Note: Descriptions are shown in the official language in which they were submitted.
~ 2~970
wo 92/11294 Pcr/~Ps2~o~7
Hepar 1 n derl vat 1 ve s
Background and Summary of th,e Invention
The pre~ent invention concerns new heparin
derivatives with pharmacological properties which ar~ ,
modified with respect to those of the heparin
preparations currently available on the market and used
in anticoagulant therapies. The relationship between the
anti-thrombin activity and the activity on the Xa factox a~d the
platelet factor 4 (PF4) of the new derivatives diffexs
from that of heparin. In other words, their considerable
affinity for PF4 is accompanied by reduced activity on
the reaction between anti-thrombin III and thrombin,
catalyzed by heparin.
In parallel, the reaction between antith_ombin III
and the.Ya factor, catalyzed by heparin itself, is even more
sensitive to the new derivatives. Because of their
different ratio of activity from that of heparin, on PF4
and on the reactions activated by thrombin and Xa factGr,
the derivatives of the invention are able to pravent -'
thrombosis without the risk of hemorrhage which
accompanies the anti-thrombotic action of heparin. It
,~ must be noted that hemorrhagic activity can be caused by '''
an excessive action of thrombin inhibition. The ability '
of the new derivatives of the invention to bind PF4, to
catalyze at low concentrations the reaction activated by the
Xa factor, and only at higher concentrations reactions
catalyzed by thrombin, make these compounds suitable zs
drugs for ~he treab~t of arterial and venous thro~kosis.
; The new heparin derivatives and their salts are
obtained by
(1) prolonged treatment of an alkylating
- (etherifying) agent derived from a hydrocarbon with more
than 6 carbon atoms, at room temperature or slightly '~
over, with a quaternary ammonium salt of heparin,
dissolved in a heterocyclic organic solvent chosen from
; N-hydrocarbyl-pyrrolidine-2-one or
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N-hydrocarbyl~piperidine-2-one, not substituted or
substituted by lower alkyl groups, and their derivatives
interrupted in the heterocyclic ring ~y a heteroatom or
hetero group chosen from 0-, -S- or -NH- or in a
concentrated solution of one such compound in an aprokic
solvent,
(2) treating the reaction product at a high
temperature with an organic or inorganic base in a~eous
solution,
(3) isolating the heparin derivatives obtained ln
free form or their alkaline salts, and, if desired,
(4) converting the resulting compounds into the
free form or into the alkaline salts or as salts of
other metals or organic bases.
The new heparin derivatives, whose aforesaid
pharmacological properties are different from those of
heparin, also have a modified chemical structure and
different molecular weight; their chemical structure has
- not yet been completely defined. It has however been
ascertained that they:
a) constitute a mixture of mostly depolymerized
products and therefore have a medium-to-low
molecular weight compared the
starting products, which are rich in fragments
similar to one another and with molecular weights
coming within the same narrow range, and with a
minimum of fragments which deviate from this mean.
b) Compared to their starting products, they have a
notably diminished ratio between the iduronic-type
saccharide unit and the glucuronic-type unit.
Other modifications of the chemical structure, with
respect to heparin, which can be found in the new
derivatives, are in part also those which have already
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been described for some heparin fragments obtained by
alkaline treatment of heparin esters, for example
desulfation and the presence of unsaturated monosic
units.
The identification of the new derivatives can be
carried out, a~t from said chemical tests, by
spectroscopy, especially nuclear magnetic resonance
(NMR), which allows in particular to differentiate the
new derivatives from heparin fragments already described
in the literature.
The treatment of a quaternary ammonium salt of
heparin with an alkylating agent chosen from those
commonly used, that is, derived from a lower hydrocarbon,
such as a lower alkyl halide, for example ethyl bromide~
in a normally used solvent, such as dimethylsulfoxide or
dimethylformamide, leads to heparin esters, such as those
described in British patent No. 1501095. If these esters
undergo alkaline treatment, such as that included in the
second phase of the procedure of the present invention,
different heparin fragments are obtained, as can be
demonstrated by NMR analysis.
The new low-molecular-weight heparin derivative descr:~d in
the examples, constitutes a novel product, which can
be characterized mainly on the basis of NMR spectroscopic
25 data at high frequency (400-500 MHz for the proton analysis;
100-125 MHz for C-13 analysis), utilizing traditional
one-dimensional spectres in FT (Fourier Transformation),
corroborated with the latest two-dimensional techniques
(H-H and C-H correlation), optionally resorting to more
recent methods (e~g. TOCSY - total correlation
spectroscopy), wher2 necessary for the study of such
complex products.
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WO92/1129~ PCr/EP~3/02479 ~;
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From an overall examination of the results
obtainable by the cited methods, it can be determined
that:
1) The new "low-molecular-weight heparin" of the
invention is a complex mixture of oligosaccharides,
sulfated both at N and at O, reflecting the
heterogeneity of the starting heparin. Its
fundamental constituents IdoA and GlcnAc,
differently sulfated, and secondary constituents
(essentially GlcA) link together, giving rise to
different sequential combinations.
2) With respect to the starting heparin, the product
which is the object of the present invention shows a
considerable decrease in the IdoA/GlcN ratio, this
decrease being detectable by NMR. Accordingly, the
signals of the anomeric sites of the typical IdoA
seguence (2SO3)-GlcNSO3) (6SO3) (102 and 99.5 ppm for
carbon; 5.18 and 5.32 ppm for the proton) prove to be
drastically diminished. Such transformations cannot
be detected in the known, low-molecular-weight
; heparins, or if they can, it is to a far lesser
degree. Concomitantly, markedly intense siynals
appear of a new unsaturated monosaccharide unit (see
point 4).
3) Another characteristic fact is the appearance of new
signals (at 54.2 and S3.3 ppm for carbon,
correlatable to protons at 3.73 and 3.68 ppm
respectively) which is compatible, in the present
context, only with the 2-position of a GlcN residue,
with no SO3H groups at the nitroqen). This
characteristic, dete~ctable by 2-D NMR, is
- practically absent in samples of Xnown,
low-molecular-weight heparins.
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WO92/1129~ PC'r/~ 79
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4) Typically, most of the aforesaid oligosaccharides
terminate with an unsaturated monosaccharide unit,
three-substituted in positions 1-2, as can be shown
by NMR (signals at 147 and 172 ppm in C-13: doublet
at 6 ppm for the proton). These signals are absent
or very reduced in the other known,
low-molecular-weight heparins.
It must therefore be concluded that as early as the
first step of the procedure of the present inventionj
what happens is not just a simple esterification, but a
more complex chemical reaction.
It can therefore be assumed from these findings that
the heparin-like products of the present invention are
new and different from the heparin fragments already
described in the literature. For example, they are
different from the fragments described in European patent
No. 0 302 034, obtained by alkaline treatment of heparin
esters, these fragments being suitable for the formation
of complexes with copper ions having an angiogenic
action. In the same way, the heparin fragments described
in U.S. patent No. 4,440,926, obtained starting from
well-defined heparin esters by treatment with NaOH
(0.1-0.5 N) between 20 and 60C, are not identical to
the products of the present invention.
The aforesaid qualities of the new heparin
derivatives of the present invention can be demonstrated
by the following experiments, performed on the produck
described in the illustrative Examples and abridged to the
code name PE. In the experiments, unfractionated heparin
(UFH) and a low-molecular-weight heparin derivative (CY
2l6 = Fraxiparine~) are used as comparison products. Both
these products are commercially available for the
treatment of thrombosis.
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WO92/11294 PCT/EP9~/0~7
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Brief Description of the Drawinqs
Figure l shows the anticoagulant activity in vitro
of PE compared to UFH and CY 216 in terms of thrombin
time (sec.). The data obtained are mean values of 7-l0
replications for each test product.
Figure 2 shows the ex vlvo anticoagulant activity of
PE, compared to UFH and CY 216, according to the e~fect
on thrombin time after i.v. administration. The data are
mean values from experiments on 5 rabbits for each test,
product.
Figure 3 illustrates the ex vivo anticongulant
activity of PE, compared to UFH and CY 216, based on
anti-FXa activity (disappearance kinetics after s.c.
administration). The data are mean values from
experiments on 5 rabbits per test product.
Figure 4 shows the ex vivo activity for PF4 after
i.v. administration of PE, in comparison to UFH and CY
216.
.
1. ANTICOAGULANT ACTIVITY IN VITRO ON RECONSTRUCTED
HUMAN PLASMA
Materials and Methods
Test products (solubilization and concentrations)
The following products were tested:
- heparin derivative PE
- unfractionated heparin (UFH)
- low-molecular-weight heparin derivative (CY 216 -
Fraxiparine~)
The test products were dissolved in sterile saline
and tested at concentrations ranging from 0.5 to
t2.5 ~g/ml.
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Parameters
1. Thrombin time (as an indicator of anticoagulant
activity)
2. Anti-FXa activity (as an ~cator of anticoagulant
activity)
Results
l. Thrombin time
From Fig. l it is clear that:
- PE has anticoagulant activity starting at
concentrations of l.25 ~g/ml
- The anticoagulant activity of PE is considerably ~.
inferior to that of unfractionated heparin UFH
(thrombin time increases at concentrations about
4-5 times greater than UFH).
No significant differences from CY 216 can be
observed.
2. Anti-FXa activity
In Table l, the preliminary data indicate
that: .
- PE has anti-FXa activity starting from
concentrations of 3 ~g/ml
- the anti-FXa activity is distinctly inferior to
that of heparin.
It must also be noted that this activity is also
inferior to that of CY Zl6; in this case, however,
the difference is less e~ident than that observed
in comparison to UFH.
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Table 1
ANTI-FXa ACTIVITY IN VITRQ OF PE IN COMPARISON TO
UNFRACTIONATED HEPARIN AND CY 216
5 CONCENTRATIONS UF~ CY 216PE
(~g/ml)
0~5 0.336 - -
1 - 0.3310.342
1.5 0.281
2.5 0.212
3 - 0.2610.296
3.5 0.179
- 0.2000.245
7 - 0.1580.201
The data are means of 3 experiments per concentratio~,
20 one for each test product.
`; '
2. ANTICOAGULANT ACTIVITY EX VIVO ON R~BBIT PLAS~
AFTER I.V. AND S.C. ADMINISTRATION
.
The anticoagulant activity o~f the new heparin
derivative PE was assessed by ex vivo methods on rabbit
plasma. By "ex vivo" is meant in vitro measurement of ~: :
blood withdrawn from an animal to which the chemical
substance has been administered. Examined in particular
were the fol'lowing:
a) thrombin time, on arterial blood after acute bolus
intravenous administration (i.v.) of the test
products;
b) anti-FXa activity, on arterial blood after acute
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subcutaneous administration (s.c.).
This method, being highly sensitive, i5
indicative of the "bioavailability" of the test
products, since it allows the kinetics of the
disappearance of the test products to be
monitored after subcutaneous administration.
The test products were dissolved in sterile saline
solution and administered acutely at 0.86 mg/kg by the
i.v. route and at 1-2 mg/kg by the s.c. route.
Test description
a? Thrombin time
- This test assesses thrombin time on samples of
arterial blood from rabbits at various time
intervals (2-5-10-20-30-40 minutes) after i.v.
administration of a bolus of test product.
b) Anti-FXa activitv
By measuring the anti-FXa activity (by a chromogenic
test which is sensitive at very low concentrations of
compound) it is possible to monitor the presence of the
test products in the circulation for a very long timeO
Samples of arterial blood (from the central artery of the
ear) were taken at various intervals (1-2-3-4-5-6-7-8
hrs) after s.c. administration.
: 25
Results
- a) Thrombin time
: From Fig. 2 it is clear that:
- PE has a low anticoagulant activity at 0.86 mg/kg
i.v., confirming the in vitro data
- the anticoagulant activity of PE is distinctly
inferior to that of UFH, but does not differ
significantly from that ~F Cy 2 1 6 .
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b) anti-FXa activity
Fig. 3 shows that:
- PE (after s.c. administratiun at 1 mg/kg) remains
present in the circulation for longer than UFH,
administered at twice the dose, that is, at 2
mg/kg s.c. -
The disappearance kinetics of the heparin
derivative PE is therefore slower than that of
UFH, but similar to that of CY 216.
` 10
3. EX VIVO AFFINITY FOR PLATELET FACTOR 4 (PF4)
By the experiments described hereafter, the affinity
o~ the new heparin derivative PE of the present invention
lS was assessed for human PF4, i.e. the ~binding properties"
of such products for PFb.
The ex vivo technique used monitored the kinetics of
the disappearance of PF4 from the circulation in the
absence and in the presence of heparin.
It is well known that heparin or glycosaminoglycans
(GAGs), administered before PF4, markedly enhance its
kinetics (G. Cella et al., "Human platelet factor 4 and
protamine sulphate interaction with glycosaminoglycans in
the rabbit", Eur. J. Clin. Invest., Vol. 17, pp. 548-554
(1987~.
Compounds able to bind PF4 should therefore greatly
prolong its presence in the circulation. This effect is
; proportional to the quantity of GAG injected and to its
` affinity for PF4.
The test products were dissolved in sterile saline
solution and administered at 0.86 mg/kg i.v.
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DescriPtion of the test
Administrati.on of the test products was acute
(intravenous bolus), at a dose of 0.86 mg/kg. Samples of
arterial blood were taken 2-3 mins later from the central
artery of the ear (baseline sample). 5 minutes after
administration of the first bolus, a second bolus of
purified human PF4 was given (30 ~g/kg).
Samples were taken 1.5-2.5-5-10-20-30 minutes after
administration of PF4. These were centrifuged, stored and
assessed by the RIA test (commercially available kit) for
human PF4.
Results
As reported in Fig. 4, the data obtained indicate
that:
- PE has affinity for platelet factor PF4 and
gradually disappears from the circulation reaching
low values about 30 minutes after administration.
- The affinity of PE for PF4 i~ much lower (about 60%)
than that of UFH and the kinetics of the
disappearance of the complex with PF4 is far more
rapid than that of UFH, while it is similar to
that of CY 2160
4. ANTITHROMBOTIC ACTIVITY IN VIVO IN A MODEL OF
ARTERIAL THROMBOSIS IN RABBIT
The experiment described hereafter assessed the
antithrombotic activity in vivo of the new heparin
: 30 derivative PE. The objective was to test the
efficacy of the products in preventing the formation
of àn arterial thrombus in an acute model of
arterial thrombosis in the rabbit carotid artery, following
endothelial damage and reduction of the vascular
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WO92/11294 2 ~ 7 ~ ~ 7 ~ pcrlEp9l/o2~7~J~
diameter.
The test products were dissolved in sterile saline
and administered by the i.v~ route at concentrations
ranging from 1.2 to 6 mg/kg.
Descri~tion of the model
The experiments were performed on rabbits
anesthetized with Nembutal (30 mg/kg i.v., bolus) arld
tracheotomized for forced breathing. A tube was inserted
into the femoral vein and artery for the continuous
administration of anesthetic and drug. Systemic blood
pressure was monitored and the carotid was fixed with a
flowmeter and snare. When the system stabilized, stenosis
was p~ormed. After suitable intervals tnot less than 20
minutes), mechanical damage was induced with surgical
forceps and sténosis was performed above the damaged
vessel. The decrease in flow was moni~ored until it
re~ched the value zero (0), corresponding to occlusion of the
vessel. The controlateral carotidwas prepared in the same
way. The test products were ~ infused i.v. for ~ight
minutes in all, with mechanical damage being performed
after two minutes of infusion. The flow was monitored
constantly until at least one hour after the end of
treatment. The animal was sacrificed by an overdose of
anesthetic at the end of the experiment.
:
Results
As reported in Table 2, the data obtained indicate
that:
- PE is effective in completely preventing the
` formation of an occlusive thrombus. The effect is
' evident at a dose of 6 mg/kg i.v.;
; - The antithrombotic efficac~ of PE is inf erior
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(about 5x) to that of unfractionated heparin UFH
(which can ~e seen to inhibik the formation of
occlusive thrombi at a dose as low as l.2 mg/kg), the
pharmacological efficacy of PE is similar to
that of CY 216.
Table 2
Antithrombotic activity of PE in comparison to
unfractionated heparin (UFH) and CY 216 in a model
of arterial stenosis in rabbit
Products l.v. dose No. occluded animals
No. treated animals
UFH l.2 mg/kg 2/12
PE 6 mg/kg 2/12
3 mg/kg 6/8
CY 216 6 mg/kg 2/12
3 mg/kg 5/9
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5. ANTITHROMBOTIC ACTIVITY IN A MODEL OF VENOUS
THROMBOSIS IN VIVO
: The experiment described hereafter was performed to assess
the antithrombotic efficacy in vivo of PE in a
model of venous thrombosis in the rat.
The venous stasis technique was used, by occluding
: 30 the vena cava inferior, as this method causes the
- formation of a mainly fibrinic thrombus due to the
`. variation in blood flow at the level of the bifurcation
o the vena cava with the left renal vein.
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W092/12294 æ o ~ ~ ~ 7 PCI/EP~/024Y9
The model under examination therefore proves to be
sensitive to the activity of anticoagulant drugs and in
particular to the antithrombotic potential of heparin
(after administration and stasis).
The test products were dissolved in sterile saline
and administered intravenously at doses ranging from 0~5
to 3 mg/kg i.v. (15 minutes before stasis induction).
Description of the test
The experiments were performed on male CD-COBS rats,
weighing 175-200 grams. The animals were anesthetised
with sodium pentobarbital (40 mg/kg i.p.) and their
undersides were disinfected with alcohol. A 4-cm incision
was made along the middle of the abdomen starting from
the ribs. The vena cava was isolated from the aorta to
the common iliac artery and tied with a cotton thread
immediately underneath the fork with the left renal vein~
The double occlusion was such as to exert constant ~Id continuous
pressure for 5 seconds. The abdominal wall was then
closed. Two hours later the abdomen was reopened to verify
thrombus formation. After closing off the vena cava, in
correspondence to the common iliac arteries, and the
collateral circulation with a hemostat, the vascular
; section below the occlusion was cut longitudinally with a
needle. The thrombus was removed and immersed in distilled
water, blotted dry on paper and then placed in a dry box
for 24 hours to determine the dry residue
expressed in mg.
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Results
The data relative to the weight (mg) of the thrombi
(Table 3) and incidence (~) of the thrombi (Table 4) two
hours after venous stasis, show that:
- PE has antithrombotic efficacy; its protective
effect (pre-treatment) is significant at a dose
of 1.5 mg/kg i.v.
- the efficacy of PE is inferior to that of
unfractionated heparin UFH (which already has a
lo significant effect at 0.5 mg/kg i.v.)
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Table 3
Antithrombotic effect in vivo of PE compared to UFH
in a venous stasis model: weight ~mg) of the thrombi
obtained 2 hrs after venous stasis (effec.ted 15
minu~es after i.v. administration of the test
products)
.
Doses (mg/kg i.v.)
Products 0 0.5 1. 0 1.8 2 .0 3 . O
n = 1~ n = 14 n= 24 n = 20 n = 14 n = 14
`.
PE 2.2 1.4 1.5 0.8*0.0**0.0**
UFH 2.2 0.7* 0.5** 0.02** - -
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* p < 0.05; ** p < O.Ol Dunnett's test
n = number of animals
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W092~1~294 PCr/EPgl/0247g
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Table 4
Antithrombotic effect in vivo of PE compared to UFH in
a venous stasis model: incidence (%) of thrombi obtained
2 hours after venous stasis (effected 15 minutes after
5 i.v. administration of the test products)
_
Doses (mg/kg i.v.)
___
Products 0 0.5 1.0 1.5 2.0 3.0
n = 15n = 14n = 24 n = 20n = 14 n = 14
_
PE 93.3 85.7 62.5 60.0 0.0 0.0
UFH 93.3 57.1 50.0 14.3 - -
-
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6. EFFECT ON BLEEDING TIME IN RAT
The experiment described hereafter assessed the
effect of PE on bleeding time 15 minutes after
intravenous administration of the test products.The test
products were dissolved in sterlle saline and
administered i.v. at concentrations ranging from 1 to 3
mg/kg.
Description of the test
The experiments were performed on male, CD-COBS rats
(17S-200 grams) in which the test substances were
administered acutely by the intravenous route.The
bleeding time (in seconds) was assessed 15 minutes later
by the standard template method of cutting the tail
(Dejana et al., "sleeding time in rats: A comparison of
different experimental conditions", Thromb. HaemostatO,
Vol. 48, pp. 108-111 (1982)).
Results
The data obtained (Table 5) show that:
- The effect of PE on "bleeding time" is
significantly increased at a dose of 2.0 mg/kg
i .v .
- the effect of PE is notably inferior to that
obtained with unfractionated heparin UF~ (in
which the bleeding value already increases
significantly at a dose of 1 mg/kg i.v.), but it
is comparable to that of CY 216 (data not
reported)`.
` 30 It is interesting to note that in order to obtain
the same valuès for the bleeding time it is necessary to
administer a dose of PE which is at least 3 times
greater than that of UFh.
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Table_5
The effects of PE and UFH on bleeding time
(sec) 15 minutes after i.v. administration of the
test products
Doses (mg/kg e.v.)
Products 0 1.0 1.5 2.0 3O0
n = 14n = 14 n = 14n = 14 n = 14
-
PE 130 146 147 250* 3~9**
UFH 130 413** 487** - -
-
* p < 0.05; ** p < 0.01 Dunnett's test
n = number of animals
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7. IN VIVO TOL~RANCE AFTER I.V. AND S.C.
ADMINIST~TION IN MOUSE
The present experiment assessed the maximum
tolerated dose of the compound PE after
administration by the intravenous and subcutaneous
route in the mouse.
The test products were dissolved-in sterile saline
and tested at concentrations ranging between 125
and 2000 mg/kg i.v. and s.c.
Descri~tion of the test
The experiment was performed on male and female
CD-l mice (C. River) (25-35 grams) and comprised two
steps.
l) Screening. Groups of one male mouse and one
female mouse were treated with one of the
following doses: 125, 250, 500, 1000 and 2000
mg/kg. Mortality within 7 days of treatment was
monitored.
2~ Groups of five male and five female mice were
treated (for each compound and each
administration route) with the highest
: tolerated dose found on screening. The animals
were observed for 14 days, during which
mortality and the presence of altered behaviour and
general toxicity symptoms were observed. In
the case of mortality a lower dose was used,
until the maximum tolerated dose was found.
Where possible, autopsy was performed on the
deceased animals.
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Results
1. Intravenous tolerance dose
As reported in Tables 6 and 7, the maximum
tolerated dose of the test compounds proved to
be the following:
- PE: 1000 mg/kg i.v.
- unfractionated heparin UFH: 125 mg/kg i.v.
- CY 216: 1000 mg/kg i.v.
It must be noted therefore that PE has greater tolerance
lo dose than unfractionated heparin UFH.
Autopsy of the animals which died after
treatment with unfractionated heparin UFH at 500
and 250 mg/kg, reve~led no macroscopic findings
except, in some animals, the presence of
probable residues of internal hemorrhage in the
intestine.
2. Subcutaneous tolerance dose
The results obtained, as reported in Tables 8 and 9,
show that the maximum tolerated doses of the test
products are as follows:
- PE: 2000 mg/kg s.c.
- unfractionated heparin UFH: 125 mg/kg s.c.
- CY 216: 2000 mg/kg s.c.
25 As with the intravenous route, therefore, the
.tolerance dose of PE is far greater than that of
unfractionated heparin (UFH), while it is similar
to that of CY 216. It is however important to highlight
that:
- In all the animals (10/10~ treated with CY 216 (2000
mg/kg s.c.) a circular area of skin affected by
necrosis was observed on the back, corresponding to
the injection site, the size of which differed
from animal to animal (maximum diameter about 1 cm).
.
. . . .
,: , . -
.. . .
WO 92/11294 2 0 7 S 9 7 ~ I'CI/EP91/0~47') ~
-22- !
.
Autopsy revealed necrosis to be limited to the skin,
it did not therefore affect the underlying muscle~
- No anomalies were observed in animals treated with
PE (2000 mg/kg s.c.).
- The animals which died after treatment with UFH (250
mg/kg) presented extensive bruising in the
subcutaneous dorsal region, where treatment had been
performed.
The subcutaneous tolerance data therefore show a
lo difference between the compoun~s PE and CY 216.
Indeed, although the maximum tolerance doses are the
same, in the case of CY 216 a marked area of
necrosis of the skin is present at the injection
site, which indicates that CY 216 is tolerated to a
lesser degree locally than PE (which presented no
anomalie,).
'' ' , .
. .
. : ,
. :
.. . .
:. .
- - . . : :
07~97~
O92/11294 P~!EP~]/02479
-23-
Tabl e 6
Lethality of compounds PE, CY 216 and unfractionated
heparin UFH after a single intravenous administration
(screening)
, . . _ . .
Treatment Deceased animals/Treated animals
(mg/kg i.v.) males females
10 PE 2000 0/1 0/1 : :
PE 1000 0/1 0/1
PE 500 0/1 0/1
PE 250 0/1 0/1
PE 125 0/1 0/1
CY216 2000 1/1 0/1
CY216 1000 0/1 0/1
CY216 500 0/1 0/1
Heparin UFH 2000 1/1 1/1
Heparin UFH 1000 1/1 1/1
Heparin UFH 500 0/1 0/1
Heparin UFH 250 0/1 0/1
Heparin UFH 125 0/1 0/1
; ~ .,. : ' ,
''''' ' ~, ' '', ~ ' ' ., , , ' :, . ' . . .
'. : '. . ' :'
: ,
. . .
-
207597~ 1
WO92/11294 PCT/EP'31/0247')~
-2~-
Table 7
Lethality of compounds PE, CY 216 and unfractionated heparin
(UFH) after a single intravenous administration
. . _ ~
Treatment Deceased animals/Treated animals Tlme of death
(mg/kg i.v~)males females days after
~ treatment
immëdiate* 1 2 3 4
1 0
PE 2000 3/7 1/5 2 - 1 - 1
PE 1000 0/5 o/5 - - - -
CY216 10000/5 0/5
Heparin 5000/5 2/5 - 2
UFH 250 3/5 0/5 - 3
125 o/5 0/5
*Within minutes of tre~tment
~ ' .
., .
. :
' ~'~' ~ ''"' '' ' '' ''` ' '' " ' '. ,' ' ' ' ' ' ' ' " '
. ', ' ' ' ' ' '
' ~ ' ', . '. ' '' ' ' . ,''
'
~ ~O92~11294 ~ 7 ~ 9 ~ ~ pcr/~ 79
-25-
Table 8
Lethality of the compounds PE, CY 216 and
unfractiona~ed heparin UFH after a single
subcutaneous administration (screening)
_
Treatmen~ Deceased animals/Treated animals
tmg/kg s.c.) males females
PE 2000 0/1 0/1
PE 1000 0/1 0/1
PE 500 0/1 0/1
PE 250 0/1 0/1
PE 125 0/1 0/1
CY216 2000 0/1 0/
CY216 1000 0/1 0/
CY216 500 0/1 - o/
Heparin UFH 2000 1/1 1/1
~eparin UFH 1000 1/1 0/1 .
Heparin UFH 500 1/1 0/1
Heparin UFH 250 0/1 0/1
Heparin UFH 125 0/1 0/1
- ., - - . . ..
. -- - -, .. . : ,. . .
, - ,- - ... . ..
. - - : - : ., ... :. -:. : : , . .
: - . : . : , . . .
- - - .. ... : .. " .. , .: : : ... : . . . .:.
. . .. :.: . .. ~ - . - , ..
2~7~70
WO92/11294 PCr'/~P91/~2~'7~)
-2s- .
'rabl e 9
Lethality of compounds PE, CY 216 and unfractionated heparin
(UFH) after a single subcutaneous administration
Treatment Deceased animals/Treated animals Time of deat~
(mg/kg i.v.) males females treatment
immediate 1 2 3
PE 2000 0/5 o/5 _ _ _ _ _
CY216 2000 0/5 0/5 -
Heparin 250 1/5 2/5 - 2
15 UFH 125 0/5 0/5 ~
~ .
.
' '' ~ . ' '': ' ' . :
. . . . . .
." ' ' ; ,", . .
. . ~ ,
.. . .
-W092/11294 PC~ 10~75
-27-
CONCLUSIONS
The in vitro and in vivo results previously
described outline an interesting pharmacological profile
of the heparin derivative PE, setting it apart from
unfractionated heparin (UXF) and rendering its
pharmacological activities more valuable.
The activity of PE differs slightly ~rom that of CY
216, which i5 one of the best low-molecular-weight
heparins currently on sale in France, and shows in
particular better local tolerability after s.c.
administration in the mouse. Indeed, although the
maximum tolerated doses are the same, all animals treated
with CY 216 (2000 mg/kg s.c.) had evident necrosis
of the skin at the injectidn site, while the skin of the
animals treated with PE was unaffected.
In the case of pharmacological activity in vitro and
in vivo, it is interesting to note that PE has:
- anticoagulant activity, far inferior to that of
UFH;
- bioavailability, a~ter s.c. administration, much
greater than that of UFH, thereby allowing fewer
daily administrations for the same pharmacological
effect;
- lesser side effects, with regard to UFH, as shown
by the reduced bleeding time and by the absence of
skin necrosis at the injection site, with regard
to CY 216 (the best low-molecular-weight heparin
; on sale in France) which caused a local
intolerance phenomena (necrosis) in all the
animals treated;
- antithrombotic activity in vivo, as shown in both
the model of venous stasis in the rat and in the m~del of
arterial thrombosis in the rabbit. The product is
therefore efficacious in preventing these
.
.
'; :,, , . , . ,, ,, .' ,' , .- . - : : ,,
- : . :. :. . .. . : ...
~,arl~7 ~
WO92/11294 pcr/E~9l/o2~7s
28-
pathologies at dosages of 1.5 and 6 mg/kg i.v.,
respectively, which do not induce any signif icant
variations in the side effects.
In particular, with regard to the ven~us antithrombotic
activity, the derivative PE, at
a dose of 1.5 mg/kg i.v., does not induce:any
alterations in bleeding time, unlike UFH;
- The new, low-molecular-weight heparin derivatives
according to the present invention can therefore be used
in the place of unfractionated heparin in all its
indications, as an anticoagulant and thrombocyte
aggregation inhibitor. The dose is adapted to each
particular case, and is normally around 1-7 mg/kg per
day, by the intravenous route.
The heparins to be used in the previously described
procedure of the present invention can be of any type and
of various origin, for example heparins extracted from
the intestine of pigs, cattle, and sheep and from ox
; 20 heart, especially any one of the products which are
commercially available or described in the literature.
Such products have widely ranging molecular weights, for
example between 2,000 and 30,000 daltons, especially
unfractionated heparin UFH, and also low-molecular-weight
heparins obtained by fractionation of standard heparins
(2,000-lO,000 daltons) and heparin fragments with a
molecular weight of 500-lO,000 daltons obtained by
~ partial depolymerization of standard heparins by chemical
: or enzymatic means. The quaternary ammonium salts to be
used as starting material for said procedure can be
~` prepared in the known way, for instance by ion exchange
of the heparin in aqueous solution as a sodium or
` potassium salt with resins based on quaternary ammonium
salts, for example a salified sulfonic resin with a
.
- , , . , . : . .
, ~ ~. . '
~ 2~97~
WO 92/11294 PC-r/EP91/024'7g
--29--
quaternary ammonium base. The quaternary ammonium salt
can be obtained by freeze-drying the eluate. As
quaternary ammonium salts, tetraalkylammonium salts
derived from lower alkyls may be advantageously used,
particularly tetraalkylammonium salts with alkyl groups
having a maximum of 6 carbon atoms. Optionally,
alkyl-aryl-ammonium salts may also be used, for example
those having long-chained alkyl groups. of the
tetraalkylammonium salts, tetrabutylammonium salts are
preferably used. The quaternary ammonium salts to be used
as starting substance for the procedure of the present
invention are those obtainable by ion exchange i~ the
aforesaid manner using alkaline salts, for example sodium
or potassium salts, of heparin of the type used
commercially, that is, neutral salts. They are reacted
with an excess of quaternary ammonium ions so as to
obtain also the corresponding neutral salts.
Quaternary ammonium salts are soluble both in the
aforesaid heterocyclic solvents, and in aprotic organic
solvents, such as dimethylsulfoxide and
dimethylfor~amide. The reaction with khe alkylating
; agent, according to the first step of the procedure, can
be performed both in the aforesaid heterocyclic solvents,
or in a solution of the same in one of the aforesaid
aprotic organic solvents, prefexably in a concentrated
solution. Of the aforesaid heterocyclic solvents, the
preferred ones are above all those which are not
substituted in the cyclic structure, that is,
N-alkyl-o~aryl-2-pyrrolidone, or their derivatives
containing a heterocyclic atom in the cyclic structure,
such as the corresponding derivatives of imidazoline,
piperazine or morpholine. The N-alkyl groups of
; heterocyclic solvents are derived preferably from a lower
~ alkyl with a maximum of 6 carbon atoms, and the aryl
'~:
.. . .. . .
~,. . . ,., ' '" ' ~ ,. ' '
,. - ~ , . . , , ~ .:
- . : . , . , ~ :
.
'': .,~.. , -, ' , ' : :. ,
20~970 ~ I
WO 92/11294 PCr/EP(~/02~'79
-30-
group is primarily a pheny1 group, optionally substituted
by 1 to 3 lower alkyl groups, especially methyl groupsO
Preferably, N-methyl-2-pvrrolidone is used.
An alkylating agent derived from a hydrocarbon
having from 6 to 30 carbon atoms, preferably from 8 tQ 18
carbon atoms, is a compound easily capable of yielding
the corresponding hydrocarbyl groups, such as a reagent
ester group of an acid with an alcohol with a
corxesponding number of carbon atoms, which can be an
aliphatic or araliphatic alcohol having preferably a
maximum of 18 carbon atoms, for example a hexyl, heptyl,
octyl or nonyl alcohol. The reagent esters can be derived
from inorganic or organic acids, such as hydracids,
sulfuric or sulfurous acid, or alkyl- or aryl-sulfonic
l~ acids, for example methane-sulfonic or p-toluenesulfonic
acid. The esters of the hydracids are in particular
chlorides, bromides and iodides. The reaction between the
quaternary ammonium salt of the aforesaid alkylating
agent is performed at room temperature (20C.)or slightly
higher, for example at a temperature of from 30 ~ 35Co
but not exceeding 60C, and is maintained for a prolonged
period of time of about 5 to 20 hours, typically about 16
hours.
The reaction product can be isolated and
subsequently submitted to the second step of the
procedure, that is, to alkaline treatment, or it can be
converted directly into the final product, performing the
alkaline treatment directly on the solution, optionally
concentrated, of the reaction product of the first step.
3 0 It is also possible to evaporate the solvent under bland
(mildj conditions after the first step of the procedure
and to perform alkaline treatment on the residue. It is
therefore possible to perform the procedure of the
~ invention as a "one-pot" process. The basic treatment of
:'
-,., . ~. ~ :
-'WO92/1129~ ~ O~ PCT/~ Z47
-31~
the reaction product between the quaternary ammonium salt
of heparin with the alkylating agent is performed at a
temperature of from 5O to 120 C ., suitably at a
temperature between 50C and 120C, preferably about
70OC, for about two hours. Alkaline hydrates are used~
such as NaOH or KOH, or other inorganic and/or organic
bases, preferably at a concentration of between 0.l and
lM. The bases are used in aqueous or alcoholic solutio~s/
but other solvents can be present which are miscible with
water or with an aqueous-alcoholic mixture, such as, for
example, the residue solvents used in the first step of
the procedure in the case of the aforesaid "one-pot"
process.
If it is desired to isolate the reaction product of
the first step of the proc-dure, it can be precipitated
by the addition of an organic polar solvent, preferably
an aliphatic alcohol such as methyl or ethyl alcohol, to
which a basic buffer has preferably been added, such as
especially a basic salt of a carboxylic acid, e.g., an
acetate or propionate of sodium or potassium. Other basic
buffers can be used, such as alkaline bicarbonates or
alkaline phosphates. The precipitated product is
preferably washed with the polar solvent used in the
precipitation, for example methanol. It is advisable to
purify the precipitated product further by
reprecipitating it once more or several times, that is,
by dissolving the product in water and precipitating it
with an alcohol.
- The reaction product, comprising the
low-molecular-weight heparin derivative and having the
previously described properties, can be released in free
form either from a metal salt or from an organic base in
the conventional way. The alkaline salt corresponding to
the alkaline hydrate used in the second step of the
.
~` . ' " ' ' ~ ~ '
~ ' ' ~ , , ' . , .
. ~ .~ , ~, ' , ' ' ' .
207~7~ f~7;,
WO92/1l294 PCI/EP~1/02~7l)
-32-
procedure may be obtained by neutralizing the solution
with an acid, e.g., 2M hydrochloric acid, purifying the
solution, extracting it with an organic solvent which
cannot be mixed with water, for example methylene
chloride, and repeating this operation several timesO
The salt is then dialyzed with distilled water and sodlum
chloride and freeze-dried.
The products of the invention are used mainly in the
form of their metal salts or organic base salts. If it ls
desired to obtain metal salts other than alkaline sal~s
corresponding to the ions of the alkaline hydrates used
in the aforesaid alkaline treatment, it is possible ko
use ion exchange methods by known t~chniques.
Alternatively, it is possible to isolate the heparin
product in its acid form, adding the alkaline salt
obtained with the calculated quantity of a diluted strong
acid, such as hydrochloric acid or sulfuric acid,
extracting the product with a suitable organic solvent,
isolating the heparin compound in ~ree form and then
converting it into the desired salt.
Of the metal or organic base salts which can be used
according to the present invention, of particular
importance are those which are therapeutically
acceptable, such as alkali and alkaline-earth metals, for
example sodium, potassium, ammonium, calcium, and
magnesium salts, or heavy metal salts, such as copper and
iron salts. The salts of organic bases can be derived
~rom primary, secondary or tertiary aliphatic, aromatic
or heterocyclic amines, such as methylamine, ethylamine,
propylamine, piperidine, morpholine, ephedrine,
furfurylaminecholine, ethylenediamine and aminoethanolO
Salts which cannot be directly used in therapy,
which are also part of the invention, are for example
those which can be used for the purification of the new
'
:
: ,
~ .
, ~- ~ ' ' ''' ' .
.,, ~ " , - .
3 9 7 ~ 1
' W092/11294 PCr/EP~1/02~79
-33-
heparin derivatives of the invention, such as some of the
heavy metal salts.
The invention also includes pharmaceutical
preparations containing as active substance a new
low-molecular-weight heparin derivative obtainable by the
procedure described herein, especially in the form o~
their therapeutically acceptable salts. Such
preparations can be for parenteral use, for example for
subcutaneous or intravenous administration, or for
topical use, for example in the form of creams or
ointments, or suppositories, or nose sprays. They can
therefore be formulated as solutions of the active
compounds or as freeze-dried powders of the active
compounds to be mixed with one or more pharmaceutically
lS acceptable excipients or diluents which are convenient
for the aforesaid uses and with an osmolarity which is
compatible with physiological fluids.The invention
includes, besides the new low-molecular-weight heparin
derivatives, the aforesaid procedure for their
p:eparation. Included in the invention is also the
performance of the single steps of the preparation
procedure, that is, the reaction of a quaternary ammonium
salt of heparin with an alkylating agent as defined above
in on of said solvents and the alkaline treatment of the
1 25 reaction product.
The invention is illustrated in the following
Examples which, however, are not to be considered as
limiting.
' ..
.
. .
- ~ . - .. - . - . . .
.. . . .. .. . ..
, . , ~ .:
:, . -. . , ~ . : ~ .
.... . . ... : , , : , .
- : , - : , , . ; . . : . .,
: :. ,, . : . :: .
: . : , . . . .
.. . . . . . .. ... .. . .
: - , . . .. . , :. ~ :
- .. . ... : . ,, : ,. .: . .
. . .. . . . .
207597~ ~ '
W092/11294 PC~'/EP91/0247~ i
-34-
1) F RMULATIONS FOR INJECTION
1 . 1
PE mg 10 20 30 40
water for injection in ml 0.1 0.2 0.3 004
Package with ready-to-use syringe containing
sterile solution for subcutaneous admlnistration~
: 1.2
PE mg 150 300
other components:
water for injection in ml 4
Package with vial containing sterile solution for
intravenous administration.
.
15 1.3
PE mg 10 20 30 40
other components:
sodium chloride mg 0.3 0.6 0.9 1.2
- water for injection in ml 0.1 0.2 0.3 0.4
Package with ready-to-use syringe containing
sterile solution for suboutaneous administration.
. . - ' ' ' '
. . .
~ , . . .
.. - I .
-
.: - ., . . . . :
:', ': . . ' -
- W092/11~94 PCT/~P~1/02~7
1.4
PE mg 150 300
other components:
sodium chloride mg 35 70
water for injection in ml 5 10
; Package with vial containing sterile solution ~or
intravenous administration.
1.5
PE mg 150 300
other components:
sodium chloride mg 35 70
sodium metabisulfite mg 5 10 ~ .
chlorbutanol (*) . mg 25 50
; 15 water for injection in ml 5 10
Package with vial containing sterile solution for
multidose intravenous administration.
(*) in Example 1.5 the preservative chlorbutanol can
20 be substituted by:
benzyl alcohol mg 50 100
or by:
chlorocresol mg 5 10
~ ,,
.
,, , . , ,, ., ~. " . ", -
,- . . .. : ,.. . .. , : , . ... . .. . .. .. .. . . . . .
.,: .. .. . , , . , , : . . ,., : ,, ., ",... . , .: . :. . : . ., . , : ,-
. - .. ,: : : .
.. : -:: . : .: : .,::: . .. : .. . . .
: , :- -. : .... , . : . .. .
.: - . , ~ : . -- ,
.. .. - , , , - "
,: , .,, : . , , :
WO92/11294 2 0 ~ ~ 9 7 0 -36- PCr/EY~1/1)2~7~)
2) FORMULATIONS FOR ORAL ADMINISTRATION
2.l (pill or capsule)
PE mg 3060 120
other components:
phosphatidylcholine ~
~ phosphatidylserine (*) mg 80160 320
: lactose mg 50l00 200
microcrystalline
cellulose mg l020 40
talc mg 3 5 12
colloidal silica mg 3 6 12
(*) the ratios between the phosphatidylcholine and
phosphatidylserine present in the formulations may
vary
2.2 (pill or capsule)
PE mg 3060 120
other components:
phosphatidylcholine +
phosphatidylserine (*) mg 80160 320
cholesterol mg 2 4 8
lactose mg 50l00 200
- microcrystalline
cellulose mg l020 40
talc mg 3 6 12
colloidal silica mg 3 6 12
; ~*) the ratios between the phosphatidylcholine and
phosphatidylserine present in the formulations may
30 vary.
-' ,
~: . -,. , . , . . :
, ~ . , , .
. ':, ,,: ': ' , . ,~ .
. . .. .... . . . .
.~ : , . . : . " ,, . : . . :
~ 092/11294 2 ~ ~ ;3 9 ~ Opcr/E~)l/o2479
-37-
2.3 (packet of powder)
PE mg 60120 180
other components:
phosphatidylcholine ~
phosphatidylserine (*) mg250 500 750
cholesterol mg S lo 15
lactose mg 100 200 300
fla~oring and
sweetener as required
sucrose (**) to mg20004000 6000
(*) the ratios between the phosphatidylcholine and
phosphatidylserine present in the formulations may
vary
(**) sucrose may be substituted by fructose
2.4 (retard effect tablet)
PE mg 30 60 120
other components:
phosphatidylcholine +
phosphatidylserine (*) mg80 160 320
lactose mg 30 60 120
microcrystalline
;; 25 cellulose mg 5 10 20
hydroxypropyl-
methylcellulose mg 10 20 40
talc mg 3 6 12
colloidal silica mg 3 6 12
(*~ the ratios between the phosphatidylcholine and
phosphatidylserine present in the formulations may
vary
- ' :
''
,
.
,
. .
.~: . .. ,, . . ,, . . , : . . .
: ~ . ,, . - ,........ .: . . : .
- , .: . . . : .. . .. ~ :
~: . , : , .
.~ , . . . .
WO92/1]294 2 0 l 5 !~ 7 ~ pc~/Epl)l/n247t) ~;
-38- ~
. . ,
2.5 (gastroresistant tablet)
PE mg3060 120
other components:
phosphatidylcholine +
phosphatidylserine (*) mg 80 160 320
lactosa mg 50 100 200
microcrystalline
cellulose mg 10 20 40
talc mg 30 60 120
colloidal silica mg 3 6 12
methacrylic acid
copolymer mg 15 30 60
(*~ the ratios between the phosphatidylcholine and
phosphatidylserine present in the formulations may
vary
.
2.6 (gastroresistant, retard effect tablet)
PE mg 30 60 120
other components:
phosphatidylcholine +
phosphatidylserine (*) mg80 lS0 320
: lactose mg 50 100 200
mi~rocrystalline
cellulose mg 10 20 40
hydroxypropyl_
methylcellulose mg 5 10 20
talc mg 30 60 120
colloidal silica mg ~ 6 12
. 30 methacrylic acid
copolymer mg 15 30 60
~` (*) the ratios between the phosphatidylcholine and
phosphatidylserine present in the formulations may
Yary
`
wo92/1129~ 207~ i3 7 o pcr/Ep~ ItU~79
- 3 g -
2.7 (solution to be administered orally as drops)
PE mg 30 60120
other components:
citric acid mg 7.5 15 30
purified water to ml 0.250.5
2.8 tgranules *)
PE mg 30 60120
other components:
citric acid mg 7.5 15 30
sucrose mg 30 60 60
corn starch mg 12.5 25 25
talc mg 5 10 10
polyvinylpyrrolidone mg 4 8 8
(*? 3 granules administered in a hard gelatin capsule
2.9 (gastroresistant granules *)
PE mg 30 60 120
other components:
citric acid mg 7.5 15 30
sucrose mg 30 60 60
corn starch mg 12.5 25 25
- talc mg 5 10 lO
polyvinylpyrrolidone mg 4 8 8 .
methacrylic acid .
; copol~ner mg 10 15 15
.', , . : ,
(*) 3 granules administered in a hard gelatin capsule :
. .
' -
, . , ., , ' , . ' ' ' , ~':
,
, , :.. ..
, . . , . :
' ':
WO 92/11294 2 0 7 ~ ~ 7 0 Pcr/E~l/~2~79
--40--
3. NASAL OR PUL~ONARY FORMULATIONS
3.1
PE fine white powder mg 20 40
The active principle, as a fine white powder, is
contained in a gelatin capsule to be broken open
when ready for use and loaded into suitable
apparatus for inhalation.
3.2 (powder for inhalation)
PE fine white powder mg 20 40
phosphatidylcholine +
phosphatidylserine * mg 30 60
'
The active principle, as a fine white powder, is
contained in a gelatin capsule to be broken open
when ready for use and loaded into suitable
apparatus for inhalation.
(*) the ratios between the phosphatidylcholine and
phosphatidylserine present in the formulations may
vary
4. FORMULATIONS FOR RECTAL ADMINISTRATION
4.1
PE mg 30 120
semisynthetic
glyceride mg1940 1880
4.2
PE mg 30 120
phosphatidylserine +
phosphatidylcholinemg 100 200
semisynthetic
glyceride mg1840 1680
.
.. . . . .. . .. :
. ~. , . ., . .. . :
~. ,
:: ',' ' ' , , .
. : .
092/11294 PCT/EP~1/Oz479
-4l~
In summary, the low molecular weight heparin
derivatives and salts ~hereof according to the present
invention are obtainable by treatment of a quaternary
ammonium salt of a heparin, dissolved in a heterocyclic
organic solvent chosen from the group formed by N-alkyl
pyrrolidine-2-one, N-alkyl-piperidine-2-one,
unsubstituted or substituted by lower alkyl groups, and
their derivatives interrupted in the heterocyclic ring by
another heteroatom or heterogroup selected from -o-, -s-,
and -NH-, or in a concentrated solution of said compound
in an aprotic solvent, with an alkylating (etherifying)
agent derived from a hydrocarbon having from 6 to 30
carbon atoms, preferably from 8 to 18 carbon atoms, at a
temperature of from about 20 (room temperature) to 60~C.
for a prolonged period of time, e.g., about 5 to 20
hours. The resulting reaction product is then treated at
a temperature of from 5 to 120C., preferably about
70C., with an inorganic or organic base in an aqueous
solution. The product resulting from this alkaline
treatment is then isolated in free form or as an alkali
metal or alkaline earth metal salt thereof. Salts of
other metals or organic base salts may be obtained
therefrom by conversion from one to the other.
Preferably, in the second step of the procedure, the
reaction product is treated with an alkaline hydroxide in
aqueous solution. The resulting low molecular weight
heparin derivatives and salts thereof are useful
therapeutically, for example, as anti-thrombotic agents.
- ~ .
- : ,.':~. ., , . ~ . ,
. .:
.:
. ,,:,
~ . ~
