Characterization of Potentially Inappropriate Medications That Need Special Attention in the Elderly with Dementia by Analyzing Pharmacy Claims Data.

Community pharmacists may play a key role in promoting deprescribing of potential inappropriate medications (PIMs) that are highly prevalent among community-dwelling elderly with dementia. To characterize PIMs categories that need a special attention for dementia patients, in the present study, we analyzed the anonymized pharmacy claims data of patients aged 65 years and older (n = 333869) who visited nationwide 905 community-based pharmacies of Sugi Pharmacy Co., Ltd. during December 1-31, 2019. A dementia group was defined as patients who received typical dementia medications marketed in Japan, i.e., donepezil, galantamine, memantine or rivastigmine, and a non-dementia group was defined as patients who received no such medications. After propensity score matching on the basis of patients' age, gender and home healthcare insurance usage, the data of 11486 patients in each group were subjected to logistic regression analyses, to identify PIMs categories particularly important for dementia patients. Univariate analysis indicated that the proportions of dementia patients who received 1 and 2≤ of PIMs were significantly (p < 0.001) greater than those of non-dementia patients (odds ratios were 1.35 and 1.47, respectively). Multivariate analyses identified 5 categories of PIMs that were significantly more frequently prescribed in dementia patients, i.e., 'H2 blockers,' 'drugs for overactive bladder,' 'anti-diabetes drugs' and 'sulpiride' listed as PIMs categories for non-specific cases (adjusted odds ratios (aORs): 1.29, 1.91, 1.17, and 1.38, respectively), in addition to 'antipsychotics' listed only for dementia patients (aOR: 4.29). These results provide useful information to establish strategies for pharmacist-led deprescribing of PIMs in dementia patients.

Assessment of hepatic prostaglandin E2 level in carbamazepine induced liver injury.

Objective. Carbamazepine (CBZ), a widely used antiepileptic drug, is one major cause of the idiosyncratic liver injury along with immune reactions. Conversely, prostaglandin E2 (PGE2) demonstrates a hepatoprotective effect by regulating immune reactions and promoting liver repair in various types of liver injury. However, the amount of hepatic PGE2 during CBZ-induced liver injury remains elusive. In this study, we aimed to evaluate the hepatic PGE2 levels during CBZ-induced liver injury using a mouse model. Methods. Mice were orally administered with CBZ at a dose of 400 mg/kg for 4 days, and 800 mg/kg on the 5th day. Results. Plasma alanine transaminase (ALT) level increased in some of mice 24 h after the last CBZ administration. Although median value of hepatic PGE2 amount in the CBZ-treated mice showed same extent as vehicle-treated control mice, it exhibited significant elevated level in mice with severe liver injury presented by a plasma ALT level >1000 IU/L. According to these results, mice had a plasma ALT level >1000 IU/L were defined as responders and the others as non-responders in this study. Even though, the hepatic PGE2 levels increased in responders, the hepatic expression and enzyme activity related to PGE2 production were not upregulated when compared with vehicle-treated control mice. However, the hepatic 15-hydroxyprostaglandin dehydrogenase (15-PGDH) expression and activity decreased significantly in responders when compared with control mice. Conclusions. These results indicate that elevated hepatic PGE2 levels can be attributed to the downregulation of 15-PGDH expression under CBZ-induced liver injury.

Species differences in liver microsomal hydrolysis of acyl glucuronide in humans and rats.

Acyl glucuronides (AGs) are known as one of the causes of idiosyncratic drug toxicity (IDT). Although AGs can be enzymatically hydrolysed by ß-glucuronidase and esterase, much information on their characteristics and species differences is lacking. This study was aimed to clarify species differences in AG hydrolysis between human and rat liver microsomes (HLM and RLM).To evaluate the AG hydrolysis profile, and the contribution of ß-glucuronidase and esterase towards AG hydrolysis in HLM and RLM, nonsteroidal anti-inflammatory drugs (NSAIDs) were used. AGs were incubated with 0.1 M Tris-HCl buffer (pH 7.4) and 0.3 mg/mL HLM or RLM in the absence or presence of ß-glucuronidase inhibitor, D-saccharic acid 1,4-lactone (D-SL) and esterase inhibitor, phenylmethylsulfonyl fluoride (PMSF).AGs of mefenamic acid (MEF-AG) and etodolac (ETO-AG) showed significantly higher AG hydrolysis rates in RLM than in HLM. Esterases were found to serve as AG hydrolases dominantly in HLM, whereas both esterases and ß-glucuronidase equally contribute to AG hydrolysis in RLM. However, MEF-AG and ETO-AG were hydrolysed only by ß-glucuronidase.We demonstrated for the first time that the activity of AG hydrolases towards NSAID-AGs differs between humans and rats.

Stereoselective Covalent Adduct Formation of Acyl Glucuronide Metabolite of Nonsteroidal Anti-Inflammatory Drugs with UDP-Glucuronosyltransferase.

A reactive metabolite of nonsteroidal anti-inflammatory drugs (NSAIDs), acyl-ß-D-glucuronide (AG), covalently binds to endogenous proteins. The covalent adduct formation of NSAIDs-AG may lead to the dysfunction of target proteins. Therefore, it is important to clarify the detailed characterization of the formation of covalent protein adducts of NSAID-AG. UDP-glucuronosyltransferase (UGT) catalyzes the conversion of NSAIDs to NSAIDs-AG. The aim of this study was to perform a quantitative analysis of the covalent adduct formation of NSAIDs-AG with UGT. Diclofenac-AG and ketoprofen-AG formed covalent adducts with organelle proteins. Next, the number of covalent adducts formed between NSAIDs-AG and UGT isoforms (UGT1A1, UGT1A9, UGT2B4, and UGT2B9) was determined. The capacity of diclofenac-AG to form covalent adducts with UGT1A9 or UGT2B7 was approximately 10 times higher than that of mefenamic acid-AG. The amounts of covalent adducts of AG of propionic acid derivative NSAIDs with UGT2B were higher than those with UGT1A. Stereoselectivity was observed upon covalent binding to UGT. A significant negative correlation between the half-lives of NSAIDs-AG in phosphate buffers and the amount of covalent adduct with UGT2B7 was observed, suggesting the more labile NSAID-AG forms higher irreversible bindings to UGT. This report provides comprehensive information on the covalent adduct formation of NSAIDs-AGs with UGT.

Slc25a39 and Slc25a40 Expression in Mice with Bile Duct Ligation or Lipopolysaccharide Treatment.

SLC25A39/40, involved in mitochondrial GSH (mGSH) import from the cytoplasm, is essential for protection against oxidative stress and mitochondrial dysfunction. We examined the effects of cholestasis, through bile duct ligation (BDL) and lipopolysaccharide (LPS)-induced inflammation in mice, on Slc25a39/40 expression. Additionally, we used human clear cell renal carcinoma (KMRC-1) cells to elucidate the mechanism of regulation of SLC25A39/40 expression in the kidneys after LPS treatment. BDL resulted in a decrease in Slc25a39 mRNA in the liver and a decrease in Slc25a39/40 mRNA and protein in the kidneys. Consequently, there was a significant decrease in mGSH levels in the kidneys of BDL mice compared with those in sham mice. LPS treatment resulted in increased Slc25a40 expression in the kidneys. In KMRC-1 cells, the combination treatment of LPS-RS or FPS-ZM1 with LPS suppressed the LPS-induced increase in SLC25A40, suggesting that SLC25A40 expression could be regulated by the signaling pathway via toll-like receptor 4 and the receptor for advanced glycation end products, respectively. Our findings contribute to understanding the role of mGSH in the maintenance of the mitochondrial redox state. To the best of our knowledge, this is the first study that demonstrates the changes in Slc25a39/40 expression in mice with cholestasis-associated renal injury and LPS-induced inflammation.

Diclofenac-Induced Cytotoxicity in Direct and Indirect Co-Culture of HepG2 Cells with Differentiated THP-1 Cells.

Non-steroidal anti-inflammatory drugs (NSAIDs) such as diclofenac (DIC) frequently induce drug-induced liver injury (DILI). It is unclear whether macrophages such as M1 and M2 participate in NSAID-associated DILI; elucidating this relationship could lead to a better understanding of the detailed mechanism of DILI. We co-cultured human hepatoma HepG2 cells with M1 or M2 derived from human monocytic leukemia THP-1 cells to examine the roles of M1 and M2 in DIC-induced cytotoxicity. DIC was added to the direct or indirect co-cultures of HepG2 cells with M1 or M2 (HepG2/M1 or HepG2/M2, respectively) at cell ratios of (1:0, 1:0.1, 1:0.4, and 1:1). In both direct and indirect HepG2/M2 co-cultures (1:0.4), there was lower lactate dehydrogenase release compared with HepG2/M1 co-cultures. Other NSAIDs as well as DIC showed similar protective effects of DIC-induced cytotoxicity. There were only slight differences in mRNA levels of apoptosis- and endoplasmic reticulum stress-associated factors between M1 and M2 after DIC treatment, suggesting that other factors determined the protective effects of M2 on DIC-induced cytotoxicity. Levels of high mobility group box 1 (HMGB1) in the medium and the mRNA expression levels of HMGB1 receptors were different between M1 and M2 after DIC treatment. Increased HMGB1 concentrations and expression of toll-like receptor 2 mRNA in M1 were observed compared with M2 after DIC treatment. In conclusion, these results suggested that the HMGB1/TLR2 signaling axis can be suppressed in M2 but not M1, leading to the different roles of M1 and M2 in NSAID-induced cytotoxicity.

Protein Kinase N Family Negatively Regulates Constitutive Androstane Receptor-Mediated Transcriptional Induction of Cytochrome P450 2b10 in the Livers of Mice.

In receptor-type transcription factors-mediated cytochrome P450 (P450) induction, few studies have attempted to clarify the roles of protein kinase N (PKN) in the transcriptional regulation of P450s. This study aimed to examine the involvement of PKN in the transcriptional regulation of P450s by receptor-type transcription factors, including the aryl hydrocarbon receptor, constitutive androstane receptor (CAR), and pregnane X receptor. The mRNA and protein levels and metabolic activity of P450s in the livers of wild-type (WT) and double-mutant (D) mice harboring both PKN1 kinase-negative knock-in and PKN3 knockout mutations [PKN1 T778A/T778A; PKN3 -/-] were determined after treatment with activators for receptor-type transcription factors. mRNA and protein levels and metabolic activity of CYP2B10 were significantly higher in D mice treated with the CAR activator phenobarbital (PB) but not with 1,4-bis((3,5-dichloropyridin-2-yl)oxy)benzene compared with WT mice. We examined the CAR-dependent pathway regulated by PKN after PB treatment because the extent of CYP2B10 induction in WT and D mice was notably different in response to treatment with different CAR activators. The mRNA levels of Cyp2b10 in primary hepatocytes from WT and D mice treated with PB alone or in combination with Src kinase inhibitor 1 (SKI-1) or U0126 (a mitogen-activated protein kinase inhibitor) were evaluated. Treatment of hepatocytes from D mice with the combination of PB with U0126 but not SKI-1 significantly increased the mRNA levels of Cyp2b10 compared with those from the corresponding WT mice. These findings suggest that PKN may have inhibitory effects on the Src-receptor for activated C kinase 1 (RACK1) pathway in the CAR-mediated induction of Cyp2b10 in mice livers. SIGNIFICANCE STATEMENT: This is the first report of involvement of PKN in the transcriptional regulation of P450s. The elucidation of mechanisms responsible for induction of P450s could help optimize the pharmacotherapy and improve drug development. We examined whether the mRNA and protein levels and activities of P450s were altered in double-mutant mice harboring both PKN1 kinase-negative knock-in and PKN3 knockout mutations. PKN1/3 negatively regulates CAR-mediated induction of Cyp2b10 through phosphorylation of a signaling molecule in the Src-RACK1 pathway.

Prediction of Hepatic Clearance of Stereoselective Metabolism of Carvedilol in Liver Microsomes and Hepatocytes of Sprague-Dawley and Cytochrome P450 2D-Deficient Dark Agouti Rats.

Hepatic clearance (CLh) of carvedilol (CAR), which is eliminated via stereoselective metabolism by the CYP2D subfamily of cytochromes P450 (CYPs), was predicted using liver microsomes and hepatocytes from Sprague-Dawley (SD) rats and CYP2D-deficient Dark Agouti (DA) rats to determine the usefulness of prediction method. Plasma concentrations of CAR following intravenous injection to DA rats were higher than those in SD rats. The volume of distribution at steady state and total clearance (CLtot) of S-CAR were approximately two times greater than those of R-CAR in both strains. CLh predicted from in vitro studies using DA rat liver microsomes was different from that obtained from in vivo studies. In contrast, in vitro CLh prediction using DA rat hepatocytes was nearly identical to the CLh observed in DA rats in vivo, and was lower than that in SD rats. The predicted CLh in vitro using hepatocytes correlated well with the observed CLtot in vivo, which is expected to be nearly the same as CLh. These results suggest that in vitro metabolic studies using hepatocytes are more relevant with regard to stereoselectively predicting CLh of CAR than those using liver microsomes.

Decrease in Multidrug Resistance-associated Protein 2 Activities by Knockdown of Phosphatidylinositol 4-phosphate 5-kinase in Hepatocytes and Cancer Cells.

PURPOSE: The plasma membrane localization and transport activity of multidrug resistance- associated protein 2 (MRP2/ABCC2) and P-glycoprotein (P-gp/ABCB1) efflux transporters are governed by transporter-associated proteins. Phosphatidylinositol 4,5-bisphosphate (PIP2) formed by phosphatidylinositol 4-phosphate 5-kinase type 1 (PIP5K1) activates the linker function of radixin for efflux transporters. Radixin is involved in the plasma membrane localization of efflux transporters. We examined whether PIP5K1 could be a target for the modulation of transporter activities in hepatocytes and cancer cells. METHODS: The effects of PIP5K1 depletion by siRNA in mouse primary hepatocytes, PANC1 human pancreatic carcinoma cells, and HepG2 human hepatocellular carcinoma cells on the intracellular accumulation of MRP2 and P-gp substrates were examined. RESULTS: PIP5K1A depletion resulted in increased intracellular accumulation of carboxydichlorofluorescein, a MRP2 fluorescent substrate, in mouse primary hepatocytes, PANC1 cells, and HepG2 cells. In PANC1 and HepG2 cells, the transport activities of MRP2 were significantly decreased by PIP5K1C depletion. However, the transport activities of P-gp were unchanged by PIP5K1 depletion. PIP2 levels were unchanged between control and PIP5K1A- or PIP5K1C-depleted HepG2 cells. MRP2 mRNA levels showed few changes in HepG2 cells following PIP5K1A or PIP5K1C depletion. The expression of phosphorylated radixin was decreased by PIP5K1A and PIP5K1C depletion, although total radixin levels were unchanged. CONCLUSIONS: These data suggest that PIP5K1A and PIP5K1C could be target proteins for modulating MRP2 function, partly because of the resulting changes of the linker function of radixin.

Changes in transporters and metabolizing enzymes in the livers of rats with bile duct ligation.

PURPOSE: Bile duct ligation (BDL) in experimental animals is widely used as an animal model of liver cholestasis and fibrosis. The transcriptional process and plasma membrane localization of transporters are regulated by nuclear receptors and scaffold proteins, respectively. However, the detailed changes of these factors in the livers of BDL rats remain unclear. To clarify the effects of BDL on the levels of transporters and metabolizing enzymes, nuclear receptors, and scaffold proteins, we investigated changes in mRNA and protein levels of livers from BDL rats. METHODS: Membrane proteins and microsomes were prepared from rats with BDL. The mRNA levels of transporters and nuclear receptors in livers of control and BDL rats were examined by real-time reverse transcription polymerase chain reaction. The protein levels of transporters, metabolizing enzymes and scaffold proteins in membrane proteins and microsomes were determined by liquid chromatography-tandem mass spectrometry-based targeted proteomics. RESULTS: Mdr1a mRNA was significantly decreased at 1 and 2 weeks of BDL. The mRNA levels of MRP2 were significantly decreased. The mRNA levels of nuclear receptors were significantly decreased in livers of 1-week BDL rats. The protein levels of P-gp were significantly increased by BDL. Regarding scaffold proteins, the protein levels of ezrin, moesin and EBP50 were significantly decreased at 2 weeks of BDL. The protein levels of radixin were significantly increased at 1 week of BDL. In 1-week BDL rats, the protein levels of metabolizing enzymes such as CYP and UGT were significantly decreased. CONCLUSIONS: This study reports the comprehensive changes of transporters, metabolizing enzymes, nuclear receptors, and ezrin/radixin/moesin proteins in the livers of BDL rats. The expression levels of nuclear receptors and radixin that regulate the transcription and localization of CYP and/or transporters were decreased by BDL.

Involvement of diclofenac acyl-ß-d-glucuronide in diclofenac-induced cytotoxicity in glutathione-depleted isolated murine hepatocytes co-cultured with peritoneal macrophages.

Direct hepatotoxic effects of drugs can occur when a parent drug and/or its reactive metabolites induces the formation of reactive oxygen species. Reactive metabolites of diclofenac (DIC) such as DIC acyl-ß-d-glucuronide (DIC-AG) bind covalently to proteins, potentially decreasing protein function or inducing an immune response. However, it is unclear whether the macrophages and GSH depletion participate in DIC-induced cytotoxicity. Mouse hepatocytes (Hep) co-cultured with peritoneal macrophages (PMs) were used to clarify the effects of presence of PM with GSH depletion on DIC-induced cytotoxicity in Hep. DIC-AG but not hydroxy-DIC concentrations in medium were significantly increased in Hep co-cultured with PM with GSH depletion. Depletion of GSH resulted in significantly higher LDH leakage. Interestingly, LDH leakage in Hep/PM (1:0.4) with GSH depletion was significantly higher than in Hep/PM (1:0 and 1:0.1) with BSO. It is likely that macrophages with GSH depletion could facilitate DIC-induced cytotoxicity.

Stereoselective Pharmacokinetics and Chiral Inversion of Ibuprofen in Adjuvant-induced Arthritic Rats.

2-Arylpropionic acid (2-APA) nonsteroidal anti-inflammatory drugs are commonly used in racemic mixtures (rac) for clinical use. 2-APA undergoes unidirectional chiral inversion of the in vivo inactive R-enantiomer to the active S-enantiomer. Inflammation causes the reduction of metabolic activities of drug-metabolizing enzymes such as cytochrome P450 (P450) and UDP-glucuronosyltransferase. However, it is unclear whether inflammation affects the stereoselective pharmacokinetics and chiral inversion of 2-APA such as ibuprofen (IB). We examined the effects of inflammation on the pharmacokinetics of R-IB and S-IB after intravenous administration of rac-IB, R-IB, and S-IB to adjuvant-induced arthritic (AA) rats, an animal model of inflammation. The plasma protein binding of rac-IB, glucuronidation activities for R-IB and S-IB, and P450 contents of liver microsomes in AA rats were determined. Total clearance (CLtot) of IB significantly increased in AA rats, although the glucuronidation activities for IB, and P450 contents of liver microsomes decreased in AA rats. We presumed that the increased CLtot of IB in AA rats was caused by the elevated plasma unbound fraction of IB due to decreased plasma albumin levels in AA rats. Notably, CLtot of R-IB but not S-IB significantly increased in AA rats after intravenous administration of rac-IB. These results suggested that AA could affect drug efficacies after stereoselective changes in the pharmacokinetics of R-IB and S-IB.

Inhibition of Methotrexate Uptake via Organic Anion Transporters OAT1 and OAT3 by Glucuronides of Nonsteroidal Anti-inflammatory Drugs.

Combination therapy of non-steroidal anti-inflammatory drugs (NSAIDs) and methotrexate (MTX) sometimes triggers adverse effects, such as liver injury, renal failure, gastrointestinal disorders, and myelosuppression, owing to the reduction of MTX clearance. Previous reports have suggested that NSAIDs inhibit renal MTX uptake via organic anion transporters (OATs) and reduced folate transporter (RFC)-1 and efflux via multidrug resistance-associated proteins (MRPs). Recently, our laboratory found inhibitory effects of NSAIDs-glucuronide (NSAIDs-Glu), a major metabolite of NSAIDs, on MRP-mediated MTX transport as a new site of interaction between MTX and NSAIDs. However, it remains unclear that whether NSAIDs-Glu inhibit renal uptake of MTX. Therefore, the present study aimed to evaluate inhibitory effects of several NSAIDs-Glu (diclofenac, R- and S-ibuprofen, R- and S-flurbiprofen, and R- and S-naproxen) on human OAT1 and OAT3-mediated MTX transport. In this study, [3H]MTX uptake was observed by using human OAT1 and OAT3-overexpressing HEK293 cells in the presence or absence of NSAIDs-Glu. All examined NSAIDs-Glu exhibited concentration-dependent inhibitory effects on MTX uptake via OAT1 and OAT3. Our results indicated that NSAIDs-Glu are more potent (5- to 15-fold) inhibitors of OAT3 than OAT1. Moreover, stereoselective inhibitory effects of NSAIDs-Glu on OATs-mediated MTX uptake were not observed, unlike on MRPs-mediated transport. These findings suggest that inhibition of OAT1 and OAT3-mediated renal uptake of MTX by plasma NSAIDs-Glu may be one of the competitive sites underlying complex drug interaction between MTX and NSAIDs.

Stereoselective hepatic disposition of ibuprofen in the perfused liver of rat with adjuvant-induced arthritis.

1. The effects of adjuvant-induced arthritis (AA) on the stereoselective hepatic disposition and chiral inversion of "profens" have scarcely been investigated. Ibuprofen (IB) undergoes unidirectional chiral inversion from R-IB to S-IB and is metabolized to IB-glucuronide (IB-Glu). 2. We used an in situ perfused rat liver system to clarify the effects of inflammation on the metabolic activities and chiral inversion of IB without protein binding. 3. After dosing of R-IB, AA had minimal effect on the elimination of R-IB from the perfusate. Larger amounts of S-IB-Glu than R-IB-Glu were observed in the bile at the dose of 2.4 and 4.8 µmol. However, after dosing of S-IB, the elimination of S-IB from the perfusate in AA rats was delayed, indicating a significant decrease in the hepatic clearance in AA rats. The cumulative biliary excretion of S-IB-Glu in AA rats was promoted after dosing with S-IB. There was little difference between the chiral inversion ratios of the control and AA rats. 4. The present study demonstrated that AA results in the delayed elimination of S-IB, the active form, without changes to the chiral inversion ratio. Thus, further attention to the altered stereoselective pharmacokinetics of IB during inflammation is required.

Involvement of Reactive Metabolites of Diclofenac in Cytotoxicity in Sandwich-Cultured Rat Hepatocytes.

BACKGROUND AND OBJECTIVES: Diclofenac (DIC) is metabolized to reactive metabolites such as diclofenac acyl-ß-d-glucuronide (DIC-AG). It is possible that such reactive metabolites could cause tissue damage by formation of covalent protein adducts and other modification of cellular proteins or by induction of immune responses against its covalent protein adducts. However, the detailed mechanisms of idiosyncratic drug-induced liver injury (DILI) have been unclear. The objective is to clarify the involvement of DIC-AG and 4'hydroxydiclofenac (4'OH-DIC) in acute DILI. METHODS: We examined the effects of inhibiting DIC-AG and 4'OH-DIC production on covalent protein adduct formation and lactate dehydrogenase leakage using sandwich-cultured rat hepatocytes (SCRHs). RESULTS: After pretreatment of SCRH with (-)-borneol (BOR, a uridine diphosphate (UDP)-glucuronosyltransferase inhibitor) or sulfaphenazole (SUL, a cytochrome P450 2C9 inhibitor) for 30 minutes, intracellular concentrations of DIC, DIC-AG, and 4'OH-DIC were determined after further treating cells with 300 µM DIC for 3 hours. The decreased levels of reactive metabolites caused by BOR or SUL pretreatment resulted in decreased lactate dehydrogenase leakage from SCRH, although the formation of covalent protein adducts was not affected. CONCLUSION: These results suggested that both DIC-AG and 4'OH-DIC may be involved in acute cytotoxicity by DIC.

Stereoselective Inhibition of Methotrexate Excretion by Glucuronides of Nonsteroidal Anti-inflammatory Drugs via Multidrug Resistance Proteins 2 and 4.

Combined administration of methotrexate (MTX) and nonsteroidal anti-inflammatory drugs (NSAIDs) can result in a decreased systemic clearance of MTX. To date, inhibition of renal uptake via organic anion transporters and efflux via multidrug resistance-associated protein (MRPs) by NSAIDs has been recognized as possible sites of drug interaction between MTX and NSAIDs. Although most NSAIDs are glucuronidated in kidney tissue and excreted mainly as glucuronide conjugates, it is not fully known whether the glucuronides of NSAIDs (NSAIDs-Glu) inhibit MTX excretion via MRP2 and MRP4. The purpose of this study was to investigate the inhibitory effects of the glucuronides of several NSAIDs (diclofenac, R- and S-ibuprofen, R- and S-flurbiprofen, and R- and S-naproxen), as well as the parent NSAIDs on MTX uptake using human MRP2- and MRP4-expressing inside-out vesicles. Results confirm that all NSAIDs and NSAIDs-Glu examined exhibited stereoselective and concentration-dependent inhibitory effects on MTX uptake via MRP2 and MRP4. Notably, NSAIDs-Glu potently inhibited MTX uptake via MRP2 and MRP4 compared with the corresponding parent NSAIDs except for naproxen in MRP2 and S-flurbiprofen in MRP4. The present results support that the glucuronides of NSAIDs, as well as the parent NSAIDs, are involved in the inhibition of urinary excretion of MTX via MRP2 and MRP4.

Pravastatin Modulate Niemann-Pick C1-Like 1 and ATP-Binding Cassette G5 and G8 to Influence Intestinal Cholesterol Absorption.

PURPOSE: Niemann-Pick C1-like 1 (NPC1L1), ATP-binding cassette (ABC)G5, and ABCG8 mediate intestinal cholesterol absorption. It is unclear whether pravastatin (PR) or ezetimibe (EZ) affect expression of these transporters. We examined the effects of PR and EZ on NPC1L1, ABCG5, and ABCG8 expression in human hepatoma HepG2 cells and the murine small intestine. We also assessed expression of the transcription factors liver X receptor (LXR)a, LXRb and sterol regulatory element-binding protein. METHODS: Transporter mRNA levels were determined in murine small intestines 6 and 24 h after oral PR and EZ administration by real-time reverse-transcriptase polymerase chain reaction (RT-PCR). In PR- and EZ-treated HepG2 cells, transporter and transcription factor mRNA and protein levels were examined by RT-PCR and western blot, respectively. RESULTS: Significant decreases in NPC1L1, ABCG5, and ABCG8 mRNA expression were observed in the duodenum, but not jejunum and ileum, of mice 24 h after treatment with PR, but not EZ. In HepG2 cells, PR but not EZ treatment for 24 h also significantly decreased NPC1L1 protein and ABCG5, and ABCG8 mRNA expression, while increasing LXRa mRNA levels. CONCLUSION: PR but not EZ treatment reduced duodenal cholesterol transporter expression in mice. PR-induced increases in LXRa mRNA levels may be involved in attenuation of NPC1L1 expression, subsequently decreasing intestinal cholesterol absorption.

Age-related changes in mRNA levels of hepatic transporters, cytochrome P450 and UDP-glucuronosyltransferase in female rats.

Hepatic transporters and metabolic enzymes affect drug pharmacokinetics. Limited information exists on the alteration in mRNA levels of hepatic transporters and metabolic enzymes with aging. We examined the effects of aging on the mRNA levels of representative hepatic drug transporters and metabolic enzymes by analyzing their levels in 10-, 30- and 50-week-old male and female rats. Levels of mRNA of drug transporters including multidrug resistance protein (Mdr)1a, multidrug resistance-associated protein (Mrp)2, breast cancer resistance protein (Bcrp) and organic anion-transporting polypeptide (Oatp)1a1, and the metabolic enzymes cytochrome P450 (CYP)3A1, CYP3A2 and UDP-glucuronosyltransferase (UGT)1A1 were analyzed using real-time reverse transcriptase polymerase chain reaction. The mRNA levels of transporters in male rats did not decrease with age, while the mRNA levels of Bcrp and Oatp1a1 in female rats decreased with age. The mRNA levels of CYP3A1 and CYP3A2 in male rats were higher than those in female rats. The mRNA levels of metabolic enzymes decreased with age in female but not male rats. In particular, the mRNA levels of UGT1A1 in 10-week-old female rats were higher than those in male rats. mRNA expression of hepatic transporters and metabolic enzymes are more susceptible to aging in female than male rats. The age-related decreases in the mRNA levels of Bcrp, Oatp1a1, CYP3A1 and CYP3A2 in female rats may affect the metabolism and transport of substrates. This study showed that aging affected the mRNA expression of hepatic transporters and metabolic enzymes in rats.

Alteration in plasma protein binding properties of propranolol and flurbiprofen during development of adjuvant-induced arthritis in rats.

Adjuvant-induced arthritis (AA) in the rat is used as a model for rheumatoid arthritis. In AA rats, the pharmacokinetics of various drugs is affected due to the alterations of plasma protein binding of drugs. We choose propranolol (PL) and flurbiprofen (FP) as model basic and acidic drugs, respectively, and investigated the effect of AA induction on their plasma protein binding at each developing stage of inflammation. The plasma protein binding of PL and FP was dramatically changed due to reduced albumin and increased α1-acid glycoprotein levels for at least 21 days after adjuvant treatment. Moreover, we illustrated the differences in protein binding in AA between both the drugs in each developing stage of inflammation. These results suggest that the changed plasma protein levels in AA rats accompanying the altered protein binding of drugs affect the pharmacokinetics of drugs which extensively bind to plasma protein under inflammatory condition.

Relationship between the risk of idiosyncratic drug toxicity and formation and degradation profiles of acyl-glucuronide metabolites of nonsteroidal anti-inflammatory drugs in rat liver microsomes.

Acyl glucuronides (AGs) are considered to cause idiosyncratic drug toxicity (IDT), and evaluating the chemical instability of AGs may be useful for predicting the IDT risk of novel drug candidates. However, AGs show variations in their chemical instability, degree of formation, and enzymatic hydrolysis. Therefore, we evaluated the degree of AG formation, enzymatic hydrolysis, and chemical instability in liver microsomes and their relationship with IDT risk. Nonsteroidal anti-inflammatory drugs (NSAIDs) were classified into three categories in terms of their IDT risk as parent drugs: safe (SA), warning (WA), and withdrawn (WDN). To evaluate the enzymatic and non-enzymatic degradation of AG, the parent drugs were incubated with rat liver microsomes in the absence or presence of AG hydrolase inhibitors. The degree of AG formation and disappearance was considered as the rate constant. For all NSAIDs investigated, the number of AGs formed notably increased following addition of AG hydrolase inhibitors. Particularly, AG was produced by WDN drugs at a lower level than that produced by WA and SA drugs in the absence of AG hydrolase inhibitors but was significantly increased after adding AG hydrolase inhibitors. The rate constants of AG formation and non-enzymatic AG disappearance did not significantly differ among the WDN, WA, and SA drugs, whereas the rate constant of enzymatic AG disappearance of WDN drugs tended to be higher than those of WA and SA drugs. In conclusion, we evaluated the enzymatic degradation and chemical instability of AG by simultaneously producing it in liver microsomes. This method enables evaluation of AG degradation without preparing AG. Moreover, we determined the relationship between enzymatic AG degradation in rat liver microsomes and IDT risk.