tretinoin and liarozole

The ichthyoses constitute a large and heterogeneous group of disorders characterised by varying degrees of skin scaling. Most forms are rare but between them, they affect tens of thousands of people worldwide. Treatment presently consists primarily of topical keratolytic agents, aimed at removing the ichthyotic scales, and emollients. If an ichthyosis cannot be controlled by topical therapy alone, retinoids can be used. Although effective in several forms of ichthyosis, side effects and teratogenicity severely limit their use. The recent development of retinoic acid metabolism blocking agents (RAMBAs) offers new possibilities. With these drugs, retinoid effects may be obtained with less side effects and a shorter post-treatment teratogenicity period. This review discusses the RAMBAs that are now in clinical trials and outlines possible future developments.

Topics: Animals; Drug Design; Humans; Ichthyosis; Imidazoles; Keratolytic Agents; Molecular Structure; Tretinoin

Retinoids play a crucial role in cellular differentiation and proliferation of epithelial tissue and their utility in oncology and dermatology is well documented. This mini review focuses on the role of all-trans-retinoic acid (ATRA or RA), the principal endogenous retinoid and its metabolism in cancer therapy. ATRA has been used successfully in differentiating therapy of acute promyelecytic leukemia and other types of cancers. However, its usefulness is limited by the rapid emergence of ATRA resistance due (in part) to ATRA - induced acceleration of ATRA metabolism. A novel strategy to subjugate the limitation associated with exogenous ATRA therapy has been to modulate and/or increase the levels of endogenous ATRA by inhibiting the cytochrome P450-dependent ATRA-4-hydroxylase enzyme(s) responsible for ATRA metabolism. These inhibitors are also referred to as retinoic acid metabolism blocking agents (RAMBAs). This review highlights development in the design, synthesis and evaluation of RAMBAs since 1987. Major emphasis is given to liarozole, the most studied and only RAMBA to undergo clinical investigation and also the recently developed novel and highly potent 4-azoly retinoids. The potential role of a new family of cytochrome P450 enzymes, CYP26, with specificity towards ATRA is also discussed.

Topics: Animals; Antineoplastic Agents; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Drug Design; Enzyme Inhibitors; Humans; Imidazoles; Retinoic Acid 4-Hydroxylase; Tretinoin

Although significant advances have been made in the treatment of some malignancies, the prognosis of patients with metastatic tumors remains poor. Differentiating agents redirect cells toward their normal phenotype and therefore may reverse or suppress evolving malignant lesions or prevent cancer invasion. In addition, they offer a potential alternative to the classic cytostatic drugs.. The purpose of this review was to examine the current and potential future roles of differentiating agents in the treatment of cancer.. Initial studies with differentiating agents focused on retinoid therapy. Although retinoids have shown some clinical success, their widespread use has been limited by resistance and, in the chemopreventive setting, toxicity. This has led to the synthesis of a number of new retinoids that currently are undergoing clinical investigation. A further approach to overcoming the drawbacks associated with exogenous retinoids has been to increase the levels of endogenous retinoic acid (RA) by inhibiting the cytochrome P450-mediated catabolism of RA using a novel class of agents known as retinoic acid metabolism blocking agents (RAMBAs). Liarozole, the first RAMBA to undergo clinical investigation, preferentially increases intratumor levels of endogenous RA resulting in antitumor activity.. Although studies using exogenous retinoids in this setting have not yet fulfilled their initial promise, studies with a growing set of synthetic retinoids are ongoing. Furthermore, modulation of endogenous retinoids may offer a significant new potential treatment for cancer.

Topics: Antineoplastic Agents; Cell Differentiation; Chemoprevention; Cytochrome P-450 Enzyme Inhibitors; Drug Resistance, Neoplasm; Enzyme Inhibitors; Humans; Imidazoles; Neoplasms; Phenotype; Receptors, Retinoic Acid; Retinoids; Tretinoin

Metastatic prostate adenocarcinoma is a leading cause of cancer-related deaths among men. First line treatment is primarily aimed at blocking the synthesis and action of androgens. As primary endocrine treatment, androgen deprivation is usually achieved by orchidectomy or LHRH analogues, frequently combined with androgen receptor antagonists in order to block the residual adrenal androgens. However, nearly all the patients will eventually relapse. Available or potential second line therapies include, among others, alternative endocrine manipulations and chemotherapy. Cytochrome P450-dependent enzymes are involved in the synthesis and/or degradation of many endogenous compounds, such as steroids and retinoic acid. Some of these enzymes represent suitable targets for the treatment of prostate cancer. In first line therapy, inhibitors of the P450-dependent 17,20-lyase may achieve a maximal androgen ablation with a single drug treatment. Ketoconazole at high dose blocks both testicular and adrenal androgen biosynthesis but its side-effects, mainly gastric discomfort, limit its widespread use. A series of newly synthesized, more selective, steroidal 17,20-lyase inhibitors related to 17-(3-pyridyl)androsta-5,16-dien-3beta-ol, may open new perspectives in this field. In prostate cancer patients who relapse after surgical or medical castration, therapies aiming at suppressing the remaining adrenal androgen biosynthesis (ketoconazole) or producing a medical adrenalectomy (aminoglutethimide+hydrocortisone) have been used, but are becoming obsolete with the generalization of maximal androgen blockade in first line treatment. The role of inhibition of aromatase in prostate cancer therapy, which was postulated for aminoglutethimide, could not be confirmed by the use of more selective aromatase inhibitors, such as formestane. An alternative approach is represented by liarozole fumarate (LIA), a compound that blocks the P450-dependent catabolism of retinoic acid (RA). In vitro, it enhances the antiproliferative and differentiation effects of RA in cell lines that express RA metabolism, such as F9 teratocarcinoma and MCF-7 breast carcinoma cells. In vivo, monotherapy with LIA increases RA plasma levels and, to a greater extent, endogenous tissue RA levels leading to retinoid-mimetic effects. In the rat Dunning prostate cancer models, it inhibits the growth of androgen-independent as well as androgen-dependent carcinomas relapsing after castration. Concurrently, changes i

Topics: Adenocarcinoma; Aldehyde-Lyases; Androgen Antagonists; Androgens; Animals; Antineoplastic Agents, Hormonal; Aromatase Inhibitors; Cattle; Clinical Trials as Topic; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Drug Design; Drug Screening Assays, Antitumor; Enzyme Inhibitors; Female; Humans; Imidazoles; Ketoconazole; Male; Mice; Neoplasm Proteins; Prostatic Neoplasms; Rats; Rats, Wistar; Salvage Therapy; Steroid 17-alpha-Hydroxylase; Tretinoin; Tumor Cells, Cultured

Liarozole reduced tumor growth in the androgen-dependent Dunning-G and the androgen-independent Dunning MatLu rat prostate carcinoma models as well as in patients with metastatic prostate cancer who had relapsed after orchiectomy. In vitro, liarozole did not have cytostatic properties, as measured by cell proliferation in breast MCF-7 and prostate DU145 and LNCaP carcinoma cell lines. It did not alter the metabolism of labeled testosterone i.e. the 5 alpha-reductase in cultured rat prostatic cells. In mouse F9 teratocarcinoma cells liarozole did not show any retinoid-like properties but enhanced the plasminogen activator production induced by retinoic acid. Furthermore, liarozole and retinoic acid similarly reduced the growth of the androgen-dependent Dunning-G tumor in nude mice and inhibited tumor promotion elicited by phorbol ester in mouse skin. These data have raised the hypothesis that the antitumoral properties of liarozole may be related to inhibition of retinoic acid degradation, catalyzed by a P-450-dependent enzyme that is blocked by the drug.

Topics: Animals; Antineoplastic Agents; Cell Division; Humans; Imidazoles; Neoplasms, Experimental; Plasminogen Activators; Testosterone; Tetradecanoylphorbol Acetate; Tretinoin

Lamellar ichthyosis is a keratinization disorder caused by TGM1, Ichthyin and several other gene mutations. A new treatment option is liarozole, which blocks the cytochrome P450 (CYP26)-mediated catabolism of endogenous all-trans retinoic acid. This study focuses on the expression of retinoid-related genes in ichthyotic epidermis before and after treatment with oral liarozole. We first compared the mRNA expression of cellular retinoic acid binding protein II (CRABPII), keratin (KRT) 2 and 4, CYP26A1 and B1, and two markers of inflammation (interleukin-1alpha and tumours necrosis factor (TNF)-alpha) in shave biopsies from 11 genetically defined, untreated patients and 12 age- and sex-matched healthy controls, finding no overt differences between the groups, besides elevated CRABPII expression. We then studied the biomarkers before and after 4 weeks of treatment with liarozole (75 or 150 mg/day), which produced a better therapeutic response in patients with Ichthyin (n=3) than in those with TGM1 (n=6) mutations. A significant decrease in the mRNA expression of KRT2 and TNF-alpha, and trends toward increased expression of KRT4 and CYP26A1 were observed in liarozole-treated patients, consistent with an increased retinoid stimulation of epidermis. However, there were no dose-related responses and the results of the immunostaining did not always parallel the mRNA findings. The results suggest that liarozole exerts a therapeutic effect in lamellar ichthyosis by mildly affecting the expression of retinoid- regulated genes in epidermis.

Topics: Administration, Oral; Adolescent; Adult; Aged; Biomarkers; Dermatologic Agents; Double-Blind Method; Female; Humans; Ichthyosis, Lamellar; Imidazoles; Interleukin-1alpha; Male; Middle Aged; Polymerase Chain Reaction; Receptors, Retinoic Acid; RNA, Messenger; Skin; Tretinoin; Tumor Necrosis Factor-alpha

Liarozole is a 1-substituted imidazole derivative that inhibits cytochrome P450 activity and increases endogenous plasma concentrations of retinoid acid (RA). We have previously demonstrated that RA down-modulates transforming growth factor (TGF)-alpha and epidermal growth factor receptor (EGFR) levels in head and neck squamous cell carcinoma by decreasing the transcription rate of these two genes. Previous reports suggest that RA receptor (RAR)-beta levels are down-modulated in head and neck cancer and are restored by RA therapy. Cellular RA-binding protein (CRABP)-II is up-regulated by RA and appears to modulate intracellular RA metabolism. In conjunction with a Phase I clinical trial, total intact RNA was extracted from oral cavity mucosa biopsied from 17 patients with advanced malignancies, before and after treatment with a 4-week course of liarozole. To analyze these limited quantities of total RNA (as little as 0.6 microg/sample), a quantitative reverse transcription-PCR assay was developed using delayed dropping of the 5' beta-actin primer to amplify the highly abundant beta-actin gene as an internal control. We used this method to determine the expression levels of TGF-alpha, EGFR, RAR-beta, and CRABP-II before and after treatment. There was a trend toward elevation of RAR-beta levels in oral mucosa after liarozole therapy (P = 0.107), whereas TGF-alpha, EGFR, and CRABP-II were not modulated by systemic liarozole treatment. These results suggest that liarozole may up-regulate RAR-beta in tissues from cancer patients and that expression levels of potential intermediate biomarkers may be determined in small tissue biopsies using a quantitative reverse transcription-PCR assay.

Topics: Actins; Antineoplastic Agents, Hormonal; Biomarkers; Carcinoma, Squamous Cell; DNA Primers; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; ErbB Receptors; Humans; Imidazoles; Mouth Mucosa; Mouth Neoplasms; Receptors, Retinoic Acid; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor alpha; Tretinoin; Up-Regulation

Retinoids derived from retinol or beta-carotene are inactivated, among other ways, by enzymes belonging to the P450 cytochrome group. Liarozole, an imidazole-containing compound, is known to be a potent inhibitor of the cytochrome P450-mediated metabolism of all-trans retinoic acid. As a result, increased levels of this retinoid are found in skin and plasma. Therefore, in the treatment of psoriasis, therapeutic effects may be expected with liarozole which are similar to those observed with synthetic retinoids. In an open study, oral liarozole was given at a daily dose of 75 mg b.i.d., for 12 weeks to 31 patients with severe psoriasis. After 1 month, this dosage could be increased to 150 mg b.i.d. if there was no improvement or only moderate improvement. Initially, the effect of liarozole was mainly on scaling. A decrease in the Psoriasis Area and Severity Index (PASI) score of 45% at week 4, of 69% at week 8 and of 77% at week 12 was obtained, compared with baseline. A further decrease in the PASI score of up to 87% was observed in the 16 patients who were allowed to continue treatment for a maximum period of 12 months. An excellent or good improvement was noted in 77% of the patients within 12 weeks of starting treatment. This response rate had increased to 88% by the last follow-up visit. Nearly all patients (29 of 31) experienced adverse reactions, such as dry oral mucosa, headache and itching. These were mostly mild and transient, but four patients dropped out of the study because of an adverse event. Haematological, biochemical and cardiovascular parameters were not significantly influenced by liarozole. Six patients showed an increase in triglycerides, which normalized in three of four patients during further treatment. The results of this pilot study suggest that, at doses of 75-150 mg b.i.d., liarozole is an active antipsoriatic drug, and may be a useful addition to the existing therapeutic armamentarium. Controlled studies should be performed to compare liarozole with standard oral treatments.

Topics: Adult; Aged; Aged, 80 and over; Antimetabolites; Arthritis, Psoriatic; Female; Humans; Imidazoles; Male; Middle Aged; Psoriasis; Severity of Illness Index; Treatment Outcome; Tretinoin

Continuous oral dosing with all-trans retinoic acid (RA) is associated with a progressive decrease in plasma drug concentrations that has been linked to relapse and retinoid resistance in patients with acute promyelocytic leukemia (APL). Since oxidation by cytochrome P-450 enzymes is critical in the catabolism of this drug, we evaluated whether pretreatment with an inhibitor of this system, liarozole, could attenuate this phenomenon. A total of 20 patients with solid tumors completed a 4-week course of all-trans RA therapy. On days 1, 2, 28, and 29, serial plasma samples were obtained from these patients after ingestion of a single oral dose (45 mg/m2) of all-trans RA. On days 2 and 29, liarozole was given 1 h prior to ingestion of all-trans RA at single doses ranging from 75 to 300 mg. The areas under the plasma RA concentration x time curves (AUCs) were then compared in the presence and absence of pretreatment. Following continuous oral treatment, the mean day-28 AUC of all-trans RA was significantly lower than the group mean level on day 1 (504 vs 132 ng h-1 ml-1; P = 0.05). This decline in plasma concentrations on day 28 was partially reversed by liarozole, which increased the mean plasma all-trans RA AUC on day 29 to 243 ng h-1 ml-1 (P = 0.004). The lowest dose of liarozole that reliably produced this effect was 300 mg. No enhanced toxicity was associated with liarozole administration. We conclude that liarozole at a dose of 300 mg effectively attenuates the induced decline in all-trans RA plasma concentrations that occurs with continuous treatment. This combination may be useful in attenuating or reversing retinoid resistance.

Topics: Antineoplastic Agents; Humans; Imidazoles; Neoplasms; Tretinoin

Mast cells (MCs) mature locally, thus possessing tissue-dependent phenotypes for their critical roles in both protective immunity against pathogens and the development of allergy or inflammation. We previously reported that MCs highly express P2X7, a receptor for extracellular ATP, in the colon but not in the skin. The ATP-P2X7 pathway induces MC activation and consequently exacerbates the inflammation. Here, we identified the mechanisms by which P2X7 expression on MCs is reduced by fibroblasts in the skin, but not in the other tissues. The retinoic-acid-degrading enzyme Cyp26b1 is highly expressed in skin fibroblasts, and its inhibition resulted in the upregulation of P2X7 on MCs. We also noted the increased expression of P2X7 on skin MCs and consequent P2X7- and MC-dependent dermatitis (so-called retinoid dermatitis) in the presence of excessive amounts of retinoic acid. These results demonstrate a unique skin-barrier homeostatic network operating through Cyp26b1-mediated inhibition of ATP-dependent MC activation by fibroblasts.

Topics: Adenosine Triphosphate; Animals; Cell Degranulation; Cytochrome P-450 Enzyme System; Dermatitis; Fibroblasts; Imidazoles; Immunity, Innate; Mast Cells; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Microbiota; Receptors, Purinergic P2X7; Retinoic Acid 4-Hydroxylase; Skin; Toll-Like Receptor 2; Tretinoin

In the brain, retinoic acid (RA) concentrations are under tight spatio-temporal control. Here, we show that challenge of primary mouse microglia with lipopolysaccharide (LPS) results in increased release of nitric oxide (NO) and tumor necrosis factor-α (TNF-α). Co-administration of RA attenuated microglial activation. Similarly, pretreatment with RA-metabolism inhibitor liarozole potently reduced NO and TNF-α release. Conversely, activated microglia showed increased protein expression of RA-degrading cytochromes CYP26A1, CYP26B1, CYP3A4 and CYP2C. Correspondingly, RA catabolism by activated microglia was significantly increased. Our results indicate that RA reduces microglial activation, but also, conversely, that the activation state of microglia influences RA metabolism.

Topics: Analysis of Variance; Animals; Animals, Newborn; Brain; Cells, Cultured; Cytochrome P-450 Enzyme System; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Gene Expression Regulation; Imidazoles; Lipopolysaccharides; Mice; Mice, Inbred C57BL; Microglia; Nitric Oxide; Receptors, Retinoic Acid; Retinoid X Receptors; RNA, Messenger; Time Factors; Tretinoin; Tumor Necrosis Factor-alpha

To study the influence of liarozole on leiomyoma cell proliferation and extracellular matrix (ECM) gene expression in immortalized leiomyoma cells.. Laboratory study.. University hospital.. None.. Tissue culture, real-time reverse transcription-polymerase chain reaction, Western blot.. Proliferation, messenger RNA (mRNA), and ECM protein expression.. Proliferation of leiomyoma cells was inhibited by treatment with liarozole at suprapharmacologic concentrations. The mRNA and protein expression of COL1A1, COL4A2, versican, fibromodulin, and fibronectin was increased in untreated leiomyoma cells compared with untreated patient-matched myometrial cells. Extracellular matrix mRNA expression was decreased in a dose-dependent manner in leiomyoma cells treated with pharmacologic concentrations of liarozole. In addition, myometrial cells treated with liarozole demonstrated no statistically significant alteration in ECM regulation.. Liarozole inhibited ECM protein production at pharmacologic concentrations in immortalized human leiomyoma cells. Retinoic acid metabolic blocking agents represent a potential therapeutic drug family for human leiomyomas.

Topics: Antineoplastic Agents, Hormonal; Cell Proliferation; Dose-Response Relationship, Drug; Extracellular Matrix; Extracellular Matrix Proteins; Female; Humans; Imidazoles; Leiomyoma; Tretinoin; Tumor Cells, Cultured; Uterine Neoplasms

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

The biosynthesis of retinoic acid (RA) from retinol is controlled by several enzymes, e.g. dehydrogenases (RalDH2, RoDH-4) and retinol-esterifying enzyme (LRAT), whereas its degradation mainly involves CYP26 enzymes. In keratinocytes, RA activates the nuclear retinoid-receptors inducing the transcription of many genes. Here, we examined the effects of RA and the CYP26 inhibitors, liarozole and talarozole, on retinoid metabolism and RA-regulated genes in organotypic epidermis. RA induced the expression of CYP26 enzymes already after 8 h, whereas LRAT exhibited a later response and peaked at 48 h, indicating a feedback induction of retinol esterification. In line with a reduced biosynthesis of RA from retinol after exogenous RA, the expression of RDH16 reduced 80% in response to exogenous RA. The mRNA expression of RA-regulated genes (KRT2, KRT4, CRABPII and HBEGF) was altered within 24 h after RA exposure. In contrast, the CYP26 inhibitors caused only minor effects, except for a clear-cut induction of CYP26A1 only when combined with minute amounts of exogenous RA. Cellular accumulation of exogenous [3H]RA was higher after talarozole than after liarozole, probably indicating a greater CYP26-inhibitory potency of the former drug. The present study shows that CYP26A1 expression is extremely sensitive to both exogenous RA and increased endogenous RA levels, i.e. due to CYP26 inhibition, and thus an excellent biomarker for retinoid signalling in organotypic epidermis.

Topics: Acyltransferases; Alcohol Oxidoreductases; Androgen Antagonists; Benzothiazoles; Biomarkers; Cells, Cultured; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Epidermis; Feedback, Physiological; Gene Expression Regulation, Enzymologic; Heparin-binding EGF-like Growth Factor; Humans; Imidazoles; Intercellular Signaling Peptides and Proteins; Keratin-2; Keratin-4; Keratinocytes; Receptors, Retinoic Acid; Retinal Dehydrogenase; Retinoic Acid 4-Hydroxylase; Tretinoin; Triazoles; Vitamin A

Methodology previously described by our group was applied to the preparation of a series of 4-alkyl/aryl-substituted 1-[benzofuran-2-yl-phenylmethyl]-1H-triazoles. The [1,2,4]-triazole derivatives were prepared for a range of alkyl and aryl substituents, and for the 4-methyl, 4-ethyl, 4-(i)propyl, 4-(t)butyl, 4-phenyl and 4-chlorophenyl derivatives, the minor [1,3,4]-triazole isomer also isolated. All the triazole derivatives were evaluated for CYP26A1 inhibitory activity using a MCF-7 cell-based assay. The 4-ethyl and 4-phenyl-1,2,4-triazole derivatives displayed inhibitory activity (IC(50) 4.5 and 7 microM, respectively) comparable with that of the CYP26 inhibitor liarozole (IC(50) 7 microM). Using a CYP26A1 homology model (based on CYP3A4) template, docking experiments were performed with MOE with multiple hydrophobic interactions observed in addition to coordination between the triazole nitrogen and the haem transition metal.

Topics: Benzofurans; Binding Sites; Cell Line, Tumor; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Enzyme Activation; Humans; Imidazoles; Ligands; Models, Molecular; Molecular Structure; Protein Conformation; Retinoic Acid 4-Hydroxylase; Stereoisomerism; Structure-Activity Relationship; Tretinoin; Triazoles; Tumor Cells, Cultured

The remarkable toxicity of (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthylenyl)-1-propenyl] benzoic acid (TTNPB) compared to all trans-retinoic acid (tRA) is due to multiple factors, including reduced affinities for cytosolic binding proteins (CRABPs), resistance to metabolism, and prolonged nuclear receptor activation. To further investigate the role of half-life in retinoid toxicity, experiments were performed to determine whether, and to what extent, inhibition of tRA metabolism by liarozole increased its toxicity comparable to that of TTNPB in the mouse limb bud system. Liarozole is a known inhibitor of tRA 4-hydroxylation (CYP26). In the absence of liarozole, the IC50 for inhibition of chondrogenesis by tRA was 140 nM compared to 0.3 nM for TTNPB, a 467-fold difference. Following the addition of liarozole (10(-6) M) to limb bud cultures, the potency of tRA to inhibit chondrogenesis was increased approximately 14-fold (IC50 of 9.8 nM). Although liarozole markedly increased toxicity of tRA in mouse limb bud micromass cultures, tRA metabolism was inhibited only about 10%. These results indicate that a relatively minor decrease in the metabolism of tRA in the mouse limb bud system is associated with a marked enhancement of toxicity that is likely related to the prolongation of tRA half-life during a critical period of development. Thus, the prolonged half-life of TTNPB is the most significant factor contributing to the remarkable teratogenicity of this synthetic aromatic retinoid.

Topics: Animals; Antineoplastic Agents; Benzoates; Cells, Cultured; Chondrogenesis; Chromatography, High Pressure Liquid; Drug Synergism; Enzyme Inhibitors; Imidazoles; Limb Buds; Mice; Retinoids; Structure-Activity Relationship; Tretinoin

All-trans-retinoic acid is a potent inhibitor of cell proliferation and inducer of differentiation. However, the clinical use of all-trans-retinoic acid in the treatment of cancer is significantly hampered by its toxicity and the prompt emergence of resistance, believed to be caused by increased all-trans-retinoic acid metabolism. Inhibitors of all-trans-retinoic acid metabolism may therefore prove valuable in the treatment of cancer. In this study, we characterize R116010 as a new anticancer drug that is a potent inhibitor of all-trans-retinoic acid metabolism. In vitro, R116010 potently inhibits all-trans-retinoic acid metabolism in intact T47D cells with an IC(50)-value of 8.7 nM. In addition, R116010 is a selective inhibitor as indicated by its inhibition profile for several other cytochrome P450-mediated reactions. In T47D cell proliferation assays, R116010 by itself has no effect on cell proliferation. However, in combination with all-trans-retinoic acid, R116010 enhances the all-trans-retinoic acid-mediated antiproliferative activity in a concentration-dependent manner. In vivo, the growth of murine oestrogen-independent TA3-Ha mammary tumours is significantly inhibited by R116010 at doses as low as 0.16 mg kg(-1). In conclusion, R116010 is a highly potent and selective inhibitor of all-trans-retinoic acid metabolism, which is able to enhance the biological activity of all-trans-retinoic acid, thereby exhibiting antitumour activity. R116010 represents a novel and promising anticancer drug with an unique mechanism of action.

Topics: Animals; Antineoplastic Agents; Benzothiazoles; Breast Neoplasms; Cell Division; Chromatography, High Pressure Liquid; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Dose-Response Relationship, Drug; Female; Humans; Imidazoles; Mammary Neoplasms, Experimental; Mice; Mice, Inbred Strains; Mixed Function Oxygenases; Retinoic Acid 4-Hydroxylase; Reverse Transcriptase Polymerase Chain Reaction; Thiazoles; Tretinoin; Tumor Cells, Cultured

Topics: Antineoplastic Agents, Hormonal; Biomarkers; Biopsy; Clinical Trials as Topic; Health Services Research; Humans; Imidazoles; Mouth; Mucous Membrane; Neoplasms; Treatment Outcome; Tretinoin

Embryonal carcinoma cell lines (F9 EC and P19 EC) were stably transfected with 1.8 kb promoter sequence of RARbeta2 coupled to the lacZ gene as a system for measuring active retinoids. These stable transfectants, designated F9-1.8 and P19-1.8, were used as reporter cell lines to investigate different retinoids for their ability to activate the reporter gene. F9-1.8 cells showed similar EC(50) values for the acidic retinoids all-trans retinoic acid (RA), 4-oxo RA, 9-cis RA, and 13-cis RA, in the range of 1-7 nM, while P19-1.8 cells were less sensitive. Retinal showed decreased activity compared to the RA isomers in both lines. However, P19-1.8 cells hardly showed beta-gal activity after treatment with retinol, while the lacZ reporter in F9-1.8 cells was still inducible by this retinoid. In addition, the reporter system was used to investigate RA metabolism and its inhibition by P450 inhibitors. A combination of RA and liarozole showed a 10 times greater induction of the RARbeta2-lacZ reporter in P19-1.8 cells, but not in F9-1.8 cells. The EC(50) value for 4-oxo RA, however, was not altered, indicating that metabolic conversion of RA to 4-oxo RA is the target for inhibition by liarozole in P19-1.8 cells. HPLC analysis revealed nearly complete inhibition of RA metabolism after liarozole treatment in P19-1.8 cells, resulting in higher levels of RA. Finally, the F9-1.8 cells were used to detect active retinoids during different stages of chick limb bud development, demonstrating that it is the limb bud mesenchyme which generates RA and not the epidermis, with a twofold higher level of RA in the posterior half than in the anterior half.

Topics: Animals; Carcinoma, Embryonal; Chick Embryo; Chromatography, High Pressure Liquid; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Dose-Response Relationship, Drug; Gene Expression Regulation, Developmental; Genes, Reporter; Imidazoles; Isomerism; Ketoconazole; Limb Buds; Mesoderm; Mice; Promoter Regions, Genetic; Receptors, Retinoic Acid; Retinoids; Time Factors; Transcriptional Activation; Transfection; Tretinoin; Tumor Cells, Cultured

The clinical use of all-trans-retinoic acid (ATRA) in the treatment of cancer is significantly hampered by the prompt emergence of resistance, believed to be caused by increased ATRA catabolism. Inhibitors of ATRA catabolism may therefore prove valuable for cancer therapy. Liarozole-fumarate is an anti-tumour drug that inhibits the cytochrome P450-dependent catabolism of ATRA. ATRA, but also its naturally occurring catabolites, 4-oxo-ATRA and 5,6-epoxy-ATRA, as well as its stereoisomers, 9-cis-RA and 13-cis-RA, show significant antiproliferative activity in MCF-7 human breast cancer cells. To further elucidate its mechanism of action, we investigated whether liarozole-fumarate was able to enhance the antiproliferative activity of ATRA catabolites and isomers. Liarozole-fumarate alone up to a concentration of 10(-6) M had no effect on MCF-7 cell proliferation. However, in combination with ATRA or the ATRA catabolites, liarozole-fumarate (10(-6) M) significantly enhanced their antiproliferative activity. On the contrary, liarozole-fumarate (10(-6) M) was not able to potentiate the antiproliferative activity of the ATRA stereoisomers, most probably because of the absence of cytochrome P450-dependent catabolism. Together, these findings show that liarozole-fumarate acts as a versatile inhibitor of retinoid catabolism in that it not only blocks the breakdown of ATRA, but also inhibits the catabolic pathway of 4-oxo-ATRA and 5,6-epoxy-ATRA, thereby enhancing their antiproliferative activity.

Topics: Antineoplastic Agents; Antineoplastic Agents, Hormonal; Breast Neoplasms; Cell Division; Chromatography, High Pressure Liquid; DNA Replication; DNA, Neoplasm; Dose-Response Relationship, Drug; Drug Synergism; Female; Humans; Imidazoles; Stereoisomerism; Tretinoin; Tumor Cells, Cultured

We report on the isolation of a cytochrome P450 (CYP)-like retinoic acid (RA) 4-hydroxylase cDNA from T-47D human breast cancer cells that is identical to the recently cloned hCYP26, which is involved in the metabolic breakdown of RA. Northern analysis showed that this novel human CYP26 is induced within 1 h upon RA treatment in RA-sensitive T-47D breast carcinoma cells but not in RA-resistant MDA-MB-231 breast cancer cells and HCT 116 colon cancer cells. Stable introduction of different RA receptor (RAR) subtypes in HCT 116 cells showed that CYP26 expression is dependent on RARalpha and RARgamma and, to a lesser extent, on RARbeta and closely paralleled RA metabolism, suggesting that it represents the major RA 4-hydroxylase in these human cells. Furthermore, stable introduction of all three RAR subtypes in HCT 116 cells resulted in restored RA sensitivity as assayed by growth inhibition. Interestingly, CYP26 activity was efficiently inhibited by liarozole, an inhibitor of RA metabolism, leading to enhanced growth inhibition by RA. The RA-induced CYP26 was shown to be highly specific for the hydroxylation of all-trans-RA and did not recognize the 13-cis and 9-cis isomers. This substrate specificity is promising for finding retinoids that are not recognized by this enzyme and, therefore, could be more effective in growth inhibition of susceptible cancer cells.

Topics: Blotting, Northern; Blotting, Western; Breast Neoplasms; Chromatography; Colonic Neoplasms; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; DNA; Enzyme Induction; Female; Humans; Hydroxylation; Imidazoles; Microsomes; Receptors, Retinoic Acid; Retinoic Acid 4-Hydroxylase; Retinoic Acid Receptor alpha; Retinoic Acid Receptor gamma; Substrate Specificity; Transfection; Tretinoin; Tumor Cells, Cultured

The synthesis of LIAZAL (compound 9, R085246) is described. LIAZAL inhibits all-trans-retinoic acid metabolism and thereby exerts retinoid-like effects in vivo.

Topics: Animals; Antineoplastic Agents; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Dermatologic Agents; Enzyme Inhibitors; Humans; Imidazoles; Magnetic Resonance Spectroscopy; Molecular Structure; Rats; Retinoic Acid 4-Hydroxylase; Tretinoin

Treatment with all-trans-retinoic acid (ATRA) induces complete remission in many acute promyelocytic leukemia patients. However, plasma drug levels progressively decrease following prolonged treatment with oral ATRA. This decrease is due, at least in part, to the induced cytochrome P-450-dependent metabolism of ATRA. To investigate if incorporation of ATRA in liposomes could alter its metabolism, we compared the cellular metabolism of liposomal-ATRA (L-ATRA) with free drug. Microsomes isolated from the rat liver metabolized L-ATRA to a significantly lower extent than they did free-ATRA. Similarly, in F9 cells, L-ATRA was metabolized at a slower rate than the free drug. These results suggest that L-ATRA may have important clinical implications in terms of slowing down the rate of ATRA metabolism and producing long-term remission in APL patients.

Topics: Animals; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Drug Carriers; Enzyme Inhibitors; Imidazoles; Liposomes; Male; Microsomes, Liver; Rats; Teratocarcinoma; Testicular Neoplasms; Tretinoin; Tumor Cells, Cultured

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

Cytochrome P450-dependent oxidation is a pathway for all-trans-retinoic acid (all-trans-RA) catabolism. Induction of this catabolic pathway was studied in MCF-7 breast cancer cells. MCF-7 cells showed low constitutive all-trans-RA catabolism. Concentration-dependent induction was obtained by preincubation of the cells with all-trans-RA (10(-9) to 10(-6) M). Onset of induction was fast, being detectable within 60 min, with maximal induction (45-fold) obtained after 16 h. Enzymatic characterization of induced all-trans-RA catabolism showed an estimated Km value (Michaelis-Menten constant) of 0.33 microM and a Vmax value (maximal velocity of an enzyme-catalysed reaction) of 54.5 fmol polar all-trans-RA metabolites 10(6) cells(-1) h(-1). These kinetic parameters represent the overall formation of polar metabolites from all-trans-RA. Induction of all-trans-RA catabolism was also obtained with other retinoids, CH55 >> 13-cis-RA = all-trans-RA > 9-cis-RA > 4-keto-all-trans-RA > 4-keto-13-cis-RA > retinol. The potency of the retinoids to induce all-trans-RA catabolism was correlated to their retinoic acid receptor affinity (Crettaz et al, 1990; Repa et al, 1990; Sani et al, 1990). Induction of all-trans-RA catabolism was inhibited by actinomycin D. Furthermore, all-trans-RA did not increase cytosolic retinoic acid-binding protein (CRABP) mRNA levels. These data suggest that induction of all-trans-RA catabolism in MCF-7 cells is a retinoic acid receptor-mediated gene transcriptional event. Induced all-trans-RA catabolism was inhibited by various retinoids with decreasing potency in the order: all-trans-RA > 4-keto-all-trans-RA > 13-cis-RA > 9-cis-RA > 4-keto-13-cis-RA > retinol > CH55. The antitumoral compound liarozole-fumarate inhibited all-trans-RA catabolism with a potency similar to that of all-trans-RA.

Topics: Antineoplastic Agents, Hormonal; Breast Neoplasms; Cytosol; Female; Humans; Imidazoles; Keratolytic Agents; Oxidation-Reduction; Reproducibility of Results; Retinoids; Retinol-Binding Proteins; Tretinoin; Tumor Cells, Cultured

Cellular retinoic acid binding protein II (CRABP II) mRNA is selectively induced by all-trans retinoic acid in human skin and dermal fibroblasts. In order to determine whether this response can be used as a reliable measure of retinoid potency and activity, we treated human skin fibroblasts for 24 h with increasing concentrations of several natural and synthetic retinoids. CRABP II mRNA levels were measured by quantitative Northern blotting and compared, when possible, with those obtained after topical application of the same retinoids to human skin. All eight active retinoids tested induced a concentration-dependent CRABP II mRNA response in the fibroblast assay. In contrast, one known inactive retinoid (meta-carboxy TTNPB), differing from the active form only in the position of the carboxyl substituent, failed to evoke a response. The fibroblast and human skin bioassays agreed with respect to relative potency and response amplitude for three of the three retinoids tested. Retinoic acid was approximately 10-fold more potent than retinal in both assays, suggesting that oxidation to retinoic acid underlies the activity of retinol in fibroblasts as well as in intact skin. In support of this hypothesis, treatment with liarozole, an inhibitor of P450-mediated retinoic acid oxidative catabolism, significantly increased fibroblast CRABP II mRNA levels and potentiated the effects of retinol by 1.5-fold at concentrations at which it had no effect on its own. Taken together, these results identify the fibroblast CRABP II response as a reproducible measure of retinoid bioactivity with promise as a predictor of human skin responses and further suggest that metabolism is an important determinant of retinoid bioactivity in vivo.

Topics: Biological Assay; Cells, Cultured; Cytochrome P-450 Enzyme Inhibitors; Fibroblasts; Humans; Imidazoles; Receptors, Retinoic Acid; Retinaldehyde; Retinoids; RNA, Messenger; Skin; Tretinoin

We studied the enzymatic characteristics of the oxidative catabolism of retinoic acid (RA) and its inhibition by liarozole-fumarate in homogenates of rat Dunning R3327G prostate tumors. Homogenates of rat liver were used as reference material. Both tumor and liver homogenates were able to catabolize retinoic acid. HPLC analysis revealed only very polar metabolites in tumors, while in the liver both metabolites with intermediate polarity and more polar metabolites were found. Kinetic analysis of retinoic acid catabolism showed a K(m) of 1.7 +/- 0.7 microM and a Vmax of 4.2 +/- 4.4 pmol polar RA metabolites/mg protein/hr for Dunning G tumor homogenates. In liver homogenates a K(m) value of 4.3 +/- 0.5 microM and a Vmax value of 290 +/- 120 pmol polar RA metabolites/mg protein/hr were obtained. Liarozole-fumarate inhibited retinoic acid catabolism in Dunning tumors and liver with IC50 values of 0.26 +/- 0.16 microM and 0.14 +/- 0.05, respectively. The results suggest that rat Dunning R3327G tumors are able to metabolize retinoic acid in a manner similar to that found in rat liver but with a lower metabolizing capacity.

Topics: Animals; Antineoplastic Agents; Chromatography, High Pressure Liquid; Enzyme Inhibitors; Female; Imidazoles; Kinetics; Liver; Male; NADP; Oxidation-Reduction; Prostatic Neoplasms; Rats; Rats, Wistar; Tretinoin

Metabolic inactivation of all-trans retinoic acid to 4-hydroxy retinoic acid occurs via a cytochrome P-450 enzyme. We investigated the effects of liarozole on the retinoic acid 4-hydroxylase activity of human epidermis and its ability to modify in vivo human skin responses to retinoic acid and all-trans retinol. Retinoic acid 4-hydroxylase activity induced in vivo by 4 d treatment with retinoic acid (0.1%) was inhibited in vitro by liarozole in a concentration-dependent manner. Comparable micromolar concentrations of liarozole were extracted from stratum corneum-free epidermis treated with 3% liarozole. Retinoic acid levels in liarozole-treated skin increased to 19 +/- 5 ng/g wet wt (mean +/- SEM, p < 0.002, n = 17) at 18 h and to 6 +/- 2 ng/g wet wt (p = 0.38, n = 17) at 48 h as compared to vehicle (not detectable). At 48 h, retinoic acid 4-hydroxylase activity was induced 9-fold over vehicle (p < 0.03, n = 8). At 96 h, no significant erythema or increased epidermal thickness was found when either retinoic acid (0.001%), all-trans retinol (0.0250%), or liarozole (3%) was applied individually, but when 0.001% retinoic acid and 3% liarozole were applied together, both erythema and increased epidermal thickness occurred. In contrast, 0.025% all-trans retinol and 3% liarozole together caused increased epidermal thickness but no erythema. These data demonstrate that, at doses used here, liarozole, although an effective inhibitor of retinoic acid 4-hydroxylase, cannot function alone like a retinoid in vivo, probably because of retinoic acid 4-hydroxylase induction. In the presence of a low dose retinoic acid or all-trans retinol, however, liarozole can amplify human skin responses to each retinoid in a manner characteristic of the retinoid at a higher dose (erythema and hyperplasia with retinoic acid; no erythema but hyperplasia with all-trans retinol).

Topics: Androgen Antagonists; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Dose-Response Relationship, Drug; Drug Synergism; Epidermis; Humans; Imidazoles; Retinoic Acid 4-Hydroxylase; Skin; Tretinoin; Vitamin A

Beta-carotene, canthaxanthin and retinoic acid (RA) inhibited growth of human DU145 prostate cancer cells by 45, 56 and 18%, respectively. Lycopene was also found to inhibit cell growth. Other carotenoids including xanthophyll (lutein), cryptoxanthin and zeaxanthin were less effective. Liarozole (a novel imidazole-derived inhibitor of intracellular RA catabolism) had a modest effect upon cell growth, this drug significantly amplified the pro-apoptotic actions of beta-carotene and RA. RA-induced expression of thymosin beta-10, an apoptotic accelerant, was associated with increased nuclear DNA nicking as measured using TUNEL. Liarozole enhanced the proapoptotic actions of RA upon DNA fragmentation in a dose-dependent manner. These actions were accompanied by inhibition of the cell survival factor bcl-2. Liarozole may thus prove useful as a novel chemotherapeutic/chemopreventive agent by boosting retinoid-induced apoptosis in the prostate.

Topics: Antineoplastic Agents; Apoptosis; beta Carotene; Blotting, Northern; Cell Division; DNA Fragmentation; Drug Synergism; Genes, bcl-2; Humans; Imidazoles; Male; Prostatic Neoplasms; Retinoids; RNA, Messenger; Thymosin; Tretinoin; Tumor Cells, Cultured

Liarozole inhibits cytochrome P-450-dependent enzymes that play a key role in all-trans-retinoic acid (ATRA) catabolism. In MCF-7 cells, liarozole potentiates the antiproliferative effects of ATRA. The present study demonstrates this synergistic effect on cell differentiation of MCF-7 cell cultures as measured by immunocytochemistry for cytokeratins 8, 18, and 19, actin, E-cadherin, desmoglein and desmoplakins I & II. ATRA concentration-dependently (10(-8) M-10(-6) M) induced changes in actin stress fibers and cytokeratin intermediate filaments. These changes were accompanied by a more obvious interaction of these filaments with junctional complexes. Surface area and volume of the MCF-7 cells increased markedly after ATRA exposure, with extensive filopodia formation. Liarozole (10(-6) M) alone had no effect on cell morphology, cytokeratin or actin organization, or on cellular junctions. In combination with ATRA (10(-9) M and 10(-8) M), liarozole potentiated the ATRA-induced effects. The MCF-7 cell cultures used showed morphological heterogeneity, consisting of at least two cellular subpopulations. This was reflected in the staining for E-cadherin, desmoglein and desmoplakins I & II. ATRA increased E-cadherin staining at cell-cell contact sites, but had no influence on the staining patterns of desmoglein and desmoplakins I & II. Similar to what has been observed for the cytoskeletal differentiation parameters, liarozole alone had no influence on E-cadherin, desmoglein or desmoplakins I & II expression, but in combination with ATRA again intensified the effects on E-cadherin distribution. These effects on MCF-7 cells agree with previously obtained observations concerning the inhibition of ATRA catabolism by liarozole. Furthermore, our data support the hypothesis that the antiproliferative properties of the drug are accompanied by induction of differentiation.

Topics: Antineoplastic Agents, Hormonal; Breast Neoplasms; Carcinoma; Cell Adhesion Molecules; Cell Division; Cytoskeletal Proteins; Cytoskeleton; Dose-Response Relationship, Drug; Drug Synergism; Female; Humans; Imidazoles; Neoplasm Proteins; Tretinoin

Liarozole has been reported to inhibit P450 enzymes responsible for the catabolism of retinoic acid. This suggests that it may increase the effectiveness of cancer chemopreventive agents, such as retinoic acid, and pro-vitamin A carotenoids, such as beta-carotene, which may yield retinoids. To test this we have utilized the 10T1/2 cell assay system of neoplastic transformation. Simultaneous treatment with Liarozole (10(-5) M) potentiated by a factor of 1000 the ability of low concentrations of retinoic acid (10(-10) M) to inhibit carcinogen-induced neoplastic transformation, to up-regulate gap junctional communication and to increase connexin43 expression. When tested under conventional culture conditions, Liarozole itself partially suppressed neoplastic transformation and up-regulated gap junctional communication; this ability appears due to the presence of retinol in the serum component of cell culture medium. When assays for junctional communication and of connexin43 expression were performed under defined conditions, in the absence of serum, Liarozole was ineffective alone, yet still augmented the effects of retinoic acid. HPLC analysis of cell culture medium demonstrated that Liarozole (10(-5) M) completely protected retinoic acid (10(-6) M) from catabolism over a 48 h period. Potential effects of Liarozole on the activities of carotenoids were also examined. Inhibition of neoplastic transformation by the pro-vitamin A carotenoid beta-carotene, but not by the non-pro-vitamin A carotenoid canthaxanthin, was moderately potentiated by Liarozole. The augmentation of response to beta-carotene was more apparent when tested under defined conditions; here Liarozole strongly increased junctional communication and Cx43 expression. In contrast, Liarozole did not potentiate the activity of canthaxanthin under defined conditions, while increasing the activity of 4-keto retinoic acid, a likely metabolite. Liarozole also elevated connexin43 expression in early passage human fibroblasts. These data indicate that rapid catabolism of retinoic acid limits its in vitro activities, that a portion of the action of beta-carotene as a cancer preventive agent is due to its conversion to retinoic acid and that canthaxathin exerts its chemopreventive action largely independent of conversion to 4-keto retinoic acid.

Topics: Animals; Anticarcinogenic Agents; Antineoplastic Agents; beta Carotene; Carotenoids; Cell Communication; Cell Transformation, Neoplastic; Cells, Cultured; Connexin 43; Drug Synergism; Fibroblasts; Gap Junctions; Humans; Imidazoles; Mice; Mice, Inbred C3H; Skin; Tretinoin; Up-Regulation

Liarozole showed antitumoral activity in the Dunning AT-6sq, an androgen-independent rat prostate carcinoma. To investigate its potential mechanism of action, the effects of the drug doses (ranging from 3.75 to 80 mg/kg b.i.d.) on endogenous plasma and tissue all-trans-retinoic acid levels and on the differentiation status of the tumor cells were evaluated. To follow modulation of differentiation, cytokeratins were localized in the (un)treated tumors by immunocytochemistry and quantitatively determined by immunoblotting. Results showed that liarozole statistically significantly reduced tumor weight from 30 mg/kg upwards and induced accumulation of all-trans-retinoic acid both in plasma and tumors. In the tumors, a statistically significant accumulation was already noted from 7.5 mg liarozole/kg upwards. Concomitantly, the differentiation status shifted from a keratinizing towards a non-keratinizing squamous carcinoma, which was further confirmed by the cytokeratin profile of the carcinoma (presence of CK 8, 10, 13, 14, 18, 19). Immunoblotting revealed an overall decrease in cytokeratin content, except for CK 8. These findings suggest that the antitumoral properties of liarozole might be related to an increase in the degree of tumor differentiation through accumulation of all-trans-retinoic acid.

Topics: Animals; Antibodies, Monoclonal; Antineoplastic Agents; Carcinoma, Squamous Cell; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Imidazoles; Immunoblotting; Immunohistochemistry; Keratins; Male; Neoplasm Transplantation; Organ Size; Prostate; Prostatic Neoplasms; Random Allocation; Rats; Rats, Inbred F344; Tretinoin; Tumor Cells, Cultured; Vimentin

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

Liarozole is an imidazole-containing compound that inhibits the cytochrome P-450-dependent metabolism of all-trans-retinoic acid (RA). In vitro, liarozole (IC50, 2.2 microM) suppressed the P-450-mediated conversion of RA to more polar metabolites by hamster liver microsomes. In vivo, it enhanced the plasma level of RA from mostly undetectable values (less than 0.5 ng/ml) in control rats to 1.4 +/- 0.1 and 2.9 +/- 0.1 ng/ml in animals treated p.o. with 5 and 20 mg/kg of liarozole, respectively. Moreover, liarozole possessed antikeratinizing activity: when dosed subchronically (5-20 mg/kg, once daily for 3 days) to ovariectomized rats, the compound reversed the vaginal keratinization induced in these animals by estrogenic stimulation. Dose response experiments indicated that the antikeratinizating effect of liarozole was as potent as that of RA. One-dimensional electrophoresis and immunoblotting of extracted vaginal epithelia showed that liarozole shared with RA the ability to inhibit the synthesis of high molecular weight (57-60 kDa) keratin proteins, and to enhance the expression of the 45 to 47 kDa keratin polypeptides. Furthermore, we found that antikeratinizing doses of liarozole doubled the RA concentration in the vagina of ovariectomized rats: the mean amount of RA extracted from 200 mg of vaginal tissue was increased from 1.1 +/- 0.1 ng in vehicle-treated animals to 2.2 +/- 0.2 and 2.6 +/- 0.2 ng after treatment with 5 and 20 mg/kg of liarozole, respectively. These findings indicate that liarozole, an inhibitor of RA metabolism and RA produce similar morphologic and biochemical effects on the differentiation process of rat vaginal epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Antineoplastic Agents; Chromatography, High Pressure Liquid; Cricetinae; Female; Imidazoles; Injections, Subcutaneous; Keratins; Mesocricetus; Microsomes, Liver; Ovariectomy; Rats; Rats, Inbred Strains; Tretinoin; Vagina

Liarozole is a new imidazole derivative with antitumoral properties. Effects of the compound alone and in combination with all-trans-retinoic acid on proliferation of MCF-7 human breast cancer cells were examined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay. Following 9 days of drug exposure, MCF-7 cell growth was concentration dependently inhibited by all-trans-retinoic acid (drug concentration resulting in 50% growth inhibition, 2 x 10(-8) M), while liarozole at 10(-5) M inhibited cell growth by only 35%. When MCF-7 cells were incubated with a combination of all-trans-retinoic acid and liarozole, the antiproliferative effect of all-trans-retinoic acid was clearly enhanced. This enhancement was dependent on the liarozole concentration and was more than 10-fold. A combination of 10(-8) M all-trans-retinoic acid and 10(-6) M liarozole resulted in a greater antiproliferative effect than that obtained with 10(-7) M all-trans-retinoic acid alone. When MCF-7 cells were incubated for 4 h with [3H]all-trans-retinoic acid, the radioactivity in the supernatant consisted of unaltered retinoid. However, when cells had been pretreated with 10(-6) M all-trans-retinoic acid overnight, they were able to substantially metabolize [3H]all-trans-retinoic acid during a subsequent 4-h incubation. High-performance liquid chromatography analysis of the supernatants revealed that the reaction products consisted mainly of very polar metabolites. Liarozole inhibited the metabolism of all-trans-retinoic acid in MCF-7 cells with 10(-5) M liarozole reducing the amount of polar metabolites by 87%. It is concluded that the enhancement by liarozole of the antiproliferative effects of retinoic acid on MCF-7 human breast cancer cells is probably due to inhibition of retinoic acid metabolism. Further research into these effects in MCF-7 cells as well as in other cancer cell lines will provide more information concerning the exact mechanism of action of liarozole and the use of inhibitors of retinoid metabolism in cancer treatment.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Division; Cytochrome P-450 Enzyme Inhibitors; Drug Synergism; Female; Humans; Imidazoles; Kinetics; Tretinoin; Tritium; Tumor Cells, Cultured

This study examines the effects of ketoconazole, R 75 251 and some other cytochrome P-450 inhibitors on the in vivo metabolism of all-trans-retinoic acid (RA) in normal rats. Oral treatment with ketoconazole or R 75 251 (40 mg/kg, -1 hr) reduced the elimination rate of i.v. injected RA from plasma: the half-life of RA increased from 27 min in control-treated animals to 43 min and 76 min after dosing with ketoconazole and R 75 251, respectively. However, neither drug had an effect on the distribution volume of the retinoid. Two hours after i.v. injection of RA, residual plasma levels of the retinoid were 11.2 ng/ml in ketoconazole and 22.7 ng/ml in R 75 251-treated rats. The other P-450 inhibitors, aminoglutethimide, cimetidine, itraconazole, metyrapone and saperconazole, showed no sparing effect on RA elimination: plasma levels of the acid were below 1 ng/ml, as in control-treated animals. Administration of ketoconazole or R 75 251 (40 mg/kg, -2 hr) to rats also enhanced endogenous plasma concentrations of RA. Levels of the retinoid were raised from mostly undetectable values (less than 0.5 ng/ml) to 1.3 +/- 0.1 and 2.5 0.1 ng/ml after treatment with ketoconazole and R 75 251, respectively. These data are indicative of the important contribution of the cytochrome P-450 enzyme system to the in vivo metabolic process of RA. In vivo inhibition of the P-450 pathway not only increased the biological half-life of exogenously administered RA, but also enhanced the endogenous plasma level of this vitamin A derivative.

Topics: Animals; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Imidazoles; Ketoconazole; Male; Rats; Rats, Inbred Strains; Tretinoin