Adam Jeston Dudley

P-Glycoprotein Deficient Mouse in situ Blood-Brain Barrier Permeability and its Prediction using an in combo PAMPA Model

The purpose of the study was to assess the permeability of mouse blood-brain barrier (BBB) to a diverse set of compounds in the absence of P-glycoprotein (Pgp) mediated efflux, to predict it using an in combo PAMPA model, and to explore its role in brain penetration classification (BPC). The initial brain uptake (K(in)) of 19 compounds in both wild-type and Pgp mutant [mdr1a(-/-)] CF-1 mice was determined by the in situ brain perfusion technique. PAMPA measurements were performed, and the values were used to develop an in combo model, including Abraham descriptors. Published rodent K(in) values were used to enhance the dataset and validate the model. The model predicted 92% of the variance of the training set permeability. In all, 182 K(in) values were considered in this study, spanning four log orders of magnitude and where Pgp decreased brain uptake by as much as 14-fold. The calculated permeability-surface area (PS) values along with literature reported brain tissue binding were used to group molecules in terms of their brain penetration classification. The in situ BBB permeability can be predicted by the in combo PAMPA model to a satisfactory degree, and can be used as a lower-cost, high throughput first-pass screening method for BBB passive permeability.

Development and SAR of functionally selective allosteric modulators of GABAA receptors

Positive modulators at the benzodiazepine site of α2- and α3-containing GABA(A) receptors are believed to be anxiolytic. Through oocyte voltage clamp studies, we have discovered two series of compounds that are positive modulators at α2-/α3-containing GABA(A) receptors and that show no functional activity at α1-containing GABA(A) receptors. We report studies to improve this functional selectivity and ultimately deliver clinical candidates. The functional SAR of cinnolines and quinolines that are positive allosteric modulators of the α2- and α3-containing GABA(A) receptors, while simultaneously neutral antagonists at α1-containing GABA(A) receptors, is described. Such functionally selective modulators of GABA(A) receptors are expected to be useful in the treatment of anxiety and other psychiatric illnesses.

EEG-β/γ spectral power elevation in rat: a translatable biomarker elicited by GABAAα2/3-positive allosteric modulators at nonsedating anxiolytic doses

Benzodiazepine drugs, through interaction with GABA(Aα1), GABA(Aα2,3), and GABA(Aα5) subunits, modulate cortical network oscillations, as reflected by a complex signature in the EEG power spectrum. Recent drug discovery efforts have developed GABA(Aα2,3)-subunit-selective partial modulators in an effort to dissociate the side effect liabilities from the efficacy imparted by benzodiazepines. Here, we evaluated rat EEG and behavioral end points during dosing of nine chemically distinct compounds that we confirmed statistically to selectively to enhance GABA(Aα2,3)-mediated vs. GABA(Aα1) or GABA(Aα5) currents in voltage clamped oocytes transfected with those GABA(A) subunits. These compounds were shown with in vivo receptor occupancy techniques to competitively displace [(3)H]flumazenil in multiple brain regions following peripheral administration at increasing doses. Over the same dose range, the compounds all produced dose-dependent EEG spectral power increases in the β- and and γ-bands. Finally, the dose range that increased γ-power coincided with that eliciting punished over unpunished responding in a behavioral conflict model of anxiety, indicative of anxiolysis without sedation. EEG γ-band power increases showed a significant positive correlation to in vitro GABA(Aα2,3) modulatory intrinsic activity across the compound set, further supporting a hypothesis that this EEG signature was linked specifically to pharmacological modulation of GABA(Aα2,3) signaling. These findings encourage further evaluation of this EEG signature as a noninvasive clinical translational biomarker that could ultimately facilitate development of GABA(Aα2,3)-subtype-selective drugs for anxiety and potentially other indications.

P-glycoprotein Potentiates CYP3A4-mediated Drug Disappearance during Caco-2 Intestinal Secretory Detoxification

Human intestinal Caco-2 cell monolayers grown in the presence of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) were used to test the hypothesis that drugs which interact with the apical efflux pump P-glycoprotein (Pgp) may enhance CYP3A4-mediated disappearance of substrates. 6β-Hydroxytestosterone production, a marker of CYP3A4 activity, was approximately 3- and 7-fold greater in 1,25(OH)2D3-treated cells compared to untreated cells when incubated with 50 and 500 μM testosterone, respectively, and was unaffected by the addition of digoxin to reduce Pgp activity. In the presence of digoxin, secretory transport of vinblastine and erythromycin, substrates for both Pgp and cytochrome P450 3A4 (CYP3A4), was significantly reduced, whereas absorptive transport was unaffected. In contrast, no directional transport of testosterone, a substrate for CYP3A4 only, was observed, either in the presence or absence of digoxin. Over 2 h, disappearance of erythromycin and vinblastine from the incubation medium was significantly greater from the basolateral than from the apical compartments. In the presence of digoxin, disappearance of both compounds from the basolateral, but not from the apical compartments, was significantly reduced. In contrast, disappearance of testosterone was unaffected by the addition of digoxin, demonstrating that the effect of digoxin on erythromycin and vinblastine disappearance was via inhibition of Pgp function, rather than on CYP3A4 activity. Thus, evidence is provided for Pgp/CYP3A4 co-substrates, Pgp potentiates CYP3A4-mediated drug disappearance during intestinal secretory detoxification.