JP2677544B2 - Radioactive metal element-bonded polymer compound and radiopharmaceutical containing the compound - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to an amino group and a bifunctional compound.
Reactive polymer compounds having ligands and their use, especially
(A) an amino group capable of binding to a physiologically active substance and (b) a release group
Reaction with a bifunctional ligand capable of binding to a reflective metal element
Polymer and radioactive polymer of the reactive polymer
Regarding use as a medicine. The reactive polymer compound of the present invention has not been published in the literature.
It is a new substance that visualizes specific organs, detects specific diseases,
Nuclear medicine applications such as kinetic testing of physiologically active substances and treatment of diseases
A stable radiopharmaceutical labeled with a radioactive metal element
Can be used for manufacturing. Imaging of specific organs, detection of specific diseases, dynamics
For inspection and treatment of diseases using radioisotopes
In the field of nuclear medicine, conventionally labeled with iodine-131
Bioactive substances have been widely used. For example, blood circulation
Iodine-131 labeled histamine used for system visualization and dynamic examination
Iodine-1 for the treatment of serum albumin and cancer
31-labeled cancer-specific antibody and the like. However
, Iodine-131 has a long half-life of about 8 days, and
Suitable as a diagnostic agent because it emits beta rays in addition to
I can't say I'm doing it. Also, in the application to treatment
Iodine-131 undergoes deiodination reaction in vivo and
There is a drawback of giving radiation exposure to tissues other than the nest. Therefore, depending on the purpose of nuclear medicine,
More suitable radioactive metal element with suitable physical properties
Useful radioactivity introduced into bioactive substances by chemical methods
Attempts to obtain pharmaceuticals continue. For example
Diethylenetriamine pentaacetic acid (DTPA), 3-oxobu
Chiral bis (N-methylthiosemicarbazone carvone
Each of bifunctional ligand compounds such as acid and deferoxamine
Strong chelate-forming ability for seed metals and their bifunctionality
Amino group or carboxy present at the end of the ionic ligand compound
Based on the reactivity of various groups to various physiologically active substances,
Through these bifunctional ligand compounds, radioactive metal elements and
And a method of binding a physiologically active substance has been proposed.
The labeled compounds obtained by these methods are relatively stable.
Moreover, since it retains the activity of physiologically active substances,
It is a drug of great interest in the medical field. But
However, the radiopharmaceuticals obtained by these methods are
High molecular weight bioactive substances such as thrombosis and cancer
Used for the diagnosis and treatment of
Uses ibrinogen and immune antibody (IgG) with a molecular weight of about 150,000
If it is, obtain the one with high specific activity required for diagnosis and treatment.
There are drawbacks that cannot be avoided. DISCLOSURE OF THE INVENTION The present inventors have conducted various studies
As a result, the polyfunctional amine compound was combined with the bifunctional ligand.
Bioactive substance-binding polymer in which a substance and a bioactive substance are bound
Is the compound useful as a carrier for radioactive metal elements?
Radioactive metal element binding
Compound compounds are available as radiopharmaceuticals that overcome the above-mentioned drawbacks.
I found the fact that it was for business. 1 minute for this radiopharmaceutical
It has a large number of bifunctional ligands per child.
A radioactive metal that binds per molecule without any problem
Use of the conventional bifunctional ligand compound itself with the number of elements
It means much more than. And in the present invention
By using reactive polymer compounds in
High ratio without denaturing or degrading the active substance
It is now possible to provide radioactive radiopharmaceuticals.
Generally, when a physiologically active substance with a large molecular weight is administered to the human body,
If the antigenicity is taken into consideration, the dose should be as low as possible.
It is desirable to set the amount. Therefore, the radiation obtained here
High specific activity of sex drugs is extremely advantageous in this respect
It is. [0006] Provided by the present invention
There are four new polymer compounds: 1. Polya having at least 3 amino groups in the molecule
Min compound (1) and bifunctional ligand compound (2)
The latter has at least two molecules per child, and amide bonds
At least one bonded through a group (-CONH-)
The reactive polymer compound (A) having a free amino group. 2. Polya having at least 3 amino groups in the molecule
Physiological activity on at least one amino group of Min compound (1)
Bioactive substance-bound polymer compound formed by binding substance (3)
(B '). 3. Of the free amino group present in the reactive polymer compound (A)
Physiology consisting of at least one bioactive substance (3) bound
An active substance-bound polymer compound (B). 4. A radioactive metal element is added to the bioactive substance-binding polymer compound (B).
Radioactive metal consisting of element (4) bound via chelate bond
Element-bound polymer compound (C). The reactive polymer compound (A) is a polyamine compound.
(1) and a bifunctional ligand compound (2) are combined to form
Things. At least polyamine compound (1) in the molecule
Also needs to have 3 amino groups.
The larger the number, the better. Less of those amino groups
Both of them are consumed for binding with the bifunctional ligand compound (2).
And at least one other is in the reactive polymer (A).
It remains free and is later bound to the physiologically active substance (3).
It is useful in the case. As described above, the polyamine compound (1) is
Since it is desirable that the number of amino groups present in the molecule is large,
For example, a high molecular polymer having a free amino group in the side chain
Is preferably used. Its molecular weight is the physiology to be bound later
Taking into account the physical and chemical properties of the active substance (3)
It may be selected appropriately. Preferred polyamination
As a specific example of the compound (1), the molecular weight is about 500 to 1,000,000.
Polylysine, polyimine with a molecular weight of about 500-500,000,
There is. On the other hand, the bifunctional ligand compound (2) (bifunctional
As a chelating agent), it is strong against the radioactive metal element (4).
It forms a solid chelate bond and is capable of forming porosity under relatively mild conditions.
Functional group capable of reacting with the amino group of the Liamine compound (1)
Eg carboxyl groups or the reactivity derived from them
A group having a group) is used. Such a bifunctional composition
Specific examples of the ligand compound (2) include compounds represented by the formula: Diethylenetriamine pentaacetic acid cyclic
Acid anhydride, formula: Ethylenediaminetetraacetic acid succinimide represented by
And so on. To produce the reactive polymer compound (A),
For example, polyamine compound (1) and bifunctional ligand compound
The (2) is reacted by a conventional method, and the dialysis method, salting out method, gel
Filtration method, column chromatography, high performance liquid chromatography
Purify by conventional means such as topography.
No. The by-products and unreacted products in the above reaction are
Reactive polymer compound (A) and physiologically active substance to be performed later
Unless it has an inhibitory effect on the reaction with (3),
No special application is necessary. Of the bifunctional ligand compound (2)
Due to differences in functional groups, reaction conditions, etc., polyamine compounds
(1) A molecule of a bifunctional ligand compound (2) that binds to one molecule
The number is different, but generally 2 or more, especially 5 or more.
Or more is preferable. However, obtained in this reaction
In the polyamine compound (1) part of the reactive polymer compound (A)
At least one amino group in the
It should remain free due to the binding of Commercially available polyline as polyamine compound (1)
Gin (lysine units of about 3 to 2000, preferably 3 to 500
Reactive polymer compound
The structure of (A) is shown in formula as follows: [In the formula, X represents a carbonyl from a bifunctional ligand compound (2).
Residue after removal of the group, p is an integer from 2 to about 2000, q is 1 to about 20
Represents an integer of 00. However, p + q is an integer from 3 to 2000
You. The reactive polymer compound (A) has a small amount in its molecule.
Since there is at least one free amino group, the amino
Physiology with or without suitable cross-linking agents
The active substance (3) is combined with it, and if necessary, the conventional procedure described above.
Carrier for radiopharmaceutical preparation by purification in stages
Provide a bioactive substance-binding polymer compound (B) useful as −
Can be offered. The physiologically active substance (3) mentioned here means a suitable substance.
Accumulate or identify in organs or tissues or in specific lesions
Means a substance that exhibits unique behavior in response to the physiological state of
You. Specific examples of the physiologically active substance (3) include blood proteins
(Eg human serum albumin, fibrinogen), fermentation
Element (e.g., urokinase, streptokinase),
Mon (e.g. adrenocorticotropic hormone, thyroid stimulating hormone
Mon), immune antibodies (eg IgG and its fragments F (a
b ') _Two , Fab ', Fab), antibiotics (eg Bleomycin)
, Mitomycin), neurotransmitters, sugars, fatty acids,
Examples include amino acids. These bioactive substances are
Ordinary molecular weight of 10,000 or more, especially 100,000 or more is preferable.
Good. Physiological activity against reactive polymer compound (A)
To bind the substance (3), for example, carbodiimid
De, maleimide, active ester compound, glutaral
This is preferably done with a suitable cross-linking agent such as dehydrate.
New Introduced per molecule of reactive polymer (A)
The number of molecules of the physiologically active substance (3) depends on the cross-linking agent, reaction conditions, etc.
More different but usually 10 or less, especially 3 or
Is preferably less than that. Reactive polymer generated here
Compound (A) and physiologically active substance (3), ie, physiologically active substance
The substance-binding polymer compound (B) is a polymer compound as necessary.
Column chromatography applied to quality, gel filtration
Method, dialysis method, etc.
Good. A commercially available polyamine as the polyamine compound (1)
Gin (lysine units of about 3 to 2000, preferably 3 to 500
) Is used as an example and the amount of bioactive substance-bound
The structure of the child compound (B) can be represented by the following formula: [In the formula, X represents a carbonyl from a bifunctional ligand compound (2).
The residue from which the group has been removed, Y is a physiologically active substance (3) or a physiologically active substance.
Residue of crosslinker-crosslinker residue conjugate, p is between 2 and about 2000
Number, q is an integer from 1 to about 2000, r is an integer from 0 to about 2000
You. However, p + q and p + q + r are 3 to 2000, respectively.
It is an integer. The physiologically active substance-bound polymer compound (B) is also
Combine the polyamine compound (1) with the physiologically active substance (3)
In addition, the bioactive substance-bound polymer compound obtained here
To attach the bifunctional ligand compound (2) to (B ')
Therefore, it can be prepared. Front-stage connection and second-stage connection
And the reactive polymer compound (A) and the physiological activity
Of functional substance (3) and polyamine compound (1) and bifunctional
This may be performed according to the binding of the ionic ligand compound. The physiologically active substance-bound polymer compound (B) emits radiation.
It is useful as a carrier for preparing a sex drug. You
That is, the reactive polymer compound (A) is added to the conjugate.
There are multiple bifunctional ligand compounds (2).
Can capture multiple radioactive metal elements (4)
Possible, physiologically active substance (3) Radioactive substance per unit
Providing radiopharmaceuticals with extremely high dose and specific activity
Can be done. As a carrier for preparing a radiopharmaceutical,
The physiologically active substance-bound polymer compound (B) is stored in the form of a solution.
May be present, but usually freeze-drying method, low temperature vacuum evaporation method
It is converted into a powder state by such as and stored, and it is ready for use and is sterile.
It is dissolved in water, saline, buffer, etc. Powder state
In addition, it is necessary for the physiologically active substance-bound polymer compound (B) after dissolution.
If desired, a pharmaceutically acceptable solubilizing agent (eg organic
Solvents), pH regulators (eg acids, bases, buffers), stabilizers
(Eg ascorbic acid), preservatives (eg benzoic acid)
Sodium), isotonicity agents (eg sodium chloride), etc.
Reduction for adjusting the valence state of radioactive metal element (4)
Agents and oxidants may be added. The amount of the carrier for preparing a radiopharmaceutical is
The labeling rate of the finally manufactured radiopharmaceutical is a practical obstacle.
The amount is high enough that there is no
It must be within the acceptable range. Radiopharmaceutical
For preparing radiopharmaceuticals using a preparative carrier
Is a radiopharmaceutical preparation which may contain the additives mentioned above.
A carrier for water and an appropriate form of radioactive metal element (4)
It may be contacted in the medium. Usually less than both
Both are made into an aqueous solution beforehand, and the other is added to it.
You. The radioactivity of the radioactive metal element (4) to be contacted is arbitrary
However, sufficient information is available when conducting nuclear medicine diagnosis.
And the radiation exposure of the subject.
Hopefully the radioactivity range will be as low as possible
Good. On the other hand, when the purpose of treatment is
It needs enough radioactivity to
Minimize radiation exposure to normal organs and tissues
It is desirable to be in the range of radioactivity. As the above-mentioned radioactive metal element (4),
It is a metal element with radioactivity and is useful for nuclear medicine diagnosis and treatment.
Has suitable physical and chemical properties and is bifunctional
Easily captured by the ligand structure of the ligand compound (2)
Is used. Specific examples include the purpose of diagnosis
Gallium-67, gallium-6
8, Thallium-201, Indium-111, Techneti
Um-99m, copper-62, etc.
Yttrium-90, palladium-
109, Rhenium-186, Gold-198, Bismuth-2
12 and the like. These are usually salts, especially water soluble
It is used in the form of a salt and binds bioactive substances in an aqueous medium.
The labeling is performed by contacting with the polymer compound (B).
However, the radioactive metal element (4) forms a stable chelate complex.
When there is a valence state that can occur (for example, gallium
-67, indium-111), other reagents in the reaction system
Does not need to be present, but forms a stable chelate complex
If you need to change the valence state in order to
(Eg technetium-99m), reducing agent in the reaction system
Would require the presence of an oxidant. As an example of a reducing agent
Divalent tin salts (for example, tin halides and sulfuric acid sulfates)
, Tin nitrate, tin acetate, tin citrate).
You. Specific examples of the oxidizing agent include hydrogen peroxide.
For example, as a radioactive metal element (4), technetium-99
When m is used, a bioactive substance-binding polymer compound (B)
In the presence of stannous salt as a reducing agent in an aqueous solvent,
Treat with technetium-99m in the form of technetate
The technetium-99m labeled polymer compound was prepared by
Can be manufactured. In the above preparation, the mixing order of each reagent
There is no particular restriction on the introduction, but it is usually the best in an aqueous medium.
Avoid mixing stannous salt and pertechnetate for the first time.
It is desirable to have a digit. Stannous salt fills pertechnetate
It is preferable to use it in an amount that can be reduced. The radioactive metal element bond thus obtained
Synthetic polymer (C) is useful as a radiopharmaceutical
To achieve this, a sufficient amount of radioactivity that enables diagnosis or treatment and
It is necessary to have a radioactive concentration. For example radioactive
When technetium-99m is used as the metal element (4)
In the case of administration, about 0.1 to 50 mCi per about 0.5 to 5.0 ml
It is desirable to have a radioactive concentration. Also like this
The radioactive metal element-bonded polymer compound (C) is thrown immediately after preparation.
Although it may be given, it is preferably resistant to storage for a suitable time after preparation.
It is desirable to have sufficient stability. Nao
In addition, the radioactive metal element-bonded polymer compound (C) is not necessary.
The same solubilizer (eg organic solvent), pH regulator (eg
Acids, alkalis, buffers), stabilizers (eg ascorbine)
Acid), preservatives (eg sodium benzoate), tonicity agents
(For example, sodium chloride) may be added. Useful as a radiopharmaceutical according to the invention
When preparing the radioactive metal element-bonded polymer compound (C)
Specific examples of polyamine compound (1) are polylysine,
Diethylenetriamine pentavine as the active ligand compound (2)
Acid cyclic acid anhydride (CADTPA), physiologically active substance
(3) Human serum albumin (HSA), radioactive metal element
Indium-111 (as element (4) ¹¹¹ In)
The following is a description of the case. First, polylysine
And CADTPA to bind to polylysine-diethylene
Liamine pentaacetic acid (DTPA) conjugate was prepared and the conjugate
And HSA with N- (γ-maleimidobutyryloxy)
HSA-Poly
A gin-DTPA conjugate is obtained. This conjugate and trivalent in
In the form of Zium ion ¹¹¹ Contact with an aqueous solution containing In
With, stable and high radioactivity ¹¹¹ In labeled HSA-poly
A lysine-DTPA conjugate is obtained. The labeled conjugate thus obtained was subjected to high-performance liquid chromatography.
The matographic behavior is similar to that of HSA.
You. In addition, the behavior of this labeled conjugate in rats is
It is almost the same as the conventional iodine-131 labeled HSA. Obedience
Obtained by the next method ¹¹¹ In-labeled HSA-CADTPA binding
The specific activity of the coalescence is about 7 mCi / mgHSA, while
Obtained by the present invention ¹¹¹ In-labeled HSA-polylysine-
That of the DTPA conjugate is above 35 mCi / mgHSA
You. To administer the radiopharmaceutical of the present invention to the human body
Is usually performed intravenously, but the physiology of the radiopharmaceutical
Suitable for active substance (3) part to express its activity after administration
And, as long as it is advantageous, is particularly limited to this
Instead, any other suitable method may be employed. that's all
As is clear from the above point, the radiopharmaceutical according to the present invention
The preparative carrier is an aqueous solution containing radioactive metal ions.
High specific release is achieved by the extremely simple method of contacting with liquid.
It is possible to provide radioactive radiopharmaceuticals. Moreover, it is profitable
Radiopharmaceuticals that are used are bioactive substances that make up them (3)
A feature that substantially retains the physiological activity derived from the part.
Have signs. Currently, as a radiopharmaceutical, nuclear medicine diagnosis
Know not only what is intended, but also what is intended for treatment
Have been. The basic principle of therapeutic radiopharmaceuticals is radiation
It is based on the destructive action of cells and tissues in the disease area by
So, as a practical example, iodine-1 used for goiter
31 labeled sodium iodide, inside body cavity such as abdomen and chest
Gold-198 colloid used for malignant tumors on the surface
, Beta-emitting nuclides with a relatively short half-life are used.
You. Recently, various diseases including monoclonal antibodies
Development of physiologically active substances that can be expected to accumulate specifically in nests
According to these are beta and alpha emitting nuclides,
Radiation labeled with a nuclide that undergoes electron capture or nuclear isomer transition
The possibility of cancer treatment with sex drugs has been suggested. Departure
The radioactive metal element-bound polymer compound (C) of Ming
It is suitable for various therapeutic purposes, especially per molecule
Can bind many radioactive metal elements (4)
For effective treatment with high activity and high specific activity.
There are possible advantages. The present invention is further illustrated by the following examples.
This will be described specifically. EXAMPLES The present invention will be described more concretely with reference to the following examples.
explain. Example 1 Human serum albumin-polylysine-diethylene triamie
Containing a pentaacetic acid (HSA-Poly Lys-DTPA) conjugate
Preparation of composition (1):-with 200 mg of polylysine hydrochloride having a molecular weight of about 5,000
Dissolved in 10 ml of 0.4 M phosphate buffer (pH = 8.0)
Diethylene while stirring with a magnetic stirrer
148 mg of triaminepentaacetic anhydride are added. Overnight at room temperature
After stirring and reacting, take 0.05 ml of the reaction solution and
0.1M citrate buffer 0.45 ml, indium chloride
( ¹¹¹ In) 0.25 ml, (0.5 mCi) was added, and thin layer chromatography was performed.
Graphography method (silica gel thin layer plate, methanol 10% acetic acid
Ammonium solution (3: 1 solution) is used to analyze 1 molecule of polylysine
Diethylenetriaminepentaacetic acid (DT
PA) The number of molecules was calculated. Rf of unreacted DTPA 0.5-0.6 Polylysine DTPA Original point The binding rate under the above reaction conditions was 5.4 DTP.
A / polylysine. Polylysine obtained here-
DTPA (Poly (Lys-DTPA) 5.4) N-to 1.5 ml
(γ-maleimidobutyloxy) succinimide (GM
BS) dimethyl sulfoxide solution (33.6 mg / ml)
Add 0.075 ml and stir with a magnetic stirrer.
While reacting at room temperature for 15 minutes. Next in the middle of this reaction
1.38 ml of body was taken and 90 mg / ml human serum albumi
Phosphate buffer (pH = 7.5) (0.5 ml) was added and the mixture was not stirred.
Furthermore, the mixture is further reacted at room temperature overnight. Cut off reaction
Put in 10,000 dialysis tube and add 1M sodium chloride solution.
After dialysis against the cephad, it was equilibrated with physiological saline.
Unreacted polymer on the G-75 column (22 x 300 mm)
− Was removed and purified. Of the above operations other than measurement of binding rate
Is aseptic, and all equipment used is 18
Heat treatment at 0 ° C for 4 hours and burn with pyrodiene
Wash with distilled water for injection and sterilize with autoclave before use
Was. Also, the buffer solution should be adjusted using distilled water for injection.
Sterilized by the filter sterilization method using a filter
Was. Resin for column chromatography is dilute alkaline solution
After washing with, it was dealkalized with physiological saline for injection. This
The purified human serum albumin-Poly (Lys-D thus obtained)
TPA) 5.4 1.1mg / ml of 0.45 ml was taken and DTPA
10 ^-6 mole / ml, 0.2 ml, 0.1M citrate buffer (pH
= 6.0) 0.35 ml, indium chloride ( ¹¹¹ In) 2mCi / m
Add 1, 0.5 ml and analyze by electrophoresis under the following conditions
And a polymer bound to one molecule of human serum albumin
The number of molecules was calculated. Support: Cellulose acetate membrane running buffer: 0.06 M veronal buffer pH = 8.6 Running conditions: 1 mA / cm 20 minutes The binding rate under the above reaction conditions is about 1 molecule of Poly (Lys). -D
TPA) 5.4 / human serum albumin. Get here
HSA-Poly Lys-DTPA with 0.1M citric acid
Dilute with buffer solution (pH = 6.0) to a concentration of 1 mg / ml and
Sterile vials of 1 ml each while filtering with a blanc filter.
And the desired composition was obtained. [0027] Example 2 (HSA-Poly Lys-DTPA)- ¹¹¹ Manufacture of In injection
And properties (distribution in the body):-Commercially available chloride in the vial containing the composition obtained in Example 1.
Dium ( ¹¹¹ In) Injectable solution 2mCi / ml 1.0ml
An injection solution was obtained. The above operation is performed aseptically. here
Take 25 μl of the injection solution obtained in step 1, and use the high-speed solution under the following conditions
Of dimers as analyzed by body chromatography
Is less than 1%, unreacted polymer, DTPA is below the detection limit
it was under. The retention time of the main component is about 23 minutes,
The average molecular weight calculated from a calibration curve obtained separately was about 7
It was 0,000. Column: Toyo Soda TSK-2000SW
Column (0.75 × 60 cm) Eluent: 0.1 M citrate buffer pH = 6.0 Elution rate: 0.75 ml / min Also labeled with 0.2 ml was administered through the tail vein of SD female rats.
Then, the biodistribution rate was measured 1 hour after the administration. Conclusion
The results are shown in Table 1. Directly added to human serum albumin as a control
HSA-DTPA- with bound DTPA ¹¹¹ In de
-Shows As is clear from the results, Poly (Lys-D
Introduction of TPA) 5.4 into HSA
No denaturation was observed and almost the same distribution was observed. [Table 1] Rat biodistribution test carrier HSA-Poly (Lys-DTPA) 5.4 * ¹ HSA-DTPA * ¹ Organ blood * ^Two 83.0 88.0 Liver 9.4 9.0 Kidney 2.7 2.0 Lung 1.8 3.0 Bladder 0.7 0.7 Note: * 1 Unit is% / organ. * 2 Whole blood was 6.4% of body weight. [0030] Example 3 Human serum albumin-polylysine-diethylene triamie
A composition containing pentaacetic acid (HSA-Poly Lys-DTPA)
(2): HSA-Poly (Lys-DT) obtained in the same manner as in Example 1.
PA) is diluted with 0.9% physiological saline to obtain the amount of protein.
Adjust to 2 mg / ml. Stannous chloride (Sn
Cl _Two ) Is added to 1 mM, and a membrane filter-
Dispense 1.5 ml each into a sterile vial while filtering with
A composition was obtained. Perform all the above operations aseptically
Was. [0031] Example 4 Preparation of (HSA-Poly Lys-DTPA) -99mTc injection
Construction and properties: -A vial containing the composition obtained in Example 3 was filled with 20 mCi / ml.
1 injection of sodium pertechnetate (99mTc) injection
Add ml (HSA-Poly Lys-DTPA) -99mTc injection
A liquid was obtained. The labeled body obtained here is shown in Example 1.
Labeling rate by thin-layer chromatography and electrophoresis
When calculated, high values of 90% or more were obtained for all
Was. [0032] Example 5 Anti-Myosin Antibody Fab-Polylysine-Diethylenetriami
A group containing a pentapentaacetic acid (Fab-Poly Lys-DTPA) conjugate
Manufacture of product (3):-Poly (Lys-DTPA) 5.4,3 ml obtained in Example 1
Then, add to this 3- (2-pyridyldithio) propionic acid N
-Dihydroxy hydroxysuccinimide ester (SPDP)
Add chill sulfoxide solution 40mg / ml, 0.12ml
3 hours at room temperature while stirring with a magnetic stirrer
To react for a while. Mercaptoethanol 0.01 in the reaction solution
Add 3 ml and react for an additional 1 hour. Cut reaction liquid
Off Place in a dialysis tube at 3,500 and release 0.04M phosphate buffer.
After dialysis against buffer solution-1mM EDTA solution,
Sephadex G-25 column equilibrated with one buffer
(22 × 300mm) to remove unreacted SPDP
-DTPA) -SH is obtained. Apart from this, anti-myosin anti-
Body Fab 10mg was dissolved in 0.4M phosphate buffer pH = 7.0.
Then, make 10 mg / ml solution and N- (γ-
Maleimidobutyloxy) succinimide (GMBS)
4.2 mg / ml-DMSO solution 0.02 ml was added and the mixture was allowed to stand at room temperature for 1 hour.
Stir for 5 minutes to react. This reaction liquid was mixed with P prepared above.
oly- (Lys-DTPA) -SH, 1.7 × 10 ^-6 mole / m
l, 2.4 ml was added, and the mixture was stirred overnight at room temperature for reaction.
The reaction mixture was placed in a dialysis tube with a cutoff of 10,000 and 1M salt was added.
Sodium chloride solution, followed by 0.9% physiological saline solution
Dialyze. After dialysis, the reaction solution was flattened with 0.9% physiological saline solution.
Balanced Sephadex G-50 column (22 x 300 m
Unreacted Poly (Lys-DTPA) -SH was removed with m)
Body Fab-Poly (Lys-DTPA) can be purified. The above operation
All the work is done aseptically, as well as the equipment and reagents used here.
Aseptic Pyrodienf was prepared by the method described in Example 1.
It was used after being re-formed (SPF). Anti obtained here
Body Fab-Poly (Lys-DTPA) 0.8 mg / ml solution 0.3 ml
Take DTPA10 ^-7 mole / ml citrate buffer (pH =
6.0) 0.2 ml and indium chloride ( ¹¹¹ In) Injection solution 2mC
Add i / ml and 0.2 ml and analyze by electrophoresis under the following conditions:
The number of polymer molecules bound to one antibody molecule was calculated. Support: Cellulose acetate membrane running buffer: 0.06 M veronal buffer pH = 8.6 Running conditions: 1 mA / cm, 20 minutes The binding rate obtained by the analysis of the above reaction conditions is 0.9 molecule
Poly (Lys-DTPA) 5.4 / antibody Fab. here
The amount of protein was Fab-Poly (Lys-DTPA) obtained in
0.1M citrate buffer pH =
Dilute with 6.0 and sterilize through a membrane filter.
Fill 0.5 ml each into a sterile vial to obtain the desired composition.
I got something. [0034] Example 6 (Fab-Poly Lys-DTPA)- ¹¹¹ Manufacture of In injection
And properties: -a commercially available vial containing the composition obtained in Example 5
Dium ( ¹¹¹ In) Injection solution (2 mCi / ml)
A target injection solution was obtained. The above operation was performed aseptically.
Take 25 μl of the labeled product obtained here, and
When analyzed by high performance liquid chromatography,
Abundance is 10% or less, unreacted polymer and DTP
No radioactivity was detected in the A fraction. Column: Toyo Soda TSK-3000SW
Column (0.75 × 60 cm) Eluent: 0.1 M citrate buffer pH = 6.0 Elution rate: 0.75 ml / min Also, the retention time of the main component was about 23 minutes, and a separately obtained
The molecular weight calculated from the calibration curve was 60,000. This
Fab-Poly (Lys-DTP) prepared by the method described above
A)- ¹¹¹ For In, radio with cardiac myosin as an antigen
10 when the antibody activity was measured by the metric assay method
⁸ ・ M ^-1 The affinity constant of was obtained. Clear from the above results
Poly (Lys-DTPA) was introduced into the antibody Fab
However, the immune activity of the antibody was not lost. [0036] Example 7 Antitumor Antibody 19-9 Fab'-Polylysine-Diethyleneto
Liamine pentaacetic acid conjugate (19-9 Fab'-Poly Lys-D
Preparation of a composition containing TPA):-Poly (Lys-DTPA) 5.4,20 m obtained in Example 1
To 1.5 ml of g / ml phosphate buffer, N- (γ-maleimide)
Butyloxy) succinimide (GMBS) 2.52 mg was added.
Then, react at room temperature for 15 minutes. 1.2 ml of this reaction solution
, Containing anti-tumor antibody 19-9 Fab'18.7 mg.
04M phosphate buffer-1mM EDTA solution (pH = 6.0) 3.
5 ml was added and reacted at room temperature for 18 hours. Reaction solution is 1M
Dialyze against saline solution and 0.9% saline solution,
Sephadex G-75 color equilibrated with physiological saline
It was purified using All the above operations are performed aseptically.
In addition, all the equipment and reagents used are shown in Example 1.
The SPF was used by the method. Obtained here (19-9 Fab '
-Poly Lys-DTPA) was diluted with physiological saline to 0.5 mg.
/ Ml (protein amount) and dispense into sterile vials
The desired composition was obtained. [0037] Example 8 (19-9 Fab'-Poly Lys-DTPA)- ¹¹¹ In injection
Preparation and properties of the liquor: -Commercially available chloride of chloride in the vial containing the composition obtained in Example 7.
Dium ( ¹¹¹ In) Add 1 ml of injection solution (2 mCi / ml) aseptically.
To obtain an injection solution. Anti-labeled in this way
Imitation using beads with immobilized 19-9 antigen per body
The immunological activity was measured by a nomometric assay.
3 x 10 ⁸ M ^-1 The affinity constant (Ka) was obtained. By the way
Is a difunctional ligand directly on 19-9 Fab '
Fab'-DTPA directly bound with tritriaminepentaacetic acid
Ka value is about 3 × 10 ⁸ M ^-1 Met. [0038] Example 9 Polyethyleneimine-diethylenetriaminepentaacetic acid (P
Preparation of EI-DTPA) conjugates: -Polyethyleneimine with side chains of average molecular weight about 70,000
(PEI) 10% aqueous solution was added to 0.2M phosphate buffer (pH 7.
Dilute with 8) to prepare a 0.1% aqueous solution. Mol to this liquid
10 times the amount of diethylenetriaminepentaacetic acid
Cucic anhydride was added, and the mixture was stirred at room temperature overnight. Then PEI
Measure the number of DTPA molecules bound per molecule
For reaction, take 200 μl of the reaction solution and add 0.1M
Add 100 μl of acid buffer (pH 6.0) and mix to 2 mCi
/ Ml of indium chloride ( ¹¹¹ In) Add 100 μl of aqueous solution and add
I knew it. One hour after labeling, the following thin layer chromatography
Method for PEI-DTPA- ¹¹¹ In (near the origin) and free
¹¹¹ In-DTPA (Rf value 0.5 to 0.7) is separated and
The radioactivity of each was measured and the binding rate was calculated. Thin layer plate: Silica gel G thin layer plate (Merck)
Developing solvent: methanol / 10% sodium acetate solution (1 /
1) Development time: about 1 hour As a result, PEI1 in the above reactive polymer compound
It was confirmed that 9 DTPAs were bound per molecule.
Was. [0040] Example 10 Polyethyleneimine-diethylenetriaminepentaacetic acid (P
Determination of the average molecular weight of the (EI-DTPA) conjugate: -For the PEI-DTPA obtained in Example 9,
High performance liquid chromatography to determine the average molecular weight.
It was measured. Column: TSK-3000SW Solvent: 0.1M citrate buffer pH = 6.0 Pressure: 380 psi Flow rate: 0.75 ml / min Absorption wavelength: 280 nm The retention time of PEI-DTPA in this system is about 24 minutes, and the retention time is about 24 minutes.
The retention time of DTPA at release was about 35 minutes. Molecular weight
From the standard curve obtained using known standard protein,
The average molecular weight of PEI-DTPA is calculated to be about 100,000
Was. As is apparent from the above, the present invention
Polymer compound of the invention, ie reactive polymer compound
(A), bioactive substance-bound polymer compound (B) and radioactive
The metal element-bonded polymer compound (C) is a new substance.
Yes, reactive polymer compound (A) and bioactive substance binding
Considering the chemical structure of polymer compound (B),
Radiopharmaceutical containing a relatively large number of radioactive metal elements
Radioactive metal-bonded polymer compound (C) useful as a product
Can be provided.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display area A61K 39/44 A61K 43/00 45/00 49/02 A 37/04 (72) Inventor Nobuo Ueda 14 (2), 11-11, Nagaura Station, Sodegaura-cho, Kimitsu-gun, Chiba Prefecture (56) References JP-A-60-55077 (JP, A) JP-A-57-143325 (JP, A) JP-A-53-44560 (JP, A) Japanese Patent Sho 60-47287 (JP, B2) Japanese Patent Sho 60-43360 (JP, B2)

Claims (1)

(57) [Claims] To a bioactive substance-bonded polymer compound (B) comprising a bioactive substance (3) bound to at least one free amino group present in a reactive polymer compound (A) having at least one free amino group Radioactive metal element-bound polymer compound in which radioactive metal element (4) is bound via chelate bond
(C) (however, the reactive polymer compound (A) is a polylysine having a molecular weight of 500 to 1,000,000 and a molecular weight of 5).
A polyamine compound (1) having at least three amino groups in a molecule selected from polyimines of 00 to 500,000 and a bifunctional ligand compound (2) having a carboxyl group or a reactive group derived therefrom. ) And the former 1
An amide bond (--CO) formed between the former amino group and the latter carboxyl group or a reactive group derived therefrom in a ratio of at least two molecules per molecule.
NH-). 2. To a bioactive substance-bonded polymer compound (B) comprising a bioactive substance (3) bound to at least one free amino group present in a reactive polymer compound (A) having at least one free amino group Radioactive metal element-bound polymer compound in which radioactive metal element (4) is bound via chelate bond
A radiopharmaceutical containing (C) as an essential component (provided that the reactive polymer compound (A) has a molecular weight of 500 to 1,000,000).
A polyamine compound (1) having at least three amino groups in a molecule selected from polylysine having a molecular weight of 0 and polyimine having a molecular weight of 500 to 500,000, and a bifunctional compound having a carboxyl group or a reactive group derived therefrom. The ligand compound (2) forms an amide bond (-CONH) formed between the former amino group and the latter carboxyl group or a reactive group derived therefrom in a ratio of the latter at least 2 molecules per molecule of the former. (-) Through a ())).