Comparison and characterisation of ribozyme delivery in cultured glial and neuronal cells in vivo and in vitro

Ribozymes are short strands of RNA that possess a huge potential as biological tools for studying gene expression and as therapeutic agents to down-regulate undesirable gene expression. Successful application of ribozymes requires delivery to the target site in sufficient amounts for an adequate duration. However, due to their large size and polyanionic character ribozymes are not amenable to transport across biological membranes. In this study a chemically modified ribozyme with enhanced biological stability, targeted against the EGFR mRNA has been evaluated for cellular delivery to cultured glial and neuronal cells with a view to developing treatments for brain tumours. Cellular delivery of free ribozyme was characterised in cultured glial and neuronal cells from the human and rat. Delivery was very limited and time dependent with no consistent difference observed between glial and neuronal cells in both species. Cellular association was largely temperature and energy-dependent with a small component of non-energy dependent association. Further studies showed that ribozyme cellular association was inhibited with self and cross competition with nucleic and non-nucleic acid polyanions indicating the presence of cell surface ribozyme-binding molecules. Trypsin washing experiments further implied that the ribozyme binding surface molecules were protein by nature. Dependence of cellular association on pH indicated that interaction of ribozyme with cell surface molecules was based on ionic interactions. Fluoresence studies indicated that, post cell association, ribozymes were sequestered in sub-cellular vesicles. South-Western blots identified several cell surface proteins which bind to ribozymes and could facilitate cellular association. The limited cellular association observed with free ribozyme required the development and evaluation of polylactide-co-glycolide microspheres incorporating ribozyme for enhanced cellular delivery. Characterisation of microsphere mediated delivery of ribozyme in cultured glial and neuronal cells showed that association increased by 18 to 27-fold in all cell types with no differences observed between cell lines and species. Microsphere mediated delivery was temperature and energy dependent and independent of pH. In order to assess the potential of PLGA micro spheres for the CNS delivery of ribozyme the distribution of ribozyme entrapping microspheres was investigated in rat CNS after intracerebroventricular injection. Distribution studies demonstrated that after 24 hours there was no free ribozyme present in the brain parenchyma, however microsphere entrapped ribozyme was found in the CNS. Microspheres remained in the ventricular system after deposition and passed from the lateral ventricles to the third and fourth ventricle and in the subarachnoid space. Investigation of the influence of microsphere size on the distribution in CNS demonstrated that particles up to 2.5 and O.5f.lm remained in the ventricles around the choroid plexus and ependymal lining.

Spontanoeusly resolved macroaneurysm associated with a congenital anomalous retinal artery

Purpose: To report the outcome of retinal artery macroaneurysm associated with a congenital anomalous retinal artery. Methods: The patient was examined with ophthalmoscopy and fluorescein angiography. Results: A 60-year-old woman presented with visual acuity of 20/80 in her right eye. She was found to have an abnormal retinal vessel emerging from the optic disk, passing toward the center of the macula, and looping back toward the disk. The center of the loop had a leaking macroaneurysm, which was treated conservatively. At the 4-month follow-up visit, the visual acuity in the right eye had improved to 20/20 with resolution of most of the macular edema, exudates, and hemorrhage. Conclusion: This case demonstrates that retinal artery macroaneurysm can be successfully managed with a conservative approach, even when associated with a congenital anomalous retinal vessel.

The SECURE study:long-term safety of Ranibizumab 0.5 mg in neovascular age-related macular degeneration

Objective - To evaluate long-term safety of intravitreal ranibizumab 0.5-mg injections in neovascular age-related macular degeneration (nAMD). Design - Twenty-four–month, open-label, multicenter, phase IV extension study. Participants - Two hundred thirty-four patients previously treated with ranibizumab for 12 months in the EXCITE/SUSTAIN study. Methods - Ranibizumab 0.5 mg administered at the investigator's discretion as per the European summary of product characteristics 2007 (SmPC, i.e., ranibizumab was administered if a patient experienced a best-corrected visual acuity [BCVA] loss of >5 Early Treatment Diabetic Retinopathy Study letters measured against the highest visual acuity [VA] value obtained in SECURE or previous studies [EXCITE and SUSTAIN], attributable to the presence or progression of active nAMD in the investigator's opinion). Main Outcome Measures - Incidence of ocular or nonocular adverse events (AEs) and serious AEs, mean change in BCVA from baseline over time, and the number of injections. Results - Of 234 enrolled patients, 210 (89.7%) completed the study. Patients received 6.1 (mean) ranibizumab injections over 24 months. Approximately 42% of patients had 7 or more visits at which ranibizumab was not administered, although they had experienced a VA loss of more than 5 letters, indicating either an undertreatment or that factors other than VA loss were considered for retreatment decision by the investigator. The most frequent ocular AEs (study eye) were retinal hemorrhage (12.8%; 1 event related to study drug), cataract (11.5%; 1 event related to treatment procedure), and increased intraocular pressure (6.4%; 1 event related to study drug). Cataract reported as serious due to hospitalization for cataract surgery occurred in 2.6% of patients; none was suspected to be related to study drug or procedure. Main nonocular AEs were hypertension and nasopharyngitis (9.0% each). Arterial thromboembolic events were reported in 5.6% of the patients. Five (2.1%) deaths occurred during the study, none related to the study drug or procedure. At month 24, mean BCVA declined by 4.3 letters from the SECURE baseline. Conclusions - The SECURE study showed that ranibizumab administered as per a VA-guided flexible dosing regimen recommended in the European ranibizumab SmPC at the investigator's discretion was well tolerated over 2 years. No new safety signals were identified in patients who received ranibizumab for a total of 3 years. On average, patients lost BCVA from the SECURE study baseline, which may be the result of disease progression or possible undertreatment.