Discovery of CYR715: A novel carboxylic acid-containing soluble guanylate cyclase stimulator.

Soluble guanylate cyclase (sGC) is a clinically validated therapeutic target in the treatment of pulmonary hypertension. Modulators of sGC have the potential to treat diseases that are affected by dysregulation of the NO-sGC-cGMP signal transduction pathway. This letter describes the SAR efforts that led to the discovery of CYR715, a novel carboxylic acid-containing sGC stimulator, with an improved metabolic profile relative to our previously described stimulator, IWP-051. CYR715 addressed potential idiosyncratic drug toxicity (IDT) liabilities associated with the formation of reactive, migrating acyl glucuronides (AG) found in related carboxylic acid-containing analogs and demonstrated high oral bioavailability in rat and dose-dependent hemodynamic pharmacology in normotensive Sprague-Dawley rats.

Multidrug Resistance-Associated Protein 3 Is Responsible for the Efflux Transport of Curcumin Glucuronide from Hepatocytes to the Blood.

Curcumin, a major polyphenol present in turmeric, is predominantly converted to curcumin-O-glucuronide (COG) in enterocytes and hepatocytes via glucuronidation. COG is a principal metabolite of curcumin in plasma and feces. It appears that the efflux transport of the glucuronide conjugates of many compounds is mediated largely by multidrug resistance-associated protein (MRP) 3, the gene product of the ATP-binding cassette, subfamily C, member 3. However, it is currently unknown whether this was the case with COG. In this study, Mrp3 knockout (KO) and wild-type (WT) mice were used to evaluate the pharmacokinetics profiles of COG, the liver-to-plasma ratio of COG, and the COG-to-curcumin ratio in plasma, respectively. The ATP-dependent uptake of COG into recombinant human MRP3 inside-out membrane vesicles was measured for further identification, with estradiol-17ß-d-glucuronide used in parallel as the positive control. Results showed that plasma COG concentrations were extremely low in KO mice compared with WT mice, that the liver-to-plasma ratios of COG were 8-fold greater in KO mice than in WT mice, and that the ATP-dependent uptake of COG at 1 or 10 µM was 5.0- and 3.1-fold greater in the presence of ATP than in the presence of AMP, respectively. No significant differences in the Abcc2 and Abcg2 mRNA expression levels were seen between Mrp3 KO and WT mice. We conclude that Mrp3 is identified to be the main efflux transporter responsible for the transport of COG from hepatocytes into the blood. SIGNIFICANCE STATEMENT: This study was designed to determine whether multidrug resistance-associated protein (Mrp) 3 could be responsible for the efflux transport of curcumin-O-glucuronide (COG), a major metabolite of curcumin present in plasma and feces, from hepatocytes into the blood using Mrp3 knockout mice. In this study, COG was identified as a typical Mrp3 substrate. Results suggest that herb-drug interactions would occur in patients concomitantly taking curcumin and either an MRP3 substrate/inhibitor or a drug that is predominantly glucuronidated by UDP-glucuronosyltransferases.

Chemopreventive efficacy of oral curcumin: a prodrug hypothesis.

Oral consumption of curcumin, a natural polyphenol, is associated with reduced incidence of cancer. Yet, a significant amount of the orally dosed compound is eliminated in the feces, and a major fraction of the absorbed compound is metabolized to inactive glucuronides, resulting in poor bioavailability (<1%). It is not known how oral curcumin exhibits chemopreventive activity. We propose curcumin glucuronide is an inflammation-responsive natural prodrug that is converted back to curcumin on demand at the site of action. Our studies show elevated levels of ß-glucuronidase, an enzyme that hydrolyzes the glycosidic bond of glucuronides to generate the parent compound, in human breast cancer. Oral administration of curcumin in mouse tumor models generated significant tumor levels of the polyphenol. Intravenous administration of the glucuronide resulted in the formation of curcumin in the tumor tissue. Chronic daily oral curcumin dosing led to tumor accumulation of curcumin and inhibition of tumor growth in tumor models with high ß-glucuronidase activity. Overall, the study presented here provides preliminary evidence for a novel mechanism of action for orally administered curcumin.-Liu, G., Khanna, V., Kirtane, A., Grill, A., Panyam, J. Chemopreventive efficacy of oral curcumin: a prodrug hypothesis.

Pharmacokinetics, Pharmacodynamics, and PKPD Modeling of Curcumin in Regulating Antioxidant and Epigenetic Gene Expression in Healthy Human Volunteers.

Curcumin is a major component of the spice turmeric ( Curcuma longa), often used in food or as a dietary supplement. Many preclinical studies on curcumin suggest health benefits in many diseases due to its antioxidant/anti-inflammatory and epigenetic effects. The few human studies and curcumin's unfavorable pharmacokinetics (PK) have limited its potential, leading researchers to study and develop formulations to improve its PK. The purpose of this clinical study is to describe the acute pharmacokinetics and pharmacodynamics (PK/PD) of commercially marketed curcumin in normal, healthy human volunteers. Twelve volunteers received a 4 g dose of curcumin capsules with a standard breakfast. Plasma samples were collected at specified time points and analyzed for curcumin and its glucuronide levels. RNA was extracted from leukocytes and analyzed for expression of select antioxidant and epigenetic histone deacetylase (HDAC) genes. Plasma levels of parent curcumin were below the detection limit by HPLC-ITMS/MS/MS. However, curcumin-O-glucuronide (COG), a major metabolite of curcumin, was detected as soon as 30 min. These observations of little to no curcumin and some levels of metabolite are in line with previous studies. PD marker antioxidant genes NRF2, HO-1, and NQO1 and epigenetic genes HDAC1, HDAC2, HDAC3, and HDAC4 were quantified by qPCR. COG PK is well-described by a one-compartment model, and the PK/PD of COG and its effect on antioxidant and epigenetic gene expression are captured by an indirect response model (IDR). A structural population PK model was sequentially established using a nonlinear mixed-effect model program (Monolix Lixoft, Orsay, France). Physiologically based pharmacokinetic modeling (PBPK) and simulation using Simcyp correlated well with the observed data. Taken together, these results show that the bioavailability of the parent curcumin compound is low, and oral administration of curcumin can still deliver detectable levels of curcumin glucuronide metabolite. But most importantly, it elicits antioxidant and epigenetic effects which could contribute to the overall health beneficial effects of curcumin.

Liquid chromatography-tandem mass spectrometry assay for the simultaneous determination of three major flavonoids and their glucuronidated metabolites in rats after oral administration of Artemisia annua L. extract at a therapeutic ultra-low dose.

The traditional antimalarial herb Artemisia annua L., from which artemisinin is isolated, is widely used in endemic regions. It has been suggested that artemisinin activity can be enhanced by flavonoids in A. annua; however, how fast and how long the flavonoids are present in the body remains unknown. In the present study, a rapid and sensitive liquid chromatography with tandem mass spectrometry method was developed and validated for the simultaneous determination of three major flavonoids components, i.e. chrysosplenol D, chrysoplenetin, and artemetin and their glucuronidated metabolites in rats after oral administrations of A. annua extracts at a therapeutic ultra-low dose. The concentration of the intact form was determined directly, and the concentration of the glucuronidated form was assayed in the form of flavonoids aglycones, after treatment with ß-glucuronidase/sulfatase. The method was linear in the range of 0.5-300.0 ng/mL for chrysoplenetin and artemetin, and 2-600 ng/mL for chrysosplenol D. All the validation data conformed to the acceptance requirements. The study revealed a significantly higher exposure of the flavonoid constituents in conjugated forms in rats, with only trace intact from. Multiple oral doses of A. annua extracts led to a decreased plasma concentration levels for three flavonoids.

Antidiabetic Effect of Cyclocarya paliurus Leaves Depends on the Contents of Antihyperglycemic Flavonoids and Antihyperlipidemic Triterpenoids.

Cyclocarya paliurus has been used commonly to treat diabetes in China. However, the effective components and the effect of plant origin remain unclear. In this study, C. paliurus leaves with different chemical compositions were selected from five geographical locations, and their effects on streptozotocin (STZ)-induced diabetic mice were evaluated with both ethanol and aqueous extracts. Glucose levels, lipid levels, and biomarkers of liver and kidney function were measured. The principal components of both C. paliurus ethanol and aqueous extracts from different geographical locations differed quantitatively and qualitatively. Results showed that C. paliurus extracts with better antihyperglycemic effects were characterized by higher contents of total flavonoids, especially quercetin-3-O-glucuronide and kaempferol-3-O-glucuronide. Furthermore, significantly negative correlations were found between triterpenoids contents and lipid levels. These results revealed the potential antihyperglycemic capacity of C. paliurus flavonoids and the antihyperlipidemic effect of C. paliurus triterpenoids. Thus, we suggest that the composition of C. paliurus compounds might help to design therapeutic alternatives for the treatment of diabetes mellitus. However, geographic origins and the extraction solvents can also affect the effectiveness of the treatment as these factors influence the chemical compositions and thereby the biological activities.

Randomized Trial of Food Effect on Pharmacokinetic Parameters of ABX464 Administered Orally to Healthy Male Subjects.

ABX464 is an antiviral that provides a novel approach to the reduction and control of HIV infection. Investigation of food influence is important in the optimization of treatment. An open-label, food effect, randomized study which included 2 groups of 24 subjects each was carried out to assess the bioavailability and safety of single (group 1) and repeated (group 2) oral doses of ABX464 (50 mg) under fed or fasted conditions. The maximum concentration (Cmax) and the area under the concentration-time curve from time zero to infinity (AUC0-∞) of ABX464 were demonstrated to increase with food after a single dose of ABX464 (219% and 188%, respectively). The apparent terminal elimination half-lives (t1/2s) under fed and fasted conditions were comparable, at about 0.80 h. The median time to maximum concentration (Tmax) was delayed from 1.5 to 2.8 h, and the ratio of the AUC0-∞ obtained under fed conditions to the AUC0-∞ obtained under fasted conditions (Frel) was 2.69. Comparable results were obtained on day 1 and day 10 in group 2. The increases in Cmax and AUC0-∞ of the metabolite ABX464-N-glucuronide (ABX464-NGlc) were, however, much more limited when ABX464 was given with food. The t1/2s were also comparable under the two conditions (around 100 h). Between day 1 and day 10, the Cmax increased by 5% under the fasted condition and by 25% under the fed condition. The most common related treatment-emergent adverse events were headaches, vomiting, and nausea. It was concluded that food has a significant impact on the levels of ABX464 in plasma with a delay in absorption and increased relative bioavailability, with a lesser impact on its biotransformation into ABX464-NGlc. ABX464 was well tolerated under both fasted and fed conditions. (This study has been registered at ClinicalTrials.gov under registration no. NCT02731885.).

Curcumin ß-D-Glucuronide Plays an Important Role to Keep High Levels of Free-Form Curcumin in the Blood.

Curcumin, a polyphenol derived from the rhizome of the naturally occurring plant Curcuma longa, has various pharmacological actions such as antioxidant and anti-inflammatory effects. In this paper, we evaluated the role of its internal metabolite, curcumin ß-D-glucuronide (curcumin monoglucuronide, CMG), by investigating curcumin kinetics and metabolism in the blood. Firstly, we orally administered highly bioavailable curcumin to rats to elucidate its kinetics, and observed not only the free-form of curcumin, but also, curcumin in a conjugated form, within the portal vein. We confirmed that curcumin is conjugated when it passes through the intestinal wall. CMG, one of the metabolites, was then orally administered to rats. Despite its high aqueous solubility compared to free-form curcumin, it was not well absorbed. In addition, CMG was injected intravenously into rats in order to assess its metabolic behavior in the blood. Interestingly, high levels of free-form curcumin, thought to be sufficiently high to be pharmacologically active, were observed. The in vivo antitumor effects of CMG following intravenous injection were then evaluated in tumor-bearing mice with the HCT116 human colon cancer cell line. The tumor volume within the CMG group was significantly less than that of the control group. Moreover, there was no significant loss of body weight in the CMG group compared to the control group. These results suggest that CMG could be used as an anticancer agent without the serious side effects that most anticancer agents have.

Oral Morphine Pharmacokinetic in Obesity: The Role of P-Glycoprotein, MRP2, MRP3, UGT2B7, and CYP3A4 Jejunal Contents and Obesity-Associated Biomarkers.

The objective of our work was to study the association between the jejunal expression levels of P-gp, MRP2, MRP3, UGT2B7, CYP3A4, the ABCB1 c.3435C > T polymorphism, and several obesity-associated biomarkers, as well as oral morphine and glucuronides pharmacokinetics in a population of morbidly obese subjects. The pharmacokinetics of oral morphine (30 mg) and its glucuronides was performed in obese patients candidate to bariatric surgery. A fragment of jejunal mucosa was preserved during surgery. Subjects were genotyped for the ABCB1 single nucleotide polymorphism (SNP) c.3435C > T. The subjects were 6 males and 23 females, with a mean body mass index of 44.8 (35.4-61.9) kg/m(2). The metabolic ratios AUC0-inf M3G/morphine and AUC0-inf M6G/morphine were highly correlated (rs = 0.8, p < 0.0001) and were 73.2 ± 24.6 (34.7-137.7) and 10.9 ± 4.1 (3.8-20.6). The pharmacokinetic parameters of morphine and its glucuronides were not associated with the jejunal contents of P-gp, CYP3A4, MRP2, and MRP3. The jejunal content of UGT2B7 was positively associated with morphine AUC0-inf (rs = 0.4, p = 0.03). Adiponectin was inversely correlated with morphine Cmax (rs = -0.44, p = 0.03). None of the factors studied was associated with morphine metabolic ratios. The interindividual variability in the jejunal content of drug transporters and metabolizing enzymes, the ABCB1 gene polymorphism, and the low-grade inflammation did not explain the variability in morphine and glucuronide exposure. High morphine metabolic ratio argued for an increased morphine glucuronidation in morbidly obese patients.

In vivo-formed versus preformed metabolite kinetics of trans-resveratrol-3-sulfate and trans-resveratrol-3-glucuronide.

Metabolites in safety testing have gained a lot of attention recently. Regulatory agencies have suggested that the kinetics of preformed and in vivo-formed metabolites are comparable. This subject has been a topic of debate. We have compared the kinetics of in vivo-formed with preformed metabolites. trans-3,5,4'-Trihydroxystilbene [trans-resveratrol (RES)] and its two major metabolites, resveratrol-3-sulfate (R3S) and resveratrol-3-glucuronide (R3G) were used as model substrates. The pharmacokinetics (PK) of R3S and R3G were characterized under two situations. First, the pharmacokinetics of R3S and R3G were characterized (in vivo-formed metabolite) after administration of RES. Then, synthetic R3S and R3G were administered (preformed metabolite) and their pharmacokinetics were characterized. PK models were developed to describe the data. A three-compartment model for RES, a two-compartment model for R3S (preformed), and an enterohepatic cycling model for R3G (preformed) was found to describe the data well. These three models were further combined to build a comprehensive PK model, which was used to perform simulations to predict in vivo-formed metabolite kinetics. Comparisons were made between in vivo-formed and preformed metabolite kinetics. Marked differences were observed in the kinetics of preformed and in vivo-formed metabolites.

Development of population PK model with enterohepatic circulation for mycophenolic acid in patients with childhood-onset systemic lupus erythematosus.

AIM: This study aimed to develop a population pharmacokinetic (PK) enterohepatic recycling model for MPA in patients with childhood-onset systemic lupus erythematosus (cSLE). METHODS: MPA concentration-time data were from outpatients on stable oral mycophenolate mofetil (MMF) and collected under fasting conditions, with standardized meals (1 and 4 h post-dose). Sampling times were pre-dose, 20, 40 min, 1, 1.5, 2, 3, 4, 6 and 9 h, post dose. The population PK analysis simultaneously modelled MPA and 7-O-MPA-ß-glucuronide (MPAG) concentrations using nonlinear mixed effect modelling. RESULTS: PK analysis included 186 MPA and MPAG concentrations (mg l(-1)) from 19 patients. cSLE patients, age range 10-28 years, median 16.5 years were included. Mean ± SD disease duration was 3.8 ± 3.7 years. The final PK model included a gallbladder compartment for enterohepatic recycling and bile release time related to meal times, with first order absorption and single series of transit compartments. The PK estimates for MPA were CL(1) /F 25.3 l h(-1), V(3) /F 20.9 l, V(4) /F 234 l and CL(2) /F 19.8 l h(-1). CONCLUSION: The final model fitted the complex processes of absorption and enterohepatic circulation (EHC) in those treated with MMF for cSLE and could be applied in Bayesian dose optimization algorithms.

Method for predicting human intestinal first-pass metabolism of UGT substrate compounds.

1. As intestinal glucuronidation has been suggested to generate the low oral bioavailability (F) of drugs, estimating its effects would be valuable for selecting drug candidates. Here, we investigated the absorption and intestinal availability (F(a)F(g)) in animals, and intrinsic clearance via UDP-glucuronosyltransferase (UGT) in intestinal microsomes (CL(int,UGT)) for three drug candidates possessing a carboxylic acid group, in an attempt to estimate the impact of intestinal glucuronidation on F and select potential drug candidates with high F in humans. 2. The F(a)F(g) values of the three test compounds were low in rats and monkeys (0.16-0.51), and high in dogs (≥0.81). Correspondingly, the CL(int,UGT) values were high in rats and monkeys (101-731 µL/min/mg), and low in dogs (≤ 59.6 µL/min/mg). A good inverse correlation was observed between F(a)F(g) and CL(int,UGT), suggesting that intestinal glucuronidation was a major factor influencing F(a)F(g) of these compounds. 3. By applying this correlation to F(a)F(g) in humans using human CL(int,UGT) values (26.9-114 µL/min/mg), compounds 1-3 were predicted to have relatively high F(a)F(g). 4. Our approach is expected to be useful for estimating the impact of intestinal glucuronidation on F in animals and semiquantitatively predicting human F for drug candidates.

Population pharmacokinetic modeling of trans-resveratrol and its glucuronide and sulfate conjugates after oral and intravenous administration in rats.

PURPOSE: To develop a population pharmacokinetic (PK) model which allowed the simultaneous modeling of trans-resveratrol and its glucuronide and sulfate conjugates. METHODS: Male Sprague-Dawley rats were administered i.v. and p.o. with 2, 10 and 20 mg·kg(-1) of trans-resveratrol. Blood was collected at different times during 24 h. An integrated PK model was developed using a sequential analysis, with non-linear mixed effect modeling (NONMEM). A prediction-corrected visual predictive check (pcVPC) was used to assess model performance. The model predictive capability was also evaluated with simulations after the i.v. administration of 15 mg·kg(-1) that were compared with an external data set. RESULTS: Disposition PK of trans-resveratrol and its metabolites was best described by a three-linked two-compartment model. Clearance of trans-resveratrol by conversion to its conjugates occurred by a first-order process, whereas both metabolites were eliminated by parallel first-order and Michaelis-Menten kinetics. The pcVPC confirmed the model stability and precision. The final model was successfully applied to the external data set showing its robustness. CONCLUSIONS: A robust population PK model has been built for trans-resveratrol and its glucuronide and sulfate conjugates that adequately predict plasmatic concentrations.

Development of a LC-MS/MS method for determination of propofol-glucuronide in hair and preliminary study on relationships between dose and hair concentration.

Propofol abuse has been reported worldwide, suggesting the need to establish analytical methods for human biological samples to investigate the abuse of propofol. This study aimed to investigate the relationship between dose and hair concentration using a simple and rapid analytical method developed and validated in this study. In the sample preparation, hair samples were washed with distilled water and methanol and extracted in methanol during 16h at room temperature. After centrifugation and evaporation, the residue was reconstituted and filtered through a 0.22µm membrane filter before LC-MS/MS analysis. The precursor-to-product ion transitions were 353 → 175, 113 for propofol glucuronide and m/z 370 → 175, 113 for internal standard(propofol glucuronide-d17). The calibration curves were satisfactory (R2=0.9997) and the limits of detection and quantification were 2 and 5pg/mg, respectively. In addition, this study collected the history of propofol use from subjects using a questionnaire and analyzed subjects' hair samples using a validated analytical method. As a result, the concentrations of propofol glucuronide ranged from 7 to 122pg/mg (mean : 51pg/mg). There were cases of positive relationships, but generally there was no correlation between dose and hair concentration.

Targeting 1,25(OH)2D-mediated calcium absorption machinery in proximal colon with calcitriol glycosides and glucuronides.

High intestinal calcium (Ca) absorption efficiency is associated with high peak bone mass in adolescents and reduced bone loss in adulthood. Transepithelial intestinal Ca absorption is mediated by 1,25-dihydroxyvitamin D (1,25(OH)2D, calcitriol) through the vitamin D receptor (VDR). Most research on Ca absorption focuses on the proximal small intestine but evidence shows that large intestine plays a crucial role in whole body Ca homeostasis. We directly assessed and compared Ca absorption capacity at the proximal colon and duodenum using in situ ligated loops (2 mM Ca, 10 min). In C57BL/6 J mice, the proximal colon (26.2 ±â€¯3.7 %) had comparable ability to absorb Ca as the duodenum (30.0 ±â€¯6.7 %). In VDR knockout (KO) mice, Ca absorption efficiency was reduced by 67 % in duodenum and 48 % in proximal colon. These data suggest that large intestine could be targeted to improve Ca absorption and protect bone in at risk-groups (e.g. bariatric patients). Glycoside forms of calcitriol found in Solanum Glaucophyllum (Sg) leaf are biologically inert but can be activated in the colon upon bacterial cleavage of the glycosides. We conducted a study to test whether Sg leaf, as well as a novel, synthetic 1,3-diglucuronide form of calcitriol (1,3-diG) could target the proximal colon and upregulate genes involved in Ca absorption (i.e. Trpv6, S100g). 13-week-old female C57BL6/J mice were fed AIN93 G diet containing increasing levels of one of the two compounds for 2 weeks (delivering 0, 0.25, 0.5, 1, or 2 ng calcitriol equivalent per day). Both compounds induced a dose-dependent upregulation of Cyp24a1 and Trpv6 gene expression in the proximal colon. 1,3-diG also induced S100g gene expression in the proximal colon. Duodenal expression of Trpv6 was upregulated at higher doses of 1,3-diG but not Sg leaf. These data suggest that both glycosylated and glucuronidated calcitriol could be used to target the proximal colon but that dosing must be optimized to limit systemic effects that could cause hypercalcemia. Future studies will test the translational potential of these compounds to determine if they can increase Ca absorption at proximal colon and whether this can help protect bone.

Determination of raltegravir and raltegravir glucuronide in human plasma and urine by LC-MS/MS with application in a maternal-fetal pharmacokinetic study.

Raltegravir (RAL) is a HIV-integrase inhibitor recommended for treatment of HIV type 1 infection during pregnancy. The elimination of RAL to RAL glucuronide (RAL GLU) is mediated primarily by UDP glucuronosyltransferase 1A1 (UGT1A1). The present study shows the development and validation of 4 different methods for the analysis of RAL and RAL GLU in plasma and in urine samples. The methods were applied to evaluate the maternal-fetal pharmacokinetics of RAL and RAL GLU in a HIV-infected pregnant woman receiving RAL 400 mg twice daily. The sample preparation for RAL and RAL GLU analysis in 25 µL plasma and 100 µL diluted urine (10-fold with water containing 0.1% formic acid) were carried out by protein precipitation procedure. RAL and RAL GLU generate similar product mass fragments and require separation in the chromatographic system, so a suitable resolution was achieved for unchanged RAL and RAL GLU employing Ascentis Express C18 (75 × 4.6 mm, 2.7 µm) for both plasma and urine samples. The methods showed linearities at the ranges of 0.1-13.5 µg/mL RAL and 0.15-19.5 µg/mL RAL GLU in urine and 10-2000 ng/mL RAL and 2.5-800 RAL GLU in plasma. Precise and accurate evaluation showed coefficients of variation and relative errors ≤ 15%. The methods have been successfully applied in a maternal-fetal pharmacokinetic study.

Roles of diclofenac and its metabolites in immune activation associated with acute hepatotoxicity in TgCYP3A4/hPXR-humanized mice.

Diclofenac (DCF) is a widely used nonsteroidal anti-inflammatory drug, but it comes with a high risk of drug-induced liver injury (DILI). Despite the quinone-imine adduct pathways, the immunotoxicity is recently considered as another factor for DILI. However, such immune responses are still elusive. In the present study, investigation of the immune response in the acute hepatotoxicity model of TgCYP3A4/hPXR-humanized mice was conducted by administration of DCF and DCF metabolites, respectively. In a single dose intraperitoneal injection of 80 mg/kg DCF, the pharmacokinetic results showed the major DCF metabolites, including 4'-hydroxy-diclofenac (4'-OH-DCF), 5-hydroxy-diclofenac (5-OH-DCF) and diclofenac glucuronide (DCF-G) were generated after DCF treatment. Not only DCF, but those DCF metabolites could also directly cause different degrees of acute liver injury as significantly increased the serum ALT levels in a short time period in the TgCYP3A4/hPXR-humanized mice. Furthermore, the three DCF metabolites could directly stimulate the significant elevation of serum immune-related factors in varying degrees. Transcriptome analysis revealed the differentially expressed genes in the liver of DCF-G treated mice were mostly involved with the "immune system process" and "cell death" and related to "IL-17 signaling pathway" and "TNF-α signaling pathway", but 5-OH-DCF had little effect on the expressions of those genes. These results indicate that the metabolite DCF-G plays an important role in the activation of the hepatic immune system, which might be involved in the pathogenesis of DCF-induced acute liver injury.

Metabolic disposition of [14C]bazedoxifene in healthy postmenopausal women.

Bazedoxifene is a selective estrogen receptor modulator under development for the prevention and treatment of osteoporosis. The disposition of [(14)C]bazedoxifene was determined in six healthy postmenopausal women after administration of a single oral dose of 20 mg (200 microCi). After dosing, blood was collected at frequent intervals, and urine and fecal samples were collected for up to 10 days. Aliquots of plasma, blood, urine, and fecal homogenates were analyzed for concentrations of radioactivity. Bazedoxifene metabolite profiles in plasma and feces were determined by high-performance liquid chromatography with radioactivity flow detection; metabolite structures were confirmed by liquid chromatography-mass spectrometry. Bazedoxifene was rapidly absorbed, exhibiting a mean peak plasma concentration of 3.43 ng/ml at 1.2 h postdose. The total mean recovery of the radioactive dose in excreta was 85.6%, with the majority recovered in feces (84.7%) and only a small fraction (0.81%) in urine. Radiochromatograms of plasma revealed that glucuronidation was the major metabolic pathway; little or no cytochrome P450-mediated metabolism was evident. The majority of circulating radioactivity was constituted by metabolites, with bazedoxifene-5-glucuronide being the predominant metabolite (up to 95%). Bazedoxifene-4'-glucuronide was a minor metabolite (up to 20%), and unchanged bazedoxifene represented 0 to 13% of the radioactivity in most plasma samples. Unchanged bazedoxifene was the major radioactive component in feces, however, reflecting unabsorbed drug and/or glucuronides that were hydrolyzed by intestinal bacterial enzymes. [(14)C]Bazedoxifene was generally well tolerated. These findings demonstrated that, after oral administration in healthy postmenopausal women, bazedoxifene was rapidly absorbed, metabolized via glucuronidation, and excreted predominantly in feces.

Pharmacokinetic role of protein binding of mycophenolic acid and its glucuronide metabolite in renal transplant recipients.

Mycophenolic acid (MPA), the active compound of mycophenolate mofetil (MMF), is used to prevent graft rejection in renal transplant recipients. MPA is glucuronidated to the metabolite MPAG, which exhibits enterohepatic recirculation (EHC). MPA binds for 97% and MPAG binds for 82% to plasma proteins. Low plasma albumin concentrations, impaired renal function and coadministration of cyclosporine have been reported to be associated with increased clearance of MPA. The aim of the study was to develop a population pharmacokinetic model describing the relationship between MMF dose and total MPA (tMPA), unbound MPA (fMPA), total MPAG (tMPAG) and unbound MPAG (fMPAG). In this model the correlation between pharmacokinetic parameters and renal function, plasma albumin concentrations and cotreatment with cyclosporine was quantified. tMPA, fMPA, tMPAG and fMPAG concentration-time profiles of renal transplant recipients cotreated with cyclosporine (n = 48) and tacrolimus (n = 45) were analyzed using NONMEM. A 2- and 1-compartment model were used to describe the pharmacokinetics of fMPA and fMPAG. The central compartments of fMPA and fMPAG were connected with an albumin compartment allowing competitive binding (bMPA and bMPAG). tMPA and tMPAG were modeled as the sum of the bound and unbound concentrations. EHC was modeled by transport of fMPAG to a separate gallbladder compartment. This transport was decreased in case of cyclosporine cotreatment (P < 0.001). In the model, clearance of fMPAG decreased when creatinine clearance (CrCL) was reduced (P < 0.001), and albumin concentration was correlated with the maximum number of binding sites available for MPA and MPAG (P < 0.001). In patients with impaired renal function cotreated with cyclosporine the model adequately described that increasing fMPAG concentrations decreased tMPA AUC due to displacement of MPA from its binding sites. The accumulated MPAG could also be reconverted to MPA by the EHC, which caused increased tMPA AUC in patients cotreated with tacrolimus. Changes in CrCL had hardly any effect on fMPA exposure. A decrease in plasma albumin concentration from 0.6 to 0.4 mmol/l resulted in ca. 38% reduction of tMPA AUC, whereas no reduction in fMPA AUC was seen. In conclusion, a pharmacokinetic model has been developed which describes the relationship between dose and both total and free MPA exposure. The model adequately describes the influence of renal function, plasma albumin and cyclosporine co-medication on MPA exposure. Changes in protein binding due to altered renal function or plasma albumin concentrations influence tMPA exposure, whereas fMPA exposure is hardly affected.

Tissue deconjugation of urolithin A glucuronide to free urolithin A in systemic inflammation.

Urolithin A (Uro-A) is an anti-inflammatory and cancer chemopreventive metabolite produced by the gut microbiota from the polyphenol ellagic acid. However, in vivo conjugation of Uro-A to Uro-A glucuronide (Uro-A glur) dramatically hampers its activity. We describe here for the first time the tissue deconjugation of Uro-A glur to Uro-A after lipopolysaccharide (LPS)-induced inflammation, which could explain the systemic in vivo activity of free Uro-A in microenvironments subjected to inflammatory stimuli.