RP-HPLC separation of the diastereomers of technetium-99m labelled tropanes and identity confirmation using radio-LC-MS.

99mTc-TRODAT-1 (technetium(V)-oxo-2-[[2-[[[3-(4-chlorophenyl)-8-methyl-8-azabicyclo[3.2.1]oct-2-yl]methyl](2-mercaptoethyl)amino]ethyl]]amino]-ethanethiolato(3-)) and 99mTc-TRODAT-M, the 4-methylphenyl derivative of 99mTc-TRODAT-1, are at this moment being evaluated in clinical trials as imaging agents for the central nervous dopamine transporter system. Both compounds are formed as a mixture of two major diastereomers. As the tracer concentration in preparations for clinical investigations is very low (30-150 pmol/ml), identification of these 99mTc-complexes was, up to now, carried out indirectly using X-ray diffraction analysis of the corresponding rhenium complexes which can be synthesized in gram amounts. In this study, we developed a convenient and practical reversed phase HPLC-method for purification and isolation of the respective diastereomers of 99mTc-TRODAT-1 and three of its derivatives using mixtures of solvents which are compatible with biological studies, i.e. aqueous buffers and ethanol. Furthermore, direct identity confirmation of the 99mTc-complexes using radio-LC-MS was successfully elaborated.

An efficient HPLC method for the analysis of isomeric purity of technetium-99m-exametazime and identity confirmation using LC-MS.

99mTc-exametazime (99mTc-d,l-HMPAO, 99mTc-d,l-hexamethylpropyleneamine oxime) is a neutral rather unstable complex of short-lived 99mTc (t(1/2)=6 h) with the d,l-isomer (mixture of D,D- and L,L-isomers) of a bis-amine bis-oxime tetraligand. It is widely used for measurement of regional cerebral perfusion in nuclear medicine. The meso-isomer (D,L-form) should not be present in a preparation as it is not retained in brain and thus does not provide clinically useful information. Meso-HMPAO is removed from the ligand during the synthesis procedure by repeated recrystallization, but can still be present as impurity in d,l-isomer. Due to the lack of a suitable chromatographic method for analysis of the isomeric purity of 99mTc-exametazime preparations, United States Pharmacopoeia 25 (USP 25) prescribes a biological test in rats for quality control purpose. In this study, we developed a suitable high-performance liquid chromatography (HPLC) method which allows to demonstrate the relative amounts of d,l- and meso-isomer in 99mTc-exametazime and so obviates the need for a biodistribution test in animals as part of the quality control. Due to the low concentrations in which 99mTc-d,l-HMPAO is obtained (typically 2-6 ng/ml), confirmation of the identity of 99mTc-d,l-HMPAO in the monograph of the European Pharmacopoeia is now performed only indirectly by TLC and assessment of its retention time on RP-HPLC. To investigate the potential of radio-LC-MS for assessment of the identity of 99mTc-exametazime, 99mTc-d,l-HMPAO and 99mTc-meso-HMPAO prepared using a Tc-rich eluate were analyzed using a radio-LC-MS system equipped with a time-of-flight mass spectrometer with electrospray ionization. The main peak in the radiometric channel coincided with the molecular ion mass of 99mTc-d,l-HMPAO in the mass spectrometer channel and the measured accurate mass differed only by 0.26 ppm from the theoretical mass. The identity of 99mTc-meso-HMPAO was also confirmed. Thus, radio-LC-MS allowed to obtain strong evidence for the structure of 99mTc-d,l-HMPAO and 99mTc-meso-HMPAO at nanomolar concentration. It is concluded that radio-LC-MS can become a sensitive aid in quality control of "no carrier added" radiopharmaceutical preparations.

In vitro affinity of 99Tcm-labelled N2S2 conjugates of chrysamine G for amyloid deposits of systemic amyloidosis.

To date, systemic amyloidosis is diagnosed histologically in vitro using Congo red staining or in vivo using iodine-123 serum amyloid P component (123I-SAP) scintigraphy. 99Tcm-labelled derivatives of chrysamine G (CG), a lipophilic analogue of Congo red, were synthesized as potential tracer agents for direct and quantitative scintigraphic evaluation of amyloid deposits. To determine the affinity of 99Tcm-MAMA-CG, 99Tcm-Me4MAMA-CG and 99Tcm-MAMA-CG diethyl ester for amyloid, in vitro autoradiography was performed on sections of human kidney biopsy cylinders from kidneys with amyloid deposits (types AA, Alambda and Akappa) or control kidney tissue after incubation with the respective tracer agents. The binding of 99Tcm-MAMA-CG and its tetramethyl derivative was higher to kidney biopsy material with amyloid deposits of the AA, Alambda or Akappa type compared with control kidney tissue. This higher binding was prevented by the presence of 10 microM Congo red in the incubation medium. The diethyl ester of 9Tcm-MAMA-CG did not demonstrate increased binding to Congo red-positive kidney tissue. In conclusion, 99Tcm-MAMA-CG and 99Tcm-Me4MAMA-CG localize specifically to amyloid deposits in human kidney tissue, suggesting that these tracer agents may be applicable as specific targeting agents for diagnostic purposes in clinical amyloidosis.

99mTc-MAMA-chrysamine G, a probe for beta-amyloid protein of Alzheimer's disease.

Chrysamine G (CG), an analogue of Congo red, is known to bind in vitro to the beta-amyloid protein (Abeta 10-43) and to homogenates of several regions of the brain of Alzheimer's disease (AD) patients. We synthesised a conjugate of 2-(acetamido)-CG with a bis-S-trityl protected monoamide-monoaminedithiol (MAMA-Tr(2)) tetraligand, which was efficiently deprotected and labelled with a 75% yield with technetium-99m, to obtain (99m)Tc-MAMA-CG. In mice, (99m)Tc-MAMA-CG was cleared mainly by the hepatobiliary system, resulting in a fast blood clearance. Brain uptake of (99m)Tc-MAMA-CG was low. Co-injection with the blood pool tracer iodine-125 human serum albumin ((125)I-HSA) demonstrated a brain/blood activity ratio for (99m)Tc-MAMA-CG that was significantly higher than that for (125)I-HSA (t test for dependent samples, P<0.02), indicating the ability of (99m)Tc-MAMA-CG to cross the blood-brain barrier. In vitro autoradiography demonstrated pronounced binding of (99m)Tc-MAMA-CG to beta-amyloid deposits in autopsy sections of the parietal and occipital cortex of an AD patient as compared with controls. Adding 10 microM Congo red during incubation displaced the binding of (99m)Tc-MAMA-CG. Congo red staining and autoradiography identified the same lesions. (99m)Tc-MAMA-CG seems to bind selectively to beta-amyloid deposition in human brain parenchyma and blood vessels in vitro and thus might be a lead compound for further development of a useful tracer agent for the in vivo diagnosis of Alzheimer's disease.

99Tcm-dimercaptopropionyl-HSA prepared from a labelling kit: preliminary investigations as a blood pool agent.

The blood retention of 99Tcm-dimercaptopropionyl human serum albumin (99Tcm-DMP-HSA), prepared from a kit, was compared with that of five other 99Tcm-labelled blood pool tracers in two healthy volunteers. 99Tcm-DMP-HSA showed an almost identical behaviour to in vitro labelled red blood cells (RBCs), which are generally considered the reference standard for blood pool agents. The mean apparent blood mass of 99Tcm-DMP-HSA was 2.1% higher 10 min post-injection (p.i.) than that of in vitro 99Tcm-RBCs, 2.0% higher 30 min p.i., 4.7% higher 60 min p.i. and 2.3% higher 120 min p.i. In vivo labelling of RBCs yielded a labelling efficiency of 75-98%, depending on the stannous agent used. About 20 min after pertechnetate administration, the intravascular activity as a percentage of injected dose stabilized at levels close to that of in vitro labelled RBCs. One commercially available 99Tcm-HSA kit was found to be unsuitable as a blood pool tracer. As 99Tcm-DMP-HSA offers the same practical advantages as 99Tcm-HSA, but better biological characteristics, it shows promise as a new tracer for radionuclide ventriculography and further large-scale investigations are warranted.

Complexes of technetium-99m with tetrapeptides, a new class of 99mTc-labelled agents.

Tetrapeptides are a class of N4-tetraligands that can efficiently bind 99mTc. In fact, tetrapeptides can be considered as derivatives of mercaptoacetyltriglycine (MAG3) in which the mercaptoacetyl moiety is replaced by a more stable and easier to handle aminoacyl group. Direct labelling of tetrapeptides with 99mTc in alkaline medium (pH > or = 11) in the presence of stannous ions gave a high yield (> 95%) of one or two (probably isomeric) radiochemical species. Exchange labelling at different pH values in the presence of stannous tartrate resulted in lower yields of the same 99mTc-labelled products as those formed during direct labelling. In addition, other radiochemical species were formed of which one was characterized as an oxotechnetium-complex with the cyclisized tetrapeptide. Tetrapeptides with a chiral centre in the first amino acid yield upon labelling with 99mTc two radiochemical species, probably the two diastereomers with an oxotechnetium core respectively syn and anti with respect to the substituent on the amino acid. Only one diastereomer was observed when the chiral carbon atom is located in the second or third amino acid. Electrophoresis indicated that these new 99mTc-labelled complexes are neutral in acidic medium and negatively charged in neutral and alkaline conditions. This correlates with a complex in which an oxotechnetium(V) group is bound to the ligand through three deprotonated nitrogen atoms of the amide functions and the free electron pair of the amine nitrogen atom. Biodistribution in mice showed for all studied 99mTc-labelled tetrapeptides a rapid clearance from the blood mainly by the renal system. The presence of a methyl substituent in the tetrapeptide increased the urinary excretion. 99mTc-labelled L-glycylalanylglycylglycine showed in mice a urinary excretion comparable to that of 99mTc-MAG3. Further rise of lipophilicity by introduction of a dimethyl, isopropyl or isobutyryl group leads to increased hepatobiliary handling. It is concluded that tetrapeptides are an interesting group of technetium complexing agents which can easily be labelled with 99mTc at room temperature in alkaline medium. This class offers the possibility of a wide variety of derivatives, just by substituting one or more amino acids. This group of ligands thus opens a new research field of 99mTc-complexes with potential usefulness in several areas.

First experience in healthy volunteers with technetium-99m L,L-ethylenedicysteine, a new renal imaging agent.

Animal studies have indicated that technetium-99m L,L-ethylenedicysteine (99mTc-L,L-EC) may be a promising tracer agent for renal function studies. We have performed a paired study with 99mTc-mercaptoacetyltriglycine (99mTc-MAG3) and 99mTc-L,L-EC in six male volunteers. In both cases, iodine-131-labelled o-iodohippurate was co-injected as an internal biological standard. The analog images between 0 and 30 min p.i. were of identical diagnostic value for both tracer agents. The two renograms were similar in all volunteers. The mean 1-h plasma clearance for 99mTc-MAG3 and 99mTc-L,L-EC was significantly different, respectively 382.9 +/- 17.1 ml/min per 1.73 m2 versus 460.2 +/- 47.7 ml/min per 1.73 m2 (P < 0.003). The urinary excretion after 30 min p.i. was 69.4% +/- 5.6% of the injected dose for 99mTc-MAG3 versus 66.5% +/- 2.5% for 99mTc-L,L-EC (P > 0.05) and after 60 min p.i. respectively 83.1% +/- 3.9% versus 79.8% +/- 4.3% (P > 0.05). 99mTc-L,L-EC has a very low plasma protein binding (31% +/- 6.8%) as compared to 99mTc-MAG3 (88% +/- 5.2%) and a larger volume of distribution. Although the exact mechanism responsible for the high plasma clearance of 99mTc-L,L-EC is not yet fully known, we conclude that this new agent merits further clinical evaluation in patients to establish its value as a renal radiopharmaceutical.

Technetium-99m-L,L-ethylenedicysteine: a renal imaging agent. I. Labeling and evaluation in animals.

L,L-ethylenedicysteine (L,L-EC) can be labeled efficiently with 99mTc at pH 12 to obtain a highly pure and very stable tracer agent (99mTc-L,L-EC). The biological behavior of 99mTc-L,L-EC was studied in mice and a baboon. In mice, 99mTc-L,L-EC demonstrated a more rapid urinary excretion and less retention in the kidneys, the liver, the intestines, and the blood than did 99mTc-MAG3 at 10 and 60 min p.i. Urinary excretion decreased in probenecid pretreated mice, which indicates active tubular transport. In the baboon, the renograms for 99mTc-MAG3 and 99mTc-L,L-EC were comparable. Plasma-protein binding of 99mTc-L,L-EC was lower than that of 99mTc-MAG3 while its distribution volume and 1-hr plasma clearance were clearly higher. The promising results of the animal experiments suggest that 99mTc-L,L-ethylenedicysteine may be a useful alternative to 99mTc-MAG3 for renal function studies in humans.

Evaluation of the renal excretion characteristics of technetium-99m mercaptoacetylglycyl-D-alanylglycine in healthy volunteers.

During a study of C-methyl derivatives of technetium-99m mercaptoacetyltriglycine (99mTc-MAG3) it was found that the isomer of 99mTc-mercaptoacetylglycyl-D-alanylglycine (99m-TC-MAGAG-DA) is superior to 99mTc-MAG3 with regard to its renal excretion characteristics in both mice and the baboon. We have now compared the renal handling of 99mTc-MAGAG-DA and 99mTc-MAG3 in 6 healthy volunteers in a paired study. Renograms of 99mTc-MAGAG-DA show a shorter time to renal maximum and a lower residual renal activity at 30 min post-injection (p.i.). The urinary excretion of 99mTc-MAGAG-DA is higher at both 30 and 60 min p.i. The plasma concentration of 99mTc-MAGAG-DA is lower than that of 99mTc-MAG3 at each moment up to 60 min p.i., and the plasma clearance is accordingly higher. It is concluded that 99mTc-MAGAG-DA is excreted more efficiently than 99mTc-MAG3, but the preparation of 99mTc-MAGAG-DA requires a HPLC purification step, and this limits its practical clinical usefulness.