Oral administration of retinol (50 mg/kg) to NMRI mice on day 11 of gestation (vaginal plug = day 0) led to the metabolic formation of high quantities of all-trans retinoic acid and all-trans-4-oxoretinoic acid, both known as potent teratogenic agents in the mouse. A 96% reduction of the area under the concentration-versus-time-curve (AUC) of metabolically generated all-trans retinoic acid in maternal plasma, and an 84% decrease in the embryonic AUC were observed when mice had been pretreated with the alcohol dehydrogenase inhibitor 4-methylpyrazole. A similar reduction was observed for the major metabolite of all-trans retinoic acid in the mouse, all-trans-4-oxoretinoic acid. However, 4-methylpyrazole pretreatment decreased the AUC of retinol by 10% in maternal plasma and 15% in embryo. Treatment with retinol alone resulted in 55.6%, 43.9% and 56.0% skeletal anomalies of the forelimbs, hindlimbs and craniofacial structures, respectively. Pretreatment with 4-methylpyrazole lowered the retinol induced skeletal defects to 31.3%, 24.0% and 31.3%, respectively, in the forelimb, hindlimb and craniofacial region. Typical retinoid-induced malformations for gestational day 11, e.g. bent or reduced zeugopod or stylopod elements, or cleft palate, were significantly reduced by 4-methylpyrazole pretreatment but were still detected in significantly higher prevalence than in control mice. These data suggest that the teratogenic activity of a single high dose of vitamin A in mouse is partially but not exclusively dependent on the metabolic activation of retinol to all-trans retinoic acid. Thus it could be hypothesized that retinol is either a proximate teratogen or a coteratogen with all-trans retinoic acid.
Retinoic acids (RA) are active metabolites of vitamin A which affect the expression of many genes involved in embryonic development, cell differentiation, and homeostasis. One important target gene for RA is matrix metalloproteinase (MMP-1, collagenase), the only enzyme active at neutral pH that can degrade interstitial collagen, a major component of extracellular matrix. Using a cell line of normal rabbit synovial fibroblasts, HIG82 cells, as a model, we report that both all-trans- and 9-cis-RA inhibit collagenase synthesis. This inhibition occurs at a transcriptional level and is ligand-dependent. Constitutive levels of retinoic acid receptor (RAR) mRNA levels are low, but are increased by all-trans and by 9-cis RA. In contrast, constitutive levels of retinoid X receptor (RXR) mRNA are higher and are not affected by RA. To measure DNA/protein interactions, we used a gel mobility shift assay with oligonucleotides containing either an AP-1 site or a 40 bp region between -182/-141, nuclear extracts from RT-treated cells, and antibodies to RARs and RXRs. We found that both RARs and RXRs interact with these regions of the collagenase promoter, perhaps as part of a complex with other proteins. Our results suggest that heterodimers between RARs and RXRs mediate suppression of the collagenase gene by RA, and that RAR is a limiting factor in this negative regulation.
Plasma concentrations of retinyl esters, retinol, retinol-binding protein and the polar retinol metabolites all-trans-retinoic acid, 13-cis-retinoic acid, all-trans-4-oxoretinoic acid and 13-cis-4-oxoretinoic acid were measured for six male volunteers who received 0.46 mg retinyl palmitate per kilogram body weight as oily drops (equivalent to 0.25 mg retinol per kilogram body weight) once daily over a 20-d period. Retinol and retinol-binding protein levels remained virtually constant throughout the study. Following absorption of vitamin A, retinyl esters as well as all-trans-retinoic acid and 13-cis-retinoic acid were transiently increased in plasma. 13-cis-4-Oxoretinoic acid increased gradually to a steady state level present on d 10 or 20. All-trans-4-oxoretinoic acid was not detected in plasma of the volunteers, with the exception of one on d 10 of the study. Plasma pharmacokinetic profiles of retinyl esters and polar metabolites of retinol displayed great interindividual differences (peak concentrations, time to peak, area-under-the-concentration-time curve values) among the volunteers. Because of the relatively high and consistent steady state concentrations of plasma 13-cis-4-oxoretinoic acid, we suggest that this compound be further investigated as a biochemical marker of vitamin A uptake in humans.
Two compounds, obtained by random screening, and displaying micromolar activities on the mu opiate receptor were used as starting points for optimization. In that work, the traditional concept of the activity of a compound (related to one or a few targets) was extended to the comprehensive pharmacological profile of that compound on more than 70 receptors, transporters, and channels relevant to a CNS-oriented project. Using the two complementary design strategies based on two similarity concepts described in the previous paper, we have obtained analogues with IC(50) values ranging between 0.9 nM and a few micromolar on the mu receptor and displaying qualitatively different profiles. We discuss here, both on a case-by-case basis and from a statistical standpoint, the pharmacological profiles in light of the two similarity concepts.
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