Theragnostic Radionuclide Pairs for Prostate Cancer Management: 64Cu/67Cu, Can Be a Budding Hot Duo.

Prostate cancer (PCa) is one of the preeminent causes of mortality in men worldwide. Theragnostic, a combination of therapy and diagnostic, using radionuclide pairs to diagnose and treat disease, has been shown to be a promising approach for combating PCa. In PCa patients, bone is one of the most common sites of metastases, and about 90% of patients develop bone metastases. This review focuses on (i) clinically translated theragnostic radionuclide pairs for the management of PCa, (ii) radionuclide therapy of bone metastases in PCa, and (iii) a special emphasis on emerging theragnostic radionuclide pair, Copper-64/Copper-67 (64Cu/67Cu) for managing the disease.

Imaging urothelial bladder cancer: A VPAC PET targeted approach.

INTRODUCTION Accurate staging of urothelial bladder cancer (UBC) with imaging, which guides effective bladder cancer treatment, remains challenging. This investigation is to validate a hypothesis that targeting Vasoactive intestinal and pituitary adenylate cyclase activating peptide (VPAC) receptors using 64Cu-TP3805 can PET image UBC efficiently. MATERIALS AND METHODS: Nineteen patients (44-84 years of age) scheduled for radical cystectomy, underwent VPAC positron emission tomography (PET) imaging prior to surgery. Sixteen had completed neoadjuvant chemotherapy prior to imaging. All 19 received 64Cu-TP3805 (148 % ± 10% MBq) intravenously, and were imaged 60 to 90 minutes later. Standard uptake value (SUV)max for malignant lesions and SUVmean for normal tissues were determined and mean +/-SEM recorded. Following radical cystoprostatectomy, pelvic lymphadenectomy and urinary diversion imaging, results were compared with final surgical pathology. RESULTS: 64Cu-TP3805 had no adverse events, negligible urinary excretion and rapid blood clearance. UBC PET images for residual disease were true positive in 11 patients and true negative in four. Of remaining 4, one had false positive and 3 had false negative scans, equating to 79% sensitivity (95%, CI 49%-95%), 80% specificity (95%, CI 28%-100%), 92% positive predictive value (95%, CI 62%-100%) and 57% negative predictive value (95%, CI 18%-90%). CONCLUSIONS: These first in man results, in a group, heavily pretreated with neoadjuvant chemotherapy, indicate that VPAC PET imaging can identify UBC effeiciently and suggest, that VPAC PET can diagnose UBC in a treatment naïve cohort for accurate staging, guide biopsy and treatment in patients with suspected metastasis and determine response to therapy. Further investigation of this molecular imaging approach is warranted.

Targeting VPAC1 Receptors for Imaging Glioblastoma.

PURPOSE: Scintigraphic imaging of malignant glioblastoma (MG) continues to be challenging. We hypothesized that VPAC1 cell surface receptors can be targeted for positron emission tomography (PET) imaging of orthotopically implanted MG in a mouse model, using a VPAC1-specific peptide [64Cu]TP3805. PROCEDURES: The expression of VPAC1 in mouse GL261 and human U87 glioma cell lines was determined by western blot. The ability of [64Cu]TP3805 to bind to GL261 and U87 cells was studied by cell-binding. Receptor-blocking studies were performed to validate receptor specificity. GL261 tumors were implanted orthotopically in syngeneic T-bet knockout C57BL/6 mouse brain (N = 15) and allowed to grow for 2-3 weeks. Mice were injected i.v., first with ~ 150 µCi of 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) then 24 h later with ~ 200 µCi of [64Cu]TP3805. In another set of tumor-bearing mice, (N = 5), ionic [64Cu]Cl2 was injected as a control. Mice were imaged at a 2-h post-injection using an Inveon micro-PET/CT, sacrificed and % ID/g of [64Cu]TP3805 and [64Cu]Cl2 were calculated in a tumor, normal brain, and other tissues. For histologic tissue examination, 3-µm thick sections of the tumors and normal brain were prepared, digital autoradiography (DAR) was performed, and then the sections were H&E stained for histologic examination. RESULTS: Western blots showed a strong signal for VPAC1 on both cell lines. [64Cu]TP3805 cell-binding was 87 ± 1.5 %. Receptor-blocking reduced cell-binding to 24.3 ± 1.5 % (P < 0.01). PET imaging revealed remarkable accumulation of [64Cu]TP3805 in GL261 MG with a negligible background in the normal brain, as compared to [18F]FDG. Micro-PET/CT image analyses and tissue distribution showed that the brain tumor uptake for [64Cu]TP3805 was 8.2 ± 1.7 % ID/g and for [64Cu]Cl2 2.1 ± 0.5 % ID/g as compared to 1.0 ± 0.3 % ID/g and 1.4 ± 0.3 % ID/g for normal mouse brains, respectively. The high tumor/normal brain ratio for [64Cu]TP3805 (8.1 ± 1.1) allowed tumors to be visualized unequivocally. Histology and [64Cu]TP3805 DAR differentiated malignant tumors from healthy brain and confirmed PET findings. CONCLUSION: Targeting VPAC1 receptors using [64Cu]TP3805 for PET imaging of MG is a promising novel approach and calls for further investigation.

Evaluating Ga-68 Peptide Conjugates for Targeting VPAC Receptors: Stability and Pharmacokinetics.

PURPOSE: In recent years, considerable progress has been made in the use of gallium-68 labeled receptor-specific peptides for imaging oncologic diseases. The objective was to examine the stability and pharmacokinetics of [68Ga]NODAGA and DOTA-peptide conjugate targeting VPAC [combined for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP)] receptors on tumor cells. PROCEDURES: A VPAC receptor-specific peptide was chosen as a model peptide and conjugated to NODAGA and DOTA via solid-phase synthesis. The conjugates were characterized by HPLC and MALDI-TOF. Following Ga-68 chelation, the radiochemical purity of Ga-68 labeled peptide conjugate was determined by radio-HPLC. The stability was tested against transmetallation using 100 nM Fe3+/Zn2+/Ca2+ ionic solution and against transchelation using 200 µM DTPA solution. The ex vivo and in vivo stability of the Ga-68 labeled peptide conjugate was tested in mouse plasma and urine. Receptor specificity was determined ex vivo by cell binding assays using human breast cancer BT474 cells. Positron emission tomography (PET)/X-ray computed tomography (CT) imaging, tissue distribution, and blocking studies were performed in mice bearing BT474 xenografts. RESULTS: The chemical and radiochemical purity was greater than 95 % and both conjugates were stable against transchelation and transmetallation. Ex vivo stability at 60 min showed that the NODAGA-peptide-bound Ga-68 reduced to 42.1 ± 3.7 % (in plasma) and 37.4 ± 2.9 % (in urine), whereas the DOTA-peptide-bound Ga-68 was reduced to 1.2 ± 0.3 % (in plasma) and 4.2 ± 0.4 % (in urine) at 60 min. Similarly, the in vivo stability for [68Ga]NODAGA-peptide was decreased to 2.1 ± 0.2 % (in plasma) and 2.2 ± 0.4 % (in urine). For [68Ga]DOTA-peptide, it was decreased to 1.4 ± 0.3 % (in plasma) and 1.2 ± 0.4 % (in urine) at 60 min. The specific BT474 cell binding was 53.9 ± 0.8 % for [68Ga]NODAGA-peptide, 25.8 ± 1.4 % for [68Ga]-DOTA-peptide, and 18.8 ± 2.5 % for [68Ga]GaCl3 at 60 min. Inveon microPET/CT imaging at 1 h post-injection showed significantly (p < 0.05) higher tumor to muscle (T/M) ratio for [68Ga]NODAGA-peptide (3.4 ± 0.3) as compared to [68Ga]DOTA-peptide (1.8 ± 0.6). For [68Ga]GaCl3 and blocked mice, their ratios were 1.5 ± 0.6 and 1.5 ± 0.3 respectively. The tissue distributions data were similar to the PET imaging data. CONCLUSION: NODAGA is superior to DOTA in terms of radiolabeling kinetics. The method of radiolabeling was reproducible and yielded higher specific activity. Although both agents have relatively low in vivo stability, PET/CT imaging studies delineated BC tumors with [68Ga]NODAGA-peptide, but not with [68Ga]DOTA-peptide.

A glance at imaging bladder cancer.

PURPOSE: Early and accurate diagnosis of Bladder cancer (BCa) will contribute extensively to the management of the disease. The purpose of this review was to briefly describe the conventional imaging methods and other novel imaging modalities used for early detection of BCa and outline their pros and cons. METHODS: Literature search was performed on Pubmed, PMC, and Google scholar for the period of January 2014 to February 2018 and using such words as "bladder cancer, bladder tumor, bladder cancer detection, diagnosis and imaging". RESULTS: A total of 81 published papers were retrieved and are included in the review. For patients with hematuria and suspected of BCa, cystoscopy and CT are most commonly recommended. Ultrasonography, MRI, PET/CT using 18F-FDG or 11C-choline and recently PET/MRI using 18F-FDG also play a prominent role in detection of BCa. CONCLUSION: For initial diagnosis of BCa, cystoscopy is generally performed. However, cystoscopy can not accurately detect carcinoma insitu (CIS) and can not distinguish benign masses from malignant lesions. CT is used in two modes, CT and computed tomographic urography (CTU), both for dignosis and staging of BCa. However, they cannot differentiate T1 and T2 BCa. MRI is performed to diagnose invasive BCa and can differentiate muscle invasive bladder carcinoma (MIBC) from non-muscle invasive bladder carcinoma (NMIBC). However, CT and MRI have low sensitivity for nodal staging. For nodal staging PET/CT is preferred. PET/MRI provides better differentiation of normal and pathologic structures as compared with PET/CT. Nonetheless none of the approaches can address all issues related for the management of BCa. Novel imaging methods that target specific biomarkers, image BCa early and accurately, and stage the disease are warranted.

VPAC1-targeted PET/CT scan: improved molecular imaging for the diagnosis of prostate cancer using a novel cell surface antigen.

PURPOSE: Current approaches to prostate cancer screening and diagnosis are plagued with limitations in diagnostic accuracy. There is a compelling need for biomolecular imaging that will not only detect prostate cancer early but also distinguish prostate cancer from benign lesions accurately. In this topic paper, we review evidence that supports further investigation of VPAC1-targeted PET/CT imaging in the primary diagnosis of prostate cancer. METHODS: A non-systematic review of Medline/PubMed was performed. English language guidelines on prostate cancer diagnosis and management, original articles, and review articles were selected based on their clinical relevance. RESULTS: VPAC1 receptors were overexpressed 1000 times more in prostate cancer than benign prostatic stromal tissue. In vitro and in vivo studies showed that Copper-64 labeled analogs of VPAC1 ligands can be synthesized with high radiochemical efficiency and purity. The radioactive probes had excellent VPAC1 receptor binding specificity and affinity. They had good biochemical stability in vitro and in mouse and human serum. They had minimal urinary excretion, which made them favorable for prostate cancer imaging. Initial feasibility study in men with prostate cancer showed that the probes were safe with no reported adverse reaction. 64Cu-TP3805 PET/CT detected 98% of prostate cancer lesions and nodal metastasis as confirmed with whole mount histopathological evaluation. CONCLUSIONS: VPAC1 receptors are promising targets for biomolecular imaging of primary prostate cancer that can distinguish malignant from benign lesions non-invasively. Further investigations are warranted to validate initial findings and define the clinical utilities of VPAC1-targeted PET imaging for prostate cancer diagnosis and management.

VPAC1 Targeted 64Cu-TP3805 kit preparation and its evaluation.

INTRODUCTION: Previously, our laboratory has shown that 64Cu-TP3805 can specifically target VPAC1 receptors and be used for positron emission tomography (PET) imaging of breast (BC) and prostate cancer (PC) in humans. Present work is aimed at the formulation of a freeze-dried diaminedithiol-peptide (N2S2-TP3805) kit and it's evaluation for the preparation of 64Cu labeled TP3805. Parameters such as pH, temperature and incubation time were examined that influenced the radiolabeling efficiency and stability of the product. METHODS: Kits were prepared under different conditions and radiolabeling efficiency of TP3805 kit was evaluated for a range of pH3.5-8.5, after addition of 64Cu in 30µl, 0.1M HCl. Incubation temperature (37-90°C) and time (30-120min.) were also investigated. Kits were stored at -10°C and their long term stability was determined as a function of their radiolabeling efficiency. Further, stability of 64Cu-TP3805 complex was evaluated in presence of fetal bovine serum and bovine serum albumin by using SDS polyacrylamide gel electrophoresis. Kits were then used for PET imaging of BC and PC following eIND (101550) and institutional approvals. Specificity of 64Cu-TP3805 for VPAC1 was examined with digital autoradiography (DAR) of prostate tissues obtained after prostatectomy, benign prostatic hyperplasia (BPH) tissue, and benign and malignant lymph nodes. Results were compared with corresponding tissue histology. RESULTS: Radiolabeling efficiency was ≥95% at final pH ~7.2 when incubated at 50°C for 90min. Kits were stable up to 18months when stored at -10°C, and 64Cu-TP3805 complex exhibited excellent stability for up to 4h at room temperature. 64Cu-TP3805 complex did not show any transchelation even after 2h incubation at 37°C in 10% FBS as well as in BSA as determined by SDS PAGE analysis. DAR identified ≥95% of malignant lesions 11 new PC lesions, 20 high grade prostatic intraepithelial neoplasia, 2/2 ejaculatory ducts and 5/5 urethra verumontanum not previously identified The malignant lymph nodes were correctly identified by DAR and for 3/3 BPH patients, and 5/5 cysts, DAR was negative. In human BC (n=19) and PC (n=26) were imaged with 100% sensitivity. CONCLUSION: Availability of ready to use N2S2-peptide kits for 64Cu labeling is convenient and eliminates possible day to day variation during its routine preparation for clinical use.

Development of a voided urine assay for detecting prostate cancer non-invasively: a pilot study.

OBJECTIVE: To validate a hypothesis that prostate cancer can be detected non-invasively by a simple and reliable assay by targeting genomic VPAC receptors expressed on malignant prostate cancer cells shed in voided urine. PATIENTS/SUBJECTS AND METHODS: VPAC receptors were targeted with a specific biomolecule, TP4303, developed in our laboratory. With an Institutional Review Board exempt approval of use of de-identified discarded samples, an aliquot of urine collected as a standard of care, from patients presenting to the urology clinic (207 patients, 176 men and 31 women, aged ≥21 years) was cytospun. The cells were fixed and treated with TP4303 and 4,6-diamidino-2-phenylindole (DAPI). The cells were then observed under a microscope and cells with TP4303 orange fluorescence around the blue (DAPI) nucleus were considered 'malignant' and those only with a blue nucleus were regarded as 'normal'. VPAC presence was validated using receptor blocking assay and cell malignancy was confirmed by prostate cancer gene profile examination. RESULTS: The urine specimens were labelled only with gender and presenting diagnosis, with no personal health identifiers or other clinical data. The assay detected VPAC positive cells in 98.6% of the men with a prostate cancer diagnosis (141), and none of the 10 men with benign prostatic hyperplasia. Of the 56 'normal' patients, 62.5% (35 patients, 10 men and 25 women) were negative for VPAC cells; 19.6% (11, 11 men and no women) had VPAC positive cells; and 17.8% (10, four men and six women) were uninterpretable due to excessive crystals in the urine. Although data are limited, the sensitivity of the assay was 99.3% with a confidence interval (CI) of 96.1-100% and the specificity was 100% with a CI of 69.2-100%. Receptor blocking assay and fluorescence-activated cell sorting (FACS) analyses demonstrated the presence of VPAC receptors and gene profiling examinations confirmed that the cells expressing VPAC receptors were malignant prostate cancer cells. CONCLUSION: These preliminary data are highly encouraging and warrant further evaluation of the assay to serve as a simple and reliable tool to detect prostate cancer non-invasively.

Evaluation of a PACAP Peptide Analogue Labeled with (68)Ga Using Two Different Chelating Agents.

OBJECTIVE: The authors have conjugated chelating agents (DOTA and NODAGA) with a peptide (pituitary adenylate cyclase-activating peptide [PACAP] analogue) that has a high affinity for VPAC1 receptors expressed on cancer cells. To determine a suitable chelating agent for labeling with (68)Ga, they have compared the labeling kinetics and stability of these peptide conjugates. METHODS: For labeling, (68)GaCl3 was eluted in 0.1 M HCl from a [(68)Ge-(68)Ga] generator. The influences of peptide concentration, pH, and temperature on the radiolabeling efficiency were studied. The stability was evaluated in saline, human serum, DTPA, transferrin, and metallic ions (FeCl3, CaCl2, and ZnCl2). Cell binding assay was performed using human breast cancer cells (T47D). Tissue biodistribution was studied in normal athymic nude mice. RESULTS: Optimal radiolabeling (>95.0%) of the DOTA-peptide conjugates required a higher (50°C-90°C) temperature and 10 minutes of incubation at pH 2-5. The NODAGA-peptide conjugate needed incubation only at 25°C for 10 minutes. Both radiocomplexes were stable in saline, serum, as well as against transchelation and transmetallation. Cell binding at 37°C for 15 minutes of incubation with (68)Ga-NODAGA-peptide was 34.0% compared to 24.5% for (68)Ga-DOTA-peptide. Tissue biodistribution at 1 hour postinjection of both (68)Ga-labeled peptide conjugates showed clearance through the kidneys. CONCLUSIONS: NODAGA-peptide showed more convenient radiolabeling features than that of DOTA-peptide.

CCR5 receptor antagonists block metastasis to bone of v-Src oncogene-transformed metastatic prostate cancer cell lines.

Src family kinases (SFK) integrate signal transduction for multiple receptors, regulating cellular proliferation, invasion, and metastasis in human cancer. Although Src is rarely mutated in human prostate cancer, SFK activity is increased in the majority of human prostate cancers. To determine the molecular mechanisms governing prostate cancer bone metastasis, FVB murine prostate epithelium was transduced with oncogenic v-Src. The prostate cancer cell lines metastasized in FVB mice to brain and bone. Gene expression profiling of the tumors identified activation of a CCR5 signaling module when the prostate epithelial cell lines were grown in vivo versus tissue cultures. The whole body, bone, and brain metastatic prostate cancer burden was reduced by oral CCR5 antagonist. Clinical trials of CCR5 inhibitors may warrant consideration in patients with CCR5 activation in their tumors.

Fluorescence detection of KRAS2 mRNA hybridization in lung cancer cells with PNA-peptides containing an internal thiazole orange.

We previously developed reporter-peptide nucleic acid (PNA)-peptides for sequence-specific radioimaging and fluorescence imaging of particular mRNAs in cells and tumors. However, a direct test for PNA-peptide hybridization with RNA in the cytoplasm would be desirable. Thiazole orange (TO) dye at the 5' end of a hybridization agent shows a strong increase in fluorescence quantum yield when stacked upon a 5' terminal base pair, in solution and in cells. We hypothesized that hybridization agents with an internal TO could distinguish a single base mutation in RNA. Thus, we designed KRAS2 PNA-IGF1 tetrapeptide agents with an internal TO adjacent to the middle base of the 12th codon, a frequent site of cancer-initiating mutations. Our molecular dynamics calculations predicted a disordered bulge with weaker hybridization resulting from a single RNA mismatch. We observed that single-stranded PNA-IGF1 tetrapeptide agents with an internal TO showed low fluorescence, but fluorescence escalated 5-6-fold upon hybridization with KRAS2 RNA. Circular dichroism melting curves showed ∼10 °C higher Tm for fully complementary vs single base mismatch TO-PNA-peptide agent duplexes with KRAS2 RNA. Fluorescence measurements of treated human lung cancer cells similarly showed elevated cytoplasmic fluorescence intensity with fully complementary vs single base mismatch agents. Sequence-specific elevation of internal TO fluorescence is consistent with our hypothesis of detecting cytoplasmic PNA-peptide:RNA hybridization if a mutant agent encounters the corresponding mutant mRNA.

Determining efficacy of breast cancer therapy by PET imaging of HER2 mRNA.

INTRODUCTION: Monitoring the effectiveness of therapy early and accurately continues to be challenging. We hypothesize that determination of Human Epidermal Growth Factor Receptor 2 (HER2) mRNA in malignant breast cancer (BC) cells by positron emission tomography (PET) imaging, before and after treatment, would reflect therapeutic efficacy. METHOD: WT4340, a peptide nucleic acid (PNA) 12-mer complementary to HER2 mRNA was synthesized together with -CSKC, a cyclic peptide, which facilitated internalization of the PNA via IGFR expressed on BC cells, and DOTA that chelated Cu-64. Mice (n = 8) with BT474 ER+/HER2+ human BC received doxorubicin (DOX, 1.5mg/kg) i.p. once a week for six weeks. Mice (n = 8) without DOX served as controls. All mice were PET imaged with F-18-FDG and 48 h later with Cu-64-WT4340. PET imaging were performed before and 72 h after each treatment. Standardized uptake values (SUVs) were determined and percent change calculated. Animal body weight (BW) and tumor volume (TV) were measured. RESULTS: SUVs for Cu-64-WT4340 after DOX treatment declined by 54% ± 17% after the second dose, 41% ± 15% after the fourth dose, and 29% ± 7% after the sixth dose, compared with 42% ± 22%, 31% ± 18%, and 13% ± 9% (p<0.05) for F-18-FDG. In untreated mice, the corresponding percent SUVs for Cu-64-WT4340 were 145% ± 82%, 165% ± 39%, and 212% ± 105% of pretreatment SUV, compared with 108% ± 28%, 151% ± 8%, and 152% ± 35.5%, (p<0.08) for F-18-FDG. TV in mice after second dose was 114.15% ± 61.83%, compared with 144.7% ± 64.4% for control mice. BW of DOX-treated mice was 103.4% ± 7.6% of pretreatment, vs. 100.1% ± 4.3% for control mice. CONCLUSION: Therapeutic efficacy was apparent sooner by molecular PET imaging than by determination of reduction in TV.

Consistent surgeon evaluations of three-dimensional rendering of PET/CT scans of the abdomen of a patient with a ductal pancreatic mass.

Two-dimensional (2D) positron emission tomography (PET) and computed tomography (CT) are used for diagnosis and evaluation of cancer patients, requiring surgeons to look through multiple planar images to comprehend the tumor and surrounding tissues. We hypothesized that experienced surgeons would consistently evaluate three-dimensional (3D) presentation of CT images overlaid with PET images when preparing for a procedure. We recruited six Jefferson surgeons to evaluate the accuracy, usefulness, and applicability of 3D renderings of the organs surrounding a malignant pancreas prior to surgery. PET/CT and contrast-enhanced CT abdominal scans of a patient with a ductal pancreatic mass were segmented into 3D surface renderings, followed by co-registration. Version A used only the PET/CT image, while version B used the contrast-enhanced CT scans co-registered with the PET images. The six surgeons answered 15 questions covering a) the ease of use and accuracy of models, b) how these models, with/without PET, changed their understanding of the tumor, and c) what are the best applications of the 3D visualization, on a scale of 1 to 5. The six evaluations revealed a statistically significant improvement from version A (score 3.6±0.5) to version B (score 4.4±0.4). A paired-samples t-test yielded t(14) = -8.964, p<0.001. Across the surgeon cohort, contrast-enhanced CT fused with PET provided a more lifelike presentation than standard CT, increasing the usefulness of the presentation. The experienced surgeons consistently reported positive reactions to 3D surface renderings of fused PET and contrast-enhanced CT scans of a pancreatic cancer and surrounding organs. Thus, the 3D presentation could be a useful preparative tool for surgeons prior to making the first incision. This result supports proceeding to a larger surgeon cohort, viewing prospective 3D images from multiple types of cancer.

Effects of hypoxanthine substitution in peptide nucleic acids targeting KRAS2 oncogenic mRNA molecules: theory and experiment.

Genetic disorders can arise from single base substitutions in a single gene. A single base substitution for wild type guanine in the twelfth codon of KRAS2 mRNA occurs frequently to initiate lung, pancreatic, and colon cancer. We have observed single base mismatch specificity in radioimaging of mutant KRAS2 mRNA in tumors in mice by in vivo hybridization with radiolabeled peptide nucleic acid (PNA) dodecamers. We hypothesized that multimutant specificity could be achieved with a PNA dodecamer incorporating hypoxanthine, which can form Watson-Crick base pairs with adenine, cytosine, thymine, and uracil. Using molecular dynamics simulations and free energy calculations, we show that hypoxanthine substitutions in PNAs are tolerated in KRAS2 RNA:PNA duplexes where wild type guanine is replaced by mutant uracil or adenine in RNA. To validate our predictions, we synthesized PNA dodecamers with hypoxanthine, and then measured the thermal stability of RNA:PNA duplexes. Circular dichroism thermal melting results showed that hypoxanthine-containing PNAs are more stable in duplexes where hypoxanthine-adenine and hypoxanthine-uracil base pairs are formed than single mismatch duplexes or duplexes containing hypoxanthine-guanine opposition.

VPAC1 receptors for imaging breast cancer: a feasibility study.

UNLABELLED: VPAC1 encodes G-protein-coupled receptors expressed on all breast cancer (BC) cells at the onset of the disease, but not on benign lesions. Our extensive preclinical studies have shown that (64)Cu-TP3805 has a high affinity for VPAC1, is stable in vivo, and has the ability to distinguish spontaneously grown malignant BC masses from benign lesions. Our long-term goal is to develop (64)Cu-TP3805 as an agent to perform in vivo histology, to distinguish malignant lesions from benign masses noninvasively and thereby avoid patient morbidity and the excess economic costs of benign biopsies. METHODS: (18)F-FDG obtained commercially served as a control. (64)Cu-TP3805 was prepared using a sterile kit containing 20 µg of TP3805. Radiochemical purity and sterility were examined. Nineteen consenting women with histologically proven BC were given 370 MBq of (18)F-FDG. One hour later, 6 of these patients were imaged with PET/CT and 13 with positron emission mammography (PEM). Two to 7 d later, 6 PET/CT patients received 111 MBq (± 10%) (n = 2), 127 MBq (± 10%) (n = 2), or 148 MBq (± 10%) (n = 2) of (64)Cu-TP3805 and were imaged 2 and 4 h later. Thirteen PEM patients received 148 MBq (± 10%) of (64)Cu-TP3805 and were imaged 15 min, 1 h, 2 h, and 4 h later. Standardized uptake value (SUV) was calculated for PET/CT patients, and PUV/BGV (PEM uptake value/background value) was calculated for PEM patients. Tumor volume was also calculated. RESULTS: The radiochemical purity of (64)Cu-TP3805 was 97% ± 2%, and specific activity was 44.4 GBq (1.2 Ci)/µmol. In 19 patients, a total of 24 lesions were imaged (15 invasive ductal carcinoma, 1 high-grade mammary carcinoma, 3 lobular carcinoma, 1 invasive papilloma, and 4 sentinel lymph nodes). All lesions were unequivocally detected by (64)Cu-TP3805 and by (18)F-FDG. The average tumor volume as determined by PET/CT with (64)Cu-TP3805 was 90.6% ± 16.1% of that with (18)F-FDG PET/CT, and the average SUV was 92% ± 26.4% of that with (18)F-FDG. For PEM, the tumor volume with (64)Cu-TP3805 was 113% ± 37% of that with (18)F-FDG and the PUV/BGV ratio was 97.7% ± 24.5% of that with (18)F-FDG. CONCLUSION: (64)Cu-TP3805 is worthy of further investigation in patients requiring biopsy of suggestive imaging findings, to further evaluate its ability to distinguish malignant lesions from benign masses noninvasively.

Molecular determinants of epidermal growth factor binding: a molecular dynamics study.

The epidermal growth factor receptor (EGFR) is a member of the receptor tyrosine kinase family that plays a role in multiple cellular processes. Activation of EGFR requires binding of a ligand on the extracellular domain to promote conformational changes leading to dimerization and transphosphorylation of intracellular kinase domains. Seven ligands are known to bind EGFR with affinities ranging from sub-nanomolar to near micromolar dissociation constants. In the case of EGFR, distinct conformational states assumed upon binding a ligand is thought to be a determining factor in activation of a downstream signaling network. Previous biochemical studies suggest the existence of both low affinity and high affinity EGFR ligands. While these studies have identified functional effects of ligand binding, high-resolution structural data are lacking. To gain a better understanding of the molecular basis of EGFR binding affinities, we docked each EGFR ligand to the putative active state extracellular domain dimer and 25.0 ns molecular dynamics simulations were performed. MM-PBSA/GBSA are efficient computational approaches to approximate free energies of protein-protein interactions and decompose the free energy at the amino acid level. We applied these methods to the last 6.0 ns of each ligand-receptor simulation. MM-PBSA calculations were able to successfully rank all seven of the EGFR ligands based on the two affinity classes: EGF>HB-EGF>TGF-α>BTC>EPR>EPG>AR. Results from energy decomposition identified several interactions that are common among binding ligands. These findings reveal that while several residues are conserved among the EGFR ligand family, no single set of residues determines the affinity class. Instead we found heterogeneous sets of interactions that were driven primarily by electrostatic and Van der Waals forces. These results not only illustrate the complexity of EGFR dynamics but also pave the way for structure-based design of therapeutics targeting EGF ligands or the receptor itself.

Fluorescent peptide-PNA chimeras for imaging monoamine oxidase A mRNA in neuronal cells.

Monoamine oxidases (MAO) catalyze the oxidative deamination of many biogenic amines and are integral proteins found in the mitochondrial outer membrane. Changes in MAO-A levels are associated with depression, trait aggression, and addiction. Here we report the synthesis, characterization, and in vitro evaluation of novel fluorescent peptide-peptide nucleic acid (PNA) chimeras for MAOA mRNA imaging in live neuronal cells. The probes were designed to include MAOA-specific PNA dodecamers, separated by an N-terminal spacer to a µ-opioid receptor targeting peptide (DAMGO), with a spacer and a fluorophore on the C-terminus. The probe was successfully delivered into human SH-SY5Y neuroblastoma cells through µ-opioid receptor-mediated endocytosis. The K(d) by flow cytometry was 11.6 ± 0.8 nM. Uptake studies by fluorescence microscopy showed ∼5-fold higher signal in human SH-SY5Y neuroblastoma cells than in negative control CHO-K1 cells that lack µ-opioid receptors. Moreover, a peptide-mismatch control sequence showed no significant uptake in SH-SY5Y cells. Such mRNA imaging agents with near-infrared fluorophores might enable real time imaging and quantitation of neuronal mRNAs in live animal models.

Three dimensional projection environment for molecular design and surgical simulation.

We are developing agents for positron emission tomography (PET) imaging of cancer gene mRNA expression and software to fuse mRNA PET images with anatomical computerized tomography (CT) images to enable volumetric (3D) haptic (touch-and-feel) simulation of pancreatic cancer and surrounding organs prior to surgery in a particular patient. We have identified a novel ligand specific for epidermal growth factor receptor (EGFR) to direct PET agent uptake specifically into cancer cells, and created a volumetric haptic surgical simulation of human pancreatic cancer reconstructed from patient CT data. Young's modulus and the Poisson ratio for each tissue will be adjusted to fit the experience of participating surgeons.

Physiologically based pharmacokinetics of molecular imaging nanoparticles for mRNA detection determined in tumor-bearing mice.

Disease detection and management might benefit from external imaging of disease gene mRNAs. Previously we designed molecular imaging nanoparticles (MINs) based on peptide nucleic acids complementary to cancer gene mRNAs. The MINs included contrast agents and analogs of insulin-like growth factor 1 (IGF-1). Analysis of MIN tumor uptake data showed stronger binding in tumors than in surrounding tissues. We hypothesized that MINs with an IGF-1 analog stay in circulation by binding to IGF-binding proteins. To test that hypothesis, we fit the tissue distribution results of several MINs in xenograft-bearing mice to a physiological pharmacokinetics model. Fitting experimental tissue distribution data to model-predicted mass transfer of MINs from blood into organs and tumors converged only when the parameter for MINs bound to circulating IGF-binding proteins was set to 10%-20% of the injected MIN dose. This result suggests that previous mouse imaging trials used more MINs than necessary. This prediction can be tested by a ramp of decreasing doses.

Imaging human pancreatic cancer xenografts by targeting mutant KRAS2 mRNA with [(111)In]DOTA(n)-poly(diamidopropanoyl)(m)-KRAS2 PNA-D(Cys-Ser-Lys-Cys) nanoparticles.

95% of patients with ductal pancreatic cancer carry 12th codon activating mutations in their KRAS2 oncogenes. Early whole body imaging of mutant KRAS2 mRNA activation in pancreatic cancer would contribute to disease management. Scintigraphic hybridization probes to visualize gene activity in vivo constitute a new paradigm in molecular imaging. We have previously imaged mutant KRAS2 mRNA activation in pancreatic cancer xenografts by positron emission tomography (PET) based on a single radiometal, (64)Cu, chelated to a 1,4,7,10-tetra(carboxymethylaza)cyclododecane (DOTA) chelator, connected via a flexible, hydrophilic spacer, aminoethoxyethoxyacetate (AEEA), to the N-terminus of a mutant KRAS2 peptide nucleic acid (PNA) hybridization probe. A peptide analogue of insulin-like growth factor 1 (IGF1), connected to a C-terminal AEEA, enabled receptor-mediated endocytosis. We hypothesized that a polydiamidopropanoyl (PDAP) dendrimer (generation m), with increasing numbers (n) of DOTA chelators, extended via an N-terminal AEEA from a mutant KRAS2 PNA with a C-terminal AEEA and IGF1 analogue could enable more intense external imaging of pancreatic cancer xenografts that overexpress IGF1 receptor and mutant KRAS2 mRNA. ([(111)In]DOTA-AEEA)(n)-PDAP(m)-AEEA(2)-KRAS2 PNA-AEEA-IGF1 analogues were prepared and administered intravenously into immunocompromised mice bearing human AsPC1 (G12D) pancreatic cancer xenografts. CAPAN2 (G12 V) pancreatic cancer xenografts served as a cellular KRAS2 mismatch control. Scintigraphic tumor/muscle image intensity ratios for complementary [(111)In](n)-PDAP(m)-KRAS2 G12D probes increased from 3.1 +/- 0.2 at n = 2, m = 1, to 4.1 +/- 0.3 at n = 8, m = 3, to 6.2 +/- 0.4 at n = 16, m = 4, in AsPC1 (G12D) xenografts. Single mismatch [(111)In](n)-PDAP(m)-KRAS2 G12 V control probes showed lower tumor/muscle ratios (3.0 +/- 0.6 at n = 2, m = 1, 2.6 +/- 0.9 at n = 8, m = 3, and 3.7 +/- 0.3 at n = 16, m = 4). The mismatch results were comparable to the PNA-free [(111)In]DOTA control results. Simultaneous administration of nonradioactive Gd(n)-KRAS2 G12 V probes (n = 2 or 8) increased accumulation of [(111)In](8)KRAS2 G12 V probes 3-6-fold in pancreatic cancer CAPAN2 xenografts and other tissues, except for a 2-fold decrease in the kidneys. As a result, tissue distribution tumor/muscle ratios of (111)In uptake increased from 3.1 +/- 0.5 to 6.5 +/- 1.0, and the kidney/tumor ratio of (111)In uptake decreased by more than 5-fold from 174.8 +/- 17.5 to 30.8 +/- 3.1. Thus, PDAP dendrimers with up to 16 DOTA chelators attached to PNA-IGF1 analogs, as well as simultaneous administration of the elevated dose of nonradioactive Gd(n)-KRAS2 G12 V probes, enhanced tumor uptake of [(111)In](n)KRAS2 PNA probes. These results also imply that Gd(III) dendrimeric hybridization probes might be suitable for magnetic resonance imaging of gene expression in tumors, because the higher generations of the dendrimers, including the NMR contrast Gd(n)-KRAS2 G12 V probes, improved tumor accumulation of the probes and specificity of tumor imaging.