Synthesis and biological evaluation of new chlorin derivatives as potential photosensitizers for photodynamic therapy

Three new water-soluble chlorin derivatives 3, 5 and 8 for potential use as photosensitizers in photodynamic therapy (PDT) for cancer were synthesized from photoprotoporphyrin IX dimethyl ester (1). The in vivo biodistribution and clearance of chlorin derivatives 3, 5 and 8 were investigated in tumor-bearing mice. Iminodiacetic acid derivative 8 showed the greatest tumor-selective accumulation among the new chlorin derivatives with maximum accumulation in tumor tissue at 3 h after intravenous injection and rapid clearance from normal tissues within 24 h after injection. The in vivo therapeutic efficacy of PDT using 8 was evaluated by measuring tumor growth rates in tumor-bearing mice with 660 nm light-emitting diode irradiation at 3 h after injection of 8. Tumor growth was significantly inhibited by PDT using 8. These results indicate that iminodiacetic acid derivative 8 is useful as a new photosensitizer to overcome the disadvantages of photosensitizers that are currently in clinical use.     

Radioiodinated ligands for the estrogen receptor: Tissue distribution of 17α-[125I]iodovinylestradiol derivatives in normal and tumor-bearing adult female rats

A series of three ¹²⁵I-labeled 17α-iodovinylestradiol derivatives previously demonstrated to have a selectivity for estrogen receptor tissues in immature female rats were selected for further evaluation in adult female rats. In normal adult female rats, the iodovinyl analogs of moxestrol (IVβME₂) and moxestrol-3-O methyl ether (IVβME₂-3-OMe) demonstrated high uterine uptake (0.296–0.437 and 0.135–0.199%ID-kg/g) and selectivity (10–18:1) over the 6 h time period. Subsequent evaluation in two tumor models indicated that [¹²⁵I]VβME₂ also possessed the highest tumor uptake and selectivity in the adult female rats bearing the estrogen responsive 9,10-dimethyl-1,2-benzanthracene(DMBA)-induced mammary tumors and that this was receptor mediated. An estrogen independent tumor, the transplanted Walker 256 mammary adenocarcinoma, showed no selectivity of radioligand uptake compared with nontarget tissues. The results suggest the applicability of this agent to the in vivo detection and characterization of estrogen responsive tumors in man.     

Synthesis and characterization of 64Cu- and Cy5.5-labeled tetraiodothyroacetic acid derivatives for tumor angiogenesis imaging

It was previously reported that tetraiodothyroacetic acid (tetrac) inhibits angiogenesis by binding to the cell surface receptor for thyroid hormone on integrin α_Vβ₃. Therefore, we synthesized and evaluated two ⁶⁴Cu-labeled tetrac derivatives and a Cy5.5-labeled tetrac derivative for tumor angiogenesis imaging. Tetrac was structurally modified to conjugate with 1,4,7,10-tetraazacyclododecane-N,N′,N″,N″′-tetraacetic acid (DOTA) via its hydroxy or carboxylic acid end, and the resulting DOTA-conjugated tetrac derivatives were then labeled with ⁶⁴Cu. Tetrac was also conjugated with Cy5.5 via its carboxylic acid end. All three tetrac derivatives (1–3) exhibited greater inhibitory activity than tetrac against endothelial cell tube formation. The U87MG cell binding of [⁶⁴Cu]2 showed a time-dependent increase over 24 h and it was inhibited by 38% at 4 h in the presence of tetrac, indicating specificity of [⁶⁴Cu]2 to the thyroid hormone receptor site on integrin α_Vβ₃. Positron emission tomography (PET) images of U87MG tumor-bearing mice injected with [⁶⁴Cu]1 and [⁶⁴Cu]2 revealed that high radioactivity accumulated in the tumors, and that the tumor uptake and tumor-to-nontarget uptake ratio were higher in small tumors than in large tumors. In addition, the Cy5.5-labeled tetrac derivative (3) displayed a strong near-infrared (NIR) signal in the tumors. Taken together, these results suggest that these ligands hold promise as imaging agents for visualization of tumor angiogenesis.     

Uptake of biotin by human hepatoma cell line,Hep G2: A carrier-mediated process similar to that of normal liver

Little is known about the cellular and molecular regulation of the uptake process of the water-soluble vitamin biotin into liver cells, the major site of biotin utilization and metabolism. Such studies are best done using a highly viable and homogeneous cellular system that allows examination of prolonged exposure to an agent(s) or a particular condition(s) on the uptake process. Isolated hepatocytes when maintained in primary culture lose their ability to transport biotin by the specialized carrier system. The aim of the present study was, therefore, to examine the mechanism(s) of biotin uptake by the cultured human-derived liver cells, Hep G₂. Uptake to biotin by Hep G₂ cells was appreciable and linear for up to 10 min of incubation. The uptake process was Na⁺ gradient-dependent as indicated by studies of Na⁺ replacement and pretreatment of cells with gramicidin and ouabain. Biotin uptake was also dependent on both incubation temperature and intracellular energy. Unlabeled biotin and the structural analogs with free carboxyl groups (thioctic acid, desthiobiotin) but not those with blocked carboxyl group (biocytin, biotin methyl ester, and thioctic amide) caused significant inhibition of ³H-biotin uptake at 37°C but not 4°C. Initial rate of biotin uptake was saturable as a function of concentration at 37°C but was lower and linear at 4°C. Pretreatment of Hep G₂ cells with sulfhydryl group inhibitors (e.g., p-chloromer-curibenzene sulfonate) led to a significant inhibition in biotin uptake; this inhibition was effectively reversed by reducing agents (e.g., dithiothreitol). Biotin uptake was also inhibited by the membrane transport inhibitors probenecid (noncompetitively), DIDS and furosemide but not by amiloride. Pretreatment of Hep G₂ cells with valinomycin did not alter biotin uptake. The stoichiometric ratio of biotin to Na⁺ uptake in Hep G₂ cells was also determined and found to be 1:1. These findings demonstrate that biotin uptake by these cultured liver cells is mediated through a specialized carrier system that is dependent on Na⁺-gradient, temperature, and energy and transports the vitamin by an electroneutral process. These findings are similar to those seen with native liver tissue preparations and demonstrate the suitability of Hep G₂ cells for in-depth investigations of the cellular and molecular regulation of biotin uptake by the liver. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work, and as such, is in the public domain in the United State of America

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Fluorescent fructose derivatives for imaging breast cancer cells

Breast cancer cells are known to overexpress Glut5, a sugar transporter responsible for the transfer of fructose across the cell membrane. Since Glut5 transporter is not significantly expressed in normal breast cells, fructose uptake can potentially be used to differentiate between normal and cancerous cells. Fructose was labeled with two fluorophores at the C-1 position: 7-nitro-1,2,3-benzadiazole (NBD) and Cy5.5. The labeling site was chosen on the basis of the presence and substrate specificity of the key proteins involved in the first steps of fructose metabolism. Using fluorescence microscopy, the uptake of the probes was studied in three breast cancer cell lines: MCF 7, MDA-MB-435, and MDA-MB-231. Both fluorescent fructose derivatives showed a very good uptake in all tested cell lines. The level of uptake was comparable to that of the corresponding glucose analogs, 2-NBDG and Cy5.5-DG. Significant uptake of 1-NBDF derivative was not observed in cells lacking Glut5 transporter, while the uptake of the 1-Cy5.5-DF derivative was independent of the presence of a fructose-specific transporter. While 1-NBDF showed Glut5-specific accumulation, the coupling of a large fluorophore such as Cy5.5 likely introduces big structural and electronic changes, leading to a fructose derivative that does not accurately describe the uptake of fructose in cells.     

A novel concept of radiosynthesis of a 99mTc-labeled dimeric RGD peptide as a potential radiotracer for tumor imaging

Radiolabeled Arg-Gly-Asp (RGD) peptides are promising agents for non invasive imaging of α_vβ₃ expression in malignant tumors. The integrin α_vβ₃ binding affinity and consequent tumor uptake could be improved when a dimeric RGD peptide is used as the targeting moiety instead of a monomer. Towards this, a novel approach was envisaged to synthesize a ^99mTc labeled dimeric RGD derivative using a RGD monomer and [^99mTcN]⁺² intermediate. The dithiocarbamate derivative of cyclic RGD peptide G₃-c(RGDfK) (G₃ = Gly-Gly-Gly, f = Phe, K = Lys) was synthesized and radiolabeled with [^99mTcN]⁺² intermediate to form the ^99mTcN-[G₃-c(RGDfK)]₂ complex in high yield (～98%). Biodistribution studies carried out in C57/BL6 mice bearing melanoma tumors showed good tumor uptake [4.61 ± 0.04% IA/g at 30 min post-injection] with fast clearance of the activity from non-target organs/tissue. Scintigraphic imaging studies showed visible accumulation of activity in the tumor with appreciable target to background ratio.     

A novel d-glucose derivative radiolabeled with technetium-99m: Synthesis,biodistribution studies and scintigraphic images in an experimental model of Ehrlich tumor

A d-glucose-MAG₃ derivative was successfully synthesized and radiolabeled in high labeling yield. Biodistribution studies and scintigraphic images in Ehrlich tumor-bearing mice were performed. This compound showed high accumulation in tumor tissue with high tumor-to-muscle ratio. Thus, d-glucose-MAG₃ could be considered as agent for tumor diagnosis.     

Solute accumulation and electrical membrane potential in Agrobacterium tumefaciens-induced crown galls in Kalanchoë daigremontiana leaves

Leaves of Kalanchoë daigremontiana were infected with Agrobacterium tumefaciens strain C58 in order to determine the relative contributions of energy-dependent ion uptake to the observed higher solute concentrations in crown-gall tissue compared with unaffected tissue. The tumor tissue exhibited the following characteristics with respect to unaffected tissue: the content of most solutes was much higher: Na⁺, K⁺, Cl^–, soluble P_i, total N, protein, soluble amino acids, and soluble sugars. Yet NH⁻₄ and starch content was less. Malate did not fluctuate in a typical CAM rhythm and was lower. The respiration rate on a cytoplasmic volume basis was similar. Photosynthetic rates were much lower. The cytoplasmic ATP concentration was even less, while that of NAD(P)H was higher. Electrical membrane potential was lower (− 184 mV tumor vs. − 223 mV control) and was composed of a higher energy-independent component (− 125 vs. − 98 mV) and a smaller energy-dependent component (− 59 vs. − 125 mV). The response of the membrane potential upon addition of neutral, acidic and basic amino acids including the opines octopine and nopaline was similar in both tissue types. It is suggested that the stronger accumulation of solutes in tumor vs. mesophyll cells cannot be due to thermodynamically more favourable conditions at the plasmalemma, but more probably to a hormone-regulated solute transport across the tonoplast.     

Simulation of Tumor-Specific Delivery of Radioligand Comparison of One Step,Two Step,and Genetic Transduction Systems

A mathematical model simulation was performed to estimate the amount of radioactivity in plasma, normal tissues, and tumor tissue through three delivery approaches: one step radiolabeled monoclonal antibody (MAb) CC49 i.v. bolus injection, two step method with biotin conjugated CC49 i.v. bolus injection followed 72 hours later by i.v. bolus radiolabeled streptavidin injection, and gene therapy method to express biotin on the tumor cell surface followed by i.v. bolus radiolabeled streptavidin injection. The mathematical model was built based on a system of ordinary differential equations consisting of inputs and outputs of model components in plasma, normal tissues, and tumor tissue. Through computer modeling, we calculated concentrations of each component for plasma, tumor and normal tissues at various time points. Radioactivity ratios of tumor to plasma and tumor to normal tissues increased with time. The increase of tumor to normal tissue ratios was much faster for the gene therapy approach than for single step and two step approaches, e.g., a ratio of 24.26 vs. 2.06 and 6.24 at 72 hours after radioligand injection. Radioactivity ratios predicted by the model varied with the amount of radioactivity injected and the time interval between injections. The model could be used to evaluate different radioimmunotherapy strategies and to predict radioactivity biodistribution using other receptor-ligand systems.     

Synthesis and in vitro binding properties of halogenated analogues of GBR as new dopamine uptake carrier ligands

We present the original synthesis of two halogenated analogues of the diphenyl piperazine GBR, bromo-GBR and iodo-GBR, as new dopamine uptake carrier ligands. The derivatives were purified by HPLC and chemically characterized. Bromo-GBR and iodo-GBR are potent inhibitors of [³H]GBR 12935 binding to rat striatal membrane, with K_i values of 116 and 113 nM, respectively. We prepared iodo-GBR labeled with iodide-125 from the brominated derivative and concluded that [¹²³I]iodo-GBR could be a potential tool to explore the in vivo dopamine uptake carrier.     

Bombesin derivative radiolabeled with technetium-99m as agent for tumor identification

A bombesin derivative was successfully radiolabeled in high labeling yield. Biodistribution studies and scintigraphic images in Ehrlich tumor-bearing mice were performed. This compound showed high accumulation in tumor tissue with high tumor-to-muscle and tumor-to-blood ratios. Thus, ^99mTc-HYNIC-Bombesin_(7–14) could be used as an agent for tumor diagnosis.     

Nitroimidazole conjugates of bis(thiosemicarbazonato)Cu(II) - Potential combination agents for the PET imaging of hypoxia

Combination agents comprising two different pharmacophores with the same biological target have the potential to show additive or synergistic activity. Bis(thiosemicarbazonato)copper(II) complexes (e.g. ⁶⁴Cu-ATSM) and nitroimidazoles (e.g. ¹⁸F-MISO) are classes of tracer used for the delineation of tumor hypoxia by positron emission tomography (PET). Three nitroimidazole-bis(thiosemicarbazonato)copper(II) conjugates were produced in order to investigate their potential as combination hypoxia imaging agents. Two were derived from the known bifunctional bis(thiosemicarbazone) H₂ATSM/A and the third from the new precursor diacetyl-2-(4-N-methyl-3-thiosemicarbazone)-3-(4-N-ethylamino-3-thiosemicarbazone) - H₂ATSM/en. Oxygen-dependent uptake studies were performed using the ⁶⁴Cu radiolabelled complexes in EMT6 carcinoma cells. All the complexes displayed appreciable hypoxia selectivity, with the nitroimidazole conjugates displaying greater selectivity than a simple propyl derivative used as a control. Participation of the nitroimidazole group in the trapping mechanism is indicated by the increased hypoxic uptake of the 2- vs. the 4-substituted ⁶⁴Cu-ATSM/A derivatives. The 2-nitroimidazole derivative of ⁶⁴Cu-ATSM/en demonstrated superior hypoxia selectivity to ⁶⁴Cu-ATSM over the range of oxygen concentrations tested. Biodistribution of the radiolabelled 2-nitroimidazole conjugates was carried out in EMT6 tumor-bearing mice. The complexes showed significantly different uptake trends in comparison to each other and previously studied Cu-ATSM derivatives. Uptake of the Cu-ATSM/en conjugate in non-target organs was considerably lower than for derivatives based on Cu-ATSM/A.     

Construction and Application of Elastin Like Polypeptide Containing IL-4 Receptor Targeting Peptide

Various human solid tumors highly express IL-4 receptors which amplify the expression of some of anti-apoptotic proteins, preventing drug-induced cancer cell death. Thus, IL-4 receptor targeted drug delivery can possibly increase the therapeutic efficacy in cancer treatment. Macromolecular carriers with multivalent targeting moieties offered great advantages in cancer therapy as they not only increase the plasma half-life of the drug but also allow delivery of therapeutic drugs to the cancer cells with higher specificity, minimizing the deleterious effects of the drug on normal cells. In this study we designed a library of elastin like polypeptide (ELP) polymers containing tumor targeting AP1 peptide using recursive directional ligation method. AP1 was previously discovered as an atherosclerotic plaque and breast tumor tissue homing peptide using phage display screening method, and it can selectively bind to the interleukin 4 receptor (IL-4R). The fluorescently labeled [AP1-V₁₂]₆, an ELP polymer containing six AP1 enhanced tumor-specific targeting ability and uptake efficiency in H226 and MDA-MB-231 cancer cell lines in vitro. Surface plasmon resonance analysis showed that multivalent presentation of the targeting ligand in the ELP polymer increased the binding affinity towards IL-4 receptor compared to free peptide. The binding of [AP1-V₁₂]₆ to cancer cells was remarkably reduced when IL-4 receptors were blocked by antibody against IL-4 receptor further confirmed its binding. Importantly, the Cy5.5-labeled [AP1-V₁₂]₆ demonstrated excellent homing and longer retention in tumor tissues in MDA-MB-231 xenograft mouse model. Immunohistological studies of tumor tissues further validated the targeting efficiency of [AP1-V₁₂]₆ to tumor tissue. These results indicate that designed [AP1-V₁₂]₆ can serve as a novel carrier for selective delivery of therapeutic drugs to tumors.     

A novel reactive ester derivative of biotin with reduced membrane permeability for in vivo biotinylation experiments

The in vivo perfusion of rodent models of disease with biotin derivatives and the subsequent comparative proteomic analysis of healthy and diseased tissues represent a promising methodology for the identification of vascular accessible biomarkers. A novel, triply charged biotinylation reagent, NHS‐β‐Ala‐(L ‐Asp)₃‐biotin, was synthesized and validated in terms of its applicability for in vivo protein biotinylation. Compared to sulfo‐NHS‐LC‐biotin, NHS‐β‐Ala‐(L ‐Asp)₃‐biotin exhibited a reduced membrane permeability and a preferential labeling of proteins localized in compartments readily accessible in vivo from the vasculature.     

Pharmacokinetic evaluation of C-3 modified 1,8-naphthyridine-3-carboxamide derivatives with potent anticancer activity: lead finding

Abstract

To develop naphthyridine derivatives as anticancer candidates, pharmacokinetic (PK) evaluations of 10 novel derivatives of 1,4-dihydro-4-oxo-1-proparagyl-1,8-naphthyridine-3-carboxamide, with potent anticancer activity were done using in vitro ADME (absorption, distribution, metabolism, excretion) and pharmacokinetic--pharmcodynamic (PK/PD) assays. Only derivatives 5, 6, 9 and 10 showed better metabolic stability, solubility, permeability, partition coefficient and cytochrome P450 (CYP) inhibition values. PK of derivatives 5, 6, 9 and 10 in rat showed comparable PK profile for derivative 5 (C₀?=?6.98?µg/mL) and 6 (C₀?=?6.61?µg/mL) with no detectable plasma levels for derivatives 9 and 10 at 5.0?mg/kg i.v. dose. PK/PD assay of derivatives 5 and 6 in tumor-bearing mice (TBM) showed comparable PK but tumor plasma index (TPI) of derivative 6 (4.02) was better than derivative 5 (2.50), suggesting better tumor uptake of derivative 6. Derivative 6, as lead compound, showed highest tumor growth inhibition (TGI) value of 33.6% in human ovary cancer xenograft model.     

Tissue distribution of 99mic-labeled liposomes prepared from synthetic amphiphiles containing amino acid residues

Abstract

The tissue distribution of ^99mTc-labeled liposomes prepared from synthetic amphiphiles containing amino acid residues was investigated for application to radiopharmaceuticals. The amphiphiles used were N,N-didodecyl-N α-[6-(trimethylammoniohexanoyl]-L-ala-ninamide bromide (N+C₅Ala2C₁₂), N,N-didodecyl-N_α-{6-[dimethyl(2-carboxyethyl)ammonio]hexanoyl}-L-alaninamide bromide (CAC₂N⁺C₅Ala2C₁₂) and S-{l-carboxy-2-([2,3-bis (he xadecyloxy)propoxy]carbony1)ethyl}homocy ste ine. These liposomes were stable in saline and 50% serum at 37° for at least 24h in comparison with the liposomes prepared from phosphatidylcholine and cholesterol (1:1). Most of the radioactivity of N+C₅Ala2C₁₂ and CAC₂N+C₅Ala2C₁₂ liposomes was firmly bound to Ehrlich ascites tumor cells in vitro. But the accumulation of three liposomes into the tumor of Ehrlich solid tumor-bearing mice after intravenous injection was low and most of the liposomes was taken up highly in liver and spleen which belong to the reticuloendothelial system (RES). Some approaches were made to reduce the RES uptake of N+C5Ala2C12 liposomes as follows: (1) the pretreatment of dextran sulfate depressed the uptake of the liposomes in the liver accompanied by increasing uptake in tumor and other tissues except stomach, (2) the modification of the liposomes with n-dodecyl glucoside or n-dodecyl sucrose depressed the uptake in liver and spleen, resulting in an increase in blood and other tissues such as tumor, duodenum and kidney, (3) the modification of the liposomes with ganglioside GM3 or GM1 reduced the uptake in liver and spleen, but increased scarcely the uptake in blood and tumor because of the rapid excretion into urine, (4) the intraperitoneal injection reduced the uptake of the liposomes in liver and increased significantly the accumulation in pancreas.     

The assembly of lipid-linked oligosaccharides in plant and animal membranes

Membrane preparations from growing regions of pea stems and activelydividing mouse L-cells form lipid-linked saccharides from GDP-mannose and UDP-N-acetylglucosamine. These lipids have properties which are consistent with those of mono-and di-phosphoryl polyisoprenyl derivatives. In experiments using plant membranes, the monophosphoryl derivative labeled with GDP-(¹⁴C) mannose contains mannose only, while the diphosphoryl derivative labeled with the same nucleotide sugar is heterogeneous, containing oligosaccharides corresponding to mannosaccharides of 5, 7, and 9-12 residues. Only the diphosphoryl polyisoprenyl derivatives are labeled with UDP-(¹⁴C)glucosamine and these contain predominantly chitobiose and N-acetylglucosamine itself. Unlabeled GDP-mannose added after UDP-N-acetyl (¹⁴C)glucosamine results in the formation of higher lipid-linked oligosaccharides which are apparently the same as those which are labeled with GDP-(¹⁴C)mannose alone. Incubation of the membranes with GDP-(¹⁴C)mannose in the presence of Mn²⁺, unlabeled UDP-glucose or unlabeled UDP-N-acetylglucosamine results in marked changes in the accumulation of both the polyisoprenyl monophosphoryl mannose and polyisoprenyl diphosphoryl oligosaccharides. Animal cell membranes synthesise lipid-linked oligosaccharides when incubated with UDP-N-acetylglucosamine and GDP-mannose. These oligosaccharides are similar in size to those synthesised by the plant membranes but their formation is more efficient. The potential roles of these compounds in glycoprotein biosynthesis in both plant and animal tissues is discussed.     

Interaction of amphiphilic chlorin-based photosensitizers with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine monolayers

The drawbacks of the presently used photosensitizers include their relatively low selectivity toward cancer cells, and long-lasting accumulation in healthy tissues. Our recent results indicate that conjugating a photosensitizer with folic acid both enhances the active uptake by cells, and decreases the accumulation in healthy tissue. Here, the interaction between 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) monolayers used as model membranes, and three different photosensitizers were studied; the derivatives were the non-conjugated meta-tetrahydroxyphenylchlorin (m-THPC, CHL1) and tris(3-hydroxyphenyl)-4-carboxyphenylchlorin (CHL2), as well as a folic acid-conjugated m-THPC-like molecule (CHL3). The results obtained indicate that the folate moiety present in the conjugated derivative CHL3 is involved in the interaction with the phospholipid polar heads. This interaction may be responsible for a better miscibility of CHL3 with the DPPC films compared to CHL1 and CHL2, while elimination of CHL3 from the tissue may be due rather to specific, biological processes and not to its polarity.     

An optimized antibody-chelator conjugate for imaging of carcinoembryonic antigen with indium-111

A monoclonal antibody to carcinoembryonic antigen showing minimal cross-reactivity with blood cells and normal tissues was derivatized with benzylisothiocyanate derivatives of EDTA and DTPA. Seven chelators per immunoglobulin could be incorporated without loss of immunoreactivity. The resulting conjugates, labeled with indium-111, showed low liver uptake in animals. A cold kit, comprising the DTPA conjugate at a molarity of antibody bound chelator exceeding 1 x 10⁻⁴M, gave radiochemical yields of indium labeled antibody of ⩾95% and was stable for 1 yr.     

Synthesis of Tc-99m labeled 1,2,3-triazole-4-yl c-met binding peptide as a potential c-met receptor kinase positive tumor imaging agent

The mesenchymal-epithelial transition factor (c-Met), which is related to tumor cell growth, angiogenesis and metastases, is known to be overexpressed in several tumor types. In this study, we synthesized technetium-99m labeled 1,2,3-triazole-4-yl c-Met binding peptide (cMBP) derivatives, prepared by solid phase peptide synthesis and the ‘click-to-chelate’ protocol for the introduction of tricarbonyl technetium-99m, as a potential c-Met receptor kinase positive tumor imaging agent, and evaluated their in vitro c-Met binding affinity, cellular uptake, and stability. The ^99mTc labeled cMBP derivatives ([^99mTc(CO)₃]12, [^99mTc(CO)₃]13, and [^99mTc(CO)₃]14) were prepared in 85-90% radiochemical yields. The cold surrogate cMBP derivatives, [Re(CO)₃]12, [Re(CO)₃]13, and [Re(CO)₃]14, were shown to have high binding affinities (0.13 μM, 0.06 μM, and 0.16 μM, respectively) to a purified cMet/Fc chimeric recombinant protein. In addition, the in vitro cellular uptake and inhibition studies demonstrated the high specific binding of these ^99mTc labeled cMBP derivatives ([^99mTc(CO)₃]12–14) to c-Met receptor positive U87MG cells.