Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1998-09-24
2001-09-04
Low, Christopher S. F. (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C530S324000, C530S325000, C530S326000, C530S327000, C514S015800
Reexamination Certificate
active
06284734
ABSTRACT:
The present invention relates to inhibitors against prostate-specific antigen.
BACKGROUND OF THE INVENTION
Human prostate-specific antigen essentially has been well known as a diagnostic marker in blood for patients with prostate carcinoma or hyperplasia, but recently, there has been advocated a new theory that human prostate-specific antigen acts to degrade specifically IGF (insulin-like growth factor) binding protein 3 to thereby cause increases in TGF levels in the prostate tissue, resulting in onset or progression of prostate carcinoma or development of prostate hyperplasia.
Accordingly, there exist great expectations that a drug being capable of inhibiting the said antigen would be effective in the treatment of prostate carcinoma and prostate hyperplasia.
Prostate-specific antigen (hereinafter referred to briefly as “PSA”) was isolated in the form of &ggr;-seminoprotein from human seminal plasma by Hara et al. in 1971 (Jp. J. Legal Med., 25: 322, 1971), and was thereafter found to be identical to a substance purified from the prostate tissue by Wang et al., while the said antigen was also observed to be expressed specifically in the prostate (Invest. Urol., 17: 159-163, 1979 and Oncology, 99: 1, 1982), thus leading i-o establishment of the unified appellation “prostate-specific antigen”. And Wang et al. discovered that patients with prostate hyperplasia or carcinoma show a raised blood level of PSA (Prostate, 2: 89-95, 1981), and currently, PSA has been put in wide use as a diagnostic marker in blood for such diseases.
PSA was long left unclassified for most of its actions in the living body, but Watt et al. determined its complete amino acid sequence and demonstrated that PSA is a protease of the kallikrein family which belongs to a kind of serine proteases (Proc. Natl. Acad. Sci. USA, 88: 3166-3170, 1986), They also clarified that PSA exhibits chymotrypsin activity.
Recently, Cohen et al. and Lee et al. found that the substrate in the living body for PSA is IGF binding protein 3 (J. Endocrinol., 142: 407-415, 1994, and J. Clin. Endocrinol, Metab., 7 1367-1372, 1994), and from this finding, it has come to be inferred that when rises in PSA levels in the prostate tissue is brought about by prostate carcinoma, etc., there takes place selective degradation of IGF binding protein 3, with the resultant increases in free IGF levels, which in turn may induce diseases, such as prostate carcinoma and prostate hyperplasia. When it is feasible to inhibit the protease activity of PSA, accordingly, this would offer a possibility for providing a therapeutic drug for prostate carcinoma, prostate hyperplasia, etc., but such attempt has never been made so far in the past.
In recent years, the number of patients with prostate carcinoma or prostate hyperplasia has been on an increasingly upward trend, and in view of the fact that there has been no effective therapeutic drug available, the development of a highly specific drug without adverse effects is strongly demanded. Under these circumstances, the present inventors, taking the view that development of an inhibitor against PSA would be a means for achieving the above-mentioned objective, decided to conduct investigation into the said PSA inhibitor.
As a result, the present. inventors determined the site where PSA cleavages IGF bidning protein 3 and subsequently carried out intensive research on amino acid sequences surrounding such cleavage site, and these led to the finding that a peptide consisting of Gly-Phe-Tyr-Lys-Lys-Lys-Gln-Ser-Arq elicits inhibitory activity against PSA, resulting in establishment of this research work and completion of the present invention.
SUMMARY OF THE INVENTION
Namely, the present invention relates to PSA inhibitors which contain as a minimum unit a peptide consisting of Gly-Phe-Tyr-Lys-Lys-Lys-Gln-Ser-Arg.
DETAILED DESCRIPTION OF THE INVENTION
Using PSA as purified from human urine or seminal plasma, the present inventors identified the cleavage site for recombinant human IGF binding protein 3 (hereinafter referred to briefly as “r-IGFBP3”) utilized as a substrate by the following procedure: in the first place, 50 &mgr;g of PSA and 20 &mgr;g of r-IGFBP3 were dissolved in 0.1M Tris hydrochloride buffer (pH 8.0), followed by reaction at 37° C. for 16 hrs.
1
and the reaction solution was subjected to reverse-phase HPLC to thereby fractionate peptide fragments split by PSA. Then, the fractionated peptide fragments were determined for their amino acid sequences, leading to the finding that cleavage took place at five sites of Arg97, Arg132, Lys198, Lys220 and Arg230. In order to give proved certainty to such consequence, the following peptide having the amino acid sequences vicinal to those of five cleavage sites, followed by investigation into PSA cleavage activities on such peptide fragments, with the result that PSA splits all of these fragments, and this finding made sure that the above five locations serve as the specific recognition and cleavage sites for PSA. In the experiment, the below-described peptides were used, while there was adopted the procedure which comprised dissolving 10 &mgr;g of PSA and 10 nmol each of the peptides in 0.1M Tris; hydrochloride buffer (pH 8.0), and allowing the reaction to proceed at 37° C. for 16 hrs., followed analysis by reverse-phase HPLC
Peptide 1: val(88)-Asn-Ala-Ser-Ala-Val-Ser-Arg-Leu-Arg-Ala-Tyr-Leu-Leu-Pro(102);
Peptide 2: Val(127)-Ser-Ser-Thr-His-Arg-Val-Ser-Asp-Pro-Lys-Phe(138);
Peptide 3: Leu(194)-Asn-His-Leu-Lys-Phe-Leu-Asn-val-Leu(203);
Peptide 4: Gly(217)-Phe-Tyr-Lys-Lys-Lys-Gln-Ser-Arg(225); and
Peptide 5: Pro(226)-Ser-Lys-Gly-Arg-Lys-Arg-Gly-Phe-Met(235).
Then, investigation was conducted into whether or not these peptides act to inhibit the PSA activity. Using as the substrate for assaying the PSA. activity Pro-Phe-Arg-MCA (4-methylcoumarin amide) which is utilized in the assay of kallikrein, as is described in detail below in the Example, determination was made of the residual activities in the coexistence of each of the peptides, with the resultant finding that only Peptide 4 inhibited specifically the PSA activity and therefore would possibly act as a specific inhibitor against PSA.
Among the sites where PSA cleavages r-IGFBP3, the peptide moieties corresponding to Peptides 1 and 2 were previously reported by Fielder et al. (Growth Regulation, 1: 164-172, 1994), whereas the those corresponding to Peptides 3, 4 and 5 were for the first time discovered by the present inventors.
With particular reference to Peptide 4 consisting of 9 amino acid residues, the peptide derivatives generated by having two residues deleted from Peptide 4 orL the side of the N-terminus or the c-terminus showed fairly decreased inhibitory activity, thus suggesting that the said nine amino acid residues are essential for the inhibitory activity.
And PSA degrades r-IGFB3 into fragments, whereby its degradation process can be confirmed by means of SDS-electrophoresis and Western blotting. In the degradation reaction system, addition of Peptide 4 was found to suppress significantly the degradation of r-IGFEIP3, and this finding demonstrated that the said pepetide, by blocking the PSA activity, can actually suppress the degradation of r-IGFBP3.
Generally speaking, processed peptide preparations after administration to the living body often undergo degradation by peptidases, and in administering to patients the peptides according to the present invention, it is preferable to modify through acylation &agr;-amino groups in the N-terminal amino acids, for example, with benzyloxycarbonyl (2), acetyl, t-butoxycarbonyl (Boc) and 9-tluorenylmethoxycarbonyl (Fmoc) groups or to amidate or esterify carboxyl groups in the C-terminal amino acids.
REFERENCES:
patent: 5569742 (1996-10-01), Kirby et al.
patent: 5854206 (1998-12-01), Twardzik et al.
Ngo et al., ‘Computational Complexity, Potein Structure Prediction, and the Levinthal Paradox,’ The Protein Folding Problem and Tertiary Structuer Prediction. Ed. K. Merz and L. Le Grand. BirkHauser, Boston MA. pp. 491-495, 1994.*
Rudinger, J. (1976).
Hirano Kazuyuki
Kajihara Jun-ichi
Shibata Kozue
Gupta Anish
Jacobson & Holman PLLC
JCR Pharmaceuticals Co. Ltd.
Low Christopher S. F.
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