The presence of lead decreases the availability of meso-2, 3-dimercaptosuccinic acid for analysis in the monobromobimane assay.

meso-2,3-Dimercaptosuccinic acid is a suitable chelating agent for routine pharmacotherapy of lead poisoning in children. Administration of meso-2,3-dimercaptosuccinic acid presumably permits complexation of lead in vivo, allowing excretion through urine or feces. Quantification of the lead is achieved independently from the analysis of meso-2,3-dimercaptosuccinic acid and metabolites from the monobromobimane assay. To date, no direct chemical characterization of the Pb species excreted in urine has been successful. Pharmacokinetic correlation of lead excretion with excretion of meso-2,3-dimercaptosuccinic acid and metabolites has been utilized as an indirect method to draw conclusions regarding the identity of the active chelating agent. In this study, we hypothesized that the Pb-coordinated thiols are not reactive with respect to monobromobimane, and thus, the active chelator contained in the lead complex escapes detection. We performed variations of the assay and found that (1) the fluorescence detector response for the meso-2,3-dimercaptosuccinic acid-monobromobimane adduct was clearly attenuated as a function of added Pb, (2) when meso-2, 3-dimercaptosuccinic acid and monobromobimane were mixed prior to the addition of lead, the lead had no effect on detector response, (3) the addition of dithiothreitol does not affect the ability of Pb to react with meso-2,3-dimercaptosuccinic acid and verifies that oxidation of meso-DMSA had not occurred, and (4) the addition of ethylenediaminetetraacetic acid to the assay reverses the result found in point 1, presumably through trans chelation of the Pb-DMSA complex. Indirect quantification of the Pb-DMSA complexes found in urine might be accomplished through modification of the standard monobromobimane assay for analysis of meso-2,3-dimercaptosuccinic acid.

Regional distribution of 203PbCl2 in the mouse after intravenous injection.

UNLABELLED: Lead is a known neurotoxicant, and concentrations of lead in the CNS after acute exposure to low doses have not been studied in detail. In this investigation, the temporal distribution of lead ([203Pb]), injected intravenously at no-carrier-added (NCA) (0.6-0.7 mumol/kg) and at carrier-added (7.0-62.3 mumol/kg) levels was determined in mice. Concentrations of [203Pb] were measured in major organs and in different regions of the brain. Ex-vivo autoradiographic visualization was used to confirm and extend the brain distribution findings. RESULTS: The highest concentrations of NCA [203Pb] were observed initially in the kidneys (52% of the injected dose (ID)/g at 1 hr) and liver (10.5% ID/g at 30 min). Uptake into bone occurred gradually, reaching 25% ID/g at 24 hr. In accord with previous reports, excretion of the tracer was very slow. Approximately 80% of total ID remained in the body after 24 hr and 68% at 48 hr. Interestingly, in the mouse brain, the highest levels of [203Pb] were noted in the area of the hypothalamus. At all times between 30 min and 16 hr postinjection, and at all Pb dose levels injected, the accumulation of [203Pb] in the hypothalamic region exceeded that in all other brain regions examined. Autoradiography performed at the 16 hr time point confirmed the high uptake and strong retention of [203Pb] by the hypothalamus. These studies afford new insight into the distribution of acutely administered lead in the brain, and may have implications for the understanding of some of the neurotoxic effects of lead.

High-affinity no-carrier-added 99mTc-labeled chemotactic peptides for studies of inflammation in vivo.

Nalpha-for-Nle-Leu-Phe-Nle-Tyr-Lys, a chemotactic peptide that binds with high affinity to the chemoattractant receptor on granulocytes and monocytes, was labeled with 99mTc using the diaminedithiol (DADT) chelating system to coordinate the Tc. 99mTc labeling of the DADT-coupled peptide was accomplished in 84% overall yield (room temperature for 10 min) using [99mTc]glucoheptonate as the donor of prereduced Tc. HPLC analysis showed two major 99mTc-labeled peptide peaks, 99mTc-DADT-Pep-I and 99mTc-DADT-Pep-II, were obtained in a ratio of 1:0.85. Using an iodoacetamide-derivatized gel to remove unlabeled peptide from the 99mTc labeling mixtures, essentially no-carrier-added (nca) high-specific activity 99mTc-labeled chemotactic peptides were obtained. The 99Tc analogues of the peptides were synthesized (72% yield) in a similar fashion and correlated with 99mTc complexes I and II by HPLC. In vitro competitive receptor binding assays of the isolated 99Tc analogues were performed against the tritiated chemotactic peptide [3H]N-for-Met-Leu-Phe ([3H]fMLF) using isolated granulocytes. The 99Tc-derivatized peptides showed similar binding affinities to the chemoattractant receptor as the unlabeled Nalpha-for-Nle-Leu-Phe-Nle-Tyr-Lys. The nca 99mTc-labeled peptides gave high contrast images of experimental inflammation in rabbits without causing neutropenia. Thus, it is feasible to attach the Tc-DADT chelate to low-molecular weight receptor binding chemotactic peptides and retain substantial binding to the receptor. Chemotactic peptides labeled with 99mTc via the DADT ligand system have the potential for imaging focal sites of inflammation without toxic effects, an important consideration in the successful utilization of chemotactic peptide agonists.

Validation and application of a solid phase chemistry method for preparation of high effective specific activity technetium-99m radiopharmaceuticals.

Solid phase chemistry with UltraLink Iodoacetyl gel was investigated as a method for removing excess ligand present in preparations of diaminedithiol (DADT) 99mTc radiopharmaceuticals. The process was optimized and tested under 99mTc labeling conditions with 2,2,4,9,9-pentamethyl-4,7-diaza-1,10-decanedithiol (N-methyl-DADT), 1, and 4-(1-(2'-methoxyphenyl) piperazinylbutyl)-2,2,9,9-tetramethyl-4,7-diaza-1,10 -decanedithiol (DADT-4C-MPP), 2, a serotonin receptor-binding ligand. The results illustrated that this gel removed > or = 99.9% excess ligand used in labeling reactions, and was comparable to the level of purification usually obtained by the more labor intensive method of HPLC.

Preparation of radioactive lead complexes utilizing Chelex methodology.

The use of Chelex resin for the synthesis of radioactive lead complexes has been explored. The process involved immobilization of 203Pb on the resin and subsequent elution of complexed lead by chelating agents. 203Pb complexes derived from meso- and racemic dimercaptosuccinic acid (meso-DMSA, rac-DMSA) were prepared and assessed for stability in vitro.

Rate of binding of various inhibitors at the dopamine transporter in vivo.

The rate of entry of drugs into brain is thought to be a factor in their abuse liability. In this investigation, we have examined the rate of entry and binding at dopamine transporters in mouse striatum for a variety of dopamine transporter inhibitors. The method utilized was based on measuring the displacement of 3H-WIN 35,428 from striatal dopamine transporter sites in vivo at different times. Eleven cocaine analogs (RTI-31, RTI-32, RTI-51, RTI-55, RTI-113, RTI-114, RTI-117, RTI-120, RTI-121, WIN 35,065-2, and WIN 35,428) as well as other dopamine uptake site blockers (bupropion, nomifensine, and methylphenidate) were compared with (-) cocaine for their rates of displacement of 3H-WIN 35,428 binding in vivo. The drugs that displayed the fastest occupancy rates were bupropion, (-) cocaine, nomifensine, and methylphenidate. RTI-51, RTI-121, RTI-114, RTI-117, RTI-120, RTI-32, RTI-55, and RTI-113, showed intermediate rates, whereas RTI-31, WIN 35,065-2, and WIN 35,428 exhibited the slowest rates of displacement. While many of the cocaine analogs have proven to be behaviorally and pharmacologically more potent than (-) cocaine, their rates of entry and binding site occupancy were slower than that for (-) cocaine. Earliest times of transporter occupancy by the different drugs were correlated (although weakly) with their degree of lipophilicity (r = 0.59; P < 0.02). Kinetic effects and metabolism of the compounds could complicate the interpretations of these data.(ABSTRACT TRUNCATED AT 250 WORDS)

Pulmonary accumulation of neutral diamine dithiol complexes of technetium-99m.

Twenty-two neutral, lipid-soluble 99mTc complexes have been synthesized from diamine dithiol (DADT) ligands which vary in alkyl substitution pattern on nitrogen and carbon. The logarithm of the partition coefficients (log PC), as well as the capacity factor k', of the purified complexes increased linearly with molecular weight. The biodistribution of these complexes was determined in normal mice, and several of the complexes selectively accumulated in the lungs as compared to the liver or other organs. Pulmonary accumulation varied greatly with subtle changes in structure, and a 30-fold range of lung uptake (1-31% of the injected dose/organ) was observed for isomeric technetium complexes which have identical molecular weights and similar log PC. Further, a parabolic relationship between lung uptake and log PC was observed for a subset of the complexes which are derived from a homologous series of tetramethyl-DADT ligands. Neutral and lipophilic radiopharmaceuticals labeled with technetium can therefore be developed which exhibit structurally specific uptake in the lung.

Bifunctional chelator for facile preparation of neutral technetium complexes.

The diaminedithiol (DADT) ligand system has proven to be useful as a carrier of technetium-99m in the preparation of a wide variety of site-specific radiopharmaceuticals. To expand the utility of the ligand system, we have designed and synthesized a bifunctional chelating agent based on the ligand system whose adducts generate a neutral technetium complex core and therefore can penetrate intact membranes. We have evaluated both the coupling of the thiolactone reactive moiety of the bifunctional chelate to benzylamine as a model as well as subsequent labeling with technetium-99m. Reaction with benzylamine was complete at room temperature within 2 h, producing the adduct in 74% isolated yield. On coordination of the benzylamine adduct to technetium-99m, one major product was obtained in high yield (> 90%). The product was stable in serum and physiologic saline at 37 degrees C over a 20-h study period. The partition coefficient of the technetium complex was 101 +/- 6.2, indicating that the complex was lipophilic. Biodistribution studies in mice showed that the brain concentration at 5 min postinjection was 0.91 +/- 0.09% injected dose/g indicating that the complex penetrates the intact blood-brain barrier. This is further evidence that the complex is neutral and lipophilic. This bifunctional chelate should facilitate the incorporation of technetium-99m into molecules of biological interest such as drugs, small peptides, and metabolic substrates.

Novel technetium ligands with affinity for the muscarinic cholinergic receptor.

The synthesis and preliminary biological characterization of two isomeric technetium labeled complexes (2,5,5,9-tetramethyl-4,7-diaza-7-(3' (R)-quinuclidinylcarboxymethyl)-2,9-decanedithiolato oxo 99/99mtechnetium(V), [99/99mTc]-1 and [99/99mTc]-2) designed to exhibit affinity to muscarinic cholinergic receptors are described. In vitro binding assays were conducted in mouse brain homogenates (whole brain-cerebellum) at 37 degrees C by the centrifugation method, where non-specific binding was defined by atropine (1 microM). The measured affinity (KD) of [99Tc]-1 for mAChR was 1.9 +/- 0.5 microM (mean +/- SEM; n = 3) and [99Tc]-2 was 4.5 +/- 0.5 microM (mean +/- SEM; n = 3). Scatchard analysis indicated that Bmax values were 10.6 +/- 0.5 and 16.9 +/- 0.5 pmol/mg tissue, respectively. In competition assays, [99Tc]-1 exhibited an apparent affinity (KI) of 16.5 microM (n = 2) against [125I] iododexetimide, whereas [99Tc]-2 exhibited an affinity (KI) of 105 microM. In vivo, 0.3% of the injected dose of [99mTc]-1 and [99mTc]-2 accumulated in the brain at 5 min after injection. These values indicate technetium analogues of neuroreceptor binding ligands can be synthesized and retain some affinity for the receptor.

Synthesis and in vivo evaluation of a 99m/99Tc-DADT-benzovesamicol: a potential marker for cholinergic neurons.

The diaminodithiol (DADT) ligand has been conjugated to the neuromuscular blocking agent benzovesamicol (BVM) in the 5-position. DADT-BVM 1 was synthesized by coupling of 5-aminomethylbenzovesamicol with a BCA thiolactone reagent. 99mTc radiolabeling of 1 with [99mTc]glucoheptonate gave a 4.7:1 mixture of two 99mTc complexes as determined by HPLC. Biodistribution data of the major [99mTc]-1 complex in CD-1 mice (n = 4-5) showed very little uptake and no regional selectivity in the mouse brain. At all time points examined, the lung and liver showed the highest uptake. For whole brain, the % injected dose values were 0.27, 0.12, 0.04 and 0.01% at t = 1, 5, 30 and 240 min. The major [99mTc]-1 product exhibited a log P = 3.13 +/- 0.06 (SD) with an IC50 = 140-280 nM for the corresponding [99Tc]-1 vs (-)-N-[3H]methyl-5-aminobenzovesamicol. The low brain uptake of [99mTc]-1 vs 5-iodobenzovesamicol is attributed to its higher molecular weight (752) and lower binding affinity.

Structure of a neutral N-alkylated diaminedithiol (dadt) 99TcV complex: syn[99TcO(NEt-tmdadt)].

syn-(4-Ethyl-2,9-dimethyl-4,7-diaza-2,9-decanedithiolato)oxo (99Tc) technetium(V), syn-[99TcO(NEt-tmdadt)], [Tc(O)(C12H25N2S2)], Mr = 375, monoclinic, P2(1)/n, a = 9.638(2), b = 14.371(5), c = 11.893(3) A, beta = 100.79(2) degrees, V = 1618.15(75) A3, Z = 4, Dx = 1.54 g cm-3, lambda(Mo K alpha) = 0.71069 A, mu = 11.0 cm-1, F(000) = 776, T = 293 K, R = 0.028, wR = 0.042 for 2788 unique observed reflections with I greater than sigma(I). This study is the first which conclusively establishes the structure of the major complex derived from the reaction of an N-substituted diaminedithiol ligand with reduced pertechnetate. An intramolecular repulsion exists between the ethyl substituent and the oxo-metal core, resulting in a larger-than-expected angle for N(2)-Tc-O, 104.7(1) degrees.

Synthesis of a diaminedithiol bifunctional chelating agent for incorporation of technetium-99m into biomolecules.

The synthesis of a bifunctional chelating agent (BCA), 1, based on the diaminedithiol (DADT) ligand system, is described. The six-step synthetic sequence has been accomplished in 16% overall yield, affording 1, which contains a thiolactone as a reactive moiety, which permits direct coupling to nucleophiles without the formation of byproducts. The reactivity of 1 toward benzylamine and subsequent labeling of the ligand with technetium-99m has been evaluated as a model for preparation of various bioconjugates. Both coupling and exchange labeling occur in high yield under mild conditions, and competition reactions with diethylenetriaminepentaacetic acid (DTPA) indicate the superior stability of the technetium-99m-DADT complex. Preparation of BCA 1 thus provides a new avenue into technetium-labeled radiopharmaceuticals.

99mTc labeling of proteins: initial evaluation of a novel diaminedithiol bifunctional chelating agent.

The initial evaluation of a novel thiolactone bifunctional chelating agent, 1, for facile introduction of the diaminedithiol (DADT) ligand system onto proteins for subsequent labeling with 99mTc under mild conditions is presented. For human serum albumin and a monoclonal anti-colon carcinoma antibody (B72.3), time-dependent coupling was achieved at pH 7-9 at room temperature. The coupled proteins could be labeled in a direct fashion (Sn2+/99mTcO4-) and by exchange labeling (with preformed 99mTc-glucoheptonate) in good to excellent yields at pH 7. In contrast, the unmodified proteins could be labeled only by direct methods with maximum yields of 30%. The labeled, coupled proteins were purified by high performance liquid chromatography and found to be stable in vitro over a 20-h period of study with no evidence for the loss of label. This exceptional stability is due to the chelation of 99mTc by the DADT ligand since 66% (3.5 h) and 47% (20 h) of the label were lost from directly labeled unmodified human serum albumin and the unmodified antibody, respectively. Biodistribution studies in normal mice and rabbits confirmed the stability of the 99mTc-DADT-labeled proteins compared to the 99mTc-labeled unmodified proteins in vivo. Thus, the novel thiolactone 1 is a useful bifunctional chelating agent for introducing the DADT ligand system onto a variety of proteins for subsequent incorporation of technetium under mild conditions such that the resultant labeled products are stable in vitro and in vivo.

Comparison of technetium-99m aminoalkyl diaminodithiol (DADT) analogs as potential brain blood flow imaging agents.

N-ethyl piperidinyl diaminodithiol (NEP-DADT), complexed with 99mTc has been developed as an agent for the measurement of brain blood flow using SPECT. Studies in patients have shown that 99mTc NEP-DADT enters rapidly into the brain, but also clears rapidly (t1/2 = 17 min). In this study nine new aminoalkyl DADT derivatives were synthesized, labeled with 99mTc and tested in mice with the aim of developing an agent with increased retention in the brain. In addition, relationships between chemical properties of the derivatives and their in vivo localization were investigated. The results were as follows: (a) the R-group and its isomeric configuration has a profound influence on the biodistribution; (b) 99mTc aminoalkyl DADT derivatives with apparent pKa values of greater than 6.9 show poor brain uptake (less than 0.40% dose at 5 min); (c) lengthening of the chain between the DADT moiety and the amino-R group from ethyl to hexyl generally increases the apparent pKa and consequently lowers brain uptake; (d) a correlation (r = 0.71) exists between initial brain uptake and the octanol-buffer partition coefficient; (e) 99mTc-4'-methyl NEP-DADT has the highest partition coefficient, relatively high uptake, and longest retention in the mouse brain. This complex has characteristics suited for brain blood flow measurements.

Comparison of 99Tcm complexes (NEP-DADT, ME-NEP-DADT and HMPAO) with 123IAMP for brain SPECT imaging in dogs.

Several new lipophilic 99Tcm complexes have recently been described as alternatives to N-isopropyl (123I) iodoamphetamine (123IAMP) for measurement of regional cerebral blood flow (RCBF). In this study we have compared brain uptake and blood clearance of 99Tcm-N-ethylpiperidine-diamino dithiol (99Tcm-NEP DADT), its 4-methylated derivative (99Tcm-Me-NEP-DADT) and 99Tcm-hexamethyl-propylene-amine-oxime (99Tcm-HMPAO) with that of 123IAMP in two dogs. Single photon emission tomography (SPECT) was employed to measure brain accumulation and retention of the four radiopharmaceuticals. Cerebral uptake of the 99Tcm complexes (0.8-1.1%) was lower than that of 123IAMP (1.6% of the injected dose). There was considerable extracerebral activity in the dog's head, especially in the olfactory and snout regions. Because of slow blood clearance 99Tcm-HMPAO showed high uptake in these regions. Brain uptake of 99Tcm-HMPAO reached a plateau 5 to 10 min after intravenous injection and remained constant for the entire study period (1 h). 99Tcm-NEP-DADT, on the other hand, showed significant clearance from the brain after reaching maximal uptake at 10 to 15 min after injection. However, brain imaging with these agents was possible during the first 20 min. The mechanism of brain uptake, as well as the relationship between brain uptake and RCBF need to be evaluated for each of the four radiopharmaceuticals.

Brain imaging agents: synthesis and characterization of (N-piperidinylethyl) hexamethyl diaminodithiolate oxo technetium(V) complexes.

Two 99mTc complexes of (N-piperidinylethyl) hexamethyl diaminodithiol (NEP-DADT) have shown high brain uptake in rodents and lower primates. One of these 99mTc complexes has given positive images of the brain in man which are qualitatively related to regional brain blood flow (rCBF). In order to determine the structure of these 99mTc products, the corresponding 99Tc(NEP-DADT) complexes were prepared and characterized by HPLC, TLC, fast-atom-bombardment mass spectrometry (FAB MS) and other analytical techniques. These results indicate that the two 99Tc (NEP-DADT) complexes are syn and anti isomers (i.e. one isomer has the N-piperdinylethyl side chain located syn to the technetium oxo core while the other has this side chain located anti to the technetium oxo core).

Design, preparation, and biodistribution of a technetium-99m triaminedithiol complex to assess regional cerebral blood flow.

A new ligand (N-piperidinylethyl-DADT, 5) has been prepared which forms two complexes with 99mTc when stannous chloride is used as a reducing agent for [99mTc] pertechnetate. Biodistribution studies of one of the complexes in mice showed that 2.2% of the injected dose of the tracer was in the brain at 5 min postintravenous injection with 0.53% of the dose remaining in the brain at 30 min postinjection. Brain-to-blood ratios at these times were 5.3 and 3.0, respectively. Biodistribution studies of the other complex showed similar behavior with a slightly lower initial uptake by and faster clearance from the brain. Imaging studies of the more promising of the two complexes were conducted in a monkey and a baboon. In both cases, rapid uptake of the tracer in the brain was observed and clear brain images were obtained. Time-activity curves showed peak uptake in the brain at approximately 5 to 7 min postintravenous injection followed by a plateau of about 11 min. The half-lives for clearance of the tracer from the brains of the monkey and baboon were found to be 63 and 58 min, respectively. These results suggest that this tracer may be useful for brain imaging in humans.