Metabolomics as read-across tool: An example with 3-aminopropanol and 2-aminoethanol.

Read-across and grouping is one of the most commonly used alternative approaches for data gap filling in registrations submitted under the REACH Regulation as defined by the European Chemicals Agency (ECHA) in their 'Read-Across Assessment Framework' (RAAF, 2017). At the same time, the application of read-across is rejected by ECHA frequently due to various reasons. As a major reason hereof, applicants fail to reduce the level of 'remaining uncertainty' intrinsical to every read-across approach compared to testing a substance experimentally. Recently, the use of metabolomics to support read-across cases with biological information has been reported in a case study with phenoxy herbicides (Ravenzwaay et al., 2016). In the present case-study a 'weight-of-evidence' read-across approach from 2-aminoethanol (MEA = 'source') to 3-aminopropanol (3AP = 'target') with metabolomics as 'supporting evidence' reducing the remaining uncertainties is reported. We demonstrate the high structural similarity of the two analogous substances based on the available data and we report how metabolome data add confidence concerning mechanistic similarity in this read-across approach. Finally, the herein described read-across case supported by metabolomics is used to cover the data gaps in repeated dose and reproductive toxicity endpoint of 3AP via weight of evidence for the REACH-registration.

Safety Assessment of Hydroxypropyl Bis(N-Hydroxyethyl-p-Phenylenediamine) HCl as Used in Cosmetics.

The Cosmetic Ingredient Review Expert Panel (CIR Panel) reviewed the safety of hydroxypropyl bis(N-Hydroxyethyl-p-Phenylenediamine) HCl, which functions as an oxidative hair dye ingredient. The Panel considered relevant animal and human data provided in this safety assessment and concluded that hydroxypropyl bis(N-hydroxyethyl-p-phenylenediamine) HCl is safe for use in oxidative hair dye formulations as described in this safety assessment.

High-throughput screening of drug-binding dynamics to HERG improves early drug safety assessment.

The use of computational models to predict drug-induced changes in the action potential (AP) is a promising approach to reduce drug safety attrition but requires a better representation of more complex drug-target interactions to improve the quantitative prediction. The blockade of the human ether-a-go-go-related gene (HERG) channel is a major concern for QT prolongation and Torsade de Pointes risk. We aim to develop quantitative in-silico AP predictions based on a new electrophysiological protocol (suitable for high-throughput HERG screening) and mathematical modeling of ionic currents. Electrophysiological recordings using the IonWorks device were made from HERG channels stably expressed in Chinese hamster ovary cells. A new protocol that delineates inhibition over time was applied to assess dofetilide, cisapride, and almokalant effects. Dynamic effects displayed distinct profiles for these drugs compared with concentration-effects curves. Binding kinetics to specific states were identified using a new HERG Markov model. The model was then modified to represent the canine rapid delayed rectifier K(+) current at 37°C and carry out AP predictions. Predictions were compared with a simpler model based on conductance reduction and were found to be much closer to experimental data. Improved sensitivity to concentration and pacing frequency variables was obtained when including binding kinetics. Our new electrophysiological protocol is suitable for high-throughput screening and is able to distinguish drug-binding kinetics. The association of this protocol with our modeling approach indicates that quantitative predictions of AP modulation can be obtained, which is a significant improvement compared with traditional conductance reduction methods.

Effective inhibition of acid and neutral ceramidases by novel B-13 and LCL-464 analogues.

Induction of apoptosis mediated by the inhibition of ceramidases has been shown to enhance the efficacy of conventional chemotherapy in several cancer models. Among the inhibitors of ceramidases reported in the literature, B-13 is considered as a lead compound having good in vitro potency towards acid ceramidase. Furthermore, owing to the poor activity of B-13 on lysosoamal acid ceramidase in living cells, LCL-464 a modified derivative of B-13 containing a basic ω-amino group at the fatty acid was reported to have higher potency towards lysosomal acid ceramidase in living cells. In a search for more potent inhibitors of ceramidases, we have designed a series of compounds with structural modifications of B-13 and LCL-464. In this study, we show that the efficacy of B-13 in vitro as well as in intact cells can be enhanced by suitable modification of functional groups. Furthermore, a detailed SAR investigation on LCL-464 analogues revealed novel promising inhibitors of aCDase and nCDase. In cell culture studies using the breast cancer cell line MDA-MB-231, some of the newly developed compounds elevated endogenous ceramide levels and in parallel, also induced apoptotic cell death. In summary, this study shows that structural modification of the known ceramidase inhibitors B-13 and LCL-464 generates more potent ceramidase inhibitors that are active in intact cells and not only elevates the cellular ceramide levels, but also enhances cell death.

Safety and tolerability of once-daily controlled-release carvedilol 10-80 mg in Japanese patients with chronic heart failure.

BACKGROUND: The aim of the present study was to assess the safety and tolerability of the controlled-release (CR) formulation of the ß-blocker carvedilol in Japanese patients with chronic heart failure (HF). METHODS AND RESULTS: In this multicenter, randomized, open-label, phase I/II dose-escalation study, 41 patients receiving standard therapy for chronic HF were randomized in a ratio of 1:1 to carvedilol CR or immediate-release (IR) carvedilol. The primary objective was to evaluate the tolerability and safety of escalating doses of carvedilol CR (10-40 mg/day), with a reference arm of 5-20 mg/day of carvedilol IR. In addition, the tolerability and safety of titration to a carvedilol CR dose up to 80 mg/day were examined, as were plasma concentrations of carvedilol and changes in vital signs. The proportions of patients who completed 40-mg/day carvedilol CR and 20-mg/day carvedilol IR were 42% (8/19) and 50% (11/22), respectively. In the CR group, 7/19 (37%) attained a dose of 80 mg. During the primary evaluation period, 7/19 (37%) and 4/22 (18%) patients experienced drug-related adverse events in the CR and IR groups, respectively, the characteristics of which were similar between groups. CONCLUSIONS: No new safety issues emerged in Japanese chronic HF patients treated with carvedilol CR in contrast to those known in carvedilol IR.

Synthesis, biological evaluation and mechanistic studies of totarol amino alcohol derivatives as potential antimalarial agents.

Herein we report on the semisynthesis and biological evaluation of ß-amino alcohol derivatives of the natural product totarol and other simple aromatic systems. All ß-amino alcohol derivatives of totarol exhibited higher antiplasmodial activity than totarol [IC(50): 11.69 µM (K1, chloroquine and multi-drug resistant strain), and 11.78 µM (D10, chloroquine sensitive strain)]-12e was the most active [IC(50): 0.63 µM (K1), and 0.61 µM (D10)]. The phenyl and naphthyl ß-amino alcohol derivatives were much less active than their corresponding totarol equivalents. The majority of the ß-amino alcohol derivatives of totarol were more active against K1 than the D10 strains of Plasmodium falciparum, a trend similar to the inverse relationship observed with the established aryl-amino alcohol antimalarial mefloquine. Selected compounds were shown to affect erythrocyte morphology, inhibit erythrocyte invasion and trigger CQ accumulation.

Novel aminopeptidase N (APN/CD13) inhibitors derived from chloramphenicol amine.

Aminopeptidase N (APN) is involved in different physiological and pathological processes of tumor cells, including proliferation, invasion, apoptosis and metastasis. Herein one series of compounds derived from commercially available (1S,2S)-2-amino-1-(4-nitrophenyl) propane-1,3-diol have been designed and synthesized. Furthermore, preliminary activity evaluation showed that some compounds elicited moderate inhibitory activity against APN with compounds 10e (IC(50)=6.1±0.5 µM) possessing the best efficacy, which could be used as the lead compound in the future for anticancer agents research.

Final amended report on safety assessment on aminomethyl propanol and aminomethyl propanediol.

Aminomethyl propanol and aminomethyl propanediol are substituted aliphatic alcohols that function as pH adjusters in cosmetic products at concentrations less than 10%; additionally, aminomethyl propanediol is a fragrance. Extensive oral toxicity data are reviewed, with fewer inhalation toxicity data. Dermal toxicity data are presented that demonstrate, for example, that a mascara with 1.92% aminomethyl propanediol does not cause dermal irritation or allergic contact sensitization, suggesting that the maximum reported use concentration of 2% in mascara would be safe. Although these ingredients are primary amines that are not substrates for N-nitrosation, they may contain secondary amines as impurities in finished products that may undergo N-nitrosation. These ingredients should not be included in cosmetic formulations containing N-nitrosating agents. The Cosmetic Ingredient Review Expert Panel concludes that aminomethyl propanol and aminomethyl propanediol are safe as cosmetic ingredients in the practices of use and concentrations as described in this safety assessment.

Effect of diisopropanolamine upon choline uptake and phospholipid synthesis in Chinese hamster ovary cells.

Aminoalcohols differ in mammalian toxicity at least in part based upon their ability to alter the metabolism of phospholipids and to cause depletion of the essential nutrient choline in animals. This study examined the incorporation of diisopropanolamine (DIPA) into phospholipids (PLs) and effects of DIPA upon choline uptake and phospholipid synthesis in Chinese hamster ovary (CHO) cells. Results were compared to those of a related secondary alcohol amine, diethanolamine (DEA), whose systemic toxicity is closely associated with its metabolic incorporation into PLs and depletion of choline pools. DIPA caused a dose-related inhibition of (3)H-choline uptake by CHO cells that was approximately 3-4 fold less potent, based upon an IC50, than that reported for DEA. DIPA, in contrast to DEA, did not cause changes in the synthesis rates of (33)P-phosphatidylethanolamine, (33)P-phosphatidylcholine or (33)P-sphingomyelin at either non-toxic or moderately toxic concentrations. Only approximately 0.004%, of administered (14)C-DIPA was metabolically incorporated into PLs, over 30-fold less than the incorporation of (14)C-DEA under similar conditions. Overall, these data and previous pharmacokinetic and toxicity data obtained in vivo suggests that DIPA is distinct from DEA and lacks significant choline and PL metabolism related toxicity in animals.

Steroid and beta-adrenergic receptor modifications in target organs of broiler chickens fed with a diet containing beta2-adrenergic agents.

The illegal use of beta2-agonists as repartitioning agents to improve production performance and carcass composition can induce changes in various organs of exposed animals. The aim of the present study was to evaluate the effects induced by dietary beta-agonists on beta-AR, AnR and GR in male broiler target organs. Fifty-four male broiler chickens (Ross 508), 26 days old, were randomly divided into three homogeneous experimental groups and fed for 21 days with a standard diet containing placebo (group 1, control), 1ppm of clenbuterol (group 2) and 1ppm of cimaterol (group 3). Tissue samples of heart, lung, brain, testicle, spleen, thymus and Bursa of Fabricius were collected post-mortem then cytosol fractions were used for AnR (testicles) and GR (spleen, thymus and Bursa of Fabricius), and membrane fractions for beta-AR (all tissues but testicles) determination by binding assays. The dietary administration of beta-adrenergic agents as repartitioning agents induced a significant decrease in AnR concentration in the testicle, in GR levels in the lymphoid tissues and in beta-AR concentrations of different target organs of male chickens. Present data confirm those observed in female chickens and suggest that in poultry the regulation exerted by adrenergic stimulation on steroid receptor concentrations produces different effects than in mammals.

Effect of carvedilol on the production of reactive oxygen species by HL-60 cells.

OBJECTIVES: The generation of reactive oxygen species (ROS) by phagocytes is one of the irreplaceable microbicidal tools of innate immunity. It has been reported in our previous studies that short-term treatment by carvedilol ex vivo inhibits ROS generation. The purpose of this study was to investigate the long-term effect of carvedilol on phagocytes. METHODS: Human leukemia HL-60 cells differentiated into granulocyte-like cells were used as the model. Final concentrations of carvedilol were 0.1-100 micromol/l. The production of ROS by HL-60 cells was measured using luminol-enhanced chemiluminescence (CL). RESULTS: Carvedilol in concentrations 0.1-10 micromol/l did not exhibit any toxic effect on cells (measured using bioluminescent bacteria Photorhabdus luminescens subsp. thracensis). One hour's treatment with 10 micromol/l carvedilol significantly decreased both spontaneous and activated CL of cells. Conversely, no inhibitory effects on CL were observed in 10 micromol/l carvedilol after 48 h incubation; lower concentrations of carvedilol even slightly increased the CL activity of HL-60 cells. A significant increase in spontaneous CL activity was detected in cells incubated with 10 micromol/l carvedilol in comparison with the control. Powerful antioxidative properties of carvedilol against peroxyl radical (ORAC assay) were proved. No scavenging of nitric oxide (electrochemical method) was observed. CONCLUSIONS: Long-term influence of carvedilol can induce an increase in the generation of phagocyte-derived ROS and potentially also other inflammatory mediators. The increased ROS production is compensated for by antioxidative properties of carvedilol although the increased production of inflammatory mediators could affect the proper function of immune system.

Mechanisms of carvedilol-induced [Ca2+] i rises and death in human hepatoma cells.

The effect of the cardiovascular drug carvedilol on cytosolic free Ca2+ concentrations ([Ca2+]i) and viability has not been explored in human hepatoma cells. This study examined whether carvedilol altered [Ca2+]i and caused cell death in HA59T cells. [Ca2+]i and cell viability were measured using the fluorescent dyes fura-2 and WST-1, respectively. Carvedilol at concentrations >or=1 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 20 microM. The Ca2+ signal was reduced partly by removing extracellular Ca2+. Carvedilol induced Mn2+ quench of fura-2 fluorescence, implicating Ca2+ influx. The Ca2+ influx was sensitive to La3+, econazole, nifedipine, and SKF96365. In Ca2+-free medium, after pretreatment with 1 muM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), carvedilol-induced [Ca2+]i rises were abolished; and conversely, carvedilol pretreatment inhibited a major part of thapsigargin-induced [Ca2+]i rises. Inhibition of phospholipase C with 2 microM U73122 did not change carvedilol-induced [Ca2+]i rises. At concentrations between 1 and 50 microM, carvedilol killed cells in a concentration-dependent manner. The cytotoxic effect of 1 microM (but not 30 microM) carvedilol was fully reversed by prechelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). Apoptosis was induced by 30 (but not 1) microM carvedilol. Collectively, in HA59T hepatoma cells, carvedilol induced [Ca2+]i rises by causing Ca2+ release from the endoplasmic reticulum in a phospholipase-C-independent manner and Ca2+ influx via store-operated Ca2+ channels. Carvedilol-caused cytotoxicity was mediated by Ca2+ and apoptosis in a concentration-dependent manner.

A cardiovascular monitoring system used in conscious cynomolgus monkeys for regulatory safety pharmacology. Part 2: Pharmacological validation.

INTRODUCTION: This project addresses the validation study design of a test system using a telemetered non-human primate model for cardiovascular safety pharmacology evaluation. METHODS: In addition to non-pharmacological validation including installation and operation qualifications, performance qualification (locomotor activity and cardiovascular evaluations) was completed on free-moving cynomolgus monkeys by quantifying the degree of cardiovascular response measured by the telemetric device to various positive control drugs following their intravenous administration. Remifentanil (0.0005, 0.001, 0.002, 0.004, 0.008 and 0.016 mg/kg) was given to induce bradycardia and hypotension. Medetomidine (0.04 mg/kg) was used to induce an initial phase of hypertension followed by hypotension and bradycardia. Esmolol (0.5, 1.0 and 2.0 mg/kg) was used to induce bradycardia. Dopamine (0.002, 0.008, 0.01, 0.02, 0.03 and 0.05 mg/kg/min) was infused over 30 min to induce an increase in arterial and pulse pressures and tachycardia. Amiodarone (0.4, 0.8 and 1.6 mg/kg/min) was infused over 10 min to induce QT interval prolongation. Potassium chloride (0.08 mEq/kg/min) was infused for periods of less than 30 min to induce electrocardiographic (EKG) changes characteristic of hyperkalemia. Reliability was evaluated over 60 days. RESULTS: Monitoring with a reference methodology and the telemetry system was important in order to evaluate precision and accuracy of the test system. Positive control drugs produced a wide range of cardiovascular effects with different amplitudes, which were useful in identification of the limits of the test system. DISCUSSION: Reference monitoring methods and selection of a battery of positive control drugs are important to ensure proper test system validation. Drugs inducing not only QT prolongation but also positive and negative chronotropic effects, positive and negative systemic arterial pressure changes and ECG morphology alterations were useful to identify test system limitations during performance qualification. ECG data processing at significantly elevated heart rates revealed that a trained observer should review all cardiac cycles evaluated by computer.

Repeated dose toxicity and developmental toxicity of diisopropanolamine to rats.

The repeated dose oral and dermal toxicity of diisopropanolamine (DIPA) was evaluated in rats and compared to the reported toxicity of the related secondary alcohol amine, diethanolamine (DEA). Fischer 344/DuCrl rats were given up to 750 mg/kg/day by dermal application, 5 days/week, for 4 weeks; or up to 1,000 mg DIPA/kg/day by drinking water for 13 weeks to evaluate potential toxic effects. Time-mated female CRL:CD(SD) rats were given up to 1,000 mg/kg/day by gavage on gestation days (GD) 6-20 for evaluation of maternal and fetal effects. In the dermal toxicity study, no adverse treatment-related in-life effects other than mild irritation at the site of dermal application at >or= 500 mg/kg/day were observed. There were no systemic effects in rats given up to 750 mg/kg/day. In the subchronic oral toxicity study, the most significant effects were an increase in absolute and relative kidney weights, unaccompanied by histopathologic changes, at >or= 500 mg/kg/day DIPA. The latter effect was ameliorated following a 4-week recovery period. In the developmental toxicity study, there were no maternal or developmental effects at any dose level evaluated. The toxicity of DIPA contrasts with that of DEA which has been shown to affect a number of organ systems when repeatedly administered orally or dermally at similar or lower dosages.

Phototoxic assessment of a sunscreen formulation and its excipients: An in vivo and in vitro study.

BACKGROUND: Cosmetic preservatives are used to protect cosmetic formulations and improve its shelf-life. However, these substances may exert phototoxic effects when used under sunlight. OBJECTIVE: To assess safety, efficacy and putative phototoxic effects of a sunscreen formulation SPF 30 and its excipients. MATERIALS/METHODS: Irradiation was performed with solar simulated light (SSL) and the sunscreen from the School of Pharmacy/UFRJ/Brazil. We used albino hairless mice in different groups (control (G1), only irradiated (G2), sunscreen plus irradiation (G3) and vehicle plus irradiation (G4) for morphological assessment and immunefluorescence detection to OKL38. In vitro analyses were with a Saccharomyces cerevisiae (SC) strain plus SSL in the presence of methylparaben, propylparaben, imidazolidinyl urea, aminomethyl propanol and their association. RESULTS: G3 and G4 displayed photosensitization leading to thickening of the epidermis and increased dermal cellularity. G4 displayed strong OKL38 labeling when compared with other groups. Aminomethyl propanol, methylparaben and propylparaben are endowed with phototoxic activity against SC. Propylparaben displayed the highest phototoxic effect, followed by excipients association. CONCLUSIONS: The sunscreen's vehicle is endowed with phototoxic activity. Propylparaben was the most phototoxic agent, increasing the overall phototoxicity of excipient association, pointing to a critical concern regarding vehicle associations intended to cosmetic purposes.

Microbial detoxification of carvedilol, a ß-adrenergic antagonist, by the filamentous fungus Cunninghamella echinulata.

Beta adrenergic antagonists like carvedilol are typical environmental pollutants detected in wastewater and surface water. Human metabolism of carvedilol is well investigated, while its environmental fates are still unknown. In recent years, there have been appearing reports on high toxicity of ß-blockers toward aquatic organisms. In this paper the ability of the filamentous fungus C. echinulata to eliminate the ß-blocker has been described for the first time. An 83% loss of carvedilol was observed after 120 h incubation of the tested fungus with the compound, where hydroxylated carvedilol metabolites were identified as the major biotransformation products. Carvedilol degradation by C. echinulata was proceeded by hydroxylation and conjugation reactions similar to its mammalian metabolism. Glucose conjugate was found in the fungi cultures, whereas glucuronide conjugates were detected in mammals. The impact of carvedilol on the functionality of fungal cells was also evaluated. A 2-fold decrease in the PC/PE ratio was noticed in the C. echinulata cell membrane after the exposition to carvedilol compared to control mycelium incubated without the ß-blocker. The change can denote perturbation of fungal cell membrane integration by carvedilol. Moreover, 2.8-fold lower toxicity of postcultures supernatants toward D. magna were shown in contrast to abiotic control.

Blockade of ß2-adrenoceptor, rather than ß1-adrenoceptor, deteriorates cardiac anaphylaxis in isolated blood-perfused rat hearts.

BACKGROUND: Cardiac anaphylaxis is one of the features of anaphylactic hypotension. Patients treated with propranolol, a nonselective ß-adrenoceptor (AR) antagonist, develop severe anaphylaxis, but the mechanism remains unknown. Under examination were the effects of ß1- and ß2-AR antagonist on anaphylaxis-induced coronary vasoconstriction and cardiac dysfunction in isolated blood-perfused rat hearts. METHODS: Isolated hearts from ovalbumin-sensitized Wistar rats were subjected to coronary perfusion with blood at a constant pressure and measurements were made of coronary blood flow and left ventricu-lar (LV) pressure. Following pretreatment with selective ß2-AR antagonist ICI118,551 or selective ß1-AR antagonist atenolol, cardiac anaphylaxis was induced by intracoronary injections of ovalbumin antigen. LV contractility was evaluated by the maximum increasing rate of systolic LV pressure (dP/dtmax). RESULTS: In response to antigen administrations, ICI118,551 pretreated hearts showed a greater de-crease in coronary blood flow and consequently a greater increase in coronary vascular resistance than the atenolol pretreated hearts. Pretreatment with ICI118,551 caused a greater decrease in dP/dtmax than those with atenolol. CONCLUSIONS: Cardiac anaphylaxis-induced contractile dysfunction and coronary spasm are severe in b2-, rather than ß1-AR antagonist, pretreated isolated blood-perfused rat hearts.

Liver tumors induced by a new beta-adrenoreceptor blocking agent in female rats.

A newly synthesized beta-adrenoreceptor blocking agent, DL-1-(2-nitro-3-methyl-phenoxy)-3-tert-butylamino-propan-2-ol (ZAMI 1305), administered intragastrically for 6 months induced hepatocellular carcinomas in female outbred Wistar rats in a dose-dependent fashion but was harmless to the livers of male outbred Wistar rats. Experiments with castrated animals confirmed the dependence of the carcinogenic effect of ZAMI 1305 on the hormonal status of the rat. When an isomer of ZAMI 1305, DL-1-(2-nitro-5-methyl-phenoxy)-3-tert-butylamino-propan-2-ol (ZAMI 1327), and DL-practolol, which have pharmacologic activities similar to those of ZAMI 1305, were administered by the same experimental technique as used for ZAMI 1305, no liver tumors appeared either in male or female rats.

Safety and efficacy of esmolol (ASL-8052: an ultrashort-acting beta-adrenergic blocking agent) for control of ventricular rate in supraventricular tachycardias.

Esmolol (ASL-8052) is a new intravenous beta-adrenergic blocking agent that has exhibited both cardiac selectivity and an extremely short half-life in animal studies. To assess its clinical efficacy, 16 patients were studied with a rapid ventricular rate associated with atrial flutter (n = 2), atrial fibrillation (n = 10), atrial tachycardia (n = 2) and multifocal atrial tachycardia (n = 2). During a 30 minute control period of observation, the ventricular rate ranged from 121 to 150 beats/min (mean 133.2 +/- 10.6). Using a double blind crossover method, esmolol was infused intravenously for a maximum of 60 minutes. Infusions of 50, 100, 150, 200, 250 and 300 micrograms/kg per min were given in consecutive 5 minute periods (during the first minute of each period, a loading dose of 500 micrograms/kg was given). Not all patients received the maximal dose. A response was defined as conversion to sinus rhythm or a 20% reduction in ventricular rate. One patient with atrial fibrillation associated with the Wolff-Parkinson-White syndrome did not respond. In the remaining 15 patients, their highest esmolol infusion rate was maintained for an additional 30 minutes. This resulted in a reduction in ventricular rate to a mean of 97.8 +/- 12.9 beats/min (range 72 to 119) (p less than 0.001). Conversion from flutter/fibrillation to sinus rhythm occurred in two patients. During the infusion, six had transient asymptomatic hypotension that was mild and manageable. After infusion, ventricular rate and blood pressure returned rapidly toward control values within 25 minutes in patients without conversion to sinus rhythm.(ABSTRACT TRUNCATED AT 250 WORDS)