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The previously reported 6-month angiographic and 12-month clinical outcomes of the CONSEQUENT trial demonstrated the safety and efficacy of a novel paclitaxel–resveratrol-coated balloon for the treatment of lesions in the femoropopliteal segment. The purpose of this report is to present the 2-year results including a cost-benefit analysis for Germany. Patients with symptomatic peripheral artery occlusive disease in femoropopliteal lesions were randomized either to drug-coated balloon (DCB, n = 78) or plain old balloon angioplasty (POBA, n = 75). As secondary endpoints, the 2-year clinical results consisting of target lesion revascularization (TLR), patency and increase in walking distance were recorded. Based on the Kaplan–Meier analyses for TLR and other adverse events, a cost-benefit analysis was conducted for the German DRG system. There were no additional TLRs in both groups between 14 and 24 months so that the corresponding rates remained significantly different between the treatment groups (DCB: 19.1 vs. POBA 40.6%, p = 0.007). At 2 years, the patency rate was significantly higher in the DCB group (72.3 vs. 48.4%, p = 0.006). The walking distance increase was also significantly higher after DCB angioplasty (172 ± 103 vs. 52 ± 136 m, p = 0.001). We estimated 2-year cost savings of € 1111.97 per patient treated with DCB instead of POBA. The use of paclitaxel–resveratrol matrix-coated peripheral balloons compared to POBA was associated with a significantly reduced TLR rate, superior patency and substantial cost savings at 2 years. ClinicalTrials.gov Identifier NCT01970579.

The quantification of magnetic iron oxide nanoparticles in biological systems (cells, tissues and organs) is of vital importance in the development of novel biomedical applications such as magnetofection, drug targeting or hyperthermia. Among several techniques established to detect iron in tissue, the recently developed technique of magnetic particle spectroscopy (MPS) provides signals that are specific for magnetic nanoparticles. MPS utilizes the non-linear response of an MNP sample to a strong sinusoidal excitation field of up to 25 mT amplitude and 25 kHz. We demonstrate the feasibility of this technique to quantify nanoparticle uptake in cells using a commercial magnetic particle spectrometer (Bruker BioSpin).

Typically, magnetic iron oxide nanoparticles (MNP) with core diameter in the range of about 16 nm to 22 nm are accessible by magnetorelaxometric (MRX) measurements at room temperature whereas the relaxation of smaller particles is too fast to be observed with a conventional MRX setup. In order to extend the size limitation towards smaller particles, we suggest applying temperature dependent magnetorelaxometry (TMRX). In this study, we outline and validate the procedure experimentally for temperatures between 5 K and 200 K on in-vitro preparations of MNP using a conventional MPMS SQUID magnetometer. On this basis, we applied TMRX for the in-vitro quantification of small sized MNP uptake by tumor cells, i.e. on HeLa and Jurkat tumor cell lines, reaching a detection limit of about 100 ng. We further showed that TMRX signals are characteristic for particular MNP preparations, opening the possibility to observe changes in the particle size distribution during the uptake of MNP by a biological system.

Within the hemocompatibility testing portfolio of medical devices a range of dynamic models were established in recent years. In contrast to the static hemocompatibility testing method the dynamic models allow considering the impact of hemorheological and hemodynamic blood characteristics on the hemocompatibility of medical devices. Unfortunately the EN DIN ISO 10993-4 for the biological evaluation of medical devices for interaction with blood gives no hints towards the period of time during which the medical devices should be exposed to the blood in these tests. To examine whether different exposure times impact the comparability of hemocompatibility test results low density polyethylene (LD-PE) tubes and nitinol stents were tested exemplarily in a closed loop model for changes of the fibrinogen content, the prothrombin time, the thrombin time, and the C5a activity after 30 and 90 min exposure to the blood. Low density polyethylene was used as negative control because it is one of the European reference materials for hemocompatibility testing. After 90 min blood exposure to the LD-PE tubing and the nitinol stents the prothrombin time was significantly longer and the fibrinogen content significantly lower (p < 0.05) than after 30 min. In contrast the thrombin time and the C5a were comparable after 30 and 90 min blood exposure time. These results might recommend to an initial 30 min exposure time, which is followed by a 90 min exposure time in cases of unclear results.

The detection of pathological tissue alterations by manual palpation is a simple but essential diagnostic tool, which has been applied by physicians since the beginnings of medicine. Recently, the virtual “palpation” of the brain has become feasible using magnetic resonance elastography, which quantifies biomechanical properties of the brain parenchyma by analyzing the propagation of externally elicited shear waves. However, the precise molecular and cellular patterns underlying changes of viscoelasticity measured by magnetic resonance elastography have not been investigated up to date. We assessed changes of viscoelasticity in a murine model of multiple sclerosis, inducing reversible demyelination by feeding the copper chelator cuprizone, and correlated our results with detailed histological analyses, comprising myelination, extracellular matrix alterations, immune cell infiltration and axonal damage. We show firstly that the magnitude of the complex shear modulus decreases with progressive demyelination and global extracellular matrix degradation, secondly that the loss modulus decreases faster than the dynamic modulus during the destruction of the corpus callosum, and finally that those processes are reversible after remyelination.

The aim of the study is to evaluate in vivo fluorescence imaging of experimental inflammatory joint disease by applying two different near-infrared (NIR) dyes in a model of Borrelia-induced Lyme arthritis.Forty mice, 20 with Lyme arthritis and 20 controls, were examined. Two nonspecific NIR carbocyanine dyes, indocyanine green (ICG) and a hydrophilic carbocyanine derivative (1,1'-bis-[4-sulfobutyl] indotricarbocyanine-5,5'-dicarboxylic acid diglucamide monosodium salt [SIDAG]), were administered intravenously at two doses. Fluorescence images were acquired before and during 120 seconds after injection of cyanine dyes. For both dyes, the area under the curve (AUC) was determined for the interval between 40 and 80 seconds after injection. In addition, the slope of the signal decrease was compared among animal groups. Results were compared with histological findings.The general temporal fluorescence intensity course for ICG was characterized by a rapid increase, with a peak at 40-50 seconds followed by a decrease; conversely for SIDAG, by a slow increase. AUC analysis for both dyes showed that the fluorescence signal differed significantly between controls and arthritic animals (P.05). Within these groups, there were significant differences between the two doses investigated. ICG differed significantly between control and arthritic animals in the slope of the signal decrease for both doses investigated (P.05). Histological examination showed early stages of inflammation in arthritic animals.NIR fluorescence imaging based on the pharmacokinetic behavior of ICG or SIDAG is a promising approach to detect inflammatory joint changes of experimental arthritis. Moreover, SIDAG is suited to differentiate inflammatory and noninflammatory joints 24 hours after dye application.

Using 15 rats with collagen-induced arthritis (30 joints) and 7 control rats (14 joints), we correlated the intensity of near-infrared fluorescence (NIRF) of the nonspecific dye tetrasulfocyanine (TSC) with magnetic resonance imaging (MRI), histopathology, and clinical score. Fluorescence images were obtained in reflection geometry using a NIRF camera system. Normalized fluorescence intensity (INF) was determined after intravenous dye administration on different time points up to 120 min. Contrast-enhanced MRI using gadodiamide was performed after NIRF imaging. Analyses were performed in a blinded fashion. Histopathological and clinical scores were determined for each ankle joint. INF of moderate and high-grade arthritic joints were significantly higher (p

We present a highly selective approach for the targeting of inflammation with a multivalent polymeric probe. Dendritic polyglycerol was employed to synthesize a polyanionic macromolecular conjugate with a near-infrared fluorescent dye related to Indocyanine Green (ICG). On the basis of the dense assembly of sulfate groups which were generated from the polyol core, the resulting polyglycerol sulfate (molecular weight 12 kD with ~70 sulfate groups) targets factors of inflammation (IC(50) of 3-6 nM for inhibition of L-selectin binding) and is specifically transported into inflammatory cells. The in vivo accumulation studied by near-IR fluorescence imaging in an animal model of rheumatoid arthritis demonstrated fast and selective uptake which enabled the differentiation of diseased joints (score 1-3) with a 3.5-fold higher fluorescence level and a signal maximum at 60 min post injection. Localization in tissues using fluorescence histology showed that the conjugates are deposited in the inflammatory infiltrate in the synovial membrane, whereas nonsulfated control was not detected in association with disease. Hence, this type of polymeric imaging probe is an alternative to current bioconjugates and provides future options for targeted imaging and drug delivery.

Introduction Fluorochromatic labeling of organic nanoparticles is often used to investigate their biodistribution in animal models. However, this technique is descriptive in nature and does not allow for signal quantification. Furthermore, fluorochromatic labeling changes chemical and physical properties and is therefore thought to change the distribution characteristics of nanoscaled particles. We therefore hypothesized that radioactive labeling facilitates a quantifiable determination of the biodistribution of nanoparticles. Material and Methods 47 NMRI-mice received 35S- labeled, 7 ± 1,5 nm sized dendritic Polyglycerol Sulfate (dPGS) or unlabelled dPGS intravenously or subcutaneously. Radioactivity from tissues at different times up to 21 days was analyzed by surface-counts, autoradiography, liquid-scintillation, imager-survey and histological photoemulsion. Results Radioactivity measurements allowed for a tissue- and time-dependent quantification of the labelled dPGS. Radioactively-labelled dPGS were still quantifiable in liver and spleen after 21 days following intravenous injection. Subcutaneous application resulted in a similar but delayed distribution kinetic. Discussion The biodistribution of dPGS was quantitatively determined by all methods used for radioactivity testing. This approach should provide an innovative, sensitive and adaptable method to detect nanoparticles without changing their biorelevant properties.

We investigated a molecular imaging approach for the detection of collagen-induced arthritis in rats by targeting the extra domain B (ED-B) of the extracellular matrix protein fibronectin. ED-B is a highly conserved domain (identical in human and rats) that is produced by alternative splicing during embryonic development and during vascular remodeling such as angiogenesis. The hallmark of rheumatoid arthritis is synovitis leading to both angiogenesis in the synovium and the promotion of cartilage and bone disruption. For in vivo diagnostics, the ED-B-binding single-chain antibody fragment AP39 was used as a targeting probe. It was covalently linked to the near-infrared dye tetrasulfocyanine (TSC) to be visualized by near-infrared fluorescence imaging. The resulting AP39-TSC conjugate was intravenously administered to rats with collagen-induced arthritis and the respective controls. Ovalbumin-TSC was used as control conjugate. Optical imaging over a time period of 24 hours using a planar imaging setup resulted in a clear enhancement of fluorescence intensity in joints with moderate to severe arthritis compared with control joints between 3 and 8 hours postinjection. Given that AP39 is a fully human antibody fragment, this molecular imaging approach for arthritis detection might be translated to humans.

We have developed a flying-spot scanner for fluorescence imaging of rheumatoid arthritis in the near infrared (NIR) spectral range following intravenous administration of contrast agents. The new imaging system has been characterized with respect to linearity, dynamic range and spatial resolution with the help of fluorescent phantoms. In vivo experiments were performed on an animal model of rheumatoid arthritis. Finally, NIR-fluorescence images of early stages of joint inflammation have been compared with findings from contrast enhanced MR imaging and histology.

An in vivo near-infrared fluorescence (NIRF) imaging technique is described for therapy monitoring of ankle joints affected by collagen-induced arthritis, a model of human rheumatoid arthritis. Arthritis was induced in rats by intradermal injections of collagen and Freund's incomplete adjuvant. For in vivo imaging, the nonspecific NIR dye tetrasulfocyanine (TSC) was used. Prior to and after treatment with a nonsteroidal anti-inflammatory drug, meloxicam, or analgesic drug, tramadol hydrochloride (which served as no-therapy control), normalized fluorescence intensities of each ankle joint were measured. Additionally, each ankle joint was characterized by clinical arthritis scoring and histopathology. Over a 3-week treatment period, a significant difference in disease progression between animals treated with meloxicam and tramadol hydrochloride was detected. A statistically significant improvement in ankle joint pathology from high- or moderate-grade to moderate- or low-grade upon meloxicam therapy, as determined by clinical evaluation, translated into a significant decrease in fluorescence intensity. In contrast, all arthritic joints of the no-therapy control group deteriorated to high-grade arthritis with high-fluorescence intensities in NIRF imaging.

Background Assessment of arthritis using fluorescence optical imaging (FOI) in combination with the fluorophore indocyanine green (ICG) is now clinically established [1]. Beyond imaging of inflammation, FOI also provides the opportunity to monitor drug targeting of biological therapeutics into inflamed joints. Quantitative information of drug targeting would allow for patient selection and individual treatment schedules. Objectives We report the first synthesis of etanercept-ICG-conjugates and the quantitative monitoring of drug targeting into inflamed joints of collagen II induced arthritis using a modified fluorescent camera system (Xiralite). Methods Inflammatory arthritis was induced by twice injections of bovine collagen II (day 0, day 7) in female Lewis rats. Rats were monitored for joint swelling, pain, redness and mobility (score 0 to 3). The TNFa binding protein etanercept (Enbrel®, Pfizer) was chemically conjugated with an ICG derivative suited for labeling of proteins. The etanercept-6S-ICG (Eta-6S-ICG) conjugate was photophysically characterized and tested for binding affinity The fluorescence camera system Xiralite (mivenion GmbH, Germany) [1] was modified to enable imaging of rats. On day 14 after first injection of collagen, Eta-6S-ICG (n=6), IgG-6S-ICG (n=3), or the inflammation-targeted macromolecule polyglycerolsulfate-6S-ICG (dPGS-6S-ICG, n=5) [2] were s.c. or i.v. injected in a dose of 10 nMol/kg b.w. Fluorescence measurement was performed before and 1 h, 3 h, 24 h and 48 h after injection. Fluorescence intensity (FI) was measured in ROI over the tibio-tarsal and interphalangeal articulations and compared with healthy reference ROI. In addition, targeting was studied in rat tissues 3 or 24 h post injection of Eta-6S-, IgG-6S- or dPGS-6S-indocarbocyanine conjugates using fluorescence histology. Results Binding affinity of etanercept was not attenuated by chemical conjugation with 6S-ICG. Eta-6S-ICG exerts high fluorescence quantum yield compared with ICG. FI was detected already 24 hours after administration of Eta-6S-ICG and further increased until 48 hours after injection. Significant FI at 48 hours after injection of Eta-6S-ICG was measured in tibio-tarsal joints (score 1: 15.8±2.9; score 3: 31.0±4.6) while FI in interphalangeal joints was low (score 1: 6.3±1.0; score 3: 7.7±1.2). In contrast, dPGS-6S-ICG exhibited high FI in both tibio-tarsal joints (score 1: 23.5±4.9; score 3: 69.8±17.5) and interphalangeal joints (score 1: 20.9±5.8; score 3: 37.0±4.7). Both Eta-6S- ICG and dPGS-6S-ICG show strong targeting to inflamed synovial tissue and cartilage compared with IgG-6S-ICG. Conclusions FOI and labeling of macromolecular therapeutics with novel ICG derivatives provide a novel rationale for sensitive and quantitative monitoring of arthritis therapy. Further studies have to determine the effect of varying molecular size on the degree of targeting of inflamed joints. References Werner S et al. Inflammation assessment in patients with arthritis using a novel in vivo fluorescence optical imaging technology. Ann Rheum Dis doi:10.1136/ard.2010.148288. Licha K et al. Fluorescence imaging with multifunctional polyglycerol sulfates: novel polymeric near-IR probes targeting inflammation. Bioconjug Chem; doi:10.1021/bc2002727 Disclosure of Interest P. Welker Employee of: mivenion GmbH, K. Licha Shareholder of: mivenion GmbH, I. Gemeinhardt: None Declared, M. Bahner Shareholder of: mivenion GmbH, J. Berger Employee of: mivenion GmbH, D. Mangoldt Employee of: mivenion GmbH, J. Schnorr: None Declared, M. Schirner Shareholder of: mivenion GmbH