Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2000-05-08
2003-11-11
Low, Christopher S. F. (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S009100, C514S012200, C514S015800, C514S016700, C424S059000, C530S317000, C530S324000, C530S327000, C530S328000, C530S333000, C530S344000, C530S300000, C435S213000, C435S219000
Reexamination Certificate
active
06645934
ABSTRACT:
The present invention relates to a peptide having a radioprotective effect.
A peptide of this kind is known, for example, from the publication of K. Dittmann et al. (1995): “Bowman-Birk Protease Inhibitor (BBI) modulates radiosensitivity and radiation-induced differentiation of human fibroblasts in culture,” Radiotherapy and Oncology 34, pages 137-143.
A “radioprotective effect” of a peptide is understood to mean its protective activity for cells, tissue, or organisms with respect to harmful or injury-causing radiation. For living organisms in particular, harmful radiation is ionizing radiation as well as UV radiation, i.e. energy-rich types of radiation. A peptide exhibits a radioprotective effect if the injury brought about by radiation is reduced by that peptide. The underlying mechanisms of a radioprotective effect are at present still entirely unexplained.
The injury brought about by energy-rich radiation includes, for example, alteration of DNA (i.e. mutagenesis) that can result in tumor formation, but also the degeneration, atrophy, fibrosis, or necrosis of tissues exposed to high levels of radiation.
For example, the occurrence of malignant melanoma is promoted by high levels of sunlight on the skin.
The human body is confronted with particularly high radiation intensities not only when exposed to a great deal of sunlight, but also during X-ray diagnosis or in the context of radiation therapy of tumor diseases.
Protection against UV radiation is offered, for example, by UV-filtering substances such as those contained, for example, in suntan lotion. In order to protect against ionizing radiation, body parts that are not to be irradiated are shielded, and the radiation is applied in as precisely localized a fashion as possible.
It has recently been recognized that there are peptides which can exert a radioprotective effect. One such peptide is the aforementioned Bowman-Birk Protease Inhibitor (BBI), an inhibitor of the serine proteases trypsin and chymotrypsin that has been known for some time and that is present in large quantities in soybeans.
The amino acid sequence of BBI is known, and by 1984 the corresponding soybean gene had already been cloned (R. W. Hammond (1984): “Molecular Cloning and Analysis of a Gene Coding for the Bowman-Birk Protease Inhibitor in Soybean,” J.Biol.Chem. 269, pages 9883-9890).
BBI comprises 71 amino acids and has a molecular weight of approximately 8,000 daltons. One of the characteristics of BBI is the presence of fourteen cysteine residues, which form seven disulfide bonds and thus make a substantial contribution to determining the folding or secondary structure of BBI.
By chemical and enzymatic cleavage with cyanogen bromide (CNBr) and the protease pepsin, BBI is split into two halves, one of which exhibits the trypsin-inhibiting activity, and the other the chymotrypsin-inhibiting activity (S. Odani and T. Ikenaka (1978): “Studies on Soybean Trypsin Inhibitors,” J. Biochem. 83, pages 747-753).
In addition to the protease inhibitor function, two additional physiological activities of BBI have been demonstrated, namely an anticarcinogenic effect and the radioprotective effect already mentioned.
In the publication of B. H. St.Clair, (1990): “Suppression of Dimethylhydrazine-induced Carcinogenesis in Mice by Dietary Addition of the Bowman-Birk Protease Inhibitor,” Cancer Research 50, pages 580-586, it was shown that BBI has an anticarcinogenic effect. In vitro studies with cultivated cells demonstrated that the chymotrypsin-inhibiting domain could prevent malignant transformation of the cells. In vivo studies, on the other hand, in which tumors were induced in mice using carcinogens and in which BBI was administered orally, showed that the trypsin-inhibiting domain of BBI is necessary in order to suppress tumor formation. This anticarcinogenic effect is therefore attributed directly to the protease-inhibiting effect of BBI. It was further demonstrated in the in vivo studies that BBI which had been autoclaved, i.e. thermally denatured, no longer possessed an anticarcinogenic effect.
The aforementioned radioprotective effect of BBI was described in the publication of Dittmann et al. cited above. It was demonstrated in this instance that BBI decreases the radiation-induced mortality of cultivated human fibroblasts (connective tissue cells that occur in large numbers, for example, in the skin).
U.S. Pat. No. 5,376,373 has proposed a method in which the weight loss and hair loss caused by radiation is inhibited by oral administration of a “concentrate,” obtained from soybeans, that contains BBI. Isolation of the BBI concentrate involves repeated fragmentation, precipitation, ultrafiltration, dilution, and reconcentration of a soybean extract in order to obtain the radioprotective product. It is not known which additional constituents, for example additional protease inhibitors or the like, are contained in the concentrate.
One problem involving oral intake of BBI with food is the fact that it has been demonstrated in rats that large quantities of trypsin inhibitors result in hypertrophy and hyperplasia of pancreatic cells, and in loss of body weight. Rats that ingested trypsin inhibitors from soybeans for long periods in fact developed pancreatic tumors.
A further problem with the administration of serine protease inhibitors, especially with intravenous administration, is the fact that blood clotting, in which serine proteases play a substantial role, can be disturbed.
In view of the above, it is an object of the present invention to make available a peptide having a radioprotective effect and which can be produced with little effort and in which the disadvantages of the previously known radioprotective products are avoided.
This object is achieved, according to the present invention, in that a peptide having a radioprotective effect comprises a modified form and/or an optionally modified fragment of the Bowman-Birk Protease Inhibitor.
Specifically, the inventors have found, surprisingly, that structurally modified forms and even fragments of BBI also possess a radioprotective activity. It is now therefore no longer necessary to make BBI available in its native form in order to obtain a peptide having the desired radioprotective effect.
In this context, modified is understood for the purposes of the invention to mean any change in the structure or conformation of BBI and any alteration in its amino acid sequence, whether by chemical or enzymatic insertion or removal of individual groups of amino acids or by the exchange of individual amino acids. A modified form of BBI also encompasses a peptide in which further amino acids are appended onto the N-terminal or C-terminal end of the BBI, for example domains of further proteins or peptides which facilitate purification of the BBI and/or enhance its radioprotective effect even further.
A fragment for the purposes of the invention is understood to mean any portion of the BBI in which either only individual amino acids or larger amino acid segments of the BBI are missing. Such fragments comprise, for example, individual domains of the BBI. According to the present invention, such fragments can also be modified in the sense discussed above.
Modified forms of BBI or BBI fragments can be produced either by treatment of the native (i.e. unaltered) BBI with chemicals or enzymes, or by synthesis with chemical or molecular biological methods.
The fact that modified forms or fragments of BBI can also have a radioprotective effect was not to be expected, since modifications, especially if they involve the conformation of the peptide, usually destroy or at least greatly reduce the physiological activity of a peptide.
In addition, with respect to the anticarcinogenic activity of BBI it had been shown that impairment of the BBI structure by thermal denaturing caused its anticarcinogenic effect to be completely lost.
Modified forms or fragments of BBI can be handled without difficulty, since there is no need to ensure, during either manufacture or storage, that any contact with modifying agents, proteases, or the
Dittmann Klaus
Güven Nûri
Mayer Claus
Rodemann Hans Peter
Eberhard-Karls-Universität Tübingen
Kam Chih-Min
Low Christopher S. F.
LandOfFree
Peptide with radio protective effect does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Peptide with radio protective effect, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Peptide with radio protective effect will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3134585