Development of an optimized activatable MMP-14 targeted SPECT imaging probe.

Matrix metalloproteinase-14 (MT1-MMP or MMP-14) is a membrane-associated protease implicated in a variety of tissue remodeling processes and a molecular hallmark of select metastatic cancers. The ability to detect MMP-14 in vivo would be useful in studying its role in pathologic processes and may potentially serve as a guide for the development of targeted molecular therapies. Four MMP-14 specific probes containing a positively charged cell penetrating peptide (CPP) d-arginine octamer (r(8)) linked with a MMP-14 peptide substrate and attenuating sequences with glutamate (8e, 4e) or glutamate-glycine (4eg and 4egg) repeating units were modeled using an AMBER force field method. The probe with 4egg attenuating sequence exhibited the highest CPP/attenuator interaction, predicting minimized cellular uptake until cleaved. The in vitro MMP-14-mediated cleavage studies using the human recombinant MMP-14 catalytic domain revealed an enhanced cleavage rate that directly correlated with the linearity of the embedded peptide substrate sequence. Successful cleavage and uptake of a technetium-99m labeled version of the optimal probe was demonstrated in MMP-14 transfected human breast cancer cells. Two-fold reduction of cellular uptake was found in the presence of a broad spectrum MMP inhibitor. The combination of computational chemistry, parallel synthesis and biochemical screening, therefore, shows promise as a set of tools for developing new radiolabeled probes that are sensitive to protease activity.

Whole-body 18F-FDG PET in recurrent or metastatic nasopharyngeal carcinoma.

UNLABELLED: The aim of this retrospective study was to evaluate the sensitivity and prognostic significance of whole-body (18)F-FDG PET for nasopharyngeal carcinoma (NPC) patients for whom there was a suspicion of recurrence or metastasis by conventional radiologic or clinical findings during their follow-up examinations. METHODS: Whole-body (18)F-FDG PET examinations were performed on 64 Taiwanese NPC patients (14 female, 50 male; mean age +/- SD, 45.8 +/- 13.0 y; age range, 16-75 y) 4-70 mo (mean +/- SD, 14.1 +/- 13.5 mo) after radiotherapy or induction chemotherapy followed by concurrent chemoradiotherapy from February 1997 to May 2001. The accuracy of (18)F-FDG PET detection for each patient was determined by the histopathologic results or other clinical evidence. RESULTS: The sensitivity, specificity, accuracy, positive predictive value, and negative predictive value of (18)F-FDG PET images in the diagnosis of NPC recurrence or metastases and secondary primary cancers were 92%, 90%, 92%, 90%, and 91%, respectively. Furthermore, the presence of (18)F-FDG hypermetabolism was highly correlated with the survival time of NPC patients. CONCLUSION: Whole-body (18)F-FDG PET is a sensitive follow-up diagnostic tool for the evaluation of NPC recurrences and metastases. It is also an effective prognostic indicator for NPC patients. To determine the optimized utilization of (18)F-FDG PET in the follow-up for NPC patients, further cost-effectiveness analysis of (18)F-FDG PET in combination with conventional management is necessary.

Biliary hamartomas with delayed 99mTc-diisopropyl iminodiacetic acid clearance.

We report a 61-year-old asymptomatic man who had a hepatic lesion whose nature was undetermined, based on biochemistry, serology, ultrasonography, computed tomography, and magnetic resonance imaging studies. Biliary hamartomas were diagnosed by echo-guided needle biopsy. Functional studies of bile excretion, using 99mTc-diisopropyl iminodiacetic acid (99mTc-DISIDA) scintigraphy liver single-photon emission computed tomography (SPECT) showed the delayed transit of tracers from hepatocytes to the biliary hamartomas and the delayed emptying of tracers to the neighboring bile ducts. Therefore, biliary hamartomas should be included in the differential diagnosis of single or multiple hepatic lesions whose nature is undetermined, and a liver biopsy would be warranted for further treatment plans. We believe this is the first functional study of bile excretion in biliary hamartomas using radionucleotide studies; such studies may have prognostic implications.

Early detection of splenic metastasis of lung cancer by 18F-2-fluoro-2-deoxyglucose positron emission tomography.

Diagnosis of splenic involvement by lung cancer in the early stage is helpful for introducing proper treatment. We report a case of pulmonary adenocarcinoma with splenic metastasis which was first detected by 18F-2-fluoro-2-deoxyglucose (18F-FDG) positron emission tomography (PET). This 56-year-old male underwent left pneumonectomy, extensive lymph node dissection and postoperative adjuvant chemotherapy and radiotherapy. Because progressive elevation of carcinoembryonic antigen was detected 2 years after the operation, whole-body 18F-FDG PET was performed and revealed an 18F-FDG hypermetabolic spot in the splenic hilum. Although subsequent computed tomography (CT) did not reveal any lesions in the spleen, the second and third CT scans performed 10 and 13 months later confirmed the previous PET finding of splenic metastasis. This case suggests that 18F-FDG PET may be useful for early detection of splenic metastasis not visible on CT.

Using 18-fluoro-2-deoxyglucose positron emission tomography in detecting infectious endocarditis/endoarteritis: a preliminary report.

RATIONALE AND OBJECTIVES: We evaluated the effectiveness of positron emission tomography (PET) with 18-fluoro-2-deoxyglucose (FDG) in the detection of infectious endocarditis/endoarteritis. MATERIALS AND METHODS: For this study, we recruited 6 patients (4 women, 2 men; age range, 35 - 78 years; mean age, 55.8 +/- 16.8 years) who were clinically diagnosed as having infective endocarditis/endoarteritis by their echocardiographic findings and by Duke criteria. RESULTS: For all 6 patients, we also found increased FDG uptakes in the corresponding areas detected in echocardiography. CONCLUSION: FDG-PET appears to be a promising tool in diagnosing infective endocarditis/endoarteritis, and further prospective studies on a large scale to fully exploit the usefulness of FDG-PET for infective endocarditis/endoarteritis are needed.

Monitoring molecular-specific pharmacodynamics of rapamycin in vivo with inducible Gal4->Fluc transgenic reporter mice.

Rapamycin (Rap), a small-molecule inhibitor of mTOR, is an immunosuppressant, and several Rap analogues are cancer chemotherapeutics. Further pharmacologic development will be significantly facilitated if in vivo reporter models are available to enable monitoring of molecular-specific pharmacodynamic actions of Rap and its analogues. Herein we present the use of a Gal4→Fluc reporter mouse for the study of Rap-induced mTOR/FKBP12 protein-protein interactions in vivo with the use of a mouse two-hybrid transactivation strategy, a derivative of the yeast two-hybrid system applied to live mice. Upon treatment with Rap, a bipartite transactivator was reconstituted, and transcription of a genomic firefly luciferase reporter was activated in a concentration-dependent (K(d) = 2.3 nmol/L) and FK506-competitive (K(i) = 17.1 nmol/L) manner in cellulo, as well as in a temporal and specific manner in vivo. In particular, after a single dose of Rap (4.5 mg/kg, i.p.), peak Rap-induced protein-protein interactions were observed in the liver at 24 hours post treatment, with photon flux signals 600-fold over baseline, which correlated temporally with suppression of p70S6 kinase activity, a downstream effector of mTOR. The Gal4→Fluc reporter mouse provides an intact physiologic system to interrogate protein-protein interactions and molecular-specific pharmacodynamics during drug discovery and lead characterization. Imaging protein interactions and functional proteomics in whole animals in vivo may serve as a basic tool for screening and mechanism-based analysis of small molecules targeting specific protein-protein interactions in human diseases.

Dual-color click beetle luciferase heteroprotein fragment complementation assays.

Understanding the functional complexity of protein interactions requires mapping biomolecular complexes within the cellular environment over biologically relevant time scales. Herein, we describe a set of reversible multicolored heteroprotein complementation fragments based on various firefly and click beetle luciferases that utilize the same substrate, D-luciferin. Luciferase heteroprotein fragment complementation systems enabled dual-color quantification of two discrete pairs of interacting proteins simultaneously or two distinct proteins interacting with a third shared protein in live cells. Using real-time analysis of click beetle green and click beetle red luciferase heteroprotein fragment complementation applied to ß-TrCP, an E3-ligase common to the regulation of both ß-catenin and IκBα, GSK3ß was identified as a candidate kinase regulating IκBα processing. These dual-color protein interaction switches may enable directed dynamic analysis of a variety of protein interactions in living cells.

Advances in bioluminescence imaging of live animal models.

Many of the obligate steps of physiology and disease are dynamic in time and space, and thus, end-point assays do not always provide a full understanding of these processes. Comprehensive understanding of the functional complexity of protein interactions and cell trafficking requires mapping of cellular and molecular function within complex systems over biologically relevant time scales. New approaches to bioluminescence imaging of cell migration, signaling pathways, drug action, and interacting protein partners in vivo allow the study of biology and disease within the context of living animals.

Biodistributions of 177Lu- and 111In-labeled 7E11 antibodies to prostate-specific membrane antigen in xenograft model of prostate cancer and potential use of 111In-7E11 as a pre-therapeutic agent for 177Lu-7E11 radioimmunotherapy.

INTRODUCTION: Prostate-specific membrane antigen is a transmembrane glycoprotein highly expressed in many prostate cancers and can be targeted with radiolabeled antibodies for diagnosis and treatment of this disease. To serve as a radioimmunotherapeutic agent, a kinetically inert conjugate is desired to maximize tumor uptake and tumor radiation dose with minimal nonspecific exposure to bone marrow and other major organs. MATERIALS AND METHODS: In this study, we assessed the pharmacokinetics and biodistribution of the 7E11 monoclonal antibody (MAb) radiolabeled with the lutetium-177 ((177)Lu)-tetraazacyclododecanetetraacetic acid conjugate system ((177)Lu-7E11) versus those of the 7E11 MAb radiolabeled with the indium-111 ((111)In)/glycyl-tyrosyl-(N,-diethylenetriaminepentaacetic acid)/lysine hydrochloride conjugate system ((111)In-7E11, also known as ProstaScint) to determine the feasibility of using (111)In-7E11 as a pre-therapeutic agent for (177)Lu-7E11 radioimmunotherapy. Pharmacokinetic and biodistribution studies of (177)Lu-7E11 in lymph node cancer of the prostate (LNCaP) xenograft mice were performed at 2, 8, 12, 24, 72, and 168 h after radiopharmaceutical administration. For (111)In-7E11, pharmacokinetic and biodistribution studies were performed at 8, 24, and 72 h. Parallel studies of (177)Lu-7E11 in non-tumor-bearing mice at 8, 24, and 72 h post-injection served as controls. Gamma scintigraphy was performed, followed by autoradiography and tissue counting, to demonstrate and quantify the distributions of radioconjugated MAb in the tumor and normal tissues. RESULTS AND DISCUSSION: Both (177)Lu- and (111)In-7E11 conjugates demonstrated an early blood pool phase in which uptake was dominated by the blood, lung, spleen and liver, followed by uptake and retention of the radiolabeled antibody in the tumor which was most prominent at 24 h. Total accumulation of radioconjugated MAb in tumor at 24 h was greater in the case of (177)Lu-7E11 in comparison to that of (111)In-7E11. Continued accumulation in tumor was observed for the entire time course studied for both (177)Lu-7E11 and (111)In-7E11. The liver was the only major organ demonstrating a significant difference in accumulation between the two conjugates. In conclusion, pharmacokinetic and biodistribution studies of (177)Lu-7E11 in LNCaP xenograft mouse models support its potential application as a radioimmunotherapeutic agent targeting prostate cancer, and the distribution and tumor uptake of (111)In-7E11 appear to be similar to those of (177)Lu-7E11, supporting its use as a pre-therapeutic tool to assess the potential accumulation of (177)Lu-7E11 radioimmunotherapeutic at sites of prostate cancer. However, the different accumulation patterns of the (111)In and (177)Lu immunoconjugates in liver will likely prevent the use of (111)In-7E11 as a true dosimetry tool for (177)Lu-7E11 radioimmunotherapy.

Early restaging whole-body (18)F-FDG PET during induction chemotherapy predicts clinical outcome in patients with locoregionally advanced nasopharyngeal carcinoma.

PURPOSE: This study was undertaken to evaluate the utility of whole-body (18)F-FDG PET in monitoring therapeutic effect during induction chemotherapy (IC) and in predicting prognosis in patients with locoregionally advanced nasopharyngeal carcinoma (NPC). METHODS: Fifty patients who had histologically proven, locoregionally advanced NPC without distant metastasis and had received IC were recruited in this study. The study cohort consisted of 19 females and 31 males (age 17-72 years, mean 45.9+/-11.9). Whole-body (18)F-FDG PET was performed in each patient after completion of one (33 patients) or two (17 patients) courses of IC. Each patient was restaged on the basis of the (18)F-FDG PET results. Patients who were downstaged to stage I or II were classified as major responders; the rest were classified as non-major responders. RESULTS: Only 1 of the 23 major responders subsequently developed local recurrence. At the time of data analysis, all major responders were alive; by contrast, of the 27 non-major responders, 15 had locoregional recurrence or distant metastasis and nine had died (seven of NPC and two of treatment-related complications). Kaplan-Meier survival analysis showed significantly longer recurrence-free survival and overall survival in major responders (56.4+/-9.2 and 58.1+/-2.2 months) as compared with non-major responders (33.7+/-23.2 and 44.7+/-20.0 months), with p<0.0001 and p=0.0024, respectively. CONCLUSION: The results of this study suggest that early restaging by a single whole-body (18)F-FDG PET scan after the first or second course of IC is useful for predicting therapeutic response and outcome in patients with locoregionally advanced NPC.

18-fluoro-2-deoxyglucose positron emission tomography in detecting residual/recurrent nasopharyngeal carcinomas and comparison with magnetic resonance imaging.

BACKGROUND: It is known that 18-fluoro-2-deoxyglucose positron emission tomography (FDG-PET) is effective in the early detection of residual/recurrent nasopharyngeal carcinomas (NPC). To compare FDG-PET with the conventional magnetic resonance imaging (MRI) for the detection of residual/recurrent NPC, the authors studied 67 follow-up cases of patients with NPC using both FDG-PET and MRI. METHODS: From February 1997 to February 2001, 67 NPC patients (14 women, 53 men; age range, 16-67 years; mean age, 46.6 +/- 12.5 years) were recruited. Both FDG-PET and MRI of the head and neck area for each patient were performed at least 4 months (duration range, 4-70 months; mean, 14 +/- 13.5 months) after radiotherapy or radiotherapy with concurrent chemotherapy. The final diagnosis was confirmed by biopsy or clinical follow-up for at least 6 months. RESULTS: The sensitivity, specificity, accuracy, positive predictive value (PPV), and negative predictive value (NPV) of FDG-PET images were 100%, 93.4%, 95.5%, 87.5%, and 100%, respectively. In contrast, the sensitivity, specificity, accuracy, PPV, and NPV of the MRI scans were 61.9%, 43.5%, 49.3%, 33.3%, and 70.0%, respectively. CONCLUSIONS: The results of the current study suggest that FDG-PET is much more effective than MRI in detecting residual/recurrent NPC.