The bivalent ligand approach is very promising and can give rise to molecular probes with enhanced conformational flexibility for tissue selective modulation. In this study we describe the synthesis of a homodimeric 5-HT1A receptor ligand by incorporating two identical pharmacophores , 1-(2methoxyphenyl)piperazine, linked through DTPA. This bivalent derivative was efficiently synthesized and characterized by 1 H, 13 C NMR and mass spectroscopy. 99m Tc-labeling was performed with a high radiolabeling yield (>95%) and radiochemical purity (>98%) using very low ligand concentration. In vitro studies were carried out on hippocampal cultures and PC3 cell lines which express 5-HT1A receptors. The selectivity of the complex for 5-HT1A receptors is 1000 times more than it is for 5-HT2A receptors, with K d in picomolar range. Hill coefficients between 1.9 and 2.3 were observed, indicating that the homodimeric binding pharmacophores appear to be essential for the cooperative binding mode. In vitro binding assays in rat hippocampal cultures demonstrated the high affinity of the complex for 5-HT1A receptors. Further studies include in vivo organ distribution and gamma scintigraphy carried out in rat and rabbit. Plasma clearance rate (C R ) revealed a value of 4.86 mg mL À1 min À1 in normal rabbit. Dynamic imaging performed in rabbit showed the beginning of radiolabeled uptake in brain as early as 2 min. The tumor-to-contralateral muscle tissue ratio of 99m Tc-DTPA-bis(MPBA) in athymic mice with PC3 xenograft was found to be 65 AE 3.3 at 1 h. Significant accumulation was seen in mice and rat brain at 10 min with 2.07 AE 0.76% ID g À1 and 2.81% ID g À1 respectively. The high uptake in hippocampus and cerebral cortex was accredited to the 5-HT1A receptor rich regions in post mortem rat brain. This imaging agent holds a promising future in imaging 5-HT1A receptors for the effective diagnosis of neuropathological disorders.

A new macrocyclic system 2,2'-(12-amino-11,13-dioxo-1,4,7,10-tetraazacyclotridecane-4,7-diyl)diacetic acid (ATRIDAT) was designed for coordinating metals in +2 and +3 oxidation states particularly Ga(III), for PET imaging. ATRIDAT was conjugated to d-biotin for pretargeting via biotin-avidin interaction. This model provides high tumor targeting efficiency and stability to biotinidase activity leading to modest signal amplification at the tumor site. Cyclization of triethylenetetramine with protected diethylamino malonate resulted in the formation of 13 membered diamide ring. d-Biotin was then anchored on the pendant amine rendering α-methyne carbon to the biotinamide bond which blocks the biotinidase enzyme activity. Biotinidase stability assay showed remarkable stability toward the action of biotinidase with ∼95% remaining intact after treatment following 4 h. Binding affinity experiments such as HABA assay, competitive displacement studies with d-biotin and CD showed high binding affinity of the molecule with avidin in nanomolar range. Biotin conjugate was successfully radiolabeled withGa(III) with radiolabeling efficiency of ∼70% and then purified to get 99.9% radiochemical yield. IC of the compound was found to be 2.36 mM in HEK cell line and 0.82 mM in A549 as assessed in MTT assay. In biodistribution studies, the major route of excretion was found to be renal. Significant uptake of 4.15 ± 0.35% was observed in tumor in the avidin pretreated mouse at 1 h. μPET images also showed a high tumor to muscle ratio of 26.8 and tumor to kidney ratio of 1.74 at 1 h post-injection after avidin treatment.

We investigated the potential of DTPA-bis(Methionine), a target specific amino acid based probe for detection of L-type amino acid transporters (LAT1) known to over express in proliferating tumours using multimodality imaging. The ligand, DTPA-bis(Met) was readily converted to lanthanide complexes and was found capable of targeting cancer cells using multimodality imaging. DTPA-bis(Met) complexes were synthesized and characterized by mass spectroscopy. MR longitudinal relaxivity, r₁ = 4.067 ± 0.31 mM⁻¹s⁻¹ and transverse relaxivity, r₂ = 8.61 ± 0.07 mM⁻¹s⁻¹ of Gd(III)-DTPA-bis(Met) were observed at pH 7.4 at 7 T. Bright, localized fluorescence of Eu(III)-DTPA-bis(Met) was observed with standard microscopy and displacement studies indicated ligand functionality. K(D) value determined for Eu(III)-DTPA-bis(Met) on U-87 MG cells was found to be 17.3 pM and showed appreciable fluorescence within the cells. Radio HPLC showed a radiochemical purity more than 95% (specific activity = 400-500 MBq/μmol, labelling efficiency 78 %) for ⁶⁸Ga(III)-DTPA-bis(Met). Pre-treatment of xenografted U-87 MG athymic mice with ⁶⁸Ga(III)-DTPA-bis(Met) following unlabelled L-methionine administration reduced tumour uptake by 10-folds in Micro PET. These data support the specific binding of ⁶⁸Ga(III)-DTPA-bis(Met) to the LAT1 transporter. To summarize, this agent possesses high stability in biological environment and exhibits effective interaction with its LAT1 transporters giving high accumulation in tumour area, excellent tumour/non-tumour ratio and low non-specific retention in vivo.

An efficient approach in the design and synthesis of a multi-functional chelating agent based on 1-(2-methoxyphenyl)piperazine for targeting 5-HT1A receptors in brain was envisaged.