Uptake of the aromatic retinoids Ro 10-9359 (etretinate) and Ro 10-1670 (acitretin), its main metabolite, delivered to cultured human fibroblasts by serum proteins. Lack of evidence for perturbation of LDL catabolism

Etretinate or acitretin are efficiently delivered to cultured human fibroblasts in the presence of low density lipoproteins, high density lipoproteins or human serum albumin. In contrast to acitretin, delivery of etretinate to fibroblasts is more efficiently achieved with human serum albumin than with lipoproteins. The uptake of etretinate and acitretin via low density lipoproteins delivery, does not take place via the low density lipoprotein-receptor endocytotic pathway but mostly through a passive exchange with the plasma membrane. However, in contrast to acitretin, the exchange of etretinate seems to occur alter binding of etretinate-loaded low density lipoproteins to the apolipoprotein B receptors. No differences are observed in binding, internalization and degradation of native, etretinate-loaded low density lipoproteins and acitretin-loaded low density lipoproteins, suggesting that the presence of these retinoids in low density lipoproteins does not alter their processing by the cells. Furthermore, the presence of these retinoids in the cells does not notably affect, under our experimental conditions, the catabolism of native low density lipoproteins.     

Retinoid-induced inhibition of eosinophil LTC4 production

Naturally occurring and synthetic retinoids demonstrate a marked antiinflammatory effect when employed in such disorders as acne and psoriasis. This effect may result in part from their inhibition of release of potent mediators (e.g. eicosanoids) by inflammatory cells. In this study, we examined the effect of eight retinoids (tretinoin, isotretinoin, retinol, retinal, acitretin, retinyl palmitate, etretinate, Ro 15-0778) on the release of leukotriene (LT)C4, an important lipid mediator generated by eosinophils. Tretinoin, isotretinoin, retinol, retinal, and acitretin at 10(-5) M or 10(-4) M concentrations inhibited LTC4 release by A23187-stimulated horse eosinophils in vitro; 10(-4) M retinyl palmitate was also inhibitory. However, 10(-5) M etretinate augmented A23187-induced LTC4 release, and the arotinoid Ro 15-0778 had no effect on LTC4 production. These data suggest that selected retinoids may have potential use in the reduction of LTC4 generation by eosinophils. This inhibition could be beneficial in the therapy of such diseases as bronchial asthma in which release of LTC4 may be involved in the inflammatory process.     

Behavioral effects and benzodiazepine antagonist activity of Ro 15-1788 (flumazepil) in pigeons

The selective benzodiazepine receptor antagonist, Ro 15-1788, produced behavioral effects in pigeons at doses at least 100 times lower than those previously reported to possess intrinsic pharmacological activity in mammals. In contrast to its effects in mammalian species, in pigeons, Ro 15-1788 does not exhibit partial agonist activity. Key-peck responses of pigeons were studied under a multiple fixed-interval 3-min, fixed-interval 3-min schedule in which the first response after 3-min produced food in the presence of red or white keylights. In addition, every 30th response during the red keylight produced a brief electric shock (punishment). Under control conditions, punished responding was suppressed to 30% of unpunished response levels. Ro 15-1788 (0.01 mg/kg, i.m.) increased unpunished response rates by 33% without affecting rates of punished responding. Doses of 0.1 to 1.0 mg/kg Ro 15-1788 produced dose-related decreases in both punished and unpunished responding. As is characteristic of other benzodiazepines, midazolam (0.1 and 0.3 mg/kg, i.m.) markedly increased punished responding but had little effect on rates of unpunished responding. Ro 15-1788 antagonized the increases in punished responding and also reversed the rate-decreasing effects of higher doses of midazolam. However, the effectiveness of Ro 15-1788 as a benzodiazepine antagonist was limited by its intrinsic activity: rate-decreasing doses of Ro 15-1788 were unable to completely reverse behavioral effects of midazolam. Midazolam was an effective antagonist of the behavioral effects of Ro 15-1788 (up to 0.1 mg/kg) but midazolam did not influence the rate-decreasing effects of 1.0 mg/kg Ro 15-1788 across a 100-fold dose range. In the pigeon, the behavioral effects of relatively low doses of Ro 15-1788 (0.01-0.1 mg/kg) appear to be related to benzodiazepine receptor mechanisms, whereas other systems appear to be involved in the effects of higher doses.     

Lipoproteins secreted by cultured rat hepatocytes contain the antioxidant 1-alk-1-enyl-2-acylglycerophosphoethanolamine

Monolayer cultures of rat hepatocytes have been examined for their ability to secrete ethanolamine plasmalogen as a component of nascent lipoproteins. In culture medium from these cells, ethanolamine plasmalogen comprises approx. 20-30% of total ethanolamine glycerophospholipids when measured either as phospholipid mass or by the incorporation of [1-3H]ethanolamine. An approximately equal distribution of the plasmalogen was found throughout all lipoprotein density fractions. The content of plasmalogen in whole rat serum, was 36% of total ethanolamine glycerophospholipids. In contrast, in rat liver and cultured hepatocytes the amount of ethanolamine plasmalogen was 5-fold lower than in serum or culture medium (approx. 5% of total ethanolamine phospholipids). Normal human plasma also contains ethanolamine plasmalogen in relatively large amounts (approx. 50% of total ethanolamine phospholipids). Thus, a major function of plasmalogen biosynthetic enzymes in liver may be the provision of ethanolamine plasmalogen for secretion into lipoproteins. Previous studies (e.g., Zoeller, R.A. et al. (1988) J. Biol. Chem. 263, 11590-11596) have suggested that ethanolamine plasmalogen may function as an antioxidant for the protection of lipid and protein membrane components against oxidation. Oxidized, but not native, low-density lipoprotein is rapidly taken up by macrophages with the formation of foam cells characteristic of atherosclerotic lesions (Steinbrecher, U.P. et al. (1984) Proc. Natl. Acad. Sci. USA 81, 3883-3887). Thus, the presence of plasmalogen as part of newly secreted lipoprotein particles may prevent their oxidation and subsequent uptake by macrophages.     

Ro 15-1788 antagonizes the discriminative stimulus effects of diazepam in rats but not similar effects of pentobarbital

The benzodiazepine antagonist properties of Ro 15-1788 were evaluated in rats trained to discriminate between saline and either 1.0 mg/kg of diazepam or 10 mg/kg of pentobarbital in a two-choice discrete-trial shock avoidance procedure. When administered alone, 1.0 mg/kg of diazepam and 10 mg/kg of pentobarbital produced comparable amounts of drug-appropriate responding (> 84%), whether rats were trained to discriminate between diazepam or pentobarbital and saline. Ro 15-1788 (3–32 mg/kg, p.o.), administered 10 min before diazepam or pentobarbital, produced a dose-related blockade of the discriminative effects of diazepam in both groups of rats, but was completely ineffective in blocking the discriminative effects of pentobarbital. The dose-effect curve for the discriminative effects of diazepam was shifted to the right in a parallel fashion 3- and 13-fold by 10 and 32 mg/kg of Ro 15-1788, respectively, indicating that Ro 15-1788 acts as a surmountable, competitive antagonist of diazepam. When administered alone, Ro 15-1788 (32–100 mg/kg, p.o.) produced primarily saline-appropriate responding, although 100 mg/kg of Ro 15-1788 produced drug-appropriate responding in one out of eight rats. When administered orally 30 min after diazepam, Ro 15-1788 (32 mg/kg) completely reversed within 10 min the discriminative effects of diazepam. The blockade of diazepam's discriminative effects by 32 mg/kg of Ro 15-1788 appeared to last at least as long (approximately 2 hr) as the effects of diazepam alone.     

Antiobesity effects of a novel lipid synthesis inhibitor (Ro 22-0654)

The present studies were conducted to determine the antiobesity effects of a novel inhibitor of hepatic fatty acid synthesis, 4-amino-5-ethyl-3-thiophene-carboxylic acid methyl ester hydrochloride (Ro 22-0654/001). Weight gain was decreased by treatment with Ro 22-0654 in Sprague Dawley and in lean or obese Zucker rats. Food intake was only transiently suppressed during the first few days of each study. The decreased weight gain in treated rats could be accounted for almost entirely by a decrease in total body lipid levels. Hepatic fatty acid synthesis was inhibited following two months of treatment, in rats fed either a 1% or 10% corn oil diet. It is suggested that the inhibition of hepatic fatty acid synthesis may account, in part, for the reduced weight gain.     

Calcification inhibitors in rat and human serum and plasma

Rat and human serum and plasma were shown to contain considerable amounts of calcium phosphate precipitation inhibitors. Two general classes of inhibiting molecules were observed for both species: high molecular weight (approx. 30 000-200 000) and low molecular weight (less than 1000). The high molecular weight components eluted from a Bio-Gel P-200 column in two peaks, one at approx. 158 000 and a broader peak at approx. 43 000. The identity of these inhibitors is unknown at present. Low molecular weight inhibitors include magnesium, pyrophosphate, and citrate ions and at least one unidentified component that coelutes with pyrophosphate and citrate on a Bio-Gel P-4 column. Quantitatively, most of the inhibitor activity resides in the high molecular weight components and it is possible that it is this activity which is responsible for maintaining the metastability of the circulating fluids. The role of the low molecular weight components may be to regulate calcification at sites inaccessible to high molecular weight molecules.     

Assessing the function of the Ro ribonucleoprotein complex using Caenorhabditis elegans as a biological tool.

The Ro ribonucleoprotein complex (Ro RNP) was initially described as an autoimmune target in human diseases such as systemic lupus erythematosus and Sj?gren's syndrome. In Xenopus and human cells, its general structure is composed of one major protein of 60 kDa, Ro60, that binds to one of four small RNA molecules, designated Y RNAs. Although no function has been assigned to the Ro RNP, Ro60 has been shown to bind mutant 5S ribosomal RNA (rRNA) molecules in Xenopus oocytes, suggesting a role for Ro60 in 5S rRNA biogenesis. Ro60 has also been shown to participate in the regulation of the translational fate of the L4 ribosomal protein mRNA by interacting with the 5' untranslated region, again suggesting its possible implication in ribosome biogenesis. To identify the function of Ro RNP, we have taken a genetic approach in the nematode Caenorhabditis elegans. As such, we characterized the gene encoding the protein ROP-1, the homologue of the human Ro60 protein. Here, we review the phenotypic analysis of C. elegans rop-l(-) mutants and integrate these results into a model for the function of the Ro RNP particle.     

外周苯二氮(艹卓)受体激动剂Ro5-4864抑制大鼠心肌细胞线粒体通透性转换

线粒体通透性转换（mitochondrial permeability transition,MPT）导致线粒体氧化应激性损伤。近年研究认为,位于线粒体外膜的外周苯二氮节受体（peripheral benzodiazepine receptor,PBR）参与了线粒体的重要生理功能。本研究在心肌细胞线粒体水平探讨激动PBR能否抑制Ca^2＋诱发的MPT。分离Sprague—Dawley大鼠心肌细胞线粒体,将PBR激动剂Ro5-4864（50、100、200μmol／L）和线粒体孵育,利用150μmol／L Ca^2＋诱发MPT,部分线粒体在与100μmol／L Ro5-4864孵育前5min加入MPT孔道开放剂苍术苷（atractyloside,ATR）。采用分光光度法观察线粒体膨胀情况：Westernblot检测线粒体细胞色素C（cytochrome C,CytoC）释放;利用荧光探针JC-1在激光共聚集显微镜下观察线粒体膜电位的变化。50、100、200μmol／L Ro5-4864均显著抑制Ca^2＋诱发的520nm处线粒体吸光度的下降,而且抑制Ca^2＋引起的线粒体CytoC释放和线粒体膜电位下降,但ATR可阻断R05—4864的上述作用。结果提示,PBR激动剂可抑制大鼠心肌MPT,保持线粒体CytoC含量和稳定线粒体膜电位,减轻线粒体损伤。PBR的激活可能成为减轻心肌细胞应激性损伤及心肌保护的新方法。     

In vivo binding of (3H)Ro 15-1788 in mice: comparison with the in vivo binding of (3H)flunitrazepam

Benzodiazepine binding sites have generally been labelled with benzodiazepine agonists: (3H)flunitrazepam and (3H)diazepam in vivo. We studied the in vivo binding of the antagonist (3H)Ro 15-1788 in mice and compared it to the in vivo binding of (3H)flunitrazepam. For this in vivo labelling, mice were injected with labelled and unlabelled ligands. Animals were then sacrificed and bound radioactivity was measured after homogenization of the excised brain and filtration of the homogenate. (3H)Ro 15-1788 is a better tool than (3H)flunitrazepam for in vivo labelling of benzodiazepine receptors since 1) it labels specifically the central type binding sites, 2) injection of 4 times less (3H)Ro 15-1788 (50 microCi/kg) than (3H)flunitrazepam (200 microCi/kg) produced the same amount of bound radioactivity, 3) 70-90% of the total (3H)Ro 15-1788 present in the brain is membrane bound instead of 45-55% with (3H)flunitrazepam, 4) maximal binding of (3H)Ro 15-1788 is reached within 3 min, 5) only 5% of the membrane bound (3H)Ro 15-1788 is nonspecific instead of 15% for (3H)flunitrazepam.     

Ethanol-induced limb defects in mice: effect of strain and Ro15-4513

It is now thought that ethanol exerts many of its behavioral effects in the CNS by interaction with the gamma-aminobutyric acid (GABA) receptor, and it has been shown that the benzodiazepine reverse agonist Ro15-4513 reverses some of the CNS effects produced by ethanol. The hypothesis was tested that ethanol exerts its teratogenic effects through interaction with a putative embryonic GABA receptor by determining whether Ro15-4513 reverses ethanol-induced forelimb ectrodactyly in C57BL/6 mice. First, pregnant C57BL/6 dams were injected twice i.p. with ethanol (2.9 g/kg body weight, 4 hr apart) on day 10 of gestation: 49% of the fetuses were resorbed or dead and 46% of the survivors showed forelimb ectrodactyly. In contrast, when SWV mice were treated with ethanol, embryolethality was only 11.9% and no forelimb ectrodactyly was observed. In a second experiment, when ethanol (2.6 g/kg x 2) was administered to C57BL/6 mice, 34% resorptions and 31% forelimb ectrodactyly were observed. Ectrodactyly induced by ethanol was primarily of the forelimb and exclusively postaxial. Ethanol produced an unusual forelimb defect in a small number of instances where there was a postaxial autopod reduction defect coupled with a preaxial zeugopod reduction defect. Ro15-4513 administered alone (50 mg/kg x 2) was neither embryolethal nor teratogenic in C57BL/6 mice. To attempt to reverse the teratogenic effect of ethanol, dams that were injected 5 min before each ethanol administration with Ro15-4513 (0.5, 1, 2.5, 5, 10 mg/kg twice) showed no significant change in frequency of forelimb ectrodactyly compared to embryos treated with ethanol alone. However, resorptions increased significantly to 77% and 62% with the 5 and 10 mg/kg doses of Ro15-4513. Thus there appears to be an embryolethal interaction of Ro15-4513 with ethanol. Nevertheless, since Ro15-4513 did not reverse the teratogenic effect induced by ethanol, these results do not support the hypothesis that the teratogenic mechanism of ethanol is mediated through a putative embryonic GABA receptor.     

Disposition and metabolism of Ro 24-4736 in the rat

Ro 24-4736, a new platelet activating factor antagonist, is currently under preclinical and clinical development. The tissue distribution of the ¹⁴C-label in male rats following a single intravenous dose of 1.0 mg/kg of ¹⁴C-Ro 24-4736 indicated appreciable uptake by the liver, kidney, heart and gastrointestinal tract. Peak plasma and tissue concentrations were seen at 5 minutes after dosing except for the small intestine (4 hrs) and abdominal fat, stomach and large intestine (4 hrs). Thereafter, the ¹⁴C-label rapidly declined in all tissues. At 48 hours, only 3.5% of the dose was present in the tissues, and 6.1% in the lumen of the gastrointestinal tracts. The excretion of ¹⁴C was essentially completed; 94% of the administered ¹⁴C was excreted in the feces and 4.0% in the urine. Overall recoveries of the administered ¹⁴C label ranged from 96 to 116%. The purified major ¹⁴C-labelled component in the fecal extracts yielded essentially the same NMR spectrum as authentic Ro 24-4736 which accounted for 11% of the dose. In vitro incubations of Ro 24-4736 with rat liver 9S supernatant in an NADPH generating system produced two metabolites. NMR spectra indicated that one metabolite was hydroxylated at carbon-1 while the other one contained a hydroxyl at carbon-10 of the parent molecule. Interestingly, the sites of hydroxylation were at carbons C₁, and C₁₀ bearing the protons guarding the bay area of the phenanthrenoid ring, rather than carbons of the phenyl-methyl-thienotriazolodiazepine moiety.     

Blood pressure response to Ro15-1788, a benzodiazepine antagonist

The effects of Ro15-1788, a benzodiazepine antagonist, on heart rate and blood pressure were studied in chloralose anesthetized cats. In previously untreated controls, Ro15-1788 lowered both systolic and diastolic arterial pressure about 15 mm Hg, and slightly decreased heart rate. In cats that had been given a single acute dose of diazepam or flurazepam, Ro15-1788 increased blood pressure about 40 mm Hg. A similar increase was measured in cats that were tolerant and physically dependent after 5 weeks of chronic flurazepam treatment. High spinal (C-1) section abolished all Ro15-1788 effects. It is suggested that the observed drug actions occur within the CNS rather than in the periphery, and that it might be useful to study further the cardiovascular actions of benzodiazepine agonists and antagonists.     

Retinoid-induced inhibition of bosinophil LTC4 production

Naturally occuring and synthetic retinoids demonstrate a marked antiinflammatory effect when employed in such disorders as acne and psoriastis. This effect may result in part from their inhibition of release of potent mediators (e.g. eicosanoids) by inflammatory cells. In this study, we examined the effect of eight retinoids (tretinoin, isotretinoin, retinol, retinal, acitretin, retinyl palmitate, etretinate, Ro 15–0778) on the release of leukotriene (LT)C₄, an important lipid mediator generated by eosinophils. Tretinoin, isotretinoin, retinol, retinal, and acitretin at 10⁻⁵ M or 10⁻⁴ M concentrations inhibited LTC₄ release by A23187-stimulated horse eonsinophils in vitro; 10⁻⁴ M retinyl palmitate was also inhibitory. However, 10⁻⁵ M etretinate augmented A23187-induced LTC₄ release, and the arotinoid Ro 15–0778 had no effect on LTC₄ production. These data suggest that selected retinoids may have potential use in the reduction of LTC₄ generation by eosinophils. This inhibition could be beneficial in the theraphy of such diseases as bronchial asthma in which release of LTC₄ may be involved in the inflammtory process.     

Acitretin embryopathy: a case report

BACKGROUND: Acitretin is an aromatic retinoid analog of vitamin A. Drugs of this group are well-known teratogenic agents. Nevertheless, acitretin embryopathy has been described only in fetuses. CASE: An infant was exposed to 10 mg/day of acitretin from the beginning of pregnancy until the 10th gestational week. At term, the newborn showed the following abnormalities: microcephaly, epicanthal folds, low nasal bridge, high palate, cup-shaped ears, anteverted nostrils, atrial septal defect, and bilateral sensorineural deafness. At 18 months of age, the patient showed microcephaly and neurodevelopmental delay. CONCLUSIONS: Our patient shows a pattern of anomalies resembling that observed in isotretinoin- and etretinate-exposed children. After ingestion, acitretin is partially converted into etretinate, and etretinate is partially metabolized into acitretin. A similar phenotype would therefore be expected after prenatal exposure to either drug. Moreover, in the present case, teratogenic effects were observed even though the dose was lower than in the previously reported acitretin embryopathy cases. Therefore, we propose that different retinoids, acitretin included, produce only one malformation pattern with variable phenotypic expression.     

The effect of the beta-adrenoreceptor antagonist Ro03-7894 on rat atrial tension development and (-)-[3H]dihydroalprenolol binding to cardiac and lung membranes

The ability of 1-(5-chloracetylaminobenzfuran-2-yl)-2-isopropylaminoethanol (Ro03-7894) to irreversibly inactivate beta-adrenoreceptors was studied. In isolated rat atria Ro03-7894 (500 microM) depressed and shifted the tension development curve for isoproterenol to the right. After a 2 hour washout period the dose response curve for isoproterenol was further depressed. At a lower dose of Ro03-7894 (50 microM), the isoproterenol dose response curve was also depressed and shifted to the right although after a 2 hour washout, the sensitivity to isoproterenol was restored but the maximum response was still depressed. Ro03-7894 (50 microM) also depressed the tension development response to increasing concentrations of external calcium. The concentration of Ro03-7894 that inhibited (-)-[3H]dihydroalprenolol (DHA) binding by 50% in cardiac and lung membranes was 20 microM. Incubation of rat ventricular or lung membranes for 1 hour with 100 microM Ro03-7894 followed by washing did not change the concentration of beta-adrenoreceptors or the KD values for [3H]DHA binding. Furthermore, neither the concentration of beta-adrenoreceptors nor the KD for [3H]DHA binding was changed in cardiac and lung membranes at 4 or 24 hours after an i.p. injection of 20 mg/kg of Ro03-7894. The results suggested that Ro03-7894 was a relatively weak beta-adrenoreceptor antagonist which under the conditions used did not irreversibly inactivate the receptor but probably depressed tension development in intact atria nonspecifically.     

Interaction of a cyclic peptide, Ro09-0198, with phosphatidylethanolamine in liposomal membranes

Ro09-0198 is a cyclic peptide isolated from Streptoverticillium griseoverticillatum. This peptide caused permeability increase and aggregation of liposomes containing phosphatidylethanolamine. Liposomes containing phosphatidylserine, phosphatidylinositol or cardiolipin instead of phosphatidylethanolamine were, however, not appreciably reactive with the peptide. Among the structural analogs of phosphatidylethanolamine, dialkylphosphatidylethanolamine and 1-acylglycerophosphoethanolamine incorporated into liposomes could interact with Ro09-0198 to cause a permeability increase, whereas liposomes consisting of alkylphosphoethanolamine or phosphatidyl-N-monomethylethanolamine were insensitive to the peptide. These findings indicate that a glycerol backbone and a primary amino group of phosphatidylethanolamine are necessary for interaction with Ro09-0198 to cause membrane damage. Ro09-0198 induced a selective permeability change on liposomes. Glucose and umbelliferyl phosphate were effluxed significantly, but sucrose was only slightly permeable and inulin could not be released. Consequently, the permeability increase induced by Ro09-0198 is rather specific to molecules smaller than sucrose. Line broadening of electron spin resonance signals of spin-labeled phosphatidylethanolamine was observed upon treatment of liposomes with Ro09-0198. It was suggested from these results that Ro09-0198 can alter the physical organization of phosphatidylethanolamine in membranes, thus providing a basis for changes in membrane permeability.     

The imidazobenzodiazepine Ro 15-4513 antagonizes methoxyflurane anesthesia

Parenteral administration of the imidazobenzodiazepine Ro 15-4513 (a high affinity ligand of the benzodiazepine receptor with partial inverse agonist qualities) produced a dose dependent reduction in sleep time of mice exposed to the inhalation anesthetic, methoxyflurane. The reductions in methoxyflurane sleep time ranged from approximately 20% at 4 mg/kg to approximately 38% at 32 mg/kg of Ro 15-4513. Co-administration of the benzodiazepine receptor antagonist Ro 15-1788 (16 mg/kg) or the inverse agonists DMCM (5-20 mg/kg) and FG 7142 (22.5 mg/kg) blocks this effect which suggests that the reductions in methoxyflurane sleep time produced by Ro 15-4513 are mediated via occupation of benzodiazepine receptors. Moreover, neither DMCM (5-20 mg/kg) nor FG 7142 (22.5 mg/kg) reduced methoxyflurane sleep time which suggests this effect of Ro 15-4513 cannot be attributed solely to its partial inverse agonist properties. These observations support recent findings that inhalation anesthetics may produce their depressant effects via perturbation of the benzodiazepine/GABA receptor chloride channel complex, and suggest that Ro 15-4513 may serve as a prototype of agents capable of antagonizing the depressant effects of inhalation anesthetics such as methoxyflurane.     

A newly discovered xenobiotic metabolic pathway: ethyl ester formation.

Formation of etretinate, ethyl ester of acitretin, can be confirmed in vitro and in vivo using acitretin as the substrate. Etretinate was identified by LC/MS. The in vitro incubation was performed using rat and human liver 12,000 g supernatant, and the in vivo experiment was conducted in rats after oral dosing of acitretin. The ethyl ester formation was greatly enhanced by addition of or dosing with ethanol.