Distribution, teratogenicity, and embryonic delivered dose of retinoid Ro 23-9223.

Ro 23-9223 is a highly lipophilic aromatic retinoid with antiproliferative and sebum supressive effects in preclinical disease models of acne. To investigate the relation between Ro 23-9223 developmental toxicity, drug distribution, and transplacental transfer, groups of pregnant hamsters were given oral doses of 50-500 mg/kg Ro 23-9223 on days 8 and 9 of gestation. The teratogenic phenotype induced at doses greater than 125 mg/kg per day was similar to that found after exposure to doses of 13-cis-retinoic acid (isotretinoin, Accutane) greater than 37.5 mg/kg per day. Oral bioavailability of Ro 23-9223 was very low compared to 13-cis-retinoic acid. The highest concentrations of Ro 23-9223 were found in maternal liver, lung, adipose tissue, cardiac muscle, and placenta, whereas only little of the compound crossed the blood-brain barrier. Based on embryo AUC, Ro 23-9223 had a 30- to 50-fold greater embryo:maternal concentration ratio than 13-cis-retinoic acid plus its bioactive metabolites following similar doses of the two retinoids. In preclinical pharmacology studies, oral doses of Ro 23-9223 (5 mg/kg per day) and 13-cis-retinoic acid (10 mg/kg per day) produced comparable gland size reductions in the hamster ear sebaceous gland reduction assay. Under these conditions, Ro 23-9223 plasma AUC was 40 times smaller than that of 13-cis-retinoic acid plus its bioactive metabolites. Assuming that the near linear dose-exposure relationship of Ro 23-9223 extends beyond the dose range of this study, embryo AUCs of Ro 23-9223 and 13-cis-retinoic acid (plus metabolites) would be near identical following pharmacologically equivalent doses. A comparison of embryo retinoid AUCs suggests a 4-fold lower teratogenic potency of Ro 23-9223 compared to with 13-cis-retinoic acid. Despite high embryo levels in hamsters, the data suggest an improved therapeutic index for Ro 23-9223 compared with 13-cis-retinoic acid in a preclinical acne disease model.

19F-NMR studies of retinol transfer between cellular retinol binding proteins and phospholipid vesicles.

The cellular retinol binding proteins, CRBP and CRBP II, are implicated in the cellular uptake of retinol and intracellular trafficking of retinol between sites of metabolic processing. 19F-NMR studies of retinol transfer between CRBP and CRBP II and phospholipid vesicles, using either fluorine-labeled ligand or protein, demonstrated that there was significantly more transfer of retinol from CRBP II to lipid vesicles than from CRBP. Differences in how readily protein-bound retinol is released to lipid bilayers may lead to differences in how these two proteins modulate intracellular retinol metabolism.

NMR studies of fluororetinol analogs complexed to two homologous rat cellular retinol-binding proteins.

Comparative 19F-NMR studies of fluororetinol analogs with rat cellular retinol binding protein II (CRBP II) and rat cellular retinol-binding protein (CRBP) were performed to probe differences in the binding interactions of these two homologous proteins. Line shape analyses of 19F-NMR spectra of (E,E,Z,E)-6-fluoro-9-(4-methoxy-2,3,6-trimethylphenyl)-3,7-dimethyl- 2,4,6,8-nonatetren-1-ol (ligand 1), (E,E,Z,E)-6-fluoro-9-(2,2' dimethyl-6-methylcyclohexenyl)-3,7- dimethyl-2,4,6,8-nonatetren-1-ol (ligand 2), (E,Z,E,E)-5-fluoro-9-(2,2'- dimethyl-6-methylcyclohexenyl)-3,7-dimethyl-2,4,6,8-nonatetren+ ++-1-ol (ligand 3), when complexed with CRBP II at temperatures ranging from 0-45 degrees C, revealed that the 19F resonances corresponding to the bound ligand were in slow chemical exchange between two resonance frequencies. This was further supported by a 2D-NOESY exchange experiment. The kex at 25 degrees C was estimated from spectral simulation and fitting analyses to be 887 s-1, 1010 s-1 and 771 s-1 for CRBP II complexed 1, 2, and 3, respectively. In contrast, only a single absorption was observed for bound ligands complexed with rat CRBP over this temperature range, suggesting that the conformational dynamics of retinol binding are different for these two closely homologous proteins.

Differential binding of retinol analogs to two homologous cellular retinol-binding proteins.

A comparative study of the interactions of rat cellular retinol-binding protein (CRBP) and cellular retinol-binding protein II (CRBP II) with a number of synthetic phenyl-substituted analogs of all-trans-retinol was performed using fluorescence and nuclear magnetic resonance analysis. These studies indicate that CRBP II is more sensitive to modifications of the ring moiety than CRBP. Removal of the two methyl substituents on the ring which are ortho to the polyene chain abolishes binding to CRBP II. Conformational analysis of the ligands indicates that these two methyl groups influence the planarity of the ligand. The identification of monospecific ligands may prove useful for studying the physiological roles of these two proteins.

Retinoic acid receptors and retinoid X receptors: interactions with endogenous retinoic acids.

The binding of endogenous retinoids and stereoisomers of retinoic acid (RA) to the retinoid nuclear receptors, RA receptor (RARs) and retinoid X receptors (RXRs), was characterized using nucleosol preparations from transiently transfected COS-1 cells. Among several stereoisomers of RA tested, including 7-cis-, 9-cis-, 11-cis-, 13-cis-, and all-trans-RA, only 9-cis-RA effectively competes with 9-cis-[3H]RA binding to the RXRs. Additionally, the endogenous retinoid trans-didehydro-RA (t-ddRA) does not interact with RXRs, whereas the 9-cis form of ddRA competes effectively. RXRs (alpha, beta, and gamma) bind 9-cis-RA with dissociation constants (Kd) of 15.7, 18.3, and 14.1 nM, respectively. In contrast to the selectivity of RXRs for 9-cis-RA, RARs bind both t-RA and 9-cis-RA with high affinity, exhibiting Kd values in the 0.2-0.7 nM range for both ligands. Unlike RARs, the cellular RA binding proteins CRABPI or CRABPII bind t-RA but do not bind 9-cis-RA. Consistent with the binding data, 9-cis-RA and 9-cis-ddRA transcriptionally activate both GAL4-RXR and GAL4-RAR chimeric receptors with EC50 values of 3-20 nM for 9-cis-RA and 9-cis-ddRA, whereas t-RA and t-ddRA efficiently activate only GAL4-RAR chimeric receptors. Thus, 9-cis forms of endogenous retinoids can contribute to the pleiotropic effects of retinoids by interacting with both the RARs and RXRs.

9-cis retinoic acid stereoisomer binds and activates the nuclear receptor RXR alpha.

Vitamin A (retinol) and its natural derivatives are required for many physiological processes. The activity of retinoids is thought to be mediated by interactions with two subfamilies of nuclear retinoic acid receptors, RAR and RXR. The RARs bind all-trans retinoic acid (t-RA) with high affinity and alter gene expression as a consequence of this direct ligand interaction. RXR alpha is activated by t-RA, yet has little binding affinity for this ligand. t-RA may be converted to a more proximate ligand that directly binds and activates RXR alpha, and we have developed a method of nuclear receptor-dependent ligand trapping to test this hypothesis. Here we report the identification of a stereoisomer of retinoic acid, 9-cis retinoic acid, which directly binds and activates RXR alpha. These results suggest a new role for isomerization in the physiology of natural retinoids.

Anti-inflammatory and immuno-modulatory effects of novel retinoid-like 2,4,6,8-nonatetraenoic acids (NTA) in adjuvant-induced arthritis.

Prophylactic treatment (p.o.) of rats with adjuvant-induced arthritis (AA) with two retinoid-like 2,4,6,8-nonatetraenoic acids (NTA), Ro 23-6457 and Ro 23-2895, significantly reduced hind paw swelling between days 10-23 and the level of plasma fibrinogen (MED approximately 25 mumoles/kg). When given therapeutically (75 mumoles/kg between day 21 and 28) either NTA arrested the progression of the disease (MED, 25-75 mumoles/kg). Unseparated and adherent cell (AC) depleted spleen cells from rats with AA (day 12-15) responded poorly to the T cell mitogen, Con A (2.5 micrograms/ml) and the B cell mitogen, LPS (10 micrograms/ml). The responses were partially restored (approximately 30% of normal responses) in AC-depleted (but not unseparated) spleen cells from Ro 23-6457 treated rats (75 and 250 mumoles/kg/day). These data demonstrate an immunomodulatory effect of Ro 23-6457 in the adjuvant rat which may contribute to its anti-inflammatory activity in AA.

Fluorinated retinoic acids and their analogues. 3. Synthesis and biological activity of aromatic 6-fluoro analogues.

Several analogues (15a--e) of methyl (E,E,Z,E)-3,7-dimethyl-6-fluoro-9-(4-methoxy-2,3,6-trimethylphenyl)nonatetraenoate (15f), which had been found to cause a marked regression of chemically induced skin papillomas in mice, were prepared. Two synthetically versatile methods leading to these derivatives are described. The key intermediate, ethyl (Z)-2-fluoro-3-methyl-4,4-dimethoxy-2-butenoate (8), was elaborated to the C10 aldehyde ester, methyl (2E,4E,6Z)-3-methyl-6-fluoro-7-formyl-2,4,6-octatrienoate (14a), which upon Wittig condensation with the aryl-phosphonium salts 13a--e gave the (2E,4E,6Z,8E)-3,7-dimethyl-6-fluoro-9-aryl-2,4,6,8-nonatetraenoates 15a--e. Alternatively, Wittig reaction of 8 and [(4-methoxy-2,3,6-trimethylphenyl)methyl]triphenylphosphonium chloride (13f) gave a mixture of (E/Z,E)-2-fluoro-3-methyl-5-(2,3,6-trimethyl-4-methoxyphenyl)-2,4-pentadienoates 17 and 18, which was converted to 15f. The biological activity of these analogues and the 1H and 19F NMR spectral properties of the intermediates and final products are discussed.