Synthesis of Bifunctional chelating agents to label monoclonal antibodies for radioimmunodiagnosis of cancer



Synthesis of Bifunctional Chelating Agents to label monoclonal antibodies for radioimmunodiagnosis of cancer


Radiopharmaceuticals are drugs containing a radionuclide and are used routinely in nuclear medicine for diagnosis and therapy of various diseases. Diagnostic radiopharmaceuticals contain high penetrating g (gamma) emitting radionuclide while the therapeutical contains high energy a (alpha) and b (beta) radiation emitting radionuclide. The invasive biopsy techniques ‘Radiodiagnosis’ utilize either metal based radiopharmaceuticals or biomolecule based radiopharmaceuticals. In the later case, the radioactive metal is attached by using bifunctional chelating agents, the organic chemical moiety which coordinates metals at one end and bear a reactive group on other side for conjugation to biomolecules. Thermodynamic and kinetic stability of metal complex along with reactivity of functional group to attach to biomolecule are central while choosing the efficient bifunctional chelating agents. In this thesis, new aromatic bifunctional chelating agents (5a-c) are evaluated for 99mTc scintigraphy. The thesis starts with a overview of various diagnostic modalities available and essentials of designing of radiopharmaceuticals, continues with study of available bifunctional chelating agents followed by the description of synthesis of new bifunctional chelating agents, radiocomplexation studies with 99mTc radionuclide, quality control studies with complexes, biodistribution studies and tumor imaging studies using EGFr mAb and avidine-biotin bioconjugates. Further synthesis and evaluation of radiocomplex of modified antibacterial drug is described for in-vivo imaging of internal bacterial infection sites. A chemical transformation observed during the synthesis of BFCs, which lead to benzothiazine heterocycle is also included.  


The Introduction (Chapter 1) entitled “Introduction to diagnostic modalities and basic of radiopharmaceuticals” explains some of the basic principles of Nuclear Medicine, a medical specialty that deals with application of radioactive substances for diagnosis and therapy. Further details about the metaloradiopharmaceuticals designing are included. Basically there are four main diagnostic modalities (SPECT, PET, MRI, US), that are frequently used for diagnostic purpose. SPECT (Single photon emission computed tomography) and PET (Positron emission tomography) are based on the radioactive properties of elements, while MRI (Magnetic Resonance Imaging) makes use of paramagnetic property of metals.

Radiopharmaceuticals are of two types: metal essential and biomolecules based. In metal essential radiopharmaceuticals, the biodistribution and diagnosis are dependent on the metal complex, while the biodistribution and diagnosis is determined by bioligands in case of biomolecules based radiopharmaceuticals. In the latter case, various bioligands viz. Antibodies, peptides, steroids receptor ligands, folate receptor ligand, somatostatin analogues, CCK analogues have been evaluated for tumor diagnosis. Designing of radiopharmaceuticals depends upon the type of tumor or malignancy to be diagnosed or treated, type of metal to be used, purity of radiopharmaceuticals, stability of metal complex in in vivo and biodistribution of radiopharmaceutical. Keeping these factors in view, synthesis of new chelating agents to radiolabel the monoclonal antibodies and avidin-biotin system (for pretargeting strategy) was planned for this study. Besides that radiolabeling of antibacterial drugs (after suitable modification) for infection imaging was kept objective for this study.


Chapter 2 entitled “Synthesis and evaluation of bifunctional chelating agents and their bioconjugation with anti-EGFr for tumor imaging” includes the synthesis of new bifunctional chelating agents, qualitiy control studies with 99mTc radionuclide and evaluation in vivo for scintigraphic application for tumor imaging with anti-EGFr antibodies.

Bifunctional chelating agents (BFCs) are chemical entities that have a coordinating sphere for complexation of metals on one end and a reactive functional group for conjugation with biomolecules at other end. The selection of bifunctional chelating agents depends on the radiometal chemistry, coordination chemisty of metal and pharmacological property requirement of radiopharmaceutical. For the efficient in vivo stability of metal complexes, denticity requirement, bonding unit and donor atoms, the macrocyclic effect of coordinating sphere and ligand framework of BFCs is considered. Various chelating agents e.g. DTPA, DOTA, TETA, NOTA, HYNIC etc. are being used in the clinical applications. A brief review of synthetic strategies reported for the synthesis of these chelating agents is described. Taking lead from the previous reported studies, macrocyclic BFCs based on the Bis(2-aminophenyl thio) alkane moiety were designed and synthesized. Bifunctional chelatig agnets 5a-c were synthesized by macrocyclization of bis (2-aminophenyl thio) alkanes with 3,5-bis-bromomethylnitrobenzene followed by reduction of nitro group using Zn/NH2NH2. The  bifunctional chelating agents were characterized on the basis of spectroscopic analysis (FT-IT, NMR and mass spectra). Radiocomplexes formed by these chelating agents 5a-c with radioactive 99mTc were obtained in high yield. Among the three chelating agents used, 5c showed maximum complexation with 98% purity followed by 5b (92-94%) with lowest being of 5a (82%). 99mTc-5b and 99mTc-5c are stable in serum under physiological conditions. The blood clearance of complexes was slower, leaving 14% of activity in the blood after 8h. The bioconjugation of BFC 5b to monoclonal antibody anti-EGFr was done using 2-hydroxyacetaldehyde by Amadori rearrangement. The bioconjugate anti-EGFr-5b labeled with 99mTc showed high labeling with stability of radiobioconjugate as was observed with the BFC 5b. The EAT (Eherlic Ascite Tumor) developed in right thigh of mice was imaged using this conjugate under gamma camera which showed good localization of radiobioconjugate in tumor site. This clearly contrasted with non-tumor site of left thigh where no or negligible radio activity was present and thus indicated the successful utilization of aromatic BFCs for scintigraphic applications.


Chapter 3 entitled “Pretargeting Approach: Conjugation and evaluation of Avidin-Biotin system for tumor imaging” describe the pretargeting strategy for tumor imaging by using Avidine (or Streptavidine) Biotin system. The concept of pretargeting has been utilized for the enhancing the contrast (Signal to background radiation) in imaging of different tumors. The BFC-5b-c were conjugated with NHS ester of biotin to obtain Biotin-5b conjugate (B5bC) and Biotin-5c conjugate (B5cC). The radiolabeling procedure followed was essentially similar to that standardized for unconjugated chelating agents. 99mTc-5b-biotin (99mTcB5bC) was prepared with more than 95 percent labeling purity while 99mTc-5c-biotin (99mTc-B5cC) was obtained in more than 98 percent radiochemical purity. The uptake of radiopharmaceuticals in EAT (Ehrlich Ascite Tumors) cells was calculated as the ratio of the radiopharmaceutical/free 99mTcO4Na. The mean ratio of 99mTcB5bC/Na99mTcO4 was 50:1. EAT tumor generated in right thigh of mice was taken for evaluation in vivo. The avidin was injected subcutaneously followed by i.v. injection of 99mTc-B5bC through tail of tumor bearing mice. At the interval of 4 hours, major distribution in tumor was observed as indicated by biodistribution studies and in vivo images. The BFCs thus could be successfully utilized for pretargeting approach.


Chapter 4 entitled “Synthesis and evaluation of 4-aminosalicyclic acid derivatives for infection imaging” describe the derivatization of a commercially available antibacterial drug for the radiolabeling with 99mTc radionuclide and subsequently utilization as infection imaging agent. PAS or p-aminosalicylic acid on derivatization with DTPA dianhydride yielded BPAS-DTPA (N1,N3-bis{N-(3-hydroxy-4-carboxyphenyl) methylenecarboxamide} diethylenetriamine-N1,N2,N3-triacetic acid).  BPAS-DTPA was radiolabeled with 99mTc in presence of reducing agent SnCl2. After quality control studies the complex was used for imaging the infections in mice (in vivo). As such complex was distributed in liver and kidneys in major amount and thus had renal route of excretion as indicated by in vivo imaging in Rabbit. The infections of S. aureus and P. aureogonosa developed in thigh muscle of mice were imaged using above complex. The complex showed maximum localization of after 1 hours of injection with clear contrast from non-infectious site leaving a future perspective for further evaluation of the drug.


Chapter 5 entitled “Synthesis of 3-Oxo-3,4-dihydro-2H-1,4-benzothiazine” describes a transformation observed during the synthesis of macrocyclic chelating agents. The varied transformation was observed during the cyclization of bis(2-aminophenylthio)propane with bromoacetylbromide where the product obtained was 3-oxo-3,4-dihydro-2H-1,4-benzothiazine (1) instead of expected macrocycle. Further evaluation with other 2-aminophenyl thioalkanes also gave the 1 as sole product. This thus established a general transformation of 2-aminophenylthioalkanes with bromoacetylbromide to form 3-oxo-3,4-dihydro-2H-1,4-benzothiazine (1) heterocycle.