Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
w ~ CA 02485268 2004-11-08
' VOSSIUS 8z PARTNER
Patentanwalte
SIEBERTSTRASSE 3 ~ 81675 M~7NCHEN
TEL.: +49-89-41 30 40 ~ FAX:+49-89-41 30 41 11 ~ FAX (Marken-Trademarks): +49-
89-41 30 44 00
E-MAIL: infoQvossiusa~~ru~:.oornn-HOMEPAGE: www.vassiusandpatu~a:oom
PCT/EP03/04680
RHEINISCH-WESTFALISCHE TECHNISCHE HOCHSCHULE AACHEN
Our Ref.: H 1722 PCT
English translation of the PCT-application as originally filed on 3 June 2003
Resorbable Pharmaceutical Formulation
The present invention relates to a resorbable pharmaceutical formulation for
continuous local release of thrombin comprising thrombin embedded in
resorbable
spheres of polymers, the spheres being incorporated in a collagen sponge. The
formulation according to the invention provides a resorbable hemostyptic for
use in
surgery, particularly in dental surgery/dento-alveolar operations.
Due to most varied diseases, there is an increased number of patients in need
of
therapeutic treatment of blood clotting inhibition (anticoagulation). These
include, for
example, patients with artificial prosthetic valves or patients who suffered a
stroke
or a vein thrombosis.
In nearly all cases, therapeutically-induced anticoagulation is achieved by
daily
administration of vitamin K-antagonists (coumarin derivatives such as e.g.
phenprocoumon in the form of Marcumar~ or Falithrom~ or coumadin derivatives
in
the form of Warfarin~). Due to inhibition of blood clotting, even small
surgical
operations such as e.g. tooth extractions are linked with a high risk of
haemorrhage.
For this reason, the patients concerned are usually hospitalised for
treatment, which
is cost-intensive. In this case, permanent oral anti-coagulation is replaced
by
administering heparin intravenously, which also results in an inhibition of
blood
clotting. However, heparin has a significantly shorter half-life, which allows
a better
control of the anti-coagulation in the case of bleeding complications whilst
not
having to dispense with the necessary thrombosis protection. Apart from these
forms of therapeutically-induced blood clotting inhibition, post-operative
secondary
bleeding is also due to the plasmin system. On a systemic level, the plasmin
system, the physiological antagonist of the blood clotting system, provides a
dissolution of blood clots. This function, however, is not desired in the case
of an
increased risk of post-operative haemorrhage.
CA 02485268 2004-11-08
2
VOSSIUS & PARTNER
Apart from the indications mentioned above, there are further groups of
patients
who, due to an innate disorder of the blood clotting system such as e.g. lack
of
protein C, protein S or antithrombin, need therapeutic anticoagulation with
vitamin K
antagonists, but also an innate tack of factors such as e.g. haemophilia A or
B or
von Willebrand's syndrome or an isolated lack of factors within the
prothrombin
complex can, even in the case of small, local surgeries, lead to considerable
post-
operative secondary bleedings. In order to avoid post-operative secondary
bleedings after tooth extractions or dento-surgical operations without risk of
losing
the necessary protection against thrombosis, the following procedures
according to
the state of the art have become established. However, even with these two
therapy
regimens it is not possible to avoid possible secondary bleedings:
L) Changing the oral permanent anticoagulation by vitamin K-antagonists to an
intravenous permanent heparinisation in the so-called therapeutic range (PTT
twice or three times the norm). Due to the relatively short duration/half-life
of
heparin, secondary bleedings which are partly caused by the heparin
anticoagulation (PTT over 80 sec) are to be controlled by reduction of the
heparin dosage. In this case, however, a therapeutic heparinisation as
protection against thrombembolic complications does not exist any more (PTT
< twice the norm). In addition, this procedure involves considerable costs for
the insurers due to the temporary hospitalisation of the patients.
II.) Continuing the oral permanent medication by vitamin K antagonists and
carrying out small dento-surgical operations in the upper limit of the oral
anticoagulation (Quick value of 25% to 35%). In this case, however, the
problem arises that patients who need strict anticoagulation (Quick maximum
25% with an artificial prosthetic valve in mitralis position) cannot be
treated by
this concept. Furthermore, the physiological, perioperative use of the
residual
blood clotting factors present in the circulation leads to a further reduction
of
the supply of endogenous factors, since these are reproduced only to an
insufficient extent during the continued administration of vitamin K-
antagonists.
Thus, the PTT decreases further and post-operative haemorrhage
complications occur. At times, the frequency of secondary bleedings increases
to a tenfold high.
CA 02485268 2004-11-08
3
VOSrSIUS & PARTNER
The optimal local wound care is carried out identically in both concepts:
after the
extraction of the teeth or after a smaller dental surgery was carried out the
bone
wound was tamponed with a collagen fleece (e.g. Lyostypt) or a gelatine sponge
(e.g. Topostatin). The mechanical compression of the bone wound thus achieved
can lead to an initial arrest of bleeding. If a stronger bleeding from the
site of the
tooth occurs intra-operatively, a local hemostyptic on a thrombin base or a
fibrin
glue preparation can additionally be applied onto the bone wound.
In order to reduce the fibrinolytic influence of the sputum, the mucosal wound
is
subsequently closed sputum-tight with atraumatic sutures. It is for the same
reason
that a plastic plate protecting the wound is fastened onto the patient's
remaining
teeth.
The result of an own, prospective study by the inventors could identify the
following
reasons for post-operative bleeding in dental surgery with ongoing
marcumarisation
(Gerhards et al., Gerhards F., Wagner W.: "Zum Blutungsrisiko nach
zahnartzlich-
chirurgischen Eingriffen unter fortgefuhrter Marcumarisierung". Dtsch.
Zahnarztl. Z.
(1997) 52:53-56):
1. Consumption of the residual coagulation factors circulating in the
blood circulation system
and
2. their additional inactivation by the intravenous administration of
heparin (inactivation of the complete prothrombin complex).
Thus, the problem underlying the present invention is to provide a
pharmaceutical
formulation with which it is possible to provide the end product of the blood
coagulation cascade, thrombin, over a limited period of time. Thus, the phase
until a
stable intra-oral wound healing is achieved is considered the required period
of
time. This corresponds to 7 to 10 days. After this period of time, a stable
wound
healing has occurred. As a result external mechanical influences on the wound
can
no longer have an effect.
In order to achieve this, thrombin is embedded in resorbable spheres,
preferably
from commercially available polymers which are already used for other
medicinal
products (e.g. surgical suture material) (cf. figure 1).
CA 02485268 2004-11-08
4
VOS'SIUS & PARTNER
According to the invention, the term "spheres" includes particles, capsules or
liposomes in which the active ingredient is embedded, enclosed, dispersed or
- dissolved, such as described in Voigt, Pharmazeutische Technologie,
Deutscher
Apothekerverlag Stuttgart, 2000, pages 467-471.
- Under the term "spheresu according to the present invention, particularly
micro or
nano particles are understood which contain an active ingredient embedded in a
polymer matrix without the formation of a separate capsule skin.
Here, spheres having a size of 0.1 to 1,000 Vim, more particularly having a
size of
to 150 wm are preferred.
The loading of the spheres can, depending on the corresponding thrombin
preparation, be 0.1 to 20% wherein the loading of the collagen sponge can be
0.1 to
50%. The overall dosage should be 1 I.U. to 2,000 I.U., preferably 250 I.U. to
1,000
I.U. thrombin per implant.
The spheres can be prepared with the help of known methods, in this respect
particularly emulsion techniques (w/o; o/w; w/o/w-emulsion-evaporation or
extraction method) or spray drying have to be mentioned. The methods are
described in the prior art. Thus, EP 0 330 180 describes micro spheres of the
polylactic acid type containing physiologically active substances and methods
for
their preparation. Other methods for preparation are inter alia disclosed in
Muller.
R.H. and Hildebrand G.E., Pharmazeutische Technologies Moderne Arzneiformen,
Wissenschaftliche Verlagsgesellschaft Stuttgart, 1998, p. 243-258 and S. 339-
355,
Eldrige J. et al., J. Controlled Release 1990, 11, 205-214; Jeffrey J. et al.
Pharm.
Res. 1993, 10, 362-368 and Pavanetto et al., Int. J. of Pharmaceutics 1992,
84,
152-159.
Resorbable polymers used according to the invention for the production of the
resorbable spheres include polysugars and their derivatives, polyvinyl alcohol
(PVA), polyvinyl pyrrolidine (PVP), polylactic acid (PLA), polylactides,
polyhydroxybutyrate (PHB), poly-L-lysine, their homologues and co-condensates,
lactic acid glycol acid co-polymer (PLG), particularly Resomer RG 504 and 505
by
Boehringer Ingelheim.
Other usable polymers are amongst others disclosed in Muller R.H. and
Hildebrand
G.E., Pharmazeutische Technologies Moderne Arzneiformen, Wissenschaftliche
Verlagsgesellschaft Stuttgart, 1998, p. 243-258 and p. 339 to 355.
CA 02485268 2004-11-08
VOSSIUS & PARTNER
The spheres thus obtained are, in turn, incorporated into resorbable sponges,
particularly collagen sponges. This is, e.g., carried out by freeze drying of
an
aqueous collagen or an aqueous collagen suspension or gelatine solution in
which
the spheres are uniformly dispersed. The production of collagen sponges is,
e.g.,
described in US 4,5'15,637, EP 562862 or by Chvapil, J. Biomed. Mater. Res.
11,
721-74i (1977).
Collagen
The collagen used according to the present invention should preferably be
soluble
in vivo by enzymatic degradation or by other biological processes. Native
collagen
in its potentially soluble form or natural insoluble collagen which is
inherently cross-
linked and which is either soluble in an acidic or alkaline medium such as
e.g.
described in US 4,515,637 are preferred.
The collagen used is preferably type 1 collagen.
The origin of the collagen for use according to the present invention is not
subject to
any special limitation. In general, collagen is used which is derived from the
skin,
bone, cartilage, tendons, inner organs etc. of a mammal such as e.g. a human,
horse, cow, pig, sheep, rabbit, mouse. Collagen-like protein derived from
birds, fish
and the like can also be used. Moreover, collagen prepared by genetic
engineering
can also be used, the production of which by means of genetic engineering is
at
present worked at (e.g. ZymoGenetics, WA, U.S.A.).
Thrombin
The thrombin of the invention can be derived from a great number of sources
such
as pooled human or animal plasma. Bovine thrombin is e.g. available from a
great
number of commercial sources. Moreover, according to the invention,
recombinant
thrombin can also be used (e.g. ZymoGenetics, WA, U.S.A.).
This can be obtained from a great number of recombinant sources such as e.g.
transformed host cells (bacteria, yeasts or mammalian cells). Moreover,
thrombin-
iike compounds such as proteolytic snake venoms and thrombin precursors such
as
prothrombin can be used as sources for thrombin.
Thus, the term "thrombin" as used herein also comprises thrombin precursors
and
thrombin-like compounds and refers to all proteins and amino acid polymers of
natural and synthetic origin which are capable of catalysing the formation of
fibrin
clots from fibrinogen and/or of activating the blood platelets.
CA 02485268 2004-11-08
6
V~S,SIUS &, PARTNER
Moreover, other coagulation factors such as e.g. factor VIII can also be used.
- This formulation of the invention has various advantages as, on the one
hand, the
existing therapy concept is only slightly changed due to the use of collagen
sponges
- for the mechanical wound compression and the collagen-induced aggregation of
the
thrombocytes, and, on the other hand, the wound healing is supported over a
period
of time of 7 to 10 days by means of the thrombin released. The aggregation of
the
thrombocytes caused by the collagen leads to the activation of the plasmatic
blood
coagulation via the release of mediators.
To sum up, it can be stated that the combination of the invention of collagen
with its
thrombocyte aggregation-supporting properties and a temporary - for the period
of
time sufficient for wound healing - local release of the coagulation factor
Ila
(thrombin), makes a local hemostatis which is independent of systemic
influences
possible over a longer period of time. The release is carried out 0 to 14
days,
particularly preferred 7 to 10 days.
Moreover, the preparation can be supplemented by adding further therapy-
supporting antifibrinolytics which are incorporated into the resorbable
spheres, such
as e.g. tranexamic acid, E-aminocaprinic acid, 4-(aminomethyl)benzoic acid,
aprotinine, EPO, acetaminonaphtone, thromboplastin, menadione sodium
bisulphate, adrenochrome monoaminoguanidine methanesulfonate and
carbazochromosodium sulphate. By this means, it is possible to balance
additional
thrombolytic influences by the plasmin system and the fibrinolytic effect of
the
sputum until the wound healing is completed. Moreover, the spheres of the
invention can contain further active ingredients such as antibiotics, anti-
infectants or
local anaesthetics.
The release of active thrombin from resorbable polylactic acid co-glycolid
spheres
has been examined and detected in vitro by means of a chromogenic substrate.
Figure 2 shows the release profile of thrombin from poly(DL-lactic-co-
glycolid) 50:50
(Boehringer Ingelheim RG 504) at 37°C in PBS buffer. The time-dependent
cumulative thrombin release is shown, determined by means of the Bradford test
in
vitro measured over the release period of 28 days.
CA 02485268 2004-11-08
7
VOSSIUS 8r. PARTNER
Figure 3 shows a release profile of thrombin from poly (DL-lactid-co-glycolid)
50:50
(Boehringer Ingelheim RG 504) at 37°C in PBS buffer. The time-dependent
cumulative thrombin release is shown which is measured in vitro by means of
the
cleavage of p-nitroaniline from the chromogenic substrate over the release
period of
28 days. 1 NIH thrombin corresponds to 0.324+/- 0.073 pg thrombin. The amount
of
p-nitrolanilin released is proportional to the active thrombin, the thrombin
used had
an activity of 50 NIH/mg.
The present invention moreover provides the use of the pharmaceutical
formulation
of the invention as local hemostyptic, particularly for local hemostasis in
patients
where the coagulation is inhibited or who have impaired coagulation. The cause
for
the inhibition of the coagulation can in this respect be iatrogenic, i.e. due
to an
inhibition of the coagulation due to the administration of vitamin K
antagonists such
as phenoprocoumon or coumadin, inhibitors of thrombocyte aggregation such as
acetylsalicylic acid, tirofibran, dipyramidol, ticlopidine or eptifibatid or
is endogenous
such as in the case of patients with a mild form of an innate defect in blood
coagulation, e.g. hemophilia A/B, von Willebrand syndrome and an isolated lack
in
factors within the prothrombin complex.
As is known from the literature and as has already been discussed above, the
frequency of the postoperative secondary bleeding in patients who have
impaired
coagulation is approximately 30% and the duration of the hospitalisation
amounts to
several days (on the average 5 days). The clinical use of the pharmaceutical
formulation of the invention considerably decreases the frequency of secondary
bleeding and thus shortens the duration of hospitalisation. Moreover, it makes
it
possible to carry out certain operations on an out-patient basis. Apart from a
markedly reduced risk for the patients, this also means a considerable saving
of
costs in the health sector.
