RP463: a stabilized technetium-99m complex of a hydrazino nicotinamide derivatized chemotactic peptide for infection imaging.

A HYNIC-conjugated chemotactic peptide (fMLFK-HYNIC) was labeled with (99m)Tc using tricine and TPPTS as coligands. The combination of fMLFK-HYNIC, tricine, and TPPTS with (99m)Tc produced a ternary ligand complex [(99m)Tc(fMLFK-HYNIC)(tricine)(TPPTS)] (RP463). RP463 was synthesized either in two steps, in which the binary ligand complex [(99m)Tc(fMLFK-HYNIC)(tricine)(2)] (RP469) was formed first and then reacted with TPPTS, or in one step by direct reduction of [(99m)Tc]pertechnetate with stannous chloride in the presence of fMLFK-HYNIC, tricine, and TPPTS. The radiolabeling yield for RP463 was usually >/=90% using 10 microg of fMLFK-HYNIC and 100 mCi of [(99m)Tc]pertechnetate. Unlike RP469, which decomposed rapidly in the absence of excess tricine coligand, RP463 was stable in solution for at least 6 h. [(99)Tc]RP463 was prepared and characterized by HPLC and electrospray mass spectrometry. In an in vitro assay, [(99)Tc]RP463 showed an IC(50) of 2 nM against binding of [(3)H]fMLF to receptors on PMNs. [(99)Tc]RP463 also induces effectively the superoxide release of polymorphonuclear leukocytes (PMNs) with an EC(50) value of 0.2 +/- 0.2 nM. The localization of RP463 in the infection foci was assessed in a rabbit infection model. RP463 was cleared from the blood faster than RP469 and was excreted mainly through the renal system. As a result of rapid blood clearance and increased uptake, the target-to-background ratios continuously increased from 1.5 +/- 0.2 at 15 min postinjection to 7.5 +/- 0.4 at 4 h postinjection. Visualization of the infected area could be as early as 2 h. A transient decrease in white blood cell count of 35% was observed during the first 30 min after injection of the HPLC-purified RP463 in the infected rabbit. This suggests that future research in this area should focus on developing highly potent antagonists for chemotactic peptide receptor or other receptors on PMNs and monocytes.     

Preparation and properties of 99mTc(CO)3+-labeled N,N-bis(2-pyridylmethyl)-4-aminobutyric acid

Labeling biomolecules with (99m)Tc(CO)(3)(+) ((99m)Tc tricarbonyl) is attracting increasing attention. Although histidine is often considered an ideal bifunctional chelator for (99m)Tc (or (188)Re) tricarbonyl, the family of dipicolylamine carboxylate chelators may be a useful alternative because of the expected ease of synthesis and because the structure provides a pendent carboxylate for potential conjugation to biomolecules. The dipicolylamine chelator N,N-bis(2-pyridylmethyl)-4-aminobutyric acid (BPABA) was synthesized using 4-aminobutyric acid in place of glycine or aminopropionic acid in the literature, to avoid possible involvement of the carboxylate in the complex formation process by forming five- or six-membered chelation rings. Using a commercial tricarbonyl kit (Mallinckrodt), the complex formation properties of both BPABA and commercial histidine with (99m)Tc tricarbonyl were investigated, and the in vitro complex stabilities in saline and in serum were compared. Stability in vivo was also examined following i.v. administration to normal mice. BPABA was synthesized simply and quantitatively by reacting picolyl chloride with aminobutyric acid in one step. On RP HPLC, the product eluted essentially in one peak and the structure was confirmed by ESI-MS. After labeling, both BPABA and histidine were shown by RP HPLC to form tricarbonyl complexes. In both cases, after incubation at 100 degrees C for 20 min, only one predominant peak of (99m)Tc(CO)(3)(+)-histidine or (99m)Tc(CO)(3)(+)-BPABA was apparent, and both complexes were stable at room temperature in saline for at least 24 h. After incubation for 24 h in 37 degrees C serum, by SE HPLC, 20% of the (99m)Tc(CO)(3)(+)-histidine was bound to serum protein compared to less than 10% for (99m)Tc(CO)(3)(+)-BPABA. A 5000 molar excess of histidine at 100 degrees C for 6 h was unable to dissociate (99m)Tc(CO)(3)(+)-BPABA. By contrast, BPABA easily dissociated (99m)Tc(CO)(3)(+)-histidine under the same conditions. Both complexes were stable in vivo in mice, and (99m)Tc(CO)(3)(+)-BPABA showed rapid and specific hepatobiliary clearance while (99m)Tc(CO)(3)(+)-histidine was cleared through the kidneys. In conclusion, BPABA was easily synthesized and was shown to possess properties comparable to histidine for labeling of biomolecules with (99m)Tc tricarbonyl. However, it was found that the chelator concentration required for quantitative (99m)Tc tricarbonyl labeling with both BPABA and histidine were 2 orders higher than that required with more conventional labeling using MAG(3). Finally, the complex (99m)Tc(CO)(3)(+)-BPABA itself was found to clear exclusively via the hepatobiliary pathway and may have value as a potential hepatobiliary imaging agent.     

99mTc-labeling of a hydrazinonicotinamide-conjugated vitronectin receptor antagonist useful for imaging tumors.

This report describes the (99m)Tc labeling of a HYNIC-conjugated vitronectin receptor antagonist (SQ168 = [2-[[[5-[carboonyl]-2-pyridinyl]hydrazono]methyl]benzenesulfonic acid]-Glu(cyclo[Lys-Arg-Gly-Asp-D-Phe])-cyclo[Lys-Arg-Gly-Asp-D-Phe]). The ternary ligand complex [(99m)Tc(SQ168)(tricine)(TPPTS)] (RP593) was prepared using a non-SnCl(2)-containing formulation. The corresponding (99)Tc analogue, [(99)Tc]RP593, was also prepared and characterized by HPLC and LC-MS. A HPLC concordance experiment using RP593 and [(99)Tc]RP593 showed that the same technetium complex was prepared at both the tracer and macroscopic levels. The LC-MS data is completely consistent with the 1:1:1:1 composition for Tc:SQ168:tricine:TPPTS and provides direct evidence that the two radiometric peaks in the radio-HPLC chromatogram of RP593 are indeed due to the resolution of diastereomers. In an in vitro receptor binding assay, [(99)Tc]RP593 was shown to have comparable binding affinity for the vitronectin receptor to that of SQ168 itself.     

Development and characterization of annexin V mutants with endogenous chelation sites for (99m)Tc

[(99m)Tc]Annexin V can be used to image organs undergoing cell death during cancer chemotherapy and organ transplant rejection. To simplify the preparation and labeling of annexin V for nuclear-medicine studies, we have investigated the addition of peptide sequences that will directly form endogenous chelation sites for (99m)Tc. Three mutant molecules of annexin V, called annexin V-116, -117, and -118, were constructed with N-terminal extensions of seven amino acids containing either one or two cysteine residues. These molecules were expressed cytoplasmically in Escherichia coli and purified to homogeneity with a final yield of 10 mg of protein/L of culture. Analysis in a competitive binding assay showed that all three proteins retained full binding affinity for erythrocyte membranes with exposed phosphatidylserine. Using SnCl(2) as reducing agent and glucoheptonate as exchange agent, all three proteins could be labeled with (99m)Tc to specific activities of at least 50-100 microCi/microg. The proteins retained membrane binding activity after the radiolabeling procedure, and quantitative analysis indicated a dissociation constant (K(d)) of 7 nmol/L for the annexin V-117 mutant. The labeling reaction was rapid, reaching a maximum after 40 min at room temperature. The radiolabeled proteins were stable when incubated with phosphate-buffered saline or serum in vitro. Proteins labeled to a specific activity of 25-100 microCi/microg were injected intravenously in mice at a dose of 100 microg/kg, and biodistribution of radioactivity was determined at 60 min after injection. Uptake of radioactivity was highest in kidney and liver, consistent with previous results obtained with wild-type annexin V. Cyclophosphamide-induced apoptosis in vivo could be imaged with [(99m)Tc]annexin V-117. In conclusion, annexin V can be modified near its N-terminus to incorporate sequences that form specific chelation sites for (99m)Tc without altering its high affinity for cell membranes. These annexin V derivatives may be useful for in vivo imaging of cell death.     

A novel ternary ligand system useful for preparation of cationic (99m)Tc-diazenido complexes and (99m)Tc-labeling of small biomolecules

This report describes a novel ternary ligand system composed of a phenylhydrazine, a crown ether-containing dithiocarbamate (DTC), and a PNP-type bisphosphine (PNP). The combination of three different ligands with (99m)Tc results in cationic (99m)Tc-diazenido complexes, [(99m)Tc(NNAr)(DTC)(PNP)]+, with potential radiopharmaceuticals for heart imaging. Synthesis of cationic (99m)Tc-diazenido complexes can be accomplished in two steps. For example, the reaction of phenylhydrazine with (99m)TcO4- at 100 degrees C in the presence of excess stannous chloride and 1,2-diaminopropane-N,N,N',N'-tetraacetic acid (PDTA) results in the [(99m)Tc(NNPh)(PDTA)n] intermediate, which then reacts with sodium N-(dithiocarbamato)-2-aminomethyl-15-Crown-5 (L4) and N,N-bis[2-(bis(3-ethoxypropyl)phosphino)ethyl]ethoxyethylamine (PNP6) at 100 degrees C for 15 min to give the complex, [(99m)Tc(NNPh)(L4)(PNP6)]+ in high yield (>90%). Cationic complexes [(99m)Tc(NNPh)(DTC)(PNP)]+ are stable for > or = 6 h. Their composition was determined to be 1:1:1:1 for Tc:NNPh:DTC:PNP using the mixed-ligand experiments on the tracer ((99m)Tc) level and was further confirmed by the ESI-MS spectral data of a model compound [Re(NNPh)(L4)(L6)]+. It was found that both DTCs and bisphosphines have a significant impact on the lipophilicity of their cationic (99m)Tc-diazenido complexes. Results from a (99m)Tc-labeling efficiency experiment showed that 4-hydrazinobenzoic acid (HYBA) might be useful as a bifunctional coupling agent for (99m)Tc-labeling of small biomolecules. However, the (99m)Tc-labeling efficiency of HYBA is much lower than that of 6-hydrazinonicotinic acid (HYNIC) with tricine and trisodium triphenylphosphine-3,3',3'-trisulfonate (TPPTS) as coligands.     

Radio-LC-MS for the characterization of 99mTc-labeled bioconjugates

This report describes the first example of using radio-LC-MS for determining the composition of (99m)Tc radiopharmaceuticals at the tracer level. The in-line radiometric detector is a useful addition to a standard LC-MS and provides direct correlation between the MS data and the radioactive species in a radiopharmaceutical kit. Complexes [(99m)Tc(HYNICtide)(tricine)(L)] (RP444, L = TPPTS; RP445, L = TPPDS; and RP446, L = TPPMS) were prepared using a decayed generator eluant. All the ternary ligand (99m)Tc complexes show the expected monoprotonated molecular ions, (M + 1)(+), and diprotonated molecular ions, (M + 2)(2+). The LC-MS spectral data support the proposed structure and are consistent with those obtained for their corresponding (99)Tc analogues. Ternary ligand complexes [(99m)Tc(HYNICtide)(tricine)(L)] (L = ISONIC-HE and ISONIC-Sorb) are neutral, and the molecular weights are also lower than that of RP444. Using a fresh generator eluant (24 h prior elution), only 1-2 mCi of (99m)Tc [(7 x 10(-)(12))-(1.5 x 10(-)(11)) mol of technetium complex] are required to obtain a reasonably clean mass spectrum. Radio-LC-MS is a quick and accurate analytical tool for characterization of (99m)Tc radiopharmaceuticals at the tracer level.     

99mTc-Labeling of a hydrazinonicotinamide-conjugated LTB(4) receptor antagonist useful for imaging infection and inflammation

This report describes the (99m)Tc labeling of a hydrazinonicotinamide (HYNIC)-conjugated LTB(4) receptor antagonist (SG380). The ternary ligand technetium complex [(99m)Tc(SG38)(tricine)(TPPTS)] (RP517) was prepared using a non-SnCl(2)-containing formulation ((2001) J. Pharm. Sci. 90, 114-123). Unlike other HYNIC-conjugated small biomolecules, SG380 is lipophilic and has low solubility in the kit matrix. Using a combination of a solubilizing agent (Lysolecithin) and a cosolvent (ethanol), we have developed a new formulation for routine preparation of RP517. Using this formulation, RP517 can be prepared in high radiochemical purity (RCP > 90%) and remains stable in the kit matrix for at least 6 h. We also prepared the corresponding (99)Tc analogue, [(99)Tc]RP517. An HPLC concordance experiment using RP517 and [(99)Tc]RP517 showed that the same technetium complex was prepared at both the tracer and macroscopic levels. The LC-MS data are completely consistent with the 1:1:1:1 composition for Tc:SG380:tricine:TPPTS.     

A Tc-99m-labeled long chain fatty acid derivative for myocardial imaging

C-11- and I-123-labeled long chain fatty acid derivatives have been reported as useful radiopharmaceuticals for the estimation of myocardial fatty acid metabolism. We have reported that Tc-99m-labeled N-[[[(2-mercaptoethyl)amino]carbonyl]methyl]-N-(2-mercaptoethyl)-6-aminohexanoic acid ([(99m)Tc]MAMA-HA), a medium chain fatty acid derivative, is metabolized by beta-oxidation in the liver and that the MAMA ligand is useful for attaching to the omega-position of fatty acid derivatives as a chelating group for Tc-99m. On the basis of these findings, we focused on developing a Tc-99m-labeled long chain fatty acid derivative that reflected fatty acid metabolism in the myocardium. In this study, we synthesized a dodecanoic acid derivative, MAMA-DA, and a hexadecanoic acid derivative, MAMA-HDA, and performed radiolabeling and biodistribution studies. [(99m)Tc]MAMA-DA and [(99m)Tc]MAMA-HDA were prepared using a ligand-exchange reaction. Biodistribution studies were carried out in normal mice and rats. Then, a high initial uptake of Tc-99m was observed, followed by a rapid clearance from the heart. The maximum heart/blood ratio was 3.6 at 2 min postinjection of [(99m)Tc]MAMA-HDA. These kinetics were similar to those with postinjection of p-[(125)I]iodophenylpentadecanoic acid. Metabolite analysis showed [(99m)Tc]MAMA-HDA was metabolized by beta-oxidation in the body. In conclusion, [(99m)Tc]MAMA-HDA is a promising compound as a long chain fatty acid analogue for estimating beta-oxidation of fatty acid in the heart.     

Synthesis, characterization, and biological evaluation of neutral nitrido technetium(V) mixed ligand complexes containing dithiolates and aminodiphosphines. A novel system for linking technetium to biomolecules

A new biomolecule labeling method that utilizes the [(99m)Tc(N)(PNP)](2+) metal fragment is presented. Thus, a series of nitrido mixed-ligand M(V) complexes (M = (99m)Tc, (99g)Tc, Re), [M(N)(Ln)(PNP)], where Ln is the dianionic form of a dithiolate or substituted-dithiolate ligand and PNP is an aminodiphosphine, is described. (99m)Tc complexes can be prepared using either a two-step or a three-step procedure starting from generator-eluted pertechnetate through a prereduced mixture of [(99m)Tc(N)]-containing species, followed by sequential or contemporary addition of the relevant dithiolate and aminodiphosphine. The reactions of 2,3-dimercaptopropionic acid (H(2)L1) with [Tc(N)(PNP)](2+) were investigated in detail. It was found that this bidentate ligand coordinated the metal fragment through the [S(-),S(-)] donor atom pair, to yield neutral mixed-ligand complexes [(99m)Tc(N)(L1)(PNP)] in high specific activity. The additional carboxylic functional group was not involved in metal coordination, thus remaining available for conjugation to target-specific molecules. Dithiolates incorporating pendant functional group(s) gave rise to a 1:1 diastereoisomeric mixture of syn-[M(N)(Ln)(PNP)] and anti-[M(N)(Ln)(PNP)] derivatives, depending on the relative orientation of the dithiolate substituent(s) with respect to the terminal nitrido group, and no isomeric conversion was detected. (99m)Tc species had been proven to be identical with the (99g)Tc complexes prepared at the macroscopic level by comparison of the corresponding radiometric and UV/vis HPLC profiles. Challenge experiments with cysteine or glutathione indicated that these physiological agents had no effect on the stability of this class of mixed-ligand (99m)Tc-complexes. Biodistribution studies in rats of selected (99m)Tc-complexes showed a rapid clearance from the blood and tissues after 60 min pi.     

Necrosis avidity of (99m)Tc(CO)3-labeled pamoic acid derivatives: synthesis and preliminary biological evaluation in animal models of necrosis

In a search for an infarct avid tracer agent with improved properties, we have observed that bis-DTPA derivatives of pamoic acid have a high avidity for necrotic tissue. Here, we report the synthesis, radiolabeling, and preliminary evaluation in normal mice and rats with hepatic infarction of the (99m)Tc-tricarbonyl complexes of N, N'-bis(diethylenetriaminopentaacetato)-4,4'-methylene bis(2-hydroxy-3-naphthoic hydrazide) ( (99m)Tc(CO) 3-bis-DTPA-pamoate) and [ N-(5-aminopentyl)pyridin-2-yl-methylamino]methylacetato-4,4'-methylene-2-hydroxy-3-napthalenecarboxamide-(2'-hydroxy-3'-naphthoic acid methyl ester) ( (99m)Tc(CO) 3 -12). Radiolabeling with (99m)Tc(CO) 3 (+) was achieved with a radiochemical yield of over 95% for both tracer agents. In normal mice, the polar (99m)Tc(CO) 3-bis-DTPA-pamoate was cleared from plasma via both the liver and the kidneys, while the more lipophilic (99m)Tc(CO) 3 -12 was rapidly cleared via the liver. Blood clearance in mice was faster for (99m)Tc(CO) 3 -12 (0.1% injected dose per gram at 4 h postinjection) than for (99m)Tc(CO) 3-bis-DTPA-pamoate (9.3% injected dose per gram at 4 h postinjection). Affinity and specificity of the tracers for necrotic tissue was studied in rats with hepatic infarction and ethanol-induced necrosis of the liver or muscles. Activity ratios of infarct to viable liver tissue of (99m)Tc(CO) 3-bis-DTPA-pamoate quantified by autoradiography of tissue slices ranged from 4 to 18, depending on the necrosis model and time postinjection of the tracer. Infarcts were also visualized in vivo by (99m)Tc(CO) 3-bis-DTPA-pamoate planar gamma imaging. After injection of (99m)Tc(CO) 3-bis-DTPA-pamoate, in vivo and ex vivo images correlated well with histochemical staining with triphenyltetrazolium chloride and hematoxylin and eosin. (99m)Tc(CO) 3 -12 on the other hand showed no uptake in necrotic tissue. Stability of the tracers was determined in vitro after storage at room temperature and by histidine challenge experiments, and in vivo in mouse plasma and in urine (for (99m)Tc(CO) 3-bis-DTPA-pamoate). (99m)Tc(CO) 3-bis-DTPA-pamoate was unstable in vitro to histidine challenge, while (99m)Tc(CO) 3 -12 was 98% stable in vitro in the same conditions. Both tracers showed good in vivo stability. (99m)Tc(CO) 3-bis-DTPA-pamoate shows high specificity for necrotic tissue and merits further evaluation as a necrosis avid imaging agent. (99m)Tc(CO) 3 -12 is not useful for visualization of necrotic tissue.     

Synthesis and biological evaluation of diethylenetriamine pentaacetic acid-polyethylene glycol-folate: a new folate-derived, (99m)Tc-based radiopharmaceutical

(99m)Technetium-labeled diethylenetriamine pentaacetic acid-polyethylene glycol-folate (DTPA-PEG-folate) was synthesized and tested as a radiopharmaceutical agent, which targeted the lymphatic system with metastatic tumor. Folic acid was reacted with H2N-PEG-NH2 to yield H2N-PEG-folate. After purification by anion-exchange chromatography, the product was reacted with cyclic DTPA. By removal of unreacted DTPA by size-exclusion chromatography, DTPA-PEG-Folate was obtained. Fluorescein-5-isothiocyanate (FITC)-labeled DTPA-PEG-folate and DTPA-PEG-OCH3 were prepared via a dicyclohexylcarbodiimide-mediated coupling. In vitro competitive binding test showed that the uptake of [125I] folic acid was inhibited by DTPA-PEG-folate and the 50% inhibitory concentration was 4.37 pmol/L (R2 = 0.9922). The relative affinity of DTPA-PEG-FITC was 0.18 for human folate receptor comparing with folic acid. In cultured tumor cells, uptake of fluorescence-labeled DTPA-PEG-folate was found to increase significantly in folate-deficient medium compared with that of untargeted DTPA-PEG-OCH3 and FITC-ethylenediamine. The competition with free folic acid blocked the cell uptake of DTPA-PEG-folate. These results confirmed the DTPA-PEG-folate entered into KB cells through the folate receptor endocytosis pathway in vitro. The radiolabeled yield of [(99m)Tc] DTPA-PEG-folate was in excess of 98%, and specific activities of 7.4 kBq (0.2 microCi/microg) were achieved. After subcutaneous injection, [(99m)Tc] DTPA-PEG-folate exhibited an initial increase and successive decline of accumulation in popliteal nodes in normal Wistar rats. Expect for the kidney, uptake by other tissues was rather low. In a normal rabbit imagine study, the lymphatic vessels were readily visualized by single-photon-emission computed tomography following subcutaneous injection of [(99m)Tc] DTPA-PEG-folate. In conclusion, the [(99m)Tc] DTPA-PEG-folate conjugate may have a potential as a lymphatic tumor-targeted radiopharmaceutical.     

Synthesis and evaluation of two technetium-99m-labeled peptidic 2-nitroimidazoles for imaging hypoxia.

The presence of hypoxic cells in solid tumors is a marker for therapy-resistant, aggressive disease. The noninvasive detection of hypoxic cells in tumors by radiolabeled 2-nitroimidazoles is a diagnostic technique under current evaluation. Two peptidic agents, dimethylglycyl-L-seryl-L-cysteinyl-lysyl{N(epsilon)-[1-(2-nitro-1H -im idazolyl)acetamido]}glycine (RP435) and dimethylglycyl-tert-butylglycyl-L-cysteinyl-glycine-[2-(2-ni tro-1H-im idazolyl)ethyl]amide (RP535) have been synthesized. Both agents contain an N(3)S class chelator for (99m)Tc and Re and a 2-nitroimidazole group which can be enzymatically reduced and selectively trapped in cells under hypoxic conditions. Two isomers of (99m)TcO-RP435, which are assumed to be syn and anti conformations, were observed on HPLC analysis. The interconversion of the two isomers in aqueous solution was investigated. In contrast, RP535 chelated (99m)Tc to form a single isomer and no conversion to its counterpart has been observed on HPLC analysis. The tert-butyl group on the chelator may inhibit the formation and interconversion of the syn and anti isomers of (99m)TcO-RP535. Both tracers showed a significant degree of hypoxia-specific accumulation in an in vitro assay, with (99m)TcO-RP535 showing higher selectivity for hypoxic cells than (99m)TcO-RP435. These results suggest that (99m)TcO-RP535 represents a lead compound worthy of further investigation as an agent for imaging hypoxia in tumors.     

99mTc-labeling of hydrazones of a hydrazinonicotinamide conjugated cyclic peptide.

Eight HYNICtide hydrazones (three with aliphatic substituents and five with aromatic groups) were studied for their potential use as the final intermediate for preparation of RP444, a new radiopharmaceutical under development for imaging thrombosis. The goal of this study is to screen various hydrazones through stability testing and radiolabeling and find those which are able to remain stable without significant degradation in the manufacturing process and at the same time are reactive to produce enough free hydrazine in situ for successful (99m)Tc-labeling. In an initial screening study, only hydrazones 6 and 8, which contain aliphatic substituents, gave satisfactory (>/=90%) yields of RP444 using 50 degrees C and 30 min of heating. However, their solution instability excludes them from being used as commercial reagents. Hydrazones 1 and 4 gave >/=90% yields when the reaction mixtures were heated at 80 degrees C for 30 min. Both hydrazone 1 and hydrazone 4 can be used as the final intermediate for preparation of RP444. The combination of 40 mg of tricine, 1-10 mg of TPPTS, 20-40 microg of hydrazone 1 or 4 for 50 mCi of [(99m)Tc]pertechnetate, 20-50 microg of stannous chloride, pH 4.5 +/- 0.5, and heating at 80 degrees C for 30 min gives the best yield for RP444. It is surprising that hydrazones 1 and 4 have both the solution stability with respect to decomposition and to reaction with aldehydes and ketones and yet are able to hydrolyze in situ to produce enough free HYNICtide for the (99m)Tc-labeling.     

Evaluation of 99mTc-labeled cyclic RGD dimers: impact of cyclic RGD peptides and 99mTc chelates on biological properties

The main objective of this study is to explore the impact of cyclic RGD peptides and (99m)Tc chelates on biological properties of (99m)Tc radiotracers. Cyclic RGD peptide conjugates, HYNIC-K(NIC)-RGD(2) (HYNIC = 6-hydrazinonicotinyl; RGD(2) = E[c(RGDfK)](2) and NIC = nicotinyl), HYNIC-K(NIC)-3G-RGD(2) (3G-RGD(2) = Gly-Gly-Gly-E[Gly-Gly-Gly-c(RGDfK)](2)), and HYNIC-K(NIC)-3P-RGD(2) (3P-RGD(2) = PEG(4)-E[PEG(4)-c(RGDfK)](2)), were prepared. Macrocyclic (99m)Tc complexes [(99m)Tc(HYNIC-K(NIC)-RGD(2))(tricine)] (1), [(99m)Tc(HYNIC-K(NIC)-3G-RGD(2))(tricine)] (2), and [(99m)Tc(HYNIC-K(NIC)-3P-RGD(2))(tricine)] (3) were evaluated for their biodistribution and tumor-targeting capability in athymic nude mice bearing MDA-MB-435 human breast tumor xenografts. It was found that 1, 2, and 3 could be prepared with high specific activity (～111 GBq/μmol). All three (99m)Tc radiotracers have two major isomers, which show almost identical uptake in tumors and normal organs. Replacing the bulky and highly charged [(99m)Tc(HYNIC)(tricine)(TPPTS)] (TPPTS = trisodium triphenylphosphine-3,3',3″-trisulfonate) with a smaller [(99m)Tc(HYNIC-K(NIC))(tricine)] resulted in less uptake in the kidneys and lungs for 3. Surprisingly, all three (99m)Tc radiotracers shared a similar tumor uptake (1, 5.73 ± 0.40%ID/g; 2, 5.24 ± 1.09%ID/g; and 3, 4.94 ± 1.71%ID/g) at 60 min p.i. The metabolic stability of (99m)Tc radiotracers depends on cyclic RGD peptides (3P-RGD(2) > 3G-RGD(2) ～ RGD(2)) and (99m)Tc chelates ([(99m)Tc(HYNIC)(tricine)(TPPTS)] > [(99m)Tc(HYNIC-K(NIC))(tricine)]). Immunohistochemical studies revealed a linear relationship between the α(v)β(3) expression levels and tumor uptake or tumor/muscle ratios of 3, suggesting that 3 is useful for monitoring the tumor α(v)β(3) expression. Complex 3 is a very attractive radiotracer for detection of integrin α(v)β(3)-positive tumors.     

Influence of the denticity of ligand systems on the in vitro and in vivo behavior of (99m)Tc(I)-tricarbonyl complexes: a hint for the future functionalization of biomolecules

Functionalization of biologically relevant molecules for the labeling with the novel fac-[(99m)Tc(OH(2))(3)(CO)(3)](+) precursor has gained considerable attention recently. Therefore, we tested seven different tridentate (histidine L(1)(), iminodiacetic acid L(2)(), N-2-picolylamineacetic acid L(3)(), N, N-2-picolylaminediacetic acid L(4)()) and bidentate (histamine L(5)(), 2-picolinic acid L(6)(), 2,4-dipicolinic acid L(7)()) ligand systems, with the potential to be bifunctionalized and attached to a biomolecule. The ligands allowed mild radiolabeling conditions with fac-[(99m)Tc(OH(2))(3)(CO)(3)](+) (30 min, 75 degrees C). The ligand concentrations necessary to obtain yields of >95% of the corresponding organometallic complexes 1-7 ranged from 10(-)(6) to 10(-)(4) M. Complexes of the general formula "fac-[(99m)TcL(CO)(3)]" (L = tridentate ligand) and "fac-[(99m)Tc(OH(2))L'(CO)(3)]" (L' = bidentate ligand), respectively, were produced. Challenge studies with cysteine and histidine revealed significant displacement of the ligands in complexes 5-7 but only little exchange with complexes 1-4 after 24 h at 37 degrees C in PBS buffer. However, no decomposition to (99m)TcO(4)(-) was observed under these conditions. All complexes showed a hydrophilic character (log P(o/w) values ranging from -2.12 to 0.32). Time-dependent FPLC analyses of compounds 1-7 incubated in human plasma at 37 degrees C showed again no decomposition to (99m)TcO(4)(-) after 24 h at 37 degrees C. However, the complexes with bidentate ligands (5-7) became almost completely protein bound after 60 min, whereas the complexes with tridentate coordinated ligands (1-4) showed no reaction with serum proteins. The compounds were tested for their in vivo stability and the biodistribution characteristics in BALB/c mice. The complexes with tridentate coordinated ligand systems (1-4) revealed generally a good and fast clearance from all organs and tissues. On the other hand, the complexes with only bidentate coordinated ligands (5-7) showed a significantly higher retention of activity in the liver, the kidneys, and the blood pool. Detailed radiometric analyses of murine plasma samples, 30 min p.i. of complex fac-[(99m)TcL(1)(CO)(3)], 1, revealed almost no reaction of the radioactive complex with the plasma proteins. By contrast, in plasma samples of mice, which were injected with complex fac-[(99m)Tc(OH(2))L(5)(CO)(3)](+), 5, the entire radioactivity coeluded with the proteins. On the basis of these in vitro and in vivo experiments, it appears that functionalization of biomolecules with tridentate-chelating ligand systems is preferable for the labeling with fac-[(99m)Tc(OH(2))(3)(CO)(3)](+), since this will presumably result in radioactive bioconjugates with better pharmacokinetic profiles.     

Determination and characterization of cysteine, glutathione and phytochelatins (PC₂₋₆) in Lolium perenne L. exposed to Cd stress under ambient and elevated carbon dioxide using HPLC with fluorescence detection

Metal-binding thiols, involved in detoxification mechanisms in plant and other organism under heavy metal stress, are receiving more and more attentions, and various methods have been developed to determine related thiols such as cysteine (Cys), glutathione (GSH) and phytochelatins (PCs). In present study, an HPLC method was established for simultaneous determination of Cys GSH and PC(2-6) after treatment with disulfide reductant of tris (2-carboxyethyl) phosphine hydrochloride (TCEP) and thiolyte reagent of monobromobimane (mBBr). The separation of thiol derivatives was performed on an Agilent Zorbax Eclipse XDB-C18 column (4.6 mm × 30 mm, 1.8 μm) with a linear gradient elution of 0.1% (v/v) trifluoroacetic acid (TFA)-acetonitrile (ACN) at 0.8 mL min(-1). The temperature of the column was maintained at 25°C. The excitation and emission wavelengths were set at 380 and 470 nm, respectively. The thiol derivatives were well separated in 19 min, and the total analysis time was 30 min. The established method was proved selective, specific and reproducible, and could be applicable to determine Cys, GSH and PC(2-6) and to evaluate their roles in detoxification mechanisms in Cd-treated Lolium perenne L. under ambient and elevated carbon dioxide (CO(2)). It was found that the total SH contents and proportions of thiols in roots and shoots were dependent on Cd concentration, whereas the total SH contents decreased and the proportions of thiols altered without significance at elevated CO(2) level.     

Phosphine-containing HYNIC derivatives as potential bifunctional chelators for (99m)Tc-labeling of small biomolecules

Two prototype phosphine-containing HYNIC chelators, HYNIC-Kp-DPPB and HYNIC-Ko-DPPB (HYNIC = 6-hydrazinonicotinamide; K = lysine; and DPPB = diphenylphosphine-benzoic acid), have been synthesized and characterized by NMR ((1)H, (13)C, and (31)P) and LC-MS. Macrocyclic (99m)Tc complexes, [(99m)Tc(HYNIC-Ko-TPPB)(tricine)] and [(99m)Tc(HYNIC-Kp-DPPB)(tricine)], were prepared by reacting the phosphine-containing HYNIC chelator with (99m)TcO(4)(-) in the presence of excess tricine and stannous chloride. Results from this study clearly demonstrated that both HYNIC-Kp-DPPB and HYNIC-Ko-DPPB are able to form highly stable macrocyclic (99m)Tc complexes, [(99m)Tc(HYNIC-Ko-TPPB)(tricine)] and [(99m)Tc(HYNIC-Kp-DPPB)(tricine)], when tricine is used as the coligand. Radio-HPLC data suggest that the complex [(99m)Tc(HYNIC-Kp-DPPB)(tricine)] exists as only one detectable isomer in solution while the complex [(99m)Tc(HYNIC-Ko-DPPB)(tricine)] has three isomers. It was also found that three isomers of [(99m)Tc(HYNIC-Ko-DPPB)(tricine)] interconvert at elevated temperatures, suggesting that the presence of these isomers might be due conformational changes in the macrocyclic Tc chelate. The LC-MS data for both macrocyclic (99m)Tc complexes are completely consistent with the proposed composition. The phosphine-containing HYNIC chelators described in this study may have the potential as bifunctional chelators for (99m)Tc labeling of small biomolecules.     

Novel Tat-peptide chelates for direct transduction of technetium-99m and rhenium into human cells for imaging and radiotherapy

Rapid and efficient delivery of radioactive metal complexes to the cell interior would enable novel applications in medical imaging and radiotherapy. Membrane permeant peptide conjugates incorporating HIV-1 Tat transactivation protein sequences (GRKKRRQRRR) and an appropriate peptide-based motif (epsilon-KGC) that provides an N(3)S donor core for chelating technetium and rhenium were synthesized. Oxotechnetium(V) and oxorhenium(V) Tat-peptide complexes were prepared by facile transchelation reactions with permetalates, tin(II) chloride and sodium glucoheptonate. RP-HPLC showed two major [(99m)Tc]Tat-peptide species (4) that differed in retention time by approximately 2 min corresponding to two [Re]Tat-peptide species (7) shown to have identical mass, consistent with formation of two isomers, likely the oxo-metal diastereomers. [(99m)Tc]Tat-peptides were stable to transchelation in vitro. In human Jurkat cells, [(99m)Tc]Tat-peptide 4 showed concentrative cell accumulation (30-fold greater than extracellular concentration) and rapid uptake kinetics (t(1/2) < 2 min) in a diastereomeric-comparable manner. Paradoxically, uptake was enhanced in 4 degrees C buffer compared to 37 degrees C, while depolarization of membrane potential as well as inhibition of microtubule function and vesicular trafficking showed no inhibitory effect. Cells preloaded with 4 showed rapid washout kinetics into peptide-free solution. Modification of [(99m)Tc]Tat-peptide by deletion of the N-terminus Gly with or without biotinylation minimally impacted net cell uptake. In addition, the C-terminus thiol of the prototypic Tat-peptide was labeled with fluorescein-5-maleimide to yield conjugate 8. Fluorescence microscopy directly localized conjugate 8 to the cytosol and nuclei (possibly nucleolus) of human Jurkat, KB 3-1 and KB 8-5 tumor cells. Preliminary imaging studies in mice following intravenous administration of prototypic [(99m)Tc]Tat-peptide 4 showed an initial whole body distribution and rapid clearance by both renal and hepatobiliary excretion. Analysis of murine blood in vivo and human serum ex vivo revealed >95% intact complex, while murine urine in vivo showed 65% parent complex. Thus, these novel Tat-peptide chelate conjugates, capable of forming stable [Tc/Re(V)]complexes, rapidly translocate across cell membranes into intracellular compartments and can be readily derivatized for further targeted applications in molecular imaging and radiotherapy.     

Synthesis and comparative assessment of a labeled RGD peptide bearing two different ⁹⁹mTc-tricarbonyl chelators for potential use as targeted radiopharmaceutical

During the past decade radiolabeled RGD-peptides have been extensively studied to develop site-directed targeting vectors for integrins. Integrins are heterodimeric cell-surface adhesion receptors, which are upregulated in cancer cells and neovasculature during tumor angiogenesis and recognize the RGD aminoacid sequence. In the present study, we report the synthesis and development of two derivatives of the Nε-Lys derivatized cyclic Arg-Gly-Asp-D-Phe-Lys peptide, namely of cRGDfKHis and cRGDfK-CPA (CPA: 3-L-Cysteine Propionic Acid), radiolabeled via the [(99m)Tc(H(2)O)(3)(CO)(3)](+) metal aquaion at a high yield even at low concentrations of 10-5M (>87%) for cRGDfK-10-5M (>93%) for cRGDfK-CPA. Radiolabeled peptides were characterized with regard to their stability in saline, in His/Cys solutions, as well as in plasma, serum and tissue homogenates and were found to be practically stable. Internalization and efflux assays using αvβ3-receptor-positive MDA-MB 435 breast cancer cells showed a good percentage of quick internalization (29.1 ± 9.8% for (99m)Tc-HiscRGDfK and 37.0 ± 0.7% for (99m)Tc-CPA-cRGDfK at 15 min) and no retention of radioactivity for both derivatives. Their in vivo behavior was assessed in normal mice and pathological SCID mice bearing MDA-MB 435 ανβ3 positive breast tumors. Both presented fast blood clearance and elimination via both the urinary and hepatobiliary systems, with (99m)Tc-His-cRGDfK remaining for a longer time than (99m)Tc-CPA-cRGDfK in all organs examined. Tumor uptake 30 min pi was higher for (99m)Tc-CPAcRGDfK (4.2 ± 1.5% ID/g) than for (99m)Tc-His-cRGDfK (2.8 ± 1.5% ID/g). Dynamic scintigraphic studies showed that the tumor could be visualized better between 15 and 45 min pi for both radiolabeled compounds but low delineation occurred due to high abdominal background. It was finally noticed that the accumulated activity on the tumor site was depended on the size of the experimental tumor; the smaller the size, the higher was the radioactivity concentration.     

Radiosynthesis, biodistribution and micro-SPECT imaging study of dendrimer-avidin conjugate

Partially acetylated generation five polyamidoamine (PAMAM) dendrimer (G5-Ac) was reacted with biotin and 2-(p-isothiocyanatobenzyl)-6-methyl-diethylenetria minepentaacetic acid (1B4M-DTPA), respectively to form the complex Bt-G5-Ac-1B4M which was further conjugated with avidin to give the conjugate Av-G5-Ac-1B4M. Then both of the conjugates were radiolabeled with technetium-99m ((99m)Tc), respectively. Their in vitro cellular uptake study shows that the conjugate of Av-G5-Ac-1B4M-(99m)Tc exhibits much higher cellular uptake in HeLa cells than that of Bt-G5-Ac-1B4M-(99m)Tc. Accordingly the following evaluation such as in vitro/in vivo stability, biodistribution and micro-SPECT imaging was observed only for the conjugate of Av-G5-Ac-1B4M-(99m)Tc.