CA1258850A - Radioactive chemical product for use as a diagnostic agent - Google Patents

Abstract

Abstract:
The invention provides a chemical product which comprises (1) a unit of a polyformyl compound having at least three formyl groups per molecule, (2) at least two units of an amino group-containing chelating compound bonded to the polyformyl compound via a methyleneimine linkage (-CH=N-) or a methyleneamine linkage (-CH2NH-) formed by the condensation between a formyl group in the polyformyl compound and the amino group in the chelating compound, optionally followed by reduction, (3) at least one unit of an amino group-containing physiologically active substance bonded to the polyformyl compound via a methyleneimine linkage or a methyleneamine linkage formed by the condensation between a formyl group in the polyformyl compound and the amino group in the physiologically active substance, optionally followed by reduction, and (4) at least two radioactive metallic elements of which each is bonded to the chelating compound through a chelating bond. The product can be used as a diagnostic agent for non-invading nuclear medical diagnosis.

Description

~ZS~85~

Radioactive chemical product for use as a diagnostic agent The present application has been divided out of Canadian Patent Application Serial No. 442,833 filed December ~, 1983.
The present invention relates to a chemical product for use as a radioactive diagnostic agent.
For the purpose of a non-invading nuclear medical diagnosis such as recording, dynamic study and quantitative measurement of the blood circulation system, detection of physiological abnormalities or localization of abnormalities by imaging, physiologically active substances labeled with ; iodine-131 (131I) have been widely used, such as 131I-labeled serum albumin and 131I-labeled fibrinogen. However, 131I has a long half life of about 8 days and emits beta-rays so that the patient administered therewith is exposed to a large quantity of radiation.
In order to overcome this drawback of 131I-labeled physiologically active substances, attempts have been made to provide radioactive diagnostic agents which combine physiologically active substances and radioactive metallic elements having more favorable physical properties ~2~

( - 2 -than iodine-131. Among such attempts, a labeling method is known wherein a physiologically active substance is treated directly with a radioactive metal salt to make a chelate compound, which may be used as a radioactive diagnostic agent. For instance, human serum albumin has been treated with an aqueous solution oontaining technetium-99m (99mTc~ in the form of pertechnetate in the presence of a reducing agent to give 99mTc-labeled human serum albumin~ Further, for example, bleomycin has been treated ~ith an aqueous solution containing indium-lll ~ In) in the form of indium chloride to give lllIn-labeled bleomycin. ~owever, the chelate forming property of these physiologically ac~ive substances is not very great and the chelating bond, once formed~ is readily broken. In fact, 99mTc-labeled serum albumin and In-labeled bleomycin have low stability after administration into living bodies, so that the behavior o~ the radioactivity in such bodies does no~ necessarily coincide with that of the serum albumin or the bleomycin used as the physiologically active substance. This is a very serious disadvantage for nuclear medical diagnosis which is based on exact tracing of the behavior of the radioactivity on the assumption that it coincides with the behavior of the physiologically active substance.
In recent years, attention has been drawn to some chelating compounds which show, on the one hand, a strong 1'~5885~

( - 3 -chelate forming property with a variety of metals and have, on the other hand, an amino group or a carboxyl group which is highly reactive with various physiologically active substances, and by utilization of these character-istic features, attempts have been made to link both a radioaetive metallic element and a physiologically aetive substance to them. Examples of such chelating compounds are diethylenetriamine-pentaacetic aeid, ethylenediamine-triacetic aeid, 3-oxobu~yral-bis(N-methylthiosemicarba-zone)carboxylic acid, deferoxamine, 3-aminomethylene-

2,4-pentanedione-bis(thiosemiearbazone) derivatives, l-~p-aminoalkyl)phenylpropane-1,2-dione-bis(N-methyLthio-semicarbazone) derivatives, ete. [grejcarek: Biochemical & Biophysieal Research Comm, Vol. 77, 2, 581-585 (1977?;
lS Leurg: Int. J. AppL. Radiation & Isotopes, Vol. 29, 687 692 (1978); Japanese Patent Publn. (unexamined) Nos. 56-34634, 56-125317, 57-102820, ete.]. Since the resulting produets are stable and retain the aetivities of the physiologically active substanees contained there-~0 in, they are suitable for diagnostie use. However, sueh products whieh include physiologieally aetive substanees of larqe moleeular weight, such as fibrinogen (molecular weight, about 340,000) and IgG (molecular weight, about 160,000), do not usually provide a sufieiently high radioactivity for satisfaetory diagnosis.
In order to overcome the above drawback, a ~LZ5~3l350 physiologically active substance may be combined with many chelating compounds and the resulting product can be bonded to many radioactive metallic elements. ~hiLe this method will assure a high radioactivity, the resulting S physiologically active substance may be unfavorably de-natuced or its physiological activity may be undesirably decreased or Lost.
Besides, physiologically active substances of high molecular weight are preerably administered to human beings in small doses in view o their antigen properties.
In view of this, the physiologically active substance should have a high radioactivity.
As a result of an extensive study, it has now been found that ~he use o~ a ~ormyl group-containing chelating substance comprising a unit of a polyEormyl compound and a unit of an amino group-containing c~elating compound in combination as a carrier for a physioLogically active substance and a radioac~ive metallic element can pcovide a radioactive diagnostic agent having a relatively high radioactivity pec molecule without causing any de-terioration or decrease o~ the physiologicaL activity inherent to the physiologically ac~ive substan~e~

~ ..

~S8~350 According to one aspect of the invention there is provided a chemical product which comprises tl) a unit of a polyformyl compound having at least three ormyl groups per molecule, (2) at least two units of an amino group-containing chelating compound bonded to the polyformyl compound via a ; methyleneimine linkage (-CH=N-) or a methyleneamine linkage (-CH2NH-) formed by the condensation between a formyl group in the polyformyl compound and the amino group in the chelating compound, optionally followed by reduction, (3) at least one unit of an amino group-containing physiologically active substance bonded to the polyformyl compound via a methyleneimine linkage or a methyleneamine linkage formed by the condensation between a formyl group in the polyformyl compound and the amino group in the physiologically active substance, optionally followed by reduction, and (4) at least two radioactive metallic elements of which each is bonded to the chelating compound via a chelating bond.
The polyformyl compound (1) is required to have at least three formyl groups in the molecule and preferably has more. Of these formyl groups, at least two are to be combined with the corresponding number of molecules of the amino group-containing chelating compound (2), and at least one is to be combined with the physiologically active subst~nee (3). Specific examples of the polyformyl compound (1) are polyaerolein, polymethacrolein, etc. Preferred are ; polyaeroleins of the formu].a:
-(CH2-fH)p-CHO

wherein p is usually from 3 to ~,000, preferably from 10 ~s~s~

( 6 -to 500. Such polyacroleins may be prepared, for example, by subjecting acrolein to Redox polymerization [Schulz et al.: Makromol. Chem., Vol. 24, page 141 (1975)]. Other specific examples are poly(diaLdehydosaccharides), typical of which is dialdehydostarch of the formula:

C~2H
/1H-O \
C~-o) p, -CHO CHO

wherein p' is usually from 2 to 1000, and preferably from 10 to 500. These may be prepared, for example, by oxidizing polysaccharides (e.g. starch, amylose, dextran, purdan) with an oxidizing agent (e.g. sodium periodate) so as to ~orm two formyl groups ~rom each saccharide unit.
~ ny amino group containing chelating compound (2) may be used which shows a strong chelate ~orming property to a radioactive metallic element and has an amino group capable o~ reacting ~ith a formyl group in the polyformyl compound (1~ under relatively mild conditions. Speci~ic examples are deferoxamine (i.e. l-amino 6,17~di-hydroxy-7,10,18,21-tetra~xo-27-~N-acetyl-hydroxylamino)-6,11,17,22-tetraazeheptaeioosane~ [The Merck Index, 9th Ed., page 374 2~ (1976)1, 3-aminomethylene-2,4-pentanedione bis-(thiosemi-carbazone) derivatives o~ the ~ormula:

:~ZS~350 ( ~ 7 -CH3-C=N-NH-C-NH-R
H2N-CE~=f CH3-C=N-NH-fi-NH-R2 wherein Rl and R2 are each a hydrogen atom, a Cl-C3 alkyl group or a phenyl group [EP A-0054920], l-(p-amino-alkyl~phenylpropane-1,2-dione-bis (thiosemicarbazone) S derivati~es of the formula:
S
H2N- (C~12 ) n~C-N-NH-C-NHR3 ~=N-NH-C-NHR

wherein R3 and R4 are each a hydrogen atom or a Cl-C3 alkyl group and n is 0 or an integer of 1 to

3 ~Australian patent 533722~, etc. Any compound which ha~ a metal capturing property suitable to form a che-late and does not have an amino group but can be readily modif ied so as to include an amino group or an amino group-containing function is also suitable as the chelating compound (2) ater such modification. For example, a compound bearing a carboxyl group may be reacted with hexanediamine to convert it to a compound containing an aminohexylaminocarbonyl group, which can be r.eadily condensed wi th a ~ormyl group. Specific ; examples are diethylenetriaminepentaacetic acid, ethylene-diaminetriacetic acid, 2-oxopropionaldehyde-bis (thiosemi-: carbazone) derivatives of the ~ormula:

88S~

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

' ~ R7-C=N-NH_C_NH_R8 Il . ~ S
I ~ wherein R5, R6, R7 and R8 are each a hydrogen atom or a Cl-C3 alkyl group [U.S. patent 4287362], ete.
The term "physiologieally aetive substanee" whieh is used to describe the constituent (3) is intended to mean any substanee which shows a speci~ic accumulability in a certain organ or tissue or a certain diseased locus or which exhibits a speei~ie behavior correspondlng to a certain ~physiologieal state. Tracing of the behavior o~
~U sueh substance in the living body can provide information useful for diagnosis. Physiologically aetive substances having an amino group capable of belng condensed with a formyl group under relatively mild conditions are advantageous in this invention. Even when an amino lS group is not present, however, the substance may be used as the physiologically active substance ~3) a~ter chemical modi~ication to provide an amino group or an amino group-containing function. Speci~ic examples of suitable physiologically active substances are blood proteins (e.g. human serum albuminr ~ibrinogen), enzymes (e~g.
urokinase, streptokinase), hormones (e.g. thyroid ~LZ58~50 g stimulating hormone, parathyroid hoemone), immune an~i-bodies (e.g. IgG), monoclonal antibodies, antibiotics (e.g. bleomycin, kanamycin), saccharides, fa~ty acids, amino acids, etc. In general, this invention is advan-S tageously applicable to physiologically active substances ; having molecular weights of not less than about 100,000.
~he term "radioactive metallic element~ used to describe the constituent (4) is intended to mean any metal~ic element having radioactivity, and which has physical characteristics suitable for nuclear medical diagnosis and can be readily captured with the chelate forming structure in the chelating compound (2). Speci-fic examples of suitable radioactive metallic elements are gallium-67 (67Ga), gallium-6~ (68Ga), thallium-201 ( 01Tl), indium~ I In), technetium-99m ( 9 Tc), etc. They are normally employed in their salt form, particularly in their water-soluble salt forms.

~Z5~385(:~

A ~urther app].ication divided out oE Canadian Patent Application ~erial No. ~2,833 is directed to a chemical product which comprises (1) a unit of a polyformyl compound having at least three formyl groups per molecule, and (2) at least two units of an amino group-containing chelating compound bonded to the polyformyl compound via a methyleneimine linkage (-CH=N-) or a methyleneamine linkage (-CH2NH-) formed by the condensation between a formyl group in the polyformyl compound and the amino group in the chelating compound, optionally followed by reduction. This chemical product functions as a non-radioactive carrier and for its preparation, the polyformyl compound (1) and the chelating compound (2) are subjected to condensation to form a methyleneimine linkage between the formyl group in the former and the amino group in the latter, optionally followed by reduction of the methyleneimine linkage to the methyleneamine linkage. Depending on the kinds of the reactants, the reaction conditions, etc., the number of units of the chelating compound (2) to be introduced into ~zs~so the polyformyl compound (1) may vary and generally not less than about 5 units, and especially not less than about 10 units, o~ the chelating compound (2) per mole-cule of the polyformyl compound (1) should be introduced, S but at least one formyl group in the polyformyl compound (1) should be left ~or oombination with the physiologically active substance ~3).
~he resulting condensation (or condensation-reduction) product of the polyformyl compound (1) and the chelating compound ~2) (hereinafter re~erred to as "the ~ondensation or condensation-reduction product~) used as the non-radioactive carrier is then condensed with the physiologically active substance (3), optionally followed by reduction so as -to form a methyleneimine group or a methyleneamine group, by reaction between a formyl group in the polyformyl compound (1) moiety o the former and the amino group of the latter to give a physiologically active su~stance-G~mbined condensation or condensation-reduction product. The number of units of the physio-logically active substance (3) to be introduced into the ~condensation or condensation-reduc~ion product varies with the kinds of the reactants, the reaction conditions, etc., and usually a small number o~ not more than about 10 units, preferably o~ not more than 3 units, of the physiologically active substance (3) per molecule o~ the polyformyl compound (1) is desirable.

~L258~50 Alternatively, the physiologically active substance-combined oondensation or condensation-reduction product may be prepared by first condensing the polyformyl compound (l) with the physiologically active substance (3) to orm a methyleneimine linkage between a formyl group in the former and an amino group in the latter, optionally ~ollowed by reduction of the methyleneimine linkage to a methyleneamine linkage, to give a physiologically active substance-combined polyformyl compound, which is then condensed with the chelating compound ~2) to orm a methylene~imine linkage between a formyl group in the polyformyl compound moiety of the physiologically active substance-combined polyformyl compound and an amino group in the chelating compound (2), optionally followed by reduction of the methyleneimine linkage to a methylene-amine linkage, whereby a physioLogically active substance- -combined condensation or condensation-reduction product is ; obtained. As for the number of the units of the chelating compound (2) and of the physiologically active substance ~3), the same comments as stated above apply~
In the above preparation procedures, the reduction optionally carried out after the condensation may be accom-plished in a single step at the final stageu Further, each of the reactions, such as the oondensation and the reduc-tion, may be carried out by per se conventional procedures.
Furthermore, during the reduction, a formyl group may be ~Z5~3~350 converted into a hydroxymethyl group simultaneously with the conversion of a methyLeneimine linkage into a methyleneamine linkage. Usually, the condensation proceeds easily a~ room temperature. For ~he reduction, a reductive metal hydride compound such as sodium boro hydride is desirably employed as the reducing agent.
At any stage in the above preparation procedures, ~he reaetion product may optionally be purified by per se conventional methods, such as column chromatography, gel permeation and dialysis.
The thus obtained physiologically active substanee-eombined condensation or condensation-reduction produet may be then labeled with the radioactive metallic element (4) to give a radioactive metallic element-labeled, physiologically active substance-combined condensation or condensation-reduction product, i.e. a radioactive diag-nostie agent according to the invention.
One of two different labeling procedures may be employed depending upon the kind or state of the radio-active metallie element (4)7 When the radioactive metallic element (4) is in a valency state which can form a stable chelate compound, the physiologically active substance-combined oondensation or condensation-reduction product may be contacted with the radioactive metallic element

(4) in an aqueous medium to form the radioactive metallic element-labeled, physiologically active substance-combined 1~5~3~50 condensation or condensation-reduction product. This labeling manner may be applied to 67Ga, lllIn, etc.
~hen the radioactive metallic element (4) is in a valency s~ate which has to be changed for the ~ormation of a S stable chelate compound, the physiologically active substance-~ombined oondensation or condensation-reduction product may be con~acted with the radioactive metallic element (4) in an aqueous medium in the presence of a r~ducing agent or an oxidizing agent to form the radio-active metallic element-labeled, physiologically active substance-combined condensation or condensation-reduction product. This labeling manner may be applied to 99mTc, etc.
.. Examples of suitable reducing a~ents are stannous salts, i.e. salts of divalent tin ion (Sn~+). Specific examples are stannous ha~ides (e.g. stannous chloride, stannous fluoride), stannous sulfate, stannous nitrate, stannous acetate, stannous citrate, etc. Sn+~ ion-kearing resins, e.g. ion-exchange resins charged with Sn~+ ion, are also suitable.
When r for example, the radioactive metallic element (4) is 99mTc, the physiologically active substance-combined condensation or condensation-reduction product may be ~treated with 99mTc in the ~orm o a pertechnetate in an aqueous medium in the presence o~
a reducing agent, e.~. a stannous salt. There is no ~s~

( particular requirement concerning the order of the intro-duction o~ 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.
S The stannous salt may be used in an amount that can su~~
~iciently reduce the pertechnetate.
The resulting radioactive diagnostic agent should have sufficient radioactivity and radioactivity concen-~ration to assure reliable diagnosi~. For example, the radioactive metallic element 99mTc may be used in an amount of 0.1 to 50 mCi in about O.S to 5.0 ml at the time o~ administration. The amount of the physiologically active su~stance-combined condensation or condensation-reduction product should be suf~icient to form a stable chelate compound with the radioactive metallic element (4).
T~e thus produced radioaetive metallic element-labeLed, p~ysiologieally active substance-combined condensation or oondensation-reduction product used as a radioactive diagnostic agent is quite stable, and therefore it may be stored as such and supplied on demand. When desired, ~he radioactive diagnvstie agent may contain any suitable additive such as a pH
controlling agent (e.g. an aeid, a base, a buffer), a stabilizer ~e~g. ascorbic aeid) or an isotonizing agent (e.g. sodium chloride).
The radioactive metallic element-labeled, :~S~5~

( - 16 -physiolocially active substance-combined condensation or condensation-reduction product of this invention is useful for nuclear medical diagnosis. For example, a 99mTc or 67Ga-labeled streptokinase-combined oondensation or condensation-reduction product may be used for recording and functional measuremen~ of m~ocardium. Also, for example, a 99mTc-labeled~ human serum albumin-combined condensation or condensation-reduction product can be used for recording, dynamic s~udy and quantitative measurement of the blood circulation system by intravenous administra-tion to the human body. Further, for example, a 99mTc-labeled, fibrinogen or urokinase-combined condensation or condensation-reduction product may be used for detection and recording of thrombosis as well as the localization of thrombosis, since this product accumulates at t~e locus of throm~osis. Furthermore, for exampler a 99mTc-labeled, st~eptokinase-combined condensation or condensation-reduction product is useful for determination of the locus of a myocardial infarction. Moreover, a 99mTc-labeled, thyroid stimulating hormone~combined condensation or condensation-reduction product is useful ~or the detection and recording of a cancer at the thyroid gland.
The radioactive diagnostic agent of this invention may be administered to a patien~ in an amount suf~icient 2S to produce the radioactivity necessary for examination of a particular organ or tissue, by any appropriate route, ~L~S~S~

usually via an intravenous route. For example, the intravenous administration to a patient of a 99mTc-labeled radioactive diagnostic agent in an amount o~ about ]. to 3 ml by volume having a radioactivity of about 1 to 20 mCi is quite suitable ~or diagnostic purposes.
The advantages of the physiologically active substance-combined condensation or condensation-reduction product, i.eO the physiologically active substance-combined non-radioactive carrier, may be summarized as follows: (a~
it is stable over a long period of time after manu~acture;
(b) since it can be produced under mild conditions, no unfavorable side reactions such as inactivation, denaturation or decomposition are caused in the physiologically active substance; (c) any physiologically active substance having an amino group can be used as the starting material; (d) even when an amino group is not present, the introduction of such a group into a physiologically active substance makes it suitable as the starting material; (e) a radioactive metallic element-labeled, physiologically active condensation or condensation-reduction product can be formed by a very simple procedure, e.g. by merely contacting the physiologically active substance-combined condensation or condensation-reduction product with a radioactive metallic element in an aqueous medium. The advantages of the radioactive metallic element-labeled, physîologically active substance-combined condensation or condensation-reduction product used as a radioactive diagnostic agent may be also s~
- 17a -summarized as follows: (a) it is stable over a long period of time after manufacture; (b) the labeling efflciency with the radioactive metallic element is extremely high (nearly 100%); (c) since the labeling operation is quite simple, no unfavourable side reactions such as inactivation, denaturation or decomposition are caused in the physiologically active substance bonded to the condensation or condensation-reduction product; (d) the most suitable for a particular diagnostic purpose of various radioactive metallic elements may be chosen so that the diagnosis can be improved not only in quantity but also in quality while obtaining reduction of the exposure dose.
Practical and presently preferred embodiments of the invention described herein as well as the invention described in Canadian Patent Application Serial No. 442,833 from which the present application was divided and a further co-pending divisional application are illustratively shown in the following Examples.

5~35~

( Reference Examlple 1 Preparation of polyacrolein:-Water (50 ml) was charged in a 1ask and heated under refLux while introducing nitrogen gas therein. After cooling below 20C, potassium peroxodisulfate (0.475 g) and acrolein (purity, more than 95 %) (10 ml) were added thereto. After acrolein was dissolved, a solution of silver nitrate (0.296 g) in water (~ ml) was dropwise added thereto in about l minute while vigorous agita~ion. The reac ion was continued for 2.5 hours, during which care was taken to avoid the elevation of the temperature above 20C. After the reackion was completed, khe reaction mix~ure was added to water ~50 ml) r whereby the produced polyacrolein was precipitated. The precipitate was collected by filtration, washed with water two times and dispersed in a solution of sodium thiosulfate (0.5 g) in water lSO ml), followed by stirring for 1 hour. The dispersion was filtered to collect the solid material, which was ~ashed with water several times and dried under reduced pressure overniqht to obtain polyacrolein.
Polyacrolein (50 mg~ as prepared above was dissoLved in dimethylsulfoxide (10 ml), sodium borohydride (3 mg) was added thereto~ and stirring was continued at room ~emperature for 1 hour. To the resulting mixture, ethyl acetate (10 ml) w~s added to precipitate partialLy reduced polyacrolein. The precipitate was collected by filtration, dissolved in water and subjected to measurement of molecular weight by hiqh speed liquid chromatography under the follow-~2S~35~

( ing conditions:
Column: TSK-3000SW
Solvent: 0.05M Tris-0.15M sodium chloride-hydrochloric acid buffer (pH, 7.4) Flow rate: 1.0 ml/min Since the partially reduced polyacrolein was eluted at a retention volume of 23.2 ml, the molecular weight of polyacrolein was determined to be about 21,000.
Reference Example 2 ~reparation of dialdehydodextran:-To a solution of dextran (average molecular weight, 10,000~ (3.24 g) in O.lM sodium acetate solution (pH~ 4.2; 200 ml~, O.lM ~odium periodate solution (40 ml~
was added, and the resultant mixture was stirred at a dark place overnight. The reaction mixture was admi~ted in a cellulose tube and dialyzed to water for 2 days, followed by lyophilization to obtain dialdehydodextran.
A~out 50 mg of the abGve prepared dialdehydo-dextran was weighed precisel~ and dissolved in O.~lM
phosphoric acid-0.15M sodium chloride buffer (100 mll. The resulting solution (abou~ 5 ml) was precisely measured, 1/100 N iodine solution (5 ml) was added thereto, and further 0.15M sodium carbonate solution (1 ml~ was added thereto, ~ollowed by allowing to stand at room temperature ~or 1.5 hours. ~fter addition of 0.2 N sulfuric acid (~
ml), titration was carried out with l/lO0 N sodium thio-sulfa~e soLution until a colorless, transparent solution was obtained. This titration value was taken as A. In the same ~ S8850 manner as above, O.OlM phosphoric acid-O.lSM sodium chloride buffer (5 ml) was titrated with l/lO0 N sodium thiosulfate solution, and the resulting titration value was taken as B.
The content of aldehyde groups in 1 mg of the product was calculated according to the following equation:
Aldehyde groups ~nole/mg) = (A-B)xlO/2W wherein W is the amount of dialdehydodextran (mg) contained in 5 ml o~ the sample. As the result, the aldehyde group content in the dialdehydodextran as prepared above was determined to be 5.1 ~ ole/mg.
ExamPle 1 ~ A) Preparation of the polyacrolein-deferox~nine condensation~reduction product as a non-radioactive carrier:-.. Polyacrolein (molecular weight, 21,000) (~00 mg~
was dissol~ed in dimethylsulfoxide ~10 ml), and the resultant soIution wa~ admixed with a solution of deferox-amir.e (420 mg~ in dimethylsulfoxide (lO ml). The reaction was continued at room temperature for 3 hours. To the reaction mixture, sodium borohydride ~100 mg) was added, and s-tirring was con~inued at room temperature for l hour. The resultant mixture was subjected to dialysis to water over-ni~ht, followed by gel chromatography under the ~ollowing conditions:
. 25 Carrier: Sephadex*G-50 Sol~ent: Water Column: diameter, 4.5 cm; height, 50 cm Flow rate: 2.s ml/min * Trade Mark ~l2S~1~5C~

( ~ he polyacrolein-deferoxamine condensation-reduction product was eLuted at a volume of 270 400 ml, while the unreacted deferoxamine was eluted at a volume of 550 to 600 ml. ~he eluate containing the poLyacrolein-deferoxamine condensation-reduction product was subjec~ed to lyophilization~
The polyacrolein-deferoxamine condensation-reduction product thus obtained was dissolved in water~
~erric ch~oride was add~d thereto, and the res~ltant solution was analyzed ~y high sp~ed liquid chroma~ography under the following conditions to determine a retention volume of 21.2 ml:
Column: TSK-3000SW
Solvent: 0.05M Tris-0~15M sodium chloride-hydrochloric acid buffer (pH, 7.4) Flow rate: l.0 ml/min Absorptive w~velPngth: 420 nm No free deferoxamine was detected. ~The retention volume of deferoxamine in the above system is 32.8 ml.) A definite amount the polyacrolein-deferoxamine condensation-reduction product as obtained above was ~issolved in water, and a sufficient amount of an aqueous ferric chloride solu~ion was added thereto to make a 1 : 1 complex between the deferoxamine moiety in said conden-sation-reduction product and Fe(III) in said ferric chloride. The reaction mixture was allowed to stand for 1 hour and ~hen subjec~ed to measurement of absorbance at 420 nm, whereby ~he number of the deferoxamine moieties in said ~2~3l350 22 _ condensation-reduction product was confirmed to be 18.3 per one molecule of polyacrolein. The average molecular weight of said condensation-reduction product was thus caLculated ~o be about 32,000.
still, deferoxamine and Fe(III) can form a 1 : 1 complex having a maximum abso~ption at 420 nm, and the ~max value of the complex at 420 nm is 2.63 x 103.
Example 2 ~A~ Preparation of the polyacrolein-hexane-diamine:3-oxobutyral-bis~N-methylthiosemicarbazone)-carboxylic acid condensate condensation-reduction product as a non-radioactive carrier:-A solution of 3-oxobutyralbis(N-methylthiosemi-carbazone)carboxylic acid (hereinafter referred to as I'KTS" ) : 15 (132 mg) in dry dioxane (5 ml3 was cooled to abou~ 10C.
Tri-n-bu.tyl~mine (0.12 ml) and isobutyl chloroformate (64 ~1) were added thereto. The resultant mixture was stirred at the same temperature as above for about 50 minutes to obtain a mixed acid anhydride solution. To this solution, a solution of N-tert-butyloxycarbonyl-l,6-hexanediamine (104 mg) in dry dioxane (2 ml) was added, and the resultant mixture was s~irred at 10C for abou~ 15 hours to produce N-tert-butyloxycarbonyl-l,6-hexanediamine:KTS condensate. A
~ew drops o~ conc. hydrochloric acid we~e added ~hereto to - 25 ~ake a pH of abou~ 2, whereby the N-tert-butyloxycarbonyl group was eliminated to give a solution of hexanediamine-KTS

condensa~e.
The above solution was added to a solution of :lZS13~5~
-- 21 _ polyacrolein (200 mg) in dimethylsulfoxide (S ml), sodium borohydride (17.2 mg~ was added thereto, and the resultant mixture was reacted at room temperature for 3 hours. The reaction mixture was subjected to dialysis by a conventional procedure for 30 hours to eliminate the unreacted reagents and lyophilized to obtain the polyacrolein-hexanediamine:KTS
condensate condensation-reduction product useful as a non-radioactive carrier.
The polyacrolein-hexanediamine:KTS condensate condensation-reduction product as above obtained was dissolved in water to ma~e a concen~ration of 3 mg/ml, and the resulting solution was subjected to measurement of absorbance at 334 nm using water as the control, whereby the number of the KTS moieties in said condensation-reduction product was confirmed to be 21.3 per one molecule of poly-acrolein. The average molecular weight of said conden-sation-reduction product was thus calculated to he about 29,600.
Still, the hexanediamine:KTS condensate had a maximum absorption at 334 nm, and i~s ~max value was 4.37 x 104.
(B) Preparation of the fibrinogen-combined polyacrolein-hexanediamine:K~s condensate condensation-reduction product (a fibrinogen-combined non-radioactive . 25 carrier):-A solution of the non-radioactive carrier as obtained in (A) (before lyophilization) (5 ml) was added to ~s~s~

a solution af human ibrinogen (250 mg) in 0.OlM phosphate bu~er-0~15M aqueous sodium chloride mixture (pH~ 8~4) (50 ml), followed by stirring at room temperature for about 3 hours. Sodium borohydride (12.9 mg) was added thereto. ~he resultant mixture was stirred for about l hour. The reac-tion mixture was dialyzed ~o O.OlM glucose-0.35~ sodium citrate solution at 0 to 4C for 24 hours and then passed ~hrough a column of S~pharose*4s (diameter, 4.4 cm; height, 50 cm) using 0.OlM glucose-0.35M sodium citrate solution as an eluting solvent~ The eluate was lyophilized to give the polyacrolein-hexanediamine:XTS condensate condensation-reduction product as cotton-like crystals.
~ he cotton-like crystals (100 mg) were dissolved in deoxygenated water (160 ml), and 1 mM stannous chloride solution (10 ml) and sodium ascorbate (0.6 g) were added there~o to make a clear solution. The solution was passed through a filter having a pore diameter of 0.45 ~m, and the filtrate (1.5 ml) was fiLled in a vial flushed with nitrogen gas to obtain a fibrinogen-combined non-radioactive carrier.
The above operativns were effected under a sterile condi-tion.
The ~ibrinogen-combined non-radioactive carrier as above obtained was a pale yellow, clear solution.
(C) Preparation of the 99mTc-labeled, fibrinogen~
combined polyacrolein-hexanediamine:KTS condensate conden-sation-reduction product as a radioactive diagnostic agent:-To the ~ibrinogen-combined non-radioactive carrier (1.5 ml) as obtained in (B), a physiological saline solution * Trade Mark (1.5 ml~ containing Tc (3.3 mCi) in the Çorm of sodium pertechnetate was added to obtain the 99mTc-labeled, figrinogen-combined polyacrolein-hexanediamine:KTS conden-sate condensation-reduction product useful as a radioactive s diagnos~ic agent.
This solution was pale yellow, transparent.
(D) Properties of the radioactive diagnostic agent as obtained in (C):
The radioactive ~iagnostic agent as obtained in (C) was subjected to electrophoresis (1.7 mA/cm; 15 minutes) using Veronal*buffex (pH, 8.6) as a developing solvent as~d a cellulose acetate membrane as an electrophoretic membrane, and scanning was carried out by the use of a radiochromato-scanner. The radioacti~ity was recognized as a single peak at.the locus o 0.5 cm distant from the original line towards the negative side. This locus was the same as that of ~he coloring band of fibrinogen with Ponceau 3R.
From the above result, it may be said that the radioactive diagnostic agent has a labeling ef~iciency of nearly 100 ~ and its electric charge is substantially the same as that of fibrinogen.
To the radioactive diagnostic agent as obtained in (C), O.lM sodium diethylbarbiturate hydrochloride bu,fer (pH, 7.3~ containing 0.05 ~ calci~ chloride was added to make a fibrinogen concentration oÇ 1 mg/ml. Thrombin (l~0 units/ml; 0.1 ml) was added th2reto. The resultant mixture was allowed to stand in an ice bath for 30 minutes. The produced fibris~ogen clots were completely separated s~rom the * Trade Mark ~2S~S~

( liquor, and radioactivi~y was measured on the clots and also on the liquor. From the obtained results, it was detemined that the clottability of the radioactive diagnostic agent is 93 ~ based on the starting fibrinogen.
Ex-ample 3 (A) Preparation of the polyacrolein-deferoxamine condensation produc~ as a non-radioctive carrier:-To a solution of polyacrolein (125 mg~ in dimethylsulfoxide ~2.5 ml~, a solution of deferoxamine ~105 mg) in dimethylsulfoxide (2.5 ml) was added, and the resultant mixture was agitated at room temperature ~or 3 hours to produce a solution containing the poLyacrolein-deferoxamine condensation produc~, which is use~ul as a non-radioactive carrier.
(B) Preparation of the fibrinogen-combined, poly-acrolein-deferoxamine condensation product (a fibrinogen-combined non-radioactive carrier):-The non-radioactive carrier (5 ml) as obtained above was added to a so~ution of human fibrinogen (200 mg~ !
20 in 0. OlM phosphate buffer-OOlSM aqueous sodium chloride mixture ~p~, 8.4) at 0 to 4C, followed by stirring at the same temperature as above for about 3 hours. The reaction mixture was dialyzed to O.OlM glucose-0.35M sodium citrate solution at 0 to 4C for 24 hours and then passed through a column of Sepharose*4B (diameter, 4.4 cm; height, 50 cm~
using O.OlM gLucose-0.35M sodium citrate solution as an elu~iny solvent. The elua~e containing the fibrinogen-* Trade ~ark ~51~S~

combined polyacrolein~deferoxamine condensation product was diluted with O.OlM glucose-0.3SM sodium citrate solution to make a fi~rinogen concentration of 1 mg/ml, and sodium ascorbate was added thereto to make a concentration of 30 mM. The resultant solution (3 ml) was admitted into a vial, followed by lyophilization to obtain a fibrinogen-combined non-radioactive carrier as a cotton-like product. The above opera~ions were effected under a s~erile condition.
Example 4 (A) Preparation of the polyacrolein-deferoxamine condensation product (a non-radioctive carrier):-To a solution of polyacrolein (125 mg) in dimethylsul~oxide (2~5 ml), a solution of deferoxamine (105 mg) in dimethylsulfoxide (2.5 ml) was added, and the resultant mixture was agitated at room temperature for 3 hours to obtain a solution containing the poLyacrolein-de~eroxamine condensation product useful as a non-radio-active carrier.
(B) Preparation o~ the fibrinogen-combined polyacrolein-de~eroxamine condensation-reduction product (a fibrinogen-combined non radioactive carrier):-~ he non-radioactive carrier (S ml) as obtained above wa~ added to a solution of human fibrinogen (200 mg) in O.OlM phosphate buf~er-0.15M aqueous sodium chLoride 25 mixture (pH, 8.4) at 0 to 4C, followed by stirring a~ the same temperature as above ~or about 3 hours. To the resulting mixture, sodium borohydride (7.0 mg) was added, and stirring was continued at 0 to 4C for about l hour.

~zs~so To a portion of the reaction mixture, a solution containing 67Ga (1 mCi) in the form of gallium chloride was added for labelinq, and the resultant solution was sub jected to high speed liquid chromatography under the following conditions:
CQ lumn: TSK-300OSW
Solvent: 0.05M Tris-O.lSM sodium chloride-hydrochloric acid buffer (p~ 7.4) Pressure: 100 kg/cm~
Flow rate: 1.0 ml/min Detection was made on the radioactivity of 67GaO
As the result, the eluted pa~tern gave three peaks attri-butable to 67Ga-labeled fibrinogen, 67~a-labeled poly-acrolein-deferoxamine condensation-reduction product and lS 67Ga-labeled deferoxamine~ From the area ratio of the peak due to 67Ga-labeled polyacrolein-deferoxamine condensation-reduc~ion product and the peak due to 67~a-labeled deferox-amine, 18.9 of the deferoxamine moieties were confirmed to combine to one molecule of polyacrolei~. Since the number of the deferoxamine moie~ies in the fi~rinogen-combined polyacrolein-deferoxamine condensation-reduction product was confirmed to be 14.8 per one molecule of fibrino~en, the number of fibrinogen bonded ~o one molecule of polyacrolein was calculated to be about 0.8.
~5 The remainder of the reaction mixture was dialyzed to O.OlM glucose-0.35M sodium cintrate solution at 0 to 4C
for 24 hours and then passed through a column of Sepharose*

* Trade Mark .... . ....

:12S~~5~

4~ (diameter, 4.4 cm; height, 50 cm) as an eluting solvent.
The eluate containing the fibrinogen-combined polyacrolein-de~eroxamine condensation-reduction product was diluted with O.OlM glucose 0.35M sodium citrate solution to make a S fibrinogen concentration of 1 mg/ml, and sodium ascorbate was added thereto to make a concentra~ion of 30 mM. The resultant solution 13 ml) was admit~ed into a vial, followed ~y lyophilization to obtain a fibrinogen-combined non-radio-active carriex as a cotton-like product. The above operations were effected under a s~erile condition.
The fibrinogen-combined non-radioactive carrier as obtained above was dissolved in sterile water to make a fibrinogen concentration of l mg/ml, and a suficient amount of an aqueous ferric chloride solution was added thereto to make a l : l complex between the deferoxamine moiety in said non-radioactive carrier and Pe(III) in said ferric chloride solution. The reaction mixture was allowed to stand for l hour and then subjected to measurement of absor~ance at 420 nm using a solution of said non-radioactive carrier in sterile water as the control, whereby the number of the deferoxamine moieties in said non-radioactive carrier was confirmed to be 14.8 per one molecule of fibrinogen.
IC) Preparation of the 67Ga-labeled, fibrinogen-combined polyacrolein-deferoxamine condensation-reduction product as a radioactive diagnostic agent:-To the fibrinogen-combined non-radioactive carrier as obtained in (B), an a~ueous solution (2 ml~ containing 67Ga (2 mCi) in the form of gallium citrate was added to ~25~35(~

( 67 obtain the Ga-labeled, fibrinogen combined polyacrolein-deferoxamine condensation-reduction product as a radio-active diagnostic agent.
This solution was pale yellow, transparent and had a pH of about 7.8.
(C') Preparation of the 67Ga-labeled, fibrinogen-combined polyacrolein deferoxamine condensation-reduction product as a radioactive diagnostic agent:-The fibrinogen-labeled non-radioactive carrier obtained in (B) was dissolved in sterile water, and human fibrinogen (0.5, 0.75, 1.0, 1.5, 2.0 or 3. n mg) dissolved in O.OlM phosphate bu~fer-O.lSM aqueous ~odium chloride mixture tpH, 8.4) and 1 ml of an aqueous solution containing 67Ga (1 mCil in the form o~ gallium citrate were added thereto. The lS resulting mix~ure was allowed to stand at room temperature for 1 hour and then subjected to measurement o lebeling rate. In the same manner as above, the labeling rate of 67Ga-labeLed, fibrinogen-combined deferoxamine as prepared ~y labeling 67Ga on~o ~ibrinogen combined deferoxamine was also measured. The results are shown in Table 1.

~2S~ 35(~

Table 1 (Labeling eficiency with 67Ga~
~ ~ . .
Fibrinogen (mg) LaheLing rate (~) Sample 1 1) 1 Sample 2 2) ~ _ . .
0.5 59 3 5 ~.75 ~3 2 1.0 97.8 17.0 1. 5 ~--100 2.0 ~ 100 35.2 3.0 ~vlO0 10 6.3 _ 41 4 12.6 _ 70 9 18.8 _ 80.6 25.1 _ ~3~5 _ ........ _ _ , ___ . _ Note: *l) Radioactive diagnostic agent accord-ing to the invention.
*2) 67Ga-labeled fibrinogen-combined deferoxamine As understood from the above, the non-radioactive carrier of the invention could be labeled with 97.8 % of 67Ga ~1 mCi) within 1 hour when 1 mg of fibxinogen was used.
~he conventional non-radioactive carrier ~i.e. fibrinogen-combined deferoxaminej could be labeled only with 17.0 % of 67Ga under th~ same condition as above. Even when 25.1 mg of fibrinogen were used, the conventional non-radioactive carrier was labeled with 83.5 ~ of 67Ga at the most~ It is thus appreciated that the non-radioactive carrier of the inven~ion can afford a radioactive diagnostic agent having a higher relative radioactivity. Further, the radioactive diagnostic agent is useful in nuclear medical diagnosis aiming at detection of thrombosis.
(D) Properties of the radioactive diagnostic agent as obtained in (C):-. . .

~zs~sv - ~2 -The radioactive diagnostic agent as obtained in tC) was subjected to electrophoresis (1.7 mA/cm; 15 minutes) using Veronal buffer (pH, 8.6) as a developing salvent and a cellulose acetate membrane as an electrophoretic membrane, S and scanning was carried out by the use of a radiochromato-scanner. The radioactivity was recognized as a single pea~
at the locus of 0.5 cm distant from the original line towards the negative side. This locus was the same as that of the coloring band of fibrinogen with Ponceau 3Ro From the above result, i~ may be said that the radioactive diagnostic agent has a labeling efficiency of nearly 100 % and its electric charge is substantially the same as that o~ fibrinogen.
To the radioactive diagnostic a~ent as obtained in (C); O.lM sodium diethylbarbiturate hydrochloride buffer (p~, 7.3) containing 0.05 ~ calci~n chLoride was added to make a fibrinogen concentration of 1 mg/ml. Thrombin (100 units/ml; 0.1 ml) was add~d th~reto. The resultant mixture W25 ailowed to stand in an ice bath for 30 minutes. The 20 produced fibrinogen clots were comple~ely separated ~rom the liquor, and radioactivity was measured on the clots and also on the liquor. From the obtained results, it was determined that the clottability o the radioactive diagnostic agent is 86 ~ based on the starting fibrinogen.
(E~ Behaviors of the radioactive diagnostic agent as obtained in ~C) in rats:-The radioactive diagnostic agent as obtained in ~C) ~0.2 ml) was administered intravenously to each of lZ513~350 emale rats o SD strain, and the variations of the blood level and the organ distribution with the lapse of time were recorded. The results are shown in Tabl~ 2.
Table 2 (Distribution in rat body; ~/g) Organs ~ime after administration (min) . _____ _ -'--1 _ _ Blood 8.33 6.82 6.22 4.81 Liver 1.47 1.62 1.73 1.78 Heart 0.85 0.88 1.03 0.96 Spleen 1.21 1.15 1.32 1.34 Large intestine 0.11 0.18 0.15 0.20 Small intestine 0~24 0~35 0.36 0.41 ._ _ The extremely high blood level over a long period of time and the figure of distribution into various organs of the radioactive diagnostic agent as shown in Table 2 are quite similar to those of 131I-labeled fibrinogen as conventionally employed.
(F) Behaviors of the raclioactive diagnostic agent as obtained in (C) in thrombosed rabbits:-2~ Thrombosis was produced in rabbits at the femoral part by the formalin application procedure. To the rabbits, the radioactive diagnostic agent (0.5 ml) as obtained in (C) was administered through the ear vein. After 24 hours from the administration, a constant amount of the blood was sampled, and the locus of thrombosis was taken out. Radio-activity was measured on the blood and the locus of thrombosis. The radioactivity ratio of the locus of thrombosis to the blood or the same weight was 7.44 _ 3.41 (average in 10 animals + S.D. value).

~;2S~35~) ( From the above results, it is understood that the radioactive diagnostic agent as obtained in (C) has the nearly same physiological activity as fibrinogen does.
Thus, the radioactive diagnostic agent is useful for nuclear medical diagnosis.
(G) Toxicity of the radioactive diagnostic agent as obtained in (C):-The radioactive diagnostic agent as obtained in~C) was subjected to attenuàtion of the radioactivity ~o an appropriate extent, and the resultant product was ad-ministered intravenously to groups of male and female rats o SD strain, each group consisting of five animals, at a dose o~ 1 ml per 100 grams of the bodyweight (corresponding to 600 times the expec~ed dose to h~man ~eings7 and also to groups of male and female mice of ICR strain, each group consisting of five animals, a~ a dose of 0.5 ml per 1.0 gram of the bodyweight (corresponding to 3,000 times the expected dose to human beings~. As the control, the same ~rolume o a physiolo~ical saline solution as a~ove was intravenousl~
administered to the separate groups of the same animals as abo~e. The animals were fertilized for 10 days, and the varia~ion in bodyweight during that period was recorded~ No significant difference was recognized between the medicated groups and the control groups.
2S After 10 days ~rom the administration, all the animals were sacrificed and subjected to observation of the abnormality in various organs. But, no abnormality was seen in any of the animals.

~S8~50 ( From the above results, it may be said that the toxicity of the non-radioactive carrier of the invention i5 extremely low.
Example 5 (~) Prepara~ion of the dialdehydostarch-deferox-amine condensation product as a non-radioactive carrier:-Dialdehydostarch (average moLecular weight, 7000;
oxidation rate, 80 %) (1 g) was dissolved in water (40 ml).
Separately, deferoxamine (2.4 g) was dissolved in water (30 ml), an equimolar amount of triethylamine (388 mg) was added thereto, and the resultant solution was stirred at room temperature for 10 minutes. Both solutions were combined together and stirred at room temperature for 15 minutes.
The reaction mixture was sl~bjected to gel chromatography under the ~ollowing conditions:
Carrier: Sephade~ G-S0 Solvent: Water Columno diameter, ~.5 cm; height, 50 cm Flow rate: 2.5 ml/min The dialdehydostarch-deferoxamine condensation product was eluted at a volume of 270 - 430 ml, while the unreacted deferoxamine was eluted a~ a volume of 550 to 600 ml. The eluate containing ~he dialdehydostarch-deferoxamine ~ondensation product was subjected to lyophilization.
. 25 The dialdehydostarch-de~eroxamine condensation product thus obtained was subjected to analysis by high speed liquid chromatography under the ~ollowing conditions:

Column: TS~-3000SW

* Trade Mark _13Z 5 ~ ~ 5 Solvent: O.OSM Tris-0.15M sodium chloride-hydrochloric acid buffer ~pH, 7.4) Pressure: 100 kg/cm2 Flow rate: 1.0 ml/min s Absorptive wavelength: 280 nm No free deferoxamine was detected. (The retention volume of deferoxamine in the above system is 32.8 ml.) Example 6 (A) Preparation of the dialdehydostarch-deferox-]0 amine condensa~ion-reduction product as a non-radioacti~e carrier:-Dialdehydostarch (average molecular weight, 7000;oxidation rate, 80 %) (1 g~ was dissol~ed in water ~40 ml).
Separately, deferoxamine (2.4 g~ was dissolved in water (30 lS ml)t an equimolar amount o triethylamine (388 mg) was added thereto, and the resultant solution was stirred at room temperature for 10 minutes. Both solutions were combined together and stirred at room temperature for 15 minutes.
Sodium borohydride (167 mg) was added thereto, and stirring was continued a~ room temperature for about 1 hour. The reaction mixture was subjected to gel chromatography under the following conditions:
Carrier: Sephadex*G-50 Solvent: Water Column: diameter, 4.5 cm; height, S0 cm Flow rate: 2.5 ml/min The dialdehydostarch-deferoxamine condensation-* Trade ~ark reduction pxoduct was eluted at a volume of 300 - 450 ml, while the unreacted deferoxamine was eluted at a volume of 550 to 600 ml. The eluate containing the dialdehydostarch-deferoxamine condensation~reduction product was subjected to lyophilization.
The dialdehydostarch-deferoxamine condensation reduction product thus obtained was subjected to analysis by high speed liquid chromatography under the following conditions:
Column: TS~-3000SW
Solvent: 0~05M Tris-0.15M sodium chloride-hydrochloric acid buffer (p~, 7.4) Pressure: 100 kg/cm~
Flow rate~ 1.0 ml/min Absorptive wavelength: 280 nm No free deferoxami.e was detected. (The r~tention volume of deferoxamine in the above system is 32.8 ml.) A ~efinite amount the dialdehydostarch-deferox-amine condensation-reduction product W2S disso'ved in water, and a sufficient amount o an aqueous ~erric chloride solution was added thereto to make a 1 : 1 complex between the deferoxamine moiety in said condensation-reduction product and Fe(III) in said ferric chloride solution. The reaction mixture was allowed to stand for 1 hour and then - 25 subjected to measurement of absorbance at 420 nm, whereby it was confirmed that the number of the deferoxamine moieties in said condensation-xeduction product is 19.6 per one molecule of dialdehydostarch. The number average molecular ~l2S~SO

weight of said condensation-reduction product was thus calculated to ~e about 18~000.
Still, deferoxamine and Fe(III) can form a 1 : l complex having a maximum absorption at 420 nm, and the ~max S value of the complex at 420 nm is 2.63 x 103.
Example 7 (A) Preparation of the dialdehydostarch-hexane-diamine: KTS condendate condensation-reduction product as a non-radioactive carrier:-A solution of KTS (132 mg) in dry dioxane ~5 ml~
was cooled to about 10C. Tri-n-butylamine (0.12 ml~ and isobutyl chloroformate (64 ml) were added thereto. The resultant mixture was s~irred at the same tempera~ure as above for about 50 minutes to obtain a mixed acid anhydride solution. To this solution, a solution of N-tert-butyloxy-carbon~l-1,6-hexanediamine (104 mg) in dry dioxane (2 ml) was added, and the resultan~ mixture ~as stirred at 10C for about 15 hours to produce N-tert-butyloxycarbonyl-1,6-hexanediamine:KTS condensate. A few drops of co~c. hydro-chloric acid were added thereto to make a pH of about 2,whereby the N-tert-butyloxycarbonyl group was eliminated to give a solution of hexanediamine-KTS condensate.
The above solution was added to a solution of dialdehydostarch (200 mg) in dimethylsulfoxide (5 ml~, sodium borohydride (17.2 mg) was added ~hereto, and the resultant mixture was reacted at room temperature for 3 hours. The reaction mixture was subjected to dialysis by a ~zs~so conventional procedure for 30 hours to eliminate the unreacted reagents and lyophilized to obtain dialdehydo-starch-hexanediamine:KTS condensate condensation-reduction product.
The dialdehydostarch-hexanediamine KTS condensate condensation-reduction product as obtained above was dissolved in water to ma~e a concentration of 3 mg/ml, and the resulting solution was subjected to measurement of absorbance at 334 nm using water as the control, whereby it was confirmed tha~ the number of the KTS moieties in said condensation-reduction product is 11~2 per one molecule of dialdehydostarch. The avexage molecular weight of said condensation-reduction product was thus calculated to be about 11~500.
Still, the hexanediamine:KTS condensate had a maximum absorption at 334 nmr and its ~max value was 4.37 x 104.
IB) Preparation of the ~ibrinogen-combined ~ial~ehydostarch-hexanediamine:KTS condensate condensation-reduction produc~ (a fibrinogen-combined non-radioactive carrier):-The hexanediamine:KTS condensa~e solution asobtained in (A) ~as added to a solution of dialdehydostarch (200 mg) in dimethylsulfoxide (S ml), and the resultant mixture was stirred at room temperature ~or about 3 hours.
The resulting solution containing the dialdehydostarch-hexanediamine:R~S condensate con~ensation product (S mli was added to a solution of fibrinogen t250 mg) in O.OlM phos~

:~25~3850 phate bufer-n.lSM aqueous sodium chloride mixture ~pH, 8.4) (50 ml~, followed by stirring at room temperature ~or about 3 hours. Sodium borohydride (12.9 mg) was added thereto. The resultant mixture was stirred for about 1 S hour. The reaction mixture was dialyzed to O.OlM
glucose-0.35M sodium citrate solution at 0 to 4C for 24 hours and then passed through a column of Sepharose*4B
(diameter, 4.4 cm; height, 50 cm) using O.OlM glucose-0.35M
sodium citrate solution as an eluting solvent. The eluate was lyophili~ed to give the dialdehydostarch-hexanedi-amine~KTS condensate condensation reduction product as cotton-like crystals. The cotton-like crystals (100 mgl were dissolved in deoxygenated water (160 ml), and 1 mM stannous chloride solu~ion (10 ml) and sodium ascorbate (0.6 g) were added thereto to make a clear solution. The solution was passed through a filter having a pore diameter o~ 0.22 ~m, and the filtrate (1.5 ml) was filled in a vial ~lushed with nitrogen gas to obtain a ~ibrinoqen combined non-radioactive carrier as a pale yellow, transparent solu~ion. The above operations were e~ected under a sterile condition.
(C) Preparation of the 99mTc-labeled, ~ibrinogen-com~ined dialdehydostarch-hexanediamine:K~S
condensate condensation-reduction product as a radioactive diagnostic agent:-To the ibrinogen-combined non-radioactive carrier tl.5 ml) as obtained in (B), there was added a physiological saline solution (1.5 ml3 containing 99mTc (3.3 mCi) in the * Trade Mark ~2~ Sl~

form of sodium pertechnetate, followed by stirring ~or 15 minutes to obtain the 99mTc-labeled, figrinogen-combined polyacrolein-hexanediamine: KTS condensate condensation-reduction product useful as a radioactive diagnostic agent.
This solution was pale yellow, transparent.
(D~ Properties of the radioac~ive diagnostic agent as obtained in (C):-The radioactive diagnostic agent as obtained in(C1 was subjected to electrophoresis (1O7 mA/cm; 15-minutes) using a Veronal buffer (pH, 8.6) as a developing solvent and a cellulose acetate membrane as an electrophoretic membrane, and scanning was carried out by the use of a radiochromato-scanner. The radioactivity was recognized as a single peak at the locus of 0.5 cm distant from the original line towards the negative side. This locus was the same as that of the coloring band of fibrinogen with Pon~eau 3R.
From the above result, it may be said that the radioactive diagnostic agent as obtained in (C) has a labeling eficiency of nearly 100 % and its electric charge is substantially the same as that of fibrinogen.
To the radioactive diagnostic agent as obtained in (C), 0.1 M sodium diethylbarbi~urate hydrochloride buffer (pH, 7.31 containing O.OS ~ calcium chloride to make a fibrinogen concentration of 1 mg/ml. Thrombin (lO0 units/ml; O.l ml) was added there~o. The resultant mixture was allowed to stand in an ice bath for 30 minutes. The produced fibrinogen clots were completely separated Çrom the liquor, and radioactivity was measured on the clots and also ~ZS8~

on the liquor. From the obtained results, it was determined that the clottability o~ the radioactive diagnostic agent is 91 % based on the starting fihrinogen.
Example 8 (A) Preparation of the dialdehydostarch-deferoxamine condensation product as a non-radioactive carrier:-To a solution of deferoxamine (130 mg) in O.OlM
phosphoric acid-0.15M sodium chloride buffer (1.5 mr), triethylamine (99 % solution; 27.9 ul) was added, and the resultant mixture was agitated at room temperature ~or 5 minutes. An aqueous solution of dialdehydostarch (25 mg/ml; 2 ml) was added thereto. The resulting mixture was stirred at room temperature for 15 minutes to obtain a solu ion containing the dialdehydostarch-deferoxamine condensation product which is useful as a non-radioacti~e carrier.
(B) Preparation of the ibrinogen-combined dialdehydostarch-deeroxamine conden~ation product (a fibrinogen-combined non-radioactive carrier):-The non-radioactive carrier (5 ml) as obtained in ~A~ was added to a solution o human fibrin~gen (~00 mg) in O.OlM phosphate buffer-0.15M aqueous sodium chloride mixture (pH, 8.4) (30 ml) at 0 ~o 4C, followed by stirring at the 25 same temperature as above for about 3 hours. The reaction mixture was dialyzed to 0.01~ glucose-0.35M sodium citrate solution at 0 to 4C for 24 hours and then passed through a column o~ Sepharose~4B (diameter, 4.4 cm; height, 50 cm) * ~l~rade Mark ~s~so ~ ~3 -using O.OlM glucose-0.35M sodium citrate solution as an eluting solvent.
The eluate containing the fibrinogen-combined dialdehydostarch-deferoxamine condensation product was diluted with 0.OlM glucose-0.35M sodium citrate solution to make a fibrinogen concentration of 1 mg/ml, and sodium ascorbate was added thereto to make a concentra~ion of 30 ~M. The resultant solution ~3 ml) was admitted into each vial, fo~lowed by lyophilization to obtain a fibrinogen-combined, non-radioactive carrier as a cotton-like product.
~he above opera~ions were effected under a sterile condi-tion.
Example 9 (A) Preparation of the dialdehydostarch-deferox-lS amine condensation product as a non-radioctive carrier:-To a solution of deferoxamine ~130 mg) in O.OlMphosphate buffer-O.lSM aqueous sodium chloride solution (1.5 ml~, triethylamine (99 ~ solution) (27.9 ~1) was added, and the resultant mixture was agitated at room temperature for 5 minutes. An aqueous solution of dialdehydostarch t25 mg/ml; 2 ml) was added thereto, and stirring was continued at room temperature for 15 minutes to obtain a solution containing the dialdehydostarch-deferoxamine condensation product which is useful as a non-radioactive carrier.
(B) Preparation of the fibrinogen-combined dialdehydostarch-deferoxamine condensation-reduction product la fibrinogen-combined non-radioactive carrier):

~Z58850 - ~4 -~ he non-radioactive carrier (3.5 ml) as obtained above was added to a solution of ~ibrinogen ~00 mg) in O.OlM phosphate-0.15M aqueous sodium chloride mixture (pE~, 8.4) t30 ml) at 0 to 4C, followed by stirring at the same temperature as above for about 3 hours. To the resulting mixture, sodium borohydride (12~9 mg~ was added, and stirring was continued at 0 to 4C for about 1 hour for reduction.
To a portion of the reaction mixture, a solution of gallium citrate containing 67Ga (1 mCi3 was added for labeling, and the resultant solution was subjected to high speed liquid chromatography under the following conditions~
Column: TSK-3000SW
Solvent: 0.05M Tris-0.15M sodium chloride-. hydrochloric acid ~uffer (pH 7.4) Pressuxe: 100 kg/cm2 Flow rate: 1.0 ml/min Detection was made on the radioactivity of 67Ga.
~5 the result, the eluted pattern ga~e three peaks attri-Z0 butable to 67Ga-labeled fibrinogen, the 67Ga-labeled dialdehydostarch-deferoxamine condPnsation-reduction product and ~7Ga-labeled deferoxamine. From the area ratio of the peak due to 67Ga-labeled dialdehydostarch-deferox-amine condensation reduction product and the peak due to 67Ga-labelled defsroxamine, it was confirmed that 17.4 of the deferoxamine moieties are combined to one moLecule of dialdehydos~arch. Since the number of deferoxamine moieties in the fibrinogen-combined dialdehydostarch-deferoxamlne ~Z513135~

condensation-reduction product was confirmed to be 15.2 per one molecule of fibrinogen, the number of fibrinogen bonded to one molecule of dialdehydostarch was calculated as about 0.9.
S The remainder of the rea~tion mixture was dialyzed to O.OlM glucose-0.35M sodium citrate solution at 0 to 4C
for 24 hours and then passed through a column of Sepharose*
4B (diameter, 4.4 cm; height, 50 cm) using the same solution as above as an eluting solvent. The eluate containing the fi~rinogen-combined dialdehydostarch-deferoxamine conden-sation-reduction product was diluted with O.OlM glucose-0.35M sodium citrate solu~ion to make a fibrinogen concen-tration of 1 mg/ml, and sodium ascorbate was added thereto to make a concentration of 30 m~l. The resultant solution (3 ml) was filled in a vial, followed by lyophilization to obtain a fibrinogen-combined non-radioactive carrier as a cotton-like product. The above operations were effected under a sterile condition.
The ~ibrinogen-combined non-radioactiYe carrier was 2~ dissolved in sterile water to make a fibrinogen concen-tration of 1 mg/ml, and a su~ficient amount of an aqueous ferric chloride solution was added thereto to make a 1 : 1 complex between the deferoxamine moiety in said non-radioactive carrier and Fe(III) in said ferric chloride 2S solution. The reaction mix~ure was allowed to stand for 1 hour and then subjected to measurement of absorbance at 420 nm using a solution o~ said non radioactive carrier in sterile water as control, whereby ~he number o~ the ~ Trade Mar~

~2S8~350 ~ 46 -deferoxamine moieties in said non-radioactive carrier was confirmed to be 15.2 per one molecule of fibrinogen.
(C) Preparation of the 67Ga-labeled, fibrinogen-combined dialdehydostarch-deferoxamine condensation-S reduction product as a radioactive diagnostic agent:-To the fibrinogen-combined non-radioactive carrier as obtained in (B), an aqueous solution (2 ml) containing 67Ga (2 mCi) in the form of gallium citrate was added to obtain the 67Ga-labeled, fibrinogen-combined dialdeXydo-starch deferoxamine condensation-reduction product as a radioactive diagnostic agent. This soLu~ion was pale yellow, transparent and had a pH of about 7.8.
(C') Preparation of the 67Ga-labeled, fîbrinogen-combined dialdehydostarch-deferoxamine condensation-reduc-tion product as a radioactive diagnostic agent:-The non-radioactive carrier obtained in (B) was dissolved in sterile water, and human fibrinogen (0.5, 0.75, 1.0, 1.5, 2.0 or 3.0 mg) dissolved in O.OlM phosphoric acid-O.lSM sodium chloride buffer ~pH, 8.4) and 1 ml o~ an aqueous solution containing 67Ga (1 mCi) in the form of gallium citrate were added thereto. The resulting mixture was allowed to stand at room temperature ~or 1 hour and then subjected to measurement of lebeling rate. In the same manner as above, the labeling rate of 67Ga-labeled, ~5 fibrinogen-combined de~eroxamine as prepared by labeling 67Ga onto ibrinogen-combined deferoxamine was also measured. The resul~s are shown in T~ble 3.

~2S~1!35~
q7 -( 67 Table 3 (Labeling efficiency with Ga) .. _ Fibrinogen (mg)Labeling rate (~) Sample 1*l) Sample 2 2~) ,.
0.5 68.4 S 0.75 85.4 1.0 ~ lO0 17.0 1.5 ~- lO0 3 0 r- lO0 35.2

6.3 _ 41.4 - 12.6 ~ 70.9 18.8 _ 80.6 ~5.1 ~ _ 83.5 Note: ~1) Radioactive diagnostic agent accord-ing to the invention.

*2) 67Ga-labeled fibrinogen-combined deferoxamine As understood from the above, the non-radioactive carrier of the invention could be labeled with 100 % o 67Ga Z0 (1 mCi) within 1 hour when l mg of fibrinogen was used. The conventional non-radioactive carrier (i.e. fibrinogen-combined deLeroxamine) could be labeled only with 17.0 ~ of 67Ga under the same condition as above. Even when 25.1 mg or fibrinogen were used, the conventional non-radioactive carrier is labeled with ~3,5 ~ of 67Ga at the most. It is thus appreciated that the non-radioactive carrier of the invention c~n afford a radioactive diagnostic agent having a higher relative radioactivity. Further, the radioactive diagnostic agent is useful in nuclear medical diagnosis aiming at detection of thrombosis.

~ D) Properties of the radioactive diagnostic agent a5 obtained in (C):-~s~s~

The radioactive diagnostic agent as obtained in (C) was subjected to electrophoresis (1.7 mA/cm; 15 minutes) using a Veronal buffer (pH, 8.6) as a developing solvent and a cellulose acetate membrane as an electrophoretic membrane, S and scanning was carried out by the use of a radiochromato-scanner. The radioactivity was recognized as a single peak at the locus of 0.5 cm distan~ from the original line towards the negative side. This locus was the same as that of the coloring band o, fibrinogen with Ponceau 3R.
iO From the above result, it may be said that the radioactive diagnostic agent as obtained in (C) has a labeling efficiency of nearly 100 % and its electric charge is substantially the same as that of fibrinogen.
To the radioactive diagnostic agent as obtained in (C)~ O.lM sodi-~m diethylbarbiturate hydrochloride buffer (pH, 7.3~ containing 0.05 ~ calcium chloride was added to make a fibrinogen concentration of 1 mg/ml. Thrombin (100 units/ml; 0.1 ml) was added thereto. The resultant mixture w~s allowed to 5tand in an ice bath for 30 minutes. ~he produced ibrinogen clots were completely separated from the liquor, and rad.ioactivity was measured on the clots and also on the liquor. From the obtained results, it was determined that the clottability of the radioactive diagnostic agent is 89 ~ based on the starting ~ibrinogen~
(E) Behaviors o the radioactive diagnostic agent obtained in (C) in rats:-The radioactive dia~nostic agenk as o~tained in ~C) ~0.2 ml) was administered intravenously to each o~

8l~5~
- 4g -female rats o~ SD strain, and the variations of the blood level and the organ distribution with the lapse of time were recorded. The results are shown in Table 4.
Table 4 (Distribution in rat body; ~/g) Organs Time after administration (min) _ _ - 5 30 60 ~ 18-0 -Blood 8.74 7008 6.62 5.34 Liver 1.45 1.32 1.05 1.03 Heart 0.90 0.89 1.40 0.98 Spleen 0.92 0.52 1.89 0.84 Large intestine 0.18 0.11 0.17 0.29 Small intestine 0.25 1.75 0.44 0.45 The extremely high blood level over a long period of time and the figure of distribution into various organs o the radioactive diagnostic agent as shown in Table 4 are quite similar to those of 131~-labeled fibrinogen as conven-tionally employed.
(F) Behaviors of t:he radioactive diaqnostic agent obtained in (C) in thrombosed rabbits:-Thrombosis was produced in rabbits at the ~emoral part by the formalin application procedure. To the rabbits, the radioactive diagnostic a~ent (0.5 ml) obtained in (C) was administered through the ear vein. After 24 hours from the administration, a constant amount of the blood was ~5 sampled, and the locus of thrombosis was taken out. Radio-activity was measured on the blood and the locus of thrombosis. The radioacitivity ratio of the locus or thrombosis to the blood for the same weight was 8.63 + 3.83 (average in 10 animals ~ S.D. value).

~L258850 -- so --From th~ above results, it is understood that the radioactive diagnostic agent obtained in (C) has tha nearly same physiological activity as fibrinogen does. Thus, the radioactive diagnostic agent is useul for nuclear medical diagnosis.
(G) Toxicity of the radioactive diagnostic agent obtained in (C):-The radioactive diagnostic agent obtained in (C)was subjected to attenuation of the radioactivity to an appropriate extent, and the resultant product was ad-ministered intravenously to groups of male and female rats of SD strain, each group consisting of five animals, at a dose of 1 ml per 100 grams o~ the bodyweigh~ (corresponding to 600 times the expected dose to human beings) and also to groups of male and female mice of ICR strain, each group consisting of five animals, at a dose o~ 0.5 ml per 1.O gram of the bodyweight (correspondin~ ~o 3,000 times the expected dose to hum2n beings). As the con~~ol, the same volume of a physiological saline solution as above was intravenously administered to the separate groups of the same animals as above. The animals were fertilized for 10 days, and the variation in bodyweight during that period was recorded. No signi~icant difference was reco~nized between the medicated groups and the control groups.
~5 After 10 days ~rom the administration~ all the animals were sacrificed and subjected to observation of the abnormality in various organs. But, no abnormality was seen in any of the animals.

lZS~3~3S~

From the above results, it may be said that the toxicity of the non-radioactive carrier o the invention is extremely low.
Example 10 (A) Preparation of dialdehydodextran-deferoxamine condensation-reduction product as ~ non-radioactive carrier:-To a solution of deferoxamine (2.8 g) in water (30 ml), an equimolar amount o~ triethylamine (432 mg) was added, followed by stirring at.room temperature for 10 minutes. The resultant solution was added to a solution of dialdehydodextran (1 g; aldehyde group content, 5~1 ~mole/mg) in water (40 ml~, followed by stirring at room temperature for 15 minutes. To the reaction mixture, sodium borohydride (167 mg) was added, and stirring was continued at room tempera~ure for about 1 hour. The resulting solu-tion was admitted in a cellulose tube and dialyzed to water or 3 days, followed by gel chrom~tography under the follow-ing conditions:
Carrier: Sephadex~G=50 Solvent: water Column: diameter, 4.5 cm; height, S0 cm Flow rate: 2.5 ml/min The dialdehydodextran-deferoxamine condensation-reduction product was eluted at a volume o 300 to 450 ml, while the unreacted deferoxamine was eluted at a volume of 550 to 600 ml. 1`he eluate cont.aining said condensation~
reduction product was lyophilized~

* Trade Mark ~25~3~35~

The lyophilized product was sub~ected to analysis with high speed liquid chromatography under the following conditions:
Column: TSK-3000SW
S Solvento 0.05M ~ris-0.15~ sodium chloride-hydrochloric acid buffer (pH, 7.4) Pressure: 100 kg/cm2 Flow rate: 1.0 ml/min Absorptive wavelength: 280 nm As the result, said condensation-reduction produc~
was confirmed to show a retention volume of 27.3 ml. No free deferoxamine was de~ectPd. (The retention volume of deferoxamine in the above system is 32.8 ml.) (B) Preparation of ~he fibrinogen-combined dia}dehydodextran-deferoxamine condensation-reduction product (a fibrinogen-combined, non-radioctiv~ carrier):-Into a solution of dialdehydodextran (127 mg) in0.01~ phosphate bufer-O.lSM aqueous sodium chloride mixture t5 ml), defexoxamine (370 mg) was dissolved, and triethyl-amine (78.9 lul) was added thereto, followed by stirring at10 to 15C ~or 20 minutes. The resulting solution was added to a solution of fibrinogen (400 mg) in O.OlM phosphate buff~r-0.15M aqueous sodium chloride solution (40 ml) at 10 to 15C, ollowed by sitrring at the same temperature as ~5 above ~or abo1lt 2 hours. To the reaction mixture, sodium borohydride (12.3 mg) was added, and stirring was continued a~ 10 to 15C or about 1 hour.

12S8~5~

The resulting mixture was dialyzed to O.OlM
glucose-0.35M sodium citrate solution at 0 to 4C for 3 days and then passed through a column of Sepharose CL6B (dia-meter, 4.4 cm; height, 100 cm) using the same solution as above as a eluting solvent. The eluate was diluted with O.OlM glucose-0.35M sodium citrate solution to make a fibrinogen concentration of 1 mg/ml, and sodium ascorbate was added to make a concentration of 30mM. The resultant solution (3 ml) was filled in a vial, followed by lyophi-lization to obtain a cotton-like product, which is useful as a fibrinogen-combined non-radioactive carrier. The above operations were effected under a sterile condition.
~ C) Preparation of the 67Ga-labeled, fibrinogen-combined dialdehydodex~ran-deferoxamine condensation-reduction product as a radioactive diagnostic agent:-To the fibrinogen-combined non-radioactive carrier obtained in (B), an aqueous solution (2 ml) containing 67Ga (2 mCi) in the orm of gallium citrate was added to obtain the 67Ga-labeled, fibrinogen-combined dialdehydostarch deferoxamine condensation-reduction product useful as a radioactive diagnostic agent. This solution was pale yellow, transparent and had a pH of about 7.8.
(D) Properties of the radioactive diagnostic agent as obtained in (C):-The radioactive diagnostic agent as obtained in (C) was subjected to electrophoresis (1.7 m~/cm; 15 minutes) using a Veronal*bu~er ~pll, 8.6) as a developing solvent and a cellulose ace~ate membrane as an electrophoretic membrane, * Trade Mark ~2S885~

and scanning was carried out by the use of a radiochromato-scanner. The radioactivity was recognized as a single peak at the locus of 0.5 cm distant from the original line towards the negative side. This locus was the same as that of the coloring band of fibrinogen with Ponceau*3R.
From the above result, it may be said that the 67Ga-labeled, fibrinogen-combined dialdehydodextran-deferox-amine condensation-reduction product has a labeling effi-ciency of nearly 100 % and its electric charge is substan-tially the same as that of human fibrinogen.
To the radioactive diagnostic agent as obtained in(C), O.lM sodium diethylbarbiturate hydrochloride buffer (p~, 7.3) containing 0.05 ~ calcium chloride was added to make a fibrinogen concentration of 1 mg/ml. Thrombin (100 units/ml; 0.1 ml) was added thereto~ ~he resultant mixture was allowed to stand in an ice bath for 30 minutes. The produced fibrinogen clots were completely separated ,rom tne liquor, and radioactivity was measured on the clots and also on the liquor. From ~he obtained re~ults, it was determined that the clottability o the radioactive diagnostic agent is 84 % based on the s~arting fibrinogen.
Example 11 ~A) Preparation of the dialdehydostarch-deferox-amine condensation product as a non-radioactive carrier:-2S Into a solution o~ dialdehydo$tarch (10 mg~ in 0.03M phosphate bu~er-0.15M aqueous sodium chloride mixture (1.0 ml~, deferoxamine ~23 mg) was dissolved a~ room ~emperature. ~ter addition o triethylamine (5.2 ~1), Trade Mark so stirring was continued at 12 to 15C for 20 minutes to obtain a solution containing the dialdehydostarch-deferox-amine condensation product useful as a non-radioactive carrier.
(B) Preparation of the 19-9 F(ab')2 fragment-combined dialdehydostarch-deferoxamine condensation product (a 19-9 F(ab')2 fragment-com~ined non~radioactive carrier):-The non-radioactive carrier (0.42 ml) as obtained in (A) was added to a physiological saline solution of 19-9 F(ab')2 fragment (i.e. F(ab')2 fragment of monoclonal anti-human colorectal carcinoma antibody 19-9; concentration, 18 mg/ml) (0.55 ml), followed by stirring at 4 to 6C for about 2 hours. After addition of sodium borohydride (3 mg), stirring was continued at 4 to 6C for about 1 hour. The reaction mixture was dialyzed to 0.05M phosphate buffer-0.15M aqueous sodium chloride mixture (pH, 5.5) at 4 to 6C
~or 24 hours and then passed through a column OL 5ephadex G-150 Superfine (diameter, 2.2 cm; height, 30 cm) using 0.05M ~hosphate buffer-0.15M a~ueous sodium chloride mixture as an eluting solvent. The resultant solution was diluted with the same solution as the eluting solvent to make a 19-9 F(ab')2 fragment concentration o~ 0.5 mg/ml, and sodium ascorbate was added thereto to make a concentration of 100 mM, whereby a 19-9 F(ab')2 fragment-combined non-radioactive -- 2S carrier was obtained as a pale yellow transparent solution.
(C~ Preparation of the 67Ga-labeled, 19-9 ~(ab')2 ~ragmen~ combined dialdehydostarch-deferoxamine condensation product as a radioactive diagnostic agent:-8~350 ( To the 19-9 F(ab')2 fra~ment-combined non-radioactive carrer ~1 ml) as obtained in ~A), a solution (0.5 ml) containing Ga (0.5 mCi) in the form of gallium citrate was added to obtain the 67Ga-labeled, 19-9 F(ab')2 fragment-combined dialdehydostarch-deferoxamine condensation product as a pale yellow transparent solution, which is useful as a radioactive diagnostic agent.
(D) Properties of the radioactive diagnostic agent as obtained in (C):-The radioactive diagnostic agent as obtained in (C) was subjected to electrophoresis (1 mA/cm, 30 minutes) using Beronal buffer (pH, 8.6) as a developing solvent and a cellulose acetate membrane as an electrophoretic membrane, and scanning was carried out with a radiochromato-scanner.
Radioactivity was recognized as a single peak at the locus of 1.1 cm distant from the original line towards the negative side. This locus was the same as the coloring band o~ the 19-9 F(ab')2 fragment with Ponceau 3R. I
~rom the above result, it is understood that the ~0 radioactive diagnos~ic agen~ has a labeling rate of nearly 100 ~ and its electrostatic state is substantially equal to that of the 19-9 F~ab')2 fragment.

Claims (7)

Claims:

1. A chemical product which comprises (1) a unit of a polyformyl compound having at least three formyl groups per molecule, (2) at least two units of an amino group-containing chelating compound bonded to the polyformyl compound via a methyleneimine linkage (-CH=N-) or a methyleneamine linkage (-CH2NH-) formed by the condensation between a formyl group in the polyformyl compound and the amino group in the chelating compound, optionally followed by reduction, (3) at least one unit of an amino group-containing physiologically active substance bonded to the polyformyl compound via a methyleneimine linkage or a methyleneamine linkage formed by the condensation between a formyl group in the polyformyl compound and the amino group in the physiologically active substance, optionally followed by reduction, and (4) at least two radioactive metallic elements of which each is bonded to the chelating compound via a chelating bond.

2. A chemical product according to claim 1, wherein the polyformyl compound is a polyacrolein.

3. A chemical product according to claim 2, wherein the polyacrolein comprises 3 to 4,000 units of acrolein.

4. A chemical product according to claim 1, wherein the polyformyl compound is a poly(dialdehydosaccharide).

5. A chemical product according to claim 4, wherein the poly(dialdehydosaccharide) is dialdehydostarch.

6. A chemical product according to claim 4, wherein the poly(dialdehydosaccharide) is dialdehydodextran.

7. A chemical product according to claim 4, wherein the poly(dialdehydosaccharide) is dialdehydoamylose.