Sridhar Nimmagadda

Radiohalogenated Prostate-Specific Membrane Antigen (PSMA)- Based Ureas as Imaging Agents for Prostate Cancer

To extend our development of new imaging agents targeting the prostate-specific membrane antigen (PSMA), we have used the versatile intermediate 2-[3-(5-amino-1-carboxy-pentyl)-ureido]-pentanedioic acid (Lys-C(O)-Glu), which allows ready incorporation of radiohalogens for single photon emission computed tomography (SPECT) and positron emission tomography (PET). We prepared 2-[3-[1-carboxy-5-(4-[(125)I]iodo-benzoylamino)-pentyl]-ureido]-pentanedioic acid ([(125)I]3), 2-[3-[1-carboxy-5-(4-[(18)F]fluoro-benzoylamino)-pentyl]-ureido]-pentanedioic acid ([(18)F]6), and 2-(3-[1-carboxy-5-[(5-[(125)I]iodo-pyridine-3-carbonyl)-amino]-pentyl]-ureido)-pentanedioic acid ([(125)I]8) in 65-80% (nondecay-corrected), 30-35% (decay corrected), and 59-75% (nondecay-corrected) radiochemical yields. Compound [(125)I]3 demonstrated 8.8 +/- 4.7% injected dose per gram (%ID/g) within PSMA(+) PC-3 PIP tumor at 30 min postinjection, which persisted, with clear delineation of the tumor by SPECT. Similar tumor uptake values at early time points were demonstrated for [(18)F]6 (using PET) and [(125)I]8. Because of the many radiohalogenated moieties that can be attached via the epsilon amino group, the intermediate Lys-C(O)-Glu is an attractive template upon which to develop new imaging agents for prostate cancer.

68Ga-Labeled Inhibitors of Prostate-Specific Membrane Antigen (PSMA) for Imaging Prostate Cancer

Gallium-68 is a generator-produced radionuclide for positron emission tomography (PET) that is being increasingly used for radiolabeling of tumor-targeting peptides. Compounds [(68)Ga]3 and [(68)Ga]6 are high-affinity urea-based inhibitors of the prostate-specific membrane antigen (PSMA) that were synthesized in decay-uncorrected yields ranging from 60% to 70% and radiochemical purities of more than 99%. Compound [(68)Ga]3 demonstrated 3.78 +/- 0.90% injected dose per gram of tissue (%ID/g) within PSMA+ PIP tumor at 30 min postinjection, while [(68)Ga]6 showed a 2 h PSMA+ PIP tumor uptake value of 3.29 +/- 0.77 %ID/g. Target (PSMA+ PIP) to nontarget (PSMA- flu) ratios were 4.6 and 18.3, respectively, at those time points. Both compounds delineated tumor clearly by small animal PET. The urea series of imaging agents for PSMA can be radiolabeled with (68)Ga, a cyclotron-free isotope useful for clinical PET studies, with maintenance of target specificity.

2-(3-{1-Carboxy-5-[(6-[18F]fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-pentanedioic acid, [18F]DCFPyL, a PSMA-based PET Imaging Agent for Prostate Cancer

Purpose: We have synthesized and evaluated in vivo 2-(3-{1-carboxy-5-[(6-[18F]fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-pentanedioic acid, [18F]DCFPyL, as a potential imaging agent for the prostate-specific membrane antigen (PSMA). PSMA is upregulated in prostate cancer epithelia and in the neovasculature of most solid tumors. Experimental Design: [18F]DCFPyL was synthesized in two steps from the p-methoxybenzyl (PMB) protected lys-C(O)-glu urea precursor using 6-[18F]fluoronicotinic acid tetrafluorophenyl ester ([18F]F-Py-TFP) for introduction of 18F. Radiochemical synthesis was followed by biodistribution and imaging with PET in immunocompromised mice using isogenic PSMA PC3 PIP and PSMA- PC3 flu xenograft models. Human radiation dosimetry estimates were calculated using OLINDA/EXM 1.0. Results: DCFPyL displays a Ki value of 1.1 ± 0.1 nmol/L for PSMA. [18F]DCFPyL was produced in radiochemical yields of 36%–53% (decay corrected) and specific radioactivities of 340–480 Ci/mmol (12.6–17.8 GBq/μmol, n = 3). In an immunocompromised mouse model [18F]DCFPyL clearly delineated PSMA+ PC3 PIP prostate tumor xenografts on imaging with PET. At 2 hours postinjection, 39.4 ± 5.4 percent injected dose per gram of tissue (%ID/g) was evident within the PSMA+ PC3 PIP tumor, with a ratio of 358:1 of uptake within PSMA+ PC3 PIP to PSMA− PC3 flu tumor placed in the opposite flank. At or after 1 hour postinjection, minimal nontarget tissue uptake of [18F]DCFPyL was observed. The bladder wall is the dose-limiting organ. Conclusions: These data suggest [18F]DCFPyL as a viable, new positron-emitting imaging agent for PSMA-expressing tissues. Clin Cancer Res; 17(24); 7645–53. ©2011 AACR.

The intricate role of CXCR4 in cancer.

Chemokines mediate numerous physiological and pathological processes related primarily to cell homing and migration. The chemokine CXCL12, also known as stromal cell-derived factor-1, binds the G-protein-coupled receptor CXCR4, which, through multiple divergent pathways, leads to chemotaxis, enhanced intracellular calcium, cell adhesion, survival, proliferation, and gene transcription. CXCR4, initially discovered for its involvement in HIV entry and leukocytes trafficking, is overexpressed in more than 23 human cancers. Cancer cell CXCR4 overexpression contributes to tumor growth, invasion, angiogenesis, metastasis, relapse, and therapeutic resistance. CXCR4 antagonism has been shown to disrupt tumor-stromal interactions, sensitize cancer cells to cytotoxic drugs, and reduce tumor growth and metastatic burden. As such, CXCR4 is a target not only for therapeutic intervention but also for noninvasive monitoring of disease progression and therapeutic guidance. This review provides a comprehensive overview of the biological involvement of CXCR4 in human cancers, the current status of CXCR4-based therapeutic approaches, as well as recent advances in noninvasive imaging of CXCR4 expression.

Molecular Imaging of CXCR4 Receptor Expression in Human Cancer Xenografts with [64Cu]AMD3100 Positron Emission Tomography

The chemokine receptor CXCR4 and its cognate ligand CXCL12 are pivotal for establishing metastases from many tumor types. Thus, CXCR4 may offer a cell surface target for molecular imaging of metastases, assisting diagnosis, staging, and therapeutic monitoring. Furthermore, noninvasive detection of CXCR4 status of a primary tumor may provide an index of the metastatic potential of the lesion. Here, we report the development and evaluation of [(64)Cu]AMD3100, a positron-emitting analogue of the stem cell mobilizing agent plerixafor to image CXCR4 in human tumor xenografts preselected for graded expression of this receptor. This imaging method was evaluated in lung metastases derived from human MDA-MB-231 breast cancer cells. Ex vivo biodistribution studies, performed to validate the in vivo imaging data, confirmed the ability of [(64)Cu]AMD3100 to image CXCR4 expression. Our findings show the feasibility of imaging CXCR4 by positron emission tomography using a clinically approved agent as a molecular scaffold.

Hypoxia regulates CD44 and its variant isoforms through HIF-1α in triple negative breast cancer.

Background The CD44 transmembrane glycoproteins play multifaceted roles in tumor progression and metastasis. CD44 expression has also been associated with stem-like breast cancer cells. Hypoxia commonly occurs in tumors and is a major cause of radiation and chemo-resistance. Hypoxia is known to inhibit differentiation and facilitates invasion and metastasis. Here we have investigated the effect of hypoxia on CD44 and two of its isoforms in MDA-MB-231 and SUM-149 triple negative human breast cancer cells and MDA-MB-231 tumors using imaging and molecular characterization. Methods and Findings The roles of hypoxia and hypoxia inducible factor (HIF) in regulating the expression of CD44 and its variant isoforms (CD44v6, CD44v7/8) were investigated in human breast cancer cells, by quantitative real-time polymerase chain reaction (qRT-PCR) to determine mRNA levels, and fluorescence associated cell sorting (FACS) to determine cell surface expression of CD44, under normoxic and hypoxic conditions. In vivo imaging studies with tumor xenografts derived from MDA-MD-231 cells engineered to express tdTomato red fluorescence protein under regulation of hypoxia response elements identified co-localization between hypoxic fluorescent regions and increased concentration of 125I-radiolabeled CD44 antibody. Conclusions Our data identified HIF-1α as a regulator of CD44 that increased the number of CD44 molecules and the percentage of CD44 positive cells expressing variant exons v6 and v7/8 in breast cancer cells under hypoxic conditions. Data from these cell studies were further supported by in vivo observations that hypoxic tumor regions contained cells with a higher concentration of CD44 expression.

Sequential SPECT and Optical Imaging of Experimental Models of Prostate Cancer with a Dual Modality Inhibitor of the Prostate-Specific Membrane Antigen

We describe a platform for dual modality (radionuclide/optical) imaging of prostate cancer (PCa) based on targeting the prostate-specific membrane antigen (PSMA). An example provided demonstrates that after a single intravenous (IV) injection of tracer amounts (0.1 nmol) of imaging agent to a tumor-bearing mouse, both single photon emission computed tomography (SPECT) and near-infrared fluorescence (NIRF) imaging were capable of delineating tumor specifically. That such small injected amounts could identify tumor in vivo suggests that optical agents, as has long been known for radiopharmaceuticals, may obey the tracer principle, enabling more rapid clinical translation.

Immunoimaging of CXCR4 expression in brain tumor xenografts using SPECT/CT

Chemokine receptor 4 (CXCR4) is expressed in a variety of cancers, including breast, brain, ovarian, and prostate. CXCR4–CXCL12 interactions are critical for tumor development, growth, and metastasis. Compared with normal tissue, neoplastic tissue (including metastases) expresses high levels of CXCR4. Previous clinical and preclinical observations suggest that CXCR4 levels could be used as a predictive marker of metastatic potential. Here we report the results of SPECT/CT of CXCR4 expression levels in experimental brain tumors using 125I-labeled anti-CXCR4 monoclonal antibodies (mAbs). Methods: hCXCR4 antibody 12G5 and control IgG2A antibody were radiolabeled. Radio-mAbs were obtained in 40%–60% yield, with 1.4–1.9 MBq/μg specific radioactivities and greater than 95% purity. Severe combined immunodeficient mice harboring U87 xenografts were used for ex vivo biodistribution and imaging studies. Surface CXCR4 expression levels on U87 tumor–derived cells were analyzed by flow cytometry. Results: Biodistribution and imaging studies showed a specific accumulation of 125I-12G5 in U87 tumors, with tumor-to-muscle uptake ratios reaching 15 ± 3 at 48 h after injection. The tumor-to-tumor uptake ratio for 125I-12G5 and 125I-IgG2A was 2.5 at 48 h after injection. Flow cytometry analysis of tumor-derived cells showed a 2- to 7-fold increase in CXCR4 expression relative to inoculums, accounting for the high mAb uptake observed in the tumors. Conclusion: Our data demonstrate the feasibility of imaging CXCR4 expression in experimental brain tumors. The elevated CXCR4 levels observed may have been, in part, due to the hypoxic tumor microenvironment.

64Cu-Labeled Inhibitors of Prostate-Specific Membrane Antigen for PET Imaging of Prostate Cancer

Prostate-specific membrane antigen (PSMA) is a well-recognized target for identification and therapy of a variety of cancers. Here we report five 64Cu-labeled inhibitors of PSMA, [64Cu]3–7, which are based on the lysine–glutamate urea scaffold and utilize a variety of macrocyclic chelators, namely NOTA(3), PCTA(4), Oxo-DO3A(5), CB-TE2A(6), and DOTA(7), in an effort to determine which provides the most suitable pharmacokinetics for in vivo PET imaging. [64Cu]3–7 were prepared in high radiochemical yield (60–90%) and purity (>95%). Positron emission tomography (PET) imaging studies of [64Cu]3–7 revealed specific accumulation in PSMA-expressing xenografts (PSMA+ PC3 PIP) relative to isogenic control tumor (PSMA– PC3 flu) and background tissue. The favorable kinetics and high image contrast provided by CB-TE2A chelated [64Cu]6 suggest it as the most promising among the candidates tested. That could be due to the higher stability of [64Cu]CB-TE2A as compared with [64Cu]NOTA, [64Cu]PCTA, [64Cu]Oxo-DO3A, and [64Cu]DOTA chelates in vivo.

Imaging of Musculoskeletal Bacterial Infections by [124I]FIAU-PET/CT

Background Traditional imaging techniques for the localization and monitoring of bacterial infections, although reasonably sensitive, suffer from a lack of specificity. This is particularly true for musculoskeletal infections. Bacteria possess a thymidine kinase (TK) whose substrate specificity is distinct from that of the major human TK. The substrate specificity difference has been exploited to develop a new imaging technique that can detect the presence of viable bacteria. Methodology/Principal Findings Eight subjects with suspected musculoskeletal infections and one healthy control were studied by a combination of [124I]FIAU-positron emission tomography and CT ([124I]FIAU-PET/CT). All patients with proven musculoskeletal infections demonstrated positive [124I]FIAU-PET/CT signals in the sites of concern at two hours after radiopharmaceutical administration. No adverse reactions with FIAU were observed. Conclusions/Significance [124I]FIAU-PET/CT is a promising new method for imaging bacterial infections.