TARGET OF RAPAMYCIN signaling plays a role in Arabidopsis growth promotion by Azospirillum brasilense Sp245.

Azospirillum brasilense colonizes plant roots and improves productivity, but the molecular mechanisms behind its phytostimulation properties remain mostly unknown. Here, we uncover an important role of TARGET OF RAPAMYCIN (TOR) signaling on the response of Arabidopsis thaliana to A. brasilense Sp245. The effect of the bacterium on TOR expression was analyzed in the transgenic line TOR/tor-1, which carries a translational fusion with the GUS reporter protein, and the activity of TOR was assayed thought the phosphorylation of its downstream signaling target S6K protein. Besides, the role of TOR on plant growth in inoculated plants was assessed using the ATP-competitive inhibitor AZD-8055. A decrease in growth of the primary root correlates with an improved branching and absorptive capacity via lateral root and root hair proliferation 6 days after transplant to different concentrations of the bacterium (103 or 105 CFU/mL). Bacterization increased the expression of TOR in shoot and root apexes and promoted phosphorylation of S6K 3 days after transplant. The TOR inhibitor AZD-8055 (1 µM) inhibited plant growth and cell division in root meristems and in lateral root primordia, interfering with the phytostimulation by A. brasilense. In addition, the role of auxin produced by the bacterium to stimulate TOR expression was explored. Noteworthy, the A. brasilense mutant FAJ009, impaired in auxin production, was unable to elicit TOR signaling to the level observed for the wild-type strain, showing the importance of this phyhormone to stimulate TOR signaling. Together, our findings establish an important role of TOR signaling for the probiotic traits elicited by A. brasilense in A. thaliana.

Molecular dynamics simulation and 3D-pharmacophore analysis of new quinoline-based analogues with dual potential against EGFR and VEGFR-2.

Epidermal growth factor and vascular endothelial growth factor-2 are important targets of tyrosine kinase for the treatment of various cancerous diseases. Combination of inhibition of both targets to produce synergy in the signal pathway is a critical approach to identify novel tyrosine kinase inhibitors. In this study, a series of new compounds derived from the 4-aminoquinoline as dual inhibitors were synthesized. The obtained results of cytotoxicity assay against human carcinoma cell lines indicated 0.8 µM for 4c against A549 showing its high efficiency in comparison to erlotinib. Pharmacophore modeling as a structure-based method was investigated on dual inhibitors and 4c which was compared with co-crystallized in the active site of EGFR and VEGFR-2. They have shown the same binding orientation as vandetanib, erlotinib and sorafenib. Molecular dynamics simulation results approved that Met769, Lys721, Asp1046, and Lys868 are key residues in two binding sites for dual activity. Ala1050 and Pro968 were identified as new amino acid interaction sites for dual inhibition. 4c showed more favorable stability than vandetanib in VEGFR-2 receptor for a 50 ns dynamic simulation. The high correlation between essential pharmacophoric features of designed compounds and lead inhibitors interactions provided a deeper insight into the structural basis of 4-aminoquinoline inhibition.

Evaluation of [18F]pitavastatin as a positron emission tomography tracer for in vivo organic transporter polypeptide function.

INTRODUCTION: To understand the pathways involved in drug clearance from the body, quantitative evaluations of the hepatobiliary transport of drugs are important. The organic anion transporting polypeptide (OATP) family transporter, particularly OATP1B1 and 1B3, are considered to play an important role in hepatic uptake of organic anion compounds. Pitavastatin is a substrate of OATP, and it includes a fluorine group. Therefore, it represents an acceptable positron-emission tomography (PET) tracer using fluorine-18 to image in vivo hepatic transporter functions. METHOD: [18F]Pitavastatin was synthesized using the method we previously reported. To evaluate the potential of [18F]pitavastatin in PET imaging of in vivo OATP functions, we investigated the hepatic uptake with/without rifampicin as an OATP inhibitor after administration in normal SD rats. [18F]Pitavastatin metabolite was evaluated using reverse-phase thin-layer chromatography (TLC) autoradiography. We subsequently analyzed the PET image results and demonstrated that [18F]pitavastatin selectively accumulated in the liver post-administration. Result and discussion In metabolite analysis using reverse-phase TLC, we found that the radioactivity detected in the plasma, liver (>90% intact), and bile mostly originated from the parent pitavastatin of the PET study (~40 min). [18F]pitavastatin's hepatic uptake decreased (approximately 76%) with rifampicin co-administration in PET analysis. Because [18F]pitavastatin has lower clearance in rats than other previously reported OATP1B PET s, it holds the potential of an imaging tracer that has a higher sensitivity in monitoring hepatic OATP1B function's changes. CONCLUSION: Compared with the previously reported OATP imaging tracers, [18F]pitavastatin is more suitable for the sensitive detection of functional changes in OATP transporters. We believe that [18F]pitavastatin enables quantitative analysis of the hepatobiliary transport system for organic anion compounds.

Utilizing comprehensive and mini-kinome panels to optimize the selectivity of quinoline inhibitors for cyclin G associated kinase (GAK).

We demonstrate an innovative approach for optimization of kinase inhibitor potency and selectivity utilising kinase mini-panels and kinome-wide panels. We present a focused case study on development of a selective inhibitor of cyclin G associated kinase (GAK) using the quin(az)oline inhibitor chemotype. These results exemplify a versatile, efficient approach to drive kinome selectivity during inhibitor development programs.

Autolytic enzymes are responsible for increased melanization of carbon stressed Aspergillus nidulans cultures.

Melanization of carbon stressed Aspergillus nidulans cultures were studied. Melanin production showed strong positive correlation with the activity of the secreted chitinase and ß-1,3-glucanase. Deletion of either chiB encoding an autolytic endochitinase or engA encoding an autolytic ß-1,3-endoglucanase, or both, almost completely prevented melanization of carbon stressed cultures. In contrast, addition of Trichoderma lyticase to cultures induced melanin production. Synthetic melanin could efficiently inhibit the purified ChiB chitinase activity. It could also efficiently decrease the intensity of hyphal fragmentation and pellet disorganization in Trichoderma lyticase treated cultures. Glyphosate, an inhibitor of L-3,4-dihydroxyphenylalanine-type melanin synthesis, could prevent melanization of carbon-starved cultures and enhanced pellet disorganization, while pyroquilon, a 1,8-dihydroxynaphthalene-type melanin synthesis inhibitor, enhanced melanization, and prevented pellet disorganization. We concluded that cell wall stress induced by autolytic cell wall hydrolases was responsible for melanization of carbon-starved cultures. The produced melanin can shield the living cells but may not inhibit the degradation and reutilization of cell wall materials of dead hyphae. Controlling the activity of autolytic hydrolase production can be an efficient approach to prevent unwanted melanization in the fermentation industry, while applying melanin synthesis inhibitors can decrease the resistance of pathogenic fungi against the chitinases produced by the host organism.

Antagonism of the adenosine A2A receptor attenuates akathisia-like behavior induced with MP-10 or aripiprazole in a novel non-human primate model.

Akathisia is a subset of the larger antipsychotic side effect profile known as extrapyramidal syndrome (EPS). It is associated with antipsychotic treatment and is characterized as a feeling of inner restlessness that results in a compulsion to move. There are currently no primate models available to assess drug-induced akathisia; the present research was designed to address this shortcoming. We developed a novel rating scale based on both the Barnes Akathisia Rating Scale (BARS) and the Hillside Akathisia Scale (HAS) to measure the objective, observable incidence of antipsychotic-induced akathisia-like behavior in Cebus apella non-human primates (NHPs). To induce akathisia, we administered the atypical antipsychotic aripiprazole (1 mg/kg) or the selective phosphodiesterase 10A (PDE10A) inhibitor MP-10 (1-3 mg/kg). Treatment with both compounds produced significantly greater akathisia scores on the rating scale than vehicle treatment. Characteristic behaviors observed included vocalizations, stereotypies, teeth grinding, restless limb movements, and hyperlocomotion. Adenosine A2A receptor antagonists have previously been shown to be effective in blocking antipsychotic-induced EPS in primates. The selective A2A receptor antagonist, SCH 412348 (10-30 mg/kg), effectively reduced or reversed akathisia-like behavior induced by both aripiprazole and MP-10. This work represents the first NHP measurement scale of akathisia and demonstrates that NHPs are responsive to akathisia-inducing agents. As such, it provides a useful tool for the preclinical assessment of putative antipsychotics. In addition, these results provide further evidence of the utility of A2A receptor antagonists for the treatment of antipsychotic-induced movement disorders.

Inhibitors of reactive oxygen species accumulation delay and/or reduce the lethality of several antistaphylococcal agents.

Perturbation of hydroxyl radical accumulation by subinhibitory concentrations of 2,2'-bipyridyl plus thiourea protects Escherichia coli from being killed by 3 lethal antimicrobial classes. Here, we show that 2,2'-bipyridyl plus thiourea delays and/or reduces antimicrobial killing of Staphylococcus aureus by daptomycin, moxifloxacin, and oxacillin. While the protective effect of 2,2'-bipyridyl plus thiourea varied among strains and compounds, the data support the hypothesis that hydroxyl radical enhances antimicrobial lethality.

Reevaluation of the microsomal metabolism of montelukast: major contribution by CYP2C8 at clinically relevant concentrations.

According to published in vitro studies, cytochrome P450 3A4 catalyzes montelukast 21-hydroxylation (M5 formation), whereas CYP2C9 catalyzes 36-hydroxylation (M6), the primary step in the main metabolic pathway of montelukast. However, montelukast is a selective competitive CYP2C8 inhibitor, and our recent in vivo studies suggest that CYP2C8 is involved in its metabolism. We therefore reevaluated the contributions of different cytochrome P450 (P450) enzymes, particularly that of CYP2C8, to the hepatic microsomal metabolism of montelukast using clinically relevant substrate concentrations in vitro. The effects of P450 isoform inhibitors on montelukast metabolism were examined using pooled human liver microsomes, and montelukast oxidations by human recombinant CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5 were investigated. The results verified the central role of CYP3A4 in M5 formation. The CYP2C8 inhibitors gemfibrozil 1-O-ß glucuronide and trimethoprim inhibited the depletion of 0.02 µM montelukast and formation of M6 from 0.05 µM montelukast more potently than did the CYP2C9 inhibitor sulfaphenazole. Likewise, recombinant CYP2C8 catalyzed montelukast depletion and M6 formation at a 6 times higher intrinsic clearance than did CYP2C9, whereas other P450 isoforms produced no M6. On the basis of depletion of 0.02 µM montelukast, CYP2C8 was estimated to account for 72% of the oxidative metabolism of montelukast in vivo, with a 16% contribution for CYP3A4 and 12% for CYP2C9. Moreover, CYP2C8 catalyzed the further metabolism of M6 more actively than did any other P450. In conclusion, CYP2C8 plays a major role in the main metabolic pathway of montelukast at clinically relevant montelukast concentrations in vitro.

Blockade of tachykinin NK3 receptor reverses hypertension through a dopaminergic mechanism in the ventral tegmental area of spontaneously hypertensive rats.

BACKGROUND AND PURPOSE: Intracerebroventricularly injected tachykinin NK(3) receptor (R) antagonists normalize mean arterial blood pressure (MAP) in spontaneously hypertensive rats (SHR). This study was pursued to define the role played by NK(3)R located on dopamine neurones of the ventral tegmental area (VTA) in the regulation of MAP in SHR. EXPERIMENTAL APPROACH: SHR (16 weeks) were implanted permanently with i.c.v. and/or VTA guide cannulae. Experiments were conducted 24 h after catheterization of the abdominal aorta to measure MAP and heart rate (HR) in freely behaving rats. Cardiovascular responses to i.c.v. or VTA-injected NK(3)R agonist (senktide) and antagonists (SB222200 and R-820) were measured before and after systemic administration of selective antagonists for D(1)R (SCH23390), D(2)R (raclopride) or non-selective D(2)R (haloperidol), and after destruction of the VTA with ibotenic acid. KEY RESULTS: I.c.v. or VTA-injected SB222200 and R-820 (500 pmol) evoked anti-hypertension, which was blocked by raclopride. Senktide (10, 25, 65 and 100 pmol) elicited greater increases of MAP and HR when injected in the VTA, and the cardiovascular response was blocked by R-820, SCH23390 and haloperidol. VTA-injected SB222200 prevented the pressor response to i.c.v. senktide, and vice versa, i.c.v. senktide prevented the anti-hypertension to VTA SB222200. Destruction of the VTA prevented the pressor response to i.c.v. senktide and the anti-hypertension to i.c.v. R-820. CONCLUSIONS AND IMPLICATIONS: The NK(3)R in the VTA is implicated in the maintenance of hypertension by increasing midbrain dopaminergic transmission in SHR. Hence, this receptor may represent a therapeutic target in the treatment of hypertension.

Antimutagenic effects of lycopene and tomato purée.

Several health benefits, including protection from tumors at various anatomic sites, such as the lungs, stomach, and prostate gland, have been attributed to tomatoes and tomato-based products. Among tomato carotenoids, lycopene is the most active antioxidant, although it has many other biological effects, but data on its antimutagenic effects are scarce and often discrepant. The aim of our work was to determine the protective effects of lycopene, with regard to mutagenicity, via two indirect mutagens/carcinogens-2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and aflatoxin B1 (AFB1) and the direct mutagen/carcinogen N-nitroso-N-methylurea (MNU)--using the Ames and micronucleus tests. The significant, dose-dependent, antimutagenic effects of two concentrations of lycopene (30 µg and 300 µg per plate) were demonstrated at various concentrations of both AFB1 and IQ in two strains of Salmonella typhimurium (TA98 and TA100). The protective effects of lycopene relative to MNU were lower in comparison to its protective effects relative to AFB1 and IQ. Mice treated for 3 days with different doses of lycopene (either 25 or 50 mg/kg of body weight) prior to administration of individual mutagens resulted in a significant reduction of micronuclei numbers in the micronucleus test. Tomato purée (tested using the Ames test and AFB(1)) revealed a much stronger, dose-dependent, antimutagenic effect compared with corresponding doses of pure lycopene. Results indicate that lycopene has antimutagenic effects, although the effects are lower than that of tomato purée, which contains a complex mixture of bioactive phytochemicals. The antimutagenic effect is connected with the chemoprotective role of lycopene, tomatoes, and tomato products in the prevention of carcinogenesis.

ARQ-197, an oral small-molecule inhibitor of c-Met for the treatment of solid tumors.

ARQ-197 is an oral, selective c-Met inhibitor under development by ArQule Inc, in partnership with Daiichi Sankyo Co Ltd and Asian licensee Kyowa Hakko Kirin Co Ltd, for the potential treatment of solid tumors, including NSCLC, hepatocellular carcinoma and pancreatic cancer, as well as microphthalmia transcription factor-driven tumors. c-Met, a key cell surface receptor tyrosine kinase involved in diverse regulatory functions, is often aberrantly activated in human cancers. While the precise mechanism of action of ARQ-197 remains undefined, data from preclinical studies have demonstrated that ARQ-197 inhibits c-Met activation in numerous human tumor cell lines and specifically targets c-Met in various cancer types; uniquely, ARQ-197 inhibits c-Met in a non-ATP-competitive manner. Phase I/II clinical trials demonstrated promise in terms of both tolerability and tumor response. Intriguingly, dose-limiting adverse effects were hematological in nature. Combinational trials are also ongoing to take advantage of the signaling crosstalk between c-Met and other oncogenic signaling systems. Prioritization of the clinical development of c-Met inhibitors, such as ARQ-197, among different tumor disease types is a key challenge at present; an improved understanding of the prediction of molecular determinants in tumors with respect to c-Met kinase as the driver oncogenic receptor, and of the prediction of tumor response, is still urgently needed.

Larvicidal activity of sterol carrier protein-2 inhibitor in four species of mosquitoes.

A previous report has shown that mosquito sterol carrier protein-2 inhibitors (SCPIs) are larvicidal to larvae of the yellowfever mosquito, Aedes aegypti (L.) (J. Lipid Res. 46: 650-657, 2005). In the current study, we tested SCPI-1 in an additional four mosquito species for larvicidal activities: Culex pipiens pipiens, Anopheles gambiae, Culex restuans, and Aedes vexans. Cholesterol accumulation in SCPI-treated Ae. aegypti fourth instars was examined. SCPI-1 is lethal to all tested mosquito species, with the LC50 value ranging from 5.2 to 15 microM when treatments started at the first to third instar. However, LC50 values increase to from 5.2 to 38.7 microM in treatments started at first and fourth instar, respectively. The results indicate that the lethal effect of SCPI-1 decreases with the growth of larvae, which suggests that SCPI-1 is more effective before the larvae reach final growth period (the last instar). SCPI-1 suppressed cholesterol uptake in Ae. aegypti fourth instars, suggesting that one of the modes of action of SCPI-1 is via reduction in cholesterol absorption.

Pitavastatin up-regulates the induction of iNOS through enhanced stabilization of its mRNA in pro-inflammatory cytokine-stimulated hepatocytes.

Studies have indicated that protective effects of statins (HMG-CoA reductase inhibitor) are associated with the regulation of endothelial nitric oxide synthase (eNOS) or inducible NOS (iNOS) in heart and liver diseases. Statins have been reported to enhance hepatic NO production and decrease the vascular tone in patients with cirrhosis. However, it is unclear which NOS contributes to the increased NO production. We hypothesized that statins are involved in the up-regulation of iNOS in inflammatory liver, resulting in decreased hepatic resistance. Primary cultured rat hepatocytes were treated with pro-inflammatory cytokine interleukin (IL)-1beta in the presence or absence of pitavastatin. Pretreatment of cells with pitavastatin resulted in up-regulation of iNOS induction by IL-1beta, followed by increased NO production. Pitavastatin had no effects on the degradation of IkappaB or activation of NF-kappaB. However, pitavastatin super-induced the up-regulation of type I IL-1 receptor (IL-1RI), which is essential for iNOS induction in addition to the IkappaB/NF-kappaB pathway. Mevalonate and geranylgeranylpyrophosphate blocked the stimulatory effects of pitavastatin on iNOS and IL-1RI induction. Transfection experiments revealed that pitavastatin increased the stability of iNOS mRNA rather than its promoter transactivation. In support of this observation, pitavastatin increased the antisense-transcript corresponding to the 3'-UTR of iNOS mRNA, which stabilizes iNOS mRNA by interacting with the 3'-UTR- and RNA-binding proteins. These findings demonstrate that pitavastatin up-regulates iNOS by the stabilization of its mRNA, presumably through the super-induction of IL-1RI and antisense-transcript. This implies that statins may contribute to a novel potentiated treatment in liver injuries including cirrhosis.

Rifampicin reduces plasma concentrations of moxifloxacin in patients with tuberculosis.

BACKGROUND: The long duration of the current tuberculosis (TB) treatment is demanding and warrants the development of new drugs. Moxifloxacin shows promising results and may be combined with rifampicin to shorten the duration of TB treatment. Rifampicin induces the phase II metabolic enzymes that are involved in the biotransformation of moxifloxacin. Therefore, the interaction between rifampicin and moxifloxacin should be investigated. PATIENTS AND METHODS: Nineteen Indonesian patients with pulmonary TB who were in the last month of their TB treatment completed a 1-arm, 2-period, fixed-order pharmacokinetic study. In phase 1 of the study, they received 400 mg of moxifloxacin every day for 5 days in addition to 450 mg of rifampicin and 600 mg of isoniazid 3 times per week. In phase 2 of the study, after a 1-month washout period, patients received moxifloxacin for another 5 days (without rifampicin and isoniazid). A 24-h pharmacokinetic curve for moxifloxacin was recorded on the last day of both study periods, and its pharmacokinetic parameters were evaluated for an interaction with rifampicin, using a bioequivalence approach. RESULTS: Coadministration of moxifloxacin with rifampicin and isoniazid resulted in an almost uniform decrease in moxifloxacin exposure (in 18 of 19 patients). The geometric means for the ratio of phase 1 area under the curve to phase 2 area under the curve and for the ratio of phase 1 peak plasma concentration to phase 2 peak plasma concentration were 0.69 (90% confidence interval, 0.65-0.74) and 0.68 (90% confidence interval, 0.64-0.73), respectively. The median time to reach peak plasma concentration for moxifloxacin was prolonged from 1 h to 2.5 h when combined with rifampicin and isoniazid (P=.003). CONCLUSIONS: Coadministration of moxifloxacin with intermittently administered rifampicin and isoniazid results in reduced moxifloxacin plasma concentrations, which is most likely the result of induced glucuronidation or sulphation by rifampicin. Further studies are warranted to evaluate the impact of the interaction on the outcome of TB treatment.

CYP2C76-mediated species difference in drug metabolism: a comparison of pitavastatin metabolism between monkeys and humans.

The monkey is often used to predict metabolism of drugs in humans since it generally shows a metabolic pattern similar to humans. However, metabolic profiles different from humans are occasionally seen in monkeys for some drugs including pitavastatin. Recently, we have successfully identified a monkey-specific cytochrome P450 (CYP) 2C76, which possibly accounts for a species difference between monkeys and humans because of its sequence and functional uniqueness. The present study on the role of CYP2C76 and other monkey CYP2Cs in pitavastatin metabolism, as an example, has revealed that CYP2C76 is important for the metabolism of the lactone form, indicating a major role of CYP2C76 for the difference in the metabolism of pitavastatin and possibly other drugs between monkeys and humans. The current investigation on the involvement of CYP2C76 in the metabolism of other drugs is expected to reveal further the further importance of this monkey-specific drug-metabolizing enzyme.

Participation of ATP in nonadrenergic, noncholinergic relaxation of longitudinal muscle of wistar rat jejunum.

A role of ATP in nonadrenergic, noncholinergic (NANC) relaxations was examined in the Wistar rat jejunum. Electrical field stimulation (EFS) induced NANC relaxation of longitudinal muscle of the jejunal segments in a frequency-dependent manner. A purinoceptor antagonist, adenosine 3'-phosphate 5'-phosphosulfate (A3P5PS, 100 muM) inhibited the relaxation: relaxations induced by EFS at lower or higher frequencies were either completely or partially inhibited, respectively. After the jejunal segments had been desensitized to ATP, the relaxations were decreased to the same extent as those inhibited by A3P5PS. An inhibitor of small conductance Ca(2+)-activated K(+) channels (SK channels), apamin (100 nM), completely inhibited EFS-induced relaxations. Treatment of the segments with an inhibitor of sarcoplasmic reticulum Ca(2+)-ATPase, thapsigargin (1 muM), significantly inhibited the relaxations. The exogenous ATP-induced relaxation of longitudinal muscle occurred with a concomitant decrease in intracellular Ca(2+) levels. Apamin and thapsigargin abolished these ATP-induced responses. A3P5PS significantly inhibited the inhibitory junction potentials which were induced in the longitudinal muscle cells. In addition, apamin significantly inhibited the hyperpolarization that was induced by exogenous ATP in the cells. These findings in the Wistar rat jejunum suggest that ATP participates in the NANC relaxation via activation of SK channels induced by Ca(2+) ions that are released from the thapsigargin-sensitive store site.

Biphasic firing response of nucleus accumbens neurons elicited by THPB-18 and its correlation with DA receptor subtypes.

AIM: To investigate the possibility whether THPB-18 (l-12-shloroscoulerine) possesses the D1 agonist-D2 antagonist action on meso-accumbens-mPFC DA system. METHODS: Single unit spontaneous firing activity was recorded in the nucleus accumbens (NAc) neurons of naive and unilateral-6-hydroxydopamine (6-OHDA)-lesioned Sprague-Dawley rats. The effects of drugs applied intravenously or iontophoretically were determined by the change of firing rates. RESULTS: Under normal conditions, the systemic administration of THPB-18 produced a decrease-increase biphasic firing pattern in the NAc neurons during cumulative doses. High dose of THPB-18 was capable of reversing the inhibition induced by both D2 agonist LY171555 and D1/D2 agonist APO on NAc firing activity. Spiperone pretreatment could not block the high dose of THPB-18-induced firing rate increase, which was reversed by the D1 selective antagonist SCH23390. The tested NAc neurons were effectively inhibited by iontophoretically applied THPB-18 in 90 % of 6-OHDA-lesioned rats, while THPB-18 caused variable effects on the firing of NAc neurons in the neurons of unlesioned rats. The inhibitory effect of THPB-18 was blocked by iontophoretic application of SCH23390, but not D2 antagonist spiperone. CONCLUSION: Similar to L-stepholidine, THPB-18 also possesses the D1 agonistic-D2 antagonistic dual action on the VTA-NAc DA system.

Pitavastatin, a potent hydroxymethylglutaryl coenzyme a reductase inhibitor, increases cholesterol 7 alpha-hydroxylase gene expression in HepG2 cells.

BACKGROUND: The effect of pitavastatin on the mRNA levels of apolipoprotein (apo) A-I, peroxisome proliferator-activated receptor alpha (PPARalpha), cholesterol 7alpha-hydroxylase (CYP7A1), and farnesoid X receptor (FXR) in HepG2 cells was examined to establish whether pitavastatin affects bile acid synthesis and if so, to determine a possible molecular mechanism. METHODS AND RESULTS: HepG2 cells were cultured in serum-free Dulbecco's modified Eagle medium for 18 h before drug treatment. Total RNA was extracted at set times and mRNA levels were quantified by reverse transcription-real time polymerase chain reaction. Pitavastatin at 0.1, 1, 5, and 10 micromol/L increased the mRNA levels of apo A-I, PPARalpha, CYP7A1, and FXR in a dose-dependent manner. The mRNA levels of apo A-I, PPAR alpha, CYP7A1, and FXR similarly increased with increasing doses of pitavastatin. Coincubation of mevalonate (4 mmol/L) with pitavastatin (5 micromol/L) reversed the inductive effects of pitavastatin on the mRNA levels of these genes, indicating that the inductive effects of pitavastatin were related to its inhibition of HMG-CoA reductase. CONCLUSIONS: Pitavastatin increased the mRNA levels of CYP7A1 in HepG2 cells, suggesting that increased conversion of cholesterol to bile acids may be the mechanism for its potent low-density lipoprotein cholesterol-lowering effects.

Metabolic stability and uptake by human hepatocytes of pitavastatin, a new inhibitor of HMG-CoA reductase.

To gain a better understanding of the metabolic stability and transport of pitavastatin (CAS 147526-32-7), a new and potent 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, experiments were conducted using human hepatocytes and oocytes of Xenopus laevis expressing human organic anion transporting polypeptide-2 (OATP2), respectively. Almost the entire radioactivity was from the unchanged substance or lactone form in human hepatocytes, and the cytochrome P450 (CYP)-mediated metabolism of pitavastatin was negligible. The results suggested that CYPs are not critically involved in determining the metabolic fate of pitavastatin. The hepatic uptake of pitavastatin reached saturation with a Km of 2.99 +/- 0.79 micromol/L. Also, the uptake of pitavastatin was mediated by OATP2 expressed in oocytes with a Km of 5.53 +/- 1.70 micromol/L. These results indicated that OATP2 plays a major role in the distribution of pitavastatin in liver. Furthermore, to elucidate the increase in the plasma concentration of pitavastatin in a clinical setting, the inhibitory effect of ciclosporin (cyclosporin A, CAS 59865-13-3) on the uptake of pitavastatin was examined. The uptake of pitavastatin was inhibited in the presence of cyclosporin A and the apparent IC50 value was 2.91 +/- 0.78 micromol/L. This result may at least partly explain the drug-drug interaction between pitavastatin and cyclosporin A. In conclusion, the characterization of transporters needs to be taken into account to avoid transporter-mediated drug-drug interaction.

Precautions for use and adverse effects of vesnarinone: potential mechanisms and future therapies.

This article reviews the precautions and adverse effects associated with vesnarinone use, and the potential mechanisms responsible for these complications as well as suggested treatment strategies. Vesnarinone, a quinolinone derivative, improves the haemodynamics and quality of life in patients with congestive heart failure (CHF); however, it is associated with the adverse effects of increased sudden cardiac death and neutropenia. These adverse effects have limited the application of vesnarinone to the general population but perhaps with continued research into vesnarinone-induced neutropenia and advances in arrhythmia management, the risk/ benefit ratio of vesnarinone may become favourable. For now, the use of vesnarinone should be limited to patients with CHF who have demonstrated a poor response to other cardiac medications and devices. These patients should be closely monitored for both cardiac and non-cardiac adverse effects.