Preparation, Radiolabeling with 68Ga/177Lu and Preclinical Evaluation of Novel Angiotensin Peptide Analog: A New Class of Peptides for Breast Cancer Targeting.

AIM: Angiotensin II (AngII) is known to play a significant part in the development of breast cancer by triggering cell propagation of breast cancer, tumor angiogenesis, and regulating tumor invasion and cell migration. AngII arbitrates its action via two G-protein-coupled receptors, AngII type 1 receptor (AT1) and AngII type 2 receptor (AT2). Overexpression of the AT1 receptor in breast cancer cells seems to promote tumor growth and angiogenesis, thus targeting the AT1 receptor using AngII peptide would facilitate the detection of breast carcinoma. We developed an AngII peptide intending to assess whether the peptide of the renin-angiotensin system holds the ability to target AT1 receptor-overexpressing breast cancer in vivo. METHODS: DOTA-coupled AngII peptide was synthesized by conventional solid-phase peptide synthesis according to Fmoc/HATU chemistry. 68Ga/177Lu labeled AngII peptide was evaluated for its binding with TNBC MDA-MB-231 and ER+ MCF7 cell lines. Pharmacokinetics was studied in healthy balb/c mice and in vivo tumor targeting in nude mice with MDA-MB-231 tumors xenografts. RESULTS: DOTA-AngII peptide was labeled efficiently with 68Ga/177Lu with high labeling efficiency (≥90%). The stability of the radiopeptide in human plasma was found to be high. The AngII peptide analog showed nanomolar (<40 nM) AT1 receptor-specific binding affinity. The radioactivity internalized into MDA-MBA-231 and MCF7 cells were 14.97% and 11.75%, respectively. In vivo, biodistribution in balb/c mice exhibited efficient clearance of 68Ga/177Lu-DOTA-AngII peptide from the blood and elimination predominantly by the renal system due to its hydrophilic nature. A low amount of radioactivity was seen in the major organs including lungs, liver, stomach, spleen, and intestines (<3% ID/g) except the kidneys. A high renal-urinary excretion was observed for the radiotracer. In the TNBC MDA-MB-231 xenografts model, radiolabeled AngII peptide exhibited specific and effective AT1-based targeting in vivo. A rapid and efficient tumor targeting (2.18% ID/g at 45 min p.i.) together with fast renal excretion (~67% ID) highlights the tumor-targeting potential of the radiotracer. The AT1 receptor specificity of the radiotracer was validated by blocking assays. Furthermore, PET imaging provided sufficient visualization of MDA-MB-231 tumors in nude mice. CONCLUSION: Our findings suggest that 68Ga/177Lu-DOTA-AngII peptide can be useful for the theranostic application of breast carcinomas. This study suggests the potential of this innovative class of peptides for rapid and efficient targeting of tumors and warrants further evaluation.

Development of new copper-64 labeled rhodamine: a potential PET myocardial perfusion imaging agent.

BACKGROUND: Myocardial perfusion imaging (MPI) is one of the most commonly performed investigations in nuclear medicine procedures. Due to the longer half-life of the emerging positron emitter copper-64 and its availability from low energy cyclotron, together with its well-known coordination chemistry, we have synthesized 64Cu-labeled NOTA- and 64Cu-NOTAM-rhodamine conjugates as potential cardiac imaging agents using PET. RESULTS: 64Cu-NOTA- and 64Cu-NOTAM-rhodamine conjugates were synthesized using a traightforward and one-step simple reaction. Radiochemical yields were greater than 97% (decay corrected), with a total synthesis time of less than 25 min. Radiochemical purities were always greater than 98% as assessed by TLC and HPLC. These synthetic approaches hold considerable promise as a simple method for 64Cu-rhodamine conjugates synthesis, with high radiochemical yield and purity. Biodistribution studies in normal Fischer rats at 60 min post-injection, demonstrated significant heart uptake and a good biodistribution profile for both the radioconjugates. However, the 64Cu-NOTAM-rhodamine conjugate has shown more heart uptake (~ 10% ID/g) over the 64Cu-NOTA-rhodamine conjugate (5.6% ID/g). CONCLUSIONS: These results demonstrate that these radioconjugates may be useful probes for the PET evaluation of MPI.

Synthesis, Radiolabeling, and Preclinical Evaluation of 68Ga/177Lu-Labeled Leuprolide Peptide Analog for the Detection of Breast Cancer.

Objectives: The expansion of novel and potent tumor receptor binding peptides is a promising approach for the precise targeting of various cancer. Leuprolide is a 9-residue peptide analog of gonadotropin-releasing hormone and is extensively used in the treatment of sex hormone-dependent tumors, including prostate, breast, and ovarian cancer. This preclinical study was undertaken to prepare a new radiolabeled leuprolide peptide for the detection of breast carcinoma. Methods: A 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA)-coupled 9-amino acid leuprolide peptide was synthesized after typical 9-fluorenylmethyl-oxycarbonyl-based solid-phase peptide synthesis and radiolabeled with both 68Ga and 177Lu radionuclides for theranostic use. The systemic pharmacokinetics was done in healthy balb/c mice. The in vitro tumor cell binding affinity was determined on MCF7, T47D, and MDA-MB-231 breast cancer cell lines. In vivo tumor targeting and micro positron-emission tomography imaging was performed on nude mice with MCF7 breast tumor xenografts. Results: The leuprolide peptide was conveniently synthesized by solid-phase synthesis strategy and its identity and purity were validated by mass spectrometry and high-performance liquid chromatography. The peptide radiolabeled efficiently (˃94%) with both diagnostic (68Ga) and therapeutic (177Lu) radionuclides and displayed nanomolar binding potency to all three tested MCF7, T47D, and MDA-MB-231 cell lines. Fast and favorable pharmacokinetics was observed for 68Ga/177Lu-leuprolide in healthy Balb/c mice. In nude mice, 68Ga-leuprolide peptide exhibited rapid clearance from the blood circulation with low to moderate (up to 5% ID/g) uptake/retention by the major body organs. The accumulation in the estrogen receptor-positive MCF7 tumor was 2.24% ± 0.62% ID/g at 45 min p.i, with good tumor to blood and muscle uptake ratios. The radiolabeled peptide was excreted primarily through the renal pathway. Conclusion: The encouraging results of this initial study demonstrate that additional testing of this leuprolide peptide seems to be indicated because of its convincing potential to be a new agent for the management of breast carcinoma.

IAEA Activities on 67Cu, 186Re, 47Sc Theranostic Radionuclides and Radiopharmaceuticals.

Despite interesting properties, the use of 67Cu, 186Re and 47Sc theranostic radionuclides in preclinical studies and clinical trials is curtailed by their limited availability due to a lack of widely established production methods. An IAEA Coordinated Research Project (CRP) was initiated to identify important technical issues related to the production and quality control of these emerging radionuclides and related radiopharmaceuticals, based on the request from IAEA Member States. The international team worked on targetry, separation, quality control and radiopharmaceutical aspects of the radionuclides obtained from research reactors and cyclotrons leading to preparation of a standard recommendations for all Member States. The CRP was initiated in 2016 with fourteen participants from thirteen Member States from four continents. Extraordinary results on the production, quality control and preclinical evaluation of selected radionuclides were reported in this project that was finalized in 2020. The outcomes, outputs and results of this project achieved by participating Member States are described in this minireview.

Development of the Tumor-Specific Antigen-Derived Synthetic Peptides as Potential Candidates for Targeting Breast and Other Possible Human Carcinomas.

The human epidermal growth factor receptor 2 (HER2) represents one of the most studied tumor-associated antigens for cancer immunotherapy. The receptors for HER2 are overexpressed in various human cancers, such as breast and ovarian cancer. The relatively low expression of this antigen on normal tissues makes it a clinically useful molecular target for tumor imaging and targeted therapy. HER2 overexpression is correlated with aggressive tumor behavior and poor clinical outcomes. Thus, HER2 has become an important prognostic and predictive factor, as well as a potential molecular target. Due to the heterogeneity of breast cancer and possible discordance in HER2 status between primary tumors and distant metastases, assessment of HER2 expression by noninvasive imaging is important. Molecular imaging of HER2 expression may provide essential prognostic and predictive information concerning disseminated cancer and aid in the selection of an optimal therapy. Another tumor-specific antigen is MUC1, which is silent on normal tissues, but overexpressed in almost all human epithelial cell cancers, including >90% of human breast, ovarian, pancreatic, colorectal, lung, prostate, and gastric cancers and is a promising tumor antigen with diagnostic as well as the therapeutic potential of cancer. Radiolabeled small peptide ligands are attractive as probes for molecular imaging, as they reach and bind the target receptor efficiently and clear from blood and non-target organs faster than bulky antibodies. In this study, HER2 and MUC1-based peptides were synthesized and preclinically evaluated in an effort to develop peptide-based SPECT radiopharmaceuticals derived from tumor-associated antigens for the detection of breast cancer. Our findings demonstrate that the tumor antigen peptides radiolabeled efficiently with 99mTc and showed high metabolic stability in human plasma in vitro. The data from breast tumor cell binding confirmed the high affinity (in low nanomolar range) towards respective breast cancer cell lines. In healthy mice, 99mTc-labeled peptides displayed favorable pharmacokinetics, with high excretion by the renal system. In tumor xenografts nude mice models, good uptake by the SKBR3, MCF7, and T47D tumors were found, with good tumor-to-blood and tumor to muscle ratios. Additionally, tumor lesions can be seen in γ-camera imaging. Our data suggest that based on its ability to detect HER2- and MUC1-positive breast cancer cells in vivo, 99mTc-HER2 and 99mTc-MUC1-targeted peptides may be promising tumor imaging probes and warrant further investigation.

A convenient and efficient total solid-phase synthesis of DOTA-functionalized tumor-targeting peptides for PET imaging of cancer.

INTRODUCTION: An efficient and cost-effective synthesis of the metal chelating agents that couple to tumor-targeting peptides is required to enhance the process of preclinical research toward the clinical translation of molecular imaging agents. DOTA is one of the most widely used macrocyclic ligands for the development of new metal-based imaging and therapeutic agents owing to its ability to form stable and inert complexes under physiological conditions. Although solid-phase synthesis compatible DOTA-tris-(t-Bu ester) is a commercial product, it is expensive and contain chemical impurities. There is a need to explore new and cost-effective methods for the preparation of metal chelating agents, i.e., DOTA, directly on solid support to facilitate rapid, cost-effective, and high purity preparation of DOTA-linked peptides for imaging and therapy. In the present study, we describe a facile synthetic strategy of DOTA preparation and its linkage to peptides directly on solid-phase support. METHODS: Bombesin (BN) peptides were functionalized with DOTA chelator prepared from cyclen precursor on solid-phase and from commercial DOTA-tris and radiolabeled with 68Ga. In vitro BN/GRP receptor binding affinities of the corresponding radiolabeled peptides were determined by saturation binding assays on human breast MDA-MB-231, MCF7, T47D, and PC3 prostate cancer cells. Pharmacokinetics were studied in Balb/c mice and in vivo tumor targeting in MDA-MB-231 tumor-bearing nude mice. RESULTS: DOTA was prepared successfully from cyclen on solid-phase support, linked specifically to BN peptides and resultant DOTA-coupled peptides were radiolabeled efficiently with 68Ga. The binding affinities of all the six BN peptides were comparable and in the low nanomolar range. All 68Ga-labeled peptides showed high metabolic stability in plasma. These radiopeptides exhibited rapid pharmacokinetics in Balb/c mice with excretion mainly through the urinary system. In nude mice, MDA-MB-231 tumor uptake profiles were slightly different; the BN peptide with Ahx spacer and linked to DOTA through cyclen exhibited higher tumor uptake (2.32% ID/g at 1 h post-injection) than other radiolabeled BN peptides investigated in this study. The same leading BN peptide also displayed favorable pharmacokinetic profile in Balb/c mice. The PET images clearly visualized the MDA-MB-231 tumor. CONCLUSIONS: DOTA prepared from cyclen on solid-phase support showed comparable potency and efficiency to DOTA-tris in both in vitro and in vivo evaluation. The synthetic methodology described here allows versatile, site-specific introduction of DOTA into peptides to facilitate the development of DOTA-linked molecular imaging and therapy agents for clinical translation.

Preparation and In Vitro and In Vivo Characterization of the Tumor-specific Antigen-derived Peptide as a Potential Candidate for Targeting Human Epidermal Growth Factor Receptor 2-positive Breast Carcinomas.

BACKGROUND/AIM: The human epidermal growth factor receptor (HER2) is considered as one of the most well-characterized tumor-associated antigens for cancer therapy and plays an important role in the growth and progression of breast cancer. Overexpression of HER2 in various cancers and the availability of its extracellular region makes it a clinically useful target for the development of tumor-antigen specific agents. In this study, we have prepared a HER2-targeted hybrid peptide as a single-photon emission computed tomography (SPECT) imaging probe and evaluated its tumor targeting potential in subcutaneous HER2-positive breast cancer xenograft models. MATERIALS AND METHODS: The HER2-targeted hybrid peptide was prepared by solid-phase peptide synthesis and radiolabeled with 99mTc by the ligand exchange method. In vitro tumor cell binding properties of 99mTc-HER2 were evaluated in HER2-positive (SKBR3) and ER-positive (MCF7 and T47D) breast cancer cell lines. In vivo tumor targeting characteristics were investigated in both SKBR3 (HER2-positive) and MDA-MB-231 (HER2-negative) xenografted animal models. RESULTS: A high labeling efficiency of greater than 95% was achieved when HER2 peptide was radiolabeled with 99mTc by the standard ligand exchange method. 99mTc-HER2 displayed a high binding affinity (Kd=49.95±14.11 nM) to HER2-positive SKBR3 cell line whereas in the case of the ER-positive cell lines (MCF-7 and T47D), the binding affinity was found to be 2-3-fold lower than SKBR3. In vivo tumor uptake in nude mice with SKBR3 tumor xenografts was 2.81±0.79% ID/g as early as 60 min p.i. The uptake in SKBR3 tumors was always higher than the uptake in the blood and muscle, with good tumor-to-blood and tumor-to-muscle ratios. In contrast, low accumulation in ER-positive tumors (MCF7 and T47D) was observed compared to HER2-positive SKBR3 tumor mice. A low to moderate (less than 5% ID/g) accumulation and retention of 99mTc-HER2 was found in most of the major organs excluding the kidneys in both healthy and tumor-bearing mice. CONCLUSION: In view of its ability to detect HER2-positive breast cancer cells in vivo, 99mTc-HER2-targeted peptide may be a promising tumor imaging probe and warrants further investigation.

Preparation and evaluation of the tumor-specific antigen-derived synthetic mucin 1 peptide: A potential candidate for the targeting of breast carcinoma.

PURPOSE: The goal of this study was to prepare a synthetic peptide derived from breast tumor associated antigen and to evaluate its potential as a breast cancer imaging agent. METHODS: A mucin 1 derived peptide was synthesized by solid-phase peptide synthesis and examined for its radiochemical and metabolic stability. The tumor cell binding affinity of (99m)Tc-MUC1 peptide was investigated on MUC1-positive T47D and MCF7 breast cancer cell lines. In vivo biodistribution was studied in normal Balb/c mice and in vivo tumor targeting and imaging in MCF7 and T47D tumor-bearing nude mice. RESULTS: The synthesized MUC1-derived peptide displayed high radiochemical and metabolic stability. In vitro tumor cell-binding on T47D and MCF7 cell lines demonstrated high affinity of (99m)Tc-MUC1 peptide towards human breast cancer cells (binding affinities in nanomolar range). Pharmacokinetic studies performed on Balb/c mice are characterized by an efficient clearance from the blood and excretion predominantly through the urinary system. In vivo tumor uptake in nude mice with MCF7 tumor xenografts was 2.77±0.63% ID/g as early as 1h p.i. whereas in nude mice with T47D human ductal breast epithelial cancer cells, the accumulation in the tumor was found to be 2.65±0.54% ID/g at 1h p.i. Also tumor lesion was detectable in γ-camera imaging. The tumor uptake values were always higher than the blood and muscle uptake, with good tumor retention and good tumor-to-blood and tumor-to-muscle ratios. A low to moderate (<5% ID/g) accumulation and retention of (99m)Tc-MUC1 was found in the major organs (i.e., lungs, stomach, liver, intestines, kidneys, etc.) in both normal and tumor-bearing mice. CONCLUSION: This study suggests that (99m)Tc-MUC1 tumor-antigen peptide may be a potential candidate for the targeted imaging of MUC1-positive human tumors and warrants further investigation.

Novel synthesis and initial preclinical evaluation of (18)F-[FDG] labeled rhodamine: a potential PET myocardial perfusion imaging agent.

Myocardial perfusion imaging is one of the most commonly performed investigations in nuclear medicine studies. Due to the clinical importance of [(18)F]-fluoro-2-deoxy-D-glucose ([(18)F]-FDG) and its availability in almost every PET center, a new radiofluorinated [(18)F]-FDG-rhodamine conjugate was synthesized using [(18)F]-FDG as a prosthetic group. In a convenient and simple one-step radiosynthesis, [(18)F]-FDG-rhodamine conjugate was prepared in quantitative radiochemical yields, with total synthesis time of nearly 20 min and radiochemical purity of greater than 98%, without the need for HPLC purification, which make these approaches amenable for automation. Biodistribution studies in normal rats at 60 min post-injection demonstrated a high uptake in the heart (>11% ID/g) and favorable pharmacokinetics. Additionally, [(18)F]-FDG-rhodamine showed an extraction value of 27.63%±5.12% in rat hearts. These results demonstrate that [(18)F]-FDG-rhodamine conjugate may be useful as an imaging agent for the positron emission tomography evaluation of myocardial perfusion.

Development and pre-clinical evaluation of new 68Ga-NOTA-folate conjugates for PET imaging of folate receptor-positive tumors.

In an attempt to develop new folate radiotracers with favorable biochemical properties for detecting folate receptor-positive cancers, we synthesized 68Ga-NOTA- and 68Ga-NOTAM-folate conjugates using a straightforward and a one-step simple reaction. Radiochemical yields were greater than 95% (decay-corrected) with total synthesis time of less than 20 min. Radiochemical purities were always greater than 98% without high-performance liquid chromatography (HPLC) purification. These synthetic approaches hold considerable promise as a rapid and simple method for 68Ga-folate conjugate preparation with high radiochemical yield in a short synthesis time. In vitro tests on the KB cell line showed that significant amounts of the radioconjugates were associated with cell fractions. Biodistribution studies in nude mice bearing human KB xenografts, demonstrated a significant tumor uptake and favorable biodistribution profile for 68Ga-NOTA-folate over the 68Ga-NOTAM-folate conjugate. The uptake in the tumors was blocked by excess injection of folic acid, suggesting a receptor-mediated process. These results demonstrate that the 68Ga-NOTA-folate conjugate may be useful as a molecular probe for detection and staging of folate receptor-positive cancers, such as ovarian cancer and their metastasis, as well as monitoring tumor response to treatment.

Preparation and evaluation of bombesin peptide derivatives as potential tumor imaging agents: effects of structure and composition of amino acid sequence on in vitro and in vivo characteristics.

OBJECTIVES: Among the many clinically relevant peptide receptor systems, bombesin (BN) receptors have attracted enormous attraction due to their overexpression in various frequently occurring human tumors including breast and prostate, thus making such receptors promising targets with radiolabeled BN analogs. The present study describes the preparation and evaluation of a series of new BN derivatives as potential tumor imaging agents. METHODS: Several new BN derivatives with the common structure MAG(3)-X-BN(1-14 or 6-14), where X=Asp or Asp-Asp, were synthesized by solid-phase peptide synthesis. S-benzoylmercaptoacetic acid was incorporated at the end of synthesis via manual conjugation to yield MAG(3)-BN conjugates. Radiolabeling with (99m)Tc was accomplished by ligand exchange method. The receptor-binding affinity assays were performed in MDA-MB-231, MCF-7, T47-D and PC-3 cancer cell lines. In vivo biodistribution and clearance kinetics were assessed in Balb/c mice, and tumor targeting efficacy was determined in nude mice bearing breast tumor xenografts. RESULTS: The peptides were prepared conveniently and radiolabeled efficiently with (99m)Tc (up to 95% labeling efficiency). In vitro cell binding assays demonstrated high affinity (values in the nanomolar range) of (99m)Tc peptides towards breast and prostate cancer cell lines. In addition, the radioconjugates displayed significant internalization (values ranged between 19% and 35%) in tumor cells. In vivo biodistribution and biokinetics are characterized by efficient clearance from the blood and variable degrees of excretion through the renal pathway. In vivo tumor targeting studies displayed variable uptake capacity of different BN derivatives, underlining the influence of specific amino acid sequence on tumor targeting profiles. Tumor uptake was always higher than the radioactivity in the blood and muscle, with good tumor retention and good tumor-to-blood and tumor-to-muscle ratios, indicating the potential of these agents for targeting tumors in vivo. CONCLUSIONS: The combination of favorable in vitro and in vivo properties may render these BN peptides as potential candidates for targeting BN/GRP receptor-positive tumors. They deserve further evaluation to determine their real strength. The present data indeed provide useful information regarding peptide structure-pharmacologic activity relationship, which might be useful in designing and developing new BN-like peptides for efficient targeting of tumors in vivo.

Synthesis and evaluation of a technetium-99m labeled cytotoxic bombesin peptide conjugate for targeting bombesin receptor-expressing tumors.

Conjugation of the cytotoxic drugs to receptor-binding peptides is an attractive approach for the targeted delivery of cytotoxic peptide conjugates to tumor cells. In an attempt to develop an efficient peptide-based radiopharmaceutical for targeting bombesin (BN) receptor-expressing tumors (i.e., breast and prostate), we have prepared by solid-phase peptide synthesis, a novel BN analog derived from the universal sequence of BN and conjugated to a widely characterized antineoplastic agent, methotrexate (MTX). MTX-BN, after radiolabeling with (99m)Tc via stannous-tartrate exchange, showed a good stability against cysteine and histidine transchelation as well as a high in vitro metabolic stability in human plasma. In vitro cell-binding and internalization on MDA-MB-231, MCF-7, T47-D breast cancer and PC-3 prostate cancer cell lines demonstrated high affinity and specificity of (99m)Tc-MTX-BN towards both human breast and prostate cancer cells (binding affinities in nanomolar range). In addition, the radioconjugate displayed a significant internalization (values ranged between 19-35%) into the tumor cells. In vivo biodistribution and clearance kinetics in Balb/c mice are characterized by an efficient clearance from the blood and excretion mainly through the renal-urinary pathway with some elimination via the hepatobiliary system. In vivo tumor uptake in nude mice bearing MDA-MB-231 cells was 2.70+/-0.44% ID/g at 1 h, whereas in nude mice with human epidermoid KB cells the accumulation in the tumor was found to be 1.48+/-0.31% ID/g at 1 h post injection. The tumor uptake was always higher than in the blood and muscle, with good tumor retention and good tumor-to-blood and tumor-to-muscle ratios. The accumulation/retention in the major organs (i.e., lungs, stomach, liver, intestines, etc.) was low to moderate (<6% ID/g) in both healthy and tumor-bearing mice. However, the uptake/retention in the kidneys was rather high (up to 11.05+/-1.80% ID/g), which is of a concern, particularly for radionuclide therapy. This initial study towards the development of a novel cytotoxic BN conjugate suggest that the combination of favorable in vitro and in vivo properties may render (99m)Tc-MTX-BN a potential candidate for the targeted imaging and eventually for radionuclide therapy (when labeled with an appropriate radionuclide) of BN receptor-positive tumors and deserves further evaluation.

Design, synthesis, radiolabeling and in vitro and in vivo characterization of tumor-antigen- and antibody-derived peptides for the detection of breast cancer.

HER2/neu and MUC1-based synthetic peptides were prepared and evaluated in an effort to develop peptide-based radiopharmaceuticals derived from tumor-associated-antigens for the detection of breast cancer. The receptors for HER2/neu and MUC1 are overexpressed in various human cancers, such as breast and ovarian cancer. The relatively low expression of these antigens on normal tissues makes them attractive targets for tumor imaging. In addition, antitumor-antibody-derived peptides based on the Glu-Pro-Pro-Thr (EPPT) sequence were prepared for the detection of breast cancer. It has been shown that the EPPT peptide has high affinity for the MUC1-derived peptide. The peptides were prepared by solid-phase synthesis and radiolabeled efficiently with (99m)Tc via ligand exchange. They exhibited good stability in vitro in human plasma and against cysteine and histidine challenge. The peptides displayed high affinities (in nanomolar range) for MCF-7, MDA-MB-231 and T47-D breast cancer cell lines in vitro. Additionally, they exhibited a rapid internalization into tumor cells. In vivo biodistribution in mice showed rapid and efficient blood clearance and excretion mainly through the renal-urinary route, with some elimination via the hepatobiliary pathway. However, the extent of urinary excretion was found to be variable for radiopeptides, with the highest being observed for antitumor-antibody-derived peptide. The peptides showed moderate tumor uptake (up to 2.2+/-0.98% ID/g) in nude mice carrying breast tumor xenografts. The uptake in the tumor was always higher than in the blood and muscle. A fast clearance from the blood and low accumulation (<6% ID/g) by the major organs was obtained in nude mice resulting in favorable tumor/blood and tumor/muscle ratios as early as 1 h after injection. The combination of favorable in vitro and in vivo characteristics makes this new and interesting class of peptides potential candidates for the diagnosis of breast cancer in vivo.

Preparation and in vitro and in vivo evaluation of technetium-99m-labeled folate and methotrexate conjugates as tumor imaging agents.

The cell-membrane folic acid (FA) receptors are known to be responsible for cellular accumulation of FA and FA analogs, such as methotrexate (MTX), and are overexpressed on several tumor cells. Folate, as well as antifolates (i.e., MTX), possess high affinity for the folate-receptor positive cells and tissues and were deemed useful for diagnostic imaging. We have prepared and evaluated technetium-99m (99mTc)- labeled FA and MTX analogs using MAG3 and MAG2 chelating agents in an attempt to develop folate-receptor targeting radiopharmaceuticals. Folate and MTX-conjugates after labeling with 99mTc by ligand exchange method displayed high in vitro stability in human plasma. In vitro cell binding and internalization on MCF-7 and MDA-MB-231 cells indicated the affinity and specificity of the radioconjugates toward human breast cancer cells. In mice, all radioconjugates showed rapid clearance from the blood and excretion mainly through the renal/urinary pathway, with some elimination by way of the biliary route. There was no significant accumulation of radioactivity observed in other organs, with the exception of the intestines. Uptake in the breast tumor was moderate in nude mice. These findings could be of potential diagnostic interest in designing and developing FA/MTX-based radiopharmaceuticals for tumor imaging.

Peptide-based radiopharmaceuticals: future tools for diagnostic imaging of cancers and other diseases.

Small synthetic receptor-binding peptides are the agents of choice for diagnostic imaging and radiotherapy of cancers due to their favorable pharmacokinetics. Molecular modification techniques permit the synthesis of a variety of bioactive peptides with chelating groups, without compromising biological properties. Various techniques have been developed that allow efficient and site-specific labeling of peptides with clinically useful radionuclides such as (99m)Tc, (123)I, (111)In, and (18)F. Among them, (99m)Tc is the radionuclide of choice because of its excellent chemical and imaging characteristics. Recently, many (99m)Tc-labeled peptides have proven to be useful imaging agents. Beside (99m)Tc-labeled peptides, several peptides radiolabeled with (111)In and (123)I have been prepared and characterized. In addition, (18)F-labeled peptides hold clinical potential due to their ability to quantitatively detect and characterize a variety of human diseases using positron-emission tomography. The availability of this wide range of peptides labeled with different radionuclides offers multiple diagnostic and therapeutic applications. Various receptors are over-expressed in particular tumor types and peptides binding to these receptors can be used to visualize tumor lesions scintigraphically. Thus, radiolabeled peptides have potential use as carriers for the delivery of radionuclides to tumors, infarcts, and infected tissues for diagnostic imaging and radiotherapy. Many radiolabeled peptides are currently under investigation to determine their potential as imaging agents. These peptides are designed mainly for thrombus, tumor, and infection/inflammation imaging. This article presents recent developments in small synthetic peptides for imaging of thrombosis, tumors, and infection/inflammation.