Pharmacokinetics of a sustained release formulation of PDGFβ-receptor directed carrier proteins to target the fibrotic liver

Liver fibrogenesis is associated with excessive production of extracellular matrix by myofibroblasts that often leads to cirrhosis and consequently liver dysfunction and death. Novel protein-based antifibrotic drugs show high specificity and efficacy, but their use in the treatment of fibrosis causes a high burden for patients, since repetitive and long-term parenteral administration is required as most proteins and peptides are rapidly cleared from the circulation. Therefore, we developed biodegradable polymeric microspheres for the sustained release of proteinaceous drugs. We encapsulated the drug carrier pPB-HSA, which specifically binds to the PDGF beta R that is highly upregulated on activated myofibroblasts, into microspheres composed of hydrophilic multi-block copolymers composed of poly(L-lactide) and poly ethylene glycol/poly(is an element of-caprolactone), allowing diffusion-controlled release. Firstly, we estimated in mice with acute fibrogenesis induced by a single CCl4 injection the half-life of I-125-labeled pPB-HSA at 40 min and confirmed the preferential accumulation in fibrotic tissue. Subsequently, we determined in the Mdr2-/- mouse model of advanced biliary liver fibrosis how the subcutaneously injected microspheres released pPB-HSA into both plasma and fibrotic liver at 24 h after injection, which was maintained for six days. Although the microspheres still contained protein at day seven, pPB-HSA plasma and liver concentrations were decreased. This reduction was associated with an antibody response against the human albumin-based carrier protein, which was prevented by using a mouse albumin-based equivalent (pPB-MSA). In conclusion, this study shows that our polymeric microspheres are suitable as sustained release formulation for targeted protein constructs such as pPB-HSA. These formulations could be applied for the long-term treatment of chronic diseases such as liver fibrosis

Polymeric microspheres for the sustained release of a protein-based drug carrier targeting the PDGFβ-receptor in the fibrotic kidney

Injectable sustained release drug delivery systems are an attractive alternative for the intravenous delivery of therapeutic proteins. In particular, for chronic diseases such as fibrosis, this approach could improve therapy by reducing the administration frequency while avoiding large variations in plasma levels. In fibrotic tissues the platelet-derived growth factor receptor beta (PDGFβR) is highly upregulated, which provides a target for site-specific delivery of drugs. Our aim was to develop an injectable sustained release formulation for the subcutaneous delivery of the PDGFβR-targeted drug carrier protein pPB-HSA, which is composed of multiple PDGFβR-recognizing moieties (pPB) attached to human serum albumin (HSA). We used blends of biodegradable multi-block copolymers with different swelling degree to optimize the release rate using the model protein HSA from microspheres produced via a water-in-oil-in-water double emulsion evaporation process. The optimized formulation containing pPB-HSA, showed complete release in vitro within 14 days. After subcutaneous administration to mice suffering from renal fibrosis pPB-HSA was released from the microspheres and distributed into plasma for at least 7days after administration. Furthermore, we demonstrated an enhanced accumulation of pPB-HSA in the fibrotic kidney. Altogether, we show that subcutaneously administered polymeric microspheres present a suitable sustained release drug delivery system for the controlled systemic delivery for proteins such as pPB-HSA

The antifibrotic potential of a sustained release formulation of a PDGFβ-receptor targeted rho kinase inhibitor

Rho kinase activity in hepatic stellate cells (HSCs) is associated with activation, transformation and contraction of these cells, leading to extracellular matrix production and portal hypertension in liver cirrhosis. Inhibition of rho kinase activity can reduce these activities, but may also lead to side effects, for instance systemic hypotension. This can be circumvented by liver-specific delivery of a rho kinase inhibitor to effector cells. Therefore, we targeted the rho kinase inhibitor Y27632 to the key pathogenic cells in liver fibrosis, i.e. myofibroblasts including activated HSCs that highly express the PDGF beta-receptor, using the drug carrier pPB-MSA. This carrier consists of mouse serum albumin (MSA) covalently coupled to several PDGF beta-recognizing moieties (pPB). We aimed to create a prolonged release system of such a targeted construct, by encapsulating pPB-MSA-Y27632 in biodegradable polymeric microspheres, thereby reducing short-lasting peak concentrations and the need for frequent administrations. Firstly, we confirmed the vasodilating potency of PDGF beta-receptor targeted Y27632 in vitro in a contraction assay using HSCs seeded on a collagen gel. We subsequently demonstrated the in vivo antifibrotic efficacy of pPB-MSA-Y27632-loaded microspheres in the Mdr2 - / - mouse model of progressive biliary liver fibrosis. A single subcutaneous microsphere administration followed by organ harvest one week later clearly attenuated liver fibrosis progression and significantly suppressed the expression of fibrosis related genes, such as several collagens, profibrotic cytokines and matrix metalloproteinases. In conclusion, we demonstrate that polymeric microspheres are suitable as drug delivery system for the sustained systemic delivery of targeted protein constructs with antifibrotic potential, such as pPB-MSA-Y27632. This formulation appears suitable for the sustained treatment of liver fibrosis and possibly other chronic diseases

Looking back, looking forward: Methodological challenges and future directions in research on persons with profound intellectual and multiple disabilities

Within the context of the Special Interest Research Group (SIRG) on Persons with Profound Intellectual and Multiple Disabilities (PIMD), researchers often discuss the methodological problems and challenges they are confronted with. The aim of the current article was to give an overview of these challenges. The challenges are centred on six topics. These reflect the main components of a study's design: (a) participant demarcation, (b) participant recruitment, (c) data collection and instruments, (d) data analysis, (e) ethics/including the “voice” of persons with PIMD and (f) theoretical models. Next, to describing the specific challenges, possible solutions and pathways to address them are discussed. These are illustrated by recent studies by the authors and other researchers in the field. The current contribution wants to stimulate further discussion and ex-change of ideas, and the development of creative research techniques

Antiviral TRIMs: friend or foe in autoimmune and autoinflammatory disease?

The concept that viral sensing systems, via their ability to drive pro-inflammatory cytokine and interferon production, contribute to the development of autoimmune and autoinflammatory disease is supported by a wide range of clinical and experimental observations. Recently, the tripartite motif-containing proteins (TRIMs) have emerged as having key roles in antiviral immunity — either as viral restriction factors or as regulators of pathways downstream of viral RNA and DNA sensors, and the inflammasome. Given their involvement in these pathways, we propose that TRIM proteins contribute to the development and pathology of autoimmune and autoinflammatory conditions, thus making them potential novel targets for therapeutic manipulation

In vitro degradation and biocompatibility of poly(DL-lactide-epsilon-caprolactone) nerve guides

Bridging nerve gaps by means of autologous nerve grafts involves donor nerve graft harvesting. Recent studies have focused on the use of alternative methods, and one of these is the use of biodegradable nerve guides. After serving their function, nerve guides should degrade to avoid a chronic foreign body reaction. The in vitro degradation, in vitro cytotoxicity, hemocompatibility, and short-term in vivo foreign body reaction of poly(65/35 (85/15L/D) lactide-ϵ-caprolactone) nerve guides was studied. The in vitro degradation characteristics of poly(DLLA-ϵ-CL) nerve guides were monitored at 2-week time intervals during a period of 22 weeks. Weight loss, degree of swelling of the tube wall, mechanical strength, thermal properties, and the intrinsic viscosity of the nerve guides were determined. Cytotoxicity was studied by measuring the cell proliferation inhibition index (CPII) on mouse fibroblasts in vitro. Cell growth was evaluated by cell counting, while morphology was assessed by light microscopy. Hemocompatibility was evaluated using a thrombin generation assay and a complement convertase assay. The foreign body reaction against poly(DLLA-ϵ-CL) nerve guides was investigated by examining toluidine blue stained sections. The in vitro degradation data showed that poly(DLLA-ϵ-CL) nerve guides do not swell, maintain their mechanical strength and flexibility for a period of about 8–10 weeks, and start to lose mass after about 10 weeks. Poly(DLLA-ϵ-CL) nerve guides were classified as noncytotoxic, as cytotoxicity tests demonstrated that cell morphology was not affected (CPII 0%). The thrombin generation assay and complement convertase assay indicated that the material is highly hemocompatible. The foreign body reaction against the biomaterial was mild with a light priming of the immunesystem. The results presented in this study demonstrate that poly(65/35 (85/15L/D) lactide-ϵ-caprolactone) nerve guides are biocompatible, and show good in vitro degradation characteristics, making these biodegradable nerve guides promising candidates for bridging peripheral nerve defects up to several centimeters