Intestinal flora induces the expression of Cyp3a in the mouse liver

1. In order to determine the effects of intestinal flora on the expression of cytochrome P450 (CYP), the mRNA expression of CYP was compared between specific pathogen-free (SPF) and germ-free (GF) mice.

2. Most of the major CYP isozymes showed higher expression in the livers of SPF mice compared with GF mice.

3. Nuclear factors such as pregnane ? receptor (PXR) and constitutive androstane receptor (CAR), as well as transporters and conjugation enzymes involved in the detoxification of lithocholic acid (LCA), also showed higher expression in SPF mice.

4. The findings suggest that in the livers of SPF mice, LCA produced by intestinal flora increases the expression of CYPs via activation of PXR and CAR.

5. Drugs such as antibiotics, some diseases and ageing, etc. are known to alter intestinal flora. The present findings suggest that such changes also affect CYP and are one of the factors responsible for individual differences in pharmacokinetics.

     

Statin-induced myopathy in the rat: relationship between systemic exposure,muscle exposure and myopathy

1. Rare instances of myopathy are associated with all statins, but cerivastatin was withdrawn from clinical use due to a greater incidence of myopathy.

2. The mechanism of statin-induced myopathy with respect to tissue disposition was investigated by measuring the systemic, hepatic, and skeletal muscle exposure of cerivastatin, rosuvastatin, and simvastatin in rats before and after muscle damage.

3. The development of myopathy was not associated with the accumulation of statins in skeletal muscle. For each statin exposure was equivalent in muscles irrespective of their fibre-type sensitivity to myopathy. The low amount of each statin in skeletal muscle relative to the liver does not support a significant role for transporters in the disposition of statins in skeletal muscle. Finally, the concentration of cerivastatin necessary to cause necrosis in skeletal muscle was considerably lower than rosuvastatin or simvastatin, supporting the concept cerivastatin is intrinsically more myotoxic than other statins.

     

Impact of SLCO1B1 (OATP1B1) and ABCG2 (BCRP) genetic polymorphisms and inhibition on LDL-C lowering and myopathy of statins

1. Statins are the preferred class of drugs for treating patients with atherosclerosis and related coronary heart disease. Treatment with statins leads to significant low-density lipoprotein cholesterol (LDL-C) lowering, resulting in reductions in major coronary and vascular events. Statins are generally well tolerated and safe; however, their use is complicated by infrequent, but often serious, muscular adverse events.

2. For many statins, both efficacy and risk of adverse muscle events can be influenced by membrane transporters, which are important determinants of statin disposition. Genetic polymorphisms and drug–drug interactions (DDIs) involving organic anion-transporting polypeptide 1B1 and breast cancer resistance protein have shown the capacity to reduce the activity of these transporters, resulting in changes in LDL-C lowering by statins, as well as changes in the frequency of adverse muscle events associated with their use.

3. This review presents evidence for how reduced transporter activity impacts the safety and pharmacology of statins. It expands on the scope of other recent statin reviews by providing recommendations on in vitro evaluation of statin interaction potential, discussing how reduced transporter activity impacts statin management during drug development, and proposing ideas on how to evaluate the impact of DDI on statin efficacy during clinical trials. Furthermore, the potential clinical consequences of perturbing statin efficacy via DDI are discussed.

     

Species difference in the regulation of cytochrome P450 2S1: lack of induction in rats by the aryl hydrocarbon receptor agonist PCB126

1. CYP2S1 is an evolutionarily conserved, mainly extra-hepatic member of the CYP2 family and proposed to be regulated by the aryl hydrocarbon receptor (AhR).

2. The present study explores AhR’s regulation of CYP2S1 in male Sprague Dawley rats using PCB126 (3,3′,4,4′,5-pentachlorobiphenyl), the most potent AhR agonist among the PCBs. Additionally, CYP2S1 expression was examined after treatments with the classic CYP-inducers β-naphthoflavone (β-NF, AhR activator), phenobarbital (PB, CAR activator) and dexamethasone (Dex, PXR activator). CYP2S1 and CYP1A1/2, CYP1B1, CYP2B and CYP3A mRNAs were measured in liver, lung, spleen, stomach, kidney, and thymus at different time points.

3. Constitutive CYP2S1 was expressed at comparable levels to other CYPs with the highest expression levels in stomach, kidney and lung. CYP2S1 mRNA was only non-significantly elevated by β-NF in liver tissues. PCB126 did not increase CYP2S1 mRNA in any organ and at any time point examined despite a significant induction of CYP1 genes. PCB126 reduced CYP2S1 mRNA by 40% (not significant) from the 7th post-exposure day in thymus. PB and Dex had no effect on CYP2S1 mRNA levels.

4. These observations show that in this model CYP2S1 is not, or only weakly, regulated by AhR and not induced by CAR or PXR activators.

     

Regulation of human pregnane X receptor and its target gene cytochrome P450 3A4 by Chinese herbal compounds and a molecular docking study

1. The pregnane X receptor (PXR) plays a critical role in the regulation of human cytochrome P450 3A4 (CYP3A4) gene. In this study, we investigated the effect of an array of compounds isolated from Chinese herbal medicines on the activity of PXR using a luciferase reporter gene assay in transiently transfected HepG2 and Huh7 cells and on the expression of PXR and CYP3A4 in LS174T cells. Furthermore, molecular docking was performed to investigate the binding modes of herbal compounds with PXR.

2. Praeruptorin A and C, salvianolic acid B, sodium danshensu, protocatechuic aldehyde, cryptotanshinone, emodin, morin, and tanshinone IIA significantly transactivated the CYP3A4 reporter gene construct in either HepG2 or Huh7 cells. The PXR mRNA expression in LS174T cells was significantly induced by physcion, protocatechuic aldehyde, salvianolic acid B, and sodium danshensu. However, epifriedelanol, morin, praeruptorin D, mulberroside A, tanshinone I, and tanshinone IIA significantly down-regulated the expression of PXR mRNA in LS174T cells.

3. All the herbal compounds tested can be readily docked into the ligand-binding cavity of PXR mainly through hydrogen bond and aromatic interactions with Ser247, Gln285, His407, and Arg401.

4. These findings suggest that herbal medicines can significantly regulate PXR and CYP3A4 and this has important implication in herb–drug interactions.

     

Influence of capsaicin on fluctuation of digoxin pharmacokinetics in lipopolysaccharide-treated rats

1. In the present study, we investigated the influence of Cap on digoxin pharmacokinetics in lipopolysaccharide (LPS)-treated rats.

2. After the oral administration of digoxin (0.1?mg/kg), the area under the plasma concentration-time curve (AUC) of digoxin increased significantly until day 3 after LPS treatment. In the LPS + Cap group, the recovery period of AUC was shortened to 3 days. On days 5 and 7, the maximum plasma concentrations decreased significantly as compared to the control group. The bioavailability of digoxin in LPS group was higher than that in the LPS + Cap group.

3. The hepatic cytochrome P450 (CYP) 3A2 content decreased significantly until day 5 after LPS administration, but it returned to the control level until 5 days in the LPS + Cap group. Hepatic CYP3A2 mRNA expression of LPS group decreased significantly until day 3, but it returned to the control level on day 3 and increased significantly until day 7 in the LPS + Cap group.

4. The DNA-binding activity of pregnane X receptor (PXR) was increased on days 3–7 in the Cap and LPS + Cap group.

5. Cap decreased the absorption of digoxin by inducing CYP3A2 mRNA expression via indirect activation of PXR in LPS-treated rats.

     

Comparison of the hepatic and thyroid gland effects of sodium phenobarbital and pregnenolone-16α-carbonitrile in wild-type and constitutive androstane receptor (CAR)/pregnane X receptor (PXR) knockout rats

1. The hepatic and thyroid gland effects of the constitutive androstane receptor (CAR) activator sodium phenobarbital (NaPB) and the pregnane X receptor (PXR) activator pregnenolone-16α-carbonitrile (PCN) were examined in male Sprague-Dawley wild-type (WT) and knockout (KO) rats lacking both hepatic CAR and PXR receptors (CAR KO/PXR KO rats).

2. The treatment of WT rats for 7?d with 500?ppm NaPB in the diet and 100?mg/kg/d PCN by gavage resulted in increased relative liver weight, hepatocyte hypertrophy, increased hepatocyte replicative DNA synthesis (RDS) and induction of cytochrome P450 CYP2B and CYP3A subfamily enzymes. NaPB and PCN also induced thyroid gland follicular cell RDS and hepatic microsomal UDP-glucuronosyltransferase activity towards thyroxine as substrate. These effects were not observed in the liver and thyroid gland of CAR KO/PXR KO rats.

3. Male C57BL/6?J (WT) and CAR KO/PXR KO mice were given 1000?ppm NaPB in the diet for 7?d. In WT, but not in CAR KO/PXR KO, mice NaPB treatment resulted in liver hypertrophy and induction of hepatocyte RDS and Cyp2b enzymes.

4. These results suggest that the CAR KO/PXR KO rat and mouse models are useful experimental models for mode of action studies with rodent CAR activators.

     

Characterization of the porcine constitutive androstane receptor (CAR) and its splice variants

1. Porcine constitutive androstane receptor (CAR; NR1I3) was cloned and compared for homology and activity with mouse and human CAR (mCAR, hCAR). Porcine CAR (pgCAR) was 86% and 75% homologous to hCAR at the nucleotide and protein levels.

2. Five alternatively spliced variants of pgCAR were identified, each of which generated a truncated protein product. Real-time polymerase chain reaction (PCR) analyses showed that these variants were present in pig liver cDNA samples from 4.61% to 9.20% of total pgCAR.

3. pgCAR and hCAR responded similarly to more ligands than did hCAR and mCAR. The known hCAR agonist (6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime (CITCO) activated pgCAR, while the murine agonist 1,4 bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) had no effect. 5β-dihydrotestosterone was identified as a novel inverse agonist of both pgCAR and hCAR.

4. pgCAR splice variant 2 (SV2) had a dose-dependent dominant negative effect on the activity of wild-type pgCAR in dual luciferase assays. SV2 had no effect against pgPXR (pregnane X receptor) or pgFXR (farnesoid X receptor) activity when using PXR- or FXR-specific reporters.

     

Altered expression of CYP in TSOD mice: a model of type 2 diabetes and obesity

1. To investigate the pharmacokinetic characteristics in TSOD (Tsumura, Suzuki, obese, diabetes) mice, a model of type 2 diabetes and obesity, the expressions of major hepatic CYP enzymes in TSOD and TSNO (Tsumura, Suzuki, non-obesity; control) mice were compared.

2. The 7-month-old TSOD mice, which represented severe obesity/diabetes-related pathophysiology, showed higher expressions of Cyp2c and Cyp3a compared with TSNO mice, while those of Cyp1a and Cyp2e were lower. Cyp3a metabolic activity was also higher in TSOD mice.

3. In the 7-month-old liver, pregnane X receptor (PXR) (nuclear receptor) and peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) (cofactor) mRNA expression were higher in TSOD mice, possibly playing a role in the altered expression of Cyp3a.

4. This specifically altered CYP expression in TSOD mice suggests that the biotransformation of drugs metabolized by these CYP enzymes differs from that in normal animals. Based on these findings, further investigation on the relationship between altered CYP expression and pathophysiology may be useful in elucidating changes in pharmacokinetics in obese/diabetic patients.

     

o-Nitroanilines as major metabolic products of anti-Trypanosoma cruzi 5-phenylethenylbenzofuroxans in microsomal and cytosolic fractions of rat hepatocytes and in whole parasitic cells

1. The metabolism of six anti-Trypanosoma cruzi 5-phenylethenylbenzofuroxans (PhEBfx) was studied in vitro using rat hepatic microsomal and cytosolic fractions as a mammalian model and whole cells of T. cruzi as a parasitic model.

2. Some of the expected metabolites were synthesized to provide authentic chromatographic standards.

3. The metabolites were identified using high-performance liquid chromatography (HPLC) in comparison with the authentic standards and their proportions were determined. Their structures were confirmed using mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy.

4. The behaviour of the six PhEBfx in the three different systems was similar. The main metabolites, formed by reductive processes, were the corresponding o-nitroanilines.

5. Two of the test compounds were studied for extended time periods in the rat liver preparations and their terminal metabolites were identified as o-phenylendiamine derivatives.

     

Studies on induction of lamotrigine metabolism in transgenic UGT1 mice

1. A transgenic ‘knock-in’ mouse model expressing a human UGT1 locus (Tg-UGT1) was recently developed and validated. Although these animals express mouse UGT1A proteins, UGT1A4 is a pseudo-gene in mice. Therefore, Tg-UGT1 mice serve as a ‘humanized’ UGT1A4 animal model.

2. Lamotrigine (LTG) is primarily metabolized to its N-glucuronide (LTGG) by hUGT1A4. This investigation aimed at examining the impact of pregnane X receptor (PXR), constitutive androstane receptor (CAR) and peroxisome proliferator-activated receptor (PPAR) activators on LTG glucuronidation in vivo and in vitro. Tg-UGT1 mice were administered the inducers phenobarbital (CAR), pregnenolone-16α-carbonitrile (PXR), WY-14643 (PPAR-α), ciglitazone (PPAR-γ), or L-165041 (PPAR-β), once daily for 3 or 4 days. Thereafter, LTG was administered orally and blood samples were collected over 24?h. LTG was measured in blood and formation of LTGG was measured in pooled microsomes made from the livers of treated animals.

3. A three-fold increase in in vivo LTG clearance was seen after phenobarbital administration. In microsomes prepared from phenobarbital-treated Tg-UGT1 animals, 13-fold higher CL_int (V_max/K_m) value was observed as compared with the untreated transgenic mice. A trend toward induction of catalytic activity in vitro and in vivo was also observed following pregnenolone-16α-carbonitrile and WY-14643 treatment. This study demonstrates the successful application of Tg-UGT1 mice as a novel tool to study the impact of induction and regulation on metabolism of UGT1A4 substrates.

     

Alternative Designs for Clinical Trials

Abstract

Background: The classical two-period crossover and two-parallel-groups designs for clinical drug trials are unable to answer many current scientific questions, and are sometimes ethically or financially impossible.

Objectives: To identify what a classical clinical trial cannot manage, to summarize and discuss alternative trial designs that are helpful for such purposes.

Results: What a classical clinical txkI cannot manage

1. assess multimodal therapies

2. account historical data

3. safeguard ethics and efficacy during the course of long-term trials

4. study drugs, before well-established toxicity information is available

5. account the possibility of therapeutic equivalence between test and reference treatment

6. study multiple treatments in one trial

7. adjust change scores for baseline levels.

Alternative designs helpful for such purposes are respectively

1. factorial designs

2. historical controls designs

3. group-sequential interim analysis designs

4. sequential designs for continuous monitoring

5. therapeutic equivalence designs

6. multiple crossover-periods / multiple parallel-groups design

7. increased precision designs through multivariate adjustment.

Main problems include the increased risks of type I/II errors, the loss of validity criteria.

Conclusions: Non-classical trial designs are reviewed. They offer relevant scientific, ethical, and financial advantages. The increased risks of type MI errors should be accounted for in the design stage of the trial.     

In vivo metabolism and kinetics of ethylene glycol monobutyl ether and its metabolites, 2-butoxyacetaldehyde and 2-butoxyacetic acid,as measured in blood,liver and forestomach of mice

1. Ethylene glycol monobutyl ether (EGBE) causes forestomach hyperplasia and neoplasia in mice when administered chronically by inhalation.

2. The study was initiated to test the physiologically based pharmacokinetic (PBPK) model prediction that 2-butoxyacetaldehyde (BAL), a transient, labile intermediate in the oxidation of EGBE to butoxyacetic acid (BAA), is unlikely to achieve concentrations sufficient to cause DNA damage in target tissues.

3. Male and female B6C3F1 mice were administered a high oral dose of EGBE (600?mg?kg^?1), and tissues were collected at 5, 15, 45 and 90?min following the dose. The tissues were processed for determination of EGBE, BAL and BAA by gas chromatography-mass spectrometry.

4. BAL was detected at low concentrations in all tissues sampled and at all time points following EGBE administration (about 0.3–33?μM). BAL concentrations were highest in the initial samples (5?min) in all tissues and declined from that point.

5. BAL concentrations in liver and forestomach tissues corresponded to the peak concentrations predicted by an already published PBPK model, and are higher than BAL concentrations that could be achieved by inhalation exposure to EGBE.

6. Mouse inhalation exposure to EGBE is therefore unlikely to generate BAL concentrations in tissues sufficient to initiate a carcinogenic response.

     

Identification of Three Novel Natural Product Compounds that Activate PXR and CAR and Inhibit Inflammation

Purpose

To investigate the effects of three natural product compounds, carapin, santonin and isokobusone, on the activity of pregnane X receptor (PXR) and constitutive androstane receptor (CAR) in induction of drug-metabolizing enzymes and inhibition of inflammation.

Methods

The monkey kidney-derived fibroblast (CV-1) cells and human embryonic kidney HEK293 cells were used for transient transfection and luciferase reporter gene assays. Human primary hepatocytes and primary hepatocytes from wild type, PXR?/?, and hPXR transgenic mice were used to study the induction of drug-metabolizing enzymes and the implication of these compounds in inflammation.

Results

Carapin, santonin and isokobusone activated both PXR and CAR in transient transfection and luciferase reporter gene assays. Mutagenesis studies showed that two amino acid residues, Phe305 of the rodent PXR and Leu308 of the human PXR, are critical for the recognition of these compounds by PXR. Importantly, the activation of PXR and CAR by these compounds induced the expression of drug-metabolizing enzymes in primary human and mouse hepatocytes. Furthermore, activation of PXR by these compounds inhibited the expression of inflammatory mediators in response to lipopolysaccharide (LPS). The effects of these natural compounds on drug metabolism and inflammation were abolished in PXR?/? hepatocytes.

Conclusions

Our results show that carapin, santonin and isokobusone activate PXR and CAR and induce drug-metabolizing enzymes. In addition, these compounds inhibited the expression of inflammatory mediators in response to LPS through the activation of PXR.     

Baicalin,an emerging multi-therapeutic agent: pharmacodynamics,pharmacokinetics, and considerations from drug development perspectives

1. Baicalin was extensively researched for utility in a number of therapeutic areas owing to its anti-inflammatory, anti-oxidant, anti-bacterial, and anti-cancer properties.

2. A number of preclinical studies, in vitro work, and mechanistic studies were performed to understand the absorption, distribution, metabolism, and excretion profiles of baicalin.

3. The absorption of baicalin involved several complexities: the restriction to two distant sites; the conversion of baicalin to baicalein; the possible role of transporter(s); and enhanced absorption due to breakdown of conjugates by beta-glucuronidase.

4. Limited distribution data suggest that baicalin reached several sites such as the brain, eye lens, thymus, etc. Hepatobiliary recycling also served as a distribution phase for sustained delivery of baicalin.

5. Metabolism data suggest the rapid conversion of baicalin to baicalein, which was extensively subjected to Phase 2 metabolism, conjugates baicalein glucuronide/sulfate have been identified.

6. Limited excretion data suggest involvement of renal and faecal routes—glucuronide and sulfate conjugates were excreted in urine and faeces (via biliary excretion).

7. The published data on baicalin suggest imminent challenges for developing baicalin and/or during co-administration with other agents. These challenges are absorption related (transporter or changes in the microenvironment), metabolism related (CYP2B6 induction and/or CYP2E1 inhibition), and excretion/efflux related (competitive biliary pathway and/or OATP1B1 transport).