CA1222692A - Tc99m-phenida, radioscintigraphic agent for diagnosis of hepatobiliary disease - Google Patents

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:

wherein R1 is selected from the group consisting of:

or

Description

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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.

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- 2 - 16757 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 :12~2~

_ 3 _ 16757 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.

2 ~

~ 4 ~ 16757 ~ 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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~ 5 ~ 16757 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 2~

- 6 - 16757 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 ~2~6Yi;~

~ 16757 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.

6~

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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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) ~ lfi757 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~

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.

2~

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.

Claims (16)

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:

wherein R1 is selected from the group consisting of:

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-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:

wherein R1 is selected from the group consisting of:
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:

wherein R1 is selected from the group consisting of:

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