JP2002293796A - Reagent for contrastradiography and detection of cytochrome c by labeled peptide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a compound prepared by labeling a small synthetic peptide to be specifically bonded to cytochrome C with a radionuclide and to provide a new method for organism assay using the same. SOLUTION: In preparing a peptide compound for cytochrome C detection by subjecting a peptide having a specific bond part to cytochrome C and a bond part to the radionuclide to a covalent bond, a peptide comprising a specific amino acid sequence is used as the peptide and technetium-99m, etc., is used as the radionuclide.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention
The present invention relates to a radiolabeling reagent and a contrast agent used for detection.

[0002]

2. Description of the Related Art Cytochrome is a generic term for all heme proteins except for hemoglobin, myoglobin, peroxidase and catalase among the heme proteins present in living cells. , B, C, and D. These cytochromes have an important function as an intermediate electron carrier in the intracellular redox reaction utilizing the reversible redox reaction of heme iron. Usually, multiple cytochromes are present in large particles such as mitochondria in one cell, and they cooperate in cell respiration. Therefore, by measuring cytochrome in cells, not only respiratory activity but also physiological activity related to respiration can be evaluated.

[0003] In the field of nuclear medicine, there is a method of administering a small amount of a radiolabeled tracer compound to a living body to evaluate a biorelated component localized or distributed in the living body. Detection methods using these radioactive compounds are known as radiographic methods. In radiolabeling, the labeling substance is a gamma-ray nucleus, and the distribution of the radiolabeled tracer compound is measured using a gamma-ray detection camera. The presence or absence of the biological substance or the amount thereof can be determined based on whether or not the radiation tracer is localized at a specific portion of the living body.

There are many factors that must be considered in radiographic imaging of a living body. First, in order to maximize the detection effect, it is desirable to use a nuclide that emits gamma energy of 100 to 200 keV. On the other hand, in order to minimize the amount of radiation absorbed into the living body, it is desirable that the half-life of the radionuclide be as short as possible in the imaging and detection steps. In addition, it is desired to label with a radionuclide that can be easily used so that imaging and detection can be performed at any time. Many such radionuclides are known so far, for example, ⁶⁷ gallium,
^99m technetium, ¹¹¹ iodine, ¹²³ iodine, ¹²⁵ iodine,
¹⁶⁹ ytterbium or ¹⁸⁶ rhenium. Technetium-99m, which emits 140 keV gamma rays and has a half-life of 6 hours, is a particularly preferred nuclide. Such radiographic methods, especially gamma scintigraphy, provide an excellent method for non-invasive measurement of living cells. A radiation tracer that binds to cytochrome preferentially over various components in a living body is extremely useful as a scintigraphy for externally measuring a physiological activity such as respiration of a living body.

Some of such radiographic tracers for imaging are known in the prior art. For example, Kinght (1990
Sem. Nucl. Med., Vol. 20: 52-67)
Autologous transplanted platelets labeled with ¹¹¹ indium or ^99m technetium and fibrinogen labeled with ¹²³ or ¹²⁵ iodine are mentioned as radiotracers for contrast thrombus. In addition, plasmin, plasminogen activator, heparin, fibronectin, fibrin fragment E1, and anti-fibrin and anti-platelet monoclonal antibodies can also be compounds that specifically target thrombus.

[0006] EP0063002 discloses a radiolabeled protein selected from fragment E1 isolated from cross-linked fibrin, fragment E2 isolated from cross-linked fibrin, and a proteolytic fragment having an amino acid sequence intermediate between fragments E1 and E2. Degradate fragments are shown. Further, WO8807685 discloses a method of administering a labeled / attenuated thrombus protein to a living body, in which the labeled compound binds to a thrombus protein site other than the fibrin-binding domain, and detects the distribution pattern of the labeled thrombus protein. .

[0007] WO8912680 discloses a method for determining the position of a thrombus in a living body using enzymatically inactivated radiolabeled tissue plasminogen activator.

Some peptides that specifically recognize biologically relevant substances such as thrombus binding ability are also known. For example, USP 4578079 describes a peptide having a sequence of X-Arg-Gly-Asp-RY (where X and Y are H or amino acids, and R is Thr or Cys), and the peptide has a thrombotic ability. have. Further, USP4792525 discloses Arg-Gly-Asp-X (where X is Ser, Thr or Cys) as a peptide having thrombus binding ability. In addition, WO8901010, WO891
0135, EP0410537, EP0410539, EP0410540, EP0410541,
EP0422937, EP0422938, EP0425212, WO9101331, WO9115
515 and JP-A-10-291939 also disclose amino acid sequences recognizing biological substances.

On the other hand, the use of chelating agents for radiolabeled polypeptides and the method of labeling peptides and polypeptides with technetium-99m are known in the prior art. For example, USP653012, USP807062, USP871282, USP88
6752, USP893981, USP955466, USP019864, WO9213572,
WO9310747, WO9317806, and the like.

[0010]

SUMMARY OF THE INVENTION Cytochrome in living organisms
Imaging and detection reagents for in vivo and non-invasive measurement will be new biometric methods such as visualization of biological activity.
An object of the present invention is to provide a compound in which a radionuclide is labeled on a small synthetic peptide that specifically binds to cytochrome C, thereby enabling a novel biometric method.

[0011]

The peptide compound for detecting cytochrome C according to the present invention has a peptide having a specific binding site to cytochrome C, and a binding portion to a radionuclide. A peptide compound for detecting cytochrome C, which is covalently bound to the binding moiety, wherein the peptide has the following amino acid sequences (1) and (2) and specific binding of these amino acid sequences to cytochrome C: A peptide compound for detecting cytochrome C, comprising an amino acid sequence selected from amino acid sequences in which one to several amino acids have been deleted, substituted or added within a range not to impair the sex. Amino acid sequence (1) (SEQ ID NO: 1): (N-terminal) Trp-Pro-Thr-Asn-Trp-Trp-Pro-As
p-Asp-Leu-Phe-Pro amino acid sequence (2) (SEQ ID NO:
2): (N-terminal) Trp-Pro-Thr-Ser-Ser-Leu-Thr-Leu-Pr
o-Trp-Thr-Phe The reagent for imaging and detecting cytochrome C according to the present invention is characterized by containing the peptide compound having the above constitution.

Further, the scintigraphy contrast agent according to the present invention comprises a technetium-99m
Are obtained by combining

[0013] Further, the present invention relates to a cytochrome C imaging method according to the present invention.
The kit for detection is characterized by containing the peptide compound having the above constitution, a radionuclide to be bound to the peptide compound, and a reducing agent for these reactions.

The contrast analysis method for cytochrome C according to the present invention is a method for contrast analysis of cytochrome C in a sample. The contrast agent having the above constitution is incorporated into the sample, and the cytochrome C in the sample is analyzed. A method for contrast analysis of cytochrome C, comprising the step of contrast analysis of presence.

According to the present invention, a radiolabeling reagent useful as a raw material for a scintigraphic contrast agent can be provided.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION The peptide compound according to the present invention comprises a first portion comprising a peptide having a site that specifically binds to cytochrome C in vivo and the like, and a radionuclide covalently bonded to the first portion. And a second portion for coupling.

The first part is a peptide consisting of the above amino acid sequence (1) or (2), or a peptide within the range that does not impair the specific binding of these amino acid sequences to cytochrome C. A deletion of one to several amino acids,
A peptide consisting of an amino acid sequence obtained by substitution or addition is used. Thus, by using a small peptide having a molecular weight of about 3000 daltons or less,
Chemical or commercial benefits arise. A peptide consisting of 12 amino acids can be easily synthesized, has no antigenicity, and disappears quickly after a certain period of time even when administered to a living body. The properties of such small peptides are useful for rapidly imaging and detecting biological substances such as cytochromes. On the other hand, a large peptide has a high synthesis cost and may be toxic to a living body due to its antigenicity. Furthermore, since it disappears slowly from the living body, further improvement is required as a contrast / detection reagent.

The second portion has a binding site for a radionuclide, and preferably forms a neutral complex with the radionuclide. As a structure forming such a neutral complex, the following formula (1) or (2):

[0019]

Embedded image

A structure comprising picolinic acid or picolylamine and forming a complex with a radionuclide under a reducing atmosphere can be suitably used.

Further, a preferable bonding form between the second portion having the above structure and the first portion is represented by the following formula (3) or (4):

[0022]

Embedded image

(In the above formula,-(amino acid)-is 0 to 1)
Shown is a bond through 0 amino acids. ) Can be mentioned.

Examples of the reducing agent for forming the complex include a dithionite ion, a stannous ion, and a ferrous ion.
More than one species can be used in combination. Alternatively, the peptide compound may be labeled by reducing the radionuclide in advance and forming a complex with the second moiety by ligand exchange.

In the structure represented by the above formula (3) or (4), a first portion composed of a peptide that specifically binds to cytochrome C and a second portion (picolinic acid or Picolylamine) is 0
It is linked through ~ 10 amino acids. That is,-
(Amino acid)-is a direct bond (in formula (3), -CO-C
O-, the formula (4) in showing the -CH ₂ NH-NH-. )
Or an amino acid sequence consisting of up to 10 amino acids.

The first portion composed of a peptide strongly binds to a specific site of cytochrome C, thereby achieving specific binding. However, since the radionuclide or its binding site is bulky, it depends on the specific binding mode. May be inhibited by steric hindrance. In such a case, an appropriate spacer may be provided between the first portion and the binding site of the radionuclide. As the spacer compound, a methylene chain,
An oxyethylene chain, a sugar chain, a polyester, a polyamide, or the like can be used. Among them, a peptide in which a plurality of amino acids are bound has characteristics such as high biocompatibility and low toxicity, and is preferable as a spacer. In order to prevent the peptide forming the spacer from becoming a new specific binding compound for cytochrome C,
Care must be taken in the amino acid sequence.

A reagent for imaging and detecting cytochrome C can be obtained by using the peptide compound having the above-mentioned structure and a carrier or a diluent used as necessary. Furthermore, a kit for imaging / detection of cytochrome C can be supplied by combining this imaging / detection reagent with a radionuclide as a label and a reagent used for binding it to a peptide compound.

For example, a kit for labeling a peptide compound of the above formula (3) or (4) with a radionuclide is a sealed kit containing a sufficient amount of a reducing agent to label a predetermined amount of the peptide compound with a radionuclide. Made of vials.

As described above, the present invention provides a method for producing a reagent for imaging and detecting cytochrome C by chemical synthesis in vitro. Preferably, the 12 amino acid sequences that form the binding site for cytochrome C are synthesized by solid phase peptide synthesis. Further, the distribution of cytochrome C in a living body can be visualized in vivo by the imaging / detection reagent and the gamma scintigraphy imaging method of the present invention.
Specifically, this is achieved by administering an imaging / detection reagent labeled with a radionuclide to a living body, and detecting gamma rays generated from the radionuclide of the reagent bound to cytochrome C.

As the radionuclide, ^99m technetium, ¹¹¹ indium, ⁶⁸ gallium and the like can be used, and among them, ^99m technetium is preferable.

The nuclide and radioactivity characteristics of the radioisotope are related to sensitivity, selectivity, and radiation exposure in scintigraphic imaging. Among them, the ^99m technetium-labeled cytochrome C imaging and detection reagent has a single photon energy of 140 keV and a radioactive half-life of about 6 hours, and is easily available from a ⁹⁹ molybdenum- ^99m technetium generator. is there. Another feature is that the biotoxicity is lower than that of ¹²⁵ iodine or the like.

On the other hand, the 12 amino acids in the amino acid sequence (1) or (2) may be either L-form or D-form. Furthermore, the amino acids constituting the binding site between the peptide and the radionuclide in the above formulas (3) and (4) include L-form or D-form amino acids, natural or artificial amino acids, and the like.

A peptide that specifically binds to cytochrome C can be efficiently synthesized in vitro, for example, using an amino acid synthesizer. Further, in this synthesis process, the binding site of the radionuclide is bound to the specific binding peptide directly or via a spacer. Furthermore, during peptide synthesis, a radiolabel binding site can be introduced into a specific peptide or a peptide that is a spacer. Compound consisting of picolinic acid [(Pic-); for example, Pi
For c-Gly-Cys (protecting group)-], the radiolabeled binding site may be synthesized as the last amino-terminal residue during the synthesis. The picolinic acid binding site is located at the E-
AN (Fmoc) -Lys-EN [Pic-Gly-Cys (protecting group)] that can be covalently bonded to an amino group and introduced into any position in the peptide chain
Can also be configured.

Similarly, the sequence [-Cys (protecting group) -Gl
By including y-], the binding site of picolylamine (Pica) [-Cys (protecting group) -Gly-Pica] during peptide synthesis
Can also be synthesized. It is also possible to activate the carboxyl terminus of the specific peptide or spacer peptide to couple picolylamine.However, in this method, all reactive side chain functional groups are protected, and picolylamine is not a terminal carboxylic acid. And must not react.

Taking the radioactive technetium-99m as an example,
In forming a complex between radioactive technetium-99m and the reagent of the present invention, the technetium complex, preferably pertechnetate, is reacted with the reagent in the presence of a reducing agent. Preferred reducing agents are dithionite ion, stannous ion and ferrous ion, and the most preferred reducing agent is stannous chloride. Means for synthesizing such a complex include a sealed vial containing a predetermined amount of the reagent of the present invention to be labeled and a sufficient amount of a reducing agent to label the reagent with technetium-99m. It is conveniently provided in kit form.

As one specific example of the present invention, there is a kit for producing a reagent for imaging and detecting cytochrome C. An appropriate amount of the reagent is placed in a vial containing an amount of reducing agent sufficient to label the reagent with technetium-99m. Also,
The kit can also contain conventional pharmaceutical additives such as, for example, pharmaceutically acceptable salts for adjusting the osmotic pressure, buffers, preservatives and the like. The components of the kit may be in liquid, frozen or dried form. In a more preferred embodiment, the kit components are provided in lyophilized form.

The radiolabeled scintigraphic contrast agent provided by the present invention has an appropriate dose of radioactivity. In the formation of technetium-99m radioactive complex, 1
Approximately 0.01 millicurie (mCi) to 100 mCi per ml
It is generally preferred to form the radioactive complex in a solution containing a concentration of radioactivity.

The cytochrome contrast / detection reagent labeled with technetium-99m can be used for visualizing cytochrome C in a living body. According to the present invention, the labeling reagent is administered directly to the organism in a single unit injection dose. Generally, the unit dose administered will be from about 0.01 mCi to about 10 mCi.
It has a radioactivity of 0 mCi, preferably 1 mCi to 20 mCi. The solution to be injected in a unit dose is about 0.01 mL to about 10 mL. After administration, in vivo imaging occurs within minutes. However, if desired, imaging occurs for several hours or even longer after administration of the radiolabeled reagent to an organism. In most cases, when a sufficient amount of dose is administered, about 0.5.
It accumulates in an area to be imaged within one hour and enables comma-ray imaging.

Although various embodiments according to the present invention have been specifically described above, it is needless to say that the present invention is not limited to these embodiments.

[0040]

EXAMPLES The present invention will be described in detail with reference to the following Examples, which do not limit the present invention in any way.

Example 1 (solid phase peptide synthesis)
Using 9-fluorenylmethyloxycarbonyl (Fmoc) amino end protection, using p-hydroxymethylphenoxy-methyl polystyrene as the carboxyl terminal acid or using linkamide resin as the carboxyl terminal amide, Applied Biosystems. Solid phase synthesis of the peptide of amino acid sequence (1) or (2) was performed on a 0.25 mmol scale using a model 431A peptide synthesizer. Products bound to the resin were routinely cleaved using a solution of trifluoroacetic acid, water, prepared in a ratio of 100: 5.

A picolinic acid group was introduced by using picolinic acid as the last residue in peptide synthesis.
Alternatively, picolylamine groups were introduced by coupling picolylamine to the precursor peptide using diisopropylcarbodiimide and N-hydroxysuccinimide.

The crude peptide was purified by preparative high performance liquid chromatography (HPLC) using a Waters Delta Pak C18 column and a gradient eluent using acetonitrile-0.1% trifluoroacetic acid. Acetonitrile was distilled off from the eluted fraction and freeze-dried. The identity of each product was confirmed by fast atom bombardment mass spectrometry.

Example 2 (Method of radiolabeling with technetium-99m) 0.1 mg of the peptide reagent prepared in Example 1 was used.
0.1 ml water or 1: 1 ethanol: water or phosphate buffered saline or potassium phosphate buffer (pH =
5.6 or 7.4). Glucoscan vials (EIDuPont de Nem
technetium containing up to 200 mCi
Regenerate with 1.0 ml of sodium pertechnetate containing -99m and leave it at room temperature for 15 minutes to remove technetium-9
9m gluceptate was prepared. Next, 25 μl of technetium-99m glyceptate was added to the peptide and the reaction was allowed to proceed for 15-30 minutes at room temperature or 100 ° C.
Filtered through a μm filter.

The purity of the technetium-99m-labeled peptide was measured by high performance liquid chromatography. Radioactive components were detected by an in-line dose detector connected to an integrating recorder. Technetium-99m gluceptate and technetium-99m containing pertechnetate were eluted under these conditions in 1 to 4 minutes, whereas the technetium-99m labeled peptide eluted much later.

Example 3 (Contrast and Detection of Cytochrome C) Cytochrome C is dissolved in a phosphate buffer (pH 7.0) and its concentration is adjusted to 1 μg / ml. 10 μl of this solution was added dropwise to a polystyrene well, and cytochrome C was fixed to the well by adsorption. The wells were thoroughly washed with a phosphate buffer (pH 7.0) to wash away non-immobilized cytochrome C.

Imaging of cytochrome C synthesized in Example 2
The detection reagent was prepared so as to have a radioactivity of 1 mCi, and added dropwise to a well on which cytochrome C was immobilized or a well on which no protein was immobilized (control well). After standing at room temperature for about 10 minutes, each well was further washed with a phosphate buffer (pH 7.0) to wash away excess imaging and detection reagents. The gamma dose from each well was imaged with a gamma camera, and almost no gamma rays were observed from the control wells, whereas gamma rays were observed from the wells on which cytochrome C was fixed, and cytochrome C was selectively detected by the imaging and detection reagents. It can be seen that the image can be contrasted and detected.

[0048]

According to the present invention, a reagent for scintigraphy suitable for imaging and detection of cytochrome C in vivo is provided.

[0049]

[Sequence List] SEQUENCE LISTING <110> Canon INC. <120> A constant and measurement reagent for cyctochrome C by a labeled p eptide <130> 40 12037 <160> 2 <210> 1 <211> 12 <212> PRT <213 > Artificial Sequence <220> <223> Peptide for detecting cytochrome C <400> 1 Trp Pro Thr Asn Trp Trp Pro Asp Asp Leu Phe Pro 1 5 10 <210> 2 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Peptide for detecting cytochrome C <400> 2 Trp Pro Thr Ser Ser Leu Thr Leu Pro Trp Thr Phe 1 5 10

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 2G045 AA40 DA36 FA11 FA19 FA40 FB02 FB10 4C085 HH03 JJ02 KA29 KB09 LL09 4H045 AA10 AA30 BA16 BA51 BA71 EA34 EA50 FA20 FA33 FA58

Claims (12)

[Claims] 1. A cytochrome C having a peptide having a specific binding site to cytochrome C and a binding portion to a radionuclide, wherein the peptide and the binding portion are covalently bound.
A peptide compound for detection, wherein the peptide has one to several amino acids within the following amino acid sequences (1) and (2) and within a range that does not impair the specific binding of these amino acid sequences to cytochrome C: A peptide compound for detecting cytochrome C, comprising an amino acid sequence selected from amino acid sequences in which amino acids have been deleted, substituted or added.
Amino acid sequence (1): (N-terminal) Trp-Pro-Thr-Asn-Trp-
Trp-Pro-Asp-Asp-Leu-Phe-Pro amino acid sequence (2): (N
-Terminal) Trp-Pro-Thr-Ser-Ser-Leu-Thr-Leu-Pro-Trp-Thr
-Phe 2. The peptide compound according to claim 1, wherein the radionuclide is technetium-99m. 3. The peptide compound according to claim 1, wherein the radionuclide and the binding site bind by forming a neutral complex. 4. The bonding moiety according to claim 1, wherein the bonding moiety has a structure represented by the following formula (1) or (2): The peptide compound according to claim 3, which has a structure consisting of picolinic acid or picolylamine represented by the following formula, and the neutral complex is formed by these structures. 5. The method according to claim 1, wherein the bonding moiety has a structure represented by the following formula (3) or (4): (Wherein-(amino acid)-represents a bond via 0 to 10 amino acids). 6. Contrast-enhancing cytochrome C comprising the peptide compound according to any one of claims 1 to 5.
Detection reagent. 7. A scintigraphic contrast agent for detecting cytochrome C, which is obtained by binding a radionuclide to the peptide compound according to any one of claims 1 to 5. 8. The scintigraphic contrast agent according to claim 7, wherein the compound containing the radionuclide is reacted with the peptide compound in the presence of a reducing agent. 9. The scintigraphic contrast agent according to claim 8, wherein the reducing agent comprises at least one of dithionite ion, stannous ion and ferrous ion. 10. The scintigraphic contrast agent according to claim 7, wherein a radionuclide is bonded to the peptide compound to form a neutral complex by ligand exchange of the complex of the radionuclide that has been reduced in advance. 11. An imaging method for cytochrome C, comprising: the peptide compound according to claim 1; a radionuclide to be bound to the peptide compound; and a reducing agent for these reactions. Kit for detection. 12. A method for performing contrast analysis of cytochrome C in a sample, wherein the contrast agent according to claim 7 is incorporated into the sample, and the presence of cytochrome C in the sample is analyzed by contrast analysis. A contrast analysis method for cytochrome C.