Discovery of novel ketoxime ether derivatives with potent FXR agonistic activity, oral effectiveness and high liver/blood ratio.

The farnesoid X receptor (FXR) is a promising therapeutic target for nonalcoholic steatohepatitis (NASH) and other bile acid related diseases because it plays a critical role in fibrosis, inflammation and bile acid homeostasis. Obeticholic acid (OCA), a FXR agonist which was synthesized from chenodeoxycholic acid, showed desirable curative effects in clinical trials. However, the pruritus which was the main side effect of OCA limited its further applications in NASH. Although pruritus was also observed in the clinical trials of non-steroidal FXR agonists, the proportion of patients with pruritus was much smaller than that of OCA. Thus, we decided to develop non-steroidal FXR agonists and discovered a series of novel FXR agonists which were synthesized from GW4064 by replacing the stilbene group with ketoxime ether. Encouragingly, in the following biological tests, our target compounds 13j and 13z not only showed potent FXR agonistic activities in vitro, but also effectively promoted the expression of target genes in vivo. More importantly, in the pharmacokinetic experiments, compounds 13j and 13z displayed high liver/blood ratio characteristics which were helpful to reduce the potential side effects which were caused by prolonged systemic activation of FXR. In summary, our compounds were good choices for the development of non-steroidal FXR agonists and were deserved further investigation.

Preparation and Characterization of Cellulose Ether Liposomes for the Inhibition of Prion Formation in Prion-Infected Cells.

Prion accumulation in the brain and lymphoreticular system causes fatal neurodegenerative diseases. Our previous study revealed that cellulose ethers (CE) have anti-prion activities in vivo and in prion-infected cells when administered at high doses. This study aims to improve the bioavailability of a representative CE using a liposomal formulation and characterized CE-loaded liposomes in cultured cells. The liposomal formulation reduced the EC50 dose of CE by <1/200-fold in prion-infected cells. Compared to empty liposomes, CE-loaded liposomes were taken up much more highly by prion-infected cells and less by macrophage-like cells. Phosphatidylserine modification reduced the uptake of CE-loaded liposomes in prion-infected cells and did not change the anti-prion activity, whereas increased the uptake in macrophage-like cells. Polyethylene glycol modification reduced the uptake of CE-loaded liposomes in both types of cells and reduced the anti-prion activity in prion-infected cells. These results suggest that a liposomal formulation of CE is more practical than unformulated CE and showed that the CE-loaded liposome uptake levels in prion-infected cells were not associated with anti-prion activity. Although further improvement of the stealth function against phagocytic cells is needed, the liposomal formulation is useful to improve CE efficacy and elucidate the mechanism of CE action.

Dose- and time-dependent pharmacokinetics of apigenin trimethyl ether.

Apigenin trimethyl ether (5,7,4'-trimethoxyflavone, ATE), one of the key polymethoxyflavones present in black ginger (rhizome of Kaempferia parviflora) possesses various health-promoting activities. To optimize its medicinal application, the pharmacokinetics of ATE was assessed in Sprague-Dawley rats with emphases to identify the impacts from dose and repeated dosing on its major pharmacokinetic parameters. Plasma ATE levels were monitored by liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Upon single intravenous administration (2 mg/kg), plasma levels of ATE declined through an apparent first-order process while dose-escalation to 4 and 8 mg/kg led to its non-linear disposition, which could be described by the Michaelis-Menten model. Similarly, dose-dependent oral pharmacokinetics was confirmed and when the dose was escalated from 5 to 15 and 45 mg/kg, much longer mean residence time (MRT0→last), higher dose-normalized maximal plasma concentration (Cmax/Dose) and exposure (AUC/Dose) were observed at 15 and/or 45 mg/kg. One-week daily oral administration of ATE at 15 mg/kg caused its accelerated elimination and the plasma exposure (AUC) after intravenous (2 mg/kg) and oral administration (15 mg/kg) dropped ~40 and 60%, respectively. As ATE displayed both dose- and time-dependent pharmacokinetics, caution is needed in the medicinal applications of ATE and/or black ginger.

Synthesis, anticancer evaluation and pharmacokinetic study of novel 10-O-phenyl ethers of dihydroartemisinin.

Twenty hybrid compounds, tethering dihydroartemisinin (DHA) with diaryl-pyrazoline/diaryl-pyrazole through ether linkage, were synthesized based on hybridization strategy and assessed for their anticancer activity. The representative compound 6f exhibited significantly elevated antiproliferative activity compared with DHA against a panel of cancer cell lines. Unexpected sensitivity of 6f in Adriamycin-resistant MCF-7 cells (MCF-7/Adr) inspired subsequent research on anticancer activity of these DHA derivatives against breast cancer cell lines. All the novel compounds exhibited potent activity in three breast cancer cells including MDA-MB-231, MCF-7 and MCF-7/Adr. Most of them exerted almost 10-fold higher potency in MCF-7/Adr than in MCF-7 and MDA-MB-231 cells. Compound 5f, the most potent compound against MCF-7/Adr (GI50 = 18 nM), was used for mechanism research which revealed that compound 5f arrested MCF-7 and MCF-7/Adr cells in G0/G1 phase with decreased levels of cyclin D1 and increased levels of p27. Preliminary pharmacokinetic properties of 5f and 6f were investigated in rats after a single intravenous administration. The high sensitivity of MCF-7/Adr indicates that these compounds have potential to be developed as therapeutic agents to treat drug-resistant cancer.

Structure activity relationship of brevenal hydrazide derivatives.

Brevenal is a ladder frame polyether produced by the dinoflagellate Karenia brevis. This organism is also responsible for the production of the neurotoxic compounds known as brevetoxins. Ingestion or inhalation of the brevetoxins leads to adverse effects such as gastrointestinal maladies and bronchoconstriction. Brevenal shows antagonistic behavior to the brevetoxins and shows beneficial attributes when administered alone. For example, in an asthmatic sheep model, brevenal has been shown to increase tracheal mucosal velocity, an attribute which has led to its development as a potential treatment for Cystic Fibrosis. The mechanism of action of brevenal is poorly understood and the exact binding site has not been elucidated. In an attempt to further understand the mechanism of action of brevenal and potentially develop a second generation drug candidate, a series of brevenal derivatives were prepared through modification of the aldehyde moiety. These derivatives include aliphatic, aromatic and heteroaromatic hydrazide derivatives. The brevenal derivatives were tested using in vitro synaptosome binding assays to determine the ability of the compounds to displace brevetoxin and brevenal from their native receptors. A sheep inhalation model was used to determine if instillation of the brevenal derivatives resulted in bronchoconstriction. Only small modifications were tolerated, with larger moieties leading to loss of affinity for the brevenal receptor and bronchoconstriction in the sheep model.

Cytoprotective effect of hydroxytyrosyl alkyl ether derivatives after oral administration to rats in a model of glucose-oxygen deprivation in brain slices.

This study was designed to determine whether the oral administration of hydroxytyrosol (HT) alkyl ether derivatives has a neuroprotective effect in rats. The animals were treated for 7 days with HT or ethyl, butyl, hexyl, octyl, and dodecyl HT ether. A method of in vitro hypoxia-reoxygenation in brain slices was used. Hexyl, octyl, and dodecyl HT derivatives reduced brain cell death (LDH efflux). Lipid peroxidation and nitrite concentrations were inhibited most by hexyl, octyl, and dodecyl derivatives. Concentrations of 3-nitrotyrosine were reduced by HT butyl, hexyl, octyl, and dodecyl ether derivatives. Interleukin-1ß was significantly reduced in brain slices from rats treated with all HT ether derivatives. LDH efflux showed a linear correlation with brain concentrations of lipid peroxides, nitrites plus nitrates, and interleukin 1ß. The reduction in oxidative and nitrosative stress and decreased production of pro-inflammatory interleukins may be the basis for the observed neuroprotective effects.

Incorporation and controlled release of silyl ether prodrugs from PRINT nanoparticles.

Asymmetric bifunctional silyl ether (ABS) prodrugs of chemotherapeutics were synthesized and incorporated within 200 nm × 200 nm particles. ABS prodrugs of gemcitabine were selected as model compounds because of the difficulty to encapsulate a water-soluble drug within a hydrogel. The resulting drug delivery systems were degraded under acidic conditions and were found to release only the parent or active drug. Furthermore, changing the steric bulk of the alkyl substituents on the silicon atom could regulate the rate of drug release and, therefore, the intracellular toxicity of the gemcitabine-loaded particles. This yielded a family of novel nanoparticles that could be tuned to release drug over the course of hours, days, or months.

Nanobody-shell functionalized thermosensitive core-crosslinked polymeric micelles for active drug targeting.

The aim of this study was to develop poly(ethylene glycol)-b-poly[N-(2-hydroxypropyl) methacrylamide-lactate] (mPEG-b-p(HPMAm-Lac(n))) core-crosslinked thermosensitive biodegradable polymeric micelles suitable for active tumor targeting, by coupling the anti-EGFR (epidermal growth factor receptor) EGa1 nanobody to their surface. To this end, PEG was functionalized with N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP) to yield a PDP-PEG-b-p(HPMAm-Lac(n)) block copolymer. Micelles composed of 80% mPEG-b-p(HPMAm-Lac(n)) and 20% PDP-PEG-b-p(HPMAm-Lac(n)) were prepared and lysozyme (as a model protein) was modified with N-succinimidyl-S-acetylthioacetate, deprotected with hydroxylamine hydrochloride and subsequently coupled to the micellar surface. The micellar conjugates were characterized using SDS-PAGE and gel permeation chromatography (GPC). Using the knowledge obtained with lysozyme conjugation, the EGa1 nanobody was coupled to mPEG/PDP-PEG micelles and the conjugation was successful as demonstrated by western blot and dot blot analysis. Rhodamine labeled EGa1-micelles showed substantially higher binding as well as uptake by EGFR over-expressing cancer cells (A431 and UM-SCC-14C) than untargeted rhodamine labeled micelles. Interestingly, no binding of the nanobody micelles was observed to EGFR negative cells (3T3) as well as to14C cells in the presence of an excess of free nanobody. This demonstrates that the binding of the nanobody micelles is indeed by interaction with the EGF receptor. In conclusion, EGa1 decorated (mPEG/PDP-PEG)-b-(pHPMAm-Lac(n)) polymeric micelles are highly promising systems for active drug targeting.

Anti-osteosarcoma effects and mechanisms of 4-O-amino-phenol-4'-demethylepipodophyllotoxin ether.

The purpose of this study was to investigate the anti-osteosarcoma effects and mechanisms of 4-O-amino-phenol-4'-demethylepipodophyllotoxin ether (ODE), a new derivative of podophyllotoxin. The results showed that ODE inhibited proliferation of K562, OS-9901, CNE, BGC-823 and Tca-8113 cells in a time- and concentration-dependent manner as determined by microculture tetrazolium (MTT) assay. OS-9901 and K562 cells treated with ODE for 24 h showed cell cycle arrest at G(2)/M and a parallel decrease in G(0)/G(1) and S phase as detected by flow cytometry (FCM). Meanwhile, a fraction of cells with hypodiploid DNA content representing apoptosis were detected by FCM. Morphology observation also revealed typical apoptotic features, including shrinkage of cellular and nuclear membranes, condensed heterochromatin around the nuclear periphery and cytoplasmic vacuolation in OS-9901 cells. Under a confocal laser scanning microscope, intracellular Ca2+ and Mg2+ concentrations were greatly increased whereas the pH value, mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) were markedly reduced in OS-9901 cells after treatment with ODE. Taken together, these results suggest that the anti-osteosarcoma mechanisms of ODE are attributed to apoptosis through increasing intracellular Ca2+ and Mg2+ concentrations, and reducing pH value, MMP and ROS.

Short-term oral toxicity of pentyl ether, 1,4-diethoxybutane, and 1,6-dimethoxyhexane in male rats.

Pentyl ether (PE) and two newly synthesized polyoxy ethers, 1,4-diethoxybutane (DEB) and 1,6-dimethoxyhexane (DMH), have been proposed as candidate diesel fuel additives. To characterize and compare their toxicity and to provide information for risk assessment, a 4-week oral study was conducted on these compounds. Male Sprague-Dawley rats (288 +/- 20 g) were divided into groups of seven animals each, and were administered by gavage low (2 mg/kg body weight), medium (20 mg/kg body weight), or high (200 mg/kg body weight) doses of PE, DEB, or DMH, respectively, 5 days/week for 4 weeks. Animals in the control group received the vehicle (corn oil, 1 ml/100 g body weight) only. At the end of the exposure period, relative testis and thymus weights were reduced by 30 and 46%, respectively, in animals treated with the high dose of DMH. Significant reductions in serum lactate dehydrogenase (LDH), serum uric acid, and blood platelet counts were also observed in the high dose of DMH. Serum corticosterone was significantly depressed in the high doses of PE and DEB and in the low dose of DMH. Serum thiobarbituric acid-reactive substances (TBARS) were decreased (p < 0.05) in all DMH treatment groups and in the medium and high dose PE and DEB groups, while liver TBARS were unaffected by treatment. In the liver, increased glutathione (GSH) level and glutathione-S-transferases activity were detected in the high dose DMH group. Urinary ascorbic acid levels were markedly increased in animals receiving the high doses of PE, DEB, and DMH. Urinary formic acid was increased by 13 times in the high dose PE and DEB groups. Testes of all animals receiving the high dose of DMH showed a moderate to marked degree of degeneration of the seminiferous tubules, including a mild degree of vacuolation. At the same time, the epididymis of these animals had substantially reduced sperm density with prominent presence of spermatid giant cells. Mild histological changes were seen in the liver at all dose levels for all three chemicals. Thyroid effects were also observed in the high dose PE and DEB groups and in the medium and high dose DMH groups. It was concluded that DMH is the most toxic of the three ethers tested, with testicular, epidiymal, and thymic effects being the most prominent at 200 mg/kg. Other significant changes included depressed platelet counts and serum biochemical changes. Increased production of formic acid, an ocular toxin, from PE and DEB treatments may also be of toxicological concern.

A comparison of the effects of prolonged (>10 hour) low-flow sevoflurane, high-flow sevoflurane, and low-flow isoflurane anaesthesia on hepatorenal function in orthopaedic patients.

This study compared the effects of low-flow sevoflurane, high-flow sevoflurane and low-flow isoflurane on hepatorenal function during and after more than 10 hours of anaesthesia. Twenty-five patients scheduled for elective orthopaedic surgery were categorized into three groups; low-flow sevoflurane (fresh gas flow at 1 litre/min, n = 9), high-flow sevoflurane (5 l/rmin, n = 7), or low-flow isoflurane (1 l/min, n=9). Inspiratory compoundA concentrations were measured. The groups had similar duration of anaesthesia and exposure to anaesthetic agents. The area under the curve of concentration (mean, SD) of compound A in the low-flow sevoflurane group (359.8, 106.1 ppm.h) was greater than that in the high-flow sevoflurane group (61.1, 29.3 ppm.h; P<0.01). All groups showed normal plasma creatinine and creatinine clearance, and transient postoperative increases in plasma alanine aminotransferase and alpha glutathione-S-transferase, as well as urinary glucose and alpha glutathione-S-transferase, with no significant differences between groups. There were no significant relationships between the area under the curve of concentration of compound A and the biomarkers. These findings suggest that prolonged anaesthesia with low-flow sevoflurane has similar effects on hepatorenal function to prolonged anaesthesia with high-flow sevoflurane and low-flow isoflurane.

[Twenty-eight day repeated dose oral toxicity test of synergist of a pyrethroid insecticide, 2,3,3,3,2',3',3',3'-Octachlorodipropyl ether (S-421) in rats].

2,3,3,3,2',3',3',3'-Octachlorodipropyl ether (Abbreviation; S-421) is originally developed as synergist of a pyrethroid insecticide. In recent years, S-421 is used widely at home, for a mosquito-repellent incense, electric mosquito-repellent, an insect-killing spray, a vacuum cleaner paper pack, etc. as well. On the other hand, S-421 has been detected in vacuum cleaner dust samples as well as human milk samples in Japan indicating that our living environment is already contaminated by this compound. Long term toxicity studies including a carcinogenesis study have been performed and NOEL of chronic toxicity has been settled. However, it is clear that S-421 is used in close proximity so that acute or subacute exposure at relatively higher dose levels than chronic NOEL values are easily assumed, such as use of a spray in an ill-ventilated room, etc. This study, 28 day repeated oral dose toxicity study of S-421 was performed to monitor the outcome of acute and subacute exposure assuming possible exposure accidents mentioned above. The protocol is as follows; Groups of 10 rats of each sex(5 week-old), were treated with intragastric administration of S-421 with a dose of 0 (olive oil, control), 10, 40, 160 or 640 mg/kg body weight. For recovery test, 14 day after the last treatment, the control and 640 mg/kg groups were examined, respectively. All animals of all groups in both sexes survived. In the 640 mg/kg groups of the both sexes, all animals were set to drowsiness from about 5 hours after administration, however, they recovered by the next morning. In the hematology examination, Hb, MCH, MCHC, WBC values were significantly decreased and MCV value was significant increased in the 640 mg/kg group of both sexes. In the serum biochemistry, items increased in the 640 mg/kg groups of both sexes returned to normal level after 14 days recovery period. Absolute and relative liver weight increase seen in the 160 mg/kg and above also returned to control level after recovery. Histopathologically, slight hepatocellular swelling was observed in the 160 mg/kg groups and severe hepatocellular swelling with vacuolization and slight necrosis was seen in the 640 mg/kg group. In conclusion, the no-observed-effect levels (NOEL) of S-421 under these conditions was judged to be 40 mg/kg/day.

Human cytochrome P450 isozymes in metabolism and health effects of gasoline ethers.

To reduce the production of carbon monoxide and other pollutants in motor vehicle exhaust, methyl tert-butyl ether (MTBE*), ethyl tert-butyl ether (ETBE), and tert-amyl methyl ether (TAME) are added to gasoline as oxygenates for more complete combustion. Among them, MTBE is the most widely used. The possible adverse effect of MTBE in humans is a public concern, but the human enzymes responsible for metabolism of these gasoline ethers and the causes or factors for increased sensitivity to MTBE in certain individuals are totally unknown. This information is important to understanding the health effects of MTBE in humans and to assessing the human relevance of pharmacokinetics and toxicity data obtained from animals. In the present study, we demonstrated that human liver is active in metabolizing MTBE to tert-butyl alcohol (TBA), a major circulating metabolite and an exposure marker of MTBE. The activity is localized in the microsomal fraction but not in the cytosol. Formation of TBA in human liver microsomes is NADPH-dependent and is significantly inhibited by carbon monoxide, which inhibits cytochrome P450 (CYP) enzymes. These results provide strong evidence that CYP enzymes play a critical role in the metabolism of MTBE in human livers. Human liver is also active in the oxidative metabolism of 2 other gasoline ethers, ETBE and TAME. We observed a large interindividual variation in metabolizing these gasoline ethers in 15 microsomal samples prepared from normal human livers. The activity level (pmol metabolite/min/mg) ranged from 204 to 2,890 for MTBE; 179 to 3,134 for ETBE; and 271 to 8,532 for TAME. The microsomal activities in metabolizing MTBE, ETBE, and TAME correlated highly with each other (r = 0.91 to 0.96), suggesting that these ethers are metabolized by the same enzyme(s). Correlation analysis of the ether-metabolizing activities with individual CYP enzyme activities in the human liver microsomes showed that the highest degree of correlation was with CYP isoform 2A6 (CYP2A6)+ (r = 0.94 for MTBE, 0.95 for ETBE, and 0.90 for TAME), which is constitutively expressed in human livers and known to be polymorphic. CYP2A6 displayed the highest turnover number in metabolizing gasoline ethers among a battery of human CYP enzymes expressed in human B-lymphoblastoid cells. CYP2A6 coexpressed with human CYP reductase by a baculovirus expression system was also more active than CYP isoform 2E1 (CYP2E1) in the metabolism of MTBE, ETBE, and TAME. Kinetic studies on MTBE metabolism with human liver microsomes (n = 3) exhibited an apparent Michaelis constant (Km) of 28 to 89 microM and a maximum rate of metabolism (Vmax) of 215 to 783 pmol/min/mg. Metabolism of MTBE, ETBE, and TAME by human liver microsomes was inhibited by coumarin, a known substrate of human CYP2A6, in a concentration-dependent manner. Monoclonal antibody against human CYP2A6 caused a significant inhibition (75% to 95%) of the metabolism of MTBE, ETBE, and TAME in human liver microsomes. Taken together, these results clearly indicate that, in human liver, CYP2A6 is a major enzyme responsible for metabolism of MTBE, ETBE, and TAME. Although CYP2E1 metabolizes diethyl ether and was previously suggested to be involved

Renal toxicity with sevoflurane: a storm in a teacup?

The inhaled anaesthetic sevoflurane is metabolised into two products that have the potential to produce renal injury. Fluoride ions are produced by oxidative defluorination of sevoflurane by the cytochrome P450 system in the liver. Until recently, inorganic fluoride has been thought to be the aetiological agent responsible for fluorinated anaesthetic nephrotoxicity, with a toxic concentration threshold of 50 micromol/L in serum. However, studies of sevoflurane administration in animals and humans have not shown evidence of fluoride-induced nephrotoxicity, despite serum fluoride concentrations in this range. Compound A (fluoromethyl-2,2-difluoro-1-[trifluoromethyl] vinyl ether) is a breakdown product of sevoflurane produced by its interaction with carbon dioxide absorbents in the anaesthesia machine. The patient then inhales compound A. Compound A produces evidence of transient renal injury in rats. The mechanism of compound A renal toxicity is controversial, with the debate focused on the role of the renal cysteine conjugate beta-lyase pathway in the biotransformation of compound A. The significance of this debate centres on the fact that the beta-lyase pathway is 10- to 30-fold less active in humans than in rats. Therefore, if biotransformation by this pathway is responsible for the production of nephrotoxic metabolites of compound A, humans may be less susceptible to compound A renal toxicity than are rats. In three studies in human volunteers and one in surgical patients, prolonged (8-hour) sevoflurane exposures and low fresh gas flow rates resulted in significant exposures to compound A. Transient abnormalities were found in biochemical markers of renal injury measured in urine. These studies suggested that sevoflurane can result in renal toxicity, mediated by compound A, under specific circumstances. However, other studies using prolonged sevoflurane administration at low flow rates did not find evidence of renal injury. Finally, there are substantial data to document the safety of sevoflurane administered for shorter durations or at higher fresh gas flow rates. Therefore, the United States Food and Drug Administration recommends the use of sevoflurane with fresh gas flow rates at least 1 L/min for exposures up to 1 hour and at least 2 L/min for exposures greater than 1 hour. We believe this is a rational, cautious approach based on available data. However, it is important to note that other countries have not recommended such limitations on the clinical use of sevoflurane and problems have not been noted.

Contraceptive progestins. Various 11-substituents combined with four 17-substituents: 17alpha-ethynyl, five- and six-membered spiromethylene ethers or six-membered spiromethylene lactones.

Norethisterone (NET) is a 19-nortestosterone derivative with progestagenic and some androgenic activity, which was used in the first generation of contraceptives. NET was succeeded by levonorgestrel (LNG) and later on by desogestrel (DSG) and gestodene (GSD). Although these latter two progestins had increased potency, there was still androgenicity with gestodene and to a lesser extent with desogestrel. New progestins were synthesized in order to further enhance progestagenic and to reduce androgenic activity. Four different chemical moieties were introduced in position 17 of 19-nortestosterone, viz. 17alpha-ethynyl, five- and six-membered spiromethylene ethers, and a six-membered-spiromethylene lactone. In combination with these structures seven different substituents were added at position 11, i.e. methylene, methyl, ethyl, ethenyl, ethynyl, 2-propenyl and 1-propynyl. All substituents except for methylene occupied the 11beta-position. All these 32 compounds were synthesized and analysed in vitro and in vivo against etonogestrel (ETG, 3-keto-desogestrel), the biologically active metabolite of desogestrel. Their relative binding potency to progesterone (PR), androgen (AR) and estrogen (ER) receptors were determined in cell lysates of human breast tumor MCF-7 cells and to glucocorticoid (GR) receptors in that of human leukemic IM-9 cells. Moreover, their relative agonistic activities were assessed in Chinese hamster ovary cell-based transactivation assays. All in vivo activities were determined in McPhail (progestagenic), ovulation inhibition (progestagenic and estrogenic), Hershberger (androgenic), hormone screening (glucocorticoid and estrogen) and Allen-Doisy (estrogenic) tests after oral and for the McPhail test also after subcutaneous administration. The progestagenic binding and transactivation potencies of all compounds in the three 17-spiro series were higher than those of the corresponding analogues in the 17alpha-ethynyl series. None of the compounds showed estrogenic or clear androgenic binding and transactivation potential except for a six-membered-spiromethylene lactone with a propynyl group. This compound showed strong androgenic binding. The glucocorticoid binding and transactivation were very low for the compounds with the 17alpha-ethynyl and the five-membered-spiromethylene ether groups, whereas both six-membered-spiro series showed, clearly with methyl and ethynyl substituents, and less pronounced with methylene and ethenyl, higher binding and transactivation values. For the 17alpha-ethynyl series, the McPhail test showed high potencies with methylene, methyl and ethenyl substituents after oral treatment or with propenyl after subcutaneous administration. The introduction of the spiro substituents in position 17 led to high potencies for other 11-substituents as well. Besides methyl, also ethyl, ethynyl and propynyl were potent substituents. With ovulation inhibition tests, the ethyl, ethenyl and ethynyl substituents were the more potent compounds in all four series. However, compounds with methyl or ethynyl additions appeared to be glucocorticoidal in the hormone screening test irrespective of the 17-substituent, while with the three spiro series even methylene and ethenyl groups became active. Androgenicity was only observed at dose levels at or above 5 mg/kg, which is 2.5-fold weaker than ETG. Moreover, estrogenicity appeared negligible with the three spiro series, while with the 17alpha-ethynyl series methyl, ethyl, ethenyl and ethynyl substituents, a very high estrogenic potential was assessed. Based on the high efficacy and low side-effects, the following compounds show a high selectivity: 17alpha-ethynyl with ethyl, ethenyl and 2-propenyl substituents, six-membered spiromethylene ether with ethyl and six-membered-spiromethylene lactone with ethyl, 2-propenyl or 1-propynyl substituents. (ABSTRACT TRUNCATED)

Prevention of DNA photodamage by vitamin E compounds and sunscreens: roles of ultraviolet absorbance and cellular uptake.

Topical application of alpha-tocopherol (alphaTH), the most prominent naturally occurring form of vitamin E, inhibits ultraviolet (UV) B-induced photocarcinogenesis and DNA photodamage in C3H mice in vivo. In this study, we compared alphaTH with other vitamin E compounds and with three commercial sunscreen compounds for their ability to inhibit DNA photodamage in C3H mouse skin in vivo. When applied in a 5% dispersion in a neutral cream vehicle, alpha-tocopherol (alphaTH), gamma-tocopherol (gammaTH), and delta-tocopherol (deltaTH) each produced a statistically significant inhibition of thymine dimer formation, whereas alpha-tocopherol acetate (alphaTAc) and alpha-tocopherol methyl ether (alphaTOMe) did not. Application of 5% dispersions of the commercial sunscreen agent octylmethoxycinnamate also inhibited dimer formation, whereas ethylhexyl salicylate and oxybenzone did not, despite their considerably greater UVB absorbances than alphaTH. To test the hypothesis that cellular uptake and distribution are necessary for optimal photoprotection by tocopherols, photoprotection was studied in mouse 308 keratinocyte cells in vitro. Preincubation of 308 cells with 1 microM alphaTH for at least 2 h before exposure to 2.5 J/m2/s UVB for 10 min significantly (P < 0.05) attenuated thymine dimer formation. Pre-incubation with 1 microM gammaTH, deltaTH, alphaTAc, or alphaTOMe for 2 h did not inhibit thymine dimer formation significantly. Uptake of alphaTH was measured after incubation with 1 microM [2H3]alphaTH (d3-alphaTH) and resulted in a time-dependent increase in alphaTH levels. Use of d3-alphaTH allowed separate, simultaneous measurement of added d3-alphaTH and unlabeled endogenous alphaTH by gas chromatography-mass spectrometry. Accumulation of 167 +/- 62 pmol d3-alphaTH/mg protein was measured within 1 h in whole-cell fractions. d3-AlphaTH in the nuclear fraction reached levels of 15 +/- 4 pmol d3-alphaTH/mg protein at 2 h. Accumulation of alphaTH in the whole cell and nuclei corresponded temporally with significant protection against DNA photodamage. The kinetics of accumulation of the three tocopherols in whole cells and in nuclei were similar. Although only alphaTH conferred significant protection compared with irradiated controls at 2 h, the differences between individual tocopherols were not statistically significant. This work suggests that incorporation of tocopherol compounds into sunscreen products confers protection against procarcinogenic DNA photodamage and that cellular uptake and distribution of tocopherol compounds is necessary for their optimal photoprotection.

Acute toxicity of ethylene glycol mono-n-butyl ether in the guinea pig.

Acute toxicity values, such as oral and percutaneous LD50s, are often used as the basis for classifying chemicals into toxicity categories, and their subsequent regulation. Such values obtained for ethylene glycol mono-n-butyl ether (EGBE; 2-butoxyethanol) in rats and rabbits indicate that it is moderately toxic. However, the cause of death in these acute studies appeared to be secondary to acute intravascular haemolysis, an effect for which guinea pigs and humans are much less sensitive than rats, mice and rabbits. Recently-conducted acute toxicity studies in the guinea pig resulted in an acute oral LD50 of 1400 mg/kg, an acute percutaneous LD50 of greater than 2000 mg/kg, and a 1-hr LC50 greater than 633 ppm. These data are compared with published acute toxicity values, and indicate that the predicted acute toxicity of EGBE in humans, based on data from the guinea pig, would be less than that observed in other animal species. Based in part on the guinea pig data, EBGE is no longer classified as a poisonous substance by either the United Nations or US Department of Transportation.

[Low flow anesthesia using a fresh gas flow of 600 ml.min-1 for 5 hours].

Low flow anesthesia (LFA) using a fresh gas flow (FGF) of 600 ml.min-1 with oxygen and nitrous oxide flow each set at 300 ml.min-1, and dial setting of sevoflurane 3% was administered to 30 patients for a duration of 5 hours. There were no problems such as unsuitable concentrations of nitrous oxide and sevoflurane in inspired and expired gases or low FIO2 below 0.3 during anesthesia in 15 patients of group A. Their body weight was 53 +/- 5 kg. FIO2 decreased below 0.29 at about 4 hours in 7 patients of group B weighing 62 +/- 6 kg, and at about 1 h in 8 patients of group C weighing 71 +/- 7 kg. In group A and B, the sum of concentrations of oxygen, nitrous oxide and sevoflurane in inspired gas decreased for a moment and recovered as anesthesia progressed, but in group C, it kept decreasing without recovery. The body weight was significantly different among the 3 groups (P < 0.05). It was suggested that in group A the FGF per body weight was suitable; in group B though oxygen flow was larger than oxygen consumption, hypoxia occurred due to saturation of nitrous oxide in the body; and in group C the FGF was insufficient. The compound A was detected in the breathing circuit, and the concentration was around 20 ppm and it did not depend on the duration of LFA. It was concluded in this study that LFA using the FGF of 600 ml.min-1 with setting of 3% sevoflurane, 50% oxygen and nitrous oxide, could be performed safely without risks such as hypoxia and severe delay of induction for patients weighing 53 +/- 5 kg for a duration of 5 hours.

Effects of low-flow sevoflurane anesthesia on renal function: comparison with high-flow sevoflurane anesthesia and low-flow isoflurane anesthesia.

BACKGROUND: The safety of low-flow sevoflurane anesthesia, during which CF2=C(CF3)-O-CH2F (compound A) is formed by sevoflurane degradation, in humans has been questioned because compound A is nephrotoxic in rats. Several reports have evaluated renal function after closed-circuit or low-flow sevoflurane anesthesia, using blood urea nitrogen (BUN) and serum creatinine as markers. However, these are not the more sensitive tests for detecting renal damage. This study assessed the effects of low-flow sevoflurane anesthesia on renal function using not only BUN and serum creatinine but also creatinine clearance and urinary excretion of kidney-specific enzymes, and it compared these values with those obtained in high-flow sevoflurane anesthesia and low-flow isoflurane anesthesia. METHODS: Forty-eight patients with gastric cancer undergoing gastrectomy were studied. Patients were randomized to receive sevoflurane anesthesia with fresh gas flow of 1 l/min (low-flow sevoflurane group; n = 16) or 6-10 l/min (high-flow sevoflurane group; n = 16) or isoflurane anesthesia with a fresh gas flow of 1 l/min (low-flow isoflurane group; n = 16). In all groups, the carrier gas was oxygen/nitrous oxide in the ratio adjusted to ensure a fractional concentration of oxygen in inspired gas (FiO2) of more than 0.3. Fresh Baralyme was used in the low-flow sevoflurane and low-flow isoflurane groups. Glass balls were used instead in the high-flow sevoflurane group, with the fresh gas flow rate adjusted to eliminate rebreathing. The compound A concentration was measured by gas chromatography. Gas samples taken from the inspiratory limb of the circle system at 1-h intervals were analyzed. Blood samples were obtained before and on days 1, 2, and 3 after anesthesia to measure BUN and serum creatinine. Twenty-four-hour urine samples were collected before anesthesia and for each 24-h period from 0 to 72 h after anesthesia to measure creatinine, N-acetyl-beta-D-glucosaminidase, and alanine aminopeptidase. RESULTS: The average inspired concentration of compound A was 20 +/- 7.8 ppm (mean +/- SD), and the average duration of exposure to this concentration was 6.11 +/- 1.77 h in the low-flow sevoflurane group. Postanesthesia BUN and serum creatinine concentrations decreased, creatinine clearance increased, and urinary N-acetyl-beta-D-glucosaminidase and alanine aminopeptidase excretion increased in all groups compared with preanesthesia values, but there were no significant differences between the low-flow sevoflurane, high-flow sevoflurane, and low-flow isoflurane groups for any renal function parameter at any time after anesthesia. CONCLUSIONS: The only difference between the low-flow and high-flow sevoflurane groups was compound A formation, and postanesthesia laboratory data showed no significant effects of compound A formation during sevoflurane anesthesia on renal function. No significant effects on renal function were observed in either the low-flow or high-flow sevoflurane groups compared with the low-flow isoflurane group.

Role of adenosine in the ethanol-induced potentiation of the effects of general anesthetics in rats.

Acetate, derived from ethanol metabolism in the liver, is released into the circulation and utilized in many tissues including the brain. The subsequent metabolism of acetate results in the production of adenosine that has a number of effects in the central nervous system. The purpose of the present studies, therefore, was to investigate the contribution of metabolically generated adenosine to the ethanol-induced potentiation of the inhalational agents isoflurane and sevoflurane. Changes in the anesthetic requirement for isoflurane and sevoflurane were determined in rats using the tail-clamp procedure. Both ethanol and sodium acetate reduced anesthetic requirement for isoflurane and sevoflurane in a dose-dependent fashion. The effect of acetate on anesthetic requirement was completely blocked by the administration of the adenosine receptor blocker, 8-phenyltheophylline. The ethanol-induced reduction in anesthetic requirement, however, was only partially blocked by 8-phenyltheophylline. Direct intracerebroventricular (i.c.v.) administration of the water-soluble adenosine receptor blocker, 8-sulfophenyltheophylline, also completely blocked the effect of acetate and partially blocked the effect of ethanol. This i.c.v. administration demonstrates that the actions of ethanol and acetate on anesthetic requirement are a central nervous system effect. The i.c.v. administration of the adenosine A1 receptor subtype agonist, R-phenylisopropyl adenosine, potentiated the anesthetic effects of isoflurane and suggests that the A receptor mediates the observed potentiation of anesthetic effect. This is further supported by the concomitant administration of 5-N-ethylcarboxamido adenosine, a non-selective adenosine agonist, with the selective A1 antagonist, 8-cyclopentyltheophylline, showing A1 receptor potentiation of anesthetic requirements. The studies show that (1) acetate potentiates the anesthetic effects of the inhalational anesthetics, sevoflurane and isoflurane; (2) acetate contributes in part to the effect of ethanol on anesthetic potency through metabolically generated adenosine; (3) these effects are likely mediated via adenosine A1 receptor subtypes.