99mTc Labeled L-Tryptophan Conjugated Macrocyclic Chelating Agent: Synthesis, Characterization and In Vitro & In Vivo Biological Studies

Target specific delivery of the radiolabeled compound to cancer cell to avoid possible uptake/damage to the surrounding normal healthy cells is an important aspect of tumour imaging and therapy. Fictionalisation of molecules with amino acid analogues has been proved a diverse and useful class of PET and SPECT tracers that records amino acid transportation and uptake exhibited by many tumour cells i.e. brain, neuroendocrine, prostate cancer [1-5].


Introduction
Target specific delivery of the radiolabeled compound to cancer cell to avoid possible uptake/damage to the surrounding normal healthy cells is an important aspect of tumour imaging and therapy. Fictionalisation of molecules with amino acid analogues has been proved a diverse and useful class of PET and SPECT tracers that records amino acid transportation and uptake exhibited by many tumour cells i.e. brain, neuroendocrine, prostate cancer [1][2][3][4][5].
Tumor cells show property of unlimited malignant growth and they require amino acid for proliferation thus amino acid transport generally increased malignant transportation. Increase rate for amino acid uptake is one of the earliest and most important events associated with proliferation. There are many evidences show that IDO (indoleamine 2, 3-dioxygenase) is over expressed in a variety of human tumors, including lung tumors resulting increase of abnormal tryptophan metabolism via the kynurenine pathway. L-Tryptophan is a substrate of IDO [5][6][7][8]. Intracellular IDO activity may result in the trapping of polar tryptophan (or their derivatives) metabolites or indirect accumulation via intracellular L-tryptophan depletion [9,10].
For the development of target specific amino acid based imaging system that can be utilized for imaging techniques viz. PET, SPECT and magnetic resonance imaging (MRI), the amino acids are conjugated with macrcyclic chelating agents which have ability to bind with metal ions to yield thermodynamically stable and kinetically inert metal chelates. For complexation with metal ions, macrocyclic polyamines, particularly tetraazacy cloalkanes, have gained a great deal of importance due to their ligating and biological properties [11][12][13]. Their strong affinity and selective binding with certain metals make them very useful in the field of radioactive diagnosis. An alternative to the conventional N-substituted macrocyclic frameworks is the functionalization of the carbon skeleton of the macro cycle which restores the reactivity of the secondary amines making them available for reaction with other coordinating groups [14]. 99 mTc is one of the most desirable radionuclide for imaging in diagnostic radiopharmaceuticals because of its optimal nuclear-physical properties and easy availability. The emission of gamma rays of optimal energy (140keV), a suitable halflife (6h), and availability from 99 Mo-99 mTc generator systems, excellent imaging properties, favourable dosimetry, and high specific activity makes 99 mTc a logical choice for labelling [15]. Taking into account these features of the high stability and kinetic inertness and our interest for the development of radiolabeled complexes suitable for SPECT imaging, we have synthesized and characterized the tetraaza macrocyclic chelator 16-[methyl-3-(1H-indol-3-yl)-2 (2(amino)acetamido)] 3,6,9,12-tetraazabicyclo-[12.3.1] octadeca-1,14,16-triene-2,13dione, 16Trp4A. Biological studies of this radiolabeled chelator were performed to explore their application as SPECT tumor imaging agent.

Synthesis of methyl-2-(2-chloro acetamido)-3-(1Hindol-3-yl) propanoate (2):
The methyl ester of L-tryptophan was dissolved in water (20mL). The chloro-acetylchloride (1.2eq.) in dichloromethane (20mL) and K 2 CO 3 solution in water (1.2eq.) were added slowly to the stirring solution at 00C using a pressure equalized dropping funnel. The resulting reaction mixture was stirred at room temperature overnight. After completion of the reaction, the pale yellow viscous oil was washed with H 2 O (2x20mL), 0.1M HCl (2x20mL) and brine (2x20mL). The organic layer was dried over MgSO 4 , filtered and the solvent was removed under reduced pressure to yield pure chloroacetylated product (2)

In vitro cytotoxicity determination
Cell culture: The human cancer cell lines U-87 and U-373 (glioblastoma) were procured from the National Centre for Cell Science (NCCS), Pune, India. The cells (glioblastoma U-87, U373, cervical U756 and HeLa) were cultured and maintained in complete Dulbecco's modified Eagle's medium (DMEM, Invitrogen) as per supplier's specifications supplemented with 10% heat inactivated fetal bovine serum (FBS) and 1% antibiotic cocktail (Himedia, India) at 37°C in a humidified atmosphere with 5% CO 2 .
Cytotoxicity assay: MTT assay was performed to assess the cytotoxicity. For measurement of the cell proliferation, exponentially growing cells (7,000 cells/well) were seeded in 96-well microtitre plate in triplicate and cultured in complete Dulbecco's modified Eagle's medium (DMEM). The cells were treated with different concentration of the ligand (TBPD and OPTT individually dissolved in deionized water) for cytotoxicity determination. Following exposure to the ligands, cell viability was assessed by using 200μg/mL of 3-(4, 5-dimethyl-2thiazoyl)-2,5-diphenyltetrazoliumbromide (Sigma-Aldrich) as per manufacturer's protocol. For measurement of the cell proliferation, cells (U-87, U-373, SW756 and HeLa) were seeded in 96-well plates (7,000 cells/well) in triplicate and cultured in complete Dulbecco's modified Eagle's medium (DMEM). Cells were treated with varying concentrations (0.001mM to 1.0mM) of ligands for different time intervals (24,48,72h). After treatment, the cells were incubated with MTT for 4h. The yellow tetrazolium salt (MTT) is reduced in metabolically active cells to form insoluble purple formazan crystals, which were solubilised by the addition of DMSO. Absorbance was measured at 570nm in a multi-well plate reader (BIO-RAD model 680 micro plate reader version 1.70), and the absorbance values of treated cells were compared with the absorbance values of untreated cells.

Radio labeling:
The compound was radiolabeled with 99 mTc using SnCl 2 as reducing agent. All the labeling parameters such as pH, concentration of reducing agent etc. were standardized, to achieve the maximum labeling efficiency. In vitro and in vivo stability of the labelled complexes was checked and the complexes were found to be stable for 24h under physiological conditions. The labelling yield was found to be greater than 95%, as determined chromatographically by different solvent systems. The reaction mixture was kept in saline for different time intervals to carry out in vitro stability studies. Percentage radio labeling was calculated for 0, 2, 4, 6, and 24h. The metal binding assay confirmed the radiochemical purity to be >99%. Radio chromatogram showed a single peak for the ligand which was found intact over 6h.
The developed target specific biologically active molecule conjugated with macrocyclic ligand was labeled with 99 mTc through a facile radio labeling protocol with greater than 95% labeling efficiency and high radiochemical purity. The compound was radiolabeled with 99 mTc using SnCl 2 as reducing agent. All the labeling parameters such as pH, concentration of reducing agent etc. were standardized, to achieve the maximum labeling efficiency. Biodistribution studies in mice revealed significant uptake in blood, lung, liver, kidney, spleen and intestine initially, which decreases as time passes up to 24h.