Note: Descriptions are shown in the official language in which they were submitted.
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Applicant: Gundula Wiemann
"Use of a fibrinogen receptor-antagonist for preventing disseminated
intravascu-
lar coagulation"
Our Ref: W 1620 - py / js
Description
The present invention relates to the use of fibrinogen receptor-antagonists
for
preventing disseminated intravascular coagulation (DIC) related to sepsis or
systemic inflammatory response syndrome (SIRS) in humans.
An appropriate fibrinogen receptor-antagonist for the use according to the
present invention is a platelet specific immunoglobulin fragment directed
against
the human glycoprotein gp Ilb/Illa receptor present on platelets, comprising
an
antigen binding region derived from monoclonal antibody 7E3. An example is the
Fab fragment of a chimeric mouse-human antibody which is disclosed in WO
89/11538, and commercially available as the pharmaceutically effective com-
pound Abciximab in the pharmaceutical composition ReoProT"", which has been
developed by Centocor and is provided by Beiersdorf-Lilly GmbH.
It has been shown that the main fibrinogen receptor on the surface of
platelets
is the glycoprotein (gp) Ilb/Illa complex, which is a member of the integrin
superfamily (Fitzgerald, L. A. et al. (1987) J. Biol. Chem. 262, 3936-3939;
Poncz, M. et al. (1987) J. Biol. Chem. 262, 8476-8482). The gp Ilb/Illa
complex
is composed of one molecule of gp Ilb (mol. wt. =140,000) which consists of a
large (mol. wt. =125,000) and a small (mol. wt. = 25,000) polypeptide chain
which are linked by one or more disulfide bonds, and one molecule of gp Illa
(mol. wt. =105,000) which is a single polypeptide chain (Carrell, N. A. et al.
(1979) J. Biol. Chem. 260, 1743). A normal platelet contains about 50,000 to
80,000 gp Ilb/Illa complexes which represent about 1 % to 2% of the total
platelet protein. Only about 70% of the gp 1Ibl111a complexes are randomly
distributed on the exposed platelet surface (Isenberg, W. M. et al. (1987) J.
Cell
Biol. 104, 1655), while the remaining so-called "cryptic" gp Ilb/Illa is
distributed
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2
between the membranes of the surface-connected canalicular system (Woods,
V. L. et al. (1986) Am. J. Path. 124, 324) and the membranes of the cytoplas-
mic a-granules which release their contents upon platelet stimulation (Wencel-
Drake, J. D. et al. (1985) J. Biol. Chem. 260, 1743). Thus, the gp Ilb/Illa
recep-
for is of main importance for the binding of fibrinogen to platelets and plays
a
key role in platelet aggregation. The symmetric fibrinogen molecule thereby
functions as a link between the gp Ilb/Illa receptors of two adjacent
platelets.
Fibrinogen receptor-antagonists inhibit platelet aggregation by blocking the
binding site of fibrinogen on the surface of activated platelets. Various ant-
agonists of the receptor complex are disclosed in the prior art. These include
e.g. monoclonal antibodies (WO 89/11538, WO 90/06134, WO 95/12412),
(cyclicl peptides comprising Arg-Gly-Asp (RGD) or Lys-Gly-Asp (KGD) amino acid
motifs, respectively (WO 91 /1 1458; WO 92/17492), or structural analogs of
such motifs (EP-A 0 454 651; WO 94/29349), the dodecapeptide His-His-Leu-
Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val (HHLGGAKC1AGDV) and analogues therof
(Timmons et al. ( 1989) Biochemistry 28, 2919-2922), bicyclic compounds
comprising two condensed six membered rings (WO 96/18602), and non-pepti-
de compounds isolated from snake venom samples (WO 90/15620).
A focus of prior art studies has been the monoclonal anitbody 7E3 which was
initially characterized by its ability of binding to the gp 1Ibl111a receptors
of
resting as well as activated platelets (Coller, B. S. et al. ( 1991 ) Ann. NY
Acad.
Sci. 614, 193-213). By means of in vitro assays it has been shown that c7E3
which represents a mouse-human chimeric variant of the antibody, inhibits the
gp Ilb/Illa dependent aggregation of platelets in a dosage dependent manner.
This inhibition was independent of the tested stimuli for platelet aggregation
such as ADP, collagen, thrombin, adrenalin, etc. In order to establish the
relative
extend of binding of 7E3 and of fragments thereof (Fab, Fab', F(ab')2) to
plate-
lets, direct binding studies using '251-labeled antibodies were performed. All
forms of the 7E3 antibody bound to human platelets in a saturable fashion.
Platelets from different individuals exhibited different levels of 7E3
binding.
However, an average of approximately 80,000 molecules of 7E3 Fab as well as
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_ 3
7E3 Fab' are bound per platelet, in contrast to approximately 40,000 molecules
of 7E3 IgG or 7E3 F(ab'12 fragments (Jordan, R. et al. (1991 ) Thromb.
Haemost.
65, 828). Similar studies also showed that approximately 80,000 molecules of
the 'z51-labeled Fab' fragment of the mouse-human chimeric 7E3 (c7E3) are
bound per platelet.
Therefore, the 7E3 antibody and its mouse-human chimeric variant c7E3 as well
as Fab, Fab' or F(ab)2 fragments thereof have been proposed as agents for
antithrombotic therapy of patients at risk for cardiac failure (Coller, B. S.
(1995)
Circulation 92, 2373-2380).
This has led to the development of the drug ReoProT"" by Centocor, which is
available from Beiersdorf-Lilly GmbH. ReoProT"" contains Abciximab as the
pharmceutically effective compound, which is the Fab fragment of a mouse-
human chimeric immunoglobulin comprising an antigen binding region derived
from the highly variable region of the mouse antibody 7E3 and constant regions
derived from human origin. According to the product description of ReoProT""
its
use, in addition to heparin and acetylsalicylic acid, is restricted to the
prevention
or reduction of reocclusion following thrombolytic therapy and to accelerate
the
rate of thrombolyses in patients treated by percutane transluminal coronary
angioplasty.
According to the product description, due to the increased bleeding tendency,
the treatment with ReoProT"" should be further restricted to patients being at
high
risk for suffering from acute coronary thrombosis provided that the patient
displays at least one of the following criteria:
1. Angina pectoris with the following symptomes:
therapy resistent angina at quiescent state along with ischemic ST
modifications,
- therapy resistent, repeatedly appearing angina along with ischemic
ST modifications,
- therapy resistent angina post infarction along with ischemic ST
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4
modifications during the first 7 days post myocard infarction.
2. Acute transmural myocard infarction, which appeared not more than 12
hours before, and which deserves the following treatment:
- direct interventional procedure,
- necessary measure after unsuccesful thrombolytic therapy.
3. Angiographically defined lesions within the coronary circuit narrowing the
vascular volume:
- two features of a type B lesion,
- one feature of a type C lesion,
- women elder than 65 years of age displaying one feature of a type
B lesion and/or suffering from diabetes mellitus.
It is further known that septic multiple organ dysfuction syndrome (MODS) and
multiple organ failure (MOF) are still the major cause of death in post
operative
and post traumatic intensive care patients (Machiedo, G. W. et al. (1981) Ann.
Int. Med. 152, 757-759; Fourrier, F. et al. (1192) Chest 101, 816-832). Espe-
cially prominent among the reasons for these complications are sepsis related
to
infections and a systemic inflammatory reaction which is referred to as
"system-
is inflammatory response syndrome" (SIRS) related to various clinical
conditions
such as shock, trauma, ischemia, pancreatitis, burn injury, surgery, etc.
(Bone,
R. C. et al. (1992) Chest 101, 1644-1655). MODS and MOF, respectively, are
thought to be a result of the excessive activation of humoral and cellular
casca-
de systems by endotoxins or related substances released into the blood stream
(Bone, R. C. et al. ( 1991 ) Ann. Int. Med. 115, 457-469; Lowry, S. F. et al.
(1993) Arch. Surg. 128, 1235-1241 ).
It has been shown that MODS and MOF can be due to organ malperfusion
caused by microcirculation disturbances. This often lethal complication is fre-
quently found in cases of e.g. gram negative sepsis and characterized by the
so-
called disseminated intravascular coagulation (DIC). DIC is an early
complication
of sepsis, and characterized by the development of microclots in small vessels
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of the large arterial circulation system. In these patients there is a
tendency for
bleeding as a consequence of an uncontrolled consumption of platelets and
coagulation factors. The fibrin formation in the vessels causes ischemia which
in turn results in functional disturbances of the organs. Moreover,
intravascular
5 fibrin formation can contribute to the accumulation of leukocytes in the
lungs,
and thrombin leads to an increased translucence of the vessels. A developing
DIC can thus represent a factor for triggering an acute dyspnea syndrome.
Despite the fact that activation of neutrophils is in itself not directly
triggering
the coagulation process, it may induce an increased release of cytokines such
as
interleukin-1 (IL-1 ), interleukin-6 (IL-6) and tumor necrosis factor (TNF) in
pa
tients suffering from sepsis, which promotes (a) the kallikreine/kinine system
and
(b) the complement system in addition to (c) the coagulation system, thus
promoting disturbance in microcirculation and not at least formation of micro
clots.
Finally, it can be stated that the activation of host defence system leads, on
the
one hand, to a generalized release of proinflammatory cytokines and, on the
other hand, to a parallel down-regulation and consumption of natural
inhibitors.
These changes ultimately produce an overspill of proinflammatory mediators
(Bone, R. C. et al. (1992) Crit. Care Med. 20, 891-898) that may result in
widespread endothelial damage and following platelet activation known to be a
central mechanism in the pathogenesis of MODS and MOF, respectively (Gawaz,
M. et al. (1995) Eur. J. Clin. Invest. 25, 843-851 ).
When blood vessels are damaged by an injury or other causative factors, plate-
lets adhere to the disrupted subendothelial surface. The adherent platelets
subsequently release biologically active constituents, and aggregate. Aggrega-
tion is initiated by the binding of agonists, such as thrombin, epinephrine,
or
ADP to specific platelet membrane receptors. Stimulation by agonists results
in
exposure of latent fibrinogen receptors on the platelet surface, and binding
of
fibrinogen to the glycoprotein Ilb/Illa receptor complex.
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6
Attempts to prevent MODS and MOF, respectively, have been made with anti-
thrombin III (AT III), the most important natural inhibitor of thrombin
activity as
well as of many other serine proteases to rise inhibitors of the coagulation
system (Wilson, R. F. et al. (1989) Am. Surg. 55, 450-456).
Despite the fact that AT III is consumed, and low AT III plasma activities
correla-
to with high mortality, not all clinical trials showed beneficial results
under AT III
substitution.
There is a long list of attempts to modulate coagulation related to sepsis and
SIRS. For example, synthetic proteinase inhibitors, hirudin and thrombin
inhibi-
tors have been used by Zawilska, K. et al. (Thromb. Res. (1995) 80, 99-104).
Recombinant soluble thrombomodulin has been used by Aoki, Y. et al. (Thromb.
Haemost. (1994) 71, 452-455). Factor Xa inhibition has been used by Hara, T.
et al. (Thromb. Haemost. ( 1995), 74, 635-639), who could not demonstrate an
effect on bleeding time, etc.
However, all these studies failed to show clinical efficacy in well controlled
phase II and III clinical trials.
The following list demonstrates the high all-cause mortality rate in 12
prospecti-
ve controlled randomized, double-blind, multicenter trials involving 6266 pa-
tients, including all important trials in the last years:
Methylprednisolone (placebo, n =190, mortality 25 %; agent, n =191, mortality
34%) in: Bone, R. C. et al. (1987) New Engl. J. Med. 317, 653-658.
Methylpredinsolone (placebo, n =11 1, mortality 22%; agent, n =112, mortality
21 %) in: Veterans Administration Systemic Sepsis Cooperative Group (1987)
New Engl. J. Med. 317, 659-665.
HA-1 A (anti-I.PS) (placebo, n = 281, mortality 43%; agent, n = 262, mortality
39%) in: Ziegler, E. J. et al. (1991) New Engl. J. Med. 324, 429-436.
E5 (anti-LPS) (placebo, n = 239, mortality 43%; agent, n = 247, mortality 40%)
in: Greenman, R. L. et al. (1991 ) J. Am. Med. Assoc. 266, 1097-1 102.
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7
Platelet-activating factor receptor-antagonist (placebo, n =150, mortality 51
%;
agent, n=132, mortality 42%) in: Dhainaut, J-F. A. et al. (1994) Crit. Care
Med. 22, 1720-1728.
Recombinant human II-1 ra (placebo, n = 302, mortality 34%; agent, n = 591,
mortality 30%) in: Fisher, C. J. et al. (1994) J. Am. Med. Assoc. 271, 1836-
1843.
E5 /anti-LPS) (placebo, n=412, mortality 26%; agent, n=418, mortality 28%)
in: Bone, R. C. et al. (1995) Crit. Care Med. 23, 994-1006.
Anti-TNF monoclonal antibody (placebo, n = 326, mortality 33%; agent, n = 655,
mortality 30%) in: Abraham, E. et al. (1995) J. Am. Med. Assoc. 273, 934-941.
Anti-TNF monoclonal antibody (placebo, n =167, mortality 40%; agent, n =386,
mortality 37%) in: Cohen, J. et al. (1996) Crit. Care Med. 24, 1431.1440.
TNF-R: Fe fusion proteins (placebo, n = 33, mortality 30%; agent, n =108,
mortality 44%) in: Fisher, C. J. et al. (1996) New Engl. J. Med. 334, 1697-
1702.
P55 TNF-R: Fe fusion proteins (placebo n =140, mortality 39%; agent, n = 358,
mortality 35%) in: Abraham, E. et al. (1997) 277, 1531-1538.
Ibuprofen (placebo, n = 231, mortality 40%; agent, n = 224, mortality 37%) in:
Bernard, G. R. et al. (1997) New Engl. J. Med. 336, 912-918.
It has been suggested that the fibrinogen related platelet-platelet inteaction
represents the so-called "final step" of platelet activation regardless of the
pathway that leads to it, and that it plays also a role in DIC development. As
a
consequence of the activation, a conformational change of the gp Ilb/Illa
recptor
complex leads to the presentation of the entirety of the about 50,000 to
80,000
fibrinogen receptors contained within each platelet on their exposed surface.
In experiments utilizing an animal model of E. coli infection, F(ab')2
fragments of
monoclonal antibody 7E3 were used to investigate the influence of this fibrino-
gen receptor-antagonist on microvascular changes (Taylor, F. B: et al. (1997)
Blood 89, 4078-4084). In this study, baboons were infected with E, coli and co-
treated with C4b-binding protein (C4bBP). The group treated with 7E3 Flab')Z
fragments showed a positive effect on the survival rate. However, an extra-
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potation of these data to humans is impossible for several reasons. For
example,
the animals were pretreated with the antibody fragment prior to infection.
Second, the pathophysiology of the animal model and comparable human
disorders is strikingly different. Moreover, the co-treatment of the baboons
with
the C4b-binding protein (C4bBP) in addition to the bacteria results in an
artificial
pathophysiological state which is never encountered in the treatment of human
patients.
Therefore, the technical problem underlying the present invention is to
provide
a novel system for the prevention of disseminated intravascular coagulation
(DIC) related sepsis or systemic inflammatory response syndrome (SIRS) in
humans.
The solution to the above technical problem is provided by the emodiments
characterized in the claims.
In particular, the present invention relates to the use of at least one
fibrinogen
receptor-antagonist for the preparation of a pharmaceutical composition for
preventing disseminated intravascular coagulation (DIC) related to sepsis or
systemic inflammatory response syndrome (SIRS), by blocking the fibrinogen
receptors on platelets in humans.
The term "fibrinogen receptor-antagonist" comprises a molecule which is charac-
terized by its ability to prevent fibrinogen from binding to its receptor. The
receptor may be present on the surface of platelets. In a preferred embodiment
of the present invention the receptor is the glycoprotein Ilb/Illa receptor.
In a further preferred embodiment of the present invention the fibrinogen
recep-
tor-antagonist is selected from the group consisting of Tirofiban and
pharmaceu-
tically effective derivatives thereof, Lamifiban and pharmaceutically
effective
derivatives thereof, Integrilin and pharmaceutically effective derivatives
thereof,
Fradafiban and pharmaceutically effective derivatives thereof, Xemilofiban and
pharmaceutically effective derivatives thereof, Orbofiban and pharmaceutically
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effective derivatives thereof, Lefradafiban and pharmaceutically effective
deri-
vatives thereof, Clopidogrel and pharmaceutically effective derivatives
thereof,
peptides comprising the amino acid motif RGD and pharmaceutically effective
derivatives thereof, peptides comprising the amino acid motif KGD and pharma-
ceutically effective derivatives thereof, peptides comprising the amino acid
sequence HHLGGAKQAGDV and pharmaceutically effective derivatives thereof,
and immunoglobulins or biologically active fragments thereof. Preferably, the
immunoglobulin is a mouse-human chimeric monoclonal antibody. It is further
preferred that the chimeric mouse-human monoclonal antibody comprises the
antigen binding region of the monoclonal antibody 7E3. An example of such a
mouse-human chimeric antibody is c7E3. More preferably, the fibrinogen recep
tor-antagonist is the Fab fragment of- the chimeric mouse-human monclonal
antibody as defined before. An example of such an Fab fragment is the Fab
fragment of c7E3 (Abciximab) which is contained in the drug ReoProT"" as the
pharmaceutically effective ingredient.
The term "disseminated intravascular coagulation" means the formation of
microclots in small vessels of the large arterial circulation system.
The term "sepsis" means the entirety of life-threatening clinical disorders
and
pathophysiological changes as a consequence of the action of pathogens and/or
products thereof which invade from the place of infection into the blood
stream,
activating general biological cascades and special cellular systems, and
triggering
the release of humoral and cellular mediators. In a preferred embodiment of
the
present invention sepsis may be caused by an infection with pathogens selected
from the group constisting of gram negative bacteria, gram positive bacteria,
viruses, fungi and Plasmodia spec. Plasmodia spec. may comprise e.g. P. mala-
riae, P. vivax, P, ovate, and P, falciparum.
According to the present invention, sepsis may be diagnosed according to one
or more of the following criteria systems:
1. Criteria for sepsis according~to R. C. Bone et al. (Crit. Care Med. (1989)
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17, 389-3931: clinically evident infection, plus:
- body temperature of 38,3°C or more, or of 35,6°C or less, when
measured rectal,
- tachycardia of 90/min or more
5 - tachypnoe of 20/min or more, and
- evidence of at least one parameter of inadequate organ perfusion
or organ dysfunction:
- change of mental state,
- pa02 of 75 mmHg at room air,
10 - increased lactate serum concentration, and/or
- diuresis of 30 ml/h or less.
2. Criteria for sepsis according to the Veterans Administration Systemic
Sepsis Coorperation Group (New Engl. J. Med. (1987) 317, 659-665):
clinical suspection for sepsis and at least four of the symptomes selected
from the group consisting of
- chill and/or a body temperature of 38,9°C or more, or of
35,5°C or
less, when measured rectal,
- tachypnoe of 28/min or more, or hypocapnia of 32 mmHg paC02 or
less,
- tachycardia of 100/min or more,
- systolic hypotension of 90 mmHg or less,
- leukocyte count of 3,500/mm3 or less, or of 15,000/mm3 or more,
- thrombocytopenia of 100,000/mm3 or less,
- surgery intervention during the past 48 hours, and/or
- obviously present primary source of sepsis.
3. Criteria according to Ziegler, E. J. et al. (New Engl. J. Med. (1991) 324,
429-436): suspection for gram negative infection, plus:
- body temperature of 38,3°C or more, or of 35,6°C or less, when
measured rectal, and
- tachycardia of 90/min or more, and tachypnoe (20/min or more, or
necessity for mechanical ventilation by a respirator),
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11
or the patient further displays at least two of the symptomes indicating a
systemic toxination or a peripheral hypoperfusion selected from the group
consisting of
- metabolic acidosis of unknown origin as evidenced by a pH 7,3
base excess (BE) of -5 mmol/L or more, or by hyperlactacidemia,
- arterial hypoxemia of 75 mmHg pa02 or less, or of a quotient
pa02/Fi02 of 250 or less,
- acute renal failure with a diuresis of 0,5 ml/kg body weight/hour or
less
- thrombocytopenia of less than 100,000/mm3 or indicated by a
reduction at least to the half of the number of platelets/mm3 than
the normal range, or prolonged prothrombin time, or PTT,
- sudden reduced consciousness,
- increased cardiac index of 4 liters/min or more, accompanied by a
total peripheral resistence of 800 dyn'*s/cms.
4. In addition to the criteria systems 1. to 3., the patient further displays
one
or more of the biochemical symptomes selected from the group consisting
of
- increased tumor necrosis factor (TNF) serum concentration
- increased serum concentration of one or more of interleukines 1 to
6 (IL-1 to IL-6),
- increased serum concentration of activated complement factors
(C3a; C5a)
- reduced antithrombin III (AT III) serum activity,
- reduced 2-plasmin inhibitor serum activity,
- increased serum concentration of arachidonic acid metabolites
(thromboxanes, prostaglandines, leukotrienes)
- detection of platelet activating factor (PAF) in the serum, and/or
- detection of free oxygen radicals, peroxide, superoxide and/or
hydroxyl in the serum.
The term "systemic inflammatory response syndrome (SIRS)" means a systemic
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_ 12
inflammatory response resulting in the same processes as defined above for the
term "sepsis", but which can not be related to an infection by a pathogen,
i.e.
no positive blood culture.
According to a preferred embodiment of the present invention SIRS may be
caused by shock, trauma, ischemia, pancreatitis, burn injury, and/or surgery.
SIRS may be diagnosed according to the criteria for sepsis as defined above.
A further embodiment of the present invention relates to a method for treating
a human patient at risk for suffering from DIC caused by sepsis or SIRS com-
prising administering a pharmaceutical composition comprising a
pharmceutically
effective amount of at least one fibrinogen receptor-antagonist, and thereby
blocking the fibrinogen receptors on platelets. This means that the human
patient should exhibit at least one of the above defined criteria for sepsis
or SIRS
before administering the pharmaceutical composition according to the present
invention.
According to a preferred embodiment of the method of the present invention
sepsis may be caused by infection with the pathogens as defined above.
According to a further preferred embodiment of the method of the present
invention SIRS may be caused by the conditions as defined above.
According to a further preferred embodiment of the method of the present
invention, the pharmaceutically effective amount of the fibrinogen receptor-
antagonist is sufficient for blocking of about 80% or less of the fibrinogen
receptors contained within the platelets of the human patient.
According to a further preferred embodiment of the method of the present
invention, the fibrinogen receptor-antagonist may be selected from the group
as
defined above.
According to a preferred embodiment of the method of the present invention,
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_ 13
the pharmaceutical composition is administered by bolus injection comprising
an
amount of the Fab fragments of about 0.25 mg/kg body weight, followed by
infusion of an amount of the Fab fragments of about 10 ,ug/min over 12 h or
less.
The use of a fibrinogen receptor-antagonist according to the present invention
is described hereinafter by the following non-limiting example.
EXAMPLE
The example uses the Fab fragments of the mouse-human chimeric antibody
derived from monoclonal antibody 7E3 (also referred to as Abciximab) specific
for gp Ilb/Illa as the fibrinogen receptor-antagonist.
Surprisingly, Abciximab exhibits an especially high efficacy in the prevention
of
microcirculation disturbances and in the improvement of microcirculation after
an
occurrence of microcirculation disturbances, respectively, as they are caused
by
sepsis or SIRS. Thus, the risk for MOF is advantageously reduced.
According to the product description of ReoProT"", initially a dosage is
suggested
comprising an intravenous bolus injection (0.25 mg/kg body weigth) followed by
continuously intravenous infusion at a rate of 10 ,ug/min.
Therefore, the application may be generally carried out by continuously in-
travenous infusion of a dosage of not more than 10 ,ug Abciximab/min, or by
bolus injection of 0.25 mg/kg body weight, followed by infusion of 10,ug/min
over 12 hours or less.
By this treatment it is possible to adjust the binding kinetics so as to
minimally
interfere with actually occurring coagulation procedures in wound and/or
surgery
areas.
Therefore, the proposed dosage relates to the known value from the product
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14
description of ReoProT"", which is sufficient for blocking of about 80% of the
fibrinogen receptors present on the exposed surfaces of the activated
platelets
of the patient. However, the dosage may be less if there is just a moderate
platelet activation.
Especially important for the use according to the present invention is the mo-
ment for administering the fibrinogen receptor-antagonist, since the proposed
dosage does not achieve dissolving of existing platelet aggregates, but su-
presses the formation of new platelet aggregates. As far as platelet
aggregation
has been already initialized, this will be reduced and suppressed in the
further
course of treatment.
In general, two regimens are suitable:
1. Prophylactical administration of the antibody immediately after the diagno-
sis of sepis or SIRS. In this case, the dosage may remain below the above
proposed dosage, provided that the degree of platelet activation and
platelet count do not differ substantially from their normal range.
2. Administration of the antibody as soon as an increased platelet activation
and a simultaneous reduction of circulating platelets is recognized. In this
case, the dosage proposed above can be regarded as a recommended
dosage which can shortly be exceeded until the values of the above
parameters, or at least their tendencies, are stabilized.
The platelet activity is measured by means of flow cytometric analysis of the
patient's blood sample using one or more of the following markers specific for
molecules on the surface of activated platelets:
- RUU-SP 2.41 IgG 1 (Heynen, H. F. G. et al. ( 1994) J. Clin. Invest. 94,
1098-1112), kindly provided by Dr. Nieuwenhuis, and FITC-labeled by
resarch laboratory of the University of Leipzig, Germany; RUU-SP 2.41
IgG 1 does not react with resting platelets;
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- FITC-labeled P2 IgG 1 (Coulter-Immunotech); reacts with gp Illa;
- FITC-labeled P-selectin (CD 62-p) (Coulter-Immunotech); specific for GMP-
140 of activated platelets; and/or
- FITC-labeled PAC-1; does only react with a ligand induced binding site of
5 activated platelets (Shattil, S. J. et al. ( 1985) J. Biol. Chem. 260, 1 1
107-
11114).
The flow cytometric analyses are carried out using a Coulter XL Epics Flow
Cytometer (Coulter-Immunotech).
Blood samples are collected into a Sarsted tube every 2 hours from patients
who
showed at least one of the sepsis criteria 1. to 4. as described above.
Simulta-
neously, haemodynamic monitoring, blood-gas analysis, and measurement of
ventilation parameters are carried out. An aliquot of blood is mixed with PBS
(phosphate budffered saline), fixed and stained for a two-colour analysis
method
according to Koksch, M., and Wittig, K. (Ann. Haematol. (1997) 173).
Flow cytometric analysis of an aliquot of blood sample is carried out with
each
of the platelet activation markers given above by measuring the mean intensity
of the FITC fluorescence of each FITC-labeled marker.
In each case the flow-cytometric signal detected in the patient's blood sample
is compared with that of a mean value of a group of controls.
