Novel composition of matter of antithrombin III bound to a heparin fragment

Novel chemical compounds which are heparin fragments with a molecular weight of from 2,000 to 5,500 covalently bound to antithrombin III, and their use in medicine.

FIELD OF THE INVENTION 
The present invention relates to new chemical compounds consisting of 
heparin fragments covalently bound to antithrombin III, methods for their 
preparation, pharmaceutical compositions containing them and their use in 
therapy. 
BACKGROUND OF THE INVENTION 
Heparin, a sulfate-containing polysaccharide, is widely used clinically as 
a parenterally administered agent for the treatment and prevention of 
thrombosis. However, a very significant problem at heparin therapy is that 
the half life of heparin in blood is short, or about 1.5 hours. Because of 
this, heparin must ordinarily be administered by continuous intravenous 
infusion or by subcutaneous injection two to three times per 24 hours. 
Presence of the plasma protein antithrombin III is a necessary prerequisite 
for the anticoagulation activity of heparin. Antithrompin III inhibits 
most of the coagulation enzymes which are formed at the blood coagulation. 
But these inhibition reactions are slow and insufficient to prevent blood 
from coagulating. When heparin is present, it is bound to antithrombin III 
and activates said antithrombin III to form an inhibitor with a greatly 
increased reactivity which is sufficient to prevent the coagulation. The 
heparin-antithrombin bound in this inhibitor is not covalent bound but is 
reversible. 
Collen et al, Abstracts VIII Int. Congr. Thromb. Haemostasis, Thrombos. 
Haemostas. 46, 185 (1981), describe a product obtained by covalent 
coupling of standard heparin to antithrombin III. The products obtained 
had the properties of rapidly inhibiting the coagulation enzymes thrombin 
and activated Factor X. The products were shown in tests on rabbits to 
have a half life in blood which was two to three times longer than the 
half life of standard heparin. However, even though this represents a step 
forward, there is a need for heparin products with longer half life in 
blood and accordingly with longer duration of therapeutic activity. The 
present invention provides such heparin products with a very long half 
life in blood and correspondingly long duration of anticoagulation 
activity. 
DESCRIPTION OF THE INVENTION 
It has been found according to the present invention that novel compounds 
consisting of heparin fragments with a molecular weight less than 5,500, 
covalently bound to antithrombin III, have a half life in blood which is 
up to 30 times longer than the half life of standard heparin and about 10 
times longer than the antithrombin III-standard herparin product described 
by Collen et al in the prior art. The novel compounds of the present 
invention rapidly inactivate activated coagulation Factor X, which 
indicates a high anticoagulant activity. 
The heparin fragments contained in the novel compounds of the invention 
have a molecular weight of 5,500 or less, suitably from 2,000 to 5,500. 
Such fragments are prepared in known manner, for example by nitrous acid 
degradation of standard heparin, as is described for example in European 
patent publications No. 0 014 184 and No. 0 048 231. 
The novel compounds of the present invention may be prepared in a three 
step process as follows. 
In a first step, amino groups are introduced into the heparin fragments. 
This can be done by reacting carboxylic groups which are present in the 
heparin fragments with a suitable amine such as hexamethylenediamine. This 
reaction is carried out in the presence of a suitable coupling agent such 
as a carbodiimide, for example 
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. Care must be taken so that 
not all of the carboxylic groups react. The resulting modified heparin 
fragments will contain on average between 1 and 2 NH.sub.2 groups per 
fragment. 
Alternatively, amino groups can be introduced into the heparin fragment by 
limited N-desulfation or by converting the heparin fragments so that they 
will contain aldehyde functions. 
In a second step, the amino groups introduced on the heparin fragments are 
reacted with a bifunctional reagent which is suitable to give substituted 
heparin fragments capable of reacting with the amino groups in 
antithrombin III. A suitable such bifunctional reagent is 
tolylene-2,4-diisothiocyanate. This reagent is suitably used in excess. 
The use of tolylene-2,4-diisothiocyanate will give a reactive 
isothiocyanate substituted heparin fragment. 
In the third step the reactive substituted heparin fragment obtained in the 
second step is reacted with antithrombin III. In this reaction, the amino 
groups contained in the antithrombin III molecule will react with the 
isothiocyanate groups in the isothiocyanate substituted fragments and 
yield a reaction product which is a stable, identifiable, and novel 
chemical compound. In this third step, the heparin fragments will bind to 
antithrombin III with a 1:1 stoichiometry. 
It will be understood that the skilled worker will be able to vary and 
choose the particular reagents used in the above reaction sequence without 
departing from the scope of the invention. For example, among coupling 
agents to be used in the first reaction step may be mentioned bromcyan 
activation of heparin before addition of diamine. Coupling of heparin 
fragment directly to antithrombin III through formation of Schiff base 
between the terminal aldehyde group of the heparin fragment and amino 
groups in antithrombin III can also be visualized. 
In clinical practice, the novel compounds of the inventions will be used 
generally in the same manner and in the same form of pharmaceutical 
preparations as commercially available heparin for clinical use. Thus, the 
novel heparin derivatives of the present invention may be incorporated in 
aqueous solution for injection or in ointment preparations for 
administration via the skin and mucuous membranes. 
The intermediate products obtained consisting of heparin fragments 
containing amino groups are also novel. They represent an additional 
aspect of the invention. Also the aldehyde form of the heparin fragments 
are novel compounds and represent an aspect of the invention. In the said 
aldehyde form, one terminal group in the heparin fragments contains an 
aldehyde function.

The invention will be further illustrated by the following working 
examples. 
Preparation of Heparin Fragments Used as Starting Material by 
Depolymerization of Standard Heparin with Nitrous Acid 
EXAMPLE 1 
Heparin (0.5 g), isolated from the intestines of swine and dissolved in 150 
ml water, was cooled to +4.degree. C. and passed through a 3.times.7 cm 
column, Dowex.RTM.50 W-X8 (H.sup.+ -form), 200-400 mesh. The column was 
thereafter washed with 100 ml water, whereafter the washing liquid was 
combined with the eluate containing purified heparin. To the combined 
fluid was added 250 ml dimetoxietan (glyme), cooled to -20.degree. C., and 
10 ml isoamylnitrit, and the mixture, which had a temperature of about 
+10.degree. C., was allowed to stand for two minutes. Thereafter the 
reaction was interrupted by adding 10 ml 10% Na.sup.+ -acetate. After 
addition of 5.2 liter ethanol, precipitated carbohydrate (heparin 
derivative) was collected by centrifugation. The product was dissolved in 
500 ml 0.05M NaCl-0.05M Tris-HCl, pH 7.4. This solution was fractionated 
in 100 ml portions by affinity chromatography on a column containing 75 ml 
antithrombin-agarose-Sepharose.RTM. (Pharmacia Fine Chemicals, Uppsala), 
about 5 mg protein per ml gel. The column was eluted with a salt gradient 
(500 ml 0.05M NaCl-0.05M Tris-HCl in the mixing vessle; 500 ml 3M 
NaCl-0.05M Tris-HCl in the reservoar, whereby the major part of the 
applied material either passes, unretarded through the column or is eluted 
at low ion strength (0.4M NaCl); this material lacks biological activity. 
The active components (purified heparin derivatives) are eluted in a broad 
fraction between 0.5M NaCl and 3M NaCl corresponding to about 4% of the 
starting material. These fractions were pooled, concentrated and desalted 
by gel chromatography. Heparin derivatives, prepared and purified in this 
manner, had a molecular weight of between 3,000-5,000. 
EXAMPLE 2 
Covalent Coupling of Heparin Fragments to Antithrombin III 
Step I 
Amino groups were introduced into heparin fragments obtained as described 
above by dissolving 15 mg heparin fragments, prepared as described above 
in 4.5 ml water, followed by addition of 1 ml hexametylendiamine solution 
(21 mg/ml) and 2 ml of a solution of 
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (7.3 mg/ml). 
The pH of the solution was adjusted to 4.75 and the reaction mixture was 
allowed to stand during stirring for 20 minutes while pH was kept constant 
by addition of 0.1M HCl. The reaction was stopped by increasing pH to 9.5 
using 2M NaOH. The reaction mixture was diluted to 3 times the original 
volume using 0.1M NaHCO.sub.3 buffer, pH 9.5, and was then dialyzed 
against 0.05M phosphate buffer pH 7.5 containing 0.05M NaCl. 
Step II 
The material obtained in Step I was separated in a low affinity fraction 
and a high affinity fraction by affinity chromatography on matrix bound 
antithrombin III in the same way as described in Example 1. Thereafter was 
added to 2.2 mg of the high affinity fraction 4 ml 
N,N-dimethyl-N-allylamine buffer pH 9.2 and 100 mg 
tolylene-2,4-diisothiocyanate. Thereafter nitrogen gas was bubbled through 
the solution for 1 minute, and the solution was incubated for one hour at 
45.degree. C. Thereafter 2 ml water was added and the suspension was 
extracted 4 times with 4 ml benzene and 3 times with heptane/ethylacetate 
(2/l) in order to remove excessive reagent. 
Step III 
The water phase obtained in Step II was thereafter immediately added to 20 
ml 0.1M NaHCO.sub.3 buffer, pH 8.6, containing 50 mg antithrombin III. The 
mixture obtained was incubated for one hour at 30.degree. C. under 
stirring and was then dialyzed against 0.02M imidazol-HCl buffer, 7.35 
over night. The products formed were then purified by having the dialyzed 
reaction mixture pass a column, of DEAE-Sephadex, which then was eluted 
with a salt gradient 0-0.5M NaCl. The heparin fragment-antithrombin 
compound obtained were separated from unreacted antithrombin by affinity 
chromatography on heparin-Ultrogel column, equilibrated with 0.1M Tris-HCl 
buffer pH 7.6. The elution was made by a salt gradient (0.1-1.0M NaCl). 
The purified product obtained was homogenous in 
polyacrylamid-electrophores in the presence of sodium dodecyl sulphate. 
The molecular weight of the product obtained was from 65,000-80,000, with 
an average about 70,000, obtained in comparison with the molecular weight 
of proteins with known molecular weight. The product obtained inhibited 
activated Factor X with a second order rate constant of 
0.92.times.10.sup.6 M.sup.-1 S.sup.-1. The yield with respect to heparin 
fragment-antithrombin III-compound was 24%. 
Biological Half Life for the Compounds of the Invention 
Two mg of the heparin fragment-antithrombin III compound obtained as 
described in Example 2, and where the heparin fragment was labelled with 
3H, was dissolved in 1 ml 0.1M NaCl solution and injected into a vein in 
the ear of a rabbit. Thereafter a series of blood samples were taken 
during the next ten hours and radioactivity and inhibition of activated 
Factor X were measured. In this way, the biological half life for the 
tested compound was obtained. This half life was then compared with the 
half life for standard heparin, administered in the same manner. 
The result of the test was that the half life of the heparin 
fragment-antithrombin compound according to the invention was 7.8 hours. 
This value was obtained using radioactivity measurements as well as 
biological activity measurements, that is Factor X.sub.a -inhibition. The 
half life for standard heparin measured in the same way is 0.3 hours. 
Thus, the half life for the heparin fragment-antithrombin III compound was 
26 times longer than the half life for standard heparin. 
The great increase in biological half life which was obtained for the novel 
compounds of the present invention is very valuable clinically. In the 
present treatment with heparin, it is normal that two or three injections 
must be given per day at prophylactic treatment of post-operative 
thrombosis. Using the novel compounds of the present invention, it would 
be sufficient with one injection every two or every three days. This is a 
great improvement from the point of view of the patient and also from the 
point of view of the clinic because of the practical and economical 
advantages which follow from better utilized care resources. 
Studies on antithrombotic effects of the heparin-fragment-antithrombin III 
Compound of the invention on the Wessler rabbit stasis model show that 
intravenous injection of the compound effectively prevents thrombus 
formation in the rabbits.