Process for obtaining low molecular weight heparins endowed with elevated pharmacological properties, and product so obtained

The present invention relates to a process for obtaining low molecular weight heparins endowed with pharmacological and therapeutic properties of outstanding interest, starting from normal heparin. The said process is characterized in that it comprises the following steps: PA1 (a) acidification of normal heparin to obtain heparinic acid, PA1 (b) depolymerization of said heparinic acid by heating in autoclave in the presence of peroxides, to obtain a low molecular weight heparamine, PA1 (c) sulphation of said heparamine to obtain the corresponding low molecular weight heparin. A further object of the invention is the low molecular weight heparins, or LMW heparins, so obtained, and their utilization in the field of therapeutics.

The subject matter of the present invention is a process for obtaining low 
molecular weight heparins endowed with pharmacological and therapeutic 
properties of outstanding interest, starting from normal heparin. 
Heparin is a well-known and stable uni- or bivalent salt of the unstable 
heparinic acid. It is a polymer of a disaccharide unit formed by hexuronic 
(D-glucuronic and L-iduronic) acids and glucosamine 60- and N-sulphate 
linked by alpha 1-4 glycoside linkages. 
Its anti-clotting properties have been known for many years, since its 
discovery and isolation from tissues in 1917. 
Heparin is present in different forms in many tissues and cells, more or 
less loosely bound to a protein moiety. In the skin and, partially, in the 
lungs of different species heparin is present in a high molecular form, 
which is sensitive to the action of ascorbic acid or of some enzymes 
thought to be present in the intestinal mucosa. After treatment with 
ascorbic acid or with intestinal mucosa homogenates, these macromolecular 
forms of lung or skin heparin can be reduced to the same molecular size as 
the heparin that can be isolated from the intestinal mucosa. 
The heparin isolated from intestinal mucosa having a mean molecular weight 
(MW) of 15,000 Dalton, or heparins from other sources reduced to this MW 
by some of the conventional methods as above described, represent the 
minimal size of the natural molecules of heparin. According to the present 
invention, such type of heparin is defined as normal heparin. In fact, 
there exist no enzymes in the body that can further split the heparin from 
the intestinal mucosa into lower molecular weight fractions, nor have any 
chemical methods so far been developed able to depolymerize the normal 
heparin molecule without total loss of its biological activity. 
Only certain microbial enzymes, e.g. Heparinase from Flavobacterium 
heparinum are able wholly to dissociate the heparin molecule into modified 
disaccharide units. This method has been applied in structural studies but 
has proved unsuitable for obtaining any new biologically active molecules. 
One purpose of the present invention is therefore to provide a process such 
as makes it possible, starting from normal heparin, to obtain lower 
molecular weight heparin fractions endowed with elevated pharmacological 
activity. 
To achieve the said purpose the present invention proposes a process 
characterized in that it comprises the following steps: 
(a) acidification of normal heparin to obtain heparinic acid, 
(b) depolymerization of said heparinic acid by heating in an autoclave in 
the presence of peroxides, to obtain a low molecular weight heparamine, 
(c) sulphation of said heparamine, to obtain the corresponding low 
molecular weight heparin. 
A scheme of reaction according to the process proposed by the present 
invention is described below. 
##STR1## 
wherein m&lt;n, and 7&lt;m&lt;22 when n.apprxeq.26.7, which is the n value 
corresponding to heparin of MW.apprxeq.15,000 D. 
It is calculated as follows: 
##EQU1## 
wherein 562 is the MW of the disaccharide unit (of chemical formula 
C.sub.12 H.sub.15 O.sub.16 NS.sub.2 Na.sub.3) represented by the formulas 
(I) and (IV), reported above. 
According to the above scheme of reaction, the formula I indicates the 
sodium salt of normal heparin; for the sake of simplicity, said formula 
shows only D-glucuronic acid and not the part of the molecule which 
contains L-iduronic acid. Similarly, any other univalent or divalent 
cation, can be present in place of the sodium cation. 
The said heparin I is acidified to obtain its free acid, the heparinic acid 
of formula II, according to the step a shown previously. 
The heparinic acid of formula II is depolymerized in the step b, with loss 
of the majority of the N-sulphate groups and with formation of a 
heparamine of formula III. 
Finally, the heparamine of formula III is treated according to step c by 
reconstitution of the N-sulphate groups, to obtain the final product 
according to the invention, i.e. the heparins of formula IV, which are 
defined as LMW (low Molecular Weight) heparins or RD (Reconstituted 
Depolymerized) heparins. 
According to a more specific description of the process of the invention 
the said heparinic acid is obtained from normal heparin I by simple 
acidification or, preferably, by treatment with cationic exchange resins. 
The heparinic acid II then goes through the step b of depolymerization, 
which is carried out in an autoclave in the presence of a peroxide, for 
example H.sub.2 O.sub.2, by heating at a pressure preferably between 1 and 
2 atm.; the auto-claving can be stopped at preselected times (for instance 
at 15, 30, 60, 120, 240 minutes). In this way it is possible to obtain 
final products, i.e. LMW or RD.Heparins, with different mean molecular 
weights (MW) in a range approximately between 12,000 and 4,000 D, starting 
from normal heparin of MW=15,000 D. The yields of said depolymerization 
are the same (about 65% by weight) in all instances. The decrease in MW is 
followed by titration of the end reducing groups (Somogji's method) or by 
decrease of the specific viscosity. 
The products of said depolymerization are low molecular weight heparamines 
III, the molecular weight of which is lower than that of the corresponding 
starting heparin I down to about one third, according to the preselected 
different times of autoclaving. 
Since in said depolymerization step, and as has been previously stated, the 
majority of the sulphate groups bound to the nitrogen are lost, the 
heparamine III is finally--after cooling of the reaction mixture and 
raising of the pH--subjected to reconstitution of the active sulphuric 
groups to obtain the final product IV according to the said step c by 
known methods, such as for example by reaction with sulphotrioxides of 
nitrogen-containing bases (e.g. pyridine sulphotrioxide, trimethylamine 
sulphotrioxide; etc.) in the presence of alkaline carbonates, or by 
reaction with chlorosulphonic acid and nitrogen-containing bases. 
The yields of the final products, no matter of the time of autoclaving, are 
in all cases the same, in terms of weight, as referred to the starting 
amount of heparin.

For a better comprehension of the invention, some examples of actuation 
thereof are reported, which are not, however, to be considered limiting. 
EXAMPLE 1 
A quantity of 20 g of sodium or calcium heparin (anticlotting activity=150 
IU/mg) was dissolved in 100 ml of deionized water and placed into a column 
containing 100 ml of Amberlite .RTM. IRC 120 in H.sup.+ FORM. 
The column was washed with 100 ml of deionized water and the liquids were 
collected. The pH of the solution was 1-1.5. 20 ml of a saturated solution 
of hydrogen peroxide (36% were added and the liquid was heated in an 
autoclave for 15 minutes at 1 atm. After cooling to room temperature, the 
pH was raised to 7.2 with 2N NaOH; 16 ml of pyridine and 16 g of pyridine 
sulphotrioxide were added under stirring. 
The reaction lasted for 12 hours, with small addition of pyridine to 
maintain the pH between 5 and 6. The pH was then raised to 8 with NaOH and 
the solution was concentrated in vacuo to 100 ml to remove the excess of 
pyridine. Salts were removed in a mixed-bed ion exchange column and the 
solution was sterile-filtered and freeze-dried. 
Yield: 65% as a white powder with an anti-clotting activity of 85-100 IU/mg 
and a MW (Somogji method) which was 2/3 of the original. 
EXAMPLE 2 
20 g of sodium or calcium heparin, 150 IU/mg, were dissolved in 100 ml of 
water for injection and decationized on a cation exchange resin in H.sup.+ 
form. 
To the solution (pH=1-1.5) addition was made of 20 ml of peracetic acid 
(40% solution) and the whole was then autoclaved for 60 minutes at 1 atm. 
After cooling and raising the pH to 7.2, 20 g of trimethylamine 
sulphotrioxide+20 g of Na.sub.2 CO.sub.3 were added. The reaction mixture 
was stirred for many hours at a temperature not exceeding 60.degree. C., 
then passed through a Dowex Retardion .RTM. 11 A 8 column. The pH of the 
solution was adjusted to 6, the solution was cooled and 3 volumes of 
ethanol were added. The white precipitate was collected, washed with 
ethanol or methanol or acetone and dried in vacuo. 
The final product possesses an anti-clotting activity of 35-50 IU/mg and a 
MW (Somogji method) which is 1/3 of the original. 
EXAMPLE 3 
By operating as specified in EXAMPLE 1, but with the following operative 
conditions: autoclaving for 30 minutes at 2 atm., a final product is 
obtained with an anti-clotting activity of 35-50 IU/mg and a MW which is 
about 1/3 of the original. 
It is firstly stated that the product IV has a chemical composition 
(content of sulphates, hexosamines, hexuronic acids) and certain 
physico-chemical properties (specific optical rotation) which are 
identical to those of the purified heparin I, but gives a different value 
in the titration of the end reducing groups (Somogji method), a different 
electrophoretic-mobility and shifted wavelength absorption maxima in the 
complexes formed with basic dyes, e.g. with toluidine blue. 
The LMW heparins (RD heparin) obtained according to the invention show very 
interesting pharmacological and therapeutic properties as compared with 
normal heparin I. The most important of these is a modified ratio, either 
in vitro or in vivo (in experimental animals and in humans), between the 
anti-thrombotic and the total anti-clotting activities, as measured by the 
ratio 
##EQU2## 
where: Anti-Xa test=Yin's test; APTT=Activated Partial thromboplastin 
time. 
The different RD heparins show increasing values of the ratio 
##EQU3## 
with decreasing MW although the specific anti-clotting activity decreases 
with decreasing MW. 
In any case, normal heparin with MW=15,000, has a total anti-clotting 
activity.gtoreq.150 IU/mg and a ratio anti-Xa/APTT=1, while RD heparins of 
the invention have a total anti-clotting activity&lt;150 IU/mg and an 
Anti-Xa/APTT&gt;1. In particular, the ratio is almost double for compound IV 
as compared with compound I. This means that, on the basis of the same 
anti-clotting activity, the anti-thrombotic activity of compound IV is 
twice that of normal heparin I; in other words, in terms of anti-clotting 
activity, half a dose of compound IV, or even less, is required to produce 
the same anti-thrombotic action as is produced with a whole dose of normal 
heparin. 
These data have been confirmed in experimental animals by measuring the 
protective action of intravenously administered compounds I and IV against 
the formation of thrombin and thromboplastin-induced thrombi. 
Therefore, in the family of RD Heparins that can be obtained with the 
present method, it is possible to choose the most suitable product, in 
terms of specific anti-clotting activity and 
##EQU4## 
ratio, for peculiar uses in the field of the protection against 
thrombosis. 
For instance: a patient in thrombogenic state, with very shortened clotting 
time, could require an anti-thrombotic agent with a fairly high 
anti-clotting activity, whereas a patient to be submitted to a surgical 
intervention, where both the risks of hemorrhage and post-operative 
thrombosis exist, could require an anti-thrombotic agent with low 
anti-clotting activity. 
Additionally, after subcutaneous administration in humans, compounds IV 
remain in the bloodstream longer than compound I. Different tests, namely 
recalcification time, activated partial thromboplastin time (APTT), 
thrombin time, Anti-Xa activity, were used to check how long compounds IV 
remains in the circulation. 
Moreover, compound IV shows some oral absorption in certain animal species 
(rats, mice, dogs), whereas compound I is not orally absorbed.