Quantitative Model Analysis and Simulation of Pharmacokinetics and Metastasis-Associated Lung Adenocarcinoma 1 RNA Knockdown Effect After Systemic Administration of Cholesterol-Conjugated DNA/RNA Heteroduplex Oligonucleotide Crossing Blood-Brain Barrier of Mice.

The cholesterol-conjugated heteroduplex oligonucleotide (Chol-HDO) is a double-stranded complex; it comprises an antisense oligonucleotide (ASO) and its complementary strand with a cholesterol ligand. Chol-HDO is a powerful tool for achieving target RNA knockdown in the brains of mice after systemic injection. Here, a quantitative model analysis was conducted to characterize the relationship between the pharmacokinetics (PK) and pharmacodynamics (PD), non-coding RNA metastasis-associated lung adenocarcinoma 1 (Malat1) RNA, of Chol-HDO, in a time-dependent manner. The established PK model could describe regional differences in the observed brain concentration-time profiles. Incorporating the PD model enabled the unique knockdown profiles in the brain to be explained in terms of the time delay after single dosing and enhancement following repeated dosing. Moreover, sensitivity analysis of PK exposure/persistency, target RNA turnover, and knockdown potency identified key factors for the efficient and sustained target RNA knockdown in the brain. The simulation of an adequate dosing regimen quantitatively supported the benefit of Chol-HDO in terms of achieving a suitable dosing interval. This was achieved via sufficient and sustained brain exposure and subsequent strong and sustained target RNA knockdown in the brain, even after systemic injection. The present study provides new insights into drug discoveries and development strategies for HDO in patients with neurogenic disorders. SIGNIFICANCE STATEMENT: The quantitative model analysis presented here characterized the PK/PD relationship of Chol-HDO, enabled its simulation under various conditions or assumptions, and identified key factors for efficient and sustained RNA knockdown, such as PK exposure and persistency. Chol-HDO appears to be an efficient drug delivery system for the systemic administration of desired drugs to brain targets.

Relationship of MATE1 Inhibition and Cytotoxicity in Nephrotoxicity: Application for Safety Evaluation in Early Drug Discovery.

Accumulation of toxic endogenous and/or exogenous substances can trigger tissue injury. Multidrug and toxin extrusion proteins (MATEs) are transporters at renal proximal tubules involved in the secretion of hydrophilic substances into urine. Multidrug and toxin extrusion protein inhibition can lead to nephrotoxicity via accumulation of toxic substances; however, case studies demonstrating causality are rare, except for drug-drug interaction studies. To explore the involvement of MATE inhibition in nephrotoxicity, MATE1 inhibition, cytotoxicity, and mitochondrial toxicity (MT) of 38 in-house compounds that showed toxicity were assessed in in vivo safety evaluations using rats, dogs, and monkeys and compared considering unbound exposures at minimal steady-state concentration (C24h,u) between nephrotoxicity positive and negative compounds. Logarithmic-corrected means of C24h,u normalized by MATE1 IC50 or cytotoxicity EC50 (C24h,u/IC50 and C24h,u/EC50) were higher for nephrotoxic compounds. An exposure cutoff of C24h,u/IC50 > 0.01 filtered nephrotoxicity with a 54% positive predictive value. Of 7 cases filtered with this cutoff, all the cases showed pathological changes at renal proximal tubules expressing MATE1. Furthermore, all cases with > 0.01 reliable exposure for MATE1 inhibition and cytotoxicity exhibited nephrotoxicity. Although compounds potent for MATE1 inhibition and cytotoxicity without and with MT (potentials of 10, 30, and 40 µM, respectively) were correctly classified as nephrotoxic by evaluation of in vitro potency alone, without considering exposures, these results suggest that MATE1 inhibition potency and cytotoxicity can be used to assess nephrotoxicity, especially at proximal tubules, and could be used for safety assessment in early drug discovery.

Mechanism-Based Pharmacokinetic/Pharmacodynamic Modeling of the Glucagon-Like Peptide-1 Receptor Agonist Exenatide to Characterize Its Antiobesity Effects in Diet-Induced Obese Mice.

In addition to their potent antidiabetic effects, glucagon-like peptide-1 (GLP-1) analogs lower body weight in humans. Hence, agonistic targeting of the GLP-1 receptor could be a valid approach to target obesity. However, quantitative analyses of the pharmacokinetic/pharmacodynamic (PK/PD) relationship between GLP-1 analogs and their antiobesity effect have not been reported in either animals or humans. Therefore, the present study was performed to establish a mechanism-based PK/PD model of GLP-1 receptor agonists using the GLP-1 analog exenatide for the development of promising new antiobesity drugs. Exenatide was administered to high-fat diet-induced obese C57BL/6J mice via subcutaneous bolus and continuous infusion. Food intake and body-weight reductions were observed and depended on the plasma concentrations of exenatide. The homeostatic feedback model, in which food intake is assumed to be regulated by appetite control signals, described the relationship among the plasma concentration-time profile of exenatide, food intake, and body weight. The estimated IC50 of exenatide against food intake was 2.05 pM, which is similar to the reported KD value of exenatide in rat brain and the estimated EC50 value for augmentation of insulin secretion in humans. The PK/PD model simulation indicated that subcutaneous infusion would show a stronger effect on body-weight reduction than bolus dosing would. This novel, quantitative PK/PD model could be used for antiobesity research and development of GLP-1 analogs, GLP-1 secretagogues, GLP-1 degradation inhibitors, and combinations thereof by allowing the estimation of appropriate pharmacokinetic profiles and dosing regimens.

Regulation of multidrug resistance protein 2 (MRP2, ABCC2) expression by statins: involvement of SREBP-mediated gene regulation.

Multidrug resistance protein 2 (MRP2, ABCC2) is localized to the apical membrane of hepatocytes and played an important role in the biliary excretion of a broad range of endogenous and xenobiotic compounds and drugs, such as pravastatin. However, the effects of statins on MRP2 in the liver and the precise mechanisms of their actions have been obscure. The goal of this study was to determine the regulatory molecular mechanism for statin-induced MRP2 expression in hepatocytes. In vitro and in vivo studies suggested that pitavastatin increased MRP2 expression. Pitavastatin promoted liver X receptor (LXR) α/ß translocation from the cytosol to nuclei, resulting in LXR activation. Deletion and mutational analysis suggested that the potential sterol regulatory element (SRE) played a major role in the observed modulation of MRP2 expression by pitavastatin. Furthermore pitavastatin increased the protein-DNA complex, and when SRE was mutated, stimulation of the protein-DNA complex by pitavastatin was decreased. It was demonstrated that pitavastatin upregulated MRP2 expression by an SREBP regulatory pathway in hepatocytes and that the actions of statins may lead to improve the biliary excretion of MRP2 substrates.

[Effects of acidification and alkalinization agents on statins-induced muscle toxicity].

The aim of this study was to determine the effects of α-cyano-4-hydroxycinnamic acid (CHC), a lactate efflux inhibitor, and citrate, an alkaline reagent, on statin-induced muscle injury using a human prototypic embryonal rhabdomyosarcoma cell line (RD) as a model of in vitro skeletal muscle and on statin-induced muscle damage in an in vivo study. Statin-induced reduction of cell viability and apoptosis was measured by the 3-(4,5-dimethylthiazol-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT) assay and caspase assay. In an in vivo study, plasma creatine phosphokinase (CPK) level was examined in cerivastatin-treated rats. CHC increased growth inhibition of RD cells induced by cerivastatin, a lipophilic statin, but not these induced by pravastatin, a hydrophilic statin. On the other hand, citrate suppressed cerivastatin-, simvastatin- and atorvastatin-induced reduction of cell viability and caspase activation in RD cells. Moreover, citrate prevented cerivastatin-induced increase in CPK concentration in a concentration-dependent manner. This is first study to evaluate CHC or citrate-induced exacerbation or improvement of statin-induced muscle damage.

Regulation mechanism of ABCA1 expression by statins in hepatocytes.

ATP-binding cassette transporter A1 (ABCA1) is predicted to be involved in the control of apolipoprotein AI-mediated cholesterol efflux: biosynthesis of high-density lipoprotein (HDL). However, the effects of HMG-CoA reductase inhibitors (statins) on ABCA1 in the liver and the precise mechanisms of their actions have been obscure. The aims of this study were to determine whether statins (atorvastatin (Ato) and pitavastatin (Pit)) affect hepatic ABCA1 expression and to clarify the mechanisms of their actions using HepG2 cells and the rat liver. We examined alterations in mRNA and protein levels of ABCA1 and peroxisome proliferator-activated receptors (PPARs) by quantitative real-time polymerase chain reaction (PCR) and Western blot analysis, respectively. In vitro and in vivo studies suggested that Pit increases ABCA1 mRNA level, but not Ato. Pit greatly increased Abca1 mRNA level and also increased the amount of plasma HDL and the mRNA level of PPARα. Clofibrate (PPARα agonist) increased ABCA1 expression in HepG2 cells and rat primary hepatocytes more than did PPAR ß/δ and γ agonists. Pit-induced ABCA1 expression alteration was blocked by GW6471 (PPARα antagonist) and by PPARα knockdown. In this study, we demonstrated that Pit affect ABCA1 expression via PPARα in hepatocytes. The strategy to target a PPARα agonist in the liver can lead to increases in ABCA1 expression and HDL level.

Liver X receptor regulates expression of MRP2 but not that of MDR1 and BCRP in the liver.

Liver X receptors (LXRs) belong to the nuclear hormone receptor superfamily. Multidrug resistance-associated protein 2 (MRP2), multidrug resistance 1 (MDR1) and breast cancer resistance protein (BCRP) play an important role in the efflux of a broad range of endogenous and xenobiotic compounds from hepatocytes. Since the effects of LXR activation on there transporters have been obscure, we investigated the effects of LXR agonists, TO901317 and 25-hydroxycholesterol, on MRP2, MDR1, BCRP expression in HepG2 cells and the rat liver. In an in vitro study, TO901317 increased ABCA1, an LXR target gene, and MRP2 mRNA and protein levels. On the other hand, TO901317 had little effect on MDR1 and BCRP mRNA levels. In an in vivo study, Abca1 and Mrp2 mRNA and protein levels were increased by TO901317, but TO901317 had no effect on Mdr1a and Bcrp mRNA levels in the rat liver. Moreover, TO901317-induced MRP2 mRNA expression was blocked by LXRalpha knockdown. In this study, we demonstrated that LXR activation induced expression of MRP2 but not that of MDR1 and BCRP in hepatocytes. The results suggest that agonists for LXR activate transcription of the MRP2 gene in order to promote excretion of endogenous and xenobiotic compounds from hepatocytes into bile.

Association between risk of myopathy and cholesterol-lowering effect: a comparison of all statins.

In the present study, we examined the mechanisms underlying the cytotoxicity of pitavastatin, a new statin, and we compared the in vitro potencies of muscle cytotoxicity using a prototypic embryonal rhabdomyosarcoma cell line (RD cells), a typical side effect of statins and compared the cholesterol-lowering effects of statins using Hep G2 hepatoma cells. Pitavastatin reduced the number of viable cells and caused caspase-9 and -3/7 activation in a time- and concentration-dependent manner. The comparison of cytotoxities of statins showed that statins significantly reduced cell viability and markedly enhanced activity of caspase-3/7 in concentration-dependent manner. On the other hand, the effects of hydrophilic statins, pravastatin, rosuvastatin were very weak. The rank order of cytotoxicity was cerivastatin > simvastatin acid> fluvastatin > atorvastatin > lovastatin acid > pitavastatin >> rosuvastatin, pravastatin. Statin-induced cytotoxicity is associated with these partition coefficients. On the other hand, the cholesterol-lowering effect of statins did not correlate with these partition coefficients and cytotoxicity. Thus, it is necessary to consider the association between risk of myopathy and cholesterol-lowering effect of a statin for precise use of statins.