Synthetic derivatives of the antifungal drug ciclopirox are active against herpes simplex virus 2.

We previously showed that the anti-fungal drug ciclopirox olamine effectively inhibits replication of herpes simplex virus (HSV)-1 and HSV-2. Given the rise of HSV strains that are resistant to nucleos(t)ide analog treatment, as well as the incomplete efficacy of nucleos(t)ide analogs, new inhibitory compounds must be explored for potential use in the treatment of HSV infection. In the present study, we analyzed 44 compounds derived from the core structure of ciclopirox olamine for inhibitory activity against HSV. Thirteen of these derivative compounds inhibited HSV-2 replication by > 1000- to ∼100,000-fold at 1 µM and displayed EC50 values lower than that of acyclovir, as well as low cytotoxicity, indicating their strong therapeutic potential. Through structural comparison, we also provide evidence for the importance of various structural motifs to the efficacy of ciclopirox and its derivatives, namely hydrophobic groups at R4 and R6 of the ciclopirox core structure. Like ciclopirox, representative analogs exhibit some oral bioavailability but are rapidly cleared in vivo. Together, these results will guide further development of N-hydroxypyridones as HSV therapeutics.

Optimization of the Urea Linker of Triazolopyridazine MMV665917 Results in a New Anticryptosporidial Lead with Improved Potency and Predicted hERG Safety Margin.

Cryptosporidiosis is caused by infection of the small intestine by Cryptosporidium parasites, resulting in severe diarrhea, dehydration, malabsorption, and potentially death. The only FDA-approved therapeutic is only partially effective in young children and ineffective for immunocompromised patients. Triazolopyridazine MMV665917 is a previously reported anti-Cryptosporidium screening hit with in vivo efficacy but suffers from modest inhibition of the hERG ion channel, which could portend cardiotoxicity. Herein, we describe our initial development of structure-activity relationships of this novel lead series with a particular focus on optimization of the piperazine-urea linker. We have discovered that piperazine-acetamide is a superior linker resulting in identification of SLU-2633, which has an EC50 of 0.17 µM, an improved projected margin versus hERG, prolonged pharmacokinetic exposure in small intestine, and oral efficacy in vivo with minimal systemic exposure. SLU-2633 represents a significant advancement toward the identification of a new effective and safe treatment for cryptosporidiosis.

Sedentary Behavior and Body Weight and Composition in Adults: A Systematic Review and Meta-analysis of Prospective Studies.

BACKGROUND: The cumulative effect of too much sedentary behavior may contribute to weight gain and obesity. OBJECTIVE: The aim of this study was to conduct a systematic review and meta-analysis of prospective cohorts and randomized controlled studies to determine the association between sedentary behavior and body weight and obesity in adults. DATA SOURCES AND STUDY SELECTION: Relevant studies were identified from searches of the MEDLINE, Embase, AMED and PubMed databases up to May 2017, and by manual searches of in-text citations. Studies that evaluated the association in adults between sedentary behavior and body weight or obesity, while controlling for physical activity, were included. Overall, 31 publications met the eligibility criteria, including 23 prospective cohort studies with data that could be extracted for a quantitative meta-analysis, and a single randomized controlled trial. RESULTS: There were no significant associations between sedentary behavior and any measure of body weight or obesity, with the exception of waist circumference. For the latter outcome, over a 5-year follow-up period, each 1 h per day increase-from baseline to follow-up-in sedentary behavior was associated with a 0.02 mm [95% confidence interval (CI) 0.01-0.04; p = 0.001) increase in waist circumference. The odds ratio of becoming overweight or obese was 1.33 (95% CI 1.11-1.60; p = 0.001) in the highest compared with lowest categories of sedentary behavior. CONCLUSIONS: Meta-analysis of data from prospective cohort studies showed small, inconsistent and non-significant associations between sedentary behavior and body weight.

Influence of HIV antiretrovirals on methadone N-demethylation and transport.

Drug interactions involving methadone and/or HIV antiretrovirals can be problematic. Mechanisms whereby antiretrovirals induce clinical methadone clearance are poorly understood. Methadone is N-demethylated to 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) by CYP2B6 and CYP3A4 in vitro, but by CYP2B6 in vivo. This investigation evaluated human hepatocytes as a model for methadone induction, and tested the hypothesis that methadone and EDDP are substrates for human drug transporters. Human hepatocyte induction by several antiretrovirals of methadone N-demethylation, and CYP2B6 and CYP3A4 transcription, protein expression and catalytic activity, and pregnane X receptor (PXR) activation were evaluated. Methadone and EDDP uptake and efflux by overexpressed transporters were also determined. Methadone N-demethylation was generally not significantly increased by the antiretrovirals. CYP2B6 mRNA and activity (bupropion N-demethylation) were induced by several antiretrovirals, as were CYP3A4 mRNA and protein expression, but only indinavir increased CYP3A activity (alfentanil dealkylation). CYP upregulation appeared related to PXR activation. Methadone was not a substrate for uptake (OCT1, OCT2, OCT3, OATP1A2, OATP1B1, OATP1B3, OATP2B1) or efflux (P-gp, BCRP) transporters. EDDP was a good substrate for P-gp, BCRP, OCT1, OCT3, OATP1A2, and OATP1B1. OATP1A2- and OCT3-mediated EDDP uptake, and BCRP-mediated EDDP efflux transport, was inhibited by several antiretrovirals. Results show that hepatocyte methadone N-demethylation resembles expressed and liver microsomal metabolism more than clinical metabolism. Compared with clinical studies, hepatocytes underreport induction of methadone metabolism by HIV drugs. Hepatocytes are not a good predictive model for clinical antiretroviral induction of methadone metabolism and not a substitute for clinical studies. EDDP is a transporter substrate, and is susceptible to transporter-mediated interactions.

Significance of lipid composition in a blood-brain barrier-mimetic PAMPA assay.

Endothelial cells forming the blood-brain barrier limit drug access into the brain, due to tight junctions, membrane drug transporters, and unique lipid composition. Passive permeability, thought to mediate drug access, is typically tested using porcine whole-brain lipid. However, human endothelial cell lipid composition differs. This investigation evaluated the influence of lipid composition on passive permeability across artificial membranes. Permeability of CNS-active drugs across an immobilized lipid membrane was determined using three lipid models: crude extract from whole pig brain, human brain microvessel lipid, and microvessel lipid plus cholesterol. Lipids were immobilized on polyvinylidene difluoride, forming donor and receiver chambers, in which drug concentrations were measured after 2 h. The log of effective permeability was then calculated using the measured concentrations. Permeability of small, neutral compounds was unaffected by lipid composition. Several structurally diverse drugs were highly permeable in porcine whole-brain lipid but one to two orders of magnitude less permeable across human brain endothelial cell lipid. Inclusion of cholesterol had the greatest influence on bulky amphipathic compounds such as glucuronide conjugates. Lipid composition markedly influences passive permeability. This was most apparent for charged or bulky compounds. These results demonstrate the importance of using species-specific lipid models in passive permeability assays.

Mechanism of autoinduction of methadone N-demethylation in human hepatocytes.

BACKGROUND: There is considerable interindividual and intraindividual variability in methadone metabolism and clearance. Methadone dosing is particularly challenging during initiation of therapy, because of time-dependent increases in hepatic clearance (autoinduction). Although methadone N-demethylation is catalyzed in vitro by cytochrome P4502B6 (CYP2B6) and CYP3A4, and clearance in vivo depends on CYP2B6, mechanism(s) of autoinduction are incompletely understood. In this investigation, we determined mechanism(s) of methadone autoinduction using human hepatocytes. METHODS: Fresh human hepatocytes were exposed to 0.1 to 10 µM methadone for 72 hours. Cells were washed and methadone N-demethylation assessed. CYP2B6, CYP3A4, and CYP3A5 messenger RNA (mRNA), protein expression (by gel-free high-performance liquid chromatography mass spectrometry) and catalytic activity (bupropion hydroxylation and alfentanil dealkylation for CYP2B6 and CYP3A4/5, respectively) were measured. Mechanisms of CYP induction were characterized using pregnane X receptor and constitutive androstane receptor reporter gene assays. RESULTS: Methadone (10 µM) increased methadone N-demethylation 2-fold, CYP2B6 and CYP3A4 mRNA 3-fold, and protein expression 2-fold. CYP3A5 mRNA was unchanged. CYP2B6 and CYP3A4/5 activities increased 2-fold. Induction by methadone enantiomers (R-methadone versus S-methadone) did not differ. Induction was relatively weak compared with maximum induction by phenobarbital and rifampin. Lower methadone concentrations had smaller effects. Methadone was an agonist for the pregnane X receptor but not the constitutive androstane receptor. CONCLUSIONS: Methadone caused concentration-dependent autoinduction of methadone N-demethylation in human hepatocytes, related to induction of CYP2B6 and CYP3A4 mRNA expression, protein expression, and catalytic activity. Induction was related to pregnane X receptor but not constitutive androstane receptor activation. These in vitro findings provide mechanistic insights into clinical autoinduction of methadone metabolism and clearance.

P-glycoprotein is a major determinant of norbuprenorphine brain exposure and antinociception.

Norbuprenorphine is a major metabolite of buprenorphine and potent agonist of µ, δ, and κ opioid receptors. Compared with buprenorphine, norbuprenorphine causes minimal antinociception but greater respiratory depression. It is unknown whether the limited antinociception is caused by low efficacy or limited brain exposure. Norbuprenorphine is an in vitro substrate of the efflux transporter P-glycoprotein (Mdr1), but the role of P-glycoprotein in norbuprenorphine transport in vivo is unknown. This investigation tested the hypothesis that limited norbuprenorphine antinociception results from P-glycoprotein-mediated efflux and limited brain access. Human P-glycoprotein-mediated transport in vitro of buprenorphine, norbuprenorphine, and their respective glucuronide conjugates was assessed by using transfected cells. P-glycoprotein-mediated norbuprenorphine transport and consequences in vivo were assessed by using mdr1a(+/+) and mdr1a(-/-) mice. Antinociception was determined by hot-water tail-flick assay, and respiratory effects were determined by unrestrained whole-body plethysmography. Brain and plasma norbuprenorphine and norbuprenorphine-3-glucuronide were quantified by mass spectrometry. In vitro, the net P-glycoprotein-mediated efflux ratio for norbuprenorphine was nine, indicating significant efflux. In contrast, the efflux ratio for buprenorphine and the two glucuronide conjugates was unity, indicating absent transport. The norbuprenorphine brain/plasma concentration ratio was significantly greater in mdr1a(-/-) than mdr1a(+/+) mice. The magnitude and duration of norbuprenorphine antinociception were significantly increased in mdr1a(-/-) compared with mdr1a(+/+) mice, whereas the reduction in respiratory rate was similar. Results show that norbuprenorphine is an in vitro and in vivo substrate of P-glycoprotein. P-glycoprotein-mediated efflux influences brain access and antinociceptive, but not the respiratory, effects of norbuprenorphine.

Interaction of porphyrins with human organic anion transporting polypeptide 1B1.

The existence of a porphyrin uptake transporter in hepatocytes has been hypothesized in recent years, but to date it has not been identified. While the linear tetrapyrrole bilirubin has been shown to be a substrate for the organic anion transporting polypeptide 1B1 (OATP1B1), similar studies have not been conducted for the cyclic tetrapyrroles (porphyrins). The aim of this study was to determine the structural features of linear and cyclic tetrapyroles necessary for interaction with OATP1B1. The interaction was quantified using HEK cells stably expressing OATP1B1 and measuring the inhibition of OATP1B1-mediated uptake of estradiol 17beta-d-glucuronide in the presence or absence of various linear and cyclic tetrapyrroles. Ditaurine-conjugated bilirubin was the most potent inhibitor of uptake, with an IC50 of 5 nM, while the substitution of the taurine side chains with methyl ester eliminated the inhibition of estradiol 17beta-d-glucuronide uptake. Hematoporphyrin, a cyclic tetrapyrrole with carboxyalcohol side chains at positions C-3 and C-8 and carboxyethyl side chains at positions 13 and 17 had an IC50 of 60 nM, while porphyrins lacking charged side chains such as etioporphyrin I and phthalocyanine did not inhibit OATP1B1. Chlorin e6 and hematoporphyrin were shown to be competitive inhibitors of OATP1B1-mediated uptake of bromosulfophthalein with Kis of 5.8+/-0.3 and 1.6+/-0.3 microM, respectively. While these studies do not provide direct evidence, they do support the assumption that tetrapyrroles are transported by OATP1B1. Additionally, these findings offer a possible explanation for the clinical observation that patients suffering from certain porphyrietic diseases have a reduced ability to excrete organic anions.

Ibogaine, a noncompetitive inhibitor of serotonin transport, acts by stabilizing the cytoplasm-facing state of the transporter.

Ibogaine, a hallucinogenic alkaloid with purported anti-addiction properties, inhibited serotonin transporter (SERT) noncompetitively by decreasing V(max) with little change in the K(m) for serotonin (5-HT). Ibogaine also inhibited binding to SERT of the cocaine analog 2beta-2-carbomethoxy-3-(4-[(125)I]iodophenyl)tropane. However, inhibition of binding was competitive, increasing the apparent K(D) without much change in B(max). Ibogaine increased the reactivity of cysteine residues positioned in the proposed cytoplasmic permeation pathway of SERT but not at nearby positions out of that pathway. In contrast, cysteines placed at positions in the extracellular permeation pathway reacted at slower rates in the presence of ibogaine. These results are consistent with the proposal that ibogaine binds to and stabilizes the state of SERT from which 5-HT dissociates to the cytoplasm, in contrast with cocaine, which stabilizes the state that binds extracellular 5-HT.

Role of transporters in the disposition of the selective phosphodiesterase-4 inhibitor (+)-2-[4-({[2-(benzo[1,3]dioxol-5-yloxy)-pyridine-3-carbonyl]-amino}-methyl)-3-fluoro-phenoxy]-propionic acid in rat and human.

The role of transporters in the disposition of (+)-2-[4-({[2-(benzo[1,3]dioxol-5-yloxy)-pyridine-3-carbonyl]-amino}-methyl)-3-fluoro-phenoxy]-propionic acid (CP-671,305), an orally active inhibitor of phosphodiesterase-4, was examined. In bile duct-exteriorized rats, a 7.4-fold decrease in the half-life of CP-671,305 was observed, implicating enterohepatic recirculation. Statistically significant differences in CP-671,305 pharmacokinetics (clearance and area under the curve) were discernible in cyclosporin A- or rifampicin-pretreated rats. Considering that cyclosporin A and rifampicin inhibit multiple uptake/efflux transporters, the interactions of CP-671,305 with major human hepatic drug transporters, multidrug resistance protein 1 (MDR1), multidrug resistance-associated protein 2 (MRP2), breast cancer resistant protein (BCRP), and organic anion-transporting polypeptide (OATPs) were evaluated in vitro. CP-671,305 was identified as a substrate of MRP2 and BCRP, but not MDR1. CP-671,305 was a substrate of human OATP2B1 with a high affinity (Km = 4 microM) but not a substrate for human OATP1B1 or OATP1B3. Consistent with these results, examination of hepatobiliary transport of CP-671,305 in hepatocytes indicated active uptake followed by efflux into bile canaliculi. Upon examination as a substrate for major rat hepatic Oatps, CP-671,305 displayed high affinity (Km = 12 microM) for Oatp1a4. The role of rat Mrp2 in the biliary excretion was also examined in Mrp2-deficient rats. The observations that CP-671,305 pharmacokinetics were largely unaltered suggested that compromised biliary clearance of CP-671,305 was compensated by increased urinary clearance. Overall, these studies suggest that hepatic transporters play an important role in the disposition and clearance of CP-671,305 in rat and human, and as such, these studies should aid in the design of clinical drug-drug interaction studies.

Differential interaction of 3-hydroxy-3-methylglutaryl-coa reductase inhibitors with ABCB1, ABCC2, and OATP1B1.

The present study examined the interaction of four 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (atorvastatin, lovastatin, and simvastatin in acid and lactone forms, and pravastatin in acid form only) with multidrug resistance gene 1 (MDR1, ABCB1) P-glycoprotein, multidrug resistance-associated protein 2 (MRP2, ABCC2), and organic anion-transporting polypeptide 1B1 (OATP1B1, SLCO21A6). P-glycoprotein substrate assays were performed using Madin-Darby canine kidney (MDCK) cells expressing MDR1, and the efflux ratios [the ratio of the ratio of basolateral-to-apical apparent permeability and apical-to-basolateral permeability between MDR1 and MDCK] were 1.87, 2.32/4.46, 2.17/3.17, and 0.93/2.00 for pravastatin, atorvastatin (lactone/acid), lovastatin (lactone/acid), and simvastatin (lactone/acid), respectively, indicating that these compounds are weak or moderate substrates of P-glycoprotein. In the inhibition assays (MDR1, MRP2, Mrp2, and OATP1B1), the IC50 values for efflux transporters (MDR1, MRP2, and Mrp2) were >100 microM for all statins in acid form except lovastatin acid (>33 microM), and the IC50 values were up to 10-fold lower for the corresponding lactone forms. In contrast, the IC50 values for the uptake transporter OATP1B1 were 3- to 7-fold lower for statins in the acid form compared with the corresponding lactone form. These data demonstrate that lactone and acid forms of statins exhibit differential substrate and inhibitor activities toward efflux and uptake transporters. The interconversion between the lactone and acid forms of most statins exists in the body and will potentially influence drug-transporter interactions, and may ultimately contribute to the differences in pharmacokinetic profiles observed between statins.

Inhibition of human organic anion transporting polypeptide OATP 1B1 as a mechanism of drug-induced hyperbilirubinemia.

OATP1B1 (a.k.a. OATP-C, OATP2, LST-1, or SLC21A6) is a liver-specific organic anion uptake transporter and has been shown to be a higher affinity bilirubin uptake transporter than OATP1B3. Using human embryonic kidney (HEK 293) cells stably transfected with OATP1B1, we have studied the effects of indinavir, saquinavir, cyclosporin A, and rifamycin SV on human OATP1B1 transport function. These drugs are potent inhibitors of OATP1B1 transport activity in vitro. We further provide evidence that the calculated fraction of OATP1B1 inhibited at the clinical exposure level correlated very well with the observed hyperbilirubinemia outcome for these drugs in humans. Our data support the hypothesis that inhibition of OATP1B1 is an important mechanism for drug-induced unconjugated hyperbilirubinemia. Inhibition of OATPs may be an important mechanism in drug-drug and drug-endogenous substance interactions.