Note: Descriptions are shown in the official language in which they were submitted.
- 1 -
SHORT CHAINED OLIGOSACCHARIDES HAVING BIOLOGICAL PROPERTIES,
A PROCESS FOR MAKING THE SAME AND THE USE THEREOF AS DRUGS
The invention relates to short chained oligosaccharides,
having biological properties enabling -them particularly -to con-trol
only certain stages of blood coagulation.
It also relates to a process for obtaining -the same and to
their uses as active principles of drugs.
The inventors have already described oligosaccharides
having biological properties of -the type mentioned above.
When depolymerizing heparin by a chemical or an enzymatic
route, the inventors have in particular obtained octasaccharides
of great value corresponding to the sequence A-B-C-D-E-F-G-H (denoted
below by the abreviation A-H).
COO OR OR COO OR rF<
H~ ~O~b~0~V~~~OU `
0503 NHS03 OH NHAc OH NHS03 OS03 ~ IS03
.. . . .
A B C D E F G H
in which R represents a hydrogen atom or a -S03 group.
These octasaccharides are observed to be particularly
valuable because of their high specificity with respect to the
activated X factor or Xa factor of the blood (measured according
to the Yin-Wessler test) whilst their activity on the total coagula-
tion (measured according to the USP or APTT -test) is very low.
The references regarding these tests are given in the
Examples.
~q~
In pursuing their work in this field, the inventors were interested,
particularly, in the production of olisosaccharides with shorter
chains than these octasaccharides, bu-t possessing however an advanta-
geous ratio of their Yin-~essler titer to their USP or APTT titer.
They have thus been led to observe that by operating
under well-determined conditions, it was possible to selectively
shorten the active chains of the A-H octasaccharides and to obtain
compositions of high homogeneity in active oligosaccharides with
short chains.
It is therefore an object of the invention to provide
novel oligosaccharides with shorter chains than the oc-tasaccharides,
advantageously possessing at least the biological propertires of
thes octasaccharides.
It is another object of the invention to provide an easy
practice enabling elimination highly selectively of the A-H octasac-
charide units not taking part essentially in the biological action
concerned of these products. It is still another object to provide
means for obtaining an enzyme particularly suited to the practising
of this process. It is also another object to provide active princi-
ples of drugs and the drugs themselves capable of inhibiting the
Xa factor~ when i-t is present in the blood, with a high degree of
selectivity whist possessing very low activi-ty on total coagulation,
and useful, -through this fact, for antithrombotic treatments without
hemorragic risk for the patient.
The oligosaccharide compositions of high homogeneity
3~ according to the invention, are characterised in that they are formed
essentially of hexasaccharides possessing the sequence C'DEFGH (deno-
ted below by the abreviation C'-H).
.,~
z
CO~- 0~ coo- r"~ rOR
o ~ ~~ ~ o H
OH ~HAc OH NHSO3 0503 NHSO3
C' D ,.,
in which R represen-ts a hydrogen atom or the group S03 .
The hexasaccharides of the invention are characterised by
a high affinity for ATIII.
They are also, characterised by NMR spectra comprising,
among others, a signal in the region of the carbon a-t the 2 position
ofthe N-sulfate-glucosamine residues, which does not appear
with heparin. This signal can be attributed to the presence
of a substituent on the oxygen atom at the 3 position, and
more particularly to a 3-0-sulfate group on an N-sulfate-D-
glucosamine residue, (unit F of the diagram).
The hexasaccharides of the invention are, moreo-
ver, characterised by Yin-Wéssler titers largely superior
to those of heparin. More especially, hexasaccharide compo-
sitions of the invention may have Yin-Wessler titers, hi-
gher than 2000 u/mg, capable of attaining even about 2500
u/mg. Advantageously, their APTT or USP titer is observed
to be particularly low, of -the order of 10, which corres-
ponds to co~positions having ratios of their Yin-Wessler
titer to their USP or APTT titer as high as 200, even a-
bout 250.
Advantageous hexasaccharides are formed from C'-H
hexasaccharides in which at least one or two of the R groups
represent a -S03- group.
The invention is also aimed at a process for obtai-
~fu~
-- 4 --
.
ning the abovementioned hexasaccharide compositions.
According to this process, there are subjected, under pre-
determined conditions, A-H octasaccharides1 possibly octasaccharide
compositions formed to a major extent by these A H octasac-
charides, to the action of an enzyme.
Unexpectedly, it is observed that the A-H octasac-
charides, which may be themselves obtained by the action of
an enzyme on the heparin chains, are however still degra-
dable by the enzymatic route. By operating under predeter-
mined condi-tions, it is then possible to eliminate units
not taking part essentially in the activity of these pro-
ducts, and this with a high specificity, which enables
hexasaccharide compositions of great structural homogenei-
ty to be obtained.
The process according to the inven-tion is hence
characterised in that the abovementioned A-H octasacchari-
des, or octasaccharide compositions formed to a large ex-
tent from these octasaccharides, having a high ratio of the
Yin-Wessler titer to their APTT or USP ti-ter, are contacted
with an enzymatic agent under conditions adjusted so as to
fragment these octasaccharides specifically in order to
remove the A-B units not taking part essentially in the
activity concerned, this specificity leading to the produc-
tion of mixtures formed practically entirely of active he-
xasaccharides.
According to a feature of the invention, these
conditions comprise advantageously the application of the
enzyme at high concentrations of the order of 0.25 to l mg
per mg of octasaccharides treated, preferably of the order
of 0.5 mg of enzyme per mg of octasaccharide.
In order to provide hexasaccharide compositions
highly homogeneous in C'-~ hexasaccharides, recourse is
advantageously had to a treatment enabling the separation
from the degradation mixture of a least the major portion
of the C'-H active hexasaccharides.
A suitable treatment comprises a fractiona-
tion carried out in order to remove the proteins resulting
from the enzymatic reaction and the unreacted reagents.
This fractionation may be carried out, for example, by gel
permeation according to the molecular weight and~or the
ionic density of the products.
In a preferred embodiment of the invention,
the A-H octasaccharide is subjected to the action of an
enzyme.
As a suitable enzyme for obtaining selective
removal of the AB units of the A~H octosaccharide, recourse
is advantageously had to heparinase, more specifically
a heparinase of bacterial origin.
Such a heparinase is advantageously of the
type of heparinases which can be obtained from Flavo-
bacterium heparinum bacteria~
For the satisfactory practising of the
process of the-invention for obtaining C'-H hexasaccha-
rides, recourse is more especially had to a heparinase
~5 such as obtained according to a process including steps,
notably of cultivating bacteria of Flavobacterium he-
parinum from the extraction of crude heparinase from
these bacteria, and purifications~ carried out so as to
obtain a purified heparinase, having sufficiently high
activity to effect the desired removal of the A-B units
with satisfactory yields.
Advantageously, the heparinase applied
possesses a heparinasic activity of at least about
90 000 units, preferably higher than 100,000 units,
particularly of the order of 110,000 to 140,000 units.
The activity of the enzyme is evaluated,
with respect to the increase in the absorption of a
heparin titrating at least ~15 iu/mg at 230 nm.
By units, is then meant, in the description
and the claims, the amount of enzymes which results
in the appearence of an increase of one thousandth of
an optical density unit per minute.
As already indicated, 0.25 to 1 mg of
enzyme (having an activitv of the order of at least
90 000 units) was employed per mg of octosaccharide
preferably about 0.5 mg of enzyme.
Study of the action of the enzyme on the
octasaccharide has shown that it was appropriate to
work at a temperature above room temperature, particu-
larly between 35 and 40C, preferably of the orderof 37~C.
Under these conditions, a total duration
of about 24 hours appeared satisfactory.
Advantageously, the operation is carried out
in a buf~er medium, preferably of a pH of the order of 6
to 8, in particular close to neutrality.
Taklng into account the moderate stability
of the enzyme, it is pre~erable, in order to increase
its efficacity, to add it portion by portion, particular-
ly at regular intervals during this lapse of time.
It will be noted that, advantageously,
the process of the invention uses a starting product,
namely the A-H octasaccharide of high quality : homo-
geneous, and highly specific.
Under the conditions developed by the
inventors, this octasaccharide is hence observed to be
degradable and this, selectively, leading to shorter
chains preserving the sequence responsible for the
activity of these products and hence possessing advan-
tageously biological properties at least as great,
even greater than those of the octasaccharide.
Preferably, the process of the invention
is applied with a heparinase obtained by induction
from Flavobacterium heparinum bacteria, extraction
of the crude heparinase from the bacteria, frac-tio-
na-tion of the crude heparinase extract obtained
followed by several purifications of the fractions
possessing the desired heparinasic activity.
The preparation of heparinase is effec-
ted from a Flavobacterium heparinum culture madeunder the conditions described by Payza et Coll.
(~ Biol. Chem. 1956, 223, p.853-858).
By operating at a temperature close to
room temperature, with aeration and average stirring,
a culture period of the order o~ 25 to 30 hours appea-
red satisfactory.
Bacteria were then recovered from the
culture medium, for example, by centrifugation, prefe-
rably carried out at low temperature, particularly
below 10C, preferably of the order of 4C.
Before extracting the heparinase, it is
advantageous to resuspend the bacteria, then after
having subjected them to a dispersing operationl -to
lyophilise them.
The bacteria were then subjected to a
treatment with a view to the extraction of the hepa-
rinase. This treatment advantageously comprises
grinding them then their recovery for example, by
centrifugation.
In order to obtain satisfactory extraction,
it was convenient to carry out several ~rindings,
for example, firstly in the dry and then in a buffer
medium of pH of the order of 6 to 8, advantageously 7
or close to 7.
The crude heparinase extract recovered
~7
by centrifugation was then subjected,for purification
purposes, to a fractionation step. To provide a hepari-
nase possessing the desired activity and properties,
additional operations of purification of the successi-
vely obtained fractions advantageously followed.
Study of these fractionation and purification processes
has shown that it was preferable to operate at a tempe-
rature below room temperature, particularly below
10C, preferably of about ~ 4C.
The fractionation step is advantageously
carr;ed out particularly according to an exclusion chro-
matographic process by means of DEAE cellulose, in the
presence of ammonium sulfate.
In a first batch step, the DEAE cellulose
is advantageously used in the proportion of about at
least 3 g per g of bacterial cells, preferably of about
5 g.
The DEAE cellulose is advantageously equili-
brated previously by means of a buffer of pH of the order
of 6 to 8, preferably of about 7.
The ammonium sulfate is utili~ed in the pro-
portion of about at least 3 g/l, preferably of about
6 g/l.
The suspension thus formed is filtered and
the filtrate is collected, supplemented advantageously
with rinsing solutions frorn the DEAE cellulose. In
a second step, the previously obtained solution is
subjected to an additional fractionation by means of
DEAE cellulose, advantageously, preequilibrated by
means o the previously used buffer.This s~ep is
carried out with advantage by chromatography in a
column, the filtrates being percolated at a rate of
about 40 to 60 ml/h.
From the effluent, the heparinase was
recovered, for example, by precipitation, particularly by
- 9
means of ammonium sulfa-te.
The heparinase obtained at the conclusion of
this fractionation opera-tion which, under -the particular
conditions reported above, occurs in the form of a sulfo-
ammoniacal precipitate, is t'nen advan-tageously purified
by a process including at least one placing in contact
with an agarose of -the Sepharose type, more especially
tha-t known under the registered trademark (CM SEPHAROSE
CL6B", followed by contacting the collected purified.
fractions with heparinasic activity, with an agarose
of the type marketed under the registered trademark
"ULTRAGEL ACA54".
The purification step by means of an agarose
of the CM Sepharose CL 6 B type is advantageously carried
out in a chromatographic column.
It appears desirable to dissolve -the heparinase
(which occurs in the form of a sulfoammoniacal precipi-
tate), in distilled water, so as to obtain a solution
having a conductivity of the order of 6000 ~icromhos and
to adjust its pH to about 6.
The agarose applied is advan-tageously equili-
brated previously by means of a bu~fer of pH of the order
of 5 to 7, preferably close to 6.
After having washed the column preferably by
means of the washing buffer, the chroma-tography of the
heparinase solution is carried out and the heparinase
is recovered by elution by a linear gradient obtained
with the buffer used for washing and this same buffer
is brought to a higher ionic s-trength.
The fractions possessing the desired heparinasic
activity are recovered to collect the heparinase they
contain. For example, a precipitation particularly by
means of arnmonium sulfate is carried ou-t, followed by
j~
~ .
-- 10 --
centrifugation.
As already indicated, it is advantageous -to
proceed with an additional purification of the heparinase
collected by operating once again a placing in contact
with agarose, preferably, an agarose such as ULTROGEL ACA
54. This operation is, preferably carried out in a column
equilibrated by means oE a buffer of pH of the order of
6 to 8, particularly of the order of 7.
This buffer is advantageously used to develop
the column.
In this way fractions with a high heparinasic
activity were recovered.
As already described by the inventors, the A-H
octasaccharide employed in the process of the invention
can be obtained by contacting the heparin (or heparinic
fractions) possessing anticoagulant activity and chains
having a molecular weight of the order of abou-t 2000 to
50 000, with an enzymatic agent, preferably a purified
heparinase, more especially, of bacterial origin possess-
ing an activity of the order of 45 000 units, it beingunderstood -that the assay is carried out with a heparin
titrating at least 215 iu/mg. The conditions used for
carrying out this step are adjusted so as to obtain a
depolymerisation mixture containing octasaccharide chains
having an anti-Xa activity (Yin-Wessler) and comprising
a sequence responsable for -the specific anti-Xa activity
of these products.
The enzyme causes the cleavage of -the heparinic
chains between the anomeric carbon of the N-sulfa-te=
glucosamine residue and the following uronic acid unit.
The biologically active octasaccharides are then
separared from the depolymerisation mixture by adsorp-tion
on antithrombin III (ATIII) fixed to a suppor-t such as
agarose, under conditions enabling the oc-tasaccharides
having an a~inity for the ATIII to be fixed or retained
on the ATIII.
This s-tep is advantageously followed by the
elution of the re-tained or adsorbed products in order
to recover them and by their fractionation, Eor example
by gel filtration in order to isolate them.
These oli~osaccharides appear capable of exerting
a powerful antithrombotic activity. By reason of their
low and even practically zero anticoagulant ac-tivity,
the risks of hemorrhage are advantageously practically
eliminated. It has been moreover observed that this type
of oligosaccharide does not cause any reactivity of the
blood platelets.
The short chain hexasaccharides of the invention
15 are devoid of ~oxicity. Administration of 10 000 u/kg
(Yin-Wessler titer) of C'-H does not cause any toxic
reaction in the rabbi-t, nor pyrogenic effect in the
pyrogenicity test in the rabbit according to the French
Pharmacopoes.
Administration to mice of doses as large as
3200 mg/kg has not permi-tted the determination of the
LD50
~he invention hence relates to pharmaceutical
preparations which include said hexasaccharides with high
anti-Xa activity.
It relates more particularly -to pharmaceutical
preparations devoid of pyrogenic substances containing
an effective amount of active principle in association
with pharmaceutical excipients.
It relates also to compositions in which the
pharmaceutical vehicle is suitable for administration by
the oral route. Suitable administrative forms of the
invention for administration by the oral route can be
advantageously gastroresistant capsules, pellets or
~1
)2~2
tablets, pills, or again ln the form of liposomes.
O-ther pharmaceutical compositions comprise these
oligosaccharides in association with suitable excipients
for administration by the rectal route. Corresponding
administrative forms are constituted by suppositories.
Otner administrative forms of the invention are
constituted by aerosols or pomades.
The invention also relates to injectable, sterile
or sterilisable pharmaceutical compositions.
These solutions advan~ageously contain 1000 to
100 000 u tYin-Wessler)/ml of oligosaccharides, preferably
from 5000 to 50 000, for example 25 000 u/ml, when these
solutions are in-tended for injection subcutaneously.
They can contain, for example, from 500 to 10 000,
lS particularly 5000 u/ml of oligosaccharides when they
are intended for injection intraveneously or by perfusion.
Advantageously, such pharmaceutical preparations
are offered in the form of non~recoverable syringes,
ready for use.
The invention also relates to pharmaceutical
eompositions eontaining said oligosaeeharides in asso-
ciation with another aetive principle, useful in par-ti-
cular for prophylaxis and treatment of thrombosis, such
as a venotonic agent such as dihydroergotamine, a nico-
tinie aeid salt or a thrombolytie agent like urokinase.
The short-chain oligosaccharides of the invention
are advantageously in the form of salts of at least one
physiologieally aeeeptable metal sueh as sodium and/or
ealeium and/or magnesium.
The pharmaeeutical compositions of the invention
are partieularly adapted for the control (preventive or
curative) of certain steps in the coagula-tion of the
blood in man or in the animal, particularly in the case
where the patient is subjeeted to risks of hypercoagula-
,~
.?;~2
13
bili-ty resulting from a distur~ance in -the intrinsic
phase, for example consequen-t upon a release by the
organism of thromboplas-tins, for example, tissular
thromboplas-tins, (surgical operations, atheromateous
processes, development of tumeurs and disorders of
coagulation by bacterial or enzymatic activators, etc).
In order to illustrate the invention, there is
indicated, below, an example of the dosage usable in man :
this posology comprises, for example, the administration
to the patient of 1000 to 25 000 u (Yin and Wessler)
subcutaneously, twice or three times daily, according to
the degree of hypercoagulability risk or the thrombotic
condition of the patient, or from 1000 to 2S 000 u/24
hours intraveneously, in discontinuous administrations
at regular intervals, or continuously by perfusion, or
again from 1000 to 25 000 u (three times weekly) intra-
muscularly or subcutaneously (these titers are expressed
in Yin-Wessler units). These doses may naturally be
adjusted for each patient according to the results and
blood analyses carried out previously, the na-ture of the
disease from which he suffers, and, generally, his state
of health.
The 7nvention also relates to the use of the
oligosaccharides of the invention and of frac-tions
containing them, to the constitution of biological
reagents, useful in the laboratory, particularly as
comparison elements for -the study of other substances
whose anticoagulant ac-tivity it is desired to test,
particularly at the level of inhibition of the Xa factor.
It is directed also at the use of the fractions
and oligosaccharides in nuclear medecine, as radiophar-
maceutical products. The ilogosaccharides and the frac-
tions defined above are labelled by tracers selected
from among those currently used in this field, and
ff~12~2
- 14 -
particularly by means of technetium 99 m.
For this purpose, the -techne-tium 99 m obtained
from commercial generators, in the form of unreactive
sodium pertechnetate of valency 7 is conver-ted into a
reduced technetium of valency 4 which would be -the most
reactive form of technetium. This conversion is carried
out by means of a reducing system effected from tin
salts (s-tannous chloride), iron salts (ferrous sulfate),
titanium salts (titanium trichloride) or other salts.
Most of the time, this mere reduc-tion of the
technetium is enough to fix the technetium to the mole-
cule concerned under given conditions of pH.
It is possible to use the products of the in-
vention, which constitutes in a way a support, at doses
of the order of 100 to 200 u Yin-Wessler.
For developing these radiopharmaceutical reagents,
it is possible to operate in accordance with the method
of P.V.
~ ~ ~f~
- 15 -
KULKARNI et al. in The Journal of Nuclear Medicine 21,
N 2, p. 177-121.
The so-labelled products are advantageously
used in ~n v~vo tests for the detection and diagnosis of
5 extended thromboses and thrombotic conditions.
THE DRAWINGS
In Figures 1 and 2 are shown the elution
graphs obtained by recording the OD at 280 nm of re-
spectively (1~ an heparinase preparation chromatog-
raphied on CM sepharose CL6B wherein portion D corre-
sponds to the fractions with a high heparinase activity,
and (2) the centrifugation culot recovered from D,
chromatographied on ULT~OGEL ACA 54,
in Figure 3 is shown the elution diagram of a de-
polymerization mixture of octasaccharide A-H chromatog-
raphied on a superfine SEPHADEX G-50 column, and
in Figure 4 are given the graphs obtained by measuring
O.D at 230 nm of eluted fractions obtained by fragment-
ing an hexasaccharide fraction with HNO2 (curve a in
continuous lines), the content in 2,5-anhydromannose
groups of said fragments (curve b with dotted lines),
their content in uronic acids (curve c with dashed lines
- - - -) and in N-acetylglucosamine groups (curve d with
dashed dotted lines -.-.-.-.).
.~
- 16
EXAMPLE 1.
Process for producing ac-tive C'-H hexasaccharides by the
_
action of heparinase onA-~I oc-tasaccharide.
_ _ _
This process includes the following -three steps
1 to 3 :
1) the preparation of the heparinase ;
2) the action of -the heparinase on the A-H octasaccharide
for the purposes of selective degradation, followed by
3) fractionation of the degradation mixture by filtration
on gel, and recovery of the fractions containing the
desired hexasaccharides.
These steps are carried out as follows :
1) Preparation of the heparinase.
Enzymes derived from the cultivation of Flavobac-
terium heparinum obtained by the following procedure wereused :
In an 1~ liter fermenter, of the type marketed
under the trademark BIOLAFFITE, the cultiva-tion of Flavo~
bact~erium heparinum ATCC 13125 was carried out, for 26
hours in a culture broth corresponding to the following
composition (in grams per liter of distilled water).
Flavobacterium heparinum culture bro-th
which has reached the stationary phase : 500 ml
Monosodium sodium phospha-te : 2,5
25 Disodium sodium phosphate : 25
Ammonium sulfate : 1
K Cl : 0.1
Sodium heparinate of -ti-ter equal or greater
than 150 iu/mg of Codex quality : 3
30 Calcium chloride : 0,01
Ferric chloride : 0.01
Magnesium sulfate : 0.01
Manganese chloride : 0.01
Sodium molybdate : 0,01
- 17 -
The pH of the broth was Einally adjusted to 7.0
with phosphoric acid or soda.
This culture was carried out at a temperature of
+24C with aeration and medium s-tirring.
Af-ter 26 hours of cultivation, the medium was
cooled to -~ 4C in a time interval of about 2 hours. The
bacteria were recovered by centrifugation at 50,000 rpm,
on a centrifuge of -the type of the SHARPLESS pneumatic
type T 313 A type and this for 2 hours. The centrifugation
culot was taken up again in 1 liter cold distilled water,
subjected to dispersion by an ULTRA-TURAX turbine at
maximum speed for 5 minutes, then freeze-dried. The total
duration of this operation was about 36 hours. Under these
conditions, 4.1 grams of cells were obtained.
- Extraction of the cells.
... . . . . _
The lyophilised cells, obtained by the preceding
step, were vigorously ground dry, in a mortar, in the
presence of 2 g of calcined alumine, for 1 hour, 10 ml of
buffer 1 was then added (0.1 M sodium acetate buffer,
pH 7). The grinding in the mortar o~ the paste then
obtained is continued for 30 minutes at + 4C. 450 ml
of buffer was then added cold and the whole was allowed
to stand with stirring for 1 hour at + 4C. The suspension
obtained was centrifuged on a centrifuge of the SORVALL
RC2 B type, at 18,000 rpm at + 4C, for 20 minutes. The
centrifugation culots composed of alumina and cell debris
were discarded. The supernatant liquor (orange yellow and
viscous) was collected, which constituted the crude cell
extract, and corresponds to a volume of 470 ml. The rest
o~ the manipulations were carried ou-t at + 4C.
- Exclusion chromatography by DEAE cellulose.
With stirring, at t 4C, 2.82 g of ammonium
sulfate was added to the previously obtained supernatant
liquor, and then 21 g of DEAE cellulose previously
~,~
.~. ,~
1~81~0~g~
- 18 -
equilibrated with buffer 2) (0.1 M sodium acetate buffer,
pH 7 containing 6 g/l of ammonium sulfate). The whole
was subjected to stirring for 2 hours at ~ 4C wi-th
control and adjustment if necessary of the pH 7Ø Then
the DEAE cellulose was separated by filtra-tion on a
Buchner funnel and it was washed in the cold with buffer
2), until the absence of proteins in the wash solutions.
The whole filtrate and wash solutions (680 ml~ were passed
over a column of 400 ml (23 = 5 cm) of DEAE cellulose
preequilibrated in buffer 2)~ at + 4C, at the flow rate
of 50 ml/h. The column was finally rinsed with buffer 2),
until the absence of protein in the rinse solutions. The
column effluents and the rinse solutions were combined,
which corresponds to a volume of 1100 ml and 715 g of
ammonium sulfa-te was added with stirring. The precipitated
proteins were collected by centrifugation at 7000 rpm and
this for 30 minutes, on acentrifuge of the SORVALL type.
2.5 g of very wet precipitate which constitutes the hepa-
rinase was collected. This precipitate can be stored at
- 20C for several weeks.
- Chromatography on CM Sepharose CL 6 B.
The previously obtained sulfoammonical precipi-
tate was dissolved in cold distilled water used in a
sufficient amount -to obtain a final conductivity of 6000
micromhos. The pH of the solution obtained was adjusted
to 6.0 by acetic acid or 2 N soda. The final volume was
440 ml. The solution was then percolated at + 4C over
a column of 70 ml (15 x 2.6 cm) of CM Sepharose CL 6 B
previously equilibrated with buffer 3) (0.1 M sodium
acetate buffer, 0.22 M NaCl, pH 6.0), at a flow rate
of 25 ml/h. The column was rinsed by buffer 3) until
the absence of proteins in the effluent. The column
effluents were discarded.
The heparinase was then eluted at 60 ml/h by
-- 19 --
means of a linear gradient formed from 600 ml of buffer
3) and 600 ml of buffer 3) adjusted to 0.34 M NaCl.
The proteins emerging from the column were
detected by con-tinuous recording of the optical density
(O.D.) at 280 nm and the eluate was collected by frac-tions
of 5 ml by means of a fraction collec-tor.
In Figure 1, is shown the elution graph ob-tained
by recording the O.D. at 280 nm of the column effluent.
The heparinase ac-tivi-ty of each fraction was
assayed. The fractions 45 to 48 ~portion D of the graph)
with a high heparinase content were grouped. These frac-
tions correspond to a volume of 45 ml.
The proteins were precipitated by the addition
of 30 g of ammonium sulfate and they were recovered by
centrifugation at ~ 4C at 15,000 rpm, for 10 minutes.
- Gel filtration on ULTROGEL ACA 5~.
The centrifugation culot previously obtained
was dissolved incold distilled water and a final volume
of 5 ml was obtained. This solution was placed at the
top of a column (1 m x 26 mm) of ULT~OGEL ACA 54 equili-
brated with 0.1 M of sodium acetate buffer, 0.33 M NaCl,
pH 7. The column was developed by this same buffer at
a flow rate 15 ml per hour. As previously the pro-teins
emerging from thecolumn were detected at 280 nm, and the
column effluent was collected by fractions of 5 ml. The
elution graph is shown in Figure 2. The heparinase acti-
vity of each fraction was assayed. The fraction 32 to 37
(portion F of the graph), which contained the heparinase
activity, were grouped together, which corresponds to a
volume of 60 ml. This solution contains 7 mg of purified
heparinase, having an activity of the order to 100 000
to 110 000 units/mg. (There was taken as an enzyme unit,
the amount of enzyme which causes the appearance at 231
nm, of one thousandth of an optical density unit per
-- 20 --
minute when -the heparinase is contac-ted a-t 38C with
heparin titrating at least 215 iu/mg 0.065 % in 0.125 M
tris-HCl buffer, pH 7, into which calcium chloride CaC12
is added).
The solution ob-tained is preserved and frozen
at - 20C.
2) Degradation of the octasaccharide hy the heparinase.
10 mg of A-H octasaccharide (batch BC IV 135),
obtained according to the process described in Pa-tent
Application filed by Applicant in CANAD~ on October 6,
1980, No 361 600. having an anti-Xa (~in-Wessler) activity
of 2100 u/mg, was dissolved in 10 ml of 0.1 M sodium
acetate buffer calcium chloride, pH 7.2.
The solution was incubated at + 37C. The heparin-
ase addition was carried out as follows.
At time t = O, 17 ml of the heparinase solutionpreviously obtained (namely 2 mg of heparinase) was added :
at time tl = 8 hours, 17 ml of heparinase solution was
again added : at time t2 = 16 hours, there was added
finally 8.5 ml of heparinase solution. At time t3 = 24
hours, the evaporation to dryness under vacuum at 40C
of 52.5 ml of solution followed in an appara-tus of -the
Rotavapor B~CHI -type.
3) Fractiona-tion of the degradation mixture by filtration
on gel.
The mixture obtained on emerging from the degra-
dation step was placed at the top of a superfine Sephadex
G-50 column (registered tradernark) (200 x 2.5 cm). Elu-
tion of the products followed by means of 0.2 M sodium
chloride. The products were detected by their absorption
at 230 nm. In Figure 3 is shown the elution diagram
ob-tained. Three principle fractions are distinguished.
The first is constituted by undegraded starting material
(4 mg), the second contains the desired hexasaccharides
2~:
-- 21 --
(7 mg) and the -third disaccharides. A less important
fraction is also distinguished in the region of te-tra-
saccharides between the peaks of -the hexa and of the
disaccharides.
The hexa~accharide fractions are collected -toge~
ther, -the salts were removed and they were freeze-dried.
- S-tudy of the structure o~ the hexasaccharides of the
fractions collected.
A study of the structure of these :Eractions by
colorimetric analysis of the fragments obtained by degra-
dation by means of nitrous acid followed by gel filtra-tion,
ensued. The degradation, by means of nitrous acid, was
carried out according to the method of SHIVELY and CONRAD
described in Biochemistry, vol. 15 N 12, 1976, O. 3932
to 3942.
The action of nitrous acid is manifested by
severance of the glycoside bonds between the N-sulfate-
glucosamine units and the following uronic acid and
converts the sulfate-glucosamine units in-to 2,5-anhydro-
mannose residues.
The hexasaccharide was then converted into te-
trasaccharide and into disaccharide. These two oligosaccha-
rides were separated by filtration on a column of Super-
fine G-50 Sephadex (registered trademark) (200 x 0.~ cm),
eluted with 0.2 M sodium chloride. In figure 4, are plot-ted
the values of the optical density measured a-t 230 nm, of
the eluted fractions (curve a in continuous lines) as well
as their content of 2.5 anhydromannose groups (curbe b in
dotted lines ... ), uronic acids (curve c in dashed lines
30 -----) and N-acetyl-glucosamine (curve d in dashed - dotted
lines -.-.-.) (for the determination of this latter yroup,
measurements were carried out before and after acid
hydrolysis, the difference obtained corresponding to the
content of N-acetyl-glucosamine groups).
U~2
- 22 -
It is to be seen on examining Figure 4 (-the arrow
in Figure 4 indicates -the elution volume for the original
hexasaccharide) that the unsaturated uronic acid units
which absorb the light at 230 nm are contained in the
tetrasaccharide fraction whilst -the disaccharide fraction
is practically entirely devoid of such compounds.
In the same way, it appears tha-t the N-acetyl-
glucosamine groups are only pre~ent, as anticipated, in
the tetrasaccharide fraction.
The content of uronic acid groups appears twice
as high in the tetrasaccharide fraction as in the disaccha-
ride fraction.
It is observed, also, that the 2,5-anhydromannose
groups are present in equivalent manner in the two frac-
tions, which implies that the disaccharides and thetetrasaccharides are obtained in a molar ratio 1/1.
These results show that a cleavage has been ef~ected on
the hexasaccharide, giving rise to the disaccharide not
bearing a double bond and the tetrasaccharide (cleavage
between F and G).
In addition, these results establish that the
hexasaccharide fraction is almost exclusively constituted
by a single species which carries an N-sulfate-glucosamine
unit at its reducing end, and an unsaturated uronic acid
unit at its non-reducing end.
Comparison with the starting octasaccharide
structure permits the conclusion that two other glucosa-
mine units of which one is N-acetylated and the other
N-sulfated-3-0-sulfated, and two other uronic acid
residues (one glucuronic and one iduronic-2-0-sulfa-ted)
complete the hexasaccharide sequence.
- Study of the in vitro and in vlvo biological activity
of the hexasaccharide fraction obt ned according to
the above-described process~
~,'
- 23 -
The an-ti-Xa activity was de-termined by the Yin-
Wessler test described by its authors in J. Lab. Clin.
Med. 1976, 81, 298-300.
The overall anticoagulant activity was rneasured
by the USP test or the APTT method.
The USP test is described in ~Pharmacopea of
the Uni-ted States of America", XIX, pages 229-230 (see
also the second supplement USP-NF, page 62, and -the
fourth supplement USP, page 90, respectively intitled
~Drug substances~ and "Dosage forms").
The APTT titer is measured by the method of
J. CA~N & al. in "Hemos-tase, expansion scientific
française", Paris, 1968, pages 133-135.
The in vivo antithrombotic activity was studied
by using the method of Wessler & al. described in J. of
appl. physiol. 1959, 14, 943-946, using a different
thrombogenic stimulant.
- Anti-Xa activity (Yin-Wessler) : 2400 u/mg.
- USP titer or APTT titerless than 10 u/mg.
The activity of this hexasaccharide fraction
was studied in vivo in the rabbit according to the
Wessler model. The administration of 250 u anti-Xa per
kg before administration of 25 u/kg of a thrombogenic
complex (concentrated prothrombin complex sold under
the name Konyn by Cutter Laboratories, U.S.A.) prevents
the formation of a thrombus.
