In vitro studies with two human organic anion transporters: OAT2 and OAT7.

1. Penciclovir, ganciclovir, creatinine, para-aminohippuric acid (PAH), ketoprofen, estrone 3-O-sulfate (E3S), dehydroepiandrosterone 3-O-sulfate (DHEAS) and cyclic guanosine monophosphate (cGMP) were screened as substrates of human liver organic anion transporters OAT2 and OAT7. 2. For OAT7, high uptake ratios (versus mock transfected HEK293 cells) of 29.6 and 15.3 were obtained with E3S and DHEAS. Less robust uptake ratios (≤3.6) were evident with the other substrates. OAT2 (transcript variant 1, OAT2-tv1) presented high uptake ratios of 30, 13, ∼35, ∼25, 8.5 and 9 with cGMP, PAH, penciclovir, ganciclovir, creatinine and E3S, respectively. No uptake was observed with DHEAS. 3. Although not a substrate of either transporter, ketoprofen did inhibit transfected OAT2-tv1 (IC50 of 17, 22, 23, 24, 35 and 586 µM; creatinine, ganciclovir, penciclovir, cGMP, E3S and prostaglandin F2α, respectively) and penciclovir uptake (IC50 = 27 µM; >90% inhibition) by plated human hepatocytes (PHH). 4. It is concluded that penciclovir and ketoprofen may serve as useful tools for the assessment of OAT2 activity in PHH. However, measurement of OAT7 activity therein will prove more challenging, as high uptake rates are evident with E3S and DHEAS only and both sulfoconjugates are known to be substrates of organic anion transporting polypeptides.

Evaluation of Ketoconazole and Its Alternative Clinical CYP3A4/5 Inhibitors as Inhibitors of Drug Transporters: The In Vitro Effects of Ketoconazole, Ritonavir, Clarithromycin, and Itraconazole on 13 Clinically-Relevant Drug Transporters.

Ketoconazole is a potent CYP3A4/5 inhibitor and, until recently, recommended by the Food and Drug Administration (FDA) and the European Medicines Agency as a strong CYP3A4/5 inhibitor in clinical drug-drug interaction (DDI) studies. Ketoconazole sporadically causes liver injury or adrenal insufficiency. Because of this, the FDA and European Medicines Agency recommended suspension of ketoconazole use in DDI studies in 2013. The FDA specifically recommended use of clarithromycin or itraconazole as alternative strong CYP3A4/5 inhibitors in clinical DDI studies, but many investigators have also used ritonavir as an alternative. Although the effects of these clinical CYP3A4/5 inhibitors on other CYPs are largely established, reports on the effects on the broad range of drug transporter activities are sparse. In this study, the inhibitory effects of ketoconazole, clarithromycin, ritonavir, and itraconazole (and its CYP3A4-inhibitory metabolites, hydroxy-, keto-, and N-desalkyl itraconazole) toward 13 drug transporters (OATP1B1, OATP1B3, OAT1, OAT3, OCT1, OCT2, MATE1, MATE2-K, P-gp, BCRP, MRP2, MRP3, and BSEP) were systematically assessed in transporter-expressing HEK-293 cell lines or membrane vesicles. In vitro findings were translated into clinical context with the basic static model approaches outlined by the FDA in its 2012 draft guidance on DDIs. The results indicate that, like ketoconazole, the alternative clinical CYP3A4/5 inhibitors ritonavir, clarithromycin, and itraconazole each have unique transporter inhibition profiles. None of the alternatives to ketoconazole provided a clean inhibition profile toward the 13 drug transporters evaluated. The results provide guidance for the selection of clinical CYP3A4/5 inhibitors when transporters are potentially involved in a victim drug's pharmacokinetics.

Behavioral effects and mechanisms of migraine pathogenesis following estradiol exposure in a multibehavioral model of migraine in rat.

Migraine is one of the most common neurological disorders, leading to more than 1% of total disability reported and over 68 million visits to emergency rooms or physician's offices each year in the United States. Three times as many women as men have migraine, and while the mechanism behind this is not well understood, 17ß-estradiol (estradiol) has been implicated to play a role. Studies have demonstrated that exposure to estrogen can lead to activation of inflammatory pathways, changes in sodium gated channel activity, as well as enhanced vasodilation and allodynia. Estradiol receptors are found in trigeminal nociceptors, which are involved in signaling during a migraine attack. The purpose of this study was to investigate the role of estradiol in migraine pathogenesis utilizing a multibehavioral model of migraine in rat. Animals were surgically implanted with a cannula system to induce migraine and behavior was assessed following exposure to a proestrus level of estradiol for total locomotor activity, light and noise sensitivity, evoked grooming patterns, and enhanced acoustic startle response. Results demonstrated decreased locomotor activity, increased light and noise sensitivity, altered facial grooming indicative of allodynia and enhanced acoustic startle. Further examination of tissue samples revealed increased expression of genes associated with inflammation and vasodilation. Overall, this study demonstrates exacerbation of migraine-like behaviors following exposure to estradiol and helps further explain the underlying mechanisms behind sex differences found in this common neurological disorder.

Exposure to bisphenol A exacerbates migraine-like behaviors in a multibehavior model of rat migraine.

Migraine is a common and debilitating neurological disorder suffered worldwide. Women experience this condition 3 times more frequently than men, with estrogen strongly implicated to play a role. Bisphenol A (BPA), a highly prevalent xenoestrogen, is known to have estrogenic activity and may have an effect in migraine onset, intensity, and duration through estrogen receptor signaling. It was hypothesized that BPA exposure exacerbates migraine symptoms through estrogen signaling and downstream activation of nociception related pathways. Utilizing a multibehavior model of migraine in ovariectomized female rats, changes in locomotion, light and sound sensitivity, grooming, and acoustic startle were examined. Furthermore, changes in the expression of genes related to estrogen (ERα, GPR30), and nociception (extracellular signal regulated kinase, ERK, sodium gated channel, Nav1.8, and fatty acid amide hydrolase, FAAH) were studied following behavioral experiments. The following results were obtained: BPA treatment significantly exacerbated migraine-like behaviors in rats. Rats exposed to BPA demonstrated decreased locomotion, exacerbated light and sound aversion, altered grooming habits, and enhanced startle reflexes. Furthermore, BPA exposure increased mRNA expression of estrogen receptors, total ERK mRNA and ERK activation, as well as Nav1.8, and FAAH mRNA, indicative of altered estrogen signaling and altered nociception. These results show that BPA, an environmentally pervasive xenoestrogen, exacerbates migraine-like behavior in a rat model and alters expression of estrogen and nociception-related genes.

Catechol and aldehyde moieties of 3,4-dihydroxyphenylacetaldehyde contribute to tyrosine hydroxylase inhibition and neurotoxicity.

Parkinson's disease (PD) is a progressive neurodegenerative disorder which leads to the selective loss of dopaminergic neurons. This causes a decrease in the important neurotransmitter dopamine (DA), which is essential for coordinated movement. Previous studies have implicated the monoamine oxidase metabolite of DA, 3,4-dihydroxphenylacetaldehyde (DOPAL), in the pathogenesis of PD and have shown it to be a reactive intermediate capable of protein modification. DOPAL also has demonstrated the ability to cause mitochondrial dysfunction and lead to significant inhibition of the rate-limiting enzyme in DA synthesis, tyrosine hydroxylase (TH). The current study was undertaken to investigate four analogs of DOPAL, including a novel nitrile analog, to determine how the structure of DOPAL is related to its toxicity and inhibition of TH. Both mitochondrial function and inhibition of TH in cell lysate were investigated. Furthermore, a novel whole cell assay was designed to determine the consequence to enzyme action when DOPAL levels were elevated. The results presented here demonstrate that changes to DOPAL structure lead to a decrease in toxicity and inhibition of enzyme activity as compared to the parent compound. Furthermore, the production of superoxide anion but not hydrogen peroxide increased in the presence of elevated DOPAL. These results reveal the toxicity of DOPAL and demonstrate that both the catechol and aldehyde are required to potently inhibit TH activity.