ABSTRACT
Porphyria cutanea tarda (PCT), the most common of the human porphyrias, arises from a deficiency of uroporphyrinogen decarboxylase. Studies have shown a high prevalence of hepatitis C virus (HCV) infection in patients with PCT. While these observations implicate HCV infection as a risk factor for PCT pathogenesis, the mechanism of interaction between the virus and porphyrin metabolism is unknown. This study aimed to assess the effect of HCV core protein on intracellular porphyrin metabolism to elucidate the link between HCV infection and PCT. The accumulation and excretion of porphyrins after treatment with 5-aminolevulinic acid, a porphyrin precursor, were compared between cells stably expressing HCV core protein and controls. Cells expressing HCV core protein had lower amounts of intracellular protoporphyrin IX and heme and had higher amounts of excreted coproporphyrin III, the oxidized form of coproporphyrinogen III, compared with controls. These observations suggest that HCV core protein affects porphyrin metabolism and facilitates the export of excess coproporphyrinogen III and/or coproporphyrin III, possibly via porphyrin transporters. Real-time PCR analysis revealed that the presence of HCV core protein increased the mRNA expression of porphyrin exporters ABCG2 and FLVCR1. Western blot analysis showed a higher expression level of FLVCR1, but not ABCG2, as well as a higher expression level of mature ALAS1, which is the rate-limiting enzyme in the heme synthesis pathway, in HCV core protein-expressing cells compared with controls. The data indicate that HCV core protein induced abnormal intracellular porphyrin metabolism, with an over-excretion of coproporphyrin III. These findings may partially account for the susceptibility of HCV-infected individuals to PCT development.
Subject(s)
Carcinoma, Hepatocellular/metabolism , Hepacivirus/physiology , Hepatitis C/metabolism , Liver Neoplasms/metabolism , Porphyrins/metabolism , Aminolevulinic Acid/pharmacology , Biosynthetic Pathways/drug effects , Carcinoma, Hepatocellular/complications , Carcinoma, Hepatocellular/pathology , Carcinoma, Hepatocellular/virology , Cells, Cultured , Hep G2 Cells , Hepacivirus/drug effects , Hepatitis C/complications , Hepatitis C/pathology , Humans , Liver Neoplasms/complications , Liver Neoplasms/pathology , Liver Neoplasms/virologyABSTRACT
3-Deoxyglucosone (3-DG) is a highly reactive carbonyl intermediate in glycation reaction (also known as Maillard reaction) and plays an important role in diabetic complications. We investigated the potential involvement of 3-DG in doxorubicin (DXR)-induced cardiotoxicity. Male Crl:CD(SD) rats received intravenous injections of DXR at 2mg/kg, once weekly, for 6 weeks, with/without daily intraperitoneal treatment with 3-DG scavenging agents, i.e., aminoguanidine (AG, 25mg/kg/day) and pyridoxamine (PM, 60mg/kg/day). Cardiac levels of 3-DG, thiobarbituric acid reactive substances (TBARS), fructosamine, and pentosidine, plasma glucose levels and cardiac troponin I (cTnI), echocardiography, and histopathology were assessed at 4 and 6 weeks after treatment. Cardiac 3-DG levels were significantly increased by DXR treatment at 4 and 6 weeks. Cardiac fructosamine levels and plasma glucose were not altered by DXR; however, TBARS levels in the heart were significantly increased at 4 and 6 weeks, suggesting that the enhanced generation of 3-DG is not attributed to any abnormal glycemic status, but may be related to oxidative stress by DXR. An advanced glycation end-product, pentosidine, was significantly increased by DXR treatment at 6 weeks. Intervention by AG and PM ameliorated the DXR-induced echocardiographic abnormalities, increased cTnI in plasma, and histopathological lesion as well as normalizing the elevation of 3-DG and pentosidine levels. These results suggest that 3-DG is generated by DXR and involved, at least in part, in the pathogenesis of DXR-cardiotoxicity through glycation reaction.
ABSTRACT
Bile acid (BA) retention within hepatocytes is an underlying mechanism of cholestatic drug-induced liver injury (DILI). We previously developed an assay using sandwich-cultured human hepatocytes (SCHHs) to evaluate drug-induced hepatocyte toxicity accompanying intracellular BA accumulation. However, due to shortcomings commonly associated with the use of primary human hepatocytes (e.g., limited availability, lot-to-lot variability, and high cost), we examined if the human hepatic stem cell line, HepaRG, might also be applicable to our assay system. Consequently, mRNA expression levels of human BA efflux and uptake transporters were lower in HepaRG cells than in SCHHs but higher than in HepG2 human hepatoma cells. Nevertheless, HepaRG cells and SCHHs showed similar toxicity responses to 22 selected drugs, including cyclosporine A (CsA). CsA (10 µM) was cytotoxic toward HepaRG cells in the presence of BAs and also reduced the biliary efflux rate of [(3)H]taurocholic acid from 38.5% to 19.2%. Therefore, HepaRG cells are useful for the evaluation of BA-dependent drug toxicity caused by biliary BA efflux inhibition. Regardless, the prediction accuracy for cholestatic DILI risk was poor for HepaRG cells versus SCHHs, suggesting that our DILI model system requires further improvements to increase the utility of HepaRG cells as a preclinical screening tool.
Subject(s)
Bile Acids and Salts/metabolism , Chemical and Drug Induced Liver Injury/metabolism , Hepatocytes/drug effects , Hepatocytes/metabolism , Biological Transport/drug effects , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Line , Chemical and Drug Induced Liver Injury/genetics , Hep G2 Cells , Humans , RNA, Messenger/geneticsABSTRACT
Herbal medicines are currently in high demand, and their popularity is steadily increasing. Because of their perceived effectiveness, fewer side effects and relatively low cost, they are being used for the management of numerous medical conditions. However, they are capable of affecting the pharmacokinetics and pharmacodynamics of coadministered conventional drugs. These interactions are particularly of clinically relevance when metabolizing enzymes and xenobiotic transporters, which are responsible for the fate of many drugs, are induced or inhibited, sometimes resulting in unexpected outcomes. This article discusses the general use of herbal medicines in the management of several ailments, their concurrent use with conventional therapy, mechanisms underlying herb-drug interactions (HDIs) as well as the drawbacks of herbal remedy use. The authors also suggest means of surveillance and safety monitoring of herbal medicines. Contrary to popular belief that "herbal medicines are totally safe," we are of the view that they are capable of causing significant toxic effects and altered pharmaceutical outcomes when coadministered with conventional medicines. Due to the paucity of information as well as sometimes conflicting reports on HDIs, much more research in this field is needed. The authors further suggest the need to standardize and better regulate herbal medicines in order to ensure their safety and efficacy when used alone or in combination with conventional drugs.
Subject(s)
Herb-Drug Interactions , Pharmaceutical Preparations/metabolism , Plant Preparations/adverse effects , Plants, Medicinal/adverse effects , Animals , Herbal Medicine , Humans , Plant Preparations/pharmacologyABSTRACT
The risk of drug-induced liver injury (DILI) is of great concern to the pharmaceutical industry. It is well-known that metabolic activation of drugs to form toxic metabolites (TMs) is strongly associated with DILI onset. Drug-induced mitochondrial dysfunction is also strongly associated with increased risk of DILI. However, it is difficult to determine the target of TMs associated with exacerbation of DILI because of difficulties in identifying and purifying TMs. In this study, we propose a sequential in vitro assay system to assess TM formation and their ability to induce mitochondrial permeability transition (MPT) in a one-pot process. In this assay system, freshly-isolated rat liver mitochondria were incubated with reaction solutions of 44 test drugs preincubated with liver microsomes in the presence or absence of NADPH; then, NADPH-dependent MPT pore opening was assessed as mitochondrial swelling. In this assay system, several hepatotoxic drugs, including benzbromarone (BBR), significantly induced MPT in a NADPH-dependent manner. We investigated the rationality of using BBR as a model drug, since it showed the most prominent MPT in our assay system. Both the production of a candidate toxic metabolite of BBR (1',6-(OH)2 BBR) and NADPH-dependent MPT were inhibited by several cytochrome P450 (CYP) inhibitors (clotrimazole and SKF-525A, 100µM). In summary, this assay system can be used to evaluate comprehensive metabolite-dependent MPT without identification or purification of metabolites.
Subject(s)
Benzbromarone/toxicity , Chemical and Drug Induced Liver Injury/etiology , Liver/drug effects , Membrane Potential, Mitochondrial/drug effects , Mitochondria, Liver/drug effects , Activation, Metabolic , Animals , Benzbromarone/metabolism , Chemical and Drug Induced Liver Injury/metabolism , Chemical and Drug Induced Liver Injury/pathology , Cytochrome P-450 CYP2C9 Inhibitors/pharmacology , Cytochrome P-450 CYP3A/metabolism , Cytochrome P-450 CYP3A Inhibitors/pharmacology , Cytochrome P-450 Enzyme System/metabolism , Kinetics , Liver/metabolism , Liver/pathology , Male , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , Mitochondria, Liver/metabolism , Mitochondria, Liver/pathology , Mitochondrial Swelling/drug effects , NADP/metabolism , Rats, Sprague-DawleyABSTRACT
The bile salt export pump (BSEP or Bsep) functions as an apical transporter to eliminate bile acids (BAs) from hepatocytes into the bile. BSEP or Bsep inhibitors engender BA retention, suggested as an underlying mechanism of cholestatic drug-induced liver injury. We previously reported a method to evaluate BSEP-mediated BA-dependent hepatocyte toxicity by using sandwich-cultured hepatocytes (SCHs). However, basal efflux transporters, including multidrug resistance-associated proteins (MRP or Mrp) 3 and 4, also participate in BA efflux. This study examined the contribution of basal efflux transporters to BA-dependent hepatocyte toxicity in rat SCHs. The apical efflux of [(3)H]taurocholic acid (TC) was potently inhibited by 10 µM cyclosporine A (CsA), with later inhibition of basal [(3)H]TC efflux, while MK571 simultaneously inhibited both apical and basal [(3)H]TC efflux. CsA-induced BA-dependent hepatocyte toxicity was 30% at most at 10 µM CsA and â¼60% at 50 µM, while MK571 exacerbated hepatocyte toxicity at concentrations of ≥50 µM. Quinidine inhibited only basal [(3)H]TC efflux and showed BA-dependent hepatocyte toxicity in rat SCHs. Hence, inhibition of basal efflux transporters as well as Bsep may precipitate BA-dependent hepatocyte toxicity in rat SCHs.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Bile Acids and Salts/metabolism , Hepatocytes/metabolism , Multidrug Resistance-Associated Proteins/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 11 , Animals , Cell Culture Techniques , Cells, Cultured , Cholestasis , Cyclosporine/pharmacology , HEK293 Cells , Humans , Propionates/pharmacology , Quinidine/pharmacology , Quinolines/pharmacology , Rats, Sprague-DawleyABSTRACT
Patients with long-lasting hepatitis C virus (HCV) infection are at major risk of hepatocellular carcinoma (HCC). Iron accumulation in the livers of these patients is thought to exacerbate conditions of oxidative stress. Transgenic mice that express the HCV core protein develop HCC after the steatosis stage and produce an excess of hepatic reactive oxygen species (ROS). The overproduction of ROS in the liver is the net result of HCV core protein-induced dysfunction of the mitochondrial respiratory chain. This study examined the impact of ferric nitrilacetic acid (Fe-NTA)-mediated iron overload on mitochondrial damage and ROS production in HCV core protein-expressing HepG2 (human HCC) cells (Hep39b cells). A decrease in mitochondrial membrane potential and ROS production were observed following Fe-NTA treatment. After continuous exposure to Fe-NTA for six days, cell toxicity was observed in Hep39b cells, but not in mock (vector-transfected) HepG2 cells. Moreover, mitochondrial iron ((59)Fe) uptake was increased in the livers of HCV core protein-expressing transgenic mice. This increase in mitochondrial iron uptake was inhibited by Ru360, a mitochondrial Ca(2+) uniporter inhibitor. Furthermore, the Fe-NTA-induced augmentation of mitochondrial dysfunction, ROS production, and cell toxicity were also inhibited by Ru360 in Hep39b cells. Taken together, these results indicate that Ca(2+) uniporter-mediated mitochondrial accumulation of iron exacerbates hepatocyte toxicity caused by the HCV core protein.
Subject(s)
Iron Overload/metabolism , Iron/metabolism , Mitochondria, Liver/metabolism , Viral Core Proteins/genetics , Animals , Cell Line, Tumor , Cell Survival/drug effects , Ferric Compounds , Hep G2 Cells , Humans , Iron Overload/chemically induced , Membrane Potential, Mitochondrial/drug effects , Mice, Inbred C57BL , Mice, Transgenic , Nitrilotriacetic Acid/analogs & derivatives , Reactive Oxygen Species/metabolismABSTRACT
Multidrug resistance-associated protein 2 (MRP2)/ATP-binding cassette protein C2 (ABCC2) and multidrug resistance protein 1 (MDR1)/ABCB1 are well-known efflux transporters located on the brush border membrane of the small intestinal epithelia, where they limit the absorption of a broad range of substrates. The expression patterns of MRP2/ABCC2 and MDR1/ABCB1 along the small intestinal tract are tightly regulated. Several reports have demonstrated the participation of ERM (ezrin/radixin/moesin) proteins in the posttranslational modulation of MRP2/ABCC2 and MDR1/ABCB1, especially with regard to their membrane localization. The present study focused on the in vivo expression profiles of MRP2/ABCC2, MDR1/ABCB1, ezrin, and phosphorylated ezrin to further elucidate the relationship between the efflux transporters and the ERM proteins. The current results showed good correlation between the phosphorylation status of ezrin and Mrp2/Abcc2 expression along the gastrointestinal tract of rats and between the expression profiles of both ezrin and Mdr1/Abcb1 in the small intestine. We also demonstrated the involvement of conventional protein kinase C isoforms in the regulation of ezrin phosphorylation. Furthermore, experiments conducted with wild-type (WT) ezrin and a T567A (Ala substituted Thr) dephosphorylated mutant showed a decrease in membrane surface-localized and total expressed MRP2/ABCC2 in T567A-expressing vs. WT ezrin-expressing Caco-2 cells. In contrast, T567A- and WT-expressing cells both showed an increase in membrane surface-localized and total expressed MDR1/ABCB1. These findings suggest that the phosphorylation status and the expression profile of ezrin differentially direct MRP2/ABCC2 and MDR1/ABCB1 expression, respectively, along the small intestinal tract.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B/metabolism , ATP-Binding Cassette Transporters/metabolism , Cytoskeletal Proteins/metabolism , Intestinal Mucosa/metabolism , Intestine, Small/metabolism , ATP Binding Cassette Transporter, Subfamily B/genetics , ATP-Binding Cassette Transporters/genetics , Animals , Caco-2 Cells , Cytoskeletal Proteins/genetics , Humans , Intestine, Small/cytology , Multidrug Resistance-Associated Protein 2 , Mutation, Missense , Phosphorylation , Protein Kinase C/genetics , Protein Kinase C/metabolism , Protein Processing, Post-Translational , Protein Transport , Rats , Rats, Sprague-DawleyABSTRACT
Test compound A ((5Z)-6-[(2R,3S)-3-({[(4-Chloro-2-methylphenyl)sulfonyl]amino}methyl) bicyclo[2.2.2]oct-2-yl]hex-5-enoic acid) was withdrawn from premarketing clinical trials due to severe liver injury. Intracellular accumulation of lipids (steatosis) has been observed in human-derived cells and may account for the severe hepatotoxicity. Mitochondrial ß-oxidation and ketogenesis play a fundamental role in energy homeostasis. Mitochondrial dysfunction can therefore cause severe deficiency in fatty acid oxidation and apoptosis which finally triggers the hepatocellular injury. Some of hepatotoxic drugs (e.g., salicylic acid, diclofenac and troglitazone) are known to induce mitochondrial dysfunction. This study therefore examined the effect of compound A on the mitochondrial permeability transition (MPT) and membrane potential in mitochondria isolated from mouse, rat and monkey livers. The incubation of rat and monkey mitochondria energized by succinate in the presence of Ca(2+) (20µM) and compound A (2.5-10µM) resulted in cyclosporin A (CsA)-sensitive MPT pore opening and a decline in mitochondrial membrane potential in a concentration-dependent manner. However, mouse mitochondria showed low susceptibility to compound A-induced dysfunction. Rat mitochondrial expression of cyclophilin D (CyPD) was about twice that of mouse mitochondria, but the expression levels of other MPT pore proteins (adenine nucleotide translocator and voltage-dependent anion channel) were comparable in both species. An assessment of the effect of compound A on CyPD knockdown cells demonstrated that mitochondrial susceptibility to compound A was attenuated in CyPD knockdown cells. These results suggest that an interspecies difference in the susceptibility to mitochondrial dysfunction induced by compound A exists as a result of species-specific discrepancies in CyPD expression.
Subject(s)
Bridged Bicyclo Compounds/toxicity , Caproates/toxicity , Cyclophilins/metabolism , Mitochondria, Liver/drug effects , Animals , Peptidyl-Prolyl Isomerase F , HeLa Cells , Humans , In Vitro Techniques , Macaca fascicularis , Male , Membrane Potential, Mitochondrial/drug effects , Mice , Mice, Inbred ICR , Mitochondria, Liver/physiology , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Permeability Transition Pore , Rats , Rats, Sprague-Dawley , Species SpecificityABSTRACT
PURPOSE: Methotrexate (MTX)-induced intestinal mucositis limits the use of the drug. We previously reported that MTX-dependent production of reactive oxygen species is an initiating signal leading to neutrophil migration and intestinal barrier dysfunction. Moreover, alterations of zonula occludens (ZO)-1, an integral component of tight junctions (TJs), contribute to its dysfunction. This study aimed to clarify the identity of inflammatory mediators in the intestine of MTX-treated rats and to evaluate MTX-stimulated alterations in the expression of TJ proteins other than ZO-1 (e.g., occludin and claudins). METHODS: Male Wistar rats were administrated MTX (15 mg kg(-1)) orally once daily for 4 days. Tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, macrophage inflammatory protein (MIP)-2, cytokine-induced neutrophil chemoattractant-2, Toll-like receptor 4 (TLR4), and occludin were determined by real-time RT-PCR. Expression, distribution, and interactions of TJ proteins were evaluated by Western blotting, immunohistochemistry, and immunoprecipitation. RESULTS: MTX increased the mRNA levels of TNF-α, IL-1ß, MIP-2, and TLR4 in the small intestine, as well as the protein expression of claudin-2. Increased claudin-2 and decreased claudin-4 immunostaining were also observed. Occludin mRNA levels were significantly diminished by MTX administration, whereas occludin protein levels and the interaction between ZO-1 and occludin were unaltered; however, the interaction between ZO-1 and claudin-4 was significantly compromised. CONCLUSIONS: These results indicate that elevated levels of inflammatory cytokines and chemokines in the small intestine of MTX-treated rats may contribute to the inhibition of ZO-1/claudin-4 binding, and that inhibition of ZO-1/claudin-4 binding may in turn lead to a reduction in claudin-4 expression.
Subject(s)
Claudin-4/metabolism , Inflammation/chemically induced , Methotrexate/toxicity , Mucositis/chemically induced , Zonula Occludens-1 Protein/metabolism , Animals , Antimetabolites, Antineoplastic/toxicity , Chemokines/metabolism , Cytokines/metabolism , Gene Expression Regulation/drug effects , Inflammation/pathology , Inflammation Mediators/metabolism , Intestine, Small/drug effects , Intestine, Small/pathology , Male , Mucositis/pathology , Occludin/genetics , RNA, Messenger/metabolism , Rats , Rats, Wistar , Reverse Transcriptase Polymerase Chain Reaction , Tight Junctions/metabolism , Tight Junctions/pathologyABSTRACT
BACKGROUND: Previously, we reported that hepatic transporters were down-regulated consistent with intestinal injury in indomethacin (IDM)-treated rats. AIM: The purpose of this study was to characterize this mechanism of the down-regulation of hepatic transporters in IDM-treated rats. METHODS: Hepatic nuclear receptor expressions, oxidative stress condition and the expression of hepatic transporters were evaluated in rats with IDM-induced intestinal injury with or without the administration of mucosal protectant ornoprostil, a prostaglandin E1 analogue, or aminoguanidine (AG), an iNOS inhibitor. RESULTS: All the nuclear receptors examined in the present study, which regulates hepatic transporters, were decreased by the administration of IDM. Hepatic glutathione, an indicator of oxidative stress, was significantly reduced compared with control. We then determined the expression of hepatic transporters by semi-quantitative real-time RT-PCR and Western blot analysis in IDM-treated rats with or without the administration of ornoprostil or AG. Ornoprostil recovered the gene expression of Oatp1a1, Oatp1b2 and Mrp2 and protein expression of Mrp2 while it had no effect on Oatp1a1 and Oatp1b2 proteins. These results indicated that the gene expression of hepatic transporters was down-regulated in association with the intestinal injury. On the other hand, there is no effect of AG on the reduced gene expression of hepatic Oatp1a1, Oatp1b2 and Mrp2. In protein expression, AG slightly recovered Mrp2 expression accompanied by a partial decrease in portal NO levels. CONCLUSIONS: We suggest that the transcriptional process influenced by a dysfunction of hepatic nuclear receptors as well as the effect of NO on the post-transcriptional process due to intestinal injury are partially involved in the down-regulation of hepatic transporters.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/adverse effects , Down-Regulation/drug effects , Indomethacin/adverse effects , Intestinal Mucosa/drug effects , Liver/drug effects , ATP-Binding Cassette Transporters/metabolism , Alprostadil/analogs & derivatives , Alprostadil/pharmacology , Animals , Biomarkers/metabolism , Blotting, Western , Glutathione/metabolism , Guanidines/pharmacology , Intestinal Mucosa/metabolism , Liver/metabolism , Male , Nitric Oxide/blood , Organic Anion Transporters, Sodium-Independent/metabolism , Oxidative Stress/drug effects , Protective Agents/pharmacology , Rats , Rats, Sprague-Dawley , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Solute Carrier Organic Anion Transporter Family Member 1B3ABSTRACT
Previously, we reported a long-lasting inhibition of transport mediated by organic anion-transporting polypeptides (OATPs) in humans and rats by cyclosporin A (CsA). In the present study, we examined the effects of several other compounds on OATP1B1-mediated transport, with a focus on long-lasting inhibition. Effects of coincubation, preincubation, or preincubation plus coincubation of 12 compounds on uptake of estrone 3-sulfate (E1 S) in OATP1B1-expressing HEK293T cells were examined. The OATP1B1 inhibitors used in the present study inhibited OATP1B1-mediated uptake of E1 S in a concentration-dependent manner. Among them, saquinavir and ritonavir in addition to CsA exhibited long-lasting inhibitory effects on OATP1B1-mediated transport of E1 S at ≥ 5 and 25 µM, respectively, even after they were washed out from the incubation buffer. After preincubation with saquinavir, its inhibitory effect on OATP1B1 remained for at least 6 h, whereas the effect of ritonavir did not remain. Protein expression of OATP1B1 was not altered by preincubation with 25 µM saquinavir or ritonavir. The present study firstly showed that saquinavir and ritonavir as well as CsA have long-lasting inhibitory effects on OATP1B1. But, at plasma unbound concentrations of saquinavir and ritonavir in clinical situations, they may not cause long-lasting inhibition of OATP1B1.
Subject(s)
Estrone/analogs & derivatives , HIV Protease Inhibitors/pharmacology , Organic Anion Transporters/antagonists & inhibitors , Organic Anion Transporters/metabolism , Ritonavir/pharmacology , Saquinavir/pharmacology , Biological Transport/drug effects , Estrone/metabolism , Estrone/pharmacokinetics , Gene Expression/drug effects , HEK293 Cells , Humans , Liver-Specific Organic Anion Transporter 1 , Organic Anion Transporters/geneticsABSTRACT
Vernonia amygdalina (VA), Carica papaya (CP), and Tapinanthus sessilifolius (ML) are widely used in some countries as medicinal herbs to treat ailments including malaria, cancer, and diabetes. We previously reported the inhibitory effects of these herbs on permeability glycoprotein (P-gp) in Caco-2 cell monolayers. This study used ex vivo and in vivo models to investigate the likelihood of P-gp-mediated herb-drug interactions occurring. The study utilized excised rat intestinal tissues mounted in Ussing chambers to predict changes in drug absorption and an in vivo study in rats using digoxin as the P-gp substrate. Apparent permeability values and pharmacokinetic parameters of digoxin were compared to determine if co-administration of digoxin with ML, CP, or VA modulated the activity of P-gp. When VA was co-administered, the total area under the plasma concentration-time curve was significantly higher (2.1-fold) than when digoxin was administered alone. Co-administration of ML, VA, and CP significantly increased the mean digoxin apparent permeability in the mucosal-to-serosal direction by 7.8, 43.3, and 54.5%, respectively, in comparison to when digoxin was administered alone. These findings suggest that VA increases intestinal absorption of digoxin in vivo by inhibiting P-gp and may also modulate the pharmacokinetic disposition of other p-gp substrate drugs.
Subject(s)
Digoxin/metabolism , Herb-Drug Interactions , Plant Extracts/pharmacology , ATP Binding Cassette Transporter, Subfamily B/antagonists & inhibitors , ATP Binding Cassette Transporter, Subfamily B/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Animals , Carica/chemistry , Digoxin/pharmacokinetics , Intestinal Absorption/drug effects , Loranthaceae/chemistry , Male , Rats , Rats, Sprague-Dawley , Vernonia/chemistryABSTRACT
Phenformin causes lactic acidosis in clinical situations due to inhibition of mitochondrial respiratory chain complex I. It is reportedly taken up by hepatocytes and exhibits mitochondrial toxicity in the liver. In this study, uptake of phenformin and [(14)C]tetraethylammonium (TEA) and complex I inhibition by phenformin were examined in isolated liver and heart mitochondria. Uptake of phenformin into isolated rat liver mitochondria was higher than that into heart mitochondria. It was inhibited by several cat ionic compounds, which suggests the involvement of multispecific transport system(s). Similar characteristics were also observed for uptake of TEA; however, uptake of phenformin into mitochondria of organic cation/carnitine transporter 1 (OCTN1) knockout mice was lower than that in wild-type mice, whereas uptake of TEA was comparable between the two strains, suggesting the involvement of distinct transport mechanisms for these two cations in mitochondria. Inhibition by phenformin of oxygen consumption via complex I respiration in isolated rat liver mitochondria was greater than that in heart mitochondria, whereas inhibitory effect of phenformin on complex I respiration was similar in inside-out structured submitochondrial particles prepared from rat livers and hearts. Lactic acidosis provoked by iv infusion of phenformin was weaker in octn1(-/-) mice than that in wild-type mice. These observations suggest that uptake of phenformin into liver mitochondria is at least partly mediated by OCTN1 and functionally relevant to its inhibition potential of complex I respiration. This study was, thus, the first to demonstrate OCTN1-mediated mitochondrial transport and toxicity of biguanide in vivo in rodents.
Subject(s)
Carnitine/metabolism , Electron Transport Complex I/antagonists & inhibitors , Mitochondria, Heart/metabolism , Organic Cation Transporter 1/physiology , Phenformin/pharmacokinetics , Animals , Electron Transport Complex I/physiology , HEK293 Cells , Humans , In Vitro Techniques , Mice , Mitochondria, Heart/drug effects , Organic Cation Transporter 1/metabolism , Oxygen Consumption , Phenformin/pharmacology , RatsABSTRACT
Organic anion transporting polypeptide (OATP) family transporters accept a number of drugs and are increasingly being recognized as important factors in governing drug and metabolite pharmacokinetics. OATP1B1 and OATP1B3 play an important role in hepatic drug uptake while OATP2B1 and OATP1A2 might be key players in intestinal absorption and transport across blood-brain barrier of drugs, respectively. To understand the importance of OATPs in the hepatic clearance of drugs, the rate-determining process for elimination should be considered; for some drugs, hepatic uptake clearance rather than metabolic intrinsic clearance is the more important determinant of hepatic clearances. The importance of the unbound concentration ratio (liver/blood), K(p,uu) , of drugs, which is partly governed by OATPs, is exemplified in interpreting the difference in the IC(50) of statins between the hepatocyte and microsome systems for the inhibition of HMG-CoA reductase activity. The intrinsic activity and/or expression level of OATPs are affected by genetic polymorphisms and drug-drug interactions. Their effects on the elimination rate or intestinal absorption rate of drugs may sometimes depend on the substrate drug. This is partly because of the different contribution of OATP isoforms to clearance or intestinal absorption. When the contribution of the OATP-mediated pathway is substantial, the pharmacokinetics of substrate drugs should be greatly affected. This review describes the estimation of the contribution of OATP1B1 to the total hepatic uptake of drugs from the data of fold-increases in the plasma concentration of substrate drugs by the genetic polymorphism of this transporter. To understand the importance of the OATP family transporters, modeling and simulation with a physiologically based pharmacokinetic model are helpful.
Subject(s)
Liver/metabolism , Organic Anion Transporters/metabolism , Pharmaceutical Preparations/metabolism , Animals , Biological Transport , Blood-Brain Barrier/metabolism , Computer Simulation , Humans , Intestinal Absorption , Liver-Specific Organic Anion Transporter 1 , Models, Biological , Organic Anion Transporters/genetics , Organic Anion Transporters, Sodium-Independent/genetics , Organic Anion Transporters, Sodium-Independent/metabolism , Polymorphism, Genetic , Solute Carrier Organic Anion Transporter Family Member 1B3 , Tissue DistributionABSTRACT
ETHNOPHARMACOLOGICAL RELEVANCE: Several herbal medicines are concomitantly used with conventional medicines with a resultant increase in the recognition of herb-drug interactions. The phytomedicines Vernonia amygdalina Delile (VA), family Asteraceae; Azadiractha indica A. Juss (NL), family Meliaceae; Morinda lucida Benth (MLB), family Rubiaceae; Cymbopogon citratus Stapf (LG), family Poaceae; Curcuma longa L. (CUR), family Zingiberaceae; Carica papaya L. (CP), family Caricaceae and Tapinanthus sessilifolius Blume (ML), family Loranthaceae are used in African traditional medicine for the treatment of malaria. They are also used in several regions world over in managing other ailments like cancer and diabetes. This study investigated their interaction with digoxin (DIG) with a view to predict the potential of P-glycoprotein (p-gp) mediated drug-herb interactions occurring with p-gp substrate drugs. MATERIALS AND METHODS: To assess p-gp mediated transport and inhibition, bidirectional transport studies were carried out on Caco-2 cell monolayers using digoxin (DIG) as a model p-gp substrate. Cell functionality was demonstrated using the determinations of transepithelial electric resistance (TEER), cell cytotoxicity testing utilizing the MTT assay as well as the inclusion of inhibition controls. RESULTS: Under the conditions of this study, extracts of ML, VA and CP showed significant inhibition to (3)H-Digoxin basolateral-to-apical (B-A) transport at 0.02-20mg/mL; the concentrations examined. Their apical-to-basolateral (A-B) transport was further investigated. Increases in the mean A-B transport and significant decreases in the B-A transport and efflux ratio values were observed. The apparent permeability coefficient and efflux ratio were computed providing an estimate of drug absorption. CONCLUSION: The findings show that extracts of ML, VA and CP significantly inhibit p-gp in vitro and interactions with conventional p-gp substrate drugs are likely to occur on co-administration which may result in altered therapeutic outcomes.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Anti-Arrhythmia Agents/metabolism , Digoxin/metabolism , Herb-Drug Interactions , Medicine, African Traditional , Plant Extracts/pharmacology , Biological Transport , Caco-2 Cells , Cell Survival/drug effects , Humans , Magnoliopsida , Plant Bark , Plant LeavesABSTRACT
A solid dispersion (SPD) of carbamazepine (CBZ) with hydroxypropyl methylcellulose acetate succinate (HPMC-AS) was prepared by the spray drying method. The apparent solubility (37 °C, pH 7.4) of CBZ observed with the SPD was over 3 times higher than the solubility of unprocessed CBZ. The supersaturated solution was stable for 7 days. A higher concentration of CBZ in aqueous medium was also achieved by mixing with Poloxamer 407 (P407), a solubilizing agent. From permeation studies of CBZ using Caco-2 monolayers and dialysis membranes, we observed improved CBZ permeation across the membrane in the supersaturated solution of CBZ/HPMC-AS SPD. On the contrary, the CBZ-solubilized P407 solution exhibited poor permeation by CBZ. The chemical shifts of CBZ on the (1)H NMR spectrum from CBZ/HPMC-AS SPD solution were not altered significantly by coexistence with HPMC-AS. In contrast, an upfield shift of CBZ was observed in the CBZ/P407 solution. The spin-lattice relaxation time (T(1)) over spin-spin relaxation time (T(2)) indicated that the mobility of CBZ in the HPMC-AS solution was much lower than that in water. Meanwhile, the mobility of CBZ in P407 solution was significantly higher than that in water. NMR data indicate that CBZ does not strongly interact with HPMC-AS. CBZ mobility was suppressed due to self-association and microviscosity around CBZ, which do not affect permeation behavior. Most of the CBZ molecules in the CBZ/P407 solution were solubilized in the hydrophobic core of P407, and a few were free to permeate the membrane. The molecular state of CBZ, as evaluated by NMR measurements, directly correlated with permeation behavior.
Subject(s)
Anticonvulsants/chemistry , Carbamazepine/chemistry , Cell Membrane Permeability , Magnetic Resonance Spectroscopy , Methylcellulose/analogs & derivatives , Anticonvulsants/administration & dosage , Caco-2 Cells , Carbamazepine/administration & dosage , Chemical Phenomena , Chromatography, High Pressure Liquid , Dialysis , Dosage Forms , Drug Compounding , Humans , Membranes, Artificial , Methylcellulose/administration & dosage , Methylcellulose/chemistry , Solubility , X-Ray DiffractionABSTRACT
Antimalarials are widely used in African and Southeast Asian countries, where they are combined with other drugs for the treatment of concurrent ailments. The potential for P-glycoprotein (P-gp)-mediated drug-drug interactions (DDIs) between antimalarials and P-gp substrates was examined using a Caco-2 cell-based model. Selected antimalarials were initially screened for their interaction with P-gp based on the inhibition of rhodamine-123 (Rho-123) transport in Caco-2 cells. Verapamil (100 µM) and quinidine (1 µM) were used as positive inhibition controls. Lumefantrine, amodiaquin, and artesunate all showed blockade of Rho-123 transport. Subsequently, the inhibitory effect of these antimalarials on the bi-directional passage of digoxin (DIG) was examined. All of the drugs decreased basal-to-apical (B-A) P-gp-mediated DIG transport at concentrations of 100 µM and 1 mM. These concentrations may reflect therapeutic doses for amodiaquin and artesunate. Therefore, clinically relevant DDIs may occur between certain antimalarials and P-gp substrates in general.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/physiology , Antimalarials/pharmacology , Caco-2 Cells , Drug Interactions , Humans , Rhodamine 123/metabolismABSTRACT
The purpose of the present study is to examine the long-lasting inhibition of intestinal organic anion transporting polypeptides (Oatps) by cyclosporin A (CsA) in rats using fexofenadine (FEX) as a probe drug. We examined the pharmacokinetics of FEX after its intravenous or oral administration to rats at 3 or 24 h after the oral administration of CsA. When FEX was administered at 3 h after the administration of CsA, its plasma concentration increased regardless of whether it was administered intravenously or orally. When FEX was intravenously administered at 24 h after the oral administration of CsA, its plasma concentration was increased; however, that observed after its oral administration was not significantly different from the vehicle-treated control. When FEX was administered at 3 h after the administration of CsA, the hepatic availability (F(h)) and the fraction absorbed in the intestine as an unchanged form (F(a)·F(g)) of FEX were increased, resulting in increased bioavailability (=F(a)·F(g)·F(h)). At 24 h after the administration of CsA, the F(h) of FEX was increased, whereas its bioavailability was decreased, suggesting that its F(a)·F(g) was decreased because of the long-lasting inhibition. In conclusion, CsA has long-lasting inhibitory effects on Oatps in the rat intestine as well as in the liver.
Subject(s)
Anti-Allergic Agents/pharmacokinetics , Antifungal Agents/pharmacology , Cyclosporine/pharmacology , Intestinal Absorption/drug effects , Organic Anion Transporters/antagonists & inhibitors , Terfenadine/analogs & derivatives , Administration, Oral , Animals , Anti-Allergic Agents/administration & dosage , Anti-Allergic Agents/blood , Antifungal Agents/administration & dosage , Antifungal Agents/blood , Cyclosporine/administration & dosage , Cyclosporine/blood , Gene Expression Regulation/drug effects , Injections, Intravenous , Intestinal Mucosa/metabolism , Intestines/drug effects , Organic Anion Transporters/genetics , RNA, Messenger/genetics , Rats , Rats, Sprague-Dawley , Terfenadine/administration & dosage , Terfenadine/blood , Terfenadine/pharmacokineticsABSTRACT
Multidrug resistance-associated protein 2 (MRP2) is a member of a family of efflux transporters that are involved in biliary excretion of organic anions from hepatocytes. Disrupted canalicular localization and decreased protein expression of MRP2 have been observed in patients with chronic cholestatic disorder and hepatic failure without a change in its mRNA expression. We have previously demonstrated that post-transcriptional regulation of the rapid retrieval of rat MRP2 from the canalicular membrane to the intracelluar compartment occurs under conditions of acute (~30min) oxidative stress. However, it is unclear whether MRP2 expression is decreased during its sustained internalization during chronic oxidative stress. The present study employed buthionine sulfoximine (BSO) to induce chronic oxidative stress in the livers of Sprague-Dawley rats and then examined the protein expression and localization of MRP2. Canalicular MRP2 localization was altered by BSO treatment for 2h without changing the hepatic protein expression of MRP2. While the 8h after exposure to BSO, hepatic MRP2 protein expression was decreased, and the canalicular localization of MRP2 was disrupted without changing the mRNA expression of MRP2. The BSO-induced reduction in MRP2 protein expression was suppressed by pretreatment with N-benzyloxycarbonyl (Cbz)-Leu-Leu-leucinal ( MG-132), a proteasomal inhibitor. Furthermore, the modification of MRP2 by small ubiquitin-relatedmodifier 1 (SUMO-1) was impaired in BSO-treated rat liver,while that by ubiquitin (Ub) and MRP2 was enhanced. Taken together, the results of this study suggest the sustained periods of low GSH content coupled with altered modification of MRP2 by Ub/SUMO-1 were accompanied by proteasomal degradation of MRP2.