Native T1 and ECV of Noninfarcted Myocardium and Outcome in Patients With Coronary Artery Disease.

BACKGROUND: Coronary artery disease (CAD) remains the major cause of cardiac morbidity and mortality worldwide, despite the advances in treatment with coronary revascularization and modern antiremodeling therapy. Risk stratification in CAD patients is primarily based on left ventricular volumes, ejection fraction (LVEF), risk scores, and the presence and extent of late gadolinium enhancement (LGE). The prognostic role of T1 mapping in noninfarcted myocardium in CAD patients has not yet been determined. OBJECTIVES: This study sought to examine prognostic significance of native T1 mapping of noninfarcted myocardium in patients with CAD. METHODS: A prospective, observational, multicenter longitudinal study of consecutive patients undergoing routine cardiac magnetic resonance imaging with T1 mapping and LGE. The primary endpoint was all-cause mortality. Major adverse cardiocerebrovascular events (MACCE) (cardiac mortality, nonfatal acute coronary syndrome, stroke, and appropriate device discharge) are also reported. RESULTS: A total of 34 deaths and 71 MACCE (n = 665, males n = 424, median age [interquartile range] 57 [22] years; 64%; median follow-up period of 17 [11] months) were observed. Native T1 and extracellular volume were univariate predictors of outcome. Native T1 and LGE were stronger predictors of survival and MACCE compared with extracellular volume, LVEF, cardiac volumes, and clinical scores (p < 0.001). Native T1 of noninfarcted myocardium was the sole independent predictor of all-cause mortality (chi-square = 21.7; p < 0.001), which was accentuated in the absence of LGE or LVEF ≤35%. For MACCE, native T1 and LGE extent were joint independent predictors (chi-square = 25.6; p < 0.001). CONCLUSIONS: Characterization of noninfarcted myocardium by native T1 is an important predictor of outcome in CAD patients, over and above the traditional risk stratifiers. The current study's results provide a basis for a novel risk stratification model in CAD based on a complementary assessment of noninfarcted myocardium and post-infarction scar, by native T1 mapping and LGE, respectively.

PEGylation enables the specific tumor accumulation of a peptide identified by phage display.

Peptides are excellent alternatives to small molecules and proteinaceous drugs. Their high medicinal potential for diagnostic and therapeutic applications has prompted the development of tumor targeting peptides. Despite its excellent tumor binding capacity, FROP-DOTA (H-Glu-Asn-Tyr-Glu-Leu-Met-Asp-Leu-Leu-Ala-Tyr-Leu-Lys(DOTA)-NH2), a peptide that we had identified in phage display libraries, revealed slow binding kinetics. Consequently, biodistribution studies showed that its excretion forestalled a significant tumor accumulation. The aim of this study was to investigate whether the conjugation of PEG to FROP-DOTA resulted in a derivative with a prolonged residence time in the blood. A synthetic method for the PEGylation of the tumor specific peptide FROP-DOTA was developed. Thereafter, binding studies were done in vitro and a biodistribution was performed in tumor bearing animals. These were compared to the data obtained with FROP-DOTA. The binding kinetics of the PEGylated FROP-DOTA was even slower than that of FROP-DOTA. Biodistribution studies of the labeled conjugate in mice bearing human FRO82-2 tumors showed a time dependent increased uptake of the PEGylated peptide with a high retention (at 24 h p.i. 76% of the maximal activity concentration persisted in the tumor). The highest uptake values were determined at 120 min p.i. reaching 2.3%ID/g tumor as compared to 0.06%ID/g observed for the non-PEGylated derivative at 135 min p.i. Apparently, PEGylation provides a substantially improved stabilization in the circulation which allowed a stable tumor accumulation.

Postprandial walking but not consumption of alcoholic digestifs or espresso accelerates gastric emptying in healthy volunteers.

BACKGROUND: Postprandial consumption of alcoholic beverages with high ethanol concentration (so-called digestifs) is a widespread custom to alleviate dyspeptic symptoms after comprehensive meals. Alcoholic beverages preprandially ingested inhibit gastric emptying rate of solid meals. However, the effect of a postprandial intake has never been studied in a controlled manner. METHODS: In 10 healthy male subjects gastric emptying was repeatedly studied by ultrasonography after the intake of a 576 kcal meal. Immediately after the meal subjects received in a randomized order 40 ml of the following liquids: brandy, herb flavored liqueur, Williams pear brandy, aquavit (each 40 % (v/v) ethanol concentration), espresso, water, 40% (v/v) ethanol and 70% (w/v) glucose. Postprandial satiety, fullness and bloating were determined on a visual analogue scale every 10 minutes. On another occasion subjects received 40 ml of water and walked afterwards slowly (4 km/h) on a treadmill. RESULTS: Gastric half emptying time (t 1/2) of the meal with water was 123 +/- 5 min, while with brandy (119 +/- 9 min), herb flavored liqueur (123 +/- 10 min), aquavit (125 +/- 9 min), Williams pear brandy (126 +/- 6 min) or espresso (125 +/- 9 min) t(1/2) it was not significantly different. Postprandial walking accelerated t(1/2) significantly (107 +/- 5 min, p=0.02). Dyspeptic symptoms were unchanged. Blood ethanol concentrations were under the level of detection (< 5 mg/dl). CONCLUSIONS: Postprandial consumption of alcoholic digestifs did not affect gastric emptying rate of a solid meal nor postprandial dyspeptic complaints. However, postprandial walking accelerated gastric emptying of the meal but this had no effect on dyspeptic symptoms.

Influence of chelate conjugation on a newly identified tumor-targeting peptide.

UNLABELLED: The transfer of peptide sequences identified by screening of phage-displayed libraries to clinical application is often difficult. This study investigated whether coupling of a new peptide, FROP-1, to the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) resulted in structural restriction and, consequently, improved binding and stability. METHODS: The peptide FROP-1 was coupled to the chelator DOTA and labeled with (111)In. The structural changes caused by the addition of the chelator were determined by circular dichroism. The properties of this modified peptide were investigated in in vitro binding assays and monitored for kinetics, competition, and internalization as well as serum stability. A cell-type binding profile was established and the in vivo biodistribution was evaluated in a nude mouse model. RESULTS: When compared with the free peptide without chelator, FROPDOTA revealed different cellular uptake kinetics, reaching a maximum at 2 h in vitro. The cells completely accumulated the tracer, and competition experiments revealed that 99.4% (FRO82-2 cells), 98.6% (MCF-7 cells), or 99.3% (average for 3 primary head and neck tumor cell lines) of tracer accumulation could be suppressed, revealing the specificity of this process. The internalization kinetics determined in MCF-7 cells supported this finding: After an incubation time of 180 min, the major fraction of FROPDOTA was trapped intracellularly. Serum stability experiments revealed an increase in stability due to the chelator, with a half-life of 71 min. Circular dichroism measurements indicated a fixed alpha-helix structure of FROPDOTA representing a strong change in secondary structure. In competition binding experiments, the binding constant (K(D)) to FRO82-2 cells was determined to be 494 nM. Despite this avid binding affinity, the binding kinetics were found to be too slow to induce an uptake in vivo before clearance. Consequently, the biodistribution revealed a rapid renal and hepatobiliary clearance, with blood levels dropping from 5.48 +/- 0.26 %ID/g (percentage injected dose per gram) 5 min after injection to 0.77 +/- 0.15 %ID/g at 135 min after injection. CONCLUSION: This study revealed that peptides that are identified by display techniques may be underrated. Careful alteration of their structure will permit going beyond the possibilities that the limited pool of naturally occurring peptides provide for tumor targeting.

Identification and evaluation of a new tumor cell-binding peptide, FROP-1.

UNLABELLED: Peptides are useful tools for the targeted delivery of radionuclides or chemotherapeutic drugs to their site of action within an organism. Given that the peptide receptor is overexpressed at the tumor, therapeutically active doses can be delivered to the tumor with reduced side effects. Because currently known peptides are restricted to a small number of tumors, new molecules and their corresponding receptors have to be identified to enlarge the spectrum of malignancies that can be diagnosed or treated using tumor-targeting peptides. METHODS: A 12-amino-acid peptide phage display system was applied to identify a new peptide binding to follicular thyroid carcinoma cells. The properties of the radiolabeled peptide were assessed in binding, competition, and internalization experiments in a variety of tumor cell lines including FRO82-2 and MCF-7 cells, and the pharmacokinetic behavior of the radiolabeled peptide was evaluated in tumor-bearing mice. Peptide stability was studied in human serum. RESULTS: After 5 selection rounds, the new peptide, FROP-1 (EDYELMDLLAYL), was identified. It showed binding to follicular thyroid carcinoma as well as anaplastic thyroid carcinoma, mammary carcinoma, cervix carcinoma, prostate carcinoma, and cell lines derived from head and neck tumors, and low affinity could be observed to control cells such as human umbilical vein endothelial cells or immortalized keratinocytes. In MCF7 cells, 78% and 86% of the bound activity was internalized after 10 and 60 min of incubation, respectively. Stability experiments in human serum showed the appearance of a degradation product after 15 min. Tumor uptake of the radioactive labeled peptide increased for 45 min in nude mouse models, reaching an accumulation level of approximately 3.6 percentage injected dose (%ID)/g for FRO82-2 tumors or approximately 3.8 %ID/g for MCF-7 tumors. CONCLUSION: The target of FROP-1 is most likely a molecule found generally in tumors, making this peptide highly attractive for diagnostic or therapeutic applications. However, modifications are needed to increase stability and affinity.

Characterization and development of a peptide (p160) with affinity for neuroblastoma cells.

UNLABELLED: Drug-targeting strategies can increase the efficacy and reduce the side effects and toxicity of conventional chemotherapy or may lead to new radiolabeled molecules useful for diagnosis and therapy. To identify and characterize new carrier molecules, we evaluated a peptide that had been identified by phage display technology. METHODS: The peptide p160 (VPWMEPAYQRFL) was prepared by solid-phase peptide synthesis and radiolabeled with (125)I or (131)I. The radiolabeled peptide and derivatives of it were used to study binding and internalization in vitro and to assess their distribution in tumor-bearing mice. RESULTS: Cell-binding assays on the human neuroblastoma cell line WAC 2 indicated the affinity and specificity of (125)I-labeled p160 toward neuroblastoma cells. Binding of the (125)I-labeled p160 was inhibited up to 95% by the unlabeled peptide. Furthermore, 50% of the total bound activity was internalized into the neuroblastoma cells. Biodistribution studies on nude mice showed a higher tracer accumulation in tumors than in most organs. Perfusion of the animals reduced uptake in all tissues, whereas tumor uptake remained constant. Fluorescence-activated cell-sorting studies with fluorescein isothiocyanate-labeled p160 demonstrated an increased fluorescence signal. Investigation of the binding properties of the fragments p160-8-1, p160-8-2, and p160-8-3 indicated that the sequence EPAYQR might be of significance for the binding of p160. CONCLUSION: These data indicate that the p160 peptide is an attractive candidate for the development of a neuroblastoma-specific vector that can be used for drug targeting or radiopeptide-based diagnosis and therapy.

Preclinical evaluation of the breast cancer cell-binding peptide, p160.

PURPOSE: Selective delivery of drugs into the target tissue is expected to result in high drug concentrations in the tissue of interest and therefore enhanced drug efficacy. To develop a peptide-based radiopharmaceutical, we investigated the properties of a peptide with affinity for human breast cancer, which has been selected through phage display. EXPERIMENTAL DESIGN: The bioactivity of the p160 peptide (VPWMEPAYQRFL) was evaluated in vitro and in vivo. The specific binding to human breast cancer MDA-MB-435 cells was confirmed in competition experiments. Internalization of the peptide was investigated with confocal microscopy. Furthermore, the biodistribution of (131)I-labeled p160 was studied in tumor-bearing mice. In vivo stability was evaluated at different periods after tracer administration using high-performance liquid chromatography analysis. RESULTS: The binding of (125)I-labeled p160 was inhibited up to 95% by the unlabeled peptide with an IC(50) value of 0.6 micromol/L. In addition, 40% of the total bound activity was found to be internalized into the human breast cancer cells. Although a rapid degradation was seen, biodistribution studies in nude mice showed a higher uptake in tumor than in most of the organs. Perfusion of the animals caused a reduction of the radioligand accumulation in the healthy tissues, whereas the tumor uptake remained constant. A comparison of [(131)I]p160 with a (131)I-labeled Arg-Gly-Asp peptide revealed a higher tumor-to-organ ratio for [(131)I]p160. CONCLUSIONS: p160 has properties that make it an attractive carrier for tumor imaging and the intracellular delivery of isotopes or chemotherapeutic drugs.

Identification of a new prostate-specific cyclic peptide with the bacterial FliTrx system.

UNLABELLED: Peptides are useful tools for directing radioisotopes into tumors. We evaluated the ability of a bacterial peptide display system to isolate new prostate tumor-specific peptides. METHODS: We used the bacterial FliTrx system to identify a new cyclic peptide that binds to prostate carcinoma. Serum stability and binding affinities of the (125)I-labeled peptide were tested. Furthermore, the (131)I-labeled peptide was used to evaluate its biodistribution. RESULTS: Several peptides showing a potential consensus motif were identified. The new peptide MM-2 is stable in serum for up to 24 h. It binds to PC-3 cells, and this binding can be inhibited more than 70% with the unlabeled peptide. Binding to human umbilical vein endothelial cells (HUVECs) and PNT-2 cells is weaker, and competition (27%) in HUVECs is less efficient. The biodistribution showed moderate accumulation in tumor. CONCLUSION: Bacterial peptide display, an alternative to phage peptide display, can allow the identification of specific binding and stable peptides.

Effects of Pax8 and TTF-1 thyroid transcription factor gene transfer in hepatoma cells: imaging of functional protein-protein interaction and iodide uptake.

UNLABELLED: The thyroid transcription factors TTF-1 and Pax8 cooperate in the transcriptional activation of thyroid-specific genes such as thyroglobulin (Tg), thyroperoxidase (TPO), and sodium/iodide symporter (NIS). METHODS: Dog TTF-1 (dTTF-1) and the human Pax8 (hPax8) gene were transfected in Morris hepatoma (MH3924A) cells to investigate (a) the possible visualization of functional protein-protein interaction and (b) the induction of thyroid-specific gene expression. In MH3924A cell lines expressing dTTF-1, hPax8, or both, the activation of human Tg (hTg), human TPO (hTPO), or rat NIS (rNIS) promoter/enhancer was measured using firefly luciferase reporter constructs. Furthermore, the possible induction of thyroid-specific genes was investigated in iodide uptake and reverse transcription polymerase chain reaction (RT-PCR) experiments. RESULTS: Low transcriptional activation of these constructs was observed in cells expressing either hPax8 or dTTF-1 alone. In contrast, the hTg and hTPO and, to a lesser extent, the rNIS regulatory region were significantly activated in cell lines expressing both transcription factors. Imaging the transcriptional activation of the thyroid-specific regulatory regions by Pax8 and TTF-1 was possible in nude mice implanted with MHhPax8dTTF-1 cells using a cooled charge-coupled device camera. Na(125)I uptake experiments and RT-PCR showed no effect of hPax8 and dTTF-1 on endogenous thyroid-specific gene expression in genetically modified cells. CONCLUSION: The activation of thyroid-specific promoter/enhancer elements in Morris hepatoma cells depends on the functional interaction of hPax8 and dTTF-1. The cooperation of these 2 transcription factors can be visualized in vitro as well as in vivo. With regard to a possible application for radioiodine therapy, further modifications are required.

A new prostate carcinoma binding peptide (DUP-1) for tumor imaging and therapy.

PURPOSE: Prostate carcinomas belong to the most widespread tumors, and their number is increasing. Imaging modalities used for diagnosis, such as ultrasound, computed tomography, and positron emission tomography, often produce poor results. Radiolabeled peptides with high sensitivity and specificity for prostate cancer would be a desirable tool for tumor diagnosis and treatment. EXPERIMENTAL DESIGN: We used phage display and the prostate-specific membrane antigen-negative cell line DU-145 to identify a peptide. The isolated DUP-1 was tested in vitro for its binding specificity, kinetics, and affinity. Internalization of the peptide was evaluated with confocal microscopy. The tumor accumulation in a nude mouse model was analyzed with 131I-labeled DUP-1 in PC-3 and DU-145 prostate tumors as well as in the rat prostate tumor model AT-1. RESULTS: The synthesized peptide showed rapid binding kinetics peaking at 10 minutes. It shows specific binding to prostate carcinoma cells but low binding affinity to nontumor cells. Peptide binding is competed with unlabeled DUP-1, and a time-dependent internalization into DU-145 cells was shown. Biodistribution studies of DUP-1 in nude mice with s.c. transplanted DU-145 and PC-3 tumors showed a tumor accumulation of 5% and 7% injected dose per gram, and bound peptide could not be removed by perfusion. The rat prostate tumor model showed an increase of radioactivity in the prostate tumor up to 300% in comparison with normal prostate tissue. CONCLUSIONS: DUP-1 holds promise as a lead peptide structure applicable in the development of new diagnostic tracers or anticancer agents that specifically target prostate carcinoma.

Increased rates of spontaneous sister chromatid exchange in lymphocytes of BRCA2+/- carriers of familial breast cancer clusters.

Heterozygous carriers of germ-line mutations of the BRCA2 breast cancer susceptibility gene are predisposed to breast, ovarian, pancreatic and other cancers. The BRCA2 protein is implicated in the maintenance of chromosome stability through its essential function in double-strand DNA repair and recombination. Our previous studies had revealed multiple intrachromosomal rearrangements, duplications, inversions and deletions on 9p23-24 in lymphocytes and fibroblasts of BRCA2+/- members from independently ascertained familial breast cancer clusters. In pursuit of evaluating if there is a subtle genomic instability in BRCA2+/- individuals, we have determined frequencies of spontaneous sister chromatid exchanges (SCEs) in BRCA2 wild-types and BRCA2 mutation carriers of two familial breast cancer clusters. Here, we demonstrate an average increase of 65% of spontaneous SCEs in BRCA2+/- versus BRCA2+/+ family members. In one cluster, the number of metaphases with multiple SCEs was 5-times higher in BRCA2+/- compared to wild-type members, while in the second cluster BRCA2+/- members had 8.9% of metaphases with multiple SCEs compared to a level below detection in BRCA2 wild types. To investigate the correlation between SCE and genomic instability in 9p, we performed fluorescence detection of SCEs and FISH analysis with 9p probes. The frequency of SCE in 9p of BRCA2 mutation carriers was 3-4 fold (P = 0.005) higher compared to BRCA2 wild-types. Collectively, the increased rates of SCE in BRCA2 heterozygous mutation carriers indicate a BRCA2 haploinsufficiency, which might be an important factor for the accumulation of structural chromosomal alterations with the consequence of damage in as yet unidentified genes.

Iodide kinetics and dosimetry in vivo after transfer of the human sodium iodide symporter gene in rat thyroid carcinoma cells.

UNLABELLED: Transfer of the human sodium iodide symporter (hNIS) has been proposed as a new principle of cancer gene therapy. This study evaluates the iodide kinetics and dosimetry of iodide in hNIS-expressing thyroid carcinoma cells under optimized conditions. METHODS: Using a bicistronic retroviral vector for the transfer of the hNIS and the hygromycin resistance gene, hNIS-expressing rat thyroid carcinoma cell lines were generated. Afterward, Na(125)I uptake and efflux were determined in genetically modified and wild-type cells in the presence or absence of modulators of iodide transport. In addition, the (131)I distribution in thyroid-ablated nude mice bearing wild-type and genetically modified thyroid carcinomas was monitored after intraperitoneal administration of (131)I with and without coadministration of lithium carbonate. RESULTS: hNIS-expressing cell lines accumulated up to 49 times more iodide than did noninfected cells, with a maximal iodide uptake after 30 min of incubation. However, a 90% efflux of the radioactivity occurred 20 min after replacement of the medium. In mice, the hNIS-expressing tumors accumulated up to 23 and 19.5 times more iodide than did the wild-type tumors in lithium-treated and control animals, respectively. However, efflux of the radioactivity was also observed in vivo: After 24 h, hNIS-expressing tumors lost 82.5% and 80.4% of the initial activity. Dosimetric calculations showed that 1,650 MBq of (131)I per square meter resulted in 5.4 and 5.2 Gy in hNIS-expressing tumors and 0.24 and 0.26 in wild-type tumors. CONCLUSION: Transduction of the hNIS gene in rat thyroid carcinoma cells induces iodide transport, which is associated with rapid efflux. Application of (131)I in clinically relevant amounts did not result in therapeutically useful absorbed doses in hNIS-expressing tumors in vivo, even under optimized conditions of thyroid ablation and treatment with lithium carbonate.

Increased MIBG uptake after transfer of the human norepinephrine transporter gene in rat hepatoma.

UNLABELLED: The transport of MIBG by the human norepinephrine transporter (hNET) seems to be the critical step in the treatment of MIBG-concentrating tumors. Therefore, we investigated whether the accumulation of MIBG may be induced by retroviral transfection of the hNET gene in Morris hepatoma cells. METHODS: A bicistronic retroviral vector for the transfer of the hNET coding sequence and the hygromycin resistance gene was generated. Morris hepatoma cells (MH3924A) were infected with the respective retroviral particles, and hNET-expressing cell lines MHhNEThyg1 to MHhNEThyg9 were obtained through hygromycin selection. The uptake of (3)H-norepinephrine or (131)I-MIBG and the efflux of (131)I-MIBG were determined in transfected and wild-type cells. In addition, the (131)I-MIBG distribution was monitored in nude mice and rats bearing wild-type and hNET-expressing hepatomas. RESULTS: hNET-expressing hepatoma cell lines accumulated up to 36 times more norepinephrine than did wild-type cells and 8 times more than did hNET-expressing neuroblastoma cell line SK-N-SH. The addition of nisoxetine, a selective inhibitor of noradrenaline uptake, inhibited norepinephrine uptake. Maximal (131)I-MIBG accumulation was observed 2 h after incubation and was followed by 43% efflux within 4 h after the (131)I-MIBG-containing medium had been removed. In vivo experiments performed with nude mice bearing both hNET-expressing and wild-type tumors showed a 10-fold-higher accumulation of (131)I-MIBG in transfected tumors than in wild-type tumors. The ex vivo calculations revealed doses of 605 and 75 mGy in hNET-expressing and wild-type tumor tissues, respectively. CONCLUSION: Transduction of the hNET gene enables Morris hepatoma cells to accumulate norepinephrine and MIBG. However, the retention of MIBG is brief; therefore, the absorbed dose of radiation in vivo is not expected to be therapeutically effective.

Arginine-glycine-aspartic acid (RGD)-peptide binds to both tumor and tumor-endothelial cells in vivo.

Targeting tumor cells or tumor vasculature by peptides is a promising strategy for delivering cytotoxic drugs for cancer therapy. The identification of efficient targeting peptides depends on the availability of informative methods for determining cellular binding specificities. Here, we have used fluorescence-activated cell-sorting (FACS) analysis in combination with an isopentane freezing method to show targeted binding of the Arg-Gly-Asp (RGD)-4C-peptide labeled with FITC, not only to endothelial cells but also to tumor cells in human breast cancer xenografts grown in nude mice. Nontumorous cells showed only background binding. This study suggests, that the RGD-4C-peptide can target tumor endothelial cells as well as tumor cells. Consequently, it should be possible to design a combination therapy approach against both targets.