17β-Estradiol Delivered in Eye Drops: Evidence of Impact on Protein Networks and Associated Biological Processes in the Rat Retina through Quantitative Proteomics

Abstract

To facilitate the development of broad-spectrum retina neuroprotectants that can be delivered through topical dosage forms, this proteomics study focused on analyzing target engagements through the identification of functional protein networks impacted after delivery of 17β-estradiol in eye drops. Specifically, the retinae of ovariectomized Brown Norway rats treated with daily eye drops of 17β-estradiol for three weeks were compared to those of vehicle-treated ovariectomized control animals. We searched the acquired raw data against a composite protein sequence database by using Mascot, as well as employed label-free quantification to detect changes in protein abundances. Our investigation using rigorous validation criteria revealed 331 estrogen-regulated proteins in the rat retina (158 were up-regulated, while 173 were down-regulated by 17β-estradiol delivered in eye drops). Comprehensive pathway analyses indicate that these proteins are relevant overall to nervous system development and function, tissue development, organ development, as well as visual system development and function. We also present 18 protein networks with associated canonical pathways showing the effects of treatments for the detailed analyses of target engagements regarding potential application of estrogens as topically delivered broad-spectrum retina neuroprotectants. Profound impact on crystallins is discussed as one of the plausible neuroprotective mechanisms.

Retina-Targeted Delivery of 17β-Estradiol by the Topically Applied DHED Prodrug

“…In vivo substantiation of the ex vivo and in vitro findings (Table 1 and Figure S1) was done by instilling eye drops (0.1% w/v DHED) into the eyes of OVX rats, similarly to our earlier studies [3,19], and following the formation of the parent hormone at the target site (retina) as well as in the blood. As shown in Figure 3, the topically applied prodrug produced the expected rapid formation of E2 in the retina, while this was absent in the blood as DHED remained inert in the circulation.…”

“…As shown in Table 1, approximately 10-fold higher J ss values were determined for DHED compared to those of the parent compound in all species studied, while saline vehicle (Vehicle A) was used in the donor chamber of the perfusion apparatus. When the vehicle was 20% w/v HPβCD in saline (Vehicle B) [3,19,35], permeabilities expectedly increased, as cyclodextrins are well-known permeability enhancers and solubilizers utilized to increase ocular bioavailability [39]. The increase was markedly higher for the highly lipophilic parent estrogen (5-to 10-fold) than that of its much more hydrophilic bioprecursor prodrug (2-3-fold) and, therefore, the steady-state transcorneal flux differences between DHED versus E2 decreased to 3-5-fold (Table 1).…”

“…Solutions for eye drops (0.1% w/v) in 20% w/v HPβCD in saline were prepared as reported before [3,19,35]. Animals (n = 4 per treatment group) received a single eye drop (10 µL for rats and 40 µL for rabbits) in each eye.…”

“…E2 eye drop treatment for three weeks did, however, result in a large E2 concentration in the retina and provided profound neuroprotection by preventing the deterioration of visual acuity. Through mass spectrometry-based proteomics, we have also identified several protein targets in numerous protein networks implicated in retina health upon this topical E2 treatment schedule [19]. On the other hand, treatment with eye drops containing the hormone also produced high circulating E2 level and, therefore, triggered unwanted side-effects such as adverse impact on the uterus of experimental animals [3].…”

“…The retina is part of the CNS, and it is also impaired in patients with neurodegeneration in the brain [27]. Therefore, potential retina-selective delivery of E2 by DHED is a plausible hypothesis from our previous work [10,19,22]. 10β,17β-dihydroxyestra-1,4-dien-3-one (DHED) is a bioprecursor prodrug converted to 17β-estradiol (E2) in neuronal environments through NADPH-dependent enzymatic reduction [22,23].…”