BACKGROUND:Mouse strain differences in teratologic response are well documented. However, because retinoids cause similar malformation syndromes across many species, the strain differences may be predicted to be minimal. The goals of this study were to characterize and explain the differences between the C57BL/6N and SWV mouse strains in terms of all-trans-retinoic acid (RA)-induced teratologic effects at the time of gestation that cause postaxial forelimb ectrodactyly.METHODS:Visceral and skeletal malformations were determined by Wilson's sectioning and double-staining techniques, respectively; developmental staging was performed according to the somite count; and retinoid concentrations were assessed by HPLC.RESULTS:C57BL/6N mice were more susceptible than SWV mice to induction of embryolethality, cardiovascular defects, and forelimb ectrodactyly, whereas the opposite was true for the induction of ear, thymus, and tail agenesis, and cleft palate, gastroschisis, and anal atresia. As determined by somite counts, 1 strain intercross was developmentally advanced compared to the parental strains and the reciprocal cross. Retinoid susceptibility was equivalent between the reciprocal crosses for some malformations and determined by the maternal genotype for others. Toxicokinetic experiments showed that whole-embryo peak retinoid concentrations did not differ between the strains, but the area under the curve (AUC) for all-trans-RA was 1.3 times higher in C57BL/6N than in SWV embryos.CONCLUSIONS:The malformation spectrum induced by RA was strain-specific, and the strain sensitivity for forelimb ectrodactyly was consistent with all previously tested teratogenic agents (i.e., C57BL/6N was more sensitive than SWV). The strain differences in teratologic effects were not explained by developmental timing differences or toxicokinetic differences at the whole-embryo level.

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.

Compound 1 obtained by random screening and displaying a micromolar activity on the mu opiate receptor was chosen as a starting point for optimization. Two complementary concepts of similarity were used for the design of analogues and compared. These are based, respectively, on a computer-aided comparison of pharmacophoric patterns and on topological similarity. The structure-activity relationships are discussed in light of both similarity concepts. Compound 40, an N-methyl-3-(4-oxo-1-phenyl-1,3,8-triazaspiro[4.5]decyl)acetamide derivative, designed by combining the structure-activity relationships enlightened by each method, has a subnanomolar affinity for mu (h) receptor (IC(50) = 0.9 nM). It is a promising lead, allowing the design of a new series of analogues substituted at the N-3 of the spirocycle moiety.

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.

BACKGROUND Vitamin A and its metabolites have been shown to be teratogenic in animals and humans producing defects of neural crest derived structures that include abnormalities of the craniofacial skeleton, heart, and thymus. Our prior studies with retinoic acid have established that gestational day (gd) 9 is a sensitive embryonic age in the mouse for inducing craniofacial and thymic defects. METHODS We exposed pregnant mice to variable doses of vitamin A (retinyl acetate) on gd 9 and embryos were evaluated for changes in developing pharyngeal arch and pouch morphology, neural crest cell migration and marker gene expression. Additionally, we investigated whether a single organ system was more sensitive to low doses of vitamin A and could potentially be used as an indicator of vitamin A exposure during early gestation. RESULTS High (100 mg/kg) and moderate (50 and 25 mg/kg) doses of vitamin A resulted in significant craniofacial, cardiac outflow tract and thymic abnormalities. Low doses of vitamin A (10 mg/kg) produced craniofacial and thymic abnormalities that were mild and of low penetrance. Exposed embryos showed morphologic changes in the 2nd and 3rd pharyngeal arches and pouches, changes in neural crest migration, abnormalities in cranial ganglia, and altered expression of Hoxa3. CONCLUSIONS These animal studies, along with recent epidemiologic reports on human teratogenicity with vitamin A, raise concerns about the potential for induction of defects (perhaps subtle) in offspring of women ingesting even moderate to low amounts of supplemental vitamin A during the early gestational period.

Retinyl palmitate (RP) is a known laboratory animal teratogen inducing abnormalities of the second visceral arch when administered on day 9 of gestation in the rat. However, there are significant problems when attempting to extrapolate this result to the human. A combined in vivo/in vitro model was developed to assist in human risk assessment. The in vitro teratogenic threshold concentration of a number of retinyl palmitate metabolites was established. Serum concentrations of retinyl palmitate metabolites following a single teratogenic dose of RP in the pregnant rat were also measured. These dosed sera were also used to culture rat embryos. Our hypothesis was that malformations would only be induced by the dosed sera in vitro if the threshold concentration(s) of one or more metabolites was exceeded. Using this approach, it was determined that the teratogenicity of the sera were best predicted by serum retinol levels, with some indication that all-trans-retinoic acid and 4-oxo-all-trans-retinoic acid could be involved in some cases. The available human data suggest that threshold concentrations of these retinoids were unlikely to be exceeded following vitamin A supplements of 25,000 IU/day. While the proposed model does not take into account species differences, protein binding, and transfer to the embryo, it does have potential for human risk assessment.

All-trans-retinoic acid (all-trans-RA) is required in normal embryogenesis and both deficiency and excess are teratogenic. Isotretinoin (13-cis-RA) is teratogenic in all species examined; based on administered dose, humans appear most sensitive, followed by (in order or decreasing sensitivity) monkey, rabbit, hamster, mouse, and rat. Identification of the teratogenic threshold in these species is difficult because RAs are normal physiologic constituents. The rabbit no-observed-adverse-effect-level (NOAEL) and lowest-observed-adverse-effect-level (LOAEL) administered doses (3 and 15 mg/kg/day, respectively, on gestation Days 8-11) are less than the corresponding values in hamster (7.5 and 37.5 mg/kg/day, respectively, on gestation Days 7 and 8), but drawing conclusions from administered dose alone ignores differences in absorbed, metabolized, and embryonic delivered dose. Therefore, distribution and metabolism studies of 13-cis-RA at the NOAEL and LOAEL in pregnant hamsters were performed and plasma and tissue concentrations of parent compound and metabolites were compared to those found in rabbits. Metabolites of 13-cis-RA common to all species include three RAs (all-trans-RA, all-trans-4-oxoRA, 13-cis-4-oxoRA) and the glucuronide conjugate of 13-cis-RA (13-cis-RAG). As in rabbits, we found 13-cis-4-oxoRA also to be the major metabolite of 13-cis-RA in hamster plasma, peripheral tissues, and embryo. Of maternal tissues, peak 13-cis-RA concentrations were highest in liver. Total concentration of RA (13-cis-RA + 13-cis-4-oxoRA + all-trans-RA + all-trans-4-oxoRA) per gram of wet tissue was greatest in maternal liver, followed by that in lung, adipose tissue, muscle, kidney, and brain. At the NOAEL, total RA plasma Cmax in hamster was 6 times that in rabbit; at the LOAEL, hamster plasma total RA Cmax was 4 times that in rabbit. Hamster absorbed and metabolized dose (as AUC of plasma total RA) at the NOAEL and LOAEL was 2.6 and 2.4 times that in rabbit, respectively. In the embryo, hamster total RA Cmax was 2.7 times (at NOAEL) and 2.6 times (at LOAEL) that in rabbit. However, embryonic delivered dose (total RA AUC in hamster and rabbit embryo, respectively) at the NOAEL (2.08 and 2.14 microg . hr.g-1) and LOAEL (5.34 and 5.54 microg . hr . g-1) was virtually identical. Embryonic AUCs in hamster and rabbit for all-trans-RA and all-trans-4-oxoRA, metabolites which transactivate directly the nuclear RA receptors (RARs), were also very similar at the NOAEL (0.66 and 0.81 microg . hr g-1) and at the LOAEL (1.14 and 1.32 microg . hr g-1). Based on embryonic delivered dose, we suggest that 13-cis-RA is an equipotent teratogen in hamster and rabbit.

To investigate the relationship between sulfon arotinoid biotransformation and teratogenic activity, the potency of the ethyl (Ro 15-1570) and methyl (Ro 14-9706) arotinoid sulfones and their in vivo disposition in pregnant hamsters were studied. Administration of Ro 15-1570 was teratogenic, but Ro 14-9706 showed no such activity. Total absorbed doses of the ethyl and methyl sulfones (measured as maternal plasma AUC) were very similar. Total delivered dose of Ro 14-9706 to liver and lung was 120-160% that of Ro 15-157, and Ro 14-9706 was transferred in greater amounts to the embryo as well. Placenta AUC for parent sulfon arotinoids was 160-250% that in the embryo. Plasma analyses by HPLC suggested that the ethyl sulfone was oxidized and appeared in maternal plasma as the corresponding sulfinic (Ro 14-9572) and sulfonic (Ro 14-3899) acids, amounting to 10% and 16%, respectively, of the mean maternal ethyl sulfone Cmax value. The concentrations of sulfinic and sulfonic metabolites were always less than the analytical limit of detection in placenta and embryo after maternal ethyl sulfone intubation. Neither the sulfinic nor the sulfonic acid were ever detected in maternal circulation, placenta, or embryo after methyl sulfone intubation. Comparisons of their binding affinities found that neither the ethyl nor the methyl arotinoid sulfone could act as a ligand for cellular retinoic acid-binding protein (CRABP), nor could these compounds bind retinoid nuclear receptors (RAR). Transcriptional activation of RARs was weak and similar for both compounds. The sulfinic and sulfonic acid arotinoids bind and transactivate RARs, and bind CRABP with efficiencies similar to all-trans-retinoic acid. Furthermore, they are active in cultured limb bud chondrocytes. The results suggest that the methyl sulfone (in accord with its lack of activity in cultured limb bud chondrocytes) is of no toxicologic significance in hamster embryo--even after relatively high delivered dose. Teratogenicity of the ethyl sulfone (which shows marked inhibition of chondrogenesis in cultured limb bud) does not appear to depend on measurable concentrations of these sulfinic/sulfonic acid metabolites in the hamster embryo.

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 inhition 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. (C) 1995 Wiley-Liss, Inc.

The intravenous pharmacokinetics of 13-cis-, all-trans-, 13-cis-4-oxo, and all-trans-4-oxo retinoic acid (RA) were determined in nonpregnant female cynomolgus monkeys. All-trans- and 13-cis-RA were injected at two doses (0.25 or 0.0125 mg/kg) and all-trans-4-oxo RA and 13-cis-4-oxo RA at 0.25 mg/kg. Total body clearance, volume of distribution, and volume of distribution at steady state of all-trans-RA were dose-dependent with greater values at the lower dose. Elimination half-life was longer for the cis-compounds and not dose-dependent (N = 1 for 13-cis-4-oxo RA, N = 3 for other compounds, harmonic mean +/- pseudostandard deviation, min): 13-cis-4-oxo RA (837) > or = 13-cis-RA (301 +/- 204) > all-trans-RA (38 +/- 3) > all-trans-4-oxo RA (11 +/- 2). Secondary plasma peaks were noted only after administration of 13-cis-4-oxo RA. The low area under the time concentration curves for observable metabolites after intravenous injection of the oxidated compounds suggests further metabolism plays a minimal role in the elimination of these compounds from the monkey. Plasma-time concentration curves were fitted to multicompartmental models and suggested < 30% of each compound was available in the central compartment for elimination in the postdistribution phase. A comparison of the kinetics of the isomers indicated oxidation of all-trans-RA to all-trans-4-oxo RA increased mean total body clearance values 4-fold.(ABSTRACT TRUNCATED AT 250 WORDS)

Background: Vitamin A ingestion raises plasma levels of several potentially teratogenic retinoic acids (RAs) with all-trans and 13-cis configuration, but definitive data concerning the consequences of topical administration of all-trans-RA are lacking.Objective: The study objective was to investigate the potential for inducing systemic activity after topical administration of 0.025% all-trans-RA by measuring plasma retinoid levels.Methods: Plasma levels of all-trans-RA, 13-cis-RA, and 4-oxo-13-cis-RA were measured in four healthy subjects before, during, and after 14 daily topical applications of all-trans-RA.Results: Topical administration of all-trans-RA did not significantly increase plasma levels of all-trans-RA, 13-cis-RA, and 4-oxo-13-cis-RA. Significant decreases in levels of these RAs observed during the night may reflect diurnal variations of retinoid metabolism or lowered absorption of dietary vitamin A.Conclusion: Diurnal and nutritional factors influence plasma levels of endogenous retinoids to a greater extent than topical administration of all-trans-RA at doses used for acne therapy, which on the basis of these results appears unlikely to induce systemic effects.

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

A solid-phase extraction technique followed by automated high-performance liquid chromatography sample elution was successfully used to evaluate the effect of three pharmaceutical parameters on the plasma profile of various forms of vitamin A after an oral dose to cynomolgus monkeys. The three parameters evaluated were the chemical form of vitamin A (retinol versus retinyl acetate), the vehicle (acetone/Tween 20/water versus acetone/soybean oil), and the retinol dose (2, 10, and 50 × 103 retinol equivalents/kg). The form of the administered compound, retinol or retinyl acetate, appeared to have no major effect on the formation of nonpolar retinoids. The ester profile in plasma differed depending on whether the dose was administered in the water-based or the oil-based vehicle. Irrespective of the vehicle type the predominant retinoid formed was retinyl palmitate/ oleate. However, retinol doses in the water-based vehicle formed relatively high concentrations of retinyl laurate and retinyl my-ristate but no retinyl linolenate. The retinol dose in the oil-based vehicle formed consistent, but relatively minor, concentrations of retinyl linolenate, higher relative concentrations of retinyl linoleate, and no retinyl laurate or myristate. The dose of retinol administered had an impact on the diversity of the nonpolar retinoid profile. The low dose led to the presence of almost exclusively retinyl palmitate/oleate and retinyl stearate, whereas at higher doses the other retinyl esters became major retinol constituents. These findings are consistent with the hypothesis that, at low doses, the principle esterification pathway is via lecithin:retinol acyltransferase (LRAT) which produces retinyl palmitate, but at higher doses acyl-CoA:retinol acyltransferase is a prominent esterification enzyme, due to the saturation of the LRAT pathway, producing a more diverse profile of retinyl esters.

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.

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.

Using a sensitive HPLC method, plasma concentrations of vitamin A and metabolites were measured from 6 female volunteers who had taken once daily 0,46 mg/kg BW retinolpalmitat (Vitadral(R)) for 10 days. The metabolites all-trans- and 13-cis-retinoic acid were increased significantly (2- and 5-fold, resp.) 6 h after the ist intake. 13-cis-4-oxo retinoic acid the 10th intake in the morning of the 10th day (9,22 +/- 2.77 ng/ml, 4-fold increase). The results show continous increase of retinoic acids, which have to be considered as potential teratogens, after administration of vitamin A. The plasma concentration of retinol itself did not change, whereas only short-term increases were observed for retinol esters.

It has been reported that fractionated doses of 13-cis-retinoic acid are disproportionately more embryotoxic in pregnant mice than is the same dose given in a single bolus. Here, we examined limited pharmacokinetic profiles of a single (100 mg/kg dose given to NMRI mice on day 11 of gestation) versus multiple (3 x 100 mg/kg, 4 h apart) doses in an effort to assess the relative contribution to teratogenicity made by the drug and/or its metabolites. The major plasma metabolite of 13-cis-retinoic acid in the mouse was 13-cis-retinoyl-beta-glucuronide, followed by the 4-oxo metabolites and all-trans-retinoic acid. Transfer to the mouse embryo was very efficient for all-trans-retinoic acid, whereas, it was tenfold less efficient for 13-cis-retinoic acid and 100-fold less efficient for 13-cis-retinoyl-beta-glucuronide. The isomer all-trans-retinoic acid was found in the placenta at concentrations two- to three-fold higher than in the plasma, suggesting placental accumulation as well as placental cis/trans isomerization. Since 13-cis-retinoyl-beta-glucuronide and 13-cis- and all-trans-retinoic acid were detected in the embryo after this multiple dosing schedule, any of the three or their combinations may have been involved in the induction of malformations, but all-trans-retinoic acid, a well-known potent teratogen detected at concentrations of between 590 and 80 ng/g for 10 critical hours during gestation, could have been the major component.