Sirtuin 3 regulates mouse pancreatic beta cell function and is suppressed in pancreatic islets isolated from human type 2 diabetic patients.

AIMS/HYPOTHESIS: Sirtuin (SIRT)3 is a mitochondrial protein deacetylase that regulates reactive oxygen species (ROS) production and exerts anti-inflammatory effects. As chronic inflammation and mitochondrial dysfunction are key factors mediating pancreatic beta cell impairment in type 2 diabetes, we investigated the role of SIRT3 in the maintenance of beta cell function and mass in type 2 diabetes. METHODS: We analysed changes in SIRT3 expression in experimental models of type 2 diabetes and in human islets isolated from type 2 diabetic patients. We also determined the effects of SIRT3 knockdown on beta cell function and mass in INS1 cells. RESULTS: SIRT3 expression was markedly decreased in islets isolated from type 2 diabetes patients, as well as in mouse islets or INS1 cells incubated with IL1ß and TNFα. SIRT3 knockdown in INS1 cells resulted in lowered insulin secretion, increased beta cell apoptosis and reduced expression of key beta cell genes. SIRT3 knockdown also blocked the protective effects of nicotinamide mononucleotide on pro-inflammatory cytokines in beta cells. The deleterious effects of SIRT3 knockdown were mediated by increased levels of cellular ROS and IL1ß. CONCLUSIONS/INTERPRETATION: Decreased beta cell SIRT3 levels could be a key step in the onset of beta cell dysfunction, occurring via abnormal elevation of ROS levels and amplification of beta cell IL1ß synthesis. Strategies to increase the activity or levels of SIRT3 could generate attractive therapies for type 2 diabetes.

The multidrug transporter ABCG2 (BCRP) is inhibited by plant-derived cannabinoids.

BACKGROUND AND PURPOSE: Cannabinoids are used therapeutically for the palliation of the adverse side effects associated with cancer chemotherapy. However, cannabinoids also inhibit both the activity and expression of the multidrug transporter, P-glycoprotein in vitro. Here we address the interaction of cannabinol (CBN), cannabidiol (CBD) and delta 9-tetrahydrocannabinol (THC) with the related multidrug transporter, ABCG2. EXPERIMENTAL APPROACH: Cannabinoid inhibition of Abcg2/ABCG2 was assessed using flow cytometric analysis of substrate accumulation and ATPase activity assays. The cytotoxicity and chemosensitization by cannabinoids was determined with cell viability assays. Expression of cannabinoid and vanilloid receptors was assessed using reverse transcriptase polymerase chain reaction, and cannabinoid modulation of ABCG2 expression was examined using immunoblotting. KEY RESULTS: CBN, CBD and THC increased the intracellular accumulation of the Abcg2/ABCG2 substrate, mitoxantrone, in an over-expressing cell line. The THC metabolite, (-)-11-nor-9-carboxy-delta 9-THC was much less potent. The plant cannabinoids inhibited both basal and substrate stimulated ATPase activity of human ABCG2. Cannabinoid cytotoxicity occurred in the absence of known cannabinoid cell surface receptors, and only at concentrations higher than those required for Abcg2/ABCG2 inhibition. Sub-toxic concentrations of the cannabinoids resensitized the overexpressing cell line to the cytotoxic effect of Abcg2/ABCG2 substrates, mitoxantrone and topotecan. This occurred in the absence of any effect on ABCG2 expression. CONCLUSIONS AND IMPLICATIONS: Cannabinoids are novel Abcg2/ABCG2 inhibitors, reversing the Abcg2-mediated multidrug-resistant phenotype in vitro. This finding may have implications for the co-administration of cannabinoids with pharmaceuticals that are ABCG2 substrates.

The effects of cannabinoids on P-glycoprotein transport and expression in multidrug resistant cells.

Cannabis is the most widely used illicit drug in the world. Cannabinoids are used therapeutically by some patients as they have analgesic, anti-emetic and appetite stimulant properties which palliate adverse symptoms. Use of these agents in an oncology setting raises the question of whether they act to modulate the effectiveness of concurrently administered anti-cancer drugs. The transporter, P-glycoprotein (P-gp) confers multiple drug resistance (MDR) by effluxing a diverse array of anti-cancer agents. This study was undertaken to examine the effect of cannabinoids on P-gp. Unlike the known P-gp inhibitor, PSC833, short 1h exposure to three plant-derived cannabinoids, cannabinol (CBN), cannabidiol (CBD) and Delta(9)-tetrahydrocannabinol (THC) and the synthetic cannabinoid receptor agonist, WIN55, 212-2 (WIN) did not inhibit the efflux of the P-gp substrate Rhodamine 123 (Rh123) in either a drug-selected human T lymphoblastoid leukaemia cell line (CEM/VLB(100)) or in a mouse fibroblast MDR1 transfected cell line (77.1). However, in CEM/VLB(100) cells, prolonged 72 h exposure to the cannabinoids, THC and CBD, decreased P-gp expression to a similar extent as the flavonoid, curcumin (turmeric). This correlated with an increase in intracellular accumulation of Rh123 and enhanced sensitivity of the cells to the cytotoxic actions of the P-gp substrate, vinblastine. Taken together, these results provide preliminary evidence that cannabinoids do not exacerbate P-gp mediated MDR. Further, plant-derived cannabinoids are moderately effective in reversing MDR in CEM/VLB(100) cells by decreasing P-gp expression.

Social-emotional characteristics of preschool-aged children referred for Child Find screening and assessment: a comparative study.

Differences in parent ratings of social-emotional behavior among young children referred for Child Find screening and assessment and nonreferred children were examined. Participants included 64 preschool-aged children referred for Child Find screening and assessment (CF group) and 64 preschool-aged children without such referrals or identified disabilities (Comparison group). The Comparison group was matched to the CF group by gender and age, using a randomized block procedure. Social-emotional behavior of the participants was assessed using parent ratings on the Preschool and Kindergarten Behavior Scale (PKBS), a social skills and problem-behavior rating scale for use with children aged 3-6. PKBS scores were found to classify the participants into their respective groups with a substantial degree of accuracy. Significant differences were found between the two groups in social skills and problem behavior scores, with the CF participants evidencing greater social skills deficits and problem behavior excesses than the participants in the Comparison group. An inspection of frequency distributions of the two groups revealed that children referred for Child Find screening were approximately four times as likely to have significant social deficits, and approximately six times as likely to have significant problem-behavior excesses than their nonreferred comparison peers. New validity evidence for the PKBS is provided, along with recommendations for future research and clinical practice with the Child Find population.

Social-emotional behavior of preschool-age children with and without developmental delays.

Differences in parent and teacher ratings of social-emotional behavior among young children with developmental delays and those without significant developmental problems were examined. Participants included 198 preschool-age children identified as having a developmental delay (DD group) and 198 preschool-age children without significant developmental problems (Comparison group) who were matched to the DD group by age and gender, using a randomized block procedure. Parent and teacher perceptions of social-emotional behavior of the participants were assessed using the Preschool and Kindergarten Behavior Scale (PKBS), a social skills and problem behavior rating scale for the use with young children. PKBS scores were found to classify the participants into their respective groups with a substantial degree of accuracy. Statistically significant differences in social skills and problem behavior scores between the two groups were found, with the DD participants evidencing greater social skills deficits and problem behavior excesses than the Comparison group. Individuals in the DD group were found to be four to five times more likely to have significant social skills deficits and problem behavior excesses than individuals in the Comparison group. The critical social-emotional behaviors separating the two groups appeared to be social interaction and independence skills, and socially withdrawn and isolated behavior patterns. New validity evidence for the PKBS is discussed, as are future needs pertaining to research and clinical practice in the area of social-emotional behavior of young children with developmental delays.

Effect of the oxytocin antagonist atosiban (1-deamino-2-D-tyr(OET)-4-thr-8-orn-vasotocin/oxytocin) on nocturanl myometrial contractions, maternal cardiovascular function, transplacental passage, and fetal oxygenation in the pregnant baboon during the last third of gestation.

The oxytocin antagonist, atosiban (1-deamino-2-D-tyr(OET)-4-thr-8-orn-vasotocin/oxytocin), was infused i.v. to chronically instrumented pregnant baboons in the last third of pregnancy. Atosiban (6 microg/kg per min) inhibited myometrial electromyographic activity associated with spontaneous myometrial contractions that occurred around the onset of darkness between 134 and 162 days gestation (term 180 days gestation). The effect of atosiban on maternal heart rate was minimal. Maternal blood pressure remained unaltered during atosiban infusion. Fetal carotid arterial PO2 was unchanged during a 2-h infusion of atosiban. Transplacental passage of atosiban from mother to fetus was assessed at cesarean section under halothane anesthesia in four baboons and in two chronically instrumented fetuses in the absence of anesthesia. The maternal:fetal concentration gradient ranged from 9.2 to 22.8. Maternal atosiban clearance rates were 9.2-16.9 ml/kg per min. In conclusion, atosiban was very effective at inhibiting spontaneously occurring nocturnal myometrial contractions during the last third of gestation in the pregnant baboon. Although atosiban crosses the placenta relatively freely, there was no effect on fetal oxygenation.

The pharmacokinetics of the oxytocin antagonist atosiban in pregnant women with preterm uterine contractions.

OBJECTIVE: Our purpose was to evaluate the pharmacokinetics of atosiban, an oxytocin antagonist, during and after intravenous infusion in pregnant patients having at least six contractions per hour. The relationship between atosiban infusion and uterine activity was also assessed. STUDY DESIGN: Plasma samples from eight pregnant patients treated with intravenous atosiban (300 micrograms/min for 6 to 12 hours) were analyzed for atosiban concentration by a specific radioimmunoassay procedure. Contraction rate data were obtained by external tocodynamometry for 1 hour before the infusion and during the subsequent infusion. RESULTS: The average steady-state plasma concentrations of patients receiving intravenous atosiban were 442 +/- 73 ng/ml (mean +/- SD), with steady state achieved by 1 hour after the start of the infusion. After the completion of the infusion, plasma concentrations declined rapidly in a biexponential manner with initial and terminal half-life estimates of 13 +/- 3 and 102 +/- 18 minutes, respectively. The effective half-life was 18 +/- 3 minutes. The plasma clearance of atosiban was relatively high (42 L/hr) and the volume of distribution (approximately 18 L) was consistent with distribution into extracellular fluid. Of the seven patients evaluated for uterine activity, the mean contraction rate decreased by 75% during the third hour of treatment and remained low until treatment termination. CONCLUSION: On the basis of earlier published reports, the pharmacokinetics of atosiban in pregnant patients are similar to those in nonpregnant women. Although the patient population was small, a consistent reduction in uterine activity was observed during atosiban infusion.

Placental passage of the oxytocin antagonist atosiban.

OBJECTIVE: We wanted to determine the degree of placental transfer of atosiban (Antocin), an oxytocin antagonist, in pregnant women at term. We also assessed the effects of the infusion on umbilical cord blood gases at birth and the maternal hematocrit drop after cesarean section. STUDY DESIGN: Eight women undergoing elective cesarean section at term were studied. Each received an infusion of 300 micrograms/min of atosiban over 208 to 443 minutes; the infusion was continued up to the time of cord clamping. Uterine vein and umbilical blood samples were obtained simultaneously. They were assayed by specific radioimmunoassay. Cord blood gases were obtained and compared with those from a control group of women undergoing elective cesarean section. RESULTS: The mean (+/- SD) maternal uterine vein concentration was 331.9 +/- 42.9 ng/ml, compared with 42 +/- 13 ng/ml in the umbilical vein (p < 0.05). The mean maternal/fetal was 12 +/- 0.03, which was not affected by the length of infusion. There was no significant difference in the hematocrit drop between the cesarean delivery groups: 5.9 +/- 0.4 for the control group versus 5.8 +/- 1.1 for the atosiban group (p > 0.1). The mean cord pH was 7.27 for the atosiban group versus 7.27 for the control group (n = 141) (p > 0.1). One year follow-up of the infants (n = 7) was normal. CONCLUSIONS: Our results show minimal placental transfer of atosiban. Drug levels did not increase with longer infusions, and no effect was seen on umbilical cord gases. Administration of atosiban even at high doses up to the time of delivery did not increase maternal blood loss at cesarean section.

Pharmacokinetics and bioavailability of topiramate in the beagle dog.

Male and female beagle dogs showed rapid absorption following oral administration of single oral gavage (40 mg/kg) and single or multiple (15 days) oral capsule (10, 40, and 150 mg/kg) doses of the novel anticonvulsant drug, topiramate, with the peak plasma concentration (Cmax) occurring between 0.6 and 3.8 hr. The absolute bioavailability of an oral dose of topiramate was estimated to be in the range of 27-59%, depending on the formulation. The mean topiramate Cmax values increased in a dose-proportional manner for both single (9.2-137.7 micrograms/ml) and multiple (10.3-145.2 micrograms/ml) oral capsule administrations, whereas the corresponding area under the plasma concentration vs. time curve (AUC) values increased in a dose-related but nonproportional manner for both single (51-1131 micrograms.hr/ml) and multiple (54-858 micrograms.hr/ml) doses. Over the 10-150 mg/kg dosing range, oral plasma clearance and terminal half-life values were found to be 2.4-3.6 ml/min/kg and 2.6-3.7 hr following a single oral administration, and 3.0-4.2 ml/min/kg and 2.0-3.8 hr after multiple doses. There were no significant differences between the pharmacokinetic parameters calculated following the first and fifteenth daily doses of topiramate at the 10 and 40 mg/kg levels, indicating that there was no accumulation and no autoinduction or inhibition of enzymes that metabolize topiramate resulting from multiple dosing at these levels. A slight (24%) decrease in AUC was observed at the 150 mg/kg level after the fifteenth daily dose.(ABSTRACT TRUNCATED AT 250 WORDS)

Maternal and fetal cardiovascular effects and placental transfer of the oxytocin antagonist atosiban in late-gestation pregnant sheep.

OBJECTIVES: Atosiban is a synthetic oxytocin antagonist that is currently undergoing dose-ranging clinical trials. To date, no data are available on the cardiovascular effects of combined oxytocin and vasopressin blockade during late pregnancy. Our aims were (1) to determine the effects of atosiban infusion on the maternal and fetal cardiovascular system and on uterine blood flow and (2) to determine the maternal pharmacokinetics and the rate of placental transfer of atosiban. STUDY DESIGN: Five chronically catheterized pregnant sheep were treated with a 2-hour infusion of atosiban (300 micrograms.min-1) at 116 to 126 days' gestation. Maternal and fetal blood pressure and heart rate and uterine blood flow were measured before and during the infusion. Maternal and fetal arterial blood samples were obtained throughout the experiment for measurement of plasma atosiban levels and blood gas values. RESULTS: No significant change in maternal cardiovascular parameters or uterine blood flow were observed. Similarly, no changes in fetal blood pressure and arterial blood gases were present during the infusion of the atosiban. Maternal plasma levels of atosiban reached a maximum of 585.2 +/- 82.2 (ng/ml mean +/- SD) at the end of the infusion and decreased biexponentially with a mean t1/2 alpha of 17 minutes and a mean t1/2 beta of 2.2 hours. Fetal plasma levels of atosiban were at or below the detection limit. CONCLUSION: Atosiban does not significantly affect maternal or fetal cardiovascular parameters when it is administered for 2 hours in late-pregnant sheep. Although significant levels were measured in maternal blood, negligible transfer to the fetus occurred.

High-performance liquid chromatographic assay with ultraviolet detection for the determination of etoperidone and two active metabolites, 5-(1-hydroxyethyl) etoperidone and 1-(3-chlorophenyl)piperazine, in plasma.

A selective and sensitive high-performance liquid chromatographic assay with ultraviolet detection for the determination of the antidepressant drug etoperidone and two active metabolites in plasma is described. The drug, metabolites and internal standard are isolated from plasma using a two-step liquid-liquid extraction procedure. The resulting sample is chromatographed on a C18 column (10 cm x 2.1 mm I.D.) with ultraviolet detection at 254 nm. Standard curves are linear for each compound over the concentration range 2-1000 ng/ml. The accuracy and precision of the assay, expressed as the percentage deviation of measured values from the true value and the relative standard deviation (inter-run), are less than or equal to 10% at all concentrations except the minimum quantification limit. Using an automated injector and computerized data acquisition, eighty samples can be routinely processed in one day. The assay has been successfully used for the analysis of plasma samples from pharmacokinetic studies in mice, rats, dogs and humans.

Urinary profiles of organic acids and volatile metabolites during the starvation process in rats.

Capillary gas chromatographic procedures were used to quantify the volatile and acidic compound profiles in the urinary samples of Sprague-Dawley rats during the starvation and refeeding periods. Numerous metabolites, identified through mass spectrometry, showed significant variations due to these physiological processes. Correlations are attempted with the previously studied biochemical processes in diabetic animals.

Automated capillary gas chromatographic assay using nitrogen-phosphorus ionization detection for the determination of bepridil in human plasma.

A highly sensitive and specific capillary gas chromatographic method for the determination of the antianginal drug bepridil in plasma is described. The capillary gas chromatograph and nitrogen-selective detector combination provides excellent sensitivity for clinical samples. The lowest concentration of bepridil which can be measured accurately and precisely in a 1-ml plasma sample is 1 ng/ml. Standard curves are linear over the concentration range 1-60 ng/ml. Accuracy and precision of the assay, expressed as relative deviation from the true value and relative standard deviation (inter-run) are less than 15% at all concentrations in the linear range. No interfering peaks are observed. Using an automatic injector and a laboratory computer system, sixty samples can be analyzed routinely in one day. The present assay has been successfully cross-validated with a published high-performance liquid chromatographic assay.

Chromatographic profiling of urinary volatile and organic acid metabolites of normal and diabetic C57BL/Ks mice.

Capillary gas chromatography and gas chromatography-mass spectrometry were used to examine the urinary volatile and organic acid metabolites in normal and diabetic C57BL/Ks male mice. Quantitative differences in the excretion of these metabolites were assessed in the animals from 5 to 24 weeks of age. Statistically significant differences were examined with respect to known metabolic abnormalities in the diabetic animals and to the possible toxic effects of elevated levels of certain metabolites. A number of aldehyde metabolites and aromatic acids, as well as most other organic acids, were found at consistently higher levels in diabetic urine. Several ketone metabolites, linalool, and 2-sec.-butylthiazoline were found at consistently low levels throughout the study.

Excretion of urinary volatile metabolites in response to alloxan induced diabetes of short duration in rats.

Alterations in urine volatile metabolites due to the induction of alloxan diabetes in the rat were examined by capillary gas chromatography and gas chromatography--mass spectrometry for the five days immediately following the onset of chronic hyperglycemia. Elevations of a number of metabolites were observed including several short chain ketones, acetophenone, 2-acetylfuran and indole. The value of urine volatile metabolic profiles as characteristic indicators of the diabetic condition is demonstrated through profiles obtained from a diabetic animal which spontaneously reverted to normal.