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; Shirin Tavakoli; Blanca Arango-Gonzalez; Marius Ueffing; Elisa Toropainen; Marika Ruponen; Marco G Casteleijn; Arto Urtti

doi:10.3390/pharmaceutics12070611

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

Pharmaceutics. 2020 Jun 30;12(7):611. doi: 10.3390/pharmaceutics12070611.

Authors

Jaakko Itkonen¹, Ada Annala^{2

3}, Shirin Tavakoli¹, Blanca Arango-Gonzalez⁴, Marius Ueffing⁴, Elisa Toropainen², Marika Ruponen², Marco G Casteleijn^{1

5}, Arto Urtti^{1

2

6}

Affiliations

¹ Drug Research Program, Faculty of Pharmacy, University of Helsinki, Viikinkaari 5 E, 00790 Helsinki, Finland.
² School of Pharmacy, University of Eastern Finland, Yliopistonranta 1, 70211 Kuopio, Finland.
³ Utrecht Institute for Pharmaceutical Science, Utrecht University, David de Wiedgebouw, Universiteitsweg 99, 3584 CG Utrecht, The Netherlands.
⁴ Institute for Ophthalmic Research, Centre for Ophthalmology, University of Tübingen, Elfriede-Aulhorn-Strasse 7, D-72076 Tübingen, Germany.
⁵ VTT Technical Research Centre of Finland Ltd., Solutions for Natural Resources and Environment, Tietotie 2, Espoo, P.O. Box 1000, FI-02044 VTT, Finland.
⁶ Laboratory of Biohybrid Technologies, Institute of Chemistry, St. Petersburg State University, Universitetskii pr. 26, Peterhoff, 198504 St. Petersburg, Russia.

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.

Keywords: CNTF; intravitreal delivery; neuroprotection; protein aggregation; retinal penetration; stability.

Abstract

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