Developing a Standard Set of Patient-Centred Outcomes for Inflammatory Bowel Disease—an International, Cross-disciplinary Consensus

Success in delivering value-based healthcare involves measuring outcomes that matter most to patients. Our aim was to develop a minimum Standard Set of patient-centred outcome measures for inflammatory bowel disease (IBD), for use in different healthcare settings.An international working group (n=25) representing patients, patient associations, gastroenterologists, surgeons, specialist nurses, IBD registries and patient-reported outcome measure (PROM) methodologists participated in a series of teleconferences incorporating a modified Delphi process. Systematic review of existing literature, registry data, patient focus groups and open review periods were used to reach consensus on a minimum set of standard outcome measures and risk adjustment variables. Similar methodology has been used in 21 other disease areas (www.ichom.org).A minimum Standard Set of outcomes was developed for patients (aged ≥16) with IBD. Outcome domains included survival and disease control (survival, disease activity/remission, colorectal cancer, anaemia), disutility of care (treatment-related complications), healthcare utilisation (IBD-related admissions, emergency room visits) and patient-reported outcomes (including quality of life, nutritional status and impact of fistulae) measured at baseline and at 6 or 12 month intervals. A single PROM (IBD-Control questionnaire) was recommended in the Standard Set and minimum risk adjustment data collected at baseline and annually were included: demographics, basic clinical information and treatment factors.A Standard Set of outcome measures for IBD has been developed based on evidence, patient input and specialist consensus. It provides an international template for meaningful, comparable and easy-to-interpret measures as a step towards achieving value-based healthcare in IBD

An Orthotopic Model of Glioblastoma Is Resistant to Radiodynamic Therapy with 5-AminoLevulinic Acid

Radiosensitization of glioblastoma is a major ambition to increase the survival of this incurable cancer. The 5-aminolevulinic acid (5-ALA) is metabolized by the heme biosynthesis pathway. 5-ALA overload leads to the accumulation of the intermediate fluorescent metabolite protoporphyrin IX (PpIX) with a radiosensitization potential, never tested in a relevant model of glioblastoma. We used a patient-derived tumor cell line grafted orthotopically to create a brain tumor model. We evaluated tumor growth and tumor burden after different regimens of encephalic multifractionated radiation therapy with or without 5-ALA. A fractionation scheme of 5 × 2 Gy three times a week resulted in intermediate survival [48-62 days] compared to 0 Gy (15-24 days), 3 × 2 Gy (41-47 days) and, 5 × 3 Gy (73-83 days). Survival was correlated to tumor growth. Tumor growth and survival were similar after 5 × 2 Gy irradiations, regardless of 5-ALA treatment (RT group (53-67 days), RT+5-ALA group (40-74 days), HR = 1.57, p = 0.24). Spheroid growth and survival were diminished by radiotherapy in vitro, unchanged by 5-ALA pre-treatment, confirming the in vivo results. The analysis of two additional stem-like patient-derived cell lines confirmed the absence of radiosensitization by 5-ALA. Our study shows for the first time that in a preclinical tumor model relevant to human glioblastoma, treated as in clinical routine, 5-ALA administration, although leading to important accumulation of PpIX, does not potentiate radiotherapy

Developing a Standard Set of Patient-Centred Outcomes for inflammatory Bowel Disease-an international, cross-disciplinary consensus

Background and Aims: Success in delivering value-based healthcare involves measuring outcomes that matter most to patients. Our aim was to develop a minimum Standard Set of patient-centred outcome measures for inflammatory bowel disease [IBD], for use in different healthcare settings. Methods: An international working group [n = 25] representing patients, patient associations, gastroenterologists, surgeons, specialist nurses, IBD registries and patient-reported outcome measure [PROM] methodologists participated in a series of teleconferences incorporating a modified Delphi process. Systematic review of existing literature, registry data, patient focus groups and open review periods were used to reach consensus on a minimum set of standard outcome measures and risk adjustment variables. Similar methodology has been used in 21 other disease areas [www.ichom.org]. Results: A minimum Standard Set of outcomes was developed for patients [aged =16] with IBD. Outcome domains included survival and disease control [survival, disease activity/remission, colorectal cancer, anaem

Optical Molecular Imaging of Ultrasound-mediated Drug Delivery

The goal of this PhD project was to develop optical molecular imaging methods to study drug delivery facilitated by ultrasound waves (US) and hyperthermia. Fibered confocal fluorescence microscopy (FCFM), together with dedicated image analysis, was used in vitro on a cell monolayer, and in vivo at the tissue scale, to monitor in real time and assess model drug and drug distribution. To this end, setups were designed that allowed ultrasound exposure or hyperthermia conditions, and that would present geometrical constraints in conventional optical imaging systems. However, these were largely overcome by the fiber based design of the microscope. In chapter 2, the feasibility to monitor in real-time US- and microbubble-mediated uptake of a cell-impermeable fluorescent model drug, i.e., SYTOX Green, is evaluated. An in vitro setup was designed that combined a mono-element US transducer, a cell culture chamber containing a monolayer of tumor cells together with SonoVue® microbubbles, and FCFM. The sequences showed a remaining plasma membrane permeability after the end of US exposure. To improve the accuracy of uptake kinetic parameter estimates of SYTOX Green, a post-processing method including cell tracking was presented in chapter 3. Using a two-compartment model representing the extracellular space and the cellular compartment, separated by a plasma membrane, the statistical analysis of the population kinetic data showed a median time constant of 2 minutes 19 seconds. Using the setup described in chapter 2 and the post-processing pipeline developed in chapter 3, we investigated in chapter 4 whether endocytosis is involved in US- and microbubbles- mediated delivery of small molecules using chlorpromazine, an inhibitor of clathrin-mediated endocytosis, or genistein, an inhibitor of caveolae-mediated endocytosis. During the real-time monitoring of SYTOX Green uptake, the cells in the presence of SonoVue® microbubbles were exposed to 1.4 MHz US waves at a 0.2 MPa peak-negative pressure. Both inhibitors were observed to slow down the US-mediated uptake of SYTOX Green, with a significant 2.5-fold increase of the uptake time constant with chlorpromazine, and a 1.1-fold increase with genistein. The impact of photobleaching on uptake rate estimates measured by FCFM was evaluated in chapter 5 to correct for it and improve the accuracy of pharmacokinetic parameter estimates. To model photobleaching of SYTOX Green, a photobleaching rate was added to the current two-compartment model describing cell uptake. Using this three-compartment model, an uptake rate of 6.0 10-3 s-1, independent of the applied laser power, was measured. In chapter 6, a feasibility study is described to evaluate tissue penetration of doxorubicin after its intravascular release from the thermo-sensitive liposomes ThermodoxTM using FCFM, and in a normal physiological environment. To this end, rat R1 rhabdomyosarcoma tumor pieces were implanted subcutaneously in the hind leg of 9 nude rats. A setup combining a water bath with a platform was designed to create local hyperthermia and thus trigger the release of doxorubicin from ThermodoxTM injected intravenously. In the tumor microenvironment, real-time simultaneous monitoring of doxorubicin penetration (488-nm excitation channel) succeeded in 2 of the 9 rats. A strong heterogeneity of doxorubicin distribution in the tumor was observed, which likely limited the success rate of the real-time monitoring

Microbubble-Assisted Ultrasound-Induced Transient Phosphatidylserine Translocation

Microbubble-assisted ultrasound (sonopermeabilization) results in reversible permeabilization of the plasma membrane of cells. This method is increasingly used in vivo because of its potential to deliver therapeutic molecules with limited cell damage. Nevertheless, the effects of sonopermeabilization on the plasma membrane remain not fully understood. We investigated the influence of sonopermeabilization on the transverse mobility of phospholipids, especially on phosphatidylserine (PS) externalization. We performed studies using optical imaging with Annexin V and FM1-43 probes to monitor PS externalization of rat glioma C6 cells. Sonopermeabilization induced transient membrane permeabilization, which is positively correlated with reversible PS externalization. This membrane disorganization was temporary and not associated with loss of cell viability. Sonopermeabilization did not induce PS externalization via activation of the scramblase. We hypothesize that acoustically induced membrane pores may provide a new pathway for PS migration between both membrane leaflets. During the membrane-resealing phase, PS asymmetry may be re-established by amino-phospholipid flippase activity and/or endocytosis, along with exocytosis processes

Cell Tracking for Assessment of In-Vitro Uptake Kinetics in Ultrasound-Mediated Drug Delivery using Fibered Confocal Fluorescence Microscopy

Local drug delivery in oncology aims at depositing high doses of anticancer agents while limiting their toxic side effects. Biological barriers, such as cell plasma membranes, hinder their delivery and requires strategies to address this challenge. Previously [1], we demonstrated the feasibility to monitor in real-time, with dynamic fluorescence microscopy, the in-tracellular delivery of a hydrophilic model drug mediated by ultrasound (US), and to quantify the pharmacokinetic parameters derived from a two-compartment model. We evaluate here the impact of the photobleaching (PB) effect experimentally , and compute the PB-corrected uptake kinetics

CORRECTION OF PHOTOBLEACHING FOR THE ASSESSMENT OF PHARMACOKINETIC PARAMETERS USING DYNAMIC FLUORESCENCE MICROSCOPY

Local drug delivery in oncology aims at depositing high doses of anticancer agents while limiting their toxic side effects. Biological barriers, such as cell plasma membranes, hinder their delivery and requires strategies to address this challenge. Previously [1], we demonstrated the feasibility to monitor in real-time, with dynamic fluorescence microscopy, the in-tracellular delivery of a hydrophilic model drug mediated by ultrasound (US), and to quantify the pharmacokinetic parameters derived from a two-compartment model. We evaluate here the impact of the photobleaching (PB) effect experimentally , and compute the PB-corrected uptake kinetics

Tracking of Cell Nuclei for Assessment of In Vitro Uptake Kinetics in Ultrasound-Mediated Drug Delivery using Fibered Confocal Fluorescence Microscopy

International audiencePurpose: Previously, we demonstrated the feasibility to monitor in real-time the ultrasound-mediated uptake of a cell-impermeable model drug with fibered confocal fluorescence microscopy. Here we present a complete post-processing methodology in order to improve the accuracy of the measured pharmacokinetic parameters.Procedures: After the detection of the nuclei based on a radial symmetry transform algorithm, a frame-by-frame tracking allowed for the monitoring of each individual uptake. The resulting pharmacokinetic parameters were derived from a two-compartment model.Results: With the tracking, 93% of the 370 nuclei showed a fluorescence signal variation that was well described by a two-compartment model. For each kinetic parameter, the tracking allowed for more homogeneous distributions.Conclusions: This post-processing methodology improved the accuracy of the uptake pharmacokinetic parameters assessed in a cell population. This study extends the proof of concept of designing an in vitro setup for the real-time monitoring of an US-mediated model drug uptake

Recruitment of endocytosis in sonopermeabilization-mediated drug delivery : a real-time study

Microbubbles (MBs) in combination with ultrasound (US) can enhance cell membrane permeability, and have the potential to facilitate the cellular uptake of hydrophilic molecules. However, the exact mechanism behind US- and MB-mediated intracellular delivery still remains to be fully understood. Among the proposed mechanisms are formation of transient pores and endocytosis stimulation. In our study, we investigated whether endocytosis is involved in US- and MB-mediated delivery of small molecules. Dynamic fluorescence microscopy was used to investigate the effects of endocytosis inhibitors on the pharmacokinetic parameters of US- and MB-mediated uptake of SYTOX Green, a 600 Da hydrophilic model drug. C6 rat glioma cells, together with SonoVue (R) MBs, were exposed to 1.4 MHz US waves at 0.2 MPa peak-negative pressure. Collection of the signal intensity in each individual nucleus was monitored during and after US exposure by a fibered confocal fluorescence microscope designed for real-time imaging. Exposed to US waves, C6 cells pretreated with chlorpromazine, an inhibitor of clathrin-mediated endocytosis, showed up to a 2.5-fold significant increase of the uptake time constant, and a 1.1-fold increase with genistein, an inhibitor of caveolae-mediated endocytosis. Both inhibitors slowed down the US- mediated uptake of SYTOX Green. With C6 cells and our experimental settings, these quantitative data indicate that endocytosis plays a role in sonopermeabilization-mediated delivery of small molecules with a more predominant contribution of clathrin-mediated endocytosis

Correction for Photobleaching in Dynamic Fluorescence Microscopy: Application in the Assessment of Pharmacokinetic Parameters in Ultrasound-Mediated Drug Delivery

Purpose. We have previously demonstrated the feasibility of monitoring ultrasound-mediated uptake of a hydrophilic model drug in real time with dynamic confocal fluorescence microscopy. In this study, we evaluate and correct the impact of photobleaching to improve the accuracy of pharmacokinetic parameter estimates.Procedures. To model photobleaching of the fluorescent model drug SYTOX Green, a photobleaching process was added to the current two-compartment model describing cell uptake. After collection of the uptake profile, a second acquisition was performed when SYTOX Green was equilibrated, to evaluate the photobleaching rate experimentally.Results. Photobleaching rates up to 5.0 10-3 s-1 were measured when applying power densities up to 0.2 W.cm-2. By applying the three-compartment model, the model drug uptake rate of 6.0 10-3 s-1 was measured independent of the applied laser power.Conclusions. The impact of photobleaching on uptake rate estimates measured by dynamic fluorescence microscopy was evaluated. Subsequent compensation improved the accuracy of pharmacokinetic parameter estimates in the cell population subjected to sonopermeabilization