TECHNETIUM-99m COMPLEX FOR EXAMINING THE RENAL FUNCTION

Abstract: The present invention relates to a technetium-99m complex of general formula (I), wherein Z is a sulphur atom or an amino group of the general formula R17-N-(R18)<k>, in which k is 0 or 1 and R17 and R18 have the same meanings as the symbols R1-R16; each of the symbols R1-R16 is individually selected from the group consisting of hydrogen, straight or branched, unsubstituted or substituted alkyl having 1-4 carbon atoms, and ACOOH, wherein A is a straight or branched, unsubstituted or substituted alkyl group having 0-4 carbon atoms; and R5 together with R6 or R9 together with R10 additionally may form an oxygen atom; Tc represents technetium-99m; t is 0 or 1; and n is 0 or 1; with the provisos that (a) if R15, R16, R17 and/or R18 are/is ACOOH, A is a straight or branched, unsubstituted or substituted alkyl group having 1-4 carbon atoms; (b) at least one of the symbols R1-R18 is ACOOH; and (c) if t is 1, at least two of the symbols R1-R18 is ACOOH; or a pharmaceutically acceptable salt of this compound. The present invention further relates to a radiopharmaceutical composition comprising said complex, to the use of this composition for examining the renal function, and to a kit for preparing such a composition.

Description: TECHNETIUM-99m COMPLEX FOR EXAMINING THE RENAL FUNCTION The present invention relates to a technetium-99m complex and to a method of preparing the complex. The present invention further relates to a radiopharmaceutical composition comprising the complex, to the use of the composition for examining the renal function, and to a kit for preparing such a composition. Radioactive labelled compounds are used for the examination of patients, for example, into deviations in shape and function of internal organs and into the presence and location of pathological processes in the body. For this purpose, a composition in which the radioactive compound is present is administered to the patient, for example, in the form of an injectable liquid. By means of suitable detection apparatus, e.g. a gamma camera, images can be obtained of, for example, the organ or the pathological process in which the radioactive compound has been incorporated, by recording the emitted radiation. Compounds which are generally used for examining the renal function are radioactive iodo-Hippuran^ and Tc99m- diethylene tria ine pentaacetic acid (DTPA) , which will be discussed hereinafter. In addition to glomerular filtration, an active tubular secretion- also takes place in the kidneys. The functioning of the kidneys is determined to a considerable extent by the functioning of the kidney tubes. In an adult person approximately 125 ml of blood plasma per minute is purified by glomerular filtration. This means: the clearance is 125 ml per minute. The total clearance which can be effected by the kidneys is 600 to 700 ml of plasma per minute. It appears from the clearance of 100 ml of blood plasma per minute which is found for the above- mentioned .chelate of DTPA that the chelate is eliminated entirely or substantially entirely by glomerular filtration and hence is not very suitable for examining the renal function. An example of a radioactive iodo-Hippuran s compound generally used for examining the renal function is iodo- 131-Hippur n ® which, as is generally known, is secreted actively tubularly and hence is very suitable for examining the renal function as regards organ specificity. There exists a great need for a suitable composition for examining the renal function which is permanently available, in particular for kidney transplantation patients, accident victims and patients after large vascular operations. The above-mentioned iodo-131-Hippuran would be excellently suitable for these applications, also due to its ready availability. Like all iodo-131 compounds, iodo- 131-Hippuran e , however, constitutes a serious radiation burden for the patient. Therefore, said iodo-131 compound can be administered to the patient only in restricted doses, as a result of which the resulting information is insufficient to obtain statistically reliable images of the renal function by means of a gamma camera. Another radioactive iodo-Hippur n ® compound frequently used for examining the renal function is iodo-123-Hippuran s' which iε excellently suitable as regards the organ specificity and the restricted radiation burden. Iodo- 123-containing compositions, however, have only -a restricted availability due to the short half-life, namely 13.3 hours, and because the production of iodo-123 must necessarily be carried out in a cyclotron. Technetium-99m complexes which show a tubular secretion which is comparable to that if iodo-Hippuran ® are known from European Patent Application 173424. This application discloses inter alia the preparation of Tc- 99m-mercaptoacetyltriglycine (Tc99m-MAG3) , which complex iε secreted by the kidneys selectively and approximately equally rapidly as iodo-Hippuran s . However, the organ specificity of said complexes still leaves something to be desired. In practice this is considered to be a disadvantage, the more so because these compounds are used for function examination. chemically related compounds having an improved organ specificity are the subject of the recently published European patent application 250013. In connection with the comparatively short half-life of radionuclides it is often hardly possible or impossible to deliver the ready-to-use labelled product to the user. In such cases it is desirable to place the various reaction components at the user's disposal in a so-called kit. By meanε of this kit, the user himself can carry out the labelling reaction with the radionuclide in the clinical hospital or laboratory at any desired moment. This is favorable in particular for preparing technetium-99m- labelled products, because most modern clinical hospitals or laboratories have at their disposal a molybdenu - technetium generator, from which the desired quantity of technetium-99m can very easily be obtained in the form of a pertechnetate solution. It will be obvious that the user must be capable of preparing the technetiuπι-99m-labelleά product from the supplied kit with a few simple manipulations, without laborious operations, and by using the facilities which are at his disposition in the clinic. Furthermore, the stability of the labelled product is of great importance. In fact, if the stability is not satisfactory, there is insufficient opportunity to be able to prepare and perform the renal function examination in patients carefully. Moreover, there iε a constant risk that if the shelf life is exceeded, a contaminated composition may be administered to the patient and the results of the examination will no longer be reliable. It has now been ' found that the self life of technetium-99m complexes described in the European patent applications mentioned hereinbefore is at moεt a few hours, depending on the complex-forming ligands and the labelling method used. In practice this is often insufficient because it is desired to have a suitable composition available immediately at any instant of the day. Moreover, it is advantageous that a radioactive composition need be prepared only once daily. Furthermore the reaction conditions in which the user has to prepare the labelled product from the kit are not very favorable. In fact, in order to rpeare the technetium-99m complexes described in the European patent applications, the kit constituents must be heated for at least approximately 5 minutes with the eluate from a molybdenum-technetium generator on a boiling water bath to produce the desired reaction resulting in the formation of the technetium-99m complex. In carrying out this operation, the possibility of accidents in which radioactive material is released are very possible. It is the object of the present invention to provide a technetium-99m complex suitable for examining the renal function which complex has a high organ specificity and an improved stability, and which is better suitable for the preparation from a kit than the above known complexes. This object can be achieved by meanε of a technetium- 99m complex according to the invention which satiεfieε the general formula wherein Z is a sulphur atom or an amino group of the general formula R 17 -N-( lβ )k, in which k is 0 or 1 and R 17 and R ιe have the same meanings aε the symbols i-R 16 ; each of the symbols R α -R 16 is individually selected from the group consisting of hydrogen, straight or branched, unsubstituted or substituted alkyl having 1-4 carbon atoms, and ACOOH, wherein A is a straight or ■ branched, unsubstituted or substituted alkyl group having 0-4 carbon atoms; and R δ together with R ^ or _j together with R 1L additionally may form an oxygen atom; - Tc represents technetium-99m; - t is 0 or 1; and - n os 0 or 1; with the provisos that (a) if R 1B , R 16 , R 17 and/or R 1B are/is ACOOH, A is a straight or branched, unsubstituted or substituted alkyl group having 1-4 carbon atoms; (b) at least one of the symbols Ri-R 1B is ACOOH; and (c) if t is 1, at least two of the symbols Rj- ιε is ACOOH; or a pharmaceutically acceptable salt of this compound. When the above symbols k and/or t are/is 1, there iε a coordinative bond between the amino-N and Tc. The cordinative bonds in the above formula I are also denoted by solid structures where N-R 15 has been exchanged with N- (R 1£ ) t or S has been exchanged with Z. If the above symbols represent or include substituted alkyl groups, such substituents are preferably selected from hydroxy groups and acid groups; wherein examples of suitable acid groups are carboxy groups. Pharmaceutically acceptable salts may be salts with various acids, for example, hydrochloric acid, sulfuric acid, phosphoric acid, perchloric acid or organic acids such as citric acid, tartaric acid, and the like. The new technetium-99m complexes will usually occur in stereoisomeric configurations which, as will become apparent from the examples, may differ in the biological properties. By starting from the stereochemically most suitable complex-fo ' rming ligands, stereoisomeric technetium complexes can be prepared having properties which are most favorable for the intended purpose, as will be described in more detail hereinafter. Chemically related technetium-99m complexes are described in the recently published European patent application 279417. It has been found that these compounds which are destined for brain scintigraphy, are not suitable for examining the renal function. In view of easy accessibility and biological properties a technetium-99m complex is to be preferred with satisfies the general formula wherein each of the symbols R- ' , R 3 ' , R 5 ' , R , R ^ ', R 13 ' , R 1S ' and R 16 ' is individually selected from the ' group consisting of hydrogen, methyl and (CH 2 ) g COOH, wherein q is 0 or 1, Tc represents technetium-99m, and t is 0- or 1, with the provisos that (a) if R 15 ' and/or R i6 ' are/is (CH 2 ) q COOH, q is 1, (b) at least one of the symbols R , R 3 ' , R s ', R , R X1 ' , *-__ ' , R»' and R 16 ' is (CH 2 )qC00H, (c) at most four of the symbols R_ ' , R 3 ', R 5 ', R , R αι ', R__ ' 1 R 13 ' and R 16 " are (CH 2 ) q COOH, and (d) if t is 1, at least two of the symbols R , R 3 ', R 5 ', R,', R, , R 13 ', R 15 ' and R ιe ' are (CH 2 ) q COOH, or a pharmaceutically acceptable salt of this compound. Examples of technetium-99m complexes according to the invention are the technetium99m complexes of N,N'-biε(l- carboxy-2-mercaptoethyl)ethylene diamine and N,N'-bis(2- mercaptoethyl) diamino succinic acid, which compounds may occur in the LL-, LD- or DD-configurations, and of N,N'- [biε(2-mercaptoethyl) ]-N,N'-ethylenediamino-diacetic acid. A technetium-99m complex according the invention is generally used in the form of a compoεition which iε suitable for examining the renal function. In- addition to the radioactive complex, such a radiopharmaceutical composition will usually comprise a liquid, pharmaceutically acceptable carrier material, preferably a physiological saline solution. A radiodiagnostic examination can be performed with such a composition by administering the composition to a warm-blooded living being, in particular a primate, in a quantity of 0.1 to 30 mCi, preferably of a 0.5 to 10 mCi, per 70 kg of body weight, and by then recording the radioactive radiation emitted by the living being by means of, for example, a gamma camera. The invention further relates to a method of preparing a technetium-99m complex according to the invention by reacting technetium-99m in the form of a pertechnetate in the preserice of a reducing agent and optionally a εuitable chelator with a diaminothio compound of the general formula wherein - the symbols n and R J -R K , have the meanings given hereinbefore, - Y is hydrogen atom or a suitable protecting group, - Z'is a sulphur atom or an amino group of the general formula R 17 -N-R lβ , wherein R l7 and R 18 alεo have the above meanings, and - m is 0 or 1, with the provisos that, if Z' is a sulphur atom, m = 1 and if __ ' is a amino group, = 0. Examples of suitable protective groups Y for the mercapto group are: acetyl, trifluoroacetyl, hydroxyacetyl, carboxyacetyl, acetamidomethyl, benzoyl, benzyl, benεoyl- aminomethyl and the like. The reducing agent serves to reduce the Tc-99m pertechnetate which in a physiological saline solution is eluted from a molybdenu -technetium generator. Suitable reducing agents are, for example, dithionite, formamidine sulphinic-. acid, diaminoethane disulphinate or suitable metallic reducing agents such as Sn(II), Fe(II), Cu(I , Ti(III) or Sb(III); wherein Sn(II) has proved to be particularly suitable. For the above-mentioned complex-forming reaction, technetium-99m is presented to the above-mentioned diaminothio compound as a salt or in the form of a chelate bound to comparatively weak chelators; in the latter case the desired technetium-99m complex is formed by ligand exchange. Examples of suitable chelators for the radionuclide are dicarboxylic acids, polycarboxylic acids or hydroxy carboxylic acids, such as oxalic acid, malonic acid, succinic acid, maleic acid, orthophthalic acid, malic acid, lactic acid, tartaric acid, citric acid, ascorbic acid, salicylic acid or derivatives of these acids; phosphorus compounds such as pyrophosphates; or enolates. Citric acid, tartaric acid, ascorbic acid, glucoheptonic acid or a derivative thereof are particularly suitable chelators for thiε purpose, because it appears that a chelate of technetium-99m with of one these chelators particularly easily undergoes the desired ligand exchange. It has been found that the above-mentioned complex- forming reaction occurs quantitatively at room temperature i.e. with a radiochemical yield exceeding 98%. So heating of the reaction mixture iε not neceεεary to reach a full conversion to the deεired technetium-99m complex. Since the radiopharmaceutical compoεition according to the invention can be prepared so easily and simply, the preparation can be carried out particularly readily by the user himself. The invention therefore also relates to a so-called kit, comprising (1) in an optionally dry condition a diaminothio compound of the above general formula III, wherein the symbols have the meanings given hereinbefore and to which optionally an inert, pharmaceutically acceptable carrier and/or auxiliary substances have/has been added, (2) a reducing agent and optionally a chelator, ingredients (1) and (2) being optionally combined, and (3) if desired, instructions for use with a prescription for carrying out the above- described method by reacting ingredients (1) and (2) with technetium-99m in the form of a pertechnetate solution. Examples of suitable reducing agents and chelators for the above kit have been given hereinbefore. The pertechnetate solution can simply be obtained by the user himself from a molybdenum-technetium generator which is available to him. The above-mentioned ingredients (1) and (2) may be combined, provided they are compatible. Such a monocomponent kit, in which the combined ingredients are preferably lyophilized, is excellently suitable to be reacted by the user with the pertechnetate solution in a simple manner. The constituent (1) of the above kits may be delivered aε a solution, for example, in the form of a physiological saline solution, or in some buffer solution, but is preferably present in a dry condition, for example in a lyophilized condition. When used as a component for an injection liquid, it should be sterile, in which, if. the constituent is present in a dry condition, the user should use a εterile physiological saline solution as a solvent. If desired, the above-mentioned constituent may be stabilized in a usual manner with εuitable εtabilizerε εuch aε aεcorbic acid, gentisic acid or saltε of theεe acidε, or it may be provided with other auxiliary meanε such aε fillerε, e.g. glucose, lactose, mannitol, inositol, and the life. The kit according to the invention preferably comprises a diaminodithio compound of the general formula wherein the symbols have the meanings given hereinbefore. These complex-forming ligands are readily acceεsible and can very easily be converted into the desired technetium- 99m complexes. The stereoche ical configuration of the technetium- 99m complex iε determined by the configuration of the starting diaminothio compound of the above general formula III or IV. Different εtereoiεomerε of theεe diaminothio compounds can be separated from each other by using techniques known for this purpose such as recrystallization and/or chromatographic methods. If desired, for the separation the stereoisomer mixture may be converted with a stereochemically pure D- or L-isomer of a suitable amine, carboxylic acid, and the like, after which the isomer separation is carried out, succeeded by eliminating the used amine, carboxylic acid, etc. An alternative, also particularly suitable method of preparing stereochemically pure diaminothio compounds, consists in using for the synthesis a starting material which is already stereochemically pure and which is easily available or obtainable as a stereoisomer, and in ensuring that during the synthesis of the intended diaminothiol the stereochemical purity is not lost, i.e. that no racmisation occurs. The invention will now be described in greater detail with reference to the ensuing specific examples. EXAMPLE I Preparation of N.N'-bisfl-carboxy-2-mercaptoethyltethylene diamine The L,L'-isomer of the title compound iε prepared by reductive dimerisation of L-thiazolidine-4-carboxylic acid under the influence of sodium in liquid ammonia, as described by Blondeau et al in Can. J. Chem. 45 . (1), 49- 52 (1967). The corresponding D,D'-isomer and the meso form (L,L'-isomer) are prepared in a corresponding manner from D-thiazolidine-4-carboxylic acid and racemic thiazolidine- 4-carboxylic acid, reεpectively. These thiazolidine carboxylic acids are obtained by reaction of L-,D- or DL- cyεtein with formaldehyde according to Nagaεawa et al, J. Med. Chem. 22, 591 (1984). EXAMPLE II ' - Preparation of N.N'-biε-f2-benzoγlmercaptoethyl ethylene diamine-N r N f -diacetic acid N,N -1.2 ethylenediyl bis-S-benzylcyεteamine To a solution of S-benzylcyεteamine (0.482 mol , 93 g) and 1,2-dibromoethane (0.241 mol, 45.27 g, 20.7 ml) in 500 ml CH 2 C1 2 waε added 1.2 mol triethylamine (121.9 g) . The mixture waε ' refluxed for 2h hours and then stored in a refrigerator for 48 hours. The clear solution. was washed 3 times with 200 ml water and evaporated under reduced pressure. A yellow oil was obtained. The oil was dissolved in 400 ml methanol and saturated with hydrogen chloride gas for 30 minutes. The resulting slurry was cooled for 1 hour at 0°C, then filtered and washed with 200 ml methanol, 200 ml acetone and 200 ml diethylether. It was recrystalized from 250 ml methanol to yield 56 g of white crystals (64.5%). CH; • CH, NEt -CH 2 -S-CH 2 -CH 2 -NH 2 B 5 B, -CH 2 -S-CH 2 -CH 2 -NH-CH 2 -CH 2 -S-CH 2 -CH 2 -NH-CH 2 B) N.N'-bis (2-benzylmercaptoethyll ehtylene dia ine- N,n'-diacetic acid N,N'-1.2 ethylenediyl bis-S-benzylcysteamine (0.005 M, 1.8 g) was dissolved in 20 ml water and NaOH 4N waε added to pH 12.8. Bromoacetic acid (1.39 g, 0.01 M) was added in 20 ml ethanol and the mixture was stirred for 16 hours at room temperature. The pH was kept at pH 12 with NaOH 4N. The ethanol was then evaporated and the water layer extracted twice with 40 ml diethylether. The water layer was acidified with HCL 2N. After standing in the refrigerator for' 16 hours, the crystals were filtered off and washed with ethanol and diethylether to give 2.3 g white crystals (40%). 2 Br-Ch 2 -COOH Q)-CH 2 -S-CH 2 -CH 2 -NH-CH 2 (NaOH) j > -CH 2 -S-CH 2 -CH 2 -NH-CH 2 C) N f N'-bis (2-benzoylmercaptoethyn ethylenediamine- N.N'-diacetic acid To a solution of 5.77 g (10 mmol) of N,N'-bis (2- benzylmercaptoethy1) ethylenediamine-N,N'-diacetic acid in 100 ml of liquid ammonia, sodium was added until the blue color persisted for 10 minutes. After addition of ammoniumchloride the solution was allowed to evaporate. To the residue was added 30 ml of water. The clear solution was cooled to 0-5°C and a solution of bezoylchloride (0.02 M, 2.81 g) in 30 ml of dioxane was added while the pH was maintained at pH 10 with NaOH 4N. The solution was kept at pH 20 for half an hour and then evaporated under reduced pressure. 50 ml water was added and the water layer was washed twice with 20 ml of diethylether. After acidification to pH 2 a slurry was obtained. After standing for 15 hours at 0°C the slurry was filtered off, washed successively with water, acetone and diethylether. After drying 3.9 g of a white powder iε obtained (67.6%). ,CH 2 -COOH -CH,-S-CH,-CH,-N-CH, Na/NH 3 -CH 2 -S-CH 2 -CH 2 -N-CH 2 CH 2 -COOH CH 2 -COO " * S-CH 2 -CH 2 -N-CH 2 2 < ) -COCl -> NMR analysis: "C <5(DMSO): 23.9(CH 2 -S), 39.6(N-CH 2 -CH 2 -N) 49.6(CH 2 -COOH) , 53.0(N-CH 2 -CH 2 -S) 127.2, 129, 134.1(-phenyl) 169.2(-CO-phenyl) , 180.5(-COOH) EXAMPLE III Preparation of N .N / -bis-f2-mercaptoethyl -diaminosuccinic adid Into a three-necked 500 ml flask, equipped with a gas inlet of argon, a magnetic stirrer and a dry-ice condenser, 13 g (0.1 mol) of 2-carboxy-thiazolidine (prepared as described in the literature, Lalezari et al, J. Med. Chem. (1982) pp 1427-1429) was dissolved in 125 ml liquid ammonia. 5 g of sodium spheres were added until a persistent blue color was obtained for 10 minutes. The reaction was quenched with solid ammonium chloride, the condenser removed and the ammonia evaporated under an argon stream. The white solid residue was dissolved in 125 ml of water and concertrated hydrochloric acid was added up to pH 2. The slurry waε cooled to 0 C C for i hour, then filtered and washed with 2 x 100 ml water, 50 ml ethanol and 50 ml diethylether. After drying in a desεiccator under vacuum 5 g of a white powder was obtained. HOOC COOH i I HC- CH NMR analysis: ~ R fS(NaOD)-: 2.3(m,8H,2xCH 2 -CH 2 ) , 2.9(s,2H,2xCH). EXAMPLE IV Preparation of 1.2 ethylene dicysteine 1.2 dicarboxylic acid L-thiazolidine-2,4-dicarboxylic acid was prepared starting from L-cysteine and glyoxylic acid by a decribed procedure (Lalezare et al, J. Med. Chem. (1988) p. 1427). This compound was converted to the desired product by the same method as described for N,N'-bis-(2-mercaptoethyl)- diaminoεuccinic acid. (See EXAMPLE III) . NMR analysiε : ' H «5 ( NaOD ) : 2 . 3 ( m , 4H , ( 2xCH-SH ) , 2.8(m,4H(4xCH-COOH) EXAMPLE V Preparation of 1.2 ethylene-biε-homocγsteine To a solution of DL-tetrahydrol-1,3-thiazine-4- carbόxylic acid hydrochloride in 150 ml of liquid ammonia, (prepared as described in the literature, Lewis er al, J. Med. Chem. ____ , (1978) p. 1070), sodium was added until blue color persisted for 30 minutes. After the addition', of ammonium chloride, the solution was allowed to evaporate. The residue was dissolved in 250 ml of water and acidified to pH 2. A precipitate was separated after cooling the solution to 0°C. The precipitate was then filtered off and washed with water, ethanol and ether, to yield 240 mg; mp 258-259°C (decomp). NMR analysis: *H <5(D 2 0 and NaOD) : 1.8(m, 4,2 CH-CH 2 ) , 2.4(m,8,2CH 2 ,SH,2CH 2 -NH) , 3.1(t,2,2CH . --C 5COM: 22(CH 2 SH), 42(CH 2 -CH), 50(CH 2 -NH), 64 (CH), 177(COOH). EXAMPLE VI Labelling of N ,N -bis(l-carboxγ-2-raercaptoethγl1 ethylene diamine with technetium-99m 20 mg of [L r L / ]-N,N'-bis(l-carboxy-2-mercaptoethyl) ethylene diamine are dissolved in 4 ml of 0.5 N sodium hydroxide solution while stirring and flushing with nitrogen. The pH of the solution is successively reduced to 10 with 0.5 N hydrochloric acid and to 7.5 with 0.1 N hydrochloric acid. After diluting with water to 10 ml, vials are dispensed with 0.5 ml solutions under nitrogen or in vacuo. These vials are stored at -20°C or lyophilized. For the labelling with technetium-99m the content of a vial is allowed to reach ambient temperature, after which 100 μg of SnCl 2 .2H 2 0, dissolved in 25 μl 0.05 N hydrochloric acid, and 1-2 ml of a sodium pertechnetate solution, obtained from a molybdenum-technetium generator and comprising 10-100 mCi Tc-99m, are added successively. The resulting technetium-99m-labelled N,N'-bis(l- carboxy-2-mercaptoethyl)ethylene diamine has a radiochemical purity of > 98%. The stability of the labelled compound is determined by measuring until 8 hours after preparation the radiochemical purity with TLC or HPLC. The Tc-99m complexes according to the invention prove to be completely stable for at least 8 hours at room temperature. Starting from an optically pure iεomer reεultε in a technetium-99m complex of an unambiguouε εtereoisomer. When a mixture of εtereόiεomeric dia inodithiolε iε used as the starting material, the reεulting technetium-99m complex may be resolved in itε stereoisomers (LL-,DD-,LD-iεomer) by means of HPLC. This will be described in example VII. EXAMPLE VII Purification by means of HPLC The product labelled according to Example VI is applied in a quantity of 30 to 150 μl on a column filled with Hypersil ® C8 (3 μm) . Gradient elution with 100% 0.0125 M phosphate buffer (pH 2.5) to 0.0125 M phosphate buffer-ethanol mixture (70:30) yields the desired pure stereoisomers. Detection is performed radiometrically by passing the eluate over a scintillation detector connected to a one-channel analyzer and an integrator. After collection of the main fraction, this may be diluted with physiological saline solution for intravenous administration. EXAMPLE VIII Biodistribution studies in mice Each- time 5 male mice are injected with 0.5 μCi of a Tc-99m-labelled N,N'-biε(l-carboxy-2-mercaptoethyl) ethylene diamine (LL, DD or DL-isomer) according to the invention. For the validation of the examination, iodo- 131-Hippuran ® is used as an internal biological standard. For comparison, 0.5 μCi Tc-99m-mercaptoacetyl triglycin (Tc99m-MAG3) known from European patent application 173424 mentioned hereinbefore, is also tested. After 10 minutes the mice are sacrificed and the radioactivity in the various organs is determined. The accumulated radioactivity in various organs and in urine ("organs") in comparison with that of Tc99m-MAG3 is recorded in the table below. TABLE Uptake in organs of mice after 10 minutes as a percentage of the Tc99m-MAG3 value. Orσan urine kidneys liver Froir. the above results it appearε that the co poundε according to the invention εhow conεiderably less liver activity, in which especially the LL-isomer shows very favorable characteriεticε. Further, from an enhanced activity in the urine and a considerably reduced activity in the -kidneys it appears that the plasma clearance and urinary excretion of the compoundε according to the invention are very faεt in comparison with those of Tc99m- MAG3. EXAMPLE IX Labelling with Tc99m and biodistribution in mice To a solution of 2 mg of N,N'-bis-(2-mercaptoethyl) succinic acid in 1 ml of water ar pH 12 was added 50 μg of SnCl 2 .2H 2 0 in 25 μl of HCl 0.05 N and then 10 mCi of (Tc99m)-NaTcθ < aε generator eluate (2 ml). The mixture iε incubated for 10 minutes and then purified by HPLC on a Hypersil ® ODS 5 μm column (25 cm x 4.6 mm), eluted with a gradient mixture of phosphate buffer 0.05 N, pH 2.5 and ethanol (0% to 30% ethanol in 20 minutes). The main peak is isolated, diluted with saline to a concentration of 10 μCi/ml and the biodistribution is εtudied in 5 mice at 30 minutes after injection. Biodistribution in five mice expressed as a ratio between organ uptake of studied compound and organ uptake of Tc99m-MAG3 at 30 minutes after injection, urine 1.008 kidneys 0.94 liver 1.64 intestines 1.56. EXAMPLE X Plasma clearance in a primate A quantity of 0.5 mCi of Tc-99m N,N'-bis (1-carboxy- 2-mercaptoethyl)ethylene diamine, LL- or DD-isomer, iε administered intravenously to a male baboon, sedated with Ketalar ® and pentobarbital (1-131 Hippuran s as an internal biological standard) . Through an intra-arterial puncture 0.5 l-blood samples are taken at regular intervals during 60 minutes. The radioactivity of the samples is determined and the plasma clearance is then calculated. For the LL- isomer this. value iε 119.6% with respect to the plasma clearance of Tc99m-MAG3 and for the DD-isomer 145.1%. After the injection of 1 mCi the radioactivity is recorded at the region of the kidneys by means of a gamma camera. The maximum kidney accumulation of Tc99m-MAG3, the LL-isomer, the DD-isomer and the LD-isomer of Tc-99m N,N'- biε(l-carboxy-2-mercaptoethyl)ethylene diamine doeε net differ essentially. For Tc99m-MAG3 the maximum activity in the kidneys is reached after 3.5 minutes, for the iεomers according to the invention already after 2.5, 2.5 and 2.0 minuteε, respectively. This also clearly indicates a more rapid plasma clearance of the Tc99m-complexes according to the invention compared with the known product. EXAMPLE XI Biodistribution studies of Tc-99m rLL ~ l-N,N'-bisfl-carbo y- 2—mercaptoethyl")ethylene diamine compared with Tc99m-MAG3 in a human being The above mentioned labelled diaminodithiol is administered intravenouεly in a human being in a quantity of 0.5 mCi (1-131 Hippuran ® aε an internal biological standard) . The radioactivity at the region of the kidneys is recorded by means of gamma camera equipped with a high- εenεitive collimator. The maximum renal activity iε achieved after 2.5 minuteε both for the LL-isomer according to the invention and for Tc99m-MAG3. This activity is slightly higher for the Tc99m-LL-isomer than for T.c99m- MAG3. The renogram obtained with the title compound is approximately identical to the renogram obtained with Tc99m-MAG3 in the same person. The liver accumulation is determined after 40 minuteε. For the tested Tc99m-LL-iεomer it amounts to 2.9%, for Tc99m-MAG3 it amountε to ' 4.0% of the injected dose. From this experiment it appears that for the tested Tc99m complex according to the invention considerably lesε liver accumulation occurs in the human being than for the known Tc99m-MAG3. EXAMPLE XII Renograms of Tc-99m rPDI-N,N'-bis (l-carboxy-2- mercapto&thyl)ethylene diamine compared with Tc99m-MAG3 in human beings In the same manner as indicated in Example XI, the title renograms are obtained in two human volunteerε. The maximum renal activity (mean of 2 kidneyε) for the title compound is reached after 2.5 and 3.24 minuteε for the two volunteers respectively, as compared to 5.0 and 4.0 minutes respectively for Tc99m-MAG3.

Claims: What is claimed is: Technetium-99m complex " of the general formula wherein - Z is a sulphur atom or an amino group of the general formula R 17 -N-(R ιa ) Λ , in which k iε 0 or 1 and R 17 and R ιa have the same meanings aε the εymbolε R_ - - each of the symbols R t - R 16 is individually selected from the group consisting of hydrogen; a . straigtit or branched, unsubstituted or substituted alkyl having 1 - 4 carbon atoms; and ACOOH, wherein A is a straight or branched, unsubstituted or substituted alkyl group having 0 - 4 carbon atoms; - s together with « or R, together with R 1C may form an- oxygen atom; - Tc represents technetium-99m; - t is 0 or 1; and - n is 0 or 1; with the provisos that (a) if at least one of R 15 , R 16 , R 17 and R lβ is ACOOH, then A is a straight or branched, unsubstituted or substituted alkyl group having 1 - 4 carbon atoms; (b) at least one of the symbols R_ - R 18 is ACOOH; and (c) if t is 1, then at least two of the symbols Rj - R 18 are ACOOH; or a pharmaceutically acceptable salt of this compound. 2. A complex according to claim 1, of the general formula wherein - each of the symbols R.' , R 3 ' , R 5 ' , R β ' , R 1: ' , R 13 ' , R 15 ' , and R 16 ' , is individually selected from the group consisting of hydrogen, methyl and (CH 2 ) q COOH wherein q is 0 or 1; - Tc represents technetium-99m; and - t is 0 or 1; with the provisos that (a) if at least one of R 15 ' , and ;6 ' is (CH 2 ) q C00H, then q is 1; (b) at least one of the symbols R_', R , R 5 ' , R , R M ' , R13' Ris' , and R 16 ', is (CH 2 ) q COOH; (c) at most four of the symbols R_ ' , R. ' , R. ' , . ' , Rn'', . ' , Ri 5 ', and R„', are (CH 2 ) q COOH; and (d) if t is 1, then at least two of the symbols R. ' , R 3 ', R s ', R,', R ', R 13 ', R__ ' , and R 16 ', are (CH 2 ) q C00H; or a pharmaceutically acceptable salt of this compound. 3. A radiopharmaceutical composition for examining the renal function which, in addition to a liquid, pharmaceutically acceptable carrier material, comprises a technetium-99m complex, characterized in that the composition comprises a complex according to claim 1 or 2 as the technetium-99 complex. 4. A method of performing a renal function examination, characterized in that a composition according to claim 3 is administered to a warm-blooded living being in a quantity of 0.1 to 30 mCi, per 70 kg of body weight, and the radioactive radiation emitted by the living being is then recorded. 5. A method according to claim 4, wherein the quantity of the composition administered is 0.5 to 10 " mCi per 70 kg of body weight. 6. Technetium-99m complex.of the general formula wherein - Z is a sulphur atom or an amino group of the general formula R ι7 -N-(R 18 ) k , in which k is 0 or 1 and R 17 and R X8 have the same meanings as the symbols R x - R. ' - each of the symbols R x - R 16 is individually selected from the group consisting of hydrogen; a straight or branched, unsubstituted or substituted alkyl having 1 - 4 carbon atoms; and ACOOH, wherein A is a straight or branched, unsubstituted or substituted alkyl group having 0 - 4 carbon atoms; - R s together with , or R, together with R 10 may form an oxygen atom; - Tc represents technetium-99m; - t is 0 or 1; and - n is 0 or 1; with the proviso that at least two of the symbols R_ - R l8 are ACOOH, wherein A is a straight or branched, unsubstituted or substituted alkyl group having 1 - 4 carbon atoms; or a pharmaceutically acceptable salt of this compound. 7. A complex according to claim 6, of the general formula wherein - each of the symbols R , R 3 ' , R 5 ' , R,' , R, , R 13 ', R ιs ' , and R 16 ' , is individually selected from the group consisting of hydrogen, methyl and (CH 2 ) q COOH wherein q is 0 or 1; - Tc represents technetium-9 m, and - t is 0 or 1; with the provisos that (a) at least two of the symbols R ' , R , R 5 ', R , R u ', Ri 3 Λ , Ris' and R 16 ', is (CH 2 ) q COOH, wherein q iε 1; and (b) at most four of the symbols R , R 3 ' , R 5 ', R 9 ' , R u ', χ 3 7 Rχ 5 ', and R 16 ', are (CH 2 ) ς COOH; or a pharmaceutically acceptable salt of this compound. 8. A radiopharmaceutical composition for examining the renal function which, in addition to a liquid, pharmaceutically acceptable carrier material, comprises a technetium-99m complex, characterized in that the composition comprises a complex according to claim 6 or 7 as the technetium-99m complex. 9. A method of performing a renal function examination, characterized in that a composition according to claim 8 is administered to a warm-blooded living being in a quantity of 0.1 to 30 mCi per 70 kg of body weight, and the radioactive radiation emitted by the living being iε then recorded. 10. A method according to claim 9, wherein the quantity of the composition administered is 0.5 to 10 mCi per 70 kg of body weight. 11. Technetium-99m complex of the general formula wherein - Z is a sulphur atom or an amino group of the general formula R 17 -N-(R 18 ) X , in which k iε 0 or 1 and R 17 and R lβ have the εame meaningε as the symbols R. - R__; - each of the symbols R- - R 16 is individually selected from the group consisting of hydrogen; a straight or branched, unsubstituted or substituted alkyl having 1 - 4 carbon atoms, and COOH; - R s together with R, or R, together with R 1C may form an oxygen atom; - Tc represents technetium-99m; - t is 0 or 1; and - n is 0 or 1; with the provisos that (a) at least one of the symbols R t - R lβ is COOH; and (b) if t is 1, then at least two of the symbols R. - R 18 are COOH, or a pharmaceutically acceptable salt of this compound. 12. A complex according to claim 11 of the general formula wherein - each of the symbols R-' , R 3 ' , R 5 ' , R , R. , R I3 ' , R 1£ ', and R 16 ' , is individually selected from the group consisting of hydrogen, methyl and COOH; - Tc represents technetium-99m; and - t is 0 or 1; with the provisos that (a) at least one of the symbols R , R,' , R 5 ', R,' , Rii', R._ ' , Ris', and R 1£ ', is COOH; (b) at most four of the symbols R , R,' , R 5 ' , R 9 ' , •Rn'f -Ri3 , » Ris' / and R lt ', are COOH; and (c) if t is 1, then at least two of the symbols R_' , R 3 ', s ', ^, R^', R i3 ', ^', and R 16 ', are COOH; or a pharmaceutically acceptable salt of this compound. 13. A radiopharmaceutical composition for examining the renal function which, in addition to a liquid, pharmaceutically acceptable carrier material, comprises a technetium-99m complex, characterized in that the composition comprises a complex according to claim 11 or 12 as the technetium-99m complex. 14. A method of performing a renal function examination, characterized in that a composition according to claim 13 is administered to a warm-blooded living being in a quantity of 0.1 to 30 mCi per 70 kg of body weight, and the radioactive radiation emitted by the living being is then recorded. 15, A method according to claim 14, wherein the quantity of the composition administered is 0.5 to 10 mCi per 70 kg of body weight. 16. A radiopharmaceutical composition for examining the renal function, comprising a liquid, pharmaceutically acceptable carrier material and a technetium-99m complex of the general formula wherein - Z is a sulphur atom or an amino group of the general formula ^-N-fR ^ ),,, in which k iε 0 or 1 and R 17 and R 18 have the same meanings as the symbols R x - - each of the symbols R. - R X6 is individually selected from the group consisting of hydrogen; a straight or branched, unsubεtituted or εubεtituted alkyl having 1 - 4 carbon atoms; and ACOOH, wherein A is a straight or branched, unsubstituted or substituted alkyl group having 0 - 4 carbon atoms; - R, together with I or E, together with R 1C may form an oxygen atom; - Tc represents technetium-99m; - t is 0 or 1; and - n is 0 or 1; with the provisos that (a) if at least one of R 15 , R 16 , R 17 , and R 18 is ACOOH, then A is a straight or branched, unsubstituted or substituted alkyl group having 1 - 4 carbon atoms; (b) at least one of the symbolε R. - R 18 iε ACOOH; and (c) if t is 1, then at least two of the symbols R x - R 18 are ACOOH; or a pharmaceutically acceptable salt of this compound. 17. A method of performing a renal function examination, characterized in that a composition according to claim 16 is administered to a warm-blooded living being in a quantity of 0.1 to 30 mCi per 70 kg of body weight, and.the radioactive radiation emitted by the living being is then recorded. 18. A method according to claim 17, wherein the quantity of the composition administered is 0.5 to 10 mCi per 70 kg of body weight. 19. A method of preparing a technetium-99m complex of the general formula wherein - Z is a sulphur atom or an amino group of the general formula R 37 -N-(R 18 ) k , in which k is 0 or 1 and R 17 and R 18 have the same meanings as the symbols R_ - Rι«' - each of the symbols R x - R 16 is individually selected from the group consisting of hydrogen; a straight or branched, unsubstituted or substituted alkyl having 1 - 4 carbon atoms; and ACOOH, wherein A is a straight or branched, unsubεtituted or substituted alkyl group having 0 - 4 carbon atoms; - g together with R, or R, together with R 10 may form an oxygen atom; - Tc represents technetium-99m; - t is 0 or 1; and - n is 0 or 1; with the provisos that (a) if at least one of R 15 , R lβ , R 17 , and R 18 is ACOOH, then A is a straight or branched, unsubstituted or substituted alkyl group having 1 - 4 carbon atoms; (b) at least one of the symbols Rj - R 1S is ACOOH; and (c) if t is 1, then at least two of the symbols R_ - R 1B are ACOOH; or a pharmaceutically acceptable salt of this compound; the method comprising reacting technetium-99m in the form of pertechnetate with a diaminothio compound of the general formula wherein - the symbols n and R x - R 16 have the meanings given above; - Y is a hydrogen atom or a suitable protecting group; - Z' is a sulphur atom or an amino group of the general formula R 17 -N-R 1B , wherein R i7 and R 18 also have the meanings given above; and - is 0 or 1; with the provisos that if Z' is a sulphur atom, thenm is 1; and if Z' is an amino group, then m is 0; and wherein the reaction is carried out in the presence of a reducing agent. 20. A method according to claim 19, wherein the reaction is carried out in the presence of a chelator. 21. A kit for preparing a radiopharmaceutical composition comprising a technetium-99m complex of the general formula wherein - Z is a sulphur atom or an amino group of the general formula in which k is 0 or 1 and R 17 and R 18 have the same meanings as the symbols R. - - each of the symbols R. - R 16 is individually selected from the group consisting of hydrogen; a straight or branched, unsubstituted or substituted alkyl having 1 - 4 carbon atoms; and ACOOH, wherein A is a straight or branched, unsubstituted or subεtituted alkyl group having 0 - 4 carbon atoms; - Rj together with « or R, together with R 1C may form an oxygen atom; - Tc represents technetium-99m; - t is 0 or 1; and - n is 0 or 1; with the provisos that (a) if at least one of R 16 , R 16 , R 17 , and R 18 is ACOOH, then A is a straight or branched, unsubstituted or substituted alkyl group having 1 - 4 carbon atoms; (b) at least one of the symbols R - R lβ is ACOOH; and (c) if t is 1, then at least two of the symbols R 3 - R lβ are ACOOH; or a pharmaceutically acceptable salt of this compound; the kit comprising ( 1 ) a diaminothio compound of the general formula wherein - the symbols n and R x - R 16 have the meanings given above; - Y is a hydrogen atom or a suitable protecting group; - Z r is a sulphur atom or an amino group of the general formula R 17 -N-R 18 , wherein R 17 and R ιa also have the meanings given above; and - is 0 or 1; with the provisos that if Z' is a sulphur atom, then m is 1; and if Z r is an amino group, then m is 0; and (2) a reducing agent. 22. A kit according to claim 21, wherein the diaminothio compound is in a dry condition. 23. A kit according to claim 21, wherein the kit further includes a pharmaceutically acceptable carrier. 24. A kit according to claim 21, wherein the kit further includes a chelator. 25. A kit according to claim 21, wherein the kit further includes instructions for use with a prescription for reacting " ingredients (1) and (2) with technetium-99m in the form of a pertechnetate solution. 26. A kit according to claim 21, wherein the diaminothio compound has the general formula wherein each of the symbols R 3 ', R 3 ' , Rs' , R,' , R αι ', R 13 ' , R :5 ' and R 16 ' is individually selected from the group consisting of hydrogen, methyl and wherein q iε 0 or 1; and Y is a hydrogen atom or a suitable protecting group.

Inventors: NOSCO DENNIS L, VERBRUGGEN ALFONS M, NOSCO, DENNIS, L., VERBRUGGEN, ALFONS, M.
Applicants: MALLINCKRODT INC, MALLINCKRODT, INC.
Application year: 1989
Main classification: A61K49
IPC codes: A61K51, C07F13, G01N33