Complex-bound inhibitors of metabolic enzymes capable of being activated, useful as molecular markers for diagnostic and therapy monitoring purposes

This invention relates to the use of an inhibitor of an activatable metabolic enzyme, which inhibitor is bound to a high molecular weight carrier, as a molecular marker for determining the activation of this enzyme for the diagnosis of the enzyme. The invention relates in particular to the use of a thrombin inhibitor, which is bound to a high molecular weight carrier, as a molecular marker for determining clotting activation in clotting diagnosis and therapy monitoring. The invention preferably relates to the use of dextran-hirudin or PEG-coupled hirudin as a molecular marker for clotting diagnosis and therapy monitoring.

The invention relates to the use of inhibitors of activatable or activated 
metabolic enzymes, which inhibitors are bound to high molecular weight 
carriers, as molecular markers for determining the activation of the 
enzyme in order to indirectly diagnostically and therapeutically monitor 
the enzyme. 
This invention in particular relates to the use of an inhibitor of an 
activation product of the blood clotting cascade or of an activated 
fibrinolysis enzyme, which inhibitor is bound to a high molecular weight 
carrier, as a molecular marker to indirectly monitor an activation product 
of the blood clotting cascade or an activated fibrinolysis enzyme. The 
invention preferably relates to the use of a thrombin inhibitor, which is 
bound to a high molecular weight carrier, as a molecular marker for 
determining clotting activation in clotting diagnosis and therapy 
monitoring. The invention relates in particular to complex-bound hirudin 
(CBH) as a molecular marker for determining clotting activation. 
Many metabolic physiological processes are regulated via cascade 
mechanisms, which are often branched, and in which an initiator or a chain 
reaction is intensified via a series of activatable enzymes. For example, 
mechanisms such as these are effective in the regulation of glycogen 
metabolism, in the transmission of extracellular signals and in blood 
clotting in particular. On a molecular level, sequential phosphorylation 
of the factors involved in a cascade mechanism is often to be found here. 
The intensification of an initiator by this mechanism is due to the 
enzymes which participate in the different stages of the mechanism being 
capable of modifying a plurality of substrate molecules, which themselves 
are often enzymes also. The activated enzymes of a mechanism such as this 
are suitable as molecular markers for determining the activation reaction 
which is proceeding in the body in each case. In a procedure such as this, 
specific, high-affinity inhibitors of the respective activated key enzyme 
are used which are to be fed as such to a permanent diagnosis line. This 
so-called molecular marker principle has only been developed 
unsatisfactorily hitherto, however. 
The search for suitable molecular markers for the determination of 
intravasal activation reactions of blood clotting has been intensively 
pursued for some years. In the course of this work a series of metabolic 
products of clotting activation has emerged as molecular markers. These 
comprise prothrombin F1+2 fragments, platelet factor IV and 
fibrinopeptides A and B, as well as some cleavage products of fibrin 
decomposition (e.g. d-dimers) which are produced by fibrinolysis, and also 
comprise complexes formed between natural antithrombins and the serine 
protease thrombin, which are known as TAT complexes. 
The usability of these molecular markers has been analysed in large-scale 
clinical studies. It has generally been ascertained that it is possible to 
determine blood levels of molecular markers of this type which are 
definitely increased or which persist during thrombotic occurrences or 
thrombo-embolytic diseases. The efficiency of these markers leaves very 
much to be desired, however. In clinical patients who suffered from venous 
thromboses or from arterial thrombotic occlusion diseases, the response 
capacity of the most sensitive marker for clotting, namely prothrombin 
fragment F1+2, was less than 20%. Moreover, no correlation could be found 
between the severity of the thrombo-embolytic disease or thrombotic 
occurrence and the magnitude of the blood level of this and other 
molecular markers. 
From experience with persons suffering from diseases of this type, it can 
be deduced that in haemostaseology there has hitherto not been a principle 
of molecular measurement which enables conclusions to be drawn on the 
intensity of clotting activation by determining the actual blood level of 
the marker. The cause of this is that the molecular markers, as metabolic 
products of clotting enzymes which occur naturally in the body, are 
removed more or less rapidly from the circulation by elimination 
mechanisms. In this respect, it has to be taken into consideration that 
the markers are metabolised more or less rapidly, depending on the 
function of the organ concerned, particularly in the area of liver 
metabolism. 
The underlying object of the present invention is thus to identify 
sensitive markers for the detection of an early phase of metabolic 
activation. 
More particularly, an underlying object of the present invention is thus to 
identify sensitive markers for the detection of an early phase of clotting 
activation. 
Moreover, the molecular marker should have a wide diagnostic range for the 
determination of intravasal activation reactions and more specifically for 
determining blood clotting reactions. In this respect, the marker should 
act in the organism independently of metabolic processes or elimination 
reactions. It must be ensured that the marker is distributed rapidly, 
exclusively in the blood circulation, is not metabolised or is only 
metabolised to a slight extent, and is only eliminated slowly. 
This object is achieved by the use of a specific inhibitor of an enzyme 
involved in metabolism, which inhibitor is bound to a high molecular 
weight carrier, and thereby functions as an indirect marker for monitoring 
the activation or activity of this enzyme, e.g., an enzyme involved in the 
blood clotting cascade or fibrinolysis. 
Surprisingly, it has now been found that inhibitors of an activatable 
metabolic enzyme, which inhibitors are bound to a high molecular weight 
carrier, i.e. complex-bound inhibitors of an activation product of the 
blood clotting cascade or of an activated fibrinolysis enzyme, are 
exclusively and rapidly distributed in the blood circulation, are only 
decomposed or eliminated slowly in the organism, and still have almost the 
same affinity for the enzyme as do the free, un-bound inhibitors. This 
principle can be employed for all activatable key enzymes for which 
high-affinity and specific inhibitors are available. It is particularly 
suitable for the activation products of the clotting cascade, such as 
thrombin, activated factor VII or activated factor X, and is also suitable 
for activated fibrinolysis enzymes, e.g. tissue plasminogen activator 
(tPA) or plasmin. Examples of other key enzymes involved in metabolism 
which can be inhibited by high-affinity specific inhibitors include 
angiotensin-converting enzyme (involved in blood pressure regulation) and 
elastase (involved in shock reactions). 
The description given below relates to a preferred embodiment of the 
invention, namely the use of thrombin inhibitors. However, it should be 
understood that the molecular marker principle which is illustrated in 
this example for the diagnostic monitoring of blood can be employed 
correspondingly for any combination of an activatable enzyme and a 
high-affinity, specific inhibitor, which is bound to a high molecular 
weight carrier substance. 
It has surprisingly been found that thrombin inhibitors which are bound to 
high molecular weight carriers (complex-bound thrombin inhibitors) are 
exclusively and rapidly distributed in the blood circulation, are only 
decomposed or eliminated slowly in the organism, and also always have the 
same affinity for thrombin as do free thrombin inhibitors and are thereby 
suitable for diagnostic purposes. It is not possible to use un-bound 
thrombin inhibitors for diagnoses of this type, because these substances 
are distributed in the body as a whole, not only in the blood, and when 
consumed they become redistributed without a detectable decrease in their 
concentration in the blood. 
High-affinity natural thrombin inhibitors. e.g. hirudin, and also all other 
direct-binding synthetic thrombin inhibitors which have a high affinity 
for thrombin, can be used as thrombin inhibitors. Examples thereof include 
PEG-bonded 4-amidinophenylalanine (see Peptide Research 8, No. 2, 78-85 
(1995)). Natural or synthetic substances can be used as the high molecular 
weight carriers. Examples include polyethylene glycol, dextran, and also 
blood proteins which occur naturally in the body. Other examples thereof 
include albumin, .gamma.-globulins, and also ferritin, succinylated 
gelatine, crosslinked polypeptides, and polyhydroxy-starch. 
A dextran-bound (DP) hirudin is suitable for use, as is hirudin which is 
coupled to polyethylene glycol (PEG), or hirudin which has been bound to 
human body proteins. Due to their molecular size, these proteins 
(albumin-bound hirudin or hirudins which are bound to defined 
gamma-globulins) only undergo a very slow biological elimination process. 
Complex-bound hirudins of this type have the considerable advantage that 
they are distributed almost exclusively in the blood and exhibit no 
extravasation into the extracellular fluid space. Even small amounts of 
this marker, when distributed in the blood, can thereby function as a 
high-activity inhibitor, which binds rapidly and strongly, for 
intravasally activated thrombin or activated intermediates of the 
prothrombin-thrombin transformation. Due to the binding of the activated 
enzyme to the complex-bound thrombin inhibitor, the amount of "free" 
complexed hirudin is reduced by the extent to which active thrombin 
becomes available in the circulation. 
Hirudin, and also hirudin which is bound to macromolecules, has high 
affinity for the serine protease thrombin. Its singular specificity 
exclusively for thrombin species allows this marker only to become active 
for this serine protease in the organism. Binding to other enzymes is not 
possible and is not known. 
If the key enzyme of the clotting system, namely the serine protease 
thrombin, becomes available in the blood in the organism due to a 
permanent clotting activation, as the final product of intrinsic or 
extrinsic clotting, it is immediately bound and deactivated by the CBH 
molecular marker which is present in the blood. The amount of 
complex-bound hirudin, namely of the free inhibitor, decreases in flowing 
blood by the extent to which thrombin-hirudin complexes are formed. 
The "free" molecular marker, namely complex-bound hirudin, is determined 
with the aid of a sensitive, specific method of detection for free hirudin 
which can rapidly be carried out. With the aid of this method of 
detection, discrete clotting activation effects can also be detected and 
quantified at an earlier point in time in the organism by repeated 
monitoring. 
The molecular markers according to the invention are employed at a dose of 
0.005-0.5 mg/kg, preferably 0.01-0.05 mg/kg, most preferably 0.01-0.02 
mg/kg, with respect to the body weight of the patient. They are 
administered parenterally, preferably intravenously. Oral application, in 
which resorption of the marker is ensured by a corresponding formulation, 
is also possible. For this purpose, the molecular markers according to the 
invention are employed, together with customary adjuvant substances and 
carrier substances, in a formulation which is suitable for this method of 
administration. Thus, for example, complex-bound hirudin preparations can 
be produced either in a freeze-dried formulation (to be dissolved in 5 ml 
water; PEG-hirudin, albumin-hirudin, .gamma.-globulin-hirudin) or as a 
ready-to-use injection solution (e.g. 5 ml; gelatine-hirudin, 
hydroxy-starch-hirudin), wherein the content of hirudin is appropriately 5 
mg hirudin/ampoule. The requisite dose for the application is calculated 
according to the formula 
kg body weight: 20= ml application volume and is administered as an 
intravenous bolus.

The invention is explained in more detail with reference to the following 
example. 
Example: Complex-bound Hirudin as a Molecular Marker 
Patients from the group at risk were given an intravenously administered 
dose of complex-bound hirudin corresponding to their body weight. After a 
short distribution period (10-15 minutes) a constant blood level of this 
CBH was reached. The elimination half-life for CBH was known as a 
comparison quantity from corresponding control investigations on healthy 
test volunteers. 
Small amounts of citrate whole blood (0.5 ml) were taken at short time 
intervals from patients at risk from thrombosis. The free circulating CBH 
was determined with the aid of a specific detection method for hirudin, 
namely the ecarin clotting time (ECT, European Patent No. 93 903 232.2). 
The ecarin clotting time is a method of determining activity and is 
extremely sensitive for the detection of free hirudin, but not for 
hirudin-thrombin complexes. There was a direct correlation between the 
decrease in the blood level of CBH and the intensity of thrombin 
liberation in the blood circulation. A quantification of the liberation of 
thrombin in the bloodstream with time was obtained due to the possibility 
of mathematically modelling the "rate of disappearance" or the more 
pronounced decrease in the blood level of free CBH. By using this method, 
it is even possible to detect early phases of clotting activation, which 
could not hitherto be achieved diagnostically. 
In further tests, these molecular thrombin "probes" were checked for their 
efficiency in a modelling study applied to experiments on animals. 
The experimental animals used here were rats (HAN-WISt, Central 
Experimental Animal Unit of the University of Jena) and rabbits 
(Chinchilla bastards, Savo, Bad Kislegg). Both male and female animals, 
conforming to the SPF livestock standard, were used from both species. The 
rats were narcotised with ethylurethane (1.5 g/kg subcutaneously), and the 
rabbits were narcotised with pentobarbital (25 mg/kg intravenously). Two 
different methods of detection were used as methods of determining 
hirudins in blood. In addition to the ecarin clotting time, a chromogenic 
substrate method was also used. In this method, chromogenic substrate 
(Chromozym TM, Pentapharm Basle) and ecarin (25 Eu/ml) or thrombin (1.5 
NIH U/ml) were added to the plasma (diluted 1:10), and the extinction was 
measured in a spectrophotometer after two minutes (NIH U: International 
Standard for Thrombin Clotting Activity; NIH=National Institute of 
Health). 
The complex-bound hirudin was used in two different hirudin preparations: 
1. Dextran-hirudin (Dextran 150 kDa) was bound to hirudin by means of a 
method according to Walsmann et al. The specific activity was 971 ATU/mg 
(ATU=anti-thrombin units=International Standard for Direct Thrombin 
Inhibitors). 
2. PEG-coupled hirudin. Commercial preparations supplied by Knoll were used 
(PEG-hirudin/144 or PEG/153). 
1. Effect of Thrombin on the Plasma Level of PEG Hirudin in Rats 
Experimental Design: 
Narcotised rats were given PEG-hirudin in a dosage of 140 ATU/kg body 
weight, administered intravenously as a bolus (ATU=anti-thrombin units). 
10 minutes later the rats were given an infusion of thrombin and the 
control animals were given the same infusion volume of saline. 0.5 ml of 
citrate blood was taken from the rats at 60 minute intervals via a 
permanent catheter in the jugular vein, so as thereby to monitor the blood 
level of the molecular marker. Thrombin was infused into the rats in the 
following concentrations over 360 minutes: 35, 70, 140, 250 and 500 NIH 
U/kg.times.hour.sup.-1. The results are illustrated in FIG. 1. It can be 
seen that after a short initial distribution period a relatively constant 
blood level of PEG-hirudin was detected in the plasma. The approximate 
half-lives were about 12-14 hours. For an infusion of 35 NIH 
U/kg.times.hour.sup.-1 thrombin, the time-dependence of the blood level of 
PEG-hirudin was almost identical to that of the control group (saline 
infusion). At 70 NIH U/kg.times.hour.sup.-1 thrombin, a significantly 
accelerated decrease in the blood level of PEG-hirudin was detected even 
after 120 minutes. At higher doses of thrombin, free PEG-hirudin 
disappeared very rapidly from the blood circulation. At 250 or 500 NIH U 
thrombin, PEG-hirudin could no longer be detected in the plasma even after 
120 minutes. 
2. Effect of Repeated Thrombin Application on the PEG-hirudin Level of Rats 
The PEG-hirudin (PEG153) which was used in these tests exhibited a 
time-dependence of its distribution and blood level which was almost the 
same as that in the test example described above. After a brief 15 minute 
infusion of 100 NIH U/kg thrombin in each case, only a discrete 
influencing of the PEG-hirudin level was detected after 120, 180 and 240 
minutes, whereas on the application of 250 NIH-U/kg thrombin a more 
pronounced decrease in the PEG-hirudin blood level occurred after 3 hours; 
on increasing the dose to 500 NIH U/kg thrombin, this decrease was even 
greater. 
3. Effect of Thrombokinase on the Plasma Level of Dextran-hirudin in 
Rabbits 
The rabbits were pre-treated with 5000 ATU/kg of dextran-hirudin. From the 
monitoring of the dextran-hirudin blood level it could be seen that a 
constant hirudin blood level in the rabbits first detected after 24 hours. 
On the infusion of a purified thrombokinase solution (1 ml/kg/hour) over 6 
hours, a more pronounced decrease in the dextran-hirudin blood level was 
detected. For these investigations, the results of five separate tests 
were combined. 
It follows from these investigations that dextran-hirudin has a very long 
distribution phase (24 hours) in rabbits, due to the interactions of the 
dextran with surface structures of the endothelium cells, with the RES of 
the liver and with the corpuscular constituents of the blood. For this 
species of animal, dextran-hirudin was only of limited suitability for 
marker investigations. It could not be identified from these 
investigations whether the dextran-hirudin was also distributed in deeper 
compartments of the rabbit organism. In contrast, the PEG-coupled hirudins 
(PEG 144 and 153) proved to be suitable for corresponding molecular marker 
modelling in the tests on rats which are presented here. A relatively 
constant distribution equilibrium was attained in the circulation of rats, 
even after 10 minutes, and modelling of intravasal clotting activation by 
means of the continuous infusion of small amounts of thrombin, or by the 
discontinuous application of thrombin, could be followed and measured by a 
corresponding disturbance of the PEG-hirudin blood level. 
It can be deduced from these modelling investigations of animal experiments 
that complex-bound hirudins are suitable as molecular markers for 
intravasal clotting activation. The advantage of the molecular markers 
which are presented here is that high molecular weight hirudin complexes 
of this type are not subject to any elimination function in the organism. 
They are permanently available for binding active thrombin, which can 
occur permanently in the circulation as a final activation product of 
clotting activation. A definite clotting activation can be quantified by a 
sensitive method of detecting the level of free marker.