Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing – Stabilized enzymes or enzymes complexed with nonenzyme
Reexamination Certificate
2000-05-08
2004-10-26
Weber, Jon P. (Department: 1653)
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
Stabilized enzymes or enzymes complexed with nonenzyme
C435S069100, C435S069600, C435S183000, C514S002600, C514S822000, C530S350000, C530S380000, C930S010000
Reexamination Certificate
active
06808706
ABSTRACT:
INDUSTRIAL FIELD OF APPLICATION
The present invention relates to a method for maintaining the quality of aqueous injection preparation of thrombomodulin in a non-frozen or non-freeze-dried liquid form over its storage/transportation and to an aqueous injection preparation of thrombomodulin stable over its storage/transportation.
PRIOR ART
Thrombomodulin (in the following, abbreviated sometimes as TM) is a substance having a function of coupling specifically with thrombin and accelerating the activation of protein C by thrombin remarkably. Protein C consists of a vitamin K dependent protein which plays an important role in the coagulation fibrinolysis system and is activated by the action of thrombin into activated protein C. It has been known that activated protein C will inactivate the activated coagulation factors V and VIII in the blood coagulation system of living body and that it participates in the production of plasminogen activator which reveals a thrombolytic function (See Koji Suzuki, “Igaku-no Ayumi (Progress in Medical Science)”, Vol. 125, 901 (1983))
Thus, it has been recognized that thrombomodulin accelerates activation of protein C by thrombin to produce a large amount of activated protein C exhibiting inhibitive action to blood coagulation and thrombolytic function and is useful as an anticoaglant and as a thrombolytic agent. It has heretofore been expected to use thrombomodulin for therapy and prophylaxis of, such as acute coronary syndrome (ACS), for example, myocardiac infarction and unstable angina, for reconstruction of coronary circulation; for therapies and prophylaxes of thromboses, such as acute and chronic cerebral thromboses and acute and chronic arterial and venous peripheral thromboses; peripheral blood vessel obstruction, such as Buerger disease and Raynaud disease; obstructive arteriosclerosis; blood vessel inflammation, such as systemic lumpus erythematodes (SLE), Becet disease and Kawasaki disease; and for therapies and prophylaxes of functional disorders pursuant to a cardiac surgery, complications pusuant to an organ transplantation, disease of intravascular coagulation (DIC), angina pectoris, transient cerebral ischemic attack, gestational toxicosis, diabetes, liver veno-occlusive diseases (VOD), such as venoocclusive diseases following fulminant hepatitis or after bone marrow transplantation, and deep venous thrombosis (DVT).
In the past, thrombomodulin was confirmed and isolated as a glycoprotein appearing on the endothelial cells of blood vessel of animals, including human. Our group of inventors of the present invention had succeeded to clone it for the first time. Thus, the inventors had performed cloning of genes of a precursor of a human thrombomodulin having a signal peptide from the human lung cDNA library using a technique of genetic engineering, whereby the entire gene sequence of the thrombomodulin was analyzed and its amino acid sequence having 575 residues including a signal peptide of 18 amino acid residues was made clear (See Japanese Patent Kokai Sho 64-6219 A). It has been known that a mature thrombomodulin with the signal peptide being cut is composed of five domains, namely, the NH
2
-terminal domain (1st to 226th amino acid residue, in the order from the NH
2
-terminus), the domain having 6 EGF-like structures (227th to 462nd residue), the O-glycosylation site-rich domain (463rd to 498th residue), the transmembrane domain (499th to 521st residue) and the cytoplasmic domain, enumerated respectively in the sequential order from the NH
2
-terminus, wherein the segment exhibiting the activity corresponding to that of the full length thrombomodulin, namely, the least active unit thereof, is constituted of the segemnt consisting of the fourth, fifth and sixth EGF-like structures in the domain having 6 EGF-like structures as enumerated in the order from the NH
2
-terminus (See M. Zushi et al, J. Biol. Chem., 246, 10351-10353 (1989)).
It has been confirmed that at least such a thrombomodulin which is prepared so as to eliminate the transmembrane domain has a nature of being clearly dissolved in water even without using any surfactant (in the following, referred to sometimes as “soluble thrombomodulin”) and, for example, a thrombomodulin composed of only the three domains, i.e. the NH
2
-terminal domain, the domain having 6 EGF-like structures and the O-glycosylation site-rich domain, namely, composed of the amino acid sequence from the 19th to 516th amino acid residues of the sequence listing SEQ ID NO:1, can be obtained by applying a gene recombination technology and that the so-obtained recombinant thrombomodulin has an activity which is the same as that of natural thrombomodulin (Japanese Patent Kokai Sho 64-6219 A).
By the way, as recognized in many cases, genes may suffer from natural and artificial mutations due to inspection work upon, for example, their isolation etc., without exception for human in which also a polymorphic mutation had been discovered, wherein two mutants of the precursor of human thrombomodulin constituted of the amino acid sequence composed of the above-mentioned 575 amino acid residues were confirmed, in which the amino acid residue at the 473rd site consists of Val, for the one, and in which this amino acid residue consists of Ala, for the other. This corresponds, in the base sequence coding such amino acids, to mutations of T and C of the 1418th site, respectively (Wen et al, Biochemistry, 26, 4350-4357 (1987)). They do reveal, however, no difference in the activity and in the physical properties from each other and can be regarded as being substantially identical. Therefore, the above-mentioned human thrombomodulin constituted of the amino acid sequence of the sequence listing SEQ ID NO:1 is regarded as one of polymorphs of the peptide of thrombomodulin composed of the amino acid sequence of sequence listing SEQ ID NO: 2 and both should be judged as substantially identical with each other.
On the other hand, thrombomodulin preparations have currently been distributed steadily for practical use in a form of freeze-dried medicinal pharmaceutical. Meanwhile, it had been discovered that, in the process of freeze-drying of a thrombomodulin-containing aqueous solution, a part of thrombomodulin is converted, though in a minute amount, into a polymeric matter due to a denaturization to form a polymer in which several molecules of thrombomodulin are held in association. For resolving this problem, the inventors made sound researches, whereby they reached the discovery that denaturization of thrombomodulin upon freeze-drying thereof could be prevented, of which invention was previously applied for a patent (Japanese Patent Kokai Hei 6-321805 A).
THEME TO BE SOLVED BY THE INVENTION
Despite of the above circumstances, requests had been raised for providing a novel preparation in a non-frozen or non-freeze-dried form which can be used simply and easily and can be produced at a lower production cost.
MEANS FOR SOLVING THE THEME
The inventors of the present invention had investigated the possibility of realizing an aqueous injection preparation of thrombomodulin which can afford to avoid incorporation of freeze-drying process and to eliminate necessity of dissolution procedure upon practical use, under an attempt of developing a new preparation other than the freeze-dried preparation. While it is requested at first for an aqueous injection preparation that its residual potency after storage over a long period of time at 5° C. to room temperature should not be decreased to a considerable extent (i.e. a long term stability), investigations have shown that it is not easy to attain such a long term stability. To our surprise, it was further found that an aqueous injection preparation may in some cases become turbid by shaking it. It is expected enough that a liquid preparation maybe subjected to a shaking motion, though at different intensity levels, on its distribution, such as transportation. Such a problem is not encountered for conventional freeze-dried powdery preparation. Thus, a new and unexpected problem wa
Murata Tomoyo
Shimizu Hirotomo
Tsuruta Kazuhisa
Yokozawa Akira
Yui Masaki
Asaki Kasei Pharma Corporation
Schnizer Holly
Weber Jon P.
Young & Thompson
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