JPS58194820A - Radioactive labeling method for proteins by technetium-99m - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method of tagging proteins and various chelating agents with technetium-99m to obtain compositions.

It is well known to utilize compositions that emit several levels of externally detectable radiation after administration to the human body.

These compositions are used to visually capture and/or monitor the functions of various parts of the human body, or to target specific antigens, antibodies,
It is used to detect tumors related to carcinoembryonic antigens, which diagnose the presence and location of hormones, etc. US Special IF No. 46
No. 63,684, No. 5, 867.565 and No. S, 9
As disclosed in each specification of No. 24195, ■11
Antibodies against carcinoembryonic antigens tagged with 1 or 11m6 have been used to detect tumors that have developed carcinoembryonic antigens or tumors associated with said antigens. Furthermore, a diagnostic agent (dl
It is well known that protein molecules can be tagged with technetium-99m in order to obtain (mgnomtia ag@nt) proteins, chelating agents, Technetium-99m has been utilized to radioactively tag bone scanning phosphonate compositions and the like. In the above method, pertechnetate is contacted with a reducing agent, preferably stannous chloride, and the protein to be radioactively tagged.
Technetium is reduced to a +3, +4 or +5 valence oxidation state in the presence of a chelating agent or the like. In order to maintain the chemical bond between the technetium molecule and the substrate to be tagged,
It is necessary, however, to keep the reduced technetium in a reduced state. However, it is also necessary that the reduced technetium is not hydrolyzed after reduction. When administered to a patient, these substances automatically deposit in the liver, bone marrow, and/or spleen, rather than depositing in other parts of the body where a concentration of technetium is desired.Furthermore, It is also necessary that the technetium be tightly bound to the protein to the extent that the reduced technetium does not migrate to other proteins such as buffer molecules or small molecules in the patient's blood.

Otherwise, upon administration, technetium will not be transferred to the desired location in the human body. Finally, the technetium bond must have sufficient strength so that the technetium label does not transfer to other molecules before reaching the desired location in the human body.

Prior to the present invention, it was very difficult to maintain the stability of substrates tagged with technetium-99m. This is primarily due to problems associated with oxidation during tagging substrate formation and/or the use of buffer compositions that cause reducible stannous ions to precipitate out of the tagging solution. Additionally, conventional compositions containing technetium-99m and a substrate to be radiotagged are generally unstable and fail to maintain radiochemical purity during storage or after administration to a patient. Therefore, we provide a substrate composition that is highly stable and can be tagged with technetium-99m in such a way that both the complex state and oxidation state of technetium-99m can be properly maintained when radioactively tagged with technetium-99m, and therefore stability can be guaranteed. It is strongly desired to do so. Traditionally, some methods of labeling proteins alter or reduce the ability of proteins involved in various immune reactions to change the biological properties of proteins, or in the case of immune brepurins or antibodies, they There were also methods to reduce reactivity with antigens.

According to the present invention, the substrate to be radioactively tagged with Technetium A-9pm is mixed with a buffered stannous chloride composition having a pH of about 4.5 to about 8.5, wherein the stannous chloride is non-oxidized. It is obtained from a tin source, oxygen is removed from the buffered stannous chloride, and the buffering agent is an alkali metal cyphthalate and an alkali metal tartrate i! It contains a mixture of salts. Alternatively, the buffering agent is an alkali metal borium.
It may contain a salt or gentisin salt tolerant salt. Stannous chloride is prepared by reacting concentrated hydrochloric acid with substantially oxygen-free tin to form a substantially oxygen-free solution of stannous chloride. For this purpose, it is advantageous to purge all solutions and the working environment with inert gas or nitrogen. The stannous chloride solution is then mixed with a buffer and the resulting mixture is neutralized with sodium hydroxide. This neutralized solution is finally mixed with a substrate that is radiotagged with technetium-99m.

This solution is left to age at room temperature for several hours (usually 15 hours or more) in the absence of oxygen. It is possible to raise or lower the storage temperature, and the storage time changes accordingly. In other words, if the storage temperature is set to 35°C, the storage time is approximately 1
/2, whereas if the device temperature were increased to 15° C., the time would have to be doubled. In any case, the temperature at which the compound is left to be used must not cause deterioration of the tin-pretreated compound. This solution is then mixed with sodium pertechnetate-Tc99m, avoiding the introduction of atmospheric oxygen, to obtain the radioactive tag material. The substrate-stannous chloride-buffer solution can also be lyophilized prior to contacting with sodium pertetaneate and reconstituted when radiotagging is required. The present invention also provides a kit, ie, a kit, which includes a buffer, stannous chloride, and a substrate. This kit contains technetium +
For chromatographs containing substances capable of binding a reducing agent that reduces 7 to technetium +4 to +5 and at the same time binding technetium as pertechnetate, reductively hydrolyzed technetium, colloidal technetium or weakly complexed technetium. May include columns.

The present invention will be explained in more detail below.

The stannous chloride reducing agent can be prepared by contacting an unoxidized solid tin pellet with concentrated hydrochloric acid, or it can be prepared from other raw materials such as stannous chloride or stannous fluoride. In the latter case, the resulting stannous chloride solution is freed from oxygen before being added to the substrate. Removal of oxygen can be achieved by introducing an inert gas such as nitrogen or helium into the stannous chloride solution in the form of a bubble. When using an unoxidized solid tin source for one time, the above removal step may not be performed.

The buffering agents are alkali metal cyphthalates and alkali metal tartares v1. The preferred salt solution is sodium borate or sodium gentisate. Preferred sifthal salts are sifthalic acid salt, sodium sifthalate, or mixtures thereof; preferred tartrates include sodium tartrate, sodium gentisate; Potassium tartrate or mixtures thereof may be mentioned. The molar ratio of cyphthalate to tartrate ranges from about 1 to about 10, preferably from about 2 to about 3. This particular &! shock agent composition It has been found that when using R, little or no reaction with stannous chloride occurs.

Therefore, di(11) pentachloride is free to act as a reducing agent for technetium. After the buffer composition and the stannous chloride composition are mixed, the mixed composition is neutralized by adding lithium hydroxide to a pH in the range of about 4.5 to about 85.

The substrate to be radiotagged with technetium-99,n in a subsequent step is added to the buffered reducing agent and left in the absence of ambient oxygen for at least about 15 hours, preferably about 21 hours, at about room temperature. . If desired, the standing time can be shortened by heating this solution appropriately. The solution is then lyophilized and reconstituted later when mixed with pertechnetate.
Alternatively, it can be mixed directly with pertechnetate solution to form a tagging substrate. If desired, the radioactively tagged substrate can be further purified to separate the tagged substrate from free technetium, such as by a Sephadex column pretreated with a saturated solution of stannous cyphthalate or by leumatograph analysis in a Sephalase column. It is. However, the above final step is not always necessary. In any case, the purification process is also carried out while avoiding or minimizing the introduction of atmospheric oxygen. Table 1 shows typical results obtained with the present invention.

Table 1 Protein Yield % of 99m Te-99m tagged protein purified by 'l'c-chromatographic method reduced on Sephadex column Human serum albumin-6299!3 200m C5 of Tc-99m (Millicuries) Bovine gamma globulin-7899.9 2.0 m of Tc-99m CI (Millicuries) Bovine gamma globulin-8899.7 200 m of TC-99m Ct (Millicuries) The source of technetium-99m is preferably an alkali metal. Water-soluble substances such as pertechnetate.

Technetium is normal 99 M o / 99 m T
It can be obtained as sodium pertechnetium from the c-generated material. Pharmaceutically acceptable technetium-99m
All proteins, chelating agents, and other substrates that are normally tagged with technetium-99m can be radiotagged according to the present invention. A typical and suitable substrate is IgG.
, Fab7 fragment of IgG, human villous gonad-stimulating polmon (hca), anti-hCG or anti-beta type hC
G, antibodies against anti-hCG or anti-beta hCG
Antibodies against, fipurinogen, urokinase, alpha-fetoprotein
tein), carcinoembryonic antigen (CEA), anti-CEA, human serum albumin (nSA), and other proteins.

The present invention further provides kits or devices that enable a user to prepare and administer the compositions of the present invention relatively quickly after preparation. This kit contains the substrate in lyophilized, frozen, or liquid form with appropriate ionic strength and pH, with or without stannous chloride or fluoride-m reducing agent. It can also be +1. If not premixed with stannous chloride or stannous fluoride, it can be mixed with stannous chloride or stannous fluoride in a separate container in the substrate kit. can. The stannous chloride can be mixed with a buffer, or the buffer can be lyophilized and stored in a separate container. In use, it is preferable to store the substrate, stannous chloride, and buffer together with the neutralizing agent in a lyophilized state in a common container. When this composition is regenerated with sodium pertechnetate-Tc99m, its reducing pH ranges from about 6 to about 55 when the composition is bound to technetium-99m as pertechnetate. This renders the tagged substrate solution suitable for patient administration. The present invention further provides novel compositions comprising protein products treated with stannous ions, wherein the stannous ion concentration is at least about 70%.
The concentration of technetium-99m is high enough to bind strongly. As shown in Figure 1,
In order to obtain such technetium binding strength, the presence of at least a6 milliequivalent of stannous ions is required. Also shown in Figure 1, the preferred range of stannous ion concentration is from about 15 meq to about 12.5. It is between t-equivalent. If the amount of stannous ions is high (some colloids will be formed).

Although undesirable, stiff colloids are easily r
Can remove excess.

According to another embodiment of the invention, technetium-99m
It may also contain a material such as Sephadex, Sepharose or cellulose, which captures or binds the substance. The column of this material may also contain a reducing agent for the technetium, or a reducing agent may be added to the column if it is desired to reduce the technetium.

The tagged substrate is administered by intravenous injection as a sterile, pyrogen-free, pharmaceutically acceptable saline solution. A suitable dose is usually about 5 to 30 milliquiries, preferably about 10 to 20 milliquiries of technetium-99m substrate for a normal patient weighing 70 degrees. The patient may then be scanned using conventional scintillation analysis within about 1 to about 24 hours after administration of the tagged protein.

Hereinafter, the present invention will be explained with reference to Examples, but the present invention is not limited to these Examples.Example 1 This example describes the details of the present invention for tagging immunoglobulin a (T gG ) k. This is an explanation.

IKG is obtained from immunized animals such as sheep, goats, mice or humans.

40mM potassium cyphthalate/10mM sulfur/lium or potassium tartrate solution (pH 5,6) 20R1 with concentrated HC
1 (12M) in a solution of [15M stannous chloride].
2 #I/ was added. This geochemical-gr was prepared by adding concentrated hydrochloric acid to 5nC12 non-oxidized pellets that did not contain dull stannous oxide on the w-plane. IOMN a OR
was added to bring the total pH of the solution to 56. Put 52 PILl of this solution into a test tube, and then add IgG (0.9%N
a solution of 10 Da/IIl in Cl) 0.48 m/I was added. The test tube was sealed to prevent introduction of atmospheric acid violet and left to stand for about 1 hour. After leaving, 99rnTc21rl was added to the above mixture α as sodium pertechnetium.
32.1 and the resulting mixture was left open for an additional 30 minutes to equilibrate the labeling reaction. Technetium-99m50
fflCI bound to less than 150 proteins, and more than 90% of the radioactivity was bound to IgG.

The advantages of the method of the invention are as follows.

a. Labeling is quick.

b. High specific activity in small amounts.

C0 is a formulation suitable for administration to humans.

d. Contamination by technetium that is not bound to the labeling material is lower than in the conventional method.

e There is sufficient bonding strength to prevent technetium from transferring to other molecules.

Example 2 This example describes a direct tagging method for forming tagged anti-beta hCG (anti-beta human chorionic gonadotropin) or anti-hCG. Anti-hcG or anti-beta hCG was obtained from 5erono Lab, Inc.

Sodium pertechnetate is New F:nglan
Obtained from company d.

50yy+ adjusted to pH 5.50 with 5omM tartaric acid
M sodium-potassium tartrate buffer (p r5.s
, 10.511/l) a4yd, 1 ONNmOH
50mM potassium cyphthalate buffer (pH 5,50, IQ, 211/l) adjusted to pH 5,50 with
6txl was added.

5 M Sn C11-HC1 (9
4,8Ji'/1, concentrated HCI) 0.02d was added.

Add 10 NNa0)IO,02ILl to this solution and back titrate the pT(pT) to 565 to 0.05.
The pT of this solution was adjusted to 5.65±(105) using H.
〃lp protein/#1!・0.3 ml of salt aqueous solution (salt water)
was added. The reaction vessel was left at room temperature for about 24 hours. Then, NaTc0 with an activity of about 20 millicuries
, 0.24 d was added to the protein-containing composition and allowed to stand for approximately 1 hour to provide substantially complete protein tagging prior to use.

The resulting product was passed through a Sephadex column to remove free technetium from the tagged protein product.

Example ■ According to the method of Example ■, IgG F was used instead of whole antibody.
eb fragment was replaced. Sephadex G-2
The yield by chromatography using a 5-80 column was approximately 8% of the Fab bound to technetium-99ff+.
It showed that 0% was recovered. F a with tag
b Antibody fragments can be converted into kits for immediate technetium tagging.

Fab1509 (0.115m of labeled mixture before freeze-drying)
A single-administration kit containing 1) was prepared.

Fab kit analysis was performed using Sephacryl S-200 (Saphacryl S-2) using a 2.5 x 952 column.
00) by high-speed chromatography. 1f
A 10-71M sodium gentisate solution with a pH of 7 was used as a fi agent. Since the chromatographic method was performed in an environment of 4°C, deterioration of the technetium tag was prevented due to the antioxidant effect of sodium gentisate. Three Fab kits were remade for each elution, each containing 50 mCl equivalent of decayed technetium (Tc-99).
) α5ILl and 50 microCurie Tc-99m tracers were included. After mixing the contents, each kit was allowed to stand for 1' hour and tested prior to the four-mad graph method using the method described below.

1. After remanufacturing, use only the contents of the Fab kit.

2. Immediately after chromatography, add 3 ml of 1% H8A (major serum albumin) to the Fab kit.

5. Immediately after the p-madography method, pass the P''abab 1 hour through a cuprous-Sephadex filter.

4. After gift reconstitution and addition of 1% HSA, pass the contents of the Fab kit through a Tin-1 Sephadex filter.

Chromatographic methods allowed reliable quantification of colloidal xIsA material, non-colloidal) ISA and Fab fragments.

The table below shows the yields of the three methods.

Table I■ Yes No 15.8 -
1482.8 No Yes! an 4
゜5 2.6! L5 59.6 Yes Yes
25.1 &5 2.2 2.4
64.8 As shown in Table ■, using this method, IgG
Te99m-tagged fragments (7 fragments such as Fab) can be prepared and there is relatively little contamination with colloidal or soluble H8A (human serum albumin). In the absence of carrier HSA, the loss of the labeled 7 fragment due to the filter is large. Low yield of Tc99m-Fab (15,8-33.5
%) is due to an artifact of the Sephacryl column chromatography required to separate the Fab from the H8A7 lactyone. Cephacryl reacts with Tc-Fab,
Approximately 2/3 of the product is lost on the column.

EXAMPLE ■ Method of Example I (Pre-Tannization Method), U.S. Pat. No. 4,057.
Abra described in the specification No. 617 (1977)
ynoviei and Ermana method (basic reduction method) and U.S. Pat. No. 4.042.626 (1977)
Molingki and W cm described in the specification
@-.

+1 Beef gamma puddings were prepared by nimki's method (sodium tartrate method) and analyzed by a Sephadex G150 column cupadographic method using 50mM phthalate buffer saturated with stannous phthalate as the elution solvent. I again. The results are shown in Table ■.

Table ■ Yield Attack Colloidal exchangeable Tc pre-stannation method 75 98 17
2B basic reduction 60 85 2
2 61 Tin-tartrate method 59 99
As shown in Table 2, the present invention has the following advantages: 0 1 High total radiochemical yield.

2. To can be reduced virtually completely (98%).

3. Contains less radiochemical impurities such as colloidal 'fc-99m.

4. The percentage of exchangeable Tc-99tH is low.

As shown in FIG. 1, in the pre-copperization method, the yield of radioactively tagged protein can be optimized by changing the total amount of stannous ions to obtain the optimum four equivalents of B n t%.

Similarly, as shown in FIG. 2, in the pretinization process, the standing or aging time, ie, the pretinization period at 25° C., is about 21 hours or more.

Furthermore, as shown in FIG. 3, in the pre-tination method, the optimum pH for labeling bovine gamma globulin is in the range of 55 to 5.6.

[Brief explanation of drawings]

Figure 1 shows the relationship between tin concentration and final technetium-tagged protein product, Figure 2 shows the relationship between pre-tannization ripening time and percent technetium bound to protein, and Figure 3 shows the relationship between pH and the final technetium-tagged protein product. FIG. 3 is a diagram showing the relationship between the percentage of technetium bound to protein and the percentage of technetium bound to protein. For procedural amendment (method) 1975, September 16 [-1 Commissioner of the Japan Patent Office Kazuo Wakasugi Display of Lordship f71 1982 Patent Application No. 75577'
;',. Patent application (Name: University Patent Incorporated (1 other person) Agent address: 〒Ii+3 □ Same address: 1-8), I-14- 1 August 31, 1980 → Type H - Inventive disease of Sodeichi's subject application ``JIYI of applicant'' by Mutsu H. -゛6'Door'-゛''゛-do departure homme makumei 1 kasen tomo + i = power of attorney and its translation Drawings for each 1 copy Contents of amendments An engraving of the same side as shown in the attached sheet (no changes to the content)

Claims (9)

[Claims] (1) A reducing composition suitable for radiolabeling with technetium-99m, comprising a protein and at least about 6
a buffer composition comprising milliequivalents of stannous ions and a mixture of an alkali metal cyphthalate and an alkali metal tartrate having a pH of about 4.5 to about F1.5 and substantially free of oxygen. , the reducing composition prepared by allowing the mixture of the protein, stannous ion, and buffer composition to stand for at least about 15 hours. (2) The composition according to claim 1, which is freeze-dried. (3) The composition according to any one of claims 1 to 2, wherein the protein is radioactively tagged with technetium-99m. (4) A diagnostic kit suitable for preparing a composition to be administered to a patient, comprising a stannous ion source and an alkali metal siftal! a gauze composition comprising a mixture of Rm and an alkali metal tartrate and having a pH of about 4.5 to 8.5; and a protein suitable for radiolabeling with technetium-99m. and a second sterile packaging container containing. (5) The composition contained in the first sterilized packaging container is freeze-dried! ? The kit according to claim 4. (6) A method for producing a reducing composition suitable for radiolabeling with technetium-99m, the composition comprising a mixture of an alkali metal cyphthalate and an alkali metal tartrate and having a pH of about 4.5 to 85. A method comprising allowing a mixture of a stannous ion source and a protein in the composition to stand for at least about 15 hours at a temperature that does not denature the protein. (7) Claim #l comprising allowing said mixture to stand for at least about 21 hours! The method described in Section 6. (8) The composition according to claim 1, wherein the protein is anti-hCG. (9) The composition according to claim 1, wherein the protein is anti-beta hCG.