Bioanalytical method development, validation and quantification of dorsomorphin in rat plasma by LC-MS/MS.

The present investigation describes the development and validation of a sensitive liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) method for the estimation of dorsomorphin in rat plasma. A sensitive LC-MS/MS method was developed using multiple reaction monitoring mode, with the transition of m/z (Q1/Q3) 400.2/289.3 for dorsomorphin and m/z (Q1/Q3) 306.2/236.3 for zaleplon. Chromatographic separation was achieved on a reverse phase Agilent XDB C18 column (100 × 4.6 mm, 5 µm). The mobile phase consisted of acetonitrile and 5 mm ammonium acetate buffer (pH 6.0) 90:10 v/v, at a flow rate of 0.8 mL/min. The effluence was ionized in positive ion mode by electrospray ionization (ESI) and quantitated by mass spectrometry. The retention times of dorsomorphin and internal standard were found to be 2.13 and 1.13 min, respectively. Mean extraction recovery of dorsomorphin and internal standard in rat plasma was above 80%. Dorsomorphin calibration curve in rat plasma was linear (r(2) ≥ 0.99) ranging from 0.005 to 10 µg/mL. Inter-day and intra-day precision and accuracy were found to be within 85-115% (coefficient of variation). This method was successfully applied for evaluation of the oral pharmacokinetic profile of dorsomorphin in male Wistar rats.

Effects of 1alpha,25-dihydroxyvitamin D3 on transporters and enzymes of the rat intestine and kidney in vivo.

1alpha,25-Dihydroxyvitamin D3 (1,25(OH)2D3), the natural ligand of the vitamin D receptor (VDR), was found to regulate bile acid related transporters and enzymes directly and indirectly in the rat intestine and liver in vivo. The kidney is another VDR-rich target organ in which VDR regulation on xenobiotic transporters and enzymes is ill-defined. Hence, changes in protein and mRNA expression of nuclear receptors, transporters and enzymes of the rat intestine and kidney in response to 1,25(OH)2D3 treatment (0 to 2.56 nmol/kg/day intraperitoneally in corn oil for 4 days) were studied. In the intestine, protein and not mRNA levels of Mrp2, Mrp3, Mrp4 and PepT1 in the duodenum and proximal jejunum were induced, whereas Oat1 and Oat3 mRNA were decreased in the ileum after 1,25(OH)2D3 treatment. In the kidney, VDR, Cyp24, Asbt and Mdr1a mRNA and protein expression increased significantly (2- to 20-fold) in 1,25(OH)2D3-treated rats, and a 28-fold increase of Cyp3a9 mRNA but not of total Cy3a protein nor Cyp3a1 and Cyp3a2 mRNA was observed, implicating that VDR played a significant, renal-specific role in Cyp3a9 induction. Additionally, renal mRNA levels of PepT1, Oat1, Oat3, Ostalpha, and Mrp4, and protein levels of PepT1 and Oat1 were decreased in a dose-dependent manner, and the approximately 50% concomitant reduction in FXR, SHP, HNF-1alpha and HNF-4alpha mRNA expression suggests the possibility of cross-talk among the nuclear receptors. It is concluded that the effects of 1,25(OH)2D3 changes are tissue-specific, differing between the intestine and kidney which are VDR-rich organs.

Expression and regulation of the bile acid transporter, OSTalpha-OSTbeta in rat and human intestine and liver.

The regulation of the OSTalpha and OSTbeta expression was studied in the rat jejunum, ileum, colon and liver and in human ileum and liver by ligands for the farnesoid X receptor (FXR), pregnane X receptor (PXR), vitamin D receptor (VDR) and glucocorticoid receptor (GR) using precision cut tissue slices. The gradient of protein and mRNA expression in segments of the intestine for rOSTalpha and rOSTbeta paralleled that of rASBT. OSTalpha and OSTbeta mRNA expression, quantified by qRT-PCR, in rat jejunum, ileum, colon and liver, and in human ileum and liver was positively regulated by FXR and GR ligands. In contrast, the VDR ligand, 1,25(OH)2D3 decreased the expression of rOSTalpha-rOSTbeta in rat intestine, but had no effect on human ileum, and rat and human liver slices. Lithocholic acid (LCA) decreased the expression of rOSTalpha and rOSTbeta in rat ileum but induced OSTalpha-OSTbeta expression in rat liver slices, and human ileum and liver slices. The PXR ligand, pregnenolone-16alpha carbonitrile (PCN) had no effect. This study suggest that, apart from FXR ligands, the OSTalpha and OSTbeta genes are also regulated by VDR and GR ligands and not by PXR ligands. This study show that VDR ligands exerted different effects on OSTalpha-OSTbeta in the rat and human intestine and liver compared with other nuclear receptors, FXR, PXR, and GR, pointing to species- and organ-specific differences in the regulation of OSTalpha-OSTbeta genes.

1alpha,25-Dihydroxyvitamin D(3) triggered vitamin D receptor and farnesoid X receptor-like effects in rat intestine and liver in vivo.

1alpha,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), a natural ligand of the vitamin D receptor (VDR), was found to increase the rat ileal Asbt and bile acid absorption. The effects of VDR, whose expression is low in liver, on hepatic transporters and enzymes are unknown. Protein and mRNA levels of target genes in the small intestine, colon and liver after intraperitoneal dosing of 1,25(OH)(2)D(3) (0-2.56 nmol/kg/day for 4 days) to the rat were determined by Western blotting and qPCR, respectively. The 1,25(OH)(2)D(3) treatment increased total Cyp3a protein and Cyp3a1 mRNA expressions in the proximal small intestine, and the short heterodimer partner (SHP), the fibroblast growth factor 15 (FGF15), organic solute transporter (Ostalpha and Ostbeta) mRNA and Asbt protein expressions in the ileum. About 50% higher portal bile acid concentration (65.1+/-14.9 vs 41.9+/-7.8 microm, p<0.05) and elevated expressions of the hepatic farnesoid X receptor (FXR) and SHP mRNA resulted with 1,25(OH)(2)D(3) treatment. Increased Bsep and Ostalpha mRNA expressions in liver and a>50% reduction in the Cyp7a1 protein level (p<0.05) and cholesterol metabolism in rat liver microsomes (p=0.002), likely consequences of the bile acid-FXR-SHP cascade and activation of the signaling pathway for Cyp7a1 inhibition by FGF15, were found. Increased hepatic multidrug resistance-associated protein (Mrp3) and multidrug resistance protein 1a (Mdr1a) mRNA and P-gp protein were also observed. It was concluded that the changes in hepatic transporters and enzymes in the rat were indirect, secondary effects of the liver FXR-SHP cascade due to increased intestinal absorption of bile acids and elevated levels of FGF15, events that led to the activation of FXR.

Characterization of 1-Aminobenzotriazole and Ketoconazole as Novel Inhibitors of Monoamine Oxidase (MAO): An In Vitro Investigation.

BACKGROUND AND OBJECTIVES: 1-Aminobenzotriazole, a known time-dependent inhibitor of cytochrome P450 (CYP) enzymes, and ketoconazole, a strong inhibitor of the human CYP3A4 isozyme, are used as standard probe inhibitors to characterize the CYP and/or non-CYP-mediated metabolism of xenobiotics. In the present investigation, 1-Aminobenzotriazole and ketoconazole are characterized as potent monoamine oxidase (MAO) inhibitors in vitro using mouse, rat and human liver microsomes and S9 fractions. METHODS: Inhibition potential of 1-aminobenzotriazole and ketoconazole was studied in mice, rat and human liver microsomes, S9 fractions, MAO-A and MAO-B expressed enzymes by monitoring the formation of 4-hydroxyquinoline (4-HQ) from kynuramine, a specific substrate of MAO by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Mechanism of MAO inhibition was studied by incubating varying concentration of kynuramine with mouse, rat and human S9 fractions at varying concentration of 1-aminobenzatriazole and ketoconazole and monitoring the formation of 4-HQ. RESULTS: 1-aminobenzotriazole and ketoconazole inhibited both MAO isozymes (MAO-A and MAO-B) with more specificity towards MAO-B. Kynuramine substrate kinetics in mouse, rat and human S9 fractions with varying 1-aminobenzotriazole and ketoconazole concentrations showed decreased maximum rate (V max) for 4-HQ formation without affecting the Michaelis-Menten constant (K m). A non-competitive inhibition model was constructed and inhibition constants (K i) for 1-aminobenzotriazole (7.87 ± 0.61, 8.61 ± 0.92, 65.2 ± 1.61 µM for mice, rat and humans, respectively) and ketoconazole (0.12 ± 0.01, 2.04 ± 0.08, 5.52 ± 0.47 µM for mice, rat and humans, respectively) were determined. CONCLUSIONS: 1-Aminobenzotriazole and ketoconazole are characterized as non-competitive inhibitors of mice, rat and human MAO in vitro and the extent of their MAO inhibition potential is species specific. 1-Aminobenzotriazole or ketoconazole can be used as a probe inhibitor in vitro for screening the involvement of MAO-dependent metabolism of new chemical entities (NCE) in early drug discovery.

The role of lithocholic acid in the regulation of bile acid detoxication, synthesis, and transport proteins in rat and human intestine and liver slices.

The effects of the secondary bile acid, lithocholic acid (LCA), a VDR, FXR and PXR ligand, on the regulation of bile acid metabolism (CYP3A isozymes), synthesis (CYP7A1), and transporter proteins (MRP3, MRP2, BSEP, NTCP) as well as nuclear receptors (FXR, PXR, LXRα, HNF1α, HNF4α and SHP) were studied in rat and human precision-cut intestine and liver slices at the mRNA level. Changes due to 5 to 10 µM of LCA were compared to those of other prototype ligands for VDR, FXR, PXR and GR. LCA induced rCYP3A1 and rCYP3A9 in the rat jejunum, ileum and colon, rCYP3A2 only in the ileum, rCYP3A9 expression in the liver, and CYP3A4 in the human ileum but not in liver. LCA induced the expression of rMRP2 in the colon but not in the jejunum and ileum but did not affect rMRP3 expression along the length of the rat intestine. In human ileum slices, LCA induced hMRP3 and hMRP2 expression. In rat liver slices, LCA decreased rCYP7A1, rLXRα and rHNF4α expression, induced rSHP expression, but did not affect rBSEP or rNTCP expression; whereas in the human liver, a small but significant decrease was found for hHNF1α expression. These data suggests profound species differences in the effects of LCA on bile acid transport, synthesis and detoxification. An examination of the effects of prototype VDR, PXR, GR and FXR ligands showed that these pathways are all intact in precision cut slices and that LCA exerted VDR, PXR and FXR effects. The LCA-induced altered enzymes and transporter expressions in the intestine and liver would affect the disposition of drugs.

Regulation of VDR expression in rat and human intestine and liver--consequences for CYP3A expression.

The vitamin D receptor (VDR) regulates the expression of drug metabolizing enzymes and transporters in intestine and liver, but the regulation of VDR expression in intestine and liver is incompletely understood. We studied the regulation of VDR mRNA expression by ligands for VDR, farnesoid X receptor (FXR), glucocorticoid receptor (GR) and protein kinase C alpha (PKCalpha) in rat and human ileum and liver using precision-cut slices. 1,25(OH)(2)D(3) induced VDR expression in rat ileum and liver, and human ileum but not in liver. Chenodeoxycholic acid (CDCA), but not lithocholic acid (LCA) and GW4064 induced VDR mRNA expression in rat ileum and liver. The PKCalpha activator, phorbol-12-myristate-13-acetate (PMA) induced the expression of VDR in the rat liver, and the induction of VDR by 1,25(OH)(2)D(3) and CDCA was inhibited by the PKCalpha inhibitor, bisindolyl maleimide I (Bis I). These results show that the expression of VDR is likely to be regulated by PKC but not by FXR or VDR activation at least in the rat liver. The VDR mediated induction of its target genes CYP3A1 and CYP3A2 by 1,25(OH)(2)D(3) or LCA in the rat ileum was strongly reduced in the presence of CDCA despite the higher VDR expression. Thus, CDCA might potentiate the toxicity of LCA by inhibiting its metabolism.

Comparison of effects of VDR versus PXR, FXR and GR ligands on the regulation of CYP3A isozymes in rat and human intestine and liver.

In this study, we compared the regulation of CYP3A isozymes by the vitamin D receptor (VDR) ligand 1 alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) against ligands of the pregnane X receptor (PXR), the glucocorticoid receptor (GR) and the farnesoid X receptor (FXR) in precision-cut tissue slices of the rat jejunum, ileum, colon and liver, and human ileum and liver. In the rat, 1,25(OH)(2)D(3) strongly induced CYP3A1 mRNA, quantified by qRT-PCR, along the entire length of the intestine, induced CYP3A2 only in ileum but had no effect on CYP3A9. In contrast, the PXR/GR ligand, dexamethasone (DEX), the PXR ligand, pregnenolone-16 alpha carbonitrile (PCN), and the FXR ligand, chenodeoxycholic acid (CDCA), but not the GR ligand, budesonide (BUD), induced CYP3A1 only in the ileum, none of them influenced CYP3A2 expression, and PCN, DEX and BUD but not CDCA induced CYP3A9 in jejunum, ileum and colon. In rat liver, CYP3A1, CYP3A2 and CYP3A9 mRNA expression was unaffected by 1,25(OH)(2)D(3), whereas CDCA decreased the mRNA of all CYP3A isozymes; PCN induced CYP3A1 and CYP3A9, BUD induced CYP3A9, and DEX induced all three CYP3A isozymes. In human ileum and liver, 1,25(OH)(2)D(3) and DEX induced CYP3A4 expression, whereas CDCA induced CYP3A4 expression in liver only. In conclusion, the regulation of rat CYP3A isozymes by VDR, PXR, FXR and GR ligands differed for different segments of the rat and human intestine and liver, and the changes did not parallel expression levels of the nuclear receptors.

Pharmacological and pharmacokinetic evaluation of celecoxib prodrugs in rats.

This study demonstrates the utility of an in vitro - in vivo correlative approach in the selection and optimization of a prodrug candidate of celecoxib (CBX), a COX(2) inhibitor. As an initial screening step, a comparative single oral dose pharmacokinetic study was conducted in rats for CBX and its three aliphatic acyl water-soluble prodrugs viz., CBX-acetyl (CBX-AC), CBX-propionyl (CBX-PR) and CBX-butyryl (CBX-BU) at high equimolar dose, 100 mg/kg. Only CBX-BU and CBX-PR converted rapidly to CBX and yielded approximately five-fold greater systemic exposure of CBX than CBX alone or CBX-AC. Rank order of systemic exposure of prodrugs in its intact form was CBX-AC >CBX-PR >CBX-BU. Further in vitro hydrolysis studies of CBX prodrugs in intestinal mucosal suspensions and liver homogenates indicated that CBX-BU is rapidly and completely converted to CBX, whereas CBX-PR and CBX-AC require longer incubation period for complete conversion to CBX. There was a very good correlation of the in vitro and in vivo data supporting CBX-BU as the prodrug of choice. Further in vitro pharmacological studies showed that COX(2) selective inhibition is improved for CBX-BU as compared to CBX-AC and CBX-PR. Dose proportionality in pharmacokinetic studies of CBX-BU and CBX at equimolar oral doses confirmed that relative oral bioavailability of CBX was improved following CBX-BU administration and there was linearity in pharmacokinetics of CBX over a wide dose range (10-100 mg/kg), whereas CBX in its conventional form showed poor bioavailability and lack of dose linearity in pharmacokinetics. The oral bioavailability of CBX from CBX-BU was dose independent and was in the range 78-96%. At a 50% reduced molar dose, CBX-BU showed an equivalent efficacy to that of CBX in the in vivo carrageenan model. Based on the study, water-soluble CBX-BU prodrug can be considered for clinical development in view of its potential advantages.