Accumulation of toxic lipofuscin pigment has been implicated in pathogenesis of atrophic AMD and recessive Stargardt disease. It was suggested that formation of cytotoxic lipofuscin bisretinoids occurs in the retina in a nonenzymatic manner and can be considered a byproduct of a properly functioning visual cycle.
9,10 This model assumes that all-trans-retinaldehyde (formed in the outer segments in a light-dependent manner) serves as a direct precursor for A2E synthesis, implying that biogenesis of lipofuscin fluorophores is light-dependent. Recent studies, however, showed no significant difference in lipofuscin and A2E levels between dark- and cyclic light–reared mice,
41 indicating a more complex relationship between the visual cycle and bisretinoid synthesis. While the detailed mechanisms of bisretinoid formation in the retina may require additional elucidation, two pharmacologic classes of compounds have been shown unequivocally to reduce bisretinoid formation in the
Abca4−/− model of enhanced lipofuscinogenesis. The first class comprises direct inhibitors of key visual cycle enzymes, such as isomerohydrolyze (RPE65) and 11-cis-retinol dehydrogenase. This pharmacologic class is exemplified by isotretinoin,
12 farnesyl isoprenoids,
29 a group of retinylamine derivatives,
13,42,43 and a synthetic RPE65 antagonist ACU-4429 of undisclosed structure.
43 The second class of compounds with proven ability to inhibit bisretinoid formation comprises RBP4 antagonists capable of inhibiting retinol-stimulated RBP4-TTR interaction. Until recently, fenretinide was the only representative of this class. It was shown to induce the disruption of the tertiary retinol-RBP4-TTR complex in circulation, with subsequent lowering in serum RBP4 and retinol levels.
11,44 Additionally, it was proven experimentally that fenretinide-induced serum retinol lowering is associated with partial depletion of visual cycle retinoids and accompanied by significant reduction of bisretinoid production in the
Abca4 −/− model.
11 As fenretinide safety profile may be incompatible with chronic dosing in individuals with atrophic AMD and Stargardt disease, identification of new structural classes of RBP4 antagonists is highly important. A1120, a potent nonretinoid RBP4 antagonist, originally was developed as a potential treatment for diabetes
27 based on the observation that pharmacologic downregulation or genetic ablation of RBP4 enhance insulin sensitivity.
45 However, administration of A1120 to diet-induced obese mice did not improve insulin sensitivity,
27 while re-evaluation of
Rbp4 −/− phenotype resulted in a significantly diminished support for RBP4 as a diabetes drug target.
27 Even though A1120 is unlikely to become a diabetes treatment, it may provide a starting point for developing a novel structural class of drugs for atrophic AMD and Stargardt disease. In our study, we conducted a head-to-head comparison of A1120 and fenretinide in a battery of in vitro assays. In competitive binding experiments with RBP4 (
Fig. 1) we established that A1120 that demonstrates IC
50 of 14.8 nM is approximately 4 times more potent than fenretinide (IC
50 = 56 nM). To compare the ability of A1120 and fenretinide to antagonize retinol-dependent RBP4-TTR interaction, we conducted compound titrations in the TR-FRET RBP4-TTR interaction assay in the presence of 1 μM retinol. As shown in
Figure 3, both compounds are capable of antagonizing retinol-dependent RBP4-TTR interaction. However, under our assay conditions A1120 behaves as a much more potent antagonist (IC
50 = 155 nM), while significantly higher concentrations of fenretinide are required to antagonize retinol-induced RBP4-TTR interaction (IC
50 = 4.5 μM). As it has been suggested that fenretinide's adverse effects may be mediated by its action as a ligand of a nuclear receptor RAR,
18,23,35,36 we tested fenretinide along with A1120 in two assays capable of detecting RARα agonistic activity. We established an in vitro TR-FRET assay for agonist-induced RARα interaction with a synthetic fragment of the transcriptional co-activator SRC1. We showed that A1120 does not promote RARα interaction with a SRC1 fragment, STC2-2 peptide, in this assay, which indicates the lack of RARα agonistic activity for A1120 (
Fig. 4B). To the contrary, fenretinide was capable of inducing RARα-SRC2-2 interaction, confirming that at higher concentrations it can act as an RARα agonist (
Fig. 4B). To assess RARα agonistic activity of test compounds in a more physiologic cellular context, we established a mammalian two-hybrid assay that probes agonist-sensitive apo-RARα interaction with transcriptional co-repressor NCoR. We showed that fenretinide was able to inhibit RARα-NCoR interaction in a concentration-dependent manner (
Fig. 4D) confirming its ability to act as an RAR agonist. Consistent with the lack of RARα agonistic activity, A1120 did not affect RARα-NCoR interaction (
Fig. 4D). The experiments conducted in two assay formats established clearly that A1120 cannot act as an RARα agonist, indicating that this compound is unlikely to exhibit RAR-mediated adverse effects, which are typical for fenretinide. Attempting to establish the ability of A1120 to inhibit formation of cytotoxic lipofuscin fluorophores (A2E, A2-DHP-PE, and all-trans retinol dimer-PE) in the retina, we administered the compound at the daily 30 mg/kg dose to
Abca4 −/− mice for a period of 6 weeks. As shown in
Figure 6C, administration of A1120 reduces the production of A2E, A2-DHP-PE, and atRAL di-PE in A1120-treated
Abca4 −/− mice compared to the vehicle-treated
Abca4 −/− animals by approximately 50%. This result demonstrated clearly that A1120 can inhibit in vivo accumulation of lipofuscin bisretinoids in the animal model of enhanced lipofuscinogenesis. Analysis of blood samples collected from the treatment and control groups at different time points revealed 64% decrease in serum RBP4 level at day 21 of compound dosing, while a 75% decrease of serum RBP4 in the treatment group was accomplished by day 42 of A1120 administration (
Fig. 5). To establish a mechanistic link between serum RBP4 reduction and inhibition of bisretinoid formation, we studied the A1120 effect on visual cycle retinoids. A 12-day A1120 administration in wild-type mice induced significant (∼30%–50%) reduction in steady-state light adapted 11-cis retinaldehyde and all-trans retinyl palmitate levels, while the effect on all-trans retinaldehyde was much less pronounced (see Table). These data proved the correlation between A1120-induced reduction in serum RBP4 and partial depletion of ocular retinyl palmitate, a storage form of vitamin A in the retina, and established the ability of A1120 to reduce 11-cis retinal significantly, which may be a direct
41 or indirect precursor of lipofuscin bisretinoids.