Retinoid Agonists in the Targeting of Heterotopic Ossification.

Retinoids are metabolic derivatives of vitamin A and regulate the function of many tissues and organs both prenatally and postnatally. Active retinoids, such as all trans-retinoic acid, are produced in the cytoplasm and then interact with nuclear retinoic acid receptors (RARs) to up-regulate the transcription of target genes. The RARs can also interact with target gene response elements in the absence of retinoids and exert a transcriptional repression function. Studies from several labs, including ours, showed that chondrogenic cell differentiation and cartilage maturation require (i) the absence of retinoid signaling and (ii) the repression function by unliganded RARs. These and related insights led to the proposition that synthetic retinoid agonists could thus represent pharmacological agents to inhibit heterotopic ossification (HO), a process that recapitulates developmental skeletogenesis and involves chondrogenesis, cartilage maturation, and endochondral ossification. One form of HO is acquired and is caused by injury, and another severe and often fatal form of it is genetic and occurs in patients with fibrodysplasia ossificans progressiva (FOP). Mouse models of FOP bearing mutant ACVR1R206H, characteristic of most FOP patients, were used to test the ability of the retinoid agonists selective for RARα and RARγ against spontaneous and injury-induced HO. The RARγ agonists were found to be most effective, and one such compound, palovarotene, was selected for testing in FOP patients. The safety and effectiveness data from recent and ongoing phase II and phase III clinical trials support the notion that palovarotene may represent a disease-modifying treatment for patients with FOP. The post hoc analyses showed substantial efficacy but also revealed side effects and complications, including premature growth plate closure in some patients. Skeletally immature patients will need to be carefully weighed in any future regulatory indications of palovarotene as an important therapeutic option in FOP.

Effectiveness and mode of action of a combination therapy for heterotopic ossification with a retinoid agonist and an anti-inflammatory agent.

Heterotopic ossification (HO) consists of ectopic cartilage and bone formation following severe trauma or invasive surgeries, and a genetic form of it characterizes patients with Fibrodysplasia Ossificans Progressiva (FOP). Recent mouse studies showed that HO was significantly inhibited by systemic treatment with a corticosteroid or the retinoic acid receptor γ agonist Palovarotene. Because these drugs act differently, the data raised intriguing questions including whether the drugs affected HO via similar means, whether a combination therapy would be more effective or whether the drugs may hamper each other's action. To tackle these questions, we used an effective HO mouse model involving subcutaneous implantation of Matrigel plus rhBMP2, and compared the effectiveness of prednisone, dexamathaosone, Palovarotene or combination of. Each corticosteroid and Palovarotene reduced bone formation at max doses, and a combination therapy elicited similar outcomes without obvious interference. While Palovarotene had effectively prevented the initial cartilaginous phase of HO, the steroids appeared to act more on the bony phase. In reporter assays, dexamethasone and Palovarotene induced transcriptional activity of their respective GRE or RARE constructs and did not interfere with each other's pathway. Interestingly, both drugs inhibited the activity of a reporter construct for the inflammatory mediator NF-κB, particularly in combination. In good agreement, immunohistochemical analyses showed that both drugs markedly reduced the number of mast cells and macrophages near and within the ectopic Matrigel mass and reduced also the number of progenitor cells. In sum, corticosteroids and Palovarotene appear to block HO via common and distinct mechanisms. Most importantly, they directly or indirectly inhibit the recruitment of immune and inflammatory cells present at the affected site, thus alleviating the effects of key HO instigators.

Inverse agonists and antagonists of retinoid receptors.

Nuclear receptors (NRs) are ligand-inducible transcription factors that regulate a plethora of cell biological phenomena, thus orchestrating complex events like development, organ homeostasis, immune function, and reproduction. Due to their regulatory potential, NRs are major drug targets for a variety of diseases, including cancer and metabolic diseases, and had a major societal impact following the development of contraceptives and abortifacients. Not surprisingly in view of this medical and societal importance, a large amount of diverse NR ligands have been generated and the corresponding structural and functional analyses have provided a deep insight into the molecular basis of ligand action. What we have learnt is that ligands regulate, via allosteric conformational changes, the ability of NRs to interact with different sets of coregulators which in turn recruit enzymatically active complexes, the workhorses of the ligand-induced epigenetic and transcription-regulatory events. Thus, ligands essentially direct the communication of a given NR with its intracellular environment at the chromatin and extragenomic level to modulate gene programs directly at the chromatin level or via less well-understood extranuclear actions. Here we will review our current structural and mechanistic insight into the functionalities of subsets of retinoid and rexinoid ligands that act generically as antagonists but follow different mechanistic principles, resulting in "classical" or neutral antagonism, or inverse agonism. In addition, we describe the chemical features and guidelines for the synthesis of retinoids/rexinoids that exert specific functions and we provide protocols for a number of experimental approaches that are useful for studies of the agonistic and antagonistic features of NR ligands.

All-trans retinoic acid ameliorates trinitrobenzene sulfonic acid-induced colitis by shifting Th1 to Th2 profile.

Inflammatory bowel disease is characterized with uncontrolled immune response in inflamed mucosa, with dominance of Th1 cells. Recently, all-trans retinoic acid has been shown that can lead T-cell response by suppressing Th17 development via retinoic acid receptor (RAR), but it is still unknown whether all-trans retinoic acid can modulate Th1 response of inflammatory bowel disease. In the experiment, we investigated the effect of all-trans retinoic acid on trinitrobenzene sulfonic acid (TNBS)-induced murine colitis, and the possible mechanism. Mice were intraperitoneally treated daily with all-trans retinoic acid (the agonist of RAR-alpha) or LE135 (the antagonist of RAR-alpha) or medium, and sacrificed 6 days later. Colon was collected for histological analysis and myeloperoxidase (MPO) activity measurement. Lamina propria mononuclear cells (LPMCs) were isolated, cultured, and assayed for the expressions of T-bet and GATA-3 by the use of Western blot and for cytokine levels by the use of ELISA. All-trans retinoic acid treatment inhibited inflammatory responses as shown by lower histological inflammatory scores and MPO activity, compared with LE135 and medium groups. Furthermore, in LPMCs culture supernatants, the levels of Th1 cytokines (INF-gamma, IL-12, and TNF-alpha) were decreased while those of Th2 cytokines (IL-4 and IL-10) were increased significantly in all-trans retinoic acid-treated mice. In addition, T-bet expression in LPMCs was inhibited and GATA-3 expression was up-regulated in all-trans retinoic acidtreated mice. On the contrary, LE135 showed the reverse effects in colon inflammation and cytokine profile. By shifting Th1 to Th2 profile in inflamed mucosa, all-trans retinoic acid down-regulates inflammatory response and ameliorates acute TNBS-induced colitis, which suggests the ligand of RAR-alpha-based pharmaceutical strategies for managing inflammatory bowel disease.

Retinoic acid receptor stimulation protects midbrain dopaminergic neurons from inflammatory degeneration via BDNF-mediated signaling.

Functions of retinoic acid receptors (RARs) in adult CNS have been poorly characterized. Here we investigated potential neuroprotective action of tamibarotene (Am80), an RARalpha/beta agonist available for the treatment of acute promyelocytic leukemia, on midbrain dopaminergic neurons. Am80 protected dopaminergic neurons in rat midbrain slice culture from injury mediated by lipopolysaccharide-activated microglia, without affecting production of nitric oxide, a key mediator of cell injury. The effect of Am80 was mimicked by another RAR agonist, TAC-101, but not by a retinoid X receptor agonist, HX630, and HX630 did not synergize with Am80. We observed neuronal expression of RARalpha and RARbeta in midbrain slice culture and also found that Am80 increased tissue level of brain-derived neurotrophic factor (BDNF) mRNA. Exogenous BDNF prevented dopaminergic neurodegeneration, and the neuroprotective effect of Am80 was suppressed by a TrkB inhibitor, K252a, or by anti-BDNF neutralizing antibody. These results reveal a novel action of RARs mediated by enhancement of BDNF expression. Finally, oral administration of Am80 prevented dopaminergic cell loss in the substantia nigra induced by local injection of lipopolysaccharide in mice, indicating that RARs are a promising target of therapeutics for neurodegenerative disorders.

Tratamiento del linfoma cutáneo de células T con ligandos selectivos del receptor retinoide X: alteraciones endocrinológicas y metabólicas / Treatment of cutaneous T-cell lymphoma with retinoid receptor x- selective ligands: endocrine and metabolic disorders

Los ligandos selectivos del receptor retinoide X se utilizan actualmente en el tratamiento de estadios avanzados del linfoma cutáneo de células T resistentes a otros tratamientos sistémicos, siendo el bexaroteno el primer fármaco de este grupo aprobado en Europa. Se han descrito numerosos efectos adversos asociados a su utilización, entre los que destacan las alteraciones endocrino-metabólicas. Presentamos a 2 pacientes con linfoma cutáneo de células T, tratados con bexaroteno, que desarrollaron hipotiroidismo central y dislipemia de forma precoz tras su inicio. Se describen también la respuesta de estas alteraciones al tratamiento y su remisión completa tras suspender el bexaroteno (AU)

The retinoid X receptor-selective ligands has been used for advanced stages of cutaneous T-cell lymphoma refractory to previous systemic therapy, being bexarotene the first drug in this group approved in Europe. Multiple drug-related adverse events has been reported such as endocrine-metabolic disorders. We report 2 patients with cutaneous T-cell lymphoma, treated with bexarotene, that developed central hypothyroidism and dyslipidaemia immediately after the beginning of this treatment. We also showed the successfully treatment response of these alterations and the total clinical remission after discontinuing the drug (AU)

Alteraciones endocrino-metabólicas inducidas por bexaroteno en el tratamiento del linfoma cutáneo de células T / Bexarotene-induced endocrine-metabolic alterations in the treatment of cutaneous T-cell lymphoma

Introducción: La utilización de bexaroteno, agonista selectivo del receptor de retinoide X (RXR), en pacientes con linfoma cutáneo de células T (CTLC), ha puesto de manifiesto la aparición de dos complicaciones frecuentes, como son el hipotiroidismo central y la hipertrigliceridemia. Nos proponemos comprobar las repercusiones en las hormonas tiroideas y los lípidos plasmáticos en un pequeño grupo de pacientes con CTLC tratados con bexaroteno. Pacientes y método: Presentamos a 6 pacientes (2 mujeres, 4 varones) con CTLC, en distintos estadios de la enfermedad y resistentes por lo menos a un tratamiento sistémico previo, que recibieron tratamiento con bexaroteno a dosis de 300 mg/m2. Se evaluó la TSH basal y la T4 libre y los lípidos plasmáticos (colesterol y triglicéridos) al inicio del tratamiento, a las 4 y las 8 semanas y, en los pacientes en que se suspendió el tratamiento, 4 semanas después de finalizado. Resultados: En 4 de 6 pacientes, la función tiroidea antes del tratamiento con bexaroteno era normal, en un paciente se desconocía y otro había sido diagnosticado previamente de hipotiroidismo primario autoinmunitario subclínico. A las 4 semanas de iniciado el tratamiento, todos mostraron una disminución de las concentraciones de tirotropina (TSH) y tiroxina (LT4), y 4 precisaron tratamiento con dosis sustitutivas de levotiroxina. Todos los pacientes presentaron elevaciones en las concentraciones plasmáticas de colesterol y/o triglicéridos que requirieron tratamiento con estatinas o fenofibrato. En aquellos en que se suspendió el bexaroteno, las hormonas tiroideas y los lípidos retornaron a cifras similares a las basales. Conclusiones: El tratamiento con bexaroteno en pacientes con CTLC induce la aparición de alteraciones endocrino-metabólicas, como hipotiroidismo central y dislipemia mixta, que requieren tratamiento con dosis sustitutivas de levotiroxina y fármacos hipolipemiantes en la mayoría de las ocasiones. Dichas alteraciones son reversibles tras la suspensión del fármaco

Introduction: Bexarotene, a synthetic retinoid receptor X (RXR)-selective ligand recently approved for the treatment of cutaneous T-cell lymphoma (CTCL), often produces two complications: central hypothyroidism and hypertriglyceridemia. The aim of this study was to analyze the effects of bexarotene on thyroid hormones and plasma lipids in a small group of patients with CTLC treated with this drug. Patients and method: We assessed a cohort of 6 patients (2 women and 4 men) who were in different stages of the disease and resistant to at least one previous systemic treatment. The patients received bexarotene at a dose of 300 mg/m2. Levels of thyroid-stimulating hormone (TSH), free thyroxine (LT4), cholesterol, and triglycerides were evaluated at baseline and at weeks 4 and 8. In patients who suspended treatment, these levels were evaluated 4 weeks after treatment was stopped. Results: Pretreatment thyroid function was normal in four out of the 6 patients and was unknown in one; subclinical autoimmune hypothyroidism had previously been diagnosed in the remaining patient. After 4 weeks of treatment, TSH and LT4 were reduced in all patients, and thyroid hormone replacement therapy was required in 4. Plasma cholesterol and/or triglyceride levels were increased in all patients, and additional treatment with statins or fenofibrate was required. In patients who discontinued bexarotene treatment, thyroid hormone and lipid levels returned to baseline values. Conclusions: Bexarotene treatment in patients with CTCL produces endocrine-metabolic alterations such as central hypothyroidism and mixed dyslipidemia, which usually require treatment with additional doses of levothyroxine and lipid-lowering agents. These changes are reversible after discontinuing the drug

N-retinoyl-D-glucosamine, a new retinoic acid agonist, mediates topical retinoid efficacy with no irritation on photoaged skin.

BACKGROUND: Chronic ultraviolet (UV) radiation from sunlight induces wrinkle formation. Retinoic acid (RA) can markedly improve wrinkles, although RA does have some side-effects, such as skin irritation. As the efficacy and cytotoxicity of RA has been traced to its free carboxylic acid, we synthesized a new molecule, N-retinoyl-D-glucosamine (GRA), in which a glucosamine has been attached to the polar end group of all-trans retinoic acid. OBJECTIVES: To analyse the effect of topical GRA in wrinkle repair and anti-irritation in photoaged mice compared with topical RA, as well as to determine retinoic acid receptor (RAR) and retinoid X receptor (RXR) transactivation activity in vitro. METHODS: Hairless mice were irradiated with 60 mJ cm-2 of UVB for 10 weeks, and then topically treated with 0.05% GRA or 0.05% RA for 8 weeks. An in vitro transcriptional assay was performed and the activity of GRA in 293 cells transfected with RAR-alpha or RXR-alpha expression plasmid and luciferase reporter plasmid then determined. RESULTS: Topical GRA and RA brought about almost complete disappearance of the wrinkles caused by UVB irradiation. The two ligands promoted both a wide repair zone histologically, and the expression of type 1 collagen in the skin. In contrast, topical GRA treatment did not produce irritation such as erythema or roughness, or alteration of transepidermal water loss values, compared with RA. In the in vitro luciferase assay, GRA resulted in significant dose-dependent RAR transactivation activity in a 100 times higher concentration range than RA. GRA did not mediate RXR transactivation activity at all. CONCLUSIONS: Topical GRA appears to be able to repair photoaged skin damage without any of the irritation caused by topical RA, probably via RAR transactivation activity.

The transcription factor NGFI-B (Nur77) and retinoids play a critical role in acute neuroleptic-induced extrapyramidal effect and striatal neuropeptide gene expression.

Despite extensive investigation, the cellular mechanisms responsible for neuroleptic actions remain elusive. We have previously shown that neuroleptics modulated the expression of some members of the ligand-activated transcription factors (nuclear receptors) including the nerve-growth factor inducible gene B (NGFI-B or Nur77) and retinoid X receptor (RXR) isoforms. Using genetic and pharmacological approaches, we investigated the role of NGFI-B and retinoids in acute behavioral and biochemical responses to dopamine antagonists. NGFI-B knockout (KO) mice display a profound alteration of haloperidol-induced catalepsy and striatal neuropeptide gene expression. Haloperidol-induced increase of striatal enkephalin mRNA is totally abolished in NGFI-B KO mice whereas the increase of neurotensin mRNA expression is reduced by 50%. Interestingly, catalepsy induced by raclopride, a specific dopamine D(2)/D(3) antagonist is completely abolished in NGFI-B-deficient mice whereas the cataleptic response to SCH 23390, a dopamine D(1) agonist, is preserved. Accordingly, the effects of haloperidol on striatal c-fos, Nor-1, and dynorphin mRNA expression are also preserved in NGFI-B-deficient mice. The cataleptic response and the increase of enkephalin mRNA expression induced by haloperidol can also be suppressed by administration of retinoid ligands 9-cis retinoic acid and docosahexaenoic acid. In addition, we demonstrate that haloperidol enhances colocalization of NGFI-B and RXRgamma1 isoform mRNAs, suggesting that both NGFI-B and a RXR isoform are highly coexpressed after haloperidol administration. Our data demonstrate, for the first time, that NGFI-B and retinoids are actively involved in the molecular cascade induced by neuroleptic drugs.

Retinoid agonist isotretinoin ameliorates obstructive renal injury.

PURPOSE: Interstitial fibrosis is a major cause of end stage renal failure. Retinoids, which are involved in tissue repair and fibrosis, inhibit inflammatory and proliferative pathways. Therefore, we studied the dose dependent effects of the retinoid receptor agonist isotretinoin 13-cis retinoic acid in the unilateral ureteral obstruction model (UUO). MATERIALS AND METHODS: Sham operated control rats were compared with UUO rats treated with vehicle (UUO-Veh), or low (5 mg/kg body weight (UUO-LD) or high (25 mg/kg) (UUO-HD) dose isotretinoin. Kidneys were evaluated using reverse transcriptase-polymerase chain reaction and immunohistology 7 days after UUO. Renal injury and fibrosis were quantified by immunostaining and expression measurements of the genes involved in renal fibrosis. RESULTS: In UUO-Veh kidneys the interstitial area was expanded 5-fold but only 3-fold in UUO-HD and 3.5-fold in UUO-LD rats. Interstitial cell counts were 3-fold higher in UUO-Veh rats but significantly less in UUO-HD or UUO-LD animals. Tubular and interstitial cell proliferation was significantly higher in UUO-Veh rats compared with sham operated control plus vehicle animals but less so in UUO-LD and UUO-HD rats. In UUO-Veh rats interstitial infiltration by monocytes/macrophages was higher compared with unobstructed controls. It was significantly less after isotretinoin treatment. In UUO-Veh rats mRNA for procollagen I, and transforming growth factor-beta1 and II receptor was significantly increased. It was significantly less after treatment with isotretinoin. Fibronectin and collagen I immunostaining was also decreased by isotretinoin. CONCLUSIONS: Since isotretinoin limits proliferation, inflammation and fibrosis after UUO, retinoids should be further investigated as potentially promising therapeutic agents for renal disease.

Arginine of retinoic acid receptor beta which coordinates with the carboxyl group of retinoic acid functions independent of the amino acid residues responsible for retinoic acid receptor subtype ligand specificity.

The biological actions of retinoic acid (RA) are mediated by retinoic acid receptors (RARalpha, RARbeta, and RARgamma) and retinoid X receptors (RXRalpha, RXRbeta, and RXRgamma). Consistent with the X-ray crystal structures of RARalpha and RARgamma, site-directed mutagenesis studies have demonstrated the importance of a conserved Arg residue (alphaArg(276), betaArg(269), and gammaArg(278)) for coordination with the carboxyl group of RA. However, mutation of Arg(269) to Ala in RARbeta causes only a 3- to 6-fold increase in the K(d) for RA and EC(50) in RA-dependent transcriptional transactivation assays while the homologous mutation in either RARalpha or RARgamma causes a 110-fold and a 45-fold increase in EC(50) value, respectively. To further investigate the nature of this difference, we prepared mutant RARs to determine the effect of conversion of betaR269A to a mutant which mimics either RARalpha ligand selectivity (betaA225S/R269A) or RARgamma ligand selectivity (betaI263M/R269A/V338A). Our results demonstrate that in RARbeta mutants that acquire either RARalpha or RARgamma ligand specificity the Arg(269) position responsible for coordination with the carboxyl group of retinoids continued to function like that of RARbeta. Furthermore, three mutant receptors (betaA225S/R269A, betaA225S/F279, and alphaF286A) were found to have a greater than wild-type affinity for the RARalpha-selective ligand Am580. Finally, a homology-based computer model of the ligand binding domain (LBD) of RARbeta and the X-ray crystal structures of the LBD of both RARalpha and RARgamma are used to describe potential mechanisms responsible for the increased affinity of some mutants for Am580 and for the difference in the effect of mutation of Arg(269) in RARbeta compared to its homologous Arg in RARalpha and RARgamma.

Retinoids cause apoptosis in pancreatic cancer cells via activation of RAR-gamma and altered expression of Bcl-2/Bax.

All-trans-retinoic acid and 9-cis-retinoic acid have been reported to have inhibitory effects on pancreatic adenocarcinoma cells and we have shown that this is partly due to induction of apoptosis. In this study, the mechanisms whereby 9-cis-retinoic acid induces apoptosis in these cells were investigated. An involvement of the Bcl-2 family of proteins was shown, such that 9-cis-retinoic acid causes a decrease in the Bcl-2/Bax ratio. Overexpression of Bcl-2 also resulted in inhibition of apoptosis induced by 9-cis-retinoic acid. Furthermore, two broad-range caspase inhibitors blocked DNA fragmentation induced by 9-cis-retinoic acid, but had no effect on viability defined by mitochondrial activity. Using synthetic retinoids, which bind selectively to specific retinoic acid receptor subtypes, we further established that activation of retinoic acid receptor-gamma is essential for induction of apoptosis. Only pan-retinoic acid receptor and retinoic acid receptor-gamma selective agonists reduced viability and a cell line expressing very low levels of retinoic acid receptor-gamma is resistant to the effects of 9-cis-retinoic acid. A retinoic acid receptor-beta/gamma selective antagonist also suppressed the cytotoxic effects of 9-cis-retinoic acid in a dose-dependent manner. This study provides important insight into the mechanisms involved in suppression of pancreatic tumour cell growth by retinoids. Our results encourage further work evaluating the clinical use of receptor subtype selective retinoids in pancreatic carcinoma.

Retinoids--which dermatological indications will benefit in the near future?

Retinoids are compounds with pleiotropic functions and a relatively selective targeting of certain skin structures. They are vitamins, because retinol (vitamin A) is not synthesized in the body and must be derived from diet, but also hormones with intracrine activity, because retinol is transformed into molecules that bind to nuclear receptors, exhibit their activity, and are subsequently inactivated. Retinoids exert their effects on target cells by binding and activating nuclear retinoid receptors. Retinoid receptors bind their ligands in form of dimers. Heterodimers can be formed between two different retinoid receptor molecules but also between retinoid X receptors and the vitamin D receptor as well as the triiodothyronin receptor. This fact indicates complex interactions between retinoids and further hormonal signal transduction molecules. Interaction of retinoid receptors with transcriptional factors activated by other signal transduction mechanisms, e.g. AP-1, may provide dissociation of the retinoid effects. Retinoids can exhibit agonistic activity but also be neutral antagonists and inverse agonists. Topical and oral retinol, tretinoin, isotretinoin, and bexarotene, topical alitretinoin, retinaldehyde, motretinide, adapalene, tazarotene, and systemin acitretin compose the list of launched retinoids. Psoriasis and related disorders, congenital disorders of keratinization, acne, photoaging and hypovitaminosis A are classical approved indications of retinoid treatment, whereas cutaneous T-cell lymphoma, AIDS-associated Kaposi's sarcoma, acute promyelocytic leukemia and actinic lentigines were currently confirmed. In addition, retinoids have been successfully used in several other dermatoses, e.g. epithelial precanceroses and tumors, seborrhea, rosacea and acneiform dermatoses, lichen planus, eosinophilic folliculitis, condylomata accuminata, lichen sclerosus and atrophicus. Highly receptor selective molecules, retinoic acid receptor-beta-inducers, AP-1 complex antagonists, and inverse agonists will be probably lead the retinoid development in the near future. New, more effective and less toxic retinoids, alone or in combination with other drugs and new delivery systems may provide therapeutic solutions for benign and malignant proliferative skin diseases, such as psoriasis and non-melanoma tumors, cancer chemoprevention and differentiation therapy.

Structure-Activity study of retinoid agonists bearing substituted dicarba-closo-dodecaborane. Relation between retinoidal activity and conformation of two aromatic nuclei.

We have investigated the structure activity relationships of the potent retinoid agonist, 4-[4-(2-propyl-1,2-dicarba-closo-dodecaboran-l-yl)phenylamino]benzoic acid (BR403), which we have previously reported. Substitution of a methyl group on the aromatic nucleus or a methyl group on the nitrogen atom, or replacement of the amino group with ether, methylene, carboxyl or 1,1-ethylene greatly decreased the activity. The relatively planar conformation at the phenyl-N-phenyl moiety seems to play a critical role in the appearance of the biological activity.

Cell type and gene-specific activity of the retinoid inverse agonist AGN 193109: divergent effects from agonist at retinoic acid receptor gamma in human keratinocytes.

Retinoids are important regulators of epithelial differentiation. AGN 193109 is a high-affinity antagonist and inverse agonist for the nuclear retinoic acid receptors (RARs). Paradoxically, both AGN 193109 and retinoid agonists inhibit the expression of the differentiation marker MRP-8 in normal human keratinocytes (NHKs). TTNPB, an RAR agonist, and AGN 193109 mutually antagonize MRP-8 inhibition at both mRNA and protein levels. We find that this antagonism, which is greatest at an AGN 193109:TTNPB ratio of about 10:1, is absent when either compound is in significant excess. The potent RARalpha-specific agonist, AGN 193836, has no effect on MRP-8 regulation. These data indicate that inverse agonists and agonists suppress MRP-8 in NHKs through RARgamma using distinct and mutually inhibitory mechanisms. The activity of AGN 193109 on MRP-8 is cell type specific. In differentiating ECE16-1 cervical cells, TTNPB inhibits while AGN 193109 induces MRP-8 mRNA levels. The effect of AGN 193109 on genes inhibited by retinoid agonists in NHKs is also selective; expression of the differentiation markers transglutaminase 1 and keratin 6 is not down-regulated by AGN 193109 whereas stromelysin-1 expression is suppressed. These results show a complex gene and cell context-specific interplay between agonist and inverse agonist for the regulation of gene expression.

Regulation of retinoidal actions by diazepinylbenzoic acids. Retinoid synergists which activate the RXR-RAR heterodimers.

In human HL-60 promyelocytic leukemia cells, diazepinylbenzoic acid derivatives can exhibit either antagonistic or synergistic effects on the differentiation-inducing activities of natural or synthetic retinoids, the activity depending largely on the nature of the substituents on the diazepine ring. Thus, a benzolog of the retinoid antagonist LE135 (6), 4-(13H-10,11,12,13-tetrahydro-10, 10,13,13,15-pentamethyldinaphtho[2,3-b][1,2-e]diazepin-7-yl) benzoic acid (LE540, 17), exhibits a 1 order of magnitude higher antagonistic potential than the parental LE135 (6). In contrast, 4-[5H-2,3-(2,5-dimethyl-2,5-hexano)-5-methyldibenzo[b,e] [1,4]diazepin-11-yl]-benzoic acid (HX600, 7), a structural isomer of the antagonistic LE135 (6), enhanced HL-60 cell differentiation induced by RAR agonists, such as Am80 (2). This synergistic effect was further increased for a thiazepine, HX630 (29), and an azepine derivative, HX640 (30); both synergized with Am80 (2) more potently than HX600 (7). Notably, the negative and positive effects of the azepine derivatives on retinoidal actions can be related to their RAR-antagonistic and RXR-agonistic properties, respectively, in the context of the RAR-RXR heterodimer.

Receptor-selective retinoid agonists and teratogenic activity.

Retinoid mechanism of action is dependent upon interaction with the retinoid nuclear RAR and RXR subfamilies of receptors. Study of ligands selective for the different receptors or which modify that interaction may provide insight into which receptors play roles in or contribute to retinoid teratogenesis. The retinoids considered here include in the RAR alpha-selective arylcarboxamidobenzoic acid CD 336 (Am580), the RAR beta/gamma-selective naphthalenecarboxylic acid CD 135 and the adamantyl-phenylcarboxamidobenzoic acid CD 394, the RAR-selective tetrahydrotetra-methylanthracenylbenzoic acid (TTAB) SRI 3961, the carboxyphenylretinamide SRI 7167-67, and the RXR-selective diarylisopropylidene SR 11217. CD 135 has a 3-fold higher affinity for RAR beta when compared with RAR gamma, whereas CD 394 has a 3-fold higher affinity for RAR gamma when compared with RAR beta. A separate investigation into potential amelioration of retinoid teratogenesis by concomitant administration of the cyclohexanetrione (Ro 31-0521) was also conducted. When pregnant hamsters were given an oral bolus of CD 336 or CD 135 during the early primitive streak stage of gestation, these retinoids proved 60-100 times more potent teratogens than all-trans-retinoic acid. Intubation of CD 394 resulted in production of terata similar to that seen after an equivalent dose of all-trans-retinoic acid. Administration of SRI 3961 found this compound 8000 times more potent than all-trans-retinoic acid, while SRI 7167-67 failed to show any evidence for developmental toxicity even after exposure to 105 mg/kg. Studies with the RXR-selective SR 11217 found it to be far less potent than all-trans-retinoic acid. These data point to the conclusion that those retinoids which have no affinity for retinoid nuclear receptors also have little potential for induction of developmental toxicity at doses which do not also provoke maternal intoxication. Comparing in vitro transcriptional activation of wild-type human RAR for the supertoxic TTNBP (Ro 13-7410) and TTAB (SRI 3961) with their relative teratogenic potency in hamster found that the more toxic congener also had the lower in vitro EC50 transactivation value (at ratios approximating their differential toxicities measured as administered dose). The RAR beta/gamma-selective CD 135 (TTNN) was not as efficient as TTNBP (Ro 13-7410) or TTAB (SRI 3961) in hRAR transactivation and CD 135 was less toxic than either Ro 13-7410 or SRI 3961. Although the RXR-selective SR 11217 failed to elicit terata after moderate doses, malformations consistent with those induced by high doses of retinoic acid could be produced following a single large bolus of SR 11217. Under the conditions here, simultaneous administration of Ro 31-0521 with all-trans-retinoic acid appeared to reduce the total percentage of abnormal fetuses seen after exposure to retinoic acid alone, but fetal body weights remained depressed and the numbers of dead embryos remained elevated, suggesting only limited influence of the cyclohexanetrione on retinoid developmental toxicity.

Retinoid agonist activities of synthetic geranyl geranoic acid derivatives.

Micromolar concentrations of 4,5- didehydro geranyl geranoic acid (GGA) were able to induce up-regulation of retinoic acid receptor-beta gene expression in human hepatoma-derived cell line, HuH-7, to the same extent as all-trans RA. In chloramphenicol acetyl transferase (CAT) assay with retinoic acid response element-beta, GGA and 4,5-didehydro GGA were both positive, but 2,3-dihydro GGA was negative, even though these GGA derivatives have been reported to be all potent ligands for cellular retinoic-acid-binding protein(CRABP). However, 10,11,14,15- tetrahydro- 4,5- didehydro GGA, a compound without any affinity for CRABP, transactivated CAT gene expression. On the other hand, only GGA and 4,5-didehydro GGA were active to induce CAT gene expression through retinoid X response element of cellular retinol binding protein, type II gene. We show for the first time that chemically synthesized acyclic organic acids are potential agonists of natural retinoids.

[Novel synthetic retinoid agonists and antagonists].

Retinoic acid acts as a specific modulator of cellular differentiation and proliferation. Its natural and synthetic analogs, classified as retinoids, can be applied to the chemotherapy in the field of dermatology and oncology. Various benzoic acid derivatives exhibited the specific biological responses of retinoic acid and were named retinobenzoic acids. Especially, the aromatic amides such as Am80 and Am580 have better therapeutic effects than retinoic acid. N-Methylation of these highly active aromatic secondary amides caused the disappearance of the activity due to the change of the amide conformation from trans into cis. From such observations, the conformation of the linking group between alkyl-substituted benzene ring and benzoic acid moiety is an important factor for the activity. Some retinobenzoic acids do not bind to the cellular-retinoic acid-binding protein, but bind to nuclear retinoic acid receptors (RARs) with the binding affinity corresponding to the potency of their biological activities. Among them, Am80 can bind to two of the three RAR subtypes (RAR alpha and beta). The selectivity is favorable for the clinical application of retinoid since it has possibility to elicit a part of a number of the biological activities of retinoic acid.