Note: Descriptions are shown in the official language in which they were submitted.
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SUBSTANCES WITH HEPARIN-LIKE STRUCTURE AND TH~IR METHOD OF
PROI)UCTION
Natural glucosaminoglycans obtained by extraction, (GAG),
such as the Heparansulfate (HS), have been isolated and
characterized (Jorpes, J. - Gardell, S.JBC 176, 267-276). Their
structure is similar to the Heparin one, varying fundamentally in
the degree of N-acetilation: in the Heparin, the group -NH of the
glucosamine is preferably sulfated, instead, in the HS, is N-
acetilated.
As a consequence of said structural difference, the HS shows
peculiar biological characteristics, different from those of the
Heparin:
1) It has low anticlottig activity tested in vitro (APTT,
anti-X).
2) It shows proPibrinolitic activity (release of the
plasminogen tissular activator), when used in humans or in
laboratory animals.
3) It presents a superior bioavailability to that of
Heparin.
ZO Therefore, said substances are specially relevant to
cardiovascular medicine, mainly, in long-lasting therapies
intended to prevent the formation of microthrombus, as well as
to contribute to their dissolution. This can be achieved with
little risk of hemorrhages and with reduced side ePfects.
Obviously, these natural mucopolysaccharides cannot be
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extracted in large scale, due to their low concentration in the
tissular mass. This can limit significatively its extensive use in
the therapeutical field.
Now, in accordance with the patent application, a process
has been developed, which permits to obtain a pure N-acetilated
compound with a Heparin-like structure with good yields,
starting from Heparin as raw material.
The process of the invention is based upon a controlled
hidrolysis of the Heparin with N-desulfatation without
depolimerization, followed by the introduction of the acetyl
group in the specific position of the aminic N, through a
reaction using acetic anhydride.
The Heparin desulfatation is achieved specifically on the
amino group, without hydrolizing the group O-sulfate and without
producing hidrolysis of the glycosidic bond or other parasitic
reactions. This has already been described by Inoue and Nagasawa
(1975~ "Inoue, Y. - Nagasawa, K. Carbohyd, Res. 46, 87-95
(1976)". However, this method cannot be easily conducted at large
scale, since it works on a heparin-piridynic complex dissolved in
zO acqueous or methanolic dimetilsulfoxide with its associated
problems, for example: the separation of the complex from the
reacting solvent, the quantitative ellmination of the pyridine,
the recovery o~ expensive solvents, etc.
On the contrary starting from calcium Heparin, due to the
contra-ion size, a selective N-desulfatation in an acid acqueous
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medium can be achieved, without causing structural alterations
and without affecting the medium MW.
Particularly, the browning of the product and the hydrolysis
of the O-sulfate group are avoided.
Then, the partially N-desulfated Heparin (Intermediate A),
is subjected to the acetylation reaction.
The Heparin acetylation has been described by Danishefsky
(Danishefsky, N. Meth. Carb. Chem. ~, (85), 11) and Nagasawa as
well (Inoue, Y. - Nagasawa, K. Carbohyd, Res. 46, 87-95 (1976)).
Both of them work with great quantities of acetic anhydride,
pH-6,5-7,o and a wide range of temperatures.
In these conditions, and working at low temperatures lO-
5 C), a parasitic O-acetylation is produced over the free -OH of
the disaccharidic unit.
It has been discovered, and it is the object of the present
invention, that when using a quantity practically stoichiometric
of the acetic anhydride (1-1,1 moles (CH,CO) =O/Mol of the free-
amino N, previously determined by the titration curve and at a pH
between to 9,3 and 9,5 (maximun limit compatible whit the acetic
anhydride hydrolisis), the N-acetylation is exclusively produced,
even working at 25-30C.
At this temperature, the reaction is fast and complete
within 15 minutes. Even if the reaction is lasted for a longer
time, it does not produce secondary reactions.
The obtained product (final product B) has an extremely
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constant composition and is represented by a molecule chemically
well characterised, with a precise analitical description, as
defined in the following table and figures.
To better identify the product object of the present
invention the 3C-NMR characterisation of the product is hereby
described.
The NMR spectrum of the final product 8 (figure 1), was
determined in D20 solution, at 75 MHz, by means of a Brucker Mod.
CxP-300 spectrometer. For comparison purposes also the spectra of
a standard Heparin (figure 2) ad of Heparansulfate tHS, figure 3)
are enclosed; these spectra correspond to those known in the
literature (B. Casu et al, Arzneim-Forsch. (Drug Research) 33,
135-142, 1983).
In figure 1, the peaks in the region 90-95 ppm (due to C-l
anomeric carbon) and in the region 53-61 ppm (due to C-2 of amino
sugar units) are particularly suitable in order to identify the
different uronic units, to measure the amount of said units and
to determine the sulfate group distribution on the uronic and
hexosamine units.
In the present text the following abbreviations have been
adopted:
L-iduronyl-2-sulfate = Ido A2S03
D-glucuronic acid = GlcA
Glucosamine N-sulfate = GlcNS03
25 N-acetyl glucosamine = GlcAc
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Glucosamine with no substituted amine groups = GlcNH2
r = reducing terminal groups, mainly referred to hexosaminic
units.
The following relative percentages were calculated by
considering the areas under the characteristic peaks of different
groups:
IdoA2S03 = 68.2%; GlcA = 31.8% referred to total uronic acids;
GlcNS03 = 37.8%; GlcNAc bound to IdoA2S03=37.8%: GlcNAc bound to
GlcA = 11.0%; reducing GlcN = 13.4%, with respect to total
hexosamines (C-1 peaks evaluated).
From the area under C-2 and CH3 (N-acetyl) peaks the
GlcNS03 groups percentage amounts to 36.2%, and the percentage of
total GlcNAc units amounts to 53%.
The product is analogous to Heparin (because of high
IdoA2S03 unit content 71-91% of total uronic acid in Heparin)
and to Heparansulfate (because of high N-acetyl group 58% content
of total glucosamine Heparansulfate); but it differs in a
considerable measure from abovementioned glycosaminoglicaes,
because of predominant GlcNAc-IdoA2S~3 sequences which are in
minor amount in both aforesaid GAG.
The example which follows, describes the original process
Por the production of Heparin-like N-acetylated compound (f~nal
product B) obtained from calcium Heparin. Based on laboratory
models this compound shows fibrinolytic and antithrombotic
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activity, and lacks anticlotting activity. Therefore, the
hemorraghic risk is low. The antithrombotic activity has been
tested using a model on a rat's tail, according to Bekemeyer
(Bekemeyer H. - Hirschelmann, "Agents and Actions". Vol.16 pag.
446 (1985)).
The fibrinolytic activity ex-vivo was tested on the plasma
of rabbits $njected with the product through the evalutation of
FDP (Fibrin Depolymerization Peptides), using the Bohringer
Diagnostica Kit.
The method of production, as well as the complexes obtained
and their use as fibrinolytic and protecting agents of
thrombosis, are part of this invention.
The purification of the final product B and of the
intermediate A are carried out following the general techniques,
lS using ammonium quaternary salts and ethanol in saline solution,
as described by Scott (Scott, J. Meth. Bioch. Analysis VIII 145-
197 (1960)).
EXAMPLE
1) Preparation o~ the calcium Neparin:
100 g of Heparin sodium 150 IU/mg are dissolved in distilled
water, a quantity sufficient for 1 liter of solution (10%). It is
heated at 50 C and, in agitation, 120 g of cetiltrimethylammonium
bromide (CTAB) dissolved in 1 liter of distilled water, are
slowly added.
The precipitated is left still for 1 hour at said
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temperature ad is separated by cetrifugation. It is washed twice
with 100 ml each time, of 1% solution of CTAB in water, and
finally the solid substance obtained is dissolved in 1 liter of
2M CaCl2 solution in water. Then, the polysaccharide is
precipitated adding 1 volume of ethanol 96. It is settled during
all night, the supernatant is put aside and the solid substance
obtained is redissolved in 1 liter of 2 M CaC12 in acqueous
solution.
The clear solution is precipitated again with a volume of
ethanol 96. The solid substance is settled, redissolving for a
second time in 1 liter 2 M CaC12. The rests of CTAB are
eliminated by adding bentonite according to Scott ( Scott, J.
Meth. Bioch. Analysis VIII, 145-197 (1960)). The bentonite is
eliminated by filtration and the clear liquid is precipitated for
the last time with 1 volume of ethanol.
The solid substance is anhydrated with ethanol and dried in
a vacuum drying chamber.
Then, 90 g of calcium Heparin are obtained with a degree of
sodium inferior to 0.1%.
2) Preparation of the intermediate A:
The 90 g of calcium polysaccharide are dissolved by flowing
back in HCl 0.25 M, a quantity sufficient to obtain a 10~
solution, heating at 80 C. After 2 hours, the N-desulfatation
reaction is practically completed, which is evidenced by the
absence of anticlotting activity of the liquid in reaction, and
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the appearance of a strong calcium sulfate precipitate.
When the action desired is completed, the reaction is
stopped adjusting the pH to 7 by adding a 10 M NaOH solution.
A sufficient quantity of 10% w/v C03Na2 solution is added to
complete precipitation of CO~Ca, and the precipitate obtained is
eliminated by filtration.
The polysaccharide N-desulfated is isolated from the liguid
adding 1 vol. ethanol and it is settled during the whole night.
The heparaminic material is anhydrated and dried in vacuum drying
10 stoves.
Then, 60 g of material are obtained containing 50% of amino
free groups (Intermediate A).
3) Preparation of the final product B:
g of intermediate A are dissolved in distilled H20, a
sufficient quantity for 1 liter of solution (5% w/v) and the pH
is adjusted to 9,5 with 10 M NaOH solution. The temperature is
raised to 25-28 C, and under agitation 5 ml of acetic anhydride
are added drop by drop, maintaining the pH in 9,3 - 9,5 with a
NaOH solution. The aggregate of acetic anhydride takes
approximately 5 minutes and the reaction time under agitPtion is
minutes, after which 10 g of NaCl are added. Then, all the
liquid is precipitated in 1 vol. ethanol and settled for the
whole night.
After it has been precipitated and settled, the paste is
dissolved in 500 ml of sterile water, 5 g of NaCl are added, the
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pH is adjusted to 6,0 with 6 M HCl and filtration is started
using depyrogenic plates and sterilizing membranes of o,8 - 0,22
U. The liquid filtered is precipitated with 1 vol. ethanol
filtered, it is anhydrated and dried in vacuum drying stoves at
60 C, during 12 hours.
g of N-acetylated product are obtained, without
free amino groups, but with a chemical composition (see titration
curve), analytical characteristics, and biological and
pharmacological properties similar as a whole to the ones of the
natural HS.
The analytical tipification of the original Heparin, the
intermediate A complex and the final product B are shown here
below.
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Ana]ytical Determ. Sodium Heparin Intermediate A Final Prod. B
APPT Activity150 IU/mg10 IU/mg 10 IU/mg
Anti-X Activity150 lU/mg10 IU/mg 10 IU/mg
N-acetyl 0,8% 0,9% 4.3%
Uronic acid 30,0% 35,0% 31,0X
Hexosamines 30.0% 35'0% 32,0%
Organic sulfate (as S) 11,0%8,2% 7,8%
Specific rotation + 36 ~ 40 1 40
Molar Relation uronic/
-hexosamine/S/acetyl 1:1:2,2:0,1 1:1:1,7:0,1 1:1:1,7:0,5
NH/N total 0,0 o,5 0,0
The figures 1 to 3 are the 13C-NMR spectra of the following
compounds:
fig. 1: final product B; fig 2: Heparin; Fig. 3: Heparansulfate.
The figures 4 to 7 represent evalutations as per the
following details:
fig. 4,6,7: B-A= carboxylic uronic groups
A= sulfate groups
fig. 5 : B-A= carboxylic uronic groups
C-tB-A)= sulfate group~
C-B= free amino groups
Therefore, being specially described and determined the
nature of this patent application, and the corresponding way to
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put the same into effect, it is hereby claimed its exclusive
right and property.
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