U.S. patent application number 14/109769 was filed with the patent office on 2014-05-08 for molecular probe for imaging of pancreatic islets and use of the same.
This patent application is currently assigned to ARKRAY, INC.. The applicant listed for this patent is ARKRAY, INC., Kyoto University. Invention is credited to Nobuya Inagaki, Hiroyuki Kimura, Yu Ogawa, Hideo Saji, Kentaro Toyoda.
Application Number | 20140127128 14/109769 |
Document ID | / |
Family ID | 43649136 |
Filed Date | 2014-05-08 |
United States Patent
Application |
20140127128 |
Kind Code |
A1 |
Inagaki; Nobuya ; et
al. |
May 8, 2014 |
Molecular Probe for Imaging of Pancreatic Islets and Use of the
Same
Abstract
To provide a molecular probe for imaging of pancreatic islets. A
molecular probe for use in imaging of pancreatic islets is
provided. The molecular probe includes any one of the following
polypeptides: polypeptides represented by the following formulae
(1), (5), and (9); and polypeptides having homology with the
foregoing polypeptides: TABLE-US-00001
Z-DLSXQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH.sub.2 (1)
Z-DLSKQMEEEAVRLFIEWLXNGGPSSGAPPPS-NH.sub.2 (5)
B-DLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH.sub.2 (9) where X in the
formulae (1) and (5) and B- in the formula (9) indicate that an
amino group is labeled with a group represented by the formula (I)
below having an aromatic ring, ##STR00001## wherein A represents
either an aromatic hydrocarbon group or an aromatic heterocyclic
group, R.sup.1 represents a substituent that contains radioactive
iodine, R.sup.2 represents either a hydrogen atom or a substituent
different from that represented by R.sup.1, and R.sup.3 represents
any one of a bond, a methylene group, and an oxymethylene
group.
Inventors: |
Inagaki; Nobuya; (Kyoto,
JP) ; Saji; Hideo; (Kyoto, JP) ; Toyoda;
Kentaro; (Kyoto, JP) ; Kimura; Hiroyuki;
(Kyoto, JP) ; Ogawa; Yu; (Kyoto, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ARKRAY, INC.
Kyoto University |
Kyoto
Kyoto |
|
JP
JP |
|
|
Assignee: |
ARKRAY, INC.
Kyoto
JP
KYOTO UNIVERSITY
Kyoto
JP
|
Family ID: |
43649136 |
Appl. No.: |
14/109769 |
Filed: |
December 17, 2013 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12872837 |
Aug 31, 2010 |
8642008 |
|
|
14109769 |
|
|
|
|
61272282 |
Sep 8, 2009 |
|
|
|
Current U.S.
Class: |
424/1.69 ;
530/324 |
Current CPC
Class: |
A61K 51/088 20130101;
A61P 3/10 20180101 |
Class at
Publication: |
424/1.69 ;
530/324 |
International
Class: |
A61K 51/08 20060101
A61K051/08 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 4, 2009 |
JP |
2009-204769 |
Mar 16, 2010 |
JP |
PCT/JP10/54450 |
Claims
1. A molecular probe for use in imaging of pancreatic islets, the
molecular probe comprising any one of the following polypeptides: a
polypeptide represented by any one of the following formulae (1) to
(12); a polypeptide obtained by deletion, insertion, or
substitution of one to several amino acids with respect to a
polypeptide represented by any one of the following formulae (1) to
(12), the polypeptide being capable of binding to pancreatic
islets; and a polypeptide having a homology of 80% or higher with
any one of the amino acid sequences of polypeptides represented by
the following formulae (1) to (12), the polypeptide being capable
of binding to pancreatic islets, TABLE-US-00015 (SEQ ID NO. 1)
Z-DLSXQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH.sub.2 (1) (SEQ ID NO. 2)
Z-LSXQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH.sub.2 (2) (SEQ ID NO. 3)
Z-SXQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH.sub.2 (3) (SEQ ID NO. 4)
Z-XQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH.sub.2 (4) (SEQ ID NO. 5)
Z-DLSKQMEEEAVRLFIEWLXNGGPSSGAPPPS-NH.sub.2 (5) (SEQ ID NO. 6)
Z-LSKQMEEEAVRLFIEWLXNGGPSSGAPPPS-NH.sub.2 (6) (SEQ ID NO. 7)
Z-SKQMEEEAVRLFIEWLXNGGPSSGAPPPS-NH.sub.2 (7) (SEQ ID NO. 8)
Z-KQMEEEAVRLFIEWLXNGGPSSGAPPPS-NH.sub.2 (8) (SEQ ID NO. 9)
B-DLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH.sub.2 (9) (SEQ ID NO. 10)
B-LSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH.sub.2 (10) (SEQ ID NO. 11)
B-SKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH.sub.2 (11) (SEQ ID NO. 12)
B-KQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH.sub.2 (12)
where in the foregoing formulae (1) to (8), Z- indicates that an
.alpha.-amino group at an N-terminus is either not modified, or is
modified with a modifying group having no electric charge, and X
represents a lysine residue, an amino group of a side chain of the
lysine residue being labeled with a group represented by the
formula (I) below having an aromatic ring, in the formulae (9) to
(12), B- indicates that an .alpha.-amino group at an N-terminus is
labeled with a group represented by the formula (I) below having an
aromatic ring, and in the foregoing formulae (1) to (12),
--NH.sub.2 indicates that a carboxyl group at a C-terminus is
amidated, ##STR00015## wherein A represents either an aromatic
hydrocarbon group or an aromatic heterocyclic group, R.sup.1
represents a substituent that contains any one of .sup.123I,
.sup.124I, .sup.125I, and .sup.131I, R.sup.2 represents either a
hydrogen atom, or one or more substituents different from that
represented by R.sup.1, and R.sup.3 represents any one of a bond, a
methylene group, and an oxymethylene group.
2. The molecular probe for imaging of pancreatic islets according
to claim 1, wherein the group having an aromatic ring is a group
represented by the following formula (II): ##STR00016## wherein
R.sup.1 represents a substituent that contains any one of
.sup.123I, .sup.124I, .sup.125I, and .sup.131I.
3. A kit for performing imaging of pancreatic islets, comprising
the molecular probe for imaging of pancreatic islets according to
claim 1.
4. The kit according to claim 3, wherein the molecular probe for
imaging of pancreatic islets included in the kit is in a form of a
parenteral solution.
5. A reagent for performing imaging of pancreatic islets,
comprising the molecular probe for imaging of pancreatic islets
according to claim 1.
6. A method for imaging of pancreatic islets comprising detecting a
signal of the molecular probe for imaging of pancreatic islets
according to claim 1 bound to pancreatic islets preliminarily.
7. The method for imaging of pancreatic islets according to claim
6, further comprising determining a state of pancreatic islets from
results of the imaging of pancreatic islets using the molecular
probe for imaging of pancreatic islets.
8. A method for determining an amount of pancreatic islets,
comprising: detecting a signal of the molecular probe for imaging
of pancreatic islets according to claim 1, the molecular probe
being bound to pancreatic islets preliminarily; and calculating an
amount of pancreatic islets from the detected signal of the
molecular probe for imaging of pancreatic islets.
9. The method for determining an amount of pancreatic islets
according to claim 8, further comprising presenting the calculated
amount of pancreatic islets.
10. A method for producing the molecular probe for imaging of
pancreatic islets according to claim 1, comprising labeling and
deprotecting a precursor of the molecular probe for imaging of
pancreatic islets, wherein the precursor of the molecular probe for
imaging of pancreatic islets includes any one of the following
polypeptides: a polypeptide represented by any one of the following
formulae (13) to (24); a polypeptide obtained by deletion,
insertion, or substitution of one to several amino acids with
respect to a polypeptide represented by any one of the following
formulae (13) to (24), the polypeptide being capable of binding to
pancreatic islets after being labeled and deprotected; and a
polypeptide having a homology of 80% or higher with any one of the
amino acid sequences of polypeptides represented by the following
formulae (13) to (24), the polypeptide being capable of binding to
pancreatic islets after being labeled and deprotected,
TABLE-US-00016 (SEQ ID NO. 13) *-DLSKQMEEEAVRLFIEWLK*
NGGPSSGAPPPS-NH.sub.2 (13) (SEQ ID NO. 14) *-LSKQMEEEAVRLFIEWLK*
NGGPSSGAPPPS-NH.sub.2 (14) (SEQ ID NO. 15) *-SKQMEEEAVRLFIEWLK*
NGGPSSGAPPPS-NH.sub.2 (15) (SEQ ID NO. 16) *-KQMEEEAVRLFIEWLK*
NGGPSSGAPPPS-NH.sub.2 (16) (SEQ ID NO. 17) *-DLSK*
QMEEEAVRLFIEWLKNGGPSSGAPPPS-NH.sub.2 (17) (SEQ ID NO. 18)
*-LSK*QMEEEAVRLFIEWLKNGGPSSGAPPPS-NH.sub.2 (18) (SEQ ID NO. 19)
*-SK*QMEEEAVRLFIEWLKNGGPSSGAPPPS-NH.sub.2 (19) (SEQ ID NO. 20) *-K*
QMEEEAVRLFIEWLKNGGPSSGAPPPS-NH.sub.2 (20) (SEQ ID NO. 21) DLSK*
QMEEEAVRLFIEWLK* NGGPSSGAPPPS-NH.sub.2 (21) (SEQ ID NO. 22) LSK*
QMEEEAVRLFIEWLK* NGGPSSGAPPPS-NH.sub.2 (22) (SEQ ID NO. 23) SK*
QMEEEAVRLFIEWLK* NGGPSSGAPPPS-NH.sub.2 (23) (SEQ ID NO. 24) K*
QMEEEAVRLFIEWLK* NGGPSSGAPPPS-NH.sub.2 (24)
wherein in the foregoing formulae (13) to (20), *- indicates that
an .alpha.-amino group at an N terminus is either protected by a
protecting group or modified by a modifying group having no
electric charge, and in the foregoing formulae (13) to (24), K*
indicates that an amino group of a side chain of a lysine is
protected by a protecting group, and --NH.sub.2 indicates that a
carboxyl group at a C-terminus is amidated.
11. The method for producing the molecular probe for imaging of
pancreatic islets according to claim 10, wherein the labeling of
the precursor of the molecular probe for imaging of pancreatic
islets includes labeling of the precursor with a labeling compound
having a group represented by the following formula (I) having an
aromatic ring: ##STR00017## wherein A represents either an aromatic
hydrocarbon group or an aromatic heterocyclic group, R.sup.1
represents a substituent that contains any one of .sup.123I,
.sup.124I, .sup.125I, and .sup.131I, R.sup.2 represents either a
hydrogen atom, or one or more substituents different from that
represented by R.sup.1, and R.sup.3 represents any one of a bond, a
methylene group, and an oxymethylene group.
12. A method for radioactively labeling a peptide having a
plurality of amino acids having radioactively-labelable functional
groups on side chains, the method comprising: synthesizing a
peptide using a protected amino acid in which an .alpha.-amino
group at an N-terminus and a functional group of a side chain are
protected by protecting groups; deprotecting a functional group by
removing a protecting group therefrom, wherein the functional group
to be deprotected is a functional group that is not to be
radioactively labeled, among the radioactively labelable functional
groups of the side chains of the amino acids of the synthesized
peptide; protecting, again, the deprotected functional group of the
side chain of the amino acid by a protecting group different from
that removed upon the deprotecting; deprotecting, by removing
protecting groups, the other functional groups than the functional
group of the side chain of the amino acid that is again protected,
so as to obtain a peptide to be radioactively labeled;
radioactively labeling the obtained peptide with a labeling
compound; and deprotecting the radioactively-labeled peptide by
removing protecting groups.
13. A method for producing a radioactively-labeled peptide, wherein
the peptide has a plurality of amino acids having radioactively
labelable functional groups on side chains, the method comprising:
synthesizing a peptide to be radioactively labeled, using protected
amino acids in each of which an .alpha.-amino group at an
N-terminus and a functional group of a side chain are protected by
protecting groups; deprotecting a functional group by removing a
protecting group therefrom, wherein the functional group to be
deprotected is a functional group that is not to be radioactively
labeled, among the radioactively labelable functional groups of the
side chains of the amino acids of the synthesized peptide;
protecting, again, the deprotected functional group of the side
chain of the amino acid by a protecting group different from that
removed upon the deprotecting; deprotecting, by removing protecting
groups, the other functional groups than the functional group of
the side chain of the amino acid that is again protected, so as to
obtain a peptide to be radioactively labeled; radioactively
labeling the obtained peptide with a labeling compound; and
deprotecting the radioactively-labeled peptide by removing
protecting groups.
Description
TECHNICAL FIELD
[0001] The present invention relates to a molecular probe for
imaging of pancreatic islets, and relates to use of the same.
BACKGROUND ART
[0002] Today, type-II diabetics are continuously increasing in
Japan, and the estimated number of the same exceeds 8,200,000. As a
measure against this increase, interventions for preventing
diabetes from developing have been made based on the glucose
tolerance test, resulting, however, in unsatisfactory effects. The
cause is as follows: at such a borderline stage that functional
abnormalities are found by the glucose tolerance test, disorders of
pancreatic islets have already advanced to a high degree, and this
stage possibly is too late as a time for starting
interventions.
[0003] More specifically, in the diabetes developing process, the
amount of pancreatic islets (particularly, the amount of pancreatic
.beta.-cells) decreases prior to the occurrence of glucose
tolerance abnormalities. Therefore, when functional abnormalities
are detected or there are subjective symptoms, diabetes has already
reached the stage where it is too difficult to be treated. On the
other hand, if a decrease in the amount of pancreatic islets and/or
the amount of pancreatic .beta.-cells can be detected at an early
stage, there is a possibility for the prevention and treatment of
diabetes. Therefore, a noninvasive technique of imaging of
pancreatic islets, particularly a noninvasive technique for imaging
of pancreatic islets for determining the amount of the pancreatic
islets and/or the amount of pancreatic .beta.-cells, has been
desired for the prevention and diagnosis of diabetes. Among these,
a molecular probe that enables imaging of pancreatic islets,
preferably pancreatic .beta.-cell imaging, and noninvasive
determination of an amount of pancreatic .beta.-cells has been
desired in particular.
[0004] In designing a molecular probe for imaging of pancreatic
islets, various target molecules in pancreatic islet cells,
particularly functional proteins specific in the .beta.-cells, are
being researched. Among these, GLP-1R (glucagon-like peptide-1
receptor) is being researched as a target molecule; GLP-1R is
distributed in pancreatic .beta.-cells, and is a
seven-transmembrane G protein coupled receptor.
[0005] As a molecular probe for pancreatic .beta.-cells imaging
that uses GLP-1R as a target molecule, a molecular probe obtained
by labeling a derivative of exendin-4(9-39) as a GLP-1R antagonist
with [.sup.18F] fluorine has been researched (e.g., H. Kimura et
al. Development of in vivo imaging agents targeting glucagons-like
peptide-1 receptor (GLP-1R) in pancreatic islets. 2009 SNM Annual
Meeting, abstract, Oral Presentations No. 326 (Document 1)).
[0006] Further, a molecular probe for imaging a GLP-1R-positive
tumor has been researched as an imaging probe targeting GLP-1R as a
target molecule. Examples of the molecular probe for imaging a
GLP-1R-positive tumor include a molecular probe obtained by
labeling a derivative of exendin-4 as an GLP-1R agonist with
[.sup.111In] indium via diethylenetriaminepentaacetic acid (DTPA);
and a molecular probe obtained by labeling a derivative of
exendin-4(9-39) as a GLP-1R antagonist with [.sup.111In] indium via
DTPA (e.g., M. Beche et al. Are radiolabeled GLP-1 receptor
antagonists useful for scintigraphy? 2009 SNM Annual Meeting,
abstract, Oral Presentations No. 327 (Document 2)).
[0007] However, another molecular probe for imaging of pancreatic
islets is ultimately preferred that enables noninvasive
three-dimensional imaging of pancreatic islets.
DISCLOSURE OF INVENTION
[0008] The present invention provides a molecular probe for imaging
of pancreatic islets that enables noninvasive three-dimensional
imaging of the pancreatic islets.
[0009] The present invention relates to a molecular probe for use
in imaging of pancreatic islets, the molecular probe comprising any
one of the following polypeptides:
[0010] a polypeptide represented by any one of the following
formulae (1) to (12);
[0011] a polypeptide obtained by deletion, insertion, or
substitution of one to several amino acids with respect to a
polypeptide represented by any one of the following formulae (1) to
(12), the polypeptide being capable of binding to pancreatic
islets; and
[0012] a polypeptide having a homology of 80% or higher with any
one of the amino acid sequences of polypeptides represented by the
following formulae (1) to (12), the polypeptide being capable of
binding to pancreatic islets,
TABLE-US-00002 (SEQ ID NO. 1)
Z-DLSXQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH.sub.2 (1) (SEQ ID NO. 2)
Z-LSXQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH.sub.2 (2) (SEQ ID NO. 3)
Z-SXQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH.sub.2 (3) (SEQ ID NO. 4)
Z-XQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH.sub.2 (4) (SEQ ID NO. 5)
Z-DLSKQMEEEAVRLFIEWLXNGGPSSGAPPPS-NH.sub.2 (5) (SEQ ID NO. 6)
Z-LSKQMEEEAVRLFIEWLXNGGPSSGAPPPS-NH.sub.2 (6) (SEQ ID NO. 7)
Z-SKQMEEEAVRLFIEWLXNGGPSSGAPPPS-NH.sub.2 (7) (SEQ ID NO. 8)
Z-KQMEEEAVRLFIEWLXNGGPSSGAPPPS-NH.sub.2 (8) (SEQ ID NO. 9)
B-DLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH.sub.2 (9) (SEQ ID NO. 10)
B-LSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH.sub.2 (10) (SEQ ID NO. 11)
B-SKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH.sub.2 (11) (SEQ ID NO. 12)
B-KQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH.sub.2 (12)
where,
[0013] in the foregoing formulae (1) to (8),
[0014] Z- indicates that an .alpha.-amino group at an N-terminus is
either not modified, or is modified with a modifying group having
no electric charge, and
[0015] X represents a lysine residue, an amino group of a side
chain of the lysine residue being labeled with a group represented
by the following formula (I) having an aromatic ring, and
[0016] in the formulae (9) to (12),
[0017] B- indicates that an .alpha.-amino group at an N-terminus is
labeled with a group represented by the following chemical formula
(I) having an aromatic ring, and
[0018] in the foregoing formulae (1) to (12), --NH.sub.2 indicates
that a carboxyl group at a C-terminus is amidated,
##STR00002##
wherein
[0019] A represents either an aromatic hydrocarbon group or an
aromatic heterocyclic group,
[0020] R.sup.1 represents a substituent that contains any one of
.sup.123I, .sup.124I, .sup.125I, and .sup.131I,
[0021] R.sup.2 represents either a hydrogen atom, or one or more
substituents different from that represented by R.sup.1, and
[0022] R.sup.3 represents any one of a bond, a methylene group, and
an oxymethylene group.
[0023] The present invention enables imaging of pancreatic islets,
preferably three-dimensional imaging of pancreatic islets, and more
preferably noninvasive imaging of pancreatic islets by, for
example, positron emission tomography (PET) or single photon
emission computed tomography (SPECT).
BRIEF DESCRIPTION OF DRAWINGS
[0024] FIGS. 1A and 1B show exemplary graphs showing variations
with time of biodistribution of a molecular probe for imaging of
pancreatic islets according to Example 1.
[0025] FIGS. 2A and 2B show exemplary graphs showing variations
with time of biodistribution of a molecular probe according to
Comparative Example.
[0026] FIG. 3 shows a graph showing exemplary results of a blocking
experiment using a molecular probe according to Example 1.
[0027] FIG. 4 shows exemplary results of image analysis of a
pancreatic islet section using a molecular probe for imaging of
pancreatic islets according to Example 1.
[0028] FIGS. 5A and 5B show exemplary graphs showing variations
with time of biodistribution of a molecular probe for imaging of
pancreatic islets according to Example 2.
[0029] FIG. 6 shows exemplary results of image analysis of a
pancreas section using a molecular probe for imaging of pancreatic
islets according to Example 2.
[0030] FIGS. 7A to 7C show exemplary SPECT images obtained by using
a molecular probe for imaging of pancreatic islets according to
Example 3.
DESCRIPTION OF THE INVENTION
[0031] The diameter of a pancreatic islet is, for example,
approximately 50 to 500 .mu.m in the case of a human. In order to
noninvasively image or quantify such pancreatic islets in vivo, a
molecular probe, for example, is considered to be necessary that
can show specific uptake in pancreatic islets, thereby making
contrast between pancreatic islets and surrounding organs. Various
researches and developments of molecular probes therefore have been
made.
[0032] For example, Document 2 (M. Beche et al.) reports the
research on the affinity of Lys.sup.40(Ahx-DTPA-.sup.111In)
Exendin-(9-39) with respect to GLP-1R in GLP-1R-positive tumors and
pancreatic islet cells. According to this document, the following
was proven consequently: the uptake of
Lys.sup.40(Ahx-DTPA-.sup.111In) Exendin-(9-39) in pancreatic islets
was about 0.4%, and the uptake thereof in the GLP-1R-positive tumor
cells was about 7.5%. In other words, the results show that
Lys.sup.40(Ahx-DTPA-.sup.111In) Exendin-(9-39) has a low affinity
with respect to GLP-1R.
[0033] In addition to these, the detection of pancreatic
.beta.-cells by targeting GLP-1R as a target molecule is attempted
with use of a commercially available [.sup.125I]
Bolton-Hunter-labeled Extendin (9-39) (Perkin Elmer Inc.)(E. Mukai
et al. Non-invasive imaging of pancreatic islets targeting
glucagon-like peptide-1 receptors, 44.sup.th EASD Annual Meeting
Rome 2008, abstract, Presentation No. 359 (Document 3). The
following results have been obtained: when [.sup.125I]
Bolton-Hunter-labeled Exendin (9-39) (hereinafter referred to also
as "BH-labeled probe") was administered to mice via tail veins
thereof, the uptake of the BH-labeled probe in the pancreas was
highest among those in the other organs except for the lungs during
a time period from the point of 60 minutes to the point of 120
minutes after the administration. On the other hand, according to
the results of experiments by the inventors of the present
invention, after [.sup.125I] Bolton-Hunter-labeled Exendin (9-39)
was administered, uptake of the BH-labeled probe in the neck (the
thyroid grand) increased with time (see, for example, FIGS. 2A and
2B of the present application). The increase of uptake in the neck
(the thyroid grand) means that radioactive iodine was eliminated in
vivo from the BH-labeled probe administered, and the radioactive
iodine thus eliminated accumulated in the thyroid gland. It is
known that the radioactive iodine such as .sup.125I easily
accumulates in the thyroid gland, and once it accumulates in the
thyroid gland, it causes a thyroid gland cancer, etc. Therefore, a
molecular probe for imaging use in which radioactive iodine is used
as a radioactive nuclide preferably exhibits small accumulation of
eliminated radioactive iodine in the neck (the thyroid gland); that
is, a molecular probe that exhibits small elimination of
radioactive iodine in vivo, thereby having excellent biological
stability, is preferred.
[0034] Therefore, currently, a new molecular probe is sought for
that is capable of accumulating specifically in pancreatic islets,
thereby generating contrast with surrounding organs, and that is
characterized in that the elimination of radioactive iodine from
the molecular probe in vivo is suppressed.
[0035] The present invention is based on the finding that the
molecular probe for imaging of pancreatic islets, labeled with the
group represented by the chemical formula (I) above enables
noninvasive three-dimensional imaging of pancreatic islets by, for
example, PET, or SPECT, and that the elimination of radioactive
iodine from the probe is suppressed. In other words, the present
invention preferably achieves an effect of enabling noninvasive
three-dimensional imaging of pancreatic islets. Further, since the
molecular probe of the present invention preferably can accumulate
more specifically in pancreatic islets, as compared with the
molecular probes disclosed by Documents 1, 2, and 3, the present
invention can achieve an effect of enabling the imaging of
pancreatic islets for quantification.
[0036] Further, as described above, it is known that in the
diabetes developing process, the amount of pancreatic islets
decreases prior to the occurrence of glucose tolerance
abnormalities. Therefore, by performing imaging of pancreatic
islets and/or determining the amount of pancreatic islets, for
example, minute changes in pancreatic islets can be found in a
state prior to the development of diabetes or in an initial stage
of the same, whereby the ultra-early detection and diagnosis of
diabetes are enabled by the molecular probe for imaging of
pancreatic islets of the present invention. Thus, the molecular
probe for imaging of pancreatic islets of the present invention is
useful for the prevention, early detection, and diagnosis of
diabetes, preferably for the ultra-early detection and diagnosis of
diabetes.
[0037] More specifically, the present invention relates to the
following:
[1] A molecular probe for use in imaging of pancreatic islets, the
molecular probe comprising any one of the following
polypeptides:
[0038] a polypeptide represented by any one of the following
formulae (1) to (12);
[0039] a polypeptide obtained by deletion, insertion, or
substitution of one to several amino acids with respect to a
polypeptide represented by any one of the following formulae (1) to
(12), the polypeptide being capable of binding to pancreatic
islets; and
[0040] a polypeptide having a homology of 80% or higher with any
one of the amino acid sequences of polypeptides represented by the
following formulae (1) to (12), the polypeptide being capable of
binding to pancreatic islets:
TABLE-US-00003 (SEQ ID NO. 1)
Z-DLSXQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH.sub.2 (1) (SEQ ID NO. 2)
Z-LSXQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH.sub.2 (2) (SEQ ID NO. 3)
Z-SXQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH.sub.2 (3) (SEQ ID NO. 4)
Z-XQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH.sub.2 (4) (SEQ ID NO. 5)
Z-DLSKQMEEEAVRLFIEWLXNGGPSSGAPPPS-NH.sub.2 (5) (SEQ ID NO. 6)
Z-LSKQMEEEAVRLFIEWLXNGGPSSGAPPPS-NH.sub.2 (6) (SEQ ID NO. 7)
Z-SKQMEEEAVRLFIEWLXNGGPSSGAPPPS-NH.sub.2 (7) (SEQ ID NO. 8)
Z-KQMEEEAVRLFIEWLXNGGPSSGAPPPS-NH.sub.2 (8) (SEQ ID NO. 9)
B-DLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH.sub.2 (9) (SEQ ID NO. 10)
B-LSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH.sub.2 (10) (SEQ ID NO. 11)
B-SKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH.sub.2 (11) (SEQ ID NO. 12)
B-KQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH.sub.2 (12)
where
[0041] in the foregoing formulae (1) to (8),
[0042] Z- indicates that an .alpha.-amino group at an N-terminus is
either not modified, or is modified with a modifying group having
no electric charge, and
[0043] X represents a lysine residue, an amino group of a side
chain of the lysine residue being labeled with a group represented
by the chemical formula (I) below having an aromatic ring,
[0044] in the formulae (9) to (12), B- indicates that an
.alpha.-amino group at an N-terminus is labeled with a group
represented by the chemical formula (I) below having an aromatic
ring, and
[0045] in the foregoing formulae (1) to (12), --NH.sub.2 indicates
that a carboxyl group at a C-terminus is amidated,
##STR00003##
wherein
[0046] A represents either an aromatic hydrocarbon group or an
aromatic heterocyclic group,
[0047] R.sup.1 represents a substituent that contains any one of
.sup.123I, .sup.124I, .sup.125I, and .sup.131I,
[0048] R.sup.2 represents either a hydrogen atom, or one or more
substituents different from that represented by R.sup.1, and
[0049] R.sup.3 represents any one of a bond, a methylene group, and
an oxymethylene group;
[2] The molecular probe for imaging of pancreatic islets according
to [1], wherein the group having an aromatic ring is a group
represented by the following chemical formula (II):
##STR00004##
wherein R.sup.1 represents a substituent that contains any one of
.sup.123I, .sup.124I, .sup.125, and .sup.131I; [3] A kit for
performing imaging of pancreatic islets, comprising the molecular
probe for imaging of pancreatic islets according to [1] or [2]; [4]
The kit according to [3], wherein the molecular probe for imaging
of pancreatic islets included in the kit is in a form of a
parenteral solution; [5] A reagent for performing imaging of
pancreatic islets, comprising the molecular probe for imaging of
pancreatic islets according to [1] or [2]; [6] A method for imaging
of pancreatic islets comprising detecting a signal of the molecular
probe for imaging of pancreatic islets according to [1] or [2] from
an analyte to which the molecular probe has been administered; [7]
The method for imaging of pancreatic islets according to [6],
further comprising determining a state of pancreatic islets from
results of the imaging of pancreatic islets using the molecular
probe for imaging of pancreatic islets; [8] A method for
determining an amount of pancreatic islets, comprising;
[0050] detecting a signal of the molecular probe for imaging of
pancreatic islets according to [1] or [2] from an analyte to which
the molecular probe has been administered; and
[0051] calculating an amount of pancreatic islets from the detected
signal of the molecular probe for imaging of pancreatic islets;
[9] The method for determining an amount of pancreatic islets
according to [8], further comprising presenting the calculated
amount of pancreatic islets; [10] A method for producing the
molecular probe for imaging of pancreatic islets according to [1]
or [2], comprising labeling and deprotecting a precursor of the
molecular probe for imaging of pancreatic islets,
[0052] wherein the precursor of the molecular probe for imaging of
pancreatic islets includes any one of the following
polypeptides:
[0053] a polypeptide represented by any one of the following
formulae (13) to (24);
[0054] a polypeptide obtained by deletion, insertion, or
substitution of one to several amino acids with respect to a
polypeptide represented by any one of the following formulae (13)
to (24), the polypeptide being capable of binding to pancreatic
islets after being labeled and deprotected; and
[0055] a polypeptide having a homology of 80% or higher with any
one of the amino acid sequences of polypeptides represented by the
following formulae (13) to (24), the polypeptide being capable of
binding to pancreatic islets after being labeled and
deprotected,
TABLE-US-00004 (SEQ ID NO. 13) *-DLSKQMEEEAVRLFIEWLK*
NGGPSSGAPPPS-NH.sub.2 (13) (SEQ ID NO. 14) *-LSKQMEEEAVRLFIEWLK*
NGGPSSGAPPPS-NH.sub.2 (14) (SEQ ID NO. 15) *-SKQMEEEAVRLFIEWLK*
NGGPSSGAPPPS-NH.sub.2 (15) (SEQ ID NO. 16) *-KQMEEEAVRLFIEWLK*
NGGPSSGAPPPS-NH.sub.2 (16) (SEQ ID NO. 17) *-DLSK*
QMEEEAVRLFIEWLKNGGPSSGAPPPS-NH.sub.2 (17) (SEQ ID NO. 18) *-LSK*
QMEEEAVRLFIEWLKNGGPSSGAPPPS-NH.sub.2 (18) (SEQ ID NO. 19) *-SK*
QMEEEAVRLFIEWLKNGGPSSGAPPPS-NH.sub.2 (19) (SEQ ID NO. 20) *-K*
QMEEEAVRLFIEWLKNGGPSSGAPPPS-NH.sub.2 (20) (SEQ ID NO. 21) DLSK*
QMEEEAVRLFIEWLK* NGGPSSGAPPPS-NH.sub.2 (21) (SEQ ID NO. 22) LSK*
QMEEEAVRLFIEWLK* NGGPSSGAPPPS-NH.sub.2 (22) (SEQ ID NO. 23) SK*
QMEEEAVRLFIEWLK* NGGPSSGAPPPS-NH.sub.2 (23) (SEQ ID NO. 24) K*
QMEEEAVRLFIEWLK* NGGPSSGAPPPS-NH.sub.2 (24)
where
[0056] in the foregoing formulae (13) to (20),
[0057] *- indicates that an .alpha.-amino group at an N terminus is
either protected by a protecting group or modified by a modifying
group having no electric charge, and
[0058] in the foregoing formulae (13) to (24),
[0059] K* indicates that an amino group of a side chain of a lysine
is protected by a protecting group, and
[0060] --NH.sub.2 indicates that a carboxyl group at a C-terminus
is amidated;
[11] The method for producing the molecular probe for imaging of
pancreatic islets according to [10],
[0061] wherein the labeling of the precursor of the molecular probe
for imaging of pancreatic islets includes labeling of the precursor
with a labeling compound having a group represented by the
following chemical formula (I) having an aromatic ring:
##STR00005##
wherein
[0062] A represents either an aromatic hydrocarbon group or an
aromatic heterocyclic group,
[0063] R.sup.1 represents a substituent that contains any one of
.sup.123I, .sup.124I, .sup.125I, and .sup.131I,
[0064] R.sup.2 represents either a hydrogen atom, or one or more
substituents different from that represented by R.sup.1, and
[0065] R.sup.3 represents any one of a bond, a methylene group, and
an oxymethylene group.
[12] A method for radioactively labeling a peptide having a
plurality of amino acids having radioactively-labelable functional
groups on side chains, the method comprising:
[0066] synthesizing a peptide using a protected amino acid in which
an .alpha.-amino group at an N-terminus and a functional group of a
side chain are protected by protecting groups;
[0067] deprotecting a functional group by removing a protecting
group therefrom, wherein the functional group to be deprotected is
a functional group that is not to be radioactively labeled, among
the radioactively labelable functional groups of the side chains of
the amino acids of the synthesized peptide;
[0068] protecting, again, the deprotected functional group of the
side chain of the amino acid by a protecting group different from
that removed upon the deprotecting;
[0069] deprotecting, by removing protecting groups, the other
functional groups than the functional group of the side chain of
the amino acid that is again protected, so as to obtain a peptide
to be radioactively labeled;
[0070] radioactively labeling the obtained peptide with a labeling
compound; and
[0071] deprotecting the radioactively labeled peptide by removing
protecting groups.
[13] A method for producing a radioactively-labeled peptide,
wherein the peptide has a plurality of amino acids having
radioactively labelable functional groups on side chains, the
method comprising:
[0072] synthesizing a peptide to be radioactively labeled, using
protected amino acids in each of which an .alpha.-amino group at an
N-terminus and a functional group of a side chain are protected by
protecting groups;
[0073] deprotecting a functional group by removing a protecting
group therefrom, wherein the functional group to be deprotected is
a functional group that is not to be radioactively labeled, among
the radioactively labelable functional groups of the side chains of
the amino acids of the synthesized peptide;
[0074] protecting, again, the deprotected functional group of the
side chain of the amino acid by a protecting group different from
that removed upon the deprotecting;
[0075] deprotecting, by removing protecting groups, the other
functional groups than the functional group of the side chain of
the amino acid that is again protected, so as to obtain a peptide
to be radioactively labeled;
[0076] radioactively labeling the obtained peptide with a labeling
compound; and
[0077] deprotecting the radioactively-labeled peptide by removing
protecting groups.
[0078] [Imaging of Pancreatic Islets]
[0079] In the present specification, the "imaging of pancreatic
islets" refers to "molecular imaging of pancreatic islets", and
includes the imaging of in-vivo spatial and/or time distribution of
pancreatic islets. Further, in the present invention, the imaging
of pancreatic islets preferably images pancreatic .beta.-cells as
target molecules, from the viewpoint of the prevention, treatment,
and diagnosis of diabetes. Still further, in the present invention,
the imaging of pancreatic islets preferably noninvasive
three-dimensional imaging, from the viewpoint of the quantification
of the amount of pancreatic islets, and the application of this
imaging to humans. The method of imaging is not limited
particularly, if it is a method that enables noninvasive imaging of
pancreatic islets. Examples of the method include methods utilizing
positron emission tomography (PET), single photon emission computed
tomography (SPECT), etc. Among these methods, PET and SPECT are
preferred, from the viewpoint of quantifying the amount of
pancreatic islets using the molecular probe of the present
invention.
[0080] [Molecular Probe for Imaging of the Present Invention]
[0081] The molecular probe for imaging according to the present
invention is a molecular probe for imaging of pancreatic islets
including a polypeptide used in imaging of pancreatic islets, the
polypeptide being represented by any one of the above-mentioned
formulae (1) to (12). Preferably the molecular probe for imaging
according to the present invention consists of any one of the
following polypeptides: a polypeptide represented by any one of the
above-mentioned formulae (1) to (12); a polypeptide obtained by
deletion, insertion, or substitution of one to several amino acids
with respect to a polypeptide represented by any one of the
foregoing formulae (1) to (12), the polypeptide being capable of
binding to pancreatic islets; and a polypeptide having a homology
of 80% or higher with any one of the amino acid sequences of
polypeptides represented by the foregoing formulae (1) to (12), the
polypeptide being capable of binding to pancreatic islets.
[0082] Amino acid sequences of polypeptides of the foregoing
formulae (1) to (12) are the amino acid sequences according to SEQ
ID NOS. 1 to 12 shown in the Sequence Listing, respectively. Each
of the following amino groups is labeled with a group represented
by the chemical formula (I) above having an aromatic ring: the
amino group of the side chain of a lysine at position 4 in the
polypeptide of the formula (1) above; the amino group of the side
chain of a lysine at position 3 in the polypeptide of the formula
(2) above; the amino group of the side chain of a lysine at
position 2 in the polypeptide of the formula (3) above; and the
amino group of the side of a lysine at position 1 in polypeptide of
the formula (4) above. Each of the following amino groups is
labeled with a group represented by the formula (I) having an
aromatic ring: the amino group of the side chain of a lysine at
position 19 in the polypeptide of the formula (5) above; the amino
group of the side chain of a lysine at position 18 in the
polypeptide of the formula (6) above; the amino group of the side
chain of a lysine at position 17 in the polypeptide of the formula
(7) above; and the amino group of the side chain of a lysine at
position 16 in the polypeptide of the formula (8) above. The
.alpha.-amino group at an N-terminus of the polypeptide of each of
the formulae (9) to (12) is labeled with a group represented by the
chemical formula (I) having an aromatic ring. The .alpha.-amino
group at an N-terminus of the polypeptide of each of the formulae
(1) to (8) is either not modified, or is modified with a
modification group having no electric charge. The carboxyl group at
a C-terminus of the polypeptide of each of the formulae (1) to (12)
is amidated with an amino group from the viewpoint of improving the
affinity to the pancreatic .beta.-cell.
[0083] Here, the amino acid sequences of the foregoing formula (1)
(SEQ ID NO. 1 in the Sequence Listing) and the foregoing formula
(5) (SEQ ID NO. 5 in the Sequence Listing) are identical to the
amino acid sequence of exendin (9-39) except for the group
represented by the chemical formula (I) above having an aromatic
ring, which is bonded to the amino group of the side chain of a
lysine, and the modifying group bondable to an .alpha.-amino group
at an N-terminus. Further, the amino acid sequence of the foregoing
formula (9) (SEQ ID NO. 9 in the Sequence Listing) is identical to
the amino acid sequence of exendin (9-39) except for the group
represented by the chemical formula (I) above having an aromatic
ring, which is bonded to an .alpha.-amino group at an N-terminus.
It is known that exendin (9-39) is bonding to GLP-1R (glucagon-like
peptide-1 receptor) expressed on the pancreatic .beta.-cell. The
molecular probe for imaging of pancreatic islets according to the
present invention also is capable of binding to pancreatic islets,
and preferably the pancreatic .beta.-cells.
[0084] In the present specification, the description of "being
capable of binding to pancreatic islets" means the following: from
the viewpoint of applying the present invention to the
quantification of the pancreatic islets and a use of the
examination and diagnosis, the molecular probe for imaging
according to the present invention preferably is capable of binding
to the pancreatic .beta.-cells, more preferably is at least
specific to the pancreatic .beta.-cells in the pancreas, and
further more preferably is at least specific to such an extent that
a signal thereof does not overlap a signal of another organ/tissue
in the signal detection in the noninvasive imaging with respect to
humans.
[0085] In an another exemplary embodiment, the molecular probe for
imaging according to the present invention includes a polypeptide
used in imaging of pancreatic islets that is obtained by deletion,
insertion, or substitution of one to several amino acids with
respect to any one of the polypeptides of the foregoing formulae
(1) to (12), and that is capable of binding to pancreatic islets.
Here, exemplary ranges expressed by the foregoing description of
"one to several" include the following ranges: 1 to 10; 1 to 9; 1
to 8; 1 to 7; 1 to 6; 1 to 5; 1 to 4; 1 to 3; 1 to 2; and 1. In the
molecular probe for imaging according to this embodiment of the
present invention, in the case of a polypeptide obtained by
deletion, insertion, or substitution of one to several amino acids
with respect to any one of the polypeptides of the foregoing
formulae (1) to (8), it is preferable that the polypeptide includes
one lysine labeled with a group represented by the chemical formula
(I) above having an aromatic ring, that a carboxyl group at a
C-terminus is amidated, and that the .alpha.-amino group at the
N-terminus may be either not modified or modified by a modifying
group having no electric charge. In the case where the polypeptide
is a polypeptide that is obtained by deletion, insertion, or
substitution of one to several amino acids with respect to any one
of the polypeptides of the foregoing formulae (9) to (12), it is
preferable that the .alpha.-amino group at a N-terminus is labeled
with a group represented by the chemical formula (I) above having
the aromatic ring and does not include any other labeling group,
and further, that a carboxyl group at a C-terminus is amidated.
Polypeptide obtained by deletion, insertion, or substitution of one
to several amino acids with respect to a polypeptide represented by
any one of the following formulae (1) to (12) preferably has a
working effect identical to those of the polypeptides of the
formulae (1) to (12), and more preferably has a working effect
identical to those of the polypeptides of the formula (1) or the
polypeptide of the formula (9).
[0086] In still another embodiment, the molecular probe for imaging
according to the present invention may include a polypeptide used
in imaging of pancreatic islets that has a homology of 80% or
higher with any one of the amino acid sequences of polypeptides
represented by the following formulae (1) to (12), and is capable
of binding to pancreatic islets. Here, the "homology" may be any
value calculated by an algorithm usually used by those skilled in
the art, for example, BLAST or FASTA, or alternatively, it may be
based on a value obtained by dividing the number of identical amino
acid residues existing in two polypeptides compared, by the number
of amino acids of an entire length of one of the polypeptides.
Exemplary ranges of the homology may include the following ranges:
not less than 85%; not less than 90%; and not less than 95%. In the
molecular probe for imaging according to the present embodiment of
the present invention, as well, in the case of a polypeptide having
a homology of 80% or higher with any of the polypeptides of the
formulae (1) to (8), the polypeptide preferably includes one lysine
labeled with a group represented by the chemical formula (I) having
an aromatic ring, and a carboxyl group at a C-terminus preferably
is amidated, while an .alpha.-amino group at an N-terminus may be
either not modified, or modified with a modifying group having no
electric charge. In the case of a polypeptide having a homology of
80% or higher with an amino acid sequence of any of the
polypeptides of the foregoing formulae (9) to (12), it is
preferable that an .alpha.-amino group at an N-terminus is labeled
with a group represented by the aforementioned chemical formula (I)
having an aromatic ring, and does not have any other labeling
group, and that a carboxyl group at a C-terminus is amidated.
Polypeptide having homology of 80% or higher with any one of the
amino acid sequences of polypeptides represented by the following
formulae (1) to (12) preferably has a working effect identical to
those of the polypeptides of the formulae (1) to (12), and more
preferably has a working effect identical to that of the
polypeptide of the formula (1) or the polypeptide of the formula
(9).
[0087] The molecular probe for imaging of the present invention, as
described above, can be used in imaging of pancreatic islets, and
from the viewpoint of the application of the same to the
examination and diagnosis for a human, preferably is used in
noninvasive imaging of pancreatic islets, more preferably is used
in imaging of pancreatic .beta.-cells, and further preferably is
used in imaging of GLP-1 receptor of pancreatic .beta.-cells. From
the same viewpoint, the molecular probe for imaging of the present
invention preferably is used in imaging of pancreatic islets for
quantifying the amount of the pancreatic islets, more preferably is
used in imaging of pancreatic .beta.-cells for quantifying the
amount of the pancreatic islets, and further preferably is used in
imaging of GLP-1 receptor of pancreatic .beta.-cells for
quantifying the amount of the pancreatic islets. Further, the
molecular probe for imaging of pancreatic islets according to the
present invention preferably is used in imaging of pancreatic
islets for the prevention treatment or diagnosis of diabetes. Such
imaging of pancreatic islets may be performed by, for example, PET
or SPECT.
[0088] [Group to Label Amino Group of Side Chain of Lysine Residue
or .alpha.-Amino Group at N-Terminus]
[0089] In the molecular probe for imaging of the present invention,
the amino group of the side chain of the lysine residue represented
by X in the amino acid sequences of the polypeptides of the
aforementioned formulae (1) to (8), and the .alpha.-amino group at
the N-terminus of each of the polypeptides of the aforementioned
formulae (9) to (12), are labeled with groups represented by the
following chemical formula (I) having an aromatic ring:
##STR00006##
[0090] In the foregoing chemical formula (I), A represents an
aromatic hydrocarbon group or an aromatic heterocyclic group. The
aromatic hydrocarbon group preferably is an aromatic hydrocarbon
group having 6 to 18 carbon atoms, and examples of the same include
phenyl group, o-tolyl group, m-tolyl group, p-tolyl group,
2,4-xylyl group, p-cumenyl group, mesityl group, 1-naphthyl group,
2-naphthyl group, 1-anthryl group, 2-anthryl group, 9-anthryl
group, 1-phenanthryl group, 9-phenanthryl group, 1-acenaphthyl
group, 2-azulenyl group, 1-pyrenyl group, 2-triphenylenyl group,
o-biphenylyl group, m-biphenylyl group, p-biphenylyl group, and
terphenyl group. The aromatic heterocyclic group preferably is a 5
to 10-membered heterocyclic group having one or two of a nitrogen
atom, an oxygen atom, or a sulfur atom, and examples of the same
include triazolyl group, 3-oxadiazolyl group, 2-furyl group,
3-furyl group, 2-thienyl group, 3-thienyl group, 1-pyrrolyl group,
2-pyrrolyl group, 3-pyrrolyl group, 2-pyridyl group, 3-pyridyl
group, 4-pyridyl group, 2-pyradyl group, 2-oxazolyl group,
3-isoxazolyl group, 2-thiazolyl group, 3-isothiazolyl group,
2-imidazolyl group, 3-pyrazolyl group, 2-quinolyl group, 3-quinolyl
group, 4-quinolyl group, 5-quinolyl group, 6-quinolyl group,
7-quinolyl group, 8-quinolyl group, 1-isoquinolyl group,
2-quinoxalynyl group, 2-benzofuryl group, 2-benzothienyl group,
N-indolyl group, and N-carbazolyl group. A preferably is, among
these, phenyl group, triazolyl group, or pyridyl group, and more
preferably, phenyl group.
[0091] In the aforementioned chemical formula (I), R.sup.1
represents a substituent that contains any of .sup.123I, .sup.124I,
.sup.125I, and .sup.131I. Examples of R.sup.1 include [.sup.123I]
iodine atom, [.sup.124I] iodine atom, [.sup.125I] iodine atom,
[.sup.131I] iodine atom, [.sup.123I] iodine atom substituted
C.sub.1-C.sub.3 alkyl groups, [.sup.124I] iodine atom-substituted
C.sub.1-C.sub.3 alkyl groups, [.sup.125I] iodine atom-substituted
C.sub.1-C.sub.3 alkyl groups, [.sup.131I] iodine atom-substituted
C.sub.1-C.sub.3 alkyl groups, [.sup.123I] iodine atom-substituted
C.sub.1-C.sub.3 alkoxy groups, [.sup.124I] iodine atom-substituted
C.sub.1-C.sub.3 alkoxy groups, [.sup.125I] iodine atom-substituted
C.sub.1-C.sub.3 alkoxy groups, and [.sup.131] iodine
atom-substituted C.sub.1-C.sub.3 alkoxy groups. In the present
specification, the "C.sub.1-C.sub.3 alkyl group" refers to an alkyl
group that has 1 to 3 carbon atoms, and examples of the same
include methyl group, ethyl group, and propyl group. In the present
specification, the "[.sup.123I] iodine atom-substituted,
[.sup.124I] iodine atom-substituted, [.sup.125I] iodine
atom-substituted, or [.sup.131I] iodine atom-substituted
C.sub.1-C.sub.3 alkyl group" refers to an alkyl group that has 1 to
3 carbon atoms and in which one hydrogen atom is substituted with
[.sup.123I] iodine atom, [.sup.124I] iodine atom, [.sup.125I]
iodine atom, or [.sup.131I] iodine atom. In the present
specification, the "C.sub.1-C.sub.3 alkoxy group" refers to an
alkoxy group that has 1 to 3 carbon atoms, and examples of the same
include methoxy group, ethoxy group, and propoxy group. In the
present specification, the "[.sup.123I] iodine atom-substituted,
[.sup.124I] iodine atom-substituted, [.sup.125I] iodine
atom-substituted, or [.sup.131I] iodine atom-substituted
C.sub.1-C.sub.3 alkoxy group" refers to an alkoxy group that has 1
to 3 carbon atoms and in which one hydrogen atom is substituted
with [.sup.123I] iodine atom-substituted, [.sup.124I] iodine
atom-substituted, [.sup.125I] iodine atom-substituted, or
[.sup.131I] iodine atom-substituted. From the viewpoint of
performing PET, R.sup.1 preferably is a substituent containing
.sup.124I that emits positron. From the viewpoint of performing
SPECT, R.sup.1 preferably is a substituent containing .sup.123I or
.sup.125I that emits .gamma.-rays. From the viewpoint of an amount
of energy emitted, R.sup.1 preferably is a substituent containing
.sup.123I. R.sup.1 preferably is [.sup.123I] iodine atom,
[.sup.124I] iodine atom, methyl [.sup.123I] iodide group, methyl
[.sup.124I] iodide group, methoxy [.sup.123I] iodide group, or
methoxy .sup.[124I] iodide group, and R.sup.1 more preferably is
[.sup.123I] iodine atom, or [.sup.124I] iodine atom. In R.sup.1 in
the aforementioned chemical formula (I), preferably a hydrogen atom
at any one of an ortho-position, a meta-position, and a
para-position is substituted, from the viewpoint of quantification,
and more preferably, at a meta-position or a para-position.
[0092] In the aforementioned chemical formula (I), R.sup.2
represents a hydrogen atom or one or more substituents different
from that represented by R.sup.1. R.sup.2 may be a hydrogen atom or
a substituent, but preferably, it is a hydrogen atom. In other
words, in the aforementioned chemical formula (I), A preferably
does not have a substituent other than R.sup.1. In the case where
R.sup.2 represents a plurality of substituents, these substituents
may be identical or different. Examples of the substituent include
hydroxyl group, electron attractive groups, electron donative
groups, C.sub.1-C.sub.6 alkyl groups, C.sub.2-C.sub.3 alkenyl
groups, and C.sub.2-C.sub.6 alkynyl groups. Examples of the
electron attractive group include cyano group, nitro group, halogen
atoms, carbonyl group, sulfonyl group, acetyl group, and phenyl
group. Examples of the halogen atom include fluorine atom, chlorine
atom, bromine atom, and iodine atom. In the present specification,
the "C.sub.1-C.sub.6 alkyl group" refers to an alkyl group having 1
to 6 carbon atoms, and examples of the same include methyl group,
ethyl group, propyl group, isopropyl group, butyl group, isobutyl
group, sec-butyl group, tert-butyl group, pentyl group, isopentyl
group, and hexyl group. In the present specification, the
"C.sub.2-C.sub.6 alkenyl group" refers to an alkenyl groups having
2 to 6 carbon atoms, and examples of the same include vinyl group,
1-propenyl group, 2-propenyl group, isopropenyl group, 1-butenyl
group, 2-butenyl group, and 3-butenyl group. In the present
specification, the "C.sub.2-C.sub.6 alkynyl group" refers to an
alkynyl group having 2 to 6 carbon atoms, and examples of the same
include ethynyl group, 1-propynyl group, 2-propynyl group,
1-butynyl group, 2-butynyl group, and 3-butynyl group. Among these,
the substituent preferably is hydroxyl group or an electron
attractive group.
[0093] In the aforementioned chemical formula (I), R.sup.3
represents any one of a bond, a methylene group, and an
oxymethylene group. Among these, a bond or a methylene group is
preferred, and a bond is preferred further.
[0094] In the molecular probe for imaging according to the present
invention, the group represented by the chemical formula (I) above
having an aromatic ring preferably is the group represented by the
chemical formula (II) below. In the chemical formula (II) below,
R.sub.1 is as described above.
##STR00007##
[0095] [Modifying Group]
[0096] In the molecular probe for imaging of the present invention,
an .alpha.-amino group at an N-terminus in any one of the
polypeptides of the above-described formulae (1) to (8) may be
modified with a modifying group having no electric charge, from the
viewpoint of canceling a positive charge of the .alpha.-amino group
at the N-terminus thereby suppressing accumulation in kidneys of
the molecular probe for imaging the present invention. Examples of
such a modifying group having no electric charge include
9-fluorenylmethyloxycarbonyl group (Fmoc), tert-butoxycarbonyl
group (Boc), benzyloxycarbonyl group (Cbz),
2,2,2-trichloroethoxycarbonyl group (Troc), allyloxycarbonyl group
(Alloc), 4-methoxytrityl group (Mmt), amino group, alkyl groups
having 3 to 20 carbon atoms, 9-fluoreneacetyl group,
1-fluorenecarboxylic acid group, 9-fluorenecarboxylic acid group,
9-fluorenone-1-carboxylic acid group, benzyloxycarbonyl group,
xanthyl group (Xan), trityl group (Trt), 4-methyltrityl group
(Mtt), 4-methoxy-2,3,6-trimethylbenzenesulfonyl group (Mtr),
mesitylene-2 sulfonyl group (Mts), 4,4-dimethoxybenzohydryl group
(Mbh), tosyl group (Tos), 2,2,5,7,8-pentamethylchroman-6 sulfonyl
group (Pmc), 4-methylbenzyl group (MeBzl), 4-methoxybenzyl group
(MeOBzl), benzyloxy group (BzlO), benzyl group (Bzl), benzoyl group
(Bz), 3-nitro-2-pyridinesulfenyl group (Npys),
1-(4,4-dimethyl-2,6-diaxocyclohexylidene)ethyl group (Dde),
2,6-dichlorobenzyl group (2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl
group (2Cl-Z), 2-bromobenzyloxycarbonyl group (2Br-Z),
benzyloxymethyl group (Bom), cyclohexyloxy group (cHxO),
t-butoxymethyl group (Burn), t-butoxy group (tBuO), t-butyl group
(tBu), acetyl group (Ac), trifluoroacetyl group (TFA),
o-bromobenzyloxycarbonyl group, t-butyldimethylsilyl group,
2-chlorobenzyl (Cl-z) group, cyclohexyl group, cyclopentyl group,
isopropyl group, pivalyl group, tetrahydropyran-2-yl group, and
trimethylsilyl group. Among these, preferably, the modifying group
is acetyl group, benzyl group, benzyloxymethyl group,
o-bromobenzyloxycarbonyl group, t-butyl group, t-butyldimethylsilyl
group, 2-chlorobenzyl group, 2,6-dichlorobenzyl group, cyclohexyl
group, cyclopentyl group, isopropyl group, pivalyl group,
tetrahydropyran-2-yl group, tosyl group, trimethylsilyl group, or
trityl group. More preferably, the modifying group is acetyl
group.
[0097] [Imaging Method]
[0098] Another aspect of the present invention relates to a method
for imaging of pancreatic islets that includes imaging pancreatic
islets using the molecular probe for imaging of the present
invention. Still another aspect of the present invention relates to
a method for imaging of pancreatic islets that includes detecting a
signal of the molecular probe for imaging of the present invention
from an analyte to which the molecular probe has been administered,
or detecting a signal of the molecular probe for imaging of the
present invention that has been bound to pancreatic islets
preliminarily. In the imaging method, a signal of the molecular
probe for imagine of the present invention from an analyte to which
the molecular probe has been administered in an enough amount for
imaging, or a signal of the molecular probe for imaging of the
present invention that in an enough amount for imaging has been
bound to pancreatic islets preliminarily is detected preferably.
The imaging of pancreatic islets is as described above. The method
for imaging islets according to the present invention preferably is
a method for imaging pancreatic .beta.-cells from the viewpoint of
the application of the same to the examination and diagnosis.
[0099] The detection of a signal of a molecular probe for imaging
of the present invention can be performed by, for example, the
determination by means of PET and/or the determination by means of
SPECT. The determination by means of PET and the determination by
means of SPECT include, for example, photographing an image, and
determining an amount of pancreatic islets. The method for imaging
of the present invention may include reconfiguring the detected
signal so as to convert the same into an image, and further may
include displaying the image.
[0100] The determination by means of SPECT includes, for example,
determining, with use of a gamma camera, .gamma.-rays emitted from
a subject having pancreatic islets to which the molecular probe for
imaging of the present invention has been bound preliminarily/an
analyte to which the molecular probe for imaging of present
invention has been administered. The determination with use of the
gamma camera includes, for example, measuring radiation
(.gamma.-rays) emitted from the radioactive iodine used for
labeling the molecular probe for imaging of the present invention
during a certain time unit, and preferably includes determining a
direction in which the radiation is emitted and a radiation dose
during a certain time unit. The method for imaging according to the
present invention further may include presenting the determined
distribution of the molecular probe for imaging of the present
invention obtained by the measurement of the radiation as a
cross-sectional image, and reconfiguring the obtained
cross-sectional image. Examples of the subject (analyte) include
humans and/or mammals other than humans.
[0101] The determination by means of PET include, for example,
simultaneously measuring a pair of annihilation radiations
generated upon the coupling between a positron and an electron,
with use of a detector for PET, from a subject having pancreatic
islets to which the molecular probe for imaging of the present
invention has been bound preliminarily/an analyte to which the
molecular probe for imaging of the present invention has been
administered, and further may include figuring a three-dimensional
distribution of positions of radioactive iodine emitting positrons,
based on the measurement results.
[0102] In the method for imaging according to the present
invention, the determination by means of X-ray CT or MRI may be
performed, together with the determination by means of SPECT or the
determination by means of PET. This makes it possible to obtain,
for example, a fusion image obtained by fusion of an image obtained
by SPECT or an image obtained PET (functional image), with an image
obtained by CT or an image obtained by MRI (morphological
image).
[0103] The method for imaging of the present invention may include
determining a state of pancreatic islets based on the results of
the imaging of pancreatic islets with use of the molecular probe
for imaging of the present invention. Determining a state of
pancreatic islets based on the results of the imaging of pancreatic
islets with use of the molecular probe includes, for example,
determining the presence/absence of pancreatic islets by analyzing
an image of the imaging of pancreatic islets, and determining an
increase/decrease in the amount of pancreatic islets.
[0104] The method for imaging of the present invention may include
administering the molecular probe for imaging of the present
invention to a subject, and it is preferable to administer the
molecular probe for imaging of the present invention in an enough
amount for obtaining a desired contrast for imaging. The detection
of a signal of the molecular probe for imaging of the present
invention preferably is carried out after a certain lapse of time
since the administration of the molecular probe. Examples of the
subject include humans and/or mammals other than humans. The
administration to a subject may be local administration or systemic
administration. A path for administration may be determined
appropriately according to a state of a subject and the like, and
it may be, for example, intravenous, intraarterial, intradermal,
and intraabdominal injection or infusion. The molecular probe for
imaging of the present invention preferably is administered
together with a carrier. Examples usable as the carrier include
aqueous solvents and non-aqueous solvents. Examples of the aqueous
solvent include potassium phosphate buffer solution, physiologic
saline, Ringer's solution, and distilled water. Examples of the
non-aqueous solvent include polyethylene glycol, vegetable fats and
oils, ethanol, glycerol, dimethyl sulfoxide, and propylene glycol.
The amount of the molecular probe of the present invention for
imaging of pancreatic islets or determining an amount of pancreatic
islets may be set to be, for example, not more than 1 .mu.g. The
time period from the administration to the determination may be
decided appropriately according to, for example, a time that the
molecular probe takes to bind to pancreatic islets, the type of the
molecular probe, the decomposition time of the molecular probe,
etc.
[0105] [Method for Determining Amount of Pancreatic Islets]
[0106] Still another aspect of the present invention relates to a
method for determining an amount of pancreatic islets, including
detecting a signal of the molecular probe for imaging of the
present invention that has been bound to pancreatic islets
preliminarily, and calculating an amount of the pancreatic islets
from the detected signal of the molecular probe. The method for
determining an amount of pancreatic islets according to the present
invention may include performing imaging of pancreatic islets using
the molecular probe for imaging of the present invention. The
imaging of pancreatic islets is as described above. The calculation
of an amount of pancreatic islets from results of imaging of
pancreatic islets using the molecular probe may be performed by,
for example, analyzing an image obtained by imaging of pancreatic
islets. The quantification of a subject of the imaging from results
of the imaging can be performed easily by any person skilled in the
art, using a calibration curve, an appropriately program, or the
like. The method for determining an amount of pancreatic islets
according to the present invention preferably is a method for
determining an amount of pancreatic .beta.-cells from the viewpoint
of the application of the same to the examination and
diagnosis.
[0107] Still another aspect of the present invention relates to a
method for determining an amount of pancreatic islets, detecting a
signal of the molecular probe for imaging of the present invention
from an analyte to which the molecular for imaging of the present
invention that has been administered and/or a signal of the
molecular probe for imaging of the present invention that has been
bound to pancreatic islets preliminarily, and calculating an amount
of the pancreatic islets from the detected signal of the molecular
probe for imaging.
[0108] The method for determining an amount of pancreatic islets
according to the present invention may include presenting the
calculated amount of pancreatic islets. Presenting the calculated
amount of pancreatic islets includes, for example, storing the
calculated amount of pancreatic islets or outputting the same to
the outside. Outputting the same to the outside includes, for
example, displaying the same on a monitor and printing the
same.
[0109] [Methods for Prevention, Treatment, and Diagnosis of
Diabetes]
[0110] Still another aspect of the present invention relates to a
method for prevention, treatment, or diagnosis of diabetes. As
described above, in the diabetes developing process, the amount of
pancreatic islets (particularly, the amount of pancreatic
.beta.-cells) decreases prior to the occurrence of glucose
tolerance abnormalities, and therefore, when functional
abnormalities are detected or there are subjective symptoms,
diabetes has already reached the stage where it is too difficult to
be treated. With the imaging method using the molecular probe for
imaging of the present invention and/or the method for determining
an amount of the pancreatic islets using the same, however, a
decrease in the amount of the pancreatic islets and/or the amount
of the pancreatic .beta.-cells can be detected at an early stage,
and further, new methods for prevention, treatment, and diagnosis
of diabetes can be created. Examples of a subject on which
prevention, treatment, and diagnosis of diabetes is carried out
include humans and/or mammals other than humans.
[0111] A method for diagnosis of diabetes according to the present
invention may include performing imaging of pancreatic islets with
use of the molecular probe for imaging of the present invention,
determining a state of the pancreatic islets based on the obtained
image of the pancreatic islets or the obtained amount of the
pancreatic islets, and performing diagnosis of diabetes based on
the determination results. The determination of a state of
pancreatic islets includes, for example, determining an
increase/decrease, or a change, in the amount of pancreatic islets
by comparing the obtained image of pancreatic islets with an image
of pancreatic islets as a reference, or comparing the obtained
amount of pancreatic islets with an amount of pancreatic islets as
a reference. Further, the determination of a state of pancreatic
islets may be carried out using an information processing device.
When it is determined that the amount of pancreatic islets has
decreased, preferably this information is presented, and when it is
determined that the amount of pancreatic islets has increased or
has been maintained, preferably this information is presented. The
diagnosis of diabetes on the basis of the determination results
includes, for example, determining a risk of development of
diabetes, judging it to be diabetes, and determining a degree of
development of diabetes.
[0112] A method for treatment of diabetes of the present invention
includes performing imaging of pancreatic islets with use of the
molecular probe for imaging of the present invention, determining a
state of pancreatic islets on the basis of the obtained image of
the pancreatic islets or the obtained amount of the pancreatic
islets so as to perform diagnosis of diabetes, and treating
diabetes on the basis of the diagnosis. The determination of a
state of pancreatic islets and the diagnosis of diabetes can be
performed in the same manner as those in the method for diagnosis
of diabetes according to the present invention. The method for
treatment of diabetes according to the present invention may
include evaluating an effect of treatment such as medication and
diet performed on a subject, focusing on a change in an amount of
pancreatic islets.
[0113] A method for prevention of diabetes of the present invention
includes performing imaging of pancreatic islets with use of the
molecular probe for imaging of the present invention, and
determining a state of pancreatic islets on the basis of the
obtained image of the pancreatic islets or the obtained amount of
the pancreatic islets so as to determine a risk of development of
diabetes. The method for prevention of diabetes of the present
invention may include regularly determining an amount of pancreatic
islets, and checking presence/absence of a tendency of a decrease
in the amount of pancreatic islets.
[0114] Still another preferable aspect of the present invention
relates to a method for ultra-early diagnosis of diabetes. The
method for ultra-early diagnosis of diabetes of the present
invention may include, for example, imaging pancreatic islets or
determining an amount of pancreatic islets by the method of the
present invention in comprehensive or ordinary medical examination,
and determining a state of the pancreatic islets on the basis of
the obtained image of the pancreatic islets or the determined
amount of the pancreatic islets. Further, a method for treatment of
diabetes of the present invention may include imaging pancreatic
islets and/or determining an amount of pancreatic islets by the
method of the present invention, and evaluating functional recovery
of the pancreatic islets on the basis of the obtained image of the
pancreatic islets and/or the determined amount of the pancreatic
islets.
[0115] [Kit of the Present Invention]
[0116] Still another aspect of the present invention also relates
to a kit including the molecular probe for imaging of the present
invention. Examples of embodiments of the kit of this aspect
include a kit for performing the imaging method of the present
invention, a kit for performing the method for determining an
amount of pancreatic islets according to the present invention, and
a kit for prevention, treatment, or diagnosis of diabetes according
to the present invention. Preferably, in each of these embodiments,
the kit includes an instruction manual suitable for the
embodiment.
[0117] In the kit of the present invention, the molecular probe for
imaging of the present invention included in the kit preferably is
in a form of a parenteral solution. Therefore, the kit of the
present invention preferably includes a parenteral solution that
contains the molecular probe for imaging of the present invention.
The parenteral solution may contain the molecular probe for imaging
of the present invention as an effective ingredient, and further,
for example, a medicinal additive such as a carrier. In the present
specification, the "medicinal additive" refers to a compound that
has obtained authorization as a medicinal additive in the Japanese,
U.S. and/or European pharmacopoeias. Examples of the carrier
include aqueous solvents and non-aqueous solvents. Examples of the
aqueous solvent include potassium phosphate buffer solution,
physiologic saline, Ringer's solution, and distilled water.
Examples of the non-aqueous solvent include polyethylene glycol,
vegetable fats and oils, ethanol, glycerol, dimethyl sulfoxide, and
propylene glycol. The kit of the present invention further may
include a container for containing the molecular probe for imaging
of the present invention, and the container may be filled with the
molecular probe for imaging of the present invention or a
parenteral solution that contains the molecular probe for imaging
of the present invention. Examples of the container include a
syringe and a vial.
[0118] The kit of the present invention may further include, for
example, a component used for preparing a molecular probe, such as
a buffer or an osmotic regulator, and an instrument used in
administration of a molecular probe, such as a syringe.
[0119] [Reagent for Imaging of the Present Invention]
[0120] Still another aspect of the present invention relates to a
reagent for imaging that contains the molecular probe for imaging
of the present invention. The reagent for imaging according to the
present invention may contain the molecular probe for imaging of
the present invention as an effective ingredient, and further, a
medicinal additive such as a carrier. The carrier is as described
above.
[0121] [Method for Preparation of Molecular Probe for Imaging of
the Present Invention]
[0122] The molecular probe for imaging according to the present
invention can be prepared by labeling a molecular probe precursor
containing a polypeptide represented by any one of the formulae
(13) to (24) below with a labeling compound having a group
represented by the chemical formula (I) above having an aromatic
ring, and thereafter deprotecting the molecular probe precursor by
removing a protecting group. Through this labeling operation, an
amino group of a side chain of a lysine to which no protecting
group is bonded, or an .alpha.-amino group at an N-terminus to
which no protecting group or modifying group is not bonded, can be
labeled.
TABLE-US-00005 (SEQ ID NO. 13) *-DLSKQMEEEAVRLFIEWLK*
NGGPSSGAPPPS-NH.sub.2 (13) (SEQ ID NO. 14) *-LSKQMEEEAVRLFIEWLK*
NGGPSSGAPPPS-NH.sub.2 (14) (SEQ ID NO. 15) *-SKQMEEEAVRLFIEWLK*
NGGPSSGAPPPS-NH.sub.2 (15) (SEQ ID NO. 16) *-KQMEEEAVRLFIEWLK*
NGGPSSGAPPPS-NH.sub.2 (16) (SEQ ID NO. 17) *-DLSK*
QMEEEAVRLFIEWLKNGGPSSGAPPPS-NH.sub.2 (17) (SEQ ID NO. 18) *-LSK*
QMEEEAVRLFIEWLKNGGPSSGAPPPS-NH.sub.2 (18) (SEQ ID NO. 19) *-SK*
QMEEEAVRLFIEWLKNGGPSSGAPPPS-NH.sub.2 (19) (SEQ ID NO. 20) *-K*
QMEEEAVRLFIEWLKNGGPSSGAPPPS-NH.sub.2 (20) (SEQ ID NO. 21) DLSK*
QMEEEAVRLFIEWLK* NGGPSSGAPPPS-NH.sub.2 (21) (SEQ ID NO. 22) LSK*
QMEEEAVRLFIEWLK* NGGPSSGAPPPS-NH.sub.2 (22) (SEQ ID NO. 23) SK*
QMEEEAVRLFIEWLK* NGGPSSGAPPPS-NH.sub.2 (23) (SEQ ID NO. 24) K*
QMEEEAVRLFIEWLK* NGGPSSGAPPPS-NH.sub.2 (24)
where
[0123] in the foregoing formulae (13) to (20),
[0124] *- indicates that an .alpha.-amino group at an N terminus is
either protected by a protecting group or modified by a modifying
group having no electric charge,
[0125] in the foregoing formulae (13) to (24),
[0126] K* indicates that an amino group of a side chain of a lysine
is protected by a protecting group, and
[0127] --NH.sub.2 indicates that a carboxyl group at a C-terminus
is amidated.
[0128] The molecular probe precursor of the present invention can
be synthesized by peptide synthesis in accordance with a typical
method such as the Fmoc method, and the peptide synthesis method is
not limited particularly.
[0129] [Protecting Group]
[0130] The protecting group is intended to protect the other amino
group of the molecular probe precursor than a specific amino group
of the molecular probe for imaging according to the present
invention while the specific amino group is being labeled, and a
known protecting group that can perform such a function can be
used. As the protecting group, any known protecting group capable
of performing such a function can be used. The protecting group is
not limited particularly, and examples of the same include
9-fluorenylmethyloxycarbonyl group (Fmoc), tert-butoxycarbonyl
group (Boc), benzyloxycarbonyl group (Cbz),
2,2,2-trichloroethoxycarbonyl group (Troc), allyloxycarbonyl group
(Alloc), 4-methoxytrityl group (Mmt), amino group, alkyl groups
having 3 to 20 carbon atoms, 9-fluoreneacetyl group,
1-fluorenecarboxylic acid group, 9-fluorenecarboxylic acid group,
9-fluorenone-1-carboxylic acid group, benzyloxycarbonyl group,
xanthyl group (Xan), trityl group (Trt), 4-methyltrityl group
(Mtt), 4-methoxy2,3,6-trimethyl-benzenesulfonyl group (Mtr),
mesitylene-2-sulfonyl group (Mts), 4,4-dimethoxybenzohydryl group
(Mbh), tosyl group (Tos), 2,2,5,7,8-pentamethylchroman-6-sulfonyl
group (Pmc), 4-methylbenzyl group (MeBzl), 4-methoxybenzyl group
(MeOBzl), benzyloxy group (BzlO), benzyl group (Bzl), benzoyl group
(Bz), 3-nitro-2-pyridinesulfenyl group (Npys),
1-(4,4-dimethyl-2,6-diaxocyclohexylidene)ethyl group (Dde),
2,6-dichlorobenzyl group (2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl
group (2-Cl--Z), 2-bromobenzyloxycarbonyl group (2-Br--Z),
benzyloxymethyl group (Bom), cyclohexyloxy group (cHxO),
t-butoxymethyl group (Bum), t-butoxy group (tBuO), t-butyl group
(tBu), acetyl group (Ac), and trifluoroacetyl group (TFA). From the
viewpoint of handleability, Fmoc or Boc is preferred. The
deprotecting methods with respect to these protecting groups are
known, and any person skilled in the art is able to perform the
deprotecting appropriately.
[0131] The labeling can be performed using a labeling compound
having a group represented by the foregoing chemical formula (I)
having an aromatic ring. The labeling compound used in the labeling
preferably is a succinimidyl ester compound in which the group
represented by the foregoing chemical formula (I) is bonded with
succinimide via ester bond, more preferably, a succinimidyl ester
compound represented by the chemical formula (III) shown below, and
further more preferably, a succinimidyl ester compound represented
by the chemical formula (IV) shown below. In the chemical formula
(III) below, A, R.sup.1, R.sup.2, and R.sup.3 represent the same as
those in the case of the foregoing chemical formula (I). In the
chemical formula (IV) below, R.sup.1 represents the same as that in
the case of the foregoing chemical formula (I).
##STR00008##
[0132] A labeling compound used in the labeling preferably in
particular is [.sup.123I]N-succinimidyl 3-iodobenzoate,
[.sup.124I]N-succinimidyl 3-iodobenzoate, or
[.sup.131I]N-succinimidyl 3-iodobenzoate, in which R.sup.1 in the
foregoing chemical formula (IV) is [.sup.123I] iodine atom,
[.sup.124I] iodine atom, or [.sup.131I] iodine atom,
respectively.
[0133] Still another aspect of the present invention relates to a
method for producing a molecular probe for imaging according to the
present invention, comprising labeling and deprotecting the
molecular probe precursor for imaging. In the method for producing
the molecular probe for imaging according to the present invention,
the molecular probe precursor for imaging preferably consists of
any one of the following polypeptides:
[0134] a polypeptide represented by any one of the formulae (13) to
(24) above;
[0135] a polypeptide obtained by deletion, insertion, or
substitution of one to several amino acids with respect to a
polypeptide represented by any one of the formulae (13) to (24)
above, the polypeptide being capable of binding to pancreatic
islets after being labeled and deprotected; and
[0136] a polypeptide having a homology of 80% or higher with any
one of the amino acid sequences of polypeptides represented by the
formulae (13) to (24) above, the polypeptide being capable of
binding to pancreatic islets after being labeled and
deprotected.
[0137] In the method for producing a molecular probe for imaging
according to the present invention, the labeling of the molecular
probe precursor for imaging preferably includes the labeling with a
labeling compound including a group represented by the following
chemical formula (I) having an aromatic ring:
##STR00009##
where A, R.sup.1, R.sup.2, and R.sup.3 are as described above. The
labeling compound having the group represented by the foregoing
chemical formula (I) above having an aromatic ring preferably is a
succinimidyl ester compound in which the group represented by the
chemical formula (I) above is bonded with succinimide via ester
bond, more preferably, a succinimidyl ester compound represented by
the chemical formula (III) mentioned above, and further more
preferably, a succinimidyl ester compound represented by the
chemical formula (IV) mentioned above.
[0138] In the method for producing a molecular probe for imaging of
pancreatic islets according to the present invention, the synthesis
of a labeling compound having a group represented by the chemical
formula (I) above having an aromatic ring may be carried out by an
automatic synthesizing device. Alternatively, both of the following
may be carried out by a single automatic synthesizing device: the
synthesis of the labeling compound having the group represented by
the foregoing chemical formula (I) having an aromatic ring; and the
labeling and deprotecting of a molecular probe precursor for
imaging in which the foregoing labeling compound is used.
[0139] Examples of the above-described molecular probe precursor
include a precursor of a molecular probe for imaging that can be a
precursor for the molecular probe for imaging of the present
invention, wherein the precursor consists of any one of the
following polypeptides: a polypeptide obtained by deletion,
insertion, or substitution of one to several amino acids with
respect to a polypeptide represented by any one of the following
formulae (13) to (24), the polypeptide being capable of binding to
pancreatic islets after being labeled and deprotected; and a
polypeptide having a homology of 80% or higher with any one of the
amino acid sequences of polypeptides represented by the following
formulae (13) to (24), the polypeptide being capable of binding to
pancreatic islets after being labeled and deprotected.
[0140] Therefore, still another aspect of the present invention
provides a molecular probe precursor for imaging of pancreatic
islets, consisting of any one of the following polypeptides: a
polypeptide represented by any one of the formulae (13) to (24)
above; a polypeptide obtained by deletion, insertion, or
substitution of one to several amino acids with respect to a
polypeptide represented by any one of the formulae (13) to (24)
above, the polypeptide being capable of binding to pancreatic
islets after being labeled and deprotected; and a polypeptide
having a homology of 80% or higher with any one of the amino acid
sequences of polypeptides represented by the formulae (13) to (24)
above, the polypeptide being capable of binding to pancreatic
islets after being labeled and deprotected. With use of the
molecular probe precursor for imaging according to the present
invention, including the polypeptide of any one of the formulae
(13) to (24) above, an effect of easily providing the molecular
probe for imaging according to the present invention can be
achieved.
[0141] [Another Aspect of the Kit of the Present Invention]
[0142] Still another aspect of the present invention relates to a
kit including the aforementioned molecular probe precursor for
imaging. Exemplary embodiments of the kit including the molecular
probe precursor for imaging according to the present invention
include a kit for preparing the molecular probe for imaging
according to the present invention, a kit for performing the method
for imaging of the present invention, a kit for performing the
method for determining an amount of pancreatic islets according to
the present invention, and a kit for prevention, treatment, or
diagnosis of diabetes according to the present invention.
Preferably, in each of these embodiments, the kit including the
molecular probe precursor for imaging according to the present
invention includes an instruction manual suitable for each
embodiment.
[0143] The kit including the molecular probe precursor for imaging
may include, for example, a labeling compound used in the labeling
of the precursor of the molecular probe for imaging, the labeling
compound having the group represented by the aforementioned
chemical formula (I) having an aromatic ring. The labeling compound
having the group represented by the chemical formula (I) having an
aromatic ring preferably is a succinimidyl ester compound in which
the group represented by the foregoing chemical formula (I) is
bonded with succinimide via ester bond, more preferably, a
succinimidyl ester compound represented by the aforementioned
chemical formula (III), and further more preferably, a succinimidyl
ester compound represented by the aforementioned chemical formula
(IV). The kit of the present embodiment more preferably includes,
in particular, [.sup.123I]N-succinimidyl 3-iodobenzoate,
[.sup.124I]N-succinimidyl 3-iodobenzoate, or
[.sup.131I]N-succinimidyl 3-iodobenzoate as the labeling compound.
The kit of the present embodiment further may include, for example,
an instruction manual that describes the method for labeling the
precursor of the molecular probe for imaging of the present
invention in which the above-described labeling compound is
used.
[0144] The kit including the molecular probe precursor for imaging
preferably further includes a starting material for the
above-described described labeling compound. Examples of the
starting material include 2,5-dioxopyrrolidin-1-yl
3-(tributylstannyl)benzoate.
[0145] The kit including the molecular probe precursor for imaging
further may include, for example, a reagent to be used for
deprotecting the molecular probe precursor for imaging and/or a
reagent to be used for the labeling.
[0146] The kit including the molecular probe precursor for imaging
further may include, for example, an automatic synthesizing device
for synthesizing the labeling compound, and an instruction manual
that describes a method for synthesizing the labeling compound
having a group represented by the aforementioned chemical formula
(I) having an aromatic ring, using the foregoing automatic
synthesizing device for synthesizing the labeling compound. The
automatic synthesizing device may be capable of synthesizing the
labeling compound, and further, for example, capable of labeling
and deprotecting the precursor of the molecular probe for imaging
in which the synthesized labeling compound is used. The kit further
may include, for example, a reagent containing a radioactive iodine
to be used in synthesizing the labeling compound. Examples of the
reagent containing a radioactive iodine include reagents containing
radioactive isotopes such as .sup.123I, .sup.124I, .sup.125I, and
.sup.131I.
[0147] Still another aspect of the present invention relates to a
kit that includes an automatic peptide synthesizing device for
synthesizing the molecular probe precursor for imaging, and the
labeling compound having the group represented by the
aforementioned chemical formula (I) having an aromatic ring, and/or
an automatic synthesizing device for synthesizing the labeling
compound. The automatic synthesizing device may be capable of
synthesizing the labeling compound, and further, for example,
capable of labeling and deprotecting the molecular probe precursor
for imaging in which the synthesized labeling compound is used. The
kit may include an instruction manual that describes a method for
synthesizing the molecular probe precursor for imaging. The
instruction manual further may describe, for example, a method for
synthesizing the labeling compound having a group represented by
the aforementioned chemical formula (I) having an aromatic ring, a
labeling method using the same, and a deprotecting method using the
same. The kit further may include a reagent containing radioactive
iodine to be used in synthesis of a labeling compound.
[0148] Still another aspect of the present invention relates to a
kit that includes the following: an automatic synthesizing device
that performs the synthesis of the molecular probe precursor for
imaging, the synthesis of the aforementioned labeling compound, and
the labeling and deprotecting of the aforementioned molecular probe
precursor for imaging in which the aforementioned labeling compound
is used; and an instruction manual that describes a method for
producing a molecular probe for imaging of the present invention
with use of the foregoing automatic synthesizing device. The
instruction manual preferably describes, for example, a method for
synthesizing the molecular probe precursor, a method for
synthesizing the aforementioned labeling compound, and a method for
labeling and deprotecting the molecular probe precursor for imaging
in which the aforementioned labeling compound is used. The kit
further may include a reagent containing radioactive iodine to be
used in synthesis of the labeling compound.
[0149] [Method for Labeling a Peptide According to the Present
Invention]
[0150] Still another aspect of the present invention relates to a
method for labeling a peptide. The method for labeling a peptide
according to the present invention is a method for radioactively
labeling a peptide having a plurality of amino acids having
radioactively-labelable functional groups on side chains, and the
method includes: synthesizing a peptide using protected amino acids
in which an .alpha.-amino group at an N-terminus and a functional
group of a side chain are protected by protecting groups;
deprotecting a functional group by removing a protecting group
therefrom, the functional group to be deprotected being a
functional group that is not to be radioactively labeled, among the
radioactively labelable functional groups of the side chains of the
amino acids of the synthesized peptide; protecting, again, the
deprotected functional group of the side chain of the amino acid by
a protecting group different from that removed upon the
deprotecting; deprotecting, by removing protecting groups, the
other functional groups than the functional group of the side chain
of the amino acid that is again protected, so as to obtain a
peptide to be radioactively labeled; radioactively labeling the
obtained peptide with a labeling compound; and deprotecting the
radioactively-labeled peptide by removing protecting groups.
[0151] The method for labeling a peptide according to the present
invention, as another aspect thereof, relates to a method for
labeling a peptide, the method including the steps of: peptide
synthesis; substitution of protecting group; deprotection by
removing protecting groups; and radioactive labeling.
[0152] The step of peptide synthesis include synthesizing a peptide
using protected amino acids, and the protected amino acids are
selected from the group consisting of a protected amino acid in
which an .alpha.-amino group at an N-terminus is protected by a
protecting group X; a protected amino acid in which an
.alpha.-amino group at an N-terminus is protected by a protecting
group X and a functional group a of a side chain is protected by a
protecting group Y1; a protected amino acid in which an
.alpha.-amino group at an N-terminus is protected by a protecting
group X and a functional group b of a side chain is protected by a
protecting group Y2; and a protected amino acid in which an
.alpha.-amino group at an N-terminus is protected by a protecting
group X and a functional group c of a side chain is protected by a
protecting group Y3. The functional group a is a functional group
of a side chain of an amino acid to be radioactively labeled, the
functional group is a radioactively-labelable functional group of a
side chain of an amino acid that is not to be radioactively
labeled, and the functional group c is a functional group of a side
chain of an amino acid other than the functional group a and the
functional group b.
[0153] The step of substitution of a protecting group includes
deprotecting the functional group b by removing the protecting
group Y2, and thereafter protecting the functional group b by a
protecting group Z different from the protecting group Y2. The step
preferably includes deprotecting the functional group b by removing
the protecting group Y2 without deprotecting the functional groups
a and c, and thereafter protecting the functional group b by a
protecting group Z different from the protecting group Y2.
[0154] The step of deprotection by removing protecting groups
include deprotecting the functional groups a and c by removing the
protecting groups Y1 and Y3, respectively. The step preferably
includes deprotecting the functional groups a and c by removing the
protecting groups Y1 and Y3, respectively, without deprotecting the
functional group b.
[0155] The step of radioactive labeling includes radioactively
labeling the functional group a of the peptide after the step of
deprotection, using a radioactive labeling compound, and
deprotecting the functional group b and the .alpha.-amino group at
the N-terminus by removing the protecting group Z and the
protecting group X, respectively.
[0156] In the labeling method of the present invention, the
radioactive labeling of a peptide with a labeling compound is
conducted to the peptide in a state in which a
radioactively-labelable functional group (functional group b) of a
side chain of an amino acid other than a functional group to be
radioactively labeled (functional group a) is protected by a
protecting group (protecting group Z). More specifically in the
labeling method of the present invention, the radioactive labeling
of a peptide with a labeling compound is conducted to a peptide in
a state in which the functional group b is protected again by the
protecting group Z after the protecting group Y2 is removed, while
the functional group a to be radioactively labeled is deprotected.
Therefore, with the labeling method of the present invention, it is
possible to selectively label an intended functional group
(functional group a) alone. With the labeling method of the present
invention, it is possible to improve the labeling efficiency, and
to improve the yield of a desired peptide that is radioactively
labeled.
[0157] [Peptide Synthesis]
[0158] The peptide synthesis is conducted by a peptide synthesizing
method using protected amino acids in which an .alpha.-amino group
at an N-terminus and/or a functional group of a side chain
(functional group a, b, or c) are protected by protecting
groups.
[0159] The peptide synthesis can be carried out by for example, a
known organic-chemical peptide synthesis method. For example, the
peptide synthesis can be carried out according to the descriptions
in "Seikagaku Jikken Koza" ("Biochemical Experiment Seminar")
edited by the Japanese Biochemical Society Vol. 1, "Protein IV",
pages 207 to 495 (published by Tokyo Kagaku Dojin, 1977), and "Shin
Seikagaku Jikken Koza" ("New Biochemical Experiment Seminar")
edited by the Japanese Biochemical Society, Vol. 1, "Protein VI",
pages 3 to 74 (published by Tokyo Kagaku Dojin, 1992), etc.
[0160] Examples of the organic-chemical peptide synthesis method
include the peptide solid-phase synthesis method, and the peptide
liquid-phase synthesis method, among which the peptide solid-phase
synthesis method is preferred. In the present specification, the
"peptide solid-phase synthesis method" refers to a method in which
a C-terminus of an amino acid or a peptide is fixed to a
solid-phase carrier via a linker, and amino acids are extended one
by one toward an N-terminus. Examples of the peptide solid-phase
synthesis method include the Fmoc method and the Boc method, among
which the Fmoc method is preferred. In the present specification,
the "Fmoc method" refers to a method wherein amino acids in which
the .alpha.-amino group at the N-terminus is protected by Fmoc
(9-fluorenylmethyloxycarbonyl group) are used, and they are
condensed, so as to synthesize a peptide. More specifically an
amino acid corresponding to a C-terminus of a peptide to be
synthesized, or a peptide including an amino acid corresponding to
the C-terminus of a peptide to be synthesized, is bonded to a
solid-phase carrier such as a resin, the deprotection of an
.alpha.-amino group at a N-terminus by removing the Fmoc group as a
protecting group for an .alpha.-amino group at a N-terminus and the
washing, and the condensation of the protected amino acids and the
washing, are carried out repeatedly, whereby a peptide chain is
extended. In the end, a final deprotection reaction is caused,
whereby an intended peptide can be synthesized. In the present
specification, the "Boc method" refers to a method wherein amino
acids in which an .alpha.-amino group at an N-terminus is protected
by Boc (tert-butoxycarbonyl group) are used, and they are
condensed, so as to synthesize a peptide. The peptide synthesis may
be conducted with use of an automatic peptide synthesizing device.
Examples of the automatic peptide synthesizing device include the
A443A type (produced by Applied Biosystems), and PSSM8 (produced by
Shimadzu Corporation).
[0161] Examples of the protected amino acid include a protected
amino acid in which an .alpha.-amino group at an N-terminus is
protected by a protecting group X; a protected amino acid in which
an .alpha.-amino group at an N-terminus is protected by a
protecting group X and a functional group a of a side chain is
protected by a protecting group Y1; a protected amino acid in which
an .alpha.-amino group at an N-terminus is protected by a
protecting group X and a functional group b of a side chain is
protected by a protecting group Y2; and a protected amino acid in
which an .alpha.-amino group at an N-terminus is protected by a
protecting group X and a functional group c of a side chain is
protected by a protecting group Y3.
[0162] The functional group a is a functional group of a side chain
of an amino acid to be radioactively labeled. Examples of the
functional group a include an amino group or a group having an
amino group.
[0163] The functional group b is a functional group not to be
radioactively labeled, among radioactively-labelable functional
groups of side chains of an amino acid. The functional group b is,
for example, a functional group of the same type as that of the
functional group a, and preferably, a functional group of a side
chain of the same amino acid as the amino acid to which the
functional group a belongs to.
[0164] The functional group c is a functional group of a side chain
of an amino acid other than the functional groups a and b.
[0165] The protecting group X is a protecting group for an
.alpha.-amino group at an N-terminus of an amino acid used in the
synthesis of a peptide. Examples of the protecting group X include
the above-described protecting groups, and the protecting group X
may be determined appropriately depending on the peptide
synthesizing method. In the case where the peptide synthesizing
method is the Fmoc method, the protecting group X is usually Fmoc;
in the case where the peptide synthesizing method is the Boc
method, the protecting group X is usually Boc.
[0166] The protecting groups Y1 to Y3 are protecting groups for
functional groups of side chains of amino acids used in the
synthesis of a peptide. The protecting group Y1 is a protecting
group for the functional group a, the protecting group Y2 is a
protecting group for the functional group b, and the protecting
group Y3 is a protecting group for the functional group c. Examples
of the protecting groups Y1 to Y3 include the above-described
protecting groups, and the protecting groups Y1 to Y3 may be
determined appropriately depending on the type of the functional
group and the peptide synthesizing method. Preferably, the
protecting groups Y1 to Y3 different from the protecting group
(protecting group X) for the .alpha.-amino group at an N-terminus
are used.
[0167] The protecting group Y2 is preferably different from the
protecting group Y1 for the functional group a, in view of that the
protecting group Y2 for the functional group b has to be removed
selectively upon deprotection, that is, only the protecting group
Y2 is removed while the protecting group Y1 for the functional
group a is not removed upon deprotection. Further, the protecting
group Y2 is more preferably different from both of the protecting
groups Y1 and Y3 for the functional groups a and c, in view of that
only the protecting group Y2 is removed while the protecting groups
Y1 and Y3 for the functional groups a and c are not removed upon
deprotection. When the functional groups a and b are amino groups,
the protecting group Y2 for the functional group b is a protecting
group of a trityl type from the viewpoint of selective
deprotection. Furthermore preferably the protecting group Y2 for
the functional group b is a protecting group of a trityl type, and
the protecting group Y1 for the functional group a is a protecting
group of a carbamate type. Examples of the protecting group of the
trityl type include Mmt, Trt, Mtt, and Mtr. From the viewpoint, of
more selective deprotection, Mmt and Mtt are preferred. Examples of
the protecting group of the carbamate type include Fmoc, Boc, Cbz,
Alloc, and Troc, among which Boc is preferred particularly.
[0168] [Substitution of Protecting Group]
[0169] The substitution of a protecting group is to substitute the
protecting group for the functional group b, which includes
deprotecting the functional group b by removing the protecting
group Y2, and thereafter protecting the functional group b by a
protecting group Z different from the protecting group Y2.
[0170] The deprotection by removal of the protecting group Y2 may
be determined appropriately depending on the type of the protecting
group Y2. The deprotection by removal of the protecting group Y2 is
preferably carried out by deprotecting the functional group b by
removing the protecting group Y2, without deprotection of the
functional groups a and c, from the viewpoint of selective
labeling. The protecting group Z is a protecting group for
protecting the functional group b after the functional group b is
deprotected by removing the protecting group Y2 therefrom, and it
is different from the protecting group Y2. The protecting group Z
can be selected appropriately from the above-described protecting
groups, and preferably is the same as the protecting group for the
.alpha.-amino group at the N-terminus (protecting group X) in the
synthesis of a peptide. When the peptide synthesis is conducted by
the Fmoc method, the protecting group Z is preferably Fmoc.
[0171] [Deprotection by Removing Protecting Groups]
[0172] The step of deprotection by removing protecting groups
includes deprotecting functional groups by removing protecting
groups, the functional groups being those other than the functional
group b of the side chain of the amino acid that is protected
again, so as to obtain a peptide to be radioactively labeled. More
specifically the step includes deprotecting the functional groups a
and c removing the protecting groups Y1 and Y3, respectively. As a
result, a peptide to be radioactively labeled is obtained. The
peptide has the functional group b protected by the protecting
group Z.
[0173] The deprotection of the functional groups a and c by
removing the protecting groups Y1 and Y3, respectively, is
preferably conduced without deprotection of the functional group b.
The deprotection of the functional groups a and c by removing the
protecting groups Y1 and Y3 may be determined appropriately
depending on the types of the protecting groups. The deprotection
of the functional groups a and c by removing the protecting groups
Y1 and Y3, respectively, may be carried out upon the excision of
the peptide from a solid-phase carrier.
[0174] The step may include a step of washing and/or isolating and
purifying the obtained peptide, which is conducted after
deprotection by removing the protecting groups. The isolation and
purification can be carried out by a known separating operation for
purification of a peptide or a protein. Examples of the separating
operation include the ion-exchange chromatography, the hydrophobic
chromatography, the reversed phase chromatography and the high
precision liquid chromatography (HPLC), and some of these may be
used in combination as required. The purified peptide may be
isolated by, for example, concentrating and/or freeze-drying the
same, depending on the intended final form.
[0175] [Labeling of Peptide]
[0176] The labeling is carried out using a peptide in which the
functional group b is protected by the protecting group Z and the
functional group a is deprotected by removing the protecting group
Y1 therefrom. The peptide to be labeled is preferably a peptide in
which the functional group b is protected by the protecting group Z
and the functional groups a and c are deprotected by removing the
protecting groups Y1 and Y3, respectively from the viewpoint of the
selective labeling; and the peptide is more preferably a peptide in
which the functional group b is protected by the protecting group
Z, the .alpha.-amino group at the N-terminus is protected by the
protecting group X, and the functional groups a and c are
deprotected by removing the protecting groups Y1 and Y3,
respectively. This makes it possible to conduct the selective
labeling. As a labeling compound, a known compound used in
radioactive labeling can be used, and the examples of the same
include those described above.
[0177] An exemplary peptide to be synthesized is a peptide having
two or more amino acids each of which has an amino group on a side
chain. Examples of the amino acid having an amino group on a side
chain include lysine. Though the length of the peptide to be
synthesized is not limited particularly, the length is, for
example, 5 or more amino acid residues, preferably 10 to 150 amino
acid residues, and more preferably 20 to 80 amino acid residues.
The peptide to be synthesized may have, for example, one amino acid
having the functional group a on a side chain, or two, three, or
more of such amino acids. The peptide to be synthesized is
preferably a polypeptide expressed by any one of the following
formulae (25) to (28), form the viewpoint that a GLP-1R imaging
molecular probe, or more preferably a molecular probe of the
present invention is to be obtained:
TABLE-US-00006 (SEQ ID NO. 25) DLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (25)
(SEQ ID NO. 26) LSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (26) (SEQ ID NO. 27)
SKQMEEEAVRLFIEWLKNGGESSGAPPPS (27) (SEQ ID NO. 28)
KQMEEEAVRLFIEWIANGGPSSGAPPPS (28)
[0178] Further, the labeling method of the present invention, as
still another aspect thereof, relates to a method for radioactively
labeling a peptide in which a functional group to be labeled is an
.alpha.-amino group at an N-terminus. The radioactive labeling
method of the present aspect can be conducted in the same manner as
described above except the following:
[0179] the step of peptide synthesis is conducted using the
following: [0180] as the amino acid positioned at the N-terminus, a
protected amino acid in which an .alpha.-amino group at the
N-terminus is protected by a protecting group Y4; a protected amino
acid in which an .alpha.-amino group at the N-terminus is protected
by a protecting group Y4 and a functional group b on a side chain
is protected by a protecting group Y2; or a protected amino acid in
which an .alpha.-amino group at the N-terminus is protected by a
protecting group Y4 and a functional group c on a side chain is
protected by a protecting group Y3; and [0181] as the other amino
acid, a protected amino acid selected from the group consisting of
a protected amino acid in which an .alpha.-amino group at the
N-terminus is protected by a protecting group X; a protected amino
acid in which an .alpha.-amino group at the N-terminus is protected
by a protecting group X and a functional group b on a side chain is
protected by a protecting group Y2; and a protected amino acid in
which an .alpha.-amino group at the N-terminus is protected by a
protecting group X and a functional group c on a side chain is
protected by a protecting group Y3;
[0182] the step of deprotection by removing a protecting group
includes deprotecting the .alpha.-amino group at the N-terminus and
the functional group c by removing the protecting groups Y4 and Y3,
respectively; and
[0183] the step of radioactive labeling is conducted by
radioactively labeling the .alpha.-amino group at the N-terminus
with a radioactive labeling compound, and deprotecting the
functional group b by removing the protecting group Z. The
protecting group Y4 is a protecting group for the .alpha.-amino
group of the amino acid positioned at the N-terminus of the
peptide, and is preferably different from the protecting groups X
and Y2. The step of deprotection by removing the protecting groups
preferably include deprotecting them-amino group at the N-terminus
and the functional group c by removing the protecting groups Y4 and
Y3, respectively, without deprotection of the functional group
b.
[0184] [Peptide Producing Method of the Present Invention]
[0185] The present invention, as still another aspect thereof,
relates to a method for producing a radioactively labeled peptide,
wherein the peptide has a plurality of amino acids having
radioactively labelable functional groups on side chains, the
method including: synthesizing a peptide to be radioactively
labeled, using protected amino acids in each of which an
.alpha.-amino group at an N-terminus and a functional group of a
side chain are protected by protecting groups; deprotecting
functional groups by removing protecting groups therefrom, the
functional groups to be deprotected being functional groups that
are not to be radioactively labeled, among the radioactively
labelable functional groups of the side chains of the amino acids
of the synthesized peptide; protecting, again, the deprotected
functional groups of the side chains of the amino acids by
protecting groups different from those removed upon the
deprotecting; deprotecting, by removing protecting groups, the
other functional groups than the functional groups of the side
chains of the amino acids that are again protected, so as to obtain
a peptide to be radioactively labeled; radioactively labeling the
obtained peptide with a labeling compound; and deprotecting the
radioactively-labeled peptide by removing protecting groups. The
method for producing a radioactively labeled peptide according to
the present invention, as another aspect thereof, includes
synthesis of a peptide, the deprotection by removing protecting
groups, and the radioactive labeling, using the aforementioned
labeling method of the present invention.
[0186] According to the method for producing a peptide according to
the present invention, for example, only an intended functional
group can be labeled selectively. Therefore, it is possible to
produce a desired peptide that is radioactively labeled, at a high
yield. Further, according to the method for producing a peptide
according to the present invention, for example, the molecular
probe for imaging according to the present invention can be
produced efficiently and preferably a high-purity molecular probe
for imaging according to the present invention can be produced.
[0187] In the peptide producing method of the present invention,
the functional groups and the protecting groups are the same as
those in the labeling method of the present invention, and the
peptide synthesis, the deprotection, the radioactive labeling, and
the like can be conducted in the same manner as those of the
labeling method of the present invention.
[0188] The labeling method of the present invention and the peptide
producing method of the present invention are explained below, with
reference to an exemplary case where the peptide to be labeled was
the polypeptide of the formula (25) shown above and the peptide
synthesis was carried out by the peptide solid-phase synthesis
method. In the peptide, the functional groups a and b are amino
groups, the amino acid having, on a side chain, a functional group
(functional group a) to be labeled is a lysine at position 4; the
amino acid having, on a side chain, a functional group (functional
group b) not to be labeled is a lysine at position 19; and an amino
acid having the functional group c is asparaginic acid, serine,
glycin, glutamine, arginine, asparagine, or tryptophan. The
following describes merely one example, and needless to say the
present invention is not limited to this example.
[0189] (1) First, the peptide synthesis is carried out using
protected amino acids in which the .alpha.-amino group at the
N-terminus and functional groups on side chains are protected by
protecting groups.
[0190] The peptide synthesis can be conducted by, for example, the
Fmoc method. Specifically, the synthesis can be carried out by
fixing a carboxyl group of serine as an amino acid at a C-terminus
via a linker to a resin, and binding amino acids one by one from
the C-terminus toward the N-terminus.
[0191] As the protected amino acids used in the peptide synthesis,
a Fmoc-amino acid derivative used in a common Fmoc-peptide
synthesis method can be used. Specifically, as an amino acid having
a functional group On a side chain (Asp, Ser, Lys, Gln, Arg, Asn,
Trp), an amino acid in which the functional group is protected by a
protecting group depending on the type of the functional group and
the .alpha.-amino group at the N-terminus is protected by Fmoc can
be used, and as the other amino acid, an amino acid in which the
.alpha.-amino group at the N-terminus is protected by Fmoc can be
used. As the protecting groups (protecting groups Y1 and Y3) for
the functional groups a and c it is preferable that protecting
groups that can be removed for deprotection under the same
conditions are selected.
[0192] From the viewpoint of selective deprotection, preferably
used as the lysine at position 19 that is not to be radioactively
labeled is a lysine in which an amino group (functional group b) on
a side chain is protected by a protecting group Y2 different from
the protecting group Y1 for the amino group (functional group a) on
a side chain of the lysine at position 4 to be radioactively
labeled. For example, a lysine in which the amino group on the side
chain is protected by a carbamate-type protecting group other than
Fmoc may be used as the lysine at position 4, and a lysine in which
the amino group on the side chain is protected by a trityl-type
protecting group may be used as the lysine at position 19.
[0193] (2) Next in the synthesized peptide, the amino group
(functional group b) at the side chain of the lysine at position 19
is deprotected by removing the protecting group Y2, and protected
by the protecting group Z.
[0194] The deprotection is preferably carried out by deprotecting
the amino group (functional group b) on the side chain of the
lysine at position 19 by removing the protecting group Y2, without
deprotecting the amino group (functional group a) on the side chain
of the lysine at position 4 and the functional group c,
respectively. The deprotection can be carried out appropriately
depending on the type of the protecting group Y2 for the functional
group b. In the case where the protecting group is a trityl-type
protecting group, for example, it can be removed under weak acid
conditions. A reagent making the weak acid conditions is, for
example, a reagent containing trifluoroacetic acid.
[0195] The protecting group Z is a protecting group for the amino
group (functional group b) on the side chain of the lysine at
position 19 deprotected, and it is different from the protecting
group before the deprotection (protecting group Y2). Though the
protecting group Z is not limited particularly as long as it is
different from the protecting group before the deprotection
(protecting group Y2), it is preferably Fmoc as the protecting
group (protecting group X) for the .alpha.-amino group at the
N-terminus. Fmoc can be introduced to the functional group b by,
for example, causing a reaction between the functional group b and
N-(fluorenylmethyloxycarbonyloxy)succinimide(FmocOSu) under the
presence of amine.
[0196] (3) Subsequently, the functional groups other than the
.alpha.-amino group at the N-terminus and the amino group
(functional group b) on the side chain of the lysine at position 19
are deprotected, or more specifically, the amino group (functional
group a) on the side chain of the lysine at position 4 and the
functional group c are deprotected. This causes the following
peptide to be obtained: a peptide in which the .alpha.-amino group
at the N-terminus and the amino group (functional group b) on the
side chain of the lysine at position 19 are protected by protecting
groups, while the amino group (functional group a) on the side
chain of the lysine at position 4 is deprotected. This peptide
corresponds to the molecular probe precursor of the present
invention described above.
[0197] The deprotection can be conducted in accordance with a known
method depending on the type of the protecting group removed for
deprotection. This deprotection is carried out upon the excision of
the peptide from a solid-phase carrier, and for example, the
above-described deprotection by removing the protecting group may
be carried out under the condition for the excision of the
peptide.
[0198] (4) The obtained peptide is radioactively labeled with use
of a labeling compound.
[0199] In the peptide, the amino group (functional group a) on the
side chain of the lysine at position 4 to be radioactively labeled
is deprotected, and the amino group (functional group b) on the
side chain of the lysine at position 19 not to be radioactively
labeled is protected by the protecting group Z; and from the
viewpoint of reducing the deprotecting operation after the
radioactive labeling, preferably the functional groups c and a are
deprotected by removing the protecting groups Y3 and Y1, while the
functional group b is protected by the protecting group Z. Using
such a peptide, it is possible to selectively radioactively label
only the intended site (the amino group on the side chain of the
lysine at position 4).
[0200] The radioactive labeling can be conducted in accordance with
a known method depending on the type of the peptide to be
radioactively labeled. Though the labeling compound is not limited
particularly, it may be, for example, the labeling compound having
a group represented by the chemical formula (I), or a chelate
compound bondable to a metal radioactive isotope (metal nuclide).
Examples of the metal nuclide include .sup.64Cu, .sup.67Ga,
.sup.68Ga, .sup.82Rb, .sup.99mTc, .sup.111In, and .sup.186Re.
Examples of the chelate compound include
diethylenetriaminepentaacetic acid (DTPA), 6-hydrazinoeulysin-3
carboxylic acid (HYNIC), tetraazacyclododecanetetraacetic acid
(DOTA), dithisosemicarbazone (DTS), diaminedithiol (DADT),
mercaptoacetylglycylglycylglycine (MAG3), monoamidemonoaminedithiol
(MAMA), diamidedithiol (DADS), and propylene diamine dioxime
(PnAO). From the viewpoint of production of the molecular probe for
imaging according to the present invention, the labeling compound
is preferably the labeling compound having a group represented by
the chemical formula (I), more preferably, the succinimidyl ester
compound represented by the chemical formula (III) shown above, and
further more preferably a succinimidyl ester compound represented
by the chemical formula (IV) shown above.
[0201] (5) Subsequently, the other protecting groups of the thus
radioactively labeled peptide are removed for deprotection. As a
result, the peptide in which the intended functional group a (amino
group on the side chain of the lysine at position 4) is
radioactively labeled can be produced.
[0202] In this step, the other protecting groups bonding to the
peptide, that is, the protecting group of the amino group at the
N-terminus, and the amino group (functional group b) on the side
chain of the lysine at position 19 not to be radioactively labeled
are removed for deprotection. The deprotection can be conducted by
a known method depending on the type of the protecting group. In
the case where the protecting group is Fmoc, the deprotection can
be carried out, for example, under the piperidine conditions. Thus,
the molecular probe of the present invention can be produced.
[0203] From the viewpoint for the production of a high-purity
radioactively-labeled peptide, a purification step may be conducted
additionally. The purification step can be carried out for example,
between the step (3) of deprotection and the step (4) of
radioactive labeling.
[0204] Further, a step of modifying the .alpha.-amino group at the
N-terminus with a modifying group having no electric charge, or a
step of amidating the carboxyl group at the C-terminus may be
included additionally.
[0205] Hereinafter, the present invention will be described further
by way of Examples and Comparative Examples. It should be noted
that the present invention is, when interpreted, not limited to the
following Examples.
[0206] In the description of the present specification, the
following abbreviations are used.
OBu: butyl ester group Boc: butoxycarbonyl group Trt: trityl group
Pdf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl group Mmt:
4-methoxytrityl group Fmoc: 9-fluorenylmethyloxycarbonyl group
EXAMPLES
Binding Assay
[0207] Binding assay analysis was conducted using a molecular probe
of the formula (29) below (SEQ ID NO. 29) in which an amino group
on a side chain of a lysine residue at position 4 was labeled with
[.sup.127I] 3-iodobenzoyl group, and a molecular probe of the
formula (30) below (SEQ ID NO. 30) in which an .alpha.-amino group
at an N-terminus was labeled with [.sup.127I] 3-iodobenzoyl
group.
##STR00010##
[0208] The above-mentioned molecular probes of the formulae (29)
and (30) were prepared by the same method as that for Examples 1
and 2 described below except that [.sup.127I]N-succinimidyl
3-iodobenzoate was used in place of [.sup.125I]N-succinimidyl
3-iodobenzoate.
[0209] Pancreatic islets isolated from a mouse was recovered in a
50 ml-tube and after it was subjected to centrifugation (2000 rpm,
2 minutes), it was washed once with 20 mL, of cold PBS. To this, 15
mL of trypsin-EDTA (which was prepared by adding 12 mL of
PBS-containing 0.53 mM EDTA (pH 7.4 (NaOH) to 3 mL of trypsine-EDTA
(0.05%/0.53 mM) was added. This was incubated at 37.degree. C. for
one minute while shaken, then immediately placed on ice.
Subsequently after it was pipetted vigorously 20 times with a 10 mL
pipette dropper without being foamed, the cold PBS was added so
that the final amount would be 30 mL. After centrifugation (3000
rpm, 2 minutes), it was washed twice with 30 mL of cold PBS. The
supernatant was removed, whereby a pancreatic islet cells sample
was obtained. The obtained pancreatic islet cells sample was
reserved at -80.degree. C.
[0210] The pancreatic islet cells sample was suspended in a buffer
(20 mM, HEPES (pH 7.4), 1 mM MgCl.sub.2, 1 mg/ml bacitracin, 1
mg/ml BSA) so as to make 100 .mu.L/tube. Then, 800 .mu.L of the
buffer and 10 .mu.L of a solution including the molecular probe of
the above formula (29) or the molecular probe of the above formula
(30) (final concentration of molecular probe: 0, 1.times.10.sup.-6
to 1.times.10.sup.-12M), and 10 .mu.L of a solution including
[.sup.125I] Bolton-Hunter labeled Exendin (9-39) (prepared by
adding 90 .mu.L of a buffer to 10 .mu.L of [.sup.125I]
Bolton-Hunter labeled Exendin (9-39) (product code: NEX335, 1.85
MBq/mL=50 .mu.Ci/mL, 22.73 pmol/mL=76.57 ng/mL, manufactured by
Perkin Elmer) were added thereto, which was incubated for 60
minutes at room temperature. Here, the final concentration of the
.sup.[125I] Bonton-Hunter labeled Exendin (9-39) was set to 0.05
.mu.Ci/tube. Next, after B/F separation by aspirating with use of
an aspirator to which a moistened glass fiber filter (Whatman GF/C
filter) was attached, the filter was washed three times with 5 ml
of an ice-cooled PBS. The filter was set in the tube, and the
radioactivity measurement was carried out with a gamma counter.
[0211] Both of the molecular probe of the above formula (29) in
which an amino group on a side chain of a lysine residue at
position 4 was labeled with [.sup.127I]3-iodobenzoyl group, and the
molecular probe of the above formula (30) in which an .alpha.-amino
group at an N-terminus was labeled with [.sup.127I] 3-iodobenzoyl
group inhibited in a concentration-dependent manner the binding
between the GLP-1R and the [.sup.125I] Boton-Hunter labeled Exendin
(9-39). The IC.sub.50 of the molecular probe of the above formula
(29) was 1.6.times.10.sup.-9 M, and the IC.sub.50 of the molecular
probe of the above formula (30) was 1.4.times.10.sup.-9M. Thus,
both of the molecular probes of the above formulae (29) and (30)
exhibited a high affinity with respect to the GLP-1 receptor of
pancreatic islets. Moreover, both of the IC.sub.50 of the molecular
probe of the above formula (29) and the IC.sub.50 of the molecular
probe of the above formula (30) were approximate to that of Exendin
(9-39) (IC.sub.50:1.4.times.10.sup.-9M), and thus, with respect to
the GLP-1 receptor of pancreatic islets, both of the molecular
probes of the above formulae (29) and (30) are considered to have
an affinity comparable to that of Exendin (9-39) as a GLP-1
receptor antagonist. Therefore, it was confirmed that both of the
molecular probe of the above formula (29) in which an amino group
on a side chain of a lysine residue at position 4 was labeled with
[.sup.127I]3-iodobenzoyl group, and the molecular probe of the
above formula (30) in which an .alpha.-amino group at an N-terminus
was labeled with [127I] 3-iodobenzoyl group had sufficient ability
of binding to a GLP-1 receptor, particularly to a GLP-1 receptor of
pancreatic islets.
Example 1
[0212] Using the molecular probe of the formula (31) below (SEQ ID
NO. 31), having a configuration in which an amino group of a side
chain of a lysine residue at position 4 was labeled with
[.sup.125I] 3-iodobenzoyl group (hereinafter referred to also as
"[.sup.125I]IB label") and a carboxyl group at a C-terminus was
amidated in the sequence of SEQ ID NO. 1, biodistribution of the
same in a mouse was determined. First, the molecular probe of the
formula (31) below was prepared in the following manner.
##STR00011##
[0213] [Preparation of Molecular Probe]
[0214] Polypeptide synthesis was performed by using an automatic
peptide synthesizer (Model 433A) manufactured by Applied
Biosystems, in accordance with the attached software. For the amino
acids having functional groups at the side chains, Asp(OBu),
Ser(OBu), Lys(Boc), Gln(Trt), Glu(OBu), Arg(Pbf), Asn(Trt) and
Trp(Boc) were used respectively. For a lysine at position 19,
Lys(Mmt) was used. Rink Amide MBHA (0.125 mmol, 0.34 mmol/g) was
employed as the starting resin, the amino acids were extended
serially according to the sequence, whereby the polypeptide having
the sequence of the following formula (32) was obtained. In the
following formula (32), the protecting groups of the side chains
other than Lys(Mmt) were not recited.
TABLE-US-00007 (SEQ ID NO. 32)
Fmoc-DLSKQMEEEAVRLFIEWLK(Mmt)NGGPSSGAPPPS-protective peptide resin
. . . (32)
[0215] By a typical process using 1.5% TFA--5% TIS--93.55%
CH.sub.2Cl.sub.2, the protecting group (Mmt group) of the side
chain at a lysine residue at position 19 was removed from the
polypeptide of the above formula (32), and the amino group of the
side chain at the free lysine residue at position 19 was
Fmoc-bonded. Subsequently, removal of all of the protecting groups
other than the Fmoc group of the lysine residue at position 19, and
excision of peptide from the resin, were carried out by a typical
process using 92.5% TFA--2.5% TIS--2.5% H.sub.2O--2.5% ethanediol.
After completion of the reaction, the carrier resin was removed by
filtration, and dry ether was added thereto for precipitating the
crude product, which was then filtered. The thus obtained crude
product was purified in a linear gradient system of
CH.sub.3CN--H.sub.2O containing 0.1% TFA, using a liquid
chromatograph LC8A manufactured by Shimadzu Corp. (ODS column 3
cm.times.25 cm). Then, intended fractions were collected by using a
fraction collector, and thus the molecular probe precursor of the
following formula (33) was obtained as a lyophilized white
powder.
TABLE-US-00008 (SEQ ID NO. 33)
Fmoc-DLSKQMEEEAVRLFIEWLK(Fmoc)NGGPSSGAPPPS-NH.sub.2 (33)
[0216] The thus obtained molecular probe precursor (950 .mu.g) of
the above-described formula (33) was dissolved in borate buffer (pH
7.8). [.sup.125I] N-succinimidyl 3-iodobenzoate (SIB) was added
thereto so that pH of the reaction solution was adjusted to 8.5 to
9.0. Thus, the precursor was labeled. Thereafter, piperidine was
added thereto so as to cause a deprotecting reaction, whereby the
intended molecular probe of the above-described formula (31)
(molecular probe having a configuration in which the lysine residue
at position 4 was labeled in the sequence of SEQ ID NO. 1) was
obtained. It should be noted that the .alpha.-amino group at the
N-terminus is not modified in the molecular probe of the foregoing
formula (31).
[0217] [Biodistribution]
[0218] The molecular probe thus prepared (0.57 .mu.Ci) of the
aforementioned formula (31) was administered to unanesthetized
6-week old ddY mice (male, weight: 30 g) by intravenous injection
(through the tail vein). At points of 5 minutes, 15 minutes, 30
minutes, 60 minutes, and 120 minutes after the administration,
organs were dissected out of the mice, respectively (n=5). The
weight and the radioactivity of each organ were determined, and an
accumulation amount (% dose/g) of the molecular probe was
calculated from the radioactivity per unit weight. Exemplary
results are shown in Table 1 below, FIGS. 1A and 1B. FIG. 1A is a
graph showing how the accumulation of the molecular probe in each
organ varied with time, and FIG. 1B is a graph of an enlarged part
of FIG. 1A.
TABLE-US-00009 TABLE 1 Time after administration 5 min 15 min 30
min 60 min 120 min Pancreas 17.53 25.43 45.37 27.49 23.02 (3.43)
(5.09) (5.87) (12.49) (4.74) Blood 9.62 5.73 4.03 1.84 1.54 (0.99)
(0.43) (0.57) (0.46) (0.42) Heart 4.55 2.91 2.46 1.03 0.74 (0.29)
(0.42) (0.19) (0.16) (0.08) Lung 39.67 51.64 67.29 44.74 41.21
(4.48) (12.04) (13.73) (15.20) (9.63) Stomach 2.69 4.24 9.61 4.08
11.30 (0.77) (0.63) (10.24) (0.95) (6.28) Intestine 2.24 2.95 4.36
3.08 13.16 (0.30) (0.47) (2.23) (0.76) (16.82) Liver 15.79 8.89
6.66 3.05 2.56 (0.93) (0.98) (0.72) (0.69) (0.33) Spleen 3.73 2.86
2.28 1.11 0.71 (0.62) (0.48) (0.54) (0.33) (0.16) Kidney 18.24
19.22 18.36 9.72 8.25 (1.76) (1.73) (4.53) (1.35) (1.41) Thyroid
gland 4.06 2.91 2.81 1.63 2.32 (0.85) (0.54) (0.52) (0.42) (0.71)
Each numerical value indicates an average (SD) of 5 mice.
[0219] As shown in Table 1 above, FIGS. 1A and 1B, the accumulation
of the molecular probe of the above-described formula (31) into the
pancreas was 17.5% dose/g at a point of 5 minutes after the
administration, 25.4% dose/g at a point of 15 minutes after the
administration, and 45.4% dose/g at a point of 30 minutes after the
administration. During a time period from the point of 15 minutes
to the point of 120 minutes after the administration, the molecular
probe of the foregoing formula (31) accumulated most in the
pancreas among the organs other than the lungs, and the
accumulation of the molecular probe in the pancreas was maintained
at a level exceeding 20% dose/g. During a time period from the
point of 15 minutes to the point of 60 minutes after the
administration, the accumulation amount of the molecular probe in
the pancreas was not less than 4.5 times as much as that in the
stomach, not less than 9 times as much as that in the intestine,
and not less than 2.5 times as much as that in the liver.
Particularly during a time period from the point of 30 minutes to
the point of 120 minutes, the accumulation amount of the molecular
probe in the pancreas was not less than 6.5 times as much as that
in the liver. In other words, it can be concluded that the
molecular probe of the formula (31) accumulated specifically in the
pancreas. Further, no great change was seen in the accumulation in
the thyroid gland, and this suggests that the molecular probe of
the formula (31) above was not subjected to deiodization metabolism
in vivo. Therefore, the molecular probe of the formula (31) above
is considered suitable for the pancreatic .beta.-cell imaging,
particularly noninvasive pancreatic .beta.-cell imaging.
Comparative Example
[0220] For Comparative Example, using a molecular probe of the
following formula (34) (SEQ ID NO. 34), in which an amino group of
a side chain of a lysine residue at position 4 was labeled with
[.sup.125I] 3-(3-iodo-4-hydroxyphenyl)propanoyl group (hereinafter
referred to also as "[.sup.125I]BH label") and a carboxyl group at
a C-terminus was amidated in the sequence of SEQ ID NO. 1,
biodistribution of the same in a mouse was determined. The
preparation of the molecular probe of the formula (34) below was
carried out in the same manner as that in Example 1, except that
the labeling was carried out using the Bolton-Hunter Reagent
(produced by Perkin Elmer Inc.). The determination of
biodistribution was carried out in the same manner as that in
Example 1. Exemplary results of the same are shown in Table 2
below, FIGS. 2A and 2B.
##STR00012##
[0221] Incidentally, it is known that in [.sup.125I]
Bolton-Hunter-labeled Extendin (9-39) produced by Perkin Elmer
Inc., a lysine residue at position 12 (an amino group of a side
chain of a lysine at position 4 in the amino acid sequence of SEQ
ID NO. 1) is labeled, like in the molecular probe of the formula
(34) above (Suleiman Al-Sabah et al., The positive charge at
Lys-288 of the glucagon-like peptide-1 (GLP-1) receptor is
important for binding the N-terminus of peptide agonists, FEBS
Letters 553 (2003) 342-346).
TABLE-US-00010 TABLE 2 Time after administration 2 min 5 min 15 min
30 min 60 min 120 min Pancreas 5.69 9.25 11.99 19.91 23.81 21.54
(2.27) (1.52) (3.60) (2.32) (3.98) (3.00) Blood 3.53 3.57 2.40 2.48
2.05 1.36 (1.63) (0.30) (0.83) (0.28) (0.26) (0.10) Heart 2.28 2.94
1.58 2.27 1.86 1.11 (1.20) (0.56) (0.69) (0.28) (0.32) (0.13) Lung
33.32 50.91 52.34 73.37 91.46 61.35 (17.61) (5.33) (14.78) (16.98)
(29.44) (13.28) Stomach 1.06 1.86 1.86 2.28 3.89 9.30 (0.58) (0.18)
(0.94) (0.58) (0.52) (3.23) Intestine 0.82 1.11 1.13 1.85 3.42 5.50
(0.36) (0.10) (0.44) (0.45) (0.63) (0.54) Liver 22.13 34.45 21.01
23.22 18.02 11.09 (9.19) (3.78) (7.09) (2.80) (2.69) (0.53) Spleen
1.03 1.23 1.08 0.93 0.90 0.60 (0.41) (0.19) (0.58) (0.16) (0.32)
(0.15) Kidney 6.01 11.43 10.69 15.99 13.95 9.57 (1.89) (0.60)
(3.25) (1.88) (2.57) (1.05) Thyroid gland 1.98 2.45 3.27 4.57 7.94
18.29 (0.75) (0.37) (1.19) (1.34) (1.72) (1.32) Each numerical
value indicates an average (SD) of 5 mice.
[0222] As shown in Tables 1 and 2 above. FIGS. 1A and 1B, and 2A
and 2B, the molecular probe of the formula (31) of Example 1
labeled with a [.sup.125I] 3-iodobenzoyl group accumulated much in
the pancreas in every time period, as compared with the molecular
probe of the formula (34) above of Comparative Example labeled with
[.sup.125I]3-(3-iodo-4-hydroxyphenyl)propanoyl group. Particularly,
the accumulation amount of the molecular probe of the formula (31)
above in the pancreas at a point of 30 minutes after the
administration was substantially twice as much as the accumulation
amount of the molecular probe of the formula (34) above of
Comparative Example in the pancreas.
[0223] Further, as shown in Table 2 above, FIGS. 2A and 2B, the
accumulation of the molecular probe of the formula (34) of
Comparative Example in the thyroid gland increased as time passed,
and this suggests that the molecular probe was subjected to
deiodization metabolism in vivo. On the other hand, the molecular
probe of the formula (31) above did not exhibit increased
accumulation in the thyroid gland as shown in Table 1 above, FIGS.
1A and 1B, that is, it was not subjected to deiodization metabolism
in vivo. Therefore, the molecular probe of the formula (31) above
labeled with [.sup.125I] 3-iodobenzoyl group is considered more
suitable for the pancreatic .beta.-cell imaging, particularly
noninvasive pancreatic .beta.-cell imaging, as compared with the
molecular probe of the formula (34) above of Comparative Example
labeled with [.sup.125I]3-(3-iodo-4-hydroxyphenyl)propanoyl
group.
[0224] Based on the accumulation amount obtained by the
biodistribution experiments on the molecular probe in Example 1 and
the molecular probe in Comparative Example, the ratio of
pancreas/liver (`accumulation amount in pancreas`/`accumulation
amount in liver`) for each probe is shown in Table 3 below, and the
ratio of pancreas/kidney (`accumulation amount in
pancreas`/`accumulation amount in kidney`) for each probe is shown
in Table 4 below.
TABLE-US-00011 TABLE 3 Pancreas/Liver Ratio Time after
administration 5 min 15 min 30 min 60 min 120 min Example 1 1.11
2.91 6.84 8.73 9.05 (0.21) (0.71) (0.78) (2.21) (1.83) Com. Example
0.27 0.57 0.86 1.32 1.94 Example 2 0.54 1.09 1.66 2.30 3.52 (0.13)
(0.39) (0.22) (0.54) (0.92)
TABLE-US-00012 TABLE 4 Pancreas/Kidney Ratio Time after
administration 5 min 15 min 30 min 60 min 120 min Example 1 0.97
1.34 2.54 2.75 2.79 (0.21) (0.29) (0.42) (0.90) (0.30) Com. Example
0.81 1.12 1.25 1.71 2.25 Example 2 1.18 1.89 3.15 3.61 4.53 (0.29)
(0.39) (0.65) (0.80) (0.49)
[0225] As shown in the above Tables 3 and 4, the ratio of
pancreas/liver and the ratio of pancreas/kidney for the molecular
probe of Example 1 (the molecular probe of the above formula (31))
were high in comparison with the molecular probe of Comparative
Example. Particularly in a time period from the point of 30 minutes
to the point of 60 minutes after the administration, the ratio of
pancreas/liver of the molecular probe of Example 1 (the molecular
probe of the above formula (31)) exceeded 6 times as much as that
of the molecular probe of Comparative Example. Thus, it was
suggested that clear images of pancreas can be obtained at the time
of imaging with the molecular probe of Example 1 where the ratio of
accumulation amount in the pancreas to the surrounding organs of
the pancreas is high and the accumulation amount in the surrounding
organs of the pancreas is low.
[0226] [Blocking Experiment]
[0227] A blocking experiment was performed by using a molecular
probe prepared in Example 1 (the molecular probe of the formula
(31)). For the mice, 6-week-old ddY mice (male, weight: about 30 g)
were used.
[0228] First, non-labeled exendin(9-39) (cold probe, SEQ ID NO. 37)
was administered (0.1 mL of 0.5 nag/mL solution) preliminarily by
intravenous injection to unanesthetized mice. At a point of 30
minutes after the foregoing preliminary administration, the
prepared molecular probe of the formula (31) (5 .mu.Ci) was
administered by intravenous injection. Then, at a time of 30
minutes after the administration of the molecular probe, the organs
were dissected out, respectively (n=5). The weight and the
radioactivity of each organ were determined, and an accumulation
amount (% dose/g) was calculated from the radioactivity per unit
weight. Exemplary results are shown in FIG. 3.
TABLE-US-00013 (SEQ ID NO. 37)
H.sub.2N-DLSKQMEEKAVRLFIEWLKNGGPSSGAPPPS-NH.sub.2 (37)
[0229] As a control, without preliminary administration of a cold
probe, the prepared molecular probe (5 .mu.Ci) of the formula (31)
was administered to unanesthetized mice by intravenous injection.
Then, at a time of 30 minutes after the administration, the
respective organs were dissected (n=5). The weight and the
radioactivity of each organ were determined, and an accumulation
amount (% dose/g) of the molecular probe was calculated from the
radioactivity per unit weight. Exemplary results are shown in FIG.
3, together with the exemplary results for the case including the
preliminary administration.
[0230] FIG. 3 is a graph showing an accumulation amount (% dose/g)
for the case including the preliminary administration and an
accumulation amount (% does/g) for the control (without preliminary
administration). As shown in FIG. 3, it was observed that, the
binding with a GLP-1 receptor was inhibited by preliminary
administration of a cold probe, whereby about 92.2% of the uptake
of the molecular probe of the formula (31) was inhibited.
Therefore, it can be concluded that the molecular probe of the
formula (31) above bound to the GLP-1 receptor, particularly the
GLP-1 receptor of pancreatic islets.
[0231] [Two-Dimensional Imaging Analysis]
[0232] Two-dimensional imaging analysis was performed using
transgenic mice that have a genetic background of C57BL/6 mice and
express GFP (green fluorescent protein) under regulation of MIP
(mouse insulin I gene promoter) (hereinafter these mice are
referred to as "MIP-GFP mice"). First, the molecular probe thus
prepared of the aforementioned formula (31) (1 .mu.Ci) was
administered to unanesthetized MIP-GFP mice (male, weight: 20 g) by
intravenous injection, and at points of 30 minutes and 60 minutes
after the administration, the pancreases were dissected out of the
mice, respectively (n=2). Sections were cut out of the dissected
pancreases, and each section was placed on a slide glass, covered
with a cover glass. Fluorescence and radioactivity
(autoradiography) of each section were determined using an image
analyzer (trade name: Typhoon 9410, produced by GE Health Care
Inc.) (exposure time: 14 hours). Exemplary results of the same are
shown in FIG. 4.
[0233] Non-labeled exendin (9-39) (cold probe) was administered
(0.1 mL of 0.5 mg/mL solution) preliminarily by intravenous
injection to unanesthetized MIP-GFP mice (male, weight: 20, and
these mice were used as controls 1-1. At a point of 30 minutes
after the foregoing preliminary administration, the molecular probe
of the formula (31) (1 .mu.Ci) was administered by intravenous
injection. Then, at a time of 30 minutes after the administration
of the molecular probe of the formula (31), the pancreases were
dissected out of the mice, respectively (n=2). Sections were cut
out of the dissected pancreases, and fluorescence and radioactivity
of each section were determined in the same manner as described
above.
[0234] DPP (dipeptidyl peptidase) IV inhibitor (0.1 mL of 6 mg/mL
solution) was administered by intravenous injection to
unanesthetized MIP-GFP mice (male, weight: 20 g), and these mice
were used as controls 1-2. At a point of 30 minutes after the
administration of the DPP-IV inhibitor, GLP-1 (0.1 mL of 0.5 mg/mL
solution) was administered to these mice, and immediately after
this, the molecular probe of the formula (31) (1 .mu.Ci) was
administered thereto. At a point of 30 minutes after the
administration of the molecular probe of the formula (31), the
pancreases were dissected out of the mice, respectively (n=2).
Sections were cut out of the dissected pancreases, and fluorescence
and radioactivity of each section were determined in the same
manner as described above. Exemplary results of the controls 1-1
and 1-2 are shown in FIG. 4 together with the results of Example
1.
[0235] FIG. 4 illustrates exemplary results of the imaging analysis
of the pancreas sections of the MIP-GFP mice to which the molecular
probe of the formula (31) was administered. The images shown
therein are images showing a fluorescence signal (a) and a
radioactivity signal (b) of each of the sections at the point of 30
minutes after the administration of the molecular probe of the
formula (31), the sections at the point of 60 minutes after the
administration of the molecular probe of the formula (31), the
sections of the controls 1-1, and the sections of the controls
1-2.
[0236] As shown in (a) of FIG. 4, a fluorescence GFP signal was
observed by an image analyzer in each of the pancreas sections of
the MIP-GFP mice. As shown in (b) of FIG. 4, substantially no
radioactivity signal was detected from the sections of the controls
1-1 to which the cold probe was administered before the molecular
probe of the formula (31) was administered, and the controls 1-2 to
which GLP-1 was administered. From this observation, it was found
that the binding with a receptor was inhibited by administration of
a cold probe or GLP-1, whereby the uptake of the molecular probe of
the formula (31) was inhibited. Further, as shown in (a) and (b) of
FIG. 4, the localization of the radioactivity signal detected from
the labeled molecular probe of the formula (31) was consistent with
that of the GFP signal. From this, it was confirmed that the
molecular probe of the formula (31) accumulated specifically in the
pancreatic .beta.-cells.
[0237] Here, all of .sup.125I, .sup.123I, and .sup.131I were
.gamma.-ray emitting nuclides. Still further, .sup.125I and
.sup.123I have the same numbers of nuclear spins. In view of these,
it can be presumed that even a molecular probe obtained by
replacing the radioactive iodine atom (.sup.125I) used in the
labeling of the molecular probe of the formula (31) with .sup.123I
or .sup.131I will exhibit behaviors substantially identical to
those of the molecular probe of the formula (31). Further, it also
can be presumed that even a molecular probe obtained by replacing
the radioactive iodine atom (.sup.125I) with .sup.124I will exhibit
behaviors substantially identical to those of the molecular probe
of the formula (31). Thus, it was suggested that using the
molecular probe obtained by replacing .sup.125I of the molecular
probe of the formula (31) with .sup.123I, .sup.124, or .sup.131I,
the noninvasive three-dimensional imaging of pancreatic
.beta.-cells by SPECT, PET, or the like is enabled, and preferably,
the quantification of pancreatic .beta.-cells is enabled.
Example 2
[0238] Using the molecular probe of the formula (35) below (SEQ ID
NO. 35), having a configuration in which an .alpha.-amino group at
an N-terminus was labeled with [.sup.125I]3-iodobenzoyl group and a
carboxyl group at a C-terminus is amidated in the sequence of SEQ
ID NO. 5, biodistribution of the same in a mouse was determined.
The molecular probe of the formula (35) below was prepared in the
same manner in Example 1 except that an amino group labeled was an
.alpha.-amino group at an N-terminus.
##STR00013##
[0239] [Biodistribution]
[0240] The molecular probe thus prepared of the formula (35) (0.58
.mu.Ci) was administered to unanesthetized 6-week-old ddY mice
(male, weight: 30 g) by intravenous injection (through the tail
vein). At points of 5 minutes, 15 minutes, 30 minutes, 60 minutes,
and 120 minutes after the administration, organs were dissected out
of the mice, respectively (n=5). The weight and the radioactivity
of each organ were determined, and an accumulation amount (%
dose/g) of the molecular probe was calculated from the
radioactivity per unit weight. Exemplary results are shown in Table
5 below, FIGS. 5A and 5B. FIG. 5A is a graph showing how the
accumulation of the molecular probe in each organ varied with time,
and FIG. 5B is a graph of an enlarged part of 5A.
TABLE-US-00014 TABLE 5 Time after administration 5 min 15 min 30
min 60 min 120 min Pancreas 14.34 22.01 27.92 27.47 29.04 (2.49)
(5.49) (5.10) (3.04) (4.81) Blood 6.53 4.80 3.45 3.10 2.24 (0.38)
(0.22) (0.49) (0.59) (0.29) Heart 3.55 2.40 1.99 1.54 1.20 (0.60)
(0.48) (0.17) (0.44) (0.19) Lung 68.73 79.05 72.66 82.90 80.59
(14.19) (29.24) (14.51) (10.50) (10.19) Stomach 3.21 5.35 9.95 5.00
7.52 (1.21) (2.27) (10.68) (1.42) (2.35) Intestine 1.94 2.31 2.91
2.44 2.83 (0.38) (0.46) (0.48) (0.43) (0.36) Liver 27.06 20.72
16.84 12.35 8.45 (3.28) (2.24) (2.33) (2.37) (1.15) Spleen 2.67
1.92 1.62 1.37 0.88 (0.77) (0.20) (0.29) (0.36) (0.23) Kidney 12.54
11.58 8.94 7.79 6.39 (2.06) (0.93) (0.92) (1.05) (0.56) Thyroid
gland 5.12 3.58 4.15 4.11 5.20 (0.75) (0.20) (0.94) (1.23) (0.86)
Each numerical value indicates an average (SD) of 5 mice.
[0241] As shown in above Table 5, FIGS. 5A and 5B, the accumulation
of the molecular probe of the above-described formula (35) into the
pancreas was 14.3% dose/g at a point of 5 minutes after the
administration, 22.0% dose/g at a point of 15 minutes after the
administration, 27.9% dose/g at a point of 30 minutes after the
administration, 27.5% dose/g at a point of 60 minutes after the
administration, and 29% dose/g at a point of 120 minutes after the
administration. During a time period from the point of 15 minutes
to the point of 120 minutes after the administration, the molecular
probe of the foregoing formula (35) accumulated most in the
pancreas among the organs other than the lungs, and the
accumulation of the molecular probe in the pancreas was maintained
at a level exceeding 20% dose/g. During any time period, the
accumulation amount of the molecular probe in the pancreas was not
less than 3 times as much as that in the stomach, and not less than
11 times as much as that in the intestine. During a time period
from the point of 60 minutes to the point of 120 minutes after the
administration, the accumulation amount in the pancreas was not
less than 2 times as much as that in the liver. In other words, it
can be concluded that the molecular probe of the formula (35)
accumulated specifically in the pancreas. Further, no great change
was seen in the accumulation in the thyroid gland, and this
suggests that the molecular probe of the formula (35) above was not
subjected to deiodization metabolism in vivo. Therefore, the
molecular probe of the formula (35) above is considered suitable
for the pancreatic .beta.-cell imaging, particularly noninvasive
pancreatic .beta.-cell imaging.
[0242] As shown in the above Tables 3 and 4, the ratio of
pancreas/liver and the ratio of pancreas/kidney for the molecular
probe of Example 2 (the molecular probe of the above formula (35))
were high in comparison with the molecular probe of Comparative
Example. Thus, it was suggested that Clear images of pancreas can
be obtained at the time of imaging with the molecular probe of
Example 2 where the ratio of accumulation amount in the pancreas to
the surrounding organs of the pancreas is high and the accumulation
amount in the surrounding organs of the pancreas is low.
[0243] [Two-Dimensional Imaging Analysis]
[0244] The molecular probe thus prepared of the aforementioned
formula (35) (0.8 .mu.Ci) was administered to unanesthetized
MIP-GFP mice (male, weight: 20 g) by intravenous injection, and at
points of 30 minutes and 60 minutes after the administration, the
pancreases were dissected out of the mice, respectively (n=2).
Sections were cut out of the dissected pancreases, and each section
was placed on a slide glass, covered with a cover glass.
Fluorescence and radioactivity of each section (autoradiography)
were determined using an image analyzer (trade name: Typhoon 9410,
produced by GE Health Care Inc.) (exposure time: 18 hours).
Exemplary results of the same are shown in FIG. 6.
[0245] Unlabeled exendin (9-39) (cold probe) (0.1 mL of 0.5 mg/mL
solution) was preliminarily administered by intravenous injection
to unanesthetized MIP-GEP mice (male, weight: 20 g), and these mice
were used as controls 2. At a point of 30 minutes after the
foregoing preliminary administration, the molecular probe of the
formula (35) (0.8 .mu.Ci) was administered by intravenous
injection. Then, at a time of 30 minutes after the administration
of the molecular probe of the formula (35), the pancreases were
dissected out of the mice, respectively (n=2). Sections were cut
out of the dissected pancreases, and fluorescence and radioactivity
of each section thus obtained were determined in the same manner as
described above. Exemplary results thereof are shown in FIG. 6,
together with the results of Example 2 described above.
[0246] FIG. 6 illustrates exemplary results of the imaging analysis
of the pancreas sections of the MIP-GFP mice to which the molecular
probe of the formula (35) was administered. The images shown
therein are images showing a fluorescence signal (a) and a
radioactivity signal (b) of each of the sections at the point of 30
minutes after the administration of the molecular probe of the
formula (35), the sections at the point of 60 minutes after the
administration of the molecular probe of the formula (35), and the
sections of the controls 2.
[0247] As shown in (a) of FIG. 6, a fluorescence GFP signal was
observed by an image analyzer in each of the pancreas sections of
the MIP-GFP mice. As shown in (b) of FIG. 6, substantially no
radioactivity signal was detected from the sections of the control
2 to which a cold probe was administered before the molecular probe
of the formula (35) was administered. From this observation, it was
found that the binding with a GLP-1 receptor was inhibited by
preliminary administration of a cold probe, whereby the uptake of
the molecular probe of the formula (35) was inhibited. Further, as
shown in (a) and (b) of FIG. 6, the localization of the
radioactivity signal detected from the labeled molecular probe of
the formula (35) was consistent with that of the GFP signal. From
this, it was confirmed that the molecular probe of the formula (35)
accumulated specifically in the pancreatic .beta.-cells.
[0248] Here, all of .sup.125I, .sup.123I, and .sup.131I were
.gamma.-ray emitting nuclides. Still further, .sup.125I and
.sup.123I have the same numbers of nuclear spins. In view of these,
it can be presumed that even a molecular probe obtained by
replacing the radioactive iodine atom (.sup.125I) used in the
labeling of the molecular probe of the formula (35) with .sup.123I
or .sup.131I will exhibit behaviors substantially identical to
those of the molecular probe of the formula (35). Further, it also
can be presumed that even a molecular probe obtained by the
radioactive iodine atom (.sup.125I) with .sup.125I will exhibit
behaviors substantially identical to those of the molecular probe
of the formula (35). Thus, it was suggested that using the
molecular probe obtained by replacing .sup.125I of the molecular
probe of the formula (35) with .sup.123I, .sup.124I, .sup.131I, for
example, the noninvasive three-dimensional imaging of pancreatic
.beta.-cells by SPECT, PET, or the like is enabled, and preferably,
the quantification of pancreatic .beta.-cells is enabled.
[0249] The foregoing results suggest that the molecular probe for
imaging according to the present invention enables noninvasive
three-dimensional imaging of the pancreas, and particularly,
noninvasive three-dimensional imaging of pancreatic .beta.-cells,
in humans.
Example 3
[0250] Using a molecular probe of the formula (36) below (SEQ ID
NO. 36), having a configuration in which an amino group of a side
chain of a lysine residue at position 4 was labeled with
3-([.sup.123I] iodobenzoyl group (hereinafter referred to also as
"[.sup.125I]IB label") and a carboxyl group at a C-terminus is
amidated in the sequence of SEQ ID No. 1, biodistribution of the
same in a mouse was determined. The molecular probe of the formula
(36) below was prepared in the same manner as in the case of the
molecular probe of the aforementioned formula (31) using the
molecular probe precursor of the aforementioned formula (31) except
that [.sup.123I]SIB was used in place of [.sup.125I]SIB.
##STR00014##
[0251] [Three-Dimensional Imaging]
[0252] Using the prepared molecular probe of the aforementioned
formula (36), SPECT imaging was carried out. The prepared molecular
probe of the formula (36) (498 .mu.Ci) was administered to
anesthetized 5-week-old ddy mice (male, weight: 25 g) by
intravenous injection, and the SPECT imaging was carried out. The
SPECT imaging was cared out under the following imaging conditions
for 21 minutes starting at the point of 30 minutes after the
administration, with use of a gamma camera (product name: SPECT
2000H-40, manufactured by Hitachi Medical Corporation). Images
obtained were reconfigured under the following reconfiguration
conditions.
Imaging Conditions
[0253] Collimator: LEPH pinhole collimator Collecting angle of
detector: 360.degree. at 11.25.degree./40 sec Collecting time: 40
sec.times.32 frames, 21 minutes
Reconfiguration Condition
[0254] Pretreatment filter: Butterworth filter (order 10, cutoff
frequency: 0.12)
[0255] Exemplary results are shown in FIGS. 7A to 7C. The images
were taken at 30 minutes after the administration of the molecular
probe (integrating time: 20 minutes). FIG. 7A shows a transverse
view, FIG. 7B shows a coronal view, and FIG. 7C shows a sagittal
view. In FIGS. 7A to 7C, the positions of the pancreas are
indicated by arrows. It should be noted that the images of FIGS. 7A
to 7C are at the same contrast.
[0256] As shown in FIGS. 7A to 7C the position of the pancreas was
confirmed noninvasively in mice with use of the molecular probe of
the formula (36) above. In other words, it was confirmed that
molecular probe of the present invention enables the noninvasive
three-dimensional imaging of the pancreas.
[0257] Thus, view of that the position of the pancreas was
confirmed noninvasively in a mouse that has the pancreas in a
smaller size than that at a human and in which the organs are
present more densely than in a human, this suggests that in a human
that has the pancreas in a greater size than that of a mouse and in
which the organs are present not as densely as in a mouse, the
position of the pancreas and the size of the pancreas can be
determined more clearly, and moreover, an amount of expression of
the molecular probe in the pancreas can be determined. Therefore,
it was suggested that the molecular probe for imaging of the
present invention should enable noninvasive three dimensional
imaging of the pancreas in a human, particularly noninvasive
three-dimensional imaging if pancreatic .beta.-cells.
INDUSTRIAL APPLICABILITY
[0258] As described above, the present invention is useful in, for
example, the medical field, the molecule imaging field, and the
field relating to diabetes.
Sequence Listing Free Text
[0259] SEQ ID NOS. 1 to 12: the amino acid sequences of the
molecular probes for imaging of pancreatic islets according to the
present invention
[0260] SEQ ID NOS. 13 to 24: the amino acid sequences of the
molecular probe precursors for imaging of pancreatic islets
according to the present invention
[0261] SEQ ID NOS. 25 to 28: the amino acid sequences of the
peptides used in the labeling method according to the present
invention
[0262] SEQ ID NOS. 29 to 30: the amino acid sequences of the
molecular probes used in the binding assay
[0263] SEQ ID NO. 31: the amino acid sequence of the molecular
probe for imaging according to Example 1
[0264] SEQ ID NO. 32: the amino acid sequence of polypeptide used
in the manufacture of the molecular probe for imaging according to
Example 1
[0265] SEQ ID NO. 33: the amino acid sequence of the molecular
probe precursor used in the manufacture of the molecular probe for
imaging according to Example 1
[0266] SEQ ID NO. 34: the amino acid sequence of the molecular
probe of Comparative Example
[0267] SEQ ID NO. 35: the amino acid sequence of the molecular
probe for imaging according to Example 2
[0268] SEQ ID NO. 36: the amino acid sequence of the molecular
probe for imaging according to Example 3
[0269] SEQ ID NO. 37: the amino acid sequence of Exendin-(9-39)
Sequence CWU 1
1
37131PRTArtificial SequenceA molecular probe for imaging of
pancreatic islets 1Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg
Leu Phe Ile Glu 1 5 10 15 Trp Leu Lys Asn Gly Gly Pro Ser Ser Gly
Ala Pro Pro Pro Ser 20 25 30 230PRTArtificial SequenceA molecular
probe for imaging of pancreatic islets 2Leu Ser Lys Gln Met Glu Glu
Glu Ala Val Arg Leu Phe Ile Glu Trp 1 5 10 15 Leu Lys Asn Gly Gly
Pro Ser Ser Gly Ala Pro Pro Pro Ser 20 25 30 329PRTArtificial
SequenceA molecular probe for imaging of pancreatic islets 3Ser Lys
Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu 1 5 10 15
Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser 20 25
428PRTArtificial SequenceA molecular probe for imaging of
pancreatic islets 4Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile
Glu Trp Leu Lys 1 5 10 15 Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro
Pro Ser 20 25 531PRTArtificial SequenceA molecular probe for
imaging of pancreatic islets 5Asp Leu Ser Lys Gln Met Glu Glu Glu
Ala Val Arg Leu Phe Ile Glu 1 5 10 15 Trp Leu Lys Asn Gly Gly Pro
Ser Ser Gly Ala Pro Pro Pro Ser 20 25 30 630PRTArtificial SequenceA
molecular probe for imaging of pancreatic islets 6Leu Ser Lys Gln
Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp 1 5 10 15 Leu Lys
Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser 20 25 30
729PRTArtificial SequenceA molecular probe for imaging of
pancreatic islets 7Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe
Ile Glu Trp Leu 1 5 10 15 Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro
Pro Pro Ser 20 25 828PRTArtificial SequenceA molecular probe for
imaging of pancreatic islets 8Lys Gln Met Glu Glu Glu Ala Val Arg
Leu Phe Ile Glu Trp Leu Lys 1 5 10 15 Asn Gly Gly Pro Ser Ser Gly
Ala Pro Pro Pro Ser 20 25 931PRTArtificial SequenceA molecular
probe for imaging of pancreatic islets 9Asp Leu Ser Lys Gln Met Glu
Glu Glu Ala Val Arg Leu Phe Ile Glu 1 5 10 15 Trp Leu Lys Asn Gly
Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser 20 25 30 1030PRTArtificial
SequenceA molecular probe for imaging of pancreatic islets 10Leu
Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp 1 5 10
15 Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser 20 25 30
1129PRTArtificial SequenceA molecular probe for imaging of
pancreatic islets 11Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe
Ile Glu Trp Leu 1 5 10 15 Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro
Pro Pro Ser 20 25 1228PRTArtificial SequenceA molecular probe for
imaging of pancreatic islets 12Lys Gln Met Glu Glu Glu Ala Val Arg
Leu Phe Ile Glu Trp Leu Lys 1 5 10 15 Asn Gly Gly Pro Ser Ser Gly
Ala Pro Pro Pro Ser 20 25 1331PRTArtificial SequenceA precursor of
a molecular probe for imaging of pancreatic islets 13Asp Leu Ser
Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu 1 5 10 15 Trp
Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser 20 25 30
1430PRTArtificial SequenceA precursor of a molecular probe for
imaging of pancreatic islets 14Leu Ser Lys Gln Met Glu Glu Glu Ala
Val Arg Leu Phe Ile Glu Trp 1 5 10 15 Leu Lys Asn Gly Gly Pro Ser
Ser Gly Ala Pro Pro Pro Ser 20 25 30 1529PRTArtificial SequenceA
precursor of a molecular probe for imaging of pancreatic islets
15Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu 1
5 10 15 Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser 20 25
1628PRTArtificial SequenceA precursor of a molecular probe for
imaging of pancreatic islets 16Lys Gln Met Glu Glu Glu Ala Val Arg
Leu Phe Ile Glu Trp Leu Lys 1 5 10 15 Asn Gly Gly Pro Ser Ser Gly
Ala Pro Pro Pro Ser 20 25 1731PRTArtificial SequenceA precursor of
a molecular probe for imaging of pancreatic islets 17Asp Leu Ser
Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu 1 5 10 15 Trp
Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser 20 25 30
1830PRTArtificial SequenceA precursor of a molecular probe for
imaging of pancreatic islets 18Leu Ser Lys Gln Met Glu Glu Glu Ala
Val Arg Leu Phe Ile Glu Trp 1 5 10 15 Leu Lys Asn Gly Gly Pro Ser
Ser Gly Ala Pro Pro Pro Ser 20 25 30 1929PRTArtificial SequenceA
precursor of a molecular probe for imaging of pancreatic islets
19Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu 1
5 10 15 Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser 20 25
2028PRTArtificial SequenceA precursor of a molecular probe for
imaging of pancreatic islets 20Lys Gln Met Glu Glu Glu Ala Val Arg
Leu Phe Ile Glu Trp Leu Lys 1 5 10 15 Asn Gly Gly Pro Ser Ser Gly
Ala Pro Pro Pro Ser 20 25 2131PRTArtificial SequenceA precursor of
a molecular probe for imaging of pancreatic islets 21Asp Leu Ser
Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu 1 5 10 15 Trp
Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser 20 25 30
2230PRTArtificial SequenceA precursor of a molecular probe for
imaging of pancreatic islets 22Leu Ser Lys Gln Met Glu Glu Glu Ala
Val Arg Leu Phe Ile Glu Trp 1 5 10 15 Leu Lys Asn Gly Gly Pro Ser
Ser Gly Ala Pro Pro Pro Ser 20 25 30 2329PRTArtificial SequenceA
precursor of a molecular probe for imaging of pancreatic islets
23Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu 1
5 10 15 Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser 20 25
2428PRTArtificial SequenceA precursor of a molecular probe for
imaging of pancreatic islets 24Lys Gln Met Glu Glu Glu Ala Val Arg
Leu Phe Ile Glu Trp Leu Lys 1 5 10 15 Asn Gly Gly Pro Ser Ser Gly
Ala Pro Pro Pro Ser 20 25 2531PRTArtificial SequenceA polypeptide
for use in labeling or preparation of labelled polypeptide 25Asp
Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu 1 5 10
15 Trp Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser 20
25 30 2630PRTArtificial SequenceA polypeptide for use in labeling
or preparation of labelled polypeptide 26Leu Ser Lys Gln Met Glu
Glu Glu Ala Val Arg Leu Phe Ile Glu Trp 1 5 10 15 Leu Lys Asn Gly
Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser 20 25 30 2729PRTArtificial
SequenceA polypeptide for use in labeling or preparation of
labelled polypeptide 27Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu
Phe Ile Glu Trp Leu 1 5 10 15 Lys Asn Gly Gly Pro Ser Ser Gly Ala
Pro Pro Pro Ser 20 25 2828PRTArtificial SequenceA polypeptide for
use in labeling or preparation of labelled polypeptide 28Lys Gln
Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys 1 5 10 15
Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser 20 25
2931PRTArtificial SequenceA molecular probe for use in Binding
Assay 29Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile
Glu 1 5 10 15 Trp Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro
Pro Ser 20 25 30 3031PRTArtificial SequenceA molecular probe for
use in Binding Assay 30Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val
Arg Leu Phe Ile Glu 1 5 10 15 Trp Leu Lys Asn Gly Gly Pro Ser Ser
Gly Ala Pro Pro Pro Ser 20 25 30 3131PRTArtificial SequenceA
molecular probe for imaging of pancreatic islets 31Asp Leu Ser Lys
Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu 1 5 10 15 Trp Leu
Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser 20 25 30
3231PRTArtificial SequenceA polypeptide for preparation of a
precursor of a molecular probe for imaging of pancreatic islets
32Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu 1
5 10 15 Trp Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser
20 25 30 3331PRTArtificial SequenceA precursor of a molecular probe
for imaging of pancreatic islets 33Asp Leu Ser Lys Gln Met Glu Glu
Glu Ala Val Arg Leu Phe Ile Glu 1 5 10 15 Trp Leu Lys Asn Gly Gly
Pro Ser Ser Gly Ala Pro Pro Pro Ser 20 25 30 3431PRTArtificial
SequenceA molecular probe of comparative example 34Asp Leu Ser Lys
Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu 1 5 10 15 Trp Leu
Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser 20 25 30
3531PRTArtificial SequenceA molecular probe for imaging of
pancreatic islets 35Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg
Leu Phe Ile Glu 1 5 10 15 Trp Leu Lys Asn Gly Gly Pro Ser Ser Gly
Ala Pro Pro Pro Ser 20 25 30 3631PRTArtificial SequenceA molecular
probe for imaging of pancreatic islets 36Asp Leu Ser Lys Gln Met
Glu Glu Glu Ala Val Arg Leu Phe Ile Glu 1 5 10 15 Trp Leu Lys Asn
Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser 20 25 30
3731PRTArtificial SequenceExendin-(9-39) 37Asp Leu Ser Lys Gln Met
Glu Glu Glu Ala Val Arg Leu Phe Ile Glu 1 5 10 15 Trp Leu Lys Asn
Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser 20 25 30
* * * * *