FR2622450A1 - Heparin/dermatan sulphate combination - Google Patents

Abstract

The combination of the invention is composed essentially of a mixture of: a) heparin or one of its derivatives possessing an anti-factor IIa activity at least equal to that of heparin, and b) dermatan sulphate, in which combination each of the components is present in a quantity in which it is only slightly active or is even inactive per se, as an antithrombotic, the aforementioned combination having a synergistic effect. This combination is useful as an active principle of medicaments for the prevention and treatment of thromboembolic disease.

Description

HEPARIN / DERMATANE SULFATE ASSOCIATION
The present invention relates to a combination based on glucosaminoglycans essentially formed by a mixture of heparin or heparin derivatives having an anti-factor IIa activity at least equal to that of heparin, and of dermatan sulfate, the compositions pharmaceuticals containing it and their application as a medicament, in particular with antithrombotic activity.

Heparin is a glycosaminoglycan, well known in the literature, which exerts an anticoagulant activity by inhibiting various serine coagulation proteases, in particular by potentiating the inhibitory activity of a plasma protein, antithrombin
III.

A lot of work has been done on heparin both in terms of its structure and its activity. In particular, numerous heparin fractionation studies have been carried out for structure / activity studies. Heparin consists of polysaccharide chains alternately composed of
D-glucosamines and uronic acids (D-glucuronic acid and L-iduronic acid) substituted by various functional groups, in particular sulfate groups, and more particularly N-sulfate and O-sulfate.

 Heparin also exhibits antithrombotic activity, but in doses likely to cause a certain risk of haemorrhage. The use of low molecular weight heparin fractions and fragments lacking activity on global coagulation has partially overcome this drawback, but their use in certain high-risk surgeries is still difficult.

Dermatan sulfate also belongs to the family of glycosaminoglycans. It consists of polysaccharide chains in which alternate
D-galactosamines and L-iduronic acids, substituted by various functional groups, in particular sulfate groups. It is often present in very small proportions (approx. 1 to 5) in pharmaceutical heparin.

 Its activity with regard to coagulation is exerted via another plasma protein, the heparin cofactor II. Various studies have shown that dermatan sulfate, like heparin, has antithrombotic activity, but this activity is exerted at doses approximately five times higher than those of heparin. Based on the data in the literature, a possible antithrombotic treatment based solely on dermatan sulfate would require the use of large quantities of product, which would make treatment difficult and expensive.

 There is therefore a great interest in having a product which makes it possible to reduce or even completely eliminate the risk of bleeding, in particular for the treatment of high-risk patients, while being easy to use.

 Unexpectedly, it has been found that by combining heparin at a weakly active, even inactive dose from an antithrombotic point of view and dermatan sulfate at a dose substantially devoid of antithrombotic activity, therefore using a relatively small amount. of dermatan sulfate, it is possible to obtain a strong antithrombotic activity, which demonstrates a synergistic effect of the two compounds of the association.

This association with synergistic effect has the advantage of reducing, or even eliminating, any hemorrhagic risk, which allows its use in cases where hemorrhagic effects are to be feared.

It has also been observed that fractions or fragments of heparin of low molecular mass, as described in the literature fractions or fragments which have little or no anti-factor activity
IIa, do not have this synergistic activity in association with dermatan sulfate.

 Additional work has made it possible to discover that the synergistic activity of heparin with respect to dermatan sulfate is due to the presence of a majority of molecular species exhibiting an antifactor IIa activity, that is to say with sufficient length, as described in the article Oosta et al., Proc.

Natl. Acad. Sci. USA, 78, 1981, 829-833: Multiple functional domains of the heparin molecule: "The smallest complex carbohydrate fragment that accelerated the inactivation of thrombin by antithrombin had 14 residues" (see Abstract, p. 829).

 The invention relates to a combination based on glycosaminoglycans, with activity in particular antithrombotic, characterized in that it is essentially formed of a mixture a) of heparin or one of its derivatives having an anti-factor IIa activity at least equal to that of heparin, and b) of dermatan sulfate, in which each of the components is present in a weakly active, or even inactive, amount in itself, as an antithrombotic, said combination having a synergistic effect.

 The term “heparin derivative having an anti-factor IIa activity at least equal to that of heparin” means any glycosaminoglycan of heparin type whatever its mode of production, the majority of molecular species of which have a sufficient length for cause strong anti-factor IIa activity.

 A preferred combination consists of a mixture of components a) and b).

 Advantageously, a quantity of components a) and b) is used in the combination in a component a) / component b) weight ratio of 1/1 to 1/25, preferably 1/10; in particular, amounts of heparin and dermatan sulfate are preferably used in a ratio of i of component a) to 2.5 of component b). This latter ratio is likely to vary depending on the characteristics of the products used, in particular with regard to dermatan sulfate.

 The invention also relates to pharmaceutical compositions characterized in that they contain, as active principle, an association as defined above, in a pharmaceutically acceptable vehicle or excipient.

 The pharmaceutical compositions according to the invention are preferably in sterile or sterilizable injectable form in a pharmaceutically acceptable excipient for administration by the intravenous, subcutaneous or intramuscular route. They can also be presented in a form suitable for oral administration, advantageously in the form of gastro-resistant capsules, tablets, tablets or pills.

 Advantageously, the pharmaceutical compositions of the invention contain per dosage unit an amount of 2.5 to 25 mg, preferably 5 to 15 mg of heparin or one of its derivatives, and 2, 5 to 50 mg, preferably 10 to 35 mg of dermatan sulfate, and are presented either in the form of sterilized ampoules, vials or syringes, in injectable solutions, or combined with excipients suitable for oral administration under form of capsules, tablets, tablets, pills.

 The invention also relates to the use of such a combination as a drug with antithrombotic activity.

Advantageously for the preparation of the combination according to the invention, use is made of a heparin of conventional injectable pharmaceutical quality, preferably in the form of sodium salt, having a molecular weight of 2,000 to 50,000 daltons (average molecular weight 13,000 da.), and having specifications in accordance with those indicated in the Pharmacopoeia
European (1984, monograph 333). Such a product is commercially available. It is also possible to use a heparin derivative having an average molecular mass equal to or greater than 6,000-7,000 daltons.

 The dermatan sulfate used may be commercial dermatan sulfate.

It can also be prepared by using, for example, a raw material such as an animal tissue containing it, in particular mucus or intestine, lung or skin of mammal, preferably beef or pig, using for example a process such as that described by L. Roden, JR Baker, JC Cifonelli and M.Bo. Mathews,
Methods in enzymology, 26 (1972), 73-140.

 Dermatan sulfate has the following characteristics - it is formed from a mixture of chains with a molecular weight ranging from 10,000 to 60,000 daltons, the average molecular weight being approximately 25,000 da.

- activities. anti-factor Xa: <1 u / mg
anti-factor IIa: <2 u / mg
APTT: <5 u / mg - sulfate (S) level: between 1.50 and 2.40
Carboxyl content (C): between 1.60 and 2.00
S / C: between 0.95 and 1.40
The average molecular mass was determined by GPC (gel permeation chromatography) on a TSK 3000 SW column using the following products as standards: hyaluronic acid (230,000 da.), Dermatan sulfate (45,000 da.), Chondroitin-6-sulfate (29,000 da.), chondroitin-4-sulfate (15,000 da.), heparin (16,000 da., 13,000 da. and 7,700 da.), oligpsaccharides preferably prepared from heparin by degradation with heparinase (di-, tetra-, hexa-, octa- and decasaccharides). The anti-factor xa and anti-factor IIa activities were measured by a chromogenic method (Stago,
France) using standard heparin as a standard.

APTT activity (Actin FS, Dade, USA) was measured using a KC4 device (AHS, Dade, USA). The sulphate and carboxyl levels were determined by conductimetry.

 It is also possible to use a commercial dermatan sulfate, such as that sold by Sigma, USA (Catalog no. C4259 from porcine pine).

 At the doses administered, the pharmaceutical compositions according to the invention did not cause any toxic effect and no prolongation of the bleeding time was observed in the animals treated.

 The invention is not limited to the particular aspects which have just been described, on the contrary it encompasses all the variants thereof and it will be easier to understand with the aid of the application examples which will follow.

EXAMPLE 1: Product activity in a model of
experimental thrombosis
The products tested were as follows
- dermatan sulfate (DS) having the characteristics
described above,
- standard heparin (SH) commercially available
form of sodium salt, in accordance with
European Pharmacopoeia.

 The doses of products to be injected were determined by a dose-effect relationship study by choosing doses of each of the products having only a weak or insignificant antithrombotic action when administered separately.

 The products were administered alone or in combination.

- Experimental model
Male New Zealand rabbits (2 to 2.5 kg) are anesthetized with sodium pentobarbital (30 mg / kg) administered intraperitoneally. Then each jugular vein is isolated between two loose sutures. 10 curies of fibrinogen labeled at 1.125 l are then injected, followed by the desired dose of the glycosaminoglycans to be tested alone or in combination, via a catheter introduced into the carotid artery. Three minutes later, the human brain thromboplastin is injected into the carotid artery (National Reference Laboratory for Anticoagulant
Reagents, Manchester), at a dose of 1 mg / kg in 30 seconds. Thirty seconds later, the proximal and distal sutures are tightened to occlude the two segments of jugular veins. 15 minutes later, these segments are opened and the size of the thrombi is measured by comparing their radioactivity to that of a ml of blood.

 For each product or combination of products, groups of 5 to 11 animals are used per dose in order to calculate the percentage reduction in the size of the thrombus relative to the values obtained in control animals to which physiological saline has been injected. (For more details, see Carter et al.

Thromb. Res., 21, 1981, 169). The statistical significance of the differences between control and treated animals was calculated using the Wilcoxon test (it is represented by the P value).

The results are given in the following table
Dose Equivalence n Prevention P
administered anti-Xa u / kg of thrombus
ug / kg
DS 25 0 10 17 + 12 NS
DS 35 0 7 32 + 14 <0.05 SH 10 1.6 Il 12 + 11 NS
SH 25 4 9 49 t 13 <0.05
DS 25 + SH 10 1.6 11 54 + 7 <0.01
DS 25 + SH 25 4 7 76 + 10 <0.01
DS 35 + SH 10 1.6 7 73 + 8 <0.01
DS 35 + SH 25 4 7 78 + 5 <0.01 n = number of animals treated
NS = not significant
These results show that by combining two
respectively inactive doses of dermatan sulfate,
25 pg / kg, and heparin, 10 pg / kg, we obtain, by
combining, a strong antithrombotic activity (inhibit
tion of thrombus formation greater than 50% by
witness report), which demonstrates a synergistic effect
of the two components of the association.

Experiments with dermatan
sulfate of another quality (Sigma, USA) gave
comparable results using a higher dose
aunt of dermatan sulfate, i.e. 50 to 75 pg / kg
for heparin doses of 10 and 25 pg / kg.

EXAMPLE 2
In complementary experiments using the experimental model described in Example 1, the association of dermatan sulfate was tested with either fractions or fragments of heparin prepared so as to have chains of lower molecular mass.

 The product CY 216 is a fraction of heparin of low molecular mass (average molecular weight 4,500 da.), Prepared by depolymerization as described in French Patent 2,572,080. The ratio of anti-factor Xa activity / anti-factor activity It is equal to 4.

 The product PS is a synthetic pentasaccharide corresponding to the minimum sequence for the binding of heparin to antithrombin III, the synthesis of which is described in European patent application 0 084 999 (Product No. 50). This product is completely devoid of anti-factor IIa activity.

The results are given in the following table
Dose Equivalence n Prevention P
administered anti-Xa u / kg of thrombus cig / kg
DS 25 0 10 17 + 12 NS
DS 35 0 7 32 + 14 <0.05
CY 216 50 6 7 11 t 12 NS
CY 216 100 12 5 41 + 15 <0.05
PS 14 4 6 1Q + 10 NS
PS 34 10 6 55 t 15 <0.01
DS 25 + CY 216 50 6 8 18 + 16 NS
DS 25 + CY 216 100 12 8 39 + 14 <0.05
DS 35 + CY 216 50 6 8 44 t 13 <0.01
DS 35 + CY 216 100 12 8 32 + 16 NS
DS 25 + PS 14 4 8 5 t 14 NS
DS 25 + PS 34 10 5 59 t 9 <0.01
These results show that the combination of respectively inactive amounts of dermatan sulfate and CY 216 or of dermatan sulfate and pentasaccharide does not make it possible to obtain an antithrombotic activity greater than that obtained with the dose considered for each of the products separately.

In addition, when using quantities of
CY 216 or pentasaccharide having a certain antithrombotic activity when administered alone, their association with dermatan sulfate even leads to a decrease in antithrombotic activity. For example, 100 pg / kg of CY 216 show a reduction in the incidence of thrombosis by 41%, while the combination with 35 pg / kg of dermatan sulfate results in an inhibition of only 32%, like dermatan sulfate alone at the same dose. We therefore do not observe the synergistic effect obtained with heparin.

EXAMPLE 3
Using the experimental model described in Example 1, the activity of dermatan sulfate was tested in combination either with a heparin fraction of high affinity for AT III (HA Hep), or with a fraction of low affinity (LA Hep). These fractions were obtained by affinity chromatography on an AT III-Sepharose column, as described in Hook et al.,
Febs Lett., 66, 1976, 90-93.

The results are given in the following table
Dose Equivalence n Prevention P
administered anti-Xa u / kg of thrombus
ug / kg%
DS 25 0 10 17 + 12 NS
DS 35 0 7 32 + 14 <0.05
HA Hep 5 2.8 8 25 + 14 NS
LA Hep 10 0.07 8 0 + 13 NS
DS 25 + HA Hep 5 2.8 8 52 t 12 <0.01
DS 25 + LA Hep 10 0.07 7 9.1 + 17 NS
DS 35 + HA Hep 5 2.8 7 66 t 7 <0.01
DS 35 + LA Hep 10 0.07 7 52-t 12 <0.01
It is observed that the association of an inactive or weakly active amount of dermatan sulfate with an inactive amount of high affinity heparin results in significant antithrombotic activity, demonstrating a synergistic effect of this association, while this effect is not obtained with low affinity heparin.

 All of these results show that in order to obtain a strong antithrombotic activity in the model described, resulting from a synergistic effect, the presence of the AT III binding site is important, since there is no not observe this effect with low affinity heparin which does not have it. However, the presence of this site is not sufficient since this effect does not occur with the pentasaccharide, nor with a fraction of heparin of low molecular weight like CY 216 which both have it.

 These observations show that the synergistic effect is due to the presence in heparin and its derivatives of molecular chains responsible for the anti-factor IIa activity.

EXAMPLE 4 Pharmaceutical Formulations
Standard Heparin from the Pharmacopoeia
Commercial dermatan sulfate
Formula 1: subcutaneous use
Heparin, sodium salt 10 mg
Dermatan sulfate 25 mg
Water for preparation
injectable, qs 0.5 ml
Formula 2: subcutaneous use
Heparin, sodium salt 10 mg
. Dermatan sulfate ........... 50 mg
Water for preparation
injectable qs 0.5 ml
Formula 3: intravenous route
Heparin, sodium salt ..... 20 mg
. Dermatan sulfate .......... 200 mg
Water for preparation
injectable qs 5 ml
EXAMPLE 5 Treatment method
The pharmaceutical compositions of the invention can be administered as an antithrombotic medicament as a preventive measure, for example in the case of surgical interventions in the case of patients at high risk of haemorrhage. The doses administered are 1 to 3 ampoules per day of one of the above formulas, depending on the condition of the patient and the opinion of the attending physician.

 In general, the pharmaceutical compositions of the invention can be used as a medicament for the prevention and treatment of thromboembolic disease.

Claims (10)

 1. Association based on glycosaminoglycans, in particular antithrombotic activity, characterized in that it is essentially formed of a mixture a) of heparin or of a derivative thereof having an anti-factor lIa activity at least equal to that of heparin, and b) of dermatan sulfate, in which each of the components is present in a weakly active, or even inactive, amount in itself, as an antithrombotic, said combination having a synergistic effect.  2. Association according to claim 1, characterized in that it consists of a mixture of components a) and b).  3. Association according to claims 1 and 2, characterized in that the weight ratio component a) / component b) is from 1/1 to 1/25.  4. Association according to claim 3, characterized in that the weight ratio of the quantities of the components is 1 of component a) for 10 of component b).  5. Association according to claim 3, characterized in that the weight ratio of the quantities of the components is 1 of component a) to 2.5 of component b).  6. Pharmaceutical compositions containing as active principle a combination according to any one of claims 1 to 5, in a pharmaceutically acceptable vehicle or excipient.  7. Pharmaceutical compositions according to claim 6, characterized in that they comprise the combination of heparin or one of its derivatives and of dermatan sulfate in a pharmaceutically acceptable excipient for administration by the intravenous, subcutaneous or intramuscular.  8. Pharmaceutical compositions according to claim 6, characterized in that they comprise the combination of heparin or one of its derivatives and dermatan sulfate in a form suitable for administration by the oral route, advantageously in the form of gastro capsules. resistant, tablets, tablets or pills.  9. Pharmaceutical compositions according to any one of claims 6 to 8, characterized in that they contain per dosage unit an amount of 2.5 to 25 mg, preferably 5 to 20 mg of heparin or d one of its derivatives, and from 10 to 100 mg, preferably from 10 to 35 mg of dermatan sulfate.  10. Use of a pharmaceutical composition according to any one of claims 6 to 9 as a medicament for the prevention and treatment of thromboembolic disease.