Characterization, Stability, and in Vivo Efficacy Studies of Recombinant Human CNTF and Its Permeation into the Neural Retina in ex Vivo Organotypic Retinal Explant Culture Models.

Jaakko Itkonen,Ada Annala,Marius Ueffing,Arto Urtti,Shirin Tavakoli,Elisa Toropainen,Marika Ruponen,Blanca Arango-Gonzalez,Marco G Casteleijn

doi:10.3390/pharmaceutics12070611

Abstract

Ciliary neurotrophic factor (CNTF) is one of the most studied neuroprotective agents with acknowledged potential in treating diseases of the posterior eye segment. Although its efficacy and mechanisms of action in the retina have been studied extensively, it is still not comprehensively understood which retinal cells mediate the therapeutic effects of CNTF. As with therapeutic proteins in general, it is poorly elucidated whether exogenous CNTF administered into the vitreous can enter and distribute into the retina and hence reach potentially responsive target cells. Here, we have characterized our purified recombinant human CNTF (rhCNTF), studied the protein’s in vitro bioactivity in a cell-based assay, and evaluated the thermodynamic and oligomeric status of the protein during storage. Biological activity of rhCNTF was further evaluated in vivo in an animal model of retinal degeneration. The retinal penetration and distribution of rhCNTF after 24 h was studied utilizing two ex vivo retina models. Based on our characterization findings, our rhCNTF is correctly folded and biologically active. Moreover, based on initial screening and subsequent follow-up, we identified two buffers in which rhCNTF retains its stability during storage. Whereas rhCNTF did not show photoreceptor preservative effect or improve the function of photoreceptors in vivo, this could possibly be due to the used disease model or the short duration of action with a single intravitreal injection of rhCNTF. On the other hand, the lack of in vivo efficacy was shown to not be due to distribution limitations; permeation into the retina was observed in both retinal explant models as in 24 h rhCNTF penetrated the inner limiting membrane, and being mostly observed in the ganglion cell layer, distributed to different layers of the neural retina. As rhCNTF can reach deeper retinal layers, in general, having direct effects on resident CNTF-responsive target cells is plausible.

Highlights

Progressive diseases of the posterior eye segment, and in particular those affecting the retina, are among the most common causes of visual impairment and blindness [1]
Fractions containing recombinant human CNTF (rhCNTF) were pooled, concentrated, and subjected to SEC purification, with rhCNTF eluting from the column as a mostly solitary major peak at 56 mL (Figure 1), corresponding to an estimated molecular weight of approximately 26 kDa and in accordance with published literature and earlier SDS-PAGE analysis [42], with void volume aggregates and smaller oligomPehrasrPmehaalcruemutateiccedsu2tai0css202d,012i0s2,t16i2n1,1c6t11minor peaks and readily discarded
Four separ1a0t1eo0fbo31fa3t1ches of rhCNTF were purified in high purity, on average yielding 70 mg of protein per liter of culture

Summary

Introduction

Progressive diseases of the posterior eye segment, and in particular those affecting the retina, are among the most common causes of visual impairment and blindness [1]. For instance, are the two leading causes of blindness globally, whereas age-related macular degeneration (AMD) is the most common culprit for legal blindness in the aged population in high-income countries, affecting approximately 10% of the population over 60 years of age [2,3]. Degenerative diseases of the posterior eye segment, including conditions such as diabetic retinopathy and retinitis pigmentosa (RP), are often age-related, and their prevalence is growing in the aging populations. Pharmaceuticals that target retinal disease pathogenesis are currently available only for the treatment of diabetic macular edema and the wet form of AMD, conditions that feature pathological angiogenesis in the retina, which is driven by the overexpression of pro-angiogenic factors [3]. VEGF-blocking as a treatment strategy is most commonly achieved with therapeutic proteins that bind and neutralize VEGF [1]

Methods

Results

Conclusion