Challenges in Pharmacokinetic Modelling of [18F]fluoro-PEG-folate PET/CT Imaging in Epithelial Ovarian Cancer Patients.

PURPOSE: To describe the pharmacokinetic properties of the [18F]fluoro-polyethylene glycol(PEG)-folate radiotracer in PET/CT imaging of patients with advanced stage epithelial ovarian cancer (EOC). PROCEDURES: In five patients with advanced EOC (FIGO stage IIIB/IIIC, Fédération Internationale de Gynécologie et d'Obstétrique), a 90-min dynamic PET acquisition of the pelvis was performed directly after i.v. administration of 185 MBq [18F]fluoro-PEG6-folate. Arterial blood samples collected at nineteen timepoints were used to determine the plasma input function. A static volume of interest (VOI) for included tumor lesions was drawn manually on the PET images. Modelling was performed using PMOD software. Three different models (a 1-tissue compartment model (1T2k) and two 2-tissue compartment models, irreversible (2T3k) and reversible (2T4k)) were compared in goodness of fit with the time activity curves by means of the Akaike information criterion. RESULTS: The pharmacokinetic analysis in the pelvic area has proven to be much more challenging than expected. Only four out of 22 tumor lesions in five patients were considered suitable to perform modelling on. The remaining tumor lesions were inapt due to either low tracer uptake, small size, proximity to other [18F]fluoro-PEG6-folate -avid structures and/or displacement by abdominal organ motion in the dynamic scan. Data from the four analyzed tumor lesions suggest that the irreversible 2T3k may best describe the pharmacokinetics. All 22 lesions were immunohistochemically stained positive for the folate receptor alpha (FRα) after resection. CONCLUSION: Performing pharmacokinetic analysis in the abdominal pelvic region is very challenging. This brief article describes the challenges and pitfalls in pharmacokinetic analysis of a tracer with high physiological accumulation in the intestines, in case of lesions of limited size in the abdominal pelvic area.

Comparison of two hybrid sentinel node tracers: indocyanine green (ICG)-99mTc-nanocolloid vs. ICG-99mTc-nanoscan from a nuclear medicine and surgical perspective.

BACKGROUND: Lymph node (LN) metastasis is a relevant predictor for survival in patients with a.o. penile cancer (PeCa), malignant melanoma. The sentinel node (SN) procedure comprises targeted resection of the first tumour-draining SNs. Here, the hybrid tracer indocyanine green (ICG)-99mTc-nanocolloid has been used for several years to combine optical and nuclear detection. Recently, the resource of the nanocolloid precursor stopped production and the precursor was replaced by a different but chemically comparable colloid, nanoscan. Our aim was to study the performance of ICG-99mTc-nanoscan compared to ICG-99mTc-nanocolloid from a nuclear and surgical perspective. METHODS: Twenty-four patients with either PeCa or head-and-neck (H&N) melanoma and scheduled for a SN procedure were included. The initial group (n = 11) received ICG-99mTc-nanocolloid until no longer available; the second group (n = 13) received ICG-99mTc-nanoscan. Tracer uptake was assessed on lymphoscintigraphy and single-photon emission (SPECT). Intraoperatively, SNs were identified using gamma tracing and fluorescence imaging. Ex vivo (back-table) measurements were conducted to quantify the fluorescence emissions. Chemical analysis was performed to compare the ICG assembly on both precursors. RESULTS: The mean tracer uptake in the SNs was similar for ICG-99mTc-nanocolloid (2.2 ± 4.3%ID) and ICG-99mTc-nanoscan (1.8 ± 2.6%ID; p = 0.68). 3 SNs (interquartile range (IQR) 3-4) were detected on lymphoscintigraphy in PeCa patients receiving ICG-99mTc-nanoscan compared to 2 SNs (IQR 2-3) in PeCa patients receiving ICG-99mTc-nanocolloid (p = 0.045), no differences were observed in H&N patients. Back-table measurements of resected SNs revealed a lower total fluorescence intensity in the ICG-99mTc-nanoscan group (24*109 arbitrary units (A.U) IQR 1.6*109-14*109 in the ICG-99mTc-nanocolloid group versus 4.6*109 A.U. IQR 2.4*109-42*109 in the ICG-99mTc-nanoscan group, p = 0.0054). This was consistent with a larger degree of "stacked" ICG observed in the nanoscan formulation. No tracer-related adverse events were reported. CONCLUSIONS: Based on this retrospective analysis, we can conclude that ICG-99mTc-nanoscan has similar capacity for SN identification as ICG-99mTc-nanocolloid and can safely be implemented in SN procedures.

Microspheres as a Carrier System for Therapeutic Embolization Procedures: Achievements and Advances.

The targeted delivery of anti-cancer drugs and isotopes is one of the most pursued goals in anti-cancer therapy. One of the prime examples of such an application is the intra-arterial injection of microspheres containing cytostatic drugs or radioisotopes during hepatic embolization procedures. Therapy based on the application of microspheres revolves around vascular occlusion, complemented with local therapy in the form of trans-arterial chemoembolization (TACE) or radioembolization (TARE). The broadest implementation of these embolization strategies currently lies within the treatment of untreatable hepatocellular cancer (HCC) and metastatic colorectal cancer. This review aims to describe the state-of-the-art TACE and TARE technologies investigated in the clinical setting for HCC and addresses current trials and new developments. In addition, chemical properties and advancements in microsphere carrier systems are evaluated, and possible improvements in embolization therapy based on the modification of and functionalization with therapeutical loads are explored.

Design and validation of a specific scavenger receptor class AI binding peptide for targeting the inflammatory atherosclerotic plaque.

OBJECTIVE: Scavenger receptor A (SR-A) is abundantly expressed by macrophage and plays a critical role in foam cell formation and atherogenesis. In search of selective SR-AI antagonists, we have used affinity selection of a phage displayed peptide library on the synthetic extracellular domain of SR-AI. METHODS AND RESULTS: Phage selection led to an almost 1,000-fold enrichment of SR-AI binding phage, which bound avidly to human THP-1 cells. A 15-mer corresponding to the peptide insert of the major SR-AI binding phage (PP1) displaced phage binding to SR-AI. Peptides, docked to a streptavidin scaffold, were effectively internalized by macrophages in an SR-AI-dependent manner. The enriched phage pool and streptavidin bound PP1 exhibited marked uptake by hepatic macrophages in mice. Importantly, PP1 significantly increased streptavidin as well as particulate accumulation in advanced aortic plaques, and in particular intraplaque macrophage, of apolipoprotein E(-/-) mice. CONCLUSIONS: We have identified a novel peptide antagonist selective for SR-AI; this antagonist could be a valuable tool in SR-AI targeted imaging of atherosclerotic lesions.

Efficient degradation-aided selection of protease inhibitors by phage display.

In this report, we describe a novel phage display strategy for the identification of dedicated protease inhibiting peptides, based on degradation-aided enrichment of protease resistant phages. Phages were directly incubated with a range of phage-degrading proteases, after which non-degraded phages were used for the next selection round. For proteinase-K we identified after only four selection rounds a peptide (VLIMPVLLGIPLLC) that inhibits proteinase-K activity with an inhibition constant of 4 microM. In analogy, we identified a peptide capable of inhibiting substrate degradation by cathepsin-S (VWNCERITISRLIN), which showed functional inhibition of cathepsin-S induced sprouting of endothelial cells. We envision that the pursued strategy of degradation-aided selection of protease inhibitors (DASPI) represents an effective approach in the design of new protease inhibitors but also of new strategies to render gene and drug vectors protease resistant.

P-selectin as a candidate target in atherosclerosis.

P-selectin is of critical importance in early atherogenesis by initiating leukocyte rolling at the site of endothelial injury. In order to validate P-selectin as a candidate target for the development of anti-atherogenic strategies, we wanted to obtain quantitative information on P-selectin expression, and identify novel peptide-based lead structures that interact with P-selectin. P-selectin mRNA expression in the aortic arch and in other tissues of apoE-deficient (apoE-/-) mice was determined by real-time PCR technology. P-selectin mRNA expression of apoE-/- mice increased steadily with age to levels 14-fold higher than that of control animals. The onset and level of P-selectin expression correlated well with the extent of lesion development, and was more specific for atherosclerotic tissue as compared with other adhesion molecules. Phage display technology was used to obtain novel P-selectin antagonists. Phage display selections resulted in the isolation of a highly P-selectin-specific phage clone. Synthetic peptide-equivalents of this clone displaced the binding of the parent phage and antagonized the binding of a sialyl Lewis(x) analogue to P-selectin. In conclusion, P-selectin expression correlates with early and advanced atherosclerotic lesion development. P-selectin ligands, like the lead structure we have developed here, can therefore be considered as promising tools to identify, target or antagonize P-selectin function within the chronically inflamed arterial wall.

Rational optimization of a short human P-selectin-binding peptide leads to nanomolar affinity antagonists.

P-selectin plays an important role in the development of various diseases, including atherosclerosis and thrombosis. In our laboratory we recently identified a number of specific human P-selectin-binding peptides containing a Glu-Trp-Val-Asp-Val consensus motif, displaying a low micromolar affinity for P-selectin (IC(50) = 2 microm). In search of more potent antagonists for P-selectin, we have optimized the EWVDV pentapeptide core motif via a two-step combinatorial chemistry approach. A dedicated library of peptide derivatives was generated by introducing seven substituents at the N and C termini of the motif. In particular, pentapeptides with gallic acid or 1,3,5-benzenetricarboxylic acid substituents at the N terminus proved to be considerably more potent inhibitors of P-selectin binding than the parental peptide. After removal of the N-terminal glutamic acid from the core sequence, which appeared to be replaceable by a carboxamide function without loss of affinity, a second library was synthesized to map the chemical moieties within the gallic acid or 1,3,5-benzenetricarboxyl acid groups responsible for the enhanced P-selectin binding. Moreover, by varying the length and rigidity of the connective spacer, we have further optimized the spatial orientation of the N-terminal substituent. The combined use of phage display and subsequent combinatorial chemistry led to the design of a number of gallic acid- containing peptides with low nanomolar affinity for P-selectin both under static and dynamic conditions (IC(50) = 15.4 nm). These small synthetic antagonists, which are equally as potent as the natural ligand P-selectin glycoprotein ligand-1, are promising leads in anti-atherothrombotic therapy.

Targeting of peptides to restenotic vascular smooth muscle cells using phage display in vitro and in vivo.

Restenosis after angioplasty occurs in 30-40% of the treated patients. To develop a strategy to deliver drugs to restenotic lesions, we selected phages that bind to proliferating vascular smooth muscle cells (VSMC), from a random constraint 15-mer peptide phage display library. Phages were selected for binding to cultured primary aortic VSMC (in vitro biopanning) and selected for binding to denudated carotid arteries in mice (in vivo biopanning). In vitro biopanning did not result in a consensus sequence, but recurring FLGW and LASR amino acid motifs were identified. In vivo biopanning resulted in two consensus peptides 5G6 (CNIWGVVLSWIGVFPEC) and 5E5 (CESLWGGLMWTIGLSDC). Surprisingly, these two sequences were recovered after both in vitro and in vivo biopanning, but predominantly in vivo. Moreover, a strong recurring motif, IGR, was identified in the in vivo clones. The consensus phages 5G6 and 5E5 bind selectively to VSMC compared to other cell types. Furthermore, they bind preferentially to proliferating VSMC compared to VSMC that were growth arrested, and are effectively internalized by their target cells. The specific binding capacities of 5G6 and 5E5 phages suggest that these peptide sequences can be used for targeting of restenotic lesions, in which proliferating VSMC are the dominant cell type.

The effect of TGF-beta receptor binding peptides on smooth muscle cells.

TGF-beta1 is a potent regulator of vascular smooth muscle cell (VSMC) proliferation, migration, and extracellular matrix (ECM) synthesis. In this study, we selected two peptides, IM-1 and IM-2, that bind to the TGF-beta type II receptor (TGF-beta RII) using phage display. IM-1 and IM-2 bind to the TGF-beta RII, with a K(d) of 1 microM. Like TGF-beta, IM-1 induced VSMC chemotaxis and PAI-1 mRNA expression, as determined using Boyden chambers and real time quantitative PCR. In contrast, IM-2 had no effect on VSMC chemotaxis or PAI-1 induction. Induction of ECM synthesis, involving proteins such as osteopontin and alpha-smooth muscle actin, was determined by ELISA. Osteopontin expression was inhibited by both peptides, but TGF-beta-induced alpha-smooth muscle actin expression could only be inhibited by IM-1. In conclusion, IM-1 activity on VSMC is agonistic with TGF-beta, except for ECM synthesis, whereas the IM-2 peptide is antagonistic for some examined TGF-beta functions.