Note: Descriptions are shown in the official language in which they were submitted.
Radioactiv _diagnostic agent and its preparation
The present invention relates to a radioactive
diagnostic agent and its preparation. More particularly,
it relates to a radioactive element-labeled compound for
nuclear medical diagnosis, particularly for diagnosis of
ailments of the brain, and its preparation process.
Radioactive diagnostic agents for nuclear medical
diagnosis of ailments of the brain, such as imaging of
the brain and brain functional study, are required to
have the following properties: (1) they must be able to
pass through the blood-brain barrier to reach the brain;
and t2) they must accumulate in the brain at a high con-
centration within a short time and skay there over the
period of time required for clinical study. Various
studies have been carried out in an attempt to dis-
cover radioactive diagnostic agents satisfyiny these
requirements. Of the materials studied, 18F-labeled
deoxyglucose (Gallagher et al.: J. Nuclear Medicine,
~ol. 19, pages 1154-1161 (1978)) and 123I-labeled
phenylalkylamines (Winchell et al.: J. Nuclear Medicine,
Vol. 21, pages 940~946 (1980)) are notable from the
practical viewpoint. These compounds can pass through
the blood-brain barrier and accumulate in the brain and
are evaluated as practically useful for the purpose of
imaging of brain and brain functional study.
-- 2
However, 18F is a positron emitting nuclide, and
special apparatus, such as a positron camera, is needed
for imaging. Thus, ordinary scintillation cameras, which
are widely employed in the field of nuclear medicine, are
not usable. Further, the half life of 18F is only 1.8
hours, and therefore there are severe limitations imposed
on the time available for manufacture, transportation
and supply of the radioactive element or the labeled
diagnostic agent. These drawbacks exist inherently in
18F-labeled deoxyglucose.
On the other hand, 123~-labeled phenylalkylamines
can not give a sufficiently clear image upon the use of
a collimator for low energy gamma-rays, which is most
frequently employed in scintillation cameras. Further,
lS 1 3I is relatively expensive, and the use of 123I-
labeled phenylalkylamines in amounts sufficient for
diagnosis is uneconomical.
As a result of an extensive study seeking for any
substance suitable as a carrier for radioactive elements
in the field of nuclear medicine, it has now been found
that glucosone bis(thiosemicarbazone) (hereinafter re-
ferred to as "GBT") of the formula:
H-C=N-NH-CS-NH
C=N-NH-CS-NH
HO-C-H
H-C-OH
H-C-OH
CH2OH
can form stable chelate compounds with various radioactive
elements and the resulting chelate compounds (i.e. the
radioactive element-labeled compounds) can pass through
the blood-brain barrier. ~t has also been found that the
radioactive element-labeled GBT can be used as a radio-
active diagnostic agent which malces possible highly
reliable diagnosis, particularly in the brain.
According to one aspect of the present invention, there
.. .
t~ 7
-- 3 --
is provided a non-radioactive carrier, which comprises as
the essential component glucosone-bis(thiosemicarbazone)
of the formula:
~-C=N-N~-CS-NH2
C=N-NH CS-NH2
HO-C-H
H-C-OH
H-C-OH
CH20H,
said carrier being of pharmaceutically acceptable purity.
There is also provided a radioactive diagnostic agent
which comprises a radioactive element and the said
non-radioactive carrier.
~GT can be produced, for example, by oxidizing
~-D-glucose with cupric acetate to form a carbonyl group
at the 2-position and reacting the resultant glucosone
with thiosemicarbazide to introduce thiosemicarbazone
groups into the 1- and 2~positions.
GBT may be used as a carrier in two different ways
depending upon the kind or state of the radioactive element
to be carried. When the radioactive element is in a
valency state which is not required to be reduced or
oxidized for the formation of a stable chelate compound,
GBT is contacted with the radioactive element in an aqueous
medium to form a radioactive element-labeled BGT as a
chelate compound. This labeling manner may be applied to
Gallium-67, Indium-lll, etc. When the radioactive element
is in a valency state which is required to be reduced or
oxidized for the formation of a stable chelate compound,
GBT is contacted with the radioactive element in an aqueous
medium in the presence of a reducing agent or an oxidiz-
ing agent to form a radioactive element-labeled G~T as a
chelate compound. This labeling manner may be applied to
Technetium-99m, etc.
Therefore, the non-radioactive carrier of the invention
may comprise GBT optionally with a reducing agent or an
oxidizing agent for the radioactive element to be used for
labeling.
-- 4 --
A stannous salt, i.e. a salt of the divalent tin ion
(Sn++), may be employed, for example, as the reducing
agent. Specific examples are stannous halides (eOg.
stannous chloride, stannous fluoride), stannous sulfate,
stannous nitrate, stannous acetate, stannous citrate,
etc. Sn ion-bearing resins, e.g. ion-exchange resins
charged with the Sn++ ion, are also usable.
In addition to GBT as the essential component and a
reducing agent or an oxidizing agent as the optional com-
ponent, the carrier of the invention may comprise anyother additive(s) when desired. Bxamples of such
additive(s) are a pH controlling agent e.g. an acid, a
base or a buffering substance, a reductive stabilizer e.g.
ascorbic acid, erythorbic acid or gentisic acid or a salt
thereof, an isotonizing agent e.g. sodium chloride, a
preserving agent e.g. benzyl alcohol, etc.
When preparing the non-radioactive carrier of the
invention, GBT and, if used, other additives including
a reducing agent or an oxidizing agent, may be mixed in
any optional order. The carrier may be formulated in the
form of a powder, particularly a lyophilized powder, or in
the form of liquid preparation, particularly an aqueous
solution.
For preparation of a radioactive diagnostic agent of
the invention, a radioactive element may be contacted with
the non-radioactive carrier, usually in an aqueous medium,
whereby the radioactive element-labeled radioactive diag-
nostic agent is prepared in s1tu. The radioactive element
is usually employed in the form of salt, preferably a
water-soluble salt, and is normally used as an aqueous
solution, which may additionally comprise any conventional
additive(s) eOg. an isotonizing agent (e.g. sodium
chloride) or a preserving agent (e.g. benzyl alcohol).
For instance, technetium-99m is usually available in the
form of a pertechnetate (wherein 9gmTc is heptavalent)
and may be employed as an aqueous solution. When such an
p~
-- 5 --
aqueous solution is combined with the non-radioactive
carrier comprising a reducing agent, e.g. a stannous
salt, 99mTc is reduced by the reducing agent to a lower
valency (i.e. tetravalent) state, and a 99mTc-labeled
radioactive diagnostic agent is obtained comprising a
chelate compound between GBT and 99mTc in a stable stateO
When the reducing agent in a water-insoluble form, e.g. an
ion-exchange resin charged with the Sn++ ion, is used,
it is eliminated from the resulting radioactive diagnostic
agent by an appropriate separation procedure, e.g.
filtration prior to its administration.
The radioactive element in the radioactive diagnostic
agent should have sufficient radioactivity and radioactiv-
ity concentration to insure reliable diagnosis, although
there are no particular predetermined limitations. For
instance, when the radioactive element is 99mTc, the
amount of the radioactive diagnostic agent to be admin-
istered to a human adult may be from about 0.5 to 5.0 ml,
which usually displays a radioactivity of 0.1 to 50 mCi.
The radioactive diagnostic agent of this invention
is useful for nuclear medical diagnosis, particularly for
imaging of the brain and brain functional study.
Practical and presently preferred embodiments of
the invention are illustratively shown in the following
Examples wherein percentages are by weight, unless
otherwise defined.
-- 6
Exam~le 1
Preparation of glucosone:-
A solution of cupric acetate (20 g) in methanol
(250 ml) was added to a solution of ~-D-glucose (~.5 g) in
water (10 ml) and the resultant mixture was heated on a
water bath for 1 hour. The reaction mixture was cooled,
and the precipitate cuprous oxide was eliminated by
filtration. Hydrogen sulfide gas was introduced into the
filtrate for about 1 minute to precipitate the unreacted
cupric acetate in the form of cupric sulfide. After
elimination of the precipitate b~ filtration, the filtrate
was treated with a small amount of activated charcoal and
concentrated under reduced pressure to give glucosone in
a syrupy state.
Example 2
Preparation of glucosone-bis(thiosemicarbazone)
(GBT):-
~ solution of thiosemicarbazide (4.5 g) in water
(50 ml) was added dropwise to a solution of glucosone
obtained in Example 1 in 0.1 N acetic acid (6 ml), and the
resulting mixture was refluxed for about 1 hour. The
reaction mixture was cooled with ice, and the precipitated
crystals were collected by filtration and recrystallized
from water to give glucosone-bis(thiosemicarbazone) (5 g).
M.P. 225C (decomp.). Elementary analysis for C8H16O4N6S2
(%): Calcd.: C, 29.62; H, 4O97; O, 19.73; N, 25.91; S,
19.77~ Found: C, 29.57; EI, 4.88; O, 19.46; N, 26.14; S,
19.70.
L~
s
-- 7 --
Example 3
Preparation of the non-radioactive carrier:-
Glucosone-bis(thiosemicarbazone) obtained in
Example 2 was dissolved in a 0.1 ~ acetate buffer (pH,
5.0) from which dissolved oxygen was previously elimin-
ated, to produce a concentration o 10 3 M. This
solution was filtered through a microfilter to eliminate
bacteria and transferred to an ampoule. After the
addition of benzyl alcohol as a preservative thereto to
produce a 0.9 ~ concentration, the air above the solution
in the ampoule was replaced by nitrogen gas, and the
ampoule was sealed.
Example 4
Preparation of the non-radioactive carrier:-
Glucosone-bis(thiosemicarbazone) obtained in
Example 2 was dissolved in a 0.1 M acetate buffer (pH,
5.0) from which dissolved oxygen had been previously
eliminated, to produce a concentration of 10 3 M. ~n
aqueous solution of stannous chloride (4ug/ml; 10 ml) was
added to the resulting solution (10 ml), and the resultant
mixture was passed through a microfilter and transferred
to an ampoule. Benzyl alcohol as a preservative was added
thereto to produce a concentration of 0.9 %. After
replacement of the air above the solution in the ampoule
by nitrogen gas, the ampoule was sealed.
Example _
Preparation of the non-radioactive carrier:-
Glucosone-bis(thiosemicarbazone) obtained in
Example 2 was dissolved in a 0.1 M acetate buffer (pH,
5.0) to a concentration of 10 3 M. An ion-exchange
resin containing adsorbed Sn++ ions (5.5 ~g of tin ion
per mg of the resin; 4 mg) was added to the resulting
solution (10 ml) and the resultant mixture was placed
in an ampoule. After replacement of the air above the
solution in the ampoule by nitrogen gas, the ampoule was
sealed.
."i ,~
. , ~, ,
Example 6
Preparation of the radioactive diagnostic agent:-
The non-radioactive carrier obtained in Example
3 (l ml) was admixed with an aqueous solution of gallium
chloride-67Ga solution (l mCi/ml; pH, about 2) (l ml)
under aseptic conditions and the resultant mixture was
passed through a microfilter and placed in a vial. After
replacement of the air above the solution in the vial by
nitrogen gas, the vial was sealed.
The radioactive diagnostic agent as prepared
above was subjected to paper chromatography (Toyo Filter
Paper No. 51) using 80 ~ methanol as the developing sol-
vent. After development, scanning was carried out with a
radiochromato-scanner. In the radioactivity chart, a main
peak was observed at an Rf value of about 0.6, and a small
peak probably due to unlabeled gallium chloride-67 Ga
was present near the original point. By the radiochroma-
togram and the coloring method with a cuprous salt
solution, it was con~irmed that nearly all of the radio-
isotope formed a chelate compound with GBT.
Exam~
Preparation of the radioactive diagnostic agent:-
The non-radioactive carrier obtained in Example 5
(l ml) was admixed with an aqueous solution of sodium per-
technetate-99mTc solution tlO mCi/ml; pH, 5.5) (l ml)
under aseptic conditions and the resultant mixture was
passed through a microfilter and placed in a vial. After
replacement of the ai~ above the solution in the vial by
nitrogen gas, the vial was sealed.
The radioactive diagnostic agent as prepared
above was subjected to thin layer chromatography using
silica gel (Merck ~, 0.25 mm thick) as the adsorbent
and 80 % acetone as the developing solvent~ After the
development, scanning was carried out with a radiochroma-
scanner. In the radioactivity chart, a main peak was
-
D~5~
_ g _
observed at an Rf value of about 0.9. Besides, a small
peak probably due to 99mTc-labeled tin colloid was seen
at the original point, and a small peak due to an unident-
ified compound was present at an Rf value of about 0.7.
By the radiochromatogram and the coloring method with a
cuprous salt so]ution, i-t was confirmed that nearly all
of the radioisotope formed a chelate compound with GBT.
Example 8
Relationship between the amount o~ Sn++ ion
in the non~radioactive carrier and the property of the
99m~c-labeled radioactive diagnostic agent prepared by
the use of said non-radioactive carrier:-
Non-radioactive carriers were prepared in the
same manner as in Example 5 but using different amounts
lS of the Sn++ ion~ Using those non-radioactive carriers,
99mTc-labeled radloactive diagnostic agents were prepared
in the same manner as in Example 7. Chromatographic exam-
ination was carried out in the same manner as in Example
7. On the thus prepared 99mTc~labeled radioactive
diagnostic agents. The results are shown in Table 1
wherein the numerals indicate the relative radioactivity
values (%)~
Table 1
. , __
\Amount of Sn
~ ~-ecl (~g) 1) 0.5 1.01.5 ~ 5.5 11 55 550
.__~ --r - ~ ~
Rf = 0.9 86.995.7 93.3 19.3 187.578.0 72.4
_ _ . - __
Rf = 0.7 1.91.9 2.3 3.4 ¦ 4.1 4.4 3.7
.. ~ . .
Original point11.2 2.34.4 6.3 ¦ 8.4 117.6 23.9
Note: *l) Amo~nt of Sn++ ion per ml of the
non-radioactive carrier calculated
from the amount of Sn++ ion
adsorbed on the ion-exchange resin.
~,
2~5
- 10 -
From the above results, it can be seen that when
the amount of the Sn++ ion in the non-radioactive carrier
is at least from 0.5 to 550 ~9 per ml of the lO 3 M GBT
solution, the 99mTc-labeled radioactive diagnostic agent
can be produced with a good efficiency. Taking the produc-
tion rate of the major component into consideration, the
amount of Sn+~ ion is preferably from 1 to 5.5 ~g.
Exam~le 9
Distribution of the 99mTc-labeled radioactive
diagnostic agent in rabbit:-
The 99mTc-labeled radioactive diagnostic agent
(containing the radioactivity of l mCi) (0.2 ml) was
administered to each of several nembutal-anesthetized
rabbits at the auricular vein, and continuous imaging
with a scintillation camera was carried out. Regions of
interest were provided in the brain, heart, left kidney
and lung, and the relative values of the radioactivities
in brain and in other organs were measured. The results
are shown in Table 2.
Table 2
..... _ _~ _ ,
\Time after admini- lO 20 30 60 120 240
\~ =,_~
Item _ _
Brain/Heart 5.3 3.1 l.9 1.0 0.6 0.5
Brain/Left kidney41.0 15.2 4.0 2.3 1.7 l~0
Brain/Lung 1.5 3.3 2.5 2.7 2.4 2.0
From the above results, it can be seen that the
radioactive diagnostic agent of the invention can pass
through the blood-brain barrier immediately after adminis-
tration to accumulate in the brain, and the amount of
accumulation in the brain is much higher than that in
other organs. Thus, it is quite useful for imaging of
the brain as well as dynamic study of the brain.
, ,~.~.'''~'
Example 10
Toxicity of the 67Ga- or 99mTc~labeled
radioactive diagnostic agent:-
The 67Ga- and 99mTc-labeled radioactive diagnostic
agent obtained in Examples 6 and 7 were attenuated radio-
actively to an appropriate extent and then administered
intravenously to groups of SD strain male and female rats,
each group consisting of 10 rats, at a dose o~ 1 ml per 100
g of the bodyweight (which corresponds to 300 times the
amount usually administered to human beings) or to groups
of ICR strain male and female mice, each group consisting
of 10 mice, at a dose of 0.5 ml per 10 g of the bodyweight
(which corresponds to 1500 times the amount usually
administered to human beings). For the control groups, the
same volume of physiologically saline solution as above was
intravenously administered. All the groups were bred for
10 days, and the variation of the bodywei~ht was recorded
every day. No significant difference was observed between
the medicated groups and the control groups. After the
observation over 10 days, all the animals were sacrificed,
and no abnormality was observed on any organ taken out
from them. Thus, it is understood that the toxicity of
the 67Ga- or 99mTc-labeled radioactive diagnostic
agent is extremely low.
