Melanoma-targeting properties of (99m)technetium-labeled cyclic alpha-melanocyte-stimulating hormone peptide analogues.

Preliminary reports have demonstrated that (99m)technetium (Tc)-labeled cyclic [Cys(3,4,10), D-Phe7]alpha-MSH(3-13) (CCMSH) exhibits high tumor uptake and retention values in a murine melanoma mouse model. In this report, the tumor targeting mechanism of 99mTc-CCMSH was studied and compared with four other radiolabeled alpha-melanocyte stimulating hormone (alpha-MSH) peptide analogues: 125I-(Tyr2)-[Nle4, D-Phe7]alpha-MSH [125I-(Tyr2)-NDP]; 99mTc-CGCG-NDP; 99mTc-Gly11-CCMSH; and 99mTc-Nle11-CCMSH. In vitro receptor binding, internalization, and cellular retention of radiolabeled alpha-MSH analogues in B16/F1 murine cell line demonstrated that >70% of the receptor-bound radiolabeled analogues were internalized together with the receptor. Ninety % of the internalized 125I-(Tyr2)-NDP, whereas only 36% of internalized 99mTc-CCMSH, was released from the cells into the medium during a 4-h incubation at 37 degrees C. Two mouse models, C57 mice and severe combined immunodeficient (Scid) mice, inoculated s.c. with B16/F1 murine and TXM-13 human melanoma cells were used for the in vivo studies. Tumor uptake values of 11.32 and 2.39 [% injected dose (ID)/g] for 99mTc-CCMSH at 4 h after injection, resulted in an uptake ratio of tumor:blood of 39.0 and 11.5 in murine melanoma-C57 and human melanoma-Scid mouse models, respectively. Two strategies for decreasing the nonspecific kidney uptake of 99mTc-CCMSH, substitution of Lys11 in CCMSH with Gly11 or Nle11, and lysine coinjection, were evaluated. The biodistribution data for the modified peptides showed that Lys11 replacement dramatically decreased the kidney uptake, whereas the tumor uptakes of 99mTc-Nle11- and 99mTc-Gly11-CCMSH were significantly lower than that of 99mTc-CCMSH. Lysine coinjection significantly decreased the kidney uptake (e.g., from 14.6% ID/g to 4.5% ID/g at 4 h after injection in murine melanoma-C57 mice) without significantly changing the value of tumor uptake of 99mTc-CCMSH. In conclusion, the compact cyclic structure of 99mTc-CCMSH, its resistance to degradation, and its enhanced intracellular retention are the major contributing factors to the superior in vivo tumor targeting properties of 99mTc-CCMSH. Lys11 residue in 99mTc-CCMSH is critical to the tumor targeting in vivo, and lysine coinjection rather than lysine replacement can significantly decrease the nonspecific renal radioactivity accumulation without impeding the high melanoma-targeting properties of 99Tc-CCMSH. The metal-cyclized CCMSH molecule displays excellent potential for the development of melanoma-specific diagnostic and therapeutic agents.

The single-pass cerebral extraction and capillary permeability-surface area product of several putative cerebral blood flow imaging agents.

We have determined cerebral blood flow (CBF) and the single-pass cerebral extraction (E) of several putative agents for external imaging of CBF. Simultaneous measurements of blood flow and extraction were performed in 106 rats. For all agents, comparison of linear and exponential regressions of E on CBF indicates that this relationship can be described as linear over the range of flows studied. Analysis of covariance indicates that the extraction of 123I-IMP, 67Cu-PTSM and 99mTc-HMPAO is higher than that of 99mTc-Cl(DMG)3(2MP) and 99mTc-ECD, particularly at flows above the normal range. Accordingly, for 123I-IMP, 67Cu-PTSM and 99mTc-HMPAO, the slope of the linear regression equation for the relationship between brain capillary permeability surface area product (PS) and CBF is higher than that for 99mTc-Cl(DMG)(3)2MP and 99mTc-ECD. PS varies as a linear function of CBF over the range of flows studied. At a CBF level that corresponds to normal regional CBF for human cortex, 0.5 ml/g/min, all the agents have a single-pass extraction of approximately 70% or greater. While all the agents detected changes in CBF in the normal to ischemic range, at higher flows 123I-IMP, 67Cu-PTSM and 99mTc-HMPAO showed substantially greater fidelity to true CBF than 99mTc-Cl(DMG)(3)2MP and 99mTc-ECD.

Evaluation of an (111)In-DOTA-rhenium cyclized alpha-MSH analog: a novel cyclic-peptide analog with improved tumor-targeting properties.

UNLABELLED: The aim of this study was to examine the effect of rhenium-mediated peptide cyclization on melanoma targeting, biodistribution, and clearance kinetics of the alpha-melanocyte-stimulating hormone (alpha-MSH) analog 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) coupled ReO-cyclized [Cys(3,4,10),D-Phe(7)]alpha-MSH(3-13) (DOTA-ReCCMSH). METHODS: DOTA-ReCCMSH was compared with its reduced nonmetalated linear homolog, DOTA-CCMSH, and an analog in which rhenium cyclization was replaced by disulfide bond cyclization, DOTA-[Cys(4,10),D-Phe(7)]alpha-MSH(4-13) (CMSH). DOTA was also conjugated to the amino terminus of one of the highest-affinity alpha-MSH receptor-binding peptides, [Nle(4),D-Phe(7)]alpha-MSH (NDP), as a linear peptide standard. The DOTA-conjugated alpha-MSH analogs were radiolabeled with (111)In and examined for their in vitro receptor-binding affinity with B16/F1 murine melanoma cells, and their in vivo biodistribution properties were evaluated and compared in melanoma tumor-bearing C57 mice. RESULTS: The tumor uptake values of (111)In-DOTA-ReCCMSH were significantly higher than those of the other closely related (111)In-DOTA-alpha-MSH conjugates. Even at 24 h after injection, a comparison of the tumor uptake values for (111)In-DOTA-coupled ReCCMSH (4.86 +/- 1.52 percentage injected dose [%ID]/g), CCMSH (1.91 +/- 0.56 %ID/g), CMSH (3.09 +/- 0.32 %ID/g), and NDP (2.47 +/- 0.79 %ID/g) highlighted the high tumor retention property of ReCCMSH. Rhenium-coordinated cyclization resulted in less renal radioactivity accumulation of (111)In-DOTA-ReCCMSH (8.98 +/- 0.82 %ID/g) than of (111)In-DOTA-CCMSH (63.2 +/- 15.6 %ID/g), (111)In-DOTA-CMSH (38.4 +/- 3.6 %ID/g), and (111)In-DOTA-NDP (12.0 +/- 1.96 %ID/g) at 2 h after injection and significantly increased its clearance into the urine (92 %ID at 2 h after injection). A high radioactivity uptake ratio of tumor to normal tissue was obtained for (111)In-DOTA-ReCCMSH (e.g., 489, 159, 100, and 49 for blood, muscle, lung, and liver, respectively, at 4 h after injection). CONCLUSION: The novel ReO-coordinated cyclic structure of DOTA-ReCCMSH contributes significantly to its enhanced tumor-targeting and renal clearance properties and makes DOTAReCCMSH an excellent candidate for melanoma radiodetection and radiotherapy.

Radiochemical studies of 99mTc complexes of modified cysteine ligands and bifunctional chelating agents.

The synthesis of four novel ligands using the amino-acid cysteine and its ethyl carboxylate derivative is described. The synthetic method involves a two-step procedure, wherein the intermediate Schiff base formed by the condensation of the amino group of the cysteine substrate and salicylaldehyde is reduced to give the target ligands. The intermediates and the final products were characterized by high resolution nuclear magnetic resonance spectroscopy. Complexation studies of the ligands with 99mTc were optimized using stannous tartrate as the reducing agent under varying reaction conditions. The complexes were characterized using standard quality control techniques such as thin layer chromatography, paper electrophoresis, and paper chromatography. Lipophilicities of the complexes were estimated by solvent extraction into chloroform. Substantial changes in net charge and lipophilicity of the 99mTc complexes were observed on substituting the carboxylic acid functionality in ligands I and II with the ethyl carboxylate groups (ligands II and IV). All the ligands formed 99mTc complexes in high yield. Whereas the complexes with ligands I and II were observed to be hydrophilic in nature and not extractable into CHCl3, ligands III and IV resulted in neutral and lipophilic 99mTc complexes. The 99mTc complex with ligand II was not stable and on storage formed a hydrophilic and nonextractable species. The biodistribution of the complexes of ligands I and II showed that they cleared predominantly through the kidneys, whereas the complexes with ligands III and IV were excreted primarily through the hepatobiliary system. No significant brain uptake was observed with the 99mTc complexes with ligands III and IV despite their favorable properties of neutrality, lipophilicity, and conversion into a hydrophilic species. These ligands offer potential for use as bifunctional chelating agents.

In vivo evaluation of 99mTc/188Re-labeled linear alpha-melanocyte stimulating hormone analogs for specific melanoma targeting.

Radiolabeled alpha-melanocyte stimulating hormone (alpha-MSH) analogs were examined in melanoma-bearing mice to determine the effects of peptide length, structure, and radiometal chelation chemistry on tumor targeting and in vivo biodistribution. The linear alpha-MSH analogs [Nle4,D-Phe7]alpha-MSH (NDPMSH) and [D-Phe7]alpha-MSH(5-10) (DPMSH) were radiolabeled with 99mTc and 188Re via the addition of tetrafluorophenyl mercapto-acetylglycylglycyl-gamma-aminobutyrate (MAG2) or tetrapeptide Ac-Cys-Gly-Cys-Gly (CGCG) chelation moieties. 125I-Tyr2-NDPMSH was obtained by direct iodination of the Tyr2 residue. Tumor uptake of 99mTc-labeled CGCG- and MAG2-NDPMSH analogs at 30 min postinjection were 6.52 +/- 1.11 %ID/g and 4.17 +/- 1.34 %ID/g, respectively, resulting in a significantly higher tumor-to-blood uptake ratio than that of 125I-NDPMSH or a shorter alpha-MSH analog, 99mTc-CGCG-DPMSH. The combination of radiolabeling efficacy and in vivo tumor uptake highlights the potential of 99mTc-CGCG-NDPMSH as a melanoma imaging agent.

Rhodium-105 tetrathioether complexes: radiochemistry and initial biological evaluation.

105Rhodium(III) complexes with three different acyclic tetrathioether ligands containing pendant carboxylic acid groups have been synthesized and characterized. The complexes were evaluated for stability under physiological conditions and the most promising complexes were evaluated in vivo in normal mice. The primary route of clearance for these complexes was the renal/urinary system, consistent with the presence of pendant carboxylate groups. The results indicate that the cis-[Rh(III)Cl2(2,5,8,11-tetrathiadodecane-1,12-dicarboxylic acid)]+ complex shows the most promising in vivo characteristics on which to base a potential therapeutic radiopharmaceutical.

[99mTc] Teboroxime and [99mTc]Cl(DMG)3B2MP: binding characteristics andmetabolism of two [99mTc]BATOs in blood and tissues.

Studies were performed in vitro and in vivo to evaluate the binding properties and metabolism of [99mTc]Cl(CDO)3BMe (Teboroxime) and [99mTc]Cl(DMG)3B2MP in blood and target tissues of rats. Both radiopharmaceuticals displayed rapid binding (within 1-3 min) with high affinity to plasma proteins and blood cells. The amounts of radioactivity associated with blood components became progressively greater with time of exposure to either compound. There was a higher proportion of the radiopharmaceuticals associated with blood components during in vivo conditions, likely due, at least in part, to clearance of the free fraction from the plasma pool. Exposure of both compounds to blood results in axial ligand exchange of the chloro atom to a hydroxyl. The results suggest that it is the free species that is extracted primarily by tissues.

Technetium-99m complexes of polydentate amine-pyrrole and amine-thiophene ligands.

Novel polydentate amine-pyrrole and amine-thiophene ligands were synthesized and characterized by 1H and 13C NMR spectroscopy. Radiochemical studies with 99mTc were carried out at 0.1-100 microM of technetium. Complexation yields were estimated from thin layer chromatography (TLC), paper electrophoresis, and solvent extraction studies. The 99mTc complexes formed were found to be neutral and lipophilic. Complexes with the corresponding imine-ligands were formed in lower yields. Biodistribution studies of the 99mTc complexes of these ligands showed no significant uptake in brain or heart, and the clearance was mainly through the hepatobiliary system.

Synthesis of heptadentate (N4O3) amine-phenol ligands and radiochemical studies with technetium-99m.

Heptadentate amine-phenol ligands with N4O3 donor atoms for coordination were synthesized by condensing tris(2-aminoethyl)amine with salicylaldehyde or acetophenone and reducing the Schiff bases formed with NaBH4. The ligands were characterized by 1H and 13C nuclear magnetic resonance spectroscopy. Radiochemical studies were carried out with no-carrier-added 99mTc and 99mTc spiked with 0.1-100 microM of 99Tc. Complexation yields were estimated from thin layer chromatography, paper electrophoresis, and solvent extraction studies. 99mTc complexes were formed in yields better than 90% with the amine-phenol ligands. The complexes were found to be neutral and lipophilic. Biodistribution studies of the 99mTc complexes showed that clearance was mainly through the hepatobiliary system.

Boronic acid adducts of technetium dioxime (BATO) complexes derived from quinuclidine benzilate (QNB) boronic acid stereoisomers: syntheses and studies of their binding to the muscarinic acetylcholine receptor.

We have investigated the possibility of using BATO complexes derivatized with the muscarinic acetylcholine receptor (mAChR) antagonist, quinuclidinyl benzilate (QNB), for mAChR imaging. The BATO complexes, TcCl(DMG)3B-QNB, were prepared using QNB derivatives containing a 4'-boronic acid substituent on one of the benzilic benzene rings (QNB-boronic acid). The QNB-boronic acid molecule has two chiral centers, and all four QNB-BATO stereoisomers were made and evaluated. When studied using in vitro receptor binding assays based on tissue from rat brain caudate-putamen (which contains primarily M1 and M4 mAChR) and rat heart (M2 mAChR), the QNB-boronic acid stereoisomers had binding affinities (KA) in the range 2 x 10(5)-1 x 10(8), at least 10-fold lower than the KA for QNB (ca 2 x 10(9)). The stereochemistry of both centers had some influence on the affinity constant. When the TcCl(DMG)3B-QNB complexes were studied, none of the stereoisomeric complexes displayed measurable specific binding (KA < 10(6)), but all showed high non-specific binding. In vitro autoradiography with rat brain slices confirmed the absence of specific binding in these tracers. In vivo, the 99mTcCl(DMG)3B-QNB complexes displayed minimal brain uptake, and modest heart uptake; the latter was unlikely to be related to uptake by the mAChR. In light of these findings, we conclude that the interaction between the TcCl(DMG)3B-QNB complexes and biological membranes is dominated by the hydrophobicity of the BATO moiety. The TcCl(DMG)3B-QNB complexes, therefore, have little potential for mAChR imaging.

An Rh-105 complex of tetrathiacyclohexadecane diol with potential for formulating bifunctional chelates.

1,5,9,13-Tetrathiacyclohexane-3,11-diol (16S4-diol), a sulfur crown ether analog, was studied as a potential chelating agent to complex no-carrier-added (NCA) grade 105Rh(III) in high yield at low ligand concentrations. trans-[RhCl2(16S4-diol)]chi (chi = Cl, PF6) was prepared using nonradioactive RhCl3.3H2O and characterized by UV-Vis, nuclear magnetic resonance (NMR) and X-ray crystallography. It was shown to have a +1 charge with the Rh(III) metal center coordinated to the four S atoms equatorially and two Cl atoms in trans axial positions. The 105Rh-16S4-diol complex prepared with NCA 105Rh(III)-chloride reagent was found to exhibit identical chromatographic properties as trans-[Rh(III)Cl2(16S4-diol)]+ (including silica and C-18 thin-layer chromatography [TLC] and electrophoresis). The preparation of 105Rh-16S4-diol complex formation optimized for conditions of pH, temperature, time, % ethanol and quantity of 16S4-diol resulted in yields > 90%. Very low quantities of 16S4-diol (3 nmol) complex NCA 105Rh(III) under relatively mild reaction conditions (heating at 64 degrees C for 90 min) in the presence of ethanol (10%), yielded the high specific activity 105Rh-16S4-diol complex as a single cationic species. The 105Rh-16S4-diol complex was shown to be stable for > or = 4 days in physiological buffers at room temperature and in human serum at 37 degrees C.

BATO complexes derived from dimethoxy dioximes: synthesis, characterization and biodistribution.

To prepare less lipophilic BATO complexes, two new methoxy-substituted dioximes were synthesized: cis-4,5-dimethoxycyclohexane-1,2-dione dioxime (DMCDO) and 1,4-dimethoxybutane-2,3-dione dioxime (DMDMG). 99mTcCl(DMCDO)3BMe (BMe = methylboronic acid) was prepared and characterized. Reversed-phase HPLC analyses of 99mTcCl(DMCDO)3BMe and 99mTcCl(DMCDO)3-p-TBA (p-TBA = p - tolylboronic acid) indicated that both of these complexes were mixtures of four enantiomeric pairs of diastereomers. Attempted preparation of a BATO complex from DMDMG gave a mixture of products. In rats, 99mTcCl(DMCDO)3BMe displayed more rapid liver and renal clearance than 99mTcCl(CDO)3BMe, but 99mTcCl(DMCDO)3BMe and 99mTcCl(DMCDO)3-p-TBA displayed low uptake in both heart and brain.

Development of an in vitro model for assessing the in vivo stability of lanthanide chelates.

An in vitro model was developed to evaluate the in vivo stability of lanthanide polyaminocarboxylate complexes. The ligand-to-metal ratios for the chelates EDTA, CDTA, DTPA, MA-DTPA (monoamide-DTPA) and DOTA with the lanthanides lanthanum, samarium, and lutetium were optimized to achieve > or = 98% complexation yield for the resultant radiolanthanide complexes. The exchange of the radiolanthanides from their EDTA, CDTA, DTPA, MA-DTPA and DOTA complexes with Ca(2+) was determined by in vitro adsorption and in vitro column studies using hydroxyapatite (HA), an in vitro bone model. In vitro serum stability of these radiolanthanide complexes was used as an additional indicator of in vivo stability, although the mechanism of instability in serum will be different than with bone. The in vitro studies were consistent with the expected findings that the smallest lanthanide (Lu) formed the most stable complexes. In vivo studies were done to validate the in vitro model. Biodistribution studies in normal CF-1 mice showed that in vivo stability of the complex (i.e., the more lanthanide remaining in complex form) could be assessed by a combination of the urinary, bone and liver uptake. For example, biodistribution studies demonstrate that high urinary excretion correlated with complex stability, while high liver plus bone uptake correlated with complex instability. The urinary excretion of the EDTA complexes decreased from (177)Lu to (140)La indicating a loss in stability in the direction of (140)La, consistent with the in vitro studies. The more stable a lanthanide complex is, the lower its exchange with HA in vitro will be, and the lower its combined bone plus liver uptake and higher its urinary excretion will be in vivo. This investigation indicates that the in vivo stability can be determined by a screening method that measures the degree of exchange from the lanthanide chelate with hydroxyapatite (HA) and its serum stability.

Current and potential therapeutic uses of lanthanide radioisotopes.

In the last 25 years, diagnostic nuclear medicine has come to depend on the versatile chemistry of a single radioisotope, technetium-99m (Tc-99m). Different chelating molecules can be used to guide Tc-99m through various physiological pathways in the body to gain information about disease states. No single radioisotope similarly dominates therapeutic applications. In the field of radioisotope therapy, much discussion and debate have focused on what radioisotope might be "ideal" for treatment of malignant tumors. The ideal may not be a single radioisotope, but rather the class of very closely related radiolanthanides and lanthanide-like radioisotopes. These radioisotopes possess strikingly similar chemistries and thus all may be conjugated to biomolecules using a single chelate, the DOTA moiety (and its chemical analogs). They also provide a wide range of physical characteristics, such as half-lives and beta energies, that can be chosen to match the biological properties of the conjugated biomolecule and the malignant tumor. Thus, the radiolanthanide-DOTA bioconjugate model provides a set of physically diverse, but chemically very similar, therapeutic radiopharmaceutical agents, the individual members of which can be tailored to treat specific types of cancers.

Selenium-72 formation via nat Br(p,x) induced by 100 MeV protons: steps towards a novel 72Se/72As generator system.

Selenium-72 production by the proton bombardment of a natural NaBr target has been successfully demonstrated at the Los Alamos National Laboratory Isotope Production Facility (LANL-IPF). Arsenic-72 (half life 26 h) is a medium-lived positron emitting radionuclide with the major advantage of being formed as the daughter of another "generator" radioisotope (Se-72, 8.5 d). A (72)Se/(72)As generator would be the preferred mechanism for clinical utilization of (72)As for positron emission tomography (PET). No portable (72)Se/(72)As generator system has been demonstrated for convenient, repeated (72)As elution ("milking"). In this work, we describe (72)Se production and recovery from irradiated NaBr targets using a 100 MeV proton beam. We also introduce an (72)As generator principle based on (72)Se chelation followed by liquid-liquid extraction, which will be transferred to a solid-phase sorption/elution system.

Radiometal complexes: characterization and relevant in vitro studies.

Radiometals are, and will continue to be, very important to diagnostic and therapeutic nuclear medicine applications as they predominantly possess the most suitable nuclear properties for these types of applications. This article attempts to give the reader an overview of key aspects that need to be considered in the design and synthesis of a radiopharmaceutical using the commonly known and employed radionuclides, such as technetium, rhenium, the lanthanides and copper. While it is important to understand each radiometal ion has its own specific coordination chemistry requirements, there are several issues that are critical to all radiometal ions for their incorporation into a radiopharmaceutical. 1) The route of production and the presence of long lived contaminating radionuclides and or of naturally occurring metal ions that will interfere with the efficient and optimum radiolabelling of their ligand of choice as well as the final specific activity of the product; 2) the significant differences between the chemistry at the macroscopic (mM and higher concentrations) and radiotracer levels (uM and lower concentrations for the high specific activity radionuclides); 3) the rate of complexation and of dissociation of the radiometal ion vs the competing reaction of radiometal hydrolysis; 4) natural biological pathway of the radio-metal ion and therefore the design of the appropriate and relevant in vitro tests to assess the stability of the radiometal complex. These are a selection of critical factors that need to be considered in the design of a successful radiopharmaceutical, whether it is used for imaging or therapy. However, one should consider tailoring their investigations to suit the radiometal under investigation, and to be mindful where the technology is to be applied (e.g. imaging organs or disease).