Discovery of phenylpyrrolidine derivatives as a novel class of retinol binding protein 4 (RBP4) reducers.

Retinol-binding protein 4 (RBP4) is a potential drug target for metabolic and ophthalmologic diseases. A high-throughput screening of our compound library has identified a small-molecule RBP4 reducer 7a, as a hit compound. Aiming to provide a suitable tool for investigating the pharmacological effects of RBP4 reducers, we conducted a structure-activity relationship study of 7a. Exploration of the aryl head, oxazole core, and propanoic acid tail of 7a resulted in the discovery of novel, potent, and orally available phenylpyrrolidine derivatives 43b and 43c. Compound 43b had a potent and long-lasting blood RBP4-level-reducing effect when orally administered to mice at a dose as low as 0.3 mg/kg.

Retinol binding protein 4 antagonists and protein synthesis inhibitors: Potential for therapeutic development.

Retinol-binding protein 4 (RBP4) is a serum protein that transports Vitamin A. RBP4 is correlated with numerous diseases and metabolic syndromes, including insulin resistance in type 2 diabetes, cardiovascular diseases, obesity, and macular degeneration. Recently, RBP4 antagonists and protein synthesis inhibitors are under development to regulate the effect of RBP4. Several RBP4 antagonists, especially BPN-14136, have demonstrated promising safety profiles and potential therapeutic benefits in animal studies. Two RBP4 antagonists, specifically tinlarebant (Belite Bio) and STG-001 (Stargazer) are currently undergoing clinical trials. Some antidiabetic drugs and nutraceuticals have been reported to reduce RBP4 expression, but more clinical data is needed to evaluate their therapeutical benefits. As regulating RBP4 levels or its activities would benefit a wide range of patients, further research is highly recommended to develop clinically useful RBP4 antagonists or protein synthesis inhibitors.

Identification of Transthyretin Tetramer Kinetic Stabilizers That Are Capable of Inhibiting the Retinol-Dependent Retinol Binding Protein 4-Transthyretin Interaction: Potential Novel Therapeutics for Macular Degeneration, Transthyretin Amyloidosis, and Their Common Age-Related Comorbidities.

Dissociation of transthyretin (TTR) tetramers may lead to misfolding and aggregation of proamyloidogenic monomers, which underlies TTR amyloidosis (ATTR) pathophysiology. ATTR is a progressive disease resulting from the deposition of toxic fibrils in tissues that predominantly presents clinically as amyloid cardiomyopathy and peripheral polyneuropathy. Ligands that bind to and kinetically stabilize TTR tetramers prohibit their dissociation and may prevent ATTR onset. Drawing from clinically investigated AG10, we designed a constrained congener (14) that exhibits excellent TTR tetramer binding potency, prevents TTR aggregation in a gel-based assay, and possesses desirable pharmacokinetics in mice. Additionally, 14 significantly lowers murine serum retinol binding protein 4 (RBP4) levels despite a lack of binding at that protein's all-trans-retinol site. We hypothesize that kinetic stabilization of TTR tetramers via 14 is allosterically hindering all-trans-retinol-dependent RBP4-TTR tertiary complex formation and that the compound could present ancillary therapeutic utility for indications treated with RBP4 antagonists, such as macular degeneration.

Discovery of Bispecific Antagonists of Retinol Binding Protein 4 That Stabilize Transthyretin Tetramers: Scaffolding Hopping, Optimization, and Preclinical Pharmacological Evaluation as a Potential Therapy for Two Common Age-Related Comorbidities.

Accumulation of cytotoxic lipofuscin bisretinoids may contribute to atrophic age-related macular degeneration (AMD) pathogenesis. Retinal bisretinoid synthesis depends on the influx of serum all-trans-retinol (1) delivered via a tertiary retinol binding protein 4 (RBP4)-transthyretin (TTR)-retinol complex. We previously identified selective RBP4 antagonists that dissociate circulating RBP4-TTR-retinol complexes, reduce serum RBP4 levels, and inhibit bisretinoid synthesis in models of enhanced retinal lipofuscinogenesis. However, the release of TTR by selective RBP4 antagonists may be associated with TTR tetramer destabilization and, potentially, TTR amyloid formation. We describe herein the identification of bispecific RBP4 antagonist-TTR tetramer kinetic stabilizers. Standout analogue (±)-44 possesses suitable potency for both targets, significantly lowers mouse plasma RBP4 levels, and prevents TTR aggregation in a gel-based assay. This new class of bispecific compounds may be especially important as a therapy for dry AMD patients who have another common age-related comorbidity, senile systemic amyloidosis, a nongenetic disease associated with wild-type TTR misfolding.

Comparative pharmacokinetics and pharmacodynamics of the advanced Retinol-Binding Protein 4 antagonist in dog and cynomolgus monkey.

Accumulation of lipofuscin bisretinoids in the retina contributes to pathogenesis of macular degeneration. Retinol-Binding Protein 4 (RBP4) antagonists reduce serum retinol concentrations thus partially reducing retinol delivery to the retina which decreases bisretinoid synthesis. BPN-14136 is a novel RBP4 antagonist with good in vitro potency and selectivity and optimal rodent pharmacokinetic (PK) and pharmacodynamic (PD) characteristics. To select a non-rodent species for regulatory toxicology studies, we conducted PK and PD evaluation of BPN-14136 in dogs and non-human primates (NHP). PK properties were determined following oral and intravenous administration of BPN-14136 in beagle dogs and cynomolgus monkeys. Dynamics of plasma RBP4 reduction in response to compound administration was used as a PD marker. BPN-14136 exhibited favorable PK profile in both species. Dose-normalized exposure was significantly higher in NHP than in dog. Baseline concentrations of RBP4 were considerably lower in dog than in NHP, reflecting the atypical reliance of canids on non-RBP4 mechanisms of retinoid trafficking. Oral administration of BPN-14136 to NHP induced a strong 99% serum RBP4 reduction. Dynamics of RBP4 lowering in both species correlated with compound exposure. Despite adequate PK and PD characteristics of BPN-14136 in dog, reliance of canids on non-RBP4 mechanisms of retinoid trafficking precludes evaluation of on-target toxicities for RBP4 antagonists in this species. Strong RBP4 lowering combined with good PK attributes and high BPN-14136 exposure achieved in NHP, along with the biology of retinoid trafficking that is similar to that of humans, support the choice of NHP as a non-rodent safety species.

Design, Synthesis, and Preclinical Efficacy of Novel Nonretinoid Antagonists of Retinol-Binding Protein 4 in the Mouse Model of Hepatic Steatosis.

Retinol-binding protein 4 (RBP4) serves as a transporter for all- trans-retinol (1) in the blood, and it has been proposed to act as an adipokine. Elevated plasma levels of the protein have been linked to diabetes, obesity, cardiovascular diseases, and nonalcoholic fatty liver disease (NAFLD). Recently, adipocyte-specific overexpression of RBP4 was reported to cause hepatic steatosis in mice. We previously identified an orally bioavailable RBP4 antagonist that significantly lowered RBP4 serum levels in Abca4-/- knockout mice with concomitant normalization of complement system protein expression and reduction of bisretinoid formation within the retinal pigment epithelium. We describe herein the discovery of novel RBP4 antagonists 48 and 59, which reduce serum RBP4 levels by >80% in mice upon acute oral dosing. Furthermore, 59 demonstrated efficacy in the transgenic adi-hRBP4 murine model of hepatic steatosis, suggesting that RBP4 antagonists may also have therapeutic utility for the treatment of NAFLD.

RBP4 regulates trophoblastic cell proliferation and invasion via the PI3K/AKT signaling pathway.

Insufficient trophoblast invasion is associated with preeclampsia (PE) development. Retinol-binding protein 4 (RBP4) is important for regulating cell differentiation, migration and invasion. The aim of the present study was to determine RBP4 expression and function in the human placenta and to examine the underlying mechanisms. In the present study, RBP4 expression was determined in serum samples from 35 pregnant women with PE and 30 healthy pregnant women using enzyme-linked immunosorbent assays. Cell proliferation was assessed by Cell Counting Kit-8 assays, and cell invasion was examined with transwell assays. RBP4 concentrations were significantly lower in the PE group when compared with the control group. RBP4 overexpression enhanced HTR8/SVneo cell proliferation and invasion, and the levels of phosphorylated (p-) phosphoinositide 3-kinase (PI3K) and p-protein kinase B (AKT) in HTR8/SVneo cells. RBP4 knockdown significantly inhibited HTR8/SVneo cell proliferation and invasion, and repressed the expression of matrix metalloproteinases. In addition, RBP4 knockdown significantly reduced the levels of p-PI3K and p-AKT in HTR8/SVneo cells. Taken together, the results of the present study demonstrated that RBP4 overexpression increased HTR8/SVneo cell proliferation and invasion by suppressing PI3K/AKT signaling and RBP4 knockdown induced the opposite effects.

A non-retinoid antagonist of retinol-binding protein 4 rescues phenotype in a model of Stargardt disease without inhibiting the visual cycle.

A primary pathological defect in the heritable eye disorder Stargardt disease is excessive accumulation of cytotoxic lipofuscin bisretinoids in the retina. Age-dependent accumulation of lipofuscin in the retinal pigment epithelium (RPE) matches the age-dependent increase in the incidence of the atrophic (dry) form of age-related macular degeneration (AMD) and therefore may be one of several pathogenic factors contributing to AMD progression. Lipofuscin bisretinoid synthesis in the retina depends on the influx of serum retinol from the circulation into the RPE. Formation of the tertiary retinol-binding protein 4 (RBP4)-transthyretin-retinol complex in the serum is required for this influx. Herein, we report the pharmacological effects of the non-retinoid RBP4 antagonist, BPN-14136. BPN-14136 dosing in the Abca4-/- mouse model of increased lipofuscinogenesis significantly reduced serum RBP4 levels and inhibited bisretinoid synthesis, and this inhibition correlated with a partial reduction in visual cycle retinoids such as retinaldehydes serving as bisretinoid precursors. BPN-14136 administration at doses inducing maximal serum RBP4 reduction did not produce changes in the rate of the visual cycle, consistent with minimal changes in dark adaptation. Abca4-/- mice exhibited dysregulation of the complement system in the retina, and BPN-14136 administration normalized the retinal levels of proinflammatory complement cascade components such as complement factors D and H, C-reactive protein, and C3. We conclude that BPN-14136 has several beneficial characteristics, combining inhibition of bisretinoid synthesis and reduction in retinaldehydes with normalization of the retinal complement system. BPN-14136, or a similar compound, may be a promising drug candidate to manage Stargardt disease and dry AMD.

Transgenic Mice Over-Expressing RBP4 Have RBP4-Dependent and Light-Independent Retinal Degeneration.

Purpose: Transgenic mice overexpressing serum retinol-binding protein (RBP4-Tg) develop progressive retinal degeneration, characterized by microglia activation, yet the precise mechanisms underlying retinal degeneration are unclear. Previous studies showed RBP4-Tg mice have normal ocular retinoid levels, suggesting that degeneration is independent of the retinoid visual cycle or light exposure. The present study addresses whether retinal degeneration is light-dependent and RBP4-dependent by testing the effects of dark-rearing and pharmacological lowering of serum RBP4 levels, respectively. Methods: RBP4-Tg mice reared on normal mouse chow in normal cyclic light conditions were directly compared to RBP4-Tg mice exposed to chow supplemented with the RBP4-lowering compound A1120 or dark-rearing conditions. Quantitative retinal histological analysis was conducted to assess retinal degeneration, and electroretinography (ERG) and optokinetic tracking (OKT) tests were performed to assess retinal and visual function. Ocular retinoids and bis-retinoid A2E were quantified. Results: Dark-rearing RBP4-Tg mice effectively reduced ocular bis-retinoid A2E levels, but had no significant effect on retinal degeneration or dysfunction in RBP4-Tg mice, demonstrating that retinal degeneration is light-independent. A1120 treatment lowered serum RBP4 levels similar to wild-type mice, and prevented structural retinal degeneration. However, A1120 treatment did not prevent retinal dysfunction in RBP4-Tg mice. Moreover, RBP4-Tg mice on A1120 diet had significant worsening of OKT response and loss of cone photoreceptors compared to RBP4-Tg mice on normal chow. This may be related to the very significant reduction in retinyl ester levels in the retina of mice on A1120-supplemented diet. Conclusions: Retinal degeneration in RBP4-Tg mice is RBP4-dependent and light-independent.

Involvement of RBP4 in all­trans retinoic acid induced cleft palate.

The current study was designed to elucidate the mechanism of retinol binding protein 4 (RBP4) in cleft palate induced by all­trans retinoic acid (atRA). To establish a cleft palate model in C57BL/6J mice, pregnant mice were administered atRA (100 mg/kg) by gavage at the tenth embryonic stage (E10.0). Control groups were given the equivalent volume of corn oil. Pregnant mice were dissected at E12.5, E13.5 and E14.5 to obtain the embryonic palates. The expression levels of RBP4 in the embryonic palatal mesenchyme (EPM) were determined by immunohistochemistry, reverse transcription­quantitative polymerase chain reaction (RT­qPCR) and western blotting. Human embryonic palatal mesenchymal cells were exposed to atRA to detect the variation in RBP4 induced by atRA in vitro. Small interfering RNA was used to suppress the expression of RBP4, and a plasmid overexpressing RBP4 was used to examine upregulated expression. The cell counting kit­8 assay was used to evaluate the effect of RBP4 on cell proliferation. The expression levels of p27 and cyclin D1 were determined by RT­qPCR and western blotting, while the expression levels of extracellular signal­related kinase (ERK) 1/2 and protein kinase B (AKT) were assessed by western blotting. At E14.5, RBP4 was strongly expressed in the EPM, while it was downregulated following atRA treatment, which induced cleft palate in vivo. In vitro experiments indicated that atRA suppressed the expression of RBP4 and altered the expression of p27 and cyclin D1 to cause growth inhibition. Knockdown of RBP4 resulted in decreased expression of cyclin D1 and increased p27, and suppressed proliferation. Overexpression of RBP4 reversed the inhibitory effect of atRA and promoted proliferation via the ERK1/2 and AKT signaling pathways. These results suggested that RBP4 was involved in cleft palate induced by atRA and it can be suppressed by atRA to cause growth inhibition in the embryonic palate.

Altered retinoid signaling compromises decidualization in human endometriotic stromal cells.

Decidualization alters multiple molecular pathways in endometrium to permit successful embryo implantation. We have reported that paracrine factors, including retinoids, secreted from progesterone-treated endometrial stromal cells, act on nearby epithelial cells to induce the estradiol metabolizing enzyme HSD17B2. This same induction is not seen in endometriotic stromal cells. We have also shown significant differences in retinoid uptake, metabolism and action in endometriotic tissue and stromal cells compared to normal endometrium. Here, we characterize retinoid signaling during decidualization in these cells. Endometrial and endometriotic cells were isolated, cultured and incubated and decidualized. Genes involved in retinoid metabolism and trafficking were examined using RT-PCR and Western blotting. Prolactin, a decidualization marker, was also examined. We found that both endometrial and endometriotic stromal cells express all intracellular proteins involved in retinoid uptake and metabolism. Decidualization significantly reduced the expression of the genes responsible for retinoid uptake and shuttling to the nucleus. However, expression of CRBP1, an intracellular carrier protein for retinol, increased, as did RBP4, a carrier protein for retinol in the blood, which can function in a paracrine manner. Secreted RBP4 was detected in the media from decidualized endometrial cells but not from endometriotic cells. We believe that retinoid trafficking in endometrial stromal cells during decidualization may shift to favor paracrine rather than intracrine signaling, which may enhance signaling to the adjacent epithelium. There is blunting of this signaling in endometriotic cells. These alterations in retinoid signaling may help explain the decidualization defects and deficient estradiol inactivation (via HSD17B2) seen in endometriosis.

Aptamer-Conjugated Calcium Phosphate Nanoparticles for Reducing Diabetes Risk via Retinol Binding Protein 4 Inhibition.

OBJECTIVES: Inhibition of the binding of retinol to its carrier, retinol binding protein 4, is a new strategy for treating type 2 diabetes; for this purpose, we have provided an aptamer-functionalized multishell calcium phosphate nanoparticle. METHODS: First, calcium phosphate nanoparticles were synthesized and conjugated to the aptamer. The cytotoxicity of nanoparticles releases the process of aptamer from nanoparticles and their inhibition function of binding retinol to retinol binding protein 4. RESULTS: After synthesizing and characterizing the multishell calcium phosphate nanoparticles and observing the noncytotoxicity of conjugate, the optimum time (48 hours) and the pH (7.4) for releasing the aptamer from the nanoparticles was determined. The half-maximum inhibitory concentration (IC50) value for inhibition of retinol binding to retinol binding protein 4 was 210 femtomolar (fmol). CONCLUSIONS: The results revealed that the aptamer could prevent connection between retinol and retinol binding protein 4 at a very low IC50 value (210 fmol) compared to other reported inhibitors. It seems that this aptamer could be used as an efficient candidate not only for decreasing the insulin resistance in type 2 diabetes, but also for inhibiting the other retinol binding protein 4-related diseases.

Sitagliptin down-regulates retinol-binding protein 4 and reduces insulin resistance in gestational diabetes mellitus: a randomized and double-blind trial.

Gestational diabetes mellitus (GDM) is a condition that affects increasing number of pregnant women worldwide. Sitagliptin was reported to alleviate symptoms of type 2 diabetes mellitus by reducing serum levels of retinol-binding protein 4 (RBP-4). We investigated the effectiveness of sitagliptin on insulin sensitivity parameters in GDM patients. Pregnant GDM women in the 2nd trimester were recruited for this study. Participants were then assigned randomly to sitagliptin treatment group or placebo treatment group, and administered sitagliptin or placebo daily for 16 weeks. Glucose and insulin profiles, as well as serum RBP-4 level, were measured at both baseline and end of the study. After 16 weeks of treatment, participants in the STL group exhibited significantly improved levels of fasting plasma glucose and serum insulin, homeostasis model of assessment of ß cell function (HOMA-ß) and insulin resistance (HOMA-IR), compared with those in the placebo group. Serum levels of RBP-4 were also markedly decreased in the sitagliptin treatment group, and more importantly it was positively correlated with improved insulin resistance parameters. Our study supports a potentially promising role of sitagliptin in improving insulin resistance by decreasing RBP-4 in GDM-affected women.

Bicyclic [3.3.0]-Octahydrocyclopenta[c]pyrrolo Antagonists of Retinol Binding Protein 4: Potential Treatment of Atrophic Age-Related Macular Degeneration and Stargardt Disease.

Antagonists of retinol-binding protein 4 (RBP4) impede ocular uptake of serum all-trans retinol (1) and have been shown to reduce cytotoxic bisretinoid formation in the retinal pigment epithelium (RPE), which is associated with the pathogenesis of both dry age-related macular degeneration (AMD) and Stargardt disease. Thus, these agents show promise as a potential pharmacotherapy by which to stem further neurodegeneration and concomitant vision loss associated with geographic atrophy of the macula. We previously disclosed the discovery of a novel series of nonretinoid RBP4 antagonists, represented by bicyclic [3.3.0]-octahydrocyclopenta[c]pyrrolo analogue 4. We describe herein the utilization of a pyrimidine-4-carboxylic acid fragment as a suitable isostere for the anthranilic acid appendage of 4, which led to the discovery of standout antagonist 33. Analogue 33 possesses exquisite in vitro RBP4 binding affinity and favorable drug-like characteristics and was found to reduce circulating plasma RBP4 levels in vivo in a robust manner (>90%).

Design, synthesis, and evaluation of nonretinoid retinol binding protein 4 antagonists for the potential treatment of atrophic age-related macular degeneration and Stargardt disease.

Accumulation of lipofuscin in the retina is associated with pathogenesis of atrophic age-related macular degeneration and Stargardt disease. Lipofuscin bisretinoids (exemplified by N-retinylidene-N-retinylethanolamine) seem to mediate lipofuscin toxicity. Synthesis of lipofuscin bisretinoids depends on the influx of retinol from serum to the retina. Compounds antagonizing the retinol-dependent interaction of retinol-binding protein 4 (RBP4) with transthyretin in the serum would reduce serum RBP4 and retinol and inhibit bisretinoid formation. We recently showed that A1120 (3), a potent carboxylic acid based RBP4 antagonist, can significantly reduce lipofuscin bisretinoid formation in the retinas of Abca4(-/-) mice. As part of the NIH Blueprint Neurotherapeutics Network project we undertook the in vitro exploration to identify novel conformationally flexible and constrained RBP4 antagonists with improved potency and metabolic stability. We also demonstrate that upon acute and chronic dosing in rats, 43, a potent cyclopentyl fused pyrrolidine antagonist, reduced circulating plasma RBP4 protein levels by approximately 60%.

Structure-assisted discovery of the first non-retinoid ligands for Retinol-Binding Protein 4.

Retinol-Binding Protein 4 (RBP4) is a plasma protein that transports retinol (vitamin A) from the liver to peripheral tissues. This Letter highlights our efforts in discovering the first, to our knowledge, non-retinoid small molecules that bind to RBP4 at the retinol site and reduce serum RBP4 levels in mice, by disrupting the interaction between RBP4 and transthyretin (TTR), a plasma protein that binds RBP4 and protects it from renal excretion. Potent compounds were discovered and optimized quickly from high-throughput screen (HTS) hits utilizing a structure-based approach. Inhibitor co-crystal X-ray structures revealed unique disruptions of RBP4-TTR interactions by our compounds through induced loop conformational changes instead of steric hindrance exemplified by fenretinide. When administered to mice, A1120, a representative compound in the series, showed concentration-dependent retinol and RBP4 lowering.

CRF type 2 receptors mediate the metabolic effects of ghrelin in C2C12 cells.

OBJECTIVE: Ghrelin is known to regulate appetite control and cellular metabolism. The corticotropin-releasing factor (CRF) family is also known to regulate energy balance. In this study, the links between ghrelin and the CRF family in C2C12 cells, a mouse myoblast cell line was investigated. DESIGN AND METHODS: C2C12 cells were treated with ghrelin in the presence or absence of CRF receptor antagonists and then subjected to different metabolic analyses. RESULTS: Ghrelin enhanced glucose uptake by C2C12 cells, induced GLUT4 translocation to the cell surface and decreased RBP4 expression. A CRF-R2 selective antagonist, anti-sauvagine-30, blocked ghrelin-induced glucose uptake, Ghrelin upregulated CRF-R2 but not CRF-R1 levels. Moreover, ghrelin-treated C2C12 cells displayed a cAMP and pERK activation in response to Ucn3, a CRF-R2 specific ligand, but not in response to CRF or stressin, CRF-R1 specific ligands. Ghrelin also induced UCP2 and UCP3 expression, which were blocked by anti- sauvagine-30. Ghrelin did not induce fatty acids uptake by C2C12 cells or ACC expression. Even though C2C12 cells clearly exhibited responses to ghrelin, the known ghrelin receptor, GHSR1a, was not detectable in C2C12 cells. CONCLUSION: The results suggest that, ghrelin plays a role in regulating muscle glucose and, raise the possibility that suppression of the CRF-R2 pathway might provide benefits in high ghrelin states.

A1120, a nonretinoid RBP4 antagonist, inhibits formation of cytotoxic bisretinoids in the animal model of enhanced retinal lipofuscinogenesis.

PURPOSE: Excessive accumulation of lipofuscin is associated with pathogenesis of atrophic age-related macular degeneration (AMD) and Stargardt disease. Pharmacologic inhibition of the retinol-induced interaction of retinol-binding protein 4 (RBP4) with transthyretin (TTR) in the serum may decrease the uptake of serum retinol to the retina and reduce formation of lipofuscin bisretinoids. We evaluated in vitro and in vivo properties of the new nonretinoid RBP4 antagonist, A1120. METHODS: RBP4 binding potency, ability to antagonize RBP4-TTR interaction, and compound specificity were analyzed for A1120 and for the prototypic RBP4 antagonist fenretinide. A1120 ability to inhibit RPE65-mediated isomerohydrolase activity was assessed in the RPE microsomes. The in vivo effect of A1120 administration on serum RBP4, visual cycle retinoids, lipofuscin bisretinoids, and retinal visual function was evaluated using a combination of biochemical and electrophysiologic techniques. RESULTS: In comparison to fenretinide, A1120 did not act as a RARα agonist, while exhibiting superior in vitro potency in RBP4 binding and RBP4-TTR interaction assays. A1120 did not inhibit isomerohydrolase activity in the RPE microsomes. A1120 dosing in mice induced 75% reduction in serum RBP4, which correlated with reduction in visual cycle retinoids and ocular levels of lipofuscin fluorophores. A1120 dosing did not induce changes in kinetics of dark adaptation. CONCLUSIONS: A1120 significantly reduces accumulation of lipofuscin bisretinoids in the Abca4(-/-) animal model. This activity correlates with reduction in serum RBP4 and visual cycle retinoids confirming the mechanism of action for A1120. In contrast to fenretinide, A1120 does not act as a RARα agonist indicating a more favorable safety profile for this nonretinoid compound.

Investigation of oral fenretinide for treatment of geographic atrophy in age-related macular degeneration.

BACKGROUND: Excessive accumulation of retinol-based toxins has been implicated in the pathogenesis of geographic atrophy (GA). Fenretinide, an orally available drug that reduces retinol delivery to the eye through antagonism of serum retinol-binding protein (RBP), was used in a 2-year trial to determine whether retinol reduction would be effective in the management of geographic atrophy. METHODS: The efficacy of fenretinide (100 and 300 mg daily, orally) to slow lesion growth in geographic atrophy patients was examined in a 2-year, placebo-controlled double-masked trial that enrolled 246 patients at 30 clinical sites in the United States. RESULTS: Fenretinide treatment produced dose-dependent reversible reductions in serum RBP-retinol that were associated with trends in reduced lesion growth rates. Patients in the 300 mg group who achieved serum retinol levels of ≤ 1 µM (≤ 2 mg/dL RBP) showed a mean reduction of 0.33 mm in the yearly lesion growth rate compared with subjects in the placebo group (1.70 mm/year vs. 2.03 mm/year, respectively, P = 0.1848). Retinol-binding protein reductions <2 mg/dL correlated with further reductions in lesion growth rates (r = 0.478). Fenretinide treatment also reduced the incidence of choroidal neovascularization (approximately 45% reduction in incidence rate in the combined fenretinide groups vs. placebo, P = 0.0606). This therapeutic effect was not dose dependent and is consistent with anti-angiogenic properties of fenretinide, which have been observed in other disease states. CONCLUSION: The findings of this study and the established safety profile of fenretinide in chronic dosing regimens warrant further study of fenretinide in the treatment of geographic atrophy.

Suppression of retinol-binding protein 4 with RNA oligonucleotide prevents high-fat diet-induced metabolic syndrome and non-alcoholic fatty liver disease in mice.

Conflicting data have been reported regarding the role of retinol-binding protein (RBP4) in insulin resistance, obesity, type 2 diabetes and non-alcoholic fatty liver disease (NAFLD). In this study, we used pharmacological methods to investigate the role of RBP4. RNA oligonucleotide against RBP4 (anti-RBP4 oligo) was transfected into 3T3-L1 adipocytes. RT-PCR analysis showed that RBP4 mRNA expression decreased by 55% (p<0.01) compared with control cells. Validated RNA oligo was used in an in vivo study with high fat diet (HFD) fed - mice. 14 weeks of HFD feeding increased RBP4 expression (associated with elevated serum levels measured with immunoblotting and ELISA) by 56% in adipose tissue (p<0.05) and 68% in the liver (p<0.01). Adipose RBP4 levels were significantly reduced after 4 weeks treatment with anti-RBP4 oligo (25mg/kg, p<0.01) and rosiglitazone (RSG, 10mg/kg, p<0.05) compared with scrambled RNA oligo (25mg/kg) treated mice. Only anti-RBP4 oligo significantly inhibited RBP4 protein (p<0.01) and mRNA expression (p<0.01) in the liver and reduced serum RBP4 levels. Anti-RBP4 oligo and RSG showed comparable effects on impaired glucose tolerance, hyperinsulinaemia and hyperglycaemia. Anti-RBP4 oligo significantly enhanced adipose-GLUT4 expression (p<0.01) but did not increase muscle-GLUT4. Both RSG and anti-RBP4 oligo significantly reduced hepatic phosphoenolpyruvate carboxykinase expression (both p<0.05). Histological analysis revealed that anti-RBP4 oligo ameliorated hepatic steatosis and reduced lipid droplets associated with normalized liver function. Histological and pharmacological results of this study indicate that RBP4 is not only an adipocytokine, but also a hepatic cytokine leading to metabolic syndrome, NAFLD and type 2 diabetes.