Comparative Preclinical Evaluation of HYNIC-Modified Designed Ankyrin Repeat Proteins G3 for the 99mTc-Based Imaging of HER2-Expressing Malignant Tumors.

HER2 status determination is a necessary step for the proper choice of therapy and selection of patients for the targeted treatment of cancer. Targeted radiotracers such as radiolabeled DARPins provide a noninvasive and effective way for the molecular imaging of HER2 expression. This study aimed to evaluate tumor-targeting properties of three 99mTc-labeled DARPin G3 variants containing Gly-Gly-Gly-Cys (G3C), (Gly-Gly-Gly-Ser)3-Cys ((G3S)3C), or Glu-Glu-Glu-Cys (E3C) amino acid linkers at the C-terminus and conjugated to the HYNIC chelating agent, as well as to compare them with the clinically evaluated DARPin G3 labeled with 99mTc(CO)3 using the (HE)3-tag at the N-terminus. The labeling of DARPin G3-HYNIC variants provided radiochemical yields in the range of 50-80%. Labeled variants bound specifically to human HER2-expressing cancer cell lines with affinities in the range of 0.5-3 nM. There was no substantial influence of the linker and HYNIC chelator on the binding of 99mTc-labeled DARPin G3 variants to HER2 in vitro; however, [99mTc]Tc-G3-(G3S)3C-HYNIC had the highest affinity. Comparative biodistribution of [99mTc]Tc-G3-G3C-HYNIC, [99mTc]Tc-G3-(G3S)3C-HYNIC, [99mTc]Tc-G3-E3C-HYNIC, and [99mTc]Tc-(HE)3-G3 in healthy CD1 mice showed that there was a strong influence of the linkers on uptake in normal tissues. [99mTc]Tc-G3-E3C-HYNIC had an increased retention of activity in the liver and the majority of other organs compared to the other conjugates. The tumor uptake of [99mTc]Tc-G3-(G3S)3C-HYNIC and [99mTc]Tc-(HE)3-G3 in Nu/j mice bearing SKOV-3 xenografts was similar. The specificity of tumor targeting in vivo was demonstrated for both tracers. [99mTc]Tc-G3-(G3S)3C-HYNIC provided comparable, although slightly lower tumor-to-lung, tumor-to spleen and tumor-to-liver ratios than [99mTc]Tc-(HE)3-G3. Radiolabeling of DARPin G3-HYNIC conjugates with 99mTc provided the advantage of a single-step radiolabeling procedure; however, the studied HYNIC conjugates did not improve imaging contrast compared to the 99mTc-tricarbonyl-labeled DARPin G3. At this stage, [99mTc]Tc-(HE)3-G3 remains the most promising candidate for the clinical imaging of HER2-overexpressing cancers.

[99mTc]Tc-labeled HYNIC conjugated chlorambucil as a tumor targeting Agent: Synthesis, characterization and ex-vivo evaluation.

Chlorambucil is an alkylating drug that finds application towards chemotherapy of different types of cancers. In order to explore the possibility of utilization of this drug as an imaging agent for early diagnosis of solid tumors, attempt was made to synthesize a 99mTc complex of chlorambucil and evaluate its potential in tumor bearing small animal model. HYNIC-chlorambucil was synthesized by conjugation of HYNIC with chlorambucil via an ethylenediamine linker. All the intermediates and final product were purified and characterized by standard spectroscopic techniques viz. FT-IR, 1H/13C-NMR as well as by mass spectrometry. HYNIC-chlorambucil conjugate was radiolabeled with [99mTc]Tc and found to be formed with > 95 % radiochemical purity via RP-HPLC studies. The partition coefficient (Log10Po/w) of the synthesized complex was found to be -0.78 ± 0.25 which indicated the moderate hydrophilic nature for the complex. Biological behaviour of [99mTc]Tc-HYNIC-chlorambucil, studied in fibrosarcoma bearing Swiss mice, revealed a tumor uptake of about 4.16 ± 1.52 %IA/g at 30 min post-administration, which declined to 1.91 ± 0.13 % IA/g and 1.42 ± 0.14 %IA/g at 1 h and 2 h post-administration, respectively. A comparison of different [99mTc]Tc-chlorambucil derivatives (reported in the contemporary literature) formulated using different methodologies revealed that tumor uptake and pharmacokinetics exhibited by these agents strongly depend on the lipophilicity/hydrophilicity of such agents, which in turn is dependent on the bifunctional chelators used for formulating the radiolabeled chlorambucils.

Synthesis and Bioevaluation of Novel Technetium-99m-Labeled Complexes with Norfloxacin HYNIC Derivatives for Bacterial Infection Imaging.

To seek a novel 99mTc-labeled quinolone derivative for bacterial infection SPECT imaging that aims to lower nontarget organ uptake, a novel norfloxacin 6-hydrazinoicotinamide (HYNIC) derivative (HYNICNF) was designed and synthesized. It was radiolabeled with different coligands, such as tricine, trisodium triphenylphosphine-3,3',3″-trisulfonate (TPPTS), sodium triphenylphosphine-3-monosulfonate (TPPMS), and ethylenediamine-N,N'-diacetic acid (EDDA), to obtain three 99mTc-labeled norfloxacin HYNIC complexes, namely, [99mTc]Tc-tricine-TPPTS-HYNICNF, [99mTc]Tc-tricine-TPPMS-HYNICNF, and [99mTc]Tc-EDDA-HYNICNF. These complexes were purified (RCP > 95%) and evaluated in vitro and in vivo for targeting bacteria. All three complexes are hydrophilic, maintain good stability, and specifically bind Staphylococcus aureus in vitro. The biodistribution in mice with bacterial infection demonstrated that [99mTc]Tc-EDDA-HYNICNF showed a higher abscess uptake and lower nontarget organ uptake and was able to distinguish bacterial infection and sterile inflammation. Single photon emission computed tomography (SPECT) image study in bacterial infection mice showed there was a visible accumulation in the infection site, suggesting that [99mTc]Tc-EDDA-HYNICNF is a potential radiotracer for bacterial infection imaging.

Radiolabeling of Platelets with 99mTc-HYNIC-Duramycin for In Vivo Imaging Studies.

Following the in vivo biodistribution of platelets can contribute to a better understanding of their physiological and pathological roles, and nuclear imaging methods, such as single photon emission tomography (SPECT), provide an excellent method for that. SPECT imaging needs stable labeling of the platelets with a radioisotope. In this study, we report a new method to label platelets with 99mTc, the most frequently used isotope for SPECT in clinical applications. The proposed radiolabeling procedure uses a membrane-binding peptide, duramycin. Our results show that duramycin does not cause significant platelet activation, and radiolabeling can be carried out with a procedure utilizing a simple labeling step followed by a size-exclusion chromatography-based purification step. The in vivo application of the radiolabeled human platelets in mice yielded quantitative biodistribution images of the spleen and liver and no accumulation in the lungs. The performed small-animal SPECT/CT in vivo imaging investigations revealed good in vivo stability of the labeling, which paves the way for further applications of 99mTc-labeled-Duramycin in platelet imaging.

Development and Characterization of 99mTc-scFvD2B as a Potential Radiopharmaceutical for SPECT Imaging of Prostate Cancer.

Previously, we demonstrated that the 177Lu-labeled single-chain variable fragment of an anti-prostate-specific membrane antigen (PSMA) IgG D2B antibody (scFvD2B) showed higher prostate cancer (PCa) cell uptake and tumor radiation doses compared to 177Lu-labeled Glu-ureide-based PSMA inhibitory peptides. To obtain a 99mTc-/177Lu-scFvD2B theranostic pair, this research aimed to synthesize and biochemically characterize a novel 99mTc-scFvD2B radiotracer. The scFvD2B-Tag and scFvD2B antibody fragments were produced and purified. Then, two HYNIC derivatives, HYNIC-Gly-Gly-Cys-NH2 (HYNIC-GGC) and succinimidyl-HYNIC (S-HYNIC), were used to conjugate the scFvD2B-Tag and scFvD2B isoforms, respectively. Subsequently, chemical characterization, immunoreactivity tests (affinity and specificity), radiochemical purity tests, stability tests in human serum, cellular uptake and internalization in LNCaP(+), PC3-PIP(++) or PC3(-) PCa cells of the resulting unlabeled HYNIC-scFvD2B conjugates (HscFv) and 99mTc-HscFv agents were performed. The results showed that incorporating HYNIC as a chelator did not affect the affinity, specificity or stability of scFvD2B. After purification, the radiochemical purity of 99mTc-HscFv radiotracers was greater than 95%. A two-sample t-test of 99mTc-HscFv1 and 99mTc-HscFv1 uptake in PC3-PIP vs. PC3 showed a p-value < 0.001, indicating that the PSMA receptor interaction of 99mTc-HscFv agents was statistically significantly higher in PSMA-positive cells than in the negative controls. In conclusion, the results of this research warrant further preclinical studies to determine whether the in vivo pharmacokinetics and tumor uptake of 99mTc-HscFv still offer sufficient advantages over HYNIC-conjugated peptides to be considered for SPECT/PSMA imaging.

Synthesis and Evaluation of 99mTc-Labeled PSMA-Targeted Tracers Based on the Lys-Urea-Aad Pharmacophore for Detecting Prostate Cancer with Single Photon Emission Computed Tomography.

Prostate-specific membrane antigen (PSMA) is a well-validated prostate cancer marker but reported PSMA-targeted tracers derived from the Lys-urea-Glu pharmacophore including the clinically validated [99mTc]Tc-EDDA/HYNIC-iPSMA have high off-target uptake in kidneys, spleen, and salivary glands. In this study, we synthesized and evaluated three novel 99mTc-labeled PSMA-targeted tracers and investigated if the tracers derived from the Lys-urea-Aad pharmacophore could have minimized uptake in off-target organs/tissues. In vitro competition binding assays showed that compared with HYNIC-iPSMA, the three novel ligands had slightly weaker PSMA binding affinity (average Ki = 3.11 vs. 8.96-11.6 nM). Imaging and ex vivo biodistribution studies in LNCaP tumor-bearing mice showed that [99mTc]Tc-EDDA/HYNIC-iPSMA and the three novel tracers successfully visualized LNCaP tumor xenografts in SPECT images and were excreted mainly via the renal pathway. The average tumor uptake at 1 h post-injection varied from 5.40 to 18.8%ID/g, and the tracers derived from the Lys-urea-Aad pharmacophore had much lower uptake in the spleen and salivary glands. Compared with the clinical tracer [99mTc]Tc-EDDA/HYNIC-iPSMA, the Lys-urea-Aad-derived [99mTc]Tc-EDDA-KL01127 had lower background uptake and superior tumor-to-background contrast ratios and is therefore promising for clinical translation to detect prostate cancer lesions with SPECT.

Can MDCT Enhancement Patterns Be Helpful in Differentiating Secretory from Non-Functional Adrenal Adenoma?

Background and Objectives: Primary adrenal tumors (AT) are a heterogeneous group of neoplasms due to their functional heterogeneity, which results in the diverse clinical presentation of these tumors. The purpose of this study was to examine cross-sectional imaging characteristics using multi-detector computed tomography (MDCT) to provide insight into the lesion characterization and functional status of these tumors. The radionuclide imaging using Technetium-99m radiolabeled hydrazinonicotinylacid-d-phenylalanyl1-tyrosine3-octreotide (99mTc-HYNIC-TOC), was also used in the diagnostic evaluation of these tumors. Materials and Methods: This cross-sectional study included 50 patients with confirmed diagnoses of AT (21 hormone-secreting and 29 non-functional) at the University Clinical Center, Kragujevac, Serbia, during the 2019-2022 year period. The morphological and dynamic characteristics using MDCT were performed, using qualitative, semi-quantitative, and quantitative analysis. Absolute washout (APW) and relative washout (RPW) values were also calculated. A semi-quantitative analysis of all visual findings with 99mTc-HYNIC-TOC was performed to compare the tumor to non-tumor tracer uptake. Results: A statistically significant difference was found in the MDCT values in the native phase (p < 0.05), the venous phase (p < 0.05), and the delayed phase (p < 0.001) to detect the existence of adrenal tumors. Most of these functional adrenocortical lesions (n = 44) can be differentiated using the delayed phase (p < 0.05), absolute percentage washout (APW) (p < 0.05), and relative percentage washout (RPW) (p < 0.001). Furthermore, 99mTc-HYNIC-TOC could have a high diagnostic yield to detect adrenal tumor existence (p < 0.001). There is a positive correlation between radionuclide imaging scan and APW to detect all AT (p < 0.01) and adrenocortical adenomas as well (p < 0.01). Conclusions: The results can be very helpful in a diagnostic algorithm to quickly and precisely diagnose the expansive processes of the adrenal glands, as well as to learn about the advantages and limitations of the mentioned imaging modalities.

Head-to-Head Comparison between Peptide-Based Radiopharmaceutical for PET and SPECT in the Evaluation of Neuroendocrine Tumors: A Systematic Review.

We compared head-to-head the most used radiolabeled peptides for single photon computed emission tomography (SPECT) and positron emission tomography (PET) imaging of neuroendocrine tumors (NETs). A comprehensive literature search was performed in PubMed, Web of Science, and Scopus databases. The following words, coupled two by two, were used: 68Ga-DOTATOC; 68Ga-DOTATATE; 68Ga-DOTANOC; 99mTc-EDDA/HYNIC-TOC; 64Cu-DOTATATE; and 111In-DTPA-octreotide. Moreover, a second-step search strategy was adopted by using the following combined terms: "Somatostatin receptor imaging,"; "Somatostatin receptor imaging" and "Functional,"; "Somatostatin receptor imaging" and "SPECT,"; and "Somatostatin receptor imaging" and "PET". Eligible criteria were: (1) original articles focusing on the clinical application of the radiopharmaceutical agents in NETs; (2) original articles in the English language; (3) comparative studies (head-to-head comparative or matched-paired studies). Editorials, letters to the editor, reviews, pictorial essays, clinical cases, or opinions were excluded. A total of 1077 articles were found in the three electronic databases. The full texts of 104 articles were assessed for eligibility. Nineteen articles were finally included. Most articles focused on the comparison between 111In-DTPA-Octreotide and 68Ga-DOTATOC/TATE. Few papers compared 64Cu-DOTATATE and 68Ga-DOTATOC/TATE, or SPECT tracers. The rates of true positivity were 63.7%, 58.5%, 78.4% and 82.4%, respectively, for 111In-DTPA-Octreotide, 99mTc-EDDA/HYNIC-TOC, 68Ga-DOTATATE/TOC and 64Cu-DOTATATE. In conclusion, as highly expected, PET tracers are more suitable for the in vivo identification of NETs. Indeed, in comparative studies, they demonstrated a higher true positive rate than SPECT agents.

Design, Synthesis and Preclinical Assessment of 99mTc-iFAP for In Vivo Fibroblast Activation Protein (FAP) Imaging.

Fibroblast activation protein (FAP) is expressed in the microenvironment of most human epithelial tumors. 68Ga-labeled FAP inhibitors based on the cyanopyrrolidine structure (FAPI) are currently used for the detection of the tumor microenvironment by PET imaging. This research aimed to design, synthesize and preclinically evaluate a new FAP inhibitor radiopharmaceutical based on the 99mTc-((R)-1-((6-hydrazinylnicotinoyl)-D-alanyl) pyrrolidin-2-yl) boronic acid (99mTc-iFAP) structure for SPECT imaging. Molecular docking for affinity calculations was performed using the AutoDock software. The chemical synthesis was based on a series of coupling reactions of 6-hidrazinylnicotinic acid (HYNIC) and D-alanine to a boronic acid derivative. The iFAP was prepared as a lyophilized formulation based on EDDA/SnCl2 for labeling with 99mTc. The radiochemical purity (R.P.) was verified via ITLC-SG and reversed-phase radio-HPLC. The stability in human serum was evaluated by size-exclusion HPLC. In vitro cell uptake was assessed using N30 stromal endometrial cells (FAP positive) and human fibroblasts (FAP negative). Biodistribution and tumor uptake were determined in Hep-G2 tumor-bearing nude mice, from which images were acquired using a micro-SPECT/CT. The iFAP ligand (Ki = 0.536 nm, AutoDock affinity), characterized by UV-Vis, FT-IR, 1H-NMR and UPLC-mass spectroscopies, was synthesized with a chemical purity of 92%. The 99mTc-iFAP was obtained with a R.P. >98%. In vitro and in vivo studies indicated high radiotracer stability in human serum (>95% at 24 h), specific recognition for FAP, high tumor uptake (7.05 ± 1.13% ID/g at 30 min) and fast kidney elimination. The results found in this research justify additional dosimetric and clinical studies to establish the sensitivity and specificity of the 99mTc-iFAP.

[99mTc]Tc-PSMA-T4-Novel SPECT Tracer for Metastatic PCa: From Bench to Clinic.

Despite significant advances in nuclear medicine for diagnosing and treating prostate cancer (PCa), research into new ligands with increasingly better biological properties is still ongoing. Prostate-specific membrane antigen (PSMA) ligands show great potential as radioisotope carriers for the diagnosis and therapy of patients with metastatic PCa. PSMA is expressed in most types of prostate cancer, and its expression is increased in poorly differentiated, metastatic, and hormone-refractory cancers; therefore, it may be a valuable target for the development of radiopharmaceuticals and radioligands, such as urea PSMA inhibitors, for the precise diagnosis, staging, and treatment of prostate cancer. Four developed PSMA-HYNIC inhibitors for technetium-99m labeling and subsequent diagnosis were subjected to preclinical in vitro and in vivo studies to evaluate and compare their diagnostic properties. Among the studied compounds, the PSMA-T4 (Glu-CO-Lys-L-Trp-4-Amc-HYNIC) inhibitor showed the best biological properties for the diagnosis of PCa metastases. [99mTc]Tc-PSMA-T4 also showed effectiveness in single-photon emission computed tomography (SPECT) studies in humans, and soon, its usefulness will be extensively evaluated in phase 2/3 clinical trials.

Preparation and assessment of a new radiotracer technetium-99m-6-hydrazinonicotinic acid-tyrosine as a targeting agent in tumor detecting through single photon emission tomography.

The goal of investigation was to bring up an impressive way to synthesize technetium-99 m-6-hydrazinonicotinic acid-tyrosine (99mTc-HYNIC-Tyr), a newfound radiotracer, and to assess the capacity of being as a tumor scintigraphy agent. The conjugate was prepared by solid phase method using tritely chloride resin. The precursor HYNIC-Tyr was labeled with 99mTc which was accomplished at 100 °C through the coligands tricine and EDDA. Furthermore, the serum albumin binding, cellular attachment, organs uptake and tumor accumulation were measured. C6 glioma cells were used for cellular and tumor uptake studies. 99mTc-HYNIC-Tyr was prepared with labeling yield of >99% (n = 3). Radiotracer showed stability in serum proteins in incubates temperature. Specific cellular attachment of radiotracer was noticeable in C6 glioma cells with dissociation constant in nano molar range (21.03 ± 1.54 nM). The amount of uptake in C6 rat glioma xenograft was 2.61 ± 0.12 percent of injection dose per gram after 30 min. In whole-body scintigraphy, C6 glioma tumor was easy to be traced and interpreted at 1 h after administration of radiotracer. Our data suggest that 99mTc-HYNIC-Tyr, a new radiotracer based on amino acid, efficiently differentiates the tumor cells and goes into them. Our results indicate that this radiotracer has excellent capacity to detect tumor cells in rat and to be included as a radiopharmaceutical for detecting cancer tumors.

Study of the 99m Tc-labeling conditions of 6-hydrazinonicotinamide-conjugated peptides from a new perspective: Introduction to the term radio-stoichiometry.

Specific tumor uptake of peptide radiopharmaceuticals depends on tumor binding affinity and their radiochemical purity. Several important parameters that influence the 99m Tc-labeling and consequently the radiochemical purity of 6-hydrazinonicotinamide (HYNIC)-conjugated peptide are radionuclide activity, the amount of peptide, the amount of coligands, and the amount of reducing agents (stannous ion). In this review article, we have attempted studying these parameters in the HYNIC-conjugated peptides (somatostatin, cholecystokinin/gastrin, bombesin, and RGD analogs) from a new perspective to obtain most used and optimized radio-stoichiometric relationships. One of the most important results in this review is that for 99m Tc-labeling of HYNIC-conjugated peptides, it is better to consider the most calculated mole ratio between technetium-99m and the peptide (mole ratio of technetium-99m to the peptide 1:200-400). The statistical results also show that among these 99m Tc-labeled peptides, the most used and favorable coligand is tricine/EDDA with two to one (2:1) mole ratio. These optimized radio-stoichiometric relationships, favorable coligand mole ratio, and applicable radiolabeling points can greatly improve the labeling process of the HYNIC-conjugated peptides, by reducing trial and error, increasing specific activity, and saving materials.

THE ROLE OF TUMOR-SEEKING RADIOPHARMACEUTICALS IN THE DIAGNOSIS AND MANAGEMENT OF ADRENAL TUMORS.

CONTEXT: The variety of tumor-seeking radiopharmaceuticals, which are currently in clinical use, may have a potential role as imaging agents for adrenal gland tumors, due to physiological characteristics of this organ. OBJECTIVE: The purpose of this study was to evaluate the diagnostic potential of 99mTc-HYNIC-TOC, 99mTc(V)-DMSA, and 99mTc-MIBI in the assessment of adrenal tumors, by correlating with imaging findings and histopathologic results. DESIGN: The research is designed as a cross-sectional prospective study. PATIENTS AND METHOD: The study included 50 patients with adrenal tumors (19 hormone-secreting and 31 nonfunctioning) and 23 controls without adrenal involvement. In all patients, single-photon emission computed tomography (SPECT) was performed, using qualitative and semiquantitative analysis. The tumor to non-tumor tracer uptake was conducted by using a region-of-interest technique. Adrenal to background (A/B) ratio was calculated in all cases. RESULTS: 99mTc-HYNIC-TOC scintigraphy showed a high statistical significance between A/B ratios, while other two tracers resulted in a lower sensitivity, specificity and accuracy. Futhermore, 99mTc-HYNIC-TOC could have a high diagnostic yield to detect adrenal tumors (the receiver-operating-characteristic curve analysis, A/B ratio cut-off value of 8.40). CONCLUSION: A semiquantitative SPECT analysis showed that 99mTc-HYNIC-TOC is a highly sensitive tumor-seeking agent for the accurate localization of adrenal tumors.

An improved kit formulation for one-pot synthesis of [99m Tc]Tc-HYNIC-E[c(RGDfK)]2 for routine clinical use in cancer imaging.

Radiolabeled Arg-Gly-Asp (RGD) peptide derivatives have immense potential for non-invasive monitoring of malignancies overexpressing integrin αv ß3 receptors. Easy availability of suitable radiotracers would augment the utility of this class of molecular imaging agents. Towards this, the present article describes the development of an improved lyophilized kit for the routine clinical formulation of [99m Tc]Tc complex of HYNIC-conjugated dimeric cyclic RGD peptide derivative E-[c(RGDfK)]2 (E = glutamic acid, f = phenyl alanine, K = lysine) without using Sn2+ and systematic evaluation of its efficacy. Five batches of the kits were prepared, and [99m Tc]Tc-HYNIC-E[c(RGDfK)]2 radiotracer was synthesized with high radiochemical purity (98.6 ± 0.5%) and specific activity (124.8 GBq/µmol) using the kits. Biodistribution studies in C57BL/6 mice bearing melanoma tumor exhibited significant accumulation of the radiotracer in tumor (5.32 ± 0.56 %ID/g at 60 min p.i.), and this uptake was also found to be receptor-specific by blocking studies. Preliminary human clinical investigations carried out in 10 breast cancer patients revealed high radiotracer uptake in the tumor along with good tumor-to-background contrast. The developed kit formulation showed an exceptionally high shelf-life of at least 18 months. These results demonstrated promising attributes of the developed kit formulation and warrant more extensive clinical investigations.

Preparation and comparative evaluation of 99m Tc-HYNIC-cNGR and 99m Tc-HYNIC-PEG2 -cNGR as tumor-targeting molecular imaging probes.

The tripeptide sequence asparagine-glycine-arginine (NGR) specifically recognizes aminopeptidase N (APN or CD13) receptors highly expressed on tumor cells and vasculature. Thus, NGR peptides can precisely deliver therapeutic and diagnostic compounds to CD13 expressing cancer sites. In this regard, 2 NGR peptide ligands, HYNIC-c(NGR) and HYNIC-PEG2 -c(NGR), were synthesized, radiolabeled with 99m Tc, and evaluated in CD13-positive human fibrosarcoma HT-1080 tumor xenografts. The radiotracers, 99m Tc-HYNIC-c(NGR) and 99m Tc-HYNIC-PEG2 -c(NGR), could be prepared in approximately 95% radiochemical purity and exhibited excellent in vitro and in vivo stability. The radiotracers were hydrophilic in nature with log P values being -2.33 ± 0.05 and -2.61 ± 0.08. The uptake of 2 radiotracers 99m Tc-HYNIC-c(NGR) and 99m Tc-HYNIC-PEG2 -c(NGR) was similar in nude mice bearing human fibrosarcoma HT-1080 tumor xenografts, which was significantly reduced (P < .05) during blocking studies. The 2 radiotracers being hydrophilic cleared rapidly from blood, liver, and intestine and were excreted through renal pathway. The pharmacokinetics of 99m Tc-labeled HYNIC peptide could not be modulated through introduction of PEG2 unit, thus posing a challenge for studies with other linkers towards enhanced tumor uptake and retention.

Development of new PTK7-targeting aptamer-fluorescent and -radiolabelled probes for evaluation as molecular imaging agents: Lymphoma and melanoma in vivo proof of concept.

Aptamers are single-stranded oligonucleotides that recognize molecular targets with high affinity and specificity. Aptamer that selectively bind to the protein tyrosine kinase-7 (PTK7) receptor, overexpressed on many cancers, has been labelled as probes for molecular imaging of cancer. Two new PTK7-targeting aptamer probes were developed by coupling frameworks from the fluorescent dye AlexaFluor647 or the 6-hydrazinonicotinamide (HYNIC) chelator-labelled to 99mTc. The derivatizations via a 5'-aminohexyl terminal linker were done at room temperature and under mild buffer conditions. Physicochemical and biological controls for both imaging agents were performed verifying the integrity of the aptamer-conjugates by HPLC. Recognition of melanoma (B16F1) and lymphoma (A20) mouse cell lines by the aptamer was studied using cell binding, flow cytometry and confocal microscopy. Finally, in vivo imaging studies in tumour-bearing mice were performed. The new probes were able to bind to melanoma and lymphoma cell lines in vitro, the in vivo imaging in tumour-bearing mice showed different uptake behaviours showing for the fluorescent conjugate good uptake by B cell lymphoma while the radiolabelled conjugate did not display tumour uptake due to its high extravascular distribution, and both showed rapid clearance properties in tumour-bearing mice.

99m Tc-labeled NGR-chlorambucil conjugate, 99m Tc-HYNIC-CLB-c(NGR) for targeted chemotherapy and molecular imaging.

Targeted delivery of chemotherapeutic drug at the tumor site enhances the efficacy with minimum systemic exposure. Towards this, drugs conjugated with peptides having affinity towards a particular molecular target are recognized as affective agents for targeted chemotherapy. Thus, in the present study, tumor-homing asparagine-glycine-arginine (NGR) peptide ligand was conjugated to DNA alkylating nitrogen mustard, chlorambucil (CLB). The peptide-drug conjugate (PDC), CLB-c(NGR), was radiolabeled with 99m Tc-HYNIC core to trace its pharmacokinetics and biodistribution pattern. In vitro cell-binding studies of 99m Tc-HYNIC-CLB-c(NGR) were conducted in murine melanoma B16F10 cells. The cytotoxicity studies conducted by incubation of the peptide/drug/PDC with B16F10 cells demonstrated enhanced cytotoxic effect of PDC in comparison to either the peptide or the drug alone. In vivo biodistribution studies in C57BL6 mice bearing melanoma tumor showed maximum tumor uptake at 30 minutes pi (2.45 ± 0.28% ID/g), which reduced to 0.77 ± 0.1% ID /g at 3 hours pi. The radiotracer being hydrophilic cleared rapidly from the heart, lungs, liver, and muscle. The tumor-to-blood and tumor-to-muscle ratios improved with time. This study opens avenues for conjugation of other targeting peptides with the drug CLB for enhanced toxicity at the diseased site.

SPECT imaging of interleukin-6 receptor in ovarian tumor xenografts with a novel radiotracer of 99mTc-HYNIC-Aca-LSLITRL.

Growing evidences have shown that the IL-6/IL-6R signal pathway promotes the tumor growth, angiogenesis, invasion and migration in various cancers, especially for epithelial ovarian cancer. Hence, including anti-IL-6 antibody (Siltuximab) and anti-IL-6R antibody (Tocilizumab), more and more therapeutic drugs targeting IL-6/IL-6R pathway were developed to block their activity. The molecular imaging of IL-6R is a significant factor for predicting tumor response to IL-6/IL-6R targeted drugs. However, few probes targeting IL-6R were designed and used for the specific detection. The purpose of this study was to develop and evaluate a novel radiotracer, (99m)Tc-HYNIC-Aca-LSLITRL, for SPECT imaging of interleukin-6 receptor. The expression of IL-6R was determined by western blot, immunofluorescence and immunohistochemistry. HYNIC-Aca-LSLITRL and HYNIC-Aca-TLQASIL were synthesized, and then were labeled with 99mTc. The stability and the cell-binding assay were performed. Ovarian tumor xenografts were established and subjected to SPECT imaging after injection of these two radiopharmaceuticals with or without excess primary peptides. The biodistribution of these two radiotracers was performed in nude mice bearing C13K tumors. (99m)Tc-HYNIC-Aca-LSLITRL and (99m)Tc-HYNIC-Aca-TLQASIL were obtained in >95 % labeling yield with favorably stability. In vitro studies demonstrated that the interleukin-6 receptor was overexpressed in ovarian cancer C13K cells. The SPECT imaging of interleukin-6 receptor and biodistribution studies showed that (99m)Tc-HYNIC-Aca-LSLITRL had higher tumor uptake and significantly lower kidney accumulation compared to (99m)Tc-HYNIC-Aca-TLQASIL. (99m)Tc-HYNIC-Aca-LSLITRL could be a promising agent for SPECT imaging of interleukin-6 receptor of ovarian cancer especially for those anti-IL-6R drugs under clinical trials, such as tocilizumab.

(99m)Tc-bioorthogonal click chemistry reagent for in vivo pretargeted imaging.

Metal-free click chemistry has become an important tool for pretargeted approaches in the molecular imaging field. The application of bioorthogonal click chemistry between a pretargeted trans-cyclooctene (TCO) derivatized monoclonal antibody (mAb) and a (99m)Tc-modified 1,2,4,5-tetrazine for tumor imaging was examined in vitro and in vivo. The HYNIC tetrazine compound was synthesized and structurally characterized, confirming its identity. Radiolabeling studies demonstrated that the HYNIC tetrazine was labeled with (99m)Tc at an efficiency of >95% and was radiochemically stable. (99m)Tc-HYNIC tetrazine reacted with the TCO-CC49 mAb in vitro demonstrating its selective reactivity. In vivo biodistribution studies revealed non-specific liver and GI uptake due to the hydrophobic property of the compound, however pretargeted SPECT imaging studies demonstrated tumor visualization confirming the success of the cycloaddition reaction in vivo. These results demonstrated the potential of (99m)Tc-HYNIC-tetrazine for tumor imaging with pretargeted mAbs.

99mTc-HYNIC-TOC increased uptake can mimic malignancy in the pancreas uncinate process at somatostatin receptor SPECT/CT.

OBJECTIVE: The aim of this study was to assess the occurrence and frequency of increased physiologic uptake of 99mTc-HYNIC-TOC by the uncinate process of the pancreas in SPECT/CT images. METHODS: Forty-six scans of 41 patients were evaluated retrospectively. The uptake of 99mTc-HYNIC-TOC was considered to be physiologic in patients with normal findings at dedicated abdominal CT or MR and lack of neoplastic lesions in clinical follow-ups. The intensity of uncinate process uptake was compared to the uptake of the normal liver. RESULTS: Focal uptake was attributed to the presence of pancreatic NET in 5 patients. Among the 36 patients without any evidence of malignancy in CT, MR and follow-up, 7 (19.4 %) showed increased uptake in the uncinate process. The intensity of uptake was lesser in 3 (8.3 %), similar in 3 and greater than the normal liver in 1 (2.8 %) case. CONCLUSION: Increased 99mTc-HYNIC-TOC uptake occurred in 19.4 % of those subjects without any evidence of neuroendocrine tumor in the uncinate process.