[99mTc]pyrophosphate and [125I]phenylphosphonate behavior in the infarcted rat myocardium

The concentration of [¹²⁵I]phenylphosphonate ([¹²⁵I]φPA), was compared to that of [^99mTc]pyrophosphate ([^99mTc]Pyro) in a rat infarct model. Twenty-three hours after ligation of the anterior descending coronary artery, [¹²⁵I]φPA was given intravenously (i.v.) to 30 adult male rats. Five minutes later, 5 of these animals received [^99mTc]Pyro IV and the content of ¹²⁵I and ^99mTc was then determined at 2 h for normal (N), and infarcted (INF) heart segments. A similar investigation was completed in 5 of the animals at each of the following times post MI; 2, 3, 4, 7 and 75 days. Data were expressed as the mean (± SEM) of (% uptake/g infarct)/(% uptake/g normal) for both tracers at each study time. These data suggest that the myocardial cells which exhibit constant uptake of [^99mTc]Pyro are similar to those exhibiting chronic retention of [¹²⁵I]φPA. The myocardial infarct to normal ratio of [^99mTc]Pyro on [¹²⁵I]φPA showed excellent correlation (r = 0.928) and suggests that persistent positive pyrophosphate scans are likely due to residual damaged tissue from the acute episode of necrosis, rather than the involvement of newly injured tissue.     

Iodine heterocycles: [125I] and [131I] ortho-iodosophenylphosphoric acid

Since organic molecules tagged with radioiodine are often subject to dehalogenation, techniques are needed for "protecting" the iodine. A suggested approach was the incorporation of iodine directly into a heterocyclic compound as one of the ring's heteroatoms. Such a compound, orthoiodosophenylphosphoric acid, was synthesized with I-125 and I-131. Upon i.v. administration to dogs and rabbits, most of the radiolabel was excreted in the urine. There was no evidence of the appearance of free iodide. The renal elimination of orthoiodosophenylphosphoric acid was contrasted with the biliary excretion of another iodine heterocycle, diphenyleneiodonium. Iodine heterocycles, with appropriate substituents, may represent a useful class of compounds for biologic studies.     

Characteristics of the binding of N-isopropyl-p-[125I]iodoamphetamine in the rat brain synaptosomal membranes

N-isopropyl-p-[125I]iodoamphetamine (125I-IMP) binding to crude synaptosomal membranes of rat brain was saturable and pharmacologically displacable. Scatchard analysis of binding data revealed an apparent dissociation constant, KD of 56.2 +/- 5.2 microM, and the estimated maximum number of binding sites, Bmax of 7.5 +/- 1.1 nmoles mg-1 protein; and competition studies demonstrated structure activity relationships among structural analogues of arylalkylamines. These findings suggest that the retention mechanism of IMP in the brain is probably associated with saturable binding of IMP to extreme high-density, relatively low-affinity binding sites rather than any of the well-known amine receptors.     

Marking hypoxia in rat prostate carcinomas with beta-D-[125I]azomycin galactopyranoside and.

The purpose of this study was to determine, with a rodent tumor model, if microelectrode measurements of unmodulated tumor oxygenation predict for the avidity of hypoxic markers to tumor tissue. METHODS: The rapidly growing, anaplastic variant of the Dunning rat prostate carcinoma cell line (R3327-AT) was implanted subcutaneously on the upper backs of Fischer X Copenhagen rats. Approximately 100 measurements of PO2 were obtained from tumors of 5-10 g in animals that were restrained and then subjected to different anesthetic procedures. Values of median PO2 (in mm Hg) and percentage of measurements <5 mm Hg obtained from individual tumors were used to define tumor oxygenation status. The radiodiagnostic hypoxic markers beta-D-iodinated azomycin galactopyranoside (IAZGP) and [99mTc]HL-91 were simultaneously administered to 26 animals whose tumor oxygen levels had been measured. Six hours after marker administration, the animals were killed; tumor, blood, and muscle tissues were sampled; and percentage injected dose per gram (%ID/g*), tumor/blood ratio (T/B), and tumor/muscle ratio (T/M) parameters were determined. Parameters of marker avidity to individual tumors were linearly correlated with microelectrode measurements of tumor oxygenation to determine the significance of inverse associations. RESULTS: The median PO2 of 41 tumors varied from 2.0 to 20.9 mm Hg, with an average value of 7.5 +/- 1.4 mm Hg. Six tumors had unusually high values; that is, >10 mm Hg, and when these were excluded from the analysis, the average median PO2 of the remaining 35 was 4.3 +/- 0.7 mm Hg. When electrode measurements of tumor oxygenation were obtained under conditions of halothane anesthesia with the animals breathing O2, carbogen, or air, median PO2 values increased significantly (P = 0.001). When animals were deeply anesthetized by intraperitoneal injection of ketamine-xylazine, median PO2 values were not significantly different (P = 0.13) from those obtained while the animals were restrained and breathing air. There was no inverse correlation of significance between the electrode measurements of median PO2 and the avidity of beta-D-IAZGP nor [99mTc]HL-91 in this tumor model. The range of median PO2 values in these tumors was at least 3 mm Hg, and the range of hypoxic marker avidity was less than twofold. CONCLUSION: These data demonstrate that microelectrode measurements of rat tumor oxygenation did not correlate with the avidity of the two hypoxic markers, at least in this tumor model. The larger dynamic range of tumor oxygen measurements obtained with microelectrodes might be biased to low values by their necrotic fractions, the zones within solid tumors that contain dead cells and debris that will not be labeled by bioreducible hypoxic markers. Hypoxic marker avidity to individual tumors will have to be validated by other assays that can predict for their radiosensitivity.     

Dual-label studies with [125I]-3(R)/[131I]-3(S)-BMIPP show similar metabolism in rat tissues.

Biodistribution studies with the radioiodinated 3(R)- and 3(S)-isomers of 15-(p-iodophenyl)-3-methylpentadecanoic acid (BMIPP) in rats have shown that 3(R)-BMIPP has 20%-25% higher heart uptake than 3(S)-BMIPP (15-180 min). In contrast, the 3(S)-isomer has slightly higher liver uptake, and uptake in other tissues examined is similar. METHODS: To evaluate the possible differences in metabolic fate of the two isomers, a mixture of [125I]-3(R)/[131I]-3(S)-BMIPP was administered to fasted female Fisher rats. Groups of rats (3 per group) were killed 15, 60 and 180 min after administration. Urine and feces were collected from a fourth group (n = 3) over 7 d. Samples of blood, heart, liver, lungs, kidney and urine were Folch extracted. The distributions of 125I and 131I in the organic (lipid), aqueous and pellet samples were determined. The lipid samples as well as the organic fractions from base-hydrolyzed triglyceride (TG) fractions and acid-hydrolyzed urine samples were then analyzed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). RESULTS: The relative distributions of 125I and 131I in the lipid, aqueous and pellet samples were similar for both isomers. Distribution of 125I and 131I in the various components of the lipid extracts observed by TLC (hexane:ether:HOAc, 70:30:1) was also similar, with principal incorporation into the free fatty acid (FFA) and TG pools. HPLC analyses (C18) of the FFA fraction showed similar 125I and 131I profiles, corresponding to BMIPP, and the alpha-methyl-C14 (14-(p-iodophenyl)-3-(R,S)-methyltetradecanoic acid) and C12, C10 and C6 carbon chain-length catabolites. By TLC, radioactive components of 125I and 131I in the urine had the same TLC mobility as hippuric acid. HPLC analyses (C18) of acid-hydrolyzed urine gave a single 125I/131I component with the same relative retention time as 2-(p-iodophenyl)acetic acid, which is the final alpha/beta-oxidative BMIPP catabolite. Unexpectedly, HPLC of lipids from base-hydrolyzed TG from the heart tissue showed 125I/131I components with the same retention times as shorter-chain fatty acids, similar to the FFA fraction, with only low levels of activity detected in BMIPP. CONCLUSION: These results show that 3(R)-BMIPP and 3(S)-BMIPP are metabolized similarly in rat tissues and that higher myocardial extraction observed for 3(R)-BMIPP may reflect differences in the relative membrane transport of the two isomers.     

Preparation of 1-[125I]iodo-[114C]dodecane

1-[¹²⁵I]Iodo-[1¹⁴C]dodecane was prepared using the phosphorus halide/alcohol-reaction. Since the conventional synthesis of iodoalkanes requires apparatus and procedures which do not conform to radioprotection standards, a new apparatus was also constructed. Apparatus and method can also be used for the synthesis of other halogen-alkanes.     

Site-specific incorporation of [125I]iododeoxyuridine into DNA

A procedure for the incorporation of [¹²⁵I]IdU into specific sites in DNA is described. The approach depends upon attachment of radioiododeoxyuridine to a controlled pore glass support which is then used for automated synthesis of an oligomer. The resulting oligomer, containing a terminal 3′[¹²⁵I]iododeoxyuridine, is used as a primer during DNA synthesis catalyzed by the Taq polymerase employing thermal cycling. The product formed includes the radioiodonucleotide at a single internal site determined by the length of the oligomer.     

Subcellular distribution of [125I]iodoaryl beta-methyl fatty acids

Subcellular distribution studies of two aryl branched-chain and one aryl straight-chain iodinated fatty acids were carried out as part of a continuing effort to determine if such acids are metabolized by beta-oxidation. For the omega-iodoaryl fatty acids, a change in subcellular radioactivity location was observed which was chain-length dependent. Chain lengths of 15 carbons, straight and branched, were largely found in the nuclear-membrane fraction, whereas a chain length of 8 carbons was largely located in the cytosol. No unequivocal evidence for metabolic trapping in the mitochondria was observed for omega-iodoaryl branched-chain fatty acids using the centrifugation technique employed in this study.     

Human pancreas scintigraphy using iodine-123-labeled HIPDM and SPECT

The pancreatic affinity of iodine-123-labeled HIPDM (N,N,N'-trimethyl-N'-(2-hydroxy-3-methyl-5-iodobenzyl)-1,3-propane diamine) ([123I]HIPDM) was studied in 18 cases (5 normal volunteers, 7 cases with pancreas cancer, and 6 with chronic pancreatitis). In the normal cases, the pancreas was visualized in the planar images as early as 3 hr, and again at 20 hr postinjection. Single-photon emission computed tomography (SPECT) performed following 3-hr planar scintigraphy, provided excellent pancreas images without an overlap of activity in the liver or spleen. The mean pancreas-to-liver (P/L) ratio was 1.26 +/- 0.22 in normal controls. With the exception of one case of massive calcification in the pancreas, the entire pancreas could be observed in the cases with chronic pancreatitis, but the P/L ratio was 0.74 +/- 0.15, significantly lower than that of normal cases. Defective areas of the distal portion of the pancreas were clearly seen in those with cancer of the pancreas. The results of our study indicate that [123I] HIPDM may have clinical potential as a human pancreas imaging agent.     

In vitro and in vivo characterization of 4-[125I]iododexetimide binding to muscarinic cholinergic receptors in the rat heart

4-[125I]iododexetimide binding to muscarinic cholinergic receptors (mAChR) was evaluated in the rat heart. 4-[125I]iododexetimide displayed high in vitro affinity (Kd = 14.0 nM) for rat myocardial mAChR. In vivo, there was high accumulation of 4-[125I]iododexetimide in the rat atrium and ventricle which could be blocked by approximately 60% by preinjection of atropine. In contrast, accumulation of the radiolabeled stereoisomer, 4-[125I]iodolevetimide, was 63% lower than 4-[125I]iodolevetimide and was not blocked by atropine. The blood clearance of 4-[125I]iododexetimide was rapid, providing heart-to-blood ratios of up to 14:1; however, heart-to-lung and heart-to-liver ratios were below unity. The data indicate that 4-[125I]iododexetimide binds potently to rat mAChR. However, since nonspecific binding is relatively high, it is not clear whether iododexetimide labeled with 123I will be useful in SPECT imaging studies of myocardial mAChR. Further studies in humans are indicated.     

Synthesis and in vitro examination of [124I]-, [125I]- and [131I]-2-(4-iodophenylamino) pyrido[2,3-d]pyrimidin-7-one radiolabeled Abl kinase inhibitors

The pyridopyrimidinones are a potent class of inhibitors of c-Abl kinase and Bcr-Abl kinase, the causative fusion protein in chronic myelogenous leukemia and Src family kinases. A novel method for routine, high-yield no-carrier-added synthesis of [(124)I]-, [(125)I]- and [(131)I]-6-(2,6-dichlorophenyl)-2-(4-iodophenylamino)-8-methyl-8H-pyrido[2,3-d]pyrimidin-7-one has been developed. The 4'-trimethylstannyl- or 4'-tri-n-butylstannyl-pyridopyrimidinone precursors were prepared from the aryl bromide via a palladium-mediated coupling with hexaalkylditin (dioxane/microwave irradiation/10 min at 160 degrees C). The radioiodination of 4'-stannylpyridopyrimidinones was found to optimally occur via an iododestannylation with Na(124)I, Na(125)I or Na(131)I in the presence of an oxidant [30% H(2)O(2)/HOAc (1:3)/10 min] in 79-87% radiochemical yield with >99% radiochemical purity. The total radiosynthesis time was 30 min. The 4-iodophenylpyridopyrimidinone 2 inhibited recombinant Abl kinase activity with an IC(50) of 2.0 nM. Cell proliferation of K562 and A431 cells was inhibited with an IC(50) of 2.0 and 20 nM, respectively. Rapid cellular uptake and equilibrium were observed within 10-15 min using [(131)I]-4-iodophenylpyridopyrimidinone 6c in K562 and A431 cells and demonstrated a 2.8-fold uptake selectivity for the Bcr-Abl-expressing K562 cells at 60 min. These results suggest that pyridopyrimidinone radiotracers may be useful in imaging Abl-, Bcr-Abl- or Src-expressing malignancies.     

Mitochondrial avid radioprobes. Preparation and evaluation of 7'(Z)-[125I]iodorotenone and 7'(Z)-[125I]iodorotenol

The loss of mitochondrial function has been implicated in a number of maladies such as Huntington's disease, Parkinson's disease (PD), cancer and cardiovascular disease. The objective of this research was to develop a radiolabeled mitochondrial probe. Two tracers, 7'-Z-iodorotenol and 7'-Z-iodorotenone, analogs of rotenone a natural product that inhibits Complex I of the mitochondrial electron transport chain, have been labeled with iodine-125 in 45-85% yield in a single step from the corresponding tributylstannyl precursor. In vivo distribution in adult male Sprague-Dawley rats for both compounds showed high accumulation in the heart (1.7-3.7 %ID/g at 1 h), a tissue with high mitochondrial content. Z-Iodorotenol did not washout of most tissues between 1 and 2 h postinjection, whereas Z-iodorotenone showed moderate washout (7-26%) over the same period. By 24 h, there was significant loss of both compounds from most tissues including the heart. Heart-to-blood, -lung and -liver ratios for Z-iodorotenone of 28.9, 10.7 and 2.4, respectively, were two- to fourfold higher than the Z-iodorotenol ratios. Compared to the current clinical perfusion tracers, 99mTc-sestamibi and 99mTc-tetrofosmin, Z-iodorotenone demonstrates similar 1 h heart accumulation and significantly higher heart-to-lung ratio (P<.001). Z-Iodorotenone heart-to-liver ratio is equivalent to 99mTc-sestamibi. 7'-Z-Iodorotenone possesses distribution characteristics of an improved tracer for SPECT perfusion studies.     

[125I] Radioiodinated metaraminol: a new platelet-specific labeling agent

In our search for a platelet-specific labeling agent, metaraminol (MA), a low-toxic pharmaceutical for the treatment of hypotension and cardiogenic shock, attracted our attention. Its active incorporation and accumulation by platelets have been recognized. At first, the preparation of ¹²⁵I radioiodinated metaraminol (¹²⁵I-MA) was carried out using the chloramine-T method. Then, upon the harvest of platelets as platelet-rich plasma (PRP), their labeling with this new radiopharmaceutical was easily performed by incubation for 10 min at 37° C. The cell-labeling efficiency was dependent on cell density, reaching 63.0%±3.1% at 2.4x10⁹ cells/ml. The specific incorporation of ¹²⁵I-MA by an active transport system similar to that of 5-hydroxytryptamine (5-HT) as well as by passive diffusion was demonstrated. In in vitro studies, the unaltered state of ¹²⁵I-MA-labeled platelets with their cellular functions fully retained was estimated. In vivo studies carried out in rabbits with induced thrombi in the femoral artery showed a rather rapid disappearance of the radioactivity from circulating blood, reaching a high thrombus-to-blood activity ratio of 19.8±4.3 within 30 min of the administration of ¹²⁵I-MA-labeled autologous platelets. Thus, with the potential availability of ¹²³I, ¹²³I-MA-labeled platelets appear to be a promising agent for thrombus imaging using single-emission computed tomography (CT) studies.     

Cytotoxicity of [125I]iodoHoechst 33342: contribution of scavengeable effects

PURPOSE: The incubation of the DNA minor-groove binder [125I]iodoHoechst 33342 (125IH) with plasmid DNA leads to the production of one double-strand break (dsb) per decay, both in the presence and absence of dimethylsulfoxide (DMSO). In contrast, when 125I is incorporated into mammalian cell DNA as an iodinated pyrimidine base, DMSO decreases the dsb yield and enhances survival. Because these variations in radioprotective effects may be due either to the location of 125I vis-à-vis the DNA helix or to differences in DNA architecture, the toxicity of 125IH and its modification by DMSO were examined in mammalian cells. METHODS: Uptake and retention of 125IH in V79 cells were measured, and survival was determined after accumulation of 125I decays at 0.3 degrees C +/-10% DMSO. RESULTS: A linear-quadratic survival curve was obtained both in the absence [D37 = 114+/-36 decays/cell, alpha = (5.39 1.17) x10(-3) cell/decay] and presence [D37 = 211+/-65 decays/cell, alpha = (1.27+/-0.52) x10(-3) cell/decay] of DMSO. The dose modification factor for the linear component of the survival curve was 4.25+/-1.97, indicating the predominance of indirect mechanisms. This value is similar to that obtained with DNA-incorporated 125I (4.05+/-1.72) and for the initial slope (alpha) of 137Cs gamma-rays (4.43+/- 1.41). CONCLUSIONS: Cytotoxicity resulting from the decay of the Auger electron emitter 125I in the mammalian cell nucleus is caused mainly by indirect mechanisms.     

In vitro characterization of the influx of 3-[125I]iodo-L-alpha-methyltyrosine and 2-[125I]iodo-L-tyrosine into U266 human myeloma cells: evidence for system T transport

The aim of this study was to investigate the cellular uptake mechanisms responsible for the accumulation of 3-[(125)I]iodo-L-alpha-methyltyrosine ((125)I-3-IMT) and 2-[(125)I]iodo-L-tyrosine ((125)I-2-IT), two radiotracers for metabolic tumor imaging, using single-photon emission tomography, into U266 human myeloma cancer cells. Time course and concentration dependency of (125)I-3-IMT uptake was assessed. Kinetic parameters were calculated using an Eadie Hofstee plot. A set of competitive inhibitors of the main amino acid transport systems was used for the discrimination of the transporters responsible for the uptake of (125)I-3-IMT and (125)I-2-IT. Protein incorporation of both tracers was determined using acid precipitation. The measured maximum velocity for (125)I-3-IMT transport was 4.199 nmol per mg protein 20 s(-1), and the Michaelis constant was 107.9 microM. Addition of 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH), a competitive inhibitor of System L, reduced the influx by 39.0+/-3.3% for (125)I-3-IMT and 66.3+/-0.9% for (125)I-2-IT. The BCH-insensitive influx was further reduced by Tryptophan (Trp) by 43.8+/-3.5% for (125)I-3-IMT and 15.3+/-1.3% for (125)I-2-IT. This suggests involvement of System T transport. We measured <2% of radioactivity in the acid precipitable fractions of both tracers with no increase in time. We conclude that the influx of (125)I-3-IMT and (125)I-2-IT into U266 human myeloma cells is mediated by both System L and System T amino acid transporters. The kinetic parameters suggest that elevated plasma levels of aromatic amino acids will reduce (123)I-3-IMT uptake in myeloma patients. Both tracers do not enter protein synthesis significantly.     

Selective binding of 2-[125I]iodo-nisoxetine to norepinephrine transporters in the brain

A radioiodinated ligand, (R)-N-methyl-(2-[(125)I]iodo-phenoxy)-3-phenylpropylamine, [(125)I]2-INXT, targeting norepinephrine transporters (NET), was successfully prepared. A no-carrier-added product, [(125)I]2-INXT, displayed a saturable binding with a high affinity (K(d)=0.06 nM) in the homogenates prepared from rat cortical tissues as well as from LLC-PK(1) cells expressing NET. A relatively low number of binding sties (B(max)=55 fmol/mg protein) measured with [(125)I]2-INXT in rat cortical homogenates is consistent with the value reported for a known NET ligand, [(3)H]nisoxetine. Competition studies with various compounds on [(125)I]2-INXT binding clearly confirmed the pharmacological specificity and selectivity for NET binding sites. Following a tail-vein injection of [(125)I]2-INXT in rats, a good initial brain uptake was observed (0.56% dose at 2 min) followed by a slow washout from the brain (0.2% remained at 3 hours post-injection). The hypothalamus (a NET-rich region) to striatum (a region devoid of NET) ratio was 1.5 at 3 hours post-i.v. injection. Pretreatment of rats with nisoxetine significantly inhibited the uptake of [(125)I]2-INXT (70-100% inhibition) in locus coeruleus, hypothalamus and raphe nuclei, regions known to have a high density of NET; whereas escitalopram, a serotonin transporter ligand, did not show a similar effect. Ex vivo autoradiography of rat brain sections of [(125)I]2-INXT (at 3 hours after an i.v. injection) displayed an excellent regional brain localization pattern corroborated to the specific NET distribution in the brain. The specific brain localization was significantly reduced by a dose of nisoxetine pretreatment. Taken together, the data suggest that [(123)I]2-INXT may be useful for mapping NET binding sites in the brain.