Radioisotopic purity and imaging properties of cyclotron-produced 99mTc using direct 100Mo(p,2n) reaction.

Evaluation of the radioisotopic purity of technetium-99m (99mTc) produced in GBq amounts by proton bombardment of enriched molibdenum-100 (100Mo) metallic targets at low proton energies (i.e. within 15-20 MeV) is conducted. This energy range was chosen since it is easily achievable by many conventional medical cyclotrons already available in the nuclear medicine departments of hospitals. The main motivation for such a study is in the framework of the research activities at the international level that have been conducted over the last few years to develop alternative production routes for the most widespread radioisotope used in medical imaging. The analysis of technetium isotopes and isomeric states (9xTc) present in the pertechnetate saline Na99mTcO4 solutions, obtained after the extraction/purification procedure, reveals radionuclidic purity levels basically in compliance with the limits recently issued by European Pharmacopoeia 9.3 (2018 Sodium pertechnetate (99mTc) injection 4801-3). Moreover, the impact of 9xTc contaminant nuclides on the final image quality is thoroughly evaluated, analyzing the emitted high-energy gamma rays and their influence on the image quality. The spatial resolution of images from cyclotron-produced 99mTc acquired with a mini-gamma camera was determined and compared with that obtained using technetium-99m solutions eluted from standard 99Mo/99mTc generators. The effect of the increased image background contribution due to Compton-scattered higher-energy gamma rays (E γ > 200 keV), which could cause image-contrast deterioration, was also studied. It is concluded that, due to the high radionuclidic purity of cyclotron-produced 99mTc using 100Mo(p,2n)99mTc reaction at a proton beam energy in the range 15.7-19.4 MeV, the resulting image properties are well comparable with those from the generator-eluted 99mTc.

Comparison of in vitro and in vivo properties of [99mTc]cRGD peptides labeled using different novel Tc-cores.

AIM: The alfa(v)beta(3) integrin is involved in angiogenesis and tumor metastasis. Arginine-glycine-aspartic acid (RGD)-peptides bind with high affinity to this integrin. This study compares the influence of (99m)Tc-labeling applying novel Technetium-cores on imaging characteristics of the radiolabeled peptide. METHODS: Different peptide conjugates based on the cyclic pentapeptide c(RGDyK) (cRGD) were prepared and characterized (HYNIC-, Cys-, L2- and Pz1-cRGD). Radiolabeling experiments using different coligands for HYNIC-cRGD, the (99m)Tc(CO)(3) metal fragment for PZ-1-cRGD (pyrazolyl-derivative), the Tc-nitrido-core using a phosphine-coligand (PNP) for Cys-cRGD and an isonitrile-conjugate (L2-cRGD) together with a NS(3)-coligand (4+1 concept) were performed and showed labeling yields >90% at high specific activities. RESULTS: A high in vitro stability was observed, plasma protein binding and lipophilicity varied considerably between different radiolabeled cRGD conjugates. Experiments on biological activity of the radiolabeled peptides using alfa(v)beta(3) positive (M21) and negative (M21L) tumor cells did show specific uptake of various conjugates. Studies in tumor bearing animals revealed significant differences between different conjugates concerning pharmacokinetic behavior (predominant renal excretion to considerable hepatobiliary clearance) as well as tumor uptake (0.2-2.7%ID/g). Highest specific tumor uptake and tumor/background values were found for [(99m)Tc]EDDA/HYNIC-c(RGDyK), [(99m)Tc]Nitrido-PNP-Cys-c(RGDyK) and [(99m)Tc(CO)(3)]-Pz1-c(RGDyK). CONCLUSIONS: Using novel Tc-cores such as the (99m)Tc(CO)(3) metal fragment, Tc-nitrido- and the 4+1 concept peptides could be labeled with [(99m)Tc]technetium at high specific activities resulting in complexes with high stability, but binding moieties have to be optimized especially concerning hydrophilicity resulting in renal rather than hepatobiliary excretion. This comparative study underlines that peptide labeling strategies using (99m)Tc have to be properly selected and optimized. Different in vitro assays are necessary to predict targeting properties in vivo.

The [(99m)Tc(N)(PNP)](2+) metal fragment: a technetium-nitrido synthon for use with biologically active molecules. The N-(2-methoxyphenyl)piperazyl-cysteine analogues as examples.

The incorporation of a bioactive molecule into a nitrido-containing (99m)Tc-complex has been successfully achieved by using the [TcN(PNP)](2+) metal fragment. In this strategy, the strong electrophilic [TcN(PNP)](2+) metal fragment efficiently reacts with bifunctional chelating ligands having a pi-donor atom set, such as N-functionalized O,S-cysteine. The 2-methoxyphenylpiperazine (2-MPP) pharmacophore, which displays preferential affinity for 5HT(1A) receptors, was conjugated to the amino group of cysteine to obtain 2-MPPP-cys-OS, where 2-MPPP is 3-[4-(2-methoxyphenyl)piperazin-1-yl]propionate. The asymmetric Tc(V)-nitrido complexes, [(99g/99m)Tc(N)(PNP)(2-MPPP-cys-OS)] (PNP = PNP3, PNP4), were obtained in high yield (95%), by simultaneous addition of PNP and 2-MPPP-cys-OS ligand to a solution containing a starting (99g)/(99m)Tc-nitrido precursor. A mixture of syn and anti isomers was observed, the latter being the thermodynamically favored species. In vitro challenge experiments using the anti isomers with glutathione and cysteine indicated that no transchelation reaction occurs. Assessment of the in vitro 5HT(1A) receptor-affinity of the technetium complexes revealed that only the anti-PNP4 complex possesses some affinity for the receptor, but displayed negligible brain uptake in biodistribution studies in rats in vivo.

Biological effects of stannous chloride, a substance that can produce stimulation or depression of the central nervous system.

It was demonstrated that tin, as stannous chloride (SnCl(2)), can facilitate the neuromuscular transmission by accelerating the transmitter release from the nerve terminals in the mouse. When this salt is injected into laboratory animals, it can produce stimulation or depression of the central nervous system. Because calcium (Ca(2+)) influx into the cytoplasm is indispensable to release the transmitter, it would be possible that SnCl(2) increases the Ca(2+) influx at the nerve terminals but not by blocking the K(+) channels. SnCl(2) is known to inhibit the immune response in rodents and to induce tumor generation in thyroid gland. There is no general agreement regarding its genotoxicity and it was discussed that the effects of this salt might depend on the physicochemical conditions and the route of its administration. SnCl(2) has been used in many sectors of human interest, such as food industry and nuclear medicine. This salt is directly administered to human beings endovenously, when it is used as a reducing agent to prepare 99mTc-radiopharmaceuticals which are also used for cerebral studies. SnCl(2) is capable to promote the generation of reactive oxygen species (ROS) that are responsible for the oxidative stress. Oxidative stress has been related with aging and other neurological diseases. So, it is relevant to evaluate other biological effects of SnCl(2). We decided to study these effects using Escherichia coli mutant strains, deficient in DNA repair genes, and supercoiled plasmid DNA. We evaluated the influence of medicinal plants, metal chelating agents, and ROS scavengers against the SnCl(2) deleterious effects. Our results show that SnCl(2) produced lesions in vitro as well as in vivo. This inactivation may be due to the production of ROS. We observed that the genotoxic effect of SnCl(2) was partly inhibited or disappeared, when the treatments were done in the presence of medicinal plants, metal chelating agents, and ROS scavengers. In conclusion, these findings suggest that the SnCl(2) biological effects may be associated with the generation of ROS. Moreover, we can speculate that ROS could be associated with the detrimental effects in the brain due to exogenous or endogenous metals.

A class of asymmetrical nitrido 99mTc heterocomplexes as heart imaging agents with improved biological properties.

Asymmetrical heterocomplexes containing a terminal technetium-nitrogen multiple bond coordinated to one diphosphine ligand (PNP) and one dithiocarbamate ligand (DBODC), were obtained through a simple two-step procedure under controlled conditions. The resulting complexes [99mTc(N)(PNP)(DBODC)]+ are monocationic, and possess a distorted square-pyramidal geometry where the Tc triple bond N multiple bond occupies an apical position and the diphosphine and dithiocarbamate ligands span the residual four coordination positions on the basal plane through the two phosphorus atoms and the two sulfur atoms, respectively. Biodistribution data in rats demonstrated that these complexes were rapidly extracted by the myocardium, and retained in this region for a prolonged time. After a few minutes post-injection, lung uptake became negligible, and liver washout was extremely rapid and quantitative. Analysis of heart/liver uptake ratios for these complexes revealed that their values increased exponentially in time, and after 60 min post-injection liver activity was almost completely eliminated into the intestine. Comparison with heart/liver ratios determined for 99mTc sestamibi and 99mTc tetrfosmin showed that values for these latter compounds were approximately 10 times lower than those measured for [99mTc(N)(PNP)(DBODC)]+ complexes at 60 min post-injection. In conclusion, the monocationic tracers [99mTc(N)(PNP)(DBODC)]+ exhibit high myocardial uptake in rats and dramatically high heart/lung and heart/liver ratios, suggesting that this novel class of perfusion agents could be conveniently employed to obtain heart images with superior imaging quality.

A novel approach to the high-specific-activity labeling of small peptides with the technetium-99m fragment [99mTc(N)(PXP)]2+ (PXP = diphosphine ligand).

A new labeling approach for incorporating bioactive peptides into a technetium-99m coordination complex is described. This method exploits the chemical properties of the novel metal-nitrido fragment [99mTc(N)(PXP)]2+, composed of a terminal Tc[triple bond] N multiple bond bound to an ancillary diphosphine ligand (PXP). It will be shown that this basic, molecular building block easily forms in solution as the dichloride derivative [99mTc(N)(PXP)Cl2], and that this latter complex selectively reacts with monoanionic and dianionic, bidentate ligands (YZ) having soft, pi-donor coordinating atoms to afford asymmetrical nitrido heterocomplexes of the type [99mTc(N)(PXP)(YZ)]0/+ without removal of the basic motif [99mTc(N)(PXP)]2+. The reactions of the amino acid cysteine was studied in detail. It was found that cysteine readily coordinates to the metal fragment [99mTc(N)(PXP)]2+ either through the [NH2, S-] pair of donor atoms or, alternatively, through the [O-, S-] pair, to yield the corresponding asymmetrical complexes in very high specific activity. Thus, these results were conveniently employed to devise a new, efficient procedure for labeling short peptide sequences having a terminal cysteine group available for coordination to the [99mTc(N)(PXP)]2+ fragment. Examples of the application of this novel approach to the labeling of the short peptide ligand H-Arg-Gly-Asp-Cys-OH (H(2)1) and of the peptidomimetic derivative H-Cys-Val-2-Nal-Met-OH (H2) will be discussed.

Synthesis of a novel class of nitrido Tc-99m radiopharmaceuticals with phosphino-thiol ligands showing transient heart uptake.

A novel class of technetium-99m radiopharmaceuticals showing high heart uptake is described. These complexes were prepared through a simple and efficient procedure, and their molecular structure fully characterized. They are formed by a terminal Tc(triple bond)N multiple bond and two bidentate phosphine-thiol ligands [R(2)P-(CH(2))(n)SH, n=2,3] coordinated to the metal ion through the neutral phosphorus atom and the deprotonated thiol sulfur atom. The resulting geometry was trigonal bipyramidal. Biodistribution studies were carried out in rats. The complexes exhibited high initial heart uptake and elimination through liver and kidneys. The washout kinetic from heart was dependent on the nature of the lateral R groups on the phosphine-thiol ligands. When R=phenyl, heart activity was rapidly eliminated within 10-20 min. Instead, when R=tolyl, cyclohexyl, persistent heart uptake was observed. Extraction of activity from myocardium tissue showed that no change of the chemical identity of the tracer occurred after heart uptake. On the contrary, metabolization to more hydrophilic species occurred in liver and kidneys.

Synthesis of hybrid distamycin-cysteine labeled with 99mTc: a model for a novel class of cancer imaging agents.

The synthesis of a hybrid constituted by distamycin A and cysteine labeled with the gamma-emitting radionuclide 99mTc to afford the conjugate complex 5 is reported. This new radiopharmaceutical is of potential interest as tumor imaging agent in diagnostic nuclear medicine. The preparation of the hybrid distamycin A-cysteine 4 has been achieved by coupling deformyldistamycin A and Boc-Dmt-OH. Compound 4 was then successfully labeled with 99mTc by reaction with the novel, high-electrophilic, metal-containing fragment [99mTc(N)(PP)]2+ (PP = diphosphine ligand) yielding the 1:1 complex 5.

An alternative approach to the preparation of (188)Re radiopharmaceuticals from generator-produced [(188)ReO(4)](-): efficient synthesis of (188)Re(V)-meso-2,3-dimercaptosuccinic acid.

A new efficient approach for the preparation of (188)Re radiopharmaceuticals starting from [(188)ReO(4)](-), produced at a carrier-free level through the (188)W/(188)Re generator system, is described. The reaction procedure was based on the combined action of different reagents and has been applied in detail to the preparation of the therapeutic agent (188)Re(V)-DMSA (H(2)DMSA [meso-2,3-dimercaptosuccinic acid]). The most efficient combination required the use of SnCl(2), oxalate ions, and gamma-cyclodextrin. These were reacted with [(188)ReO(4)](-) and H(2)DMSA to afford the final radiopharmaceutical in high radiochemical purity, at room temperature, and in weakly acidic solution. The role played by the various reagents in the reaction was investigated. It was found that SnCl(2) behaved as the actual reducing agent, whereas oxalate and gamma-cyclodextrin greatly enhanced the ease of reduction of [(188)ReO(4)](-) through the action of two hypothetical mechanisms. In the first step of the reaction, oxalate ions gave rise to the formation of Re(VII) complexes with the concomitant expansion of the coordination sphere of the metal. This process strongly favored the electron transfer between Sn(2+) and Re(+7) centers, giving rise to intermediate reduced rhenium complexes. These species were further stabilized by the formation of transient host-guest aggregates with gamma-cyclodextrin and finally converted into (188)Re(V)-DMSA through simple replacement of the coordinated ligands by H(2)DMSA.

Design and synthesis of a redox-active Tc-99m radiopharmaceutical with ferrocenedithiocarboxylate [FcCS = Fe(C5H4CS2)(C5H5)-].

The synthesis, at tracer level, of two Tc-99m complexes having the same chemical composition and structure, but differing by one electron in the total electron counting, is reported. These compounds have been prepared by reacting [99mTcO4]- with the piperidinium salt of the ligand ferrocenedithiocarboxylate {[Fe(II)(C5H4CS2)(C5H5)]- = FcCS}, in the presence of N-methyl S-methyldithiocarbazate as donor of N3-groups, and triphenylphosphine or SnCl2 as reducing agents. The formation of the neutral complex [99mTc(N)(FcCS)2] (compound A) and of the monocationic, mixed-valence complex [99mTc(N)(FcCS) (FcCS)]+ (compound B) {FcCS = [Fe(III)(C5H4CS2)(C5H5)]} was obtained in high yield. Both complexes comprise a terminal Tc triple bond N multiple bond and two FcCS ligands coordinated to the metal center through the two sulfur atoms of the -CS2 group, but they differ in the oxidation state of one of the two iron atoms of the coordinated FcCS ligands. In complex A, the two Fe atoms are both in the +2 oxidation state, while in B, one Fe atom is in the +2 and the other is in the +3 oxidation state. Thus, B is a mixed-valence Fe(II)-Fe(III) complex. B is easily converted into A by one-electron exchange with various reductants such as triphenylphosphine and excess SnCl2. Biodistribution studies in rats showed that complexes A and B are mostly retained in lungs and liver without any significant uptake in organs such as heart and brain.

Labeling and biodistribution studies of Tc-99m radiopharmaceuticals with (o-hydroxyphenyl)diphenylphosphine.

New Tc-99m radiopharmaceuticals with the ligand (o-hydroxyphenyl)diphenylphosphine have been prepared and their biodistributions evaluated in rats. The monoxo Tc(V) complex [99mTc(O)Cl(PO)2], the Tc(IV) complex [99mTc(OH)2(PO)2], the Tc(III) complex [99mTc(PO)3], and the nitrido Tc(V) complex [99mTc(N)(PO)2] have been characterized by TLC and HPLC chromatography, and their chemical structure elucidated by comparison with the corresponding complexes obtained using the beta-emitting isotope Tc-99g. Biodistribution studies of these complexes have been carried out in rats.

Clearance of technetium 99m N-NOET in normal, ischemic-reperfused, and membrane-disrupted myocardium.

BACKGROUND: Technetium 99m-labeled bis(N-ethoxy, N-ethyl dithiocarbamato) nitrido technetium(v) (99mTcN-NOET) is a new neutral cardiac perfusion imaging agent that has been shown to have very high uptake and retention in vitro. The purpose of this study was to determine the clearance kinetics of 99mTcN-NOET in control, ischemic-reperfused, and membrane-disrupted myocardium. METHODS AND RESULTS: After a 100 microCi (3.7 x 10(6) Bq) bolus of 99mTcN-NOET was injected, myocardial clearance was monitored for 1 hour by the use of a sodium iodide detector in 30 isolated, Krebs-Henseleit (KH) perfused rat hearts. Seven hearts were used as controls (group 1). In seven ischemic-reperfused hearts, tracer administration and uptake was followed by 30 minutes of no flow and 1 hour of reflow (group 2). In six additional ischemic-reperfused hearts, tracer administration was followed by deprivation of flow for 1 hour followed by 1 hour of reflow (group 3). Six hearts were perfused with a 0.5% Triton X-100 KH perfusate for 1 hour (group 4). Four hearts were perfused with KH for 10 minutes, followed by cyanide for 10 minutes (group 5). This cycle was repeated three times. Activities remaining in each heart at the end of each experiment were quantitated, and activity at peak uptake was calculated. The 99mTcN-NOET myocardial clearance was near linear in the control (0.6 +/- 0.4) and both ischemic-reperfused groups with virtually no fractional clearance (1.2% +/- 0.6% and 2.1% +/- 0.6%, respectively; p = NS). In the Triton X-100 membrane-disrupted hearts, clearance was substantial (94.2% +/- 4.0%; p < 0.0001 compared with the control and ischemic-reperfused groups). Cyanide treatment produced rapid clearance, which was arrested by a return to the standard KH perfusate. Peak uptake as a percentage of injected dose was 74.9% +/- 1.4% for all groups combined. CONCLUSION: Thus 99mTcN-NOET has extremely high myocardial retention after 1 hour in normal myocardium and is not significantly affected by ongoing myocardial ischemia or reperfusion injury in this model. Clearance is increased markedly in extreme conditions of membrane disruption. These data are consistent with the concept that 99mTc-NOET is localized predominantly in or on cell membranes. 99mTcN-NOET is a promising, new myocardial perfusion imaging agent that exhibits a stable myocardial distribution in the setting of acute developing injury.

Subcellular distribution of technetium-99m-N-NOEt in rat myocardium.

UNLABELLED: The aim of this study was to determine the subcellular distribution of bis(N-ethoxy N-ethyl)dithiocarbamato nitrido technetium(V) (99mTcN-NOEt) in rat heart by differential centrifugation techniques. Extraction of the activity from homogenized rat heart tissue was also performed to assess whether myocardial retention might induce changes in the chemical identity of the complex. METHODS: Anesthetized rats were intravenously injected with 99mTcN-NOEt, the heart tissue was extracted and homogenized and tissue fractions were obtained by differential centrifugation. The efficiency of organelle separation was determined by assay of each centrifugal fraction using enzyme markers. Lactate dehydrogenase (LDH), acid phosphatase (ACP), alkaline phosphatase (ALP) and 5'-nucleotidase (5'ND) activities were assayed using standard spectrophotometric methods. Succinic dehydrogenase (SDH) activity was determined using a p-iodo-nitrotetrazolium-linked assay. Severe cell membrane and organelle disruption were induced by prolonging the homogenization time and their effect on the subcellular distribution of 99mTcN-NOEt was studied. The activity from homogenized heart tissue was extracted using the Folch technique and analyzed by TLC and HPLC. RESULTS: Most of the 99mTcN-NOEt activity was found to be associated with the hydrophobic components of the cell. No evidence of specific association of activity with the cytosolic and mitochondrial components was observed. Organelle and membrane cleavage did not cause release of activity into the cytosol. Approximately 90% of 99mTcN-NOEt activity was extracted from ventricular tissue and the chemical nature of 99mTcN-NOEt was not altered by uptake by myocardium. CONCLUSION: Cell membranes are the most apparent site of localization of 99mTcN-NOEt in heart tissue.

Synthesis and biodistribution of nitrido technetium-99m radiopharmaceuticals with dithiophosphinate ligands: a class of brain imaging agents.

The symmetrical complexes [99mTc][TcN(R2PS2)2] [R = CH3, CH2CH3, CH2CH2CH3, CH2(CH3)2], and the unsymmetrical complex [99mTc][TcN(Me2PS2)(Et2PS2)] have been prepared, at tracer level, through a two-step procedure involving the preliminary formation of a prereduced technetium nitrido intermediate followed by substitution reaction onto this species by the appropriate dithiophosphinate ligand [R2PS2]Na. The chemical identity of the resulting complexes have been established by comparison with the corresponding 99Tc-analogs prepared, at macroscopic level, by reacting the complex [99TcNCl4] [n-Bu4N] (n-Bu = n-butyl) with an excess of ligand in methanol, and characterized by elemental analyses and spectroscopic techniques. The complexes are neutral and lipophilic, and possess a square pyramidal geometry, with an apical Tc identical to N group and two dithiophosphinate ligands spanning the four positions on the basal plane through the four sulfur atoms of the > PS2 group. In vitro studies showed that these radiopharmaceuticals are stable in solution and that their chemical identity was not altered after incubation with rat blood. Biodistribution studies have been carried out in rats and primates. The results demonstrate that these compounds are significantly retained into the brain of these animals for a prolonged time. Planar gamma camera images have been obtained in monkeys showing a good visualization of the cerebral region. However, the existence of persistent blood activity yields a brain/blood ratio lower than that observed with other 99mTc-based brain perfusion imaging agents.

Bis(dithiocarbamato) nitrido technetium-99m radiopharmaceuticals: a class of neutral myocardial imaging agents.

UNLABELLED: The synthesis and biodistribution in various animal models (rat, dog, pig and monkey) of 99mTc radiopharmaceuticals containing the Tc = N multiple bond are reported. METHODS: The complexes are represented by the general formula 99mTcN(L)2, where L is the monoanionic form of a dithiocarbamate ligand of the type [R1(R2)-N-C(=S)S]-, and R1 and R2 are variable, lateral groups. The preparations were carried out, both as a liquid and freeze-dried formulation, through a simple procedure involving the initial reaction of [99mTcO4]- with S-methyl N-methyl dithiocarbazate [H2NN(CH3)C(=S)SCH3], in the presence of tertiary phosphines or Sn2+ ion as reductants, followed by the addition of the sodium salt of the ligand (NaL) to afford the final product. The chemical identity of the resulting complexes was determined by comparing their chromatographic properties with those of the corresponding 99Tc analogs characterized by spectroscopic and x-ray crystallographic methods. The complexes are neutral and possess a distorted, square pyramidal geometry. RESULTS: No decomposition of the complexes, in physiological solution, was observed over a period of 6 hr. Imaging and biodistribution studies demonstrated that these radiopharmaceuticals localize selectively in the myocardium of rats, dogs and primates, but that they failed to visualize the pig heart. The kinetics of heart uptake and clearance were studied in rats and dogs, and found to be strongly influenced by variation of the lateral R1 and R2 groups. CONCLUSION: The high quality of myocardial images obtained in dogs and monkeys demonstrates that the derivative 99mTcN[E-t(EtO)NCS2]2 [99mTcN(NOEt)] exhibits the most favorable distribution properties for further studies in humans.