fenretinide and Vitamin-A-Deficiency

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 β-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 β-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 β-carotene in plasma (P < 0.01) and several tissues. These effects persisted in RBP4-deficient mice and were independent of changes in intestinal β-carotene absorption, suggesting that fenretinide inhibits vitamin A synthesis in mice. Using Bco1

Topics: Animals; beta Carotene; Body Weight; Dioxygenases; Fenretinide; Intestinal Absorption; Intestines; Liver; Mice, Inbred C57BL; Models, Biological; Retinol-Binding Proteins, Plasma; Vitamin A; Vitamin A Deficiency; Vitamin E

The retinamide, N-(4-hydroxyphenyl)retinamide (4-HPR), has shown promising anti-tumor activity, but it is unclear whether this compound is hydrolyzed to all-trans retinoic acid (atRA) and if so, whether this plays any role in its chemotherapeutic activity. To address this issue, the ability of 4-hydroxybenzylretinone (4-HBR), a carbon-linked analog of 4-HPR, to support growth in vitamin A-deficient (VAD) animals and to activate an atRA-responsive gene in vivo was compared to 4-HPR and atRA. Further, the non-hydrolyzable 4-HBR analog was used to determine whether the presence of the labile amide linkage in 4-HPR is essential for the induction of apoptosis in cultured MCF-7 breast cancer cells. Studies in VAD rats showed that 4-HPR, like atRA, supports animal growth and induces CYP26B1 mRNA expression in lung whereas 4-HBR does not. Analysis of plasma from 4-HPR- and atRA-treated VAD animals revealed the presence of atRA whereas it was not detected in plasma from animals given 4-HBR. To determine whether hydrolysis to atRA is necessary for apoptosis induced by 4-HPR in MCF-7 breast cancer cells, morphological and biochemical assays for apoptosis were performed. 4-HBR, like 4-HPR, induced apoptosis in MCF-7 cells. Apoptosis was not induced even at high concentrations of atRA, showing that 4-HPR and 4-HBR act in cells via a distinct signaling pathway. These results show that although limited hydrolysis of 4-HPR occurs in vivo, the ability to liberate atRA is not required for these 4-hydroxyphenyl retinoids to induce apoptosis in MCF-7 breast cancer cells. Thus the non-hydrolyzable analog, 4-HBR, may have significant therapeutic advantage over 4-HPR because it does not liberate atRA that can contribute to the adverse side effects of drug administration in vivo.

Topics: Administration, Oral; Animals; Apoptosis; Body Weight; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Fenretinide; Humans; Hydrolysis; Male; Rats; Rats, Sprague-Dawley; Tretinoin; Vitamin A; Vitamin A Deficiency

The cellular distribution of enzymes that esterify retinol and hydrolyze retinyl esters (RE) was studied in liver of vitamin A-sufficient, -deficient, and deficient rats treated with retinoic acid or N-(4-hydroxyphenyl)-retinamide. Livers were perfused and cell fractions enriched in hepatocytes, and nonparenchymal cells were obtained for assays of RE and enzyme activity. The specific activity of lecithin:retinol acyltransferase (LRAT) was approximately 10-fold greater in the nonparenchymal cell than the hepatocyte fraction from both vitamin A-sufficient and retinoid-treated rats. Total RE mass, newly synthesized [3H]RE and LRAT activity were positively correlated in liver and isolated cells of both normal (P < 0.0001) and retinoid-treated rats (P < 0.0002). In nonparenchymal cells, these three constituents were nearly equally enriched as evaluated by their relative specific activity values (RSA, defined as the percentage of recovered activity divided by the percentage of recovered protein), which were each significantly greater than 1.0, with values of 4.3 for total RE mass (P < 0.05), 3.6 for newly synthesized [3H]RE (P < 0.01) and 3.8 for LRAT activity (P < 0.01). In contrast, the specific activities of neutral and acid bile salt-independent retinyl ester hydrolases (REH) did not vary with vitamin A status, and their RSA values were close to 1.0 in both hepatocytes and nonparenchymal cells. These data show that LRAT and REH are differentially regulated by retinoids and that these enzymes also differ in their spacial distribution between liver parenchymal and nonparenchymal cells.

Topics: Acyltransferases; Animals; Carboxylic Ester Hydrolases; Female; Fenretinide; Liver; Male; Rats; Rats, Inbred Lew; Tretinoin; Vitamin A; Vitamin A Deficiency

The synthetic retinoid 4-HPR has been shown to markedly lower the plasma concentration of both retinol and RBP in rats and humans. We have studied the effect of 4-HPR on the secretion of retinol-RBP from liver cells in vivo and in vitro. In rats maintained with a normal diet, a vitamin A-deficient diet or a normal diet supplemented with 4-HPR, chylomicrons [3H]retinyl esters were rapidly cleared from the plasma. The secretion of chylomicron-derived [3H]retinol from tissues to the circulation, however, was different. In control rats, the lymph-derived [3H]retinol peaked after about 2 hr, whereas 4-HPR treatment effectively reduced this peak of [3H]retinol. Our results suggest that 4-HPR inhibits secretion of retinol-RBP from the liver. Therefore, we decided to study the effect of 4-HPR on the secretion of RBP using the human hepatoma cell line HepG2. Retinol and 4-HPR were found to induce the secretion of RBP. The medium from cells treated with 4-HPR was immunoprecipitated with antibodies against human RBP. HPLC analysis of the precipitated RBP revealed the presence of 4-HPR. When the medium from cells incubated with either 4-HPR or retinol was applied to a TTR affinity column, we found that RBP from cells incubated with 4-HPR had a considerably reduced affinity for TTR. We conclude that 4-HPR binds RBP and thereby induces secretion of RBP in HepG2 cells, and that the secreted 4-HPR-RBP complex has a reduced affinity for TTR. This observation may explain the 4-HPR-induced reduction of plasma retinol and RBP observed in in vivo studies.

Topics: Animals; Carcinoma, Hepatocellular; Chromatography, High Pressure Liquid; Chylomicrons; Culture Media; Diet; Fenretinide; Humans; Liver; Liver Neoplasms; Male; Prealbumin; Protein Binding; Rats; Rats, Wistar; Retinol-Binding Proteins; Retinol-Binding Proteins, Plasma; Vitamin A Deficiency

N-(4-Hydroxyphenyl)retinamide (HPR; Fenretinide), a synthetic retinoid possessing antitumor activity, depresses plasma retinol and retinol-binding protein (RBP) concentrations. In study 1, the ability of retinol or HPR to induce RBP secretion from the livers of vitamin A-deficient rats was compared. A large apoRBP pool accumulated in the liver rough microsomes of these rats. Following retinol repletion, 80% of the accumulated RBP was rapidly secreted into the plasma. In contrast, HPR treatment only induced two-thirds of the RBP secretion observed with retinol. Prior colchicine treatment caused a large RBP accumulation in the Golgi-enriched fraction following retinol repletion. HPR plus colchicine treatment produced significantly less accumulation of RBP in the Golgi-enriched fraction than did retinol. In study 2, HPR treatment of vitamin A-adequate rats caused RBP to accumulate in the liver rough microsomes. When vitamin A-adequate rats were treated with colchicine, the concentration of RBP in the Golgi-enriched fraction increased 2.9-fold. However, significantly less RBP accumulated in the Golgi following HPR treatment. These studies demonstrate that HPR will induce liver RBP secretion, but to a lesser degree than retinol. Further, more RBP remained in the rough microsomes of HPR treated, vitamin A-adequate rats, indicating that HPR depressed the amount of RBP secreted.

Topics: Animals; Antineoplastic Agents; Biological Transport; Cell Compartmentation; Colchicine; Fenretinide; Liver; Male; Prealbumin; Rats; Rats, Sprague-Dawley; Retinol-Binding Proteins; Retinol-Binding Proteins, Plasma; Vitamin A; Vitamin A Deficiency

N-(4-Hydroxyphenyl)-retinamide (4-HPR; Fenretinide) is a synthetic retinoid which is undergoing investigation as a cancer chemopreventive agent. However, 4-HPR alters vitamin A kinetics and reduces the concentration of plasma retinol. We have conducted studies to examine the effects of 4-HPR on the activity of the enzyme lecithin:retinol acyltransferase (LRAT). This enzyme is implicated in the absorption and storage of vitamin A and is regulated, in liver, by vitamin A nutritional status. To determine whether 4-HPR, like retinoic acid, is able to induce liver LRAT activity, vitamin A-deficient rats having negligible liver LRAT activity were treated with single doses of 4-HPR (0.02-2.5 mg) and liver homogenates were assayed for LRAT activity using 3H-retinol bound to the cellular-retinol binding protein, CRBP, as substrate. Treatment with 4-HPR resulted in a dose- and time-dependent increase in liver LRAT activity which reached a maximum at 24 h. The activity of LRAT assayed in vitro and of hepatic 3H-retinyl ester content determined after an in vivo pulse of 3H-retinol were highly correlated (r = 0.802, P < 0.0002). When vitamin A-sufficient rats were fed a 4-HPR-supplemented diet for 30 d, LRAT activity differed significantly from control values in the liver (P < 0.0001) but not the small intestines. Changes in hepatic retinol metabolism which favor the esterification of vitamin A may be related to the mechanism by which 4-HPR alters vitamin A kinetics in vivo.

Topics: Acyltransferases; Animals; Antineoplastic Agents; Dose-Response Relationship, Drug; Female; Fenretinide; Intestine, Small; Liver; Male; Rats; Rats, Inbred Lew; Time Factors; Vitamin A; Vitamin A Deficiency

Previous studies of topical retinoic acid for treatment of ocular surface disease met with limited success due to instability and irritancy of the retinoid and lack of efficacy in keratoconjunctivitis sicca. There has, however, been continued interest in the treatment of mucin deficiency and cicatrizing conjunctival diseases, such as ocular cicatricial pemphigoid (OCP), topically with retinoids. In this study the biological activity of stable, water-soluble, synthetic retinoid, N-(4-hydroxyphenyl) retinamide-O-glucuronide (4-HPROG) was investigated in vivo and in vitro using conjunctival and corneal epithelium and fibroblasts. Vitamin A-deficient rabbits with stage 3-4 corneal xerosis and squamous metaplasia confirmed by conjunctival impression cytology were treated with topical 0.1% 4-HPROG in an artificial tear vehicle for 3 weeks. Impression cytology was repeated at 2 and 3 weeks and at 3 weeks conjunctival biopsies were fixed for histology. Growth curves were generated using conjunctival fibroblasts of rabbits and humans (normals and patients with cicatrizing conjunctival disease including OCP and Stevens-Johnson syndrome) cultured in the 10(-8)-10(-6) M 4-HPROG. In vivo, corneal xerosis cleared in three days. A normal conjunctival epithelium was restored by 2 weeks and goblet cells were present by 3 wk, with no change in vehicle-treated controls. No ocular irritation occurred. In vitro, 10(-6) M 4-HPROG inhibits growth of rabbit conjunctival fibroblasts. The retinoid had no effect on proliferation of conjunctival fibroblasts from normal humans but the doubling time of cells from patients with OCP increased significantly, from 50.9 +/- 10.01 h (control) to 61.5 +/- 8.95 h (retinoid). Proliferation of conjunctival fibroblasts from a patient with Stevens-Johnson syndrome was also inhibited. N-(4-hydroxyphenyl) retinamide-O-glucuronide is biologically active and merits further study to determine its efficacy in controlling conjunctival fibrosis and treating ocular surface squamous metaplasia.

Topics: Animals; Cell Division; Cicatrix; Conjunctiva; Conjunctival Diseases; Cornea; Eye Diseases; Fenretinide; Fibroblasts; Glucuronates; Humans; Pemphigoid, Benign Mucous Membrane; Rabbits; Stromal Cells; Vitamin A Deficiency

The chemopreventive retinoid N-(4-hydroxyphenyl)retinamide (HPR) depresses serum retinol and retinol-binding protein (RBP) concentrations. To study long-term effects of HPR on serum proteins, rats were fed a control diet or a diet containing HPR (737 mumol/kg diet) for 14 d. Serum retinol and RBP of HPR-treated rats decreased to 42 and 41%, respectively, of initial concentrations. Transthyretin, albumin and transferrin did not differ between treatments. Previous studies found that HPR decreased secretion of the retinol-RBP complex into plasma. To investigate acute effects of HPR on RBP metabolism, vitamin A-deficient rats were injected with HPR (51 mumol/kg body wt), retinol (0.52 mumol/rat) or Tween carrier only. Liver RBP concentrations in HPR- and retinol-treated rats were 45 and 18%, respectively, of concentrations in Tween-treated rats, indicating rapid RBP secretion. Tween- and HPR-treated rats maintained relatively constant serum RBP concentrations, whereas retinol-replete rats had 12-fold higher serum RBP after 150 min. Rats treated with HPR and rats treated with retinol had 29- and eightfold higher kidney RBP concentrations, respectively, than Tween-treated rats, indicating rapid clearance of RBP from plasma. We conclude that HPR affects RBP metabolism by inducing secretion of liver RBP into the bloodstream and rapid RBP accumulation in the kidney.

Topics: Analysis of Variance; Animals; Disease Models, Animal; Fenretinide; Kidney; Liver; Male; Prealbumin; Radioimmunoassay; Rats; Rats, Sprague-Dawley; Retinol-Binding Proteins; Retinol-Binding Proteins, Plasma; Serum Albumin; Time Factors; Transferrin; Vitamin A; Vitamin A Deficiency

The metabolism and bioactivity of N-(4-hydroxyphenyl)-all-trans-retinamide (HPR) and of various O-alkyl and ester derivatives of HPR were investigated in rodents. The principal metabolite of HPR in tissues is N-(4-methoxyphenyl)-all-trans-retinamide. N-(4-methoxyphenyl)all-trans-retinamide is equipotent to HPR in reversing keratinization of retinoid-deficient hamster trachea in vitro. Another nonpolar metabolite of HPR is also present in tissue and (although not positively identified) is thought to be a long-chain fatty acid ester of HPR. HPR is excreted into rat bile as numerous polar retinamides, including HPR-O-glucuronide. The rate of hydrolysis of HPR esters by rat serum and hepatic enzymes in vitro is inversely related to the length of the esterified acid side group. After a 30-min incubation at 37 degrees C in serum, percentage of hydrolysis for acetyloxy, propionyloxy, butyryloxy, pivaloyloxy and octanoyloxy esters of HPR is 41, 20, 7.5, 1.9 and 1.5, respectively. In contrast, hydrolysis by hepatic esterases is more rapid, particularly for the pivaloyloxy ester. Potency of the various HPR esters in the tracheal organ culture bioassay decreases as the length of the esterified side group increases; the acetyloxy ester is at least 5 times more potent than the octanoyloxy ester.

Topics: Animals; Biotransformation; Cricetinae; Fenretinide; Hydrolysis; In Vitro Techniques; Keratins; Liver; Male; Rats; Rats, Inbred Strains; Trachea; Tretinoin; Vitamin A Deficiency