Journal of Pharmacology and Experimental Therapeutics METABOLISM, TRANSPORT, AND PHARMACOGENOMICS

[METABOLISM, TRANSPORT, AND PHARMACOGENOMICS] Radiodefluorination of 3-Fluoro-5-(2-(2-[18F](fluoromethyl)-thiazol-4-yl)ethynyl)benzonitrile ([18F]SP203), a Radioligand for Imaging Brain Metabotropic Glutamate Subtype-5 Receptors with Positron Emission Tomography, Occurs by Glutathionylation in Rat Brain

2008-11-12

Metabotropic glutamate subtype-5 receptors (mGluR5) are implicated in several neuropsychiatric disorders. Positron emission tomography (PET) with a suitable radioligand may enable monitoring of regional brain mGluR5 density before and during treatments. We have developed a new radioligand, 3-fluoro-5-(2-(2-[¹⁸F](fluoromethyl)thiazol-4-yl)ethynyl)benzonitrile ([¹⁸F]SP203), for imaging brain mGluR5 in monkey and human. In monkey, radioactivity was observed in bone, showing release of [¹⁸F]-fluoride ion from [¹⁸F]SP203. This defluorination was not inhibited by disulfiram, a potent inhibitor of CYP2E1. PET confirmed bone uptake of radioactivity and therefore defluorination of [¹⁸F]SP203 in rats. To understand the biochemical basis for defluorination, we administered [¹⁸F]SP203 plus SP203 in rats for ex vivo analysis of metabolites. Radio-high-performance liquid chromatography detected [¹⁸F]fluoride ion as a major radiometabolite in both brain extract and urine. Incubation of [¹⁸F]SP203 with brain homogenate also generated this radiometabolite, whereas no metabolism was detected in whole blood in vitro. Liquid chromatography-mass spectrometry analysis of the brain extract detected m/z 548 and 404 ions, assignable to the [M + H]⁺ of S-glutathione (SP203Glu) and N-acetyl-S-l-cysteine (SP203Nac) conjugates of SP203, respectively. In urine, only the [M + H]⁺ of SP203Nac was detected. Mass spectrometry/mass spectrometry and multi-stage mass spectrometry analyses of each metabolite yielded product ions consistent with its proposed structure, including the former fluoromethyl group as the site of conjugation. Metabolite structures were confirmed by similar analyses of SP203Glu and SP203Nac, prepared by glutathione S-transferase reaction and chemical synthesis, respectively. Thus, glutathionylation at the 2-fluoromethyl group is responsible for the radiodefluorination of [¹⁸F]SP203 in rat. This study provides the first demonstration of glutathione-promoted radiodefluorination of a PET radioligand.

[METABOLISM, TRANSPORT, AND PHARMACOGENOMICS] Involvement of ST1B Subfamily of Cytosolic Sulfotransferase in Kynurenine Metabolism to Form Natriuretic Xanthurenic Acid Sulfate

Senggunprai, L., Yoshinari, K., Shimada, M., Yamazoe, Y. — 2008-11-12

Natriuretic substances are a group of molecules affecting sodium homeostasis in the body. Recently, two new molecules having natriuresis effects, xanthurenic acid 8-O-β-d-glucoside and xanthurenic acid 8-O-sulfate (XA sulfate), have been isolated from human urine. In the present study, we have investigated the sulfation of xanthurenic acid (XA) in mouse tissues to assess the contribution of specific sulfotransferases (STs) to the reaction. Cytosols from tissues of both sexes of C57BL/6N mice (liver, stomach, jejunum, colon, and kidney) were capable of forming XA sulfate, with various K_m values. Jejunum cytosol showed the lowest K_m value, and its V_max/K_m value was much greater than those of other tissues. The kinetic analyses with recombinant mouse (m) STs (Sult1a1, Sult1b1, Sult1c2, and Sult1d1) showed the lowest K_m value for mSult1b1, and the value was comparable with that for jejunum cytosol. The highest expression of mSult1b1 in small intestine was confirmed at the mRNA and protein levels. mSult1b1 is thus suggested as a major enzyme responsible for XA sulfation in jejunum. Similar to mSult1b1, human SULT1B1 and rat Sult1b1 mediated XA sulfation efficiently. Thus, XA is likely to be an endogenous substrate for ST1B members. In contrast to XA, an XA-related compound, kynurenic acid strongly inhibited mSult1b1-mediated sulfations, with IC₅₀ values at a micromolar range. These results indicate the functional role of ST1B subfamily of ST in XA sulfate formation in the body.

[METABOLISM, TRANSPORT, AND PHARMACOGENOMICS] Transport of Angiotensin-Converting Enzyme Inhibitors by H+/Peptide Transporters Revisited

2008-10-17

Angiotensin-converting enzyme (ACE) inhibitors are often regarded as substrates for the H⁺/peptide transporters (PEPT)1 and PEPT2. Even though the conclusions drawn from published data are quite inconsistent, in most review articles PEPT1 is claimed to mediate the intestinal absorption of ACE inhibitors and thus to determine their oral availability. We systematically investigated the interaction of a series of ACE inhibitors with PEPT1 and PEPT2. First, we studied the effect of 14 ACE inhibitors including new drugs on the uptake of the dipeptide [¹⁴C]glycylsarcosine into human intestinal Caco-2 cells constitutively expressing PEPT1 and rat renal SKPT cells expressing PEPT2. In a second approach, the interaction of ACE inhibitors with heterologously expressed human PEPT1 and PEPT2 was determined. In both assay systems, zofenopril and fosinopril were found to have very high affinity for binding to peptide transporters. Medium to low affinity for transporter interaction was found for benazepril, quinapril, trandolapril, spirapril, cilazapril, ramipril, moexipril, quinaprilat, and perindopril. For enalapril, lisinopril, and captopril, very weak affinity or lack of interaction was found. Transport currents of PEPT1 and PEPT2 expressed in Xenopus laevis oocytes were recorded by the two-electrode voltage-clamp technique. Statistically significant, but very low currents were only observed for lisinopril, enalapril, quinapril, and benazepril at PEPT1 and for spirapril at PEPT2. For the other ACE inhibitors, electrogenic transport activity was extremely low or not measurable at all. The present results suggest that peptide transporters do not control intestinal absorption and renal reabsorption of ACE inhibitors.

[METABOLISM, TRANSPORT, AND PHARMACOGENOMICS] Increasing Systemic Exposure of Methotrexate by Active Efflux Mediated by Multidrug Resistance-Associated Protein 3 (Mrp3/Abcc3)

Kitamura, Y., Hirouchi, M., Kusuhara, H., Schuetz, J. D., Sugiyama, Y. — 2008-10-17

The aim of this study was to investigate the functional importance of multidrug resistance-associated protein (Mrp)3/Abcc3 and Mrp4/Abcc4 in the pharmacokinetics of methotrexate. Compared with the corresponding wild-type mice, the plasma concentrations of methotrexate given orally were similar in Abcc4^-/- mice and were significantly lower in Abcc3^-/- mice. Pharmacokinetic parameters related to hepatobiliary transport were determined under steady-state conditions in wild-type and Abcc3^-/- mice that were given a constant intravenous infusion of methotrexate. The biliary clearance, based on the plasma concentration, was 1.6-fold greater in Abcc3^-/- mice than in wild-type mice (23 and 15 ml/min/kg, respectively, P < 0.05). Because the basolateral uptake and canalicular efflux clearances of methotrexate were similar in wild-type and Abcc3^-/- mice, this result suggests that the basolateral efflux clearance of methotrexate is decreased in the liver of Abcc3^-/- mice. Furthermore, a lower fraction of absorption of methotrexate (F_a F_g) was suggested in Abcc3^-/- mice (0.49 and 0.29 in wild-type and Abcc3^-^/^- mice, respectively). The mucosal-to-serosal transport rate of methotrexate, determined in vitro using everted sacs, was highest in the duodenum and was significantly decreased in Abcc3^-/- mice compared with wild-type mice. This is ascribed to the reduced intrinsic efflux clearance of methotrexate across the serosal membrane (22 and 5.3 µl/min/sac in wild-type and Abcc3^-^/^- mice, respectively, P < 0.05). These results suggest that Mrp3 mediates basolateral efflux of methotrexate in the liver and duodenum, thereby serving to increase systemic exposure, whereas Mrp4 is likely to play only a limited role in the systemic methotrexate exposure.

[METABOLISM, TRANSPORT, AND PHARMACOGENOMICS] A Novel Human Multidrug Resistance Gene MDR1 Variant G571A (G191R) Modulates Cancer Drug Resistance and Efflux Transport

Yang, Z., Wu, D., Bui, T., Ho, R. J. Y. — 2008-10-17

The human multidrug resistance gene MDR1 encodes a membrane-bound transporter P-glycoprotein (Pgp) that confers the drug resistance of cancer cells by mediating an ATP-dependent drug efflux transport. We and others have reported a number of functionally significant MDR1 variants, including G1199A and G1199T, that modulate cancer drug resistance and intracellular levels of antivirals. In this report, we describe a novel G571A variant of MDR1 detected in 6.4% of leukemia patients. Because this nucleotide modification gives rise to an amino acid change from Gly to Arg at the 191 amino acid position of Pgp, we have developed and characterized the functional affect of the G571A variant in stable, recombinant cells. Using six chemotherapeutic drugs, doxorubicin HCl, daunorubicin HCl, vinblastine sulfate, vincristine sulfate, taxanes (paclitaxel), and epipodophyllotoxin (etoposide, VP-16), we found that the MDR1_571A variant selectively reduced the degree of Pgp-mediated resistance in drug-dependent manner. Although there was a minimal effect on doxorubicin and daunorubicin, the MDR1-dependent resistance on vinblastine, vincristine, paclitaxel, and etoposide was reduced by approximately 5-fold. The increased drug sensitivity in MDR1_571A, compared with MDR1_wt, paralleled the intracellular drug levels. These data suggest that individuals with this novel MDR1 variant, the 571A genotype, may be more sensitive to the specific anticancer drugs that are Pgp substrates.

[METABOLISM, TRANSPORT, AND PHARMACOGENOMICS] The Inhibitory Effect of 2-Halo Derivatives of D-Glucose on Glycolysis and on the Proliferation of the Human Malaria Parasite Plasmodium falciparum

van Schalkwyk, D. A., Priebe, W., Saliba, K. J. — 2008-10-17

The intraerythrocytic stage of the human malaria parasite Plasmodium falciparum relies on glycolysis for ATP generation, and because it has no energy stores, a constant supply of glucose is necessary for the parasite to grow and multiply. The 2-substituted glucose analogs 2-deoxy-d-glucose (2-DG) and 2-fluoro-2-deoxy-d-glucose (2-FG) have been previously shown to inhibit the in vitro growth of P. falciparum and have been suggested to do so by inhibiting glycosylation in the parasite. In this study, we have investigated the antiplasmodial mechanism of action of 2-DG and 2-FG and compared it with that of other 2-substituted-glucose analogs. The compounds tested inhibited parasite growth to varying degrees, with 2-FG being the most effective. The antiplasmodial activity of some, but not all, of the analogs could be altered by varying the glucose concentration in the culture medium, increasing the antiplasmodial activity of the analogs as the glucose concentration is reduced. A trend was observed between the antiplasmodial activity of these analogs and their ability to inhibit glucose accumulation, glucose phosphorylation by hexokinase, and cytosolic pH regulation within the intraerythrocytic stage of the parasite. Our data are consistent with inhibition of glycolysis being a primary mechanism by which 2-DG and 2-FG inhibit parasite growth, and they validate the early steps in glycolysis as viable drug targets.

[METABOLISM, TRANSPORT, AND PHARMACOGENOMICS] The Endocannabinoid Anandamide Is a Substrate for the Human Polymorphic Cytochrome P450 2D6

2008-10-17

Members of the cytochrome P450 (P450) family of drug-metabolizing enzymes are present in the human brain, and they may have important roles in the oxidation of endogenous substrates. The polymorphic CYP2D6 is one of the major brain P450 isoforms and has been implicated in neurodegeneration, psychosis, schizophrenia, and personality traits. The objective of this study was to determine whether the endocannabinoid arachidonoylethanolamide (anandamide) is a substrate for CYP2D6. Anandamide is the endogenous ligand to the cannabinoid receptor CB1, which is also activated by the main psychoactive component in marijuana. Signaling via the CB1 receptor alters sensory and motor function, cognition, and emotion. Recombinant CYP2D6 converted anandamide to 20-hydroxyeicosatetraenoic acid ethanolamide and 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acid ethanolamides (EET-EAs) with low micromolar K_m values. CYP2D6 further metabolized the epoxides of anandamide to form novel dioxygenated derivatives. Human brain microsomal and mitochondrial preparations metabolized anandamide to form hydroxylated and epoxygenated products, respectively. An inhibitory antibody against CYP2D6 significantly decreased the mitochondrial formation of the EET-EAs. To our knowledge, anandamide and its epoxides are the first eicosanoid-like molecules to be identified as CYP2D6 substrates. Our study suggests that anandamide may be a physiological substrate for brain mitochondrial CYP2D6, implicating this polymorphic enzyme as a potential component of the endocannabinoid system in the brain. This study also offers support to the hypothesis that neuropsychiatric phenotype differences among individuals with genetic variations in CYP2D6 could be ascribable to interactions of this enzyme with endogenous substrates.

[METABOLISM, TRANSPORT, AND PHARMACOGENOMICS] Contribution of Down-Regulation of Intestinal and Hepatic Cytochrome P450 3A to Increased Absorption of Cyclosporine A in a Rat Nephrosis Model

2008-10-17

This study examined the contribution of changes in regulation of intestinal and hepatic cytochrome P450 3A (CYP3A) and multidrug resistance transporter 1 (Mdr1) to absorption of cyclosporine A (CsA) in a rat nephrosis model. Interleukin (IL)-6 was also measured. Puromycin aminonucleoside at a dose of 20 mg/100 g was administered intravenously. Tissue samples were dissected out from the upper and middle intestines and liver after development of nephrosis to measure the expression levels of mRNA and protein. CsA at a dose of 0.5 mg/100 g was administered into a closed loop of the upper and middle intestines. Blood from the inferior vena cava (IVC) and portal vein was taken until 30 min after administration. The expression levels of CYP3A decreased markedly, whereas those of Mdr1 showed large interindividual variations for all of the tissues in the nephrotic rats. Plasma concentrations of CsA reached higher levels in the nephrotic than in the control rats and were higher when administered from the upper than the middle intestine in both the portal vein and IVC. IL-6 increased in urine in the nephrotic rats. In summary, intestinal and hepatic CYP3A were down-regulated in the nephrosis model accompanying the increased levels of IL-6. Consistent results were not obtained for the regulation of Mdr1. In conclusion, these findings suggest that the down-regulation of CYP3A in the upper intestine and liver predominantly contributes to the increase in CsA absorption, and Mdr1 showed less contribution in this rat nephrosis model.

[METABOLISM, TRANSPORT, AND PHARMACOGENOMICS] Targeting of VX2 Rabbit Liver Tumor by Selective Delivery of 3-Bromopyruvate: A Biodistribution and Survival Study

2008-09-19

The aim of this study was to determine the biodistribution and tumor targeting ability of ¹⁴C-labeled 3-bromopyruvate ([¹⁴C]3-BrPA) after i.a. and i.v. delivery in the VX2 rabbit model. In addition, we evaluated the effects of [¹⁴C]3-BrPA on tumor and healthy tissue glucose metabolism by determining ¹⁸F-deoxyglucose (FDG) uptake. Last, we determined the survival benefit of i.a. administered 3-BrPA. In total, 60 rabbits with VX2 liver tumor received either 1.75 mM [¹⁴C]3-BrPA i.a., 1.75 mM [¹⁴C]3-BrPA i.v., 20 mM [¹⁴C]3-BrPA i.v., or 25 ml of phosphate-buffered saline (PBS). All rabbits (with the exception of the 20 mM i.v. group) received FDG 1 h before sacrifice. Next, we compared survival of animals treated with i.a. administered 1.75 mM [¹⁴C]3-BrPA in 25 ml of PBS (n = 22) with controls (n = 10). After i.a. infusion, tumor uptake of [¹⁴C]3-BrPA was 1.8 ± 0.2% percentage of injected dose per gram of tissue (%ID/g), whereas other tissues showed minimal uptake. After i.v. infusion (1.75 mM), tumor uptake of [¹⁴C]3-BrPA was 0.03 ± 0.01% ID/g. After i.a. administration of [¹⁴C]3-BrPA, tumor uptake of FDG was 26 times lower than in controls. After i.v. administration of [¹⁴C]3-BrPA, there was no significant difference in tumor FDG uptake. Survival analysis showed that rabbits treated with 1.75 mM 3-BrPA survived longer (55 days) than controls (18.6 days). Intra-arterially delivered 3-BrPA has a favorable biodistribution profile, combining a high tumor uptake resulting in blockage of FDG uptake with no effects on healthy tissue. The local control of the liver tumor by 3-BrPA resulted in a significant survival benefit.

[METABOLISM, TRANSPORT, AND PHARMACOGENOMICS] Nonlinear Pharmacokinetics of ({+/-})3,4-Methylenedioxymethamphetamine (MDMA, "Ecstasy") and Its Major Metabolites in Squirrel Monkeys at Plasma Concentrations of MDMA That Develop After Typical Psychoactive Doses

Mueller, M., Peters, F. T., Maurer, H. H., McCann, U. D., Ricaurte, G. A. — 2008-09-19

At certain doses, the psychoactive drug (±)3,4-methylenedioxymethamphetamine (MDMA, "Ecstasy") destroys brain serotonin axon terminals. By causing increases in plasma MDMA concentrations that exceed those predicted by the increase in dose, nonlinear pharmacokinetics has the potential to narrow the range between safe and neurotoxic doses of MDMA. The present study sought to determine whether the pharmacokinetics of MDMA in nonhuman primates are nonlinear and, if they are, to identify plasma concentrations of MDMA at which nonlinear accumulation of MDMA occurs. Four different oral doses of MDMA were tested in the same six squirrel monkeys in random order. At each dose, pharmacokinetic parameters for MDMA and its metabolites 3,4-dihydroxymethamphetamine (HHMA), 4-hydroxy-3-methoxymethamphetamine (HMMA), and 3,4-methylenedioxyamphetamine were determined. Doses were selected to be equivalent to 0.4, 0.8, 1.6, and 2.8 mg/kg doses in humans. The maximal concentration (C_max) and area under the curve (AUC) of MDMA increased nonlinearly with dose, whereas the C_max and AUC of the metabolites HHMA and HMMA remained relatively constant. Nonlinear MDMA pharmacokinetics occurred at plasma MDMA concentrations of 100 to 300 ng/ml and above. The half-life (T_1/2) of MDMA and its metabolites also increased with dose. These results firmly establish nonlinear pharmacokinetics for MDMA in squirrel monkeys and indicate that nonlinear MDMA accumulation occurs at plasma MDMA concentrations that develop in humans taking typical doses. By raising MDMA concentrations and prolonging its action, nonlinear pharmacokinetics and T_1/2 prolongation, respectively, may influence the likelihood and severity of MDMA toxicities (including brain serotonin neurotoxicity).

[METABOLISM, TRANSPORT, AND PHARMACOGENOMICS] Apparent High CYP3A5 Expression Is Required for Significant Metabolism of Vincristine by Human Cryopreserved Hepatocytes

Dennison, J. B., Mohutsky, M. A., Barbuch, R. J., Wrighton, S. A., Hall, S. D. — 2008-09-19

Vincristine is metabolized to one primary metabolite, M1, by cDNA-expressed CYP3A4 and CYP3A5 and by CYP3A enzymes in human liver microsomes. For both systems, CYP3A5 is predicted to mediate approximately 80% of the CYP3A metabolism for individuals with high CYP3A5 expression (at least one CYP3A5^*1 allele). In the current study, the role of CYP3A5 was quantified in the metabolism of vincristine with human cryopreserved hepatocytes. The hepatocytes were genotyped for common CYP3A5 allelic variants (CYP3A5^*3, CYP3A5^*6, and CYP3A5^*7) to predict CYP3A5 expression. For each hepatocyte preparation, the rates of vincristine depletion and metabolite formation were quantified. Whereas human hepatocytes with predicted low CYP3A5 expression did not detectably metabolize vincristine, human hepatocytes with predicted high CYP3A5 expression metabolized vincristine to one primary metabolite, M1. In paired experiments using cryopreserved hepatocytes from the same donor, vincristine was incubated with intact cells and cell lysates supplemented with NADPH. The rates of M1 formation were 4 to 69-fold higher for the cell lysates compared with the intact cells. For one representative donor, the intact cells had a 3-fold higher K_m value and a 3-fold lower V_max value for M1 formation compared with the cell lysates. Thus, the rate of M1 formation in the hepatocytes may be influenced by the rate of vincristine translocation across the plasma membrane. We conclude that genetically determined CYP3A5 expression in human cryopreserved hepatocytes plays a major role in vincristine metabolism.