Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2001-04-11
2004-06-22
Nickol, Gary B. (Department: 1642)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C530S300000, C424S184100, C424S185100, C528S332000
Reexamination Certificate
active
06753310
ABSTRACT:
TECHNICAL FIELD
The present invention relates to peptide molecules homing specifically to neovascular tissues and, more particularly, to angiogenesis-specific (neovascular-specific) peptides which function as ligands to neovascular endothelial cells of cancer tissues, for instance, and are useful as molecular drugs and applicable to drug delivery system (DDS) preparations enabling selective drug delivery to target tissues and can contribute to improvements in the therapeutic effects on cancer.
BACKGROUND ART
One of the factors which make cancer chemotherapy difficult to do successfully are the fact that the drug administered kills or damages not only the target cancer tissues but also normal tissues, causing adverse effects. As means of minimizing such side effects and achieving improvements in the efficacy of anticancer agents, drug delivery systems (DDSs) have attracted attention in the field of cancer therapy.
The DDSs mentioned above which are targeted at cancer may be classified into two types, namely passive targeting type and active targeting type. Since neovascular tissues show increased vascular permeability as compared with pre-existing vessels, a preparation of the long retention-in-blood type is gradually accumulated in cancer tissues. Passive targeting is the targeting utilizing that property. A passive targeting preparation in which liposomes are used has already been used in Europe and America in the treatment of Kaposi's sarcoma. On the other hand, an active targeting preparation is designed, by modifying the drug with an antibody or some other ligand capable of binding to a cell surface marker, such as a protein, highly expressed in cancer cells or tissues surrounding the same, so that the drug can be delivered actively and selectively to cancer cells, without causing harmful effects on normal tissues.
In the field of current cancer therapy, angiogenesis has become a focus of attention. The term “angiogenesis” refers to the development of blood vessels within cancer tissues which parallel the growth of tumors in the procession of cancer. Thus, for active proliferation of cancer cells and growth and metastasis of cancer tissues, it is important that blood vessels, which are organs serving to feed nutrients and oxygen and eliminate metabolites and waste materials, be newly constructed. In this respect, the growth of cancer tissues can be highly dependent on angiogenesis.
It is considered that when this angiogenesis is inhibited, the growth and metastasis of cancer tissues, might be prevented. From this point of view, it is desired in the art that a cancer therapy targeted on neovascular tissues, in particular an active targeting preparation (DDS preparation), be developed.
DISCLOSURE OF INVENTION
A substance capable of serving as a ligand for neovascular endothelial cells in a cancer tissue, if identified, isolated and made available, will lead to its application in DDS preparations and to further improvements in the efficiency of cancer therapy.
It is an object of the present invention to provide such a novel ligand.
Another object of the invention is to provide a substance capable of inhibiting angiogenesis.
In the course of intensive investigations made for the above purposes, the inventors obtained the findings mentioned below. Thus, the inventors first induced the formation of tumor neovascular tissues in the mouse dorsum by the chamber ring method (Folkman, J., et al., J. Exp. Med., 133, 275-288 (1971)). Then, a random peptide-displaying phage constructed by inserting random DNAs into the phage+ coat protein pIII gene to thereby enable the expression of random peptides having a 15-amino-acid sequence on the phage shell was administered to the mice. Thereafter, the mice were frozen with liquid nitrogen, skin portions bearing neovascular tissues were dissected, homogenized in a culture medium containing a protease inhibitor, washed and centrifuged, and the phage was thus recovered from neovascular tissues. The phage was infected into
Escherichia coli
, which was mass-cultured. After isolation and purification, there was obtained a phage capable of expressing a peptide to be accumulated in the neovascular tissue endothelium and serve as a ligand. For a plurality of phages obtained in that manner, the peptides expressed by them were sequenced.
Then, for selecting a phage expressing a peptide having high affinity for neovascular tissues, each phage obtained in the above manner was administered into the tail vein of tumor-bearing mice prepared by tumor cell implantation. The mice were frozen in the same manner as above, tumor tissues were dissected, and the phages were isolated and purified from the materials obtained and used to infect
Escherichia coli
, followed by cultivation. And, for each phage, colony-forming units were counted, with the phage before selection being used as a control. The affinity for neovascular tissues was calculated in terms of the ratio of number of phages administered to the tail vein to number of accumulated phages per 100 mg of tumor tissue. In this way, candidate peptides for ligands having high affinity for neovascular tissues were obtained.
Further, the inventors synthesized the above peptides, dendrimers thereof, partial peptides thereof and the like, and confirmed that these peptides actually show antitumor effects and, at the same time, confirmed that liposomes modified with the peptides, in particular these peptides which contains the sequence Trp-Arg-Pro and the sequence Pro-Arg-Pro, show significantly higher levels of distribution in the tumor as compared with the control.
The present invention was accomplished on the basis of these findings.
The invention provides an angiogenesis-specific peptide selectively homing to neovascular tissues, which comprises one of the members listed below under (a) and (b):
(a) a peptide having one of the amino acid sequences shown in SEQ ID NO: 1 to 11, or a dendrimer thereof,
(b) a peptide having an amino acid sequence derived from any of the amino acid sequences of the peptide defined above under (a) by substitution, deletion or addition of one or a plurality of amino acid residues and having affinity for neovascular tissues, or a dendrimer thereof.
In particular, the invention provides an angiogenesis-specific peptide as mentioned above which is a peptide having one of the amino acid sequences shown in SEQ ID NO: 1 to 11, or a dendrimer thereof; more preferably, an angiogenesis-specific peptide as mentioned above which is a peptide having one of the amino acid sequences shown in SEQ ID NO: 1, 5 and 6, or a dendrimer thereof; an angiogenesis-specific peptide as mentioned above which is a dendrimer comprising a plurality of peptides which are the same or different and have one of the amino acid sequences shown in SEQ ID NO: 1 to 11; an angiogenesis-specific peptide as mentioned above which is a peptide having one of the amino acid sequences shown in SEQ ID NO: 12 to 17, or a dendrimer thereof; and an angiogenesis-specific peptide as mentioned above which is a peptide having one of the amino acid sequences shown in SEQ ID NO:19, 21, 23-25 and 28-32, or a dendrimer thereof.
The invention further provides an angiogenesis-specific peptide as mentioned above which homes selectively to neovascular tissues developed in cancer/tumor tissues, for example sarcoma or melanoma.
The invention still further provides an anticancer composition and a cancer metastasis inhibitor composition, each of which comprises, as an active ingredient, at least one of the above angiogenesis-specific peptides, preferably at least one peptide having one of the amino acid sequences shown in SEQ ID NO:1, 5, 6, 13-17, 19, 21, 23-25 and 28-32 or dendrimer thereof, together with a pharmaceutical carrier therefor.
The invention further provides a liposome preparation which comprises, as active ingredients, at least one of the above angiogenesis-specific peptides, preferably at least one peptide having one of the amino acid sequences shown in SEQ ID NO:15-17, or dendrimer thereof, and an anticancer agent or cancer m
Ishikawa Dai
Ogino Koichi
Oku Naoto
Taki Takao
Tanaka Michinori
Nickol Gary B.
Otsuka Pharmaceutical Co. Ltd.
Sughrue & Mion, PLLC
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