Method for obtaining biologically active mucopolysaccharides of high purity, by controlled depolymerization of heparin

Method for obtaining biologically active mucopolysaccharides by controlled depolymerization of heparin, wherein the quantity of products generating nitrous acid is selected so that those products are totally consumed when the desired depolymerization degree is reached.

The invention relates to a novel method for obtaining biologically active 
mucopolysaccharide compositions of high purity, by the controlled 
depolymerization of heparin. 
It relates more particularly to a process of the type concerned enabling 
the production of a mixture of high homogeneity in mucopolysaccharides 
(these products will be denoted below by the abbreviation MPS) capable 
especially of controlling, in a highly specific manner, certain stages of 
blood coagulation and, in particular, possessing a more selective activity 
than heparin with respect to a smaller number of factors of coagulation, 
particularly of the activated factor X or factor Xa of the blood. 
Taking into account their properties, these MPS constitute valuable 
medicaments, particularly antithrombotic. It is then possible to 
understand the advantage, for the therapeutic uses contemplated, of having 
available MPS of high purity, in particular practically completely freed 
of the contaminants that the reaction medium can introduce, whilst 
resorting only, for their production, to operational steps which are easy 
to put into practice whose cost and realisation permit exploitation on the 
industrial scale. 
For the production of compounds of this type, the inventors are more 
especially interested in the controlled depolymerization of heparin under 
the effect of a chemical agent capable of fragmenting the chains which 
constitute it, in particular nitrous acid. 
It will be noted that the term heparin is used in the description and the 
claims in its broadest sense, to denote indifferently a commercial heparin 
preparation of pharmaceutical quality or a crude heparin such as obtained 
by extraction from biological materials, in particular from mammalian 
tissues. 
Now, in a general manner, the depolymerization processes proposed until now 
lead to depolymerization mixtures containing numerous contaminants, 
particularly nitrites and nitrates derived from the unreacted nitrous 
acid. It then appears necessary to purify with care, in the course of 
additional steps, the MPS compositions of these mixtures before using them 
in therapeutics. 
In addition, the practising of the reaction to the desired degree of 
depolymerization requires strict control of the progress of the 
depolymerization in order to arrest the latter to prevent too extensive a 
depolymerization. 
The research by the inventors for means enabling the production of the 
desired MPS more satisfactorily has led them to study different conditions 
of realisation of a controlled depolymerization of the heparin. 
The studies carried out then showed that in selecting one of the parameters 
of the reaction as a function of the other parameters used, it was 
possible to obtain with high yields, a depolymerization mixture with a 
high anti-Xa (YW) activity, generally higher than 200 u, even 250, 
practically free of contaminants of the nitrite-nitrate type and to avoid, 
during the reaction, the checking of the depolymerization to arrest the 
latter when the desired degree is reached. 
It is hence an object of the invention to provide a novel process enabling 
the production of particularly valuable MPS with regard to the desired 
biological properties, whilst being practically free of any contaminants 
such as nitrites and nitrates and which are hence thereby found to be 
produced in a particularly suitable form for their uses in therapeutics. 
It was also an object to provide a process enabling the production of these 
MPS without having to exert strict checking of the reaction time to arrest 
the latter as soon as the desired degree of depolymerization is reached, 
the degree of depolymerization being adjusted at will. 
It is aimed also at providing a process whose practising on the industrial 
scale is rendered extremely easy by reason of the particular conditions 
selected for the controlled depolymerization of the heparin. 
The process for obtaining biologically active MPS compositions according to 
the invention is characterized by the fact that heparin and nitrous acid 
or, preferably, the reagents used to generate it in situ, are used in 
respective concentrations such that when the desired degree of 
depolymerization is reached, the nitrous acid, or as the case may be, the 
reagents used to generate it in situ, are entirely consumed, these 
concentrations of reagents and the conditions of the reaction other than 
these concentrations being advantageously adjusted as a function of one 
another so as to permit the production of a controlled depolymerization of 
the starting heparin chains leading to the desired MPS and, after stopping 
by itself of the reaction, there are separated from the reaction medium 
the products of the type of those which are precipitable by an alcoholic 
solvent and they are recovered. 
The advantage of these features is found to be particularly valuable with 
respect to the biological applications contemplated for the products. In 
fact, the MPS compositions recovered from the depolymerization mixture are 
practically entirely free of toxic substances. In particular, these 
compositions do not contain nitrites nor nitrates. Considering the total 
consumption of the nitrous acid and the removal in gaseous form of the 
nitrogen-containing substances, such derivatives can be formed, which 
enables the avoidance, after the reaction, of any contamination, by toxic 
nitrated compounds, of the compositions produced. 
This process presents, in addition, a considerable advantage at the level 
of the production technique. 
In fact, due to the features used, the depolymerization reaction is, in 
away, "self-regulating", stopping itself when the desired depolymerization 
degree is obtained. 
The handicap of checking the duration of the reactions which is made 
necessary in the prior techniques in order to avoid too extensive a 
depolymerization of the chains of the heparin, is thus avoided. 
According to the invention, instead of conforming with the reaction 
kinetics, arising from the parameters used until now and, particularly, 
from the concentrations of reagents, the concentration of nitrous acid 
generating substances so as to obtain the desired depolymerization is 
selected so that the depolymerization stops due to the fact of the 
disappearance, by total consumption, of one of the reagents, when the 
desired products are obtained. 
The amount of nitrous acid utilised, or of products used to generate it in 
situ, must be sufficient to obtain a satisfactory yield of 
depolymerization products of the desired quality, but must not exceed a 
limit beyond which, taking into account the amount of heparin utilised, 
the nitrous acid would not be fully consumed. 
According to a preferred embodiment of the invention, there is utilised 
about 0.02M to 0.1M, particularly from 0.03M to 0.05M approximately of 
NaNO.sub.2 with a heparin solution at about 8 to 12% by weight. 
The above features are advantageously applied in a controlled heparin 
depolymerization process in which, the conditions of the reaction other 
than the concentrations of reagents, enable more especially the production 
of MPS having an anti-Xa activity (Yin-Wessler) of at least about 200 
u/mg, advantageously higher than 250 u/mg, a USP or APTT activity less 
than about 30 iu advantageously less than 20 iu or even about 10 iu and a 
ratio of their Y-W/USP titer or of their YW/APTT titer of about at least 
3, advantageously higher than about 6, even 10 or more, formed to a major 
extent of species having a molecular weight of about 2,000 to 8,000 
daltons (determined by the HPLC method). 
It is recalled that the Yin-Wessler titer corresponds more specifically to 
the capacity of the active fractions to potentiate the inhibition of the 
Xa factor of the blood by antithrombin III in the corresponding test, 
measured according to the technique of these authors which is described in 
"J. Lab. Clin. Med.", 1976, 81,298-300. 
The USP titer which enables the measurement of an overall coagulation 
intensity under well-determined conditions is also well known. It is 
determined as described in the Pharmacopoea of the United States XIX, pp 
229-230 (see also Second Supplement USP-NF, p. 62, and the Fourth 
Supplement USP-NF, page 90, respectively entitled "Drug Substances and 
Dosage Forms" (medicinal substances and administrative forms)). 
The APTT titer (abbreviation of the English term "activated partial 
thromboplastin time") also measures the overal anticoagulant activity. 
This titer is the equivalent of the Cephalin-Kaolin time measured 
according to J. Caen et al "l'hemostase", edition extension scientifique, 
1976, p. 169-170. 
Preferentially, the raw material applied in the process of the invention is 
constituted by a heparin having a molecular weight of about 2,000 to 
50,000. 
It may be a heparin of conventional injectable pharmaceutical quality, or a 
crude heparin, such as is obtained at the end of extraction operations of 
this active principle from mammalian tissues or organs, particularly from 
intestinal or lung mucus, for example from pork or from beef. It may also 
be constituted by the products which are normally put aside in the 
purification of heparin in order to produce a heparin of injectable 
quality and of higher specific activity. 
The heparin applied is subjected, under controlled conditions, to the 
actions of a chemical agent, more particularly nitrous acid. This acid may 
be added to the heparin or formed in situ by the addition, in controlled 
amounts, of an acid, to a derivative of nitrous acid, in particular, a 
salt, an ether-salt, more especially an alkali or alkaline-earth salt. 
In an advantageous embodiment of the invention, there is used as salt of 
nitrous acid, a water soluble salt, more especially an alkali salt such as 
sodium nitrite NaNO.sub.2. 
To generate the nitrous acid in situ, controlled amounts of an acid 
advantageously containing biologically acceptable anions, such as acetic 
acid and more especially hydrochloric acid, are added. 
The action of the nitrous acid on the heparin is advantageously carried out 
in a physiologically acceptable medium, thus enabling the avoidance of any 
problem which may result through the elimination of a solvent detrimental 
for the contemplated biological applications. In this respect, water 
constitutes the preferred reaction medium. 
In accordance with the invention, the concentrations of heparin and of 
nitrous acid of the solution are selected so that when the desired degree 
of depolymerization is reached, the nitrous acid is entirely consumed, the 
other parameters coming into play for the realisation of this reaction, in 
particular temperature and pH, being in addition also adjusted with 
respect to one another in order to obtain the desired products under the 
most satisfactory experimental conditions. 
The study of these experimental conditions by the inventors has shown that 
it is advantageous to apply the heparin and the products generating the 
nitrous acid in amounts leading to a final concentration of at least 8% by 
weight of the solution in heparin, advantageously of the order of 8 to 
12%, especially close to 10%, the final concentration in sodium nitrite 
then being from about 0.02M to 0.1M and preferably 0.05M and even better 
0.055M. Taking into account the high concentrations of heparin, the 
volumes to be handled for the practising of the process according to the 
invention, are considerably reduced, by a factor of at least 5 with 
respect to the usual chemical techniques. 
The tests carried out have shown that it was appropriate to operate from 
about 0.degree. to 30.degree. C. Temperatures lower than 10.degree. C. may 
be used, for the production of the desired products. Advantageously, 
operations are at room temperature. The acid is used in amounts enabling 
the production of the desired pH advantageously about 2 to 3, preferably 
close to 2.5. 
As already stressed, these conditions enable strict checking of the 
duration of the reaction to be avoided. They permit, in fact, 
self-regulation of the controlled depolymerization reaction according to a 
principle which is distinguished from that applied in known processes, 
which is manifested by a difference at the level of the results, namely 
the absence of nitrites and nitrates in the reaction medium at the end of 
the reaction, the nitrogenous compounds formed being volatile. 
These conditions enable, in addition, the specific production of products 
corresponding to a given profile which will be specified below. 
When the nitrous acid applied is exhausted, the desired depolymerization 
degree is reached and from the depolymerization medium are precipitated 
products of the type of those which are precipitable by alcohol. 
Previously, the pH is advantageously readjusted to a value close to 
neutral. 
For the precipitation operation, it is advantageously resorted to absolute 
ethanol. Tests carried out have enabled the observation that by using 
about 2 volumes of ethanol, the desired precipitation of the biologically 
active MPS is produced. The low volume of solvent used for this 
precipitation will be noted, which presents numerous advantages at the 
handling level. 
The precipitate formed is recovered and, for its use, washed and dried. 
The application of the foregoing features enables the production of 
products characterised particularly by the following points. 
These products, like the MPS described in French Patent Application FR No. 
78 31357 of 6.11.1978 in the name of Applicant, are soluble in a 
water-alcohol medium (water-methanol), having a titer of 
55.degree.-61.degree. GL, preferably 58.degree. GL; they tend towards 
insolubility in a water-ethanol medium whose alcohol content is higher; 
they are insoluble in pure alcohol. They have, in addition, Y-W titers of 
about at least 200 u/mg advantageously higher than about 250 u/mg and USP 
or APTT titers less than at least 50 iu, advantageously 30 iu, even 20, 
and ratios of U-W to USP or APTT titers at least 3, preferably at least 
higher than about 6, particularly higher than about 10; they include 
terminal units having the basic 2,5-anhydro-D-manno basic structure whose 
primary alcohol function at the 6 position is substituted or not by a 
--SO.sub.3.sup.- group. 
This terminal unit corresponds to the following formula: 
##STR1## 
where R.sub.1 can represent a functional group selected particularly from 
among the groups aldehyde, alcohol or carboxylic acid or their 
derivatives, particularly acetals, amides, ethers, esters or corresponding 
salts and R.sub.2 a hydrogen atom or an SO.sub.3.sup.- group. The 
functional groups of R.sub.1 other than the aldehyde group are introduced 
advantageously by the method described by Applicant in the application for 
the second certificate of addition FR Pat. No. 80 06282 of Mar. 20, 1980 
to patent application FR Pat. No. 78 31357 of Nov. 6, 1978. 
In an advantageous aspect of the invention, R.sub.1 is constituted by an 
aldehyde group. In another aspect R.sub.1 is constituted by a carboxylic 
acid group or again alcohol, these groups having particularly the 
advantage of high stability. 
Again preferably, the products obtained according to the invention are 
constituted by a major part of species of molecular weight of about 2,000 
to 8,000 daltons, which corresponds to structures having from about 8 to 
40 saccharide entities (the molecular weights are measured as indicated 
above by the HPLC method, by means, for example, of a 0.5M sodium sulphate 
buffer). 
As obtained, these products are devoid of contaminants of the 
nitrate-nitrite type. 
In general, these products are particularly valuable by reason of their 
pharmacological properties of great interest. They possess, in fact, an 
anti-Xa (YW) activity well above that of the starting heparin whilst their 
USP or APTT titer (which manifest the overall anticoagulant activity) may 
be very much less than that of heparin, which facilitate their use and 
reduces the risk of hemorrhagic accidents. 
They are hence advantageously usable as medicaments, particularly as 
antithrombotic agents. 
In addition, their low molecular weight with respect to that of the 
starting heparin confers on them a delayed pharmacokinetic action of great 
value in therapeutics. They may in addition be advantageously utilised 
orally. 
The conditions reported above of practising the process of the invention 
may lead advantageously to compositions practically no longer containing 
normal heparinic chains, that is to say chains from the starting heparin. 
It is, in fact, a process with a high yield. 
It is hence thus possible to have availabe MPS compositions of high 
homogeneity of molecular weight comprised within the above-indicated 
range, containing advantageously at least about 98% of MPS as stated 
above. 
According to the starting compound, this homogeneous nature may however be 
modified. 
By operating according to other embodiments of this invention, particularly 
under more dilute conditions as regards the reaction medium, MPS 
compositions are obtained, which can contain up to 5 and even up to about 
10% by weight of heparinic chains of molecular weight higher than 10,000 
daltons, manifesting less depolymerization of the starting chains. 
By applying, for example, dilute heparin solutions in the proportion of a 
factor of about 5 with respect to the conditions specified and nitrous 
acid solutions, or more especially the derivatives generating it in situ, 
diluted in the proportion of a factor of about 10, MPS compositions of 
about 3,000 to 6,000 daltons are obtained which can contain from about 5 
to 10% of chains with a higher molecular weight, of the order of 10,000 or 
more and whose YW titers are from about 100 to 200 u/mg, the USP titers 
less than about 50 iu and more customarily comprised between about 40 and 
50, with YW/USP titer ratios of the order of about 5 to 6. 
Other features and advantages of the invention appear in the Examples which 
follow.

EXAMPLE 1 
Self-Regulated Depolymerization of Heparin and Production of MPS of Low 
Molecular Weight 
Into 15 liters of distilled water at +20.degree. C., 1,500 grams of 
commercial heparin having a YW/USP ratio in the vicinity of 1 and a USP 
titer of 160 iu, are dissolved. 51.8 g of sodium nitrate dissolved in 300 
ml of distilled water are added, and immediately the pH is lowered to 2.5 
by pure hydrochloric acid. 
The reaction then takes place and its progress is checked until the absence 
of nitrous ions. After 40 minutes, the presence or absence of nitrous ions 
is checked at regular intervals in the reaction medium. Starch-iodine 
paper, for example, is used, checking every 5 minutes. After about 60 
minutes of reaction, the nitrous acid had been entirely consumed and no 
more NO.sub.2.sup.- ions remained in the reaction medium. The pH was then 
adjusted to 7 with pure caustic soda, and the products of the reaction 
were recovered by the addition of 31 liters of pure ethanol (2 volumes). 
The precipitate formed was collected by centrifugation, washed with 
ethanol and dried at 60.degree. C. under vacuum. 1,200 g of products 
having the following characteristics were collected: 
USP titer: 19 iu/mg 
APTT titer: 13 iu/mg 
Yin and Wessler titer: 202 u/mg 
Nitrites/nitrates content: &lt;4 ppm 
EXAMPLE 2 
By proceeding as in Example 1, starting from a 10% heparin solution (100 
g/l), MPS are obtained having YW/USP ratios according to the amount of 
NaNO.sub.2 employed, namely: 
______________________________________ 
YW Titers over USP 
Value of the Ratio 
NaNO.sub.2 
Titers of the MPS 
of the Titers 
______________________________________ 
0.03 M (140/51) 2.7 
0.04 M (214/26) 8 
0.05 M (250/19) 13 
0.06 M (194/12) 16 
0.07 M (201/9) 22 
0.08 M (132/6.4) 19 
0.1 M (110/2) 44 
______________________________________ 
Preferred MPS correspond to those obtained by using from 0.03 to 0.07M, 
particularly from 0.03 to 0.05M and more especially those obtained at 
0.05M. 
EXAMPLE 3 
In 10 liters of distilled water, at room temperature (15.degree.-20.degree. 
C.) 1,000 grams of commercial injectable heparin having a USP titer of 170 
iu/mg and a YW titer of 160 u/mg, are dissolved. 38 g of sodium nitrite 
(final molarity 0.055M) dissolved in 200 ml of water, is added. The pH is 
immediately lowered to 2.5 by pure hydrochloric acid. The reaction is 
checked as in Example 1 at regular intervals of time (5-10 minutes). After 
30 minutes, NO.sub.2.sup.- ions are no longer detected in the reaction 
medium. The pH is then adjusted to 7 with 5N soda; the products of the 
reaction are recovered by the addition of 21 liters of pure ethanol (2 
volumes). The precipitate formed is collected by centrifugation, washed 
with ethanol and dried at 60.degree. C. under vacuum. 
Finally there are obtained 780 grams of white coloured powder having the 
following characteristics: 
USP Titer: 22 iu/mg 
Yin and Wessler Titer: 260 u/mg 
APTT Titer: 10 iu/mg 
Content of nitrites-nitrates: 5 ppm 
Average molecular weight: less than 6,000 
Percentage of species whose molecular weight exceeds 10,000: less than 1%