Note: Descriptions are shown in the official language in which they were submitted.
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BACKGROIJND O~' TllJi` INVEN'TION
Radiopharmaceutical imaging agents have been utilized
heretofore for the cxternal imaging of various portions of the
anatomy. Only radiopharmaceuticals which emit gamma-photons
are suitable for this utility. The field of application is
restricted due to the fact that of the radionuclides which emit
- ga~-na rays, very few meet the additional requirements imposed
by the inherent limitations of exiting imaging systems and by
the necessity of keeping the radiation dose as low as possible.
Among these requirements are the need for a simple gamma
spectrum, a high yield of photons having an energy sufficiently
low to permit effective collimation and efficient detection and
a half-life sufficiently short to permit the administration of
millicurie quantities without an excessive post-test radiation
- dose.
The usual method of external imaging generally com-
prises labeling or tagging an organic compound suitable for
administration to a patient with a suitable radio-isotope.
More particularly, a biological agent known to localize in the
particular organ or anatomical section to be imaged is labeled
to a small extent with a radio-isotope. The thus labeled
biological agent then permits external imaging of the desired ,
organ utilizing conventional radio scanning techniques.
The problems associated with prior art attempts in
this direction center mainly on combining the requirements (1)
that the biological agent be specific to the organ to be imaged
. .
(2) that a suitable radionuclide be employed as the labeling
agent (3) that the labeled agent is sufficiently stable ln vivo
to permit effective imaging and (4) that the labeled biological
ager,t retains its organ specificity.
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It is an object of the present invention to provide
a xadiolabeled biological agent having a high degree of in vlvo
stability and which is highly organ-selective. It is a further
-object of t}le invention to provide a method of external imaging
employing said agent. It is still a further object of the
invention to provide a method for the preparation of said agent.
SUMM~RY OF THE INVENTION
The above objects are achieved by providing a radio-
labeled diagnostic agent which combines the high target organ
specificity of various drugs and biochemicals with the excellent
nuclear imaging properties of the radiometals technetium-99m,
cobalt-57, gallium-67, gallium-68, indium-lll or indium-113m.
The invention is predicated on the discovery that
chelates of the above radiometals with a substituted iminodiacetic
acid or an 8-hydroxyquinoline have a high degree of in vivo
stability, are highly specific to certain organs or anatomical
sections and possess excellent nuclear imaging properties.
The above chelates may be prepared by reacting the
desired radio-isotope with the chelating agent.
DETAILED DESCRIPTION OF THE INVENTION
Technetium-99m is commercially available either from
an isotope generator as a daughter product of molybdenum-99 or
as a direct product from a commercial supplie~ It is also
available as a solvent extraction product from molybdenum-99
solutions generally as alkali metal pertechnetate solutions at
5-100 mCi. A further discussion of preparative methods appears
in U. S. Patents 3,468,808 and 3,382,152.
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The technetium-99Jn chelate is most preferably prepared
by reducing a solution of a pertechnetate, e.g., an alkali metal
pertechnetate in the presence of the chelating agent. The
reduction is preferably effected utilizing stannous chloride as
a reducing agent. ~ny suitable reducing agent may be employed
- inclu~ing other stannous salts such as stannous pyrophosphate.
As a result of this reduction step, the product will,also contain
a significant proportion of the stannous chelate. It is to be
understood that the present invention includes the product
mixture containing both the radiometal chelate and the correspond~
ing stannous chelate.
Indeed, the composition of the invention is most
conveniently provided as a sterile kit consisting of non-radio-
active chemicals for mixing with ~he radiometal source prior to
use. The kit preferably contains a stannous salt solution,
chelating agent solution, pH buffer solution or combinations
thereof. Using sterile reagents and aseptic techniques, the
respective solutions would be mixed with each other in any
desired order and then with the radiometal source solution. The
resulting solution containing the radiometal chelate, the
stannous chelate and any free chelate may then be employed-
directly for imaging purposes.
Generally, a solution adapted for intravenous
administration containing up to 15 mCi of radioactivity is
administered to the patient. Generally, this may be accomplished
by administering 0.2-1 ml of a solution containing from about 2
to about 100 mg of combined chelate product. Radioassay of the
radio-isotope in the desired organ may be accomplished utilizing
conventional equipment, such as a scintillation camera, etc.
Organ specificity is determined by the particular
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chelating a~ent employed. ~11 of the chelates accordin~ to
the present invention, however, are cleared throu~h either the
kidneys or liver. Therefore, the chelates of the abovc radio-
metals with most substituted iminodiacetic acids and 8-hydroxy-
quinolines may be utilized for the imaging of these organs.
Preferab]y, the chelating agents are of the formula
, ' ~ .
Cll ~ COOH
R - N or ~R
; CH2 - COOH ~ -
H
wherein R may be alkyl of up to about 2~ carbon atoms preferably
about 1~ carbon atoms, alkenyl, ary] alkyl or cyclo-aliphatic
groups substituted Witll halogen, hydroxy, carboxy, nitro, amino,
keto or heterocycl;c groups. The groups may be interrupted by
ether or thio-ether linka~es.
The most preferred chelating agents are the substituted
iminodiacetic acid and 8-hydroxyquinoline analogs of drugs and
biochemicals whose organ specificity characteristics are known.
Other specific chelating agents suitable for use in
the practice of the invention are N-methyl-iminodi~cetic acid,
N-(10-carboxydecyl) iminodiacetic acid, N-[N'-(2,6-dimethyl-
phenylj carbamoylmeth~l] iminodiacetic acid, N-(o-bromobenzyl)
iminodiacetic acid, N-[3-(1-naphthyloxy)-2-hydroxypropyl]
iminodiacetic acid, nitrilotriacetic acid, or 5,7-diiodo-8-
hydroxyquinoline.
It is to be understood that the term "substituted
iminodiacetic acid" is intended to include those compounds
wherein R in the above structural f~rmula combines with each
methylene group to form a heterocyclic ring. An example of
such an acid is 2,6-pyridinedicarboxylic acid.
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The gallium and indium chelates are prepared by the
add;.tion of either GaC13 or indium chloride in .05 M EICL to thc
appropriate chelating agent at pH 3.5. After a 25-minute
incubation period, the pH is raised to between 5 and 7.
The invention is illustrated by the following non-
limiting examples.
EX~MPLE 1
2 grams (~01 moles) of alpha-chloro-2~6-Acetylxylidide
and 2 grams (0.01 moles) of iminodiacetic acid (disodium salt)
; 10 were refluxed in 200 ml of a 3:1 ETOH/H2O mixture for 48 hours.
The mixture was evaporated to dryness to yield a yellow residue.
25 ~1 of H2O were added to the residue. That which failed to go
into solution was collected by vacuum filtration. To the filtrate
concentrated hydrochloric acid was added drop-wise and the pH
monitored. At pH 3 the clear solution became cloudy and was
cooled overnight. An off-white precipitate was collected which
was recrystallized from boiling water. The product was identified
as N-[N'-(2,6-dimethylphenyl) carbamoylmethyl] iminodiacetic
` acid. m.p. 201-203. Percent yield 20% of theoretical.
NMR:DMSO-d6 ~ = 7.11 (s,3, aromatic protons)
= 3.63 (s,4,CH2-COO-)
= 3.57 (s,2,-CH2-N =)
~ = 2.20 (s,6,CH3)
CHN: 57.13 C 6.16 H 9.52N Theor
57.10 C 6.23 H 9.43N Exp
EXAMPLE 2
The N-[N'-(2,6-dimethylphenyl) carbamoylmethyl]
iminodiacetic acid prepared according to Example 1 in an amount
of 150 mg.(0.51 mmoles) was dissolved in 3 ml of 0.1 N NaOH.
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The pll of the solu~ion was adjusted to 3.5 with 1 N ~ICl.
Ext~ia 0.lN NaO1I was added thereto to compensate for the
acidic SnC12 solution which follows. 0.3 cc of a solution
of SnC12 (20 mg. 0.11 mmole in 10 ml of 1 N HCl) was added.
After a five-minute wait 80 microcuries of technetium-99m as
sodium pertechnetate was added. The product was chromato-
graphed in saline and recorded on a radiochromatogram scanner.
The resulting graph showed a peak at the solvent front, Rf=l
due to the chelated compound. There was little colloid for-
mation. There was substantially no free technetium-99m (TRf=.75).
EXAMPLE 3
Methyl iminodiacetic acid in an amount of 150 mg was
dissolved in 3 ml of 0.1 N NaOH. The pH of the solution was
adjusted to 3.5 with 1 N HCl. Extra 0.1 N NaOH was added there-
to to compensate for the acidic SnC12 solution which follows.
0.3 cc of a solution of SnC12 (20 mg. 0.11 mmole in 10 ml of
1 N HCl) was added. After a five-minute wait 80 microcuries of
technetium-99m as sodium pertechnetate was added. The product
was chromatographed in saline and recorded on a radiochromato-
gram scanner. The resulting graph showed a ~eak at the solvent
front, Rf=l due to the chelated compound. There was litt~e
; colloid formation. There was substantially no free technetium-
, 99m (TRf = .75).
EXAMPLE 4
2 ~ Ci (technetium-99m) of the product of Example 2
were injected intravenously into mice. The animals were
sacrificed serially after injection and the activities in
major organs were determined by counting multiple samples from
each organ in a scintillation counter. The ln vivo distribution
of the product of Example 2 in the mice were plotted as a
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function of time. See Fig. 1.
EXAMPLE 5
The procedure of Example ~ was followed utilizing
the pxoduct of ~xample 3. The in vivo distribution of this
,.............................................. .
product in mice as a function of time were plotted. See Fig.
2.
EXAMPLE 6
4 mCi (technetium-99m) of the product of ~xample 2
were intravenously injected into laboratory dogs. One animal
was selected for imaging at various time intervals utilizing
a scintillation camera. Camera images were obtained in multiple
: exposures and demonstrated the localization of technetium-99m
in the liver. See Fig. 3, which depicts anterior imaging studies
and demonstrates the rapid uptake by the liver which is clearly
identified at 5 minutes. (Frame A). The gall bladder appears
as a cold defect. Sequential images taken at 25, 40 and 50
minutes are shown in Frames B, C, and D, in which clearance
from the liver is demonstrated with progressive accumulation of
the radiopharmaceutical in the gall b~adder. Less than 10~ and
3~ of the injected dose remained in the blood at 10 minutes and
60 minutes, respectively. Sufficient cholecystokinin was in-
jected into the dog intravenously to effect contraction of the
gall bladder. Sequential studies revealed radiopharmaceutical
activity progressing through the small intestines. See Fig. 4.
Within 1 minute of the injection of cholecystokinin the
technetium-99m labeled product is seen leaving the gall bladder
(Frame E). Frames F, G and H taken at 5, 10 and 35 minutes
show a bolus of activity moving progressively through a small
intestine. The images were obtained using a gamma scintillation
camera (Pho Gamma III) and a parallel hole high sensitivity
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collimator.
XAMPLE 7
The procedure of Example 6 was carried out and the
results compared with those obtained following injection of the
: same dog at a later time with I-131 Rose Bengal. Both before
and after plasma loading with bromosulphthalein (BSP) to
simulate hyperbilirubinemia, BSP levels of 4-7 mg percent did
not substantially alter the plasma clearance or imaging
characteristlcs of the technetium-99m labeled product. These
images were of much better quality when compared to those ob-
tained subsequently in the same dog using I-131 Rose Bengal.
See Fig. 5.
EXAMPLE 8
The procedure of Examples 2 and 3 was followed to
prepare the technetium-99m chelate of 8-hydroxyquinoline,
employing a 7 m-molar solution of 8-hydroxy~uinoline and an
acidic stannous chloride reducing solution. The chelate
was recovered by chloroform extraction at a yield greater than
. 90~. .
Biodistribution studies were undertaken utilizing the
procedure of Example 4. 2 ~ Ci (technetium-99m) of the above
chelate were injected intravenously into 25 g mice. The
animals were sacrificed after 60 minutes and the activities in
major organs were determined by counting multiple samples from
each organ in a scintillation counter. It was determined that
on an average, 40% of the injected dose appeared in the liver
: and 20% in the intestines.
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EXA~PLE 9
; . The gallium-67 chelate of 8-hydroxy~uinoline was
prepared by adding Ga67Cl3 in O.OSM HCl to an a~ueous 7
m-molar 8-hydroxyquinoline solution having a pH of 3.5.
Following a 25 minute incubation period the pH is raised to 6.
Chloroform extraction of the reaction product produced a >90%
... .
yield of the chelate. Biodistribution studies were undertaken
according to the procedure outlined in Example 8. Following
intravenous injection of the chelate into 25 g mice, 25% of
the injected dose was found in the liver, 13% in ~he intestines
and 20% in the blood after 60 minutes.
EXAMPLE 10
The technetium-99m chelate of nitrilotriacetic acia
was prepared according to the stannous chloride reduction
method outlined in Examples 2, 3 and 8. The chelate is water-
soluble with >95% migration in saline employing paper chromato-
graphy. Biodistribution studies were carried out according to
the procedure outlined in Example 8. The chelate was found to
rapidly clear through the kidneys to urine (40% eliminated in
urine after 60 minutes) with less than 5% of the injected dose
found in the liver and intestines.
EXAMPLE ll
':`' ' .
The cobalt-57 chelate of N-[N'-(2,6-dimethylphenyl)
carbamoylmethyl] iminodiacetic acid was prepared by heating
2-5 ~ Ci of Co5 C12 in the presence of l ml (20 mg/ml) of a
solution of the compound (pH 4-5) for l hour at 100C. The
chelate was chromatographed an~ biodistribution studies
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carried out using the procedure of Example 8. At 30 minutes,
28% of the injectcd dose appears in the liver and 12% in the
intestines.
EXAMPLE 12
The technetium-99M chelate of 10-carboxydecylimino-
diacetic acid was preparcd according to the stannous chlorïde
reduction method of Examples 2, 3 and 8. The product was
chromatoyraphed in saline. >98% the material had an Rf=l.
Biodistribution studies of the chelate according to Example 8
in ten 25 g mice showed rapid blood clearance with less than 6%
of the injected dose remaining in the blood.at 60 minutes.
Radioactivity was eliminated through both kidneys and liver with
! persistent activity noted in the Iiver and lungs.
., .
EXAMPLE 13
The technetium-99m chelate of N-(o-bromobenzyl)
iminodiacetic acid was prepared by the stannous chloride
reduction method described in Examples 2, 3 and 8. The product
; 20 was paper chromatographed in saline (98% had an Rf=l.)
Biodistribution studies carried out on twelve 25 g mice accord-
ing to the procedure of Example 8 showed rapid blood clearance
(less than 5% remaining at 60 minutes) with a h~igh uptake in the
liver (40%) and intestines (30%) at 30 minutes.
~` EX~MPLE 14
.`
~ The procedure of Example 11 was followed to prepare
j the cobalt-57 chelate of methyliminodiacetic acid.
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~ ~he procedurc of Examplc 9 was ol'10wed to prepare
; the gallium-67 chclatc o~ mcthyliminodiacct;c acid.
Biodistribution studics carried out according to the procedure
' of Example 8 showed rapid renal clearance.
EX~MPLE 16
The stannous chloride re~uct,ion procedllre of Examples
2, 3 and 8 was employcd to prepare the technetium--99m chelate
of 5,7-diiodo-8-hydroxyquilloline.
EXAM~LE ~7
The stannous chloride reduction method of Examples
2, 3 and 8 was used to prepare the technetium-99m chelate of
2,6-pyridinedicar~oxylic acid.
' This appli~cation is a division of Canadian Patent
Application Serial No. 242,8-5 filed December 31, 1975.
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