Note: Descriptions are shown in the official language in which they were submitted.
- 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
-- 4
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~
5 --
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~
-- 7 --
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~
-- 8
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
H3 S
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~
- 10 -
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~
-- 11 --
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~
- 13 -
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
- 15 -
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~
- 18 -
(
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~
- 19 -
(
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~
- 28 -
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~
- 37 -
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
104
~ 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
-- 43 --
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~
- 44 -
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.
- 47 -
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~
- 49 -
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~
- 54 -
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~
- 56 -
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.
