CA1340556C

CA1340556C - High molecular compound comprising unit of asialoglyco-protein acceptor-directing compound and unit of chelate-forming compound chemically bonded thereto, and its utilization

Info

Publication number: CA1340556C
Application number: CA000555608A
Authority: CA
Inventors: Komei Washino; Miki Kurami; Nobuo Ueda
Original assignee: Nihon Medi Physics Co Ltd
Current assignee: Nihon Medi Physics Co Ltd
Priority date: 1986-12-30
Filing date: 1987-12-30
Publication date: 1999-05-25
Anticipated expiration: 2016-05-25
Also published as: ES2053517T3; AU601536B2; DK693987A; DK172391B1; KR880007567A; EP0273452A3; EP0273452B1; EP0273452A2; US5032678A; US5118798A; US5089604A; KR960001742B1; DK693987D0; AU8314387A; DE3789432D1; DE3789432T2

Abstract

A high molecular weight compound useful as a non-radioactive carrier, which comprises at least one unit of (1) an asialoglycoprotein acceptor-directing compound and at least one unit of (2) a chelate-forming compound chemically bonded thereto, and which may be labeled with a radioactive metallic element to give a radioactive metallic element-labeled product useful as a radioactive diagnostic or therapeutic agent for the treatment of liver disorders.

Description

- l - 13~0~S~

HIGH MOLECULAR WEIGHT COMPOUND COMPRISING A UNIT OF
ASIALOGLYCOPROTEIN ACCEPTOR-DIRECTING COMPOUND AND A UNIT OF
CHELATE-FORMING COMPOUND CHEMICALLY BONDED THERETO, AND ITS
UTILIZATION
The present invention relates to a high molecular weight compound comprising a unit of an asialoglycoprotein acceptor-directing compound and a unit of a chelate-forming compound chemically bonded thereto, and its utilization.
More particularly, it relates to a non-radioactive carrier for a radioactive metallic element which comprises a high molecular weight compound comprising a unit of an asialoglyco-protein acceptor-directing compound and a unit of a chelate-forming compound chemically bonded thereto, and a nuclear medicine, e.g. a radioactive diagnostic or therapeutic agent prepared therefrom.
An asialoglycoprotein acceptor is a protein having a molecule-distinguishing ability and is called "animal lectinn. It is widely present in animal cells, particularly hepatic cells. An asialoglycoprotein acceptor isolated from human hepatic cells constitutes a single polypeptide having a molecular weight of about 40,000 and can recognize a glyco-protein having a galactose residue at the non-reductive terminal position of the saccharide chain (i.e. asialoglyco-protein).
While the physiological functions of asialoglycoproteinacceptors are still uncertain, it is known that acceptors such as those existing at the surfaces of hepatic cells combine with a : r,~

.. . ... ~ . . ~ . .

glycoprotein in the liver blood stream to form a complex, which is taken into and transported through the cells, during which it is dissociated in a lysosome. Thus, it is believed that an asialoglycoprotein acceptor would partici-pate in the metabolism of a glycoprotein. In fact, anincrease in the level of asialoglycoprotein in the blood is observed in the case of hepatic diseases, e.g. chronic hepatitis, liver cirrhosis and hepatic cancer. Further, a decrease in the quantity of asialoglycoprotein acceptor is observed in an experimental model of hepatic disorders induced by the administration of chemicals. In view of these phenomena, it may be possible to diagnose hepatic diseases through an assessment of the quantity and quality of an asialoglycoprotein acceptor determined by the use of an asialoglycoprotein-like substance, i.e. an asialoglyco-protein acceptor-directing compound.
In the field of nuclear medicine, there have been widely used physiologically active substances labeled with iodine-131 (131I), e.g. 131I-labeled serum albumin~

131I-labeled fibrinogen and 131I-labeled tumor specific antibody for the purpose of imaging specific organs, detection of physiological abnormalities, dynamic study of certain body systems, radiation therapy of tumors, etc.
However, iodine-131 has a long half life of about 8 days and 25 emits beta-rays so that the patient treated therewith is exposed to a large radiation dose. In addition, iodine-131 is apt to be deiodinated from physiologically active substances in living bodies so that normal organs may be , ., ~, ... .... . . . . . ~ . . . . ..

~ 3 ~ 13~0550 damaged by radiation.
In order to overcome the above drawbacks in I-labeled physiologically active substances, attempts have been made to provide radiopharmaceuticals comprising 5 physiologically active substances and radioactive metallic elements having more favorable physical properties than iodine-131 combined thereto. Among such attempts, there is known a labeling method wherein a physiologically active substance is treated directly with a radioactive metal salt to make a chelate compound, which may be used as a radio-active diagnostic agent. For instance, human serum albumin is treated with an aqueous solution containing technetium-99m (99mTc) in the form of pertechnetate in the presence of a reducing agent to give 99mTc-labeled human serum albumin.
Further, for instance, bleomycin is treated with an aqueous solution containing indium-111 (111In) in the form of indium chloride to give 111In-labeled bleomycin. However, the chelate-forming properties of these physiologically active substances is not sufficient, and the once formed chelating bond is readily broken. In fact, 99mTc-labeled serum albumin and 111In-labeled bleomycin are low in stability after administration into living bodiessuch that the behavior of the radioactivity in such bodies does not coincide with that of serum albumin or bleomycin as the physiologically active substance. This is a fatal defect for the nuclear medical diagnosis based on the exact trace of the behavior of the radioactivity which should coincide with the behavior of the physiologically active substance.

.. .. ,~ .. ......... . .. ~, _ 4 _ 13405~

Attempts have been made to label an asialogalacto-protein acceptor-directing compound as a physiologically active substance with a radioactive metallic element such as technetium-99m according to conventional labeling procedures. For instance, neogalactoalbumin (galactose-combined serum albumin; NGA) was labeled with technetium-99m through the residue of cysteine, lysine, glutamic acid or the like in its molecule. However, the labeling rate of the labeled product as well as the stability of such product in vitro and in vivo are not satisfactory. With the progress of the instability, there are produced such impurities as colloidal technetium dioxide (99mTco2). These impurities are taken into the reticuloendothelial system of the liver so that correct assessment of the behavior of neogalactoalbumin becomes difficult or impossible. While the stability is surely enhanced by the use of a larger amount of a stannous salt as a reducing agent on the labeling, the stannous salt is apt to form a colloidal substance atthe acidic pH region under which labeling is effected, and such a c~llo;~l sub-stance makes precise imaging difficult. The use of neo-galactoalbumin in an excessive amount is effective to combine free stannous salt thereto so that the precise imaging may be made possible, but in such a case,the radio-activity concentration becomes lower.
As a result of extensive study, it has been found that a high molecular weight compound comprising a unit of an asialoglycoprotein acceptor-directing compound and a unit of a chelate-forming compound chemically bonded thereto is ~ .. .. .
~ . .. . . .. . .. ... ... . ~ . , 1340.S56 useful as a non-radioactive carrier for a radioactive metallic element. A radioactive metallic element-labeled high molecular weight compound obtained by labeling said high molecular weight compound with a radioactive metallic element 5 shows a high radioactivity with a high stability in vitro and in vivo. Therefore, it can assure a satisfactory diagnosis or treatment with the use of a relatively small amount.
Thus, said labeled product is useful as a nuclear medicine, e.g. a radioactive diagnostic or therapeutic agent, parti-10 cularly for the diagnosis or treatment of liver diseases.Quite advantageously, this technique is widely applicable to asialoglycoprotein acceptor-directing compounds, i.e. not only those having a structure capable of bonding a radioactive metallic element directly thereto (e.g. neogalactoalbumin) 15 but also those not having said structure in itself.
According to the present invention, there is provided (A) a high molecular weight compound useful as a non-radio-active carrier for a radioactive metallic element, which comprises at least one unit of (1) an asialoglycoprotein 20 acceptor-directing compound and at least one unit of (2) a chelate-forming compound chemically bonded thereto as well as (B) a radioactive metallic element-labeled high molecular weight compound useful as a radioactive diagnostic or thera-peutic agent, which comprises said high molecular weight 25 compound (B) and (3) a radioactive metallic element chelate-bonded thereto.
The asialoglycoprotein acceptor-directing compound (1) is a compound having a binding affinity to an asialo-glycoprotein acceptor in a living body, of which a typical .

., , example is neogalactoalbumin. Neogalactoalbumin can be separated from natural sources and can also be synthesized from serum albumin and galactose, both being obtainable commer-cially in highly pure states. Other examples are asialo-5 glycoproteins (e.g. asialoorosomucoid, asialofetuin,asialocelluloplasmin, asialohaptoglobin), galactose-bonded polylysine, galactose-bonded polyglucosamine, etc.
As the chelate-forming compound (2), there may be used any one which has a functional group (e.g. amino, 10 carboxyl, formyl, mercapto) capable of reacting with the asialoglycoprotein acceptor-directing compound (1) under relatively mild con~i~ons and a structure capable of forming a strong chelate bond with radioactive metallic element (3). Those whichha~ a bifunctional moiety capable of 15 combining with a radioactive metallic element through a chelate bond are especially preferred. Specific examples are deferoxamine, diethylenetriaminepentaacetic acid of the formula:

HOOCCH2 ~CH2COOH

and its cyclic anhydride, ethylenediaminetetraacetic acid succinimide ester of the formula:
HOOCCH2 f H2COOH

N-CH2CH2-N ~'~

2-propionaldehyde-bis(thiosemicarbazone) derivatives of the . _ . .. ... . .

1340~

formula:
Rl S

HOOC-C-C=N-NH-C-NH-R

R3-C=N-NH-C-NH-R
S

wherein Rl, R2, R3 and R4 are each a hydrogen atom or a Cl-C3 alkyl group, 3-aminomethylene-2,4-pentadione-bis(thiosemicarbazone) derivatives of the formula:

CH3 f =N-NH-C-NH-R
NH2-CH=C
CH3-C=N-NH-C-NH-R
ll wherein R5 is a hydrogen atom, a C1-C3 alkyl group or a phenyl group, l-(p-aminoalkyl)phenylpropane-1,2-dione-bis(thiosemicarbazone) derivatives of the formula:

S

NH2-(CH2)n ~ C=N-NH-C-NH-R
C=N-NH-c_NH_R6 wherein R6 is a hydrogen atom or a Cl-C3 alkyl group and n is an integer of 0 to 3, etc.
Even compounds .having a metal capturing property to form a chelate but not a functional group capable of reacting with the asialoglycoprotein acceptor-direct-ing compound (1) under mild conditions may be used as the chelate-forming compound (2) after modification to intro-. . ... , . " .. .. ... , .. ,, ~ ...... . ~ ., .

1 3 4 0 ~ ~ ~

duce the functional group therein. Examples of such compounds include dimercaptoacetylethylenediamine, bisamino-ethanethiol, N,N'-bis(2-hydroxyethyl)ethylenediamine, etc.
The above chelate-forming compounds (2) are all of relatively low molecular weight and have only a single chelate-forming structure; namely, they are "lower" molecular weight compounds. When the asialoglycoprotein acceptor-directing compound (1) has many functional groups reactive to the chelate-forming compound (2), the high molecular weight compound (A) as obtained can also have many units of the chelate-forming compound (2) so that a sufficiently high radioactivity concentration as necessary for the diagnosis or therapy will usually be retained. When, however, the asialoglycoprotein acceptor-directing compound (1) has only a single or a few functional groups, the high molecular weight compound (A) can also have only a single or few units of the chelate-forming compound (2) so that the high radioactivity concentration required for diagnosis or therapy is difficult to maintain. In order to overcome this drawback, the chelate-forming compound (2) may be constructed from a polymeric compound having a number of functional groups and a number of said chelate-forming compounds of lower molecular weight chemically bonded thereto, whereby the resulting high molecular weight compound (A) can retain a high radioactivity concentration. In this case, the chelate-forming compounds (2) are of relatively high molecular weight and have a number of chelate-forming structures; namely, they are "higher" molecular weight compounds.

13~0j'~o Examples of polymeric compounds having a number of functional groups usable for production of the chelate-forming compound (2) of higher molecular weight are polysaccharides, e.g. pentosanes, hexosanes, polyglyco-samines, polyuronic acids, glucosaminoglycanes, glycourono-glycanes and heterohexosamines. More specifically, there are exemplified amylose, amylopectin, dextrane, cellulose, inulin, pectinic acid, prurane derivatives, etc. Other examples are polyacrolein derivatives, polysuccinimide derivatives, polyamine derivatives, polylysinepolyimine derivatives, etc.

For production of the high molecular weight compound ~A), the asialoglycoprotein acceptor-directing compound (1) and the chelate-forming compound (2) may be subjected to a chemical reaction directly or through a crosslinking agent by a per _ conventional procedure, followed by purification (e.g. dialysis, salting out, gel filtration, ion exchange chromatography, electrophoresis) in a per se conventional manner. The number of molecules of the chelate-forming compound (2) to be combined to one molecule of the asialo-glycoprotein acceptor-directing compound (1) is not limitative as long as the desirable physicological properties of the latter are substantially maintained. It is usually preferred to be 30 or less. Particularly, when the chelate-forming compound (2) is a higher molecular weight compound as explained above, the number of molecules of the chelate-forming compound (2) may be 10 or less per one molecule of the asialoglycoprotein acceptor-directing compound ~1).

13~0~5~

Alternatively, the high molecular weight compound (A) may be produced by reacting a portion of the asialo-glycoprotein acceptor-directing compound (1) with the chelate-forming compound (2) optionally in the presence of a 5 crosslinking agent and then reacting the resultant product with the remaining portion of the asialoglycoprotein acceptor-directing compound (1).
Taking neogalactoalbumin as an example of the asialo-glycoprotein acceptor-directing compound (1) and diethylene-10 triaminepentaacetic acid cyclic anhydride as an example ofthe chelate-forming compound (2), a typical procedure for preparation of a neogalactoalbumin (NGA)-diethylenetriamine-pentaacetic acid (DTPA) combined product as the high molecular weight compound (A) will be hereinafter explained in detail.
To human serum albumin (HSA; a commercially available injectionable preparation of human serum albumin preparation), phosphate buffer and DTPA cyclic anhydride are added, followed by stirring at room temperature for several minutes. Borate buffer is added thereto to adjust the pH to give a solution 20 of an HSA-DTPA combined product. Separately, a methanolic solution of sodium methoxide is added to cyanomethyl-thio-galactose, followed by stirring at room temperature for about hours. Evaporation of the methanol gives 2-imino-methoxy-l-thiogalactose. The above prepared HSA-DTPA solution 25 is added thereto and the mixture is allowed to stand at room temperature for 24 hours. Acetic acid is added thereto to interrupt the reaction, and pH is adjusted to give a solution , ~

.. . . . . . . .

1340~S~

of an NGA-DTPA combined product. The thus prepared NGA-DTPA
combined product is considered to have the following chemical structure:

(Galactose)n-(HSA)-(DTPA)M
wherein m and n each indicate an integer of 1 to 50 but m + n is an integer of 2 to 50. NGA
The thus obtained high molecular weight compound (A) is useful as a non-radioactive carrier for a radioactive metallic element and may be labeled with any appropriate radioactive metallic element to give a radioactive metallic element-labeled high molecular weight compound (B), which is itself useful as a radioactive diagnostic or therapeutic agent. Namely, the high molecular weight compound (A) has a chelate-forming structure originated from the chelate-forming compound (2), and such structure can firmly capture a radioactive metallic element (3) by a chelate bond. There-fore, even such an asialoglycoprotein acceptor-directing compound (1) as has itself not been labeled with a radio-active metallic element can be well labeled to give a stable labeled product.
The radioactive metallic element (3) covers any metallic element having radioactivity, which has physical and/or chemical characteristics suitable for nuclear medical diagnosis or therapy and can be readily captured with the chelate-forming structure in the chelate-forming compound (2). Specific examples of the radioactive metallic element for diagnostic purposes are gallium-67 (67Ga), gallium-68 - 12 - 1340~6 (63Ga) thallium 201 (201T1) indium 111 (1~
technetium-99m ( 9mTc), copper-62 (62Cu), etc. Specific examples of the radioactive metallic element for therapeutic p ~ oses are yttrium-90 (90Y), palladium-109 ( Pd), rhenium-186 (186Re), gold-198 (198Au) bi (212Bi), etc. They are normally employed in their salt forms, particularly in their water-soluble salt forms.

Depending upon the type or state of the radioactive metallic element (3), two different labeling procedures may be adopted. When the radioactive metallic element (3) is in a valency state which can form a stable chelate compound, the high molecular weigh- compound (A) may be contacted with the radioactive metallic element (3) in an aqueous medium to form the radioactive meta;lic element-labeled high molecular weight compound (B). This labeling manner may be applied to 67Ga, lllIn, etc. ~hen the radioactive metallic element (3) is in a valency state which has to be changed for the formation of a stab'e chelate compound, the high molecular weight compound (A) may be contacted with the radioactive metallic element (3) in an aqueous medium in the presence of a reducing agent or an oxidizing agent to form the radioactive metallic element-labeled high molecular weight compound (B). This labeling manner may be applied to 99mTc, etc.
25' Examples of the reducing agent are stannous salts, i.e. salts of divalent tin ion (Sn ). Specific examples are stannous halides (e.g. stannous chloride, stannous fluoride), stannous sulfate, stannous nitrate, stannous , . ~, . .. , , . , , , . . , . . . . , . , ~ , . .. ..

1340.55~

acetate, stannous citrate, etc. Sn++ ion-bearing resins, e.g. ion-exchange resins charged with Sn ion, are also suitable. Examples of the oxidizing agent are hydrogen peroxide, etc.
When, for example, the radioactive metallic element (3) is 99mTc, the high molecular weight compound (A) may be treated with 99mTc in the form of a pertechnetate in an aqueous medium in the presence of a reducing agent, e.g. a stannous salt. There is no particular requirement concerning 10 the order of the introduction of the above reagents into the reaction system. Usually, however, initial mixing of the stannous salt with the pertechnetate in an aqueous medium should be avoided. The stannous salt may be used in an amount that can sufficiently reduce the pertechnetate.
The high molecular weight compound (A) and the radio-active metallic element-labeled high molecular weight compound (B) above obtained are useful as a non-radioactive carrier and as a radioactive diagnostic or therapeutic agent, respec-tively. They are sufficiently stable and therefore may be stored as such and supplied on demand. In the most practical manner, the high molecular weight compound (A) is stored as such or in the form of an aqueous solution or a lyophilized powder and, in use, is combined with the radioactive metallic element (3) in an aqueous medium to make the radioactive metallic element-labeled high molecular weight compound (B).
When desired, the non-radioactive carrier as well as the radioactive diagnostic or therapeutic agent may contain any suitable additive, for example a pH controlling agent (e.g.

1340~6 an acid, a base, a buffer), a stabilizer (e.g. ascorbic acid) or an isotonizing agent (e.g. sodium chloride) in addition to the major component.
The radioactive metallic element-labeled high molecular weight compound (B) is useful for nuclear medical diagnosis or therapy, particularly in the diagnosis and treatment of liver diseases. For this purpose, the radioactive metallic element-labeled high molecular weight compound (B) is usually administered to living bodies through an intravenous route in an amount sufficient to produce radioactivity effective for diagnostic or therapeutic purposes. However, any other route which is advantageous for the exhibition of its physical activity may be adopted. For instance, the intravenous administration of a 99mTc-labeled 15 product in an amount of about 0.5 to 5 ml, particularly about 1 to 3 ml, having a radioactivity of about 0.1 to 50 mCi, particularly about 1 to 20 mCi, to a patient is quite suitable for diagnostic purposes.
The advantages of the high molecular weight compound (A) of this invention, which is useful as a non-radioactive carrier, may be summarized as follows: (a) it is stable over a long period of time after manufacture; (b) since it can be produced under mild conditions, no unfavorable side reactions, e.g. inactivation, denaturation or decomposition are caused; (c) any asialoglycoprotein acceptor-directing compound can be used as the starting material; (d) the radioactive metallic element-labeled high molecular weight compound (B) can be formed by a very simple procedure, e.g.

.. . . . .. . .... .. . .. .... . .. . .. .. . . .. .

1340~S6 by merely contacting with a radioactive metallic element in an aqueous medium. The advantages of the radioactive metallic element-labeled high molecular weight compound (B) useful as a radioactive diagnostic agent may also be summarized as follows: (a) it is stable over a long period of time after manufacture; (b) the labeling efficiency with the radioactive metallic element is extremely high; (c) since the labeling operation is quite simple, no unfavorable side reactions, e.g. inactivation, denaturation or decomposition are caused; (d) among various radioactive metallic elements, the most suitable one for diagnostic or therapeutic purposes may be chosen; (e) high and stable radioactivity can be obtained in a relatively small amount.
The radioactive metallic element-labeled high molecular weight compound (B) is particularly useful as a diagnostic agent for the imaging of an organ or tissue having an asialoglycoprotein acceptor, detection of a disease producing any modification of the quantity and/or quality of an asialoglycoprotein acceptor and dynamic examination of an asialoglycoprotein acceptor. When, for instance, it comprises 99mTc as the radioactive metallic element (3) and is used as a liver-imaging agent, 99mTc is firmly bonded through a chelate-bond so that it is accumulated in the liver in a stable state for properly a long time, during which specto-imaging photography can be readily made. Yet, the radioactivity is excreted from the human body so quickly so as not to afford any unfavorable influence on the human body and does not prevent the purpose of diagnosis. In addition, .. .. .~ .

1340~

it is advantageous that the toxicity and the antigenicity are sufficiently low.
Practical and presently preferred embodiments of the invention are illustratively shown in the following Examples 5 wherein % is by weight unless otherwise indicated.
The examples make reference to the appended figures so for the sake of convenience the figures will be introduced briefly as follows:
Fig. 1 is a graph plotting the distribution 10 behaviour of an lllIn-labeled NGA-DTPA combined product administered intravenously to a female rat over time;
Fig. 2 is a graph plotting the distribution behaviour of 123I-labeled NGA administered intravenously to a female rat over time; and Fig. 3 is a graph plotting the distribution behaviour of a 99mTc-labeled NGA-DTPA(Sn) combined product administered to a female rat over time.

Example 1 Preparation of a non-radioactive carrier compris-ing a neogalactoalbumin-diethylenetriaminepenta-acetic acid combined product:-To 20 % human serum albumin (HSA) solution (50 ml), 0.1 % phosphate buffer (pH, 8.0; 117 ml) was added, and diethylenetriaminepentaacetic acid cyclic anhydride (521 mg) was added thereto while stirring with the aid of a magnetic stirrer at 4~C for about S minutes. To 0.05 ml of the resultant mixture, 1 N sodium hydroxide solution (10 mll and 0.6 M borate buffer (pH, 8.5; 23 ml) were added to give a HSA-DTPA solution.
To 0.05 ml of the resulting mixture, 0.1 M citrate buffer (0.1 ml) was added, and 0.1 ml of the resultant mixture was added to a vial where 1 mrl indium chloride (0.3 ml), indium chloride (lllIn) (2 mCi/ml; 0.4 ml) and 0.1 M
citrate buffer (0.6 ml) were previously charged. The mixture was allowed to stand at room temperature for 30 minutes. To the resulting mixture, 1 mM diethylenetriaminetetraacetic acid (DTPA) solution (0.3 ml) was added, and HSA-DTPA- 1 In and free In-DTPA were separated by electrophoresis under the following conditions, and their radioactivities were determined:
Support: cellulose acetate membrane;
Buffer: 0.06 M Barbital buffer (pH, 8.6);
Conditions: 1 mA/cm, 30 minutes.
The result as obtained was calculated according to the following formula to obtain the binding rate (P) of DTPA

~ . , . ~ . . .

per one molecule of HSA:

P = 0.2055 x A/W
wherein W is the amount (mg) of HSA added to the vial and A
is the percentage (~) of In-labeled HSA-DTPA. The 5 binding rate was about 5.
Separately, cyanomethyl-thiogalactose (10 g) was dissolved in dry methanol (250 ml) at 50~C, and sodium methoxide (270 mg) was added thereto, followed by stirring at room temperature for 48 hours. After evaporation of the 10 methanol under reduced pressure, the concentrated product was added to the HSA-DTPA solution, and the resultant mixture was allowed to stand at 4~C overnight to give an NGA-DTPA combined product, which was purified by high partition liquid chromatography under the following condi-tions:
Column: TSK-3000SW column (0.75 x 60 cm);
Eluting solution: 0.1 M sodium chloride solutioni Eluting speed: 0.75 ml/min.
All of the above operations other than measurement of the binding rate were carried out aseptically. All reaction vessels and instruments were previously subjected to heat treatment at 180~C for 4 hours, or subjected to washing with injectionable distilled water and sterilization in an autoclave. The buffer was prepared using injectionable distilled water and filtered through a membrane filter for sterilization. The column was washed with a sodium hypochlorite solution and then e~uilibrated with 0.1 M

sodium chloride solution.

.~ .

.... , .~ . ~ ~ . . . ..
~ . ... . ...... . ..

13405~6 The thus obtained NGA-DTPA combined product was diluted with 0.1 M citrate buffer to a concentration of 1 mg/ml. The diluted solution was filtered through a membrane filter and charged into vials in an amount of 1 ml per vial to give a non-radioactive carrier containing an NGA-DTPA combined product.

Example 2 Preparation of a radioactive diagnostic agent comprising a lllIn-labeled NGA-DTPA combined product:-An injectionable solution of indium (lllIn) chloride (2 mCi/ml; 1.0 ml) was added to the vial containing the NGA-DTPA combined product as obtained in ~xample 1 to make a radioactive diagnostic agent comprising a lllIn-labeled NGA-DTPA combined product.

1~ Analysis was carried out on 25 ul of the above radioactive diagnostic agent according to high partition liquid chromatography under the following conditions,whereby it was confirmed that the dimer is present in an amount of 1 % and unreacted DTPA is not detected:
~0 ~olumn: TSK-3000S manufactured by Toyo Soda (0.75 x 60 cm);
Eluting solution: 0.1 ~ sodium chloride solution;
Eluting speed: 0.75 ml/min.
The maior component sho~led a retention time of about 25 minutes, and its average molecular weight as calculated from the calibration curve was about 75,000.
The above prepared rad.ioactive diagnostic asent comprising an In-labeled NGA-DTPA combined product (380 ~ ~ .. ... ... ...

1340~

ug) was administered intravenously to an SD strain female rat, and the distribution behavior in the animal's body with the lapse of time after the administration was observed.
The results are shown in Fig. 1 of the accompanying drawings (wherein LIV: liver; FEC: feces; LIT: large intestine;
URN: urine; SIT: small intestine; BLD: blood; STM:
stomach), while the results with 3I-labeled NGA as control are shown in Fig. 2. As understood from the comparison, 123I-combined NGA is taken into the liver through an asialoglycoprotein acceptor and deiodinated therein to give free iodine, which accumulates in the stomach or is excreted quickly in the urine. On the other hand, lllIn-labeled NGA-DTPA iS excreted mainly into the intestinal canal from the liver and is metabolized through an asialoglycoprotein acceptor.
Example 3 Preparation of a non-radioactive carrier compris-ing an NGA-DTPA(Sn) combined product:-The NGA-DTPA combined product as obtained in Example 1 was diluted with physiological saline to a concentration of 15 mq/ml. To the dilute solution, stannous chloride (0.4 mM) and ascorbic acid (1.5 mM) were added, and the pH was adjusted, with aqueous hydrochloric acid range of 3 to 5. The resulting solution was filtered through a membrane filter and charged into vials in an amount of 1 ml per vial to give a non-radioactive carrier containing an NGA-DTPA(Sn) combined product.
Example 4 Preparation of a radioactive diagnostic agent .. .... ..

comprising a 99mTc-labeled NGA-DTPA(Sn) combined product:-An injectionable solution of sodium pertechnetate (50 mCi/ml; 1 ml) was added to the vial containing the NGA-DTPA(Sn) combined product as obtained in Example 3 to give a radioactive diagnostic agent comprising a 99mTc-labeled NGA-DTPA(Sn) combined product.

The distribution behavior of said labeled product in a female rat was examined as in Example 2. The results are shown in Fig. 3 of the accompanying drawings. As understood from the results as shown therein, the labeled product is quickly taken into the liver and excreted mainly through the intestinal canal; the behavior in the animal's body is stable.
Also, electrophoresis was carried out on the labeled product as in Example 1 to measure the labeling rates 1 hour, 4 hours and 24 hours after labelling.
The results are shown in Table 1, from which it is under-stood that Tc-labeled NGA-DTPA (Sn)is clearly more stable and gives a higher labeling rate than 99mTc-labeled NGA.
Table 1 \ Labeling rate (%) Time lapsed after labeling (Hrs) Labeled product \ 1 4 24 Tc-labeled NGA-DTPA 81 94 94 99mTc-labeled NGA 74 77 77 ~., .. . .. . . . . ~ ~ .. .

- 22 - 1 3 4 Oj,5 B
Example 5 Preparation of a non-radioactive carrier composi-tion comprising an NGA-Amylose (Amyl)-deferoxamine ~DFO) combined product:-Deferoxamine (DFO) (15 mg) was dissolved in 0.03 M
phosphate buffer (pH 7.0) (1 ml), and triethylamine (3.2 ul) was added thereto, followed by stirring at room temperature.
An aqueous solution of dialdehydoamylose (25 mg/ml; 1 ml) was added thereto, and the resultant mixture was stirred at room temperature for 30 minutes to give solution (A).
Separately, cyanomethyl-thiogalactose (1 g) was dissolved in dry methanol (25 ml) at 50~C, and sodium methoxide (27 mg) was added thereto, followed by stirring at room temperature for 48 hours. After the evaporation of methanol under reduced pressure, 0.2 M borate buffer (20 ml) containing HSA (1 g) was added thereto, and the reaction was effected at 4~C overnight to give an NGA solution as solution (B).
Solution (A) (2 ml) and Solution (B) (2 ml) were combined together, and the resultant mixture was stirred at room temperature for about 6 hours. For the reduction reaction hydrated sodium borate (1.5 mg) was added thereto, followed by stirring at room temperature for about 1 hour.
The reaction mixture was dialyzed against 1 M sodium chloride solution and then subjected to column chromatography on Sephacryl* S-200 (diameter, 2.2 cm; length, 50 cm) using 0.03 M phosphate buffer (pH 7.0) as an eluting solvent for purification of the produced NGA-Amvl-DFO combined * Trade mark 13~05~6 product.
The thus obtained NGA-Amyl-DFO combined product was diluted with 0.03 M phosphate buffer to a concen-tration of 1 mg/ml. The diluted solution was filtered through a membrane filter and charged into vials in an amount of 1 ml per vial to give a non-radioactive carrier comprising an NGA-Amyl-DFO combined product.
The amounts of amylose and DFO in said co~ined product were analyzed by electrophoresis. Namely, a portion of the reaction mixture after the above reduction was admixed with an injectionable solution of gallium (67Ga) citrate (2 mCi) for labeling. Based on the amounts of Ga-labeled NGA-Amyl-DFO, Ga-labeled Amyl-DFO and Ga-labeled DFO as determined by electrophoresis, the numbers of the DFO molecule and the amylose molecule in said combined product were respectively calculated to be 11.5 and 0.7 per one molecule of NGA.

Example 6 Preparation of a radioactive diagnostic composition comprising a 67Ga-labeled NGA-Amyl-DFO combined product:-An injectionable gallium (67Ga) citrate solution(2mCi) was added to a vial containing an NGA-Amyl-DFO
combined product to give a radioactive diagnostic agent comprising a 67Ga-labeled NGA-Amyl-DFO combined product.
The operation was carried out aseptically. The labelled product was confirmed, by electrophoresis, to be of high purity.

;

- 24 - 1340~S6 Example 7 Preparation of a non-radioactive carrier compris-ing a polylysine (PolyLys)-DTPA-galactose (Gal) combined product:-Polylysine hydrobromide (average molecular weight, about 8,000) (77 mg) was dissolved in 0.2 M borate buffer (pH, 8.5; 2 ml), and diethylenetriaminepentaacetic acid cyclic anhydride (25 mg) was added thereto while stirring.
The resultant mixture was stirred at room temperature for 5 minutes. An appropriate amount of 2 N sodium hydroxide solution and 0.2 M borate buffer (pH, 8.5; 1 ml) were added to make a PolyLys-DTPA combined product.
To 0.1 ml of the above obtained PolyLys-DTPA
product, 0.1 M citrate buffer (0.2 ml) and indium (lllIn) chloride solution (2 mCi/ml; 0.1 ml) were added, and the resultant mixture was allowed to stand for 30 minutes for labeling. The labeled product was analyzed by electr-ophoresis to determine the amounts of In-labeled PolyLys-DTPA and lllIn-labeled DTPA, from which the number of DTPAmolecules combined to one molecule of PolyLys was calculated to be about 3.
Separately, cyanomethyl-thiogalactose (2 g) was added to a mixture of methanol (50 ml) and sodium methoxide (54 mgJ, and stirring was continued at room temperature for 48 hours. After the evaporation of methanol under reduced pressure,thetotal amount of the above prepared PolyLys-DTPA
combined product was added thereto, and stirring was continued at 35 to 40~C for 1.5 hours to give a PolyLys-.. .

- 25 - I 3 4 0 5~ 6 DTPA-Gal combined product (wherein DTPA and Gal are respectively combined to PolyLys), which was purified by gel filtration chromatography under the following conditions:
Gel: Cellophane* GC-25m (column, 2.2 cm x S0 cm);
Eluting solution: 0.1 M citrate buffer (pH, 5.7) The thus obtained PolyLys-DTPA-Gal combined product was diluted with 0.1 M citrate buffer (pH, 5.7) to a concentration of 1 mg/ml. The diluted solution was filtered through a membrane filter and charged into vials in an amount of 1 ml per vial to give a non-radioactive carrier.
Example 8 Preparation of a radioactive diagnostic agent comprising a In-labeled PolyLys-DTPA-Gal combined product:-An injectionable indium (lllIn) chloride solution(2 mCi; 1.0 ml) was added to a vial containing PolyLys-DTPA-Gal combined product as prepared in Example 7 to make an injectionable composition comprising a lIn-labeled PolyLys-DTPA-Gal combined product useful as a radioactive diagnostic agent.
The injectionable composition was intravenously administered to a female rat (SD strain; bodyweight, 380 g) through the tail vein to examine the distribution behavior of the lllIn-labeled PolyLys-DTPA-Gal product in the animal. The results are shown in Table 2, from which it is understood that the labeled product is quickly taken up *Trade mark ~ .. .. . . .

- 26 - 1340~i5~

into theliver through the asialoglycoprotein acceptor therein and then gradually excreted. Thus, it is useful for assess-ment of the asialoglycoprotein acceptor.
Table 2 Organ Weight percent to the amount administered After 10 After 30 After 1 minutes minutes hour Liver S2.80 72.33 60.60 Spleen 0.57 0.52 0.52 Stomach 0.37 0.35 0.25 Small 1.58 3.31 3.31 intestine Large 1.14 0.43 0.63 intestine Lung 0.52 0.31 0.37 Heart 0.19 0.41 0.14 Kidney 7.99 4.06 5.75 Blood 3.35 1.10 1.55 Bone 29.30 10.68 13.00 Urine 2.20 6.76 13.87 , .,,,~
,~.,

Claims

1. A high molecular compound useful as a non-radioactive carrier for a radioactive metallic element, which comprises at least one unit of an asialoglycoprotein acceptor-directing compound (1) selected from neogalacto-albumin, asialoglycoproteins, galactose-bonded polylysine and galactose-bonded polyglucosamine and at least one unit of a chelate-forming compound (2), which comprises at least one unit of diethylenetriaminepentaacetic acid of the formula:

being directly or through a crosslinking agent and/or as a part of a higher molecular weight compound bonded to (1), wherein the higher molecular weight compound cosists of a polymeric compound having a number of functional groups, and at least a unit of diethylenetriaminepentaacetic acid bonded to said polymeric compound.

2. The high molecular compound according to claim 1, wherein the asialoglycoprotein acceptor-directing compound (1) is neogalactoalbumin and the chelate-forming compound (2) is diethylenetriaminepentaacetic acid.

3. The high molecular compound according to claim 1, wherein the chelate-forming compound (2) is directly bonded diethylenetriaminepentaacetic acid.

4. The high molecular compound according to claim 1, wherein the chelate-forming compound (2) consists of a unit of a higher molecular weight compound having at least two functional groups and a unit of diethylenetriaminepentaacetic acid.

5. The high molecular compound according to claim 1, wherein the chelate-forming compound (2) consists of one unit of a higher molecular weight compound having at least three functional groups and at least two units of diethylenetriaminepentaacetic acid.

6. A radioactive metallic element-labeled high molecular compound useful as a nuclear medicine, which comprises at least one unit of (1) an asialoglycoprotein acceptor-directing compound selected from neogalacto-albumin, asialoglycoproteins, galactose-bonded polylysine and galactose-bonded polyglucosamine, at least one unit of a chelate-forming compound (2), which comprises at least one unit of diethylenetriaminepentaacetic acid of the formula:

being directly or through a crosslinking agent and/or as a part of a higher molecular weight compound bonded to (1), wherein the higher molecular weight compound cosists of a polymeric compound having a number of functional groups, and at least a unit of diethylenetriaminepentaacetic acid bonded to said polymeric compound, and at least one radioactive metallic element (3) chelate-bonded to said chelate-forming compound.

7. The compond according to claim 6, wherein the asialoglycoprotein acceptor-directing compound (1) is neogalactoalbumin and the chelate-forming compound (2) is diethylenetriaminepentaacetic acid.

8. The labeled high molecular compound according to claim 6, wherein the chelate-forming compound (2) is directly bonded diethylenetriaminepentaacetic acid.

9. The labeled high molecular compound according to claim 6, wherein the chelate-forming compound (2) consists of a unit of a higher molecular weight compound having at least two functional groups and a unit of diethylenetriaminepentaacetic acid.

10. The labeled high molecular compound according to claim 6, wherein the chelate-forming compound (2) consists of one unit of a higher molecular weight compound having at least three functional groups and at least two units of diethylenetriaminepentaacetic acid.

11. A method for preparing the high molecular compound according to claim 1, which comprises reacting the asialoglycoprotein acceptor-directing compound (1) with the chelate-forming compound (2).

12. A method for preparing the radioactive metallic element-labeled high molecular compound according to claim 6, which comprises reacting the high molecular compound according to claim 1 with the radioactive metallic element (3).

13. A method for improvement of the labeling rate of an asialoglycoprotein acceptor-directing compound with a radioactive metallic element and the labeling stability of the labeled product between the asialoglycoprotein acceptor-directing compound and the radiocative metallic element, said asialoglycoprotein acceptor-directing compound being chosen from neogalactoalbumin, asialoglycoproteins, galactose-bonded polylysine and galactose-bonded polyglucosamine, characterized in that the asialoglycoprotein acceptor-directing compound is directly or through a crosslinking agent chemically combined with at least one unit of the chelate-forming compound, diethylenetrimainepentaacetic acid, so that labeling of the resulting combined product with the radioactive metallic element is made through the portion of the chelate-forming compound in the resulting combined product.

14. The method according to claim 13, wherein the asialoglycoprotein acceptor-directing compound is neogalactoalbumin.