tretinoin and 4-oxoretinoic-acid

Isotretinoin (13-cis-retinoic acid; 13-cRA) is a differentiation inducer used to treat minimal residual disease after myeloablative therapy for high-risk neuroblastoma. However, more than 40% of children develop recurrent disease during or after 13-cRA treatment. The plasma concentrations of 13-cRA in earlier studies were considered subtherapeutic while 4-oxo-13-cis-RA (4-oxo-13-cRA), a metabolite of 13-cRA considered by some investigators as inactive, were greater than threefold higher than 13-cRA. We sought to define the metabolic pathways of 13-cRA and investigated the anti-tumour activity of its major metabolite, 4-oxo-13-cRA.. Effects of 13-cRA and 4-oxo-13-cRA on human neuroblastoma cell lines were assessed by DIMSCAN and flow cytometry for cell proliferation, MYCN down-regulation by reverse transcription PCR and immunoblotting, and neurite outgrowth by confocal microscopy. 13-cRA metabolism was determined using tandem MS in human liver microsomes and in patient samples.. Six major metabolites of 13-cRA were identified in patient samples. Of these, 4-oxo-13-cRA was the most abundant, and 4-oxo-13-cRA glucuronide was also detected at a higher level in patients. CYP3A4 was shown to play a major role in catalysing 13-cRA to 4-oxo-13-cRA. In human neuroblastoma cell lines, 4-oxo-13-cRA and 13-cRA were equi-effective at inducing neurite outgrowth, inhibiting proliferation, decreasing MYCN mRNA and protein, and increasing the expression of retinoic acid receptor-β mRNA and protein levels.. We showed that 4-oxo-13-cRA is as active as 13-cRA against neuroblastoma cell lines. Plasma levels of both 13-cRA and 4-oxo-13-cRA should be evaluated in pharmacokinetic studies of isotretinoin in neuroblastoma.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Central Nervous System Neoplasms; Chromatography, High Pressure Liquid; Cytochrome P-450 CYP3A; Humans; Isotretinoin; Mice, Inbred BALB C; Microsomes; N-Myc Proto-Oncogene Protein; Neurites; Neuroblastoma; Nuclear Proteins; Oncogene Proteins; Receptors, Retinoic Acid; RNA, Messenger; Tandem Mass Spectrometry; Tretinoin

A double-blind, placebo-controlled, randomized study using single ascending oral doses of 5 mg, 15 mg, 40 mg, 80 mg, and 150 mg of 9-cis-retinoic acid was performed to assess the single-dose pharmacokinetics, tolerability, and pharmacodynamic effects of 9-cis-retinoic acid in healthy men. Forty participants received treatment (six taking the active treatment and two taking placebo for each dose level). The pharmacokinetics of 9-cis-retinoic acid were linear over the dose range studied. Peak plasma concentrations were achieved within 3 to 4 hours on average. The half-life was in the range of 1.3 to 2.4 hours. Metabolism was the major pathway of elimination. 4-Oxo-9-cis-retinoic acid, one of four metabolites measured, which included all-trans-retinoic acid and 13-cis-retinoic acid, was the main metabolite in plasma, achieving peak plasma levels of 41% to 83% of those of 9-cis-retinoic acid. Dose-/concentration-dependent reductions of retinol in plasma, with a maximum of 30% from baseline, were observed 24 hours after administration. Baseline levels were recovered after 5 days. Concentrations of retinol binding protein remained unchanged. Overall, the drug was well tolerated at all dose levels. Adverse events observed were consistent with findings of other retinoids (all-trans-retinoic acid and 13-cis-retinoic acid) and included headache and xeroderma at high dose levels.

Topics: Adult; Alitretinoin; Antineoplastic Agents; Double-Blind Method; Humans; Isotretinoin; Male; Retinol-Binding Proteins; Retinol-Binding Proteins, Plasma; Tretinoin; Vitamin A

Retinoic acid (RA) receptor (RAR) agonists are potential teratogens to various vertebrates. Their contamination has been detected in municipal wastewater in different countries. This study involved field investigations and laboratory batch treatment experiments to elucidate the removal characteristics by activated sludge treatment of RAs (all-trans RA and 13-cis RA) and 4-oxo-RAs (4-oxo-all-trans RA and 4-oxo-13-cis RA), which were identified as major RAR agonists in municipal wastewater. Results obtained in this study show that currently employed activated sludge treatments can remove RAs, 4-oxo-RAs and overall RAR agonist contamination effectively from municipal wastewater in general, although high RAR agonistic activity might sometimes remain in the effluent. Laboratory experiments revealed that RAs were removed rapidly from the aqueous phase by adsorption to the sludge, after which they were removed further by biological and/or chemical degradation. Aside from adsorption to the sludge, 4-oxo-RAs were also apparently removed by biological and chemical degradation. Biodegradation contributed greatly to the removal. Results of additional experiments indicated that novel non-identifiable RAR agonists can occur through the biodegradation of 4-oxo-RAs by activated sludge and that they can persist for a long period.

Topics: Sewage; Time Factors; Tretinoin; Waste Disposal, Fluid; Water Pollutants, Chemical

Topics: Animals; Humans; Japan; Receptors, Retinoic Acid; Sewage; Tretinoin; Two-Hybrid System Techniques; Waste Disposal, Fluid; Water Pollutants, Chemical

13-cis Retinoic acid (13cisRA, isotretinoin) is an important drug in both dermatology, and the treatment of high-risk neuroblastoma. 13cisRA is known to undergo cytochrome P450-mediated oxidation, mainly by CYP2C8, but phase II metabolic pathways have not been characterized. In the present study, the glucuronidation activities of human liver (HLM) and intestinal microsomes (HIM), as well as a panel of human UDP-glucuronosyltransferases (UGTs) toward both 13cisRA and the 4-oxo metabolite, 4-oxo 13cisRA, were compared using high-performance liquid chromatography. Both HLM and, to a greater extent, HIM catalyzed the glucuronidation of 13cisRA and 4-oxo 13cisRA. Based on the structures of 13cisRA and 4-oxo 13cisRA, the glucuronides formed are conjugated at the terminal carboxylic acid. Further analysis revealed that UGT1A1, UGT1A3, UGT1A7, UGT1A8, and UGT1A9 were the major isoforms responsible for the glucuronidation of both substrates. For 13cisRA, a pronounced substrate inhibition was observed with individual UGTs and with HIM. UGT1A3 exhibited the highest rate of activity toward both substrates, and a high rate of activity toward 13cisRA glucuronidation was also observed with UGT1A7. However, for both substrates, K(m) values were above concentrations reported in clinical studies. Therefore, UGT1A9 is likely to be the most important enzyme in the glucuronidation of both substrates as this enzyme had the lowest K(m) and is expressed in both the intestine and at high levels in the liver.

Topics: Glucuronides; Glucuronosyltransferase; Humans; In Vitro Techniques; Intestinal Mucosa; Isoenzymes; Isotretinoin; Kinetics; Microsomes; Microsomes, Liver; Substrate Specificity; Tretinoin

We show here the role of retinoic acid receptor (RAR) beta and alpha signalling in proliferation and differentiation of endogenous adult forebrain neural progenitor cells (NPCs). RARbeta activation stimulates Sonic hedgehog signalling (Shh), and induces the proliferation of the NPCs. They can be induced to become Doublecortin (DCX) expressing migrating neuroblasts by RARalpha signalling, some of which differentiate into cholinergic neurons. The same signalling pathways cause the proliferation of embryonic forebrain NPCs. These cells express glial fibrillary acidic protein (GFAP) and are predominantly uni/bipolar, two characteristics of neuronal progenitor cells. We further show that fibroblast growth factor (FGF) signalling, induces the expression of the retinoic acid degrading enzyme cytochrome P450 (cyp) 26a1, and that one of its products, 4-oxo-RA, mimics the action of the RARalpha agonist in the differentiation of the NPCs into cholinergic neurons.

Topics: Animals; Cell Differentiation; Cells, Cultured; Doublecortin Domain Proteins; Doublecortin Protein; Enzyme Inhibitors; Fibroblast Growth Factors; Hedgehog Proteins; Imidazoles; Microtubule-Associated Proteins; Neurons; Neuropeptides; Prosencephalon; Pyrroles; Rats; Rats, Wistar; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Signal Transduction; Stem Cells; Tretinoin

We previously demonstrated that 4-oxoretinol (4-oxo-ROL) activated retinoic acid receptors (RARs) in F9 stem cells. We showed that 4-oxo-ROL inhibited the proliferation of normal human mammary epithelial cells (HMECs). To understand the mechanisms by which 4-oxo-ROL regulates HMEC growth we examined gene expression profiles following 4-oxo-ROL or all-trans retinoic acid (tRA). We also compared growth inhibition by tRA, 4-oxo-ROL, or 4-oxo-RA. All three retinoids inhibited HMEC proliferation. Gene expression analyses indicated that 4-oxo-ROL and tRA modulated gene expression in closely related pathways. The expression of many genes, e.g. ATP-binding cassette G1 (ABCG1); adrenergic receptorbeta2 (ADRB2); ras-related C3 botulinum toxin substrate (RAC2); and short-chain dehydrogenase/reductase 1 gene (SDR1) was changed after 4-oxo-ROL or tRA. Metabolism of these retinoids was analyzed by high-performance liquid chromatography (HPLC). In 1 microM tRA treated HMECs all of the tRA was found intracellularly, and tRA was the predominant intracellular retinoid. In 1 microM 4-oxo-ROL treated HMECs most 4-oxo-ROL was esterified to 4-oxoretinyl esters, no tRA was detected, and 4-oxo-ROL and 4-oxo-RA were observed intracellularly. In 1 microM 4-oxoretinoic acid (4-oxo-RA) treated HMECs little intracellular 4-oxo-RA was detected; most 4-oxo-RA was in the medium. Our results indicate that: (a) 4-oxo-ROL regulates gene expression and inhibits proliferation of HMECs; (b) 4-oxo-ROL and tRA regulate some of the same genes; (c) more tRA is found in cells, as compared to 4-oxoretinoic acid, when each drug is added at the same concentration in the medium; and (d) the mechanism by which 4-oxo-ROL exerts its biological activity does not involve intracellular tRA production.

Topics: Biological Transport; Cell Proliferation; Cells, Cultured; Chromatography, High Pressure Liquid; Epithelial Cells; Female; Gene Expression Profiling; Gene Expression Regulation; Humans; Mammary Glands, Human; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors; Transcription, Genetic; Tretinoin; Vitamin A

Isotretinoin is effective in the treatment of severe acne and rosacea. Both parent drug and its main metabolite 4-oxo-isotretinoin are potentially teratogenic compounds and contain a carboxylic acid moiety. In the presence of ethanol, naturally occurring as well as synthetic retinoids also containing a carboxylic acid moiety are capable of undergoing an ethyl esterification with the metabolic formation of more lipophilic compounds with a much longer terminal half-life.. To determine if isotretinoin (13-cis-RA), its main metabolite 4-oxo-isotretinoin (4-oxo-13-cis-RA), and other possible metabolites in the presence or absence of ethanol are converted to their corresponding ethyl derivatives in patients with severe acne or rosacea after multiple isotretinoin dosing. In addition, pharmacokinetic parameters of the parent drug and its 4-oxo metabolite were determined.. Eleven patients with severe acne or rosacea were treated with isotretinoin daily for 3 months and investigated pharmacokinetically during 24 h after 1 month of treatment and for up to 28 days after discontinuation of therapy. A possible influence of ethanol was evaluated using a simple self-administered questionnaire and by measuring serum ethanol levels during treatment. The concentrations of isotretinoin, 4-oxo-isotretinoin and possible ethylated and nonethylated metabolites were measured by reverse-phase high-performance liquid chromatography.. Although seven of 11 patients had a considerable weekly alcohol intake, no endogenous synthesis of ethyl derivatives of isotretinoin, the main 4-oxo metabolite or the all-trans compounds was chromatographically detectable in any of the patients' plasma samples during the treatment period. Multiple dose pharmacokinetic data for the parent drug and its main metabolite were comparable to previous studies.. The metabolism and pharmacokinetics of isotretinoin and its main metabolites are not influenced by ethanol during long-term isotretinoin treatment. After ceasing long-term isotretinoin therapy the recommended period of 1 month for using anticonceptive measures in fertile women seems adequate.

Topics: Acne Vulgaris; Adult; Alcohol Drinking; Chromatography, High Pressure Liquid; Dermatologic Agents; Ethanol; Female; Humans; Isotretinoin; Male; Middle Aged; Rosacea; Surveys and Questionnaires; Tretinoin; Young Adult

Retinoid signaling is essential for development of vertebrate embryos, and its action is mainly through retinoic acid (RA) binding to its RA receptors and retinoid-X receptors, while the critical concentration and localization of RA in embryos are determined by the presence and activity of retinal dehydrogenases (for RA synthesis) and cytochrome P450 RAs (Cyp26s) (for degradation of RA). Previously, we identified a novel cyp26 gene (cyp26d1) in zebrafish that is expressed in hindbrain during early development. Using reverse-phase HPLC analyses, we show here that zebrafish Cyp26D1 expressed in 293T cells could metabolize all-trans RA, 9-cis RA, and 13-cis RA, but could not metabolize retinol or retinal. The metabolites of all-trans RA produced by Cyp26D1 were the same as that produced by Cyp26A1, which are mainly 4-hydroxy-all-trans-RA and 4-oxo-all-trans-RA. Performing mRNA microinjection into zebrafish embryos, we demonstrated that overexpression of Cyp26D1 in embryos not only caused the distance between rhombomere 5 and the first somite of the injected embryos to be shorter than control embryos but also resulted in left-right asymmetry of somitogenesis in the injected embryos. These alterations were similar to those caused by the overexpression of cyp26a1 in zebrafish embryos and to that which resulted from treating embryos with 1 microm 4-diethylamino-benzaldehyde (retinal dehydrogenase inhibitor), implying that cyp26d1 can antagonize RA activity in vivo. Together, our in vitro and in vivo results provided direct evidence that zebrafish Cyp26D1 is involved in RA metabolism.

Topics: Amino Acid Sequence; Animals; Conserved Sequence; Cytochrome P-450 Enzyme System; Embryo, Nonmammalian; Molecular Sequence Data; Protein Structure, Tertiary; Retinaldehyde; Retinoic Acid 4-Hydroxylase; Sequence Homology, Amino Acid; Substrate Specificity; Tissue Distribution; Tretinoin; Vitamin A; Zebrafish Proteins

We have investigated the relative efficacy of a range of natural and synthetic retinoids on the induction of alveolar regeneration in a dexamethasone-treated mouse model. The aim was to explore the roles of the different retinoic acid receptors using receptor-selective agonists and to determine whether other natural retinoids in addition to all-trans-retinoic acid (tRA) were effective. Dexamethasone treatment of newborn pups led to a reduced lung surface area and increased mean chord length. Subsequently, tRA induced alveolar repair, improved mean chord length, and improved the lung surface area to volume ratio. We found that 4-oxo-RA and a retinoic acid receptor (RAR) alpha-selective compound were as effective as tRA at inducing alveolar regeneration, with neither showing a significantly better efficacy. An RARbeta-selective compound was also effective, whereas a RARgamma-selective compound was not. Other retinoids, such as 9-cis-RA, 13-cis-RA, retinol, and a pan retinoid X receptor (RXR) agonist, do not induce significant responses. Neither did granulocyte colony-stimulating factor. We also showed that an RARbeta-null mutant mouse line responded to dexamethasone by failing to develop alveoli appropriately and that tRA induced alveolar regeneration, suggesting that RARbeta was not required for the regenerative response.

Topics: Animals; Animals, Newborn; Animals, Outbred Strains; Dexamethasone; Female; Injections, Subcutaneous; Male; Mice; Mice, Knockout; Pregnancy; Pulmonary Alveoli; Receptors, Retinoic Acid; Regeneration; Retinoic Acid Receptor alpha; Retinoids; Tretinoin

Retinoic acid isomers, 4-oxo-retinoic acid isomers and retinol are present in the serum of mammals. In this study a high-performance liquid chromatography (HPLC) separation, sample preparation and tandem mass spectrometry (MS/MS) method was established for quick and easy sample preparation and sensitive determination of retinoids such as all-trans-4-oxo-retinoic acid, 13-cis-4-oxo-retinoic acid, 13-cis-retinoic acid, 9-cis-retinoic acid, all-trans-retinoic acid and retinol in serum and cell extracts. Serum samples were simply treated with three times the volume of isopropanol, dried under vacuum, taken up in the HPLC solvent and immediately put into the autosampler for an automated single-run HPLC analysis. With this MS/MS method we were able to detect 7 pg and quantify 20 pg of all-trans-retinoic acid, 4-oxo-all-trans-retinoic acid and retinol directly on-column and were able to determine a concentration as low as 0.2 ng/mL in ethanolic standards and in biological samples. This method allows ultra-sensitive detection, excellent selectivity and a very simple sample preparation to determine retinoic acids, 4-oxo-retinoic acids and retinol in serum and cell extracts for the study of endogenous retinoids.

Topics: Atmospheric Pressure; Chromatography, High Pressure Liquid; Humans; Leukocytes, Mononuclear; Reproducibility of Results; Sensitivity and Specificity; Tandem Mass Spectrometry; Tretinoin; Uncertainty; Vitamin A

Retinoic acid exerts a variety of effects on gene transcription that regulate growth, differentiation, and inflammation in normal and neoplastic skin cells. Because there is a lack of information regarding the influence of metabolic transformation of retinoids on their pharmacologic effects in skin, we have analyzed the functional activity of all-trans-, 9-cis-, and 13-cis-retinoic acid and their 4-oxo-metabolites in normal human epidermal keratinocytes (NHEKs) and dermal fibroblasts using gene and protein expression profiling techniques, including cDNA microarrays, two-dimensional gel electrophoresis, and MALDI-MS. It was previously thought that the 4-oxo-metabolites of RA are inert catabolic end-products but our results indicate instead that they display strong and isomer-specific transcriptional regulatory activity in both NHEKs and dermal fibroblasts. Microarray and proteomic analyses identified a number of novel genes/gene products that are influenced by RA treatment of NHEKs or fibroblasts, including genes for enzymes catalyzing biotransformation of retinoids, corticosteroids, and antioxidants and structural and transport proteins known to be essential for homeostasis. Our results expand current knowledge regarding retinoic acid action within skin cells and the target tissue/cell regulatory systems that are important for modulating the physiological and pharmacological effects of this important class of dermatological drugs.

Topics: Alitretinoin; Electrophoresis, Gel, Two-Dimensional; Fibroblasts; Fluorescent Antibody Technique; Gene Expression Regulation; Humans; In Vitro Techniques; Isotretinoin; Keratinocytes; Male; Oligonucleotide Array Sequence Analysis; Polymerase Chain Reaction; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tretinoin

The cellular retinoic acid (RA) binding proteins I and II (CRABPI and CRABPII), intracellular proteins which bind retinoic acid with high affinity, are involved in the actions of RA, though their exact roles are not fully understood. We have generated several genetically engineered AB1 cell lines in which both alleles of the CRABPI gene have been deleted by homologous recombination. We have used these CRABPI knockout cell lines to examine the consequences of functional loss of CRABPI on RA-induced gene expression and RA metabolism in the murine embryonic stem cell line, AB1, which undergoes differentiation in response to RA. Complete lack of CRABPI results in decreased intracellular [3H]RA concentrations under conditions in which external concentrations of [3H]RA are low (1-10nM) and in an altered distribution of [3H] polar metabolites of [3H]RA in the cell and in the medium. Fewer [3H] polar metabolites are retained within the CRABPI(-/-) cells compared to the wild-type cells. These data suggest that CRABPI functions to regulate the intracellular concentrations of retinoic acid and to maintain high levels of oxidized retinoic acid metabolites such as 4-oxoretinoic acid within cells.

Topics: Animals; Cell Differentiation; Cells, Cultured; Embryo, Mammalian; Gene Deletion; Gene Expression Regulation, Developmental; Genetic Engineering; Homozygote; Kinetics; Mice; Receptors, Retinoic Acid; Recombination, Genetic; RNA, Messenger; Stem Cells; Tretinoin

Recent studies from our laboratory have indicated that the metabolism of vitamin A (retinol) to retinyl esters, carried out primarily by the enzyme lecithin:retinol acyltransferase (LRAT), is greatly reduced in human carcinoma cell lines of the oral cavity, skin, breast, and kidney as compared with their normal epithelial counterparts. These studies suggest that human carcinoma cells are retinoid-deficient relative to normal epithelial cells. In this study, we examined the metabolism of [(3)H]retinol and [(3)H]retinoic acid (RA) in human prostate cancer lines and in primary cultures of human prostate epithelial cells. Normal cells esterified all of the [(3)H]retinol added to the cultures. In contrast, all seven prostate cancer cell lines and four primary cultures derived from prostatic adenocarcinomas metabolized only trace amounts of [(3)H]retinol to [(3)H]retinyl esters. Correlated with this relative lack of esterification of [(3)H]retinol by the cancer cells was loss of expression of LRAT protein, whereas normal cells expressed abundant levels of LRAT protein by Western analysis. The metabolism of [(3)H]RA was also examined in these prostatic cells. Two of the prostate cancer tumor lines, DU 145 and PJ-1, exhibited rapid metabolism of [(3)H]RA; in contrast, the other tumor lines or primary cultures metabolized [(3)H]RA at a much slower rate. We also found that the immortalization of normal human prostatic epithelial cells by SV40 T antigen led to a reduction in LRAT protein expression and esterification of [(3)H]retinol. Further transformation to tumorigenicity with the ras oncogene resulted in loss of detectable LRAT expression. Finally, we analyzed LRAT protein expression in tissue sections from six prostatectomy specimens by immunohistochemistry. LRAT protein was predominantly expressed in the basal cells of normal prostatic epithelium, whereas its expression was lost in prostate cancer. Collectively, these data implicate aberrant retinoid metabolism in the process of prostatic carcinogenesis.

Topics: Acyltransferases; Antineoplastic Agents; Cell Division; Epithelial Cells; Humans; Male; Prostatic Neoplasms; Tretinoin; Tumor Cells, Cultured; Vitamin A

Long-term and excessive ethanol intake results in decreased plasma and hepatic levels of retinoic acid (RA), the most active derivative of vitamin A. The decrease of RA by ethanol treatment has been proposed to be a cytochrome P450 enzyme (CYP)-dependent process. However, the role of the major ethanol-induced CYP, CYP2E1, in the metabolism of RA has not been defined.. In vitro incubations of RA with microsomal fractions of liver tissue containing CYPs from either ethanol-exposed or non-ethanol-exposed rats were carried out using chemical inhibitors and antibodies against various CYPs. In vivo, both ethanol-exposed and non-ethanol-exposed rats were treated with or without chlormethiazole, a specific CYP2E1 inhibitor, for 1 month. RA and its catabolic metabolites were analyzed by high-performance liquid chromatography and spectral analysis.. Incubation of RA with the liver microsomal fraction from ethanol-exposed rats resulted in greater disappearance of RA and increased appearance of 18-hydroxy-RA and 4-oxo-RA compared with control rat liver microsomal fractions. The enhancement of RA catabolism by ethanol was inhibited by both CYP2E1 antibody and specific inhibitors (allyl sulfide and chlormethiazole) in a dose-dependent fashion, whereas the metabolism of RA into polar metabolites was abolished completely by nonspecific CYP inhibitors (disulfiram and liarozole). Furthermore, treatment with chlormethiazole in ethanol-fed rats in vivo restored both hepatic and plasma RA concentrations to normal levels.. Ethanol-induced CYP2E1 plays a major role in the degradation of RA, which may provide a possible biochemical mechanism for chronic and excessive ethanol intake as a risk for both hepatic and extrahepatic cell proliferation and carcinogenesis.

Topics: Allyl Compounds; Animals; Antibodies; Antioxidants; Cell Fractionation; Central Nervous System Depressants; Chlormethiazole; Cytochrome P-450 CYP2E1; Enzyme Activation; Ethanol; GABA Modulators; In Vitro Techniques; Liver; Male; Microsomes; Rats; Rats, Sprague-Dawley; Sulfides; Tretinoin

The metabolism of all-trans retinoic acid (ATRA) has been reported to be partly responsible for the in vivo resistance to ATRA seen in the treatment of human acute promyelocytic leukemia (APL). However, ATRA metabolism appears to be involved in the growth inhibition of several cancer cell lines in vitro. The purpose of this study was to evaluate the in vitro activity of the principal metabolites of ATRA [4-hydroxy-retinoic acid (4-OH-RA), 18-hydroxy-retinoic acid (18-OH-RA), 4-oxo-retinoic acid (4-oxo-RA), and 5,6-epoxy-retinoic acid (5,6-epoxy-RA)] in NB4, a human promyelocytic leukemia cell line that exhibits the APL diagnostic t(15;17) chromosomal translocation and expresses the PML-RAR alpha fusion protein. We established that the four ATRA metabolites were indeed formed by the NB4 cells in vitro. NB4 cell growth was inhibited (69-78% at 120 h) and cell cycle progression in the G1 phase (82-85% at 120 h) was blocked by ATRA and all of the metabolites at 1 microM concentration. ATRA and its metabolites could induce NB4 cells differentiation with similar activity, as evaluated by cell morphology, by the nitroblue tetrazolium reduction test (82-88% at 120 h) or by the expression of the maturation specific cell surface marker CD11c. In addition, nuclear body reorganization to macropunctated structures, as well as the degradation of PML-RAR alpha, was found to be similar for ATRA and all of its metabolites. Comparison of the relative potency of the retinoids using the nitroblue tetrazolium reduction test showed effective concentrations required to differentiate 50% of cells in 72 h as follows: ATRA, 15.8 +/- 1.7 nM; 4-oxo-RA, 38.3 +/- 1.3 nM; 18-OH-RA, 55.5 +/- 1.8 nM; 4-OH-RA, 79.8 +/- 1.8 nM; and 5,6-epoxy-RA, 99.5 +/- 1.5 nM. The ATRA metabolites were found to exert their differentiation effects via the RAR alpha nuclear receptors, because the RAR alpha-specific antagonist BMS614 blocked metabolite-induced CD11c expression in NB4 cells. These data demonstrate that the principal ATRA Phase 1 metabolites can elicit leukemia cell growth inhibition and differentiation in vitro through the RAR alpha signaling pathway, and they suggest that these metabolites may play a role in ATRA antileukemic activity in vivo.

Topics: Antineoplastic Agents; Cell Cycle; Cell Differentiation; Cell Division; Dose-Response Relationship, Drug; Granulocytes; Humans; Integrin alphaXbeta2; Leukemia, Promyelocytic, Acute; Neoplasm Proteins; Oncogene Proteins, Fusion; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Time Factors; Tretinoin; Tumor Cells, Cultured

Clinical and preclinical studies suggest that retinoids can inhibit the growth of a small percentage of human renal cancers (RCs), although the majority of RCs both in vitro and in vivo are retinoid resistant. Our recent studies indicate that the metabolism of retinol to retinyl esters is greatly reduced in human carcinoma cell lines of the oral cavity, skin, and breast as compared with their normal epithelial counterparts, suggesting that human carcinoma cells are retinoid deficient relative to normal epithelial cells. We considered whether retinoid resistance in RCs was related to an abnormality in retinoid metabolism. The metabolism of [3H]retinol and of [3H]retinoic acid (RA) was examined in RC cell lines and normal human kidney (NK) epithelial cells cultured in media, in RA, or in RA plus IFN-alpha. The expression of LRAT (lecithin:retinol acyltransferase) was assessed by Northern and Western analysis. Retinol and retinyl ester levels were determined in tissue samples of normal human kidney and renal cell carcinoma. NK cells esterified all of the 50 nM [3H]retinol in which they were cultured. In contrast, six of the seven RC cell lines metabolized only trace amounts of [3H]retinol to [3H]retinyl esters. Consistent with this relative lack of [3H]retinol esterification by the tumor cells, the tumor cells exhibited LRAT transcripts of aberrantly low sizes relative to those in normal epithelial cells. Moreover, the NK cells expressed abundant levels of LRAT protein by Western analysis, whereas the RC cells did not express LRAT protein. When samples of human kidney tumor tissue were compared with samples of normal kidney tissue from patients who had undergone surgery for primary RC, the normal kidney tissues contained much higher levels of retinol and retinyl esters (approximately 0.5-2 microg/gram wet weight) than the tumor tissues in all seven patients examined. Culture of the RC lines in IFN-alpha plus all-trans-RA, a combination therapy used clinically, resulted in higher intracellular levels of [3H]retinol and [3H]retinyl esters. The metabolism of [3H]RA was also examined in these RC lines versus NK cells. Although the NK epithelial cells metabolized [3H]RA, the majority of the RC lines metabolized [3H]RA at a much slower rate. Most of the RC lines metabolized only 10-30% of the 50 nM [3H]RA over 6 h of culture. These data indicate that RCs both in vitro and in vivo are retinol and retinyl ester deficient relative to the normal human kidney, and they

Topics: Acyltransferases; Aged; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Renal Cell; Cell Division; Cytochrome P-450 Enzyme System; Esters; Female; Gene Expression; Humans; Interferon alpha-2; Interferon-alpha; Kidney Neoplasms; Kidney Tubules, Proximal; Male; Middle Aged; Recombinant Proteins; Retinoic Acid 4-Hydroxylase; Tretinoin; Tumor Cells, Cultured; Vitamin A

Significant differences between the metabolism of retinoic acid by different tissues might be an important determinant of the effectiveness of a systemically administered inhibitor at a particular tissue site. Here the metabolism of retinoic acid has been studied in microsomal fractions from different tissues (liver, kidney, intestinal mucosa, lung, skin, brain) of the male rat to determine their relative metabolic activity. Kinetic analysis revealed major differences between the activity of different tissue microsomes. This is shown by the Vmax values for the metabolism of retinoic acid-liver (102+/-39.0 pmol (mg protein)(-1) min(-1)) was 100 times more active than the lung (1+/-0.03 pmol (mg protein)(-1) min(-1)), which was the least active. The range of Km values for microsomes from the different tissues was narrow (0.48-1.40 microM). Taking into account the mass of the tissue, the gross activity ranking for metabolism of retinoic acid was liver >> skin = kidney > brain > intestinal mucosa >> lung. It is concluded that metabolism of administered retinoic acid occurs mainly in the liver but that cellular retinoic acid levels in some other tissues (skin, kidney, brain) could be reduced (metabolized) to such an extent that higher levels might be observed after the use of inhibitors of retinoic acid metabolism.

Topics: Animals; Antineoplastic Agents; Brain; Intestinal Mucosa; Kidney; Liver; Lung; Male; Microsomes; Rats; Rats, Wistar; Skin; Tretinoin

HaCaT keratinocytes differ from normal human epidermal keratinocytes (HEK) by constitutive expression of differentiation markers which are normally suppressed by vitamin A. In search of an explanation for this discrepancy we compared the vitamin A content, the expression of retinoid-binding proteins, and the vitamin A metabolism in the two cell types. The concentrations of retinol and 3,4-didehydroretinol in cultured HaCaT cells were less than one-fifth those in HEK, and the content of fatty acyl esters was even lower. Similarly, the concentrations of cellular retinol-binding protein and cellular retinoic acid-binding protein (CRBPI and CRABPII, respectively) were 10-30 times lower in HaCaT cells than in HEK corresponding to a reduced mRNA expression of these proteins. Unexpectedly, HaCaT cells expressed RARbeta in addition to RARalpha, RARgamma and RXRalpha, which are nuclear receptors normally found in HEK. Radioactive retinol added to the culture medium appeared only transiently in HaCaT cells, and pulse labeling confirmed a defective cellular retention of retinyl esters. After 24 h of incubation with [3H]retinol, cell-associated radioactivity corresponding to retinol, 3,4-didehydroretinol, all-trans-retinoic acid and 3,4-didehydroretinoic acid was found in both HaCaT cells and HEK. [3H]Retinoic acid showed a more rapid metabolism to 4-hydroxy/4-keto-retinoic acid in HaCaT cells than in HEK, which could be explained by a higher expression of cytochrome p450RAI in the former cells. In conclusion, the abnormal uptake of vitamin A and low levels of retinoid binding proteins in HaCaT cells, linked with an aberrant metabolism of retinol, may help to explain why these cells differentiate also in the presence of retinoids.

Topics: Biomarkers; Carrier Proteins; Cell Differentiation; Cell Line; Cytochrome P-450 Enzyme System; Diterpenes; Humans; Keratinocytes; Receptors, Retinoic Acid; Reference Values; Retinoic Acid 4-Hydroxylase; Retinoids; Retinyl Esters; Tretinoin; Vitamin A

Vitamin A is an intrinsic modulator of proliferation and differentiation in human epidermis, and may be destroyed by ultraviolet radiation (UVR) impinging on the skin. To identify the deleterious effects of a perturbed cellular vitamin A status, we investigated the endogenous retinoid concentrations and the metabolism of [3H]retinol and all-trans [3H]retinoic acid in cultured human keratinocytes and melanocytes exposed to UVR, using high performance liquid chromatography. Before UVR the retinoid content was similar in keratinocytes and melanocytes, but the uptake of [3H]retinol was three-fold higher and the uptake of [3H]retinoic acid was 10-fold higher in the melanocytes. In both cell types, UVR (UVA 360 mJ/cm2 plus UVB 140 mJ/cm2) instantaneously reduced the concentration of retinol by about 50% and that of 3,4-didehydroretinol by about 20%. The retinoid concentrations returned to normal within 1-2 days post-irradiation, despite there being no overt increase in the uptake of [3H]retinol or the biosynthesis of 3,4-didehydroretinol. However, in both types of irradiated cells, the accumulation of the biologically most active metabolite, all-trans [3H]retinoic acid, was about 60% higher than in control cells. Furthermore, the metabolism of authentically supplied [3H]retinoic acid was reduced, especially in irradiated keratinocytes, which probably contributed to the restoration of retinoid levels after UV exposure.

Topics: Cells, Cultured; Humans; Isomerism; Keratinocytes; Melanocytes; Time Factors; Tretinoin; Tritium; Ultraviolet Rays; Vitamin A

Isotretinoin for oral therapy in severe acne conglobata and acne nodulocystica represents a significant achievement; however, the drug exerts several mucocutaneous and systemic adverse effects, besides its teratogenic potency.. The aim of this study was to investigate the plasma levels of isotretinoin and of 4-oxo-isotretinoin over long-term treatment of severe acne and to assess any correlation with the given dose, the clinical improvement and the occurrence of side effects.. Forty-one patients with severe acne and acne-related disorders were studied under long-term oral intake of isotretinoin. Therapeutic effects and side effects were evaluated prior, during and at the end of therapy. The plasma levels of isotretinoin and of its major metabolite 4-oxo-isotretinoin were measured by reversed-phase HPLC and were correlated with the administered oral dose and the number and frequency of side effects.. Dose-dependent plasma levels of isotretinoin and its metabolite were observed. At a mean dosage of 0.75-1.0 mg/kg/day, 404 +/- 142 ng/ml were measured, whereas the plasma levels of 4-oxo-isotretinoin were 1-2x higher. The plasma levels correlated well with the orally administered dose of isotretinoin and the observed mucocutaneous side effects.. The study demonstrates that measuring of the plasma levels may be a helpful tool to monitor the individual therapeutic dose regimen in patients with severe acne in order to minimize undesired side effects and to control oral intake.

Topics: Acne Vulgaris; Administration, Oral; Adolescent; Adult; Chromatography, High Pressure Liquid; Drug Monitoring; Female; Humans; Isotretinoin; Male; Middle Aged; Tretinoin

Isotretinoin is the most potent human teratogen on the market. Women for whom contraception fails may conceive during or soon after discontinuing isotretinoin therapy, making its elimination kinetics a crucial determinant of fetal safety. The steady-state pharmacokinetics of isotretinoin and its major 4-oxo metabolite were studied in 16 adult patients treated for acne who were receiving doses that ranged from 0.47 to 1.7 mg/kg daily. This is the first study of the pharmacokinetics of isotretinoin in women of childbearing age (n = 11). The clinical efficacy and tolerability of isotretinoin was investigated, and the correlation between these data and steady-state serum concentrations of isotretinoin was tested. The concentration-time data best fitted a two-compartment open model with linear elimination. There was no correlation between efficacy and tolerability of isotretinoin and steady-state serum concentrations. There was no correlation between dose of isotretinoin and steady-state concentration, due to the large variability in apparent clearance. Values for elimination half-life (t1/2) of isotretinoin and its metabolite were 29+/-40 hours and 22+/-10 hours, respectively. These data suggest a longer elimination t1/2 of the parent drug than previously reported. This is probably due to the longer sampling time used in this study (as long as 28 days). This study suggests that a greater variability exists in the safe time after discontinuation of the drug for onset of conception.

Topics: Abnormalities, Drug-Induced; Acne Vulgaris; Adolescent; Adult; Area Under Curve; Chromatography, High Pressure Liquid; Female; Half-Life; Humans; Isotretinoin; Keratolytic Agents; Male; Pregnancy; Teratogens; Tretinoin

The testicular gene expression of the retinoic acid receptors, RAR alpha, -beta, and -gamma, was studied in normal mice and in vitamin A-deficient mice after the administration of all-trans-retinoic acid (ATRA). All three types of RARs were expressed in normal and/or vitamin A-deficient testes. Only the expression of RAR beta messenger RNA was transiently induced within 24 h after ATRA injection. ATRA-induced RAR beta expression was also found in purified Sertoli cells, suggesting that these cells mediate at least part of the effect of retinoids on germ cells. When an equimolar amount of retinol was administered instead of ATRA, no induction of RAR beta was seen at the point of maximal induction by ATRA, suggesting that the effect of retinol was delayed and probably less. The related nuclear receptors, RXR alpha, -beta, and, for the first time, gamma, were also shown to be present in the mouse testis. Upon administration of ATRA, messenger RNA expression of RXR alpha and -beta did not change significantly. The expression of RXR gamma was too low to allow quantification. Finally, the effect of the retinoid metabolism inhibitor liarozole on ATRA-induced proliferation of A spermatogonia was examined. The labeling index of A spermatogonia, 24 h after the administration of 0.25 mg ATRA, was significantly lowered by liarozole due to a shift of the maximal 5-bromo-deoxyuridine incorporation to an earlier point (20 h). This indicates that liarozole delays retinoid metabolism, thereby increasing the actual ATRA concentration, and more importantly, that ATRA by itself is an active retinoid in spermatogenesis. Apparently, ATRA does not need to be metabolized to 4-oxo-RA, which was previously shown to be a more potent inducer of spermatogonial proliferation than ATRA, to be effective.

Topics: Animals; Gene Expression Regulation; Imidazoles; Male; Mice; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoic Acid Receptor gamma; Retinoid X Receptors; RNA, Messenger; Spermatogenesis; Testis; Transcription Factors; Tretinoin

A fully automated isocratic high-performance liquid chromatographic method for the determination of 9-cis-retinoic acid, 13-cis-retinoic acid, all-trans-retinoic acid, 4-oxo-13-cis-retinoic acid and 4-oxo-all-trans-retinoic acid, has been developed using on-line solid-phase extraction and a column switching technique allowing clean-up and pre-concentration in a single step. A 500-microliter sample of serum was diluted with 750 microliters of a solution containing 20% acetonitrile and the internal standard 9,10-dimethylanthracene. About 1000 microliters of this mixture was injected on a 20 x 4.6 mm I.D. poly ether ether ketone (PEEK) pre-column with titanium frits packed with Bondapak C18, 37-53 microns, 300 A particles. Proteins and very polar compounds were washed out to waste, from the pre-column, with 0.05% trifluoroacetic acid (TFA)-acetonitrile (8.5:1.5, v/v). More than 200 aliquots of diluted serum could be injected on this pre-column before elevated back-pressure enforces replacement. Components retained on the pre-column were backflushed to the analytical column for separation and detection at 360 nm. Baseline separation was achieved using a single 250 x 4.6 mm I.D. Suplex pKb-100 column and a mobile phase containing 69:10:2:16:3 (v/v) of acetonitrile-methanol-n-butanol-2% ammonium acetate-glacial acetic acid. A total time of analysis of less than 30 min, including sample preparation, was achieved. Recoveries were in the range of 79-86%. The limit of detection was 1-7 ng/ml serum and the precision, in the concentration range 20-1000 ng/ml, was between 1.3 and 4.5% for all five compounds. The method was applied for the analysis of human serum after oral administration of 60 mg Roaccutan. The method is well suited for pharmacological studies, while the endogenous levels of some retinoic acid isomers are below the limit of quantitation.

Topics: Acetonitriles; Alitretinoin; Autoanalysis; Chromatography, High Pressure Liquid; Drug Stability; Humans; Isotretinoin; Linear Models; Reproducibility of Results; Retinoids; Sensitivity and Specificity; Tretinoin; Trifluoroacetic Acid

Cytochrome P450 expression in liver is influenced by several factors, including sex and strain. Whereas little is known about their metabolic capabilities, Hairless rats are widely used for the studies of tropical agents. We compared Sprague-Dawley and Hairless rat metabolic behavior to validate the use of Hairless rats in pharmacokinetic and metabolic studies of topically applied drugs. Liver microsomes of male and female rats of both strains were used to investigate the in vitro metabolism of three retinoic acid (RA) isomers: all-trans-RA, 13-cis-RA, and 9-cis-RA. In all cases, a major isomerization of the tested isomer in the two others was observed. This process was independent of the presence of NADPH, but depended on the presence of microsomal proteins. In addition, we observed, to a lesser extent, the formation of 4-oxo metabolites (4-oxo-all-trans-RA, 4-oxo-13-cis-RA, and 4-oxo-9-cis-RA), with the rate of formation of each of these compounds varying with the nature of the isomer incubated. The 4-oxo metabolites formed were statistically greater in male than in female rats in the two strains studied. No significant difference in RA biotransformation was observed between Sprague-Dawley and Hairless rats. In addition, no major difference was observed between the two strains concerning the expression of the different cytochrome P450 isoforms studied. In conclusion, phase I metabolism of RAs characterized by C4-hydroxylation varied with sex, but not within the two strains studied in rats. These results strengthen the relevance of the use of Hairless rats in pharmacokinetic and metabolic studies of topical agents, including retinoids.

Topics: Alitretinoin; Animals; Cytochrome P-450 Enzyme System; Cytochromes b5; Female; In Vitro Techniques; Isoenzymes; Isotretinoin; Male; Microsomes, Liver; Rats; Rats, Sprague-Dawley; Sex Factors; Species Specificity; Stereoisomerism; Tretinoin

Previous studies have shown that all-trans-retinoic acid (RA) inhibits in vitro proliferation of hormone-dependent human breast cancer cells but not the growth of hormone-independent cells. Here we report on RA metabolism in breast cancer cells as examined by high performance liquid chromatography analysis and found a correlation with sensitivity to growth inhibition by RA. RA-sensitive T-47D and MCF-7 cells exhibited high rate metabolism to polar metabolites, whereas RA-resistant MDA-MB-231 and MDA-MB-468 cells metabolized RA to a much lesser extent, and almost no polar metabolites could be detected. The high metabolic rate in RA-sensitive cells appears to be the result of autoinduction of RA metabolism, whereas RA-resistant cells showed no such induction of metabolism. We observed furthermore that transfection with retinoic acid receptor-alpha expression vectors in RA-resistant MDA-MB-231 cells resulted in increased RA metabolism and inhibition of cell proliferation. Metabolism of RA, however, seems not to be required to confer growth inhibition of human breast cancer cells. The biological activity of the polar metabolites formed in RA-sensitive cells was found to be equal or lower than that of RA, indicating that RA itself is the most active retinoid in these cells. Together our data suggest that RA-sensitive cells contain mechanisms to activate strongly the catabolism of RA probably to protect them from the continuous exposure to this active retinoid.

Topics: Antineoplastic Agents; Biotransformation; Breast Neoplasms; Cell Division; Cell Polarity; Chromatography, High Pressure Liquid; Drug Resistance, Neoplasm; Female; Humans; Tretinoin; Tumor Cells, Cultured

Retinoids can reverse potentially premalignant lesions and prevent second primary tumours in patients with head and neck squamous cell carcinoma (HNSCC). Furthermore, it has been reported that acquired resistance to all-trans retinoic acid (RA) in leukaemia is associated with decreased plasma peak levels, probably the result of enhanced retinoid metabolism. The aim of this study was to investigate the metabolism of retinoids and relate this to growth inhibition in HNSCC. Three HNSCC cell lines were selected on the basis of a large variation in the all-trans RA-induced growth inhibition. Cells were exposed to 9.5 nM (radioactive) for 4 and 24 h, and to 1 and 10 microM (nonradioactive) all-trans RA for 4, 24, 48 and 72 h, and medium and cells were analysed for retinoid metabolites. At all concentrations studied, the amount of growth inhibition was proportional to the extent at which all-trans-, 13- and 9-cis RA disappeared from the medium as well as from the cells. This turnover process coincided with the formation of a group of as yet unidentified polar retinoid metabolites. The level of mRNA of cellular RA-binding protein II (CRABP-II), involved in retinoid homeostasis, was inversely proportional to growth inhibition. These findings indicate that for HNSCC retinoid metabolism may be associated with growth inhibition.

Topics: Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Division; Chromatography, High Pressure Liquid; Culture Media; Head and Neck Neoplasms; Humans; Receptors, Retinoic Acid; Retinoids; Tretinoin; Tumor Cells, Cultured

A reversed-phase high-performance liquid chromatographic method for the simultaneous analysis of retinol, all-trans-retinoic acid, 13-cis-retinoic acid and 13-cis-4-oxoretinoic acid in human plasma and cell culture medium is described. Sample preparation involves precipitation of proteins and extraction of retinoids with 60% acetonitrile. After centrifugation, the acetonitrile content of the supernatant is reduced to 45%, allowing on-column concentration of analytes. Injection volumes up to 2.0 ml (equivalent to 0.525 ml of sample) can be used without compromising chromatographic resolution of all-trans-retinoic acid and 13-cis-retinoic acid. Retinoids were stable in this extract and showed no isomerization when stored in the dark in a cooled autosampler, allowing automated analysis of large series of samples. Recoveries from spiked plasma samples were between 95 and 103%. Although no internal standard was used, the inter-assay precision for all retinoids was better than 6% and 4% at concentrations of 30 nM and 100 nM, respectively. The method is a valuable tool for the study of cellular metabolism of all-trans-retinoic acid, as polar metabolites of this compound can be detected with high sensitivity in cell culture media.

Topics: Chromatography, High Pressure Liquid; Drug Stability; Humans; Isotretinoin; Solubility; Tretinoin; Tumor Cells, Cultured; Vitamin A

A highly sensitive HPLC method with automated column switching was developed for the simultaneous determination of endogenous levels of 13-cis-retinoic acid (isotretinoin), all-trans-retinoic acid (tretinoin) and their 4-oxo metabolites in plasma samples from man, Cynomolgus monkey, rabbit, rat and mouse. Plasma (0.4 ml) was deproteinated by adding ethanol (1.5 ml) containing the internal standard acitretin. After centrifugation, 1.4 ml of the supernatant were directly injected onto the precolumn packed with LiChrospher 100 RP-18 (5 microm). 1.25% ammonium acetate and acetic acid-ethanol (8:2, v/v) was used as mobile phase during injection and 1% ammonium acetate and 2% acetic acid-ethanol (102:4, v/v) was added, on-line, to decrease the elution strength of the injection solution. After backflush purging of the precolumn, the retained components were transferred to the analytical column in the backflush mode, separated by gradient elution and detected at 360 nm. Two coupled Superspher 100 RP-18 endcapped columns (both 250x4 mm) were used for the separation, together with a mobile phase consisting of acetonitrile-water-10% ammonium acetate-acetic acid: (A) 600:300:60:10 (v/v/v/v), (B) 950:20:5:20 (v/v/v/v), and (C) 990:5:0:5 (v/v/v/v). The method was linear in the range 0.3-100 ng/ml, at least, with a quantification limit of 0.3 ng/ml. The mean recoveries from human plasma were 93.2%-94.4% and the mean inter-assay precision was 2.8%-3.2% (range 0.3-100 ng/ml). Similar results were obtained for animal plasma. The analytes were found to be stable in the plasma of all investigated species stored at -20 degrees C for 4.3 months and at -80 degrees C for 9 months, at least. At this temperature, human plasma samples were even stable for 2 years. The method was successfully applied to more than 6000 human and 1000 animal plasma samples from clinical and toxicokinetic studies. Endogenous levels determined in control patients and pregnant women were similar to published data from volunteers.

Topics: Adolescent; Adult; Aged; Animals; Chromatography, High Pressure Liquid; Drug Stability; Female; Humans; Isotretinoin; Macaca fascicularis; Mice; Middle Aged; Pregnancy; Rabbits; Rats; Sensitivity and Specificity; Spectrophotometry, Ultraviolet; Tretinoin

Following a single oral dose of all-trans retinoic acid (RA) (0.167 mmole) in corn oil to 6 healthy human subjects, the mean serum retinol (ROL) level fell by approximately 20% within 1 h and remained depressed for 24 h. After dosing, RA appeared in the blood within 30 min, peaked at 0.3-0.5 mumol/l, and then declined to very low concentrations after 7 h. All-trans retinoyl beta-glucuronide (RAG) appeared simultaneously with RA in the plasma, albeit more sporadically, whereas only traces of 4-oxoretinoic acid (4-oxoRA) were detected. Some possible physiologic consequences of therapeutic uses of all-trans RA are discussed.

Topics: Adult; Female; Humans; Kinetics; Male; Middle Aged; Tretinoin; Vitamin A

Vitamin A deficiency leads to an arrest of spermatogenesis and a loss of advanced germ cells in male mice. In the present study, the effects of several retinoids and carotenoids on these mouse testis were investigated. First, the proliferative activity of the growth-arrested A spermatogonia in vitamin A-deficient (VAD) mice testis was determined, 20, 24, or 28 h after administration of 0.5 mg all-trans-retinoic acid (RA). The bromodeoxy-uridine (BrdU) labeling index of A spermatogonia in control VAD testis was 5 +/- 1% (n = 4, mean +/- SD). When RA was injected (ip), the highest labeling index was found 24 h after RA administration; 49 +/- 5%. When various concentrations of RA, all-trans-4-oxo-retinoic acid (4-oxo-RA) or all-trans-retinol acetate (ROAc), ranging from 0.13-1 mg, were injected, the labeling index of A spermatogonia always increased in comparison with the VAD situation. A maximum index at 24 h was found when 0.5 mg 4-oxo-RA was injected: 56 +/- 3%. This labeling index was even higher than those after injection of RA or ROAc, 49 +/- 5% and 34 +/- 6% respectively. The increase of the BrdU labeling index was dose dependent. After an initial increase of the labeling indices with increasing retinoid doses, the labeling indices decreased at a higher concentration. This decrease is likely due to a concentration dependent timeshift of the optimum of BrdU labeling to shorter time intervals after retinoid administration because a labeling index of 66 +/- 1% was found 20 h after injection of 1 mg RA. At 24 h, this labeling index was halved: 33 +/- 2%. These indices show that the degree of synchronization of spermatogenesis is also dependent on the retinoid dose. When the dimers of RA and 4-oxo-RA, respectively beta-carotene (beta C) and canthaxanthin, were given, 24 h after administration BrdU-labeling indices comparable with the VAD value were found. Repeated injection of beta C twice a week did induce a reinitiation of spermatogenesis, but compared with RA, the activity of beta C was lower and delayed. It is concluded that 4-oxo-RA is active in adult mammals in vivo. It is at least as potent as RA in the induction of the differentiation and subsequent proliferation of growth-arrested A spermatogonia in VAD mice testis. Furthermore, the degree of synchronization of spermatogenesis is influenced by the retinoid dose. Finally, carotenoids were shown to act in the induction of spermatogonial cell proliferation too but with a lower and delayed activity.

Topics: Animals; beta Carotene; Bromodeoxyuridine; Carotenoids; Cell Differentiation; Cell Division; Male; Mice; Retinoids; Spermatogonia; Testis; Tretinoin; Vitamin A Deficiency

Retinoids are a large family of natural and synthetic compounds related to vitamin A that have pleiotropic effects on body physiology, reproduction, immunity, and embryonic development. The diverse activities of retinoids are primarily mediated by two families of nuclear retinoic acid receptors, the RARs and RXRs. Retinoic acids are thought to be the only natural ligands for these receptors and are widely assumed to be the active principle of vitamin A. However, during an unbiased, bioactivity-guided fractionation of Xenopus embryos, we were unable to detect significant levels of all-trans or 9-cis retinoic acids. Instead, we found that the major bioactive retinoid in the Xenopus egg and early embryo is 4-oxoretinaldehyde, which is capable of binding to and transactivating RARs. In addition to its inherent activity, 4-oxoretinaldehyde appears to be a metabolic precursor of two other RAR ligands, 4-oxoretinoic acid and 4-oxoretinol. The remarkable increase in activity of retinaldehyde and retinol as a consequence of 4-oxo derivatization suggests that this metabolic step could serve a critical regulatory function during embryogenesis.

Topics: Animals; Binding, Competitive; Cell Line; Female; Ligands; Receptors, Retinoic Acid; Retinaldehyde; Retinoid X Receptors; Retinoids; Transcription Factors; Transfection; Tretinoin; Vitamin A; Xenopus

Retinoic acid and its isoforms are considered to be endogenous compounds which regulate embryonic development. In the work reported here we have determined which retinoids are present in zebrafish embryos and how their levels change throughout development and into adulthood. All-trans-RA is present and its level does not change significantly during embryogenesis. We failed to detect other retinoic acid isomers such as 9-cis-RA and 4-oxo-RA, but we did observe a rapid rise in the level of didehydroretinol after gastrulation. The most striking result is that the zebrafish embryo, like Xenopus and tunicates, contains a vast excess of t-retinal whereas the embryos of higher vertebrates have an excess of t-retinol. However, as the zebrafish grows, the levels of t-retinol rise so that by adulthood t-retinol and t-retinal concentrations are more equivalent, indicating a changing pattern of retinoid metabolism with growth. To examine the significance of the use of t-retinal as a precursor of t-RA we treated embryos with disulphiram, an inhibitor of retinaldehyde dehydrogenase. This resulted in embryos with an undulating notochord and correspondingly abnormal somites and ventral floor plate. In contrast to this effect, 4-methylpyrazole, which inhibits alcohol dehydrogenases, had no effect on development. This effect of disulphiram suggests that t-RA may be involved in the establishment of the anteroposterior axis of the embryo.

Topics: Alcohol Dehydrogenase; Aldehyde Oxidoreductases; Animals; Antimetabolites; Disulfiram; Enzyme Inhibitors; Fomepizole; Immunohistochemistry; Pyrazoles; Retinal Dehydrogenase; Retinaldehyde; Retinoids; Stereoisomerism; Tretinoin; Vitamin A; Zebrafish

Topics: Animals; Antioxidants; Blotting, Northern; Canthaxanthin; Carotenoids; Cell Communication; Chromatography, High Pressure Liquid; Gap Junctions; Glyceraldehyde-3-Phosphate Dehydrogenases; Mice; Mice, Inbred C3H; Plasmids; Rats; Retinoids; Spectrophotometry, Ultraviolet; Tretinoin

Patients with retinitis pigmentosa have been suggested to benefit from treatment with moderate doses of retinyl palmitate. Retinyl palmitate is not an active retinoid in itself but is metabolised to active components in the body. To find out which metabolites of retinyl palmitate were formed and at which concentrations, we measured the concentrations of retinol, retinyl palmitate, retinoic acids and tocopherol in serum of patients treated with oral retinyl palmitate for retinitis pigmentosa.. Nine male patients and one female diagnosed as having retinitis pigmentosa after a complete ophthalmological examination including a full-field electroretinogram were given vitamin A at their own request as one daily morning dose of 16600 IU vitamin A. Blood samples were obtained before and after > 2 weeks of treatment. The concentrations of retinoids and tocopherol were measured with established methods.. The patients were not deficient in vitamin A or vitamin E as judged from the serum vitamin concentrations. Treatment with retinyl palmitate significantly increased the serum concentration of retinyl palmitate and of 13-cis-retinoic acid but not of retinol, tocopherol or all-trans-retinoic acid.. Neither retinyl palmitate nor 13-cis-retinoic acid, are known to be biologically active. However, 13-cis-retinoic acid can isomerise to the active vitamin A derivative, all-trans-retinoic acid. It is suggested that patients may be treated with a small dose of 13-cis-retinoic acid instead, to avoid the relatively long metabolic detour from retinyl palmitate.

Topics: Administration, Oral; Adult; Aged; Anticarcinogenic Agents; Chromatography, High Pressure Liquid; Diterpenes; Electroretinography; Female; Humans; Male; Middle Aged; Retinitis Pigmentosa; Retinoids; Retinyl Esters; Tretinoin; Vitamin A; Vitamin E

A reporter cell line was established from F9 mouse teratocarcinoma cells containing the RAR beta 2 promoter coupled to the lacZ (beta-galactosidase) reporter gene. All-trans-, 9-cis-, and all-trans-4-oxoretinoic acid were equipotent in inducing cell differentiation at 1 microM, determined by induction of collagen IV mRNA expression, of morphological changes, as well as of beta-galactosidase enzyme activity. By the same criteria, beta-carotene at 10 microM also induced differentiation, but less strongly and more slowly than the retinoic acids. In contrast, the oxocarotenoid (or xanthophyll) canthaxanthin, at 10 microM, had little effect on differentiation, unless preincubated in culture medium, from which 4-oxoretinoic acid was recovered and identified as a decomposition product. This indicates that canthaxanthin can act as an effective inducer of differentiation only after breakdown to active metabolites. Likewise, beta-carotene probably also acts subsequent to breakdown to retinoic acid. Throughout these experiments the response of the RAR beta promoter-lacZ reporter gene correlated well with other parameters of differentiation, making this cell line a useful system for examination of inducers of embryonal carcinoma cell differentiation.

Topics: Animals; beta Carotene; Canthaxanthin; Carotenoids; Cell Differentiation; Collagen; Gene Expression Regulation, Neoplastic; Genes, Reporter; Lac Operon; Mice; Promoter Regions, Genetic; Receptors, Retinoic Acid; RNA, Messenger; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured

The activity of 4-oxoretinoic acid as an inducer of gap junctional communication was investigated in C3H/10T1/2 murine fibroblasts. Two isomers of this retinoid, all-trans- and 13-cis-4-oxoretinoic acid, enhance gap junctional communication. This is accompanied by increased expression of connexin43 mRNA. Decomposition fractions of canthaxanthin were isolated by preparative high-performance liquid chromatography and shown to be active in the cell-cell communication assay. Two of the decomposition compounds were identified as all-trans- and 13-cis-4-oxoretinoic acid. Therefore, it is concluded that the biological activity of canthaxanthin regarding cell-cell communication is at least in part due to the formation of active decomposition products such as 4-oxoretinoic acid.

Topics: Animals; Canthaxanthin; Cell Communication; Cell Line; Chromatography, High Pressure Liquid; Connexin 43; Fibroblasts; Gap Junctions; Gene Expression; Mice; Mice, Inbred C3H; RNA, Messenger; Tretinoin

Therapy with all-trans retinoic acid (ATRA) achieves complete remission in a high proportion of patients with acute promyelocytic leukemia (APL), but the efficacy is reported to relate to plasma ATRA level after oral administration. The pharmacokinetics of ATRA and 4-oxo all-trans retinoic acid (4-oxo ATRA), a metabolite of ATRA, were studied in four children with APL at the time of initial oral administration. After administration of ATRA at a dose of 30 mg/m2, the peak plasma ATRA level was 20-741 ng/ml and was reached at 60-120 min. The patient with the lowest peak plasma level did not achieve complete remission and had a very high 4-oxo ATRA level compared to the patients with complete remission. These findings suggest that accelerated metabolism of ATRA plays a role in the failure of this agent in the patients without remission.

Topics: Administration, Oral; Adolescent; Antineoplastic Agents; Child; Chromatography, High Pressure Liquid; Humans; Leukemia, Promyelocytic, Acute; Male; Tretinoin

The introduction of substituents at position 3 of methyl 4-oxoretinoate can be effected in good yields by alkylating the lithium dienolate. A second substituent can be introduced also, but the resulting 3,3-disubstituted-4-oxoretinoates were isolated in lower yields. Evidence was obtained for a slower rate of alkylation at the alpha-position (carbon 14) of the ester group. Some of these 4-oxoretinoic acid analogues showed high activity in assays in vivo for the inhibition of ornithine decarboxylase activity and carcinogen-induced papillomas in mouse skin.

Topics: Alkylation; Animals; Anticarcinogenic Agents; Cricetinae; Drug Stability; Enzyme Induction; Female; Magnetic Resonance Spectroscopy; Mice; Mice, Inbred Strains; Ornithine Decarboxylase; Papilloma; Skin; Skin Neoplasms; Structure-Activity Relationship; Tetradecanoylphorbol Acetate; Tretinoin

No embryotoxic or teratogenic effects, considered to be treatment related, were observed in rabbits after daily oral doses of 3 mg/kg of 13-cis-retinoic acid (13-cis-RA) from Day 8 to Day 11 of gestation. In contrast, treatment with 15 mg/kg/day significantly increased the rate of fetal resorptions (22%) and 13 out of 68 surviving fetuses (16%) were malformed. Pharmacokinetic studies with both dosing regimens of 13-cis-RA in pregnant rabbits showed that on Day 11 of gestation, high concentrations of parent compound, 13-cis-RA, and its major metabolite, 13-cis-4-oxoRA, existed in maternal plasma. Much lower concentrations were found for all-trans-4-oxoRA and all-trans-RA. The area under the concentration-time curve (AUC) of all-trans-RA following the 15 mg/kg/day dosing regimen of 13-cis-RA was only 1.2% that of parent compound 13-cis-RA. At this dose, embryo levels of 13-cis-RA, 13-cis-4-oxoRA, and all-trans-4-oxoRA were 2.5-, 4.7-, and 3.6-fold higher by AUC comparison (24-hr period of Day 11) compared with the dose of 3 mg/kg. However, embryo levels of all-trans-RA were virtually identical at both doses and were, in fact, somewhat lower than endogenous concentrations measured in untreated rabbit embryos. In contrast to mice, where isomerization from 13-cis- to all-trans-RA was suggested to be crucial for the teratogenic action of 13-cis-RA, we found that the teratogenic action of 13-cis-RA (15 mg/kg/day) in rabbits is characterized by increased whole embryo concentrations of 13-cis-RA, 13-cis-4-oxoRA, and all-trans-4-oxoRA, but not of all-trans-RA.

Topics: Abnormalities, Drug-Induced; Administration, Oral; Animals; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Embryo, Mammalian; Female; Fetus; Gestational Age; Isotretinoin; Maternal-Fetal Exchange; Placenta; Pregnancy; Rabbits; Stereoisomerism; Tretinoin

The metabolism of 4-keto-all-trans-retinoic-acid (4-keto-RA), a biologically active oxygenated metabolite of all-trans-retinoic (RA), has been examined. In vitro, incubation of [14C]4-keto-RA with hamster liver microsomes in the presence of NADPH produced two major radioactive metabolites which were more polar than the parent compound. Following isolation, appropriate derivatization and analysis by GC-MS, these compounds were tentatively identified as 2-hydroxy- and 3-hydroxy-4-ketoretinoic acid. Formation of both hydroxy-keto derivatives was suppressed by the imidazole-containing P450 inhibitor liarozole fumarate (IC50, 1.3 microM). In vitro, an i.v. injection of 4-keto-RA (20 micrograms) into rats was followed by rapid disappearance of the retinoid from plasma with a half-life of 7 min. Pretreatment with liarozole fumarate (40 mg/kg, -60 min) reduced the elimination rate of 4-keto-RA: it prolonged the plasma half-life of the retinoid to 12 min, without affecting its distribution volume. These results indicate the important role of the P450 enzyme system in the metabolism of 4-keto-RA both in vitro and in vivo. The inhibitory effect of liarozole fumarate on this metabolic process may contribute to the reported retinoid-mimetic activity of this drug.

Topics: Androgen Antagonists; Animals; Antineoplastic Agents; Cricetinae; Imidazoles; Male; Mesocricetus; Microsomes, Liver; Tretinoin

Previous studies suggested that the rabbit is much more susceptible to the teratogenic action of 13-cis-retinoic acid (13-cis-RA) than the mouse or the rat, while the teratogenicity of all-trans-RA was comparable in these species. In the present study we investigated if pharmacokinetics can explain these species- and structure-related differences. The embryotoxic and teratogenic potential of all-trans-retinoic acid (all-trans-RA) and 13-cis-RA were evaluated in the Swiss hare rabbit after oral administration of daily doses of the two drugs throughout organogenesis, from gestation day (GD) 6 to 18 (plug day = GD 0). All-trans-RA was given at dose levels of 0.7, 2 or 6 mg/kg body weight per day and 13-cis-RA at 3, 7.5 or 10 mg/kg per day. The doses needed to elicit a minimum teratogenic response were found to be 6 mg/kg per day for all-trans-RA and 10 mg/kg per day for 13-cis-RA. Using these doses, transplacental pharmacokinetics of all-trans- and 13-cis-RA were performed. Pregnant rabbits were treated once daily from GD 7 to 12 and plasma and embryo samples were collected for HPLC analysis at various time intervals after the final dose. The main plasma metabolites of all-trans- and 13-cis-RA were all-trans-beta-glucuronide (all-trans-RAG) and 13-cis-4-oxo-RA, respectively. The elimination of 13-cis-RA and its metabolites from maternal plasma were much slower than of all-trans-RA resulting in accumulation of the 13-cis-isomers in plasma. Marked differences in the placental transfer of the two drugs and their metabolites were observed. All-trans-RA and all-trans-4-oxo-RA were efficiently transferred to the rabbit embryo, reaching concentrations similar to the plasma levels. On the contrary, the 13-cis-isomers reached the embryo to a lesser extent. Despite its limited placental transfer, a considerable embryonic exposure to 13-cis-RA and 13-cis-4-oxo-RA was noticed after treatment with isotretinoin, as indicated by their area-under-the-concentration-time-curve (AUC) values in the embryo, which were in the same range as the corresponding AUC value of all-trans-RA after treatment with the all-trans-isomer.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Animals; Embryo, Mammalian; Female; Isomerism; Isotretinoin; Male; Maternal-Fetal Exchange; Pregnancy; Rabbits; Structure-Activity Relationship; Teratogens; Tretinoin

Topical all-trans retinoic acid (RA) modulates growth and differentiation of skin and is used in the treatment of various dermatological disorders. RA is metabolized to 4-hydroxy RA, 4-oxo RA and 5,6-epoxy RA, which are believed to be markedly less active than RA. 3,4-didehydroretinoic acid (ddRA) is a metabolite of 3,4-didehydroretinol which is present in skin. ddRA is biologically active and acts as a morphogen. We have determined the relative biological activity of ddRA, 4-hydroxy RA, 4-oxo RA and 5,6-epoxy RA as assessed by three retinoid responsive systems relevant to skin. RA, ddRA, 4-hydroxy RA, 4-oxo RA and 5,6-epoxy RA (10-100 nM) reduced epidermal transglutaminase activity in human keratinocytes to similar extents, and inhibited alpha-melanocyte-stimulating hormone-isobutylmethylxanthine-inducible tyrosinase activity in Cloudman S-91 mouse melanoma cells by 67, 39, 48, 51 and 19%, respectively, at 100 nM. Daily topical application of the retinoids to hairless mouse skin for 4 days resulted in dose-dependent changes in epidermal thickness and global histological score. The relative potencies of RA, ddRA, 4-hydroxy RA, 4-oxo RA and 5,6-epoxy RA, as calculated by parallel line assay, were 1.0, 0.60, 0.34, 0.29 and 0.18, respectively, for epidermal hyperplasia and 1.0, 0.78, 0.23, 0.14 and 0.08, respectively, for global histological score. Interestingly, the compounds exhibited a similar rank order of potency with respect to induction of cellular retinoic acid binding protein-II mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adult; Animals; Carrier Proteins; Cells, Cultured; Epidermis; Humans; Keratinocytes; Male; Melanoma, Experimental; Mice; Mice, Hairless; Monophenol Monooxygenase; Receptors, Retinoic Acid; RNA, Messenger; Skin; Transglutaminases; Tretinoin; Tumor Cells, Cultured

Retinoids (vitamin A and its metabolites) are suspected of regulating diverse aspects of growth, differentiation, and patterning during embryogenesis, but many questions remain about the identities and functions of the endogenous active retinoids involved. The pleiotropic effects of retinoids may be explained by the existence of complex signal transduction pathways involving diverse nuclear receptors of the retinoic acid receptor (RAR) and retinoid X receptor (RXR) families, and at least two types of cellular retinoic acid binding proteins (CRABP-I and -II). The different RARs, RXRs, and CRABPs have different expression patterns during vertebrate embryogenesis, suggesting that they each have particular functions. Another level at which fine tuning of retinoid action could occur is the metabolism of vitamin A to active metabolites, which may include all-trans-retinoic acid, all-trans-3,4-didehydroretinoic acid, 9-cis-retinoic acid, and 14-hydroxy-4,14-retroretinol. Formation of the metabolite all-trans-4-oxo-retinoic acid from retinoic acid was considered to be an inactivation pathway during growth and differentiation. We report here that, in contrast, 4-oxo-retinoic acid is a highly active metabolite which can modulate positional specification in early embryos. We also show that this retinoid binds avidly to and activates RAR beta, and that it is available in early embryos. The different activities of 4-oxo-retinoic acid and retinoic acid in modulating positional specification on the one hand, and growth and differentiation on the other, interest us in the possibility that specific retinoid ligands regulate different physiological processes in vivo.

Topics: Animals; Chromatography, High Pressure Liquid; Embryo, Nonmammalian; Embryonic Development; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Retinoid X Receptors; Transcription Factors; Tretinoin; Xenopus laevis

In the new context of the use of retinoic acid (RA) therapy as an inducer of leukemic differentiation and a selective inhibitor of human myeloid leukemia cell growth, we undertook to explore the potential physiological role of retinoids on the proliferation and differentiation of normal bone marrow myeloid progenitors. The effects of continuous exposure of all-trans-RA, its naturally occurring isomer, 13-cis-RA, and its metabolite 4-oxo-all-trans-RA were studied on the growth of normal human bone marrow cells in soft agar, directly and after liquid culture. Retinoids enhanced the total number of granulocytic colony and macrocluster formation in the presence of exogenous colony-stimulating factor (n = 9). Dose-response curve were bell-shaped, with a maximal increment between concentration of 0.5 and 0.05 nM. In all cases, a concomitant decrease of macrophagic colonies was noted. The positive effect on granulocytic colony formation was observed with each of the retinoids tested (all-trans, 13-cis and 4-oxo-all-trans) (n = 5). On erythroid colony formation, all-trans-RA had the opposite effect. Constant suppression of CFU-E and BFU-E colony formation and coloring was observed in a dose-related fashion from 0.1 to 10 microM (n = 5). Thus, in granulocytic, as in erythroid colony formation, retinoids affected both proliferation and differentiation parameters. However, after short-term suspension culture in the presence of all-trans-RA, an increase of both CFU-GM and BFU-E colonies, was observed. These results suggest a specific effect of retinoids on late myeloid precursors and places retinoids as possible candidates for enhancement of normal granulocytic differentiation.

Topics: Bone Marrow Cells; Cell Differentiation; Cell Division; Cells, Cultured; Erythroid Precursor Cells; Granulocytes; Hematopoietic Stem Cells; Humans; Isotretinoin; Tretinoin

The CRABP-I and CRABP-II proteins are high affinity cytoplasmic retinoic acid-binding proteins. In undifferentiated F9 teratocarcinoma stem cells, only the CRABP-I protein is expressed at detectable levels. We have previously shown that overexpression of the CRABP-I protein in stably transfected F9 stem cell lines results in a lower sensitivity to a given external concentration of retinoic acid relative to that of untransfected F9 cells; in contrast, reduced CRABP-I expression in CRABP-I cDNA anti-sense transfected lines is associated with increased sensitivity of these lines to retinoic acid. These three types of cell lines were cultured in the presence of 50 nM [3H]retinoic acid, and the metabolism of retinoic acid was followed over the next 24 h. The results demonstrate that CRABP-I has the ability to alter both the levels and types of RA metabolites produced in the cytoplasm of differentiating embryonic stem cells. Moreover, the level of CRABP-I determines the rate of RA metabolism to 4-oxo-RA such that the higher the CRABP-I level, the faster the metabolism of [3H]retinoic acid. This is the first reported connection between the level of CRABP-I expression and intracellular RA metabolism.

Topics: Carrier Proteins; Chromatography, High Pressure Liquid; DNA; Receptors, Retinoic Acid; Teratoma; Tretinoin; Tumor Cells, Cultured

In cultured rat conceptuses, intraamniotic microinjections of 2500 ng/mL of 4-oxo-13-cis-retinoic acid, 600 ng/mL 4-oxo-all-trans-retinoic acid or 4000 ng/mL all-trans-retinoyl-beta-glucuronide, produce qualitatively and quantitatively similar patterns of dysmorphogenesis as those reported after the intraamniotic microinjection of 250 ng/mL all-trans-retinoic acid [Lee et al., Teratology 44: 313-323, 1991; Creech Kraft et al., Teratology 45: 259-270, 1992]. In the present study, we utilized HPLC techniques to analyze retinoid levels in cultured rat conceptuses, 1.5 hr after intraamniotic microinjections of 4-oxo-13-cis-retinoic acid (2500 ng/mL), 4-oxo-all-trans-retinoic acid (600 ng/mL) or all-trans-retinoyl-beta-glucuronide (4000 ng/mL). Our findings show that, after the microinjections of 4-oxo-all-trans-retinoic acid or 4-oxo-13-cis-retinoic acid (at these selected concentrations), 4-oxo-all-trans-retinoic acid was predominant in the embryos proper at concentrations of about 200 nM. This was roughly equivalent to the levels of all-trans-retinoic acid assayed after microinjections of all-trans-retinoyl-beta-glucuronide (4000 ng/mL). We conclude from these studies that both 4-oxo-all-trans-retinoic acid and all-trans-retinoic acid behave as ultimate or proximate dysmorphogens.

Topics: Animals; Biotransformation; Embryo, Mammalian; Isotretinoin; Microinjections; Models, Biological; Morphogenesis; Organ Culture Techniques; Rats; Rats, Inbred Strains; Stereoisomerism; Time Factors; Tretinoin

4-Oxo-all-trans-retinoic acid, 4-oxo-13-cis-retinoic acid and all-trans-retinoyl-beta-glucuronide were intraamniotically microinjected in rat embryos on day 10 of gestation and cultured until day 11.5. A comparison of the concentration-effect relationships showed that the dysmorphogenic effects produced by these metabolites were qualitatively similar to those of parent all-trans-retinoic acid. Compared with all-trans-retinoic acid (300 ng/ml), the dysmorphogenic effects were elicited by a 2-fold higher concentration of 4-oxo-all-trans-retinoic acid, an approximately 10-fold higher concentration of 4-oxo-13-cis-retinoic acid and a 16-fold higher concentration of all-trans-retinoyl-beta-glucuronide. A surplus of uridine 5'-diphospho-glucuronic acid, microinjected together with 300 ng/ml all-trans-retinoic acid, decreased the observed embryo-toxicity of all-trans-retinoic acid, suggesting the possibility of glucuronidation in tissues of the conceptus per se. The results of the study provide further support for the hypothesis that 4-oxo-all-trans-retinoic acid and all-trans-retinoic acid are, in contrast to the corresponding cis-isomers and glucuronides, ultimate dysmorphogenic retinoids.

Topics: Amnion; Animals; Dose-Response Relationship, Drug; Embryo, Mammalian; Microinjections; Organ Culture Techniques; Rats; Rats, Inbred Strains; Teratogens; Tretinoin

The potential systemic availability of retinoids from topically applied isotretinoin was assessed in 12 men with acne vulgaris. Isotretinoin 0.05% gel was applied to patients at a daily dose of 20 gm (equivalent to 10 mg of isotretinoin) over a 1900 cm2 surface area of skin on the face, back, and chest for 30 days. Blood samples were collected throughout the study and up to 48 hours after the last topical application; they were assayed for isotretinoin, tretinoin, and 4-oxo-isotretinoin by specific high-performance liquid chromatography. Plasma concentrations of isotretinoin, tretinoin, and 4-oxo-isotretinoin were not measurable (less than 20 ng/ml) at any time. Most adverse experiences were cutaneous; a few systemic adverse experiences were judged to be remotely related to topical drug administration. The lack of measurable plasma concentrations of isotretinoin, tretinoin, or 4-oxo-isotretinoin and systemic adverse experiences indicates negligible systemic availability of retinoids even after multiple application of isotretinoin 0.05% gel at doses approximately 12 times greater than normal daily use.

Topics: Acne Vulgaris; Administration, Cutaneous; Adult; Chromatography, High Pressure Liquid; Gels; Humans; Isotretinoin; Male; Tretinoin

Plasma concentrations of retinyl esters, retinol, retinol-binding protein and the polar retinol metabolites all-trans-retinoic acid, 13-cis-retinoic acid, all-trans-4-oxoretinoic acid and 13-cis-4-oxoretinoic acid were measured for six male volunteers who received 0.46 mg retinyl palmitate per kilogram body weight as oily drops (equivalent to 0.25 mg retinol per kilogram body weight) once daily over a 20-d period. Retinol and retinol-binding protein levels remained virtually constant throughout the study. Following absorption of vitamin A, retinyl esters as well as all-trans-retinoic acid and 13-cis-retinoic acid were transiently increased in plasma. 13-cis-4-Oxoretinoic acid increased gradually to a steady state level present on d 10 or 20. All-trans-4-oxoretinoic acid was not detected in plasma of the volunteers, with the exception of one on d 10 of the study. Plasma pharmacokinetic profiles of retinyl esters and polar metabolites of retinol displayed great interindividual differences (peak concentrations, time to peak, area-under-the-concentration-time curve values) among the volunteers. Because of the relatively high and consistent steady state concentrations of plasma 13-cis-4-oxoretinoic acid, we suggest that this compound be further investigated as a biochemical marker of vitamin A uptake in humans.

Topics: Administration, Oral; Adult; Chromatography, High Pressure Liquid; Diterpenes; Humans; Male; Retinol-Binding Proteins; Retinol-Binding Proteins, Plasma; Retinyl Esters; Stereoisomerism; Tretinoin; Vitamin A

The effects of isotretinoin (IR) and its primary metabolite (in the human), 4-oxo-isotretinoin (4-OIR or OIR), on isolated chick neural crest cells (NCC's) in culture were studied. NCC's were found to be deficient in both superoxide dismutase (SOD) and catalase, two of the enzymes known to function in the "scavenging" (dismutation) of toxic radical oxygen species (ROS) such as the superoxide anion and hydrogen peroxide. The addition of IR or OIR to the culture medium significantly depressed the viability of the NCC's when compared to untreated cells. OIR was more potent in this regard than IR. In the presence of either IR or OIR, NCC's generated superoxide anions (O2.), hydrogen peroxide (H2O2), and hydroxyl anions (OH.). OIR was again more potent. The cytotoxicity of IR or OIR was demonstrated by the "leakage" of radioactive chromium from prelabeled cells. The latter is suggestive of a primary surface membrane defect, most logically via the induction of lipid peroxides by the retinoids. The latter is accompanied by an increase in membrane permeability and porosity as evidenced by the fact that various fluorescently labeled molecules, including BSA-FITC (MW 69,000), gain entrance into the cytoplasm of the retinoid treated cells. No label was seen in the cytoplasm of similarly treated control cells. When SOD (200 units/ml) or catalase (400 units/ml) was added to the culture media of IR- or OIR-treated NCCs, cell viability was increased and the concentration of the various ROS generated was decreased. Membrane leakiness to chromium and FITC-BSA was also decreased in the presence of these enzymes. Free radicals, when not inactivated (dismutated), are known to be pathobiotic to most cells. Cell membranes are at a particular high risk from ROS which induce structural, physiological, and biochemical alterations in the cell membrane. The latter can have a negative effect on cell permeability, maintenance of normal ionic gradients, membrane enzyme activity, cell-to-cell communication, etc. Such defects can ultimately culminate in hypoplasia, aplasia, and cell necrosis. This study has shown that NCC's may be overtly sensitive to ROS, especially since these undifferentiated cells apparently lack inherent SOD and/or catalase activity. From this study it appears as if both IR and OIR perturb the normal functional state of NCC's by "triggering" the generation of ROS. This may certainly explain the teratogenicity of these drugs as related to the viability of neural cre

Topics: Animals; Catalase; Cell Survival; Cells, Cultured; Chick Embryo; Fluorescein-5-isothiocyanate; Fluoresceins; Fluorescent Dyes; Free Radical Scavengers; Free Radicals; Hydrogen Peroxide; Hydroxides; Hydroxyl Radical; Isotretinoin; Neural Crest; Oxygen; Serum Albumin, Bovine; Superoxide Dismutase; Superoxides; Tretinoin

The concentrations of retinoic acid compounds were monitored by a newly developed highly sensitive HPLC procedure in plasma of six volunteers who received 833 IU vitamin A per kg body weight per day during a 20-day period. There was a significant increase of all-trans-retinoic acid (two-fold), 13-cis-retinoic acid (7-fold) and 13-cis-4-oxoretinoic acid (5-fold) over endogenous plasma levels of these retinoids. The same compounds had previously been found after treatment with the teratogenic drug isotretinoin (Roaccutan, Accutane). Our results raise the possibility that high vitamin A intake may carry a teratogenic risk attributable to increased levels of retinoic acid compounds generated from retinol by metabolic processes.

Topics: Adult; Chromatography, High Pressure Liquid; Humans; Isotretinoin; Male; Teratogens; Tretinoin; Vitamin A

Human plasma was analyzed by high performance liquid chromatography for the presence of retinoic acid and 4-oxoretinoic acid isomers. Peaks that coeluted with the reference compounds all-trans-retinoic acid, 13-cis-retinoic acid, and 13-cis-4-oxoretinoic acid were routinely observed in human plasma. These retinoids were unequivocally identified by the following methods: comigration with reference compounds under several high performance liquid chromatographic conditions; comparison of ultraviolet spectra with those of reference compounds; derivatization with diazomethane and coelution of the methyl esters with reference compounds in a high performance liquid chromatographic system as well as in a gas chromatography system with a mass selective detector. In vitro formation of 13-cis-retinoic acid and 13-cis-4-oxoretinoic acid as artifacts during the analytical procedure was excluded by control experiments. The mean plasma concentrations of the vitamin A metabolites in ten male volunteers were: all-trans-retinoic acid: 1.32 +/- 0.46 ng/ml; 13-cis-retinoic acid: 1.63 +/- 0.85 ng/ml; and 13-cis-4-oxoretinoic acid: 3.68 +/- 0.99 ng/ml. After oral dosing with vitamin A (833 IU/kg body weight) in five male volunteers, mean plasma all-trans-retinoic acid increased to 3.92 +/- 1.40 ng/ml and 13-cis-retinoic acid increased to 9.75 +/- 2.18 ng/ml. Maximal plasma 13-cis-4-oxoretinoic acid concentrations (average 7.60 +/- 1.45 ng/ml) were observed 6 h after dosing which was the last time point in this study. Concentrations of all-trans-4-oxoretinoic acid were low or not detectable. Our findings suggest that, in addition to all-trans-retinoic acid, 13-cis-retinoic acid and 13-cis-4-oxoretinoic acid are present in normal human plasma as metabolites of vitamin A.

Topics: Chromatography, Gas; Chromatography, High Pressure Liquid; Diazomethane; Humans; Isotretinoin; Male; Spectrophotometry, Ultraviolet; Tretinoin; Vitamin A

Pregnant hamsters were given a single oral dose (35 mumol/kg) of all-trans-retinoic acid, 13-cis-retinoic acid, all-trans-4-oxo-retinoic acid, 9-cis-retinal or all-trans-retinyl acetate during the early primitive streak stage of development. The radioactivity associated with the acidic retinoids was distributed to all tissues sampled (including placenta and fetus), with the largest accumulation in the liver and the least accumulation in fat. Radioactivity from 9-cis-retinal or retinyl acetate concentrated in the liver and lung. The all-trans-retinoic acid was oxidized in vivo to all-trans-4-oxo-retinoic acid and isomerized to 13-cis-retinoic acid: 13-cis-retinoic acid was oxidized to 13-cis-4-oxo-retinoic acid and isomerized to all-trans-retinoic acid. No parent 9-cis-retinal or retinyl acetate could be detected in maternal plasma. Plasma concentrations of the parent acidic retinoids reached their maxima within 60 min and then followed exponential decay. Of all the retinoids examined here, 13-cis-retinoic acid showed the largest area under the plasma curve, the slowest clearance and the longest elimination t1/2. Total plasma radioactivity, consisting of unidentified metabolites, remained elevated at 4 days after dosing. Maternal peak circulating concentrations of the parent retinoids, total radioactivity, plasma pharmacokinetic parameters or the total concentrations of residual radioactivity in fetal tissues could not be correlated with the differential teratogenic potencies of these retinoids.

Topics: Animals; Cricetinae; Diterpenes; Female; Mesocricetus; Permeability; Placenta; Pregnancy; Retinaldehyde; Retinoids; Retinyl Esters; Tissue Distribution; Tretinoin; Vitamin A

Retinoic acid is a natural vitamin A derivative that undergoes oxidative metabolism in the body to yield several metabolites, which apparently represent the products of a detoxification pathway. To assess if such metabolic conversions diminished teratogenic potency, one of the major metabolites (4-oxo-all-trans-retinoic acid) was tested for its teratogenic activity in pregnant ICR mice and further investigated for its pharmacokinetic features to determine if it accumulated in the embryo in concentrations sufficient to elicit a teratogenic response. Administration of single oral doses (10, 25, 50, or 100 mg/kg) of the compound to ICR mice on day 11 of gestation (plug day = day 0) produced dose-dependent frequencies of serious fetal anomalies of the type usually associated with the use of retinoic acid and other retinoids. The metabolite was equivalent in teratogenic potency to retinoic acid, and, in the instance of cleft palate frequency, it was even more active. Concentrations of 4-oxo-all-trans-retinoic acid and its 13-cis isomer were measured in the maternal plasma and whole embryos at 30 min to 10 hr after administration of the lowest (10 mg/kg) and the highest (100 mg/kg) teratogenic dose of 4-oxo-all-trans-retinoic acid by means of high-performance liquid chromatography methodology. Distribution of the compound in the maternal system and transfer to the embryo occurred rapidly with either dose. Peak concentration in the maternal plasma and the embryo persisted for 3-4 hr after the higher dose but not with the lower dose; however, elimination kinetics for the two dose levels were similar.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Administration, Oral; Animals; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Embryo, Mammalian; Female; Male; Maternal-Fetal Exchange; Mice; Mice, Inbred ICR; Pregnancy; Stereoisomerism; Teratogens; Tretinoin

13-cis-Retinoic acid (isotretinoin) is teratogenic in humans at therapeutic doses (0.5-1.5 mg/kg) but only marginally teratogenic in the mouse at a high dose of 100 mg/kg. Previous results explained why the cis isomer of retinoic acid was much less teratogenic than the trans isomer in mice. It was found that the placental transfer of all-trans retinoic acid to the mouse embryo was far greater than that of the 13-cis isomer. Since our previous study had been performed with exceedingly high doses (100 mg/kg) of 13-cis-retinoic acid and all-trans-retinoic acid, we have now performed additional experiments with 10-fold lower doses. Studies were also done with the main metabolites of the two retinoids (the 4-oxo-derivatives) to elucidate the metabolism, pharmacokinetics, and teratogenicity of each single compound. It was shown that all-trans-retinoic acid and 4-oxo-all-trans-retinoic acid were extremely teratogenic, whereas their corresponding cis isomers caused only 2% cleft palate. Embryonic exposure to the trans isomers was likewise higher than that to the cis isomers, as shown by the far higher embryonic peak concentrations and by the 30-fold higher areas under the concentration-time curve values reached for the trans isomers compared with the cis isomers. At 8 hr, embryo/maternal plasma ratios were higher than 1 after administration of the all-trans compounds. Concentrations found in the placenta and yolk sac were higher for the trans forms than for the cis forms. We propose a model for a facilitated transport of the all-trans forms to the developing embryo and suggest that the conversion to the trans isomer and trans metabolite could play a major role in the teratogenicity of 13-cis-retinoic acid in humans.

Topics: Administration, Oral; Animals; Female; Gestational Age; Maternal-Fetal Exchange; Mice; Pregnancy; Stereoisomerism; Teratogens; Tretinoin

A fully automated gradient high-performance liquid chromatographic method for the determination of isotretinoin, tretinoin and their 4-oxo metabolites in plasma was developed, using the column-switching technique. After dilution with an internal standard solution containing 20% acetonitrile, 0.5 ml of the sample was injected onto a precolumn (17 X 4.6 mm I.D.), filled with C18 Corasil 37-53 micron. Proteins and polar plasma components were washed out using 1% ammonium acetate-acetonitrile (9:1, v/v) as mobile phase 1. After valve switching, the retained components were transferred to the analytical column in the backflush mode, separated by gradient elution and detected at 360 nm by UV detection. Using two coupled reversed-phase columns (125 mm long), the separation of cis and trans isomers was possible, and all four compounds could be quantified down to 2 ng/ml of plasma. The inter-assay precision in the concentration range 20-100 ng/ml was between 1.0 and 4.7% for all compounds.

Topics: Animals; Chromatography, High Pressure Liquid; Haplorhini; Humans; Indicators and Reagents; Isotretinoin; Retinoids; Specimen Handling; Spectrophotometry, Ultraviolet; Tretinoin

Retinoids are being used increasingly in dermatologic practice. Fetal malformation is a major form of toxicity associated with certain retinoids. In this study, the developmental toxicity of isotretinoin, its metabolites, and a structurally related analog, tretinoin, were evaluated using the sea urchin model. The American sea urchin, Arbacia punctulata, completes its major developmental stages within 24 hr and has been previously utilized for screening human teratogens. The parent compound, isotretinoin, induced dose-dependent delayed rather than dysmorphic development of the sea urchin embryo. In contrast, its metabolites, 4-oxo-isotretinoin and 4-oxo-tretinoin, and the analog tretinoin induced strikingly dysmorphic development. This may indicate that the metabolites of isotretinoin, rather than the parent compound, may be responsible for the fetal abnormalities observed in the "isotretinoin teratogen syndrome." Therefore, the sea urchin model might serve as a discriminating and rapid screening test for identifying other potential developmentally toxic retinoids.

Topics: Animals; Female; Isotretinoin; Male; Ovum; Retinoids; Sea Urchins; Teratogens; Time Factors; Tretinoin

Isotretinoin (13-cis-retinoic acid) is used in the treatment of severe cystic acne. Adverse ocular reactions, including blepharoconjunctivitis and dry eye symptoms, are frequent side effects of this drug. Our previous observation that retinol is present in tears and lacrimal gland fluid suggests that isotretinoin may also be secreted by the lacrimal gland. Rabbits were treated with isotretinoin, and lacrimal gland fluid was collected from the cannulated lacrimal gland duct. Tears were collected from patients who were being treated with isotretinoin. Lacrimal gland fluid and tears were analyzed by reverse-phase high-pressure liquid chromatography and a peak eluted from each sample, which was identified as isotretinoin. We conclude that the lacrimal gland is able to secrete isotretinoin in addition to retinol and that, in animals and patients treated systemically with isotretinoin, the ocular surface is exposed to the drug via the tear film.

Topics: Acne Vulgaris; Adult; Animals; Chromatography, High Pressure Liquid; Female; Humans; Isomerism; Isotretinoin; Lacrimal Apparatus; Male; Rabbits; Tears; Time Factors; Tretinoin

A simple, precise, and specific high-performance liquid chromatography (HPLC) method was developed for the simultaneous measurement of retinol (ROH), 13-cis-retinoic acid (13-cRA), and 4-oxo-13-cRA. The average recovery of ROH from serum or plasma was 95%, and the precision of the assay was less than 5%. With this HPLC method, a series of studies was carried out to evaluate the stability of ROH in various matrices. ROH was stable under our HPLC assay conditions as well as in plasma- and in serum-enriched culture media; however, ROH was not stable in aqueous matrices. Serum or heparinized plasma may be routinely used for measurement of ROH concentrations, providing EDTA, oxalate, and citrate are not used as anticoagulants. Because of ROH stability, blood samples can be kept on ice in the dark for at least 24 hours prior to separation of plasma. In addition, plasma samples containing ROH can be stored for up to 1 year at -20 degrees C without loss of stability.

Topics: Animals; Anticoagulants; Chromatography, High Pressure Liquid; False Negative Reactions; Isotretinoin; Male; Rats; Rats, Inbred Strains; Time Factors; Tretinoin; Vitamin A

Previous observations have indicated that isotretinoin (IT), a drug in common use for therapy of cystic acne, is teratogenic in humans but possesses low embryotoxicity in pregnant mice, probably because of its shorter half-life and limited placental transfer in rodents. In human volunteers and patients, one major blood metabolite of IT is 4-oxo-isotretinoin (4-oxo-IT) which undergoes slower elimination than IT and may itself be a participant in teratogenesis. To investigate the problem of species differences displayed by IT and the role of its metabolism, embryotoxic effects of 4-oxo-IT were examined after its single or repeated intubations into pregnant ICR mice and compared with the effects of a similar regimen of IT. The two compounds were also tested for their relative ability to suppress chrondrogenesis in the in vitro cell and organ culture assays. We found that a single dose of 4-oxo-IT, 100 mg/kg, given on day 11 of gestation (plug day = day 0 of gestation) produced a moderate incidence of limb reduction defects and cleft palate (39% and 27% of surviving fetuses, respectively), while a dose of 150 mg/kg affected virtually every fetus. IT, on the other hand, produced no defects in fetuses exposed to similar dose levels. Repeated intubations with IT, however, resulted in increasing the frequencies of limb reduction defects and cleft palate to levels obtained after 4-oxo-IT administration. We found that a 3-hour interval between IT intubations was more effective in this regard than an 8-hour interval. Repeated IT intubations also uncovered sharper stage-dependency of limb and palatal defects than obtained otherwise.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Abnormalities, Drug-Induced; Animals; Cartilage; Cleft Palate; Female; In Vitro Techniques; Isotretinoin; Limb Deformities, Congenital; Male; Mice; Mice, Inbred ICR; Pregnancy; Teratogens; Tretinoin

Twelve healthy male subjects received 80, 160, 240, and 320 mg doses of oral isotretinoin as multiples of 40 mg capsules separated by 2-week washout periods in a randomized, crossover design. Blood samples were drawn at specific times over a 72-hour period after dosing. Blood concentrations of isotretinoin as well as its major metabolite, 4-oxo-isotretinoin, were determined by a specific HPLC method. In addition to the normal laboratory battery of tests, serum triglyceride levels were determined before the first dose and again 72 hours after each of the four doses. Mean (+/- SD) maximum concentrations after 80 to 320 mg doses were 366 +/- 159, 820 +/- 474, 1056 +/- 547, and 981 +/- 381 ng/ml, whereas the respective AUC0-infinity values were 3690 +/- 1280, 7030 +/- 4140, 9780 +/- 6080, and 9040 +/- 2900 ng X hr/ml. The observed apparent elimination t1/2 remained approximately the same (14.7 hours) for each dose. The maximum concentration and AUC values for isotretinoin appear to be dose proportional from 80 to 240 mg but plateau at the 320 mg dose level. Therefore, because isotretinoin blood concentrations may not increase with higher doses in the fasting state, single, oral doses in excess of 240 mg should be used with caution. The data also suggest that elevated triglyceride levels are not a simple function of isotretinoin blood concentrations across the entire study population and dose range studied, but that in subjects with triglyceride levels in excess of the normal range triglyceride levels were positively related to isotretinoin blood concentrations.

Topics: Administration, Oral; Adult; Dose-Response Relationship, Drug; Half-Life; Humans; Isotretinoin; Kinetics; Male; Tretinoin; Triglycerides

All-trans [11-3H]4,4- difluororetinyl acetate was synthesized by treating methyl all-trans [11-3H]4- oxoretinoate with diethylaminosulfurtrifluoride , followed by reduction and acetylation of the product. After oral administration of the radioactive difluoro analog in oil to rats, difluororetinol , difluororetinyl palmitate and related esters, 4- oxoretinol , 4- oxoretinoic acid and polar conjugated derivatives were identified in the intestine, liver, kidney and/or blood. The major metabolic products were difluororetinyl palmitate and related esters, which were stored in the liver. The presence of the difluoro analog in liver oil from treated rats was confirmed by 19F-NMR spectroscopy. Neither retinol nor retinyl esters were detected as products of the metabolism of the difluoro analog. Nonetheless, all-trans difluororetinyl acetate showed 26 +/- 12% of the biological activity of all-trans retinyl acetate in the rat growth assay. Presumably, the difluoro analog is active per se in growth rather than by conversion to retinol or to one of its known growth-promoting metabolites. In general, however, the difluoro analog was metabolized in a manner very similar to vitamin A. The vitamin A moiety of administered difluororetinyl acetate and retinyl acetate was poorly stored (1.8-3.3%) in the liver of vitamin A-depleted rats, confirming and extending past reports that the liver storage mechanism is severely impaired when initial liver stores are very low.

Topics: Animals; Biological Assay; Body Weight; Diterpenes; Feces; Kinetics; Liver; Magnetic Resonance Spectroscopy; Male; Rats; Rats, Inbred Strains; Retinyl Esters; Tissue Distribution; Tretinoin; Vitamin A

A pharmacokinetic profile of isotretinoin and its major dermatologically active blood metabolite, 4-oxo-isotretinoin, was developed following a single 80 mg oral suspension dose of isotretinoin to 15 normal male subjects. Blood samples were assayed for isotretinoin and 4-oxo-isotretinoin using a newly developed reverse-phase HPLC method. Following rapid absorption from the suspension formulation, isotretinoin is distributed and eliminated with harmonic mean half-lives of 1.3 and 17.4 h, respectively. Maximum concentrations of isotretinoin in blood were observed at 1 to 4 h after dosing. Maximum concentrations of the major blood metabolite of isotretinoin, 4-oxo-isotretinoin, are approximately one-half those of isotretinoin and occur at 6 to 16 h after isotretinoin dosing. The ratio of areas under the curve for metabolite and parent drug following the single dose suggests that average steady-state ratios of metabolite to parent drug during a dosing interval will be approximately 2.5. Both isotretinoin and its metabolite can be adequately described using a single linear pharmacokinetic model.

Topics: Administration, Oral; Adult; Humans; Isotretinoin; Kinetics; Male; Middle Aged; Tretinoin

The multiple dose pharmacokinetics of isotretinoin and its major blood metabolite, 4-oxo-isotretinoin, were studied in 10 patients with cystic acne and 11 patients with various keratinization disorders. Blood samples were obtained at predetermined times following the first dose, interim doses and the final dose. Blood concentrations of isotretinoin and 4-oxo-isotretinoin were measured by a specific and sensitive HPLC method. A lag time was usually observed prior to the onset of absorption following oral administration of the drug in a soft elastic gelatin capsule. Absorption then proceeded rapidly and maximum blood concentrations usually occurred within 4 h of drug administration. The harmonic mean half-life for the elimination of isotretinoin by the cystic acne patients was approximately 10 h after the initial dose and did not change significantly following 25 days of 40 mg b.i.d. dosing. Steady-state blood concentrations remained relatively constant after the fifth day of dosing. The harmonic mean elimination half-life in the patients with various disorders of keratinization was about 16 h. The results of the 2 studies suggest that no significant changes in the pharmacokinetics of isotretinoin occur during multiple dosing and that the multiple dose pharmacokinetic profile is predictable and can be described using a linear pharmacokinetic model. This suggests that the steady-state concentrations of isotretinoin can be predicted from single dose data.

Topics: Adult; Female; Humans; Isotretinoin; Kinetics; Male; Tretinoin

A high-performance liquid chromatography (HPLC) method for the quantitation of 13-cis-retinoic acid (13-cis-RA) and its major metabolite, 4-oxo-13-cis-RA, in human blood has been developed. The method includes extraction of 1 ml of blood with diethyl ether at pH 6 and the analysis of the extract by reversed-phase HPLC with solvent programming and detection at 365 nm. The quantitation ranges for 13-cis-RA and 4-oxo-13-cis-RA are 10--2000 and 50--2000 ng/ml of blood, respectively. The method also provides estimates of the concentrations of all-trans-RA and 4-oxo-all-trans-RA. The mean intra- and inter-assay variabilities for all four compounds were 6% or less. The method separates 13-cis-RA and 4-oxo-13-cis-RA from 9-cis-RA, all-trans-RA, 4-oxo-all-trans-RA, and some other possible metabolites, such as hydroxy and epoxy retinoic acids. The method has been successfully applied to the analyses of over 1200 blood samples from four 13-cis-RA clinical studies.

Topics: Acne Vulgaris; Blood Preservation; Chromatography, High Pressure Liquid; Humans; Isotretinoin; Reference Standards; Tretinoin

The clinical pharmacokinetic profiles of two orally administered retinoids, isotretinoin and etretinate, are discussed and compared. The pharmacokinetic profile of isotretinoin is predictable and can be described using linear pharmacokinetic theory. The drug is rapidly absorbed following oral administration, is highly bound to plasma protein, and is metabolized to 4-oxo-isotretinoin. The apparent half-lives of elimination of isotretinoin and 4-oxo-isotretinoin following the oral administration of isotretinoin range from 10 to 20 hours and 24 to 29 hours, respectively. Steady-state pharmacokinetic profiles in patients are consistent with the single-dose pharmacokinetics in normal subjects. Following oral administration, etretinate undergoes significant first-pass biodegradation to its corresponding carboxylic acid; the acid appears rapidly in the circulation, often earlier than the parent drug, and its plasma concentration is usually comparable to, or greater than, that of the parent drug. The apparent elimination rates of drug and metabolite are similar (6-13 hours) following a single dose, suggesting that metabolite elimination may be formation-rate limited. During multiple dosing of etretinate, a very slow terminal elimination phase is observed which is not detected after single-dose administration. The prolonged half-life of this phase suggests accumulation in a deep tissue compartment. Differences between the two retinoids reflect their differing physicochemical properties.

Topics: Acne Vulgaris; Etretinate; Humans; Isomerism; Isotretinoin; Kinetics; Male; Tretinoin

Administration of either all-trans-[3H]- or 13-cis-[3H]retinoic acid to hamsters fed a normal diet results in the formation of a number of polar metabolites. At least one of these metabolites has been shown to be common to both isomers of retinoic acid and can be generated in a hamster liver 10,000 X g supernatant system using 13-cis-retinoic acid as substrate. It has been identified as 13-cis-4-oxoretinoic acid by mass spectral, ultraviolet absorption, and proton NMR characteristics, as well as by its co-migration with synthetic 13-cis-4-oxoretinoic acid in two different high pressure liquid chromatographic systems. In addition, its metabolic precursor, 13-cis-4-hydroxyretinoic acid, has been tentatively identified. These compounds are believed to be early metabolites in the elimination pathway of retinoic acid from the body.

Topics: Animals; Chromatography, High Pressure Liquid; Cricetinae; Isotretinoin; Liver; Magnetic Resonance Spectroscopy; Mass Spectrometry; Spectrophotometry, Ultraviolet; Stereoisomerism; Tretinoin; Tritium

Incubation of [3H]retinoic acid in the presence of hamster liver 10000g supernatant produces several metabolites that are more polar than the parent compound. Two of these metabolites are identical with synthetic all-trans-4-hydroxyretinoic acid and all-trans-4-oxoretinoic acid both in ultraviolet absorption and mass spectral characteristics and in migration rates on two different reverse-phase high-pressure liquid chromatographic systems. The metabolites produced in a cell-free liver incubation reaction also migrate on a high-pressure liquid chromatography column together with metabolites isolated from a tracheal organ culture system. Both the metabolites and the synthetic standards show less biological activity than the parent all-trans-retinoic acid in a tracheal organ culture assay.

Topics: Animals; Biological Assay; Chromatography, High Pressure Liquid; Cricetinae; Female; Liver; Male; Mass Spectrometry; Spectrophotometry, Ultraviolet; Trachea; Tretinoin; Vitamin A; Vitamin A Deficiency

Topics: Animals; Biological Assay; Body Weight; Carotenoids; Fatty Acids, Unsaturated; Intestinal Mucosa; Ketones; Liver; Magnetic Resonance Spectroscopy; Male; Oxidation-Reduction; Rats; Spectrophotometry, Infrared; Spectrophotometry, Ultraviolet; Tretinoin; Vitamin A; Vitamin A Deficiency