Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1998-08-31
2003-06-24
Low, Christopher S. F. (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S012200, C514S008100, C530S350000, C530S395000, C530S324000, C435S069200, C536S023500
Reexamination Certificate
active
06583108
ABSTRACT:
FIELD OF THE INVENTION
The compositions of the invention relate to the field of proteins which inhibit serine protease activity. The invention also relates to the field of nucleic acid constructs, vectors and host cells for producing serine protease inhibiting proteins, pharmaceutical compositions containing the protein, and methods for their use.
BACKGROUND OF THE INVENTION
Problem Addressed
Blood loss is a serious complication of major surgeries such as open heart surgery and other complicated procedures. Cardiac surgery patients account for a significant proportion of transfused donor blood. Blood transfusion carries risks of disease transmission and adverse reactions. In addition, donor blood is expensive and demands often exceed supply. Pharmacological methods for reducing blood loss and the resultant need for transfusion have been described (reviewed by Scott et al., Ann. Thorac. Surg. 50: 843-851, 1990).
Protein Serine Protease Inhibitor
Aprotinin, a bovine serine protease inhibitor of the Kunitz family is the active substance in the medicament Trasylol®. Aprotinin (Trasylol®) has been reported as being effective in reducing perioperative blood loss (Royston et al., Lancet ii: 1289-1291, 1987; Dietrich et al., Thorac. Cardiovasc. Surg. 37: 92-98, 1989; Fraedrich et al., Thorac. Cardiovasc. Surg. 37: 89-91, 1989); W. van Oeveren et al. (1987), Ann Thorac. Surg. 44, pp 640-645; Bistrup et al., (1988) Lancet I, 366-367), but adverse effects, including hypotension and flushing (Bohrer et al., Anesthesia 45: 853-854, 1990) and allergic reactions (Dietrich et al., Supra) have been reported. Use of aprotinin in patients previously exposed to it is not recommended (Dietrich et al., Supra). Trasylol® has also been used for the treatment of hyperfibrinolytic hemorrhages and traumatic hemorrhagic shock.
Aprotinin is known to inhibit several serine proteases including trypsin, chymotrypsin, plasmin and kallikrein, and is used therapeutically in the treatment of acute pancreatitis, various states of shock syndrome, hyperfibrinolytic hemorrhage and myocardial infarction (Trapnell et al., (1974) Brit J. Surg. 61: 177; J. McMichan et al., (1982) Circulatory Shock 9: 107; Auer et al., (1979)Acta Neurochir. 49: 207; Sher (1977) Am J. Obstet. Gynecol. 129: 164; Schneider (1976), Artzneim.-Firsch. 26: 1606). It is generally thought that Trasylol® reduces blood loss in vivo through inhibition of kallikrein and plasmin. It has been found that aprotinin (3-58, Arg15, Ala17, Ser42) exhibits improved plasma kallikrein inhibitory potency as compared to native aprotinin itself (WO 89/10374).
Problems With Aprotinin
Because aprotinin is of bovine origin, there is a finite risk of inducing anaphylaxis in human patients upon re-exposure to the drug. Thus, a human functional equivalent to aprotinin, by virtue of a lower risk of anaphylaxis, would be most useful and desirable to have.
Aprotinin is also nephrotoxic in rodents and dogs when administered repeatedly at high dose (Bayer, Trasylol®, Inhibitor of proteinase; Glasser et al., in “Verhandlungen der Deutschen Gesellschaft fur Innere Medizin, 78. Kongress”, Bergmann, Munchen, 1972 pp. 1612-1614). One hypothesis ascribes this effect to the accumulation of aprotinin in the negatively charged proximal tubules of the kidney, due to its high net positive charge (WO 93/14120).
Accordingly, an object of the present invention is to identify human proteins with functional activity similar to aprotinin. It was also an object of the instant invention to identify human proteins, that would be less charged, yet exhibit the same, highly similar, or improved protease specificities as found for aprotinin, especially with respect to the potency of plasmin and kallikrein inhibition. Such inhibitors could then be used repeatedly as medicaments in human patients with reduced risk of adverse immune response and reduced nephrotoxicity.
BRIEF SUMMARY OF THE INVENTION
The instant invention provides for a purified human serine protease inhibitor which can specifically inhibit kallikrein, that has been isolated from human placental tissue via affinity chromatography.
The instant invention provides a newly identified human protein herein called human placental bikunin that contains two serine protease inhibitor domains of the Kunitz class. In one particular embodiment, the instant invention embodies a protein having the amino acid sequence:
ADRERSIHDF CLVSKVVGRC RASMPRWWYN VTDGSCQLFV YGGCDGNSNN
50
(SEQ ID NO:1)
YLTKEECLKK CATVTENATG DLATSRNAAD SSVPSAPRRQ DSEDHSSDMF
100
NYEEYCTANA VTGPCRASFP RWYFDVERNS CNNFIYGGCR GNKNSYRSEE
150
ACMLRCFRQQ ENPPLPLGSK VVVLAGAVS
179
In a prefered embodiment the instant invention provides for native human placental bikunin protein having the amino acid sequence:
ADRERSIHDF CLVSKVVGRC RASMPRWWYN VTDGSCQLFV YGGCDGNSNN
50
(SEQ ID NO:52)
YLTKEECLKK CATVTENATG DLATSRNAAD SSVPSAPRRQ DSEDHSSDMF
100
NYEEYCTANA VTGPCRASFP RWYFDVERNS CNNFIYGGCR GNKNSYRSEE
150
ACMLRCFRQQ ENPPLPLGSK
170
In one aspect, the biological activity of the protein of the instant invention is that it can bind to and substantially inhibit the biological activity of trypsin, human plasma and tissue kallikreins, human plasmin and Factor XIIa. In a preferred embodiment, the present invention provides for a native human placental bikunin protein, in glycosylated form. In a further embodiment the instant invention encompasses native human bikunin protein which has been formed such that it contains at least one cysteine-cysteine disulfide bond. In a preferred embodiment, the protein contains at least one intra-chain cysteine-cysteine disulfide bond formed between a pair of cysteines selected from the group consisting of CYS11-CYS61, CYS20-CYS44, CYS36-CYS57, CYS106-CYS156, CYS115-CYS139, and CYS131-CYS152, wherein the cysteines are numbered according to the amino acid sequence of native human placental bikunin. One of ordinary skill will recognize that the protein of the instant invention may fold into the proper three-dimensional conformation, such that the biological activity of native human bikunin is maintained, where none, one or more, or all of the native intra-chain cysteine-cysteine disulfide bonds are present. In a most preferred embodiment, the protein of the instant invention is properly folded and is formed with all of the proper native cysteine-cysteine disulfide bonds.
Active protein of the instant invention can be obtained by purification from human tissue, such as placenta, or via synthetic protein chemistry techniques, as illustrated by the Examples below. It is also understood that the protein of the instant invention may be obtained using molecular biology techniques, where self-replicating vectors are capable of expressing the protein of the instant invention from transformed cells. Such protein can be made as non-secreted, or secreted forms from transformed cells. In order to facilitate secretion from transformed cells, to enhance the functional stability of the translated protein, or to aid folding of the bikunin protein, certain signal peptide sequences may be added to the NH2-terminal portion of the native human bikunin protein.
In one embodiment, the instant invention thus provides for the native human bikunin protein with at least a portion of the native signal peptide sequence intact. Thus one embodiment of the invention provides for native human bikunin with at least part of the signal peptide, having the amino acid sequence:
AGSFLAWLGSLLLSGVLA
−1
(SEQ ID NO:2)
ADRERSIHDFCLVSKVVGRCRASMPRWWYNVTDGSCQLFVYGGCDGNSNN
50
YLTKEECLKKCATVTENATGDLATSRNAADSSVPSAPRRQDSEDHSSDMF
100
NYEEYCTANAVTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNSYRSEE
150
ACMLRCFRQQENPPLPLGSKVVVLAGAVS
179
In a prefered embodiment the instant invention provides for a native human placental bikunin protein with part of the leader sequence intact, having the amino acid sequence of SEQ ID NO: 52 with an intact leader segment having the amino acid sequence:
MAQLCGL RRSRAFLALL GSLLLSGVLA−1 (SEQ ID NO: 53)
In another embodiment, the instant invention provides f
Davis Gary
Delaria Katherine A.
Marlor Christopher W.
Muller Daniel K.
Tamburini Paul P.
Bayer Corporation
Bugaisky Gabriele E
Low Christopher S. F.
McDonnell & Boehnen Hulbert & Berghoff
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