Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing – Hydrolases
Reexamination Certificate
2001-07-25
2004-10-12
Prouty, Rebecca E. (Department: 1652)
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
Hydrolases
Reexamination Certificate
active
06803038
ABSTRACT:
The present invention relates to the use of bromelaine proteases, preferably basic bromelaine proteases, notably for inhibiting the blood coagulation system, especially for stimulating the production of plasmin, for inhibiting the production of fibrin and for inhibiting the adhesion of human thrombocytes to endothelium cells.
Bromelaine is a mixture of quite different proteins that may be isolated from plants of the family Bromeliaceae, the exact composition of which could so far not yet be completely characterized due to the complexity and variety of the components contained therein. It could, however, be shown that bromelaine contains different phosphatases, cellulases, glycosidases, cysteine proteases and the peptide inhibitors thereof, as well as additional not yet more closely identified components. The material and quantitative composition of bromelaine, however, varies in response to the origin and the isolation procedure from the respective source, so that different methods for isolating the raw product, for standardizing the same as well as for purifying specific components contained therein, have been developed. As used herein, the term “bromelaine” or other like terms is meant to be interchangeable with term “bromelain.”
Some of the components in bromelaine have already been identified more closely. Thus, it is reported by Murachi et al. in The Journal of Biological Chemistry 1 (1960), 99-107, that bromelaine contains at least 5 similarly acting proteases with a different substrate specificity and a different pH optimum.
During studies performed with bromelaine it has, moreover, been found that said mixture can also be used as a medicament for treating different states of diseases.
Thus, DE 41 30 221 proposes the use of papain and/or trypsin, specific proteolytic enzymes derived from the bromelaine mixture, for the production of a medicament, which is to be suitable for treating autoimmune diseases. According to said patent, the papain, or the trypsin respectively acts on proteins participating in the development of autoimmune diseases, which comprise a C
H
2-domain.
The use of bromelaine as a mixture for cancer therapy and/or metastasis prophylaxis is moreover disclosed in DE 43 02 060, in which it is assumed that bromelaine acts on CD44, a strongly glycosylized surface protein present on different cells of the organism, which is said to play a role in the development of tumors.
The isolation and characterization of a protease from the bromelaine mixture is explained in WO 95/00169, which acts on the synthetic pathway of cyclic nucleotides. The enzyme designated as “Stem Bromelaine Protease” comprises 213 amino acids and is to obviate diseases, such as the formation of tumors, atherosclerosis or bacterial infections.
Due to the development in the field of purification techniques it has been possible to isolate and partially also characterize additional components from the bromelaine mixture. Thus, it was disclosed by Harrach et al. in The Journal of Protein Chemistry 14 (1995), 41-52, that bromelaine contains at least 8 basic proteases, which could be fractioned by means of FPLC-cation exchange-chromatography. Also, the existence of two forms of acidic proteases could be shown (Maurer et al., Journal of Protein Chemistry 17 (1998), 351-361).
Although different medical fields of application for bromelaine have been found, there is a need to find additional applications for bromelaine. It would thereby be desirable, due to the not yet completely understood interactions of the individual components in the mixture, not to use the mixture itself in the respective field of application, but only the component of the mixture responsible for the respective purpose. A problem arises in this respect, however, as it cannot be predicted whether individual components are effective by themselves in an isolated state without other additional substances present in the bromelaine mixture, or whether they rather require additional components present in the bromelaine mixture as auxiliary substances, which have so far not yet been identified.
It is an object of the invention to provide additional possibilities to use bromelaine, especially the components thereof.
Another object of the invention resides in identifying the component(s) responsible for the respective medical use, and in providing access thereof to a medical use.
The inventors have carried out extensive studies and have surprisingly found, that an inhibition of blood coagulation can be achieved solely with the proteases present in the bromelaine mixture, without the other components present in said mixture.
Consequently, the above-mentioned problem is solved by using the proteases present in the bromelaine mixture for inhibiting blood coagulation.
It has been shown that especially the production of plasmin is stimulated by the bromelaine proteases, while the formation of fibrin and the adhesion of thrombocytes on endothelium cells—all of which are processes playing a significant role in blood coagulation—are inhibited.
In a preferred embodiment of the invention especially basic proteases are applied for the indicated purpose, preferably the bromelaine proteases obtained as fractions F4, F5 or, more preferably, F9 in accordance with the method described by Harrach et al. in the Journal of Protein Chemistry 14 (1995), 41-52.
The protease contained in fraction F4 has a molecular weight of about 24.4 KDa and an optimal activity at a pH in the range of about 4 to 5.5. The protease further comprises the following amino acid sequence:
Val Pro Gln Ser Ile Asp Trp Arg Asp Tyr
Gly Ala Val Thr Ser Val Lys Asn Gln Asn
The protease contained in fraction F5 has a molecular weight of about 24.5 KDa and an optimal activity at a pH in the range of about 3.5 to 5. The protease further comprises the following amino acid sequence:
Val Pro Gln Ser Ile Asp Trp Arg Asp Tyr
Gly Ala Val Thr Ser Val Lys Asn Gln Asn
The protease contained in fraction F9 has a molecular weight of about 23.4 KDa and an optimal activity at a pH in the range of about 6 to 8. The protease further comprises the following amino acid sequence:
Val Pro Gln Ser Ile Asp Trp Arg Asp Ser
Gly Ala Val Thr Ser Val Lys Asn Gln Gly
It has surprisingly shown that an effective inhibition of blood coagulation can be achieved by using bromelaine proteases, and that said inhibition can be obtained merely with the proteases isolated from said bromelaine mixture, without other additional components present in the bromelaine mixture playing a role.
The proteases can be administered to a subject in a manner already known in connection with the bromelaine mixture, i.e. by intravenous or intraperitoneal or preferably by oral administration, wherein the active substances are then formulated with excipients commonly used in the prior art, for passing the proteases through the gastrointestinal tract in an active form so as to guarantee a systemic availability.
The proteases can be isolated in accordance with conventional methods. Especially a purification as indicated by Harrach et al. in the Journal of Protein Chemistry 14 (1995), 41-52 and by Maurer et al. in the Journal of Protein Chemistry 17 (1998), can be applied. Upon purification, said proteases can be initially sequenced, and the corresponding gene can be isolated from the genome of e.g. the pineapple by means of molecular-biological methods. By means of molecular-biological methods a recombinant protein can then be provided in a conventional manner.
The invention will now be explained in more detail by means of the following examples, which merely are explanatory and are not to be construed to limit the present invention.
The proteases used in the present invention, especially the basic proteases, are isolated according to Harrach et al., The Journal of Protein Chemistry 14 (1995), 41-52 and according to Maurer et al., The Journal of Protein Chemistry 17 (1998). The contents of said publications are herewith entirely included in the contents of disclosure of the present application.
As disclosed in Harrach (1995), crude bromelain extra
Eckert Klaus
Eschmann Klaus
Grabowska Edyta
Maurer Rainer
Bell Boyd & Lloyd LLC
Prouty Rebecca E.
Ursapharm Arzneimittel GmbH
Walicka Malgorzata A.
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