Abstract
Cytochrome P450 3A4 is the predominant isoform in liver, and it metabolizes more than 50% of the clinical drugs commonly used. However, CYP3A4 is also responsible for metabolic activation of drugs, leading to liver injury. Benzodiazepines are widely used as hypnotics and sedatives for anxiety, but some of them induce liver injury in humans. To clarify whether benzodiazepines are metabolically activated, 14 benzodiazepines were investigated for their cytotoxic effects on HepG2 cells treated with recombinant CYP3A4. By exposure to 100 μM flunitrazepam, nimetazepam, or nitrazepam, the cell viability in the presence of CYP3A4 decreased more than 25% compared with that of the control. In contrast, in the case of other benzodiazepines, the changes in the cell viability between CYP3A4 and control Supersomes were less than 10%. These results suggested that nitrobenzodiazepines such as flunitrazepam, nimetazepam, and nitrazepam were metabolically activated by CYP3A4, which resulted in cytotoxicity. To identify the reactive metabolite, the glutathione adducts of flunitrazepam and nimetazepam were investigated by liquid chromatography-tandem mass spectrometry. The structural analysis for the glutathione adducts of flunitrazepam indicated that a nitrogen atom in the side chain of flunitrazepam was conjugated with the thiol of glutathione. Therefore, the presence of a nitro group in the side chain of benzodiazepines may play a crucial role in the metabolic activation by CYP3A4. The present study suggested that metabolic activation by CYP3A4 was one of the mechanisms of liver injury by nitrobenzodiazepines.
Drug-induced hepatotoxicity is one of the major causes of liver injury and is classified into intrinsic and idiosyncratic types. Intrinsic drug reactions can occur in a dose-dependent manner in any individual and are reproducible in preclinical studies. In contrast, idiosyncratic drug reactions do not occur in most patients at any dose, and they are often referred to as rare, with a typical incidence of from 1/100 to 1/100,000 (Uetrecht, 1999). Because idiosyncratic drug reactions are difficult to spot during drug development, some drugs launched on the market were later withdrawn because of idiosyncratic hepatotoxicity. Such drugs withdrawn for hepatotoxicity are known to produce reactive metabolites (Guengerich and MacDonald, 2007). The generation of reactive metabolites may relate to the formation of free radicals, oxidation of thiol, and covalent binding with endogenous macromolecules, resulting in the oxidation of cellular components or inhibition of normal cellular function (Guengerich and Liebler, 1985).
The generation of a reactive metabolite catalyzed by drug-metabolizing enzymes such as cytochrome P450 (P450) is defined as metabolic activation. P450 is the major drug-metabolizing enzyme that is highly expressed in human liver. CYP3A4 is the predominant isoform in liver (Shimada et al., 1994) and metabolizes more than 50% of the clinical drugs commonly used (Guengerich, 1995). However, CYP3A4 is also responsible for the formation of reactive metabolites of flutamide (Berson et al., 1993), trazodone (Kalgutkar et al., 2005), and troglitazone (Yamamoto et al., 2002). It is suggested that the reactive metabolites of flutamide, trazodone, and troglitazone cause the idiosyncratic hepatotoxicity in humans.
Prediction of the metabolic activation and the cytotoxicity of drug candidates is necessary in drug development. Human hepatocarcinoma HepG2 cells are commonly used for predicting hepatotoxicity in vitro. However, low expression levels of P450s in HepG2 cells may be responsible for the fact that 30% of the compounds were falsely classified as nontoxic (Rodriguez-Antona et al., 2002; Wilkening et al., 2003; Hewitt and Hewitt, 2004). In a recent study, a useful in vitro cell-based assay made by combining recombinant CYP3A4 with HepG2 cells was established (Vignati et al., 2005). It was demonstrated that hepatotoxicants whose reactive metabolites were generated by CYP3A4 exhibited cytotoxicity to the HepG2 cells. This assay system could be applied to screen for hepatotoxicity by drugs.
Benzodiazepines have been used extensively as hypnotics and sedatives for anxiety throughout the world. The mechanism of their efficacy is to amplify the action of γ-aminobutyric acid by acting as agonists at γ-aminobutyric acid receptors (Costa et al., 2002). Many benzodiazepines have been launched on the market and used in clinical practice. Two of the major benzodiazepines, flunitrazepam and nitrazepam, are widely used as hypnotic and anesthetic premedications in Europe and Japan. In 2001, it was announced by the Ministry of Health, Labor and Welfare of Japan that flunitrazepam induced hepatotoxicity. Chronic administration of antidepressant drugs including nitrazepam was reported to induce severe hepatic disorders (Seki et al., 2008). Clonazepam is one of the benzodiazepines used as an anxiolytic and anticonvulsant in clinical practice. Hepatic injury was reported to occur after treatment with clonazepam for 6 weeks in Ethiopia (Olsson and Zettergren, 1988).
The purpose of the present study was to clarify whether the metabolic activation of benzodiazepines by P450 occurs, leading to the hepatotoxicity. We investigated the cell viability in HepG2 cells in the presence or absence of CYP3A4 after exposure to 14 commercially available benzodiazepines (Fig. 1). There are many structural analogs of benzodiazepines, and the chemical structures and cytotoxicity in HepG2 cells were compared.
Materials and Methods
Materials. Clonazepam, clobazam, diazepam, lorazepam, nimetazepam, nitrazepam, and oxazepam were obtained from Wako Pure Chemicals (Osaka, Japan). Bromazepam, chlordiazepoxide, desmethyldiazepam, flunitrazepam, flurazepam, norfludiazepam, and temazepam were purchased from Sigma-Aldrich (St. Louis, MO). Human CYP2C9, 2C19, and 3A4 Supersomes (recombinant cDNA-expressed P450 enzymes prepared from a baculovirus insect cell system) and control Supersomes were purchased from BD Gentest (Woburn, MA). These microsomes coexpressed NADPH-cytochrome P450 reductase and cytochrome b5. All other reagents used in this study were of the highest or analytical grade commercially available.
Cell Culture. Human hepatocarcinoma cell line HepG2 was obtained from Riken Gene Bank (Tsukuba, Japan). The cells were cultured in Dulbecco's modified Eagle's medium (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% fetal bovine serum (Invitrogen, Melbourne, Australia) and 0.1 mM nonessential amino acids (Invitrogen) at 37°C in an atmosphere of 5% CO2 and 95% air.
Cell Viability Assay. HepG2 cells were seeded at a density of 1 × 104 cells/well in 96-well plates with medium containing 3% fetal bovine serum, benzodiazepines, 8 nM human CYP2C9, CYP2C19, CYP3A4, or control Supersomes and 1 mM NADPH and then incubated at 37°C for 24 h. In the preliminary study, we investigated the cell viability in HepG2 cells with various P450 concentrations and incubation time. The 8 nM P450 and 24-h incubation were enough to detect cytotoxicity in this assay system. The final concentration of organic solvent (dimethyl sulfoxide) in medium was less than 0.2%. Cell viability after a 24-h incubation was evaluated by the intracellular ATP concentration using a CellTiter-Glo Luminescent Cell Viability Assay (ATP assay; Promega, Madison, WI) and 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) activities using a CellTiter-Blue Cell Viability Assay (MTT assay; Promega). According to the protocols of the manufacturer, the luminescence of the generated oxyluciferin was measured in the ATP assay and the fluorescence of the generated resorufin was detected fluorometrically (excitation: 338 nm, emission: 458 nm) in the MTT assay by using a 1420 ARVO MX luminometer (PerkinElmer Wallac, Turku, Finland).
Caspase Assay. HepG2 cells were seeded under the same conditions and incubated at 37°C for 24 h. After incubation, the caspase 3/7 activity was measured using a Caspase-Glo 3/7 Assay (Promega) according to the protocol of the manufacturer. The luminescence of the generated aminoluciferin was measured using a 1420 ARVO MX luminometer.
Detection of Glutathione Adducts. A typical reaction mixture (final volume of 0.25 ml) contained 50 nM human CYP3A4 Supersomes, 100 mM potassium phosphate buffer (pH 7.4), an NADPH-generating system consisting of 0.775 mM nicotinamide adenine dinucleotide phosphate (oxidized form), 0.165 mM glucose 6-phosphate, 0.165 mM MgCl2, 0.2 unit/ml glucose-6-phosphate dehydrogenase, 10 mM glutathione (reduced form), and 100 μM benzodiazepines (flunitrazepam, nimetazepam, nitrazepam, bromazepam, or temazepam). The final concentration of dimethyl sulfoxide in the reaction mixture was less than 1%. Incubation was performed at 37°C for 60 min and terminated by adding 0.75 ml of ice-cold methanol. After centrifugation at 15,000g, the supernatant was subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) (API 4000; Applied Biosystems, Foster City, CA). An LC-10 liquid chromatograph (Shimadzu, Kyoto, Japan) was used with an Inertsil ODS-3 analytical column (2.1 × 100 mm, 3 μm; GL Science, Tokyo, Japan). The column temperature was 40°C. The mobile phase was 10 mM ammonium acetate buffer (pH 4.0) (A) and acetonitrile (B). The conditions for elution were as follows: 5 to 90% B (0–6 min), 90% B (6–11 min), 90 to 5% B (11–11.01 min), and 5% B (11.01–15 min). Linear gradients were used for all solvent changes. The flow rate was 0.2 ml/min. The liquid chromatograph was connected to an API 4000 mass spectrometer operated in the negative electrospray ionization mode. The turbo gas was maintained at 450°C. Air was used as the nebulizing and turbo gas at 60 psi. Nitrogen was used as the curtain gas at 20 psi. The collision energy was –50 V. The m/z 300 to 850 was scanned at the precursor ion (m/z 272; major mass spectrum fragment of glutathione).
Identification of Glutathione Adducts. Liquid chromatography ion trap and time-of-flight mass spectrometry (LCMS-IT-TOF) (Shimadzu) was used to identify the structures of the glutathione adducts of the nitrobenzodiazepines. The incubation mixture was the same as that described above except for CYP3A4 Supersomes (100 nM). Flunitrazepam and nimetazepam were used as test compounds. After centrifugation at 15,000g for 5 min, the supernatant was subjected to LCMS-IT-TOF using an Inertsil ODS-3 analytical column (2.1 × 100 mm, 3 μm). The LC conditions were the same as described earlier. The turbo gas was maintained at 450°C. Air was used as the nebulizing and turbo gas at 60 psi. Nitrogen was used as the curtain gas at 20 psi. The collision energy was 50 V. Structure analysis of the glutathione adducts of flunitrazepam and nimetazepam was performed by scanning at the product ion (m/z 621 and m/z 603, respectively) in the positive electrospray ionization mode.
Statistics. Data are expressed as mean ± S.D. (n = 3). Two groups were compared with a two-tailed Student's t test. P < 0.05 was considered statistically significant.
Results
Cell Viability of HepG2 Cells Treated with CYP3A4 and Benzodiazepines. HepG2 cells were incubated for 24 h with the 14 benzodiazepines at 50, 100, 200, and 400 μM in the presence of CYP3A4 or control Supersomes and then the cell viability was measured by the ATP and MTT assays. With exposure to 100 μM flunitrazepam, nimetazepam, and nitrazepam, cell viability in the presence of CYP3A4 Supersomes decreased more than 25% than with control Supersomes (Fig. 2). Although clonazepam could be dissolved up to 100 μM in the reaction mixtures, the viability of HepG2 cells treated with CYP3A4 Supersomes and 100 μM clonazepam exhibited 57 and 35% decreases in the ATP and MTT assays, respectively, compared with viability with control Supersomes (Supplemental Fig. 2). Flunitrazepam, nimetazepam, nitrazepam, and clonazepam are nitrobenzodiazepines that have a nitro group at the 7-position (Fig. 1). In contrast, for the other 10 benzodiazepines (bromazepam, chlordiazepoxide, clobazam, desmethyldiazepam, diazepam, flurazepam, lorazepam, norfludiazepam, oxazepam, and temazepam) at 100 μM, the changes in cell viability between CYP3A4 and control Supersomes were less than 10% and much smaller than those for the nitrobenzodiazepines (Fig. 2 and Supplemental Fig. 1). Moreover, 25% effective concentrations (EC25) of nitrobenzodiazepines were less than 100 μM, and EC25 values of all other benzodiazepines were more than 300 μM in the ATP assay (Supplemental Table 1). Desmethyldiazepam, diazepam, flurazepam, lorazepam, norfludiazepam, and oxazepam exhibited concentration-dependent cytotoxicity in HepG2 cells incubated both with and without CYP3A4 (Fig. 2 and Supplemental Fig. 1).
Cell Viability on HepG2 Cells Treated with CYP2Cs and Nitrobenzodiazepines. It has been reported that CYP2C9 and CYP2C19 are involved in the metabolism of flunitrazepam (Hesse et al., 2001; Kilicarslan et al., 2001). Therefore, we investigated whether CYP2C9 and CYP2C19 affect the cytotoxicity caused by nitrobenzodiazepines in HepG2 cells. As shown in Fig. 3, the differences in the cell viability between CYP2Cs and control Supersomes when exposed to 100 μM nitrobenzodiazepines were less than 10%.
Caspase 3/7 Activity in HepG2 Cells Treated with CYP3A4 and Nitrobenzodiazepines. As a key factor of apoptosis, the caspase 3/7 activity was measured in HepG2 cells treated with CYP3A4 and the nitrobenzodiazepines for 24 h. Flunitrazepam, nimetazepam, and nitrazepam significantly increased the caspase 3/7 activities in HepG2 cells in the presence of CYP3A4 Supersomes (Fig. 4, A–C). In contrast, bromazepam as the negative control had no effects on the caspase 3/7 activities both with and without CYP3A4 (Fig. 4D).
Detection of Glutathione Adducts of Benzodiazepines. The glutathione adducts of benzodiazepines were investigated by the negative ion mode of LC-MS/MS. The nitrobenzodiazepines (flunitrazepam, nimetazepam, and nitrazepam) and the negative controls (bromazepam and temazepam) were measured. As shown in Fig. 5, the glutathione adducts of flunitrazepam and nimetazepam were detected in the presence of CYP3A4 Supersomes by precursor ion scans at m/z 619 and m/z 601 ([M – H]–), respectively. In contrast, there were no adducts of flunitrazepam and nimetazepam when they were used in the control Supersomes (data not shown). In nitrazepam, bromazepam, and temazepam, glutathione adducts were not detected in the presence and absence of CYP3A4.
Identification of Glutathione Adducts of Flunitrazepam and Nimetazepam. The structures of the glutathione adducts of flunitrazepam and nimetazepam were estimated by the positive ion mode of LCMS-IT-TOF. For of the glutathione adduct of flunitrazepam, the product ion mass spectrum of m/z 621 ([M + H]+) gave fragment ions at m/z 284.1, m/z 348.1, and m/z 492.1. The molecule weight of the [M + H]+ fragment ion (m/z 492.1) meant that it was produced by the molecule weight of the compound (491) and that of a hydrogen ion (H+; 1). The possible structure of the glutathione adduct of flunitrazepam is shown in Fig. 6. A reactive metabolite of flunitrazepam, in which the nitro group might be metabolized into the amino group, was conjugated to the 7-substituent group by glutathione.
On the other hand, the [M + H]+ ion of the glutathione adduct of nimetazepam (m/z 603) gave fragment ions at m/z 266.4 and m/z 474.2 (Supplemental Fig. 3). The fragment ions at m/z 266.4 and m/z 474.2 were [M + H – 337]+ and [M + H – 129]+, respectively, corresponding to the fragment ions at m/z 284.1 and m/z 492.1 obtained from the glutathione adduct of flunitrazepam (m/z 621).
Discussion
In the present study, 14 benzodiazepine analogs were investigated for cytotoxic effects resulting from metabolic activation by CYP3A4. The major metabolic pathways of diazepam are 3-hydroxylayion by CYP3A4 and N-desmethylation by CYP2C9 (Schwartz et al., 1965; Ono et al., 1996). Thus, desmethyldiazepam, temazepam, and oxazepam are metabolites of diazepam (Fig. 1). In addition, norfludiazepam and nitrazepam would be the metabolites of flurazepam and nimetazepam, respectively.
The cytotoxicity of flunitrazepam, nimetazepam, nitrazepam, and clonazepam was observed in the presence of CYP3A4 Supersomes in HepG2 cells (Fig. 2 and Supplemental Fig. 1), suggesting that these three drugs are metabolically activated by CYP3A4. Flunitrazepam, nimetazepam, nitrazepam, and clonazepam are classified as nitrobenzodiazepines that have a nitro group in the side chain. In contrast, the other 10 benzodiazepines exhibited less cytotoxicity than the nitrobenzodiazepines (Fig. 2 and Supplemental Fig. 1; Supplemental Table 1). In the present study, we first clarified that the presence of a nitro group in the side chain of benzodiazepines may play a crucial rule in the metabolic activation by CYP3A4. To prevent the cytotoxicity by reactive metabolites in the medium, the effects of 200 μM or 1 mM glutathione (reduced form) were measured in this cell viability assay as a preliminary experiment. The glutathione recovered 10% of cell viability in HepG2 cells treated with CYP3A4 and 100 μM flunitrazepam (data not shown). This finding suggested that the reactive metabolites of nitrobenzodiazepines may bind to glutathione. However, glutathione did not completely protect against the cytotoxicity; thus, there may be another cytotoxic effect that could not be detoxified by glutathione trapping.
In humans, the major metabolites of flunitrazepam are N-desmethylflunitrazepam in plasma and 3-hydroxyflunitrazepam and 7-aminoflunitrazepam in urine (Fukazawa et al., 1978). CYP3A4 is the major P450 involved in flunitrazepam 3-hydroxylation and N-desmethylation, but CYP2C9 and CYP2C19 also catalyze the N-desmethylation of flunitrazepam (Hesse et al., 2001; Kilicarslan et al., 2001). The reductive metabolite of flunitrazepam, 7-aminoflunitrazepam, is catalyzed by NADPH-cytochrome P450 reductase in HepG2 cells (Peng et al., 2004). Nimetazepam is metabolized to N-desmethylnimetazepam and 3-hydroxylnimetazepam (Dainippon Sumitomo Pharma, unpublished data). Nitrazepam is metabolized to 7-aminonitrazepam and 3-hydroxynitrazepam (Rieder, 1965). Although which P450 isoform mediates nimetazepam and nitrazepam metabolism has not been revealed, nimetazepam and nitrazepam may be metabolized by CYP2C9, CYP2C19, and CYP3A4. In our study, when HepG2 cells were exposed to 100 μM nitrobenzodiazepines, the differences in the cell viability between CYP2Cs and control Supersomes were less than 10% (Fig. 3), indicating that the contribution of CYP2Cs to the cytotoxicity of nitrobenzodiazepines was much lower than that of CYP3A4. Therefore, the metabolic activations of the nitrobenzodiazepines were CYP3A4-specific reactions.
Caspase 3 and 7 are classified as effector caspases. Active effector caspases mediate the cleavage of an overlapping set of protein substrates, resulting in the morphological features of apoptosis and the demise of the cell (Nuñez et al., 1998). Flunitrazepam, nimetazepam, and nitrazepam significantly increased the caspase 3/7 activities in HepG2 cells in the presence of CYP3A4 Supersomes (Fig. 4). Therefore, apoptosis after caspase 3 and 7 activation is one of the cytotoxic pathways of the reactive metabolites of flunitrazepam, nimetazepam, and nitrazepam.
The maximum plasma concentrations of nitrobenzodiazepines after a single administration in humans have been reported as follows: 0.04 μM after an oral dose of 2 mg of flunitrazepam (Fukazawa et al., 1978), 0.05 μM after an oral dose of 5 mg of nimetazepam (Dainippon Sumitomo Pharma, unpublished data), 0.3 μM after an oral dose of 10 mg of nitrazepam (Rieder, 1973), and 0.05 μM after an oral dose of 2 mg of clonazepam (Cavedal et al., 2007). Flunitrazepam was reported to induce hepatotoxicity by the Ministry of Health, Labor and Welfare of Japan, and nitrazepam and clonazepam were reported to cause drug-induced liver injury (Olsson and Zettergren, 1988; Seki et al., 2008). Although it is very difficult to extrapolate from an in vitro study to in vivo in humans, we may pay attention to the metabolic activation of nitrobenzodiazepines by CYP3A4.
The metabolism of a nontoxic drug to reactive metabolites is thought to initiate a variety of adverse reactions (Park, 1986; Parke, 1987). Glutathione is an important intracellular peptide that can detoxify reactive metabolites by conjugation (Lu, 1999). Previous studies reported that reactive metabolites of flutamide (Kang et al., 2007), trazodone (Kalgutkar et al., 2005), and troglitazone (Kassahun et al., 2001) formed by CYP3A4 were detoxified by glutathione conjugations. As shown in Fig. 5, glutathione adducts of flunitrazepam and nimetazepam were detected by LC-MS/MS, suggesting the production of reactive metabolites of flunitrazepam and nimetazepam catalyzed by CYP3A4. In the present study, the structure of the glutathione adduct of flunitrazepam was estimated by LCMS-IT-TOF as shown in Fig. 6. It seemed that a nitrogen atom in the side chain of flunitrazepam was conjugated with the thiol of glutathione. The structure of the glutathione adduct of nimetazepam may be similar to that of flunitrazepam because the fragment ions, [M + H – 337]+ and [M + H – 129]+, corresponded to those of flunitrazepam (Supplemental Fig. 3). The glutathione adducts of nitrazepam could not be detected either with or without CYP3A4 in our detection system. However, the cytotoxicity of nitrazepam to HepG2 cells treated with CYP3A4 Supersomes (Fig. 2) suggested that metabolic activation might occur. One of the reasons for this discrepancy may be the sensitivity of the detection.
Nitroaromatic drugs such as flutamide, nimesulide, and tolcapone have been associated with idiosyncratic liver injury (Boelsterli et al., 2006). In the reductive pathways from nitro to the fully reduced amine catalyzed by P450 and/or reductase, several reactive metabolites including nitroso and N-hydroxylamine derivatives could be produced. Such reactive metabolites seem to bind covalently to nucleophilic targets of proteins and nucleic acids, leading to the cytotoxic effects (Biaglow et al., 1986; Rickert, 1987; Kedderis and Miwa, 1988; Kedderis et al., 1989). On the other hand, arylamines are metabolically activated by P450-mediated N-hydroxylation. Electrophilic N-hydroxylamine reacts with intracellular molecules, which induce various types of toxicity including hepatotoxicity (Kato and Yamazoe, 1994). Flutamide induced severe hepatic dysfunction. Ohbuchi et al. (2008) suggested that CYP3A4 catalyzed the N-oxidation of the amino metabolite of flutamide, which had hepatotoxic effects. Although the bioactivation pathways of nitrobenzodiazepines still remain unclear, they may undergo metabolic activation similar to that of other drugs. Further study is needed to clarify the mechanism of metabolic activation concerning nitrobenzodiazepines.
In conclusion, we revealed that nitrobenzodiazepines, such as flunitrazepam, nimetazepam, and nitrazepam, were metabolically activated by CYP3A4, resulting in cytotoxicity in HepG2 cells. The CYP3A4 metabolites of flunitrazepam and nimetazepam were conjugated with glutathione at a nitrogen atom in the side chain. This finding suggested that metabolic activation by CYP3A4 may be one of the mechanisms in liver injury. Moreover, we established a simple assay system in which the cytotoxicity in HepG2 cells incubated with recombinant P450s and the drug was observed with high sensitivity. This assay system was useful for detecting metabolic activation by P450s and would be beneficial for predicting drug-induced cytotoxicity in preclinical drug development.
Acknowledgments
We acknowledge Brent Bell for reviewing the article.
Footnotes
-
This study was supported by Young Scientists of the Ministry of Education, Culture, Sports, Science and Technology of Japan [Grant 19790120]; and Ministry of Health, Labor, and Welfare, Health and Labor Science Research [Grant H20-BI-001].
-
K.M. and M.K. contributed equally to this work.
-
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
-
doi:10.1124/dmd.108.024521.
-
ABBREVIATIONS: P450, cytochrome P450; MTT, 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; LC, liquid chromatography; MS/MS, tandem mass spectrometry; MS, mass spectrometry; LCMS-IT-TOF, liquid chromatography ion trap and time-of-flight mass spectrometry.
-
↵ The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material.
-
↵1 Current affiliation: Faculty of Pharmacy, Meijo University, Tempaku-ku, Nagoya, Japan.
- Received September 15, 2008.
- Accepted November 10, 2008.
- The American Society for Pharmacology and Experimental Therapeutics