U.S. patent application number 13/105528 was filed with the patent office on 2011-09-01 for mixtures of polysaccharides derived from heparin, their preparation and pharmaceutical compositions containing them.
This patent application is currently assigned to AVENTIS PHARMA S.A.. Invention is credited to Vesna Biberovic, Luc Grondard, Pierre Mourier, Christian VISKOV.
Application Number | 20110212915 13/105528 |
Document ID | / |
Family ID | 32912652 |
Filed Date | 2011-09-01 |
United States Patent
Application |
20110212915 |
Kind Code |
A1 |
Biberovic; Vesna ; et
al. |
September 1, 2011 |
MIXTURES OF POLYSACCHARIDES DERIVED FROM HEPARIN, THEIR PREPARATION
AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM
Abstract
The present invention relates to mixtures of polysaccharides
derived from heparin having a mean molecular weight of 1500 to 3000
Daltons and an anti-Xa/anti-IIa ratio greater than 30, their method
of preparation and pharmaceutical compositions containing them.
Inventors: |
Biberovic; Vesna;
(Aulnay-Sous-Bois, FR) ; Grondard; Luc;
(Courcouronnes, FR) ; Mourier; Pierre; (Charenton
La Pont, FR) ; VISKOV; Christian; (Combs la Ville,
FR) |
Assignee: |
AVENTIS PHARMA S.A.
Antony Cedex
FR
|
Family ID: |
32912652 |
Appl. No.: |
13/105528 |
Filed: |
May 11, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11882607 |
Aug 2, 2007 |
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13105528 |
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10680934 |
Oct 8, 2003 |
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11882607 |
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60430095 |
Dec 2, 2002 |
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Current U.S.
Class: |
514/56 |
Current CPC
Class: |
A61P 7/02 20180101; A61P
9/00 20180101; C08B 37/0078 20130101; A61P 9/10 20180101 |
Class at
Publication: |
514/56 |
International
Class: |
A61K 31/727 20060101
A61K031/727; A61P 7/02 20060101 A61P007/02; A61P 9/10 20060101
A61P009/10 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 10, 2002 |
FR |
0212584 |
Claims
1-53. (canceled)
54. A mixture of sulfated oligosaccharides having the following
characteristics: a mean molecular weight of from about 1500 daltons
to about 3000 daltons, an anti-Xa activity of between 150.5 IU/mg
and 200 IU/mg, an anti-IIa activity of less than about 10 IU/mg,
and an anti-Xa activity/anti-IIa activity ratio of greater than
about 30; wherein the constituent oligosaccharides of the mixture:
contain 2 to 26 saccharide units, have a 4,5-unsaturated uronic
acid 2-0-sulfate unit at one of their ends, contain a
hexasaccharide fraction, wherein from about 8% to about 15% of the
hexasaccharide fraction of the mixture is the hexasaccharide
.DELTA.IIa-IIs-Is of formula: ##STR00006## and are in the form of
at least one salt chosen from alkali and alkaline earth metal
salts.
55. The mixture of claim 54, wherein the alkali or alkaline-earth
metal salt is selected from sodium, potassium, calcium and
magnesium salts.
56. The mixture of claim 54, wherein the mixture exhibits an
anti-IIa activity of less than 5 IU/mg.
57. The mixture of claim 54, wherein the mixture exhibits an
anti-IIa activity of from about 0.5 IU/mg to about 3.5 IU/mg.
58. The mixture of claim 54, wherein the mixture exhibits an
anti-Xa activity/anti-IIa activity ratio of greater than about
50.
59. The mixture of claim 54, wherein the mixture exhibits an
anti-Xa activity/anti-IIa activity ratio of greater than about
100.
60. The mixture of claim 54, wherein the mixture has a mean
molecular weight of between 2000 and 3000 Daltons.
61. The mixture of claim 54, wherein the mixture has a mean
molecular weight of between 2400 and 2650 Daltons.
62. The mixture of claim 54, wherein the mixture exhibits an
anti-Xa activity of between 150.5 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 Daltons.
63. The mixture of claim 54, wherein the hexasaccharide fraction
represents from about 15% to about 25% of the mixture of
oligosaccharides.
64. The mixture of claim 54, wherein the mixture is prepared by a
process comprising: depolymerising a quaternary ammonium salt of a
benzyl ester of heparin in a reaction medium comprising an organic
solvent and an organic base with a pKa greater than 20, wherein the
organic base is selected from phosphazenes, wherein the base:ester
molar ratio is from 0.6 to 2, controlling the water content of the
reaction medium to be 0.2% or less by measuring the water content
and determining whether adjustment is necessary; wherein the
depolymerization and/or water control steps are performed under
conditions such that a mixture of sulfated oligosaccharides having
an anti-Xa activity of between 150.5 IU/mg and 200 IU/mg is
produced.
65. The mixture of claim 64, wherein the organic base with a pKa
greater than 20 is
2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,2,3-diazaphosphor-
ine.
66. The mixture of claim 64, wherein the organic solvent is
dichloromethane.
67. The mixture of claim 64, wherein said quaternary ammonium salt
of the benzyl ester of heparin is the benzethonium, cetylpyridinium
or cetyltrimethylammonium salt.
68. A pharmaceutical composition comprising the mixture of claim 54
and an inert excipient.
69. A method for the treatment of at least one condition selected
from venous thrombosis, arterial thrombosis, deep vein thrombosis,
pulmonary embolism, unstable angina, myocardial infarction, and
cardiac ischemia, the method comprising administering to a patient
in need thereof a pharmaceutical composition comprising an
effective amount of the mixture of claim 54.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority from French
Patent Application No. 0212584, filed Oct. 10, 2002, as well as the
benefit of U.S. Provisional Application No. 60/430,095, filed Dec.
2, 2002.
FIELD OF THE INVENTION
[0002] The present invention relates to mixtures of polysaccharides
derived from heparin, their method of preparation and
pharmaceutical compositions containing them.
BACKGROUND OF THE INVENTION
[0003] Heparin is a mixture of sulfated mucopolysaccharides of
animal origin which is used in particular for its anticoagulant and
antithrombotic properties.
[0004] Heparin nevertheless has disadvantages which limit the
conditions for its use. In particular, its high anticoagulant
activity (anti-IIa activity) can cause hemorrhages.
[0005] Low-molecular-weight heparins obtained by basic
depolymerization of heparin esters have been proposed (EP40144);
however, these products still have a high anti-IIa (aIIa)
activity.
[0006] Very-low-molecular-weight heparins have also been described
in U.S. Pat. No. 6,384,021. However, the anti-Xa (aXa) 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.
[0007] In WO-0208295, very-low-molecular-weight heparins are
prepared by a method different from that of U.S. Pat. No. 6,384,021
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.
[0008] A constant need however exists in this class of medicament
to improve the anti-Xaactivities, in particular, in obtaining
activities greater than 150 IU/mg, as well as the anti-Xa/anti-IIa
ratio; and therefore to develop novel generations of heparin
derivatives.
SUMMARY OF THE INVENTION
[0009] The invention, therefore, provides products having improved
anti-Xa activity and anti-Xa/anti-IIa ratio by modifying the
methods described in the prior art, in particular, by controlling
the percentage of water during the depolymerization step. The
modified heparins thus obtained exhibit an excellent antithrombotic
activity and possess an aXa activity similar to that of heparin,
while reducing the risks of hemorrhage with a very low aIIa
activity. Likewise, the products of the invention exhibit half-life
periods which are markedly greater than that of heparin.
[0010] The invention therefore provides novel mixtures of
polysaccharides derived from heparin possessing a more selective
activity toward activated factor X (factor Xa) and toward activated
factor II (factor IIa) than heparin.
[0011] It is understood that the mixtures of polysaccharides having
a mean molecular weight in the range of about 1500 to 3000 Da can
be termed oligosaccharides.
DETAILED DESCRIPTION OF THE INVENTION
[0012] More particularly, the present invention provides mixtures
of sulfated oligosaccharides having the general structure of the
constituent polysaccharides of heparin and having the following
characteristics:
[0013] they have a mean molecular weight of about 1500 to 3000
daltons, an anti-Xa activity of about 120 to 200 IU/mg, an anti-IIa
activity of less than about 10 IU/mg and an anti-Xa
activity/anti-IIa activity ratio of greater than about 30,
[0014] the constituent oligosaccharides of the mixtures contain 2
to 26 saccharide units, have a 4,5-unsaturated uronic acid
2-O-sulfate unit at one of their ends, and contain the
hexasaccharide .DELTA.IIa-IIs-Is_of formula:
##STR00001##
[0015] The hexasaccharide .DELTA.IIa-IIs-Is contained in the
mixture of oligosaccharides described in the present invention is a
sequence which has a high affinity for ATIII and is characterized
by an aXa activity greater than 740 U/mg.
[0016] The mixture of oligosaccharides described in the present
invention is in the form of an alkali or alkaline-earth metal
salt.
[0017] As the alkali or alkaline-earth metal salt, the sodium,
potassium, calcium and magnesium salts are preferred.
[0018] The mean molecular weight is determined by high-pressure
liquid chromatography using two columns in series, for example
those marketed under the names TSK G3000 XL and TSK G2000 XL. The
detection is carried out 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 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).
[0019] The anti-Xa activity is measured by the amidolytic method on
a chromogenic substrate described by Teien et al., Thromb. Res.,
10, 399-410 (1977), with, as standard, the first international
standard for low-molecular-weight heparins. See Barrowcliffe, T.
W., A. D. Curtis, et al. (1985). "Standardization of Low Molecular
Weight Heparins: A Collaborative Study." Thrombosis and Haemostasis
54: 675-679; and Barrowcliffe, T. W. (1988). "An International
Standard for Low Molecular Weight Heparin." Thrombosis and
Haemostasis 60: 1-7.
[0020] The anti-IIa activity is measured by the technique described
by Anderson L. O. et al., Thromb. Res., 15, 531-541 (1979), with,
as standard, the first international standard for
low-molecular-weight heparins.
[0021] The hexasaccharide fraction preferably represents from about
15 to 25% of the mixture of oligosaccharides.
[0022] Preferably, the mixtures according to the invention contain
from about 8 to 15% of the hexasaccharide .DELTA.IIa-IIs-Is in the
hexasaccharide fraction of the mixture of oligosaccharides.
[0023] 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 hexasaccharide 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 ACA202.sup.R (Biosepra). The mixture is eluted
with a sodium hydrogen carbonate solution. Preferably, the sodium
hydrogen carbonate solution is a 0.1 mol/l to 1 mol/l solution.
Still more preferably, the separation is carried out at a
concentration of 1 mol/l. The determination is carried out by UV
spectrometry (254 nm). After fractionation, the hexasaccharide
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 filtration funnel. The hexasaccharide mixture
obtained may be analyzed by HPLC (High-Performance Liquid
Chromatography) in order to determine the content of hexasaccharide
.DELTA.IIa-IIs-Is. Hexasaccharide .DELTA.IIa-IIs-Is may be isolated
by preparative HPLC chromatography or by affinity chromatography on
an antithrombin III sepharose column according to the 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)).
[0024] Most preferably, the mixtures according to the invention
have an anti-Xa activity of between about 150 IU/mg and 200
IU/mg.
[0025] Preferably, the mixtures according to the invention have an
anti-IIa activity of less than about 5 IU/mg, and, most preferably,
of about 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.
[0026] Preferably, the mixtures exhibit an anti-Xa
activity/anti-IIa activity ratio greater than about 50 and most
preferably greater than about 100.
[0027] Preferably, the mixtures according to the invention have a
mean molecular weight of between about 2000 and 3000 Daltons, and
most preferably a mean molecular weight of between 2400 and 2650
Da.
[0028] The present invention therefore preferably provides the
mixtures as defined above, having an anti-Xa activity of between
about 150 and 200 IU/mg, an anti-IIa activity of between about 0.5
and 3.5 IU/mg and a mean molecular weight of between about 2400 and
2650 Da.
[0029] 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 an organic medium, by means of a
strong organic base with a pKa preferably greater than 20
(properties preferably similar to the family of phosphazenes
defined, for example, according to R. Schwesinger et al., Angew.
Chem. Int. Ed. Engl. 26, 1167-1169 (1987), R. Schwesinger et al.,
Angew. Chem. 105, 1420 (1993)), 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 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.
[0030] 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 mixture during the depolymerization
step.
[0031] 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 possible to depolymerize
the heparin while preserving as much as possible the sequences with
an affinity for ATIII such as the hexasaccharide .DELTA.IIa-IIs-Is
described herein. This critical step of the method makes it
possible to obtain the polysaccharides according to the
invention.
[0032] 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 phosphazene bases which have a pKa greater than 20, a very high
steric hindrance and a weak nucleophilicity.
[0033] 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 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 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 measured and
controlled water content, this effect can be seen to be fully
expressed.
[0034] The following table summarizes the impact of the water
content on the selectivity of the depolymerization. (Only this
parameter is variable in the trials: the stoichiometry of the
reagents, the dilutions, and 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-dimethylperhydro-1,2,3-diazaphosphor-
ine):
TABLE-US-00001 Water content % 0.05% 0.1% 0.2% 0.3% 0.4% 0.57% 1.8%
2.5% aXa 192 177 161 132 122 120 105 99 IU/mg aIIa 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 aIIa
[0035] 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 of less than about 0.6% and, most
preferably, less than about 0.3% when 1 molar equivalent of
phosphazene base is used relative to the benzyl ester of heparin,
benzethonium salt.
[0036] The method of the invention is therefore most particularly
characterized by 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, wherein a base of the
family of phosphazenes, particularly in dichloromethane solution
containing a percentage of water of less than 0.6%, is used.
Preferably, this percentage of water should be less than 0.3% and,
most preferably, less than 0.2%.
[0037] Advantageously, the strong base/ester mol ratio is between
0.2 and 5, preferably between 0.6 and 2 and most preferably between
0.8 and 1.2. The use of the equimolar ratio therefore forms part of
the preferred embodiments of the invention.
[0038] Other aprotic solvents known to persons skilled in the art
may be used, such as THF or DMF.
[0039] The quaternary ammonium salt of the benzyl ester of heparin
is preferably the benzethonium, cetylpyridinium or
cetyltrimethylammonium salt.
[0040] The bases of the family of phosphazenes are preferably those
of formula:
##STR00002##
in which the radicals R1 to R7 are identical or different and
represent alkyl radicals.
[0041] In the preceding formulae, the alkyl radicals contain 1 to 6
carbon atoms in the form of a straight or branched chain.
[0042] The invention therefore provides a method for preparing the
mixtures of oligosaccharides of the invention, this method
comprising the following steps: [0043] a) transalification of
sodium heparin by the action of benzethonium chloride, [0044] b)
esterification of benzethonium heparinate by the action of benzyl
chloride, [0045] c) transalification of the benzyl ester obtained
to a quaternary ammonium salt, [0046] d) depolymerization of the
quaternary ammonium salt of the benzyl ester of heparin by the
method as defined above, [0047] e) conversion of the quaternary
ammonium salt to a sodium salt, [0048] f) optionally,
saponification of the heparin by the action of a base such as
sodium hydroxide, [0049] g) optionally, purification in particular
by the action of an oxidizing agent such as hydrogen peroxide.
[0050] The following reaction scheme illustrates the method of the
present invention:
##STR00003## ##STR00004##
[0051] 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 acetate and preferably with a 10%
solution of sodium acetate in methanol (weight/volume), at a
temperature of between 15 and 25.degree. C. The equivalent by
weight of acetate added is preferably 3 times greater than that of
the quaternary ammonium salt of the benzyl ester of heparin used in
the depolymerization reaction.
[0052] 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 20.degree. C. and preferably between
0 and 10.degree. 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.
[0053] The final product may be optionally purified (step g) by any
known method of purifying depolymerized heparins (for example, that
disclosed in EP 0037319B1). Preferably, the purification is carried
out by means of hydrogen peroxide, in an aqueous medium, at a
temperature of 10 to 50.degree. C. Preferably, this operation is
carried out at a temperature between 20 and 40.degree. C.
[0054] The quaternary ammonium salt of the benzyl ester of heparin
may be prepared according to the following steps:
[0055] a) conversion of the heparin to the form of a sodium salt by
means of benzethonium chloride in order to obtain benzethonium
heparinate (transalification),
[0056] b) esterification of the benzethonium salt obtained in step
a) 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,
[0057] c) transalification of the sodium salt of the benzyl ester
of heparin to a quaternary ammonium salt and preferably to a
benzethonium, cetylpyridinium or cetyltrimethylammonium salt.
[0058] 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.degree. C. Advantageously,
the salt/sodium heparin molar ratio is between 3 and 4.
[0059] 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 method described in patent
FR2663639.
[0060] The esterification of step b) is preferably carried out in a
chlorinated organic solvent (for example chloroform or methylene
chloride (dichloromethane)), at a temperature of between 25 and
45.degree. C. and preferably between 30 and 40.degree. C. 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 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 salt of heparin. Likewise, preferably the reaction
time will be between 10 and 35 hours.
[0061] The transalification step c) is carried out by means of a
quaternary ammonium chloride and preferably by means of
benzethonium chloride, cetylpyridinium chloride or
cetyltrimethylammonium chloride, in an aqueous medium, at a
temperature of between 10 and 25.degree. C. Advantageously, the
quaternary ammonium chloride/sodium salt of the benzyl ester of
heparin mol ratio is between 2 and 3.
[0062] 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 another is optionally
achieved using the method described in patent FR 73 13 580.
[0063] The mixtures of oligosaccharides of the present invention
are not toxic and may thus be used as medicaments.
[0064] The mixtures of oligosaccharides of the present invention
may be used as antithrombotic agents. In particular, they are
useful for the treatment or 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 and treatment of the proliferation of
smooth muscle cells, atheriosclerosis and arteriosclerosis, for the
treatment and prevention of cancer by modulating angiogenesis and
growth factors, and for the treatment and prevention of diabetic
disorders such as diabetic retinopathies and nephropathies.
[0065] The present invention also relates to pharmaceutical
compositions containing, as an active ingredient, a mixture of
formula (I), optionally in combination with one or more inert
excipients.
[0066] The pharmaceutical compositions are, for example, solutions
which can be injected by the subcutaneous or intravenous route.
They are also useful for administration by the pulmonary route
(inhalation) or by the oral route.
[0067] The dosage may vary according to the age, weight and state
of health of the patient. For an adult, it is, in general, between
about 20 and 100 mg per day by the intramuscular or subcutaneous
route.
[0068] The following examples illustrate the invention without,
however, limiting its scope.
EXAMPLE A
Preparation of the Benzethonium Salt of Benzyl of Heparinate
[0069] Benzethonium Heparinate
[0070] 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 product is filtered, washed
with water and dried.
[0071] Benzyl Ester of Heparin (Sodium Salt)
[0072] 16 ml of benzyl chloride are added to a solution of 20 g of
benzethonium heparinate in 80 ml of methylene chloride. The
solution is heated at a temperature of 30.degree. 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 of a
sodium salt whose degree of esterification is 77%.
[0073] Benzyl Ester of Heparin (Benzethonium Salt)
[0074] 36 g (0.0549 mol) of benzyl ester of heparin (sodium salt)
and 540 ml of distilled water are introduced into a 2-litre
Erlenmeyer flask A. After homogenization at a temperature of about
20.degree. 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
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
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 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 filtration funnel. The cake is then washed with 4
times 200 ml of distilled water. The wet white solid is drained and
then dried under reduced pressure (2.7 kPa) at a temperature of
about 60.degree. C. After drying for 12 hours, 87.5 g of benzyl
heparinate, benzethonium salt, are obtained. The yield obtained is
94.9%.
EXAMPLE B
Description of the Hexasaccharide (.DELTA.IIa-IIs-Is)
##STR00005##
[0076] Proton spectrum in D.sub.2O, 500 MHz, T=298 K, .delta. in
ppm: 1.97 (3H, s), 3.18 (1H, dd, 10 and 3 Hz), 3.30 (1H, t, 8 Hz),
3.37 (1H, dd, 10 and 3 Hz), 3.60 (2H, m), between 3.65 and 3.85
(6H, m), 3.87 (2H, m), 3.95 (1H, d, 8 Hz), 4.03 (1H, d, 8 Hz),
between 4.05 and 4.13 (4H, m), between 4.16 and 4.45 (8H, m), 4.52
(1H, d, 8 Hz), 4.67 (1H, m), 5.06 (1H, d, 6 Hz), 5.10 (1H, d, 3
Hz), 5.33 (1H, d, 4 Hz), 5.36 (1H, d, 3 Hz), 5.46 (1H, d, 3 Hz),
5.72 (1H, d, 4 Hz).
[0077] Decasodium salt of
4-deoxy-.alpha.-L-threo-hexenepyranosyluronic
acid-(1.fwdarw.4)-2-deoxy-2-acetamido-6-O-sulfo-.alpha.-D-glucopyranosyl--
(1.fwdarw.4)-.beta.-D-glucopyranosyluronic
acid-(1.fwdarw.4)-2-deoxy-2-sulfamido-3,6-di-O-sulfo-.alpha.-D-glucopyran-
osyl)-(1.fwdarw.4)-2-O-sulfo-.alpha.-L-idopyranosyluronic
acid-(1.fwdarw.4)-2-deoxy-2-sulfamido-6-O-sulfo-.alpha.-D-glucopyranose.
[0078] Examples 1 to 7 and 12 illustrate the influence of the water
content on the selectivity of the polymerization reaction and the
aXa and aIIa activity of the products obtained.
[0079] Examples 8 to 10 illustrate the influence of the number of
base equivalents on the aXa and aIIa activity of the product
obtained (with a water content of 0.1%).
[0080] Example 11 illustrates the use of a phosphazene base other
than
2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,2,3-diazaphosphor-
ine; that is, the use of
tert-butyliminotri(pyrrolidino)phosphorane.
EXAMPLE 1
Depolymerization and Conversion to the Sodium Salt (0.1% Water)
[0081] 70 ml 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 with stirring. The water content of the
reaction medium is adjusted to 0.1%. The solution is heated to
40.degree. C. under nitrogen. After total dissolution, the solution
is cooled to a temperature of about 20.degree. C., followed by
addition of 1.75 ml (0.006 mol) of
2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,2,3-diazaphosphor-
ine. The resulting mixture is stirred at a temperature of about
20.degree. 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
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 of about
5.degree. 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 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 sedimented precipitate, and the mixture is stirred for 5
minutes. The precipitate in suspension is then filtered through a
No. 3 sintered glass filtration funnel. The cake obtained is then
washed with 100 ml of methanol. The pale yellow wet solid is
drained and then dried under reduced pressure (6 kPa) at a
temperature of about 40.degree. 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%.
[0082] Saponification:
[0083] 2.5 g (0.0038 mol) of the crude depolymerized heparin,
sodium salt in Celite (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 filtration funnel and rinsed twice
with 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 of about
5.degree. C. After the addition, the mixture is stirred for 2
hours. The solution is neutralized by adding 1N HCl; then 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. Stirring is
then stopped, and the suspension is left to sediment for 45 minutes
at a temperature of about 5.degree. 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 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 filtration funnel. 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 of about 40.degree. C. After drying for 18 hours, 1.31
g of crude depolymerized heparin (sodium salt) are obtained. The
yield obtained is 66%.
[0084] Purification:
[0085] 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.degree. 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 .mu.m membrane, and 0.07
ml of aqueous 30% hydrogen peroxide solution is added. After
stirring for about 2 hours at a temperature of about 20.degree. 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 .mu.m membrane, and 14 ml of methanol are then poured in. The
solution is then cooled to 10.degree. 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 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 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
filtration funnel. 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 of about 40.degree. C.
After drying for 18 hours, 1.13 g of pure depolymerized heparin
(sodium salt) are obtained. The yield obtained is 87%.
[0086] The characteristics of the depolymerized heparin thus
obtained are as follows:
[0087] mean molecular weight: 2600 Daltons
[0088] anti-Xa activity: 177 IU/mg
[0089] anti-IIa activity: 1.5 IU/mg
[0090] anti-Xa activity/anti-IIa activity ratio: 118.
EXAMPLE 2
Depolymerization and Conversion to the Sodium Salt (0.2%)
[0091] 70 ml of dichloromethane are placed in an 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 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-diazaphosphor-
ine are added. The mixture is stirred for 24 hours at a temperature
of about 20.degree. C. During this time, a solution of 30 g of
anhydrous sodium acetate in 300 ml of methanol is prepared in an
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 with magnetic stirring, at a
temperature of about 5.degree. C. 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 (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 separated out and discarded (230
ml). 230 ml of methanol are added to the sedimented precipitate.
The precipitate in suspension is then filtered through a No. 3
sintered glass filter. 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 of about 40.degree.
C. After drying for 18 hours, 2.63 g of crude depolymerized heparin
in Celite (5 g) are obtained. The yield obtained is 67%.
[0092] Saponification:
[0093] 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 a 50 ml Erlenmeyer flask. The suspension is filtered
through a No. 3 sintered glass filtration funnel and rinsed twice
with 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 of about 5.degree. C.
After the addition, the mixture is stirred for 2 hours. The
solution is neutralized by adding 1N HCl; then 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 stirring for 1 hour. Stirring is then stopped
and the suspension is left to sediment for 45 minutes at a
temperature of about 5.degree. 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 in suspension is then filtered through a No. 3
sintered glass filtration funnel. 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 of about 40.degree.
C. After drying for 48 hours, 2.3 g of crude depolymerized heparin
(sodium salt) are obtained. The yield obtained is 65%.
[0094] Purification:
[0095] 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 40.degree. 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 .mu.m membrane, and 0.07
ml of aqueous 30% hydrogen peroxide solution is added. After
stirring for about 2 hours at a temperature of about 40.degree. C.,
the mixture is cooled to a temperature of about 20.degree. 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 .mu.m membrane, and 14 ml of methanol are then poured in.
The solution is then cooled to 10.degree. 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 15 minutes. The supernatant is then separated out and
discarded (40 ml). 40 ml of methanol are added to the 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 No. 3
sintered glass filtration funnel. 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 of about
40.degree. C. After drying for 18 hours, 1.2 g of pure
depolymerized heparin (sodium salt) are obtained. The yield
obtained is 86%.
[0096] The characteristics of the depolymerized heparin thus
obtained are as follows:
[0097] mean molecular weight: 2650 daltons
[0098] anti-Xa activity: 161 IU/mg
[0099] anti-IIa activity: 1.4 IU/mg
[0100] anti-Xa activity/anti-IIa activity ratio: 115.
EXAMPLE 3
Depolymerization and Conversion to the Sodium Salt (0.3% Water)
[0101] 70 ml 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 with stirring and under nitrogen pressure. The
water content of the reaction medium is adjusted to 0.3%. After
total dissolution, 1.75 ml (0.006 mol) of
2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,2,3-diazaphosp-
horine are added. The mixture is stirred at a temperature of about
20.degree. 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
separate 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 sodium acetate solution (flask B) at a temperature in
the region of 5.degree. C., with magnetic stirring. After stirring
for 5 minutes, 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 5 minutes. The
precipitate in suspension is then filtered through a No. 3 sintered
glass filtration funnel. 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 of about 40.degree. C.
After drying for 18 hours, 2.57 g of crude depolymerized heparin
are obtained, as the sodium salt in Celite (5 g). The yield
obtained is 66%.
[0102] Saponification:
[0103] 2.5 g (0.0038 mol) of the sodium salt of crude depolymerized
heparin in Celite (5 g) obtained above and 18 ml of water are
placed in a 50 ml Erlenmeyer flask. The suspension is filtered
through a No. 3 sintered glass filtration funnel, and rinsed twice
with 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 of about 5.degree. C.
After the addition, the mixture is stirred for 2 hours. The
solution is neutralized by adding 1N HCl; then 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 suspension is left to sediment for 1 hour. 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
5 minutes. The precipitate in suspension is then filtered through a
No. 3 sintered glass filtration funnel. 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 of about
40.degree. C. After drying for 48 hours, 1.42 g of crude
depolymerized heparin (sodium salt) are obtained. The yield
obtained is 62%.
[0104] Purification:
[0105] 1.4 g of crude depolymerized heparin obtained above and 14
ml of distilled water are placed in a 50 ml Erlenmeyer flask. The
mixture is brought to 40.degree. 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 .mu.m membrane, and 0.07
ml of aqueous 30% hydrogen peroxide solution is added. After
stirring for about 2 hours at a temperature of about 20.degree. 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 0.45 .mu.m membrane, and 14 ml of methanol are
then poured in. The filtrate is then cooled to 10.degree. C. and
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 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 filtration funnel. 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.degree. C. After drying for 18
hours, 1.24 g of pure depolymerized heparin (sodium salt) are
obtained. The yield obtained is 89%.
[0106] The characteristics of the depolymerized heparin thus
obtained are as follows:
[0107] mean molecular weight: 2400 daltons
[0108] anti-Xa activity: 132 IU/mg
[0109] anti-IIa activity: 1.4 IU/mg
[0110] anti-Xa activity/anti-IIa activity ratio: 94.
EXAMPLE 4
Depolymerization and Conversion to the Sodium Salt (0.4% Water)
[0111] 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. The water content of
the reaction medium is adjusted to 0.4%. The solution is heated to
30.degree. C. under nitrogen. After total dissolution, the mixture
is cooled to a temperature of about 20.degree. C., followed by
addition of 1.75 ml (0.006 mol) of
2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,2,3-diazaphosphor-
ine. The mixture is stirred at a temperature of about 20.degree. 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 separate
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 sodium acetate solution at a temperature of about
5.degree. C., 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. The precipitate in suspension
is then filtered through a No. 3 sintered glass filtration funnel.
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 of about 40.degree. C. After 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%.
[0112] Saponification:
[0113] 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 a 50 ml Erlenmeyer flask. The suspension is filtered
through a No. 3 sintered glass filtration funnel and rinsed twice
with 6 ml of water. The 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 of about 5.degree. C.
After the addition, the mixture is stirred for 2 hours. The
solution is neutralized by adding 1N HCl; then 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 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 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 filtration funnel. 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 of about 40.degree. C. After drying for 48 hours, 1.9 g
of crude depolymerized heparin (sodium salt) are obtained. The
yield obtained is 67%.
[0114] Purification:
[0115] 1.9 g of crude depolymerized heparin obtained above and 19
ml of distilled water are placed in a 50 ml Erlenmeyer flask. The
mixture is brought to 40.degree. 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 .mu.m membrane, and 0.1
ml of aqueous 30% hydrogen peroxide solution is added. After
stirring for about 2 hours at a temperature of about 20.degree. 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 .mu.m membrane, and 14 ml of methanol are then poured in, and
the mixture is then 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 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). 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 suspension is then filtered
through a No. 3 sintered glass filtration funnel. 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 of about 40.degree. C. After drying for 72 hours, 1.56
g of pure depolymerized heparin (sodium salt) are obtained. The
yield obtained is 82%.
[0116] The characteristics of the depolymerized heparin thus
obtained are as follows:
[0117] mean molecular weight: 2350 daltons
[0118] anti-Xa activity: 122 IU/mg
[0119] anti-IIa activity: 1.3 IU/mg
[0120] anti-Xa activity/anti-IIa activity ratio: 94
EXAMPLE 5
Depolymerization and Conversion to the Sodium Salt (0.57%
Water)
[0121] 140 ml of dichloromethane are placed in an 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 0.57%. After total dissolution,
3.5 ml (0.012 mol) of
2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,2,3-diazaphosphor-
ine are added. The mixture is stirred at a temperature of about
20.degree. C. for 24 hours. During this time, a solution of 60 g of
anhydrous sodium acetate in 600 ml of methanol is prepared in
another 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 of about
4.degree. 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). 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 sedimented precipitate,
and the mixture is stirred for 5 minutes. The suspension is then
filtered through a No. 3 sintered glass filtration funnel. 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 of about 50.degree. C. After drying for 18 hours,
6.66 g of crude depolymerized heparin are obtained, as the sodium
salt in Celite (10 g). The yield obtained is 85%.
[0122] Saponification:
[0123] 6.66 g (0.0101 mol) of the sodium salt of crude
depolymerized heparin in Celite (10 g) obtained above and 47 ml of
water are placed in a 50 ml Erlenmeyer flask. The suspension is
filtered through a No. 3 sintered glass filtration funnel and
rinsed twice with 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 with magnetic stirring, at a temperature of about
5.degree. C. After addition, the mixture is stirred for 2 hours.
The solution is neutralized by adding 1N HCl; then 9.5 g of sodium
chloride are added. 66 ml of methanol are added to the reaction
medium. After 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
of about 5.degree. C. The supernatant is then separated out and
discarded (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 sedimented precipitate and the mixture is
stirred for 5 minutes. The suspension is then filtered through a
No. 3 sintered glass filtration funnel. The cake obtained is then
washed twice with 50 ml of methanol. The wet solid is drained and
then dried under reduced pressure (6 kPa) at a temperature of about
40.degree. C. After drying for 18 hours, 4.53 g of crude
depolymerized heparin (sodium salt) are obtained. The yield
obtained is 74%.
[0124] Purification:
[0125] 4.53 g of crude depolymerized heparin obtained above and 45
ml of distilled water are placed in a 100 ml Erlenmeyer flask. The
mixture is brought to 40.degree. 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 .mu.m membrane, and 0.25
ml of aqueous 30% hydrogen peroxide solution is added. After
stirring for about 2 hours at a temperature of about 20.degree. C.,
the mixture is neutralized by adding 1N HCl, followed by addition
of 5.5 g of sodium chloride. The solution is then filtered through
a 0.45 .mu.m membrane, and 38 ml of methanol are then poured in, at
a temperature of about 10.degree. C. The solution is then brought
to 20.degree. C. and stirred for 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 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 filtration funnel. 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 of about
50.degree. C. After drying for 18 hours, 3.7 g of pure
depolymerized heparin (sodium salt) are obtained. The yield
obtained is 82%.
[0126] The characteristics of the depolymerized heparin thus
obtained are as follows:
[0127] mean molecular weight: 2200 daltons
[0128] anti-Xa activity: 120 IU/mg
[0129] anti-IIa activity: 1.4 IU/mg
[0130] anti-Xa activity/anti-IIa activity ratio: 86
EXAMPLE 6
Depolymerization and Conversion to the Sodium Salt (1.8% Water)
[0131] 140 ml of dichloromethane are placed in an 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 1.8%. After total dissolution,
3.5 ml (0.012 mol) of
2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,2,3-diazaphosphor-
ine are added. The mixture is stirred at a temperature of about
20.degree. C. for 24 hours. During this time, a solution of 60 g of
anhydrous sodium acetate in 600 ml of methanol is prepared in
another 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 of about
4.degree. 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). 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 sedimented precipitate and
the mixture is stirred for 5 minutes. The precipitate in suspension
is then filtered through a No. 3 sintered glass filtration funnel.
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 of about 50.degree. 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%.
[0132] Saponification:
[0133] 7.54 g (0.0101 mol) of the sodium salt of crude
depolymerized heparin in Celite (10 g) obtained above and 53 ml of
water are placed in a 50 ml Erlenmeyer flask. The solution is
filtered through a No. 3 sintered glass filtration funnel and
rinsed twice with 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 of about
4.degree. C. After addition, the mixture is stirred for 2 hours.
The solution is neutralized by adding 1N HCl; then 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, followed by stirring for 1 hour. Stirring is then stopped,
and the suspension is left to sediment for % hour at a temperature
of about 4.degree. 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 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
filtered through a No. 3 sintered glass filtration funnel. The cake
obtained is then washed twice with 50 ml of methanol. The wet solid
is drained and then dried under reduced pressure (6 kPa) at a
temperature of about 50.degree. C. After drying for 18 hours, 5.53
g of crude depolymerized heparin (sodium salt) are obtained. The
yield obtained is 80%.
[0134] Purification:
[0135] 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.degree. 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 .mu.m membrane, and 0.31
ml of aqueous 30% hydrogen peroxide solution is added. After
stirring for about 2 hours at a temperature of about 20.degree. 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 .mu.m membrane, and 49 ml of methanol are then poured in, at a
temperature of about 10.degree. C. The solution is then brought to
20.degree. C. and stirred for 15 minutes. 126 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 mixture
is stirred for 5 minutes. The precipitate 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 filtration funnel. 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 of about
55.degree. C. After drying for 18 hours, 4.53 g of pure
depolymerized heparin (sodium salt) are obtained. The yield
obtained is 82%.
[0136] The characteristics of the depolymerized heparin thus
obtained are as follows:
[0137] mean molecular weight: 2600 daltons
[0138] anti-Xa activity: 105 IU/mg
[0139] anti-IIa activity: 3.1 IU/mg
[0140] anti-Xa activity/anti-IIa activity ratio: 34.
EXAMPLE 7
Depolymerization and Conversion to the Sodium Salt (2.5% Water)
[0141] 140 ml of dichloromethane are placed in an 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-diazaphosphor-
ine are added. The mixture is stirred at a temperature of about
20.degree. C. for 24 hours. During this time, a solution of 60 g of
anhydrous sodium acetate in 600 ml of methanol is prepared in an
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 of about
4.degree. 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 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 (400 ml). The precipitate in suspension is then
filtered through a No. 3 sintered glass filtration funnel. 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.degree. 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%.
[0142] Saponification:
[0143] 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 filtration funnel and
rinsed twice with 15 ml of water. 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 of about
4.degree. C. After addition, the mixture is stirred for 2 hours.
The solution is neutralized by adding 1N HCl; then 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, followed by stirring for 1 hour. Stirring is then stopped
and the suspension is left to sediment for 3/4 hour at a
temperature of about 4.degree. 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 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
filtered through a No. 3 sintered glass filtration funnel. The cake
obtained is then washed twice with 50 ml of methanol. The wet solid
is drained and then dried under reduced pressure (6 kPa) at a
temperature of about 50.degree. C. After drying for 18 hours, 5.87
g of crude depolymerized heparin (sodium salt) are obtained. The
yield obtained is 82%.
[0144] Purification:
[0145] 5.87 g of crude depolymerized heparin obtained above and 59
ma of distilled water are placed in a 100 ml Erlenmeyer flask. The
mixture is brought to 40.degree. 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 .mu.m membrane, and 0.34
ml of aqueous 30% hydrogen peroxide solution is added. After
stirring for about 2 hours at a temperature of about 20.degree. 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 .mu.m membrane, and 49 ml of methanol are then poured in, at a
temperature of about 10.degree. C. The solution is then brought to
20.degree. C. and stirred for 15 minutes. 126 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 mixture
is stirred for 5 minutes. The precipitate 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 filtration funnel. 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 of about
55.degree. C. After drying for 18 hours, 5.21 g of pure
depolymerized heparin (sodium salt) are obtained. The yield
obtained is 89%.
[0146] The characteristics of the depolymerized heparin thus
obtained are as follows:
[0147] mean molecular weight: 3550 daltons
[0148] anti-Xa activity: 99 IU/mg
[0149] anti-IIa activity: 13.4 IU/mg
[0150] anti-Xa activity/anti-IIa activity ratio: 7.4.
EXAMPLE 8
Depolymerization and Conversion to the Sodium Salt (0.5 Equivalent
of Base)
[0151] 140 ml of dichloromethane are placed in an 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. After total dissolution
at a temperature of about 30.degree. C. and cooling to a
temperature of about 20.degree. C., 1.75 ml (0.006 mol) of
2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,2,3-diazaphosphor-
ine are added. The mixture is stirred at a temperature of about
20.degree. C. for 24 hours. During this time, a solution of 60 g of
anhydrous sodium acetate in 600 ml of methanol is prepared in an
Erlenmeyer flask B. The reaction mixture in the Erlenmeyer flask A
is poured into the methanolic sodium acetate solution at a
temperature of about 4.degree. 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 sedimented
precipitate, and the mixture is stirred for 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 is separated out
and discarded (400 ml). The precipitate in suspension is then
filtered through a No. 3 sintered glass filtration funnel. 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
(6 kPa) at a temperature of about 60.degree. C. After drying, 5.7 g
of crude depolymerized heparin are obtained, as the sodium salt.
The yield obtained is 73%.
[0152] Saponification:
[0153] 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 stirring, at a temperature of about 4.degree. C.
After addition, the mixture is stirred for 2 hours. The solution is
neutralized by adding 1N HCl; then 6 g of sodium chloride are
added. 42 ml of methanol are added to the reaction medium. After
stirring for 15 minutes, 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 of
about 4.degree. 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 5 minutes. The
precipitate 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 filtration funnel. 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
of about 60.degree. C. After drying, 3.5 g of crude depolymerized
heparin (sodium salt) are obtained. The yield obtained is
67.4%.
[0154] Purification:
[0155] 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.degree. C. with magnetic stirring. The pH
is brought to 9.6.+-.0.1 by addition of 1N sodium hydroxide. The
reaction medium is filtered through a 0.45 .mu.m membrane, and 0.18
ml of aqueous 30% hydrogen peroxide solution is added. After
stirring for about 2 hours at a temperature of about 20.degree. C.,
the mixture is neutralized by adding 1N HCl, followed by addition
of 3.6 g of sodium chloride. The solution is then filtered through
a 0.45 .mu.m membrane, and 27 ml of methanol are then poured in, at
a temperature of about 10.degree. C. The solution is then brought
to 20.degree. C. and stirred for 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 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 30 minutes. The
precipitate in suspension is then filtered through a No. 3 sintered
glass filtration funnel. The white cake obtained is then washed
twice with 30 ml of methanol. The wet solid is drained and then
dried under reduced pressure at a temperature of about 60.degree.
C. After drying, 2.8 g of pure depolymerized heparin (sodium salt)
are obtained. The yield obtained is 80%.
[0156] The characteristics of the depolymerized heparin thus
obtained are as follows:
[0157] mean molecular weight: 2900 daltons
[0158] anti-Xa activity: 146.1 IU/mg
[0159] anti-IIa activity: 5.1 IU/mg
[0160] anti-Xa activity/anti-IIa activity ratio: 28.6.
EXAMPLE 9
Depolymerization and Conversion to the Sodium Salt (0.6 Equivalent
of Base)
[0161] 280 ml of dichloromethane are placed in a three-necked flask
A. 40 g (0.024 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 of about 30.degree. C. and cooling to a
temperature of about 20.degree. C., 4.2 ml (0.014 mol) of
2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,2,3-diazaphosphor-
ine are added. The mixture is stirred at a temperature of about
20.degree. C. for 24 hours. During this time, a solution of 60 g of
anhydrous sodium acetate in 600 ml of methanol is prepared in an
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 of about 4.degree. C., with magnetic
stirring. After stirring for 1 hour, the suspension is left to
settle. The clear part 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 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 filtration funnel. 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 of
about 60.degree. C. After drying, 6 g of crude depolymerized
heparin are obtained, as the sodium salt. The yield obtained is
77%.
[0162] Saponification:
[0163] 6 g (0.0091 mol) of the sodium salt of crude 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 of about 4.degree. C.
After addition, the mixture is stirred for 2 hours. The solution is
neutralized by adding 1N HCl; then 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 is left to sediment for 30 minutes at a temperature of
about 4.degree. 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 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 glass filtration funnel. The cake
obtained is then washed twice with 30 ml of methanol. The wet solid
is drained and then dried under reduced pressure at a temperature
of about 60.degree. C. After drying, 3.6 g of crude depolymerized
heparin (sodium salt) are obtained. The yield obtained is
65.3%.
[0164] Purification:
[0165] 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.degree. C. with magnetic stirring. The pH
is brought to 9.6.+-.0.1 by addition of 1N sodium hydroxide. The
reaction medium is filtered through a 0.45 .mu.m membrane, and 0.18
ml of aqueous 30% hydrogen peroxide solution is added. After
stirring for about 2 hours at a temperature of about 20.degree. C.,
the mixture is neutralized by adding 1N HCl, followed by addition
of 3.5 g of sodium chloride. The solution is then filtered through
a 0.45 .mu.m membrane and 25 ml of methanol are then poured in, at
a temperature of about 10.degree. C. The solution is then brought
to 20.degree. C. and stirred for 15 minutes. 63 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 (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 minutes. The suspension is then filtered through a No. 3
sintered glass filtration funnel. The white cake obtained is then
washed twice with 30 ml of methanol. The wet solid is drained and
then dried under reduced pressure at a temperature of about
60.degree. C. After drying, 2.5 g of pure depolymerized heparin
(sodium salt) are obtained. The yield obtained is 71.4%.
[0166] The characteristics of the depolymerized heparin thus
obtained are as follows:
[0167] mean molecular weight: 2600 daltons
[0168] anti-Xa activity: 150.5 IU/mg
[0169] anti-IIa activity: 3.2 IU/mg
[0170] anti-Xa activity/anti-IIa activity ratio: 47.
EXAMPLE 10
Depolymerization and Conversion to the Sodium Salt (0.8 Equivalent
of Base):
[0171] 70 ml 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, with stirring. After total dissolution
at a temperature of about 30.degree. C. and cooling to a
temperature of about 20.degree. C., 1.38 ml (0.004 mol) of
2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,2,3-diazaphosphor-
ine are added. The mixture is stirred at a temperature of about
20.degree. 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
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 of about 4.degree. 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
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
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 suspension is then filtered through a No. 3 sintered
glass filtration funnel. 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 of about 40.degree.
C. After drying for 18 hours, 3.05 g of crude depolymerized heparin
are obtained, as the sodium salt. The yield obtained is 80%.
[0172] Saponification:
[0173] 3.05 g (0.0048 mol) of the sodium salt of crude
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 filtration funnel and rinsed twice with 6 ml
of water. The filtrate obtained is placed in a 150 ml Erlenmeyer
flask. 0.6 ml (0.006 mol) of 30% caustic soda is added with
magnetic stirring, at a temperature of about 4.degree. C. After
addition, the mixture is stirred for 2 hours. The solution is
neutralized by adding 1N HCl; then 4 g of sodium chloride 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 of about 4.degree.
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 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 solid is drained and then dried under reduced
pressure (6 kPa) at a temperature of about 40.degree. C. After
drying, 1.6 g of crude depolymerized heparin (sodium salt) are
obtained. The yield obtained is 57%.
[0174] Purification:
[0175] 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.degree. C. with magnetic stirring. The pH
is brought to 9.6.+-.0.1 by addition of 1N sodium hydroxide. The
reaction medium is filtered through a 0.45 .mu.m membrane, and 0.08
ml of aqueous 30% hydrogen peroxide solution is added. After
stirring for about 2 hours at a temperature of about 20.degree. 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 .mu.m membrane, and 14 ml of methanol are then poured in, at a
temperature of about 10.degree. C. The solution is then brought to
20.degree. 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
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
No. 3 sintered glass filtration funnel. The wet solid is drained
and then dried under reduced pressure (6 kPa) at a temperature of
about 40.degree. C. After drying, 1.25 g of pure depolymerized
heparin (sodium salt) are obtained. The yield obtained is 78%.
[0176] The characteristics of the depolymerized heparin thus
obtained are as follows:
[0177] mean molecular weight: 2400 daltons
[0178] anti-Xa activity: 154.3 IU/mg
[0179] anti-IIa activity: 1.6 IU/mg
[0180] anti-Xa activity/anti-IIa activity ratio: 96.4.
EXAMPLE 11
Depolymerization and Conversion to the Sodium Salt
[0181] 140 ml of dichloromethane are placed in an 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. After total dissolution
at a temperature of about 40.degree. C. and cooling to a
temperature of about 20.degree. C., 3.5 ml (0.011 mol) of
tert-butyliminotri(pyrrolidino)phosphorane are added. The mixture
is stirred at a temperature of about 20.degree. C. for 24 hours.
During this time, a solution of 60 g of anhydrous sodium acetate in
600 ml of methanol is prepared in an 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 of about 4.degree. 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). 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 sedimented precipitate and the
mixture is stirred for 5 minutes. The precipitate in suspension is
then filtered through a No. 3 sintered glass filtration funnel. 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 of about 40.degree. C. After drying, 5.39
g of depolymerized heparin (benzyl ester, sodium salt) are
obtained. The yield obtained is 69%.
[0182] Saponification:
[0183] 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 filtration funnel and rinsed twice with 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 of about 4.degree. C. After addition,
the mixture is stirred for 2 hours. The solution is neutralized by
adding 1N HCl; then 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 left to
sediment for 1 hour at a temperature of about 4.degree. 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 ml). 160 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 filtration funnel. 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 of about 40.degree. C.
After drying for 48 hours, 2.7 g of crude depolymerized heparin
(sodium salt) are obtained. The yield obtained is 59%.
[0184] Purification:
[0185] 2.6 g of crude depolymerized heparin obtained above and 25
ml of distilled water are placed in a 50 ml Erlenmeyer flask. The
mixture is brought to 40.degree. 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 .mu.m membrane, and 0.15
ml of aqueous 30% hydrogen peroxide solution is added. After
stirring for about 2 hours at a temperature of about 20.degree. C.,
the mixture is neutralized by adding 1N HCl, followed by addition
of 3 g of sodium chloride. The solution is then filtered through a
0.45 .mu.m membrane, and 21 ml of methanol are then poured in, at a
temperature of about 10.degree. C. The solution is then brought to
20.degree. 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 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 supernatant is separated out and discarded
(50 ml). The precipitate in suspension is then filtered through a
No. 3 sintered glass filtration funnel. 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 of about
40.degree. C. After drying for 18 hours, 2.35 g of pure
depolymerized heparin (sodium salt) are obtained. The yield
obtained is 90%.
[0186] The characteristics of the depolymerized heparin thus
obtained are as follows:
[0187] mean molecular weight: 2400 daltons
[0188] anti-Xa activity: 167.5 IU/mg
[0189] anti-IIa activity: 1.1 IU/mg
[0190] anti-Xa activity/anti-IIa activity ratio: 152.
EXAMPLE 12
Depolymerization and Conversion to the Sodium Salt (0.05%
Water)
[0191] 140 ml of dichloromethane are placed in an Erlenmeyer 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 0.05%, while stirring slowly for 48 h. The supernatant
is transferred under an inert atmosphere to an Erlenmeyer flask A.
After total dissolution, 3.5 ml (0.012 mol) of
2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,2,3-diazaphosphor-
ine are added. The mixture is stirred at a temperature of about
20.degree. C. for 24 hours. During this time, a solution of 60 g of
anhydrous sodium acetate in 600 ml of methanol is prepared in an
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 about 2 minutes into the
methanolic sodium acetate solution at a temperature of about
4.degree. C., with magnetic stirring. After stirring for 15
minutes, the suspension is left to 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 then filtered through a No. 3 sintered glass
filtration funnel. 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 of about 50.degree. C.
After drying for 16 hours, 5.36 g of crude depolymerized heparin
are obtained, as the sodium salt in Celite (10 g). The yield
obtained is 68.6%.
[0192] Saponification:
[0193] 5.36 g (0.00817 mol) of the sodium salt of crude
depolymerized heparin in Celite (10 g) obtained above and 50 ml of
water are placed in a 50 ml Erlenmeyer flask. The suspension is
filtered through a No. 3 sintered glass filtration funnel and
rinsed 4 times with 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 added with magnetic stirring, at a temperature of about
4.degree. C. After addition, the mixture is stirred for 3 hours.
The solution is neutralized by adding 1N HCl; then 11 g of sodium
chloride are added. 77 ml of methanol are added to the reaction
medium. After 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
of about 4.degree. C. The supernatant is then separated out and
discarded (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 sedimented precipitate and the mixture is
stirred for 10 minutes. The suspension is then filtered through a
No. 3 sintered glass filtration funnel. 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 of about
40.degree. C. After drying for 18 hours, 2.65 g of crude
depolymerized heparin (sodium salt) are obtained. The yield
obtained is 53.7%.
[0194] Purification:
[0195] 2.65 g of crude depolymerized heparin obtained above and
26.5 ml of distilled water are placed in a 100 ml Erlenmeyer flask.
The mixture is brought to 40.degree. 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 .mu.m membrane, and 0.25
ml of aqueous 30% hydrogen peroxide solution is added. After
stirring for about 2 hours at a temperature of about 20.degree. C.,
the mixture is neutralized by adding 1N HCl, followed by addition
of 3 g of sodium chloride. The solution is then filtered through a
0.45 .mu.m membrane, and 21 ml of methanol are then poured in, at a
temperature of about 10.degree. C. The solution is then brought to
20.degree. 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 left to sediment for 45 minutes. The
supernatant is then separated out and discarded (46 ml). 46 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 discarded
(50 ml). 50 ml of methanol are added and the precipitate in
suspension is then filtered through a No. 4 sintered glass
filtration funnel. 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 of about
50.degree. C. After drying for 18 hours, 2.363 g of pure
depolymerized heparin (sodium salt) are obtained. The yield
obtained is 89.1%.
[0196] The characteristics of the depolymerized heparin thus
obtained are as follows:
[0197] mean molecular weight: 2500 Daltons
[0198] anti-Xa activity: 192 IU/mg
[0199] anti-IIa activity: 1.3 IU/mg
[0200] anti-Xa activity/anti-IIa activity ratio: 148.
* * * * *