JPH0369332B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to tissue plasminogen activators, particularly pharmaceutical compositions containing tissue plasminogen activators, their manufacture and their use in human and veterinary medicine. It is believed that there is a dynamic balance that maintains a healthy open vascular bed between an enzyme system that can form blood clots - the coagulation system - and an enzyme system that can dissolve blood clots - the fibrinolytic system. To limit blood loss from injury, blood clots form in injured blood vessels. After the injury has healed naturally, excess blood clots are dissolved by the action of the fibrinolytic system. Sometimes blood clots form without trauma and can lodge in major blood vessels, resulting in partial or even total occlusion of blood flow. If this occurs in the heart, lungs, or brain, it can cause myocardial infarction, pulmonary embolism, or stroke. These combined conditions are a major cause of morbidity and mortality in industrialized countries. Blood clots consist of a fibrous network that can be dissolved by the proteolytic enzyme plasmin. This enzyme is derived from the inactive proenzyme plasminogen, which is a component of plasma, by the action of plasminogen activators. There are two immunologically distinct mammalian plasminogen activators.
Endogenous plasminogen activator, also known as urokinase, is an enzyme produced by the kidneys and can be isolated from urine. It can also be produced from many tissue culture sources. Extrinsic plasminogen activator, also known as vascular plasminogen activator and tissue plasminogen activator (t-PA), is found in significant tissue homogenates (particularly human uterus), vascular cell walls and Can be isolated from several cell cultures. In addition to these two plasminogen activators, there is streptokinase, a bacterial product produced from beta-hemolytic streptococci. The major disadvantage associated with both urokinase and streptokinase is that they are active throughout the circulatory system and not only at the site of blood clots. These destroy other blood proteins such as fibrinogen, prothrombin, Factor V and Factor, thus reducing the ability to form clots and increasing the risk of bleeding. On the contrary,
The biological activity of t-PA depends on the presence of fibrin to which it is bound and activates it. Maximum activity thus occurs only at the location of the clot, ie where there is a fibrin network to be dissolved, which significantly avoids the risk of bleeding. The main route of t-PA administration is intravascular injection;
Therefore, preparations of t-PA are also required as solutions for parenteral administration. In the case of proteins, it is preferred to provide the drug to the physician or veterinarian as a lyophilized pharmaceutical composition. This is due to its significant transport and storage advantages over liquid formulations. However, any such lyophilized preparation can be readily converted into the desired solution for parenteral administration without undue inconvenience or difficulty, and the physician or veterinarian can convert this preparation into a droplet of the solvent. It is important that the concentration required for any given situation can be obtained simply by reconstitution in the medium. For example, it is undesirable to administer large amounts of solution to a patient with cardiac or renal impairment, as this places even greater stress on the patient's heart or kidneys. Therefore, the dose volume should be kept to a minimum in such situations.
It is therefore desirable that solutions for parenteral administration can be obtained that have not only relatively low concentrations, but also high drug concentrations. Many lyophilized pharmaceutical formulations of t-PA are available in the prior art, for example EP-A-113319 and EP-A-
123304. These formulations have their PH
is produced from an aqueous saline solution of t-PA, which is approximately neutral, and has the disadvantage that the solubility of t-PA in such solutions is low without increasing the ionic concentration. Therefore, solutions for parenteral administration obtained from such lyophilized formulations contain t-PA at such low concentrations that in some situations it is necessary to administer large amounts of solution to the patient, which is undesirable. It is either hypertonic and can be harmful to red blood cells when administered. Here, the solubility of t-PA in aqueous solution is
that it can be improved by bringing the pH of the solution within the oxygen range;
It has been discovered that a lyophilized pharmaceutical formulation can be prepared from an acidic solution of t-PA and that this formulation can provide a solution for parenteral administration that does not present significant physiological problems when administered. Accordingly, the present invention provides a method for producing a lyophilized pharmaceutical formulation of t-PA, which method comprises drying a frozen aqueous solution of t-PA having a pH of 2-5. As a result of the improved solubility of t-PA, t
- at high concentrations without any substantial risk of PA precipitating out of solution.
A lyophilized pharmaceutical formulation capable of providing a solution for parenteral administration containing PA can be produced according to the present invention. Therefore, when and where required by the present invention,
The lyophilized formulation can be provided to a physician or veterinarian who can dissolve the formulation to the desired concentration using, for example, water at a neutral or acidic PH. Thus, the present invention provides a stable lyophilized pharmaceutical formulation that is extremely flexible in its handling and can be used by physicians and veterinarians, while providing a more convenient means of transport and storage. The t-PA used in the present invention can be any biologically active protein substantially corresponding to a mammalian protein, particularly human t-PA, including glycosylated and non-glycosylated forms. . This can be single chain or double chain t-PA as described in EP-A-112122, fully glycosylated human t-PA.
- In the case of PA, it has an apparent molecular weight based on polyacrylamide gels of about 70,000 and an isoelectric point of 7.5-8.0. Preferably, t-PA is about
It has a specific activity of 500000 IU/mg [IU./mg = International Units/mg; International units are WHO, National Institute Biological Standards and Control (National
Institute for Biological Standards and
Control), Holly Hill, Hampstead, London
NW 3 6 RB, activity units as defined by the United Kingdom]. The amino acid sequence of t-PA preferably substantially corresponds to the sequence set forth in FIG. That is, this sequence is identical to the sequence in Figure 1, or contains allelic or other deletions, substitutions, insertions, transpositions, or additions of one or more amino acids, and the resulting sequence is the first sequence. It has at least 80%, preferably 90% homology with the sequence shown and essentially retains the same biological and immunological properties of the protein. In particular, the t-PA sequence is either identical to the sequence in Figure 1 or serine N-
They have identical sequences except for a valine instead of a methionine at the 245th position from the end, and neither of these sequences is present in the first three amino acids, or, as the case may be,
It has an additional polypeptide N-terminal leading sequence of Gly-Ala-Arg. The amino acid sequence shown in Figure 1 has 35 cysteine residues and thus has the ability to form 17 disulfide bridges. Based on homology with other proteins whose structures have been determined in more detail, a hypothetical structure for the sequence between the amino acid at position 90 and the proline C-terminus is shown in FIG. In Figure 2, the * mark is a two-stranded t-
The cleavage site of the single chain form t-PA is shown to give PA; where the A chain contains two Kringle regions;
and the B chain contains a serine protease region. Although the structure of this N-terminal region has not been determined, several proposals have been made [Progress in fibrinolysis].
Fibrinolysis), 1983, 6 , pp. 269-273; and Proc. Nattle. Acad. Sai. (Proc.Natl.
Acad.Sci.) 1984, 81 , pp. 5355-5359]. t
-The most important feature of the structure of PA is the two cyclic parts (between amino acids 92 and 173 and between amino acids 180 and 261) that serve to bind this protein to fibrin. ) and the major region of the B chain and the serine protease region responsible for plasminogen activation. A particularly important amino acid in the serine protease region is the catalytic amino acid triad, His/Asp/Ser. t-
In PA they are located at 322nd, 371st and 463rd. Cysteine amino acid residues at positions 264 and 395 are also important;
These hold together the two chain forms of t-PA, A chain and B chain. In Figures 1 and 2, the following conventional one-letter and three-letter symbols have been used for amino acid residues: Asp D Aspartic acid Ile I Isoleucine Thr T Threonine Leu L Leucine Ser S Serine Tyr Y Tyrosine Glu E Glutamic acid Phe F Phenylalanine Pro P Proline His H Histidine Gly G Glycine Lys K Lysine Ala A Alanine Arg R Arginine Cys C Cysteine Trp W Tryptophan Val V Valine Gln Q Glutamine Met M Methionine Asn N Asparagine t-PA is available using our technology It can be obtained by any of the known or disclosed manufacturing methods.
As an example, t-PA is Biochimica et al.
Biophysica Acta), 1979, 580 , 140-153;
It can be obtained from normal or tumor cell lines of the type described in EP-A-41 766 or EP-A-113 319. However, t-PA is
For example as described in EP-A-93 619; EP-A-117 059 or EP-A-117 060.
Preferably, it is obtained from cultured transformed or transfected cell lines derived using DNA recombination techniques. Using Chinese hamster ovary (CHO) cells to produce t-PA,
Molecular and Cellular Biology, 1985.5
(7), pages 1750-1759. In this way, the cloned gene produces dihydrofluorate reductase (dhfr).
It is cotransfected by the gene encoded by dhfr-CHO cells. dhfr expressing transformants are selected on nucleoside-free medium and exposed to increasing concentrations of methotrexate. Thus, the dhfr and t-PA genes are amplified together leading to a stable cell line capable of expressing high levels of t-PA. t-PA is preferably purified using any of the methods known or disclosed in the art, such as those described in the following references: Biochimica et Biophysica Acta, 1979;
580, pp. 140-153; Journal of Biological Chemistry (J.Biol.Chem.), 1979.
254(6), pp. 1998-2003; same, 1981, 256(13) ,
Pages 7035-7041; European Journal of
Biochemistry (Eur.J.Biochem.), 1983
Year, 132 , pp. 681-686; EP-A-41 766; EP-
A-113 319; or GB-A-2 122 219. There does not appear to be any upper limit to the solubility of the t-PA used in the method of the invention in aqueous solutions. 150000000IU./ml (International unit/
Even at very high concentrations, such as greater than ml), the solution simply becomes viscous without any significant t-PA precipitation. Therefore, the concentration of t-PA in aqueous solution is within wide limits, e.g.
It can be changed from 50000 to 50000000IU/ml.
To ensure maximum benefit from the invention, t-
The concentration of PA is greater than 100000IU/ml, more especially
Greater than 500000IU/ml, more especially 1000000IU/ml
It's bigger. The concentration of t-PA is approximately 5000000IU/
ml is most preferred. The upper limit of the pH of the aqueous solution is preferably 4.5. In practice, the PH is preferably in the range 2.5 to 4.0, more preferably in the range 2.8 to 3.5, even more particularly preferably about 3.0. The desired pH of an aqueous solution is conveniently obtained using physiologically acceptable inorganic or organic acids. Examples of such acids include hydrochloric, sulfuric and nitric acids, as well as citric, tartaric and benzenesulfonic acids. Among these examples, hydrochloric acid is preferred. It is preferred that the medium for aqueous solutions be wholly or substantially aqueous, although certain physiologically acceptable co-solvents may optionally be present in addition to the water. Solutions for parenteral administration obtained from lyophilized pharmaceutical formulations can be hypertonic, hypotonic or isotonic with the patient's serum. However, in order to avoid undesirable side effects, solutions for parenteral administration are preferably isotonic, with low degrees of change having little physiological relevance. Substantially isotonic solutions for parenteral administration can be obtained by the inclusion of physiologically acceptable auxiliary agents capable of raising the tonicity of the solution to the required level. Such auxiliaries can be included in the aqueous solution to be lyophilized such that they are already present in the lyophilized pharmaceutical formulation, or they can be dissolved in such a formulation to obtain the desired solution for parenteral administration. It can also be included in water of neutral or acidic PH used for water. Examples of such auxiliaries are well known in the art;
Includes dextrose (anhydrous or monohydrate form) and sodium chloride and also mixtures thereof. Of course, the concentration of such auxiliaries in the aqueous solution or dissolution water will vary depending on the materials used. In the case of sodium chloride, this concentration is preferably between 7 and 10 mg/ml, more preferably about
8.5 mg/ml, a concentration often referred to as physiological saline or saline. In the case of anhydrous dextrose, the concentration is preferably
30 to 70 mg/ml, more preferably about 50 mg/ml. The aqueous solution can optionally contain excipients that are normally contained in lyophilized pharmaceutical preparations of this type. Examples of such include human serum albumin, binders and fillers such as mannitol, lactose and glucose. Additionally, t-PA has a tendency to adsorb to glass and plastic surfaces, so it may be desirable to include a surfactant in the aqueous solution to prevent or minimize such adsorption. . Examples of such surfactants include polyoxyethylene derivatives of fatty acid partial esters of anhydrous sorbitol, such as those sold under the tradename "Tween 80." One of the surprising advantages of the present invention is that t-
Apart from the substantially increased solubility of PA, the use of acidic parenteral administration solutions obtained by reconstitution of lyophilized pharmaceutical formulations does not exhibit any significant adverse physiological effects when administered to patients. It is in. Blood flow generally appears to raise the PH of the solution to neutral almost immediately upon contact, and t-PA is rapidly distributed within the blood stream. However, it is preferred that the process is not substantially interfered with in any way and that the solution for parenteral administration and the aqueous solution for lyophilization and also the water for reconstitution do not contain strong buffers. However, weak buffers may be included that do not significantly interfere with the process, and it is certainly true that t-PA itself acts as a weak buffer at acidic PH. Furthermore, human serum albumin can act as a weak buffering agent. Since t-PA has a substantially increased solubility in aqueous solutions with a pH of 2 to 5, the solution for parenteral administration resulting from reconstitution of this lyophilized formulation contains t-PA.
- There is no need to include any additional auxiliaries such as lysine or ornithine or mixtures thereof to enhance the solubility of PA. An aqueous solution of t-PA can be obtained by obtaining a solution of purified t-PA and then replacing it with an aqueous medium having a pH of 2 to 5;
It can be prepared by dissolving PA in an aqueous medium with a pH of 2-5. Purification of t-PA can include as a final step the elution of the protein from a chromatographic column in a solution containing a strong buffer.
As mentioned above, solutions for parenteral administration and therefore lyophilized pharmaceutical formulations and aqueous solutions do not contain strong shock absorbers. Therefore, dialysis should be used to replace the medium as a convenient means of its removal. Dialysis can be performed using dialysis tubing or an artificial kidney in which the purified solution is dialyzed against an aqueous medium having a PH of 2-5. t especially in purified solutions
−If the concentration of PA is high, first the pH of this solution is
It may be desirable to adjust the ratio to between 2 and 5.
Another means of removing strong buffers and replacing the medium is to gel filter the purified solution and then develop the column with an aqueous medium having a pH of 2-5. The precipitated solid form of t-PA is preferably obtained from a purified solution by adjusting its pH to about 5.5, cooling the solution to a temperature just above its freezing point, and then removing the protein, e.g. by centrifugation. I can do it. The precipitated solids are then treated with 2-5
It can be dissolved by conventional methods in an aqueous medium having a pH. The resulting aqueous solution is conveniently sterilized, eg by sterilization by filtration, and then dispensed into sterile plastic or glass containers, such as ampoules or vials, in volumes of eg 0.5 to 20 ml. The aqueous solution of t-PA is preferably -10 to -40°C
Freeze at a temperature of The frozen aqueous solution is then preferably maintained at this temperature until vacuum drying begins. Vacuum drying of frozen aqueous solutions can be carried out as appropriate;
For example, drying under partial or full vacuum at 0.01 to 0.1 Torr for a sufficient time to remove substantially all of the frozen liquid. The temperature at which vacuum drying is carried out is usually such that the aqueous solution is maintained in a substantially or completely frozen form.
-30 to -40°C at the beginning of the process. As the treatment progresses and water is removed, the temperature may gradually increase until room temperature is reached. 0.01 at or just above room temperature at the end of the process to remove as much of the last traces of water as possible.
Preferably, the vacuum drying is carried out under a substantial Torr vacuum. The water content of the resulting lyophilized pharmaceutical formulation is preferably 2.5% or less. Once vacuum drying is complete, the sterile plastic or glass container containing the lyophilized pharmaceutical formulation is conveniently sealed. During vacuum drying of the frozen aqueous solution, water is removed and the t-PA remains in the form of a physiologically acceptable salt. Accordingly, the present invention also provides physiologically acceptable salts of t-PA, particularly physiologically acceptable acid addition salts such as the hydrochloride. By using the present invention, t-PA can be obtained at high concentration.
A lyophilized pharmaceutical formulation can initially be obtained which can provide a solution for parenteral administration containing . Therefore,
The present invention also provides t-PA of 100,000 IU/by adding water.
more than ml, and especially more than 500000IU/ml,
Most particularly, a lyophilized pharmaceutical formulation of t-PA is provided which can be provided in concentrations greater than 1,000,000 IU/ml. To prepare a parenteral solution of t-PA for administration, the lyophilized pharmaceutical formulation obtained by the method of the invention is reconstituted with water of neutral or acidic PH. Where the aqueous solution from which the lyophilized pharmaceutical formulation is obtained is substantially isotonic, it is preferred that the water for reconstitution is also substantially tonic. t- in dissolving the fibrin network of the blood clot.
The biological activity of PA has led to its use in the treatment of thrombotic disorders [The Lancet].
Lancet), November 7, 1981, pp. 1018-1020; 1985
April 13th, pp. 842-847; The New England Journal of Medicine
New England Journal of Medicine), 1984,
310(10), pp. 609-613; same, 1985, 312(14), 932
~936 pages]. Accordingly, the present invention provides a method for the treatment of thrombotic disorders in a mammal, which method comprises treating t-
and administering to the mammal a solution for parenteral administration of PA. Alternatively, for use in humans or animals. A lyophilized pharmaceutical formulation of t-PA is provided, particularly for use in the treatment of thrombotic disorders. Specific examples of thrombotic disorders are known to those skilled in the art and include myocardial infarction, deep artery thrombosis, pulmonary embolism, and stroke. The primary route of administration of t-PA is by intravascular, particularly intravenous, injection, but other possible routes of administration can also be used, such as intramuscular administration. Intravascular injections are typically carried out using parenteral solutions contained in infusion bags or bottles or in electrically operated infusion cylinders. This solution can be given to the patient by gravity feeding from an infusion bag or bottle or by using an infusion pump. The use of weight feed infusion systems does not provide sufficient overall control over the rate of administration of solutions for parenteral administration. Therefore, the use of infusion pumps is particularly suitable for solutions containing relatively high concentrations of t-PA. However, more preferably,
An electrically operated infusion syringe is used which allows for greater overall control over the rate of administration. t- effective in the treatment of mammals with thrombotic disorders.
The amount of PA will of course vary depending on many factors, including the age and weight of the mammal, the precise condition requiring treatment and its severity, the route of administration, and ultimately is at the discretion of the attending physician or veterinarian. However, the effective amount for dissolving coronary artery thrombi, for example, is generally between 150,000 and
It is preferably in the range of 450000 IU/Kg patient weight/hour. Therefore, for an adult weighing 70 kg, the effective dose per hour is generally 10,000,000 to 3,000,000 IU,
In particular about 20,000,000 IU, this amount can be administered with or without the initial dose. moreover,
This dose is recommended for some thrombotic conditions, such as deep artery thrombosis and acute stroke, or even more for simply maintaining the patency of coronary arteries that are already reperfused. It is preferable to use a small amount. In these cases, the effective amount is generally 7000-36000 IU/Kg of patient body weight/hour. The following examples are presented to illustrate the invention and are not intended to limit it in any way. Example 1 Molecular and Cellular Biology, 1985
5(7) , pp. 1750-1759, was purified by chromatography of the purified crop of t-PA obtained from a cultured transgenic CHO cell line derived using the method of 2006, 5(7), pp. 1750-1759. t-PA is collected as an aqueous solution at PH 5.5 containing 0.17M sodium citrate and 0.01% (wt/vol) Tween 80. The pH of the solution was adjusted to 3.0 with hydrochloric acid, and the resulting solution was poured into an H-10 cartridge (Amicon Ltd., Upper Hill, Stonehouse, Gloucestershire).
Stonehouse, Gloucestershire, UK]. The concentrated aqueous solution was passed through a gel filtration column [Sephadex G-
150; Pharmacia Biotechnology, Uppsala, Sweden], 0.01
0.85% containing % (weight/volume) Tween 80
Further purify by eluting with saline at a pH of 3.0. The aqueous solution of highly purified t-PA thus obtained is concentrated once again using a disposable artificial kidney. t-PA increased the pH to 5.5 with sodium hydroxide and then the suspension to 4.
Precipitate from solution by holding at 0C for 2 hours. This solution was centrifuged at 4000 xg for 30 minutes at 4°C, and a total of 1680 x 10 ⁶ units of t-PA was collected in the precipitate. The t-PA pellets were mixed into 2300 ml of an aqueous solution of sodium chloride [0.85% (weight/volume)] containing 0.01% (weight/volume) Tween 80.
(the amount necessary to obtain a concentration of 7500000 IU/ml to 10000000 IU/ml), and the pH was adjusted to 3.0 with hydrochloric acid. The t-PA activity of this redissolved t-PA pellet was repeatedly measured and a t-PA activity of 8.09×10 ⁶ IU/ml was obtained. Add this t-PA solution to 0.01
Aqueous solution of sodium chloride containing % (wt/vol) Tween 80 [0.85% (wt/vol)] 490
ml, adjusted to PH 3.0 with hydrochloric acid, and then diluted with 930 ml of a 10% (wt/vol) mannitol solution in the same acidic saline solution. By this,
t-PA5000000IU/ml and mannitol25
3720 ml of solution containing mg/ml were obtained. The resulting solution is filter sterilized and then dispensed in 1 ml volumes into glass vials and the vials are frozen at -35°C. Apply vacuum at 0.05 torr. After about 24 hours, the temperature was gradually increased to 5°C, and at this temperature 16
Protect your time. The temperature is then increased again to 25° C. and the vacuum increased to 0.02 Torr for an additional 24 hours, after which the vial is sealed under a partial vacuum of 600 Torr of dry nitrogen. Example 2 The thrombolytic effect of the parenterally administered solution obtained from the lyophilized formulation of t-PA of Example 1 is evaluated in an in vivo model of jugular vein thrombosis. (a) Method The experimental method was first described by Collen et al. Clin. Invest. (J.Clin.Invest.), 1983, 71 , 368-376
page]. The lyophilized formulation of Example 1 was mixed with 0.01% Tween 80.
Dissolves rapidly and completely in sterile isotonic saline solution containing pH 3.0. The concentration of this reconstituted t-PA is 50000 IU/ml. A solution for parenteral administration is thus obtained for a 2 hour infusion of 500000 IU/Kg of t-PA. Injection is performed using a cannula in the right femoral vein.
Four New Zealand White rabbits are used in this experiment. After injection, evaluate the degree of thrombolysis. (b) Results The thrombolytic effect of the solution for parenteral administration obtained from the lyophilized preparation of Example 1 was expressed as % thrombolysis.
It is 28.9±4.1. Furthermore, no harmful reactions have been observed with this solution.

[Brief explanation of drawings]

Figure 1 shows a typical amino acid sequence of t-PA, and Figure 2 shows the hypothesized structure of the amino acid sequence between the 90th amino acid and the proline C-terminus of t-PA. be.

Claims (1)

[Scope of Claims] 1. A lyophilized pharmaceutical preparation of tissue plasminogen activator (t-PA) obtained by drying a frozen aqueous solution of tissue plasminogen activator (t-PA) under reduced pressure having a pH of 2 to 5. 2. The formulation according to claim 1, wherein the t-PA is single-chain or double-chain. 3 t-PA has the amino acid sequence listed in Figure 1 or 245 from the serine N-terminus.
It has the same amino acid sequence as in Figure 1 except that the amino acid No. 1 is valine instead of methionine, and optionally any of the first three amino acids are absent, or optionally Gly-Ala-Arg
3. A formulation according to any one of claims 1 or 2, having an additional polypeptide N-terminal preceding sequence. 4. The formulation according to any one of claims 1 to 3, wherein the t-PA is obtained from a cultured transformed or transfected cell line induced using recombinant DNA techniques. 5. The formulation according to any one of claims 1 to 4, wherein the concentration of t-PA in the aqueous solution is greater than 100000 IU/ml. 6. The formulation according to claim 5, wherein the concentration of t-PA is greater than 500000 IU/ml. 7. The formulation according to claim 6, wherein the concentration of t-PA is greater than 1,000,000 IU/ml. 8. The formulation according to claim 7, wherein the concentration of t-PA is 5,000,000 IU/ml. 9. The formulation according to any one of claims 1 to 8, wherein the aqueous solution has a pH of 2 to 4.5. 10. The formulation according to claim 9, which has a PH of 2.5 to 4.0. 11 Claim 10 where PH is 2.8 to 3.5
Preparations described in section. 12. The formulation of claim 11, wherein the PH is about 3.0. 13. A formulation according to claims 1 to 12, wherein the medium for aqueous solution is wholly or substantially aqueous. 14. A formulation according to any one of claims 1 to 13, wherein the medium for the aqueous solution contains physiologically acceptable auxiliaries which render the solution substantially isotonic with human serum. 15. The formulation according to claim 14, wherein the physiologically acceptable auxiliary agent is sodium chloride. 16. The formulation according to claim 14, wherein the physiologically acceptable auxiliary agent is dextrose. 17. The formulation according to any one of claims 1 to 16, wherein the aqueous solution contains a surfactant. 18. The formulation according to any one of claims 1 to 17, wherein the aqueous solution is substantially unbuffered. 19. The preparation according to any one of claims 1 to 18, wherein the aqueous solution does not substantially contain lysine or ornithine or a salt thereof. 20 Claims 1 to sterilize an aqueous solution
The formulation according to any one of paragraph 19. 21. A formulation according to claim 1 for use as a human or veterinary medicine, in particular for the treatment of thrombotic disorders.