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(11) Patent Number: 369
   
(45) Date of grant:  19/01/2010

(l2)PATENT

(51) Int.CI.7:  A 61K 31/727, C 08B 37/10

(21)Application    KE/P/2003/00305    (84) WO No.WO 2004033503  Al Number:            22/04/2004

(22) Filing Date:    08/I 0/2003       

(31)Priority Number:    0212584       

(32) Date: 10/1012002  (33) Country: FR

(73) Owner(s): Aventis Pharma S.A. of 20 Avenue Raymond Aron, F-92160 ANTONY, France           

(72) lnventor(s) BIBEROVIC, VESNA; GRONDARD, LUC; MOURIER, PIERRE and VISKOV, CHRISTIAN

(74)Agent/address for correspondence:    Kaplan & Stratton Advocates, P.O. Box 40111-00100, Nairobi   

(54) Title: HEPARIN-DERIVED POLYSACCHARIDE MIXTURES, PREPARATION THEREOF AND PHARMACEUTICAL COMPOSITlONS CONTAINING SAME

(57) Abstract: The invention concerns heparin-derived polysaccharide mixtureshaving an average molecular weight of 1500 to 3000 daltons and an anti-Xalanti-IIa ratio higher than 30, their preparation method and pharmaceutical compositions containing same.
 

HEPARIN-DERIVED  POLYSACCHARIDE  MIXTURES,

PREPARATION  THEREOF  AND  PHARMACEUTICAL  COMPOSITIONS

CONTAINING  SAME

5    The present invention relates to mixtures of poly-saccharides derived from heparin, their method of preparation and pharmaceutical compositions containing them.


10    Heparin is a mixture of sulfated mucopolysaccharides of animal origin which is used in particular for its anti-coagulant and antithrombotic properties.


Heparin  nevertheless  has  disadvantages  which   limit   the 15    conditions    for    its    use.     In    particular,    its    high anticoagulant   activity    (anti-IIa   activity)    can   cause

hemorrhages.

Low-molecular-weight    heparins    obtained    by    basic

20    depolymerization of heparin esters have been proposed (EP40144); however, these products still have a high

anti-IIa  activity.

Very-low-molecular-weight    heparins    have    also    been

25    described in US 6384021. However, the anti-Xa activity values obtained in the examples described do not exceed

120 IU, and the anti-Xa/anti-IIa ratio obtained is between 15 and 50.


30    In W0-0208295, very-low-molecular-weight heparins are prepared by a method different from US 6384021 and exhibit an activity between 100 and 150 IU with anti-Xa/anti-IIa ratios which are also very high for certain examples of application.

35

A constant need however exists in this class of medicament to improve the anti-Xa activities, in particular in obtaining activities greater than
   
    150  IU/mg,    and    the    anti-Xa/anti-IIa    ratio,    and
    thereforeto    develop    novel    generationsofheparin
    derivatives.                       
5    One  of  the  objectives  of  the  invention  is  therefore  to
    improve   the   anti-Xa   activity   and   the   anti-Xa/anti-IIa
    ratio   by  modifying   the  methods   described   in   the   prior
    art,    in   particular   by   controlling   the   percentage   of
    water   during   the   depolyrnerization   step.   The   heparins
10thus   obtained  thus    exhibit    an  excellent    anti thrombotic
    activity  and  possess  an  aXa  activity  similar  to  that  of
    heparin  while   reducing   the   risks   of   hemorrhage   with   a
    very   low  alia   activity.   Likewise,   the   products   of   the
    invention  exhibit  half-life  periods  which  are  markedly
15    greater  than  that  of  heparin.       

The subject of the invention is therefore novel mixtures of polysaccharides derived from heparin possessing a more selective activity toward activated

20    factor X (factor Xa) and toward activated factor II (factor IIa) than heparin.


It is understood that the mixtures of polysaccharides having a mean molecular weight of 1500 to 3000 Da can

25    be  termed  as  oligosaccharides.

The   subject   of   the  present   invention   is   therefore   the

mixtures of sulfated oligosaccharides having the general structure of the constituent polysaccharides of

30    heparin  and  having  the  following  characteristics:

they have a mean molecular weight of 1500 to 3000 daltons, an anti-Xa activity of 120 to 200 IU/mg, an anti-IIa activity of less than 10 IU/mg and an anti-Xa activity/anti-Ira activity ratio of greater than 30,

35 the constituent oligosaccharides of the mixtures contain 2 to 2 6 sac char ide units, have a 4, 5-unsaturated uronic acid 2-0-sulfate unit at one of their ends, and contain the hexasaccharide ~IIa-IIs-Is of formula:

L\.lla    Is

The  hexasaccharide  ~IIa-IIs-Is contained  in  the  mixture

5    of oligosaccharides described in the present invention is a sequence which has a high affinity for ATIII and characterized by an aXa activity greater than 740 U/mg.

The    mixture    of    oligosaccharides    described    in    the

10    present invention is in the form of an alkali or alkaline-earth metal salt.


As alkali or alkaline-earth metal salt, the sodium, potassium, calcium and magnesium salts are preferred.

The mean molecular weight is determined by high-pressure liquid chromatography using two columns in

series, for example those marketed under the name TSK G3000 XL and TSK G2000 XL. The detection is carried out

20    by refractometry. The eluent used is lithium nitrate and the flow rate is 0.6 ml/min. The system is calibrated with standards prepared by fractionation of enoxaparin (AVENTIS) by chromatography on agarose-polyacrylamide gel ( IBF) . This preparation is carried

25 out according to the technique described by Barrowcliffe et al., Thromb. Res., 12, 27-36 (1977-78) or D.A. Lane et al., Thromb. Res., 12, 257-271 (1977-78). The results are calculated with the GPC6 software (Perkin Elmer).

The anti-Xa activity is measured by the amidolytic method on a chromogenic substrate described by Teien et al.-,:..- Thrornb. Res., 10, 399-410 (1977), with, as

5    standard, the first international standard for low-molecular-weight heparins.


The anti-IIa activity is measured by the technique described by Anderson L.O. et al., Thrornb. Res., 15,

10    531-541 (1979), with, as standard, the first inter-national standard for low-molecular-weight heparins.

The hexasaccharide fraction preferably represents from 15 to 25% of the mixture of oligosaccharides.

15

Preferably, the mixtures according to the invention contain from 8 to 15% of the hexasaccharide ~IIa-IIs-Is in the hexasaccharide fraction of the mixture of oligo-saccharides.

20

The percentage of the hexasaccharide fraction may be analytically determined by high-pressure liquid chromatography on TSK G3000 XL and TSK G2000 XL columns or alternatively by preparative separation of the hexa-

25    saccharide fraction. The mixture is in this case chromatographed on columns filled with a polyacrylamide

agarose type gel such as that marketed under the trademark Ultrogel ACA202R (Biosepra). The mixture is eluted with a sodium hydrogen carbonate solution.

30    Preferably, the sodium hydrogen carbonate solution is a 0.1 mol/1 to 1 mol/1 solution. Still more preferably,

the separation is carried out at a concentration of 1 mol/1. The determination is carried out by UV spectrometry (254 nm) . After fractionation, the hexa-

35    saccharide fraction in solution in sodium hydrogen carbonate is neutralized with glacial acetic acid. The solution is then concentrated under reduced pressure so as to obtain a sodium acetate concentration greater than 30% by weight. The hexasaccharide fraction is

precipitated by adding from 3 to 5 volumes of methanol. The hexasaccharide fraction is recovered by filtration on No. 3 sintered glass. The hexasaccharide mixture obta~ned may be analyzed by HPLC (High-Performance

5 Liquid Chromatography) in order to determine the content of hexasaccharide diia-IIs-Is. Hexasaccharide diia-IIs-Is may be isolated by preparative HPLC chromatography or by affinity chromatography on an antithrombin III sepharose column according to the

10    techniques  used  by  persons  skilled  in  the  art   (M.  Hook,

I. Bjork, J. Hopwood and U. Lindahl, F.E.B.S letters, vol 656(1) (1976)).


Most particularly, the mixtures according to the 15 invention have an anti-Xa activity of between 150 IU/mg

and  200  IU/mg.

Preferably, the mixtures according to the invention have an anti-IIa activity of less than 5 IU/mg, and

20    most particularly of 0.5 to 3.5 IU/mg. The examples of applications described below demonstrate values of between 1.1 and 1.6 IU/mg when the preferred characteristics of the process are used.


25 Preferably, the mixtures exhibit an anti-Xa activity/anti-Ira activity ratio greater than 50 and most particularly greater than 100.


Preferably,    the   mixtures   according   to   the   invention

30    have a mean molecular weight of between 2000 and 3000 Dal tons, and most particularly a mean molecular weight of between 2400 and 2650 Da.


The    subject    of    the    invention    is    therefore    most

35    particularly the mixtures as defined above, having an anti-Xa activity of between 150 and 200 IU/mg, an anti-IIa activity of between 0.5 and 3.5 IU/mg and a mean molecular weight of between 2400 and 2650 Da.

The mixtures of oligosaccharides according to the invention may be prepared by depolymerization of a quaternary ammonium salt of the benzyl ester of heparin in aR-organic medium, by means of a strong organic base

5 with a pKa preferably greater than 20 (properties preferably similar to the family of phosphazenes defined for example according toR. Schwesinger et al., Angew. Chern. Int. Ed. Engl. 26, 1167-1169 (1987), R. Schwesinger et al., Angew. Chern. 105, 1420 (1993)),

10    conversion of the quaternary ammonium salt of the benzyl ester of the depolymerized heparin to a sodium salt, saponification of the residual esters and optionally purification. The method according to the invention repeats the main steps of the method as

15    described in WO 0208295 while adding an essential characteristic which makes it possible to obtain the mixtures of oligosaccharides according to the invention

with the physicochemical characteristics and the activities described above.

20

Indeed,   in   order   to   obtain   the   specific   mixtures   of oligosaccharides    according   to   the    invention,    it    is necessary  to   control  the   selectivity  of   the  base  by  a _  very   precise   control    of    the   water    content    of   the

25    mixture  during  the  depolymerization  step.

The method according to the present invention is indeed characterized by a control of the high selectivity of the base during the depolymerization. It makes it

30    possible to depolymerize the heparin while preserving as much as possible the sequences with an affinity for ATIII such as the hexasaccharide diia-IIs-Is described in the present invention. This critical step of the

method makes it possible to obtain the polysaccharides 3 5 according to the invent ion.

This characteristic of the method results in unexpected aXa activities in terms of the mean molecular weight of the mixtures of oligosaccharides ( 150 IU /mg < aXa <

200 IU/mg for a mean molecular weight of between 2000 Da and 3000 Da) . This selectivity is due to the very particular physicochemical characteristics of the phost3hazene bases which have a pKa greater than 20, a

5    very  high  steric  hindrance  and  a  weak  nucleophilicity.

This effect is fully expressed when the reaction medium is anhydrous. On the other hand, when the water content

of    the   reaction  medium   increases,   a   drastic   reduction

10    in the selectivity of the depolymerization is observed. The preservation of the sequences with affinity for

ATIII  decreases  and  the  consequence  is  a  large  drop  in the  aXa  activity.   In  the  presence  of  a  low  quantity  of water,   the  phosphazene  base  becomes  protonated  and  the 15    reactive     species     becomes     a     quaternary     ammonium hydroxide.   In  this  case,   the  very  high  steric  hindrance and    weak    nucleophilicity    properties    are    lost    and greatly  influence  the  quality  of  the  product  obtained.

When    depolymerization   trials   are   carried   out   with   a

20    measured and controlled water content, this effect can be seen to be fully expressed.

The  following  table  summarizes  the  impact  of  the  water content   on   the    selectivity   of    the    depolymerization 25     (only   this   parameter   is   variable   in   the   trials:   the stoichiometry    of    the    reagents,    the    dilutions,    the temperatures    remain  constant  according  to  the  criteria of   persons   skilled   in   the   art.   The   base   used   is   the phosphazene   base:   2-tert-butylimino-2-diethylamino-1,3-

30    dimethylperhydro-1,2,3-diazaphosphorine).
                   
Water    0.05%    0.1%    0.2%    0.3%    0.4%        0.57%    1. 8%    2.5%
content                                   
%                                   
axa-    192    177    161    132    122        120    105    99
IU/mg                                   
alia    1.3    1.5    1.4    1.4    1.3        1.4    3.1    13.4
IU/mg                                   
aXa/    148    118    115    94    94        85.7    34    7. 4
alia                                   

For an optimum selectivity and a maximum preservation of the sequences with affinity for ATIII, it is preferable to carry out the procedure at water contents

5    of less than 0.6% and most particularly less than 0.3% when 1 molar equivalent of phosphazene base is used

relative to the benzyl ester of heparin, benzethonium salt.


10 The subject of the invention is therefore most particularly the step of depolymerization of the quaternary ammonium salt of the benzyl ester of heparin obtained according to methods known to persons skilled in the art, characterized in that a base of the family


1!2 _ of phosphazenes, in particular in dichloromethane solution containing a percentage of water of less than 0. 6%, is used. Preferably, this percentage of water should be chosen less than 0. 3% and most particularly less than 0.2%.


20

Advantageously, the strong base/ester mol ratio is between 0.2 and 5, preferably between 0.6 and 2 and most particularly between 0.8 and 1. 2. The use of the equimolar ratio therefore forms part of the preferred

25    embodiments  of  the  invention.

Other aprotic solvents known to persons skilled in the art may be used, such as THF or DMF.

The quaternary ammonium salt of the benzyl ester of heparin is preferably the benzethoniurn, cetylpyridinium or cetyltrimethylammonium salt.


5    The bases of the family of phosphazenes are preferably those of formula:


R3
R2-NI    R4
R1-N=P' -NI- R5
\
N-R6
I
R7

10 in which the radicals R1 to R7 are identical or different and represent alkyl radicals.


In the preceding formulae, the alkyl radicals contain 1 to 6 carbon atoms in the form of a straight or branched

15    chain.

The subject of the invention is therefore a method for preparing the mixtures of oligosaccharides according to the invention comprising the following steps

20    a) transalification of sodium heparin by the action of benzethonium chloride,

b)    esterification of benzethonium heparinate by the action of benzyl chloride,

c)    transalification   of   the   benzyl   ester   obtained   to   a

25    quaternary  ammonium  salt,

d)    depolymerization of the quaternary ammonium salt of the benzyl ester of heparin by the method as defined above,

e)    conversion of the quaternary ammonium salt to a 30 sodium salt,

f)    optionally saponification of the heparin by the action of a base such as sodium hydroxide,

g)    optionally purification in particular by the action of an oxidizing agent such as hydrogen peroxide.
 

The    following   reaction   scheme   illustrates   the   present

invention:



H20TA

Step•

n=X+Y+Z(me.,.......,lowelofsulflllionori!Midioacctlorido)               
X,. O.greeafoufotionafthoU.,IIMI-isreprw... ,..dbylheradiclll  H               
Y= OogrM ofsubion oflhe sll8, lhe ,.._,is ntpnooo.,..e by lho rodicol  H        •r-o;"'r:SO,Na. J   
Z= Degr.eot....-allhooibt,tho"'-is!OfnoiNII8dbylhoradicol  COCHl       
    I~-€;--:J    {       
            ~0--<r.[O-M   
    1 O(SO,No)Y    '"'ts0.""~m            0(~)';' NH(SO,Na)Z.    rn   
                "* degree of esterificmion   
    IP'dogrMol.-lltian           
    Crude benzyl ester    Pure benzyl ester, sodium salt   
                   
"A        o./"N::-0    .J~:~:l   
p,... benzyl-. sodum Ill!   +-t-0-o~        'o-       
    Bennthonlum chloride    1    O<SO,...)V   '"'<SO,~1   
    (Hy'C:l")                m•-Z6   

Pure benzyl ester, benzethonium salt

Ci~k
11j <\'                SapooifiCIIIIon       
                NaOH4'C       
Dopolyrnorizolicn                       
21 MeOHI AcONa                       
Stepdondo                       
        Crude ULMWH (residual esters)       
0    Ho    0    H           
HoOHo        0               
o~ONa ~0(50•"""~ -&"'SOJO&l~        Q"~•~+1   
O(SO,Na)Y  NH(SO...    O(S0,N2)Y    NH(SCJ,Ha)Z    m       
                O(SO,IIIa)Y  MWO,Na~)V    NH(SO:Jm   
                    m •-3   
CrudeULMWH            puno  ULMWH       

5

The conversion of the quaternary ammonium salt of the benzyl ester of the depolymerized heparin to a sodium salt (step e) is generally carried out by treating the reaction medium with an alcoholic solution of sodium

10    acetate and preferably with a 10% solution of sodium acetate in methanol (weight/volume), at a temperature of between 15 and 25°C. The equivalent by weight of

acetate added is preferably 3 times greater than the mass of quaternary ammonium salt of the benzyl ester of

15    heparin  used  in  the  depolymerization  reaction.

The saponification (step f) is generally carried out by means of an alkali metal hydroxide such as sodium hydroxide, potassium hydroxide or lithium hydroxide, in an -aqueous medium, at a temperature of between 0 and

5    20°C and preferably between 0 and 10°C. 1 to 5 molar equivalents of alkali metal hydroxide will be generally used. Preferably, the saponification will be carried out in the presence of 1 to 2 molar equivalents of alkali metal hydroxide.


10

The final product may be optionally purified (step g) by any known method of purifying depolymerized heparins (for example EP 003731981). Preferably, the purifi-cation is carried out by means of hydrogen peroxide, in

15    an aqueous medium, at a temperature of 10 to 50 °C. Preferably, this operation is carried out between 20 and 40°C.


The   quaternary   ammonium   salt   of   the   benzyl   ester   of

20    heparin may be prepared according to the following reaction scheme:

a) conversion of the heparin to the form of a sodium salt by means of benzethonium chloride in order to

_obtain  benzethonium  heparinate   (transalification),

25    b) esterification of the benzethonium salt obtained above by means of benzyl chloride and treatment with an alcoholic solution of sodium acetate in order to obtain the sodium salt of the benzyl ester of heparin,

c)    transalification   of   the   sodium   salt   of   the   benzyl

30    ester of heparin to a quaternary ammonium salt and preferably to a benzethonium, cetylpyridinium or cetyltrimethylammonium salt.


The  reaction  of  step  a)   is  carried  out  by  the  action  of

35    benzethonium chloride in excess, on sodium heparin, at a temperature in the region of 15 to 25°C. Advan-tageously, the salt/sodium heparin molar ratio is between 3 and 4.

The starting heparin used is preferably a pig heparin. The latter may be purified beforehand in order to reduce its dermatan sulfate level according to the methed described in patent FR2663639.

5

The esterification of step b) is preferably carried out in a chlorinated organic sol vent (for example chloro-form or methylene chloride), at a temperature of between 25 and 45°C and preferably between 30 and 40°C.

10    The ester in the form of a benzethonium salt is then recovered in the form of a sodium salt by precipitation by means of sodium acetate at 10% by weight in an alcohol such as methanol. 1 to 1. 2 volumes of alcohol are generally used per volume of reaction medium. The

15    quantity of benzyl chloride and the reaction time are adjusted in order to obtain a degree of esterification of between 50 and 100% and preferably between 70 and 90%. Preferably, 0. 5 to 1. 5 parts by weight of benzyl chloride are used for 1 part by weight of benzethonium

20    salt of heparin. Likewise, preferably the reaction time will be between 10 and 35 hours.

The transalification step c) is carried out by means of _ a quaternary ammonium chloride and preferably by means

25    of benzethonium chloride, cetylpyridinium chloride or cetyltrimethylammonium chloride, in an aqueous medium, at a temperature of between 10 and 25°C. Advan-tageously, the quaternary ammonium chloride/sodium salt of the benzyl ester of heparin mol ratio is between 2

30    and  3.

The mixtures according to the invention, in the form of a sodium salt, may be converted to another alkali or

alkaline-earth metal salt. The passage from one salt to 35 another is optionally achieved using the method

described  in  patent  FR  73  13  580.

The mixtures according to the invention are not toxic and may thus be used as medicaments.

The mixtures of oligosaccharides of the present invention may be used as antithrombotic agents. In particular, they are useful for the treatment or

5    prevention of venous and arterial thromboses, deep vein thrombosis, pulmonary embolism, unstable angina, myocardial infarction, cardiac ischemia, occlusive

diseases of the peripheral arteries and atrial fibrillation. They are also useful in the prevention

10    and treatment of the proliferation of smooth muscle cells, atheriosclerosis and arteriosclerosis, for the treatment and prevention of cancer by modulating angio-genesis and growth factors, and for the treatment and prevention of diabetic disorders such as diabetic

15    retinopathies  and  nephropathies.

The   present    invention   also    relates    to   the    pharma-

ceutical compositions containing, as active ingredient, a mixture of formula (I), optionally in combination

20    with  one  or  more  inert  excipients.

The pharmaceutical compositions are for example solutions which can be injected by the subcutaneous or intravenous route. They are also useful for

25    administration by the pulmonary route (inhalation) or by the oral route.


The   dosage   may  vary   according   to   the   age,   weight   and state  of  health  of  the  patient.   For  an  adult,   it  is  in 30    general    between    20    and    100  mg    per    day    by    the

intramuscular  or  subcutaneous  route.

The following examples illustrate the invention without however limiting it .

35

Example A: PREPARATION OF THE BENZETHONIUM SALT OF BENZYL OF HEPARINATE

Benzethonium  heparinate

A solution of 25 g of benzethonium chloride in 125 ml of water is added to a solution of 10 g of heparin in the- ~form of a sodium salt in 100 ml of water. The

5    product  is  filtered,   washed  with  water  and  dried.

Benzyl  ester  of  heparin   (sodium  salt)

16 ml of benzyl chloride are added to a solution of 20 g of benzethonium heparinate in 80 ml of methylene

10    chloride. The solution is heated at a temperature of 30 °C for 12 hours. 108 ml of a 10% solution of sodium acetate in methanol are then added, the mixture is filtered, washed with methanol and dried. 7.6 g of

benzyl    ester  of   heparin   are   thus   obtained  in   the   form

15    of  a  sodium  salt  whose  degree  of  esterification  is  77%.

Benzyl  ester  of  heparin   (benzethonium  salt)

36  g   (0.0549  mol)   of   benzyl   ester   of   heparin    (sodium

salt)    and  540  ml  of  distilled  water  are  introduced  into

20    a 2-litre Erlenmeyer flask A. After homogenization at a temperature of about 20°C, a pale yellow solution is obtained. A solution of 64.45 g (0.1438 mol) of benzethonium chloride and 450 ml of water is prepared,

with  magnetic   stirring,   in   a   1-litre   Erlenmeyer   flask 25    B.   The  solution  in  Erlenmeyer  B  is  poured  over  about  35 minutes    into    the    solution    in    Erlenmeyer    A,     with

stirring.      The     formation     of     an     abundant     white precipitate   is   observed.    The   Erlenmeyer   B   is   rinsed with   200  ml   of   distilled   water   and   the   wash   water   is 30     introduced  into  the  Erlenmeyer  A.   The  stirring  is  then stopped  and  the   suspension  is  allowed  to  settle   for  12 hours.   Once  this  time  has  elapsed,   the  clear  portion  of the   supernatant   is   removed   and   discarded.    560  ml   of water   are   added   to   the   sedimented   precipitate   (slurry


35    appearance) and the mixture is stirred for 20 minutes. The precipitate is allowed to resediment for about 30 minutes. The supernatant is removed and discarded (560 ml) . This operation of washing with about 560 ml of distilled water is repeated twice on the sedimented

precipitate. In the last washing operation, the precipitate is left in suspension and filtered through a No. 3 sintered glass. The cake is then washed with 4 times-200 ml of distilled water. The wet white solid is

5    drained and then dried under reduced pressure (2.7 kPa) at a temperature in the region of 60 °C. After drying for 12 hours, 87.5 g of benzyl heparinate, benzethonium salt, are obtained. The yield obtained is 94.9%.


10    Example   B:    DESCRIPTION   OF   THE   HEXASACCHARIDE   ATIII

(~IIa-II_!-IS)

Na    Na    Na
I    I    I
0    0    0
o=s'~o    o=s~o    o=s~0
Na,O~O•••~~~~O•••~~~O ... ~OH

•-,OH    •-,NH    ~OH    0. :NH    0. :o    0. :NH
    --'        o...:.s    •        o •s
    ~        I    ''    0'~'    I -    ''
    0        Na    0    1    0    Na    0
                    Na           
alia        II.§                    Is   

1~ _ Proton spectrum in 02 0, 500 MHz, T=298 K, () in ppm : 1.97 (3H, s), 3.18 (1H, dd, 10 and 3Hz), 3.30 (1H, t, 8Hz), 3. 37 (1H, dd, 10 and 3Hz), 3. 60 (2H, m), between 3.65 and 3.85 (6H, m), 3.87 (2H, m), 3.95 (1H, d, 8Hz), 4.03 (1H, d, 8Hz), between 4.05 and 4.13 (4H, m),

20    between 4.16 and 4.45 (8H, m), 4.52 (1H, d, 8Hz), 4.67 (1H, m), 5.06 (1H, d, 6Hz), 5.10 (1H, d, 3Hz), 5.33 (1H, d, 4Hz), 5.36 (1H, d, 3Hz), 5.46 (1H, d, 3Hz),

5.72   (1H,  d,   4Hz).

Decasodium    salt   of   4-deoxy-a-L-threo-hexenepyranosyl-

25    uronic    acid-(1~4)-2-deoxy-2-acetamido-6-0-sulfo-a-D-

glucopyranosyl-(1~4)-P-D-glucopyranosyluronic     acid-

(1~4)-2-deoxy-2-sulfamido-3,6-di-0-sulfo-a-D-gluco­

pyranosyl)-(1~4)-2-0-sulfo-a-L-idopyranosyluronic  acid-

(1~4)-2-deoxy-2-sulfamido-6-0-sulfo-a-D-glucopyranose.
 

30


Examples 1 to 7 and 12 illustrate the influence of the water content on the selectivity of the polymerization reaction and the aXa and alia activity of the products obtained.

5 Examples 8 to 10 illustrate the influence of the number of base equivalents on the aXa and alia activity of the product obtained (with a water content of 0.1%)

. Example 11 illustrates the use of a phosphazene base other than 2-tert-butylimino-2-diethylamino-1,3-

10    dimethylperhydro-1, 2, 3-diazaphosphorine:

Use  of  tert-butyliminotri(pyrrolidino)phosphorane

EXAMPLE  1

Depo~ymerization  and   conversion   to   the   sodium   salt

15    (0.1%  water):

7 0 m1 of dichloromethane are placed in an Erlenmeyer flask A. 10 g (0. 006 mol) of benzyl ester of heparin (degree of esterification: 75%, benzethonium salt) obtained as described in example A are added slowly

20    with stirring. The water content of the reaction medium is adjusted to 0. 1%. The solution is heated to 40 °C under nitrogen. After total dissolution, the solution is cooled to a temperature in the region of 20°C, followed by addition of 1.75 ml (0.006 mol) of 2-tert-

25    butylimino-2-diethylamino-1,3-dimethylperhydro-1,2,3-diazaphosphorine. The resulting mixture is stirred at a temperature in the region of 20°C for 24 hours. During this time, a solution of 30 g of anhydrous sodium

acetate    in    300  ml    of   methanol    is    prepared    in    an

30    Erlenmeyer flask B. After total dissolution, 5 g of Hyflo supercel Celite are added to the solution. The reaction mixture in the Erlenmeyer flask A is poured over 1 minute 30 seconds into the methanolic solution

of   sodium   acetate   at   a   temperature   in   the   region   of 35    5 °C,     with    magnetic    stirring.     After    stirring    for 5  minutes,   the  suspension  is  left  to  settle  for   1  hour 30  minutes.    The    clear    part    of    the    supernatant    is separated    out    and    discarded     (220  ml).     220  m1    of methanol   are   added   to   the   sedimented   precipitate   and
 


the mixture is stirred for 5 minutes. The precipitate is left to resediment for about 1 hour 20 minutes. The supernatant is separated out and discarded (250 ml). 250 • ml of methanol are added to the sedimented

5    precipitate and the mixture is stirred for 5 minutes. The precipitate in suspension is then filtered through a No. 3 sintered glass. The cake obtained is then washed with 100 ml of methanol. The pale yellow wet solid is drained and then dried under reduced pressure

10 (6 kPa) at a temperature in the region of 40°C. After drying for 18 hours, 2.51 g of the sodium salt of depolymerized heparin in Celite (5 g) are obtained. The yield obtained is 64%.


15    Saponification:

2.5 g (0.0038 mol) of the crude depolymerized heparin, sodium salt in Celi te ( 5 g) obtained above, and 17 ml of water are placed in a 50 ml Erlenmeyer flask. The suspension is filtered through a No. 3 sintered glass

20    and rinsed with twice 5 ml of water. The filtrate obtained is placed in a 150 ml Erlenmeyer flask. 0.4 ml

(0.004 mol) of 30% caustic soda is introduced with magnetic stirring, at a temperature in the region of 5°C. After addition, the mixture is stirred for

25    2 hours. The solution is neutralized by adding lN HCl and 3 g of sodium chloride are added. After dissolution, 21 ml of methanol are added to the reaction medium. After stirring for 15 minutes, 44 ml of methanol are added, followed by stirring for 1 hour.

30    Stirring is then stopped and the suspension is left to sediment for 45 minutes at a temperature in the region of 5°C. The supernatant is then separated out and discarded ( 90 ml) . 90 ml of methanol are added to the sedimented precipitate and the mixture is stirred for

35    5 minutes. The precipitate is left to resediment for about 20 minutes. The supernatant is separated out and discarded (80 ml). 80 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 5 minutes. The precipitate in suspension is then

filtered through a No. 3 sintered glass. The cake obtained is then washed with 50 ml of methanol. The wet solid is drained and then dried under reduced pressure (6 kPa) at a temperature in the region of 40°C. After

5    drying for 18 hours, 1.31 g of crude depolymerized heparin (sodium salt) are obtained. The yield obtained is 66%.


Purification:

10    1.3 g of crude depolymerized heparin obtained above and 13 ml of distilled water are placed in a 50 ml Erlenmeyer flask. The mixture is brought to 40°C with magnetic stirring. The pH is brought to 9. 7 ± 0. 1 by

addition  of  1N  sodium  hydroxide.   The  reaction  medium  is

15    filtered through a 0.45 pm membrane and 0.07 ml of aqueous 30% hydrogen peroxide solution is added. After stirring for about 2 hours at a temperature in the

region of 20°C, the mixture is neutralized by adding 1N HCl, followed by addition of 2 g of sodium chloride.

20    The solution is then filtered through a 0.45 pm membrane and 14 ml of methanol are then poured in. The solution is then cooled to 10°C and stirred for about 15 minutes. 36 ml of methanol are then added, followed

by   stirring   for   1  hour.   Stirring   is   then   stopped   and 25    the     suspension     is     left     to     sediment     for     about 15  minutes.   The   supernatant   is   then   separated   out   and discarded   (50  ml).   50  ml   of  methanol   are  added   to   the sedimented  precipitate   and   the   mixture   is   stirred   for 5  minutes.   The   precipitate   is   left   to   resediment   for

30    about 25 minutes. The supernatant is separated out and discarded (50 ml). The precipitate in suspension is then filtered through a No. 3 sintered glass. The white cake obtained is then washed with 50 ml of methanol.

The    wet   solid  is   drained  and  then  dried  under   reduced


The    characteristics   of   the   depolymerized   heparin   thus

obtained  are  as  follows:

mean  molecular  weight:  2600  Daltons

anti~Xa activity:  177  IU/mg

5    anti-IIa  activity:  1.5  IU/mg

anti-Xa  activity/anti-Ira  activity  ratio:   118

EXAMPLE  2

Depolymerization    and   conversion   to   the   sodium   salt

10    (0.2%)

70   ml   of   dichloromethane   are   placed   in   a   Erlenmeyer flask   A.    10   g    (0.006  mol)    of   the   benzyl   ester   of heparin    (degree   of   esterification:    75%,   benzethonium salt)    obtained   as   described   in   example   A   are   added 15    slowly,   with  stirring  and  under  nitrogen  pressure.   The water   content   of   the   reaction   medium   is   adjusted   to 0.2%.   After   total   dissolution,   1.75  ml   (0.006  mol)   of

2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,2,3-diazaphosphorine are added. The mixture is

20    stirred  for  24  hours  at  a  temperature  in  the  region  of

20°C. During this time, a solution of 30 g of anhydrous sodium acetate in 300 ml of methanol is prepared in a Erlenmeyer flask B. After total dissolution, 5 g of Hyflo supercel Celi te are added to the solution. The

25    reaction mixture in the Erlenmeyer flask A is poured over 1 minute 30 seconds into the methanolic solution

of sodium acetate with magnetic stirring, at a temperature in the region of 5 °C. After stirring for 5 minutes, the suspension is left to settle for

30    2 hours. The clear part of the supernatant is separated out and discarded (220 ml) . 220 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 5 minutes. The precipitate is left to resediment for about 2 hours. The supernatant is


drained and then dried under reduced pressure ( 6 kPa) at a temperature in the region of 40°C. After drying for 18 hours, 2.63 g of crude depolymerized heparin in Celite (5 g) are obtained. The yield obtained is 67%.

5

Saponification:

2.5 g (0.0038 mol) of the sodium salt of depolymerized heparin in Celite (5 g) obtained above and 18 ml of water are placed in 50 ml Erlenmeyer flask. The

10    suspension is filtered through a No. 3 sintered glass and rinsed with twice 5 ml of water. The filtrate obtained is placed in a 150 ml Erlenmeyer flask. 0.4 ml

(0.004 mol) of 30% caustic soda is added with magnetic stirring, at a temperature in the region of 5°C. After

15 addition, the mixture is stirred for 2 hours. The solution is neutralized by adding 1N HCl and 2 g of sodium chloride are added. 14 ml of methanol are poured

into     the     reaction     mixture.     After     stirring     for 15  minutes,   36  ml   of   methanol   are   added,   followed   by 20    stirring  for   1  hour.   Stirring  is   then   stopped  and  the suspension   is   left   to   sediment   for   45  minutes   at   a temperature   in   the   region   of   5 °C.   The   supernatant   is then   separated   out   and   discarded    (80  ml).    80  ml   of

methanol    are   added   to   the   sedimented   precipitate   and

25    the mixture is stirred for 5 minutes. The precipitate in suspension is then filtered through a No. 3 sintered glass. The cake obtained is then washed with 50 ml of methanol. The wet solid is drained and then dried under reduced pressure (6 kPa) at a temperature in the region

30    of 40°C. After drying for 48 hours, 2.3 g of crude depolymerized heparin (sodium salt) are obtained. The yield obtained is 65%.


Purification:

35    1.4 g of crude depolymerized heparin obtained above and 15 ml of distilled water are placed in a 50 ml Erlenmeyer flask. The mixture is brought to 4 0 o C with magnetic stirring. The pH is brought to 9. 7 ± 0. 1 by addition of 1N sodium hydroxide. The reaction medium is

filtered through a 0.45 pm membrane and 0.07 ml of aqueous 30% hydrogen peroxide solution is added. After stirring for about 2 hours at a temperature in the regien of 40°C, the mixture is cooled to a temperature

5    in the region of 20°C and then neutralized by addition of 1N HCl. 2 g of sodium chloride are added to the reaction medium. The solution is then filtered through a 0.45 pm membrane and 14 ml of methanol are then

poured    in.   The   solution   is   then   cooled   to   10°C   and

10    stirred for 15 minutes. 36 ml of methanol are then added, followed by stirring for 1 hour. Stirring is then stopped and the suspension is left to sediment for

15  minutes.   The   supernatant   is   then   separated   out   and discarded   (40  ml).   40  ml   of  methanol   are   added  to   the 15    sedimented    precipitate    (slurry    appearance)    and    the mixture   is   stirred   for   5  minutes.   The   precipitate   is

left     to     resediment     for     about     20  minutes.     The supernatant  is  separated  out  and  discarded   (50  ml).   The precipitate   in   suspension   is   then   filtered   through   a 20    No.  3  sintered  glass.   The   white   cake   obtained   is   then washed  with  50  ml  of  methanol.  The  wet  solid  is  drained and   then   dried   under   reduced   pressure    ( 6  kPa)    at   a temperature   in   the   region   of   40°C.   After   drying   for 18  hours,   1. 2  g   of   pure   depolymerized   heparin   (sodium


25    salt)  are  obtained.  The  yield  obtained  is  86%.

The characteristics of the depolymerized heparin thus obtained are as follows:

mean molecular weight: 2650 daltons anti-Xa activity: 161 IU/mg

30    anti-IIa  activity:  1.4  IU/mg

anti-Xa  activity/anti-Ira  activity  ratio:   115

EXAMPLE  3

Depolymerization    and   conversion   to   the   sodium   salt

35    (0. 3%  water)

70    ml   of   dichloromethane   are   placed   in   a   Erlenmeyer

flask    A.   10  g   (0.006  mol)   of   benzyl   ester   of   heparin

(degree    of    esterification:    75%,    benzethonium    salt)

obtained    as   described   in   example   A   are   added   with

stirring and under nitrogen pressure. The water content of the reaction medium is adjusted to 0.3%. After total dissolution, 1. 7S ml (0. 006 mol) of 2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,2,3-diaza-

5    phosphorine are added. The mixture is stirred at a temperature in the region of 20°C for 24 hours. During

this time, a solution of 30 g of anhydrous sodium acetate in 300 ml of methanol is prepared in a Erlenmeyer flask B. After total dissolution, S g of

10    Hyflo supercel Celi te are added to the solution. The reaction mixture in the Erlenmeyer flask A is poured over 1 minute 30 seconds into the methanolic sodium

acetate  solution  at  a  temperature  in  the  region  of  S°C, with   magnetic   stirring.   After   stirring   for   S  minutes, lS    the  suspension  is  left  to  settle  for  1  hour  10  minutes. The  clear  part  of  the  supernatant  is  separated  out  and discarded   (220  ml) .  220  ml  of  methanol  are  added  to  the sedimented  precipitate   and  the   mixture   is   stirred   for S  minutes.    The    precipitate    in    suspension    is    then


20    filtered through a No. 3 sintered glass. The cake obtained is then washed with 100 ml of methanol. The wet solid is drained and then dried under reduced pressure (6 kPa) at a temperature in the region of 40°C. After drying for 18 hours, 2.57 g of crude

25    depolymerized heparin are obtained, as the sodium salt in Celite (S g). The yield obtained is 66%.

Saponification:

2.5    g    (0.0038  mol)    of    the    sodium    salt    of    crude

30    depolymerized heparin in Celite (S g) obtained above and 18 ml of water are placed in 50 ml Erlenmeyer flask. The suspension is filtered through a No. 3 sintered glass and rinsed with twice 5 ml of water. The

filtrate    obtained   is   placed   in   a   150  ml   Erlenmeyer

35    flask. 0.4 ml (0.004 mol) of 30% caustic soda is added with magnetic stirring, at a temperature in the region of S°C. After addition, the mixture is stirred for 2 hours. The solution is neutralized by adding 1N HCl and 3 g of sodium chloride are added. 15 ml of methanol

are poured into the reaction mixture. After stirring for 15 minutes, 36 ml of methanol are added, followed by stirring for 1 hour. Stirring is then stopped and the• =-s-uspension is left to sediment for 1 hour. The

5 supernatant is then separated out and discarded (70 ml) . 70 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 5 minutes. The precipitate in suspension is then filtered through a No. 3 sintered glass. The cake obtained is then

10    washed with 50 ml of methanol. The wet solid is drained and then dried under reduced pressure (6 kPa) at a

temperature in the region of 40°C. After drying for 48 hours, 1.42 g of crude depolyrnerized heparin (sodium salt) are obtained. The yield obtained is 62%.

15

Purification:

1.4 g of crude depolyrnerized heparin obtained above and 14 ml of distilled water are placed in a 50 ml Erlenmeyer flask. The mixture is brought to 40°C with

35    magnetic stirring. The pH is brought to 9. 7 ± 0.1 by addition of 1N sodium hydroxide. The reaction medium is filtered through a 0.45 pm membrane and 0.07 ml of aqueous 30% hydrogen peroxide solution is added. After

stirring    for   about   2  hours   at   a   temperature   in   the

25    region of 20°C, the mixture is neutralized by adding 1N HCl, followed by addition of 2 g of sodium chloride. After dissolution, the solution is then filtered through a 0. 4 5 pm membrane and 14 ml of methanol are then poured in. The filtrate is then cooled to 10°C and

30    stirred for 15 minutes. 36 ml of methanol are then added, followed by stirring for about 1 hour. Stirring

is then stopped and the suspension is left to sediment for 40 minutes. The supernatant is then separated out and discarded (50 ml) . 50 ml of methanol are added to

35    the sedimented precipitate (slurry appearance) and the mixture is stirred for 5 minutes. The precipitate is left to resediment for about 25 minutes. The precipitate in suspension is then filtered through a No. 3 sintered glass. The white cake obtained is then


washed with 50 ml of methanol. The wet solid is drained and then dried under reduced pressure (6 kPa) at a temperature in the region of 40°C. After drying for 18 hemrs, 1.24 g of pure depolymerized heparin (sodium

5    salt)   are  obtained.  The  yield  obtained  is  89%.

The characteristics of the depolymerized heparin thus obtained are as follows:

mean molecular weight: 2400 daltons anti-Xa activity: 132 IU/mg

10    anti-IIa  activity:  1.4  IU/mg

anti-Xa  activity/anti-Ira  activity  ratio:   94

EXAMPLE    4

Depolymerization    and   conversion   to    the   sodium   salt

15    (0. 4%  water) :

70 ml of dichloromethane are placed in a Erlenmeyer flask A. 10 g (0. 006 mol) of benzyl ester of heparin (degree of esterification: 75%, benzethonium salt) obtained as described in example A, are added slowly,

20    with stirring. The water content of the reaction medium is adjusted to 0. 4%. The solution is heated to 30 °C under nitrogen. After total dissolution, the mixture is cooled to a temperature in the region of 20°C, followed by addition of 1.75 ml (0.006 mol) of 2-tert-

25    butylimino-2-diethylamino-1,3-dimethylperhydro-1,2,3-diazaphosphorine. The mixture is stirred at a temperature in the region of 20°C for 24 hours. During

this time, a solution of 30 g of anhydrous sodium acetate in 300 ml of methanol is prepared in a

30    Erlenmeyer flask B. After total dissolution, 5 g of Hyflo supercel Celi te are added to the solution. The reaction mixture in the Erlenmeyer flask A is poured over 1 minute 30 seconds into the methanolic sodium

acetate  solution  at  a  temperature  in  the  region  of  5°C,

35    with magnetic stirring. After stirring for 5 minutes, the suspension is left to settle for 2 hours. The clear part of the supernatant is separated out and discarded (80 ml). 80 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 5 minutes.

The precipitate is left to resediment for about 1 hour. The supernatant is separated out and discarded (80 ml). 80 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 5 minutes.

5    The precipitate in suspension is then filtered through a No. 3 sintered glass. The cake obtained is then washed with 150 ml of methanol. The pale yellow wet solid is drained and then dried under reduced pressure (6 kPa) at a temperature in the region of 40°C. After

10    drying for 18 hours, 3.25 g of crude depolymerized heparin are obtained, as the sodium salt in Celite (5 g). The yield obtained is 83%.


Saponification:

15    3.1 g (0.0018 mol) of the sodium salt of crude depolymerized heparin in Celite (10 g) obtained above

and 21 ml of water are placed in 50 ml Erlenmeyer flask. The suspension is filtered through a No. 3 sintered glass and rinsed with twice 6 ml of water. The

20    filtrate obtained is placed in a 150 ml Erlenmeyer flask. 0. 7 ml ( 0. 007 mol) of 30% caustic soda is added with magnetic stirring, at a temperature in the region

of 5°C. After addition, the mixture is stirred for 2 hours. The solution is neutralized by adding 1N HCl

25    and 4 g of sodium chloride are added. 28 ml of methanol are poured into the reaction mixture. After stirring for 15 minutes, 72 ml of methanol are added, followed by stirring for 1 hour. Stirring is then stopped and the suspension is left to sediment for 1 hour. The

30 supernatant is then separated out and discarded (90 ml). 90 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 5 minutes. The precipitate is left to resediment for about 20 minutes. The supernatant is separated out and

35    discarded ( 90 ml) . 90 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 5 minutes. The precipitate in suspension is then filtered through a No. 3 sintered glass. The cake obtained is then washed with 50 ml of methanol. The wet


solid is drained and then dried under reduced pressure ( 6 kPa) at a temperature in the region of 4 0 o C. After drying for 48 hours, 1.9 g of crude depolymerized hepa~in (sodium salt) are obtained. The yield obtained

5    is  67%.

Purification:

1.9  g  of  crude  depolyrnerized  heparin  obtained  above  and

19  ml    of    distilled    water    are    placed    in    a    50  ml

10    Erlenmeyer flask. The mixture is brought to 40 o C with magnetic stirring. The pH is brought to 9. 7 ± 0. 1 by addition of 1N sodium hydroxide. The reaction medium is filtered through a 0.45 ~m membrane and 0.1 ml of aqueous 30% hydrogen peroxide solution is added. After

15    stirring for about 2 hours at a temperature in the region of 20°C, the mixture is neutralized by adding 1N HCl, followed by addition of 2 g of sodium chloride.

The solution is then filtered through a 0.45 ~m membrane and 14 ml of methanol are then poured in, and

20    the mixture is stirred for 15 minutes. 36 ml of methanol are then added, followed by stirring for 1 hour. Stirring is then stopped and the suspension is

left to sediment for 15 minutes. The supernatant is then separated out and discarded (40 ml). 40 ml of

25    methanol are added to the sedimented precipitate and the mixture is stirred for 5 minutes. The precipitate is left to resediment for about 20 minutes. The supernatant is separated out and discarded (50 ml).

500  ml    of    methanol    are    added    to    the    sedimented

30    precipitate and the mixture is stirred for 5 minutes. The precipitate is left to resediment for about 20 minutes. The suspension is then filtered through a No. 3 sintered glass. The white cake obtained is then washed with 50 ml of methanol. The wet solid is drained

35    and then dried under reduced pressure (6 kPa) at a temperature in the region of 40°C. After drying for 72 hours, 1.56 g of pure depolymerized heparin (sodium salt) are obtained. The yield obtained is 82%.


The characteristics of the depolymerized heparin thus obtained are as follows:

mean molecular weight: 2350 daltons anti~xa activity: 122 IU/mg
5    anti-IIa  activity:   1.3  IU/mg

anti-Xa  activity/anti-Ira  activity  ratio:   94

EXAMPLE  5

Depolymerization    and   conversion   to   the   sodium   salt

10    (0. 57%  water) :

140 ml of dichloromethane are placed in a Erlenmeyer flask A. 20 g (0. 019 mol) of benzyl ester of heparin (degree of esterification: 75%, benzethonium salt) obtained as described in example A, are added slowly,

15    with stirring. The water content of the reaction medium is adjusted to 0. 57%. After total dissolution, 3. 5 ml (0.012 mol) of 2-tert-butylimino-2-diethylamino-1,3-

dimethylperhydro-1,2,3-diazaphosphorine are added. The mixture is stirred at a temperature in the region of

20    20°C for 24 hours. During this time, a solution of 60 g of anhydrous sodium acetate in 600 ml of methanol is

prepared in a Erlenmeyer flask B. After total dissolution, 10 g of Hyflo supercel Celite are added to the solution. The reaction mixture in the Erlenmeyer

25    flask A is poured over 1 minute 30 seconds into the methanolic sodium acetate solution at a temperature in the region of 4°C, with magnetic stirring. After stirring for 5 minutes, the suspension is left to settle for 30 minutes. The clear part of the


30    supernatant is separated out and discarded (400 ml). 400 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 5 minutes. The precipitate is left to resediment for about 1 hour.

The     supernatant     is     separated     out     and     discarded 35     ( 4 2 0  ml) .     4 2 0  ml     of    methanol     are     added     to     the sedimented   precipitate   and   the   mixture   is   stirred   for 5  minutes.   The   suspension   is   then   filtered   through   a No.   3  sintered  glass.   The   cake  obtained  is  then  washed with   200  ml   of  methanol.   The   pale   yellow  wet   solid   is


drained and then dried under reduced pressure ( 6 kPa) at a temperature in the region of 50 °C. After drying for 18 hours, 6.66 g of crude depolyrnerized heparin are obtained, as the sodium salt in Celite (10 g). The

5    yield  obtained  is  85%.

Saponification:

6.66 g (0.0101 mol) of the sodium salt of crude depolyrnerized heparin in Celi te ( 10 g) obtained above

10    and 4 7 ml of water are placed in a 50 ml Erlenmeyer flask. The suspension is filtered through a No. 3 sintered glass and rinsed with twice 15 ml of water. The filtrate obtained is placed in a 150 ml Erlenmeyer flask. 1.1 ml (0.011 mol) of 30% caustic soda are added

15    with magnetic stirring, at a temperature in the region of 5°C. After addition, the mixture is stirred for 2 hours. The solution is neutralized by adding 1N HCl and 9.5 g of sodium chloride are added. 66 ml of

methanol    are    added    to    the    reaction    medium.    After

20    stirring for 15 minutes, 171 ml of methanol are added, followed by stirring for 1 hour. Stirring is then stopped and the suspension is left to sediment for ~

hour   at    a    temperature    in    the    region    of    5°C.    The supernatant    is    then    separated    out    and    discarded 25     ( 160  ml) .     160  ml    of    methanol    are    added    to     the sedimented  precipitate   and   the   mixture   is   stirred   for 5  minutes.   The   precipitate   is   left   to   resediment   for about   20  minutes.   The   supernatant  is   separated  out   and discarded   (180  ml).   180  ml  of  methanol  are  added  to  the


30    sedimented precipitate and the mixture is stirred for 5 minutes. The suspension is then filtered through a No. 3 sintered glass. The cake obtained is then washed with twice 50 ml of methanol. The wet solid is drained and then dried under reduced pressure (6 kPa) at a

35    temperature in the region of 40 °C. After drying for 18 hours, 4.53 g of crude depolymerized heparin (sodium salt) are obtained. The yield obtained is 74%.


4.    53 g of crude depolyrnerized heparin obtained above and 45 ml of distilled water are placed in a 100 ml Erlenmeyer flask. The mixture is brought to 40°C with magnetic stirring. The pH is brought to 9. 7 ± 0. 1 by

5    addition of 1N sodium hydroxide. The reaction medium is filtered through a 0.45 pm membrane and 0.25 ml of aqueous 30% hydrogen peroxide solution is added. After

stirring for about 2 hours at a temperature in the region of 20°C, the mixture is neutralized by adding

10    1N HCl, followed by addition of 5. 5 g of sodi urn chloride. The solution is then filtered through a 0.45 pm membrane and 38 ml of methanol are then poured in, at a temperature in the region of 10°C. The

solution    is   then   brought   to    20°C   and   stirred   for

15    15 minutes. 100 ml of methanol are then added, followed by stirring for 1 hour. Stirring is then stopped and the suspension is left to sediment for 20 minutes. The supernatant is then separated out and discarded

(90  ml).   90  ml  of  methanol  are  added  to  the  sedimented

20    precipitate and the mixture is stirred for 5 minutes. The precipitate is left to resediment for about 25 minutes. The supernatant is separated out and discarded (100 ml). The precipitate in suspension is

then  filtered  through  a  No.  3  sintered  glass.   The  white

25    cake obtained is then washed with 50 ml of methanol. The wet solid is drained and then dried under reduced pressure (6 kPa) at a temperature in the region of 50 °C. After drying for 18 hours, 3. 7 g of pure depolymerized heparin (sodium salt) are obtained. The

30    yield  obtained  is  82%.

The characteristics of the depolyrnerized heparin thus obtained are as follows:

mean molecular weight: 2200 daltons anti-Xa activity: 120 IU/mg

35    anti-IIa  activity:  1.4  IU/mg

anti-Xa  activity/anti-Ira  activity  ratio:  86

EXAMPLE  6

Depolymerization    and   conversion   to   the   sodium   salt

(1.8%-water):

5    140 ml of dichloromethane are placed in a Erlenmeyer flask A. 20 g ( 0. 019 mol) of benzyl ester of heparin (degree of esterification: 75%, benzethonium salt) obtained as described in example A, are added slowly, with stirring. The water content of the reaction medium

10 is adjusted to 1. 8%. After total dissolution, 3. 5 ml (0.012 mol) of 2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1, 2, 3-diazaphosphorine are added. The mixture is stirred at a temperature in the region of 20°C for 24 hours. During this time, a solution of 60 g

15    of anhydrous sodium acetate in 600 rnl of methanol is prepared in a Erlenmeyer flask B. After total

dissolution,   10  g  of  Hyflo  supercel  Celite  are  added  to the   solution.   The   reaction   mixture   in   the   Erlenmeyer flask   A  is   poured   over   1  minute   30  seconds   into   the 20    m~thanolic  sodium  acetate  solution  at  a  temperature  in the    region    of    4 °C,    with    magnetic    stirring.    After stirring    for    5  minutes,    the    suspension   is    left    to settle    for    30    minutes.     The    clear    part    of    the supernatant   is   separated   out   and   discarded    (400  ml).


25 400 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 5 minutes. The precipitate is left to resediment for about 1 hour. The supernatant is separated out and discarded (420 ml). 420 ml of methanol are added to the

30    sedimented precipitate and the mixture is stirred for 5 minutes. The precipitate in suspension is then filtered through a No. 3 sintered glass. The cake obtained is then washed with 200 ml of methanol. The pale yellow wet solid is drained and then dried under

35    reduced pressure (6 kPa) at a temperature in the region of 50 °C. After drying for 18 hours, 7. 54 g of crude depolymerized heparin are obtained, as the sodium salt in Celite (10 g). The yield obtained is 96%.

Saponification:

7.54 g (0.0101 mol) of the sodium salt of crude depolymerized heparin in Celi te ( 10 g) obtained above and- =-5-3 ml of water are placed in . a 50 ml Erlenmeyer

5    flask. The solution is filtered through a No. 3 sintered glass and rinsed with twice 15 ml of water. The filtrate obtained is placed in a 150 ml Erlenmeyer flask. 1.25 ml (0.012 mol) of 30% caustic soda are added with magnetic stirring, at a temperature in the

10    region of 4°C. After addition, the mixture is stirred for 2 hours. The solution is neutralized by adding 1N HCl and 10.5 g of sodium chloride are added. 70 ml of methanol are added to the reaction medium. After

stirring    for   15  minutes,   190  ml  of  methanol   are   added,

15    followed by stirring for 1 hour. Stirring is then stopped and the suspension is left to sediment for ~ hour at a temperature in the region of 4°C. The supernatant is then separated out and discarded

(180  ml).    180  ml    of    methanol    are    added    to    the

20    sedimented precipitate and the mixture is stirred for 5 minutes. The precipitate is left to resediment for about 20 minutes. The supernatant is separated out and discarded (180 ml). 180 ml of methanol are added to the

sedimented    precipitate   and   the   mixture   is   stirred   for

25    5 minutes. The precipitate in suspension is then filtered through a No. 3 sintered glass. The cake obtained is then washed with twice 50 ml of methanol. The wet solid is drained and then dried under reduced pressure (6 kPa) at a temperature in the region of

30    50°C. After drying for 18 hours, 5.53 g of crude depolymerized heparin (sodium salt) are obtained. The yield obtained is 80%.


Purification:

35    5. 53 g of crude depolymerized heparin obtained above and 55 ml of distilled water are placed in a 100 ml Erlenmeyer flask. The mixture is brought to 40°C with magnetic stirring. The pH is brought to 9. 7 ± 0. 1 by addition of 1N sodium hydroxide. The reaction medium is


filtered through a 0.45 ~m membrane and 0.31 ml of aqueous 30% hydrogen peroxide solution is added. After stirring for about 2 hours at a temperature in the regien of 20°C, the mixture is neutralized by adding

5    1N HCl, followed by addition of 7 g of sodium chloride. The solution is then filtered through a 0.45 pm membrane and 49 ml of methanol are then poured in, at a temperature in the region of 10°C. The solution is then

    brought   to   20 °C   and   stirred  for   15  minutes.   126  ml   of
10    methanol    are    then    added,    followed    by   stirring    for
    1  hour.   Stirring  is  then  stopped  and  the  suspension  is
    left   to   sediment   for   20  minutes.   The   supernatant   is
    then   separated   out   and   discarded   {105  ml) .   105  ml   of
    methanol   are   added   to   the   sedimented   precipitate   and
15    the   mixture   is   stirred   for    5    minutes.    The    precipitate
    islefttoresedimentforabout25  minutes.The
    supernatant   is   separated   out   and   discarded    (110  ml).
    The  precipitate  in  suspension  is  then   filtered   through
    a  No.   3  sintered  glass.   The  white  cake  obtained  is  then
20    washed  with  50  ml  of  methanol.   The  wet  solid  is  drained
    and   then   dried   under   reduced   pressure    (6  kPa)    at   a
    temperature   in    the    region    of    55 °C.    After    drying    for

18 hours, 4. 53 g of pure depolymerized heparin (sodium _ salt) are obtained. The yield obtained is 82%.

25    The characteristics of the depolymerized heparin thus obtained are as follows:

mean molecular weight: 2600 daltons anti-Xa activity: 105 IU/mg

anti-IIa  activity:   3.1  IU/mg

30    anti-Xa  activity/anti-IIa  activity  ratio:   34

EXAMPLE    7

Depolymerization    and   conversion   to   the    sodium   salt

(2. 5%  water) :

35    140 ml of dichloromethane are placed in a Erlenmeyer flask A. 20 g ( 0. 019 mol) of benzyl ester of heparin {degree of esterification: 75%, benzethonium salt) obtained as described in example A, are added slowly, with stirring. The water content of the reaction medium

is adjusted to 2. 5%. After total dissolution, 3. 5 ml (0.012 mol) of 2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,2,3-diazaphosphorine are added. The mixtare is stirred at a temperature in the region of

5    20°C for 24 hours. During this time, a solution of 60 g of anhydrous sodium acetate in 600 ml of methanol is prepared in a Erlenmeyer flask B. After total dissolution, 10 g of Hyflo supercel Celite are added to the solution. The reaction mixture in the Erlenmeyer

10    flask A is poured over 1 minute 30 seconds into the methanolic sodium acetate solution at a temperature in the region of 4°C, with magnetic stirring. After stirring for 5 minutes, the suspension is left to settle for 1 hour. The clear part of the supernatant is

15    separated out and discarded (400 ml). 400 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 5 minutes. The precipitate is left to resediment for about 30 minutes. The clear

part  of  the  supernatant  is  separated  out  and  discarded 20     (400  ml).     The    precipitate    in    suspension    is    then filtered   through   a    No.   3   sintered   glass.    The   cake obtained   is   then   washed   with   200  ml   of   methanol.   The pale  yellow  wet   solid  is   drained  and  then  dried  under reduced  pressure   (6  kPa)   at  a  temperature  in  the  region

25    of 50°C. After drying for 18 hours, 7. 78 g of crude depolymerized heparin are obtained, as the sodium salt in Celite (10 g). The yield obtained is 99.6%.


Saponification:

30 7.78 g (0.0119 mol) of the sodium salt of crude depolymerized heparin in Celite (10 g) obtained above and 79 ml of water are placed in a 50 ml Erlenmeyer flask. The suspension is filtered through a No. 3 sintered glass and rinsed with twice 15 ml of water.

35    The filtrate obtained is placed in a 150 ml Erlenmeyer flask. 1.3 ml (0.012 mol) of 30% caustic soda are added
 

with magnetic stirring, at a temperature in the region of 4°C. After addition, the mixture is stirred for 2 hours. The solution is neutralized by adding 1N HCl


and 10 g of sodium chloride are added. 60 ml of methanol are added to the reaction medium. After stirring for 15 minutes, 190 ml of methanol are added, follG~ed by stirring for 1 hour. Stirring is then

5    stopped and the suspension is left to sediment for ~ hour at a temperature in the region of 4°C. The supernatant is then separated out and discarded (180 ml). 180 ml of methanol are added to the sedimented precipitate and the mixture is stirred for

10    5 minutes. The precipitate is left to resediment for about 20 minutes. The supernatant is separated out and discarded (180 ml). 180 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 5 minutes. The precipitate in suspension is then

15    filtered   through   a   No.   3   sintered   glass.    The    cake

obtained  is   then  washed  with  twice   50  ml   of  methanol. The  wet   solid  is  drained  and  then  dried  under   reduced pressure    (6  kPa)    at   a   temperature   in   the   region   of 50°C.    After    drying    for    18  hours,    5.87  g    of    crude 20    depolyrnerized   heparin   (sodium   salt)   are   obtained.   The

yield  obtained  is  82%.

Purification:

5. 87   g   of   crude   depolymerized   heparin   obtained   above 25    and   59  ml   of   distilled   water   are   placed   in   a   100  ml Erlenmeyer   flask.   The  mixture   is   brought   to   40°C   with magnetic   stirring.   The   pH   is   brought   to   9. 7  ±  0.1   by addition  of  1N  sodium  hydroxide.  The  reaction  medium  is filtered   through   a   0.45  pm   membrane   and   0.34  ml   of 30    aqueous  30%  hydrogen  peroxide  solution  is  added.   After stirring   for   about   2  hours   at   a   temperature   in   the region   of   20°C,   the   mixture   is   neutralized   by   adding 1N  HCl,   followed  by  addition  of  7  g  of  sodium  chloride.


The    solution    is    then    filtered    through    a    0.45  pm

35    membrane and 49 ml of methanol are then poured in, at a temperature in the region of 10°C. The solution is then
 

brought to 20 °C and stirred for 15 minutes. 12 6 ml of methanol are then added, followed by stirring for 1 hour. Stirring is then stopped and the suspension is
 

left to sediment for 20 minutes. The supernatant is then separated out and discarded (105 ml). 105 ml of methanol are added to the sedimented precipitate and the- =rrrixture is stirred for 5 minutes. The precipitate

5    is left to resediment for about 25 minutes. The supernatant is separated out and discarded ( 110 ml) . The precipitate in suspension is then filtered through a No. 3 sintered glass. The white cake obtained is then washed with 50 ml of methanol. The wet solid is drained

10    and then dried under reduced pressure (6 kPa) at a temperature in the region of 55°C. After drying for

18 hours, 5.21 g of pure depolymerized heparin (sodium salt) are obtained. The yield obtained is 89%.

The    characteristics   of   the   depolymerized   heparin   thus

15    obtained  are  as  follows:

mean molecular weight: 3550 daltons anti-Xa activity: 99 IU/mg

anti-IIa  activity:  13.4  IU/mg

anti-Xa  activity/anti-Ira  activity  ratio:  7.4

20

EXAMPLE  8

Depolymerization  and  conversion  to  the  sodium  salt   (0.5

equivalent  of  base) :

140   ml   of   dichloromethane   are   placed   in   a   Erlenmeyer 25    flask   A.   20   g   (0.019  mol)   of   benzyl   ester   of   heparin (degree    of    esterification:    75%,    benzethonium    salt) obtained  as   described   in   example  A,   are   added   slowly, with  stirring.  After  total  dissolution  at  a  temperature in  the   region  of   30 °C  and  cooling  to  a   temperature  in

30    the region of 20°C, 1.75 ml (0.006 mol) of 2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,2,3-diazaphosphorine are added. The mixture is stirred at a temperature in the region of 20°C for 24 hours. During

this   time,    a   solution   of   60  g   of   anhydrous   sodium 35    acetate    in    600  ml    of    methanol    is    prepared    in    a Erlenmeyer    flask  B.     The    reaction    mixture    in    the Erlenmeyer  flask  A  is  poured  into  the  methanolic  sodium acetate  solution  at  a  temperature  in  the  region  of  4°C, with  magnetic  stirring.   After  stirring  for  1  hour,   the
 

suspension is left to settle for 2 hours. The clear part of the supernatant is separated out and discarded (420 ml). 420 ml of methanol are added to the sed-imented precipitate and the mixture is stirred for

5    30 minutes. The precipitate is left to resediment for about 18 hours. The clear part of the supernatant is

separated out and discarded (400 ml). 400 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 30 minutes. The supernatant

10 is separated out and discarded (400 ml). The precipitate in suspension is then filtered through a No. 3 sintered glass. The cake obtained is then washed with twice 100 ml of methanol. The pale yellow wet solid is drained and then dried under reduced pressure

15 ( 6 kPa) at a temperature in the region of 60 °C. After drying, 5.7 g of crude depolymerized heparin are obtained, as the sodium salt. The yield obtained is 73%.


20    S~ponification:

5.7 g (0.0086 mol) of the sodium salt of crude depolymerized heparin obtained above and 53 ml of water are placed in a 100 ml Erlenmeyer flask. 0.93 ml (0.009 mol) of 30% caustic soda is added with magnetic

25    stirring, at a temperature in the region of 4°C. After addition, the mixture is stirred for 2 hours. The solution is neutralized by adding 1N HCl and 6 g of

sodium chloride are added. 42 ml of methanol are added to the reaction medium. After stirring for 15 minutes,

30    108 ml of methanol are added, followed by stirring for 30 minutes. Stirring is then stopped and the suspension is left to sediment for 30 minutes at a temperature in the region of 4 °C. The supernatant is then separated out and discarded (180 ml). 180 ml of methanol are

35    added to the sedimented precipitate and the mixture is stirred for 5 minutes. The precipitat~ is left to
 

resediment for about 30 minutes. The supernatant is separated out and discarded (170 ml). 170 ml of methanol are added to the sedimented precipitate and


the mixture is stirred for 30 minutes. The precipitate in suspension is then filtered through a No. 3 sintered glass. The cake obtained is then washed with twice 30 m:l- of methanol. The wet solid is drained and then

5    dried under reduced pressure at a temperature in the region of 60°C. After drying, 3. 5 g of crude depolymerized heparin (sodium salt) are obtained. The yield obtained is 67.4%.


10    Puri.fi.cati.on:

3.5 g of crude depolymerized heparin obtained above and 35 ml of distilled water are placed in a 100 ml Erlenmeyer flask. The mixture is brought to 40°C with magnetic stirring. The pH is brought to 9. 6 ± 0. 1 by

15    addition of 1N sodium hydroxide. The reaction medium is filtered through a 0.45 pm membrane and 0.18 ml of aqueous 30% hydrogen peroxide solution is added. After stirring for about 2 hours at a temperature in the

region    of   20°C,   the   mixture   is   neutralized   by   adding

20    1N •HCl, followed by addition of 3. 6 g of sodium chloride. The solution is then filtered through a 0.45 pm membrane and 27 ml of methanol are then poured in, at a temperature in the region of 10°C. The

solution    is   then   brought   to   20°C   and   stirred   for

25    15 minutes. 65 ml of methanol are then added, followed by stirring for 30 minutes. Stirring is then stopped and the suspension is left to sediment for 30 minutes. The supernatant is then separated out and discarded

(80  ml).   80  ml  of  methanol  are  added  to  the  sedimented

30    precipitate and the mixture is stirred for 30 minutes. The precipitate is left to resediment for about

30 minutes. The supernatant is separated out and discarded (70 ml). 70 ml of methanol are added to the sedimented precipitate and the mixture is stirred for

35    30 minutes. The precipitate in suspension is then filtered through a No. 3 sintered glass. The white cake obtained is then washed with twice 30 ml of methanol. The wet solid is drained and then dried under reduced pressure at a temperature in the region of 60°C. After
drying, 2.8 g of pure depolymerized heparin (sodium salt) are obtained. The yield obtained is 80%.

The characteristics of the depolymerized heparin thus obtained are as follows:

5    mean molecular weight: 2900 daltons anti-Xa activity: 146.1 IU/mg

anti-Ira  activity:  5.1  IU/mg

anti-Xa  activity/anti-Ira  activity  ratio:   28.6

10    EXAMPLE  9

Depo1y.merization and conversion to the sodium sa1t (0.6 equiva1ent of base) :

280  ml  of  dichloromethane  are  placed  in  a  three-necked flask   A.   40   g   ( 0. 024  mol)   of   benzyl   ester   of   heparin 15     (degree    of    esterification:    75%,    benzethonium    salt) obtained  as   described   in  example  A,   are   added   slowly, with  stirring.  After  total  dissolution  at  a  temperature in  the  region  of   30 °C  and  cooling  to  a   temperature   in the    region   of    20°C,    4.2  ml    (0.014  mol)    of    2-tert-

20    butylimino-2-diethylamino-1,3-dimethylperhydro-1,2,3-diazaphosphorine are added. The mixture is stirred at a temperature in the region of 20°C for 24 hours. During this time, a solution of 60 g of anhydrous sodium

acetate    in    600  ml    of    methanol    is    prepared    in    a

25    Erlenmeyer flask B. Half of the reaction mixture in the three-necked flask A is poured into the methanolic sodium acetate solution at a temperature in the region of 4 °C, with magnetic stirring. After stirring for 1 hour, the suspension is left to settle. The clear part

30    of the supernatant is separated out and discarded (310 ml). 310 ml of methanol are added to the

sedimented precipitate and the mixture is stirred for 1 hour. The precipitate is left to resediment for about 18 hours. The clear part of the supernatant is

35    separated out and discarded (400 ml). 400 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 1 hour. The supernatant is separated out and discarded (300 ml). The precipitate in suspension is then filtered through a No. 3 sintered


glass. The cake obtained is then washed with twice 100 ml of methanol. The pale yellow wet solid is drained and then dried under reduced pressure at a temperature in the region of 60°C. After drying, 6 g of

5    crude depolymerized heparin are obtained, as the sodium salt. The yield obtained is 77%.

Saponification:

6    g    (0.0091  mol)    of    the    sodi urn    salt    of    crude

10 depolymerized heparin obtained above and 56 ml of water are placed in a 250 ml Erlenmeyer flask. 1 ml (0.010 mol) of 30% caustic soda is added with magnetic stirring, at a temperature in the region of 4°C. After addition, the mixture is stirred for 2 hours. The

15    solution is neutralized by adding 1N HCl and 6.4 g of sodium chloride are added. 45 ml of methanol are added to the reaction medium. After stirring for 15 minutes,

115 ml of methanol are added, followed by stirring for 30 minutes. Stirring is then stopped and the suspension

20    is left to sediment for 30 minutes at a temperature in the region of 4 °C. The supernatant is then separated out and discarded (170 ml). 170 ml of methanol are

added to the sedimented precipitate and the mixture is stirred for 30 minutes. The precipitate is left to

25    resediment for about 30 minutes. The supernatant is separated out and discarded (140 ml). 140 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 30 minutes. The precipitate

in  suspension  is  then  filtered  through  a  No.   3  sintered

30    glass. The cake obtained is then washed with twice 30 ml of methanol. The wet solid is drained and then

dried under reduced pressure at a temperature in the region of 60 oc. After drying, 3. 6 g of crude

depolymerized    heparin   (sodium   salt)   are   obtained.   The

35    yield  obtained  is  65.3%.

Purification:


Erlenmeyer flask. The mixture is brought to 40°C with magnetic stirring. The pH is brought to 9. 6 ± 0. 1 by addition of lN sodium hydroxide. The reaction medium is filtered through a 0.45 pm membrane and 0.18 ml of

5    aqueous 30% hydrogen peroxide solution is added. After stirring for about 2 hours at a temperature in the region of 20°C, the mixture is neutralized by adding lN HCl, followed by addition of 3.5 g of sodium

chloride.    The    soltition   is   then    filtered    through   a

10    0.45 pm membrane and 25 ml of methanol are then poured in, at a temperature in the region of 10°C. The solution is then brought to 20°C and stirred for 15 minutes. 63 ml of methanol are then added, followed by stirring for 30 minutes. Stirring is then stopped

15    and the suspension is left to sediment for 30 minutes. The supernatant is then separated out and discarded (70 ml). 70 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 30 minutes.

The    precipitate    is    left    to    resediment    for    a    few

20    minutes. The suspension is then filtered through a No. 3 sintered glass. The white cake obtained is then washed with twice 30 ml of methanol. The wet solid is

drained   and   then   dried   under   reduced   pressure   at   a temperature  in  the  region  of   60°C.   After  drying,   2.5  g 25    of    pure    depolymerized    heparin     (sodium    salt)     are

obtained.  The  yield  obtained  is  71.4%.

The characteristics of the depolymerized heparin thus obtained are as follows:

mean  molecular  weight:  2600  daltons

30    anti-Xa activity: 150.5 IU/mg anti-IIa activity: 3.2 IU/mg

anti-Xa  activity/anti-Ira  activity  ratio:   47

EXAMPLE  10

35    Depolymerization and conversion to the sodium salt (0.8 equivalent of base) :

70 ml of dichloromethane are placed in a Erlenmeyer flask A. 10 g (0. 006 mol) of benzyl ester of heparin (degree of esterification: 75%, benzethonium salt)

obtained as described in example A, are added slowly, with stirring. After total dissolution at a temperature in the region of 30°C and cooling to a temperature in the- ~region of 20°C, 1.38 ml (0.004 mol) of 2-tert-

5    butylimino-2-diethylamino-1,3-dimethylperhydro-1,2,3-diazaphosphorine are added. The mixture is stirred at a temperature in the region of 20°C for 24 hours. During

this   time,    a   solution   of   30  g   of   anhydrous    sodium acetate    in    300  ml    of    methanol    is    prepared    in    a 10    Erlenmeyer    flask  B.     The    reaction    mixture    in    the Erlenmeyer   flask   A  is   poured  over   1  minute   15  seconds into    the    methanolic    sodium   acetate    solution    at    a temperature    in    the    region    of    4°C,    with    magnetic stirring.   After  stirring   for   5  minutes,   the  suspension


15    is left to settle for 1 hour. The clear part of the supernatant is separated out and discarded (190 ml). 190 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 5 minutes.

The    precipitate   is   left   to   resediment   for   about   30

20    minutes. The clear part of the supernatant is separated out and discarded (190 ml). 190 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 30 minutes. The supernatant is separated

out    and    discarded    ( 190    ml) .    The    precipitate    in

25    suspension is then filtered through a No. 3 sintered glass. The cake obtained is then washed with 150 ml of methanol. The pale yellow wet solid is drained and then

dried under reduced pressure ( 6 kPa) at a temperature in the region of 40°C. After drying for 18 hours,

30    3. 05 g of crude depolymerized heparin are obtained, as the sodium salt. The yield obtained is 80%.

Saponification:

3.05    g    (0.0048  mol)    of    the    sodium   salt    of    crude

35    depolymerized heparin obtained above and 21 ml of water are placed in a 100 ml Erlenmeyer flask. The solution is filtered through a No. 3 sintered glass and rinsed with twice 6 ml of water. The filtrate obtained is placed in a 150 ml Erlenmeyer flask. 0.6 m1 (0.006 mol)


of 30% caustic soda is added with magnetic stirring, at a temperature in the region of 4°C. After addition, the mixture is stirred for 2 hours. The solution is neutralized by adding 1N HCl and 4 g of sodium chloride

5    are added. 28 ml of methanol are added to the reaction medium. After stirring for 15 minutes, 72 ml of methanol are added, followed by stirring for 1 hour.

Stirring is then stopped and the suspension is left to sediment for 30 minutes at a temperature in the region

10    of 4°C. The supernatant is then separated out and discarded (80 ml). 80 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 5 minutes. The precipitate is left to resediment for about 30 minutes. The supernatant is separated out and

15    discarded (80 ml). 80 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 30 minutes. The precipitate in suspension is then filtered through a No. 3 sintered glass. The cake obtained is then washed with 50 ml of methanol. The wet

20    solid is drained and then dried under reduced pressure {6 kPa) at a temperature in the region of 40°C. After

drying, 1. 6 g of crude depolymerized heparin (sodium salt) are obtained. The yield obtained is 57%.

25    Purification:

1.6 g of crude depolymerized heparin obtained above and 16 ml of distilled water are placed in a 50 ml Erlenmeyer flask. The mixture is brought to 40°C with

magnetic    stirring.   The   pH   is   brought   to   9.6  ±  0.1   by

30    addition of 1N sodium hydroxide. The reaction medium is filtered through a 0.45 pm membrane and 0.08 ml of aqueous 30% hydrogen peroxide solution is added. After stirring for about 2 hours at a temperature in the

region    of   20°C,   the   mixture   is   neutralized   by   adding

35    lN HCl, followed by addition of 2 g of sodium chloride. The solution is then filtered through a 0.45 pm membrane and 14 ml of methanol are then poured in, at a temperature in the region of 10°C. The solution is then brought to 20 °C and stirred for 15 minutes. 36 ml of
 
methanol are then added, followed by stirring for 1 hour. Stirring is then stopped and the suspension is left to sediment for 30 minutes. The supernatant is then~-separated out and discarded (50 ml). 50 ml of

5    methanol are added to the sedimented precipitate and the mixture is stirred for 5 minutes. The precipitate is left to resediment for about 30 minutes. The supernatant is separated out and discarded (50 ml). The

precipitate    in   suspension   is   then   filtered   through   a

10    No. 3 sintered glass. The wet solid is drained and then dried under reduced pressure ( 6 kPa) at a temperature in the region of 40°C, After drying, 1.25 g of pure depolymerized heparin (sodium salt) are obtained. The yield obtained is 78%.

15    The characteristics of the depolyrnerized heparin thus obtained are as follows:

mean molecular weight: 2400 daltons anti-Xa activity: 154.3 IU/mg

anti-IIa  activity:  1.6  IU/mg

20    anti-Xa  activity/anti-Ira  activity  ratio:   96.4

EXAMPLE  11

Depolymerization  and  conversion  to  the  sodium  salt:

140    ml   of   dichloromethane   are   placed   in   a   Erlenmeyer

25    flask A. 20 g (0.019 mol) of benzyl ester of heparin (degree of esterification: 75%, benzethonium salt)

obtained as described in example A, are added slowly, with stirring. After total dissolution at a temperature in the region of 40°C and cooling to a temperature in

30    the region of 20°C, 3.5 ml (0.011 mol) of tert-butyliminotri(pyrrolidino)phosphorane are added. The

mixture is stirred at a temperature in the region of 20°C for 24 hours. During this time, a solution of 60 g of anhydrous sodium acetate in 600 ml of methanol is

35 prepared in a Erlenmeyer flask B. After total dissolution, 10 g of Hyflo supercel Celite are added to the solution. The reaction mixture in the Erlenmeyer flask A is poured over 1 minute 30 seconds into the methanolic sodium acetate solution at a temperature in
 




-    44  -

the region of 4°C, with magnetic stirring. After stirring for 5 minutes, the suspension is left to settle for 30 minutes. The clear part of the supernatant is separated out and discarded (400 ml).

5 400 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 5 minutes. The precipitate is left to resediment for about 1 hour 20 minutes. The supernatant is separated out and discarded (250 ml). 250 ml of methanol are added to the

10    sedimented precipitate and the mixture is stirred for 5 minutes. The precipitate in suspension is then filtered through a No. 3 sintered glass. The cake obtained is then washed with 200 ml of methanol. The

pale    yellow  wet   solid  is   drained  and  then  dried  under

15    reduced pressure (6 kPa) at a temperature in the region of 40°C. After drying, 5.39 g of depolymerized heparin (benzyl ester, sodium salt) are obtained. The yield obtained is 69%.


20    Saponification:

5 g (0.0076 mol) of the depolymerized heparin (benzyl ester, sodium salt) obtained above and 35 ml of water are placed in a 50 ml Erlenmeyer flask. The solution is filtered through a No. 3 sintered glass and rinsed with

25 twice 10 ml of water. The filtrate obtained is placed in a 250 ml Erlenmeyer flask. 1 ml (0.01 mol) of 30% caustic soda is added with magnetic stirring, at a temperature in the region of 4 °C. After addition, the mixture is stirred for 2 hours. The solution is

30 neutralized by adding 1N HCl and 6 g of sodium chloride are added. 42 ml of methanol are added to the reaction medium. After stirring for 15 minutes, 104 ml of methanol are added, followed by stirring for 15 minutes. Stirring is then stopped and the suspension is

35    left to sediment for 1 hour at a temperature in the region of 4 °C. The supernatant is then separated out and discarded (140 ml). 140 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 5 minutes. The precipitate is left to resediment


for about 45 minutes. The supernatant is separated out and discarded (160 m1). 160 ml of methanol are added to the sedimented precipitate and the mixture is stirred for- -=-5- minutes. The precipitate in suspension is then

5    filtered through a No. 3 sintered glass. The cake obtained is then washed with 100 ml of methanol. The wet solid is drained and then dried under reduced pressure (6 kPa) at a temperature in the region of 40°C. After drying for 48 hours, 2.7 g of crude


10    depolymerized heparin (sodium salt) are obtained. The yield obtained is 59%.


Pur:i.f:i.cat:i.on:

2.6  g  of  crude  depolymerized  heparin  obtained  above  and

15    25 ml of distilled water are placed in a 50 ml Erlenmeyer flask. The mixture is brought to 40°C with magnetic stirring. The pH is brought to 9. 7 ± 0. 1 by addition of 1N sodium hydroxide. The reaction medium is

filtered   through   a    0.45  pm   membrane   and   0.15  ml   of 20     aqueous   30%   hydrogen  peroxide   solution  is   added.   After stirring   for   about   2  hours   at   a   temperature   in   the region   of   20°C,   the   mixture   is   neutralized   by   adding

lN  HCl,   followed  by  addition  of  3  g  of  sodium  chloride. The    solution    is    then    filtered    through    a    0.45  pm 25    membrane  and  21  ml  of  methanol  are  then  poured  in,   at  a temperature  in  the  region  of  10°C.   The  solution  is  then brought   to   20 °C   and   stirred   for   15  minutes.   54  ml   of methanol    are    then    added,    followed    by    stirring    for 1  hour.   Stirring  is  then  stopped  and  the   suspension  is


30 left to sediment for 20 minutes. The supernatant is then separated out and discarded (50 ml). 50 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 5 minutes. The precipitate is left to resediment for about 20 minutes. The


35 supernatant is separated out and discarded (50 ml) . The precipitate in suspension is then filtered through a No. 3 sintered glass. The white cake obtained is then washed with 50 ml of methanol. The wet solid is drained and then dried under reduced pressure (6 kPa) at a
 

temperature in the region of 4 0 °C. After drying for 18 hours, 2.35 g of pure depolymerized heparin (sodium salt) are obtained. The yield obtained is 90%.

The- •characteristics   of   the   depolymerized   heparin   thus

5    obtained  are  as  follows:

mean molecular weight: 2400 daltons anti-Xa activity: 167.5 IU/mg

anti-IIa  activity:  1.1  IU/mg

anti-Xa  activity/anti-Ira  activity  ratio:   152

10

EXAMPLE  12

Depolymerization    and   conversion   to   the   sodium   salt

(0.05%  water):

14 0  ml   of   dichloromethane   are   placed   in   a   Erlenmeyer

15    flask. 20 g (0.019 mol) of benzyl ester of heparin (degree of esterification: 75%, benzethonium salt)

obtained as described in example A, are added slowly, with stirring. 20 g of 4 A molecular sieve are added to the reaction medium and the water content is brought to

20    0.05%, while stirring slowly for 48 h. The supernatant is transferred under an inert atmosphere to a Erlenmeyer flask A. After total dissolution, 3.5 ml (0.012 mol) of 2-tert-butylimino-2-diethylamino-1,3-

dimethylperhydro-1,2,3-diazaphosphorine   are   added.   The

25    mixture is stirred at a temperature in the region of 20°C for 24 hours. During this time, a solution of 60 g of anhydrous sodium acetate in 600 ml of methanol is prepared in a Erlenmeyer flask B. After total dissolution, 10 g of Hyflo supercel Celite are added to

30    the solution. The reaction mixture in the Erlenmeyer flask A is poured over about 2 minutes into the methanolic sodium acetate solution at a temperature in the region of 4°C, with magnetic stirring. After stirring for 15 minutes, the suspension is left to

35    settle for 1 hour. The clear part of the supernatant is separated out and discarded ( 420 ml) . 420 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 15 minutes. The precipitate is left to resediment for about 1 hour. The supernatant
 

is separated out and discarded (450 ml). 450 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 15 minutes. The suspension is -t-h-en filtered through a No. 3 sintered glass. The

5    cake obtained is then washed with 200 ml of methanol. The pale yellow wet solid is drained and then dried under reduced pressure (6 kPa) at a temperature in the region of 50°C. After drying for 16 hours, 5.36 g of crude depolymerized heparin are obtained, as the sodium

10    salt  in  Celite  (10  g).  The  yield  obtained  is  68.6%.

Saponification:

5.36    g (0.00817  mol) of   the    sodium   salt   of   crude

depolymerized    heparin   in   Celi te   ( 10  g)   obtained   above

15    and 50 ml of water are placed in a 50 ml Erlenmeyer flask. The suspension is filtered through a No. 3 sintered glass and rinsed with 4 times 15 ml of water. The filtrate obtained is placed in a 150 ml Erlenmeyer flask. 1.01 ml (0.0122 mol) of 35% caustic soda are

20    added with magnetic stirring, at a temperature in the region of 4 oc. After addition, the mixture is stirred for 3 hours. The solution is neutralized by adding 1N HCl and 11 g of sodium chloride are added. 77 ml of

methanol    are    added    to    the    reaction    medium.    After

25    stirring for 15 minutes, 200 ml of methanol are added, followed by stirring for 1 hour. Stirring is then stopped and the suspension is left to sediment for 1 hour at a temperature in the region of 4°C. The

supernatant    is    then    separated    out    and    discarded 30     (240  ml).     240  ml    of    methanol    are    added    to    the sedimented  precipitate   and   the   mixture   is   stirred   for 10  minutes.   The   precipitate   is   left   to   resediment   for about  30  minutes.   The   supernatant  is  separated  out  and discarded   (225  ml) .  225  ml  of  methanol  are  added  to  the

36    sedimented precipitate and the mixture is stirred for 10 minutes. The suspension is then filtered through a No. 3 sintered glass. The cake obtained is then washed with twice 50 ml of methanol. The wet solid is drained and then dried under reduced pressure (6 kPa) at a

temperature in the region of 40°C. After drying for 18 hours, 2.65 g of crude depolymerized heparin (sodium salt) are obtained. The yield obtained is 53.7%.


5    Purification:

2. 65 g of crude depolymerized heparin obtained above and 2 6. 5 ml of distilled water are placed in a 100 ml Erlenmeyer flask. The mixture is brought to 40°C with magnetic stirring. The pH is brought to 9. 7 ± 0. 1 by

10    addition of lN sodium hydroxide. The reaction medium is filtered through a 0.45 pm membrane and 0.25 ml of aqueous 30% hydrogen peroxide solution is added. After stirring for about 2 hours at a temperature in the region of 20°C, the mixture is neutralized by adding

15    lN HCl, followed by addition of 3 g of sodium chloride. The solution is then filtered through a 0.45 pm

membrane  and  21  ml  of  methanol  are  then  poured  in,   at  a temperature  in  the  region  of  l0°C.   The  solution  is  then brought   to   20 °C   and   stirred   for   15  minutes.   54  ml   of 20    methanol    are   then   added,    followed   by   stirring   for 1  hour.   Stirring  is  then  stopped  and  the  suspension  is left   to   sediment   for   4 5  minutes.   The   supernatant   is then   separated   out   and   discarded    (46  ml).    46  ml   of

methanol   are   added   to   the   sedimented   precipitate   and 25    the   mixture   is   stirred   for   5  minutes.   The   precipitate is    left    to    resediment    for    about    30  minutes.    The supernatant   is   separated   out   and   discarded    (50  ml).

50  ml   of   methanol   are   added   and   the   precipitate   in suspension   is   then   filtered   through   a   No.   4   sintered 30    glass.   The   white   cake   obtained   is   then   washed   with   2 portions  of  10  ml  of  methanol.  The  wet  solid  is  drained and   then   dried   under   reduced   pressure    (6  kPa)    at   a temperature   in   the   region   of   50°C.   After   drying   for 18  hours,   2.363  g  of  pure  depolymerized  heparin   (sodium

35    salt)   are  obtained.  The  yield  obtained  is  89.1%.

The characteristics of the depolymerized heparin thus obtained are as follows:

mean molecular weight: 2500 Daltons anti-Xa activity: 192 IU/mg

anti-IIa  activity:   1.3  IU/mg

anti-Xa  activity/anti-Ira  activity  ratio:   148


CLAIMS

1)    -A:- -mixture   of   sulfated   oligosaccharides   having   the

general    structure•of  the  constituent  polysaccharides  of

5    heparin  and  having  the  following  characteristics:

- they have a mean molecular weight of 1500 to 3000 daltons, an anti-Xa activity of 120 to 200 IU/mg, an anti-IIa activity of less than 10 IU/mg and an anti-Xa activity/anti-Ira activity ratio of greater

10    than  30,

the constituent oligosaccharides of the mixtures contain 2 to 26 saccharide units,

have a 4,5-unsaturated uronic acid 2-0-sulfate unit at one of their ends,

15    contain  the  hexasaccharide  ~IIa-IIs-Is of  formula:

and are in the form of an alkali or alkaline-earth 2 0 met a 1 sa 1 t .


2)    The  mixture   of   oligosaccharides   as   claimed   in  claim

1, characterized in that the alkali or alkaline-earth metal salt is chosen from sodium, potassium, calcium
25    and  magnesium  salts.

3)    The  mixture   of   oligosaccharides   as   claimed   in  claim

1    or    2,    characterized    in    that    it    contains    a
 

hexasaccharide fraction representing from 15 to 25% of the mixture of oligosaccharides.

4)    The  mixture  of  oligosaccharides  as   claimed  in  claim 5     3,   characterized  in   that   it   contains   from   8  to   15%   of the   hexasaccharide   ~IIa-IIs-Is  in   the   hexasaccharide

fraction  of  the  mixture  of  oligosaccharides.

5)    . The mixture of oligosaccharides as claimed in any 10 one of claims 1 to 4, characterized in that it exhibits
an  anti-Xa  activity  of  between  150  IU/mg  and  200  IU/mg.

6)    The mixture of oligosaccharides as claimed in any one of claims 1 to 5, characterized in that it has an
15    anti-IIa activity of less than 5 IU/mg and in particular of 0.5 to 3.5 IU/mg.


7)    The   mixture   of   oligosaccharides   as   claimed   in   any

one  of  claims  1  to  6,   characterized  in  that  it  exhibits

20    an anti-Xa activity/anti-Ira activity ratio greater than 50 and in particular greater than 100.

8)    The   mixture   of   oligosaccharides   as   claimed   in   any

one    of   claims   1   to   7,   characterized   in   that   it   has   a

25    mean molecular weight of between 2000 and 3000 Daltons and in particular from 2400 to 2650 Da.

9)    The   mixture   of   oligosaccharides   as   claimed   in   any

one  of  claims  1  to  8,   characterized  in  that  it  exhibits

30    an anti-Xa activity of between 150 and 200 IU/mg, an anti-IIa activity of between 0.5 and 3.5 IU/mg, and a mean molecular weight of between 2400 and 2650 Da.


10)    A method for preparing mixtures of oligo-

35    saccharides as claimed in any one of claims 1 to 9, during which the quaternary ammonium salt of the benzyl ester of heparin is depolymerized in an organic medium in the presence of a strong organic base with a pKa greater than 20, characterized in that the organic base


used consists of the family of phosphazenes, in particular in a dichloromethane solution, contains a percentage of water of less than 0.6%.


5    11) The method as claimed in claim 10 for preparing mixtures of oligosaccharides as claimed in claim 1, characterized in that the water content is less than 0.3% and preferably less than 0.2%.


10    12) The method of preparation as claimed in claim 10 or 11, characterized in that the quaternary ammonium salt of the benzyl ester of heparin is the benzethonium, cetylpyridinium or cetyltrimethylarnmonium salt.


15    13) The method of preparation as claimed in claim 10 or 11, characterized in that the bases of the family of phosphazenes are those of formula:

R3
I
R2-N\    IR4
R1-N=P-N-R5
\
N-R6
I
R7

20.-

in which the radicals R1 to R7 are identical or different and represent linear or branched alkyl radicals containing from 1 to 6 carbon atoms.


25    14) The method of preparation as claimed in claim 10 or 11, characterized in that the strong base/ester mol ratio is between 0.2 and 5, and preferably between 0.6 and 2.



30    15) A method for preparing oligosaccharides as claimed in any one of claims 1 to 9 from heparins, in which the following operations are carried out:

a)    transalification of sodium heparin by the action of benzethonium chloride,
b)    esterification of benzethonium heparinate ob-::ained by the action of benzyl chloride,

c)    transalification   of   the   benzyl   ester   obtained   and

-    ~taining of  the  quaternary  ammonium  salt,

5    d) depolymerization of the quaternary ammonium salt of the benzyl ester of heparin by the method as defined in claim 10 or 11,

e)    conversion   of   the   quaternary   ammonium   salt   to   a

sodium  salt,

10    f) optionally saponification of the heparin by the action of a base such as sodium hydroxide,

g)    optionally purification in particular by the action of an oxidizing agent such as hydrogen peroxide.

15    16)    The  method  as  claimed  in  claim  15,    characterized    in
    that   the   reaction   of   step   a)    is   carried   out   by   the
    action   of   benzethonium   chloride   in   excess,   on   sodium
    heparin,   at  a   temperature  in  the  region  of  15  to  25°C,
    with   a   salt/   sodium   heparin   molar   ratio   of   between   3
20    and    4.       
    17)   The  method  as  claimed  in  claim  15,   characterized  in
    that  the  esterification  of  step  b)   is  carried  out  in  a
    chlorinatedorganicsolventsuchas    chloroform    or

25    methylene chloride, at a temperature of between 25 and 45°C, preferably between 30 and 40°C, and the ester in the form of a sodium salt is then recovered by precipitation by means of sodium acetate at 10% by weight in an alcohol such as methanol in a proportion

30    of 1 to 1.2 volumes of alcohol per volume of reaction medium.


18)    The method as claimed in claim 15 or 17,

characterized    in   that   the   degree   of   esterification   of

35    the quaternary ammonium salt of the benzyl ester of heparin is between 50 and 100%, and preferably between 70 and 90%.

19)    The   method   as   claimed   in   claim   15, 17    o~  18,

characterized in that 0. 5 to 1. 5 parts by weight of benzyl chloride per 1 part by weight of benzethonium salt'- -of heparin are used with a reaction time which

5    will  be  between  10  and  35  h.

20)    The  method  as  claimed  in  claim  15,   characterized  in

that  the  transalification  of  step  c)   is  carried  out  by means  of  a  quaternary  ammonium  chloride,   and  preferably 10    by    means    of    benzethonium    chloride,    cetylpyridinium chloride    or    cetyltrimethylammonium    chloride,    in    an

aqueous  medium  at  a  temperature  between  10  and  25°C.

21)    The  method  as  claimed  in  claim  20,   characterized  in

15    that the quaternary ammonium chloride/ sodium salt of the benzyl ester of heparin mol ratio is between 2 and

3.

22)   The  method  as  claimed  in  claim  15,   characterized  in 20    that  the  conversion  to  a   sodium  salt  of  the  quaternary ammonium   salt   of   the   benzyl   ester   of   depolymerized heparin    (step    e)    is    carried    out    by    treating    the reaction   medium   with   an   alcoholic   solution   of   sodium acetate,   and  preferably  with   a   10%   solution   of   sodium 25    acetate   in   methanol   (weight/volume),   at   a   temperature

between  15  and  25°C.

23) The method as claimed in claim 15, characterized in that the saponification (step f) is carried out by

30    means of an alkali metal hydroxide such as sodium hydroxide, potassium hydroxide or lithium hydroxide, in an aqueous medium, at a temperature between 0 and 20°C, and preferably 0 and l0°C.


35    24) The method as claimed in claim 23, characterized in that l to 5 molar equivalents of alkali metal hydroxide and preferably l to 2 molar equivalents of sodium hydroxide are used.


25)    The  method  as  claimed  in  claim  15,   characte~ized i~

that the purification (step g) is carried out by means of hydrogen peroxide, in an aqueous medium, a~ a temperature of 10 to 50°C and preferably between 20 and

5    40°C.

26) As a medicament, the oligosaccha~ides as claimed in any one of claims 1 to 9.


10    27) As a medicament having an antithrombotic activity, the oligosaccharides as claimed in any one of claims 1 to 9.


28)    The  medicaments   as   claimed   in   claim  26   or   27,   for

15    the prevention or treatment of venous and arterial thromboses, deep vein thrombosis, pulmonary embolism, unstable angina, myocardial infarction, cardiac ischemia, occlusive diseases of the peripheral arteries and atrial fibrillation, the proliferation of smooth

20    muscle cells, atheriosclerosis and arteriosclerosis, cancer by modulating angiogenesis and growth factors, and diabetic disorders such as diabetic retinopathies and nephropathies.


25    29) A pharmaceutical composition containing at least one medicament as defined in claim 2 6 and one or more pharmaceutically inert excipients or vehicles or additives.



30    30) The pharmaceutical composition as claimed in claim 29, characterized in that it consists of solutions for injection by the subcutaneous or intravenous route.


31)    The   pharmaceutical   composition  as   claimed  in   claim

35    29, characterized in that it consists of a formulation for inhalation intended for the pulmonary route.
 

32)    The pharmaceutical composition as claimed i.n c::..a.:.r.. 29, characterized in that it consists of a formu::..atior. for administration intended for the oral route.

5    33) A mixture of polysaccharides as defined in any one of claims 1 to 9, which can be obtained by the method as defined in claim 15.

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