Specific inhibition of the subunits of ribonucleotide reductase as a new approach to combination chemotherapy.
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Animals↗
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Release of CuPTSM from human serum albumin after addition of fatty acids.
Copper-62 labeled pyruvaldehyde bis (N4-methyl-thiosemicarbazonato) copper(II), CuPTSM, has been used to probe tissue perfusion by means of positron emission tomography. Despite promising results from animals, problems have been encountered in the use of 62CuPTSM to quantitate regional myocardial blood flow in humans. Ultrafiltration and plasma/erythrocyte partitioning studies with radiotracer have previously shown that CuPTSM is bound much more strongly by human serum albumin (HSA) than by dog serum albumin (DSA), limiting its ability to freely diffuse from blood into tissue. In this study, it is confirmed by electron spin resonance (ESR) that CuPTSM strongly binds to HSA with an apparent gparallel value of 2.12 and an apparent Aparallel value of 186 G. It is also shown that both spin-labeled stearic acid (5-SASL) and nonspin-labeled stearate inhibit CuPTSM binding to HSA. CuPTSM is completely released from HSA when the ratio of 5-SASL to HSA is 5:1. When pure sodium stearate is used, the binding of CuPTSM significantly decreased, about 73% of CuPTSM is released with a ratio of 4:1 stearate to HSA. These results highlight a means of liberating CuPTSM from HSA.
Electron Spin Resonance Spectroscopy↗
Changes in deoxynucleoside triphosphate pools induced by inhibitors and modulators of ribonucleotide reductase.
Changes in dNTP pools have been studied by a number of investigators, in a wide range of cell types. The in vitro pertubations in dNTP pool levels induced, in particular, by deoxynucleosides which act as allosteric modulators, are not totally consistent with current 'in vitro models' of ribonucleotide reductase function. This problem has also been addressed by Henderson et al. (1980) who stress the profusion of such models. Possible explanations, apart from the technical problems of the range of different experimental conditions (e.g. concentration of modulator used, time of incubation, etc.) for the various cell lines include: Modulators presumably have unpredictable 'network' effects by inhibiting or stimulating many other enzymes involved in the de novo and salvage synthesis of purines and pyrimidines. It is possible there are two separate forms of ribonucleotide reductase, one specifically reducing CDP/UDP, the other ADP/GDP. This, in particular, would explain the lack of decrease in dCTP levels after elevation of the dATP pool. There may be variations in ribonucleotide function which in vivo are cell specific, e.g. in thymic-derived compared with non-T-cell types. Peculiarities of T-cells include: Their ability to elevate their dNTP pools on exposure to very low exogenous concentration of deoxynucleoside. This may reflect very low rates of dNTP catabolism. The biological response of T-cells to elevation of the dATP or dGTP pool is reflected by a G1 block compared to an S phase block in cell-cycle progression in non-T-cell lines. The possibility that, in thymic cells, ribonucleotide reduction is restricted to ADP/GDP while pyrimidine dNTPs are synthesized by salvage pathways. As well, possible variation in the pool localization of dNTPs depending on production by either de novo or salvage synthesis could produce dNTP pool changes not clearly in accord with in vitro models. Clearly, the solution to these problems (although not easy) requires systematic comparative study, using cells of various origin (particularly T vs non-T), of dNTP pool responses to deoxynucleoside modulators, with an attempt to explore the factors described above. However, in the detailed pursuit of such an analysis the concept, that these variations in the control of nucleotide metabolism in T and non-T-cell systems may reflect quite significant differences in growth control and cell-cycle progression, should not be lost.
Animals↗
Effect of N-methylisatin-beta-4':4'-diethylthiosemicarbazone on intracellular Moloney leukemia virus constituents.
N-Methylisatin-beta-4':4'-diethylthiosemicarbazone (M-IBDET) inhibits intracellular production of viral constituents in a mouse cell line, 3T3/MLV, chronically infected with Moloney leukemia virus. Electron microscopic observations confirmed that inhibition of virus production by the drug was not associated with any structural changes in the cell morphology or any damage to the plasma membrane, the site of viral assembly and 'budding'. Treatment of the cells with 17 microM M-IBDET for 6 h inhibited extracellular virus production by 80% but did not affect the level of viral RNA in the cytoplasm or in the plasma membrane. Intracellular reverse transcriptase activity and levels of viral structural proteins were significantly inhibited. Thus, although the drug did not affect viral RNA, it reduced viral protein synthesis.
Animals↗
Combined modality of 2-formylpyridine monothiosemicarbazonato copper(II) and radiation.
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Animals↗
In the procurement of stable 99mTc labeled protein using bifunctional chelating agent.
Recently, technetium-99m (99mTc) labeling of proteins through the use of a bifunctional chelating agent (BCA) has been reported. In previous work, a BCA containing a di(N-methylthiosemicarbazone) (DTS) moiety for 99mTc has offered good potential; however, conjugation with bulky proteins has induced some steric interference in the 99mTc labeling step using the stannous chloride method. In order to minimize this effect, changes in the hydrocarbon chain length (spacer) between the DTS and the protein binding site (carboxyl group), as well as the chemical state of the reducing agent, stannous chloride, were considered of interest. DTS molecules with variable spacer length (n = 0, 2, 4) were synthesized, coupled with human serum albumin and labeled with 99mTc. Also, stannous chloride prepared in different media was evaluated. Validity of the present approach is tested and discussed.
Animals↗
Search for 99mTc labeled DTS bifunctional radiopharmaceutical: role of functional groups in myocardial accumulation.
Development of 99mTc-bifunctional radiopharmaceutical (BR) is attracting the interest of various research groups. In the present paper, various molecules containing a neutral 99mTc-dithiosemicarbazone (DTS) structure as the technetium chelating site, along with various functional groups (amino, carboxyl or isobutyl group with diverse charge) are tested for their chemical or biological functions. The study on the effect of those functional groups is carried out in vitro and in vivo. The validity of introducing an amino group along with the technetium chelating site DTS for myocardial accumulation is discussed.
Animals↗
A remote system for the synthesis of copper-62 labeled Cu(PTSM).
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Copper Radioisotopes↗
Design of bifunctional radiopharmaceutical for the development of 99mTc complexes for myocardial imaging agents.
Several bifunctional radiopharmaceuticals (BR), molecules containing a neutral 99mTc-dithiosemicarbazone (DTS) structure as a chelating site, along with a functional amino group (primary tertiary and quaternary amino group) are tested for their chemical or biological functionalities as myocardial agents. Investigation of these amino compounds is carried out in vitro and in vivo. Mice (ddY) distribution studies show a high radioactivity distribution in heart with every tested derivative, but the highest heart to blood ratio of 2.92 (1 h) is achieved with the quaternary amino DTS. Also, this derivative displays low lung uptake inducing a proper target/non-target ratio for myocardial agent. The role conveyed by the various amino containing side chain in the biodistribution and validity of DTS derivative as BR are discussed.
Animals↗
A potential copper radiopharmaceutical for imaging the heart and brain: copper-labeled pyruvaldehyde bis(N4-methylthiosemicarbazone).
An investigation of the biodistribution of lipophilic copper-64 (half-life = 12.7 h) compounds has been initiated in order to screen potential tracers that could be used to measure regional cerebral and/or myocardial blood flow when labeled with generator-produced 62Cu. The 64Cu complex of pyruvaldehyde bis(N4-methylthiosemicarbazone), [64Cu]Cu-PTSM, was prepared and found to be lipophlic (octanol/saline partition coefficient, log p = 1.97 +/- 0.03). Biodistribution studies following i.v. injection of [64Cu]Cu-PTSM into rats show tracer uptake by the brain and heart. At 1, 5, and 15 min post-injection 3.3, 3.0 and 2.7% of the injected dose was found in the brain. Corresponding brain to blood ratios (per gram) were 3.2, 3.6 and 4.0 respectively. Heart to blood ratios of 7.6, 7.6 and 7.3 were observed at these same time points.
Animals↗
Correlation of Cu(PTSM) localization with regional blood flow in the heart and kidney.
In a dog model of myocardial ischemia, i.v. injection of [67Cu]Cu(PTSM) leads to an excellent linear correlation for both the heart and kidney between regional tissue radiocopper concentration and regional blood flow measured by radioactive micropheres. The 62Cu-labeled complex may serve as a generator-produced position-emitting perfusion tracer for these organs.
Animals↗
Structural characterization of a metal-based perfusion tracer: copper(II) pyruvaldehyde bis(N4-methylthiosemicarbazone).
Copper(II) pyruvaldehyde bis(N4-methylthiosemicarbazone), Cu(PTSM), has been obtained as a dark red crystalline solid from EtOH-DMSO solvent mixture and structurally characterized by x-ray crystallography. The molecule possesses the expected pseudo-square planar N2S2 metal coordination sphere; however, the copper center also interacts through its axial coordination site with the sulfur atom of an adjacent Cu(PTSM) molecule in the crystal lattice. The structure of this compound is compared with the structures of other metal complexes that have been proposed in the nuclear medicine literature as perfusion tracers.
Crystallography↗
Investigation of copper-PTSM as a PET tracer for tumor blood flow.
Copper-PTSM has been shown in previous studies to act as a fluid microsphere and to be useful in quantitating blood flow in brain, myocardium, and kidneys. In this study we have evaluated this agent as a PET tumor blood flow agent. 64Cu- or 67Cu-labeled Cu-PTSM was administered (i.v.) to Golden Syrian hamsters with colorectal carcinoma cell implants (GW39). One minute prior to sacrifice (10-60 min after Cu-PTSM was administered) 125I-iodoantipyrine (125I-IAP), an agent known to measure tumor blood flow, was administered intravenously by a 3-stage, 1 min ramp infusion. Following sacrifice, samples of tumor and brain were removed (within 40s) and the tumor and brain levels of Cu-PTSM and iodoantipyrine determined. Since the brain uptake of both Cu-PTSM and IAP is perfusion rate limited, the brain was used as a reference organ to normalize tumor levels of the two tracers. The plot of Cu-PTSM versus 125I-IAP tumor/brain ratios showed a good linear correlation (r value of 0.97), suggesting that Cu-PTSM could be used to quantify tumor blood flow. Since the mechanism of Cu-PTSM trapping is likely to be due to glutathione levels in the tissue, and because tumor tissue glutathione levels might vary, the temporal uptake of Cu-PTSM was investigated by PET imaging both the tumor-bearing hamsters and approximately 300 g Copenhagen rats bearing R3227 prostate tumors. The tumors were clearly visualized and the retained copper radioactivity in the tumor was constant over the 30 min imaging period.
Animals↗
The effects of glutathione depletion on the biodistribution of Cu(PTSM) in rats.
The tissue retention of radiocopper afforded by intravenous injection of the proposed blood flow imaging agent, 62Cu-labeled copper(II) pyruvaldehyde bis(N4-methylthiosemicarbazone) [Cu(PTSM)], is thought to result from reductive decomposition of the copper(II) complex by intracellular sulfhydryls (e.g. glutathione, GSH). To determine if the tissue uptake and retention of this tracer adequately measures perfusion in tissues containing altered GSH concentrations, the biodistribution of copper-67 labeled Cu(PTSM) was determined in GSH-depleted rats. Despite treatment to induce relatively large reductions in tissue GSH levels, it was found that only very small changes in the biodistribution of copper-labeled Cu(PTSM) occurred in the treated rats compared to untreated controls.
Animals↗
Subcellular distribution of tissue radiocopper following intravenous administration of 67Cu-labeled Cu-PTSM.
The subcellular distribution of radiocopper in the brain and liver of rats has been determined following i.v. administration of Cu-PTSM, pyruvaldehyde bis(N4-methylthiosemicarbazonato)copper(II), labeled with copper-67. Homogenized tissue samples were separated by differential centrifugation into four subcellular fractions: (I) cell membrane + nuclei; (II) mitochondria; (III) microsomes; and (IV) cell cytosol. Upon sacrifice at 10 min post-Cu-PTSM injection, brain fractions, I, II, III and IV contain 35 +/- 12, 11 +/- 3, 2.8 +/- 1.3 and 51 +/- 7% of brain activity, respectively (n = 4). In animals sacrificed 24 h post-injection the subcellular fractions of brain tissue show little change from the radiocopper distribution seen at 10 min post-injection, although the mitochondrial fraction may contain slightly more tracer and the cytosolic fraction slightly less (I, 40 +/- 10%; II, 18 +/- 5%; III, 3.4 +/- 1.5%; and IV, 38 +/- 5%; n = 5). Subcellular fractions I, II, III and IV of liver contain 25 +/- 5, 12 +/- 3, 17 +/- 4 and 46 +/- 6% of 67Cu tracer in animals sacrificed 10 min post-Cu-PTSM injection. An identical subcellular distribution of 67Cu, was found in the liver following i.v. administration of ionic radiocopper (as Cu-citrate). The liver and brain cytosolic fractions at 10 min post-injection were further separated by Sephadex column chromatography. In liver cytosol, three different radiocopper components with molecular weights of about 140,000, 41,000-46,000 and 10,000-16,000 Da were found. In the brain supernatant fraction, most of the radiocopper was bound to a single low molecular weight cytosolic component (14,000-16,000 Da).(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
62Cu-labeled bifunctional radiopharmaceuticals with metabolizable ester groups.
Generator-produced positron emitting 62Cu-labeled bifunctional radiopharmaceuticals with metabolizable ester groups were synthesized and evaluated for their bifunctionality: the Cu chelating ability and the biochemical reactivity of the ester groups. In vitro studies showed high stability of the Cu chelate site (4,4-dimethyldithiosemicarbazone structure) and enzymatic hydrolysis of the ester site (ethylester or acetylester). The 62Cu-labeled compounds showed in vivo behavior which may be due to their ester hydrolysis.
Animals↗
Application of the new zinc-62/copper-62 generator: an effective labeling method for 62Cu-PTSM.
A potential PET flow tracer, 62Cu-labeled pyruvaldehyde bis(N4-methylthiosemicarbazone) (62Cu-PTSM), was prepared using a new 62Zn/62Cu generator. With this 62Cu-labeling method based on a ligand exchange reaction, 62Cu-PTSM was quantitatively obtained by simple mixing of the generator eluate, 62Cu-glycine and PTSM solution for a few seconds. The glycine contained in the 62Cu-PTSM injectate had no significant effect in mouse biodistribution studies. The PET studies of 62Cu-PTSM showed brain images that were not visualized by 62Cu-labeled albumin (a plasma pool tracer), in the dog cranium.
Animals↗
Development and validation of a solvent extraction technique for determination of Cu-PTSM in blood.
The partitioning of [67Cu]Cu-PTSM between plasma and red blood cells (RBC) was investigated in vitro with human, rat, pig and dog blood. Significant inter-species variability is observed in the plasma/RBC partitioning of tracer, ranging from c. 75% association with plasma in human blood to only c. 35% association with plasma in dog blood. This inter-species difference results from selective association of the [67Cu]Cu-PTSM tracer with human albumin. When [67Cu]Cu-PTSM is mixed with human blood in vitro at 37 degrees C the fraction of 67Cu-radioactivity that remains plasma-associated decreases with time, apparently due to the expected intracellular decomposition of the Cu-PTSM complex by RBC; however, this process is sufficiently slow that it should have limited influence on [62Cu]CU-PTSM biodistribution following intravenous injection. Octanol extraction of blood was found to be an effective technique for quantitating the amount of intact [67Cu]Cu-PTSM complex in blood samples. When imaging with [62Cu]Cu-PTSM, octanol extraction may be useful for determining the [62Cu]Cu-PTSM content of arterial blood samples to establish a true radiotracer input function.
Animals↗