TC99M-PHENIDA, RADIOSCINTIGRAPHIC AGENT FOR DIAGNOSIS OF HEPATOBILIARY DISEASE

TC99M-PHENIDA, AGENT RADIOSCINTIGRAPHIQUE POUR LE DIAGNOSTIC DES MALADIES DU SYSTEME HEPATO-BILIAIRE

English Abstract

ABSTRACT OF THE DISCLOSURE
This invention relates to complexes of techne-
tium 99m, stannous ion and novel derivatives of imino
diacetic acid of the formula:
<IMG>
wherein R1 is selected from the group consisting of:
<IMG>

- 2 -
<IMG> or <IMG>
wherein X is halo (chloro, bromo or iodo) and R7 is
lower alkyl of from 1-5 carbon atoms. It also
relates to a novel diagnostic kit for hepatobiliary
imaging comprising ingredients employed in the
intravenous injection of a complex of technetium 99m,
and an imino diacetic acid of the above formula. It
also relates to a process for preparing said
compounds by reaction of nitrilotriacetic acid
anhydride and an amine of the formula R1NH2 wherein
R1 is as defined hereinabove. The compounds of the
present invention are useful as radioscintigraphic
agents for diagnosis of hepatobiliary disease.

Claims

The embodiments of the invention in which an
exclusive property or privilege is claimed are defined
as follows:
1. A mixture of a stannous salt and a deriva-
tive of imino diacetic acid of the formula:
<IMG>
wherein R1 is selected from the group consisting of:
<IMG>
wherein X is halo and R2 is loweralkyl of from 1-5
carbon atoms.
2. A mixture according to Claim 1, wherein
the stannous salt is stannous chloride.
3. A mixture according to Claim 2, wherein
the imino diacetic acid derivative is N-[N'-(2-benzoyl-
13

4-chlorophenyl)carbamoylmethyl]imino diacetic acid
(PHENIDA).
4. A mixture according to Claim 1 which
includes technetium 99m.
5. A mixture according to Claim 2 which
includes technetium 99m.
6. A mixture according to Claim 3 which
includes technetium 99m.
7. A composition for the preparation of an
injectable solution incorporating technetium 99m which
comprises a lyophilized mixture of a compound of the
formula:
<IMG>
wherein R1 is selected from the group consisting of:
<IMG>
14

wherein X is halo and R2 is loweralkyl of from 1-5
carbon atoms, and a water-soluble stannous salt.
8. A composition according to Claim 7,
wherein the water-soluble stannous salt is stannous
chloride.
9. A composition according to Claim 8,
wherein the imino diacetic acid is N-[N'-(2-benzoyl-4-
chlorophenyl)carbamoylmethyl]imino diacetic acid
(PHENIDA).
10. A composition according to Claim 9,
wherein the weight ratio of the components is from
10-100 parts by weight of N-[N'-(2-benzoyl-4-chloro-
phenyl)carbamoylmethyl]imino diacetic acid and 1 part
of tin as stannous chloride dihydrate.
11. A molecular complex comprising a mixture
of technetium 99m, a compound of the general formula:
<IMG>
wherein R1 is selected from the group consisting of:

<IMG> or <IMG>
wherein X is halo and R2 is loweralkyl of from 1-5
carbon atoms, and a water-soluble stannous salt.
12. A molecular complex according to Claim 11,
wherein the water-soluble stannous salt is stannous
chloride.
13. A molecular complex according to Claim 12,
wherein the imino diacetic acid is N-[N'-(2-benzoyl-4-
chlorophenyl)carbamoylmethyl]imino diacetic acid.
14. A diagnostic kit for the preparation of
an injectable solution according to Claim 7 which is
useful for imaging the hepatobiliary system.
15. A diagnostic kit for the preparation of
an injectable solution according to Claim 8 which is
useful for imaging the hepatobiliary system.
16

16. A diagnostic kit for the preparation of
an injectable solution according to Claim 10 which is
useful for imaging the hepatobiliary system.
17

Description

'Z~
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This is a divisional of application serial
number 434,827 filed on August 17, 1983, Richard E.
Rolleston, inventor.
BACKGROUND OF THE TNv~NTIO~
In radiopharmacy, technetium ~9m is obtained
as an aqueous solution of sodium pertechnetate, where
Tc has the oxidation state ~VII. Several reducing
agents have been employed to reduce the pertechnetate
t~ a lower oxidation state. The stannous ion in the
form of a water soluble salt is the most commonly
used agent, particularly as the stannous chloride,
In the reduced form, Tc ~orms cations which
are complexed with a number of complexing agents,
such as diethylene triamine pentacetic acid,
, methylenediphosphonic acid, and imino diacetic
acids. The Tc II to Tc V cations are hydrated oxo
ions, and in this oxidation state ions are generally
referred to ~n the radiopharmaceutical literature as
"reduced pertechnetate - Tc99mn.
~i,.~.,~

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The use of substituted iminodiacetic acids
and substituted imiho diacetic acids ~omplexed with a
form of technetium 99m and stannous ion are disclosed
to be useful as agents ~or imaging the hepatobiliary
system by ~berg et al. in U.S. Patent 4,017,596.
Other related compounds are reported bv Subram~nian
J. Nucl. Med. 18, 624 (1977). ~hese compounds are
substituted nitrilotriacetic acid mono amides. Other
substituted iminodiacetic acids are disclosed in U~X.
Patent specification 1,545,437 as useful in
diagnosing hepatobiliary function using com~lexes
with technetium 99m; in U.K. patent specification
1,308,793 as useful chelating agents. Other
iminodiacetic acids useful as chelating agents with
technetium 99m are disclosed as follows:
1. Radiopharmaceuticals II p 587, 59~
2. Journal of Nuclear Medicine 17, 633-8 (1976)
3. ~ " n ll 18~ ~24 (1977)
4. n n n ~ 18, 455 (1977~
5. n n ~l 18~ 997 (1977)
: 6. Ra~iology 12R, 793 ~1978
7. European Journal of
Nuclear ~edicine 4, 445 ~197ql
8. Journal of Pharmaceutical
Sciences ~8, 317 (1979)
g n n ~I n ~9~ 731 (19~0)
It is known from these publications that
chelates of certain of the above notefl substituted
iminodiacetic acids with technetium 99m are excreted
throuqh both the urine and the biliary ducts. One of
the drawbacks of using these prior art compounds in
visualizing the hepatobiliary system is that in the

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case of liver disease the visualization of the hepato-
biliary system is unsatisfactory. This is believed
to be ~rue because of improper functioning of the
biliary ducts in which case excretion of the compounds
is accomodated by way of the urine.
Substituted iminodiacetic acids, as defined
hereinabove, are capable of forming molecular
complexes with te~hnetium 99m in the reduced form~
These radiolabelled biological agents have a high
degree of in vivo stability and are hiqhlY selective
for the hepatobiliary system.
It is an object of the invention to provide
such complexes useful in diaqnosing hepatobiliary
function in the presence of liver disease. It is a
further object of the invention to provide a novel
diagnostic kit for hepatobiliary imaging comprising a
freeze-dried mixture of a soluble stannous salt and a
novel iminodiacetic acid of the formula given above.
A still further object of the present invention is to
provide a process for the preparation of said com-
pounds by reaction of nitrilotriacetic acid anhydride
and an amine of the formula RlNH2 wherein ~1 is
as defined above.
SI~MA~Y OF THE TNVENTION
The above objects are achieved ~y providinq
a radiolabelled diagnostic agent which combines the
high target organ specificity of the substituted imino-
diacetic acids mentioned hereinabove with the combined
ability, even in the presence of liver disease, to accum-
ulate in the hepatobiliary system ~ven in systems in
which the common bile duct is partially blocked, as for
example bv ligation of the duct in laboratory animals.

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~ he i~ventlon i6 base~ on thc discovery that
the ~ub~tituted im~nodia~etlc aci~s ~efined
hereinabDve ~nd te~hnetlum 99m in the redu~ed ~tate
form co~p~exe6 whi~h are highly ~pec~fic to ~he
hepatob~ ry ~tem and have excellent ~magin~
propert~e6 even in ~he Prese~ce of li~er di~e~e.
In accordance with the subject invention there
are provided the substituted iminodiacetic acids which
are useful for the purposes of the present inventionr
corresponding to the formula:
3 ~2~00~
Rl-N~ CR21a
CH~OOO~
whereln R i~ ~elected ~rom t~e ~rouD ~onsi~ting of:
XJ~ ~,IL R
~ )
R ~ , or X - ~L

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wherein X is halo (chloro bromo or iodo) and R2 is
lower alkyl of from-1-5 carbon atoms preferably
methyl. Another aspect of ~he present invention is
the provision of a process for the preparation of
said substituted iminodiacetic acids. In accordance
with the process of preparinq said substituted
iminodiacetic acids, nitrilotriacetic acid anhydride
is contacted in solution with a substituted amine in
accordance with the following structural equation:
CH COOH
1l ~CH2~OOH
~o~ NH~ Rl-NH-C-C~-N
15 O ~ O~ ~ CH~COOH
wherein Rl is defined as hereinabove~ The process
is carried out by contacting the two reactants,
prefera~ly in a solvent for the reactants. The
solvent ~elected should be relatively inert under the
reaction ~onditions and aromatic hydrocarbons
especially toluene are preferred. ~he reaction
mixture is heated to 40-100 for a period of 3D
minutes to about 6 hours, preferably to 90-lQ0 for
approximately 1 hour. Following the reaction, the
solvent is removed by evaporation and the residue
containing the desired product is ~lurried with
dilute aqueous alkali, filtered and the filtrate
containin~ the product is washed with a solvent such
as chloroform and ether and purified ~y treatment
with charcoal and precipitated hy treatment with
acid. ~urther purification of the product is
accomplished hy recrystallization from a solvent for

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the product. It al60 relates to a novel Aiagnostic
kit for heptaobiliary imaging comprising ingredients
employed in the intravenous injection of a complex of
technetium g9m, stannous ion, and an iminodiacetic
acid of the above formula. It also relates to a
process for preparing said compoun~s by reaction of
nitrilotriacetic acid anhydride and an amine of the
formula RlNH2.
It also relates to a meth~d of imaginq the
hepatobiliary system of a patient (human or domestic
animal) which comprises the intravenous
administration of a molecular complex of technetium
99m, stannous ion and a substituted imin~diacetic
acid as defined hereinabove and visualizing the
hepatobiliary system hy use of an imagin~ device such
as a qamma camera to record the areas in which the
radioactive m~lecular complex accumulates for
dia~nostic purposes.
The complexes of applicant's com~ounds with
technetium 99m and stannous chloride are readily
prepared from freeze-dried mixtures of the specified
iminodiacetic acid and stannous chloride by reaction
with a s~lution of technetium 99m in the form of the
pertechnetate.
In accordance with the present i~vention,
there is provided a diagnostic kit suitahle for use
in scintigraphic studies of the hepatobiliary
system. ~he kit ~rdinarily contains sufficient
f material for more than one dose. It comprises a
freeze-dried mixture of the components suitable for
reconstitution with a solution of sodium
pertechnetate. The present kit employs individual
vials each containing a reducing agent and the
substituted iminodiacetic acid for use in the

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preparatlon of ~n-ln~e~ta~le hepatob~llary lmaging
asent. One such klt compr~e~ ~ freeze-drlea mixture
of a ~ater ~oluble ~tanncus ~alt and the preferred
compound N-[Nt-(2-benzoyl-4-chlorophenyl~
carba~oylmethy~miho~di~oetic acid (PHENIDA).
~ n the proce~s of preparin~ the in~tant
diagno~t~c kit it ~ essential that a single vial ~e
prepared Dbservin~ a eptic techni~ues and using
norm~l ~aline ~olution ~s ~ dl~uent 80 that the
ingredients when recon~tituted wlth technet~um 99m in
the reduced form are compatible w~th body fluid and
may be lntr~venously lnjected wlthout further
tre~t~ent after mixing. Anot~er important ~eature of
the pre~ent inventlon i6 the ratio of the amounts of
the sub~tituted im~nodia~etic ~cid and the stannous
salt employed as the reducin~ ~gent. V~ually the
ratio of ~ubstituted iminodiacetic acid to reducing
agent i6 from 10-100 pArts by weight of ~ubstituted
imino~ia~etlc ~cl~ to 1 p~rt by weight of reducing
agent. ~t i~ ~m~ortant to the pre~ent lnvent~on that
the we~g~t rat~o of PHENIDA to ~tannous ~alt i~ a~out
60~1. In preparing the component~ vf the present ~lt
the f~r~t ~mDo~ent is prepared by diss~lving 60
part~ by weight of PHENIDA sr the equivalent amount
of ~ noluble ~alt and one part by weight o stannous
chlorl~e d~hydrate ln water made ~lightly acid with
hydroehloric acld and ad~u~ting to pH 4-8. The
~olutlon is ~luted with water to a concentration of
approximately 30 mg/ml of PHENIDA by weight ~nd
subdi~id1ng the bulk 801uti~n ~nto lndlv~dua~ dosage
amount~ and aseptl~lly freeze-dried to prov~de a
read1ly soluble mixture of 60 mg PHENIDA and 1 mg
stannous chloride a5 the ~ihvdrate at pn 4-8.

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The kit comprifiing the freeze-dried mixture
of PHENIDA and stanhous chloride i6 readily employed
as a diagnostic tool for hepatobiliary imaging in the
following manner. To the freeze-dried mixture of
PHENIDA and stannous chloride is added a solution of
2-8 ml of a solution containing approximately 10-200
millicures of sodium pertechnetate Tc99m. The
resulting in~ectable solu~ion of PHENIDA-stannous
complex labelled with Tc99m can be used immediately
without further treatment.
In utilizing the instant kit for
hepatobiliary imaging, an aqueous solution of ~rom
2-8 ml of the required amount of sodium pertechnetate
Tc99m (available as instant technetium 99m or from a
ste~ile generat~r of the type described in U~S.
Patent 3,369,121) is mixed ~ith a ~reeze-dried
mixture of PHENIDA and stannous chloride to form a
solution of reduced pertechnetate ion bound to the
PHENIDA compound which solution is ready for
injection after standing ~or 1~ minutes to e~sure
maximum binding. Intravenous iniection of
approximately 10 millicures of the ~c99m
PHENIDA-stannous complex is followed by serial
imaging of the hepatobiliary system and gut starting
at 30 minutes. The Present improved kit is highly
satisfactory because of its simplicit~y and because of
the advantage that it may be readily employed to
image the ~epatobiliary system in patients suffering
from liver disease, as for instance, liver disease
indicated by elevated serum bilirubin levels~

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EXAMPLE 1
Preparation of PHEN~DA
Step 1: Preparati~n of 2,6-diketo-N carboxymethyl
morpholine (NTA anhydr _ e) __
A round bottom flask is charged with
dimethylformamide 72 q, acetic anhydride 25 ~,
pyridine 2 y, and nitrilotriacetic acid 38.2 g and
the suspension is nitrogen purged ~or several
mînutes. The flask is stopPered and the mixture is
stirred at room temperature for 3 davs. A small
amount of unreacted NTA is filtere~ out. ~he bulk of
the solvent 79 ml is removed in vacuo at a bath
temperature of 60-70~C. The resultinq viscous
solution is twice roto-vacued after two successive
additions of 40 ml dimethylformamide.
Step 2 Preparation of N-~N'-(2-benzoyl~4-chloro-
phenyl)carbamoylmethyl]iminodiacetic acid
PHENIDA
To the viscous solution of NTA anhydride is
added 300 ml toluene~ The mixture is stirred at room
temperature until uniform. Then 46.3 qm of
2-amino 5-chlorobenzophen~ne dissolved in 3~ ml
toluene is added to the stirred solution of NTA
anhydride and heated at 90-100C for 1 h~ur. After
cooling~ the reaction mixture is ~lashed to dryness.
The residue is taken up in 400 ml of lN sodium
hydroxide and filtered. The filtrate is extracted
with chloroform, ether and charcoaled. ~he charcoal
is removed by filtration. The product is
precipitated from the filtrate by the careful
addition of 6N HCl~ The precipitate is removed by
filtration and recrystallize~ from hot ~ethanol/water.

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M.P. 180-186C (decomposition).
N~R in D~S0-d~ with ~MS
= 3.23 singlet N-CH2C-N
= 3.33 singlet -N- ~ 2 ~ C
0 S c 7092 doublet
aromatic protons
S = 7.52 multiplet
H
~ = 10.3 variable broad singlet -N-
The procedure of Step 2 is repeated using an
equi~alent amount of the indicated amine reactant in
place of the 2-amin~-5-chlorobenzophenone with
production of the products shown in the following
table:
Amine Reac~nt Product ~.P.C
2-amino-4-chloro- N-lN'-~4-chloro- 210-13 dec
25 benzothlazole benzothia~ol-2-yl)
carbamoylmethyl]imino
diacetic acid ~A)
2-amino-6-ethoxy N-¦N'-(6-ethoxybenzo- 223-26 dec
30 benzothiazole thiazol-2-yl)carbamoyl-
methyl]iminodiacetic
acid (B)

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Amine eactant Product M.P.C
2-(4-amino- N- IN'-(4- (h-methyl- 245-50 dec.
phenyl)-6- benzothiazol 2-yl)phenyl)
methyl-ben~o- carbamoylmethyllimino
thiazole diacetic ~cid (C~
2-amino-2',5 N-[N'- ~2-(2-chlorobenzoyl~- 185-88 dec.
dichorobenzo- 4-chloropheny~ carbamoyl-
10 phenone methylliminodiacetic acid (D~
EXAMPLE 2
Preparation of kit containing a freeze-dried mixture
of 60 mg of PHENIDA and 1 mg stannous chloride
dihydrate ~er viaI
A solution is prepared by dissolvin~ 6000 mg
of PHENIDA in 100 ml 0.2N sodium hydroxide and a~ding
100 mg stannous chloride dihydrate dissolved in 10 ml
acidulated water. The pH of the solution is adjusted
to 5.5 using dilute hydrochloric acid and/or dilute
aqueous sodium hydroxide solution. Finally the
solution volume i5 adjusted to ~OD ml usinq ~terile
distilled water and aseptically filtered using a
~terilizing membrane.
The ~olution is sub-divided into two ml
portions and filled into 10 ml vials. The
sub-divided ~olutions are then aseptically ~reeze-
dried to provide a readily soluble freeze-dried
mixture of 60 mg PHENIDA and 1 mg stannvus chloride
dihydrate in each vial and stored in a nitrogen
atmosphere. The procedure of this Example is
repeated for each of the products A, ~, C and D to
prepare freeze-dried mixtures of stannous chloride
and each of product A, B, C and D.

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EXAMPLE 3
Use of ~it in Prepa~ing Diagno~tic Liver and Bile
Duct Ima ing Solution
Approximately 2-8 ml of the sterile saline
solution of from 20-200 millicures of sodium
pertechnate Tc9~m is aseptically added to the
contents of one of the vials described in the
previous example. ~he volume is adjusted to 10 ml
with sterile saline ~olution if desired. The
! 10 resulting mixture is then shaken to provide the final
dosage for Tc99m-PHENIDA-stannous complex suitable as
an agent for imaging human or animal hepatobiliary
systems. This final form usually contains more than
enough for one intravenous dose, ordinarily 3-5 doses
containing approximately 10 millicures per dose.
The above procedure is repeated using a
freeze-dried mixture of stannous chloride and product
A, B, C or ~ prepared in accordance with ~xample 2.