Seppo Rönkkö

Matrix metalloproteinases and their inhibitors in the chamber angle of normal eyes and patients with primary open-angle glaucoma and exfoliation glaucoma

BackgroundIn glaucoma, extensive pathological changes occur in the trabecular meshwork (TM) and juxtacanalicular tissue of the chamber angle. Aqueous humor drainage is disturbed due to the accumulation of extracellular matrix (ECM) material in the outflow system. Matrix metalloproteinases (MMPs) remodel ECM material and, thus, they may have a role in regulating outflow facility and intraocular pressure (IOP). This study examined the expression of MMPs and tissue inhibitors of MMPs (TIMPs) in the chamber angle of normal eyes and in primary open-angle glaucoma (POAG) and in exfoliation glaucoma (ExG).MethodsTM tissues were isolated from healthy donor eyes for corneal transplantation. Specimens of the inner wall of Schlemm’s canal and the juxtacanalicular tissue were collected from patients with POAG or ExG during deep sclerectomy operation. Monoclonal antibodies against MMPs (MMP-1, -2, -3, and -9) and antibodies against TIMPs (TIMP-1, -2, and -3) were used for immunohistochemical stainingResultsImmunoreactivity for MMP-2, TIMP-2, or TIMP-3 was observed in human normal TM and in the inner wall of Schlemm’s canal. In general, immunoreactions for all of the tested MMPs were more intense in POAG samples than in ExG samples or in the control group. The only exception was the MMP-2 level, which was the highest in the control group. The staining intensity of MMP-1 or MMP-3 was significantly higher in POAG when compared to ExG. TIMP-1 was significantly increased in POAG compared with ExG and there were no marked differences in the levels of TIMP-2 or TIMP-3 between POAG and ExG. The ratios of MMP-1/TIMP-1 and MMP1+2+3+9 and TIMP1+2+3 were significantly higher in samples from POAG compared to those of ExG.ConclusionsOur results reveal an expression imbalance between MMPs and their endogenous tissue inhibitors in tissue samples from patients with POAG and ExG. Differences in immunohistochemical reactions reflect discrete local pathogenic mechanisms involved in POAG and ExG. With respect to the proposed role of MMPs in the remodeling of ECM material, this may point to a weaker reactivity to the accumulation of ECM material in TM in ExG than POAG eyes.

Nanocrystal-based per-oral itraconazole delivery: superior in vitro dissolution enhancement versus Sporanox® is not realized in in vivo drug absorption.

Nanoscience holds true promise in enabling efficient formulation development and in vivo delivery of poorly water soluble drugs. The objective of this study was to formulate solid oral nanocrystal delivery systems of itraconazole, and thus enhance the oral bioavailability of the very poorly soluble drug. Nanocrystal suspensions were prepared by a rapid wet milling technique, after which the suspensions were transformed into solid dosage forms by both freeze drying and granulating. Finally, the obtained nanocrystalline powders were capsule-packed as well as compacted to tablets. After in vitro analysis, the formulations (nanocrystal suspension (NPs), freeze dried NPs, granulated NPs) were tested in vivo in a rat model, and compared with commercial itraconazole formulation (Sporanox). Importantly, the results indicated rapid dissolution of the nanocrystalline itraconazole with enhanced bioavailability compared to physical mixture. Drug dissolution in vitro was immediate from NPs and freeze dried powder, and differed significantly from the marketed product (P=0.004 and 0.002, correspondingly) until 30min. Freeze drying was detected to be especially advantageous for the solid dosage forms. It is possible to maintain the original character of the nanocrystals, e.g. rapid dissolution, even after tableting of the nanocrystalline powders. Interestingly, the marketed product out-performed the nanocrystalline formulations in vivo, even though the nanocrystals provided reasonable bioavailability of itraconazole absorption as well. The efficient in vitro dissolution enhancement of the nanocrystalline formulations compared to Sporanox® was not realized in in vivo drug absorption.

Brinzolamide nanocrystal formulations for ophthalmic delivery: reduction of elevated intraocular pressure in vivo.

Nanocrystal-based drug delivery systems provide important tools for ocular formulation development, especially when considering poorly soluble drugs. The objective of the study was to formulate ophthalmic, intraocular pressure (IOP) reducing, nanocrystal suspensions from a poorly soluble drug, brinzolamide (BRA), using a rapid wet milling technique, and to investigate their IOP reducing effect in vivo. Different stabilizers for the nanocrystals were screened (hydroxypropyl methylcellulose (HPMC), poloxamer F127 and F68, polysorbate 80) and HPMC was found to be the only successful stabilizer. In order to investigate both the effect of an added absorption enhancer (polysorbate 80) and the impact of the free drug in the nanocrystal suspension, formulations in phosphate buffered saline (PBS) at pH 7.4 and pH 4.5 were prepared. Particle size, polydispersity (PI), solid state (DSC), morphology (SEM) as well as dissolution behavior and the uniformity of the formulations were characterized. There was rapid dissolution of BRA (in PBS pH 7.4) from all the nanocrystal formulations; after 1 min 100% of the drug was fully dissolved. The effect was significantly pronounced at pH 4.5, where the dissolved fraction of drug was the highest. The cytotoxicity of nanocrystal formulations to human corneal epithelial cell (HCE-T) viability was tested. The effects of the nanocrystal formulations and the commercial product on the cell viability were comparable. The intraocular pressure (IOP) lowering effect was investigated in vivo using a modern rat ocular hypertensive model and elevated IOP reduction was seen in vivo with all the formulations. Notably, the reduction achieved in experimentally elevated IOP was comparable to that obtained with a marketed product. In conclusion, various BRA nanocrystal formulations, which all showed advantageous dissolution and absorption behavior, were successfully formulated.

Neural retina limits the nonviral gene transfer to retinal pigment epithelium in an in vitro bovine eye model

We investigated the permeation of liposomal and polymeric gene delivery systems through neural retina into retinal pigment epithelium (RPE) and determined the roles of various factors in permeation and subsequent uptake of the delivery systems by RPE. Anterior parts and vitreous of fresh bovine eyes were removed. Retina was left intact or peeled away. Complexes of ethidium monoazide (EMA)-labeled plasmid DNA and cationic carriers (polyethyleneimine, poly-L-lysine, DOTAP liposomes) were pipetted on the retina or RPE. Two hours later the neural retina was removed, if present, and the RPE cells were detached. Contaminants were removed by sucrose centrifugation, and the RPE cells were analyzed for DNA uptake by flow cytometry. Cellular uptake of FITC-dextrans (molecular weight [mw] 20 000, 500 000 and 2 000 000), FITC-poly-L-lysine (mw 20 000), FITC-labeled oligonucleotide (15-mer), and naked EMA-labeled plasmid DNA was determined after pipetting the solutions on the RPE or neural retina. Location of the fluorescent materials in the retina was visualized with fluorescence microscopy. Neural retina decreased the cellular uptake of DNA complexes by an order of magnitude, the uptake of FITC-dextrans slightly, whereas delivery of polycationic FITC-poly-L-lysine to RPE was almost completely inhibibited. Neural retina decreased the cellular uptake of FITC-oligonucleotides, while the uptake of uncomplexed plasmid was always negligible. conclusions from FACS and fluorescence microscopy were similar: delivery of polymeric and liposomal DNA complexes into RPE are limited by the neural retina. This is due to the size and positive charge of the complexes.

The role of cell cycle on polyplex-mediated gene transfer into a retinal pigment epithelial cell line

Retinal pigment epithelium (RPE) maintains the function of photoreceptors and eyesight and is an important target for gene delivery. Since in some diseases RPE cells proliferate uncontrollably, we investigated the role of cell cycle in non‐viral gene delivery into a RPE cell line (D407).

Long-Lasting Secretion of Transgene Product from Differentiated and Filter-Grown Retinal Pigment Epithelial Cells After Nonviral Gene Transfer

Purpose: The purpose of this study was to investigate the extent, duration, and direction of transgene expression after nonviral gene transfer to differentiated retinal pigment epithelial (RPE) cells. Methods: Polarized human RPE cells (ARPE-19) were transfected with nonviral vectors [DOTAP/DOPE with and without protamine sulfate (PS), DOTAP, PEI (polyethyleneimine), DHP-12] using secreted alkaline phosphatase (SEAP) as a reporter gene. Cellular uptake was studied by flow cytometry. Results: Up to 80-fold differences were observed in the peak reporter gene expression. The highest peak levels and the longest lifetime of SEAP expression (> 69 days) were obtained with DOTAP/DOPE/PS/pDNA complexes. With PEI, higher expression was seen to the apical side than to the basolateral side. Conclusions: In contrast to most differentiated epithelial cells, the differentiated RPE cells can be transfected at high and prolonged levels with selected lipoplexes.

Synthesis of Cellulose Nanocrystals Carrying Tyrosine Sulfate Mimetic Ligands and Inhibition of Alphavirus Infection

We present two facile approaches for introducing multivalent displays of tyrosine sulfate mimetic ligands on the surface of cellulose nanocrystals (CNCs) for application as viral inhibitors. We tested the efficacy of cellulose nanocrystals, prepared either from cotton fibers or Whatman filter paper, to inhibit alphavirus infectivity in Vero (B) cells. Cellulose nanocrystals were produced by sulfuric acid hydrolysis leading to nanocrystal surfaces decorated with anionic sulfate groups. When the fluorescent marker expressing Semliki Forest virus vector, VA7-EGFP, was incubated with CNCs, strong inhibition of virus infectivity was achieved, up to 100 and 88% for cotton and Whatman CNCs, respectively. When surface sulfate groups of CNCs were exchanged for tyrosine sulfate mimetic groups (i.e. phenyl sulfonates), improved viral inhibition was attained. Our observations suggest that the conjugation of target-specific functionalities to CNC surfaces provides a means to control their antiviral activity. Multivalent CNCs did not cause observable in vitro cytotoxicity to Vero (B) cells or human corneal epithelial (HCE-T) cells, even within the 100% virus-inhibitory concentrations. Based on the similar chemistry of known polyanionic inhibitors, our results suggest the potential application of CNCs as inhibitors of other viruses, such as human immunodeficiency virus (HIV) and herpes simplex viruses.

Extracellular and intracellular factors influencing gene transfection mediated by 1,4-dihydropyridine amphiphiles

Double-charged 1,4-dihydropyridine (1,4-DHP) amphiphiles have been shown to condense DNA and efficiently transfect it into cells in vitro [Hyvönen et al., Biochim. Biophys. Acta 1509 (2000) 451]. Alkyl chain length and buffering capacity at endosomal pH range (5.0-7.4) affected complexation and transfection activity. In this study we examined how those chemical modifications of amphiphile-DNA complexes (amphiplexes) affect their interactions with extracellular polyanions (glycosaminoglycans, albumin) and lipid bilayers, their cellular uptake and intracellular distribution. To evaluate cellular uptake, CV1-P cells were incubated with labeled DNA-amphiphile complexes and analyzed by flow cytometry. Confocal laser fluorescence microscopy was used to investigate the intracellular distribution of amphiplexes. The results showed that biophysical properties of compounds can be changed by slight structural modifications. These factors determine the intracellular kinetics and transfection efficacy of the compounds. Some extracellular glycosaminoglycans and serum interfere with 1,4-DHP-amphiphile-mediated transfection by destabilizing the amphiplexes. Neither high cellular uptake, membrane destabilizing activity nor buffering capacity alone is adequate for high transfection efficacy. The activity results from complex interplay of various factors that determine intracellular kinetics and, consequently, transfection.

Early Retinal Function Deficit without Prominent Morphological Changes in the R6/2 Mouse Model of Huntington’s Disease

Huntington’s disease (HD) is an inherited neurodegenerative disorder that primarily affects the medium-size GABAergic neurons of striatum. The R6/2 mouse line is one of the most widely used animal models of HD. Previously the hallmarks of HD-related pathology have been detected in photoreceptors and interneurons of R6/2 mouse retina. Here we aimed to explore the survival of retinal ganglion cells (RGCs) and functional integrity of distinct retinal cell populations in R6/2 mice. The pattern electroretinography (PERG) signal was lost at the age of 8 weeks in R6/2 mice in contrast to the situation in wild-type (WT) littermates. This defect may be attributable to a major reduction in photopic ERG responses in R6/2 mice which was more evident in b- than a-wave amplitudes. At the age of 4 weeks R6/2 mice had predominantly the soluble form of mutant huntingtin protein (mHtt) in the RGC layer cells, whereas the aggregated form of mHtt was found in the majority of those cells from the 12-week-old R6/2 mice and onwards. Retinal astrocytes did not contain mHtt deposits. The total numbers of RGC layer cells, retinal astrocytes as well as optic nerve axons did not differ between 18-week-old R6/2 mice and their WT controls. Our data indicate that mHtt deposition does not cause RGC degeneration or retinal astrocyte loss in R6/2 mice even at a late stage of HD-related pathology. However, due to functional deficits in the rod- and cone-pathways, the R6/2 mice suffer progressive deficits in visual capabilities starting as early as 4 weeks; at 8 weeks there is severe impairment. This should be taken into account in any behavioral testing conducted in R6/2 mice.

The Effects of 5-Fluorouracil on Ocular Tissues In Vitro and In Vivo after Controlled Release from a Multifunctional Implant

PURPOSE To evaluate the effects of 5-fluorouracil (5-FU) on ocular cells in vitro and the effects of degradable 5-FU-loaded poly(DL-lactide-co-glycolide; PDLGA) 50:50 implant in the rabbit eye in vivo. METHODS Cytotoxicity was assessed with a tetrazolium salt WST-1 cell proliferation/viability test and a lactate dehydrogenase (LDH) leakage test in rabbit corneal stromal fibroblasts (SIRCs), bovine corneal endothelial cells (BCECs), human conjunctival epithelial cells (IOBA-NHCs), human retinal pigment epithelial cells (ARPE-19), and human corneal epithelial cells (HCECs). The 5-FU-loaded PDLGA implants were surgically placed in rabbit eyes with a deep sclerectomy technique and the histopathology of the eyes was examined. RESULTS In vitro, 5-FU affected cell proliferation and survival in a time- and dose-dependent manner. In the WST-1 test, adverse effects in serum-free conditions started from 0.0005 mg/mL 5-FU in SIRCS and HCECs, whereas in other cell types, 0.005 mg/mL 5-FU hindered cell proliferation. In serum-free conditions 72-hour 5 mg/mL 5-FU treatment decreased cell viability to 40% in BCECs and to 10% to 15% in other cell types. 5-FU had no or very minor effects on LDH leakage. In vivo, the 5-FU implant showed no signs of toxicity in cornea and retina, whereas in the conjunctival stroma near the implantation site, some inflammatory cells and a marked subepithelial condensation of stromal connective tissue was observed during the postoperative period of 4 weeks. CONCLUSIONS 5-FU had a broad therapeutic range, and the 5-FU implant showed only minor tissue reactions in conjunctiva at the surgical site. 5-FU is a possible candidate for controlled drug release.