Growth modulatory effects of fenretinide encompass keratinocyte terminal differentiation: a favorable outcome for oral squamous cell carcinoma chemoprevention.

Oral squamous cell carcinoma (OSCC) is worldwide health problem associated with high morbidity and mortality. From both the patient and socioeconomic perspectives, prevention of progression of premalignant oral intraepithelial neoplasia (OIN) to OSCC is clearly the preferable outcome. Optimal OSCC chemopreventives possess a variety of attributes including high tolerability, bioavailability, efficacy and preservation of an intact surface epithelium. Terminal differentiation, which directs oral keratinocytes leave the proliferative pool to form protective cornified envelopes, preserves the protective epithelial barrier while concurrently eliminating growth-aberrant keratinocytes. This study employed human premalignant oral keratinocytes and an OSCC cell line to evaluate the differentiation-inducing capacity of the synthetic retinoid, fenretinide (4HPR). Full-thickness oral mucosal explants were evaluated for proof of concept differentiation studies. Results of this study characterize the ability of 4HPR to fulfill all requisite components for keratinocyte differentiation, i.e. nuclear import via binding to cellular RA binding protein-II (molecular modeling), binding to and subsequent activation of retinoic acid nuclear receptors (receptor activation assays), increased expression and translation of genes associated with keratinocyte differentiation [Reverse transcription polymerase chain reaction (RT-PCR), immunoblotting] upregulation of a transglutaminase enzyme essential for cornified envelope formation (transglutaminase 3, functional assay) and augmentation of terminal differentiation in human oral epithelial explants (image-analyses quantified corneocyte desquamation). These data build upon the chemoprevention repertoire of 4HPR that includes function as a small molecule kinase inhibitor and inhibition of essential mechanisms necessary for basement membrane invasion. An upcoming clinical trial, which will assess whether a 4HPR-releasing mucoadhesive patch induces histologic, clinical and molecular regression in OIN lesions, will provide essential clinical insights.

Examination of aminophenol-containing compounds designed as antiproliferative agents and potential atypical retinoids.

Retinoic acid (RA, 1), an oxidized form of vitamin A, binds to retinoic acid receptors (RAR) and retinoid X receptors (RXR) to regulate gene expression and has important functions such as cell proliferation and differentiation. Synthetic ligands regarding RAR and RXR have been devised for the treatment of various diseases, particularly promyelocytic leukemia, but their side effects have led to the development of new, less toxic therapeutic agents. Fenretinide (4-HPR, 2), an aminophenol derivative of RA, exhibits potent antiproliferative activity without binding to RAR/RXR, but its clinical trial was discontinued due to side effects of impaired dark adaptation. Assuming that the cyclohexene ring of 4-HPR is the cause of the side effects, methylaminophenol was discovered through structure-activity relationship research, and p-dodecylaminophenol (p-DDAP, 3), which has no side effects or toxicity and is effective against a wide range of cancers, was developed. Therefore, we thought that introducing the motif carboxylic acid found in retinoids, could potentially enhance the anti-proliferative effects. Introducing chain terminal carboxylic functionality into potent p-alkylaminophenols significantly attenuated antiproliferative potencies, while a similar structural modification of weakly potent p-acylaminophenols enhanced growth inhibitory potencies. However, conversion of the carboxylic acid moieties to their methyl esters completely abolished the cell growth inhibitory effects of both series. Insertion of a carboxylic acid moiety, which is important for binding to RA receptors, abolishes the action of p-alkylaminophenols, but enhances the action of p-acylaminophenols. This suggests that the amido functionality may be important for the growth inhibitory effects of the carboxylic acids.

Fenretinide combines perturbation of signaling kinases, cell-extracellular matrix interactions and matrix metalloproteinase activation to inhibit invasion in oral squamous cell carcinoma cells.

Basement membrane invasion defines malignant transformation of surface premalignancy. Treatment of oral squamous cell carcinoma (OSCC) cells with the synthetic vitamin A derivative, fenretinide (4HPR), induces numerous cancer-preventive effects including suppression of basement membrane invasion, elimination of anchorage-independent growth, disruption of actin cytoskeletal components and inhibition of the invasion-enabling focal adhesive kinase. The purpose of this study was to elucidate 4HPR's effects on additional invasion-relevant mechanisms including matrix metalloproteinase (MMP) activation and function, cell-extracellular matrix (ECM) attachments and interaction with a kinase that is essential for the epithelial-myoepithelial transformation i.e. c-Jun NH2-terminal kinase (JNK). Our data revealed that 4HPR binds with high affinity to the ATP-binding site of all three JNK isoforms with concurrent suppression of kinase function. Additional studies showed 4HPR treatment inhibited both OSCC cell-ECM adhesion and MMP activation and function. JNK downregulation and induced expression studies confirmed that the JNK3 isoform conveyed that largest impact on OSCC migration and invasion. Biodegradable polymeric implants formulated to preserve 4HPR's function and bioavailability were employed to assess 4HPR's chemopreventive impact on an OSCC tumor induction model. These studies revealed 4HPR local delivery significantly inhibited OSCC tumor size, mitotic indices and expression of the endothelial marker, erythroblast transformation-specific-related gene with concurrent increases in tumor apoptosis (cleaved caspase-3). Collectively, these data show that 4HPR suppresses invasion at multiple sites including 'outside-in' signaling, cell-ECM interactions and suppression of MMPs. These functions are also essential for physiologic function. Regulation is therefore essential and reinforces the pharmacologic advantage of local delivery chemopreventive formulations. .

N-(4-Hydroxyphenyl) Retinamide Suppresses SARS-CoV-2 Spike Protein-Mediated Cell-Cell Fusion by a Dihydroceramide Δ4-Desaturase 1-Independent Mechanism.

The membrane fusion between the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and host cells is essential for the initial step of infection; therefore, the host cell membrane components, including sphingolipids, influence the viral infection. We assessed several inhibitors of the enzymes pertaining to sphingolipid metabolism, against SARS-CoV-2 spike protein (S)-mediated cell-cell fusion and viral infection. N-(4-Hydroxyphenyl) retinamide (4-HPR), an inhibitor of dihydroceramide Δ4-desaturase 1 (DES1), suppressed cell-cell fusion and viral infection. The analysis of sphingolipid levels revealed that the inhibition efficiencies of cell-cell fusion and viral infection in 4-HPR-treated cells were consistent with an increased ratio of saturated sphinganine-based lipids to total sphingolipids. We investigated the relationship of DES1 with the inhibition efficiencies of cell-cell fusion. The changes in the sphingolipid profile induced by 4-HPR were mitigated by the supplementation with exogenous cell-permeative ceramide; however, the reduced cell-cell fusion could not be reversed. The efficiency of cell-cell fusion in DES1 knockout (KO) cells was at a level comparable to that in wild-type (WT) cells; however, the ratio of saturated sphinganine-based lipids to the total sphingolipids was higher in DES1 KO cells than in WT cells. 4-HPR reduced cell membrane fluidity without any significant effects on the expression or localization of angiotensin-converting enzyme 2, the SARS-CoV-2 receptor. Therefore, 4-HPR suppresses SARS-CoV-2 S-mediated membrane fusion through a DES1-independent mechanism, and this decrease in membrane fluidity induced by 4-HPR could be the major cause for the inhibition of SARS-CoV-2 infection. IMPORTANCE Sphingolipids could play an important role in SARS-CoV-2 S-mediated membrane fusion with host cells. We studied the cell-cell fusion using SARS-CoV-2 S-expressing cells and sphingolipid-manipulated target cells, with an inhibitor of the sphingolipid metabolism. 4-HPR (also known as fenretinide) is an inhibitor of DES1, and it exhibits antitumor activity and suppresses cell-cell fusion and viral infection. 4-HPR suppresses membrane fusion through a decrease in membrane fluidity, which could possibly be the cause for the inhibition of SARS-CoV-2 infection. There is accumulating clinical data on the safety of 4-HPR. Therefore, it could be a potential candidate drug against COVID-19.

High-fat diet exacerbates cognitive and metabolic abnormalities in neuronal BACE1 knock-in mice - partial prevention by Fenretinide.

Objective: The ß-site APP-cleaving enzyme 1 (BACE1) is a rate-limiting step in ß-amyloid (Aß) production in Alzheimer's disease (AD) brains, but recent evidence suggests that BACE1 is also involved in metabolic regulation. Here, we aimed to assess the effects of highfat diet (HFD) on metabolic and cognitive phenotypes in the diabetic BACE1 knock-in mice (PLB4) and WT controls; we additionally examined whether these phenotypes can be normalized with a synthetic retinoid (Fenretinide, Fen) targeting weight loss.Methods: Five-month old male WT and PLB4 mice were fed either (1) control chow diet, (2) 45%-saturated fat diet (HFD), (3) HFD with 0.04% Fen (HFD + Fen) or (4) control chow diet with 0.04% Fen (Fen) for 10 weeks. We assessed basic metabolic parameters, circadian rhythmicity, spatial habituation (Phenotyper) and working memory (Y-maze). Hypothalami, forebrain and liver tissues were assessed using Western blots, qPCR and ELISAs.Results: HFD feeding drastically worsened metabolism and induced early mortality (-40%) in otherwise viable PLB4 mice. This was ameliorated by Fen, despite no effects on glucose intolerance. In HFD-fed WT mice, Fen reduced weight gain, glucose intolerance and hepatic steatosis. The physiological changes induced in WT and PLB4 mice by HFD (+/-Fen) were accompanied by enhanced cerebral astrogliosis, elevated PTP1B, phopsho-eIF2α and altered hypothalamic transcription of Bace1, Pomc and Mc4r. Behaviourally, HFD feeding exacerbated spatial memory deficits in PLB4 mice, which was prevented by Fen and linked with increased full-length APP, normalized brain Aß*56 oligomerization and astrogliosis.Conclusions: HFD induces early mortality and worsened cognition in the Alzheimer's-like BACE1 mice- partial prevention was achieved with Fenretinide, without improvements in glucose homeostasis.

Dihydroceramide Δ4-Desaturase 1 Is Not Involved in SARS-CoV-2 Infection.

Dihydroceramide Δ4-desaturase 1 (DEGS1) enzymatic activity is inhibited with N-(4-hydroxyphenyl)-retinamide (4-HPR). We reported previously that 4-HPR suppresses severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry through a DEGS1-independent mechanism. However, it remains unclear whether DEGS1 is involved in other SARS-CoV-2 infection processes, such as virus replication and release. Here we established DEGS1 knockout (KO) in VeroE6TMPRSS2 cells. No significant difference was observed in virus production in the culture supernatant between wild-type (WT) cells and DEGS1-KO cells, although the levels of dihydroceramide (DHCer), a DEGS1 substrate, were significantly higher in DEGS1-KO cells than WT cells. Furthermore, the virus-induced cytopathic effect was also observed in DEGS1-KO cells. Importantly, the EC50 value of 4-HPR in DEGS1-KO cells was almost identical to the value reported previously in WT cells. Our results indicated the lack of involvement of DEGS1 in SARS-CoV-2 infection.

Fenretinide Reduces Intestinal Mucin-2-Positive Goblet Cells in Chronic Alcohol Abuse.

INTRODUCTION: Alcohol-induced thickening of the gut mucosal layer and increased expression of goblet cell gel-forming mucins, such as mucin-2 (MUC2) are associated with disruptions to the gut barrier in alcoholic liver disease (ALD). Interest in drugs that can target gut mucins in ALD has grown; however to date, no studies have examined the properties of drugs on expression of gut mucins in models of ALD. We previously demonstrated that at 10 mg/kg/day, the drug fenretinide (N-[4-hydroxyphenyl] retinamide [Fen]), a synthetic retinoid, mitigates alcohol-associated damage to the gut barrier and liver injury in a murine model of ALD. METHODS: In this study, we specifically sought to examine the effects of Fen on gut goblet cells, and expression of mucins, including MUC2 using a 25-day Lieber-DeCarli model of chronic alcohol intake. RESULTS: Our results show that chronic alcohol intake increased gut-mucosal thickening, goblet cell numbers, and mRNA and protein expression of MUC2 in both the ileum and colon. Alcohol intake was associated with marked decreases in ileal and colonic Notch signaling, levels of Notch ligands Dll1 and Dll4, and increases in the expression of Notch-associated genes indispensable for goblet cell specification, including Math1 and Spdef. Interestingly, ileal and colonic expression of KLF4, which is involved in terminal differentiation of goblet cells, was reduced in mice chronically fed alcohol. Coadministration of alcohol with Fen at 10 mg/kg/day significantly reduced alcohol-associated increases in ileal and colonic mucosal thickening, ileal Muc2, colonic Muc2, Muc5ac and Muc6 mRNAs, and goblet cell numbers. We also found that Fen strongly prevented alcohol-mediated suppression of the Notch ligand Dll1, Notch signaling, and alcohol-induced increases in expression of Notch-associated goblet cell specification genes in both the ileum and colon. In the absence of alcohol, Fen treatments alone at 10 mg/kg/day had no effects on any of the goblet cell-related endpoints. CONCLUSION: These data show for the first time that the drug Fen possesses mucosal layer-modulating properties in response to chronic alcohol abuse. These data warrant further preclinical examination of Fen given the need for anti-ALD drugs and emerging evidence of a role for intestinal goblet cell mucins in the progression of ALD.

Fenretinide in Cancer and Neurological Disease: A Two-Face Janus Molecule.

Recently, several chemotherapeutic drugs have been repositioned in neurological diseases, based on common biological backgrounds and the inverse comorbidity between cancer and neurodegenerative diseases. Fenretinide (all-trans-N-(4-hydroxyphenyl) retinamide, 4-HPR) is a synthetic derivative of all-trans-retinoic acid initially proposed in anticancer therapy for its antitumor effects combined with limited toxicity. Subsequently, fenretinide has been proposed for other diseases, for which it was not intentionally designed for, due to its ability to influence different biological pathways, providing a broad spectrum of pharmacological effects. Here, we review the most relevant preclinical and clinical findings from fenretinide and discuss its therapeutic role towards cancer and neurological diseases, highlighting the hormetic behavior of this pleiotropic molecule.

One-Step, Low-Cost, Operator-Friendly, and Scalable Procedure to Synthetize Highly Pure N-(4-ethoxyphenyl)-retinamide in Quantitative Yield without Purification Work-Up.

It is widely reported that N-(4-hydroxyphenyl)-retinamide or fenretinide (4-HPR), which is a synthetic amide of all-trans-retinoic acid (ATRA), inhibits in vitro several types of tumors, including cancer cell lines resistant to ATRA, at 1-10 µM concentrations. Additionally, studies in rats and mice have confirmed the potent anticancer effects of 4-HPR, without evidencing hemolytic toxicity, thus demonstrating its suitability for the development of a new chemo-preventive agent. To this end, the accurate determination of 4-HPR levels in tissues is essential for its pre-clinical training, and for the correct determination of 4-HPR and its metabolites by chromatography, N-(4-ethoxyphenyl)-retinamide (4-EPR) has been suggested as an indispensable internal standard. Unfortunately, only a consultable old patent reports the synthesis of 4-EPR, starting from dangerous and high-cost reagents and using long and tedious purification procedures. To the best of our knowledge, no article existed so far describing the specific synthesis of 4-EPR. Only two vendors worldwide supply 4-ERP, and its characterization was incomplete. Here, a scalable, operator-friendly, and one-step procedure to synthetize highly pure 4-EPR without purification work-up and in quantitative yield is reported. Additionally, a complete characterization of 4-EPR using all possible analytical techniques has been provided.

Rethinking Breast Cancer Chemoprevention: Technological Advantages and Enhanced Performance of a Nanoethosomal-Based Hydrogel for Topical Administration of Fenretinide.

Herein, we developed an ethosomal hydrogel based on three types of ethosomes: simple, mixed (surfactant-based micelles and lipid vesicles) or binary (comprising two type of alcohols). Ethanol injection was employed for vesicles preparation, and sodium alginate, as gelling agent. We purposed the local-transdermal administration of the off-the-shelf retinoid fenretinide (FENR) for chemoprevention of breast cancer. Rheograms and flow index values for alginate dispersion (without ethosomes) and hydrogels containing simple, mixed or binary ethosomes suggested pseudoplastic behavior. An increase in the apparent viscosity was observed upon ethosome incorporation. The ethosomal hydrogel displayed increased bioadhesion compared to the alginate dispersion, suggesting that the lipid vesicles contribute to the gelling and bioadhesion processes. In the Hen's Egg Test-Chorioallantoic Membrane model, few spots of lysis and hemorrhage were observed for formulations containing simple (score of 2) and mixed vesicles (score 4), but not for the hydrogel based on the binary system, indicating its lower irritation potential. The binary ethosomal hydrogel provided a slower FENR in vitro release and delivered 2.6-fold less drug into viable skin layers compared to the ethosome dispersion, supporting the ability of the gel matrix to slow down drug release. The ethosomal hydrogel decreased by ~ five-fold the IC50 values of FENR in MCF-7 cells. In conclusion, binary ethosomal gels presented technological advantages, provided sustained drug release and skin penetration, and did not preclude drug cytotoxic effects, supporting their potential applicability as topical chemopreventive systems.

A Lipid-Based In Situ-Forming Hexagonal Phase for Prolonged Retention and Drug Release in the Breast Tissue.

In this study, the addition of monoolein to phosphatidylcholine (PC), tricaprylin, and propylene glycol (PG) mixtures was studied to produce fluid precursor formulations (FIPs) that could transform into hexagonal phase (resistant to aqueous dilution) in vitro and in vivo. The overall goal was to obtain FIPs that could incorporate chemopreventive drugs for subcutaneous administration in the mammary tissue to inhibit the development and/or recurrence of breast cancer. Increasing PG content reduced FIP viscosity up to ~ 2.5-fold, while increases in PC (over monoolein) increased the formation of emulsified systems. The hexagonal phase was observed at 20% of water and higher, with the minimum amount of water necessary for this formation increasing with PG content. The selected FIP formed a depot in vivo after ~ 24 h of administration; its structure was compatible with the hexagonal phase and it remained in the mammary tissue for at least 30 days, prolonging the permanence of a fluorescent probe. In vitro, the release of the synthetic retinoid fenretinide was slow, with ~ 9% of the drug released in 72 h. Consistent with this slow release, fenretinide IC50 in breast cancer cells was ~ 100-fold higher in the selected FIP compared to its solution. The FIP reduced cell migration and presented higher cytotoxicity towards tumor compared to non-tumor cells. Given the limited number of options for pharmacological prevention of breast cancer development and recurrences, this formulation could potentially find applicability to reduce the frequency of administration and improve local concentrations of chemopreventive drugs.

Microemulsion for Prolonged Release of Fenretinide in the Mammary Tissue and Prevention of Breast Cancer Development.

The need of pharmacological strategies to preclude breast cancer development motivated us to develop a non-aqueous microemulsion (ME) capable of forming a depot after administration in the mammary tissue and uptake of interstitial fluids for prolonged release of the retinoid fenretinide. The selected ME was composed of phosphatidylcholine/tricaprylin/propylene glycol (45:5:50, w/w/w) and presented a droplet diameter of 175.3 ± 8.9 nm. Upon water uptake, the ME transformed successively into a lamellar phase, gel, and a lamellar phase-containing emulsion in vitro as the water content increased and released 30% of fenretinide in vitro after 9 days. Consistent with the slow release, the ME formed a depot in cell cultures and increased fenretinide IC50 values by 68.3- and 13.2-fold in MCF-7 and T-47D cells compared to a solution, respectively. At non-cytotoxic concentrations, the ME reduced T-47D cell migration by 75.9% and spheroid growth, resulting in ∼30% smaller structures. The depot formed in vivo prolonged a fluorochrome release for 30 days without producing any sings of local irritation. In a preclinical model of chemically induced carcinogenesis, ME administration every 3 weeks for 3 months significantly reduced (4.7-fold) the incidence of breast tumors and increased type II collagen expression, which might contribute to limit spreading. These promising results support the potential ME applicability as a preventive therapy of breast cancer.

Vorinostat and fenretinide synergize in preclinical models of T-cell lymphoid malignancies.

T-cell lymphoid malignancies (TCLMs) are in need of novel and more effective therapies. The histone deacetylase (HDAC) inhibitors and the synthetic cytotoxic retinoid fenretinide have achieved durable clinical responses in T-cell lymphomas as single agents, and patients who failed prior HDAC inhibitor treatment have responded to fenretinide. We have previously shown fenretinide synergized with the class I HDAC inhibitor romidepsin in preclinical models of TCLMs. There exist some key differences between HDAC inhibitors. Therefore, we determined if the pan-HDAC inhibitor vorinostat synergizes with fenretinide. We demonstrated cytotoxic synergy between vorinostat and fenretinide in nine TCLM cell lines at clinically achievable concentrations that lacked cytotoxicity for non-malignant cells (fibroblasts and blood mononuclear cells). In vivo, vorinostat + fenretinide + ketoconazole (enhances fenretinide exposures by inhibiting fenretinide metabolism) showed greater activity in subcutaneous TCLM xenograft models than other groups. Fenretinide + vorinostat increased reactive oxygen species (ROS, measured by 2',7'-dichlorodihydrofluorescein diacetate dye), resulting in increased apoptosis (via transferase dUTP nick end labeling assay) and histone acetylation (by immunoblotting). The synergistic cytotoxicity, apoptosis, and histone acetylation of fenretinide + vorinostat was abrogated by the antioxidant vitamin C. Like romidepsin, vorinostat combined with fenretinide achieved synergistic cytotoxic activity and increased histone acetylation in preclinical models of TCLMs, but not in non-malignant cells. As vorinostat is an oral agent and not a P-glycoprotein substrate it may have advantages in such combination therapy. These data support conducting a clinical trial of vorinostat combined with fenretinide in relapsed and refractory TCLMs.

The role of essential fatty acids in cystic fibrosis and normalizing effect of fenretinide.

Cystic fibrosis (CF) is the most common autosomal-recessive disease in Caucasians caused by mutations in the CF transmembrane regulator (CFTR) gene. Patients are usually diagnosed in infancy and are burdened with extensive medical treatments throughout their lives. One of the first documented biochemical defects in CF, which predates the cloning of CFTR gene for almost three decades, is an imbalance in the levels of polyunsaturated fatty acids (PUFAs). The principal hallmarks of this imbalance are increased levels of arachidonic acid and decreased levels of docosahexaenoic acids (DHA) in CF. This pro-inflammatory profile of PUFAs is an important component of sterile inflammation in CF, which is known to be detrimental, rather than protective for the patients. Despite decades of intensive research, the mechanistic basis of this phenomenon remains unclear. In this review we summarized the current knowledge on the biochemistry of PUFAs, with a focus on the metabolism of AA and DHA in CF. Finally, a synthetic retinoid called fenretinide (N-(4-hydroxy-phenyl) retinamide) was shown to be able to correct the pro-inflammatory imbalance of PUFAs in CF. Therefore, its pharmacological actions and clinical potential are briefly discussed as well.

Increasing Sphingolipid Synthesis Alleviates Airway Hyperreactivity.

Impaired sphingolipid synthesis is linked genetically to childhood asthma and functionally to airway hyperreactivity (AHR). The objective was to investigate whether sphingolipid synthesis could be a target for asthma therapeutics. The effects of GlyH-101 and fenretinide via modulation of de novo sphingolipid synthesis on AHR was evaluated in mice deficient in SPT (serine palmitoyl-CoA transferase), the rate-limiting enzyme of sphingolipid synthesis. The drugs were also used directly in human airway smooth-muscle and epithelial cells to evaluate changes in de novo sphingolipid metabolites and calcium release. GlyH-101 and fenretinide increased sphinganine and dihydroceramides (de novo sphingolipid metabolites) in lung epithelial and airway smooth-muscle cells, decreased the intracellular calcium concentration in airway smooth-muscle cells, and decreased agonist-induced contraction in proximal and peripheral airways. GlyH-101 also decreased AHR in SPT-deficient mice in vivo. This study identifies the manipulation of sphingolipid synthesis as a novel metabolic therapeutic strategy to alleviate AHR.

Synergism of 4HPR and SAHA increases anti-tumor actions in glioblastoma cells.

Glioblastoma is the most malignant and prevalent brain tumor in adults. It can grow and spread quickly causing harm to the brain health. One of the major challenges in treatment of glioblastoma is drug resistance. Use of synergistic combination of two drugs with different anti-tumor effects is nowadays highly considered in the development of effective therapeutic strategies for many malignancies. In the present study, we showed synergistic therapeutic efficacies of two chemical compounds, N-(4-hydroxyphenyl) retinamide (4HPR) and suberoylanilide hydroxamic acid (SAHA), for significant reduction in cell viability of rat C6 and human T98G glioblastoma cells. These compounds (4HPR and SAHA) were used alone or in synergistic combination for evaluating their various anti-tumor effects. The results showed that combination of 4HPR and SAHA significantly induced morphological and molecular features of astrocytic differentiation in C6 and T98G glioblastoma cells. Combination of 4HPR and SAHA proved to be an important therapeutic strategy for inhibiting cell growth and inducing differentiation in glioblastoma cells. Furthermore, combination of the two drugs showed more efficacies than either dug alone in reducing in vitro cell invasion (transwell assay), cell migration (wound healing assay), and angiogenesis (tube formation assay) due to down regulation of the molecules involved in these processes. The ultimate of goal of using this combination of drugs was induction of apoptosis. The results showed that these drugs in synergistic combination contributed highly to increases in morphological and molecular features of apoptotic death in the tumor cells. The results from molecular studies indicated that cell death occurred via activation of the extrinsic and intrinsic pathways of apoptosis in both C6 and T98G cells. The drugs in combination also contributed to dramatic inhibition of histone deacetylase 1, an important epigenetic player in promoting growth in glioblastoma cells. This novel combination of drugs should also be considered as a promising therapeutic strategy for the treatment of glioblastoma in vivo.

Treatment of Allergic Asthma with Fenretinide Formulation (LAU-7b) Downregulates ORMDL Sphingolipid Biosynthesis Regulator 3 (Ormdl3) Expression and Normalizes Ceramide Imbalance.

Zona pellucida binding protein 2 (Zpbp2) and ORMDL sphingolipid biosynthesis regulator 3 (Ormdl3), mapped downstream of Zpbp2, were identified as two genes associated with airway hyper-responsiveness (AHR). Ormdl3 gene product has been shown to regulate the biosynthesis of ceramides. Allergic asthma was shown to be associated with an imbalance between very-long-chain ceramides (VLCCs) and long-chain ceramides (LCCs). We hypothesized that Fenretinide can prevent the allergic asthma-induced augmentation of Ormdl3 gene expression, normalize aberrant levels of VLCCs and LCCs, and treat allergic asthma symptoms. We induced allergic asthma by house dust mite (HDM) in A/J WT mice and Zpbp2 KO mice expressing lower levels of Ormdl3 mRNA than WT. We investigated the effect of a novel formulation of Fenretinide, LAU-7b, on the AHR, inflammatory cell infiltration, mucus production, IgE levels, and ceramide levels. Although lower Ormdl3 expression, which was observed in Zpbp2 KO mice, was associated with lower AHR, allergic Zpbp2 KO mice were not protected from inflammatory cell infiltration, mucus accumulation, or aberrant levels of VLCCs and LCCs induced by HDM. LAU-7b treatment protects both the Zpbp2 KO and WT mice. The treatment significantly lowers the gene expression of Ormdl3, normalizes the VLCCs and LCCs, and corrects all the other phenotypes associated with allergic asthma after HDM challenge, except the elevated levels of IgE. LAU-7b treatment prevents the augmentation of Ormdl3 expression and ceramide imbalance induced by HDM challenge and protects both WT and Zpbp2 KO mice against allergic asthma symptoms. SIGNIFICANCE STATEMENT: Compared with A/J WT mice, KO mice with Zpbp2 gene deletion have lower AHR and lower levels of Ormdl3 expression. The novel oral clinical formulation of Fenretinide (LAU-7b) effectively lowers the AHR and protects against inflammatory cell infiltration and mucus accumulation induced by house dust mite in both Zpbp2 KO and WT A/J mice. LAU-7b prevents Ormdl3 overexpression in WT allergic mice and corrects the aberrant levels of very-long-chain and long-chain ceramides in both WT and Zpbp2 KO allergic mice.

Efficacy of Optimized Treatment Protocol Using LAU-7b Formulation against Ovalbumin (OVA) and House Dust Mite (HDM) -Induced Allergic Asthma in Atopic Hyperresponsive A/J Mice.

PURPOSE: To assess the efficacy of the novel clinical formulation of fenretinide (LAU-7b) for the treatment of allergic asthma. To study the association between LAU-7b treatment in allergic asthma and the modulation of very long chain ceramides (VLCC). METHODS: We used two allergens (OVA and HDM) to induce asthma in mouse models and we established a treatment protocol with LAU-7b. The severity of allergic asthma reaction was quantified by measuring the airway resistance, quantifying lung inflammatory cell infiltration (Haematoxylin and eosin stain) and mucus production (Periodic acid Schiff satin). IgE levels were measured by ELISA. Immunophenotyping of T cells was done using Fluorescence-activated cell sorting (FACS) analysis. The analysis of the specific species of lipids and markers of oxidation was performed using mass spectrometry. RESULTS: Our data demonstrate that 10 mg/kg of LAU-7b was able to protect OVA- and HDM-challenged mice against increase in airway hyperresponsiveness, influx of inflammatory cells into the airways, and mucus production without affecting IgE levels. Treatment with LAU-7b significantly increased percentage of regulatory T cells and CD4+ IL-10-producing T cells and significantly decreased percentage of CD4+ IL-4-producing T cells. Our data also demonstrate a strong association between the improvement in the lung physiology and histology parameters and the drug-induced normalization of the aberrant distribution of ceramides in allergic mice. CONCLUSION: 9 days of 10 mg/kg of LAU-7b daily treatment protects the mice against allergen-induced asthma and restores VLCC levels in the lungs and plasma.

Fenretinide reduces angiogenesis by downregulating CDH5, FOXM1 and eNOS genes and suppressing microRNA-10b.

Angiogenesis is a new vessel formation process that plays a role in various physiological and pathological conditions. This process is controlled by the balance between pro-angiogenic and anti-angiogenic mediators in the organism. Angiogenesis is needed for the growth and metastasis of solid tumors. Therefore, the anti-angiogenic treatment approach is seen as an interesting option in cancers. Fenretinide, a synthetic retinoic acid analog, is an effective agent on angiogenesis. In this study, we aimed to investigate the effects of the fenretinide on some miRNAs involving in angiogenesis process and on the expression of CDH5, FOXM1 and eNOS genes upregulated in angiogenesis. In addition, it was shown the effects of this agent on cell proliferation, cell migration and capillary-like tube formation. In our study, the data were analyzed using Kruskal-Wallis and Dunn's test. Fenretinide applied to the cells for 24 and 48 h periods reduced cell proliferation (P < 0.001) and cell migration, and suppressed tube formation (P < 0.001) as a dose dependent manner. Endothelial cells were cultured in growth-inducing media containing a variety of growth factors such as VEGF, FGF, IGF and EGF. As a result of simultaneous PCR analysis, we found that angiogenesis-promoting miR-10b was effectively suppressed (P < 0.001) and interestingly angiogenesis-modulating miR-126 was slightly increased (P < 0.05), but other miRNAs, including miR-31, miR-21, miR-101, miR-340, miR-29c, miR-206 and miR-146a were not affected. Besides, a significant decrease was observed in the levels of some angiogenesis-inducing genes, CDH5 (P < 0.001), FOXM1 (P < 0.001) and eNOS (P < 0.01 and P < 0.001) in endothelial cells treated with fenretinide. Our results have shown that fenretinide exhibited anti-angiogenic activity through the down-regulation of CDH5, FOXM1 and eNOS genes, and suppression of miR-10b.

Visual cycle modulators versus placebo or observation for the prevention and treatment of geographic atrophy due to age-related macular degeneration.

BACKGROUND: Age-related macular degeneration (AMD) is a highly prevalent condition in an ever-increasing elderly population. Although insidious in the early stages, advanced AMD (neovascular and atrophic forms) can cause significant visual disability and economic burden on health systems worldwide. The most common form, geographic atrophy, has no effective treatment to date, whereas neovascular AMD can be treated with intravitreal anti-vascular endothelial growth factor (anti-VEGF) injections. Geographic atrophy has a slow disease progression and patients tend to have preserved central vision until the final stages. This tendency, coupled with the use of modern imaging modalities, provides a large window of opportunity to intervene with validated methods to assess treatment efficacy. As geographic atrophy is an increasingly common condition with no effective intervention, many treatments are under investigation, one of which is visual cycle modulators. These medications have been shown to reduce lipofuscin accumulation in pre-clinical studies that have led to several clinical trials, reviewed herein. OBJECTIVES: To assess the efficacy and safety of visual cycle modulators for the prevention and treatment of geographic atrophy secondary to AMD. SEARCH METHODS: We searched the Cochrane Central Register of Controlled Trials (CENTRAL) (which contains the Cochrane Eyes and Vision Trials Register) (2020, Issue 1); MEDLINE Ovid; Embase Ovid; Web of Science Core Collection; Scopus; Association for Research in Vision and Ophthalmology (ARVO) website; ClinicalTrials.gov and the WHO ICTRP to 11 January 2020 with no language restrictions. We also searched using the reference lists of reviews and existing studies and the Cited Reference Search function in Web of Science to identify further relevant studies. SELECTION CRITERIA: We included randomised controlled trials (RCTs) and quasi-randomised clinical studies (if available) that compared visual cycle modulators to placebo or no treatment (observation) in people diagnosed with AMD (early, intermediate or geographic atrophy). DATA COLLECTION AND ANALYSIS: Two authors independently assessed risk of bias in the included studies and extracted data. Both authors entered data into RevMan 5. We resolved discrepancies through discussion. We graded the certainty of the evidence using the GRADE approach. MAIN RESULTS: We included three RCTs from the USA; one of these had clinical sites in Germany. Two studies compared emixustat to placebo while the other compared fenretinide to placebo. All assigned one study eye per participant and, combined, have a total of 821 participants with a majority white ethnicity (97.6%). All participants were diagnosed with geographic atrophy due to AMD based on validated imaging modalities. All three studies have high risk of attrition bias mainly due to ocular adverse effects of emixustat and fenretinide. We considered only one study to be adequately conducted and reported with high risk of bias in only one domain (attrition bias). We considered the other two studies to be poorly reported and to have high risk of attrition bias and reporting bias. People with geographic atrophy treated with emixustat may not experience a clinically important change in best-corrected visual acuity (BCVA) between baseline and 24 months compared to people treated with placebo (mean difference (MD) 1.9 Early Treatment Diabetic Retinopathy Study (ETDRS) letters, 95% confidence interval (CI) -2.34 to 6.14, low-certainty evidence). Emixustat may also result in little or no difference in loss of 15 ETDRS letters or more of BCVA compared with placebo at 24 months (16.4% versus 18%) (risk ratio (RR) 0.91, 95% CI 0.59 to 1.4, low-certainty evidence). In terms of disease progression, emixustat may result in little or no difference in the annual growth rate of geographic atrophy compared with placebo (mean difference MD 0.09 mm2/year (95% CI -0.26 to 0.44, low-certainty evidence). All three studies reported adverse events of both drugs (emixustat: moderate-certainty evidence; fenretinide: low-certainty evidence). The main adverse events were ocular in nature and associated with the mechanism of action of the drugs. Delayed dark adaptation (emixustat: 54.5%; fenretinide: 39.3%) and chromatopsia (emixustat: 22.6%; fenretinide: 25.2%) were the most common adverse events reported, and were the most prevalent reasons for study dropout in emixustat trials. These effects were dose-dependent and resolved after drug cessation. No specific systemic adverse events were considered related to emixustat; only pruritus and rash were considered to be due to fenretinide. One emixustat study reported six deaths, none deemed related to the drug. None of the included RCTs reported the other pre-specified outcomes, including proportion of participants losing 10 letters or more, and mean change in macular sensitivity. We planned to investigate progression to advanced AMD (geographic atrophy or neovascular AMD) in prevention studies, including participants with early or intermediate AMD, but we identified no such studies. Two of the included studies reported an additional outcome - incidence of choroidal neovascularisation (CNV) - that was not in our published protocol. CNV onset may be reduced in those treated with emixustat but the evidence was uncertain (risk ratio (RR) 0.67, 95% CI 0.27 to 1.65, low-certainty evidence), or fenretinide (RR 0.5, 95% CI 0.26 to 0.98, low-certainty evidence) compared to placebo. A dose-dependent relationship was observed with emixustat. AUTHORS' CONCLUSIONS: There is limited evidence to support the use of visual cycle modulators (emixustat and fenretinide) for the treatment of established geographic atrophy due to AMD. The possible reduction in the incidence of CNV observed with fenretinide, and to a lesser extent, emixustat, requires formal assessment in focused studies.