Note: Descriptions are shown in the official language in which they were submitted.
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The use of anticoagulant agents in the extracorporeal treatment
of blood
The present invention relates to the use of anticoagulant agents
in the extracorporeal treatment of blood.
Blood in an extracorporeal circulation comes into contact with
exogenous surfaces. This activates the blood coagulation system,
for example factor XII and blood platelets via the intrinsic
pathway of the coagulation cascade. The blood coagulates. The
prevention of this is the task of the anticoagulants which are
conventionally administered in this situation.
In clinical practice it is virtually always heparin and
heparin-like agents which are employed for this purpose, although
there are problems with the use thereof. Patients treated with
heparin need continuous monitoring in particular because of the
generally known risk of HIT (heparin-induced thrombocytopenia),
osteoporosis, lipid metabolism disturbances and bleeding
complications. It is generally necessary to comply with a
complicated dosage regimen. Thus, after an initial bolus of 10 -
20 U/kg, the patients usually receive a further 5 - 10 U/kg/h in
order to maintain a predetermined level in the blood (Mehta R.
L., ASAIO Journal, 931 - 935 (1994)).
In view of these disadvantages there has been a search for
favorable alternatives to heparin, and the so-called low
molecular weight heparins in particular were found, and these
provide not only a prolonged half-life in the blood but also an
increased aXa/aIla ratio. Experiments with other
glycosaminoglycans, for example heparan sulfate, dermatan
sulfate, chondroitin sulfate and mixtures thereof, were aimed in
the same direction. Thus, for example, orgaran has an aXa/aIla
ratio of 22, whereas most low molecular weight heparins are in
the range from 1 to 5 (Beijering et al., Seminars in Thrombosis
and Hemostasis, Vol. 23, No. 2, 225'- 233 (1997)).
A corresponding search for substances with a prolonged half-life
was successful with hirudins. In contrast to the
glycosaminoglycans discussed above, these are peptides, for
example natural hirudin obtained from the salivary glands of the
medical leech Hirudo medicinalis, or recombinant hirudin (EP
0 158 564). In this connection too, there have been attempts to
counteract the relatively short residence time of hirudins in the
animal or human body, for example with the aid of derivatized
hirudins. In this sense, EP 0 345 616 describes dextran- and
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2
Sepharose-derivatized hirudin. EP 0 372 670 specifies sulfated
and sulfonated, optionally pegylated, hirudins. The pegylated
hirudin muteins described in EP 0 502 962 were also developed
with the aim of achieving even longer half-lives, with
undiminished activity (Esslinger H.-U., et al.: Thromb. Haemost.
77(5) (1997) 911-919; Esslinger H.-U., et al.: Ann. Hematol. 76
(Suppl. T) (1998) A97).
Because of their anticoagulant activity, the substances described
above can always be beneficial when anticoagulation is desired.
Thus, EP 0 502 962 mentions - in this case for PEG-hirudin - the
indications typically listed for anticoagulants, including
precisely their use during extracorporeal blood circulation, for
example in a hemodialysis or a cardiopulmonary bypass (Heidrich
J.P., et al.: Clinical Chemistry and Laboratory Medicine 36
(1998) 847-854). In the coronary graft area, coatings based on
polylactic acid have already been treated with PEG-hirudin
(Schmidmaier G., et al.: Journal of the American College of
Cardiology, 29/2 (1997) 354A).
Despite the effective protection during the actual dialysis,
there is.an increasing frequency of reports of a
disproportionately high incidence of vascular complications
especially in patients with chronic kidney disease. Concerning
the occurrence of serious vascular complications, statistical
surveys indicate a high risk of 20 - 30~ a year for dialysis
patients receiving long-term treatment. About 40 - 500 of all
artifical accesses (shunts) implanted as junction between
extracorporeal circulation and vascular system in the USA have to
be renewed each year because of a diminution of function (for
example through blockage). The mortality rate owing to vascular
complications in these hemodialysis patients is about 12~ a year.
This contributes to the average survival being only 6 years for
patients with chronic kidney disease, even with regular
hemodialysis. This survival corresponds to that for a
metastasizing oncosis.
The object on which the present invention is based, of more
comprehensive protection of patients with an extracorporeal
circulation and, in particular, dialysis patients receiving
long-term treatment, is achieved by the supplementary
prophylactic, and in particular the combined therapeutic and
prophylactic, use of anticoagulant agents.
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The present invention therefore relates to the use of at least
one anticoagulant agent for the prophylactic treatment of
individuals whose blood undergoes extracorporeal circulation at
times.
The prophylactic treatment serves in particular to avert (reduce)
vascular complications. The aim of the treatment is at least a
comparatively reduced risk for, and in particular reduced
occurrence of, vascular events. The treatment is especially
important when the individual's blood is not undergoing
extracorporeal circulation. The treatment is thus in a way an
after-treatment of individuals whose blood has undergone
extracorporeal circulation. It supplements the anticoagulant
protection, which is always necessary during extracorporeal
circulation, so that prophylactic protection against the
development and occurrence of vascular complications also exists
at times when the blood is not in an extracorporeal circulation.
While the treatment according to the invention can be carried out
with anticoagulant agents differing from those used during the
extracorporeal circulation, in an advantageous embodiment of the
present invention there is use of a particular anticoagulant
agent both during an extracorporeal circulation and after the
extracorporeal circulation.
The present invention therefore relates in particular to the use
of at least one anticoagulant agent for the treatment of
individuals with an extracorporeal circulation as anticoagulant
during the extracorporeal circulation and for prophylaxis of
vascular complications after the extracorporeal circulation. This
corresponds to a method for treating individuals undergoing
extracorporeal circulation where at least one anticoagulant agent
is used as anticoagulant during the extracorporeal circulation
and for prophylaxis of vascular complications after the
extracorporeal circulation.
The treatment period is divided according to the invention into
treatment phases in which the blood of the individual to be
treated passes through an extracorporeal circulation
(extracorporeal treatment phases), and into treatment phases in
which the blood is not passed through an extracorporeal
circulation (intracorporeal treatment phases).
An extracorporeal circulation means diverting the blood outside
an individual s body. The aim is usually to exclude sections of
the body from the bloodstream and/or perform an extracorporeal
treatment of the blood. The former use is indicated in particular
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in operations on the open heart or on major blood vessels, for
example for temporary disconnection of the heart by means of a
cardiopulmonary bypass (heart-lung machine). The latter use is
particularly indicated for extrarenal kidney-function treatment
of blood, for example by hemodialysis in cases of renal
insufficiency or by hemofiltration in cases of renal
insufficiency or other conditions, for example in patients
undergoing lipid apheresis.
When blood is in an extracorporeal circulation there is contact
between blood or blood constituents and surfaces of the
extracorporeal system, which may lead inter alia to an activation
of blood coagulation. From the medical viewpoint, this
circumstance makes anticoagulant measures necessary, which are
aimed in. particular at the blood in the extracorporeal system
during the extracorporeal phase. Anticoagulant agents are used
according to the invention as anticoagulant for this purpose. The
anticoagulant effect relates in this connection in particular to
the prevention of thrombus formation and, where appropriate,
diminution of thrombus growth especially in the extracorporeal
system.
Tt is additionally possible to take further expedient
anticoagulant measures during the extracorporeal phase on use of
a particular anticoagulant agent. The expediency of and necessity
for further anticoagulant measures are subject to expert
assessment. Thus, further anticoagulants in addition to a
particular anticoagulant agent may be used within the framework
of further anticoagulant measures. A particular type of further
anticoagulant measures may comprise equipping extracorporeal
systems or parts thereof with anticoagulants, for example,
coating surfaces.
The term "anticoagulant" has the generally accepted meaning for
the purpose of the invention. Accordingly, the anticoagulant
agents include accepted anticoagulants and agents with a similar
effect on blood coagulation of vertebrates, preferably mammals
and, in particular, humans.
A particular class of anticoagulant agents comprises the direct
thrombin inhibitors, for example hirudins and hirudin
derivatives, especially PEG-hirudin.
In one aspect of the present invention, anticoagulant agents with
an extended half-life in the organism to be treated are
advantageous for particular treatment regimens according to the
invention. Preferred according to the invention for this purpose
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are anticoagulant agents with a longer half-life than heparins
and, in particular, unfractionated heparins and, especially,
those with a terminal half-life after intravenous administration
of at least about 4 h, even better of at least about 5 h and, in
5 particular, of at least about 6 h. The stated terminal half-lives
relate to essentially intact kidney function, that is to say
normally a renal elimination efficiency corresponding to a
creatinine clearance CLcR of at least about 100 ml/min.
In another aspect of the present invention, anticoagulant agents
with an enduring pharmacodynamic activity in the organism to be
treated are advantageous for particular treatment regimens
according to the invention. Agents with pharmacodynamic activity
are those which according to the invention have minimal
prophylactic activity, i.e. bring about a clinically relevant
reduction of vascular complications compared with an untreated
control group. Enduring means, in particular, a time span which
extends beyond the extracorporeal phase and, specifically in the
case of a regular alternation of extra- and intracorporeal
phases, advantageously extends to the next extracorporeal phase.
The half-life and pharmacodynamics of an anticoagulant agent not
only depend on the agent chosen but may also be controlled,
within the framework of the treatment and in particular of the
mode of administration, by pharmaceutical measures for example.
Thus, agents with a short half-life or pharmacodynamic activity
per se can be administered as suitable slow-release formulation.
Anticoagulant agents with extended half-life and/or enduring
pharmacodynamic activity are described for example in EP
0 345 616, which relates to certain hirudin derivatives composed
of hirudin and soluble carriers, as agents with delayed action.
The contents of these documents and in particular the hirudin
derivatives described therein, in particular conjugates mentioned
therein of the formula I, composed of polyalkylene glycol or
polyalkylene glycol derivatives with hirudin, desulfatohirudin or
anticoagulant hirudin muteins, form part of the present
disclosure.
The use of an anticoagulant agent with an extended half-life
and/or an enduring pharmacodynamic activity offers the advantage
of being able to be used both as anticoagulant during the
extracorporeal circulation and for prophylaxis of vascular
complications after the extracorporeal circulation. Thus, it is
preferred to carry out the treatment according to the invention
with a single agent.
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The use of PEG-hirudin is particularly preferred according to the
invention.
PEG-hirudin stands for polyethylene glycol conjugates of hirudin.
The term hirudin refers here to a class of polypeptide-based
anticoagulant substances which are derived from true hirudin, the
natural polypeptide which can be isolated from the medical leech
Hirudo medicinalis. Thus, the term hirudin according to the
invention also includes recombinant variants (r-hirudin) and also
mutated variants (hirudin muteins). Preferred for the
polyethylene glycol conjugation axe the polypeptides of the
formula II described in EP 0 502 962 and, of these, in particular
the polypeptide with the sequence SEQ ID No:1 according to the
invention. The polyethylene glycols are preferably conjugated via
lysine residues, where appropriate using suitable linkers, for
example those indicated in EP 0 502 962, which are advantageously
stable under physiological conditions.
Tt 15 particularly preferred according to the invention to use
PEG-hirudin based on the polypeptide described above with the
sequence SEQ ID N0:1, to which a polyethylene glycol residue is
bound in each case to the lysine in position 27 and the lysine in
position 33. The binding can take place, for example, via a
urethane-like linker. Polyethylene glycol residues of the formula
-CO-O-CHz-CH2-[O-CH2-CH2-]nOR
in which n is an integer from 50 to 200, preferably from 75 to
150 and, in particular, frotrc 110 to 220, and R is alkyl
preferably having 1 to 4 carbon atoms. R is, in particular,
methyl. These polyethylene glycol residues are preferably bound
to the s-amino group of lysine residues. Accordingly, the term
PEG-hirudin refers to a usually heterogeneous mixture of
pegylated peptides with varying polyethylene glycol residues. The
variation in the polyethylene glycol residues is attributable in
particular to a variation in the PEG chain length,~whose
molecular weight varies in accordance with the value of n in a
range from about 2000 to about 9000, preferably from about 3000
to about 7000 and, in particular, about 5000 +/- 1000 Da.
According to one aspect of the present invention, one embodiment
of PEG-hirudin has a weight average molecular weight, determined
by exclusion chromatography (Superose 12, calibrated with PEG,
Pharmacia), of about 17,000 +/- 1000 Da.
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According to another aspect of the present invention, an
advantageous embodiment of PEG-hirudin has a specific
antithrombotic activity of about 10,000 - 14,000 ATU/mg of
protein.
There are several possibilities for connecting the extracorporeal
system to the vascular system of the individual to be treated.
Conventional alternatives are arteriovenous (AV), venovenous (W)
and venoarterial (VA) types of connection, with which in each
case the direction of blood flow is described, based on the
corporeal vascular system. For example, an arteriovenous
connection describes an extracorporeal system which takes
arterial blood from the individual s body and - if necessary
after appropriate treatment - returns it to the venous system of
the body. AV and W connections are usually preferred in the area
of hemodialysis and hemofiltration. Whereas extracorporeal W and
VA systems are usually operated with an external pump, this is
unnecessary with extracorporeal AV systems - provided the
arterial blood pressure is sufficient. The dosage of
anticoagulant agents and adjuvant anticoagulants may be different
with different types of connection, for example higher dosages
may be necessary on use of pumps.
The access to the corporeal vascular system can be achieved for
example by introducing tubular inlet lines into corporeal
vessels, Suitable examples are cannulas or catheters, whose
dimensions, that is to say in particular length and internal
diametex, can be adapted to the particular system. For example,
short and wide-lumen catheters are preferred for AV systems, and
double-lumen catheters are preferred for W systems. Normally
so-called shunts are used as appropriate access to the corporeal
vascular system, for example in the form of artificial vascular
implants or fistulas.
In certain cases, the blood is passed along or through filters or
membranes. It may be necessary to choose the membrane in
accordance with the anticoagulant agent used. The preferred use
according to the invention of PEG-hirudin is suitable for
conventional membrane and filtration systems employed in
particular in the area of hemodialysis and hemofiltration. These
include membranes of natural materials such as cellulose
derivatives, for example cellulose triacetate, and synthetic
materials, for example polysulfones, polyamides,
polyacrylonitrile. Plate filters and hollow fiber arrangements
are examples of possible geometries. One advantage of the use of
PEG-hirudin is that it is suitable both for extracorporeal
systems with HF membranes (high flux) and for those with LF
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membranes (low flux). A further advantage is that PMMA membranes,
for example the membranes made of poly(methyl methacrylate) or
poly(methyl methacrylate) copolymers described in DE 197 15 504
A1, for example the Toray membrane known for this purpose can,
because of their particular binding properties for PEG-hirudin,
be used as functional antidote for rapid elimination of
PEG-hirudin, for example in cases of intolerance reactions or
overdosage.
I0 The purpose of the use according to the invention of
anticoagulant agents is, optionally in addition to that as
anticoagulant during the extracorporeal circulation, the
prophylaxis of, in particular secondary, vascular complications
after the extracorporeal circulation.
Vascular complications include according to the invention
disturbances of the function of the cerebral, cardiac, mesenteric
and peripheral vessels and pathological states associated
therewith and symptoms thereof. These include, for example, the
formation of thrombi in the vascular system of the individual to
be treated, that is to say, in particular, venous and arterial
thromboses, in particular deep vein thromboses, peripheral
occlusive diseases, shunt thromboses, catheter thromboses,
thromboembolisms, myocardial infarct, unstable angina pectoris
and stroke. Accordingly, the use according to the invention of
anticoagulant agents has particular advantages in individuals at
increased risk of vascular complications. Risk-increasing factors
include both disorders of the coagulation system, in particular
AT-III deficits and elevated fibrinogen levels, thrombocytosis,
HIT, and hypertension and preexistent disorders such as coronary
heart diseases, diabetes or other vascular disorders.
The use according to the invention of anticoagulant agents for
the prophylaxis of vascular complications extends at least over a
period which is subsequent to the time of the extracorporeal
circulation and, according to a particular embodiment of the
present invention, follows it directly. In the case of a
multiple, i.e. periodically interrupted, extracorporeal
circulation, that is to say, in particular,.a periodic sequence
of extra- and intracorporeal phases, this period ideally extends
until the next extracorporeal phase. According to a particular
embodiment of the present invention, anticoagulant agents are
used for treatment of an individual with multiple alternation of
extra- and intracorporeal phase as anticoagulant during the
extracorporeal phases and for the prophylaxis of vascular
complications during the intracorporeal phases. For the sake of
completeness, it may be stated that the use as anticoagulant
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during the extracorporeal phase may likewise include a
prophylactic treatment of vascular complications, and this is
also usually the case.
The use according to the invention of anticoagulant agents
comprises a method within the framework of the treatment. This
entails administering to the individual to be treated, preferably
a mammal, in particular a human, agricultural animal or domestic
animal, an appropriate amount of one or more anticoagulant
agents, usually formulated in accordance with human
pharmaceutical or veterinary practice.
The administration of anticoagulant agents can take place in
accordance with a - usually necessary - systemic agent
administration. Of the possible administration routes, including
the oral route, a convenient possibility for administering an
appropriate amount of anticoagulant agents is the parenteral
route and, in particular, injection with the blood front into the
dialysis system, in particular via an introduction means.
With a view to the extracorporeal circulation, expediency of the
amount of anticoagulant agents to be administered is determined
in particular by the anticoagulant effect of the resulting blood
levels. According to one aspect of the present invention, values
in the therapeutic range are expedient. Therapeutic means here an
effect which is able to counteract the thrombotic stimuli
occurring during the extracorporeal circulation. Advantageous in
this sense are blood levels (minimum blood levels) based on
anti-IIa of at least about 400 ng/ml, preferably of at least
about 500 ng/ml and, in particular, of at, least about 600 ng/ml.
Measurement of the APTT shows an APTT prolonged advantageously at
least about 1.3-fold, preferably at least about 1.6-fold and, in
particular, at least about 1.8-fold. Measurement of the ECT shows
an ECT prolonged advantageously at least about 1.2-fold,
preferably at least about 1.6-fold and, in particular, at least
about 1.8-fold.
According to a further aspect of the present invention, expedient
values are those which keep the risk of bleeding by the treated
individual within limits. In this sense, it is a further
advantage for the blood levels to be, about 5 minutes after
administration of the anticoagulant agent, a maximum of about
2400 ng/ml, preferably a maximum of about 1700 ng/ml and, in
particular, a maximum of about 1500 ng/ml, based on anti-IIa.
Measurement of the APTT shows an APTT prolonged advantageously by
a maximum of about 5.0-fold, preferably by a maximum of about
3.3-fold and, in particular, by a maximum of about 2.7-fold,
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Measurement of the ECT shows an ECT prolonged advantageously by a
maximum of about 5.5-fold, preferably by a maximum of about
4.5-fold and, in particular, by a maximum of about 4.0-fold.
5 The abovementioned values need not - where medically justifiable
- be maintained throughout the extracorporeal phase. According to
an advantageous embodiment of the present invention, the amount
of anticoagulant agent to be administered is such that the
aforementioned minimum blood levels are obtained when the
10 extracorporeal circulation is completed. According to a further
advantageous embodiment of the present invention, the
abovementioned values apply to the period which is limited on the
one hand by the reaching of a maximum blood level, and on the
other hand by the completion of the extracorporeal phase.
The time of adminstration of an anticoagulant agent and, where
appropriate, further anticoagulants is expediently chosen so that
an anticoagulant effect is ensured even in the initial phase of
the extracorporeal circulation. For this purpose the
administration can take place before connection to the
extracorporeal system. Administration directly on connection to
the extracvrporeal system is also possible and may in this case
conveniently take place via the extracorporeal system. If
administration takes place directly on connection to the system,
this usually takes place with the blood front or - where the
residual level of anticoagulant agent in the patient permits this
from.the medical viewpoint - shortly thereafter. Administration
via the extracorporeal'system as_to_be included according to the
invention within the term parenteral administration and - in the
case of a venous connection to the extracorporeal system - in
particular within the term intravenous administration.
With a view to the treatment according to the invention after the
extracorporeal circulation, the expediency of the amount of
anticoagulant agent to be administered will be determined in
particular by the prophylactic effect of the resulting blood
levels. A prophylactic effect is in this connection an
antithrombotic effect, which can be adapted to the relatively
weak thrombotic stimulus after the extracorporeal circulation.
For the period of an intracorporeal treatment phase it is
possible and expedient usually to choose blood levels which are
lower than the blood levels obtained during the extracorporeal
circulation. According to one aspect of the present invention,
values in the range with prophylactic activity are possible -
relatively to the therapeutic blood levels obtained during the
extracorporeal circulation. Advantageous in this sense are blood
levels of anticoagulant agents, based on anti-IIa, after the
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extracorporeal circulation of at least about 150 ng/ml,
preferably of at least about 300 ng/ml and, in particular, of at
least about 400 ng/ml. Measurement of the APTT shows an APTT
prolonged advantageously at least about 1.2-fold, preferably at
least about 1.3-fold and, in particular, at least about 1.5-fold.
Measurement of the ECT shows an ECT prolonged advantageously at
least about 1.1-fold, preferably.at least about 1.3 -fold and, in
particular, at least about 1.4-fold. In particular, the blood
levels during an intracorporeal phase vary between the blood
level present on completion of the extracorporeal circulation and
the abovementioned minimum values. The blood levels normally
decrease as a function of time.
These values need not necessarily be maintained throughout the
intracorporeal phase either. According to another advantageous
embodiment of the present invention, the amount of anticoagulant
agent to be administered is such that, with a periodic sequence
of extra- and intracorporeal phases, the blood levels obtained at
the end of the intracorporeal phases are at least about
150 ng/ml, preferably at least about 300 ng/ml and, in
particular, at least about 400 ng/ml, based on anti-IIa.
Measurement of the APTT shows an APTT prolonged advantageously at
least about 1.2-fold, preferably at least about 1.3-fold and, in
particular, at least about I.5-fold. Measurement of the ECT .shows
an ECT prolonged advantageously at least about 1.1-fold,
preferably at least about I.3-fold and, in particular, at least
about 1.4-fold. On the other hand, blood levels advantageous at
this time are a maximum of about 1000 ng/ml, preferably a maximum
of about 700 ng/ml and, in particular, a maximum of about
600 ng/ml, based on anti-IIa. Measurement of the APTT shows an
APTT prolonged advantageously by a maximum of about 3.5-fold,
preferably by a maximum of about 2.8-fold and, in particular, by
a maximum of about 2.5-fold. Measurement of the ECT shows an ECT
prolonged advantageously by a maximum of about 4.0-fold,
preferably by a maximum of about 3.0-fold and, in particular, by
a maximum of about 2.5-fold.
Depending on the therapeutic blood levels present on completion
of the extracorporeal circulation, only after a certain
transitional period following the extracorporeal circulation are
subtherapeutic blood levels usually obtained. The transitional
period from therapeutic to subtherapeutic and, in particular,
prophylactic blood levels depends on the natural or, where
appropriate, artificial elimination of anticoagulant agents from
the blood of the treated individual.
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A particular aspect of the present invention comprises the
treatment of individuals with renal insufficiency. Renal
insufficiency means according to the invention that the
elimination efficiency of the kidney is inadequate or absent.
These include, in particular, individuals with a creatinine
clearance CLCR of less than 100 ml/min, especially less than
50 ml/min and, in particular, less than IO ml/min.
According to one embodiment of the present invention, individuals
with acute renal insufficiency, i.e. with the elimination
efficiency of the kidney temporarily inadequate or absent are
treated. In this case, the blood of the affected individual
undergoes extracorporeal treatment until an adquate renal
elimination efficiency is restored. The duration of the
extracorporeal phase naturally varies from case to case,
averaging several days. This type of treatment is referred to
according to the invention as continuous hemofiltration. The
treatment duration of at least about 3 days and, in particular,
of at least about 5 days represents a particular embodiment of
the present invention.
A further particular embodiment of the present invention is
directed at the treatment of individuals with chronic renal
insufficiency. These are individuals whose renal elimination
efficiency is permanently inadequate or absent. In this case, the
extracorporeal circulation is a regular event. Both the duration
of extracorporeal phases and the gaps between the extracorporeal
phases which, according to a particular embodiment of the present
invention, correspond to the intracorporeal treatment phases are
adapted to the condition of the individual, in particular taking
account of any remaining renal elimination efficiency. The
present invention is directed in particular at the treatment of
individuals with at least one extracorporeal circulation a week
and, in particular, at individuals with advanced chronic renal
insufficiency and, accordingly, on average at least about two
and, in particular, about three, extracorporeal circulations a
week. This type of treatment is referred to according to the
invention as intermittent (periodic) hemodialysis and represents,
according to a particular embodiment of the present invention, a
long-term, treatment consisting of alternate extra- and
intracorporeal treatment phases.
Within the scope of this embodiment relating to intermittent
hemodialysis it is possible for expedient blood levels to be
reached by administering an appropriate amount of anticoagulant
agent per cycle as a single dose or through a number of doses, in
particular 2, 3 or 4. According to a particular embodiment of the
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present invention, the anticoagulant agent is administered in the
form of a single dose per cycle, and thus once per hemodialysis.
A cycle is composed of an extracorporeal and an intracorporeal
phase. The administration expediently takes place, especially in
the case of a single dose, at the start of a cycle, i.e. at the
start of an extracorporeal phase. However, it may also take place
at another time during a cycle, for example after completion of
the extracorporeal circulation. Another possibility comprises
administering anticoagulant agent at the start of an
extracorporeal phase and after completion of the extracorporeal
circulation. The amount of the single dose, preferably as bolus,
can advantageously be such that a new dose of anticoagulant agent
is given at the start of the next cycle in each instance. A
possible basis for the amount of each dose, in particular a
single dose to be administered at the start of a cycle, is the
respective blood level of the anticoagulant agent measured in
particular before the start of a cycle. The corresponding blood
level is then raised through the administration of the dose. It
reaches a maximum which is within a range appropriate for the
purose of an anticoagulant measure. In the case of a single dose
to be administered at the start of a cycle, advantageous blood
levels about 5 minutes after administration are at least about
600 ng/ml, preferably at least about 700 ng/ml and, in
particular, at least about 800 ng/ml, based on anti-IIa.
Measurement of the APTT shows an APTT prolonged advantageously at
least about 1.5-fold, preferably at least about 1.9-fold and, in
particular, at least about 2.3-fold. Measurement of the ECT shows
an ECT prolonged advantageously at least about 1.5-fold,
advantageously at least about 2.0-fold and, in particular, at
least about 2.5-fold.
On the other hand, to take account of the risk of bleeding, these
maxima should be kept as low as possible. One advantage of the
use of PEG-hirudin is that these maxima can be up to about
2400 ng/ml, preferably up to about 1700 ng/ml and, in particular,
up to about 1500 ng/ml, based on anti-IIa. Thus,~the APTT can be
prolonged up to about 5.0-fold, preferably up to about 3.3-fold
and, in particular, up to about 2.7-fold, and the ECT can be
prolonged up to about 5.5-fold, preferably up to about 4.5-fold
and, in particular, up to about 4.0-fold.
The blood levels decrease as a function of time during the
extracorporeal phase. The blood levels advantageously remain in
the therapeutic range during the extracorporeal phase. The blood
levels mentioned above in this connection are advantageous here
too. On the other hand, advantageous blood levels on completion
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14
of the extracorporeal phase are a maximum of about 2000 ng/ml,
preferably a maximum of about 1500 ng/ml and, in particular, a
maximum of about 1100 ng/ml, based on anti-IIa. Measurement of
the APTT shows an APTT prolonged advantageously by a maximum of
about 4.5-fold, preferably by a maximum of about 3.0-fold and, in
particular, by a maximum of about 2.5--fold. Measurement of the
ECT shows an ECT prolonged advantageously by a maximum of about
4.0-fold, preferably by a maximum of about 3.5-fold and, in
particular, by a maximum of about 3.0-fold.
It is possible according to the invention for the single dose to
remain essentially the same per cycle on use of anticoagulant
agents in the framework of intermittent hemodialysis.
Accordingly, an amount of anticoagulant agent which remains
essentially constant from cycle to cycle is administered to an
individual. This amount can be based on individual parameters, in
particular those influencing the dosage, for example, the body
weight of the individual to be treated, but it is also possible
to use a fixed dose per individual. However, account must be
taken of the fact that adaptation to the anticoagulant agent used
according to the invention may be necessary at the start of
therapy. Thus, for example, a relatively high dose must be chosen
at the start of regular administration of PEG-hirudin to patients
with chronic renal insufficiency in order to obtain expedient
blood levels. The dosage can then be kept from cycle to cycle at
a level which remains essentially constant during the subsequent
regular administration of PEG-hirudin. The adaptation phase
usually comprises several cycles, preferably less than 15 and, in
particular, less than 10, it being possible advantageously to
choose after about 5 cycles a dosage which is a maximum of about
+/- 25% or, in particular, +/- 10% and preferably essentially at
the desired constant dosage.
If the anticoagulant agents are administered in a dosage which
remains essentially the same, the monitoring of the individual
can be confined to checking the particular blood level before an
extracorporeal phase and, where appropriate, checking the
particular blood level after administration of the single dose.
The former check serves in particular as a basis for the amount
of the necessary dosage, and the latter to avoid an increased
risk of bleeding due to any excessive maximum blood levels. It
may be mentioned in this connection that the use of PEG-hirudin
advantageously provides a possibility of eliminating PEG-hirudin
efficiency from the blood of an individual. Reference is made to
the membranes which are described above and are known for this
purpose.
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According to a particular embodiment of the present invention,
the amount of the single dose administered for, and preferably at
the start of, a hemodialysis is such that the concentration of
anticoagulant agent varies in a range from about 400 ng/ml to
5 about 2400 ng/ml, preferably in a range from about 500 ng/ml to
about 1700 ng/ml and, in particular, in a range from about
600 ng/ml to about 1500 ng/ml, based on anti-IIa, during the
hemodialysis. In this sense, the measured APTT is prolonged in a
range of about 1.3-fold to about 5.0-fold, preferably in a range
10 from about 1.6-fold to about 3.3-fold and, in particular, in a
range from about 1.8-fold to about 2.7-fold, or the measured ECT
is prolonged in a range from about 1.2-fold to about 5.5-fold,
preferably in a range from about 1.6-fold to about 4.5-fold and,
in particular, in a range from about 1.8-fold to about 4.0-fold.
According to another particular embodiment of the present
invention, the amount of the single dose administered for, and
preferably at the start of, a hemodialysis is such that the
concentration of anticoagulant agent after completion of a
hemodialysis and until the next one varies in the range from
about 2000 ng/ml to about 150 ng/ml, preferably in a range from
about 1500 ng/ml to about 300 ng/ml and, in particular, in a
range from about 1100 ng/ml to about 400 ng/ml, based on
anti-IIa. In this sense, the measured APTT is prolonged in a
range from about 4.5-fold to about 1.2-fold, preferably in a
range from about 3.0-fold to about I.3-fold and, in particular,
in a range from about 2.5-fold to about 1.5-fold, or the measured
ECT is prolonged in a range from about 4.5-fold to about
1.1-fold, preferably in a range from about 3.5-fold to about
1.3-fold and, in particular, in a range from about 3.0-fold to
about 1.4-fold.
Within the scope of the particular embodiments of the present
invention which are described above, the amount of the single
dose administered for a hemodialysis can advantageously be such
that, about 5 minutes after administration, the concentration of
anticoagulant agent is at least about 600 ng/ml, preferably at
least about 700 ng/ml and, in particular, at least about
800 ng/ml, based on anti-IIa. Measurement of the APTT shows an
APTT prolonged advantageously by at least about 1.5-fold,
preferably by at least about 1.9-fold and, in particular, by at
least about 2.3-fold. Measurement of the ECT shows an ECT
prolonged advantageously by at least about 1.5-fold, preferably
by at least about 2.0-fold and, in particular, by at least about
2.5-fold.
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16
The blood levels described above can usually be obtained with
bolus doses in the range from about 200 to about 1400 ATU/kg,
preferably from about 400 ATU/kg to about 1200 ATU/kg and, in
particular, from about 600 ATU/kg to about 1000 ATU/kg, of body
weight. After adaptation it is possible to treat individuals with
chronic renal insufficiency, with an average of three
exracorporeal circulations a week, with a dosage of about 200 to
about 1000 ATU/kg, preferably of about 200 ATU/kg to about
800 ATU/kg and, in particular, from about 400 ATU/kg to about
IO 600 ATU/kg, of body weight. The abbreviation ATU stands for'
antithrombin units based on the WHO I thrombin standard.
In particular, an individual with chronic renal insufficiency can
be treated, with an average of three extracorporeal circulations
a week, with a dosage of about 0.02 to about 1.0 mg of
PEG-hirudin and, after adaptation, with a dosage of about 0.03 to
about 0.06 mg, in each case based on kg of body weight, on use of
a PEG-hirudin with a specific activity of about 10,000 to 14,000
ATU/mg of protein and, in particular, a specific activity of
about 13,350 ATU/mg of protein.
The invention also relates to the use of anticoagulant agents for
producing medicaments, in particular pharmaceutical compositions,
for the treatment according to the invention. Thus, anticoagulant
agents are usually administered in the form of pharmaceutical
compositions which, besides the agent, comprise at least one
pharmaceutically suitable excipient. Compositions or medicaments
of this type can be produced and formulated using techniques
generally known to the skilled worker.
35
Appropriate for parenteral administration, the pharmaceutical
compositions are preferably administered as liquid pharmaceutical
form. Agent solutions in aqueous media such as water or
physiological saline are particularly preferred.
For practical use, anticoagulant agents, in particular
PEG-hirudin, can be supplied in solid, especially lyophilized,
form and, separately therefrom, the solvent. Agent and solvent
can be packed in aliquots in suitable containers, for example
vials, which makes reconstitution of a solution of known
concentration conveniently possible. Suitable with a view to the
preferred dosages described above are, for example, 2 or 10 ml
containers respectively containing 5 to 50 mg of PEG-hirudin;
vials containing 50 mg of PEG-hirudin can be supplied as
multiple-dose containers (reconstitution of the agent with a
preserved solution).
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17
The term blood level refers to a particular amount of
anticoagulant agents) in the blood of an individual, which, on
use of the determination methods described in the reference
examples, can be expressed by one or, where appropriate, even
several of the stated activity values.
The stated concentrations of anticoagulant agents based on
anti-ITa relate to the protein content of the PEG-hirudin used.
Equivalent amounts apply to other substances with anti-IIa
activity.
Measurement of the ECT (ecarin clotting time) refers according to
the invention to the use of direct thrombin inhibitors.
The stated blood levels represent average values which relation
to a group of at least about 10 individuals. Thus, because of the
biological variability, the value for a single individual will
usually differ from the stated statistical average within the
framework of the statistical assessment and nevertheless be
assignable to the average.
The stated blood levels are guideline values which may vary
within the scope of the accuracy of measurement even in relation
to the same measurement sample. Accuracies of measurement for the
individual determination methods are indicated in the reference
examples. This variation is expressed by the "about" prefixing
each value.
The intention of the following example is to illustrate the
invention without restricting it thereto.
Example:
Treatment of dialysis patients with PEG-hirudin
20 male and female patients between 18 and 75 years who must
regularly undergo hemodialysis were selected. After an initial
treatment with heparin (UFH=unfractionated heparin), each patient
was given an intravenous injection, immediately before the first
dialysis during PEG-hirudin treatment, of a dose of 0.08 mg/kg of
PEG-hirudin with a specific antithrombin activity of 13,354
ATU/mg of protein per kg of body weight. This was followed by
hemodialysis with an average duration of 4 hours, 3 x a week
using a Hemophan low flux membrane in a GFS plus 16 dialyzer.
When the dialysis was complete and before the subsequent dialysis
sessions, firstly the PEG-hirudin concentrations in the patient's
blood were determined. The measured values served as the basis
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18
for the amount of the PEG-hirudin doses to be administered
immediately before each hemodialysis. The residual PEG-hirudin
concentrations initially increased and allowed the dose to be
reduced from the initial 0.08 mg/kg of body weight to 0.03 to
0.05 mg/kg of body weight. It emerged that this dosage was
suitable for obtaining blood levels of PEG-hirudin in the range
from about 500 to about 1000 ng/ml of whole blood on completion
of each dialysis with three hemodialyses a week. The residual
PEG-hirudin concentration in the blood of each patient between
the hemodialysis sessions ensured prophylactic protection against
vascular complications.
The results are compiled in Tables 1 to 3.
The drawings show
Figure 1 by the example of patient 15 the PEG-hirudin doses
(bars) administered for dialyses 1 to 50, and the blood
levels of PEG-hirudin (dots) measured after some dialysis
sessions;
Figure 2 by the example of patient Z6 the PEG-hirudin doses
(bars) administered for dialyses 1 to 47, and the blood
levels of PEG-hirudin (dots) measured after some dialysis
sessions;
Figure 3 by the example of patient 18 the PEG-hirudin doses
(bars) administered for dialyses 1 to 49, and the blood
levels of PEG-hirudin (dots) measured after some dialysis
sessions;
Figure 4 by the example of patient 20 the PEG-hirudin doses
(bars) administered for dialyses Z to 31, and the blood
levels of PEG-hirudin (dots) measured after some dialysis
sessions.
45
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Table 1: APTT determination
No. of APTT APTT - APTT
the
dialysis Before dialysis Bolus 5'after admin.After dialysis
[ratio] [ratio] [ratio]
1-UFH 1.0 6.9 1.3
(4.2-8.8) (0.9-2.0)
02-UFH 1.0 1.4
(0.9-2.9)
03-UFH 1.0 1.3
(0.9-2.0)
04-PEG- 1.0 2.5 - 2.2
Hirudin (2.4-2.6) (1.8-2.6)
OS-PEG- 1.7 2.3
Hi~~n (1.5-2.0) (1.9-3.1)
06-PEG- 1.9 2.3
Hirudin (1.8-2.3) (2.0-2.7)
07-PEG- 1.9 2.2
Hirudin (1.6-2.4) (1.9-2.6)
08-PEG- 2.0 2.6 2.5
Hirudin (1.7-2.4) (2.3-3.1) (2.0-3.6)
09-PEG- 2.0 2.4
Hirudin (1,8-2.4) (2.0-3.4)
10-PEG- 2.0 2.3
Hirudin (1.8-2.4) (2.0-3.2)
2 11-PEG- 2.0 2.3
5
~~~n (1.7-2.3) (2.0-2.6)
12-PEG- 2.0 2.3
Hirudin (1.6-2.3) (L9-2.9)
13-PEG- L9 2.5 2.2
~~~n 1.5-2.2 2.3-2. 2.0-2.5
40
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Table 2: Anti-IIa activity determination
No. of Anti-IIa Anti-IIa Anti-IIa
the
dialysis Before dialysis Bolus 5'after After dialysis
admin.
[ng/ml] [n~ml] [n~~]
5 01-UFH
02-UFH
03-UFH
4-PE - 0 1298 - 818
Hirudin (925-1532) (660-958)
10 05-PEG- 275 842
Hirudin (197-322) (586-1100)
06-PEG- 426 942
Hirudin (275-539) (733-1201)
07-PEG- 432 953
15 Hirudin (237-627) (681-1242)
08-PEG- 536 2240 951
Hirudin (448-699) (910-1436) (704-1288)
09-PEG- 518 995
Hirudin (326-674) (758-1691)
20 10-PEG- 462 956
Hirudin (344-555) (685-1735)
11-PEG- 516 950
Hirtxdin (438-654) ' (648-1679)
12-PEG- 491 878
~~din (373-677) (688-1243)
13-PEG- 487 1076 834
Hirudin 276-614 789-1270 584-1133
35
45
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Table 3: ECT determination
No. of ECT ECT ECT
the
dialysis Before dialysis Bolus 5' after After dialysis
admin.
[ratio) [ratios (ratio
01-UFH
02-UFH
03-UFH
04-PEG- 1.0 3.0 2.3
Hirudin (2.7-3.9) (2.0-2.5)
IO 05-PEG- 1.6 2.4
Hirudin (1.4-1.7) (2.0-2.7)
06-PEG- 1.8 2.5
~~~n (1.6-2.0) (2.3-2.7)
07-PEG- 1.9 2.5
15 Hirudin (1.6-2.0) (2.2-2.8)
08-PEG- 1.9 3.1 2.6
Hirudin (1.8-2.1) (2.7-3.4) (2.2-2.8)
09-PEG- 2.1 2.6
~~~n (1.7-2.3) (2.4-2.9)
20
10-PEG- 2.0 2.6
Hirudin (1.8-2.2) (2.4-2.7)
11-PEG- 2.1 2.6
Hirudin. (2.0-2.2) (2.3-3.0)
12-PEG- 2.1 2.6
25 Hirudin (1.8-2.4) ' (2.4-2.8)
13-PEG- 2.0 3.1 2.6
Hirudin 1.8-2.3 2.7-3.5 2.3-2.8
30 Reference Example 1
APTT determination
The determination of the activated partial thromboplastin time
35 (~TTj is based on plasma fibrin formation induced by addition of
a partial thromboplastin (Actin FS) and calcium ions to the
plasma. Ellagic acid is used as activator.
9 volumes of venous blood + 1 volume of citrate (0.13 mol/1) are
40 cautiously mixed and centrifuged at.1600 x g and 2-10~C for
min. The sample volume is at least 450 p.1. Samples are
dispatched if necessary in the frozen state, and samples are
stored in freezers.
ø5 The controls used are control plasma in the normal range, control
plasma in the therapeutic range, control plasma in the low
therapeutic range and a quality control in the normal range, for
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22
example the controls commercially available from Dade Citrol 1,
Citrol 2, Citrol 3 and Coag Trol N.
The measurement is carried out in an ACL 3000.
The ACL 3000 is a completely automatic, microcomputer-controlled
centrifugal analysis system. After the start of the analysis
cycle, sample and Actin are pipetted separately into the
half-cuvettes of a reaction rotor made of acrylic glass with 20
cuvettes, and are mixed and then incubated. After the incubation,
calcium chloride is pipetted into the cuvettes, mixed and
measured. Measurements are carried out while the rotor is
rotating. The light source fox the nephelometric measurement is a
light-emitting diode (LED) whose light beam is directed via a
light guide system (~,=660nm) onto the measuring cuvettes. The
scattered light distribution is measured at an angle of 90~ to the
light source with the aid of a semiconductor sensor located
- underneath the rotor carrier. The measured results can also be
stated as ratio and describe the ratio of the current value to
the individual baseline value for a patient before the dialysis
with PEG-hirudin.
The accuracy of measurement is +10v to -10~.
Reference Example 2
Anti-IIa activity determination
Determination of the anti-IIa activity is based on measurement of
the activity remaining after addition of excess thrombin to the
sample. Heparin and other non-thrombin serine proteases are
neutralized before the assay by adding protamine chloride and
aprotinin to the sample. Remaining thrombin cleaves the
chromogenic substrate 52238 which is added to the sample.
9 volumes of venous blood and 1 volume of citrate (0,13 mol/1)
are cautiously mixed and centrifuged at 1600 x g and 2-10°C for 10
min. The sample volume is about 100 ~,1. Samples are dispatched if
necessary in the frozen state, and samples are stored in
freezers.
The following standards are used in the PEG-hirudin
determination:
Standard A: PEG-hirudin concentration [c] = 26.6 mg/ml; specific
activity of 11,696 ATU/mg of protein
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23
Standard B: [c] = 500 ~,g/ml
(1:53.3 dilution of standard A with 0.5% BSA)
Standard C: [c] = 50 ~,g/ml
(1:10 dilution of standard B with 0.5% BSA)
Standard D: [c] = 1000 ng/ml
(1:50 dilution of standard C with normal human citrated plasma)
Standard B - D are stored in aliquots in the frozen state before
use.
Calibration samples with concentrations of 100, 200, 400, 600 and
800 ng/ml are prepared by suitable dilution of standard D with
normal human citrated plasma.
This method can be standardized correspondingly for determination
of other anticoagulant agents.
The measurement is carried out in an ACL 3000 (incubation time:
120 s; inter-ramp interval: 3 s; delay time: 3 s; acquisition
time: 120 s; speed: 600 rpm). The extinction is measured using a
405 nm filter at a constant rotor speed.
The accuracy of measurement is +20 to -10%.
Reference Example 3
ECT determination
Determination of the ECT (ecarin clotting time) is based on the
inhibition of the coagulation activity of meizothrombin. Ecarin,
a purified fraction of Echis carinatus venom, produces
meizothrombin by cleavage of the prothromin in the plasma. The
time until fibrinogen coagulates induced by ecarin is measured.
9 volumes of venous blood and 1 volume of citrate (0,13 mol/1)
are cautiously mixed. The sample volume is about 100 ~,1. Samples
are dispatched if necessary in the frozen state, and samples are
stored in freezers.
The following standards are used in the PEG-hirudin
determination:
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24
Standard A: PEG-hirudin concentration [c] = 26.6 mg/ml; specific
activity of 11,696 ATU/mg of protein
Standard B: [c] = 500 ~,g/ml
(1:53.3 dilution of standard A with 0.5% BSA)
Standard C : [ c ] = 50 ~,g/ml
(1:10 dilution of standard B with 0.5% BSA)
Standard E: [c] = 2500 ng/ml
(1:20 dilution of standard C with normal human citrated plasma)
Standard B - E are stored in aliquots in the frozen state before
use.
Calibration samples with concentrations of 250, 500, 1500, 2000
and 2500 ng/ml are prepared by suitable dilution of standard E
with normal human citrated plasma.
This method can be standardized correspondingly for determination
of other anticoagulant agents.
The measurement is carried out in an ACL 3000 (incubation time:
120 s; inter-ramp interval: 3 s; delay time: 3 s; acquisition
time: 800 s; speed: 1200 rpm).
The measured results can also be stated as ratio and describe the
ratio of the current value to the individual baseline value for a
patient before the dialysis with PEG-hirudin.
The accuracy of measurement is +30% to -10%.
Reference Example 4
Determination of the terminal half-life i1/2
The terminal half-life i1~2 is calculated from 0.693/7~Z. ~,z
represents the terminal rate of elimination which is determined
by linear regression of a logarithmic plot of the concentration
of the relevant agent in the blood against time as terminal slope
of the concentration-time curve. For example, based on the
time-dependent change in concentration.indicated in Table 4
below, ~.Z can be calculated to be 0.086 1/h and zl~Z can be
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calculated to be 8.04 h.
Table 4
5
Time [h] Concentration [nmol/1]
0 0
0.1667 143
100.3333 256
0.5 213
0.6667 193
0.8333 171
1 139
151.483 123
2 93
4 58
6 38
7.983 23
209.983 34
11.98 30
15.97 20
24.03 9
28 8
2532.17 5
35
45
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SEQUENCE LISTING
<110> Knoll AG
<120> THE USE OF ANTICOAGULANT AGENTS IN THE
EXTRACORPOREAL TREATMENT OF BLOOD
<130> M/41004
<150> EP 00105867; USSN 60/190,103
<151> 2000-03-20
<160> 1
<170> PatentIn Ver. 2.1
<210> 1
<211> 65
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: recombinant
hiruc3in mutein
<400> 1
Val Val Tyr Thr Asp Cys Thr Glu Ser Gly Gln Asn Leu Cys Leu Cys
1 5 10 15
Glu Gly Ser Asn Val Cys Gly Gln Gly Asn Lys Cys Ile Leu Gly Ser
20 25 30
Lys Gly Glu Arg Asn Gln Cys Val Thr Gly Glu Gly Thr Pro Arg Pro
35 40 45
Gln Ser His Asn Asp Gly Asp Phe Glu Glu Ile Pro Glu Glu Tyr Leu
50 55 60
Gln
