Planning preclinical confirmatory multicenter trials to strengthen translation from basic to clinical research – a multi-stakeholder workshop report

Clinical translation from bench to bedside often remains challenging even despite promising preclinical evidence. Among many drivers like biological complexity or poorly understood disease pathology, preclinical evidence often lacks desired robustness. Reasons include low sample sizes, selective reporting, publication bias, and consequently inflated effect sizes. In this context, there is growing consensus that confirmatory multicenter studies -by weeding out false positives- represent an important step in strengthening and generating preclinical evidence before moving on to clinical research. However, there is little guidance on what such a preclinical confirmatory study entails and when it should be conducted in the research trajectory. To close this gap, we organized a workshop to bring together statisticians, clinicians, preclinical scientists, and meta-researcher to discuss and develop recommendations that are solution-oriented and feasible for practitioners. Herein, we summarize and review current approaches and outline strategies that provide decision-critical guidance on when to start and subsequently how to plan a confirmatory study. We define a set of minimum criteria and strategies to strengthen validity before engaging in a confirmatory preclinical trial, including sample size considerations that take the inherent uncertainty of initial (exploratory) studies into account. Beyond this specific guidance, we highlight knowledge gaps that require further research and discuss the role of confirmatory studies in translational biomedical research. In conclusion, this workshop report highlights the need for close interaction and open and honest debate between statisticians, preclinical scientists, meta-researchers (that conduct research on research), and clinicians already at an early stage of a given preclinical research trajectory

Experimental and Computational Study on the Microfluidic Control of Micellar Nanocarrier Properties

Microfluidic-based synthesis is a powerful technique to prepare well-defined homogenous nanoparticles (NPs). However, the mechanisms defining NP properties, especially size evolution in a microchannel, are not fully understood. Herein, microfluidic and bulk syntheses of riboflavin (RF)-targeted poly(lactic-co-glycolic acid)-poly(ethylene glycol) (PLGA-PEG-RF) micelles were evaluated experimentally and computationally. Using molecular dynamics (MD), a conventional "random"model for bulk self-assembly of PLGA-PEG-RF was simulated and a conceptual "interface"mechanism was proposed for the microfluidic self-assembly at an atomic scale. The simulation results were in agreement with the observed experimental outcomes. NPs produced by microfluidics were smaller than those prepared by the bulk method. The computational approach suggested that the size-determining factor in microfluidics is the boundary of solvents in the entrance region of the microchannel, explaining the size difference between the two experimental methods. Therefore, this computational approach can be a powerful tool to gain a deeper understanding and optimize NP synthesis. © 2021 The Authors. Published by American Chemical Society

Evaluation of radiolabeled stimuli-sensitive nanogels for application in nuclear medicine

Endogenous radiotherapy allows the direct delivery of radiation to a tumor by means of radiolabeled antibodies or minibodies as well as peptides, proteins or isotopes of halogens (like radioiodine therapy of thyroid cancer). However, still only few of these endogenous radiotherapy approaches are implemented in standard patient treatment. So far and irrespective of the strong research interest in nanomedicine, the efficacy of aqueous nanogels carrying radionuclides for endogenous radiotherapy has not been evaluated systematically and there are only few reports on the diagnostic potential of radiolabeled nanogels. Among the different types of nanocarriers (e.g. liposomes, polymeric micelles) which have been proposed as drug delivery vehicles, nanogels are rather unique due to their predominantly hydrophilic character. Nanogels can be “squeezed” and the fluctuations impede adsorption of proteins for entropic reasons. Hence, their aqueous consistence ensures minimized interaction with cells and proteins in the blood, which results in an extended circulation. The shape compliance can be tailored by the degree of crosslinking and very soft nanogels can squeeze through holes, which are much smaller than their hydrodynamic radius. This is important regarding excretion pathways as well as the passive accumulation of the nanogels into the tumor tissue by the enhanced permeability and retention (EPR) effect. The open structure is, however, in contradiction with a high loading capacity. Different from cytotoxic drugs, the dose-effect relation in diagnostic as well as therapeutic application of radionuclides is not only controlled by the concentration but also by the decay characteristics of the chosen radioisotope. While an auger electron emitter can promote cell death on a single cell level, an -emitter kills the cells only in decay proximity (radiation range 40-80 µm) and a --emitter can efficiently radiate the tumor tissue within a depths of 10-100 cells (long-range penetration with 0.1-10 mm). With respect to e.g. larger solid tumor manifestations and the intratumoral turgor effect, which limits the accessibility of the drugs to the deep tumor layers, the crossfire effect of - emitters can be a major advantage. Overall, their effective concentrations are significantly lower than those of a molecular drug and the radioisotope complexes established for medical application are mostly highly hydrophilic and can thus be incorporated in a hydrophilic nanogel without altering its stealth character. Thus, we designed stimuli-sensitive hydrophilic nanogels as carriers of radiolabeled chelators and tested their suitability in different preclinical settings. The nanogel-radionuclides were diagnostically evaluated by means of positron emitting radionuclides (68Ga and 64Cu), suitable for positron emission tomography (PET). In a general biodistribution study in healthy rodents, the radiolabeled nanoformulations showed a size dependent renal elimination and independent of the size only marginal accumulation in organs associated with the mononuclear phagocyte system (MPS). However, gel electrophoresis and size exclusion experiments indicated premature degradation in the circulation of the redox-sensitive nanogels within the first 5 h post injection (p.i.). This early degradation could be reduced by pre-treatment of the animals with buthionine sulfoximine (BSO) which significantly reduced glutathione (GSH; an antioxidant) levels in vivo (especially in liver/ spleen). This GSH reduction resulted in reduced renal excretion, however, with a slight increase in liver accumulation. The non-targeted nanogels rely on the passive accumulation into the tumor tissue by the EPR effect and upon degradation in the tumor microenvironment; an improved penetration of the released low molecular weight prepolymers was expected to yield a homogenous distribution of radioactivity throughout the tumor. However, the EPR effect has its own limitations, i.e., the tremendous heterogeneity in tumor vessel leakiness over space, time, and different types of tumors. Very crucial are variations in the blood flow to the tumor, i.e., density of vascular structures and permeability of the vasculature, as well as structural barriers imposed by the perivascular tumor cells and the extracellular matrix. Furthermore, increased interstitial fluid pressure limits the transport through the dense collagen matrix surrounding the tumor. Those limitations like the high interstitial fluid pressure in combination with drug efflux pumps resulted in a low retention of the proposed polymeric structures. To tackle this limitation receptor/transporter targeting ligands were introduced to yield radiolabeled degradation products that are suitable to actively target tumor cells. This concept was evaluated in somatostatin receptor positive pancreatic carcinoma xenografted mice and PET images indicated a significant higher retention compared to solely passively targeted nanogels as well as a deep penetration that was additionally confirmed by immunohistochemistry via staining of the radiolabeled targeting moiety. In another proof of concept study, nanogels were equipped with a cross reactive material (CRM-197) to promote receptor mediated transcytosis across the Blood Brain Barrier (BBB). Herein, an auger electron emitting radionuclide-labeled nucleoside analogue was covalently attached via enzyme cleavable substrates. The nucleoside analogue alone is not able to cross the BBB, while the nanogel can promote its delivery. The auger electron emitter reduces the crossfire effect and thus reduces damage to healthy tissue. Evaluation of the delivery of (nano)irradiation was performed in an in vitro model of the BBB in a co-culture of human cerebral endothelial cell line (hCMEC/D3), pericytes and astrocytes and/or different glioblastoma cell lines. Receptor mediated transport was confirmed via competition experiments. Upon cleavage, the nucleoside was still able to target cells and induce cell death. In summary, using different isotopes or radionuclides and different targeting moieties, the same construct of nanogels can be used for a variety of diseases and be tailored according to the scientific question

PLGA-Based Nanoparticles in Cancer Treatment

Nanomedicines can be used for a variety of cancer therapies including tumor-targeted drug delivery, hyperthermia, and photodynamic therapy. Poly (lactic-co-glycolic acid) (PLGA)-based materials are frequently used in such setups. This review article gives an overview of the properties of previously reported PLGA nanoparticles (NPs), their behavior in biological systems, and their use for cancer therapy. Strategies are emphasized to target PLGA NPs to the tumor site passively and actively. Furthermore, combination therapies are introduced that enhance the accumulation of NPs and, thereby, their therapeutic efficacy. In this context, the huge number of reports on PLGA NPs used as drug delivery systems in cancer treatment highlight the potential of PLGA NPs as drug carriers for cancer therapeutics and encourage further translational research

Tumor targeting via EPR: Strategies to enhance patient responses

The tumor accumulation of nanomedicines relies on the enhanced permeability and retention (EPR) effect. In the last 5–10 years, it has been increasingly recognized that there is a large inter- and intra-individual heterogeneity in EPR-mediated tumor targeting, explaining the heterogeneous outcomes of clinical trials in which nanomedicine formulations have been evaluated. To address this heterogeneity, as in other areas of oncology drug development, we have to move away from a one-size-fits-all tumor targeting approach, towards methods that can be employed to individualize and improve nanomedicine treatments. To this end, efforts have to be invested in better understanding the nature, the complexity and the heterogeneity of the EPR effect, and in establishing systems and strategies to enhance, combine, bypass and image EPR-based tumor targeting. In the present manuscript, we summarize key studies in which these strategies are explored, and we discuss how these approaches can be employed to enhance patient responses

Additional file 1 of Planning preclinical confirmatory multicenter trials to strengthen translation from basic to clinical research – a multi-stakeholder workshop report

Additional file 1: Figure S 1. Composition of workshop participants as per field of expertise. The organizing team mainly consists of meta-researchers with a clinical or preclinical background. Other refers to e.g., stakeholders from funding agencies that also attended the workshop. Methods S1. Multi-stakeholder workshop method-Workshop design. Glossary. Glossary of terms