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Unlabelled: The BB2 receptor subtype, of the bombesin family of receptors, has been shown to be highly overexpressed in a variety of human tumors, including prostate cancer. Bombesin (BBN), a 14-amino acid peptide, has been shown to target the BB2 receptor with high affinity. 64Cu (half-life = 12.7 h, beta+: 18%, E(beta+ max) = 653 keV; beta-: 37%, E(beta- max) = 578 keV) is a radioisotope that has clinical potential for application in both diagnostic imaging and radionuclide therapy. Recently, new chelation systems such as 1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid (CB-TE2A) have been reported to significantly stabilize the 64Cu radiometal in vivo. The increased stability of the 64Cu-CB-TE2A chelate complex has been shown to significantly reduce nontarget retention compared with tetraazamacrocycles such as 1,4,7,10-tetraazacyclodoadecane-N,N',N'',N'''-tetraacetic acid (DOTA). The aim of this study was to determine whether the CB-TE2A chelation system could significantly improve the in vivo stability of 64Cu bombesin analogs. The study directly compares 64Cu bombesin analogs using the CB-TE2A and DOTA chelation systems in a prostate cancer xenograft SCID (severely compromised immunodeficient) mouse model., Methods: The CB-TE2A-8-AOC-BBN(7-14)NH2 and DOTA-8-AOC-BBN(7-14)NH2 conjugates were synthesized and radiolabeled with 64Cu. The receptor-binding affinity and internalization profile of each metallated conjugate was evaluated using PC-3 cells. Pharmacokinetic and small-animal PET/CT studies were performed using female SCID mice bearing PC-3 xenografts., Results: In vivo BB2 receptor targeting was confirmed by tumor uptake values of 6.95 +/- 2.27 and 4.95 +/- 0.91 %ID/g (percentage injected dose per gram) at the 15-min time point for the 64Cu-CB-TE2A and 64Cu-DOTA radioconjugates, respectively. At the 24-h time point, liver uptake was substantially reduced for the 64Cu-CB-TE2A radioconjugate (0.21 +/- 0.06 %ID/g) compared with the 64Cu-DOTA radioconjugate (7.80 +/- 1.51 %ID/g). The 64Cu-CB-TE2A-8-AOC-BBN(7-14)NH2 radioconjugate demonstrated significant clearance, 98.60 +/- 0.28 %ID, from the mouse at 24 h after injection. In contrast, only 67.84 +/- 5.43 %ID of the 64Cu activity was excreted using the 64Cu-DOTA-8-AOC-BBN(7-14)NH2 radioconjugate because of nontarget retention., Conclusion: The pharmacokinetic and small-animal PET/CT studies demonstrate significantly improved nontarget tissue clearance for the 64Cu-CB-TE2A8-AOC-BBN(7-14)NH2. This is attributed to the improved in vivo stability of the 64Cu-CB-TE2A chelate complex as compared with the 64Cu-DOTA chelate complex.

Three human Escherichia coli heat-stable peptide (STh) analogues, each containing a DOTA chelating group, were synthesized by SPPS and oxidative refolding and compared in in vitro and in vivo systems. One analogue, DOTA-F19-STh(1-19), contains an N-terminal DOTA group attached via an amide bond linkage to an STh moiety which is essentially wild-type except for a Tyr to Phe alteration at position 19 of the molecule. A second analogue, DOTA-R1,4,F19-STh(1-19), differs from the first in that asparagine residues in positions 1 and 4 have been altered to arginine residues in order to examine the effect of positively charged groups in the linker domain. A third analogue, DOTA-11AUN-F19-STh(1-19), differs from the first in that it incorporates an 11-aminoundecanoic acid spacer group between the DOTA group and the first asparagine residue. In vitro competitive binding assays utilizing T-84 human colon cancer cells demonstrated that significant alterations to the N-terminal region of the STh molecule were well tolerated and did not significantly affect binding affinity of STh for the guanylyl cyclase C (GC-C) receptor. Internalization and efflux studies of the indium-labeled species demonstrated that inclusion of positive charge in the linker moiety inhibits internalization of the compound within tumor cells. The characteristics of the three analogues were compared in an in vivo model utilizing T-84 human colon cancer cell xenografts in SCID mice. Clearance of all analogues was rapid, primarily via renal excretion into the urine, with >89% ID excreted into the urine at 1 h pi for all analogues. The 111In-DOTA-R1,4,F19-STh(1-19) and 111In-DOTA-11AUN-F19-STh(1-19) analogues both had longer residence times in the blood than did the 111In-DOTA-F19-STh(1-19) analogue, probably accounting for increased %ID/g values for tumors and nontarget tissues at 1 h pi. At 4 h pi, significant differences between analogues were only seen with respect to metabolic routes of excretion, indicating that increased blood residence time did not result in increased tumor residualization. Reduction of hepatic uptake of these compounds, however, could have significance in the development of agents for the imaging of hepatic metastases. The ability to manipulate in vivo pharmacodynamics and tumor uptake of radiolabeled STh peptides through modification of linker moieties is under continuing investigation in order to produce optimal imaging and therapeutic radiopharmaceuticals.

Functionalization of perfluoro aryl azides by bifunctional chelating agents (BFCAs) capable of forming high specific activity complexes with (99m)Tc (for gamma-imaging) and (188)Re (for radiotherapy) is described. The synthesis of multidonor BFCAs containing N(2)S(2), N(4), and N(3)S donor groups containing imidazole, pyridine, and pyrazine functionalities that may be important for tuning the pharmacokinetic parameters is also described. Functionalization of perfluoro aryl azides at various sites on BFCAs yields novel bifunctional photolabile chelating agents (BFPCAs) that are useful for covalent attachment to biomolecules. A representative Re-BFPCA 8a in a model solvent, diethylamine, proceeded to give a high yield of intermolecular NH insertion product without the decomplexation of the metal ion from 8a. All products originated from the photolysis of 8a in diethylamine are characterized by analytical techniques, and a plausible mechanism of formation of different photolytic products is suggested. The high yield of intermolecular NH insertion of Re-BFPCA 8a is extended to labeling of human serum albumin (HSA) and Fab fragments under aqueous conditions. The photolabeling technology developed here offers a new way to attach diagnostically and therapeutically useful radiotracers (e.g., (99m)Tc, (188)Re) to Fab fragments for potential noninvasive imaging and therapy of cancer.

A diamido-dihydroxymethylenephosphine (N(2)P(2)) bifunction chelating agent (BFCA) was shown to form well-defined (99m)Tc- and (188)Re-chelate structures. The 4, 4-bis [bis-hydroxymethyl-phosphonyl-propylcarbonmoyl]-butyric acid bifunctional chelating agent (N(2)P(2)-BFCA) formed stable complexes with (99m)Tc and (188)Re in >95% yield with high radiochemical purity (RCP). The biodistribution of the (99m)Tc- and (188)Re-N(2)P(2)-BFCAs after intravenous injection studied in normal mice showed the activity was excreted primarily via renal-urinary pathway indicating their use for labeling peptides with (99m)Tc and (188)Re.

The water-soluble dithio-bis(hydroxymethyl)phosphine ligands (HOH2C)2P(CH2)2 S(CH2)3S(CH2)2P(CH2OH)2 and (HOH2C)2P(CH2)3S(CH2)3S(CH2)3P(CH2OH)2 were complexed with 99mTc. The 99mTc-P2S2 complexes were formed in high radiochemical purity by simple mixing of 99mTcO-4 with the ligands or by transchelation from 99mTc-citrate. The 99mTc-P2S2 complexes were stable over a wide range of pHs and did not undergo in vitro decomposition for < or = 24 h. High performance liquid chromatographic analysis indicated the formation of singular chemical species. Retention times for each of the new 99mTc-P2S2 complexes are identical to those of corresponding Re(V) complexes, suggesting similar chemical species at the tracer level. Results of this study suggest that the combination of thioether and (hydroxymethyl)phosphine donor centers in new, multidentate ligand frameworks might aid in the development of new bifunctional chelating agents for the use of radiolabeling specific biomolecules.

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.

A bifunctional chelating agent (BFCA) based on a macrocyclic amine, cyclam, was used to form specifically 99mTc labeled rabbit-anti-HSA-antibodies. This study demonstrates the feasibility of forming a highly stable cyclam-based 99mTc-BFCA with its subsequent conjugation to antibodies.

A series of stable complexes of 153Sm has been produced using multidentate acetate and phosphonate ligands. Biodistribution studies in unanesthetized rats showed varying degrees of bone and soft-tissue uptake for these complexes. Of the complexes studied, [153Sm] ethylenediaminetetramethylenephosphonate (EDTMP) showed the best combination of high bone uptake, low nonosseous uptake, and rapid blood clearance which warranted its further investigation in rabbits. Rabbit studies confirmed the [153Sm]EDTMP results obtained in rats. Blood clearance in rabbits was found to be more rapid than [99mTc] methylene diphosphonate (MDP). Scintigraphic images were virtually indistinguishable from [99mTc]MDP images. Lesion/normal bone ratios were determined from digitized images obtained using a drill hole model and found to be approximately 17:1. Based on these excellent biodistribution characteristics, [153Sm] EDTMP could be therapeutically useful in treating metastatic bone cancer.

The structure-activity relationships involved in the in vivo localization and clearance of 99mTc-chelates is reviewed. Most 99mTc-chelates used for renal studies are mixed function agents and their uptake in the kidney is affected by the presence or absence of stannous ions; only a relatively few chelates are almost exclusively cleared from the body by glomerular filtration or tubular excretion. The uptake of 99mTc-phosphates and -phosphonates in metabolically active and metabolically inactive bone and the plasma clearance rate of these complexes depend upon the structure of the complexing ligand. 99mTc-chelates considered optimal for hepatobiliary imaging have a high liver extraction efficiency, are rapidly transported through the liver and have a comparatively slow renal clearance rate. Even though the elucidation of the in vivo behavior of 99mTc-chelates continues, quantitating structure-activity relationships requires that the structure of the chelates be carefully characterized, both before and after injection.