Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues
Reexamination Certificate
2000-06-20
2002-08-27
Riley, Jezia (Department: 1656)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
C530S305000, C530S332000, C530S333000, C435S004000, C514S001000
Reexamination Certificate
active
06441131
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method for assaying human pepsin II or human pepsinogen II in the human body fluid (such as gastric juice, blood, urine etc.) as diagnostic marker of gastric diseases such as gastric cancer, gastric ulcer etc. and a peptide used as a substrate in such a method.
More detailed, the present invention relates to
1) a peptide of the formula (I)
(wherein, Q is Q
a
—(AA)n— (in which AA is L-amino acid, n is 0 or an integer of 1~15, Q
a
is hydrogen, C1~4 alkyl, an amino-protective group, D- or L-amino acid residue or NH
2
—(CH
2
)r—CO— (in which r is an integer of 2~7.).),
R
1
and R
2
are (i) hydrogen or halogen or, (ii) R
1
, R
2
and unsaturated bond together form an aromatic carbon ring may be substituted by halogen,
R
3
is hydrogen or halogen,
R
4
is hydrogen, C1~3 alkyl or hydroxymethyl,
EE is D- or L-amino acid residue,
m is 0 or 1,
Z is an aniline derivative residue, an aminocoumarine derivative residue or an aminonaphthalene derivative residue,
with the proviso that (1) when n is 2 or more, each AA is same or different, and that (2) the compounds wherein all of R
1
, R
2
and R
3
is hydrogen are excluded.)
or an acid addition salt thereof and,
2) a method for assaying human pepsinogen II or human pepsin II which is characterized by digesting a peptide of the formula (I) (wherein all the symbols are as defined hereinbefore.) described in the said 1) or an acid addition salt thereof by human pepsin II which is obtained by activation of human pepsinogen II in a sample or human pepsin II in a sample to obtain an amino acid derivative of the formula (II)
(wherein all the symbols are as defined hereinbefore.),
digesting the obtained amino acid derivative by aminopeptidase to obtain an aniline, aminocoumarine or aminonaphthalene derivative of the formula Z—H and then
detecting the obtained aniline, aminocoumarine or aminonaphthalene derivative and
3) a kit for assaying human pepsinogen II or human pepsin II which is characterized by comprising a peptide of the formula (I) (wherein all the symbols are as defined hereinbefore.) described in the said 1) or an acid addition salt thereof as a substrate and an aminopeptidase.
BACKGROUND
It is known that the pepsinogen secretion is parallel to gastric acid secretion and that human serum or urine pepsinogen levels are also parallel to gastric pepsinogen secretion. The above pepsinogen exists as pepsinogen in the body fluid such as blood or urine except for gastric juice, on the other hand, it exists as pepsin in gastric juice.
It is said that human blood or urine pepsinogen I level of the patient with atrophic gastritis decreases and that human blood or urine pepsinogen I and pepsinogen II levels increase in case of gastric ulcer (Japanese Patent Application Kokai Hei 7-304800). In addition, it is said that both pepsinogen I level and pepsinogen II/I ratio decrease in the patient with gastric cancer (Jpn. J. Cancer Res., 80, 111-114 (1989)).
Further, an attention is paid to serum pepsinogen II level and pepsinogen I/II ratio as markers of therapy for helicobacter pylori gastritis. That is to say, it is said that serum pepsinogen II level decreases significantly and pepsinogen I/II ratio increases significantly in a successful group consisting of patients in whom therapy resulted in eradication of the bacteria to compare with an unsuccessful group consisting of patients who remained infected (Prog. Med., 15, 1862-1868 (1995)).
Therefor, assaying the level of human pepsinogen II in human blood or urine may be useful for diagnosis at early stage of diseases such as gastric cancer, gastric ulcer and duodenal ulcer etc.
As for a method for assaying human pepsin which was obtained by activation of human pepsinogen, a method using human serum protein etc. in urine and serum based on its digesting activity has been known (Clin. Chem., 15, 1, 42-55 (1969)). The significance of clinical trial using such a method has been discussed, but it requires a long time. In addition, its accuracy was not good, so such a method has been of no practical use. Further, the results means the activity to digest protein, so it was reflected on the total activities of both pepsin I and pepsin II. Therefore it is impossible to determine the human serum pepsinogen II specifically.
Recently, a method for assaying human pepsinogen in urine (uropepsin) indirectly, based on inactivation of an acidic enzyme by activated pepsin was proposed (Japanese Patent Application Kokai Hei 7-155198). But the substrate used in this method did not show the specificity for pepsin II. It is said that the pepsinogen in urine is pepsinogen I. But, pepsin II may be also secreted in urine in some body condition, so it is difficult to determine the accurate level of pepsin II. It is impossible to assay the level of pepsinogen II in human serum specifically.
As for a method for assaying pepsinogen II, radio immunoassay (Kaku-igaku (in Japanese), Vol. 26, No. 9, 1217-1221 (1989)) and enzyme immunoassay (Japanese Patent Application Kokai Hei 7-304800) using a specific anti-body have been practical use, but these methods cause a radioactive pollution and require a long time and complicated procedure for assaying.
Some methods for assaying pepsin II using synthesized substrate have been proposed. For example, (A) in the paper of J. Med., Chem., 36, 2614 (1993), it was described that a peptide of the formula (A-1)
LysProAlaAlaPhePhe(NO
2
)ArgLeu (A-1) (SEQ ID NO:86)
(wherein Phe(NO
2
) is p-nitrophenylalanine.)
was used as a substrate in assaying inhibitory effect of some compounds on human pepsin II. That is to say, a peptide of the formula (A-1) was digested by human pepsin II to obtain a peptide of the formula (A-2)
Phe(NO2)ArgLeu (A-2)
(wherein Phe(NO2) is as defined hereinbefore.),
and the obtained peptide of the formula (A-2) was used in the assaying inhibitory activity on enzyme based on decrease of absorbance at 234~324 nm as an index.
But this paper did not disclose that such a peptide of the formula (A-1) may be as a substrate for human pepsin II. Therefore, it is uncertain whether this peptide has specificity for human pepsin II, or not. In addition, the chromophore of this peptide is Phe(NO
2
), so it is expected that the accuracy of the method using this peptide is one tenth or less to compare with p-nitroaniline (abbreviated as pNA). Further, it is impossible to be used in automated clinical analyzer due to detection at 234~324 nm.
(B) in the paper of J. Biochem., 265, 871-878 (1990), it was described that a peptide of the formula (B-1)
LysProValValPhePhe(NO
2
)ArgLeu (B-1) (SEQ ID NO:87)
(wherein Phe(NO
2
) is as defined hereinbefore.),
was used as a substrate in assaying inhibitory effect of some compounds on human pepsin II. That is to say, a peptide of the formula (B-1) was digested by human pepsin II to obtain a peptide of the formula (B-2)
Phe(NO2)ArgLeu (B-2)
(wherein Phe(NO
2
) is as defined hereinbefore.),
and the obtained peptide of the formula (B-2) was used in the assaying inhibitory activity on enzyme based on decrease of absorbance at 234~324 nm as an index.
But this paper did not disclose that such a peptide of the formula (B-1) may be as a substrate for human pepsin II. Therefore, it is uncertain whether this peptide has a specificity for human pepsin II, or not. In addition, the chromophore of this peptide is Phe(NO
2
), so it is expected that the accuracy of the method using this peptide is one tenth or less to compare with p-nitroaniline (abbreviated as pNA). Further, it is impossible to be used in automated clinical analyzer due to detection at 234~324 nm (There are the same problems as in paper (A).).
(C) In CS-261172, it was described that a peptide of the formula (C-1)
X—A—B-Phe-D-pNA (C-1)
(wherein X is hydrogen, C3~5 carboxylalkylcarbonyl or C1~5 alkylcarbonyl,
A is pyroglutamic acid (abbreviated as pGlu), Asp, Glu or Gly residue or 2-oxoimidazoline-1-yl-carbonyl,
B is His, Gly or Pro residue,
D is Phe, Leu, Nle, Met or S—C1~3 alkyl-Cys residue
Hayashi Akio
Matsuo Masayoshi
Ono Pharmaceutical Co. Ltd.
Riley Jezia
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