CA1258851A - Chemical product useful as a non-radioactive carrier - 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, 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. The chemical product is useful as a non-radioactive carrier in a radioactive diagnostic agent. The diagnostic agent can be used for non-invading nuclear medical diagnosis.

Description

- l - 12588~

. .
Chemical Pr~duct use~ul as a non-radioactive carrier The present application was divided out of Canadian Patent Application Serial No. 442,833 filed December 8, 1983.
The present invention relates to a chemical product useful as a non-radioactive carrier.

For the purpose of a non-invading nuclear medical diagnosis such as recording, dynamic study and quantitative measurement of the blood circulation system, detection o~
physiological abnormalities or localization of abnormali-ties by imaging, physiologically active substances labeled with iodine-L31 (131I) have been widely used, such as 3 I-labeled serum albumin and 131I-labeled fibrinogen.
~owever, 131I has a long hal~ e of about B days and emits beta-rays so that the patient administered therewith is exposed to a large quantity o~ radiation.
In ordec to overcome this drawback o~ r-labeled physiologically active substances t attempts have been made to provide cadioactive diagnostic agents which combine physiologically active substances and cadioactive metaLlic elements having more favorable physical properties 125~51

- 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 S diagnostic agent. For instance, human serum albumin has been treated with an aqueous solution containing technetium-99m (99 Tc) 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 with an aqueous solution containing indium-lll (llLIn) in the form of indium chloride to give lllIn-labeled bleomycin. However, the chelate forming property of these physiologically active 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 administra~ion into living bodies, so that the behavior of the radioactivity in SUCIl bodies does not necessarily coincide with that of the serum albumin or the bleomycin used as the physiologically active substance. This is a very serious disadvantage ~or nuclear medical diagnosis which is based on exact tracing o~ the behavior of the radioactivity on the assumption that it coincides with the behavior of the physiologically active substance.
In ~ecent yea~s, attention has been drawn to some chelating compounds which show, on ~he one hand, a strong ( ~ 3 ~ 125~851 chelate ~orming property with a variety o~ 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 radioactive metallic element and a physiologically active substance to them. Examples of such chelating compounds are diethylenetriamine-pentaacetic acid, ethylenediamine-triacetic acid, 3-oxobutyral-bis(N-methylthiosemicarba-zone)carboxylic acid, deferoxamine, 3-aminomethylene-2,4-pentanedione-bis(thiosemicarbazone) derivatives, l-~p-aminoalkyl)phenylpropane-1,2-dione-bis(N-methylthio-semicarbazone) derivatives, etc, [Krejcarek: Biochemical & Biophysical Research Comm, Vol. 77, 2, S81-585 (1977);
lS Leurg: Int. J. Appl. Radiation & Isotopes, Vol. 29, 687-692 (197B); Japanese Patent Publn. (unexamined) Nos. 56-34634, 56 1253L7, 57-102820, etc~]. Since the resulting products are stable and ~etain the activities o~ the physiologically active substances contained there-~0 in, they are suitable for diagnostic use. However, such products which include physiologically active substances o~ large molecular weight, such as ~ibrinogen (molecular weight, about 3~0tO00) and rgG (molecular weight, about 160,000), do not usually provide a su~iciently high cadioactivity or satisactory diagnosis.
In order to overcomc the above drawback, a ~L2S8851 physio309ically active substance may be combined with many chelating compounds and the resulting product can be bonded to many radioactive metallic e~ements. While this method will assure a hiqh radioactivity, the resulting physiologicaLly active substance may be unÇavorabLy de-natured or its physiologicaL ac~ivity may be undesirably decreased or Lost.
Besides, physiologically active substances oE
high molecular weight are preEerably administered to human beings in small doses in view oE their antigen properties.
In vie~ oE this, the physiologically active substance should have a high radioactivity.
As a result of an extensive study, it has now been found that the use of a Eormyl yroup-containing chelating substance comprising a unit of a polyEormyl oompound and a unit of an amino group-containing chelating compound in combination as a carrier for a physiologically active substance and a radioactive metallic element can provide a radioactive diagnostic agent havinq a relatively ZO high radioactivity per molecule without causing any de-terioration or decrease oE the physiological activity inherent to the physiologically active substance.

~2S~8S~

Accordlng to one aspect of the invention there is provided 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.
In Canadian Patent Application Serial No. 442,833 there is disclosed a chemical product useful as a radioactive diagnostic agent 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 polyEormyl 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.

- 6 ~ 5~3Sl The polyfocmyl compound (L) is réquired to have at Least three formyl groups in the moleeuLe and pcececab~y has more. Oc these formyl groups, at leas t two are to be eombined with the eocreponding numcer oF moleeules o~ the 5 amino group-eontaining ehelating eompound (2), and at least one is to be eombined with the physiologieally aetive sub-stanee (3~. Speeiie examples of the polyfocmyl eompound ~L) ace polyaerolein, polymethaerolein, ete. Preferred are polyaeroleins o~ the formula:
-(CH -CHJ
C~O
lC whecein p is usu~lly from 3 to ~,000, pre~erably fcom 10 to 500. Sueh polyaecoleins may be prepaced, for example, by subjeeting aecolein to Redox polymerization tSehulz et al.: Makcomol. Chem~, Vol. 24, page 141 (1975)]. Othec speeifie examples are poly(dialdehydosaeeharides?, typieal of whieh is diaLdehydostaceh o~ the formula:

CH20E~
o~
-( ~ / H-O~p,-CHO C~O

~X5885~

wherein p' is usually from 2 to 1000, and preferably from 10 to 500. These may be prepared, for example, by oxidizing polysacchacides (e.y. starch, amylose, dextran, purdan) wi~h an oxidizing agent le.g. sodiurn periodate) so as to form two forrnyl groups from each saccharide unit.
Any amino group-containing chelating compound (2) may be used which shows a s~rong chela~e ~orming property to a radioactive metallic element and has an amino group capable o~ reacting with a formyl group in the E~lyformyl 10 comE~ound tl) under relatively mild conditions. Specific exarnples are deferoxamine (iOe. l-amino 6,17-di-hydroxy-7,10,18,21-tetraoxo-27-(N-acetyl-hydroxylamino~-6,11,17,22-tetraazeheptaeicosane) [The Merck ~ndex, 9th Ed., page 374 (19761 ), 3-aminomethylene-2,4-pentanedione-bis-(thiosemi-l_ car~azone) derivatives of the ~ocmula:

CH3-C=N-NH-C-NH-R
H2N-cH=c CH3-C=N-NE~-Ç-NH-R2 ~25885~

wherein Rl and R2 are each a hydrogen atom, a Cl-C3 alkyl group or a phenyl group tEP-A-0054920], l-(p-amino-alkyl)phenylpropane-1,2-dione-bistthiosemicarbazone) derivatives of the formula:

H2N-(CH2)n ~ C=N-NH-C-NHR
C=N-NH-C-NHR

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

3 tAustralian patent 533722], etc. Any compound which has a metal capturing property sui~abLe to eorm a che-late and does not have an amino group but can be readily modi~ied so as to include an amino group or an amino group-~ontaining function is also suitable as the chelating oompound ~2) a~ter such snodi~ication. For example, a compound ~earing a carboxyl group may be reacted with hexanediamine to convert it to a compound containing an aminohexylaminocarbonyl group, which can be readily condensed with a formyl group. Specific examples are diethylenetriaminepentaacetic acid, ethylene-diamine~riacetic acid, 2-oxopropionaldehyde-bis(thiosemi-carbazone) decivatives o~ the eormula:

lZ58B5~
g HOOC-C C=N-NEl-C-NH-R
76~
R -C=N-NH-C-NH-R8 wherein RS, R6, ~7 and ~8 are each a hydrogen atom or a C~-C3 alkyl group lU.S. patent 4287362], etc.
The term "physiologically active substance" which S is used to describe the o~nstituent (3) is intended to mean any substance which shows a specific accumulability in a certain organ or tissue or a certain diseased locus or which exhibits a specific behavior corresponding to a certain physioLogical state. Tracing of the behavior of such substance in the Living body can provide information useful ~or diagnosis. Physiologically active substances having an amino group capable of being condensed with a formyl group under relatively mild o~nditions are advantageous in t~is invention. Even when an amino l-S group is not present, however, the substance may be used as the physiologically active substance t3) after chemical modification to provide an amino group or an amino group-containing func~ion. Specific examples of suitable physiologically active substances are blood pro~eins ~a (e.g. human serum albumin, ~ibrinogen), enzymes (e.g.
urokinase, streptokinase), hormones (e.g. ~hyroid 1~S~385~

stimulating hormone, parathycoid hormone), immune anti-bodies (e.g. IgG), monoclonal antibodies, antibiotics (e.g~ bleomycin, kanamycin), saccharides, atty acids, amino acids, etc. In general, this invention is advan-S tageously applicable to physiologically active substances having molecular weights of not Less than ahout 100,000.
The term "radioactive metallic element" used to describe the oonstituent (4) is intended to mean any metallic element having radioa~tivity, and which has physical characteristics suitable or nuclear medical diagnosis and can be readily captured with the chelate forming structure in the chelating compound (2~. Speci-ic examples of suitable radioactive metallic elements are gallium-67 (67Ga), gallium-68 (6~Ga), thallium-201 (201~ indium-lll (lllIn), technetium-99m (99 Tc~, etc. They are normalLy employed in their salt form, particularly in their water-soluble salt forms.

For the preparation of the chemical product of the present invention useful as a non~radioactive carrier, 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 methylene-imine linkage to the methyleneamine linkage. Depending on the kinds Oe reactants, the reaction conditions, etc., the number of units oE the chelating compound (2) to be introduced into ~25885~

the polyormyl compound (1) may vary and generally not less than about S units, and especially not less than about 10 units, of 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 for combination with the physiologically active substance (3).
The resulting cond~nsation (or condensation-reduction) product of the polyformyl compound ~11 and the chelating compound (2) (hereinafter re~erred ~o as "the condensation or condensation-reduction producti) used as the non-radioactive carrier is then oondensed 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 polyeormyl compound (L~ moiety of the former and the amino group o~ the latter to give a physiologicalLy active substance-combined condensation or condensation-redu~tion product. The number of units Oe the physio-logically active substance (3) to be introduced into the condensation or condensation-reduction product varies with the kinds of the reactants, the reaction oanditions, etc., and usually a small number o~ not more than about 10 units, preferably o~ not more than 3 units, o~ the physioLogically active substance (3) per molecule o~ the poly~ormyl compound (1) is desirable.

( - 12 - 125~5~

Alternatively, the physiologically active substance~combined condensation or condensation-reduction product may be prepared by first condensing the poLyformyl compound ~1) with the physiologically active substance (3) to form a methyleneimine linkage between a formyl group in the former and an amino group in the latter, optionally followed by reduction o~ the methyleneimine linkage to a methyleneamine linkage, to give a physiologieally aetive substanee-combined polyformyl compound, whieh is then condensed with the chelating eompound (2) to ~orm a methyleneimine linkage between a formyl yroup in the polyformyl eompound moiety of the physiologieally active substanee-eombined polyformyl eompound and an amino group in the ehelating eompound (2), optionally ~ollowed by reduetion of the methyleneimine linkage to a methylene-amine linkage, whereby a physiologieally aetive substance-eombined eondensation or eondensation-reduction product is obtained. As or the number of the units of the ehelating eompound (2~ and of the physiologieally aetive substance (3)l the same ~omments as stated above apply.
~n the above preparation proeedures, the reduetion optionally carried out a~ter the eondensa~ion may be aeeom-plished in a single step at the ~inal stage~ Further, eaeh o~ the reaetions, such as the condensation and the redue-tion, may be.earried out by ~ se conventional proeedures.
Furthermore, during the reduction, a ~ormyl group may be ~L~S~385~

converted into a hydroxymethyl group simultaneously with the conversion of a methyleneimine linkage into a methyleneamine linkage. Usually, the condensation proceeds easily at coom temperature. For the 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, the reaction product may optionally be purified by ~ se conventional methods, such as column chromatography, geL
permeation and dialysis.

The thus obtained physiologically active substance-combined condensation or condensation-reduction product 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 diagnostlc agent according to the invention described in parent application Canadian Patent Application Serial No. 442,833.

One oF two di~erent labeling procedures may be employed depending upon the kind or state of the radio-active metallic element (4). When the radioactive metallic element (4) is in a valency state which can ~ocm a stable chelate compound, the physiologically active substance-combined condensation or condensation-reduction product may be contac~ed with the radioactive metallic element

(4) in an aqueous medium to ~o~m the radioactive metallic elemen~-labeled, physiologically ac~ive substance-combined ~258851 condensation or condensation-reduction product. ThiS
labeling manner may be applied to 67Ga, lll~n, etc.
When the radioactive metallic eLement (4) is in a valency state which has to be changed for the ~ormation o~ a S stable chelate compound, the physiologically active substance-combined condensation or condensation-reduction product may be contacted with the radioactive metallic element (4~ in an aqueous medium in the presence of a reducing agent or an oxidizing agent to form the radio-active ~netallic element labeledl physiologically actîve substance-combined condensation or condensation-reduction product. This labeling manner may be applied to 99mTc, etc.
Examples of suitable reducing agents are stannous salts, i.e. salts of divalent tin ion (snf+). Specific examples are stannous halides (e.g. stannous chloride, stannous fluoride), stannous sulfate,.stannous nitrate, stannous acetate, stannous citrate, etc. Sn+~ ion-~earing resins, e.g. ion-exchanye resins charged with snff ion, are also suitable.
When, ~or example, the radioactive metallic element ~4) is 99mTc, the physiologically active substance-combined condensation or condensation-reduction product may be tceated with 99mTc in the orm o a pertechnetate in an a~ueous medium in the presence o a reducing agent, e.g. a stannous salt. There is no ~L25&1851 particular requirement concerning the order o~ the intro-duction 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.
S The stannous salt may be used in an amount that can suf-~iciently reduce the pertechnetate.
The resulting radioactive diagnostic agent should have sufficient radioactivity and radioactivity concen-tration to assure reliable diagnosis. For example, the radioactive metallic element 99mTc may be used in an amount of 0.1 to 50 mCi in about 0.5 to S.0 ml at the time of administration. The amount of the physiologically active substance-combined condensation or condensation-reduction product should be sufficient to form a stable chelate compound with the radioactive metallic element (4).
The thus produced ~adioactive metallic element-labeled, physiologically active substance-combined condensation or oondensation-rèduction product used as a radioacti~e diagnostic agent is quite stable, and therefore it may be stored as such and supplied on demand. When desired, the radioactive diagnostic agent may contain any suitable additive such as a pH
control~ing asent (e.g. an acid, a base, a buffer), a stabiliæer (e.g. ascorbic acid) or an isotonizing agent (e.g. sodium chloride)~
The radioactive metallic element-labeled, ~25~3851 ! - 16 -physiolveially active substance-combined condensation or condensation-ceduction pcoduct is useful ~or nuclear medicaL diagnosis~ For example, a 99mTc or 67Ga-labeled streptokinase combined condensation or S eondensation-reduction produc~ may be used or recording and funetional measurement of myoeardium. Also, ~or example, a 99mTc-labeled, human serum albumin-eombined eondensation or eondensation-reduetion produet ean be used ~or ~eording, dynamie study and quantitative measurement of the blood eireulation system by intravenous administra-tion to the human body. Further, or example, a 99mTe-labeLed, ~ibcinogen or urokinase-eombined cond~nsation or eondensation-reduetion produet may be used for deteetion and reeording of thrombosis as well as the loealization of LS thrombosis, sinee this produet aeeumulates at the loeus of ~hrombosis. Furthermore, ~or example, a 99mTe-labeled, streptokinase-combined eondensation or condensation-reduetion produet is useful ~or determina~ion of the loeus of a myoeardial infaretion. Moreover, a ~9mTe-labeled, thyroid stimulating hormone-eombined eondensation or eondensation-reduction pcoduet is useful for the deteetion and reeording of a eaneer at the thyroid gland.
The radioaetive diagnostie agent may be administered to a patient in an amount su~ieient to produee tlle radioactivity neeessary for examination o~
a pactieular o~gan or tissue, by any a~p~opria~e route, ~12S~85 ! - 17 -usually via an intravenous route. Foc example, the intcavenous administration to a patient o~ a Tc-labeled radioactive diagnostic agent in an amount of about 1 to 3 ml by volume having a radioactivity o~ about 1 to S ~0 mCi is quite s-uitable or diagnostic pucposes.
The advan~ages of the physiologically active substance-combined condensation or condensation-reduction product, i.e. the physiologically active substance-combined non-radioactive carrier, may be sumrnari~ed as follows: (a) it is stable over a long period of time after manu~acture; tb) since it can be produced under mild ~onditions, no unfavorable side reaetions such as inactivation, denaturation or de-oomposition 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 gcoup is not present, the introduction of sueh a group into a physiologically active su~stance makes it suitable as the starting material; ~e) a radio-active metallic element-labeled, physiologieally active condensation or eondensation-reduction product can be formed by a very simple pcocedure, e.g. by merely con-tacting the physiologicalLy active substance-combined condensation or oondensation-ceduction product with a radioactive metallic element in an aqueous medium. The advantages o~ the cadioactivc metallic element-labeled, ~2S13~35~
- 17a -physiologically active substance-combined condensation or condensation-reduction product used as a radioactive diag-nostic agent may be also s~lmarized as ollows: (a) it is ~table over a long period of time after manufacture;
S (b) the Labeling efficiency 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 decompo-sition are caused in the physiologically active substance bonded to the condensa~ion 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 lS the exposure dose.

Practical and presently ~referred embodiments of the present invention as well as the invention described in Canadian Patent Application Seri~l No. 442,833 from which the present application was divided and a further copending divisional application are illustratively shown in the following examples.

~5885~

( Referen Preparation of polyacrolein:~
Water ~50 ml) was charged in a flask and heated under reflux while introducing nitrogen gas therein. After cooling below 20C, potassium peroxodisulfate (0.47s g) and acrolein (purity, more than 95 ~) (10 ml) were added thereto. After acrolein was dissolved, a solution of silver nitrate (0.296 g1 in water (6 ml1 was dropwise added thereto in about 1 minute while vigorous agitation. The reaction 1~ was continued ~or 2.5 hours, during which care was ta~en to avoid the elevation of the temperature above 20C. After the reaction was completed, the reaction mixture was added to water (50 ml), whereby the produced polyacrolein was precipitated. The precipitate was collected by iltration, washed with water two times and dispersed in a solution of sodium thiosul~ate (0.5 g) in water (50 ml), followed by stirring ror 1 hour. The dispersion wac filtered to collect the solid material, which was ~ashed with water several times and dried under reduced pressure overnight to obtain polyacr~lein.
Polyacrolein (50 mg) as prepared a~ove was dissolved in dimethylsulfoxide (10 ml), sodium borohydride (3 mg) was àdded thereto, and stirring was continued at room temperature for 1 hour. To the resulting mixture, ethyl acetate (10 ml1 was added to precipitate partialLy reduced polyacrolein. ~he precipitate was collected by ~iltration, dissolved in water and subjected to measurement of molecular weigh~ by high speed liquid chroma~ography under the ollow-~L~588S~

( ing conditions:
Column: TSK-3000SW
Solvent: 0.05M Tris-0.15M sodium chloride-hydrochloric acid buffer (p~, 7.4) ~low rate: 1.0 ml/min Since the partially reduced polyacrolein was eluted at a retention volume of 23.2 rnl, the molecular weight of polyacrolein was determined to be about 21,000.
Re~erence Example 2 P~eparation 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), 0.1~5 sodium periodate solution (40 ml) was added, and the resultant mixture was stirred at a dark place overnight. The reaction mix~ure was admitted in a cellulose tube and dialyzed to water for 2 days, followed by lyophilization to obtain dialdehydodextran.
~ bout 50 mg of ~he abGve prcp~red dialdehydo-dextran was weighed precisely and dissolved n O.~lM
phosphoric acid-O.lSM sodium chloride buf~er (100 ml). The resulting solukion (about 5 ml) was precisely measured, 1/100 N iodine solution ~5 ml) was added thereto, and ~urther 0.15~S sodium carbonate solution (1 ml) was added thereto, followed by allowing to stand at room temperature ~or 1.5 hours. After addition of 0.2 N suluric acid ( ml), titratioo was carried out with 1/100 N sodium thio-sul~a~e solution until a colorless, tr~nsparent solution was obtaincd. This titra~ion value was ~ken as ~. Irl the same - 20 _ 1~5885~

manner as above ? O.OlM phosphoric acid-0.15M sodium chloride buffer (5 ml) was titrated with ltloo ~ 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 (~mole/mg) c (A-B)xlO/2W wherein W is the amount of dialdehydodextran (mg~ contained in 5 ml of t~e samp~eu As the result, the aldehyde group content in the dialdehydodextran as prepared above was determined to ~e 5.1 ~ oLe~mg.
Example 1 (A) Preparation o f the polyacrolein-deferoxamine coAdensation-reduction product as a non-radioactive carrier:-~olyacrolein (molecular weight, 21,000) (500 mg) was dissolved in dimethylsulfoxide (10 ml), and the resultant solution was admixed with a solution of deferox-amir,e (~ mg) in dimethylsulfoxide (10 ml). ~he reaction was continued at rocm temperature for 3 hours. To the 2~ reaction mixture, sodium borohydride ~100 mg) was added, and stirring was continued at room temperature for 1 hour. ~he r~sultant mixture was subjected to dialysis to water over-ni~ht, followed by gel chromatography under the following conditions:
Carrier: Sephadex*G-50 Solvent: Water ~Column: diameter, 4.5 cm; height, 50 cm Flow rate: 2.5 rnl/min * ~rade t~ark - 21 _ ~258~1 ( The polyacrolein-deferoxamine condensation-reduction product was eluted at a volume of 270 - 400 ml, while the unreacted deferoxamine was eluted at a vol~ne of 550 to 600 ml. The eluate containing the polyacrolein-deferoxamine condPnsation-reduction product was subjected to lyophili2ation.
The polyacrolein-deferoxamine condensation-reduction product thus obtained was dissolved in water, ~erric chloride was added thereto, and the resultant solution was analyzed by high speed liquid chromatography under the following conditions to determine a retention volume o~ 21.2 ml:
Column: TSK-3000SW
Solvent: 0.05M ~ris-0.15M sodium chloride-lS . hydrochloric acid ~uffer (pH, 7.4) Flow ra~e: 1.0 ml/min Absorptive wavelength: 420 nm No free deferoxamine was detected. (The retention volume of de~eroxamine in the abov~ system is 32.8 ml.) A de~inite amount the polyacrolein-de~eroxamine condensation-reduction product as obtained above was dissolved in water, and a su~ficient amount o~ an aqueous ferric chloride solution was added thereto to make a 1 : 1 complex between the deferoxamine moiety in said conden-2S sation-reduction product and Fe(III) in said ~erric chloride. The reaction mixture was aLlowed to stand ~or l hour and then subjected to measurement o~ absorbance at 4~0 nm, whereby the number o~ the de~eroxamine moieties in said - 22 _ 1~58~5~

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 to be about 32,000.
Still, deferoxamine and Fe~III) can form a 1 : 1 complex having a maximum absorption 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)-ca~boxylic acid condensate condensation-reduction product as a non-radioactive carrier:-A solution of 3-oxobutyralbis(N-methylthiosemi-carbazone)carboxylic acid (hereinafter referred to as "KTS"~
1132 mg~ in dry dioxane (5 ml1 was cooled to about 10C.
Tri-n-butylamine ~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 o~ N-tert-butyloxycarbonyl-1,6-hexanediamine (104 mg) in dry dioxane (2 ml) was add~d, and the resultant mixture was stirred at 10C for about 15 hours to produce N-tert-butyloxycarbonyl-1,6-hexanediamine:KTS condensate. A
few drops of conc. hydrochloric acid were added thereto to make a pH of about 2, whereby the N-~er~-butyloxycarbonyl group was eliminated to give a solution of hexanediamine-KTS
condensate.
The above solution was added ~o a solution o~

- ~3 - ~25885~

poLyacrolein (200 mg) in dimethylsulfoxide (5 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 concentration of 3 mg/ml, and the re~ulting solution was subjec~ed to measurement of absorbance at 334 nm using water as the control, whereby ~he 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 weigh~ of said conden-sation-reductioll product was thus calculated to be about 29,600.
Still, the hexanediamine: ~TS condensate had a maximum absorption at 334 nm, and its ~max value was 4.37 x 10~ .
(B) Preparation of the fibrinogen-combined polyacrolein-hexanediamine:~TS condensate condensation-reduction product (a Cibrinogen-combined non-radioactive carrier):-A solution o~ the non-radioactive carrier as obtained in (A) (be~ore lyophiLization) (S ml) was added to - 24 - l~5~S~
( a solution of human fibrinogen (250 mg) in O.OlM phosphate buffer-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 ~hereto. The resultant mixture was stirred for about 1 hour. The reac-tion mixture was dialyzed to 0.0 lM glucose-0.35M sodium citrate solution at 0 to 4C for 24 hours and then passed through a column of S~pharose*4B (diameter, 4.4 cm; height, 50 cm) using O.OlM glucose-0. 35M sodium citrate solution as an eluting solvent. The eluate was lyophilized to give the polyacrolein-hexanediamine:~TS condensate condensation-reduction product as cotton-like crystals.
The 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 thereto 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-radioacti~e carrier.
The above operations were effected under a sterile condi-tion.
The fibrinogen-combined non-radioactive carrier as above obtained was a pale yellow, clear solution.
~ C) Preparation of the 99 ~ c-labeled, fibrinogen-combined polyacrolein-hexanediamine: KTS condensate conden-sation-reduction product as a radioac~ive diagnostic agent:-To the fibrinogen-combined non radioactive carrier ~1.5 ml) as obtained in (B~, a physiological salinc solution * Trade Mark - 25 - ~ ~5885~

(1.5 ml) containing mTc (3.3 mCi) in the form of sodium pertechnetate was added to obtain the 9 Tc-labeled, figrinogen-combined polyacrolein-hexanediamine: KTS conden-sate condensation-reduction product useful as a radioactive

5 diagnostic agent.
This solution was pale yellow, transparent.
(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 mA/cm; 15 minutes)using Veronal*buffer (pH, 8.6) as a developing solvent and a cellulose acetate mel~brane 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 ~rom 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 radioactive diagnostic agent 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 sodium diethylbarbiturate hydrochloride bu'fer (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 there~o. The resultant mixture was allowed ~o stand in an ice bath for 30 minutes. The produced ~ibrinoqen clots were completely sep~rated from the Trade Mark - 26 - ~25B851 !

liquor, and radioactivity 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.
Example 3 (A) Preparation of the polyacrolein-deferoxamine condensation product 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 fox 3 hours to produce a solution containing the polyacrolein-deferoxamine condensation product, which is useful as a non-radioactive carrier.
(B) Preparation of the fibrinogen-combined, poly-acrolein-deferoxamine condensation product (a ~ibrinogen-combined non-radioactive carrier):-The non-radioactive carrier (5 ml) as obtained above was added to a solution of human fibrinogen (200 mg) in O.OlM phosphate buer-0.15M aqueous sodium chloride mixture (pH, 8.4) at 0 to 4C, followed by stirring at the same temperature as above for abou~ 3 hours. The reaction mixture was dialyzed ~o O.OlM glucose-0.35M sodium citrate solution a~ 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 containing the ibrino~en-* Trade ~ark - 27 _ ~ 2 5 ~ 85~

combined polyacrolein-deferoxamine condensation product was diluted with O.OlM glucose-0.35M sodium citrate solution to make a ibrinogen concentration o 1 mg/ml, and sodium ascorbate was added thereto to make a concentration of 30 S 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 operations were effected under a sterile condition.
Example 4 (A~ Preparation of the polyacrolein-deferoxamine condensation product (a non-radioctive carrier):-To a solution of polyacrolein (125 mg) in dimethylsulfoxide (2.5 ml), a solution of deferoxamine (lOS
mg) in dimethylsulfoxide (2.5 ml) was added, and the resultant mixture was agitated at room temperature for 3 hours to obtain a solu~ion containing the polyacrolein-deferoxamine condensation product useful as a non-radio-active carrier.
(B) Preparation of the Eibrinogen-combined polyacrolein-deferoxamine condensation-reduction product (a fi~rinogen-combined non~radioactive carrier):-The non-radioactive carrier (5 ml) as obtained above was added to a solution of human fibrinogen (200 mg) in O.OlM phosphate buffer-O. lSM aqueous sodium chloride mixture (pH, 8.4) at O to 4C, followed by stirring at the same ~emperature as above for about 3 hours. To the resulting mixture, sodium borohydride (7.0 mg) was added, and s~irring was con~inued at O to 4C or about 1 hour.

~L25885~

To a portion of the reaction mixture, a solution containing 67Ga ll mCi) in the form of gallium chloride 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 buffer (pH 7.4) Pressure: 100 kg/cm2 Flow rate: 1.0 ml/min Detection was made on the radioactivity of 67Ga.
As the result, the eluted pattern gave three peaks attri-butable to 67Ga-labeled ibrinogen, 67Ga-labeled poly-acxolein-deferoxamine condensation-reduction product and 67Ga-labeled deferoxamine. From the area ra~io of the peak due to 67Ga labeled polyacrolein-deferoxamine condensation-reduction product and the peak due to 67Ga-labeled deferox-amine, 18.9 o~ the deferoxamine moieties were confirmed to combine to one molecule o~ polyacrolein. Since the number of the deferoxamine moieties in the fibrinogen-combined polyacrolein-deferoxamine condensation-reduction product was confirmed to be 14.8 per one molecule o fibrinogen, the number of ~ibrinogen bonded to one molecule o~ palyacrolein was calculated to be about 0.8.

The remainder o~ the reaction mixture was dialyzed to O.OlM glucose-0.35M sodium c-ntrate solution at 0 to 4C
for 24 hours and then passed ~hrough a column of Sepharose*

* Trade Mark - 29 - ~ ~5885~

4B (diameter, 4.4 cm; height, 50 cm) as an eluting solvent.
The eluate containing the fibrinogen-combined polyacrolein-deferoxamine condensation-reduction product 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 thereto to make a concentration of 30 mM. The resultant solution (3 ml) was admitted into a vial, ollowed by lyophilization to obtain a fibrinogen-combined non-radio-active carrier as a cotton-like product. The above operations were ef~ected under a sterile condition.
The fibrinogen-combined non-radioactive carrier as obtained above was dissolved in sterile water to make a fibrinogen concentration of 1 mg/ml, and a sufficient 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 solu~ion. The reaction mixture ~as allowed to stand for 1 hour and then subjected to measurern~nt of absorbance 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 o~ fibrinogen.
(C) Preparation of the 67Ga-labeled, fibrinogen-combined polyacrolein-de~eroxamine condensation-reduction produc~ as a radioactive diagnostic agent:-To the ~ibrinogen-combined non-radioactive carrier as obtained in (B), an aqueous solution t2 ml) containing 67Ga (2 mCi) in ~he ~orm o~ gallium citrate was added ~o ... . . . .. .

_ 30 _ ~ ~S8851 obtain the 67Ga-labeled, ibrinogen-combined polyacrolein-deferoxamine condensation-reduction product as a radio-active diagnostic ayent.
This solution was pale yellow, transparent and had a pH of about 7.8.
(C'~ Preparatlon of the 67Ga-labeled, fibrinogen-conbined polyacrolein-deferoxamine condensation-reduction product as a radioactive diagnostic agent:-The ibrinogen-labeled non-radioacti~e carrier 1~ obtained in (B) was dissolved in sterile water, and human fibrinogen (O.S, 0.75, 1.0, l.S, 2.0 or 3.0 mg) dissolved in O.OlM phosphate buffer-O.lSM aqueous sodium chloride mixture (pH, B.4) and 1 ml of an aqueous solution containing 67Ga (1 mci) in the fornt of gallium citrate were added thereto. The l.S resulting mixture was allowed to stand at room temperature for 1 hour and then subjected to measurement of lebeling rate. In the same manner as above~ the labeling rate of 67Ga-labeled, ~ibrinogen-combined deferoxamine as prepared by labeling 67Ga onto fibrinogen-combined deferoxamine was also m~asured. The results are shown in Table 1.

- 31 - 1~5~85~

Table 1 tLabeling efficiency with 67Ga) ~ . . . _. .
Fibrinogen (mg) Labeling rate (~) l Sample l ll ~ Sample 2 2j _ . . I ._ ._ _ .
0.5 59.3 5 0.75 83.2 l.0 97.8 17.0 l.5 ~-lO0 2.0 ~ 100 35.2 3.0 ~lO0 lO 6.3 _ ~1.4 12.6 _ 70.9 18.8 _ 80.6 25.1 _ I83.5 , __ _. . ~
Note: *l) ~adioactive diagnostic agent accord-ing to the invention.
*2) 67Ga-labeled fibrinogen-combined deferoxa~ine ~ s understood from the above, the non-radioactive carrier of the invention could be labeled ~ith 97.8 ~ of 67Ga (l ~Ci) within l hour when l mg of fibrinogen was used.
The conventional non-radioactive carrier (i.e. fibrinogen-combined deferoxamine) could be labeled only with 17.0 % of 67~a under the same condition as above. Even when 25.l mg of fibrinogen were used, ~he conventional non-radioactive carrier was labeled with 83.5 ~ O~ 67Ga at the most. It is thus app~eciated that the non-radioactive carrier of t~e invention can a~ford a radioactive diagnostic agent having a higher relative radioactivity. Further, the radioactive diagnostic agent is use~ul in nuclear medical diagnosis aiming at detection of thrombosis.
(D) Properties of the radioactive diagnostic agent as obtai.ned in (C):~

~.~588S~
- 32 ~

The radioactive diagnostic agent as obtained in (C) was subjected to electrophoresis (1.7 mA/cm; lS minutes~
using 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 recogni~ed as a single peak at the locus of O.S 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 radioactive diagnostic agent has a labeling efficiency o~
nearly 100 % and its electric charge is substantially the same as that of fibrinogen.
To the radioactive diagnostic agent as obtained in lS (C); O.lM sodium diethylbarbiturate hydrochloride buffer (pH, 7.3) containing O.OS ~ calcium chloride was added to m~ke a fibrinogen concentration of 1 mg/ml. Thrombin (100 units/ml; 0.1 ml) was added thereto. The resultant mixture was ailowed to s~and in an ice bath ror 30 minutes. The 2-0 produced fibrinogen clo~s were completely separated from the liquor, and radioactivity was measured on the clots and also on the liquor. From the obtained resuLts, it was determined that th~ clottability of the radioactive diagnostic agent is 86 ~ based on the starting fibrinogen.
(E) sehaviors of the radioactive diagnostic agent as obtained in (C) in rats~
The radioactive diagnostic agent as obtained in ~C) ~0.2 ml) was adminis~ered in~ravenously to each of - 31 _ ~2588~1 femaLe rats of 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 2.
Table 2 (Distribution in rat body; ~/g) ._ __ 5 Organs Time after administration (min) , _ !
~ 5 I 30 60 180 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 extre~ely high blood level over a long period of time and the figure o~ 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 radioactive 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 admini.stration, 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 ofthrombosis. The radioactivity ratio of the locus of thrombosis to the bLood ~or the same weight was 7.44 ~ 3.41 (avera~c in 10 animals ' S.D. value).

- 3~ _ ~25885~
( 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 S medical diagnosis.
(G~ Toxicity of the radioactive diagnostic agent as obtained in (C):-~ he radioactive diagnostic agent as 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 pex 100 grams of the bodyweight (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 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 ~olume Oc a physiological saline solution as a~ove was intravenousl~
administered to the separate groups o~ the same animals as above. The animals were fertilized for 10 days, and the variation in bodyweight during that period was recorded. No significant difference was recognized between the medicated groups and the control groups.
After 10 days from the administration, all the animals were sacrificed and subjected to observation of the abnormality in various organs. But, no abnormality was seen in any o~ the animals.

~L25~38Sl ~ 35 -( From the above results, it may be said that the toxicity of the non-radioactive carrier of the invention is extremely low.
Example 5 (A) Preparation 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 watex (30 mlJ, 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 subjected to gel chromatography under the ~ollowing conditions:
Carrier: Sephade~ G-50 Solvent: Water Column: diameter, ~.5 cm; height, 50 cm Flow rate: 2.5 ml/min The dialdehydos~arch~deferoxamine condensation product was eluted at a volume of 270 - 430 ml, while the unreacted deferoxamine was eluted a~ a volume of S50 to 600 ml. The eluate containing the dialdehydostarch-deferoxamine cosldensation 2roduct was subjected to lyophilization.
The dialdehydostarch-deferoxamine condensation product thus obtained was subjected to analysis by high speed liquid chromatography under the following conditions:

Column: TSK-3000SW

* ~rade Mark - 36 ~ 2 S ~ 851 Solvent: O.O5M Tris-0.15M sodium chloride-hydrochloric acid buf~er (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 o~ deferoxamine in the above system is 32.8 ml.3 Example 6 ~A) Preparation of ~he dialdehydostarch-deferox-].0 amine condensation-reduction product as a non-radioactive carrier:-Dialdehydostarch ~average molecular weight, 7000;oxidation rate, 80 %~ (1 g) was dissolved in water (40 ml).
Separately, de~eroxamine (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.
Sodium borohydride (167 mg~ was added thereto, and st rring 2~ was continued at 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.S cm; height, S0 cm Flow rate: 2.5 ml/min The dialdehydosta.rch-deero~amine condensation-* Trade .~ark 1~5885~

reduction product was eluted at a volume of 300 - 450 ml, while the unreacted deferoxamine was eluted at a volume of S50 to 600 ~1. The eluate containing the dialdehydos~arch-deferoxamine condensation-reduction product was subjected to S lyophilization.
The dialdehydostarch-deferoxamine condensation reduc~ion product thus obtained was subjected to analysis by high speed liquid chromatography under the following conditions:
Column: ~SK-3000SW
Solvent: 0.05M Tris-O.lSM sodit~n chloride-hydrochloric acid buffer (pH, 7.4) Pressure: 100 kg/cm Flow rate: 1.0 ml/min Absorptive wavelength: 280 nm No free deferoxamine was detected. (The retention volume of deferoxamine in the above system is 32.8 ml.) A definite amount the dialdehydostarch-deferox-amine condensation-reduction product W25 dissolved in water, ~nd a sufficient amount of an aqueous ferric 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 subjected to measurement of absorbance at 420 nm, whereby it was confirme~ that the number of the deferoxamine moieties in said condensation-reduction product is 19.6 per one molecule of dialdehydostarch. The number a~erage molecular - ~8 - ~2~88S~

weight of said condensation-reduction product was thus calculated to be about 18,000.
Still, deferoxamine and Fe(III) can form a l : 1 complex having a maximum absorption at 420 nm, and the ~max value of the complex at 420 nm is 2~63 x 103.
Xxample 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 stirred at the same temperature as above ~or about 50 minutes to obtain a mixed acid anhydride solution~ To this solution, a solu~ion o N-tert-butyloxy-carbonyl-1,6-hexanediamine (104 mg) in dry dioxane (2 ml) was added, and the resultant mixture was stirred ~t 10C for about lS hours to produce N-tert-butyloxycarbonyl-1,6-hexanediamine:KTs condensate. A rew drops of conc. hydro-chloric acid were added thereto ~o 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 thereto, and the resultant mixture was reacted at room temperature ~or 3 hours. The reactio~ mixture was subjected to dialysis by a _ 39 _ 1~58851 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. XTS condensate condensation-reduction product as obtained above was dissolved in water to ma~e a concen~ration of 3 mg/ml, and ~he resulting solution was subjected to measurement of absorbance at 334 nm using water as the control, whereby it was con~irmed that the number o~ the KTS moieties in said condensation-reduction product is 11.2 per one molecule of dialdehydostarch. The average molecular weight o said condensa~ion-reduction product was thus calcuLated to be about 11,500.
Still, the hexanediamine:KTS condensate had a maximum absorption at 334 nm, and its max value was 4.37 x ~ B) Preparation of the fibrinogen-combined ~ial~ehydo~tarch-hexanediamine: ~TS condensate condensation-reduction product (a fibrinogen-combined non-radioactive carrier):-The hexanediamine:~TS condensate solution asobtained in (A1 s~as added to a solution o~ dialdehydostarch (200 mg) in dimethyLsul~oxide ~S ml), and the resultant mixture was s~irred at room temperature ~or about 3 hours.
rhe resulting solution containing the dialdehydostarch hexanediamine:KTS condensate condcnsation product (S ml) was added to a solution ot ~ibrinogen.~250 mg) in O.OlM phos-- 40 _ ~ ~S8~51 phate buffer-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. The resultant mixture was stirred for about 1 hour. The reaction mixture was dialyæed to 0.01~
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~ing solvent. The eluate was lyophilized to give the dialdehydostarch-hexanedi-amine:KTS condensate condensation reduction product as cotton-like crystals. The 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 thereto to make a clear solution. The solution was passed through a filter having a pore diameter of 0.22 ~m, and the filtrate (1.5 ml) was filled in a vial flushed with nitrogen gas to obtain a fibrinogen-combined non-radioactive carrier ~s a pale yellow, transparent solution. The a~ove operations were effected under a sterile condition.
(C) Preparation of ~he 99mTc-labeled, ~ibrinogen-combined`dialdehydostarch-hexanediamine:KTS
condensate condensation-reduction product as a radioactive diagnostic agent:-To the ~ibrinogen-combined non-radioactive carrier (1.5 ml) as obtained in ~B), there was added a physiological saline soLu~ion (1.5 ml) containing 99m~c (3.3 mCi) in the * Trade Mark - 41 - ~25~8Sl form of sodium pertechnetate, followed by stirring for L5 minutes to obtain the 99 Tc-labeLed, figrinogen-combined polyacrolein-hexanediamine:KTS condensate condensation~
reduction product useful as a radioactive diagnostic agent.
s This solution was pale yellow, transparent.
(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 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 ou~ by the use of a radiochromato-scanner. The radioactivity was recog~ized 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 radioactive diagnostic agent as obtained in (C) has a labeling efEiciency o~ nearly 100 % and its electric charge is substantially the same as that of fibrinogen.
To the radioactive diagnos~ic agent as obtained in ~C), 0.1 M sodium diethylbarbiturate hydrochloride buf~er (pH, 7.3) containing 0.05 ~ calcium chloride to make a fibrinogen concentration oE 1 mg/ml. Thrombin (100 units/mL; 0.1 ml) was added thereto. The resultant mixture was allowed to stand in an ice bath for 30 minutes. The produced ibrinogen clots were completely separated ~rom the liquor, and radioactivity was measured on the clots and also - 42 - ~X58851 on the liquor. From the obtained results, it was determined that the clottability of the radioactive diagnostic agent is 91 ~ based on the starting fibrinogen.
Exa~
(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-O.lSM sodium chloride buffer (1.5 mI), triethylamine (99 ~ solution; 27.9 u13 was added, and the resultant mixture was agitated at room temperature for 5 minutes. An aqueous solution of dialdehydostar~h (25 mg/ml; 2 ml) was added thereto. ~he resulting mixture was stirred 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 ~ialdehydostarch-defero~amine conden~ation product (a fibrinogen-combined non-radioactive carrier):-The non-radioactive carrier (5 ml) as obtained in (A) was added to a solution of human fibrino~en (~00 mg) in O.OlM phosphate buffer-0.l5M aqueous sodium chloride mixture (pH, 8.4) (30 ml) at 0 to 4C, followed by stirring at the 2S same temperature as above for abou~ 3 hours. The reaction mixture was dialyzed to O.OlM glucose-0.35M sodium citrate solu~ion at O to ~C ~or 24 hour~ and then passed through a column o~ Sepharose~4B ~di~me~er, 4.4 cm; height, 50 cm) ~ rl~rade Mark using 0. OlM glucose-0.35M sodium citrate solution as an eluting solvent.
The eluate containing the fibrinogen-combined dialdehydostarch-deeroxamine condensation product was S diluted with O.OlM glucose-0.35M sodium citrate solution to ma~e a fibrinogen concentration of 1 mg/ml, and sodium ascorbate was added thereto to make a concentration o 30 mM. ~he resultant solution (3 ml) was admitted into each vial, followed by lyophilization to obtain a fibrinogen-combinedl non-radioactive carrier as a cotton like product.
~he above operations were effected under a s~erile condi-tion.
Example 9 ~ A) P,reparation of the dialdehydostarch-deferox-lS amine condensation product as a nvn-radioctive carrier:-To a solution o~ deferoxamine (130 mg) in OoOlMphosphate bu~fer-0.15M aqueous sodi~m chloride solution ~1.5 ml), triethylamine (99 ~ solution) (27.9 lul) was added, and the resultant mi~ture was aqita~ed at room temperature ~or 5 Z0 minutes. An aqueous solution o~ dialdehydostarch (25 mg/ml; 2 ml) was added thereto, and stirring was continued at room temperature for lS minutes ~o obtain a solution containing the dialdehydostarch-deferoxamine condensation product which is useful as a non-radioactive carrier.
(B) Preparation of the ~ibrinogen-combined dialdehydostarch-dceroxamine condensation-reduction product ~a ~i~rinogen-combined non-radioactive carrier~:-~L~S~

The non-radioactive carrier (3.5 ml) as obtained above was added to a solution of fibrinogen (200 m~) in O.OlM phosphate-0.15M aqueous sodium chloride mixture tpH, 8.4) (30 ml) at 0 to 4C, followed by stirring at the same temperature as abo~e for about 3 hours. To the resulting mixture, sodium borohydride (12.g mg) was added, and stirring was continued at 0 to 4C ~or about 1 hour for reduction.
To a portion of ~he reaction mixture, a solution of gallium citrate containing 67Ga (1 mCi) was added for labeling, and the resultant solution was subjected to high speed liquid chromatography under the following conditions:
CoLumn: TSK-3000S~
Sol~ent: 0.OSM Tris-0.15M sodium chloride-. hydrochloric acid buffer (pH 7.4) Pressure: 100 kg/cm2 Flow rate: 1.0 ml/min Detection was made on the radioact~vity of 67Ga.
~.5 the result, the eluted pattern gave three peaks a~tri-butable to 67Ga-labeled fibrinogen, the 67Ga-labeled dialdehydostarch-deferoxamine condensation-reduction product and 67Ga-labeled deferoxamine~ From the area ratio of the pea~ due ~o 67Ga-labeled dialdehydostarch-deferox-amine condensation reduction product and the peak due to 67Ga-labelled de~eroxamine, it was confirmed that 17.4 of the deferoxamine moieties are combined to one molecule of dial.dehydostarch. Since the number o~ de~eroxamine moieties in the fibrinogen~combined dialdehydostarch-deferoxamine _ 45 _ ~258~5~

condensation-reduction product was confirmed to be 15.2 per one molecule of fibrinogen, the number o~ fibrinogen bonded to one molecule of dialdehydostarch was calculated as about 0.9.
S The remainder of 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 (diame~er, 4.4 cm; height, 50 cm) using the same solution as ahove as an eluting solvent. 1'he eluate containing the fibrinogen-combined dialdehydostarch-deferoxamine conden-sation reduc~ion product was diluted with O.OlM glucose-0.35M sodium citrate solution to make a fibrinogen concen-tration o 1 mg/ml, and sodium ascorbate was added thereto to make a concentration of 30 m~l. The resultant solution ml) was filled in a vial, ollowed by lyophilization to obtain a fibrinogen-combined non-radioactive carrier as a cotton-like product. The above operations were eff~cted under a sterile condition.
The fibrinogen-combined non-radioacti~e carrier was 2~ dissolved in s~erile water to ma~e a fibrino~en concen-tration o~ 1 mq/ml, and a sufficient amount of an aqueous ~erric chloride solution was added thereto ~o make a 1 : 1 complex between the deferoxamine moiety in said non-radioactive carrier and Fe(III) in said ferric chloride solution. The reaction mixture was allowed to stand ~or 1 hour and then subjected to measurement oE absorbance at 420 nm using a solution of said non-radioactive carrier in s~crilc wa~er ~s control, whereby the number o~ the ~ Trade ~arK

- 46 - ~ ~58~5~

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-~ombined dialdehydostarch-deferoxamine condensation-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 dialdehydo-starch deeroxamine condensation-reduction product as a radioactive diagnostic agent. This solution was pale yellow, transparent and had a pH of about 7.8.
(C') Preparation of the 67Ga-labeled, fibrinogen-combined dialdehydostarch-deferoxamine condensation-reduc-LS tion product as a radioactive diagnostic agent:-The non-radioactive carrier obtained in (B) was dissolved in sterile water, and human fibrinogen (O.S, 0.75, 1~0, l.S, 2.0 or 3.0 mg) dissolved in O.OlM phosphoric acid-0.15M 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 for l hour and then subjected to measurement of lebeling rate. In ~he same manner as above, the labeling rate of 67Ga-labeled, fibrinogen-combined deferoxamine as prepared by labeling 67Ga onto fibrinogen-combined deferoxamine was also measured. The results are shown in Table 3.

125~3~5~
( 67 Table 3 (Labeling efficiency with Ga~

Fihrinogen (mg) Labeling rate (~) i ___ _ Sample 1 1) Sample 2 2) O.S 68.4 0.75 ~5.4 _ 1.0 ~ 100 17.0 1.5 ~- 100 3 0 ~_ 100 35.2

6 3 _ 41.4 12.6 _ 70.9 18.8 _ 80.6 , 25.1 Note: *1) Radioactive diagnos~ic agent accord-L5 ing to the invention.

*2~ 67Ga-labeled f~brinogen-combined deferoxamine As understood from the above, the non-radioactive carrier of the invention could be labeled with 100 % of 67~a (1 mCi) within 1 hour when 1 mg of fibrinogen was used. The conventional non-radioac~ive carrier (i.e. fibrinogen-combined de.eroxamine) could be labeled only with 17.0 % of 67Ga under the same condition as above. Even when 25.1 mg of fibrinogen were used, the conventional non-radioac~ive 25 carrie~r is labeled with 83.5 ~ of 67Ga at the most. It is th~s 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 o~ ~he radioactive diagnostic agent as obtained in ~C):~

- 48 - l~S8~51 The radioactive diagnostic agent as obtained in (C) was subjected to electrophoresis (1.7 mA/cm; lS 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 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 substantiall~ the same as that of fibrinogen.
~ o the radioactive diagnostic agent as obtained in lS (C) r 0. lM sodium diethylbarbiturate hydrochloride buffer (pH, 7.3~ containing O.OS ~ calcium chloride was added to make a fibrinogen concentration of 1 my/ml. Thrombin (100 units/ml; 0.1 ml) was added thereto~ The resultant mixture was allowed to stand in an ice bath for 30 minutes. The produced fibrinogen clots were completely 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 of the radioactive diagnostic agent is 89 ~ based on the starting fibrinogen.
2S (E) Behaviors of the radioactive diagnostic agent obtained in (C) in rats:-~he radioactive diagnostic agent as obtained in (C) (0.2 ml~ was administered intravenously to each o~

1258~

female rats of 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~
~ _ .
5 Organs Time after administration (min) _ ~3-0 - 60 180 -Blood 8.74 7.08 6.62 Liver 1.45 1.32 1.05 1.03 Heart 0.90 0.89 1.40 0.98 10 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 of the radioactive diagnostic agent as shown in Table 4 are quite similar to those of 131I-labeled fibrinogen as conven-tionally employed.
(F) Behaviors of the radioactive diagnostic agent obtained in (C) in thrombosed rabbits:-Thrombosis was produced in rabbits at the ~cnmoral part by the formalin application procedure. To the rabbits, the radioactive diagnostic agent (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 ~or the same weight was 8.63 ~ 3.83 (average in 10 animals ~ S.D. value)~

- so - ~ 2s88sl From the above results r it is understood that the radioactive diagnostic agent 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 obtained in (C):-The radioactive diagnostic agent obtained in (C)was subjected to attenuation o~ 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 of the bodyweight (corresponding to 600 times the expected dose to human beings) and also to groups of male and emale 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 volume of a physiological saline solution as above was intravenously administered to the separate groups of the same animals as above. The animals were ertilized for 10 days, and the varia~ion in bodyweight during that period was recorded. No significant diference was recognized between the medicated qroups and the control yroups.
~5 A~ter 10 days from the administration, all the animals were sacri~iced and subjected to observation o~ the abnormality in various organs. ~ut, no abnormality was seen in any o~ the animals.

lZ5~38S3L
,..
From the above results, it may be said that the toxicity of the non-radioactive carrier of the invention is extremely low.
Example 10 ~A) Preparation of dialdehydodextran-deferoxamine condensation-reduction product as a non-radioactive carrier:-To a solution of deferoxamine (2.~ g) in water (30 ml), an equimolar amount of 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 temperature for about 1 hour. The resulting solu-tion was admitted in a cellulose tube and dialyzed to water for 3 days, followed by gel chromatography under the follow-ing conditions:
Carrier: Sephadex~G-50 Solvent: water Column: diameter, 4.5 cm; height, 50 cm Flow rate: 2.5 ml/min The dialdehydodextran-deferoxamine condensation-25 reduction product was eluted at a volume o 300 to 450 ml, while the unreacted de~eroxamine was eluted at a volume o 550 to 600 ml. The eluate containing said condensation-reduction product was lyophilized * Trade Mark -- 52 - 12~885~

~he lyophilized product was subjected to analysis with high speed liquid chromatography under the following conditions:
Column: TSK-3000SW
S Solvent: 0.05M Tris-0.15M sodium chloride-hydrochloric acid buffer (pH, 7.4!
Pressure: 100 kg/cm Flow rate: 1~0 ml/min Absorptive wavelength: 280 nm As the result, said condensation-reduction product was confirmed to show a retention volume of 27.3 ml. No free deferoxamine was detected. (The retention volume of deferoxamine in the above system is 32.8 ml.) (B) Preparation of the fibrinogen-combined lS dia~dehydodextran-deferoxamine condensation-reduction product (a fibrinogen-combined, non-radioctive carrier):-Into a solution of dialdehydodextran (127 mg) inO.nl~ phosphate buffer-O.lSM aqueou~ sodium chloride mixture ~5 ml), de~eroxamine (370 mg) was dissolved, and triethyl-amine (7~.9 ~1) was added thereto, followed by stirring at10 to 15C for 20 minutes. ~he resul~ing solution was added to a solution o~ fibrinogen (400 mg) in O.OlM phosphate buffer-o~lsM aqueous sodium chloride solution (40 ml) at 10 to 15C, followed by sitrring at the same temperature as above for about 2 hours. To the reac~ion mixture, sodium borohydride (12.3 mg) was added, and stirring was continue~
at 10 to 15C ~or about 1 hour.

- 53 - ~2S8~

The resulting mixture was dialyzed to O.OlM
glucose-0.35M sodiu~n 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.
(C1 Preparation of the ~7Ga-labeled, fibrinogen-combined dialdehydodextran-deferoxamine condensation-reduction product as a radioactive diagnostic agent:-To the fibrinogen-comhined non-radioactive carrier obtained in (B), an aqueous solution (2 ml) containing 67Ga ~2 mCi1 in the form of gallium citrate was added to obtain the 67Ga-labeled, fibrinogen-combined dialdehydostarch deferoxamine condensation-reduction product useful as a radioactive dia~nostic agent. This solu~ion 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 mA/cm; 15 minutes) using a Veronal*bu~er (pH, 8.6) as a developing solvent and a cellu1ose acetate membrane as an electrophoretic memhrane, ~rade Mark `" ~L2S~38S~

and scanning was carried out by the use of a radiochromato-scanner. The radioactivity was recogni~ed as a single peak at the locus of O.S cm distant from the original line towards the negative side. This locus was the same as that o~ the coloring band of fibrinogen with Ponceau*3R.
From the above result r 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 tha~ of human fibrinogen.
To the radioactive diagnostic agent as obtained in(C), O. lM sodium diethylbarbiturate hydrochloride buffer (pH, 7.3~ containing O.OS ~ calcium chloride was added to ma~e a fibrinogen concentration of 1 mg/ml. Thrombin ~100 units/ml; 0.1 ml) was added thereto. The resultant mixture was allowed to stand in an ice bath for 30 minutes. The produced fibrinogen clots were completely separated Crom tne liquor, and radioactivity was measured on the clots and also on the liquor. From the obtained resu7ts, it was determined that the clot-tability of the radioactive diagnostic agent is 84 % based on the starting fibrinogen.
Example 11 (A) Preparation of the dialdehydostarch-deferox-amine condensation product as a non-radioactive carrier:-Into a solution of dialdehydostarch (10 mg) in 0.03M phosphate buffer-O.lSM aqueous sodium chloride mixture ~1.0 ml), deferoxamine ~23 mq~ was dissolved at room t~mperature. After addition of triethylamine (5.2 ~1), Trade Mark ~5885~

stirring was continued at 12 to 15C for 20 minutes to obtain a soiution containing the dialdehydostarch-deferox-amine condensation product useful as a non-radioactive carrier.
S (B1 Preparation of the 19-9 F(ab')2 fragment-combined dialdehydostarch-deferoxamine condensation product (a 19-9 F~ab')2 fragment-combined 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 ~or about 2 hours. After addition of sodium borohydride t3 mg), stirring was continued at 4 ~o 6C for about 1 hour. The reaction mixture was dialyzed to O.OSM phosphate buffer-0.15M aqueous sodium chloride mixture (pH, 5.5) at 4 to 6C
~or 24 hours and then passed through a column of Sephadex G-lSO Superfine ~diameter, 2.2 cm; hei~ht, 30 cm) using 0.05~ ~hosphate buffer-0.15M a~ueous sodium chloride mixture as an eiuting solventv The resultant solution was diluted with the same solution as the elu~ing solvent to make a 19-9 F(ab')z fragment concentration of 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 F(ab')~
~ragment-combined dialdehydostarch-de~eroxamine condensation product as a radioactive diagnostic agent:-3L~ 385~

To the 19-9 F(ab')2 fragment-combined non-radioactive carrer (1 ml) as obtained in (A), a solution (0.5 ml) containiny Ga (0.5 mCi) in the form of gallium citrate was added to obtain the Ga-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 în (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 color~ng band of the 19-9 F(ab')2 fragment with Ponceau 3R.
From the above result, it is understood that the ~0 radioactive diagnostic agent has a labeling rate of nearly 100 ~ and its electrostatic state is substantia~l~ e~ual to that of the 19-9 F(ab')2 ~ragment.

Claims (8)

Claims:

1. 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.

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 4000 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 polydialdehydosaccharide) is dialdehydostarch.

6. A chemical product according to claim 4, wherein the polydialdehydosaccharide is dialdehydodextran.

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

8. A process for preparing a chemical product as defined in claim 1 which comprises condensing a polyformyl compound having at least three formyl groups per molecule with an amino group-containing chelating compound to form an iminomethylene linkage between a formyl group in the former and the amino group in the chelating compound, optionally followed by reduction of the iminomethylene linkage to an aminomethylene linkage.