Inhibition of ABCG2/BCRP-mediated transport-correlation analysis of various expression systems and probe substrates.

BCRP / ABCG2 is a key determinant of pharmacokinetics of substrate drugs. Several BCRP substrates and inhibitors are of low passive permeability, and the vesicular transport assay works well in this permeability space. Membranes were prepared from BCRP-HEK293, MCF-7/MX, and baculovirus-infected Sf9 cells with (BCRP-Sf9-HAM), and without (BCRP-Sf9) cholesterol loading. Km values for three substrates - estrone-3-sulfate, sulfasalazine, topotecan - correlated well between the four expression systems. In contrast, a 10-20-fold range in Vmax values was observed, with BCRP-HEK293 membranes possessing the largest dynamic range. IC50 values of the different test systems were similar to each other, with 94.4% of pairwise comparisons being within 3-fold. Substrate dependent inhibition showed somewhat greater variation, as 81.4% of IC50 values in the BCRP-HEK293 membranes were within 3-fold in pairwise comparisons. Overall, BCRP-HEK293 membranes demonstrated the highest activity. The IC50 values showed good concordance but substrate dependent inhibition was observed for some drugs.

Membrane Assays to Characterize Interaction of Drugs with ABCB1.

ATP-binding cassette sub-family B member 1 (ABCB1) [P-glycoprotein (P-gp), multidrug resistance protein 1 (MDR1)] can affect the pharmacokinetics, safety, and efficacy of drugs making it important to identify compounds that interact with ABCB1. The ATPase assay and vesicular transport (VT) assay are membrane based assays that can be used to measure the interaction of compounds with ABCB1 at a lower cost and higher throughput compared to cellular-based assays and therefore can be used earlier in the drug development process. To that end, we tested compounds previously identified as ABCB1 substrates and inhibitors for interaction with ABCB1 using the ATPase and VT assays. All compounds tested interacted with ABCB1 in both the ATPase and VT assays. All compounds previously identified as ABCB1 substrates activated ABCB1-mediated ATPase activity in the ATPase assay. All compounds previously identified as ABCB1 inhibitors inhibited the ABCB1-mediated transport in the VT assay. Interestingly, six of the ten compounds previously identified as ABCB1 inhibitors activated the basal ATPase activity in activation assays suggesting that the compounds are substrates of ABCB1 but can inhibit ABCB1 in inhibition assays. Importantly, for ATPase activators the EC50 of activation correlated with the IC50 values from the VT assay showing that interactions of compounds with ABCB1 can be measured with similar levels of potency in either assay. For ATPase nonactivators the IC50 values from the ATPase inhibition and VT inhibition assay showed correlation. These results demonstrate the utility of membrane assays as tools to detect and rank order drug-transporter interactions.

The expressions of ABCC4 and ABCG2 xenobiotic transporters in human keratinocytes are proliferation-related.

Xenobiotic transporters of the ATP-binding cassette (ABC) protein superfamily play important roles in maintaining the biochemical barrier of various tissues, but their precise functions in the skin are not yet known. Screening of the expressions of the known xenobiotic transporter genes in two in vitro keratinocyte differentiation models revealed that the ABCC4 and ABCG2 transporters are highly expressed in proliferating keratinocytes, their expressions decreasing along with differentiation. Abrogation of the ABCC4 and ABCG2 protein functions by siRNA-mediated silencing and chemical inhibition did not affect the proliferation of HaCaT cells. In contrast, disruption of the ABCG2 function had no effect on normal human epidermal keratinocyte proliferation, while the inhibition of ABCC-type transporters by probenecid resulted in a striking decrease in the proliferation of the cells. These results indicate that, besides their possible therapy-modulating effects, xenobiotic transporters may contribute significantly to other keratinocyte functions, such as cell proliferation.

Estradiol-induced synaptic remodeling of tyrosine hydroxylase immunopositive neurons in the rat arcuate nucleus.

Gonadal steroids induce synaptic plasticity in several areas of the adult nervous system. In the arcuate nucleus of adult female rats, 17beta-estradiol triggers synaptic remodeling, resulting in a decrease in the number of inhibitory synaptic inputs, an increase in the number of excitatory synapses, and an enhancement of the frequency of neuronal firing. In the present paper, we studied the specificity of hormonal effects by determining the changes in synaptic connectivity of tyrosine hydroxylase (TH) immunoreactive (IR) neurons in the arcuate nucleus. We combined pre-embedding TH and post-embedding gamma-aminobutyric acid (GABA) immunostaining, and performed unbiased stereological measurements in gonadectomized and 17beta-estradiol-treated rats. We conclude that the synaptic connectivity of the TH-IR neurons is different from the other, nonlabeled population, and the response to estradiol is not uniform. TH-IR (dopaminergic) arcuate neurons of both male and female rats have more GABAergic (inhibitory) axosomatic inputs than the nondopaminergic population. Our study shows that the effect of 17beta-estradiol is sex and cell specific in the sense that not all arcuate neurons are affected by the structural synaptic remodeling. In ovariectomized females hormone treatment decreased the numerical density of GABAergic axosomatic synapses on TH-IR, but not on nondopaminergic, neurons, whereas in orchidectomized males, 17beta-estradiol treatment increased inhibitory synapses onto nondopaminergic neurons but did not affect the number of inhibitory terminals onto TH-IR neurons. The hormone-induced plastic changes in synaptic connectivity of TH-IR neurons may serve as the morphological basis for the cyclical regulation of the anterior pituitary.