Dihydroceramide desaturase (DES) – Fenretinide (4-HPR) inhibits DES activity in a dose- and time-dependent manner in CCRF-CEM leukemia cells. [1]

In several T-ALL cell lines, fenretinide (4-HPR) exhibits both short-term and long-term anticancer action. In CCRF-CEM leukemia cells, fenretinide suppresses DES activity in a dose- and time-dependent manner, increasing endogenous cellular dhCer levels in the process. In CCRF-CEM and Jurkat cells, fenretinide (3 μM) causes an accumulation of dhCer [1]. Insulin signaling is protected by fenretinide's inhibition of ceramide. Insulin-stimulated glucose absorption is prevented from decreasing by lipids when fenretinide is present [2]. At concentrations above 1 microM, fenretinide decreases OVCAR-5 cell survival and proliferation; at 10 microM, it suppresses growth by 70–90%. After three days of preincubation, fenretinide (1 microM) dramatically reduced OVCAR-5 invasion. After being exposed to 1 µM 4-HPR, endothelial cells did not form tubes; instead, they produced tiny cell aggregates [4].

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In human CCRF-CEM and Jurkat acute lymphoblastic leukemia cells, Fenretinide (4-HPR) induced acute loss of viability in a dose- and time-dependent manner; after 48 hours at 3 µM, viable CCRF-CEM cells decreased to 15.3% ± 6.3 and Jurkat cells to 21.1% ± 10.2, and cell death was nearly total with 10 µM as confirmed by annexin V-propidium iodide staining. [1]
Fenretinide (4-HPR) decreased clonogenic capacity similarly in both cell lines at concentrations as low as 0.5 µM (p>0.05). [1]
Fenretinide (4-HPR) inhibited dihydroceramide desaturase (DES) activity in CCRF-CEM cells in a dose (p<0.01) and time (p<0.05) dependent manner, leading to a concomitant increase in endogenous dihydroceramide (dhCer) content; DES inhibition occurred within the first hour of treatment at concentrations as low as 0.5 µM and was sustained. [1]
LC-MS analysis revealed that Fenretinide (4-HPR) induced dihydroceramide accumulation (but not ceramide) in both CCRF-CEM and Jurkat cells at sub-lethal and cytotoxic concentrations; increased dihydrosphingosine (dhSph) was also observed in CCRF-CEM cells at ≥1 µM, and slight sphingosine (Sph) accumulation in CCRF-CEM cells, but no increase in endogenous ceramide levels. [1]
Preincubation with myriocin (SPT inhibitor) blocked Fenretinide (4-HPR)-induced dhCer accumulation but did not prevent loss of viability or increase in intracellular ROS production; myriocin treatment did not prevent 4-HPR-mediated cytotoxicity in either cell line at 16 h or 24 h at 1 µM or 3 µM (p>0.05). [1]
Ascorbic acid and vitamin E significantly decreased Fenretinide (4-HPR) (3 µM)-triggered ROS production (p<0.01), though complete inhibition was only achieved with ascorbic acid; vitamin E provided sustained protection against 4-HPR-mediated cell death (by XTT and annexin V-PI staining), while ascorbic acid protection lasted no longer than 24 hours. [1]
Antioxidants (ascorbic acid, vitamin E, Trolox, Trolox-methyl ether) did not block endogenous dhCer accumulation nor substantially modify ceramide levels upon Fenretinide (4-HPR) treatment. [1]
Fenretinide (4-HPR) generated a clear dose- and time-dependent increase in lipid peroxidation in both cell lines; ascorbic acid and vitamin E both prevented 4-HPR-mediated lipid peroxidation at 6 hours, but only vitamin E retained the capacity to buffer lipid peroxidation after 24 hours of exposure. [1]
NDGA (lipoxygenase inhibitor), but not baicalein, effectively blocked general oxidative stress (CM-H2DCFDA oxidation) driven by Fenretinide (4-HPR) (3 µM) in both cell lines and protected from 24-hour 4-HPR-induced lipid peroxidation, but did not prevent cell death; NDGA also showed almost complete prevention of 4-HPR-triggered mitochondrial superoxide production, whereas vitamin E showed partial or null effect. [1]
Fenretinide (4-HPR) reduced clonogenic capacity without acute cytotoxicity in Jurkat cells; cell cycle analysis following 24-48 h exposure to 0.5 µM 4-HPR revealed no significant differences between treated and untreated cells. [1]
Fenretinide (4-HPR)-driven cell death may occur even in the absence of dihydroceramide or ROS accumulation. [1]

In male C57Bl/6 mice fed a high-fat diet, fenretinide (4-HPR) (10 mg/kg, ip) selectively prevents the build-up of ceramides. Tests for insulin and glucose tolerance show that fenretinide treatment increases insulin sensitivity and glucose tolerance [2]. The addition of 25 mg/kg ketoconazole to fenretinide raised the plasma levels of 4-HPR in NOD/SCID mice [3].

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A single intraperitoneal injection of Fenretinide (10 mg/kg) in high-fat-fed mice 12 h before sacrifice caused a roughly 6-fold increase in dihydroceramides in soleus muscle and a 2-fold increase in liver, with a non-significant tendency to decrease ceramides (20–30%). [2]

Chronic treatment: Male C57Bl/6 mice were fed a high-fat diet (HFD) for 16 weeks, with half receiving Fenretinide in drinking water (10 μg/ml, containing 0.5% ethanol) during the final 4 weeks. Fenretinide treatment improved glucose tolerance (intraperitoneal glucose tolerance test, 1 g/kg) and insulin sensitivity (insulin tolerance test, 0.75 units/kg) compared to HFD alone. HOMA-IR (calculated from fasting glucose and insulin) was significantly reduced in HFD+FEN mice. Body weight was not significantly affected by fenretinide. [2]

Lipid analysis of soleus and liver from these mice showed that fenretinide normalized ceramide levels (reduced to levels similar to standard diet controls) and stepwise increased dihydroceramides compared to HFD alone. Fenretinide also significantly reduced hepatic TAG levels and reversed hepatic steatosis as assessed by Oil red O staining, while having no effect on hepatic DAG levels. [2]

High-fat feeding markedly increased Des1 transcript and protein levels in the liver, and to a lesser extent in muscle. Chronic fenretinide treatment reduced hepatic Des1 expression (mRNA and protein) and significantly reduced Des1 mRNA in muscle. [2]

Dihydroceramide desaturase (DES) activity was measured using a synthetic dihydroceramide analogue (D-erythro-C12-dihydroceramide; C12-dhCCPS). Cells were incubated with the synthetic analogue for 1 hour prior to adding Fenretinide (4-HPR) at indicated concentrations. After 1 or 6 hours of exposure, cells were collected and levels of the synthetic substrate (C12-dhCCPS) and its desaturated product (C12-CCPS) were analyzed by LC/MS. DES activity was estimated as the percentage of desaturated product relative to total C12-Pyr (C12-PyrdhCer plus C12-PyrCer) in cells. [1]

Metabolic activity (cell viability) was determined by XTT assay. Cells were plated in 96-well plates at 750,000 cells/ml and 100 µl/well. After 4 hours, treatments were added to obtain a final density of 500,000 cells/ml and final volume of 150 µl/well. XTT reagent mixture was added 4 hours before the end of the selected treatment period and absorbance at 490 nm was measured. For myriocin or antioxidant studies, cells were seeded on 60 mm culture dishes, myriocin or antioxidants added after 4 hours, Fenretinide (4-HPR) added 2 hours later, and then cells plated in quadruplicates in 96-well plates. [1]
Clonogenic capacity assay: Cells were exposed to selected Fenretinide (4-HPR) concentrations for 16 hours (5×10^6 cells/sample). 6-well plates were covered with 2 ml/well of pre-warmed 1.2% methylcellulose medium, and a layer of 0.6% methylcellulose medium containing 50,000 cells was placed on top. Plates were maintained at 37°C in a humidified 5% CO2 incubator for 7-9 days until significant colony growth. Clonogenic capacity was determined by counting colonies (>50 cells) in 3 random areas/well using an inverted microscope. [1]
Apoptotic cell death was determined by annexin V-propidium iodide staining according to the manufacturer's RAPID protocol. 10,000 cells/sample were analyzed by flow cytometry. Cells were classified as: healthy cells (PI- and FITC-), early apoptotic (PI- and FITC+), late apoptotic/necrotic (PI+ and FITC+). Results were analyzed by WinMDI 2.8 and Summit v4.3 software. [1]
General oxidative stress was measured using CM-H2DCFDA. Cells were seeded in red phenol-free RPMI1640-based complete culture media (5×10^6 cells per treatment). After treatment, approximately 2×10^6 cells were resuspended in PBS containing 10 µM CM-H2DCFDA and incubated for 25 minutes at 37°C in darkness. Fluorescence intensity was measured by plate reader or flow cytometer. Probe-free cells were used as internal negative control and freshly prepared H2O2 (200 µM, 15 min) as positive control. [1]
Mitochondrial superoxide production was estimated by MitoSOX oxidation. After treatment, approximately 2×10^6 cells were resuspended in Hank's Buffered Salt Solution containing 5 µM MitoSOX and incubated for 15 minutes at 37°C in darkness. Samples were washed with PBS and fluorescence intensity measured by plate reader or flow cytometer. [1]
Intracellular lipid peroxidation was assayed using BODIPY 581/591 C11 (undecanoic acid). Cells were incubated with 10 µM BODIPY for 3 hours at 37°C in darkness for proper internalization. Treatments were added before or after the probe depending on exposure time. The culture medium was replaced by 1 ml PBS/sample before measuring fluorescence. Flow cytometric analysis of lipid peroxidation was performed with 10,000 cells per sample. [1]
Endogenous sphingolipid species were analyzed by LC/MS. After treatment, cells were washed twice with PBS to avoid external sphingolipid contamination. Total values were normalized to inorganic phosphates by an adapted Bligh and Dyer lipid extraction. [1]
Cell cycle analysis was performed by propidium iodide labeling following 24-48 hour exposure to Fenretinide (4-HPR) (0.5 µM). [1]

Male C57Bl/6 mice (6 weeks old) were fed standard chow or high-fat diet (D12492) for up to 16 weeks. For acute single-dose injections, mice received intraperitoneal injections of Fenretinide (10 mg/kg) dissolved in DMSO and resuspended in warmed PBS; mice were sacrificed 12 h post-injection. Soleus muscle and liver were collected for lipid analysis. [2]

For chronic intervention, HFD-fed mice (from 5 to 17 weeks) received Fenretinide in drinking water (10 μg/ml) for 4 weeks. Fenretinide was first dissolved in 100% ethanol and diluted in water to final concentration (0.5% ethanol). Water was prepared under low-light conditions and administered in light-protective bottles, replaced every 1–2 days. Control water contained equal amount of ethanol (0.5%). After 4-week treatment, mice underwent intraperitoneal glucose tolerance test (1 g/kg glucose after 6 h fast) and insulin tolerance test (0.75 units/kg insulin after 6 h fast). Blood glucose was measured using a glucose meter; insulin levels were measured by ELISA. HOMA-IR was calculated from fasting glucose and insulin. At sacrifice, soleus and liver were collected for lipid extraction (LC-MS/MS), histology (Oil red O staining of frozen liver sections), quantitative real-time PCR (TRIzol extraction, cDNA synthesis with oligo(dT), SYBR Green PCR, normalized to β-actin), and Western blot (tissue extracts resolved by SDS-PAGE, transferred to nitrocellulose, immunoblotted with antibodies against Des1, detected by infrared imaging). [2]

Metabolism / Metabolites
Known metabolites of fenritinib include (2S,3S,4S,5R)-6-[4-[[(2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)non-2,4,6,8-tetraenoyl]amino]phenoxy]-3,4,5-trihydroxyoxacyclohexane-2-carboxylic acid.

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Fenretinide (4-HPR) shows a favorable systemic toxicity profile compared to other retinoids, which allows and supports continuity of in vivo studies and further application in clinical trials. [1]

[1]. Dihydroceramide accumulation and reactive oxygen species are distinct and nonessential events in 4-HPR-mediated leukemia cell death. Biochemistry and Cell Biology (2012), 90(2), 209-223.

[2]. Fenretinide Prevents Lipid-induced Insulin Resistance by Blocking Ceramide Biosynthesis. Journal of Biological Chemistry (2012), 287(21), 17426-17437.

[3]. Fenretinide metabolism in humans and mice: utilizing pharmacological modulation of its metabolic pathway to increase systemic exposure. Br J Pharmacol. 2011 Jul;163(6):1263-75.

[4]. Action of fenretinide (4-HPR) on ovarian cancer and endothelial cells. Anticancer Res. 2005 Jan-Feb;25(1A):249-53.

4-Hydroxyphenyl retinamide is a retinoid formed by the condensation of the carboxyl group of all-trans retinoic acid and the aniline group of 4-hydroxyaniline. It is a synthetic retinoid agonist with antiproliferative, antioxidant, and anticancer activities and a long in vivo half-life. Its apoptotic mechanism appears to differ from that of "classic" retinoids. It possesses antitumor and antioxidant effects. It is a retinoid and monocarboxylic acid amide functionally related to all-trans retinoic acid. A synthetic retinoid, it can be taken orally for the prevention of prostate cancer and for the prevention of contralateral breast cancer in women at risk. It also has antitumor activity. Fentrinib is an orally effective synthetic phenylretinamide retinoid (vitamin A) analog with potential antitumor and chemopreventive activities. Fentrinib binds to and activates retinoic acid receptors (RARs), thereby inducing differentiation and apoptosis in certain tumor cell types. The drug can also inhibit tumor growth by modulating angiogenesis-related growth factors and their receptors, exhibiting retinoid receptor-independent apoptotic properties. (NCI04)
A synthetic retinoid, taken orally for the prevention of prostate cancer and also for the prevention of contralateral breast cancer in women at risk. It is also an effective anti-tumor drug.
Drug Indications

Studied for the treatment of macular degeneration.
Mechanism of Action

Fentrinib inhibits the growth of various human cancer cell lines through retinoic acid receptor-dependent and non-retinoic acid-dependent mechanisms.¹In vivo, fenritinib selectively accumulates in mammary tissue, particularly effectively inhibiting the development of breast cancer in rats.¹An important characteristic of fenritinib is that it inhibits cell growth by inducing apoptosis rather than differentiation, which is quite different from the mechanism of action of vitamin A.¹Unlike tamoxifen, which only inhibits estrogen receptor (ER)-positive tumors, fenritinib can induce apoptosis in both ER-positive and ER-negative breast cancer cell lines.²All these properties make fenritinib an ideal candidate drug for the treatment of breast cancer. Chemoprophylaxis.

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Fenretinide (4-HPR) is being tested against several cancers in vitro and in vivo as well as in clinical trials, including for neuroblastoma, breast cancer, and bladder cancer. [1]
Fenretinide (4-HPR) exerts its cell growth inhibitory effect mainly by inducing cell death rather than differentiation, unlike vitamin A and ATRA. [1]
Fenretinide (4-HPR) can be applied against retinoic acid-resistant cancers. [1]
In T-ALL cell lines (CCRF-CEM, Jurkat), Fenretinide (4-HPR) induces intrinsic mitochondrial apoptotic cell death characterized by early increase in ROS and modulation of sphingolipid levels. [1]
The study demonstrates that early changes (sphingolipid modulation and ROS production) are mechanistically independent events upon Fenretinide (4-HPR) treatment, and cell death may occur even in the absence of dihydroceramide or ROS accumulation. [1]

C26H33NO2

391.5457

391.251

65646-68-6

5288209

Light yellow to yellow solid powder

1.1±0.1 g/cm3

597.6±42.0 °C at 760 mmHg

162-163°C

315.2±27.9 °C

0.0±1.8 mmHg at 25°C

1.607

7.41

2

2

6

29

726

0

CC1=C(C(CCC1)(C)C)/C=C/C(=C/C=C/C(=C/C(=O)NC2=CC=C(C=C2)O)/C)/C

AKJHMTWEGVYYSE-FXILSDISSA-N

InChI=1S/C26H33NO2/c1-19(11-16-24-21(3)10-7-17-26(24,4)5)8-6-9-20(2)18-25(29)27-22-12-14-23(28)15-13-22/h6,8-9,11-16,18,28H,7,10,17H2,1-5H3,(H,27,29)/b9-6+,16-11+,19-8+,20-18+

(2E,4E,6E,8E)-N-(4-hydroxyphenyl)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-en-1-yl)nona-2,4,6,8-tetraenamide

4-HPR McNR-1967 McNR1967 McNR 1967 HPR Fenretinide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 130 mg/mL (~332.01 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.38 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.38 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.38 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)