Reexamination of tetradecenyl acetates in oak leaf roller sex pheromone and in plants.

The chemistry of the oak leaf roller sex pheromone is shown by means of microozonolysis and computerized gas chromatography-mass spectrometry to be dominated by an approximate 70 to 30 ratio of E- and Z-11-tetradecenyl acetates. Tetradecenyl acetates are undetectable in highly purified oak leaf, apple leaf, and corn extracts analyzed by gas chromatography-mass spectrometry. These results reflect negatively on previous reports and on the hypothesis that plant components might govern insect chemical communication systems.

Evidence for origin of insect sex pheromones: presence in food plants.

Compounds identified as sex attractant pheromones in a number of phytophagous insects were found in a variety of host plants. These agents vary in chemical composition in different plant species, which suggests that dietary factors may provide an evolutionary mechanism for diversification of certain insect species. A theoretical framework to explain this phenomenon is postulated on the basis of experiments with the oak leaf roller moth.

Posthypoxic treatment with felbamate is neuroprotective in a rat model of hypoxia-ischemia.

Felbamate, a novel dicarbamate anticonvulsant that blocks the glycine site of the N-methyl-D-aspartate receptor and protects the hippocampal slice from hypoxic damage, shows remarkably low toxicity in animals and in humans. Since most treatment of human cerebral ischemia will have to be delivered after the insult, we investigated the neuroprotective potency of post hoc felbamate in rat pups with bilateral carotid ligations exposed to an atmosphere of 6.5% O2 for 1 hour. Brain temperature was unaffected by surgery, hypoxia, or felbamate. Neuroprotection was greatest at 300 mg/kg, less effective at 200 and 400 mg/kg, and ineffective at 100 mg/kg. Post hoc felbamate (300 mg/kg) reduced the volume of infarction from 67% +/- 7% of neocortex in unmedicated rats to 32% +/- 8%, 51% +/- 12%, 38% +/- 19%, and 53% +/- 10% when given 0, 1, 2, and 4 hours after hypoxic exposure, respectively. By 6 hours, post hoc protection was no longer significant. Delayed neuronal necrosis in hippocampal granule cells was reduced from 156 +/- 33 neurons to 12 +/- 7 (0 hours, p < 0.01) and 37 +/- 17 (1 hour, p < 0.05). These effects were obtained at plasma concentrations (60 to 120 mg/ml) that have occasionally been reached without serious toxicity in human anticonvulsant trials. These data suggest that, in this animal model, felbamate given after a hypoxic-ischemic insult is effective in reducing cerebral infarction and extremely effective in preventing delayed neuronal necrosis, but that the window of opportunity for post hoc treatment is only 1 to 4 hours.

23,24,25-Trihydroxyvitamin D3, 24,25,26-trihydroxyvitamin D3, 24-keto-25-hydroxyvitamin D3, and 23-dehydro-25-hydroxyvitamin D3: new in vivo metabolites of vitamin D3.

Four new in vivo metabolites of vitamin D3 were isolated from the blood plasma of chicks given large doses of vitamin D3. The metabolites were isolated by methanol-chloroform extraction and a series of chromatographic procedures. By use of mass spectrometry, ultraviolet absorption spectrophotometry, and specific chemical reactions, the metabolites were identified as 23,24,25-trihydroxyvitamin D3, 24,25,26-trihydroxyvitamin D3, 24-keto-25-hydroxyvitamin D3 and 23-dehydro-25-hydroxyvitamin D3.

Simultaneous determination of felbamate and four metabolites in rat cerebrospinal fluid by high-performance liquid chromatography.

An isocratic liquid chromatographic method for direct sample injection has been developed for the quantitation of felbamate and four metabolites in rat cerebrospinal fluid. The method uses 0.050- or 0.025-ml aliquots of cerebrospinal fluid diluted with equal volumes of internal standard. Chromatography is performed on a 150 mm x 4.6 mm I.D. Spherisorb ODS2, 3-microns HPLC column eluted with a phosphate buffer-acetonitrile-methanol (820:120:60, v/v/v) mobile phase and ultraviolet absorbance detection at 210 nm. The linear quantitation ranges are: felbamate and the 2-hydroxy metabolite 0.195-200 micrograms/ml, the propionic acid metabolite 0.195-50.0 micrograms/ml, the p-hydroxy metabolite 0.781 to 50.0 micrograms/ml, and the monocarbamate metabolite 0.098-50.0 micrograms/ml.

Simultaneous determination of felbamate and three metabolites in rat and dog plasmas by high-performance liquid chromatography.

An isocratic liquid chromatographic method employing one extraction step and a 150 mm x 4.6 mm I.D. Spherisorb ODS2, 3-microns HPLC column using UV-absorbance detection at 210 nm has been developed for the quantitation of felbamate and three felbamate metabolites in 0.100-ml aliquots of rat and dog plasmas. The linear quantitation range in rat plasma is 0.195-200 micrograms/ml for felbamate; 1.563-200 micrograms/ml for the p-hydroxy metabolite; 0.391-200 micrograms/ml for the 2-hydroxy metabolite; and 0.098-200 micrograms/ml for the monocarbamate metabolite. The linear quantitation range in dog plasma is 0.195-200 micrograms/ml for felbamate; 0.781-200 micrograms/ml for the p-hydroxy metabolite; 0.195-200 micrograms/ml for the 2-hydroxy metabolite; and 0.098-200 micrograms/ml for the monocarbamate metabolite.

Pharmacokinetics of the new antiasthma and antiallergy drug, azelastine, in pediatric and adult beagle dogs.

The plasma concentrations and relative bioavailability of azelastine hydrochloride (AZ) and its desmethyl metabolite (DAZ) after a single and 10 once-a-day oral doses of 2.5 mg kg-1 AZ were determined in adult male and female and pediatric male and female beagle dogs. The pediatric and adult dogs were 4-6 weeks and 1-2 years of age, respectively. An analysis of variance (ANOVA) was performed on the bioavailability parameters among all groups and between the first and last doses. No statistically significant (p < 0.05) sex-related differences in the bioavailability parameters were observed. The peak concentration (Cmax) of AZ, especially after the first dose, was significantly higher in pediatric dogs than that in adult dogs, whereas following the multiple AZ administrations, the bioavailability parameters for the last dose were similar in the two age groups. The apparent volume of distribution (Vss) of AZ suggested that the drug was extensively distributed into tissue in adult as well as in pediatric dogs. The Vss was considerably smaller in pediatric dogs, which may explain the higher Cmax in this age group. The bioavailability of the metabolite in adult dogs after multiple administration of AZ was higher than that in pediatric dogs. Although some differences in the parameters among the groups are statistically significant, they do not appear to have any biological significance. Therefore, similar AZ dosages may be required in pediatric and in adult populations to achieve optimum therapeutic drug levels.

High performance liquid chromatographic determination of azelastine and desmethylazelastine in guinea pig plasma and lung tissue.

Analytical methods have been developed for the simultaneous quantitation of azelastine (AZ) and desmethylazelastine (DAZ) in guinea pig plasma and lung tissue. The methods require a 1.00 mL plasma sample and a minimum 0.100 g lung sample. Both methods employ liquid/liquid organic extraction and back-extraction into dilute acid. Quantitation is performed by high performance liquid chromatography on a 2 x 250 mm 5 microns Hypersil CPS column using fluorescence detection. The linear quantitative ranges for AZ.HCl and DAZ.HBr in plasma are 0.156-160 ng/mL and 0.313-160 ng/mL, respectively. The linear quantitative ranges for AZ.HCl and DAZ.HBr in lung tissue are 0.039-20 micrograms/g for both.

Determination of the anticonvulsant felbamate in beagle dog plasma by high-performance liquid chromatography.

An automated internal standard method for the determination of felbamate in 0.1 ml of plasma from pediatric and adult beagle dogs was developed. Plasma proteins are precipitated with acetonitrile and after centrifugation the supernatant is directly injected on a Spherisorb ODS2, 3 microns, 150 mm x 4.6 mm I.D. column with 25% acetonitrile in aqueous phosphate buffer, pH 6.50, as mobile phase with ultraviolet detection at 210 nm. The run time is 10 min, the linear range is 0.150-150 micrograms/ml felbamate, and the lower limit of quantitation is 0.150 microgram/ml.

Pharmacokinetics of felbamate in pediatric and adult beagle dogs.

The relative bioavailability and pharmacokinetics of felbamate (FBM) after a single oral dose and after 10 once-daily oral doses of 60 mg/kg were investigated in adult and pediatric dogs of both sexes. The pediatric and adult dogs were aged 4-6 weeks and 1-2 years, respectively. Analysis of variance (ANOVA) was performed on the bioavailability parameters among all groups and between the first and last doses. No sex-related differences in bioavailability and pharmacokinetic parameters were observed. The bioavailability of FBM in pediatric dogs was significantly less as compared with that in adult dogs. Rapid overall elimination of the drug in pediatric dogs appears to be responsible for the lower bioavailability. The bioavailability of FBM after the last dose was also significantly lower than after the first dose for both age groups. No major differences in the rate constant of FBM absorption (ka) and volume of distribution at steady state (VSS) were observed between the two age groups. As with other clinically useful antiepileptic drugs (AEDs), higher doses of FBM may be required in pediatric populations to achieve optimum drug levels, assuming that age-related changes in FBM disposition will also be confirmed in humans.

Distribution of the anticonvulsant felbamate to cerebrospinal fluid and brain tissue of adult and neonatal rats.

The concentrations of felbamate (FBM) and its three metabolites were determined in plasma, cerebrospinal fluid (CSF), and brain tissue of adult and neonatal rats that received single oral doses of FBM at amounts varying from 250 to 2000 mg/kg for adults and from 100 to 500 mg/kg for neonates. The increase in plasma Cmax and AUC0-24 with the dose was less than proportional in both age groups. The highest plasma Cmax in adults dosed with 2000 mg/kg was 140.3 micrograms/ml; in neonates dosed with 500 mg/kg it was 257.0 micrograms/ml. The maximum brain concentrations for these dosages were 90.9 and 220.4 micrograms/g, respectively. The average brain/plasma, CSF/plasma, and brain/CSF partition coefficients for the drug were 0.64, 0.55, and 1.16 for adults and 0.83, 0.67, and 1.23 for neonates, respectively. No statistically significant change of the partition coefficients with time or dose was observed (p < 0.05). Very good linear correlations between FBM plasma concentrations and concentrations in CSF and brain tissue were obtained (r2 > 0.98). Only the 2-hydroxy metabolite was present in considerable amounts in plasma and brain tissue of the high-dose groups of both ages.

Felbamate protects CA1 neurons from apoptosis in a gerbil model of global ischemia.

BACKGROUND AND PURPOSE: Felbamate, a novel anticonvulsant that binds to the glycine site of the N-methyl-D-aspartate receptor, has been shown to have neuroprotective properties in vitro and in vivo. In a rat pup model of hypoxia-ischemia, felbamate selectively reduced delayed death in hippocampal granule cells. The present study explores its neuroprotective potential in a gerbil model of global ischemia, in which good evidence exists that ischemia triggers apoptosis of CA1. METHODS: Gerbils were subjected to bilateral carotid occlusion for 5 minutes and then treated with felbamate (100 or 200 mg/kg IV) or vehicle. They were killed 3 days later, and the numbers of live and dead neurons in the CA1 sector of the hippocampus were counted at sterotaxically defined levels. RESULTS: Felbamate (200 mg/kg IV) administered after the release of carotid clamping did not change brain temperature but reduced neuronal death in CA1 from 332 +/- 60 cells per section of dorsal hippocampus in unmedicated gerbils to 62 +/- 12 cells in felbamate-treated animals (P<.001). A lower dose of felbamate (100 mg/kg post hoc) showed only a nonsignificant reduction of neuronal death. In the 200-mg/kg group, felbamate serum concentrations peaked at 162 microg/mL and were above 100 microg/mL for at least 3 hours, and brain levels reached 150 microg/mL at 1 hour. In the 100-mg/kg group, blood serum levels were well below 100 microg/mL. CONCLUSIONS: These results suggest that felbamate given post hoc is remarkably effective in preventing delayed apoptosis secondary to global ischemia but that effective neuroprotection requires doses higher than those used for anticonvulsant treatment.

Metabolism and binding properties of 24,24-difluoro-25-hydroxyvitamin D3.

To evaluate possible functional roles for 24,25-dihydroxyvitamin D3, 24,24-difluoro-25-hydroxyvitamin D3 has been synthesized and shown to be equally as active as 25-hydroxyvitamin D3 in all known functions of vitamin D. The use of the difluoro compound for this purpose is based on the assumption that the C-F bonds are stable in vivo and that the fluorine atom does not act as hydroxyl in biological systems. No 24,25-dihydroxyvitamin D3 was detected in the serum obtained from vitamin D-deficient rats that had been given 24,24-difluoro-25-hydroxyvitamin D3, while large amounts were found when 25-hydroxyvitamin D3 was given. Incubation of the 24,24-difluoro compound with kidney homogenate prepared from vitamin D-replete chickens failed to produce 24,25-dihydroxyvitamin D3, while the same preparations produced large amounts of 24,25-dihydroxyvitamin D3 from 25-hydroxyvitamin D3. Kidney homogenate prepared from vitamin D-deficient chickens produced 24,24-difluoro-1,25-dihydroxyvitamin D3 from 24,24-difluoro-25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 from 25-hydroxyvitamin D3. In binding to the plasma transport protein for vitamin D compounds, 24,24-difluoro-25-hydroxyvitamin D3 is less active than 25-hydroxyvitamin D3 and 24R,25-dihydroxyvitamin D3. In binding to the chick intestinal cytosol receptor, 24,24-difluoro-25-hydroxyvitamin D3 is more active than 25-hydroxyvitamin D3 which is itself more active than 24R,25-dihydroxyvitamin D3. The 24,24-difluoro-1,25-dihydroxyvitamin D3 is equal to 1,25-dihydroxyvitamin D3, and both are 10 times more active than 1,24R,25-trihydroxyvitamin D3 in this system. These results provide strong evidence that the C-24 carbon of 24,24-difluoro-25-hydroxyvitamin D3 cannot be hydroxylated in vivo, and, further, the 24-F substitution acts similar to H and not to OH in discriminating binding systems for vitamin D compounds.

Role of kidney tissue in the production of 25-hydroxyvitamin D3-26,23-lactone and 1 alpha, 25-dihydroxyvitamin D3-26,23-lactone.

Labeled 25-hydroxyvitamin D3-26,23-lactone was isolated from the serum of vitamin D-repleted rats given [3 alpha-3H]-25-hydroxyvitamin D324 hr prior to sacrifice. The metabolite was identified by cochromatography with the authentic lactone on straight-phase and reversed-phase high-performance liquid chromatography. Production of the lactone was abolished by nephrectomy indicating that the kidney is the site of synthesis. Homogenate of kidneys from chickens given large doses of vitamin D can carry out in vitro production of the lactone from 25-hydroxyvitamin D3. When 1 alpha, 25-dihydroxyvitamin D3 was used as substrate, this system produced only traces of a compound believed to be 1 alpha, 25-dihydroxyvitamin D3-26,23-lactone. However, incubation of rachitic chicken kidney homogenates with 25-hydroxyvitamin D3-26,23-lactone produced substantial amounts of a compound that has been identified by mass spectrometry as 1 alpha, 25-dihydroxyvitamin D3-26,23-lactone. Thus, the development of a functional group on C-26 and eventual lactone formation takes place in kidney by a system acting on 25-hydroxyvitamin D3.

Isolation and identification of 23,25-dihydroxyvitamin D3, an in vivo metabolite of vitamin D3.

Vitamin D supplemented rats produce a metabolite of 25-hydroxy[3 alpha-3H]vitamin D3 that is easily separated from known metabolites by using high-performance liquid chromatography. The production of this metabolite in vivo as well as 1,25-dihydroxyvitamin D3, 24(R),25-dihydroxyvitamin D3, and 25-hydroxyvitamin D3 26,23-lactone is largely if not totally eliminated by nephrectomy. Kidney homogenates from vitamin D supplemented chickens incubated with 25-hydroxyvitamin D3 produce significant quantities of the new, unknown metabolite. This metabolite was isolated in pure form from such incubation mixtures by using both straight-phase and reversed-phase high-performance liquid chromatography. This metabolite has been positively identified as 23,25-dihydroxyvitamin D3 by ultraviolet absorption spectrophotometry, mass spectrometry, and derivatization. This structure was confirmed by chemical synthesis of both C-23 stereoisomers. Although the natural product exactly comigrates with one of the synthetic isomers, the exact stereochemistry of the natural product remains unknown. It is possible that this new metabolite is an intermediate in the biosynthesis of 25-hydroxyvitamin D3 26,23-lactone.

Simultaneous determination of felbamate and three metabolites in human plasma by high-performance liquid chromatography.

An isocratic liquid-chromatographic method employing one extraction step and a 4.6 mm x 150 mm Spherisorb ODS2, 3 microns high-performance liquid chromatography (HPLC) column using ultraviolet (UV)-absorbance detection at 210 nm has been developed for quantitation of felbamate (FBM) and three felbamate metabolites in 0.100-ml aliquots of human plasma. The linear quantitation range for FBM and the two hydroxy metabolites is 0.781-200 micrograms/ml, and that for the monocarbamate metabolite is 0.391-200 micrograms/ml.

Determination of valproate in the presence of felbamate in human plasma by narrow bore capillary gas chromatography.

A narrow bore capillary gas chromatographic method with one extraction step has been developed for quantitation of valproate (VPA) in the presence of felbamate (FBM) in human plasma. The method uses 0.250-ml aliquots of human plasma and one internal standard (IS). Chromatographic conditions include a DBWAX, 0.25 mm ID x 15 m, 0.25-micron film thickness column; splitless injection; and flame ionization detection. The linear quantitation range for VPA is 1.00-256 micrograms/ml.

25-Hydroxyvitamin D3 26,23-lactone: a new in vivo metabolite of vitamin D.

A major vitamin D metabolite was isolated in pure form from the blood plasma of chicks either maintenance levels or large doses of vitamin D3. The isolation involved methanol-chloroform extraction and five column chromatographic procedures. The metabolite purification and elution position on these columns were followed by a competitive protein binding assay. The metabolite was identified, using high- and low-resolution mass spectrometry, 270-MHz proton nuclear magnetic resonance spectrometry, ultraviolet absorption spectrophotometry, Fourier transform infrared spectrophotometry, and specific chemical reactions, as 3 beta,-25-dihydroxy-9,10-seco-5,7,10(19)-cholestatrieno-26,23-lactone. The trivial names 25-hydroxyvitamin D3 26,23-lactone or calcidiol 26,23-lactone are suggested for this compound.

1,25-Dihydroxyvitamin D3 stimulated increase of 7,8-didehydrocholesterol levels in rat skin.

A convenient, accurate assay was developed for determining skin cholesta-5,7-dien-3 beta-ol (7,8-didehydrocholesterol) concentrations. Ultraviolet spectrophotometry provided quantitation of the sterol from rat skins following saponification and chromatography on Lipidex and high-performance liquid chromatography. Correction for recoveries was accomplished by using 7,8-didehydro[3 alpha-3H]cholesterol as an internal standard. Chronic dosing of vitamin D-deficient rats with 1,25-dihydroxyvitamin D3 caused a 4-fold increase in skin 7-dehydrocholesterol content. This rise was not the result of changes in food consumption, body weight, or plasma calcium. Cholesterol concentrations were not significantly elevated although some of the other nonsaponifiable lipid components found in the high-performance liquid chromatogram appeared to be increased by the treatment. These results suggest that the vitamin D hormone 1,25-(OH)2D3 may exert a positive feedback regulation on the production of vitamin D3 in skin.

Metabolism of 25-hydroxyvitamin D3 by rat kidney cells in culture: isolation and identification of cis- and trans-19-nor-10-oxo-25-hydroxyvitamin D3.

A primary confluent culture of epithelial cells from rat kidney has been developed. These cells possess a 3.2-3.4 S high-affinity, low-capacity binding protein for 1,25-dihydroxyvitamin D3. They metabolize 25-hydroxyvitamin D3 to at least five metabolites. Two have been identified as 1,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3. Two others have been identified by means of physical data and cochromatography as trans 19-nor-10-oxo-25-hydroxyvitamin D3 and the other as its cis isomer. These two "metabolites" have not been observed in vivo, but one of them (cis) comigrates with 1,25-dihydroxyvitamin D3 on straight-phase high-performance liquid chromatography. Thus, mere cochromatography on high-performance liquid chromatography is not sufficient to identify critical vitamin D metabolites.