Evaluation of the effect of a cell penetrating peptide (TAT) towards tailoring the targeting efficacy and tumor uptake of porphyrin.

Cell penetrating peptides (CPPs) are known to possess a unique capacity to penetrate biological membranes and translocate various molecules into the cells. Therefore, porphyrin-CPP conjugates could be envisaged to boost the intracellular delivery of porphyrins thereby providing an improved tool for the development of agents for multi-modal applications for cancer management. Working in this direction, an unsymmetrically substituted porphyrin derivative was conjugated with a transactivating transcriptional activator peptide (TAT) and various in vitro and in vivo studies were carried out in order to study the effect of adding a CPP to the porphyrin derivative. MTT assay revealed the preferential light dependent toxicity of the porphyrin derivative which was further enhanced upon peptide conjugation. Fluorescence and flow cytometry studies revealed the relatively higher cellular internalization of the porphyrin-TAT conjugate in comparison with the porphyrin derivative. The elevated light dependent cell toxicity of the porphyrin-TAT conjugate along with its capability of generating cytotoxic singlet oxygen indicated the advantages of using the porphyrin-TAT conjugate for PDT applications. Also, porphyrin and the porphyrin-peptide conjugate were radiolabelled with 68Ga to investigate their possible potential as PET agents. In vivo biodistribution studies revealed a higher tumor uptake for the 68Ga-porphyrin-TAT conjugate (6.32 ± 1.24% IA per g) than for 68Ga-porphyrin (2.45 ± 0.88% IA per g) at 60 min post-administration. However, the observation of a higher non-target retention of the radiolabelled agents during in vivo studies might pose a limitation on their possible application in PET imaging.

Antibody-based Radiopharmaceuticals as Theranostic Agents: An Overview.

Since the inception of antibodies as magic bullets for targeting antigens with high specificity for various in vitro and in-vivo detection and therapy applications, the field has evolved, and remarkable success has been achieved not only in the methods of development of these targeting agents but also in their applications. The utilization of these moieties for the development of antibody-based radiopharmaceuticals for diagnostic and therapy (theranostic) purposes has resulted in the availability of various cancer-targeting agents suitable for clinical applications. The high affinity and specificity of antibodies towards the target antigens overexpressed on tumors render them an excellent carrier molecules for radionuclide delivery. Although intact antibodies have high potential as imaging and therapeutic agents, a major drawback of intact antibody-based radionuclide targeting is their slow pharmacokinetics and poor penetration into solid tumors. In contrast to large intact antibodies, engineered antibody fragments, such as minibodies, diabodies, single-chain variable region fragments (scFvs), nanobodies, and non-antibody protein scaffolds-based moieties, retain the specificities and affinities of intact antibodies in addition to improved pharmacokinetics for imaging and therapy of solid tumors. These engineered carrier molecules are not only amenable for simple and robust radiolabeling procedures but also provide high contrast images with minimal radiotoxicity to vital organs. However, in various instances, rapid clearance with sub-optimal tumor accumulation, limiting renal dose, and cross-reactivity of these radiolabeled engineered smaller molecules have also been observed. Herein, we review current knowledge of the recent methods for the development of antibody-based targeting moieties, the suitability of various engineered formats for targeting tumors, and radiolabeling strategies for the development of radioformulations. We discuss promising antibody-based and non-antibody- based affibody radiopharmaceuticals reported for clinical applications. Finally, we highlight how emerging technologies in antibody engineering and drug development can be amalgamated for designing novel strategies for cancer imaging and therapy.

Effect of structural variation on tumor targeting efficacy of cationically charged porphyrin derivatives: Comparative in-vitro and in-vivo evaluation for possible potential in PET and PDT.

tetracationic (TMPyP) and tricationic porphyrin (TriMPyCOOHP) derivatives were synthesized, characterized and investigated for binding with DNA by Isothermal Titration Calorimetry as well as by UV-Vis spectroscopy in order to study the effect of structural variation on tumor targeting efficacy of cationically charged porphyrin derivatives. Fluorescence cell imaging studies performed in cancer cell lines corroborated the findings of aforementioned studies. Photocytotoxicity experiments in A549 cell lines revealed relatively higher light dependent cytotoxic effects exerted by TMPyP compared to TriMPyCOOHP. In-vivo experiments in tumor bearing animal model revealed relatively longer retention of 68Ga-TMPyP in tumorous lesion compared to that of 68Ga-TriMPyCOOHP. The study reveals that removal of one of the positive charges of the tetracationic porphyrin derivatives significantly reduces their DNA binding ability and cytotoxicity as well as brings changes in the pharmacokinetic pattern and tumor retention in small animal model.

Targeted Tumor Therapy with Radiolabeled DNA Intercalator: A Possibility? Preclinical Investigations with 177Lu-Acridine.

OBJECTIVE: A DNA intercalating agent reversibly stacks between the adjacent base pairs of DNA and thus is expected to exhibit preferential localization in the tumorous lesions as tumors are associated with enhanced DNA replication. Therefore, radiolabeled DNA intercalators are supposed to have potential to be used in targeted tumor therapy. Working in this direction, an attempt was made to radiolabel 9-aminoacridine, a DNA intercalator, with 177Lu, one of the most useful therapeutic radionuclides, and study the potential of 177Lu-acridine in targeted tumor therapy. Experiments. 9-Aminoacridine was coupled with p-NCS-benzyl-DOTA to facilitate radiolabeling, and the conjugate was radiolabeled with 177Lu. Different reaction parameters were optimized in order to obtain 177Lu-acridine complex with maximum radiochemical purity. In vitro stability of the radiolabeled complex was studied in normal saline and human blood serum. Biological behavior of the radiolabeled agent was studied both in vitro and in vivo using the Raji cell line and fibrosarcoma tumor-bearing Swiss mice, respectively. RESULTS: 177Lu-acridine complex was obtained with ~100% radiochemical purity under the optimized reaction conditions involving incubation of 1.5 mg/mL of ligand with 177Lu (1 mCi, 37 MBq) at 100°C at pH ~5 for 45 minutes. The complex maintained a radiochemical purity of >85% in saline at 6 d and >70% in human serum at 2 d postpreparation. In vitro cellular study showed uptake of the radiotracer (5.3 ± 0.13%) in the Raji cells along with significant cytotoxicity (78.06 ± 2.31% after 6 d). Biodistribution study revealed considerable accumulation of the radiotracer in tumor 9.98 ± 0.13 %ID/g within 1 h postadministration and retention therein till 6 d postadministration 4.00 ± 0.16 %ID/g with encouraging tumor to nontarget organ uptake ratios. CONCLUSIONS: The present study, although preliminary, indicates the potential of 177Lu-acridine and thus radiolabeled DNA intercalators in targeted tumor therapy. However, further detailed evaluation is required to explore the actual potential of such agents in targeted tumor therapy.

Preparation and Preliminary Evaluation of 68Ga-Acridine: An Attempt to Study the Potential of Radiolabeled DNA Intercalator as a PET Radiotracer for Tumor Imaging.

INTRODUCTION: Acridine is a well-known DNA intercalator and thereby gets easily inserted within DNA. As uncontrolled rapid cell division is one of the primary characteristics of the tumors, it is expected that acridine or its suitable derivatives will have preferential accumulation in the tumorous lesions. Therefore, an attempt was made to radiolabel an acridine derivative with 68Ga and study the potential of the 68Ga-acridine complex as a PET agent for tumor imaging. METHODS: 9-aminoacridine was coupled with p-NCS-benzyl-DOTA to render it suitable for labeling with 68Ga. The purified acridine-DOTA conjugate was radiolabeled with 68Ga, eluted from a 68Ge/68Ga radionuclide generator. Various radiolabeling parameters were optimized and the stability of the radiolabeled preparation was studied. The biological behavior of the 68Ga-acridine complex was studied both in vitro and in vivo using Raji cell line and fibrosarcoma tumor bearing Swiss mice, respectively. RESULTS: 68Ga-acridine complex was obtained with ~100% radiochemical purity under the optimized reaction conditions involving incubation of 2mg/mL of ligand at 100°C for 30 minutes. The complex maintained a radiochemical purity of >95% in normal saline and >65% in human blood serum at 3h post-incubation. In vitro cellular study showed (3.2±0.1)% uptake of the radiotracer in the Raji cells. Biodistribution study revealed significant tumor accumulation [(11.41±0.41)% injected activity in per gram] of the radiotracer within 1h postadministration along with uptake in other non-target organs such as, blood, liver, GIT kidney etc. Conclusion: The present study indicates the potential of 68Ga-acridine as a PET agent for imaging of tumorous lesions. However, further detailed evaluation of the agent is warranted to explore its actual potential.