Evaluation of a new series of copper-64-nota-bombesin targeted radiopharmecuticals with PET imaging potential [abstract]

Abstract only availableTissue-specific radiopharmaceuticals having promise as diagnostic and therapeutic targeting vectors for human cancers is of significant interest. In recent years, our group and many others have focused upon design and development of targeting vectors having high selectivity and affinity for the gastrin-releasing peptide receptor (GRPr). GRP receptors are known to be over-expressed on a variety of malignancies including breast, lung, pancreatic, and prostate cancers. Bombesin (BBN), an amphibian analogue of mammalian GRP, has demonstrated the ability to bind with high affinity and specificity to the GRP receptor. In this study, we report the synthesis of the bifunctional chelating agent (BFCA) 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and conjugation to the biologically active BBN targeting vector. Bombesin conjugates were derived of the form, NOTA-X-BBN (X = GGG, SSS, ßAla, 5Ava, and 8Aoc). The unligated BBN [7-14] peptides with spacer groups were synthesized by solid-phase peptide synthesis (SPPS) and purified by reverse phase-high performance liquid chromatography (RP-HPLC). NOTA chelator was conjugated to the N-terminal primary amine of BBN manually. The final NOTA-X-BBN derivatives were purified by RP-HPLC and their chemical constitution verified. [64Cu-NOTA-X-BBN]-conjugates were prepared by addition of 64CuCl2 to a buffered solution containing the conjugate followed by purification via RP-HPLC. In vivo studies of [64Cu-NOTA-8-Aoc-BBN]-conjugates in normal CF-1 mice showed receptor-specific uptake in normal pancreatic tissue, an organ known to express the GRPr in very high numbers. Furthermore, studies in human-prostate tumor-bearing mice have demonstrated the ability of [64Cu-NOTA-8-Aoc-BBN] to undergo receptor-specific tumor uptake. Diagnostic positron emission tomography (PET) imaging using this conjugate was successful in resolving xenografted tumors in a mouse model. Both normal and tumor-bearing mice exhibited low liver accumulation of Cu-64, a known complication of 64Cu-containing radiopharmaceuticals. Further studies are needed to verify the efficacy of [64Cu-NOTA-8-Aoc-BBN] to target other human malignancies.Life Sciences Undergraduate Research Opportunity Progra

The magic bullet: Creating Indium-111 bombesin targeting vectors for use in diagnostic imaging of prostate and breast cancer [abstract]

Abstract only availableBackground: According to the American Cancer Society, over 68,000 men and women will die from prostate and breast cancer in this year alone. Prostate, breast and other cancers have been shown to express the BB2 receptor. For the past decade the Hoffman laboratory has been synthesizing radiopharmaceutical conjugates based on the Bombesin (BBN) peptide (Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2) that target the BB2 receptor for diagnosis and treatment of cancer. The radioconjugates are composed of a bombesin targeting vector, linking group, chelation moiety and a radioactive metal. One focus of our group is to investigate the efficacy of new Bombesin Targeting Vectors (BTV) which are derivatives of the BBN peptide. In the sixth position of the BTV is a D-phenylalanine amino acid. Our hypothesis is that the D-phenylalanine is responsible for significantly reducing kidney retention. Reduction of kidney retention is crucial for clinical radiotherapeutic applications because the kidney is often the dose limiting organ. In order to understand the structure function relationship of the D-phenylalanine in the BTV targeting vectors, we synthesized and evaluated the BTV peptide with the L-phenylalanine in the sixth position to determine what effect the stereochemistry has upon the in vitro receptor binding and in vivo pharmacokinetic properties of the peptide. Methods: The peptides were synthesized using solid phase peptide synthesis, purified using RP-HPLC, and characterized using electrospray mass spectrometry. Radiolabeling of the peptides was performed using 111InCl3. In vitro cell binding assays and internalization and efflux studies were performed using the PC-3 human cancer cell line. In vivo pharmacokinetic studies were performed using CF-1 mice. Micro-SPECT (single photon emission computed tomography) imaging studies were performed in PC-3 SCID mice. Results: In vivo pharmacokinetic studies at 15 min post-injection gave 39.85 ± 5.07 %ID/g in the BB2 receptor expressing mouse pancreas for the L-Phe-BTV radioconjugate compared to 10.30 ± 0.34 for the D-Phe-BTV. Surprisingly, the kidney clearance for both radioconjugates was statistically identical. Conclusion: Incorporation of the L-Phe instead of the D-Phe into the sixth position of the BTV had no statistically significant effect upon the renal clearance of the radioconjugate. However, the change in stereochemistry from the L to the D-form had significant effects upon the in vivo uptake and retention of the radioconjugate. Further investigations will be conducted to understand the mechanism responsible for the difference in uptake and retention of the two Bombesin radioconjugates

Using Indium-111 labeled radiopharmaceuticals to target the BB2 receptor on human prostate cancer cells [abstract]

Abstract only availableThe BB2 receptor, belonging to the Bombesin receptor family, has been shown to be highly over expressed in a variety of cancer cell lines, including human prostate cancer. Our laboratory have been involved, for over a decade, in synthesizing Bombesin analogues that target the BB2 receptor for the purpose of developing radiopharmaceuticals for diagnostic and/or therapeutic treatment of cancer. Radiopharmaceuticals based on Bombesin are typically composed of a chelator, isotope, linking group and targeting vector [See Bifunctional Conjugate Design [figure below]. Previous studies by our group and others have shown that variations in linking groups affect the retention time of the bifunctional conjugate in prostate cancer (PC-3) cells. Higher retention time allows for more efficacious therapeutic benefits and enhanced diagnostic imaging capabilities. In this study, we seek to determine the pharmacokinetic benefits achieved in altering the linking group using aliphatic and aromatic linking groups. In-vitro analysis of the radiopharmaceuticals studied found that the Bombesin derivative with the aliphatic linking group demonstrated a slightly higher affinity for the BB2 receptor compared to the Bombesin analogs containing aromatic linking groups. In vivo pharmacokinetic and imaging studies were performed using pre-clinical models of prostate cancer. The tumor uptake of the Bombesin derivatives with the aromatic linking groups were found to be significantly higher compared to that of the Bombesin derivative with the aliphatic linking group. In contrast, the aromatic Bombesin analogs also exhibited higher amounts of undesirable accumulation in the kidneys and other non-target tissues. In conclusion, we found that the aliphatic compounds were more appropriate for diagnostic imaging of prostate cancer due to the reduced non-target retention. The Bombesin analogs with aromatic linking groups showed potential for use as therapeutic agents for prostate cancer treatment.National Institutes of Health Molecular Imaging Progra

Overexpression of the RNA-binding protein HuR impairs tumor growth in triple negative breast cancer associated with deficient angiogenesis [abstract]

Breast cancer is the second most common cancer in women and causes the death of 519,000 people worldwide. Many cancer genes are posttranscriptionally regulated by RNA-binding proteins (RBPs) and microRNAs. The RBP HuR binds to the AU-rich (ARE) regions of labile mRNAs, such as proto-oncogenes, stabilizing their mRNA and facilitating their translation into protein. HuR has been described to control genes in multiple areas of the acquired capabilities model of cancer and has been hypothesized to be a tumor maintenance gene, allowing for cancers to proliferate once they are established. We investigated the role of HuR in aggressive and difficult to treat triple-negative breast cancer

Targeting the BB2 receptor on human prostate cancer cells using Indium-111 labeled radiopharmaceutical [abstract]

Abstract only availableFaculty Mentor: Dr. Timothy Hoffman, Internal MedicineThe BB2 receptor, belonging to the Bombesin receptor family, has been shown to be highly over expressed in a variety of cancer cell lines, including human prostate cancer. Over expression of the BB2 receptor offers an appealing target for the design of targeted radiopharmaceuticals.  The Hoffman laboratory and others have been involved, for over a decade, in synthesizing Bombesin analogues that target the BB2 receptor for the purpose of developing a viable radiopharmaceutical for diagnostic or therapeutic treatment of cancer. Radiopharmaceuticals based on Bombesin analogues are typically composed of a targeting vector, radioisotope, chelator and linking group [See Bifunctional Conjugate Design figure below]. Previous studies have shown that variations in linking groups may affect the retention time of the bifunctional conjugate in prostate cancer (PC-3) cells.  Higher retention time allows for more efficacious therapeutic benefits and enhanced diagnostic imaging capabilities.  In the work presented, we designed and synthesized a 111In-Bombesin analogue with a phenyl linker group in order to determine if the phenyl linker group would provide higher retention times in prostate cancer.  In-vitro analysis of the radiopharmaceutical was performed using PC-3 cells to determine the affinity of the new compound for the BB2 receptor to be 1.09 nM. In-vivo studies of the radiopharmaceutical were also conducted by injection of the radiopharmaceutical into CF-1 (“normal”) mice, as well as SCID (Severe Combined Immunodeficient) mice bearing 2-3 week old PC-3 tumors. Experimental results on SCID mice revealed uptakes of 6.36, 3.34, 2.42 and 1.69 % Injected Dose of radiopharmaceutical per gram of tumor tissue at 0.25, 1, 4 and 24 hours, respectively. Imaging using Micro-SPECT (Single-Photon Emission Computed Tomography) was performed to track the dispersion of the radiopharmaceutical throughout the mouse model and confirmed the targeted uptake of the radiopharmaceutical

In vitro/in vivo assessment of novel 99mTc-bombesin conjugates in human cancer tissue [abstract]

Abstract only availableReceptor-specific, radiolabeled peptides are becoming increasingly popular as targeting vectors for the development of new diagnostic radiopharmaceuticals. The over-expression of certain receptors such as the gastrin releasing peptide receptor (GRPr) on human cancer cells makes this method of drug development a viable tool for tumor targeting in vivo. Breast, pancreatic, prostate, gastric, colon, and small-cell lung cancer have demonstrated GRPr expression. In this project, we have conjugated a diaminoproionic acid (DPR) bifunctional chelator to bombesin (BBN) peptide targeting vector by solid phase peptide synthesis. BBN is an analogue of human gastrin releasing peptide (GRP) that binds to the GRPr with high affinity and specificity. Conjugates of the general structure [DPR-(X)-BBN(7-14)NH2] (X = a series of amino acid pharmacokinetic modifiers) were purified by reverse-phase high-performance liquid chromatography and characterized by electrospray-ionization mass spectrometry. Radiolabeling investigations of with fac-[99mTc(CO)3(H2O)3]+ (Isolink®) provided for metallated conjugates of the following general structure: [99mTc(CO)3-DPR-(X)-BBN(7-14)NH2]. These new conjugates demonstrated the ability to target specific human tumors in rodent models. Subsequent radiolabeling studies of [DPR-(X)-BBN(7-14)NH2] with fac-[188Re(CO)3(H2O)3]+, the therapeutic surrogate precursor of Tc-99m, have given us the potential to treat specific human tumors via these new targeting vectors. Detailed radiolabeling protocols, in vitro cell binding studies, and in vivo biodistribution assays will be reported.Harry S. Truman Memorial VA Hospita

Performancee evaluation of a dual Micro-SPECT detector system [abstract]

Abstract only availableFaculty Mentor: Dr. Timothy Hoffman, Internal MedicineThe ability to conduct radiopharmaceutical research in vivo is largely dependent on nuclear imaging hardware and is subject to its limitations. The inability of clinical instrumentation to conduct non-invasive tracer bio-kinetics has spurred the development of dedicated pre-clinical imaging systems such as Micro-SPECT. Pixelated NaI(Tl) detectors are a relatively new attempt to further increase Micro-SPECT viability in conducting longitudinal research. The goal of this experiment is to evaluate a dual pixelated NaI(Tl) gamma-ray Micro-SPECT system and its ability to conduct routine preclinical studies. The SPECT detectors each have an area of 150 mm x 150 mm composed of 4624 (2 mm x 2 mm x 10 mm) NaI(Tl) scintillators coupled to position sensitive photomultiplier tubes. Various tungsten pinhole collimators are used depending on the amount of radioactivity in the SPECT field of view. The Micro-SPECT images are reconstructed using an OSEM routine with sub-voxel capabilities. The sensitivities and efficiencies of the SPECT detectors were determined for Tc-99m and In-111. The practical and optimum SPECT system resolutions were determined using commercial phantoms and evaluated in a Tc-99m-MDP SPECT/CT scan. Longitudinal SPECT/CT studies were performed on tumor bearing models using a receptor targeted radiopharmaceutical at 1, 4, 24, 48 and 72 hours post injection. System sensitivities of 340 cps/MBq and efficiencies of 0.03% were achieved at 25 mm from the 2 mm pinhole aperture. The spatial resolution of the SPECT was determined to optimally be 1.6 mm and practically 2.4 mm using a hot-rod reconstructed Tc-99m phantom scanned for 16 hours and 30 minutes respectively. Bone and tumor SPECT studies revealed excellent target tissue/organ visualization. Longitudinal Micro-SPECT/CT studies were conducted successfully over a 72 hour period post injection. These findings suggest that pixelated NaI(Tl) detector technology is capable of repeated imaging in the same subject.The ability to conduct radiopharmaceutical research in vivo is largely dependent on nuclear imaging hardware and is subject to its limitations.  The inability of clinical instrumentation to conduct non-invasive tracer bio-kinetics has spurred the development of dedicated pre-clinical imaging systems such as Micro-SPECT.  Pixelated NaI(Tl) detectors are a relatively new attempt to further increase Micro-SPECT viability in conducting longitudinal research.  The goal of this experiment is to evaluate a dual pixelated NaI(Tl) gamma-ray Micro-SPECT system and its ability to conduct routine preclinical studies.  The SPECT detectors each have an area of 150 mm x 150 mm composed of 4624 (2 mm x 2 mm x 10 mm) NaI(Tl) scintillators coupled to position sensitive photomultiplier tubes.  Various tungsten pinhole collimators are used depending on the amount of radioactivity in the SPECT field of view.  The Micro-SPECT images are reconstructed using an OSEM routine with sub-voxel capabilities.  The sensitivities and efficiencies of the SPECT detectors were determined for Tc-99m and In-111.  The practical and optimum SPECT system resolutions were determined using commercial phantoms and evaluated in a Tc-99m-MDP SPECT/CT scan.  Longitudinal SPECT/CT studies were performed on tumor bearing models using a receptor targeted radiopharmaceutical at 1, 4, 24, 48 and 72 hours post injection.  System sensitivities of 340 cps/MBq and efficiencies of 0.03% were achieved at 25 mm from the 2 mm pinhole aperture.  The spatial resolution of the SPECT was determined to optimally be 1.6 mm and practically 2.4 mm using a hot-rod reconstructed Tc-99m phantom scanned for 16 hours and 30 minutes respectively.  Bone and tumor SPECT studies revealed excellent target tissue/organ visualization.  Longitudinal Micro-SPECT/CT studies were conducted successfully over a 72 hour period post injection.  These findings suggest that pixelated NaI(Tl) detector technology is capable of repeated imaging in the same subject

In Vitro and In Vivo Evaluation of Alexa Fluor 680-Bombesin[7–14]NH Peptide Conjugate, a High-Affinity Fluorescent Probe with High Selectivity for the Gastrin-Releasing Peptide Receptor

Gastrin-releasing peptide (GRP) receptors are overexpressed on several types of human cancer cells, including breast, prostate, small cell lung, and pancreatic cancers. Bombesin (BBN), a 14–amino acid peptide that is an analogue of human GRP, binds to GRP receptors with very high affinity and specificity. The aim of this study was to develop a new fluorescent probe based on BBN having high tumor uptake and optimal pharmacokinetics for specific targeting and optical imaging of human breast cancer tissue. In this study, solid-phase peptide synthesis was used to produce H 2 N-glycylglycylglycine-BBN[7–14]NH 2 peptide with the following general sequence: H 2 N-G-G-G-Q-W-A-V-G-H-L-M-(NH 2 ). This conjugate was purified by reversed-phase high-performance liquid chromatography and characterized by electrospray-ionization mass spectra. The fluorescent probe Alexa Fluor 680-G-G-G-BBN[7–14]NH 2 conjugate was prepared by reaction of Alexa Fluor 680 succinimidyl ester to H 2 N-G-G-G-BBN[7–14]NH 2 in dimethylformamide (DMF). In vitro competitive binding assays, using 125 I-Tyr 4 -BBN as the radiolabeling gold standard, demonstrated an inhibitory concentration 50% value of 7.7 ± 1.4 nM in human T-47D breast cancer cells. Confocal fluorescence microscopy images of Alexa Fluor 680-G-G-G-BBN[7–14]NH 2 in human T-47D breast cancer cells indicated specific uptake, internalization, and receptor blocking of the fluorescent bioprobe in vitro. In vivo investigations in SCID mice bearing xenografted T-47D breast cancer lesions demonstrated the ability of this new conjugate to specifically target tumor tissue with high selectivity and affinity

99mTc-DPR-SSS-BBN for diagnosis of human cancers [abstract]

Abstract only availableReceptor-specific, radiolabeled peptides have become increasingly popular as targeting vectors for the development of new diagnostic radiopharmaceuticals. The over-expression of certain receptors such as the gastrin releasing peptide receptor (GRPr) on human cancer cells makes this method of drug development a viable tool for tumor targeting in vivo. Breast, pancreatic, prostate, gastric, colon, and small-cell lung cancer have demonstrated GRPr expression. In this project, we have conjugated a diaminoproionic acid (DPR) bifunctional chelator to bombesin (BBN) peptide targeting vector by solid phase peptide synthesis. BBN is an analogue of human gastrin releasing peptide (GRP) that binds to the GRPr with high affinity and specificity. A conjugate, [DPR-SSS-BBN(7-14)NH] was purified by reverse-phase high-performance liquid chromatography and characterized by electrospray-ionization mass spectrometry. Radiolabeling investigations of with fac-[99mTc(CO)3(H2O)3]+ (Isolink®) provided for the metallated conjugate [99mTc(CO)3-DPR-SSS-BBN(7-14)NH2]. This new conjugate demonstrated the ability to target specific human tumors in rodent models. In vitro cell binding studies, and in vivo biodistribution assays will be reported.Life Sciences Undergraduate Research Opportunity Progra