Tumor Hypoxia Imaging Agents in Nuclear Medicine

Hypoxia is due to imbalance in oxygen supply and oxygen demand compromising biological functions of cells. Since tumor hypoxia results in angiogenesis, apoptosis, metastasis, tumor aggressiveness and treatment failure, in vivo measurement is required. Nuclear imaging can provide information about tissue oxygen levels. 2-nitroimidazole containing compounds selectively accumulate in hypoxic cells. They have been radiolabeled with 18F, 123/124I, and 99mTc and used in clinical trial stages using PET and SPECT techniques. 62/64Cu-ATSM is a non-imidazole imaging agent, which is trapped in hypoxic cells. There is a great interest in the development of 99mTc-labeled 2-nitroimidazole compounds. Though novel compounds based on molecular mechanisms of hypoxia would be developed in future.HighlightsTumor hypoxia results in angiogenesis, apoptosis, metastasis, tumor aggressiveness, and treatment failure.Nuclear imaging can provide information about tissue oxygen levels.2-nitroimidazole compounds selectively accumulate in hypoxic cells.At present a few PET radiopharmaceuticals as hypoxia imaging agents are in clinical trial stages

Synthesis and Labeling of Two Fibrin-Targeted Peptides (HYNICGPRPILE, HYNIC-GPKGAAD) Using Technetium-99m and In Vitro Evaluation of Fibrin Binding and Platelet Aggregation: Fibrin-targeted peptides for molecular imaging of thrombus

Early detection of thrombus and its location in the body are critical factors for the treatment of thrombosis related diseases. Fibrin is the main component of thrombus, abundant in all thrombi, and is not found in non-pathological conditions. The presence of fibrin in all types of thrombi and its low concentration in blood makes it a sensitive and specific target for imaging studies of thrombus. Fibrin also accumulates in malignant tumors. Thus, fibrin imaging can be used in oncology, atherosclerosis, and thrombosis-related pathologies such as pulmonary emboli and deep vein thrombosis. Different compounds such as antibodies, nanoparticles, and peptides have been studied for fibrin imaging. Among them, peptides are more attractive because of better pharmacokinetics, simple and cheap preparation, and better radiolabeling methods. In this study, two peptides (HYNIC-GPRPILE, HYNIC-GPKGAAD) designed to target fibrin were synthesized.  The peptides were identified by LC-MS. The stability and platelet aggregation of peptides were determined. Peptides were radiolabeled with 99mTc using HYNIC as chelating agent. The release of 99mTc and fibrin binding of radiopeptides were evaluated. Based on the results, peptides were stable in human plasma for at least 6 h and had no effect on platelet aggregation. Peptides were radiolabeled with pertechnetate at 80°C in 30 min. Radiochemical purity was over 95%. Radiopeptides were stable in human plasma and there was less than 5% release of 99mTc. The fibrin binding of radiopeptides was 70%>. Since peptides had no platelet binding activity, it can be concluded that binding of radiopeptides to fibrin is specific. HIGHLIGHTS Molecular imaging of fibrin used in oncology, atherosclerosis, and thrombosis related pathologies. Invasive early detection of thrombus using radiolabeled fibrin targeted peptides. Radiolabeling of peptides with 99mTc using HYNIC as chelating agent and EDDA and tricine as co-ligand. &nbsp

Synthesis, Radiolabeling and Stability Studies of Peptide HYNIC-LIKKP-Pyr-F with 99mTc as an Apoptosis Imaging Agent : Radiolabeling of an apoptosis imaging agent

A non-invasive method for detecting phosphatidylserine (PS) exposure on the outer surface of plasma membranes, such as nuclear imaging, could aid in the diagnosis and treatment of diseases associated with apoptosis. Annexin V has been the most researched imaging agent for apoptosis to date. Due to Annexin V's limitations, additional agents, such as small peptides and molecules, have been introduced, including LIKKPF developed by Burtea et al. In this study, HYNIC-LIKKP-pyr-F, a derivative of LIKKPF was prepared using the 9-fluoroenylmethoxycarbonyl (fmoc) method, radiolabeled with Technetium-99m (99mTc) with the use of Stannous chloride (SnCl2) as a reducing agent and ethylenediamine diacetate (EDDA) and tricine as co-ligands. Radiochemical purity, labeling efficiency, and stability of radiopeptide in normal saline and human plasma were determined using thin layer chromatography (TLC). The partition coefficient of radiolabeled peptide was measured in a combination of PBS (pH 7.4) and n-octanol. Specific activity was also measured. LC-MS was used to examine the synthesized peptide. Peptide was stable in human serum for at least 4 hr. Peptide was radiolabeled with 99mTc with radiochemical purity and labeling efficiency over 95% and 90%, respectively. Radiopeptide was stable in saline and human serum for at least 4 hours. The radiolabeled peptide has a great deal of potential as an apoptosis imaging agent for in vitro and in vivo experiments. HIGHLIGHTS A noninvasive method for apoptosis imaging is the usage of radiolabeled affinity ligands. A new derivative of LIKKPF with affinity for phosphatidyl serine was synthesized. The LIKKPF peptide was radiolabeled with 99

Piperine-loaded electrospun nanofibers, an implantable anticancer controlled delivery system for postsurgical breast cancer treatment

Tumorectomy followed by radiotherapy, hormone, and chemotherapy, are the current mainstays for breast cancer treatment. However, these strategies have systemic toxicities and limited treatment outcomes. Hence, there is a crucial need for a novel controlled release delivery system for implantation following tumor resection to effectively prevent recurrence. Here, we fabricated polycaprolactone (PCL)-based electrospun nanofibers containing piperine (PIP), known for chemopreventive and anticancer activities, and also evaluated the impact of collagen (Coll) incorporation into the matrices. In addition to physicochemical characterization such as morphology, hydrophilicity, drug content, release properties, and mechanical behaviors, fabricated nanofibers were investigated in terms of cytotoxicity and involved mechanisms in MCF-7 and 4T1 breast tumor cell lines. In vivo antitumor study was performed in 4T1 tumor-bearing mice. PIP-PCL75-Coll25 nanofiber was chosen as the optimum formulation due to sustained PIP release, good mechanical performance, and superior cytotoxicity. Demonstrating no organ toxicity, animal studies confirmed the superiority of locally administered PIP-PCL75-Coll25 nanofiber in terms of inhibition of growth tumor, induction of apoptosis, and reduction of cell proliferation compared to PIP suspension, blank nanofiber, and the control. Taken together, we concluded that PIP-loaded nanofibers can be introduced as a promising treatment for implantation upon breast tumorectomy

Design, Synthesis and Radiolabeling of Peptide GPRPILE with 18FDG as Fibrin Imaging Agent for Thrombosis Detection

Background: The radionuclide of choice for routine clinical PET imaging is 18-F. As direct fluorination of peptides with 18-F is not possible, indirect methods using fluorinated prosthetic groups have been developed. Due to the availability of 18FDG in most PET centers, there is potential for 18FDG as a fluorinated prosthetic group. In this study, the linear peptide GPRPILE with an aminooxy group was designed, synthesized and radiolabeled with 18FDG as fibrin imaging agent. Material and Methods: Docking studies were conducted done using AutoDock 4.1 and HEX software programs. Aoe-GPRPILE peptide was designed, synthesized through Fmoc method and radiolabeled with 18FDG. The radiochemical purity, stability of radiolabeled and cold peptide in PBS and human plasma was determined using chromatographic methods. The solubility ratio of the radiolabeled peptide in lipid to water (LogP) was determined. Results: Docking and pharmacophore studies using HEX software revealed high affinity of designed peptide to fibrin (E Total=-0.01). The identity and structure of peptide were determined by LC-Mass. Peptide was stable over 24 hr in human plasma and PBS buffer. The optimum conditions of radiolabeling were 0.2 mg peptide, 1 mCi 18FDG, 90°C for 30 min, pH=5. The radiochemical purity was over 95%. The stability of radiolabeled peptide in human plasma for 2 hr was over 95%. The partition coefficient (LogP) was 1.5. Conclusion: 18FDG has a high potential to be used as a prosthetic group for radiolabeling of peptides with 18-F. In this study, peptide Aoe-GPRPILE with aminooxy was synthesized and labeled with 18FDG with high yield and radiochemical purity. Aminooxy is conjugated to peptide sequence as a prosthetic group in the last step with minimal effect on peptide properties and selectively forms stable oxime bond with the aldehyde group of 18FDG

Initial In Vitro and In Vivo Evaluation of a Novel CCK2R Targeting Peptide Analog Labeled with Lutetium-177

Targeting of cholecystokinin-2 receptor (CCK2R) expressing tumors using radiolabeled minigastrin (MG) analogs is hampered by rapid digestion of the linear peptide in vivo. In this study, a new MG analog stabilized against enzymatic degradation was investigated in preclinical studies to characterize the metabolites formed in vivo. The new MG analog DOTA-DGlu-Pro-Tyr-Gly-Trp-(N-Me)Nle-Asp-1Nal-NH2 comprising site-specific amino acid substitutions in position 2, 6 and 8 and different possible metabolites thereof were synthesized. The receptor interaction of the peptide and selected metabolites was evaluated in a CCK2R-expressing cell line. The enzymatic stability of the 177Lu-labeled peptide analog was evaluated in vitro in different media as well as in BALB/c mice up to 1 h after injection and the metabolites were identified based on radio-HPLC analysis. The new radiopeptide showed a highly increased stability in vivo with >56% intact radiopeptide in the blood of BALB/c mice 1 h after injection. High CCK2R affinity and cell uptake was confirmed only for the intact peptide, whereas enzymatic cleavage within the receptor specific C-terminal amino acid sequence resulted in complete loss of affinity and cell uptake. A favorable biodistribution profile was observed in BALB/c mice with low background activity, preferential renal excretion and prolonged uptake in CCK2R-expressing tissues. The novel stabilized MG analog shows high potential for diagnostic and therapeutic use. The radiometabolites characterized give new insights into the enzymatic degradation in vivo

In-house Optimization Radiolabeling of Recombinant scFv with 99mTc-Tricarbonyl and Stability Studies: Radiolabeling scFv with technetium tricarbonyl

His-tagged scFv fragments of monoclonal antibodies have better pharmacokinetic properties than whole antibodies. Radiolabeled scFvs are considered for targeted imaging and treatment. Technetium tricarbonyl provides radiolabeling of scFvs without losing its biological activity in a fast and easy procedure. Technetium tricabonyl was prepared as follows: A freshly eluted solution of Na99mTcO4 was added to a mixture containing sodium carbonate, sodium potassium tartarate, boranocarbonate, sodium borohydride. The mixture was heated for 30 min at 100°C. Radiochemical purity was determined using radio thin lyer chromatography. Then, technetium tricarbonyl was added to a solution of scFv in PBS buffer and incubated for 2 h at 50°C, purified by PD-10 column and radiochemical purity was determined. Results showed that radiochemical purity of technetium tricarbony was over 98%. The best conditions for radiolabeling of scFv was: scFv concentration >2 mg/mL, PBS buffer, 2 h incubation at 50°C, pH 8-9, and high activity concentration of tricarbonyl. The best radiochemical purity of scFv was 70% before purificarion. Radiolabeled scFv was stable in PBS for 24 h incubation and there was no release of technetium in competition with histidine. In this study, we optimized radiolabeling of a scFv with technetium tricarbonyl using house made boranocarbonates. The results are promising and will be used for future studies. HIGHLIGHTS Radiolabeling of scFv was done directly by 99mTc-tricarbonyl. 99mTc-tricarbonyl was prepared in house from boranocarbonate. 99mTc-Radiolabeled scFv can be used for radioimmunoscintigraphy

A novel anti-CD22 scFv–apoptin fusion protein induces apoptosis in malignant B-cells

Abstract CD22 marker is a highly internalizing antigen which is located on the surface of B-cells and is being used as a promising target for treatment of B cell malignancies. Monoclonal antibodies targeting CD22 have been introduced and some are currently under investigation in clinical trials. Building on the success of antibody drug conjugates, we developed a fusion protein consisting of a novel anti-CD22 scFv and apoptin and tested binding and therapeutic effects in lymphoma cells. The recombinant protein was expressed in E. coli and successfully purified and refolded. In vitro binding analysis by immunofluorescence and flow cytometry demonstrated that the recombinant protein specifically binds to CD22 positive Raji cells but not to CD22 negative Jurkat cells. The cytotoxic properties of scFv–apoptin were assessed by an MTT assay and Annexin V/PI flow cytometry analysis and showed that the recombinant protein induced apoptosis preferentially in Raji cells with no detectable effects in Jurkat cells. Our findings indicated that the recombinant anti-CD22 scFv–apoptin fusion protein could successfully cross the cell membrane and induce apoptosis with high specificity, make it as a promising molecule for immunotherapy of B-cell malignancies

Novel agonists of benzodiazepine receptors: Design, synthesis, binding assay and pharmacological evaluation of diphenyl-1,2,4-triazole derivatives

Benzodiazepines (BZD) are widely used in neurological disorders. The use of classical benzodiazepines is limited due to side effects. In this study, based on the structure–activity relationship (SAR) of Benzodiazepine receptors, new derivatives of 4-amino-3,5-diphenyl-1,2,4-triazole as benzodiazepine agonists with selective effects were designed and synthesized. Docking studies showed that pharmacophore groups of the designed structures and zolpidem, a benzodiazepine receptor agonist, are properly matched and are located well in the GABA receptor. The triazole group of the compound 4j, N N-(3,5-diphenyl-4H-1,2,4-triazol-4-yl)-2-((4-fluorobenzyl)amino)acetamide, was near the nitrogen moiety of the imidazole ring of zolpidem providing the hydrogen bond acceptor in the suitable direction in the BDZ-binding site of GABAA receptor model (α1β2ϒ2). The compounds were synthesized with acceptable yield and in-vitro affinity for the BZD receptor was determined. Compound 4j had the best affinity for the BZD site of action on GABAA receptor complex (Ki = 2.56 nM and IC50 = 6.10 nM). In addition, the sedative-hypnotic effect, the locomotor activity, and evaluated memory of the novel compounds were assessed by pentobarbital-induced sleeping, open field, and passive avoidance tests respectively. Most of the novel compounds showed significant hypnotic activity with no impairment on learning and memory performance in the mouse. The pharmacological effects of the compounds were antagonized by flumazenil, a BZD antagonist, which confirms the involvement of BZD receptors in the biological effects