JPH09143200A - 111-In labeled large agglutinating human serum albumin, its preparation method and its use - Google Patents

Abstract

(57) Abstract: A pulmonary ventilation agent using a 99m-Tc labeled substance, and a 111-In labeled pulmonary blood flow radioactive diagnostic agent applicable to a binuclear administration method are provided. SOLUTION: A labeling method in which macroaggregating human serum albumin and 111-In chloride are mixed and incubated in the presence of 111-In labeled macroaggregating human serum albumin and sodium acetate buffer, sodium acetate In the presence of a buffer or a citrate buffer, a labeling method in which 111-In and macroaggregating human serum albumin are mixed and incubated via a chelating agent such as DTPA, and labeled with 111-In prepared from the method A pulmonary blood flow radioactive diagnostic agent containing the large agglutinating human serum albumin and the large agglutinating human serum albumin labeled with 111-In.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to 111-In labeled macroaggregating human serum albumin used for pulmonary blood flow imaging such as pulmonary embolism, a method for preparing the same, and 111-In prepared from the method. And a pulmonary blood flow radioactive diagnostic agent containing the macroaggregative human serum albumin labeled with 111-In.

[0002]

2. Description of the Related Art Conventionally, in clinical settings, lungs using 131-I labeled macroaggregating human serum albumin (hereinafter referred to as 131-I-MAA) have been used for patients suspected of having pulmonary embolism and the like. Blood flow diagnosis has been carried out, but in recent years, 131-I-MAA has been replaced by 131-I-MAA because of its physical characteristics such as gamma ray emission energy of 140 KeV, half-life of 6 hours, and absence of major beta ray emission. 99m
Large-aggregating human serum albumin labeled with -Tc [hereinafter referred to as 99m-Tc-MAA (Mitsumasa Oyanagi et al., Nuclear Medicine, 16 , 927, 1979)] is widely used as a pulmonary blood flow diagnostic agent. .

In clinical practice, 99m-Tc-MA is used for the purpose of diagnosing an accurate lesion site, lesion area, etc. in a patient who is suspected to have a disease such as pulmonary embolism at the first visit.
The pulmonary blood flow diagnosis using A and the lung ventilation diagnosis such as 81m-Kr are generally performed by the binuclear administration method.

81m-K used as a lung ventilation agent
Since r has a short half-life of 13 seconds, it has problems such as limitation of transportation range. Therefore, 99m-Tc-DTPA aerosol, 99m-T, with suitable physical properties.
C-gas and the like have been studied as lung ventilation agents.

However, 99m-Tc labeled pulmonary blood flow agents and pulmonary ventilators, which have their preferred physical properties, are
Since the released energies are the same, it was impossible to use it for the binuclear administration method which continuously diagnoses pulmonary blood flow and pulmonary ventilation.

[0006]

DISCLOSURE OF THE INVENTION The present invention, in view of the above-mentioned tendency of pulmonary ventilator to shift to 99m-Tc labeled substance due to the problem of the transport range of 81m-Kr used as lung ventilator, Can be used in combination with 99m-Tc-labeled pulmonary ventilator as a pulmonary blood flow agent for dinuclide administration 111-
In-labeled macroaggregating human serum albumin, method for preparing the same, 111-In-labeled macroaggregating human serum albumin prepared by the method, and 111-In-labeling macroaggregating human serum It is intended to provide a pulmonary blood flow radioactive diagnostic agent containing albumin.

[0007]

[Means for Solving the Problems] In order to solve the above problems, the present inventors have developed a DTPA labeled with 99m-Tc.
Of pulmonary embolism and other lung diseases by the binuclide administration method with 111-In, which has physical and chemical properties similar to 99m-Tc The present inventors have completed the present invention by discovering that labeled macroaggregating human serum albumin is possible.
That is, the present invention is macroaggregating human serum albumin labeled with 111-In. Macroaggregating human serum albumin is labeled with 111-In via a chelating agent,
Alternatively, it is directly labeled with 111-In. Direct 111-
In-labeled macroaggregating human serum albumin is obtained by a labeling method in which macroaggregating human serum albumin and 111-In chloride are mixed and incubated in the presence of a sodium acetate buffer. In addition, the macroaggregating human serum albumin labeled with 111-In via the chelating agent is prepared by converting the macroaggregating human serum albumin bound with the chelating agent and the chloride of 111-In into a sodium acetate buffer or a citrate buffer. It can be obtained by a labeling method of mixing and incubating in the presence of a liquid or the like. The 111-I
The radioactive diagnostic agent containing macroaggregated human serum albumin labeled with n is effectively used as a pulmonary blood flow radioactive diagnostic agent.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION Is the large agglutinating human serum albumin (hereinafter referred to as MAA) used in the present invention produced from human serum albumin by a method well known to those skilled in the art (Japanese Patent Laid-Open No. 48218/1988)? Alternatively, commercially available MAA or the like may be used.

The labeling of MAA with 111-In is MA
The complex in which A or MAA is bound to the chelating agent is
It is possible to label with 111-In directly or indirectly by mixing in a weakly acidic aqueous solution containing -In ions. When the MAA is directly labeled with 111-In, a predetermined amount of MAA is mixed with a sodium acetate buffer solution adjusted to pH 5 to 6. Then 111-I
n-chlorides were mixed and incubated at room temperature for 10 to 200 minutes to obtain 111-In labeled MAA.
Can be prepared. Incubation is preferably completed in 10 to 20 minutes. As the buffer solution, sodium acetate-acetic acid buffer solution is preferably used.
The preferred pH of the sodium acetate buffer in the preparation method is 4
To 6 and particularly preferably pH 5 to 6.

The chelating agent for indirectly labeling MAA with 111-In may be a pharmaceutically acceptable polyaminocarboxylic acid, preferably ethylenediaminetetraacetic acid (hereinafter referred to as EDTA) and diethylenetriaminepentaacetic acid. (Hereinafter referred to as DTPA), 1, 4, 7,
10-Tetraazacyclododecane-1,4,7,10-
Tetraacetic acid (hereinafter referred to as DOTA), 1, 4, 8, 1
1-tetraazacyclotetradecane-1,4,8,11
-Tetraacetic acid (hereinafter referred to as TETA) or a derivative thereof, particularly preferably DTPA or a derivative thereof.

MAA was added to 111-In using a chelating agent.
The method of labeling with is as follows, for example. A predetermined amount of MAA is dissolved in 0.1M sodium hydrogen carbonate. Then, the chelating agent dispersed in dimethylsulfoxide was mixed with a 0.1M aqueous solution of MAA in which the chelating agent was dissolved in the chelating agent / MAA = 200/1 to 50/1 at a ratio of a large excess of the chelating agent. 5 minutes to 1
Incubate for 0 minutes at room temperature. After the incubation, the unreacted chelating agent is centrifuged to take out the chelating agent-MAA. In this case, when the chelating agent is used in the form of an acid anhydride, the chelating agent binds more effectively. Then, the chelating agent-MAA and the chloride of 111-In were added,
Sodium acetate-acetic acid buffer, sodium citrate-sodium hydroxide buffer, or potassium dihydrogen citrate-
1 at room temperature in a buffer such as sodium hydroxide buffer
By incubating for 0 to 200 minutes,
It is possible to prepare 1-In-DTPA-MAA. Incubation is preferably completed in 10 to 20 minutes. The preferred pH of the buffer solution of the preparation method is 4 to 6, particularly preferably 5 to 6.

MAA labeled with 111-In of the present invention
When used as a pulmonary blood flow radioactive diagnostic agent, impurities and unreacted 111-
In ions are removed and used.

The M-labeled with 111-In of the present invention
AA and a complex of chelate and MAA are pharmaceutically acceptable stabilizers such as ascorbic acid and p-aminobenzoic acid, pH adjusting agents such as aqueous buffers, excipients such as D-mannitol, and radiation. In addition to citric acid, tartaric acid, malonic acid, etc., which are useful for improving chemical purity, they can be provided in the form of an injection and a ready-to-use kit.

A pulmonary blood flow radioactive diagnostic agent containing a MAA complex containing a chelating agent such as 111-In labeled MAA or 111-In labeled DTPA is generally used for intravenous administration and the like. The radioactivity considered to be capable of imaging is determined in consideration of the weight and age of the patient, an appropriate radiation imaging device and the like.

Normally, 37 MBq to 111 MB for a normal person
The range is q, preferably 55 MBq to 74 MBq is applied. Hereinafter, the present invention will be described more specifically with reference to Examples. In the present specification, M
AA labeled directly with 111-In
1-In-MAA and MAA labeled with DTPA are referred to as 111-In-DTPA-MAA. or,
To demonstrate the efficacy of the 111-In labeled macroaggregating human serum albumin of the present invention in a binuclear dosing regimen, 9
Although it is preferable to use a lung ventilation agent labeled with 9m-Tc, in this example, since it is performed using a mouse, it is changed to a lung ventilation agent, and 99m-Tc used as a liver diagnostic agent is used. Performed with labeled phytic acid.

[0016]

【Example】

(Example 1) Preparation of 111-In-MAA Macrokit ^™ (manufactured by Dynabot) containing 2.0 mg of MAA, 0.23 mg of stannous chloride, and 9 mg / ml of benzyl alcohol. 0 ml was centrifuged (4 ° C., 3000 rpm) for 1 minute to separate the supernatant of the centrifuge tube from the bottom solid. Then, the separated solid is treated with a phosphate buffer solution (hereinafter referred to as PBS) consisting of 1.4 mM potassium dihydrogen phosphate, 4.3 mM disodium hydrogen phosphate, 2.7 mM potassium chloride, and 137 mM sodium chloride. Then, it was washed three times and divided into two. While containing 1.0 mg of this MAA, 136 g of sodium acetate trihydrate was dissolved in 1000 ml of purified water to obtain 1.0
Mix with 500 μl of 0.1 M sodium acetate, pH adjusted to 5.8 with M acetic acid. The other containing 1.0 mg of MAA was used as a negative control and
Mix with 0 μl. Next, 500 mL of indium chloride (111-In) was added to each of the two MAA mixed solutions.
Mix 1 μl at room temperature for 15, 30, 60 and 18 minutes
Incubate for 0 minutes, then at room temperature for 10 minutes, 150
After centrifuging at 00 rpm, the radioactivity of the supernatant containing 111-In and the solid at the bottom containing 111-In-MAA was measured by a well type gamma counter, and 1
The labeling rate of 11-In-MAA was evaluated. The labeling rate (%) was calculated by {(count of solids / min) / [(count of solids / min) + (count of supernatant / min)]} × 100. Following are 15 minutes of MAA in 0.1 M sodium acetate buffer, pH 5.8 and MAA in PBS, 30 min.
The labeling rates for minutes, 60 minutes and 180 minutes are shown in Table 1.

From Table 1, it can be seen that the pH of 5.8 containing MAA was 0.
Indium chloride (111) in 1M sodium acetate buffer
-In) incubated and labeled 111-In
-MAA is 9 minutes after 15 minutes of incubation.
The labeling rate was as high as 6.0% or more. In addition, the labeling rate of 96.0% or higher was maintained 180 minutes after the start of incubation. From the above, the effectiveness of the method for preparing 111-In-MAA of the present invention by incubating MAA and indium chloride (111-In) in a 0.1 M sodium acetate buffer solution of pH 5.8 of the present invention was shown.

[0018]

[Table 1] * PH 5.8, 0.1M sodium acetate buffer ** PBS

Example 2 Introduction of Bifunctional Crosslinking Agent (DTPA) into MAA Macrokit ^™ containing 2.0 mg MAA, 0.23 mg stannous chloride, and 9.0 mg / ml benzyl alcohol. The solution (manufactured by Dynabot) was centrifuged for 1 minute (4 ° C., 3000 rpm) to separate the supernatant from the bottom solid, and the obtained solid was dissolved in 1000 μl of 0.1 M sodium hydrogen carbonate. Next, DTP dissolved in 50 μl of dimethyl sulfonic acid (hereinafter referred to as DMSO)
A acid anhydride and the previously obtained MAA solution were combined with DTPA: MA
The mixture was mixed at a molar ratio of A = 100: 1 and incubated at room temperature for 5 minutes. DTP without introduction of MAA
MAA chelated by A and DTPA (hereinafter, DTPA-
MAA) is separated by centrifugation for 1 minute (4 ° C, 3
000 rpm) and the bottom solid containing DTPA-MAA is pH 5. consisting of 93.5 mM trisodium citrate dihydrate and 6.5 mM citric acid monohydrate.
8 was dissolved in 1.0 ml of 0.1 M citrate buffer.

(Example 3) Preparation of 111-In-DTPA-MAA DTPA-MAA obtained in Example 2 was mixed with 93.5 m
M trisodium citrate dihydrate and 6.5 mM citric acid monohydrate dissolved in 1.0 ml of 0.1 M citrate buffer solution having a pH of 5.8, and carrier-free indium chloride (111-In). ) After mixing with 1.0 ml, it was incubated at room temperature for 30 minutes and 180 minutes. Then, the mixture was centrifuged at room temperature for 10 minutes at 15000 rpm, and the radioactivity of the supernatant containing 111-In, 111-In-DTPA and the bottom solid containing 111-In-DTPA-MAA was measured by well type gamma. It measured with the counter and evaluated the labeling rate of 111-In-DTPA-MAA. Labeling rate (%) is {(solid count /
Min) / [(count of solids / min) + (count of supernatant / min)]} × 100. Table 2 shows the results. From Table 2, DTPA-M in the presence of citrate buffer
111-In-DTPA-MAA obtained by mixing AA and indium chloride (111-In) and incubating
Showed a labeling rate of 97.0% or more 30 minutes after the start of incubation. In addition, 18
The labeling rate of 96.0% or more was maintained even at the 0 minute point. From the above, preparation of 111-In-DTPA-MAA in which the complex of macroaggregating human serum albumin bound with a chelating agent and 111-In chloride are mixed and incubated in the presence of the citrate buffer of the present invention The effectiveness of the method was demonstrated.

[0021]

[Table 2] * PH 5.8, 0.1M citrate buffer

(Example 4) In vitro stability of 111-In-MAA 111-In-MAA obtained in Example 1 was treated with human normal serum albumin (hereinafter referred to as HSA) or PBS.
The resulting mixture was mixed with, incubated at room temperature for 15 minutes and 180 minutes, and then centrifuged for 10 minutes at 15000 rpm to obtain a supernatant containing 111-In, MAA and HSA, and a bottom solid containing 111-In-MAA. Was measured by a well-type gamma counter for 111
The stability of -In-MAA was evaluated. 111-I
The stability (%) of n-MAA is {(solid count /
Min) / [(count of solids / min) + (count of supernatant / min)]} × 100. Table 3 shows the results. From Table 3, the 111-In-MAA prepared by the direct labeling method of 111-In of the present invention was prepared by using HSA or P
Even when incubated with BS for 180 minutes, a high labeling rate of 93.0% or higher was maintained, and 111-In
It was suggested that there was almost no transfer of the compound to HSA or PBS, and the high stability of 111-In-MAA of the present invention was confirmed.

[0023]

[Table 3]

(Example 5) In vitro stability of 111-In-DTPA-MAA The 111-In-DTPA-MAA obtained in Example 3 was mixed with HSA or PBS and allowed to stand at room temperature for 15 minutes and 18 minutes.
Incubate for 0 minutes, then 10 minutes, 1500
Centrifuged under the condition of 0 rpm, 111-In, 111-
Supernatant containing In-DTPAMAA and HSA, 11
The radioactivity of the bottom solid containing 1-In-DTPA-MAA was measured by a well type gamma counter, and 111
An evaluation of the stability of -In-DTPA-MAA was performed.
The stability (%) of 111-In-DTPA-MAA is
{(Solid count / min) / [(solid count / min)
+ (Count of supernatant / min)]} × 100. Table 4 shows the results. From Table 4, 111-In prepared by the indirect labeling method via the chelating agent of the present invention.
-DTPA-MAA is 18 with HSA or PBS
A high labeling rate of 94.0% or higher was maintained even after 0 minute incubation, and 111-In HSA or PB was maintained.
It was suggested that almost no transition to S was observed, and the high stability of 111-In-DTPA-MAA of the present invention was confirmed.

[0025]

[Table 4]

(Example 6) Pharmacokinetics of 111-In-MAA in normal mice 111-In-MAA (37MBq) obtained in Example 1
/ Mg / ml, incubation time: 30 minutes) 7
0 μl was administered to 6-week-old female Balb / c mice (n = 8) via the tail vein. One group (n = 4) was given general anesthesia 15 minutes after administration, exsanguinated from the heart and sacrificed. In addition, the other group (n = 4) was sacrificed in the same manner as the previous group 180 minutes after the administration. Then, in each of the two groups, the weight of each organ was measured and the radioactivity was measured by a well type gamma counter. The pharmacokinetics were evaluated by calculating the% ID / g of the organ (Table 5) and the organ / blood ratio (Table 6) converted to a standard body weight of 20 g.

In Table 5, 15 minutes after administration and 180
The% ID / g of the organ of the equinox is 653.1% ID /
g, and 645.5% ID / g, showing high lung specificity. Also, in Table 6, 15 minutes and 18 points after administration.
The organ / blood ratios at 0 minutes were 728.8 and 51, respectively.
1.1 and 0.41 of the liver / blood ratio at 15 minutes and 180 minutes after administration, which is the background of the lung, and 0.
Compared with 78, it showed a very high organ / blood ratio. From the above, the effectiveness of 111-In-MAA of the present invention as a pulmonary blood flow agent was shown. The organ / blood ratio is (% I of organ
D / g) / (% ID of blood / g).

[0028]

[Table 5]

[0029]

[Table 6]

(Example 7) 111-In-DTPA-MA using a normal mouse
Pharmacokinetics of A 111-In-DTPA-MAA obtained in Example 3
(37 MBq / mg / ml, incubation time:
100 μl of (30 minutes) was administered to 6-week-old female Balb / c mice (n = 8) via the tail vein. One group (n
= 4), 15 minutes after the administration, general anesthesia was applied, and the blood was discharged from the heart and sacrificed. Also, the other groups (n = 4)
180 minutes after the administration, the animals were sacrificed in the same manner as the previous group. Then, in each of the two groups, the weight of each organ was measured and the radioactivity was measured by a well type gamma counter. The pharmacokinetics was evaluated by the% ID of the organ converted to a standard body weight of 20 g.
/ G (Table 7) and organ / blood ratio (Table 8) were obtained and evaluated.

In Table 7, 15 minutes and 180 minutes after administration
% ID / g of organs at equinox are 727.0% ID / g each
g, and 753.0% ID / g, showing high lung specificity. Also, in Table 7, 15 minutes after administration and 180 minutes
The equilibrium organ / blood ratios are 1153.9 and 876.
4, which was an extremely high organ / blood ratio, as compared to the liver / blood ratios of 0.49 and 0.56 at the 15-minute and 180-minute points after administration, which became the background of the lung. From the above, the effectiveness of 111-In-DTPA-MAA of the present invention as a pulmonary blood flow agent was shown. The organ / blood ratio was calculated by (% ID of organ / g) / (% ID of blood / g).

[0032]

[Table 7]

[0033]

[Table 8]

(Example 8) Imaging of normal mouse with 111-In-MAA 111-In-MAA (37 MBq obtained in Example 1)
/ Mg / ml, incubation time: 30 minutes) 7
0 μl was administered via the tail vein of 6-week-old female Balb / c mice. Fifteen minutes after the administration, whole-body imaging was performed with a ZLC7500 gamma camera (manufactured by Siemens) equipped with a medium energy collimator, and processed with Cintipack 700 (manufactured by Shimadzu) equipped with 128 × 128 matrix. In addition, 99m-Tc-Futic acid (3
50 μl of 7 MBq / ml) solution was administered to the same mouse,
Whole-body imaging was performed with the above-mentioned device. Then
The image of 99m-Tc-Futic acid was subtracted from the overlapping image of 111-In-MAA and 99m-Tc-Futic acid to correct the image.
It was confirmed that n-MAA clearly images mouse lungs at 15 minutes after administration. The energy window is 111-In (245 keV) and 99 m-Tc.
(141 keV) was performed at 10%.

(Example 9) Imaging of normal mouse with 111-In-DTPA-MAA 111-In-DTPA-MAA obtained in Example 3
100 μl of (37 MBq / mg / ml) was administered via the tail vein of 6-week-old female Balb / c mice. 15 minutes after administration ZLC7 with medium energy collimator
Whole body imaging was performed with a 500 gamma camera (manufactured by Siemens) and processed with a Cintipack 700 (manufactured by Shimadzu) equipped with a 128 × 128 matrix. Furthermore,
In order to correct the position of the lungs of the mouse, 100 μl of a 99m-Tc-futic acid (37 MBq / ml) solution for liver imaging was administered to the mouse, and whole-body imaging was performed using the above-mentioned device. Then, 111-In-DT
The image of 99m-Tc-Futic acid was subtracted from the overlapping image of PA-MAA and 99m-Tc-Futic acid, and the image was corrected. As a result, 111-In-DT
It was confirmed that PA-MAA clearly images mouse lungs at 15 minutes after administration. The energy window is 111-In (245 keV) and 99 m-T.
Performed at 10% of c (141 keV).

[0036]

INDUSTRIAL APPLICABILITY According to the present invention, 111m-In-labeled macroaggregating human serum albumin and sodium acetate buffer which can be effectively used in a binuclide administration method together with a pulmonary ventilation agent using a 99m-Tc-labeled substance. In the presence of a liquid, a labeling method in which 111-In chloride and macroaggregating human serum albumin are mixed and incubated, and in the presence of a sodium acetate buffer or a citrate buffer, a macroaggregating property is achieved via a chelating agent such as DTPA. Labeling method in which human serum albumin and 111-In chloride are mixed and incubated, 111-In-labeled macroaggregating human serum albumin prepared from the method, and 111-In-labeling macroaggregating human It has become possible to provide a pulmonary blood flow radioactive diagnostic agent containing serum albumin.

[Brief description of the drawings]

FIG. 1 111-In-MA using a normal mouse
Fig. 3 is a systemic scintigram (form of organism) of 111-In-MAA at 15 minutes after administration by A and a binuclear administration method of 99m-Tc-futic acid.

FIG. 2 111-In-MA using normal mouse
FIG. 7 is a systemic scintigram (form of organism) of Tc-99m-futic acid at 15 minutes after administration by A and a binuclear administration method of 99m-Tc-futic acid.

FIG. 3 111-In-DT using a normal mouse
111-In-D at 15 minutes after administration by PA-MAA and binuclear administration method of 99m-Tc-Futic acid
It is a whole body scintigram (form of organism) of TPA-MAA.

FIG. 4 111-In-DT using a normal mouse
It is a whole body scintigram (form of organism) of 99m-Tc-Futic acid 15 minutes after administration by PA-MAA and a binuclide administration method of 99m-Tc-Futic acid.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical display location C07M 5:00

Claims (9)

[Claims] 1. A macroaggregating human serum albumin labeled with 111-In. 2. The method according to claim 1, wherein 111-In is bound to macroaggregating human serum albumin via a chelating agent.
Macroaggregating human serum albumin labeled with 11-In. 3. The 111-In-labeled macroaggregating human serum albumin according to claim 2, wherein the chelating agent is diethylenetriaminepentaacetic acid. 4. A macroaggregating human serum albumin is mixed with 111-In chloride in the presence of a sodium acetate buffer,
The method according to claim 1, wherein the incubation is performed.
A method for preparing 11-In-labeled macroaggregating human serum albumin. 5. The sodium acetate buffer solution having a pH of 4 to 6
The method for preparing 111-In-labeled macroaggregating human serum albumin according to claim 4, wherein 6. A chelating agent-conjugated complex of human serum albumin and 111-In chloride are mixed and incubated in the presence of a sodium acetate buffer or a citrate buffer. The method for preparing macroaggregating human serum albumin labeled with 111-In according to claim 1, 2 or 3. 7. The method according to claim 6, wherein the pH of the sodium acetate buffer or citrate buffer is in the range of 4 to 6.
-A method for preparing In-labeled macroaggregating human serum albumin. 8. A 111-In-labeled macroaggregating human serum albumin prepared by the method according to claim 4, 5, 6 or 7. 9. The 111-I according to claim 1, 2, 3 or 8.
A pulmonary blood flow radioactive diagnostic agent containing macroaggregating human serum albumin labeled with n.