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.2018 Mar 27;115(13):E2997-E3006.
doi: 10.1073/pnas.1718792115. Epub 2018 Mar 12.

Combination of cGMP analogue and drug delivery system provides functional protection in hereditary retinal degeneration

Affiliations

Combination of cGMP analogue and drug delivery system provides functional protection in hereditary retinal degeneration

Eleonora Vighi et al. Proc Natl Acad Sci U S A..

Abstract

Inherited retinal degeneration (RD) is a devastating and currently untreatable neurodegenerative condition that leads to loss of photoreceptor cells and blindness. The vast genetic heterogeneity of RD, the lack of "druggable" targets, and the access-limiting blood-retinal barrier (BRB) present major hurdles toward effective therapy development. Here, we address these challenges (i) by targeting cGMP (cyclic guanosine- 3',5'-monophosphate) signaling, a disease driver common to different types of RD, and (ii) by combining inhibitory cGMP analogs with a nanosized liposomal drug delivery system designed to facilitate transport across the BRB. Based on a screen of several cGMP analogs we identified an inhibitory cGMP analog that interferes with activation of photoreceptor cell death pathways. Moreover, we found liposomal encapsulation of the analog to achieve efficient drug targeting to the neuroretina. This pharmacological treatment markedly preserved in vivo retinal function and counteracted photoreceptor degeneration in three different in vivo RD models. Taken together, we show that a defined class of compounds for RD treatment in combination with an innovative drug delivery method may enable a single type of treatment to address genetically divergent RD-type diseases.

Keywords: CNG channel; PKG; apoptosis; calpain; in vivo imaging.

Copyright © 2018 the Author(s). Published by PNAS.

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Conflict of interest statement

Conflict of interest statement: A. Rentsch, H.-G.G., P.G., V.M., P.E., and F.P.-D. have filed for three patents on the synthesis and use of cGMP analogues (PCTWO2016/146669A1, PCT/EP2017/066113, and PCT/EP2017/071859) and have obtained a European Medicine Agency orphan drug designation for the use of CN03 for the treatment of retinitis pigmentosa (EU/3/15/1462). H.-G.G., P.G., V.M., P.E., and F.P.-D. are shareholders of, or have other financial interest in, the company Mireca Medicines, which intends to forward clinical testing of CN03.

Figures

Fig. 1.
Fig. 1.
Structures of mono- and dimeric cGMP analogs and flow chart for biological activity testing. (A) Illustration of monomers is reduced to the modifications introduced. R1 + R2 (yellow highlight) include the PET modifications, R3 (green) refers to the 8 position (C-8) of cGMP, and a sulfur atom at R4 (red) establishes theRP-configurated phosphorothioate. (B) Stepwise compound screening in systems of increasing biological complexity. At each step compounds were filtered out based on photoreceptor-specific readouts, with only CN03 and its liposomal formulation (LP-CN03) reaching the in vivo stage.
Fig. 2.
Fig. 2.
Screening of cGMP analogs in vitro. cGMP analogs were tested in retinal neurosphere-derived rod photoreceptor-like cell cultures (A–E) and organotypic retinal explant cultures (F andG). NT, not treated. (A)rd1 mutant photoreceptor-like cell cultures treated with 50 µM of the respective compound until DIV11 (red, ethidium homodimer staining; blue, nuclear counter staining). (B) Percentage of ethidium homodimer-labeled, dying photoreceptor-like cells [green, WT; red,rd1; yellow, different analog treatments;n = 5–62 cell culture samples obtained from two to seven independent preparations; red asterisks indicate toxic effect (i.e., more dying cells compared with NT)]. (C andD) Dose–response curves for CN02–CN07 starting at attomolar concentration. (E) EC50 values for CN02–CN07 calculated from dose–response curves. (F) TUNEL assay (red) on vertical sections from P11 retinal cultures NT or treated with 50 µM of the respective compound. (G) Ratio (treated/NT) of TUNEL-labeled cells (same color code as inB, WT,n = 9 separate retinal explant cultures;rd1: NT, 22; CN02, 8; CN03, 8; CN04, 8; CN05, 6; CN06, 4; CN07, 8). Error bars: SEM. Statistical comparisons:B andG, NT-rd1 vs. treatedrd1 using one-way ANOVA with Holm–Šidak’s multiple-comparisons test; ONL, outer nuclear layer containing rod and cone photoreceptor nuclei. (Scale bars:A, 100 µm;F, 50 µm.) *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.
Fig. 3.
Fig. 3.
Effects of cGMP analogs on cone photoreceptor Ca2+ dynamics. (A) Vertical section of a mouse retina, expressing the TN-XL Ca2+ biosensor (below: magnification of cone terminals, indicated by box in top image). (Scale bar: 20 µm.) (B) Representative Ca2+ responses to light flashes before (green) and during application of 50 µM of the compound indicated below (yellow). (C) Compound-induced change in Ca2+ baseline (Rbase) measured inn = 23 cones in slices obtained from three animals (23/3) for CN02 (CN03, 18/3; CN04, 15/2; CN05, 11/2). Wash shown in gray. (D andE) Compound-induced changes in area under the curve, with negative values indicating larger response size (D), and peak height, with negative values indicating higher peaks (E; arbitrary units, a.u.). Statistical comparisons:C–E, not-treated WT vs. treated WT using Wilcoxon matched pairs, signed rank test. ΔR, amplitude; GCL, ganglion cell layer; INL, inner nuclear layer containing interneurons; IS, inner segments containing organelles of photoreceptors; ONL, outer nuclear layer containing rod and cone photoreceptor nuclei; OPL, outer plexiform layer; OS, outer segments of photoreceptor cells. n.s.,P > 0.05; *P ≤ 0.05, ***P ≤ 0.001.
Fig. 4.
Fig. 4.
CN03 acts on PKG and CNGC and reduces photoreceptor death in three different in vitro RD models. (A–D) Photoreceptor-like cell cultures derived fromrd1 mice (NT, red bars) and effects of 50 µM CN03 (yellow bars) on degeneration markers (n = 9–15 cell cultures obtained from three to five independent preparations). (A) CN03 reduced the phosphorylation at serine 239 of the PKG-target VASP (pVASP, red). (B) Fluo-4, AM Ca2+ imaging (green, arrow indicates a cell with high Ca2+) indicated reduction in intracellular Ca2+. (C) Calpain protease activity assay brightly labeled condensed dying cells (arrows). Mean calpain labeling intensity was reduced after CN03 treatment. (D) AIF (red) translocation from mitochondria to nuclei (blue) and cell death, as assessed by the TUNEL assay (green), was restrained by CN03 treatment (triple-stained nuclei shown in white; see arrows). (E) CN03 (50 µM) had no adverse effects in P11 organotypic explants derived from WT (top row). In P11rd1, P19rd2, and P18rd10 explants CN03 reduced the number of dying cells in the ONL. (F) Quantification of the data fromE; ratio (treated/NT) of TUNEL-labeled cells (NT WT,n = 2 separate retinal explant cultures; CN03 WT, 4; each NT mutant, 4; each CN03 treated mutant, 8;rd1 data from Fig. 2G shown for comparison). Error bars inA–D andF: SEM. Statistical comparisons: Student’st test (unpaired, two-tailed). NT, not treated. (Scale bars:A–C, 50 µm;D, 20 µm;E, 50 µm.) *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.
Fig. 5.
Fig. 5.
CN03 liposomal formulation improves neuroprotective efficacy. (A) Electron microscopy of GSH-PEG liposomes loaded with CN03 showing a vesicle size of ∼100 nm. (B) Neuroprotective effects of CN03 (yellow; EC50 = 400 ± 5 nM) and LP-CN03 (blue; EC50 = 3.5 ± 1 nM) inrd1 mutant photoreceptor-like cell cultures (n = 9 cell cultures from three independent preparations per concentration/treatment). (C) Cell death in NTrd1 photoreceptor-like cells (red bar), empty liposome treatment (LP, orange), coadministration of LP with free CN03 (LP+CN03, 1 µM; yellow/orange checkered), and LP-CN03 (1 µM).n = 8–62 different cell cultures from two to three independent preparations. (D) PK study in adult rats comparing in vivo half-life of free CN03 (10 min; yellow) vs. LP-CN03 (24 h; blue).n = 6 rats per group. (E) In vivo imaging using SLO. P10 WT mice were injected i.p. with carboxyfluorescein-loaded liposomes (LP-CF) and analyzed at P14. Fluorescent label was present mostly in retinal vasculature. (F) Cross-sections of P14 retinae from such LP-CF–treated animals displayed fluorescent labeling of choroidal blood vessels (chor.) but also of photoreceptors in ONL. (G–I) Three different in vivo RD mouse models were treated with CN03 (yellow), LP only (orange), or LP-CN03 (blue), beginning at the onset of degeneration (P10,rd1; P14,rd2 andrd10; cf.Table S2). Photoreceptor survival was assessed on ex vivo retinal tissue sections by counting photoreceptor rows in ONL. WT (green) shown for comparison.n = 4–8 animals (one eye per animal) per time point and genotype. (G) Progression of photoreceptor degeneration inrd1 animals (red) compared withrd1 animals treated with free CN03 (yellow) and LP-CN03 (blue). (H) Progression of degeneration in NT vs. LP-CN03–treatedrd2 mice. (I) Progression of degeneration in NT (red) or LP-treated (orange) or CN03-treated (yellow) vs. LP-CN03 treated (blue)rd10 animals. Note differenty axis scales inG–I to account for different degeneration kinetics. Error bars: SEM. Statistical comparisons:C, one-way ANOVA and Newman–Keuls multiple comparison test;G–I, Student’st test (unpaired, two-tailed). INL, inner nuclear layer; NT, not treated. (Scale bars:A, 100 nm;E, 200 µm;F, 25 µm.) *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.
Fig. 6.
Fig. 6.
LP-CN03 partly preservesrd10 retinal morphology and cone photoreceptor function at P30. (A) P30 sagittal retinal cross-sections from NTrd10 (red) and LP-CN03–treated (blue)rd10 animals, with insets showing higher magnification. (B) Quantification of photoreceptor rows (P30) along the dorsal–ventral axis, from center (optic nerve = 0°) to periphery (90°). Colors as inA; empty liposome condition (LP) shown in orange. (C) Representative ERG responses (30.0 cd⋅s/m2, dark-adapted) inrd10 animals; NT (red), LP-treated (orange), and LP-CN03–treated (blue). Congenic, age-matched WT traces (green) shown for comparison. (D) Summary plots for a- and b-wave maximum amplitudes over different light stimulus intensities. (E) Rod and cone flicker ERG responses elicited by either 5-Hz scotopic flicker (0.000122 cd⋅s/m2) or 10-Hz photopic flicker (30 cd⋅s/m2). (F) Comparison of Fourier-transformed scotopic- and photopic-flicker ERGs. Error bars: SEM. WT:n = 4 animals;rd10:n = 8–12 per condition (one eye per animal). Statistical comparisons:B, Student’st test (unpaired, two-tailed) comparing LP-CN03 vs. LP treatment;D andF, one-way ANOVA with Tukey’s honestly significant difference post hoc test; NT, not treated. (Scale bars:A, 200 µm;A,Inset, 50 µm.) *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.
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