Structural modifications of amino acid sequences of radiolabeled peptides for targeted tumor imaging.

Molecular imaging techniques are increasingly being used in localization, staging and therapy control of cancer. Due to their unique target specificity for the endogenous receptors, radiopeptides have been used widely for the development of radiopharmaceuticals for targeted tumor imaging in nuclear oncology. It is necessary to modify radiolabeled peptides in order to achieve more effective agents. Structural modifications of amino acid chains have significant effect on the metabolic stability, biological activity and efficiency of peptide conjugates that are currently applied as imaging tracers. There are several ways to modify the peptide chain but the most common strategies include amino acid substitutions, cyclization and multimerization. In this review, we have focused on studies involving these kind of modifications on amino acid sequences of radiolabeled peptides and we have provided an overview of the effects of these chemical modifications on the in vitro and in vivo properties of these radioconjugates and their potential as SPECT (Single photon emission computed tomography) and PET (positron emission tomography) imaging agents.

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.

[99mTc]-labeling and evaluation of a new linear peptide for imaging of glioblastoma as a αvß3-positive tumor.

PURPOSE: In this study, we designed a new linear 6-Hydrazinonicotinamide (HYNIC)-conjugated peptide (HYNIC-KRWrNM) (M-6) and labeled with technetium-99m for gamma imaging of glioblastoma as a αvß3-positive tumor. We evaluated tumor targeting ability of this radio-peptide and compared with previous 99mTc-labeled HYNIC-conjugated RGD analogue peptides. PROCEDURES: One new linear peptide (HYNIC-KRWrNM) (M-6) was designed and labeled with technetium-99m in the presence of 2-[[1,3-dihydroxy-2-(hydroxymethyl) propan-2-yl] amino] acetic acid (Tricine)/Ethylenediamine-N,N'-diacetic acid (EDDA) as co-ligand system. Then, this 99mTc-labeled peptide ([99mTc]Tc-M-7) was evaluated for in vitro stability in saline and serum, specific binding assay, internalization, and binding affinity (Kd). In addition, we performed biodistribution study and planar imaging on nude mice bearing U87-MG xenograft as a αvß3-positive tumor. RESULTS: The radiochemical yield of [99mTc]Tc-M-7 was obtained ˃95%. This 99mTc-labeled peptide remained stable and intact in saline solution after 24 h incubation. In addition, metabolic stability of this 99mTc-labeled peptide was obtained ˃60% after 4 h incubation in serum. The Kd value for [99mTc]Tc-M-7 was obtained 5.2 ± 1.0 nM. Based on biodistribution results in nude mice bearing U87-MG xenograft, tumor/muscle activity ratio was 6.22 and decreased to 1.89 in blocking group at the same time point (4 h p.i.). The blocking experiment results also indicated that tumor uptake and kidney uptake were αvß3-mediated. In comparison with previous HYNIC-conjugated RGD analogue peptides, kidneys had the highest uptake of this 99mTc-labeled peptide (52.29 ± 11.48 at 1.5 h p.i. and 27.04 ± 0.66%ID/g at 4 h p.i.). Finally, similar to previous 99mTc-labeled HYNIC-conjugated RGD analogue peptides, [99mTc]Tc-M-7 showed acceptable tumor uptake after 4 h post-injection (based on ROI technique, target-to-background activity ratio = 3.80). CONCLUSIONS: This small linear 99mTc-labeled peptide, with high affinity to αvß3 integrin, desirable water solubility, and cost efficient, demonstrates a potent tumor targeting ability as well as previous HYNIC-conjugated RGD analogue peptides. Hence, [99mTc]Tc-M-7 can be of service to as a new candidate for early detection of αvß3-positive tumors.

The Influence of Different Spacers on Biological Profile of Peptide Radiopharmaceuticals for Diagnosis and Therapy of Human Cancers.

BACKGROUND: Cancer is the leading cause of death worldwide. Early detection can reduce the disadvantageous effects of diseases and the mortality in cancer. Nuclear medicine is a powerful tool that has the ability to diagnose malignancy without harming normal tissues. In recent years, radiolabeled peptides have been investigated as potent agents for cancer detection. Therefore, it is necessary to modify radiopeptides in order to achieve more effective agents. OBJECTIVE: This review describes modifications in the structure of radioconjugates with spacers who have improved the specificity and sensitivity of the peptides that are used in oncologic diagnosis and therapy. METHODS: To improve the biological activity, researchers have conjugated these peptide analogs to different spacers and bifunctional chelators. Many spacers of different kinds, such as hydrocarbon chain, amino acid sequence, and poly (ethyleneglycol) were introduced in order to modify the pharmacokinetic properties of these biomolecules. RESULTS: Different spacers have been applied to develop radiolabeled peptides as potential tracers in nuclear medicine. Spacers with different charge and hydrophilicity affect the characteristics of peptide conjugate. For example, the complex with uncharged and hydrophobic spacers leads to increased liver uptake, while the composition with positively charged spacers results in high kidney retention. Therefore, the pharmacokinetics of radio complexes correlates to the structure and total charge of the conjugates. CONCLUSION: Radio imaging technology has been successfully applied to detect a tumor in the earliest stage. For this purpose, the assessment of useful agents to diagnose the lesion is necessary. Developing peptide radiopharmaceuticals using spacers can improve in vitro and in vivo behavior of radiotracers leading to better noninvasive detection and monitoring of tumor growth.

Evaluation of a New 99mTc-labeled GnRH Analogue as a Possible Imaging Agent for Prostate Cancer Detection.

INTRODUCTION: Prostate cancer is a serious threat to men's health so it is necessary to develop technics for early detection of this malignancy. The purpose of this research was the evaluation of a new99mTc-labeled GnRH analogue as an imaging probe for tumor targeting of prostate cancer. METHODS: 99mTc-labeled-DLys6-GnRH analogue was prepared based on HYNIC as a chelating agent and tricine/ EDDA as coligands for labeling with 99mTc. HYNIC was coupled to epsilon amino group of DLys6 through aminobutyric acid (GABA) as a linker. Radiochemical purity and stability in normal saline and serum, were determined by TLC and HPLC methods. Furthermore, calculation of protein-binding and partition coefficient constant were carried out for 99mTc labeled peptide. The cellular experiments including receptor binding specificity and affinity were studied using three prostate cancer cell lines LN-CaP, DU-145 and PC-3. Finally, the animal assessment and SPECT imaging of radiolabeled GnRH analogue were evaluated on normal mice and nude mice bearing LN-CaP tumor. RESULTS: The GnRH conjugate was labeled with high radiochemical purity (~97%). The radiolabeled peptide showed efficient stability in the presence of normal saline and human serum. The in vitro cellular assays on three prostate cancer cell lines indicated that the radiotracer was bound to LN-CaP cells with higher affinity compared to DU-145 and PC-3 cells. The Kd values of 99mTc- HYNIC (tricine/ EDDA)-Gaba-D-Lys6GnRH were 89.39±26.71, 93.57±30.49 and107.3±18.82 in LN-CaP, PC-3 and DU-145 cells respectively. The biodistribution studies in normal mice and LN-CaP tumor-bearing nude mice showed similar results including rapid blood clearance and low radioactivity accumulation in non-target organs. High kidney uptake proved that the main excretion route of radiopeptide was through the urinary system. The tumor uptake was 1.72±0.45 %ID/g at 1h p.i. decreasing to 0.70±0.06%ID/g at 4h p.i. for 99mTc-HYNIC-Gaba-D-Lys6GnRH. The maximum tumor/ muscle ratio was 2.30 at 1h p.i. Pre-saturation of receptor using an excess of unlabeled peptide revealed that the tumor uptake was receptor mediated. The results of the SPECT image of LN-CaP tumor were in agreement with the biodistribution data. CONCLUSION: Based on this study, we suggest LN-CaP as a favorable cell line for in vivo studies on GnRH analogues. Moreover, this report shows that 99mTc-HYNIC (tricine/EDDA)-Gaba-D-Lys6GnRH may be a suitable candidate for further evaluation of prostate cancer.

In vitro/in vivo assessment of the targeting ability of [99mTc] Tc-labeled an aptide specific to the extra domain B of fibronectin (APTEDB) for colorectal cancer.

OBJECTIVE: The APTEDB is an aptide specific to the extra domain B (EDB) of fibronectin with high affinity for EDB, which is expressed in malignant tumors including brain cancer (U87MG) and colorectal cancer (HT-29). Aim of this study was to evaluate the [99mTc] Tc-APTEDB potential as an imaging probe for colorectal cancer. METHODS: Radiochemical purity was evaluated by HPLC and radio-isotope TLC scanner. Blocking study for specific binding assay and affinity calculation (Kd) on HT-29 cell lines were also carried out. Planar imaging and bio-distribution studies were performed in HT-29 tumor-bearing mice. RESULTS: The APTEDB was efficiently labeled with technetium-99m in high radiochemical yield (up to 97%). Cellular binding study demonstrated specific binding of the [99mTc] Tc-APTEDB in cultured HT-29 cells. The Kd value was found to be 40.46 ± 13.39 nM. The tumor-to-muscle ratio was ~ 1.5 in ex vivo bio-distribution study at 1 h after injection. Planar imaging study showed higher activity accumulation in EDB expressing HT-29 tumor relative to muscle (used as control) (~ 1.7) at 1 h. CONCLUSIONS: Although more studies are required to find out the full potential of this radio-ligand as an imaging probe, the present results nevertheless provide useful information about [99mTc] Tc-APTEDB, which might be beneficial in design and development of new [99mTc] Tc-APTEDB for efficient targeting of tumor in vivo.

Preparation and evaluation of 99m Tc-HYNIC-D (TPPE) as a new targeted imaging probe for detection of colon cancer: Preclinical comparison with 99m Tc-HYNIC-EPPT.

The aim of this study was to prepare radiolabeled peptide-based agents for imaging of colon cancer. According to the incorporation of HYNIC for radiolabeling with technetium-99m, two analogs were designed and compared: an antitumor-antibody-derived peptide based on the EPPT sequence and a novel retro-inverso peptidomimetic derivative D (TPPE) structurally modified by replacing the L-amino acids with D-amino acids and reversing the primary amino acid sequence of EPPT. The HYNIC-conjugated peptides were labeled with 99m Tc using tricine/EDDA coligand with more than 98% radiochemical yield and showed high metabolic stability. Kd values of 41.77 ± 7.34 nM and 37.33 ± 8.37 nM for 99m Tc-HYNIC-EPPT and 99m Tc-HYNIC-D (TPPE) confirmed high affinity of both peptides for cell surface antigen MUC1. These radiotracers demonstrated no significant differences in the cellular uptake and internalization value, but the biodistribution profile of 99m Tc-HYNIC-D (TPPE) was more favorable than that of 99m Tc-HYNIC-EPPT as a result of better tumor-to-non-target ratios for the examined tissues and organs. HT29 tumors were visualized more clearly in scintigraphic images with 99m Tc-HYNIC-D (TPPE) in comparison with 99m Tc-HYNIC-EPPT. The results showed the retro-inverso analog to be a more promising radiotracer as a probe for in vivo targeting of HT-29 tumors than the parent peptide.

99mTc-D(RGD): molecular imaging probe for diagnosis of αvß3-positive tumors.

OBJECTIVE: Arginine-glycine-aspartic acid (RGD) peptide with its specific binding affinity to integrin αvß3, is widely investigated for the development of molecular imaging probes for diagnosis of αvß3-positive tumors. The aim of this work was to evaluate the ability of Tc- HYNIC-D(RGD), a novel retro-inverso peptidomimetic derivative for U87MG tumor (αvß3-positive) imaging. METHODS: HYNIC-D(RGD) labeled with Tc using tricine/EDDA as an exchange coligands. Single-photon emission computed tomography imaging and biodistribution study were performed in nude mice bearing U87MG xenograft tumor. RESULTS: The labeling yield was >95%. The radiopeptide showed high uptake value in the U87MG tumor relative to muscle after 2 hours (1.43 ± 0.05 vs. 0.22 ± 0.11 %ID/g). The tumor/muscle ratio was 6.5. Blocking experiment showed specific binding towards tumor. Single-photon emission computed tomography imaging study revealed that radiopeptide had prominent uptake in U87MG tumor. CONCLUSION: The novel Tc HYNIC- D(RGD) was demonstrated to be a useful radiotracer for the assessment of αvß3-positive tumor in animal model. Therefore, further clinical and preclinical studies are required.

The Effects of the Spacer on Radiochemical and Biological Properties of New Radiolabeled Bombesin(7-14) Derivative.

OBJECTIVE: The aim of this study was to develop 99mTc-[HYNIC-X-D-Phe13]-BBN(7-14)NH2 derivatives using two different tripeptidic spacer groups (X=GGG and X=SSS) in order to improve its pharmacokinetics, in vitro stability, specific binding, and affinity. BACKGROUND: Bombesin (BBN), a 14-aminoacid amphibian peptide homolog of mammalian gastrinreleasing peptide (GRP), has demonstrated the ability to bind with high affinity and specificity to GRP receptor, which is overexpressed on a variety of human cancers. METHODS: Peptide conjugates labeled with 99mTc using tricine-EDDA and radiochemical purity was assessed by TLC and HPLC. The stability of radio conjugates was evaluated in the presence of saline and human serum. Affinity, internalization, and also dissociation Constant was evaluated using MDAMB- 231 and PC-3 cell line. Biodistribution study was performed in BALB/C mice. RESULTS: Labeling yield of ˃95% was obtained. The change introduced in the BBN sequence increased plasma stability. In vitro blocking studies showed that binding and internalization of both radiolabeled peptides are mediated by their receptors on the surface of MDA-MB-231 and PC-3 cells. Biodistribution results demonstrated a rapid blood clearance, with predominantly renal excretion. Specific binding in GRP receptor-positive tissues, such as pancreas was confirmed with a blocking study. CONCLUSION: The introduction of the spacer sequence between chelator and BBN(7-14) led to improved bidistribution. Analog with tri-Gly spacer is the more promising radiopeptide for targeting GRP receptors than Ser conjugates. Therefore, these analogs can be considered as a candidate for the identification of bombesin-positive tumors.

99m Tc-(EDDA/tricine)-HYNIC-GnRH analogue as a potential imaging probe for diagnosis of prostate cancer.

Prostate cancer is a serious threat to men's health, so it is necessary to develop the techniques for early detection of this malignancy. Radiolabeled peptides are the useful tools for diagnosis of prostate cancer. In this research, we designed a new HYNIC-conjugated GnRH analogue and labeled it by 99m Tc with tricine/EDDA as coligands. We used aminohexanoic acid (Ahx) as a hydrocarbon linker to generate 99m Tc-(tricine/EDDA)-HYNIC-Ahx-[DLys6 ]GnRH. The radiopeptide exhibited high radiochemical purity and stability in solution and serum. Two human prostate cancer cell lines LN-CaP and DU-145 were used for cellular experiments. The binding specificity and affinity of radiopeptide for LN-CaP were superior to DU-145 cells. The Kd values for LN-CaP and DU-145 cells were 41.91 ± 7.03 nM and 55.96 ± 10.56 nM, respectively. High kidney uptake proved that the main excretion route of radiopeptide was through the urinary system. The tumor/muscle ratio of 99m Tc-HYNIC-Ahx-[DLys6 ]GnRH was 4.14 at 1 hr p.i. that decreased to 2.41 at 4 hr p.i. in LN-CaP tumor-xenografted nude mice. The blocking experiment revealed that the tumor uptake was receptor-mediated. The lesion was visualized clearly using 99m Tc-[DLys6 ]GnRH at 1 hr p.i. Accordingly, this research highlights the capability of 99m Tc-(tricine/EDDA)-HYNIC-Ahx-[DLys6 ]GnRH peptide as a promising agent for GnRHR-expressing tumor imaging.

Tumor targeting with 99m Tc radiolabeled peptides: Clinical application and recent development.

Targeting overexpressed receptors on the cancer cells with radiolabeled peptides has become very important in nuclear oncology in the recent years. Peptides are small and have easy preparation and easy radiolabeling protocol with no side-effect and toxicity. These properties made them a valuable tool for tumor targeting. Based on the successful imaging of neuroendocrine tumors with 111 In-octreotide, other receptor-targeting peptides such as bombesin (BBN), cholecystokinin/gastrin analogues, neurotensin analogues, glucagon-like peptide-1, and RGD peptides are currently under development or undergoing clinical trials. The most frequently used radionuclides for tumor imaging are 99m Tc and 111 In for single-photon emission computed tomography and 68 Ga and 18 F for positron emission tomography imaging. This review presents some of the 99m Tc-labeled peptides, with regard to their potential for radionuclide imaging of tumors in clinical and preclinical application.

New neurotensin analogue radiolabeled by 99m-technetium as a potential agent for tumor identification.

It has been shown that more than 75% of ductal pancreatic adenocarcinomas overexpressed neurotensin (NT) receptors. Overexpression of NT receptors has been reported in various human tumor types. Hence, a non-invasive diagnosis and staging method could be very beneficial. In this work, we describe radiolabeling and evaluation of new neurotensin analogues to target neurotensin receptor-positive tumors such as pancreatic carcinoma. Radiolabeling was performed at 95°C for 10 min using 99m Tc in the presence of tricine/EDDA exchange labeling. Radiochemical yield analysis involved ITLC and HPLC methods. A binding assay test was carried out in nine different concentrations of labeled neurotensin analogues in HT-29 cells. Radiopeptide-specific binding and internalization were studied in NT receptors expressing HT-29 cells. Biodistribution studies were performed in tumor-free BALB/c mice and HT-29 xenografted tumor-bearing nude mice. The peptide was efficiently labeled by 99m Tc with high radiochemical yields (>98%). The radioconjugate was thoroughly stable in the solution and human serum even for 24 hr. The radiolabeled peptide showed high affinity (32.66 ± 4.01 nm) and specificity internalization (>%18 after 4 hr) to HT-29 cells. The radiopeptide efficiently showed tumor size and location in tumor-bearing nude mice. In biodistribution, a receptor-specific uptake of radiopeptide was observed in neurotensin receptor-positive organs such as intestine. Uptake in the tumor was 4.59 ± 0.23% ID/g after 2 hr. Owing to excellent stability, high affinity, rapid blood clearance, low accumulation in non-target organs, and high uptake in tumor, the 99m Tc-HYNIC-peptide is a potential agent for targeting of NTR-overexpressing tumor cells in clinical surroundings. When successfully executed in the clinical surrounding, non-invasive imaging of NTR-positive tumors with 99m Tc-labeled new neurotensin analogues could facilitate therapy procedure and monitoring.

99mTc labeled D(LPR): A novel retro-inverso peptide for VEGF receptor-1 targeted tumor imaging.

INTRODUCTION: The aim of this study was to evaluate the ability of D(LPR), a novel retro-inverso peptidomimetic derivative for imaging colon cancer. METHODS: Two different D(LPR) analogs were designed and compared based on conjugation of HYNIC at peptide's C or N terminal and then labeled with technetium-99m using tricine/EDDA as an exchange coligands. The radiolabeled conjugates were assessed for in vitro stability in saline and serum. The VEGFR-1 and NRP-1 receptors affinity, in vitro internalization and also dissociation Constance was evaluated. SPCET imaging and biodistribution studies were performed in nude mice bearing HT-29 xenograft tumors. RESULTS: Both peptides labeled with technetium-99m in high radiochemical yield (˃97%). Peptide stability studies indicated a high metabolic stability of the radiopeptides in solution and serum. In vitro blocking studies demonstrated specific binding and internalization of [99mTc]Tc-HYNIC-peptides in cultured HUVEC cells. The Kd value for 99mTc-peptide 1 and 99mTc-peptide 2 were found to be 56.8 ±â€¯12.9 nM and 71.6 ±â€¯17.9 nM respectively. The tumor to muscle ratio was significant at 0.5 and 1 h after injection (4.5 and 4 for 99mTc-peptide 1 and 4.9 and 4.4 for 99mTc-peptide 2 at 0.5 and 1 h p.i. respectively). SPECT imaging studies revealed that both radioconjugates had prominent activity accumulation in VEGFR-1 and NRP-1 expressing HT-29 tumors. CONCLUSION: This study is the first instance of using a radiolabeled retro-inverso peptide for tumor imaging which is a promising tool to improve the performance of fragile peptide probes in vivo as imaging agents and warrant further investigations in other peptide-target systems.

Evaluation of the Effect of Resveratrol and Doxorubicin on 99mTc-MIBI Uptake in Breast Cancer Cell Xenografts in Mice.

BACKGROUND AND OBJECTIVE: Doxorubicin (DOX), despite having antitumor properties, also exhibits cardiotoxicity. Resveratrol has antitumor property for breast cancer cells. 99mTc-MIBI has higher absorption rate in human breast cancer cell line MCF-7. In the present study, the authors intend to investigate the effect of DOX and resveratrol on the absorption of 99mTc-MIBI in breast cancer cell xenografts in mice. MATERIALS AND METHODS: Sixteen xenograft models in nude mice were divided into four groups. Group I (S, control) received 2% DMSO in 0.9% saline, group II (D) 2.5 mg/kg DOX, group III (D + R) 20 mg/kg/d resveratrol with 2.5 mg/kg DOX (total dose of 15 mg/kg in six injections), and group IV (R) 20 mg/kg/d resveratrol for 2 weeks. Single-photon emission computed tomography (SPECT) images were taken for the determination of 99mTc-MIBI absorption. Mice were sacrificed, and the percentage of injected dose per gram (%ID/g) of the heart, liver, tumor, and muscle was measured using a gamma counter. Hematoxylin-eosin staining and Masson's trichrome staining were used for investigation of histopathological changes. RESULTS: The %ID/g of tumor was lowest in group D + R. The severity of tumor necrosis or apoptosis was highest in group D + R, but there is no significant difference in pathological injuries and %ID/g of tumor between the group D + R and group D. In addition to the results of the %ID/g, the severity of pathological injuries to the liver and heart cells in group D + R was higher compared with group D. There is a significant difference in the %ID/g of the liver between the group D + R and group D. SPECT images showed that the lowest amount of %ID/g was observed in the tumor of group D + R. CONCLUSIONS: According to the results of pathology, biodistribution study, and imaging, the combination of DOX and resveratrol has shown higher antitumor effect; hence, 99mTc-MIBI can be used to evaluate their antitumor effect.

Human Fibronectin Extra-Domain B (EDB)-Specific Aptide (APTEDB) Radiolabelling with Technetium-99m as a Potent Targeted Tumour-Imaging Agent.

BACKGROUND: Human fibronectin extra-domain B (EDB) is particularly expressed during angiogenesis progression. It is, thus, a promising marker of tumour growth. Aptides are a novel class of peptides with high-affinity binding to specific protein targets. APTEDB is an antagonist-like ligand that especially interacts with human fibronectin EDB. OBJECTIVE: This study was the first attempt in which the hydrazinonicotinamide (HYNIC)-conjugated APTEDB was labelled with technetium-99m (99mTc) as an appropriate radiotracer and tricine/EDDA exchange labeling. METHODS: Radiochemical purity, normal saline, and serum stability were evaluated by HPLC and radio-isotope TLC scanner. Other examinations, such as protein-binding calculation, dissociation radioligand binding assay, and partition coefficient constant determination, were also carried out. The cellular-specific binding of 99mTc- HYNIC-conjugated APTEDB was assessed in two EDB-positive (U87MG) and EDB-negative (U373MG) cell lines. Bio-distribution was investigated in normal mice as well as in U87MG and U373MG tumour-bearing mice. Eventually, the radiolabelled APTEDB was used for tumour imaging using planar SPECT. RESULTS: Radiolabelling was achieved with high purity (up to 97%) and accompanied by high solution (over 90% after overnight) and serum (80% after 2 hours) stability. The obtained cellular-specific binding ratio was greater than nine-fold. In-vivo experiments showed rapid blood clearance with mainly renal excretion and tumour uptake specificity (0.48±0.03% ID/g after 1h). The results of the imaging also confirmed considerable tumour uptake for EDB-positive cell line compared with the EDB-negative one. CONCLUSION: Aptides are considered to be a potent candidate for biopharmaceutical applications. They can be modified with imaging or therapeutic agents. This report shows the capability of 99mTc-HYNIC-APTEDB for human EDB-expressing tumours detection.

Radiochemical Evaluation and In Vitro Assessment of the Targeting Ability of a Novel 99mTc-HYNIC-RGD for U87MG Human Brain Cancer Cells.

BACKGROUND: Labeled RGD peptide that specifically targets ανß3 integrin has great potential for the early diagnosis of malignant tumors.αvß3 integrin receptors appear specifically more on the surface of glioblastoma (malignant glioma) cells rather than normal cells. OBJECTIVE: The aim of this study was to identify a novel RGD that can be radiolabeled with99mTc with in vitro assessment of its targeting ability for U87MG human brain cancer cells. METHOD: Novel RGD was designed by Amino Acid retro-inversion technique. The peptide HYNIC conjugate was radiolabeled with 99mTc at 95°C for 10 min and radiochemical analysis was performed using ITLC and HPLC methods. The stability of the radiopeptide was checked in the presence of human serum at 37°C up to 24 h. Binding properties and internalization were studied with U87MG cells. RESULTS: Novel HYNIC-RGD has shown high radiochemical purity over 98%. Radioconjugate binding and internalization in U87MG cells were high and specific (13.96% and 12.38% at 4 h respectively). The radiolabeled peptide revealed good affinity for glioblastoma cells (Kd =1.46 ±0.26nM). CONCLUSION: The in vitro study demonstrated the targeting ability of novel 99mTc-HYNIC-RGD for glioblastoma cells. Therefore, more in vivo studies are required.