Co-administration of succinylated gelatine with a (99m)Tc-bombesin analogue, effects on pharmacokinetics and tumor uptake.

The bombesin analogue, [(99m)Tc-GGC]-(Ornithine)3-BN(2-14), (99m)Tc-BN-O, targeting gastrin releasing peptide receptors (GRPrs) on the surface of tumors, was pre-clinically investigated as potential imaging agent for single photon emission computed tomography (SPECT). In addition, the improvement of its pharmacokinetic profile (PK) was investigated through the co-administration of a succinylated gelatin plasma expander (Gelofusine), aiming to reduce its kidney accumulation and enhance its tumor-to-normal tissue contrast ratios. Biodistribution data were collected from normal mice and rats, and PC-3 tumor bearing mice, in reference to its PK, metabolism and tumor uptake. Imaging data were also collected from PC-3 tumor bearing mice. Biodistribution and imaging experiments showed that (99m)Tc-BN-O was able to efficiently localize the tumor (5.23 and 7.00% ID/g at 30 and 60min post injection, respectively), while at the same time it was rapidly cleared from the circulation through the kidneys. HPLC analysis of kidney samples, collected at 60min p.i. from normal mice and rats, showed that the majority of radioactivity detected was due to intact peptide i.e. 56% for mice and 73% for rats. Co-administration of (99m)Tc-BN-O with Gelo resulted in the reduction of kidney uptake in both animal models. The integrated area under the curve (AUC30-60 min) from the concentration-time plots of kidneys was decreased in both mice and rats by 25 and 50%, respectively. In PC-3 tumor bearing mice, an increase of tumor uptake (AUCtumor increased by 69%) was also observed with Gelo. An improvement in tumor-to-blood and tumor-to-normal tissue ratios was noted in all cases with the exception of the pancreas, which normally expresses GRPr. The results of this preclinical study may also be extended to other similar peptides, which are utilized in prostate cancer imaging and present similar PK profile.

Evaluation of ανß3-mediated tumor expression with a 99mTc-labeled ornithine-modified RGD derivative during glioblastoma growth in vivo.

In this study, a novel way of distinguishing the intrinsic relationship between ανß3 integrin targeting and detection of tumor growth by using a radiolabeled tracer based on a cyclic Arg-Gly-Asp (RGD) peptide was provided. The potential of the in vivo scintigraphic imaging of the developing vasculature from the early stage of tumor growth was evaluated. Alongside with the scintigraphic images, biodistribution studies were performed at distinct time points to validate this noninvasive imaging approach. The ability to noninvasively assess the tumor growth of ανß3 integrin-positive glioblastoma tumors provides a method to better understand tumor angiogenesis in vivo and allows for a direct assessment of anti-integrin treatment efficacy.

(99m)Tc-labeled aminosilane-coated iron oxide nanoparticles for molecular imaging of ανß3-mediated tumor expression and feasibility for hyperthermia treatment.

HYPOTHESIS: Dual-modality imaging agents, such as radiolabeled iron oxide nanoparticles (IO-NPs), are promising candidates for cancer diagnosis and therapy. We developed and evaluated aminosilane coated Fe3O4 (10±2nm) as a tumor imaging agent in nuclear medicine through 3-aminopropyltriethoxysilane (APTES) functionalization. We evaluated this multimeric system of targeted (99m)Tc-labeled nanoparticles (NPs) conjugated with a new RGD derivate (cRGDfK-Orn3-CGG), characterized as NPs-RGD as a potential thermal therapy delivery vehicle. EXPERIMENTS: Transmission Electron Microscopy (TEM) and spectroscopy techniques were used to characterize the IO-NPs indicating their functionalization with peptides. Radiolabeled IO-NPs (targeted, non-targeted) were evaluated with regard to their radiochemical, radiobiological and imaging characteristics. In vivo studies were performed in normal and ανß3-positive tumor (U87MG glioblastoma) bearing mice. We also demonstrated that this system could reach ablative temperatures in vivo. FINDINGS: Both radiolabeled IO-NPs were obtained in high radiochemical yield (>98%) and proved stable in vitro. The in vivo studies for both IO-NPs have shown significant liver and spleen uptake at all examined time points in normal and U87MG glioblastoma tumor-bearing mice, due to their colloidal nature. We have confirmed through in vivo biodistribution studies that the non-targeted (99m)Tc-NPs poorly internalized in the tumor, while the targeted (99m)Tc-NPs-RGD, present 9-fold higher tumor accumulation at 1h p.i. Accumulation of both IO-NPs in other organs was negligible. Blocking experiments indicated target specificity for integrin receptors in U87MG glioblastoma cells. The preliminary in vivo study of applied alternating magnetic field showed that the induced hyperthermia is feasible due to the aid of IO-NPs.

Polymeric micelles and vesicles: biological behavior evaluation using radiolabeling techniques.

The application of combined diagnosis and therapy through nanotechnology applications is attracting increasing attention worldwide. Polymeric self-assembled nanoparticles (NPs) have been studied for this purpose. Micelles and vesicles with or without a magnetic core can efficiently carry diagnostic and/or therapeutic agents to a desired target. The biological behavior of these NPs has been evaluated in this study, after radiolabeling with (99m)Tc. In vitro stability, in media that mimic the environment of the living body, was better for vesicles than for micelles at 1 h and decreased for both as time passed. After administration to healthy animals, all NPs presented major uptake at liver and spleen as expected. Biodistribution and imaging studies confirmed the higher uptake in these organs for the hybrid NPs and at higher extent for the ones with larger size, indicating that the magnetic load and size play an important role on in vivo distribution.

Radiochemical and radiobiological assessment of a pyridyl-S-cysteine functionalized bombesin derivative labeled with the (99m)Tc(CO)3(+) core.

Bombesin is a neuropeptide widely studied due to its ability to target various types of cancers. Technetium-99m on the other hand is ideal for diagnostic tumor targeting. The aim of the present study is the investigation of the coupling of the ligand (S)-(2-(2'-pyridyl)ethyl)-d,l-cysteine with the BN-peptide Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met(CONH2) through the spacer aminohexanoic acidand the labeling of the resulting derivative MBN with the synthon [M(CO)3(H2O)3](+) (M=(99m)Tc, Re). The peptide was synthesized according to the SPPS method, purified and characterized by ESI-MS. The new (99m)Tc-labeled biomolecule was stable in vitro, showed high affinity for the human GRP receptor expressed in PC3 cells and the rate of internalization was found to be time-dependent tissue distribution of the radiopeptide was evaluated in normal mice and in prostate cancer experimental models and significant radioactivity uptake was observed in the pancreas of normal mice as well as in PC3 tumors. Dynamic studies of the radiopeptide showed satisfactory tumor images.

Structural modifications of 99mTc-labelled bombesin-like peptides for optimizing pharmacokinetics in prostate tumor targeting.

PURPOSE: The main goal of the present study was to investigate the importance of the addition of a positively charged aa in the naturally occurring bombesin (BN) peptide for its utilization as radiodiagnostic agent, taking into consideration the biodistribution profile, the pharmacokinetic characteristics and the tumor targeting ability. METHODS: Two BN-derivatives of the general structure [M-chelator]-(spacer)-BN(2-14)-NH(2), where M: (99m)Tc or (185/187)Re, chelator: Gly-Gly-Cys-, spacer: -(arginine)(3)-, M-BN-A; spacer: -(ornithine)(3)-, M-BN-O; have been prepared and evaluated as tumor imaging agents. RESULTS: The peptides under study presented high radiolabelling efficiency (>98%), significant stability in human plasma (>60% intact radiolabelled peptide after 1h incubation) and comparable receptor binding affinity with the standard [(125)I-Tyr(4)]-BN. Their internalization rates in the prostate cancer PC-3 cells differed, although the amount of internalized peptide was the same. The biodistribution and the dynamic γ-camera imaging studies in normal and PC-3 tumor-bearing SCID mice have shown significant tumor uptake, combined with fast blood clearance, through the urinary pathway. CONCLUSION: The addition of the charged aa spacer in the BN structure was advantageous for biodistribution, pharmacokinetics and tumor targeting ability, because it reduced the upper abdominal radioactivity levels and increased tumor/normal tissue contrast ratios.

Synthesis and comparative assessment of a labeled RGD peptide bearing two different 99mTc-tricarbonyl chelators for potential use as targeted radiopharmaceutical.

During the past decade radiolabeled RGD-peptides have been extensively studied to develop site-directed targeting vectors for integrins. Integrins are heterodimeric cell-surface adhesion receptors, which are upregulated in cancer cells and neovasculature during tumor angiogenesis and recognize the RGD aminoacid sequence. In the present study, we report the synthesis and development of two derivatives of the Nε-Lys derivatized cyclic Arg-Gly-Asp-D-Phe-Lys peptide, namely of cRGDfKHis and cRGDfK-CPA (CPA: 3-L-Cysteine Propionic Acid), radiolabeled via the [(99m)Tc(H(2)O)(3)(CO)(3)](+) metal aquaion at a high yield even at low concentrations of 10-5M (>87%) for cRGDfK-10-5M (>93%) for cRGDfK-CPA. Radiolabeled peptides were characterized with regard to their stability in saline, in His/Cys solutions, as well as in plasma, serum and tissue homogenates and were found to be practically stable. Internalization and efflux assays using αvß3-receptor-positive MDA-MB 435 breast cancer cells showed a good percentage of quick internalization (29.1 ± 9.8% for (99m)Tc-HiscRGDfK and 37.0 ± 0.7% for (99m)Tc-CPA-cRGDfK at 15 min) and no retention of radioactivity for both derivatives. Their in vivo behavior was assessed in normal mice and pathological SCID mice bearing MDA-MB 435 ανß3 positive breast tumors. Both presented fast blood clearance and elimination via both the urinary and hepatobiliary systems, with (99m)Tc-His-cRGDfK remaining for a longer time than (99m)Tc-CPA-cRGDfK in all organs examined. Tumor uptake 30 min pi was higher for (99m)Tc-CPAcRGDfK (4.2 ± 1.5% ID/g) than for (99m)Tc-His-cRGDfK (2.8 ± 1.5% ID/g). Dynamic scintigraphic studies showed that the tumor could be visualized better between 15 and 45 min pi for both radiolabeled compounds but low delineation occurred due to high abdominal background. It was finally noticed that the accumulated activity on the tumor site was depended on the size of the experimental tumor; the smaller the size, the higher was the radioactivity concentration.

Structural assessment and biological evaluation of two N3S bombesin derivatives.

The aim of the present study is the evaluation of the (99m)Tc complexes of two bombesin-like peptides: Gly1'-Gly2'-Cys3'-Aca-BN[2-14] (BN-1.1) and Gly1'-Gly2'-Cys3'-Aca-BN[7-14] (BN-1.1p). The BN derivatives were synthesized according to the solid phase peptide synthesis method, and characterized by ESI-MS and NMR. (185/187)Re-BN-1.1 and (185/187)Re-BN-1.1p were also identified by ESI-MS and NMR. The (99m)Tc complexes were stable over time in human plasma, while they degraded rapidly in kidney-liver homogenates. The peptides and their (99m)Tc complexes showed high affinity for the human GRP receptors expressed in PC-3 cells. The rate of internalization of these radiolabeled biomolecules was found to be time-dependent. Also, it was found that there was no long-term retention of the radioactive metabolites into the cells. Tissue distribution of the radiopeptides was evaluated in normal mice and in prostate cancer experimental models. Significant uptake of radioactivity was observed in the pancreas of PC-3 tumor-bearing SCID mice. Dynamic studies of both radiopeptides showed satisfactory tumor images.

Spacer site modifications for the improvement of the in vitro and in vivo binding properties of (99m)Tc-N(3)S-X-bombesin[2-14] derivatives.

It has been shown that gastrin releasing peptide receptors (GRPRs) are overexpressed in various types of cancer cells. Bombesin is an analogue of the mammalian GRP that binds with high specificity and affinity to GRPRs. Significant research efforts have been lately devoted to the design of radiolabeled 8 or 14 aminoacid bombesin (BN) peptides for the detection (either with gamma or positron emitting radionuclides) and therapy (with beta(-) emitting radionuclides) of cancer. The specific aim of the present study was to further investigate the radiolabeled peptide structure and to determine whether the total absence of a linker or the use of a basic diverse amino acid linker could influence the biodistribution profile of the new compounds for specific targeting of human prostate cancer. Thus, two new derivatives with the structure Gly-Gly-Cys-X-BN[2-14], where linker X is either zero (I) or Orn-Orn-Orn (Orn: ornithine) (II) were designed and synthesized. The corresponding (99m)Tc-BN derivatives were obtained with high radiochemical yield (>98%) and had almost identical retention times in RP-HPLC with the (185/187)Re complexes, which were also characterized by ESI-MS. Metabolic stability was found to be high in human plasma, moderate in PC-3 cells, and rather low in mouse liver and kidney homogenates for both BN derivatives studied. The BN derivative without the spacer was less stable in cell culture and liver homogenates. A satisfactory binding affinity to GRPRs, in the nanomolar range, was obtained for both BN derivatives as well as for their Re complexes, with BN (II) demonstrating the highest one. In vitro internalization/externalization assays indicated that approximately 6% of BN (I) and approximately 25% of BN (II) were internalized into PC-3 cells. In vivo evaluation in normal Swiss mice and in tumor bearing SCID mice showed that BN (II) presented higher tumor and pancreas uptake than BN (I). Small animal SPECT dynamic imaging, carried out after an injection of BN (II) in mice bearing PC-3 tumors, resulted in PC-3 tumor delineation with low background activity. Overall, this study performed for two new N(3)S-X-BN[2-14] derivatives indicated that hydrophilicity and charge strongly affected the in vitro and in vivo binding properties and the biodistribution pattern. This finding is confirmed by SPECT imaging of BN (II), which is under further in vivo evaluation for detecting cancer-positive GRPRs.

Evaluation of a series of new 99mTc-labeled bombesin-like peptides for early cancer detection.

Bombesin (BN) is a peptide exhibiting a high affinity for the gastrin-releasing peptide (GRP) receptor, which is overexpressed by a variety of tumors, including breast or prostate cancer. The aim of the present study was the investigation of the complexes formed between a series of BN-like peptides and the nuclides (185/187)Re and 99mTc. The (185/187)Re complexes were formed via the precursor Regluconate. The radiolabeling of the derivatives with 99mTc was performed using either 99mTc-gluconate or 99mTc-MDP as the intermediate complex. For the in vitro evaluation of the new peptides, the cancer cell line PC3 was used. The in vivo behavior of the 99mTc-labeled BN-like peptides was evaluated in normal mice. All the derivatives showed specific uptake in the pancreas, an organ rich in BN receptors and high affinity for the cancer cell line PC3. The above preliminary results indicated that the new BN derivatives are promising for human cancer studies.

Comparative evaluation of linear and cyclic 99mTc-RGD peptides for targeting of integrins in tumor angiogenesis.

Cell adhesion molecules, such as integrins, play a vital role in angiogenesis, a key pathway for tumor growth, invasion and metastasis. The integrin alpha(v)beta(3), which recognizes the RGD sequence (Arg-Gly-Asp), may provide a target for in vivo tumor imaging. A linear and a cyclic RGD peptide derivative (RGDfK-His and cRGDfK-His, respectively), labelled via the precursor [99mTc(H2O)3(CO)3]+, were comparatively evaluated and their radiobiological properties were assessed in normal and tumor-bearing mice. Biodistribution studies showed non-specific uptake in all organs, rapid blood clearance and elimination via the hepatobiliary and urinary systems. Tumor uptake was higher for the cyclic radiolabelled derivative, as the both biodistribution and imaging studies suggested. The cRGDfK-His, labelled via the fac-[99mTc(CO)3]-core, may prove to be a useful tool for early tumor detection.

Structural study by NMR of an oxorhenium-RGD decapeptide complex for application in radiotherapy.

The decapeptide Arg-Gly-Asp-Ser-Cys-Arg-Gly-Asp-Ser-Tyr, which contains two Arg-Gly-Asp (RGD) moieties in its sequence, has been successfully labeled with radioactive rhenium (Re-188) yielding a single, stable oxorhenium complex. This complex is being evaluated for possible application in oncology as a target-specific radiotherapeutic agent, because its radioactive technetium-99m analogue has already been applied for the scintigraphic detection of malignant melanoma in humans. For structural characterization purposes, the complex of the decapeptide was synthesized at the macroscopic level using nonradioactive rhenium (Re-185/Re-187). NMR and mass spectral analysis of the nonradioactive oxorhenium complex revealed that the decapeptide coordinates to the oxorhenium core through the N(amide) of Asp3, the N(amide) of Ser4, and the N(amide) and S(thiolate) atoms of Cys5 to form a complex of the ReO[N(3)S] type.

Role of 99mTc-bombesin scan in diagnosis and staging of prostate cancer.

Aim of this work was to asses whether a novel 99mTc labeled Bombesin (BN) can play a clinical role in diagnosis and staging of prostate cancer. 14 patients were studied with trans-rectal ultrasonography-guided biopsy, CT and MRI and with 99mTc BN Scintigraphy. Five patients were also imaged by 111In Octreotide (O) scan. All the patients but one were submitted to surgery and final diagnosis was reached by pathology, taken as the gold standard method. Two patients showed benign adenoma and 12 patients showed cancer at biopsy. 99mTc BN SPECT was positive in all 12 patients with cancer. Four of these patients also showed pelvic focal uptake, referred to inguinal lymph-nodal involvement. MRI and CT provided similar findings in only three cases. Pathologic evaluation after operation confirmed the invasion of nodes in all four subjects. Both 99mTc BN and 111In O scans provided normal findings in the two subjects affected by benign adenoma, while 111In O was positive in only two of three patients with cancer and was always unable to detect nodal invasion. These preliminary data suggest that 99mTc BN SPECT scan could be useful to detect primary prostate cancer and to reveal loco-regional node involvement.

Biodistribution and scintigraphic studies of 153Sm-labeled anti-CEA monoclonal antibody for radioimmunoscintigraphy and radioimmunotherapy.

The anti-CEA monoclonal antibody, which selectively localizes in colon cancer, was labeled with Samarium-153 (153Sm). 153Sm is mainly a beta-emitter which can be used for therapeutic purposes, while its gamma-ray facilitates imaging studies. Labeling was achieved using the bicyclic anhydride of DTPA as chelator for Sm-153 tagging onto the antibody. [153Sm]anti-CEA was biologically evaluated in nude mice bearing tumors of different weight (0.5-2.5 g), at diverse time intervals (4-72 hours), by anatomic and imaging methods. Biodistribution studies showed slow blood clearance and high retention in the liver, kidneys and lungs. In nude mice bearing tumors of about the same weight, uptake increased with time, from 4 to 72 hours post injection (p.i.). Highest uptake was observed in 0.5-0.8 g tumors compared to those of 1.5-2.5 g. The results agreed with imaging studies performed on a gamma camera at 4 to 72 hours p.i. Tumor uptake depended on time and tumor weight. The tumor can be visualized 24 hours p.i. but, due to the high background, it can be clearly distinguished at 72 hours p.i.

99mTc-bombesin detects prostate cancer and invasion of pelvic lymph nodes.

Biopsy is the standard method for the diagnosis of prostate cancer; however, it is inadequate for the assessment of lymph node invasion. Radionuclide imaging might be useful for both diagnosis and N staging, but it requires high uptake of radiotracers in order to overcome difficulties arising from the anatomy of the region. The aim of this study was to assess whether or not technetium-99m labelled bombesin (99mTc-BN) scan is able to detect prostate cancer and invasion of pelvic lymph nodes. Ten patients were studied with 99mTc-BN, transrectal ultrasonography, biopsy, computed tomography and magnetic resonance imaging. All the patients with cancer were operated on. Planar dynamic scintigraphy and single-photon emission tomography (SPET) were performed after administration of 185 MBq 99mTc-BN. Two patients showed benign adenoma and eight showed cancer at biopsy. The average Gleason's score was 7.5+/-1.3. 99mTc-BN dynamic planar scan showed hot spots in the prostatic fossa in two of the eight patients with cancer, both of whom had a prostate-specific antigen level higher than 20 ng/ml. In these patients, high uptake inside the prostatic fossa was detected as early as 1 min after injection, before the arrival of radioactivity in the bladder. True positive SPET scans were obtained in all eight patients with cancer. Invasion of the obturator nodes was detected by SPET in three patients, and in all three was confirmed at surgery. Our preliminary data encourage further studies on the prostate with 99mTc-BN. If the high sensitivity of 99mTc-BN SPET is confirmed, this method may play an important role in diagnosing and staging prostate cancer.