8, 82.0 + 1.1

SERVA 40 74.2 + 3, 5 75, 8 + 8.6

18688 80 80.1 + 3.3 160 63.7 + 6.4

Dextran 5 94.2 + 3.2

(MG 9000) 10 91, 5 + 1.5

20 99.3 + 3.3 87.4 + 1.6

SIGMA 40 88, 9 + 3, 0 83.1 + 0, 7

53-F-0240 80 91.5 + 3.6 160 109.9 + 2.7

Dextran 20 78.2 + 5.5

(MG 40,000) 40 51.2 + 6,. 5 86, 8 + 2.0

SIGMA 80 70.9 + 7, 1 _ _

114-F-0335 100 - - 96, 7 + 2, 9

160 65.3 + 13 Substance dose cell count + SE (% of control)

Cμg / m 1] SMC EC

Heparin 10 28.5 + 2.1 26.6 + 0

MG 19000 20 25.2 + 3.5 24.2 + 0.5

SIGMA 40 15.1 + 0.1 22.4 + 0.9

34-F-0357

Heparin 10 30, 7 + 0, 6 31, 7 + 0, 7

Fragments 20 26, 3 + 3.4 27.3 + 0, 6

MG 5000 40 17, 8 + 0.2 25, 6 + 0.4

HEPAR

RD 10183

2. Rat ballooning model

In this in vivo model, the mechanical injury to the endothelium induces an intimal thickening that consists almost entirely of smooth muscle cells.

Approximately 6-month-old male rats weighing approximately 380-450 g were "balloonized" by pulling an inflated balloon catheter 5 times through the abdominal aorta. This procedure largely removes the endothelium (Clowes, AW et al., In Lab. Invest., 49 (3), 327-333; 1983). - 8th -

Dextran sulfate and dextran were dissolved in phys. NaCl solution s.c. , or orally administered once a day for 21 days.

The i.v. Administration was only over a period of 8 days. However, both the control animals and the treated groups were only killed after 21 days.

The extent of intimal hyperplasia was determined by the morphometric measurement of histological thin sections.

Substance dosage N intimal hyperplasia

Δ mm + SD% io ^"2 )

•

Dextran sulfate control SC 15 15.85 + 5. 95 100

MW 5,000 0.9% NaCl

2.0 mg / kg s.c. 12 9.89 * + 5.71 62

4.0 II 14 6.88 * + 3.89 43

Ch. 65F-0216 8.0 It 15 5.40 * + 4.41 34

SIGMA 16.0 II 14 4.83 * + 2.80 30

Dextran sulfate control i.v. 16 14.28 + 5, 30 100

MW 5,000 0.9% NaCl

1.0 mg / kg IV 15 10.48 * + 4.43 73

2.0 11 15 9.85 * + 3.89 69

Ch. 65F-0216 4.0 II 15 9.83 * + 4.98 69

SIGMA 8.0 II 15 5.58 * + 3.45 39 9 -

Substance dosage N intimal hyperplasia

Dextransulf t control p.o. 18 11, 38 + 5.00 100

MG 5,000 aqua dest.

250 mg / kg p.o. 15 7.08 * + 3, 84 62

500 15 8.27 * + 3.45 73

65F-0216 1000 13 4, 75 * + 3, 59 42

SIGMA 2000 10 3.52 * + 2.67 31

Dextran sulfate control sc, 16 10.24 + 4.71 100 MG 6000 0.9% NaCl 2.0 mg / kg sc, 15 7.31 * + 3, 38 75 4.0 15 4.80 * + 2 , 41 49

Ch. N-2645 8, 0 15 5, 72 * + 4, 01 58 M.SANGYO 16.0 15 4.13 * + 1.98 42

22.5 13 3, 91 * + 0.28 40

Dextran sulfate control SC, 15 14.58 + 7.11 100 MW 8000 0.9% NaCl

2.0 mg / kg s.c, 15 11.89 + 4.55 82

4, 0 15 11, 32 + 6.23 78

Ch. 74F-0630 8, 0 12 8.27 * + 3, 04 57 SIGMA 16, 0 13 5, 08 * + 2.77 35 - 10 -

Substance dosage N intimal hyperplasia

(x! 0 ^{~ 2} )

Dextran control s.c. 15 13.53 + 4.18 100 MW 5,000 0.9% NaCl

2 mg / kg sc 14 12.18 + 5.13 90 ^■■ "15 13.26 + 4.87 98

Ch, 18688 8 "15 9.07 * + 3, 74 67 SERVA 16" 15 8.03 * + 5.15 59

Dextran control s.c. 18 12.43 + 5.53 100 MW 40,000 0.9% NaCl

2.0 mg / kg s.c. 14 10, 33 + 4.45 83

4.0 II 14 13.13 + 4.24 106

8.0 II 15 10, 53 + 5.40 85

Ch. 115F-0193 16.0 II 14 10.46 + 6.65 84

SIGMA 22.5 II 14 12.92 + 5, 79 104

Heparin control s.c, 16 14.10 + 5.25 100 0.9% NaCl

0.25 mg / kg s.c. 12 15.25 + 7.46 108

0.5 "10 19.67 + 9.52 140

1.0 "9 16, 62 + 5, 40 118

2.0 "10 14.37 + 7.17 102

4.0 "11 5.10 * + 2.86 36

8.0 "8 6.80 * + 7.99 48

significant at p = 0.05 - 11 -

In the in vitro proliferation test, dextran sulfate and heparin show a strong growth-inhibiting effect on smooth muscle cells. In contrast, non-sulfated dextrans show a much weaker antiproliferative effect.

In the ballooning model on the rat, dextran sulfates significantly reduce intimal thickening (up to 70% inhibition).

The newly found antiproliferative effect of dextran sulfate could not be predicted, since it is known from the literature (GOTO, M. et al. In Exp. Cell Res., 82 (2):

367-374, 1973) that, although dextran sulf (MG 60,000 and MG 6,000) inhibits the proliferation of transformed Zeil lines, the growth of untransformed fibroblasts is not influenced, although it is known (OCHIAI, K. et al. In Pharmacodynamics , 70: 18770, 1969) that dextran sulfate inhibits the formation of atherosclerotic plaques in hypercholesterolemic rabbits. However, this effect known from the literature must be strictly differentiated from the newly found antiproliferative effect of dextran sulfate for the following reasons:

In human atherosclerosis, both proliferation of smooth muscle cells and lipid deposition in the vascular wall play a decisive role. However, the two animal models used in atherosclerosis research cover only one of the partial aspects mentioned and thus differ fundamentally in their approach.

Model 1:

In the hypercholesterolemic rabbit model, the formation of atherosclerotic plaques is induced by feeding a cholesterol diet. Resulting from this also the plaques mainly from deposited lipid and from lipid-filled monocytes (foam cells) (see T. WATA-NABE et al., in Lab. Investigation 53 (1): 80-90, 1985 and HC STARY in Artery 8: 205-207, 1980).

Model 2:

In this animal model, the endothelial layer is mechanically injured by pulling through a balloon catheter (in the case of another model, by electrical stimulation) and there is a narrowing of the vessels which is caused by an intimal thickening. Intimal hyperplasia consists largely of smooth muscle cells.

Since in the previously cited publication by OCHIAI, K. et al. Model 1 was used and the proliferation of smooth muscle cells is not recorded in this model, it is the "antiatherosclerotic see"

Effect of dextran sulfate around a cholesterol-lowering effect, which is also described in the literature (ISO et al., In Nich dai Igaku Jasshi, 1971, 30 (10): 823-837 Japan).

As used according to the present invention model ^'2, the lipid-lowering effect is not construed er¬ of dextran sulfate, but only the inhibition of smooth muscle cells.

The application of the non-toxic dextrans mentioned at the beginning or their salts with a compatible base inhibits the formation of intimal hyperplasia, but in particular the proliferation of the smooth muscle cells.

The dextran sulfates used according to the invention are well tolerated, especially since they have an acute toxicity similar to that of heparin (WW Kennet et al. In Brit. J. Pharmacol. 9, 1 (1954)). The LDcg ^be i ^mouse is for

Heparin at 2.03 g / kg and

Dextran sulfate (MG.7500) 2.12 g / kg.

In addition, the side effects occurring in thrombosis prophylaxis with high molecular weight dextran 40,000 should not occur when using the low molecular weight dextran sulfate. Furthermore, the anticoagulant effect of dextran sulfate (MW 5,000) is 10-20 times less than that of heparin (Genichiro, 0. et al., In Thro b. Res. 37, 361-72 (1986)) and so on. With dextran sulfate, fewer side effects (especially the risk of bleeding) can also be expected.

Because of the above-mentioned biological properties of the dextran sulfates used according to the invention, they are suitable as therapeutic agents for preventing vasoconstriction and vascular occlusion, which are caused by hyperplasia of the intima. According to the invention, the application of dextran sulfate or its salt with a physiologically compatible base is indicated for all injuries to the endothelial layer, especially since these endothelial injuries are unavoidable in all vascular reconstructions. These occur particularly after vascular reconstruction (recanalization, anastosis) due to atherosclerosis-related vascular narrowing or vascular occlusions such as after bypass operations, eg after coronary artery bypass, bypass of the arteria fe oralis / vena femoralis or the arteria femoralis / arteria popli - tea, after coronary angioplasty and after carotid endarteriecto ie. Other indications with endothelial damage or loss include cardiac valve surgery (repair or replacement), heart transplants, kidney transplants and arteriovenous shunts for the purpose of dialysis. The dextran sulfates are expediently administered subcutaneously or intravenously. In the case of subcutaneous or intravenous administration, the daily dose required to achieve a corresponding effect in humans is between 1 and 100 mg / kg body weight, but preferably between 5 and 20 mg / kg body weight, distributed over 1 to 2 doses per day.

For pharmaceutical use, the above-mentioned dextran sulfates and their physiologically tolerable salts can be incorporated into the customary soluble pharmaceutical forms of administration such as ampoules or solutions, in particular in the form of their isotonic solutions.

The following examples are intended to explain the invention without restricting it.

- 15 -

Example 1

Ampoules with 500 mg dextran sulfate (M.G .: 1,000) per 2 ml

Composition:

Dextran sulfate (M.G .: 1,000) 500.00 mg sorbitol 20.00 mg

Water for injections ad 2.00 ml

Manufacturing:

Dextran sulfate and sorbitol are dissolved in water for injections and sterile filtered, e.g. using a sterile filter from Millipore (pore size: 0.2 μmm). It is then filled into ampoules.

Example 2

Ampoules with 1,000 mg dextran sulfate (M.G .: 1,000) per 2 ml

Composition: Dextran sulfate (M.G.: 1,000) 1,000.00 mg

Water for injections ad 2.00 ml

Manufacturing:

The production takes place analogously to example 1.

Example 3

Ampoules with 500 mg dextran sulfate (M.G.: 5,000) per 0.5 ml

Composition:

Dextran sulfate (M.G.: 5,000) 500.00 mg

Cooking salt 3.00 mg

Water for injections ad 0.50 ml - 16 -

Manufacturing:

The production takes place analogously to example 1.

Example 4

Ampoules with 500 mg dextran sulfate (M.G.: 5,000) per 0.5 ml

Composition:

Dextran sulfate (M.G.: 5,000) 500.00 g

Sorbitol 18.70 mg

Water for injections ad 0.50 ml

Manufacturing:

The production takes place analogously to example 1.

Example 5

Ampoules with 1,000 mg dextran sulfate (M.G.: 5,000) per 1.0 ml

Composition:

Dextran sulfate (M.G.: 5,000) 1,000.00 mg table salt 6.00 mg

Water for injections ad 1.00 ml

Manufacturing:

The production takes place analogously to example 1. - 17 -

At game 6

Ampoules with 1,000 mg dextran sulfate (M.G.: 5,000) per 1.0 ml

Composition:

Dextran sulfate (M.G.: 5,000) 1,000.00 mg sorbitol 37.40 mg

Water for injections ad 1.00 ml

Manufacturing:

The production takes place analogously to example 1.

Example 7

Ampoules with 500 mg dextran sulfate (M.G .: 10,000) per 1.0 ml

Composition:

Dextran sulfate (M.G.: 10,000) 500.00 mg

Cooking salt 7, 40 mg

Water for injections ad 1.00 ml

Manufacturing:

The production takes place analogously to example 1.

Example 8

Ampoules with 500 mg dextran sulfate (M.G .: 10,000) per 1.0 ml

Composition: dextran sulfate (M.G.: 10,000) 500.00 mg

Sorbitol 46.00 mg

Water for injections ad 1.00 ml - 18 -

Manufacturing:

The production takes place analogously to example 1.

Example 9

Ampoules with 1,000 mg dextran sulfate (M.G .: 10,000) per 2.0 ml

Composition:

Dextran sulfate (M.G.: 10,000) 1,000.00 mg

Cooking salt 14.80 mg

Water for injections ad 2.00 ml

Manufacturing:

The production takes place analogously to example 1.

Example 10

Ampoules with 1,000 mg dextran sulfate (M.G.: 10,000) per 2.0 ml

Composition:

Dextran sulfate (M.G.: 10,000) 1,000.00 mg sorbitol 92.00 mg

Water for injections ad 2.00 ml

Manufacturing:

The production takes place analogously to example 1.

At this point it should be mentioned that, of course, all dextran sulfates and their physiologically tolerable salts, in which at least 1, 3 of the 3 hydroxy groups per glucose unit are each replaced by a hydroxysulfonyloxy radical, can be incorporated as "active substance" in the examples mentioned above.

Claims

1. Medicament for the treatment and prophylaxis of vascular constrictions and vascular occlusions which are caused by intimal hyperplasia, containing a non-toxic dextran sulfate or its salts with a physiologically compatible base.

2. Medicament according to claim 1, characterized in that the dextran sulfate used has a sulfur content of at least 10% based on its sodium salt.

3. Medicament according to claim 1, characterized in that the dextran sulfate used has a sulfur content of 11.9 to 20% based on its sodium salt.

4. Medicament according to claim 1, characterized in that the dextran sulfate used has a sulfur content of 12 to 19.5% based on its sodium salt.

5. Medicament according to claim 1, 2, 3 or 4, characterized ge indicates that the sodium salt of dextran sulfate used has a molecular weight of up to 15,000.

6. Medicament according to claim 1, 2, 3 or 4, characterized in that the sodium salt of the dextran sulfate used has a molecular weight of 1,000 to 10,000.

7. Medicament according to claim 1, 2, 3 or 4, characterized ge indicates that the sodium salt of dextran sulfate used has a molecular weight of 2,500 to 8,000.

8. Use of non-toxic dextran sulfate or its salts with a physiologically compatible base for the treatment and prophylaxis of vasoconstrictions and vascular occlusions which are caused by intimal hyperplasia. - 20 -

9. Use of non-toxic dextran sulfate or its salts with a physiologically compatible base for the manufacture of a medicament for the treatment and prophylaxis of vasoconstrictions and vascular occlusions which are caused by intimal hyperplasia.

10. A method for producing a medicament according to at least one of claims 1 to 7, characterized gekennzeich¬ net that a non-toxic dextran sulfate or its salts with a physiologically compatible base is worked into one or more inert carriers and / or diluents.