Development of a novel injectable drug delivery system for subconjunctival glaucoma treatment.

In this study we present the development of an injectable polymeric drug delivery system for subconjunctival treatment of primary open angle glaucoma. The system consists of hyaluronic acid sodium salt (HA), which is commonly used in ophthalmology in anterior segment surgery, and an isocyanate-functionalized 1,2-ethylene glycol bis(dilactic acid) (ELA-NCO). The polymer mixtures with different ratios of HA to ELA-NCO (1/1, 1/4, and 1/10 (v/v)) were investigated for biocompatibility, degradation behavior and applicability as a sustained release system. For the latter, the lipophilic latanoprost ester pro-drug (LA) was incorporated into the HA/ELA-NCO system. In vitro, a sustained LA release over a period of about 60days was achieved. In cell culture experiments, the HA/ELA-NCO (1/1, (v/v)) system was proven to be biocompatible for human and rabbit Tenon's fibroblasts. Examination of in vitro degradation behavior revealed a total mass loss of more than 60% during the observation period of 26weeks. In vivo, LA was continuously released for 152days into rabbit aqueous humor and serum. Histological investigations revealed a marked leuko-lymphocytic infiltration soon after subconjunctival injection. Thereafter, the initial tissue reaction declined concomitantly with a continuous degradation of the polymer, which was completed after 10months. Our study demonstrates the suitability of the polymer resulting from the reaction of HA with ELA-NCO as an injectable local drug delivery system for glaucoma therapy, combining biocompatibility and biodegradability with prolonged drug release.

New concepts for glaucoma implants--controlled aqueous humor drainage, encapsulation prevention and local drug delivery.

Glaucoma is a common cause of blindness in industrialized countries and is the most frequent cause of irreversible blindness worldwide. Since raised intraocular pressure (IOP) has been implicated as the major risk factor, the main goal of all glaucoma treatment is to reduce IOP sufficiently to prevent continuous irreversible retinal ganglion cell damage and progression of visual field loss. Pharmacological reduction of IOP is first-line therapy, followed by laser treatment of the trabecular meshwork and filtering glaucoma surgery, and cyclophotocoagulation of the ciliary body or allogenic implants. The most important glaucoma implants are presented (MOLTENO, AHMED, BAERVELDT, KRUPIN) together with more recent developments (Ex-Press, Eyepass, iStent, Gold micro shunt). Drainage into the suprachoroidal space is a promising option, but is also limited by scarring of the new created outflow route due to proliferation and adhesion of fibroblasts. A deeper understanding of fibroblasts in the related eye compartments is required. Characterization of scleral, choroidal, and, as a reference, Tenon fibroblast subtypes, is possible based on gene expression patterns. Alongside mitomycin C and 5-fluorouracil, newer drugs to prevent fibrosis have been proposed, offering effects that are more specific and more physiological. Effectors involved in wound healing phases and signaling pathways are potential targets for pharmaceutical intervention. Downregulation of growth factors like TGF-ß and their downstream effectors may suppress proliferation and differentiation of fibroblasts, extracellular matrix deposition, wound contraction, and neovascularization. Furthermore, current approaches to local drug delivery in glaucoma implant technology are briefly summarized.

Different fibroblast subpopulations of the eye: a therapeutic target to prevent postoperative fibrosis in glaucoma therapy.

The aim of this study is the characterization of fibroblasts mainly responsible for fibrosis processes associated with trabeculectomy or microstent implantation for glaucoma therapy. Therefore we isolated human primary fibroblasts from choroidea, sclera, Tenon capsule, and orbital fat tissues. These fibroblast subpopulations were analysed in vitro for expression of the extracellular matrix components which are responsible for postoperative scarring in glaucoma therapy. For scarring the proteins of the collagen family are predominant and so we focused on the expression of collagen I, collagen III and collagen VI in every fibroblast subpopulation. Also, the extracellular matrix protein fibronectin which crosslinks collagen fibres or other extracellular matrix components and cell surfaces, was analyzed. Collagen I, III and VI were prominent in every fibroblast subpopulation. The highest amounts of collagen III were found in hCF and hOF, whereas the signal in hSF and hTF was negligible. Additionally, there is a link between scarring processes and proliferating potential of fibroblasts, in case of microstent implantation triggered through the infiltration of inflammatory cells. Thus we analyzed fibroblast subpopulations for the presence of TGF-ß1 which is one of the most important cytokines involved in proliferation processes. TGF-ß1 was prominent in all fibroblast subpopulations with lowest expression in hCF cultures. To prevent postoperative fibroblast proliferation we analyzed in vitro the proliferation-inhibitors paclitaxel and mitomycin C which are potential candidates in drug eluting drainage systems on ocular fibroblast subpopulations. These inhibitors arrest fibroblast proliferation and viability, being, however, not very specific and have a cytotoxic potential also on healthy tissues surrounding the microstent outflow area. Significant differences in protein synthesis of fibroblasts subpopulations which could be specific targets for inhibition may help to find out fibroblast specific inhibitors to prevent postoperative scarring and could prevent patients from secondary surgery after microstent implantation.

Decreased renal corin expression contributes to sodium retention in proteinuric kidney diseases.

Patients with proteinuric kidney diseases often have symptoms of salt and water retention. It has been hypothesized that dysregulated sodium absorption is due to increased proteolytic cleavage of epithelial sodium channels (ENaCs) and increased Na,K-ATPase expression. Microarray analysis identified a reduction in kidney corin mRNA expression in rat models of puromycin aminonucleoside-induced nephrotic syndrome and acute anti-Thy1 glomerulonephritis (GN). As atrial natriuretic peptide (ANP) resistance is a mechanism accounting for volume retention, we analyzed the renal expression and function of corin; a type II transmembrane serine protease that converts pro-ANP to active ANP. Immunohistochemical analysis found that corin colocalized with ANP. The nephrotic and glomerulonephritic models exhibited concomitant increased pro-ANP and decreased ANP protein levels in the kidney consistent with low amounts of corin. Importantly, kidneys from corin knockout mice had increased amounts of renal ß-ENaC and its activators, phosphodiesterase (PDE) 5 and protein kinase G II, when compared to wild-type mice. A similar expression profile was also found in cell culture suggesting the increase in PDE5 and kinase G II could account for the increase in ß-ENaC seen in nephrotic syndrome and GN. Thus, we suggest that corin might be involved in the salt retention seen in glomerular diseases.

Mechanisms of tubular volume retention in immune-mediated glomerulonephritis.

Glomerulonephritis is characterized by hematuria, proteinuria, hypertension, and edema, but the mechanisms contributing to volume disorders are controversial. Here we used the rat anti-Thy1 model of mesangioproliferative glomerulonephritis to test the hypothesis that disturbed salt and water homeostasis is based on tubular epithelial changes that cause salt retention. In this model there was an early onset of pronounced proteinuria and lipiduria associated with reduced fractional sodium excretion and a lowering of the renin-angiotensin-aldosterone system. The glomerular filtration rate and creatinine clearance were decreased on day 6. There was a reduced abundance of the major salt and water transport proteins on the proximal tubular brush border membrane and which paralleled cellular protein overload, enhanced membrane cholesterol uptake and cytoskeletal changes. Alterations in thick ascending limb were moderate. Changes in the collecting ducts were characterized by an enhanced abundance and increased subunit cleavage of the epithelial sodium channel, both events consistent with increased sodium reabsorption. We suggest that irrespective of the proximal tubular changes, altered collecting duct sodium reabsorption may be crucial for volume retention in acute glomerulonephritis. We suggest that enhanced proteolytic cleavage of ion transporter subunits might be a novel mechanism of channel activation in glomerular diseases. Whether these proteases are filtered or locally secreted awaits determination.

Analysis of pregnancy and other factors on detection of human papilloma virus (HPV) infection using weighted estimating equations for follow-up data.

Generalized estimating equations have been well established to draw inference for the marginal mean from follow-up data. Many studies suffer from missing data that may result in biased parameter estimates if the data are not missing completely at random. Robins and co-workers proposed using weighted estimating equations (WEE) in estimating the mean structure if drop-out occurs missing at random. We illustrate the differences between the WEE and the commonly applied available case analysis in a simulation study. We apply the WEE and reanalyse data of a longitudinal study of pregnancy and human papilloma virus (HPV) infection. We estimate the response probabilities and demonstrate that the data are not missing completely at random. Upon use of the WEE, we are able to show that pregnant women have an increased odds for an HPV infection compared with non-pregnant women after delivery (p=0.027). We conclude that the WEE are useful for dealing with monotone missing data due to drop-outs in follow-up data.