Note: Descriptions are shown in the official language in which they were submitted.
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F I L E, PAN-tN~T H I S A M~#B~EB
#f~ TRANSLATION
A New Use of Antit.hrombin zzI
The invention relates to a new use of antithrombin
ZIZ.
Ant~.thrombin III (AT III) is a plasma protein which
acts to inhibit coagulation by inhibiting thrombin,
factors IXa, Xa, XTs, XIIa and plasmin_
AT III deficiency (or hereditary thromboph:ilia) is
an autosomal-dominant hereditary disease with a
thx-ombosis and embolism tendency because of a reduced
formation of AT III.
An acquired AT III deficier~.cy may, a . g.., occur in
disseminated intravasal cvagulopathies (DIC), sepsis,
cirrhosis of the liver or with nephx~otic syndrome_
Likewise, AT III deficiency symptoms may occur in
case of cardiovalvular prostheses, postoperative,
thromboembolic complications, in. estrogen therapy or in
asparaginase therapy.
1~t present, all these syndrome are treated i.a. by
administering AT rII, application always having been
effected intravenously (cf. von Kries et al., Eur. J.
Pediatr. 144, 191-194 (19BS)).
Heparin is a coagulation-inhibiting polymer
occurring in the body (lungs, liver, thymus, spleen and
basophilic mast cells) and obtained from p-glucuronic
acid and D-glucoaamine, and it comprises several
molecules of su7.furic acid per structural un~.t_ Heparin
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inhibits the activity of thrombin vn e.g. fibrinogen by
binding to AT III, and therefore heparin is also called .
a ao-factor for AT III. Furthermore, heparin also
inhibits the activity of thrambolcinase and, thus, the
conversion of prothrombin to thrombin as well ass the
agglomeration. of thrombocytes and the blood coagulation
reaction.
Heparin/AT IIr has an inhibiting effect on the
arterial blood coagulation factors Va, IXa, Xa az~.d xlla
and has an activating effect on lipoprotein lipase_
Heparin treatment may, however, also have serious
side effects. It is assumed to be the cause of heparin-
induced thrombocytopenia and a consequent risk of
thrombosis of immunologieal reactions which cannot be
foreseen. Due to an a.mhibition of platelet aggregation,
hemorrhages also occur Frequently in the course of a
heparin therapy:
Clinically, heparines are routinely administered
primarily fox the prevention but also for the therapy
of thromboses and embolisms. Primarily ~.f heparins are
administered subcutaneously, which is often experienced
by patients to be very painful, frequently hematomas
( "bruises ~~ ) occur .
rlethods of recovering heparin hare been described
in large numbers; one example for a heparin preparation
suitable for subcutaneous application is disclosed in
CA patent 1,108,989.
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rt is the object. of the present invention to
provide an improved prevention and therapeutical
treatment of thromboses anal embolisms which above all
can be used routinely in hospitals az~.d which no longer
has the. disadvantages of conventional heparizz
administration.
According to the invention, this object is achieved
by using AT III fox producing a pharmaceutical
preparation for preventing hemorrhages during an
anticoagulation therapy, in particular for producing a
pharmaceutical preparation for thrombosis prevention ox
treatment.
Although the use of AT TII particularly for the
prevention of hemorrhages during the therapy with
lr~eparin as such ie a paradox because of the known
intensifying anticoagulative effect of heparins on AT
zzr (because as such an intensified coagulation-
inhibiting action had to be expected), the use
according to the invention surprisingly has yielded
extremely positive effects:
Thus, the formation of hematomas - as compared tv
conventional heparin admi.nistratzon - could be greatly
reduced, subcutanous injection was also much less
painful arid therebeyond, the treatment according to the
invention also exhibited a depot effect wh~.ch was not
to be expected, whereby it was possib7.e to highly
increase the functional half life of AT III and
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heparin, primarily in case of subcutaneous izxjection.
Thus it has become possible for the first time to carry
out a sufficient thrombosis prevention not by means of
the common daily injection of hepdril~., hut to carry out
the treatments at longer time intervals, e.g. at
intervals of 2, 3, 4 days up to one week, depending on
the dose to be admi.nistared.
According to the invention, furthermore, lIl this
manner the inhibition of platelet aggregation can be
prevented or treated, respectively, and also the
bleeding time caxl be shortened.
The rielk of a thrombocytopenia a~ well as of
undesired hemorrhages in the course of an anticoagulant
therapy with the preparation of the invention or the
use thereof, respectively, is markedly reduced. It
could be demonstrated in vitro that thrombocyte
aggregation is not affected by AT III/heparin, whereas
heparin does clearly show an inhibitory effect.
with the indication for AT III according to the
ixwentio~n, for the first time a pharmaceutical
preparation comprising AT III as an active component
has been provided which can be provided for
subcutaneous admini.sl.ration, i . a . the use of A'T zII for
produc~.ng a medicament which is to be administered
~subcutaneously.
Thus, the present ~.nvention also relates tv a
pharmaceutical preparation for subcutaneous injection,
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which prepa..ration comprises AT IzI as the or rune of the
essential active component(s). The subcutaneous
preparation comprises AT zzs at a relatively high
concentration, corresponding to at :Least a two-fvJ.d
concentration of that common for in vitro-products.
Usually, a concentration ranging from 100 to 5,000 U of
AT III/ml will be chosen, preferably between 500 and
3,000 U of AT III/ml, depending on the purity of the
protein used. Be9ide9, AT III is also suggested as an
i.v. or i.m. product to be used according to the
invention.
AT III used according to the invention may be
contained in various preparations, e.g. in combination
with heparins or heparinoids having a high affinity or
having an affinity for AT III, respectirrely, preferably
as an AT III-heparin (or heparinoid)-complex
(heparinoids being a collective term for naturally
occurring, semisynthelric and synthetic mucopoly-
saccharides of heparin-like action).
A preferred production method for such a complex
has been described in AT patent 379 310, wherein human
plasma or AT m-containing plasma fractions are
admixed with heparin or heparinvi.d, respectively, and
the heparin- or heparinoid-complex, respectively,
formed is purified by adsorption on an anion exchanger.
Such a prepartion is commercially available under the
name Atheplexa (from Baxter, AT). This method may, of
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course, alBO be carried out with starting solutions
prepared by means of recombinant D1~TA technology.
preferably, the use of A'f III acaoxding to the
invention takes place within the frame of an
anticoagulation therapy comprising a heparin treatment.
As it has been mentioned, the pain associated with
the heparin injection (as a side effect of the
anticoagulation therapy) can be greatly reduced or even
prevented by the combined administration of AT ITI and
heparin or of AT III camplexed on heparin. seside9,
hematoma fvarmation at the site of injection is clearly
reduced.
In particular, patients having a risk of heparin-
induced thrombocytopenia can be treated according to
the invention.
The preparation according to the invention. for the
subcutaneous administration may comprise different
types of heparin_ For instance, hepaxins in low-
molecular form having a molecular weight of
approximately 7.,5po to 10,000 Daltons or in, high-
molecular ~vrm having a molecular weight of
approximate~.y 10,000 try 30,000 Daltons may be
GQntain~:d .
The preparation according to tre invention has a
surprisingly high functional half-life_ Due to its
novel resorption and pharmaco>cinetics, the preparation
according to the invention is characterized by an
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extraordinary bivavailability of AT III and heparin,
respectively. By selecting the heparine or the
heparinoids and their AT III affinzty. respectively,
the components of the preparation preferably are mixed
and adminisCered such that the heparins or hepa..r.i.noids,
reepectively, contained have a functional half-life in
Wvo of more than 10 hours, in particular more than 20
hours, in certain cases also more than 30 hours_
The pharmaceutical preparation according to the
invention is produced by finishing a puri.f.ied AT IIZ
and, optionally, heparin-containing preparation
according to methods known per ee, by optionally
combining it with suitable buffer, auxiliary,
preservative and/or stabilizing substances and/or
protease inhibitors, respectively, and filling it into
containers in a form suitable for administration and
preferably packing 1t in storage-stable, optionally
lyophilized or frozen state.
When being used, the effective dosage of the
preparation will depend quite individually on the
respective patient tsiae, body weight, condition, blood
COUnt,...).
Preferably, the AT IIT concentration in the
preparation of the invention or for the use acooxding
to the invention will be chosen such that an
administration of a dose of from 5 to 10,000 U of
AT 2II/><g body weight in a volume of less than 20 ml,
-
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in particular less than 10 ml, preferably less than
ml, most preferred less than 2 ml, will be enabled.
Preferably, the preparation accord~.zlg to the
invention is subjected to a method for irxactivating
infectious or pathogenic material, in particular
viruses. This inactivation treatment is preferably
ensured by a tenside and/or heat treatment, e.g. by a
heat treatment in the solid grate, in particular a
vapor treatment according to EP-o 159 311, or EP-
0 ~i9 901 ar EP-0 674 531.
Further treatments for inactivating viruses also
include the treatment with chemical or
chemical/physical methods, e.g. with chaotropic
substances according to W094/13329, DE 44 34 538 car Fp-
0 131 740 (solvents) or photoinactivation.
Irradiation oz~ nanofiltration also is a preferred
phys~.cal method for depleting viruses withixi the scope
of the present invention.
Finally, the inventiaxi relates to a kit for
subcutaneous injection comprising an AT III-containing
pharmaceutical preparation, instructions for using the
preparation for subcutaneous injection, as well ae
optionally an application device suitable fOr
subcutaneous injection, preferably a syringe. Compared
to i.m. syringes, this syringe has a very thin needle
so as to facilitate penetration below the skin.
Preferably, the pharmaceutical preparation in the
_ g
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kit is provided in a container in lyophilized foam, it
may, however, also be provided in liquid form. in which
case, however, it is preferably stored in deep-frozen
form.
The invention will now be explained in more detail
by way of the following Examples and the drawing
figures to wh~.ch, however; it shall not be restricted.
Figs. 2A and 1$ show the local tolerance of heparin
and Atheplexa in mouse;
Fig_ 2 shows the pain-triggering et:~ect of heparin
in the rat paw model;
Fig. 3 shows the pain-tr~.ggering effet of Atheplex~'
in the rat model;
Fig_ 4 shows the comparison of the Rlstoce~-irl-
dependent platelet aggregation of heparin and
Atheplex~; and
Fig. 5 shows the determination of the half-life of
heparin and Atheplex°.
E x a m p 1 a s .
g x a m p 1 a 1 . $ematoma for~$tiosx on mice after
subcut~eous injeati,oa of heparia aad of the
preparation acco:cding to the inveatioa. respectsvely
(at present cQneidercd by Applicant to be the beet mode
of carrysug out the inverWion)
In three runs, each using a total of z5 male NMRI
mice havi,mg a weight of fxwm 25 to 3o g are treated
with heparin (IMMT1N0 AG, "Vienna) or with the
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preparation according to the invention, respec.~ively
(Atheplexm, obtainable from IMMUNO AG,,Vienna), as
follows
1.5 IU of heparin 1.5 heparin units of Atheplexe
15 IU of heparin 25 heparin units of Atheplex~
Treatment (0.5 ml) was effected subcutaneously in
the xegion of the abdomen. The mice were examined daily
for 4 days for a discoloration of the subcutis. changes
were e~raluated as follows:
SGOre
Syze
none presexxt 0
up to 0.5 cm'
0.6 - 4 cm' 2
4 cm~ 3
L~iscolorativn
none
slight intensity 1
middle intensity
high intensity
Swelling
none o
low degree
middle degree 2
high degree
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On the 4th day, the animals were sacri.Liced, and
the subcutaneous changes were pathologically-
anatomically evaluated.
mean results are apparent from Table 1 and 1~ig. 1.
TABLE lA
Score
1 5 IU i 2 3 4 Da.v
Heparin 2.86 2.66 2.26 1.73
At ex~ 1.20 0.40 0.20 0.0
TABLE 1B
.SC:OrPr.
i5 IU 1 2 3 4
Heparin 3_66 4.13 3.33 2.33
Athet~lex~ 2 86 1 80 1.40 1.33
As apparent (Figs. 3.A, B) . at 7..5 and ~.5 heparin
units there i.s a relati~rely clear difference between
the two tested preparations in tavor of Atheplex~_
control animals which had received analogously
isotonic saline did not show hematomas at any point of
time.
pi.saection confirmed the respective visual
evaluation of the 4th day.
E x a m p 1 a 2 . Testing of the pain-triggering
effect of a subcutaneous injection of hepari~a and of
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the preparation according to the invention in the. rat
paw model
this effect was determined on the izzflamed rat paw
by determining the pain threshold according to Randall
& Selitto (Arch. Int. Pharmacodyn. 111 (1957), 409-
419 ) .
Fvr this purpose, a mercury manometer was connected
with a l0 ml syringe whose piston was equipped with a
short, projectile-type peg. with this syringe, an
increasing pressure was exerted on the rat paw (26.7
mbar/s). The pain threshold was defined ag that
pressure in mbar which is necessary to trigger a
defense reaction of the animal.
For the tests, female Sprague-Dawley rats having
weight between 250 and 35o g wexe used. The pain
threshold was measured each prior to injection (initial
value) and hourly up to 6 hours after intraplantar
injection of heparin (several doses) or Ath.eplexe
(several doses), respectively, or isotonic saline
(100 ~tl, negative control) . The values after injection
of test or control substances were c-expressed in peroent
of the initial value.
The results are apparent from Fig. 2 (heparin) and
Fig. 3 (Atheplex~) (x ~ sg). On the oxdinate, the pain
threshold expressed in percent. is entered: 100 % = no .
pain; (7 % = maximum pain. The abscissa represents the
time axis (hours after intraplantar injection). The
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uppermost Curve (triangles) in each case z~epresmic_s the
control animals (isotonic saline, n= 25; no pain)_
Heparin (Fig. 2) was tested on 10 animals each, at.
the doses 1, 3, 10, 40 and 130 IU. Starting from ZO IU,
a progressive, strong pain occurs, no further dose
dependence being recognizable.
AtYieplex~ (Fig. 3) was tested at the dosis 10, 40,
130 and 200 a 'n units (n = 10 in each case). Here,
too, pain occurs initially which, however, decreases
again in the course of time. The point pf time and the
extent of such decrease are dose-dependent:
- 7.0 heparin units: reversion after the 2nd hour,
complete freedom from pain after 6 hours.
- 200 heparin units: reversion after the 5th hour,
residual pain at the 6th hour: 46.9 ~ 3.0 °s.
These results show a markedly lower pain
development by the preparation according to the
invention in contrast to the conventional heparin
therapy.
S x a m p 1 a 3 . Z'n virs~o teetiug' of the
inhibition of platelet aggregation by Heparin and by
the preparation aacorda.ng to the in~rention
The influence of the substances on platelet
aggregation is caused by means of a turbidometric
method by using an aggregometer (from Chronology. To
400 ~1 of a formaldehyde-fixed thrombocyte preparation
(250, 000 cells per ~C1) , 25 ~.l of a FVIII-vWF-
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preparation and 25 ~.1 of the sample to be tested, or of
buffer as control, respectively, are admixed. After an
incubation of 3 min at 37°C, aggregation is sr~trted
with SO ul of Ristocetin sulfate (10 mg/ml). Evaluation
of the aggregation ie effected via the rise of the
aggregation curve. The reference value is the
aggregation with buffer addition.
In contrast to heparin, the RisGocetin sulfate-
induced platelet aggregation was not inhibited by AT
III-complexed heparin. The tests were also repeated
with a purified, recombinant van Willebrand Factor. The
results with the vGlF-dependent aggregation are
identical with the Fvzzr-vWF-dependent reaction. Both
can be reduced by the adda.tion of heparin, yEt not by
the addition of .Atheplex~.
Factorial Evaluation
Uriits of heparin in
5d0 1 test mixture Heparin Atheplexe
2.5 0.47 1.01
0.25 0.84 0.99
0.025 1_09 1_07
Buffer corxtrvl (without hepar7_n) 1
The results show that heparin inhibts the
aggregation in dote-dependence, urhereas the reaction is
not yr not substantially influenced by Atheplexe.
8 x a m p 1 a g . Determ~.natioYl raf the half-life
pf gy~~~staneoualy ix~jeated heparin axed subcutan~ously
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inj Bated preparation aacord~.ng to tk~e inventir~n
To & awake rabbits each (White New Zealasld, ca. 2_5
kg, o~ Dither sex) in the heparin group, E~,000 units r~~
unfractionated heparin per kg are administered
subcutaneouely. In. ChB comparison group, 6,000 units of_
heparin are subcutaneously injected with 1,000 units of
AT III, as a complex (Atheplex°). rltrated blood was
drawn from the ear ve~.zl 6, 22, 18, 24, 3Q, 36, 42, 48,
54, 60 hours after application. mhe heparin level is
determined a.n the recovered plasma via an adapted
thrombin inhibition determination by means of
chromogenic substrate (from Coa-Chrom Diagnoetika,
Chromogenix}. The calculations are carried out
according to bioStatiG Criteria. All the tests of the
heparin-6000 L1'/kg and Atheplex'~-applicatiora 1000 u/I~g
(~ 60a0 heparin units/kg) in the rabbit test were
included in the calculation.
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Heparin 6000 U/kg AthepJ.er. iono IT/kg
subcutaneous Bubautaneous
Hep.det.(zza-Inh) Hep.det.
(IIa-Inh.) AT III(Aa).
Lag' time (h) O.OOOS 6.02 10.7
Adsorption (1/h) 0.15 0.27 0.42
Sl~.mination (1/h) 0.15 O.OS2 0.031
T/2 adscarption (h) 4.76 4.1 1.66
as of application 2.4 11.5 17.8
t/2 elimination (h) 4.59 11_2 22.7
as of application 16.9 31.7 42.4
AUC (U/ml.h) 1939 2850
Maximum (U/ml) 4.1 8.2 4.2
Time maximum (h) 12 1$ Z8
The present data demonstrate the superiority of
complexed heparin with AT III over the non~comp7.exed
heparin. An equal superiority is also shown. with an
i.v. administration of AthepJ.ex° as regards halt-life
arid recovez~y.
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