Novel lipid nanovesicle-loaded dissolving microarray patches for fenretinide in breast cancer chemoprevention.

The retinoid fenretinide (FENR) is a promising compound for preventing breast cancer recurrence but faces challenges due to poor solubility and low bioavailability. This study explores the development of dissolving microneedles (MNs) containing FENR-loaded ethosomes for minimally invasive breast cancer chemoprevention, aiming to enhance local drug distribution. Ethosomes were formulated using ethanol, propylene glycol, soya lecithin, water, and polysorbate 80 micelles. MNs were created from poly(vinyl alcohol) and poly(vinylpyrrolidone) hydrogels by adding polymer powder directly into ethosomes suspensions, reducing manufacturing time and cost. Two methods were used to load ethosomes into high-density moulds: 1) only in the needle area, and 2) in both the needle area and baseplate. Dynamic light scattering confirmed nanostructures in the hydrogels and MNs. Micelle-based ethosomes dissolved MNs in 15 min, compared to 30 min for other MNs. Skin deposition studies showed greater drug deposition (up to 10 µg/patch) and enhanced skin permeation of FENR (up to 40 µg) with Method 2. In-vivo studies in rats demonstrated that oral administration resulted in plasma FENR levels below 10 ng/g in the first three hours, whereas MN administration delayed delivery, reaching a maximum plasma concentration of 52 ng/g at 48 h. Skin deposition of FENR from MNs decreased from 3 µg/g on day 1 to <0.3 µg/g by the last day. This study indicates that MNs are a potential minimally invasive dosage form for delivering FENR, offering a new approach for breast cancer chemoprevention.

Encapsulation of 4-oxo-N-(4-hydroxyphenyl) retinamide in human serum albumin nanoparticles promotes EZH2 degradation in preclinical neuroblastoma models.

Neuroblastoma is the most prevalent and aggressive solid tumor that develops extracranially in children between the ages of 0-14 years, which accounts for 8-10% of all childhood malignancies and ∼15% of pediatric cancer-related mortality. The polycomb repressive complex 2 (PRC2) protein, EZH2, is overexpressed in neuroblastoma and mediates histone H3 methylation at lysine 27 (K27) positions through its methyl transferase activity and is a potential epigenetic silencer of many tumor suppressor genes in cancer. Phosphorylation of EZH2 decreases its stability and leads to proteasomal degradation. The 4-oxo-N-(4-hydroxyphenyl) retinamide (4O4HPR) promotes EZH2 degradation via activation of PKC-δ, but its limited solubility and physiological instability limit its application. In the current study, the encapsulation of 4O4HPR in Human Serum Albumin Nanoparticles (HSANPs) enhanced the solubility and physiological stability of the nanoformulation, leading to improved therapeutic efficacy through G2-M cell cycle arrest, depolarization of mitochondrial membrane potential, generation of reactive oxygen species and caspase 3 mediated apoptosis activation. The molecular mechanistic approach of 4O4HPR loaded HSANPs has activated caspase 3, which further cleaves PKC-δ into two fragments wherein the cleaved fragment of PKC-δ possesses the kinase activity that phosphorylates EZH2 and decreases the protein stability leading to its further ubiquitination in SH-SY5Y cells. Co-immunoprecipitation experiments revealed the direct interaction between PKC-δ and EZH2 phosphorylation, followed by ubiquitination. Moreover, 4O4HPR loaded HSANPs demonstrated improved in vivo biodistribution, greater dispersibility, and biocompatibility and exhibited enhanced protein instability and degradation of EZH2 in the neuroblastoma xenograft mouse model.

Nano-fenretinide demonstrates remarkable activity in acute promyeloid leukemia cells.

Acute promyelocytic leukemia (APL) is characterized by rearrangements of the retinoic acid receptor, RARα, which makes all-trans retinoic acid (ATRA) highly effective in the treatment of this disease, inducing promyelocytes differentiation. Current therapy, based on ATRA in combination with arsenic trioxide, with or without chemotherapy, provides high rates of event-free survival and overall survival. However, a decline in the drug activity, due to increased ATRA metabolism and RARα mutations, is often observed over long-term treatments. Furthermore, dedifferentiation can occur providing relapse of the disease. In this study we evaluated fenretinide, a semisynthetic ATRA derivative, encapsulated in nanomicelles (nano-fenretinide) as an alternative treatment to ATRA in APL. Nano-fenretinide was prepared by fenretinide encapsulation in a self-assembling phospholipid mixture. Physico-chemical characterization was carried out by dinamic light scattering and spectrophotometry. The biological activity was evaluated by MTT assay, flow cytometry and confocal laser-scanning fluorescence microscopy. Nano-fenretinide induced apoptosis in acute promyelocytic leukemia cells (HL60) by an early increase of reactive oxygen species and a mitochondrial potential decrease. The fenretinide concentration that induced 90-100% decrease in cell viability was about 2.0 µM at 24 h, a concentration easily achievable in vivo when nano-fenretinide is administered by oral or intravenous route, as demonstrated in previous studies. Nano-fenretinide was effective, albeit at slightly higher concentrations, also in doxorubicin-resistant HL60 cells, while a comparison with TK6 lymphoblasts indicated a lack of toxicity on normal cells. The results indicate that nano-fenretinide can be considered an alternative therapy to ATRA in acute promyelocytic leukemia when decreased efficacy, resistance or recurrence of disease emerge after protracted treatments with ATRA.

Fenretinide in Young Women at Genetic or Familial Risk of Breast Cancer: A Placebo-Controlled Biomarker Trial.

Fenretinide, a retinoid with a low-toxicity profile that accumulates in the breast, has been shown to prevent second breast cancer in young women. Fenretinide exhibits apoptotic and antiinvasive properties and it improves insulin sensitivity in overweight premenopausal women with insulin resistance. This study aimed to further characterize its role in cancer prevention by measuring circulating biomarkers related to insulin sensitivity and breast cancer risk.Sixty-two women, ages 20 to 46 years, healthy or who had already undergone breast cancer surgery, with a known BRCA1/2 mutation or a likelihood of mutation ≥20% according to the BRCAPRO model, were randomly assigned to receive fenretinide (200 mg/day) or placebo for 5 years (trial registration: EudraCT No. 2009-010260-41). Fasting blood samples were drawn at baseline, 12 and 36 months, and the following biomarkers were analyzed: retinol, leptin, adiponectin, retinol-binding protein 4 (RBP-4), total cholesterol, high-density lipoprotein (HDL) and low-density lipoprotein (LDL) cholesterol, triglycerides, glucose, insulin, insulin-like growth factor (IGF-1), IGF-binding protein 3, sex hormone binding globulin (SHBG), testosterone, and vascular endothelial growth factor (VEGF).After 12 months of treatment, we observed a favorable effect of fenretinide on glucose (decrease; P = 0.005), insulin (decrease; P = 0.03), homeostatic model assessment index (decrease; P = 0.004), HDL cholesterol (increase; P = 0.002), even though these effects were less prominent after 36 months. Retinol and retinol-binding protein 4 markedly decreased (P < 0.0001) throughout the study. None of the other measured biomarkers changed. PREVENTION RELEVANCE: Fenretinide exhibits beneficial effects on the metabolic profile, supporting its clinical use in breast cancer prevention especially in premenopausal women with a positive family history and pathogenic variants in BRCA1/2 genes. This finding requires further investigations in larger trials to confirm its role in breast cancer prevention.

Lipid-Restricted Culture Media Reveal Unexpected Cancer Cell Sensitivities.

Cancer cell culture models frequently rely on fetal bovine serum as a source of protein and lipid factors that support cell survival and proliferation; however, serum-containing media imperfectly mimic the in vivo cancer environment. Recent studies suggest that typical serum-containing cell culture conditions can mask cancer dependencies, for example, on cholesterol biosynthesis enzymes, that exist in vivo and emerge when cells are cultured in media that provide more realistic levels of lipids. Here, we describe a high-throughput screen that identified fenretinide and ivermectin as small molecules whose cytotoxicity is greatly enhanced in lipid-restricted media formulations. The mechanism of action studies indicates that ivermectin-induced cell death involves oxidative stress, while fenretinide likely targets delta 4-desaturase, sphingolipid 1, a lipid desaturase necessary for ceramide synthesis, to induce cell death. Notably, both fenretinide and ivermectin have previously demonstrated in vivo anticancer efficacy despite their low cytotoxicity under typical cell culture conditions. These studies suggest ceramide synthesis as a targetable vulnerability of cancer cells cultured under lipid-restricted conditions and reveal a general screening strategy for identifying additional cancer dependencies masked by the superabundance of medium lipids.

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.

Phase I trial of intravenous fenretinide (4-HPR) plus safingol in advanced malignancies.

PURPOSE: Fenretinide (4-HPR) is a synthetic retinoid that induces cytotoxicity through dihydroceramide production. Safingol, a stereochemical-variant dihydroceramide precursor, exhibits synergistic effects when administered with fenretinide in preclinical studies. We conducted a phase 1 dose-escalation clinical trial of this combination. METHODS: Fenretinide was administered as a 600 mg/m2 24-h infusion on Day 1 of a 21-day cycle followed by 900 mg/m2/day on Days 2 and 3. Safingol was concurrently administered as a 48-h infusion on Day 1 and 2 using 3 + 3 dose escalation. Primary endpoints were safety and maximum tolerated dose (MTD). Secondary endpoints included pharmacokinetics and efficacy. RESULTS: A total of 16 patients were enrolled (mean age 63 years, 50% female, median three prior lines of therapy), including 15 patients with refractory solid tumors and one with non-Hodgkin lymphoma. The median number of treatment cycles received was 2 (range 2-6). The most common adverse event (AE) was hypertriglyceridemia (88%; 38% ≥ Grade 3), attributed to the fenretinide intralipid infusion vehicle. Other treatment-related AEs occurring in ≥ 20% of patients included anemia, hypocalcemia, hypoalbuminemia, and hyponatremia. At safingol dose 420 mg/m2, one patient had a dose-limiting toxicity of grade 3 troponinemia and grade 4 myocarditis. Due to limited safingol supply, enrollment was halted at this dose level. Fenretinide and safingol pharmacokinetic profiles resembled those observed in monotherapy trials. Best radiographic response was stable disease (n = 2). CONCLUSION: Combination fenretinide plus safingol commonly causes hypertriglyceridemia and may be associated with cardiac events at higher safingol levels. Minimal activity in refractory solid tumors was observed. TRIAL REGISTRATION NUMBER: NCT01553071 (3.13.2012).

Reactive oxygen species are associated with the inhibitory effect of N-(4-hydroxyphenyl)-retinamide on the entry of the severe acute respiratory syndrome-coronavirus 2.

N-(4-hydroxyphenyl)-retinamide (4-HPR) inhibits the dihydroceramide Δ4-desaturase 1 (DEGS1) enzymatic activity. We previously reported that 4-HPR suppresses the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) spike protein-mediated membrane fusion through a decrease in membrane fluidity in a DEGS1-independent manner. However, the precise mechanism underlying the inhibition of viral entry by 4-HPR remains unclear. In this study, we examined the role of reactive oxygen species (ROS) in the inhibition of membrane fusion by 4-HPR because 4-HPR is a well-known ROS-inducing agent. Intracellular ROS generation was found to be increased in the target cells in a cell-cell fusion assay after 4-HPR treatment, which was attenuated by the addition of the antioxidant, α-tocopherol (TCP). The reduction in membrane fusion susceptibility by 4-HPR treatment in the cell-cell fusion assay was alleviated by TCP addition. Furthermore, fluorescence recovery after photobleaching analysis showed that the lateral diffusion of glycosylphosphatidylinositol-anchored protein and SARS CoV-2 receptor was reduced by 4-HPR treatment and restored by TCP addition. These results indicate that the decrease in SARS-CoV-2 spike protein-mediated membrane fusion and membrane fluidity by 4-HPR was due to ROS generation. Taken together, these results demonstrate that ROS production is associated with the 4-HPR inhibitory effect on SARS-CoV-2 entry.

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.

Pretreatment of human retinal pigment epithelial cells with sterculic acid forestalls fenretinide-induced apoptosis.

The ratio of saturated to monounsaturated fatty acids, thought to play a critical role in many cellular functions, is regulated by stearoyl-CoA desaturase (SCD), a rate-limiting enzyme in the biosynthesis of monounsaturated fatty acids. Previously, we observed a decrease in both SCD protein and enzymatic activity in apoptosis induced by fenretinide, a synthetic analog of retinoic acid, in the human retinal pigment epithelial (RPE) cell line ARPE-19. Here, we investigated the effect of pretreating ARPE-19 with sterculic acid, a cyclopropenoic fatty acid inhibitor of SCD, on preventing fenretinide-induced apoptosis, given the role of SCD in cell proliferation and apoptosis. We show that sterculic acid pretreatment prevents the effects of fenretinide-induced apoptosis shown by changes in cell morphology, viability, and caspase-3 activation. Analysis of endoplasmic reticulum (ER)-associated proteins shows that sterculic acid pretreatment reduced the fenretinide-induced upregulation of heme oxygenase-1, ATF3 and GADD153 expression that are in response to reactive oxygen species (ROS) generation. Sterculic acid is as effective as allopurinol in inhibition of xanthine oxidase (XDH), and this may play a role in reducing the potential role of XDH in fenretinide-induced ROS generation. Sterculic acid pretreatment also results in a reduction in SOD2 mRNA expression. Dihydroceramide accumulation, compared to ceramide, and ROS generation indicate that a ceramide-independent pathway mediates fenretinide-induced apoptosis, and ROS mediation is borne out by activation of the NF-κBp50 and NF-κBp65 downstream signaling cascade. Its prevention by sterculic acid pretreatment further indicates the latter's antioxidant/anti-inflammatory effect. Taken together, our results suggest that sterculic acid pretreatment can mitigate ROS-mediated fenretinide-induced apoptosis. Thus, sterculic acid may serve as a potential antioxidant and therapeutic agent. These effects may be independent of its effects on SCD activity.

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.

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. .

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.

Fenretinide inhibits vitamin A formation from ß-carotene and regulates carotenoid levels in mice.

N-[4-hydroxyphenyl]retinamide, commonly known as fenretinide, a synthetic retinoid with pleiotropic benefits for human health, is currently utilized in clinical trials for cancer, cystic fibrosis, and COVID-19. However, fenretinide reduces plasma vitamin A levels by interacting with retinol-binding protein 4 (RBP4), which often results in reversible night blindness in patients. Cell culture and in vitro studies show that fenretinide binds and inhibits the activity of ß-carotene oxygenase 1 (BCO1), the enzyme responsible for endogenous vitamin A formation. Whether fenretinide inhibits vitamin A synthesis in mammals, however, remains unknown. The goal of this study was to determine if the inhibition of BCO1 by fenretinide affects vitamin A formation in mice fed ß-carotene. Our results show that wild-type mice treated with fenretinide for ten days had a reduction in tissue vitamin A stores accompanied by a two-fold increase in ß-carotene in plasma (P < 0.01) and several tissues. These effects persisted in RBP4-deficient mice and were independent of changes in intestinal ß-carotene absorption, suggesting that fenretinide inhibits vitamin A synthesis in mice. Using Bco1-/- and Bco2-/- mice we also show that fenretinide regulates intestinal carotenoid and vitamin E uptake by activating vitamin A signaling during short-term vitamin A deficiency. This study provides a deeper understanding of the impact of fenretinide on vitamin A, carotenoid, and vitamin E homeostasis, which is crucial for the pharmacological utilization of this retinoid.

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.

Fenretinide Beneficial Effects on Amyotrophic Lateral Sclerosis-associated SOD1G93A Mutant Protein Toxicity: In Vitro and In Vivo Evidences.

Amyotrophic lateral sclerosis (ALS) is the most frequent motor neuron disease for which effective treatment options are still lacking. ALS occurs in sporadic and familial forms which are clinically indistinguishable; about 20% of familial ALS cases are linked to mutations of the superoxide dismutase 1 (SOD1) gene. Fenretinide (FEN), a cancer chemopreventive and antiproliferative agent currently used in several clinical trials, is a multi-target drug which also exhibits redox regulation activities. We analyzed the effects of FEN on mutant SOD1 (mSOD1) toxicity in motoneuronal (NSC34) and a muscle (C2C12) cell lines and evaluated the impacts of chronic administration of a new nanomicellar fenretinide formulation (NanoMFen) on ALS disease progression in the SOD1G93A mouse model. The results showed that FEN significantly prevents the toxicity of mSOD1 expression in NSC34 motor neuron; furthermore, FEN is able to partially overcome the toxic effect of mSOD1 on the myogenic program of C2C12 muscle cells. Administration of NanoMFen ameliorates the disease progression and increases median survival of mSOD1G93A ALS mice, even when given after disease onset; beneficial effects in ALS mice, however, is restricted to female sex. Our data support the therapeutic potential of FEN against ALS-associated SOD1G93A mutant protein toxicity and promote further studies to elucidate specific cellular targets of the drug in ALS. Furthermore, the sex-related efficacy of NanoMFen in mSOD1G93A ALS mice strengthens the importance, in the perspective of a precision medicine approach, of gender pharmacology in ALS research.

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.

A phase I study of intravenous fenretinide (4-HPR) for patients with malignant solid tumors.

BACKGROUND: Fenretinide is a synthetic retinoid that can induce cytotoxicity by several mechanisms. Achieving effective systemic exposure with oral formulations has been challenging. An intravenous lipid emulsion fenretinide formulation was developed to overcome this barrier. We conducted a study to establish the maximum tolerated dose (MTD), preliminary efficacy, and pharmacokinetics of intravenous lipid emulsion fenretinide in patients with advanced solid tumors. METHODS: Twenty-three patients with advanced solid tumors refractory to standard treatments received fenretinide as a continuous infusion for five consecutive days in 21-day cycles. Five different dose cohorts were evaluated between doses of 905 mg/m2 and 1414 mg/m2 per day using a 3 + 3 dose escalation design. A priming dose of 600 mg/m2 on day 1 was introduced in an attempt to address the asymptomatic serum triglyceride elevations related to the lipid emulsion. RESULTS: The treatment-related adverse events occurring in ≥ 20% of patients were anemia, hypertriglyceridemia, fatigue, aspartate aminotransferase (AST)/alanine aminotransferase (ALT) increase, thrombocytopenia, bilirubin increase, and dry skin. Five evaluable patients had stable disease as best response, and no patients had objective responses. Plasma steady-state concentrations of the active metabolite were significantly higher than with previous capsule formulations. CONCLUSION: Fenretinide emulsion intravenous infusion had a manageable safety profile and achieved higher plasma steady-state concentrations of the active metabolite compared to previous capsule formulations. Single-agent activity was minimal but combinatorial approaches are under evaluation.