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In the context of pharmaceutical development today, studies for pediatric drug approval are requested more and more often by the regulatory authorities. The developing lung represents a potential target in juvenile toxicity studies. Due to physiological differences in prenatal and postnatal development between humans and standard animal models, experimental methods have to be modified to assess pulmonary function, and basic data on respiratory parameters need to be provided. Daily nose-only inhalation exposure from postnatal days 4 to 21 using a model substance (verapamil HCl) and plethysmographic measurements between postnatal days 2 and 50 were performed noninvasively in conscious juvenile Wistar (WU) rats. The methods proved to be feasible and did not interfere with normal growth and development of the animals. Both techniques therefore permit new insights to support human neonatal risk assessment and therefore these animal models are suitable for regulatory studies.

FR-900098 is an inhibitor of 1-deoxy-d-xylulose-5-phosphate (DXP) reductoisomerase, the second enzyme in the non-mevalonate isoprenoid biosynthesis pathway. In previous studies, FR-900098 was shown to possess potent antimalarial activity in vitro and in a murine malaria model. In order to provide a basis for further preclinical and clinical development, we studied the acute toxicity and genotoxicity of FR-900098. We observed no acute toxicity in rats, i.e. there were no clinical signs of toxicity and no substance-related deaths after the administration of a single dose of 3000 mg/kg body weight orally or 400 mg/kg body weight intravenously. No mutagenic potential was detected in the Salmonella typhimurium reverse mutation assay (Ames test) or an in vitro mammalian cell gene mutation test using mouse lymphoma L5178Y/TK+/− cells (clone 3.7.2C), both with and without metabolic activation. In addition, FR-900098 demonstrated no clastogenic or aneugenic capability or significant adverse effects on blood formation in an in vivo micronucleus test with bone marrow erythrocytes from NMRI mice. We conclude that FR-900098 lacks acute toxicity and genotoxicity, supporting its further development as an antimalarial drug.

DNAzymes are single-stranded catalytic DNA molecules that bind and cleave specific sequences in a target mRNA molecule. Their potential as novel therapeutic agents has been demonstrated in a variety of disease models. However, no studies have yet addressed their toxicology and safety pharmacology profiles in detail. Here we describe a detailed toxicological analysis of inhaled hgd40, a GATA-3-specific DNAzyme designed for the treatment of allergic bronchial asthma. Subacute toxicity, immunotoxicity, and respiratory, cardiovascular, and CNS safety pharmacology were analyzed in rodents and non-rodents, and genotoxicity was assessed in human peripheral blood. Overall, hgd40 was very well tolerated when delivered by aerosol inhalation or slow intravenous infusion. Only marginal reversible histopathological changes were observed in the lungs of rats receiving the highest dose of inhaled hgd40. The changes consisted of slight mononuclear cell infiltration and alveolar histiocytosis, and moderate hyperplasia of bronchus-associated lymphoid tissue. No local or systemic adverse effects were observed in dogs. No compound-related respiratory, cardiovascular, or CNS adverse events were observed. The only relevant immunological findings were very slight dose-dependent changes in interleukin-10 and interferon-gamma levels in bronchoalveolar lavage fluid. Taken together, these results support direct delivery of a DNAzyme via inhalation for the treatment of respiratory disease.

The study presented was conducted following the reproductive study guideline OECD Guideline 416 Two-Generation Reproduction Toxicity Study. Sprague-Dawley rats were exposed to 2000, 10,000 and 50,000ppm of HFC-245fa. There was an unexpected mortality of lactating dams in the medium and high dose group beginning at day 10 of lactation. Statistically significant histopathological alterations were observed in the cerebellum of a total of 9/30 females of the high dose group of the F0-generation and in 10/27 females of the high dose group of the F1-generation. In contrast there were no brain lesions found in males or non-pregnant females of all dose groups. Neuronal necrosis and degeneration in the cerebellar cortex were observed as the most severe finding. Furthermore vacuolation of the neuropil in different degrees was diagnosed in 7/30 females of the F0-generation and in 9/30 females of the F1-generation. Acute hemorrhages - in particular perivascular - occurred in 5/30 f emales of the F0- and in 5/30 females of the F1-generation indicating a disturbed vascular integrity. The main lesions found in the cerebrum were glial scars in the corpus callosum and restricted to 2/30 females of the F0-generation of the high dose group. The increased incidence of myocardial fibrosis and mononuclear cell infiltration in males - indicating myocarditis - was only seen in the F0-generation of the high dose group. Females of the F1-generation of the high dose group showed an increased incidence of minimal myocardial fibrosis. In summary, histopathology revealed that the brain, particularly the cerebellum, and to a minor degree the heart turned out to be the toxicological target organs of the substance. Presumably substance-related energy deprivation may be responsible for the observed changes. One of the metabolites, 3,3,3-trifluoropropanoic acid has been shown to be capable of causing this effect.

Scope:trans-Resveratrol has been shown to improve insulin sensitivity and to enhance cellular glucose uptake. Evidence from recent studies indicates that these effects depend on SIRT1-pathways. Methods and results: Since ingestion of resveratrol leads to the presence of resveratrol and resveratrol metabolites in the body, we aimed at investigating (i) whether a daily dose of 300 mg resveratrol/kg body weight in healthy male Wistar rats for a period of 8 wk affects the selected parameters of glucose and lipid metabolism and (ii) whether the resulting plasma concentrations of resveratrol metabolites were effective in modulating SIRT1 expression. The dietary dose was based on the results from preceding toxicity studies. The results from the feeding experiment revealed plasma concentrations of resveratrol and its metabolites below 1 μmol/L and showed that fasting glucose and insulin levels were decreased by 35 and 41%, respectively, in the resveratrol group compared with controls. Insulin sensitivity was enhanced by 70%, whereas liver SIRT1 protein expression was not affected. Treatment of HepG2 cells with 10 μM resveratrol (1.49-fold) or its diglucuronides (1.21-fold) increased SIRT1 expression. Conclusion: These results suggest that the improved insulin sensitivity after dietary administration of 300 mg resveratrol/kg body weight does not involve increased protein expression of SIRT1.

A Type III Built-up Roofing Asphalt (BURA) fume condensate was evaluated for subchronic systemic toxicity and reproductive/developmental toxicity screening in Wistar rats, by OECD protocol 422 and OECD cytogenetic protocol 474. Animals were exposed by nose-only inhalation to target concentrations of 30, 100 and 300 mg/m(3) total hydrocarbons (actual concentrations, 30.0, 100.1 and 297.3 mg/m(3)). The study was performed to assess potential hazards from asphalt fumes to which humans could be exposed during application. No adverse effects were seen for spermology, reproductive or developmental parameters or early postnatal development of offspring from day 1 to 4 postpartum. BURA fume condensate did not induce any significant increases in micronucleus frequency in polychromatic erythrocytes of rat bone marrow nor was neurobehavioral toxicity observed at any dose. Systemic effects were slight and seen at doses above those measured at work sites. The systemic NOAEC of 100 mg/m(3) for males was based on decreased body weight gain, food consumption and increased absolute and relative lung wet weight correlated with slight histological changes in the lung, primarily adaptive in nature at 300 mg/m(3). The female NOAEC of 30 mg/m(3) was based on a statistically significant increase in relative wet lung weight at higher doses, correlated with slight histopathologic effects in the lungs at the highest dose. However, no increase in relative lung weight was seen in breeding females at 100 mg/m(3).

Ciclesonide, an inhaled corticosteroid (ICS) with prolonged anti-inflammatory activity, is being developed for the treatment of asthma. Fatty acid conjugation of ICS is thought to be related to prolonged ICS activity. In vitro studies demonstrated that ciclesonide is converted to an active metabolite, desisobutyryl-ciclesonide (des-CIC), which undergoes reversible fatty acid conjugation. We tested the in vivo metabolism of ciclesonide in the lung by exposing rats to inhaled ciclesonide (0.16 mg/kg/day) for 1h daily over 4 weeks. Lungs (n=6 per time point) were extracted with ethanol 2, 5, and approximately 27 h after the final treatment. Ciclesonide and des-CIC concentrations were determined using solid-phase extraction and reverse-phase high-performance liquid chromatography with tandem mass spectrometry (LC/MS/MS). Concentrations of fatty acid ester conjugates were indirectly assessed using enzymatic de-esterification before LC/MS/MS. At 2 and 5 h, fatty acid conjugates of des-CIC were the major metabolites (61 and 55%, respectively). Ciclesonide, des-CIC, and fatty acid conjugates of des-CIC were clearly present in lung samples the day after the last inhalation. This in vivo study confirmed ciclesonide activation to des-CIC and formation of fatty acid conjugates. The presence of des-CIC fatty acid conjugates at >24 h after dosing suggests that ciclesonide is appropriate for once-daily dosing.

Aim of the study was to investigate the potential toxic effects of di-n-butylamine (DBA), a known skin and eye irritating compound, on the respiratory tract after inhalation exposure for up to 91 days in male and female rats [Crl:(WI)WU BR]. To check whether and to what degree the no-observed-(adverse)-effect level (NO(A)EL) decreases with increasing study duration, serial sacrifices were performed after 3 and 28 days, respectively. Based on two dose range-finding studies, the concentrations for this study were determined with 0 (clean air), 50, 150, and 450 mg/m(3). Animals were exposed for 3 days (6 h/day) 28, and 91 days (5 days/wk, 6 h/day), respectively, and immediately sacrificed thereafter. The results show clear irritating effects only in the upper part of the respiratory tract, that is, the nasal cavities. While after 3 and 28 days effects were found only in the high-dose group, slight adaptive effects, expressed as mucous (goblet) cell hyperplasia, could be diagnosed in the medium- and low-dose groups after 91 days of exposure. Pathological changes were most prominent after 3 days of exposure. In the lung, only marginal effects could be observed (increased relative lung weight only in females of the high concentration after 28 days, slight, not statistically significant histopathological effects in the high concentration after 3 days, no effects on parameters of bronchoalveolar lavage fluid), while no effects were found in the remaining groups.

A study was performed in Sprague-Dawley rats (Crl:CD BR) to differentiate between effects of hydrofluorocarbon 123 (HCFC-123) on the lactating dam or on the fetus using fostering and cross-fostering of the offspring. Pregnant and/or lactating dams without the pups present were exposed to the test substance (1000 ppm) or clean air by whole-body inhalation for 6 h/day from day 6 to 19 post conceptionem (p. c.) and from day 5 to 21 post partum (p. p.). Pups were cross-fostered to new dams within the first 2 days after birth. Treatment of the mothers with HCFC-123 led to decreases in serum glucose, cholesterol, and triglycerides and increases in absolute and relative maternal liver weights. Decreased litter and individual pup weight and decreased serum triglycerides were observed in the pups of treated foster mothers. Treatment of the mothers with HCFC-123 did not influence milk production based on the body weight difference of the dam before suckling and 60 min. after beginning of suckling using 12-pup "standard litters" of untreated dams. Total fat, glucose, and protein contents in the milk were also not influenced by the treatment. Trifluoroacetic acid (TFA), a main metabolite of HCFC-123, was observed in urine samples of standard litters that had been nursed by treated dams. In conclusion, the effects on offspring due to HCFC-123 treatment consisted of decreased pup weight and decreased serum triglycerides at weaning. All effects were due to treatment of the lactating dams, as no prenatally induced effects were found. Since milk production and nutritional constituents of the milk were not influenced, but significant amounts of the main metabolite were found in pup urine, an effect of HCFC-123 or its metabolite on the pups via maternal milk is considered to be a possible cause for their decreased weight gain.