Drug Metabolism and Disposition most recent issues

Phenacetin Pharmacokinetics in CYP1A2-Deficient Beagle Dogs [Short Communication]

Whiterock, V. J., Morgan, D. G., Lentz, K. A., Orcutt, T. L., Sinz, M. W. — 2012-01-18T08:48:26-08:00

Phenacetin is widely used as an in vitro probe to measure CYP1A2 activity across species. To investigate whether phenacetin can be used as an in vivo probe substrate to phenotype CYP1A2 activity in dogs, beagle dogs previously genotyped for a single nucleotide polymorphism that yields an inactive CYP1A2 protein were selected and placed into one of three groups: CC (wild-type), CT (heterozygous), or TT (homozygous mutants). The dogs were dosed with phenacetin orally at 5 and 15 mg/kg and intravenously at 15 mg/kg. Plasma samples were analyzed by liquid chromatography-tandem mass spectrometry, and phenacetin and its primary metabolite, acetaminophen, were monitored. After intravenous dosing, all groups showed similar exposure of phenacetin irrespective of genotype. After oral dosing at 15 mg/kg, the exposure of phenacetin in CC and CT dogs was similar, but phenacetin exposure was 2-fold greater in TT dogs. Exposure of the metabolite, acetaminophen, was similar in all groups; however, the mean acetaminophen/phenacetin ratio in TT dogs was 1.7 times less than that observed in CC dogs. Similar trends between the groups of dogs with respect to phenacetin exposure were also observed after a lower 5 mg/kg p.o. dose of phenacetin; however, a proportionally greater amount of acetaminophen was generated. Although oral exposure of phenacetin was 2-fold higher and acetaminophen exposure was 2-fold lower in CYP1A2-deficient (TT) dogs, these results were considered modest and suggest that phenacetin is not a selective or robust in vivo probe to measure CYP1A2 enzyme activity in the dog.

Prenatal and Postnatal Expression of Glutathione Transferase {zeta} 1 in Human Liver and the Roles of Haplotype and Subject Age in Determining Activity with Dichloroacetate [Articles]

2012-01-18T08:48:26-08:00

Glutathione transferase 1 (GSTZ1), also known as maleylacetoacetate isomerase, catalyzes the penultimate step of tyrosine catabolism and metabolizes several α-halocarboxylic acids, including dichloroacetic acid (DCA), an investigational drug used for lactic acidosis and, recently, solid tumors. Age-related differences have been suggested in DCA pharmacotoxicology, but no information is available on GSTZ1 ontogeny in humans. Here, we investigated the cytosolic GSTZ1 developmental expression pattern and the influence of haplotype on GSTZ1 activity with DCA by using human livers from donors between 10 weeks gestation and 74 years. GSTZ1 expression was very low in fetal livers (<2 pmol of GSTZ1/mg cytosol). The expression began to increase after birth in an age-dependent manner until age 7 years. GSTZ1 was then sustained at stable, yet variable, levels (median, 20.0 pmol/mg cytosol; range, 4.8–47.3 pmol/mg cytosol) until age 74 years. GSTZ1 activity with DCA was strongly associated with haplotype and expression level. Samples homozygous or heterozygous for GSTZ1A exhibited ~3-fold higher DCA dechlorinating activity than samples carrying other alleles at a given level of expression. The correlations (r²) between activity and expression were 0.90 and 0.68, respectively, for GSTZ1A carriers (n = 11) and noncarriers (n = 61). GSTZ1 is expressed in mitochondria in addition to cytosol. The GSTZ1A allele exhibited similar effects in the mitochondrial fraction by conferring a higher activity with DCA. In summary, we report a neonatal onset and an age-related increase in GSTZ1 protein expression during human liver development. Haplotype influenced GSTZ1 activity with DCA but not protein expression.

Human UDP-Glucuronosyltransferase Isoforms Involved in Haloperidol Glucuronidation and Quantitative Estimation of Their Contribution [Articles]

Kato, Y., Nakajima, M., Oda, S., Fukami, T., Yokoi, T. — 2012-01-18T08:48:26-08:00

A major metabolic pathway of haloperidol is glucuronidation catalyzed by UDP-glucuronosyltransferase (UGT). In this study, we found that two glucuronides were formed by the incubation of haloperidol with human liver microsomes (HLM) and presumed that the major and minor metabolites (>10-fold difference) were O- and N-glucuronide, respectively. The haloperidol N-glucuronidation was catalyzed solely by UGT1A4, whereas the haloperidol O-glucuronidation was catalyzed by UGT1A4, UGT1A9, and UGT2B7. The kinetics of the haloperidol O-glucuronidation in HLM was monophasic with K_m and V_max values of 85 μM and 3.2 nmol · min^–1 · mg^–1, respectively. From the kinetic parameters of the recombinant UGT1A4 (K_m = 64 μM, V_max = 0.6 nmol · min^–1 · mg^–1), UGT1A9 (K_m = 174 μM, V_max = 2.3 nmol · min^–1 · mg^–1), and UGT2B7 (K_m = 45 μM, V_max = 1.0 nmol · min^–1 · mg^–1), we could not estimate which isoform largely contributes to the reaction. Because the haloperidol O-glucuronidation in a panel of 17 HLM was significantly correlated (r = 0.732, p < 0.01) with zidovudine O-glucuronidation, a probe activity of UGT2B7, and the activity in the pooled HLM was prominently inhibited (58% of control) by gemfibrozil, an inhibitor of UGT2B7, we surmised that the reaction would mainly be catalyzed by UGT2B7. We could successfully estimate, using the concept of the relative activity factor, that the contributions of UGT1A4, UGT1A9, and UGT2B7 in HLM were approximately 10, 20, and 70%, respectively. The present study provides new insight into haloperidol glucuronidation, concerning the causes of interindividual differences in the efficacy and adverse reactions or drug-drug interactions.

An Unusual Metabolic Pathway of Sipoglitazar, a Novel Antidiabetic Agent: Cytochrome P450-Catalyzed Oxidation of Sipoglitazar Acyl Glucuronide [Articles]

2012-01-18T08:48:26-08:00

Animal pharmacokinetic studies of sipoglitazar, a novel antidiabetic agent, showed that the deethylated metabolite (M-I) and the glucuronide conjugate of sipoglitazar (sipoglitazar-G) appeared to be the key metabolites in the elimination process. M-I was also measured as the main metabolite in the plasma of humans administered sipoglitazar. In vitro metabolic studies were performed to investigate the metabolic pathways from sipoglitazar to M-I in humans. The metabolic profile with human hepatocytes and hepatic microsomes indicated that M-I was not formed directly from sipoglitazar and that sipoglitazar-G was involved in the metabolism from sipoglitazar to M-I. Further studies of the metabolism of sipoglitazar-G revealed that the properties of the glucuronide conjugate and its metabolism are as follows: high-performance liquid chromatography, liquid chromatography-tandem mass spectrometry, and NMR analyses showed that sipoglitazar-G was composed of two glucuronides, sipoglitazar-G1, a β-1-O-acyl glucuronide, and sipoglitazar-G2, an α-2-O-acyl glucuronide. The stability study of these glucuronides suggested that sipoglitazar-G1 could be converted to sipoglitazar-G2 and sipoglitazar, but sipoglitazar-G2 could not be converted to sipoglitazar-G1. The oxidative metabolic study of sipoglitazar-G1 and -G2 with human hepatic microsomes and cytochrome P450-expressing microsomes revealed that M-I was formed only from sipoglitazar-G1, not from sipoglitazar-G2, and that CYP2C8 was mainly involved in this process. From these results, it is shown that the metabolic pathway from sipoglitazar to M-I is an unusual one, in which sipoglitazar is initially metabolized to sipoglitazar-G1 by UDP-glucuronosyltransferase and then sipoglitazar-G1 is metabolized to M-I by O-dealkylation by CYP2C8 and deconjugation. Sipoglitazar-G2 is sequentially formed by the migration of the β-site of sipoglitazar-G1.

UDP-Glucuronosyltransferase Expression in Mouse Liver Is Increased in Obesity- and Fasting-Induced Steatosis [Articles]

Xu, J., Kulkarni, S. R., Li, L., Slitt, A. L. — 2012-01-18T08:48:26-08:00

UDP-glucuronosyltransferases (Ugt) catalyze phase II conjugation reactions with glucuronic acid, which enhances chemical polarity and the elimination from the body. Few studies have addressed whether Ugt expression and activity are affected by liver disease, such as steatosis. The purpose of this study was to determine whether steatosis induced by obesity or fasting could affect liver Ugt mRNA expression and activity. Male C57BL/6J and Lep^ob/ob (ob/ob) mice were fed ad libitum or food was withheld for 24 h. In steatotic livers of ob/ob mice, Ugt1a1, -1a6, -1a9, -2a3, -3a1, and -3a2 mRNA expression increased. Fasting, which also induced steatosis, increased hepatic Ugt1a1, -1a6, -1a7, -1a9, -2b1, -2b5, -2a3, -3a1, and -3a2 mRNA expression in mouse liver. Likewise, acetaminophen glucuronidation increased by 47% in hepatic microsomes from ob/ob mice compared with that in C57BL/6J mice, but not after fasting. In both steatosis models, Ugt induction was accompanied by increased aryl hydrocarbon receptor, constitutive androstane receptor (CAR), peroxisome proliferator-activated receptor (PPAR)-α, pregnane X receptor, nuclear factor (erythroid-derived 2)-like 2 (Nrf2), and peroxisome proliferator-activated receptor- coactivator-1α mRNA expression. In addition, fasting increased CAR, PPAR, and Nrf2 binding activity. The work points to hepatic triglyceride concentrations corresponding with nuclear receptor and Ugt expression. The findings indicate that steatosis significantly alters hepatic Ugt expression and activity, which could have a significant impact on determining circulating hormone levels, drug efficacy, and environmental chemical clearance.

Characterization of Aldehyde Oxidase Enzyme Activity in Cryopreserved Human Hepatocytes [Articles]

2012-01-18T08:48:26-08:00

Substrates of aldehyde oxidase (AO), for which human clinical pharmacokinetics are reported, were selected and evaluated in pooled mixed-gender cryopreserved human hepatocytes in an effort to quantitatively characterize AO activity. Estimated hepatic clearance (Cl_h) for BIBX1382, carbazeran, O⁶-benzylguanine, zaleplon, and XK-469 using cryopreserved hepatocytes was 18, 17, 12, <4.3, and <4.3 ml · min^–1 · kg^–1, respectively. The observed metabolic clearance in cryopreserved hepatocytes was confirmed to be a result of AO-mediated metabolism via two approaches. Metabolite identification after incubations in the presence of H₂¹⁸O confirmed that the predominant oxidative metabolite was generated by AO, as expected isotope patterns in mass spectra were observed after analysis by high-resolution mass spectrometry. Second, clearance values were efficiently attenuated upon coincubation with hydralazine, an inhibitor of AO. The low exposure after oral doses of BIBX1382 and carbazeran (~5% F) would have been fairly well predicted using simple hepatic extraction (f_h) values derived from cryopreserved hepatocytes. In addition, the estimated hepatic clearance value for O⁶-benzylguanine was within ~80% of the observed total clearance in humans after intravenous administration (15 ml · min^–1 · kg^–1), indicating a reasonable level of quantitative activity from this in vitro system. However, a 3.5-fold underprediction of total clearance was observed for zaleplon, despite the 5-oxo metabolite being clearly observed. These data taken together suggest that the use of cryopreserved hepatocytes may be a practical approach for assessing AO-mediated metabolism in discovery and potentially useful for predicting hepatic clearance of AO substrates.

Identification of UDP-Glucuronosyltransferases Responsible for the Glucuronidation of Darexaban, an Oral Factor Xa Inhibitor, in Human Liver and Intestine [Articles]

2012-01-18T08:48:26-08:00

Darexaban maleate is a novel oral direct factor Xa inhibitor, which is under development for the prevention of venous thromboembolism. Darexaban glucuronide was the major component in plasma after oral administration of darexaban to humans and is the pharmacologically active metabolite. In this study, we identified UDP-glucuronosyltransferases (UGTs) responsible for darexaban glucuronidation in human liver microsomes (HLM) and human intestinal microsomes (HIM). In HLM, the K_m value for darexaban glucuronidation was >250 μM. In HIM, the reaction followed substrate inhibition kinetics, with a K_m value of 27.3 μM. Among recombinant human UGTs, UGT1A9 showed the highest intrinsic clearance for darexaban glucuronidation, followed by UGT1A8, -1A10, and -1A7. All other UGT isoforms were inactive toward darexaban. The K_m value of recombinant UGT1A10 for darexaban glucuronidation (34.2 μM) was comparable to that of HIM. Inhibition studies using typical UGT substrates suggested that darexaban glucuronidation in both HLM and HIM was mainly catalyzed by UGT1A8, -1A9, and -1A10. Fatty acid-free bovine serum albumin (2%) decreased the unbound K_m for darexaban glucuronidation from 216 to 17.6 μM in HLM and from 35.5 to 18.3 μM in recombinant UGT1A9. Recent studies indicated that the mRNA expression level of UGT1A9 is extremely high among UGT1A7, -1A8, -1A9, and -1A10 in human liver, whereas that of UGT1A10 is highest in the intestine. Thus, the present results strongly suggest that darexaban glucuronidation is mainly catalyzed by UGT1A9 and UGT1A10 in human liver and intestine, respectively. In addition, UGT1A7, -1A8, and -1A9 play a minor role in human intestine.

Characterization of a Human Keratinocyte HaCaT Cell Line Model to Study the Regulation of CYP2S1 [Articles]

McNeilly, A. D., Woods, J. A., Ibbotson, S. H., Wolf, C. R., Smith, G. — 2012-01-18T08:48:26-08:00

CYP2S1 is an extrahepatic cytochrome P450 (P450) that shows marked individuality in constitutive and inducible expression. CYP2S1 mRNA expression is increased in psoriasis and by treatments for psoriasis, including retinoids and UV radiation, although endogenous substrates remain poorly characterized. Because previous model systems have overexpressed modified CYP2S1 in bacteria, human HaCaT keratinocyte cells were screened for constitutive and regulatable CYP2S1 expression and CYP2S1 activity in HaCaT cells compared with a novel Chinese hamster ovary (CHO)-based cell line engineered to stably coexpress CYP2S1 and NADPH cytochrome P450 reductase. Constitutive mRNA expression for CYP2S1 and additional P450s, retinoid acid receptors (RARα, RARβ, RAR), and retinoid X receptors (RXRα, RXRβ and RXR) was assessed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis in HaCaT cells. Cells were then exposed to retinoids or to UV radiation (UVR), and changes in CYP2S1 mRNA abundance were further examined by qRT-PCR analysis. P450 expression in HaCaT cells was similar to human skin, with abundant CYP2S1 expression. RARα and RAR (but not RARβ) and all RXR isoforms were also detectable. All-trans retinoic acid (atRA) induced CYPS1 mRNA expression more potently than 9-cis RA or 13-cis RA. P450-dependent atRA metabolism was demonstrated in HaCaT cells, with a very similar metabolite profile to that produced by our CYP2S1-expressing CHO cells. CYP2S1 mRNA expression was also induced by UVR, more potently than CYP1B1, a known UVR-inducible P450. Our results demonstrate regulatable and functional CYP2S1 expression in HaCaT cells, thus identifying a human cell line model with utility for further analysis of CYP2S1 regulation and substrate specificity.

Pharmacokinetics of Lisdexamfetamine Dimesylate after Targeted Gastrointestinal Release or Oral Administration in Healthy Adults [Articles]

2012-01-18T08:48:26-08:00

The purpose of this work was to assess the pharmacokinetics and safety of lisdexamfetamine dimesylate (LDX) delivered and released regionally in the gastrointestinal (GI) tract. In this open-label, randomized, crossover study, oral capsules and InteliSite delivery capsules containing LDX (50 mg) with radioactive marker were delivered to the proximal small bowel (PSB), distal SB (DSB), and ascending colon (AC) during separate periods. Gamma scintigraphy evaluated regional delivery and GI transit. LDX and d-amphetamine in blood were measured postdose (≤72 h). Treatment-emergent adverse events (TEAEs) were assessed. Healthy males (n = 18; 18–48 years) were enrolled. Mean (S.D.) maximal plasma concentration (C_max) was 37.6 (4.54), 40.5 (4.95), 38.7 (6.46), and 25.7 (9.07) ng/ml; area under the concentration-time curve to the last measurable time point was 719.1 (157.05), 771.2 (152.88), 752.4 (163.38), and 574.3 (220.65) ng · h · ml^–1, respectively, for d-amphetamine after oral, PSB, DSB, and AC delivery of LDX. Median time to C_max was 5, 4, 5, and 8 h, respectively. Most TEAEs were mild to moderate. No clinically meaningful changes were observed (laboratory, physical examination, or electrocardiogram). LDX oral administration or targeted delivery to small intestine had similar d-amphetamine systemic exposure, indicating good absorption, and had reduced absorption after colonic delivery. The safety profile was consistent with other LDX studies.

Oxidative Modification of Rat Sulfotransferase 1A1 Activity in Hepatic Tissue Slices Correlates with Effects on the Purified Enzyme [Articles]

Dammanahalli, J. K., Duffel, M. W. — 2012-01-18T08:48:26-08:00

Mammalian cytosolic sulfotransferases (SULTs) catalyze the sulfation of xenobiotics as well as numerous endogenous molecules. The major aryl (phenol) SULT in rat liver, rSULT1A1, has been used extensively as a model enzyme for understanding the catalytic function of SULTs. Previous studies showed that purified rSULT1A1 displays significant catalytic changes in the presence of GSSG and other oxidants. In the present study, the effects of diamide [1,1'-azobis(N,N-dimethylformamide)] and tert-butyl hydroperoxide (TBHP) on the activity of rSULT1A1 in rat hepatic slices were compared with the effects of these oxidants on a homogeneous preparation of the enzyme. Precision-cut hepatic slices were incubated with 10 μM 7-hydroxycoumarin (7-HC) in the presence of varied concentrations of either diamide or TBHP. Analysis of the 7-hydroxycoumarin sulfate released into the incubation medium indicated that both oxidants significantly increased the sulfation of 7-HC, and this occurred at optimal concentrations of 5 and 10 μM, respectively. Cellular GSH and GSSG levels in the hepatic slices were not significantly altered from control values at these concentrations of diamide and TBHP. Exposure of homogeneous rSULT1A1 to diamide or TBHP also increased the rate of sulfation of 7-HC, although the optimal concentrations of diamide and TBHP were lower (50- and 100-fold, respectively) than those required for effects with the hepatic slices. These results indicate that both diamide and TBHP may modify the rSULT1A1 in intact cells in a manner similar to that observed with the homogeneous purified enzyme.

Use of Baculovirus BacMam Vectors for Expression of ABC Drug Transporters in Mammalian Cells [Articles]

Shukla, S., Schwartz, C., Kapoor, K., Kouanda, A., Ambudkar, S. V. — 2012-01-18T08:48:26-08:00

ATP-binding cassette (ABC) drug transporters ABCB1 [P-glycoprotein (Pgp)] and ABCG2 are expressed in many tissues including those of the intestines, the liver, the kidney and the brain and are known to influence the pharmacokinetics and toxicity of therapeutic drugs. In vitro studies involving their functional characteristics provide important information that allows improvements in drug delivery or drug design. In this study, we report use of the BacMam (baculovirus-based expression in mammalian cells) expression system to express and characterize the function of Pgp and ABCG2 in mammalian cell lines. BacMam-Pgp and BacMam-ABCG2 baculovirus-transduced cell lines showed similar cell surface expression (as detected by monoclonal antibodies with an external epitope) and transport function of these transporters compared to drug-resistant cell lines that overexpress the two transporters. Transient expression of Pgp was maintained in HeLa cells for up to 72 h after transduction (48 h after removal of the BacMam virus). These BacMam-baculovirus-transduced mammalian cells expressing Pgp or ABCG2 were used for assessing the functional activity of these transporters. Crude membranes isolated from these cells were further used to study the activity of these transporters by biochemical techniques such as photo-cross-linking with transport substrate and adenosine triphosphatase assays. In addition, we show that the BacMam expression system can be exploited to coexpress both Pgp and ABCG2 in mammalian cells to determine their contribution to the transport of a common anticancer drug substrate. Collectively, these data demonstrate that the BacMam-baculovirus-based expression system can be used to simultaneously study the transport function and biochemical properties of ABC transporters.

Mass Balance Study of [14C]Eribulin in Patients with Advanced Solid Tumors [Articles]

2012-01-18T08:48:26-08:00

This mass balance study investigated the metabolism and excretion of eribulin, a nontaxane microtubule dynamics inhibitor with a novel mechanism of action, in patients with advanced solid tumors. A single approximately 2 mg (approximately 80 μCi) dose of [¹⁴C]eribulin acetate was administered as a 2 to 5 min bolus injection to six patients on day 1. Blood, urine, and fecal samples were collected at specified time points on days 1 to 8 or until sample radioactivity was ≤1% of the administered dose. Mean plasma eribulin exposure (627 ng · h/ml) was comparable with that of total radioactivity (568 ng Eq · h/ml). Time-matched concentration ratios of eribulin to total radioactivity approached unity in blood and plasma, indicating that unchanged parent compound constituted almost all of the eribulin-derived radioactivity. Only minor metabolites were detected in plasma samples up to 60 min postdose, pooled across patients, each metabolite representing ≤0.6% of eribulin. Elimination half-lives for eribulin (45.6 h) and total radioactivity (42.3 h) were comparable. Eribulin-derived radioactivity excreted in feces was 81.5%, and that of unchanged eribulin was 61.9%. Renal clearance (0.301 l/h) was a minor component of total eribulin clearance (3.93 l/h). Eribulin-derived radioactivity excreted in urine (8.9%) was comparable with that of unchanged eribulin (8.1%), indicating minimal excretion of metabolite(s) in urine. Total recovery of the radioactive dose was 90.4% in urine and feces. Overall, no major metabolites of eribulin were detected in plasma. Eribulin is eliminated primarily unchanged in feces, whereas urine constitutes a minor route of elimination.

Prediction of In Vivo Hepatic Clearance and Half-Life of Drug Candidates in Human Using Chimeric Mice with Humanized Liver [Articles]

2012-01-18T08:48:27-08:00

Accurate prediction of pharmacokinetics (PK) parameters in humans from animal data is difficult for various reasons, including species differences. However, chimeric mice with humanized liver (PXB mice; urokinase-type plasminogen activator/severe combined immunodeficiency mice repopulated with approximately 80% human hepatocytes) have been developed. The expression levels and metabolic activities of cytochrome P450 (P450) and non-P450 enzymes in the livers of PXB mice are similar to those in humans. In this study, we examined the predictability for human PK parameters from data obtained in PXB mice. Elimination of selected drugs involves multiple metabolic pathways mediated not only by P450 but also by non-P450 enzymes, such as UDP-glucuronosyltransferase, sulfotransferase, and aldehyde oxidase in liver. Direct comparison between in vitro intrinsic clearance (CL_{int,in vitro}) in PXB mice hepatocytes and in vivo intrinsic clearance (CL_{int,in vivo}) in humans, calculated based on a well stirred model, showed a moderate correlation (r² = 0.475, p = 0.009). However, when CL_{int,in vivo} values in humans and PXB mice were compared similarly, there was a good correlation (r² = 0.754, p = 1.174 x 10^–4). Elimination half-life (t_1/2) after intravenous administration also showed a good correlation (r² = 0.886, p = 1.506 x 10^–4) between humans and PXB mice. The rank order of CL and t_1/2 in human could be predicted at least, although it may not be possible to predict absolute values due to rather large prediction errors. Our results indicate that in vitro and in vivo experiments with PXB mice should be useful at least for semiquantitative prediction of the PK characteristics of candidate drugs in humans.

Brain-Penetrating IgG-Iduronate 2-Sulfatase Fusion Protein for the Mouse [Articles]

Zhou, Q.-H., Boado, R. J., Lu, J. Z., Hui, E. K.-W., Pardridge, W. M. — 2012-01-18T08:48:27-08:00

Mucopolysaccharidosis (MPS) type II (Hunter's syndrome) is caused by mutations in the iduronate 2-sulfatase (IDS) fusion protein. MPS-II affects the brain, and enzyme replacement therapy is not effective in the brain, because the enzyme does not cross the blood-brain barrier. To treat mouse models of MPS-II with brain-penetrating IDS, the lysosomal enzyme was reengineered as an IgG-IDS fusion protein. The mature human IDS was fused to the carboxyl terminus of both heavy chains of the chimeric monoclonal antibody (MAb) against the mouse transferrin receptor (TfR), and the fusion protein is designated cTfRMAb-IDS. The purity and identity of the fusion protein was confirmed by electrophoresis and Western blotting with antibodies to mouse IgG and human IDS. The EC₅₀ of binding of the cTfRMAb-IDS fusion protein to the mouse TfR (0.85 ± 0.15 nM) was comparable to the EC₅₀ of binding of the cTfRMAb (0.78 ± 0.05 nM). The IDS enzyme activity of the cTfRMAb-IDS fusion protein was 126 ± 1 nmol · h^–1 · μg^–1 protein. After intravenous injection in the mouse, the cTfRMAb-IDS fusion protein was rapidly removed from plasma and distributed to tissues, including brain and spinal cord. The uptake of the fusion protein by brain or spinal cord was 1.3 ± 0.1 and 2.2 ± 0.2% injected dose/g, respectively, which is 100-fold greater than the brain uptake of IDS alone. This work shows that a lysosomal sulfatase can be reengineered as an IgG-enzyme fusion protein that rapidly penetrates the brain after intravenous administration.

UDP-Glucuronosyltransferase (UGT) 1A9-Overexpressing HeLa Cells Is an Appropriate Tool to Delineate the Kinetic Interplay between Breast Cancer Resistance Protein (BRCP) and UGT and to Rapidly Identify the Glucuronide Substrates of BCRP [Articles]

Jiang, W., Xu, B., Wu, B., Yu, R., Hu, M. — 2012-01-18T08:48:27-08:00

The interplay between phase II enzymes and efflux transporters leads to extensive metabolism and low bioavailability for flavonoids. To investigate the simplest interplay between one UDP-glucuronosyltransferase isoform and one efflux transporter in flavonoid disposition, engineered HeLa cells stably overexpressing UGT1A9 were developed, characterized, and further applied to investigate the metabolism of two model flavonoids (genistein and apigenin) and excretion of their glucuronides. The results indicated that the engineered HeLa cells overexpressing UGT1A9 rapidly excreted the glucuronides of genistein and apigenin. The kinetic characteristics of genistein or apigenin glucuronidation were similar with the use of UGT1A9 overexpressed in HeLa cells or the commercially available UGT1A9. Small interfering (siRNA)-mediated UGT1A9 silencing resulted in a substantial decrease in glucuronide excretion (>75%, p < 0.01). Furthermore, a potent inhibitor of breast cancer resistance protein (BCRP), 3-(6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-3-yl)-propionic acid tert-butyl ester (Ko143), caused, in a dose-dependent manner, a substantial and marked reduction of the clearance (74–94%, p < 0.01), and a substantial increase in the intracellular glucuronide levels (4–8-fold, p < 0.01), resulting in a moderate decrease in glucuronide excretion (19–59%, p < 0.01). In addition, a significant, albeit moderate, reduction in the fraction of genistein metabolized (f_met) in the presence of Ko143 was observed. In contrast, leukotriene C₄ and siRNA against multidrug resistance protein (MRP) 2 and MRP3 did not affect excretion of flavonoid glucuronides. In conclusion, the engineered HeLa cells overexpressing UGT1A9 is an appropriate model to study the kinetic interplay between UGT1A9 and BCRP in the phase II disposition of flavonoids. This simple cell model should also be very useful to rapidly identify whether a phase II metabolite is the substrate of BCRP.

Enzymatic Characterization and Elucidation of the Catalytic Mechanism of a Recombinant Bovine Glycine N-Acyltransferase [Articles]

Badenhorst, C. P. S., Jooste, M., van Dijk, A. A. — 2012-01-18T08:48:27-08:00

Glycine conjugation, a phase II detoxification process, is catalyzed by glycine N-acyltransferase (GLYAT; E.C. 2.3.1.13). GLYAT detoxifies various xenobiotics, such as benzoic acid, and endogenous organic acids, such as isovaleric acid, which makes GLYAT important in the management of organic acidemias in humans. We cloned the open reading frame encoding the bovine ortholog of GLYAT from bovine liver mRNA into the bacterial expression vector pColdIII. The recombinant enzyme was expressed, partially purified, and enzymatically characterized. Protein modeling was used to predict Glu²²⁶ of bovine GLYAT to be catalytically important. This was assessed by constructing an E226Q mutant and comparing its enzyme kinetics to that of the wild-type recombinant bovine GLYAT. The Michaelis constants for benzoyl-CoA and glycine were determined and were similar for wild-type recombinant GLYAT, E226Q recombinant GLYAT, and GLYAT present in bovine liver. At pH 8.0, the E226Q mutant GLYAT had decreased activity, which could be compensated for by increasing the reaction pH. This suggested a catalytic mechanism in which Glu²²⁶ functions to deprotonate glycine, facilitating nucleophilic attack on the acyl-CoA. The recombinant bovine GLYAT enzyme, combined with this new understanding of its active site and reaction mechanism, could be a powerful tool to investigate the functional significance of GLYAT sequence variations. Eventually, this should facilitate investigations into the impact of known and novel sequence variations in the human GLYAT gene.

Methylation of Catechins and Procyanidins by Rat and Human Catechol-O-Methyltransferase: Metabolite Profiling and Molecular Modeling Studies [Articles]

2012-01-18T08:48:27-08:00

Catechins and procyanidins are major polyphenols in plant-derived foods. Despite intensive studies in recent years, neither their biochemical nor their toxicological properties have been clarified sufficiently. This study aimed to compare the methylation of catechins and procyanidins by the enzyme catechol-O-methyltransferase (COMT) in vitro. We conducted incubations with rat liver cytosol and human placental cytosol including S-adenosyl-l-methionine. The set of substrates comprised the catechins (–)-epicatechin (EC) and (+)-catechin (CAT), the procyanidin dimers B1, B2, B3, B4, B5, and B7 as well as procyanidin trimer C1. After extraction, metabolites were analyzed by means of liquid chromatography-electrospray ionization-mass spectrometry and liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry. EC and CAT were converted to two monomethylated metabolites each by human and rat COMT, with the 3'-O-methyl derivatives being consistently the main metabolites. Furthermore, the flavanyl units of procyanidins were methylated consecutively, leading to monomethylated and dimethylated dimeric metabolites as well as monomethylated, dimethylated, and trimethylated C1 metabolites. The methylation status of each flavanyl unit was determined by means of mass spectrometric quinone-methide fragmentation patterns. In addition, molecular modeling studies were performed with the aim to predict the preferred site of methylation and to verify the experimental data. In conclusion, our results indicate that the degree and position of methylation depend clearly on the three-dimensional structure of the entire substrate molecule.

Effects of PEGylation and Immune Complex Formation on the Pharmacokinetics and Biodistribution of Recombinant Interleukin 10 in Mice [Articles]

2012-01-18T08:48:27-08:00

Interleukin 10 (IL-10) is a potent cytokine homodimer with multiple immunoregulatory functions. Here, we have characterized the effects of PEGylation and formation of human IL-10 (hIL-10)/humanized anti-human IL-10 (hαhIL-10) immune complexes in the pharmacokinetics, biodistribution, and biotransformation of IL-10 in mice. To assess the fate of native, PEGylated, and antibody-bound IL-10; we implemented an analytical set of fluorescence emission-linked assays. Plasma size exclusion chromatography analysis indicated that fluoro-labeled native and PEGylated murine IL-10 (PEG-mIL-10) are stable in the circulation. PEGylation of IL-10 resulted in a 21-fold increased exposure, 2.7-fold increase in half-life, and 20-fold reduction in clearance. Kidney is the major organ of disposition for both native and PEGylated mIL-10 with renal uptake directly related to systemic clearance. The fluorescence signal in the kidneys reached tissue/blood ratios up to 150 and 20 for native and PEG-mIL-10, respectively. hIL-10/hαhIL-10 immune complexes are detectable in the circulation without evidence of unbound or degraded hIL-10. The exposure of hIL-10 present in immune complexes versus that of hIL-10 alone increased from 0.53 to 11.28 μg · day/ml, with a half-life of 1.16 days and a 23-fold reduction in clearance. Unlike hIL-10 alone, antibody-bound hIL-10 was targeted mainly to the liver with minimal renal distribution. In addition, we found an 11-fold reduction (from 9.9 to 113 nM) in binding to the neonatal Fc receptor (FcRn) when the hαhIL10 antibody is conjugated to hIL-10. The potential changes in FcRn binding in vivo and increased liver uptake may explain the unique pharmacokinetic properties of hIL-10/hαhIL-10 immune complexes.

Paraoxonase 1 as a Major Bioactivating Hydrolase for Olmesartan Medoxomil in Human Blood Circulation: Molecular Identification and Contribution to Plasma Metabolism [Articles]

2012-01-18T08:48:27-08:00

Olmesartan medoxomil (OM) is a prodrug-type angiotensin II type 1 receptor antagonist. The OM-hydrolyzing enzyme responsible for prodrug bioactivation was purified from human plasma through successive column chromatography and was molecularly identified through N-terminal amino acid sequencing, which resulted in a sequence of 20 amino acids identical to that of human paraoxonase 1 (PON1). Two recombinant allozymes of human PON1 (PON1_192QQ and PON1_192RR) were constructed and were clearly demonstrated to hydrolyze OM; hydrolysis by the latter allozyme was slightly faster than that by the former. In addition, we evaluated the contribution of PON1 to OM bioactivation in human plasma. Enzyme kinetic studies demonstrated that OM was hydrolyzed more effectively by the recombinant PON1 proteins than by purified albumin. The OM-hydrolyzing activities of the recombinant PON1 proteins and diluted plasma were greatly reduced in the absence of calcium ions. Immunoprecipitation with anti-PON1 IgG completely abolished the OM-hydrolyzing activity in human plasma, whereas the activity was partially inhibited with anti-albumin IgG. The distribution pattern of the OM-hydrolyzing activity in human serum lipoprotein fractions and lipoprotein-deficient serum was examined and showed that most of the OM-hydrolyzing activity was located in the high-density lipoprotein fraction, with which PON1 is closely associated. In conclusion, we identified PON1 as the OM-bioactivating hydrolase in human plasma on a molecular basis and demonstrated that PON1, but not albumin, plays a major role in OM bioactivation in human plasma.

Increased Systemic Exposure to Rhizoma Coptidis Alkaloids in Lipopolysaccharide-Pretreated Rats Attributable to Enhanced Intestinal Absorption [Articles]

2012-01-18T08:48:27-08:00

Rhizoma coptidis is a rhizome commonly used in traditional Chinese medicine. After oral administration of rhizoma coptidis extract, the plasma concentrations of its effective alkaloid constituents are so low that their systemic therapeutic actions cannot be explained. This study aimed to investigate the influence of lipopolysaccharide (LPS) on the pharmacokinetics of the rhizoma coptidis alkaloids. Pharmacokinetic experiments were performed with rats; both in vitro absorption and efflux experiments were carried out with everted rat gut sacs, whereas in vitro metabolism experiments were conducted with rat liver microsomes and intestinal S9 fractions. Mucosal changes were evaluated with light microscopy and transmission electron microscopy. The results showed that, in rat plasma, LPS pretreatment increased systemic alkaloid exposure. LPS pretreatment increased the in vitro absorption of the alkaloids and decreased their efflux. The efflux of vinblastine and rhodamine 123, P-glycoprotein substrates, also was decreased. The absorption of fluorescein isothiocyanate-labeled dextran (average molecular mass, 4 kDa), a gut paracellular permeability probe, was not influenced. Obvious damage was observed in the mucosa, but the tight junctions between epithelial cells remained intact. Intestinal, rather than hepatic, alkaloid metabolism was decreased. These findings indicated that LPS pretreatment increased systemic exposure to the alkaloids through enhancement of their absorption, which was related to decreased intestinal efflux and metabolism. The results add to the understanding of why rhizoma coptidis is active despite the low plasma concentrations of the rhizoma coptidis alkaloids measured in normal subjects and experimental animals.

Assessment of the Impact of CYP3A Polymorphisms on the Formation of {alpha}-Hydroxytamoxifen and N-Desmethyltamoxifen in Human Liver Microsomes [Articles]

Mugundu, G. M., Sallans, L., Guo, Y., Shaughnessy, E. A., Desai, P. B. — 2012-01-18T08:48:27-08:00

Tamoxifen, an antiestrogen used in the prevention and treatment of breast cancer, is extensively metabolized by cytochrome P450 enzymes. Its biotransformation to α-hydroxytamoxifen (α-OHT), which may be genotoxic, and to N-desmethyltamoxifen (N-DMT), which is partially hydroxylated to 4-hydroxy-N-DMT (endoxifen), a potent antiestrogen, is mediated by CYP3A enzymes. However, the potential contribution of CYP3A5 and the impact of its low-expression variants on the formation of these metabolites are not clear. Therefore, we assessed the contributions of CYP3A4 and CYP3A5 and examined the impact of CYP3A5 genotypes on the formation of α-OHT and N-DMT, by using recombinant CYP3A4 and CYP3A5 and human liver microsomes (HLM) genotyped for CYP3A5 variants. We observed that the catalytic efficiency [intrinsic clearance (CL_int)] for α-OHT formation with recombinant CYP3A4 was 5-fold higher than that with recombinant CYP3A5 (0.81 versus 0.16 nl · min^–1 · pmol cytochrome P450^–1). There was no significant difference in CL_int values between the three CYP3A5-genotyped HLM (*1/*1, *1/*3, and *3/*3). For N-DMT formation, the CL_int with recombinant CYP3A4 was only 1.7-fold higher, relative to that with recombinant CYP3A5. In addition, the CL_int for N-DMT formation by HLM with CYP3A5*3/*3 alleles was approximately 3-fold lower than that for HLM expressing CYP3A5*1/*1. Regression analyses of tamoxifen metabolism with respect to testosterone 6β-hydroxylation facilitated assessment of CYP3A5 contributions to the formation of the two metabolites. The CYP3A5 contributions to α-OHT formation were negligible, whereas the contributions to N-DMT formation ranged from 51 to 61%. Our findings suggest that polymorphic CYP3A5 expression may affect the formation of N-DMT but not that of α-OHT.

Predicting Phenolic Acid Absorption in Caco-2 Cells: A Theoretical Permeability Model and Mechanistic Study [Articles]

Farrell, T. L., Poquet, L., Dew, T. P., Barber, S., Williamson, G. — 2012-01-18T08:48:27-08:00

There is a considerable need to rationalize the membrane permeability and mechanism of transport for potential nutraceuticals. The aim of this investigation was to develop a theoretical permeability equation, based on a reported descriptive absorption model, enabling calculation of the transcellular component of absorption across Caco-2 monolayers. Published data for Caco-2 permeability of 30 drugs transported by the transcellular route were correlated with the descriptors 1-octanol/water distribution coefficient (log D, pH 7.4) and size, based on molecular mass. Nonlinear regression analysis was used to derive a set of model parameters a', β', and b' with an integrated molecular mass function. The new theoretical transcellular permeability (TTP) model obtained a good fit of the published data (R² = 0.93) and predicted reasonably well (R² = 0.86) the experimental apparent permeability coefficient (P_app) for nine non-training set compounds reportedly transported by the transcellular route. For the first time, the TTP model was used to predict the absorption characteristics of six phenolic acids, and this original investigation was supported by in vitro Caco-2 cell mechanistic studies, which suggested that deviation of the P_app value from the predicted transcellular permeability (P_app^trans) may be attributed to involvement of active uptake, efflux transporters, or paracellular flux.

Differential Modulation of Cytochrome P450 Activity and the Effect of 1-Aminobenzotriazole on Hepatic Transport in Sandwich-Cultured Human Hepatocytes [Accelerated Communication]

2012-01-18T08:48:27-08:00

Sandwich-cultured human hepatocytes (SCHH) have been widely used for in vitro assessments of biliary clearance. However, the modulation of metabolism enzymes has not been fully evaluated in this system. The present study was therefore undertaken to determine the activity of cytochrome P450 (P450) 1A2, 2C8, 2C9, 2C19, 2D6, and 3A and to evaluate the impact of 1-aminobenzotriazole (ABT) on hepatic uptake and biliary excretion in SCHH. The SCHH maintained integrity and viability as determined by lactate dehydrogenase release and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assays conducted over the culture period. Although all assessed P450 activity decreased in day 2 SCHH, the extent of the decrease and the subsequent rebound in activity varied across the different isoforms. Day 5 CYP1A2 activity was approximately 2.5-fold higher than day 1 activity, whereas the CYP3A and CYP2C9 activities were 90 and 60% of the day 1 levels, respectively. In contrast, the initial CYP2C8, CYP2C19, and CYP2D6 activity losses did not rebound over the 5-day culture period. Furthermore, ABT was not found to have an effect, whether directly or indirectly as a P450 inactivator, with respect to the hepatic transport of rosuvastatin, atrovastatin, and midazolam in SCHH. Taken together, these results suggest that the SCHH model is a reliable tool to characterize hepatic uptake and biliary excretion. Due to the differential modulation of P450 activity, SCHH may not be considered a suitable tool for metabolic stability assessments with compounds predominantly cleared by certain P450 enzymes.

Human Hepatic Cytochrome P450-Specific Metabolism of the Organophosphorus Pesticides Methyl Parathion and Diazinon [Short Communication]

Ellison, C. A., Tian, Y., Knaak, J. B., Kostyniak, P. J., Olson, J. R. — 2011-12-16T10:09:46-08:00

Organophosphorus pesticides (OPs) are a public health concern due to their worldwide use and documented human exposures. Phosphorothioate OPs are metabolized by cytochrome P450s (P450s) through either a dearylation reaction to form an inactive metabolite, or through a desulfuration reaction to form an active oxon metabolite, which is a potent cholinesterase inhibitor. This study investigated the rate of desulfuration (activation) and dearylation (detoxification) of methyl parathion and diazinon in human liver microsomes. In addition, recombinant human P450s were used to determine the P450-specific kinetic parameters (K_m and V_max) for each compound for future use in refining human physiologically based pharmacokinetic/pharmacodynamic (PBPK/PD) models of OP exposure. The primary enzymes involved in bioactivation of methyl parathion were CYP2B6 (K_m = 1.25 μM; V_max = 9.78 nmol · min^–1 · nmol P450^–1), CYP2C19 (K_m = 1.03 μM; V_max = 4.67 nmol · min^–1 · nmol P450^–1), and CYP1A2 (K_m = 1.96 μM; V_max = 5.14 nmol · min^–1 · nmol P450^–1), and the bioactivation of diazinon was mediated primarily by CYP1A1 (K_m = 3.05 μM; V_max = 2.35 nmol · min^–1 · nmol P450^–1), CYP2C19 (K_m = 7.74 μM; V_max = 4.14 nmol · min^–1 · nmol P450^–1), and CYP2B6 (K_m = 14.83 μM; V_max = 5.44 nmol · min^–1 · nmol P450^–1). P450-mediated detoxification of methyl parathion only occurred to a limited extent with CYP1A2 (K_m = 16.8 μM; V_max = 1.38 nmol · min^–1 · nmol P450^–1) and 3A4 (K_m = 104 μM; V_max = 5.15 nmol · min^–1 · nmol P450^–1), whereas the major enzyme involved in diazinon detoxification was CYP2C19 (K_m = 5.04 μM; V_max = 5.58 nmol · min^–1 · nmol P450^–1). The OP- and P450-specific kinetic values will be helpful for future use in refining human PBPK/PD models of OP exposure.

A Semiphysiologically Based Pharmacokinetic Modeling Approach to Predict the Dose-Exposure Relationship of an Antiparasitic Prodrug/Active Metabolite Pair [Articles]

2011-12-16T10:09:46-08:00

Dose selection during antiparasitic drug development in animal models and humans traditionally has relied on correlations between plasma concentrations obtained at or below maximally tolerated doses that are efficacious. The objective of this study was to improve the understanding of the relationship between dose and plasma/tissue exposure of the model antiparasitic agent, pafuramidine, using a semiphysiologically based pharmacokinetic (semi-PBPK) modeling approach. Preclinical and clinical data generated during the development of pafuramidine, a prodrug of the active metabolite, furamidine, were used. A whole-body semi-PBPK model for rats was developed based on a whole-liver PBPK model using rat isolated perfused liver data. A whole-body semi-PBPK model for humans was developed on the basis of the whole-body rat model. Scaling factors were calculated using metabolic and transport clearance data generated from rat and human sandwich-cultured hepatocytes. Both whole-body models described pafuramidine and furamidine disposition in plasma and predicted furamidine tissue (liver and kidney) exposure and excretion profiles (biliary and renal). The whole-body models predicted that the intestine contributes significantly (30–40%) to presystemic furamidine formation in both rats and humans. The predicted terminal elimination half-life of furamidine in plasma was 3- to 4-fold longer than that of pafuramidine in rats (170 versus 47 h) and humans (64 versus 19 h). The dose-plasma/tissue exposure relationship for the prodrug/active metabolite pair was determined using the whole-body models. The human model proposed a dose regimen of pafuramidine (40 mg once daily) based on a predefined efficacy-safety index. A similar approach could be used to guide dose-ranging studies in humans for next-in-class compounds.

CPY3A4-Mediated Lopinavir Bioactivation and Its Inhibition by Ritonavir [Articles]

Li, F., Lu, J., Ma, X. — 2011-12-16T10:09:46-08:00

The combination of lopinavir (LPV) and ritonavir (RTV) is one of the preferred regimens for the treatment of HIV infection with confirmed efficacy and relatively low toxicity. LPV alone suffers the poor bioavailability due to its rapid and extensive metabolism. RTV boosts the plasma concentration of LPV by suppressing its metabolism and thus increasing LPV efficacy. In the current study, we found that RTV also inhibits LPV bioactivation. LPV bioactivation was investigated in human liver microsomes and cDNA-expressed human cytochromes P450. Twelve GSH-trapped reactive metabolites of LPV were identified by using a metabolomic approach. Semicarbazide-trapped reactive metabolites of LPV were also detected. RTV effectively suppressed all pathways of LPV bioactivation via CYP3A4 inhibition. Our data together with previous reports suggest that LPV plus RTV is an ideal combination because RTV not only boosts LPV plasma concentration, but it decreases LPV bioactivation.

In Vitro Glucuronidation of the Antibacterial Triclocarban and Its Oxidative Metabolites [Articles]

Schebb, N. H., Franze, B., Maul, R., Ranganathan, A., Hammock, B. D. — 2011-12-16T10:09:46-08:00

Triclocarban (3,4,4'-trichlorocarbanilide; TCC) is widely used as an antibacterial in bar soaps. During use of these soaps, a significant portion of TCC is absorbed by humans. For the elimination from the body, glucuronidation plays a key role in both biliary and renal clearance. To investigate this metabolic pathway, we performed microsomal incubations of TCC and its hydroxylated metabolites 2'-OH-TCC, 3'-OH-TCC, and 6-OH-TCC. Using a new liquid chromatography-UV-mass spectrometry method, we could show a rapid glucuronidation for all OH-TCCs by the uridine-5'-diphosphate-glucuronosyltransferases (UGT) present in liver microsomes of humans (HLM), cynomolgus monkeys (CLM), rats (RLM), and mice (MLM). Among the tested human UGT isoforms, UGT1A7, UGT1A8, and UGT1A9 showed the highest activity for the conjugation of hydroxylated TCC metabolites followed by UGT1A1, UGT1A3, and UGT1A10. Due to this broad pattern of active UGTs, OH-TCCs can be efficiently glucuronidated in various tissues, as shown for microsomes from human kidney (HKM) and intestine (HIM). The major renal metabolites in humans, TCC-N-glucuronide and TCC-N'-glucuronide, were formed at very low conversion rates (<1%) by microsomal incubations. Low amounts of N-glucuronides were generated by HLM, HIM, and HKM, as well as by MLM and CLM, but not by RLM, according to the observed species specificity of this metabolic pathway. Among the human UGT isoforms, only UGT1A9 had activity for the N-glucuronidation of TCC. These results present an anomaly where in vivo the predominant urinary metabolites of TCC are N and N'-glucuronides, but these compounds are slowly produced in vitro.

Bupropion Hydroxylation as a Selective Marker of Rat CYP2B1 Catalytic Activity [Articles]

Pekthong, D., Desbans, C., Martin, H., Richert, L. — 2011-12-16T10:09:46-08:00

Benzyloxyresorufin-O-dealkylation (BROD) is usually used as a marker of cytochrome P450 (P450) 2B1 in rat. However, some reports show that CYP1A2 is also highly implicated. The purpose of the present study was to establish bupropion (BUP) hydroxylation, but not BROD, as a selective in vitro marker of CYP2B1 catalytic activity. IC₅₀ for BROD and BUP hydroxylation were equivalent (40.8 ± 4.6 and 41.8 ± 3.4 μM, respectively) when using liver microsomes from β-naphthoflavone-pretreated rats in the presence of metyrapone (CYP2B1 inhibitor). When using the same microsomes in the presence of CYP1A1/2-selective inhibitor α-naphthoflavone, we found an IC₅₀ of 2.5 x 10^–3 ± 0.8 x 10^–3 μM for BROD and >100 μM for BUP hydroxylation. These results suggest that CYP2B1 is similarly involved in both activities, whereas CYP1A2 is involved in BROD activity but not in BUP hydroxylation. BUP hydroxylation was assessed in microsomes from baculovirus-infected insect cells coexpressing NADPH-P450 oxidoreductase, and 14 rat P450s and kinetic parameters (K_m and V_max) were determined. BUP hydroxylation was predominantly catalyzed by CYP2B1 (75% of total hydroxybupropion formation), low activity was detected with CYP2E1 and CYP2C11 (10.9 and 8.7% of total hydroxybupropion, respectively), and activity was almost undetectable with the other P450 isoforms at saturating substrate concentrations (2500 μM), thereby validating the use of BUP as a diagnostic in vitro marker of CYP2B1 catalytic activity in rat.

Effects of Chronic Renal Failure on Brain Drug Transporters in Rats [Articles]

2011-12-16T10:09:46-08:00

Studies demonstrated that chronic renal failure (CRF) affects the expression and activity of intestinal, hepatic, and renal drug transporters. Such drug transporters are expressed in brain cells and at the blood-brain barrier (BBB), where they limit the entry and distribution of drugs in the brain. Perturbations in brain drug transporter equilibrium by CRF could lead to central drug toxicity. This study evaluates how CRF affects BBB drug transporters using a 5/6 nephrectomized rat model. Protein and mRNA expression of influx transporters [organic anion-transporting polypeptide (Oatp), organic anion transporter (Oat)] and efflux transporters [P-glycoprotein (P-gp), multidrug resistance-related protein (Mrp), breast cancer resistance protein (Bcrp)] were measured in CRF and control rat brain. Intracerebral accumulation of radiolabeled benzylpenicillin, digoxin, doxorubicin, and verapamil was used to evaluate BBB drug permeability. Protein expression of the transporters was evaluated in rat brain endothelial cells (RBECs) and astrocytes incubated with control and CRF rat serum. We demonstrated significant decreases (30–50%) in protein and mRNA levels of Bcrp, Mrp2 to -4, Oat3, Oatp2 and -3, and P-gp in CRF rat brain biopsies, as well as in astrocytes and RBECs incubated with CRF serum. These decreases did not correlate with in vivo changes because BBB permeability of benzylpenicillin was decreased by 30% in CRF rats, whereas digoxin, doxorubicin, and verapamil permeabilities were unchanged. It thus seems that even with decreased drug transporters, BBB integrity and function is conserved in CRF.

Prediction of CYP2D6 Drug Interactions from In Vitro Data: Evidence for Substrate-Dependent Inhibition [Articles]

VandenBrink, B. M., Foti, R. S., Rock, D. A., Wienkers, L. C., Wahlstrom, J. L. — 2011-12-16T10:09:46-08:00

Predicting the magnitude of potential drug-drug interactions is important for underwriting patient safety in the clinical setting. Substrate-dependent inhibition of cytochrome P450 enzymes may confound extrapolation of in vitro results to the in vivo situation. However, the potential for substrate-dependent inhibition with CYP2D6 has not been well characterized. The inhibition profiles of 20 known inhibitors of CYP2D6 were characterized in vitro against four clinically relevant CYP2D6 substrates (desipramine, dextromethorphan, metoprolol, and thioridazine) and bufuralol. Dextromethorphan exhibited the highest sensitivity to in vitro inhibition, whereas metoprolol was the least sensitive. In addition, when metoprolol was the substrate, inhibitors with structurally constrained amino moieties (clozapine, debrisoquine, harmine, quinidine, and yohimbine) exhibited at least a 5-fold decrease in inhibition potency when results were compared with those for dextromethorphan. Atypical inhibition kinetics were observed for these and other inhibitor-substrate pairings. In silico docking studies suggested that interactions with Glu216 and an adjacent hydrophobic binding pocket may influence substrate sensitivity and inhibition potency for CYP2D6. The in vivo sensitivities of the clinically relevant CYP2D6 substrates desipramine, dextromethorphan, and metoprolol were determined on the basis of literature drug-drug interaction (DDI) outcomes. Similar to the in vitro results, dextromethorphan exhibited the highest sensitivity to CYP2D6 inhibition in vivo. Finally, the magnitude of in vivo CYP2D6 DDIs caused by quinidine was predicted using desipramine, dextromethorphan, and metoprolol. Comparisons of the predictions with literature results indicated that the marked decrease in inhibition potency observed for the metoprolol-quinidine interaction in vitro translated to the in vivo situation.

Potential Contribution of Cytochrome P450 2B6 to Hepatic 4-Hydroxycyclophosphamide Formation In Vitro and In Vivo [Articles]

2011-12-16T10:09:46-08:00

Results from retrospective studies on the relationship between cytochrome P450 (P450) 2B6 (CYP2B6) genotype and cyclophosphamide (CY) efficacy and toxicity in adult cancer patients have been conflicting. We evaluated this relationship in children, who have faster CY clearance and receive different CY-based regimens than adults. These factors may influence the P450s metabolizing CY to 4-hydroxycyclophosphamide (4HCY), the principal precursor to CY's cytotoxic metabolite. Therefore, we sought to characterize the in vitro and in vivo roles of hepatic CYP2B6 and its main allelic variants in 4HCY formation. CYP2B6 is the major isozyme responsible for 4HCY formation in recombinant P450 Supersomes. In human liver microsomes (HLM), 4HCY formation correlated with known phenotypic markers of CYP2B6 activity, specifically formation of (S)-2-ethyl-1,5-dimethyl-3,3-diphenyl pyrrolidine and hydroxybupropion. However, in HLM, CYP3A4/5 also contributes to 4HCY formation at the CY concentrations similar to plasma concentrations achieved in children (0.1 mM). 4HCY formation was not associated with CYP2B6 genotype at low (0.1 mM) or high (1 mM) CY concentrations potentially because CYP3A4/5 and other isozymes also form 4HCY. To remove this confounder, 4HCY formation was evaluated in recombinant CYP2B6 enzymes, which demonstrated that 4HCY formation was lower for CYP2B6.4 and CYP2B6.5 compared with CYP2B6.1. In vivo, CYP2B6 genotype was not directly related to CY clearance or ratio of 4HCY/CY areas under the curve in 51 children receiving CY-based regimens. Concomitant chemotherapy agents did not influence 4HCY formation in vitro. We conclude that CYP2B6 genotype is not consistently related to 4HCY formation in vitro or in vivo.

{gamma}-Hydroxybutyrate Blood/Plasma Partitioning: Effect of Physiologic pH on Transport by Monocarboxylate Transporters [Articles]

Morse, B. L., Felmlee, M. A., Morris, M. E. — 2011-12-16T10:09:46-08:00

The drug of abuse -hydroxybutyrate (GHB) displays nonlinear renal clearance, which has been attributed to saturable renal reabsorption by monocarboxylate transporters (MCTs) present in the kidney. MCT1 is also present in red blood cells (RBCs); however, the significance of this transporter on the blood/plasma partitioning of GHB is unknown. The purpose of this research was to characterize the transport of GHB across the RBC membrane and assess GHB blood/plasma partitioning in vivo in the presence and absence of a competitive MCT inhibitor, l-lactate. In vitro experiments were performed using freshly isolated rat erythrocytes at pH values of 6.5 and 7.4. Inhibition with p-chloromercuribenzene sulfonate and 4,4'-diisothiocyanostilbene-2,2'-disulfonate were used to determine the contribution of MCT1 and band 3, respectively, on GHB uptake. For in vivo experiments, rats were administered GHB (400–1500 mg/kg) with and without l-lactate. In vitro experiments demonstrated that GHB is transported across the RBC membrane primarily by MCT1 at relevant in vivo concentrations. The K_m for MCT1 was lower at pH 6.5 than that at pH 7.4, 2.2 versus 17.0 mM, respectively. The in vivo blood/plasma partitioning of GHB displayed linearity across all concentrations. l-Lactate coadministration increased GHB renal clearance but had no effect on the blood/plasma ratio. Unlike its MCT-mediated transport in the intestine and kidneys, GHB blood/plasma partitioning appears to be linear and is unaffected by l-lactate. These findings can be attributed, at least in part, to differences in physiologic pH at different sites of MCT-mediated transport.

Debrisoquine Metabolism and CYP2D Expression in Marmoset Liver Microsomes [Articles]

Cooke, B. R., Bligh, S. W. A., Cybulski, Z. R., Ioannides, C., Hall, M. — 2011-12-16T10:09:46-08:00

The objective of this study was to define CYP2D enzymes in marmoset (Callithrix jacchus) liver microsomes, both at the activity level using debrisoquine as the model substrate and at the protein level using antibodies raised to human CYP2D6. Marmoset liver microsomes were incubated with [¹⁴C]debrisoquine, and the structure of the generated metabolites was determined using liquid chromatography-tandem mass spectrometry and NMR. Marmoset liver microsomes were very effective in hydroxylating debrisoquine at various positions. Although 4-hydroxydebrisoquine was formed, in contrast to rat and human it was only a minor metabolite. Debrisoquine was more extensively hydroxylated in the 7, 5, 6, and 8 positions. In addition to the monohydroxylated metabolites, a dihydroxy metabolite, namely 6,7-dihydroxydebrisoquine, was identified. Finally, metabolites that had undergone ring opening were also detected but were not investigated further. Antibodies to CYP2D6 immunoreacted with protein in marmoset and human but not rat hepatic microsomes. In conclusion, we demonstrate that marmoset liver microsomes are effective in hydroxylating debrisoquine at various positions and that they contain a protein that is immunorelated to human CYP2D6.

Prediction of Human Metabolism of FK3453 by Aldehyde Oxidase Using Chimeric Mice Transplanted with Human or Rat Hepatocytes [Articles]

Sanoh, S., Nozaki, K., Murai, H., Terashita, S., Teramura, T., Ohta, S. — 2011-12-16T10:09:46-08:00

During drug development, it is important to predict the activities of multiple metabolic enzymes, not only cytochrome P450 (P450) but also non-P450 enzymes, such as conjugative enzymes and aldehyde oxidase (AO). In this study, we focused on prediction of AO-mediated human metabolism and pharmacokinetics (PK) of 6-(2-amino-4-phenylpyrimidine-5-yl)-2-isopropylpyridazin-3(2H)-one (FK3453) (Astellas Pharma Inc.), the development of which was suspended due to extremely low exposure in human, despite good oral bioavailability in rat and dog. We examined species difference in oxidative metabolism of the aminopyrimidine moiety of FK3453, catalyzed by AO, using human-chimeric mice with humanized liver (h-PXB mice) and rat-chimeric mice (r-PXB mice) transplanted with rat hepatocytes. AO activity of h-PXB mouse hepatocytes was higher than that of r-PXB mouse hepatocytes. Moreover, higher concentrations of human-specific AO-generated FK3453 metabolite A-M were detected in urine and feces after administration of FK3453 to h-PXB mice versus r-PXB mice. The total clearance of h-PXB mice was 2-fold higher than that of r-PXB mice. These results agreed reasonably well with the metabolism and PK profiles of FK3453 in human and rat. Our results indicated that h-PXB mice should be helpful for predicting the metabolic profile of drugs in humans, and the use of both h-PXB and r-PXB mice should be helpful for evaluation of species differences of AO metabolic activity.

Simultaneous Absolute Protein Quantification of Transporters, Cytochromes P450, and UDP-Glucuronosyltransferases as a Novel Approach for the Characterization of Individual Human Liver: Comparison with mRNA Levels and Activities [Articles]

2011-12-16T10:09:46-08:00

The purpose of the present study was to determine the absolute protein expression levels of multiple drug-metabolizing enzymes and transporters in 17 human liver biopsies, and to compare them with the mRNA expression levels and functional activities to evaluate the suitability of the three measures as parameters of hepatic metabolism. Absolute protein expression levels of 13 cytochrome P450 (P450) enzymes, NADPH-P450 reductase (P450R) and 6 UDP-glucuronosyltransferase (UGT) enzymes in microsomal fraction, and 22 transporters in plasma membrane fraction were determined using liquid chromatography/tandem mass spectrometry. CYP2C9, CYP2E1, CYP3A4, CYP2A6, UGT1A6, UGT2B7, UGT2B15, and P450R were abundantly expressed (more than 50 pmol/mg protein) in human liver microsomes. The protein expression levels of CYP3A4, CYP2B6, and CYP2C8 were each highly correlated with the corresponding enzyme activity and mRNA expression levels, whereas for other P450s, the protein expression levels were better correlated with the enzyme activities than the mRNA expression levels were. Among transporters, the protein expression level of organic anion-transporting polypeptide 1B1 was relatively highly correlated with the mRNA expression level. However, other transporters showed almost no correlation. These findings indicate that protein expression levels determined by the present simultaneous quantification method are a useful parameter to assess differences of hepatic function between individuals.

Absolute Quantification and Differential Expression of Drug Transporters, Cytochrome P450 Enzymes, and UDP-Glucuronosyltransferases in Cultured Primary Human Hepatocytes [Articles]

2011-12-16T10:09:46-08:00

The levels of metabolizing enzymes and transporters expressed in hepatocytes are decisive factors for hepatobiliary disposition of most drugs. Induction via nuclear receptor activation can significantly alter those levels, with the coregulation of multiple enzymes and transporters occurring to different extents. Here, we report the use of a targeted liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method for concurrent quantification of multiple cytochrome P450 (P450), UDP-glucuronosyltransferase (UGT), and transporter proteins in cultured primary human hepatocytes. The effects of culture format (i.e., sandwich culture versus conventional culture) and of dexamethasone (DEX) media concentrations on mRNA, protein, and activity levels were determined for three donors, and protein expression was compared with that in liver. In general, P450 and UGT expression was lower in hepatocyte cultures than that in liver, and CYP2C9 was found to be the most abundant P450 isoform expressed in cultured hepatocytes. The sandwich culture format and 0.1 μM DEX in media retained the protein expression in the hepatocytes closest to the levels found in liver. However, higher in vitro expression was observed for drug transporters, especially for multidrug resistance protein 1 and breast cancer resistance protein. Direct protein quantification was applied successfully to study in vitro induction in sandwich cultured primary hepatocytes in a 24-well format using the prototypical inducers rifampicin, omeprazole, and phenobarbital. We conclude that targeted absolute LC-MS/MS quantification of drug-metabolizing enzymes and transporters can broaden the scope and significantly increase the impact of in vitro drug metabolism studies, such as induction, as an important supplement or future alternative to mRNA and activity data.

Comparison of Cryopreserved HepaRG Cells with Cryopreserved Human Hepatocytes for Prediction of Clearance for 26 Drugs [Articles]

Zanelli, U., Caradonna, N. P., Hallifax, D., Turlizzi, E., Houston, J. B. — 2011-12-16T10:09:46-08:00

Prediction of clearance in drug discovery currently relies on human primary hepatocytes, which can vary widely in drug-metabolizing enzyme activity. Potential alternative in vitro models include the HepaRG cell (from immortalized hepatoma cells), which in culture can express drug-metabolizing enzymes to an extent comparable to that of primary hepatocytes. Utility of the HepaRG cell will depend on robust performance, relative to that of primary hepatocytes, in routine high-throughput analysis. In this study, we compared intrinsic clearance (CL_int) in the recently developed cryopreserved HepaRG cell system with CL_int in human cryopreserved pooled hepatocytes and with CL_int in vivo for 26 cytochrome P450 substrate drugs. There was quantitative agreement between CL_int in HepaRG cells and human hepatocytes, which was linear throughout the range of CL_int (1–2000 ml · min^–1 · kg^–1) and not dependent on particular cytochrome P450 involvement. Prediction of CL_int in HepaRG cells was on average within 2-fold of in vivo CL_int (using the well stirred liver model), but average fold error was clearance-dependent with greater underprediction (up to at least 5-fold) for the more highly cleared drugs. Recent reporting of this phenomenon in human hepatocytes was therefore confirmed with the hepatocytes used in this study, and hence the HepaRG cell system appears to share an apparently general tendency of clearance-limited CL_int in cell models. This study shows the cryopreserved HepaRG cell system to be quantitatively comparable to human hepatocytes for prediction of clearance of drug cytochrome P450 substrates and to represent a promising alternative in vitro tool.

Frequency of Undetected CYP2D6 Hybrid Genes in Clinical Samples: Impact on Phenotype Prediction [Articles]

Black, J. L., Walker, D. L., O'Kane, D. J., Harmandayan, M. — 2011-12-16T10:09:46-08:00

Cytochrome P450 2D6 (CYP2D6) is highly polymorphic. CYP2D6-2D7 hybrid genes can be present in samples containing CYP2D6*4 and CYP2D6*10 alleles. CYP2D7-2D6 hybrid genes can be present in samples with duplication signals and in samples with homozygous genotyping results. The frequency of hybrid genes in clinical samples is unknown. We evaluated 1390 samples for undetected hybrid genes by polymerase chain reaction (PCR) amplification, PCR fragment analysis, TaqMan copy number assays, DNA sequencing, and allele-specific primer extension assay. Of 508 CYP2D6*4-containing samples, 109 (21.5%) harbored CYP2D6*68 + *4-like, whereas 9 (1.8%) harbored CYP2D6*4N + *4-like. Of 209 CYP2D6*10-containing samples, 44 (21.1%) were found to have CYP2D6*36 + *10. Of 332 homozygous samples, 4 (1.2%) harbored a single CYP2D7-2D6 hybrid, and of 341 samples with duplication signals, 25 (7.3%) harbored an undetected CYP2D7-2D6 hybrid. Phenotype before and after accurate genotyping was predicted using a method in clinical use. The presence of hybrid genes had no effect on the phenotype prediction of CYP2D6*4- and CYP2D6*10-containing samples. Four of four (100%) homozygous samples containing a CYP2D7-2D6 gene had a change in predicted phenotype, and 23 of 25 (92%) samples with a duplication signal and a CYP2D7-2D6 gene had a change in predicted phenotype. Four novel genes were identified (CYP2D6*13A1 variants 1 and 2, CYP2D6*13G1, and CYP2D6*13G2), and two novel hybrid tandem structures consisting of CYP2D6*13B + *68x2 + *4-like and CYP2D6*13A1 variant 2 + *1xN were observed.

Cytoprotective Effect of 20(S)-Rg3 on Benzo[a]pyrene-Induced DNA Damage [Articles]

2011-12-16T10:09:46-08:00

Benzo[a]pyrene (BaP) is a polycyclic aromatic hydrocarbon ubiquitously existing in the environment. Its metabolites have been shown to cause DNA damage and cellular dysfunction in humans. Panax ginseng C.A. Meyer is a Chinese medicinal herb, and ginsenosides are the main active constituent of ginseng. Accumulating evidence had indicated that ginseng extract and ginsenosides possess cytoprotective effects. In this study, the protective effect of ginsenosides on BaP-induced DNA damage in human dermal fibroblasts (HDFs) and HepG2 cells was investigated. The genotoxic effect of BaP was measured by the comet assay. Results showed that tail moment was increased in BaP-treated cells, but cotreatment of ginsenoside 20(S)-Rg3 can significantly decrease BaP-induced DNA damage. A downstream mechanistic study revealed that 20(S)-Rg3 increased the gene expression of an important phase II detoxifying enzyme NAD(P)H:quinine oxidoreductase 1. The effect was also associated with the activation of protein kinase B (Akt) and nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2). These results indicated that 20(S)-Rg3 might protect HDFs from BaP-induced DNA damage through the activation of the phosphatidylinositol 3-kinase/Akt/Nrf2 pathway. Our results also demonstrated that 20(S)-Rg3 is a functional ligand of pregnane X receptor (PXR), a nuclear receptor that mediates the induction of drug clearance pathways. Subsequent knockdown of PXR expression by small interfering RNA confirmed the involvement of PXR on the protective effects of 20(S)-Rg3 against BaP-induced DNA damage. In summary, ginsenoside 20(S)-Rg3 can protect against BaP-induced genotoxicity in human cells, suggesting that ginseng may serve as a natural cytoprotective agent against environmental carcinogens.

In Vitro Inhibition of the Bile Salt Export Pump Correlates with Risk of Cholestatic Drug-Induced Liver Injury in Humans [Articles]

Dawson, S., Stahl, S., Paul, N., Barber, J., Kenna, J. G. — 2011-12-16T10:09:46-08:00

Inhibition of the activity of the human bile salt export pump (BSEP: ABCB11) has been proposed to play a role in drug-induced liver injury (DILI). To enhance understanding of the relationship between BSEP inhibition and DILI, inhibition of human BSEP (hBSEP) and its rat ortholog (rBsep) by 85 pharmaceuticals was investigated in vitro. This was explored using assays that quantified inhibition of ATP-dependent [³H]taurocholate uptake into inverted plasma membrane vesicles from Sf21 insect cells, which expressed the proteins. Of the pharmaceuticals, 40 exhibited evidence of in vitro transporter inhibition and overall a close correlation was observed between potency values for inhibition of hBSEP and rBsep activity (r² = 0.94), although 12 drugs exhibited >2-fold more potent inhibition of hBSEP than rBsep. The median potency of hBSEP inhibition was higher among drugs that caused cholestatic/mixed DILI than among drugs that caused hepatocellular or no DILI, as was the incidence of hBSEP inhibition with IC₅₀ <300 μM. All drugs with hBSEP IC₅₀ <300 μM had molecular weight >250, ClogP >1.5, and nonpolar surface area >180Å. A clear distinction was not evident between hBSEP IC₅₀ or unbound plasma concentration (C_{max, u}) of the drugs in humans and whether the drugs caused DILI. However, all 17 of the drugs with hBSEP IC₅₀ <100 μM and C_{max, u} >0.002 μM caused DILI. Overall, these data indicate that inhibition of hBSEP/rBsep correlates with the propensity of numerous pharmaceuticals to cause cholestatic DILI in humans and is associated with several of their physicochemical properties.

Human Metabolism of Lapatinib, a Dual Kinase Inhibitor: Implications for Hepatotoxicity [Articles]

2011-12-16T10:09:46-08:00

Lapatinib (Tykerb, Tyverb) is an important orally active dual tyrosine kinase inhibitor efficacious in combination therapy for patients with progressive human epidermal receptor 2-overexpressing metastatic breast cancer. However, clinically significant liver injury, which may be associated with lapatinib metabolic activation, has been reported. We describe the metabolism and excretion of [¹⁴C]lapatinib in six healthy human volunteers after a single oral dose of 250 mg and the potential relationships between metabolism and clinical hepatotoxicity. Overall, elimination showed high intersubject variability, with fecal elimination being the predominant pathway, representing a median of 92% of the dose with lapatinib as the largest component (approximate median 27% of the dose). In plasma, approximately 50% of the observed radioactivity was attributed to metabolites. Analysis of a 4-h pooled plasma extract identified seven metabolites related by an N- and α-carbon oxidation cascade. Fecal metabolites derived from three prominent pathways: N- and α-carbon oxidation, fluorobenzyl oxidative cleavage, and hydroxypyridine formation. Several of the lapatinib metabolites can undoubtedly be linked to reactive species such as aldehydes or quinone imines. In addition to the contribution of these potentially reactive metabolites as suspects in clinical liver injury, the role of other disposition factors, including interaction with drug transporters, pharmacogenetics, or magnitude of the therapeutic dose, should not be discounted.

Interindividual Variability in Gene Expression Profiles in Human Hepatocytes and Comparison with HepaRG Cells [Articles]

Rogue, A., Lambert, C., Spire, C., Claude, N., Guillouzo, A. — 2011-12-16T10:09:46-08:00

Interindividual variations in functions other than drug metabolism activity, remain poorly elucidated in human liver. In the present study, the whole transcriptome of several human hepatocyte populations and the differentiated human HepaRG cell line have been analyzed and compared, using oligonucleotide pangenomic microarrays. We show that, although the variation in the percentages of expressed genes did not exceed 14% among the primary human hepatocyte populations, huge interindividual differences in the transcript levels of many genes were observed. These genes were related to various functions; in addition to drug metabolism, they mainly concerned carbohydrate, amino acid, and lipid metabolism. HepaRG cells expressed from 81 to 92% of the genes active in human hepatocytes and, in addition, a specific gene subset mainly related to their transformed status, some chromosomal abnormalities, and the presence of primitive biliary epithelial cells. Of interest, a relationship was evidenced between abnormal basal expression levels of some target genes and their corresponding previously reported fold changes in one of four human hepatocyte populations treated with the hepatotoxic drug troglitazone and not with other nonhepatotoxic peroxisome proliferator-activated receptor agonists (PLoS One 6:e18816, 2011). Taken together, our results support the view that HepaRG cells express most of the genes active in primary human hepatocytes and show that expression of most human hepatic genes can quantitatively greatly vary among individuals, thereby contributing to explain the huge interindividual variability in susceptibility to drugs and other environmental factors.

Prediction of Relative In Vivo Metabolite Exposure from In Vitro Data Using Two Model Drugs: Dextromethorphan and Omeprazole [Articles]

Lutz, J. D., Isoherranen, N. — 2011-12-16T10:09:46-08:00

Metabolites can have pharmacological or toxicological effects, inhibit metabolic enzymes, and be used as probes of drug-drug interactions or specific cytochrome P450 (P450) phenotypes. Thus, better understanding and prediction methods are needed to characterize metabolite exposures in vivo. This study aimed to test whether in vitro data could be used to predict and rationalize in vivo metabolite exposures using two model drugs and P450 probes: dextromethorphan and omeprazole with their primary metabolites dextrorphan, 5-hydroxyomeprazole (5OH-omeprazole), and omeprazole sulfone. Relative metabolite exposures were predicted using metabolite formation and elimination clearances. For dextrorphan, the formation clearances of dextrorphan glucuronide and 3-hydroxymorphinan from dextrorphan in human liver microsomes were used to predict metabolite (dextrorphan) clearance. For 5OH-omeprazole and omeprazole sulfone, the depletion rates of the metabolites in human hepatocytes were used to predict metabolite clearance. Dextrorphan/dextromethorphan in vivo metabolite/parent area under the plasma concentration versus time curve ratio (AUC_m/AUC_p) was overpredicted by 2.1-fold, whereas 5OH-omeprazole/omeprazole and omeprazole sulfone/omeprazole were predicted within 0.75- and 1.1-fold, respectively. The effect of inhibition or induction of the metabolite's formation and elimination on the AUC_m/AUC_p ratio was simulated. The simulations showed that unless metabolite clearance pathways are characterized, interpretation of the metabolic ratios is exceedingly difficult. This study shows that relative in vivo metabolite exposure can be predicted from in vitro data and characterization of secondary metabolism of probe metabolites is critical for interpretation of phenotypic data.

Acetaminophen-Induced Hepatotoxicity in a Liver Tissue Model Consisting of Primary Hepatocytes Assembling around an Endothelial Cell Network [Articles]

2011-12-16T10:09:46-08:00

Primary hepatocytes have been used in drug development for the evaluation of hepatotoxicity of candidate compounds. However, the rapid depression of their hepatic characters in vitro must be improved to predict toxicity with higher accuracy. We have hypothesized that a well organized tissue construct that includes nonparenchymal cells and appropriate scaffold material(s) could overcome this difficulty by remediating the viability and physiological function of primary hepatocytes. In this study, we constructed an in vitro liver tissue model, consisting of mouse primary hepatocytes assembling around an endothelial cell network on Engelbreth-Holm-Swarm gel, and examined its response to acetaminophen treatment. The increase in lactate dehydrogenase release after the exposure to acetaminophen was induced earlier in the liver tissue model than in monolayer hepatocytes alone, suggesting that the tissue model was more sensitive to an acetaminophen-induced toxicity. On the basis of our results, we conclude that liver tissue models of this kind may enhance the responses of hepatocytes against xenobiotics via the maintenance of hepatic genes and functions such as cytochrome P450s. These findings will contribute to the development of more accurate systems for evaluating hepatotoxicity.

Species-Dependent and Receptor-Selective Action of Bilobalide on the Function of Constitutive Androstane Receptor and Pregnane X Receptor [Articles]

Lau, A. J., Yang, G., Rajaraman, G., Baucom, C. C., Chang, T. K. H. — 2011-12-16T10:09:46-08:00

Bilobalide is a naturally occurring sesquiterpene trilactone with therapeutic potential in the management of ischemia and neurodegenerative diseases such as Alzheimer's disease. In the present study, we investigated the effect of bilobalide on the activity of rat constitutive androstane receptor (rCAR) and rat pregnane X receptor (rPXR) and compared that with human CAR (hCAR) and human PXR (hPXR). Bilobalide activated rCAR in a luciferase reporter gene assay and increased rCAR target gene expression in cultured rat hepatocytes, as determined by the CYP2B1 mRNA and CYP2B enzyme activity (benzyloxyresorufin O-dealkylation) assays. This increase in hepatocyte CYP2B1 expression by bilobalide was not accompanied by a corresponding increase in rCAR mRNA level. In contrast to the activation of rCAR, the activity of rPXR, hCAR, and hPXR was not influenced by this chemical in cell-based reporter gene assays. Consistent with these results, bilobalide did not alter rPXR, hCAR, or hPXR target gene expression in rat or human hepatocytes, as evaluated by the CYP3A23, CYP2B6, CYP3A4 mRNA assays and the CYP3A (testosterone 6β-hydroxylation) and CYP2B6 (bupropion hydroxylation) enzyme activity assays. Bilobalide was not an antagonist of rPXR, hCAR, or hPXR, as suggested by the finding that it did not attenuate rPXR activation by pregnenolone 16α-carbonitrile, hCAR activation by 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime, or hPXR activation by rifampicin in reporter gene assays. In conclusion, bilobalide is an activator of rCAR, whereas it is not a ligand of rPXR, hCAR, or hPXR. Likewise, it is an inducer of rat CYP2B1, but not of rat CYP3A23, human CYP2B6, or human CYP3A4.

Effects of Omeprazole and Genetic Polymorphism of CYP2C19 on the Clopidogrel Active Metabolite [Articles]

2011-12-16T10:09:46-08:00

Clopidogrel is an antiplatelet agent widely used in cardiovascular diseases and an inactive prodrug that needs to be converted to an active metabolite in two sequential metabolic steps. Several CYP450 isoforms involved in these two steps have been described, although the relative contribution in vivo of each enzyme is still under debate. CYP2C19 is considered to be the major contributor to active metabolite formation. In the current study, net CYP2C19 contribution to the active metabolite formation was determined from exposure of the active metabolite in two clinical studies (one phase I study with well balanced genetic polymorphic populations and a meta-analysis with a total of 396 healthy volunteers) at different clopidogrel doses. CYP2C19 involvements were estimated to be from 58 to 67% in intermediate metabolizers (IMs), from 58 to 72% in extensive metabolizers (EMs), and from 56 to 74% in ultrarapid metabolizers (UMs), depending on the study and the dose. For this purpose, a static model was proposed to estimate the net contribution of a given enzyme to the secondary metabolite formation. This static model was compared with a dynamic approach (Simcyp model) and showed good consistency. In parallel, in vitro investigations showed that omeprazole is a mechanism-based inhibitor of CYP2C19 with K_I of 8.56 μM and K_inact of 0.156 min^–1. These values were combined with the net CYP2C19 contribution to the active metabolite formation, through a static approach, to predict the inhibitory effect at 80-mg omeprazole doses in EM, IM, and UM CYP2C19 populations, with good consistency, compared with observed clinical values.

Phenotype of the Most Common "Slow Acetylator" Arylamine N-Acetyltransferase 1 Genetic Variant (NAT1*14B) Is Substrate-Dependent [Articles]

Millner, L. M., Doll, M. A., Cai, J., States, J. C., Hein, D. W. — 2011-12-16T10:09:46-08:00

Human arylamine N-acetyltransferase 1 (NAT1) is a phase II cytosolic enzyme responsible for the activation or deactivation of many arylamine compounds including pharmaceuticals and environmental carcinogens. NAT1 is highly polymorphic and has been associated with altered risk toward many cancers. NAT1*14B is characterized by a single nucleotide polymorphism in the coding region (rs4986782; 560G>A; R187Q). NAT1*14B is associated with higher frequency of smoking-induced lung cancer and is the most common "slow acetylator" arylamine NAT1 genetic variant. Previous studies have reported decreased N- and O-acetylation capacity and increased proteasomal degradation of NAT1 14B compared with the referent, NAT1 4. The current study is the first to investigate NAT1*14B expression using constructs that completely mimic NAT1 mRNA by including the 5'- and 3'-untranslated regions, together with the open reading frame of the referent, NAT1*4, or variant, NAT1*14B. Our results show that NAT1 14B is not simply associated with "slow acetylation." NAT1 14B-catalyzed acetylation phenotype is substrate-dependent, and NAT1 14B exhibits higher N- and O-acetylation catalytic efficiency as well as DNA adducts after exposure to the human carcinogen 4-aminobiphenyl.

Regulation of Cytochrome P450 4F11 by Nuclear Transcription Factor-{kappa}B [Articles]

Bell, J. C., Strobel, H. W. — 2011-12-16T10:09:47-08:00

Although the mechanisms that regulate CYP4F genes have been and are currently being studied in a number of laboratories, the specific mechanisms for the regulation of these genes are not yet fully understood. This study shows that nuclear factor B of the light-chain-enhancer in activated B cells (NF-B) can inhibit CYP4F11 expression in human liver carcinoma cell line (HepG2) as summarized below. Tumor necrosis factor-α (TNF-α), a proinflammatory cytokine, has been shown to activate NF-B signaling while also activating the c-Jun NH₂-terminal kinase (JNK) signaling pathway. Other studies have reported that JNK signaling can up-regulate CYP4F11 expression. The results of this study demonstrate that in the presence of TNF-α and the specific NF-B translocation inhibitor N-[3,5-bis(trifluoromethyl)phenyl]-5-chloro-2-hydroxybenzamide (IMD-0354), there is a greater increase in CYP4F11 expression than that elicited by TNF-α alone, indicating that NF-B plays an inhibitory role. Moreover, NF-B stimulation by overexpression of mitogen-activated protein kinase kinase kinase inhibited CYP4F11 promoter expression. CYP4F11 promoter inhibition can also be rescued in the presence of TNF-α when p65, a NF-B protein, is knocked down. Thus, NF-B signaling pathways negatively regulate the CYP4F11 gene.

Brain Uptake of the Drug of Abuse {gamma}-Hydroxybutyric Acid in Rats [Articles]

Roiko, S. A., Felmlee, M. A., Morris, M. E. — 2011-12-16T10:09:47-08:00

-Hydroxybutyric acid (GHB) is an endogenous compound and a substrate for the ubiquitous monocarboxylate transporter (MCT) family. GHB is also a drug of abuse due to its sedative/hypnotic and euphoric effects, with overdoses resulting in toxicity and death. The goal of this study was to characterize the distribution of GHB into the brain using in vivo microdialysis and in vitro uptake studies and to determine concentration-effect relationships for GHB in a rat animal model. GHB was administered to rats (400, 600, and 800 mg/kg i.v.), and blood, dialysate, and urine were collected for 6 h post-GHB administration. The GHB plasma and extracellular fluid (ECF) concentration-time profiles revealed that GHB concentrations in ECF closely followed plasma GHB concentrations. Sleep time increased in a dose-dependent manner (91 ± 18, 134 ± 11, and 168 ± 13 min, for GHB 400, 600, and 800 mg/kg, respectively). GHB partitioning into brain ECF was not significantly different at 400, 600, and 800 mg/kg. GHB uptake in rat and human brain endothelial cells exhibited concentration dependence. The concentration-dependent uptake of GHB at pH 7.4 was best-fit to a single-transporter model [K_m = 18.1 mM (human), 23.3 mM (rat), V_max = 248 and 258 pmol · mg^–1 · min^–1 for human and rat, respectively]. These findings indicate that although GHB distribution into the brain is mediated via MCT transporters, it is not capacity-limited over the range of doses studied in this investigation.

Identification of the Oxidative and Conjugative Enzymes Involved in the Biotransformation of Brivanib [Articles]

Gong, J., Gan, J., Iyer, R. A. — 2011-12-16T10:09:47-08:00

Brivanib alaninate, the l-alanine ester prodrug of brivanib, is currently being developed as an anticancer agent. In humans, brivanib alaninate is rapidly hydrolyzed to brivanib. Prominent biotransformation pathways of brivanib included oxidation and direct sulfate conjugation. A series of in vitro studies were conducted to identify the human esterases involved in the prodrug hydrolysis and to identify the primary human cytochrome P450 and sulfotransferase (SULT) enzymes involved in the metabolism of brivanib. Brivanib alaninate was efficiently converted to brivanib in the presence of either human carboxylesterase 1 or carboxylesterase 2. Because esterases are ubiquitous, it is likely that multiple esterases are involved in the hydrolysis. Oxidation of brivanib in human liver microsomes (HLM) primarily formed a hydroxylated metabolite (M7). Incubation of brivanib with human cDNA-expressed P450 enzymes and with HLM in the presence of selective chemical inhibitors and monoclonal P450 antibodies demonstrated that CYP1A2 and CYP3A4 were the major contributors for the formation of M7. Direct sulfation of brivanib was catalyzed by multiple SULT enzymes, including SULT1A1, SULT1B1, SULT2A1, SULT1A3, and SULT1E1. Because the primary in vitro oxidative metabolite (M7) was not detected in humans after oral doses of brivanib alaninate, further metabolism studies of M7 in HLM and human liver cytosol were performed. The data demonstrated that M7 was metabolized to the prominent metabolites observed in humans. Overall, multiple enzymes are involved in the metabolism of brivanib, suggesting a low potential for drug-drug interactions either through polymorphism or through inhibition of a particular drug-metabolizing enzyme.

Correction to "Cytochrome P450 Pig Liver Pie: Determination of Individual Cytochrome P450 Isoform Contents in Microsomes from Two Pig Livers Using Liquid Chromatography in Conjunction with Mass Spectroscopy" [Erratum]

2011-12-16T10:09:47-08:00

Correction for Nonspecific Binding to Various Components of Ultrafiltration Apparatus and Impact on Estimating In Vivo Rat Clearance for a Congeneric Series of 5-Ethyl, 5-n-Alkyl Barbituric Acids [Short Communications]

Ballard, P., Rowland, M. — 2011-11-21T06:58:10-08:00

Accurately predicting in vivo metabolic clearance from in vitro liver microsomes or hepatocytes requires a good understanding of the factors contributing to the prediction. Although much work has concentrated on deriving scaling factors and optimizing the metabolic stability techniques for consistency and rigor, it is only relatively recently that the importance of binding to microsomes and hepatocytes has been appreciated. Ultrafiltration is often used to estimate binding to plasma proteins and microsomes, but the level of nonspecific binding (NSB) to the ultrafiltration apparatus has not been adequately described. We derive an equation to correct for NSB and demonstrate that this can significantly affect the estimate of binding to microsomes and improve the accuracy of scaling to in vivo clearance for a series of barbiturates.

Effect of Hepatic Cytochrome P450 (P450) Oxidoreductase Deficiency on 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-DNA Adduct Formation in P450 Reductase Conditional Null Mice [Short Communications]

2011-11-21T06:58:10-08:00

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), formed during the cooking of foods, induces colon cancer in rodents. PhIP is metabolically activated by cytochromes P450 (P450s). To evaluate the role of hepatic P450s in the bioactivation of PhIP, we used Reductase Conditional Null (RCN) mice, in which cytochrome P450 oxidoreductase (POR), the unique electron donor to P450s, can be specifically deleted in hepatocytes by pretreatment with 3-methylcholanthrene (3-MC), resulting in the loss of essentially all hepatic P450 function. RCN mice were treated orally with 50 mg/kg b.wt. PhIP daily for 5 days, with and without 3-MC pretreatment. PhIP-DNA adducts (i.e., N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine [dG-C8-PhIP]), measured by liquid chromatography-tandem mass spectrometry, were highest in colon (1362 adducts/10⁸ deoxynucleosides), whereas adduct levels in liver were ~3.5-fold lower. Whereas no differences in PhIP-DNA adduct levels were found in livers with active POR versus inactivated POR, adduct levels were on average ~2-fold lower in extrahepatic tissues of mice lacking hepatic POR. Hepatic microsomes from RCN mice with or without 3-MC pretreatment were also incubated with PhIP and DNA in vitro. PhIP-DNA adduct formation was ~8-fold lower with hepatic microsomes from POR-inactivated mice than with those with active POR. Most of the hepatic microsomal activation of PhIP in vitro was attributable to CYP1A. Our results show that PhIP-DNA adduct formation in colon involves hepatic N-oxidation, circulation of activated metabolites via the bloodstream to extrahepatic tissues, and further activation, resulting in the formation of dG-C8-PhIP. Besides hepatic P450s, PhIP may be metabolically activated mainly by a non-P450 pathway in liver.

Production of Ibuprofen Acyl Glucosides by Human UGT2B7 [Articles]

Buchheit, D., Dragan, C.-A., Schmitt, E. I., Bureik, M. — 2011-11-21T06:58:10-08:00

UDP-glycosyltransferases (UGTs) are an important group of enzymes that participate in phase II metabolism of xenobiotics and use the cofactor UDP-glucuronic acid for the production of glucuronides. When acting on molecules bearing a carboxylic acid they can form acyl glucuronides, a group of metabolites that has gained significant interest in recent years because of concerns about their potential role in drug toxicity. In contrast, reports about the production of drug acyl glucosides (which might also display high reactivity) have been scarce. In this study, we discovered the formation of acyl glycoside metabolites of R- and S-ibuprofen (Ibu) by human liver microsomes supplied with the cofactor UDP-glucose. Subsequently, human UGT2B7*1 and UGT2B7*2 recombinantly expressed in fission yeast Schizosaccharomyces pombe could be shown to catalyze these reactions. Moreover, we could enhance the glucoside production rate in fission yeast by overexpressing the fission yeast gene SPCC1322.04, a potential UDP-glucose pyrophosphorylase (UGPase), but not by overexpression of SPCC794.10, and therefore suggest to name this gene fyu1 for fission yeast UGPase1. It was interesting to note that pronounced differences between the two polymorphic UGT2B7 variants were observed with respect to acyl glucoside production. Finally, using the metabolic precursor [¹³C₆]glucose, we demonstrated the production of stable isotope-labeled reference standards of Ibu acyl glucoside and Ibu acyl glucuronide by whole-cell biotransformation in fission yeast.

Differences in the Disposition of Silymarin between Patients with Nonalcoholic Fatty Liver Disease and Chronic Hepatitis C [Articles]

2011-11-21T06:58:10-08:00

Silymarin, derived from the milk thistle plant Silybum marianum and widely used for self-treatment of liver diseases, is composed of six major flavonolignans including silybin A and silybin B, which are the predominant flavonolignans quantified in human plasma. The single- and multiple-dose pharmacokinetics of silymarin flavonolignans were examined in patients with nonalcoholic fatty liver disease (NAFLD) or hepatitis C virus (HCV) to determine whether the disposition of silymarin and therefore its potential efficacy vary among liver disease populations. Cohorts of eight subjects with noncirrhotic liver disease were randomized 3:1 to oral silymarin or placebo (280 or 560 mg) every 8 h for 7 days. Forty-eight-hour blood sampling was conducted after the first and final doses. In general, plasma concentrations of silybin A and silybin B were higher, whereas concentrations of conjugates were lower in NAFLD compared with HCV. After adjustment of the area under plasma concentration-time curve from 0 to 8 h (AUC_{0–8 h}) for weight and dose, only silybin B and silybin B conjugates differed significantly between disease types. For NAFLD, the adjusted mean AUC_{0–8 h} was higher for silybin B (p < 0.05) but lower for silybin B conjugates (p < 0.05) compared with that for HCV. At the 280-mg dose, steady-state plasma concentrations of silybin B conjugates for NAFLD subjects were characterized by 46% lower AUC_{0–8 h} (p < 0.05) and 42% lower C_max (p < 0.05) compared with HCV subjects. Evidence of enterohepatic cycling of flavonolignans was only observed in NAFLD subjects. In summary, the efficacy of silymarin may be more readily observed in NAFLD patients because of their higher flavonolignan plasma concentrations and more extensive enterohepatic cycling compared with those in HCV patients.

Excretion, Metabolism, and Pharmacokinetics of CP-945,598, a Selective Cannabinoid Receptor Antagonist, in Rats, Mice, and Dogs [Articles]

Miao, Z., Scott, D. O., Griffith, D. A., Day, R., Prakash, C. — 2011-11-21T06:58:10-08:00

1-(8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl)-4-(ethylamino)piperidine-4-carboxamide (CP-945,598) is an orally active antagonist of the cannabinoid CB-1 receptor that progressed into phase 3 human clinical trials for the treatment of obesity. In this study, we investigated the metabolic fate and disposition of CP-945,598 in rats, Tg-RasH2 mice, and dogs after oral administration of a single dose of [¹⁴C]CP-945,598. Total mean recoveries of the radioactive dose were 97.7, 97.8, and 99.3% from mice, rats, and dogs, respectively. The major route of excretion in all three species was via the feces, but on the basis of separate studies in bile duct-cannulated rats and dogs, this probably reflects excretion in bile rather than incomplete absorption. CP-945,598 underwent extensive metabolism in all three species, because no unchanged parent compound was detected in the urine across species. The primary metabolic pathway of CP-945,598 involved N-deethylation to form an N-desethyl metabolite (M1). M1 was subsequently metabolized by amide hydrolysis, oxidation, and ribose conjugation to numerous novel and unusual metabolites. The major circulating and excretory metabolites were species-dependent; however, several common metabolites were observed in more than one species. In addition to parent compound, M1, M3, M4, and M5 in rats, M1, M3, and M4 in mice, and M1 and M2 in dogs were identified as the major circulating metabolites. Gender-related differences were also apparent in the quantitative and qualitative nature of the metabolites in rats. An unprecedented metabolite, M4, formed by deamidation of M1 or M3 (N-hydroxy-M1), but not by decarboxylation of M2, was identified in all species. M4 was nonenzymatically converted to M5.

Cardiac Arrest and Therapeutic Hypothermia Decrease Isoform-Specific Cytochrome P450 Drug Metabolism [Articles]

Zhou, J., Empey, P. E., Bies, R. R., Kochanek, P. M., Poloyac, S. M. — 2011-11-21T06:58:10-08:00

Mild therapeutic hypothermia is emerging clinically as a neuroprotection therapy for individuals experiencing cardiac arrest (CA); however, its effects combined with disease pathogenesis on drug disposition and response have not been fully elucidated. We determined the activities of four major hepatic-metabolizing enzymes (CYP3A, CYP2C, CYP2D, and CYP2E) during hypothermia after experimental CA in rats by evaluating the pharmacokinetics of their probe drugs as a function of altered body temperature. Animals were randomized into sham normothermia (37.5–38°C), CA normothermia, sham hypothermia (32.5–33°C), and CA hypothermia groups. Probe drugs (midazolam, diclofenac, dextromethorphan, and chlorzoxazone) were given simultaneously by intravenous bolus after temperature stabilization. Multiple blood samples were collected between 0 and 8 h after drug administration. Pharmacokinetic (PK) analysis was conducted using a noncompartmental approach and population PK modeling. Noncompartmental analysis showed that the clearance of midazolam (CYP3A) in CA hypothermia was reduced from sham normothermia rats (681.6 ± 190.0 versus 1268.8 ± 348.9 ml · h^–1 · kg^–1, p < 0.05). The clearance of chlorzoxazone (CYP2E) in CA hypothermia was also reduced from sham normothermia rats (229.6 ± 75.6 versus 561.89 ± 215.9 ml · h^–1 · kg^–1, p < 0.05). Population PK analysis further demonstrated the decreased clearance of midazolam (CYP3A) was associated with CA injury (p < 0.05). The decreased clearance of chlorzoxazone (CYP2E1) was also associated with CA injury (p < 0.01). Hypothermia was found to be associated with the decreased volume of distribution of midazolam (V₁), dextromethorphan (V₁), and peripheral compartment for chlorzoxazone (V₂) (p < 0.05, p < 0.05, and p < 0.01, respectively). Our data indicate that hypothermia, CA, and their interaction alter cytochrome P450-isoform specific activities in an isoform-specific manner.

Preclinical Metabolism and Disposition of SB939 (Pracinostat), an Orally Active Histone Deacetylase Inhibitor, and Prediction of Human Pharmacokinetics [Articles]

2011-11-21T06:58:10-08:00

The preclinical absorption, distribution, metabolism, and excretion (ADME) properties of Pracinostat [(2E)-3-[2-butyl-1-[2-(diethylamino) ethyl]-1H-benzimidazol-5-yl]-N-hydroxyarylamide hydrochloride; SB939], an orally active histone deacetylase inhibitor, were characterized and its human pharmacokinetics (PK) was predicted using Simcyp and allometric scaling. SB939 showed high aqueous solubility with high Caco-2 permeability. Metabolic stability was relatively higher in dog and human liver microsomes than in mouse and rat. The major metabolites formed in human liver microsomes were also observed in preclinical species. Human cytochrome P450 (P450) phenotyping showed that SB939 was primarily metabolized by CYP3A4 and CYP1A2. SB939 did not significantly inhibit human CYP3A4, 1A2, 2D6, and 2C9 (>25 μM) but inhibited 2C19 (IC₅₀ = 5.8 μM). No significant induction of human CYP3A4 and 1A2 was observed in hepatocytes. Plasma protein binding in mouse, rat, dog, and human ranged between ~84 and 94%. The blood-to-plasma ratio was ~1.0 in human blood. SB939 showed high systemic clearance (relative to liver blood flow) of 9.2, 4.5, and 1.5 l · h^–1 · kg^–1 and high volume of distribution at steady state (>0.6 l/kg) of 3.5, 1.7, and 4.2 l/kg in mouse, rat, and dog, respectively. The oral bioavailability was 34, 65, and ~3% in mice, dogs, and rats, respectively. The predicted oral PK profile and parameters of SB939, using Simcyp and allometric scaling, were in good agreement with observed data in humans. Simcyp predictions showed lack of CYP3A4 and 2C19 drug-drug interaction potential for SB939. In summary, the preclinical ADME of SB939 supported its preclinical and clinical development as an oral drug candidate.

Independent and Combined Effects of Ethanol Self-Administration and Nicotine Treatment on Hepatic CYP2E1 in African Green Monkeys [Articles]

Ferguson, C. S., Miksys, S., Palmour, R., Tyndale, R. F. — 2011-11-21T06:58:10-08:00

Cytochrome P450 2E1 metabolizes ethanol and also bioactivates many toxins and procarcinogens. Elevated levels of hepatic CYP2E1 are associated with an increased susceptibility to chemical toxicity and carcinogenesis. This study investigated the induction of hepatic CYP2E1 by ethanol and nicotine, alone and in combination, in a nonhuman primate model. Monkeys that self-administered ethanol and that received subcutaneous injections of nicotine (0.5 mg/kg b.i.d.), alone and in combination, were compared with control animals (four groups, n = 10/group). Chlorzoxazone (CZN) was used as a probe drug to phenotype in vivo CYP2E1 activity before and after chronic ethanol and/or nicotine exposure. CYP2E1 protein levels and in vitro chlorzoxazone metabolism were assessed in liver microsomes. Average daily ethanol consumption was 3.0 g/kg (blood ethanol levels 24 mM) and was unaffected by nicotine treatment. Ethanol self-administration and nicotine treatment, alone and in combination, significantly increased in vivo CZN disposition compared with that in control animals. The effect of ethanol was only observed at higher levels of intake. Ethanol and nicotine increased CYP2E1 protein levels and in vitro CZN metabolism, with combined exposure to both drugs resulting in the greatest increase. The effect of ethanol was also dependent on level of intake. Chronic exposure to ethanol and nicotine induced hepatic CYP2E1 activity and protein levels, particularly when both drugs were used in combination and when ethanol intake was high. These results have important implications for public health, given the association between elevated CYP2E1 and disease, and the large proportion of individuals who are exposed to ethanol and nicotine, often in combination.

Complex N-Acetylation of Triethylenetetramine [Articles]

2011-11-21T06:58:10-08:00

Triethylenetetramine (TETA) is an efficient copper chelator that has versatile clinical potential. We have recently shown that spermidine/spermine-N¹-acetyltransferase (SSAT1), the key polyamine catabolic enzyme, acetylates TETA in vitro. Here, we studied the metabolism of TETA in three different mouse lines: syngenic, SSAT1-overexpressing, and SSAT1-deficient (SSAT1-KO) mice. The mice were sacrificed at 1, 2, or 4 h after TETA injection (300 mg/kg i.p.). We found only N¹-acetyltriethylenetetramine (N¹AcTETA) and/or TETA in the liver, kidney, and plasma samples. As expected, SSAT1-overexpressing mice acetylated TETA at an accelerated rate compared with syngenic and SSAT1-KO mice. It is noteworthy that SSAT1-KO mice metabolized TETA as syngenic mice did, probably by thialysine acetyltransferase, which had a K_m value of 2.5 ± 0.3 mM and a k_cat value of 1.3 s^–1 for TETA when tested in vitro with the human recombinant enzyme. Thus, the present results suggest that there are at least two N-acetylases potentially metabolizing TETA. However, their physiological significance for TETA acetylation requires further studies. Furthermore, we detected chemical intramolecular N-acetyl migration from the N¹ to N³ position of N¹AcTETA and N¹,N⁸-diacetyltriethylenetetramine in an acidified high-performance liquid chromatography sample matrix. The complex metabolism of TETA together with the intramolecular N-acetyl migration may explain the huge individual variations in the acetylation rate of TETA reported earlier.

Effect of Dose Escalation on the In Vivo Oral Absorption and Disposition of Glycylsarcosine in Wild-Type and Pept1 Knockout Mice [Articles]

Jappar, D., Hu, Y., Smith, D. E. — 2011-11-21T06:58:10-08:00

This study evaluated the in vivo absorption and disposition of glycylsarcosine (GlySar), after escalating oral doses, in wild-type and peptide transporter 1 (Pept1) knockout mice. [³H]GlySar was administered to mice at doses of 1, 10, 100, 1000, and 5000 nmol/g b.wt. Serial blood samples were obtained over 480 min, the plasma was harvested, and the area under the plasma concentration-time curve (AUC) was determined. It was observed that the GlySar AUC was 60, 45, and 30% lower in knockout than wild-type mice when evaluated over 2, 4, and 8 h, respectively (p < 0.01). Plasma levels of GlySar reached a plateau at 90 min in knockout mice and then rose to a second plateau at 240 min. In wild-type mice, the plasma levels rose continuously to reach a single plateau at 90 min. When partial AUC (0–120 min) was used as an indicator for rate of absorption, there was a 60% reduction in GlySar absorption rate in knockout mice compared with wild-type animals. Tissue distribution studies were also performed after 10 nmol/g oral doses of [³H]GlySar. When sampled 1 h after dosing, GlySar tissue concentrations were significantly lower in knockout versus wild-type mice and, with the exception of intestines, reflected differences in the systemic exposure of dipeptide between these two genotypes. Overall, PEPT1 ablation in mice resulted in significant reductions, in vivo, in the rate and extent of GlySar absorption. The AUC of GlySar was proportional to dose in both genotypes over 1 to 100 nmol/g, with minor decrements at the two highest doses.

A Novel Method for the Immunoquantification of UDP-Glucuronosyltransferases in Human Tissue [Articles]

Milne, A. M., Burchell, B., Coughtrie, M. W. H. — 2011-11-21T06:58:10-08:00

Glucuronidation is a major pathway of drug and xenobiotic metabolism that is catalyzed by members of the UDP-glucuronosyltransferase (UGT) family. Predicting the contribution of individual UGTs to drug metabolism would be of considerable value in drug development and would be greatly aided by the availability of detailed absolute expression levels of these proteins; this is hampered by the lack of purified protein standards because of the hydrophobic membrane-associated nature of UGTs and the consequential difficulties in expression and purification. Here we describe a novel solution to this problem by expressing UGTs in Escherichia coli as fusion proteins with ribonuclease S-peptide, targeted to the periplasm with the pelB leader sequence. After addition of ribonuclease S-protein to membrane extracts, a functional ribonuclease is reconstituted that provides a direct and absolute quantification of the amount of UGT fusion protein; this is subsequently used to generate standard curves for immunoquantification by immunoblotting. To illustrate the value of the method, we have quantified the expression of UGT1A1 and UGT1A6 in human liver and kidney microsomes using new isoform-specific antibodies developed against peptides from these proteins. Expression levels of both proteins in liver were highly variable (28- and 20-fold, respectively) and correlated strongly with UGT enzyme activity toward the probe substrates bilirubin and 1-naphthol, respectively. The method is broadly applicable and provides a straightforward means of determining the absolute, as opposed to relative, quantities of UGT proteins present in human tissues.

Biosynthesis and Identification of an N-Oxide/N-Glucuronide Metabolite and First Synthesis of an N-O-Glucuronide Metabolite of Lu AA21004 [Articles]

Uldam, H. K., Juhl, M., Pedersen, H., Dalgaard, L. — 2011-11-21T06:58:10-08:00

This article describes the biosynthesis and identification of a new class of metabolites, a piperazine N-oxide/N-glucuronide metabolite 4-[2-(2,4-dimethyl-phenylsulfanyl)-phenyl]-1-β-d-glucuronic acid-piperazine 1-oxide (4). The metabolite was found in urine and plasma from humans and animals dosed with 1-[2-(2,4-dimethyl-phenylsulfanyl)-phenyl]-piperazine hydrobromide (Lu AA21004, 1), as a novel multimodal antidepressant under development for treatment of depression. Human liver microsomes in combination with uridine 5'-diphosphoglucuronic acid were used as an in vitro system to generate enough material of 4 to perform one- and two-dimensional ¹H and ¹³C NMR experiments for structure elucidation. Based on rotating frame Overhauser enhancement spectroscopy NMR experiments, the distance correlation between a piperazine proton and the anomeric proton of the glucuronic acid moiety is of a magnitude similar to that of the H-3' and H-5' protons and can only be explained by proximity in space and the postulated structure (4). The structural analog, the N-O-glucuronic acid conjugate 6-{4-[2-(2,4-dimethyl-phenylsulfanyl)-phenyl]-piperazin-1-yloxy}-1-β-d-glucuronic acid (3) was also observed in biological samples from humans and animals and the first organic synthesis and structural identification of this metabolite is also reported. Treatment of the glucuronide metabolites 3 and 4 with β-glucuronidase gave mainly the expected hydrolysis product, the hydroxyl amine 4-[2-(2,4-dimethyl-phenylsulfanyl)-phenyl]-piperazin-1-ol (2).

Systemic and Direct Nose-to-Brain Transport Pharmacokinetic Model for Remoxipride after Intravenous and Intranasal Administration [Articles]

2011-11-21T06:58:10-08:00

Intranasal (IN) administration could be an attractive mode of delivery for drugs targeting the central nervous system, potentially providing a high bioavailability because of avoidance of a hepatic first-pass effect and rapid onset of action. However, controversy remains whether a direct transport route from the nasal cavity into the brain exists. Pharmacokinetic modeling is proposed to identify the existence of direct nose-to-brain transport in a quantitative manner. The selective dopamine-D2 receptor antagonist remoxipride was administered at different dosages, in freely moving rats, by the IN and intravenous (IV) route. Plasma and brain extracellular fluid (ECF) concentration-time profiles were obtained and simultaneously analyzed using nonlinear mixed-effects modeling. Brain ECF/plasma area under the curve ratios were 0.28 and 0.19 after IN and IV administration, respectively. A multicompartment pharmacokinetic model with two absorption compartments (nose-to-systemic and nose-to-brain) was found to best describe the observed pharmacokinetic data. Absorption was described in terms of bioavailability and rate. Total bioavailability after IN administration was 89%, of which 75% was attributed to direct nose-to brain transport. Direct nose-to-brain absorption rate was slow, explaining prolonged brain ECF exposure after IN compared with IV administration. These studies explicitly provide separation and quantitation of systemic and direct nose-to-brain transport after IN administration of remoxipride in the rat. Describing remoxipride pharmacokinetics at the target site (brain ECF) in a semiphysiology-based manner would allow for better prediction of pharmacodynamic effects.

CYP2C19 Progress Curve Analysis and Mechanism-Based Inactivation by Three Methylenedioxyphenyl Compounds [Articles]

Salminen, K. A., Meyer, A., Imming, P., Raunio, H. — 2011-11-21T06:58:10-08:00

Several in vitro criteria were used to assess whether three methylenedioxyphenyl (MDP) compounds, the isoquinoline alkaloids bulbocapnine, canadine, and protopine, are mechanism-based inactivators of CYP2C19. The recently reported fluorometric CYP2C19 progress curve analysis approach was applied first to determine whether these alkaloids demonstrate time-dependent inhibition. In this experiment, bulbocapnine, canadine, and protopine displayed time dependence and saturation in their inactivation kinetics with K_I and k_inact values of 72.4 ± 14.7 μM and 0.38 ± 0.036 min^–1, 2.1 ± 0.63 μM and 0.18 ± 0.015 min^–1, and 7.1 ± 2.3 μM and 0.24 ± 0.021 min^–1, respectively. Additional studies were performed to determine whether other specific criteria for mechanism-based inactivation were fulfilled: NADPH dependence, irreversibility, and involvement of a catalytic step in the enzyme inactivation. CYP2C19 activity was not significantly restored by dialysis when it had been inactivated by the alkaloids in the presence of a NADPH-regenerating system, and a metabolic-intermediate complex-associated increase in absorbance at approximately 455 nm was observed. In conclusion, the CYP2C19 progress curve analysis method revealed time-dependent inhibition by these alkaloids, and additional experiments confirmed its quasi-irreversible nature. This study revealed that the CYP2C19 progress curve analysis method is useful for identifying novel mechanism-based inactivators and yields a wealth of information in one run. The alkaloids bulbocapnine, canadine, and protopine, present in herbal medicines, are new mechanism-based inactivators and the first MDP compounds exhibiting quasi-irreversible inactivation of CYP2C19.

Involvement of Different Human Glutathione Transferase Isoforms in the Glutathione Conjugation of Reactive Metabolites of Troglitazone [Articles]

2011-11-21T06:58:10-08:00

Null mutation of glutathione transferase (GST) M1 and GSTT1 was reported to correlate statistically with an abnormal increase in the plasma levels of alanine aminotransferase or aspartate aminotransferase caused by troglitazone in diabetic patients (Clin Pharmacol Ther, 73:435–455, 2003). This clinical evidence leads to the hypothesis that GSH conjugation catalyzed by GSTT1 and GSTM1 has a role in the elimination of reactive metabolites of troglitazone. However, the contribution of GST isoforms expressed in human liver to the detoxification of reactive metabolites of troglitazone has not yet been clarified. We investigated the involvement of human GST isoforms in the GSH conjugation of reactive metabolites of troglitazone using recombinant GST enzymes. Five reported GSH conjugates of reactive metabolites were produced from troglitazone after incubation with liver microsomes, NADPH, and GSH in a GSH concentration-dependent manner. Addition of human recombinant GSTA1, GSTA2, GSTM1, or GSTP1 protein to the incubation mixture further increased the GSH conjugates. However, the addition of GSTT1 did not show any catalytic effect. It is of interest that one of the reactive metabolites with a quinone structure was predominantly conjugated with GSH by GSTM1. Thus, we demonstrated that the GST isoforms contributed differently to the GSH conjugation of individual reactive metabolites of troglitazone, and GSTM1 is the most important GST isoform in the GSH conjugation of a specific reactive metabolite produced from the cytotoxic, quinone-form metabolite of troglitazone.

Monomethylarsenic Diglutathione Transport by the Human Multidrug Resistance Protein 1 (MRP1/ABCC1) [Articles]

Carew, M. W., Naranmandura, H., Shukalek, C. B., Le, X. C., Leslie, E. M. — 2011-11-21T06:58:10-08:00

The ATP-binding cassette (ABC) transporter protein multidrug resistance protein 1 (MRP1; ABCC1) plays an important role in the cellular efflux of the high-priority environmental carcinogen arsenic as a triglutathione conjugate [As(GS)₃]. Most mammalian cells can methylate arsenic to monomethylarsonous acid (MMA^III), monomethylarsonic acid (MMA^V), dimethylarsinous acid (DMA^III), and dimethylarsinic acid (DMA^V). The trivalent forms MMA^III and DMA^III are more reactive and toxic than their inorganic precursors, arsenite (As^III) and arsenate (As^V). The ability of MRP1 to transport methylated arsenicals is unknown and was the focus of the current study. HeLa cells expressing MRP1 (HeLa-MRP1) were found to confer a 2.6-fold higher level of resistance to MMA^III than empty vector control (HeLa-vector) cells, and this resistance was dependent on GSH. In contrast, MRP1 did not confer resistance to DMA^III, MMA^V, or DMA^V. HeLa-MRP1 cells accumulated 4.5-fold less MMA^III than HeLa-vector cells. Experiments using MRP1-enriched membrane vesicles showed that transport of MMA^III was GSH-dependent but not supported by the nonreducing GSH analog, ophthalmic acid, suggesting that MMA^III(GS)₂ was the transported form. MMA^III(GS)₂ was a high-affinity, high-capacity substrate for MRP1 with apparent K_m and V_max values of 11 μM and 11 nmol mg^–1min^–1, respectively. MMA^III(GS)₂ transport was osmotically sensitive and inhibited by several MRP1 substrates, including 17β-estradiol 17-(β-d-glucuronide) (E₂17βG). MMA^III(GS)₂ competitively inhibited the transport of E₂17βG with a K_i value of 16 μM, indicating that these two substrates have overlapping binding sites. These results suggest that MRP1 is an important cellular protective pathway for the highly toxic MMA^III and have implications for environmental and clinical exposure to arsenic.

Characterization of the Expression and Activity of Carboxylesterases 1 and 2 from the Beagle Dog, Cynomolgus Monkey, and Human [Articles]

2011-11-21T06:58:10-08:00

The carboxylesterases (CESs) are a family of serine hydrolases that hydrolyze compounds containing an ester, amide, or thioester. In humans, two dominant forms, CES1 and CES2, are highly expressed in organs of first-pass metabolism and play an important role in xenobiotic metabolism. The current study was conducted to better understand species-related differences in substrate selectivity and tissue expression of these enzymes. To elucidate potential similarities and differences among these enzymes, a series of 4-nitrophenyl esters and a series of gemcitabine prodrugs were evaluated using enzyme kinetics as substrates of expressed and purified CESs from beagle dog, cynomolgus monkey, and human genes. For the substrates examined, human and monkey CES2 more efficiently catalyzed hydrolysis compared with CES1, whereas CES1 was the more efficient enzyme in dog. Quantitative real-time polymerase chain reaction and Western blot analyses indicate that the pattern of CES tissue expression in monkey is similar to that of human, but the CES expression in dog is unique, with no detectable expression of CES in the intestine. Loperamide, a selective human CES2 inhibitor, was also found to be a CES2-selective inhibitor in both dog and monkey. This is the first study to examine substrate specificity among dog, human, and monkey CESs.

Utility of P-Glycoprotein and Organic Cation Transporter 1 Double-Transfected LLC-PK1 Cells for Studying the Interaction of YM155 Monobromide, Novel Small-Molecule Survivin Suppressant, with P-Glycoprotein [Articles]

Iwai, M., Minematsu, T., Li, Q., Iwatsubo, T., Usui, T. — 2011-11-21T06:58:10-08:00

1-(2-Methoxyethyl)-2-methyl-4,9-dioxo-3-(pyrazin-2-ylmethyl)-4,9-dihydro-1H-naphtho[2,3-d]imidazolium bromide (YM155 monobromide), a novel small molecule that downregulates survivin and exhibits potent antitumor activity, is hydrophilic and cationic. Although previous studies have shown that influx transporters play important roles in the uptake of YM155 into hepatocytes and possibly into cancer cells, efflux transporters have yet to be investigated. In this study, we assessed the interaction of YM155 with P-glycoprotein [multidrug resistance 1 (MDR1)/ATP-binding cassette B1] using two kinds of transcellular transport systems: Caco-2 and MDR1-expressing LLC-PK1 cells (LLC-MDR1). We also used a newly established LLC-OCT1/MDR1 cell line, which expresses basal YM155 uptake transporter organic cation transporter1 (OCT1) and apical MDR1. Direct interaction between YM155 and MDR1 and other efflux transporters was evaluated using transporter-expressing membrane vesicles. A bidirectional transporter assay using Caco-2 and LLC-MDR1 cells showed low permeability and no vectorial transport of YM155, suggesting that YM155 is not a substrate of MDR1. However, vectorial transport across LLC-OCT1/MDR1 cells was identified, which was inhibited by the MDR1 inhibitor cyclosporine A, clearly indicating that YM155 is in fact a substrate of MDR1. Insufficient expression of basal uptake transporter of YM155 in Caco-2 and LLC-MDR1 might have confounded conclusions regarding YM155 and MDR1. Using the transporter-expressing vesicles, MDR1-mediated transport was most significantly involved in YM155 transport among the efflux transporters examined. In conclusion, these findings suggest that YM155 is a substrate of MDR1, and that MDR1 may play an important role in the pharmacokinetics of YM155. Transcellular assays lacking basal uptake transporters may be inaccurate in the assessment of hydrophilic compounds that have poor membrane permeability by passive diffusion.

Intestinal Ciprofloxacin Efflux: The Role of Breast Cancer Resistance Protein (ABCG2) [Articles]

2011-11-21T06:58:10-08:00

Intestinal secretory movement of the fluoroquinolone antibiotic, ciprofloxacin, may limit its oral bioavailability. Active ATP-binding cassette (ABC) transporters such as breast cancer resistance protein (BCRP) have been implicated in ciprofloxacin transport. The aim of this study was to test the hypothesis that BCRP alone mediates intestinal ciprofloxacin secretion. The involvement of ABC transport proteins in ciprofloxacin secretory flux was investigated with the combined use of transfected cell lines [bcrp1/BCRP-Madin-Darby canine kidney II (MDCKII) and multidrug resistance-related protein 4 (MRP4)-human embryonic kidney (HEK) 293] and human intestinal Caco-2 cells, combined with pharmacological inhibition using 3-(6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6, 7,12,12a-octahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-3-yl)-propionic acid tert-butyl ester (Ko143), cyclosporine, 3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid (MK571), and verapamil as ABC-selective inhibitors. In addition, the regional variation in secretory capacity was investigated using male Han Wistar rat intestine mounted in Ussing chambers, and the first indicative measurements of ciprofloxacin transport by ex vivo human jejunum were made. Active, Ko143-sensitive ciprofloxacin secretion was observed in bcrp1-MDCKII cell layers, but in low-passage (BCRP-expressing) Caco-2 cell layers only a 54% fraction was Ko143-sensitive. Ciprofloxacin accumulation was lower in MRP4-HEK293 cells than in the parent line, indicating that ciprofloxacin is also a substrate for this transporter. Ciprofloxacin secretion by Caco-2 cell layers was not inhibited by MK571. Secretory flux showed marked regional variability in the rat intestine, increasing from the duodenum to peak in the ileum. Ciprofloxacin secretion was present in human jejunum and was reduced by Ko143 but showed marked interindividual variability. Ciprofloxacin is a substrate for human and rodent BCRP. An additional pathway for ciprofloxacin secretion exists in Caco-2 cells, which is unlikely to be MRP(4)-mediated. BCRP is likely to be the dominant transport mechanism for ciprofloxacin efflux in both rat and human jejunum.

Complex Drug Interactions of HIV Protease Inhibitors 2: In Vivo Induction and In Vitro to In Vivo Correlation of Induction of Cytochrome P450 1A2, 2B6, and 2C9 by Ritonavir or Nelfinavir [Articles]

2011-11-21T06:58:10-08:00

Drug-drug interactions (DDIs) with the HIV protease inhibitors (PIs) are complex, paradoxical (e.g., ritonavir/alprazolam), and involve multiple mechanisms. As part of a larger study to better understand these DDIs and to devise a framework for in vitro to in vivo prediction of these DDIs, we determined the inductive effect of ~2 weeks of administration of two prototypic PIs, nelfinavir (NFV), ritonavir (RTV), and rifampin (RIF; induction positive control) on the cytochrome P450 enzymes CYP1A2, CYP2B6, CYP2C9, and CYP2D6 and the inductive or inductive plus inhibitory effect of NFV, RTV, or RIF on CYP3A and P-glycoprotein in healthy human volunteers. Statistically significant induction of CYP1A2 (2.1-, 2.9-, and 2.2-fold), CYP2B6 (1.8-, 2.4-, and 4-fold), and CYP2C9 (1.3-, 1.8-, and 2.6-fold) was observed after NFV, RTV, or RIF treatment, respectively (as expected, CYP2D6 was not induced). Moreover, we accurately predicted the in vivo induction of these enzymes by quantifying their induction by the PIs in human hepatocytes and by using RIF as an in vitro to in vivo scalar. On the basis of the modest in vivo induction of CYP1A2, CYP2B6, or CYP2C9, the in vivo paradoxical DDIs with the PIs are likely explained by mechanisms other than induction of these enzymes such as induction of other metabolic enzymes, transporters, or both.

Absorption and Metabolism of Chlorogenic Acids in Cultured Gastric Epithelial Monolayers [Articles]

Farrell, T. L., Dew, T. P., Poquet, L., Hanson, P., Williamson, G. — 2011-11-21T06:58:10-08:00

Gastric absorption of feruloylquinic acid and di-O-caffeoylquinic acid analogs has never been investigated despite their potential contribution to the proposed beneficial health effects leading to reduced risk of type 2 diabetes. Using a cultured gastric epithelial model, with an acidic apical pH, the relative permeability coefficients (P_app) and metabolic fate of a series of chlorogenic acids (CGAs) were investigated. Mechanistic studies were performed in the apical to basal direction and demonstrated differential rates of absorption for different CGA subgroups. For the first time, we show intact absorption of feruloylquinic acids and caffeoylquinic acid lactones across the gastric epithelium (P_app ~ 0.2 cm/s). Transport seemed to be mainly by passive diffusion, because good linearity was observed over the incubation period and test concentrations, and we speculate that a potential carrier-mediated component may be involved in uptake of certain 4-acyl CGA isomers. In contrast, absorption of intact di-O-caffeoylquinic acids was rapid (P_app ~ 2–10 cm/s) but nonlinear with respect to time and concentration dependence, which was potentially limited by interaction with an efflux transporter and/or pH gradient dependence. For the first time, methylation is shown in gastric mucosa. Furthermore, isoferulic acid, dimethoxycinnamic acid, and ferulic acid were identified as novel gastric metabolites of CGA biotransformation. We propose that the stomach is the first location for the release of hydroxycinnamic acids, which could explain their early detection after coffee consumption.

Raloxifene Glucuronidation in Human Intestine, Kidney, and Liver Microsomes and in Human Liver Microsomes Genotyped for the UGT1A1*28 Polymorphism [Articles]

Trdan Lusin, T., Trontelj, J., Mrhar, A. — 2011-11-21T06:58:10-08:00

Raloxifene, a selective estrogen receptor modulator, exhibits quite large interindividual variability in pharmacokinetics and pharmacodynamics. In women, raloxifene is metabolized extensively by different isoforms of UDP-glucuronosyltransferase (UGT) to its glucuronides. To gain an insight into intestine, kidney, liver, and lung glucuronidation of raloxifene, human microsomes of all tested organs were used. Raloxifene-6-β-glucuronide (M1) formation followed the Michaelis-Menten kinetics in intestinal, kidney, and liver microsomes; meanwhile, raloxifene-4'-β-glucuronide (M2) formation followed the substrate inhibition kinetics. Human lung microsomes did not show any glucuronidation activity. The tissue intrinsic clearances for kidney, intestine, and liver were 3.4, 28.1, and 39.6 ml · min^–1 · kg^–1, respectively. The aim of our in vitro study was to explain the mechanism behind the observed influence of UGT1A1*28 polymorphism on raloxifene pharmacokinetics in a small-sized in vivo study (Br J Clin Pharmacol 67:437–444, 2009). Incubation of raloxifene with human liver microsomes genotyped for UGT1A1*28 showed a significantly reduced metabolic clearance toward M1 in microsomes from donors with *28 allele. On the contrary, no significant genotype influence was observed on the formation of M2 because of the high variability in estimated apparent kinetic parameters, although a clear trend toward lower glucuronidation activities was observed when UGT1A1*28 polymorphism was present. The liver intrinsic clearances of both homozygotes differed significantly, whereas the clearance of heterozygotes did not differ from the wild-type and the mutated homozygotes. In conclusion, our results show the high importance of the liver and intestine in raloxifene glucuronidation. Moreover, the significant influence of UGT1A1*28 polymorphism on metabolism of raloxifene was confirmed.

Small Molecule Quantification by Liquid Chromatography-Mass Spectrometry for Metabolites of Drugs and Drug Candidates [Articles]

Dahal, U. P., Jones, J. P., Davis, J. A., Rock, D. A. — 2011-11-21T06:58:10-08:00

Identification and quantification of the metabolites of drugs and drug candidates are routinely performed using liquid chromatography-mass spectrometry (LC-MS). The best practice is to generate a standard curve with the metabolite versus the internal standard. However, to avoid the difficulties in metabolite synthesis, standard curves are sometimes prepared using the substrate, assuming that the signal for substrate and the metabolite will be equivalent. We have tested the errors associated with this assumption using a series of very similar compounds that undergo common metabolic reactions using both conventional flow electrospray ionization LC-MS and low-flow captive spray ionization (CSI) LC-MS. The differences in standard curves for four different types of transformations (O-demethylation, N-demethylation, aromatic hydroxylation, and benzylic hydroxylation) are presented. The results demonstrate that the signals of the substrates compared with those of the metabolites are statistically different in 18 of the 20 substrate-metabolite combinations for both methods. The ratio of the slopes of the standard curves varied up to 4-fold but was slightly less for the CSI method.

Hepatic Uptake in the Dog: Comparison of Uptake in Hepatocytes and Human Embryonic Kidney Cells Expressing Dog Organic Anion-Transporting Polypeptide 1B4 [Articles]

2011-11-21T06:58:10-08:00

Although the dog is frequently used in pharmacological, pharmacokinetic, and drug safety studies, little is known about canine drug transporters. Dog organic anion-transporting polypeptide (Oatp1b4) has recently been cloned (Comp Biochem Physiol C Toxicol Pharmacol 151:393–399, 2010), but the contribution of Oatp1b4 to hepatic uptake has yet to be clarified. This study compares the transport characteristics of dog Oatp1b4 with those of human OATP1B1/1B3 and demonstrates the importance of Oatp1b4 in the uptake of anionic compounds in dog hepatocytes. Oatp1b4 is the predominant Oatp in dog liver with expression levels double and 30 times those of Oatp2b1 and Oatp1a2, respectively. Uptake of a range of typical OATP substrates by Oatp1b4-expressing HEK293 cells was compared with that in fresh dog hepatocytes. All compounds tested were transported by Oatp1b4 and uptake intrinsic clearance (CL_{int, uptake}) in dog hepatocytes in sodium-free buffer was correlated significantly with CL_{int, uptake} in Oatp1b4-expressing cells. Dog in vivo clearance for five substrates was predicted more accurately from CL_{int, uptake} than from metabolic intrinsic clearance (CL_{int, met}), indicating that uptake governs the overall in vivo hepatic clearance of these anionic compounds in dog. The substrate specificities of dog Oatp1b4 appear to be similar to those of human OATP1B1/OATP1B3, whereas the relative uptake clearance of substrates for Oatp1b4 correlate better with OATP1B3 than with the more abundant hepatic analog OATP1B1.

Analysis and Prediction of Drug Transfer into Human Milk Taking into Consideration Secretion and Reuptake Clearances across the Mammary Epithelia [Articles]

Koshimichi, H., Ito, K., Hisaka, A., Honma, M., Suzuki, H. — 2011-11-21T06:58:10-08:00

Medication use during lactation is a matter of concern due to unnecessary exposure of infants to drugs. Although some studies have predicted the extent of drug transfer into milk from physicochemical parameters, drug concentration-time profiles in milk have not been predicted or even analyzed yet. In the present study, a drug transfer model was constructed by defining secretion and reuptake clearances (CL_sec and CL_re, respectively) between milk and plasma based on unbound drug concentrations. Through the use of this model, drug concentration-time profiles were analyzed in human milk and plasma based on data collected from the literature. CL_sec and CL_re values were obtained successfully for 49 drugs. Because the CL_sec and CL_re values were in general similar for each drug, transport across the mammary epithelia was mediated by passive diffusion in most cases. This study demonstrated that the logarithmically transformed values of CL_sec and CL_re can be predicted from physicochemical parameters with adjusted R² values of 0.705 and 0.472, respectively. Moreover, 66.7 and 77.8% of predicted CL_sec and CL_re values were within 3-fold error ranges of the observed values for 45 and 27 drugs, respectively. Finally, time profiles of drug concentrations in milk were simulated from physicochemical parameters. The milk-to-plasma area under the concentration-time curve ratios also were predicted successfully within 3-fold error ranges of the observed values for 71.9% of the drugs analyzed. The method described herein therefore may be useful in predicting drug concentration-time profiles in human milk for newly developed drugs.

Inhibition of Human Liver Aldehyde Oxidase: Implications for Potential Drug-Drug Interactions [Articles]

Barr, J. T., Jones, J. P. — 2011-11-21T06:58:10-08:00

During the course of our research efforts to understand the kinetics of human aldehyde oxidase as a xenobiotic-clearing enzyme, we investigated the effect of eight different inhibitors on the oxidation of the probe substrate phthalazine. Saturation kinetic parameters for phthalazine oxidation in human liver cytosol were found to be the following: K_m = 8.0 ± 0.4 μM and V_max = 4.3 ± 0.1 nmol · min^–1 · mg protein^–1. Inhibitory potency of the inhibitors tested ranged from 0.1 to 5 μM. Of the eight different inhibitor compounds tested, seven were observed to inhibit through a mixed mode and one through a strictly competitive mode. A ratio of the K_ii and K_is values was used to assess the relative competitiveness of each inhibitor. For the mixed inhibitors, the mode of inhibition varied from mostly uncompetitive to predominantly competitive (K_ii/K_is values ranging from 0.1 to 15). The implications for potential drug-drug interactions and inhibition mechanism are discussed. We found two inhibitors, clozapine and chlorpromazine, that have a moderate predicted risk of drug-drug interactions based on the K_i value relative to the inhibitor concentration in human plasma, having a calculated [I]/K_i value of 0.4 and 0.8, respectively.

Effect of Culture Time on the Basal Expression Levels of Drug Transporters in Sandwich-Cultured Primary Rat Hepatocytes [Articles]

Tchaparian, E. H., Houghton, J. S., Uyeda, C., Grillo, M. P., Jin, L. — 2011-11-21T06:58:10-08:00

Sandwich-cultured rat hepatocytes are used in drug discovery for pharmacological and toxicological assessment of drug candidates, yet their utility as a functional model for drug transporters has not been fully characterized. To evaluate the system as an in vitro model for drug transport, expression changes of hepatic transporters relative to whole liver and freshly isolated hepatocytes (day 0) were examined by real-time quantitative reverse transcription-polymerase chain reaction for 4 consecutive days of culture. No significant differences in transporter expression levels were observed between freshly isolated hepatocytes and whole liver. Two distinct mRNA profiles were detected over time showing 1) a more than 5-fold decline in levels of uptake transporters such as Na⁺-taurocholate cotransporting polypeptide (Ntcp), organic anion transporter (Oat) 2, organic anion-transporting polypeptide (Oatp) 1a1, Oatp1a4, and Oatp1b2 and 2) a greater than 5-fold increase of efflux transporters P-glycoprotein (P-gp), breast cancer resistance protein (Bcrp), and multidrug resistance-related proteins (Mrp) 1, 2, 3, and 4. In addition, protein levels and functional activities for selected transporters were also determined. Protein levels for Mrp2, Bcrp, P-gp, Ntcp, and Oatp1a4 corresponded to changes in mRNA. Functional activities of Oatps and Oct1 exhibited a 3- and 4-fold decrease on day 2 and day 4, respectively, relative to that on day 0, whereas a more than 10-fold reduction in Oat2 activity was observed. These results indicate that the cell culture conditions used herein did not provide an optimal environment for expression of all hepatic transporters. Significant time-dependent alterations in basal gene expression patterns of transporters were detected compared with those in liver or freshly isolated hepatocytes. Further work and new strategies are required to improve the validity of this model as an in vitro tool for in vivo drug transport or biliary clearance prediction.

Variations in ATP-Binding Cassette Transporter Regulation during the Progression of Human Nonalcoholic Fatty Liver Disease [Articles]

2011-11-21T06:58:10-08:00

Transporters located on the sinusoidal and canalicular membranes of hepatocytes regulate the efflux of drugs and metabolites into blood and bile, respectively. Changes in the expression or function of these transporters during liver disease may lead to a greater risk of adverse drug reactions. Nonalcoholic fatty liver disease (NAFLD) is a progressive condition encompassing the relatively benign steatosis and the more severe, inflammatory state of nonalcoholic steatohepatitis (NASH). Here, we present an analysis of the effect of NAFLD progression on the major ATP-binding cassette (ABC) efflux transport proteins ABCC1–6, ABCB1, and ABCG2. Human liver samples diagnosed as normal, steatotic, NASH (fatty), and NASH (not fatty) were analyzed. Increasing trends in mRNA expression of ABCC1, ABCC4–5, ABCB1, and ABCG2 were found with NAFLD progression, whereas protein levels of all transporters exhibited increasing trends with disease progression. Immunohistochemical staining of ABCC3, ABCB1, and ABCG2 revealed no alterations in cellular localization during NAFLD progression. ABCC2 staining revealed an alternative mechanism of regulation in NASH in which the transporter appears to be internalized away from the canalicular membrane. This correlated with a preferential shift in the molecular mass of ABCC2 from 200 to 180 kDa in NASH, which has been shown to be associated with a loss of glycosylation and internalization of the protein. These data demonstrate increased expression of multiple efflux transporters as well as altered cellular localization of ABCC2 in NASH, which may have profound effects on the ability of patients with NASH to eliminate drugs in an appropriate manner.

Proluciferin Acetals as Bioluminogenic Substrates for Cytochrome P450 Activity and Probes for CYP3A Inhibition [Articles]

2011-11-21T06:58:10-08:00

Cytochrome P450 (P450) assays use probe substrates to interrogate the influence of new chemical entities toward P450 enzymes. We report the synthesis and study of a family of bioluminogenic luciferin acetal substrates that are oxidized by P450 enzymes to form luciferase substrates. The luciferin acetals were screened against a panel of purified P450 enzymes. In particular, one proluciferin acetal has demonstrated sensitive and selective CYP3A4-catalyzed oxidation to a luciferin ester—K_m and k_cat are 2.88 μM and 5.87 pmol metabolite · min^–1 · pmol enzyme^–1, respectively. The proluciferin acetal was used as a probe substrate to measure IC₅₀ values of known inhibitors against recombinant CYP3A4 or human liver microsomes. IC₅₀ values for the known inhibitors correlate strongly with IC₅₀ values calculated from the traditional high-performance liquid chromatography-based probe substrate testosterone. Luciferin acetals are rapidly oxidized to unstable hemi-orthoesters by CYP3A resulting in luciferin esters and, therefore, are conducive to simple rapid CYP3A bioluminescent assays.

Role of Residue 87 in the Activity and Regioselectivity of Clozapine Metabolism by Drug-Metabolizing CYP102A1 M11H: Application for Structural Characterization of Clozapine GSH Conjugates [Articles]

2011-11-21T06:58:10-08:00

In the present study, a site-saturation mutagenesis library of drug-metabolizing CYP102A1 M11H with all 20 amino acids at position 87 was applied as a biocatalyst for the production of stable and reactive metabolites of clozapine. Clozapine is an atypical antipsychotic drug in which formation of reactive metabolites is considered to be responsible for several adverse drug reactions. Reactive intermediates of clozapine can be inactivated by GSH to multiple GSH conjugates by nonenzymatic and glutathione transferase (GST)-mediated conjugation reactions. The structures of several GST-dependent metabolites have not yet been elucidated unequivocally. The present study shows that the nature of the amino acid at position 87 of CYP102A1 M11H strongly determines the activity and regioselectivity of clozapine metabolism. Some mutants showed preference for N-demethylation and N-oxidation, whereas others showed high selectivity for bioactivation to reactive intermediates. The mutant containing Phe87 showed high activity and high selectivity for the bioactivation pathway and was used for the large-scale production of GST-dependent GSH conjugates by incubation in the presence of recombinant human GST P1-1. Five human-relevant GSH adducts were produced at high levels, enabling structural characterization by ¹H NMR. This work shows that drug-metabolizing CYP102A1 mutants, in combination with GSTs, are very useful tools for the generation of GSH conjugates of reactive metabolites of drugs to enable their isolation and structural elucidation.

Studies to Further Investigate the Inhibition of Human Liver Microsomal CYP2C8 by the Acyl-{beta}-Glucuronide of Gemfibrozil [Articles]

2011-11-21T06:58:10-08:00

In previous studies, gemfibrozil acyl-β-glucuronide, but not gemfibrozil, was found to be a mechanism-based inhibitor of cytochrome P450 2C8. To better understand whether this inhibition is specific for gemfibrozil acyl-β-glucuronide or whether other glucuronide conjugates are potential substrates for inhibition of this enzyme, we evaluated several pharmaceutical compounds (as their acyl glucuronides) as direct-acting and metabolism-dependent inhibitors of CYP2C8 in human liver microsomes. Of 11 compounds that were evaluated as their acyl glucuronide conjugates, only gemfibrozil acyl-β-glucuronide exhibited mechanism-based inhibition, indicating that CYP2C8 mechanism-based inhibition is very specific to certain glucuronide conjugates. Structural analogs of gemfibrozil were synthesized, and their glucuronide conjugates were prepared to further examine the mechanism of inhibition. When the aromatic methyl groups on the gemfibrozil moiety were substituted with trifluoromethyls, the resulting glucuronide conjugate was a weaker inhibitor of CYP2C8 and mechanism-based inhibition was abolished. However, the glucuronide conjugates of monomethyl gemfibrozil analogs were mechanism-based inhibitors of CYP2C8, although not as potent as gemfibrozil acyl-β-glucuronide itself. The ortho-monomethyl analog was a more potent inhibitor than the meta-monomethyl analog, indicating that CYP2C8 favors the ortho position for oxidation and potential inhibition. Molecular modeling of gemfibrozil acyl-β-glucuronide in the CYP2C8 active site is consistent with the ortho-methyl position being the favored site of covalent attachment to the heme. Moreover, hydrogen bonding to four residues (Ser100, Ser103, Gln214, and Asn217) is implicated.

Thr302 Is the Site for the Covalent Modification of Human Cytochrome P450 2B6 Leading to Mechanism-Based Inactivation by tert-Butylphenylacetylene [Articles]

Lin, H.-l., Zhang, H., Pratt-Hyatt, M. J., Hollenberg, P. F. — 2011-11-21T06:58:10-08:00

The mechanism-based inactivation of human CYP2B6 by tert-butylphenylacetylene (BPA) in the reconstituted system was investigated. The inactivation of CYP2B6 by BPA is time-, concentration-, and NADPH-dependent and exhibits a K_I of 2.8 μM, a k_inact of 0.7 min^–1, and a t_1/2 of 1 min. The partition ratio is ~5. Unlike CYP2B1 and CYP2B4, in addition to the formation of an apoprotein adduct and a glutathione conjugate, a small heme adduct was observed when CYP2B6 was incubated with BPA. The mass increase of the adducted apoprotein and GSH conjugate is 174 Da, equivalent to the mass of one molecule of BPA plus one oxygen atom. To identify the adducted residue, BPA-inactivated CYP2B6 was digested with trypsin, and the digest was then analyzed by liquid chromatography-tandem mass spectrometry. A mass shift of 174 Da was used for the SEQUEST database search, and the identity of the modified residue was confirmed by MS/MS fragmentation of the modified peptide. Two residues, Lys274 and Thr302, were identified as having been modified. Further mutagenesis studies have demonstrated that the residue that is modified to result in inactivation is Thr302, not Lys274. Docking studies show that in the enzyme-substrate complex, Thr302 is in close contact with the triple bond of BPA with a distance of 3.8 Å between the terminal carbon of BPA and the oxygen in the hydroxyl group of Thr302. In conclusion, Thr302 of CYP2B6 is covalently modified by a reactive metabolite of BPA, and this modification is responsible for the mechanism-based inactivation.

Development of a Highly Sensitive Method Using Liquid Chromatography-Multiple Reaction Monitoring to Quantify Membrane P-Glycoprotein in Biological Matrices and Relationship to Transport Function [Articles]

2011-11-21T06:58:10-08:00

The quantification of P-glycoprotein [P-gp, ABCB1, multidrug resistance 1 (MDR1)] protein in biological matrices is considered a key factor missing for useful translation of in vitro functional data to the in vivo situation and for comparison of transporter data among different in vitro models. In the present study a liquid chromatography (LC)-mass spectrometry method was developed to quantify P-gp membrane protein levels in different biological matrices. The amount of P-gp transporter protein was measured in Caco-2 cell monolayers and in inside-out human embryonic kidney (HEK)-MDR1 vesicles. From both in vitro systems, two preparations with different functionality were used. Transporter function was determined as digoxin efflux in Caco-2 cell monolayers and N-methylquinidine (NMQ) uptake in membrane vesicles, and, in addition, mRNA expression in the Caco-2 monolayers was measured. The results showed an excellent relationship between NMQ uptake functionality in inside-out HEK-MDR1 vesicles and protein contents. Similar concordance between the digoxin efflux and P-gp content in different Caco-2 cell cultures was observed, whereas mRNA levels are indicative of increased P-gp content and activity in older Caco-2 cultures, however, not yielding the same quantitative relationship. The results from both Caco-2 and HEK-MDR1 membrane vesicles confirm that the protein content is directly related to the level of activity in the respective system. The method presented here to quantify P-gp protein by LC-multiple reaction monitoring will facilitate the development of future methodologies to bridge between expression systems and cell/tissue models and to scale from in vitro models to whole organs.