Ronnie C. Mease

Imaging Enterobacteriaceae infection in vivo with 18F-fluorodeoxysorbitol positron emission tomography.

2-[18F]-Fluorodeoxysorbitol, a synthetic PET probe, can be used to rapidly, sensitively, and specifically image Gram-negative bacterial infections in animals. Probing Bacterial infections with PET Imaging Often acquired in hospitals and notoriously resistant to many drugs, Enterobacteriaceae represent an infectious threat to human health. Pathogenic Enterobacteriaceae, including Escherichia coli and Klebsiella pneumoniae, readily metabolize the sugar sorbitol; thus, Weinstein and colleagues reasoned that a radiolabeled version of sorbitol could be used to image bacterial infections in vivo. The authors created 18F-fluorodeoxysorbitol (FDS) and administered it to mice for positron emission tomography (PET) imaging. The probe was able to distinguish E. coli infection from general inflammation as well as from infection with the Gram-positive bacteria Staphylococcus aureus. FDS was used to monitor antimicrobial efficacy in animals infected with drug-susceptible or drug-resistant E. coli. Such imaging would allow noninvasive monitoring of disease progression or regression, without repeated time-consuming cultures. Whole-body imaging of bacteria using PET and 18F-labeled probes—two common clinical tools—could provide clinicians with a sensitive and specific means of rapidly assessing infection and making treatment decisions. The Enterobacteriaceae are a family of rod-shaped Gram-negative bacteria that normally inhabit the gastrointestinal tract and are the most common cause of Gram-negative bacterial infections in humans. In addition to causing serious multidrug-resistant, hospital-acquired infections, a number of Enterobacteriaceae species are also recognized as biothreat pathogens. As a consequence, new tools are urgently needed to specifically identify and localize infections due to Enterobacteriaceae and to monitor antimicrobial efficacy. In this report, we used commercially available 2-[18F]-fluorodeoxyglucose (18F-FDG) to produce 2-[18F]-fluorodeoxysorbitol (18F-FDS), a radioactive probe for Enterobacteriaceae, in 30 min. 18F-FDS selectively accumulated in Enterobacteriaceae, but not in Gram-positive bacteria or healthy mammalian or cancer cells in vitro. In a murine myositis model, 18F-FDS positron emission tomography (PET) rapidly differentiated true infection from sterile inflammation with a limit of detection of 6.2 ± 0.2 log10 colony-forming units (CFU) for Escherichia coli. Our findings were extended to models of mixed Gram-positive and Gram-negative thigh co-infections, brain infection, Klebsiella pneumonia, and mice undergoing immunosuppressive chemotherapy. This technique rapidly and specifically localized infections due to Enterobacteriaceae, providing a three-dimensional holistic view within the animal. Last, 18F-FDS PET monitored the efficacy of antimicrobial treatment, demonstrating a PET signal proportionate to the bacterial burden. Therapeutic failures associated with multidrug-resistant, extended-spectrum β-lactamase (ESBL)–producing E. coli infections were detected in real time. Together, these data show that 18F-FDS is a candidate imaging probe for translation to human clinical cases of known or suspected infections owing to Enterobacteriaceae.

Radioiodinated DPA-713 Imaging Correlates with Bactericidal Activity of Tuberculosis Treatments in Mice

ABSTRACT Current tools for monitoring response to tuberculosis treatments have several limitations. Noninvasive biomarkers could accelerate tuberculosis drug development and clinical studies, but to date little progress has been made in developing new imaging technologies for this application. In this study, we developed pulmonary single-photon emission computed tomography (SPECT) using radioiodinated DPA-713 to serially monitor the activity of tuberculosis treatments in live mice, which develop necrotic granulomas and cavitary lesions. C3HeB/FeJ mice were aerosol infected with Mycobacterium tuberculosis and administered either a standard or a highly active bedaquiline-containing drug regimen. Serial 125I-DPA-713 SPECT imaging was compared with 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) and standard microbiology. Ex vivo studies were performed to characterize and correlate DPA-713 imaging with cellular and cytokine responses. Pulmonary 125I-DPA-713 SPECT, but not 18F-FDG PET, was able to correctly identify the bactericidal activities of the two tuberculosis treatments as early as 4 weeks after the start of treatment (P < 0.03). DPA-713 readily penetrated the fibrotic rims of necrotic and cavitary lesions. A time-dependent decrease in both tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) levels was observed with treatments, with 125I-DPA-713 SPECT correlating best with tissue TNF-α levels (ρ = 0.94; P < 0.01). 124I-DPA-713 was also evaluated as a PET probe and demonstrated a 4.0-fold-higher signal intensity in the infected tuberculous lesions than uninfected controls (P = 0.03). These studies provide proof of concept for application of a novel noninvasive imaging biomarker to monitor tuberculosis treatments, with the potential for application for humans.

OPTIMIZATION OF PEPTIDE-BASED INHIBITORS OF PROSTATE-SPECIFIC ANTIGEN (PSA) AS TARGETED IMAGING AGENTS FOR PROSTATE CANCER

Prostate-specific antigen (PSA) is a serine protease biomarker that may play a role in prostate cancer development and progression. The inhibition of PSAs enzymatic activity with small molecule inhibitors is an attractive and, as of yet, unexploited target. Previously, we reported a series of peptidyl aldehyde and boronic acid based inhibitors of PSA. In this study, the structural requirements in the P2 and P3 positions of peptide-based PSA inhibitors are explored through the substitution of a series of natural and unnatural amino acids in these positions. This analysis demonstrated a preference for hydrophobic residues in the P2 position and amino acids with the potential to hydrogen bond in the P3 position. Using this information, a peptide boronic acid inhibitor with the sequence Cbz-Ser-Ser-Gln-Nle-(boro)-Leu was identified with a K(i) for PSA of 25nM. The attachment of a bulky metal chelating group to the amino terminal of this peptide did not adversely affect PSA inhibition. This result suggests that a platform of PSA inhibitor chelates could be developed as SPECT or PET-based imaging agents for prostate cancer.

(2S)-2-(3-(1-Carboxy-5-(4-[211At]astatobenzamido)pentyl)ureido)-pentanedioic acid for PSMA-Targeted α-Particle Radiopharmaceutical Therapy

Alpha-particle emitters have a high linear energy transfer and short range, offering the potential for treating micrometastases while sparing normal tissues. We developed a urea-based, 211At-labeled small molecule targeting prostate-specific membrane antigen (PSMA) for the treatment of micrometastases due to prostate cancer (PC). Methods: PSMA-targeted (2S)-2-(3-(1-carboxy-5-(4-211At-astatobenzamido)pentyl)ureido)-pentanedioic acid (211At-6) was synthesized. Cellular uptake and clonogenic survival were tested in PSMA-positive (PSMA+) PC3 PIP and PSMA-negative (PSMA−) PC3 flu human PC cells after 211At-6 treatment. The antitumor efficacy of 211At-6 was evaluated in mice bearing PSMA+ PC3 PIP and PSMA– PC3 flu flank xenografts at a 740-kBq dose and in mice bearing PSMA+, luciferase-expressing PC3-ML micrometastases. Biodistribution was determined in mice bearing PSMA+ PC3 PIP and PSMA– PC3 flu flank xenografts. Suborgan distribution was evaluated using α-camera images, and microscale dosimetry was modeled. Long-term toxicity was assessed in mice for 12 mo. Results: 211At-6 treatment resulted in PSMA-specific cellular uptake and decreased clonogenic survival in PSMA+ PC3 PIP cells and caused significant tumor growth delay in PSMA+ PC3 PIP flank tumors. Significantly improved survival was achieved in the newly developed PSMA+ micrometastatic PC model. Biodistribution showed uptake of 211At-6 in PSMA+ PC3 PIP tumors and in kidneys. Microscale kidney dosimetry based on α-camera images and a nephron model revealed hot spots in the proximal renal tubules. Long-term toxicity studies confirmed that the dose-limiting toxicity was late radiation nephropathy. Conclusion: PSMA-targeted 211At-6 α-particle radiotherapy yielded significantly improved survival in mice bearing PC micrometastases after systemic administration. 211At-6 also showed uptake in renal proximal tubules resulting in late nephrotoxicity, highlighting the importance of long-term toxicity studies and microscale dosimetry.

Molecular imaging of inflammation in the ApoE -/- mouse model of atherosclerosis with IodoDPA

BACKGROUNDnAtherosclerosis is a common and serious vascular disease predisposing individuals to myocardial infarction and stroke. Intravascular plaques, the pathologic lesions of atherosclerosis, are largely composed of cholesterol-laden luminal macrophage-rich infiltrates within a fibrous cap. The ability to detect those macrophages non-invasively within the aorta, carotid artery and other vessels would allow physicians to determine plaque burden, aiding management of patients with atherosclerosis.nnnMETHODS AND RESULTSnWe previously developed a low-molecular-weight imaging agent, [(125)I]iodo-DPA-713 (iodoDPA), which selectively targets macrophages. Here we use it to detect both intravascular macrophages and macrophage infiltrates within the myocardium in the ApoE -/- mouse model of atherosclerosis using single photon emission computed tomography (SPECT). SPECT data were confirmed by echocardiography, near-infrared fluorescence imaging and histology. SPECT images showed focal uptake of radiotracer at the aortic root in all ApoE -/- mice, while the age-matched controls were nearly devoid of radiotracer uptake. Focal radiotracer uptake along the descending aorta and within the myocardium was also observed in affected animals.nnnCONCLUSIONSnIodoDPA is a promising new imaging agent for atherosclerosis, with specificity for the macrophage component of the lesions involved.

An improved synthesis of the radiolabeled prostate-specific membrane antigen inhibitor, [18F]DCFPyL

The radiosynthesis of [(18) F]DCFPyL on 2 distinct automated platforms with full regulatory compliant quality control specifications is described. The radiotracer synthesis was performed on a custom-made radiofluorination module and the Sofie Biosciences ELIXYS. The radiofluorination module synthesis was accomplished in an average of 66xa0minutes from end of bombardment with an average specific activity at end of synthesis (EOS) of 4.4xa0TBq/μmol (120xa0Ci/μmol) and an average radiochemical yield of 30.9% at EOS. The ELIXYS synthesis was completed in an average of 87xa0minutes with an average specific activity of 2.2xa0TBq/μmol (59.3xa0Ci/μmol) and an average radiochemical yield of 19% at EOS. Both synthesis modules produced large millicurie quantities of [(18) F]DCFPyL while conforming to all standard US Pharmacopeia Chapter <823>xa0acceptance testing criteria.

Clinical impact of PSMA-based 18F–DCFBC PET/CT imaging in patients with biochemically recurrent prostate cancer after primary local therapy

PurposeThe purpose of our study was to assess 18F–DCFBC PET/CT, a PSMA targeted PET agent, for lesion detection and clinical management of biochemical relapse in prostate cancer patients after primary treatment.MethodsThis is a prospective IRB-approved study of 68 patients with documented biochemical recurrence after primary local therapy consisting of radical prostatectomy (nxa0=xa050), post radiation therapy (nxa0=xa09) or both (nxa0=xa09), with negative conventional imaging. All 68 patients underwent whole-body 18F–DCFBC PET/CT, and 62 also underwent mpMRI within one month. Lesion detection with 18F–DCFBC was correlated with mpMRI findings and pre-scan PSA levels. The impact of 18F–DCFBC PET/CT on clinical management and treatment decisions was established after 6xa0months’ patient clinical follow-up.ResultsForty-one patients (60.3%) showed at least one positive 18F–DCFBC lesion, for a total of 79 lesions, 30 in the prostate bed, 39 in lymph nodes, and ten in distant sites. Tumor recurrence was confirmed by either biopsy (13/41 pts), serial CT/MRI (8/41) or clinical follow-up (15/41); there was no confirmation in five patients, who continue to be observed. The 18F–DCFBC and mpMRI findings were concordant in 39 lesions (49.4%), and discordant in 40 lesions (50.6%); the majority (nxa0=xa032/40) of the latter occurring because the recurrence was located outside the mpMRI field of view. 18F–DCFBC PET positivity rates correlated with PSA values and 15%, 46%, 83%, and 77% were seen in patients with PSA values <0.5, 0.5 to <1.0, 1.0 to <2.0, and ≥2.0xa0ng/mL, respectively. The optimal cut-off PSA value to predict a positive 18F–DCFBC scan was 0.78xa0ng/mL (AUCxa0=xa00.764). A change in clinical management occurred in 51.2% (21/41) of patients with a positive 18F–DCFBC result, generally characterized by starting a new treatment in 19 patients or changing the treatment plan in two patients.Conclusions18F–DCFBC detects recurrences in 60.3% of a population of patients with biochemical recurrence, but results are dependent on PSA levels. Above a threshold PSA value of 0.78xa0ng/mL, 18F–DCFBC was able to identify recurrence with high reliability. Positive 18F–DCFBC PET imaging led clinicians to change treatment strategy in 51.2% of patients.

Positron Emission Tomography Imaging with 2-[18F]F-p-Aminobenzoic Acid Detects Staphylococcus aureus Infections and Monitors Drug Response

Staphylococcus aureus is the leading cause of life-threatening infections, frequently originating from unknown or deep-seated foci. Source control and institution of appropriate antibiotics remain challenges, especially with infections due to methicillin-resistant S. aureus (MRSA). In this study, we developed a radiofluorinated analog of para-aminobenzoic acid (2-[18F]F-PABA) and demonstrate that it is an efficient alternative substrate for the S. aureus dihydropteroate synthase (DHPS). 2-[18F]F-PABA rapidly accumulated in vitro within laboratory and clinical (including MRSA) strains of S. aureus but not in mammalian cells. Biodistribution in murine and rat models demonstrated localization at infection sites and rapid renal elimination. In a rat model, 2-[18F]F-PABA positron emission tomography (PET) rapidly differentiated S. aureus infection from sterile inflammation and could also detect therapeutic failures associated with MRSA. These data suggest that 2-[18F]F-PABA has the potential for translation to humans as a rapid, noninvasive diagnostic tool to identify, localize, and monitor S. aureus infections.

Abstract 3926: Molecular imaging of VEGFR expression modulation by antiangiogenic therapy in mouse model of human breast cancer.

Antiangiogenic therapy targeting VEGF/VEGFR signaling is an important strategy for cancer treatment, which is currently used in mono-therapy or combination therapy settings. However, the emerging resistance to antiangiogenic therapy as well as varied clinical response calls for the development of early, preferably noninvasive, markers of treatment response. In this study we used a recently developed molecular imaging tracer, site-specifically 99mTc-labeled engineered single-chain VEGF derivative, to monitor the VEGF receptor expression during the course of sunitinib antiangiogenic therapy in orthotopic human breast cancer model MDA-MB-231. The treatment protocol included two cycles of therapy delivered daily for 5 days (80mg/kg, orally) with a 5-day vacation between the cycles. Micro SPECT/CT imaging was performed longitudinally up to day 16. Animals in the control group received vehicle solution only. Tumors were excised and stained for the expression of CD31 endothelial cell marker. Experimental results suggest a highly heterogeneous vascular regression response to the first round of treatment, with significantly decreased tracer uptake and CD31 prevalence in some but not all tumors. As judged by the increase in tracer uptake and CD31 prevalence, a significant vascular rebound took place in all tumors after the vacation period. Judging by the same criteria, the second round of treatment was significantly less effective than the first round. Taken together, these results suggests that noninvasive molecular imaging of VEGF receptors reflects vascular regression and rebound in individual tumors and can provide clinically relevant information for monitoring and scheduling antiangiogenic therapy. Citation Format: Wenlian Zhu, Yoshinori Kato, Ronnie C. Mease, Susanta Sarkar, Marina V. Backer, Joseph M. Backer, Dmitri Artemov. Molecular imaging of VEGFR expression modulation by antiangiogenic therapy in mouse model of human breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3926. doi:10.1158/1538-7445.AM2013-3926