Drug Metabolism and Disposition current issue

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.