Yan, an ETS-domain transcription factor, negatively modulates the Wingless pathway in the Drosophila eye.

We report the identification of yan, an ETS-domain transcription factor belonging to the Drosophila epidermal growth factor receptor (DER) pathway, as an antagonist of the Wingless signalling pathway. We demonstrate that cells lacking yan function in the Drosophila eye show increased Wingless pathway activity, and inhibition of Wingless signalling in yan(-/-) cells rescues the yan mutant phenotype. Biochemical analysis shows that Yan physically associates with Armadillo, a crucial effector of the Wingless pathway, thereby suggesting a direct regulatory mechanism. We conclude that yan represents a new and unsuspected molecular link between the Wingless and DER pathways.

Pharmacokinetics and pharmacodynamics of alfentanil in P-glycoprotein-competent and P-glycoprotein-deficient mice: P-glycoprotein efflux alters alfentanil brain disposition and antinociception.

Previous studies have indicated that P-glycoprotein (P-gp) attenuates the central nervous system penetration and central activity of some opioids. The impact of P-gp-mediated efflux on the disposition and efficacy of the synthetic opioid alfentanil currently is unknown. In this study, P-gp-competent [mdr1a(+/+)] and P-gp-deficient [mdr1a(-/-)] mice were used to investigate the impact of P-gp-mediated efflux on the systemic pharmacokinetics, brain disposition, and central activity of alfentanil. Equipotent doses of alfentanil were administered to mdr1a(+/+) and mdr1a(-/-) mice (0.2 and 0.067 mg/kg, respectively), and the time course of brain and serum concentrations as well as antinociception were determined. A pharmacokinetic-pharmacodynamic (PK-PD) model was fit to the data and used to assess the impact of P-gp on parameters associated with alfentanil disposition and action. The mdr1a(+/+) mice were less sensitive to alfentanil than mdr1a(-/-) mice, requiring a 3-fold higher dose to produce similar antinociception. PK-PD modeling revealed no differences in alfentanil systemic pharmacokinetics between P-gp expressers and nonexpressers. However, the steady-state brain-to-serum concentration ratio (K(p,brain,ss)) was approximately 3-fold lower in mdr1a(+/+) mice compared with mdr1a(-/-) mice (0.19 +/- 0.01 versus 0.54 +/- 0.04, respectively). Consistent with the approximately 3-fold lower K(p,brain,ss), the antinociception versus serum concentration relationship in mdr1a(+/+) mice was shifted approximately 3-fold rightward compared with mdr1a(-/-) mice. However, there was no difference in the antinociception versus brain concentration relationship, or in the brain tissue EC(50) (11 +/- 1.8 versus 9.2 +/- 1.7 ng/g), between mdr1a(+/+) and mdr1a(-/-) mice. These results indicate that alfentanil is an in vivo P-gp substrate and are consistent with the hypothesis that P-gp-mediated efflux attenuates antinociception by reducing alfentanil K(p,brain,ss).

Pharmacokinetics and pharmacodynamics of seven opioids in P-glycoprotein-competent mice: assessment of unbound brain EC50,u and correlation of in vitro, preclinical, and clinical data.

This study was conducted to assess the utility of unbound brain EC50 (EC50,u) as a measure of in vivo potency for centrally active drugs. Seven mu-opioid agonists (alfentanil, fentanyl, loperamide, methadone, meperidine, morphine, and sufentanil) were selected as model central nervous system drugs because they elicit a readily measurable central effect (antinociception) and their clinical pharmacokinetics/pharmacodynamics are well understood. Mice received an equipotent subcutaneous dose of one of the model opioids. The time course of antinociception and the serum and brain concentrations were determined. A pharmacokinetic/pharmacodynamic model was used to estimate relevant parameters. In vitro measures of opioid binding affinity (Ki) and functional activity [EC50 for agonist stimulated guanosine 5'-O-(3-[35S]thio)triphosphate binding] and relevant clinical parameters were obtained to construct in vitro-to-preclinical and preclinical-to-clinical correlations. The strongest in vitro-to-in vivo correlation was observed between Ki and unbound brain EC50,u (r2 approximately 0.8). A strong correlation between mouse serum and human plasma EC50 was observed (r2 = 0.949); the correlation was improved when corrected for protein binding (r2 = 0.995). Clinical equipotent i.v. dose was only moderately related to Ki. However, estimates of ED50 and EC50 (total serum, unbound serum, total brain, and unbound brain) were significant predictors of clinical equipotent i.v. dose; the best correlation was observed for brain EC50,u (r2 = 0.982). For each opioid, brain equilibration half-life in mice was almost identical to the plasma effect-site equilibration half-life measured clinically. These results indicate that the mouse is a good model for opioid human brain disposition and clinical pharmacology and that superior in vitro-to-preclinical and preclinical-to-clinical correlations can be achieved with relevant unbound concentrations.

In vivo activation of human pregnane X receptor tightens the blood-brain barrier to methadone through P-glycoprotein up-regulation.

The ATP-driven drug export pump, P-glycoprotein, is a primary gatekeeper of the blood-brain barrier and a major impediment to central nervous system (CNS) pharmacotherapy. Reducing P-glycoprotein activity dramatically increases penetration of many therapeutic drugs into the CNS. Previous studies in rat showed that brain capillary P-glycoprotein was transcriptionally up-regulated by the pregnane X receptor (PXR), a xenobiotic-activated nuclear receptor. Here we used a transgenic mouse expressing human PXR (hPXR) to determine the consequences of increased blood-brain barrier P-glycoprotein activity. P-glycoprotein expression and transport activity in brain capillaries from transgenic mice was significantly increased when capillaries were exposed to the hPXR ligands, rifampin and hyperforin, in vitro and when the mice were dosed with rifampin in vivo. Plasma rifampin levels in induced mice were comparable with literature values for patients. We also administered methadone, a CNS-acting, P-glycoprotein substrate, to control and rifampin-induced transgenic mice and measured the drug's antinociceptive effect. In rifampin-induced mice, the methadone effect was reduced by approximately 70%, even though plasma methadone levels were similar to those found in transgenic controls not exposed to rifampin. Thus, hPXR activation in vivo increased P-glycoprotein activity and tightened the blood-brain barrier to methadone, reducing the drug's CNS efficacy. This is the first demonstration of the ability of blood-brain barrier PXR to alter the efficacy of a CNS-acting drug.