Mutations in the leucine-rich repeat kinase-2 (LRRK2) gene cause autosomal-dominant Parkinson's disease (PD) and contribute to sporadic PD. LRRK2 contains Guanosine-5'-triphosphate (GTP) binding, GTPase and kinase activities that have been implicated in the neuronal degeneration of PD pathogenesis, making LRRK2, a potential drug target. To date, there is no disease-modifying drug to slow the neuronal degeneration of PD and no published LRRK2 GTP domain inhibitor. Here, the biological functions of two novel GTP-binding inhibitors of LRRK2 were examined in PD cell and mouse models. Through a combination of computer-aided drug design (CADD) and LRRK2 bio-functional screens, two novel compounds, 68: and 70: , were shown to reduce LRRK2 GTP binding and to inhibit LRRK2 kinase activity in vitro and in cultured cell assays. Moreover, these two compounds attenuated neuronal degeneration in human SH-SY5Y neuroblastoma cells and mouse primary neurons expressing mutant LRRK2 variants. Although both compounds inhibited LRRK2 kinase activity and reduced neuronal degeneration, solubility problems with 70: prevented further testing in mice. Thus, only 68: was tested in a LRRK2-based lipopolysaccharide (LPS)-induced pre-inflammatory mouse model. 68: reduced LRRK2 GTP-binding activity and kinase activity in brains of LRRK2 transgenic mice after intraperitoneal injection. Moreover, LPS induced LRRK2 upregulation and microglia activation in mouse brains. These findings suggest that disruption of GTP binding to LRRK2 represents a potential novel therapeutic approach for PD intervention and that these novel GTP-binding inhibitors provide both tools and lead compounds for future drug development.

Previous studies suggest that P-glycoprotein (P-gp) modulates the PK/PD of many compounds including opioid agonists and chemotherapeutic agents. The objective of this study was to assess the P-gp affinity status of oxycodone, the P-gp expression, and the paclitaxel's tissue distribution in oxycodone-treated rats. P-gp ATPase assay, Caco-2 transepithelial permeability studies, and mdr1a/b (−/−) mice were used to assess the P-gp affinity status of oxycodone. P-gp expression was determined by Western blot analysis while [ 14 C] paclitaxel's distributions in the liver, kidney, brain, and plasma tissues were determined by liquid scintillation counter. Oxycodone stimulated the P-gp ATPase activity in a concentration-dependant manner. The Caco-2 secretory transport of oxycodone was reduced from 3.64 ×10 −5 to 1.96 × 10 −5 cm/s (p <0.05) upon preincubation with the P-gp inhibitor, verapamil. The brain levels of oxycodone in mdr1a/b (+/+) were not detectable (<15 ng/mL) while in mdr1a/b (−/−) the average levels were 115 ± 39 ng/mL. The P-gp protein levels were increased by 1.3-4.0 folds while paclitaxel's tissue distributions were decreased by 38-90% (p <0.05) in oxycodone-treated rats. These findings display that oxycodone is a P-gp substrate, induces overexpression of P-gp, and affects paclitaxel's tissue distribution in a manner that may influence its chemotherapeutic activity.

Salvinorin A is an unregulated potent hallucinogen isolated from the leaves of Salvia divinorum. It is the only known non-nitrogenous kappa-opioid selective agonist and rivals synthetic lysergic acid diethylamide (LSD) in potency. This objective of this study was to characterize the in vitro transport, in vitro metabolism, and pharmacokinetic properties of Salvinorin A. The transport characteristics of Salvinorin A were assessed using MDCK-MDR1 cell monolayers. The P-glycoprotein (P-gp) affinity status was assessed by the P-gp ATPase assay. In vitro metabolism studies were performed with various specific human CYP450 isoforms and UGT2B7 to assess the metabolic characteristics of Salvinorin A. Cohorts (n=3) of male Sprague Dawley rats were used to evaluate the pharmacokinetics and brain distribution of Salvinorin A (10 mg/kg, intraperitonal (i.p.) over a 240 min period. A validated UV-HPLC and LC/MS/MS method was used to quantify the hallucinogen concentrations obtained from the in vitro and in vivo studies, respectively. Salvinorin A displayed a high secretory transport in the MDCK-MDR1 cells (4.07±1.34 × 10 -5 cm/s). Salvinorin A also stimulated the P-gp ATPase activity in a concentration (5-10 μm) dependent manner, suggesting that it may be a substrate of P-gp. A significant decrease in Salvinorin A concentration ranging from 14.7 ±0.80 % to 31.1±1.20 % was observed after incubation with CYP2D6, CYP1A1, CYP2C18, and CYP2E1, respectively. A significant decrease was also observed after incubation with UGT2B7. These results suggest that Salvinorin A may be a substrate of UGT2B7, CYP2D6, CYP1A1, CYP2E1 and CYP2C18. The in vivo pharmacokinetic study showed a relatively fast elimination with a halflife (t 1/2 ) of 75 min and a clearance (Cl/F) of 26 L/h/kg. The distribution was extensive (Vd of 47.1 L/kg), however the brain to plasma ratio was 0.050. Accordingly, the brain half life was relatively short, 36 min. Salvinorin A is rapidly eliminated after i.p. dosing, in accordance with its fast onset and short duration of action. Further, it appears to be a substrate for various oxidative enzymes and multi-drug resistant protein, P-gp.

Mounting evidence demonstrates that CYP2B6 plays a much larger role in human drug metabolism than was previously believed. The discovery of multiple important substrates of CYP2B6 as well as polymorphic differences has sparked increasing interest in the genetic and xenobiotic factors contributing to the expression and function of the enzyme. The expression of CYP2B6 is regulated primarily by the xenobiotic receptors constitutive androstane receptor (CAR) and pregnane X receptor (PXR) in the liver. In addition to CYP2B6, these receptors also mediate the inductive expression of CYP3A4, and a number of important phase II enzymes and drug transporters. CYP2B6 has been demonstrated to play a role in the metabolism of 2%–10% of clinically used drugs including widely used antineoplastic agents cyclophosphamide and ifosfamide, anesthetics propofol and ketamine, synthetic opioids pethidine and methadone, and the antiretrovirals nevirapine and efavirenz, among others. Significant inter-individual variability in the expression and function of the human CYP2B6 gene exists and can result in altered clinical outcomes in patients receiving treatment with CYP2B6-substrate drugs. These variances arise from a number of sources including genetic polymorphism, and xenobiotic intervention. In this review, we will provide an overview of the key players in CYP2B6 expression and function and highlight recent advances made in assessing clinical ramifications of important CYP2B6-mediated drug–drug interactions.

Conclusions based on either in vitro or in vivo approach to evaluate the P-gp affinity status of opioids may be misleading. For example, in vitro studies indicated that fentanyl is a P-gp inhibitor while in vivo studies indicated that it is a P-gp substrate. Quite the opposite was evident for meperidine. The objective of this study was to evaluate the P-gp affinity status of methadone, buprenorphine and diprenorphine to predict P-gp-mediated drug-drug interactions and to determine a better candidate for management of opioid dependence. Two in vitro (P-gp ATPase and monolayer efflux) assays and two in vivo (tissue distribution and antinociceptive evaluation in mdr1a/b (−/−) mice) assays were used. Methadone stimulated the P-gp ATPase activity only at higher concentrations, while verapamil and GF120918 inhibited its efflux (p <0.05). The brain distribution and antinociceptive activity of methadone were enhanced (p <0.05) in P-gp knockout mice. Conversely, buprenorphine and diprenorphine were negative in all assays. P-gp can affect the PK/PD of methadone, but not buprenorphine or diprenorphine. Our report is in favor of buprenorphine over methadone for management of opioid dependence. Buprenorphine most likely is not a P-gp substrate and concerns regarding P-gp-mediated drug-drug interaction are not expected.

Objective To test whether azithromycin eradicates Ureaplasma from the respiratory tract in preterm infants. Design Prospective, phase IIb randomised, doubleblind, placebo-controlled trial. setting Seven level III-IV US, academic, neonatal intensive care units (NICUs). Patients Infants 24 0 -28 6 weeks' gestation (stratified 24 0 -26 6 ; 27 0 -28 6 weeks) randomly assigned within 4 days following birth from July 2013 to August 2016. Interventions Intravenous azithromycin 20 mg/kg or an equal volume of D5W (placebo) every 24 hours for 3 days. Main outcome measures The primary efficacy outcome was Ureaplasma-free survival. Secondary outcomes were all-cause mortality, Ureaplasma clearance, physiological bronchopulmonary dysplasia (BPD) at 36 weeks' postmenstrual age, comorbidities of prematurity and duration of respiratory support. results One hundred and twenty-one randomised participants (azithromycin: n=60; placebo: n=61) were included in the intent-to-treat analysis (mean gestational age 26.2±1.4 weeks). Forty-four of 121 participants (36%) were Ureaplasma positive (azithromycin: n=19; placebo: n=25). Ureaplasma-free survival was 55/60 (92% (95% CI 82% to 97%)) for azithromycin compared with 37/61 (61% (95% CI 48% to 73%)) for placebo. Mortality was similar comparing the two treatment groups (5/60 (8%) vs 6/61 (10%)). Azithromycin effectively eradicated Ureaplasma in all azithromycin-assigned colonised infants, but 21/25 (84%) Ureaplasma-colonised participants receiving placebo were culture positive at one or more follow-up timepoints. Most of the neonatal mortality and morbidity was concentrated in 21 infants with lower respiratory tract Ureaplasma colonisation. In a subgroup analysis, physiological BPD-free survival was 5/10 (50%) (95% CI 19% to 81%) among azithromycin-assigned infants with lower respiratory tract Ureaplasma colonisation versus 2/11 (18%) (95% CI 2% to 52%) in placebo-treated infants. Conclusion A 3-day azithromycin regimen effectively eradicated respiratory tract Ureaplasma colonisation in this study. Trial registration number NCT01778634.

Ureaplasma spp. respiratory tract colonization is a significant risk factor for bronchopulmonary dysplasia (BPD), a chronic lung disorder in preterm infants. As an initial step preparatory to future clinical trials to evaluate the clinical efficacy of azithromycin to prevent BPD, we characterized the pharmacokinetics, safety, and biological effects of a single intravenous dose of azithromycin (10 mg/kg) in preterm neonates (n=12) 24–28 weeks gestation at risk for Ureaplasma infection and BPD. A two-compartment structural model with the clearance and volume of peripheral compartment (V2) allometrically scaled on body weight (WT) best described the pharmacokinetics of azithromycin in preterm neonates. The estimated parameters were: clearance [0.18 L/h × WT(Kg)0.75], intercompartmental clearance [1.0 L/h], volume of distribution of central compartment [0.93 L] and V2 [14.2 L × WT(Kg)]. There were no serious adverse events attributed to azithromycin. A single dose of azithromycin did not suppress inflammatory cytokines or myeloperoxidase activity in tracheal aspirates. Our results demonstrated the safety of azithromycin and developed a PK model that is useful for future simulation-based clinical trials for eradicating Ureaplasma and preventing BPD in preterm neonates.

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